The Killing Effect Of Chimeric ScFvEGFRvⅢ-CD28-CD3ζ Modified T Lymphocytes Against Glioma Cells

Backgroud: Glioma is the most common malignant brain tumor in adults, although there are many treatments, the largest surgical resection, radiotherapy and chemotherapy have failed to achieve satisfactory curative effect. These treatments are restricted for the nonspecific damage to healthy brain and other tissues. The shortcomings of traditional treatments have stimulated the development of novel therapies. In comparison, the immune therapy is a precise therapy which can target central nervous system(CNS) based tumors, while minimizing off-target collateral damage to normal brain. A large number of clinical evidence has demonstrated that immunotherapy has potential against B-cell malignancies and solid tumors.Chimeric antigen receptor(CAR) is a proven method, it combines the variable region of antibody with the function of T lymphocytes, expressing on the surface of T lymphocytes to mediate the antigen specific activation. The CAR shows extraordinary potential in the treatment of renal cell carcinoma, neuroblastoma, acute lymphoblastic leukemia, chronic lymphocytic leukemia. In the past two decades, the technology of CAR has evolved dramatically, the CAR has been translated from the laboratory into clinical treatment of glioma.Epidermal growth factor receptor mutation type III(EGFRvIII) expresses on the surface of malignant glioma and other malignant tumor and doesn’t express on normal tissue, It is the best target for targeted therapy. This study designed chimeric antigen receptors scFvEGFRvIII-CD28-CD3 zeta which used scFvEGFRvIII to achieve targeted immunotherapy of EGFRvIII positive glioma cells, costimulatory molecule CD28 to promote the proliferation of T lymphocytes and prolong the life of T lymphocytes, CD3 zeta to activate T lymphocyte toxicity reaction. The lentivirus vector was used to redirect T lymphocytes with EGFRvIII-CD28/CAR, making EGFRvIII-CD28/CAR+T specificly kill EGFRvIII positive glioma cells(EGFRvIII +U87MG), the method could be regarded as a new idea for glioma immunotherapy.Objective: construct the second generation of lentivirus expression vector pCDH-CD28-CAR and explore the biological effect of chimeric scFvEGFRvIII-CD28-CD3 zeta modified T lymphocytes to EGFRvIII positive U87 MG in vitro.Methods: In part one,①Hinge-TM-CD28-CD3ζ was obtained by company using gene synthesis and inserted into the BamHI and EcoRI sites of lentivirus expression vector pCDH-EF1-MCS-T2A-copGFP through molecular cloning techniques, The new plasmid was named as pCDH-CD28-CD3ζ. scFv EGFRvIII gene sequence was amplified from pCR 2.1TOPO applying PCR reaction, and it was cloned into the EcoRI site of pCDH-CD28-CD3ζ. The connection direction of the gene sequences was identified by colony PCR. After DNA sequence analysis, the new vector named pCDH-CD28-CAR was obtained. ②three plasmids(envelope protein plasmid pVSVG, packaging plasmid pDel8.9 and pCDH-CD28-CAR containing the purpose gene) were transfected 293 T packaging cells by calcium phosphate precipitation, The medium which contained lentivirus supernatant was collected and concentrated using sucrose ultracentrifugation. The lentivirus was named as LV-EGFRvIII/CD28-CAR, and its titer was determinated by limited dilution method.In part two,①Human peripheral blood CD3+T lymphocytes were separated,activated and expended by immunomagnetic beads, and then were transfected by LV-EGFRv III/CD28-CAR. ②The expression of EGFRvIII/CD28-CAR on T lymphocytes was detected by Western blot and Flow cytometry. Cytokines IFN-γ secretion in the co-culture supernatant was tested by ELISA assay, the selective killing of EGFRvIII/CD28-CAR modified T lymphocytes was tested by 51 Cr release assay.Results: In part one, DNA sequencing analysis showed that each part of the CAR including scFv EGFRvIII, Hinge, TM, CD28 and CD3 zeta sequence was connected correctly. The drops of LV-EGFRvIII/CD28-CAR was up to 1.2x108 TU/ml, the transfection efficiency of LV-EGFRv III/CD28-CAR was 73.9%.In part two, the co-culture supernatant of the effector cells and target cells detected obvious IFN-gamma secretion, EGFRvIII/CD28-CAR+T lymphocytes showed specific killing to EGFRvIII positive U87 glioma cells in vitro, The secretion of cytokines and killing effect of EGFRvIII/CD28-CAR+T lymphocytes were both in EGFRvIII dependent manner.Conclusion: EGFRvIII/CD28-CAR+T lymphocytes showed significant specific killing effect to EGFRvIII positive U87 MG in vitro and laid a certain foundation toward experiment in vivo.