鼠源抗CD47单克隆抗体的筛选及活性评价

Screening and Activity Evaluation of Mouse-derived Anti-CD47 Monoclonal Antibody

通过杂交瘤技术筛选得到了一株鼠源抗白细胞分化抗原47(CD47)单克隆抗体。采用聚乙二醇融合法,将小鼠脾细胞与SP2/0细胞融合,通过酶联免疫吸附试验(ELISA)法筛选抗CD47的杂交瘤细胞,通过Protein G亲和柱纯化抗体,利用十二烷基磺酸钠(SDS)-聚丙烯酰胺凝胶电泳(PAGE)法检测目的蛋白的相对分子质量,通过生物分子相互作用分析仪检测目的抗体的亲和力,并通过ELISA法测定抗CD47抗体的信号调节蛋白α(SIRPα)的阻断效力。用藻红蛋白(PE)标记的Raji细胞与不同浓度的抗CD47单抗共同孵育,检测抗CD47抗体促进巨噬细胞吞噬的作用。采用血凝试验对抗CD47单抗导致红细胞凝集的能力进行了分析,并进一步通过小鼠实体瘤模型,考察抗CD47单抗的抑制肿瘤生长作用。结果显示,筛选得到的一株杂交瘤细胞,即抗CD47单克隆抗体3D8,其亲和力常数值为1.29×10^(-11)mol/L,ELISA法测得其半数有效浓度(EC_(50))值为0.17 nmol/L,可有效阻断CD47与SIRPα的相互作用,增强巨噬细胞吞噬肿瘤细胞的能力,吞噬率达85%。该抗体在亲和力、阻断力、吞噬率等方面均优于在售抗体,同时不会导致红细胞凝集。在小鼠实体瘤模型中,肿瘤生长抑制率为95.44%,可明显抑制肿瘤生长。本研究为后期抗CD47抗体的人源化和抗CD47抗体的临床前研究提供了参考。

A mouse-derived cluster of differentiation 47(CD47) monoclonal antibody was screened by hybridoma technology.The polyethylene glycol fusion method was used to fuse mouse splenocytes with SP2/0 cells,and the anti-CD47 hybridoma cells were screened by enzyme-linked immunosorbent assay(ELISA).The antibody was purified by Protein G affinity column,and the relative molecular mass of the target protein was detected by sodium dodecyl sulfate(SDS)-polyacrylamide gel electrophoresis(PAGE).The affinity of the target antibody was detected by biomolecular interaction analyzer,and the blocking efficacy of signal regulatory protein α(SIRPα) of anti-CD47 antibody was determined by ELISA.Raji cells labeled with phycoerythrin(PE) were incubated with different concentrations of anti-CD47 monoclonal antibody,and the anti-CD47 antibody-promoted phagocytosis of macrophages was detected.The ability of anti-CD47 monoclonal antibody to induce red blood cell agglutination was analyzed by hemagglutination test,and the inhibitory effect of anti-CD47 monoclonal antibody on tumor growth was further investigated by mouse solid tumor model.The results showed that the screened hybridoma cell,anti-CD47 monoclonal antibody 3D8,had an affinity constant value of 1.29×10^(-11)mol/L,and its half effective concentration(EC_(50)) value measured by ELISA was0.17 nmol/L.It could effectively block the interaction between CD47 and SIRPα,and enhance the ability of macrophages to phagocytose tumor cells,with a phagocytosis rate of 85%.The anti-CD47 monoclonal antibody 3D8 was superior to the commercially available antibody in terms of affinity,blocking power,and phagocytosis rate,and did not cause red cell agglutination.In a mouse solid tumor model,the tumor growth inhibition rate of the product was 95.44%,indicating that it could significantly inhibit tumor growth.This study provided a reference for the later humanization of anti-CD47 antibody and preclinical research of anti-CD47 antibody.