Immunomodulating effects and response prediction of the combined treatment with anti-angiogenic agents and immunotherapy in preclinical models of renal cell carcinoma

Abstract

The introduction of immunotherapy revolutionized the clinical approach of Renal Cell Carcinoma (RCC). RCC is the most common type of kidney cancer, accounting for 80 to 85% of all renal neoplasms and for 2% of all cancer diagnosis worldwide. The clear cell RCC (ccRCC) is the most represented subtype, accounting for approximately 70% of all RCCs. Metastatic ccRCC has long been considered an immunogenic malignancy, susceptible to the effect of immunotherapy. However, in spite of the little success observed with the use of cytokines, the approval of anti-angiogenic tyrosine kinase inhibitors (TKIs) from 2005 followed by the development of immune checkpoint inhibitors (ICIs) represented a real breakthrough in the management of ccRCC. In addition, the demonstration that TKIs as axitinib, whose main targets is VEGFR, play a role in the immune modulation provided the rationale for the association of TKIs and ICIs and between 2019 and 2021 several of these associations were approved as first line treatment in RCC patients. In this rapidly evolving scenario, there is still scarcity of evidences concerning the best strategy to adopt in patients that progress after a first line treatment consisting of the association of TKIs and immunotherapy. To date, the second line therapy should be any VEGF target therapy that has not already be used in association with immunotherapy. Therefore, it is imperative to define new tailored approaches able to identify the better treatment to choose among further TKIs, ICIs, or also mTOR inhibitors after a previous axitinib-based first line setting. In this context, preclinical in vitro and in vivo studies could help identify immunomodulating mechanisms, revealing the molecular and immune context generated by the drug exposure in the tumor and the host. To this end, our study aims to evaluate in vitro and in vivo, in a syngeneic murine RCC model, the effect of the combination of axitinib and avelumab or pembrolizumab (anti-PD-L1 or anti-PD-1 antibodies, respectively), in terms of modulation of the immune response and anti-cancer efficacy to search for preclinical hints that could lead the future clinical choices. Our research focused on the investigation of the immunomodulatory effect of axitinib alone or in combination with anti-PD-(L)1 agents on a panel of RCC cell lines and PBMCs, both human and murine. Particularly we observed that axitinib treatment induced the expression of the immune checkpoint PD-L1 on the whole panel of tumour cells (769-P and Caki-2, human and RENCA, murine). In addition to its effect on cancer cells, axitinib exposure reduced the activity of murine T cells activity, while it diminished the surface expression of proteins involved in the down regulation of the immune response as PD-1, TIM-3 and LAG-3 on both human and murine PBMCs, thus preventing T cell exhaustion. Notably, the addition of avelumab or its murine analogue resulted in the amplification of this effect. Overall, the increase in PD-L1 induced by TKI could induce a stronger effect of the anti-PD-L1 or PD-1 antibodies, providing the biological rationale for their combination. The setting of a syngeneic co-culture system of RENCA cells and murine PBMCs allowed us to obtain a better picture of the complex landscape of interactions happening between tumor and immune cells. Under this condition we demonstrated that the addition of an anti-PD-1 agent to axitinib-treated cells produced an increase lymphocyte activity, quantified both as cytotoxic activity against tumor cells, and as IFN-g production. These findings underline the immunomodulatory role of axitinib both on tumor cells as well as on lymphocytes activation status. The second part of the research was focused on the study of the relationship between gut microbiota and responsiveness to treatment. Indeed, in the last few years, emerging data are demonstrating the role of the intestinal microbiota in response to therapy, and how gut bacteria composition could influence the clinical outcome. With regards to RCC, it has been demonstrated that among gut bacteria population, the relative abundance of a beneficial commensal, Akkermansia muciniphila, is associated with the elicitation of immune responses beneficial against cancer. On these bases we evaluated whether standard therapies could induce a taxonomic deviation of stool composition in Balb/c mice treated with a sequential approach that mimics the first- and second-line standard approaches adopted for RCC. In addition, we evaluated the impact of gut microbiota on first line standard therapies, exploring the role of different strains of Akkermansia muciniphila to ICIs therapies in avatar mouse model. Our findings demonstrated that the presence of Akkermansia strains SGB9228 and SGB9226 in gut microbiota increased mice survival in animals treated with ICIs, suggesting the beneficial role of this commensals in the response to the standard ICI treatment, and indicating that the evaluation of the gut microbiota composition could represent a valuable tool to stratify patients.