1003 Oral nano-delivery of a gut microbial metabolite enhances T cell stemness for cancer immunotherapy

Background Metabolites from the gut commensal microbes are being revealed as critical messengers to regulate the performance of immune checkpoint blockers (ICB). However, identification of key microbial metabolites, their poor pharmacokinetic profiles, and elusive mechanism remain as the major limitations for clinical translation of microbial metabolites. In particular, the role of microbial metabolites in T cell immunity and fitness is largely unknown in the context of ICB cancer therapy. Thus, the development of microbial metabolites based formulation for improved anti-tumor T cell immunity in ICB therapy is highly challenging and desirable.Methods We examined if a library of microbial metabolites can induce memory T cell differentiation and enhance T cell stemness. Using 3,4-dihydroxybenzoate (DHB), a microbial metabolite selected from a screening campaign, we studied its effects on T cell metabolism and fitness via analyses of RNA/ATAC-sequencing, metabolomics, seahorse assay, and Western blotting. Moreover, orally available DHB-based prodrug 201 emulsion formulation was prepared. We assessed the anti-tumor efficacy of prodrug 201 plus α-PD-1 therapy in various mouse syngeneic tumor models, including CT26, B16F10, and Nooc1. Tumor and tumor draining lymph nodes were collected and analyzed for phenotypic and functional assessment of memory CD8 T+ cells.Results Through an in vitro screening of gut microbial metabolites, we identified 3,4-dihydroxybenzoic acid (DHB) that improved adoptive T cell therapy and enhanced CD8+ T cell stemness by suppressing glycolysis and regulating the Akt-mTORC1-Myc pathway. To harness the potency of DHB for systemic cancer immunotherapy, we engineered a DHB prodrug nano-emulsion, significantly increasing its oral absorption and half-life. RNA-seq analysis revealed that DHB treatment upregulated pathways related with cellular growth and proliferation and genes associated with stem-like properties of CD8 + cells. We found that glycolysis inhibition was the key contributor to DHB-induced memory T cells. Notably, DHB formulated into prodrug nano-emulsion markedly improved the oral bioavailability of DHB up to 14-fold. In multiple murine tumor models, the oral nano-emulsion enhanced the expansion of antigen specific, stem-like memory CD8+ T cells, sensitizing tumors to anti-PD-1 blockade and exerting robust antitumor efficacyConclusions We have developed a novel microbial metabolite-based immunotherapy by integrating nanotechnology, which establishes a mechanistic link between gut microbiota and T cell immunity, offering a promising approach for cancer immunotherapy. We surmise that our strategy will broaden the applications of beneficial metabolites in cancer immunotherapy.