Professional Documents
Culture Documents
1078 0432.CCR 08 0157
1078 0432.CCR 08 0157
Abstract Purpose: Effective control of pancreatic cancer has been hampered primarily by the lack of tumor
specificity of current treatment modalities. The highly specific antibody-mediated delivery of
therapeutic agents to the tumor microenvironment might overcome this problem. We therefore
investigated the therapeutic efficacy of the targeted immunocytokine L19-Interleukin-2 (L19-IL2),
consisting of the human single-chain Fv antibody L19, which is highly specific for the extradomain
B (ED-B) of fibronectin, and the human cytokine IL-2, in pancreatic cancer.
Experimental Design: Therapeutic effects of L19-IL-2, IL-2, and gemcitabine on tumor growth
and metastasis were evaluated in orthotopic mouse models for pancreatic cancer. Immunohisto-
chemistry was done to define ED-B expression, tumor necrosis, apoptosis, proliferation, and
invasion of macrophages and natural killer (NK) cells. NK cells were depleted by i.v. injection of
an anti-asialo-GM-1antibody.
Results: ED-B is selectively expressed in human pancreatic cancer and in primary tumors and
metastases of the mouse models. L19-IL-2 therapy was clearly superior to untargeted IL-2 or
gemcitabine and inhibited tumor growth and metastasis with remarkable long-term tumor control.
Therapeutic effects were associated with the induction of extensive tumor necrosis and inhibition
of tumor cell proliferation. Immunohistochemistry revealed an increase of macrophages and NK
cells in the tumor tissue, suggesting immune-mediated mechanisms. The functional relevance of
NK cells for the therapeutic effect of the targeted immunocytokine L19-IL-2 was confirmed by NK
cell depletion, which completely abolished its antitumor efficacy.
Conclusions: These preclinical results strongly encourage the initiation of clinical studies using
L19-IL-2 in pancreatic cancer.
Adenocarcinoma of the pancreas represents the fifth leading chemotherapeutic, and radiation treatments have little effect on
cause of cancer related death in industrialized western countries the aggressive course of disease (4 – 7). One promising avenue
(1). The prognosis of patients diagnosed with pancreatic cancer to overcome these obstacles is the highly specific antibody-
is extremely poor with an estimated overall 5-year survival rate mediated delivery of therapeutic agents to the tumor microen-
of 1% to 4%. Surgical resection provides the only potentially vironment. In particular, vascular and/or stromal targeting
curative treatment, but locally extended or metastasized disease represents an appealing therapeutic strategy for several reasons.
precludes surgical treatment in most cases (2, 3). Due to poor First, targets, which are selectively expressed around tumor
selectivity, toxicities against nonmalignant cells, and dose vessels and/or in the tumor stroma, are easily accessible to
limitations, currently available strategies including palliative, systemically administered antibodies. Second, markers of
tumor stroma and tumor neovasculature are typically produced
by endothelial cells and/or myofibroblasts, which are geneti-
cally more stable than tumor cells. Third, as neoangiogenesis is
Authors’ Affiliations: 1Department of Hepatology and Gastroenterology, Charite¤,
CampusVirchow-Klinikum, Humboldt-University; and 2Anti-Angiogenesis Research, a prerequisite of tumor growth and metastasis, the selective
Corporate Research Oncology, Bayer-Schering Pharma AG, Berlin, Germany targeting approach of L19-Interleukin-2 (IL-2) to newly
Received 1/20/08; revised 3/6/08; accepted 3/10/08. forming tumor blood vessels should result in a therapeutical
Grant support: Deutsche Forschungsgemeinschaft grant Scho-848/1-1,1-2 benefit. Currently, one of the most selective oncofetal antigens
(A. Scholz).
The costs of publication of this article were defrayed in part by the payment of page
associated with neoangiogenesis and tumor growth is the
charges. This article must therefore be hereby marked advertisement in accordance extradomain B (ED-B) of fibronectin (6, 8). The fibronectin
with 18 U.S.C. Section 1734 solely to indicate this fact. splice variant ED-B, a small domain of 91 amino acids, which is
Note: K.Wagner and P. Schulz contributed equally to this publication. homologous from mouse to man, is usually absent in both
Current address forA. Menrad: Genzyme Europe Research, 310 Cambridge Science
plasma and tissue-fibronectin, except for some blood vessels of
Park, Cambridge CB4 0WG, United Kingdom.
Dedicated to Prof. Stefan Rosewicz (1960-2004) who contributed significantly to the regenerating endometrium and the ovaries (6, 8). However,
this work. it may become inserted in the fibronectin molecule during
Requests for reprints: Petra Schulz, Depar tment of Hepatology and active tissue remodeling associated with neoangiogenesis,
Gastroenterology, Charite¤, Campus Virchow-Klinikum, Humboldt-University, thereby accumulating around the neovasculature and in the
Augustenburger Platz 1, 13353 Berlin, Germany. Phone: 49-30-450-559-708;
Fax: 49-30-450-559-939; E-mail: Petra.Schulz@ charite.de.
stroma of malignant tumors and other tissues undergoing
F 2008 American Association for Cancer Research. angiogenesis (6, 8). Recently, a human single-chain Fv (scFv)
doi:10.1158/1078-0432.CCR-08-0157 antibody fragment L19 has been generated, which displays
picomolar binding affinity for ED-B, and has been verified to At the end of each therapy, the tumor volume was calculated using
selectively target tumor neovasculature in both experimental the formula: length width depth p / 6. The area of lymph nodes
tumor models (9) and patients with cancer (10), thus paving of the metastatic MiaPaca-A2 tumor model was calculated by
determining the largest diameter and its perpendicular diameter and
the way for a novel therapeutic approach targeting tumor
computing the product of the two measurements.
neovasculature.
Depletion of natural killer cells. Natural killer (NK) cell depletion
IL-2 has been characterized as one of the most potent was done as described previously (19). Briefly, mice received six
antitumor cytokines. However, despite being approved for the injections i.p. with 50 AL anti-asialo-GM-1 antibody (Wako Chemicals)
clinical treatment of metastatic renal cell carcinoma, systemi- every fourth day, starting 3 days before tumor cell injection. The level of
cally applied IL-2 has failed to fulfill earlier hopes. In part, this NK cell depletion was monitored by flow cytometry and cytotoxicity
is due to serious, potentially life-threatening side effects that assay.
limit dose escalation and prevent the application of sufficiently Preparation of spleen mononuclear cells. Spleens were removed
high doses (11, 12). In addition, the fast clearance of under deep general anesthesia and digested with collagenase
systemically administered IL-2 further decreases its effective- (Worthington) for 1 h at 37jC. Subsequently, contents were forced
through a 100-Am cell strainer and washed twice with HBSS.
ness. However, local administration of IL-2 has been more
Mononuclear cells were separated from the cell suspension via Ficoll-
successful and has resulted in the control of malignant
Hypaque (Amersham) density gradient centrifugation.
effusions and remission of established lesions (11, 13 – 15). AlamarBlue cytotoxicity assay. Cytotoxic activity of isolated
In this regard, a targeted accumulation of the cytokine IL-2 at murine spleen mononuclear cells against YAC-1 cells was examined
the tumor microenvironment by conjugating it to the using a 24 h AlamarBlue cytotoxicity assay (BioSource) as detailed
homodimeric scFv L19 appears to be an attractive concept to previously (20).
enhance the therapeutic index of IL-2 and at the same time Flow cytometry. Spleen mononuclear cells (1 106) were incubated
diminish its toxic side effects. This study was therefore designed with 1 Ag phycoerythrin-labeled anti-NK1.1 for 15 min at 4jC. After
to evaluate the therapeutic efficacy of the recombinant targeted washing with PBS, the presence of fluorescence-positive cells was
immunocytokine L19-IL-2 in pancreatic carcinoma. analyzed on a FACSCalibur using CellQuest software (BD).
Immunohistochemical analysis. Paraformaldehyde-fixed, 4-Am-thick
frozen sections of human and murine tissue samples were analyzed
immunohistochemically using the avidin-biotin technique. Slides were
Materials and Methods incubated for 1 h with the following primary antibodies: biotinylated
L19-IL-2 (30 Ag/mL; provided by Bayer-Schering Pharma), monoclonal
Human tissue samples. Nineteen pancreatic carcinoma, 15 chronic mouse anti-human CD31 (Dako) at 1:100 dilution, rat anti-mouse
pancreatitis, and 11 normal pancreatic tissue samples were obtained CD11b (BD Biosciences) at 1:50 dilution, biotinylated mouse anti-
from individuals who underwent surgical resection at the Department mouse CD161b/c/NK1.1 (PK136) antibody (Serotec) at 1:50 dilution,
of Surgery at Charité University Hospital. This study was approved by and biotinylated mouse anti-human pancytokeratin (C11) antibody
the local ethics committee and all patients gave written informed (Santa Cruz Biotechnology) at 1:50 dilution. Proliferating cells were
consent prior to surgery. detected with a monoclonal mouse anti-human Ki-67 antibody
Cell culture. The human pancreatic carcinoma cell lines MiaPaca (Dianova), applying the Animal Research Kit. Apoptotic cells were
(American Type Culture Collection), DanG, and the murine lymphoma identified by terminal deoxynucleotidyl transferase – mediated dUTP
cell line YAC-1 (DSMZ) were cultured as described previously (16, 17). nick end labeling assay using TumorTACS In situ Apoptosis Detection
MiaPaca cells, stably transfected with an angiopoetin-2 DNA construct, Kit (R&D Systems). Both procedures were carried out following the
were maintained as MiaPaca wild-type cells, except for the addition of manufacturer’s directions, except that 3-amino-9-ethylcarbazole was
hygromycin B (400 Ag/mL; Invitrogen). used as the substrate chromogen. Negative controls were included by
Animals. Female NMRI nude mice (age, f10 weeks; weight, 21-25 g) omitting the primary antibody (Animal Research Kit) or the terminal
were purchased from Bomholtgard. Animal care followed institutional deoxynucleotidyl transferase (terminal deoxynucleotidyl transferase –
guidelines and all experiments were approved by local animal research mediated dUTP nick end labeling). In the end, sections were counter-
authorities. stained with hematoxylin and mounted permanently. Quantitative
Tumor implantation and in vivo treatment. Three orthotopic immunohistochemical analysis for the area of vital (nonnecrotic)
xenograft mouse models of pancreatic carcinoma were established, tumor tissue was carried out by computer-aided imaging analysis using
including two nonmetastatic models by injection of wild-type cells of Axiovision 4.2 software (Zeiss). A red color threshold was interactively
the human pancreatic carcinoma cell lines DanG and MiaPaca and one set by the examiner for each antibody to select the red stained
metastatic model by implantation of MiaPaca cells stably transfected immunopositive area. All pixels that met the defined threshold were
with an angiopoetin-2 DNA construct (MiaPaca-A2).3 Orthotopic then transferred into a binary image and analyzed. For the quantifica-
transplantation was carried out as described (18). In brief, a median tion of proliferating, apoptotic, and inflammatory cells, 20 randomly
laparotomy was done under deep general anesthesia and the pancreas selected measurement areas (680 510 Am; magnification, 20) of
was exposed. Aliquots of 1 106 tumor cells were injected into the each tumor specimen were analyzed. For evaluation of ED-B fibronectin
head of the pancreas. The pancreas was replaced and the abdominal expression, 10 areas (1,390 1,000 Am; magnification, 10) were
wall was closed. Therapy was started 7 days (DanG), 40 days (MiaPaca), scanned. The results represent the positive staining as a percentage of
or 60 days (MiaPaca-A2) after tumor cell inoculation when solid measurement area (inflammatory cells and ED-B fibronectin) or
tumors and metastases (MiaPaca-A2) had formed. Groups of 8 to the ratio of positive tumor cells as a percentage of total tumor cells
12 mice were treated with vehicle (0.9% saline) or 1.43 or 4.29 MIU/kg (Ki-67 and terminal deoxynucleotidyl transferase – mediated dUTP nick
IL-2 equivalents as either untargeted IL-2 or L19-IL-2 (provided by end labeling), respectively. For quantitative analysis of necrotic tumor
Philogen) or equimolar amounts of L19 (783 Ag/kg) for 10 days or 250 area, H&E sections were scanned at low power (2.5). Total and
mg/kg gemcitabine (Lilly) once weekly. necrotic tumor areas were marked on the screen, calculated by the
image analysis system, and the percentage of necrotic/total tumor area
was determined.
Extraction of DNA and DNA PCR. Genomic DNA of mouse lymph
3
Established byA. Scholz, unpublished data. nodes was extracted using the QIAamp DNA Mini Kit (Qiagen).
Results
ED-B fibronectin is selectively expressed in human pancreatic
carcinoma. We studied the expression pattern of ED-B
fibronectin in human nontransformed pancreatic tissue,
chronic pancreatitis, and ductal adenocarcinoma (Fig. 1A, a,
c, and e). In parallel, serial sections from the same tissues were
stained with a monoclonal antibody against the endothelial
cell – specific antigen CD31 (Fig. 1A, b, d, and f). All but one
carcinoma sample analyzed (n = 19) revealed a specific
expression of ED-B fibronectin at the abluminal side of tumor
blood vessels and in the tumor stroma (Fig. 1A, e and f). By
contrast, nontransformed pancreatic tissue (n = 11; Fig. 1A,
a and b) and chronic pancreatitis (n = 15; Fig. 1A, c and d)
showed no ED-B fibronectin signal. Morphometric quantifica-
tion of ED-B fibronectin in pancreatic cancer recorded the
presence of ED-B fibronectin in 7.35 F 1.60% of total tissue
area compared with <0.1% in nontransformed pancreatic tissue
and chronic pancreatitis (Fig. 1B). Thus, ED-B fibronectin
provides the specific and selective overexpression required for
an ED-B fibronectin-based targeted therapy in pancreatic
cancer.
Development of an orthotopic mouse model for pancreatic
cancer. To investigate the therapeutic efficacy of an ED-B
fibronectin targeted therapy, we established orthotopic models
for pancreatic cancer by injection of human wild-type DanG or
MiaPaca pancreatic cancer cells or MiaPaca cells with stable
overexpression of angiopoetin-2 (MiaPaca-A2) into the
pancreas of nude mice. Orthotopic transplantation resulted
in extensive local tumor growth with invasion of adjacent
normal pancreatic tissue and neighboring organs. Moreover,
MiaPaca-A2 tumors showed metastatic dissemination into
intra-abdominal lymph nodes and liver. The immunohisto-
chemical analysis of the orthotopically grown pancreatic
tumors consistently revealed a strong cuff-like immunostaining
for ED-B fibronectin around tumor blood vessels and in the
stromal compartment (Fig. 1A, g and h, right). In comparison
with the human situation, the extent of ED-B fibronectin
Fig. 1. Expression of ED-B fibronectin in human pancreatic carcinoma and expression was higher in DanG tumors (22.33 F 0.65%;
orthotopic nude mouse models of pancreatic cancer. A, sections of frozen tissue
were stained with L19-IL-2 and a monoclonal CD31antibody. The antigen-antibody Fig. 1B) but equal in MiaPaca tumors (7.96 F 1.32%; Fig. 1B).
complexes stain red. Representative examples of ED-B fibronectin (a, c, e, g, Thus, the observed tumor growth and immunohistochemical
and h) and CD31 (b, d, and f) immunostaining of human nontransformed pancreas findings compare well with the human situation and validate
(a and b), chronic pancreatitis (c and d), and ductal adenocarcinoma (e and f)
and DanG (g) and MiaPaca (h, right margin) pancreatic cancer xenografts and the animal models as a relevant test system for therapeutic
adjacent residual nontransformed pancreatic tissue (h, left margin). Bar, 100 Am. in vivo studies with the targeted immunocytokine L19-IL2.
P, pancreas; T, tumor. B, morphometric quantification of ED-B fibronectin
expression. *, P < 0.001, versus human pancreatic carcinoma.
Targeted immunocytokine L19-IL-2 exerts significant antitumor
activity against established pancreatic cancer. To determine the
efficacy of L19-IL-2 in pancreatic cancer, mice bearing
A specific 850-bp fragment of the a-satellite region of the human orthotopic DanG tumors of 3.1 F 1.05 mm3 average volume
chromosome 17 was detected by PCR using the following primers were randomized to receive vehicle or 1.43 or 4.29 MIU/kg
5¶-GGGATAATTTCAGCTGACTAACACG-3¶ and 5¶-TTCCGTTTAGT- IL-2 equivalents of either L19-IL-2 or untargeted IL-2 for
TAGGTGCAGTTATC-3¶ as described (21). 10 days. Whereas IL-2 treatment had no effect on tumor growth
Fig. 2. Therapeutic effects of L19-IL-2 on orthotopic pancreatic cancer in nude mice. A to E, nude mice bearing orthotopic DanG pancreatic tumors were randomly
treated. A, mean F SE tumor volume of each treatment group. Note that treatment with L19-IL-2 was significantly superior to treatment with equimolar amounts of IL-2.
*, P < 0.004, versus control; +, P = 0.0003, versus equimolar amounts of IL-2. B, percentage of tumor volume from vehicle-treated controls. Note that the naked L19 antibody
achieved no therapeutic effect, whereas equimolar amounts of L19-IL-2 led to a significant decrease of the primary tumor volume. *, P V 0.004, versus control and
scFv L19. C, serum CA19-9 levels from mice bearing orthotopic DanG tumors and tumor-free animals were determined and CA19-9 level versus tumor volume was plotted for
each individual animal (n = 56, r 2 = 0.54, P < 0.0001). D, CA 19-9 levels were determined at the designated time points. Points, mean of control or treatment group; bars,
SE. *, P < 0.05. E, determination of serum lipase. Mean F SE for each treatment group. F, nude mice bearing orthotopically transplanted MiaPaca pancreatic cancer were
treated as specified. Mean F SE tumor volume of each treatment group. Of note, L19-IL-2 treatment was significantly superior to treatment with equimolar amounts of IL-2.
*, P V 0.0007, versus control; +, P = 0.0003, 1.43 MIU/kg body weight L19-IL-2 versus equimolar amounts of IL-2; b, P = 0.0513, 4.29 MIU/kg body weight L19-IL-2
versus equimolar amounts of IL-2.
in the lower dose and only a minor effect in the higher dose, therapeutic effects of the targeted immunocytokine L19-IL-2.
administration of L19-IL-2 significantly reduced tumor volume It was possible to show a significant correlation between tumor
to 21.4% (1.43 MIU/kg) and 2.7% (4.29 MIU/kg) compared volume and serum concentrations of CA 19-9 (r 2 = 0.54,
with control mice (Fig. 2A). This result clearly shows that P < 0.0001; Fig. 2C). Accordingly and in agreement with the
L19-IL-2 is superior to the untargeted cytokine. As shown in a therapeutic effects on tumor burden described above, CA 19-9
separate experiment, this was not due to an IL-2-independent levels were significantly reduced following L19-IL-2 treatment,
antitumor activity of the scFv L19 antibody fragment, because whereas no changes were visible in the IL-2-treated group (data
administration of 4.29 MIU/kg L19-IL-2 reproducibly led to a not shown). These results prompted us to evaluate the time-
significant reduction of tumor load, whereas equimolar response relationship of L19-IL-2-induced tumor growth
amounts of the naked scFv L19 were therapeutically inactive inhibition. Interestingly, a L19-IL-2-dependent tumor growth
(Fig. 2B). delay in terms of 2-fold lower CA 19-9 levels when compared
CA 19-9 as a response marker of therapy. Orthotopic tumors with controls was observed as early as day 3 after start of
do not allow the continuous evaluation of tumor size by caliper treatment. This difference continued to increase until the end of
measurements. For this reason, we established CA 19-9 as a experiment. Here, a >15-fold increase in serum CA 19-9 levels
valuable serum marker to monitor both tumor progression and was detectable in the control group, whereas CA 19-9 levels
were comparable with baseline levels (Fig. 2D) in animals 4.29 MIU/kg. In addition to the anticipated reduction of
treated with L19-IL-2. primary tumor volume, L19-IL-2 treatment also decreased the
Targeted immunocytokine L19-IL-2 does not elicit an unspecific mean lymph node area to <20% of the values obtained in
inflammatory response. Having proven the therapeutic effect of vehicle-treated controls (P = 0.0120; Fig. 3B, compare a and b).
L19-IL-2, we next ensured that IL-2 activity and subsequent To determine, if changes in lymph node area were due to
inflammatory response is confined to the tumor not provoking infiltrating tumor cells, lymph nodes were immunostained for
an acute pancreatitis in adjacent nontransformed pancreatic the presence of human cytokeratin to detect epithelial cells of
tissue. For this purpose, serum lipase levels were determined as human origin. This immunostaining with a pancytokeratin
a surrogate marker of pancreatitis in mice treated with vehicle, antibody revealed large, polymorph cells with strong immu-
IL-2, or L19-IL-2. However, neither treatment increased serum noreactivity, indicative of tumor cells, in 80% of control mice
lipase concentrations (Fig. 2E). This finding was further (Fig. 3B, c) compared with 20% of L19-IL-2-treated mice. In
corroborated by the histologic analysis of pancreatic tumors contrast, lymph nodes of L19-IL-2-treated mice more frequently
and residual nontransformed pancreatic tissue of animals contained large quantities of small, granulocyte-like cells
treated with L19-IL-2 using conventional H&E staining
and immunostaining with antibodies directed against NK1.1
(NK cells) and CD11b (predominantly macrophages), respec-
tively. Of note, no signs of acute pancreatitis were detectable
and inflammatory cells selectively accumulated in the tumor,
sparing adjacent nontransformed pancreatic tissue (data not
shown). Thus, L19-IL-2 treatment did not cause pancreatitis as
an adverse effect.
Therapeutic effects of L19-IL-2 on pancreatic cancer are not cell
type specific. To investigate whether L19-IL-2-mediated reduc-
tion of tumor growth could be reproduced in other pancreatic
cancer cell lines, mice bearing orthotopic MiaPaca pancreatic
tumors were assigned to treatment with the same conditions as
specified above. Whereas untargeted IL-2 was not therapeutic at
low dosage, equimolar amounts of L19-IL-2 reduced tumor
load by 90%. At the high dosage, untargeted IL-2 achieved a
50% inhibition of tumor growth compared with an 83%
reduction of tumor volume by L19-IL-2 (Fig. 2F).
L19-IL-2 treatment inhibits pancreatic cancer lymph node
metastasis. Having confirmed the antitumor action of
L19-IL-2 on primary pancreatic tumors, we next explored its
potency in metastatic disease using the metastatic MiaPaca-A2
pancreatic tumor. Consistently, all lymph nodes infiltrated
by cancerous cells and all liver metastases showed immunore-
activity for ED-B fibronectin (Fig. 3A, a), whereas noninfiltrated
lymph nodes (Fig. 3A, b) and liver did not.
Based on this observation, mice bearing primary tumors and
metastases were randomly treated with vehicle or L19-IL-2 at
schedules were designed and investigated. (11, 12). High and accessibility of a target molecule within the body. ED-B
concentrations of IL-2 are indeed essential for a therapeutic fibronectin fulfills these requirements because (a) this molecule
effect and have been exemplified by the application of IL-2 in is specifically up-regulated during tumor angiogenesis and
patients with advanced pancreatic cancer via arterial or portal tumor growth (6, 25, 26) and (b) it is accessible for antibody-
venous catheters in combination with polychemotherapy targeted therapies. This has been shown with the ED-B-specific
(23, 24). Unfortunately, all locoregional applications are scFv L19, which was successfully used for tumor imaging (10)
limited to tumors that are accessible from the outside without and the delivery of various effector molecules in animal studies
having the possibility to deliver the cytokine to distant (26 – 29).
metastases. This hurdle can be overcome by a highly selective This is the first study that shows a specific overexpression
compound such as the targeted immunocytokine L19-IL-2. The of ED-B fibronectin in human pancreatic carcinoma, whereas
most important prerequisite for a novel antibody-targeted no ED-B fibronectin was detectable in normal pancreas or
delivery therapy like L19-IL-2 is the tumor-specific expression chronic pancreatitis. Our immunohistochemical data strongly
Fig. 5. Effects of L19-IL-2 on tumor necrosis, apoptosis, and proliferation. DanG tumors from mice that had received either vehicle or 4.29 MIU/kg body weight L19-IL-2
were examined by conventional H&E staining (A, a and b), terminal deoxynucleotidyl transferase ^ mediated dUTP nick end labeling assay (B, a and b), or Ki-67
staining (C, a and b). Right, quantification of necrosis (A ; *, P = 0.0159), apoptosis (B), and proliferation (C ; *, P = 0.0177). Mean F SE percentages of each group. Bar,
500 Am (A) and 50 Am (B and C).
Fig. 6. Identification of the immune effector cells mediating L19-IL-2-induced tumor regression. A and B, DanG tumors from mice that had received either vehicle or
L19-IL-2 as indicated were examined with a CD11b antibody (A) and a NK1.1antibody (B). Left, representative examples of vehicle-treated (A and B, a) and L19-IL-2-treated
mice (A and B, b). Bar, 50 Am. Right, quantification of infiltrating CD11b+ cells (A) and NK cells (B). *, P = 0.0159 (A) and 0.0012 (B). C, nude mice bearing
DanG orthotopic tumors were randomly assigned to the indicated treatment groups. Columns, mean of tumor volumes of each group; bars, SE. *, P < 0.01, versus any
other group.
supported the evaluation of the therapeutic efficacy of the metastases. Here, we report the first study that addresses the
targeted immunocytokine L19-IL-2 in mouse models for this therapeutic efficacy of L19-IL-2 in a tumor grown in its natural
devastating disease. There is increasing evidence that the ectopic microenvironment. In this orthotopic setting, we could clearly
or orthotopic environment has a different effect on tumor cell show that the systemic i.v. administration of clinically relevant
protein expression, tumor growth, invasiveness, angiogenesis, doses of L19-IL-2 efficiently inhibited the growth of established
metastasis, drug delivery, and sensitivity to therapeutic agents primary pancreatic tumors and distant metastases, whereas
(30). For this reason, we developed orthotopic mouse models equivalent concentrations of untargeted IL-2 had no or only
for pancreatic cancer, which accurately recapitulate the tumor minor therapeutic effects. It is noteworthy that the therapeutic
behavior and the clinical course of this deadly disease. effect of L19-IL-2 could be shown in two independent mouse
In both models, we strongly showed an ED-B fibronectin models with different quantitative level of ED-B fibronectin
expression pattern, which is comparable with the human expression (DanG, 22.33%; MiaPaca, 7.96%). This observation
situation in both primary tumors and lymph node and liver was unexpected, yet the density of ED-B fibronectin visualized
by immunohistochemistry must not necessarily correlate tumor vasculature and the tumor cells themselves, which are
with the in vivo accumulation of the targeted immunocytokine per se insensitive to IL-2-treatment (36). Therefore, the
L19-IL-2 in the tumor tissue. It may very well be that, in considerable decrease of Ki-67-positive tumor cells in the
comparison with DanG, the apparently low target density in the tumor of L19-IL-2-treated animals can only be attributed to
MiaPaca tumor model can be compensated by a more cytokines such as IFN-g and tumor necrosis factor-a, which
functional vascular network leading to a higher accumulation are released by the large amounts of tumor-infiltrating NK
of L19-IL-2 at the tumor site. Different levels of L19-IL-2 cells and macrophages. Both IFN-g and tumor necrosis factor-
accumulation could also explain the induction of a complete a are known to directly inhibit the growth of pancreatic
and permanent remission in 40% of the animals, whereas cancer cells (37, 38).
untargeted IL-2 showed tumor regrowth after treatment. If our We could bolster this hypothesis by showing a L19-IL-
hypothesis is true, the stratification of patients who will most 2-triggered 4-fold increase of tumor-infiltrating macrophages
likely benefit from a targeted immunocytokine therapy will be and a >70-fold increase of NK cells. These data suggest that
possible using radioactively labeled L19. Future experiments in macrophages were not the main drivers for the therapeutic
this direction are currently being done. efficacy of L19-IL-2 in our study. In line with this notion, it has
In this context, it is also important to note that, until today, been established previously that depletion of macrophages
the mechanisms underlying the antitumor activity of IL-2 are could only attenuate but not abrogate the antitumor activity of
still not fully understood. In our study, one of the most IL-2, indicating the contribution of additional mechanisms
intriguing features of L19-IL-2-treated tumors was an extensive (39). In our study, we could pinpoint the NK cells as the
tumor necrosis, which was most prominent in the central part driving force, which on depletion completely abolished the
of the tumor. In this region, few leukocytes were detectable. therapeutic efficacy of L19-IL-2.
Therefore, tumor cell death caused by direct cell-to-cell contact Our findings are in perfect agreement with published data,
with NK cells, macrophages, or other immune effector cells is which report on (a) promising antitumor effects in addition to
unlikely. Indeed, recent evidence supports the occurrence of a extravasation and infiltration of tumor tissues by NK cells in
local vascular leak syndrome provoked by lymphokines and vitro and in vivo (40) and (b) NK cells that were identified as the
nitric oxide produced by IL-2-activated immune effector cells primary target cell population for IL-2 in preclinical and clinical
(31, 32). Furthermore, direct cytotoxic effects of IL-2 (12) and settings (41).
activated NK cells (33, 34) on the endothelium were reported. In conclusion, our preclinical data strongly support the
Considering that (a) endothelial cells seem to migrate along initiation of clinical studies using the targeted immunocytokine
extracellular matrix structures containing ED-B fibronectin L19-IL-2 in pancreatic cancer.
(35) and (b) L19-IL-2 is selectively accumulating within the
ED-B fibronectin-rich proangiogenic tumor microenviron- Disclosure of Potential Conflicts of Interest
ment, we postulate that L19-IL-2-triggered direct and/or
indirect mechanisms have a detrimental effect on both the No potential conflicts of interest were disclosed.
References
1. Parker SL, Tong T, Bolden S, et al. Cancer statistics, be responsible for binding to endothelial cells and ini- 20. Nociari MM, Shalev A, Benias P, et al. A novel one-
1997. CA Cancer J Clin 1997;47:5 ^ 27. tiating vascular leak syndrome. Proc Natl Acad Sci step, highly sensitive fluorometric assay to evaluate
2. Rosewicz S, Wiedenmann B. Pancreatic carcinoma. U S A 1999;96:3957 ^ 62. cell-mediated cytotoxicity. J Immunol Methods 1998;
Lancet 1997;349:485 ^ 9. 13. Den OtterW, Dobrowolski Z, Bugajski A, et al. Intra- 213:157 ^ 67.
3. Cohen SJ, Pinover WH, Watson JC, et al. Pancreatic vesical interleukin-2 in T1 papillary bladder carcinoma: 21. Becker M, Nitsche A, Neumann C, et al. Sensitive
cancer. CurrTreat Options Oncol 2000;1:375 ^ 86. regression of marker lesion in 8 of 10 patients. J Urol PCR method for the detection and real-time quantifi-
4. MacKenzie MJ. Molecular therapy in pancreatic ade- 1998;159:1183 ^ 6. cation of human cells in xenotransplantation systems.
nocarcinoma. Lancet Oncol 2004;5:541 ^ 9. 14. Baselmans AH, Koten JW, Battermann JJ, et al. The Br J Cancer 2002;87:1328 ^ 35.
5. Schneider G, Siveke JT, Eckel F, et al. Pancreatic can- mechanism of regression of solid SL2 lymphosarcoma 22. Streicher J, Fabian B, Herkner K, et al. Anticytoker-
cer : basic and clinical aspects. Gastroenterology after local IL-2 therapy. Cancer Immunol Immunother atins are a potential source of false-positive indirect
2005;128:1606 ^ 25. 2002;51:492 ^ 8. immunofluorescence assays for C-ANCA. J Clin Lab
6. Alessi P, Ebbinghaus C, Neri D. Molecular targeting 15. Krastev Z, Koltchakov V, Popov D, et al. A case of Anal 1998;12:54 ^ 9.
of angiogenesis. Biochim Biophys Acta 2004;1654: hepatocellular carcinoma (HCC): treatment with local 23. Abdel-Wahab M, El Shennawy F, Agha S, et al.
39 ^ 49. application of alcohol and interleukin 2 (IL-2). Hepato- Evaluation of cell mediated immunity in advanced
7. Reddy LH. Drug delivery to tumours: recent strate- gastroenterology 2003;50:1647 ^ 9. pancreatic carcinoma before and after treatment with
gies. J Pharm Pharmacol 2005;57:1231 ^ 42. 16. Lode HN, Xiang R, Dreier T, et al. Natural killer cell- interleukin-2 (IL-2). Hepatogastroenterology 1999;46
8. Halin C, Zardi L, Neri D. Antibody-based targeting of mediated eradication of neuroblastoma metastases to Suppl 1:1293 ^ 6.
angiogenesis. News Physiol Sci 2001;16:191 ^ 4. bone marrow by targeted interleukin-2 therapy. Blood 24. Lygidakis NJ,Vlachos L, Raptis S, et al. Consecutive
9. Viti F, Tarli L, Giovannoni L, et al. Increased binding 1998;91:1706 ^ 15. re-explorations for final resection of initially unresect-
affinity and valence of recombinant antibody frag- 17. PlathT, Peters M, Detjen K, et al. Overexpression of able pancreatic head carcinoma. Hepatogastroenter-
ments lead to improved targeting of tumoral angio- pRB in human pancreatic carcinoma cells: function in ology 1999;46:2229 ^ 39.
genesis. Cancer Res 1999;59:347 ^ 52. chemotherapy-induced apoptosis. J Natl Cancer Inst 25. Castellani P,Viale G, DorcarattoA, et al.The fibronec-
10. Santimaria M, Moscatelli G,Viale GL, et al. Immuno- 2002;94:129 ^ 42. tin isoform containing the ED-B oncofetal domain: a
scintigraphic detection of the ED-B domain of fibro- 18. Alves F, Contag S, Missbach M, et al. An orthotopic marker of angiogenesis. Int JCancer1994;59:612 ^ 8.
nectin, a marker of angiogenesis, in patients with model of ductal adenocarcinoma of the pancreas in 26. Halin C, Rondini S, Nilsson F, et al. Enhancement of
cancer. Clin Cancer Res 2003;9:571 ^ 9. severe combined immunodeficient mice representing the antitumor activity of interleukin-12 by targeted de-
11. Bubenik J, Den Otter W, Huland E. Local cytokine all steps of the metastatic cascade. Pancreas 2001; livery to neovasculature. Nat Biotechnol 2002;20:
therapy of cancer: interleukin-2, interferons and relat- 23:227 ^ 35. 264 ^ 9.
ed cytokines. Cancer Immunol Immunother 2000;49: 19. Peron JM, Couderc B, Rochaix P, et al. Treatment of 27. Carnemolla B, Borsi L, Balza E, et al. Enhancement
116 ^ 22. murine hepatocellular carcinoma using genetically of the antitumor properties of interleukin-2 by its tar-
12. Baluna R, Rizo J, Gordon BE, et al. Evidence for a modified cells to express interleukin-12. J Gastro- geted delivery to the tumor blood vessel extracellular
structural motif in toxins and interleukin-2 that may enterol Hepatol 2004;19:388 ^ 96. matrix. Blood 2002;99:1659 ^ 65.
28. Borsi L, Balza E, Carnemolla B, et al. Selective tar- 33. Albertsson PA, Basse PH, Hokland M, et al. NK cells 38. WatanabeT, Fuchimoto S, Matsubara N, et al. Anti-
geted delivery of TNFa to tumor blood vessels. Blood and the tumour microenvironment: implications for proliferative effect on human pancreatic cancer cells of
2003;102:4384 ^ 92. NK-cell function and anti-tumour activity. Trends natural human tumour necrosis factor-h combined
29. Nilsson F, Kosmehl H, Zardi L, et al. Targeted deliv- Immunol 2003;24:603 ^ 9. with natural human interferon-a or interferon-g. J Int
ery of tissue factor to the ED-B domain of fibronectin, 34. Di Carlo E, Meazza R, Basso S, et al. Dissimilar anti- Med Res 1992;20:112 ^ 20.
a marker of angiogenesis, mediates the infarction of tumour reactions induced by tumour cells engineered 39. Masztalerz A, Van Rooijen N, Den Otter W, et al.
solid tumors in mice. Cancer Res 2001;61:711 ^ 6. with the interleukin-2 or interleukin-15 gene in nude Mechanisms of macrophage cytotoxicity in IL-2 and
30. Killion JJ, Radinsky R, Fidler IJ. Orthotopic models mice. J Pathol 2000;191:193 ^ 201. IL-12 mediated tumour regression. Cancer Immunol
are necessary to predict therapy of transplantable 35. Tarli L, Balza E, Viti F, et al. A high-affinity human Immunother 2003;52:235 ^ 42.
tumors in mice. Cancer Metastasis Rev 1998;17: antibody that targets tumoral blood vessels. Blood 40. Basse PH, Whiteside TL, Herberman RB. Use of
279 ^ 84. 1999;94:192 ^ 8. activated natural killer cells for tumor immunother-
31. De Mik HJ, Koten JW, Maas RA, et al. Tumour re- 36. Rosenberg SA. Progress in the development of apy in mouse and human. Methods Mol Biol
gression by IL-2 mediated stagnation of blood flow. immunotherapy for the treatment of patients with 2000;121:81 ^ 94.
In Vivo 1991;5:679 ^ 84. cancer. J Intern Med 2001;250:462 ^ 75. 41. Janssen RA, Sleijfer DT, Heijn AA, et al. Peripheral
32. Sakkoula E, Pipili-Synetos E, Maragoudakis ME. 37. Detjen KM, Farwig K, Welzel M, et al. Interferon g blood lymphocyte number and phenotype prior to
Involvement of nitric oxide in the inhibition of an- inhibits growth of human pancreatic carcinoma cells therapy correlate with response in subcutaneously ap-
giogenesis by interleukin-2. Br J Pharmacol 1997; via caspase-1 dependent induction of apoptosis. Gut plied rIL-2 therapy of renal cell carcinoma. Br J Cancer
122:793 ^ 5. 2001;49:251 ^ 62. 1992;66:1177 ^ 9.
Updated version Access the most recent version of this article at:
http://clincancerres.aacrjournals.org/content/14/15/4951
Cited Articles This article cites by 41 articles, 12 of which you can access for free at:
http://clincancerres.aacrjournals.org/content/14/15/4951.full.html#ref-list-1
Citing articles This article has been cited by 6 HighWire-hosted articles. Access the articles at:
http://clincancerres.aacrjournals.org/content/14/15/4951.full.html#related-urls
E-mail alerts Sign up to receive free email-alerts related to this article or journal.
Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Subscriptions Department at pubs@aacr.org.
Permissions To request permission to re-use all or part of this article, contact the AACR Publications
Department at permissions@aacr.org.
Downloaded from clincancerres.aacrjournals.org on April 26, 2015. © 2008 American Association for Cancer
Research.