小鼠抗人密封蛋白18剪接变异体2(Claudin18.2)CAR-T细胞的构建及体外功能验证

Construction of mouse anti-human Claudin18.2 CAR-T cells and verification of in vitro functions

目的筛选一种抗人密封蛋白18剪接变异体2(Claudin18.2)的单克隆抗体(mAb),并基于该抗体序列构建靶向Claudin18.2的嵌合抗原受体T(CAR-T)细胞,用于开发CAR-T细胞疗法。方法用人Claudin18.2抗原免疫小鼠,取小鼠脾细胞与SP2/0细胞进行细胞融合,经筛选获得小鼠抗人Claudin18.2 mAb。以抗体序列为模板PCR扩增出重链可变区(VH)和轻链可变区(VL)序列,用接头(linker)连接成单链抗体(scFv),并用此scFv构建CAR。将CAR克隆至慢病毒表达载体中,用包装好的慢病毒感染T细胞制备出抗Claudin18.2 CAR-T细胞。结果筛选到的小鼠抗人Claudin18.2 mAb对人和鼠的Claudin18.2抗原都有结合能力,亲和力高于对照抗体。构建的CAR-T细胞在效靶比1∶9时,对过表达Claudin18.2的阳性靶细胞的杀伤率在50%~70%。结论制备的小鼠抗人Claudin18.2 mAb具有人鼠双种属特异性,组织特异性良好,亲和力高。构建的anti-Claudin18.2 CAR-T细胞对靶细胞具有良好杀伤效果。

Objective To screen a monoclonal antibody(mAb)of anti-human Claudin-18 splice variant 2(Claudin18.2)and construct chimeric antigen receptor T(CAR-T)cells targeting Claudin18.2 based on this antibody sequence for the development of CAR-T cell therapy.Methods Mice were immunized with human Claudin18.2 antigen,and then mice spleen cells were isolated and fused with SP2/0 cells to generate hybridoma cells.By hybridoma screening,we obtained the mouse against human Claudin18.2 mAb.The heavy chain variable region(VH)and light chain variable region(VL)sequences were amplified by PCR with the antibody sequence serving as the template.The linker peptide was used to link VL and VH into a single chain antibody(scFv)for CAR construction.The CAR was cloned into a lentiviral expression vector,and T cells were infected with the packaged lentivirus to prepare targeting Claudin18.2 CAR-T cells.Results The screened mouse anti-human Claudin18.2 mAb exhibited binding ability to both human and mouse Claudin18.2 antigens,with higher affinity than the control antibody.The constructed CAR-T cells showed a killing rate between 50%to 70%against Claudin18.2-overexpressing positive target cells at an effector-to-target ratio of 1∶9.Conclusion The prepared mouse anti-human Claudin18.2 mAb exhibites cross-species specificity to humans and mice antigens,with good tissue specificity and high affinity.The constructed anti-Claudin18.2 CAR-T cells show effective killing of target cells.