tmTNF-αCAR-流感疫苗双特异性T细胞过继疗法对乳腺癌的杀伤作用

Chimeric antigen receptors (CAR) combine the antigen binding site of an antibody with the signal activating machinery of TCR, freeing antigen recognition from MHC restriction. However, the artificial CARs could not mimic the natural activation of T cells and thus showed very limited clinical benefit. It has been reported that CAR-engineered EB virus specific T cells can be completely activated by interaction with APC, showing increased antitumor activity and enhanced survival. Nevertheless, it is limited to treat EBV-associated tumors. Our previous study demonstrated transmembrane TNF-α expressed by breast cancer and several different kinds of tumor, and thus it was used as a target for making a specific monoclonal antibody. This antibody has been shown in vitro and in vivo to effectively kill TNF-positive tumor cells. In this project, we'll construct single chain antibody from this monoclonal antibody and then use it to construct transmembrane TNF-α specific CAR. This CAR will be transduced into influenza vaccine-sensitized T cells, which are then transferred to tumor(breast cancer)-bearing mice. The immunization with the vaccine would induce proliferation of adoptive T cells in vivo, facilitating persistent killing of tumor and clearance of residual tumor cells. This CAR makes influenza specific T cells also direct to transmembrane TNF-α, termed bispecific T cell which does not only provide a novel target for individual treatment of tumors, but also provide a new strategy for CAR-based adoptive T cell therapy.

嵌合抗原受体（CAR）是将抗体的抗原结合部位与TCR活化结构域结合，以克服MHC对TCR识别抗原的限制性。但因人工CAR不能模拟T细胞的天然活化，使其临床抗瘤效应有限。有人将CAR转染EB病毒特异性T细胞，因可获得APC的活化信号，使其杀瘤效应提高，体内生存延长。但该法限于治疗EBV相关肿瘤。我们前期工作证实跨膜TNF-α可表达于乳腺癌等若干不同肿瘤细胞，以此为靶点，我们筛选到特异性单克隆抗体，并证实其在体内外可有效杀伤TNF+肿瘤细胞。本项目以此为基础构建跨膜TNF-α单链抗体，继而构建跨膜TNF-α特异性CAR，将之转染流感疫苗致敏T细胞，过继于乳腺癌荷瘤小鼠，通过注射疫苗诱导过继T细胞体内增殖，而利于其持续杀瘤和清除残留肿瘤细胞。这种流感疫苗-跨膜TNF-αCAR双特异性T细胞不但为肿瘤个体化治疗提供新靶向，而且还为以CAR为基础的T细胞过继疗法提供了新策略。

我们的前期工作证实跨膜型TNF-α可在白血病细胞和部分实体瘤如乳腺癌组织中高表达。本实验制备的针对跨膜型TNF-α的单克隆抗体可在体内外有效杀伤高表达跨膜型TNF-α的急性白血病细胞和乳腺癌细胞。本项目在该单克隆抗体的基础上制备单链抗体，并证实其具有与亲本抗体同样的抗原结合能力。将跨膜型TNF-α单链抗体与CD8α链跨膜段、CD28/4-1BB/CD3ζ胞内段序列连接构建成CAR，用慢病毒载体将CAR转染人脐带血T细胞或用逆转录病毒载体将CAR转染流感疫苗免疫小鼠的T细胞，分别在体外可有效杀伤高表达跨膜型TNF-α的人乳腺癌细胞株或转染人跨膜型TNF-α的小鼠乳腺癌细胞株，但对不表达跨膜型TNF-α的乳腺癌细胞株则仅有低于20%的非特异性杀伤效应; 活化的CAR-T细胞表达效应分子FasL、颗粒酶、穿孔素和CD107a明显增加。体内输入针对跨膜型TNF-α的CAR-T细胞可明显抑制荷瘤小鼠跨膜型TNF-α阳性乳腺癌的生长，延长小鼠生存期。本项目研究开发的流感疫苗-跨膜型TNF-α的CAR-T不但为肿瘤个体化治疗提供新的靶向治疗，而且还为CAR-T治疗提供了新策略。.