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

作　　者：袁园[1] 秦扬[2] 游凤涛 陈丹 邹建炫[1] 朱学军[4] 李炳宗[5] 袁磊[6] 孟会敏[1] 张波桢 安钢力[1] 杨林[1] YUAN Yuan QIN Yang YOU Fengtao CHEN Dan ZOU Jianxuan ZHU Xuejun LI Bingzong YUAN Lei MENG Huimin ZHANG Bozhen AN Gangli YANG Lin(The Cyrus Tang Hematology Center, Soochow Universi- ty, Suzhou 215123, Jiangsu, China Department of Urology, Yunnan Cancer Hospital, Kunming 650118, Yunnan, China PersonGen Biomedicine （Suzhou） Co. , Ltd, Suzhou 215123, Jiangsu, China Department of Hematology, Jiangsu Province Hospital of TCM, Nanjing 210029, Jiangsu, China Department of Hematology, The Second Affilia- ted Hospital of Soochow University, Suzhou 215004, Jiangsu, China Department of Hematology, Chinese PLA General Hospital, Beijing 100853, China)

机构地区：[1]苏州大学唐仲英血液学研究中心,江苏苏州215123 [2]云南省肿瘤医院泌尿科,云南昆明650118 [3]博生吉医药科技(苏州)有限公司,江苏苏州215123 [4]江苏省中医院血液科,江苏南京210029 [5]苏州大学附属第二医院血液科,江苏苏州215004 [6]中国人民解放军总医院血液科,北京100853

出　　处：《中国肿瘤生物治疗杂志》2016年第5期613-619,共7页Chinese Journal of Cancer Biotherapy

基　　金：国家自然科学基金资助项目(No.31471283)~~

摘　　要：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.

关 键 词：单克隆抗体 嵌合抗原受体 NK-92MI细胞 CD19 B细胞淋巴瘤 

分 类 号：R730.51[医药卫生—肿瘤] R967[医药卫生—临床医学]