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

ABSTRACT

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 19. 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.