Modulation of P2X receptor signaling in intestinal microbiota/host cross talk to improve the efficacy of immunotherapy in solid tumors.

Immune checkpoint blockade (ICB) has revolutionized the therapeutic approach to previously incurable solid tumors. However, in the majority of patients, ICB efficacy is limited by primary or acquired resistance, thereby significantly affecting the cure rate. Several studies have demonstrated that the gut microbiota affects ICB efficacy, but the mechanisms underlying this effect have not been elucidated yet. We have previously demonstrated that extracellular ATP released by bacteria in the gut modulates the generation of secretory IgA (SIgA) by limiting T follicular helper (Tfh) cells activity via the ATP-gated ionotropic receptor P2X7. Accordingly, the reduction of intestinal ATP by administration of E. coli transformants expressing phoN2 from Shigella flexnery (E. colipApyr), that encodes for a periplasmic ATP-diphosphohydrolase (apyrase), resulted in increased SIgA production. We have shown a significant enhancement of tumor growth control in both C57BL/6 and Balb/c mice bearing subcutaneous melanoma or colorectal adenocarcinoma by combining oral administration of E. colipApyr or recombinant apyrase (rApyr) with anti-PD-L1 or anti-CTLA4 with respect to standalone antibodies or associated with an E. coli expressing a loss of function isoform of the apyrase enzyme, a vector control (E. colipHND19 or E. colipBAD28) or PBS vehicle control. We correlated the tumoricidal effect to significantly increased tumor infiltration of gut-derived cytotoxic CD8+ T cells. Taking advantage of clinically relevant cancer mouse models, single-cell genomic techniques, in-vivo tracking of fluorescently-labelled immune cells and metabolomic, this project is aimed at defining how modulation of P2X receptors signalling within the intestinal microenviroment improves the efficacy of ICB therapies. In aim 1 and 2, we will define the ontogeny, differentiation trajectory and phenotype of tumor infiltrating CD8+ T cells with enhanced cytotoxic activity by studying their TCR repertoire, by addressing whether an intestinal niche for the generation of these cells is fostered, as well as formally addressing the phenotype of these gut-derived cells by direct in-vivo fluorescent labelling. In aim 3, since secretory IgA (SIgA) plays a fundamental role in the observed therapeutic effect, we will further characterize SIgA coated bacteria and perform metabolic profiling of the ileal content and portal vein blood of mice conditioned by apyrase. This approach will allow us to identify potential novel candidates to design therapies or dietary strategies aimed at enhancing the efficacy of ICB treatments and provide a mechanistic base for the observed phenotypes. As a part of the COST action CA21130, this project is of particular relevance as it aims at providing a solid proof-of-concept regarding modulation of eATP and inter-cellular signalling through P2X receptors in the context of boosting the efficacy of approved cancer immunotherapies. Furthermore, our results can provide basis for future collaborations with other working groups of this COST action to test our therapeutic strategy in other disease models.