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目的:利用鼠杂交瘤技术筛选和制备治疗性CD19单克隆抗体,探讨以CD19 scFv序列构建CD19-CAR(p CD19-CAR)修饰的NK细胞对CD19~+B细胞淋巴瘤细胞的杀伤作用。方法 :用偶联多肽免疫小鼠制备CD19单克隆抗体,然后用基因测序法获得抗体的序列。分析抗体序列,并通过基因合成和分子克隆技术构建pCD19-CAR片段,然后将其克隆到慢病毒载体上,病毒包装制备后,转染NK-92MI细胞。最后,用流式细胞术检测不同的pCD9-CAR-NK-92MI细胞对CD19~+细胞的杀伤率。结果:(1)成功筛出特异性强的CD19单克隆抗体-pCD19;(2)抗体检测结果显示CD19+的Ramos细胞的阳性率为84.3%,Raji为85.6%,与商业化抗体结果相似;(3)被pCD19-CAR修饰的CD19-CAR阳性率为28.72%的NK-92MI细胞对CD19~+的Ramos和Raji细胞的杀伤效率明显高于未被修饰的NK-92MI细胞株对Ramos和Raji细胞的杀伤率[(47.1±1.7)%vs(24.7±6.2)%和(51.8±7.9)%vs(27.6±9.6)%,均P<0.05];对CD19-细胞Jurkat,不论是未被pCD19-CAR修饰的NK-92MI或是被修饰的NK-92MI细胞,几乎都不存在特异性杀伤作用[(16.1±0.7)%vs(17.7±2.9)%,P>0.05]。结论:成功构建pCD19-CAR,被pCD19-CAR修饰的NK-92MI细胞能特异性的识别CD19抗原并杀伤CD19~+B细胞淋巴瘤细胞。
OBJECTIVE: To screen and prepare therapeutic monoclonal antibodies against CD19 using murine hybridoma technique and to investigate the killing effect of CD19-CAR (pCD19-CAR) modified NK cells on CD19 + B cell lymphoma cells by CD19 scFv. Methods: Mice were immunized with conjugate peptide to prepare CD19 monoclonal antibody, and the sequence of antibody was obtained by gene sequencing. Antibody sequences were analyzed and the pCD19-CAR fragment was constructed by gene synthesis and molecular cloning techniques. The fragment was then cloned into lentiviral vector and transfected into NK-92MI cells after virus packaging was prepared. Finally, the killing rates of CD19 ~ + cells by different pCD9-CAR-NK-92MI cells were detected by flow cytometry. Results: (1) The CD19 monoclonal antibody-pCD19 was successfully screened out. (2) The results of antibody detection showed that the positive rate of Ramos cells was 84.3% and Raji was 85.6%, which was similar to that of commercial antibodies. 3) The CD19-CAR modified CD19-CAR positive rate of 28.72% NK-92MI cells on CD19 ~ + Ramos and Raji cells was significantly higher than the efficiency of unmodified NK-92MI cells on Ramos and Raji cells (47.1 ± 1.7)% vs (24.7 ± 6.2)% and (51.8 ± 7.9)% vs (27.6 ± 9.6)%, respectively, all P <0.05] .The killing rate of Jurkat on CD19- There were almost no specific killing effects of CAR-modified NK-92MI or modified NK-92MI cells [(16.1 ± 0.7)% vs (17.7 ± 2.9)%, P> 0.05]. CONCLUSION: The constructed pCD19-CAR and NK-92MI cells modified by pCD19-CAR can specifically recognize CD19 antigen and kill CD19 + B cell lymphoma cells.