载IL-12基因/过氧化氢酶靶向M2pep纳米产氧脂质体增强肝癌射频消融术后PD-1抗体疗效

Our former articles demonstrated that tumor cells in the hypoxic microenvironment after radiofrequency ablation (RFA) showed enhanced invasive and metastatic abilities. Although the programmed death-1 (PD-1) antibody could enhance therapeutic effect against tumor, in the clinical studies, the response rates are no more than 30%, which might due to factors such as the hypoxic tumor environment, high level of ROS and the infiltration of M2-skewed tumor associated macrophage (TAM). Our pilot experiments showed infiltration of M2-skewed TAM increased in the residual tumors. Then, can we improve the therapeutic effect of PD-1 antibody by improving postoperative hypoxia and repolarizing TAM after RFA? How to avoid the inhibitory effect of ROS, which is produced by M1-skewed TAM and the hypoxic tumor environment, on PD-1 antibody? We previously synthesized nano-targeting liposomes and gene delivery nanoparticles, and we propose to synthesize the M2pep targeted nanoparticles encapsulating IL-12 pDNA and catalase in this study. The catalase is utilized to react with the ROS produced by the hypoxic environment, and drive IL-12 pDNA to repolarize M2-skewed TAM to M1-skewed TAM. The catalase could also continuously remove the ROS produced by M1-skewed TAM and produce oxygen, improving the immune microenvironment durably and enhancing the therapeutic effect of the PD-1 antibody. The success of the study can offer prospects of new comprehensive treatment for liver cancer.

我们已发表论文表明，肝癌射频消融（RFA）术后乏氧增强残癌侵袭和转移力。虽然程序性死亡分子-1（PD-1）抗体能增强肿瘤治疗效果，但由于肿瘤微环境存在乏氧、活性氧（ROS）升高和M2型肿瘤相关巨噬细胞（TAM）浸润等免疫抑制因素，使PD-1抗体临床显效率不足30%。我们预实验也发现，RFA术后残癌区域M2型TAM浸润增多。那么，能否通过改善乏氧和重极化TAM提高PD-1抗体疗效呢？如何避免M2重极化为M1后及乏氧产生的ROS对PD-1抗体的抑制作用呢？为此，我们拟在前期制备纳米靶向脂质体和载基因纳米粒基础上，制备M2pep靶向载IL-12基因/过氧化氢酶纳米粒，利用过氧化氢酶与乏氧产生的ROS作用产生氧气，促进IL-12重极化M2型TAM为M1型，并利用过氧化氢酶持续清除M1型TAM产生的ROS，不断产生氧气，持久改善免疫微环境，增强术后PD-1抗体疗效，为肝癌综合治疗探索新方法。

文献和我们预实验证明肝癌不完全射频消融术后残癌进展迅速，且残癌内免疫微环境为免疫抑制状态。单纯重极化巨噬细胞虽然可以将巨噬细胞从抑炎表型逆转为促炎表型，但是会释放ROS，ROS可通过腺苷-A2A轴引起Treg细胞的凋亡进而引起CD8 T细胞的功能抑制。因此，单纯重极化巨噬细胞难以最大程度激活抗肿瘤免疫。本项目基于构建小鼠肝癌皮下瘤不全消融模型，发现其CD8 T细胞和巨噬细胞浸润增多。随后，我们基于谷胱甘肽响应的纳米高分子化合物外壳构建了载Motolimod、过氧化氢酶和siPD-L1的多重载药纳米治疗体系，以实现同时激活巨噬细胞和T细胞的抗肿瘤免疫能力。我们的实验证明该纳米治疗体系对高浓度谷胱甘肽响应，在低浓度谷胱甘肽和PBS中均显示出较好的稳定性。在体分布实验中证实该多重载药系统能够将荧光药物富集在小鼠肿瘤组织中。进一步细胞实验中证实，该纳米治疗体系能够同时重极化肿瘤相关巨噬细胞、清除活性氧、降低肿瘤PD-L1表达水平，逆转ROS对T细胞的功能抑制。最终，在小鼠肝癌皮下瘤模型、小鼠肝癌皮下瘤不全射频消融模型中，该纳米治疗体系均能够显著抑制肿瘤生长，同时激活肿瘤微环境内抗肿瘤免疫。因此，该多重载药纳米治疗体系能够显著激活抗肿瘤免疫，为肝癌及肝癌不全射频消融术后残癌的免疫治疗提供了新方向。

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