靶向CD19的CAR修饰的NK细胞对B细胞淋巴瘤的杀伤

Killing effect of NK cells modified with pCD19-CAR on B-cell lymphoma

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 theraputic monoclonal antibody CD19 using hybridoma technique in mice, and to explore killing effect of NK cells modified with pCD19-CAR which made by CD19 scFv sequence on B-cell lymphoma cells. Methods：CD19 monoclonal antibody was prepared in mice immunized with conjugated polypeptide, then sequence of the obtained antibody was obtained by gene sequencing method. The antibody sequence was analyzed and pCD19-CAR fragments were constructed via gene synthesis and molecular cloning PCR-based gene synthesis techniques. Then the pCD19-CAR fragments were cloned into lentiviral vectors, and transfected into NK-92MI cells after packaging preparation. and cloned into lenti-virus using molecular cloning method; and then the positive hybridoma cells were analyzed for scFv sequences. One of the scFv sequence was used to construct CAR （pCD19-CAR）.Finally, the killing rates of various pCD19-CAR-NK-92MI cells on B-cell lymphoma cells were examined using Flow cytometry assay. Results： （1） CD19 monoclonal antibody pCD19 with high specificity was successfully screened out. （2） The measurement of the antibody showed that the positive rates of Ramos and Raji cells were 84.3% and 85.6% respectively, which were similar to commercial CD19 antibody. （3） The killing efficacy of pCD19-CAR-NK-92MI cells, of which modification rate was 28.72%, on CD＋Ramos cells and CD＋Raji cells was apparently higher than those of non-modified NK-92MI cells （[47.0±1.7]% vs [24.7±6.2]% and [51.8±7.9]% vs [27.6±9.3]%, all P〈0.05）. Specific killing effects of unmodified and modified NK-92 cells on CD19- Jurkat cells were not almost found （[16.1±0.7]% vs [17.7±2.9]%, P〉0.05）. Conclusion：pCD19-CAR was successfully constructed ; pCD19-CAR-NK-92MI cells could specifically recognize CD19 antigen and kill CD19＋ B-cell lymphoma cells.