新型CD47阻断型单克隆抗体的筛选及其抗结直肠癌活性观察  

Screening of a novel CD47-blocking monoclonal antibody and observation of its anti-colorectal cancer activity

作　　者：卢杨 袁向飞 张砚君 范冬梅 熊冬生 LU Yang;YUAN Xiangfei;ZHANG Yanjun;FAN Dongmei;XIONG Dongsheng(Institute of Hematology&Blood Diseases Hospital,Chinese Academy of Medical Sciences,Tianjin 300020,China;不详)

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 [3]天津市急腹症器官损伤与中西医修复重点实验室天津市南开医院,天津300100

出　　处：《山东医药》2025年第4期37-41,共5页Shandong Medical Journal

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：目的筛选靶向人CD47蛋白的新型阻断型单克隆抗体并观察其抗结直肠癌活性,为靶向人CD47靶点的免疫治疗提供新选择。方法利用单克隆抗体筛选技术获得能够稳定分泌CD47抗体的鼠源杂交瘤细胞株,制备小鼠腹水,纯化获得抗体纯品47-F。采用流式细胞术检测47-F抗体与CD47蛋白的亲和力,观察47-F抗体与商品CD47抗体B6H12.2对靶细胞结合的竞争性;采用红细胞凝集实验检测47-F抗体的红细胞毒性。分离人外周血单核细胞来源的巨噬细胞,采用细胞吞噬实验观察47-F抗体、B6H12.2抗体及同型对照抗体对人巨噬细胞吞噬结肠癌细胞HCT116和SW620能力的体外促进作用。建立小鼠皮下结肠癌移植瘤模型,采用分层随机分组法将小鼠分为抗体给药组和PBS对照组,观察47-F抗体对小鼠结肠癌移植瘤生长的体内抑制作用。结果47-F抗体能够有效结合CD47蛋白,具有较高亲和力;47-F抗体能有效竞争商品抗体B6H12.2对人CD47蛋白的结合,且具有浓度梯度依赖性。红细胞凝集实验结果显示,47-F抗体未引起红细胞凝集,红细胞毒性较低。与对照抗体比较,47-F抗体、B6H12.2抗体均提高了人巨噬细胞对SW620、hCT116细胞的吞噬率,且47-F抗体的促吞噬作用高于B6H12.2抗体(P均<0.05)。47-F抗体给药第19天,抗体给药组肿瘤体积小于PBS对照组(P<0.05),肿瘤抑制率为80.6%。结论成功筛选获得了一株新型CD47阻断型单克隆抗体47-F;该抗体具有亲和力强、促吞噬活性强、低红细胞毒性的特点,抗结直肠癌活性较强。Objective To screen a novel blocking monoclonal antibody targeting human CD47 protein and to ob-serve its anti-colorectal cancer activity,providing a new option for immunotherapy targeting the human CD47 target.Methods We obtained a murine hybridoma cell line that stably secreted CD47 antibodies using monoclonal antibody screening technology.Ascites were prepared in mice,and the purified antibody 47-F was obtained.The binding affinity of 47-F to CD47 protein was assessed using flow cytometry,and its competitive binding with the commercial CD47 antibody B6H12.2 to target cells was evaluated.Hemagglutination assay was conducted to determine the erythrocyte toxicity of 47-F.Human macrophages derived from peripheral blood mononuclear cells were isolated,and phagocytosis assay was used to observe the in vitro effects of 47-F,B6H12.2,and isotype control antibodies on macrophage-mediated phagocytosis of colorectal cancer cell lines HCT116 and SW620.A subcutaneous colorectal cancer xenograft model in mice was estab-lished,and mice were randomly assigned into the antibody treatment group and PBS control group.The in vivo inhibitory effect of 47-F on tumor growth was evaluated.Results The 47-F antibody effectively bound to CD47 protein with high af-finity and competitively inhibited the binding of the commercial antibody B6H12.2 to human CD47 protein in a concentra-tion-dependent manner.Hemagglutination assay results showed that 47-F did not induce red blood cell agglutination,indi-cating low erythrocyte toxicity.Compared with the control antibody,both 47-F antibody and B6H12.2 antibody significant-ly increased the phagocytosis rates of SW620 and HCT116 cells by human macrophages,with 47-F antibody demonstrating stronger pro-phagocytic activity than B6H12.2 antibody(P<0.05).On day 19 of antibody administration,the tumor vol-ume in the antibody-treated group was significantly smaller than that in the PBS control group(P<0.05),with a tumor inhi-bition rate of 80.6%.Conclusions A novel CD47-blocking monoclonal antibody,47-F,h

关 键 词：CD47 单克隆抗体 结直肠癌 肿瘤相关巨噬细胞 红细胞毒性 

分 类 号：R979.1[医药卫生—药品]