(Advances in Experimental Medicine and Biology 746) Tomotoshi Marumoto, Hideyuki Saya (Auth.), Ryuya Yamanaka MD, PHD (Eds.) - Glioma - Immunotherapeutic Approaches-Springer-Verlag New York (2012)

Glioma
ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY
Editorial Board:
NATHAN BACK, State University of New York at Buffalo
IRUN R. COHEN, The Weizmann Institute of Science
ABEL LAJTHA, N.S. Kline Institute for Psychiatric Research
JOHN D. LAMBRIS, University of Pennsylvania
RODOLFO PAOLETTI, University of Milan
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Glioma
Immunotherapeutic Approaches
Edited by
Ryuya Yamanaka, MD, PhD

Kyoto Prefectural University of Medicine, Kyoto, Japan
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Glioma : immunotherapeutic approaches / edited by Ryuya Yamanaka.

p. ; cm. -- (Advances in experimental medicine and biology ; v. 746)
Includes bibliographical references.
ISBN 978-1-4614-3145-9
I. Yamanaka, Ryuya. II. Series: Advances in experimental medicine and biology ; v. 746. 0065-2598
[DNLM: 1. Glioma--therapy. 2. Glioma--immunology. 3. Immunotherapy--methods. W1 AD559 v.746
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615.37--dc23
2011052010
PREFACE
Treatment of glioma is currently one of the most challenging problems in oncology,

as well as in neurosurgery. Despite major advances in our understanding of the
pathomechanism, diagnosis by imaging and the availability of powerful therapeutic tools,
the life expectancy of patients with glioblastoma has only been slightly prolonged and a
cure remains elusive. None of the currently available surgical tools, including operative
microscopes, lasers and image-guided surgery, can enable the detection and removal
of all of the tumor tissue. In recent years, however, the landscape has been changing
immeasurably, and molecular studies over the past two decades have identified a variety
of genetic aberrations that are specifically associated with individual types of gliomas. In
addition, certain molecular abnormalities have been linked to therapy responses, thereby
establishing clinical biomarkers and molecular targets, and the use of novel agents is
being investigated. These agents have been specifically engineered to exert specific
cytotoxicity against gliomas, either on their own as single agents or in combination with
other modalities. Moreover, there has been an enormous surge of interest in the area of
immunology and immunotherapy, which has been facilitated by our understanding of the
molecular basis of gliomas. Although several kinds of immunotherapeutic trials have been
undertaken, we still await a great breakthrough in terms of clinical efficacy to prolong
the survival time of glioma patients.
At present, there is no comprehensive overview of the potential and challenges of
immunotherapeutic approaches in glioma research as well as the clinical management
of glioma patients. Since progress in this field has been astonishing over the last decade,
we felt that a comprehensive volume presenting the advances in immunotherapy for
glioma was timely. In this work, we will discuss in depth the potential of these innovative
methods in the broad field of glioma immunology and immunotherapy in particular.
We will also attempt to present a book that will appeal to clinicians, clinical scientists
and basic scientists. The chapters will review the basic and clinical background, and
proceed from basic science at the bench to the bedside to give a realistic evaluation of
the immunology and immunotherapy of glioma. Internationally distinguished experts
working in basic science and clinical neurooncology have contributed comprehensive
chapters to this volume covering the major topics of the current research in the field.
v
vi PREFACE
The opening part is focused on recent progress in the neurooncology of gliomas. In

the first chapter, H. Saya provides an update on the molecular biology of gliomas. The
following chapters by M.S. Berger and S.M. Chang address recent surgical and medical
management strategies for gliomas, respectively. The second part of the book is focused on
glioma immunology. Basic concepts, immune evasion by gliomas and glioma antigens are
the topics covered by I.F. Parney, M.S. Lesniak and M. Toda, respectively. The following
part focuses on serotherapy, adoptive transfer and other strategies. A critical review of
the role of cytokine therapy is contributed by T. Wakabayashi, while A.F. Carpentier
addresses the use of CpG oligos. The chapter by K. Tsuboi discusses adoptive cell therapy,
while D.A. Mitchell reports on the current status of the clinical significance of antibody
therapy for gliomas. The fourth part of the book is focused on active immunotherapy for
gliomas. The initial chapter by Y.K. Hong discusses several problems associated with
animal models for glioma immunotherapy. Active immunotherapy approaches such as
immunogene therapy, peptide therapy, viral therapy and dendritic cell vaccine are the
topics covered by T. Lichtor, S. Izumoto, T. Todo and R. Yamanaka, respectively. The
final part of this work is specifically devoted to novel topics in glioma immunotherapy.
H. Syuku focuses on antigen-receptor gene-modified T-cell therapy, while the concept
of glioma stem cells, which may have promising future implications for targeted glioma
treatments and overcoming resistance to therapy, is discussed by J.S. Yu.
Ryuya Yamanaka, MD, PhD

ABOUT THE EDITOR...
RYUYA YAMANAKA is a Professor at Kyoto Prefectural University of Medicine,

Kyoto, Japan. He earned his MD at Niigata University, Japan, in 1982 and completed a
neurosurgical residency training at its affiliated hospitals. He received his academic degree
(Dr Med Sci) from Niigata University. Following research fellowships at the National
Institutes of Health in the United States from 1994 to 1998, he assumed the position of
Assistant Professor and Lecturer at the Department of Neurosurgery in the Brain Research
Institute at Niigata University. In 2006, he was promoted to Professor of the Research
Center of Innovative Cancer Therapy at Kurume University School of Medicine. He
joined the faculty at Kyoto Prefectural University of Medicine in 2010. His main research
interests include translational biochemical research in clinical oncology, including brain
tumors. Dr. Yamanaka is a member of international and national scientific organizations,
including the American Association for Cancer Research (AACR), American Society
of Clinical Oncology (ASCO), Japanese Cancer Association (JCA), Japanese Society
of Medical Oncology (JSMO) and Japan Neurosurgical Society (JNS). He has board
certifications for Medical Oncology, Neurosurgery, Stroke and Cerebrovascular Diseases.
vii
PARTICIPANTS
Mitchel S. Berger Roberta P. Glick

Department of Neurological Surgery Department of Neurological Surgery
University of California San Francisco Rush University Medical Center
San Francisco, California and Mount Sinai Hospital
USA Chicago, Illinois
USA
Antoine F. Carpentier
Service de Neurologie Yong-Kil Hong
Hpital Avicenne Department of Neurosurgery
Bobigny Seoul St. Marys Hospital
France The Catholic University of Korea
Seoul
Susan M. Chang Republic of Korea
Department of Neurological Surgery
University of California San Francisco Hiroaki Ikeda
San Francisco, California Department of Immuno-Gene Therapy
USA Mie University Graduate School
of Medicine
Kevin S. Chen Tsu
Duke University School of Medicine Japan
Durham, North Carolina
USA Eiichi Ishikawa
Department of Neurosurgery
Dong-Sup Chung Doctoral Program in Functional
Department of Neurosurgery and Regulatory Medical Science
Seoul St. Marys Hospital Graduate School of Comprehensive
The Catholic University of Korea Human Sciences
Seoul University of Tsukuba
Republic of Korea Tsukuba
Japan
ix
x PARTICIPANTS
Shuichi Izumoto Atsushi Natsume

Department of Neurosurgery Center for Genetics
Hyogo College of Medicine and Regenerative Medicine
Hyogo Nagoya University Hospital
Japan Nagoya
Japan
Derek R. Johnson
Department of Neurological Surgery Masasuke Ohno
University of California San Francisco Department of Neurosurgery
San Francisco, California Nagoya University School of Medicine
USA Nagoya
Japan
Koji Kajiwara
Yamaguchi University School of Medicine Tadao Ohno
Ube Faculty of Science and Engineering
Japan Waseda University
Tokyo
Chang-Hyun Kim Japan
Medical Science Research Center
Dongguk University Research Insititute Ian F. Parney
of Biotechnology Department of Neurologic Surgery
Gyeonggi-do Mayo Clinic
Republic of Korea Rochester, Minnesota
USA
Maciej S. Lesniak
Section of Neurosurgery Cleo E. Rolle
The University of Chicago Department of Surgery
Chicago, Illinois Section of Neurosurgery
USA The University of Chicago
Pritzker School of Medicine
Terry Lichtor Chicago, Illinois
Department of Neurological Surgery USA
Rush University Medical Center
and Mount Sinai Hospital Nader Sanai
Chicago, Illinois Department of Neurological Surgery
USA University of California San Francisco
San Francisco, California
Tomotoshi Marumoto USA
Department of Molecular Genetics
Division of Molecular Hideyuki Saya
and Clinical Genetics Division of Gene Regulation
Medical Institute of Bioregulation Institute for Advanced Medical Research
Kyushu University Keio University School of Medicine
Fukuoka Tokyo
Japan Japan
Duane A. Mitchell Sadhak Sengupta

Division of Neurosurgery Department of Surgery
Department of Surgery Section of Neurosurgery
The Preston Robert Tisch Brain Tumor The University of Chicago
Center at Duke Pritzker School of Medicine
Durham, North Carolina Chicago, Illinois
USA USA
PARTICIPANTS xi
Hiroshi Shiku Renata Ursu

Department of Immuno-Gene Therapy Service de Neurologie
and Hpital Avicenne
Department of Cancer Vaccine Bobigny
Mie University Graduate School France
of Medicine
Tsu Toshihiko Wakabayashi
Japan Department of Neurosurgery
Nagoya University School of Medicine
Shingo Takano Nagoya
Department of Neurosurgery Japan
Doctoral Program in Functional
and Regulatory Medical Science Qijin Xu
Graduate School of Comprehensive Maxine Dunitz Neurosurgical Institute
Human Sciences Cedars-Sinai Medical Center
University of Tsukuba Los Angeles, California
Tsukuba USA
Japan
Ryuya Yamanaka
Masahiro Toda Kyoto Prefectural University of Medicine
Department of Neurosurgery Kyoto
Keio University School of Medicine Japan
Tokyo
Japan John S. Yu
Maxine Dunitz Neurosurgical Institute
Tomoki Todo Cedars-Sinai Medical Center
Department of Neurosurgery Los Angeles, California
The University of Tokyo USA
Tokyo
Japan Xiangpeng Yuan
Maxine Dunitz Neurosurgical Institute
Koji Tsuboi Cedars-Sinai Medical Center
Proton Medical Research Center Los Angeles, California
Graduate School of Comprehensive USA
Human Sciences
University of Tsukuba
Tsukuba
Japan
CONTENTS
PART I. BASIC AND CLINICAL

ASPECT OF GLIOMA
1. MOLECULAR BIOLOGY OF GLIOMA..............................................................2

Tomotoshi Marumoto and Hideyuki Saya
Abstract......................................................................................................................................... 2
Introduction .................................................................................................................................. 2
Primary and Secondary GBMs................................................................................................... 3
Activated Growth Factor Signaling Pathways .......................................................................... 3
Dysregulation of Cell Cycle Checkpoints................................................................................... 4
Isocitrate Dehydrogenase 1 (IDH1) and IDH2 Mutations in GBMs ....................................... 5
Aberrant Functions of MicroRNAs in GBM ............................................................................. 6
Drug Resistance in GBM ............................................................................................................. 7
Conclusion .................................................................................................................................... 8
2. RECENT SURGICAL MANAGEMENT OF GLIOMAS ..................................12

Nader Sanai and Mitchel S. Berger
Abstract....................................................................................................................................... 12
Introduction ................................................................................................................................ 12
The Evolution of Cortical Mapping Strategies ....................................................................... 13
Variability in Cortical Language Localization ........................................................................ 14

............................... 14
Patient Selection and the Role of Functional Imaging for Language Localization ............. 15
Specialized Neuroanesthesia for the Awake Craniotomy ....................................................... 15
Current Intraoperative Language Mapping Techniques ....................................................... 16
Functional Outcome following Language Mapping for Dominant
Hemisphere Gliomas .......................................................................................................... 16
Tailored Craniotomies and the Value of Negative Language Mapping ................................ 17
An Evidence-Based Approach to Understanding the Value of Extent of Resection ............ 19
Conclusion .................................................................................................................................. 21
xiii
xiv CONTENTS
3. RECENT MEDICAL MANAGEMENT OF GLIOBLASTOMA ......................26

Derek R. Johnson and Susan M. Chang
Abstract....................................................................................................................................... 26
Introduction ................................................................................................................................ 26
Cytotoxic Chemotherapy for Glioblastoma Multiforme ........................................................ 27
Molecularly Targeted Therapies for Glioblastoma Multiforme ............................................ 30
Conclusion .................................................................................................................................. 36
PART II. GLIOMA IMMUNOLOGY

4. BASIC CONCEPTS IN GLIOMA IMMUNOLOGY..........................................42
Ian F. Parney
Abstract....................................................................................................................................... 42
Introduction ................................................................................................................................ 42
Basic Immunology...................................................................................................................... 43
Neuro-Immunology .................................................................................................................... 44
Glioblastoma-Associated Antigens ........................................................................................... 45
Glioblastoma-Derived Immunosuppressive Factors............................................................... 46

!"#
....................... 47
Systemic Immunosuppression in Glioblastoma Patients........................................................ 47
$"%

!

"
......................... 48
Conclusion .................................................................................................................................. 49
5. MECHANISMS OF IMMUNE EVASION BY GLIOMAS ................................53

Cleo E. Rolle, Sadhak Sengupta and Maciej S. Lesniak
Abstract....................................................................................................................................... 53
Introduction ................................................................................................................................ 53
Intrinsic Mechanisms of Immunosuppression ........................................................................ 54
Impairment of Glioma and Immune Cell Interactions .......................................................... 57
Mechanisms of Glioma-Mediated Immunosuppression ......................................................... 57
Recruitment of Immunosuppressive Lymphocytes................................................................. 64
Conclusion .................................................................................................................................. 68
6. GLIOMA ANTIGEN ..............................................................................................77

Masahiro Toda
Abstract....................................................................................................................................... 77
Introduction ................................................................................................................................ 77
Tumor Antigens Recognized by CTLs...................................................................................... 78
"

&

%#$ .......................................................... 78

$"
'*/"
#%#$ ....................... 79

$"
"#

without Using CTLs (Reverse Immunology) ................................................................... 79
CONTENTS xv
Glioma Antigens ......................................................................................................................... 79

%:**; .............................................................................. 80
Glioma Antigens Recognized by CTLs .................................................................................... 81
Glioma Antigens Recognized by Antibodies ............................................................................ 82
Neural Stem Cells and Glioma Antigens.................................................................................. 82
Conclusion .................................................................................................................................. 82
PART III. CYTOKINE, SEROTHERAPY, ADOPTIVE

TRANSFER AND OTHER STRATEGIES
7. CYTOKINE THERAPY ........................................................................................86

Masasuke Ohno, Atsushi Natsume and Toshihiko Wakabayashi
Abstract....................................................................................................................................... 86
Introduction ................................................................................................................................ 86
IL-2 .............................................................................................................................................. 87
IL-4 .............................................................................................................................................. 88
IL-13 ............................................................................................................................................ 89
TGF-` .......................................................................................................................................... 89
GM-CSF ...................................................................................................................................... 90
IFN-` ........................................................................................................................................... 91
Conclusion .................................................................................................................................. 92
8. IMMUNOTHERAPEUTIC APPROACH
WITH OLIGODEOXYNUCLEOTIDES CONTAINING
CpG MOTIFS (CpG-ODN) IN MALIGNANT GLIOMA..........................95
Renata Ursu and Antoine F. Carpentier
Abstract....................................................................................................................................... 95
Introduction ................................................................................................................................ 95
CpG Motifs ................................................................................................................................. 96
Rationale for CpG-ODNs in Gliomas and Preclinical Data ................................................... 98
Clinical Development of CpG-ODNs in Cancer and in Brain Tumors ................................. 99
Perspectives in Clinical Trials ................................................................................................. 104
Tolerance ................................................................................................................................... 104
Conclusion ................................................................................................................................ 105
9. ADOPTIVE CELL TRANSFER THERAPY

FOR MALIGNANT GLIOMAS .................................................................109
Eiichi Ishikawa, Shingo Takano, Tadao Ohno and Koji Tsuboi
Abstract..................................................................................................................................... 109
Introduction .............................................................................................................................. 110
'

#
$<
" ..........................................................................................111
$"!:

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" ................................................................................... 113
Conclusion ................................................................................................................................ 118
xvi CONTENTS
10. MONOCLONAL ANTIBODY THERAPY

FOR MALIGNANT GLIOMA ....................................................................121
Kevin S. Chen and Duane A. Mitchell
Abstract..................................................................................................................................... 121
Introduction .............................................................................................................................. 121
Antibody CNS Bioavailability................................................................................................. 122
Delivery of Antibody Therapy ................................................................................................ 123
Unarmed Antibodies ................................................................................................................ 124
Radioisotope or Toxin Conjugated Antibodies ...................................................................... 127
Immunotherapy Modulators................................................................................................... 129
Antibody-Based Innovations in Immunotherapy ................................................................. 131
Conclusion ................................................................................................................................ 133
PART IV. ACTIVE IMMUNOTHERAPY
11. ANIMAL MODELS FOR VACCINE THERAPY ...........................................143

Dong-Sup Chung, Chang-Hyun Kim and Yong-Kil Hong
Abstract..................................................................................................................................... 143
Introduction .............................................................................................................................. 143
Transplantable Tumor Models ................................................................................................ 144
Spontaneous Tumor Models .................................................................................................... 145
Monitoring of the Animal Tumors .......................................................................................... 146
Monitoring of the Immune Function in Animal Models ...................................................... 147
Chemoimmunotherapy in GL26 Glioma Model ................................................................... 147
Conclusion ................................................................................................................................ 148
12. IMMUNOGENE THERAPY .............................................................................151

Terry Lichtor and Roberta P. Glick
Abstract..................................................................................................................................... 151
Introduction .............................................................................................................................. 152
Preclinical Experimental Findings ......................................................................................... 157
Conclusion ................................................................................................................................ 161
13. PEPTIDE VACCINE ..........................................................................................166

Shuichi Izumoto
Abstract..................................................................................................................................... 166
Introduction .............................................................................................................................. 166
Peptide-Based Vaccines ........................................................................................................... 167
WT1-Peptide Vaccination........................................................................................................ 167
Personalized Peptide Vaccination ........................................................................................... 171
EGFRvIII Peptide Vaccination ............................................................................................... 172
Hurdles to Effective Peptide Vaccine ..................................................................................... 173
CONTENTS xvii
*<

# * %

&

=
................................ 174
Evaluation of Peptide Vaccine-Induced Clinical Responses ................................................ 174
Conclusion ................................................................................................................................ 174
14. ACTIVE IMMUNOTHERAPY:

ONCOLYTIC VIRUS THERAPY USING HSV-1.....................................178
Tomoki Todo
Abstract..................................................................................................................................... 178
Introduction .............................................................................................................................. 178
Genetically Engineered Oncolytic HSV-1 .............................................................................. 179
Third-Generation Oncolytic HSV-1 ....................................................................................... 180
:
"? @:=!J ........................................ 180
Oncolytic HSV-1 in Combination with Immune Gene Therapy.......................................... 181
Oncolytic HSV-1 Armed with Immunostimulatory Genes................................................... 182
Utilization of Armed Oncolytic HSV-1 Construction Systems............................................. 182
Systemic Delivery of Armed Oncolytic HSV-1 ...................................................................... 184
Conclusion ................................................................................................................................ 184
15. DENDRITIC CELL VACCINES .......................................................................187

Ryuya Yamanaka and Koji Kajiwara
Abstract..................................................................................................................................... 187
Introduction .............................................................................................................................. 187
Dendritic Cells in Immunobiology ......................................................................................... 188
Dendritic Cells in the Central Nervous System..................................................................... 190
Dendritic Cells in Tumor Immunology .................................................................................. 191
Clinical Trials of Dendritic Cell-Based Vaccines ................................................................... 193
Future Directions ..................................................................................................................... 196
Conclusion ................................................................................................................................ 197
PART V. NOVEL TOPICS
16. ANTIGEN-RECEPTOR GENE-MODIFIED

T CELLS FOR TREATMENT OF GLIOMA............................................202
Hiroaki Ikeda and Hiroshi Shiku
Abstract..................................................................................................................................... 202
Introduction .............................................................................................................................. 202
Interaction of Immune System with Central Nervous System ............................................ 203

:
#
"<
" ................................................................ 204
Active Immunotherapy of Cancer .......................................................................................... 204
Active Immunotherapy of Brain Tumor ................................................................................ 205
Passive Immunotherapy of Cancer ........................................................................................ 206

!

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Passive Immunotherapy of Brain Tumor .............................................................................. 211
Conclusion ................................................................................................................................ 211
xviii CONTENTS
17. GLIOMA STEM CELL RESEARCH

FOR THE DEVELOPMENT OF IMMUNOTHERAPY..........................216
Qijin Xu, Xiangpeng Yuan and John S. Yu
Abstract..................................................................................................................................... 216
Introduction .............................................................................................................................. 216
Glioma Stem Cells as Cancer Initiating Cells ....................................................................... 217
Targeting Brain Cancer Using Neural Stem Cell-Delivered Therapeutics ......................... 218
Immunotherapy Strategies for Gliomas and the Promise of Dendritic
Cell Vaccination................................................................................................................ 220
Glioma Stem Cell-Targeted Immunotherapy ........................................................................ 220
Conclusion ................................................................................................................................ 222
INDEX........................................................................................................................227
ACKNOWLEDGEMENTS
I would like to thank all of the authors for their enormous contributions
to this book.
xix
PART I
BASIC AND CLINICAL ASPECTS OF GLIOMA

CHAPTER 1
MOLECULAR BIOLOGY OF GLIOMA
Tomotoshi Marumoto1 and Hideyuki Saya*,2

1
Department of Molecular Genetics, Division of Molecular and Clinical Genetics, Medical Institute
of Bioregulation, Fukuoka, Japan; 2Division of Gene Regulation, Institute for Advanced Medical Research,
Keio University School of Medicine, Tokyo, Japan
*Corresponding Author: Hideyuki SayaEmail: hsaya@a5.keio.jp
Abstract: Glioblastoma (GBM) is the most aggressive form of glioma. Despite ceaseless
efforts by researchers and physicians to find new therapeutic strategies, there have
been no significant advances in the treatment of GBMs for several decades and most
patients with GBM die within one and half years of diagnosis. Undoubtedly, one
reason for this is the insufficient understanding of the initiation and progression of
GBMs at the molecular level. However, recent information regarding the genetic
and epigenetic alterations and the microRNAs that are aberrantly activated or
inactivated in GBMs has helped elucidate the formation of GBM in more detail.
Here, we describe recent advances in the understanding of the biology of GBMs.
INTRODUCTION
Gliomas are the most common central nervous system tumors in adults. They are
histologically classified as astrocytomas, oligodendrogliomas and oligoastrocytomas.
Gliomas are graded on the World Health Organization (WHO) grading system according
to their degree of malignancy. The most malignant Grade IV gliomas are termed
glioblastomas (GBMs) and exhibit advanced features of malignancy, which include
necrosis, vascular proliferation and pleomorphism. Patients with GBMs generally die
within one and half years from the time of diagnosis because of their strong resistance to
conventional therapies, which include surgery, chemotherapy and irradiation. Although
until recently chemotherapy for GBMs did not substantially improve disease outcome
when combined with other treatment methods, a randomized clinical trial showed that
combination therapy using temozolomide and radiation is superior to radiation therapy
Glioma: Immunotherapeutic Approaches, edited by Ryuya Yamanaka.

2012 Landes Bioscience and Springer Science+Business Media.
2
MOLECULAR BIOLOGY OF GLIOMA 3
alone.1 The median survival of the patients who received this combination treatment was
14.6 months.1 This is 2.5 months longer than the median survival time of patients who
received radiation therapy alone. Therefore, combination therapy using temozolomide and
radiation is now widely used worldwide; however, physicians keep seeking for a more
effective treatment for GBMs. An understanding of the genetic and epigenetic alterations
and resulting aberrant molecular pathways leading to the initiation and progression of
GBMs is essential and indispensable for the identification of new therapeutic strategies
for GBM.
PRIMARY AND SECONDARY GBMs
GBMs have been subdivided into primary or secondary GBM subtypes on the basis
of their clinical presentation. Primary GBMs are generally found in older patients, while
secondary GBMs are found in younger patients (below the age of 45). Primary GBMs are
not associated with prior symptoms or with evidence of detectable antecedent lower grade
tumors. In contrast, secondary GBMs arise from lower grade gliomas within 5-10 years
of diagnosis.2 Although they have distinctive clinical histories, primary and secondary
GBMs exhibit features that are histologically similar and are generally associated with
an equally poor prognosis.
Several genes, such as Tumor protein 53 (TP53), p16Ink4a, Phosphatase and Tensin
Homolog Deleted from Chromosome 10 (PTEN) and Epidermal Growth Factor Receptor
(EGFR), are altered in both primary and secondary GBMs.3-7 These alterations are thought
to occur in a sequential order during the malignant progression of gliomas (Fig. 1). Loss
or mutation of the PTEN tumor suppressor gene and amplification or overexpression
of the EGFR oncogene are thought to be a characteristic of primary GBMs, whereas
mutation of the TP53 tumor suppressor gene appears to be an early event during the
development of secondary GBMs8-10 (Fig. 1). Most importantly, abnormal activation of
growth factor signaling and dysregulation of cell cycle checkpoints are found in both
types of GBMs (Figs. 1 and 2).
ACTIVATED GROWTH FACTOR SIGNALING PATHWAYS
Various growth factors, such as platelet-derived growth factor (PDGF)-A, PDGF-B,

epidermal growth factor (EGF), transforming growth factor (TGF)-_, insulin growth factor
(IGF)-1 and fibroblast growth factor (FGF) are often produced and secreted in GBMs.
In addition, the various growth factor receptors corresponding to each specific growth
factor, such as EGFR, PDGFR-A, PDGFR-B and FGFR2, are often overexpressed in
GBMs. Consequently, the autocrine/paracrine loops between ligand and receptor enhance
their impact in gliomagenesis.3,9,11-13
Several important signaling pathways, such as Ras-mitogen-activated protein
kinase (MAPK), phosphoinositide 3-kinase (PI3K)AKT and Phospholipase-C gamma
(PLCa)Protein Kinase C (PKC) are activated downstream of growth factor receptors.
Recent reports using genetically engineered mice suggest that these growth factor signaling
pathways cause the formation of gliomas directly. Weissenberger et al reported that the
astrocyte-specific expression of v-src, which is one of the oncogenes expressed in GBMs,
causes the formation of gliomas.14 In their experiments, abnormal astrogliosis was found in
4 GLIOMA: IMMUNOTHERAPEUTIC APPROACHES
Figure 1. Distinct molecular alterations between primary and secondary GBMs. Molecular alterations
during the malignant progression of gliomas between primary GBMs and secondary GBMs are different.
It should be noted that amplification or overexpression of EGFR is more frequently found in primary
GBMs whereas mutation of TP53 is an early event in secondary GBMs. MDM2: murine double minute
2, LOH: loss of heterozygosity, RB: retinoblastoma, DCC: deleted in colorectal carcinoma.
all the astrocyte-specific v-src expressing mice; however, only 14.4% of mice developed
astrocytomas within 65 weeks. Therefore, the authors speculated that factors other than
v-src are needed for the malignant transformation of astrocytes.
DYSREGULATION OF CELL CYCLE CHECKPOINTS
The eukaryotic cell cycle is composed of four distinct phases: G1 phase, S phase
(DNA synthesis), G2 phase and M phase (mitosis). Through the cell cycle, the genetic
materials are correctly replicated and transmitted to the two daughter cells. Accurate
regulation of the cell cycle is crucial for the survival of cells. Each phase has to progress
in a sequential fashion and, to activate each phase, proper progression and completion
of the previous phase is required15-17 (Fig. 2).
The engine of the cell cycle is the cyclincyclin-dependent kinase (CDK) complex.
Cyclins constitute the regulatory subunit and CDK is the catalytic subunit of an activated
heterodimeric kinase complex. CDKs are inactive in the absence of cyclin. When
activated, CDKs bound to cyclin catalyze protein phosphorylation reactions that mediate
the coordination of the progression of the cell cycle.15-17 When cells receive promitotic
extracellular signals, G1 cyclinCDK complexes, such as cyclinD/cdk4,6, are activated
and promote the expression of transcription factors, which leads to the increase of S-phase
cyclin expression.15-17
The disruption of cell cycle checkpoints caused by the dysregulation of major tumor
suppressors appears to be crucial for gliomagenesis. Firstly, the TP53 gene is either
mutated or deleted in certain gliomas, particularly in secondary GBMs.3-5 Secondly, the
Figure 2. RB and TP53 pathways controlling G1-S transition. Phosphorylation of RB by CDK4,6/Cyclin

D activated by mitogenic signals leads to the activation of transcription factor E2F, which results in
the activation of S phase genes. Due to the inactivation of tumor suppressors such as TP53 and RB,
G1-S transition is dysregulated in GBMs.
retinoblastoma (RB) gene is mutated in 10-25% of high-grade astrocytomas. In addition,

RB is functionally silenced in another 15% of astrocytomas via the amplification of its
antagonist CDK4.3,5,18 Lastly, the CDKN2A locus encoding p16INK4A and p14Arf (which
positively regulate the RB and TP53 pathways, respectively) is deleted in approximately
half of high-grade astrocytomas.3,5,6 Similar effects on the RB and TP53 pathways are
also frequently found in anaplastic oligodendrogliomas.19
ISOCITRATE DEHYDROGENASE 1 (IDH1) AND IDH2 MUTATIONS

IN GBMs
Recently, a somatic mutation at amino acid 132 of the isocitrate dehydrogenase 1

(IDH1) protein was identified in more than 70% of WHO Grade II and III astrocytomas,
oligodendrogliomas and secondary GBMs.5,20 Surprisingly, tumors expressing an intact
IDH1 gene often have mutations at the analogous amino acid 172 of the IDH2 protein.
Interestingly, the R132 residue of IDH1 and the R172 residue of IDH2 are located in
the active site of these enzymes and form hydrogen bonds with the isocitrate substrate.21
A recent report showed that forced expression of a mutated IDH1 gene in cultured cells
results in the reduction of the enzyme product _-ketoglutarate (_-KG), which leads to
the increase of the levels of the hypoxia-induced factor-1_ (HIF-1_), the stability of
which is regulated by _-KG.22 Because HIF-1_ is thought to promote tumor growth at
low oxygen levels, mutation of the IDH1 gene may facilitate tumor progression in GBMs.
It should be noted that mutations in the IDH1 or IDH2 genes are identified in the vast
majority of WHO Grade II or III gliomas and secondary GBMs.20,23 It has been pointed
out that approximately 80% of anaplastic astrocytomas and GBMs with mutated IDH1
or IDH2 genes also express mutated TP53, whereas only 3% express mutated PTEN,
EGFR, or p16Ink4a.20 On the other hand, only 18% of anaplastic astrocytomas and GBMs
with intact IDH1 or IDH2 genes express mutated TP53; however, more than 70% of these
tumors exhibit mutations in PTEN, EGFR, or p16Ink4a.20 These findings suggest that gliomas
with mutated IDH genes are genetically different from gliomas with intact IDH genes.
ABERRANT FUNCTIONS OF MicroRNAS IN GBM
It has been speculated that at least one-third of all protein-coding genes in the human
genome are partially regulated by microRNAs.24 MicroRNAs, which are single-stranded
RNA molecules that comprise approximately 20-22 nucleotides, have been shown to
play a central role in biological processes and distinct patterns of microRNA expression
are seen in different normal cells and tissues.25-27
MicroRNAs are noncoding RNAs from various chromosomal origins, which include
intergenic, intronic, or exonic regions. The primary transcripts of microRNAs (Pri-miR)
are transcribed from the genome by RNA polymerase II and are processed through a
conserved mechanism to yield mature microRNAs (Fig. 3). Mature microRNAs recognize
partially complementary sequences in the 3vUTR of their target genes and decrease the
mRNA levels of the target by cleavage or by inhibiting translation initiation directly.28
Other reported mechanisms of microRNA-mediated translational repression are the
destabilization of mRNA via deadenylation of the poly-A tail and recruitment of the
Figure 3. Maturation of microRNAs in normal cells. The primary transcripts of microRNAs (Pri-miR) are
transcribed from the genome by RNA polymerase II and are processed through a conserved mechanism
to yield mature microRNAs. Initially, the hairpinloop sequences in Pri-miRs are cleaved by a nuclear
protein complex that includes Drosha, which is a Type III RNase and Pre-miRs 60-80 base pairs in
length are formed. After being exported to the cytoplasm by the Exportin 5 mediated mechanisms,
Pre-miRs are further processed by a protein complex containing Dicer, a Type III RNase, which leads
to the generation of mature microRNAs. The mature microRNAs are then loaded onto the RNA-induced
silencing complex (RISC) and target mRNAs, thus reducing protein translation.
ribosome inhibitory protein eLF6.29,30 Surprisingly, it has also been reported that certain
types of microRNAs upregulate the translation of their target genes.31 These reports on
the functions of microRNAs suggest that microRNAs have hundreds of potential targets
and regulate multiple signaling pathways.24
Various levels of microRNA expression have been observed in cancers, including
GBMs.32-34 It has been reported that microRNA-21 (miR-21) is highly expressed
in GBMs.35 Inhibition of miR-21 in glioma cell lines results in the increase of
caspase-dependent apoptosis, which suggests that miR-21 acts as an oncogene by
inhibiting apoptosis.35 MiR-21 has also been reported to promote the cellular invasion
by directly downregulating metalloprotease inhibitors, such as tissue inhibitor of
metalloproteinases-3 (TIMP3) and Reversion-inducing cysteine-rich protein with
Kazal motifs (RECK).36 A computer-based analysis suggested that miR-21 targets a
network of TP53, TGF-` and mitochondrial apoptosis factors in GBMs.37 MiR-7 is
downregulated in GBMs and targets EGFR.38 MiR-7 has also been reported to target
insulin receptor substrate-2 (IRS-2), which is an adaptor protein that mediates PI3K
signaling downstream of receptor tyrosine kinases and suppresses AKT activation.38
These facts suggest that the oncogenes responsible for GBM formation may be regulated
by microRNAs. MiR-124 and miR-137, which target CDK6, are also downregulated
in GBMs when compared with normal brain tissues.39 MiR-128 is downregulated in
GBMs and targets B-cell-specific Moloney murine leukemia virus integration site 1
(Bim-1), which is thought to be one of the oncogenes responsible for GBM formation.36
DRUG RESISTANCE IN GBM
Drug resistance in GBM is one of the major factors that render this type of tumor
incurable. EGFR is frequently amplified, overexpressed, or mutated in GBMs.9,11-13 An
activated form of EGFR mutant caused by the deletion of exons 2-7 (known as EGFRvIII)
occurs in 20-30% of GBMs; EGFRvIII reduces apoptosis and increases the proliferation
of GBM cells. In addition, EGFRvIII has the capacity to malignantly transform murine
Ink4a/Arf null neural stem cells (NSCs) or astrocytes.40,41 Therefore, EGFR is a strongly
validated molecular target for GBM treatment and the use of EGFR inhibitors is a
reasonable therapeutic strategy for this disease. However, only 10-20% of GBM patients
respond to EGFR inhibitors.42 Although the mechanism of the resistance of GBMs to
EGFR inhibitors is not known, the loss of PTEN is suggested to be associated with this
mechanism. PTEN is commonly lost in GBMs and is an inhibitor of the PI3K signaling
pathway, which is activated downstream of EGFR. It is suggested that loss of PTEN
may dissociate EGFR inhibition from the PI3K pathway inhibition, which may result in
the resistance of GBMs to EGFR inhibitors.42
Alkylating agents, which include the chloroethylnitrosoureas (nimustine (ACNU),
carmustine (BCNU) and lomustine (CCNU)), procarbazine and temozolomide, are
commonly used for the treatment of GBMs.43-45 These drugs cause DNA damage by
adding alkyl groups to DNA (i.e., alkylation) and induce apoptosis in tumor cells.
O6-methylguanine-methyltransferase (MGMT) is a DNA repair protein that can reverse
alkylation at the O6 position of guanine. The neutralization of the cytotoxic effects of
alkylating agents by the MGMT has been demonstrated in cell lines, in xenograft models
and in an MGMT transgenic mouse.46-51
High levels of MGMT activity in tumor tissues are associated with resistance to
alkylating agents and the extent of methylation of the promoter region of the MGMT
gene, which results in the decreased expression of MGMT in GBMs, correlates with the
outcome of treatment with alkylating agents.52-54
Despite the information described above, there has been no significant improvement
in overcoming the drug resistance in GBM for several decades. Therefore, the drug
resistance mechanism in GBMs still warrants investigation.
CONCLUSION
As described above, there is accumulating information regarding the molecular

mechanisms responsible for the initiation and progression of GBMs (Fig. 1); however,
the prognosis of GBMs remains dismal. Some crucial questions remain unsolved.
For example, it is well known that the loss of chromosomes 1p and 19q is frequently
associated with the prognosis of the patients with oligodendrogliomas.55 The deletion
of chromosome 1p is most commonly combined with loss of chromosome 19q (whole
arm deletion). Oligodendrogliomas carrying the codeletion of 1p/19q are sensitive to
both chemotherapy and radiotherapy, which leads to a better prognosis in patients with
this type of tumor.56,57 However, this genetic abnormality has not been modeled in mice,
which is partly because of the lack of knowledge regarding the precise genes involved
in these regions.55 The elucidation of the molecular mechanisms leading to the high
sensitivity of tumors lacking chromosomes 1p/19q to chemotherapy and radiotherapy
may provide new and improved therapies for oligodendrogliomas.
Another crucial question that remains unsolved is the cellular origin of GBMs.
It is now believed that brain tumors, including GBMs, arise from brain tumor stem/
initiating cells (BTSCs).58,59 BTSCs have been identified as a rare population of cells
in tumor tissues and have a robust tumor initiating capability when injected into
immunocompromised mice.58,59 BTSCs are positive for CD133, which is a marker
of stem or progenitor cells in various organs.60,61 CD133
cells from GBMs have the
ability to form neurospheres in vitro and as few as 100 CD133
cells are able to form
tumors in immunocompromised mice, while most human GBM cell lines require 105
to 106 cells for tumor engraftment and formation.59,62,63 Several reports from different
laboratories support this contention; however, more recent evidence questions the
reliability of CD133 as a marker for BTSCs. Wang et al showed that transplantation of
CD133< cells from human GBMs results in the formation of tumors in nude rats and that
those cells give rise to CD133
cells.58,64 Another report indicates that the expression of
CD133 in GBMs may just reflect a response of the tumor cells to environmental stress.65
Therefore, it may be necessary to identify additional markers that more consistently
represent BTSCs.
As described above, knowledge on the molecular signaling pathways responsible
for GBM formation and the BTSC hypothesis have significantly advanced our
understanding of CNS malignancies. However, as several key questions remain
unanswered, the continuous efforts of researchers and physicians are necessary to
improve the management of GBMs.
REFERENCES
1. Stupp R, Mason WP, van den Bent MJ et al. Radiotherapy plus concomitant and adjuvant temozolomide
for glioblastoma. N Engl J Med 2005; 352:987-996.
2. Kleihues P, Ohgaki H. Primary and secondary glioblastomas: from concept to clinical diagnosis. Neuro
Oncol 1999; 1:44-51.
3. Comprehensive genomic characterization defines human glioblastoma genes and core pathways. Nature
2008; 455:1061-1068.
4. Louis DN. The p53 gene and protein in human brain tumors. J Neuropathol Exp Neurol 1994; 53:11-21.
5. Parsons DW, Jones S, Zhang X et al. An integrated genomic analysis of human glioblastoma multiforme.
Science 2008; 321:1807-1812.
6. Ueki K, Ono Y, Henson JW et al. CDKN2/p16 or RB alterations occur in the majority of glioblastomas and
are inversely correlated. Cancer Res 1996; 56:150-153.
7. Wong AJ, Bigner SH, Bigner DD et al. Increased expression of the epidermal growth factor receptor gene
in malignant gliomas is invariably associated with gene amplification. Proc Natl Acad Sci USA 1987;
84:6899-6903.
8. Ohgaki H, Kleihues P. Genetic pathways to primary and secondary glioblastoma. Am J Pathol 2007;
170:1445-1453.
9. Furnari FB, Fenton T, Bachoo RM et al. Malignant astrocytic glioma: genetics, biology and paths to treatment.
Genes Dev 2007; 21:2683-2710.
10. Weber RG, Sabel M, Reifenberger J et al. Characterization of genomic alterations associated with glioma
progression by comparative genomic hybridization. Oncogene 1996; 13:983-994.
11. Huse JT, Holland EC. Genetically engineered mouse models of brain cancer and the promise of preclinical
testing. Brain Pathol 2009; 19:132-143.
12. Di Rocco F, Carroll RS, Zhang J et al. Platelet-derived growth factor and its receptor expression in human
oligodendrogliomas. Neurosurgery 1998; 42:341-346.
13. Hesselager G, Holland EC. Using mice to decipher the molecular genetics of brain tumors. Neurosurgery
2003; 53:685-694; discussion 95.
14. Weissenberger J, Steinbach JP, Malin G et al. Development and malignant progression of astrocytomas in
GFAP-v-src transgenic mice. Oncogene 1997; 14:2005-2013.
15. Hartwell L. Defects in a cell cycle checkpoint may be responsible for the genomic instability of cancer
cells. Cell 1992; 71:543-546.
16. Nurse P. Checkpoint pathways come of age. Cell 1997; 91:865-867.
17. Kastan MB, Bartek J. Cell-cycle checkpoints and cancer. Nature 2004; 432:316-323.
18. Henson JW, Schnitker BL, Correa KM et al. The retinoblastoma gene is involved in malignant progression
of astrocytomas. Ann Neurol 1994; 36:714-721.
19. Watanabe T, Yokoo H, Yokoo M et al. Concurrent inactivation of RB1 and TP53 pathways in anaplastic
oligodendrogliomas. J Neuropathol Exp Neurol 2001; 60:1181-1189.
20. Yan H, Parsons DW, Jin G et al. IDH1 and IDH2 mutations in gliomas. N Engl J Med 2009; 360:765-773.
21. Xu X, Zhao J, Xu Z et al. Structures of human cytosolic NADP-dependent isocitrate dehydrogenase reveal
a novel self-regulatory mechanism of activity. J Biol Chem 2004; 279:33946-33957.
22. Zhao S, Lin Y, Xu W et al. Glioma-derived mutations in IDH1 dominantly inhibit IDH1 catalytic activity
and induce HIF-1alpha. Science 2009; 324:261-265.
23. Watanabe T, Nobusawa S, Kleihues P et al. IDH1 mutations are early events in the development of
astrocytomas and oligodendrogliomas. Am J Pathol 2009; 174:1149-1153.
24. Lim LP, Lau NC, Garrett-Engele P et al. Microarray analysis shows that some microRNAs downregulate
large numbers of target mRNAs. Nature 2005; 433:769-773.
25. Lee RC, Feinbaum RL, Ambros V. The C. elegans heterochronic gene lin-4 encodes small RNAs with
antisense complementarity to lin-14. Cell 1993; 75:843-854.
26. Wightman B, Ha I, Ruvkun G. Posttranscriptional regulation of the heterochronic gene lin-14 by lin-4
mediates temporal pattern formation in C. elegans. Cell 1993; 75:855-862.
27. Baulcombe D. DNA events. An RNA microcosm. Science 2002; 297:2002-2003.
28. Doench JG, Sharp PA. Specificity of microRNA target selection in translational repression. Genes Dev
2004; 18:504-511.
29. Wu L, Fan J, Belasco JG. MicroRNAs direct rapid deadenylation of mRNA. Proc Natl Acad Sci USA
2006; 103:4034-9.
30. Chendrimada TP, Finn KJ, Ji X et al. MicroRNA silencing through RISC recruitment of eIF6. Nature
2007; 447:823-838.
31. Vasudevan S, Tong Y, Steitz JA. Switching from repression to activation: microRNAs can up-regulate
translation. Science 2007; 318:1931-1934.
32. Volinia S, Calin GA, Liu CG et al. A microRNA expression signature of human solid tumors defines cancer
gene targets. Proc Natl Acad Sci USA 2006; 103:2257-2261.
33. Calin GA, Ferracin M, Cimmino A et al. A MicroRNA signature associated with prognosis and progression
in chronic lymphocytic leukemia. N Engl J Med 2005; 353:1793-1801.
34. Lawler S, Chiocca EA. Emerging functions of microRNAs in glioblastoma. J Neurooncol 2009; 92:297-306.
35. Corsten MF, Miranda R, Kasmieh R et al. MicroRNA-21 knockdown disrupts glioma growth in vivo and
displays synergistic cytotoxicity with neural precursor cell delivered S-TRAIL in human gliomas. Cancer
Res 2007; 67:8994-9000.
36. Godlewski J, Nowicki MO, Bronisz A et al. Targeting of the Bmi-1 oncogene/stem cell renewal factor by
microRNA-128 inhibits glioma proliferation and self-renewal. Cancer Res 2008; 68:9125-9130.
37. Papagiannakopoulos T, Shapiro A, Kosik KS. MicroRNA-21 targets a network of key tumor-suppressive
pathways in glioblastoma cells. Cancer Res 2008; 68:8164-8172.
38. Kefas B, Godlewski J, Comeau L et al. microRNA-7 inhibits the epidermal growth factor receptor and the
Akt pathway and is down-regulated in glioblastoma. Cancer Res 2008; 68:3566-3572.
39. Silber J, Lim DA, Petritsch C et al. miR-124 and miR-137 inhibit proliferation of glioblastoma multiforme
cells and induce differentiation of brain tumor stem cells. BMC Med 2008; 6:14.
40. Holland EC, Hively WP, DePinho RA et al. A constitutively active epidermal growth factor receptor
cooperates with disruption of G1 cell-cycle arrest pathways to induce glioma-like lesions in mice. Genes
Dev 1998; 12:3675-3685.
41. Bachoo RM, Maher EA, Ligon KL et al. Epidermal growth factor receptor and Ink4a/Arf: convergent
mechanisms governing terminal differentiation and transformation along the neural stem cell to astrocyte
axis. Cancer Cell 2002; 1:269-277.
42. Mellinghoff IK, Wang MY, Vivanco I et al. Molecular determinants of the response of glioblastomas to
EGFR kinase inhibitors. N Engl J Med 2005; 353:2012-2024.
43. Kreisl TN. Chemotherapy for malignant gliomas. Semin Radiat Oncol 2009; 19:150-154.
44. Sarkaria JN, Kitange GJ, James CD et al. Mechanisms of chemoresistance to alkylating agents in
malignant glioma. Clin Cancer Res 2008; 14:2900-2908.
45. Engelhard HH. The role of interstitial BCNU chemotherapy in the treatment of malignant glioma. Surg
Neurol 2000; 53:458-464.
46. Dumenco LL, Allay E, Norton K et al. The prevention of thymic lymphomas in transgenic mice by human
O6-alkylguanine-DNA alkyltransferase. Science 1993; 259:219-222.
47. Gerson SL. Clinical relevance of MGMT in the treatment of cancer. J Clin Oncol 2002; 20:2388-2399.
48. Kaina B, Fritz G, Mitra S et al. Transfection and expression of human O6-methylguanine-DNA
methyltransferase (MGMT) cDNA in Chinese hamster cells: the role of MGMT in protection against
the genotoxic effects of alkylating agents. Carcinogenesis 1991; 12:1857-1867.
49. Baer JC, Freeman AA, Newlands ES et al. Depletion of O6-alkylguanine-DNA alkyltransferase correlates with
potentiation of temozolomide and CCNU toxicity in human tumour cells. Br J Cancer 1993; 67:1299-1302.
50. Redmond SM, Joncourt F, Buser K et al. Assessment of P-glycoprotein, glutathione-based detoxifying
enzymes and O6-alkylguanine-DNA alkyltransferase as potential indicators of constitutive drug resistance
in human colorectal tumors. Cancer Res 1991; 51:2092-2097.
51. Hermisson M, Klumpp A, Wick W et al. O6-methylguanine DNA methyltransferase and p53 status predict
temozolomide sensitivity in human malignant glioma cells. J Neurochem 2006; 96:766-776.
52. Watts GS, Pieper RO, Costello JF et al. Methylation of discrete regions of the O6-methylguanine DNA
methyltransferase (MGMT) CpG island is associated with heterochromatinization of the MGMT
transcription start site and silencing of the gene. Mol Cell Biol 1997; 17:5612-5619.
53. Hegi ME, Diserens AC, Godard S et al. Clinical trial substantiates the predictive value of
O-6-methylguanine-DNA methyltransferase promoter methylation in glioblastoma patients treated with
temozolomide. Clin Cancer Res 2004; 10:1871-1874.
54. Hegi ME, Diserens AC, Gorlia T et al. MGMT gene silencing and benefit from temozolomide in glioblastoma.
N Engl J Med 2005; 352:997-1003.
55. Reifenberger J, Reifenberger G, Liu L et al. Molecular genetic analysis of oligodendroglial tumors shows
preferential allelic deletions on 19q and 1p. Am J Pathol 1994; 145:1175-1190.
56. Cairncross JG, Ueki K, Zlatescu MC et al. Specific genetic predictors of chemotherapeutic response and
survival in patients with anaplastic oligodendrogliomas. J Natl Cancer Inst 1998; 90:1473-1479.
57. Cairncross G, Berkey B, Shaw E et al. Phase III trial of chemotherapy plus radiotherapy compared with
radiotherapy alone for pure and mixed anaplastic oligodendroglioma: Intergroup Radiation Therapy
Oncology Group Trial 9402. J Clin Oncol 2006; 24:2707-2714.
58. Zaidi HA, Kosztowski T, DiMeco F et al. Origins and clinical implications of the brain tumor stem cell
hypothesis. J Neurooncol 2009; 93:49-60.
59. Singh SK, Hawkins C, Clarke ID et al. Identification of human brain tumour initiating cells. Nature 2004;
432:396-401.
60. Kania G, Corbeil D, Fuchs J et al. Somatic stem cell marker prominin-1/CD133 is expressed in
embryonic stem cell-derived progenitors. Stem Cells 2005; 23:791-804.
61. Weigmann A, Corbeil D, Hellwig A et al. Prominin, a novel microvilli-specific polytopic membrane protein
of the apical surface of epithelial cells, is targeted to plasmalemmal protrusions of non-epithelial cells.
Proc Natl Acad Sci USA 1997; 94:12425-12430.
62. Houchens DP, Ovejera AA, Riblet SM et al. Human brain tumor xenografts in nude mice as a chemotherapy
model. Eur J Cancer Clin Oncol 1983; 19:799-805.
63. Hu B, Guo P, Fang Q et al. Angiopoietin-2 induces human glioma invasion through the activation of matrix
metalloprotease-2. Proc Natl Acad Sci USA 2003; 100:8904-8909.
64. Wang J, Sakariassen PO, Tsinkalovsky O et al. CD133 negative glioma cells form tumors in nude rats and
give rise to CD133 positive cells. Int J Cancer 2008; 122:761-768.
65. Griguer CE, Oliva CR, Gobin E et al. CD133 is a marker of bioenergetic stress in human glioma. PLoS
ONE 2008; 3:e3655.
CHAPTER 2
RECENT SURGICAL MANAGEMENT

OF GLIOMAS
Nader Sanai* and Mitchel S. Berger

Department of Neurological Surgery, University of California San Francisco, San Francisco, California, USA
*Corresponding Author: Nader SanaiEmail:sanain@neurosurg.ucsf.edu
Abstract: Refinement of neurosurgical technique has enabled safer operations with more
aggressive outcomes. One cornerstone of modern-day practice is the utilization of
intraoperative stimulation mapping. In addition to identifying critical motor pathways,
this technique can be adapted to reliable identify language pathways, as well. Given
the individual variability of cortical language localization, such awake language
mapping is essential to minimize language deficits following tumor resection. Our
experience suggests that cortical language mapping is a safe and efficient adjunct
to optimize tumor resection while preserving essential language sites, even in the
setting of negative mapping data. However, the value of maximizing glioma resections
remains surprisingly unclear, as there is no general consensus in the literature
regarding the efficacy of extent of glioma resection in improving patient outcome.
While the importance of resection in obtaining tissue diagnosis and to alleviate
symptoms is clear, a lack of Class I evidence prevents similar certainty in assessing
the influence of extent of resection. Beyond an analysis of modern intraoperative
mapping techniques, we examine every major clinical publication since 1990 on the
role of extent of resection in glioma outcome. The mounting evidence suggests that,
despite persistent limitations in the quality of available studies, a more extensive
surgical resection is associated with longer life expectancy for both low-grade and
high-grade gliomas.
INTRODUCTION
Central nervous system tumors are a major cause of morbidity and mortality with
approximately 18,000 new cases of primary intracranial tumors diagnosed each year in
the United States. This represents approximately 2% of all adult tumors in this country.

12
RECENT SURGICAL MANAGEMENT OF GLIOMAS 13
More than half of these are high-grade gliomas. These lesions are extremely aggressive
and the vast majority of patients invariably have tumor recurrence, with the median
survival time ranging from 1 to 3 years after initial diagnosis. Despite facing a better
prognosis when compared to higher grade glial tumors, 50 to 75% of patients harboring
low-grade gliomas eventually die of their disease. Median survival times have been
reported to range between 5 years and 10 years and estimates of 10-year survival rates
range from 5 to 50%.
Although a primary tenet of neurosurgical oncology is that survival can improve with
greater tumor resection, this principle must be tempered by the potential for functional
loss following a radical removal. Current neurosurgical innovations aim to improve our
anatomic, physiologic and functional understanding of the surgical region of interest in
order to prevent potential neurological morbidity during resection. Emerging imaging
technologies, as well as state-of-the-art intraoperative techniques, can facilitate extent
of resection while minimizing the associated morbidity profile. Specifically, the value
of mapping motor and language pathways is well-established for the safe resection of
intrinsic tumors.
Interestingly, controversy persists regarding prognostic factors and treatment options
for both low- and high-grade hemispheric gliomas. Among the various tumor- and
treatment-related parameters, including tumor volume, neurological status, timing of
surgical intervention and the use of adjuvant therapy, only age and tumor histology have
been identified as reliable predictors of patient prognosis. Importantly, despite significant
advances in operative technique and preoperative planning, the effect of glioma extent of
resection in prolonging tumor-free progression and/or survival remains unknown. While
the importance of glioma resection in obtaining tissue diagnosis and decompressing mass
effect are unquestionable, a lack of Class I evidence prevents similar certainty in assessing
the influence of extent of resection. Even though low-grade and high-grade gliomas are
distinct in their biologies, clinical behaviors and outcomes, understanding the effect of
surgery remains equally important for both.
THE EVOLUTION OF CORTICAL MAPPING STRATEGIES
Direct cortical stimulation has been employed in neurosurgery since 1930, first by
Foerster1 and then later, by Penfield.2-4 In recent years, the technique of intraoperative
cortical stimulation has been adopted for the identification and preservation of language
function and motor pathways. Stimulation depolarizes a very focal area of cortex which,
in turn, evokes certain responses. Although the mechanism of stimulation effects on
language are poorly understood, the principle is based upon the depolarization of
local neurons and also of passing pathways, inducing local excitation or inhibition, as
well as possible diffusion to more distant areas by way of orthodromic or antidromic
propagation.5 Studies employing optical imaging of bipolar cortical stimulation in
monkey and human cortex have shown precise local changes, within 2 to 3 mm, after
the activation of cortical tissue.6,7 With the advent of the bipolar probe, avoidance of
local diffusion and more precise mapping have been enabled with an accuracy estimated
to be approximately 5 mm.6
Language mapping techniques were historically developed in the context of epilepsy
surgery, where large craniotomies exposed the brain well beyond the region of surgical
interest in order to localize multiple cortical regions containing stimulation-induced
language and motor function, i.e., positive sites, prior to resection. Until recently, it
has been thought that such positive site controls must be established during language
mapping before any other cortical area could be safely resected. Using this tactic, awake
craniotomies traditionally identify positive language sites in 95-100% of the operative
exposures. Brain tumor surgeons, however, are now evolving towards a different standard
of language mapping, where smaller, tailored craniotomies often expose no positive
sites and tumor resection is therefore directed by the localization of cortical regions that
contained no stimulation-induced language or motor function, i.e., negative sites. This
negative mapping strategy represents a paradigm shift in language mapping technique
by eliminating the neurosurgeons reliance on the positive site control in the operative
exposure, thereby allowing for minimal cortical exposure overlying the tumor, less
extensive intraoperative mapping and a more time-efficient neurosurgical procedure.
VARIABILITY IN CORTICAL LANGUAGE LOCALIZATION
Prediction of cortical language sites through classic anatomical criteria is inadequate

in light of the significant individual variability of cortical organization,8-11 the distortion of
cerebral topography from tumor mass effect and the possibility of functional reorganization
through plasticity mechanisms.12-14 A consistent finding of language stimulation studies
has been the identification of significant individual variability among patients.9 Speech
arrest is variably located and can go well beyond the classic anatomical boundaries of
Brocas area for motor speech. It typically involves an area contiguous with the face-motor
cortex and, yet, in some cases is seen several centimeters from the sylvian fissure. This
variability has also been suggested by studies designed to preoperatively predict the
location of speech arrest based upon the type of frontal opercular anatomy15 or using
functional neuroimaging.16-22 Similarly, for temporal lobe language sites, one study of
temporal lobe resections assisted by subdural grids demonstrated that the distance from
the temporal pole to the area of language function varied from 3 to 9 cm.23 Functional
imaging studies have also corroborated such variability.24 Furthermore, because functional
tissue can be located within the tumor nidus,25 the standard surgical principle of debulking
tumor from within to avoid neurologic deficits is not always safe. Consequently, the
use of intraoperative cortical and subcortical stimulation to accurately detect functional
regions and pathways is essential for safely removing dominant hemisphere gliomas to
the greatest extent possible.
AVOIDANCE OF FUNCTIONAL LANGUAGE DEFICITS FOLLOWING

AWAKE MAPPING
Intraoperative cortical stimulation has yielded critical data regarding essential language
sites, which seem to be organized in discrete mosaics that occupy a much smaller area
of cortex than described by traditional language maps.26-28 Interestingly, the majority of
these language sites are surrounded by cortex that, when stimulated, produce no language
errors.29 In the temporal lobe, identification of speech areas within the superior and middle
temporal gyri have been documented within 3 cm of the temporal lobe tip.9 In this region,
the distance of the resection margin from the nearest language site is the most important
variable in predicting the improvement of preoperative language deficits. Accordingly, if
the distance of the resection margin from the nearest language site is greater than 1 cm,
significantly fewer permanent language deficits occur.30 Strict adherence to this principle
when operating in any region of the dominant hemisphere can substantially reduce the
risk of inadvertently resection functional tissue.
PATIENT SELECTION AND THE ROLE OF FUNCTIONAL IMAGING

FOR LANGUAGE LOCALIZATION
Because the need to preserve cortical language function must be balanced with
the goal of maximal tumor resection, intraoperative language mapping is advocated by
some as the rule, rather than the exception.31 The greatest risk of tumor recurrence is
located within 2 cm of the contrast enhancing rim on imaging studies,32,33 supporting
the concept that the resection should ideally go beyond the gross tumor margin apparent
on preoperative imaging. However, because of the infiltrating nature of gliomas, it
is more than likely that a portion of the mass will occupy, or be continuous with,
functional tissue. This, again, emphasizes the need for cortical stimulation mapping
to avoid injuring these critical areas, particularly language pathways. Although it is
classically thought that patients who are neurologically intact or minimally affected
preoperatively have their functional pathways either displaced or obliterated by
infiltrative tumors, we now know that normally functioning language, motor, or sensory
tissue can blend with tumor.25 Therefore, it is not only patients with tumors located
within the frontal operculum that benefit from intraoperative language mapping, but
also those with lesions in proximity to this region, as there is significant variability
in this regions anatomical and functional organization.15,34
Functional imaging has experienced considerable advances in both technology
and availability, raising the question of whether it may supplant intraoperative cortical
stimulation mapping. Devices such as functional magnetic resonance imaging (fMRI),
positron emission tomography (PET) and magnetoencephelography (MEG) may aid
in the preoperative planning of the surgical resection strategy, but these techniques
remain too imprecise for complex functions such as language mapping: their sensitivity
(PET, 75%; fMRI, 81%) and specificity (PET, 81%; fMRI, 53%) are suboptimal.24,35
These modalities highlight language-associated areas of indeterminate significance36
and they do not offer real-time information intraoperatively. Consequently, for the
identification of functional language pathways and guidance of safe tumor removal,
these diagnostic imaging tools are still only supplements, not substitutes, for direct
intraoperative stimulation mapping.
SPECIALIZED NEUROANESTHESIA FOR THE AWAKE CRANIOTOMY
An experienced neuroanesthesia team is of paramount importance in not only achieving

an accurate intraoperative language map, but in assuring a short and uncomplicated
postoperative recovery. As compared to asleep craniotomies, awake craniotomies are
associated with less procedural morbidity and fewer postoperative complications31a
testimony to the safety of the neuroanesthetic regimen for awake mapping.
In our practice, patients are premedicated with midazolam and monitoring, including
a blood pressure cuff and an axillary temperature probe, is applied prior to positioning.
Sedation is achieved with propofol (up to 100 +g/kg/min) and remifentanil (0.05+g/
kg/min and higher). Propofol/remifentanil boluses are also used for foley insertion
and Mayfield head holder pin application. As an additional measure, the neurosurgeon
provides scalp analgesia with generous injection of lidocaine/marcaine. Once the bone
flap is removed, all sedatives are discontinued and the patient is asked to hyperventilate
prior to dural opening. The dura is then infiltrated with lidocaine around the middle
meningeal artery to avoid the discomfort associated with dural opening. No sedatives are
administered during mapping and IV methohexital (10 mg/ml) as well as topical ice cold
Ringers solution were available for seizure suppression.37 Once mapping is complete,
sedation is achieved with dexmedetomidine (up to 1+g/kg/min) and remifentanil (0.05
+g/kg/min and higher).
CURRENT INTRAOPERATIVE LANGUAGE MAPPING TECHNIQUES
In general, a limited craniotomy should expose the tumor and up to 2 cm of surrounding

brain. Using bipolar electrodes, cortical mapping is started at a low stimulus (1.5 mA)
and increased to a maximum of 6 mA, if necessary. A constant-current generator delivers
biphasic square wave pulses (each phase, 1.25 milliseconds) in 4-second trains at 60 Hz
across 1-mm bipolar electrodes separated by 5 mm. Stimulation sites (approximately 10
to 20 per subject) can be marked with sterile numbered tickets. Throughout language
mapping, continuous electrocorticography should be used to monitor after discharge
potentials and, therefore, eliminate the chance that speech or naming errors are caused
by subclinical seizure activity. Some groups advocate the use of language mapping along
subcortical white matter pathways, as well.38,39
Speech arrest is based upon blocking number counting without simultaneous motor
response in the mouth or pharynx. Dysarthria can be distinguished from speech arrest
by the absence of perceived or visible involuntary muscle contraction affecting speech.
For naming or reading sites, cortical stimulation is applied for three seconds at sequential
cortical sites during a slide presentation of line drawings or words, respectively. All tested
language sites should be repeatedly stimulated at least three times. A positive essential
site can be defined as an inability to name objects or read words in 66% or greater of the
testing per site. In all cases, a 1 cm margin of tissue should be measured and preserved
around each positive language site in order to protect functional tissue from the resection.40
The extent of resection is directed by targeting contrast-enhancing regions for high-grade
lesions and T2-hyperintense areas for low-grade lesions.
FUNCTIONAL OUTCOME FOLLOWING LANGUAGE MAPPING

FOR DOMINANT HEMISPHERE GLIOMAS
Despite the considerable evidence supporting the use of intraoperative cortical

stimulation mapping of language function, the efficacy of this technique in preserving
functional outcome following aggressive glioma resection remains poorly understood.
Nevertheless, the long-term neurological effects after using this technique for large,
dominant-hemisphere gliomas are important to define in order to accurately advocate
its use.41
Figure 1. Temporal profile of language deficit resolution following resection of dominant hemisphere
gliomas.
Our experience with 250 consecutive dominant hemisphere glioma patients (WHO
grades II-IV) suggests that functional language outcome following awake mapping can
be favorable, even in the setting of an aggressive resection.42 Overall, 159 of these 250
patients (63.6%) had intact speech preoperatively. At 1 week postoperatively, 194 (77.6%)
remained at their baseline language function while 21 (8.4%) worsened and 35 (14.0%)
had new speech deficits. However, by 6 months, 52 (92.8%) of 56 patients with new or
worsened language deficits returned to baseline or better and the remaining 4 (7.1%) were
left with a permanent deficit. Interestingly, among these patients, any additional language
deficit incurred as a result of the surgery improved by 3 months or not all (Fig. 1). Thus,
using language mapping, only 1.6% (4 of 243 surviving patients) of all glioma patients
develop a permanent postoperative language deficit. One explanation for this favorable
postoperative language profile may be our strict adherence to the one-centimeter rule,
first described by Haglund et al, which demonstrated that, for temporal lobe tumors, a
resection margin of one centimeter or more from a language site significantly reduces
postoperative language deficits.30
TAILORED CRANIOTOMIES AND THE VALUE OF NEGATIVE

LANGUAGE MAPPING
In contrast to the classic mapping principles practiced in epilepsy surgery, where

95-100% of operative fields contain a positive language site, a paradigm shift is emerging
Figure 2. Negative language map indicating the percentage of negative stimulations per square centimeter
of the dominant cerebral hemisphere.
in brain tumor language mapping, where positive language sites are not always found prior
to resection (Fig. 2). In our practice, because of our use of tailored cortical exposures, less
than 58% of patients have essential language sites localized within the operative field.
Our experience suggests that it is safe to employ a minimal exposure of the tumor and
resect based upon a negative language map, rather than rely upon a wide craniotomy to
find positive language sites well beyond the lesion.
Negative language mapping, however, does not necessarily guarantee the absence
of eloquent sites. Despite negative brain mapping, permanent postoperative neurologic
deficits have been reported.31 In our experience with 250 consecutive dominant hemisphere
glioma patients, all 4 of our patients with permanent postoperative neurologic deficits had
no positive sites detected prior to their resections. Other cases of unexpected postoperative
deficits have also been attributed to progressive tumor infiltration into functional areas.43
Furthermore, both intraoperative stimulation and functional imaging techniques have
provided evidence for redistribution of functional neural networks in cases of stroke,13,44,45
congenital malformations,46,47 brain injury48 and tumor progression.13,14,49 Not surprisingly,
it has been hypothesized that brain infiltration by gliomas leads to reshaping or local
reorganization of functional networks as well as neosynaptogenesis.50,51 This would
explain the frequent lack of clinical deficit despite glioma growth into eloquent brain
areas,13,49,52 as well as the transient nature of many postoperative deficits. In the case of
language function located in the dominant insula, the brains capacity for compensation
of functional loss has also been associated with recruitment of the left superior temporal
gyrus and left putamen.52
AN EVIDENCE-BASED APPROACH TO UNDERSTANDING THE VALUE

OF EXTENT OF RESECTION
Microsurgical resection remains a critical therapeutic modality for all gliomas.53-56

However, there remains no general consensus in the literature regarding the efficacy of
extent of resection in improving patient outcome.57-63 With the exception of WHO Grade
I tumors, gliomas are difficult to cure with surgery alone and the majority of patients
will experience some form of tumor recurrence. Patients with glioblastomas have median
survival rates of 12.2-18.2 months,64 while those with anaplastic astrocytomas can expect
to survive 41 months, on average.65 Low-grade gliomas carry a better prognosis, though
the vast majority of patients eventually die of their disease and 5-year survival percentages
range from 42-92% in the literature.66-73
For all gliomas, the identification of universally-applicable prognostic factors and
treatment options remains a great challenge. Among the many tumor- and treatment-related
parameters, only patient age and tumor histology have been identified as reliable predictors
of patient prognosis, although functional status can also be statistically significant.
Suprisingly, despite significant advances in brain tumor imaging and intraoperative
technology during the last 15 years, the effect of glioma resection in extending tumor-free
progression and patient survival remains unknown.
Although low-grade and high-grade gliomas are distinct in their biology, clinical
behavior and outcome, understanding the efficacy of surgery remains equally important
for each. With this in mind, an examination of the modern neurosurgical literature (1990
to present) reveals clues as to the role of extent of resection in glioma patient outcome
(Fig. 3).
Figure 3. Trends in the relative numbers of studies in the neurosurgical literature since 1990 statistically
examining the impact of extent of resection on patient survival.
Low-Grade Glioma Extent of Resection Studies
Nineteen studies66-71,74-86 since 1990 have applied statistical analysis to examine the
efficacy of extent of resection in improving survival and delaying tumor progression
among low-grade glioma patients. Five of these studies included volumetric analysis of
extent of resection.78,79,83,86,87 Of the nonvolumetric studies, 12 demonstrated evidence
supporting extent of resection as a statistically significant predictor of either 5-year
survival or 5-year progression-free survival. These studies were published from 1990 to
2005 and most commonly employed a combination of multivariate and univariate analyses
to determine statistical significance. In most instances, extent of resection was defined
on the basis of gross-total versus sub-total resection. Interestingly, only 3 nonvolumetric
studies did not support extent of resection as a predictor of patient outcome. However,
none of these reports evaluated progression-free survival, but instead focused solely on
5-year survival. Of the 5 volumetric low-grade glioma studies reviewed, 4 demonstrated
statistical significance based upon 5-year survival. For their statistical analyses, each
study divided the extent of resection percentages into two categories, although the cutoff
threshold was different in each publication and varied from 75% to 100%.
High-Grade Glioma Extent of Resection Studies
Twenty-seven studies40,56,65,88-111 since 1990 have applied statistical analysis to

examine the efficacy of extent of resection in improving survival and delaying tumor
progression among high-grade glioma patients. Four of these studies included volumetric
analysis of extent of resection.40,56,65,107 Of the nonvolumetric studies, 13 demonstrated
evidence supporting extent of resection as a statistically significant predictor of
either time to tumor progression or overall survival. Although some of these reports
showed extent of resection to significantly affect both tumor progression and overall
survival, every study showed a survival benefit. Ten studies, however, demonstrated
no significant benefit based upon extent of resection. Notably, the distribution of
adjuvant chemotherapy and radiation treatment was comparable among all high-grade
glioma extent of resection studies. Echoing the nonvolumetric study results, half of all
high-grade volumetric studies showed a significant survival advantage with greater
extent of resection.
Although the high-grade studies reviewed were all modern series conducted by expert
neurosurgeons with access to comparable operative technologies, it remains difficult to
define the many inherent disparities between the cases described that may have biased
the reported findings. One factor that may distinguish various high-grade glioma studies
from one another is the distribution of WHO Grade III and IV histologies among the
study patients. After quantifying this parameter in each publication, it remains difficult to
draw any firm conclusions regarding causality. Another dimension of extent of resection
analysis that can greatly affect the reported findings is the method with which the extent
of resection is calculated. Although volumetric MRI analysis is now the gold-standard,
many centers still rely upon the surgeons report or two-dimensional analysis based
upon postoperative MR imaging. However, in examining the distribution of extent of
resection methodologies and comparing them to the findings for both low-grade and
high-grade gliomas, there appears to be a relatively even distribution of techniques for
each study category.
Quantification of Improvement in Patient Outcome
For both low- and high-grade gliomas, one can define the mean survival time
associated with sub-total versus gross-total resection in the modern neurosurgical
literature. Although the level of evidence available for each tumor category does not
permit a statistical meta-analysis, this measurement provides an overall estimation of the
additional survival time these studies suggest may be gained through a greater extent of
resection. Not surprisingly, the effect of a greater extent of resection was more pronounced
in the low-grade glioma studies, where the mean survival was extended from 61.1 to
90 months. Among the high-grade gliomas, the improvement was more modest, with
an increase from 64.9 to 75.2 months in WHO Grade III gliomas and from 11.3 to 14.5
months in Grade IV gliomas.
CONCLUSION
Intraoperative stimulation mapping is a reliable, robust method to maximize resection

and minimize morbidity, even when removing gliomas within or near adjacent language
pathways. Unlike motor function, speech and language are variably distributed and
widely represented, thus emphasizing the utility of language mapping in this particular
patient population. Using modern language mapping techniques, in conjunction with
standardized neuroanesthesia and neuromonitoring, the postoperative language resolution
profile following glioma resection may be predictable. Specifically, in our experience, any
additional language deficit incurred as a result of the surgery will improve by 3 months
or not all. Our experience also emphasizes the value of negative language mapping in
the setting of a tailored cortical exposure. The value of extent of resection, however,
remains less clear. Based upon the available studies for both low-grade and high-grade
hemispheric gliomas in the literature, there is growing evidence, however, that a more
extensive surgical resection may be associated with a more favorable life expectancy
for both low-grade and high-grade glioma patients. Because no Class I evidence exists
to support a particular management paradigm, the optimal combination of surgery, a
chemotherapeutic agent and radiation therapy remains unknown. Since it is unlikely that
a prospective, randomized study will be designed to address these issues, retrospective,
matched studies or prospective observational trials may be a more practical solution.
REFERENCES
1. Foerster O. The cerebral cortex of man. Lancet 1931; 2:309-312.

2. Penfield W, Bolchey E. Somatic motor and sensory representation in the cerebral cortex of man as studied
by electrical stimulation. Brain 1937; 60:389-443.
3. Penfield W, Erickson TC. Epilepsy and cerebral localization. A study of the mechanism, treatment and
prevention of epileptic seizures. Springfield: Charles C. Thomas, 1941.
4. Penfield W, Rasmussen T. Secondary sensory and motor representation. New York: Macmillan, 1950.
5. Ranck JB Jr. Which elements are excited in electrical stimulation of mammalian central nervous system: a
review. Brain Res 1975; 98:417-440.
6. Haglund MM, Ojemann GA, Blasdel GG. Optical imaging of bipolar cortical stimulation. J Neurosurg
1993; 78:785-793.
7. Haglund MM, Ojemann GA, Hochman DW. Optical imaging of epileptiform and functional activity in
human cerebral cortex. Nature 1992; 358:668-671.
8. Herholz K, Thiel A, Wienhard K et al. Individual functional anatomy of verb generation. Neuroimage
1996; 3:185-194.
9. Ojemann G, Ojemann J, Lettich E et al. Cortical language localization in left, dominant hemisphere. An
electrical stimulation mapping investigation in 117 patients. J Neurosurg 1989; 71:316-326.
10. Ojemann GA, Whitaker HA. Language localization and variability. Brain Lang 1978; 6:239-260.
11. Ojemann GA. Individual variability in cortical localization of language. J Neurosurg 1979; 50:164-169.
12. Ojemann JG, Miller JW, Silbergeld DL. Preserved function in brain invaded by tumor. Neurosurgery 1996;
39:253-258; discussion 258-259.
13. Seitz RJ, Huang Y, Knorr U et al. Large-scale plasticity of the human motor cortex. Neuroreport 1995;
6:742-744.
14. Wunderlich G, Knorr U, Herzog H et al. Precentral glioma location determines the displacement of cortical
hand representation. Neurosurgery 1998; 42:18-26; discussion 26-17.
15. Quinones-Hinojosa A, Ojemann SG, Sanai N et al. Preoperative correlation of intraoperative cortical
mapping with magnetic resonance imaging landmarks to predict localization of the Broca area. J Neurosurg
2003; 99:311-318.
16. Seghier ML, Lazeyras F, Pegna AJ et al. Variability of fMRI activation during a phonological and semantic
language task in healthy subjects. Hum Brain Mapp 2004; 23:140-155.
17. Tzourio-Mazoyer N, Josse G, Crivello F et al. Interindividual variability in the hemispheric organization
for speech. Neuroimage 2004; 21:422-435.
18. Turkeltaub PE, Eden GF, Jones KM et al. Meta-analysis of the functional neuroanatomy of single-word
reading: method and validation. Neuroimage 2002; 16:765-780.
19. Tzourio N, Crivello F, Mellet E et al. Functional anatomy of dominance for speech comprehension in left
handers vs right handers. Neuroimage 1998; 8:1-16.
20. Dehaene S, Dupoux E, Mehler J et al. Anatomical variability in the cortical representation of first and
second language. Neuroreport 1997; 8:3809-3815.
21. Steinmetz H, Seitz RJ. Functional anatomy of language processing: neuroimaging and the problem of
individual variability. Neuropsychologia 1991; 29:1149-1161.
22. Josse G, Herve PY, Crivello F et al. Hemispheric specialization for language: Brain volume matters. Brain
Res 2006; 1068:184-193.
23. Davies KG, Maxwell RE, Jennum P et al. Language function following subdural grid-directed temporal
lobectomy. Acta Neurol Scand 1994; 90:201-206.
24. FitzGerald DB, Cosgrove GR, Ronner S et al. Location of language in the cortex: a comparison between
functional MR imaging and electrocortical stimulation. Am J Neuroradiol 1997; 18:1529-1539.
25. Skirboll SS, Ojemann GA, Berger MS et al. Functional cortex and subcortical white matter located within
gliomas. Neurosurgery 1996; 38:678-684; discussion 684-675.
26. Ojemann GA. Models of the brain organization for higher integrative functions derived with electrical
stimulation techniques. Hum Neurobiol 1982; 1:243-249.
27. Ojemann GA. Cortical organization of language. J Neurosci 1991; 11:2281-2287.
28. Ojemann GA, Creutzfeldt OD. Nervous system V, Part 2. In: Plum F, ed. Handbook of Physiology.
Bethesda: American Physiological Society Press, 1987:675-700.
29. Ojemann GA. Organization of language cortex derived from investigations during neurosurgery. Seminars
in Neuroscience 1990; 2:297-305.
30. Haglund MM, Berger MS, Shamseldin M et al. Cortical localization of temporal lobe language sites in
patients with gliomas. Neurosurgery 1994; 34:567-576; discussion 576.
31. Taylor MD, Bernstein M. Awake craniotomy with brain mapping as the routine surgical approach to treating
patients with supratentorial intraaxial tumors: a prospective trial of 200 cases. J Neurosurg 1999; 90:35-41.
32. Hochberg FH, Pruitt A. Assumptions in the radiotherapy of glioblastoma. Neurology 1980; 30:907-911.
33. Wallner KE, Galicich JH, Krol G et al. Patterns of failure following treatment for glioblastoma multiforme
and anaplastic astrocytoma. Int J Radiat Oncol Biol Phys 1989; 16:1405-1409.
34. Ebeling U, Steinmetz H, Huang YX et al. Topography and identification of the inferior precentral sulcus
in MR imaging. Am J Roentgenol 1989; 153:1051-1056.
35. Herholz K, Reulen HJ, von Stockhausen HM et al. Preoperative activation and intraoperative stimulation of
language-related areas in patients with glioma. Neurosurgery 1997; 41:1253-1260; discussion 1260-1252.
36. Carpentier A, Pugh KR, Westerveld M et al. Functional MRI of language processing: dependence on input
modality and temporal lobe epilepsy. Epilepsia 2001; 42:1241-1254.
37. Sartorius CJ, Berger MS. Rapid termination of intraoperative stimulation-evoked seizures with application
of cold Ringers lactate to the cortex. Technical note. J Neurosurg 1998; 88:349-351.
38. Duffau H, Capelle L, Denvil D et al. Usefulness of intraoperative electrical subcortical mapping during
surgery for low-grade gliomas located within eloquent brain regions: functional results in a consecutive
series of 103 patients. J Neurosurg 2003; 98:764-778.
39. Duffau H, Capelle L, Sichez N et al. Intraoperative mapping of the subcortical language pathways using
direct stimulations. An anatomo-functional study. Brain 2002; 125:199-214.
40. Lacroix M, Abi-Said D, Fourney DR et al. A multivariate analysis of 416 patients with glioblastoma
multiforme: prognosis, extent of resection and survival. J Neurosurg 2001; 95:190-198.
41. Sanai N, Berger MS. Mapping the horizon: techniques to optimize tumor resection before and during
surgery. Clin Neurosurg 2008; 55:14-19.
42. Sanai N, Mirzadeh Z, Berger MS. Functional outcome after language mapping for glioma resection. New
Engl J Med 2008; 358:18-27.
43. Berger MS. Lesions in functional (eloquent) cortex and sub-cortical white matter. Clin Neurosurg 1993;
41:443-463.
44. Chollet F, DiPiero V, Wise RJ et al. The functional anatomy of motor recovery after stroke in humans: a
study with positron emission tomography. Ann Neurol 1991; 29:63-71.
45. Weder B, Seitz RJ. Deficient cerebral activation pattern in stroke recovery. Neuroreport 1994; 5:457-460.
46. Lewine JD, Astur RS, Davis LE et al. Cortical organization in adulthood is modified by neonatal infarct:
a case study. Radiology 1994; 190:93-96.
47. Maldjian J, Atlas SW, Howard RS 2nd et al. Functional magnetic resonance imaging of regional brain
activity in patients with intracerebral arteriovenous malformations before surgical or endovascular therapy.
J Neurosurg 1996; 84:477-483.
48. Grady MS, Jane JA, Steward O. Synaptic reorganization within the human central nervous system following
injury. J Neurosurg 1989; 71:534-537.
49. Fandino J, Kollias SS, Wieser HG et al. Intraoperative validation of functional magnetic resonance imaging
and cortical reorganization patterns in patients with brain tumors involving the primary motor cortex. J
Neurosurg 1999; 91:238-250.
50. Duffau H, Capelle L, Denvil D et al. Functional recovery after surgical resection of low grade gliomas in
eloquent brain: hypothesis of brain compensation. J Neurol Neurosurg Psychiatry 2003; 74:901-907.
51. Thiel A, Herholz K, Koyuncu A et al. Plasticity of language networks in patients with brain tumors:
a positron emission tomography activation study. Ann Neurol 2001; 50:620-629.
52. Duffau H, Bauchet L, Lehericy S et al. Functional compensation of the left dominant insula for language.
Neuroreport 2001; 12:2159-2163.
53. Black P. Management of malignant glioma: role of surgery in relation to multimodality therapy. J Neurovirol
1998; 4:227-236.
54. Yasargil MG, Kadri PA, Yasargil DC. Microsurgery for malignant gliomas. J Neurooncol 2004; 69:67-81.
55. Guthrie BL, Laws ER Jr. Supratentorial low-grade gliomas. Neurosurg Clin N Am 1990; 1:37-48.
56. Keles GE, Anderson B, Berger MS. The effect of extent of resection on time to tumor progression and survival
in patients with glioblastoma multiforme of the cerebral hemisphere. Surg Neurol 1999; 52:371-379.
57. Proescholdt MA, Macher C, Woertgen C et al. Level of evidence in the literature concerning brain tumor
resection. Clin Neurol Neurosurg 2005; 107:95-98.
58. Sawaya R. Extent of resection in malignant gliomas: a critical summary. J Neurooncol 1999; 42:303-305.
59. Grant R MS. Biopsy versus resection for malignant glioma (Review). The Cochrane Library, 2006.
60. Nazzaro JM, Neuwelt EA. The role of surgery in the management of supratentorial intermediate and
high-grade astrocytomas in adults. J Neurosurg 1990; 73:331-344.
61. Pierga JY, Hoang-Xuan K, Feuvret L et al. Treatment of malignant gliomas in the elderly. J Neurooncol
1999; 43:187-193.
62. Hess KR. Extent of resection as a prognostic variable in the treatment of gliomas. J Neurooncol 1999;
42:227-231.
63. Sanai N, Berger MS. Glioma extent of resection and its impact on patient outcome. Neurosurgery 2008;
62:753-764; discussion 264-756.
New Engl J Med 2005; 352:997-1003.
65. Keles GE, Chang EF, Lamborn KR et al. Volumetric extent of resection and residual contrast enhancement
on initial surgery as predictors of outcome in adult patients with hemispheric anaplastic astrocytoma.
J Neurosurg 2006; 105:34-40.
66. Leighton C, Fisher B, Bauman G et al. Supratentorial low-grade glioma in adults: an analysis of prognostic
factors and timing of radiation. J Clin Oncol 1997; 15:1294-1301.
67. Nakamura M, Konishi N, Tsunoda S et al. Analysis of prognostic and survival factors related to treatment
of low-grade astrocytomas in adults. Oncology 2000; 58:108-116.
68. Philippon JH, Clemenceau SH, Fauchon FH et al. Supratentorial low-grade astrocytomas in adults.
Neurosurgery 1993; 32:554-559.
69. Rajan B, Pickuth D, Ashley S et al. The management of histologically unverified presumed cerebral gliomas
with radiotherapy. Int J Radiat Oncol Biol Phys 1994; 28:405-413.
70. Shaw E, Arusell R, Scheithauer B et al. Prospective randomized trial of low- versus high-dose radiation
therapy in adults with supratentorial low-grade glioma: initial report of a North Central Cancer Treatment
Group/Radiation Therapy Oncology Group/Eastern Cooperative Oncology Group study. J Clin Oncol
2002; 20:2267-2276.
71. Yeh SA, Ho JT, Lui CC et al. Treatment outcomes and prognostic factors in patients with supratentorial
low-grade gliomas. Br J Radiol 2005; 78:230-235.
72. Shaw EG, Daumas-Duport C, Scheithauer BW et al. Radiation therapy in the management of low-grade
supratentorial astrocytomas. J Neurosurg 1989; 70:853-861.
73. Laws ER Jr, Taylor WF, Clifton MB et al. Neurosurgical management of low-grade astrocytoma of the
cerebral hemispheres. J Neurosurg 1984; 61:665-673.
74. North CA, North RB, Epstein JA et al. Low-grade cerebral astrocytomas. Survival and quality of life after
radiation therapy. Cancer 1990; 66:6-14.
75. Ito S, Chandler KL, Prados MD et al. Proliferative potential and prognostic evaluation of low-grade
astrocytomas. J Neurooncol 1994; 19:1-9.
76. Nicolato A, Gerosa MA, Fina P et al. Prognostic factors in low-grade supratentorial astrocytomas: a
uni-multivariate statistical analysis in 76 surgically treated adult patients. Surg Neurol 1995; 44:208-221;
discussion 221-203.
77. Whitton AC, Bloom HJ. Low grade glioma of the cerebral hemispheres in adults: a retrospective analysis
of 88 cases. Int J Radiat Oncol Biol Phys 1990; 18:783-786.
78. Shibamoto Y, Kitakabu Y, Takahashi M et al. Supratentorial low-grade astrocytoma. Correlation of computed
tomography findings with effect of radiation therapy and prognostic variables. Cancer 1993; 72:190-195.
79. Karim AB, Maat B, Hatlevoll R et al. A randomized trial on dose-response in radiation therapy of low-grade
cerebral glioma: European Organization for Research and Treatment of Cancer (EORTC) Study 22844.
Int J Radiat Oncol Biol Phys 1996; 36:549-556.
80. Scerrati M, Roselli R, Iacoangeli M et al. Prognostic factors in low grade (WHO grade II) gliomas of the
cerebral hemispheres: the role of surgery. J Neurol Neurosurg Psychiatry 1996; 61:291-296.
81. Lote K, Egeland T, Hager B et al. Survival, prognostic factors and therapeutic efficacy in low-grade glioma:
a retrospective study in 379 patients. J Clin Oncol 1997; 15:3129-3140.
82. Peraud A, Ansari H, Bise K et al. Clinical outcome of supratentorial astrocytoma WHO grade II. Acta
Neurochirurgica 1998; 140:1213-1222.
83. van Veelen ML, Avezaat CJ, Kros JM et al. Supratentorial low grade astrocytoma: prognostic factors,
dedifferentiation and the issue of early versus late surgery. J Neurol Neurosurg Psychiatry 1998; 64:581-587.
84. Bauman G, Pahapill P, Macdonald D et al. Low grade glioma: a measuring radiographic response to
radiotherapy. Can J Neurol Sci 1999; 26:18-22.
85. Johannesen TB, Langmark F, Lote K. Progress in long-term survival in adult patients with supratentorial
low-grade gliomas: a population-based study of 993 patients in whom tumors were diagnosed between
1970 and 1993. J Neurosurg 2003; 99:854-862.
86. Claus EB, Horlacher A, Hsu L et al. Survival rates in patients with low-grade glioma after intraoperative
magnetic resonance image guidance. Cancer 2005; 103:1227-1233.
87. Smith JS, Chang EF, Lamborn KR et al. Role of extent of resection in the long-term outcome of low-grade
hemispheric gliomas. J Clin Oncol 2008; 26:1338-1345.
88. Vecht CJ, Avezaat CJ, van Putten WL et al. The influence of the extent of surgery on the neurological
function and survival in malignant glioma. A retrospective analysis in 243 patients. J Neurol Neurosurg
Psychiatry 1990; 53:466-471.
89. Shibamoto Y, Yamashita J, Takahashi M et al. Supratentorial malignant glioma: an analysis of radiation
therapy in 178 cases. Radiother Oncol 1990; 18:9-17.
90. Curran WJ Jr, Scott CB, Horton J et al. Does extent of surgery influence outcome for astrocytoma with
atypical or anaplastic foci (AAF)? A report from three Radiation Therapy Oncology Group (RTOG)
trials. J Neurooncol 1992; 12:219-227.
91. Simpson JR, Horton J, Scott C et al. Influence of location and extent of surgical resection on survival of
patients with glioblastoma multiforme: results of three consecutive Radiation Therapy Oncology Group
(RTOG) clinical trials. Int J Radiat Oncol Biol Phys 1993; 26:239-244.
92. Dinapoli RP, Brown LD, Arusell RM et al. Phase III comparative evaluation of PCNU and carmustine
combined with radiation therapy for high-grade glioma. J Clin Oncol 1993; 11:1316-1321.
93. Jeremic B, Grujicic D, Antunovic V et al. Influence of extent of surgery and tumor location on treatment
outcome of patients with glioblastoma multiforme treated with combined modality approach. J Neurooncol
1994; 21:177-185.
94. Nitta T, Sato K. Prognostic implications of the extent of surgical resection in patients with intracranial
malignant gliomas. Cancer 1995; 75:2727-2731.
95. Barker FG 2nd, Prados MD, Chang SM et al. Radiation response and survival time in patients with
glioblastoma multiforme. J Neurosurg 1996; 84:442-448.
96. Brown PD, Maurer MJ, Rummans TA et al. A prospective study of quality of life in adults with newly
diagnosed high-grade gliomas: the impact of the extent of resection on quality of life and survival.
Neurosurgery 2005; 57:495-504; discussion 495-504.
97. Buckner JC, Schomberg PJ, McGinnis WL et al. A phase III study of radiation therapy plus carmustine with
or without recombinant interferon-alpha in the treatment of patients with newly diagnosed high-grade
glioma. Cancer 2001; 92:420-433.
98. Lamborn KR, Chang SM, Prados MD. Prognostic factors for survival of patients with glioblastoma: recursive
partitioning analysis. Neuro Oncol 2004; 6:227-235.
99. Stark AM, Nabavi A, Mehdorn HM et al. Glioblastoma multiforme-report of 267 cases treated at a single
institution. Surg Neurol 2005; 63:162-169; discussion 169.
100. Ushio Y, Kochi M, Hamada J et al. Effect of surgical removal on survival and quality of life in patients with
supratentorial glioblastoma. Neurol Med Chir 2005; 45:454-460; discussion 460-451.
101. Duncan GG, Goodman GB, Ludgate CM et al. The treatment of adult supratentorial high grade astrocytomas.
J Neurooncol 1992; 13:63-72.
102. Hollerhage HG, Zumkeller M, Becker M et al. Influence of type and extent of surgery on early results and
survival time in glioblastoma multiforme. Acta Neurochirurgica 1991; 113:31-37.
103. Huber A, Beran H, Becherer A et al. (Supratentorial glioma: analysis of clinical and temporal parameters
in 163 cases). Neurochirurgia 1993; 36:189-193.
104. Kowalczuk A, Macdonald RL, Amidei C et al. Quantitative imaging study of extent of surgical resection
and prognosis of malignant astrocytomas. Neurosurgery 1997; 41:1028-1036; discussion 1036-1028.
105. Levin VA, Yung WK, Bruner J et al. Phase II study of accelerated fractionation radiation therapy with
carboplatin followed by PCV chemotherapy for the treatment of anaplastic gliomas. Int J Radiat Oncol
Biol Phys 2002; 53:58-66.
106. Phillips TL, Levin VA, Ahn DK et al. Evaluation of bromodeoxyuridine in glioblastoma multiforme:
a Northern California Cancer Center Phase II study. Int J Radiat Oncol Biol Phys 1991; 21:709-714.
107. Pope WB, Sayre J, Perlina A et al. MR imaging correlates of survival in patients with high-grade gliomas.
AJNR 2005; 26:2466-2474.
108. Prados MD, Gutin PH, Phillips TL et al. Highly anaplastic astrocytoma: a review of 357 patients treated
between 1977 and 1989. Int J Radiat Oncol Biol Phys 1992; 23:3-8.
109. Puduvalli VK, Hashmi M, McAllister LD et al. Anaplastic oligodendrogliomas: prognostic factors for
tumor recurrence and survival. Oncology 2003; 65:259-266.
110. Sandberg-Wollheim M, Malmstrom P, Stromblad LG et al. A randomized study of chemotherapy with
procarbazine, vincristine and lomustine with and without radiation therapy for astrocytoma grades 3 and/
or 4. Cancer 1991; 68:22-29.
111. Tortosa A, Vinolas N, Villa S et al. Prognostic implication of clinical, radiologic and pathologic features
in patients with anaplastic gliomas. Cancer 2003; 97:1063-1071.
CHAPTER 3
RECENT MEDICAL MANAGEMENT

OF GLIOBLASTOMA
Derek R. Johnson and Susan M. Chang*

Department of Neurological Surgery, University of California San Francisco, San Francisco, California, USA
*Corresponding Author: Susan M. ChangEmail: changs@neurosurg.ucsf.edu
Abstract: This chapter contains an overview of standard of care and experimental chemotherapy
treatments for glioblastoma multiforme (GBM). We discuss the role of alkylating
agents, focusing primarily on temozolomide (TMZ) which, in combination with
radiation therapy, is part of the standard of care treatment for newly diagnosed
GBM. TMZ has proven both well tolerated and effective in prolonging patient
survival, but tumor recurrence remains the rule. We review the development and
use of molecularly targeted agents, low molecular weight kinase inhibitors and
monoclonal antibodies that interrupt the signaling pathways exploited by tumors
to grow, migrate and avoid apoptosis.
INTRODUCTION
Until relatively recently the treatment of GBM with chemotherapeutic agents remained
controversial due to lack of proven efficacy. Standard therapy for newly diagnosed GBM
consisted of maximum safe resection1 followed by fractionated external-beam radiotherapy.2
Chemotherapy was often given concurrently with radiation or as an adjuvant, at time of
initial diagnosis but after the completion of surgery and radiation, but its use was inconsistent
and geographically variable. This began to change in the late 1980s with the investigation
of temozolomide (TMZ), an orally administered alkylating agent. In 2005, a large Phase 3
trial of TMZ given concurrently with radiation therapy and as an adjuvant after radiation
for newly diagnosed GBM showed a significant survival benefit over radiation alone.3
This regimen has since become the standard of care for patients with newly diagnosed
GBM. Further trials are ongoing to investigate the relative contributions of concurrent
and adjuvant TMZ in treating GBM and a variety of alternative TMZ administration

26
RECENT MEDICAL MANAGEMENT OF GLIOBLASTOMA 27
schedules and sensitizing agents are being tested to maximize efficacy and minimize
side effects. Another recent approach to GBM therapy is the use of molecularly targeted
agentslow molecular weight kinase inhibitors and monoclonal antibodies that act as
specific inhibitors of the signaling pathways implicated in tumor growth and survival.
Angiogenesis has long been recognized as a key player in tumor growth and vascular
proliferation is a defining feature of GBM, so it is little surprise that therapies targeting the
vascular endothelial growth factor (VEGF) system have been met with great enthusiasm
in the neuro-oncology community. A wide variety of other signaling pathway agents are
being evaluated both as monotherapies and in combination with traditional alkylating
chemotherapy or alongside other targeted agents. These advances in medical management
of GBM, in combination with the surgical and immunotherapeutic approaches discussed
elsewhere in this volume, provide ample reason for optimism about our ability to continue
to extend the survival time of patients with GBM.
CYTOTOXIC CHEMOTHERAPY FOR GLIOBLASTOMA MULTIFORME
Temozolomide
Temozolomide (TMZ) is an orally administered methylating agent with excellent

bioavailability and blood-brain barrier penetration.4 TMZ is a prodrug which is metabolized
into 3-methyl-(triazen-1-yl)imidazole-4-carboxamide (MTIC). MTIC methylates DNA
at a number of sites, most frequently N7-guanine and N3-adenine. While the conversion
of O6-guanine to 06-methylguanine is less frequent, accounting for only about 5% of
all methylation events, it induces a futile cycle of DNA mismatch repair which leads to
double-stranded breaks and cellular apoptosis,5 thus serving as the primary mediator of
TMZ-mediated cytotoxicity.6
Temozolomide was first evaluated in glioma as a treatment of recurrent malignant
glioma following radiation therapy,7-10 where it was shown to have good antitumor
activity as a monotherapy and an acceptable side effect profile. Six-month and overall
progression free survival (PFS) were superior to procarbazine (PCB) in an open-label
Phase II trial of GBM patients at time of first relapse.11 Following the success of this
approach, TMZ was evaluated in combination with radiation therapy as a treatment for
newly diagnosed GBM.12-14 A pivotal Phase III study by the European Organization for
Research and Treatment of Cancer (EORTC) and National Cancer Institute of Canada
Clinical Trials Group (NCIC) published in 2005 randomized patients to standard radiation
therapy or radiation therapy plus TMZ at a dose of 75 mg/m2/d daily during the period
of radiation therapy followed by up to 6 cycles of adjuvant TMZ at a dose of 150-200
mg/m2/d for 5 consecutive days in each 28 day cycle.3 The addition of TMZ increased
two-year survival from 10.4% to 26.5% and this regimen quickly became the accepted
standard of care for treatment of newly diagnosed GBM. The final results of this trial were
published in 2009 and showed a durable response to treatment with an overall survival
of 9.8% at five years in patients treated with radiation plus TMZ and 1.9% in patients
treated with radiation alone.15
The ideal delivery schedule of TMZ is not yet clear. While the standard schedule
as previously described is widely adopted, the relative contributions of concurrent and
adjuvant TMZ remain to be defined. TMZ is known to enhance the response of GBM to
radiation in preclinical testing and it is possible that the action of concurrent TMZ at the
time of RT is responsible for the majority of treatment effect.16 One single-institution

case series of patients with newly diagnosed GBM described the use of radiation therapy
with concurrent daily TMZ but without adjuvant therapy following RT and reported
results similar to the standard regimen.17 Nevertheless, most neuro-oncologists believe
that adjuvant TMZ following radiation and concurrent TMZ does have additive value
and a variety of regimens are under investigation, typically involving dose-dense
cumulative monthly TMZ doses than exceed that of the standard treatment. Examples
include TMZ at 75-100 mg/m2 for 21 days of every 28 day cycle and 150 mg/m2 on a
7-days-on/7-days-off schedule.
There are several mechanisms of tumor resistance to TMZ; the two with greatest
implications for GBM therapy are the O6-methylguanine DNA methyltransferase (MGMT)
and poly(ADP-ribose) polymerase (PARP) systems. Methylation of O6-guanine, which
as previously mentioned is the primary mediator of TMZ cytotoxicity, is repaired by the
enzyme MGMT. MGMT is irreversibly inactivated in the process and de novo synthesis is
required to maintain enzyme activity.18 While MGMT is widely expressed in both normal
human tissue and neoplasms,19 MGMT protein expression is heterogeneous within tumors
due to frequent epigenetic silencing via gene promoter hypermethylation.20 As would be
predicted from its mechanism, the level of MGMT protein within tumor tissue correlates
with response to TMZ and MGMT promoter methylation predicts increased survival in
patients with malignant glioma.21-23,15 The irreversible inactivation of MGMT during DNA
repair provides an a priori basis for the potential utility of dose-dense TMZ regimens by
suggesting that it may be possible to effectively overcome this chemotherapy resistance
mechanism by designing a regimen in which increased or prolonged TMZ exposure leads to
depletion of MGMT. MGMT can also be inhibited directly by O6-benzylguanine (O6-BG)
and related agents and a number of recent studies have examined the combination of
TMZ and MGMT inhibitors.24-25 Whereas MGMT repairs 06-methylguanine, the PARP
enzyme mediates repair of TMZ-induced methylation of N7-guanine and N3-adenine and
is a large part of the reason that methylation of non-O6-guanine targets is not typically
cytotoxic.26 TMZ is currently being evaluated in combination of inhibitors of the PARP
DNA repair system.
Temozolomide is generally well-tolerated and in the EORTC-NCIC trial no decrease in
health-related quality of life resulted from the addition of TMZ to standard radiotherapy.27
Dose-dependent myelosuppression is the primary dose-limiting side effect of TMZ
therapy. In the Phase III trial by Stupp et al 7% of patients had a Grade 3 or 4 hematologic
toxic effect during the concurrent TMZ phase and 14% of patients developed a Grade 3
or 4 hematologic toxic effect, typically thrombocytopenia, during the adjuvant phase.3
When severe myelosuppression occurs following TMZ therapy, a 25% dose-reduction of
subsequent cycles is typically recommended. Other known side effects of TMZ include
nausea, fatigue, headache and constipation. Opportunistic infections in the setting of
lymphopenia have been reported during daily concurrent TMZ therapy and with some
prolonged adjuvant regimens and Pneumocystis carinii pneumonia (PCP) prophylaxis
is sometimes prescribed in these settings.28
Nontemozolomide Systemic Chemotherapy
Prior to the introduction of TMZ, the use of adjuvant chemotherapy along with
surgery and radiation was inconsistent and geographically variable. When adjuvant therapy
was employed, nitrosourea class compounds such as carmustine (BCNU) or lomustine
(CCNU) were commonly chosen for their ability to cross the blood-brain barrier. CCNU
was often combined with procarbazine, a DNA alkylating agent and vincristine, which
disrupts microtubule formation, to create the regimen known as PCV. Despite decades of
evaluation, no nitrosourea-based adjuvant treatment was unambiguously shown to confer a
survival benefit beyond that achieved by radiation therapy alone in a Phase 3 randomized
controlled trial. Several meta-analyses of have examined the available RCT data, the most
rigorous of which showed a small but significant survival benefit equivalent to a 6%
absolute increase in 1-year survival.29 While TMZ has now supplanted the nitrosoureas
as the adjuvant agent of choice as a monotherapy, combination therapies such as TMZ/
CCNU are being investigated which may offer survival benefits superior to TMZ alone.30
Carboplatin is a platinum-based compound that interferes with DNA replication by
producing cross-links within DNA strands. Intravenous carboplatin has modest activity
against recurrent malignant glioma both in chemotherapy naive patients31 and in extensively
pretreated patients.32 Low-dose carboplatin has been shown to act as a radiosensitizer
in some non-CNS solid tumors, but the addition of carboplatin to radiation therapy for
newly diagnosed malignant glioma did not increase survival time.33 Typical side effects
include myelosuppression, nausea and fatigue.
Etoposide is a topoisomerase II inhibitor that can be administered orally or
intravenously. Etoposide has been evaluated as a monotherapy34-35 and as a part of many
different multi-drug regimens.36-37 One study of oral etoposide for recurrent malignant
glioma showed a combined 42% response and stable disease rate, but disappointingly
time to progression was less than eight weeks.35
Despite the success of concurrent and adjuvant TMZ, tumor progression after
therapy remains the rule and cytotoxic chemotherapy may have an important role in
the treatment of recurrent GBM. Phase II trials of the combinations of procarbazine
(PCB) and fotemustine38 as well as BCNU and irinotecan (CPT-11)39 in the treatment of
patients with recurrent GBM following TMZ therapy have suggested antitumor activity.
Irinotecan is also often used in combination with bevacizumab, an anti-VEGF therapy
that will be discussed later.
Surgically Implanted Carmustine (BCNU) Coated Wafers (Gliadel)
The relatively minimal antitumor activity of systemically administered

nitrosourea-based chemotherapy has led to a variety of alternative approaches to
chemotherapy delivery. The most studied method is the implantation of BCNU impregnated
wafers (Gliadel) into the tumor cavity at the time of resection. These wafers then provide
a controlled release of BCNU over a period of 2 to 3 weeks. This approach has several
theoretical advantages over systemic chemotherapy, including the ability to deliver a
greater dose to residual tumor and a more benign systemic side effect profile. Gliadel has
been evaluated both as a therapy for newly diagnosed GBM40-41 and as a salvage therapy
for recurrent GBM.42 A recent meta-analysis of the use of Gliadel for high grade glioma
concluded that the addition of Gliadel to radiotherapy led to increased survival relative
to radiotherapy alone for newly diagnosed tumors, but that there was no significant
increase in survival with Gliadel therapy for recurrent disease.43 Experience has shown
that Gliadel can be used safely in patients also receiving TMZ44 and an ongoing Phase
2 trial of Gliadel plus radiation and standard concurrent and adjuvant TMZ for newly
diagnosed GBM is ongoing. Likewise, trials currently underway to evaluate the use of
Gliadel along with molecularly targeted agents for recurrent GBM.
MOLECULARLY TARGETED THERAPIES

FOR GLIOBLASTOMA MULTIFORME
Tumor Growth Factor Pathways and Agents
Epidermal Growth Factor
Epidermal growth factor (EGF) and the epidermal growth factor receptor (EGFR)
are recognized for their role in tumor growth.45 EGF binding to the extracellular receptor
domain of an EGFR causes activation of the intracellular tyrosine kinase domain and the
initiation of a large number of signaling cascades. Excessive EGFR signaling in glioma
can result from overexpression of normal EGFR due to genetic mutations leading to
polysomy or amplification46 or EGFR itself can be subject to mutation. The most common
EGFR mutation in glioma, EGFRvIII, is a deletion from the extracellular ligand binding
domain which results in activity of the intracellular tyrosine kinase in the absence of EGF
binding.47 Two EGF-triggered signaling cascades important in glioma formation are the
RAS-RAF-MEK-MAPK pathway and the PI3K-Akt-mTOR pathway.48
A number of EGFR-targeted therapies are being investigated as treatments for
malignant glioma. Antibodies to EGFR, such as cetuximab and panitumumab, have been
evaluated in preclinical testing and a Phase I/II is currently examining the addition of
cetuximab to TMZ and radiation for newly diagnosed GBM.49 Small molecule inhibitors
of the EGFR kinase domain, such as erlotinib, are further along in development. Small
molecule inhibitors have a theoretical advantage over antibodies in EGFR targeting in that
they act on the intracellular tyrosine kinase domain rather than the extracellular receptor
domain, allowing them to modulate the activity of the important EGFR-vIII mutation
of EGFR. Despite this advantage, clinical trials of erlotinib have been disappointing. A
randomized Phase II trial of erlotinib versus TMZ or carmustine showed less 6-month
progression free survival in the erlotinib arm.50 One recent Phase II trial of erlotinib in
combination with temozolomide and radiation therapy in patients with newly diagnosed
glioblastoma or gliosarcoma demonstrated improved survival relative to historical
controls51 while a similar trial did not suggest benefit.52 Further studies are ongoing to
better define treatment effect and identify any pathologically defined subpopulations
more likely to respond to therapy. Previous studies of erlotinib and gefitinib, another
small molecule EGFR inhibitor, suggested that they are of greatest benefit to patients
with tumors that coexpress EGFRvIII and PTEN.53 This data remains controversial and
awaits prospective confirmation.
The most common side effect of anti-EGFR therapy is a papulopustular rash which
is typically self-limited, although postinflammatory hyperpigmentation is often seen.
Development of the characteristic rash has been linked to tumor response to therapy and
may be a useful surrogate marker of response.54 Gastrointestinal side effects including
diarrhea, nausea and vomiting are also common following anti-EGFR therapy. Interstitial
lung disease (ILD), which can be fatal, represents a rare but severe toxicity of the EGFR
tyrosine kinase inhibitors.55
Platelet Derived Growth Factor
The platelet derived growth factor (PDGF) pathway is quite similar to the
EGFR pathway. Both PDGF and PDGFR are frequently over-expressed in malignant
gliomas. As in EGF, PDGF receptor activation leads to the initiation of multiple

downstream signaling cascades including but not limited to the PI3K-Akt-mTOR and
RAS-RAF-MEK-MAPK systems.
The prototypical anti-PDGFR therapy is imatinib, a PDGF receptor tyrosine kinase
inhibitor that also has action against Bcl-Abl and c-kit. Imatinib has been evaluated as a
therapy for glioma in Phase II clinical trials both as monotherapy56 and in combination
with hydroxyurea.57 While both approaches were well tolerated, neither was shown
effective as a therapy for unselected patients with recurrent glioblastoma. Further
Table 1. Selected molecularly targeted agents utilized in treatment of GBM

Agent Target(s) Class
Bevacizumab VEGF Angiogenesis inhibitor
Aflibercept
Cediranib VEGFR
CT-322
MK0752 Notch
Cetuximab EGFR Tumor growth factor inhibitor
Nimotuzumab
Erlotinib
Gefitinib
AMG-102 HGF/SF
Tipifarnib RAS Effector cascade inhibitors
Lonafarnib
Perifosine AKT
Sirolimus mTOR
Temsirolimus
Everolimus
Enzastaurin PKC-Beta
Cilengitide Integins Integrin inhibitor
Imatinib PDGFR, c-KIT, Abl Multi-targeted agent
Vandetanib EGFR, VEGFR, RET
Lapatinib EGFR, HER2
Sunitinib PDGFR, VEGFR, c-KIT, Flt-3
Tandutinib PDGFR, c-KIT, Flt-3
Sorafenib Raf, VEGFR, PDGFR, c-KIT, Flt-3
XL184 VEGFR, c-Met, RET
Vatalanib VEGFR, PDGFR, c-KIT
Pazopanib VEGFR, PDGFR, c-KIT
Dasatinib Src family kinases, Stat5
Table 2. Selected active trials of conventional chemotherapy for GBM*

New or Trial
Agent(s) Recurrent Phase Intervention
TMZ Recurrent II Dose-intense TMZ (dosed for the first 21 days of

each 28 day cycle)
TMZ, ABT-888 Newly I/II ABT-888 (PARP inhibitor) in addition to XRT
diagnosed with concurrent and adjuvant TMZ
TMZ, BSI-201 Newly I/II BSI-201 (PARP inhibitor) in addition to XRT
diagnosed with concurrent and adjuvant TMZ
TMZ, topotecan Recurrent I Topotecan and TMZ for recurrent malignant
glioma
TMZ, Gliadel Newly II Gliadel wafer implantation at time of resection
diagnosed followed by XRT with concurrent and adjuvant
TMZ
CYT997, carbopla- Recurrent Ib/II CYT997 (tubulin polymerization inhibitor) in
tin, etoposide combination with carboplatin and etoposide
* Information on active trials from www.clinicaltrials.gov
studies investigating imatinib in combination with other agents, such as temozolomide,

are ongoing.
Hepatocyte Growth Factor/Scatter Factor
Hepatocyte growth factor (HGF), also known as scatter factor (SF), acts upon the
receptor tyrosine kinase c-Met to trigger a group of cellular processes collectively known as
the invasive growth program. Abnormal c-Met signaling in glial tumors is associated with
a poor prognosis.58 As with EGFR and PDGFR, c-Met mediates both the PI3K-Akt-mTOR
and RAS-RAF-MEK-MAPK second messenger systems. The HGF/c-Met system also
interacts with other pathways implicated in tumorigenesis. HGF stimulation produces
EGFR activation59 and VEGF production58 in tumor cells.
AMG102 is a fully human monoclonal antibody against HGF that prevents interaction
between HGF and c-Met. A multi-center Phase II study of AMG102 for treatment of
malignant glioma is ongoing. Several small molecule inhibitors of c-Met receptor tyrosine
kinase activity are currently being evaluated as treatments for malignant glioma. One
example is XL184, which inhibits VEGFR2 and KDR in addition to c-Met and is currently
being studied in a Phase II clinical trial.
Angiogenesis Pathways and Agents
Vascular Endothelial Growth Factor
The role of angiogenesis in tumor survival and growth has been recognized since the
early 1970s.60 Tumors utilize many different mechanisms to produce their blood supply.
The prototypical example is the vascular endothelial growth factor (VEGF) system.
Gliomas, along with most other human solid tumors, express VEGF at elevated levels.
VEGF expression is triggered by a number of factors, notably hypoxia and acidosis,
which are commonly seen in high-grade tumors. VEGF then acts on endothelial cells to
trigger increased vessel formation via PLCa-PKC-Raf kinase-MEK-MAPK pathway and
phosphatidylinositol 3 kinase (PI3K)-Akt pathway mediated mechanisms.61
The first targeted anti-angiogenic agent shown to have efficacy against malignant
glioma was bevacizumab (Avastin), a monoclonal antibody against VEGF. Prior to its
use in glioma, bevacizumab was shown to improve outcome in non-CNS malignancies
such as colon cancer when given with cytotoxic chemotherapy,62 where it is thought to
lead to improved chemotherapy response by allowing normalization of blood vessels.
Given this background, initial trials of bevacizumab for GBM patients combined the
therapy with irinotecan, a conventional chemotherapeutic agent. In a Phase II trial the
combination of bevacizumab and irinotecan led to increased 6-month progression-free
survival (PFS6) relative to historical controls.63 A company-sponsored Phase II trial of
bevacizumab monotherapy versus bevacizumab in combination with irinotecan showed
a trend towards improved outcome with combination therapy, but the difference was not
statistically significant.64 There was an improvement of the response rate and 6 month
progression free survival compared to historical controls and in May 2009, bevacizumab
received accelerated approval from the Federal Drug Administration for patients with
recurrent glioblastoma. A Phase III trial will be needed to more fully define the risks
and benefits of the addition of irinotecan to bevacizumab therapy. Further studies of
bevacizumab including combination therapy trials with other targeted agents are ongoing.
While bevacizumab is the best-studied of the VEGF pathway modifiers, several other
agents are currently being evaluated. Aflibercept (VEGF Trap) is a soluble receptor that
binds circulating VEGF and placental growth factor (PlGF), a related ligand of the VEGFR.
Aflibercept has been shown to be effective against human glioma in mouse models65 and
a clinical trial of aflibercept in patients with recurrent malignant glioma is ongoing. Small
molecule kinase inhibitors aimed at VEGFR include vatalanib, pazopanib, cediranib and
CT-322. While none of these therapies has shown unequivocal survival benefit in patients
with glioma, cediranib therapy does lead to a rapid and dramatic radiographic response
due to reversible vascular normalization.66 In addition to the VEGF-specific therapies
above, several agents currently under investigation act on VEGF in addition to other
targets of glioma growth. Examples include sunutinib, vandetanib, sorafinib and axitinib.
Anti-VEGF therapy is generally well tolerated. Side effects attributable to avastin
included venous thromboembolism, hypertension, gastrointestinal perforation, bleeding and
impaired wound healing.67 Fatigue is also a commonly noted. Small molecule inhibitors of
VEGFR, such as sorafenib and sunitinib, cause mucousitis, diarrhea, hand-foot reaction
and skin rash in addition to bevacizumab-like side effects.
Protein Kinase C-`
The protein kinase C family is a group of serine/threonine kinases that phosphorylates

several targets involved in cellular signaling. Increased PKC` activity stimulates
angiogenesis through interplay with the VEGF system.68 PKC` also activates the
phosphoinositide 3-kinase (PI3K) second messenger cascade which plays an important
role in cellular survival and regulation of apoptosis.
Enzastaurin is a potent small-molecule PKC` inibitor that has been evaluated as a
therapy for a variety of tumors. In preclinical evaluation, enzastaurin is able to inhibit
Table 3. Selected active trials of molecularly targeted agents for GBM*

New or Trial
Bevacizumab Recurrent II Bevacizumab monotherapy
Sunitinib Recurrent II Sunitinib monotherapy
Everolimus Recurrent II Everolimus for recurrent malignant glioma
Perifosine Recurrent II Perifosine for recurrent malignant glioma
Bevacizumab, ertlotinib Recurrent II Bevacizumab and erlotinib
Bevacizumab, Recurrent II Bevacizumab and enzastaurin
enzastaurin
VEGF-stimulated angiogenesis and suppress growth of human glioblastoma.69 An initial

Phase II trial of enzastaurin monotherapy for recurrent glioma showed an encouraging
radiographic response rate,70 but the follow-up Phase III trial of enzastaurin versus
lomustine (CCNU) was terminated early for futility.71 Clinical trials evaluating enzastaurin
in combination with other therapies such as radiation, temozolomide and bevacizumab
are ongoing.
Thalidomide and Analogs
Thalidomide was one of the first anti-angiogenesis agents evaluated for use in the
treatment of cancer. Hepatic metabolism of thalidomide produces a metabolite that inhibits
basic fibroblast growth factor (bFGF) induced angiogenesis.72 Thalidomide also inhibits
tumor necrosis factor alpha (TNF-_) which upregulates production of bFGF and VEGF.73
Clinical trials of thalidomide as monotherapy for recurrent malignant gliomas showed
transient cytostatic activity but no significant sustained response.74 Lenalidomide is a
more potent thalidomide analog that has also been evaluated in patients with recurrent
glioblastoma with similarly disappointing antitumor efficacy.75 Given the negative results
of monotherapy trials in malignant glioma, ongoing trials are examining thalidomide and
analogs in combination with other agents.
Integrin Therapies
Integrins are transmembrane glycoproteins that interact with the extracellular matrix
and serve as receptors for extracellular ligands. Through ligand binding, integrins help
regulate many cellular processes including proliferation, migration, angiogenesis and
survival.76 Integrin signaling plays a role in a wide variety of tumors and integrin expression
is increased in both glioblastoma cells77 and tumor-associated vasculature.78 There are
many integrins, but _v3 and _v5 are the forms that have been most investigated in
glial tumors.79
Cilengitide is a peptide that competitively binds to integrins _v3 and _v5 and
disrupts normal signaling.79 In a Phase II study of two different doses of cilengitide
monotherapy in patients with newly diagnosed glioblastoma, cilengitide was well-tolerated
and antitumor activity was suggested by radiographic response in 9% of patients, with

a trend towards prolonged progression free survival (PFS) and overall survival (OS) in
the high-dose group.80 A Phase I/IIa trial of cilengitide in combination with radiation and
temozolomide showed prolonged PFS and OS in comparison with a previous cohort of
patients that had been treated with radiation and temozolomide alone. Tumor MGMT
status correlated with outcome; patients whose tumors did not express MGMT were more
likely to reach the 6 month PFS endpoint.81 Further trials of cilengitide in combination
with radiation and temozolomide are ongoing.
Intracellular Signaling Pathways and Agents
PI3K-Akt-mTOR Second Messenger System
The Phosphoinositide 3-kinase (PI3K) second messenger cascade is a downstream

mediator of several growth factor receptors. Initiation of this system leads to the activation
of Akt (also known as protein kinase B). Once activated by phosphorylation, Akt both
increases transcription of pro-survival genes and inactivates pro-apoptotic proteins.
Akt has several targets, notably mammalian target of rapamycin (mTOR). The major
negative regulator of the PI3K-Akt-mTOR pathway is PTEN (phosphatase and tensin
homolog) which deactivates Akt. Overactivation of the PI3K-Akt-mTOR pathway in
glioblastoma can occur by excessive EGFR input or through decreased PTEN inhibitory
feedback. The previously mentioned EGFR-vIII mutation of EGFR preferentially activates
the PI3K-Akt-mTOR pathway.82 The PTEN gene, located on chromosome 10q, is a
commonly mutated tumor suppressor gene in a variety of cancers. In glioblastoma, loss
of heterozygosity at 10q is found in approximately 70% of tumors and PTEN mutations
are seen in 25% of tumors.83 Loss of PTEN function is associated with an aggressive
tumor phenotype due to unopposed stimulation of Akt activation.
The best studied target within the PI3K-Akt-mTOR pathway is mTOR. Temsirolimus
and everolimus are analogs of sirolimus (rapamycin) with more favorable pharmacokinetic
properties. Phase II trials of temsirolimus in patients with recurrent glioblastoma showed
minimal efficacy as a monotherapy but little treatment-related toxicity.84 Follow-up
studies to examine temsirolimus in combination with other agents or as a monotherapy
in pathologically defined subsets of glioblastoma are ongoing. Akt itself is the target of
the small molecule inhibitor perifosine,85 which is currently being evaluated in a Phase
II trial as a therapy for recurrent malignant glioma.
Ras-Raf-MEK-MAPK Second Messenger System
Ras is a signal transduction protein that lies downstream of EGFR and PDGFR.
Ras activates a number of signaling cascades, but its action on the mitogen-activated
protein (MAP) kinases is especially important for tumor propagation and survival.
While mutations in the Ras-Raf-MEK-MAPK pathway itself are rare in glioblastoma,86
pathway activity is frequently increased due to mutation and over-expression of upstream
receptor kinases.
The activation of Ras presents an opportunity for intervention to inhibit the
Ras-Raf-MEK-MAPK pathway before it branches to exert its diverse down-stream
effects. Farnesyltransferase inhibitors (FTIs) are small molecule inhibitors of the enzyme
that activates Ras. Tipifarnib and lonafarnib are two FTIs that have been evaluated in
humans with recurrent glioblastoma multiforme or anaplastic glioma. A Phase II trial

of tipifarnib in patients with recurrent high-grade glioma demonstrated modest activity
and excellent tolerability.87 Raf can also be inhibited directly and it is one of several
targets of the small molecule sorafenib.88 Several clinical trials of sorafenib for malignant
glioma are underway.
CONCLUSION
In the past decade, the oncologists armamentarium against GBM has expanded
dramatically. Temozolomide represents the first and to date the only, cytotoxic
chemotherapeutic agent to unambiguously demonstrate prolonged survival in a
randomized Phase 3 trial of patients with newly diagnosed GBM. Ongoing investigations
of alternative TMZ dosing regimens, coupled with a more sophisticated understanding
of the role of MGMT gene silencing as a mediator of response to TMZ, may allow for
more rational and patient-specific therapy choicesultimately extending survival and
minimizing treatment-related toxicity. Molecularly targeted therapies represent an equally
promising avenue of investigation. While the first wave of agents have demonstrated
disappointing efficacy as monotherapies, the interdependence of angiogenesis and tumor
growth pathways suggests that combinations of multiple targeted therapies may have
synergistic effects that far exceed the power of single agents. Future work in these areas,
along with treatments harnessing our knowledge of tumor immunology and continued
refinement of surgical and radiotherapeutic techniques, will undoubtedly lead to further
improvements in expected survival for patients with GBM.
Table 4. Selected active trials of convention chemotherapy in combination with targeted

agents for GBM*
New or Trial
TMZ, bevacizumab Newly II XRT with concurrent and adjuvant TMZ and
diagnosed bevacizumab
TMZ, bevacizumab, Newly II XRT with concurrent TMZ followed by TMZ,
erlotinib diagnosed erlotinib and bevacizumab adjuvant therapy
TMZ, sorafenib Newly II XRT with concurrent TMZ followed by TMZ
disgnosed and sorafenib adjuvant therapy
TMZ, bevacizumab Recurrent II Bi-Weekly TMZ plus bevacizumab
Etoposide, bevaci- Recurrent II Bevacizumab and etoposide
zumab
Gliadel, bevacizumab, Recurrent II Gliadel followed by bevacizumab and irinote-
irinotecan can
REFERENCES
1. Lacroix M, Abi-Said D, Fourney DR et al. A multivariate analysis of 416 patients with glioblastoma
multiforme: prognosis, extent of resection and survival. J Neurosurg 2001; 95:190-198.
2. Laperriere N, Zuraw L, Cairncross G. Cancer care ontario practice guidelines initiative neuro-oncology
disease site group. Radiotherapy for newly diagnosed malignant glioma in adults: a systematic review.
Radiother Oncol 2002; 64:259-273.
4. Ostermann S, Csajka C, Buclin T et al. Plasma and cerebrospinal fluid population pharmacokinetics of
temozolomide in malignant glioma patients. Clin Cancer Res 2004; 10:3728-3736.
5. Roos WP, Batista LF, Naumann SC et al. Apoptosis in malignant glioma cells triggered by the
temozolomide-induced DNA lesion O6-methylguanine. Oncogene 2007; 26:186-197.
6. Newlands ES, Stevens MF, Wedge SR et al. Temozolomide: a review of its discovery, chemical properties,
preclinical development and clinical trials. Cancer Treat Rev 1997; 23:35-61.
7. Bower M, Newlands ES, Bleehen NM et al. Multicentre CRC phase II trial of temozolomide in recurrent or
progressive high-grade glioma. Cancer Chemother Pharmacol 1997; 40:484-488.
8. Yung WK, Prados MD, Yaya-Tur R et al. Multicenter phase II trial of temozolomide in patients with
anaplastic astrocytoma or anaplastic oligoastrocytoma at first relapse. Temodal Brain Tumor Group.
J Clin Oncol 1999; 17:2762-2771.
9. Brada M, Hoang-Xuan K, Rampling R et al. Multicenter phase II trial of temozolomide in patients with
glioblastoma multiforme at first relapse. Ann Oncol 2001; 12:259-266.
10. Brandes AA, Ermani M, Basso U et al. Temozolomide in patients with glioblastoma at second relapse after
first line nitrosourea-procarbazine failure: a phase II study. Oncology 2002; 63:38-41.
11. Yung WK, Albright RE, Olson J et al. A phase II study of temozolomide vs procarbazine in patients with
glioblastoma multiforme at first relapse. Br J Cancer 2000; 83:588-593.
12. Stupp R, Dietrich PY, Ostermann S et al. Promising survival for patients with newly diagnosed glioblastoma
multiforme treated with concomitant radiation plus temozolomide followed by adjuvant temozolomide.
J Clin Oncol 2002; 20:1375-1382.
13. Lanzetta G, Campanella C, Rozzi A et al. Temozolomide in radio-chemotherapy combined treatment for
newly-diagnosed glioblastoma multiforme: phase II clinical trial. Anticancer Res 2003; 23:5159-5164.
14. Athanassiou H, Synodinou M, Maragoudakis E et al. Randomized phase II study of temozolomide and
radiotherapy compared with radiotherapy alone in newly diagnosed glioblastoma multiforme. J Clin
Oncol 2005; 23:2372-2377.
15. Stupp R, Hegi ME, Mason WP et al. Effects of radiotherapy with concomitant and adjuvant temozolomide
versus radiotherapy alone on survival in glioblastoma in a randomised phase III study: 5-year analysis
of the EORTC-NCIC trial. Lancet Oncol 2009; 10:459-466.
16. Chakravarti A, Erkkinen MG, Nestler U et al. Temozolomide-mediated radiation enhancement in glioblastoma:
a report on underlying mechanisms. Clin Cancer Res 2006; 12:4738-4746.
17. Combs SE, Gutwein S, Schulz-Ertner D et al. Temozolomide combined with irradiation as postoperative
treatment of primary glioblastoma multiforme. Phase I/II study. Strahlenther Onkol 2005; 181:372-377.
18. Hegi ME, Liu L, Herman JG et al. Correlation of O6-methylguanine methyltransferase (MGMT) promoter
methylation with clinical outcomes in glioblastoma and clinical strategies to modulate MGMT activity.
J Clin Oncol 2008; 26:4189-4199.
19. Belanich M, Randall T, Pastor MA et al. Intracellular Localization and intercellular heterogeneity of the
human DNA repair protein O(6)-methylguanine-DNA methyltransferase. Cancer Chemother Pharmacol
1996; 37:547-555.
20. Watts GS, Pieper RO, Costello JF et al. Methylation of discrete regions of the O6-methylguanine DNA
methyltransferase (MGMT) CpG island is associated with heterochromatinization of the MGMT
transcription start site and silencing of the gene. Mol Cell Biol 1997; 17:5612-5619.
21. Esteller M, Garcia-Foncillas J, Andion E et al. Inactivation of the DNA-repair gene MGMT and the clinical
response of gliomas to alkylating agents. N Engl J Med 2000; 343:1350-1354.
22. Jaeckle KA, Eyre HJ, Townsend JJ et al. Correlation of tumor O6 methylguanine-DNA methyltransferase
levels with survival of malignant astrocytoma patients treated with bis-chloroethylnitrosourea: a Southwest
Oncology Group study. J Clin Oncol 1998; 16:3310-3315.
N Engl J Med 2005; 352:997-1003.
24. Quinn JA, Desjardins A, Weingart J et al. Phase I trial of temozolomide plus O6-benzylguanine for patients
with recurrent or progressive malignant glioma. J Clin Oncol 2005; 23:7178-7187.
25. Quinn JA, Jiang SX, Reardon DA et al. Phase II trial of temozolomide plus o6-benzylguanine in adults with
recurrent, temozolomide-resistant malignant glioma. J Clin Oncol 2009; 27:1262-1267.
26. Chalmers AJ. The potential role and application of PARP inhibitors in cancer treatment. Br Med Bull
2009; 89:23-40.
27. Taphoorn MJ, Stupp R, Coens C et al. Health-related quality of life in patients with glioblastoma: a
randomised controlled trial. Lancet Oncol 2005; 6:937-944.
28. Mahindra AK, Grossman SA. Pneumocystis carinii pneumonia in HIV negative patients with primary brain
tumors. J Neurooncol 2003; 63:263-270.
29. Stewart LA. Chemotherapy in adult high-grade glioma: a systematic review and meta-analysis of individual
patient data from 12 randomised trials. Lancet 2002; 359:1011-1018.
30. Glas M, Happold C, Rieger J et al. Long-term survival of patients with glioblastoma treated with radiotherapy
and lomustine plus temozolomide. J Clin Oncol 2009; 27:1257-1261.
31. Yung WK, Mechtler L, Gleason MJ. Intravenous carboplatin for recurrent malignant glioma: a phase II
study. J Clin Oncol 1991; 9:860-864.
32. Warnick RE, Prados MD, Mack EE et al. A phase II study of intravenous carboplatin for the treatment of
recurrent gliomas. J Neurooncol 1994; 19:69-74.
33. Peterson K, Harsh G 4th, Fisher PG et al. Daily low-dose carboplatin as a radiation sensitizer for newly
diagnosed malignant glioma. J Neurooncol 2001; 53:27-32.
34. Finn GP, Bozek T, Souhami RL et al. High-dose etoposide in the treatment of relapsed primary brain
tumors. Cancer Treat Rep 1985; 69:603-605.
35. Fulton D, Urtasun R, Forsyth P. Phase II study of prolonged oral therapy with etoposide (VP16) for patients
with recurrent malignant glioma. J Neurooncol 1996; 27:149-155.
36. Hellman RM, Calogero JA, Kaplan BM. VP-16, vincristine and procarbazine with radiation therapy for
treatment of malignant brain tumors. J Neurooncol 1990; 8:163-166.
37. Lassen U, Kristjansen PE, Wagner A et al. Treatment of newly diagnosed glioblastoma multiforme with
carmustine, cisplatin and etoposide followed by radiotherapy. A phase II study. J Neurooncol 1999;
43:161-166.
38. Silvani A, Lamperti E, Gaviani P et al. Salvage chemotherapy with procarbazine and fotemustine combination
in the treatment of temozolomide treated recurrent glioblastoma patients. J Neurooncol 2008; 87:143-151.
39. Brandes AA, Tosoni A, Basso U et al. Second-line chemotherapy with irinotecan plus carmustine in
glioblastoma recurrent or progressive after first-line temozolomide chemotherapy: a phase II study of
the Gruppo Italiano Cooperativo di Neuro-Oncologia (GICNO). J Clin Oncol 2004; 22:4779-4786.
40. Westphal M, Hilt DC, Bortey E et al. A phase 3 trial of local chemotherapy with biodegradable carmustine
(BCNU) wafers (Gliadel wafers) in patients with primary malignant glioma. Neuro Oncol 2003; 5:79-88.
41. Westphal M, Ram Z, Riddle V et al. Executive Committee of the Gliadel Study Group. Gliadel wafer in
initial surgery for malignant glioma: long-term follow-up of a multicenter controlled trial. Acta Neurochir
(Wien) 2006; 148:269-75; discussion 275.
42. Brem H, Piantadosi S, Burger PC et al. Placebo-controlled trial of safety and efficacy of intraoperative
controlled delivery by biodegradable polymers of chemotherapy for recurrent gliomas. The Polymer-brain
Tumor Treatment Group. Lancet 1995; 345:1008-1012.
43. Hart MG, Grant R, Garside R et al. Chemotherapeutic wafers for High Grade Glioma. Cochrane Database
Syst Rev 2008; 3:CD007294.
44. McGirt MJ, Than KD, Weingart JD et al. Gliadel (BCNU) wafer plus concomitant temozolomide therapy
after primary resection of glioblastoma multiforme. J Neurosurg 2009; 110:583-588.
45. Cohen S. The epidermal growth factor (EGF). Cancer 1983; 51:1787-1791.
46. Ekstrand AJ, James CD, Cavenee WK et al. Genes for epidermal growth factor receptor, transforming
growth factor alpha and epidermal growth factor and their expression in human gliomas in vivo. Cancer
Res 1991; 51:2164-2172.
47. Pelloski CE, Ballman KV, Furth AF et al. Epidermal growth factor receptor variant III status defines
clinically distinct subtypes of glioblastoma. J Clin Oncol 2007; 25:2288-2294.
48. Scaltriti M, Baselga J. The epidermal growth factor receptor pathway: a model for targeted therapy. Clin
Cancer Res 2006; 12:5268-5272.
49. Safety Study of Cetuximab, Radiotherapy and Temozolomide in Primary Glioblastoma Multiforme(GERT).
Available at: http://www.clinicaltrials.gov/ct2/show/NCT00311857. Accessed 04/22, 2009;
50. van den Bent MJ, Brandes AA, Rampling R et al. Randomized phase II trial of erlotinib versus temozolomide
or carmustine in recurrent glioblastoma: EORTC brain tumor group study 26034. J Clin Oncol 2009;
27:1268-1274.
51. Prados MD, Chang SM, Butowski N et al. Phase II study of erlotinib plus temozolomide during and after
radiation therapy in patients with newly diagnosed glioblastoma multiforme or gliosarcoma. J Clin Oncol
2009; 27:579-584.
52. Brown PD, Krishnan S, Sarkaria JN et al. Phase I/II trial of erlotinib and temozolomide with radiation
therapy in the treatment of newly diagnosed glioblastoma multiforme: North Central Cancer Treatment
Group Study N0177. J Clin Oncol 2008; 26:5603-5609.
EGFR kinase inhibitors. N Engl J Med 2005; 353:2012-2024.
54. Perez-Soler R. Can rash associated with HER1/EGFR inhibition be used as a marker of treatment outcome?
Oncology (Williston Park) 2003; 17(11 Suppl 12):23-28.
55. Tsuboi M, Le Chevalier T. Interstitial lung disease in patients with nonsmall-cell lung cancer treated with
epidermal growth factor receptor inhibitors. Med Oncol 2006; 23:161-170.
56. Wen PY, Yung WK, Lamborn KR et al. Phase I/II study of imatinib mesylate for recurrent malignant
gliomas: North American Brain Tumor Consortium Study 99-08. Clin Cancer Res 2006; 12:4899-4907.
57. Reardon DA, Egorin MJ, Quinn JA et al. Phase II study of imatinib mesylate plus hydroxyurea in adults
with recurrent glioblastoma multiforme. J Clin Oncol 2005; 23:9359-9368.
58. Abounader R, Laterra J. Scatter factor/hepatocyte growth factor in brain tumor growth and angiogenesis.
Neuro Oncol 2005; 7:436-451.
59. Reznik TE, Sang Y, Ma Y et al. Transcription-dependent epidermal growth factor receptor activation by
hepatocyte growth factor. Mol Cancer Res 2008; 6:139-150.
60. Folkman J. Tumor angiogenesis: therapeutic implications. N Engl J Med 1971; 285:1182-1186.
61. Kerbel RS. Tumor angiogenesis. N Engl J Med 2008; 358:2039-2049.
62. Hurwitz H, Fehrenbacher L, Novotny W et al. Bevacizumab plus irinotecan, fluorouracil and leucovorin
for metastatic colorectal cancer. N Engl J Med 2004; 350:2335-2342.
63. Vredenburgh JJ, Desjardins A, Herndon JE 2nd et al. Bevacizumab plus irinotecan in recurrent glioblastoma
multiforme. J Clin Oncol 2007; 25:4722-4729.
64. Cloughesy TF, Prados MD, Wen PY et al. A phase II, randomized, noncomparative clinical trial of the
effect of bevacizumab (BV) alone or in combination with irinotecan (CPT) on 6-month progression free
survival (PFS6) in recurrent, treatment-refractory glioblastoma (GBM) [abstract]. J Clin Oncol 2008;
26(May 20 Supplement):Abstract 2010b.
65. Gomez-Manzano C, Holash J, Fueyo J et al. VEGF Trap induces antiglioma effect at different stages of
disease. Neuro Oncol 2008; 10:940-945.
66. Batchelor TT, Sorensen AG, di Tomaso E et al. AZD2171, a pan-VEGF receptor tyrosine kinase inhibitor,
normalizes tumor vasculature and alleviates edema in glioblastoma patients. Cancer Cell 2007; 11:83-95.
67. Norden AD, Young GS, Setayesh K et al. Bevacizumab for recurrent malignant gliomas: efficacy, toxicity
and patterns of recurrence. Neurology 2008; 70:779-787.
68. Xia P, Aiello LP, Ishii H et al. Characterization of vascular endothelial growth factors effect on the
activation of protein kinase C, its isoforms and endothelial cell growth. J Clin Invest 1996; 98:2018-2026.
69. Graff JR, McNulty AM, Hanna KR et al. The protein kinase Cbeta-selective inhibitor, Enzastaurin (LY317615.
HCl), suppresses signaling through the AKT pathway, induces apoptosis and suppresses growth of human
colon cancer and glioblastoma xenografts. Cancer Res 2005; 65:7462-7469.
70. Fine HA, Kim L, Royce C et al. Results from phase II trial of Enzastaurin (LY317615) in patients with
recurrent high grade gliomas [abstract]. J Clin Oncol 2005; 23(16S, June 1 Supplement.):Abstract 1504.
71. Fine HA, Puduvalli VK, Chamberlain MC et al. Enzastaurin (ENZ) versus lomustine (CCNU) in the
treatment of recurrent, intracranial glioblastoma multiforme (GBM): A phase III study [abstact]. J Clin
Oncol 2008; 26(No 15S (May 20 Supplement)):Abstract 2005.
72. Bauer KS, Dixon SC, Figg WD. Inhibition of angiogenesis by thalidomide requires metabolic activation,
which is species-dependent. Biochem Pharmacol 1998; 55:1827-1834.
73. Sampaio EP, Sarno EN, Galilly R et al. Thalidomide selectively inhibits tumor necrosis factor alpha
production by stimulated human monocytes. J Exp Med 1991; 173:699-703.
74. Fine HA, Figg WD, Jaeckle K et al. Phase II trial of the antiangiogenic agent thalidomide in patients with
recurrent high-grade gliomas. J Clin Oncol 2000; 18:708-715.
75. Fine HA, Kim L, Albert PS et al. A phase I trial of lenalidomide in patients with recurrent primary central
nervous system tumors. Clin Cancer Res 2007; 13:7101-7106.
76. Parise LV, Lee J, Juliano RL. New aspects of integrin signaling in cancer. Semin Cancer Biol 2000; 10:407-414.
77. Gingras MC, Roussel E, Bruner JM et al. Comparison of cell adhesion molecule expression between
glioblastoma multiforme and autologous normal brain tissue. J Neuroimmunol 1995; 57:143-153.
78. Gladson CL. Expression of integrin alpha v beta 3 in small blood vessels of glioblastoma tumors. J Neuropathol
Exp Neurol 1996; 55:1143-1149.
79. Nabors LB, Mikkelsen T, Rosenfeld SS et al. Phase I and correlative biology study of cilengitide in patients
with recurrent malignant glioma. J Clin Oncol 2007; 25:1651-1657.
80. Reardon DA, Fink KL, Mikkelsen T et al. Randomized phase II study of cilengitide, an integrin-targeting
arginine-glycine-aspartic acid peptide, in recurrent glioblastoma multiforme. J Clin Oncol 2008;
26:5610-5617.
81. Stupp R, Goldbrunner R, Neyns B et al. Phase I/IIa trial of cilengitide (EMD121974) and temozolomide
with concomitant radiotherapy, followed by temozolomide and cilengitide maintenance therapy in
patients (pts) with newly diagnosed glioblastoma (GBM) [abstract]. J Clin Oncol 2007; 25(18S, (June
20 Supplement)):Abstract 2000.
82. McLendon RE, Turner K, Perkinson K et al. Second messenger systems in human gliomas. Arch Pathol
Lab Med 2007; 131:1585-1590.
83. Ohgaki H, Kleihues P. Genetic pathways to primary and secondary glioblastoma. Am J Pathol 2007;
170:1445-1453.
84. Galanis E, Buckner JC, Maurer MJ et al. Phase II trial of temsirolimus (CCI-779) in recurrent glioblastoma
multiforme: a North Central Cancer Treatment Group Study. J Clin Oncol 2005; 23:5294-5304.
85. Momota H, Nerio E, Holland EC. Perifosine inhibits multiple signaling pathways in glial progenitors and
cooperates with temozolomide to arrest cell proliferation in gliomas in vivo. Cancer Res 2005; 65:7429-7435.
86. Knobbe CB, Reifenberger J, Reifenberger G. Mutation analysis of the Ras pathway genes NRAS, HRAS,
KRAS and BRAF in glioblastomas. Acta Neuropathol 2004; 108:467-470.
87. Cloughesy TF, Wen PY, Robins HI et al. Phase II trial of tipifarnib in patients with recurrent malignant
glioma either receiving or not receiving enzyme-inducing antiepileptic drugs: a North American Brain
Tumor Consortium Study. J Clin Oncol 2006; 24:3651-3656.
88. Hahn O, Stadler W. Sorafenib. Curr Opin Oncol 2006; 18:615-621.
PART II
GLIOMA IMMUNOLOGY
CHAPTER 4
BASIC CONCEPTS IN GLIOMA IMMUNOLOGY
Ian F. Parney
Department of Neurologic Surgery, Mayo Clinic, Rochester, Minnesota, USA
Email: parney.ian@mayo.edu
Abstract: Glioblasotmas are the most common primary central nervous system tumor
and typically have a dismal prognosis. Immunotherapy has been a promising
experimental treatment. Understanding brain tumor immunobiology is critical to
designing glioblasotma immunotherapies. In this chapter, we review aspects of
basic immunology and neuro-immunology. The antigenic underpinnings of brain
tumor immunotherapy including glioma-associated and glioma-specific antigens
are discussed. Finally, the molecular and cellular facets of glioma-mediated
immunosuppression are outlined. The role of multiple cell types (glioma cells,
glioma-infiltrating monocytes, regulatory T cells and myeloid derived suppressor
cells) in mediating local and systemic immunosuppression in glioma patients is
evaluated.
INTRODUCTION
Glioblastomas are the most common and most malignant primary central nervous
system tumors. Despite aggressive treatment with surgery, radiation and chemotherapy,
average survival for glioblastoma patients remains only 14 months.1 More effective
therapies are urgently needed. Immunotherapy (stimulating the immune system to
attack the tumor) is one such promising therapy. Effective immunotherapy requires
an understanding of the basic immunobiology of these tumors. In this chapter, we will
review basic immunology and neuro-immunology and move on to a discussion of local
and systemic glioblastoma immunobiology.

42
BASIC CONCEPTS IN GLIOMA IMMUNOLOGY 43
BASIC IMMUNOLOGY
The immune system made up of the various cellular and visceral components involved
in recognizing and eliminating foreign and/or dangerous antigens. Anatomically, it is
made up of primary and secondary lymphoid organs. Functionally, it consists of both
innate and adaptive immune responses. Innate immunity deals with antigen-nonspecific
responses to foreign or dangerous molecules (Fig. 1).2,3 Some of this is accomplished
at a purely molecular level with the complement system and systemic responses to
inflammatory cytokines. At a cellular level, it is made up of macrophages, neutrophils and
natural killer cells. Activation of innate immunity is not antigen-specific but is induced in
response to molecular factors common to multiple infectious or dangerous agents termed
pathogen-associated molecular patterns (PAMPs) such as cytosine-guanine (CpG)-rich
regions of bacterial DNA. Toll-like receptors (TLRs ) are often the receptors for PAMPs
and are key in unleashing innate immune responses.
In contrast to innate immunity, adaptive immune responses are antigen-specific.
Foreign and/or dangerous antigens are scavenged from the environment, processed and
presented by professional antigen presenting cells (APCs) of the reticuloendothelial
system, including dendritic cells and macrophages. APCs form an important connection
between adaptive and innate immunity. Adaptive immunity is comprised of humoral
(antibody) mediated by B cells and cellular immune responses mediated by cytotoxic
Figure 1. Components of the innate immune system. Innate immunity can be defined as the cellular and
molecular defenses in place prior to exposure to infection. The innate immune system includes physical
barriers such as epithelial surfaces, phagocytic cells like macrophages and neutrophils, blood proteins
such as the complement systems and other inflammatory mediators and natural killer lymphocytes.
While aspects of innate immunity do respond to shared structures present on or in microbes and absent
in mammalian cells, the repertoire of antigens recognized by innate immune mechanisms is relatively
limited and there is no true antigen-specificity.
Figure 2. Components of the adaptive immune system. A) Humoral immunity. B-lymphocytes recognize
specific extra-cellular or cell surface antigens that bind their B-cell receptor. In the presence of appropriate
T-cell help (specifically, TH2 cells secreting appropriate cytokines), B cells that specifically bind to
these antigens will proliferate and begin to secrete antigen-specific antibodies. These antibodies bind
antigen and promote its elimination through phagocytes and the complement system (effector phase).
B) Cellular immunity. Cytolytic (killer) T-lymphocytes bind antigen presented to them in the context
of a MHC molecule by an antigen presenting cell (APC). These antigens are typically intracellular (like
most tumor antigens) and either result from APC infection or APC phagocytosis of cells expressing
these antigens. In the presence of appropriate T-cell help (TH1 cells and cytokines), TK cells that react
specifically to this antigen proliferate. In an effector phase, T K cell bind and lead to the destruction of
cells expressing their requisite antigen. Note: for both humoral and cellular immune responses, there is
a later memory phase (not shown) in which excess effector cells are eliminated after antigen has been
cleared but memory lymphocytes capable of rapid response to repeat antigen challenge are retained.
T cells (Fig. 2).4 Both forms of adaptive immunity require signals from helper T cells.
Antigens are presented to T cells in the context of major histocompatibility complexes
(MHC). Class I MHC are key for presentation to CD8
cytotoxic T cells while class
II MHC are important for presentation to CD4
helper T cells. In addition to antigen
presentation in the context of a MHC molecule, T-cell activation also requires a second
costimulatory signal most commonly through the T-cell costimulatory molecules B7-1
or B7-2 on APCs binding their ligand CD28 on T cells (Fig. 3).5
NEURO-IMMUNOLOGY
The brain has long been considered an immunologically privileged site owing to its
apparent inability to reject intracranial xenografts in older reports,6 separation via the blood
brain barrier and lack of obvious connections with the lymphatic system.7 However, unlike
Figure 3. T-cell activation. T-cell activation requires antigen presentation in the context of a MHC
molecule to a T cell with a T-cell receptor that specifically recognizes that antigen. However, this is
insufficient get full T-cell activation. A second signals from costimulatory molecules (most commonly
B7-1 or B7-2 on an antigen presenting cell binding to CD28 on the T cell surface) is needed. Antigen
presentation to a T cell in the absence of this second signal leads to T-cell anergy. In addition, antigen
presenting cells secrete cytokines that can influence the subsequent type of immune response generated
(i.e., TH1 vs TH2).
other immunologically privileged site like testes, no specific CNS-associated antigens

have been described that are immunogenic systemically but evade immune detection
within the brain. Furthermore, it is now clear that the blood brain barrier is only a relative
barrier to lymphocyte tracking, particularly in pathological states.8 Connections between
cerebrospinal fluid and cervical lymphatics have been documented.9 Abundant evidence
demonstrates that intracranial xenografts are actually rejected very efficiently in all but
the most immunocompromised hosts.10 Microglia, resident antigen presenting cells of
the central nervous system, play an active part in very dynamic immune responses in
the brain.11 Multiple pathological states, ranging from multiple sclerosis to Alzheimers
disease to Parkinsons disease, bear witness to the ability of immune responses to occur
within the brain.12-14 In short, it is clear that, while the central nervous system may be
an immunologically distinct environment, it is not truly immunologically privileged.
GLIOBLASTOMA-ASSOCIATED ANTIGENS
A key assumption underlying most attempts at tumor immunotherapy (particularly

strategies aimed at stimulating adaptive immune responses) is that tumors express specific
abnormal antigens that can be recognized and eliminated by the immune system. Several
lines of evidence support this assumption in cancer in general and glioblastomas in particular
that can be demonstrated by discussion of a representative antigen. In glioblastomas,
mutant epidermal growth factor receptor-viii is an excellent example of a tumor-specific
antigen.15 EGFRviii is a truncated version of the epidermal growth factor receptor (EGFR)
present in approximately 30% of glioblastomas.16 It has lost its extracellular domain and
is constitutively active. It contains specific peptide sequences that have not been described
in any normal human tissues17 and have been used to target vaccines.15,18
Almost any tumor peptide has potential to function as a tumor antigen, leading to a
bewildering number of potential targets. This begs the question: which antigens are the
most attractive targets for immunotherapy? The National Cancer Institute of the United
States has recently attempted to address this issue. They performed an in-depth review of
75 tumor-associated antigens that could be targeted by immunotherapy.19 Antigens were
ranked based on nine factors: therapeutic function, immunogenicity, role in oncogenicity,
specificity, express level in tumors, cancer stem cell expression, percentage of patients with
positive tumors and cellular location of expression. The highest ranked antigens included
WT1, MUC1, LMP2, HPV E6/E7, HER2/Neu, EGFRviii, MAGE A3 and NY-ESO-1.
While the expression of some of these antigens (e.g., EGFRviii) by glioblastomas is well
established,16 the presence or absence of others (e.g., MUC1) have not been reported at
all. Only a handful of systematic studies have been performed examining expression of
potentially immunogenic antigens in glioblastomas have been performed. Many have
included antigens that did not make the NCIs top ten (e.g., gp100, MART) or that
were not considered in the NCI analysis at all (e.g., IL13R_).20,21
GLIOBLASTOMA-DERIVED IMMUNOSUPPRESSIVE FACTORS
It has long been recognized that glioblastomas secrete multiple immunosuppressive

factors.22,23 Expression of transforming growth factor-`2 (TGF- `2; originally described
as glioblastoma-derived immunoinhibitory factor) and prostaglandin E2 (PGE2) by
glioblastoma cells were originally described more than two decades ago.24-27 These
secreted factors have multiple immunosuppressive effects, the most marked of which is
the suppressing lymphocyte proliferation. In addition to these frankly immunosuppressive
factors, glioblastomas secrete several factors that are profoundly immunomodulatory.
Some, such as interleukin-6 (IL-6) and colony stimulating factor-1 (CSF-1) have obvious
immune functions such as shifting adaptive immunity to humoral (TH2) responses that may
be less effective against solid tumors and/or attracting and stimulating monocytic cells.22,23,28
Others, such as vascular endothelial growth factor (VEGF), impact immune responses
in a more tangential fashion. Tumor-derived VEGF acts primarily as a pro-angiogenic
factor and can be targeted for anti-angiogenic therapy (e.g., bevacizumab).29 However, it
has other functions beyond this such as stimulating the proliferation of myeloid-derived
suppressor cells that have profound immunosuppressive implications.30
The molecular drivers of immunosuppression in malignant glioma cells are beginning
to be dissected. It is increasingly evident that oncogenic mutations that increase cell
division may also lead to increased immunosuppression. For example, STAT3 is a novel
oncogene that has been implicated in many tumors in both driving cellular proliferation
and inhibiting immune responses through downstream effects on immunosuppressive
cytokine secretion.31 STAT3 activation-associated immunosuppression has been recently
reported in malignant gliomas.32,33 Similarly, mutations or deletions in the PTEN tumor
suppressor gene have been recently associated with expression of the immunosuppressive
T-cell costimulatory molecule homologue B7-H1.34 B7-H1 expression is normally
limited to germinal centers at the end of immune responses. Expression by tumor cells
(including glioblastoma cells) has profound immunosuppressive effects, including
inducing apoptosis in activated T cells and stimulating the proliferation of regulatory
T cells.34-36
MODULATION OF LOCAL IMMUNE RESPONSE

BY GLIOBLASTOMA-INFILTRATING CELLS
Glioblastoma cells are not the only cells within a glioblastoma tumor that can
participate in immune responses. Infiltrating inflammatory cells may play a role as
well. Although they have relatively few tumor-infiltrating lymphocytes, glioblastomas
are heavily infiltrated with monocytes/microglia.23,37 Reports suggest that these cells
comprise between 10-30% of viable cells within the tumor mass. Data from patients
suggest that these cells likely represent infiltrating systemic macrophages, not resident
microglia.37 While early studies suggested that these cells might be associated with an
improved prognosis,38 it has become increasingly evident that these cells are not part of
an effective antitumor immune response.39 Instead, they appear to have come under the
influence of the tumor and are actively immunosuppressive.40 Data from patients has
shown that they are the major source of intratumoral immunosuppressive interleukin-10
(IL-10).41,42 In rodent models, they induce apoptosis in T cells, possibly through the
expression of Fas-Ligand.43 In our laboratory, we have recently demonstrated that
normal monocytes that come into contact with glioblastoma cells express multiple
immunosuppressive factors (IL-10, TGF-`, B7-H1), have reduced phagocytic ability
and induce apoptosis in activated T cells.44 Similarly, Gustafson and colleagues have
reported that immunosuppressive CD14
/HLA-DR- monocytes increase after exposing
normal monocytes to human malignant glioma cells.45
SYSTEMIC IMMUNOSUPPRESSION IN GLIOBLASTOMA PATIENTS
It has long been recognized that glioblastoma patients are systemically

immunosuppressed.46,47 Lymphocyte counts (particularly CD4) are reduced. T-cell function
itself is also suppressed, with impaired proliferation in response to interleukin-2 and
other nonspecific mitogens.45 Similarly, we have recently demonstrated that glioblastoma
patients have decreased circulating immature dendritic cells, suggesting defects in antigen
presenting capacity (Zhang L et al; unpublished). While some systemic immunosuppression
may reflect dexamethasone administration in many glioblastoma patients, corticosteroids
cannot account for all of these systemic alterations. Others have performed exhaustive
searches for systemically increased tumor-derived immunosuppressive factors in
glioblastoma patients serum but this has been unrewarding to date.48,49 Indeed, most
known immunosuppressive factors secreted by glioblastoma cells are either undetectable
in glioblastoma patients serum or not increased compared to healthy individuals.44,45
Alternatively, it is increasingly evident that circulating immunosuppressive cells are
increased in glioblastoma patients blood compared to healthy individuals. For example,
circulating CD4
/CD25
/FoxP3
regulatory T cells (Treg) are increased in glioblastoma
patients50,51 despite global decreases in CD4 counts that are comparable to patients
with AIDS.45 Treg have the potential to reduce T-cell proliferation in response to both
antigens and nonspecific mitogens and could underlie many of the systemic defects in
lymphocyte function seen in glioblastoma patients. The origin of Treg in glioblastoma
patients remains uncertain. It seems as unlikely that Treg proliferate systemically in
response to glioblastoma-secreted factors as it is that lymphocyte function is generally
suppressed due to these factors because these factors are not increased in glioblastoma
patients serum.44,45 It has been proposed that they may be stimulated by direct contact
with glioblastoma cells.52 However, this also seems problematic given that at least some
studies have reported tumor-infiltrating lymphocytes are relatively rare in glioblastoma
and largely limited to perivascular locations.23,37
In addition to Treg, circulating immunosuppressive monocytic cells may also play a role
in systemic immunosuppression in glioma patients. We have recently reported increased
levels of myeloid derived suppressor cells (MDSC) in glioblastoma patients blood.44
MDSC are a heterogeneous group of early myeloid lineage cells that are increased in
multiple forms of cancer.30,53 They are identified by flow cytometry as CD33
/HLA-DR-/
Lin- cells where Lin represents a cocktail of mature myeloid, lymphoid and granulocytic
markers (CD3, CD14, CD16, CD19 and CD56). A similar population of immunosuppressive
CD14
/HLA-DR- monocytes has recently been reported by our colleagues Gustafson et al
to be increased in glioblastoma patients.45 Multiple immunosuppressive properties have
been described for MDSC, including the ability to induce apoptosis in activated T cells,
to alter T-cell recognition and to stimulate Treg proliferation among nave T cells.54-56 Thus,
it is likely that MDSC and other immunosuppressive monocytic cells play a significant
role in systemic immunosuppression in malignant glioma patients. However, the origin
of increased systemic MDSC in glioma patients remains just as cryptic as the origin of
increased regulatory T cells.
TOWARDS A UNIFIED MODEL OF GLIOBLASTOMA-MEDIATED

IMMUNOSUPPRESSION
It is now clear that multiple immunosuppressive cell types are abundantly present
in glioblastoma patients, including glioblastoma cells, glioblastoma-infiltrating
monocytes, MDSC and Treg.22,34,37,44,45,50 These various cell types combine to produce
not only a potently immunosuppressive microenvironment within the tumor but also a
markedly suppressed systemic immune response. It remains uncertain to what degree
(if any) proliferation of these various immunosuppressive cells are related to one
another. However, one can hypothesize an interconnected model in which glioblastoma
cells attract circulating monocytes to tumor parenchyma, these glioma-infiltrating
monocytes adopt immunosuppressive properties and re-enter the circulation as
MDSC and circulating MDSC stimulate systemic proliferation of Treg that results in
systemic defects in lymphocyte activation (Fig. 4). This proposed model can now
be systematically interrogated, which will yield new insights into the true nature of
glioma-mediated immunosuppression. This will facilitate rational design of glioma
immunotherapy studies in the future.
Figure 4. Potential model of glioblastoma-mediated immunosuppression. Glioblastoma patients have

increases in multiple cell types that could play multiple roles in glioblastoma-mediated immunosuppression.
This schematic shows one potential model for how these cells might interact. A) Glioblastoma cells
themselves express multiple immunosuppressive molecules (TGF-`2, PGE2, B7-H1) that inhibit activated
T cells through multiple mechanisms (decreased proliferation, increased apoptosis). They also secrete
multiple factors that may serve to attract monocytes/microglia (IL-6, CSF-1). B) Glioma-infiltrating
monocytes. Glioblastomas are heavily infiltrated by monocytic cells that are a source of further
immunosuppressive molecules (IL-10, FasL). C) Myeloid-derived suppressor cells (MDSC). MDSC are
immature myeloid cells that induce apoptosis in activated T cells and stimulate proliferation of regulatory
T cells (Treg). They are increased in glioblastoma patients blood. Their origin is unknown. However,
normal monocytes cultured with glioma cells acquire MDSC-like properties in a contact-dependent
fashion, suggesting that MDSC may arise from glioma-infiltrating monocytes. D) Treg are increased in
glioblastoma patients circulation. The cells can reduce T-cell proliferation and activation. Their origin
in glioblastoma patients is not known, but could possibly reflect activity of increased circulating MDSC.
CONCLUSION
Glioblastomas continue to be a formidable challenge. Therapies based on stimulating

antitumor immune responses are promising in preclinical studies despite the relatively
unique immunological environment within the central nervous system. In keeping with
this, glioblastomas express multiple tumor-specific and tumor-associated antigens

that can be targeted in immunotherapy. However, glioblastomas are also profoundly
immunosuppressive both locally (within the tumor) and systemically. This reflects multiple
immunosuppressive factors expressed by glioblastoma cells themselves such as TGF-`,
PGE2 and B7-H1 which, in turn, may reflect underlying oncogenic molecular abnormalities
such as mutation/deletion of PTEN or aberrant STAT-3 activation. Furthermore, other
cells such as tumor-infiltrating monocytes/macrophages and circulating myeloid-derived
suppressor cells and regulatory T cells contribute significantly to both local and systemic
immunosuppression in glioblastoma patients. While the exact relationships between these
cells are not clear, it is possible that they act as an immunosuppressive cell network in
glioblastoma patients. Understanding and disrupting this network may be a key part
of developing successful clinical glioblastoma immunotherapy strategies in the future.
REFERENCES
2. Janeway CA Jr, Medzhitov R. Innate immune recognition. Annu Rev Immunol 2002; 20:197-216.
3. Medzhitov R, Janeway C Jr. Innate immunity. N Engl J Med 2000; 343:338-344.
4. Abbas AK, Lichtman AH. General properties of immune responses. Cellular and Molecular Immunology.
Philadelphia: Elsevier Saunders 2005:3-15.
5. Abbas AK, Lichtman AH. Activation of T Lymphocytes. Cellular and Molecular Immunology. Philadelphia:
Elsevier Saunders, 2005:163-188.
6. Medawar PB. Immunity to homologous grafted skin. III. The fate of skin homografts transplanted to the
brain, to subcutaneous tissue and to the anterior chamber of the eye. Br J Exp Pathol 1948; 29:58-69.
7. Bertrand I, Mannen H. Etude des reactions vasculaires dans les astrocytomes. Rev Neurol (Paris) 1960; 102:3-19.
8. Bundy GM, Merchent RE. Basic research applied to neurosurgery: lymphocyte trafficking to the central
nervous system. Neurosurg Q 1996; 6:51-68.
9. Goldmann J, Kwidzinski E, Brandt C et al. T-cells traffic from brain to cervical lymph nodes via the cribroid
plate and the nasal mucosa. J Leukoc Biol 2006; 80:797-801.
10. Sughrue ME, Yang I, Kane AJ et al. Immunological considerations of modern animal models of
malignant primary brain tumors. J Transl Med 2009; 7:84.
11. Graeber MB, Streit WJ. Microglia: biology and pathology. Acta Neuropathologica 2010; 119:89-105.
12. Siffrin V, Vogt J, Radbruch H et al. Multiple sclerosiscandidate mechanisms underlying CNS atrophy.
Trends Neurosci 2010; 33:202-210.
13. Eikelenboom P, van Exel E, Hoozemans JJ et al. Neuroinflammationan early event in both the history
and pathogenesis of Alzheimers disease. Neurodegener Dis; 7:38-41.
14. Glass CK, Saijo K, Winner B et al. Mechanisms underlying inflammation in neurodegeneration. Cell;
140:918-934.
15. Sampson JH, Archer GE, Mitchell DA et al. Tumor-specific immunotherapy targeting the EGFRvIII
mutation in patients with malignant glioma. Semin Immunol 2008; 20:267-275.
16. Gan HK, Kaye AH, Luwor RB. The EGFRvIII variant in glioblastoma multiforme. J Clin Neurosci 2009;
16:748-754.
17. Li G, Wong AJ. EGF receptor variant III as a target antigen for tumor immunotherapy. Expert Rev Vaccines
2008; 7:977-985.
18. Sampson JH, Archer GE, Mitchell DA et al. An epidermal growth factor receptor variant III-targeted vaccine
is safe and immunogenic in patients with glioblastoma multiforme. Mol Cancer Ther 2009; 8:2773-2779.
19. Cheever MA, Allison JP, Ferris AS et al. The prioritization of cancer antigens: a national cancer institute
pilot project for the acceleration of translational research. Clin Cancer Res 2009; 15:5323-5337.
20. Zhang JG, Eguchi J, Kruse CA et al. Antigenic profiling of glioma cells to generate allogeneic vaccines
or dendritic cell-based therapeutics. Clin Cancer Res 2007; 13:566-575.
21. Zhang JG, Kruse CA, Driggers L et al. Tumor antigen precursor protein profiles of adult and pediatric brain
tumors identify potential targets for immunotherapy. J Neurooncol 2008; 88:65-76.
22. Parney IF, Farr-Jones MA, Chang L-J et al. Human glioma immunobiology in vitro: implications for
immunogene therapy. Neurosurgery 2000; 46:1169-1178.
23. Hao C, Parney IF, Roa WH et al. Cytokine and cytokine receptor mRNA expression in human glioblastomas:
evidence of Th1, Th2 and Th3 dysregulation. Acta Neuropathol (Berl) 2002; 103:171-178.
24. Siepl C, Bodmer S, Frei K et al. The glioblastoma derived T-cell suppressor factor/transforming growth
factor beta 2 inhibits T-cell growth without affecting the interaction of interleukin-2 with its receptor.
Eur J Immunol 1988; 18:593-600.
25. Kuppner M, Hamou M, Sawamura Y et al. Inhibition of lymphocyte function by glioblastoma-derived
transforming growth factor beta 2. J Neurosurg 1989; 71:211-217.
26. Fontana A, Kristensen F, Dubs R et al. Production of prostaglandin E and interleukin-1 like factor by
cultured astrocytes and C6 glioma cells. J Immunol 1982; 129:2413-2419.
27. Sawamura Y, Diserens A-C, de Tribolet N. In vitro Prostaglandin E2 production by glioblastoma cells and
its effect on interleukin-2 activation of oncolytic lymphocytes. J Neurooncol 1990; 9:125-130.
28. Bender AM, Collier LS, Rodriguez FJ et al. Sleeping beauty-mediated somatic mutagenesis implicates
CSF1 in the formation of high-grade astrocytomas. Cancer Res 2010; 70:3557-3565.
29. Norden AD, Drappatz J, Wen PY. Antiangiogenic therapies for high-grade glioma. Nat Rev Neurol 2009;
5:610-620.
30. Gabrilovich D. Mechanisms and functional significance of tumour-induced dendritic-cell defects. Nat Rev
Immunol 2004; 4:941-952.
31. Yu H, Pardoll D, Jove R. STATs in cancer inflammation and immunity: a leading role for STAT3. Nat
Rev Cancer 2009; 9:798-809.
32. Hussain SF, Kong LY, Jordan J et al. A novel small molecule inhibitor of signal transducers and activators of
transcription 3 reverses immune tolerance in malignant glioma patients. Cancer Res 2007; 67:9630-9636.
33. Kong LY, Wei J, Sharma AK et al. A novel phosphorylated STAT3 inhibitor enhances T-cell cytotoxicity
against melanoma through inhibition of regulatory T-cells. Cancer Immunol Immunother 2009;
58:1023-1032.
34. Parsa AT, Waldron JS, Panner A et al. Loss of tumor suppressor PTEN function increases B7-H1 expression
and immunoresistance in glioma. Nat Med 2007; 13:84-88.
35. Dong H, Strome SE, Salomao DR et al. Tumor-associated B7-H1 promotes T-cell apoptosis: a potential
mechanism of immune evasion. Nat Med 2002; 8:793-800.
36. Wintterle S, Schreiner B, Mitsdoerffer M et al. Expression of the B7-related molecule B7-H1 by glioma
cells: a potential mechanism of immune paralysis. Cancer Res 2003; 63:7462-7467.
37. Parney IF, Waldron JS, Parsa AT. Flow cytometry and in vitro analysis of human glioma-associated
macrophages. J Neurosurg 2009; 110:572-582.
38. Frei K, Siepl C, Groscurth P et al. Antigen presentation and tumor cytotoxicity by interferon-gamma-treated
microglial cells. Eur J Immunol 1987; 17:1271-1278.
39. Watters JJ, Schartner JM, Badie B. Microglia function in brain tumors. J Neurosci Res 2005; 81:447-455.
40. Yang I, Han SJ, Kaur G et al. The role of microglia in central nervous system immunity and glioma
immunology. J Clin Neurosci 2010; 17:6-10.
41. Huettner C, Paulus W, Roggendorf W. Messenger RNA: expression of the immunosuppressive cytokine
IL-10 in human gliomas. Am J Pathol 1995; 146:317-322.
42. Wagner S, Czub S, Greif M et al. Microglial/macrophage expression of interleukin 10 in human glioblastomas.
Int J Cancer 1999; 82:12-16.
43. Badie B, Schartner J, Prabakaran S et al. Expression of Fas ligand by microglia: possible role in glioma
immune evasion. J Neuroimmunol 2001; 120:19-24.
44. Rodrigues J, Gonzalez G, Zhang L et al. Normal human monocytes cultured with human gliomas resemble
myeloid suppressor cells. Neuro Oncol 2010; 12:351-365.
45. Gustafson MP, Lin Y, New KC et al. Systemic immune suppression in glioblastoma: the interplay between
CD14
HLA-DRlo/neg monocytes, tumor factors and dexamethasone. Neuro Oncol 2010.
46. Mahaley MS Jr., Brooks WH, Roszman TL et al. Immunobiology of primary intracranial tumors. Part 1:
studies of the cellular and humoral general immune competence of brain-tumor patients. J Neurosurg
1977; 46:467-476.
47. Dix AR, Brooks WH, Roszman TL et al. Immune defects observed in patients with primary malignant
brain tumors. J Neuroimmunol 1999; 100:216-232.
48. Morford LA, Dix AR, Brooks WH et al. Apoptotic elimination of peripheral T-lymphocytes in patients
with primary intracranial tumors. J Neurosurg 1999; 91:935-946.
49. Zou JP, Morford LA, Chougnet C et al. Human glioma-induced immunosuppression involves soluble
factor(s) that alters monocyte cytokine profile and surface markers. J Immunol 1999; 162:4882-4892.
50. Fecci PE, Mitchell DA, Whitesides JF et al. Increased regulatory T-cell fraction amidst a diminished CD4
compartment explains cellular immune defects in patients with malignant glioma. Cancer Res 2006;
66:3294-3302.
51. Humphries W, Wei J, Sampson JH et al. The role of tregs in glioma-mediated immunosuppression: potential
target for intervention. Neurosurg Clin N Am 2010; 21:125-137.
52. Hussain SF, Yang D, Suki D et al. The role of human glioma-infiltrating microglia/macrophages in mediating
antitumor immune responses. Neuro Oncol 2006; 8:261-279.
53. Ostrand-Rosenberg S, Sinha P. Myeloid-derived suppressor cells: linking inflammation and cancer.
J Immunol 2009; 182:4499-4506.
54. Serafini P, De Santo C, Marigo I et al. Derangement of immune responses by myeloid suppressor cells.
Cancer Immunol Immunother 2004; 53:64-72.
55. Sinha P, Clements VK, Ostrand-Rosenberg S. Reduction of myeloid-derived suppressor cells and induction
of M1 macrophages facilitate the rejection of established metastatic disease. J Immunol 2005; 174:636-645.
56. Nagaraj S, Gupta K, Pisarev V et al. Altered recognition of antigen is a mechanism of CD8
T-cell tolerance
in cancer. Nat Med 2007; 13:828-835.
CHAPTER 5
MECHANISMS OF IMMUNE
EVASION BY GLIOMAS
Cleo E. Rolle,1 Sadhak Sengupta1 and Maciej S. Lesniak*,2

1
Department of Surgery, Section of Neurosurgery, The University of Chicago Pritzker School of Medicine, Chicago,
Illinois, USA; 2Section of Neurosurgery, The University of Chicago, Chicago, Illinois, USA
*Corresponding Author: Maciej S. LesniakEmail: mlesniak@surgery.bsd.uchicago.edu
Abstract: A major contributing factor to glioma development and progression is its ability to
evade the immune system. This chapter will explore the mechanisms utilized by
glioma to mediate immunosuppression and immune evasion. These include intrinsic
mechanisms linked to its location within the brain and interactions between glioma
cells and immune cells. Lack of recruitment of nave effector immune cells perhaps
accounts for most of the immune suppression mediated by these tumor cells. This is
enhanced by increased recruitment of microglia which resemble immature antigen
presenting cells that are unable to support T-cell mediated immunity. Furthermore,
secreted factors like TGF-`, COX-2 and IL-10, altered costimulatory molecules and
inhibition of STAT-3 all contribute to the recruitment and expansion of regulatory
T cells, which further modulate the immunosuppressive environment of glioma.
In light of these findings, multiple immunotherapeutic treatment modalities are
currently being explored.
INTRODUCTION
The brain has classically been considered an immune privileged organ isolated
from the immune system on the basis of several key observations. Seminal transplant
immunology studies conducted by Medewar found that foreign tissues implanted in the
brain were not immediately rejected.1 Later work suggested that this was due to the fact
that the brain was physical isolated from the immune system by the blood-brain barrier
(BBB). Further, it was suggested that the brain was not connected to the lymphatic
system and that lymphocytes did not traffic into the brain. However, with improvements
in techniques and assessment of immune responses the concept of immune privilege was

53
refined. Subsequent studies demonstrated the rejection of intracerebral xenografts and

allografts in immunocompetent hosts.2
It is now well established that there is exchange between the peripheral immune
compartment and the brain. Although naive T cells fail to traffic into the brain, activated
T cells are capable of traversing the BBB. Additionally, Tsugawa and colleagues
demonstrated that brain interstitial fluid drained into the cervical lymph nodes.3 Using
an animal model, dendritic cells injected into brain tumors were later detected in cervical
lymph nodes. Our laboratory and others have characterized T cells in experimental animal
models of glioma and primary patient samples.4-7 Collectively, these data indicated that
the brain was not isolated from the immune system as previously thought, rather immune
responses are tightly regulated within the brain.
Many studies regarding glioma-mediated immunosuppression have been undertaken
in attempts to develop more effective treatment strategies. Patients with glioma exhibit
a suppression of cell-mediated immunity, which has been linked to the skewing of
the immune response within the central nervous system (CNS) towards a humoral,
rather than an inflammatory or cytotoxic immune response. This chapter will discuss
the mechanisms proposed to mediate glioma immunosuppression, including intrinsic
mechanisms directly related to its location within the brain and interactions between
glioma cells and immune cells.
INTRINSIC MECHANISMS OF IMMUNOSUPPRESSION
Altered Human Leukocyte Antigen Expression
Human leukocyte antigens (HLA) are the MHC molecules in humans. The class I
antigens are comprised of three major (classical) genesHLA-A, -B and -C; and three
minor (nonclassical) genesHLA-E, -F and -G. The classical HLA class I molecules
present intracellular antigenic peptides on the surface of altered cells, thus targeting the
cells for lysis by cytotoxic CD8
T cells (CTL). They are typically expressed by most
nucleated cells in the body. Analysis of HLA class I antigens expression in 47 glioblastoma
multiforme (GBM) lesions revealed that expression was lost in approximately 50% of
the samples.8 More detailed analysis found a selective HLA-A2 antigen loss. There was
a significant positive correlation between HLA class I antigen loss and tumor grade (P
0.025). The selective loss of HLA class I antigens would suggest a defect in antigen
presentation by glioma cells and a concomitant impairment of CTL lysis. Accordingly,
Mehling et al found that antigen processing and accessory antigen presentation molecules
were also downregulated and the extent of downregulation was correlated with tumor
grade.9 Interestingly, HLA class II antigen expression was lost in only approximately
30% of the 44 lesions analyzed.8 These data would suggest that while antigen presentation
to CD8
T cells might be impaired, glioma cells retain the ability to present antigen to
CD4
T cells.
The major HLA class II antigens are HLA-DR, -DP and -DQ. The expression of
class II molecules is restricted to antigen presenting cells. HLA-DR presents antigenic
peptides to CD4
T cells. In vitro studies of HLA-DR expression by glioma cells found that
its expression was modulated by transforming growth factor-` (TGF-`).10 Interestingly,
the expression of other HLA-related molecules, including HLA class I molecules and
beta 2-microglobulin were not modulated by TGF-`. Immunohistochemical studies
MECHANISMS OF IMMUNE EVASION BY GLIOMAS 55
found that approximately 67.2-83% of glioma patient samples expressed HLA-DR.11,12

Unlike the expression of HLA class I which decreased with tumor grade, HLA-DR
expression increased as tumor grade increased. The increased expression of HLA-DR
suggests a skewing of the immune response towards helper T cells, rather than CTL.
Further, this would explain that although CTL are found in glioma lesions they appear
to be unable to lyse glioma cells.
Recently, HLA-G, a nonclassical HLA class I molecule, has been identified in
malignant tumors and glioma cell lines and may be involved in tumor immune escape.13
The normal function of HLA-G is to maintain immune tolerance at the maternal-fetal
interface by inhibiting maternal NK cell activity against fetal tissue. The major isoforms
of HLA-G are HLA-G1 and HLA-G5, which exist as membrane bound surface molecules,
as well as in a soluble form. Expression of soluble HLA-G is increased in the plasma of
patients with malignant melanoma, glioma, breast and ovarian cancer.14,15 The expression
of membrane-bound HLA-G1 and soluble HLA-G5 by tumor cells inhibited alloreactive
and Ag-specific immune responses in vitro. Ectopic expression of HLA-G1 or HLA-G5
in HLA-G-negative glioma cells (U87MG) conferred resistance to lysis and prevented
efficient priming of cytotoxic T cells. Interestingly, HLA-G expression by a few tumor
cells within a population of predominantly HLA-G-negative tumor cells was sufficient
to confer significant inhibitory effects on immune cells.16 Therefore, the aberrant
expression of HLA-G by glioma cells is a mechanism by which they are able to evade
NK cell-mediated lysis, as well as prevent T-cell activation.
Another inhibitory molecule is the nonclassical MHC class I molecule HLA-E.
It is the only known ligand for CD94/NKG2A, an inhibitory receptor, expressed on
NK cells and CD8
T cells. The over-expression of HLA-E by glioma cells would
render them resistant to NK cell and CTL cytotoxicity. Analysis of human long-term
glioma cell lines, primary ex vivo polyclonal glioblastoma cell cultures and surgical
glioblastoma specimens, revealed high expression of HLA-E.17,18 There was a massive
over-expression of HLA-E in Grade IV glioblastomas compared to normal CNS tissue.
HLA-E expression levels and immune cell infiltration were positively correlated in WHO
Grade IV glioblastomas. According to Wischhusen et al, siRNA-mediated silencing of
HLA-E or antibody-mediated blocking of CD94/NKG2A enables NKG2D-mediated
lysis of tumor cells by NK cells.17
The altered expression pattern of HLA molecules is one potential mechanism by
which glioma cells are able to evade the immune system. The coordinated down-regulation
of HLA-A and up-regulation of HLA-DR could explain the defective CTL responses
and enhanced CD4
T-cell responses observed in glioma patients. In addition to the
down-regulation of HLA-A, accessory antigen processing and presentation molecules
are also down-regulated, suggesting a mechanism by which glioma cells prevent antigen
presentation to CD8
T cells. Conceivably, HLA-E and HLA-G inhibit NK cell and T-cell
lysis of glioma cells.
The Role of Microglia
Microglia are the brains resident macrophages. They differentiate from monocytes
which migrate into the brain during embryogenesis.19 Microglia are distinguished from
macrophages based on low expression of CD45 and high expression of CD11b (CD45dim
CD11b
). Under normal physiological conditions, microglia roam the CNS, phagocytosing
debris and maintaining homeostasis. Microglia were first described in brain tumors in the
early 1920s.20 This finding was later confirmed using animal models and primary patient
samples.11,21,22 More recently, primary patient samples obtained from 9 newly diagnosed
malignant gliomas quantified microglial infiltration.23 While these studies clearly identified
microglia in gliomas, their exact role in immunosuppression was not described until much
later. Studies have suggested that the extent of microglia infiltration correlates to tumor
grade. Roggendorf et al proposed early on that tumors actively recruited migroglia.24 The
expression of MCP-1, a monocyte chemoattractant, by glioma cells was later confirmed
and shown to be functional.25,26
Although the presence of microglia in glioma has long been established, the actual
role of tumor infiltrating microglia during glioma remains controversial. This is related
to conflicting data regarding the immunostimulatory ability of microglia. On the basis
of the expression of MHC and costimulatory molecules, microglia resemble immature
antigen presenting cells. However, upon activation, microglia up-regulate the expression
of MHC and B7, making them more likely to stimulate T cells. Therefore, some studies
have found that microglia are able to prime T cells.27 Microglia derived from normal
brains are inefficient at presenting glioma-derived antigens to nave T cells and CTL in
vitro.28 In contrast, microglia isolated from normal brain and expanded in vitro with growth
factors, express MHC class II, efficiently process antigen and stimulate T cells.27 Primary
microglia isolated from human glioma samples only weakly induced T-cell cytotoxicity
against glioma cells.29These data indicate that with appropriate stimuli microglia are able
to activate T cells, however in the tumor microenvironment they are unable to support
T-cell-mediated cytotoxicity, and in some cases directly inhibit T-cell proliferation.
It is likely that microglia may play dual roles during glioma development through the
expression of cytokines. Microglia have been postulated to support glioma cell proliferation
through the production of IL-10 and IL-6.30,31 IL-10 is also an immunosuppressive cytokine
which inhibits T-cell proliferation. Furthermore, microglia express a clearly Th2 skewed
cytokine profile, which suppresses cytotoxic responses. Taken together, glioma infiltrating
microglia support glioma tumorigenesis while promoting immune evasion.
The Regulation of Th1/Th2 Cytokine Responses
The cytokine milieu of the brain ensures that immune responses are primarily humoral
responses in order to prevent damage due to inflammation. The normal humoral response
is further skewed in glioblastoma patients. Analysis of gene expressions of Th1/Th2
cytokines in 62 specimens of human glioma tissues, 4 glioma cell lines and 5 specimens
of normal adult brain tissue determined predominant expression of Th2 cytokines in
glioma cell lines (P 0.01) and specimens of human glioma tissues (P 0.01).32 The total
positive rates of Th1 and Th2 cytokines genes in human glioma cells were 14.77% and
75%, respectively.33 In addition to glioma cells, the cytokine profiles of glioma infiltrating
lymphocytes were also Th2 skewed.34 Therefore, the immunosuppressive cytokine milieu
of gliomas is not only influenced by glioma cells, but also the infiltrating lymphocytes.
IL-10 is a Th2 cytokine that is has been described in CNS responses. There was
high expression of IL-10 reported at the mRNA level in 87.5% of Grade III and IV, but
only 4% of Grade II tumors. Elevation of IL-10 serum levels was found in 11% of low
grade and in 63.6% of high grade glioma patients.30 IL-10 was found to stimulate the
proliferation of glioma cells. This glioma cell proliferation was blocked by the addition
of blocking IL-10 antibody.35 Additonally, glioma-derived IL-10 greatly downregulated
HLA-DR expression on monocytes. In vitro, IL-10 increased the proliferation and
migratory capacity in human glioma cell lines. Interestingly, blocking IL-10 antibody was
not sufficient to inhibit IL-2 production.36 These data suggest that IL-10 in the cytokine
milieu of glioma is sufficient to inhibit T-cell proliferation but does not induce T-cell
anergy. During glioma tumorigenesis, it is plausible that IL-10 plays dual roles, acting
as a growth factor for glioma cells and as an immunosuppresive factor.
Since this early work assessed IL-10 mRNA levels in patient tumor samples and not
purified glioma cells, it was proposed that IL-10 was produced by glioma cells. However,
in situ hybridization for IL-10 and CD68 immunostaining revealed that only cells of the
microglia/macrophage lineage produced IL-10.37 More recently, Samaras et al assessed
the expression of IL-10 in paraffin-embedded neoplastic tissue of 12 glioma patients.
In comparison to healthy controls, IL-10 secretion from peripheral mononuclear and
tumor tissue from glioma patients was higher (P 0.0002).38 As in the earlier study,
IL-10 expression in glioblastomas was restricted to microglia/macrophage cells. IL-10
production by microglia supports the growth and proliferation of glioma cells and
mediates immune evasion.
IMPAIRMENT OF GLIOMA AND IMMUNE CELL INTERACTIONS
Cell-to-cell contact is required for lymphocytes to lyse tumor cells. Specifically, the
release of the cytotoxic molecules, perforin and granzymes, by natural killer (NK) cells
and CTL is cell contact dependent. Glioma cells have been shown to alter the extracellular
matrix and this may have an impact on cell-to-cell contact. Recently, tenascin-C expression
has been described in glioma cells. Tenascin-C belongs to a specialized class of ECM
proteins, matricellular proteins, which function as adaptors and modulators of cell-matrix
interactions.39,40 The exact role of tenascin-C is unresolved as it has been implicated in
both cell adhesion and migration.41-43 Tenascin-C has also been shown to inhibit T-cell
proliferation and IFNa production.44,45 The infiltration of lymphocytes into tumors was
inhibited by tenascin-C.46 Through the expression of tenascin-C, glioma cells could
potentially enhance cell migration, while suppressing T-cell responses by limiting
cell-to-cell contact.
MECHANISMS OF GLIOMA-MEDIATED IMMUNOSUPPRESSION
Secreted Immunosuppressive Factors
TGF-`
Glioma cells secrete multiple factors that have been shown to suppress antitumor
immune responses, namely TGF-`. There are three isoforms of TGF-`: TGF-`1, -`2,
-`3. TGF-`2 has been shown to enhance tumor growth and invasion, angiogenesis and
immunosuppression. The expression of TGF`-1 and -2 in two glioblastoma cell lines and
newly isolated patient samples was confirmed at the mRNA level.47 However, only TGF`-2
was detected in the supernatant of glioma cell lines and in the cerebral spinal fluid of
patients with malignant glioma.48 Interestingly, TGF-`2 was originally isolated and cloned
from glioblastoma cell lines and was named for its ability to suppress T-cell growth and
IL-2 production.49,50 More recently, TGF-`1 and -`2 have been shown to induce FoxP3
expression in anti-CD3 activated CD4

T cells, resulting in inducible regulatory T cells
(Tregs), whose role in glioma-mediated immunosuppression will be discussed further.
Thus, the secretion of TGF-` by glioma cells mediates immunosuppression not only by
acting directly on T cells and NK cells, but also indirectly through the induction of Tregs.
Cyclooxygenase-2 and Prostaglandin E2
Cyclooxygenase-2 (COX-2) is an isoform of the cyclooxygenase enzyme or

prostaglandin synthase responsible for the formation of important biological mediators
called prostanoids (including prostaglandins, prostacyclin and thromboxane). COX-2 is
an inducible enzyme, becoming abundant in activated macrophages and other cells at sites
of inflammation. Inhibitors of COX-2 are used as nonsteroidal anti-inflammatory drugs
(NSAID) for treating inflammation and pain. The over-expression of cyclooxygenase-2
(COX-2), the key enzyme for the synthesis of prostaglandin E2 (PGE2) from arachidonic
acid, has also been well characterized in many types of cancers including colorectal, lung,
urinary bladder and malignant gliomas.51-55
PGE2 suppresses cell-mediated immune responses while enhancing humoral
immune responses.56,57 In macrophages the down-regulation of cell-mediated immune
responses by PGE2 is characterized by a remarkable drop in LPS-mediated TNF-_ and
IL-12 production.58,59 PGE2 modulates a variety of physiological processes, including
APC function and the production of inflammatory cytokines.60,61 PGE2 is also a potent
inducer of IL-10,62 which is produced by a variety of cells including monocytes, and
exerts suppressive effects on dendritic cell maturation and Th1 responses.63-64
PGE2, contributes to cellular immune suppression in cancer patients through an
unknown mechanism. In 2004 Akasaki et al, reported that Tr1 Tregs were induced
by CD11c
mature dendritic cells that phagocytosed allogeneic and autologous
COX-2-overexpressing glioma.65 These gliomas did not express IL-10 receptors
and recombinant IL-10 did not suppress their COX-2 expression. Exposure to
COX-2-over-expressing glioma induced mature dendritic cells (DC) to over-express
IL-10 and decreased IL-12p70 production. These dendritic cells (DC) induced a Tr1
response, which is characterized by secretion of IL-10 and TGF- with negligible IL-4
secretion by CD4
T cells and an inhibitory effect on lymphocytes. Peripheral CD4
T-cell populations isolated from a glioma patient also predominantly demonstrated a

Tr1 response. Selective COX-2 inhibition in COX-2-overexpressing gliomas at the
time of phagocytic uptake by DCs abrogated this Tr1 response and instead elicited Th1
activity. Stable transfection of COX-2 in glioma cell lines also induced a Tr1 response.
These results indicate that COX-2-overexpressing tumors induce a Tr1 response, which
is mediated by tumor-exposed, IL-10-enhanced DCs.
PGE2 levels were significantly higher in patient glioma samples compared to control
brain samples.66 Furthermore, surgical removal of malignant brain tumors resulted in
a decrease of PGE2 levels.67 PGE2 has been linked to tumor metastasis and immune
evasion, such that the inhibition of PGE2 synthesis using COX inhibitors suppressed
tumor growth.68-71 The increased biosynthesis of PGE2 in glioma cells is due to increased
expression of mPGES-1, an enzyme which catalyses the isomerization of PGH2 into PGE2.72
PGE2 has been implicated in tumorigenesis, mediating tumor growth and angiogenesis,
as well as immunosupression.
PGE2 has been shown to increase vascular permeability and edema and at physiological
concentrations blocks IL-2 production.73-75 It also inhibits the induction of CTLs, NK
cells and lymphokine activated killer cells (LAK).76 Lauro et al found that serum-free
supernatant from glioma cells inhibited the proliferation of PHA-stimulated T cells.77
The expression of PGE2 by glioma cells in vitro was sufficient to inhibit the induction of
LAK cells from PBMCs and tumor infiltrating lymphocytes, even at high concentrations
of IL-2.78
Not only do glioma cells themselves produce PGE2, but they can also stimulate tumor
infiltrating microglia to produce PGE2.79 Coculture of glioma cells with microglia or
microglia cultured in the presence of glioma conditioned media increased the expression
of mPGES1 and enhanced the production PGE2.80 Therefore, the production of PGE2 by
the glioma cells themselves and the enhancement of PGE2 production in microglia both
contribute to immunosuppression.
Induction of T-Cell Anergy
Altered B7 Expression
While T cells infiltrate gliomas, in both animal models and primary patient samples,
they often lack cytotoxic activity and fail to produce IL-2. This observed T-cell anergy
has been linked to defective antigen presentation and poor costimulation by tumor cells.
As we discussed previously, antigen presentation to CD8
T cells is decreased during
glioma tumorigenesis due to the loss of HLA class I expression. Additionally, glioma
cells express very low levels of B7 costimulatory molecules.81 Functional assays using
heterogeneous ex vivo tumor preparations or pure populations of ex vivo tumor cells and
microglia, demonstrated CD4+ T-cell activation only in the presence of exogenous B7
costimulation (provided by addition of soluble agonist anti-CD28 monoclonal antibody).
Therefore, to overcome immunosuppression in glioma, strategies need to overcome the
low levels of B7 costimulation.
STAT3
In recent years, signal transducer and activator of transcription-3 (STAT3) has been
identified as a major molecular hub of several signaling pathways in several types of cancer
including glioblastoma, breast, lung, ovarian, pancreatic, skin and prostate cancer.82,83
The binding of STAT3 to its gene targets affects proliferation, survival, differentiation
and development. It is a member of the STAT family of cytoplasmic latent transcription
factors. Receptor engagement by members of IL-6 cytokine family like IL-6, oncostatin
M and Leukemia inhibitory factor, or growth factors like platelet-derived growth factor
(PDGF), fibroblast growth factor (FGF) and epithelial growth factor (EGF) activate
STAT3 by tyrosine phosphorylation. Kinases that induce STAT3 activation are receptor
kinases like Janus kinase (JAK) family memebers associated with, FGFR, EGFR, PDGFR
or nonreceptor-associated kinases like Ret, Src or Bcl-Abl. Postactivation STAT3 works
together with other transcription factors to regulate expression of bcl2, bclxL, mcl1
and cyclinD1 among others.83 STAT3 activity is attenuated by suppressors of cytokine
signaling (SOCS) by down-regulating its upstream kinase activity, while protein inhibitors
of activated STAT (PIAS) and protein tyrosine phosphatases target STAT3 directly.84-85
Other than promoting oncogenesis, active STAT3 also enables tumor growth
by suppressing tumor recognition by the immune system.86 STAT3 promotes tumor
immune evasion by inhibiting pro-inflammatory cytokine signaling and amplifying
Tregs. Inhibiting STAT3 activity by dominant-negative or antisense STAT3 expression

increased expression of proinflammatory IFN-`, TNF-_, IL-6, RANTES and IP-10, while
activating STAT3 hindered expression of IL-6 and RANTES in experimental systems.87
STAT3 is also involved in maintaining immature dendritic cells and promoting tumor
immune tolerance. Mice lacking STAT3 had elevated levels of MHC class II, CD80
and CD86. Antitumor activity of T cells, NK cells and neutrophils was increased and
dendritic cell maturation was enhanced by inhibition of STAT3.88,89 STAT3 activity
was shown to be elevated in tumor-associated Treg cells that maintain tumor immune
evasion.88,90 In these Tregs, elevated activation of STAT3 increased proliferation and
promoted expression of FoxP3, TGF-` and IL-10, all of which inhibit CD8
T-cell
differentiation, dendritic cell maturation and promote proliferation of Tregs.86,90
In gliomas, activation of STAT3 has been associated with the inhibition of T-cell
response. Tregs have been shown in the blood and within the tumor microenvironment
of glioblastoma patients and are thought to contribute to the lack of effective immune
responsiveness against glioblastomas, although the STAT3 activation status of these cells
was not examined in these studies.29,91 In a tissue microarray using tumor samples from 129
glioma patients, Abou-Ghazal et al92 showed correlation between prognosis of activated
STAT3 residues and tumor-infiltrating Tregs. Low incidence of activated STAT3 was
observed in normal brain or low-grade astrocytomas. While the incidence of activated STAT3
varied in different grades of gliomas, it correlated with the tumor-infiltrating immune-cells
but not with that of Tregs. Glioma-infiltrating CD8
T cells were characterized as a marker
of immunosuppressive environment as they were neither activated nor proliferating.29
Using a novel small molecule inhibitor of STAT3 in monocytes from glioma patients, the
expression of costimulatory molecules such as CD80 and CD86 were up-regulated.93 This
apparently enhanced T-cell activation which is critical in overcoming immune suppression.
PDL-1
Programmed Death Ligand-1 (PDL-1) or B7-H1 is a member of the B7 family and is

a ligand for PD-1(Programmed Death-1), an extended member of CD28 family of T-cell
regulators. B7-H1 is primarily inhibitory when expressed on tumors. It is significantly
expressed on the surface of many human cancers, while undetectable in normal tissues.94
B7-H1 is found to be highly expressed in carcinomas of colon, breast, ovarian, lung and
melanoma cancer, oral squamous cell carcinoma, head and neck cancers and glioma
among others.95-98
B7-H1 induction of IFN-a and IL-10, granulocyte macrophage colony-stimulating
factor production and proliferation of T cells in vitro and the late downstream effects of
signaling initiated through the B7-H1:PD-1:unidentified receptor network result in negative
regulation of immune function.99,100 The inhibitory effects of B7-H1:PD-1 interactions in
tumor immunity were described by models using either PD-1 knockout mice or blocking
monoclonal antibodies for B7-H1 or PD-1. Both increase cytokine production and antitumor
CTL activity in experimental tumor models in mice.101-105 Another possible mechanism is
by induction of apoptosis in effector CTL cells through PD-1 after exposure to B7-H1 on
tumor cells.101 Several models in which both human and murine tumors are transfected with
B7-H1 become resistant to CTL attack, resistant to lysis and more resistant to apoptosis
induction, while actually inducing lysis of CTLs through PD-1.102,106
Expression and immune regulatory activity of B7-H1 has been recently identified
in human glioma cells in vitro and in vivo. In 12 different glioma cell lines, Wintrelle
et al observed constitutive expression of B7-H1 mRNA, although these cells lacked

B7.1/2 (CD80/86).98 Upon exposure to IFN-a, B7-H1 expression was strongly enhanced.
High B7-H1 expression was observed in all 10 malignant glioma specimens upon
immunohistochemical analysis, whereas no B7-H1 expression could be detected on
normal brain tissues. Functional significance of glioma cell-related B7-H1 expression
was elucidated by coculture experiments of glioma cells with alloreactive CD4
and
CD8
T cells. Glioma-related B7-H1 was identified as a strong inhibitor of CD4
as well
as CD8
T-cell activation as assessed by increased production of IFN-a, IL-2 and IL-10
and expression of CD69 in the presence of a neutralizing antibody against B7-H1. These
observations infer that B7-H1 may significantly influence the outcome of T-cell tumor
interactions and correspond to a novel mechanism by which glioma cells evade immune
recognition and destruction.
Inhibition of Natural Killer Cells
Natural killer (NK) cells are innate immune cells that lyse tumor cells or infected
cells. Upon activation they also participate in the initiation of adaptive immune responses,
primarily through the production of cytokines. Unlike T and B cells which express an
antigen-specific receptor and only become activated following antigen presentation via
MHC molecules, NK cells are not MHC-restricted. In fact, NK cells were named for
their ability to lyse target cells lacking MHC class I molecules.107 Natural killer cells are
cytotoxic lymphocytes, expressing perforin and granzymes, resulting in the induction
of apotosis or necrosis in target cells. The activation of NK cells is tightly regulated and
requires increased signaling via activating receptors coupled with decreased signaling
via inhibitoty receptors. Interferons, IL-2, IL-12 and IL-8, also serve to activate NK cells.
Natural killer cells express the following activating and inhibitory receptors: NKG2, Ly49,
killer cell immunoglobulin-like receptors (KIR) and leucocyte inhibitory receptors (LIR).
Engagement of NKG2D, an activating receptor, has been shown to be critical for NK
cell-mediated tumor rejection.108,109 Despite an increased fraction of NK cells in the blood
and tumor-infiltrating lymphocytes of glioma patients, MHC-unrestricted NK activity is
impaired.110 As tumor grade increases, NK cell cytotoxicity becomes impaired.111
Lectin-Like Transcript 1
Although it is well established that malignant brain tumors have significantly altered
HLA expression they are able to escape NK cell mediated rejection. This would suggest
that activating signals are overridden by inhibitory signals.112 As we discussed earlier, the
expression of TGF`, HLA-E and HLA-G by glioma cells, all contribute to inhibit NK cell
activity against malignant brain tumors. More recently, lectin-like transcript 1 (LLT1)
has been shown to inhibit NK cell activity via an interaction with CD161 on human NK
cells. Lectin-like transcript 1 was identified as a ligand for CD161 based on its sequence
homology to ligands for other CD161 receptor family members.113 Engagement of CD161
expressed on NK cells by LLT1 on target cells inhibited NK cell cytotoxicity and IFNa
secretion. The expression of LLT1 was primarily restricted to monocytes and B cells in the
peripheral blood and PBMCs, NK cells and T cells expressed it upon activation.
According to Roth et al, human glioma cells expressed LLT1 mRNA and protein in
vitro and in vivo.114 Although LLT1 was expressed by normal brain cells, it was expressed
at significantly lower levels compared to glioma cell lines and primary glioma samples
(p ) 0.0001). There was a positive correlation between WHO tumor grade and level of
LLT1 expression. Interestingly, TGF` treatment enhanced the expression of LLT1 by
glioma cells in vitro. In order to assess the role of LLT1 in glioma immunosuppression,
siRNA was used to inhibit LLT1 expression in glioma cells. Natural killer cells were able
to lyse glioma cells in which LLT1 levels were successfully knocked down. Therefore,
the expression of LLT1 by glioma cells mediates immune evasion.
Regeneration and Tolerance Factor
According to a recent report by Roth and colleagues, RTF mRNA and protein
were expressed in human glioma cells in vitro and in vivo. Originally identified in the
placenta for its role in fetal allograft survival, RTF has been implicated in the inhibition
of T-cell activation and the induction of T-cell apoptosis.115 In animal models of glioma,
suppression of RTF using RNAi promoted the lysis of glioma cells by NK cells and T
cells and by extension RTF-depleted glioma cells failed to grow tumors in nude mice.116 In
vivo, RTF-mediated tumor rejection was reversed in NK cell depleted hosts. Collectively,
these data suggests that RTF expression by glioma cells enhances immunosuppression
by inhibiting NK cells.
The Induction of Apoptosis
Fas
The induction of apoptosis is a key immune evasion strategy for multiple malignancies,
including glioma. Fas/APO-1 is a cell surface receptor that mediates apoptosis when
it reacts with Fas ligand (FasL) or anti-Fas antibody. The expression of Fas has been
demonstrated on perinecrotic glioma cells, a histological hallmark of glioblastomas,
suggesting a correlation between Fas expression and cell death. Since glioma cells
are not always able to turn off Fas expression they must use other strategies to evade
Fas-induced apoptosis. A potential endogenous antagonist of Fas is soluble Fas which
lacks the transmembrane domain. The soluble form of the Fas mRNA was detected in
one anaplastic astrocytoma and in two glioblastomas.117 Interestingly, some glioma cells
co-expressed Fas and FasL suggesting that soluble Fas expression by the glioma cells
prevented tumor cell-induced apoptosis.
The expression of FasL was observed in 10 glioblastoma cell lines and in 14 astrocytic
brain tumors (three low-grade astrocytomas and 11 glioblastomas).118 The expression
of FasL by glioma cells may act as a glioma immune evasion mechanism. Apoptosis
was induced in T cells by FasL
tumor explants and tumor cell lines.119 According to
Didenko et al, T cells that had undergone apoptosis expressed Fas and were colocalized
with FasL-expressing tumor cells.120
Since glioma cells have the ability to express both Fas and FasL, they have evolved
mechanisms to prevent tumor cell apoptosis, while simultaneously inducing apoptosis
in T cells. In a recent report by Roth et al, the expression of decoy receptor 3 (DcR3),
a soluble FasL molecule with anti-apoptotic properties, was detected in human glioma
cells and in GBM samples.121 The expression of DcR3 was correlated to tumor grade and
was not detected in WHO Grade II tumors. The protective effect of DcR3 was specific to
Fas induced cell death and did not prevent apoptosis induced by TRAIL ligands. Ectopic
expression in the rat glioma cell line 9L inhibited T-cell and microglia infiltration. These
data demonstrate a mechanism by which glioma are able to ensure tumor propagation by
evading the immune system and prevent death in tumor cells.
CD70
CD70, TNF-related cell surface ligand, is normally expressed on mature dendritic

cells, activated T and B cells.122 The binding of CD70 to its cognate receptor CD27,
a TNF receptor family member, is thought to play an important role in T-cell, B-cell
and NK cell activation. CD27 is constitutively expressed by T cells, B cells and NK
cells.123 Signaling via CD27 by activated T cells is critical for survival and memory cell
formation.124 In contrast, the ligation of CD27 has also been shown to induce apoptosis
in activated T and B cells.125 CD70-CD27 induced cell death has been implicated in
models of chronic immune activation as a means to maintain self tolerance.126 Moreover,
CD27 costimulation has been shown to enhance Treg function.127
According to a study conducted by Wischhusen et al, 11 of 12 human glioma cell
lines expressed CD70 mRNA and protein.16 CD70
glioma cells were able to induce
apoptosis in PBMC. In vitro experiments using glioma cells, in which Fas and TNF_
were blocked, revealed that CD70 was sufficient to induce apoptosis in T cells.128 The
expression of CD70 is a novel mechanism utilized by glioma cells to induce apoptosis
in T cells and consequently evade the immune response.
Gangliosides
Previous studies demonstrated the role of tumor-derived gangliosides as important

mediators of T-cell apoptosis and hence, as one mechanism by which tumors evade
immune destruction.129 Gangliosides are most highly expressed in cells of the CNS,
where they comprise approximately 5-10% of the lipids.130 Using a human glioblastoma
biopsy brain tumor nude rat xenograft model, glioma cells were shown to express
multiple gangliosides: GD3, GM2 and 3v-isoLM1.131 Ganglioside GD3 was detected
in the tumor mass while ganglioside 3v-isoLM1 was more abundantly expressed in the
periphery of the tumor, associated with areas of tumor cell invasion. The ganglioside
GM2 was only detected within the main tumor mass. According to a study conducted by
Markowska-Woyciechowska et al, high and low grade gliomas had similar ganglioside
profiles.132 The expression of gangliosides GD2 and GD3 was restricted to the cytoplasm
of highly fibrillary (gemistocytic) astrocytes in all grades of gliomas.133 Moreover, the
increase of GD3 was associated with a decrease of normal brain gangliosides and this
was correlated with a higher grade of malignancy.134 This work supports the notion that
gangliosides play a role in apoptosis and indicates that antibodies or ligands directed
against GD3 and 3v-isoLM1 might be complementary when applied in the treatment of
human glioblastomas.131
RECRUITMENT OF IMMUNOSUPPRESSIVE LYMPHOCYTES
Expression of Chemokines
Chemokines are a family of cytokines helping in the migration of responder cells by

inducing directed chemotaxis. These proteins are very small in size (8-10 kDa) and have
a characteristic two cysteine residues joined by disulfide bonds (cystine). The different
members of chemokine family share gene sequence and amino acid homology. They are
classified into several categories according to spacing of the cystine moieties that are
key to forming their 3-dimensional shape and accordingly, different families of receptors
for these molecules have been described.135 Their role has been implicated in various
processes including angiogenesis and CNS development. Most importantly, chemokines
play a central role within the immune system, as the secretion of these molecules leads
to migration of leucocytes.136-138 Chemokines appear to play a significant role in various
human diseases, including cancer.139 As chronic inflammation can predispose to cancer
formation and progression, the expression of chemokines is suspected to contribute to
this process.140 Alternatively, chemokines might elicit an intrinsic effect on tumor cells.
For instance, multiple human cancers including leukemias, lymphomas, gliomas and
various epithelial carcinomas express CXC receptor 4 (CXCR4) and respond to its ligand
CXC ligand 12 (CXCL12). This ligand-receptor interaction promotes the migration and
metastatic establishment of tumor cells.141,142
One of the initial reports of chemokines secretion by gliomas was made in 2001 by
Choi and others, where the authors demonstrated that human glioma cell lines secrete
MCP-1 (CCL2) and IL-8 on stimulation with anti-Fas antibody or soluble Fas ligand in a
time and dose-dependent manner.143 However, at that time its function was not correlated
to Treg migration and was instead attributed to proinflammatory properties.
With regards to the tumoral migration of Tregs, cancers express a series of chemokines
that promote the infiltration by these regulatory lymphocytes. For instance, chemokine
CCL22 promotes the migration of Tregs into prostate and ovarian carcinomas.144 Human
glioma cell lines have been reported to express CCL22 in addition to CCL2, although
only CCL2 was secreted by samples from GBM patients.91 This has been investigated
in the human glioma cell lines D-54, U-87, U-251 and LN-229 as well as in tumor cells
from eight patients with GBM. The authors further reported that Tregs from these brain
tumor patients had significantly higher expression of the CCL2 receptor CCR4 than the
Tregs from healthy controls. Migration experiments have suggested that Treg migration
is mediated by CCL2 and CCL22, which was blocked by antibodies to the chemokine
receptors CCR2 and CCR4.
Myeloid-Derived Suppressor Cells
Myeloid-derived suppressor cells (MDSC) were originally named natural

suppressors because they functioned independently of MHC interactions and inhibited
the antigen-induced proliferation of T cells and antibody production by B cells.145-147
MDSC were isolated from the bone marrow of tumor patients and named for their
ability to suppress T-cell proliferation.148 They lack expression of surface molecules
characteristic of T, B, macrophage, or NK cells. Myeloid-derived suppressor cells
are immature myeloid cells that have the potential to generate mature granulocytes,
macrophages and dendritic cells.149-151 In mice, they are identified based on co-expression
of CD11b and Gr-1.152,153 Human MDSC equivalents are CD34

CD33
, CD15- and
CD13
, with variable expression of CD11c and HLA-DR and were originally identified
in patients with head and neck cancer.154 Further studies of patients with tumors showed
an increased population of myeloid-derived suppressor cells in secondary lymphoid
organs, blood and tumor site. In fact, tumor-derived factors promoted MDSC recruitment
and maturation into immunosuppressive cells and inhibited the differentiation of
dendritic cells. The effect of tumor-derived factors was abrogated with neutralizing
antibodies to IL-6 and colony stimulating factor-1 (CSF-1).155,156 Interestingly, glioma
cells are known to produce both IL-6 and CSF-1, which may aid in the recruitment
and/or expansion of MDSC in patients.
Regulatory T Cells
An increased fraction of Regulatory T cells (Tregs; CD4

CD25
FOXP3
) has
been reported to infiltrate the tumor contributing to the immunodeficient status
associated with glioma.4-6,29,157 Tregs are a fraction of the T-cell population that
suppress immune activation and thereby maintain immune system homeostasis and
tolerance to self-antigens. Functional deletion of Treg cells induce autoimmunity,
facilitate transplantation tolerance and also increases immunity to tumors.5,158,159 A
lack of immune rejection of neoplastic cells is believed to be maintained by Tregs in
many malignancies including colorectal, esophageal, pancreatic, breast, lung, ovarian
and brain tumors.5,160-163 It is therefore very important to understand the biology and
function of Tregs for its potential therapeutic benefits.
Sakaguchi et al, demonstrated that a unique subset of CD4
T cells expressing CD25
(IL-2 receptor _-chain) in normal rodents displayed potent immunoregulatory functions
in vitro and in vivo.164 Treg cells consist of 1-10% of total CD4
T cells in thymus,
peripheral blood and lymphoid tissues and could conceivably recognize a wide spectrum
of self and nonself antigens.165 Treg development takes place directly in the thymus and
leave thymus as mature with defined phenotype.166 This is unlike the development of
other T-cell subsets, which are induced upon antigen exposure.
They are dependent on IL-2 stimulation for their development, peripheral expansion
and suppression function.164 Experimental mice which are deficient for IL-2 and IL-2R have
a reduced pool of Treg cells and die prematurely from a severe lymphoproliferative and
autoimmune syndrome.167-169 In IL-2R` knock-out mice, introduction of IL-2R` transgene,
which is expressed predominantly in the thymus, CD4
CD25
T-cell development is restored
indicating that an intact IL-2/IL-2R pathway is also required for thymic generation of
Treg.170 In experimental mouse models, adoptive transfer of wild-type (WT) Treg cells
prevents autoimmunity in mice lacking a functional IL-2R, but not in those lacking IL-2.
These donor Treg cells undergo rapid and extensive IL-2-dependent proliferation in lymph
nodes and spleen.168 These studies imply that IL-2 is a critical growth and differentiation
factor for Treg cells.171 Blocking the IL-2R on Treg cells leads to a loss of their regulatory
activity, suggesting a possible role for IL-2 for suppressor function.172 These studies show
that IL-2 may be required in the production of Treg and alterations in this pathway may
block Treg cell development. Functionally, IL-2 promotes proliferation and survival in T
cells either by activating the signal transducer and activator of transcription 5 (STAT5)
transcription factor or by up-regulation of antiapoptotic molecules, Bcl-2.173,174 Blocking
IL-2 signaling in Treg cells does not disrupt an essential survival pathway, whereas
abrogation of STAT5 show autoimmune pathology similar to IL-2 and IL-2R knock-out
mice and a decrease in number of CD4

CD25
Treg cells. Mice transgenic for the active
form of STAT5 possess a greater frequency of Treg cells.175-177
Tregs express forkhead box P3 (FoxP3), a transcription factor that plays a central
role in defining their function. FoxP3 is essentially expressed in CD4
CD25
CD8<
thymocytes but not in any other thymic cells.178-180 FoxP3 deficient mice suffer from
autoimmune symptoms and die from inflammatory diseases. However, this condition can
be abrogated by adoptive transfer of CD4
CD25
FOXP3
cells from immune-competent
mice.170 In humans, Type I diabetes, thyroiditis and inflammatory bowel diseases is
associated with mutations on the FoxP3 gene.
Existence of other populations of T regulatory lymphocytes has been reported in
recent publications. Unlike classic CD4
CD25
FoxP3
Tregs, these cells are induced in
the periphery via T-cell receptor (TCR)-MHC/peptide stimulation. Qiao et al, described
the existence of a different population of regulatory T cells called iTregs or induced
regulatory T cells.181 These were developed from CD4
CD25< cells when exposed
to CD4
CD25
FoxP3
Tregs and suppressive conditions like TGF-` and suppressed
responder T-cell activation by cell-to-cell contact and secreted factors. These cells
did not express Foxp3.181 Another example of nonclassical regulatory T cells are Tr1
cells which are induced in the periphery in a process that is dependent on IL-10 and
interactions with immature dendritic cells which lack the expression of costimulatory
molecules.182 These cells are characterized by the expression of CD4
CD25int/high and
mediate suppression by secreting large amounts of IL-10. In contrast to Tregs, FoxP3
is not constitutively expressed in Tr1 cells.183 Third of this kind are Th3 cells, induced
in the periphery through a TGF-` dependent process and these cells require IL-10 for
expansion. Th3 cells suppress via the secretion of TGF-`.184 Both Tr1 and Th3 cells
are implicated in oral tolerance.185 They play a role in neoplasia as a member of the
tumor infiltrating lymphocytes from gastric cancer patients.161,186
The precise means by which Tregs suppress effector T-cell-mediated immune responses
have not been definitively characterized. Some studies suggest roles of cytokines in their
regulation and others support the contribution of cell-to-cell contact with effector T cells
on APC, where membrane bound TGF-` and cytotoxic T-lymphocyte protein (CTLA-4)
plays an important role.187-189 Heme oxygenase-1 (HO-1), a rate limiting enzyme in heme
metabolism also plays a role in Treg mediated immune suppression. HO-1 is constitutively
expressed in human Tregs and is induced by FoxP3 expression.190,191 It is suggested that
HO-1 suppresses effector T cell by carbon-monoxide production.192,193
In 2006, a groundbreaking observation made by our laboratory demonstrated tumor
infiltration of Tregs in glioblastoma multiforme (GBM) patients.4 The expression of
FoxP3
Tregs was significantly higher in patients with GBM than in controls. A mean
of 24.7% of Tregs among the glioma-infiltrating lymphocytes were observed, whereas
these cells were absent from control brain specimens (p 0.01). Higher levels of FoxP3
expression were observed in regulatory T cells isolated from the tumor tissue (55.1%)
in comparison to autologous patient blood (33.4%) and blood from control individuals
(15.6%) (p 0.01). In an in vitro suppression assay Tregs inhibited T-cell proliferation
in a dose-dependent manner. Among various markers analyzed, the expression of CD62L
and CTLA-4 was elevated in the glioma infiltrating Tregs in comparison to that of the
controls. Following depletion of Tregs with anti-CD25 monoclonal antibody, in mice
with experimental brain tumors, we showed that an increased survival.
At the same time, another study was reported from the Heimberger laboratory at the
M.D. Anderson Cancer Center.29 GBM patient tumors were analyzed for glioma-infiltrating
microglia/macrophage (GIM) and their effect on antitumor immune responses. The authors
revealed that GIMs failed to induce T-cell proliferation. Although GIMs expressed MHC
class II expression, they lacked costimulatory molecules CD86, CD80 and CD40 critical
for T-cell activation. They demonstrated a corresponding lack of effector/activated T cells
and there was a prominent population of Tregs (CD4
CD25
FoxP3
) infiltrating the tumor.
Fecci et al found that absolute counts of both CD4
T cells and FoxP3
CD45RO
Tregs were greatly diminished in the peripheral pool of patients with malignant glioma,
but the Treg fraction was increased in the remaining CD4 compartment in 5 out of
the 8 patients evaluated.6 The proportion of Tregs in the peripheral blood of patients
with GBM was 2.63 times higher than that found in the blood of normal volunteers (p
0.004). Interestingly, their experiments suggested that despite the reduction in their
total number, the increased Treg fraction (p 0.0003 versus controls) was sufficient to
elicit the characteristic impairment of T-cell responsiveness in vitro. The patients with
an elevated Treg fraction showed significant CD4
T-cell lymphopenia (p 0.0001),
whereas the patients without Treg elevation possessed normally proliferating CD4
T-cell
levels. T cells from the patients bearing malignant gliomas regained their function after
Treg depletion in vitro and proliferated to levels equivalent to those of healthy controls.
Curtin et al observed that efficacy of immunotherapy using anti-CD25 depleting
antibody (PC61), although glioma progression in experimental animals was dependent
upon the tumor load.194 Systemic depletion of Tregs 15 days after tumor implantation
improved long-term survival, but Tregs depleted 24 days after tumor implantation showed
no improvement in survival. Immunotherapy was supplemented with intratumoral delivery
of oncolytic adenovirus and herpes simplex Type 1, to further enhance tumor regression
and long-time survival. It is very important to note that this observation suggests that
immunotherapy in combination with Treg depeletion is a more effective therapeutic
strategy, provided that Treg depletion occurs prior to immunotherapy and when tumor
burden is low.
Heme Oxygenase-1
Heme oxygenase (HO) is the rate-limiting enzyme in the catabolism of heme to

biliverdin, carbon monoxide (CO) and free iron. There are three isoforms of mammalian
HO that have been identified: HO-1, HO-2 and HO-3.195 HO-1 is inducible by a variety of
stimuli, particularly oxidative stress.196,197 Induction of HO-1 expression has been associated
with neuroprotection during hyperthermia in glial cells and during hypoxia.198,199 HO-1
expression has also been observed in a wide range of experimental diseases of the rodent
brain such as traumatic brain injury, ischemia and human Alzheimers disease.200-202 In brain
tumors, elevated HO-1 expression has been observed and in at least one report, it would
appear that HO-1 accumulates during oligodenodroglioma progression.203-205
The precise function of HO-1 expression in T cells is not fully understood.
Nevertheless, growing evidence has shown that human Tregs constitutively express
HO-1 and that HO-1 inhibits T-cell proliferation.190 Over-expression of HO-1 renders
T cells resistant to Fas-mediated apoptosis.206 Most recently, FoxP3 expression, which
encodes a forkhead/winged-helix transcription repressor specifically expressed in Treg,
has been shown to induce HO-1 expression.191 These studies suggest that HO-1 may be
an important effector of FoxP3-mediated immune suppression and an important target
for further clinical development.
Our group investigated the correlation between glioma infiltrating Tregs and tumor
grade, as well as the expression of HO-1. Patients with Grade IV tumors (11.54%)
showed the highest level of FoxP3 expression versus Grade III (6.74%) or Grade II
(2.53%) (p 0.05).207 Moreover, in Grade IV tumors, the frequency of HO-1 expressing
Treg cells was 11.8 ( 2.45% vs 7.42 ( 0.31% in Grade III and 2.33 ( 0.12% in Grade
II. HO-1 accumulated during glioma progression and apparently, plays a role in FoxP3
mediated immune suppression. Tumor infiltrating Tregs stained positively with anti-HO-1
antibody and the expression of HO-1 correlated with CD4
CD25
FoxP3
infiltration (r
0.966). These results suggested that the induction of HO-1 expression is linked to the
expression of FoxP3 in glioma infiltrating Tregs. Collectively, this data supports the
notion that HO-1 is a key suppressive factor for regulatory T cells during the growth of
malignant brain tumors.
CONCLUSION
The anatomical location of glioma within the CNS has many advantages for tumor
progression. First, the cytokine milieu of the CNS is intrinsically Th2 skewed in order
to protect vital CNS structures from damage due to inflammation. Gliomas exploit the
primarily humoral response to suppress cytotoxic responses, not only through the production
of Th2 cytokines by tumor cells, but also by enhancing Th2 cytokine production by TIL.
Second, entry into the CNS and immune cell activation are tightly regulated. In general,
gliomas are under less immune surveillance than tumors exposed to the peripheral
immune compartment. Furthermore, the predominant effector cells in glioma appear to
be suppressor cells, Tregs and myeloid-derived suppressor cells. These suppressor cells
act to prevent cytotoxic responses mediated by NK cells and CTL. The efficient immune
evasion and suppression mediated by glioma suggests that novel strategies to overcome
these mechanisms may be beneficial for the treatment of this disease.
In light of the studies of CNS immunobiology and gliomas, more selective
immunotherapeutic strategies have emerged and have resulted in moderate clinical
successes. Gliomas not only recruit, but also aid in the expansion, of suppressor cells. In
studies where these chemotaxic factors were blocked, either by chemotherapeutic drugs
or blocking antibodies, immune cell recruitment was also inhibited.91 In addition, TGF`
has been implicated in the expansion of Tregs and has been shown to be overexpressed in
gliomas. Antisense TGF` ODN, (AP 12009) has been used successfully to inhibit TGF`
expression in primary glioma samples, in vitro.208 Additionally, small molecule inhibitors
of TGF` signaling effectively block signaling in glioma cells209 and immune cells93 and
restore immune surveillance in animal models of glioma. There are dual therapeutic
benefits of inhibiting chemokines and cytokines in gliomas, not only inhibiting glioma
cell proliferation, but also reducing the recruitment of immunosuppressive cells into
tumors, thus preventing immunosuppressive mechanisms.
Another novel strategy that has emerged is the depletion of Tregs in tumor patients.
Treg depletion strategies have been successful in the treatment of ovarian cancer. Using
ONTAK (IL-2 fused to diptheria toxin),210 Barnett and colleagues demonstrated the
selective depletion of Tregs, with a concomitant increase of IFNa
effector cells.211 Our
laboratory and others have shown that the depeletion of Tregs improves the surivival of
glioma bearing mice.5,157 Recently, Grauer and colleagues illustrated that precisely timed
Treg depletion may enhance adoptive immunotherapy for the treatment of glioma.212
Therefore, whether used alone or in combination with other immunotherapeutic
strategies, Treg depletion may be critical for the treatment of glioma.
Despite the fact that effector cells have been detected in glioma tumor lesions, these
cells are unable to eradicate the tumor. In light of this, vaccination strategies aimed at
increasing the frequency of tumor antigen-specific T cells have been developed. One such
vaccine activates the T cells which recognize the tumor specific antigen, EGFRviii.213
In clinical trials, the EGFRviii vaccine increased the time to progression and improved
the mean survival in glioma patients.214 Additionally, DC-based vaccines are currently
being evaluated in glioma patients. Early results from the clinical trials showed improved
patient survival over historical norms. Although there is great potential for vaccination
strategies, more studies are necessary before concrete benefits are realized. An improved
understanding of the myriad of immunosuppressive mechanisms in play during glioma
progression will lead to the development of optimal treatment strategies and with hope,
should improve the prognosis of glioma patients.
REFERENCES
1. Medawar PB. Immunity to homologous grafted skin; the fate of skin homografts transplanted to the brain,
to subcutaneous tissue and to the anterior chamber of the eye. Br J Exp Pathol 1948; 29:58-69.
2. Freed WJ, Dymecki J, Poltorak M et al. Intraventricular brain allografts and xenografts: studies of survival
and rejection with and without systemic sensitization. Prog Brain Res 1988; 78:233-241.
3. Tsugawa T, Kuwashima N, Sato H et al. Sequential delivery of interferon-alpha gene and DCs to intracranial
gliomas promotes an effective antitumor response. Gene Ther 2004; 11:1551-1558.
4. El Andaloussi A, Lesniak MS. An increase in CD4
CD25
FOXP3
regulatory T-cells in tumor-infiltrating
lymphocytes of human glioblastoma multiforme. Neuro Oncol 2006; 8:234-243.
5. El Andaloussi A, Han Y, Lesniak MS. Prolongation of survival following depletion of CD4
CD25
regulatory
T-cells in mice with experimental brain tumors. J Neurosurg 2006; 105:430-437.
66:3294-3302.
7. Heimberger AB, Abou-Ghazal M, Reina-Ortiz C et al. Incidence and prognostic impact of FoxP3
regulatory
T-cells in human gliomas. Clin Cancer Res 2008; 14:5166-5172.
8. Facoetti A, Nano R, Zelini P et al. Human leukocyte antigen and antigen processing machinery component
defects in astrocytic tumors. Clin Cancer Res 2005; 11:8304-8311.
9. Mehling M, Simon P, Mittelbronn M et al. WHO grade associated downregulation of MHC class I
antigen-processing machinery components in human astrocytomas: does it reflect a potential immune
escape mechanism? Acta Neuropathol 2007; 114:111-119.
10. Zuber P, Kuppner MC, De Tribolet N. Transforming growth factor-beta 2 down-regulates HLA-DR antigen
expression on human malignant glioma cells. Eur J Immunol 1988; 18:1623-1626.
11. Rossi ML, Esiri MM, Jones NR et al. Characterization of the mononuclear cell infiltrate and HLA-Dr
expression in 19 oligodendrogliomas. Surg Neurol 1991; 36:119-125.
12. Rossi ML, Jones NR, Karr GF et al. HLA-Dr expression by tumor cells compared with survival in high
grade astrocytomas. Tumori 1991; 77:122-125.
13. Mouillot G, Marcou C, Rousseau P et al. HLA-G gene activation in tumor cells involves cis-acting epigenetic
changes. Int J Cancer 2005; 113:928-936.
14. Rebmann V, Regel J, Stolke D et al. Secretion of sHLA-G molecules in malignancies. Semin Cancer Biol
2003; 13:371-377.
15. Pistoia V, Morandi F, Wang X et al. Soluble HLA-G: Are they clinically relevant? Semin Cancer Biol
2007; 17:469-479.
16. Wiendl H, Mitsdoerffer M, Hofmeister V et al. A functional role of HLA-G expression in human gliomas:
an alternative strategy of immune escape. J Immunol 2002; 168:4772-4780.
17. Wischhusen J, Friese MA, Mittelbronn M et al. HLA-E protects glioma cells from NKG2D-mediated immune
responses in vitro: implications for immune escape in vivo. J Neuropathol Exp Neurol 2005; 64:523-528.
18. Mittelbronn M, Simon P, Loffler C et al. Elevated HLA-E levels in human glioblastomas but not in grade
I to III astrocytomas correlate with infiltrating CD8
cells. J Neuroimmunol 2007; 189:50-58.
19. Guillemin GJ, Brew BJ. Microglia, macrophages, perivascular macrophages and pericytes: a review of
function and identification. J Leukoc Biol 2004; 75:388-397.
20. Penfield W. Microglia and the process of phagocytosis in gliomas. Am J Path 1925; 1:77-97.
21. Rossi ML, Hughes JT, Esiri MM et al. Immunohistological study of mononuclear cell infiltrate in malignant
gliomas. Acta Neuropathol 1987; 74:269-277.
22. Rossi ML, Jones NR, Candy E et al. The mononuclear cell infiltrate compared with survival in high-grade
astrocytomas. Acta Neuropathol 1989; 78:189-193.
23. Parney IF, Waldron JS, Parsa AT. Flow cytometry and in vitro analysis of human glioma-associated
macrophages. J Neurosurg. 2009 Mar;110(3):572-82.
24. Roggendorf W, Strupp S, Paulus W. Distribution and characterization of microglia/macrophages in human
brain tumors. Acta Neuropathol 1996; 92:288-293.
25. Leung SY, Wong MP, Chung LP et al. Monocyte chemoattractant protein-1 expression and macrophage
infiltration in gliomas. Acta Neuropathol 1997; 93:518-527.
26. Platten M, Kretz A, Naumann U et al. Monocyte chemoattractant protein-1 increases microglial infiltration
and aggressiveness of gliomas. Ann Neurol 2003; 54:388-392.
27. Aloisi F, Ria F, Penna G et al. Microglia are more efficient than astrocytes in antigen processing and in
Th1 but not Th2 cell activation. J Immunol 1998; 160:4671-4680.
28. Flugel A, Labeur MS, Grasbon-Frodl EM et al. Microglia only weakly present glioma antigen to cytotoxic
T-cells. Int J Dev Neurosci 1999; 17:547-556.
29. Hussain SF, Yang D, Suki D et al. The role of human glioma-infiltrating microglia/macrophages in mediating
antitumor immune responses. Neuro Oncol 2006; 8:261-279.
30. Huettner C, Paulus W, Roggendorf W. Messenger RNA expression of the immunosuppressive cytokine
IL-10 in human gliomas. Am J Pathol 1995; 146:317-322.
31. Goswami S, Gupta A, Sharma SK. Interleukin-6-mediated autocrine growth promotion in human glioblastoma
multiforme cell line U87MG. J Neurochem 1998; 71:1837-1845.
32. Hu YS, Zhang QL, Tian ZG et al. [Significance of the unbalanced expression of Th1/Th2 type cytokines
in human glioma]. Zhongguo Yi Xue Ke Xue Yuan Xue Bao 2001; 23:594-598.
33. Li G, Hu YS, Li XG et al. Expression and switching of TH1/TH2 type cytokines gene in human gliomas.
Chin Med Sci J 2005; 20:268-272.
34. Roussel E, Gingras MC, Grimm EA et al. Predominance of a type 2 intratumoural immune response in
fresh tumour-infiltrating lymphocytes from human gliomas. Clin Exp Immunol 1996; 105:344-352.
35. Huettner C, Czub S, Kerkau S et al. Interleukin 10 is expressed in human gliomas in vivo and increases
glioma cell proliferation and motility in vitro. Anticancer Res 1997; 17:3217-3224.
36. Hishii M, Nitta T, Ishida H et al. Human glioma-derived interleukin-10 inhibits antitumor immune responses
in vitro. Neurosurgery 1995; 37:1160-1166; discussion 1166-1167.
37. Wagner S, Czub S, Greif M et al. Microglial/macrophage expression of interleukin 10 in human glioblastomas.
Int J Cancer1999; 82:12-16.
38. Samaras V, Piperi C, Korkolopoulou P et al. Application of the ELISPOT method for comparative analysis
of interleukin (IL)-6 and IL-10 secretion in peripheral blood of patients with astroglial tumors. Mol Cell
Biochem 2007; 304:343-351.
39. Murphy-Ullrich JE. The de-adhesive activity of matricellular proteins: is intermediate cell adhesion an
adaptive state? J Clin Invest 2001; 107:785-790.
40. Sage EH. Regulation of interactions between cells and extracellular matrix: a command performance on
several stages. J Clin Invest 2001; 107:781-783.
41. Erickson HP. Gene knockouts of c-src, transforming growth factor beta 1 and tenascin suggest superfluous,
nonfunctional expression of proteins. J Cell Biol 1993; 120:1079-1081.
42. Erickson HP. A tenascin knockout with a phenotype. Nat Genet 1997; 17:5-7.
43. Jones PL, Jones FS. Tenascin-C in development and disease: gene regulation and cell function. Matrix
Biol 2000; 19:581-596.
44. Hemesath TJ, Marton LS, Stefansson K. Inhibition of T-cell activation by the extracellular matrix protein
tenascin. J Immunol 1994; 152:5199-5207.
45. Puente Navazo MD, Valmori D, Ruegg C. The alternatively spliced domain TnFnIII A1A2 of the
extracellular matrix protein tenascin-C suppresses activation-induced T-lymphocyte proliferation and
cytokine production. J Immunol 2001; 167:6431-6440.
46. Parekh K, Ramachandran S, Cooper J et al. Tenascin-C, over expressed in lung cancer down regulates
effector functions of tumor infiltrating lymphocytes. Lung Cancer 2005; 47:17-29.
47. Bodmer S, Strommer K, Frei K et al. Immunosuppression and transforming growth factor-beta in glioblastoma.
Preferential production of transforming growth factor-beta 2. J Immunol 1989; 143:3222-3229.
48. Tada T, Yabu K, Kobayashi S. Detection of active form of transforming growth factor-beta in cerebrospinal
fluid of patients with glioma. Jpn J Cancer Res 1993; 84:544-548.
49. de Martin R, Haendler B, Hofer-Warbinek R et al. Complementary DNA for human glioblastoma-derived
T-cell suppressor factor, a novel member of the transforming growth factor-beta gene family. EMBO
J 1987; 6:3673-3677.
50. Wrann M, Bodmer S, de Martin R et al. T-cell suppressor factor from human glioblastoma cells is a 12.5-kd
protein closely related to transforming growth factor-beta. EMBO J 1987; 6:1633-1636.
51. Eberhart CE, Coffey RJ, Radhika A et al. Up-regulation of cyclooxygenase 2 gene expression in human
colorectal adenomas and adenocarcinomas. Gastroenterology 1994; 107:1183-1188.
52. Wolff H, Saukkonen K, Anttila S et al. Expression of cyclooxygenase-2 in human lung carcinoma. Cancer
Res 1998; 58:4997-5001.
53. Mohammed SI, Knapp DW, Bostwick DG et al. Expression of cyclooxygenase-2 (COX-2) in human invasive
transitional cell carcinoma (TCC) of the urinary bladder. Cancer Res 1999; 59:5647-5650.
54. Shono T, Tofilon PJ, Bruner JM et al. Cyclooxygenase-2 expression in human gliomas: prognostic
significance and molecular correlations. Cancer Res 2001; 61:4375-4381.
55. Joki T, Heese O, Nikas DC et al. Expression of cyclooxygenase 2 (COX-2) in human glioma and in vitro
inhibition by a specific COX-2 inhibitor, NS-398. Cancer Res 2000; 60:4926-4931.
56. Phipps RP, Stein SH, Roper RL. A new view of prostaglandin E regulation of the immune response.
Immunol Today 1991; 12:349-352.
57. Roper RL, Phipps RP. Prostaglandin E2 regulation of the immune response. Adv Prostaglandin Thromboxane
Leukot Res 1994; 22:101-111.
58. Kunkel SL, Spengler M, May MA et al. Prostaglandin E2 regulates macrophage-derived tumor necrosis
factor gene expression. J Biol Chem 1988; 263:5380-5384.
59. van der Pouw Kraan TC, Boeije LC, Smeenk RJ et al. Prostaglandin-E2 is a potent inhibitor of human
interleukin 12 production. J Exp Med 1995; 181:775-779.
60. Harizi H, Juzan M, Grosset C et al. Dendritic cells issued in vitro from bone marrow produce PGE(2) that
contributes to the immunomodulation induced by antigen-presenting cells. Cell Immunol 2001; 209:19-28.
61. Williams JA, Shacter E. Regulation of macrophage cytokine production by prostaglandin E2. Distinct roles
of cyclooxygenase-1 and -2. J Biol Chem 1997; 272:25693-25699.
62. Strassmann G, Patil-Koota V, Finkelman F et al. Evidence for the involvement of interleukin 10 in the
differential deactivation of murine peritoneal macrophages by prostaglandin E2. J Exp Med 1994;
180:2365-2370.
63. Fiorentino DF, Zlotnik A, Vieira P et al. IL-10 acts on the antigen-presenting cell to inhibit cytokine
production by Th1 cells. J Immunol 1991; 146:3444-3451.
64. Harizi H, Juzan M, Pitard V et al. Cyclooxygenase-2-issued prostaglandin e(2) enhances the production
of endogenous IL-10, which down-regulates dendritic cell functions. J Immunol 2002; 168:2255-2263.
65. Akasaki Y, Liu G, Chung NH et al. Induction of a CD4
T regulatory type 1 response by
cyclooxygenase-2-overexpressing glioma. J Immunol 2004; 173:4352-4359.
66. Kokoglu E, Tuter Y, Sandikci KS et al. Prostaglandin E2 levels in human brain tumor tissues and arachidonic
acid levels in the plasma membrane of human brain tumors. Cancer Lett 1998; 132:17-21.
67. Loh JK, Hwang SL, Lieu AS et al. The alteration of prostaglandin E2 levels in patients with brain tumors
before and after tumor removal. J Neurooncol 2002; 57:147-150.
68. Rozic JG, Chakraborty C, Lala PK. Cyclooxygenase inhibitors retard murine mammary tumor progression
by reducing tumor cell migration, invasiveness and angiogenesis. Int J Cancer 2001; 93:497-506.
69. Attiga FA, Fernandez PM, Weeraratna AT et al. Inhibitors of prostaglandin synthesis inhibit human prostate
tumor cell invasiveness and reduce the release of matrix metalloproteinases. Cancer Res 2000; 60:4629-4637.
70. Kundu N, Fulton AM. Selective cyclooxygenase (COX)-1 or COX-2 inhibitors control metastatic disease
in a murine model of breast cancer. Cancer Res 2002; 62:2343-2346.
71. Connolly EM, Harmey JH, OGrady T et al. Cyclo-oxygenase inhibition reduces tumour growth and
metastasis in an orthotopic model of breast cancer. Br J Cancer 2002; 87:231-237.
72. Lalier L, Cartron PF, Pedelaborde F et al. Increase in PGE2 biosynthesis induces a Bax dependent apoptosis
correlated to patients survival in glioblastoma multiforme. Oncogene 2007; 26:4999-5009.
73. Chouaib S, Bertoglio JH. Prostaglandins E as modulators of the immune response. Lymphokine Res Fall
1988; 7:237-245.
74. Goodwin JS, Ceuppens J. Regulation of the immune response by prostaglandins. J Clin Immunol 1983;
3:295-315.
75. Rappaport RS, Dodge GR. Prostaglandin E inhibits the production of human interleukin 2. J Exp Med
1982; 155:943-948.
76. Kuppner MC, Sawamura Y, Hamou MF et al. Influence of PGE2- and cAMP-modulating agents on human
glioblastoma cell killing by interleukin-2-activated lymphocytes. J Neurosurg 1990; 72:619-625.
77. Lauro GM, Di Lorenzo N, Grossi M et al. Prostaglandin E2 as an immunomodulating factor released in
vitro by human glioma cells. Acta Neuropathol 1986; 69:278-282.
78. Sawamura Y, Diserens AC, de Tribolet N. In vitro prostaglandin E2 production by glioblastoma cells and
its effect on interleukin-2 activation of oncolytic lymphocytes. J Neurooncol 1990; 9:125-130.
79. Nakano Y, Kuroda E, Kito T et al. Induction of macrophagic prostaglandin E2 synthesis by glioma cells.
J Neurosurg 2006; 104:574-582.
80. Nakano Y, Kuroda E, Kito T et al. Induction of prostaglandin E2 synthesis and microsomal prostaglandin
E synthase-1 expression in murine microglia by glioma-derived soluble factors. Laboratory investigation.
J Neurosurg 2008; 108:311-319.
81. Anderson RC, Anderson DE, Elder JB et al. Lack of B7 expression, not human leukocyte antigen expression,
facilitates immune evasion by human malignant gliomas. Neurosurgery 2007; 60(6):1129-1136; discussion
1136.
82. Bromberg J. Stat proteins and oncogenesis. J Clin Invest 2002; 109(9):1139-1142.
83. Brantley EC, Benveniste EN. Signal transducer and activator of transcription-3: a molecular hub for signaling
pathways in gliomas. Mol Cancer Res 2008; 6(5):675-684.
84. Chung CD, Liao J, Liu B et al. Specific inhibition of Stat3 signal transduction by PIAS3. Science 1997;
278(5344):1803-1805.
85. Pillemer BB, Xu H, Oriss TB et al. Deficient SOCS3 expression in CD4
CD25
FoxP3
regulatory T-cells
and SOCS3-mediated suppression of Treg function. Eur J Immunol 2007; 37(8):2082-2089.
86. Yu H, Kortylewski M, Pardoll D. Crosstalk between cancer and immune cells: role of STAT3 in the tumour
microenvironment. Nat Rev Immunol 2007; 7(1):41-51.
87. Wang T, Niu G, Kortylewski M et al. Regulation of the innate and adaptive immune responses by STAT3
signaling in tumor cells. Nat Med 2004; 10(1):48-54.
88. Kortylewski M, Kujawski M, Wang T et al. Inhibiting Stat3 signaling in the hematopoietic system elicits
multicomponent antitumor immunity. Nat Med 2005; 11(12):1314-1321.
89. Almand B, Resser JR, Lindman B et al. Clinical significance of defective dendritic cell differentiation in
cancer. Clin Cancer Res 2000; 6(5):1755-1766.
90. Kasprzycka M, Marzec M, Liu X et al. Nucleophosmin/anaplastic lymphoma kinase (NPM/ALK)
oncoprotein induces the T regulatory cell phenotype by activating STAT3. Proc Natl Acad Sci USA
2006; 103(26):9964-9969.
91. Jordan JT, Sun W, Hussain SF et al. Preferential migration of regulatory T-cells mediated by glioma-secreted
chemokines can be blocked with chemotherapy. Cancer Immunol Immunother 2008; 57(1):123-131.
92. Abou-Ghazal M, Yang DS, Qiao W et al. The incidence, correlation with tumor-infiltrating inflammation and
prognosis of phosphorylated STAT3 expression in human gliomas. Clin Cancer Res 2008; 14(24):8228-8235.
93. Hussain SF, Kong LY, Jordan J et al. A novel small molecule inhibitor of signal transducers and activators of
transcription 3 reverses immune tolerance in malignant glioma patients. Cancer Res 2007; 67(20):9630-9636.
94. Ichikawa M, Chen L. Role of B7-H1 and B7-H4 molecules in down-regulating effector phase of T-cell
immunity: novel cancer escaping mechanisms. Front Biosci 2005; 10:2856-2860.
95. Ghebeh H, Mohammed S, Al-Omair A et al. The B7-H1 (PD-L1) T-lymphocyte-inhibitory molecule is
expressed in breast cancer patients with infiltrating ductal carcinoma: correlation with important high-risk
prognostic factors. Neoplasia 2006; 8(3):190-198.
96. Tsushima F, Tanaka K, Otsuki N et al. Predominant expression of B7-H1 and its immunoregulatory roles
in oral squamous cell carcinoma. Oral Oncol 2006; 42(3):268-274.
97. Strome SE, Dong H, Tamura H et al. B7-H1 blockade augments adoptive T-cell immunotherapy for
squamous cell carcinoma. Cancer Res 2003; 63(19):6501-6505.
cells: a potential mechanism of immune paralysis. Cancer Res 2003; 63(21):7462-7467.
99. Dong H, Chen L. B7-H1 pathway and its role in the evasion of tumor immunity. J Mol Med 2003;
81(5):281-287.
100. Tamura H, Dong H, Zhu G et al. B7-H1 costimulation preferentially enhances CD28-independent T-helper
cell function. Blood 2001; 97(6):1809-1816.
101. Dong H, Strome SE, Salomao DR et al. Tumor-associated B7-H1 promotes T-cell apoptosis: a potential
mechanism of immune evasion. Nat Med 2002; 8(8):793-800.
102. Hirano F, Kaneko K, Tamura H et al. Blockade of B7-H1 and PD-1 by monoclonal antibodies potentiates
cancer therapeutic immunity. Cancer Res 2005; 65(3):1089-1096.
103. Blank C, Brown I, Peterson AC et al. PD-L1/B7H-1 inhibits the effector phase of tumor rejection by T-cell
receptor (TCR) transgenic CD8
T-cells. Cancer Res 2004; 64(3):1140-1145.
104. Blank C, Kuball J, Voelkl S et al. Blockade of PD-L1 (B7-H1) augments human tumor-specific T-cell
responses in vitro. Int J Cancer 2006; 119(2):317-327.
105. Iwai Y, Ishida M, Tanaka Y et al. Involvement of PD-L1 on tumor cells in the escape from host immune
system and tumor immunotherapy by PD-L1 blockade. Proc Natl Acad Sci USA 2002; 99(19):12293-12297.
106. Blank C, Gajewski TF, Mackensen A. Interaction of PD-L1 on tumor cells with PD-1 on tumor-specific
T-cells as a mechanism of immune evasion: implications for tumor immunotherapy. Cancer Immunol
Immunother 2005; 54(4):307-314.
107. Karre K, Ljunggren HG, Piontek G, Kiessling R. Selective rejection of H-2-deficient lymphoma variants
suggests alternative immune defence strategy. Nature 1986; 319(6055):675-678.
108. Diefenbach A, Jensen ER, Jamieson AM et al. Rae1 and H60 ligands of the NKG2D receptor stimulate
tumour immunity. Nature 2001; 413(6852):165-171.
109. Cerwenka A, Baron JL, Lanier LL. Ectopic expression of retinoic acid early inducible-1 gene (RAE-1)
permits natural killer cell-mediated rejection of a MHC class I-bearing tumor in vivo. Proc Natl Acad
Sci USA 2001; 98(20):11521-11526.
brain tumors. J Neuroimmunol 1999; 100(1-2):216-232.
111. Braun DP, Penn RD, Harris JE. Regulation of natural killer cell function by glass-adherent cells in patients
with primary intracranial malignancies. Neurosurgery 1984; 15(1):29-33.
112. Proescholdt MA, Merrill MJ, Ikejiri B et al. Site-specific immune response to implanted gliomas.
J Neurosurg 2001; 95(6):1012-1019.
113. Aldemir H, Prodhomme V, Dumaurier MJ et al. Cutting edge: lectin-like transcript 1 is a ligand for the
CD161 receptor. J Immunol 2005; 175(12):7791-7795.
114. Roth P, Mittelbronn M, Wick W et al. Malignant glioma cells counteract antitumor immune responses
through expression of lectin-like transcript-1. Cancer Res 2007; 67(8):3540-3544.
115. Boomer JS, Derks RA, Lee GW et al. Regeneration and tolerance factor is expressed during T-lymphocyte
activation and plays a role in apoptosis. Hum Immunol 2001; 62(6):577-588.
116. Roth P, Aulwurm S, Gekel I et al. Regeneration and tolerance factor: a novel mediator of
glioblastoma-associated immunosuppression. Cancer Res 2006; 66(7):3852-3858.
117. Tachibana O, Nakazawa H, Lampe J et al. Expression of Fas/APO-1 during the progression of astrocytomas.
Cancer Res 1995; 55(23):5528-5530.
118. Gratas C, Tohma Y, Van Meir EG et al. Fas ligand expression in glioblastoma cell lines and primary
astrocytic brain tumors. Brain Pathol 1997; 7(3):863-869.
119. Saas P, Walker PR, Hahne M et al. Fas ligand expression by astrocytoma in vivo: maintaining immune
privilege in the brain? J Clin Invest 1997; 99(6):1173-1178.
120. Didenko VV, Ngo HN, Minchew C et al. Apoptosis of T-lymphocytes invading glioblastomas multiforme:
a possible tumor defense mechanism. J Neurosurg 2002; 96(3):580-584.
121. Roth W, Isenmann S, Nakamura M et al. Soluble decoy receptor 3 is expressed by malignant gliomas
and suppresses CD95 ligand-induced apoptosis and chemotaxis. Cancer Res 2001; 61(6):2759-2765.
122. Hintzen RQ, Lens SM, Koopman G et al. CD70 represents the human ligand for CD27. Int Immunol
1994; 6(3):477-480.
123. Sugita K, Hirose T, Rothstein DM et al. CD27, a member of the nerve growth factor receptor family, is
preferentially expressed on CD45RA
CD4 T-cell clones and involved in distinct immunoregulatory
functions. J Immunol 1992; 149(10):3208-3216.
124. Hendriks J, Gravestein LA, Tesselaar K et al. CD27 is required for generation and long-term maintenance
of T-cell immunity. Nat Immunol 2000; 1(5):433-440.
125. La Rosa FG, Adams FS, Krause GE et al. Inhibition of proliferation and expression of T-antigen in
SV40 large T-antigen gene-induced immortalized cells following transplantations. Cancer Lett 1997;
113(1-2):55-60.
126. Tesselaar K, Arens R, van Schijndel GM et al. Lethal T-cell immunodeficiency induced by chronic
costimulation via CD27-CD70 interactions. Nat Immunol 2003; 4(1):49-54.
127. Koenen HJ, Fasse E, Joosten I. CD27/CFSE-based ex vivo selection of highly suppressive alloantigen-specific
human regulatory T-cells. J Immunol 2005; 174(12):7573-7583.
128. Chahlavi A, Rayman P, Richmond AL et al. Glioblastomas induce T-lymphocyte death by two distinct
pathways involving gangliosides and CD70. Cancer Res 2005; 65(12):5428-5438.
129. Kudo D, Rayman P, Horton C et al. Gangliosides expressed by the renal cell carcinoma cell line SK-RC-45
are involved in tumor-induced apoptosis of T-cells. Cancer Res 2003; 63(7):1676-1683.
130. Ledeen RW, Wu G. Nuclear lipids: key signaling effectors in the nervous system and other tissues. J Lipid
Res 2004; 45(1):1-8.
131. Hedberg KM, Mahesparan R, Read TA et al. The glioma-associated gangliosides 3v-isoLM1, GD3 and
GM2 show selective area expression in human glioblastoma xenografts in nude rat brains. Neuropathol
Appl Neurobiol 2001; 27(6):451-464.
132. Markowska-Woyciechowska A, Bronowicz A, Ugorski M et al. Study on ganglioside composition in brain
tumours supra- and infratentorial. Neurol Neurochir Pol 2000; 34(6 Suppl):124-130.
133. Mennel HD, Bosslet K, Geissel H et al. Immunohistochemically visualized localisation of gangliosides Glac2
(GD3) and Gtri2 (GD2) in cells of human intracranial tumors. Exp Toxicol Pathol 2000; 52(4):277-285.
134. Wagener R, Rohn G, Schillinger G et al. Ganglioside profiles in human gliomas: quantification by
microbore high performance liquid chromatography and correlation to histomorphology and grading.
Acta Neurochir (Wien) 1999; 141(12):1339-1345.
135. Ward SG, Westwick J. Chemokines: understanding their role in T-lymphocyte biology. Biochem J 1998;
333(Pt3):457-470.
136. Mantovani A. Chemokines. Introduction and overview. Chem Immunol 1999; 72:1-6.
137. Mantovani A, Allavena P, Sozzani S et al. Chemokines in the recruitment and shaping of the leukocyte
infiltrate of tumors. Semin Cancer Biol 2004; 14:155-160.
138. Rollins BJ. Chemokines. Blood 1997; 90:909-928.
139. Gerard C, Rollins BJ. Chemokines and disease. Nat Immunol 2001; 2:108-115.
140. Rollins BJ. Inflammatory chemokines in cancer growth and progression. Eur J Cancer 2006; 42:760-767.
141. Zhou Y, Larsen PH, Hao C et al. CXCR4 is a major chemokine receptor on glioma cells and mediates
their survival. J Biol Chem 2002; 277:49481-49487.
142. Li M, Ransohoff RM. Multiple roles of chemokine CXCL12 in the central nervous system: a migration
from immunology to neurobiology. Prog Neurobiol 2008; 84:116-131.
143. Choi C, Xu X, Oh JW et al. Fas-induced expression of chemokines in human glioma cells: involvement
of extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinase. Cancer Res 2001;
61:3084-3091.
144. Curiel TJ, Coukos G, Zou L et al. Specific recruitment of regulatory T-cells in ovarian carcinoma fosters
immune privilege and predicts reduced survival. Nat Med 2004; 10:942-949.
145. Mazzoni A, Bronte V, Visintin A et al. Myeloid suppressor lines inhibit T-cell responses by an NO-dependent
mechanism. J Immunol 2002; 168:689-695.
146. Duwe AK, Singhal SK. The immunoregulatory role of bone marrow. II. Characterization of a suppressor
cell inhibiting the in vitro antibody response. Cell Immunol 1979; 43:372-381.
147. Duwe AK, Singhal SK. The immunoregulatory role of bone marrow. I. Suppression of the induction
of antibody responses to T-dependent and T-independent antigens by cells in the bone marrow. Cell
Immunol 1979; 43:362-371.
148. Young MR, Newby M, Wepsic HT. Hematopoiesis and suppressor bone marrow cells in mice bearing
large metastatic Lewis lung carcinoma tumors. Cancer Res 1987; 47:100-105.
149. Bronte V, Kasic T, Gri G et al. Boosting antitumor responses of T-lymphocytes infiltrating human prostate
cancers. J Exp Med 2005; 201:1257-1268.
150. Kusmartsev S, Cheng F, Yu B et al. All-trans-retinoic acid eliminates immature myeloid cells from
tumor-bearing mice and improves the effect of vaccination. Cancer Res 2003; 63:4441-4449.
151. Kusmartsev S, Gabrilovich DI. Inhibition of myeloid cell differentiation in cancer: the role of reactive
oxygen species. J Leukoc Biol 2003; 74:186-196.
152. Bronte V, Apolloni E, Cabrelle A et al. Identification of a CD11b(
)/Gr-1(
)/CD31(
) myeloid progenitor
capable of activating or suppressing CD8(
) T-cells. Blood 2000; 96:3838-3846.
153. Gabrilovich DI, Bronte V, Chen SH et al. The terminology issue for myeloid-derived suppressor cells.
Cancer Res 2007; 67:425; author reply 426.
154. Pak AS, Wright MA, Matthews JP et al. Mechanisms of immune suppression in patients with head and
neck cancer: presence of CD34(
) cells which suppress immune functions within cancers that secrete
granulocyte-macrophage colony-stimulating factor. Clin Cancer Res 1995; 1:95-103.
155. Lin EY, Gouon-Evans V, Nguyen AV et al. The macrophage growth factor CSF-1 in mammary gland
development and tumor progression. J Mammary Gland Biol Neoplasia 2002; 7:147-162.
156. Menetrier-Caux C, Montmain G, Dieu MC et al. Inhibition of the differentiation of dendritic cells from
CD34(
) progenitors by tumor cells: role of interleukin-6 and macrophage colony-stimulating factor.
Blood 1998; 92:4778-4791.
157. Grauer OM, Nierkens S, Bennink E et al. CD4
FoxP3
regulatory T-cells gradually accumulate in gliomas
during tumor growth and efficiently suppress antiglioma immune responses in vivo. Int J Cancer 2007;
121:95-105.
158. Yong Z, Chang L, Mei YX et al. Role and mechanisms of CD4
CD25
regulatory T-cells in the induction
and maintenance of transplantation tolerance. Transpl Immunol 2007; 17:120-129.
159. Brusko TM, Putnam AL, Bluestone JA. Human regulatory T-cells: role in autoimmune disease and
therapeutic opportunities. Immunol Rev 2008; 223:371-390.
160. Ling KL, Pratap SE, Bates GJ et al. Increased frequency of regulatory T-cells in peripheral blood and
tumour infiltrating lymphocytes in colorectal cancer patients. Cancer Immun 2007; 7:7.
161. Ichihara F, Kono K, Takahashi A et al. Increased populations of regulatory T-cells in peripheral blood
and tumor-infiltrating lymphocytes in patients with gastric and esophageal cancers. Clin Cancer Res
2003; 9:4404-4408.
162. Liyanage UK, Moore TT, Joo HG et al. Prevalence of regulatory T-cells is increased in peripheral blood
and tumor microenvironment of patients with pancreas or breast adenocarcinoma. J Immunol 2002;
169:2756-2761.
163. Okita R, Saeki T, Takashima S et al. CD4
CD25
regulatory T-cells in the peripheral blood of patients
with breast cancer and nonsmall cell lung cancer. Oncol Rep 2005; 14:1269-1273.
164. Sakaguchi S, Sakaguchi N, Asano M et al. Immunologic self-tolerance maintained by activated T-cells
expressing IL-2 receptor alpha-chains (CD25). Breakdown of a single mechanism of self-tolerance causes
various autoimmune diseases. J Immunol 1995; 155:1151-1164.
165. Toda A, Piccirillo CA. Development and function of naturally occurring CD4
CD25
regulatory T-cells.
J Leukoc Biol 2006; 80:458-470.
166. Itoh M, Takahashi T, Sakaguchi N et al. Thymus and autoimmunity: production of CD25
CD4
naturally
anergic and suppressive T-cells as a key function of the thymus in maintaining immunologic self-tolerance.
J Immunol 1999; 162:5317-5326.
167. Schimpl A, Berberich I, Kneitz B et al. IL-2 and autoimmune disease. Cytokine Growth Factor Rev
2002; 13:369-378.
168. Bayer AL, Yu A, Adeegbe D et al. Essential role for interleukin-2 for CD4(
)CD25(
) T regulatory cell
development during the neonatal period. J Exp Med 2005; 201:769-777.
169. Bayer AL, Yu A, Malek TR. Function of the IL-2R for thymic and peripheral CD4
CD25
Foxp3
T
regulatory cells. J Immunol 2007; 178:4062-4071.
170. Malek TR, Yu A, Vincek V et al. CD4 regulatory T-cells prevent lethal autoimmunity in IL-2Rbeta-deficient
mice. Implications for the nonredundant function of IL-2. Immunity 2002; 17:167-178.
171. Malek TR. The main function of IL-2 is to promote the development of T regulatory cells. J Leukoc Biol
2003; 74:961-965.
172. de la Rosa M, Rutz S, Dorninger H et al. Interleukin-2 is essential for CD4
CD25
regulatory T-cell
function. Eur J Immunol 2004; 34:2480-2488.
173. Moriggl R, Topham DJ, Teglund S et al. Stat5 is required for IL-2-induced cell cycle progression of
peripheral T-cells. Immunity 1999; 10:249-259.
174. Van Parijs L, Refaeli Y, Lord JD et al. Uncoupling IL-2 signals that regulate T-cell proliferation, survival
and Fas-mediated activation-induced cell death. Immunity 1999; 11:281-288.
175. Antov A, Yang L, Vig M et al. Essential role for STAT5 signaling in CD25
CD4
regulatory T-cell
homeostasis and the maintenance of self-tolerance. J Immunol 2003; 171:3435-3441.
176. Snow JW, Abraham N, Ma MC et al. Loss of tolerance and autoimmunity affecting multiple organs in
STAT5A/5B-deficient mice. J Immunol 2003; 171:5042-5050.
177. Burchill MA, Goetz CA, Prlic M et al. Distinct effects of STAT5 activation on CD4
and CD8
T-cell
homeostasis: development of CD4
CD25
regulatory T-cells versus CD8
memory T-cells. J Immunol
2003; 171:5853-5864.
178. Hori S, Nomura T, Sakaguchi S. Control of regulatory T-cell development by the transcription factor
Foxp3. Science 2003; 299:1057-1061.
179. Fontenot JD, Gavin MA, Rudensky AY. Foxp3 programs the development and function of CD4
CD25
regulatory T-cells. Nat Immunol 2003; 4:330-336.

180. Yagi H, Nomura T, Nakamura K et al. Crucial role of FOXP3 in the development and function of human
CD25
CD4
regulatory T-cells. Int Immunol 2004; 16(:1643-1656.
181. Qiao M, Thornton AM, Shevach EM. CD4
CD25
[corrected] regulatory T-cells render naive CD4
CD25< T-cells anergic and suppressive. Immunology 2007; 120:447-455.

182. Sundstedt A, ONeill EJ, Nicolson KS et al. Role for IL-10 in suppression mediated by peptide-induced
regulatory T-cells in vivo. J Immunol 2003; 170:1240-1248.
183. Vieira PL, Christensen JR, Minaee S et al. IL-10-secreting regulatory T-cells do not express Foxp3 but
have comparable regulatory function to naturally occurring CD4
CD25
regulatory T-cells. J Immunol
2004; 172:5986-5993.
184. Miller A, Lider O, Roberts AB et al. Suppressor T-cells generated by oral tolerization to myelin basic
protein suppress both in vitro and in vivo immune responses by the release of transforming growth factor
beta after antigen-specific triggering. Proc Natl Acad Sci USA 1992; 89:421-425.
185. Zhang X, Izikson L, Liu L et al. Activation of CD25(
)CD4(
) regulatory T-cells by oral antigen
administration. J Immunol 2001; 167:4245-4253.
186. Levings MK, Gregori S, Tresoldi E et al. Differentiation of Tr1 cells by immature dendritic cells requires
IL-10 but not CD25
CD4
Tr cells. Blood 2005; 105:1162-1169.
187. Takahashi Y, Onda M, Tanaka N et al. Establishment and characterization of two new rectal neuroendocrine
cell carcinoma cell lines. Digestion 2000; 62:262-270.
188. Manzotti CN, Tipping H, Perry LC et al. Inhibition of human T-cell proliferation by CTLA-4 utilizes
CD80 and requires CD25
regulatory T-cells. Eur J Immunol 2002; 32:2888-2896.
189. Tang Q, Boden EK, Henriksen KJ et al. Distinct roles of CTLA-4 and TGF-beta in CD4
CD25
regulatory
T-cell function. Eur J Immunol 2004; 34:2996-3005.
190. Pae HO, Oh GS, Choi BM et al. Differential expressions of heme oxygenase-1 gene in CD25- and CD25
subsets of human CD4

T-cells. Biochem Biophys Res Commun 2003; 306:701-705.
191. Choi BM, Pae HO, Jeong YR et al. Critical role of heme oxygenase-1 in Foxp3-mediated immune
suppression. Biochem Biophys Res Commun 2005; 327:1066-1071.
192. Pae HO, Oh GS, Choi BM et al. Carbon monoxide produced by heme oxygenase-1 suppresses T-cell
proliferation via inhibition of IL-2 production. J Immunol 2004; 172:4744-4751.
193. Song R, Zhou Z, Kim PK et al. Carbon monoxide promotes Fas/CD95-induced apoptosis in Jurkat cells.
J Biol Chem 2004; 279:44327-44334.
194. Curtin JF, Candolfi M, Fakhouri TM et al. Treg depletion inhibits efficacy of cancer immunotherapy:
implications for clinical trials. PLoS ONE 2008; 3:e1983.
195. Maines MD, Polevoda B, Coban T et al. Neuronal overexpression of heme oxygenase-1 correlates with
an attenuated exploratory behavior and causes an increase in neuronal NADPH diaphorase staining.
J Neurochem 1998; 70:2057-2069.
196. Stocker R, Glazer AN, Ames BN. Antioxidant activity of albumin-bound bilirubin. Proc Natl Acad Sci
USA 1987; 84:5918-5922.
197. Stocker R, Yamamoto Y, McDonagh AF et al. Bilirubin is an antioxidant of possible physiological
importance. Science 1987; 235:1043-1046.
198. Ewing JF, Haber SN, Maines MD. Normal and heat-induced patterns of expression of heme oxygenase-1
(HSP32) in rat brain: hyperthermia causes rapid induction of mRNA and protein. J Neurochem 1992;
58:1140-1149.
199. Panahian N, Yoshiura M, Maines MD. Overexpression of heme oxygenase-1 is neuroprotective in a model
of permanent middle cerebral artery occlusion in transgenic mice. J Neurochem 1999; 72:1187-1203.
200. Fukuda K, Panter SS, Sharp FR et al. Induction of heme oxygenase-1 (HO-1) after traumatic brain injury
in the rat. Neurosci Lett 1995; 199:127-130.
201. Nimura T, Weinstein PR, Massa SM et al. Heme oxygenase-1 (HO-1) protein induction in rat brain
following focal ischemia. Brain Res Mol Brain Res 1996; 37:201-208.
202. Schipper HM, Cisse S, Stopa EG. Expression of heme oxygenase-1 in the senescent and Alzheimer-diseased
brain. Ann Neurol 1995; 37:758-768.
203. Hara E, Takahashi K, Tominaga T et al. Expression of heme oxygenase and inducible nitric oxide synthase
mRNA in human brain tumors. Biochem Biophys Res Commun 1996; 224:153-158.
204. Nishie A, Ono M, Shono T et al. Macrophage infiltration and heme oxygenase-1 expression correlate
with angiogenesis in human gliomas. Clin Cancer Res 1999; 5:1107-1113.
205. Deininger MH, Meyermann R, Trautmann K et al. Heme oxygenase (HO)-1 expressing macrophages/
microglial cells accumulate during oligodendroglioma progression. Brain Res 2000; 882:1-8.
206. Choi BM, Pae HO, Jeong YR et al. Overexpression of heme oxygenase (HO)-1 renders Jurkat T-cells
resistant to fas-mediated apoptosis: involvement of iron released by HO-1. Free Radic Biol Med 2004;
36:858-871.
207. El Andaloussi A, Lesniak MS. CD4
CD25
FoxP3
T-cell infiltration and heme oxygenase-1 expression
correlate with tumor grade in human gliomas. J Neurooncol 2007; 83:145-152.
208. Schlingensiepen KH, Schlingensiepen R, Steinbrecher A et al. Targeted tumor therapy with the TGF-beta
2 antisense compound AP 12009. Cytokine Growth Factor Rev 2006; 17:129-139.
209. Iwamaru A, Szymanski S, Iwado E et al. A novel inhibitor of the STAT3 pathway induces apoptosis in
malignant glioma cells both in vitro and in vivo. Oncogene 2007; 26:2435-2444.
210. Foss FM. DAB(389)IL-2 (denileukin diftitox, ONTAK): a new fusion protein technology. Clin Lymphoma
2000; (1 Suppl 1):S27-31.
211. Barnett B, Kryczek I, Cheng P et al. Regulatory T-cells in ovarian cancer: biology and therapeutic potential.
Am J Reprod Immunol 2005; 54:369-377.
212. Grauer OM, Sutmuller RP, van Maren W et al. Elimination of regulatory T-cells is essential for an
effective vaccination with tumor lysate-pulsed dendritic cells in a murine glioma model. Int J Cancer
2008; 122:1794-1802.
213. Heimberger AB, Crotty LE, Archer GE et al. Epidermal growth factor receptor VIII peptide vaccination
is efficacious against established intracerebral tumors. Clin Cancer Res 2003; 9:4247-4254.
214. Sampson JH, Archer GE, Mitchell DA et al. Tumor-specific immunotherapy targeting the EGFRvIII
mutation in patients with malignant glioma. Semin Immunol 2008; 20:267-275.
CHAPTER 6
GLIOMA ANTIGEN
Masahiro Toda
Department of Neurosurgery, Keio University School of Medicine, Tokyo, Japan
Email:todam@sc.itc.keio.ac.jp
Abstract: Because several antigenic peptides of human tumors that are recognized by
T-lymphocytes have been identified, immune responses against cancer can now
be artificially manipulated. Furthermore, since T-lymphocytes have been found
to play an important role in the rejection of tumors by the host and also to have
antigen-specific proliferative potentials and memory mechanisms, T-lymphocytes
are thought to play a central role in cancer vaccination. Although multidisciplinary
therapies have been attempted for the treatment of gliomas, the results remain
unsatisfactory. For the development of new therapies against gliomas, it is required
to identify tumor antigens as targets for specific immunotherapy. In this chapter,
recent progress in research on glioma antigens is described.
INTRODUCTION
Tumor antigens are recognized by T cells or antibodies in the immune system and
immunotherapy-targeting tumor antigens can therefore be divided into cell therapy,
in which T cells are administered, vaccine therapy, in which T cells are induced
in the body and antibody therapy. Because cytotoxic T-lymphocytes (CTLs) have
been shown to play a profound role in antitumor effects in the body, clinical studies
of cell therapy and vaccine therapy for a wide variety of cancers are currently
in progress.1-3 As a result of technical progress in methods of antibody production,
antibody therapy is now being applied clinically and because the efficacy of such
a strategy has been demonstrated against the target tumors, great expectations for
drug discovery now exist.4-6 The principal mechanisms responsible for the antitumor
effects of antibodies are antibody-dependent cell-mediated cytotoxicity (ADCC) and
complement-dependent cytotoxicity (CDC) and when the recognized antigens act as

77
functional molecules that contribute to tumor cell proliferation, etc., an antitumor

effect via molecular-targeted therapy can also be anticipated. Attempts are now
underway to identify tumor antigens for use as targets for the development of new
immunotherapies for the treatment of gliomas. In this article, the latest findings related
to glioma antigens are reviewed.
TUMOR ANTIGENS RECOGNIZED BY CTLs
CTLs play an important role in the rejection of tumors and research on tumor antigens
that are targets of CTLs has attracted a great deal of interest. Tumor cells process tumor
antigens and present epitope peptides for CTL recognition on the tumor cell surface in
the context of human leukocyte antigen (HLA) class I (Fig. 1). Methods of identifying
human tumor antigens recognized by CTLs can be divided into those that do and those
that do not utilize CTLs for antigen screening.7 For the methods that do not utilize CTLs,
candidate molecules must first be identified; then, the ability of the molecules to induce
CTLs must be analyzed to confirm that they do indeed act as tumor antigens that are
recognized by CTLs (reverse immunology).
DIRECT IDENTIFICATION OF ANTIGENIC PEPTIDE USING CTLs
When CTLs are available, one approach is the direct screening of peptides that bind
to HLA molecules on the tumor cells. After the acid extraction of tumor peptides that
Figure 1. Tumor cells and cytotoxic T cells (CTLs). CTLs specifically recognize tumor antigenic
peptides presented on HLA (human leukocyte antigen) class I through T-cell receptors.
GLIOMA ANTIGEN 79
bind to HLA class I, the peptides are fractionated by chromatography. The fractionated
peptides are then screened with CTLs and, finally, the peptides that are recognized by
CTLs are purified and their amino acid sequences are determined. Since this method
directly identifies HLA-binding peptides, it is useful for tumor antigens that have been
posttranslationally modified, although this method is technically difficult. The epitope of
a melanoma antigen, gp100,8 and a mutated peptide of the lung cancer antigen, elongation
factor 2,9 have been identified using this method.
IDENTIFICATION OF TUMOR ANTIGEN BY cDNA EXPRESSION

CLONING USING CTLs
The majority of the known tumor antigens recognized by CTLs have been identified
by screening cDNA expression libraries using CTLs. A cDNA library generated from
cancer is transfected into cells expressing an appropriate HLA. The gene products are
then processed into short peptides in the cells and these antigenic peptides are presented
on the HLA. Genes encoding tumor antigen are then identified by screening for reactivity
with CTLs. cDNA fragments of various lengths are prepared from the genes that are
identified and the position of the T-cell epitope is estimated by analyzing their reactivity
with CTLs. Peptides from the estimated site that conform to the HLA-binding motif
are then synthesized and the peptides are identified by confirming their reactivity with
CTLs. A melanoma antigen, MAGE-1,10 and a glioma antigen, GARC-1,11 have been
identified by this method.
IDENTIFICATION OF TUMOR ANTIGENS FROM CANDIDATE

MOLECULES WITHOUT USING CTLs (REVERSE IMMUNOLOGY)
Candidate molecules of tumor antigens are isolated using gene expression analysis
or SEREX (serological identification of antigens by recombinant cDNA expression
cloning) methods, in which a tumor-derived cDNA expression library is screened with
antibodies from the serum of cancer patients.12 To determine whether the candidate
molecules identified are potential tumor antigens recognized by CTLs, T-cell epitope
peptides are predicted by using both an HLA-binding motif algorithm and a proteasome
cleavage algorithm based on the amino acid sequence of the candidate molecules
(Fig. 2); the CTL inducing ability of the synthesized candidate peptides are analyzed.
A tumor antigen, NY-ESO-1,13 and a glioma antigen, SOX6,14 have been identified
using this method.
GLIOMA ANTIGENS
The central nervous system is an immunologically privileged site and access by

immune cells and the activities of intrinsic immune cells are highly restricted. However,
because CTLs have been shown to invade the brain and to be capable of reacting with
gliomas, attempts have been made to isolate and identify glioma antigens in order to
develop antigen-specific immunotherapies.
Figure 2. Selection of candidate HLA-binding peptides derived from tumor antigen. The T-cell epitope
peptides were predicted by using an HLA-binding motif algorithm and a proteasome cleavage algorithm
based on the amino acid sequences of the tumor antigens.
GLIOMA ANTIGENS IDENTIFIED USING SEREX
SEREX using patients sera has been developed and used to isolate a variety of
tumor antigens.12 The advantages of SEREX are that it does not require tumor-specific
CTLs, which are difficult to isolate and culture and that the tumor antigens identified by
SEREX are reportedly also recognized by CTLs.13
Pfreundschuh et al screened a glioma-derived cDNA library with patients sera and
isolated the TEGT gene.15 TEGT is highly expressed in gliomas and antibody reactions were
observed using the sera of glioma patients, but not of healthy donors. Meese et al screened
a glioma-derived cDNA library with patients sera in a similar manner and identified two
different glioma antigens (PHD finger protein 3, PHF3; and glioma-expressed antigen
2, GLEA2).16,17 PHF3 is an intranuclear protein that is strongly expressed in muscles as
well as gliomas. Antibody reactions with PHF3 have been observed in sera from glioma
patients but not in sera from healthy donors. GLEA2 is expressed ubiquitously, but
antibody reactions with GLEA2 were limited to sera from glioma patients.16 Because
these antigens do not exhibit expression specificity or gene mutations, the mechanism
responsible for the stronger IgG response in glioma patients, compared with in healthy
donors, remains unknown.
Okada et al performed SEREX using sera obtained after immunotherapy with IL-4 in a
rat glioma model and identified the gene of a glioma antigen, MIDA1 (mouse Id-associated
protein 1).18 MIDA1 is highly expressed in gliomas and no clear expression has been
found in normal tissue other than in the testis and thymus gland. DNA vaccine using
the MIDA gene induced an antibody reaction to MIDA1 and an antitumor effect against
gliomas in an animal model. We screened a testis-derived cDNA library with sera from
glioma patients and identified a transcription factor, SOX6.14 SOX6 was highly expressed
in glioma tissue, but no apparent expression was observed in normal adult tissues with
the exception of testis tissue. A SOX6-specific IgG antibody was found in approximately
1/3 of glioma patients but was hardly ever detected in patients with other brain diseases
GLIOMA ANTIGEN 81
or in healthy donors. Moreover, in a mouse glioma model, DNA vaccine using the SOX6
gene resulted in the induction of glioma-specific CTLs and an antitumor effect.19
Thus, because glioma antigens recognized by serum IgG were identified using
SEREX, the existence of helper T cells specific for antigens activated in glioma patients
was demonstrated. Determining whether a specific immune response against gliomas can
be clinically induced using these antigens will be an important research task.
GLIOMA ANTIGENS RECOGNIZED BY CTLs
Several glioma antigens recognized by CTLs have been identified; these antigens can
be classified as cancer-testis (CT) antigens, tissue-specific antigens, mutated antigens and
others (Table 1). CT antigens, which are expressed in a variety of human cancers but not in
normal tissues except for in the testis, represent promising targets for immunotherapeutic
approaches.13 MAGE-1 and SOX6 are both CT antigens and have been observed in gliomas
but not in normal brain tissues.20,21 In the category of tissue-specific antigens, gp100 and
TRP-2, which were originally identified as melanoma antigens, have also been shown to be
expressed in gliomas.22,23 The epidermal growth factor receptor (EGFR) is often amplified
and structurally rearranged in malignant gliomas, with the most common mutation being
EGFRvIII and an EGFRvIII-specific peptide has been identified.24 Other glioma antigens
that have been identified include IL13Ra2,25 EphA2,26 EphB6,27 AIM-2,28 HER-2,23 WT1,29
ARF4L,30 SART-3,31 SOX11,32 KIF1C and KIF3C.33 These tumor antigens could be utilized
for immunotherapy in a variety of forms (i.e., peptides, proteins, genes, etc.) using various
techniques in combination with adjuvants. So far, antigenic peptides have been generally
used in clinical studies on immunotherapy for gliomas because they are simple to synthesize
and easy to use.34,35
We have been identifying and analyzing glioma antigens using a variety of methods;
36
based on its expression specificity and immunogenicity, we think that SOX6 (which was
identified using SEREX) might be an ideal target antigen for the treatment of gliomas.14
Recently, two HLA-A24-binding peptides and one HLA-A02-binding peptide were
indentified as CTL epitopes in the glioma antigen SOX6; the stimulation of lymphocytes from
glioma patients using these peptides revealed them to be capable of inducing glioma-specific
CTLs (unpublished data). In the near future, the results of clinical studies examining
immunotherapy with glioma antigens will need to be verified and immune-induction
methods may need to be modified to enhance the antitumor effect.
Table 1. Human glioma antigens recognized by CTLs

Category Antigen
Cancertestis (CT) MAGE-1, SOX6
antigen
Tissue-specific antigen gp100, TRP-2
Mutated antigen EGFRvPPP
Others IL13Ra2, EphA2, EphB6, AIM-2, HER-2, WT1 ARF4L,
SART-3, SOX11, KIF1C, KIF3
GLIOMA ANTIGENS RECOGNIZED BY ANTIBODIES
Tenascin37 and mutant-type EGFR (EGFRvIII)38 are glioma antigens that are reportedly
recognized by antibodies. Tenascin is a glycoprotein expressed on the cell surface and it
is more highly expressed in gliomas than in normal brain tissue. Radioisotope-conjugated
antibodies have been developed and a clinical application for the treatment of gliomas
has been attempted.39 Gene mutations in cancer can result in antigens recognized by the
immune system. The expression of mutant-type EGFR (EGFRvIII) has been observed
in gliomas and the clinical application of an anti-EGFRvIII antibody is currently being
assessed.40 EGFRvIII also shows promise as a marker because of its tumor specificity,
but the frequency of this gene mutation in gliomas has been reported to be approximately
20%. In addition, an EGFRvIII-derived antigenic peptide was recently identified,24 and
its application to vaccine therapy is currently being attempted.
NEURAL STEM CELLS AND GLIOMA ANTIGENS
Gliomas and neural stem cells, from which gliomas are thought to originate, are expected
to exhibit a common gene expression pattern. The biological association between brain
tumor stem cells and neural stem cells has recently become a topic of interest and the neural
stem cell marker CD133 has been used as a brain tumor stem cell marker.41 In addition,
two other neural stem cell markers, nestin and musashi-1, have also been reported to be
useful as glioma markers.42,43 Because molecules that are highly expressed in tumors are
potential CTL targets, an analysis of the CTL epitopes of these molecules is anticipated.
CONCLUSION
Since antibodies do not cross the blood-brain barrier (BBB), the method of
administering antibodies should be modified for the treatment of brain tumors. However,
recent clinical trials combining an anti-VEGF antibody with chemotherapy have reported
encouraging response rates.44 Therefore, antibody therapy for the treatment of gliomas is
being reconsidered and has also attracted interest from the standpoint of molecular-targeted
therapy. Unlike antibodies, T cells can cross the BBB and are capable of migration within
the brain, where they can selectively attack glioma targets. Thus, immunotherapy utilizing
T cells is an ideal treatment strategy for invasive gliomas. Identifying glioma antigens
will enable a major contribution to the development of effective immunotherapies as well
as the development of new diagnostic methods as glioma markers.
REFERENCES
1. Dudley ME, Rosenberg SA. Adoptive-cell-transfer therapy for the treatment of patients with cancer. Nat
Rev 2003; 3:666-675.
2. Rosenberg SA. A new era for cancer immunotherapy based on the genes that encode cancer antigens.
Immunity 1999; 10:281-287.
3. Rosenberg SA. Progress in human tumour immunology and immunotherapy. Nature 2001; 411:380-384.
4. Pfeiffer P, Qvortrup C, Eriksen JG. Current role of antibody therapy in patients with metastatic colorectal
cancer. Oncogene 2007; 26:3661-3678.
GLIOMA ANTIGEN 83
5. Bernard-Marty C, Lebrun F, Awada A et al. Monoclonal antibody-based targeted therapy in breast cancer:
current status and future directions. Drugs 2006; 66:1577-1591.
6. Adams GP, Weiner LM. Monoclonal antibody therapy of cancer. Nat Biotech 2005; 23:1147-1157.
7. Kawakami Y, Rosenberg SA. Human tumor antigens recognized by T-cells. Immunol Res 1997; 16:313-339.
8. Cox AL, Skipper J, Chen Y et al. Identification of a peptide recognized by five melanoma-specific human
cytotoxic T-cell lines. Science 1994 Apr 29;264(5159):716-719.
9. Hogan KT, Eisinger DP, Cupp SB, 3rd et al. The peptide recognized by HLA-A68.2-restricted, squamous
cell carcinoma of the lung-specific cytotoxic T-lymphocytes is derived from a mutated elongation
factor 2 gene. Cancer Res1998; 58:5144-5150.
10. van der Bruggen P, Traversari C, Chomez P et al. A gene encoding an antigen recognized by cytolytic
T-lymphocytes on a human melanoma. J Immunol 2007 Mar 1;178(5):2617-2621.
11. Iizuka Y, Kojima H, Kobata T et al. Identification of a glioma antigen, GARC-1, using cytotoxic
T-lymphocytes induced by HSV cancer vaccine. Int J Cancer 2006; 118:942-949.
12. Sahin U, Tureci O, Pfreundschuh M. Serological identification of human tumor antigens. Curr Opin
Immunol 1997; 9:709-716.
13. Chen YT, Scanlan MJ, Sahin U et al. A testicular antigen aberrantly expressed in human cancers detected
by autologous antibody screening. Proc Natl Acad Sci USA 1997; 94:1914-1918.
14. Ueda R, Iizuka Y, Yoshida K et al. Identification of a human glioma antigen, SOX6, recognized by
patients sera. Oncogene 2004; 23:1420-1427.
15. Sahin U, Tureci O, Schmitt H et al. Human neoplasms elicit multiple specific immune responses in the
autologous host. Proc Natl Acad Sci USA 1995; 92:11810-11813.
16. Fischer U, Struss AK, Hemmer D et al. Glioma-expressed antigen 2 (GLEA2): a novel protein that can elicit
immune responses in glioblastoma patients and some controls. Clin Exp Immunol 2001; 126:206-213.
17. Struss AK, Romeike BF, Munnia A et al. PHF3-specific antibody responses in over 60% of patients with
glioblastoma multiforme. Oncogene 2001; 20:4107-4114.
18. Okada H, Attanucci J, Giezeman-Smits KM et al. Immunization with an antigen identified by cytokine
tumor vaccine-assisted SEREX (CAS) suppressed growth of the rat 9L glioma in vivo. Cancer Res
2001; 61:2625-2631.
19. Ueda R, Kinoshita E, Ito R et al. Induction of protective and therapeutic antitumor immunity by a DNA
vaccine with a glioma antigen, SOX6. Int J Cancer 2008; 122:2274-2279.
20. Kuramoto T. Detection of MAGE-1 tumor antigen in brain tumor. Kurume Med J 1997; 44:43-51.
21. Ueda R, Yoshida K, Kawakami Y et al. Expression of a transcriptional factor, SOX6, in human gliomas.
Brain Tumor Pathol 2004; 21:35-38.
22. Chi DD, Merchant RE, Rand R et al. Molecular detection of tumor-associated antigens shared by human
cutaneous melanomas and gliomas. Am J Pathol 1997; 150:2143-2152.
23. Liu G, Ying H, Zeng G et al. HER-2, gp100 and MAGE-1 are expressed in human glioblastoma and
recognized by cytotoxic T-cells. Cancer Res 2004; 64:4980-4986.
24. Heimberger AB, Crotty LE, Archer GE et al. Epidermal growth factor receptor VIII peptide vaccination
is efficacious against established intracerebral tumors. Clin Cancer Res 2003; 9:4247-4254.
25. Okano F, Storkus WJ, Chambers WH et al. Identification of a novel HLA-A*0201-restricted, cytotoxic
T-lymphocyte epitope in a human glioma-associated antigen, interleukin 13 receptor alpha2 chain. Clin
Cancer Res 2002; 8:2851-2855.
26. Hatano M, Eguchi J, Tatsumi T et al. EphA2 as a glioma-associated antigen: a novel target for glioma
vaccines. Neoplasia 2005 Aug;7(8):717-722.
27. Jin M, Komohara Y, Shichijo S et al. Identification of EphB6 variant-derived epitope peptides recognized
by cytotoxic T-lymphocytes from HLA-A24
malignant glioma patients. Oncol Rep 2008; 19:1277-1283.
28. Liu G, Yu JS, Zeng G et al. AIM-2: a novel tumor antigen is expressed and presented by human glioma
cells. J Immunother 2004; 27:220-226.
29. Hashiba T, Izumoto S, Kagawa N et al. Expression of WT1 protein and correlation with cellular proliferation
in glial tumors. Neurol Med Chir 2007; 47:165-170; discussion 170.
30. Nonaka Y, Tsuda N, Shichijo S et al. Recognition of ADP-ribosylation factor 4-like by HLA-A2-restricted
and tumor-reactive cytotoxic T-lymphocytes from patients with brain tumors. Tissue Antigens 2002;
60:319-327.
31. Murayama K, Kobayashi T, Imaizumi T et al. Expression of the SART3 tumor-rejection antigen in brain
tumors and induction of cytotoxic T-lymphocytes by its peptides. J Immunother 2000; 23:511-518.
32. Schmitz M, Wehner R, Stevanovic S et al. Identification of a naturally processed T-cell epitope derived
from the glioma-associated protein SOX11. Cancer Lett 2007; 245:331-336.
33. Harada M, Ishihara Y, Itoh K et al. Kinesin superfamily protein-derived peptides with the ability to
induce glioma-reactive cytotoxic T-lymphocytes in human leukocyte antigen-A24
glioma patients.
Oncol Rep 2007; 17:629-636.
34. Izumoto S, Tsuboi A, Oka Y et al. Phase II clinical trial of Wilms tumor 1 peptide vaccination for patients
with recurrent glioblastoma multiforme. J Neurosurg 2008; 108:963-971.
35. Yajima N, Yamanaka R, Mine T et al. Immunologic evaluation of personalized peptide vaccination for
patients with advanced malignant glioma. Clin Cancer Res 2005; 11:5900-5911.
36. Toda M. Cancer vaccine for brain tumors and brain tumor antigens. Cancer Therapy 2004; 2:21-26.
37. Zalutsky MR, Moseley RP, Benjamin JC et al. Monoclonal antibody and F(ab)2 fragment delivery to
tumor in patients with glioma: comparison of intracarotid and intravenous administration. Cancer Res
1990; 50:4105-4110.
38. Wikstrand CJ, Hale LP, Batra SK et al. Monoclonal antibodies against EGFRvIII are tumor specific and
react with breast and lung carcinomas and malignant gliomas. Cancer Res 1995; 55:3140-3148.
39. Reardon DA, Zalutsky MR, Bigner DD. Antitenascin-C monoclonal antibody radioimmunotherapy for
malignant glioma patients. Expert Rev of Anticancer Ther 2007; 7:675-687.
40. Wikstrand CJ, Cole VR, Crotty LE et al. Generation of anti-idiotypic reagents in the EGFRvIII
tumor-associated antigen system. Cancer Immunol Immunother 2002; 50:639-652.
41. Woodward WA, Sulman EP. Cancer stem cells: markers or biomarkers? Cancer Metastasis Rev 2008;
27:459-470.
42. Toda M, Iizuka Y, Yu W et al. Expression of the neural RNA-binding protein Musashi1 in human gliomas.
Glia 2001; 34:1-7.
43. Tohyama T, Lee VM, Rorke LB et al. Nestin expression in embryonic human neuroepithelium and in
human neuroepithelial tumor cells. Lab Investi 1992; 66:303-313.
44. Kreisl TN, Kim L, Moore K et al. Phase II trial of single-agent bevacizumab followed by bevacizumab
plus irinotecan at tumor progression in recurrent glioblastoma. J Clin Oncol 2009; 27:740-745.
PART III
CYTOKINE, SEROTHERAPY, ADOPTIVE TRANSFER

AND OTHER STRATEGIES
CHAPTER 7
CYTOKINE THERAPY
Masasuke Ohno,1 Atsushi Natsume2 and Toshihiko Wakabayashi*,1

1
Department of Neurosurgery, Nagoya University School of Medicine, Nagoya, Japan; 2Center for Genetics
and Regenerative Medicine, Nagoya University Hospital, Nagoya, Japan
*Corresponding Author: Toshihiko WakabayashiEmail: wakabat@med.nagoya-u.ac.jp
Abstract: Cytokines are a heterogeneous group of soluble small polypeptides or glycoproteins,

which exert pleiotropic and redundant effects that promote growth, differentiation
and activation of normal cells. Cytokines can have either pro- or anti-inflammatory
activity and immunosuppressive activity, depending on the microenvironments.
The tumor microenvironment consists of a variable combination of tumor cells,
endothelial cells and infiltrating leukocytes, such as macrophages, T-lymphocytes,
natural killer (NK) cells, B cells and antigen-presenting cells (APCs). Cytokine
production acts as a means of communication in the tumor microenvironment. In
this article, we review the cross-talk between cytokines in the tumor environment
and the cytokine therapies that have been used till date for glioma treatment.
INTRODUCTION
Cytokines are a heterogeneous group of soluble small polypeptides or glycoproteins,

which exert pleiotropic and redundant effects that promote growth, differentiation and
activation of normal cells.1 Cytokines can have either pro- or anti-inflammatory activity
and immunosuppressive activity, depending on the microenvironments. Immune cells are
the major source of cytokines but many human cells are capable of producing them and,
importantly, their production acts as a means of communication between both cells and tissue.
The brain is an immunologically privileged site, as evidenced by the relative
impermeability of the blood-brain barrier to immune cells, the lack of lymphatics in the
CNS and the relative immunoincompetence of microgliathe resident macrophages
in the CNS. However, it had recently been suggested that the interaction between the
CNS and immune system is much more complex.2 Immunosuppression by gliomas
may contribute to tumor progression and treatment resistance. It is not known when

86
CYTOKINE THERAPY 87
Figure 1. Regulatory role of tumor-derived cytokines, chemokines and growth factors in the tumor
microenvironment. TIL: tumor-infiltrating lymphocytes; DC: dendritic cells; TAM: tumor associated
macrophages.
immunosuppression occurs during tumor development, but it likely involves cross-talk

among tumor cells, tumor-associated macrophages and microglia (TAMs) and peripheral
as well as tumor-infiltrating lymphocytes (TILs).3
The tumor microenvironment consists of a variable combination of tumor cells,
endothelial cells and infiltrating leukocytes, such as macrophages, T-lymphocytes,
natural killer (NK) cells, B cells and antigen-presenting cells (APCs). A variety of
cytokines and other substances, such as chemokines and growth factors, are produced by
different cells in the local tumor environment, resulting in a complex cell interaction and
regulation of differentiation, activation, functioning and survival of multiple cell types.
The interaction between cytokines, chemokines and growth factors and their receptors
results in a comprehensive network at the tumor site, which is primarily responsible for
the overall progression of tumors, the spread of antitumor immune responses, or the
induction of tumor rejection (Fig. 1).
In this chapter, we review the cross-talk between cytokines in the tumor environment
and the cytokine therapies that have been used till date for glioma treatment.
IL-2
Interleukin-2 (IL-2) is known as the growth factor of the immune system, which
includes helper T cells, cytotoxic T cells (CTLs), B cells, NK cells and macrophages. It
also enhances the production of other cytokines such as the tumor necrosis factor (TNF),
IL-1 and interferon (IFN)-a. The exact mechanism of its antitumor activity is not known.
However, studies have shown that IL-2 enhances the mitogenesis of lymphocytes as well
as the proliferation and clonal expansion of lymphokine-activated killer cells (LAKs),
which are mainly NK cells induced into a hyperactivated state and TILs. Initial efforts
were mostly focused on the use of cytokines to increase LAKs and TILs and then use
these cells to treat tumors such as renal cell carcinoma and melanoma.1-5 In addition to its
use in adoptive immunotherapy, IL-2 was used as a pharmacological agent to be directly
administered into the patients body. However, because of the serious adverse reactions
such as hypotension, renal dysfunction, myocardial infarction, bowel perforation, seizures
and coma, clear clinical benefits were only seen in a limited number of patients with
these types of cancers.6-7
In glioma patients, the first clinical trial with IL-2 was reported in 1986. Recombinant
IL-2 or autologous LAK cells were directly injected into the tumor cavity to treat 9
patients. In this Phase I study, there were no signs of systemic toxicity or neurotoxicity
after treatment.4 IL-2 was mainly administered intracavitarily in other trials,5-7 except
in 1 trial where a combination of autologous tumor vaccination and systemic IL-2
administration was used.8 However, even in this study, the systemic IL-2 dose was reduced
from 8.8 = 106 to 3 = 106 units/day for the last 7 patients because of the considerable
discomfort caused by IL-2.
Recently, the efficacy of combination therapy with a suicide gene and the IL-2 gene
for treating recurrent glioblastoma was investigated.9 A total of 12 patients received
intratumoral injections of retroviral vector-producing cells, followed by intravenous
ganciclovir. This treatment was well tolerated: 2 patients showed a partial response;
4, minor response; 4, stable disease; and 2, progressive disease. The 6- and 12-month
progression-free survival (PFS) rates were 47% and 14%, respectively, whereas the 6-
and 12-month overall survival rates were 58% and 25%, respectively.
IL-4
IL-4 is a glycoprotein with an approximate molecular mass of 15 kDa. It is secreted

by several hematopoietic cell types, including T cells, NK cells, basophils, eosinophils
and mast cells.10-14 B cells, T cells, myeloid cells, monocytes, macrophages, mast cells,
fibroblasts, endothelial cells and some cancer cells express a high-affinity receptor that
mediates the biological activity of IL-4.15-17 IL-4R has 2 types of receptors. One consists
of IL-4R _ and IL-2R a chains and is expressed on hematopoietic cells, whereas the other
consists of IL-4R _ and IL-13R _ 1 chains.18,19 IL-4 promotes the growth and differentiation
of B cells, T cells and mast cells and mediates profound regulatory effects on macrophages.
IL-4 stimulates the growth of both normal helper and CTLs, including TILs and acts
synergistically with IL-2 to enhance the proliferation of precursor CTLs (pCTLs) and
induce their differentiation into active CTLs. Although IL-4 is generally regarded as an
inducer of Type 2 responses, recent studies clearly showed that it has pleiotropic effects
on immune cells of multiple lineages and that it plays an important role as an inducer of
Type 1 T-cell immunity. In particular, IL-4 supports dendritic cell (DC) maturation and
enhances IL-12p70 secretion from DCs.20 A strategy for using IL-4 in glioma treatment is
vaccinations with autologous glioma cells and IL-4 gene-transfected fibroblasts.21 Adult
participants with recurrent glioblastoma multiforme (GBM) or anaplastic astrocytoma
underwent gross total resection of the recurrent tumors, followed by 2 vaccinations with
CYTOKINE THERAPY 89
autologous fibroblasts retrovirally transduced with the IL-4 and thymidine kinase genes
and mixed with irradiated autologous glioma cells. The patients who were vaccinated
twice demonstrated encouraging immunological and clinical responses. Another promising
strategy is the so-called immunotoxin therapy, which takes advantage of the fact that
IL-4R is abundantly present in malignant gliomas.22 A chimeric recombinant fusion protein
composed of circularly permuted IL-4 and a truncated form of Pseudomonas exotoxin (PE)
has been evaluated as a potential therapeutic agent. In a Phase I trial of IL-4-PE (NBI-3001)
in patients with recurrent malignant glioma, 31 patients were treated with intratumoral
administration of IL-4R-PE via convection-enhanced delivery. Treatment-related adverse
effects were limited and no deaths were attributable to the treatment. Drug-related Grade
3 and 4 toxicity was seen in 39% of the patients in all the dose groups and in 22% of the
patients given the maximum tolerated dose. The median overall survival was 5.8 months
in patients with recurrent GBM and 8.2 months in all patients.
IL-13
IL-13 is a cytokine derived from Type 2 T helper cells that can bind to 2 receptor
chains, IL-13R_1 and IL-13R_2. It has low affinity for the IL-13R_1 chain and high
affinity for the IL-4R_ chain. It forms a receptor complex with the IL-4R_ chain,
which is involved in IL-13-induced signal transduction through either JAK-STAT or
phosphatidylinositol 3-kinase.23 The IL-13R_2 chain binds to IL-13 with high affinity
and internalizes it after ligand binding without the involvement of other chains. IL-13R
is found to be overexpressed in a majority of glioma cell lines and resected GBM
specimens.24 A chimeric fusion protein composed of human IL-13 and mutated PE has
been developed and shown to affect the specific cytotoxicity of glioma cell lines.24,25
IL-13-PE is reportedly more active against glioma cell lines than IL-4-targeted toxins
in vitro.25 In a Phase I trial, 51 GBM patients were administered IL-13-PE (cintredekin
besudotox, also known as IL-13-PE38QQR), via convection-enhanced delivery.26 A Phase
III study was conducted to compare the efficacy of IL-13-PE to that of Gliadel wafers.25
PFS was longer (17.7 versus 11.4 weeks) in patients treated with IL-13-PE, but there was
no significant difference in the median survival time. As a more advanced strategy, PE
has been conjugated to a smaller single-chain variable fragment for producing anti-IL13R
humanized antibodies.27 Overall, IL-13-based toxins have the potential to be utilized
in adjuvant therapy for malignant glioma but their use requires further clinical studies.
TGF-`
Transforming growth factor-beta (TGF- `) is a multifunctional regulatory

polypeptide from a ligand surperfamily that includes the TGF-`s, activins and bone
morphogenetic proteins (BMPs). TGF-` controls many aspects of cellular function,
including proliferation, differentiation, migration, apoptosis, adhesion, angiogenesis,
immune surveillance and survival.28 Three mammalian TGF-` isoformsTGF-`1,
TGF-`2 and TGF-`3have been identified. In normal adult tissues, TGF-`1 is by
far the predominant isoform, whereas the expression of TGF-`2 and TGF-`3 is much
lesser. The secreted TGF-` binds to TGF-` receptors (T-`R) and initiates a signaling
cascade via mediators of cytoplasmic signaling, called Smads, into the nucleus where the
Smad complex regulates target gene expression.29 The actions of TGF-` are dependent
on several factors, including cell type, growth conditions and the presence of other
polypeptide growth factors. Because of the dual role of TGF-` as a tumor suppressor
and pro-oncogenic factor, the members of the TGF-` signaling pathway are being
considered as predictive biomarkers of progressive tumors as well as molecular targets
for the prevention and treatment of cancer and metastasis.30
The TGF-`2 isoform is specifically overexpressed in malignant gliomas. The increased
levels of TGF-`2 are associated with disease stage and cause immunodeficiencies
in patients.31 TGF-`2 not only inhibits the proliferation of lymphocytes but also has
multiple effects on the immune system. These effects include the inhibition of immune
cell activation, the blockade of antitumor activity, a shift of cytokine balance towards
immunosuppression and the inhibition of antigen presentation. Thus, the targeted inhibition
of TGF-`2 should have these antitumor effects and allow an immune-mediated response.
Several approaches to block TGF-` function are currently under study, such as the use of
monoclonal antibodies to TGF-`, recombinant fusion proteins containing the ectodomains
of T`RII and T`III to prevent the binding of TGF-` ligands, ATP competitive inhibitors
at the ATP-binding site of the T`RI kinase and antisense oligonucleotides specific for
TGF-`2.32-37 Trabedersen (AP-12009) is a synthetic antisense oligodeoxynucleotide
designed to block the production of TGF-`2. In a Phase IIb trial, the survival of patients
with brain tumors improved when they were intratumorally administered with trabedersen,
as compared to patients receiving standard chemotherapy. An international clinical Phase
III trial is currently recruiting patients with recurrent or refractory anaplastic astrocytoma.38
GM-CSF
Granulocyte-macrophage colony-stimulating factor (GM-CSF) is secreted by a

variety of cell types such as monocytes, endothelial cells, activated T cells, fibroblasts,
mitogen-stimulated B cells and lipopolysaccharide (LPS)-stimulated macrophages.
GM-CSF binds to a high-affinity receptor composed of a GM-CSF-specific _ chain and
the conventional signal-transduction subunit, i.e., the `-subunit, which is common with the
receptors for IL-3 and IL-5. The activation of the GM-CSF receptor is known to stimulate
at least 3 pathways: the JAK-STAT pathway, MAPK pathway and PIP3K pathway. The
GM-CSF receptor is expressed by CD34
progenitor cells, all myeloid lineages and
vascular endothelial cells. GM-CSF can promote myeloid differentiation and, in fact,
it was first defined by its ability to generate both granulocyte and macrophage colonies
from bone marrow precursor cells. However, GM-CSF also plays an important role in

These pivotal roles mainly derive from the ability of GM-CSF to affect the properties and
functional status of immature and mature myeloid cells such as granulocytes, dendritic
cells (DCs), macrophages and eosinophils.39 GM-CSF has been widely used as an adjuvant
in clinical trials of vaccination with autologous tumor cells, peptides and/or dendritic
cells in different human neoplasms. This cytokine was administered subcutaneously or
intradermally either as a product of gene-transduced tumor cells or as a recombinant protein,
together with the vaccine. Autologous or allogeneic human tumor cells transduced with
the GM-CSF gene have been tested as vaccines in many cancers, such as renal cancer,
melanoma, prostate cancer, lung cancer and pancreatic tumors. A pilot clinical trial of
combined B7-2 and GM-CSF immunogene therapy for GBMs and melanomas has been
CYTOKINE THERAPY 91
reported.40 In this trial, patients with recurrent malignant gliomas were vaccinated with
irradiated autologous tumor cells transduced with the B7-2 and GM-CSF genes, using
a retroviral vector. Although vaccine preparation was attempted using 116 samples of
malignant glioma tissue, vaccine preparation was successful for only 5 GBM samples.
Minor toxicities were observed and all patients have now died. Most patients showed
evidence of an inflammatory response but specific antitumor immunity was not observed.
The GM-CSF protein is also widely used as an adjuvant at the site of vaccination to
avoid the cumbersome procedure of gene transduction and to determine the appropriate
dose of the administered cytokine for other tumors. Nineteen patients with recurrent
malignant glioma were vaccinated twice with irradiated autologous whole tumor cells,
using GM-CSF as an adjuvant. The patients then underwent leukapheresis, followed by
adoptive transfer of peripheral blood lymphocytes activated in vitro by using anti-CD3 and
IL-2. Of the 19 patients, 17 developed a delayed-type hypersensitivity (DTH) response
to vaccination, which appeared to be directed against the autologous tumor. In 8 patients,
there was radiological evidence of a response and in 5 there was evidence of clinical
improvement. The median survival time was 12 months (range, 6-28 months) and the
presence of both, a DTH response and a radiological response, correlated with survival.41
IFN-`
IFN-` has pleiotropic biological effects and has widely been used either alone
or in combination with other antitumor agents for treating malignant gliomas and
melanomas.42 In the treatment of malignant gliomas, IFN-` can act as a drug sensitizer
and it enhances the toxicity against various neoplasms when administered in combination
with nitrosourea. Combination therapy with IFN-` and nitrosourea has been used for
treating gliomas in Japan.43 Previously, we demonstrated that IFN-` markedly enhanced
chemosensitivity to temozolomide (TMZ) in an in vitro study of human glioma cells,44
this finding suggests that one of the main mechanisms by which IFN-` enhances
chemosensitivity is the downregulation of methylguanine methyltransferase (MGMT)
transcription, via p53 induction. This effect was also observed in an experimental
animal model.45 These 2 studies suggested that chemotherapy with IFN-` and TMZ
plus radiation might further improve the clinical outcome in patients with malignant
gliomas, as compared to TMZ plus radiation therapy. In 2000, a multicenter clinical
trial was conducted at Nagoya University, Japan, using cytokine gene therapy, wherein
the IFN-` gene was delivered via cationic liposomes. This gene therapy system induced
specific cytotoxic T-cell immunity against mouse glioma and NK cells.46,47 Based on
these observations, a Phase I clinical trial of IFN-` gene therapy was conducted in
5 patients with recurrent malignant glioma.48 This was a two-stage trial in which the
initial treatment comprised tumor removal and injection of liposomes containing the
human IFN-` gene into the margin of the resulting defect and delivery of subsequent
injections via an implanted catheter. The clinical toxicity was found to be minimal. At
10 weeks after treatment initiation, 2 patients showed more than 50% reduction while
others had stable disease. The median survival was longer in the treated subjects than in
the matched historical controls from our institution. After the gene therapy, significant
changes were observed in the histology and gene expression related to immunoresponse,
apoptosis and neovascularization49 (Fig. 2). This study provides the foundation for a
Phase II trial of IFN-` gene therapy. Very recently, Chiocca et al reported a Phase
Figure 2. Mechanisms of liposome-mediated IFN-` gene therapy. Histology and cDNA expression
microarray analyses revealed significant inductions in apoptosis (A) and antitumor immunoresponse
(B) and inhibition of neovascularization (C).
I clinical trial (with a dose-escalation cohort) that involved stereotactic injection of

an IFN-`-expressing adenoviral vector in 11 patients with malignant glioma. Direct
injection of the vector into the tumor and the surrounding normal areas in the brain
after surgical removal of the tumor was feasible. A reproducible increase in tumor cell
apoptosis was observed after the treatment.50
CONCLUSION
Tumors contain immune cells and a network of pro- and anti inflammatory cytokines
collaborate in the development and progression of cancer. Increasingly, cytokine-based
drugs and anticytokines are playing a crucial role in the treatment of malignant gliomas.
Research is continuing with an aim to develop new therapies, refine therapies that
CYTOKINE THERAPY 93
have already used and establish the safest and most effective dosage levels. The latest
development in the field of cytokines is the use of cytokine gene therapy for treatment
of malignant gliomas than systemic administration of cytokine. But to evaluate the
effectiveness of these approaches would be further studies.
REFERENCES
1. Borish LC, Steinke JW. 2. Cytokines and chemokines. J Allergy Clin Immunol 2003; 111:S460-475.
2. Carson MJ, Doose JM, Melchior B et al. CNS immune privilege: hiding in plain sight. Immunol Rev 2006;
213:48-65.
3. Watters JJ, Schartner JM, Badie B. Microglia function in brain tumors. J Neurosci Res 2005; 81:447-455.
4. Jacobs SK, Wilson DJ, Kornblith PL et al. Interleukin-2 or autologous lymphokine-activated killer cell
treatment of malignant glioma: phase I trial. Cancer Res 1986; 46:2101-2104.
5. Vaquero J, Martinez R. Intratumoral immunotherapy with interferon-alpha and interleukin-2 in glioblastoma.
Neuroreport 1992; 3:981-983.
6. Hayes RL, Koslow M, Hiesiger EM et al. Improved long term survival after intracavitary interleukin-2 and
lymphokine-activated killer cells for adults with recurrent malignant glioma. Cancer 1995; 76:840-852.
7. Kruse CA, Cepeda L, Owens B et al. Treatment of recurrent glioma with intracavitary alloreactive cytotoxic
T-lymphocytes and interleukin-2. Cancer Immunol Immunother 1997; 45:77-87.
8. Holladay FP, Heitz-Turner T, Bayer WL et al. Autologous tumor cell vaccination combined with adoptive
cellular immunotherapy in patients with grade III/IV astrocytoma. J Neurooncol 1996; 27:179-189.
9. Colombo F, Barzon L, Franchin E et al. Combined HSV-TK/IL-2 gene therapy in patients with recurrent
glioblastoma multiforme: biological and clinical results. Cancer Gene Ther 2005; 12:835-848.
10. Mosmann TR, Cherwinski H, Bond MW et al. Two types of murine helper T-cell clone. I. Definition
according to profiles of lymphokine activities and secreted proteins. J Immunol 1986; 136:2348-2357.
11. Yoshimoto T, Paul WE. CD4pos, NK1.1pos T-cells promptly produce interleukin 4 in response to in vivo
challenge with anti-CD3. J Exp Med 1994; 179:1285-1295.
12. Plaut M, Pierce JH, Watson CJ et al. Mast cell lines produce lymphokines in response to cross-linkage of
Fc epsilon RI or to calcium ionophores. Nature 1989; 339:64-67.
13. Moqbel R, Ying S, Barkans J et al. Identification of messenger RNA for IL-4 in human eosinophils with
granule localization and release of the translated product. J Immunol 1995; 155:4939-4947.
14. Velazquez JR, Lacy P, Mahmudi-Azer S et al. Interleukin-4 and RANTES expression in maturing eosinophils
derived from human cord blood CD34
progenitors. Immunology 2000; 101:419-425.
15. Boulay JL, Paul WE. The interleukin-4-related lymphokines and their binding to hematopoietin receptors.
J Biol Chem 1992; 267:20525-20528.
16. Cabrillat H, Galizzi JP, Djossou O et al. High affinity binding of human interleukin 4 to cell lines. Biochem
Biophys Res Commun 1987; 149:995-1001.
17. Park LS, Friend D, Sassenfeld HM et al. Characterization of the human B-cell stimulatory factor 1 receptor.
J Exp Med 1987; 166:476-488.
18. Foxwell BM, Woerly G, Ryffel B. Identification of interleukin 4 receptor-associated proteins and expression
of both high- and low-affinity binding on human lymphoid cells. Eur J Immunol 1989; 19:1637-1641.
19. Galizzi JP, Zuber CE, Harada N et al. Molecular cloning of a cDNA encoding the human interleukin 4
receptor. Int Immunol 1990; 2:669-675.
20. Okada H, Kuwashima N. Gene therapy and biologic therapy with interleukin-4. Curr Gene Ther 2002;
2:437-450.
21. Okada H, Lieberman FS, Edington HD et al. Autologous glioma cell vaccine admixed with interleukin-4
gene transfected fibroblasts in the treatment of recurrent glioblastoma: preliminary observations in a
patient with a favorable response to therapy. J Neurooncol 2003; 64:13-20.
22. Weber F, Asher A, Bucholz R et al. Safety, tolerability and tumor response of IL4-Pseudomonas exotoxin
(NBI-3001) in patients with recurrent malignant glioma. J Neurooncol 2003; 64:125-137.
23. Wynn TA. IL-13 effector functions. Annu Rev Immunol 2003; 21:425-456.
24. Debinski W, Obiri NI, Powers SK et al. Human glioma cells overexpress receptors for interleukin 13 and are
extremely sensitive to a novel chimeric protein composed of interleukin 13 and pseudomonas exotoxin.
Clin Cancer Res 1995; 1:1253-1258.
25. Mut M, Sherman JH, Shaffrey ME et al. Cintredekin besudotox in treatment of malignant glioma. Expert
Opin Biol Ther 2008; 8:805-812.
26. Kunwar S, Chang SM, Prados MD et al. Safety of intraparenchymal convection-enhanced delivery of
cintredekin besudotox in early-phase studies. Neurosurg Focus 2006; 20:E15.
27. Kioi M, Seetharam S, Puri RK. Targeting IL-13Ralpha2-positive cancer with a novel recombinant
immunotoxin composed of a single-chain antibody and mutated Pseudomonas exotoxin. Mol Cancer
Ther 2008; 7:1579-1587.
28. Massague J. TGF-beta signal transduction. Annu Rev Biochem 1998; 67:753-791.
29. Derynck R, Zhang YE. Smad-dependent and Smad-independent pathways in TGF-beta family signalling.
Nature 2003; 425:577-584.
30. Jakowlew SB. Transforming growth factor-beta in cancer and metastasis. Cancer Metastasis Rev 2006;
25:435-457.
31. Kjellman C, Olofsson SP, Hansson O et al. Expression of TGF-beta isoforms, TGF-beta receptors and
SMAD molecules at different stages of human glioma. Int J Cancer 2000; 89:251-258.
32. Uhl M, Aulwurm S, Wischhusen J et al. SD-208, a novel transforming growth factor beta receptor I kinase
inhibitor, inhibits growth and invasiveness and enhances immunogenicity of murine and human glioma
cells in vitro and in vivo. Cancer Res 2004; 64:7954-7961.
33. Hjelmeland MD, Hjelmeland AB, Sathornsumetee S et al. SB-431542, a small molecule transforming
growth factor-beta-receptor antagonist, inhibits human glioma cell line proliferation and motility. Mol
Cancer Ther 2004; 3:737-745.
34. Hau P, Jachimczak P, Schlingensiepen R et al. Inhibition of TGF-beta2 with AP 12009 in recurrent malignant
gliomas: from preclinical to phase I/II studies. Oligonucleotides 2007; 17:201-212.
35. Tran TT, Uhl M, Ma JY et al. Inhibiting TGF-beta signaling restores immune surveillance in the SMA-560
glioma model. Neuro Oncol 2007; 9:259-270.
36. Naumann U, Maass P, Gleske AK et al. Glioma gene therapy with soluble transforming growth factor-beta
receptors II and III. Int J Oncol 2008; 33:759-765.
37. Schneider T, Becker A, Ringe K et al. Brain tumor therapy by combined vaccination and antisense
oligonucleotide delivery with nanoparticles. J Neuroimmunol 2008; 195:21-27.
38. Vallieres L. Trabedersen, a TGFbeta2-specific antisense oligonucleotide for the treatment of malignant
gliomas and other tumors overexpressing TGFbeta2. IDrugs 2009; 12:445-453.
39. Parmiani G, Castelli C, Pilla L et al. Opposite immune functions of GM-CSF administered as vaccine
adjuvant in cancer patients. Ann Oncol 2007; 18:226-232.
40. Parney IF, Chang LJ, Farr-Jones MA et al. Technical hurdles in a pilot clinical trial of combined B7-2
and GM-CSF immunogene therapy for glioblastomas and melanomas. J Neurooncol 2006; 78:71-80.
41. Sloan AE, Dansey R, Zamorano L et al. Adoptive immunotherapy in patients with recurrent malignant
glioma: preliminary results of using autologous whole-tumor vaccine plus granulocyte-macrophage
colony-stimulating factor and adoptive transfer of anti-CD3-activated lymphocytes. Neurosurg Focus
2000; 9:e9.
42. Chawla-Sarkar M, Lindner DJ, Liu YF et al. Apoptosis and interferons: role of interferon-stimulated genes
as mediators of apoptosis. Apoptosis 2003; 8:237-249.
43. Wakabayashi T, Hatano N, Kajita Y et al. Initial and maintenance combination treatment with interferon-beta,
MCNU (Ranimustine) and radiotherapy for patients with previously untreated malignant glioma. Journal
of neuro-oncology 2000; 49:57-62.
44. Natsume A, Ishii D, Wakabayashi T et al. IFN-beta down-regulates the expression of DNA repair gene
MGMT and sensitizes resistant glioma cells to temozolomide. Cancer research 2005; 65:7573-7579.
45. Natsume A, Wakabayashi T, Ishii D et al. A combination of IFN-beta and temozolomide in human glioma
xenograft models: implication of p53-mediated MGMT downregulation. Cancer Chemother Pharmacol
2007; 61:653-659.
46. Natsume A, Mizuno M, Ryuke Y et al. Antitumor effect and cellular immunity activation by murine
interferon-beta gene transfer against intracerebral glioma in mouse. Gene therapy 1999; 6:1626-1633.
47. Natsume A, Tsujimura K, Mizuno M et al. IFN-beta gene therapy induces systemic antitumor immunity
against malignant glioma. Journal of neuro-oncology 2000; 47:117-124.
48. Yoshida J, Mizuno M, Fujii M et al. Human gene therapy for malignant gliomas (glioblastoma multiforme
and anaplastic astrocytoma) by in vivo transduction with human interferon beta gene using cationic
liposomes. Human gene therapy 2004; 15:77-86.
49. Wakabayashi T, Natsume A, Hashizume Y et al. A phase I clinical trial of interferon-beta gene therapy
for high-grade glioma: novel findings from gene expression profiling and autopsy. J Gene Med 2008;
10:329-339.
50. Chiocca EA, Smith KM, McKinney B et al. A Phase I Trial of Ad.hIFN-beta Gene Therapy for Glioma.
Mol Ther 2008; 16:618-626.
CHAPTER 8
IMMUNOTHERAPEUTIC APPROACH
WITH OLIGODEOXYNUCLEOTIDES
CONTAINING CpG MOTIFS (CpG-ODN) IN
MALIGNANT GLIOMA
Renata Ursu and Antoine F. Carpentier*

Service de Neurologie, Hpital Avicenne, Bobigny, France
*Corresponding Author: Antoine F. CarpentierEmail: antoine.carpentier@avc.aphp.fr
Abstract: Bacterial DNA and synthetic oligodeoxynucleotides containing CpG motifs

(CpG-ODNs) are strong activators of both innate and specific immunity, driving
the immune response towards the Th1 phenotype. In cancer patients, CpG-ODNs
can be used to activate the innate immunity and trigger a tumor-specific immune
response. Several clinical trials are on-going worldwide in various cancers. In this
chapter, we will focus on the potential applications of CpG-ODNs in glioma. So
far, CpG-ODN has mainly been used by intratumoral injections. Indeed, human
gliomas display a locally invasive pattern of growth and rarely metastasize, making
local treatment clinically relevant.
INTRODUCTION
Since the late 90s, CpG-ODNs have emerged as powerful immunostimulating agents
for both innate and specific immunity. These oligodeoxynucleotides containing CpG motifs
are derived from bacterial DNA and their biologic effects have been dramatically enhanced
by stabilization with phosphorothioate backbones.
The immunogenic properties of CpG-ODNs have been successfully used in several
experimental models of allergies, viral infections and cancers. In the last few years,
CpG-ODNs have entered numerous clinical trials with vaccines against infectious diseases
or for the treatment of allergies, asthma and cancer.

95
In cancer, CpG-ODNs have shown promising activity in multiple animal models. They
can be used as a local immunostimulating agents when used alone or as an adjuvant when
combined to tumor antigens, antitumor antibodies or dendritic cells. Several clinical trials
are ongoing, the results of which should be available in the next few years. In this chapter,
we will focus on the potential applications of CpG-ODN in glioma immunotherapy.
CpG MOTIFS
The immunogenic properties of DNA were first discovered in 1984 by a Japanese

team when DNA extracts from Mycobacter tuberculosis were reported to activate natural
killer (NK) cells.1 Subsequent works showed that the immunogenic properties were due
to the presence within the bacterial DNA of CpG sequences.2-3 Interestingly, synthetic
oligodeoxynucleotides containing CpG motifs display similar properties to bacterial DNA.
Their biological activity is further increased if they are rendered nuclease resistant by a
phosphorothioate backbone modification.4
The Toll-like receptor 9 (TLR9) plays a critical role in CpG-ODN recognition.5 The
Toll-like receptor (TLR) family is a phylogenetically conserved mediator of innate immunity
that is essential for microbial recognition.6 In contrast to most TLRs, TLR9 has not been
detected on the surface of cells and is localized in the endoplasmic reticulum prior to
stimulation.7 No specific transporter for CpG-ODN uptake inside the cells has been identified
so far. Following binding to CpG ODN, TLR9 colocalizes with CpG ODN in lysosomes.8
In murine species, TLR9 is constitutively expressed in B-lymphocytes, monocytes,
macrophages and all types of dendritic cells. In humans, this expression is mainly restricted
to B-lymphocytes and plasmacytoid dendritic cells (DCs),9 although TLR9 expression
has also been reported in neutrophils, monocytes, cluster differentiation (CD) 4 T cells
and in some hematological malignancies.10-12
CpG-ODNs display pleiotropic effects on the immune system reviewed by Krieg.13
(Fig. 1). In B cells, CpG-ODNs induce mitosis, secretion of a number of cytokines such as
interleukin (IL) 6 or IL10, prevent apoptosis triggered by several apoptotic agents and promote
immunoglobulin (Ig) secretion.3 Plasmacytoid Dendritic Cells (pDCs) are directly activated
by CpG-ODNs to express costimulatory molecules (CD40, CD80, CD86), chemokine (C-C
motif) receptor (CCR) 7 and secrete a wide variety of cytokines such as tumour necrosis
factor plan (TNF_ , interferons (IFN), (IL)-6 or IL-12.14-16 Most interestingly, CpG-ODNs are
able to license DCs to directly prime CD8 T cells by a (Th) cell-independent mechanism.17
Direct activation of NK or T cells by CpG-ODNs is unlikely. However, cytokine secretion
by dendritic cells activate NK-cells and T cells, reviewed by Klinman et al.18 Secretion of
IL12 and IFN gamma drive the T-cell differentiation towards the Th1 phenotype and can
even redirect established Th2-biased to Th1 immune responses.19
Several subclasses of CpG-ODNs have been described based on their in vitro activity.20
The classical B-class CpG-ODNs induce secretion of IL12, maturation of pDCs and
B-cell proliferation. The biological activity of B-type CpG motifs also depends upon the
3v and 5v flanking bases leading to several optimized sequences such as 5v-GTCGTT
in humans, 5v-GACGTT in murine species or 5v-AACGTT in both.21-23 The A-class is a
mixed phosphodiester-phosphorothioate CpG-ODN that preferably induces IFN alpha
secretion by DC and activation of NK-cells and gamma delta T cells. The C-class ODNs
have immune properties intermediate between the A and B classes. So far, only the
B-class has entered clinical studies.
CpG-ODN FOR GLIOMA IMMUNOTHERAPY
Figure 1. Innate and Adaptive immunity activation by CpG-ODN.

97
RATIONALE FOR CpG-ODNs IN GLIOMAS AND PRECLINICAL DATA
Glioma cells induce secretion of immunosuppressive cytokines such as prostaglandin

E2 (PGE2) or transforming growth factor beta (TGFbeta) 2, leading to a well-documented
impairment of T- and B-cell immunity in glioma patients.24 A major contributor to the
depressed immunity in these patients is an increased level of regulatory T cells,25-26
which exerts a functional inhibition on NK- and T cells.27 In vitro, this elevation in T
regulatory cells (Treg) fraction correlates with a diminished CD4 proliferation in response
to anti-CD3, which can be restored by Treg depletion.27 In several animal models, Treg
depletion can restore effective tumor immunity and sometimes lead to tumor rejection.28
CpG-ODNs, especially when used as local immunostimulating agents, offer the
unique opportunity both to trigger and to boost antitumor immunity. However, the
identification in gliomas of tumor antigens is a limiting step for the design of therapeutic
vaccines. Therefore, in most preclinical studies, CpG-ODNs have been used as a single
agent directly injected into the tumor, with the expectation that the immune system
will select the most relevant antigens. The validity of such an approach was shown in a
neuroblastoma model, in which peritumoral injections of a synthetic ODN containing
a CpG motif induced complete tumor rejection in the majority of mice and triggered a
long-term immunity.29 Promising data have been obtained in all syngenic murine glioma
models tested so far. When 9L glioma cells were inoculated subcutaneously, local treatment
with CpG-ODNs inhibited tumor growth and complete tumor rejection in a significant
subset of animals.30 Experiments with the poorly immunogenic RG2 glioma showed
similar results. Most interestingly, in an intracranial model of syngenic glioma (CNS1),
while none of the control animals survived the tumor challenge, more than 85% of the
rats treated with a single intratumoral injections of CpG-ODNs five days after the tumor
inoculation showed long term survival. Rats which were cured by CpG-ODN injections
were further protected against a new tumor challenge, showing that a long term immunity
was primed.31 Another study showed that a single intratumoral injection of CpG-ODN
inhibited glioma growth in vivo and cured 80% of glioma-bearing C57BL/6 mice.
Experiments in knockout mice revealed that the efficacy of local CpG-ODN treatment
in vivo required TLR9 expression on non tumor cells.32
The mechanisms underlying tumor rejections following CpG-ODN administration in
murine models have been partially explored. No significant cytotoxicity of CpG-ODNs
has been detected in vitro.31 Increased cellular invasion after stimulation with CpG-ODN
has been reported in vitro.33 Both innate immunity (with NK cells and macrophages)
and T-cells appear to play a critical role in achieving optimal rejection.29-30 Further
experiments demonstrated that CpG-ODNs induced maturation and migration of
DC into the draining lymph nodes in vivo.34 In a murine glioma model, CpG-ODN
appeared to enhance the antigen-presenting capacity of microglia, shift the immune
response towards CD8
T-cells and prolong the survival of mice with experimental
tumors). Furthermore, intratumoral Treg depletion was reported after treatment with
CpG-ODN.26 Antibody-dependant cell cytotoxicity (ADCC) has been suggested in a
lymphoma model,35 but not in solid tumors.
Usage of CpG-ODN as a local immunostimulating agent in brain tumors is strengthened
by recent evidences that TLR9 is expressed within the central nervous system. TLR9
expression, although not directly detected by northern blot study of whole brain extracts,
can be identified on purified subsets of cells, especially microglia and astrocytes.23,36
Furthermore, TLR9 expression within human glioma surgical samples was recently
CpG-ODN FOR GLIOMA IMMUNOTHERAPY 99
shown.37 TLR9 expression was variable among patients, which might suggest that some
patients are more likely to benefit from CpG-ODN. No significant relationship between
TLR9 expression and age, tumor histology were found. The expression of TLR9 directly
by tumor cells was reported in murine and human glioma cell lines.26 However, this
expression appears to be very low when compared to B-cell or pDCs expression37 and
it is likely that tumor-infiltrating immune cells, more than tumor cells, are the relevant
targets of CpG-ODN immunotherapy.
Altogether, the use of CpG-ODNs as a local treatment appears promising in preclinical
models and fully justify a clinical development in glioma patients. Moreover, human
gliomas display a locally invasive pattern of growth and rarely metastasize, making local
treatment clinically relevant.
CLINICAL DEVELOPMENT OF CpG-ODNs IN CANCER

AND IN BRAIN TUMORS
Four different B-Type CpG-ODNs have reached clinical trials: CpG-28,38 CpG 7909
also known as PF 3512676,39 1018 ISS and IMO 2055.40
In nonbrain tumors, most of the trials have been using CpG-ODN subcutaneously or
intravenously to stimulate systemic immunity (Table 1). Interestingly, a randomized Phase
2 in nonsmall-cell lung cancer (NSCLC) showed a higher response rate and a better survival
in the arm receiving subcutaneous CpG and chemotherapy versus chemotherapy alone.41
This data prompted two Phase 3 studies exploring the combination of CpG 7909 with
chemotherapy versus chemotherapy alone in patients with advanced non-small cell lung
carcinoma (NSCLC). Both trials showed a disappointing efficacy and a higher frequency
of serious adverse events in the CpG-ODN arm with neutropenia, thrombocytopenia and
a higher frequency of sepsis like events leading to early termination of most trials using
CpG-ODNs subcutaneously.42
In recurrent glioblastomas (GBM), based on promising preclinical data with local
injections and because the blood-brain barrier would impede any diffusion of CpG-ODN
to the brain, our clinical approach for CpG-ODN favoured intratumoral administration of
CpG-ODN. In a Phase 1 trial conducted on 24 patients with recurrent glioblastoma, local
administration of CpG ODN within the tumor was achieved by convectionenhanced
delivery (CED) around the tumor mass which was left unresected. This study showed
a good clinical tolerance and 2 minor radiological responses. The median of survival
was 7.2 months, slightly greater than would have been expected in this population of
patients (Fig. 2).38 In a subsequent Phase 2 trial, 31 patients with recurrent glioblastoma
received 20 mg CpG-28 locally by means of peri-tumoral catheters and CED. The main
side effects were manageable and consisted of fever, transient neurological worsening and
seizures. A contrast between a poor 6-month PFS (progression-free survival) and a higher
than expected long term survival (1-year survival: 24% and 2-year survival of 15%) was
observed. Using the RPA classification for recurrent glioblastoma,43 the median survival
for RPA class 7 patients (68% of the patients in our study) was 6 months, higher than
the 4.9 months that should have been expected (Ursu et al, manuscript submitted). The
presence of long term survivors suggests that this treatment might have been beneficial
in some patients, whose molecular or clinical characteristics remain to be defined. For
example, expression of TLR9 within the tumor, which is variable among glioblastomas,37
is currently under investigation.
100
Table 1. Clinical trials with CpGODN in cancer

Clinical Trial (nbr of Patients)
Cancer Drug and Administration Way Status Ref
CpG monotherapy
Recurrent glioblastoma CpG-28 Phase I (24), intratumoral Completed (2 MR; 2 SD) 38
Phase II (31), intratumoral Completed (1 PR; 3 MR and 3 SD)
Meningeal carcinomatosis Phase 1 (15), intrathecal and sc On-going
Non-Hodgkins lymphoma CpG 7909 (PF 3512676) Phase I (23), IV Completed 62
Melanoma Phase I (23), intratumoral Completed (activation of pDC in 67
lymph nodes)
Phase II (20), sc Completed (2PR, 3SD) 61
Basal cell carcinoma and Phase I (5
5), intratumoral Completed (BCC: 4 PR; MM: 1 CR) 60
metastatic melanoma
Cutaneous T-cell Phase I/II (28), sc Completed (3 CR, 6 PR) 68
Lymphoma
Renal cell carcinoma Phase I (31), sc Completed (1 PR, 9SD) 69
Chronic lymphocytic Phase I, sc or IV On-going
leukemia
Renal cell carcinoma IMO-2055 Phase II, sc On-going
CpG combined with chemotherapy
Glioblastoma Radiotherapy
Temozolomide
/ Phase II (80), intratumoral On-going
CpG-28
Taxane
Platinum +/ CpG 7909 Phase II (111), sc Completed (23% response for 41
chemotherapy vs 40% for chemo-
therapy
CpG 7909)
Taxane
Platinum +/ CpG 7909 Phase III , sc Prematurely closed 42
Permetrexed +/CpG 7909 Phase II, sc Prematurely closed
continued on next page
Table 1. Continued
Non-small-cell lung cancer Gemcitabine/Cisplatin +/CpG 7909 Phase III, sc Prematurely closed
Paclitaxel
Carboplatin
Phase II, sc Prematurely closed
bevacizumab
/ CpG 7909
Erlotinib
/ CpG 7909 Phase II, sc On-going
Erlotinib
Bevacizumab
IMO Phase I, sc On-going
2055
Melanoma CpG 7909
/ DTIC Phase II (184), intratumoral Completed (4 PR/92 in combination 70
arm)
Breast Cancer CpG 7909+ Docetaxel Phase II Prematurely closed
Colorectal cancer IMO 2055+ Irinotecan
Cetuximab Phase I, sc On-going
CpG-ODN FOR GLIOMA IMMUNOTHERAPY
1018 ISS+ Irinotecan

Cetuximab Phase I, sc On-going
CpG combined with radiotherapy
Recurrent Lymphomas CpG 7909 Phase I, intratumoral On-going
Recurrent mycosis Phase I, intratumoral On-going
fungoides
CpG combined with monoclonal antibody
Non-Hodgkins Lymphoma CpG 7909+ Rituxan Phase I (50), IV or sc Completed (24% response) 59
CpG 7909+ Rituximab
Phase I, sc On-going
Ibritumomab Tiuxetan
1018 ISS+ Rituxan Phase I (20), sc Completed (Induction of Th 1 72
cytokines )
1018 ISS+ Rituxan Phase II, sc On-going
Breast cancer CpG 7909+ Herceptin Phase I/II, sc Completed
continued on next page
101
102
Table 1. Continued
CpG in vaccines
CpG 7909+ MART1 peptide Phase I (8), sc Completed (no response) 55
CpG 7909+ Montanide
ISA 51 VG Phase I, sc On-going
Melanoma CPG 7909+ Melanoma Antigen
Montanide
CpG 7909+ MAGE-3.A1 Phase I, sc On-going
Cancers expressing CpG 7909
NY-ESO-1
Montanide Phase I (8), sc On-going (no response) 56
NY-ESO-1
CpG 7909+Montanide
/Cyclo- Phase I, sc On-going
phosphamide
/ NY-ESO-1
Esophageal cancer CpG 7909 + URLC10-177
TTK-567
Montanide
Prostate cancer CpG 7909 + NY-ESO-1 Phase I, sc On-going
Breast cancer MUC1/HER-2/Neu Peptide +/- CpG Phase I, sc On-going
sc: subcutaneous; IV: intravenous; MR: minor responses; PR: partial response; SD: stable disease; pDC: plasmacytoid dendritic cells; BCC: basal cell
carcinoma; MM: metastatic melanoma.
Figure 2. Strong line: Overall survival in patients with recurrent glioblastoma treated with local
treatment with CpG-ODN (updated survival data from the Phase I trial). Dash line: Overall survival in
an historical control group of patients from the same institution, matched one-to-one with age, KPS,
number of previous chemotherapy and time from first diagnosis to treatment.
A multicenter single-blinded randomized Phase 2 trial was simultaneously launched

in newly diagnosed GBM after surgical resection. The rationale for this approach
was to treat the patient at a time when the tumor mass is minimal and before the
patients become immuno-compromised by chemotherapy. Patients underwent large
surgical resection and CpG-28 was administrated (or not, depending on the arm)
locally around the surgical cavity. The patients were then treated with concomitant
radiotherapy and temozolomide. The primary objective of our clinical trial is 2-year
survival. As of October 2009, 80 patients with histologically documented glioblastoma
were enrolled in 7 French centers. Inclusions are temporarily closed, waiting for the
interim analysis. Depending on the results of this analysis which is now on-going,
the inclusions will be definitely closed or extended. Preliminary results should be
available by September 2009.
Finally, a Phase 1 trial is ongoing in carcinomatous meningitis. Neoplastic
meningitis is a devastating disease, with no efficient therapy available. The objective
of this dose-escalating trial is to assess tolerance of intrathecal and subcutaneous
injection of CpG-28. So far, 12 patients with neoplastic meningitis have been enrolled
in the study with subcutaneous injections ranging from 0.15 to 0.3 mg/kg/week and
intrathecal injections of 3 to 7 mg every other week. Adverse events were limited to
fever in a few patients. In patients who were treated with subcutaneous injections only,
no significant improvement in clinical or radiological symptoms was seen. In three
patients who were treated with both subcutaneous and intrathecal administrations,
clinical improvement was observed. However these patients received concomitant
treatment with spinal radiotherapy or systemic chemotherapy which might have
impacted the outcome.
PERSPECTIVES IN CLINICAL TRIALS
CpGODNs Combined with Radiotherapy (RT) or Chemotherapy
Tumor radiation up-regulates expression of inflammatory mediators, induces secretion

of inflammatory cytokines and can trigger antitumor immune responses,44 thus providing
a strong rationale for combined RT and treatment with CpG-ODN. In a murine glioma
model, promising results were also obtained when RT was associated with intratumoral
injections of CpG-ODN.45 Similar results were obtained in other tumor models.46-47 A
clinical trial combining CpG-ODNs with radiotherapy in recurrent low-grade lymphoma
and mycosis fungoides is on-going.
Similarly, several preclinical models have shown that chemotherapy can be
advantageously combined to CpG-ODN, including in gliomas, probably because they
release tumor antigens, hence triggering a more efficient antitumor response.23,48-51 Several
clinical trials are on-going in breast, colorectal cancer and lung cancer.
CpG-ODNs Combined with Antigens, Antibodies or Dendritic Cells
When a tumor antigen has been identified, vaccination protocols can be designed
using CpG-ODN combined with the purified antigen or with dendritic cells loaded with
this antigen.52-53 In glioma-bearing mice for example, successful immunization with DCs
loaded with glioma cell lysate was reported.54 Several Phase 1 trials have been launched,
mainly in melanoma (but not in human glioma), leading to occasional reports of CD8
T-cell responses.55-57 Epidermal growth factor receptor (EGFR), which is frequently
truncated in glioblastoma (EGFRv3), represents a potential tumor antigen for GBM, but
so far, no trials are combining CpG and EGFRv3.
Another potential approach combines CpG-ODNs and monoclonal antibodies
targeted against tumor antigens. CpG-ODNs stimulate NK cells and macrophages, hence
enhancing antibody-dependant cell cytotoxicity (ADCC). A combination of CpG-ODNs
with monoclonal antibodies was effective in preventing tumor growth in the 38C13 B-cell
lymphoma model.58 Clinical trials combining CpG with Rituximab,59 Bevacizumab or
Ibritumomab have been reported or are on-going in non cerebral malignancies (Table 1).
However, the blood-brain barrier impeaches the diffusion of antibodies into the brain,
hence limiting such an approach in brain tumors.
TOLERANCE
In CpG clinical trials performed to date, the most common adverse events were
reversible and consist of local site reactions (erythema), fever, fatigue, arthralgias and
myalgias.60-61 In patients injected locally into the brain tumor, fever and fatigue were
also a common finding. Transient neurological worsening and seizures, observed in
some patients, were probably related to local site reactions in the form of inflammation
seen after subcutaneous injections.38 The combination of CpG treatment with cytotoxic
chemotherapy in patients with NSCLC induced a higher frequency of sepsis like events
leading to early termination of these trials.42
A common unexpected biological event seen in CpG trials is lymphopenia.23,60-62

Lymphopenia seems counterintuitive with a drug that stimulate lymphoproliferation.
This lymphopenia peaked around 4 weeks in glioma trials and concerned equally CD4
and CD8 T-cell subsets. The Treg subset was not clearly modified (personal data).
The interpretation of these lymphopenia remains unclear and might be related to a
margination process.
The main concern about CpG-ODN, given their powerful immune effects, is
auto-immune diseases. Such activation of auto-immunity has been reported in some
animal models.63-64 First results obtained in clinical trials with CpG-ODN were reassuring,
showing sometimes a slight increase of anti-dsDNA antibodies in melanoma38,61 and
one case of Sjogrens syndrome.59 However, a recent case of Wegener disease in a
healthy patient included in a trial for hepatitis B vaccine with CpG-ODN aroused serious
concerns and led to premature stop of all clinical trials for CpG-ODNs with vaccines
in healthy volunteers. In cancer patients, the situation is clearly different, given the
prognosis of these patients and the higher benefit/risk ratio. However, careful monitoring
of autoimmunity is warranted.
CONCLUSION
In summary, the clinical trials using CpG-ODN therapy as a single agent in cancer
showed a reasonable safety profile, but the frequency of objective responses has been
relatively low so far. Ongoing clinical trials have therefore shifted to combination therapies
in an attempt to increase the clinical effectiveness of TRL9 agonists.
In addition, further studies should identify subgroups of patients that could benefit
the most from CpG, based either on individual sensitivity to CpG, as already reported in
clinical trials with CpG,65-66 or on the characteristics of the antitumor immune response
in each patient.
REFERENCES
1. Tokunaga T, Yamamoto H, Shimada S et al. Antitumor activity of deoxyribonucleic acid fraction from
Mycobacterium bovis BCG. I. Isolation, physicochemical characterization and antitumor activity. J Natl
Cancer Inst 1984; 72:955-962.
2. Kuramoto E, Watanabe N, Iwata D et al. Changes of host cell infiltration into Meth A fibrosarcoma tumor
during the course of regression induced by injections of a BCG nucleic acid fraction. Int J Immunopharmacol
1992; 14:773-782.
3. Krieg AM, Yi AK, Matson S et al. CpG motifs in bacterial DNA trigger direct B-cell activation. Nature 1995;
374:546-549.
4. Zhao Q, Temsamani J, Iadarola PL et al. Effect of different chemically modified oligodeoxynucleotides on
immune stimulation. Biochem Pharmacol 1996; 51:173-182.
5. Cornlie S, Hoebeke J, Schacht AM et al. Direct evidence that toll-like receptor (TRL9) functionally binds plasmid
DNA by specific cytosine-phosphate-guanine motif regognition. J Biol Chem 2004; 279(15):15124-15129.
6. Medzhitov R, Preston-Hurlburt P, Janeway CA. A human homologue of the Drosophila Toll protein signals
activation of adaptive immunity. Nature 1997; 388:394-397.
7. Leifer CA, Kennedy MN, Mazzoni A et al. TRL9 is localized in the endoplasmic reticulum prior to stimulation.
J Immunol 2004; 173(2):1179-1183.
8. Latz E, Verma A, Visintin A et al. Ligang-induced conformational changes allosterically activate Toll-like
receptor 9. Nat Immunol 2007; 8(7):772-779.
9. Hornung V, Rothenfusser S, Britsch S et al. Quantitative expression of toll-like receptor 1-10 mRNA in cellular
subsets of human peripheral blood mononuclear cells and sensitivity to CpG oligodeoxynucleotides. J
Immunol 2002; 168:4531-4537.
10. Henault M, Lee LN, Evans GF et al. The human Burkitt lymphoma cell line Namalwa represents a homogenous
cell system characterized by high levels of Toll-like receptor 9 and activation by CpG oligonucleotides. J
Immunol Methods 2005; 300:93-99.
11. Jego G, Bataille R, Geffroy-Luseau A et al. Pathogen-associated molecular patterns are growth and survival
factors for human myeloma cells through Toll-Like receptors. Leukemia 2006; 20(6):1130-1137.
12. Vollmer J, Krieg AM. Immunotherapeutic applications of CpG oligodeoxynucleotide TLR9 agonists. Adv
Drug Deliv Rev 2009; 61(3): 195-204.
13. Krieg AM. CpG motifs in bacterial DNA and their immune effects. Annu Rev Immunol 2002; 20:709-760.
14. Stacey KJ, Sweet MJ, Hume DA. Macrophages ingest and are activated by bacterial DNA. J Immunol 1996;
157:2116-2122.
15. Klinman DM, Yi AK, Beaucage SL et al. CpG motifs present in bacteria DNA rapidly induce lymphocytes to
secrete interleukin 6, interleukin 12 and interferon gamma. Proc Natl Acad Sci USA 1996; 93:2879-2883.
16. Sparwasser T, Koch ES, Vabulas RM et al. Bacterial DNA and immunostimulatory CpG oligonucleotides
trigger maturation and activation of murine dendritic cells. Eur J Immunol 1998; 28:2045-2054.
17. Cho HJ, Takabayashi K, Cheng PM et al. Immunostimulatory DNA-based vaccines induce cytotoxic lymphocyte
activity by a T-helper cell-independent mechanism. Nat Biotechnol 2000; 18:509-514.
18. Klinman DM, Verthelyi D, Takeshita F et al. Immune recognition of foreign DNA: a cure for bioterrorism?
Immunity 1999; 11:123-129.
19. Kline JN, Waldschmidt TJ, Businga TR et al. Modulation of airway inflammation by CpG oligodeoxynucleotides
in a murine model of asthma. J Immunol 1998; 160:2555-2559.
20. Vollmer J, Weeranta R, Payette P et al. Characterization of three CpG oligodeoxynucleotide classes with
distinct immunostimulatory activities. Eur J Immunol 2004; 34(1):251-262.
21. Hartmann G, Weeratna RD, Ballas ZK et al. Delineation of a CpG phosphorothioate oligodeoxynucleotide
for activating primate immune responses in vitro and in vivo. J Immunol 2000; 164:1617-1624.
22. Bauer S, Kirschning CJ, Hacker H et al. Human TLR9 confers responsiveness to bacterial DNA via
species-specific CpG motif recognition. Proc Natl Acad Sci 2001; 98:9237-9242.
23. Carpentier AF, Auf G, Delattre JY. CpG-oligonucleotides for cancer immunotherapy: review of the literature
and potential applications in malignant glioma. Front Biosci 2003; 8:115-127.
24. Dix AR, Brooks WH, Roszman TL et al. Immune defects observed in patients with primary malignant brain
tumors. J Neuroimmunol 1999; 100:216-232.
66(6):3294-3302.
26. El Andaloussi A, Sonabend AM, Han Y et al. Stimulation of TLR9 with CpG ODN enhances apoptosis of
glioma and prolongs the survival of mice with experimental brain tumors. Glia 2006; 54:526535.
27. Curiel TJ, Coukos G, Zou L et al. Specific recruitment of regulatory T-cells in ovarian carcinoma fosters
immune privilege and predicts reduced survival. Nat Med 2004; 10:942-949.
28. Golgher D, Jones E, Powrie F et al. Depletion of CD25
regulatory cells uncovers immune responses to shared
murine tumor rejection antigens. Eur J Immunol 2002; 32:3267-3275.
29. Carpentier AF, Chen L, Maltonti F et al. Oligodeoxynucleotides containing CpG motifs can induce rejection
of a neuroblastoma in mice. Cancer Res 1999; 59:5429-5432.
30. Auf G, Carpentier AF, Chen L et al. Implication of macrophages in tumor rejection induced by
CpG-oligodeoxynucleotides without antigen. Clin Cancer Res 2001; 7:3540-3543.
31. Carpentier AF, Xie J, Mokhtari K et al. Successful treatment of intracranial gliomas in at by oligodeoxynucleotides
containing CpG motifs. Clin Cancer Res 2000; 6:2469-2473.
32. Grauer OM, Molling JW, Bennink E et al. TRL ligands in the local treatment of established intracerebral
murine gliomas. J Immunol 2008; 181(10):6720-6729.
33. Ilvesaro JM, Merrell MA, Li L et al. Toll-like receptor 9 mediates CpG oligonucleotideinduced cellular
invasion. Mol Cancer Res 2008; 6(10):1534-1543.
34. Kawarada Y, Ganss R, Garbi N et al. NK- and CD (
) T-cell-mediated eradication of established tumors
by peritumoral injection of CpG-containing oligodeoxynucleotides. J Immunol 2001; 167(9):5247-5253.
35. Wooldridge JE, Ballas Z, Krieg AM et al. Immunostimulatory oligodeoxynucleotides containing CpG motifs
enhance the efficacy of monoclonal antibody therapy of lymphoma. Blood 1997; 89(9):2994-2998.
36. Takeshita S, Takeshita F, Haddad DE et al. Activation of microglia and astrocytes by CpG oligodeoxynucleotides.
Neuroreport 2001; 12:3029-3032.
37. Meng Y, Kujas M, Marie Y et al. Expression of TLR9 within human glioblastoma. J Neurooncol 2008; 88:19-25.
38. Carpentier A, Laigle-Donadey F, Zohar S et al. Phase 1 trial of CpG ODN for patients with recurrent
glioblastoma. Neurol Oncol 2006; 8(1):60-66.
39. Gupta K, Cooper C. A review if the role of CpG oligodeoxynucleotides as toll-like 9 agonists in prophylactic
and therapeutic vaccine development in infectious diseases. Drugs R D 2008; 9(3):137-145.
40. Agrawal S, Kandimalla ER. Synthetic agonists of Toll-like receptors 7, 8 and 9. Biochem Soc Trans 2007;
35(Pt 6):1461-1467.
41. Manegold C, Gravenor D, Woytowitz D et al. Randomized phase II trial of a toll-like receptor 9 agonist
oligodeoxynucleotide, PF-3512676, in combination with first-line taxane plus platinum chemotherapy for
advancedstage nonsmall-cell lung cancer. J Clin Oncol 2008; 26(24):3979-3986.
42. Readett DRJ, DenisL, Krieg AM et al. PF-3512676 (CPG 7909), a Toll-like receptor 9 agoniststatus of
development for nonsmall cell lung cancer (NSCLC).(abstract) 12th World Congress on Lung Cancer 2007;
2-6; Seoul, Korea, PD3-1-6.
43. Carson AK, Grossman SA, Fisher JD et al. Prognostic factors for survival in adult with recurrent glioma
enrolled onto the new approaches to brain tumor therapy CNS Consortium phase I and II clinical trials. J
Clin Oncol 2007; 25:2601-2606.
44. Friedman EJ. Immune modulation by ionizing radiation and implications for cancer immunotherapy. Curr
Pharm Des 2002; 8(19):1765-1780.
45. Meng Y, Carpentier AF, Chen L et al. Successful combination of local CpG-ODN and radiotherapy in
malignant glioma. Int J Cancer 2005; 116(6):992-997.
46. Mason KA, Ariga H, Neal R et al. Targeting toll-like receptor 9 with CpG oligodeoxynucleotides enhances
tumor response to fractionated radiotherapy. Clin Cancer Res 2005; 11(1):361-369.
47. Milas L, Mason KA, Ariga H et al. CpG oligonucleotide enhances tumor response to radiation. Cancer Res
2004; 64(15):5074-5077.
48. Weigel BJ, Rodeberg DA, Krieg AM et al. CpG oligodeoxynucleotides potentiate the antitumor effects of
chemotherapy or tumor resection in an orthotopic murine model of rhabdomyosarcoma. Clin Cancer Res
2003; 9(8):3105-3114.
49. Balsari A, Tortoreto M, Besusso D et al. Combination of a CpG-oligodeoxynucleotide and a topoisomerase I
inhibitor in the therapy of human tumor xenografts. Eur J Cancer 2004; 40(8):1275-1281.
50. Pratesi G, Petrangolini G, Tortoreto M et al. Therapeutic synergism of gemcitabine and
CpG-oligodeoxynucleotides in an oythotopic human pancreatic carcinoma xenograft. Cancer Res
2005; 65(14):6388-6393.
51. Roux S, Bernat C, Al-Sakere B et al. Tumor destruction using electrochemotherapy followed by CpG
oligodeoxynucleotide injection induces distant tumor responses. Cancer Immunol Immunother 2008;
57:1291-1300.
52. Hiraoka K, Yamamoto S, Otsuru S et al. Enhanced tumor-specific long-term immunity of hemagglutinating
(correction of hemaggluttinating) virus of Japan-mediated dendritic cell-tumor fused cell vaccination by
coadministration with CpG oligodeoxynucleotides. J Immunol 2004; 173(77):4297-4307.
53. Chamoto K, Takeshima T, Wakita D et al. Combination immunotherapy with radiation and CpG-based
tumor vaccination for the eradication of radio- and immunoresistant lung carcinoma cells. Cancer Sci
2009; 100:934-939.
54. Wu A, Oh S, Gharagozlou S et al. In vivo vaccination with tumor cell lysate plus CpG oligodeoxynucleotides
eradicates murine glioblastoma. J Immunother 2007; 30(8):789-797.
55. Speiser DE, Lienard D, Ruffer N et al. Rapid and strong human CD8
T cell responses to vaccination with
peptide, IFA and CpG oligodeoxynucleotide 7909. J Clin Invest 2005; 115:739-746.
56. Valmori D, Souleimanian NE, Tosello V et al. Vaccination with NY-ESO-1 protein and CpG in Montanide
induces integrated antibody/Th1 responses and CD8 T-cells through cross-priming. Proc Natl Acad Sci
USA 2007; 104(21):8947-8952.
57. Germeau C, Ma W, Schiavetti F et al. High frequency of antitumor T-cells in the blood of melanoma patients
before and after vaccination with tumor antigens. J Exp Med 2005; 201(2):241-248.
58. Warren TL, Dahle CE, WeinerGJ. CpG oligodeoxynucleotides enhance monoclonal antibody therapy of a
murine lymphoma. Clin Lymphoma 2000; 1:57-61.
59. Leonard JP, Link BK, Emmanouilides C et al. Phase I trial of toll-like receptor 9 agonist PF-3512676 with
and following rituximab in patients with recurrent indolent and aggressive non Hodgkins lymphoma. Clin
Cancer Res 2007; 13(20):6168-6174.
60. Hofmann MA, Kors C, Audring H et al. Phase 1 evaluation of intralesionally injected TLR9-agonist PF-3512676
in patients with basal cell carcinoma or metastatic melanoma. J Immunother 2008; 31(5):520-527.
61. Pashenkov M, Goss G, Wagner C et al. Phase II trial of a toll-like receptor 9 oligonucleotide in patients with
metastatic melanoma. J Clin Oncol 2006; 24:5716-5724.
62. Link BK, Ballas ZK, Weisdorf D et al. Oligodeoxynucleotide CpG 7909 delivered as intravenous infusion
demonstrates immunologic modulation in patients with previously treated non-Hodgkins lymphoma. J
Immunother 2006; 29(5):558-568.
63. Ichikawa HT, Williams LP, Segal BM. Activation of APCs through CD40 or Toll-like receptor 9 overcomes
tolerance and precipitates autoimmune disease. J Immunol 2002; 169(5):2781-2787.
64. Krieg AM, Vollmer J. Toll-like receptors 7, 8 and 9 linking innate immunity to autoimmunity. Immunol Rev
2007; 220:251-269.
65. Cooper CL, Davis HL, Morris ML et al. Safety and immunogenicity of CPG 7909 injection as an adjuvant
to Fluarix influenza vaccine. Vaccine 2004; 22:3136-3143.
66. Krieg AM. Therapeutic potential of Toll-like receptor 9 activation. Nat Rev Drug Discov 2006; 5:471-484.
67. Molenkamp BG, van Leeuwen PA, Meijer S et al. Intradermal CpG-B activates both plasmacytoid and myeloid
dendritic cells in the sentinel lymph node of melanoma patients. Clin Cancer Res 2007; 13(10):2961-2969.
68. Kim Y, Girardi M, McAuley S et al. Cutaneous T-cell lymphoma (CTCL) responses to a TLR9 agonist CPG
immunomodulator (CPG 7909), a phase I study. J Clin Oncol 2004; 22:582s (suppl; abstr 6600).
69. Thompson JA, Kuxel T, Bukowski F et al. Phase Ib trial of a targeted TLR9 CpG immunomodulator (CPG
7909) in advanced renal cell carcinoma (RCC). J Clin Oncol 2004; 22:417s (suppl; abstr 4644).
70. Wagner S, Weber J, Redman B et al. CPG 7909, a TLR9 agonist immunomodulator in metastatic melanoma:
A randomized phase II trial comparing two doses and in combination with DTIC (abstract). Proc Am Soc
Clin Oncol, 2005 ASCO Annual Meeting Proceedings. 23:7526.
71. Friedberg JW, Kim H, McCauley M et al. Combination immunotherapy with a CpG oligonucleotide (1018
ISS) and rituximab in patients with non-Hodgkin Lymphoma: increased interferon-alpha/beta-inducible
gene expression, without significant toxicity. Blood 2005; 105(2):489-495.
CHAPTER 9
ADOPTIVE CELL TRANSFER THERAPY

FOR MALIGNANT GLIOMAS
Eiichi Ishikawa, Shingo Takano, Tadao Ohno and Koji Tsuboi*

Proton Medical Research Center, Graduate School of Comprehensive Human Sciences, University of Tsukuba,
Tsukuba, Japan
*Corresponding Author: Koji TsuboiEmail: tsuboi@pmrc.tsukuba.ac.jp
Abstract: To date, various adoptive immunotherapies have been attempted for treatment of
malignant gliomas using nonspecific and/or specific effector cells. Since the late
1980s, with the development of rIL-2, the efficacy of lymphokine-activated killer
(LAK) cell therapy with or without rIL-2 for malignant gliomas had been tested with
some modifications in therapeutic protocols. With advancements in technology, ex
vivo expanded tumor specific cytotoxic T-lymphocytes (CTL) or those lineages were
used in clinical trials with higher tumor response rates. In addition, combinations
of those adoptive cell transfer using LAK cells, CTLs or natural killer (NK) cells
with autologous tumor vaccine (ATV) therapy were attempted. Also, a strategy of
high-dose (or lymphodepleting) chemotherapy followed by adoptive cell transfer
has been drawing attentions recently. The most important role of these clinical
studies using cell therapy was to prove that these ex vivo expanded effector cells
could kill tumor cells in vivo. Although recent clinical results could demonstrate
radiologic tumor shrinkage in a number of cases, cell transfer therapy alone has
been utilized less frequently, because of the high cost of ex vivo cell expansion,
the short duration of antitumor activity in vivo, and the recent shift of interest to
vaccine immunotherapy. Nevertheless, NK cell therapy using specific feeder cells
or allergenic NK cell lines have potentials to be a good choice of treatment because
of easy ex vivo expansion and their efficacy especially when combined with vaccine
therapy as they are complementary to each other. Also, further studies are expected
to clarify the efficacy of the high-dose chemotherapy followed by a large scale cell
transfer therapy as a new therapeutic strategy for malignant gliomas.

109
INTRODUCTION
The identification and functional characterization of a cytokine, interleukin-2

(IL-2) and the subsequent development of recombinant IL-2 (rIL-2) has enabled
us to expand cultures of lymphocytes ex vivo.1 Cultivation of the idea to utilize ex
vivo-expanded lymphocytes to kill tumors as an adoptive cell transfer therapy is thus
straightforward. In fact, the adoptive transfer of immunologically-activated effector
cells is thought to be one of the breakthrough treatments for incurable malignancies,
such as high-grade gliomas.
The adoptive cell transfer therapies that have been developed so far can be divided
into two categories: (1) nonspecific cell therapies which use either lymphokine-activated
killer (LAK) cells, natural killer (NK) cells, or natural killer T (NKT) cells and (2)
tumor-specific lymphocytes, which include either cytotoxic T-lymphocytes (CTL),
autologous tumor specific T-lymphocytes (ATTL), or tumor-infiltrated lymphocytes
(TIL). Pure CTL are considered a final effector which play a crucial role in tumor cell
killing in a major histocompatibility complex (MHC) class I restricted manner (Fig. 1).
ATTL are a mixture of CTL and helper T cells (CD4 Th) and TILs are considered to
be a mixture of CTL, Th, NK cells and regulatory T cells (Treg). These immunologic
cell transfer therapies are summarized and illustrated in Table 1 and Figure 1.
To date, various adoptive immunotherapies2-6 have been attempted for malignant
gliomas using both nonspecific (Table 2) and specific cells (Table 3) since the
milestone report of Rosenberg et al was published in 1985.7 Herein, we describe the
immunologic effector cell transfer therapies as a further prospective clinical strategy
for high grade gliomas.
Figure 1. Major effectors which play important roles in tumor immunotherapy. NK and NKT cells
are nonspecific effectors. The tumor-specific immunoreaction starts with incorporation of degraded
tumor tissue containing tumor-associated antigens into antigen-presenting dendritic cells. These
TAAs are presented in a MHC class II-restricted manner on their surface and are recognized by
CD4-positive helper T cells which sequentially activate the final effectors CD8-positive killer T
cells called cytotoxic T-lymphocytes (usually called CTL). In a narrow sense, CTL refers to only
CD8-positive killers. Autologous tumor-specific T-lymphocytes (ATTL) refer to a mixture of CD4
helpers and CD8 killers.
ADOPTIVE CELL TRANSFER THERAPY 111
Table 1. Adoptive cell transfer therapies

Nonspecific Cell Therapy Tumor Specific Cell Therapy
LAK Cell NK Cell
Effector Therapy Therapy TIL Therapy CTL or ATTL Therapy
Source Peripheral blood Peripheral Fresh tumor Peripheral blood or
blood tissue lymphnode cells (after
in vivo vaccine)
Culture IL-2 IL-2 IL-2 IL-2 (
1, 4, 6)
Target Raji, Daudi K-562, HFWT Autologous Autologous tumor cells
tumor cells
Specificity None None Autologous Autologous tumor cells
tumor cells
Major CD3
CD4
CD16
CD56
CD3
CD4
CD3
CD4
including Th
phenotype CD3 including Th
(minor CD3
CD8
(CD3
CD56) CD3
CD8
CD3
CD8
including CTL
phenotype) including
CTL
(CD56
CD3) (CD56
CD3) (CD3
CD4
CD25high)
(CD3
CD4
(CD3
CD4
CD25high) CD25high)
NONSPECIFIC IMMUNE CELL THERAPY
LAK cell therapy usually involves the focal or systemic injection of activated cells
from autologous peripheral blood mononuclear cells (PBMCs) cultured in vitro with
rIL-2, which contains a major fraction of nonspecific activated T cells and a minor
fraction of activated NK cells.
Since the late 1980s, several authors have reported the safety and efficacy of
LAK cell therapy in patients with malignant gliomas.8-16 In 1986, Jacobs et al11 first
reported 6 patients with malignant gliomas treated with the locally infused LAK cells.
Following this report, Yoshida et al15 reported 23 malignant brain tumor cases treated
with LAK and IL-2. Around the same time, Okamoto et al14 used LAK cells and IL-2 via
intrathecal (i.t.) injections for the treatment of cerebrospinal fluid (CSF) dissemination
of a medulloblastoma. This application was very reasonable to achieve the direct contact
of effectors and target tumor cells in the disseminated condition, which is very close to
in vitro coculture and the results were favorable. In 2001, Hayes et al10 reported that 6
of 28 patients with high grade primary brain tumors had long-term survival (2 years)
postreoperation with the focal injection of low doses of IL-2 and LAK cells. They
concluded that this cell therapy was well-tolerated and should be considered an option
for patients with high-grade tumors refractive to standard therapeutic approaches.10
Focal LAK therapy for 40 glioblastoma multiforme (GBM) cases verified the safety and
feasibility of the therapy. In this study, a median overall survival (mOS) from the date
of original diagnosis for 31 patients who had GBM at the time of initial diagnosis was
17.5 months versus 13.6 months for a control group of 41 contemporary GBM patients.8
112
Table 2. Clinical trials of nonspecific cell therapy for malignant gliomas

Author Year Patients Effecter Type Administration Toxicity Response
1 Jacobs 1986 6 LAK (

IL-2) Direct Tolerable (no toxicity) <
2 Okamoto 1988 6 dissemi- MB LAK
IL-2 i.t. Tolerable (no toxicity) 3 resp.
3 Merchant 1988 13 LAK
IL-2 i.c. IICP, headache, fever, etc No benefit in survival
4 Yoshida 1988 23 LAK
IL-2 i.c. Tolerable (fever) 6 resp.
5 Barba 1989 9 LAK
HD-IL-2 i.c. Edema in 9 cases 1PR
6 Lillehei 1991 20 (11) LAK/ASL
IL-2 i.c. Tolerable No benefit in survival
7 Blancher 1993 13 Cont. IL-2
/< LAK i.c. Fever, confusion, edema <
8 Boiardi 1994 9 Adherent LAK
i.c. Tolerable 1CR, 2PR, 4SD, 2PD RR:
IL-2 33%
9 Hayes 1995 19 LAK
IL-2 i.c. Tolerable 1CR, 2PR, increase in
survival
10 Sankhla 1996 10 LAK
IL-2 i.c. or i.t. Tolerable 2PR
11 Hayes 2001 28 LAK
IL-2 i.c. Tolerable Increase in survival
12 Dillman 2004 40 GBM LAK (
IL-2) i.c. Tolerable Increase in survival
13 Ishikawa 2004 9 NK i.c. or i.v. Tolerable 3PR, 2MR, 4NC, 7PD
Abbreviations: dissemi-MB: medulloblastoma with CSF dissemination; ASL: autologous stimulated lymphocyte; cont. IL-2: continuous perfusion of IL-2; HD-IL-2:
high dose IL-2; resp.: response cases; direct: direct injection into the tumor tissue; i.t.: intrathecal; i.c.: intracavital; i.v.: intravenous; i.a.: intra-arterial; SD: stable
disease; CR: complete response; PR: partial response; MR: minor response; PD: progressive disease.
Modified protocols of LAK cell therapy have been tried by several research
groups. Combination therapy with high-dose IL-2 and LAK cells resulted in a partial
tumor response in 1 of 9 patients and neurologic side effects in all the patients.17 In a
continuous intracerebral perfusion by an implanted catheter of IL-2 with and without
LAK cells of recurrent GBM, tumor progression was diagnosed in all cases, although
several toxicities, including brain edema, were observed.18 Boiardi et al19 reported that
the focal injection of autologous adherent LAK cells and IL-2 in 9 cases with recurrent
GBM was well-tolerated and the response rate was 33%, although the mOS was not
prolonged significantly.19
It is known that NK cells have strong cytotoxic activity against malignant tumor
cells.20 The function of NK cells is partially compensatory for the cytotoxic function of
CTL against tumor cells via MHC class I molecules, while NK cells play an important
role in cellular immune responses in cooperation with dendritic cells (DCs), CTL and
other cell types. NK cell therapy is defined as the clinical injection of NK cell-rich effector
cells derived from PBMCs. However, the clinical application of the therapy has been
reported in only a limited number of cases in malignant brain tumors, as well as other
neoplasms.21,22 The major reason for this is that enrichment and large-scale expansion of NK
cells has been difficult. Recently, we developed a simple method of NK cell expansion23
and subsequently reported a clinical trial of NK cell therapy for patients with malignant
gliomas.22 We first expanded NK cells from autologous PBMCs by coculturing with an
irradiated human feeder cell line (HFWT) using rIL-2. Then, the high killing activity of
the harvested NK cells was confirmed and the NK cells were injected intravenously and/or
intracranially into 9 patients (16 courses) with recurrent malignant gliomas in this study.
The clinical evaluation demonstrated [3 partial response (PR), 2 minor response (MR),
4 no change (NC) and 7 progressive disease (PD)] in a total of 16 courses of treatment.
Severe neurologic toxicity was not observed in any of the patients.
Based on these results, we have initiated a Phase I/IIa trial with activated NK cell
therapy (Table 4). In the current study, activated NK cells harvested by the above-mentioned
method were injected through peripheral veins (intravenously (i.v.)). Until December
2008, the treatments were administered to 6 patients with recurrent malignant gliomas
(2 patients with GBM and 4 patients with Grade III anaplastic gliomas). The mean age
was 50 years and ranged from 32-65 years. Two patients were males. Radiotherapy with
a total dose of 61.264.8 Gy and several chemotherapy regimens were administered to
all patients. The NK cell therapies were applied for 4 patients with 1st recurrent tumors,
1 patient with a 2nd recurrence and 1 patient with a 3rd recurrence. The mean follow-up
period after NK therapy was 20.8 months, ranging from 5-48 months. Four patients
received one injection and two patients received two injections. Clinical evaluation
demonstrated one CR (Fig. 2) and one long-NC in six cases. Neurologic toxicity after
NK cell injection was observed in one patient causing middle cerebral artery (MCA)
stenosis and one patient with a skin eruption.
TUMOR-SPECIFIC IMMUNE CELL THERAPY
Among several immune cells associated with acquired immunity, CTL have been
considered to play a crucial role in tumor rejection in vivo (Fig. 1). CTL are defined as
CD8
T-lymphocytes that kill tumor cells specifically in a MHC class I-restricted manner.
Indeed, there are many animal studies showing that immunologic cell therapy against
Figure 2. Magnetic resonance (MR) images before (A) and after (B) ATTL therapy in a 53-year-old
man with a glioblastoma are shown. After local injection of 3.5 = 107ATTL in 3 fractions, the enhanced
area decreased 50%.
gliomas via CTL is very effective. Moreover, recent analyses indicate that tumor-specific
CD4
T-lymphocytes are not only capable of killing tumor cells, but also of playing an
important role in CTL-mediated tumor cell killing.24 Therefore, the global scheme of
adoptive immunotherapy using ATTL, consisting of CD8
and CD4
, is straightforward.
A limited number of clinical trials using CTL or ATTL have been reported.25-28 Kruse
et al25 reported five patients with malignant gliomas treated by intracavitary (i.c.)
alloreactive CTL and IL-2 and they were able to stabilize tumor growth in three patients
with Grade III gliomas. Quattrocchi et al26 reported a pilot study involving the local
injection of autologous TIL combined with a low dose of adjuvant chemotherapy on
six cases of recurrent malignant gliomas with over-all responses being one CR, two PR
and three PD.26 We demonstrated a method to expand autologous ATTL ex vivo from
PBMC using autologous GBM cells without pre-injection of tumor vaccine in 199629
and a clinical trial of i.c. or i.t. injections of ATTL expanded in the same method was
carried out.27,28 In this pilot study, there were 1 CR and 4 PR in 10 cases with high-grade
gliomas without intolerable toxicity (Table 3). A patient with a GBM which showed
a PR after i.c. injections of ATTL and a patient with an anaplastic oligoastrocytoma
(AOA) which showed marked improvement in spinal dissemination after i.t. injections
are demonstrated in Figures 3 and 4.
A combination of autologous irradiated tumor vaccine (ATV) and specific cell
therapy using LAK/ATTL from PBMC or lymph nodes was attempted on patients with
malignant gliomas.30-33 Plautz et al31 treated 12 cases of newly diagnosed gliomas using
Table 3. Clinical trials of tumor specific cell therapy for malignant gliomas
Author Year Patients Effecter Type Administration Toxicity Response
1 Holladay 1996 15 ATV

LAK/ATTL i.v. Torelable (fever, nausea) -
2 Kruse 1997 5 Alloreactive CTL i.c. Torelable 3 resp.
(Grade 1-3 including nausea)
3 Quattrocchi 1999 6 TIL
IL-2 i.c. Torelable 1CR, 2PR, 3PD
(Grade 1-2 including edema)

4 Wood 2000 9 ATV
LAK/ATTL i.v. or i.a. Torelable 2 resp.
(Fever, nausea) 7 nonresp
5 Sloan 2000 19 ATV
LAK/ATTL i.v. Torelable 1CR, 7PR,
(Grade 1 including muscle spasm) 9SD, 2PD
6 (Plautz) (1998) ATV
LAK/ATTL i.v. Torelable 4PR, 2SD,
7 Plautz 2000 12 From draining LN cells (Grade 1-2 including myalgia) 5PD
8 (Tsurushima) (1999) ATTL with no ATV i.c. Torelable 1CR, 4PR,
9 Tsuboi 2003 10 (Grade 1-2 including fever) 3SD, 2PD
Abbreviations: ATV: autologous tumor vaccine; resp.: response cases; nonresp: nonresponse cases.
115
Figure 3. MR images before (A) and after (B) ATTL therapy in a 23-year-old woman with spinal
dissemination of an anaplastic oligoastrocytoma are shown. After intrathecal injection of 47.2 = 107
ATTL in 3 fractions, the dissemination decreased markedly with significant clinical improvement.
Figure 4. MR images before (A) and after (B) 2 courses of NK cell therapy in patient No. 1 (anaplastic
oligodendroglioma; Table 4) are shown.
Table 4. Summary of recurrent malignant gliomas treated with NK cell therapy (Phase I/IIa tial, on going)
Injected NK Response
Case No. Cells CD3< Activity (Evaluated mos Follow-Up Prognosis
No. Age Sex Pathology of Tx (x104) CD56
(%) (%) Complications from Tx) (mos) (KPS%)
1 46 F AO 2 6.5 83.1 85.8 MCA stenosis SD

12.9 85.3 75.7 None CR (13 m) 48 CR (70%)
2 32 M GBM 1 19.3 59.0 100.0 None Long SD (12 m) 12 SD (60%)

3 59 F AOA 1 7.3 72.8 71.4 None SD (4 m) 12 Dead
4 44 F AA 2 30.5 90.8 81.7 None SD
29.5 88.7 100.0 None PD (3 m) 25 Dead
5 65 M AO 1 9.3 89.1 100.0 None PD (4 m) 5 Dead
6 55 F GBM 1 31.5 68.1 82.1 Skin eruption PD (2 m) 5 Dead
Abbreviations: AO: anaplastic oligodendroglioma; GBM: glioblastoma multiforme; AOA: anaplastic oligoastrocytoma; AA: anaplastic astrocytoma; MCA: middle
cerebral artery; KPS: Karnofsky performance score; Tx:Therapy; mos: months.
117
LAK/ATTL (71% of which were CD 4

T cells) expanded from inguinal lymph nodes
after injection of ATV and a PR was observed in 4 of 8 cases with residual tumor.
Some authors have reported a few cases treated with a combination of high-dose (or
lymphodepleting) chemotherapy, followed by the adoptive transfer for brain tumors.34,35
Dudley34 reported a combination of lymphodepleting chemotherapy followed by the
adoptive transfer of TIL for the treatment of 35 patients with refractory metastatic
melanoma. In this study, 18 patients, including a patient with brain metastases,
experienced objective clinical responses.34
CONCLUSION
Further Clinical Strategy
The most important role of these clinical studies using cell therapy was to prove that
these effecter cells could decrease tumor volume in in vivo as well as ex vivo studies.
LAK cell therapy alone is not considered very efficient for clinical use at present, possibly
because of its low killing activity and nonspecificity, while some studies of NK cell and
TIL/CTL/ATTL therapies support the feasibility of these therapies and some degree of
effectiveness for radiologic tumor response. However, in these cell therapies, no study has
accurately proven prolongation of the mOS in glioma patients. A Phase III randomized
study with a large number of patients will be essential.
On the other hand, cell therapies have two common major problems: (1) difficulty of
ex vivo cell expansion and expense and (2) the antitumor effect is temporary because the
transferred cells frequently fail to engraft and persist. Although several authors, including
us, have simplified the expansion method in innate immune cell and acquired immune
cell therapies, the difficulty cannot be overcome completely. As for innate immune cell
therapy, NK cell therapy using specific feeder cells23 or allogeneic NK cell lines21 may be
a suitable strategy for easier expansion and numerous injections for prolongation of the
reaction span. Regarding the combination of NK and other therapies, radiation therapy36,37
and anti-angiogenic agents, as well as vaccine therapy,38 are promising candidates and
might be tried in clinical studies.
For the treatment of lung cancer patients, another cell therapy using Valpha24 natural
killer T (V_24 NKT) cells has been tried.39 The V_24 NKT cells may also be used regardless
of personal restrictions because the cells bearing an invariant V_24J_Q antigen receptor
are activated by a glycolipid ligand, _-galactosylceramide, in a CD1d-dependent manner
independent of personal antigens or MHC-I. The activated human V_24 NKT cells have
been shown to produce large amounts of cytokines, exert potent killing activity against
various tumor cell lines and stimulate several immune cells, including DC and NK cells.
Such therapy might be a new strategy for innate therapy of gliomas.
With respect to acquired immune therapy, most researchers, including us, have been
attracted to vaccine therapies,40,41 including a formalin-fixed tumor tissue vaccine,42 a
peptide-based vaccine41 and DC-based vaccines,43,44 rather than effector cell therapy. The
main reason for such an approach is that ex vivo cell expansion is relatively complicated
and expensive and the antitumor effect is temporary, despite the high response ratio in
most studies of CTL/ATTL therapies, including our experience, as shown in Table 3.
However, in these classic effector cell therapies, high-dose chemotherapy, followed
by cell therapies,34,35 will be new strategies of cell therapy because chemotherapies may
enhance immunotherapies by promoting homeostatic proliferation/activation of transferred
cells experimentally.45 Moreover, it has been reported that immunotherapy followed by
chemotherapy can significantly increase survival in malignant glioma patients in several
studies using a DC vaccine.46 Ex vivo gene delivery strategies to introduce a T-cell receptor
with known specificity and affinity against a particular tumor antigen into transferred T
cells is a promising candidate.47
In conclusion, classical cell therapy has some issues, including a short duration
of treatment response and the recent interest in vaccine therapy. However, some new
strategies, including high-dose chemotherapies followed by cell therapies, have attracted
clinical attention.
REFERENCES
1. Grimm EA, Mazumder A, Zhang HZ et al. Lymphokine-activated killer cell phenomenon. Lysis of natural
killer-resistant fresh solid tumor cells by interleukin 2-activated autologous human peripheral blood
lymphocytes. J Exp Med 1982; 155(6):1823-1841.
2. Carpentier AF, Meng Y. Recent advances in immunotherapy for human glioma. Curr Opin Oncol 2006;
18(6):631-636.
3. Jaeckle KA. Immunotherapy of malignant gliomas. Semin Oncol 1994; 21:249-259.
4. Kushen MC, Sonabend AM, Lesniak MS. Current immunotherapeutic strategies for central nervous system
tumors. Surg Oncol Clin N Am 2007; 16:987-1004, xii.
5. Mitchell DA, Fecci PE, Sampson JH. Immunol Rev 2008; 222:70-100.
6. Parney IF, Hao C, Petruk KC. Glioma immunology and immunotherapy. Neurosurgery 2000; 46:778-791.
7. Rosenberg SA, Lotze MT, Muul LM et al. Observations on the systemic administration of autologous
lymphokine-activated killer cells and recombinant interleukin-2 to patients with metastatic cancer. N Engl
J Med 1985; 313:1485-1492.
8. Dillman RO, Duma CM, Schiltz PM et al. Intracavitary placement of autologous lymphokine-activated killer
(LAK) cells after resection of recurrent glioblastoma. J Immunother 2004; 27:398-404.
9. Hayes RL, Koslow M, Hiesinger EM. Improved long term survival after intracavitary interleukin-2 and
lymphokine-activated killer cells for adults with recurrent malignant glioma. Cancer 1995; 76:840-852.
10. Hayes RL, Arbit E, Odaimi M et al. Adoptive cellular immunotherapy for the treatment of malignant
gliomas. Crit Rev Oncol Hematol 2001; 39:31-42.
11. Jacobs SK, Wilson DJ, Kornblith PL et al. Interleukin-2 and autologous lymphokine-activated killer cells
in the treatment of malignant glioma. Preliminary report. J Neurosurg 1986; 64:743-749.
12. Lillehei KO, Mitchell DH, Johnson SD et al. Long-term follow-up of patients with recurrent malignant
gliomas treated with adjuvant adoptive immunotherapy. Neurosurgery 1991; 28:16-23.
13. Merchant RE, Grant AJ, Merchant LH et al. Adoptive immunotherapy for recurrent glioblastoma multiforme
using lymphokine activated killer cells and recombinant interleukin-2. Cancer 1988; 62:665-671.
14. Okamoto Y, Shimizu K, Tamura K et al. An adoptive immunotherapy of patients with medulloblastoma
by lymphokine-activated killer cells (LAK). Acta Neurochir (Wien) 1988; 94:47-52.
15. Sankhla SK, Nadkarni JS, Bhagwati SN. Adoptive immunotherapy using lymphokine-activated killer (LAK)
cells and interleukin-2 for recurrent malignant primary brain tumors. J Neurooncol 1996; 27:133-140.
16. Yoshida S, Tanaka R, Takai N et al. Local administration of autologous lymphokine-activated killer cells
and recombinant interleukin 2 to patients with malignant brain tumors. Cancer Res 1988; 48:5011-5016
17. Barba D, Saris SC, Holder C et al. Intratumoral LAK cell and interleukin-2 therapy of human gliomas. J
Neurosurg 1989; 70:175-182.
18. Blancher A, Roubinet F, Grancher AS et al. Local immunotherapy of recurrent glioblastoma multiforme
by intracerebral perfusion of interleukin-2 and LAK cells. Eur Cytokine Netw 1993; 4:331-341.
19. Boiardi A, Silvani A, Ruffini PA et al. Locoregional immunotherapy with recombinant interleukin-2 and
adherent lymphokine-activated killer cells (A-LAK) in recurrent glioblastoma patients. Cancer Immunol
Immunother 1994; 39:193-197.
20. Bordignon C, Carlo-Stella C, Colombo MP et al. Cell therapy: Achievements and perspectives. Haematologica
1999; 84:1110-1149.
21. Arai S, Meagher R, Swearingen M et al. Infusion of the allogeneic cell line NK-92 in patients with advanced
renal cell cancer or melanoma: A phase I trial. Cytotherapy 2008; 10:625-632.
22. Ishikawa E, Tsuboi K, Saijo K et al. Autologous natural killer cell therapy for human recurrent malignant
glioma. Anticancer Res 2004; 24:1861-1871.
23. Harada H, Saijo K, Watanabe S et al. Selective expansion of human natural killer cells from peripheral
blood mononuclear cells by the cell line, HFWT. Jpn J Cancer Res 2002; 93:313-319.
24. Wang RF. The role of MHC class II-restricted tumor antigens and CD4
T-cells in antitumor immunity.
Trends Immunol 2001; 22:269-276.
25. Kruse CA, Cepeda L, Owens B et al. Treatment of recurrent glioma with intracavitary alloreactive cytotoxic
T-lymphocytes and interleukin-2. Cancer Immunol Immunother 1997; 45:77-87.
26. Quattrocchi KB, Miller CH, Cush S et al. Pilot study of local autologous tumor infiltrating lymphocytes
for the treatment of recurrent malignant gliomas. J Neuro-Oncol 1999; 45:141-157.
27. Tsuboi K, Saijo K, Ishikawa E et al. Effects of local injection of ex vivo expanded autologous tumor-specific
T-lymphocytes in cases with recurrent malignant gliomas. Clin Cancer Res 2003; 9:3294-3302.
28. Tsurushima H, Liu SQ, Tuboi K et al. Reduction of end-stage malignant glioma by injection with autologous
cytotoxic T-lymphocytes. Jpn J Cancer Res 1999; 90:536-545.
29. Tsurushima H, Liu SQ, Tsuboi K et al. Induction of human autologous cytotoxic T-lymphocytes against
minced tissues of glioblastoma multiforme. J Neurosurg 1996; 84:258-263.
cellular immunotherapy in patients with grade III/IV astrocytoma. J Neurooncol 1996; 27:179-189.
31. Plautz GE, Miller DW, Barnett GH et al. T-cell adoptive immunotherapy of newly diagnosed gliomas.
Clin Cancer Res 2000; 6:2209-2218.
glioma: Preliminary results of using autologous whole-tumor vaccine plus granulocyte-macrophage
2000; 9:e9.
33. Wood GW, Holladay FP, Turner T et al. A pilot study of autologous cancer cell vaccination and cellular
immunotherapy using anti-CD3 stimulated lymphocytes in patients with recurrent grade III/IV astrocytoma.
J Neurooncol 2000; 48:113-120.
34. Dudley ME, Wunderlich JR, Yang JC et al. Adoptive cell transfer therapy following nonmyeloablative
but lymphodepleting chemotherapy for the treatment of patients with refractory metastatic melanoma. J
Clin Oncol 2005; 23:2346-2357.
35. Peres E, Wood GW, Poulik J et al. High-dose chemotherapy and adoptive immunotherapy in the treatment
of recurrent pediatric brain tumors. Neuropediatrics 2008; 39:151-156.
36. Ferrara TA, Hodge JW, Gulley JL. Combining radiation and immunotherapy for synergistic antitumor
therapy. Curr Opin Mol Ther 2009; 11:37-42.
37. Ishikawa E, Tsuboi K, Saijo K et al. X-irradiation to human malignant glioma cells enhances the cytotoxicity of
autologous killer lymphocytes under specific conditions. Int J Radiat Oncol Biol Phys 2004; 59:1505-1512.
38. Ishikawa E, Tsuboi K, Takano S et al. Intratumoral injection of IL-2-activated NK cells enhances the
antitumor effect of intradermally injected paraformaldehyde-fixed tumor vaccine in a rat intracranial
brain tumor model. Cancer Sci 2004; 95:98-103.
39. Motohashi S, Ishikawa A, Ishikawa E et al. A phase I study of in vitro expanded natural killer T-cells in
patients with advanced and recurrent nonsmall cell lung cancer. Clin Cancer Res 2006; 12(20 Pt 1):6079-86.
40. Das S, Raizer JJ, Muro K. Immunotherapeutic treatment strategies for primary brain tumors. Curr Treat
Options Oncol 2008; 9:32-40.
41. Yamanaka R. Dendritic-cell- and peptide-based vaccination strategies for glioma. Neurosurg Rev 2009;
32:265-273.
42. Ishikawa E, Tsuboi K, Yamamoto T et al. Clinical trial of autologous formalin-fixed tumor vaccine for
glioblastoma multiforme patients. Cancer Sci 2007; 98:1226-1233.
43. de Vleeschouwer S, Rapp M, Sorg RV et al. Dendritic cell vaccination in patients with malignant gliomas:
Current status and future directions. Neurosurgery 2006; 59:988-999.
44. Yamanaka R, Homma J, Yajima N et al. Clinical evaluation of dendritic cell vaccination for patients with
recurrent glioma: Results of a clinical phase I/II trial. Clin Cancer Res 2005; 11:4160-4167.
45. Bracci L, Moschella F, Sestili P et al. Cyclophosphamide enhances the antitumor efficacy of adoptively
transferred immune cells through the induction of cytokine expression, B-cell and T-cell homeostatic
proliferation and specific tumor infiltration. Clin Cancer Res 2007; 15:644-653.
46. Liu G, Black KL, Yu JS. Sensitization of malignant glioma to chemotherapy through dendritic cell
vaccination. Expert Rev Vaccines 2006; 5:2332-47.
47. Guinn BA, Kasahara N, Farzaneh F et al. Recent advances and current challenges in tumor immunology
and immunotherapy. Mol Ther 2007; 15:1065-1071.
CHAPTER 10
MONOCLONAL ANTIBODY THERAPY

FOR MALIGNANT GLIOMA
Kevin S. Chen1 and Duane A. Mitchell*,2

1
Duke University School of Medicine; 2Division of Neurosurgery, Department of Surgery, The Preston Robert Tisch
Brain Tumor Center at Duke, Durham, North Carolina, USA
*Corresponding Author: Author NameEmail: duane.mitchell@duke.edu
Abstract: Monoclonal antibody (mAb) therapy is a rapidly evolving treatment immunotherapy

modality for malignant gliomas. Many studies have provided evidence that the blood
brain barrierboth at baseline and in the context of malignancyis permissive for
mAbs, thus providing a rationale for their use in treating intracranial malignancy.
Furthermore, techniques such as convection enhanced delivery (CED) are being
implemented to maximize exposure of tumor cells to mAb therapy. The mechanisms
and designs of mAbs are widely varying, including unarmed immunoglobulins as
well as immunoglobulins conjugated to radioisotopes, biological toxins, boronated
dendrimers and immunoliposomes. The very structure of the immunoglobulin
molecule has also been manipulated to generate a diverse armamentarium including
single-chain Fv, bispecific T-cell engagers and chimeric antigen receptors. The
targeted neutralization capacity of mAbs has been employed to modulate the
immunologic milieu in hopes of optimizing other immunotherapy platforms. Many
clinical trials have evaluated these mAb strategies to treat malignant gliomas, and
the implementation of mAb therapy seems imminent and optimistic.
INTRODUCTION
Immunotherapy in the management of malignant gliomas is rapidly developing and

has the potential to be a significant treatment paradigm alongside surgery, radiation and
chemotherapy. Monoclonal antibody (mAb) therapy has emerged as both a powerful
yet adaptive modality by which to target glioma tumor cells and other cancers. The
exquisite specificity of the antibody variable region (Fv) allows therapy to focus the
immune system onto a single antigenic epitope expressed specifically on the surface of

121
tumor cells. Antibodies can also be linked to various biological and radiological moieties
to focus toxicity at the site of the tumor while sparing normal tissue. Additionally, with
the advent of modern genetic technology, we can go beyond the use of the prototypical
immunoglobulin molecule and attach antibody binding domains to a diversity of therapeutic
biomolecules in order to create new classes of anticancer agents.
This chapter will describe the use of unarmed antibodies and antibodies conjugated
to various radio- or chemotherapies to treat malignant brain tumors, as well as discuss
methods of antibody delivery to maximize clinical efficacy. Major tumor-associated
antigens that have been targeted with mAb therapy in gliomas will also be described.
The selective neutralizing capacity of antibodies has also been used to target nontumor
antigens in strategies that aim to augment other treatment modalities through modulation
of the biological response to cancer in the treated host. Finally, this chapter concludes
with a discussion of future directions in small molecule and bioengineered therapies
based on antibody binding motifs.
ANTIBODY CNS BIOAVAILABILITY
The central nervous system (CNS) has long been regarded as an immunoprivileged site
after early work by Medawar1 showed the inability of animals to reject skin homografts to
this site. Therefore, the rationale for immunotherapy, particularly peripherally administered
mAbs in the treatment of CNS cancers, appears self-defeating. However, since the initial
description of the blood-brain barrier, many lines of evidence have demonstrated effective
surveillance of the CNS by the immune system and infiltration by immune effector cells,
with cells like astrocytes and microglia serving as antigen presenting cells.2-4 While it is
true that normal cerebrospinal fluid (CSF) contains sparse amounts of immunoglobulin,
certain inflammatory states such as multiple sclerosis, neuromyelitis optica and some
paraneoplastic disorders demonstrate markedly increased levels of immunoglobulin
in the CSF.5-7 In the context of malignant glioma, therapies such as irradiation can be
used to disrupt the blood-brain barrier, potentially allowing egress of tumor-associated
antigens and a portal of entry for immune cells and antibodies.8,9 Other techniques for
selective site-specific disruption of the blood-brain barrier at the tumor site have also been
developed such as osmotic disruption, use of bradykinin agonists and focused ultrasound
to temporarily allow access of chemotherapy or potentially larger macromolecules such as
antibodies or antibody fragments to infiltrating tumor cells sequestered within the brain.10-16
Even in the absence of inflammation or radiation, early animal experiments by Freund
showed detectable antibodies within CNS tissue and CSF after either active or passive
vaccinations to S. typhi. Levels of antibody within brain tissue extract (after perfusion
and surface rinsing) were about 0.82% after active immunization and 0.7% after passive
immunization compared to levels of antibody achieved in the serum.17 Furthermore, even
negating treatment-induced change in permeability, the blood-tumor barrier within
malignant gliomas is already more permissive than normal endothelia due to the abnormal
physiology of tumor-associated vasculature. This phenomenon has been exploited in
neuroradiology, allowing gliomas to accumulate contrast media given i.v. and studies
have shown that malignant gliomas can increase vascular permeability through secretion
of factors such as transforming growth factor `2 (TGF`2) or vascular endothelial growth
factor (VEGF), implicated in upregulating the expression of matrix metalloproteinases
that breakdown the integrity of the blood-brain barrier.18-20
MONOCLONAL ANTIBODY THERAPY FOR MALIGNANT GLIOMA 123
Perhaps the best evidence for antibody bioavailability in the CNS comes from
immunotherapy trials for Alzheimers disease. Attempts have been made to develop
therapy for Alzheimers disease targeting amyloid-` (A`), a peptide deposited as
amyloid plaques in neural tissue and implicated in the pathophysiology of Alzheimers
disease. The efficacy of such therapy and clearance of plaques appears to be mediated
largely by antibody responses.21 Several theories as to the mechanism by which elicited
antibodies decrease plaque burden are proposed including (1) binding of A` peripherally
in the blood by specific antibodies with a resultant shift in A` equilibrium to favor
egress from the CNS, (2) solubilization of existing plaques and (3) entry into CNS by
specific antibodies and direct clearance of plaques through constant region (Fc) mediated
mechanisms. Support for this last theory is provided by experiments in a murine model
of Alzheimers disease in which monoclonal antibodies against A` were given i.p. and
subsequently demonstrated detectable levels of this antibody within the CNS. Antibodies
to A` plaques were visualized after application of an antimouse secondary antibody on
cryostat sections and coculture of cryostat sections with microglia showed increased
phagocytosis of A` plaques.22
Further evidence of antibody penetration into CNS was provided by a small cohort
of 3 patients with Alzheimers disease vaccinated with a full-length A` peptide which
elicited strong humoral responses: two of three patients had obvious clearance of A` in
large areas of cortex with evidence of phagocytosis of A` by microglia.23 A subsequent
clinical trial evaluating the same A` peptide vaccine was halted early when 6% of
patients receiving vaccine developed meningoencephalitis (compared to none in the
placebo arm, p 0.03).24 It is not known whether antibodies generated in these patients
by vaccination entered the CNS and were the effective mediators of the induced CNS
toxicity. However, long-term follow-up of these patients with postmortem histologic
data showed a strong correlation between increased anti-A` antibody levels and increased
A` clearance.25 As outlined in the following discussions, initial steps in utilizing mAbs in
glioma immunotherapy have already demonstrated activity in the CNS and thus further
advance the rationale of using mAbs as a powerful treatment option in the management
of malignant brain tumors.
DELIVERY OF ANTIBODY THERAPY
Currently, mAb therapy for a variety of diseases is administered peripherally.

Despite the limitations imposed by the blood-brain barrier, many clinical trials have
used peripherally administered mAbs in the treatment of CNS tumors and demonstrated
promising clinical results, possibly due to the changes in blood-brain barrier physiology
at the tumor interface discussed previously. Peripherally administered antibodies are
detectable in intracranial tumors at higher levels than in surrounding normal brain and
have been used to diagnose and identify glioma expression patterns radiographically.26,27
Still, the amount of antibody that enters tumor tissue is an extremely small fraction of
the administered dose and this carries an added burden of potential side effects due to
accumulation of high-doses of administered mAbs in other peripheral organ sites.
Many strategies at bypassing the blood-brain barrier have been attempted. One such
alternative to i.v. administration is intrathecal administration of antibody. Administered
reagents are thus able to circulate in the cerebrospinal fluid (CSF) with reduced
antibody clearance. This method seems particularly suited for malignant gliomas with
leptomeningeal spread or for antibody-directed therapy of multifocal metastatic lesions.

However, the degree of antibody penetration into brain tissue beyond cellular surfaces
in direct contact with CSF is likely very limited and thus the overall effectiveness of this
strategy is questionable.28
Monoclonal antibodies can also be directly administered to the surgical cavity
immediately after tumor resection, an approach that has been used in several clinical trials.
Advantages of such a direct technique include the high concentration of therapeutic agent
at the site of tumor while minimizing nonspecific toxicities against normal peripheral
tissues. However, slow diffusion of macromolecules like immunoglobulins through tumor
tissue and the high interstitial pressure present in tumors opposing this diffusion presents
a barrier to passively applied mAb treatment. This diffusion barrier remains a problem for
strategies such as microdialysis or implanted biodegradable drug-releasing polymers.29
Convection enhanced delivery (CED) is a promising method to overcome the hurdles
of limited physiologic diffusion. The principle of CED is the maintenance of high fluid
pressures at the site of the drug delivery catheter in order to enhance pressure gradients
and bulk fluid flow through the tissue interstitium. This allows therapy such as mAbs to
penetrate deeper and distribute more evenly, up to 2 cm deep from the site of infusion.30,31
Work in animal models have demonstrated that administration of drug or targeted toxins
by CED appears superior to systemic routes32-34 and co-infusion with an MRI contrast
reagent allows for monitoring of infusate distribution.35,36 A recent study examined patients
with recurrent GBM (n 10) randomized to CED versus intratumoral bolus injection of
the antitenascin mAb 123I-81C6 (and crossover to the other modality during the second
infusion): a crossover effect was seen wherein the mean volume of distribution (Vd) for
those receiving bolus injection first was 9.58 ( 11.03 cm3 whereas those that received
bolus injection after CED showed a mean Vd of 19.35 ( 8.27 cm3.37 These results support
CED as a useful method for increased the penetration and availability of infused biologics.
Optimization of various parameterscatheter design, placement, positioning, etc.are
required to achieve consistent and predictable infusate tissue coverage, offering an ideal
vehicle by which drugs can be delivered intracranially.38-41
With rapidly progressing biomedical technologies, new and optimized delivery
techniques for biological therapies such as mAbs will undoubtedly come to fruition. One
critical factor for improving the efficacy of therapeutics delivered using CED will be the
capacity to accurately model and predict the distribution of drugs within the brain using
catheters placed within the resection cavity or tumor parenchyma. Studies employing
computer modeling of drug distribution using data obtained through imaging contrast
media or radiolabeled biomolecules in patients treated with CED are central to the creation
of reliable algorithms for predicting intracranial drug delivery. These delivery platforms,
as they are inevitably improved, can then be applied in combination with a diversity of
mAbs and their varied conjugates.
UNARMED ANTIBODIES
Unarmed or unconjugated mAb therapy acts by a diversity of mechanisms to

mediate tumor cell killing. The accumulation of mAbs on the cell surface bound to tumor
antigens can trigger antibody-dependent cellular cytotoxicity (ADCC) mediated by
natural killer (NK) cells and other immune effectors.42-44 Furthermore, antibody binding
to receptors can elicit receptor internalization, thus decreasing responsiveness of tumor
cells to extracellular survival and proliferative signals mediated by these receptors.45,46

mAbs are also able to bind directly to critical hormones, cytokines or cell surface receptors,
thereby inactivating their biological function or blocking key receptor-ligand interactions.
Current clinical implementation of mAb therapy for cancers is chiefly in the form
of unmodified immunoglobulin. Bevacizumab (Avastin; Genentech, Inc., South San
Francisco, CA, USA), a peripherally administered antibody directed against VEGF, is
a widely used biologic that has been approved by the Food and Drug Administration
(FDA) for treatment of recurrent glioblastoma multiforme (WHO Grade IV glioma;
GBM). GBM is a highly vascularized tumor and antibody binding to VEGF prevents
angiogenic signaling, thus limiting tumor size and survival, possibly through a number
of proposed mechanisms such as normalization of endothelia, sensitization of endothelia
to cytotoxic therapies, or even disrupting a perivascular cancer stem-cell niche.47-49
After recent clinical trials demonstrating benefit in recurrent GBM, bevacizumab given
intravenously every 2 weeks has become a central treatment option in this setting.50-52
Six-month progression free survival (6M-PFS) in the Phase II trials leading to FDA approval
ranged between 39-46% with high proportions of radiographic responses, compared to
historical data with treatment regimens achieving 6M-PFS of between 9% and 21%.53-56
In the environment of limited angiogenesis with VEGF inhibition, tumors may also
be more susceptible to co-administered chemotherapies or radiation. In fact, initial trials
used bevacizumab co-administered with irinotecana topoisomerase I inhibitorin hopes
that blocking VEGF would bolster chemotherapeutic effects. In a Phase II, single-arm,
clinical trial with recurrent GBM patients (n 35), the combination of i.v. bevacizumab
plus irinotecan every 2 weeks achieved a 6M-PFS to 46% (95%CI 32-66%), with
20/35 patients showing at least a partial radiographic response.57 A subsequent Phase
II, randomized trial with recurrent GBM patients tested bevacizumab alone (n 85)
or bevacizumab with irinotecan (n 82). The 6M-PFS was similar between the two
treatment groups (42.6%, 97.5%CI 29.6-55.5% for bevacizumab alone and 50.3%,
97.5%CI 36.8-63.9% for bevacizumab plus irinotecan) and the median OS also was not
different between the two groups (9.2 months, 95%CI 8.2-10.7 months for bevacizumab
alone and 8.7 months, 95%CI 7.8-10.9 months for bevacizumab plus irinotecan.58 Use
of bevacizumab combined with an oral metronomic dose of etoposidea topoisomerase
II inhibitorresulted in similar results as the bevacizumab-irinotecan combination for
Grade 3 gliomas (n 32)6M-PFS 41% (95%CI, 24-57%) and median OS 63.1 weeks
(95%CI, 36-' weeks)as well as patients with GBM (n 27)6M-PFS 44.4% (95%CI,
26-62%) and median OS 46.4 weeks (95%CI, 25-70 weeks).59 Bevacizumab may also
enhance radiotherapy in recurrent gliomas: a recent Phase II clinical trial enrolling patients
with recurrent GBM (n 20) or other recurrent anaplastic glioma (AG, n 5) showed
6M-PFS of 64% (95%CI, 42-79%) overall and 6M-PFS of 65% (95%CI, 40-82%) in the
GBM cohort after bevacizumab and hypofractionated stereotactic radiotherapy.60 This
translated to a median OS for the GBM cohort of 12.5 months (95%CI, 6.9-22.8 months)
and 16.5 months (95%CI, 8-'months) for the AG cohort.
An example of unarmed antibody-mediated therapy directed against a major target
expressed on tumor cells is mAbs developed against the epidermal growth factor receptor
(EGFR). Malignant gliomas frequently overexpress this receptor and binding of EGFR
prevents mitogenic signals from promoting tumor growth and induces apoptosis in tumor
cells.61 Cetuximab (Erbitux, YM BioSciences; Mississauga, Ontario, Canada)a
chimeric EGFR mAbis an antibody that showed success in preclinical trials of either
subcutaneous or intracranial tumors using i.p. administered antibody.61-63 However, a Phase
II trial enrolling patients with primary (n 33) or recurrent GBM (n 22) suggested no
impact of weekly i.v. infusion of cetuximab on PFS or overall survival (OS), regardless
of EGFR expression levels.64 Most promising results have been seen with EGFR mAbs
used in conjunction with other modalities; for example, both cetuximab and another EGFR
mAb, nimotuzumab (YM BioSciences), were shown to sensitize tumors to radiotherapy,
perhaps by synergizing with radiation effects on the tumor itself or by an effect on tumor
angiogenesis.65-67 Other EGFR mAbs, trastuzumab (Herceptin, Genentech, Inc.) and
panitumumab (Vectibix, YM Biosciences), have been applied clinically in a variety
of settings such as breast and colorectal cancer but little investigation into application in
malignant glioma has been reported to date.68-73
One major barrier in EGFR-directed mAbs is the expression of this protein in many
normal tissues,74,75 reducing the amount of free antibody to bind tumor as well as increasing
the risk of limiting toxicity. An attractive alternative is the targeting of the EGFR class
III variant (EGFRvIII), an in-frame splice variant in which exons 2-7 are lost, resulting
in a constitutively active receptor tyrosine kinase that is not expressed in normal cells.76-80
This variant contributes to unchecked proliferation in many types of cancer including
high-grade gliomas. Because this splice variant produces a unique epitope on tumor cells
not found elsewhere on normal tissues, gliomas that are EGFRvIII
(approximately a third
of high-grade gliomas) are attractive options for immunotherapy strategies such as mAbs.
An EGFRvIII targeted murine mAb, Y10, was demonstrated to have activity in a
mouse model of glioma. In these studies, mice bearing intracranial EGFRvIII+ tumors and
treated with intratumoral Y10 demonstrated a median survival extended by 488% when
compared to isotype-matched control treatment. Furthermore, 60% of treated animals
survived past 90 days with no signs of disease whereas all isotype control mice succumbed
to tumors.81 Interestingly, the mechanism of antitumor efficacy in this case required Fc
receptors and efficacy was not abrogated by depletion of complement, granulocytes, NK
cells or T cells. This success was translated to mouse xenograft models of human malignant
gliomas using mAb 806, another murine mAb against EGFRvIII.82 Even with intracranial
gliomas, mice treated with mAb 806 given peripherally i.p. showed prolonged survival of
between 15-61% when compared to isotype controls.62,83 A chimeric form of mAb 806 has
been administered by peripheral i.v. to a patient with an anaplastic astrocytoma (AA) in
Phase I clinical trials, showing localization of antibody at the tumor site.84 These studies
underscore the ability for peripherally administered antibody to accumulate specifically at
intracranial tumor sties. Clinical efficacy of this EGFRvlll-targeted paradigm now awaits
evaluation in larger clinical trials.
Other targets of unarmed mAbs also show promise. Pritumumab (Nascent Biologies,
San Diego, CA, USA), a mAb directed at a tumor-specific variant of vimentin, has been
tested in a Phase I and Phase II clinical trials and shown promising results. Monoclonal
antibodies generated against the hepatocyte growth factor and its receptor c-Met have
also shown antitumor activity in many preclinical studies.86-89 The list of antigenic targets
for unarmed mAbs in malignant gliomas continues to grow, including tumor necrosis
factor-related apoptosis-inducing ligand (TRAIL) death receptor 5, GP240, histone/DNA
complexes, (B
)-fibronectin, glycoprotein NMB, multi-drug resistance protein-3 and
many others.90,91 It is likely that combinations of antibodies against multiple antigens will
be needed for effective control of malignant gliomas due to the inherent heterogeneity of
expression characteristic of these tumors that makes it unlikely that a single target will result
in effective elimination of all residual tumor cells. As our catalogue of glioma-associated
antigens expands, so will our arsenal for targeting these tumor markers with mAb therapy.
RADIOISOTOPE OR TOXIN CONJUGATED ANTIBODIES
Beyond the capacity for antibodies to recognize a specific epitope and prevent
signaling or induce ADCC, the specificity of antibodies toward tumor cells has spurred
efforts to use mAbs as carriers for radioisotopes or cellular toxins, allowing targeted
killing. Particularly when antibodies accumulate surface receptors and are subsequently
internalized conjugated substances can be concentrated in neoplastic cells while sparing
healthy tissue.92,93
One epitope targeted for radioisotope conjugated mAb therapy is tenascin C, a large
extracellular glycoprotein that is produced in malignant glioma but not normal brain.94,95
Most clinical trials of an antitenascin C antibody have employed direct, local application
in the surgical resection cavity in order to limited systemic distribution since this protein
is also normally expressed in tissues such as liver, kidney, spleen, dermis and in areas
of inflammation.96 An early Phase I clinical trial used 131I-labeled murine antitenascin C
mAbs BC-2 and BC-4 delivered by an indwelling catheter placed in the resection cavity
at the time of surgery.97 Newly diagnosed (n 24) and recurrent GBM patients (n 26)
were enrolled to receive 131I-labeled BC-2/BC-4 delivered to tumor resection cavity via
indwelling catheter at regular intervals (1-4 month intervals) after surgery, radiation
and/or chemotherapy and continued to receive treatment at regular intervals until either
progression or nondetectability of disease by radiological imaging. Although 69% of
patients developed human antimouse antibodies (HAMA), no major side effects were
reported and these antibodies did not change the pharmacokinetics of administered mAb.
Median OS was higher than historical controls, reaching 23 months for primary GBM
patients and 18 months for recurrent patients, with patients with larger lesions (2cm
diameter) demonstrating diminished benefit (median OS 17 months). A subsequent Phase
II study enrolled primary (n 38) and recurrent GBM (n 36) patients for treatment
with 131I-conjugated BC-2/BC-4 infused every 1-6 months by indwelling catheter in
the resection cavity. In grouping both primary and recurrent patients together (n 74),
median OS was 19 months. When analysis was restricted to patients with small tumor
burden (2cm3), median OS for newly diagnosed GBM patients (n 27) was 25 months
and 21 months for recurrent GBM patients (n 16).98
Another antitenascin C murine mAb, 81C6, has also seen clinical application
with conjugation to 131I.99-101 Phase I trials enrolling patients with high grade gliomas
appeared promising,102-104 with one enrolling primary GBM (n 32) or other high grade
glioma (n 10) and administering a single dose of 131I-labeled 81C6 after surgery via
catheter placed in the resection cavity.105 Median survival was 79 weeks (95%CI, 61
to 106 weeks) for all patients and 69 weeks (95%CI, 52-106 weeks) for GBM patients.
Similar results were seen in Phase II trials: one study investigated patients with primary
GBM (n 27) or other high grade glioma (n 6) after single dose of 131I-81C6 to the
resection cavity and demonstrated median OS of 86.7 weeks (95%CI, 76.7-'weeks)
and, in the subset of GBM patients, a 79.4 week median OS (96%CI, 61.4-'weeks).106
In a separate study with primary GBM (n 15) or AA (n 6) patients, a 131I-81C6 dose
to the resection cavity was specifically tailored to each patient to give a 44 Gy boost,
showing a median survival of 90.6 weeks (95%CI, 73.3-97.1 weeks) in GBM patients
and median survival exceeding median follow-up time (124.4 weeks) for patients
with anaplastic astrocytoma.107 A Phase II study using the same treatment parameters
as above enrolled patients with recurrent GBM (n 33) or other advanced glioma
(n 9) and one patient with metastatic adenocarcinoma, showing median survival of
68.6 weeks (95%CI, 38.8 to 90 weeks) for all patients and 63.9 weeks (95%CI, 38.8-90
weeks) for patients with GBM.108
Most recent trials have utilized chimeric mAbs to avoid the problem of HAMA. This
engineered antibody shows increased activity and greater stability when compared to the
murine mAb.109 A Phase I trial was initiated with an 131I-labeled chimeric 81C6 antibody
administered by catheter placed in the resection cavity of primary (n 30) or recurrent
(n 8) GBM, or primary (n 5) or recurrent (n 4) high grade glioma of another type.110
In the newly diagnosed glioma patients, median OS was 88.6 weeks (95%CI, 72.9-128.7
weeks) and 86.1 weeks (95%CI, 70.1-99.1 weeks) in the subset of newly diagnosed GBM
patients. In recurrent disease, median OS was 65 weeks for high grade glioma (95%CI,
45.0 -142.4 weeks) and 48.9 weeks (95%CI, 30.4-83.3 weeks) in the recurrent GBM
patients. Another Phase I trial with the chimeric 81C6 administered in the tumor resection
cavity, the antibody conjugated this time to an _-emitter 211At, enrolled recurrent GBM
(n 14) or other high grade glioma (n 4) patients and demonstrated 57 week median
OS (95%CI, 47 to 87 weeks) for all patients and 52 week median OS (95%CI, 33-76
weeks) for the subset of patients with GBM.111
EGFR and EGFRvIII have also been targeted with radioisotope-linked mAbs.
For example, nimotuzumab was recently linked to 188Re and used in a Phase I trial by
administration to the surgical resection cavity. Enrolling recurrent GBM (n 8) and
recurrent high grade gliomas of other types (n 3), the study found 1 GBM and 1AA
patient with complete response, 1 GBM patient with partial response and 1 GBM and 1
AA showing stable disease, whereas other patients progressed while on therapy.112 Another
murine mAb, the monoclonal antibody 425, has been used to target EGFR, most often
in a form linked to125I.113,114 An early trial with 25 newly diagnosed high grade glioma
patients treated with peripherally infused 125I-mAb425 resulted in a projected median OS
of 15.6 months for both AA and GBM.115 A larger trial with primary GBM (n 118)
and primary AA (n 55) showed median OS 13.4 months for the GBM cases and 50.9
months for the 55 anaplastic astrocytoma cases.116
Instead of radioactive substances, cytotoxic bacterial toxins have also been targeted
against glioma cells. This strategy has the advantage of specific tumor toxicity upon
internalization of therapy without the precautions and difficulty of handling radioactive
substances. The cytokine ligands IL-4, IL-13 and TGF _although not mAbs and
outside the scope of the present discussionhave seen preliminary success when linked
to Pseudomonas exotoxin since the receptors for these cytokines are often overexpressed
in gliomas.39,117-126 A single-chain Fv (scFv) antibody to the IL-13 receptor alpha 2
(IL-13R_2) was subsequently conjugated to Pseudomonas exotoxin and was shown
to have both in vitro cytotoxic activity against IL-13R_2
glioma cell lines as well as
in vivo effect against implanted s.c. tumors in a xenograft model of glioma.127 In this
study, the Pseudomonas exotoxin-linked IL-13 ligand showed increased efficacy over the
scFv antibody product. However, a Phase III trial of recurrent GBM patients (n 276),
randomized to carmustine (Gliadel) wafers (n 93) or a toxin-linked IL-13R_2 mAb
(cintredekin besudotox) infused by CED (n 183), resulted in no significant difference
in median OS (36.4 weeks for cintredekin besudotox treated group versus 35.3 weeks
for carmustine control arm, p 0.476).40 A possible confounder was the inclusion of all
recurrent GBM patients regardless of IL-13R_2 expression, as well as the heterogeneous
expression of this receptor variant within tumors.
A different molecule using the same toxin-bound paradigm is a scFv antibody against
EGFRvIII conjugated to Pseudomonas exotoxin, called MR1-1.128,129 Preclinical studies
using intratumoral injection of subcutaneous EGFRvIII

tumors with MR1-1 showed
strong treatment effect, wherein all mice receiving MR1-1 were able to reject tumor while
all saline injected mice succumbed to tumors (p 0.0001).130 This antitumor immunity was
long lasting, allowing all treated mice to reject EGFRvIII
tumors at rechallenge 65 days
after initial tumor treatment (p 0.0001). Mice initially treated with MR1-1 for EGFRvIII
tumors were also able to reject EGFRvIII-negative tumors at rechallenge, hypothesized

to act by a mechanism of cross-presentation, in which antigens released by dying tumor
cells during immunotoxin treatment are taken up by host antigen presenting cells and
lead to the priming of a long-lived antitumor immune response. The promising results
from these studies have spurred the opening of a Phase I clinical trial to evaluate MR1-1
delivered intratumorally by CED (NCT01009866).
Another innovative antibody conjugate is the use of heavily boronated molecules
linked to mAbs for use in neutron capture therapy. The physics of boron neutron capture
therapy (BNCT) involve administration of boronated substances that are preferentially
internalized by tumor cells, followed by application of low energy thermal neutrons at the
tumor site, thus producing an _ particle and 7Li as fission products that are locally toxic
to tumor cells.131 To increase the specificity of BNCT, a boronated dendrimer has been
conjugated to cetuximab132,133 or to an anti-EGFRvlll mAb,134,135 which has demonstrated
improved efficacy over boronophenylalanine (BPA), an untargeted molecule previously
used for BNCT. In preclinical studies using a rat model of intracranial EGFR-expressing
glioma, administration by CED of boronated cetuximab resulted in median OS increases
between 45-54.5 days compared to about 40 days for those receiving i.v. BPA alone;
further, boronated cetuximab by CED seemed to be synergistic with i.v. BPA, extending
median survival to 59-70.9 days.132,133 Studies of a boronated anti-EGFRvIII mAb (L8A4)
administered by CED also showed promise, extending median OS of tumor-bearing rats
from 40.1 ( 2.2 days for i.v. BPA alone to 70.4 ( 11.1 days for CED of L8A4 and further
to 85.5 ( 15.5 days for L8A4 with i.v. BPA.136
Antibodies have also been used to direct and localize liposomes carrying various
chemotherapeutics for the targeted killing of tumor cells. In the realm of glioma, most
research has been performed with these liposomes conjugated to anti-EGFR antibodies
such as cetuximab. Immunoliposomes loaded with doxorubicin, epirubicin or vinorelbine
demonstrated significant ability to control tumor growth over untargeted liposomes in a
murine subcutaneous glioma xenograft model.137 The adaptability of the immunoliposome as
a carrier for a wide range of substances has led to experimental work with immunoliposomal
delivery of boronated chemicals for BNCT and even RNA interference gene therapy.138-140
These therapies show promising results, taking steps toward the therapeutic ideal of an
effective, selective, potent yet noninvasive antiglioma treatment.
IMMUNOTHERAPY MODULATORS
The ability for selective binding and neutralization has made mAb therapy useful
not only in directly attacking tumors, but also by indirectly enhancing other treatment
paradigms. In many immunotherapeutic strategies, mAbs are used to modify immunologic
physiology to achieve maximum immune responses. It is fairly well-established that the
immune system is suppressed in malignancy, particularly malignant gliomas.141-144 Thus,
modulation of various cytokines and other signaling molecules can help to overcome this
immunosuppression and mobilize a strong and directed antitumor response.
An important mediator of immunosuppression is the regulator T cell (T-reg). T-regs

are generally defined as CD4
/CD25
/FoxP3
lymphocytes that serve to dampen
the immune response.145-149 T-regs are hypothesized to function in limiting immune
responses, thus avoiding inflammatory and autoimmune pathologies, but are found to be
abnormally elevated in patients with high grade gliomas.150-152 Thus, the CD25 molecule
that characterizes T-regs has been targeted with mAb therapy in hopes of decreasing
this immunosuppressive population. Animal studies have demonstrated prolonged
survival in intracranial glioma models upon administration of an anti-CD25 mAb.153 The
therapeutic benefit provided by anti-CD25 mAbs may not be simply through depletion
of T-reg numbers but also via inactivation of T-reg function.154,155 Preclinical success
has led to the initiation of early phase clinical trials to evaluate the ability for anti-CD25
mAbsfor example daclizumab or basiliximabto enhance responses to immunotherapy
(NCT00626015, NCT00626483).
Another immunomodulatory target for mAbs is the cytotoxic T-lymphocyte
associated protein 4 (CTLA-4). CTLA-4 is a regulatory molecule expressed on T cells
and, structurally similar to CD28, interacts with costimulatory molecules B7.1 and
B7.2 on antigen presenting cells.156-158 In contrast to CD28, binding by CTLA-4 sends
an inhibitory signal to T cells and prevents full T-cell activation. Decreasing CTLA-4
activity may theoretically galvanize T cells to overcome the immunosuppression present
in glioma patients. In a murine model of intracranial glioma, peripheral administration
of anti-CTLA-4 mAb resulted in 80% long term survival past 90 days while all control
animals succumbed to tumor with median survival of 26 days (p 0.0022).159 The
improved tumor rejection appeared to result from lymphocyte proliferative ability that
was restored to normal levels. These experiments also demonstrated a significant role
for T-regs, since T-reg levels in treated mice were restored to normal after CTLA-4
blockade. The complex interplay between T-reg immunosuppression and costimulatory
T-cell activation is underscored by experiments in a murine intracranial glioma challenge
model which showed that pretreatment with both anti-CD25 and anti-CTLA-4 mAbs
resulted in an additive protective effect and prevented establishment of tumor from all
tumor-implanted animals.160
A central question remains unanswered as to the mechanism by which gliomas
exert their potent and systemic immunosuppressive effects. If this can be targeted at
its source, T-reg and T-cell stimulation should normalize and appropriate antitumor
responses potentially could be mobilized by the host to eradicate residual tumor cells. A
candidate for such a signal is the secreted cytokine transforming growth factor, TGF-`.
TGF-` is secreted by many types of malignant cells including glioma tumor cells and it
suppresses cytotoxic immune responses by a variety of mechanisms (including activation
of T-regs).161-165 Preclinical studies using a neutralizing anti-TGF-` mAb showed the
ability to maximize response to peptide vaccine immunotherapy toward the antigens
EphA2 and GARC-1: 6/10 mice receiving mAb plus peptide vaccine therapy survived
more than 100 days after intracranial tumor implantation whereas only 2/10 mice survived
to 100 days with peptide vaccine alone (p 0.05). Interestingly, animals treated with
anti-TGF-` alone did not demonstrate enhanced survival over controls. Furthermore, in
mice receiving mAb therapy, tumor-infiltrating T-regs were observed to be depleted,
providing additional evidence for T-reg-mediated immunosuppression being central in
glioma pathogenesis.
ANTIBODY-BASED INNOVATIONS IN IMMUNOTHERAPY
With accumulated experience using mAb therapy in cancer immunotherapy,

innovations in antibody design and creative application of recombinant molecular
biology technique has resulted in an explosion of new therapeutic options (Fig. 1).
Figure 1. Modern immunotherapy has produced a diversity of strategies based on the prototypical
monoclonal antibody structure. A) The basic structure of the immunoglobulin consists of a heavy chain
paired with a light chain, with 2 heavy-light pairs joined by disulfide bonds at the hinge region.
N-terminal domains of each chain consist of the variable domains (Fv) that specifically bind antigenic
epitopes, whereas the constant domains (Fc) remain conserved within isotype classes. B) In efforts to
reduce the development of human antimouse antibodies (HAMA), chimeric mAbs (with murine Fv
domains and human Fc domains) and humanized mAbs (with murine complementarity determining
regions engineered into a human immunoglobulin molecule) have been developed. C) Unarmed
mAbs as well as conjugation to a variety of cytotoxic substances have allowed selective targeting of
therapy to tumor-associated epitopes. D) With recombinant genetic technologies, a variety of novel
immunoglobulin-based structures have been created, including single-chain Fv, chimeric antigen receptors
(CARs) or Bispecific T-cell engagers (BiTEs).
The first challenge that has been encountered is the endogenous response to murine,
humanized or even native human antibodies. The ability to mount an immune response
to antibodies transferred from another species has long been recognized and formed the
initial understanding of humoral immunity and serum sickness.166-168 In many early clinical
trials of murine mAbs, a human antimouse antibody response (HAMA) was observed and
concern was raised over the hosts capacity to neutralize the administered treatment.90
Application of murine mAbs for gliomas, however, seemed appropriate in the context of
direct application to the tumor resection bed or intrathecal administration, since these sites
are relatively protected from a systemic humoral neutralizing response.169 So far, HAMA
responses have been detectable in many of the clinical trials described above and evidence
thus far does not suggest a significant neutralizing or clearing effect of these responses
to intracranially administered mAbs. However, HAMA can lead to allergic reactions
and potentially interfere with antibody-dependent cytotoxicity mechanisms, limiting the
utility of mAb therapy. Recombinant strategies have been utilized to combine the epitope
binding regions identified in murine antibodies with the less immunogenic human Fc
regions to reduce the incidence of HAMA.170-173 For example, chimeric antibodies fuse
the murine Fv regions with human Fc regions and humanized antibodies are fine-tuned
in selecting only the murine complementarity-determining regions to insert into a human
antibody framework. More sophisticated genetic technologies have allowed the capacity
to engineer fully human antibodies, for example a fully human mAb against EGFR.174,175
Furthermore, even the basic structure of an antibody can be altered and even transplanted
to other biomolecules to generate new classes of high-affinity and cancer-specific reagents.
Modern techniques in molecular biology have made possible the manipulation of
biomolecules and have resulted in the creative engineering of novel proteins utilizing the
fine specificity of antibody Fv regions. Rather than using the complete immunoglobulin
structure, new designs take advantage of the compartmentalized nature of the
immunoglobulin molecule. For example, taking only the variable regions of one heavy
and one light chain and re-expressing them as a single polypeptide, scFv molecules have
been utilized in antitumor therapy.176-178 Although scFv have decreased affinity when
compared to complete immunoglobulins (due to having only one epitope recognizing
domain) and are also more rapidly cleared by the kidney, their small size enables scFv to
penetrate tissue more effectively and intercalate between tumor cells.169,179-181 Utilization
of combinatorial variation and phage display libraries also allows for the rapid generation
and selection of an optimally binding scFv.182,183 As described above, the scFv has already
been used to target IL-13R_2 and EGFRvIII with some clinical success.127,130
Yet another molecule, the bispecific T-cell engager (BiTE), is a divalent molecule
in which one Fv region binds to the T-cell coreceptor CD3 and the other fragment is
engineered to bind to tumor specific antigen.184-190 The BiTE acts to facilitate accumulation
of T cells at the local tumor microenvironment. The accumulation and clustering of CD3
molecules in the proximity of tumor-specific surface markers results in cytotoxic T-cell
activation, regardless of T-cell clonality and regardless of tumor cell MHC expression
(often suppressed in malignancy). Thus, a larger population of tumor-infiltrating T cells
can be mobilized against tumor. Currently, two BiTEs are being evaluated clinically in
lymphoma/leukemia (directed against CD3 and CD19)191 as well as in lung and Gl cancers
(directed against CD3 and epithelial cell adhesion molecule).192 With a growing list of
potential glioma-specific antigens to target, prospects are exciting and it seems only a
matter of time before a BiTE is constructed for use in malignant gliomas.
An even more direct method by which to direct cytotoxic T cells against

tumor-specific antigens is though use of a chimeric antigen receptor (CAR). This
method engineers T cells to express T-cell receptors that have the MHC-binding regions
replaced with a specific antibody Fv motif.193,194 Endogenous clonally selected T cells
may have difficulty recognizing tumors since malignant cells often either present
self-antigen in their MHC molecules or have globally downregulated expression of
MHC molecules. CARs, like BiTEs, allow T cells to bypass the MHC-peptide-T-cell
receptor interaction and allow T cells to activate in direct response to tumor surface
antigen. CARs have had a great degree evaluation in preclinical models of cancer and
some clinical evaluation in ovarian cancer, renal cancer, colorectal cancer, lymphoma,
neuroblastoma and medulloblastoma.195-200 Early clinical trial results were modest,
with persistence of administered modified T cells a potential limiting factor. A recent
case of CAR-directed adoptive cellular therapy, however, utilizing the high-affinity
antibody binding domain from the humanized mAb trastuzumab (targeting Her-2/neu),
underscored the potential potency and toxicity of genetically redirected lymphocyte
therapy. A patient treated for metastatic colon cancer using adoptive T-cell transfer
of autologous lymphocytes expressing the Her-2/neu CAR experienced the sudden
onset of pulmonary edema and cytokine storm, which eventually led to cardiovascular
collapse and cardiac arrest in this patient.201 Recognition of low levels of the target
antigen in lung and heart was deemed to be the likely mediator of the resultant deadly
immune-mediated toxicity.
In the field of gliomas, a CAR was tested in which the T-cell receptor was fused to
IL-13 to target the IL-13R_2.202 This modified T cell was able to show in vivo eradication
of an intracranial glioma xenograft in all treated animals while all nontreated animals were
killed by tumor burden. A case was reported using a similar CAR (a membrane-tethered
CAR, or IL-13 zetakine) in a patient with recurrent GBM after surgical, radiological and
temozolomide therapy.203 T cells were isolated from the patients leukapheresis sample,
electroporated with the IL-13 zetakine as well as a herpes simplex virus 1 thymidine
kinase (HSVItk), expanded and subsequently infused in the tumor reresection cavity by
Rickham reservoir, eventually receiving a cumulative dose of 1 = 109 cells. After peripheral
infusion of a PET reporter probe (18F-FHBG which accumulates in cells expressing the
HSVItk), infused T cells were detectable in the tumor resection cavity as well as later
at a new tumor near the corpus callosum. Subsequent stereotactic biopsy of this second
tumor revealed malignancy with infiltrating CD8
T cells. The patient survived for
14 months after initial recurrence. Like BiTEs, it seems inevitable that the expanding
catalog of tumor-specific epitopes and antibodies will be combined with techniques in
immunogenetic engineering to create a tailored armada of cytotoxic cells utilizing CARs.
CONCLUSION
The potential for the successful and safe application of mAbs in the immunotherapy
of malignant gliomas has been clearly demonstrated. Not only is there an expanding
repertoire of tumor-associated antigens that can serve as mAb targets, these biological
homing missiles will undoubtedly play an adjunctive role in other immunotherapies.
And with the toolbox of molecular biology, only imagination and creativity limit the
potential uses of engineered molecules utilizing antibody epitope binding domains.
Furthermore, the innovative molecular prototypes that are being produced in antiglioma
therapy can be adapted to cancers of all kinds and even in areas beyond oncology. The
vast and exciting next-generation of mAb applications are only just beginning to come to
fruition and are anticipated to heavily influence the state of the art treatment and prognosis
for malignant gliomas. Significant advances in mechanisms to deliver antibodies or
antibody-derived molecules beyond the blood-brain-barrier and enhanced capacity to
understand and predict the distribution of intracranially administered reagents, will be
critical to the full realization of the utility of these classes of biomolecules in our battle
against invasive malignant brain tumors.
REFERENCES
1. Medawar PB. Immunity to homologous grafted skin; the fate of skin homografts transplanted to the brain,
to subcutaneous tissue and to the anterior chamber of the eye. Br J Exp Pathol 1948; 29(1):58-69.
2. Aloisi F, Ria F, Adorini L. Regulation of T-cell responses by CNS antigen-presenting cells: different roles
for microglia and astrocytes. Immunol Today 2000; 21(3):141-147.
3. Gehrmann J, Matsumoto Y, Kreutzberg GW. Microglia: intrinsic immuneffector cell of the brain. Brain Res
Brain Res Rev 1995; 20(3):269-287.
4. Stevens A, Kloter I, Roggendorf W. Inflammatory infiltrates and natural killer cell presence in human brain
tumors. Cancer 1988; 61(4):738-743.
5. Bhat R, Steinman L. Innate and adaptive autoimmunity directed to the central nervous system. Neuron
2009; 64(1):123-132.
6. Bradl M, Misu T, Takahashi T et al. Neuromyelitis optica: pathogenicity of patient immunoglobulin in vivo.
Ann Neurol 2009; 66(5):630-643.
7. Darnell RB, Posner JB. Paraneoplastic syndromes involving the nervous system. N Engl J Med 2003;
349(16):1543-5154.
8. Qin D, Ou G, Mo H et al. Improved efficacy of chemotherapy for glioblastoma by radiation-induced opening
of blood-brain barrier: clinical results. Int J Radiat Oncol Biol Phys 2001; 51(4):959-962.
9. Qin DX, Zheng R, Tang J et al. Influence of radiation on the blood-brain barrier and optimum time of
chemotherapy. Int J Radiat Oncol Biol Phys 1990; 19(6):1507-1510.
10. de Vries NA, Beijnen JH, Boogerd W et al. Blood-brain barrier and chemotherapeutic treatment of brain
tumors. Expert Rev Neurother 2006; 6(8):1199-1209.
11. Bidros DS, Vogelbaum MA. Novel drug delivery strategies in neuro-oncology. Neurotherapeutics 2009;
6(3):539-546.
12. Rapoport SI, Hori M, Klatzo I. Testing of a hypothesis for osmotic opening of the blood-brain barrier. Am
J Physiol 1972; 223(2):323-331.
13. Neuwelt EA, Frenkel EP, Rapoport S et al. Effect of osmotic blood-brain barrier disruption on methotrexate
pharmacokinetics in the dog. Neurosurgery 1980; 7(1):36-43.
14. Cloughesy TF, Black KL. Pharmacological blood-brain barrier modification for selective drug delivery. J
Neurooncol 1995; 26(2):125-132.
15. Inamura T, Nomura T, Bartus RT et al. Intracarotid infusion of RMP-7, a bradykinin analog: a method for
selective drug delivery to brain tumors. J Neurosurg 1994; 81(5):752-758.
16. Hynynen K, McDannold N, Vykhodtseva N et al. Noninvasive MR imaging-guided focal opening of the
blood-brain barrier in rabbits. Radiology 2001; 220(3):640-646.
17. Freund J. Accumulation of Antibodies in the Central Nervous System. J Exp Med 1930; 51(6):889-902.
18. Ishihara H, Kubota H, Lindberg RL et al. Endothelial cell barrier impairment induced by glioblastomas
and transforming growth factor beta2 involves matrix metalloproteinases and tight junction proteins. J
Neuropathol Exp Neurol 2008; 67(5):435-448.
19. Grabb PA, Gilbert MR. Neoplastic and pharmacological influence on the permeability of an in vitro
blood-brain barrier. J Neurosurg 1995; 82(6):1053-1058.
20. Schneider SW, Ludwig T, Tatenhorst L et al. Glioblastoma cells release factors that disrupt blood-brain
barrier features. Acta Neuropathol 2004; 107(3):272-276.
21. Lee M, Bard F, Johnson-Wood K et al. Abeta42 immunization in Alzheimers disease generates Abeta
N-terminal antibodies. Ann Neurol 2005; 58(3):430-435.
22. Bard F, Cannon C, Barbour R et al. Peripherally administered antibodies against amyloid beta-peptide
enter the central nervous system and reduce pathology in a mouse model of Alzheimer disease. Nat Med
2000; 6(8):916-919.
23. Nicoll JA, Barton E, Boche D et al. Abeta species removal after abeta42 immunization. J Neuropathol Exp
Neurol 2006; 65(11):1040-1048.
24. Orgogozo JM, Gilman S, Dartigues JF et al. Subacute meningoencephalitis in a subset of patients with AD
after Abeta42 immunization. Neurology 2003; 61(1):46-54.
25. Holmes C, Boche D, Wilkinson D et al. Long-term effects of Abeta42 immunisation in Alzheimers
disease: follow-up of a randomised, placebo-controlled phase I trial. Lancet 2008; 372(9634):216-223.
26. Gibbs-Strauss SL, Samkoe KS, OHara JA et al. Detecting epidermal growth factor receptor tumor activity
in vivo during cetuximab therapy of murine gliomas. Acad Radiol. 17(1):7-17.
27. Wei LH, Olafsen T, Radu C et al. Engineered antibody fragments with infinite affinity as reporter genes
for PET imaging. J Nucl Med 2008; 49(11):1828-1835.
28. Blasberg RG, Patlak C, Fenstermacher JD. Intrathecal chemotherapy: brain tissue profiles after
ventriculocistemal perfusion. J Pharmacol Exp Ther 1975; 195(1):73-83.
29. Groothuis DR. The blood-brain and blood-tumor barriers: a review of strategies for increasing drug delivery.
Neuro Oncol 2000; 2(1):45-59.
30. Bobo RH, Laske DW, Akbasak A et al. Convection-enhanced delivery of macromolecules in the brain.
Proc Natl Acad Sci USA 1994; 91(6):2076-2080.
31. Ferguson SD, Foster K, Yamini B. Convection-enhanced delivery for treatment of brain tumors. Expert
Rev Anticancer Ther 2007; 7(12 Suppl):S79-85.
32. Degen JW, Walbridge S, Vortmeyer AO et al. Safety and efficacy of convection-enhanced delivery of
gemcitabine or carboplatin in a malignant glioma model in rats. J Neurosurg 2003; 99(5):893-898.
33. Heimberger AB, Archer GE, McLendon RE et al. Temozolomide delivered by intracerebral microinfusion
is safe and efficacious against malignant gliomas in rats. Clin Cancer Res 2000; 6(10):4148-4153.
34. Kaiser MG, Parsa AT, Fine RL et al. Tissue distribution and antitumor activity of topotecan delivered by
intracerebral clysis in a rat glioma model. Neurosurgery 2000; 47(6):1391-8; discussion 1398-1399.
35. Ding D, Kanaly CW, Bigner DD et al. Convection-enhanced delivery of free gadolinium with the recombinant
immunotoxin MR1-1. J Neurooncol 2010; 98(1):1-7.
36. Ding D, Kanaly CW, Cummings TJ et al. Long-term safety of combined intracerebral delivery of free
gadolinium and targeted chemotherapeutic agent PRX321. Neurol Res 2009.
37. Sampson JH, Akabani G, Friedman AH et al. Comparison of intratumoral bolus injection and
convection-enhanced delivery of radiolabeled antitenascin monoclonal antibodies. Neurosurg Focus
2006; 20(4):E14.
38. Sampson JH, Archer G, Pedain C et al. Poor drug distribution as a possible explanation for the results of
the PRECISE trial. J Neurosurg 2009; 113:301-309.
39. Sampson JH, Akabani G, Archer GE et al. Intracerebral infusion of an EGFR-targeted toxin in recurrent
malignant brain tumors. Neuro Oncol 2008; 10(3):320-329.
40. Kunwar S, Chang S, Westphal M et al. Phase III randomized trial of CED of IL13-PE38QQR vs Gliadel
wafers for recurrent glioblastoma. Neuro Oncol 2010; 12:871-878.
41. Mueller S, Polley MY, Lee B et al. Effect of imaging and catheter characteristics on clinical outcome for
patients in the PRECISE study. J Neurooncol 2010; 101:267-277.
42. Clynes RA, Towers TL, Presta LG et al. Inhibitory Fc receptors modulate in vivo cytoxicity against tumor
targets. Nat Med 2000; 6(4):443-436.
43. Clynes R, Takechi Y, Moroi Y et al. Fc receptors are required in passive and active immunity to melanoma.
44. Stavenhagen JB, Gorlatov S, Tuaillon N et al. Fc optimization of therapeutic antibodies enhances their ability
to kill tumor cells in vitro and controls tumor expansion in vivo via low-affinity activating Fcgamma
receptors. Cancer Res 2007; 67(18):8882-8890.
45. Hens M, Vaidyanathan G, Welsh P et al. Labeling internalizing anti-epidermal growth factor receptor
variant III monoclonal antibody with (177)Lu: in vitro comparison of acyclic and macrocyclic ligands.
Nucl Med Biol 2009; 36(2):117-128.
46. Lillo AM, Sun C, Gao C et al. A human single-chain antibody specific for integrin alpha3beta1 capable of
cell internalization and delivery of antitumor agents. Chem Biol 2004; 11(7):897-906.
47. Calabrese C, Poppleton H, Kocak M et al. A perivascular niche for brain tumor stem cells. Cancer Cell
2007; 11(1):69-82.
48. Gorski DH, Beckett MA, Jaskowiak NT et al. Blockage of the vascular endothelial growth factor stress
response increases the antitumor effects of ionizing radiation. Cancer Res 1999; 59(14):3374-3378.
49. Jain RK et al. Angiogenesis in brain tumours. Nat Rev Neurosci 2007; 8(8):610-622.
50. Chamberlain MC, Johnston SK. Salvage therapy with single agent bevacizumab for recurrent glioblastoma.
J Neurooncol 2010; 96(2):259-269.
51. Narayana A, Kelly P, Golfinos J et al. Antiangiogenic therapy using bevacizumab in recurrent high-grade
glioma: impact on local control and patient survival. J Neurosurg 2009; 110(1):173-180.
52. Norden AD, Young GS, Setayesh K et al. Bevacizumab for recurrent malignant gliomas: efficacy, toxicity
and patterns of recurrence. Neurology 2008; 70(10):779-787.
53. Ballman KV, Buckner JC, Brown PD et al. The relationship between six-month progression-free survival
and 12-month overall survival end points for phase II trials in patients with glioblastoma multiforme.
Neuro Oncol 2007; 9(1):29-38.
54. FFriedman HS, Petros WP, Friedman AH et al. Irinotecan therapy in adults with recurrent or progressive
malignant glioma. J Clin Oncol 1999; 17(5):1516-1525.
55. Wong ET, Hess KR, Gleason MJ et al. Outcomes and prognostic factors in recurrent glioma patients enrolled
onto phase II clinical trials. J Clin Oncol 1999; 17(8):2572-2578.
56. Yung WK et al. A phase II study of temozolomide vs. procarbazine in patients with glioblastoma multiforme
at first relapse. Br J Cancer 2000; 83(5):588-593.
57. Vredenburgh JJ, Desjardins A, Herndon JE 2nd et al. Bevacizumab plus irinotecan in recurrent glioblastoma
multiforme. J Clin Oncol 2007; 25(30):4722-4729.
58. Friedman HS, Prados MD, Wen PY et al. Bevacizumab alone and in combination with irinotecan in recurrent
glioblastoma. J Clin Oncol 2009; 27(28):4733-4740.
59. Reardon DA, Desjardins A, Vredenburgh JJ et al. Metronomic chemotherapy with daily, oral etoposide plus
bevacizumab for recurrent malignant glioma: a phase II study. Br J Cancer 2009; 101(12):1986-1994.
60. Gutin PH, Iwamoto FM, Beal K et al. Safety and efficacy of bevacizumab with hypofractionated stereotactic
irradiation for recurrent malignant gliomas. Int J Radiat Oncol Biol Phys 2009; 75(1):156-163.
61. Eller JL, Longo SL, Hicklin DJ et al. Activity of anti-epidermal growth factor receptor monoclonal antibody
C225 against glioblastoma multiforme. Neurosurgery 2002; 51(4):1005-13; discussion 1013-1014.
62. Perera RM, Narita Y, Furnari FB et al. Treatment of human tumor xenografts with monoclonal antibody
806 in combination with a prototypical epidermal growth factor receptor-specific antibody generates
enhanced antitumor activity. Clin Cancer Res 2005; 11(17):6390-6399.
63. Banerjee D, Matthews P, Matayeva E et al. Enhanced T-cell responses to glioma cells coated with the
anti-EGF receptor antibody and targeted to activating FcgammaRs on human dendritic cells. J Immunother
2008; 31(2):113-120.
64. Neyns B, Sadones J, Joosens E et al. Stratified phase II trial of cetuximab in patients with recurrent high-grade
glioma. Ann Oncol 2009; 20(9):1596-1603.
65. Diaz Miqueli A, Rolff J, Lemm M et al. Radiosensitisation of U87MG brain tumours by anti-epidermal
growth factor receptor monoclonal antibodies. Br J Cancer 2009; 100(6):950-958.
66. Ramos TC et al. Treatment of high-grade glioma patients with the humanized anti-epidermal growth factor
receptor (EGFR) antibody h-R3: report from a phase l/ll trial. Cancer Biol Ther 2006; 5(4):375-379.
67. Eller JL, Longo SL, Kyle MM et al. Anti-epidermal growth factor receptor monoclonal antibody cetuximab
augments radiation effects in glioblastoma multiforme in vitro and in vivo. Neurosurgery 2005;
56(1):155-162; discussion 162.
68. Amado RG, Wolf M, Peeters M et al. Wild-type KRAS is required for panitumumab efficacy in patients
with metastatic colorectal cancer. J Clin Oncol 2008; 26(10):1626-1634.
69. Giusti RM, Shastri KA, Cohen MH et al. FDA drug approval summary: panitumumab (Vectibix). Oncologist
2007; 12(5):577-583.
70. Giusti RM, Cohen MH, Keegan P et al. FDA review of a panitumumab (Vectibix) clinical trial for first-line
treatment of metastatic colorectal cancer. Oncologist 2009; 14(3):284-290.
71. Hecht JR, Mitchell E, Chidiac T et al. A randomized phase IIIB trial of chemotherapy, bevacizumab and
panitumumab compared with chemotherapy and bevacizumab alone for metastatic colorectal cancer. J
Clin Oncol 2009; 27(5):672-680.
72. Mineo JF, Bordron A, Quintin-Rou I et al. Recombinant humanised anti-HER2/neu antibody (Herceptin)
induces cellular death of glioblastomas. Br J Cancer 2004; 91(6):1195-1199.
73. Pillay V, Allaf L, Wilding AL et al. The plasticity of oncogene addiction: implications for targeted therapies
directed to receptor tyrosine kinases. Neoplasia 2009; 11(5):448-458, 2 p following 458.
74. Gusterson B, Cowley G, Smith JA et al. Cellular localisation of human epidermal growth factor receptor.
Cell Biol Int Rep 1984; 8(8):649-658.
75. Nagao K, Hisatomi H, Hirata H et al. Expression of molecular marker genes in various types of normal
tissue: implication for detection of micrometastases. Int J Mol Med 2002; 10(3):307-310.
76. Choi BD, Archer GE, Mitchell DA et al. EGFRvlll-targeted vaccination therapy of malignant glioma.
Brain Pathol 2009; 19(4):713-723.
77. Ekstrand AJ, Sugawa N, James CD et al. Amplified and rearranged epidermal growth factor receptor genes
in human glioblastomas reveal deletions of sequences encoding portions of the N- and/or C-terminal tails.
78. Bigner SH, Humphrey PA, Wong AJ et al. Characterization of the epidermal growth factor receptor in
human glioma cell lines and xenografts. Cancer Res 1990; 50(24):8017-8022.
79. Nishikawa R, Ji XD, Harmon RC et al. A mutant epidermal growth factor receptor common in human
glioma confers enhanced tumorigenicity. Proc Natl Acad Sci USA 1994; 91(16):7727-7731.
80. Wikstrand CJ, McLendon RE, Friedman AH et al. Cell surface localization and density of the tumor-associated
variant of the epidermal growth factor receptor, EGFRvlll. Cancer Res 1997; 57(18):4130-4140.
81. Sampson JH, Crotty LE, Lee S et al. Unarmed, tumor-specific monoclonal antibody effectively treats brain
tumors. Proc Natl Acad Sci USA 2000; 97(13):7503-7508.
82. Luwor RB, Johns TG, Murone C et al. Monoclonal antibody 806 inhibits the growth of tumor xenografts
expressing either the de2-7 or amplified epidermal growth factor receptor (EGFR) but not wild-type
EGFR. Cancer Res 2001; 61(14):5355-5361.
83. Mishima K, Johns TG, Luwor RB et al. Growth suppression of intracranialxenografted glioblastomas
overexpressing mutant epidermal growth factor receptors by systemic administration of monoclonal antibody
(mAb) 806, a novel monoclonal antibody directed to the receptor. Cancer Res 2001; 61(14):5349-5354.
84. Scott AM, Lee FT, Tebbutt N et al. A phase I clinical trial with monoclonal antibody ch806 targeting transitional
state and mutant epidermal growth factor receptors. Proc Natl Acad Sci USA 2007; 104(10):4071-4076.
85. Glassy MC, Hagiwara H. Summary analysis of the preclinical and clinical results of brain tumor patients
treated with pritumumab. Hum Antibodies 2009; 18(4):127-137.
86. Burgess T, Coxon A, Meyer S et al. Fully human monoclonal antibodies to hepatocyte growth factor with
therapeutic potential against hepatocyte growth factor/c-Met-dependent human tumors. Cancer Res
2006; 66(3):1721-179.
87. Kim KJ, Wang L, Su YC et al. Systemic anti-hepatocyte growth factor monoclonal antibody therapy induces
the regression of intracranial glioma xenografts. Clin Cancer Res 2006; 12(4):1292-1298.
88. Li Y, Guessous F, DiPierro C et al. Interactions between PTEN and the c-Met pathway in glioblastoma
and implications for therapy. Mol Cancer Ther 2009; 8(2):376-385.
89. Martens T, Schmidt NO, Eckerich C et al. A novel one-armed anti-c-Met antibody inhibits glioblastoma
growth in vivo. Clin Cancer Res 2006; 12(20 Pt 1):6144-6152.
90. Boskovitz A, Wikstrand CJ, Kuan CT et al. Monoclonal antibodies for brain tumour treatment. Expert
Opin Biol Ther 2004; 4(9):1453-1471.
91. Fiveash JB, Gillespie GY, Oliver PG et al. Enhancement of glioma radiotherapy and chemotherapy response
with targeted antibody therapy against death receptor 5. Int J Radiat Oncol Biol Phys 2008; 71(2):507-516.
92. Derui L, Woo DV, Emrich J et al. Radiotoxicity of 1251-labeled monoclonal antibody 425 against cancer
cells containing epidermal growth factor receptor. Am J Clin Oncol 1992; 15(4):288-294.
93. Foulon CF, Reist CJ, Bigner DD, et al. Radioiodination via D-amino acid peptide enhances cellular
retention and tumor xenograft targeting of an internalizing anti-epidermal growth factor receptor variant
III monoclonal antibody. Cancer Res 2000; 60(16):4453-4460.
94. Bourdon MA, Matthews TJ, Pizzo SV et al. Immunochemical and biochemical characterization of a
glioma-associated extracellular matrix glycoprotein. J Cell Biochem 1985; 28(3):183-195.
95. Bourdon MA, Wikstrand CJ, Furthmayr H et al. Human glioma-mesenchymal extracellular matrix antigen
defined by monoclonal antibody. Cancer Res 1983; 43(6):2796-2805.
96. Reardon DA, Zalutsky MR, Bigner DD. Antitenascin-C monoclonal antibody radioimmunotherapy for
malignant glioma patients. Expert Rev Anticancer Ther 2007; 7(5):675-687.
97. Riva P et al. Local treatment of malignant gliomas by direct infusion of specific monoclonal antibodies
labeled with 1311: comparison of the results obtained in recurrent and newly diagnosed tumors. Cancer
Res 1995; 55(23 Suppl):5952s-5956s.
98. Riva P, Franceschi G, Frattarelli M et al. 1311 radioconjugated antibodies for the locoregional
radioimmunotherapy of high-grade malignant glioma-phase I and II study. Acta Oncol 1999; 38(3):351-359.
99. Zalutsky MR, Moseley RP, Coakham HB, et al. Pharmacokinetics and tumor localization of 1311-labeled
anti-tenascin monoclonal antibody 81C6 in patients with gliomas and other intracranial malignancies.
Cancer Res 1989; 49(10):2807-2813.
100. Zalutsky MR, Moseley RP, Benjamin JC et al. Monoclonal antibody and F(abv)2 fragment delivery to
tumor in patients with glioma: comparison of intracarotid and intravenous administration. Cancer Res
1990; 50(13):4105-4110.
101. Schold SC Jr, Zalutsky MR, Coleman RE et al. Distribution and dosimetry of 1-123-labeled monoclonal
antibody 81C6 in patients with anaplastic glioma. Invest Radiol 1993; 28(6):488-496.
102. Bigner DD, Brown MT, Friedman AH et al. lodine-131-labeled antitenascin monoclonal antibody
81C6 treatment of patients with recurrent malignant gliomas: phase I trial results. J Clin Oncol 1998;
16(6):2202-2212.
103. Brown MT, Coleman RE, Friedman AH et al. Intrathecal 1311-labeled antitenascin monoclonal antibody
81C6 treatment of patients with leptomeningeal neoplasms or primary brain tumor resection cavities with
subarachnoid communication: phase I trial results. Clin Cancer Res 1996; 2(6):963-972.
104. Akabani G, Cokgor I, Coleman RE et al. Dosimetry and dose-response relationships in newly diagnosed
patients with malignant gliomas treated with iodine-131-labeled anti-tenascin monoclonal antibody 81C6
therapy. Int J Radiat Oncol Biol Phys 2000; 46(4):947-958.
105. Cokgor I, Akabani G, Kuan CT et al. Phase I trial results of iodine-131-labeled antitenascin monoclonal
antibody 81C6 treatment of patients with newly diagnosed malignant gliomas. J Clin Oncol 2000;
18(22):3862-3872.
106. Reardon DA, Akabani G, Coleman RE et al. Phase II trial of murine (131)l-labeled antitenascin monoclonal
antibody 81C6 administered into surgically created resection cavities of patients with newly diagnosed
malignant gliomas. J Clin Oncol 2002; 20(5):1389-1397.
107. Reardon DA, Zalutsky MR, Akabani G et al. A pilot study: 1311-antitenascin monoclonal antibody 81
c6 to deliver a 44-Gy resection cavity boost. Neuro Oncol 2008; 10(2):182-189.
108. Reardon DA, Akabani G, Coleman RE et al. Salvage radioimmunotherapy with murine iodine-131-labeled
antitenascin monoclonal antibody 81C6 for patients with recurrent primary and metastatic malignant brain
tumors: phase II study results. J Clin Oncol 2006; 24(1):115-122.
109. He X, Archer GE, Wikstrand CJ et al. Generation and characterization of a mouse/human chimeric
antibody directed against extracellular matrix protein tenascin. J Neuroimmunol 1994; 52(2):127-137.
110. Reardon DA, Quinn JA, Akabani G et al. Novel human lgG2b/murine chimeric antitenascin monoclonal
antibody construct radiolabeled with 1311 and administered into the surgically created resection cavity
of patients with malignant glioma: phase I trial results. J Nucl Med 2006; 47(6):912-918.
111. Zalutsky MR, Reardon DA, Akabani G et al. Clinical experience with alpha-particle emitting 211 At:
treatment of recurrent brain tumor patients with 211At-labeled chimeric antitenascin monoclonal antibody
81C6. J Nucl Med 2008; 49(1):30-38.
112. Casac A, Lpez G, Garca I et al. Phase I single-dose study of intracavitary-administered Nimotuzumab
labeled with 188 Re in adult recurrent high-grade glioma. Cancer Biol Ther 2008; 7(3):333-339.
113. Emrich JG, Bender H, Class R et al. In vitro evaluation of iodine-125-labeled monoclonal antibody (MAb
425) in human high-grade glioma cells. Am J Clin Oncol 1996; 19(6):601-608.
114. Emrich JG, Hand CM, Dilling TJ et al. Biodistribution of 1251-MAb 425 in a human glioma xenograft
model: effect of chloroquine. Hybridoma 1997; 16(1):93-100.
115. Brady LW, Miyamoto C, Woo DV et al. Malignant astrocytomas treated with iodine-125 labeled
monoclonal antibody 425 against epidermal growth factor receptor: a phase II trial. Int J Radiat Oncol
Biol Phys 1992; 22(1):225-230.
116. Emrich JG, Brady LW, Quang TS et al. Radioiodinated (1-125) monoclonal antibody 425 in the treatment of
high grade glioma patients: ten-year synopsis of a novel treatment. Am J Clin Oncol 2002; 25(6):541-546.
117. Debinski W, Obiri NI, Powers SK et al. Human glioma cells overexpress receptors for interleukin 13
and are extremely sensitive to a novel chimeric protein composed of interleukin 13 and pseudomonas
exotoxin. Clin Cancer Res 1995; 1(11):1253-1258.
118. Joshi BH, Leland P, Asher A et al. In situ expression of interleukin-4 (IL-4) receptors in human brain
tumors and cytotoxicity of a recombinant IL-4 cytotoxin in primary glioblastoma cell cultures. Cancer
Res 2001; 61(22):8058-8061.
119. Kawakami M, Kawakami K, Puri RK. lnterleukin-4-Pseudomonasexotoxin chimeric fusion protein for
malignant glioma therapy. J Neurooncol 2003; 65(1):15-25.
120. Kunwar S, Chang SM, Prados MD et al. Safety of intraparenchymal convection-enhanced delivery of
cintredekin besudotox in early-phase studies. Neurosurg Focus 2006; 20(4):E15.
121. Mut M, Sherman JH, Shaffrey ME et al. Cintredekin besudotox in treatment of malignant glioma. Expert
Opin Biol Ther 2008; 8(6):805-812.
122. Vogelbaum MA, Sampson JH, Kunwar S et al. Convection-enhanced delivery of cintredekin besudotox
(interleukin-13-PE38QQR) followed by radiation therapy with and without temozolomide in newly
diagnosed malignant gliomas: phase 1 study of final safety results. Neurosurgery 2007; 61(5):1031-7;
discussion 1037-1038.
123. Oh S, Ohlfest JR, Todhunter DA et al. Intracranial elimination of human glioblastoma brain tumors in
nude rats using the bi sped fie ligand-directed toxin, DTEGF13 and convection enhanced delivery. J
Neurooncol 2009; 95(3):331-342.
124. Weber F, Asher A, Bucholz R et al. Safety, tolerability and tumor response of IL4-Pseudomonas exotoxin
(NBI-3001) in patients with recurrent malignant glioma. J Neurooncol 2003; 64(1-2):125-137.
125. Sampson JH, Akabani G, Archer GE et al. Progress report of a Phase I study of the intracerebral microinfusion
of a recombinant chimeric protein composed of transforming growth factor (TGF)-alpha and a mutated
form of the Pseudomonas exotoxin termed PE-38 (TP-38) for the treatment of malignant brain tumors.
J Neurooncol 2003; 65(1):27-35.
126. Sampson JH, Reardon DA, Friedman AH et al. Sustained radiographic and clinical response in patient
with bifrontal recurrent glioblastoma multiforme with intracerebral infusion of the recombinant targeted
toxin TP-38: case study. Neuro Oncol 2005; 7(1):90-96.
127. Kioi M, Seetharam S, Puri RK. Targeting IL-13Ralpha2-positive cancer with a novel recombinant
immunotoxin composed of a single-chain antibody and mutated Pseudomonas exotoxin. Mol Cancer
Ther 2008; 7(6):1579-1587.
128. Archer GE, Sampson JH, Lorimer IA et al. Regional treatment of epidermal growth factor receptor
vlIlex pressing neoplastic meningitis with a single-chain immunotoxin, MR-1. Clin Cancer Res 1999;
5(9):2646-2652.
129. Beers R, Chowdhury P, Bigner D et al. Immunotoxins with increased activity against epidermal growth factor
receptor vlll-expressing cells produced by antibody phage display. Clin Cancer Res 2000; 6(7):2835-2843.
130. Ochiai H, Archer GE, Herndon JE 2nd et al. EGFRvlll-targeted immunotoxin induces antitumor immunity
that is inhibited in the absence of CD4
and CD8
T-cells. Cancer Immunol Immunother 2008; 57(1):115-121.
131. Barth RF, Coderre JA, Vicente MG et al. Boron neutron capture therapy of cancer: current status and
future prospects. Clin Cancer Res 2005; 11(11):3987-4002.
132. Barth RF, Wu G, Yang W et al. Neutron capture therapy of epidermal growth factor (
) gliomas using
boronated cetuximab (IMC-C225) as a delivery agent. Appl Radiat Isot 2004; 61(5):899-903.
133. Wu G, Yang W, Barth RF et al. Molecular targeting and treatment of an epidermal growth factor
receptor-positive glioma using boronated cetuximab. Clin Cancer Res 2007; 13(4):1260-1280.
134. Yang W, Barth RF, Wu G et al. Boron neutron capture therapy of EGFR or EGFRvlll positive gliomas
using either boronated monoclonal antibodies or epidermal growth factor as molecular targeting agents.
Appl Radiat Isot 2009; 67(7-8 Suppl):S328-31.
135. Yang W, Barth RF, Wu G et al. Boronated epidermal growth factor as a delivery agent for neutron capture
therapy of EGF receptor positive gliomas. Appl Radiat Isot 2004; 61(5):981-985.
136. Yang W, Barth RF, Wu G et al. Molecular targeting and treatment of EGFRvlll-positive gliomas using
boronated monoclonal antibody L8A4. Clin Cancer Res 2006; 12(12):3792-3802.
137. Mamot C, Drummond DC, Noble CO et al. Epidermal growth factor receptor-targeted immunoliposomes
significantly enhance the efficacy of multiple anticancer drugs in vivo. Cancer Res 2005; 65(24):11631-1168.
138. Feng B, Tomizawa K, Michiue H et al. Delivery of sodium borocaptate to glioma cells using immunoliposome
conjugated with anti-EGFR antibodies by ZZ-His. Biomaterials 2009; 30(9):1746-1755.
139. Pan X, Wu G, Yang W et al. Synthesis of cetuximab-immunoliposomes via a cholesterol-based membrane
anchor for targeting of EGFR. Bioconjug Chem 2007; 18(1):101-108.
140. Zhang Y, Zhang YF, Bryant J et al. Intravenous RNA interference gene therapy targeting the human
epidermal growth factor receptor prolongs survival in intracranial brain cancer. Clin Cancer Res 2004;
10(11):3667-377.
brain tumors. J Neuroimmunol 1999; 100(1-2):216-232.
142. Roszman T, Elliott L, Brooks W. Modulation of T-cell function by gliomas. Immunol Today 1991;
12(10):370-374.
143. Morford LA, Elliott LH, Carlson SL et al. T-cell receptor-mediated signaling is defective in T-cells obtained
from patients with primary intracranial tumors. J Immunol 1997; 159(9):4415-4425.
144. Roszman TL, Brooks WH. Immunobiology of primary intracranial tumours. III. Demonstration of a
qualitative lymphocyte abnormality in patients with primary brain tumours. Clin Exp Immunol 1980;
39(2):395-402.
145. Gershon RK, Kondo K. Infectious immunological tolerance. Immunology 1971; 21(6):903-914.
146. Sakaguchi S, Sakaguchi N, Asano M et al. Immunologic self-tolerance maintained by activated T-cells
expressing IL-2 receptor alpha-chains (CD25). Breakdown of a single mechanism of self-tolerance causes
various autoimmune diseases. J Immunol 1995; 155(3):1151-1164.
147. Thornton AM, Shevach EM. CD4
CD25
immunoregulatory T-cells suppress polyclonal T-cell activation
in vitro by inhibiting interleukin 2 production. J Exp Med 1998; 188(2):287-296.
148. Jonuleit H, Schmitt E, Stassen M et al. Identification and functional characterization of human CD4(
)CD25(
)
T-cells with regulatory properties isolated from peripheral blood. J Exp Med 2001; 193(11):1285-1294.
149. Dieckmann D, Plottner H, Berchtold S et al. Ex vivo isolation and characterization of CD4(
)CD25(
)
T-cells with regulatory properties from human blood. J Exp Med 2001; 193(11):1303-1310.
150. El Andaloussi A, Lesniak MS. An increase in CD4
CD25
FOXP3
regulatory T-cells in tumor-infiltrating
lymphocytes of human glioblastoma multiforme. Neuro Oncol 2006; 8(3):234-243.
151. Fecci PE, Mitchell DA, Whitesides JF et al. Increased regulatory T-cell fraction amidst a diminished
CD4 compartment explains cellular immune defects in patients with malignant glioma. Cancer Res
2006; 66(6):3294-3302.
152. Learn CA, Fecci PE, Schmittling RJ et al. Profiling of CD4
, CD8
and CD4
CD25
CD45RO
FoxP3
T-cells in patients with malignant glioma reveals differential expression of the immunologic transcriptome
compared with T-cells from healthy volunteers. Clin Cancer Res 2006; 12(24):7306-7315.
153. El Andaloussi AY, Han, Lesniak MS. Prolongation of survival following depletion of CD4
CD25
regulatory
T-cells in mice with experimental brain tumors. J Neurosurg 2006; 105(3):430-437.
154. Kohm AP, McMahon JS, Podojil JR et al. Cutting Edge: Anti-CD25 monoclonal antibody injection
results in the functional inactivation, not depletion, of CD4
CD25
T regulatory cells. J Immunol 2006;
176(6):3301-3305.
155. Fecci PE, Sweeney AE, Grossi PM et al. Systemic anti-CD25 monoclonal antibody administration safely
enhances immunity in murine glioma without eliminating regulatory T-cells. Clin Cancer Res 2006;
12(14 Pt 1):4294-4305.
156. Linsley PS, Greene JL, Brady W et al. Human B7-1 (CD80) and B7-2 (CD86) bind with similar avidities
but distinct kinetics to CD28 and CTLA-4 receptors. Immunity 1994; 1(9):793-801.
157. Thompson CB, Allison JP. The emerging role of CTLA-4 as an immune attenuator. Immunity 1997;
7(4):445-450.
158. Walunas TL, Lenschow DJ, Bakker CY et al. CTLA-4 can function as a negative regulator of T-cell
activation. Immunity 1994; 1(5):405-413.
159. Fecci PE, Ochiai H, Mitchell DA et al. Systemic CTLA-4 blockade ameliorates glioma-induced changes
to the CD4
T-cell compartment without affecting regulatory T-cell function. Clin Cancer Res 2007;
13(7):2158-2167.
FoxP3
121(1):95-105.
161. Fontana A, Hengartner H, de Tribolet N et al. Glioblastoma cells release interleukin 1 and factors inhibiting
interleukin 2-mediated effects. J Immunol 1984; 132(4):1837-1844.
162. Wrann M, Bodmer S, de Martin R et al. T-cell suppressor factor from human glioblastoma cells is a 12.5-kd
protein closely related to transforming growth factor-beta. EMBO J 1987; 6(6):1633-1636.
163. Kuppner MC, Hamou MF, Sawamura Y et al. Inhibition of lymphocyte function by glioblastoma-derived
transforming growth factor beta 2. J Neurosurg 1989; 71(2):211-217.
164. Fontana A, Frei K, Bodmer S et al. Transforming growth factor-beta inhibits the generation of cytotoxic
T-cells in virus-infected mice. J Immunol 1989; 143(10):3230-3234.
165. Kehrl JH, Wakefield LM, Roberts A et al. Production of transforming growth factor beta by human
T-lymphocytes and its potential role in the regulation of T-cell growth. J Exp Med 1986; 163(5):1037-1050.
166. Gronski P, Seiler FR, Schwick HG. Discovery of antitoxins and development of antibody preparations
for clinical uses from 1890 to 1990. Mol Immunol 1991; 28(12):1321-1332.
167. Karelitz S, Serum sickness. Ann N Y Acad Sci 1949; 50(Art. 7):705-717.
168. von-Pirquet C, Schick B. Die serumkrankheit. Franz Deuticke, Wien und Leipzig 1905.
169. Batra SK, Jain M, Wittel UA et al. Pharmacokinetics and biodistribution of genetically engineered
antibodies. Curr Opin Biotechnol 2002; 13(6):603-608.
170. Jones PT, Dear PH, Foote J et al. Replacing the complementarity-determining regions in a human antibody
with those from a mouse. Nature 1986; 321(6069):522-525.
171. Reist CJ, Bigner DD, Zalutsky MR. Human lgG2 constant region enhances in vivo stability of anti-tenascin
antibody 81C6 compared with its murine parent. Clin Cancer Res 1998; 4(10):2495-2502.
172. Riechmann L, Clark M, Waldmann H et al. Reshaping human antibodies for therapy. Nature 1988;
332(6162):323-327.
173. Shin SU. Chimeric antibody: potential applications for drug delivery and immunotherapy. Biotherapy
1991; 3(1):43-53.
174. Green LL. Antibody engineering via genetic engineering of the mouse: XenoMouse strains are a vehicle for
the facile generation of therapeutic human monoclonal antibodies. J Immunol Methods 1999; 231(1-2):11-23.
175. Lynch DH, Yang XD. Therapeutic potential of ABX-EGF: a fully human anti-epidermal growth factor
receptor monoclonal antibody for cancer treatment. Semin Oncol 2002; 29(1 Suppl 4):47-50.
176. Barbi T, Drake PM, Drever M et al. Single-chain antigen-binding proteins. Science 1988; 242(4877):423-426.
177. Huston JS, Levinson D, Mudgett-Hunter M et al. Protein engineering of antibody binding sites: recovery
of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia coli. Proc Natl
Acad Sci USA 1988; 85(16):5879-5883.
178. Reiter Y, Brinkmann U, Kreitman RJ et al. Stabilization of the Fv fragments in recombinant immunotoxins
by disulfide bonds engineered into conserved framework regions. Biochemistry 1994; 33(18):5451-5459.
179. Begent RH, Verhaar MJ, Chester KA et al. Clinical evidence of efficient tumor targeting based on
single-chain Fv antibody selected from a combinatorial library. Nat Med 1996; 2(9):979-984.
180. Yokota T, Milenic DE, Whitlow M et al. Rapid tumor penetration of a single-chain Fv and comparison
with other immunoglobulin forms. Cancer Res 1992; 52(12):3402-3408.
181. Kuan CT, Reist CJ, Foulon CF et al. 1251-labeled anti-epidermal growth factor receptor-vlll single-chain
Fv exhibits specific and high-level targeting of glioma xenografts. Clin Cancer Res 1999; 5(6):1539-1549.
182. Schier R, McCall A, Adams GP et al. Isolation of picomolar affinity anti-c-erbB-2 single-chain Fv by
molecular evolution of the complementarity determining regions in the center of the antibody binding
site. J Mol Biol 1996; 263(4):551-567.
183. Kuan CT, Wikstrand CJ, Archer G et al. Increased binding affinity enhances targeting of glioma xenografts
by EGFRvlll-specific scFv. Int J Cancer 2000; 88(6):962-929.
184. Amann M, Brischwein K, Lutterbuese P et al. Therapeutic window of MuS110, a single-chain antibody
construct bispecific for murine EpCAM and murine CD3. Cancer Res 2008; 68(1):143-151.
185. Baeuerle PA, Reinhardt C. Bispecific T-cell engaging antibodies for cancer therapy. Cancer Res 2009;
69(12):4941-4944.
186. Bluemel C, Hausmann S, Fluhr P et al. Epitope distance to the target cell membrane and antigen size
determine the potency of T-cell-mediated lysis by BiTE antibodies specific for a large melanoma surface
antigen. Cancer Immunol Immunother 2010; 59:1197-1209.
187. Nagorsen D, Bargou R, Ruttinger D et al. Immunotherapy of lymphoma and leukemia with T-cell engaging
BiTE antibody blinatumomab. Leuk Lymphoma 2009; 50(6):886-981.
188. Schlereth B, Fichtner I, Lorenczewski G et al. Eradication of tumors from a human colon cancer cell line
and from ovarian cancer metastases in immunodeficient mice by a single-chain Ep-CAM-/CD3-bispecific
antibody construct. Cancer Res 2005; 65(7):2882-2889.
189. Schlereth B, Kleindienst P, Fichtner I et al. Potent inhibition of local and disseminated tumor growth in
immunocompetent mouse models by a bispecific antibody construct specific for Murine CD3. Cancer
Immunol Immunother 2006; 55(7):785-796.
190. Offner S, Hofmeister R, Romaniuk A et al. Induction of regular cytolytic T-cell synapses by bispecific
single-chain antibody constructs on MHC class l-negative tumor cells. Mol Immunol 2006; 43(6):763-771.
191. Bargou R, Leo E, Zugmaier G et al. Tumor regression in cancer patients by very low doses of a
T-cell-engaging antibody. Science 2008; 321(5891):974-977.
192. Brischwein K, Schlereth B, Guller B et al. MT110: a novel bispecific single-chain antibody construct with
high efficacy in eradicating established tumors. Mol Immunol 2006; 43(8):1129-1143.
193. Eshhar Z, Waks T, Gross G et al. Specific activation and targeting of cytotoxic lymphocytes through
chimeric single chains consisting of antibody-binding domains and the gamma or zeta subunits of the
immunoglobulin and T-cell receptors. Proc Natl Acad Sci USA 1993; 90(2):720-724.
194. Gross G, Eshhar Z. Endowing T-cells with antibody specificity using chimeric T-cell receptors. FASEB J
1992; 6(15):3370-3378.
195. Sadelain M, Brentjens R, Riviere I. The promise and potential pitfalls of chimeric antigen receptors. Curr
Opin Immunol 2009; 21(2):215-223.
196. Kershaw MH, Westwood JA, Parker LL et al. A phase I study on adoptive immunotherapy using
gene-modified T-cells for ovarian cancer. Clin Cancer Res 2006; 12(20 Pt 1):6106-6115.
197. Lamers CH, Sleijfer S, Vulto AG et al. Treatment of metastatic renal cell carcinoma with autologous
T-lymphocytes genetically retargeted against carbonic anhydrase IX: first clinical experience. J Clin
Oncol 2006; 24(13):e20-2.
198. Pule MA, Savoldo B, Myers GD et al. Virus-specific T-cells engineered to coexpress tumor-specific receptors:
persistence and antitumor activity in individuals with neuroblastoma. Nat Med 2008; 14(11):1264-1270.
199. Till BG, Jensen MC, Wang J et al. Adoptive immunotherapy for indolent nonHodgkin lymphoma and
mantle cell lymphoma using genetically modified autologous CD20-specific T-cells. Blood 2008;
112(6):2261-2271.
200. Ahmed N, Ratnayake M, Savoldo B et al. Regression of experimental medulloblastoma following transfer
of HER2-specific T-cells. Cancer Res 2007; 67(12):5957-5964.
201. Morgan RA, Yang JC, Kitano M et al. Case report of a serious adverse event following the administration
of T-cells transduced with a chimeric antigen receptor recognizing ERBB2. Mol Ther 2010; 18(4):843-851.
202. Kahlon KS, Brown C, Cooper LJ et al. Specific recognition and killing of glioblastoma multiforme by
interleukin 13-zetakine redirected cytolytic T-cells. Cancer Res 2004; 64(24):9160-9166.
203. Yaghoubi SS, Jensen MC, Satyamurthy N et al. Noninvasive detection of therapeutic cytolytic T-cells
with 18F-FHBG PET in a patient with glioma. Nat Clin Pract Oncol 2009; 6(1):53-58.
PART IV
ACTIVE IMMUNOTHERAPY
CHAPTER 11
ANIMAL MODELS FOR VACCINE THERAPY
Dong-Sup Chung,1 Chang-Hyun Kim2 and Yong-Kil Hong*,1

1
Department of Neurosurgery, Seoul St. Marys Hospital, The Catholic University of Korea, Seoul, Republic
of Korea; 2Medical Science Research Center, Dongguk University Research Insititute of Biotechnology,
Gyeonggi-do, Republic of Korea
*Corresponding Author: Yong-Kil HongEmail: hongyk@catholic.ac.kr
Abstract: Animal models are important for defining paradigms of tumor immunology and for
evaluating therapeutic efficacy of immunotherapy. Many animal models have been
used for evaluating in vivo characteristics of malignant gliomas and their responses
to therapy. No animal model, however, is perfect because malignant glioma has
a very heterogeneous biological behavior. There are so many parallels between
mouse and human immunology, but there are significant discrepancies in immune
system. Animal models for vaccine therapy can be classified as transplantable
tumor models and models of spontaneous tumor in genetically engineered animals.
Although transplantable tumor models have been used to test immunotherapeutic
efficacy and remain a mainstay in study of brain tumor immunology, a lot of tumor
vaccines that look promising in experimental animals have turned out to be ineffective
clinically. Recent advances of laboratory techniques and understanding of genetic
and molecular characteristics of gliomas allows for animal models of gliomas with
similar biologic characteristics. Well-designed glioma models that accurately reflect
the biology, pathology and clinical behaviors of human gliomas can provide more
useful preclinical informations to predict clinical efficacy of novel immunotherapies
and cancer vaccines.
INTRODUCTION
Animal models are important for defining paradigms of tumor immunology and for
evaluating therapeutic efficacy of immunotherapy because they provide an in vivo milieu.
Many animal models have been used for evaluating in vivo characteristics of malignant
gliomas and their responses to therapy. No animal model, however, is perfect because
malignant glioma has a very heterogeneous biological behavior. Moreover, there has been

143
144 GLIOMA: IMMUNOTHERAPEUTIC APPROACHES TITLE
a tendency to ignore differences because there are so many parallels between mouse and
human immunology, but there are significant discrepancies in immune system. Those
differences should be taken into consideration when using mice models in preclinical
testing for therapeutic efficacy of human disease. One of the major limitations to improve
the prognosis of malignant gliomas has been the lack of available animal models that
accurately reflect the biology, neuropathology and clinical behaviors of these tumors.
Animal models for vaccine therapy can be classified as transplantable tumor models and
models of spontaneous tumor in genetically engineered animals. Although transplantable
tumor models have been used to test immunotherapeutic efficacy and remain a mainstay
in study of brain tumor immunology, many of these tumor models seem to be not good
predictors for human clinical trials because a lot of tumor vaccines that look promising in
experimental animals turned out to be ineffective clinically. Recent advances of genomics
can recapitulate the casual genetic events in mice and consequent molecular evolution
of gliomas as they form in situ.
TRANSPLANTABLE TUMOR MODELS
Transplantable animal brain tumor models were usually generated by implantation

or inoculation of primary animal glioma cells into immunocompetent rats or mice
subcutaneously (under the skin) or orthotopically (into native tumor site). Transplantable
brain tumor models are characterized by synchrony and reproducibility of tumor formation,
rapid tumor development, high penetrance and commercially available immunocompetent
syngeneic recipient B6C3F1 mice. Immunotherapy protocols for patients with malignant
gliomas should be based on demonstrated efficacy in a syngeneic animal brain tumor
model. The first prerequisite of animal models for assessing immunotherapeutic responses
is syngeneicity of original glioma cells and animals since immune rejection mechanisms
in nonsyngeneic systems can mimic therapeutic efficacy.
Several syngeneic rodent models such as the 9L Fischer model are currently used for
studying immunotherapy protocols. The 9L cell line is a gliosarcoma cell line chemically
induced in CD Fischer rats. On the other hand, C6 rat glioma cells cloned from an
N-nitrosomethylurea transformed rat astrocyte cell line express a MHC which is allogeneic
to Wistar rat. So Wistar rats with C6 tumors are not suitable for studying immunotherapy
because allogeneicity of tumor cells to animals induce a vigorous immune reaction.
In a murine glioma model, although a primarily cultured cell line derived from an
astrocytoma that arose spontaneously in a mouse might have been used in generating
animal model to study primary brain tumors including antiglioma immune responses,
the GL26 or GL261 murine glioma models have usually been used extensively. The
methylcholanthrene-induced murine anaplastic ependymoblastoma, GL26, propagated by
serial subcutaneous transplantation in C57BL/6 mice is not immunogenic and is suitable
for studying immunotherapy against gliomas.
Transplantable brain tumor models that inbred animals are inoculated with passaged
tumor cells derived from the same genetic strain have been used in tumor immunology
research for a long time. This approach is convenient and has led to valuable information.
They have, however, several limitations in their applicability to human disease and these
limitations may lead to different immunological outcomes. First, most tansplantable
brain tumors that were inoculated by numerous passaged cell lines might have altered
genetics according to culture or isolation conditions. As a result, animal models for
ANIMAL MODELS FOR VACCINE THERAPY 145
brain tumors that are well circumscribed, lack histologically accurate vascularization
and lack their native tumor stroma do not resemble the histology of human gliomas.
Moreover, they may no longer be fully syngeneic with original tumors such as C6/Wista
rat intracerebral glioma model. Second, transplantable tumors generally grow very
rapidly after tumor cell inoculation compared with spontaneous human tumors. The
immune system of humans is slowly accommodated to tumors, whereas the immune
system of animals with transplanted brain tumors is abruptly exposed. So transplantable
tumor models may not reflect the characteristics of original tumor and make it hard to
recognize immunotherapeutic efficacy.
Transplantable tumors have also been derived from spontaneous tumors that arise
in genetically engineered mice. These tumors have been used in conjunction with the
spontaneous models because they are syngeneic with their spontaneous tumor counterparts.
For example, a malignant glioma transplantable model was developed and characterized
by using the cell lines Tu-9548 and Tu-2449, which were previously isolated from two
different tumors that arose spontaneously in glial fibrillary acidic protein (GFAP)-v-src
transgenic mice. These GFAP-v-src gliomas closely match characteristic features of
human malignant gliomas in the aspects of astrogliosis, dysplastic changes, transition
from low-grade to high-grade astrocytomas, diffuse infiltration, endothelial proliferation
and necrosis.
SPONTANEOUS TUMOR MODELS
Spontaneous tumor models in genetically engineered mice are appealing for tumor
study due to predictability of the tumor-initiating lesion, immunocompetence and tumor
development at the appropriate site. Such tumor formation reflects complex processes of
tumor genetic alteration, angiogenesis, tumor-host interactions and metastasis to distant
sites. Possibility of controlled manipulation of genes and resultant tumor formation
has tremendous implications for the study of tumor causality and provides ideal models
to assess the efficacy of experimental therapies for tumors. Further, these models are
advantageous for predicting preclinical efficacy of novel immunotherapeutic strategies
as the mice bearing tumors are immunocompetent and develop tumors that have the same
antigenic profile as their host organism.
Preferentially, characterization of genetic alterations in the critical steps of glioma
development is highly informative to spontaneous brain tumor model. Genetic modification
can be achieved with the gene transfer (transgenesis) using viral vectors or nonviral
transfection and targeted gene deletion through homologous recombination (genetic
knockouts). A number of genetically engineered mice are available, harboring constitutive
or conditional alleles of genes associated with malingnant glioma development.
Overexpression of EGFR and p53 mutations are mutually exclusive in the development
of primary and secondary GBM. Although EGFR or p53 expresses in gliomas frequently,
individual EGFR or p53 mutations do not lead to glioma formation in mice. Expression
of a constitutively active mutant EGFR in glial cells with tumor suppressor gene deletions
induces tumor formation like human glioma in mice. These tumors were also commonly
induced in INK4a-ARF-deficient mice. Early inactivation of p53 tumor suppressor gene
with additional NF1 loss induces malignant glioma in mice. Addition of mutant PTEN
allele to this model decreases latency and increases tumor grade.
PTEN, a tumor suppressor gene, was found to be either mutated or not expressed in
the considerable ratio of GBMs and loss of PTEN function provides an oncogenic signal
resulting in activation of Akt. Although inactivation of PTEN by itself does not lead
to glioma formation, both postnatal PTEN inactivation and EGFRvIII expression in
RasB8 mice potentiate high grade glioma development.
The Ras pathway plays a critical role in glioma development and the role of the Ras
pathways has also already been explored with transgenic mice. Combined activation of
Ras and Akt in neural progenitor cells induces glioblastoma formation in mice. Acivation
of KRas and Akt in both astrocytes and glial progenitors cooperating with deletions in
tumor suppressor locus INK4a-ARF also leads GBM formation in mice. A series of
transgenic lines has been established based on astrocyte-specific expression of activated
p21-ras using GFAP promoter to express oncogenic V(12)Ha-ras leads to the formation
of malignant astrocytoma in a transgenic mouse model.- These transgenic astrocytomas
are pathologically similar to human astrocytomas and tumor grade appears to depend
on transgene dosage. Moreover, these tumors exhibit additional molecular alterations
associated with human astrocytomas, including a decreased or absent expression of p16,
p19 and PTEN as well as overexpression of EGFR, MDM2 (p53 antagonist) and CDK4 (cell
cycle regulator). Activation of EGFRvIII in a Ras transgenic mice can induce the formation
of tumors that resemble human oligodendrogliomas and mixed oligoastrocytomas. A
similar murine model of oligodendroglioma utilizes S100` promoter to generate transgenic
mice expressing v-erbB, a transforming allele of EGFR.
Spontaneous malignant gliomas can be induced by intracerebral injection of murine
retroviral vector encoding a gene of interest leads to transgene expression in the desired
cell type such as platelet-derived growth factor.- In this approach, the avian leukosis
virus (ALV)-based replication-competent ALV splice-acceptor (RCAS) vectors were
injected into the brain of neonatal mice expressing the RCAS receptor, tv-a (a receptor
for the ALV envelope lycoprotein) from GFAP or the nestin promoter. Mouse GBM can
also be induced in adult immunocompetent mice by injecting lentiviral vectors expressing
Harvey-Ras (H-Ras) and AKT into the brain of mice.
To generate spontaneous brain tumor mouse model, transgenic mice or viral vectors
have been required because nonviral gene transfer is transient. Recently, a spontaneous
malignant glioma model was developed using the Sleeping Beauty transposable element
delivered as plasmid DNA to achieve chromosomal integration and longterm expression
of human oncogenes into endogenous brain cells of immunocompetent mice. Another
approach to generate spontaneous glioma in mice is a virally mediated glioma driven
by a constitutively active fusion receptor tyrosine kinase (RTK). In this model, a
glioblastoma-associated, ligand-independent rearrangement product of ROS (FIG-ROS)
cooperates with loss of the tumor suppressor gene locus INK4a-ARF to produce
glioblastomas in mice.
MONITORING OF THE ANIMAL TUMORS
Not all animals predictably develop tumors with 100% penetrance whether they receive
tumor cell inoculation or they have genetic modification. Therefore, novel diagnostic
tools are needed to allow feasible experiments using animal models.
Magnetic resonance imaging (MRI) have been utilized for screening and detecting
brain tumors in mice. Imaging of gliomas in mice reveals similarities to those observed
in humans with identical pathology. Bioluminescence imaging technology, which

visualizes the conversion of chemical energy into visible light by luciferase enzymes
has been used to measure cell proliferation as well as to monitor the formation of grafted
tumors in vivo. Luciferase transgene is expressed primarily in dividing cells and
mediates the release of light in the presence of the bioluminescent compound luciferin.
Consequently, rapidly dividing tumor cells release sufficient light so as to be detectable
by sensitive luminometer.
MONITORING OF THE IMMUNE FUNCTION IN ANIMAL MODELS
Because it is still not yet clearly known which are the most important immune
effectors induced after vaccination, both humoral and cellular immune responses
should be measured. For an antibody response, an established standard techniquethe
enzyme-linked immunosorbent assay (ELISA)is the most reliable assay that can be
used to look for antitumour activity. For T cells, the frequency of tumour-specific T cells
is difficult to assess using standard assays. Two sensitive in vitro assays namely IFN-a
ELISPOT and HLA-tetramers are being used. Fresh peripheral blood mononuclear cells
can be used directly in the assays. However, a single round of in vitro stimulation can
be carried out in the case of weak responses. The fact that HLA-tetramers detect both
antigen-stimulated and naive T cells and IFN-a ELISPOT only measures the number of
memory and effector cells makes the latter a more appropriate and reliable technique
for discriminating between naive and vaccine-induced T cells. Finally, the in vivo assay
DTH is being used in several clinical trials and DTH responses seem to correlate with
clinical responses.
CHEMOIMMUNOTHERAPY IN GL26 GLIOMA MODEL
Most tumors express an array of antigens that act as targets for their immune-mediated
destruction and a number of potential therapies have emerged to exploit this. The
immunotherapeutic strategy to induce an immune response against tumors is quite attractive
because it offers the potential for high tumor-specific cytotoxicity, minimal side effect
and a durable effect. Dendritic cells (DCs) are the most potent antigen-presenting cells
(APCs) in the induction of primary immune responses. Because of their central role
in controlling cell-mediated immunity, DCs hold much promise as cellular adjuvants
in therapeutic cancer vaccines. DCs based immunotherapy has been reported to induce
strong antitumor immune responses in animal experiments and in selected clinical trials
involving malignant glioma. However, its clinical effects on patients with malignancy
have not been up to the expectation because of immune tolerance, the sheer physical
burden of tumor antigens and tumor escape mechanisms from the immune surveillance
system, among others.
Multimodality treatments that combine conventional tumor therapies with
immunotherapy, such as DC-based vaccine, have emerged as a potentially plausible
antitumor approach. We previously reported that a multimodality treatment regimen
using DCs-based vaccine in combination with chemotherapeutic agent TMZ leads to an
enhanced tumor-specific CTL responses and enhanced antitumor effects, resulting in a
higher cure rate than either DC-based vaccine or TMZ alone. The combination of low-dose
TMZ chemotherapy and tumor antigen-pulsed DCs immunotherapy may have potential
as a safe and effective therapeutic strategy for the malignant gliomas with minimal tumor
burden and its enhancing antitumor mechanisms may involve cross priming mediated by
increased caireticulin (CRT) exposure and suppression of regulatory T cells (Treg) in part
(Unpublished data). Maximizing the effect of the combined protocols, treatment doses,
treatment numbers, treatment timing, administration routes and optimizing treatment
schedules of TMZ chemotherapy are goals that will be pursued.
CONCLUSION
One of the major limitations to improve the prognosis of patients with malignant
gliomas has been the lack of available animal models similar to humans. Recent advances
of laboratory techniques and understanding of genetic and molecular characteristics
of gliomas allows for animal models of gliomas with similar biologic characteristics.
Well-designed glioma models that accurately reflect the biology, pathology and clinical
behaviors of human gliomas can provide more useful preclinical informations to predict
clinical efficacy of novel immunotherapies and cancer vaccines.
ACKNOWLEDGEMENTS
Funding by National R&D Program for Cancer Control, Korea (0720330).
REFERENCES
1. Mestas J, Hughes CCW. Of mice and not men: differences between mouse and human immunology.
J Immunol 2004; 172:2731-2738.
2. Fomchenko EI, Holland EC. Mouse models of brain tumors and their applications in preclinical trials. Clin
Cancer Res 2006; 12:5288-5297.
3. Smilowitz HM, Weissenberger J, Weis J et al. Orthotopic transplantation of v-src-expressing glioma cell
lines into immunocompetent mice: establishment of a new transplatanble in vivo model for malignant
glioma. J Neurosurg 2007; 106:652-659.
4. Barth RF. Rat brain tumor models in experimental neuro-oncology: the 9L, C6, T9, F98, RG2 (D74), RT-2
and CNS-1 gliomas. J Neurooncol 1998; 36:91-102.
5. Fleshner M, Watkins LR, Redd JM et al. A 9L gliosarcoma transplantation model for studying adoptive
immunotherapy in the brains of conscious rats. Cell Transplant 1992; 1:307-312.
6. Parsa AT, Chakrabarti I, Hurley PT et al. Limitations of the C6/Wistar rat intracerebral glioma model:
implications for evaluating immunotherapy. Neurosurgery 2000; 47:993-1000.
7. Heimberger AB, Crotty LE, Archer GE et al. Bone marrow-derived dendritic cells pulsed with tumor
homogenate induce immunity against syngeneic intracerebral glioma. J Neuroimmunol 2000, 103:16-25.
8. Albright L, Madigan JC, Gaston MR et al. Therapy in an intracerebral murine glioma model, using Bacillus
Calmette-Guerin, neuramindase-treated tumor cells and 1-(2-Chloroethyl)-3-cyclohexyl- 1-nitrosourea.
Cancer Res 1975; 35:658-665.
9. Finkelstein SD, Black P, Nowak TP et al. Histological characteristics and expression of acidic and basic
fibroblast growth factor genes in intracerebral xenogeneic transplants of human glioma cells. Neurosurgery
1994; 34:136-143.
10. Ostrand-Rosenberg S. Animal models of tumor immunity, immunotherapy and cancer vaccines. Curr Opin
Immunol 2004; 16:143-150.
11. Hann B, Balmain A. Building validated mouse models of human cancer. Curr Opin Cell Biol 2001;
13:778-784.
12. Smilowitz HM, Weissenberger J, Weis J et al. Orthotopic transplantation of v-src-expressing glioma cell
lines into immunocompetent mice: establishment of a new transplatanble in vivo model for malignant
glioma. J Neurosurg 2007; 106:652-659.
13. Theurillat JP, Hainfellner J, Maddalena A et al. Early induction of angiogenetic signals in gliomas of
GFAP-v-src transgenic mice. Am J Pathol 1999; 154:581-590.
14. Weissenberger J, Steinbach JP, Malin G et al. Development and malignant progression of astrocytomas in
GFAP-v-src transgenic mice. Oncogene 1997; 14:2005-2013.
15. Aguzzi A, Brandner S, Isenmann S et al. Transgenic and gene disruption techniques in the study of
neurocarcinogenesis. Glia 1995; 15:348-364.
16. Weiss WA, Israel M, Cobbs C et al. Neuropathology of genetically engineered mice: Consensus report and
recommendations from an international forum. Oncogene 2002; 21:7453-7463.
17. Watanabe K, Tachibana O, Sata K et al. Overexpression of the EGF receptor and p53 mutations are mutually
exclusive in the evolution of primary and secondary glioblastomas. Brain Pathol 1996; 6:217-224.
18. Ding H, Shannon P, Lau N et al. Oligodendrogliomas result from the expression of an activated mutant
epidermal growth factor receptor in a RAS transgenic mouse astrocytoma model. Cancer Res 2003;
63:1106-1113.
19. Donehower LA, Harvey M, Slagle BL et al. Mice deficient for p53 are developmentally normal but
susceptible to spontaneous tumours. Nature 1992; 356:215-221.
20. Holland EC, Hively WP, DePinho RA et al. A constitutively active epidermal growth factor receptor
cooperates with disruption of G1 cell-cycle arrest pathways to induce glioma-like lesions in mice.
Genes Dev 1998; 12:3675-3685.
axis. Cancer Cell 2002; 1:269-277.
22. Zhu Y, Guignard F, Zhao D et al. Early inactivation of p53 tumor suppressor gene cooperating with NF1
loss induces malignant astrocytoma. Cancer Cell 2005; 8:119-130.
23. Kwon CH, Zhao D, Chen J et al. Pten haploinsufficiency accelerates formation of high-grade astrocytomas.
Cancer Res 2008; 68:3286-3294.
24. Stambolic V, Suzuki A, de la Pompa JL et al. Negative regulation of PKB/Akt-dependent cell survival
by the tumor suppressor PTEN. Cell 1998; 95:29-39.
25. Ohgaki H, Dessen P, Jourde B et al. Genetic pathways to glioblastoma: a population-based study. Cancer
Res 2004; 64:6892-6899.
26. Fraser MM, Zhu X, Kwon CH et al. Pten loss causes hypertrophy and increased proliferation of astrocytes
in vivo. Cancer Res 2004; 64:7773-7779.
27. Wei Q, Clarke L, Scheidenhelm DK et al. High-grade glioma formation results from postnatal pten loss
or mutant epidermal growth factor receptor expression in a transgenic mouse glioma model. Cancer
Res 2006; 66:7429-7437.
28. Holland EC, Celestino J, Dai C et al. Combined activation of Ras and Akt in neural progenitors induces
glioblastoma formation in mice. Nat Genet 2000; 25:55-57.
29. Uhrbom L, Dai C, Celestino JC et al. Ink4-Arf loss cooperates with KRas activation in astrocytes and
neural progenitors to generate glioblastomas of various morphologies depending on activated Akt.
Cancer Res 2002; 62:2065-2069.
30. Ding H, Shannon P, Lau N et al. Oligodendrogliomas result from the expression of an activated mutant
epidermal growth factor receptor in a RAS transgenic mouse astrocytoma model. Cancer Res 2003;
63:1106-1113.
31. Ding H, Roncari L, Shannon P et al. Astrocyte-specific expression of activated p21-ras results in malingnant
astrocytoma formation in a transgenic mouse model of human gliomas. Cancer Res 2001; 61:3826-3836.
32. Shannon P, Sabha N, Lau N et al. Pathological and molecular progression of astrocytomas in a GFAP:
12 V-Ha-Ras mouse astrocytoma model. Am J Pathol 2005; 167:859-869.
33. Weiss WA, Burns MJ, Hackett C et al. Genetic determinants of malignancy in a mouse for oligodendroglioma.
Cancer Res 2003; 63:1589-1595.
34. Holland EC, Varmus HE. Basic fibroblast growth factor induces cell migration and proliferation after
glia-specific gene transfer in mice. Proc Natl Acad Sci USA 1998; 95:1218-1223.
35. Uhrbom I, Hesselager G, Nister M et al. Induction of brain tumors in mice using a recombinant platelet-derived
growth factor B-chain retrovirus. Cancer Res 1998; 58:5275-5279.
36. Assanah M, Lochhead R, Ogden A et al. Glial progenitors in adult white matter are driven to form malignant
gliomas by platelet-derived growth factor-expressing retroviruses. J Neurosci 2006; 26:781-790.
37. Marumoto T, Tashiro A, Friedmann-Morvinski D et al. Development of a novel mouse glioma model
using lentiviral vectors. Nat Med 2009; 15:110-116.
38. Abdallah B, Hassan A, Benoist C et al. A powerful nonviral vector for in vivo gene transfer into the adult
mammalian brain: polyethylenimine. Hum Gene Ther 1996; 7:1947-1954.
39. Hirko AC, Buethe DD, Meyer EM et al. Plasmid delivery in the rat brain. Biosci Rep 2002; 22:297-308.
40. Wiesner SM, Decker SA, Larson JD et al. De novo induction of genetically engineered brain tumors in
mice using plasmd DNA. Cancer Res 2009; 69:431-439.
41. Charest A, Wilker EW, McLaughlin ME et al. ROS fusion tyrosine kinase activates a SH2 domain-
containing phosphatase-2/phosphatidylinositol 3-kinase/mammalian target of rapamycine signaling
axis to form glioblastoma in mice. Cancer Res 2006; 66:7473-7481.
42. Koutcher JA, Hu X, Xu S et al. MRI of mouse models for gliomas shows similarities to humans and can
be used to identify mice for preclinical trials. Neoplasia 2002; 4:480-485.
43. Uhrbom L, Nerio E, Holland EC. Dissecting tumor maintenance requiredments using bioluminescence
imaing of cell proliferation in a mouse glioma model. Nat Med 2004; 10:1257-1260.
44. Pittet MJ, Valmori D, Dunbar PR et al. High frequencies of naive Melan-A/MART-1-specific CD8(
)
T-cells in a large proportion of human histocompatibility leukocyte antigen (HLA)-A2 individuals.
J Exp Med 1999; 190:705-715.
45. Gnjatic S, Nagata Y, Jager E et al. Strategy for monitoring T-cell responses to NY-ESO-1 in patients with
any HLA class I allele. Proc Natl Acad Sci USA 2000; 97:1091710922.
46. Harris JE, Ryan L, Hoover Jr HC et al. Adjuvant active specific immunotherapy for stage II and III colon
cancer with an autologous tumor cell vaccine: Eastern Cooperative Oncology Group Study E5283. J
Clin Oncol 2000; 18:148157.
47. Porgador A, Snyder D, Gilboa E. Induction of antitumor immunity using bone marrow-generated dendritic
cells. J Immunol 1996; 156:2918-2926.
48. Ardavin C, Amigorena S, Reis e Sousa C. Dendritic cells: immunobiology and cancer immunotherapy.
Immunity 2004; 20:17-23.
49. Dunn GP, Bruce AT, Ikeda H et al. Cancer immunoediting: from immunosurveillance to tumor escape.
Nat Immunol 2002; 3:991-998.
50. Bauer C, Bauernfeind F, Sterzik A et al. Dendritic cell-based vaccination combined with gemcitabine
increases survival in a murine pancreatic carcinoma model. Gut 2007; 56:1275-1282.
51. Park SD, Kim CH, Kim CK et al. Cross-priming by temozolomide enhances antitumor immunity of
dendritic cell vaccination in murine brain tumor model. Vaccine 2007; 25:3485-3491.
CHAPTER 12
IMMUNOGENE THERAPY
Terry Lichtor* and Roberta P. Glick

Department of Neurological Surgery, Rush University Medical Center and Mount Sinai Hospital,
Chicago, Illinois, USA
*Corresponding Author: Terry LichtorEmail: terry_lichtor@rush.edu
Abstract: Antigenic differences between normal and malignant cells of the cancer patient
form the rationale for clinical immunotherapeutic strategies. Because the antigenic
phenotype of neoplastic cells varies widely among different cells within the same
malignant cell-population, immunization with a vaccine that stimulates immunity
to the broad array of tumor antigens expressed by the cancer cells is likely to be
more efficacious than immunization with a vaccine for a single antigen. A vaccine
prepared by transfer of DNA from the tumor into a highly immunogenic cell line
can encompass the array of tumor antigens that characterize the patients neoplasm.
Poorly immunogenic tumor antigens, characteristic of malignant cells, can become
strongly antigenic if they are expressed by highly immunogenic cells. A DNA-based
vaccine was prepared by transfer of genomic DNA from a breast cancer that arose
spontaneously in a C3H/He mouse into a highly immunogenic mouse fibroblast cell
line, where genes specifying tumor-antigens were expressed. The fibroblasts were
modified in advance of DNA-transfer to secrete an immune augmenting cytokine
and to express allogeneic MHC Class I-determinants. In an animal model of breast
cancer metastatic to the brain, introduction of the vaccine directly into the tumor
bed stimulated a systemic cellular antitumor immune response measured by two
independent in vitro assays and prolonged the lives of the tumor-bearing mice.
Furthermore, using antibodies against the various T-cell subsets, it was determined
that the systemic cellular antitumor immunity was mediated by CD8+, CD4+
and NK/LAK cells. In addition an enrichment strategy has also been developed
to increase the proportion of immunotherapeutic cells in the vaccine which has
resulted in the development of enhanced antitumor immunity. Finally regulatory T
cells (CD4+CD25+Fox p3+-positive) were found to be relatively deficient in the
spleen cells from the tumor-bearing mice injected intracerebrally with the enriched
vaccine. The application of DNA-based genomic vaccines for the treatment of a
variety of brain tumors is being explored.

151
INTRODUCTION
Essentials of Tumor Immunology
The function of the immune system is to protect the body. This defensive
function is performed by leukocytes (white blood cells) and a number of accessory
cells distributed throughout the body. Lymphocytes are the key cells controlling the
immune response. They specifically recognize foreign material and distinguish it
from the bodys own self components.
There are two main types of lymphocytes: B cells which produce antibodies and T
cells which have a number of functions including:
i. Helping B cells to make antibodies
ii. Recognizing and destroying virus-infected cells
iii. Activating phagocytes to destroy pathogens
iv. Controlling the level and quality of the immune response
The essential role of T-lymphocytes is to recognize antigen, through specific cell surface
antigen receptors (TCR) presented by antigen presenting cells. Antigen presenting cells
(APCs) are a group of cells which are capable of taking up antigens, partially degrading
them and presenting them to T-lymphocytes in a form they can recognize. Whereas B cells
recognize antigen in its native form, T cells only recognize antigenic peptide derivatives of
complex antigens which have become associated with major histocompatibility complex
(MHC) molecules. Thus, MHC molecules present antigen i.e., peptides to T cells. MHC
Class I molecules are found on all nucleated cells and platelets. MHC Class II molecules
(Ia antigens) required for helping B cells or making antibodies are expressed on B cells,
macrophages, monocytes, APCs and some T cells. CD8 cells (cytotoxic T cells or CTLs)
are Class I restricted, meaning they only recognize antigen presented in the context of
MHC Class I molecules, while CD4 (helper T cells) are MHC Class II restricted (Fig.
1). Antigens synthesized within a cell, such as viral polypeptides, associate preferentially
with MHC Class I molecules and present antigen directly to CD8 cells (direct pathway).
In contrast, antigens that are taken up by an APC are partially degraded (processed) and
returned to the cell surface associated with MHC Class II molecules which are recognized
by CD4 cells (indirect pathway).
Overview of the Treatment Limitations of Patients with Malignant Gliomas
Treatment Limitations of Patients with Malignant Brain Tumors
Although technical advances have resulted in marked improvements in the ability

to diagnose and surgically treat primary brain tumors, the incidence and mortality rates
of these tumors are increasing.1 Particularly affected are young adults and the elderly.
Primary malignant brain tumors are the second leading cause of death in people under the
age of 35. Furthermore in the elderly population, mortality rates from these tumors have
increased more than 5-fold since 1970.2 The present standard treatment modalities following
surgical resection including cranial irradiation and systemic or local chemotherapy each
have serious adverse side effects. The few long-term survivors are inevitably left with
cognitive deficits and other disabilities.3,4 The difficulties in treating malignant gliomas
can be attributed to several factors. Glial tumors are inherently resistant to radiation and
IMMUNOGENE THERAPY 153
Figure 1. Direct versus indirect recognition of antigenic peptides. T-lymphocytes recognize short
antigenic peptides presented in a groove formed by the external domains of major histocompatibility
complex (MHC) class I and class II molecules. Tumor cell antigens on the surface of the tumors are
recognized by cytotoxic T cells (CD8
T cells) via MHC class I presentation. Alternatively the tumor
antigens can be ingested by macrophages which can then express the antigens and stimulate CD4
T
cells via MHC class II presentation.
standard cytotoxic chemotherapies.5,6 The existence of blood-brain and blood-tumor

barriers impedes drug delivery to the tumor and adjacent brain infiltrated with tumor.
Finally the low therapeutic index between tumor sensitivity and toxicity to normal brain
severely limits the ability to systemically deliver therapeutic doses of drugs to the tumor.
Transfer of Genomic DNA from One Cell Type to Another Alters Both
the Genotype and the Phenotype of Cells That Take Up the Exogenous DNA
Classic studies indicate that transfection of genomic DNA from one cell type to another
results in integration of the transferred DNA and stable alteration of the genotype of the
recipient cells. The transferred genes are replicated as the cells divide and are expressed.
Wigler, et al7 found that the genome of adenine phosphoribosyltransferase-deficient
mouse cells was modified to express the missing enzyme by transfer of DNA from mouse
cells whose genome included the gene for the missing enzyme. Analogous findings were
observed for membrane-associated determinants. Mendersohn, et al8 transferred genomic
DNA from human cells into polio virus-receptor-negative mouse cells and the transfected
cells expressed the missing receptor. Others9,10 used this approach to identify genes involved
in metastasis. Hsu et al11 and Kavathas and Herzenberg12 generated stable transfectants of
mouse fibroblasts. The transfected cells expressed human membrane T-cell antigens, HLA
determinants and B2-microglobulin. The expression of the transferred human genes by the
transfected cells was stable and long-term (more than six months). The proportion of the
transfected mouse cells that expressed the human gene of interest was surprisingly large
in the range of 1/500. The importance of these findings for development of DNA-based
tumor vaccines is that the transfer of genomic DNA into cells resulted in the expression of
genes specifying missing enzymes, genes controlling cell proliferation and metastasis and
genes specifying membrane associated determinants. An analogous approach can be used

to prepare a vaccine for use in patients with malignant gliomas. Genes specifying tumor
associated antigens (TAAs) that fail to provoke antitumor immunity can become highly
immunogenic antigenic determinants if they are expressed by highly immunogenic cells.
Multiple Mutant/Dysregulated Genes in Cancer Cells Specify TAAs
A major rationale for the use of DNA-transfer to prepare vaccines for use in cancer
therapy is that the vaccine expresses an array of multiple altered genes which define the
malignant phenotype. Genetic instability in cancer cells is responsible for the formation of
TAAs. TAAs such as `- catenin,13 gp100, Melan A/Mart-1 and tyrosinase in melanoma14
are differentiation antigens whose expression is dysregulated in cancer cells. Mutant
genes also specify TAAs.6,15 For example Boon found that a point mutation in a gene in
P815 murine mastocytoma cells specified a tumor-rejection antigen. Thus, the malignant
cell-population is characterized by the presence of numerous TAAs, some of which are
unique and others are differentially expressed by cancer cells but all are strong potential
targets of immune-mediated attack.
DNA from the Patients Neoplasm Is the Ideal Source of Tumor Antigens
for Immunotherapy
Since the total number of different TAAs within the population of malignant cells is
large and diverse, successful therapy will depend upon the use of a vaccine that is capable
of inducing immunity to the broad array of tumor antigens that characterizes the patients
cancer. Therapy based on the induction of immunity to a single antigen, or peptide, is
less likely to be successful. Multi-epitope vaccines are expected to be more efficacious
than single-epitope vaccines. This is especially the case for malignant astrocytomas,
where clinically relevant TAAs, i.e., immunity to TAAs that leads to tumor rejection,
have not been identified.
Characteristics of the Modified Cell Line Used as the Recipient of Tumor DNA
Among other advantages of this approach, the cells chosen as DNA-recipients can
be selected for their ability to enhance the immune response. The expression of both
syngeneic and allogeneic MHC-determinants by the DNA recipient cells is important in
order to obtain an optimum antitumor response.17 The syngeneic determinants provide
a restriction element for direct presentation of TAAs to CTLs of the host. Allogeneic
antigens served as potent immune adjuvants. Numerous investigators found that the
immunogenic properties of cancer cells could be enhanced if the cells were modified to
express allogeneic MHC-determinants.18-23 The modified cells, which ordinarily proliferate
in syngeneic immunocompetent recipients, were recognized as foreign and were rejected.
In the mouse, immunization with tumor cells altered by the introduction and expression
of allogeneic Class I genes led to immune-mediated rejection of the malignant cells and
the induction of protective antitumor immunity. However, the introduction of genes
specifying allogeneic determinants into cells from a primary neoplasm is technically
challenging and not always successful. In contrast, transfer of DNA from the tumor into
highly immunogenic syngeneic/allogeneic cells is consistently and reliably achieved.
Important Advantages of Preparing a Vaccine by Transfer of DNA

from the Patients Neoplasm into Nonmalignant Fibroblasts
A vaccine prepared by transfer of DNA from the patients neoplasm into highly
immunogenic, nonmalignant human fibroblasts has a number of important advantages. A
major advantage is that the cells used as recipients of the DNA can be selected for special
properties, which will enhance the antitumor immune response. Since the recipient cells
are capable of prolonged proliferation in vitro and the transferred DNA is replicated as
the cells divide, only a small quantity of DNA from the neoplasm is required to generate
the vaccine. In addition, the number of transfected fibroblasts can be expanded as needed,
to obtain sufficient quantities for repeated immunizations of the cancer patient. The
fibroblasts used as DNA-recipients will also express allogeneic Class I determinants
which is a desirable feature since this leads to an augmented immune response. In
addition a cell line derived from the patients primary neoplasm does not have to be
established, which is the case if genes specifying cytokines, allogeneic MHC-determinants,
costimulatory molecules or other immune-augmenting properties are to be introduced
into the autologous tumor cells. The establishment of tumor cell lines, especially cell
lines derived from astrocytomas, is technically difficult, often not feasible and may not be
representative of the tumor cell population as a whole. Furthermore hybrid cell vaccines
prepared by fusion of tumor cells with antigen presenting cells pose similar concerns.24-26
Immunization with tumor cells modified to secrete immune-augmenting cytokines such
as IL-2 and GM-CSF has been investigated and shown to result in the development of
generalized MHC-restricted antitumor immune responses in animal models.40-49 However
tumor cells are also a source of immunosuppressive factors, which inhibit the antitumor
activity of the effector cells,27,28 The DNA-based vaccines are successful because a full
complement of genes is transferred to the recipient cells which results in a robust signal
for the development of antitumor immune responses.
Advantages of DNA-Based Vaccines Relative to Other Types of Vaccines
A number of different vaccination strategies are currently being evaluated.29-34 The

approaches to vaccination with TAAs include those based on: (a) defined antigens or
antigenic peptides, (b) tumor cell lysates or lysate fractions and (c) whole irradiated
tumor cells or apoptotic tumor cell bodies. Clinical trials involving vaccines prepared
using TAAs or TAA-derived epitopes presented by APCs or fed to dendritic cells (DCs)
have shown some promising results. However, defined antigens have to be identified and
purified, a tremendous effort requiring an antigen discovery approach. The quantity
of purified antigen must be increased, to enable multiple immunizations of the cancer
patient. While new TAAs are being discovered, the question of which TAA to be used in
the vaccine is uncertain and extensively debated. The heterogeneity of antigen expression
in the tumor cell population is likely to be a concern. Some tumor cells may not express
the antigen chosen for therapy. In one study for example, it was found that expression of
known tumor antigens such as gp100 and tyrosinase was variable in different melanoma
lesions in the same patient.14 Not all the malignant cells in the patients neoplasm expressed
these determinants. Since the tumor cell population is heterogeneous, tumor cells that
fail to express the defined antigen chosen for therapy are likely to escape destruction by
the activated immune system.
The major advantage of vaccines prepared by transfer of tumor DNA into

nonmalignant fibroblasts is that TAAs do not have to be purified or produced in
large quantities. In comparison with protein vaccines, DNA-based vaccines provide
prolonged expression and direct presentation of tumor antigens which results in robust
and long-lasting activation of the immune system. From a practical point of view, these
vaccines are easy and relatively inexpensive to prepare. Unlike other strategies, vaccines
can be prepared from only a limited quantity of tumor-derived DNA, which can be
obtained from small surgical specimens (vaccines can routinely prepared from 50 +g of
DNA). Furthermore, the recipient fibroblasts can be selected to meet the requirement
for rapid expansion in culture and MHC restriction. The DNA-based vaccines offer a
number of important advantages, which greatly encourage their further development
for cancer immunotherapy.
Disadvantages of Transfer of Tumor-Derived DNA Transfer into Fibroblasts

for Expression of TAAs
While vaccination based on transfer of tumor-derived DNA into highly immunogenic

cells has a number of advantages, there are concerns as well. Since the proportion of total
DNA that specifies TAAs is likely to be small, it is possible that a large number of the
transfected cells may not express TAAs or may express TAAs at low levels. This concern
is minimized, however, by preclinical data which indicate that the proportion of the cells
that take-up tumor DNA and express TAAs is sufficient to induce an effective antitumor
immune response and to significantly increase survival. Another concern related to therapy
with DNA-based vaccines is that genes specifying normal self antigens are likely to
be expressed by the DNA-transfected cells, creating a danger that autoimmune disease
might develop, although this has not been observed thus far. Inbred mice immunized
with the DNA-based vaccine or tumor-bearing mice injected with therapeutic DNA-based
vaccines failed to exhibit adverse effects. Of course, protocols that depend upon the
use of tumor cell-extracts, peptide eluates of tumor cells, fusion cells, cDNAs or RNAs
derived from tumor cells are subject to the same concern. In DNA-based vaccines,
genes encoding determinants expressed by nonneoplastic cells are likely to be present
in the largest proportion relative to genes specifying TAAs. While the use of purified
tumor antigen in the form of cDNA or polynucleotide vaccines specific for known
TAAs eliminates this concern, those types of vaccines are dependent on the selection
of the most relevant vaccinating epitope, as discussed above. It is also conceivable
that a cellular vaccine, including one using nonmalignant fibroblasts might grow in the
patient, forming a tumor. Conceivably, a transforming oncogene or a defective tumor
suppressor gene might be transferred to a normal cell, provoking a neoplasm although
this has not been observed. Overall, the disadvantages of DNA-based vaccines are few
and are certainly no more difficult to overcome than those associated with other types
of experimental tumor-vaccines.
Defects in TAA Presentation by Tumor Cells
Defects in presentation of TAAs by tumor cells have been described in both murine
as well as human tumors.35,36 They can result in tumor cell escape from host immunity.
One mechanism is the loss of MHC determinants, which results in the impaired ability of
the tumors to present TAAs. Loss of MHC antigen expression in several murine tumors
is correlated with an increase in the malignant properties of the cells.37 Melanomas that
recurred in mice treated with a vaccine prepared by transfer of DNA from murine melanoma
cells into mouse fibroblasts were deficient in expression of MHC Class I determinants.38
Primary and especially metastatic cells may have global or selective down-regulation of
Class I or Class II HLA antigens, due to mutations in `2 microglobulin or TAP genes
and thus they may fail to present TAAs in an immunogenic form to immune cells. Even
if the host generates tumor-specific CTLs, the effector cells may not be able to eliminate
the tumor. In addition to a failure to express HLA antigens, tumors may not express
costimulatory molecules resulting in an inadequate immune response to TAAs by the
host. Immunization with a DNA-based vaccine can overcome certain of these tumor
escape mechanisms.
Significance
The most compelling reason for the vaccination strategy involving DNA-based
cellular vaccines is the current lack of effective therapy for patients with malignant
gliomas. This is verified by the dismal survival statistics, which have remained essentially
unchanged for 30 years. Immunization with a vaccine that induces strong antitumor
responses is an attractive addition or possibly even an alternative to conventional
therapies. The DNA-based vaccines described in this chapter have shown remarkable
therapeutic efficiency and survival benefits in some initial murine preclinical studies.
PRECLINICAL EXPERIMENTAL FINDINGS
Treatment of Intracerebral Glioma in C57Bl/6 Mice by Immunization

with Allogeneic Cytokine-Secreting Fibroblasts
As an initial study, we measured the survival of C57Bl/6 mice injected intracerebrally

(i.c.) with a mixture of Gl261 glioma cells and cytokine secreting LM cells.39 Gl261
cells are a glioma cell-line of C57Bl/6 mouse origin (H-2b). LM fibroblasts are
derived from C3H/He mice and express H-2k determinants. We initially evaluated
the immunotherapeutic effects of single cytokine-secreting LM-IL-2 cells and double
cytokine-secreting LM-IL-2/interferon-a cells in mice bearing an i.c. glioma. A mixture
of G1261 cells and the single or double cytokine-secreting cells were injected i.c. into
the right frontal lobe of C57BL/6 mice, syngeneic with G1261 cells. Mice injected
i.c. with the mixture of glioma and LM-IL-2 cells survived significantly longer (P
0.025) than control mice injected i.c. with an equivalent number of glioma cells alone.
Somewhat more dramatic results were obtained for mice injected i.c. with a mixture of
glioma cells and LM-IL-2/interferon-a double cytokine-secreting cells. No prolongation
of survival was noted when allogeneic cytokine secreting fibroblasts mixed with tumor
cells or tumor antigens were administered subcutaneously in mice with an intracerebral
tumor even though a strong antitumor immune response was detected in the spleen cells
of the treated animals. Of special interest, mice injected i.c. with an equivalent number
Figure 2. Spleen cell mediated antitumor immunity in mice bearing an i.c. glioma treated with cytokine
secreting cells. C57BL/6 mice received a single i.c. injection of (105) glioma cells together with one
of the modified fibroblast cell-types (106 cells). Three weeks after the injection, mononuclear cells
from the spleens of the immunized mice obtained through Ficoll-Hypaque centrifugation were used
for the 51Cr-release assay. All values represent the mean ( SD of triplicate determinations. Probability
values were as follows: P 0.005 for spleen cells taken from IL-2 treated animals relative to 51Cr
release for spleen cells from animals immunized with glioma and P 0.05 relative to 51Cr release for
spleen cells from animals immunized with glioma
LM cells; P 0.025 for spleen cells taken from
animals treated with IL-2/IFN-a relative to 51Cr release for spleen cells from animals immunized with
glioma and P 0.05 relative to 51Cr release for spleen cells from animals immunized with glioma
LM-IL-2 cells. A) Cytotoxicity toward glioma cells in spleen cells from mice immunized with various
cytokine-secreting cells. B) Effect of mAbs against T-cell subsets or NK/LAK cells on the antiglioma
cytotoxic activities of spleen cells.
of LM-IL-2 cells alone lived for more than three months and showed no evidence of ill
effects or neurologic deficit. Immunocytotoxic studies (Fig. 2) demonstrate a significantly
elevated cromium release from Gl261 cells co-incubated with spleen cells from mice
injected i.c. with glioma cells and the cytokine secreting fibroblasts. This indicates that
a systemic antitumor response did develop in the mice injected intracerebrally with the
cytokine secreting cells in the presence of tumor antigens.
Treatment of Intracerebral Breast Cancer in C3H Mice by Immunization with

Syngeneic/Allogeneic Fibroblast Transfected with DNA from Breast Cancer Cells
Whether results obtained by transfer of DNA from a tumor cell line into mouse
fibroblasts can be applied to tumors that develop spontaneously is uncertain. Conclusions
based on a model system involving tumor cell lines may not apply to neoplasms that
arise spontaneously in patients. The appearance of spontaneous breast neoplasms in
C3H mice provides an opportunity to investigate this question. DNA isolated from a
breast neoplasm that arose in a C3H mouse (H-2K) was transferred into mouse fibroblasts
(H-2k). To increase their immunogenic properties and to ensure rejection, the fibroblasts
were modified to express H-2Kb determinants beforehand. H-2Kb determinants are
allogeneic in C3H mice. The results indicated that C3H mice with intracerebral breast
cancer treated solely by immunization with fibroblasts transfected with DNA from the
same spontaneous breast neoplasm survived significantly longer (p 0.005) than mice
in various control groups.40
T-Cell Mediated Toxicity Toward Intracerebral Breast Cancer in Mice

Immunized with Syngeneic/Allogeneic Transfected Fibroblasts Modified
to Secrete IL-2, GM-CSF or IL-18
An MTS cytotoxicity assay was used to detect the presence of T cells reactive with
breast cancer cells in mice injected i.c. with the mixture of SB5b cells and the modified,
DNA-transfected fibroblasts. The T cells obtained from the spleens of the injected mice
were analyzed two weeks after the i.c. injection of the cell mixture. The results indicated
that the cytotoxic response of greatest magnitude was in mice injected i.c. with the
mixture of SB5b cells and transfected fibroblasts modified to secrete IL-2 or GM-CSF.40
Lesser cytotoxic effects were present in mice injected i.c. with SB5b cells and transfected
fibroblasts modified to secrete IL-18.
The Proportion of T Cells Responsive to Tumor Cells in Mice Bearing

an Intracerebral Tumor Immunized Intracerebrally with Syngeneic/Allogeneic
Transfected Fibroblasts Modified to Secrete IL-2, IL-18 or IL-2
IL-18
An ELISPOT-IFN-a assay was used to determine the proportion of splenic T cells

reactive with SB-5b cells in mice immunized with transfected fibroblasts modified to
secrete IL-2, IL-18 or both IL-2 and IL-18. The animals were injected i.c. with a mixture
of 1.0 = 104 SB-5b breast carcinoma cells and 1.0 = 106 treatment cells consisting of
LMKbIL-2/SB5b, LMKbIL-18/SB5b , or a mixture of LMKbIL-2/SB5b and LMKbIL-18/
SB5b cells. The animals were sacrificed at two weeks and an ELISPOT assay was done
using the spleen cells to detect IFN-a secretion in the presence of SB-5b tumor cells and
antibodies against various T-cell subsets. The results indicate that the cellular antibreast
carcinoma immune response was mediated by CD4
, CD8
and NK/LAK cells.40 Although
IL-18 secreting cells did not produce a significant antitumor immune response as detected
with the ELISPOT assay, the combination of IL-2 with IL-18 secreting cells did result in
an enhancement of the antitumor responses in comparison to animals that were treated
with IL-2 secreting cells alone.
Increased Numbers of Responding T Cells Were Detected in the Spleens

and Cervical Lymph Nodes of Nave Mice or Mice with i.c. Breast Cancer
Injected into the Brain with Cells from the Immunohigh Pool
An enrichment strategy for the vaccine was developed based on the hypothesis that
if aliquots of a transfected cell population were divided into smaller populations, some
populations by chance would contain more highly immunogenic cells than others. The
populations with higher numbers of immunogenic cells could be identified by their
stronger immunogenic response against SB5b cells in C3H/He mice. Two subpools that
stimulated immunity to the greatest (immunohigh pool) and least (immunolow pool) extents
after three rounds of enrichment were selected for further study.
To determine if systemic antitumor immunity was generated in tumor-free mice
injected i.c. with cells from the immunohigh pool, cervical lymph node and spleen cells
from the injected mice were analyzed by ELISPOT IFN-a assays for responding T cells.
Nave C3H/He mice received 2 i.c. injections at weekly intervals of 1.0 = 106 cells from
the immunohigh pool. One week after the second injection, mononuclear cells from the
spleens and cervical lymph nodes of the immunized mice were analyzed for the presence
of T cells responsive to the breast cancer cells. As controls, an equivalent number of cells
from the nonselected master pool or cells from the immunolow pool were substituted for
cells from the immunohigh pool. As additional controls, the same protocol was followed
except that the mice were injected i.c. with equivalent numbers of SB5b cells, with LMKb
cells or with media. Mice injected with SB5b tumor cells received only one injection.
The results from the cervical lymph nodes (Fig. 3) indicated that the highest number of
responding cells was in mice injected i.c. with cells from the immunohigh pool (p 0.005
vs cells from mice in any of the other groups). Similar results were found in studies using
the spleen cells from these animals.41
ELISPOT IFN-a assays were also used to determine the number of responding T
cells in the spleens of mice with i.c. breast cancer injected into the tumor bed with cells
from the immunohigh pool.41 A micro cannula was placed into the right frontal lobe of
C3H/He mice. SB5b cells (1.0 = 104 in 10 +l) were introduced into the brain through the
cannula. On days two and nine following, the animals were injected through the cannula
into the tumor bed with 1.0 = 106 cells from the immunohigh pool. As controls, the same
Figure 3. Development of antitumor immunity in cervical lymph nodes from nave mice injected
intracerebrally with an enriched cellular vaccine. ELISPOT IFN- a assays for responding T cells in the
cervical lymph nodes of mice injected i.c. with cells from the immuno high pool. Nave C3H/He mice
received intracerebral injections through a small burr hole two times at weekly intervals with 1.0 = 106
cells from the immunohigh pool of transfected cells. One week after the second injection, mononuclear
cells from the cervical lymph nodes of the injected mice were analyzed by ELISPOT IFN- a assays
for responding T cells. As controls, cells from the nonenriched master pool (LMIL-2Kb/SB5b) or cells
from the immunolow pool were substituted for cells from the immunohigh pool. As additional controls,
the same protocol was followed except that the mice were injected i.c. with media or with equivalent
numbers of either SB5b or LMKb cells, or the mice were not injected. The animals injected i.c. with
SB5b cells alone were injected only once. In some instances, SB5b cells (stimulated) were added to
the cervical lymph node cell suspensions 16 hrs before the ELISPOT IFN-a assays were performed (the
ratio of spleen cells: SB5b cells 10:1). In this assay the number of IFN-a spots/106 cervical lymph
node cells is measured. Error bars represent one standard deviation. p 0.005 for the difference in the
number of spots in the group injected with high pool LMIL-2Kb/SB5b cells co-incubated with SB5b
cells versus any of the other groups.
procedure was followed except that the cells from the nonenriched master pool or cells
from the immunolow pool were substituted for cells from the immunohigh pool. As additional
controls, the tumor bearing mice were injected into the tumor bed with equivalent numbers
of non DNA-transfected LMKb cells or the mice were injected with SB5b cells alone.
The results indicate that the highest number of responding T cells were in the spleens of
tumor-bearing mice injected i.c. with cells from the immunohigh pool (p 0.05 versus the
number of responding spleen cells in mice injected with cells from the master pool and
p 0.005 versus the number of spots obtained from any of the other groups).
The effect of antibodies against various T-cell subsets on the cytotoxic response was
used to determine the types of cells activated for antitumor immunity in mice injected
into the tumor bed with cells from the immunohigh pool. The greatest inhibitory effect was
obtained when CD4
antibodies were added to the mixed cell cultures.41 Lesser effects
were observed if the spleen cells were incubated in the media containing CD8
or NK/
LAK antibodies.
T-Reg Cells Are Relatively Deficient in the Spleens of Mice with i.c. Breast
Cancer Injected into the Tumor Bed with Cells from the Immunohigh Pool
T-reg cells are potent inhibitors of natural antitumor immunity. The success of
immunotherapeutic protocols may depend upon the relative numbers of T-reg cells and
cytotoxic T-lymphocytes in tumor-bearing animals and patients. Quantitative RT-PCR
for Foxp3, a transcription factor characteristic of T-reg cells, was used to determine the
relative proportions of T-reg cells in the spleens and brains of mice with i.c. breast cancer
injected into the tumor bed with cells from the immunohigh pool of transfected cells. Nave
C3H/He mice were injected i.c. with 5.0 = 104 SB5b cells along with 1.0 = 106 cells from
the immunohigh pool of transfected cells. One week later, the animals received a second i.c.
injection of cells from the immunohigh pool through the same burr hole alone. As controls,
the same procedure was followed except that the mice were injected with equivalent
numbers of SB5b cells and cells from the nonenriched master pool or the immunolow
pool. The results indicate that CD4
/CD25
/Foxp3
T-reg cells were relatively deficient
in the spleens but not in the brains of animals injected with cells from the immunohigh
pool.41 An analysis by FACS of the spleens of the injected animals revealed a relative
deficiency of CD4
/CD25
T cells and a corresponding increase in the relative numbers
of CD8
cells in the spleens of mice injected i.c. with cells from the immunohigh pool.
CONCLUSION
Despite standard therapeutic approaches, the survival of patients with primary or

metastatic tumors to the brain has not improved significantly in more than thirty years.
There is an urgent need for new and more effective forms of treatment. Immunotherapy,
designed to stimulate immunity to the autologous tumor, is under active investigation
for a number of different histologic types of cancer. The enhanced immunotherapeutic
properties of a vaccine prepared by transfer of a cDNA expression library derived from breast
cancer cells into a mouse fibroblast cell line appears to have great potential in treatment of
intracerebral tumors. As the transferred cDNA integrates spontaneously into the genome
of the recipient cells, replicates as the cells divide and is expressed, the vaccine could be
prepared from small amounts of tumor tissue, enabling treatment at an early stage of the
disease, when tumor tissue is available in only limited amounts and the tumor is most
susceptible to immune-based therapy. However, like other cellular tumor vaccines, only
a small proportion of the transfected cell population was expected to have incorporated
cDNA fragments that specified tumor antigens. A novel enrichment strategy has also been
developed to increase the proportion of immunotherapeutic cells in the vaccine.
A number of different strategies have been attempted to develop vaccines that
generate enhanced antitumor immune responses in mice and patients with intracerebral
neoplasms involving the central nervous system. Vaccines have been prepared by feeding
antigen presenting (dendritic) cells apoptotic bodies from tumor cells or tumor cell
lysates. Introduction of tumor cell-derived RNA into dendritic cells is another approach
which has been developed. Immunization with dendritic cells fed derivatives of tumor
cells or transfected with tumor-RNA can result in the induction of immune responses
against the broad array of tumor antigens expressed by the population of malignant cells
including tumors of neuroectodermal origin.42,43 In patients, immunization with autologous
dendritic cells transfected with mRNA from malignant glioma elicited tumor-specific
CD8
cytotoxic T-lymphocyte (CTL) responses against the patients malignant cells.44
Although results of dendritic cell immunotherapy have demonstrated promise in animal
models, clinical trials have been disappointing thus far.43
Other tumor vaccination strategies have been used including modification of
neoplastic cells to generate antitumor immune responses. Immunization with tumor
cells modified to secrete immune-augmenting cytokines such as IL-2 and GM-CSF has
resulted in the development of generalized MHC-restricted antitumor immune responses
in animal models.36,45-53 Selective tumor regression was observed in experimental animals
and patients receiving immunotherapy alone, in support of the potential of this type of
treatment for patients with malignant disease.54 The effects of cytokine expression by
central nervous system tumors (CNS) were examined initially using glioma cells that were
engineered to secrete IL-4.55 In these studies it was demonstrated that IL-4 transduced
glioma cells resulted in the development of antitumor immune responses. Delivery of an
IFN-` expression plasmid by cationic liposomes to the CNS tumor site was also found to
induce significant anti-CNS tumor immunity in preclinical models.56 Use of a high-titer
adenoviral vector encoding IL-12 is another strategy that was reported to induce antitumor
responses in a glioma model.57
Previous studies indicated that transfection of genomic DNA from the malignant cells
into a fibroblast cell line resulted in stable integration and expression of the transferred
DNA. Both the genotype and the phenotype of the cells that took up the exogenous
DNA were altered as portions of the transferred DNA were expressed. Immunization
of tumor-bearing mice with the DNA-based vaccine resulted in the induction of cell
mediated immunity directed toward the type of tumor from which the DNA was obtained
and prolongation of survival, consistent with the expression of an array of TAA by the
transfected cells. This was the case for mice with melanoma, squamous cell carcinoma and
in mice with breast cancer.58 Multiple undefined genes specifying TAA that characterize
the malignant cell population were expressed by cells that took up DNA from the tumor.
The number of vaccine cells could be expanded as required for multiple immunizations.
In addition, the recipient cells can also be modified before DNA-transfer to increase
their immunogenic properties, as for example, by the introduction of genes specifying
immune-augmenting cytokines or allogeneic MHC-determinants, which act as strong
immune adjuvants. In animal models, injection of cytokine-secreting allogeneic fibroblasts
into the tumor bed of intracerebral neoplasms was partially effective in the treatment of
mice with established brain tumors.59
To be successful, every remaining tumor cell in the patient must be eliminated.
It is unlikely that a single form of therapy is capable of achieving this goal. However
immunotherapy in combination with surgery, radiation therapy and chemotherapy will
likely find a place as a new and important means of treatment for patients with brain
tumors. A major advantage of DNA-based vaccines is that they do not require protein
purification or its production and yet they are able to elicit robust and long-lasting activation
of the immune response, which results in tumor rejection. From a practical point of view,
these vaccines are easy to prepare and they are relatively inexpensive. Only a limited
quantity of tumor-derived DNA is required, which can be obtained from small surgical
specimens. The enrichment strategy enables the generation of highly immunogenic pools
of transfected cells with enhanced immunotherapeutic properties.
Thus DNA-based vaccines offer a number of advantages, which greatly encourage
their further development for cancer immunotherapy in general and specifically for
treatment of malignant brain tumors.
REFERENCES
1. Ries LAG, Kosary CL, Hankey BF et al. SEER Cancer Statistics Review, 1973-1995. National Cancer
Institute, 1988.
2. Radhakrishnan K, Mokri B, Parisi JE et al. The trends in incidence of primary brain tumors in the population
of Rochester, Minnesota. Ann Neurol 1995; 37:67-73.
3. Imperato JP, Paleologos NA, Vick NA. Effects of treatment on long-term survivors with malignant
astrocytomas. Ann Neurol 1990; 28:818-822.
4. Heimans JJ, Taphoorn MJ. Impact of brain tumour treatment on quality of life. J Neurol 2002; 249:955-960.
5. Belanich M, Randall T, Pastor MA et al. Intracellular localization and intercellular heterogeneity of the
human DNA repair protein O(6)-methylguanine-DNA methyltransferase. Cancer Chemother Pharmacol
1996; 37:547-555.
6. Hotta T, Saito Y, Fujita H et al. O6-alkylguanine-DNA alkyltransferase activity of human malignant glioma
and its clinical implications. J Neurooncol 1994; 21:135-140.
7. Wigler M, Pellicer A, Silverstein S et al. DNA-mediated transfer of the adenine phosphoribosyltransferase
locus into mammalian cells. Proc Natl Acad Sci USA 1979; 76:1373-1376.
8. Mendersohn C, Johnson B, Lionetti KA et al. Transformation of a human poliovirus receptor gene into
mouse cells. Proc Natl Acad Sci USA 1986; 83:7845-7849.
9. Barraclough R, Chen HJ, Davies BR et al. Use of DNA transfer in the induction of metastasis in experimental
mammary systems. Biochem Soc Symp 1998; 63:273-294.
10. Chen H, Ke Y, Oates AJ et al. Isolation of and effector for metastasis-inducing DNAs from a human
metastatic carcinoma cell line. Oncogene 1997; 14:1581-1588.
11. Hsu C, Kavathas P, Herzenberg LA. Cell surface antigens expressed on L cells transfected with whole
DNA from non-expressing and expressing cells. Nature 1984; 312:68-69.
12. Kavathas P, Herzenberg LA. Stable transformation of mouse LM cells (a transformed fibroblast cell
line) for human membrane T-cell differentiation antigens, HLA and B2 microglobulin: Selection by
fluorescence-activated cell sorting. Proc Natl Acad Sci USA 1983; 80:524-528.
13. Robbins PF, El-Gamil M, Li YF et al. A mutated `-catenin gene encodes a melanoma-specific antigen
recognized by tumor-infiltrating lymphoyctes. J Exp Med 1996; 183:1185-1192.
14. de Vries TJ, Fourkour A, Wobbes T et al. Heterologous expression of immunotherapy candidate proteins
gp100, MART-1 and tyrosinase in human melanoma cell lines and in human melanocytic lesions. Cancer
Res 1997; 57:3223-3229.
T-lymphocytes on a human melanoma. Science 1991; 254:1643-1647.
16. Boon T, Cerottini JC, Van den Bynde B et al. Tumor antigens recognized by T-lymphocytes. Ann Rev
Immunol 1994; 12:337-365.
17. deZoeten E et al. An optimum anti melanoma response in mice immunized with fibroblasts transfected
with DNA from mouse melanoma cells requires the expression of both syngeneic and allogeneic
MHC-determinants. Gene Therapy 2002; 9:1163-1172.
18. Hammerling GJ, Klar D, Katzav S et al. Manipulation of metastasis and tumour growth by transfection
with histocompatibility class I genes. J Immunogen 1986; 13:15-157.
19. Hui KM, Sim TF, Foo TT et al. Tumor rejection mediated by transfection with allogeneic class I
histocompatibility gene. J Immunol 1989; 143:3835-3843.
20. Ostrand-Rosenberg S, Thakur A, Clements V. Rejection of mouse sarcoma cells after transfection of MHC
class II genes. J Immunol 1990; 144:4068-4071.
21. Fearon ER, Itaya T, Hunt B et al. Induction in a murine tumor of immunogenic tumor variants by transfection
with a foreign gene. Cancer Res 1988; 48:2975-2980.
22. Gattoni-Celli S, Willett CG, Rhoads DB et al. Partial suppression of anchorage-independent growth and
tumorigenicity in immunodeficient mice by transfection of the H-2 class I gene H-2Ld into a human colon
cancer cell line (HCT). Proc Natl Acad Sci USA 1988; 85:8543-8547.
23. Nabel GJ, Gordon D, Bishop DK et al. Immune response in human melanoma after transfer of an allogeneic
class I major histocompatibility complex gene with DNA-liposome complexes. Proc Natl Acad Sci USA
1996; 93:15388-15393.
24. Gong J, Chen D, Kashiwaba M et al. Induction of antitumor activity by immunization with fusions of
dendritic and carcinoma cells. Nat Med 1997; 3:558-561.
25. Liang W, Cohen EP. Resistance to murine leukemia in mice rejecting syngeneic somatic hybrid cells.
J Immunol 1976; 116:623-628.
26. Liang W, Cohen EP. Resistance to murine leukemia in mice receiving simultaneous injections of syngeneic
hybrid and parental neoplastic cells. J Immunol 1977; 118:903-908.
27. Whiteside TL, Rabinowich H. The role of Fas/FasL in immunosuppression induced by human tumors.
28. Strand S, Galle PR. Immune evasion by tumors: involvement of the CD95 (APO-1/Fas) system and its
clinical implications. Mol Med Today 1998; 4:63-68.
29. Nestle FO, Alijagic S, Gilliet M et al. Vaccination of melanoma patients with peptide-or-tumor lysate-pulsed
dendritic cells. Nature Med 1998; 4:328-332.
30. Tighe H, Corr M, Roman M et al. Gene vaccination: plasmid DNA is more than just a blue print. Immunol
Today 1998; 19:89-97.
31. Condon C, Watkins SC, Celluzi CM et al. DNA-based immunization by in vivo transfection of dendritic
cells. Nature Med 1996; 2:1122-1128.
32. Nair SK, Boczkowski D, Morse M et al. Induction of primary carcinoembryonic antigen (CEA)-specific
cytotoxic T-lymphocytes in vitro using human dendritic cells transfected with RNA. Nat Biotechnol
1998; 16:364-369.
33. Ashley DM, Faiola B, Nair S et al. Bone marrow-generated dendritic cells pulsed with tumor extracts or tumor
RNA induce antitumor immunity against central nervous system tumors. J Exp Med 1997; 186:1177-1182.
34. Gilboa E, Nair K, Lyerly HK. Immunotherapy of cancer with dendritic cell-based vaccines. Cancer Immunol
Immunother 1998; 46:82-87.
35. Restifo NP, Esquivel F, Asher AL et al. Defective presentation of endogenous antigens by a murine sarcoma:
Implications for the failure of an anti tumor immune response. J Immunol 1991; 147:1453-1458.
36. Ohlen C, Bastin J, Ljunggren HG et al. Resistance to H-2-restricted but not to allo-H-2-specific graft and
cytotoxic T-lymphocyte responses in lymphoma mutant. J Immunol 1990; 145:52-58.
37. Cohen EP, Kim TS. Neoplastic cells that express low levels of MHC class I determinants escape host
immunity. Seminars in Cancer Biology. London: Academic Press, 1994; 5:419-428.
38. Kim TS, Cohen EP. MHC antigen expression by melanomas recovered from mice treated with
allogeneic mouse fibroblasts genetically modified for interleukin-2 secretion and the expression of
melanoma-associated antigens. Cancer Immunol Immunother 1994; 38:185-193.
39. Lichtor T, Glick RP, Kim TS et al. Prolonged survival of mice with glioma injected intracerebrally with
double cytokine-secreting cells. J Neurosurg 1995; 83:1038-1044.
40. Lichtor T, Glick RP, Lin H et al. Intratumoral injection of IL-secreting syngeneic/allogeneic fibroblasts
transfected with DNA from breast cancer cells prolongs the survival of mice with intracerebral breast
cancer. Cancer Gene Ther 2005; 12:708-714.
41. Lichtor T, Glick RP, Feldman LA et al. Enhanced immunity to intracerebral breast cancer in mice immunized
with a cDNA-based vaccine enriched for immunotherapeutic cells. J Immunother 2008; 31:18-27.
42. O I, Ku G, Ertl HCJ et al. A dendritic cell vaccine induces protective immunity to intracranial growth of
glioma. Anticancer Res 2002; 22:613-622.
43. Rosenberg SA, Yang JC, Restifo NP. Cancer immunotherapy: moving beyond current vaccines. Nat Med
2004; 10:909-915.
44. Kobayashi T, Yamanaka R, Homma J et al. Tumor mRNA-loaded dendritic cells elicit tumor-specific CD8
cytotoxic T-cells in patients with malignant glioma. Cancer Immunol Immunother 2003; 52:632-637.
45. Gansbacher B, Bannerji R, Daniels B et al. Retroviral vector-mediated gamma-interferon gene transfer
into tumor cells generates potent and long lasting antitumor immunity. Cancer Res 1990; 50:7820-7825.
46. Colombo MP, Ferrari G, Stoppacciaro A et al. Granulocyte colony-stimulating factor gene transfer suppressed
tumorigenicity of a murine adenocarcinoma in vivo. J Exp Med 1991; 173:889-897.
47. Golumbek PT, Lazenby AJ, Levitsky HI et al. Treatment of established renal cancer by tumor cells engineered
to secrete interleukin-4. Science 1991; 254:713-716.
48. Mullen CA, Coale MM, Levy AT et al. Fibrosarcoma cells transduced with the IL-6 gene exhibit reduced
tumorigenicity, increased immunogenicity and decreased metastatic potential. Cancer Res 1992;
52:6020-6024.
49. Dranoff G, Jaffee E, Lazenby A et al. Vaccination with irradiated tumor cells engineered to secrete murine
granulocyte-macrophage colony-stimulating factor stimulates potent, specific and long-lasting anti-tumor
immunity. Proc Nat Acad Sci USA 1993; 90:3539-3543.
50. Connor J, Bannerji R, Saito S et al. Regression of bladder tumors in mice treated with interleukin
2 gene-modified tumor cells. J Exp Med 1993; 177:1127-1134.
51. Cavallo F, Pierro FD, Giovarelli M et al. Protective and curative potential of vaccination with
interleukin-2-gene-transfected cells from a spontaneous mouse mammary adenocarcinoma. Cancer Res
1993; 53:5067-5070.
52. Tahara H, Zeh HJ, Storkus WJ et al. Fibroblasts genetically engineered to secrete interleukin 12 can suppress
tumor growth and induce antitumor immunity to a murine melanoma in vivo. Cancer Res 1994; 54:182-189.
53. Marincola FM, Shamamian P, Alexander RB et al. Loss of HLA haplotype and down-regulation in melanoma
cell lines. J Immunol 1994; 153:1225-1237.
54. Valmori D, Levy F, Miconnet I et al. Induction of potent antitumor CTL responses by recombinant vaccinia
encoding a melon-A peptide analogue. J Immunol 2000; 164:1125-1131.
55. Yu JS, Wei MX, Chiocca EA et al. Treatment of glioma by engineered interleukin 4-secreting cells. Cancer
Research 1993; 53:3125-3128.
56. Natsume A, Mizuno M, Ryuke Y et al. Antitumor effect and cellular immunity activation by murine
interferon-beta gene transfer against intracerebral glioma in mouse. Gene Ther 1999; 6:1626-1633.
57. Liu Y, Ehtesham M, Samoto K et al. In site adenoviral interleukin 12 gene transfer confers potent and
long-lasting cytotoxic immunity in glioma. Cancer Gene Ther 2002; 9:9-15.
58. Cohen EP. DNA-based vaccines for the treatment of canceran experimental model. Trends Mol Med
2001; 7:175-179.
59. Lichtor T, Glick RP, Tarlock K et al. Application of interleukin-2-secreting syngeneic/allogeneic fibroblasts
in the treatment of primary and metastatic brain tumors. Cancer Gene Ther 2002; 9:464-469.
CHAPTER 13
PEPTIDE VACCINE
Shuichi Izumoto
Department of Neurosurgery, Hyogo College of Medicine, Hyogo, Japan
Email: sizumoto@hyo-med.ac.jp
Abstract: Current combinations of surgical therapy, radiotherapy and chemotherapy regimens

do not significantly improve long-term survival of the patients with malignant
glioma. Cancer immunotherapy against malignant glioma is a potentially new
therapeutic strategy that primes a patients immune system to attack glioma cells.
Peptide-based vaccination appears promising as an approach to successfully induce
an antineoplastic immune response, produce clinical response and prolong survival
in patients with malignant glioma without major side effects. In this chapter, clinical
progress is reviewed in developing peptide-based vaccinations for malignant glioma
to date.
INTRODUCTION
Recent advances in molecular biology and tumor immunology have resulted in the
identification of a large number of tumor-associated antigens (TAAs) that could be used
for cancer vaccination, since their epitopes associated with HLA class I molecules were
recognized by cytotoxic T-lymphocytes (CTLs). Figure 1 shows elicitation of the cellular
immune responses against TAA in glioma cells.
On the other hand, glioma cells have been thought to be poor antigen-presenting cells
(APCs) to the immune system because of the down-regulation of molecules required to
activate the immune system1 and the secretion of immunosuppressive cytokines, such as
transforming growth factor-`,2 vascular endothelial growth factor3 and interleukin-10.4
There are increasing reports demonstrating systemic immunotherapy using dendritic
cells and peptide antigens that induce immune response against malignant gliomas within
the immunologically privileged brain.5-14 The blood-brain barrier, which was thought to be
one of the hurdles hindering the development of therapeutically effective immunotherapy

166
PEPTIDE VACCINE 167
for gliomas, does not always function effectively in cases involving malignant gliomas.6
These advances suggest the possibility of the development of a new peptide-based cancer
immunotherapy for malignant gliomas.
PEPTIDE-BASED VACCINES
Peptide-based vaccines represent a major focus of cancer vaccine research that

offers exciting clinical possibilities. Peptides used as cancer vaccines usually consist of
introducing small peptides (typically 7-14 amino acids in length) that are immunologic
and expressed by targeted cells (Fig. 2). Peptides are capable of binding to a particular
HLA class I antigen. Such peptides are processed by host antigen presenting cells (APC),
which travel to the lymph nodes and sensitize circulating T-lymphocytes, known as
cytotoxic T cells (CTL).
A peptide suitable for the individual patient is generally mixed with an adjuvant
followed by subcutaneous administration every 7-14 days to form a vaccine. CTL
recognizing a peptide on APC become activated in association with clonal expansion
in the nodes. These activated CTL come out through lymph nodes or blood circulation,
migrate and infiltrate into the brain, recognize the corresponding peptideHLA complex
on glioma cells and eliminate glioma cells, which in turn results in tumor regression.
Several clinical trials of peptide-based immunotherapy for cancer have been conducted
in the past decade, but major clinical responses were rarely obtained.15 Recently, objective
responses for peptide vaccinations including WT1 peptide, personalized peptide and
EGFRvIII peptide are obtained and reported. These clinical trials for malignant gliomas
were reviewed in this chapter.
WT1-PEPTIDE VACCINATION
One of the identified TAAs was Wilms tumor gene WT1 product.16 The WT1 gene
was isolated as a gene responsible for Wilms tumor. It encodes a zinc finger transcription
factor, which is involved in cell proliferation and differentiation, apoptosis and organ
development. Although the WT1 gene was first categorized as a tumor suppressor gene,
it was later proposed that the wild-type WT1 gene functions as an oncogene rather than
as a tumor-suppressor gene.
Recent investigations demonstrated that immune responses against WT1 protein,
both humoral and cellular, were naturally elicited in cancer patients, indicating that WT1
protein is immunogenic.17-23 According to accumulating evidences, it seems obvious that
WT1 protein-derived CTL epitopes (peptides) identified so far are highly immunogenic
in the clinical setting. It is notable that only a single kind of WT1 peptide could induce an
increase in WT1 tetramer
CD8
T-cell frequencies and/or make the peptide-specific DTH
reaction positive after vaccination, leading to the emergence of clinical responses24-28 (Fig.
1). It was also shown that only a single injection of WT1 peptide could induce an increase
in WT1 tetramer
CD8
T cells.25,29
In many reports, the wild-type WT1 gene was shown to be overexpressed in various
types of solid tumors including malignant glioma.30 The WT1 protein was found to be
an attractive target antigen for immunotherapy against these malignancies.31 Recently,
a Phase I clinical trial was performed to examine the safety of a WT1-based vaccine,
Figure 1. Figure shows elicitation of the cellular immune responses against tumor-associated antigen
(TAA) in glioma patients. Glioma cell-derived TAA is ingested by antigen-presenting cells (APCs) such
as dendritic cells and is processed in them, followed by presentation of peptide antigen in association
with HLA class I or II molecules on the surface of the APCs. Peptide antigen/HLA class I complexes
stimulate CD8
T cells to make antigen-specific cytotoxic T-lymphocytes (CTLs); killer T cells. Peptide
antigen/HLA class II complexes stimulate CD4
T cells to make antigen-specific helper T cells, which
more activate (help) CTLs. Activated antigen-specific killer T cells attack glioma cells that have
peptide antigen/HLA class I complexes on the cell surfaces.
as well as the clinical and immunological responses of patients with a variety of cancer
types, including leukemia, lung cancer and breast cancer.24 The immunization consisted
of an HLA-A*2402-restricted, modified 9 mer WT1 peptide (amino acids 235-243
CYTWNQMNL) (Multiple Peptide Systems, San Diego CA), in which Y was substituted
for M at amino acid position 2 (the anchor position) of the natural WT1 peptide. About
60% of Japanese have HLA-A*2402 which is the most common HLA Class I type in
the Japanese population. The modified 9-mer WT1 peptide was shown to induce much
stronger CTL activity against WT1-expressing tumor cells than the natural peptide.32
The authors demonstrated that WT1 peptide vaccination emulsified with Montanide
ISA51 adjuvant at 0.3, 1.0, 3.0 mg per body at 2-week intervals was safe for patients.
Furthermore, the vaccination induced peptide-specific CTLs and was associated with
clinical response. Very recently, it was confirmed that the potential toxicities of the
weekly WT1 vaccination treatment schedule (3 mg per body) with the same adjuvant
were also acceptable.33
A Phase I/II trial of weekly injection of WT1 peptide vaccine for HLA-A*2402
patients with various kinds of solid cancers was conducted. The Phase I part composed
of the first 10 patients was finished33 and vaccination related systemic toxicities were
PEPTIDE VACCINE 169
not observed. As disease-specific Phase II trial targeting glioblastoma, 21 patients with

HLA-A*2402 positive type were enrolled13(Fig. 2). In all the excluded patients who were
HLA-A*2402 negative type, the survival time from recurrence or progression to death
was investigated. The median survival time after tumor recurrence of the HLA-A2402
negative patients was 21 weeks, which was almost the same with that of the patients as a
historical control (20 weeks). The WT1 vaccinations were scheduled to be given weekly
for 12 consecutive weeks. Twelve weeks after the initial vaccination, the response was
evaluated on MR imaging. If an effect was observed after the 12 vaccinations, WT1
vaccination was continued at 1-week intervals until tumor progression was again noted.
All treated patients had a local inflammatory response with erythema at the WT1 vaccine
injection site. No Grade 3 or 4 toxicities were observed.
As a result of WT1 vaccination for 21 recurrent glioblastoma patients, two partial
response (PR) and 10 stable disease (SD) and 9 progressive disease (PD) in Response
Evaluation Criteria in Solid Tumors (RECIST)34 were obtained. The overall response rate
was 9.5%. The disease control rate, which included cases having CR, PR and SD in the
initial three months (the clinical trial period) was 57.1%. The median progression-free
survival (PFS) period was 20.0 weeks and PFS rate at 6 months was 33.3% (Fig. 3).
Median overall survival after initial vaccination was 36.7 weeks. Median overall survival
after tumor recurrence in WT1-vaccinated patients was 46 weeks. The 6-month PFS rate
was 33.3% in the patients with glioblastoma- which was higher than the 10% that was set
Figure 2. HLA-A2402 restricted WT1 peptide is injected intradermally and is processed in the APCs,
followed by presentation of WT1 peptide in association with HLA class I molecule on the surface
of the APCs. WT1 peptide/HLA class I complexes stimulate CD8
T cells to make WT1-specific
cytotoxic T-lymphocytes; killer T cells. Activated WT1-specific CTLs attack glioma cells that have
WT1 peptide/HLA class I complexes on the cell surfaces and eliminate glioma cells, which in turn
result in glioma regression.
Figure 3. Figure shows Kaplan-Meier curves for median progression-free survival (PFS) (solid line)
and overall survival (dotted line) after initial WT1 vaccination for patients with recurrent glioblastoma.
Median PFS in the 21 patients with glioblastoma who were included in the study was 20.0 weeks and
the progression-free survival rate at 6 months (26 weeks) was 33.3%. Median overall survival after
initial vaccination was 36.7 weeks. Median overall survival after tumor recurrence in WT1-vaccinated
patients was 46 weeks. Modified with permission from a figure in J Neurosug 2008; 108:963-971.13
as indicating an active level35- and, moreover, was higher than the 30% that was set as the
alternative hypothesis before the study was started. Thus, this result suggested that WT1
vaccination was active.
The frequencies of WT-1 specific CTLs before WT1 vaccination were significantly
higher in patients with glioma than in healthy controls (p 0.0019) (Fig. 4). These results
indicate that the immune-system in patients with WT1-expressing glioblastoma cells
responded to the WT1 protein derived from the tumor cells and elicited WT1-specific
CTLs that were present before WT1 vaccination; this suggests that the WT1 protein
in glioblastoma cells is naturally immunogenic. The existence of the high frequencies
of WT1-specific CTLs before WT1 vaccination may have contributed to the favorable
clinical responses in patients with glioblastoma. There was no correlation between the
induction of a clinical response and WT1-specific CTL frequencies in the PBMCs of the
patients prior to vaccination. Furthermore, the CTL frequencies did not increase after
vaccination, even in the responder patients (Fig. 4).
It was found that, in HLA-A*2402 positive glioblastoma patients, immunotherapy with
HLA-A*2402-restricted, modified 9-mer WT1 peptide vaccination had disease stabilizing,
as well as disease progression-inhibiting, effects that were at least equal or superior to
chemotherapy, with systemic toxicity that was much less than that of chemotherapy
and, thus, allowed the vaccination to be given for a long time. This is another advantage
of the vaccine: one does not need to choose a suitable combination of peptides in the
PEPTIDE VACCINE 171
Figure 4. Figure shows the frequencies of WT1-specific CTLs before WT1 vaccination, 4 and 12 weeks
after WT1 vaccination and in healthy controls. Both cases with controlled disease (PR and SD; closed
circle) and those with uncontrolled disease (PD; open circle) had a high frequency of WT1-specific
CTLs during the entire evaluation period compared to healthy controls. These results indicate that the
immune system in patients with WT1-expressing glioblastoma cells responded to the WT1 protein derived
from the tumor cells and elicited WT1-specific CTLs that were present before WT1 vaccination; this
suggests that the WT1 protein in glioblastoma cells is naturally immunogenic. Modified with permission
from a figure in J Neurosug 2008; 108:963-971.13
laboratory before vaccination. Compared to DC therapy, WT1 vaccination is simple. The

use of a more suitable adjuvant, such as BCG-CWS,36 or combination therapy involving
vaccination37 and other modalities may further enhance the clinical usefulness of this
treatment for glioblastoma patients. All the treated patients had an inflammatory response
with erythema at the WT1 vaccine injection site, but no systemic toxicities were observed.
Taken together, these findings would allow one to conclude that WT1 vaccination had
an antiglioblastoma effect, it was safe and the patients tolerated it well.38
PERSONALIZED PEPTIDE VACCINATION
Dr. Itoh, et al6,37,39 have developed the personalized peptide vaccination. This strategy
is based on the assumption that initiation of immune boosting of CTLs through peptide
vaccination could be more effective than immune priming of nave T cells with regard
to induction of prompt and strong immunity. Vaccination with peptides in patients with
higher levels of CTL precursors in prevaccination PBMCs might induce stronger and
faster activation of CTL compared to that with rare CTL precursors. Consistent with this
notion, the objective response rates of classical (nonpersonalized) peptide vaccines were
0%, whereas that of personalized vaccines was 11.1% in the total advanced cancers.37
A Phase I study of personalized peptide vaccination was conducted for patients
with advanced malignant glioma.6 Twenty-five patients with advanced malignant glioma
(eight Grade III and 17 Grade IV) were enrolled. For personalized peptide vaccination,
prevaccination PBMCs and plasma were provided to examine cellular and humoral
responses to 25 or 23 peptides in HLA-A24
or HLA-A2
patients, respectively and
then only the reactive peptides (maximum of four) were used for in vivo administration.
The peptides derived from SART3, Lck (lymphocyte-specific protein tyrosine kinase)
and multidrug resistance-associated protein 3 antigens were most frequently selected
for vaccination. The protocols were well tolerated with the major adverse effects being
a Grade 1 or 2 inflammatory skin reaction at the injection site. Increases in cellular or
humoral responses specific to at least one of the vaccinated peptides was observed in
the postvaccination (sixth) samples from 14 or 11 of 21 patients, respectively. Clinical
responses were 5 PR, 8 SD and 8 PD. The median PFS was 3 months and the PFS at
6 months was 13%. The median overall survival for patients with recurrent glioblastoma
in this study was 622 days. Personalized peptide vaccinations were recommended for
further clinical study in malignant glioma patients.40,41
EGFRvIII PEPTIDE VACCINATION
The majority of glioblastoma cases harbor mutations in the Epidermal Growth

Factor Receptor (EGFR). In particular, the epidermal growth factor receptor variant
III (EGFRvIII) is a consistent tumor-specific mutation that is widely expressed in
malignant gliomas and other neoplasms. This mutation encodes a constitutively active
tyrosine kinase that enhances tumorgenicity and migration and confers radiation and
chemotherapeutic resistance. The EGFRvIII mutation arises from an in-frame deletion/
fusion that results in constitutively active EGF signaling. This in-frame deletion mutation
splits a codon resulting in the creation of a novel glycine at the fusion junction between
normally distant parts of the molecule and producing a sequence re-arrangement which
creates a tumor-specific epitope for cellular or humoral immunotherapy in patients with
malignant gliomas. An EGFRvIII-specific peptide that spans this deletion/fusion has
shown to be an immunogenic antigen42 and was found to be an attractive target antigen
for immunotherapy against malignant gliomas.
In a Phase I clinical trial conducted at Duke University, 20 patients of malignant
glioma were enrolled and vaccinated with the EGFRvIII peptide. Following surgical
resection, each patient underwent leukapheresis to obtain peripheral blood mononuclear
cells (PBMCs) for autologous mature DC generation and baseline immunologic monitoring.
Three patients with anaplastic astrocytomas and 13 patients with glioblastomas were
vaccinated. DCs were pulsed for 2 h, with 500 +g of EGFRvIII-specific peptide, PEPvIII
(LEEKKGNYVVTDHC) (Multiple Peptide Systems, San Diego CA) which was conjugated
1:1 with keyhole limpet hemocyanin (KLH )(Biosyn, Carlsbad, CA).14 The first 3 patients
received 3 = 107 mature DCs per vaccination intradermally beginning 2 weeks following
completion of radiation therapy, the remaining patients received one-third of their total
generated DCs per injection (up to 1.1 = 108 cells).
PEPTIDE VACCINE 173
Patients in this trial demonstrated immunologic responses42 without any evidence

of adverse events aside from Grade I and II local reactions at the vaccine injection site.
Median time-to-progression (TTP) in patients with glioblastoma (n 13) was 46.9
weeks and median survival was 110.8 weeks. This compares well with other published
reports in similar patient populations using carmustine wafers43 or temozolomide cycles44
where reported median survival times were 59.6 and 63.3 weeks, respectively. These trials
demonstrated that vaccines targeting EGFRvIII are capable of inducing potent T- and
B-cell immunity in these patients and lead to an unexpectedly long survival time. Most
importantly, vaccines targeting EGFRvIII were universally successful at eliminating
tumor cells expressing the targeted antigen without any evidence of symptomatic
collateral toxicity.
In a Phase II multicenter trial45 conducted at Duke University and the University
of Texas, M.D. Anderson Cancer Center, newly diagnosed glioblastoma patients were
enrolled and vaccinated with the EGFRvIII peptide. As in the previous trial, 2 weeks after
completing standard external beam radiation therapy, patients received 3 vaccinations at
2-week intervals of 500 +g of PEPvIII-KLH (AnaSpec, Inc., San Jose, CA 95131) in 0.8
mL of saline with GM-CSF. DCs were not treated because to simplify the manufacture
of the vaccine. Unlike in the previous trial, subsequent vaccines were then continued
monthly until tumor progression was evident. Symptomatic adverse events in this trial
again consisted mostly of Grade I and II vaccine site reactions. The results confirmed the
encouraging results seen in the Phase I study.42 Humoral and cellular immune responses
that were specific for EGFRvIII were detected ex vivo after vaccination in this trial. In
combination, these results strongly suggest that vaccination with PEPvIII-KLH may
prolong survival in at least some patients with newly diagnosed glioblastoma.42
HURDLES TO EFFECTIVE PEPTIDE VACCINE
There are drawbacks that could still hinder the peptide vaccine therapy. The
mechanisms of tumor escape from immune recognition/destruction are thought to be
multifactorial.46 First, there are frequent lack of Major Histocompatibility Markers (MHC)
on glioma cells.47 Peptide-based vaccines function through the use of antigen presenting
cells which activate a CTL response by presenting antigen on MHC I. Because CTLs are
MHC I restricted, antigens that are presented in other ways fail to provoke an immune
response. In glioblastoma, it was documented that MHC I is absent in approximately
50% of cases.48,49 Glioma cell-mediated immunosuppression like those may prevent
CTL or Natural Killer (NK) cells from effectively targeting cells. Second, high levels
of interleukin-10, as well as TGFbeta2 signaling in the vicinity of tumors may lead to a
bias in T-cell differentiation towards the Th2 subtype. In addition to the observation that
quantitative levels of immune cells observed near tumors are typically depressed, a larger
fraction of those cells are regulatory T cells (Th2 phenotype) known for their ability to
further depress the immune response.50 Third, patients with recurrent glioma are usually
in an immunosuppressive state because of the advanced disease and myelosuppression
by anticancer agents.51 It should be difficult to induce antiglioma immune responses in
such a condition using immunotherapy. Fourth, peptide has problems of the potential of
tumor antigens escape and limited repertoire of using defined antigens. Peptides are HLA
class I and class II restricted and, consequently, restrict patient enrollment into clinical
trials and class I peptides are insufficient to generate a CD4

T helper response which
are required for optimal effective antitumor immunity.52
ENHANCEMENT OF CLINICAL EFFICACY AND USEFULNESS

OF PEPTIDE VACCINE
Peptide vaccination has several advantages including easy supply of good

manufacturing practice levels of materials and a reliable laboratory marker for the
prediction of clinical outcome. Although it was revealed that peptide vaccinations had
clinical efficacy and usefulness at least for some patients with malignant gloma6,13,14 and
for patients with solid and hematological cancers,24,25,28,53-56 we need to further improve
the vaccine. Strategies to immunologically enhance the power of the vaccine may include
first, to find or develop more strong adjuvant; second, to use HLA class II-restricted
helper epitopes which enhance induction/activation of CTLs and make memory CTLs
in combination with CTL epitopes57-60 and third, to use multiple CTL epitopes.61,62 In
addition to treatment using only peptide vaccine with appropriate adjuvant/cytokine,
combined usage of peptide vaccine and other kinds of drugs, including molecular
target-based drugs,61 or chemotherapy drugs,63-65 might be another strategy to develop a
novel modality for anticancer action.
EVALUATION OF PEPTIDE VACCINE-INDUCED CLINICAL RESPONSES
Chemotherapy drugs directly attack glioma cells, while peptide vaccine does not.
The latter indirectly give damage to glioma cells by the activation of immune system.
Therefore, we may need peptide vaccine-specific response evaluation criteria.66,67
It is probable that some of the peptide vaccine-treated patients survive long-term
with good QOL even if tumor regression is not obvious23,24,28 or that the tumor
growth may be stabilized after an initial increase in its size because immunotherapy
is generally not as quick-acting as chemotherapy.66,67 Therefore, when we evaluate
vaccination-induced clinical responses with RECIST, which is a gold standard in the field
of cancer chemotherapy, it may be recommended that SD is highly regarded in peptide
immunotherapy, particularly when SD persists long-term. It may also be recommended
that the stabilization of disease after initial progression of disease, which is categorized
as PD in RECIST, is also evaluated favorably, like SD after PD.
CONCLUSION
In the patients with malignant glioma, immunotherapy with peptide vaccination had
disease-stabilizing, as well as disease progression-inhibiting, effects that were equal or
superior to those of chemotherapy, with systemic toxicity that was much less than that
of chemotherapy and thus allowed the vaccinations to be given for a long time. The
use of more suitable adjuvant or combination therapy involving vaccination and other
modalities may further enhance the clinical usefulness of this treatment for patients with
malignant glioma.
PEPTIDE VACCINE 175
REFERENCES
1. Satoh J, Lee YB, Kim SU. T-cell costimulatory molecules B7-1 (CD80) and B7-2 (CD86) are expressed in
human microglia but not in astrocytes in culture. Brain Res 1995; 704:92-99.
2. Constam DB, Philipp J, Malipiero UV et al. Differential expression of transforming growth factor-beta 1, -beta
2 and -beta 3 by glioblastoma cells, astrocytes and microglia. J Immunol 1992; 148:1404-1410.
3. Gabrilovich DI, Ishida T, Nadaf S et al. Antibodies to vascular endothelial growth factor enhance the efficacy of
cancer immunotherapy by improving endogenous dendritic cell function. Clin Cancer Res 1999; 5:2963-2970.
4. Chen Q, Daniel V, Maher DW et al. Production of IL-10 by melanoma cells: examination of its role in
immunosuppression mediated by melanoma. Int J Cancer 1994; 56:755-760.
5. Yu JS, Wheeler CJ, Zelzer PM et al. Vaccination of malignant glioma patients with peptide-pulsed dendritic
cells elicits systemic cytotoxicity and intracranial T-cell infiltration. Cancer Res 2001; 61:842-847.
6. Yajima N, Yamanaka R, Mine T et al. Immunological evaluation of personalized peptide vaccination for
patients with advanced malignant glioma. Clin Cancer Res 2005; 11:5900-5911.
7. Kikuchi T, Akasaki Y, Irie M et al. Results of a Phase I clinical trial of vaccination of glioma patients with
fusions of dendritic and glioma cells. Cancer Immunol Immunother 2001; 50:337-344.
8. Yamanaka R, Abe T, Yajima N et al. Vaccination of recurrent glioma patients with tumour lysate-pulsed dendritic
cells elicits immune responses: results of a clinical Phase I/II trial. Br J Cancer 2003; 89:1172-1179.
9. Yu JS, Liu G, Ying H et al. Vaccination with tumor lysate-pulsed dendritic cells elicits antigen-specific, cytotoxic
T-cells in patients with malignant glioma. Cancer Res 2004; 64:4973-4979.
10. Kikuchi T, Akasaki Y, Abe T et al. Vaccination of glioma patients with fusions of dendritic and glioma cells
and recombinant human interleukin 12. J Immunother 2004; 27:452-459.
11. Rutkowski S, De Vleeschouwer S, Kaempgen E et al. Surgery and adjuvant dendritic cell-based tumour
vaccination for patients with relapsed malignant glioma, a feasibility study. Br J Cancer 2004; 94:1656-1662.
recurrent glioma: results of a clinical Phase I/II trial. Clin Cancer Res 2005; 11:4160-4167.
with recurrent glioblastoma multiforme. J Neurosurg 2008; 108:963-971.
14. Heimberger AB, Sun W, Hussain SF et al. Immunological responses in a patient with glioblastoma multiforme
treated with sequential courses of temolozomide and immunotherapy: Case study. Neuro Oncol 2008;
10:98-103.
15. Rosenberg SA, Yang JC, Restifo NP. Cancer immunotherapy: moving beyond current vaccines. Nat Med
2004; 10:909-915.
16. Oka Y, Tsuboi A, Elisseeva OA et al. WT1 as a novel target antigen for cancer immunotherapy. Curr Cancer
Drug Targets 2002; 2:45-54.
17. Gaiger A, Reese V, Disis ML et al. Immunity to WT1 in the animal model and in patients with acute myeloid
leukemia. Blood 2000; 96:1480-1489.
18. Gaiger A, Carter L, Greinix H et al. WT1-specific serum antibodies in patients with leukemia. Clin Cancer
Res 2001; 7:761s-765s.
19. Elisseeva OA, Oka Y, Tsuboi A et al. Humoral immune responses against Wilms tumor gene WT1 product
in patients with hematopoietic malignancies. Blood 2002; 99:3272-3279.
20. Wu F, Oka Y, Tsuboi A et al. Th1-biased humoral immune responses against Wilms tumor gene WT1 product
in the patients with hematopoietic malignancies. Leukemia 2005; 19:268-274.
21. Scheibenbogen C, Letsch A, Thiel E et al. CD8 T-cell responses to Wilms tumor gene product WT1 and
proteinase 3 in patients with acute myeloid leukemia. Blood 2002; 100:2132-2137.
22. Rezvani K, Grube M, Brenchley JM et al. Functional leukemia-associated antigen-specific memory CD8
T-cells exist in healthy individuals and in patients with chronic myelogenous leukemia before and after
stem cell transplantation. Blood 2003; 102:2892-2900.
23. Rezvani K, Yong AS, Savani BN et al. Graft-versus-leukemia effect associated with detectable Wilms tumor-1
specific T-lymphocytes after allogeneic stem-cell transplantation for acute lymphoblastic leukemia. Blood
2007; 110:1924-1932.
24. Oka Y, Tsuboi A, Taguchi T et al. Induction of WT1 (Wilms tumor gene)-specific cytotoxic T-lymphocytes by
WT1 peptide vaccine and the resultant cancer regression. Proc Natl Acad Sci USA 2004; 101:13885-13890.
25. Oka Y, Tsuboi A, Murakami M et al. Wilms tumor gene peptide-based immunotherapy for patients with overt
leukemia from myelodysplastic syndrome (MDS) or MDS with myelofibrosis. Int J Hematol 2003; 78:56-61.
26. Mailaender V, Scheibenbogen C, Thiel E et al. Complete remission in a patient with recurrent acute myeloid
leukemia induced by vaccination with WT1 peptide in the absence of hematological or renal toxicity.
Leukemia 2004; 18:165-166.
27. Kawakami M, Oka Y, Tsuboi A et al. Clinical and immunological responses to the vaccination with very
low dose (5 mg/body) of WT1 peptide in a patient with chronic myelomonocytic leukemia. Int J Hematol
2007; 85:426-429.
28. Iiyama T, Udaka K, Takeda S et al. WT1 (Wilms Tumor 1) peptide immunotherapy for renal cell carcinoma.
Microbiol Immunol 2007; 51:519-530.
29. Rezvani K, Yong ASM, Mielke S et al. Leukemia-associated antigen specific T-cell responses following
combined PR1 and WT1 peptide vaccination in patients with myeloid malignancies. Blood 2008; 111:236-242.
30. Hashiba T, Izumoto S, Kagawa N et al. Expression of WT1 protein and correlation with cellular proliferation
in glial tumors. Neurol Med Chir (Tokyo)2007; 47:165-170.
31. Oka Y, Udaka K, Tsuboi A et al. Cancer immunotherapy targeting Wilms tumor gene WT1 product. J
Immunol 2000; 164:1873-1880.
32. Tsuboi A, Oka Y, Udaka K et al. Enhanced induction of human WT1-specific cytotoxic T-lymphocytes with
a 9-mer WT1 peptide modified at HLA-A*2403-binding residues. Cancer Immunol Immunother 2002;
51:614-620.
33. Morita S, Oka Y, Tsuboi A et al. A phase I/II trial of a WT1 (Wilms tumor gene) peptide vaccine in patients
with solid malignancy: safety assessment based on the phase I data. Jpn J Clin Oncol 2006; 36:231-236.
34. Therasse P, Arbuck SG, Eisenhauer EA et al. New guidelines to evaluate the response to treatment in solid
tumors. J Natl Cancer Inst 2006; 92:205-216.
35. Hosli P, Sappino AP, de Tribolet N et al. Malignant glioma: Should chemotherapy be overthrown by
experimental treatments? Ann Oncol 1998; 9:589-600.
36. Nakajima H, Kawasaki K, Oka Y et al. WT1 peptide vaccination combined with BCG-CWS is more efficient
for tumor eradication than WT1 peptide vaccination alone. Cancer Immunol Immunother 2004; 53:617-624.
37. Ito K, Yamada A. Personalized peptide vaccines: a new therapeutic modality for cancer. Cancer Sci 2006;
97:970-976.
38. Oka Y, Tsuboi A, Oji Y et al. WT1 peptide vaccine for the treatment of cancer. Curr Opin Immunol 2008;
20:211-220.
39. Mine T, Sato Y, Noguchi M et al. Humoral responses to peptides correlate with overall survival in advanced
cancer patients vaccinated with peptides based on pre-existing peptide-specific cellular responses. Clin
Cancer Res 2004; 10:929-937.
40. Yamanaka R. Dendritic-cell- and peptide-based vaccination strategies for glioma. Neurosurg Rev 2009.
(Epub ahead of print)
41. Yamanaka R, Itoh K. Peptide-based immunotherapeutic approaches to glioma: a review. Expert Opin Biol
Ther 2007; 7:645-649.
42. Sampson JH, Archer GE, Mitchell DA et al. Tumorspecific immunotherapy targeting the EGFRvIII mutation
in patients with malignant glioma. Semin Immunol 2008; 20:267-275.
43. Westphal M, Hilt DC, Bortey E et al. A phase 3 trial of local chemotherapy with biodegradable carmustine
(BCNU) wafers (Gliadel wafers) in patients with primary malignant glioma. Neuro Oncol 2003; 5:79-88.
44. Stupp R, Mason WP, van den Bent MJ et al. Radiotherapy plus concomitant and adjuvant temozolomide for
glioblastoma. N Engl J Med 2005; 352:987-996.
45. Heimberger AB, Hussain FS, Aldape K et al. Tumor-specific peptide vaccination in newly-diagnosed patients
with GBM. Proc Am Soc Clin Oncol 2006; 24:2529.
46. Ahmad M, Rees RC, Ali SA. Escape from immunotherapy: possible mechanisms that influence tumor
regression/progression. Cancer Immunol Immunother 2004; 53:844-854.
47. Steinman RM, Dhodapkar M. Active immunization against cancer with dendritic cells: the near future. Int J
Cancer 2001; 94:459-473.
defects in astrocytic tumors. Clin Cancer Res 2005; 11:8304-8311.
49. Wiendl H, Mitsdoerffer M, Weller M. Hide-and-seek in the brain: a role for HLAG mediating immune privilege
for glioma cells. Semin Cancer Biol 2003; 13:343-351.
66:3294-3302.
51. Jacobs JF, Idema AJ, Bol KF et al. Regulatory T-cells and the PD-L1/PD-1 pathway mediate immune
suppression in malignant human brain tumors. Neuro Oncol 2008. [Epub ahead of print]
52. Knutson KL, Disis ML. Tumor antigen-specific T helper cells in cancer immunity and immunotherapy. Cancer
Immunol Immunother 2005; 54:721-728.
53. Mailaender V, Scheibenbogen C, Thiel E et al. Complete remission in a patient with recurrent acute myeloid
leukemia induced by vaccination with WT1 peptide in the absence of hematological or renal toxicity.
Leukemia 2004; 18:165-166.
54. Rezvani K, Yong ASM, Mielke S et al. Leukemia-associated antigen specific T-cell responses following
combined PR1 and WT1 peptide vaccination in patients with myeloid malignancies. Blood 2008; 111:236-242.
55. Kawakami M, Oka Y, Tsuboi A et al. Clinical and immunological responses to the vaccination with very
low dose (5 mg/body) of WT1 peptide in a patient with chronic myelomonocytic leukemia. Int J Hematol
2007; 85:426-429.
PEPTIDE VACCINE 177
56. Tsuboi A, Oka Y, Osaki T et al. WT1 peptide based immunotherapy for patients with lung cancer: report of
two cases. Microbiol Immunol 2004; 48:175-184.
57. Knights AJ, Zaniou A, Rees RC et al. Prediction of an HLA-DR-binding peptide derived from Wilms tumour
1 protein and demonstration of in vitro immunogenicity of WT1(124-138)-pulsed dendritic cells generated
according to an optimised protocol. Cancer Immunol Immunother 2002; 51:271-281.
58. Kobayashi H, Nagato T, Aoki N et al. Defining MHC class II T helper epitopes for WT1 tumor antigen.
59. Fujiki F, Oka Y, Tsuboi A et al. Identification and characterization of a WT1 (Wilms tumor gene) protein-derived
HLA-DRB1*0405-restricted 16-mer helper peptide that promotes the induction and activation of WT1-specific
cytotoxic T-lymphocytes. J Immunother 2007; 30:282-293.
60. May RJ, Dao T, Pinilla-Ibarz J et al. Peptide epitopes from the Wilms tumor 1 oncoprotein stimulate CD4
and CD8
T-cells that recognize and kill human malignant mesothelioma tumor cells. Clin Cancer Res
2007; 13:4547-4555.
61. Noguchi M, Itoh K, Suekane S et al. Phase I trial of patient-oriented vaccination in HLA-A2-positive patients
with metastatic hormone-refractory prostate cancer. Cancer Sci 2004; 95:77-84.
62. Bocchia M, Gentili S, Abruzzese E et al. Effect of a p210 multipeptide vaccine associated with imatinib
or interferon in patients with chronic myeloid leukaemia and persistent residual disease: a multicentre
observational trial. Lancet 2005; 365:657-662.
63. Ercolini AM, Ladle BH, Manning EA et al. Recruitment of latent pools of high-avidity CD8(
) T-cells to the
antitumor immune response. J Exp Med 2005; 201:1591-1602.
64. Chong G, Morse MA. Combining cancer vaccines with chemotherapy. Expert Opin Pharmacother 2005;
6:2813-2820.
65. Suzuki K, Kapoor V, Jassar AS et al. Gemcitabine selectively eliminates slpenic Gr-1
/CD11b
myeloid
suppressor cells in tumor-bearing animals and enhances antitumor immune activity. Clin Cancer Res 2005;
11:6713-6721.
66. Hori A, Kami M, Kim S-W et al. Urgent need for a validated tumor response evaluation system for use in
immunotherapy. Bone Marrow Transplant 2004; 33:255-256.
67. Hoos A, Parmiani G, Hege K et al. A clinical development paradigm for cancer vaccines and related biologics.
J Immunother 2007; 30:1-15.
CHAPTER 14
ACTIVE IMMUNOTHERAPY
Oncolytic Virus Therapy Using HSV-1
Tomoki Todo
Department of Neurosurgery, The University of Tokyo, Tokyo, Japan
Email: toudou-nsu@umin.ac.jp
Abstract: Conditionally replicating herpes simplex viruses Type 1 (HSV-1) are promising
therapeutic agents for glioma. They can replicate in situ, spread and exhibit oncolytic
activity via a direct cytocidal effect. In addition, specific antitumor immunity is
effectively induced in the course of oncolytic activities. G476 is a genetically
engineered HSV-1 with triple mutations that realized augmented viral replication in
tumor cells, strong induction of antitumor immunity and enhanced safety in normal
tissues. A clinical trial of G476 in patients with recurrent glioblastoma has started
in 2009. One of the advantages of HSV-1 is its capacity to incorporate large and/or
multiple transgenes within the viral genome. In preclinical studies, arming of an
oncolytic HSV-1 with transgenes encoding immunomodulatory molecules, such as
interleukin 12, has been shown to greatly augment the efficacy of oncolytic HSV-1
therapy. Oncolytic virus therapy using HSV-1 may be a useful treatment for glioma
that can also function as an efficient in situ tumor vaccination.
INTRODUCTION
Oncolytic virus therapy is an attractive and rapidly developing means for treating
glioma.1,2 Genetically engineered viruses, such as herpes simplex virus Type 1 (HSV-1)
and adenovirus, are designed so that virus replication is restricted to tumor cells and
therefore infection causes no harm to normal tissues. In principle, infected tumor cells
are destroyed by a direct oncolytic activity of the viruses. Importantly, oncolytic viruses
can also function as vectors that provide amplified transgene delivery.
HSV-1, especially in comparison with adenovirus, has suitable features for cancer
therapy: (i) HSV-1 infects most tumor cell types. (ii) A relatively low multiplicity

178
ACTIVE IMMUNOTHERAPY 179
of infection is needed for total cell killing. (iii) Antiviral drugs are available. (iv) A
large genome (152 kb) allows the insertion of large and/or multiple transgenes. (v)
The host immune reactions enhance antitumor effects. (vi) Circulating anti-HSV-1
antibodies do not affect cell-to-cell spread of the virus. (vii) There are HSV-1 sensitive
mouse and nonhuman primate models for preclinical evaluation. (viii) Viral DNA is
not integrated into the host genome. Whereas glioma cells grow invasively within
the brain tissue, the viral genes of HSV-1 necessary for neuropathogenicity are
identified and can be mutated, therefore the use of HSV-1 is especially advantageous
for glioma therapy.
GENETICALLY ENGINEERED ONCOLYTIC HSV-1
In order to target HSV-1 replication to tumor cells, viral genes that are essential for
viral replication in normal cells but dispensable in tumor cells are inactivated or deleted.3
G207 was the first oncolytic HSV-1 used to treat glioma patients in the United States.4
This second-generation oncolytic HSV-1 has double mutations created in the HSV-1
genome.5 G207 has deletions in both copies of the a34.5 gene, the major determinant of
HSV-1 neurovirulence.6 Gamma34.5-deficient HSV-1 vectors are considerably attenuated
in normal cells, but retain their ability to replicate within neoplastic cells. In normal cells,
HSV-1 infection induces activation of double-stranded RNA-dependent protein kinase
(PKR), which in turn leads to phosphorylation of the _-subunit of eukaryotic initiation
factor 2 and a subsequent shutdown of host and viral protein synthesis.7 The product of
the a34.5 gene antagonizes this PKR activity. However, tumor cells in general have low
PKR activities, thereby allowing a34.5-deficient HSV-1 vectors to replicate.8,9 G207
also has an insertion of the E. coli lacZ gene in the infected-cell protein 6 (ICP6) coding
region (UL39), inactivating ribonucleotide reductase, a key enzyme for viral DNA
synthesis in nondividing cells but not in dividing cells.10 Recently, it has been reported
that ICP6-defective HSV-1 can replicate in quiescent cells with homozygous genetic
mutations in p16.11
After an extensive in vivo safety evaluation using HSV-1-susceptible mice and
nonhuman primates, the G207 Phase I clinical trial was performed between 1998 and 2000
at two institutions.4 Twenty-one patients with recurrent malignant glioma were treated
and G207 was administered directly into the tumor via stereotactic inoculation. This dose
escalation study started from 106 plaque-forming units (pfu) and increased to 3 = 109 pfu,
with three patients at each dose. As a result, no acute, moderate to severe adverse events
attributable to G207 were observed. Eight of 20 patients that had serial MRI evaluations
had a decrease in tumor volume between 4 days and one month postinoculation and 2
patients survived for more than 5 years.
In the Phase Ib trial that followed, 6 patients with recurrent glioblastoma received
two doses each of G207.12 The first dose (1.5 = 108 pfu) was injected via a catheter placed
stereotactically in the tumor and, 2 to 5 days later, the second dose (1 = 109 pfu) was
injected into the wall of the resection cavity after the tumor was resected en bloc with
catheter in place. From some of the resected tumor specimens, viral RNA encoding DNA
polymerase was detected by RT-PCR indicating G207 replication. One patient developed
transient neurological deterioration, but no patient developed HSV-1 encephalitis. Median
time to progression by MRI was 3 months and median survival from G207 administration
was 6.6 months.
THIRD-GENERATION ONCOLYTIC HSV-1
The clinical trials proved the safety of G207 and hinted its efficacy for human
malignant glioma. However, in order to further improve the efficacy without compromising
its safety, a third-generation oncolytic HSV-1 termed G476 was newly created from
G207 by introducing another genetic alteration, i.e., the deletion of the _47 gene and the
overlapping US11 promoter region, in the G207 genome.13 Because the _47 gene product
inhibits transporter associated with antigen presentation, which translocates peptides across
the endoplasmic reticulum, the down-regulation of MHC class I that normally occurs in
human cells after infection with HSV-1 does not occur when the _47 gene is deleted.14
G476-infected human cells in fact presented higher levels of MHC class I expression than
cells infected with other HSV-1 vectors.13 Further, human melanoma cells infected with
G476 were better at stimulating their matched tumor-infiltrating lymphocytes in vitro
than those infected with G207. The deletion also places the late US11 gene under control
of the immediate-early _47 promoter, which results in suppression of the reduced growth
phenotype of a34.5-deficient HSV-1 mutants including G207.15 In the majority of cell
lines tested, G476 replicated better than G207, resulting in the generation of higher virus
titers and exhibiting greater cytopathic effect.13 In athymic mice bearing subcutaneous
U87MG human glioma and A/J mice bearing subcutaneous Neuro2a neuroblastoma,
G476 was significantly more efficacious than G207 at inhibiting the tumor growth when
inoculated intraneoplastically.13 G476 was also more efficacious than G207 in athymic
mice bearing intracerebral U87MG tumors (Ino Y, et al manuscript in preparation). G476
also efficiently kills cancer stem-like cells isolated from surgically resected glioblastoma.16
Nevertheless, the safety of G476 remained unchanged from G207 following injection
into the brain of HSV-1-sensitive A/J mice.13
In Japan, an open labelled, single armed, Phase I-II clinical study of G476 in recurrent
glioblastoma patients is ongoing at the University of Tokyo since November 2009.17
Each patient receives two doses of G476 starting at a total of 6 = 108 pfu per subject.
One dose is divided into 2-5 sites within the tumor and administered stereotactically and
within 14 days, the same dose is administered into the same coordinates. Three cohorts
of 3 subjects each are planed in the dose escalation phase up to 6 = 109 pfu per subject
and 12 additional subjects will be treated at the highest safe dose.
INDUCTION OF SPECIFIC ANTITUMOR IMMUNITY BY ONCOLYTIC

HSV-1
In preclinical studies using immunocompetent animals, the most remarkable finding

with G207 was that it induced systemic antitumor immunity in the course of oncolytic
activity.18,19 For example, in A/J mice bearing bilateral subcutaneous N18 (syngeneic
neuroblastoma) tumors, intraneoplastic G207 inoculation into the left tumor alone caused
growth reduction not only of the inoculated tumors but also of the noninoculated contralateral
tumors. The antitumor immunity was associated with an elevated cytotoxic T-lymphocyte
(CTL) activity specific to N18 tumor cells that persisted for at least 13 months.
Corticosteroid administration did not reduce the oncolytic activity of G207, but did
suppress the CTL-mediated immune responses, which led to delayed regrowth of tumors
that once diminished by G207 treatment.19 G207-induced systemic antitumor immunity

was also observed in tumor models using mouse CT26 colon carcinoma,20 mouse M3
melanoma20 or hamster KIGB-5 gallbladder carcinoma.21 In mice harboring subcutaneous
CT26 tumors, G207 treatment was shown to elicit CTL activities specific to the CT26
immunodominant MHC class I-restricted antigen AH1.20
In a model using poorly immunogenic murine melanoma cells transfected with the
HSV-1 entry coreceptors, Hve A and C, inoculation of intracranial tumors with oncolytic
HSV-1 induced tumor-specific cytotoxic and proliferative T-cell responses.22 However,
there was no increase in either serum tumor antibodies or virus-neutralizing antibodies
following the oncolytic HSV-1 therapy, therefore specific T-cell responses mediated the
prolongation of survival caused by the therapy. The CTL responses were directed toward
both tumor and viral antigens; however, the proliferative response was toward tumor
antigens only. Following oncolytic HSV-1 treatments, the lysis of tumor cells recruits
CD4
T cells to the tumor mass, where the cells are triggered to respond to tumor antigens
and not viral antigens. This may be partly due to the ability of the virus to block immune
recognition of infected cells through modulation of both MHC classes I and II.23-25 CD4/,
CD8/ and NK/ mice were unable to mount an immune response to prolong survival
after oncolytic HSV-1 therapy. The recruited CD4
T cells secrete cytokines and, in turn,
recruit and activate CD8
T cells. CD8
T cells, along with NK cells, lyse both infected
and uninfected tumor cells, leading to tumor destruction. Induction of the tumor-specific
CTL activities together with proliferative T cells can lead to recognition and destruction
of remote, untreated metastases.
ONCOLYTIC HSV-1 IN COMBINATION WITH IMMUNE

GENE THERAPY
Because the efficacy of an oncolytic HSV-1 depends not only on the extent of
replication capability within the tumor but also on the extent of antitumor immunity
induction,18,19 it should be augmented when combined with immune gene therapy. A
defective HSV-1 vector (dvB7Ig) expressing a soluble form of B7-1, one of the most
potent costimulatory molecules,26 has been tested in combination with G207.27 The in
vivo efficacy was tested in A/J mice harboring syngeneic, poorly-immunogenic Neuro2a
neuroblastoma under the skin or in the brain. Intraneoplastic inoculation of dvB7Ig/
G207 at a low titer successfully inhibited the growth of established subcutaneous
tumors, despite that the expression of soluble B7-1 was detected in only 1% or less
of tumor cells at the inoculation site. The dvB7Ig/G207 treatment also prolonged the
survival of mice bearing intracerebral tumors. Inoculation of dvB7Ig/G207 induced a
significant influx of CD4
and CD8
T cells in the tumor. In vivo depletion of immune
cell subsets further revealed that the antitumor effect required CD8
T cells but not
CD4
T cells. The dvB7Ig/G207 treatment conferred tumor-specific protective immunity
on cured animals.
Intraneoplastic inoculation of a defective HSV-1 vector expressing murine
interleukin 12 (IL-12) in combination with G207 showed greater efficacy than G207
alone in a subcutaneous CT26 tumor model.28 Increases in tumor-specific CTL activity
and IFN-a production by splenocytes were observed.28
ONCOLYTIC HSV-1 ARMED WITH IMMUNOSTIMULATORY GENES
One of the favorable features of HSV-1 is its capacity to incorporate large and/
or multiple transgenes within the viral genome, therefore immunostimulatory genes
can be inserted directly into the genome of oncolytic HSV-1. The so-called armed
oncolytic HSV-1 has attractive advantages over the use of nonarmed oncolytic HSV-1 in
combination with defective HSV-1 vectors carrying transgenes: (i) A continuous generation
of a high-titer, homogenous vector stock is possible, which allows manufacturing of a
large amount with a better quality control. (ii) A high transgene expression level due to
an amplified gene delivery can be obtained in vivo. A disadvantage was the need for a
time-consuming effort to create a new recombinant HSV-1, but the utilization of bacterial
artificial chromosome (BAC) and multiple DNA recombinases has lead to development of
epoch-making armed oncolytic HSV-1 generation systems as described in the next section.
Gamma34.5 -deficient HSV-1 containing the murine IL-4 gene displayed a
significantly higher antitumor activity and prolonged survival of mice with intracranial
tumors compared with its parental virus or the one expressing IL-10.29 First-generation
oncolytic HSV-1 expressing IL-12 (M002 and NV1042) showed improved in vivo efficacy
against 4C8 glioma in syngeneic B6D2F mice30 and intracerebral Neuro2a tumors in
A/J mice,31 and also against murine squamous cell carcinoma32 and murine colorectal
tumor.33 Immunohistochemical analyses of tumors treated with these IL-12-expressing
HSV-1 revealed a significant influx of CD4
, CD8
T cells and macrophages. The
antitumor efficacy of M002 was augmented when used in combination with M010,
the same backbone HSV-1 that expresses chemokine CCL2.34 The virus selected after
in vivo serial passage of M002 in tumors of a D54-MG human malignant glioma cell
line improved survival in two independent murine brain tumor models compared to the
parent M002.35 The oncolytic HSV-1 expressing IL-12 (NV1042) was more efficacious
than the one expressing granulocyte macrophage colony-stimulating factor (GM-CSF)
in the same backbone (NV1034) in mice with subcutaneous squamous cell carcinoma.32
The mice cured by NV1042 had a higher rate of rejecting rechallenged tumor cells than
those cured by NV1034.32
UTILIZATION OF ARMED ONCOLYTIC HSV-1 CONSTRUCTION

SYSTEMS
In the past, a recombinant HSV-1 was constructed by conventional homologous

recombination techniques that required selection of a correctly structured clone from
millions of candidates. It often took years until the intended HSV-1 was obtained. In order
to circumvent the time-consuming processes, we have developed an innovative, armed
oncolytic HSV-1 construction system using G476 as the backbone.36 Besides its favorable
features for human cancer therapy, G476 is especially suited as a replication-competent
backbone for expressing any foreign protein molecules, because of the wide therapeutic
window and preclusion of the shutoff of protein synthesis in the infected host cells. The
system, termed T-BAC system, utilizes BAC and two DNA recombinase systems (Cre/
loxP and FLP/FRT) (Fig. 1). It allows i) a construction of armed oncolytic HSV-1 in a
short period (usually 3-4 months), (ii) a simultaneous construction of multiple vectors,
(iii) an accurate insertion of a desired transgene into the deleted ICP6 locus, (iv) an
Figure 1. The structure of T-BAC-derived armed oncolytic HSV-1. The HSV-1 genome consists of
long and short unique regions (UL and US) each bounded by terminal (T) and internal (I) repeat regions
(RL and RS). Armed oncolytic HSV-1 created by using the T-BAC system has the backbone structure
of G476, a third-generation oncolytic HSV-1. It has triple deletions in the a34.5, ICP6 and _47 genes.
The transgene is inserted into the deleted ICP6 locus. As a marker, it also expresses the LacZ gene
driven by the ICP6 promoter.
insertion of multiple transgenes using the same effort as inserting a single transgene and
(v) a direct comparison of multiple armed oncolytic HSV-1 with the same backbone.
Using the T-BAC system, we generated a G476-backbone oncolytic HSV-1 armed
with mouse fusion-type IL-12, termed T-mfIL12 (Fig. 2). In A/J mice bearing bilateral
subcutaneous Neuro2a tumors, intraneoplastic inoculation with T-mfIL12 into the left
tumor alone led to a significantly better antitumor activity than the unarmed control
virus, T-01, not only in the inoculated left tumors but also in the noninoculated remote
tumors (Miyamoto S et al manuscript in preparation). We also created a G476-backbone
Figure 2. Concept of antitumor efficacy augmentation using oncolytic HSV-1 armed with an
immunostimulatory gene. When oncolytic HSV-1 armed with the IL-12 gene infects tumor cells, IL-12
is secreted in the course of viral replication and stimulates the immune cells. In addition to direct tumor
cell killing via viral replication and spread, tumor cells, including noninfected ones, are attacked and
destroyed by tumor-specific immune cells, resulting in enhanced antitumor activities.
HSV-1 armed with both IL-18 and soluble B7-1.36 This double-armed oncolytic HSV-1
showed a significant enhancement of antitumor efficacy via T-cell mediated immune
responses in A/J mice with subcutaneous Neuro2a tumors as well as in C57BL/6 mice
bearing subcutaneous TRAMP-C2 prostate cancer.
A similar system, termed HSVQuik system, has been also developed using a
G207-like backbone,37,38 and was used to create oncolytic HSV-1 armed with murine
IL-4, CD40 ligand or 6CK.38 In BALB/c mice bearing 4T1 breast cancer in the brain,
all of these armed HSV-1 showed better antitumor efficacy than the control virus. Using
the HSVQuik system, we also created oncolytic HSV-1 armed with IL-12, IL-18, or
soluble B7-1.37 In A/J mice harboring subcutaneous Neuro2a tumors, IL-12 was the most
efficacious among the immunostimulatory molecules investigated when expressed by
the G207-like HSV-1. The triple combination of the three armed viruses exhibited the
highest efficacy amongst all single viruses or combinations of two viruses. Combining
1 = 105 pfu each of the three armed viruses showed stronger antitumor activities than
any single armed virus at 3 = 105 pfu in inoculated tumors as well as noninoculated
remote tumors.
SYSTEMIC DELIVERY OF ARMED ONCOLYTIC HSV-1
Whereas the most common route of delivery of oncolytic HSV-1 has been a direct
intratumoral inoculation, an intravenous delivery would further broaden the clinical
application of oncolytic HSV-1 if proven effective. The main hurdle for intravenous
delivery is that only a small percentage of the administered virus reaches the tumor.
By arming of oncolytic HSV-1, a large antitumor effect can be induced from a small
number of virus that initiates replication at the tumor. We observed that intravenous
delivery of IL-12-expressing T-mfIL12 caused a significant inhibition of tumor growth
compared with mock and the unarmed control virus (T-01) treatments in A/J mice
bearing subcutaneous Neuro2a tumors (Guan Y et al manuscript in preparation). When
A/J mice bearing intracerebral tumors were treated by repeated intravenous injections,
T-mfIL12, but not T-01, significantly prolonged the survival compared with mock.
Also, in a renal cancer lung metastases model using BALB/c mice and syngeneic RenCa
cells, intravenous administrations of T-mfIL12 significantly inhibited the number of
metastases compared with mock and T-01 treatments (Tsurumaki Y et al manuscript
in preparation).
CONCLUSION
In summary, oncolytic virus therapy using HSV-1 is a promising treatment for glioma
that can also function as an efficient in situ tumor vaccination. Arming of an oncolytic
HSV-1 with transgenes encoding immunomodulatory molecules, such as interleukin 12,
can further augment the vaccination effect of oncolytic HSV-1 therapy. I foresee that, in
the future, a series of armed oncolytic HSV-1 will be developed and used differentially
or in combination according to the type and progression pattern of brain tumors.
ACKNOWLEDGEMENTS
I thank the current members of my laboratory, especially Drs. Yasushi Ino and
Hiroshi Fukuhara. Our research has been supported in part by grants to T. Todo from
the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan.
REFERENCES
1. Aghi M, Martuza RL. Oncolytic viral therapiesthe clinical experience. Oncogene 2005; 24(52):7802-7816.
2. Zemp FJ, Corredor JC, Lun X et al. Oncolytic viruses as experimental treatments for malignant gliomas:
using a scourge to treat a devil. Cytokine Growth Factor Rev 2010; 21(2-3):103-117.
3. Todo T. Oncolytic virus therapy using genetically engineered herpes simplex viruses. Front Biosci 2008;
13:2060-2064.
4. Markert JM, Medlock MD, Rabkin SD et al. Conditionally replicating herpes simplex virus mutant, G207
for the treatment of malignant glioma: results of a phase I trial. Gene Ther 2000; 7(10):867-874.
5. Mineta T, Rabkin SD, Yazaki T et al. Attenuated multi-mutated herpes simplex virus-1 for the treatment of
malignant gliomas. Nat Med 1995; 1(9):938-943.
6. Chou J, Kern ER, Whitley RJ et al. Mapping of herpes simplex virus-1 neurovirulence to gamma 34.5, a
gene nonessential for growth in culture. Science 1990; 250:1262-1266.
7. He B, Chou J, Brandimarti R et al. Suppression of the phenotype of gamma(1)34.5- herpes simplex virus 1:
failure of activated RNA-dependent protein kinase to shut off protein synthesis is associated with a
deletion in the domain of the alpha47 gene. J Virol 1997; 71(8):6049-6054.
8. Leib DA, Machalek MA, Williams BR et al. Specific phenotypic restoration of an attenuated virus by
knockout of a host resistance gene. Proc Natl Acad Sci USA 2000; 97(11):6097-6101.
9. Farassati F, Yang AD, Lee PW. Oncogenes in Ras signalling pathway dictate host-cell permissiveness to
herpes simplex virus 1. Nat Cell Biol 2001; 3(8):745-750.
10. Goldstein DJ, Weller SK. Herpes simplex virus type 1-induced ribonucleotide reductase activity is dispensable
for virus growth and DNA synthesis: isolation and characterization of an ICP6 lacZ insertion mutant. J
Virol 1988; 62(1):196-205.
11. Aghi M, Visted T, Depinho RA et al. Oncolytic herpes virus with defective ICP6 specifically replicates in
quiescent cells with homozygous genetic mutations in p16. Oncogene 2008; 27(30):4249-4254.
12. Markert JM, Liechty PG, Wang W et al. Phase Ib trial of mutant herpes simplex virus G207 inoculated
pre-and posttumor resection for recurrent GBM. Mol Ther 2009; 17(1):199-207.
13. Todo T, Martuza RL, Rabkin SD et al. Oncolytic herpes simplex virus vector with enhanced MHC class I
presentation and tumor cell killing. Proc Natl Acad Sci USA 2001; 98(11):6396-6401.
14. York IA, Roop C, Andrews DW et al. A cytosolic herpes simplex virus protein inhibits antigen presentation
to CD8
T-lymphocytes. Cell 1994; 77(4):525-535.
15. Mohr I, Gluzman Y. A herpesvirus genetic element which affects translation in the absence of the viral
GADD34 function. EMBO J 1996; 15(17):4759-4766.
16. Wakimoto H, Kesari S, Farrell CJ et al. Human glioblastoma-derived cancer stem cells: establishment of
invasive glioma models and treatment with oncolytic herpes simplex virus vectors. Cancer Res. 2009;
69(8):3472-3481.
17. Ino Y, Todo T. Clinical development of a third-generation oncolytic HSV-1 (G476) for malignant glioma.
Gene Therapy and Regulation 2010 (in press).
18. Todo T, Rabkin SD, Sundaresan P et al. Systemic antitumor immunity in experimental brain tumor therapy
using a multimutated, replication-competent herpes simplex virus. Hum Gene Ther 1999; 10(17):2741-2755.
19. Todo T, Rabkin SD, Chahlavi A et al. Corticosteroid administration does not affect viral oncolytic activity,
but inhibits antitumor immunity in replication-competent herpes simplex virus tumor therapy. Hum Gene
Ther 1999; 10(17):2869-2878.
20. Toda M, Rabkin SD, Kojima H et al. Herpes simplex virus as an in situ cancer vaccine for the induction
of specific anti-tumor immunity. Hum Gene Ther 1999; 10(3):385-393.
21. Nakano K, Todo T, Chijiiwa K et al. Therapeutic efficacy of G207, a conditionally replicating herpes
simplex virus type 1 mutant, for gallbladder carcinoma in immunocompetent hamsters. Mol Ther 2001;
3(4):431-437.
22. Miller CG, Fraser NW. Requirement of an integrated immune response for successful neuroattenuated
HSV-1 therapy in an intracranial metastatic melanoma model. Mol Ther 2003; 7(6):741-747.
23. Goldsmith K, Chen W, Johnson DC et al. Infected cell protein (ICP)47 enhances herpes simplex virus
neurovirulence by blocking the CD8
T-cell response. J Exp Med 1998; 187(3):341-348.
24. Hill A, Jugovic P, York I et al. Herpes simplex virus turns off the TAP to evade host immunity. Nature
1995; 375(6530):411-415.
25. Fruh K, Ahn K, Djaballah H et al. A viral inhibitor of peptide transporters for antigen presentation. Nature
1995; 375(6530):415-418.
26. Galea-Lauri J, Farzaneh F, Gaken J. Novel costimulators in the immune gene therapy of cancer. Cancer
Gene Ther 1996; 3(3):202-214.
27. Todo T, Martuza RL, Dallman MJ et al. In situ expression of soluble B7-1 in the context of oncolytic herpes
simplex virus induces potent antitumor immunity. Cancer Res 2001; 61(1):153-161.
28. Toda M, Martuza RL, Kojima H et al. In situ cancer vaccination: an IL-12 defective vector/
replication-competent herpes simplex virus combination induces local and systemic antitumor activity.
J Immunol 1998; 160(9):4457-4464.
29. Andreansky S, He B, van Cott J et al. Treatment of intracranial gliomas in immunocompetent mice using
herpes simplex viruses that express murine interleukins. Gene Ther 1998; 5(1):121-130.
30. Hellums EK, Markert JM, Parker JN et al. Increased efficacy of an interleukin-12-secreting herpes simplex
virus in a syngeneic intracranial murine glioma model. Neuro Oncol 2005; 7(3):213-224.
31. Parker JN, Gillespie GY, Love CE et al. Engineered herpes simplex virus expressing IL-12 in the treatment
of experimental murine brain tumors. Proc Natl Acad Sci USA 2000; 97(5):2208-2213.
32. Wong RJ, Patel SG, Kim S et al. Cytokine gene transfer enhances herpes oncolytic therapy in murine
squamous cell carcinoma. Hum Gene Ther 2001; 12(3):253-265.
33. Bennett JJ, Malhotra S, Wong RJ et al. Interleukin 12 secretion enhances antitumor efficacy of oncolytic
herpes simplex viral therapy for colorectal cancer. Ann Surg 2001; 233(6):819-826.
34. Parker JN, Meleth S, Hughes KB et al. Enhanced inhibition of syngeneic murine tumors by combinatorial
therapy with genetically engineered HSV-1 expressing CCL2 and IL-12. Cancer Gene Ther 2005;
12(4):359-368.
!"#$' <='>@\# ^_134.5
herpes simplex virus type 1 mutant that exhibits decreased neurotoxicity and prolongs survival of mice
with experimental brain tumors. J Virol 2006; 80(15):7308-7315.
36. Fukuhara H, Ino Y, Kuroda T et al. Triple gene-deleted oncolytic herpes simplex virus vector double-armed
with interleukin 18 and soluble B7-1 constructed by bacterial artificial chromosome-mediated system.
Cancer Res 2005; 65(23):10663-10668.
37. Ino Y, Saeki Y, Fukuhara H et al. Triple combination of oncolytic herpes simplex virus-1 vectors armed
with interleukin-12, interleukin-18, or soluble B7-1 results in enhanced antitumor efficacy. Clin Cancer
Res 2006; 12(2):643-652.
38. Terada K, Wakimoto H, Tyminski E et al. Development of a rapid method to generate multiple oncolytic HSV
vectors and their in vivo evaluation using syngeneic mouse tumor models. Gene Ther 2006; 13(8):705-714.
CHAPTER 15
DENDRITIC CELL VACCINES
Ryuya Yamanaka1,* and Koji Kajiwara2

1
Kyoto Prefectural University of Medicine, Kyoto; 2Yamaguchi University School of Medicine, Ube, Japan
*Corresponding Author: Ryuya YamanakaEmail: ryaman@cmt.kpu-m.ac.jp
Abstract: Despite progress in brain tumor therapy, the prognosis of malignant glioma
patients remains dismal. Among the new treatments currently being investigated,
immunotherapy is theoretically very attractive since it offers the potential for high
tumor-specific cytotoxicity. Increasing numbers of reports demonstrate that systemic
immunotherapy using dendritic cells is capable of inducing an antiglioma response.
Therefore, dendritic cell-based immunotherapy could be a new treatment modality
for patients with glioma. In this chapter, we will discuss the implications of these
findings for glioma therapy, reviewing current literature on dendritic cell-based glioma
immunotherapy. We will overview the role of dendritic cells in immunobiology, the
central nervous system and tumor immunology, before outlining dendritic cell therapy
results in clinical trials and future directions. Dendritic cell-based immunotherapy
strategies appear promising as an approach to successfully induce an antitumor
immune response in patients with glioma, where it seems to be safe and without
major side effects. The development of methods for manipulating dendritic cells
for the purpose of vaccination will enhance the clinical usefulness of these cells
for biotherapy. Its efficacy should be further determined in randomized, controlled
clinical trials.
INTRODUCTION
Despite advances in radiation and chemotherapy along with surgical resectioning,

the prognosis for patients with malignant glioma remains dismal. Among the new
treatments currently being investigated for malignant glioma, immunotherapy is
theoretically very attractive because it offers the potential for high tumor-specific
cytotoxicity.1-4 The ongoing development of immunotherapies offers new hope for more
effective, lower toxicity, targeted treatment. Previous immunotherapeutic treatments

187
for brain tumors have focused on passive, adoptive and nonspecific strategies and
failed to yield clear benefits. The central nervous system (CNS) has been considered
to be immunologically privileged based on the fact that tissue or tumor allografts
survive better in the brain than in extracerebral locations.5 Glioma cells are poor
antigen presenting cells (APCs) for the immune system because of down-regulation
of costimulatory molecules required to activate the immune system6 in glioma cells
and their secretion of immunosuppressive cytokines such as transforming growth
factor-` (TGF-`),7 vascular endothelial growth factor (VEGF)8 and interleukin (IL)-10.9
Gliomas, like other human tumors, express antigens but these do not appear to be
immunogenic. This may be due to a defect in the presentation of tumor antigens to the
immune system. A subset of T cells, termed CD4
CD25
regulatory T cells (Treg),
have been recently shown to infiltrate gliomas and have increased numbers in the blood
of glioma patients and animals with experimental brain tumors.10 These Tregs have
been shown to inhibit the actions of effector lymphocytes. However, subcutaneous
vaccination with genetically modified cytokine-secreting tumor cells and genetically
modified costimulatory signal expressing tumor cells has been demonstrated to be
efficacious against intracranial tumors in a murine model, supporting the notion
that an effective immune response can be generated against intracerebral tumors.3,11
There are increasing numbers of reports demonstrating that systemic immunotherapy
using dendritic cells (DCs) is capable of inducing an antitumor response within the
immunologically privileged brain,12-15 confirming that the CNS may not be an absolute
barrier to DC-based immunotherapy. Therefore, the possibility that the immune system
can mediate interactions with lesions in brain tumors suggests excellent potential for
investigating novel immunotherapies. For example, DC-based immunotherapy might
be a new treatment modality for patients with brain tumors. To induce an antitumor
immune response against glioma cells, APCs such as DCs may be required to efficiently
internalize, process and present glioma-specific antigens to the immune system.16-18
In this chapter, we will survey DC-based glioma immunotherapy by presenting an
overview of the DC in immunobiology, in the CNS and in tumor immunology, and by
reviewing DC therapy in clinical trials and outline future directions.
DENDRITIC CELLS IN IMMUNOBIOLOGY
The immune system has evolved as a highly complex and adaptive mechanism to
distinguish nonself and self. Extracellular pathogens are attacked primarily by humoral
immune responses, which depend on soluble antibodies produced by B lymphocytes.
Foreign cells, including host cells bearing intracellular pathogens, are recognized and
destroyed by cellular immune responses, which depend on the T-cell receptor for specific
recognition of cell-surface antigens. T cells that directly kill foreign cells are commonly
CD8
, generally recognizing a cell-surface complex of major histocompatibility complex
(MHC) Class I molecules and foreign peptides derived from intracellular proteins processed
in the foreign cells through a cytoplasmic pathway.
DCs are rare, hematopoietically derived leukocytes that form a cellular network
involved in immune surveillance, antigen capture and antigen presentation.18,19 DCs
are predominantly found in the T-cell-dependent areas of lymphoid tissue,19 as well
as in other tissues and organs. DCs can differentiate from both myeloid precursors
and peripheral blood monocytes, maturing and migrating via the afferent lymphatics
DENDRITIC CELL VACCINES 189
to T-cell-rich areas of draining lymph nodes and the spleen where they develop
antigen-presenting function. DCs that have encountered antigens have been shown
to undergo maturation that renders them capable of migrating to regional lymph
nodes and activating antigen-specific T cells.20 Activated DCs express high levels of
Class I and II MHC antigens, as well as adhesion molecules (CD11a and CD54) and
accessory molecules (CD40, CD80 and CD86)18 (Fig. 1). DCs are capable of taking
up and processing antigens before presenting immunogenic epitopes in the context of
MHC Class I or Class II molecules for recognition by T cells. During the maturation
process, they lose their ability to process and present soluble antigen and become
extremely potent stimulators of T-lymphocytes.21 It has now become clear that DCs
may play an important supportive role in promoting and maintaining antigen-specific
T cells in vivo. Although DCs play central roles in immunity, they also have a critical
role in maintaining self-tolerance.22 DCs are unique, highly potent APCs capable of
sensitizing naive CD4
/CD8
T cells.22 While CD8
T cells are critical effectors in
antitumor immune responses, CD4
T cells also have an important role.23 Possible
mechanisms by which CD4
T cells may promote antitumor cytotoxic T cells (CTLs)
Figure 1. Interactions between DCs and T cells. DCs can recognize T-cell receptors on CD8
T cells
with peptide presented in the context of MHC Class I molecules or on CD4
T cells with peptide
presented in the context of MHC Class II molecules. Costimulatory and adhesion molecules such as
lymphocyte function-associated antigen-1/intercellular adhesion molecule-1 (LFA-1/ICAM-1), CD40/
CD40L, CD80/CD28, CD86/CD28, increase cell adhesion and enhance T-cell activation. DCs induce
proliferation and differentiation of activated B cells through their CD40 antigen, as well as expression
of IL-2 and IL12. Modified from: Yamanaka R. Trends Mol Med 2008; 14:228-235;76 2008 with
permission from Elsevier.
development and function include production of cytokines such as interferon (IFN)-a,

modification of DCs and maintenance of CD8
T-cell numbers.24
Originating from CD34
bone marrow stem cells, human DC precursors are found
in the bone marrow as well as peripheral blood and, in a more mature form, in lymphoid
and nonlymphoid tissues. Three different subtypes of DC have been defined: Langerhans
cells, interstitial DCs and plasmacytoid DCs. Human skin contains the first two types of
DCs, which are generally referred to as myeloid DCs. The plasmacytoid cells are derived
from the lymphoid lineage and are found in the T-cell areas of lymphoid organs, the
thymus and in the peripheral circulation.25
DENDRITIC CELLS IN THE CENTRAL NERVOUS SYSTEM
The CNS has been recognized as an immune privileged site. The blood-brain barrier
(BBB) functions to regulate the passage of macromolecules and intravascular immune cells
from the lumen of vessels in the neural parenchyma into the extravascular compartment,
implying that the neural environment is protected from surveillance by immune cells.26
The anatomical and physiological basis of the BBB consists of junctional complexes of
the CNS parenchymal vascular endothelium, the glia limitans (consisting of pericytes and
astrocytic foot processes external to the endothelial basal lamina of the brain parenchyma),
the choroids plexus epithelium and arachnoid villi, which are characterized by special
junctional complexes essential to cerebrospinal fluid (CSF) secretion and the pia mater on
the brain surface, which acts to regulate the passage of macromolecules and immune cells
from the CSF in the subarachnoid space into the CNS parenchyma. It has been reported
that approximately 50% of brain interstitial fluid and CSF in the rat drains through the
connective tissue sheaths of the olfactory nerves, which pierce the cribriform plate of the
ethmoid bone. This allows communication with the interstitial connective tissue of the
nasal mucosa, which drains via nasal lymphatics to cervical lymph nodes.27 In pathological
conditions affecting the CNS, alterations in adhesion properties of the endothelium occur
which facilitate immune cell extravasation. Moreover, cells close to the CNS vessels, such
as astrocytes and perivascular macrophages, are capable of producing cytokines such as
tumor necrosis factor (TNF)-_ when activated, causing up-regulation of adhesion molecules
such as intercellular adhesion molecule (ICAM)-1 and E-selectin.28 Several subsets of
immune cells such as lymphocytes, monocytes, neutrophils, natural killer (NK) cells
and DCs are attracted to the site of the pathological condition by chemokines and other
molecules released by tissue cells such as astrocytes and microglia.29,30 Endothelial cells
also participate in this process by releasing chemokines or altering their adhesiveness for
circulating immune cells, and by loosening tight endothelial junctions.31 Candidate APCs in
the CNS include parenchymal cells, such as astrocytes, oligodendrocytes and endothelium
and nonparenchymal hematogenous-derived immune cells including microglia, perivascular
macrophages, other macrophage populations and DCs.32 Macrophages in the leptomeninges
and choroid plexus perform a variety of functions in common with other tissue macrophages,
such as removing tissue debris from the CSF in the subarachnoid space, which would
otherwise cause obstruction to the CSF drainage pathways.33 Recent observations indicate
that populations of MHC Class II
cells present in the choroid plexus, pial layer, arachnoid
and dura mater have dendriform morphology, which strongly suggests they are of the DC
lineage.34 Several pathological studies of experimental allergic encephalitis (EAE) have
demonstrated the accumulation of inflammatory cells in periventricular, leptomeningeal
and perivascular sites. The involvement of these sites could be a consequence of the rich
network of DCs, which may act as APCs to trafficking activated T cells. Although DCs
are absent from the CNS parenchyma under normal conditions, DCs are found in the
CNS parenchyma during inflammation. This recruitment probably depends on changes
in adhesive characteristics between mononuclear cells and the endothelium, in part due to
the release of specific cytokines which allow trans-endothelial migration of mononuclear
cells.35 Further research will be needed to clarify whether DCs in the supporting tissues of
the CNS are capable of acting as APCs within the parenchyma or in the CSF and to identify
their migration routes.
DENDRITIC CELLS IN TUMOR IMMUNOLOGY
The ability of DCs to initiate or regulate immunity has led to the study of their use
as cellular vaccine adjuvants for the immunotherapy of cancer. With techniques available
for the isolation and bulk propagation of DCs in vitro, great efforts have been made to
use DCs in various immunization strategies.36 DCs are efficient at recruiting, selecting
and expanding naive T cells with antigen specificity within lymphoid organs. These T
cells may be exported into tumor sites as immune effectors capable of directly killing
targets and releasing several cytokines that facilitate additional immune responses.
DCs can sensitize both CD4
and CD8
T cells to specific antigens, while CD4
T
cells are critical for priming CTLs. Therefore, both CD4
and CD8
T cells are equally
important in tumor immunology. Many strategies for delivering antigens into DCs have
been established in murine models and are now undergoing evaluation in clinical trials
(Fig. 2). These include the use of synthetic peptides where the tumor antigen is known,37-39
stripped peptides derived from Class I molecules from tumors,14 tumor RNA40,41 and
tumor lysates.13,42 The advantages of vaccinating with total tumor-derived material, such
as tumor cell lysates or tumor-derived mRNA, are that the identity of the tumor antigens
need not be known and the use of multiple tumor antigens reduces the risk of developing
antigen-negative escape mutants. Another promising and efficient approach is by using
cells produced by the fusion of DCs and tumor cells as a vaccine.43,44 The success of this
strategy depends on the generation and selection of hybrids that are stable and retain
the components critical for stimulating the immune system.
To accomplish such a complex series of events, DCs are equipped with a sophisticated
molecular array of cell components representing the antigen-processing machinery
(APM). The APM is essential for the uptake and processing by DCs of tumor-derived
antigens, allowing tumor-derived epitopes to be cross-presented to T cells. Immature
DCs are characterized by their ability to induce a tolerizing immune phenotype through
activation of Treg cells and inability to stimulate naive or antigen specific memory T
cells.45,46 The presence of toll-like receptor (TLR) ligands, provided by microorganisms or
stimulating cytokines, induces final maturation into fully activated mature DCs, resulting
in presentation of the internalized entities on appropriate MHC molecules. Mature DCs
express high levels of Class I and Class II molecules, have altered chemokine expression
and express costimulatory molecules.47,48 They migrate to draining lymph nodes and
activate innate and adaptive immune response.
The ability of DCs to traffic and to localize in appropriate regions of lymphatic
tissues is critical for the success of DC-based vaccines. Most clinical trials manipulate
DCs through ex vivo culture to assure accurate antigen delivery and DC activation. The
Figure 2. There are several strategies to deliver either lysates, peptides, mRNA and cDNA into DC.
Reprinted with permission from: Yamanaka R et al. Dendritic cell-based glioma immunotherapy (review).
Int J Oncol 2003; 23:5-15.
route of DC administration as well as their maturation state could affect tissue localization
of these cells. Similarly, the number and potency of the DCs is likely to influence in vivo
interactions with other cells. Therefore, the doses and routes of DC administration have
been intensely debated. DCs are typically administered intradermally, intravenously, or,
in special circumstances, intraperitoneally. Clearly, one of the critical issues for induction
of effective antigen-specific TH1-type (T-helper Type 1) immunity in patients with cancer
depends on defining a strategy for DC delivery that facilitates antigen presentation in
vivo. Furthermore, studies of DC trafficking in experimental animals using labeled DCs
have determined that only a very small percentage (0.1-2%) of DCs injected intradermally
ever reach the tissue-draining lymph nodes,49 while DCs injected intravenously are
rapidly sequestered by lung macrophages. Hence, most clinical protocols require very
high numbers of DCs for vaccination. Recent approaches to DC delivery seem to have
embraced the idea of smaller doses of highly potent DCs, which retain their functions
during migration in vivo, rapidly localize to lymph nodes and effectively interact with
CD8
and CD4
T cells. It is expected, although not yet proven, that this type of DC-based
vaccination will produce dramatically improved therapeutic results.
Human tumors express a variety of protein antigens recognizable by the immune
system and these antigens are potential targets for cancer vaccination therapy.
Unfortunately, the tumor antigens are self-derived and are generally considered weak
antigens. Selection of tumor antigen and appropriate loading of in vitro-generated DCs
with the antigen is an initial and crucial step in the development of an efficient DC-based
cancer vaccine. DC-based therapy in patients with cancer has now progressed to Phase II
trials. With the safety of DC transfers established, the challenge of the ongoing clinical
studies will be to determine effective therapeutic doses and to obtain evidence for clinical
efficacy for this form of immunotherapy. Opportunities are therefore available in the
context of these clinical trials to acquire a better understanding of how DCs mediate
antitumor effects, especially since there are still a number of questions that have to be
addressed: culture conditions for DCs must be optimized, especially if tailored subsets
of polarized DCs are to be produced; cytokine/chemokine profiles that characterize
different DC subsets must be defined; conditions for DC polarization or repolarization
toward clinically beneficial Type-1 T-cell responses must be established; and finally,
finding the means to sustain DC functions in the hostile tumor microenvironment. Studies
are necessary to be able to understand which subsets of DCs exert immunogenic versus
tolerogenic effects in vivo. In a study involving patients with Stage IV melanoma, de
Vries et al directly compared the efficacy of vaccines using immature or mature DCs
at inducing an immune response.50 They reported that delayed-type hypersensitivity
reactions (DTH) and humoral responses to keyhole limpet hemocyanin (KLH) were
observed in patients receiving mature DCs whereas those receiving immature DCs had
no DTH reactions. The plasticity of DCs and the potential for differential regulation
of their state of maturation have to be carefully handled to assure that cancer patients
receive adoptive transfers of immunogenic DCs, engineered to promote TH1- and
TC1-type (Type 1 CD8
T-cell) tumor-specific responses. These and other issues are
components of future translational research aimed at the understanding the biology of
DC subsets, their mechanism of action and their utility for immunotherapy not only
of cancer but also of other diseases.
CLINICAL TRIALS OF DENDRITIC CELL-BASED VACCINES
There are several reports concerning clinical trials of DC based vaccines for patients
with glioma (Table 1). The antigen sources in the trial varied from peptides eluted from
autologous glioma cells, fusions of DCs and tumor cells, to tumor lysates.
Yu et al51 reported a Phase I clinical trial of DCs pulsed with peptides eluted from
autologous glioma cells. Two patients had recurrent anaplastic astrocytoma (AA) and
7 glioblastoma (GBM). Peptide-pulsed DCs were injected intradermally in the deltoid
region 3 times biweekly. DC vaccination elicited systemic cytotoxicity in seven patients
and intratumoral cytotoxic and memory T-cell infiltration was detected in two patients.
DC vaccination proved to be associated with increased survival: median survival time
for the study group and the control group were 455 days and 257 days, respectively.
This Phase I study demonstrated the feasibility, safety and bioactivity of DC vaccines.
Liau et al52 also reported a Phase I clinical trial of DCs pulsed with peptides eluted
from autologous glioma cells. Twelve GBM patients were enrolled and three biweekly
intradermal vaccinations were given. There were no serious adverse events observed
and six patients developed systemic antitumor CTL responses. Unfortunately, the
induction of systemic effector cells did not translate into objective clinical responses,
particularly for patients with actively progressing tumors and/or those with tumors
expressing high levels of TGF-`. However, T-cell infiltration correlated with clinical
survival. The study group experienced four long term survivors (more than 2 years)
who had DC therapy during initial stages and received Temozolomide chemotherapy
after recurrence. Yu et al53 reported another Phase I trial of DCs pulsed with tumor
194
Table 1. Clinical trials of DC vaccination for glioma patients

Clinical
Trial Patient Number/ Immune Clinical
Author Design Population Antigen/ Adjuvant Dose Frequency Route Result Result
Yu JS51 Phase I n=9, GBM/AA, Peptides derived from 106 DC 3/2 weeks s.c. CTL, MST:455
newly ATC /Immature DC TIL vs 257 days
Liau LM52 Phase I n=12,GBM, Peptides derived from 1-10106 DC 3/ week i.d. CTL, TTP: 19.9
newly/recurrent ATC /Immature DC TIL vs 8.2 months
Yu JS53 Phase I n=14,GBM/AA, ATC lysate/Immature DC 107-108 DC 3/2 weeks i.d. CTL, MST:133 vs
newly/recurrent TIL 30 weeks
Rutkowski S54 Phase I n=12,Grade III/ ATC lysate/mature DC 2-4106 DC ~7/2-4 weeks i.d. DTH 2CR, 2PR
GBM, recurrent
Liau LM55 Phase I n=13, GBM, ATC lysate/TLR-7 1-10106 DC 3/2 weeks i.d. CTL mPFS 18.1, OS
newly 33.8 months
Kikuchi T 56,57 Phase I n=17, Grade III/ Fushion of DC and 2.4-32106 ~8/2 weeks i.d. CTL, 6PR
GBM, recurrent tumor cell /mature fused cell TIL
DC+IL-12
Yamanaka R58,59 Phase I/II n=24, Grade III/ ATC lysate/Immature 1-32106 DC ~22/3 weeks i.d. or DTH, 2 year survival:
GBM, recurrent or mature DC+KLH i.t. CTL 23.5 vs 3.7%
or OK432
Wheeler CJ60 Phase II n=34,GBM, ATC lysate/mature DC 10-40106 DC 4/2-6 weeks i.d. CTL TTP:308 vs
newly/recurrent 167 days
Vleeschouwer SD61 Phase II n=56, GBM, ATC lysate/Immature DC 0.7-25.710 6 DC 3-9/2-4 weeks i.d. n.d. mPFS 3, OS
recurrent 9.6 months
a) GBM: glioblastoma; b) AA: anaplastic astrocytoma; c) ATC: autologous tumor cell; d) TRL: toll-like receptor; e) IL-12: interleukin-12; f) KLH: keyhole lim-
pet hemocyanin; g) s.c.: subcutaneous; h) i.d.: intradermal; i) i.t.: intratumoral; j) CTL: cytotoxic T lymphocyte; k) TIL: tumor infiltrating lymphocyte; l) DTH:
delayed-type hypersensitivity reaction; m) n.d.: not determined; n) MST: median survival time; o) TTP: time to tumor progression; p) CR: complete response;
q) PR: partial response; r) mPFS: median progression free survival; s) OS: overall survival.
lysates of autologous glioma cells. Four patients had recurrent AA and 10 GBM. Six
of 10 patients demonstrated robust systemic cytotoxicity as demonstrated by IFN-a
expression by peripheral blood mononuclear cells (PBMCs) in response to tumor
lysates after vaccination. A significant CD8
T-cell infiltrate was noted intratumorally
in three of six patients who underwent re-operation. The median survival for patients
with recurrent GBM in this study was 133 weeks. Vaccination with tumor lysate pulsed
DCs was safe and no evidence of autoimmune disease was noted. Rutkowski et al54
reported a Phase I trial of DCs pulsed with tumor lysates of autologous glioma cells. One
patient had recurrent AA and 11 GBM. There were no serious adverse effects, with no
clinical or radiological evidence of autoimmune reactions in any of the patients in these
studies, with the exception of 1 patient who repetitively developed peritumoral edema.
Two of the 6 patients with complete resection are still showing a continuous complete
response (CR) after 3 years. Liau et al55 have reported a tumor lysate-pulsed DC vaccine
in combination with the TLR-7 agonist, imiquiod, following radio-chemotherapy for
newly diagnosed GBM. Thirteen patients received 3 immunizations at 2-week intervals,
following completion of a 6-week course of radio-chemotherapy. Patients without
tumor progression received booster vaccinations combined with topical administration
of the imiquiod. All immunizations were well tolerated, with only mild side effects.
Increased levels of CD8
T cells reactive against tumor antigens were detected in 5
patients. The median progression free survival (PFS) is 18.1 months and median overall
survival (OS) is 33.8 months. Newly diagnosed GBM patients are currently enrolling
for a multi-center Phase II trial to test the efficacy of their autologous DC vaccine.
Kikuchi et al56 reported on immunotherapy involving fusions of DCs and glioma cells.
Three patients had recurrent AA and 5 GBM. Clinical results showed that there were
no serious adverse effects and 2 partial responses (PRs). Kikuchi et al57 also reported
another immunotherapy with fusions of DCs and glioma cells combined with recombinant
IL-12. Nine patients had recurrent AA and 6 GBM. Clinical results showed that there
were no serious adverse effects, 4 PRs and 1 minor response (MR) in patients with AA.
Yamanaka et al58,59 reported on therapy using DCs pulsed by tumor lysates.
Twenty-four patients with recurrent malignant glioma (6 Grade III and 18 Grade IV
patients) were evaluated in a Phase I/II clinical study. DCs were injected intradermally,
or both intratumorally and intradermally every 3 weeks. The protocols were well
tolerated with only local redness and swelling at the injection site in several cases,
while clinical responses showed 1 patient with PR and 3 MRs. Increased ELISPOT
and DTH responses after vaccination provided good laboratory markers to predict the
clinical outcome of patients receiving DC vaccination. The OS of patients with GBM
was 480 days, which was significantly better than that in the control group.
Wheeler et al60 reported a Phase II trial of DCs pulsed with tumor lysates of
autologous glioma cells. Thirty-four GBM patients were enrolled. Seventeen patients
exhibited a positive vaccine response quantified by IFN-a responsiveness after three
vaccinations. Vaccine responders exhibited significantly longer posttreatment times to
tumor progression (TTP) and survival (TTS) relative to nonresponders. Vaccine-induced
responses elicited therapeutic benefits primarily by sensitizing tumors to chemotherapy.
Vleeschouwer et al61 reported a Phase II trial of DC pulsed with tumor lysates of
autologous glioma cells. Fifty-six patients with relapsed GBM were treated with at
least three vaccinations. The median PFS and OS were 3 and 9.6 months, respectively,
with a 2-year OS of 14.8%. Vaccine-related edema in one patient with gross residual
disease before vaccination was the only serious adverse event.
Although immunological monitoring including tetramer and ELISPOT assays

may detect specific antiglioma immunity, clinical efficacy has been minimal following
immunotherapies. Due to the limited sample population, further evaluation of the role
of DC immunotherapy is necessary. Critically, a number of key elements still need
to be determined: optimum doses of DCs; the appropriate routes of vaccination; the
source of tumor antigens; and finally, the methods of antigen loading.
FUTURE DIRECTIONS
From the above information, it is apparent that new therapeutic strategies for
gliomas need to take into consideration the unique molecular abnormalities, glioma
stem cells (cells with stem-like properties of self-renewal, multipotent differentiation
and tumor initiation efficiency) and glioma associated antigens. While there has been
much progress in terms of DC-based vaccines, there are significant technical hurdles that
could still hinder this therapy. To what extent these immunization protocols could be
improved remains debatable. In order to achieve better immunization efficacy, various
strategies have been proposed. Direct manipulation of professional APCs to ensure
optimal presentation or indirect antigen presentation through molecules, DC-derived
exosomes, or cellular vaccines may equally lead to sufficient immune reactions. Injection
of the vaccine into lymph nodes could potentially be more efficient because the antigens
become quickly available to professional APCs.
The other possibility is to enhance or modify the antigen presenting ability of
DCs. This could be achieved by using adjuvants to upregulate DC function. It has been
reported that the CpG motif is effective as an immunoadjuvant,62,63 activating certain
subsets of DC to promote TH1-like immune responses.64 The efficacy of combinations
of these adjuvants in DC-based glioma immunotherapy should be further investigated.
Protocols on how to modulate DCs to obtain the efficient immunostimulatory effects
need to be established. Many new subsets of DCs are being identified and their functional
diversity cannot be explained by different lineage origins, but instead depend on the
activation signals, maturation stage and local microenvironment.64 It may be more
clinically relevant to consider DCs as a mass consisting of many phenotypically and
functionally diverse cells. It will be a new challenge to exploit these differences for
the purposes of immunotherapy of glioma. A fundamental issue that should be further
clarified is whether in vitro derived DC have the capacity to migrate to the lymphoid
organ when administered back to the patients.
Several reports have provided evidence that immunotherapy was clinically effective
from the perspective of the OS of some GBM patients by DCs or a virus-modified autologous
tumor cell vaccine.51-61,65 However, such immunotherapies have several disadvantages:
limited materials for vaccination, labor intensity for preparation and difficulty finding a
reliable laboratory marker. Tumor lysates or RNA made from tumor tissue are often in
limited quantities, are an inconsistent source of antigenic material and can make immune
monitoring difficult. Furthermore, they may often be contaminated with nontarget cells such
as cells from healthy brain tissue. In contrast, peptide vaccination has several advantages
which include easy supply of good manufacturing practice (GMP) levels of materials and
a reliable laboratory marker for the prediction of clinical outcome. However, peptides have
problems associated with potential tumor escape due to a limited repertoire of defined
antigens. They are also MHC Class I and II restricted, consequently impacting patient
enrollment into clinical trials. Finally, Class I peptides alone are insufficient to generate
a CD4
T-helper response which is required for optimal effective antitumor immunity.66
The insertion of whole proteins or cDNA encoding an entire tumor antigen into DCs
is an alternative to the use of peptides for DC-based immunotherapy. A major benefit
of such protein- or gene-based vaccines compared with peptide-based approaches is
that their application does not require prior knowledge of patient MHC-type or specific
T-cell epitopes. Viral and nonviral vectors have been used to genetically modify DCs to
express tumor antigens.15,67-71 Yamanaka et al15 showed the usefulness of a self-replicating
Semliki forest virus system to deliver tumor antigen into DCs. The adoptive transfer
of these DCs was able to promote antigen-specific T-cell responses and prophylactic
as well as therapeutic cell-mediated immune responses against tumors.
The other question in improving immunotherapy is how to eliminate loss of
MHC Class I on tumor cells, seen in 50% of glioblastoma cases, which are crucial for
CTL-mediated elimination of tumor cells.71 This problem could be overcome by the
combined use of a DC vaccine and either chemotherapy or a biological therapy capable
of activating NK cells and macrophages that are not affected by MHC expression on
tumor cells. There are several reports describing sensitization of malignant glioma to
chemotherapy through vaccination.72-74 Based on these experiences, we propose a DC
vaccine combined with chemotherapy as a new treatment modality for gliomas. The
second point is how to overcome the immunosuppressive state of glioma patients.
Patients with recurrent glioma are usually in an immunosuppressive state because
of the advanced disease and myelosuppression by anticancer agents.75 It could be
difficult to induce antiglioma immunoresponses in such a condition by immunotherapy.
Therefore, prophylactic immunotherapy at initial stages of the disease may have more
merit. Alternatively, some patients who were again treated with chemotherapy might
show an improved chemoresponsiveness after vaccination, as has been suggested for
high grade recurrent glioma.73,74 DC vaccination has shown potential as a method for
overcoming chemotherapy resistance, a significant problem that is associated with high
grade glioma. A controlled prospective analysis is thus warranted to rigorously test
the prediction that combinational immune/chemotherapy is superior to either vaccine
therapy or standard chemotherapy alone and represents the best available treatment in
a larger population of GBM patients.
CONCLUSION
DC based strategies appear promising as an approach to successfully induce antitumor

immune responses and prolong survival in patients with glioma. DC therapy of glioma
seems to be safe and without major side effects, thus its efficacy should be determined in
randomized and controlled clinical trials. Every patient with glioma will be evaluated for
the molecular genetic abnormalities in their individual tumors and novel immunotherapeutic
strategies based on pharmacogenomics will be offered according to the genetic findings.
REFERENCES
1. Dranoff G, Jaffee E, Lazenby A et al. Vaccination with irradiated tumor cells engineered to secrete murine
granulocyte macrophage colony-stimulating factor stimulates potent, specific and long-lasting antitumor
immunity. Proc Natl Acad Sci USA 1993; 90:3539-3543.
2. Parney IF, Hao C, Petruk KC. Glioma immunology and immunotherapy. Neurosurgery 2000; 46:778-791.
3. Sampson JH, Archer GE, Ashley DM et al. Subcutaneous vaccination with irradiated, cytokine-producing
tumor cells stimulates CD8
cell-mediated immunity against tumors located in the immunologically
privileged central nervous system. Proc Natl Acad Sci USA 1996; 93:10399-10404.
4. Soling A, Rainov NG. Dendritic cell therapy of primary brain tumors. Mol Med 2001; 7:659-667.
5. Medawar PB. Immunity to homologous grafted skin: III. The fate of skin homografts transplanted to the
brain, to subcutaneous tissue and to the anterior chamber of the eye. Br J Exp Pathol 1948; 29:58-69.
6. Satoh J, Lee YB, Kim SU. T-cell costimulatory molecules B7-1 (CD80) and B7-2 (CD86) are expressed in
human microglia but not in astrocytes in culture. Brain Res 1995; 704:92-99.
7. Constam DB, Philipp J, Malipiero UV et al. Differential expression of transforming growth factor-beta 1,
-beta 2 and -beta 3 by glioblastoma cells, astrocytes and microglia. J Immunol 1992; 148:1404-1410.
8. Gabrilovich DI, Ishida T, Nadaf S et al. Antibodies to vascular endothelial growth factor enhance the
efficacy of cancer immunotherapy by improving endogenous dendritic cell function. Clin Cancer Res
1999; 5:2963-2970.
9. Chen Q, Daniel V, Maher DW et al. Production of IL-10 by melanoma cells: examination of its role in
immunosuppression mediated by melanoma. Int J Cancer 1994; 56:755-760.
10. Sonabend AM, Rolle CE, Lesniak MS. The role of regulatory T-cells in malignant glioma. Anticancer
Res 2008; 28:1143-1150.
11. Morioka J, Kajiwara K, Yoshikawa K et al. Vaccine therapy for murine glioma using tumor cells genetically
modified to express B7.1. Neurosurgery 2004; 54:182-190.
12. Amano T, Kajiwara K, Yoshikawa K et al. Anti-tumor effects of vaccination with dendritic cells transfected
with modified RHAMM (receptor for hyaluronan-mediated motility) mRNA for mice glioma. J Neurosurg
2007; 106:638-645.
13. Heimberger AB, Crotty LE, Archer GE et al. Bone marrow-derived dendritic cells pulsed with tumor
homogenate induce immunity against syngeneic intracerebral glioma. J Neuroimmunol 2000; 103:16-25.
14. Liau LM, Black KL, Prins RM et al. Treatment of intracranial gliomas with bone marrow-derived dendritic
cells pulsed with tumor antigens. J Neurosurg 1999; 90:1115-1124.
15. Yamanaka R, Zullo SA, Tanaka R et al. Enhancement of antitumor immune response in glioma models in
mice by genetically modified dendritic cells pulsed with Semliki forest virus-mediated complementary
DNA. J Neurosurg 2001; 94:474-481.
16. Constant S, SantAngelo D, Pasqualini T et al. Peptide and protein antigens require distinct antigen-presenting
cell subsets for the priming of CD4
T-cells. J Immunol 1995; 154:4915-4923.
17. Levin D, Constant S, Pasqualini T et al. Role of dendritic cells in the priming of CD4
T-lymphocytes to
peptide antigen in vivo. J Immunol 1993; 151:6742-6750.
18. Steinman RM. The dendritic cell system and its role in immunogenicity. Annu Rev Immunol 1991; 9:271-296.
19. Steinman RM, Pack M, Inaba K. Dendritic cells in the T-cell areas of lymphoid organs. Immunol Rev
1997; 156:25-37.
20. Reis e Sousa C, Stahl PD, Austyn JM. Phagocytosis of antigens by Langerhans cells in vitro. J Exp Med
1993; 178:509-519.
21. Banchereau J, Briere F, Caux C et al. Immunobiology of dendritic cells. Annu Rev Immunol 2000; 18:767-811.
22. Steinman RM, Turley S, Mellman I et al. The induction of tolerance by dendritic cells that have captured
apoptotic cells. J Exp Med 2000; 191:411-416.
23. Toes RE, Ossendorp F, Offringa R et al. CD4 T-cells and their role in antitumor immune responses. J Exp
Med 1999; 189:753-756.
24. Marzo AL, Kinnear BF, Lake RA et al. Tumor-specific CD4(
) T-cells have a major Post-Licensing role
in CTL mediated anti-tumor immunity. J Immunol 2000; 165:6047-6055.
25. Lipscomb MF, Masten BJ. Dendritic cells: immune regulators in health and disease. Physiol Rev 2002;
82:97-130.
26. Rowland LP. Blood-brain barrier, cerebrospinal fluid, brain edema and hydrocephalus. In: Kanel ER,
Schwartz JH eds. Principles of Neuroscience. Amsterdam, Elsevier, 1985:837-844.
27. Cserr HF, Knopf PM. Cervical lymphatics, the blood-brain barrier and the immunoreactivity of the brain:
a new view. Immunol Today 1992; 13:507-512.
28. Joo F. Insight into the regulation by second messenger molecules of the permeability of the blood-brain
barrier. Microsc Res Tech 1994; 27:507-515.
29. Gourmala NG, Buttini M, Limonta S et al. Differential and time-dependent expression of monocyte
chemoattractant protein-1 mRNA by astrocytes and macrophages in rat brain: effects of ischemia and
peripheral lipopolysaccharide administration. J Neuroimmunol 1997; 74:35-44.
30. Miyagishi R, Kikuchi S, Takayama C et al. Identification of cell types producing RANTES, MIP-1 alpha and
MIP-1 beta in rat experimental autoimmune encephalomyelitis by in situ hybridization. J Neuroimmunol
1997; 77:17-26.
31. Mesri M, Liversidge J, Forrester JV. ICAM-1/LFA-1 interactions in T-lymphocyte activation and adhesion
to cells of the blood retina barrier in the rat. Immunology1994; 83:52-57.
32. Sedgwick JD, Hickey WF. Antigen presentation in the central nervous system. In: Keane RW, Hickey WF
eds. Immunology of the Nervous System. Oxford: Oxford University Press, 1997:364-418.
33. McMenamin PG, Forrester JV. Dendritic cells in the central nervous system and eye and their associated
supporting tissues. In: Lotze MT, Thomson AW eds. Dendritic cells: Biology and Clinical Applications.
San Diego: Academic press, 1999:205-248.
34. Serot JM, Foliguet B, Bene MC et al. Ultrastructural and immunohistological evidence for dendritic-like
cells within human choroid plexus epithelium. Neuroreport 1997; 8:1995-1998.
35. Matyszak MK, Perry VH. The potential role of dendritic cells in immune-mediated inflammatory diseases
in the central nervous system. Neuroscience 1996; 74:599-608.
36. Fong L, Engleman EG. Dendritic cells in cancer immunotherapy. Annu Rev Immunol 2000; 18:245-273.
37. Celluzzi CM, Mayordomo JI, Storkus WJ et al. Peptide-pulsed dendritic cells induce antigen-specific CTL
mediated protective tumor immunity. J Exp Med 1996; 183:283-287.
38. Mayordomo JI, Zorina T, Storkus WJ et al. Bone marrow-derived dendritic cells pulsed with synthetic
tumour peptides elicit protective and therapeutic antitumour immunity. Nat Med 1995; 1:1297-1302.
39. Porgador A, Gilboa E. Bone marrow-generated dendritic cells pulsed with a class I-restricted peptide are
potent inducers of cytotoxic T-lymphocytes. J Exp Med 1995; 182:255-260.
40. Ashley DM, Faiola B, Nair S et al. Bone marrow-generated dendritic cells pulsed with tumor extracts or tumor
RNA induce antitumor immunity against central nervous system tumors. J Exp Med 1997; 186:1177-1182.
41. Boczkowski D, Nair SK, Snyder D et al. Dendritic cells pulsed with RNA are potent antigen-presenting
cells in vitro and in vivo. J Exp Med 1996; 184:465-472.
42. Aoki H, Mizuno M, Natsume A et al. Dendritic cells pulsed with tumor extract-cationic liposome complex
increase the induction of cytotoxic T-lymphocytes in mouse brain tumor. Cancer Immunol Immunother
2001; 50:463-468.
43. Gong J, Chen D, Kashiwaba M et al. Induction of antitumor activity by immunization with fusions of
dendritic and carcinoma cells. Nat Med 1997; 3:558-561.
44. Gong J, Avigan D, Chen D et al. Activation of antitumor cytotoxic T-lymphocytes by fusions of human
dendritic cells and breast carcinoma cells. Proc Natl Acad Sci USA 2000; 97:2715-2718.
45. Dhodapkar MV, Steinman RM. Antigen-bearing immature dendritic cells induce peptide-specific CD8(
)
regulatory T-cells in vivo in humans. Blood 2002; 100:174-177.
46. Wakkach A, Fournier N, Brun V et al. Characterization of dendritic cells that induce tolerance and
T-regulatory 1 cell differentiation in vivo. Immunity 2003; 18:605-617.
47. Bax M, Garca-Vallejo JJ, Jang-Lee J et al. Dendritic cell maturation results in pronounced changes in
glycan expression affecting recognition by siglecs and galectins. J Immunol 2007; 179:8216-8224.
48. Dietz AB, Bulur PA, Knutson GJ et al. Maturation of human monocyte-derived dendritic cells studied by
microarray hybridization. Biochem Biophys Res Commun 2000; 275:731-738.
49. Barratt-Boyes SM, Zimmer MI, Harshyne LA et al. Maturation and trafficking of monocyte-derived dendritic
cells in monkeys: implications for dendritic cell-based vaccines. J Immunol 2000; 164:2487-2495.
50. de Vries IJ, Lesterhuis WJ, Scharenborg NM et al. Maturation of dendritic cells is a prerequisite for inducing
immune responses in advanced melanoma patients. Clin Cancer Res 2003; 9:5091-5100.
51. Yu JS, Wheeler CJ, Zeltzer PM et al. Vaccination of malignant glioma patients with peptide-pulsed dendritic
cells elicits systemic cytotoxicity and intracranial T-cell infiltration. Cancer Res 2001; 61:842-847.
52. Liau LM, Prins RM, Kiertscher SM et al. Dendritic cell vaccination in glioblastoma patients induces systemic
and intracranial T-cell responses modulated by the local central nervous system tumor microenvironment.
Clin Cancer Res 2005; 11:5515-5525.
53. Yu JS, Liu G, Ying H et al. Vaccination with tumor lysate-pulsed dendritic cells elicits antigen-specific,
cytotoxic T-cells in patients with malignant glioma. Cancer Res 2004; 64:4973-4979.
vaccination for patients with relapsed malignant glioma, a feasibility study. Br J Cancer 2004; 91:1656-1662.
55. Liau LM, Prins RM, Odesa SK et al. Dendritic cell vaccination in combination with TLR-7 agonist, imiquiod
following radio-chemotherapy for newly diagnosed glioblastoma. Proc Am Soc Clin Oncol 2007; 25:2021.
56. Kikuchi T, Akasaki Y, Irie M et al. Results of a phase I clinical trial of vaccination of glioma patients with
fusions of dendritic and glioma cells. Cancer Immunol Immunother 2001; 50:337-344.
57. Kikuchi T, Akasaki Y, Abe T et al. Vaccination of glioma patients with fusions of dendritic and glioma
cells and recombinant human interleukin 12. J Immunother 2004; 27:452-459.
58. Yamanaka R, Abe T, Yajima N et al. Vaccination of recurrent glioma patients with tumour lysate-pulsed
dendritic cells elicits immune responses: results of a clinical phase I/II trial. Br J Cancer 2003; 89:1172-1179.
recurrent glioma: results of a clinical phase I/II Trial. Clin Cancer Res 2005; 11:4160-4167.
60. Wheeler CJ, Black KL, Liu G et al. Vaccination elicits correlated immune and clinical responses in
glioblastoma multiforme patients. Cancer Res 2008; 68:5955-5964.
61. De Vleeschouwer S, Fieuws S, Rutkowski S et al. Postoperative adjuvant dendritic cell-based immunotherapy
in patients with relapsed glioblastoma multiforme. Clin Cancer Res 2008; 14:3098-3104.
62. Weiner GJ, Liu HM, Wooldridge JE et al. Immunostimulatory oligodeoxynucleotides containing the CpG
motif are effective as immune adjuvants in tumor antigen immunization. Proc Natl Acad Sci USA 1997;
94:10833-10837.
63. Hartmann G, Weiner GJ, Krieg AM. CpG DNA: a potent signal for growth, activation and maturation of
human dendritic cells. Proc Natl Acad Sci USA 1999; 96:9305-9310.
64. Grabbe S, Kmpgen E, Schuler G. Dendritic cells: multi-lineal and multi functional. Immunol Today
2000; 21:431-433.
65. Steiner HH, Bonsanto MM, Beckhove P et al. Antitumor vaccination of patients with glioblastoma multiforme:
a pilot study to assess feasibility, safety and clinical benefit. J Clin Oncol 2004; 22:4272-4781.
66. Knutson KL, Disis ML. Tumor antigen-specific T-helper cells in cancer immunity and immunotherapy.
67. Arthur JF, Butterfield LH, Roth MD et al. A comparison of gene transfer methods in human dendritic cells.
Cancer Gene Ther 1997; 4:17-25.
68. Henderson RA, Nimgaonkar MT, Watkins SC et al. Human dendritic cells genetically engineered to express
high levels of the human epithelial tumor antigen mucin (MUC-1). Cancer Res 1996; 56:3763-3770.
69. Reeves ME, Royal RE, Lam JS et al. Retroviral transduction of human dendritic cells with a tumor associated
antigen gene. Cancer Res 1996; 56:5672-5677.
70. Szabolcs P, Gallardo HF, Ciocon DH et al. Retrovirally transduced human dendritic cells express a normal
phenotype and potent T-cell stimulatory capacity. Blood 1997; 90:2160-2167.
defects in astrocytic tumors. Clin Cancer Res 2005; 11(23):8304-8311.
72. Liu G, Akasaki Y, Khong HT et al. Cytotoxic T-cell targeting of TRP-2 sensitizes human malignant glioma
to chemotherapy. Oncogene 2005; 24:5226-5234.
73. Liu G, Black KL, Yu JS. Sensitization of malignant glioma to chemotherapy through dendritic cell
vaccination. Expert Rev Vaccines 2006; 5:233-247.
74. Wheeler CJ, Das A, Liu G et al. Clinical responsiveness of glioblastoma multiforme to chemotherapy after
vaccination. Clin Cancer Res 2004; 10:5316-5326.
FoxP3
121(1):95-105.
76. Yamanaka R. Cellular and peptide based immunotherapeutic approaches for glioma. Trends Mol Med
2008; 14:228-235.
PART V
NOVEL TOPICS
CHAPTER 16
ANTIGEN-RECEPTOR GENE-MODIFIED
T CELLS FOR TREATMENT OF GLIOMA
Hiroaki Ikeda1 and Hiroshi Shiku1,2

1
Department of Immuno-Gene Therapy, 2Department of Cancer Vaccine, Mie University Graduate School
of Medicine, Tsu, Japan
Emails: hikeda@clin.medic.mie-u.ac.jp; shiku@clin.medic.mie-u.ac.jp
Abstract: Immunological effector cells and molecules have been shown to access intracranial
tumor sites despite the existence of blood brain barrier (BBB) or immunosuppressive
mechanisms associated with brain tumors. Recent progress in T-cell biology
and tumor immunology made possible to develop strategies of tumor-associated
antigen-specific immunotherapeutic approaches such as vaccination with defined
antigens and adoptive T-cell therapy with antigen-specific T cells including
gene-modified T cells for the treatment of patients with brain tumors. An array
of recent reports on the trials of active and passive immunotherapy for patients
with brain tumors have documented safety and some preliminary clinical efficacy,
although the ultimate judgment for clinical benefits awaits rigorous evaluation in
trials of later phases. Nevertheless, treatment with lymphocytes that are engineered
to express tumor-specific receptor genes is a promising immunotherapy against
glioma, based on the significant efficacy reported in the trials for patients with other
types of malignancy. Overcoming the relative difficulty to apply immunotherapeutic
approach to intracranial region, current advances in the understanding of human
tumor immunology and the gene-therapy methodology will address the development
of effective immunotherapy of brain tumors.
INTRODUCTION
Central nervous system (CNS) has been considered as an immunological privileged

site that may provide a unique difficulty for cancer immunotherapy. However, recent
studies have clearly demonstrated that immunological maneuvers such as delivery of
effector cells and molecules could target tumor sites in CNS. Currently, advances in

202
ANTIGEN-RECEPTOR GENE-MODIFIED T CELLS FOR TREATMENT OF GLIOMA 203
cancer immunology from multiple aspects have provided strategies of antigen-specific

immunotherapy for malignancy. In this article, we briefly overview the recent progress
in the understanding of interaction of immune cells and central nerve system, review
the recent novel strategies of immunotherapy of cancer with a special focus on adoptive
therapy with gene-modified T cells and discuss how these promising strategies can be
applied to treat patients with brain tumors.
INTERACTION OF IMMUNE SYSTEM WITH CENTRAL NERVOUS

SYSTEM
Historically, the brain has been assumed as an immune-privileged site because of (1)
the presence of blood brain barrier (BBB), (2) the lack of lymphatics and conventional
dendritic cells (DC) and (3) immunosuppressive environment evidenced by the lack
of allograft rejection in the brain. However, recent studies have demonstrated that
immune cells do interact with CNS that is strongly evidenced in the diseases such as
multiple sclerosis or experimental autoimmune encephalitis. In addition, the immune
effector cells and molecules were shown to approach to intracranial tumors in numerous
preclinical studies in mouse.1
BBB, that consists with the CNS capillary endothelial cells, functions with
pericytes, parenchymal membrane, and astrocytic feet as a neurovascular unit (NVU).2
The BBB in patients with brain tumor appear to be compromised,3,4 associated with
increased edema and/or pericyte swelling. These disruptions are considered to affect
the migration of immune cells and the perfusion of effector molecules into the
parenchyma. Moreover, it is now well understood that immune cells do move across
the intact BBB.5
The primary antigen presenting cells (APC) in CNS have been referred to various
cell types including vascular endotherial cells, smooth muscle cells, astrocytes,
perivascular macrophages, choroid plexus epitherial cells, neurons and DC. Recent work
focused on CNS DC as more potent antigen presenting- and T-cell stimulating-APC
compared to CNS microglias and macrophages.6 Other reported the suppressing activity
of plasmacytoid DC, the major population of CNS DC, suggesting a regulatory role
for plasmacytoid DC in T-cell activation in CNS.7 Cervical lymph nodes have been
shown to play an important role as the major draining lymph node for DC in CNS.8,9
Immunosuppressive factors have been found in the environment associated with
brain tumor. These include soluble factors such as TGF-`1, -2 and -3,10-12 PGE2,13-15
IL-1016-18 and gangliosides.19-21 Interactions between cell surface molecules such as
Fas-FasL,22,23 PD-1-PD-L1,24,25 receptor-binding cancer antigen expressed on Sico cells,26
and CD7027,28 has been suggested to play a role in the suppression of immunological
reaction against brain tumor. These factors may play an important role for brain
tumor in their evasion from immunosurveillance and may be attractive targets for the
manipulation for effective immunotherapy.
In summary, recent works have explored the characteristics of CNS as an
immune-specialized rather than immune-privileged site. Many of the fundamental
mechanisms shown in non-CNS models seem to work also in CNS in general. Future
work will segregate the generality and specificity of immune reaction in CNS more
precisely and will help the development of effective immunotherapy of brain tumor.
DEVELOPMENT OF SPECIFIC CANCER IMMUNOTHERAPY
Immune system has been considered to protect host by eliminating exogenous agents
as nonself while keeping self-tissues intact, known as immunological tolerance. Therefore,
whether tumor cells that developed from normal tissues can be recognized by immune
system or not had been a warm debate. In 1953, Foley et al29 showed the existence of tumor
specific antigens in 3-methylcholanthrene-induced mouse tumors by demonstrating the
development of specific immunity to individual tumor in mice immunized with different
tumor lines. Burnet and Thomas30,31 formally introduced the notion that the immune system
could protect the host from neoplastic disease as the cancer immunosurveillance hypothesis.
However, following studies indicated the low immunogenisity of the naturally occurring
spontaneous tumors compared to the carcinogen-induced tumors and suggested the difficulty
of immunotherapy of human malignancies. In 1980s, technology to establish tumor-reactive
cytotoxic T cells from peripheral blood mononuclear cells or tumor infiltration lymphocytes
(TIL) has emerged. In 1991, Boon and his colleagues32 identified MAGE-1/MAGE-A1
as a tumor antigen of human melanoma recognized by a cytotoxic T-cell line established
from a patient with malignant melanoma. Since this memorial milestone, numerous tumor
antigens and their epitopes recognized by CD8
or CD4
T cells have been identified.33 Recent
studies confirmed the high incidence of tumor formation in a variety of immunodeficient
mice clearly indicating the existence of immunosurveillance of cancer.34
Identification of the tumor antigens in human tumors made possible to develop
the therapeutic approaches to enhance the specific immunity against tumors. Specific
immunotherapy of tumor consists of two major approaches. In one approach, identified
tumor antigen in many kinds of form are directly administrated into hosts as cancer vaccine
to develop specific immune response in patients, known as active immunotherapy. Another
approach use technologies to establish tumor-reacting immune cells in vitro by culturing
patients lymphocytes in order to react to tumor antigens, followed by the administration
of established immune cells into patients, known as passive immunotherapy. Recent
progress in the gene-therapy technology provided the means to endow T cells with defined
antigen specificity as well as increased functional properties.
ACTIVE IMMUNOTHERAPY OF CANCER
Initially, tumor cells and their lysates have been tested for their potential as vaccine
against tumor. Recent progress in the identification of tumor-associated antigens made
possible to utilize the synthetic peptides of antigen epitopes for T-cell recognition,
recombinant or synthetic proteins that contain multiple T-cell epitopes, or nucleic acids
that encode the antigens. In 2010, Provenge was approved by FDA for the treatment
of patients with prostate cancer as the first drug of therapeutic vaccine against tumor
in USA. This vaccine consists of in vitro cultured patients immune cells including
DC that incorporate fusion protein of PAP and GM-CSF. Oncophage utilizes the heat
shock protein manufactured from patients own tumor tissue. This heat shock protein is
considered as a molecular chaperone that contains antigenic peptides derived from the
tumor. Oncophage was approved in 2008 in the Russian Federation and subsequently
in EU for the treatment of patients with renal cancer. DCVax-Brain is a DC vaccine
utilizing patients DC pulsed with patients own tumor lysate and was approved in 2007
in Switzerland as a drug to treat brain cancer.
ACTIVE IMMUNOTHERAPY OF BRAIN TUMOR
A variety of mouse models have demonstrated that peripheral vaccinations against

intracranial tumor can be effective despite the existence of BBB and immunosuppressive
characteristics of tumor.1 Clinical trials of cancer vaccines for patients with brain tumors,
however, are in the early stages and await precise evaluation of their effectiveness in
randomized studies, although encouraging results indicated some objective clinical
responses and potential improvements of patients survival.
Besides DCVax-Brain , there have been a substantial number of studies
utilizing brain tumor cell-based vaccine approaches that have basically demonstrated
safety and preliminary efficacy.35-54 These studies utilize either irradiated or fixed
tumor cells, tumor cell lysates, DC pulsed with tumor/tumor lysates/peptides
eluted from tumor, or DC-tumor fusion cells. The whole tumor cell-based
approach has benefits as to: (1) it does not require the identification of antigens;
(2) it may contain multiple antigens to be recognized by a wide variety of immune cell
types including both CD8
and CD4
T cells. However, the whole tumor cell-based
medicinal product may have its limitation and shortcomings as to: (1) because of the
component of self antigens it may induce immunological tolerance mediated by regulatory
mechanisms such as regulatory T cells, (2) normal brain components may otherwise
induce autoimmune encephalitis, and (3) high costs, troublesome procedures, and complex
quality control issues associated with large-scale culture of autologous tumor cells may
hamper feasibility and widespread application.
Taking advantage of the identification of brain tumor-associated antigens, peptide-based
vaccine strategies including DC vaccines pulsed with antigenic peptides have been evaluated.
It has been difficult to find a tumor antigen that is widely expressed in brain tumors but
completely absent in normal tissues. Nevertheless, a variety of molecules are known to be
expressed preferentially in brain tumors and epitope peptides to elicit T-cell response were
identified (Table 1). Among these, some peptides are in the process of clinical evaluation as
therapeutic vaccines. Yajima et al55 reported a Phase I study of personalized peptide-based
vaccine in patients with recurrent malignant gliomas. In this trial, each patient was tested
for their humoral immune response against a panel of antigens prior to the enrollment. The
personalized combination of peptides was decided according to the positive immune reaction
to the peptides because the authors consider that it can be a measure of pre-existence of
sensitized T-cell population. The treatment was well tolerated and resulted in an 89-week
median survival of the treated patients. Izumoto et al56 reported a Phase II clinical trial utilizing
a Wilms Tumor (WT) 1-derived peptide. In this study, median progression-free survival was
20 weeks and possible association between the WT1 expression level and clinical responses
was reported. However, the overexpression of WT1 antigen in solid tumors including brain
tumors is controversial, and therefore the rationale for WT1-based immunotherapy for
brain tumor awaits further evaluation. Recently Okada et al57 reported a Phase I/II trial of a
vaccination with _-Type 1 polarized DC (_DC1) loaded with 4 glioma-associated antigen
epitope synthetic peptides (EphA2883-891, IL-13 R_2345-353:1A9V, YKL-40201-210, GP100209-217:M2)
and administration of polyinosinic-polycytidylic acid [poly(IC)] stabilized by lysine and
carboxymethylcellulose (polyICLC) in patients with recurrent malignant gliomas. They
reported that the regimen was well tolerated. The vaccine induced the upregulation of Type
1 cytokines including IFN_ and CXCL10. Of 22 patients enrolled, 9 achieved progression
free status lasting at least 12 months. Two patients experienced objective clinical tumor
regression. Of these two patients, one demonstrated sustained complete response.
Table 1. CTL epitopes of glioma

Antigens HLA Restriction Expression in Normal Cells References
EphA2 A*0201 Site of cell-to-cell contact 87-89
IL-13R A*0201, A*2402 Testis 90-93
gp100 16 HLA class I Melanocytic cells 94
epitopes
YKL-40 A*0201 Macrophages, Neutrophils, Serum, Blood 57,95,96
vessels, Extracellular matrix, Astrocytes
SOX2 A*0201 Testis, Neutal stem cells, Fetal brain 97-99
SOX11 A*0201 Fetal brain 98
HER2/neu 13 HLA class I Ubiquitously expressed 94
epitopes
EGFRvIII A*0201 No 100
MAGE-1 11 HLA class I Testis, Placenta 94
epitopes
TRP-2 A*0201 Melanocytis cells 101
AIM-2 A1 Testis, Liver 94
Survivin A*0201, A*2402, Undetected in most differentiated normal 102-105
A1, A3 adult tissues
SART-1 A*2402, A*2601 Testis 106,107
WT1 A*0201, Kidney, Bone marrow, Pleura, Peritonium, 108-112
A*2402, A1 Testis, Ovary, Hematopoietic stem cells
PASSIVE IMMUNOTHERAPY OF CANCER
Passive immunotherapy of cancer includes the administration of various immune

effector cells and effector molecules such as mAbs, cytokines, or receptor ligands. The
approach to administrate effector molecule is very important in the scope of manipulation
of immunological balance in the tumor-bearing hosts. However, it rather belongs to the
molecular targeted therapy. This chapter solely focuses on the administration of effector
T cells.
All of adoptive T-cell therapies remain as experimental therapies at present. However,
the strategy to administrate a large number of tumor-reactive T cells is an attractive approach
to the treatment for patients with malignancy. Indeed, adoptive immunotherapy with in
vitro expanded lymphocytes derived from patients TIL for the treatment of malignant
melanoma has demonstrated objective clinical response by RECIST criteria in around 50%
of enrolled patients in recent early phase trials.58 Initial trials to administrate tumor-reactive
T-cell line/clone were unsuccessful in both clinical response and persistence of infused
cells.59-61 Recent advances in several areas of human T-cell biology suggested that these
disappointed results might be related to two major obstacles. One obstacle comes from
immunosuppressive environment of tumor-bearing hosts.62 The second comes from the
reduced quality of T cells generated by long term in vitro culture for their expansion.63
To overcome these obstacles, recent protocols incorporate the pretreatment of patients
with lymphodepleting chemotherapy and/or irradiation. The reason for the effectiveness
of these pretreatment is not fully understood but is suggested to depend on (1) depletion
of immunosuppressive cell populations such as regulatory T cells, (2) ablation of cytokine

competition between infused and pre-existing lymphocytes, known as cytokine sinks,
(3) supply of spaces for infused cell to expand, known as homeostatic expansion, and
(4) improvement of APC function and availability.58 Recent protocols also tend to use
lymphocytes cultured in relatively short period in vitro in order to preserve T-cell quality
to maintain in vivo survival. Combination of these maneuvers with high dose IL-2
administration has reported a significant improvement of adoptive therapy with TIL for
patients with progressive malignant melanoma as up to 72% of patients demonstrated
objective clinical response of CR or PR in RECIST criteria.64 Although the combination of
conditioning of patients has shown to be effective, if every kinds of adoptive T-cell therapy
requires such intensive pretreatment and/or IL-2 administration for in vivo maintenance
of infused cells as well as successful clinical response needs further evaluation.
ANTIGEN-RECEPTOR GENE-MODIFIED T CELLS FOR TREATMENT

OF CANCER
In addition to the advances in the control of T-cell quality and fate, ex vivo
genetic manipulation has been developed to extend the availability of adoptive T-cell
therapy. Adoptive T-cell therapy has been almost exclusively applied to patients with
malignant melanoma with very limited exceptions. It is because the isolation and
expansion of tumor-reacting lymphocytes that pre-exist in patients has been difficult
in patients with other solid tumors. Moreover, the T cells with T-cell receptor (TCR)
of sufficiently high affinity are generally in very low incidence because majority of
human tumor-associated antigens are of self-antigens to some extent and are poorly
immunogenic. To overcome this problem, genetic engineering of polyclonal patients
lymphocytes by retrovius or lentivirus vector encoding tumor-reactive TCR has been
developed.65,66 In this technique, large amount of polyclonal lymphocytes can be redirected
their specificity by in vitro culture in relatively short period to a tumor-associated
antigen with considerably high affinity because the TCR is derived from a preselected
T-cell clone reactive to tumor (Figs. 1 and 3). The adoptive transfer of lymphocytes
engineered to express MART-1 specific TCR into patients with metastatic melanoma
demonstrated long-lasting maintenance in 2 out of 15 patients enrolled, both demonstrated
objective tumor regression.66 Subsequent trial with higher avidity of TCR demonstrated
objective clinical response in up to 30% of patients.67 Recent report on the usage of
artificially modified high avidity TCR reacting to NY-ESO-1 antigen demonstrated
objective clinical responses in four (60%) of six patients with synovial cell sarcomas
and five (45%) of 11 patients with melanoma.68
The existence of endogenous TCR in T cells has been reported to be associated
with the inefficient expression of transduced TCRs in T-lymphocytes. It is because
endogenous TCR competes with introduced TCR for CD3 molecules. In addition, the
introduced TCR _ and ` chains form mispaired TCRs with endogenous TCR subunits,
which not only further decrease the expression level of transduced TCR pairs but also
cause the generation of T cells with unexpected specificities including self-reactivity.69
To improve the efficacy of TCR engineering, we developed novel retroviral vectors
encoding both siRNA that down-regulate the endogenous TCR and a siRNA-resistant TCR
specific for tumor-associated antigens such as MAGE-A4 or WT1 (Fig. 2). These vectors
Figure 1. Antigen recognition of TCR-engineered CD8

T cells. Polyclonal T cells are redirected their
antigen specificity by retroviral or lentiviral transfer of tumor-reactive TCR gene. A large amount of
tumor-reactive T cells are generated by relatively short period of in vitro culture.
efficiently suppressed the endogenous TCR and enhanced the expression of transduced
tumor-associated antigen-specific TCR resulting in the enhanced tumor cytotoxicity.70
Another unique attempt to provide tumor-reacting capacity to polyclonal
lymphocytes by genetic engineering is to engineer lymphocytes to express a chimeric
antigen receptor (CAR) that consists of antigen-binding region of mAb fused with the
signal-transduction domain of CD3c or FcRIa (Fig. 3).71 Theoretical advantages of
this method are (1) independence of MHC class I expression of tumor, (2) capability
to engineer not only CD8
T cells but many of other cell types including CD4
T cells,
(3) avoidance of the influence of endogenous TCR that is one major obstacles in TCR
transduction, and (4) better persistence and penetration into tumor site compared to
mAb drug. Initial clinical trials (a trial targeting _-Folate receptor to treat ovarian
cancer,72 a trial targeting carbonic anhydrase IX to treat renal cell carcinoma,73 a
trial targeting CD20 to treat lymphoma74) demonstrated very limited persistence
of transferred cells without clear clinical responses. The absence of appropriate
costimulatory signal was considered to be responsible at least in part for these results.
To overcome this obstacle, next generation of CARs that includes signal transduction
domains of CD28 and some other costimulatory receptors such as OX40, 4-1BB have
been developed.71 Recently, the improved persistence of lymphocytes engineered
to express anti-CD19 CAR incorporating CD28 signaling fragment in patients with
lymphoma has been demonstrated.75
One alternative approach for prolonged in vivo persistence of CAR engineered
lymphocytes was demonstrated utilizing Epstain-Barr virus (EBV)-specific T cells
engineered to co-express CAR specific for GD2.76 In this trial, CAR engineered
Figure 2. Retrovirus vectors to down-regulate endogenous TCR by siRNA. Novel retrovirus vectors
that encode both siRNA to down-regulate endogenous TCR and codon-optimized TCR specific for
tumor-associated antigens were created. These vectors achieved high expression of induced TCR with
low proviral copy number in the transduced lymphocytes.
EBV-specific cytotoxic T cells persisted longer than CAR engineered polyclonal T cells
in patients with neuroblastoma. The authors reasoned that EBV-specific engineered T
cells could receive optimal costimulation by physiologic condition using EBV-specific
TCR, enhancing survival and antitumor activity.
On-target adverse events, however, have been reported for TCR gene therapies targeting
melanocyte-differentiating antigens especially when high-avidity TCRs were used.67 The
patients in the trial showed severe histological destruction in normal tissues where melanocytic
cells exist, such as skin, eyes, and inner ears. T cells engineered to express TCR specific
to carcinoembryonic antigen also induced a severe transient inflammatory colitis.77 Case
reports exploring the severe adverse events seen in the patients receiving T cell with CAR
targeting CD1978 or HER2/neu79 highlighted the potential risk in the usage of receptor
genes reactive not only to tumor cells but also a subset of normal cells. These observations
showed the potential power of T-cell therapy to overcome the immunological tolerance
in cancer patients as well as the need of careful approach in clinical trials. Interestingly,
lymphocytes engineered to express TCR specific to a cancer/testis antigen, NY-ESO-1,
did not demonstrate adverse events despite the fact that this TCR was modified to be very
high affinity,68 suggesting the importance of the selection of target antigen. Incorporation of
suicide gene might also be one of the promising ways to solve the risk of on-target toxicity.
In vitro experiments and mouse models have shown the strategy of genetic engineering
of lymphocytes to enhance their functions as well as resistance to tumor-mediated
immunosuppression through the addition of genes encoding homeostatic or pro-inflamatory
cytokines,80,81 chemokine receptors,82 anti-apoptotic molecules,83 and costimulatory
210
Figure 3. Adoptive cell therapy with antigen-receptor gene-modified lymphocytes. TCR genes derived from tumor-reactive cytotoxic T cells are transduced into
patients lymphocytes. Alternatively, single chained VH domain and VL domain derived from antibody reactive to tumor-associated antigen are fused with signal
transduction domain of CD3c to create a chimeric antigen receptor (CAR) gene. CAR gene is transduced into patients lymphocytes to generate tumor-reactive T
cells (T-body). These lymphocytes genetically engineered to become tumor-reactive are adoptively transferred into patients with tumor. Modified from Figure 6-2;
Naoko Imai et al. In: Masabumi Shibata, ed. Cancer Biology. Yodosha, 2011:260-9.
molecules84,85 as well as the silencing of coinhibitory molecules,86 although these

modifications await the validations of their concepts in clinic.
PASSIVE IMMUNOTHERAPY OF BRAIN TUMOR
Based on the above discussion on the understanding that CNS is an immune-specialized

rather than immune-privileged site, we envisage that adoptive T-cell therapy with tumor
antigen-specific T cells can be applied to the treatment of patients with brain tumors.
Several clinical trials using adoptive T-cell therapy for patients with brain tumor are
currently active according to the NIH clinical trial database (www.clinicaltrials.gov).
These include the usage of CMV-specific T cells, T cells genetically engineered to express
IL-13-Zetakine, and CAR targeting HER2. As mentioned previously, CAR targeting
GD2 expressed in EBV-specific T cells showed promising outcome in early phase trial,76
encouraging the extensive evaluation of this strategy into trials with later phases. Since
the list of tumor-associated antigens for brain tumor consistently grow (Table 1), the
evaluation of adoptive T-cell therapy against new targets will also prove useful for the
development of effective and safe therapeutic protocols for patients with brain tumor.
CONCLUSION
Passing more than a half of a century after Frank Macfarlane Burnet proposed the
concept of cancer immunosurveillance, we are facing a stage that immunotherapy is
emerging as a realistic and useful modality in the treatment for patients with cancer.
This is also unmistakably true in the challenge to fight with brain tumor. Among
the immunotherapies that are currently in development, adoptive T-cell therapy with
lymphocytes genetically engineered to express tumor antigen-specific receptor is certainly
a promising strategy to treat patients with glioma. To further overcome the multiple layers
of immuno-suppression/evasion mechanisms of tumor, the progress in basic science in
immunobiology and oncology harmonized with extensive effort in clinical studies is
indispensable. Combination of active and passive immunotherapy with manipulation of
immunologic balance in cancer patients will open a new gate for effective immunotherapy
of cancer.
References
1. Parney IF, Farr-Jones MA, Chang LJ et al. Human glioma immunobiology in vitro: implications for
immunogene therapy. Neurosurgery 2000; 46(5):1169-1177;discussion 77-78.
2. Neuwelt E, Abbott NJ, Abrey L et al. Strategies to advance translational research into brain barriers. Lancet
Neurol 2008; 7(1):84-96.
3. Davies DC. Blood-brain barrier breakdown in septic encephalopathy and brain tumours. J Anat 2002;
200(6):639-646.
4. Rascher G, Fischmann A, Kroger S et al. Extracellular matrix and the blood-brain barrier in glioblastoma
multiforme: spatial segregation of tenascin and agrin. Acta Neuropathol 2002; 104(1):85-91.
5. Galea I, Bernardes-Silva M, Forse PA et al. An antigen-specific pathway for CD8 T-cells across the blood-brain
barrier. J Exp Med 2007; 204(9):2023-2030.
6. Miller SD, McMahon EJ, Schreiner B et al. Antigen presentation in the CNS by myeloid dendritic cells
drives progression of relapsing experimental autoimmune encephalomyelitis. Ann NY Acad Sci 2007;
1103:179-191.
7. Bailey-Bucktrout SL, Caulkins SC, Goings G et al. Cutting edge: central nervous system plasmacytoid
dendritic cells regulate the severity of relapsing experimental autoimmune encephalomyelitis. J Immunol
2008; 180(10):6457-6461.
8. Dunn GP, Dunn IF, Curry WT. Focus on TILs: prognostic significance of tumor infiltrating lymphocytes
in human glioma. Cancer Immun 2007; 7:12.
9. de Vos AF, van Meurs M, Brok HP et al. Transfer of central nervous system autoantigens and presentation
in secondary lymphoid organs. J Immunol 2002; 169(10):5415-5423.
10. Olofsson A, Miyazono K, Kanzaki T et al. Transforming growth factor-beta 1, -beta 2 and -beta 3 secreted
by a human glioblastoma cell line. Identification of small and different forms of large latent complexes.
J Biol Chem 1992; 267(27):19482-19488.
11. Weller M, Constam DB, Malipiero U et al. Transforming growth factor-beta 2 induces apoptosis of murine
T-cell clones without down-regulating bcl-2 mRNA expression. Eur J Immunol 1994; 24(6):1293-1300.
12. Sasaki A, Naganuma H, Satoh E et al. Secretion of transforming growth factor-beta 1 and -beta 2 by
malignant glioma cells. Neurol Med Chir (Tokyo) 1995; 35(7):423-430.
13. Black KL, Chen K, Becker DP et al. Inflammatory leukocytes associated with increased immunosuppression
by glioblastoma. J Neurosurg 1992; 77(1):120-126.
14. Castelli MG, Chiabrando C, Fanelli R et al. Prostaglandin and thromboxane synthesis by human intracranial
tumors. Cancer Res 1989; 49(6):1505-1508.
15. Couldwell WT, Dore-Duffy P, Apuzzo ML et al. Malignant glioma modulation of immune function: relative
contribution of different soluble factors. J Neuroimmunol 1991; 33(2):89-96.
16. Nitta T, Hishii M, Sato K et al. Selective expression of interleukin-10 gene within glioblastoma multiforme.
Brain Res 1994; 649(1-2):122-128.
17. Hishii M, Nitta T, Ishida H et al. Human glioma-derived interleukin-10 inhibits antitumor immune responses
in vitro. Neurosurgery 1995; 37(6):1160-1166;discussion 6-7.
18. Huettner C, Paulus W, Roggendorf W. Messenger RNA expression of the immunosuppressive cytokine
IL-10 in human gliomas. Am J Pathol 1995; 146(2):317-322.
19. Wikstrand CJ, Fredman P, Svennerholm L et al. Detection of glioma-associated gangliosides GM2, GD2,
GD3, 3v-isoLM1 3v,6v-isoLD1 in central nervous system tumors in vitro and in vivo using epitope-defined
monoclonal antibodies. Prog Brain Res 1994; 101:213-223.
20. Fredman P, Mansson JE, Dellheden B et al. Expression of the GM1-species, [NeuN]-GM1, in a case of
human glioma. Neurochem Res 1999; 24(2):275-279.
21. Kawai K, Takahashi H, Watarai S et al. Occurrence of ganglioside GD3 in neoplastic astrocytes. An
immunocytochemical study in humans. Virchows Arch 1999; 434(3):201-205.
22. Choi C, Xu X, Oh JW et al. Fas-induced expression of chemokines in human glioma cells: involvement
of extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinase. Cancer Res 2001;
61(7):3084-3091.
23. Choi C, Gillespie GY, Van Wagoner NJ et al. Fas engagement increases expression of interleukin-6 in
human glioma cells. J Neurooncol 2002; 56(1):13-19.
cells: a potential mechanism of immune paralysis. Cancer Res 2003; 63(21):7462-7467.
25. Parsa AT, Waldron JS, Panner A et al. Loss of tumor suppressor PTEN function increases B7-H1 expression
and immunoresistance in glioma. Nat Med 2007; 13(1):84-88.
26. Nakabayashi H, Nakashima M, Hara M et al. Clinico-pathological significance of RCAS1 expression in
gliomas: a potential mechanism of tumor immune escape. Cancer Lett 2007; 246(1-2):182-189.
27. Wischhusen J, Jung G, Radovanovic I et al. Identification of CD70-mediated apoptosis of immune effector
cells as a novel immune escape pathway of human glioblastoma. Cancer Res 2002; 62(9):2592-2599.
28. Held-Feindt J, Mentlein R. CD70/CD27 ligand, a member of the TNF family, is expressed in human brain
tumors. Int J Cancer 2002; 98(3):352-356.
29. Foley EJ. Antigenic properties of methylcholanthrene-induced tumors in mice of the strain of origin. Cancer
Res 1953; 13(12):835-837.
30. Burnet FM. The concept of immunological surveillance. Prog Exp Tumor Res 1970; 13:1-27.
31. Thomas L. On Immunosurveillance in Human Cancer. Yale J Biol Med 1982; 55:329-333.
T-lymphocytes on a human melanoma. Science 1991; 254(5038):1643-1647.
33. Boon T, Old LJ. Cancer tumor antigens. Curr Opin Immunol 1997; 9(5):681-683.
34. Dunn GP, Bruce AT, Ikeda H et al. Cancer immunoediting: from immunosurveillance to tumor escape.
Nat Immunol 2002; 3(11):991-998.
cellular immunotherapy in patients with grade III/IV astrocytoma. J Neurooncol 1996; 27(2):179-189.
36. Plautz GE, Barnett GH, Miller DW et al. Systemic T-cell adoptive immunotherapy of malignant gliomas.
J Neurosurg 1998; 89(1):42-51.
37. Plautz GE, Miller DW, Barnett GH et al. T-cell adoptive immunotherapy of newly diagnosed gliomas.
Clin Cancer Res 2000; 6(6):2209-2218.
38. Wood GW, Holladay FP, Turner T et al. A pilot study of autologous cancer cell vaccination and cellular
immunotherapy using anti-CD3 stimulated lymphocytes in patients with recurrent grade III/IV astrocytoma.
J Neurooncol 2000; 48(2):113-120.
cells elicits systemic cytotoxicity and intracranial T-cell infiltration. Cancer Res 2001; 61(3):842-847.
40. Andrews DW, Resnicoff M, Flanders AE et al. Results of a pilot study involving the use of an antisense
oligodeoxynucleotide directed against the insulin-like growth factor type I receptor in malignant
astrocytomas. J Clin Oncol 2001; 19(8):2189-2200.
41. Kikuchi T, Akasaki Y, Irie M et al. Results of a phase I clinical trial of vaccination of glioma patients with
fusions of dendritic and glioma cells. Cancer Immunol Immunother 2001; 50(7):337-344.
42. Schneider T, Gerhards R, Kirches E et al. Preliminary results of active specific immunization with modified
tumor cell vaccine in glioblastoma multiforme. J Neurooncol 2001; 53(1):39-46.
43. Yamanaka R, Abe T, Yajima N et al. Vaccination of recurrent glioma patients with tumour lysate-pulsed
dendritic cells elicits immune responses: results of a clinical phase I/II trial. Br J Cancer 2003;
89(7):1172-1179.
cytotoxic T-cells in patients with malignant glioma. Cancer Res 2004; 64(14):4973-4979.
45. Steiner HH, Bonsanto MM, Beckhove P et al. Antitumor vaccination of patients with glioblastoma multiforme:
a pilot study to assess feasibility, safety, and clinical benefit. J Clin Oncol 2004; 22(21):4272-4281.
vaccination for patients with relapsed malignant glioma, a feasibility study. Br J Cancer 2004;
91(9):1656-1662.
47. Kikuchi T, Akasaki Y, Abe T et al. Vaccination of glioma patients with fusions of dendritic and glioma
cells and recombinant human interleukin 12. J Immunother 2004; 27(6):452-459.
recurrent glioma: results of a clinical phase I/II trial. Clin Cancer Res 2005; 11(11):4160-4167.
49. Liau LM, Prins RM, Kiertscher SM et al. Dendritic cell vaccination in glioblastoma patients induces systemic
and intracranial T-cell responses modulated by the local central nervous system tumor microenvironment.
Clin Cancer Res 2005; 11(15):5515-5525.
glioma: preliminary results of using autologous whole-tumor vaccine plus granulocyte-macrophage
2000; 9(6):e9.
51. Ishikawa E, Tsuboi K, Yamamoto T et al. Clinical trial of autologous formalin-fixed tumor vaccine for
glioblastoma multiforme patients. Cancer Sci 2007; 98(8):1226-1233.
52. Okada H, Lieberman FS, Walter KA et al. Autologous glioma cell vaccine admixed with interleukin-4
gene transfected fibroblasts in the treatment of patients with malignant gliomas. J Transl Med 2007; 5:67.
53. De Vleeschouwer S, Fieuws S, Rutkowski S et al. Postoperative adjuvant dendritic cell-based immunotherapy
in patients with relapsed glioblastoma multiforme. Clin Cancer Res 2008; 14(10):3098-3104.
54. Wheeler CJ, Black KL, Liu G et al. Vaccination elicits correlated immune and clinical responses in
glioblastoma multiforme patients. Cancer Res 2008; 68(14):5955-5964.
55. Yajima N, Yamanaka R, Mine T et al. Immunologic evaluation of personalized peptide vaccination for
patients with advanced malignant glioma. Clin Cancer Res 2005; 11(16):5900-5911.
with recurrent glioblastoma multiforme. J Neurosurg 2008; 108(5):963-971.
57. Okada H, Kalinski P, Ueda R et al. Induction of CD8
T-cell responses against novel glioma-associated
antigen peptides and clinical activity by vaccinations with {alpha}-type 1 polarized dendritic cells and
polyinosinic-polycytidylic acid stabilized by lysine and carboxymethylcellulose in patients with recurrent
malignant glioma. J Clin Oncol 2011; 29(3):330-336.
58. Gattinoni L, Powell DJ Jr., Rosenberg SA et al. Adoptive immunotherapy for cancer: building on success.
Nat Rev Immunol 2006; 6(5):383-393.
59. Topalian SL, Solomon D, Avis FP et al. Immunotherapy of patients with advanced cancer using
tumor-infiltrating lymphocytes and recombinant interleukin-2: a pilot study. J Clin Oncol 1988;
6(5):839-853.
60. Rosenberg SA, Packard BS, Aebersold PM et al. Use of tumor-infiltrating lymphocytes and interleukin-2
in the immunotherapy of patients with metastatic melanoma. A preliminary report. N Engl J Med 1988;
319(25):1676-1680.
61. Rosenberg SA, Yannelli JR, Yang JC et al. Treatment of patients with metastatic melanoma with autologous
tumor-infiltrating lymphocytes and interleukin 2. J Natl Cancer Inst 1994; 86(15):1159-1166.
62. Khong HT, Restifo NP. Natural selection of tumor variants in the generation of tumor escape phenotypes.
Nat Immunol 2002; 3(11):999-1005.
63. Seder RA, Darrah PA, Roederer M. T-cell quality in memory and protection: implications for vaccine
design. Nat Rev Immunol 2008; 8(4):247-258.
64. Dudley ME, Yang JC, Sherry R et al. Adoptive cell therapy for patients with metastatic melanoma: evaluation
of intensive myeloablative chemoradiation preparative regimens. J Clin Oncol 2008; 26(32):5233-5239.
65. Kessels HW, Wolkers MC, van den Boom MD et al. Immunotherapy through TCR gene transfer. Nat
Immunol 2001; 2(10):957-961.
66. Morgan RA, Dudley ME, Wunderlich JR et al. Cancer regression in patients after transfer of genetically
engineered lymphocytes. Science 2006; 314(5796):126-129.
67. Johnson LA, Morgan RA, Dudley ME et al. Gene therapy with human and mouse T-cell receptors mediates
cancer regression and targets normal tissues expressing cognate antigen. Blood 2009; 114(3):535-546.
68. Robbins PF, Morgan RA, Feldman SA et al. Tumor regression in patients with metastatic synovial cell
sarcoma and melanoma using genetically engineered lymphocytes reactive with NY-ESO-1. J Clin Oncol
2011; 29(7):917-924.
69. Bendle GM, Linnemann C, Hooijkaas AI et al. Lethal graft-versus-host disease in mouse models of T-cell
receptor gene therapy. Nat Med 2010; 16(5):565-570, 1p following 70.
70. Okamoto S, Mineno J, Ikeda H et al. Improved expression and reactivity of transduced tumor-specific TCRs
in human lymphocytes by specific silencing of endogenous TCR. Cancer Res 2009; 69(23):9003-9011.
71. Sadelain M, Brentjens R, Riviere I. The promise and potential pitfalls of chimeric antigen receptors. Curr
Opin Immunol 2009; 21(2):215-223.
72. Kershaw MH, Westwood JA, Parker LL et al. A phase I study on adoptive immunotherapy using gene-modified
T-cells for ovarian cancer. Clin Cancer Res 2006; 12(20 Pt 1):6106-6115.
73. Lamers CH, Sleijfer S, Vulto AG et al. Treatment of metastatic renal cell carcinoma with autologous
T-lymphocytes genetically retargeted against carbonic anhydrase IX: first clinical experience. J Clin
Oncol 2006; 24(13):e20-e22.
74. Till BG, Jensen MC, Wang J et al. Adoptive immunotherapy for indolent non Hodgkin lymphoma and
mantle cell lymphoma using genetically modified autologous CD20-specific T-cells. Blood 2008;
112(6):2261-2271.
75. Savoldo B, Ramos CA, Liu E et al. CD28 costimulation improves expansion and persistence of chimeric
antigen receptor-modified T-cells in lymphoma patients. J Clin Invest 2011; 121(5):1822-1826.
76. Pule MA, Savoldo B, Myers GD et al. Virus-specific T-cells engineered to coexpress tumor-specific receptors:
persistence and antitumor activity in individuals with neuroblastoma. Nat Med 2008; 14(11):1264-1270.
77. Parkhurst MR, Yang JC, Langan RC et al. T-cells targeting carcinoembryonic antigen can mediate regression
of metastatic colorectal cancer but induce severe transient colitis. Mol Ther 2011; 19(3):620-626.
78. Brentjens R, Yeh R, Bernal Y et al. Treatment of chronic lymphocytic leukemia with genetically targeted
autologous T-cells: case report of an unforeseen adverse event in a phase I clinical trial. Mol Ther 2010;
18(4):666-668.
79. Morgan RA, Yang JC, Kitano M et al. Case report of a serious adverse event following the administration of
T-cells transduced with a chimeric antigen receptor recognizing ERBB2. Mol Ther 2010; 18(4):843-851.
80. Hsu C, Hughes MS, Zheng Z et al. Primary human T-lymphocytes engineered with a codon-optimized IL-15
gene resist cytokine withdrawal-induced apoptosis and persist long-term in the absence of exogenous
cytokine. J Immunol 2005; 175(11):7226-7234.
81. Liu K, Rosenberg SA. Interleukin-2-independent proliferation of human melanoma-reactive T- lymphocytes
transduced with an exogenous IL-2 gene is stimulation dependent. J Immunother 2003; 26(3):190-201.
82. Kershaw MH, Wang G, Westwood JA et al. Redirecting migration of T-cells to chemokine secreted from
tumors by genetic modification with CXCR2. Hum Gene Ther 2002; 13(16):1971-1980.
83. Charo J, Finkelstein SE, Grewal N et al. Bcl-2 overexpression enhances tumor-specific T-cell survival.
Cancer Res 2005; 65(5):2001-2008.
84. Topp MS, Riddell SR, Akatsuka Y et al. Restoration of CD28 expression in CD28< CD8
memory effector
T-cells reconstitutes antigen-induced IL-2 production. J Exp Med 2003; 198(6):947-955.
85. Stephan MT, Ponomarev V, Brentjens RJ et al. T-cell-encoded CD80 and 4-1BBL induce auto- and
transcostimulation, resulting in potent tumor rejection. Nat Med 2007; 13(12):1440-1449.
86. Borkner L, Kaiser A, van de Kasteele W et al. RNA interference targeting programmed death receptor-1
improves immune functions of tumor-specific T-cells. Cancer Immunol Immunother 2010; 59(8):1173-1183.
87. Hatano M, Eguchi J, Tatsumi T et al. EphA2 as a glioma-associated antigen: a novel target for glioma
vaccines. Neoplasia 2005; 7(8):717-722.
88. Hatano M, Kuwashima N, Tatsumi T et al. Vaccination with EphA2-derived T-cell-epitopes promotes
immunity against both EphA2-expressing and EphA2-negative tumors. J Transl Med 2004; 2(1):40.
89. Miao H, Wei BR, Peehl DM et al. Activation of EphA receptor tyrosine kinase inhibits the Ras/MAPK
pathway. Nat Cell Biol 2001; 3(5):527-530.
90. Okano F, Storkus WJ, Chambers WH et al. Identification of a novel HLA-A*0201-restricted, cytotoxic
T-lymphocyte epitope in a human glioma-associated antigen, interleukin 13 receptor alpha2 chain. Clin
Cancer Res 2002; 8(9):2851-2855.
91. Shimato S, Natsume A, Wakabayashi T et al. Identification of a human leukocyte antigen-A24-restricted
T-cell epitope derived from interleukin-13 receptor alpha2 chain, a glioma-associated antigen. J Neurosurg
2008; 109(1):117-122.
92. Debinski W, Gibo DM, Hulet SW et al. Receptor for interleukin 13 is a marker and therapeutic target for
human high-grade gliomas. Clin Cancer Res 1999; 5(5):985-990.
93. Eguchi J, Hatano M, Nishimura F et al. Identification of interleukin-13 receptor alpha2 peptide analogues
capable of inducing improved antiglioma CTL responses. Cancer Res 2006; 66(11):5883-5891.
94. Liu G, Ying H, Zeng G et al. HER-2, gp100 and MAGE-1 are expressed in human glioblastoma and
recognized by cytotoxic T-cells. Cancer Res 2004; 64(14):4980-4986.
95. Pelloski CE, Mahajan A, Maor M et al. YKL-40 expression is associated with poorer response to radiation
and shorter overall survival in glioblastoma. Clin Cancer Res 2005; 11(9):3326-3334.
96. Nutt CL, Betensky RA, Brower MA et al. YKL-40 is a differential diagnostic marker for histologic subtypes
of high-grade gliomas. Clin Cancer Res 2005; 11(6):2258-2264.
97. Schmitz M, Temme A, Senner V et al. Identification of SOX2 as a novel glioma-associated antigen and
potential target for T-cell-based immunotherapy. Br J Cancer 2007; 96(8):1293-1301.
98. Schmitz M, Wehner R, Stevanovic S et al. Identification of a naturally processed T-cell epitope derived
from the glioma-associated protein SOX11. Cancer Lett 2007; 245(1-2):331-336.
99. Bao S, Wu Q, McLendon RE et al. Glioma stem cells promote radioresistance by preferential activation
of the DNA damage response. Nature 2006; 444(7120):756-760.
100. Wu AH, Xiao J, Anker L et al. Identification of EGFRvIII-derived CTL epitopes restricted by HLA A0201
for dendritic cell based immunotherapy of gliomas. J Neurooncol 2006; 76(1):23-30.
101. Liu G, Khong HT, Wheeler CJ et al. Molecular and functional analysis of tyrosinase-related protein (TRP)-2
as a cytotoxic T-lymphocyte target in patients with malignant glioma. J Immunother 2003; 26(4):301-312.
102. Andersen MH, Pedersen LO, Becker JC et al. Identification of a cytotoxic T-lymphocyte response to the
apoptosis inhibitor protein survivin in cancer patients. Cancer Res 2001; 61(3):869-872.
103. Andersen MH, Pedersen LO, Capeller B et al. Spontaneous cytotoxic T-cell responses against survivin-derived
MHC class I-restricted T-cell epitopes in situ as well as ex vivo in cancer patients. Cancer Res 2001;
61(16):5964-5968.
104. Uematsu M, Ohsawa I, Aokage T et al. Prognostic significance of the immunohistochemical index of
survivin in glioma: a comparative study with the MIB-1 index. J Neurooncol 2005; 72(3):231-238.
105. Blanc-Brude OP, Yu J, Simosa H et al. Inhibitor of apoptosis protein survivin regulates vascular injury.
Nat Med 2002; 8(9):987-994.
106. Imaizumi T, Kuramoto T, Matsunaga K et al. Expression of the tumor-rejection antigen SART1 in brain
tumors. Int J Cancer 1999; 83(6):760-764.
107. Shichijo S, Nakao M, Imai Y et al. A gene encoding antigenic peptides of human squamous cell carcinoma
recognized by cytotoxic T-lymphocytes. J Exp Med 1998; 187(3):277-288.
108. Oji Y, Suzuki T, Nakano Y et al. Overexpression of the Wilms tumor gene W T1 in primary astrocytic
tumors. Cancer Sci 2004; 95(10):822-827.
109. Oka Y, Elisseeva OA, Tsuboi A et al. Human cytotoxic T-lymphocyte responses specific for peptides of
the wild-type Wilms tumor gene (WT1) product. Immunogenetics 2000; 51(2):99-107.
110. Sugiyama H. Cancer immunotherapy targeting WT1 protein. Int J Hematol 2002; 76(2):127-132.
111. Oka Y, Tsuboi A, Elisseeva OA et al. WT1 as a novel target antigen for cancer immunotherapy. Curr
Cancer Drug Targets 2002; 2(1):45-54.
112. Iiyama T, Udaka K, Takeda S et al. WT1 (Wilms tumor 1) peptide immunotherapy for renal cell carcinoma.
Microbiol Immunol 2007; 51(5):519-530.
CHAPTER 17
GLIOMA STEM CELL RESEARCH FOR THE

DEVELOPMENT OF IMMUNOTHERAPY
Qijin Xu, Xiangpeng Yuan and John S. Yu*

Maxine Dunitz Neurosurgical Institute, Cedars-Sinai Medical Center, Los Angeles, California, USA
*Corresponding Author: John S. YuEmail: John.Yu@cshs.org
Abstract: Malignant gliomas are characterized by its invasiveness and dissemination, resulting in
frequent tumor recurrence after surgical resection and/or conventional chemotherapy
and radiation therapy. Various strategies of active and passive immunotherapy
in developing stages have shown promise to increase patient survival time with
little severe side effects. In recent years, glioma stem cells had been isolated from
patient tumor specimens. Biochemical and biological characterization of these
cancer-initiating cells implicated their critical roles in cancer growth, malignancy
and resistance to conventional treatments. In this chapter, we review recent research
progress in targeting brain cancer using neural stem cells-delivered cytotoxic factors
and immune regulation factor, dendritic cell-based vaccination, with special emphasis
on targeting glioma stem cells. We present evidence supporting the notion that glioma
stem cells may be preferred therapeutic targets not only for conventional therapies,
but also for immunotherapies. Future progress in glioma stem cell research may
fundamentally improve the prospect of malignant glioma treatments.
INTRODUCTION
The cancer stem cell (CSC) hypothesis has provided an alternative framework for
understanding cancer heterogeneity, tumorigenesis and cancer progression. Recent
identification of cancer-initiating stem cells in brain tumor,1,2 prostate cancer,3 colon
cancer4,5 and breast cancer6,7 suggested that CSCs may play a central role in the propagation
of several cancer types. CSCs have also been shown to be responsible for prevalent
radioresistance and chemoresistance in glioma.8 Compared to conventionally cultured
human cancer cell lines, CSCs have been shown to recapitulate human brain tumors in

216
GLIOMA STEM CELL RESEARCH DEVELOPMENT OF IMMUNOTHERAPY 217
phenotype and in cancer genetics and thus may more faithfully model mechanisms of
tumorigenesis and tumor propagation.9
The identification of glioma stem cells may have important applications in cancer
therapy for glioma patients. Therapies targeting glioma stem cells may help overcome
the persistent cancer resistance to chemotherapies and radiation therapies. In addition,
strategies targeting glioma stem cells may have positive impact on the development of
immunotherapy for malignant brain cancer patients.
In this chapter, we first introduce the concept of glioma stem cells and the status of
current research on the biology of glioma stem cells. Then we discuss the opportunity of
novel chemotherapies and radiation therapies from glioma stem cell research. Next, we
explore the potential applications of neural stem cells as gene delivery tools for glioma
therapies. Finally, we elaborate on the current immunotherapy research and clinical trials,
especially dendritic cell vaccination, for glioma patients and the possibility and hope of
targeting glima stem cells in future glioma immunotherapies.
GLIOMA STEM CELLS AS CANCER INITIATING CELLS
Glioblastoma multiforme (GBM) is the most malignant form of human primary brain
tumor and can be initiated from brain tumor stem-like cells (BTSCs).8-10 However, unlike
normal tissue stem cells, CSCs may be heterogeneous,11-13 meaning that all CSCs from
tumors of the same tissue origin and grade, or even from the same tumor, are not the same.
For example, both CD44
/CD24 and CD133
cell populations had been identified from
the same cancer in a BRCA breast cancer model,11 suggesting that one initial mechanism
may lead to diverse CSCs with different phenotypes. This conclusion may explain the
paradoxical findings from a recent study identifying nonCD133
cells as colon cancer
initiating cells,14 as opposed to results from other two studies.4,5
The capability of BTSCs to sustain brain tumor growth apparently lies in their active
self-renewal and/or suppressed cell differentiation.2 Several major signaling pathways
that are critical in brain development have also been implicated in tumorigenesis,
including: bone morphogenetic protein (BMP),15 Notch16 and Sonic Hedgehog (SHH),17,18
EGFR,19,20 PTEN/PI3K/mTOR,16,19,21 PDGFR22 and OLIG2.23 Recently, a gene expression
profiling of gliomas has shown that SHH signaling is active in a subset of gliomas.24
This study further showed that SHH signaling is essential for glioma CSC self-renewal
and CSC-initiated brain tumor growth.24 It is postulated that the relative homogenous
population of CSCs, rather than the heterogeneous tumor cells, may reveal key
mechanisms of tumor initiation and propagation of primary tumors and hence predict
clinical prognosis, therapy and drug response of tumors.
We had performed gene expression profiling of BTSCs and unveiled salient signaling
pathway signatures. We identified both SHH signaling-dependent and -independent
BTSCs that can initiate brain tumors retaining their respective characteristics of signaling
dependency. BTSC self-renewal could be abrogated in a pathway-dependent fashion in vitro
and in an intracranial tumor model in SCID mice. Furthermore, we found that hyperactive
SHH-GLI signaling in PTEN-coexpressing tumors was associated with reduced survival
times in glioblastoma patients.
The identification of both SHH signaling-dependent and independent brain tumors in
this study suggested that there are molecularly distinct subclasses of GBMs that have an
effect on progression and prognosis. These findings were reminiscent of a recent study in
which high-grade gliomas were classified into a proneural subclass and a mesenchymal
subclass, resembling two stages in neurogenesis.16 In that study, Phillips et al discovered
a prognostic model utilizing PTEN-AKT and Notch signatures to predict poor versus
better glioma prognosis, respectively.16 In our study, the SHH signaling-dependent
brain tumors showed high activities in Notch and PDGFR pathways, while the SHH
signaling-independent brain tumors showed PTEN-deficiency and high activity in
PI3K-AKT pathway. Our study emphasized that BTSCs with distinct signaling patterns
determined the different GBM phenotypes and progression.
The pivotal role of PTEN expression status in GBM development and progression
is further supported in our study using BTSCs. PTEN expression is widely lost in GBM
and other malignant tumors.7,25-28 Co-expression of PTEN with active EGFR in GBM
determines clinical cancer responses to an EGFR inhibitor.19 PTEN loss and AKT
activation is associated with more invasive and malignant cancer and poor prognosis for
GBM patients.16 Furthermore, a drug that potently inhibits GBM cell proliferation was
found to be an inhibitor of both PI3 kinase _ and mTOR.21 We found that there are both
PTEN-expressing and PTEN-deficient BTSCs and that all BTSCs tested are inhibited
by PI3K and AKT inhibitors.
Our study presented evidence indicating genetic interaction between the SHH signaling
pathway and PTEN in human glioblastoma. After analysis of a tissue array of GBM tumors
and additional frozen GBM tissues, we found that the average SHH signaling activity is
significantly higher in PTEN-expressing tumors than in PTEN-deficient tumors. While
the expression levels of SHH and GLI1 are significantly higher in PTEN-expressing
tumors, the expression level of PTCH1, a SHH receptor, is significantly lower. PTCH1 is
tumor suppressor and a negative regulator of SHH signaling in the absence of the ligands.
These results suggest that hyperactive SHH-GLI signaling is critical for brain tumor cell
self-renewal only when the tumor suppression mechanism of PTEN pathway is intact.
When PTEN expression is lost through genetic or epigenetic alteration, alternative signaling
pathways, including PI3K-AKT-mTOR pathway, are usually activated, leading to bypass
of the requirement for an active SHH-GLI signaling pathway. PTEN expression alone,
however, is not a strong indicator of malignancy in our study, as there is no association
between PTEN expression in GBM and patient survival. Finally, we have shown that
higher SHH signaling in PTEN-expressing GBM is associated with reduced survival
time, further supporting a critical role for SHH signaling in the PTEN-coexpressing
subset of GBM tumors.
TARGETING BRAIN CANCER USING NEURAL STEM CELL-DELIVERED

THERAPEUTICS
One possible method to target disseminated tumor islands is the use of NSCs, which
can display intracranial migratory activity similar to that of glioma cells.29 NSCs have
been shown to be capable of actively tracking migrating glioma cells within the brain30
and have been used to deliver the therapeutic cytokine interleukin (IL)-4 to glioma in
vivo with encouraging results.31 Therefore, the use of NSCs for the delivery of therapeutic
gene products to migratory tumor islands may represent a viable modality for targeting
these otherwise difficult to access neoplastic reservoirs. With the aim of additionally
investigating this therapeutic strategy, we reported the use of Tumor Necrosis Factor-related
Apoptosis-inducing Ligand (TRAIL)- and IL-12-secreting NSCs for the treatment
of intracranial glioma. We demonstrate that in C57Bl/6 mice bearing GL26 gliomas,
intratumoral inoculation of IL-12-secreting NSCs significantly prolonged survival and
resulted in long-term antitumor immunity.32,33 NSCs were generally found interspersed
within the main tumor mass and were also present in small tumor islands detached from the
primary tumor body. In addition, NSCs could be seen actively tracking outgrowths from
the main tumor that extended deep into adjacent normal tissue. We also demonstrated that
the prolonged survival seen in animals treated with IL-12-secreting NSCs was associated
with significantly increased intratumoral CD4
and CD8
T-cell infiltration, particularly
at the tumor/normal tissue boundary and in migrating tumor micro-satellites.
To address the issue of limited source of NSCs, we isolated neural stem-like cells from
the bone marrow of adult mice. The isolated cells were capable of producing progenies of
three lineages, neurons, astrocytes and oligodendrocytes, in vitro and tracking glioma in
vivo. By genetically manipulating bone marrowderived neural stem-like cells (BM-NSC)
to express a recently discovered cytokine, IL-23, the cells showed protective effects in
intracranial tumor-bearing C57BL/6 mice.34 Depletion of subpopulation lymphocytes
showed that CD8
T cells were critical for the antitumor immunity of IL-23expressing
BM-NSCs and that CD4
T cells and natural killer (NK) cells participated in the activity.
Furthermore, the IL-23expressing BM-NSC-treated survivors were resistant to the same
tumor rechallenge and the protection was associated with enhanced IFN-a, but not IL-17,
expression in the brain tissue. This study suggests that IL-23expressing BM-NSCs can
effectively induce antitumor immunity against intracranial gliomas. CD8
T cells are critical
for such antitumor activity; in addition, CD4
T cells and NK cells are also involved.
Dendritic cells (DCs) are potent antigen-presenting cells that play a critical role
in priming immune responses to tumor. IL-23 may act directly on DC to promote
immunogenic presentation of tumor peptide in vitro. We evaluated the combination of
bone marrowderived DC and IL-23 on the induction of antitumor immunity in a mouse
intracranial glioma model. DCs were transduced with an adenoviral vector expressing
high levels of bioactive IL-23. Intratumoral implantation of IL-23expressing DCs
produced a protective effect on intracranial tumorbearing mice. The mice consequently
gained systemic immunity against the same tumor rechallenge. The protective effect of
IL-23expressing DCs was comparable with or even better than that of IL-12expressing
DCs.34,35 IL-23transduced DC (DC-IL-23) treatment resulted in robust intratumoral
CD8
and CD4
T-cell infiltration and induced a specific TH1-type response to the tumor
in regional lymph nodes and spleen at levels greater than those of nontransduced DCs.
Moreover, splenocytes from animals treated with DC-IL-23 showed heightened levels
of specific CTL activity. In vivo lymphocyte depletion experiments showed that the
antitumor immunity induced by DC-IL-23 was mainly dependent on CD8
T cells and
that CD4
T cells and natural killer cells were also involved. In summary, i.t. injection
of DC-IL-23 resulted in significant and effective systemic antitumor immunity in
intracranial tumorbearing mice. These findings suggest a new approach to induce potent
tumor-specific immunity to intracranial tumors. This approach may have therapeutic
potential for treating human glioma.
IMMUNOTHERAPY STRATEGIES FOR GLIOMAS AND THE PROMISE

OF DENDRITIC CELL VACCINATION
Immunotherapies harness the bodys own immune system to counter brain tumors
and could overcome the difficulties in conventional treatments.36,37 Various strategies
of immunotherapy had been reported, including passive serologic immunotherapy,
adoptive immunotherapy and cytokine therapy.38 However, one of the most promising
strategies is active immunotherapy using dendritic cell (DC)-based vaccination to initiate
T-cell-mediated antitumor immunity.
A key limiting factor in developing antitumor immunity lies in presenting immunogenic
tumor antigens to T cells in vivo. Identification of brain tumor-associated antigens (TAAs)
remains a challenge, although we have previously reported expression of some TAAs
(e.g., gp100, TRP-2 and MAGE-1, etc) in gliomas. In practice, vaccination strategies
have often utilized DC pulsed with tumor-derived whole lysates/peptides as modalities
to present a broad range of tumor antigens to T cells ex vivo to stimulate effective
antitumor T-cell immunity.36 This process includes two stages: in vitro DC generation and
maturation and in vivo DC migration and licensing in the draining lymph node (DLN).
Human DCs are commonly generated from peripheral blood-derived monocytes, followed
by a differentiation step using GM-CSF and IL-4 treatments to produce immature DCs
(iDCs). The iDCs undergo maturation and antigen loading steps to produce mature DCs.
DC maturation is typically accomplished by culturing iDCs for an additional 24-48 hours
in the presence of cytokines TNF, IL-6 and IL-1`. The most common antigen loading
method in clinical trials is to incubate DCs with tumor antigens or tumor lysates. Mature
DCs loaded with tumor antigens are administrated subcutaneously into patients. The goal
is to generate ex vivo a population of tumor antigen-loaded DCs that stimulates robust and
long lasting tumor-specific CD4
and CD8
T-cell responses in the patient with cancer.
Increasing evidence suggested that DC-based vaccination could increase tumor
antigen presentation and elicit significant antitumor immune responses. Vaccination
with DCs pulsed with acid-eluted glioblastoma peptides was well tolerated and can
induce systemic antigen-specific immunity in patients with recurrent GBM.36,39,40,41 Our
earlier Phase I study demonstrated the feasibility, safety and bioactivity of an autologous
tumor lysate-pulsed DC vaccine for patients with malignant glioma.38 However, further
understanding of immune tolerance and immune regulation may contribute to improved
immunotherapy for glioblastoma.
GLIOMA STEM CELL-TARGETED IMMUNOTHERAPY
Recent studies identifying cancer stem-like cells (CSCs) as brain tumor initiating cells
may have implications for modifying GBM treatments, including DC vaccination-based
immunotherapy.1,2,42,43 Therapies targeting CSCs may prevent tumor recurrences seen after
conventional radiation and chemotherapies. Furthermore, it is likely that certain stem cell
markers expressed by CSCs may have distinct antigenicity and thus provide opportunities
for enhanced immunotherapy. Some proteins expressed by CSCs are normally seen only in
early development stages. Antibodies against the stem cell-associated antigen SOX2 was
identified in a human patient.44 CSC-associated proteins may be used for cancer vaccination.
It was reported recently that vaccination using Prostate Stem Cell Antigen induced long term
protective immune response against prostate cancer without autoimmunity.45 Even without
identification of specific antigens, CSCs can be a useful source of tumor antigens in DC

vaccination-based immunotherapy. Using a mouse GL261 glioma model, Pellegatta et al
demonstrated that vaccination with DCs loaded with glioma CSC antigens elicited robust
antitumor T-cell immunity.46 In this study, DC vaccination using CSC antigens cured up
to 80% GL261 tumors, while DC vaccination using regular GL261 antigens cured none
of the CSC-initiated tumors. However, whether using human CSC antigens may improve
the antitumor effect of DC vaccination against human cancer is unclear.
In a recent study, we investigate expression of tumor associated antigens (TAAs)
and major histocompatibility complex (MHC) molecules by GBM-derived CSCs. We
report that CSCs express MHC I and increased levels of a range of TAAs. CSCs can
be recognized by T cells generated after DC pulsed with CSC tumor antigens. DC
vaccination induced interferon (IFN) -a production is positively correlated with the number
of antigen-specific T cells generated. Finally, using a 9L CSC brain tumor model, we
demonstrate that 9L CSC-loaded DC vaccination induced higher IFN-a production than
vaccination using parent cells and differentiated daughter cells and that vaccination with
only 9L CSC-loaded DCs prolongs survival of tumor-bearing animals.
Increasing evidence supports the notion that a small subpopulation of cancer stem-like
cells is responsible for cancer progression, therapy resistance and relapse. To inhibit
tumor recurrence more effectively, it would behoove any cancer therapy, including
immunotherapy, to target the cancer stem cell. In this study, we build on our extensive
experience of DC vaccination for human GBM patients and explore the possibility of
targeting CSCs in DC vaccination, using both human samples and a syngeneic animal
brain tumor model. Our results demonstrate that GBM-derived CSCs express a range of
TAAs and class I MHC molecules that are critical for immune recognition. The expression
levels of some TAAs in CSCs are as high as over 200 fold of the levels in differentiated
daughter cells. Importantly, vaccination with DCs loaded with CSCs antigens induced
antigen-specific Th1 immune response. Finally, we tested DC vaccination using 9L CSC
tumor antigens in a 9L CSCs brain tumor model and achieved robust antitumor T-cell
immunity and a significant survival benefit.
Two recent studies using animal models demonstrated the potential of DC vaccination
targeting CSCs in cancer immunotherapy.45,47 In their study of DC vaccination using
GL261 neurospheres in a mouse brain tumor model, Finocchiaro and collegues found
that immunization with DCs loaded with GL261 neurospheres cured 60-80% animals
with glioma, while vaccines of DCs loaded with adherent GL261 cells cured only 50%
of GL261 tumors and none of the GL261 neurospheres initiated tumors.46 They also
reported robust tumor infiltration by CD8
and CD4
T-lymphocytes. Although there is
no tumor antigen characterization, antigen presentation function or other mechanistic data
in this study, it indicated the distinct potential of CSCs in inducing antitumor immunity.
In another recent study of prostate cancer, Garcia-Hernandez Mde et al vaccinated with
prostate stem cell antigen in mice bearing progressing prostate cancer and found induced
MHC expression, cytokine production, lymphocyte infiltration and long term protection
again prostate cancer.45 Both cancer vaccination studies in murine models support the
hypothesis that CSCs-derived whole lysates or CSC-associated antigens may be superior
to conventional tumor antigens in generating therapeutic antitumor effects. Consistent with
these studies, our data on cancer immunization using 9L CSCs in a rat model indicated
that vaccination with DCs loaded with only 9L CSCs antigens, but not the differentiated
daughter cell antigens, induced CTL responses against CSCs and significantly extended
survival of animals bearing 9L CSC tumors.
One key point from our study is that CSC-targeting DC vaccination appears to be
superior not only in experimental murine models, but also in human brain tumors. Due
to the known difference in cancer immunity between murine species and human, it is
important to investigate CSC-targeting DC vaccination in human cancer and compare
the results in human cancer study to those in murine models. In our study, we took
advantage of several well characterized human GBM-derived CSCs1,43 and explored
the possibility of DC vaccination using CSC antigens against human brain tumors.
Significantly, we found that these CSCs highly express a range of known TAAs as well
as MHC molecules. Immunization with apoptotic CSCs induced an antigen-specific
Th1 response. These data suggest that CSCs may be better sources of antigens for
cancer immunization that conventionally cultured tumor cells. It is to be seen whether
this CSC-targeting vaccination will generate better antitumor clinical effects in human
GBM patients. It is important that CSC-targeted DC vaccination should not lead to
immune reaction to normal cells that may express common antigens. However, multiple
mechanisms may exist that spare normal cells from such side effects. We found that
NSCs had very low expression of cell surface MHC molecules. NSCs may also evade
immune attack due to decreased expression of costimulatory proteins.48 We are in the
progress of initiating a clinical trial to study the safety and efficacy of DC vaccination
using CSC antigens.
To date there is very limited number of studies of CSC-targeting DC vaccination
in animal models or in patients. And detailed immunological analysis data on the
development of antitumor immunity after DC vaccination are not available. Questions
remain regarding mechanisms underlying the apparent superior outcomes from
CSC-targeting DC vaccination. For example, is there any difference between CSC
antigens and conventional tumor lysates in promoting DC maturation and polarization,
or in effector cell differentiation and memory T-cell generation in vivo? Although higher
expression of TAAs in CSCs, as shown in our study, may be one factor contributing to
the outcomes, it is likely other factors in addition to TAA expression levels also play
a role. Finally, outcomes of DC vaccination may be improved when it is administered
in combination with chemotherapy, radiotherapy, or other therapies.49-52
In summary, we explored the suitability of CSCs as sources of antigens for DC
vaccination again human GBM, with the aims of achieving CSC-targeting and better
antitumor immunity. We found that CSCs express increased levels of TAAs as well
MHC molecules. Furthermore, DC vaccination using CSC antigens elicited a potent
antigen-specific Th1 response. Finally, we show that vaccination with DCs loaded with
9L CSCs, but not the differentiated daughter cells or conventionally cultured 9L cells,
induced CTLs that recognized CSCs and prolonged survival animals bearing 9L CSC
tumors. Understanding how immunization with CSCs generates superior antitumor
immunity may help develop novel and more effective cancer immunotherapies.
CONCLUSION
Glioma stem cell research is currently a very active field and is bound to have
practical impact on glioma therapy. These CSCs may be the initiator of glioma growth
and malignancy. CSCs may also be the root of cancer resistance to conventional
therapies. Therefore, CSCs are the promising targets of future glioma therapies, including
immunotherapies.
DC-based vaccination appears to have great potential as effective treatment of

malignant gliomas. One important aspect of DC vaccination is to identify robust and
tumor-specific antigens. Intriguingly, work from our and other laboratories indicated
that CSCs preferentially express a group of tumor-associated antigens. In murine brain
cancer models, DCs loaded with tumor stem cell-derived antigens manifested superior
antitumor immune response, tumor inhibition and longer survival, compared with DC
vaccination with differentiated tumor cells. As for human gliomas, limited data so far
suggested that DC vaccination with CSCs induced more potent immune response than
that with differentiated cells.
Challenges are ahead to apply glioma stem cell knowledge to immunotherapy.
For one thing, efficient immunotherapies targeting glioma stem cells required better
understanding of mechanisms of immune function and regulation to optimize treatment
procedure and conditions. For another, practice of immunotherapies, including glioma
stem cell-targeted cancer vaccination, for glioma patients calls for much broader and
deeper translational research to transform our knowledge gained from basic research
into medical progress benefiting human health. Therefore, it is imperative to further
both preclinical and clinical research on glioma immunotherapies.
ACKNOWLEDGEMENTS
Research work in the authors laboratory was supported in part by NIH grants R01
NS048959 and NS048879 to J.S.Y.
REFERENCES
1. Yuan X, Curtin J, Xiong Y et al. Isolation of cancer stem cells from adult glioblastoma multiforme. Oncogene
2004; 23(58):9392-9400.
2. Singh SK, Hawkins C, Clarke ID et al. Identification of human brain tumour initiating cells. Nature 2004;
432(7015):396-401.
3. Bruggeman SW, Hulsman D, Tanger E et al. Bmi1 controls tumor development in an Ink4a/Arf-independent
manner in a mouse model for glioma. Cancer Cell 2007; 12(4):328-341.
4. OBrien CA, Pollett A, Gallinger S et al. A human colon cancer cell capable of initiating tumour growth in
immunodeficient mice. Nature 2007; 445(7123):106-110.
5. Ricci-Vitiani L, Lombardi DG, Pilozzi E et al. Identification and expansion of human colon-cancer-initiating
cells. Nature 2007; 445(7123):111-115.
6. Liao MJ, Zhang CC, Zhou B et al. Enrichment of a population of mammary gland cells that form mammospheres
and have in vivo repopulating activity. Cancer Res 2007; 67(17):8131-8138.
7. Karnoub AE, Dash AB, Vo AP et al. Mesenchymal stem cells within tumour stroma promote breast cancer
metastasis. Nature 2007; 449(7162):557-563.
8. Bao S, Wu Q, McLendon RE et al. Glioma stem cells promote radioresistance by preferential activation of
the DNA damage response. Nature 2006; 444(7120):756-760.
9. Lee EJ, Russell T, Hurley L et al. Pituitary transcription factor-1 induces transient differentiation of adult
hepatic stem cells into prolactin-producing cells in vivo. Mol Endocrinol 2005; 19(4):964-971.
10. Furnari FB, Huang HJ, Cavenee WK. Genetics and malignant progression of human brain tumours. Cancer
Surv 1995; 25:233-275.
11. Wright MH, Calcagno AM, Salcido CD et al. Brca1 breast tumors contain distinct CD44
/CD24 and
CD133
cells with cancer stem cell characteristics. Breast Cancer Res 2008; 10(1):R10.
12. Folkins C, Man S, Xu P et al. Anticancer therapies combining antiangiogenic and tumor cell cytotoxic effects
reduce the tumor stem-like cell fraction in glioma xenograft tumors. Cancer Res 2007; 67(8):3560-3564.
13. Wicha MS. Cancer stem cell heterogeneity in hereditary breast cancer. Breast Cancer Res 2008; 10(2):105.
14. Shmelkov SV, Butler JM, Hooper AT et al. CD133 expression is not restricted to stem cells and both
CD133
and CD133 metastatic colon cancer cells initiate tumors. J Clin Invest 2008; 118(6):2111-2120.
15. Piccirillo SG, Reynolds BA, Zanetti N et al. Bone morphogenetic proteins inhibit the tumorigenic potential
of human brain tumour-initiating cells. Nature 2006; 444(7120):761-765.
16. Phillips HS, Kharbanda S, Chen R et al. Molecular subclasses of high-grade glioma predict prognosis, delineate
a pattern of disease progression and resemble stages in neurogenesis. Cancer Cell 2006; 9(3):157-173.
17. Dahmane N, Sanchez P, Gitton Y et al. The Sonic Hedgehog-Gli pathway regulates dorsal brain growth
and tumorigenesis. Development 2001; 128(24):5201-5212.
18. Katayam M, Yoshida K, Ishimori H et al. Patched and smoothened mRNA expression in human astrocytic
tumors inversely correlates with histological malignancy. J Neurooncol 2002; 59(2):107-115.
EGFR kinase inhibitors. N Engl J Med 2005; 353(19):2012-2024.
axis. Cancer Cell 2002; 1(3):269-277.
21. Fan QW, Knight ZA, Goldenberg DD et al. A dual PI3 kinase/mTOR inhibitor reveals emergent efficacy
in glioma. Cancer Cell 2006; 9(5):341-349.
22. Jackson EL, Garcia-Verdugo JM, Gil-Perotin S et al. PDGFR alpha-positive B cells are neural stem cells
in the adult SVZ that form glioma-like growths in response to increased PDGF signaling. Neuron 2006;
51(2):187-199.
23. Ligon KL, Huillard E, Mehta S et al. Olig2-regulated lineage-restricted pathway controls replication
competence in neural stem cells and malignant glioma. Neuron 2007; 53(4):503-517.
24. Clement V, Sanchez P, de Tribolet N et al. HEDGEHOG-GLI1 signaling regulates human glioma growth,
cancer stem cell self-renewal and tumorigenicity. Curr Biol 2007; 17(2):165-172.
25. Choe G, Horvath S, Cloughesy TF et al. Analysis of the phosphatidylinositol 3-kinase signaling pathway
in glioblastoma patients in vivo. Cancer Res 2003; 63(11):2742-2746.
26. Backman SA, Ghazarian D, So K et al. Early onset of neoplasia in the prostate and skin of mice with
tissue-specific deletion of Pten. Proc Natl Acad Sci USA 2004; 101(6):1725-1730.
27. Cully M, Elia A, Ong SH et al. grb2 heterozygosity rescues embryonic lethality but not tumorigenesis in
pten
/ mice. Proc Natl Acad Sci USA 2004; 101(43):15358-15363.
28. Okumura K, Zhao M, Depinho RA et al. Cellular transformation by the MSP58 oncogene is inhibited by its
physical interaction with the PTEN tumor suppressor. Proc Natl Acad Sci USA 2005; 102(8):2703-2706.
29. Dirks PB. Glioma migration: clues from the biology of neural progenitor cells and embryonic CNS cell
migration. J Neurooncol 2001; 53(2):203-212.
30. Aboody KS, Brown A, Rainov NG et al. Neural stem cells display extensive tropism for pathology in
adult brain: evidence from intracranial gliomas. Proc Natl Acad Sci USA 2000; 97(23):12846-12851.
31. Benedetti S, Pirola B, Pollo B et al. Gene therapy of experimental brain tumors using neural progenitor
cells. Nat Med 2000; 6(4):447-450.
32. Ehtesham M, Kabos P, Gutierrez MA et al. Induction of glioblastoma apoptosis using neural stem cell-mediated
delivery of tumor necrosis factor-related apoptosis-inducing ligand. Cancer Res 2002; 62(24):7170-7174.
33. Ehtesham M, Kabos P, Kabosova A et al. The use of interleukin 12-secreting neural stem cells for the
treatment of intracranial glioma. Cancer Res 2002; 62(20):5657-5663.
34. Yuan X, Hu J, Belladonna ML et al. Interleukin-23-expressing bone marrow-derived neural stem-like cells
exhibit antitumor activity against intracranial glioma. Cancer Res 2006; 66(5):2630-2638.
35. Hu J, Yuan X, Belladonna ML et al. Induction of potent antitumor immunity by intratumoral injection of
interleukin 23-transduced dendritic cells. Cancer Res 2006; 66(17):8887-8896.
36. Luptrawan A, Liu G, Yu JS. Dendritic cell immunotherapy for malignant gliomas. Rev Recent Clin Trials
2008; 3(1):10-21.
37. Yamanaka R. Cell- and peptide-based immunotherapeutic approaches for glioma. Trends Mol Med 2008;
14(5):228-235.
38. Ehtesham M, Yuan X, Kabos P et al. Glioma tropic neural stem cells consist of astrocytic precursors and
their migratory capacity is mediated by CXCR4. Neoplasia 2004; 6(3):287-293.
39. Liau LM, Black KL, Martin NA et al. Treatment of a patient by vaccination with autologous dendritic cells
pulsed with allogeneic major histocompatibility complex class I-matched tumor peptides. Case Report.
Neurosurg Focus 2000; 9(6):e8.
cells elicits systemic cytotoxicity and intracranial T-cell infiltration. Cancer Res 2001; 61(3):842-847.
cytotoxic T-cells in patients with malignant glioma. Cancer Res 2004; 64(14):4973-4979.
42. Lee J, Kotliarova S, Kotliarov Y et al. Tumor stem cells derived from glioblastomas cultured in bFGF and
EGF more closely mirror the phenotype and genotype of primary tumors than do serum-cultured cell
lines. Cancer Cell 2006; 9(5):391-403.
43. Xu Q, Yuan X, Liu G et al. Hedgehog signaling regulates brain tumor initiating cell proliferation and portends
shorter survival for patients with PTEN-coexpressing glioblastomas. Stem Cells 2008; 26(12):3018-26.
44. Spisek R, Kukreja A, Chen LC et al. Frequent and specific immunity to the embryonal stem cell-associated
antigen SOX2 in patients with monoclonal gammopathy. J Exp Med 2007; 204(4):831-840.
45. Garcia-Hernandez Mde L, Gray A, Hubby B et al. Prostate stem cell antigen vaccination induces a long-term
protective immune response against prostate cancer in the absence of autoimmunity. Cancer Res 2008;
68(3):861-869.
46. Pellegatta S, Poliani PL, Corno D et al. Neurospheres enriched in cancer stem-like cells are highly effective
in eliciting a dendritic cell-mediated immune response against malignant gliomas. Cancer Res 2006;
66(21):10247-10252.
47. Manzo G. Cancer genesis: stem tumour cells as an MHC-null/HSP70very high primordial self escaping
both MHC-restricted and MHC-non-restricted immunesurveillance. Med Hypotheses 2001; 56(6):724-730.
48. Odeberg J, Piao JH, Samuelsson EB et al. Low immunogenicity of in vitro-expanded human neural cells
despite high MHC expression. J Neuroimmunol 2005; 161(1-2):1-11.
49. Liu G, Akasaki Y, Khong HT et al. Cytotoxic T-cell targeting of TRP-2 sensitizes human malignant glioma
to chemotherapy. Oncogene 2005; 24(33):5226-5234.
50. Heisel SM, Ketter R, Keller A et al. Increased seroreactivity to glioma-expressed antigen 2 in brain tumor
patients under radiation. PLoS ONE 2008; 3(5):e2164.
51. Okada H, Lieberman FS, Walter KA et al. Autologous glioma cell vaccine admixed with interleukin-4 gene
transfected fibroblasts in the treatment of patients with malignant gliomas. J Transl Med 2007; 5:67.
52. Xu Q, Liu G, Yuan X et al. Antigen-specific T cell response from dendritic cell vaccination using cancer
stem-like cell-associated antigens. Stem Cells 2009; 27(8):1734.
INDEX
Symbols Antigen 42-48, 50, 53-56, 59, 61, 64,

65, 68, 69, 77-82, 86, 87, 90, 96, 98,
1p/19q 8 102, 104, 110, 118, 119, 121, 122,
9L Fischer model 144 124, 126, 129-133, 147, 151-158,
1018 ISS 99, 101 162, 166-168, 172, 173, 180, 181,
180, 183 188, 189, 191-197, 202-211, 219-223
179-183 Antigen
presentation 44, 45, 54, 55, 59, 61,
A 90, 180, 188, 192, 196, 220, 221
Active immunotherapy 178, 204, 205, presenting cell (APC) 43-45, 53,
220 54, 56, 58, 66, 86, 87, 122, 129,
Adjuvant 13, 20, 26-29, 32, 36, 81, 130, 147, 152, 155, 166-169, 173,
89-91, 96, 114, 147, 154, 162, 167, 188-191, 196, 203, 207
168, 171, 174, 191, 194, 196
43, 44, 61, 68, 77, 79, 168,
Adoptive cell therapy 202, 203, 206, 189, 191, 192, 197, 202-204, 208,
207, 210, 211 211, 220-222
Adoptive therapy 203, 207 AP-12009 90
Alkylating agent 7, 8, 26, 29 Avastin see Bevacizumab 182, 183
Alzheimers disease 45, 67, 123
Anaplastic astrocytoma (AA) 5, 6, B
19, 62, 88, 90, 117, 126-128, 172, B7-1 44, 45, 181, 184
193-195 B7-H1 47, 49, 50, 60, 61
Animal model 54, 56, 59, 62, 68, 80, 91, |
|$
96, 98, 105, 124, 143, 144, 146-148, 182, 183
151, 155, 162, 221, 222 B cell 7, 43, 44, 61, 63, 64, 86-88, 90,
Antibody 26, 27, 30, 32, 33, 43, 44, 96, 98, 99, 104, 152, 173, 189
55-57, 59-68, 77, 79, 80, 82, 89, Bevacizumab (Avastin) 29, 31, 33, 34,
90, 96, 98, 101, 104, 105, 121-129, 36, 46, 101, 104, 125
131-134, 147, 151, 152, 159, 161, Bipolar stimulation 13, 16
179, 181, 188, 210, 220 |
| 121,
131-133
227
Blood brain barrier (BBB) 27, 29, 44, Chimeric antigen receptor (CAR) 121,
45, 53, 54, 82, 86, 99, 104, 121-123, 131, 133, 208-211
134, 166, 190, 202, 203, 205 Clinical trial 2, 27, 30-34, 36, 69, 82,
Boron neutron capture therapy (BNCT) 88, 90-92, 95, 96, 99-105, 109,
129 112-115, 121, 123-127, 129, 130,
Brain tumor 8, 14, 18, 19, 42, 54, 55, 132, 133, 144, 147, 155, 162, 167,
58, 61-68, 82, 90, 98, 99, 104, 111, 169, 172, 173, 178-180, 187, 188,
113, 118, 122, 123, 134, 143-146, 191, 193, 194, 197, 205, 208, 209,
151, 152, 163, 182, 184, 187, 211, 217, 220, 222
188, 202, 203, 205, 211, 216-218, Cluster differentiation (CD) 96, 118,
220-222 144
stem/initiating cell (BTSC) 8, 217, CNS bioavailability 122
218 Colony stimulating factor-1 (CSF-1) 46,
49, 65
C Cortical stimulation 13-16, 121, 124
CpG 43, 95-105, 196
C6 Wistar model 144 CpG-28 99, 100, 103
Cancer CpG 7909 99-102
immunosurveillance 204, 211 CpG-ODN 95-99, 103-105
initiating cell 216, 217 Craniotomy 13-18
progression 216, 221 Cross-talk 86, 87
stem cell (CSC) 46, 216, 217, Cyclin 4, 5
220-223 Cyclin-dependent kinase (CDK) 4
vaccine 143, 147, 148, 167, 192, 204, Cyclooxygenase (COX) 53, 58
205 Cytokine 43-46, 56-61, 64, 66, 68,
Carcinomatous meningitis 103 86-93, 96, 98, 101, 104, 110, 118,
Carmustine 7, 28-30, 128, 173 125, 128-130, 133, 151, 155, 157,
CD4+ T cell 54, 55, 58, 59, 65, 67, 153, 158, 162, 166, 174, 181, 188, 190,
168, 181, 189, 191, 192, 204, 205, 191, 193, 205-207, 209, 218-221
208, 219 therapy for glioma 86, 87
CD25 47, 65-68, 130, 151, 161, 188 Cytotoxic T-lymphocyte associated
CD133 8, 82, 217 protein 4 (CTLA-4) 66, 130
cDNA 79, 80, 92, 156, 161, 162, 192, Cytotoxic T lymphocyte (CTL) 43, 44,
197 54-58, 60, 66, 68, 77-82, 87, 88, 91,
Cell therapy 77, 109-119, 187, 202, 206, 109-111, 113-115, 118, 130, 132,
207, 209-211 133, 147, 152, 154, 157, 161, 162,
Central nervous system (CNS) 2, 8, 12, 166-174, 180, 181, 189, 191, 193,
29, 33, 42, 45, 49, 54-56, 63, 64, 68, 194, 197, 204, 206, 209, 210, 219,
79, 86, 98, 122, 123, 162, 187, 188, 221, 222
190, 191, 202, 203, 211
Chemoimmunotherapy 147
Chemotherapy 2, 8, 20, 26-29, 32, 33,
D
36, 42, 82, 90, 91, 99, 100, 103, 104, Decoy receptor 3 (DcR3) 62, 63
109, 113, 114, 118, 119, 121, 122,
17
125, 127, 147, 148, 152, 153, 163, Delayed-type hypersensitivity reaction
166, 170, 174, 187, 193, 195, 197, (DTH) 91, 147, 167, 193-195
206, 216, 217, 220, 222 Delivery strategy 119
INDEX 229
Dendritic cell (DC) 43, 47, 54, 58, 60, Glioma 2-7, 12-21, 27-36, 42, 46-49,
63-66, 69, 87, 88, 90, 96, 98, 102, 53-69, 77-82, 86-93, 95, 96, 98,
104, 110, 113, 118, 119, 147, 155, 99, 104, 105, 109-119, 121-123,
162, 166, 168, 171-173, 187-197, 125-130, 132-134, 143-148, 152,
203-205, 216, 217, 219-223 154, 157, 158, 162, 166-170,
Double-stranded RNA-dependent protein 172-174, 178-180, 182, 184, 187,
kinase 179 188, 192-197, 202, 205, 206, 211,
216-223
E stem cell 196, 216, 217, 220, 222,
223
Electrocorticography 16 Good manufacturing practice (GMP)
ELISPOT 147, 159, 160, 195, 196 174, 196
Epidermal growth factor (EGF) 3, 30, Granulocyte macrophage
31, 46, 59, 81, 104, 125, 172 colony-stimulating factor (GM-CSF)
receptor (EFGR) 3, 30, 46, 81, 104, 60, 90, 91, 155, 159, 162, 173, 182,
125, 172 204, 220
receptor variant III (EGFRvIII) 7, 30,
81, 82, 126, 128, 129, 132, 146,
167, 172, 173, 206
H
Epilepsy 13, 17 Helper T cell, see T helper cell
Erlotinib 30, 31, 34, 36, 101 Heme oxygenase (HO) 66-68
Extent of resection 12, 13, 16, 19-21 Herpes simplex virus (HSV) 133,
178-184
F High-grade glioma 12, 13, 19-21, 29,
36, 56, 110, 114, 126-128, 130, 146,
Forkhead box P3 (FoxP3) 47, 57, 60, 197, 218
66-68, 130, 151, 161 Human leukocyte antigen (HLA) 47, 48,
Functional outcome 16 54-56, 59, 61, 65, 78-81, 147, 153,
157, 166-170, 172-174, 206
G
^ 178, 180, 182, 183 I
G207 179-181, 184 Immunoliposome 121, 129
Gene therapy 91-93, 129, 181, 209 Immunosuppression 42, 46-50, 53, 54,
Genetic alteration 145, 180 56-59, 62, 86, 87, 90, 129, 130, 173,
Genetic engineering 207-209 209
GL26 model 144, 147, 157, 158, 219, Immunotherapy 42, 45, 46, 48, 50,
221 67, 68, 77-82, 88, 96, 99, 109, 110,
Gliadel 29, 32, 36, 89 114, 119, 121-123, 126, 129-131,
Glioblastoma 2, 19, 26, 27, 30, 31, 33-36, 133, 143, 144, 147, 148, 154, 156,
42, 45-50, 54-57, 59, 60, 62, 63, 66, 161-163, 166, 167, 170, 172-174,
88, 99, 100, 103, 104, 111, 114, 117, 178, 187, 188, 191-193, 195-197,
125, 146, 169-173, 178-180, 193, 202-206, 211, 216, 217, 220-223
194, 197, 217, 218, 220 IMO 2055 99-101
multiforme (GBM) 2-8, 26-34, 36, Infected-cell protein 6 (ICP6) 179, 182,
54, 63, 64, 66, 67, 88-91, 99, 103, 183
104, 111-114, 117, 124-128, 133, In situ tumor vaccination 178, 184
145, 146, 193-197, 217, 218,
220-222
Intercellular adhesion molecule (ICAM) Leukocyte inhibitory receptor (LIR) 61

189, 190 Lomustine 7, 28, 34
Interferon (IFN) 57, 60, 61, 68, 88, 91, Low-grade glioma 13, 19-21, 63
92, 96, 147, 157-160, 162, 181, 190, Lymphocyte 43-48, 53, 56-59, 61,
195, 219, 221 64, 66, 77, 81, 86-88, 90, 91, 96,
\ 60, 162 109, 110, 112-114, 130, 133, 152,
\_ 57, 60, 61, 68, 147, 157-160, 153, 161, 162, 166-169, 172, 180,
195, 219, 221 188-190, 194, 202, 204, 206-211,
Interleukin (IL) 46, 47, 49, 53, 56-61, 219, 221
64-66, 68, 80, 87-91, 96, 110-115, Lymphokine-activated killer cell (LAK)
128, 132, 133, 155, 157-159, 59, 88, 109-115, 118, 151, 158, 159,
162, 166, 173, 178, 181-184, 188, 161
189, 194, 195, 203, 205-207, 211,
218-220 M
IL-2 47, 57-59, 61, 65, 68, 87, 88,
91, 110-115, 155, 157-159, 162, Major histocompatibility complex
189, 207 (MHC) 44, 45, 54-56, 60, 61, 64,
IL-4 58, 80, 88, 89, 128, 162, 182, 66, 67, 110, 113, 118, 132, 133,
184, 220 144, 151-157, 162, 173, 180, 181,
IL-6 46, 49, 56, 59, 60, 65, 220 188-191, 197, 208, 221, 222
IL-10 47, 49, 53, 56-58, 60, 61, 66, Malignant glioma 27-30, 32-36,
96, 166, 173, 182 46-48, 56-58, 61, 67, 81, 89-93,
IL-12 58, 61, 96, 162, 181-184, 194, 95, 109-115, 117, 119, 121-123,
195, 219 125-127, 129, 132-134, 143-148,
IL-13 89, 128, 133, 205, 211 152, 154, 157, 162, 166, 167, 172,
Invasion 7, 57, 63, 98 174, 179, 180, 182, 187, 195, 197,
Irinotecan 29, 33, 36, 101, 125 205, 216, 220, 223
Isocitrate dehydrogenase 1/2 (IDH1/2) Methylguanine-methyltransferase
5, 6 (MGMT) 7, 8, 28, 35, 36, 91
MicroRNA 2, 6, 7
Mitogen-activated protein kinase
K (MAPK) 3, 30-33, 35, 90
Keyhole limpet hemocyanin (KLH) 172, Monitoring of the animal tumor 146
173, 193, 194 Monitoring of the immune function 147
Killer cell immunoglobulin-like receptor Monoclonal antibody (mAb) 26, 27,
(KIR) 61 32, 33, 59, 60, 66, 90, 101, 104,
Kinase inhibitor 26, 27, 30, 31, 33 121-134, 158, 206, 208
therapy 121-127, 129-132
L Monocyte 42, 47-50, 55, 56, 58, 60, 61,
88, 90, 96, 152, 188, 190, 220
Language Motor pathway 12, 13
mapping 12-18, 21 Myeloid-derived suppressor cell (MDSC)
site 12, 14-18 42, 46, 48-50, 64, 65, 68
variability 12, 14, 15
Lectin-like transcript 1 (LLT1) 61, 62
INDEX 231
N Protein kinase B (AKT) 3, 7, 30-33, 35,

146, 218
Natural killer cell (NK) 43, 55, 57, 58, Protein kinase R (PKR) 179
60-64, 68, 86-88, 91, 96, 98, 104,
109-113, 116-118, 124, 126, 151,
158, 159, 161, 173, 181, 190, 197, R
219 Radiation 2, 3, 20, 21, 26-30, 34, 35,
NBI-3001 89 42, 91, 104, 118, 121, 122, 125-127,
Negative mapping 12, 14 152, 163, 172, 173, 187, 216, 217,
Neural stem cell (NSC) 7, 82, 216-219, 220
222 Radiolabeled 124
Neuroanesthesia 15, 21 Radiotherapy 8, 26, 28, 29, 100, 101,
\ 103, 104, 113, 125, 126, 166, 222
(NSAID) 58 Recombinant virus 182
Recurrent glioblastoma 31, 33-36, 88,
O 99, 100, 103, 125, 169, 170, 172,
178-180
O6-methylguanine-methyltransferase 7, Regeneration and tolerance factor (RTF)
28 62
Oncolytic virus 178, 184 Regulatory T cell (Treg) 42, 47-50, 53,
58, 60, 63-68, 98, 105, 110, 148,
P 151, 173, 188, 191, 205, 207
p16Ink4a 3, 6 Retrovirus 209
Passive immunotherapy 202, 204, 206,
211, 216 S
Peptide 34, 46, 54, 66, 77-82, 90, Signal transducer and activator
102, 118, 123, 130, 133, 152-156, of transcription (STAT) 50, 53, 59,
166-174, 180, 188, 189, 191-194, 65, 89, 90
196, 197, 204, 205, 219, 220 Single-chain Fv (scFv) 121, 128, 131,
Personalized vaccine 172 132
Phosphatase and tensin homolog (PTEN) siRNA 55, 62, 207, 209
3, 6, 7, 30, 35, 46, 50, 145, 146, 217, SOX6 79-81
218 Speech arrest 14, 16
Phosphoinositide 3-kinase (PI3K) 3, 7, Spontaneous tumor model 145
30-33, 35, 217, 218 Stereotactic administration 92, 125, 133,
Phytohemagglutinin (PHA) 59 179, 180
Plaque-forming unit (PFU) 179 Suppressors of cytokine signaling
Plasmacytoid DC 190, 203 (SOCS) 59
Plasticity 14, 193 Survival 3, 4, 13, 19-21, 26-30, 32-36,
Platelet-derived growth factor (PDGF) 42, 59, 62, 63, 65-67, 69, 87-91, 98,
3, 30, 31, 59, 146 99, 103, 111, 112, 119, 125-127,
Procarbazine 7, 27, 29 129, 130, 156, 157, 161, 162, 166,
Programmed death (PD) 60, 112-114, 169, 170, 172, 173, 179, 181, 182,
117, 169, 171, 172, 174, 203 184, 193-195, 197, 205, 207, 209,
ligand-1 (PDL-1) 60 216-219, 221-223
Prostaglandin E2 (PGE2) 46, 49, 50, 58,
59, 98, 203
T U
T cell 42, 44, 45, 47-50, 53-69, 77-82, Unarmed 121, 122, 124, 125, 126, 131,
87, 88, 90, 91, 96, 98, 100, 104, 183, 184
105, 110, 111, 118, 119, 121, US11 180
126, 130-133, 147, 148, 151-153,
158-161, 167-169, 171, 173, 181, V
182, 184, 188-193, 195, 197,
202-211, 219-222 Vaccine 46, 69, 77, 80-82, 90, 91,
receptor (TCR) 45, 66, 78, 119, 133, 95, 98, 102, 105, 109, 111, 114,
152, 188, 189, 207-210 115, 118, 119, 123, 130, 143, 144,
Temozolomide (TMZ) 2, 3, 7, 26-30, 147, 148, 151, 153-157, 159-163,
32, 34, 35, 36, 91, 100, 103, 133, 166-174, 187, 191-193, 195-197,
147, 173, 193 204, 205, 220, 221
T helper cell (Th) 44, 55, 81, 87, 89, 96, enrichment 151, 159, 162, 163
101, 110, 111, 152, 168 Vascular endothelial growth factor
T-helper type 1 (Th1-type) 192 (VEGF) 27, 29, 31-34, 46, 82, 122,
Toll-like receptor (TLR) 43, 96, 191, 125, 188
194, 195 Viral vector 145, 146
Toxin conjugated antibody 127 Volumetric analysis 20
Transforming growth factor-` (TGF`)
7, 47, 50, 53, 54, 57-62, 66, 68, 89, W
90, 98, 122, 130, 166, 188, 193
Transplantable tumor model 143-145 World Health Organization (WHO) 2, 5,
TRL9 agonist 105 17, 19-21, 55, 62, 63, 125
Tumor WT1 46, 81, 167-171, 205-207
antigen 44, 46, 68, 77-81, 96, 98,
104, 119, 124, 147, 151, 153-158,
162, 173, 181, 188, 191, 192,
195-197, 204, 205, 211, 220, 221
associated antigen (TAA) 110,
154-157, 162, 166-168, 202, 207,
208, 210, 220-222
growth 5, 27, 30, 31, 36, 57-59, 98,
104, 114, 125, 129, 174, 180, 184,
217
immunology 36, 143, 144, 152, 166,
187, 188, 191, 202

68, 87,
88, 110, 111, 114, 115, 118, 194,
204, 206, 207
lysate 191, 193, 195, 196, 204, 205,
220, 222
necrosis factor (TNF) 34, 58, 60, 63,
87, 96, 126, 190, 219, 220
protein 53 (TP53) 3-7

113
vaccine 109, 114, 115, 143, 144, 153,
162

(Advances in Experimental Medicine and Biology 746) Tomotoshi Marumoto, Hideyuki Saya (Auth.), Ryuya Yamanaka MD, PHD (Eds.) - Glioma - Immunotherapeutic Approaches-Springer-Verlag New York (2012)

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Glioma

ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY

Recent Volumes in this Series

Ryuya Yamanaka, MD, PhD

Springer Science+Business Media, LLC

Copyright 2012 Landes Bioscience and Springer Science+Business Media, LLC

All rights reserved.

Printed in the USA.

Please address all inquiries to the publishers:

Glioma: Immunotherapeutic Approaches, edited by Ryuya Yamanaka. Landes Bioscience / Springer

Library of Congress Cataloging-in-Publication Data

Glioma : immunotherapeutic approaches / edited by Ryuya Yamanaka.

Treatment of glioma is currently one of the most challenging problems in oncology,

The opening part is focused on recent progress in the neurooncology of gliomas. In

Ryuya Yamanaka, MD, PhD

RYUYA YAMANAKA is a Professor at Kyoto Prefectural University of Medicine,

Mitchel S. Berger Roberta P. Glick

Shuichi Izumoto Atsushi Natsume

Duane A. Mitchell Sadhak Sengupta

Hiroshi Shiku Renata Ursu

PART I. BASIC AND CLINICAL

1. MOLECULAR BIOLOGY OF GLIOMA..............................................................2

2. RECENT SURGICAL MANAGEMENT OF GLIOMAS ..................................12

3. RECENT MEDICAL MANAGEMENT OF GLIOBLASTOMA ......................26

PART II. GLIOMA IMMUNOLOGY

5. MECHANISMS OF IMMUNE EVASION BY GLIOMAS ................................53

6. GLIOMA ANTIGEN ..............................................................................................77

Glioma Antigens ......................................................................................................................... 79

PART III. CYTOKINE, SEROTHERAPY, ADOPTIVE

7. CYTOKINE THERAPY ........................................................................................86

9. ADOPTIVE CELL TRANSFER THERAPY

10. MONOCLONAL ANTIBODY THERAPY

PART IV. ACTIVE IMMUNOTHERAPY

11. ANIMAL MODELS FOR VACCINE THERAPY ...........................................143

12. IMMUNOGENE THERAPY .............................................................................151

13. PEPTIDE VACCINE ..........................................................................................166

14. ACTIVE IMMUNOTHERAPY:

15. DENDRITIC CELL VACCINES .......................................................................187

PART V. NOVEL TOPICS

16. ANTIGEN-RECEPTOR GENE-MODIFIED

17. GLIOMA STEM CELL RESEARCH

BASIC AND CLINICAL ASPECTS OF GLIOMA

MOLECULAR BIOLOGY OF GLIOMA

Tomotoshi Marumoto1 and Hideyuki Saya*,2

Glioma: Immunotherapeutic Approaches, edited by Ryuya Yamanaka.

PRIMARY AND SECONDARY GBMs

ACTIVATED GROWTH FACTOR SIGNALING PATHWAYS

Various growth factors, such as platelet-derived growth factor (PDGF)-A, PDGF-B,

DYSREGULATION OF CELL CYCLE CHECKPOINTS

Figure 2. RB and TP53 pathways controlling G1-S transition. Phosphorylation of RB by CDK4,6/Cyclin

retinoblastoma (RB) gene is mutated in 10-25% of high-grade astrocytomas. In addition,

ISOCITRATE DEHYDROGENASE 1 (IDH1) AND IDH2 MUTATIONS

Recently, a somatic mutation at amino acid 132 of the isocitrate dehydrogenase 1

ABERRANT FUNCTIONS OF MicroRNAS IN GBM

DRUG RESISTANCE IN GBM

As described above, there is accumulating information regarding the molecular

RECENT SURGICAL MANAGEMENT

Nader Sanai* and Mitchel S. Berger

Glioma: Immunotherapeutic Approaches, edited by Ryuya Yamanaka.

THE EVOLUTION OF CORTICAL MAPPING STRATEGIES

VARIABILITY IN CORTICAL LANGUAGE LOCALIZATION

Prediction of cortical language sites through classic anatomical criteria is inadequate

AVOIDANCE OF FUNCTIONAL LANGUAGE DEFICITS FOLLOWING