以CD27的胞外结构域为嵌合抗原受体构建靶向CD70的CAR-T细胞  

Construction and Functional Verification of CAR-T Cells Targeting CD70 Using the Extracellular Domain of CD27 as the Chimeric Antigen Receptor

作　　者：王兵[1] 郭睿婷 赵明峰[1] WANG Bing;GUO Ruiting;ZHAO Mingfeng(Tianjin First Central Hospital,Hematology Department,Tianjin 300192,China)

机构地区：[1]天津市第一中心医院,血液科,天津300192

出　　处：《中国细胞生物学学报》2023年第7期1029-1037,共9页Chinese Journal of Cell Biology

基　　金：天津市卫生健康科技项目(批准号:TJWJ2022XK018);天津市自然科学基金(批准号:22JCQNJC00820)资助的课题。

摘　　要：该研究以CD27分子的胞外段的部分肽段为嵌合抗原受体构建靶向CD70的嵌合抗原受体T(chimeric antigen receptor T,CAR-T)细胞并在体外验证其功能。通过流式细胞术检测急性髓系白血病(acute myeloid leukemia,AML)细胞系的CD70靶抗原的表达情况,利用基因工程方法构建包含4-1BB共刺激因子的第二代Anti-CD70慢病毒表达载体,并制备相应慢病毒,感染激活的人CD3+T细胞,获得靶向CD70的第二代CAR-T细胞。流式细胞术检测靶向CD70 CAR-T细胞对AML细胞系的体外杀伤功能;CBA试剂盒测定其细胞因子(包括IL-2、IL-4、IL-6、IL-10、TNF-α、IFN-γ)的分泌水平。研究结果表明,AML细胞系均表达CD70,并且在效靶比为1?1、2?1和5?1时,CD70 CAR-T细胞都能明显且特异性地杀伤表达CD70的AML细胞系。相比于对照组,CD70CAR-T细胞在杀伤靶细胞时分泌更高水平的IL-6、IL-10、IFN-γ等细胞因子(P<0.05)。综上所述,该研究成功构建了靶向CD70的CAR-T细胞,其可高效地杀伤表达CD70的靶细胞,为以后进一步改进和优化CD70 CAR-T提供了依据。This study constructed CAR-T(chimeric antigen receptor T)cells targeting CD70 using partial peptides of the extracellular segment of CD27 molecule as chimeric antigen receptor and verified its function in vitro.The expression of CD70 targets antigen in AML(acute myeloid leukemia)cell lines was detected by flow cytometry.The second generation anti-CD70 Lentivirus expression vector containing 4-1BB costimulatory factor was constructed by genetic engineering method,and the corresponding Lentivirus was prepared to infect activated human CD3+T cells to obtain the second generation CAR-T cells targeting CD70.Flow cytometry was used to detect the killing function of CD70 CAR-T cells targeting AML cell lines in vitro;the secretion level of cytokines was measured by CBA kit(including IL-2,IL-4,IL-6,IL-10,TNF-α、IFN-γ).The research results indicated that CD70 was expressed in AML cell lines.And the CD70 CAR-T significantly and specifically killed AML cells expressing CD70 when the effector target ratio was 1׃1,2׃1 and 5׃1.Compared with the untransduced T cells,CD70 CAR-T cells killed target cells and secreted higher levels of inflammatory cytokines,such as interleukin-6,interleukin-10 and interferon-γ(P<0.05).In summary,this study the CD70 targeting CAR-T cells have been successfully constructed and have excellent killing functions,which provided the foundation for further improvement and optimization of CD70 CAR-T in the future.

关 键 词：急性髓系白血病 CD27分子 CD70分子 CAR-T 细胞治疗 

分 类 号：R733.71[医药卫生—肿瘤]