Basic And Clinical Study On CD19-CAR-T Cells For B Cell Non-hodgkin Lymphoma

Objective: To establish a system which can produce CD19-CAR-T cells.To evalulate safety and efficacy through cytotoxicity assay and mouse xenograft model on CD19-CAR-T cells for B-NHL cell line.To evalute safety and efficacy through clinical study on CD19-CAR-T cells for 9 B-NHL patients.Methods: 1.Produce CD19-CAR-T cells through some methods including plasmid amplification and extraction ? lentivirus package ? CD3+ cells extraction ?transfection and so on.2.CD19-CAR-T cells and non-transduced T cells(NT cells)were separately co-cultured with tumor cell lines(Pfeiffer?Nalm6?K562).Then,we analyzed cytotoxicity of CD19-CAR-T cells and NT cells by LDH Release Assay Kit.3.Establishing B-NHL mouse xenograft model by injecting Pfeiffer cells venously.In order to confirm if the mouse xenograft model was established successfully,we analyzed the percentage of human CD19+ cells in peripheral blood by flow cytometry.Dviding 10 mice into 2 groups randomly,then,mice in experimental group were treated with CD19-CAR-T cells,mice in control group were treated with saline.Next,we analyzed the percentage of human CD19+ cells in peripheral blood every week and recorded the lifetimes of mice.4.Establishing B-NHL mouse xenograft model by injecting Pfeiffer cells subcutaneously.Tumours,liver and spleen were analyzed in pathology.The volumes of tumours were measured by MRI.Selecting 10 mice whose volume of tumours was 100-200mm3 and dividing them into 2groups,then,mice in experimental group were treated with CD19-CAR-T cells,mice in control group were treated with saline.Next,we measured volumes of tumours every 5 days and recorded the lifetimes of mice.5.Study on CD19-CAR-T cells for 9 B-NHL patients.Evaluating efficacy of CD19-CAR-T cells by flow cytometry,MRI,PET-CT and so on.Evaluating safety of CD19-CAR-T cells by monitoring vital signs(T,BP,Sp O2),testing level of cytokines,testing level of hs-CRP,observing clinical symptoms and so on.Results: 1.A system which can produce enough and stable CD19-CAR-T cells has been established.2.In vitro,CD19-CAR-T cells can kill tumor cell lines which express CD19 by targeting to it,and the killing ability of CD19-CAR-T cells is more stronger than that of NT cells.As for killing tumor cell lines which can not express CD19,there is no difference between CD19-CAR-T cells and NT cells.3.B-NHL mouse xenograft models are established by injecting tumor cells subcutaneously and venously.4.As for B-NHL mouse xenograft model established by injecting tumor cells venously,percentage of human CD19+ cells in peripheral blood of mice which are treated with CD19-CAR-T cells decrease gradually,but in control group,percentage of human CD19+ cells still rise.The lifetimes of experimental group are longer than that of control group.5.As for B-NHL mouse xenograft model established by injecting tumor cells subcutaneously,tumours of experimental group increase slower than that of control group during the early stage,it decrease after CD19-CAR-T cells were injected 10 days.The lifetimes of experimental group are longer than that of control group.6.As for 9 B-NHL patients,four of them get CR,one of them get PR but relapse in the end.6 patients suffer from CRS.Conclusion: 1.A system which can produce enough and stable CD19-CAR-T cells has been established,in order to provide new therapy for B-NHL.2.CD19-CAR-T cells can kill B-NHL cell line in vitro.3.CD19-CAR-T cells can kill B-NHL cell line in vivo effectively and safely.4.Clinical tudy on CD19-CAR-T cells for B-NHL patients shows it is safe,effective and feasible.