摘要
目的:探讨鼠李糖(rhamnose,Rha)修饰对曲妥珠单抗(trastuzumab,Tra)Fc效应功能的影响。方法:将二硫键还原的Tra分子与含交联剂的Rha衍生物分子偶联,构建Tra-Rha偶联物。使用聚丙烯酰胺凝胶电泳、免疫印迹和高效液相分子排阻色谱对偶联物进行表征,并通过流式细胞术评估偶联物的HER2抗原亲和水平,免疫荧光和流式细胞术评估偶联物的抗Rha抗体募集能力。最后,通过在体外检测偶联物介导的补体依赖的细胞毒作用(complement-dependent cytotoxicity,CDC)和抗体依赖细胞介导的吞噬作用(antibody-dependent cell-mediated phagocytosis,ADCP),评估其Fc效应功能的增强水平。结果:成功构建了一款Tra-Rha偶联物。亲和实验和抗体募集实验表明Tra-Rha偶联物保留了良好的HER2抗原结合能力,并能将高水平抗Rha抗体选择性募集至靶细胞表面。CDC实验显示,Tra-Rha偶联物可介导显著的CDC效应对HER2阳性的SKBR3和SKOV3细胞进行杀伤,最高杀伤率分别为75%和70%,而相同条件下未修饰Rha的Tra则不能介导CDC。ADCP实验则显示,在SKBR3和SKOV3细胞中,Tra-Rha介导的ADCP水平较Tra分别提高了2.3倍和1.2倍。结论:Rha修饰可作为一种经济高效的策略,用于同时增强Tra的多种Fc效应功能,进而改善Tra的治疗指数。
Objective:To investigate whether rhamnose(Rha)modification could improve the Fc effector functions of trastuzumab(Tra).Methods:Tra-Rha conjugate was prepared by conjugating reduced Tra with Rha derivative.SDS-PAGE,Western blotting and HPLC-SEC assays were performed to identify the purified Tra-Rha conjugate.The specific binding affinity of the Tra-Rha conjugate to HER2 was evaluated by flow cytometry.The anti-Rha antibody recruiting capacity of the Tra-Rha conjugate was evaluated by immunofluorescence and flow cytometry.CDC and ADCP assays were performed to assess the level of enhanced Fc effector functions.Results:The Tra-Rha conjugate was successfully synthesized.Binding affinity and anti-Rha antibody recruiting capacity assays showed that Tra-Rha conjugate retained almost full binding affinity to HER2,and was able to selectively redirect high-level anti-Rha antibodies to target cells.In the subsequent CDC assay,Tra-Rha was shown to be capable of inducing potent cell killing in the presence of anti-Rha antibodies,with the maximum cell lysis in Tra-Rha-treated SKBR3 and SKOV3 cells being 75%and 70%,respectively.Notably,Tra failed to eliminate cells via CDC under the same conditions.Finally,the dramatically enhanced ADCP activity of the Tra-Rha conjugate was observed,with the detected phagocytosis rate in Tra-Rha treated SKBR3 and SKOV3 cells being 2.3-fold and 1.2-fold higher,respectively,compared to that of cells treated with Tra.Conclusion:Rha modification is a cost-effective strategy to generate novel Tra with enhanced effector functions,thereby improving the therapeutic index of Tra.
作者
洪皓飞
赵洁
李艳春
李丹
吴志猛
HONG Haofei;ZHAOJie;LI Yechun;LIDan;WUZhimeng(Key Laboratory of Carbohydrate Chemistry and Biotechnology of Ministry of Education,School of Biotechnology,Jiangnan University,Wuxi 214122,China)
出处
《中国生物工程杂志》
CAS
CSCD
北大核心
2024年第5期1-9,共9页
China Biotechnology
基金
国家自然科学基金(32000904、22177040)
博士后创新人才支持计划(BX20200153)
中央高校基本科研业务费专项资金(JUSRP123037)资助项目。
