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-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 Among various microbial metabolites we examined, DHB highly increased the differentiation of CD44⁺CD62L⁺ central memory CD8 T cells, the frequency of Tcf1⁺CD8⁺ T cells, as well as the T cell survival property in vitro. Bulk 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. Oral administration of DHB prodrug in combination with α-PD-1 therapy in tumor-bearing mice promoted the generation of tumor-specific CD44⁺CD62L⁺CD8⁺ central memory T cells, and CD44^-CD62L⁺Sca-1⁺PD-1^-CD8⁺ stem cell memory T cells, resulting in robust anti-tumor efficacy in multiple murine tumor models, including CT26, B16F10, and Nooc1 models.

Conclusions We have developed a novel oral prodrug formulation derived from a microbial metabolite that improves the efficacy of immune checkpoint blockers through preserving T cell stemness in memory CD8 T cells. Based on the robust and unique properties of DHB prodrug, we surmise that our strategy will broaden the applications of beneficial metabolites in cancer immunotherapy.

Acknowledgements This work was supported by NIH (R01AI127070, R01CA210273, U01CA210152, R01DK108901, R01DE026728, R01DE030691, R01DE031951) and the University of Michigan Rogel Cancer Center Support Grant (P30CA46592).