Construction And Killing Effect Of Target CD19 Allogeneic Universal CAR-T Cells

2017 is the first year of CAR-T,two products from autologous T cells approved by FDA,namely,Kymriah of Novartis and Yescarta of Kite.These CAR-T cells approved for clinical use must be generated on a custom-made basis,there are expensive and lengthy production process;Critically ill patients have the risk of not collecting enough good quality T cells and can not be administered in time;In addition,It has risk of production failure and tumor cell contamination;T cells are derived from autologous T cells,so cannot guarantee product stability and quality control.It is difficult to produce standardized products and limit large-scale clinical applications.Therefore,the preparation of allogeneic universal CAR-T cells is a better strategy to solve these problems.Objective: In this study,we constructed the lentiviral expression vector p LVX-EF1?-CAR19,transducted cord blood mononuclear cells(CBMC)derived from healthy humans to express the CAR sequence targeting human CD19 molecule,and introduced the RNP complex by electroporation using CRISPR/Cas9 gene editing technology.The TCR and HLA-I molecules were knocked out to obtain allogeneic universal CAR-T cells,and their killing effect was verified in vitro.Method: Part I Optimization of lentiviral preparation conditions and conditions for infecting human primary T cells 1.The second and third generation lentiviral packaging plasmids were used to transfect human embryonic kidney 293 FT cells by liposome Lipofectamine 3000 mediated liposome transfection to obtain recombinant lentivirus.Compare the effects of different lentiviral packaging systems,promoters,and virus preservation methods on lentivirus titers;2.CBMC were obtained by Ficoll density gradient centrifugation,CD3+ T cells were sorted from cord blood mononuclear cells with magnetic beads,and lentivirus was transducted 24 hours after activation with anti-CD3/CD28 magnetic beads.Flow cytometry was used to detect the transduction rate.Observe the effect of different magnetic beads dosage and transduction time on the virus transduction rate;Part II Construction and killing effect of targeting CD19 allogeneic universal CAR-T cells 1.The CD19-CAR sequence fragment was amplified by PCR and the lentiviral vector plasmid p LVX-EF1?-EGFP was digested with Eco RI and Mlu I to construct the p LVX-EF1?-CAR19 lentiviral expression vector,which was identified by PCR,restriction enzyme digestion and DNA sequencing.2.The recombinant lentivirus was packaged by co-transfecting the recombinant lentiviral expression plasmid p LVX-EF1?-CAR19 with p LP1,p LP2,and p LP-VSVG into 293 FT cells.3.Using CRISPR/Cas9 gene editing technique,RNP complex was introduced into T cells by electroporation to knock out TCR and HLA-I genes,and the knockout efficiency was detected by flow cytometry and knockout effect was detected by mixed lymphocyte reaction.TCR and HLA-I genes double negative cells were obtained by magnetic bead negative sorting method.4.The TCR and HLA-I genes double negative cells sorted by magnetic beads were infected with recombinant lentivirus to obtain the Allogeneic universal CAR-T cells.5.The killing effect experiment was carried out by mixing allogeneic universal CAR-T cells with NAMALWA which is CD19 positive malignant leukemia cells.The killing rate of allogeneic universal CAR-T cells to tumor cells was calculated by measuring the expression rate of luciferase in tumor cells.The concentration of IL-2 and IFN-? in the serum confirmed the killing effect of allogeneic universal CAR-T cells on tumor cells;Results: Part I Optimization of lentiviral preparation and conditions for infecting human primary T cells 1.Comparing the second generation of lentiviral packaging system consisting of p SPAX2 and p MD2.G with third generation of lentiviral packaging system consisting of p LP1,p LP2 and p LP-VSVG,the third-generation lentiviral packaging system was found to has higher virus titer than the second generation.Finally,it is determined that the third generation lentivirus packaging system will be used in subsequent experiments.2.The recombinant lentiviral expression vector of the EF-1? promoter has a higher viral titer than the CMV promoter,and the T cell infection efficiency is also higher than the CMV promoter;the virus is repeatedly frozen and thawed more than twice or stored at 4? for more than one day,the titer decreased significantly,thereby affecting the activity of the virus.The prepared virus was allowed to store at 4? for a maximum of one day and freeze-thaw one time at-80 ° C;3.T cells can be infected by lentivirus after activation;Lentivirus is infected 24 hours after T cell activation,and the infection rate is significantly improved.Part II Construction and killing effect of target CD19 allogeneic universal CAR-T cells 1.The recombinant lentiviral expression plasmid p LVX-EF1?-CAR19 was verified by PCR,restriction enzyme digestion,agarose gel electrophoresis and DNA sequencing.2.The recombinant lentivirus expressing CD19-CAR was used to transfect T cells,and the results of flow cytometry show that CD19-CAR is correctly expressed.3.The results of flow cytometry confirmed that the TCR and HLA-I genes of T cells were knocked out by CRISPR/Cas9.The results of the mixed lymphocyte reaction showed that the TCR and HLA-I gene double-deficient T cells had reduced alloreactivity and did not cause graft versus host disease.4.The Allogeneic universal CAR-T cells showed robust in vitro anti-tumor activities,such as lytic capacity,cytokine secretion and proliferation.Conclusion: Allogeneic universal CAR-T cells that targeted the CD19+ positive malignant leukemia cells were successfully constructed.The Allogeneic universal CAR-T cells showed robust in vitro anti-tumor activities,such as lytic capacity,cytokine secretion and proliferation.Thus laying basis for future preclinical and clinical studies on Allogeneic universal CAR-T cells.