Review Article

Immunotherapy for the Treatment of Urothelial Carcinoma

Nicholas M. Donin, Andrew T. Lenis, Stuart Holden, Alexandra Drakaki,
Allan Pantuck, Arie Belldegrun and Karim Chamie*
From the Department of Urology, Institute of Urologic Oncology (NMD, SH, AD, AP, AB, KC) and Department of Medicine,
Division of Hematology and Oncology (AD), David Geffen School of Medicine at UCLA and Jonsson Comprehensive Cancer
Center (AD, AP, AB, KC), University of California, Los Angeles, California

Abbreviations
and Acronyms
AE adverse event
APC antigen presenting cell
BCG bacillus Calmette-Gu
erin
CAR chimeric antigen receptor
CIS carcinoma in situ
CTA cancer testis antigen
CTLA-4 cytotoxic T-lymphocyte
antigen 4
FDA U.S. Food and Drug
Administration
HLA human leukocyte antigen
IFN interferon
IHC immunohistochemistry
MCNA Mycobacterium phlei
cell wall nucleic acid complex
MHC major histocompatibility
complex
NK natural killer
NMIBC nonmuscle invasive
bladder cancer
NSCLC nonsmall cell lung
cancer
RB retinoblastoma
RCC renal cell carcinoma
TCR T-cell receptor
TLR Toll-like receptor
TUR transurethral resection
UC urothelial cancer
Purpose: We review the biological mechanisms of action, clinical safety and ef-
ficacy of immunotherapies for urothelial carcinoma. We also describe current
areas of research in immunotherapy, and highlight ongoing trials and promising
and novel investigational agents.
Materials and Methods: Data were obtained by a search of PubMed, Clin-
icalTrials.gov and Cochrane databases for English language articles published
through February 2016. Applicable abstracts from recent Society of Urologic
Oncology, European Association of Urology, American Urological Association
and ASCO meetings were used.
Results: Bacillus Calmette-Gu
erin is one of the most successful immunother-
apies in cancer treatment and remains the gold standard of care for patients with
high risk, nonmuscle invasive bladder cancer, with initial response rates of
approximately 70%. However, with the exception of valrubicin and standard
chemotherapeutics there is a paucity of available treatment options for patients
with recurrence or progression to more advanced disease. Recently there has
been significant interest in novel immunotherapeutic agents in the management
of cases where bacillus Calmette-Gu
erin fails, as well as cases of more advanced
cancer. These investigational therapies can generally be classified into several
broad categories, including recombinant bacillus Calmette-Gu
erin and cell wall
derived therapies, cytokines, gene therapy, cancer vaccines, immune checkpoint
inhibitors, oncolytic viruses, adoptive immunotherapies and immune agonists, as
well as several additional immunomodulatory agents. The majority of these
agents are currently under investigation in phase I or II clinical trials. Recently
investigators reported evidence that inhibition of the PD-1/PD-L1 pathway has
clinical activity in patients with advanced bladder cancer. These findings, along
with successful phase III trials and U.S. Food and Drug Administration ap-
provals of other checkpoint inhibitors in melanoma, nonsmall cell lung cancer
and renal cell carcinoma, ultimately led to Food and Drug Administration
approval of atezolizumab for advanced disease, the first new treatment approved
for advanced urothelial carcinoma in 20 years.
Accepted for publication February 20, 2016.
No direct or indirect commercial incentive associated with publishing this article.
The corresponding author certifies that, when applicable, a statement(s) has been included in the manuscript documenting institutional
review board, ethics committee or ethical review board study approval; principles of Helsinki Declaration were followed in lieu of formal ethics
committee approval; institutional animal care and use committee approval; all human subjects provided written informed consent with gua-
rantees of confidentiality; IRB approved protocol number; animal approved project number.
* Correspondence: Department of Urology, David Geffen School of Medicine, University of California, Los Angeles, 300 Stein Plaza, 3rd Floor,
Los Angeles, CA 90095. e-mail: kchamie@mednet.ucla.edu

0022-5347/17/1971-0014/0 http://dx.doi.org/10.1016/j.juro.2016.02.3005

14 j www.jurology.com
THE JOURNAL OF UROLOGY
2017 by AMERICAN UROLOGICAL ASSOCIATION EDUCATION AND RESEARCH, INC.
Vol. 197, 14-22, January 2017
Printed in U.S.A.
 IMMUNOTHERAPY FOR UROTHELIAL CANCER 15

Conclusions: While bacillus Calmette-Gu
erin has demonstrated significant clinical efficacy in the treatment
of patients with bladder cancer, additional therapies are needed for those in whom bacillus Calmette-Gu
erin
fails, as well as for those with advanced disease. Immunotherapy for urothelial carcinoma remains a prom-
ising and active area of research, and numerous agents, particularly the monoclonal antibodies targeting
checkpoint inhibition pathways, are showing encouraging signs of clinical activity.

Key Words: BCG vaccine, cancer vaccines, cell cycle checkpoints, immunotherapy, urologic neoplasms

THE use of bacillus Calmette-Gu
erin in the treat- IMMUNOBIOLOGY

ment of urothelial carcinoma remains one of the
most successful stories in the history of cancer
immunotherapy, and currently represents one of
the most active and exciting areas of ongoing uro-
logical research. As early as 1891, the interaction
between the immune system and neoplasia was
recognized and exploited as a potential target for
cancer therapeutics.1 However, not until the semi-
nal article of Morales et al in 1976 revealing the
efficacy of bacillus Calmette-Gu
erin was immuno-
therapy proved effective for bladder cancer.2 Some
40 years later bacillus Calmette-Gu
erin remains the
standard of care for patients with high risk non-
muscle invasive bladder cancer, and serves as proof
of principle that immunotherapy for urothelial
carcinoma can be effective.
Immunotherapy represents a technically feasible
treatment strategy for bladder cancer for multiple
reasons. Bladder tumors possess one of the highest
rates of mutation of all human tumors, and are thus
likely to be highly antigenic.3 Other advantages
include the ability to achieve direct contact between
therapeutic agents and the tumor via intravesical
administration, the ability to deliver high concen-
trations of agents with theoretically limited systemic
exposure and the ability to monitor for treatment
response via cystoscopic surveillance, biopsy, and
noninvasive monitoring of cells and urinary bio-
markers. Nonetheless, few effective therapeutic
agents exist for the treatment of UC. A large pro-
portion of agents ultimately fail to achieve cure,
including BCG and platinum based systemic thera-
pies. Given the significant burden of morbidity and
mortality associated with the disease, alternative
therapies are needed. As such, multiple immuno-
therapeutic agents are being investigated, several of
which have demonstrated preliminary promise.
In this analysis we discuss basic cancer immu-
nology as it pertains to the mechanism of action of
currently approved and investigational agents. We
review the discovery of BCG and the data support-
ing its use, and we discuss cytokines, adoptive cell
therapies, checkpoint inhibition and additional
agents currently being investigated in patients with
UC. Ongoing clinical trials and completed seminal
trials are summarized in the supplementary table
and supplementary Appendix (http://jurology.com/).
The current understanding of antitumor cellular
immunity outlines several key events that occur
during immune destruction of tumor cells.4 First,
tumor associated antigen must be sampled, pro-
cessed and presented by APCs in the context of
MHC molecules. These cells must receive a suffi-
cient secondary activation signal (ie B7-1 or B7-2) to
achieve an immunogenic phenotype. Once acti-
vated, APCs travel to lymphoid tissue, where they
interact with T-cells. If these APCs are sufficiently
immunogenic, they can induce the production of
CD8 cytotoxic and NK effector cells. Finally, these
effector cells travel to the tumor to infiltrate the
microenvironment and carry out an antitumor
response.
These processes are subject to complex modula-
tion, and if sufficient negative regulation occurs, an
antitumor effect will not be achieved. Tumors
themselves evade this process through various
mechanisms working at multiple points along the
pathway. Secretion of paracrine mediators such as
adenosine, prostaglandin E2, TGF-b and VEGF-A
suppress activation of APCs and prevent T-cell
infiltration of the tumor bed. Cell surface ligands on
tumor cells, such as PD-L1 and PD-L2, may be up-
regulated, interacting with tumor infiltrating lym-
phocytes and suppressing their cytotoxic activity.
Tumors may down-regulate the normal antigen
processing and presentation on MHC-I molecules,
thus avoiding TCR and MHC interaction and sub-
sequent cytotoxicity. Finally, tumors are known to
recruit populations of immunomodulatory cells,
such as regulatory T-cells, tumor associated mac-
rophages and myeloid derived suppressor cells,
which can blunt the antitumor response. The pres-
ence of these immunosuppressive cell types within
tumor tissues has been shown to correlate with poor
prognosis and tumor stage.5


BACILLUS CALMETTE-GUERIN
BCG is a unique strain of mycobacterium bovis
developed by Albert Calmette and Camille Gu
erin
at the Pasteur Institute in the early 1920s as an
antituberculosis vaccine.6 Concurrent with its
development was a growing appreciation of the
connection between the immune system and
16 IMMUNOTHERAPY FOR UROTHELIAL CANCER
malignancy, and research gradually began on the
use of BCG as a cancer therapy. Early investigators
found that inoculation of mice with BCG exerted a
protective effect against subsequently implanted
tumors.7 Clinical findings in leukemia, melanoma,
and cancers of the head and neck had already been
described when in 1975 deKernion et al published
their report of a melanoma lesion metastatic to the
bladder that was successfully eradicated with an
endoscopic intralesional injection of BCG.8 This case
was followed soon after by the seminal report of
Morales et al on use of intravesical BCG in the
treatment of NMIBC.2 The initial findings were
subsequently reproduced in prospective randomized
trials.9 BCG was approved for use intravesically in
1990 and currently remains the standard of care in
patients with high risk NMIBC.
While a complete understanding of the mecha-
nisms underlying the therapeutic effect of BCG re-
mains the subject of investigation, a broad
understanding of the events underlying its activity
has emerged.10 It is clear that BCG works via acti-
vation of the immune system and induction of an
inflammatory response. This effect begins with
attachment of BCG to urothelial cells, followed by
internalization via macropinocytosis. These cells
then up-regulate MHC-II molecules and secrete
cytokines, resulting in recruitment of immune cells,
including lymphocytes, to the tumor environment.
These lymphocytes induce what is believed to be a
predominantly Th1 cytokine milieu, which ulti-
mately leads to immune mediated cytotoxicity via
CD8 lymphocytes, NK cells and granulocytes.
In the original study of Morales et al patients
with NMIBC who had frequent recurrence and pa-
tients with incompletely resected tumors were
treated with 120 mg/50 cc BCG administered
intravesically plus intradermal injections for
6 weeks.2 In the period before BCG treatment the
cohort of 9 patients experienced 22 recurrences
during 77 patient-months. Following BCG treat-
ments these same 9 patients experienced 1 recur-
rence during 41 patient-months. These findings
were followed by a randomized trial of 37 patients in
which those treated with BCG for 6 weeks exhibited
a 22% recurrence rate at 1 year, compared to 42%
recurrence in untreated controls. A Cochrane Group
meta-analysis published in 2000 of 6 randomized
trials including 585 patients revealed that BCG
decreased the odds of recurrence at 1 year by 70%
compared to TUR alone (odds ratio 0.30, CI 0.21 to
0.43).11 BCG has since been compared with multiple
chemotherapeutic agents and has demonstrated
persistent superiority for the prevention of
recurrence.12
BCG is widely accepted as the standard of care
for the treatment of CIS of the bladder. While
reported rates of progression of CIS in the pre-BCG
era were upward of 50%, BCG has been shown in
multiple trials to result in initial complete response
rates of approximately 70%.12,13 Complete response
rates appear to increase between the 3 and 6-month
evaluations and, as such, a proportion of patients
with residual CIS at the 3-month point will go on to
experience a complete response, even without
maintenance therapy.13 The benefit of maintenance
therapy remains questioned by some. While early
smaller studies of varying schedules failed to illus-
trate a benefit of BCG maintenance, the totality of
the evidence, including that from large prospective
trials and meta-analyses,12e14 reveals that the
maximal benefits of BCG require the use of main-
tenance therapy.
Several groups have attempted to modify or
create derivatives of BCG that might prove more
effective with fewer side effects. Recombinant BCG
strains expressing IL-2, IL-12, IL-18, IFN-a and
IFN-g have been developed and tested in preclinical
models in the hope of enhancing the response.
However, no clinical trials have evaluated these
agents in humans. A preparation of MCNA, which
has immunostimulatory and directly cytotoxic ac-
tivity, was evaluated in a single arm trial of 129
patients with BCG refractory or relapsing
NMIBC.15 With a 1-year disease-free survival rate
of 25% the study failed to meet its primary end
point. In addition, metastatic disease developed in
11% of patients during the trial, prompting concerns
regarding safety.
CYTOKINE AND GENE THERAPY
Interferons are a class of cytokines produced by
immune cells and tumor cells in response to the
presence of pathogens, and cause a myriad of
immunomodulatory effects. Initial studies evalu-
ated IFN-a2B as a potential intravesical mono-
therapy for NMIBC. While a small but detectable
antitumor effect has been demonstrated when given
in higher doses (80 million units or greater),16 IFN-
a2B monotherapy has consistently been observed to
be inferior to mitomycin C and BCG.17,18
IFN-a2B has also been investigated in combina-
tion with BCG, given that the 2 agents are
biocompatible and may act synergistically. A large
prospective study of combination BCG plus IFN-
a2B in 670 BCG nave patients with NMIBC failed
to find an improvement in disease-free rates
compared to BCG alone.19 Therefore, combination
therapy is not recommended for use in BCG nave
patients. IFN-a2B has also been evaluated for use in
the setting of BCG failures, and in this context
several smaller series have shown disease-free rates
of approximately 50% at 1 year.20,21 The largest
 IMMUNOTHERAPY FOR UROTHELIAL CANCER
study of combination therapy included 1,007 in-
dividuals, including BCG nave patients (53%) and
BCG failures (46%).22 In this study a recurrence-
free survival rate of 45% was noted in the BCG
failure group at a median of 24 months. As such,
BCG plus IFN-a2B is a viable alternative for pa-
tients in whom BCG monotherapy has failed.
One explanation for the limited efficacy of intra-
vesical IFN-a2B may be its transient presence
within the bladder, and thus prolonged delivery of
the cytokine within the urothelium may improve its
efficacy. An intravesical gene therapy using a re-
combinant IFN replication deficient adenovirus
system has been developed and tested in early
phase clinical trials. In a phase I multicenter study
17 patients with NMIBC in whom at least 2 cycles of
BCG had failed were treated with the investiga-
tional agent, which includes Syn3, an excipient that
enhances transduction of the urothelium.23 Pro-
longed and increased levels of IFN-a were detected
in urine. Of 14 patients treated at higher doses 6
(43%) were complete responders at 3 months and 2
(14%) were disease-free at 39 months. A phase II
study of 40 patients is ongoing (NCT01687244).
ONCOLYTIC VIRUSES
Oncolytic viruses work by selective replication
within tumor cells, causing tumor cell lysis and
release of additional oncolytic virions as well as
tumor antigens. One such agent being evaluated is
CG0070, a serotype 5 oncolytic adenovirus that
selectively replicates in and destroys RB pathway
deficient cells, and carries the gene for the immu-
nomodulatory cytokine GM-CSF. This virus is a
logical agent in patients with UC, given the sub-
stantial proportion of tumors that harbor RB gene
mutations or inactivation.24 In a phase I study 35
subjects underwent intravesical administration
using varying doses and treatment schedules.25 A
49% complete response at a median of 10.4 months
was noted, and responses were greater than 80% in
the subset of patients with borderline or high RB
status and 77% in those receiving 6 weekly doses.
An open-label phase II/III study is ongoing
(NCT01438112).
VACCINE THERAPY
Therapeutic cancer vaccines eliminate tumors by
activation of acquired cellular immunity and come
in various forms, including synthetic peptides, re-
combinant proteins and viral vectors. PANVAC-VF
is a cancer vaccine using recombinant vaccinia and
fowlpox viruses engineered to contain genes for
human CEA and MUC-1 (both known to be over-
expressed in urothelial carcinoma), along with the
17
costimulatory molecules B7.1, ICAM-1 and LFA-3.26
The virus is given as a series of subcutaneous in-
jections and is subsequently processed by APCs,
with the goal of eliciting a T-cell antigen specific
response. PANVAC-VF was studied in advanced
pancreatic cancer and failed to display a survival
advantage. It is currently being investigated in a
phase II study in patients with NMIBC in whom
BCG has failed (NCT02015104).
HS-410 is a cellular vaccine based on a human
cancer cell line that has been transfected to express
HLA-A1 and secrete gp96-Ig, an endoplasmic retic-
ulum chaperone protein known to act as an immune
stimulant, particularly to CD8 T-cells. Hypotheses
regarding the potential mechanism of action of HS-
410 include activation of dendritic cells, NK cells
and cytotoxic lymphocytes, and stimulation of anti-
gen cross-presentation via CD19 and Toll-like re-
ceptors. A phase I/II study is currently recruiting,
and will include patients who have undergone TUR
and are candidates for BCG (NCT02010203). The
FDA has granted fast track designation for HS-410.
Cancer testis antigens are a class of proteins
whose expression in normal tissues is limited to the
testis but that are aberrantly expressed in various
tumor types. As a result of this limited expression in
normal tissues, CTAs are an attractive potential
target for cancer vaccines. UC has high levels of
expression of various CTAs,27 and early phase vac-
cine trials targeting CTAs are ongoing for UC. A
dendritic cell based vaccine, in which patient
autologous dendritic cells pulsed with the CTA
MAGE-A3 were injected into patients with meta-
static bladder cancer, exhibited some efficacy in a
small pilot study.28 A phase I trial of recombinant
MAGE-A3 plus BCG given to patients following
TUR has completed accrual and the results are
pending (NCT01498172). In a phase II trial evalu-
ating the efficacy of the recombinant MAGE-A3
vaccine after cystectomy the tumor tissue of pa-
tients will be tested for MAGE-A3 expression and, if
present, a course of 13 injections will be adminis-
tered spanning 27 months (NCT01435356).
NY-ESO-1 is another CTA that has garnered in-
terest based on its high degree of immunogenicity
and high frequency of expression in several cancer
types, including UC. In a phase I study 6 patients
who underwent cystectomy for NY-ESO-1 tumors
were vaccinated postoperatively with recombinant
NY-ESO-1, GM-CSF and BCG.29 CD4 T-cell re-
sponses were found in all 6 patients, NY-ESO-1
specific antibody responses were induced in 5 and
CD8 T-cell responses were seen in 1.
HER2 is a cell surface receptor tyrosine kinase
best known for its association with aggressive forms
of breast cancer but is overexpressed or amplified in
some urothelial tumors. Overexpression appears to
18 IMMUNOTHERAPY FOR UROTHELIAL CANCER

be associated with aggressive tumors and poor clin-
ical outcomes.30 DN24-02 is an autologous cellular
immunotherapy that uses the same antigen engi-
neering technology as sipuleucel-T and targets
HER2. Patients undergo leukapheresis in which im-
mune cells are harvested and activated ex vivo with a
recombinant fusion protein that has components of
the extracellular and intracellular domains of HER2
linked to GM-CSF. NeuACT (NCT01353222) is an
open-label, randomized, phase II trial comparing
adjuvant DN24-02 with surveillance in patients with
HER2 UC at high risk for relapse following cys-
tectomy or nephroureterectomy.
CHECKPOINT INHIBITION
Immune checkpoint inhibition is one of the most
exciting and promising areas of research in cancer
therapeutics. Positive phase III clinical trials in
advanced melanoma, nonsmall cell lung cancer and
RCC have led to FDA approval of 3 checkpoint in-
hibitors. A large number of clinical studies evalu-
ating these agents in various malignancies are now
ongoing, including multiple trials in UC. Check-
point inhibition involves targeting T-cell regulatory
pathways to reduce inhibitory signaling and pro-
mote T-cell activation and enhanced antitumor ac-
tivity. It is now recognized that in addition to TCR
activation, costimulatory and inhibitory molecules
on the surface of T-cells exist that further regulate
T-cell behavior. The discovery that blockade of
inhibitory signaling pathways resulted in tumor
rejection in in vivo models ultimately led to an up-
surge in investigation and discovery in this
area.31,32
The CTLA-4 receptor is similar in structure to
the CD28 costimulatory receptor on the surface of
T-cells and is known to be a critical element in the
T-cell activation process (fig. 1). CTLA-4 expression
on T-cells is induced with T-cell activation and has
been observed to compete with CD28 for interaction
with the costimulatory B7-1 and B7-2 molecules on
APCs. However, rather than promote T-cell activa-
tion and effector functions, the B7:CTLA-4 interac-
tion inhibits T-cell activation primarily in lymphoid
tissues. Ipilimumab, a monoclonal anti-CTLA-4
antibody, blocks the B7:CTLA-4 interaction, thus
shifting T-cell equilibrium toward activation and
effector function, with subsequent antitumor ef-
fects. Successful trials of ipilimumab in metastatic
melanoma led to its FDA approval in 2011.33 Ipili-
mumab was evaluated in a small study of 12 pa-
tients with bladder cancer to investigate its
biological effects on bladder cancer tissue.34 In this
study patients scheduled for cystectomy for cT1-
T2N0M0 disease were given 2 doses of the drug
beginning approximately 7 weeks before cys-
tectomy. The majority of AEs were grade 1 or 2,
although 2 patients had a delay in surgery due to
immune related AEs. A phase II ongoing clinical
trial evaluating ipilimumab in combination with
gemcitabine and cisplatin in patients with advanced
disease was presented at the ASCO 2016 Genito-
urinary Cancers Symposium and failed to meet its
primary end point (NCT01524991).
The programmed cell death 1 receptor (PD-1) and
its ligands, PD-L1 and PD-L2, are an exciting set of
receptor ligands that are vital in T-cell immuno-
modulation and tolerance (fig. 2). Like CTLA-4, the
PD-1 receptor becomes expressed on activated

T-CELL APC INTERACTION

CTLA-4 ANTIBODY BLOCKS CTLA-4 SIGNALING

CTLA-4
Lymph nodes
B7-1, Activated
B7-2 T-cell

28
CD

Bladder
carcinoma
Antigen TCR
Presenting MHC
Cell IL-2 production
Tumor T-cell proliferation
antigen
Cytotoxic function
CTLA-4 expression

Figure 1. T-celleAPC interaction. Medical Illustration by JustinKleinCMI.com 2016.

 IMMUNOTHERAPY FOR UROTHELIAL CANCER 19

T-CELL TUMOR CELL INTERACTION

PD-L1 INHIBITORS
AND PD-1 INHIBITORS
INHIBIT THE DOWN REGULATION
OF CYTOTOXIC FUNCTION

Bladder
carcinoma
PD-1 T-cell
cytotoxic
PD-L1,
function
PD-L2

Tumor
TCR
cell
Tumor antigen
MHC
Tumor cells

Oncogenic activation, induction

from immune activity in
tumor microenvironment

Figure 2. T-celletumor cell interaction. Medical Illustration by JustinKleinCMI.com 2016.

T-cells, and PD-1/ligand interaction results in inhi-
bition of TCR mediated functions and suppression of
the T-cell effector function. While CTLA-4 activation
is generally thought to down-regulate T-cell activa-
tion in lymphoid tissues, PD-1 activity is believed to
act primarily in the tumor microenvironment,
where it restrains T-cell mediated tumor destruc-
tion. The observation of up-regulation of PD-L1 on
tumor cells indicated activation of the PD-1 pathway
as a mechanism of immune evasion.35 Immunohis-
tochemical studies have revealed that increased PD-
L1 expression is associated with increasing bladder
tumor stage and grade, suggesting that blockade
may be efficacious.36 This hypothesis was ultimately
borne out in successful phase III trials in melanoma,
squamous NSCLC and RCC.
In what is arguably the most exciting develop-
ment in bladder cancer therapeutics in decades, in
May 2016 the FDA approved atezolizumab
(MPDL3280A), a monoclonal IgG1 antibody that
binds PD-L1, for the treatment of advanced stage
bladder cancer in patients who experienced disease
progression on platinum based therapy.31,37 This
approval was based on data from seminal phase I
and phase II studies in which pathological speci-
mens from patients with advanced bladder cancer
were graded for the presence of PD-L1 before
treatment. In the phase I study 67 patients with
metastatic bladder cancer had the pathological
specimen graded for the presence of PD-L1 before
treatment. An objective response was seen in 17
patients overall (25%), with 15 partial responses
and 2 complete responses. Objective responses
occurred in 13 of 30 patients with lymphocyte IHC 2
to 3 tumors (43%), of which 2 were complete
responses, and in 4 of 35 patients with lymphocyte
IHC 0 to 1 tumors (11%). Interestingly responses to
atezolizumab were associated with the tumor-
infiltrating immune cell IHC scores but not with
tumor cell IHC scores. There were no grade 4 or 5
treatment related AEs, and only 4% of patients
experienced a grade 3 event (NCT01375842).32
More recently investigators reported results from
IMvigor 210, a phase II study of atezolizumab in
patients with metastatic or locally advanced UC
who experienced disease progression during or after
platinum based therapy.37 In 310 evaluable patients
the overall response rate was 15%, with 38 of 45
(84%) of responses ongoing at the time of data cut-
off. Response rate correlated with IHC status (ORR
26% in IHC in 2 to 3 tumors, 18% in IHC 1 to 3
tumors). Grade 3 or 4 treatment related AEs
occurred in 5 of 310 patients (16%).
Another emerging PD-1 inhibiting antibody,
pembrolizumab, is demonstrating antitumor activ-
ity in early phase clinical trials. In data presented at
ASCO 2015 a total of 33 patients with advanced UC
were treated with pembrolizumab every 2 weeks
until complete response, progression or unaccept-
able toxicity. A complete response was noted in 3 of
28 patients (11%) and a partial response in 4 (14%)
at a median of 13 months. Grade 3 to 4 drug related
AEs occurred in 15% of patients (NCT01848834).38
Combination therapy with PD-1/PD-L1 and
CTLA-4 inhibition is being investigated in a number
of malignancies, including UC (NCT01928394,
NCT02496208). Several other T-cell surface re-
ceptors that regulate cell activation and efficacy
have been identified as potential therapeutic tar-
gets, such as B7-H3 and OX40, and antibodies
20 IMMUNOTHERAPY FOR UROTHELIAL CANCER
modulating their activity are also being evaluated
in early phase clinical trials (NCT01391143,
NCT02221960, NCT02219724).
ADOPTIVE IMMUNOTHERAPY
Adoptive immunotherapy involves administering
immune cells with direct anticancer activity to a
cancer bearing host. Lymphocytes are first obtained
from the patient via biopsy of the primary or met-
astatic tumor site, or via leukapheresis. These cells
are cultured in vitro to expand the lymphocyte
population and ultimately transferred back to the
patient, typically following a nonmyeloablative
conditioning regimen. Studies of melanoma and
lymphoma have shown complete and durable re-
sponses.39 In UC adoptive immunotherapy remains
in the early stages of clinical investigation. In a
feasibility trial involving 12 patients with metasta-
tic bladder cancer lymphocytes were extracted from
metastatic lymph nodes, and tumor specific T helper
cells were expanded in vitro and subsequently
infused into patients. The process was safe and no
major AEs were noted. However, the process was
completed in only 50% of the patients due to tech-
nical limitations.40
Adoptive immunotherapy is somewhat limited by
the need to obtain tumor tissue for the harvest of
tumor-infiltrating lymphocytes. TCR T-cell therapy
is an emerging technology that circumvents this
problem by taking peripherally obtained lympho-
cytes and transfecting them with a gene for a re-
combinant TCR specifically engineered to bind a
particular CTA-MHC complex. Advantages include
access to peripheral lymphocytes, as well as the
ability to engineer a TCR that is optimized for
maximal affinity for the CTA-MHC complex. Early
reports show success with this therapy in melanoma
and synovial sarcoma, including 1 study evaluating
a TCR engineered to recognize NY-ESO-1.41 Given
that a proportion of urothelial tumors are known to
express NY-ESO-1, TCR T-cell therapy is being
investigated for use in bladder cancer in early phase
trials (NCT02457650). Some limitations of TCR
T-cell therapy are the known propensity of tumors
to down-regulate MHC and, thus, evade TCR
mediated autoimmunity, as well as the specificity of
a given TCR to only certain HLA alleles.
CAR T-cell therapy represents a similar emerging
adoptive T-cell technology that potentially circum-
vents some of the aforementioned limitations of TCR
therapy. CARs are engineered fusion complexes
consisting of a single-chain variable region of an
antibody domain that binds a specific CTA, a trans-
membrane domain, a costimulatory domain and the
CD3-zeta domain, which serves as the intracellular
signaling domain. T-cells expressing this chimeric
receptor can bind directly to the cell surface to become
activated, as opposed to the engineered TCRs, which
must bind specific allelic variants of MHC molecules.
Thus, CAR therapies are not limited to a particular
HLA subtype and circumvent the problem of MHC
down-regulation. However, they are limited in
epitope repertoire to cell surface proteins and cannot
bind intracytoplasmic derived peptides.
In CAR therapy T-cells from a patient are ob-
tained via leukapheresis, activated, transfected
with a gene coding for the fusion complex, expanded
to the desired dosage and infused back into the pa-
tient. CAR therapy has displayed notable efficacy in
patients with leukemia and lymphoma. The anti-
CD19 CAR T-cell agent CTL019 was granted
breakthrough therapy designation in July 2014.
Clinical results in UC are not yet available.
OTHER EMERGING THERAPIES
ALT-801 is a unique molecule in which an IL-2
molecule has been fused to a single-chain TCR
that binds a peptide derived from p53 when dis-
played in the context of HLA-A*0201. The purpose
of this fusion protein is to direct IL-2 activation of
T-cells to cells with aberrant p53. In a phase I trial
of 26 patients with p53 metastatic malignancies
increases in IFN-g were detected in the serum after
treatment, although in contrast to treatment with
IL-2, no TNF-a was detected.42 Of the patients 10
had stable disease at 11 weeks. Given that a
significant proportion of UCs have mutated p53,
ALT-801 is currently being investigated for UC
in the muscle invasive and metastatic settings
in combination with gemcitabine and cisplatin
(NCT01326871), as well as in the setting of BCG
failures for NMIBC (NCT01625260).
Another promising agent under investigation is a
fusion protein consisting of an IL-15 super agonist
bound to a soluble domain of the IL-15 receptor pro-
tein. This fusion complex, known as ALT-803, has
displayed 25 times the in vivo biological activity of
native IL-15. Because IL-15 is a critical factor in the
development, proliferation and activation of effector
NK and CD8T cells, it is speculated to be a prom-
ising agent. In a carcinogen induced rat NMIBC
model tumor burden was reduced by 35% and 15%
with ALT-803 and BCG, respectively.43 When the
agents were given in combination, tumor burden was
decreased by 46%. This agent is being evaluated in
clinical trials in several tumor types, including mel-
anoma, lymphoma and NSCLC, and in combination
with BCG for NMIBC (NCT02138734).
Toll-like receptors are transmembrane proteins
that function in innate and adaptive immunity.
TLRs are expressed in normal urothelium and in
NMIBC cells. Imiquimod, a small molecule of the
 IMMUNOTHERAPY FOR UROTHELIAL CANCER 21

imidazoquinoline family, is known to activate TLR7
and TLR8, a pathway that is also believed to be
activated by BCG, and has exhibited antitumor ef-
fect in basal cell carcinoma and penile CIS when
used topically. TMX-101 is a formulation of imiqui-
mod optimized for intravesical administration, and
has been evaluated in several early phase studies of
low grade disease44 and CIS (unpublished data),
totaling 33 patients. While the majority of patients
reported AEs, virtually all were grade 1 or 2, and all
patients received all 6 planned instillations.
CONCLUSIONS
Urothelial carcinoma remains a common malignancy
with few treatment advances in the last 20 years.
However, the 1990 approval of BCG for NMIBC
represents one of the first immuno-oncologic ap-
provals in solid tumors and shows that UC is an
immunosensitive tumor amenable to immune medi-
ated treatment strategies. Progressive understand-
ing of the complex regulatory mechanisms that
govern cellular immunity has led to the development
of a number of novel agents and strategies, most
notably checkpoint inhibitors, which have resulted in
overall survival benefits in patients with advanced
melanoma, NSCLC and RCC. The recent FDA
approval of atezolizumab heralds a new era in the
treatment of UC, and this agent will likely assume a
central role in the management of many cases of
advanced stage disease. There is ample reason to
believe a number of novel immunotherapeutics
currently under investigation will soon emerge as
important treatment options for patients with UC.

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