Repurposing Sunitinib with Oncolytic Reovirus as a Novel Immunotherapeutic Strategy for Renal Cell Carcinoma

In this study, we demonstrate the ability to repurpose sunitinib, a multi-tyrosine kinase inhibitor and first-line metastatic renal cell carcinoma (RCC) agent, to potentiate the immunotherapeutic efficacy of the oncolytic virus, reovirus. This novel therapeutic strategy not only improved tumor responses and overall survival in a murine model of RCC, but also, resulted in the generation of a systemic protective anti-tumor immune response. We validated our findings in a murine model of lung squamous cell carcinoma (SCC), highlighting the potential broad applicability of this treatment approach. Taken together, these results provide clear rationale to investigate this promising viroimmunotherapeutic strategy in early phase clinical trials for a broad range of tumor histologies. Abstract Purpose: In addition to their direct cytopathic effects, oncolytic viruses are capable of priming anti-tumor immune responses. However, strategies to enhance the immunotherapeutic potential of these agents are lacking. Here, we investigated the ability of the multi-tyrosine kinase inhibitor and first-line metastatic renal cell carcinoma (RCC) agent, sunitinib, to augment the anti-tumor immune response generated by oncolytic reovirus. Experimental design: In vitro , oncolysis and chemokine production were assessed in a panel of human and murine RCC cell lines following exposure to reovirus, sunitinib or their combination. In vivo , the RENCA syngeneic murine model of RCC was employed to determine therapeutic and tumor-specific immune responses following treatment with reovirus (intra-tumoral), sunitinib or their combination. Parallel investigations employing the KLN205 syngeneic murine model of lung squamous cell carcinoma (NSCLC) were conducted for further validation. Results: Reovirus mediated oncolysis and chemokine production was observed following RCC infection. Reovirus monotherapy reduced tumor burden and was capable of generating a systemic adaptive anti-tumor immune response evidenced by increased numbers of tumor-specific CD8 + IFN-γ producing cells. Co-administration of sunitinib with reovirus further reduced tumor burden resulting in improved survival, decreased accumulation of immune suppressor cells and the establishment of protective immunity upon tumor re-challenge. Similar results were observed for KLN205 tumor bearing mice, highlighting the potential broad applicability of this approach. Conclusion: The ability to repurpose sunitinib for augmentation of reovirus’ immunotherapeutic efficacy positions this novel combination therapy as an attractive strategy ready for clinical testing against a range of histologies, including RCC and NSCLC. cell populations in mediating resistance to reovirus-mediated immunity our results suggest sunitinib prevention of MDSC and Tregs accumulation as a potential mechanism for enhancing reovirus mediated adaptive immunity. Our laboratory is currently focused on elucidating the precise immune mechanism underlying the observed immunotherapeutic efficacy of reovirus-sunitinib combination therapy. Taken together, we believe these findings provide proof-of-principle for the study of this novel viroimmunotherapeutic paradigm against a broad range of malignancies, including RCC. Beyond this, as sunitinib is currently a first line mRCC approved therapeutic and reovirus is in advanced phase III clinical trials, we believe the rapid translation of these findings in the setting of a clinical trial for patients with this incurable cancer is warranted.


Translational relevance
In this study, we demonstrate the ability to repurpose sunitinib, a multi-tyrosine kinase inhibitor and first-line metastatic renal cell carcinoma (RCC) agent, to potentiate the immunotherapeutic efficacy of the oncolytic virus, reovirus. This novel therapeutic strategy not only improved tumor responses and overall survival in a murine model of RCC, but also, resulted in the generation of a systemic protective anti-tumor immune response. We validated our findings in a murine model of lung squamous cell carcinoma (SCC), highlighting the potential broad applicability of this treatment approach. Taken together, these results provide clear rationale to investigate this promising viroimmunotherapeutic strategy in early phase clinical trials for a broad range of tumor histologies.

Introduction
Despite the improvements in progression free (PFS) and overall survival (OS) associated with the use of targeted therapy, metastatic renal cell carcinoma (mRCC) remains an incurable disease. With a five-year survival rate of less than 10% this malignancy remains a significant health issue [1]. As such, the need for the development of novel therapeutic strategies for this disease is obvious. Sunitinib, a multi-tyrosine kinase inhibitor targeting VEGFR, PDGFR, C-KIT, RET, CSF-1R and FLT-3 is currently a first line therapy for mRCC [2]. While this drug has historically been associated with potent anti-angiogenic activity, recent clinical studies have highlighted its immune modulatory effects. Indeed, following two cycles of oral sunitinib therapy at 50 mg daily for 4 weeks every 6 weeks, mRCC patients display an increase in the percentage of IFN-γ producing T-cells relative to treatment naive patients [3]. Moreover, the immune suppressive type-2 T-cell cytokine response and accumulation of T regulatory cells (Treg) and myeloid derived suppressor cells (MDSC) that are characteristic of mRCC patients is reversed following sunitinib therapy [3,4].
Accordingly, this evidence has generated significant interest in targeting Tregs and MDSC with sunitinib to reverse the mRCC induced immunosuppressive microenvironment and enhance the anti-tumor immune response generated by immunotherapeutics [5]. The feasibility of this approach has been demonstrated in an immunocompetent syngeneic murine model of RCC (RENCA) in which the downregulation of MDSC and Treg by sunitinib enhanced intratumoral infiltration and activation of adoptively transferred CD8 + T-cells, leading to tumor regression [6].
Furthermore, sunitinib has also been demonstrated to reduce tumor burden and improve OS in mouse models of melanoma, hepatocellular carcinoma and colorectal metastasis in immunotherapeutic models, highlighting the efficacy and widespread utility of this approach against multiple histologies [7][8][9].
In the current study, we investigated the ability of sunitinib to enhance the antitumor immune response generated by the dsRNA oncolytic reovirus. To date, reovirus is one of the most clinically advanced oncolytic viruses (OV), demonstrating modest efficacy in phase II clinical trials as a monotherapy and in combination with platinum and taxane based chemotherapy across multiple solid malignancies [10][11]. Of the over 1,500 patients treated thus far with this agent, the maximum tolerated dose has not been reached, highlighting its tolerability and safety. As such, reovirus is one of the most promising OV currently being investigated in the clinic. Despite this however, early phase monotherapy clinical trials have failed to demonstrate appreciable clinical effectiveness in the form of objective and durable complete responses, highlighting the need to augment reovirus' therapeutic potency. Interestingly, in experimental murine models, reovirus administration is capable of priming innate and adaptive anti-tumor immune responses [12][13][14]. This anti-tumor immunity has not only been shown to contribute to reovirus' therapeutic efficacy, but has also been demonstrated to result in the generation of long-term tumor immunosurveillance [15][16][17]. Moreover, reovirusmediated anti-tumor immunity has been demonstrated clinically in a phase I trial of a single intra-prostatic injection of reovirus that involved 6 patients with localized prostate cancer [18]. In this study, despite the robust anti-viral neutralizing antibody response that was seen, a significant number of tumor infiltrating CD8 + T-cells were present following reovirus injection. Likewise, in a phase I clinical trial of intravenous reovirus administration involving patients who had previously received cytotoxic chemotherapy and radiotherapy, increased CD8 + T-and NK-cell (CD3 -/CD56 + ) numbers were observed in the peripheral blood [19]. Hence, in addition to its direct oncolytic effects, reovirus is also a potent immunotherapeutic. While a considerable amount of literature supports reovirus' immunotherapeutic potential, most pre-clinical investigations have instead focused on improving the systemic delivery of the virus with immunosuppression or histology relevant cytotoxic chemotherapy, respectively [20,21].
As such, strategies to augment reovirus mediated anti-tumor immune responses for generation of long-term protective immunity capable of improving clinical objective responses are warranted.
Herein, the ability to augment reovirus's immunotherapeutic efficacy through combination with sunitinib was explored utilizing clinically relevant syngeneic murine models. Collectively, this work highlights the promise of combining reovirus with sunitinib as a novel viroimmunotherapeutic approach, supporting the investigation of this strategy in phase II clinical trials. Grand Island, NY). All media was supplemented with 10% heat-inactivated fetal calf serum (FCS) (Invitrogen, Grand Island, NY). Cultures were free of antibiotics and negative for mycoplasma as determined by routine testing. Reovirus, Dearing strain serotype 3 was grown in L-929 cells then purified and tittered as previously described [18]. Sunitinib was purchased from Selleck chemicals (Houston, TX).

Methods
Cell Viability Assay. Cells were seeded at a density of 3 × 10 3 (A498, 786-0) or 8 × 10 3 (ACHN, RENCA) cells/well into 96 well micro-titre plates and incubated for 24 hours in 10% FCS containing media. Reovirus, sunitinib or their combination were then added to each well for 48 hours. Subsequently, drug containing media was replaced with media containing WST-1 (diluted 10:1) and absorbance was quantified utilizing a BioTek® plate reader (Winooski, VT). Percent viability was calculated as the absorbance ratio of were then freeze-thawed 3 times and to quantify progeny production, supernatants were harvested and plaque titrated on monolayers of L-929 cells in semi-solid medium for 72 hours as previously described [18].  For tumor re-challenge studies, Balb/C RENCA tumor bearing mice successfully treated with reovirus + sunitinib combination therapy were re-challenged with 1 x 10 6 RENCA cells in the contralateral hindflank and tumor burden was assessed by caliper measurement. Treatment naïve Balb/C mice were challenged in the same manner to serve as a control.
CD8 + Enrichment. Spleens were processed as described above from Balb/C or DBA/2 mice bearing RENCA or KLN205 tumors, respectively, and enriched for CD8 + T-cells Statistics. Statistical analysis was preformed utilizing Graph Pad version 6. Unpaired two-tailed t-tests and two-way ANOVA followed by Bonferroni Post Hoc tests were used to determine significance between experimental groups. Kaplan-Meier analysis together with log-rank sum test was utilized to determine significant for in vivo survival benefits. Statistical significance was defined as p-values being < 0.05 unless otherwise stated.    Reovirus has been demonstrated to initiate innate immune responses characterized by the production of pro-inflammatory cytokines including RANTES, MIP-1-α, MCP-1, KC, IP-10 and MIG across a variety of melanoma and prostate cancer cell lines in addition to its direct oncolytic effects [14,17]. Based on these findings we characterized the ability of reovirus to stimulate production of these chemokines during infection of the murine RCC RENCA cell line. This cell line was studied to provide proof-  To assess in vitro synergy between reovirus and sunitinib, combination index (CI) values as per the Chou and Talalay method were determined [23]. Dose response data for reovirus and sunitinib on RCC cells was used to calculate ED 50 values ( Figure.    The RENCA murine model of RCC is an established immunocompetent syngeneic model for studying novel therapeutics including immunotherapeutics against this disease preclinically [24]. This model was employed to investigate whether or not reovirus has therapeutic efficacy against RCC in vivo and determine the ability of sunitinib to augment this activity. Sunitinib dose response studies were conducted in vivo to determine the optimal dose to administer in combination with reovirus. A dose range of 20 to 60 mg/kg was chosen as it is clinically relevant and has previously been utilized in the RENCA model [6].  As reovirus infection results in the production of pro-inflammatory cytokines and consequent priming of innate and adaptive immunity [14][15][16][17], the anti-tumor immune response generated by reovirus treatment of RCC in vivo was determined. Mice were treated as in Figure 2A with sunitinib, reovirus or combination therapy and CD8 + splenocytes were harvested and co-cultured with RENCA cells or reovirus infected RENCA cells for antigenic stimulation prior to IFN-γ ELISA. In mice receiving reovirus monotherapy a significant IFN-γ response was demonstrated from harvested CD8 + splenocytes following stimulation with reovirus infected RENCA cells (Figure. 3A).

Reovirus replicates in human and
Interestingly, sunitinib therapy significantly augmented this reovirus generated antitumor immunity, as a robust increase in IFN-γ production (4-fold) in those mice that on July 9, 2020. © 2016 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
Author Manuscript Published OnlineFirst on May 24, 2016; DOI: 10.1158/1078-0432.CCR-  received reovirus in combination with sunitinib was observed ( Figure. 3A). In mice receiving PBS, DV or sunitinib, minimal or no IFN-γ response was seen. Notably, while CD8 + reactive splenocytes were not observed in reovirus or combination therapy treated mice by ELISA when RENCA cells were used alone as an antigenic stimulus, their presence was confirmed by the higher sensitivity ELISPOT assay ( Figure. Figure. 3E) [26]. However, as expected, CD4 depletion did not abrogate the therapeutic effects of reovirus or combination therapy. Taken together, these results suggest that reovirus generates a systemic adaptive anti-tumor immune response against RCC that is augmented by sunitinib.
As sunitinib is known to reverse tumor induced immunosuppression [4], we next immunophenotyped splenocytes and tumor-infiltrating immune populations in mice receiving reovirus, sunitinib or combination therapy. Consistent with a viroimmunotherapeutic effect, reovirus monotherapy resulted in an increased accumulation of CD8 + splenocytes, as well as, CD8 + tumor-infiltrating lymphocytes ( Figure. 4A-C). This was associated with a concomitant accumulation of splenic and intra-tumoral MDSC as well as intra-tumoral Tregs ( Figure. Figure. 4D-F), highlighting this as a potential mechanism explaining sunitinib's ability to augment reovirus-mediated adaptive immune responses ( Figure. 3A-C).
To determine whether reovirus or combination therapy resulted in the establishment of a protective immune response, adoptive transfer experiments were conducted. Here, splenocytes from mice treated with reovirus, sunitinib, or combination therapy were isolated and intravenously transferred into treatment-naive mice ( Figure   5A). These mice were then challenged with a s.c injection of RENCA cells and followed for tumor initiation and burden. Interestingly, only those mice receiving splenocytes from mice treated with combination therapy demonstrated reduced tumor growth rate relative to controls, highlighting an established protective immune response in this group (P < 0.05 between all groups by two-way ANOVA) (Figure 5B). To further support these findings tumor re-challenge experiments were conducted with mice that had achieved a complete response to combination therapy versus mice that were treatment naïve.
Indeed, this demonstrated that pre-treated cured animals were protected against tumor re-challenge whereas naïve mice formed rapidly growing tumors ( Figure 5C). Overall, these results confirmed establishment of protective immunity following combination therapy.  (Supplementary Figure. 5A-C). In vivo, combination therapy also resulted in decreased KLN205 tumor burden as well as improved overall survival relative to monotherapy ( Figure. 6A-B). Moreover, this effect was associated with increased splenic CD8 + lymphocytes with a memory phenotype [CD8 + /CCR7 + /CD62L -] ( Figure. 6C-D) and a concomitant prevention of reovirus-induced MDSC accumulation ( Figure. 6E). Furthermore, isolated CD8+ splenocytes from mice treated with combination therapy demonstrated increased effector function in co-culture cytotoxicity assays with KLN205 cells, confirming enhanced adaptive immunity (Figure. 6F). Taken together, these results establish a viroimmunotherapeutic benefit for reovirus-sunitinib combination therapy against murine lung SCC, highlighting the broad potential of this novel treatment paradigm.

Discussion
Metastatic renal cell carcinoma remains an incurable disease with an urgent need for novel therapeutics to be developed that impact overall survival.
Immunotherapeutic strategies against RCC are of particular interest given that durable complete responses have only been demonstrated in patients receiving cytokine (IL-2) immunotherapy, as well as recent evidence highlighting the efficacy of immune checkpoint inhibition of PD-1 [27][28][29]. The current study represents the first preclinical This not only positions reovirus-sunitinib combination therapy as an attractive novel treatment strategy, but further supports the growing body of literature highlighting the benefit of harnessing the immune system to improve oncolytic virotherapy.
The conducted in vitro studies demonstrate the ability of reovirus to induce oncolysis against RCC, as has been described for multiple tumor histologies ( Figure. 1A-C). Oncolysis of human RCC cell lines was seen within 48 hours of infection with all ED 50 values being less than 40 MOI, which is comparable to that seen with other solid malignancies [18,[30][31][32]. Notably, the RENCA cell line demonstrated significantly greater in vitro sensitivity relative to human RCC cells, consistent with previous reports highlighting increased murine cell line sensitivity to reovirus [33]. While the precise mechanism for the enhanced sensitivity remains unknown, this observation suggested a direct oncolytic in vivo response could be achieved. As such, the established sensitivity of the RENCA cell line coupled with the observed production of pro-inflammatory cytokines known to be involved in the priming of reovirus mediated innate and adaptive immunity (RANTES, MIP-1α, MCP-1, KC, IP-10 and MIG) supported its use for in vivo investigation of reovirus immunotherapeutic efficacy against RCC as a model to establish pre-clinical proof-of-principle [14,17] (Figure. 1D). Moreover, these results also highlight that similar to infection of melanoma and prostate cancer cells, reovirus replication induces an inflammatory cell death response against RCC [14,18]. The therapeutic utility of the in vitro studies was confirmed in vivo utilizing the RENCA immunocompetent murine model of RCC. Treatment of mice with reovirus and sunitinib resulted in a reduction in tumor burden relative to both PBS and DV controls ( Figure. 2A). Furthermore, in those mice receiving reovirus and sunitinib, tumor specific CD8 + splenocytes could be isolated ( Figure. 3A-B), highlighting the ability to generate a systemic adaptive anti-tumor immune response.
The combination of OV with relevant standard-of care treatments will likely be essential for their successful translation into clinical practice. The observation that the combination of sunitinib, a first-line mRCC therapeutic, with reovirus resulted in a significant regression in tumor burden and improved overall survival relative to either of these agents given as monotherapies, providing proof-of-principle to support the use of these agents in combination for superior anti-tumor activity ( Figure. 2A-B). The potential to broaden this treatment paradigm to other histologies was confirmed by demonstrating combination therapy efficacy in the KLN205 murine lung SCC model ( Figure. 6A-B). While the observed benefit in this model was significant, it was not as robust as seen with the RENCA model possibly due to disparities in sensitivity to reovirus oncolysis or differences in immunogenicity.
To better understand the direct cytotoxic effects of reovirus-sunitinib combination therapy on RCC, the Chou and Talalay method was utilized [23]. This line of experimentation demonstrated the potential for reovirus and sunitinib to synergistically induce cell death, highlighting enhanced direct cytotoxicity as a plausible mechanism explaining the superior therapeutic efficacy seen with these agents in combination in vivo ( Figure. 1F). Interestingly a recent report has demonstrated the ability to sunitinib on July 9, 2020. © 2016 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.
to enhance viral replication through targeting innate immune pathways such as double stranded RNA protein Kinase R (PKR) and RNaseL, shedding light on a possible mechanism for the observed in vitro synergy [3]. Moreover, as reovirus mediated apoptosis and autophagy have previously been well characterized in other cancer cell lines and ex vivo human tumor specimens [35][36][37][38][39], we hypothesize the observed synergy may reside in sunitinib's ability to sensitize RCC cells to these alternate modes of cell death.
In addition to direct synergistic effects on cell viability these results also support augmentation of anti-tumor immunity as a contributing mechanism mediating the observed in vivo synergy between reovirus and sunitinib. This is evidenced by a significant increase in the production of IFN-γ from tumor specific CD8 + splenocytes isolated from mice treated with reovirus-sunitinib combination therapy ( Figure 3A-B).
Augmentation of a therapeutic immune response by sunitinib is further established through our CD8 depletion ( Figure 3D-E vasculature has also been observed through intratumoral endothelial cell sensitization to oncolytic virotherapy following resolution of sunitinib therapy [41]. As such, it is likely that sunitinib and reovirus work through multiple independent mechanisms to achieve the observed in vivo synergy including both immune and non-immune mediated. Sunitinib also has been reported to enhance therapeutic immune responses through reversing tumor induced immune suppression in patients with mRCC [3]. This activity resides partly in its ability to downregulate the levels of circulating and intratumoral MDSC, which have been demonstrated to orchestrate mRCC immune suppression through direct T-cell inhibition as well as stimulating the upregulation of Tregs [3][4]. Given this, mechanistic investigations were focused on correlating MDSC and Treg levels with tumor specific CD8 + splenocyte IFN-γ production between monoand combination-therapy groups in vivo. Consistent with an inflammatory cell death, reovirus monotherapy administration induced a marked rise in both immune stimulatory (CD8 + and CD4 + lymphocytes) and suppressor (MDSC and Treg) populations within the spleen and tumor, similar to recent reports in the IP8 ovarian peritoneal carcinomatosis model [33] ( Figure. 4A-F). In keeping with our hypothesis, the accumulation of immune suppressor cells could be prevented by combination of reovirus with sunitinib, correlating with the observation that mice treated with combination therapy produce more tumor specific CD8 + splenocytes. These results were consistent with our findings in the KLN205 murine lung squamous cell carcinoma model, where mice treated with reovirus-sunitinib combination therapy had increased numbers of tumor reactive CD8 + cells and increased memory T cells, while splenic MDSC accumulation was prevented ( Figure. 6C-F). Given the previously documented ability of these immune suppressor cell populations in mediating resistance to reovirus-mediated immunity [42], our results suggest sunitinib prevention of MDSC and Tregs accumulation as a potential mechanism for enhancing reovirus mediated adaptive immunity. Our laboratory is currently focused on elucidating the precise immune mechanism underlying the observed immunotherapeutic efficacy of reovirus-sunitinib combination therapy.
Taken together, we believe these findings provide proof-of-principle for the study of this novel viroimmunotherapeutic paradigm against a broad range of malignancies, including RCC. Beyond this, as sunitinib is currently a first line mRCC approved therapeutic and reovirus is in advanced phase III clinical trials, we believe the rapid translation of these findings in the setting of a clinical trial for patients with this incurable cancer is warranted.

Financial Support
The Alberta Cancer Foundation supported this work through a graduate studentship award to K.A. Lawson. D.G. Morris is supported with operating grants from the Alberta Cancer Foundation and Alberta Health Innovates Solutions.   , or a combination of these agents. Sunitinib was given daily for 14 consecutive days starting on day 5 post RENCA implantation. Reovirus was administered three times (day 8,11,14). A) Tumor size was followed with caliper measurements. N = 6 mice per group. Error bars = SEM of tumors within each group. P < 0.001 (***), 0.01 (**), 0.05 (*) by two-way ANOVA. Data representative of two independent experiments. B) Kaplan-Meier plot of mice treated as per panel A. N = 8 mice per group. Analysis represents data pooled from two independent experiments involving 3 and 5 mice, respectively. P < 0.001 (***) by log-rank between PBS and RV or Sunitinib + RV. P < 0.05 (*) by log-rank between RV and Sunitinib + RV.    [17]. Briefly, mice (donor) were implanted with RENCA tumors and treated as shown in figure 2A. On day 19, splenocytes were harvested and transferred to treatment naive mice (recipient) by tail vein injection, followed by RENCA (2.5 x 10 6 cells s.c.) tumor challenge. Arrow indicates treatment start date, X indicates treatment end date. B) Tumor size in recipient mice challenged with RENCA cells s.c. N = 6 mice/group. Error bars = SEM of tumors within each group. P < 0.01 (**), by twoway ANOVA. Data representative of two independent experiments. C) Pre-treated (reovirus + sunitinib) mice demonstrating curative response (N = 3) and a cohort of treatment naïve mice (N = 5) were challenged with RENCA tumors (1 x 10 6 cells, s.c). Tumor burden over time is displayed. P < 0.001 (***), by two-tailed students t-test. Sunitinib was given daily for 14 consecutive days starting on day 6 post KLN205 implantation. Reovirus was administered four times (day 9,12,15,18). A) Tumor size was followed with caliper measurements. N = 8 mice per group. Error bars = SEM of tumors within each group. P < 0.001 (***), 0.01 (**), 0.05 (*) by two-way ANOVA. B) Kaplan-Meier plot of mice treated as per panel A. N = 4 mice per group. P < 0.05 (*) by log-rank between PBS or RV or Sunitinib and Sunitinib + RV. C-E) Splenocytes from KLN205 tumor-bearing mice treated as per panel A (sacrificed day 23) were immunophenotyped by flow cytometry. In all panels error bars = SEM of experimental triplicate. P < 0.001 (***), 0.01 (**), 0.05 (*) by two-tailed students t-test. F) CD8 + splenocytes were isolated from KLN205 tumor-bearing mice treated as per panel A and co-cultured with KLN205 cells. % KLN205 cytotoxicity was determined by LDH assay. Error bars = SEM of experimental triplicate. P < 0.05 (*) by two-tailed students t-test.