The Combination Immunotherapy of TLR9 Agonist and OX40 Agonist via Intratumoural Injection for Hepatocellular Carcinoma

BACKGROUND: The response rate of immunotherapy via immune checkpoint blockade in hepatocellular carcinoma (HCC) is limited due to multiple immune evasion mechanisms. OX40 is a T cell co-stimulating molecule which suppresses the cancer immune evasion by activating effector T cells (Teffs) and counteracting regulatory T cells (Tregs). TLR9 belongs to the toll-like receptor superfamily which promotes tumour antigen presentation by stimulating the maturation of dendritic cells. Though the combination immunotherapy of TLR9 agonist (CpG) and OX40 agonist (anti-OX40 antibody) has shown encouraging efficacy in various tumours, its effect on HCC remains unknown. MATERIALS AND METHODS: Orthotopic and ectopic HCC models were constructed by implanting Hepa1-6 cells at different body sites of the mice. Immune agents were administrated via three ways, including intratumoural injection into one site of the tumour, intraperitoneal injection, and subcutaneous injection. The anti-tumour immune response was evaluated by the regression of both the local treated tumour and distant untreated tumour. The ratio and function of CD4+ T cells, CD8+ T cells, Tregs and myeloid-derived suppressor cells (MDSCs) were analyzed by flow cytometry. RESULTS: CpG via intratumoural injection remarkably upregulated the weakly expressed OX40 of intratumoural T cells. The combination immunotherapy of CpG and anti-OX40 antibody via intratumoural injection significantly inhibited the growth of local and distant tumours, and also effectively prevented their recurrence. Excitingly, drug administration via intratumoural injection, rather than via intraperitoneal or subcutaneous injections, induced potent anti-tumour immune response. Furthermore, we demonstrated that the combination immunotherapy promoted CD8+ and CD4+ T cells, and inhibited Tregs and myeloid-derived suppressor cells, contributing to the effective inhibition on HCC. Noteworthily, the combination immunotherapy also induced an immune memory response. CONCLUSION: The intratumoural administration of combined CpG and anti-OX40 antibody serves as a promising immunotherapy against HCC.