The Construction Of CD19-CAR-T Cell And The Effect Of CD19-CAR-T Cell On CD19 Positive Hematological Malignancies

Objective With the gradual development of gene editing technology, adoptive cellular therapy plays an important role in tumor targeted therapy. Due to many limitations, adoptive immunotherapy did not have significant anti-tumor effect. But a new development of adoptive cellular therapy- chimeric antigen receptor(CAR), greatly enhances the therapeutic effects of adoptive immunotherapy; its outstanding advantage is that the CAR can overcome the limitation of MHC. The CD19 antigen has a high expression rate on B lymphocytes and some malignant tumor cells, and it is not expressed on normal hematopoietic stem cell surface. Therefore it is an ideal target for targeted therapy. We designed the second generation of chimeric antigen receptor. In this study, the pc DNA?3.1(+) was used as plasmid vector, and the mouse anti human CD19 monoclonal antibody(clone No: FMC63) was used as single chain variable region sequence, costimulatory molecule CD137 to enhance T cell activity, CD3δ to activate the T cell toxicity reaction. Then CAR-T cells were cultured in the serum free medium with or without IL-15. And we observed the CAR-T expression efficiency in different medium. At the same time, the cultured CD19-CAR-T cells were co-cultured with CD19 positive tumor cells. And we make a preliminary study of CAR-T anti CD19 positive tumor in vitro by observing the secretion of killing cytokine.Methods Firstly, we determined the CD19 monoclonal antibody single chain variable fragment, CD137 costimulation molecules and CD3 zeta chain sequence. Then we integrated them into a whole sequence by using genetic engineering method, and it was named CD19-CAR. Then the CD19-CAR fragment was cloned into lentiviral vector pc DNA?3.1(+) and the insertion of the restriction site is Nhe I/Bam HI. Then the shuttle plasmids and the packaging plasmids(p Gag/Pol、p Rev、p VSV-G) and pc DNA?3.1(+) plasmid were co-cultured and transfected into 293 T cells to produce lentiviral. The DNA sequencing and flow cytometry were used to detect whether the lentiviral sequence is correct. The CD19 lentiviral was used to transfect T cells and the CAR-T cells were cultured in the serum free medium with or without IL-15. The expression of CD19-CAR and cytokine(TNF-α、IL-2) in the transduced T cells were detected by flow cytometry.Results DNA sequencing analysis showed that each part of chimeric antigen receptor including CD19 monoclonal antibody sc Fv, hinge region, CD137 and CD3δ was correct. The titer of lentivirus CD19-CAR is about 2.0x106TU/ml. The results of flow cytometry showed that CD19-CAR was successfully constructed and expressed on the surface of cell membrane. The expression rate of CD19-CAR was 45% in the serum free medium with low concentration of IL-15, and the rate was 9% in the serum free medium without IL-15. When the CD19-CAR-T cells were co-cultured with 293 T cells, the secretion of IL-2 and TNF-αwere 1.89% and 0.2%, however, when the CD19-CAR-T cells were co-cultured with CD19-K562 cells, the cytokine secretion were 32.49% and 30.1%, respectively.Conclusion The sequence of CD19-CAR were constructed correctly and expressed successfully, the low concentration of IL-15 can promote the growth of CAR T cells. The CD19-CAR-T cells could recognize CD19 positive cells specifically and cause the release of large amounts of IL-2 and TNF-α.