基于多链结构的CD30 CAR-T细胞的抗肿瘤作用研究  

Study on the anti-tumor effect of CD30 CAR-T cells based on multi-chain structure

作　　者：宋羽佳 汪晨 王恩秀 汪波[1] Song Yujia;Wang Chen;Wang Enxiu;Wang Bo(Dept of Oncology,The Seventh Affiliated Hospital of Sun Yat-sen University,Shenzhen 518107;Nanjing KATI Medical Technology Co.Ltd.,Nanjing 210000)

机构地区：[1]中山大学附属第七医院肿瘤科,深圳518107 [2]南京卡提医学科技有限公司,南京210000

出　　处：《安徽医科大学学报》2024年第4期666-670,共5页Acta Universitatis Medicinalis Anhui

基　　金：国家自然科学基金(编号:81970177)。

摘　　要：目的基于衔接蛋白DAP12的多链嵌合抗原受体T细胞(CAR-T)开发靶向CD30的CAR-T细胞药物,研究CD30 CAR-T对霍奇金淋巴瘤肿瘤细胞的体外和体内临床前药效。方法通过基因合成和分子克隆技术,设计构建靶向CD30的CAR质粒,进行慢病毒包装,将得到的慢病毒转染T细胞,其中靶向CD30的多链CAR-T为CD30-KIRS2/Dap12-BB组,单链二代CAR-T为CD30-41BBζ组,未做病毒侵染的T细胞为NTD组,利用流式细胞术检测CAR阳性率情况,通过乳酸脱氢酶(LDH)释放检测细胞的杀伤活性,采用酶联免疫吸附试验(ELISA)检测细胞因子干扰素γ(IFN-γ)的分泌水平,进一步通过小鼠异种移植瘤模型检测CD30 CAR-T在小鼠体内抗肿瘤活性。结果靶向CD30的多链CAR-T和单链二代CAR-T进行对比,研究发现多链CAR-T与单链CAR-T的杀瘤作用相似。但值得注意的是,多链CAR-T的IFN-γ分泌水平更高(P<0.001)。更重要的是,在小鼠的肿瘤模型实验中,多链CAR-T实现了肿瘤的完全消退。结论靶向CD30的多链CAR-T在抗肿瘤活性方面优于传统单链CAR-T。Objective To develop a CD30-targeted CAR-T cell drug based on the multi-chain chimeric antigen receptor T cells(CAR-T)of the bridging protein DAP12,and to study the in vitro and in vivo preclinical efficacy of CD30 CAR-T on Hodgkin lymphoma tumor cells.Methods Through gene synthesis and molecular cloning techniques,a CAR plasmid targeting CD30 was designed and constructed,and the obtained lentivirus was packaged.The T cells were transfected with the lentivirus,where the multi-chain CAR-T targeting CD30 was the CD30-KIRS2/Dap12-BB group,the single-chain second-generation CAR-T was the CD30-41BBζgroup,and the T cells without virus infection were the NTD group.The positive rate of CAR was detected by flow cytometry,the cytotoxicity of the cells was detected by lactate dehydrogenase(LDH)release assay,the secretion level of the cytokine interferonγ(IFN-γ)was detected by enzyme-linked immunosorbent assay(ELISA),and the antitumor activity of CD30 CAR-T in mice was further detected by a mouse xenograft tumor model.Results A comparison was made between the multi-chain CAR-T targeting CD30 and the single-chain second-generation CAR-T.It was found that the antitumor effect of the multi-chain CAR-T was similar to that of the single-chain CAR-T.However,it was worth noting that the IFN-γsecretion level of the multi-chain CAR-T was higher(P<0.001).More importantly,in the mouse tumor model experiment,the multi-chain CAR-T achieved complete tumor regression.Conclusion The multi-chain CAR-T targeting CD30 is superior to the traditional single-chain CAR-T in terms of antitumor activity.

关 键 词：嵌合抗原受体修饰的T细胞 CD30 霍奇金淋巴瘤 DAP12 过继性细胞疗法 

分 类 号：R733.4[医药卫生—肿瘤]