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Abstract
Background Recent studies have suggested that certain metabolites from the gut commensal microbes, such as tryptophan, short-chain-fatty acids, and bile acids, may serve as a potential link among the gut microbiota, tumor cells, and systemic anti-tumor immunity. However, the role of many other microbial metabolites in regulating T cell immunity and memory CD8+ T cells is unclear. In addition, it remains unknown whether these beneficial metabolites (also known as postbiotics) can synergize with immune checkpoint blockers (ICBs). In this work, we have screened microbial metabolites and developed an oral prodrug formulation that can improve the stemness of CD8+ T cells and the antitumor efficacy of ICBs.
Methods We screened microbial metabolites in vitro and assessed their capabilities to induce the differentiation of central memory CD8+ T cells. Bulk RNA sequencing was conducted on OT-I CD8+ T cells treated with 3,4-dihydroxybenzoate (DHB) to assess gene expression profiles. In addition, we have developed a DHB-based prodrug 201 formulation for oral administration. In vivo anti-cancer efficacies of prodrug 201 plus α-PD-1 were evaluated in various mouse tumor models, including CT26, B16F10, and Nooc1. Peripheral blood mononuclear cells (PBMCs) were analyzed for antigen-specific CD8+ T cells by flow cytometry and enzyme-linked immunosorbent spot (ELISPOT) assay. Tumor and tumor-draining lymph nodes were analyzed for the phenotypic and functional assessment of memory CD8+ T cells.
Results Among various metabolites that 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 survival of CD8+ T cells (figure 1). Bulk RNA-seq analysis revealed that DHB treatment upregulated the pathways related to cell growth and proliferation and genes associated with stem-like properties of CD8+ T cells. In vivo studies showed that orally administered DHB improved the systemic anti-tumor efficacy of α-PD-1 antibody. DHB metabolite was further modified with lipid and engineered into a prodrug 201 formulation for oral administration. DHB prodrug 201 significantly improved the antitumor efficacy of α-PD-1 therapy in multiple tumor models and promoted the induction of stem cell-like memory CD8+ T cells in vivo.
Conclusions We have developed a novel metabolite-based prodrug formulation for cancer immunotherapy. Oral administration of DHB prodrug 201 preserves the stemness among memory CD8+ T cells and improves the antitumor efficacy of ICBs. We anticipate that our strategy will broaden the potential use of metabolites and their derivatives 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).
OT-I CD8+ T cells were cultured with different metabolites in vitro as indicated (A), shown are the frequency of CD44+CD62L+ Tcm CD8+ T cells (B), the level of survival of CD8+ T cells (Annexin V-PI-) (C), and the frequency of the Tcf1+CD8+ T cells (D). Data represent the mean ± s.e.m. (n = 5).
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