靶向微环境免疫因素提高CAR-T细胞浸润和杀伤肿瘤能力的研究

Chimeric antigen receptor T cells (CAR-T) have shown unprecedented efficacy in treatment of hemapoietic malignancies. However, CAR-T cells fail to induce responses in solid tumor frequently, due to limited infiltration and immune-suppression in tumor microenvironment. In previous reports, we have shown that immune cells and factors intimately affect T cells in tumor microenvironment, such as chemokines and their corresponding receptors determining T cell localization, and type 2 tumor-associated macrophages (M2) roughly inhibiting T cells. Therefore, we hypothesize that patients with solid tumor would benefit from CAR-T cell therapy with increasing specific chemokine receptor expression to enhance tumor-directed migration and compromising inhibitory effects of M2. To address this hypothesis, CAR-T cell modifications are tested in non-small cell lung cancer (NSCLC) model, which is the most representative tumor type with extremely high incidence in China. In NSCLC, mesothelin was exclusively and highly expressed in malignant lesions. The 3rd generation of CAR-T cells targeting mesothelin specifically lysed lung cancer cells and inhibited tumor growth in PDX model. Additionally, higher expressions of CCL4 and CCL5 were noted in tumor tissues, compared with marginal areas. And CAR-T cells with enforced expression of CCR5, the receptor cognizing CCL4 and CCL5, demonstrated improved directional movement. In situ analysis showed dense accumulations of M2 in NSCLC. IL-12 induced immune-inhibitory M2 cells repolarizing to immune-promoting M1 macrophages in vitro. Those data indicated CCR5 and IL-12 may facilitate CAR-T cells to gather and alleviate immune-suppression within tumor, bringing on greater anti-tumor responses. Further in vitro experiments including cell lysis, proliferation and chemotactic migration, and in vivo observations in PDX model, will be performed to check how transduced-CCR5 or/and -IL-12 impact the therapeutic efficiency of CAR-T cells. This project would shed a new light on improving CAR-T cells in solid tumor therapy.

嵌合抗原受体T细胞（CAR-T）在血液病治疗中取得巨大成功，但T细胞非肿瘤特异分布和肿瘤微环境免疫抑制等因素严重限制了CAR-T细胞的实体瘤治疗效果。我们曾报道趋化因子及其受体决定T细胞定位、M2肿瘤相关巨噬细胞抑制T细胞功能。前期结果显示，CCR5配体（CCL4与CCL5）在非小细胞肺癌（NSCLC）组织高表达，CAR-T细胞过表达CCR5后的定向运动能力更强；IL-12可诱导M2细胞向抑制肿瘤的M1细胞转化；间皮素在NSCLC肿瘤组织特异高表达，针对该靶点的CAR-T细胞能够杀伤表达间皮素的肺癌细胞、控制PDX肿瘤生长。本项目以NSCLC为模型，以靶向间皮素的3代CAR-T细胞为基础，通过CCR5和IL-12转基因，利用细胞杀伤、增殖、趋化等体外实验以及PDX肿瘤模型研究转基因修饰对CAR-T细胞浸润肿瘤、逆转免疫抑制及清除肿瘤能力的影响。本项目将为CAR-T细胞治疗实体瘤提供新思路。

CAR-T细胞在血液肿瘤的治疗中展现了令人印象深刻且持久的反应。但是CAR-T细胞治疗实体瘤仍面临诸多困难。CAR-T细胞浸润不足、增殖和效应功能差，以及实体瘤中的免疫抑制肿瘤微环境（TME）导致CAR-T细胞治疗无效。间皮素在间皮瘤、肺癌、食管癌等多种癌症中高度表达，靶向间皮素的CAR具有治疗多种实体瘤的潜力。因此，在本研究中我们选择靶向间皮素的CAR-T细胞（CARTmeso）。在前期的研究中，我们发现CCL4和CCL5在肿瘤组织中的表达高于癌旁组织。为了增加CAR-T细胞的迁移能力，我们改造CAR-T细胞表达CCR5（CARTmeso-CCR5）。与传统的CARTmeso相比，CARTmeso-CCR5 细胞显示出有效且相当的细胞毒性，同时具有优越的趋化能力。同时文献报道IL-12不仅能增强T细胞的抗肿瘤反应，还能将M2转化为M1型巨噬细胞。我们将 IL-12 基因插入CAR 结构使CAR-T细胞同时可以表达CAR和IL-12（CARTmeso-IL-12）。我们结果表明CARTmeso-IL-12细胞具有更好的细胞毒性和极化巨噬细胞的能力。在此，基于以上研究结果，我们构建了同时过表达CCR5和IL-12的靶向间皮素的CAR-T细胞（CARTmeso-CCR5-IL12）来解决目前实体瘤CAR-T疗法面临的各种障碍，并评估了它们的抗肿瘤潜力。与常规CAR-T细胞相比，CCR5和IL-12的强制共表达增强了 CAR-T细胞向肿瘤部位的迁移，提高了效应分子的产生，同时将M2极化为M1巨噬细胞。此外，CARTmeso-CCR5-IL12细胞抑制肿瘤生长并延长小鼠存活时间，表明CARTmeso-CCR5-IL12细胞在治疗实体瘤方面具有优异的抗肿瘤活性。本研究中我们设计了一种新的CAR结构为实体瘤的治疗提供新的策略。同时其增强的抗肿瘤疗效为后续临床试验提供了临床前证据，具有临床转化意义。我们已经完成课题相关的全部研究内容，已经整理数据和材料，并写好文章准备投稿。