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

Zhimei Zhou; Liteng Lin; Yongcheng An; Meixiao Zhan; Ye Chen; Mingyue Cai; Xiaojing Zhu; Ligong Lu; Kangshun Zhu

doi:10.2147/JHC.S301375

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

J Hepatocell Carcinoma. 2021 Jun 8:8:529-543. doi: 10.2147/JHC.S301375. eCollection 2021.

Authors

Zhimei Zhou^#¹, Liteng Lin^#¹, Yongcheng An^#¹, Meixiao Zhan², Ye Chen¹, Mingyue Cai¹, Xiaojing Zhu¹, Ligong Lu², Kangshun Zhu¹

Affiliations

¹ Laboratory of Interventional Radiology, Department of Minimally Invasive Interventional Radiology and Department of Radiology, The Second Affiliated Hospital, Guangzhou Medical University, Guangzhou, 510260, People's Republic of China.
² Zhuhai Interventional Medical Center, Zhuhai Precision Medical Center, Zhuhai People's Hospital, Zhuhai Hospital Affiliated with Jinan University, Zhuhai, Guangdong Province, 519000, People's Republic of China.

^# Contributed equally.

Abstract

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.

Keywords: combination therapy; hepatocellular carcinoma; immunotherapy; intratumoural administration; toll-like receptor.

Grants and funding

This work was supported by the National Natural Science Foundation of China (81873920, 81571774, 82001929); National Key Research and Development Program of China (2017YFA0205200); Science and Technology Program of Guangzhou (202002030135); Medical Scientific Research Foundation of Guangdong Province (A2020519). Basic and Applied Basic Research Foundation of Guangdong Province (2020A1515110654). Doctor Initiative Foundation of the Second Affiliated Hospital of Guangzhou Medical University (010G271108, 010G271103).