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. Author manuscript; available in PMC: 2015 Apr 1.
Published in final edited form as: Int J Cancer. 2013 Oct 8;134(7):1695–1705. doi: 10.1002/ijc.28488

Combination of Sunitinib with anti-tumor vaccination inhibits T cell priming and requires careful scheduling to achieve productive immunotherapy

Ritika Jaini 1, Patricia Rayman 1, Peter A Cohen 2, James H Finke 1,3, Vincent K Tuohy 1,3
PMCID: PMC3947113  NIHMSID: NIHMS527399  PMID: 24105638

Abstract

Sunitinib, a protein tyrosine kinase inhibitor is the frontline therapy for renal and gastrointestinal cancers. We hypothesized that by virtue of its well documented tumor apoptosis and immune adjuvant properties, combination of Sunitinib with anti-tumor immunotherapeutics will provide synergistic inhibition of tumor growth. The current study was designed to evaluate the impact of Sunitinib on immunotherapy mediated anti-tumor immune responses and evaluate its efficacy as a combinatorial therapy with tumor targeted immunotherapeutic vaccination. Mice immunized with recombinant α-lactalbumin, a lactation protein expressed on majority of breast tumors were treated with 1mg of Sunitinib for 7 consecutive days beginning (1) concurrently, on the day of α-lactalbumin immunization or (2) sequentially, on day 9 after immunization. 10 day lymph nodes or 21 day spleens were tested by ELISPOT assays and flow cytometry to evaluate responsiveness to α-lactalbumin immunization in presence of Sunitinib and distribution of cells involved in T cell antigen priming and proliferation in different lymphoid compartments. In addition, therapeutic efficacy of the α-lactalbumin/ Sunitinib combination was evaluated by monitoring tumor growth in the 4T1 transplanted tumor model. Our studies reveal that concurrent administration of Sunitinib with active vaccination against a targeted tumor antigen inhibits priming to the immunogen due to a drastic decrease in CD11b+CD11c+ antigen presenting cells, leading to failure of vaccination. However, sequential delivery of Sunitinib timed to avoid the priming phase of vaccination results in the desired vaccination mediated boost in immune responses.

Keywords: Sunitinib, alpha lactalbumin, immunotherapy, priming inhibition, dendritic cells

Introduction

Sunitinib (Sutent® or SU11248) an oral multi target receptor tyrosine kinase inhibitor is recognized as the most effective therapy for metastatic renal cell carcinoma and gastrointestinal stromal cancers (1–3). The drug has been approved by the Food and drug administration as first line therapy for patients of metastatic renal cell cancer and proto-oncogene c-kit+ gastrointestinal stromal tumors and has had a major therapeutic impact on treatment of these cancers (4). Most of its tumor inhibitory and immune stimulatory effects have been attributed to its inhibitory role in STAT3 signaling (5). Numerous clinical trials are ongoing to test improvement in therapeutic efficacy by treatment of various cancers with Sunitinib in combination with other chemotherapeutic drugs (6) However, in spite of its well documented role as an immune adjuvant, there are very few ongoing trials aimed at analyzing efficacy of Sunitinib in combination with immunotherapeutic strategies.

Although recent breast cancer clinical trials by Pfizer on efficacy of Sunitinib alone or in combination with paclitaxel revealed no significant increase in progression free survival when compared to established standard of care therapies; capecitabine or combination of bevacizumab and paclitaxel respectively (7), its most useful application especially for solid tumors such as breast and renal cancer could be as a combination drug with established or experimental immunotherapeutic strategies. This is especially true in view of the fact that a large majority of anti-tumor immunotherapeutic protocols are ineffective in providing adequate anti-tumor efficacy when therapeutic intervention is provided during later stages of tumor progression (8). We have recently shown with α-lactalbumin targeted immunotherapy (9) that although immunization with α-lactalbumin can provide effective inhibition of breast tumor progression in 4T1 transplanted tumors in mice, vaccination with α-lactalbumin at later stages of tumor growth is not very effective in inhibiting tumor growth, suggesting that late stage tumors require combinations of chemical and immunological strategies targeting multiple pathways or protein targets on the tumor simultaneously in order to achieve significant anti-tumor efficacy. Protein tyrosine kinase inhibitors such as Sunitinib have proven to be effective therapies and have potential to provide long lasting and effective immunotherapy in combination with such strategies.

Sunitinib has been shown to have a direct inhibitory effect on tumor growth by promoting tumor apoptosis (5) and inhibiting the tumor promoting effect of vascular endothelial growth factor (VEGF; 10). In addition, Sunitinib can indirectly inhibit tumor growth by stimulating anti-tumor immune responses (11–15). Based on its well documented tumor apoptosis and immune adjuvant properties, we hypothesize that combination of Sunitinib with anti-tumor immunotherapeutic strategies could be highly effective in providing enhanced as well as long term immunity against solid tumors such as renal and breast tumors. Although the immune stimulatory action of Sunitinib is well documented, few studies have also demonstrated Sunitinib mediated inhibition of immune responses (16) mainly by inhibition of T cell activation in vitro or reduction in CD3+CD4+ T cells in patients (17). In view of these conflicting reports the effects of Sunitinib on anti-tumor immunity are still unclear. In order to design relevant protocols of treatment with Sunitinib in combination with immunotherapeutics, it is essential to characterize the impact of Sunitinib on the anti-tumor immune response mediated and boosted by the immunotherapy and evaluate efficacy of the combinatorial therapy in providing enhanced tumor inhibition.

In the current study we administered Sunitinib in combination with our recently established immunotherapeutic α-lactalbumin targeted vaccination strategy against breast cancer in order to evaluate its effect on anti-tumor immunity mediated through direct vaccination. In addition, we also evaluated the potential of both treatment strategies to provide enhanced therapeutic efficacy against 4T1 transplanted murine tumors. We find that Sunitinib can inhibit antigen presentation and priming to the immunotherapeutic antigen if administered simultaneously with active vaccination. However, sequential delivery of Sunitinib timed to avoid the priming phase of active immunization against α-lactalbumin allows generation of a substantial vaccine specific immune response, although with a marginal enhancement in tumor inhibition in the 4T1 tumor model. Our results emphasize that combination of Sunitinib with vaccination strategies is feasible but will require careful planning and scheduling of drug administration relative to and specific for the immunotherapy protocol used for boosting anti-tumor immune responses.

MATERIAL AND METHODS

Mice

6–8 week old female BALB/cJ (H-2d) mice were purchased from the Jackson laboratory (Bar Harbor, ME) and maintained in micro-isolator cages. Animals were handled under aseptic conditions per an Institutional Animal Care and Use Committee (IACUC) approved protocol.

α-lactalbumin purification

recombinant murine α-lactalbumin was purified as described earlier (9). Briefly α-lactalbumin cDNA generated from lactating mouse mammary tissue was inserted into the pQE-82L expression vector (Qiagen, Valencia, CA) for producing a 6×-His tagged fusion protein in SG13009 E. coli (Stratagene, La Jolla, CA). Recombinant protein was isolated from bacterial lysates under denaturing conditions and purified on nickel nitrilotriacetic acid (Ni-NTA) agarose columns by selecting for the 6× His tag. Protein preparations were further purified on a C4 preparative column by high performance liquid chromatography (HPLC) to eliminate any traces of endotoxin. All our protein preparations are tested for endotoxin levels which have consistently been identified as <0.1EU/ml. All protein preparations were checked for purity on a 10% Tris-Hcl polyacrylamide gel before in vivo or ex vivo experimentation.

Tumor inoculation and measurements

4T1 mouse mammary carcinoma cell line was procured from the American Type Culture Collection (CRL-2539; Manassas, VA) and cultured at 37°C and 5% CO2 in 75 cm2 tissue culture flasks in RPMI 1640 (Mediatech Inc, Manassas, VA) containing 4.5g/l glucose and supplemented with penicillin/streptomycin, HEPES buffer, sodium pyruvate and L-glutamine. At 70–75% confluence, cells were harvested by treatment with 0.25% trypsin and 0.02% EDTA (Sigma Aldrich, St. Louis, MO), and 1×104 washed cells were inoculated s.c. in the abdominal flank of 7–8 week old BALB/c females. Tumors were measured using Vernier calipers along the longest measurements in two directions perpendicular to each other. Tumor area was calculated as length × breadth. Mice were euthanized when their tumor reached 17mm along any measurement according to IACUC specifications.

Immunization and Sunitinib treatment

Mice were immunized with 100μg of recombinant α-lactalbumin in Complete Freund’s Adjuvant (CFA) or CFA alone. All immunizations were performed 5 days after s.c. inoculation of 1×104 4T1 tumor cells per mouse. Sunitinib treatment were done at day 5 or 9 after tumor inoculation as per the experimental design and involved i.p. injection of 1mg of Sunitinib in 1ml volume of PBS per mouse daily for 7 consecutive days. All Sunitinib treatments were performed according to two schedules: (a) Concurrent administration-CFA control or α-lactalbumin in CFA immunization combined with Sunitinib administration starting on same day as immunization and continued for the following seven consecutive days (b) Sequential treatment: CFA or α-lactalbumin in CFA immunization followed by Sunitinib administration beginning at day 9 after immunization and continued for 7 consecutive days. The same Sunitinib treatment protocol was followed for all immunological and tumor inhibition studies with Sunitinib administration commencing on the day of immunization (concurrent treatment) or 9 days after immunization (sequential treatment).

Tumor infiltrating lymphocyte isolation

17mm tumors were excised from BALB/cJ female mice and trimmed of extra tissue and fur. The tumor was minced into small pieces using a scalpel and incubated in 0.2mg/ml collagenase and 50KU/ml DNAase (Sigma Aldrich, St. Louis, MO) in phosphate buffered saline for 30 minutes at 37°C. Following digestion tumor tissue was pipetted repeatedly with a wide bore pipette to loosen entrapped cells. Supernatant from digested tumor tissue was collected and centrifuged at 1500 rpm for 10 minutes to collect all suspended cells. Cell pellets obtained were re-suspended in 70% percoll (GE Healthcare Biosciences, Pittsburgh, PA) and overlayed with 30% percoll to form a discontinuous gradient. The 30%–70% percoll gradient was centrifuged at 2400 rpm for 30 minutes at 20°C. Mononuclear cell layer obtained at the interface of 30% and 70% percoll were collected, washed and resuspended in media. TILs suspended in media were further selected by incubation at 37°C in a nylon wool column for 30 minutes followed by flushing with 3–5 volumes of warm media to select for lymphocytes. TILs obtained were pelleted by centrifugation and resuspended in complete DMEM for further assays.

ELISpot assays

Capture and detection antibody pairs (AN-18 and R4-6A2 biotin respectively) for IFNγ ELISpot assays were purchased (eBioscience Inc., San Diego, CA). Draining lymph nodes and spleens were extracted at different time points according to experimental plan and teased into a single cell suspension. 2×105 lymph node cells or splenocytes (Millipore, Billerica, MA) were cultured with 25 and 50 μg/ml α-lactalbumin or grade VII OVA control (Sigma Aldrich) in IFNγ antibody pre-coated ELISpot plates (Millipore) in 200 μl/well total culture volume in supplemented DMEM (Mediatech, Manassas, VA). Spots were developed and counted after 72 hours of culture on an immunospot image analyzer using the Immunospot v4.0 software (Cellular Technologies, Cleveland, OH).

Flow cytometry analysis

Single cell suspensions were prepared from lymph nodes, spleens and tumors of tumor bearing or naïve mice treated with different combinations of Sunitinib and α-lactalbumin alone and in combination with each other as per the experimental design. 1×106 lymph node, spleen and bone marrow cells and tumor infiltrating lymphocytes were blocked with Fcγ RIII antibody and surface stained in three colors for CD3, CD4 and CD8 markers and two colors for CD11b and CD11c or CD11b and Gr1. All Flourescein Isothyocynate (FITC), Phycoerythrin (PE) and Allophycocyanin (APC) florochrome labeled antibodies were purchased from BD Biosciences. T regulatory cell staining and analysis was done using the mouse regulatory cell staining kit#1 (ebioscience) according to the manufacturers protocol. Cells stained for specific markers with appropriate positive and unstained negative controls were acquired immediately on a Becton Dickinson FACScan™ flow cytometer and data analyzed using the FlowJo version 7.6.4 software (Tree Star Inc. Ashland, OR).

Statistical Analysis

Difference between tumor progression curves was statistically evaluated using the Mann Whitney rank sum test. Differences in survival curves, cell percentage or numbers in vaccine alone vs combination therapy treated groups were evaluated by applying the student t test.

RESULTS

Combination of Sunitinib with α-lactalbumin immunization does not enhance inhibition of 4T1 tumor progression when administered during the priming phase of immunization

We evaluated the anti-tumor efficacy of combination therapy with Sunitinib and α-lactalbumin vaccination in the murine 4T1 transplanted tumor model. 6–8 week old BALB/cJ females were inoculated with 1×104 4T1 mammary tumor cells by s.c. injection. Five days after tumor inoculation, mice were randomly assigned to two separate schedules of treatment. As per the first schedule of treatment, mice were immunized with α-lactalbumin at D5 and treated concurrently with Sunitinib beginning at D5 for 7 consecutive days (figure 1a, left panel). As per the second treatment schedule mice were immunized with α-lactalbumin at D5 and treated subsequently with Sunitinib, 9 days after α-lactalbumin immunization, i.e. beginning on D13 after tumor inoculation and continued for 7 consecutive days thereafter (figure 1a, right panel). For each schedule of treatment the following treatment and controls groups were set up (1) α-lactalbumin in CFA (n=8) (2) α-lactalbumin in CFA + Sunitinib (n=8) (3) CFA alone (n=8) and (4) CFA + Sunitinib (n=8). Tumors were measured daily and tumor area plotted as length × breadth. Tumor progression curves revealed lack of any synergy in individual therapeutic effects when Sunitinib was administered at the same time as α-lactalbumin vaccination on D5 after tumor inoculation (concurrent treatment; figure 1a, left panel; p< 0.438). However, when administration of Sunitinib was delayed till after the initial priming phase of immunization to α-lactalbumin was completed (9 days after immunization, on D13 after tumor inoculation), a marginally enhanced (Mann Whitney sum of rank 144.5 for combination therapy vs 155.5 for α-lactalbumin vaccination alone) but statistically insignificant inhibition of 4T1 tumor growth was observed with combined therapy (sequential treatment; figure 1a, right panel; p< 0.76). Sunitinib administration stopped progression of the 4T1 tumor but is ineffective in regressing 4T1 tumors. Since death is not an IACUC approved end point, survival analysis was performed using a 100 mm2 tumor size as an endpoint. Data reveal a small but statistically insignificant (p≤0.59) expected survival advantage till 23 days after tumor inoculation (87.5% survival with Sunitinib alone (n=8) vs 100% survival with the combination therapy (n=8; figure 1b). Although, the survival advantage conferred by Sunitinib treatment was still evident till the end of the experiment compared to CFA or the vaccine alone, no difference was seen in survival between groups receiving α-lactalbumin+Sunitinib or Sunitinib alone.

Figure 1. Inhibition of tumor growth by combinatorial therapy with Sunitinib and α-lactalbumin.

Figure 1

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6–8 week old BALB/cJ female mice were injected s.c. with 1×104 4T1 tumor cells. Mice injected with tumors were randomly assigned to two separate treatment regiments. (1) concurrent treatment, (2) sequential treatment. Treatment groups were set up as (1) α-lactalbumin alone (n=8) (2) α-lactalbumin + Sunitinib (n=8) (3) CFA alone (n=8) (4) CFA + Sunitinib (n=8). Tumors were measured and tumor area plotted. (Figure 1a, left panel) Combination of Sunitinib with α-lactalbumin conferred no enhanced tumor inhibition when the Sunitinib was administered concurrently with α-lactalbumin immunization. (Figure 1a, right panel) Sequential administration of Sunitinib beginning after completion of priming to α-lactalbumin resulted in slightly enhanced anti-tumor effect but was not statistically significant (p <0.76). (b) Expected survival curves were plotted with a tumor size of 100 mm2 as an end point. Minor survival advantage till day 23 after tumor inoculation was observed for mice receiving combination therapy compared to Sunitinib alone (n=8 each; p≤0.59). (c) A significant drop in the percentage of CD11b+Gr1+ myeloid derived suppressor cells (MDSCs) was seen in the spleens of mice treated with combination of the α-lactalbumin vaccine and Sunitinib compared to vaccine alone both under the simultaneous and staggered treatment protocols (n=4; p<0.0001 for both treatment protocols). However no difference was seen in the percentage of MDSCs in the tumors of 4T1 tumor bearing mice treated with Sunitinib simultaneously (n=4; p<0.73) or with staggered treatment vis-a-vis the vaccination (n=4; p<0.1) (c). (d) Treatment of 4T1 tumor bearing animals with Sunitinib in combination with or without immunization resulted in normal sized spleens that were significantly smaller than spleens of mice treated with vaccination alone. (e) Decrease in number of T regulatory cells within the tumor is evident at day 22 after tumor inoculation in mice treated with combination therapy compared to vaccine alone (n=4; p≤0.08). (f) Ratio of tumor infiltrating CD4+ and CD8+ cells to T regulatory cells were slightly increased (although not statistically significant) with combination treatment with Sunitinib compared to vaccine alone (n=4; CD4/Treg: p≤0.4 and CD8/Treg: p≤0.22).

In order to determine therapeutic effect of Sunitinib using parameters other than tumor progression, we evaluated Sunitinib mediated reduction in CD11b+Gr1+ myeloid derived suppressor cells (MDSCs) in the spleens and tumor of 4T1 bearing mice. A significant drop in the percentage of CD11b+Gr1+ myeloid derived suppressor cells (MDSCs) was seen in the spleens of mice (n=4) treated with α-lactalbumin vaccine and Sunitinib under both the simultaneous and staggered treatment protocols (p<0.0001 for both treatment protocols) indicating therapeutic activity of Sunitinib administration. However no reduction was seen in the percentage of MDSCs within the 4T1 tumors (n=4) treated with Sunitinib simultaneously (p<0.73) or with staggered treatment relative to the vaccination (p<0.1) (figure 1c). Sunitinib treatment (alone or in combination with α-lactalbumin vaccination) of 4T1 tumor bearing mice resulted in a significant decrease in spleen size comparable to normal mice in contrast to highly enlarged spleens in 4T1 breast tumor bearing mice treated with α-lactalbumin or CFA alone with no Sunitinib treatment (figure 1d). The observed lack of MDSC reduction within the 4T1 tumor after Sunitinib treatment reconfirms earlier findings by our group (12) where the lack of therapeutic efficacy against 4T1 tumors was correlated with a lack of MDSC reduction within the tumor in spite of a drastic decrease in this suppressor cell population within the spleen of tumor bearing mice. Combination therapy also affected a decrease in the number of T regulatory cells within the tumors when examined at day 22 after tumor inoculation. Although statistically insignificant (n=4; p≤0.08) the decrease in T regulatory cells in tumors treated with combination therapy compared to vaccine alone was substantial as is evident in figure 1e. Treatment with Sunitinib +vaccine marginally enhanced the ratio of effector cells to T regulatory cells (figure 1f, n=4; CD4/Treg: p≤0.4 and CD8/Treg: p≤0.22).

Tumor bearing mice treated with Sunitinib starting concurrently with α-lactalbumin vaccination show lack of reactivity to α-lactalbumin

Tumor infiltrating lymphocytes (TILs) were extracted from 17mm tumors of mice immunized with α-lactalbumin and treated with Sunitinib beginning at the same time as immunization. TILs extracted from mice immunized with (1) α-lactalbumin in CFA (2) CFA alone or (3) CFA + Sunitinib treatment were used as controls. TILs from all groups mentioned were evaluated for frequency of IFNγ producing cells reactive to α-lactalbumin. Mice receiving Sunitinib treatment starting on the same day (concurrent treatment) as the immunization with α-lactalbumin showed no detectable α-lactalbumin reactive IFNγ producers in their TILs in contrast to mice immunized with α-lactalbumin alone (figure 2; n=2; p<0.01). Mice receiving CFA immunization with or without Sunitinib treatment showed background levels of IFNγ producing cells. The lack of reactivity to α-lactalbumin in mice receiving concurrent combinatorial therapy with Sunitinib and α-lactalbumin, is further evident in its failure to confer any added therapeutic advantage against tumor growth as shown in figure 1a.

Figure 2. Tumor infiltrating lymphocytes from mice treated with simultaneous Sunitinib and α-lactalbumin immunotherapy are unresponsive to α-lactalbumin.

Figure 2

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Tumor bearing BALB/cJ female mice immunized with α-lactalbumin with or without Sunitinib treatment (started simultaneously with immunization, figure 1a) were euthanized when their tumors reached 17mm. These mice showed no synergistic effect of combinatorial treatment with α-lactalbumin and Sunitinib. Tumors were excised and tumor infiltrating lymphocytes (TILs) purified by percoll gradient centrifugation. 1×105 purified TILs/ well were tested for recall responsiveness to α-lactalbumin using ELISpot analysis. Results show a significant decrease in IFNγ producing TILs responsive to α-lactalbumin stimulation in mice treated concurrently with Sunitinib and α-lactalbumin compared to those that were immunized but not treated with Sunitinib (p≤0.01).

Sunitinib inhibits the priming phase of immunization but does not affect immune responses in a primed system

To analyze the lack of reactivity to α-lactalbumin when concurrent combinatorial therapy with Sunitinib was administered, we tested the effect of Sunitinib on priming to the α-lactalbumin vaccine. Six to eight week old BALB/cJ females were immunized with 100μg of α-lactalbumin in CFA and randomly divided into two groups (1) mice administered Sunitinib by i.p. injections at the same time as the immunization according to the 7 day regimen described above (n=7) (2) mice receiving immunization alone with no additional treatment (n=7). Ten days after immunization, lymph nodes were extracted and tested for recall responsiveness to α-lactalbumin since the most significant phase of priming occurs in the lymph nodes 3–10 days after immunization. Mice that received Sunitinib simultaneously with α-lactalbumin immunization showed significantly reduced recall responses to α-lactalbumin as assessed by low frequencies of IFNγ producing cells reactive to α-lactalbumin (figure 3a; p<0.003). These data indicated a significant defect in priming in mice immunized in presence of Sunitinib. However, when the same treatment groups were studied with Sunitinib treatment beginning 9 days after immunization (i.e. completion of the priming phase of immunization), similar recall responsiveness to α-lactalbumin was observed in both groups treated with Sunitinib + vaccine or vaccine alone (n=4, figure 3b). For the sequential mode of treatment, splenic responses were tested after completion of priming and Sunitinib treatment, i.e. 18 days after α-lactalbumin immunization since the spleen is the primary site for amplification of the immune response with very few immunoreactive cell remaining in the lymph node at that time point (>10 days after immunization). Sunitinib treatment after completion of priming to α-lactalbumin did not affect immune responsiveness to α-lactalbumin as assessed by frequencies of IFNγ producing splenocytes reactive to α-lactalbumin in both Sunitinib treated and non-treated mice. These data show that presence of Sunitinib during the priming phase of immunization inhibits priming; however it does not significantly affect immune responses once priming to the immunogen has been completed.

Figure 3. Sunitinib inhibits the initial priming phase of immunization when administered concurrently.

Figure 3

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Six to eight week old BALB/cJ females were immunized with 100μg of α-lactalbumin in CFA and randomly divided into two groups (1) mice administered Sunitinib by i.p. injections at the same time as the immunization (n=7) and (2) mice administered Sunitinib by i.p. injections starting 9 days after immunization and continued for 7 consecutive days (n=4) and (3) mice receiving immunization alone with no additional treatment (n=7). Ten days after immunization, lymph nodes were extracted and restimulated with α-lactalbumin for testing efficiency of in vivo priming to the antigen. (a) Mice receiving Sunitinib simultaneously with α-lactalbumin immunization showed significantly reduced recall responses to α-lactalbumin as assessed by low frequencies of IFNγ producing cells reactive to α-lactalbumin (p≤0.003). (b) Two days after completion of Sunitinib treatment, spleens were harvested and tested for recall responses to α-lactalbumin using ELISpot assay. Sunitinib administration after completion of priming to α-lactalbumin did not affect recall responses to α-lactalbumin as assessed by similar frequencies of IFNγ producers reactive to α-lactalbumin in the spleen of both Sunitinib treated and non-treated mice.

Sunitinib treatment causes a transient decrease in antigen presenting cells

To establish the mechanism by which priming is inhibited by Sunitinib, 6–8 week old BALB/cJ mice were immunized with α-lactalbumin and treated concurrently with Sunitinib according to the 7 days treatment protocol starting on the same day as the immunization. Lymph nodes, spleen and bone marrow were harvested from both Sunitinib treated and non-treated mice (n=6 per group) at 2, 6, 8 and 10 days after immunization and beginning of Sunitinib treatment. Characterization of CD4+ and CD8+ T cells as well as CD11b+CD11c+ myeloid cell populations in different lymphoid compartments after Sunitinib treatment revealed a significant reduction with time in percentage of CD11b+CD11c+ cells within 2 days of starting Sunitinib treatment both in the lymph nodes and the spleen. Although a significant reduction in number of CD11b+CD11c+ positive cells was observed at day 2 in the lymph nodes (p≤0.005; figure 4 a left panel), a more substantial effect was observed in the spleen with significant decrease in the number of CD11b+CD11c+ antigen presenting cells beginning at day 2 (p≤ 0.02) and continuing through day 6 (p≤ 0.005), day 8 (p≤ 0.0001) and day 10 (p≤ 0.03) (figure 4 a right panel). In both the lymph node and spleen compartments the drop in percentage of CD11b+CD11c+ cells was evident within 2 days of starting Sunitinib and continued till the end of Sunitinib treatment (day 7 of consecutive treatments beginning on day of immunization). This observed decrease in frequencies of CD11b+CD11c+ cells in the lymph nodes and spleens of Sunitinib treated mice was transient and lasted till Sunitinib administration continued. Cell numbers quickly recovered to levels equivalent to naïve mice within a few days after cessation of treatment and clearance of Sunitinib from the system (day 10). Control mice that were immunized with α-lactalbumin but not treated with Sunitinib did not show any decrease in CD11b+ CD11c+ cells and in fact showed a small increase in percentage of CD11b+ CD11c+ cells in lymph nodes and spleen at some time points after immunization consistent with active immunization and antigen processing in these compartments. No change in cell numbers or percentage of CD11b+ CD11c+ cells were observed in the bone marrow compartment of both Sunitinib treated or non-treated mice (n=4; data not shown). Similar decrease was also observed in total CD11b+ cells in both the lymph nodes (n=6; Day 2: p≤0.07; Day 6–10: p≤0.03; figure 4b left panel) and the spleen (n=6; Day 2: p≤0.2; Day 6–10: p≤0.0007; figure 4b right panel). No difference in number of CD3+CD4+ and CD3+CD8+ T cells was evident in animals treated with Sunitinib compared to controls in both the lymph nodes (figure 4c, left panel) and the spleen (figure 4c, right panel).

Figure 4. Sunitinib treatment causes a transient decrease in antigen presenting cells.

Figure 4

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In order to establish the mechanism of inhibition of priming by Sunitinib, 6–8 week old BALB/cJ mice were immunized with α-lactalbumin and treated simultaneously with Sunitinib according to a 7 day treatment protocol starting on the same day as the immunization. Lymph nodes and spleens were extracted from both Sunitinib treated and non-treated mice (n=6 per group) on day 2, 6, 8 and 10 after immunization/beginning of Sunitinib treatment. Results depicted show the percentage of (a) CD11b+CD11c+, (b) total CD11b+ cells, (c) ratio of CD3+CD4+ cells to CD3+CD8+ cells in lymph nodes (left panel) and spleen (right panel) at different time points after beginning Sunitinib administration. α-lactalbumin immunized mice treated with Sunitinib showed a decrease in (a) the percentage of CD11b+CD11c+ cells in the lymph nodes at day 2 (p≤0.005; figure 4a, left panel) and at day 2 (p≤ 0.02), day 6 (p≤ 0.005), day 8 (p≤ 0.0001) and day 10 (p≤ 0.03) in the spleen (figure 4a, right panel). The decrease in CD11b+CD11c+ cells was transient and lasted till Sunitinib treatment continued. Immediate recovery in cell numbers to levels equivalent to that of naïve mice occurred after termination of Sunitinib administration. (b) Similar decrease in percentage of total CD11b+ cells was observed after combination therapy with Sunitinib compared to vaccine alone beginning at day 2 both in the lymph nodes (Day 2: p≤0.07; Day 6–10: p≤0.03; figure 4b, left panel) as well as the spleen (Day 2: p≤0.2; Day 6–10: p≤0.0007; figure 4b, right panel). (c) No differences were observed in the ratio of CD4+ to CD8+ cells both in the lymph nodes (figure 4c, left panel) or in the spleen (figure 4c, right panel) after combination of Sunitinib with the α-lactalbumin vaccine.

DISCUSSION

In the current study we have shown that Sunitinib can inhibit priming to an immunogen by causing a reduction in critical antigen presenting cells especially within the lymph nodes, the primary site of antigen presentation and priming after immunization. The current study effectively demonstrates that concurrent delivery of Sunitinib with vaccination inhibits the boost in immune responses conferred by vaccination and that sequential delivery of Sunitinib after completion of the priming phase of immunization allows effective priming to the vaccine. Therefore, combinatorial therapy of Sunitinib with vaccination targeted against tumor specific antigens is feasible and advantageous but requires careful scheduling to avoid inhibition of priming to the vaccine.

Although the efficacy of tyrosine kinase inhibitors (TKIs) like Sunitinib in mediating control of tumor growth through their pleiotropic effects on multiple receptors such as the VEGF receptor, platelet derived growth factor (PDGF) receptor, FLT-3 and proto-oncogene c-Kit among other is well established (5, 10, 18, 19), their effect on anti-tumor immunity is not clear. There are conflicting reports on both immunostimulatory as well as immunosuppressive effects of the same tyrosine kinase inhibitor by different research groups. Our group as well as others have shown that Sunitinib treatment can modulate tumor growth by reducing accumulation of immunosuppressive, myeloid derived suppressor cells (MDSCs) infiltrating the renal tumor stroma (11,12, 15). In addition, numerous studies by us and others have demonstrated a role of tyrosine kinase inhibitors in potentiating immune responses against tumors through reduction in T regulatory cells (13–15), increase in CD8+ and CD4+ infiltrating lymphocytes within tumors (20) and reduction in expression of anti-inflammatory regulatory molecules on T cells, MDSCs and dendritic cells (DCs) (15). On the other hand, some inhibitory effects of Sunitinib on immune responses mainly as a result of reduction in T cell proliferation and function (16, 17, 21), inhibition of NK cell activity and TNF-α production and enhancement of IL-10 production in vitro and in vivo (22, 23) have also been demonstrated. Therefore the role of tyrosine kinase inhibitors in anti-tumor immunity is still not clear. Determining the effect of TKIs on cells such as T cells, dendritic cells and Natural Killer cells that are critical for tumor immunity may be key to evaluating their role in tumor inhibition. More importantly, it is key to evaluating the feasibility and efficacy of their combination with immunotherapeutic strategies for possibly more effective and long lasting treatment of cancers, especially those that are refractory to current chemotherapeutics.

The current study shows that Sunitinib administration results in a drastic reduction in the number of total CD11b+ and CD11b+CD11c+ antigen presenting cells in the lymph nodes and spleen of treated animals resulting in a major impact on antigen priming in these primary locations. Conflicting findings on both inhibitory and stimulatory effects of protein tyrosine kinase inhibitors on dendritic cells and cells of the macrophage lineage have been reported in previous studies. Tyrosine kinase inhibitors have been shown to stimulate dendritic cell function by increasing the number and expression of co-stimulatory molecules (24) and enhancement of type I interferon production (24, 25). In addition a number of studies show inhibitory effects of TKIs on maturation and function of DC’s by down regulation of MHC, CD40 and CD80 co-stimulatory molecule expression (26), inhibition of DC expansion (27, 28), inhibition of antigen presentation (14) and pro-inflammatory cytokine production (29). However in these and other studies (14), Sunitinib itself has not been shown to have inhibitory effects on dendritic cells and is considered to be the most ideal TKI among all others for use as an adjuvant with immunotherapeutics. Our study is the first to show an inhibitory effect of Sunitinib on antigen priming via depletion of the antigen presenting cell (APC) pool in locations critical to antigen presentation and T cell priming. We have shown that administration of Sunitinib can cause a decrease in number of CD11b+CD11c+ dendritic cells in the spleen and more critically in the lymph nodes but not in the bone marrow. Although this decrease in DC numbers is transient and lasts till Sunitinib administration continues, it can significantly impact tumor immunity by inhibiting antigen priming during active vaccination and result in failure to generate desired T cell responses against the targeted tumor specific antigens. We have shown in the current manuscript as well as in our previous work (Jaini et al, Nature Medicine, 2010, 16; 799–803) that the response to the α-lactalbumin vaccine is an inflammatory Th1 polarized response. Simultaneous Sunitinib administration results in a dramatic reduction in the availability of DCs in the peripheral immune compartment thereby completely abolishing the predominant type 1 response to the vaccine. The number of DCs after Sunitinib treatment is so drastically reduced that no IL-4 and only few IL-12 producing DCs are detectable in the spleen (data not shown). Therefore we believe that reduction in immune responses to the vaccine occur due to An overall reduction in type 1 DCs without any detectable deviation to type-2 DCs. Although the mechanism of Sunitinib mediated decrease in APC numbers is not clear it may have an underlying mechanism similar to that proposed by us for Sunitinib mediated apoptotic deletion of MDSCs (12) in tumor bearing mice. Our study clearly demonstrates the role of Sunitinib in deletion of APCs in critical lymphoid compartments and its negative impact on tumor vaccination mediated immunity characterized by lack of priming to the targeted tumor specific antigen and resulting failure of immunotherapeutic vaccination.

Although the reduction in myeloid derived CD11b+CD11c+ cells in the lymph node and spleens of mice is evident immediately after commencement of Sunitinib administration and is observed throughout the duration of administration, its most significant impact is seen only on the inhibition of priming with no substantial inhibition of subsequent immune responses. The selective and more profound impact of decrease in dendritic cells on the efficacy of priming is to be expected considering that the availability of a substantial population of antigen presenting cells would be more critical for overcoming the initial T cell activation threshold than for subsequent immune responses in an already primed system. Based on these findings we conclude that simultaneous Sunitinib administration can potentially inhibit immunotherapy protocols involving active immunization against tumor specific targets due to their dependence on the availability of a substantial population of antigen presenting cells to achieve required T cell activation (30). We reason that immunotherapy protocols that mediate tumor specific immune activation via antigen loaded dendritic cell transfer or passive transfer of activated antigen specific T cells will not be significantly inhibited by combination with Sunitinib (31). Recent Phase II trials with combinatorial therapy of Sunitinib with dendritic cell immunotherapy AGS-003 showed immense promise with prolonged survival in unfavorable risk metastatic renal cell cancer (32, 33). Similarly, immunotherapy protocols that employ frequent booster doses and repeated immunization could also be resistant to the inhibitory effects of Sunitinib since the availability of a substantial number of DCs would be critical only during the priming phase of an active immunization protocol.

Our data show that simultaneous administration of Sunitinib with any immunotherapy can be deleterious to generation of immune responses elicited by the vaccination/ immunotherapy process. Although sequential combination therapy with Sunitinib and α-lactalbumin allows generation of adequate immune responses against tumor antigens, we did not observe synergistic inhibition of tumor growth in the current study. Although, the current manuscript is not intended to demonstrate the most effective combination treatment for breast cancer, the lack of synergy after sequential therapy warrants discussion. This lack of synergy in spite of an adequate vaccine mediated immune response can be attributed to the limited efficacy of the α-lactalbumin immunotherapy protocol as a treatment for 4T1 tumors. It has been shown by us previously (12) as well as in the current study that even when Sunitinib broadly reverses peripheral MDSC accumulation, intra-tumoral MDSC can be significantly less affected and lead to restricted therapeutic efficacy on tumors such as the 4T1. This lack of reduction in intratumoral immune suppressors might also play a role in the eventual inefficacy of the vaccination protocol later during tumor progression; thereby affecting the possibility of synergy between the two therapies. We have used a single protein, single inoculation protocol as our immunotherapy protocol in order to dissect out the subtle effects of Sunitinib on the vaccine mediated immune response. A prime and boost protocol would most probably be more effective and show the desired synergy in therapeutic effect with the combination therapy. Alternatively, we could be restricted by the immunotherapy protocol being used and the effect shown in figure 1b is the best therapeutic effect that can be achieved by combining Sunitinib with α-lactalbumin targeted immune responses. More enhanced and synergistic effects may be possible with alternate immunotherapeutic protocols and booster doses. Breast tumor models other than the 4T1 cells that demonstrate sensitivity to Sunitinib and intratumoral MDSC reduction remain to be tested.

Recent studies have shown that continuous administration of Sunitinib prior to immunotherapy can precondition a more immuno-permissive environment to maximize efficacy of combinatorial therapy (34). In contrast, our study shows that presence of Sunitinib has adverse effects on active vaccination protocols when present during the initial priming phase. These contrasting demonstrations of immunostimulatory and inhibitory effects of Sunitinib on anti-tumor immunotherapies can be attributed to significant differences in drug administration as well as active priming and boosting protocols used in both studies. Our study employs a protocol with single immunization targeting a naturally expressed differentiation antigen on the tumor with no subsequent booster doses in order maintain be close to the physiological state and highlight any inhibitory effects of Sunitinib on immune responses to the vaccine. On the other hand, the study by Farsaci et al was performed in a transgenic mouse system targeting the overexpressed carcinoembryonic antigen with virus based vaccine and booster doses. These contrasting results reemphasize the caveat that Sunitinib administration schedules in reference to immunotherapy should be carefully designed for each study taking into account the mode of transfer (active or passive transfer) of anti-tumor immunity and protocols of vaccination and booster doses to avoid inhibition of vaccination.

In light of the multidimensional effects of Sunitinib directly on the tumor as well as on anti-tumor immune responses we hypothesized that its combination with α-lactalbumin immunotherapy will improve the therapeutic efficacy against advanced or late stage breast tumors. We find that the two therapeutic modalities when combined have the potential to provide greater inhibition of tumor progression; however the timing of administration of both therapies in reference to one another is critical and should be carefully scheduled before or after the priming phase in order to allow effective priming to the immunogen.

Significance and Impact.

We show for the first time that Sunitinib inhibits antigen presentation and priming to the target antigen when administered simultaneously with active immunotherapeutic vaccination. However, delivery of Sunitinib combination therapy, timed to avoid the critical priming phase of active immunization can potentially enhance anti-tumor therapeutic efficacy. Our results emphasize that combination of Sunitinib with immunotherapeutics is feasible but requires careful scheduling of drug administration, specific for the immunotherapy protocol being used for boosting anti-tumor immune responses.

Acknowledgments

This work was supported by the U.S. National Institutes for Health grant RO1CA-150959 (JHF) and R01CA-140350 (V.K.T.).

Abbreviations used

CFA

Complete Freund’s Adjuvant

D5/9/13

Day5/9/13

DC

Dendritic cells

DMEM

Dulbecco’s modified eagle’s medium

ELISPOT

Enzyme linked immunospot

Flt-3

fms like tyrosine kinase-3

MDSC

Myeloid derived suppressor cells

MHC

Major Histocompatibility Complex

STAT3

Signal transducer and activation of transcription 3

TILs

Tumor Infiltrating Lymphocytes

TKI

Tyrosine kinase inhibitors

VEGF

Vascular endothelial growth factor

Treg

T regulatory cells

Footnotes

Conflict of interest statement: The authors declare no competing financial interests.

Contributor Information

Ritika Jaini, Email: jainir@ccf.org.

Patricia Rayman, Email: raymanp@ccf.org.

Peter A. Cohen, Email: cohenpeter@mayo.edu.

James H. Finke, Email: finkej@ccf.org.

Vincent K. Tuohy, Email: tuohyv@ccf.org.

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