In vitro construction and amplification and primary functional analysis of antiCD19 chimeric antigen receptor(CD19-CAR) modifiedTcells / 中国肿瘤生物治疗杂志

ABSTRACT

@#[Abstract] Objective: To establish a chimeric antigen receptor（CAR）modified T cells specifically targeting CD19 molecule (CD19CAR-T cells) and to testify their in vitro killing effect on target cells. Methods: CD19-CAR fragments yielded by PCR were constructed into pCDH-GFP lentiviral vectors by molecular cloning technology. The packaged lentiviral particles were transducted into CD3+ T cells of donors. Transduction efficiency was measured by flow cytometry and PCR. The in vitro cytotoxicity of obtained CD19CAR-T cells against CD19+ Ramos cells was tested by 7-AAD staining. Results: The amplification folds of CD3+ T cells increased to (78.8± 23.2) folds after in vitro culture for 10 days, and about (58.3±5.4)% cells expressing GFP.About (57.4±9.3)% CD19+Ramos cells were specifically killed by the CD19-CAR-T cells in vitro at the E∶T ratio of 5∶1. Conclusion: This study successfully established an effective method for constructing and amplifying CD19-CAR-T cells in vitro, which showed profound efficiency and specific cytotoxity against CD19+ Ramos cells.And this report might provide an experimental evidence for clinical treatment of CD19+ B cell neoplasmas.