Vascular Endothelial Growth Factor Blockade Reduces Intratumoral ...

Cancer Therapy: Preclinical

Vascular Endothelial Growth Factor Blockade Reduces Intratumoral Regulatory T Cells and Enhances the Efficacy of a GM-CSF ^ Secreting Cancer Immunotherapy Betty Li, Alshad S. Lalani,Thomas C. Harding, Bo Luan, Kathryn Koprivnikar, Guang HuanTu, Rodney Prell, Melinda J.VanRoey, Andrew D. Simmons, and Karin Jooss

Abstract

Purpose: The purpose of the present study was to evaluate granulocyte macrophage colonystimulating factor (GM-CSF) ^ secreting tumor cell immunotherapy in combination with vascular endothelial growth factor (VEGF) blockage in preclinical models. Experimental Design: Survival and immune response were monitored in the B16 melanoma and the CT26 colon carcinoma models. VEGF blockade was achieved by using a recombinant adeno-associated virus vector expressing a solubleVEGF receptor consisting of selected domains of the VEGF receptors 1 and 2 (termed sVEGFR1/R2). Dendritic cell and tumor infiltrating lymphocyte activation status and numbers were evaluated by fluorescence-activated cell sorting analysis. Regulatory Tcells were quantified by their CD4+CD25hi and CD4+FoxP3+ phenotype. Results: The present study established that GM-CSF ^ secreting tumor cell immunotherapy with VEGF blockade significantly prolonged the survival of tumor-bearing mice. Enhanced anti-tumor protection correlated with an increased number of activated CD4+ and CD8+ tumor-infiltrating T cells and a pronounced decrease in the number of suppressive regulatory T cells residing in the tumor. Conversely, overexpression of VEGF from tumors resulted in elevated numbers of regulatory Tcells in the tumor, suggesting a novel mechanism ofVEGF-mediated immune suppression at the tumor site. Conclusion: GM-CSF ^ secreting cancer immunotherapy and VEGF blockade increases the i.t. ratio of effector to regulatory T cells to provide enhanced antitumor responses. This therapeutic combination may prove to be an effective strategy for the treatment of patients with cancer.

Irradiated granulocyte-macrophage colony-stimulating factor (GM-CSF) – secreting tumor cell immunotherapies have been shown to elicit systemic tumor-specific immune responses in numerous preclinical and clinical studies (1 – 7). GM-CSF augments the function of antigen-presenting cells by inducing the maturation, activation, and recruitment of dendritic cells (8 – 10). These dendritic cells are critical in priming antigenspecific immune responses and have been shown to efficiently acquire and process antigens, migrate to secondary lymphoid organs, and up-regulate costimulatory molecules upon activation. Both CD4+ and CD8+ T cells are required for the efficacy of GM-CSF – secreting tumor cell immunotherapies, highlighting the important role of GM-CSF – activated dendritic cells in priming a broad humoral and cellular immune response (2). Given the crucial role of dendritic cells in initiating an immune Authors’Affiliation: Cell Genesys, Inc., South San Francisco, California Received 6/26/06; revised 9/5/06; accepted 9/7/06. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). Requests for reprints: Andrew D. Simmons, Cell Genesys, Inc., 500 Forbes Boulevard, South San Francisco, CA 94080. Phone: 650-266-3067; Fax: 650266-2910; E-mail: andrew.simmons@ cellgenesys.com. F 2006 American Association for Cancer Research. doi:10.1158/1078-0432.CCR-06-1558

Clin Cancer Res 2006;12(22) November 15, 2006

response, strategies to improve dendritic cell recruitment, activation, and/or maturation may further enhance the antitumor efficacy of GM-CSF – secreting tumor cell immunotherapies. There is compelling evidence that vascular endothelial growth factor (VEGF) is a major regulator of tumor growth and metastasis (11, 12). VEGF is secreted at high levels in numerous tumor types, and its production is associated with a poor prognosis (13). Although the role of VEGF in tumor angiogenesis is well recognized, it is also a key factor in promoting and sustaining the nonresponsiveness of the immune system to growing tumors (13, 14). Tumor-derived VEGF binds to the VEGFR1/FLT1 receptor on CD34+ bone marrow progenitor cells, decreasing the ability of these cells to differentiate into functional dendritic cells (15). Consistent with this observation, deficiencies in the dendritic cell population have been reported in cancer patients (16). It has also been suggested that VEGF may affect T-cell development directly (17); however, this has not been firmly established to date. The aforementioned data prompted us to perform a series of ‘‘proof-of-concept’’ studies to evaluate whether VEGF inhibition could enhance the potency of GM-CSF – secreting tumor cell immunotherapies. VEGF blockade was achieved by systemic expression of a soluble chimeric VEGF receptor, designated sVEGFR1/R2, that binds VEGF with high affinity and efficiently blocks its function (18). When animals were treated with sVEGFR1/R2 in combination with a GM-CSF – secreting tumor

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VEGF Blockade Enhances GM-CSF ^ Secreting Immunotherapy

cell immunotherapy, prolonged survival and a significant increase in the ratio of CD4+ and CD8+ tumor-infiltrating lymphocytes (TIL) was observed. Despite an overall increase in CD4+CD25+ T cells, TILs derived from animals treated with the combination therapy contained significantly fewer regulatory T cells than animals treated with the cancer immunotherapy alone. The decreased number of regulatory T cells in the tumors of animals treated with the combination therapy correlated with enhanced apoptosis of these cells and the presence of fewer immature dendritic cells. In contrast, tumors that had been modified to overexpress VEGF contained higher numbers of regulatory T cells in the TIL population than the parental tumors. These data establish that tumor-generated VEGF increases the number of regulatory T cells within the tumor, and that combining VEGF blockade with a GM-CSF – secreting tumor cell immunotherapy enhances the survival of tumorbearing animals by altering the regulatory T/effector T cell ratio at the tumor site and may prove to be an effective novel strategy for the treatment of patients with cancer.

Materials and Methods Mice and cell lines. C57BL/6 mice (Taconic, Hudson, NY), BALB/c (Taconic), and OT-1 TCR transgenic mice (The Jackson Laboratory, Bar Harbor, ME) were purchased and maintained according to NIH guidelines. Efficacy and mechanism studies were initiated with mice between 8 and 12 weeks of age. Mouse experiments were approved and done according to the Cell Genesys Animal Use and Care Committee. The B16F10 melanoma and the CT26 colon carcinoma cell lines were purchased from the American Type Culture Collection (Manassas, VA). The generation of the retrovirally transduced GM-CSF – secreting cell lines has been described previously (1). The B16.GM and the CT26.GM generates 150 ng/106 cells/24 hours and 80 ng/106 cells/24 hours of mouse GM-CSF, respectively. The murine VEGF (mVEGF) coding sequence was obtained from plasmid pBLAST49-mVEGF (Invivogen, San Diego, CA) and cloned into the retroviral transfer vector pRT43.2 (19) using standard molecular biology techniques. Vector production and transduction conditions used to generate the F10.mVEGF cell populations from B16F10 melanoma cells have been described previously (19). F10.mVEGF generates 1,000 ng/106 cells/24 hours of mVEGF. Recombinant adeno-associated virus vector construction. The creation of a sVEGFR1/R2 chimeric receptor has been described previously (18). Briefly, individual Ig-like domains of the parental VEGF receptors were PCR amplified using an Expand High Fidelity PCR kit (Roche Applied Science, Indianapolis, IN). The domains were joined using a secondary PCR amplification and cloned into the adeno-associated virus (AAV) vector plasmid pTR-CAG-VEGFR3-WPRE-BGHpA (20). A control vector containing a null insert was also cloned using standard molecular biology techniques (20). In vivo tumor treatment and mechanism experiments. AAV vectors were given i.v. to C57BL/6 mice using 1 1010 particles of the vector control or sVEGFR1/R2 virus. Eleven days after vector administration, mice were challenged with 2 105 B16F10 cells by s.c. injection at a dorsal site. Three days later, mice were vaccinated with 1 106 irradiated GM-CSF – secreting B16F10 (B16.GM) tumor cells. Animals were monitored for the formation of palpable tumors twice weekly and sacrificed if tumors became necrotic or exceeded 1,500 mm3 in size. Dendritic cell characterization, flow cytometry, and in vivo CTL. The draining lymph nodes (axillary, lateral axillary, and superficial inguinal) were collected on day 5 after challenge and mechanically dissociated using glass slides. Cells were counted and stained with conjugated antibodies (BD PharMingen, San Diego, CA). Flow


cytometry acquisition and analysis was done on a FACScan apparatus using CellQuest Pro software (BD Biosciences, San Jose, CA). To perform in vivo CTL assays, single-cell suspensions of total splenocytes from naive C57BL/6 mice were loaded with 100 Ag/mL OVA-specific peptide (SIINFEKL) for 1 hour at 37jC. After washing in HBSS, peptideloaded and naive target cells were labeled for 30 minutes with 15 and 1.5 mmol/L carboxyfluorescein succinimidyl ester (Molecular Probes, Carlsbad, CA), respectively. Cells were washed and injected i.v. at total doses of 5 to 10 106 per mouse. Approximately 24 hours later, single-cell suspensions of splenocytes were prepared and analyzed by flow cytometry. The percent of specific lysis was calculated as follows: [1 (A/B)] 100, where A = the number of unloaded targets/ number of peptide-loaded targets in naive recipient mice and B = the number of unloaded targets/number of peptide-loaded targets in experimental mice. TIL characterization. On the indicated days, tumors were removed from mice and digested in 1 mg/mL collagenase IV (Sigma, St. Louis, MO) and 0.1 mg/mL DNase for 1 hour at 37jC. Dissociated cells were filtered through a 0.3-Am filter, and leukocytes were positively selected using CD45 MACs beads according to manufacturer’s instructions (Miltenyi Biotec, Auburn, CA). Enriched leukocytes were directly stained with antibodies for phenotype characterization by fluorescence-activated cell sorting (FACS) analysis. All antibodies were obtained from BD PharMingen with the exception of the phycoerythrin-conjugated FoxP3 (eBioscience, San Diego, CA). In vitro apoptosis experiment. CD4+CD25+ regulatory T cells and CD4+CD25 naive T cells were enriched from C57BL/6 mice using a CD4+CD25+ regulatory T cell isolation kit according to manufacturer’s instructions (Miltenyi Biotec). Purity was verified by FACS analysis and was found to be >95%. Enriched populations were coincubated with a dose titration of recombinant Fas ligand (FasL; Axxora, San Diego, CA). Cells were evaluated for apoptosis using an Annexin V and 7AAD apoptosis kit (BD PharMingen) and analyzed by FACS. mVEGF overexpressing tumor studies. Mice were inoculated with 2 105 F10.mVEGF tumor cells by s.c. injection at a dorsal site. Seventeen days after tumor inoculation, tumors were removed and evaluated according to the methods described in TIL Characterization. Statistical analysis and data presentation. Multi-parameter statistics for the Kaplan-Meier survival curves were done by a log-rank test using GraphPad Prism Software. Relative differences between groups were also done using a Student’s t test and GraphPad Prism Software.

Results GM-CSF – secreting tumor cell immunotherapy in combination with VEGF inhibition significantly enhances the survival of tumor-bearing animals. The maturation and differentiation of dendritic cells is impaired by tumor-derived VEGF (13, 14, 16, 17, 21 – 23). This observation prompted us to evaluate whether VEGF blockade could improve the efficacy of a GM-CSF – secreting tumor cell immunotherapy, which is known to function through the recruitment and activation of dendritic cells. Although VEGF blockade is often done using specific monoclonal antibodies, the availability of murine anti-VEGF antibodies is restricted. Thus, an alternative approach using a recombinant AAV vector expressing a soluble VEGF receptor consisting of selected domains of the VEGFR1 and VEGFR2 (18) was employed. This chimeric VEGF receptor, designated sVEGFR1/R2, has been shown to inhibit VEGF-stimulated biological activities by efficiently binding and sequestering VEGF (18). Using sVEGFR1/R2 to block VEGF, we previously observed a reduction in tumor blood vasculature density in multiple preclinical tumor models (24). For these studies and the experiments done here, the recombinant AAV vector was

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given 10 days before tumor inoculation to allow for transgene expression to reach a plateau, generally observed after 3 weeks (25). A dose-response study of sVEGFR1/R2 was initially done in the B16F10 tumor model to identify a suboptimal dose that resulted in the prolonged survival of f20% of treated animals. Mice were injected with a single i.v. dose of 1 1010, 2 1010, or 5 1010 viral particles of AAV-sVEGFR1/R2, or 5 1010 virus particles of AAV-control vector expressing no transgene. The vector dose of 1 1010 virus particles yielded sustained sVEGR1/ R2 serum levels of f75 Ag/mL (data not shown) and afforded a broad window for the detection of potential additive or synergistic effects in animals treated with a GM-CSF– secreting tumor cell immunotherapy in combination with sVEGFR1/R2 (Fig. 1A). For all combination studies, AAV vector administration, tumor inoculation, and immunization was done as described in Materials and Methods. The control vector had no effect on tumor growth rate with a median survival time (MST) of 31 days compared with an MST of 35 days in HBSS-treated mice (Fig. 1B). Animals treated with sVEGFR1/R2 had improved survival, with a MST of 63 days. Mice receiving the B16.GM plus the control vector (B16.GM alone) also showed improved survival, with a MST of 56 days. Most importantly, animals treated with the combination therapy consisting of the B16.GM immunotherapy plus sVEGFR1/R2 exhibited a significant prolongation of survival with a MST of 93 days (*, P < 0.05, compared with B16.GM alone). Mice treated with a GM-CSF – secreting tumor cell immunotherapy in combination with VEGF blockade have increased numbers of activated dendritic cell and T cells present in the tumor. Previous reports have suggested that sustained and elevated VEGF serum levels lead to a reduction in dendritic cell number and function, resulting in an impaired immune response (13, 22, 26, 27). To investigate the effect of VEGF on dendritic cells, dendritic cells were harvested from the draining lymph nodes and the tumor and evaluated by FACS analysis on days 5 and 17 after tumor challenge. There was only a negligible difference in the number of activated dendritic cells isolated from draining lymph nodes between animals treated with B16.GM alone and animals treated with B16.GM plus sVEGFR1/ R2 (Supplementary Fig. S1). In contrast, in the tumor, the percentage of CD11c+ dendritic cells in animals treated with B16.GM plus sVEGFR1/R2 was significantly greater than in animals treated with B16.GM alone (Fig. 2A). Costaining of CD11c+ dendritic cells with antibodies recognizing the activation markers CD40 or CD80 revealed the same pattern (Fig. 2B and C). In comparison, immature dendritic cells as identified by CD11c+/CD4 /CD8 staining, were significantly reduced within the tumors of the GM-CSF – secreting tumor cell immunotherapy/sVEGFR1/R2 combination – treated groups compared with the animals receiving either monotherapy (Fig. 2D). Because dendritic cells are recognized as the most potent antigen-presenting cells and present antigens to naive T cells, it is expected that increased numbers of activated dendritic cells would correlate with a more superior T-cell response. To address this issue, tumor-infiltrating T cells were evaluated by FACS analysis on days 17 and 31 after tumor implantation in the different treatment groups. Significantly greater numbers of tumor-infiltrating CD4+ and CD8+ T cells per 1 106 tumor cells were present in animals treated with the combination therapy compared with animals that had received B16.GM alone at both time points (Supplementary Fig. S2). In addition,


Fig. 1. GM-CSF ^ secreting tumor cell immunotherapy in combination with VEGF inhibition significantly enhances the survival of B16 tumor-bearing animals. A, C57BL/6 mice (n = 10 per group) were given a single i.v. administration of varying doses of recombinant AAV-sVEGFR1/R2 (day 10) 10 days before s.c. inoculation with 2 105 B16F10 tumor cells (day 0). Mice were monitored for the formation of palpable tumors twice weekly and sacrificed if tumors became necrotic or exceeded 150 mm3 in size. The Kaplan-Meier survival curve of percentage of surviving mice was used for evaluation. B, using the suboptimal recombinant AAV-sVEGFR1/R2 dose of 1 1010 viral particles per mouse, a survival study evaluating the combination therapy was set up following the same protocol as described in (A) with the exception that the mice were immunized s.c. at the ventral site on day 3 with 1 106 irradiated GM-CSF ^ secreting B16F10 tumor cells (B16.GM). Representative of at least three experiments.

when the CD4+ T cells were costained with the CD25+ T-cell activation marker, there was also a significantly greater number of tumor-infiltrating CD4+CD25+ T cells per 1 106 tumor cells in animals treated with B16.GM plus sVEGFR1/R2. When the TILs were further characterized for IFNg expression and various cell surface activation markers (Fig. 3; Supplementary Fig. S3), there was a significantly greater percentage of IFNg-expressing

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cells in the CD8+ and CD4+ T-cell subpopulations in animals receiving the combination therapy compared with animals that received B16.GM alone (Fig. 3A). Furthermore, when the TILs were evaluated for FasL expression, a tumor necrosis factor family member that induces apoptosis in Fas-bearing cells through the Fas/FasL pathway, a significantly greater percentage of FasL-expressing T cells in the CD8+ and CD4+ T-cell subpopulations were found in animals treated with the combination therapy compared with animals that were given B16.GM alone (Fig. 3B). Moreover, in the combination therapytreated animals, CD8+ T cells were found to express significantly higher levels of CD107a, a marker for cytotoxic-associated cell degranulation (Fig. 3C). Increased T-cell function, as assessed by an in vivo CTL assay, was also observed in the combination therapy – treated group (Fig. 3D). Taken together, these data suggest that VEGF blockade, when given in combination with a GM-CSF – secreting tumor cell immunotherapy, enhances the ratio of highly activated effector T cells to tumor cells in the tumor microenvironment resulting in increased T-cell activity. VEGF inhibition combined with a GM-CSF – secreting tumor cell immunotherapy leads to reduced numbers of regulatory T cells in the tumor microenvironment. It has been well documented that tumor-reactive effector T cells accumulate in the tumor microenvironment but fail to provide an effective antitumor response (26, 28). There is also clear evidence that regulatory T cells, classified by their coexpression of the CD4 and CD25 cell surface markers, accumulate in tumors and play a critical role in the inhibition of antitumor T-cell responses (29). Although effector and regulatory T cells are both characterized by a CD4+CD25+ phenotype, it has been shown that CD4+CD25lo cells function as effector cells, whereas CD4+CD25hi cells act as regulatory T cells with suppressive properties (30, 31). Interestingly, in tumors of B16.GM plus sVEGFR1/R2 – treated animals, the CD4+CD25hi population was significantly lower than in tumors of animals treated with B16.GM alone (Fig. 4A and B). Apart from using the gating strategy to distinguish CD4+CD25hi cells, the regulatory T cell – specific transcription factor FoxP3 was used as an additional marker for regulatory T cells. In agreement with our previous data, the total number of CD4+FoxP3+ cells in tumors of animals treated with the combination therapy was significantly lower than in the group treated with the B16.GM monotherapy alone (Fig. 4B).

To verify the decrease in functional regulatory T cells observed in the B16F10 tumor model in C57BL/6 mice, the studies were replicated using the highly immunogenic CT26 colon carcinoma tumor model in BALB/c mice. It has been well documented that these two mouse strains differ significantly in their cytokine milieu; BALB/c mice display a more Th-2-biased environment, whereas C57BL/6 mice are more prone towards a Th-1 like response (32). In support of the data obtained in the B16 tumor model, the CD4+CD25hi population in the CT26.GM plus sVEGFR1/R2 – treated animals was significantly smaller than in the group treated with CT26.GM alone (Fig. 4C). These findings were further confirmed using CD4+ and FoxP3+ as markers to distinguish the regulatory T cell population within the CT26 tumors (Fig. 4C). In summary, significantly fewer regulatory T cells were present in tumors of animals receiving the combination treatment compared with animals treated with the cancer immunotherapy alone. Furthermore, the ratio of activated T cells (CD8+/IFNg+ and CD4+/IFNg+) to regulatory T cells (CD4+/FoxP3+) was augmented in tumors obtained from mice treated with the combination of immunotherapy and sVEGFR1/R2 (Fig. 4D). Regulatory T cells express high levels of Fas receptor and are highly susceptible to FasL-induced apoptosis in vivo and in vitro. It has been reported that in contrast to human effector T cells, human regulatory T cells are highly susceptible to FasLinduced apoptosis in vitro (33). Because the tumor-infiltrating effector T cells from mice treated with the combination therapy show strong FasL up-regulation, it is conceivable that they act to keep regulatory T cell numbers in check by modulating their survival via Fas-mediated killing. Therefore, in vitro and in vivo experiments were done to evaluate FasL-induced apoptosis of murine regulatory T cells. CD4+CD25+ regulatory T cells and CD4+CD25 naive T-cell populations were isolated from naive C57BL/6 mice and confirmed to be >95% pure by FACS analysis. Fas expression on the two populations was evaluated using an antibody against CD95 (Fas). Indeed, increased levels of Fas expression were observed on CD4+CD25+ regulatory T cells when compared with CD4+CD25 T cells (Fig. 5A). To evaluate FasL-induced apoptosis, CD4+CD25+ regulatory T cells and CD4+CD25 naive T cells were incubated with a dose titration of recombinant FasL for 1 and 3 hours, and cells were evaluated for apoptosis using Annexin V and 7-AAD. At both

Fig. 2. Mice treated with a GM-CSF ^ secreting tumor cell immunotherapy in combination with VEGF blockade have significantly increased numbers of activated dendritic cells in the tumor. On day 10, mice were given a single i.v. administration of 1 1010 viral particles per mouse of an AAV vector encoding for sVEGFR1/R2. Control mice were injected i.v. with the same dose of empty AAV vector (Control). On day 0, mice were challenged dorsally s.c. with 2 105 live B16F10 tumor cells. On day 3, mice were immunized s.c. with irradiated GM-CSF ^ secreting tumor cells (B16.GM). On day 17, tumors were removed, digested, enriched forTILs, and evaluated for the presence of activated dendritic cells by FACS analysis (n = 5 mice per group). Percentage of (A) CD11c+ dendritic cells, (B) CD40+ cells in the CD11c subpopulation, (C) CD80+ cells in the CD11c subpopulation, and (D) percentage of CD4 CD8 cells in the CD11c subpopulation.


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time points evaluated, a greater percentage of FasL-induced apoptosis was apparent in the CD4+CD25+ regulatory T cell population compared with the CD4+CD25 naive T cells (Fig. 5B). To assess whether this was also true in vivo and the observed reduction of regulatory T cells in the tumors of animals treated with the combination therapy was a result of increased apoptosis of these cells, tumors were harvested 17 days after inoculation and evaluated for the presence of regulatory T cells that were undergoing apoptosis by FACS analysis. A significantly greater percentage of Annexin V – positive cells were detected in the CD4+CD25hi subpopulation in animals treated with the combination therapy than in animals treated with B16.GM alone (Fig. 5C). Taken together, these data show that mice treated with the combination therapy bear higher numbers of activated, FasL-expressing effector T cells correlating with a greater percentage of regulatory T cells undergoing apoptosis in the tumor microenvironment than mice treated with B16.GM alone. Overexpression of VEGF by the tumor increases the presence of regulatory T cells in the tumor microenvironment. It is well known that tumors secrete VEGF to promote angiogenesis

(11, 12). To evaluate whether there is a correlation between VEGF overexpression and the number of regulatory T cells present in the tumor, the number of regulatory T cells was evaluated in tumors that were modified to express high levels of VEGF. B16F10 parental tumor cells were retrovirally transduced to increase expression of mVEGF. mVEGF expression was confirmed by ELISA, and the mVEGF-transduced B16F10 cells (F10.mVEGF) produced 1,000 ng/1 106 cells/24 hour in contrast to the parental B16F10 cells that generated VEGF levels below the detection limit of this assay. In this study, mice were inoculated with parental B16F10 or F10.mVEGF tumor cells (2 105) by s.c. injection. Seventeen days later, tumors were harvested and evaluated for the presence of regulatory T cells. Using identical variables for regulatory T cell evaluation as described above, a significant difference in the number of CD4+CD25hi and CD4+FoxP3+ T cells was observed between mice inoculated with parental B16F10 tumors or with F10.mVEGF tumors (Fig. 6A). When F10.mVEGF tumorbearing animals were treated with the combination therapy, significantly fewer regulatory T cells were observed in the tumor

Fig. 3. Increased numbers of tumor infiltratingT lymphocytes with effector phenotype are found in tumors of mice treated with a GM-CSF ^ secreting tumor cell immunotherapy in combination with VEGF blockade. Mice were treated as described Fig. 2 legend. On day 17 (D17), CD4+ and CD8+ Tcells isolated from the B16F10 tumors were costained for the expression of IFNg, FasL, and CD107a (n = 6 mice per group). Percentage of (A) IFNg-positive cells in the CD8 and CD4 subpopulation, (B) FasL-positive cells in the CD8 and CD4 subpopulation, and (C) CD107a-positive cells in the CD8 subpopulation. Representative of at least three experiments. T-cell activation was evaluated in the same groups described above using an in vivo CTL assay. For these studies, mice were challenged and immunized as previously described using tumor cells that were modified to express ovalbumin as a surrogate antigen. On day 17 after challenge, mice were injected with syngeneic splenocytes pulsed with carboxyfluorescein succinimidyl ester ^ labeled OVA-specific peptides. Approximately 18 hours later, splenocytes were harvested and evaluated for CTL activity by measuring the ratio of carboxyfluorescein succinimidyl ester ^ labeled cells.


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Fig. 4. GM-CSF ^ secreting tumor cell immunotherapy in combination with VEGF inhibition decreases the percentage of regulatoryTcells in the tumor of B16F10 and CT26 tumor-bearing animals. Mice were administered a single i.v. administration of 1 1010 viral particles per mouse of the recombinant AAV vector encoding for sVEGFR1/R2 on day 10. Control mice were injected i.v. with the same dose of empty AAV vector (Control). On day 0, mice were challenged s.c. with 2 105 live tumor cells, and on day 3, mice were immunized ventrally s.c. with 1 106 irradiated GM-CSF ^ secreting tumor cells. Tumors from the B16F10 tumor-bearing animals were harvested on day 17 (D17), digested, enriched forTILs, and stained for FACS analysis. A, representative dot plots of i.t. CD4- and CD25-positive events are shown in the total lymphocyte population gated according to FSC and SSC. B, percentage of i.t. CD4+CD25hi cells (left) and CD4+FoxP3+ Tcells (right) in the B16F10 tumor model in B16.GM- and B16.GM/ sVEGFR1/R2 ^ treated groups. C, percentage of i.t. CD4+CD25hi cells (left) and CD4+FoxP3+ Tcells (right) in the CT26 tumor model in CT26.GM- and CT26.GM/ sVEGFR1/R2 ^ treated groups. D, ratio of the number of activated CD8+/IFNg (left) and CD4+ IFNg (right) cells to the number of CD4+FoxP3+ regulatoryTcells in the tumor microenvironment.

microenvironment (Fig. 6B). Moreover, prolonged survival was observed in mice bearing VEGF-overexpressing tumors when treated with the combination therapy, resulting in a MST of 46 days compared with an MST of 25 days for both the vehicle-


treated (HBSS) and B16.GM monotherapy – treated animals (Fig. 6C). Again, the observed enhanced tumor protection correlated with increased numbers of IFNg and FasL-expressing, activated CD4+ CD8+ T cells (Supplementary Fig. S4).

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Interestingly, in contrast to animals bearing the parental B16F10 tumors, the animals treated with B16.GM alone showed no survival advantage over animals bearing VEGFoverexpressing tumors (Fig. 1 and Fig. 6C).

Discussion The data presented herein show that VEGF blockade improves the efficacy of GM-CSF – secreting tumor cell immunotherapies in murine tumor models. The enhanced antitumor protection correlated with an increased number of activated CD4+ and CD8+

Fig. 5. RegulatoryTcells express higher levels of Fas receptor and are more susceptible to FasL-induced apoptosis in vivo and in vitro. CD4+CD25+ regulatory Tcells and CD4+CD25 naiveTcells were enriched from naI« ve C57BL/6 mice using a CD4+CD25+ regulatoryT-cell isolation kit. A, both populations were stained with an antibody against Fas and evaluated by FACS analysis.The shaded and open histograms represent Fas expression from CD4+CD25 naive and CD4+CD25+ regulatoryTcells, respectively. B, the previously described populations were coincubated in vitro with a dose titration of recombinant FasL and evaluated for apoptosis at the time points indicated. Percentage of cells during advanced stages of apoptosis (Annexin V and 7-AAD double positive) after 1and 3 hours of coincubation with FasL. C, tumors were harvested on day 17 from animals treated with the B16.GM immunotherapy and an AAV control vector or the VEGFR1/ R2 ^ expressing AAV vector. The CD4+/CD25hi cells were harvested as described in Materials and Methods and stained for Annexin V. Percentage of Annexin V ^ positive cells from a gated CD4+CD25hi subpopulation from the indicated groups.


tumor-infiltrating effector T cells and a pronounced decrease in the number of CD4+CD25hi and CD4+FoxP3+ regulatory T cells residing in the tumor. Conversely, overexpression of VEGF from the tumor resulted in enhanced numbers of regulatory T cell within the tumors. These data indicate that VEGF blockade in combination with a cancer immunotherapy increases the ratio of effector T cells to regulatory T cells in the tumor microenvironment, thus revealing a novel mechanism of immune suppression mediated by tumor-derived VEGF. VEGF causes a deficiency in the number and functional maturation of dendritic cells from progenitors (14, 27, 34) and acts as an important mediator of tumor-associated immunodeficiency (13, 16, 22). In our studies, VEGF blockade resulted in an increase in dendritic cell numbers and/or maturation in the local tumor environment, but not systemically. One explanation for this observation is that local VEGF levels in the tumor are higher than systemic VEGF levels and must exceed a minimal threshold to affect dendritic cell numbers and/or function. Although increased systemic dendritic cell activation was not observed, an improved T-cell response was evident both systemically and in the local tumor microenvironment of the animals receiving combination therapy when compared with animals treated with B16.GM alone. In addition to augmenting activated dendritic cell numbers, sVEGFR1/R2 can affect the role dendritic cells play in the development of regulatory T cells. It has previously been shown that CD4 CD8 CD11c+ immature dendritic cells prime CD4+ regulatory T cell to suppress antitumor immunity (35). These immature dendritic cells have a less mature phenotype, secrete high levels of transforming growth factor-h, and induce the development of interleukin10 – secreting regulatory T cells. Consistent with this report, our studies show that tumors of animals treated with the combination therapy have lower numbers of immature dendritic cells than animals treated with B16.GM alone (Fig. 2D). Furthermore, the remaining dendritic cells present in the tumor express lower levels of the activation markers CD40 and CD80 and are negative for both CD4 and CD8. In addition, T cells from tumors of mice treated with the combination therapy showed minimal expression of interleukin-10 (data not shown) compared with mice treated with the B16.GM alone. Moreover, Nishikawa et al. report that IFNg controls the generation/activation of CD4+CD25+ regulatory T cells in antitumor responses (36). Although the exact relationship between IFNg expression and the generation of regulatory T cells remains elusive, there is circumstantial evidence to indicate that regulatory T cells favor Th-2 immunologic status (37, 38), and IFNg promotes a Th-1 response. In agreement with these findings, our data reveal a high percentage of IFNg-expressing T cells in animals treated with the combination therapy compared with animals treated with B16.GM alone. Although VEGF plays a role in the induction of immature dendritic cells, and immature dendritic cells in turn promote the generation of regulatory T cells in the tumor microenvironment, the precise mechanism and role of VEGF blockade in this process remains to be determined. The number of local regulatory T cells can also be modulated by effector T cells. Furthermore, it has recently been shown that an increased effector T/regulatory T cell ratio in the tumor microenvironment following treatment with a cancer immunotherapy strongly correlates with improved survival of tumor-bearing mice (39). Notably, differences in the effector T/regulatory T cell ratios were not observed in the draining

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Fig. 6. Overexpression of VEGF by the tumor increases the number of regulatory Tcells in the tumor microenvironment. On day 0, mice (n = 5 mice per group) were challenged s.c. with 2 105 of either B16F10 parental tumor cells or B16F10 cells modified to express high levels of mVEGF (1,000 ng/1 106 cells/24 hours). On day 17, tumors were harvested, enzymatically digested, and evaluated by FACS analysis. A, percentage of CD4+CD25hi-positive cells (left ; *, P < 0.001) and absolute number of CD4+FoxP3+-positive cells (right ; *, P < 0.05). To evaluate the impact of GM-CSF ^ secreting immunotherapy on the F10.mVEGF tumors, mice were given a single i.v. administration of 1 1010 viral particles per mouse of an AAV vector encoding sVEGFR1/ R2 on day 10. On day 0, mice were challenged s.c. with 2 105 live F10.mVEGF tumor cells, and on day 3, mice were immunized ventrally s.c. with 1 106 irradiated GM-CSF ^ secreting tumor cells. Tumors from the animals were harvested on day 17. B, percent CD4+CD25hi-positive cells (left ; *, P = 0.003) and absolute number of CD4+FoxP3+-positive cells (right ; *, P = 0.02). C, percent survival from treatment therapy.

lymph nodes or systemic circulation, suggesting that altering this ratio in the tumor microenvironment is crucial in eliciting antitumor immunity that results in a therapeutic benefit. The immune system uses the interaction of Apo-1/Fas (CD95) and FasL (CD95L) as a mechanism to maintain T-cell homeostasis during the contraction phase of the immune response. Our data suggest that regulatory T cells can potentially be eliminated by this same interaction in the tumor microenvironment in animals treated with the combination therapy. We show that CD4+CD25+ and CD4+FoxP3+ regulatory T cells are Fas positive and are highly sensitive to FasL-induced apoptosis. In addition, the effector T cells obtained from mice treated with the combination therapy exhibit elevated cell surface levels of FasL. Lastly, an increase in the percentage of Annexin V – positive cells was observed in the CD4+CD25hi subpopulation isolated from tumors of animals treated with the combination therapy compared with animals treated with B16.GM alone. These data support the hypothesis that high numbers of FasL-expressing CD4+ and CD8+ T cells present in tumors of animals following immunization with a potent cancer immunotherapy have the potential to keep regulatory T cells in check by modulating their survival via Fas-mediated killing, thus increasing the ratio of effector T cells to regulatory T cells within the tumor and generating a more potent local antitumor response. These ‘‘proof-of-concept’’ studies show that VEGF blockade significantly improves the efficacy of a GM-CSF – secreting tumor cell immunotherapy. Although the studies presented


here employed an AAV vector expression system to achieve a sustained and long-term expression of sVEGFR1/R2 to sequester VEGF, one can envision alternative clinical strategies for the interruption of VEGF signaling, including blocking antibodies targeted against VEGF or soluble decoy receptors that prevent VEGF from binding to its receptors. For example, bevacizumab, a humanized monoclonal antibody against VEGF that is approved by the Food and Drug Administration as the first antiangiogenic therapy for the treatment of cancer might provide a valid alternative to the recombinant AAV system. Based on the mechanism of action of sVEGFR1/R2 in combination with a GM-CSF – secreting tumor cell immunotherapy presented in this study, VEGF blockade is expected to augment the potency of alternative cancer immunotherapies, particularly those based on the activation of dendritic cells. One can postulate that VEGF blockade will target the solid tumor by preventing the sprouting of new blood vessels. At the same time, the immunotherapy will target both the primary tumor and micrometastases via a concerted effector T-cell response. Importantly, VEGF blockade will, in addition, reduce the number of regulatory T cells in the tumor microenvironment, thereby dramatically increasing the effectiveness of the T cells activated by the immunotherapy. In our study, we evaluated CD4+CD25hi and CD4+FoxP3+ regulatory T cells in the tumor microenvironment. However, there could be other immune regulatory cells present, which could exert their suppressor/regulatory activity to reduce

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the antitumoral efficacy of a GM-CSF – secreting tumor cell immunotherapy. These other regulatory cells include CD11b+/ Gr-1+ myeloid suppressor cells (40), tumor associated macrophages (41), ah TCR+ T cells (CD3+CD4 CD8 ; ref. 42), interleukin-10 – dependent CD4 helper T cells (Tr1; ref. 43), and CD8+CD28 suppressor cells (44). Each regulatory cell type is unique by their suppressive activity on the immune system, and the role of how VEGF affects these regulatory cells is currently undefined; therefore, additional studies are

warranted to further investigate the effect of VEGF blockade on these regulatory cells in the tumor microenvironment and determine how they may influence effector cell function and ultimately antitumor efficacy.

Acknowledgments We thank P. Working for critical reading of the manuscript and B. Batiste, J. Ho, T. Langer, and S. Tanciongo for their technical assistance.

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