| Objective:Chronic Myeloid Leukemia(CML)is a malignant tumor of the blood system.Imatinib(IM)has significantly increased survival of patients with CML.However,it does not eliminate the leukemia stem cells(LSC).CML is a model immune system-sensitive disease,chimeric antigen receptor T cells(CAR-T)therapy has become the most studied in recent years due to its advantages such as high specificity and strong tumor cell killing effect.Compared with solid tumor tissue,CML does not have a powerful immunosuppressive microenvironment,its tumor cells circulate in peripheral blood and bone marrow.CAR-T cells can be injected into the body which can fully contact tumor cells.Therefore,CML is a disease suitable for the treatment of CAR-T cells.The key of CAR-T is to find specific antigens on the surface of tumor cells.Previous studies have shown that CD26 antigen is highly expressed on CML-LSC and has specificity,which can be considered as a specific molecular marker on CML-LSC.Therefore,this study intends to prepare CAR-T cells targeting CD26 and explore its biological effects.Methods:Mononuclear cells(MNC)were isolated from bone marrow specimens from newly diagnosed CML patients and healthy controls.The MNC were stained by flow cytometry antibodies CD34(PE),CD38(APC),and CD26(FITC).Flow cytometry detected the expression of CD26antigen in CD34~+/CD38~-stem cells.Prokaryotic expression experiments were used to verify the affinity of humanized anti-CD26 single-chain antibody(scFv)with CD26 antigen.Constructing pET32a-CD26-scFv recombinant plasmid,induce and purify the recombinant protein CD26-scFv,and the affinity of CD26-scFv with CD26 antigen was analyzed by indirect immunofluorescence and flow cytometry.The target binding domain CD26-scFv,the hinge region and the transmembrane region CD8?,the costimulatory molecules CD28,4-1BB and the intracellular signal transduction domain CD3?sequence were used to construct a third-generation CAR Structure,and ligated it to the lentiviral vector to form the pCDH-CD26-CAR recombinant plasmid.The lentiviral three-plasmid packaging system(pCDH-CD26-CAR,pMD2.G,pSPAX2)was selected for lentivirus preparation and modified T cells to construct CD26 CAR-T cells.Finally,the in vitro biological effects of anti-CD26-CAR-T cells were explored by proliferation,apoptosis experiments,and cytokine release analysis of CAR-T cells.Results:The results of flow cytometry showed that the average expression of CD26 antigen on CD34~+/CD38~-LSC was 29.26%,while CD34~+/CD38~-HSC was 6.53%.Comparison between the two groups P<0.05,the difference was statistically significant.The prokaryotic expression experiment was used to induce and purify the recombinant protein CD26-scFv.The affinity of CD26-scFv with CD26 antigen was analyzed by indirect immunofluorescence and FCM.And the results proved the good affinity of CD26-scFv with CD26 antigen.The second-generation lentivirus system was used to prepare CD26-CAR lentivirus.CD26-CAR transduced T cells were used to construct CD26CAR-T cells.The infection efficiency was 30.06%detected by FCM.The results of CCK8 experiments showed that CAR-T cell proliferation was not significant,FCM detection of apoptosis revealed that the apoptosis rate of CD26~+T cells reached 70%after 96 hours of lentiviral transduction.Conclusion:The study identified the expression and specificity of CD26 antigen on CML-LSC,and provided target support for killing CML-LSC.However,Due to activated T lymphocytes express CD26molecules,and the TRAF signal of the co-stimulatory molecule 4-1BB up-regulates intercellular adhesion molecule 1,the third generation of CD26 CAR-T cells we constructed exhibit extensive self-antigen-driven fratricide.But the results in this article lay the foundation for improving the construction of CD26 CAR-T cells. |
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