新型抗人CD47scFv-hFc融合抗体的制备、体外活性鉴定及抗原表位分析  

A novel anti-human CD47 scFv-hFc fusion antibody:production,in vitro characterization,and epitope mapping

作　　者：卢杨 张砚君 范冬梅 熊冬生 LU Yang;ZHANG Yanjun;FAN Dongmei;XIONG Dongsheng(State Key Laboratory of Experimental Hematology,National Clinical Research Center for Blood Diseases,Tianjin Key Laboratory of Cell Therapy 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,天津市血液病细胞治疗研究重点实验室,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：《生命的化学》2025年第3期503-511,共9页Chemistry of Life

基　　金：国家自然科学基金项目(82400238);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：CD47/信号调节蛋白α(signal regulatory proteinα,SIRPα)虽为最受关注的吞噬检查点之一,但其靶向抗体却因普遍存在的红细胞毒性限制了临床应用。2C8是一株低红细胞毒性的CD47单克隆抗体,基于抗体可变区骨架序列的高度保守性设计简并引物,通过RT-PCR法从2C8杂交瘤细胞中扩增获得全新抗人CD47轻、重链可变区序列。利用真核表达系统表达并纯化获得2C8scFv-hFc融合抗体纯品。采用流式细胞术检测融合抗体亲和力。采用吞噬实验检测融合抗体对不同亚型巨噬细胞的促吞噬能力。采用红细胞凝集实验检测融合抗体红细胞毒性。结果显示,2C8scFv-hFc融合抗体亲和力良好,对M0、M1、M2亚型巨噬细胞的促吞噬作用均显著优于阳性对照抗体,不同浓度下均不引起红细胞凝集,维持了亲本全抗的低红细胞毒性优势。利用生物大分子计算平台对抗体2C8的抗原结合表位进行了初步分析,结果显示,2C8与SIRPα的关键结合表位存在竞争及空间位阻。综上,扩增获得一对全新抗人CD47可变区序列,基于此表达制备的2C8scFv-hFc嵌合人源抗体具有高亲和力、强促吞噬能力、低红细胞毒性等特性,并对其促吞噬机制从结合表位角度进行了初步分析,为后续CD47抗体靶向治疗提供新的选择。Although CD47/SIRPαis one of the most studied“don't eat me”signals,the clinical application of its targeting antibodies is limited because of universal red blood cell(RBC)toxicity.2C8 is a CD47monoclonal antibody with low RBC toxicity.Based on the highly conserved framework regions of the antibody variable region,degenerate primers were designed to amplify the full-length light and heavy chain variable region sequences from 2C8 hybridoma cells by RT-PCR.The 2C8scFv-hFc fusion antibody was expressed and purified to obtain the pure product by a eukaryotic expression system.The affinity of the fusion antibody was determined by flow cytometry.The phagocytosis-promoting ability of the fusion antibody on different subtypes of macrophages was evaluated by phagocytosis assay.The hemagglutination assay was used to assess the RBC toxicity of the fusion antibody.The results showed that the 2C8scFv-hFc fusion antibody exhibited good affinity and significantly enhanced the phagocytosis of M0,M1 and M2 macrophages compared to the positive control antibody.It did not induce RBC agglutination at various concentrations,maintaining the low RBC toxicity advantage of the parental antibody.Initial analysis of the antigen-binding epitope of antibody 2C8 using a bio-macromolecular computation platform revealed that 2C8 competes with and sterically hinders the key binding site of SIRPα.In conclusion,a novel pair of human anti-CD47 variable region sequences was amplified.The 2C8scFv-hFc chimeric humanized antibody based on this sequence exhibited high affinity,strong phagocytosis-promoting ability,and low RBC toxicity.Its phagocytosispromoting mechanism was preliminarily analyzed from the perspective of the binding epitope.This study provides a new option for future CD47 antibody-targeted therapy.

关 键 词：CD47 肿瘤相关巨噬细胞 单克隆抗体 抗原结合表位 免疫治疗 

分 类 号：R392.11[医药卫生—免疫学]