表达肿瘤血管定位信号GPI-VEGFR-2-scFv及肿瘤基质降解酶HPSE的CAIX-CAR-T细胞治疗肾癌研究

Adoptive transfer of chimeric antigen receptor (CAR)-redirected T lymphocytes (CAR-T cells) has had less striking therapeutic effects in solid tumors than in lymphoid malignancies. One of the reasons is limited infiltrating ability of CAR-T cells into tumors. We confirmed the therapeutic effects of CAR-T cells targeting CAIX for renal carcinoma previously. We will develop novel strategies to enhance the therapeutic effects of CAIX-CAR-T cells in this study: 1) direct CAR-T cells to accumulate at tumor site through the binding of anti-VEGFR-2 scFv anchored on CAR-T cells by Glycosyl-phosphatidylinositol (GPI) to VEGFR-2 over expressed on endothelial cells of tumor vessels; 2) enhance the ability of CAR-T cells to penetrate stroma-rich tumor tissue through heparanase (HPSE) over expressed in CAR-T cells, which degrades heparin sulfate proteoglycans, the main components of extracellular matrix (ECM). The two strategies will promote the killing effects of CAIX-CAR-T cells to renal carcinoma cells synergistically. We will confirm the therapeutic efficacy of the novel CAR-T cells to renal carcinoma by in vitro and in vivo experiments.

CAR-T细胞肿瘤浸润能力不足是其实体瘤疗效欠佳的主要原因之一。申请者在前期开展靶向肾癌抗原CAIX的CAR-T细胞治疗肾癌研究的基础上提出如下改进策略：1）利用GPI信号结构域将抗VEGFR-2的单链抗体（scFv）锚定于CAR-T细胞表面，利用抗VEGFR-2 scFv对肿瘤血管内皮表面VEGFR-2的识别，使CAR-T细胞靶向聚集于肿瘤部位；2）在CAR-T细胞中过表达肿瘤细胞外基质（ECM）降解酶heparanase（HPSE），通过HPSE对肿瘤ECM的降解作用增强CAR-T细胞的肿瘤浸润能力。二者共同促进靶向CAIX的CAR-T细胞对肾癌的杀伤作用。本研究将通过体外及动物实验证实这种新型CAR-T细胞的肾癌治疗效果。

肾癌恶性程度极高，根治性切除术后仍有20-30%患者发生转移。放、化疗及细胞因子治疗均不理想，近年来以酪氨酸激酶抑制剂（TKI）为代表的分子靶向治疗逐渐成为转移性肾癌的一线治疗用药，但临床客观反应率也只有30%左右，且耐药问题严重，仅原发性耐药就高达25%。因此，针对转移性肾癌急需新的治疗手段。嵌合抗原受体修饰的T细胞（CAR-T）疗法是一种新兴的肿瘤治疗技术，本项目构建了靶向肾癌抗原CAIX、以4-1BB为共刺激分子的CAR-T（CAIX-CAR-T）。通过细胞因子释放（ELISA）和细胞毒性分析（RTCA）证实CAIX-CAR-T对CAIX阳性肾癌细胞具有特异性杀伤作用；体内实验证实CAIX-CAR-T对肾癌NPG小鼠肺转移模型（OSRC-2细胞）具有显著治疗效果，能够显著延长何瘤小鼠的生存期。. 为进一步增加CAIX-CAR-T细胞的肿瘤浸润能力提高治疗效果，本项目构建了表达GPI-VEGFR-2 scFv的CAIX-CAR-T细胞（CAIX-CAR-T-VR2），拟利用抗VEGFR-2 scFv对肿瘤血管内皮表面VEGFR-2的识别，使CAR-T细胞靶向聚集于肿瘤部位增加CAR-T细胞的肿瘤浸润，提高治疗效果。本项目通过流式、免疫组化等一系列实验证实GPI-VEGFR-2 scFv能够显著提高CAR-T细胞的肿瘤浸润能力，并证实与CAIX-CAR-T细胞相比，CAIX-CAR-T-VR2对肾癌肺转移瘤模型和皮下移植瘤模型均具有更强的治疗效果。为增加肿瘤组织的通透性提高CAR-T细胞的肿瘤浸润，本项目构建了过表达Decorin的溶瘤腺病毒（OAV-Decorin），Decorin具有重塑肿瘤细胞外胶原纤维的功能，本项目通过肾癌皮下移植瘤模型证实OAV-Decorin与CAIX-CAR-T联合治疗肾癌，能够显著增加CAR-T细胞的肿瘤浸润能力，提高肿瘤抑制率。本项目的研究成果为肾癌治疗开拓了新思路，对于解决晚期肾癌无药可治的问题具有重要意义。