Research On Anti-tumor Effect And Mechanism Of Nanobody-based CD 19 CAR T Cells With PD-1 Secretion

Background:Acute Lymphoblastic Leukemia(ALL)is a malignant tumor in the blood system,mostly originates from B cells.Although the effect of chemotherapy is considerable,there are still many refractory ALL patients with high mortality.As an emerging immunotherapy method,the development of Chimeric Antigen Receptors T Cell(CAR T)technology is obvious to all.So far,the clinical research of CAR T in treating tumors is mainly focused on hematological malignancies targeting CD 19.In terms of hematological malignancies,especially in the treatment of ALL,it has achieved gratifying results.However,most of the extracellular antigen recognition domains currently used to construct CAR are single-chain antibody fragments(scFv)that recognize tumor-associated antigens(TAA),which is sticky,agglomerated,and highly immunogenic.In addition,some clinical patients are ineffective or resistant to CD 19 CAR T.Therefore,it is urgent to find a novel antibody different from the single-chain antibody epitope and prepare a new CAR T to improve the efficacy of relapse or refractory ALL.Objective:To construct a new type of anti-tumor CAR T cell that secreted PD-1 nanobodies and targeted CD19(CD19-PD-1/Nb CAR T).To verify whether the simultaneous PD-1 secretion can minimize the inhibitory effect related to the blocking of immune checkpoints and improve the T cell killing activity.To provide new evidence and strategies for immunotherapy of malignant hematological tumors.Method:1.Mock,PD-1/Nb CAR,CD19/Nb CAR,Irrelevant-PD-1/Nb CAR,CD19-PD-1/Nb CAR gene sequences were designed.The GV400 lentiviral vector was cut by the double-enzyme cutting scheme to complete the target gene and vector plasmid ligation.Lentivirus packaging and concentration were performed,and lentivirus titer was calculated by fluorescence counting.2.Human peripheral blood T lymphocytes were isolated;the harvested lentivirus was transfected into T cells to complete the construction of CAR T cells;the expression of GFP of CAR T cells was detected by a fluorescence microscope;the ratio of GFP and His tag expressed by CAR T cells was detected by flow cytometry.3.Using untransfected T cells(Utd)as control,flow cytometry was used to detect the T cells in the Utd,Mock,PD-1/Nb CAR,CD19/Nb CAR,Irrelevant-PD-1/Nb CAR,and CD19-PD-1/Nb CAR groups.The proliferation of CAR T cells after co-incubation with target cells was evaluated and the expression of surface-activating molecules CD25,CD69,memory molecules CD62L,lysosomal protein CD 107a,and intracellular IFN-γ.4.Flow cytometry was used to detect the killing effect of CD19-PD-1/Nb CAR T on Raji,K562-CD19,and K562 cells.The content of cytokines(IFN-γ,TNF-α,IL-2,IL-10)secreted in the supernatant was detected by ELISA.ELISPOT was used to count the number of CAR T cells that secret IFN-γ from CAR T cells under stimulation of target cells.5.NOD/SCID mice-bearing B-ALL hematologic model(Raji model)was constructed,and randomly divided into PBS,Utd,Mock,PD-1/Nb CAR,CD19/Nb CAR,FAP-PD-1/Nb CAR,CD19/Nb CAR+ PD-1Nb,and CD19-PD-1/Nb CAR groups.After injecting CAR T cells and untransfected T cells in each group through the tail vein,the residual number of malignant tumor cells in the peripheral blood of tumor-bearing mice was measured by flow cytometry,and the survival rate of tumor-bearing mice was respectively observed to evaluate the anti-tumor effect of CAR T cells in vivo.6.NOD/SCID mice-bearing B-ALL hematoma model(Raji model)was constructed,and randomly divided into PBS,Utd,Mock,PD-1/Nb CAR,Irrelevant-PD-1/Nb CAR,CD19/Nb CAR,CD19/Nb CAR+PD-1Nb,and CD 19-PD-1/Nb CAR group.After injection of CAR T cells and untransfected T cells in each group through the tail vein,flow cytometry was used to detect the expression of human CD3 in the spleen of tumor-bearing mice on day 7,14 and 28 of each group.Results:1.Our study successfully completed GV400 lentiviral vector cleavage and gene recombination using a double-enzyme digestion scheme.PCR method was applied to verify that the CAR genes of each group could be successfully inserted into the vector;the recombinant vector was packaged into lentivirus and concentrated,and the lentiviral titer was finally harvested up to 2×109TU/mL.2.After applying the constructed lentivirus to T cell infection,green fluorescence of GFP was observed under a fluorescence microscope.Flow cytometry showed that the GFP and His tag expression of CAR T cells in each group exceeded 40%,indicating that CAR T cells were successfully prepared.3.CD 19-PD-1/Nb CAR T cells proliferated actively after being stimulated by CD19+target cells,the proliferation frequency and multiples are significantly higher than those of the other control groups;their cell surface activating molecules CD25,CD69,memory molecules CD62L,and the expression of lysosomal protein CD 107a was higher than that of the other control groups,and the number of positive cells secreting IFN-γ was significantly increased compared with the other control groups.4.CD19-PD-1/Nb CAR T cells can significantly kill CD 19+target cells in vitro,and its killing effect is enhanced with the increase of the effect:target ratio.The killing ratio of Raji target cells with high expression of CD 19(96.6%)is higher than that of CD 19 with low expression K562-CD 19(42.2%)cells.CD19-PD-1/Nb CAR T cells have no killing effect on CD19’ tumor cells.When co-cultured with CD 19+target cells,the contents of cytokines IFN-γ,TNF-α,and IL-2 in CD 19-PD-1/Nb CAR T cell supernatants increased significantly,while the content of IL-10 did not change significantly.5.In vivo experiments observed that CD 19-PD-1/Nb CAR T cell treatment can significantly inhibit tumor development in Raji hematological transplanted tumor mice,and prolong the survival time and increase the survival rate of mice.6.GFP expression in spleen tissue of mice in CD 19-PD-1/Nb CAR group was significantly higher than that of the other control groups at 14 days after tail vein injection of CAR T cells in each group.Conclusion:The novel anti-tumor CD 19-PD-1/Nb CAR T cells were successfully constructed using lentiviral packaging technology.The constructed CD19-PD-1/Nb CAR T has certain anti-malignant hematological effects in vivo and in vitro which provides a little experimental basis for the clinical treatment of CAR T and a new strategy for adoptive treatment of malignant hematological tumors.