Optimizating The CD22 CAR Design And Studying The Ca²⁺ Signaling Dynamics In CAR-T Cells

Chimeric Antigen Receptor-T cell(CAR-T)therapy is a new type of cancer immunotherapy.After more than 20 years of optimization and improvement in CAR-T preclinical and clinical research,in 2017 U.S.FDA approved the two CD 19 targeting CAR-T products,namely KYMRIAH and YESCARTA,which are used to treat B lymphocytic leukemia and lymphoma respectively.Although CAR-T therapy has achieved remarkable clinical results in treating hematological malignancies,there are still some side effects,such as cytokine release syndrome(CRS),B cell aplasia,neurotoxicity and on-target off-tumor toxicity,threatening the lives of patients sometimes.In addition,CAR-T therapy has limited effect in treating solid tumors so far Therefore,CAR-T therapy still needs to be improved to solve the above issuesThis thesis is to optimize the design of CARs and test the functions of CAR-T cells with different designs.In vitro experiments showed that,the different connection order of VH and VL of the scFv region and the number and position of IT AM in the CD3ζ chain can affect the function of CAR-T cells targeting CD22 alone.In addition,we attempted to use the Forster resonance energy transfer(FRET)technology to investigate the Ca2+signal activation level of different CAR-T cells upon antigen stimulation and its relevance to cellular function.Finally,based on the above experimental results,we designed a well-performmed bispecific CAR targeting both CD 19 and CD22.The in vitro results selected the best CAR designs for further preclinical validations in mouse models and paved the way for future translational clinical research.