Folate-Hapten Mediated Immunotherapy Synergizes with Vascular Endothelial Growth Factor Receptor Inhibitors in Treating Murine Models of Cancer

Home , Hapten

Author Manuscript Published OnlineFirst on December 15, 2016; DOI: 10.1158/1535-7163.MCT-16-0569 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

N. Achini Bandara1, Cody D. Bates1, Yingjuan Lu2, Emily K. Hoylman1, and Philip S. Low1

1 Purdue University, Department of Chemistry, 560 Oval Dr., West Lafayette, IN 47907 2 Endocyte, Inc., 3000 Kent Ave, West Lafayette, IN 47906

Financial Support: This work was supported in part by a grant from Endocyte, Inc. awarded to Dr. Philip S. Low

Corresponding Author: Philip S. Low Purdue University Center for Drug Discovery 720 Clinic Drive West Lafayette, IN 47907-2084 Phone: 765-494-5273 E-mail: [email protected]

Running Title: Folate-hapten immunotherapy and VEGFR inhibitors

Keywords: Folate receptor, folate-hapten conjugate, cancer immunotherapy, combination immunotherapy, vascular endothelial growth factor receptor inhibitor.

Conflict of Interest: The studies reported in this publication were supported in part by a grant from Endocyte, Inc. Endocyte, Inc. is a biopharmaceutical company that develops folate-targeted therapies for the treatment of cancer and inflammatory diseases. Dr. Philip S. Low is the Founder, Chief Science Officer, and a member of the Board of Directors of Endocyte, Inc. Yingjuan Lu is the Director of Pharmacology, Inflammation Research at Endocyte, Inc.

Word Count: 3823

Number of Figures/Tables: 6

Downloaded from mct.aacrjournals.org on September 23, 2021. © 2016 American Association for Cancer Research. Author Manuscript Published OnlineFirst on December 15, 2016; DOI: 10.1158/1535-7163.MCT-16-0569 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Abstract

The over-expression of folate receptors (FR) on many human cancers has led to the development

of folate-linked drugs for the imaging and therapy of FR-expressing cancers. In a recent phase 1

clinical trial of late stage renal cell carcinoma patients, folate was exploited to deliver an

immunogenic hapten, fluorescein, to FR+ tumor cells in an effort to render the cancer cells more

immunogenic. Although >50% of the patients showed prolonged stable disease, all patients

eventually progressed, suggesting that the folate-hapten immunotherapy was insufficient by itself

to treat the cancer. In an effort to identify a companion therapy that might augment the folate-

hapten immunotherapy, we explored co-administration of two approved cancer drugs that had

been previously shown to also stimulate the immune system. We report that sunitinib and

axitinib (VEGF receptor inhibitors that simultaneously mitigate immune suppression) synergize

with the folate-hapten targeted immunotherapy to reduce tumor growth in three different

syngeneic murine tumor models. We further demonstrate that the combination therapy not only

enhances tumor infiltration of CD4+ and CD8+ effector cells, but surprisingly reduces tumor

neovasculogenesis more than predicted. Subsequent investigation of the mechanism for this unexpected suppression of neovasculogenesis revealed that it is independent of elimination of any tumor cells, but instead likely derives from a reduction in the numbers of FR+ tumor-

associated macrophages and myeloid derived suppressor cells, i.e. immunosuppressive cells that

release significant quantities of VEGF. These data suggest that a reduction in stromal cells of

myeloid origin can inhibit tumor growth by suppressing neovasculogenesis.

Introduction

Folic acid, a vitamin required for the synthesis of nucleotide bases, methylation of DNA and the post-translational modification of G proteins, enters cells via either the reduced folate carrier, the proton coupled folate transporter, or a folate receptor (1-4). While the reduced folate carrier and proton coupled folate transporter are expressed in virtually all cells, the folate receptor (FR) is present and accessible primarily on activated macrophages, the proximal tubule cells of the kidney, and certain cancers of epithelial origin, including malignancies of the ovary, endometrium, kidney, lung and breast (5-8). Because folate-linked imaging and therapeutic agents can only enter cells via the folate receptor, their uptake in normal tissues is highly limited, allowing folate to be used as a targeting ligand to deliver attached drugs to cancers and sites of inflammation (9-14).

The first folate-targeted therapy to enter human clinical trials involved the delivery of a

highly immunogenic hapten (fluorescein) to FR over-expressing tumor cells. In this strategy, the

patient was first vaccinated against fluorescein by immunization with keyhole limpet

hemocyanin (KLH)-linked to fluorescein. After establishing the presence of a high anti-

fluorescein antibody titer, the patient was injected with folate-fluorescein to decorate the surfaces

of FR-expressing cancer cells with the immunogenic hapten, thereby marking the malignant cells

for elimination by the immune system (15-19). Although the phase 1 human clinical data in

advanced renal cell carcinoma patients (>75% stage 4) with bulky disease revealed no complete

responses and few partial responses, >50% of the patients showed prolonged stable disease

(20,21), suggesting some degree of tumor suppression. Unfortunately, when more potent

immune stimulants, IFN-α and IL-2, were later added to the immunotherapy to further stimulate

immune activity, influenza-like symptoms triggered by the cytokine treatments emerged,

discouraging further clinical testing of the combination therapy (20). Nevertheless, the prolonged stable disease in the majority of patients motivated further exploration of less toxic companion therapies that might augment the ability of folate-fluorescein to label and stimulate removal of

FR+ cancers.

In this paper, we examine the efficacy of combining the aforementioned folate-

fluorescein immunotherapy with inhibitors of vascular endothelial growth factor receptor

(VEGFR). Our reasons for exploring this combination lies not only in the observation that

VEGFR inhibitors suppress unregulated neovascularization (22-24), but they also enhance tumor immunogenicity (25,26). Thus, sunitinib, a VEGFR inhibitor, has not only been shown to reduce

tumor neovascularization, but also to decrease immunosuppressive Treg and myeloid derived

suppressor cell (MDSC) populations in tumor-bearing animals (26,27). Since sunitinib is

approved for treatment of multiple malignancies, we elected to examine whether a combination

of a VEGFR inhibitor and the folate-hapten therapy might exceed the efficacies of either therapy

alone. In this paper, we report significant synergy between the VEGFR inhibitors and the folate targeted immunotherapy, not only in their abilities to suppress tumor growth, but also in their capacities to reduce angiogenesis.

Materials and Methods

Antibodies and Reagents

Folic acid, keyhole limpet hemocyanin (KLH), carboxymethylcellulose (CMC), Tween

20, and female Balb/c serum were purchased from Sigma Aldrich (St. Louis, MO). Bovine serum

albumin conjugated to fluorescein (BSA-FITC), folate-EDA-fluorescein (Folate-FITC) and the

GPI-0100 adjuvant was kindly provided by Endocyte, Inc. (West Lafayette, IN). The structure of folate-FITC has previously been published (28). Sunitinib malate and axitinib free base were purchased from LC Laboratories (Woburn, MA). Disposable PD-10 desalting columns were obtained from GE Healthcare Bio-Sciences (Pittsburgh, PA). The biotin-conjugated goat anti- mouse IgG2a antibody and streptavidin-HRP conjugate for ELISA antibody titer analysis was manufactured by Caltag Laboratories (Burlingame, CA). The Shandon™ Cryomatrix™ resin was purchased from Thermo Scientific (Waltham, MA). All fluorescently labeled antibodies for flow cytometry and confocal microscopy were obtained from either BioLegend (San Diego, CA) or eBioscience, Inc. (San Diego, CA). The special folate-deficient diet on which animals in treatment studies were maintained was purchased from Harlan Laboratories (Indianapolis, IN).

Cell Lines and Culture

L1210A cells were a generous gift from Dr. Gerrit Jansen, Department of Rheumatology at the University Hospital Vrije Universiteid (Amsterdam, Netherlands) in 2012. M109 cells were kindly provided by Dr. Alberto Gabizon, Hadassah-Hebrew University Medical Center

(Jerusalem, Israel) in 2012. Both L1210A (lymphocytic leukemia) and M109 (lung cancer) cells were selected for high folate receptor expression and have been shown to maintain these elevated

FR levels through multiple passages. M109 cells were harvested by digestion of solid tumor xenografts grown in Balb/c mice and cryopreserved at passage 0 or 1. When desired, cell lines were cultured in folate-deficient RPMI 1640 medium (Invitrogen, Grand Island, NY) supplemented with 10% heat inactivated fetal bovine serum (Sigma Aldrich, St. Louis, MO), penicillin (100 units/mL) and streptomycin (100µg/mL) at 37°C in a humidified atmosphere containing 5% CO2. Adherent M109 cells were passaged in a monolayer, while L1210A cells

were grown in suspension before implantation into syngeneic mice. Renca (renal cell carcinoma) cells were used as a folate receptor negative control.

Animals and Tumor Models

All procedures conducted on animals were carried out in accordance with protocols approved by the Purdue Animal Care and Use Committee. M109 and Renca tumors were grown in 5 to 7 week old female Balb/c mice (Harlan Laboratories, Indianapolis, IN), while L1210A tumors were implanted in 5 to 7 week old female DBA/2 mice (The Jackson Laboratory, Bar

Harbor, ME). For M109 tumor implantation, frozen cells at early passage were thawed and allowed to grow to confluence. On the day of tumor injection, M109 or Renca cells were collected and suspended in folate-deficient RPMI-1640 medium containing 1% syngeneic female

Balb/c serum. Mice were injected with 1 x 106 cells subcutaneously (s.c.), and therapy was

usually initiated between day 10 and 14, when tumors had reached 50-75 mm3. Similar procedures were followed for L1210A tumor growth, only cells were suspended in PBS immediately prior to implantation and treatment began in 7-10 days when tumors had reached

50-75 mm3. Tumor growth was monitored at 48 h intervals by measurement of size with

laboratory calipers and tumor volume was calculated using a standard two-dimensional tumor

volume equation; i.e. the length of the longest axis of each tumor (L) and the length of the axis

perpendicular to it (W) were recorded in millimeters and the corresponding tumor volume in

mm3 was determined by solving ½ (L x W2).

Immunization with KLH-FITC

Animals were vaccinated with a keyhole limpet hemocyanin (KLH) labeled with fluorescein in order to elicit an anti-fluorescein antibody response. The KLH-FITC conjugate was synthesized by reaction of KLH protein with excess FITC according to previously published procedures (15). The resulting KLH-FITC conjugate was purified on a PD-10 desalting column followed by dialysis in PBS at 4°C. KLH-FITC stock solutions were stored in the dark at -20°C until use in immunization cocktails.

Female Balb/c and DBA/2 mice were vaccinated with 35 µg KLH-FITC in 50 µg GPI-

0100 adjuvant formulated in sterile saline every two weeks for 6 weeks by subcutaneous injection alternating between the base of tail and back of the neck. Blood samples from immunized mice were collected by submandibular puncture one week after the second and third vaccinations and analyzed for anti-fluorescein titer by ELISA, as described previously (15).

Formulation of Drugs for in vivo Administration

Human cancer patients generally take sunitinib or axitinib orally (29,30). Therefore, in

order to mimic human treatment procedures, sunitinib malate was formulated in a CMC

suspension (0.5% carboxymethylcellulose, 1.8% sodium chloride, 0.4% Tween 80, and 0.9%

benzyl alcohol, pH 6) and administered to mice by oral gavage (p.o). Axitinib was dosed

similarly, but was formulated in a different CMC suspension (0.5% CMC in deionized water

acidified to pH 2-3). Both drug formulations were prepared freshly every week and stored in the

dark at 4°C. The suspension were shaken vigorously to ensure even drug distribution before

dosing. Folate-FITC stock solutions supplied by Endocyte, Inc. were diluted to the desired

concentration in sterile PBS, aliquoted, and stored in the dark at -20°C. Aliquots were thawed

completely on the day of treatment and mice in the appropriate groups were injected subcutaneously with 100 µL of the diluted conjugate.

Combination Therapy Studies

The day of tumor cell implantation was designated as day 0 for all experiments. Once tumors had reached ~50-75mm3, mice were randomized into different treatment groups: PBS

control, folate-FITC immunotherapy, sunitinib or axitinib therapy alone, or the combination of

folate-FITC plus sunitinib or axitinib. Mice in the PBS control group were injected daily with

100µL of sterile PBS (s.c.) and mice treated with folate-FITC alone were injected with 500

nmol/kg on a 5 days on 2 days off schedule (s.c.). Mice in the sunitinib alone or axitinib alone

groups were gavaged daily with 20 mg/kg sunitinib or 15 mg/kg axitinib, respectively. All mice

treated with the combination of folate-FITC plus sunitinib or axitinib were dosed as described

above for each of the individual components. Treatments were administered continually until the

PBS control tumors reached 1000-1500 mm3, at which point mice were euthanized and tumors resected for further analysis. In order to reduce serum folate levels to concentrations comparable

with folate levels in humans, all mice were placed on a special folate-deficient diet one week

following the second immunization.

Flow Cytometric Analysis of Isolated Spleen Cells

At the conclusion of each study, all animals were euthanized by CO2 asphyxiation, and

their tumors and spleens were harvested, washed in PBS, and weighed. Each tumor was then

snap frozen for immunofluorescence staining. For analysis of immune cells in the spleens,

spleens were gently mashed and pressed through a 40 µm cell strainer, after which the strainer

was carefully washed with PBS to collect any residual cells. Red blood cells were then removed using lysis buffer (Sigma Aldrich, St. Louis, MO) and the remaining splenocytes were suspended in labeling buffer (PBS containing 1% BSA). Fc receptors were blocked using a commercially available Fc blocker (BD Biosciences, San Jose, CA) to reduce non-specific labeling. Blocked cells were then incubated for 1 h at 4°C with the following antibody combinations: APC or PE conjugated anti-mouse F4/80 for macrophages; PE conjugated anti-mouse CD3 and FITC conjugated anti-mouse CD4 or FITC conjugated anti-mouse CD8 for CD4+ and CD8+ T cells, respectively; and APC conjugated anti-mouse CD11b and PE conjugated anti-mouse GR-1 for

MDSCs. Following labeling, cells were washed with cold labeling buffer and analyzed on a

Beckton Dickinson FACS Caliber instrument using CellQuest software. At least 100,000 cells were counted from each sample. All flow cytometry data were analyzed on FlowJo software.

Cryopreservation and Confocal Imaging of Tumors

Each resected tumor was embedded in Shandon™ Cryomatrix™ resin and snap frozen by partial submersion in liquid nitrogen. The frozen tumor tissues were then sectioned (10 µm) using a Shandon SME Cryotome cryostat (GMI, Inc., Ramsey, MN) and mounted on to polylysine-coated Superfrost microscopy slides (Fisher Scientific, Waltham, MA). For confocal imaging, the slides were allowed to equilibrate at room temperature for ~1h before fixation and permeabilization in ice-cold acetone (10 min). After washing in PBS, the tissue sections were incubated in PBS-Tween containing 1% BSA to block any nonspecific binding followed by washing in PBS (2x2 min). Individual slides were then incubated with a 1:50 dilution of

AlexaFluor 647 conjugated rat anti-mouse CD31 antibody (BioLegend, San Diego, CA) in a dark humidified chamber for 1h at room temperature. The stained slides were washed (3x5 min

in PBS), dried, and mounted with cover slips before examination under an Olympus FV1000 inverted confocal laser scanning microscope using a Plan Apo 10X objective. All fluorescence images were obtained under identical conditions and unlabeled tissue sections used as controls were treated in the same manner as labeled slides. Confocal microscopy data were analyzed on

FLUOVIEW Viewer software. The confocal images of tumor sections stained with CD31 were also analyzed for vascular density by calculating the percentage of each image’s surface that was occupied by a colored pixel.

Statistical Analysis

Graphing was performed using GraphPad Prism software. Statistical analyses were performed using the PROC MIXED procedure of SAS statistical software version 9.3. Data were analyzed by two-tailed analysis of variance and differences were considered statistically significant at P < 0.05. Pair-wise comparisons were performed using a Tukey-Kramer adjustment.

Results

Induction of Anti-FITC Antibody Titer in Mice

Mice were immunized 3x at two week intervals with a KLH-FITC conjugate formulated

in GPI-0100 adjuvant. Blood was collected following both the second and third immunization,

and antibody titers were assessed to ensure development of a robust anti-FITC immune response.

As shown in Supplemental Figure S1, a strong anti-FITC antibody titer was induced in all

immunized groups, and subsequent titer analysis at completion of each study further established maintenance of the titer during the treatment period.

Sunitinib Synergizes with Folate-Hapten Mediated Immunotherapy in L1210A Tumor-

Bearing Mice

VEGFR inhibitors have been observed to not only downregulate angiogenesis, but also inhibit the immunosuppressive activities of both myeloid derived suppressor cells (MDSCs) and regulatory T cells (Tregs) (26). In order to test whether these immunomodulatory properties might augment the potency of our receptor-targeted hapten immunotherapy, it was necessary to grow folate receptor-expressing tumors in immune competent mice. For this purpose, L1210A tumors were implanted subcutaneously in DBA/2 mice and the mice were treated with either sunitinib, folate-hapten immunotherapy, or a combination of the two. As seen in Fig. 1, the PBS-treated

DBA/2 mice grew tumors that expanded to 1000 mm3 in <3 weeks. Surprisingly, neither the

moderate doses of sunitinib nor folate-targeted immunotherapy used in this study exerted a

significant effect on this rapid tumor expansion. In contrast, a combination of the same doses of

sunitinib and folate-targeted immunotherapy caused a decrease in tumor volume (Fig. 1A). These

data imply that some type of synergy must exist between the tumor-targeted hapten therapy and

VEGFR inhibitor therapy.

Because L1210A cells derive from a murine lymphocytic leukemia that commonly

metastasizes to the spleen, it was also instructive to examine whether the spleen weights of each treatment group might differ as an indication of tumor cell metastasis (31). As shown in Fig. 1B, spleens became similarly enlarged in the PBS-, sunitinib- and folate-FITC- treated animals, but were relatively small in the combination therapy animals. Isolation of extractable cells from

these spleens demonstrated that their increase in size derived exclusively from accumulation of metastatic L1210A cells. These data suggest that metastasis of tumor cells to the spleen is suppressed by the combination therapy but not by either individual therapy.

Sunitinib Synergizes with Folate-Hapten Mediated Immunotherapy in the M109 Tumor-

Bearing Mice

To determine whether the above hypothesized synergy between the folate-hapten therapy and anti-VEGFR therapy might also apply to other tumor types, a second FR+ tumor was similarly examined in syngeneic mice. As seen in Fig. 2, M109 tumor-bearing mice treated with either sunitinib or folate-FITC alone responded only mildly to the individual therapy, resulting in moderately retarded tumor growth. However, mice treated with both folate-FITC and sunitinib again maintained a significantly slower tumor growth rate than either monotherapy group alone.

Combination therapy resulted in a 77% reduction in tumor volume and a 55% reduction in tumor weight compared to PBS-treated controls (P<0.05), which supports the hypothesis that a combination of folate-hapten plus anti-VEGFR therapy results in augmentation of each drug’s individual efficacy.

Axitinib Synergizes with Folate-Hapten Mediated Immunotherapy in L1210A Tumor-

Bearing Mice

To determine whether other angiogenesis inhibitors might similarly synergize with the folate-hapten immunotherapy, we explored the use of a more selective VEGFR inhibitor

(axitinib) than sunitinib in combination with our FR-targeted immunotherapy. For this purpose,

L1210A tumors were again implanted in DBA/2 mice and the affected mice were treated with

axitinib (15 mg/kg), folate-targeted immunotherapy or the combination of the two. As shown in

Figure 3, axitinib, like sunitinib, combined with folate-FITC immunotherapy to significantly slow tumor growth and prolong survival of all treated mice. The average tumor volume in the axitinib alone treated group at the end of the study (day 20) was 53% of the average tumor volume of the PBS treated mice. Mice treated with axitinib plus folate-FITC, however, displayed significantly smaller tumor volumes (23% of controls, P<0.05).

Immune Effector and Suppressor Cell Levels are Altered in Combination Therapy Treated

Mice

Studies reported in the literature propose several mechanisms for the known abilities of angiogenesis inhibitors to down-regulate immunosuppressive MDSCs and Tregs (22,25,26). To

determine whether the observed augmentation of folate-FITC immunotherapy might arise from a

reduction in either of these immune cell types, spleen cells were isolated from all mice in the

above studies and analyzed for different immune cell populations by flow cytometry. Although

no differences in Treg numbers could be detected among treatment groups (data not shown),

CD4+ and CD8+ T cells were found to be elevated in the combination therapy groups in both

L1210A and M109 tumor models (Fig. 4A and 4B). Moreover, MDSC numbers were reduced in the spleens of M109 tumor-bearing mice treated with the combination therapy (Fig. 4C), although no difference could be observed in the L1210A model. Because immunosuppressive T cells constitute only a fraction of the total T cell population (usually <3%) (32), the significant

increase in both CD4+ and CD8+ T cells argues that the combination therapy augments the cellular immune response against the tumor.

Synergistic Inhibition of Tumor Vascular Growth is Observed in Combination Therapy

Treated Mice

Since a proposed mechanism of anti-tumor activity of angiogenesis inhibitors derives from their retardation of neovascularization, we anticipated a reduction in tumor blood vessel density in sunitinib and axitinib treated animals, but no significant diminution in other treatment groups. To ascertain the extent of inhibition of neovasculogenesis, we stained tumor sections from mice in each of the treatment groups with an antibody to CD31 (i.e. an established endothelial cell marker), and examined the vascular density/distribution by confocal microscopy.

In contrast to expectations, sunitinib alone had little effect on blood vessel density in either of the tumor models, whereas the combination therapy showed a dramatic reduction in CD31 staining in both L1210A and M109 tumors (Fig. 5 Top and Center). Indeed, quantitative analysis of

CD31 staining in each image showed the vascular density of combination treated tumors to be approximately half that of tumors treated with either folate-FITC immunotherapy or sunitinib alone. These data indicate that folate-hapten therapy synergizes with VEGFR inhibitors to augment down-regulation of the tumor vasculature. However, as initially anticipated, tumors treated solely with axitinib showed a significant reduction in tumor vasculature staining (~57% of control), and the combination of axitinib plus immunotherapy further decreased this vascular density to 37% of the PBS-treated control. Representative images of anti-CD31 stained tumors derived from each treatment group as well as the corresponding graphs of a computer analysis of the vascular density are shown in Figure 5.

The unanticipated observation that folate-hapten immunotherapy can augment the ability of VEGFR inhibitors to suppress tumor neovascularization raised the question of whether inhibition of neovasculogenesis might constitute the predominant mechanism accounting for

their synergistic effect on tumor growth. To test this hypothesis, folate receptor negative Renca tumors were implanted in Balb/c mice and treated with folate-hapten immunotherapy, sunitinib, or a combination of the two. In this tumor model, any impact of the folate-targeted immunotherapy would have to be exerted on the FR+ tumor-infiltrating macrophages, since all

Renca tumor cells are FR negative. As shown in Fig. 6A, inhibition of tumor growth was minimal in both sunitinib and folate-hapten monotherapies. In contrast, the combination therapy caused a dramatic decrease in tumor growth, suggesting that the mechanism of synergy does not rely on any direct effect of folate-FITC on the cancer cells. Importantly, anti-CD31 stained sections of the same Renca tumors revealed that their vascular density was lower in the combination- than monotherapy-treated animals (Fig. 6B). These data further argue that suppression of neovascularization does not depend on prior destruction of the cancer cells, but rather that a reduction in the vasculature can independently inhibit tumor growth. Based on all of the above data, it is not unreasonable to posit that downregulation of neovasculogenesis may constitute the primary mechanism of tumor growth inhibition in the combination therapy group.

Discussion

As noted above, the folate-hapten targeted immunotherapy showed significant promise in

human clinical trials, but failed to yield complete responses probably due to inadequate

activation of the immune system (21). Attempts to augment the immunotherapy’s potency by co-

administration of cytokines also failed due to unacceptable toxicities (20). In an effort to identify

a less toxic and more effective companion therapy, we explored co-administration of VEGFR

inhibitors which are known to activate immune effector cells (22,33,34). We found a strong

synergy between the VEGFR inhibitors and the folate-hapten immunotherapy, where the

combination therapy significantly augmented suppression of tumor vascularization, reduction of

MDSCs, and recruitment of CD4+ and CD8+ T cells.

One of the more interesting results from this study was the observed synergy in

suppression of tumor neovasculogenesis. While reduction in blood vessel formation by the two

VEGFR inhibitors (i.e. sunitinib and axitinib) was anticipated, the strong augmentation of this

reduction by the FR-targeted immunotherapy was not. Contemplation of plausible mechanisms

raised the possibility that the immunotherapy-mediated elimination of tumor-associated myeloid

cells (i.e. tumor-associated macrophages (TAMs) and MDSCs) will simultaneously reduce the

levels of growth factors that they produce. Thus, recent examination of folate receptor beta

expression in a large number of human tumor samples reveals that virtually all tumor types are

characterized by significant infiltration of FR+ TAMs and MDSCs (35). Because these tumor-

associated myeloid cells are known to release VEGF, FGF, and many other angiogenic factors

(36-39), immune-mediated elimination of these TAMs/MDSCs should significantly reduce the

tumor’s supply of cytokines that impact tumor angiogenesis. Together with the blockade of

VEGF receptor signaling by sunitinib and axitinib (22,24,29), the observed synergy of the

combination therapy in suppressing vasculogenesis should probably have been predicted. If the

same reduction in vascular density can lower the probability of tumor metastasis, then the

mechanism underpinning the observed synergy in the combination therapy’s ability to eliminate

L1210A cell metastasis to the spleen might also be explained.

Taken together, our data suggest that multiple mechanisms may contribute to the efficacy

of the combination therapy comprised of the folate-hapten immunotherapy plus VEGFR

inhibitors. Not only can the targeted therapy lead to antibody opsonization and immune cell-

mediated destruction of FR+ cancer cells (16-18), but the concurrent elimination of FR+ tumor-

associated macrophages and MDSCs could reduce both the immunosuppressive microenvironment and growth of new blood vessels within the tumor. Since many human cancer cells do not express FR, but virtually all human tumors contain large numbers of FR+ myeloid cells (35,40,41), the combination therapy described here could conceivably prove useful in controlling many human tumor types.

Acknowledgements

We thank Endocyte, Inc. for providing folate-FITC (EC17) and GPI-0100. We are grateful to Dr. Leroy Wheeler for advice during design of flow cytometry experiments and to Dr.

Ahmad Gheith for assistance in interpretation of confocal images of CD31 stained tumor tissues.

The authors gratefully acknowledge the flow cytometry resources from the Purdue University

Center for Cancer Research, NIH grant P30 CA023168, and the imaging facilities of the Bindley

Bioscience Center, a core facility of the NIH-funded Indiana Clinical and Translational Sciences

Institute. This work was supported in part by a grant from Endocyte Inc.

References

1. Antony A. The biological chemistry of folate receptors. Blood 1992;79:2807-20. 2. Matherly LH, Goldman ID. Membrane transport of folates. Vitamins & Hormones 2003;66:403-56. 3. Lucock M. Folic acid: nutritional biochemistry, molecular biology, and role in disease processes. Molecular genetics and metabolism 2000;71:121-38. 4. Kim Y-I. Nutritional epigenetics: impact of folate deficiency on DNA methylation and colon cancer susceptibility. The Journal of nutrition 2005;135:2703-09. 5. Elnakat H, Ratnam M. Distribution, functionality and gene regulation of folate receptor isoforms: implications in targeted therapy. Advanced drug delivery reviews 2004;56:1067-84. 6. Parker N, Turk MJ, Westrick E, Lewis JD, Low PS, Leamon CP. Folate receptor expression in carcinomas and normal tissues determined by a quantitative radioligand binding assay. Analytical biochemistry 2005;338:284-93. 7. Weitman SD, Lark RH, Coney LR, Fort DW, Frasca V, Zurawski VR, et al. Distribution of the folate receptor GP38 in normal and malignant cell lines and tissues. Cancer research 1992;52:3396-401. 8. Weitman SD, Weinberg AG, Coney LR, Zurawski VR, Jennings DS, Kamen BA. Cellular localization of the folate receptor: potential role in drug toxicity and folate homeostasis. Cancer research 1992;52:6708-11. 9. Ayala-López W, Xia W, Varghese B, Low PS. Imaging of atherosclerosis in apoliprotein e knockout mice: targeting of a folate-conjugated radiopharmaceutical to activated macrophages. Journal of Nuclear Medicine 2010;51:768-74. 10. Turk MJ, Breur GJ, Widmer WR, Paulos CM, Xu LC, Grote LA, et al. Folate‐targeted imaging of activated macrophages in rats with adjuvant‐induced arthritis. Arthritis & Rheumatism 2002;46:1947-55. 11. Wang S, Luo J, Lantrip DA, Waters DJ, Mathias CJ, Green MA, et al. Design and synthesis of [111In] DTPA-folate for use as a tumor-targeted radiopharmaceutical. Bioconjugate chemistry 1997;8:673-79. 12. Reddy JA, Dorton R, Dawson A, Vetzel M, Parker N, Nicoson JS, et al. In Vivo Structural Activity and Optimization Studies of Folate− Tubulysin Conjugates. Molecular pharmaceutics 2009;6:1518-25. 13. Reddy JA, Low PS. Folate-mediated targeting of therapeutic and imaging agents to cancers. Critical Reviews™ in Therapeutic Drug Carrier Systems 1998;15. 14. Low PS, Henne WA, Doorneweerd DD. Discovery and development of folic-acid-based receptor targeting for imaging and therapy of cancer and inflammatory diseases. Accounts of chemical research 2007;41:120-29. 15. Lu Y, Low PS. Folate targeting of haptens to cancer cell surfaces mediates immunotherapy of syngeneic murine tumors. Cancer Immunology, Immunotherapy 2002;51:153-62. 16. Lu Y, Low PS. Immunotherapy of folate receptor-expressing tumors: review of recent advances and future prospects. Journal of Controlled Release 2003;91:17-29.

17. Lu Y, Sega E, Leamon CP, Low PS. Folate receptor-targeted immunotherapy of cancer: mechanism and therapeutic potential. Advanced drug delivery reviews 2004;56:1161-76. 18. Lu Y, Sega E, Low PS. Folate receptor‐targeted immunotherapy: Induction of humoral and cellular immunity against hapten‐decorated cancer cells. International Journal of Cancer 2005;116:710-19. 19. Lu Y, Xu L-C, Parker N, Westrick E, Reddy JA, Vetzel M, et al. Preclinical pharmacokinetics, tissue distribution, and antitumor activity of a folate-hapten conjugate– targeted immunotherapy in hapten-immunized mice. Molecular cancer therapeutics 2006;5:3258-67. 20. Amato RJ, Shetty A, Lu Y, Ellis PR, Mohlere V, Carnahan N, et al. A Phase I/Ib Study of Folate Immune (EC90 Vaccine Administered With GPI-0100 Adjuvant Followed by EC17) With Interferon-α and Interleukin-2 in Patients With Renal Cell Carcinoma. Journal of Immunotherapy 2014;37:237-44. 21. Amato RJ, Shetty A, Lu Y, Ellis R, Low PS. A phase I study of folate immune therapy (EC90 vaccine administered with GPI-0100 adjuvant followed by EC17) in patients with renal cell carcinoma. Journal of Immunotherapy 2013;36:268-75. 22. Ellis LM, Hicklin DJ. VEGF-targeted therapy: mechanisms of anti-tumour activity. Nature reviews cancer 2008;8:579-91. 23. Goodman VL, Rock EP, Dagher R, Ramchandani RP, Abraham S, Gobburu JV, et al. Approval summary: sunitinib for the treatment of imatinib refractory or intolerant gastrointestinal stromal tumors and advanced renal cell carcinoma. Clinical Cancer Research 2007;13:1367-73. 24. Hu-Lowe DD, Zou HY, Grazzini ML, Hallin ME, Wickman GR, Amundson K, et al. Nonclinical antiangiogenesis and antitumor activities of axitinib (AG-013736), an oral, potent, and selective inhibitor of vascular endothelial growth factor receptor tyrosine kinases 1, 2, 3. Clinical Cancer Research 2008;14:7272-83. 25. Ozao-Choy J, Ma G, Kao J, Wang GX, Meseck M, Sung M, et al. The novel role of tyrosine kinase inhibitor in the reversal of immune suppression and modulation of tumor microenvironment for immune-based cancer therapies. Cancer research 2009;69:2514-22. 26. Xin H, Zhang C, Herrmann A, Du Y, Figlin R, Yu H. Sunitinib inhibition of Stat3 induces renal cell carcinoma tumor cell apoptosis and reduces immunosuppressive cells. Cancer research 2009;69:2506-13. 27. Draghiciu O, Nijman HW, Hoogeboom BN, Meijerhof T, Daemen T. Sunitinib depletes myeloid-derived suppressor cells and synergizes with a cancer vaccine to enhance antigen-specific immune responses and tumor eradication. OncoImmunology 2015;4:e989764. 28. De Jesus E, Keating JJ, Kularatne SA, Jiang J, Judy R, Predina J, et al. Comparison of Folate Receptor Targeted Optical Contrast Agents for Intraoperative Molecular Imaging. Int J Mol Imaging 2015;2015:469047. 29. Motzer RJ, Rini BI, Bukowski RM, Curti BD, George DJ, Hudes GR, et al. Sunitinib in patients with metastatic renal cell carcinoma. Jama 2006;295:2516-24. 30. Rini BI, Wilding G, Hudes G, Stadler WM, Kim S, Tarazi J, et al. Phase II study of axitinib in sorafenib-refractory metastatic renal cell carcinoma. Journal of Clinical Oncology 2009;27:4462-68. 18

31. Hofer KG, Prensky W, Hughes WL. Death and metastatic distribution of tumor cells in mice monitored with 125I-iododeoxyuridine. Journal of the National Cancer Institute 1969;43:763-73. 32. Caramalho I, Lopes-Carvalho T, Ostler D, Zelenay S, Haury M, Demengeot J. Regulatory T cells selectively express toll-like receptors and are activated by lipopolysaccharide. The Journal of experimental medicine 2003;197:403-11. 33. Bose A, Taylor JL, Alber S, Watkins SC, Garcia JA, Rini BI, et al. Sunitinib facilitates the activation and recruitment of therapeutic anti‐tumor immunity in concert with specific vaccination. International Journal of Cancer 2011;129:2158-70. 34. Huang Y, Yuan J, Righi E, Kamoun WS, Ancukiewicz M, Nezivar J, et al. Vascular normalizing doses of antiangiogenic treatment reprogram the immunosuppressive tumor microenvironment and enhance immunotherapy. Proceedings of the National Academy of Sciences 2012;109:17561-66. 35. Shen J, Putt KS, Visscher DW, Murphy L, Cohen C, Singhal S, et al. Assessment of folate receptor-β expression in human neoplastic tissues. Oncotarget 2015. 36. Lewis C, Leek R, Harris A, McGee J. Cytokine regulation of angiogenesis in breast cancer: the role of tumor-associated macrophages. Journal of Leukocyte Biology 1995;57:747-51. 37. Murdoch C, Muthana M, Coffelt SB, Lewis CE. The role of myeloid cells in the promotion of tumour angiogenesis. Nature reviews cancer 2008;8:618-31. 38. Pepper MS, Mandriota SJ, Montesano R. Angiogenesis-Regulating Cytokines. In: Fan T- PD, Kohn EC, editors. The New Angiotherapy. Totowa, NJ: Humana Press; 2002. p 7-40. 39. Yang L, DeBusk LM, Fukuda K, Fingleton B, Green-Jarvis B, Shyr Y, et al. Expansion of myeloid immune suppressor Gr+CD11b+ cells in tumor-bearing host directly promotes tumor angiogenesis. Cancer Cell 2004;6:409-21. 40. Cortez-Retamozo V, Etzrodt M, Newton A, Rauch PJ, Chudnovskiy A, Berger C, et al. Origins of tumor-associated macrophages and neutrophils. Proceedings of the National Academy of Sciences 2012;109:2491-96. 41. Noy R, Pollard JW. Tumor-associated macrophages: from mechanisms to therapy. Immunity 2014;41:49-61.

Figure Legends

Figure 1. (A) Effect of folate-FITC (500 nmoles/kg, 5 days/week) and/or sunitinib (20 mg/kg daily) therapy on the growth of L1210A tumors in DBA/2 mice. Treatments started on day 7 when tumors reached 50-75 mm3. (B) Average spleen weight. Data are mean ± SEM (n=5-9).

Figure 2. (A) Effect of folate-FITC (500 nmoles/kg, 5 days/week) and/or sunitinib (20 mg/kg daily) therapy on the growth of M109 tumors in Balb/c mice. (B) Average weights of excised tumors from M109 tumor-bearing Balb/c mice dosed with the indicated therapies. Data are mean ± SEM (n=5-7). Means that do not share a letter are significantly different, P < 0.05.

Figure 3. Effect of folate-FITC and/or axitinib therapy on the growth of L1210A tumors in DBA/2 mice. Data are mean ± SEM (n=6). Means that do not share a letter are significantly different, P < 0.05.

Figure 4. (A) CD4+ and CD8+ T cell populations in the spleens of L1210A tumor-bearing DBA/2 mice and (B) M109 tumor-bearing Balb/c mice. Isolated splenocytes stained with florescent antibodies against CD3, CD4, and CD8, were analyzed by flow cytometry. (C) Percent of myeloid derived suppressor cells (MDSC) found in the spleens isolated from M109 tumor-bearing mice dosed with the indicated therapies. Isolated splenocytes labeled with fluorescent antibodies against CD11b and GR-1 were analyzed by flow cytometry. 100,000 cells were counted from each sample. Data are mean ± SEM (n=5-7). Means that do not share a letter are significantly different, P < 0.05. #P = 0.06.

Figure 5. Fluorescent staining and imaging of the vasculature within tumors harvested from treated mice. Top row shows images obtained from solid L1210A tumors treated with PBS, folate-FITC, sunitinib, or a combination of folate-FITC and sunitinib. The center row represents images obtained from solid M109 tumors treated with PBS, folate-FITC, sunitinib, or a combination of folate-FITC and sunitinib. The bottom row shows images collected from stained L1210A tumors collected from mice treated with PBS, folate-FITC, axitinib, or a combination of

folate-FITC and axitinib. Frozen tumors were sectioned and stained with an anti-CD31 antibody labeled with AlexaFluor 647 and imaged under an Olympus FV1000 confocal microscope using a 10X objective. Error bars represent SEM. Means that do not share a letter are significantly different, P < 0.05.

Figure 6. (A) Effect of folate-FITC and/or sunitinib therapy on the weight of folate receptor negative Renca tumors in Balb/c mice (n=2-3). Individual data points and means (horizontal lines) are shown. (B) Quantification of CD31 fluorescent staining and imaging of the vasculature within Renca tumors harvested from mice treated with PBS, folate-FITC, sunitinib, or a combination of folate-FITC and sunitinib. Data are from 5 tumor sections. Individual data points and means (horizontal lines) are shown. (C) Representative images of CD31 staining for above tumors. Frozen tumors were sectioned and stained with an anti-CD31 antibody labeled with AlexaFluor 647 and imaged under an Olympus FV1000 confocal microscope using a 10X objective.

Folate-Hapten Mediated Immunotherapy Synergizes with Vascular Endothelial Growth Factor Receptor Inhibitors in Treating Murine Models of Cancer

N. Achini Bandara, Cody D. Bates, Yingjuan Lu, et al.

Mol Cancer Ther Published OnlineFirst December 15, 2016.

Updated version Access the most recent version of this article at: doi:10.1158/1535-7163.MCT-16-0569

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