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.



中文翻译：

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TLR9激动剂和OX40激动剂瘤内注射联合免疫治疗肝细胞癌

背景：由于多种免疫逃避机制，通过免疫检查点阻断对肝细胞癌 (HCC) 进行免疫治疗的反应率有限。OX40 是一种 T 细胞共刺激分子，通过激活效应 T 细胞 (Teffs) 和对抗调节性 T 细胞 (Tregs) 来抑制癌症免疫逃逸。TLR9 属于 toll 样受体超家族，通过刺激树突状细胞的成熟来促进肿瘤抗原呈递。尽管 TLR9 激动剂 (CpG) 和 OX40 激动剂（抗 OX40 抗体）的联合免疫疗法在各种肿瘤中显示出令人鼓舞的疗效，但其对 HCC 的影响仍然未知。
材料和方法：通过在小鼠的不同身体部位植入Hepa1-6细胞构建原位和异位HCC模型。免疫剂通过三种方式给药，包括肿瘤内注射到肿瘤的一个部位、腹膜内注射和皮下注射。通过局部治疗的肿瘤和远处未治疗的肿瘤的消退来评估抗肿瘤免疫反应。通过流式细胞术分析CD4+ T细胞、CD8+ T细胞、Tregs和髓源性抑制细胞（MDSCs）的比例和功能。
结果：通过瘤内注射的 CpG 显着上调了瘤内 T 细胞弱表达的 OX40。肿瘤内注射CpG和抗OX40抗体的联合免疫治疗显着抑制了局部和远处肿瘤的生长，也有效地防止了它们的复发。令人兴奋的是，通过肿瘤内注射而不是通过腹膜内或皮下注射给药，诱导了有效的抗肿瘤免疫反应。此外，我们证明联合免疫疗法可促进 CD8+ 和 CD4+ T 细胞，并抑制 Tregs 和髓源性抑制细胞，有助于有效抑制 HCC。值得注意的是，联合免疫疗法还诱导了免疫记忆反应。
结论：联合 CpG 和抗 OX40 抗体的肿瘤内给药可作为一种有前途的 HCC 免疫疗法。