5-Aza-2'-Deoxycytidine Induces CD19 Antigen Expression In B Cell-derived Hematologic Malignancy And Enhancescytotoxic Effect Of T Cell With An Anti-CD19 Chimeric Receptor

Purpose:CD19-directed Chimeric antigen receptor(CAR)T cells brought substantial benefit to patients with B-cell malignancies.Despite of encouraging therapeutic efficiency,anti-CD19 CAR-T cell to treat relapsed and refractory(R/R)B cell lymphomas has a limited overall response rate in patients,which guarantees further investigation to potentiate CAR-T cell efficacy.Our study focused on improving the therapeutic efficiency and mechanism of lymphoma through combination of5-Aza-2'-Deoxycytidine(DAC)and CAR-T cell.Experimental Design:Construct CD19-directedCAR T cells and CAR T cell transfection efficiency was detected by flow cytometry.CD19 CAR T cells were co-culturedwithlymphomacelllines(RAMOS,RAJI)atdifferent ratios of effective cells(E)to target cells(T),and their killing effects were detected by flow cytometry.Lymphoma cell lines(RAMOS,RAJI)were treated with DAC at different concentrations for 48 hours.CD19 CAR-T cells and non-transfected activated T cells were added and co-cultured at different target ratios(1:5,1:1 and 5:1)for 24hours.Similarly,CAR-T cells were treated with DAC at different concentrations for 24hours,and CAR-T cells were co-cultured with RAJI at E:T ratio of 1:1 for 24 hours.After pretreated with different concentrations of DAC for 48 hours,the mean fluorescence intensity(MFI)of CD19 on the surface of RAMOS and RAJI cells was measured by flow cytometry.Meanwhile,in this study,two patients with R/R CD19~+B-cell lymphoma were further selected to observe the efficacy and safety of DAC combined with CD19 CAR-T cells for the preliminary discussion of clinical feasibility.Results:CAR-T cells were prepared successfully and they recognized CD19 expressed on lymphoma cell lines specifically.The transfection efficiency of CD19 CAR-T cells was about 30%.Cell-line study shown that compared with untreated cells,lymphoma cells pre-treated by DAC made CAR-T cells had enhanced antitumor repertoire,especially at the 5:1 E:T ratio(p<0.05).Cell-line studies further suggested that,tumor antigen expression in lymphoma cell lines was increased by DAC pre-treatment,and CAR-T cells function was not compromised(p?0.05).The expression of CD19antigen on the surface of lymphoma cells(RAMOS,RAJI)was significantly up-regulated after DAC pretreated(p<0.05).Two patients with R/R B cell lymphoma were pre-treated with DAC then treated with CAR-T,both achieved complete remission(CR).Conclusions:DAC can effectively increase the killing effection of CAR-T cells.At lower concentrations,it may be related to the up-regulation of CD19 antigen expression,while at higher concentrations,DAC has directly killing effect on tumor cells.Adoptive transfer of anti-CD19 CAR-T cells,in combination with epigenetic modifying drugs(DAC)increases expression of the surface antigen,which improves treatment of R/R lymphoma.