药学学报, 2018, 53(3): 388-395
引用本文:
马琳琳, 朱敏, 李光辉, 李延飞, 盖军伟, 万亚坤. TIM-3纳米抗体噬菌体展示文库的构建及筛选[J]. 药学学报, 2018, 53(3): 388-395.
MA Lin-lin, ZHU Min, LI Guang-hui, LI Yan-fei, GAI Jun-wei, WAN Ya-kun. Construction and screening of phage display library for TIM-3 nanobody[J]. Acta Pharmaceutica Sinica, 2018, 53(3): 388-395.

TIM-3纳米抗体噬菌体展示文库的构建及筛选
马琳琳1, 朱敏2, 李光辉2, 李延飞1, 盖军伟2, 万亚坤2
1. 上海健康医学院上海市教委分子影像重点实验室, 上海 201318;
2. 上海洛启生物医药技术有限公司, 上海 201318
摘要:
肿瘤免疫治疗是肿瘤治疗的一个重要突破口。T细胞免疫球蛋白黏蛋白-3(T cell immunoglobulin and mucin-domain-containing molecule-3,TIM-3)作为T细胞抑制性免疫检查点,在肿瘤抗体治疗中具有独特的应用优势。骆驼科动物体内存在的一种天然缺失轻链的纳米抗体(nanobody,Nb),逐渐成为新一代抗体治疗的新兴力量。本研究利用TIM-3抗原成功免疫新疆双峰驼,建立噬菌体展示文库,并通过噬菌体展示技术初步筛选出29株序列差异的TIM-3纳米抗体。另外,本研究成功构建了TIM-3稳转细胞株,利用流式细胞仪从这29株纳米抗体中筛选出10株特异性强且亲和力高的TIM-3纳米抗体。本研究将为功能性阻断型TIM-3纳米抗体的后续筛选和TIM-3全人源纳米抗体创新药物的开发奠定基础。
关键词:    T细胞免疫球蛋白黏蛋白-3      纳米抗体      噬菌体展示库     
Construction and screening of phage display library for TIM-3 nanobody
MA Lin-lin1, ZHU Min2, LI Guang-hui2, LI Yan-fei1, GAI Jun-wei2, WAN Ya-kun2
1. Key Laboratory of Molecular Imaging of Shanghai Education Commission, Shanghai University of Medicine and Health Sciences, Shanghai 201318, China;
2. Shanghai Novamab Biopharmaceuticals Co., Ltd., Shanghai 201318, China
Abstract:
Immunotherapy is a new strategy for cancer treatment that has the potential to treat all types of cancer. T cell immunoglobulin and mucin-domain-containing molecule-3 (TIM-3) is a key negative regulator of T cell activation. TIM-3 blockage using anti-TIM-3 monoclonal antibody therapy has a great appeal and special advantages. Nanobodies, derived from heavy chain fragment in camelid animals, are now proving clinical values in the development of antibody drugs. In this study, we have immunized camel with TIM-3 antigens and then constructed phage display library. Moreover, 29 nanobodies with different complementarity-determining regions sequences have been screened from the phage display library by phage display technology. In addition, we successfully constructed the cell line stably expressing TIM-3, and screened 10 TIM-3 nanobodies with high specificity and high affinity using flow cytometry. Our study will lay the foundation for the future screening and development of anti-TIM-3 whole humanized functional nanobody.
Key words:    T cell immunoglobulin and mucin-domain-containing molecule-3    nanobody    phage display library   
收稿日期: 2017-11-16
DOI: 10.16438/j.0513-4870.2017-1092
基金项目: 上海健康医学院种子基金项目(HMSF-17-21-007);上海市高校教师培训培养项目(A1-2600-17-311006);上海市教师专业发展工程项目(A1-2601-17-311001).
通讯作者: 万亚坤,Tel:86-21-20985259,E-mail:ykwan@novamab.com
Email: ykwan@novamab.com
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