靶向CD123抗原的双特异性抗体的构建及其抗急性髓系白血病的作用研究  

Preparation of a dual-specific antibody targeting human CD123 and exploration of its anti-acute myeloid leukemia effects

作　　者：周彤 陈曼玲 张楚悦 刘晓雨 王珍珍 邢海燕 唐克晶 田征 饶青 王敏 王建祥 Zhou Tong;Chen Manling;Zhang Chuyue;Liu Xiaoyu;Wang Zhenzhen;Xing Haiyan;Tang Kejing;Tian Zheng;Rao Qing;Wang Min;Wang Jianxiang(State Key Laboratory of Experimental Hematology,National Clinical Research Center for Blood Diseases,Haihe Laboratory of Cell Ecosystem,Institute of Hematology&Blood Diseases Hospital,Chinese Academy of Medical Sciences&Peking Union Medical College,Tianjin 300020,China;Tianjin Institutes of Health Science,Tianjin 301600,China)

机构地区：[1]中国医学科学院、北京协和医学院血液病医院(中国医学科学院血液学研究所),血液与健康全国重点实验室,国家血液系统疾病临床医学研究中心,细胞生态海河实验室,天津300020 [2]天津医学健康研究院,天津301600

出　　处：《中华血液学杂志》2024年第3期225-232,共8页Chinese Journal of Hematology

基　　金：国家自然科学基金重点项目(81830005);国家重点研发计划(2021YFC2500300);中国医学科学院医学与健康科技创新工程项目(2021-I2M-1-041)。

摘　　要：目的构建一种新的靶向CD123抗原的双特异性抗体(CD123 DuAb),研究CD123DuAb在急性髓系白血病(AML)治疗中的作用。方法以自主研发的CD123单克隆抗体可变区为基础,利用分子克隆技术,构建CD123 DuAb表达质粒,转染ExpiCHO-S细胞,表达该双功能抗体。通过功能实验,验证CD123 DuAb在T细胞活化及增殖中的作用,及其促进T细胞对AML细胞的杀伤作用。结果①构建了CD123 DuAb表达质粒,并通过Expi-CHO真核系统表达。②CD123 DuAb可以分别与T细胞上的CD3位点及CD123阳性肿瘤细胞上的CD123位点结合。③将1 nmol/L CD123 DuAb加入T细胞与MV4-11细胞共培养体系中,T细胞中CD69阳性表达率为68.0%,CD25阳性表达率为44.3%,均显著高于对照组(P值均<0.05)。④在CD123 DuAb浓度为1 nmol/L的条件下培养T细胞,可促进T细胞增殖,其绝对计数第1天为5×10^(5)/ml,第9天扩增至3.2×10^(6)/ml,且CFSE荧光强度显著降低。⑤CD123 DuAb能够显著激活T细胞,且激活强度与其浓度呈正相关,浓度为1 nmol/L时,T细胞CD107a表达率可达16.05%,较对照组显著升高(P<0.05)。⑥随培养体系中CD123 DuAb浓度的升高,T细胞耗竭和凋亡也随之增加,浓度为1 nmol/L时,T细胞CD8+PD-1+LAG-3+比例为10.90%,PI-AnnexinⅤ+比例为18.27%,PI+AnnexinⅤ+比例为11.43%,均较对照组显著升高(P<0.05)。⑦CD123 DuAb能够促进T细胞分泌细胞因子,浓度为1 nmol/L时,共培养体系上清中的IFN-γ和TNF-α的浓度可分别达到193.8 pg/ml和169.8 pg/ml,较对照组显著升高(P<0.05)。⑧将CD123 DuAb以1 nmol/L的浓度加入T细胞与CD123阳性肿瘤细胞共培养体系中,能显著增强T细胞对肿瘤细胞的杀伤作用,共培养3 d,CD123+MV4-11细胞、CD123+Molm13细胞和CD123+THP-1细胞的残留率分别为7.4%、6.7%和14.6%,较对照组显著降低(P<0.05)。结论本研究构建及表达了一种靶向CD123的双特异性抗体,并通过体外实验验证其可以同时结合CD123阳性肿瘤细胞和T细胞,促进T细胞�Objective To construct a novel dual-specific antibody targeting human CD123(CD123 DuAb)and study its effects in acute myeloid leukemia(AML).Methods Based on the variable region of the CD123 monoclonal antibody independently developed at our institution,the CD123 DuAb expression plasmid was constructed by molecular cloning and transfected into ExpiCHO-S cells to prepare the antibody protein.Through a series of in vitro experiments,its activation and proliferation effect on T cells,as well as the effect of promoting T-cell killing of AML cells,were verified.Results①A novel CD123 DuAb plasmid targeting CD123 was successfully constructed and expressed in the Expi-CHO eukaryotic system.②The CD123 DuAb could bind both CD3 on T cells and CD123 on CD123+tumor cells.③When T cells were co-cultured with MV4-11 cells with addition of the CD123 DuAb at a concentration of 1 nmol/L,the positive expression rates of CD69 and CD25 on T cells were 68.0%and 44.3%,respectively,which were significantly higher than those of the control group(P<0.05).④Co-culture with CD123 DuAb at 1 nmol/L promoted T-cell proliferation,and the absolute T-cell count increased from 5×10^(5)/ml to 3.2×10^(6)/ml on day 9,and CFSE fluorescence intensity decreased significantly.⑤With the increase in CD123 DuAb concentration in the culture system,T-cell exhaustion and apoptosis increased.When the CD123 DuAb was added at a concentration of 1 nmol/L to the culture system,the proportion of CD8+PD-1+LAG-3+T cells was 10.90%,and the proportion of propidium iodide(PI)−AnnexinⅤ+T cells and PI+AnnexinⅤ+T cells was 18.27%and 11.43%,respectively,which were significantly higher than those in the control group(P<0.05).⑥The CD123 DuAb significantly activated T cells,and the activation intensity was positively correlated with its concentration.The expression rate of CD107a on T cells reached 16.05%with 1 nmol/L CD123 DuAb,which was significantly higher than that of the control group(P<0.05).⑦The CD123 DuAb promoted cytokine secretion by T cells at a co

关 键 词：白血病 髓系 急性 CD123 双特异性抗体 免疫治疗 

分 类 号：R73[医药卫生—肿瘤]