A Novel Molecular Switch Design For Improving The Safety Of CAR-T Cell Therapy

Chimeric antigen receptor T(CAR-T)cell therapy is one of the fastest growing areas of tumor immunology.In recent years,it has revolutionized the immunotherapy model and,with outstanding clinical data in the treatment of B-cell leukemia and lymphoma,has become a new breakthrough in cancer treatment,opening a new door for cell therapy.But with reports of deaths due to severe cytokine release syndrome and off-target effects,thoughts on car-T safety have followed.The most critical issue is how to further improve the controland and safety of CAR-T.One achievable strategy is to use a suicide gene system to initiate CAR-T cell removal in the body when side effects occur to reduce or even avoid side effects.This paper uses a short human CD38 molecule as a "switch" mark and expression on CAR-T cells with CAR,and establishes a new CAR-T suicide gene system.In addition,the car-T construction process to obtain high-dropvirus concentration methods are compared and discussed.The main research content is as follows:First,the human CD38 molecule is cut,stitched with CAR gene,and two different genetic patterns are constructed.The gene sequence of CD38 was adjusted by NCBI gene database,and the cd38 cell and extracellular non-binding segment were shortened by cutting out the antibody recognition epitope of CD38 target drug Daratumumab.On the one hand,in order to realize the CD38 t molecule and CAR on the cell membrane,the CD38 t sequence is connected with the guide peptide and CD8 alpha cross-membrane segment,and connected to CAR by a 2A self-shearing oligopeptide,on the other hand,the common guide peptide makes CD38 t directly connected to CAR,so that CD38 and CAR are expressed "integrated".The establishment of two CD38 t expression patterns was completed on different slow virus vectors.Second,through liposome transfection of slow viral plasmids,the purpose gene is expressed instantaneously on the cell.The heK293 T cell packaging system was used to pack slow virus plasmids carrying the target gene to produce viral particles with infectious effect,collect the viral venom,and infect T-cells,and established a steady-transition cell line of expression gene,and carried out follow-up experiments on this basis.Third,in order to facilitate the construction of a human initial T-cell on the basis of the ability to kill tumor cells CD8+CAR-T,the resulting slow virus needs to be concentrated to improve titer.To this end,the two commonly used and more advantageous virus enrichment methods were compared with the titration and recovery rate,and the effective establishment of CD8+CAR-T was pre-paved.