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Expression of 5T4 extracellular domain fusion protein and preparation of anti-5T4 monoclonal antibody with high affinity and internalization efficiency.
Wang, Ruixue; Lai, Qinhuai; Lu, Ying; Zhou, Yan; Tang, Liangze; Tao, Yiran; Yao, Yuqin; Yu, Lin; Liu, Yu; Wang, Yuxi; Zhang, Ruirui; Jiang, Xiaohua; Gou, Lantu; Yang, Jinliang.
Afiliación
  • Wang R; Department of Biotherapy, Cancer Center/Collaborative Innovation Center for Biotherapy, West China Hospital, Sichuan University, Chengdu, China.
  • Lai Q; Department of Biotherapy, Cancer Center/Collaborative Innovation Center for Biotherapy, West China Hospital, Sichuan University, Chengdu, China.
  • Lu Y; Department of Biotherapy, Cancer Center/Collaborative Innovation Center for Biotherapy, West China Hospital, Sichuan University, Chengdu, China.
  • Zhou Y; The Gastroenterology Tumor and Microenvironment Laboratory, Department of Gastroenterology, The First Affiliated Hospital of Chengdu Medical College, Chengdu Medical College, Chengdu, China.
  • Tang L; Department of Biotherapy, Cancer Center/Collaborative Innovation Center for Biotherapy, West China Hospital, Sichuan University, Chengdu, China.
  • Tao Y; Department of Biotherapy, Cancer Center/Collaborative Innovation Center for Biotherapy, West China Hospital, Sichuan University, Chengdu, China.
  • Yao Y; Department of Biotherapy, Cancer Center/Collaborative Innovation Center for Biotherapy, West China Hospital, Sichuan University, Chengdu, China; Research Center for Public Health and Preventive Medicine, West China School of Public Health and Healthy Food Evaluation Research Center, NO. 4 West China
  • Yu L; Department of Biotherapy, Cancer Center/Collaborative Innovation Center for Biotherapy, West China Hospital, Sichuan University, Chengdu, China.
  • Liu Y; Department of Biotherapy, Cancer Center/Collaborative Innovation Center for Biotherapy, West China Hospital, Sichuan University, Chengdu, China.
  • Wang Y; Department of Biotherapy, Cancer Center/Collaborative Innovation Center for Biotherapy, West China Hospital, Sichuan University, Chengdu, China.
  • Zhang R; Department of Biotherapy, Cancer Center/Collaborative Innovation Center for Biotherapy, West China Hospital, Sichuan University, Chengdu, China.
  • Jiang X; Department of Biotherapy, Cancer Center/Collaborative Innovation Center for Biotherapy, West China Hospital, Sichuan University, Chengdu, China.
  • Gou L; Department of Biotherapy, Cancer Center/Collaborative Innovation Center for Biotherapy, West China Hospital, Sichuan University, Chengdu, China.
  • Yang J; Department of Biotherapy, Cancer Center/Collaborative Innovation Center for Biotherapy, West China Hospital, Sichuan University, Chengdu, China; Guangdong Zhongsheng Pharmaceutical Co., Ltd., China. Electronic address: jlyang01@163.com.
Protein Expr Purif ; 158: 51-58, 2019 06.
Article en En | MEDLINE | ID: mdl-29981846
5T4, a membrane protein, is overexpressed in many tumor tissues but rarely expressed in normal tissues. Here, CHO-5T4+ cells were generated and served as the antigen to immunize mice. Hybridoma techniques were employed to produce monoclonal antibodies (mAbs). The recombinant protein of human IgG Fc-fused extracellular domain of 5T4 (5T4 ECD-Fc) was obtained from transient expression in HEK293F cells. The fusion protein 5T4 ECD-Fc and CHO-5T4+ cells were respectively utilized to screen anti-5T4 antibodies that could bind to the native antigen. In preliminary screening, three hundred and fifty mAbs were obtained. Via surface plasmon resonance and flow cytometry screening, seven anti-5T4 mAbs stood out. Among them, H6 showed a high affinity (KD = 1.6 × 10-11 M) and internalization percentage (36% for 1 h and 80% for 4 h). The molecular weight and isoelectric point of H6 were determined by LC-MS and iCIEF. Moreover, the specific reactivity of H6 was demonstrated by western blotting, flow cytometry, and immunohistochemistry, respectively. In conclusion, we produced human recombinant protein of 5T4 extracellular domain and developed high-affinity internalizing monoclonal antibodies which may be applied in the 5T4-targeting ADC therapy and basic research.
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Proteínas Recombinantes de Fusión / Fragmentos Fc de Inmunoglobulinas / Glicoproteínas de Membrana / Anticuerpos Monoclonales de Origen Murino / Antineoplásicos Inmunológicos / Afinidad de Anticuerpos Límite: Animals / Female / Humans Idioma: En Revista: Protein Expr Purif Asunto de la revista: BIOLOGIA MOLECULAR Año: 2019 Tipo del documento: Article País de afiliación: China Pais de publicación: Estados Unidos
Texto completo
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Proteínas Recombinantes de Fusión / Fragmentos Fc de Inmunoglobulinas / Glicoproteínas de Membrana / Anticuerpos Monoclonales de Origen Murino / Antineoplásicos Inmunológicos / Afinidad de Anticuerpos Límite: Animals / Female / Humans Idioma: En Revista: Protein Expr Purif Asunto de la revista: BIOLOGIA MOLECULAR Año: 2019 Tipo del documento: Article País de afiliación: China Pais de publicación: Estados Unidos