Single-cell RNA sequencing of immune infiltrates in CT26 tumors (combined administration)
Toll like receptor 7 (TLR7) is an essential innate immunity molecule. Following the binding of TLR7 to its ligand, myeloid cells including dendritic cells (DCs) and macrophages are activated and play important roles in triggering adaptive immunity. Therefore, TLR7 agonists have been used for cancer immunotherapy. We developed DSP-0509 as a systemic injectable TLR7 agonist. In this study, we analyzed DSP-0509-induced alterations of tumor-infiltrating lymphocytes (TILs) using single-cell RNA sequencing (scRNA-seq) in the tumor-bearing mouse model. We found that DSP-0509 increased the size of immune cell populations including NK cells, CD4+T cells and CD4+ regulatory T cells (Tregs). Then, we combined IDO1 inhibitor with DSP-0509 to enhance anti-tumor efficacy by decreasing Tregs since DSP-0509 increased Tregs in tumors. We found that combination therapy reduced Treg infiltration in the tumor resulting in enhanced anti-tumor activity. Next, we combined DSP-0509 and anti-PD-1 antibody to prevent CD8+T cell exhaustion and analyzed the changes in TILs by scRNA-seq. In the combination group, we found the cluster of CD8+Tcells that strongly expressed Gzmb, Prf1, Ctla4, and Icos was increased compared to DSP-0509. Focusing on macrophage clusters, the cluster of M1-like macrophages was drastically increased in the combination group compared to DSP-0509. To verify the possibility to enhance anti-tumor efficacy by modulating myeloid cell in tumor, we combined DSP-0509 and the inhibitor of the receptor tyrosine kinase AXL. In bone marrow derived macrophages (BMDMs), AXL inhibitor further increased the level of DSP-0509-stimulated TNFalpha secretion and decreased the level of DSP-0509-stimulated IL-10 secretion. Finally, combining DSP-0509 and AXL inhibitor increased anti-tumor activity in the in vivo tumor model. In this study, we clarified the DSP-0509-induced alteration of immune activity in the tumor microenvironment. Focusing on the phenotype alteration of immune cells in tumor microenvironment by DSP-0509, we showed that multiple combination immunotherapy enhanced anti-tumor efficacy. This study provides insights that may open new avenues to the development of combination immunotherapies.