| BACKGROUND:In hematological malignant tumors,the incidence of B-cell malignant tumors is increasing year by year,but the treatment of B-cell malignant hematological tumors is still very limited.Although CD 19 CAR-T has made great progress in the treatment of CD 19-positive B-cell malignant hematological tumors,it still has a high recurrence rate after traditional CD 19 CAR-T treatment.Therefore,the preparation of a novel CAR-T anti-B-cell malignant tumors is an urgent problem.OBJECTIVE:In this study,CD19/NB CAR-T cells(CD19/NB CAR-T)based on nanobodies were constructed by using the CD 19 nano-antibodies screened by our research group.The proliferation activity of CD19/NB CAR-T cells in vitro and the killing effect on target cells were explored.The growth inhibition of CD19/NB CAR-T cells on NOD/SCID mice bearing Nalm6 cell hematoma in vivo was further studied.It provides a new strategy for the use of nanobodies in the treatment of malignant hematological tumors.METHOD:1.Human peripheral blood T lymphocytes were isolated and cultured.CD19/NB CAR-T cells were transfected with CD19/NB CAR lentivirus to obtain CD19/NB CAR-T cells.The expression of GFP in CD19/NB CAR-T cells was observed by fluorescence microscopy.The expression of GFP in CD19/NB CAR-T cells and the ratio of CD4+/CD8+in CD19/NB CAR-T cells were detected by flow cytometry.2.Flow cytometry was used to detect the proliferation of CD19/NB CAR-T cells after co-incubation with target cells;Flow cytometry was used to detect the expression of CD25,CD69,memory molecule CD62L and lysosomal protein CD107a on CD19/NB CAR-T cells after co-incubation with target cells.3.Flow cytometry was used to detect the expression of CD 19 on Nalm6,Raji,K562-CD19 and K562 cells.Flow cytometry was used to detect the killing effect of CD19/NB CAR-T cells on Nalm6,Raji,K562-CD19 and K562 cells.4.The secretion of cytokines(TNF-α,IL-2,IL-10)by CD19/NB CAR-T cells stimulated by target cells was detected by ELISA.The number of T cells secreting IFN-gamma by CD19/NB CAR-T cells stimulated by target cells was detected by ELI SPOT.5.The model of NOD/SCID mice bearing Nalm6 cell hematoma was established and randomly divided into PBS,UTD,Mock,CD105/NB CAR and CD19/NB CAR groups.Car T cells were injected into the tail vein to detect the residual number of malignant tumor cells in the blood of tumor-bearing mice and observe the survival rate of mice to explore the anti-tumor effect of CD 19/NB CAR-T cells in vivo.RESULT:1.After lentivirus transfection into T cells,GFP fluorescence was observed under fluorescence microscope.The results of flow cytometry showed that the expression rates of GFP in T cells of Mock group,CD105/NB CAR group and CD19/NB CAR group were all over 40%,reaching(40.5+2.37)%,(42.2+3.09)%and(41.7+2.86)%,respectively,indicating that CD19/NB CAR-T cells were successfully prepared.2.The proliferation of CD19/NB CAR-T cells stimulated by CD 19+target cells was significantly higher than that of the other control groups(P<0.001);the expression levels of CD25,CD69,memory molecule CD62L and lysosomal protein CD 107a on the surface of CD19/NB CAR-T cells were higher than those of the other control groups(P<0.001).3.Flow cytometry showed that the expression rates of CD 19 in K562-CD19,Nalm6 and Raji cells were 90.1%,77.3%and 69.5%respectively.K562 cells hardly expressed CD 19.CD19/NB CAR-T cells can specifically kill CD 19+target cells in vitro.4.The levels of TNF-alpha and IL-2 secreted by CD19/NB CAR-T cells stimulated by CD19+target cells(K562-CD19 cells and Nalm6 cells)were significantly higher than those of other control groups,and there was no statistical difference in the levels of IL-10 in each group.The levels of TNF-alpha,IL-2 and IL-10 secreted by CD19/NB CAR-T cells stimulated by CD19-target cells(K562 cells)were not statistically different from those of other control groups.Differences in learning.ELISPOT results showed that the number of T cells secreting IFN-gamma in CD19/NB CAR-T cells stimulated by CD19+target cells(Nalm6 cells)was significantly higher than that in other control groups.5.In vivo experimental results showed that CD19/NB CAR-T cells treated Nalm6 NOD/SICD mice had certain therapeutic effect on hematoma expressing CD 19.The growth of tumor cells in tumor-bearing mice was inhibited to some extent.The survival curve showed that the survival time of tumor-bearing mice was prolonged after treatment with CD19/NB CAR-T cells.CONCLUSION:This study successfully constructed CD 19 CAR-T cells based on nano-antibodies.CD19/NB CAR-T cells can specifically target CD19+target cells in vitro,and have anti-tumor effect in NOD/SCID mice bearing Nalm6 cells.It provides a new strategy for the use of nano-antibodies in the subsequent immunotherapy of malignant hematological tumors. |
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