针对 GD2 的嵌合单链可变片段-人免疫球蛋白 G 可结晶片段抗体用于神经母细胞瘤靶向免疫疗法

Q3 Medicine
Witida Laopajon, Nuchjira Takheaw, Kamonporn Kotemul, S. Pata, S. Hongeng, W. Kasinrerk
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引用次数: 0

摘要

目的:制备嵌合小鼠单链可变片段(scFv)和免疫球蛋白G1 (IgG1)结晶片段(Fc)抗二对话甘脂苷(GD2)抗体,用于治疗神经母细胞瘤(NB)。所生成的scFv-IgG Fc抗体缺乏重链第一恒定结构域(CH1),比天然抗体尺寸小,具有抗肿瘤活性。方法:构建scFv-IgG Fc抗体载体,在人胚胎肾293T (HEK293T)细胞系中表达scFv-IgG Fc抗体。用蛋白G亲和层析法从转染HEK293T细胞的培养上清中纯化scFv-IgG Fc抗体。采用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)、免疫印迹(WB)和免疫荧光技术验证了scFv-IgG Fc抗体的结构和结合活性。测定了抗体依赖性细胞毒性(ADCC)和抗体依赖性细胞吞噬(ADCP)的抗肿瘤活性。结果:利用质粒融合人IgG1-Fc2标签载体(pFUSE-hIgG1-Fc2),成功构建了小鼠scFv与hIgG1- fc2嵌合抗体的质粒载体。将该载体转染人HEK293T细胞,产生scFv-IgG Fc抗体。转染后的HEK293T细胞可产生嵌合的scFv-IgG Fc抗体,该抗体针对GD2,缺乏IgG重链CH1结构域,但携带CH2和CH3结构域。转染HEK293T后,在zeocin药物存在下,从培养上清中纯化出嵌合抗体。制备的GD2 scFv-IgG Fc抗体比完整抗体小,可通过ADCC和ADCP机制触发对表达GD2的NB细胞株SH-SY5Y的杀伤。结论:嵌合的scFv-hIgG Fc抗体缺乏重链CH1结构域,可介导抗体诱导的抗肿瘤活性。这种嵌合抗体体积小,可作为抗gd2抗体用于NB治疗。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Chimeric single-chain variable fragment-human immunoglobulin G crystallizable fragment antibody against GD2 for neuroblastoma targeted immunotherapy
Aim: The present study aims to generate chimeric mouse single-chain variable fragment (scFv) and immunoglobulin G1 (IgG1) crystallizable fragment (Fc) antibody against disialoganglioside (GD2) for the treatment of neuroblastoma (NB). The generated scFv-IgG Fc antibody, lacking first constant domain of heavy chain (CH1), is of a smaller size than the natural antibody and has anti-tumor activity. Methods: Vector for scFv-IgG Fc antibody was constructed and scFv-IgG Fc antibody was expressed in human embryonic kidney 293T (HEK293T) cell line. Purification of scFv-IgG Fc antibody from the culture supernatant of transfected HEK293T cells was performed by Protein G affinity chromatography. The structure and binding activity of scFv-IgG Fc antibody were verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), western blotting (WB), and immunofluorescence techniques. Anti-tumor activities by antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) were determined. Results: Using plasmid fusion-human IgG1-Fc2 tag vector (pFUSE-hIgG1-Fc2), a plasmid vector encoding chimeric mouse scFv and hIgG1 Fc antibody against GD2 was successfully constructed. This vector was transfected into human HEK293T cells to produce scFv-IgG Fc antibody. The transfected HEK293T cells could produce chimeric scFv-IgG Fc antibody against GD2, which lacks the IgG heavy chain CH1 domain but carries CH2 and CH3 domains. The chimeric antibodies could be purified from the culture supernatant of the transfected HEK293T culture in the presence of zeocin drug. The produced GD2 scFv-IgG Fc antibodies, which are smaller in size than the intact antibody, could trigger the killing of GD2 expressed NB cell line SH-SY5Y by ADCC and ADCP mechanisms. Conclusions: The results indicate that chimeric scFv-hIgG Fc antibody, lacking heavy chain CH1 domain, could mediate antibody induced anti-tumor activities. The small size of this type of chimeric antibody may be employed as anti-GD2 antibody for NB therapy.
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CiteScore
2.80
自引率
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