The Study Of CAR Modified NK-92 Cells Targeting CD20~+ Lymphoma In Vitro

Chimeric Antigen Receptor(CAR)cell immunotherapy has made breakthroughs in the field of cancer.The abnormal expression of CD20 is related to most non-Hodgkin's lymphoma(NHL),and CD20 has become an important target for the treatment of non-Hodgkin's lymphoma.To explore the effective treatment of malignant B-cell lymphoma targeting CD20,and to research and develop cell therapy drugs targeting CD20-positive malignant B-cell lymphoma.The human-mouse chimeric monoclonal antibody Rituximab against CD20 antigen was humanized to reduce the immunogenicity of its mouse-derived single-chain fragment variable(scFv).Three generations of human and wild-type NK-92 cells modified with CAR were constructed using CD20 as target and scFv as extracellular segment.The anti-tumor activity of two kinds of CAR-modified NK-92 cells was verified by various in vitro experiments.The following experiments were carried out in this study.1.On the basis of Rituximab,the pCMV-scfv-Fc of wild type anti-CD20 and the anti-CD20 pCMV-H-scfv-Fc expression vector of humanized modified Rituximab single-chain variable region were constructed by Overlapping PCR.Western Blotting results showed that all the target proteins were expressed after transient transfection.2.Anti-CD20-scfv-Fc fusion protein(scfv-Fc)was obtained by affinity chromatography.Flow cytometry or ELISA showed that scfv-Fc fusion protein had biological activity.In addition,humanized anti-CD20-H-scfv-Fc fusion protein(H-scfv-Fc)also had biological activity.3.The lentiviral vector pCDH-CARCD20(CARCD20)with wild type anti-CD20-scfv and humanized anti-CD20-scfv was constructed by overlapping PCR.After transfection into 293T cells,the lentiviral particles were collected.The titers of CARCD20 and hCARCD20 were 3.56×10~8 TU/mL and 1.785×10~8TU/mL respectively.The former was about twice as high as the latter.4.The infection efficiency of NK-92 cells infected with MOI=20 was more than97%.Flow cytometry analysis showed that the expression of CAR in NK-92 cells infected with wild-type CARCD20 virus(NK-92-CARCD20)was 59.5%,and that in NK-92 cells infected with humanized hCARCD20 virus(NK-92-hCARCD20)was35.2%.5.The results of LDH release assay showed that the killing activity of Mock,NK-92-CARCD20 and NK-92-hCARCD20 on CD20+Raji cells was higher than that of K562-CD20 cells with low expression of CD20 and negative K562 cells with the same effective-target ratio.The killing effect of effector cells on target cells was positively correlated with the effective-target ratio.When E:T=9:1,NK-92-CARCD20 and NK-92-hCARCD20 had the best killing effect on CD20+Raji cells,which were 64.36%(**P<0.01)and 48.86%(**P<0.01),respectively.At E:T=9:1,TUNEL staining and co-staining with CFSE and PI also showed that NK-92-CARCD20 and NK-92-hCARCD20 had no difference in killing Raji cells and were stronger than Mock.The results showed that NK-92-CARCD20 and NK-92-hCARCD20 had stronger cleavage effect on Raji cells than Mock.Compared with Mock,the amount of perforin and granulase released by NK-92-CARCD20 and NK-92-hCARCD20 co-cultured with Raji cells was different,suggesting that perforin and granulase enhanced NK-92-CARCD20.NK-92-hCARCD20 and NK-92-hCARCD20 were used to kill target cells.After co-culture of effector cells and target cells expressing different amounts of CD20 for 6,12 and 24 hours,the levels of IL-2 and IFN-gamma in supernatant were not different,and indicated the safety of NK-92 modified by CAR from a certain point of view.To sum up,wild-type anti-CD20-scFv and humanized anti-CD20-scFv were successfully constructed as NK-92 cell lines which modified by extracellular segment of CAR,and their anti-tumor activity in vitro was verified.When the expression of two kinds of anti-CD20-scFv in CAR was identical,they had similar killing ability to CD20+Raji cells and had certain safety.NK-92-hCARCD20 cells had low immunity and were the potential of drugs targeting CD20+cells.