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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2024, Vol. 44 Issue (2/3): 59-68    DOI: 10.13523/j.cb.2307036
技术与方法     
抗IL-5纳米抗体筛选及活性检测*
李诗洁1,代维燕2,王雪莲2,刘畅2,梁耀极2,白仲虎1,**(),陈永奇2,**()
1 江南大学粮食发酵工艺与技术国家工程实验室 无锡 214122
2 珠海瑞思普利医药科技有限公司 珠海 519040
Anti-IL-5 Nanobody Screening and Activity Detection
LI Shijie1,DAI Weiyan2,WANG Xuelian2,LIU Chang2,LIANG Yaoji2,BAI Zhonghu1,**(),CHEN Yongqi2,**()
1 National Engineering Laboratory of Cereal Fermentation Technology, Jiangnan University, Wuxi 214122, China
2 Zhuhai Resproly Pharmaceutical Technology Co., Ltd, Zhuhai 519040, China
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摘要：

白介素-5(IL-5)是一种同源二聚体细胞因子,是嗜酸性粒细胞(eosinophilic,EOS)增殖、活化和成熟的重要调节因子。抗IL-5单克隆抗体能阻断IL-5与IL-5受体亚单位α(IL-5Rα)结合,已成功用于治疗嗜酸性粒细胞哮喘。目前上市的单克隆抗体药物需要反复注射给药,严重影响了患者的依从性,而且注射给药的全身暴露率高。为获得适合吸入给药的抗体,在羊驼天然库中通过3轮淘选,挑取单克隆通过Phage ELISA初筛,共获得461个阳性克隆,其中50个为序列独特的分子,最终选择30个分子进行重组表达纯化。通过ELISA结合、ELISA阻断、FACS阻断、TF-1增殖抑制等实验对候选抗体进行体外活性检测,成功获得一个具有阻断IL-5和IL-5Rα结合的纳米抗体AIL-A96-Fc。通过与人和猴IL-5的ELISA结合实验表明,该分子具有良好人猴交叉活性,而且在FACS阻断实验和ELISA阻断实验中,AIL-A96-Fc表现出良好的阻断效果。该开发方法不仅提供了一个靶向IL-5的候选纳米抗体AIL-A96-Fc,也为后续开发更多靶向IL-5的候选纳米抗体提供了指导意义。
关键词： 白介素-5;  纳米抗体;  阻断活性;  噬菌体展示;  哮喘    
Abstract:

Interleukin-5 (IL-5), a homodimeric cytokine, is an important regulator of eosinophil (EOS) proliferation, activation and maturation. Anti-IL-5 monoclonal antibodies block the binding of IL-5 to the IL-5 receptor subunit alpha (IL-5Rα) and have been used successfully in the treatment of eosinophilic asthma. Currently available monoclonal antibody drugs require repeated administration by injection, which has a significant impact on patient compliance, and the systemic exposure rate of injection is high. To obtain nanobodies suitable for inhalation administration, monoclonal clones were selected through three rounds of panning in the natural alpaca library by phage ELISA screening. A total of 461 positive clones were obtained, of which 50 clone sequences were unique, and 30 molecules were selected for recombinant expression and purification of nanobodies. The in vitro activity of the candidate antibodies was tested by ELISA binding, ELISA blocking, FACS blocking and TF-1 proliferation inhibition assays, and a nanobody AIL-A96-Fc with the ability to block the binding of IL-5 and IL-5Rα was successfully obtained. ELISA binding assays with human and cynomolgus IL-5 showed that the molecule has good human-cynomolgus cross-species activity, and AIL-A96-Fc showed good blocking effects in FACS and ELISA blocking assays. This study not only provides a candidate nanobody (AIL-A96-Fc), but the development methodology also provides guidance for the subsequent development of additional candidate nanobodies targeting IL-5.
Key words: Interleukin-5    Nanobodies    Blocking activity    Phage display    Asthma
收稿日期: 2023-07-27 出版日期: 2024-04-03
ZTFLH:  Q511  
基金资助: *国家轻工技术与工程一流学科自主课题(LITE2018-24);珠海市产学研合作及基础与应用基础研究项目(ZH2201700220000ZPWC);2020年度珠海创新创业团队项目(2120004000325)
通讯作者: **电子信箱:baizhonghu@jiangnan.edu.cn; yqchen@resproly.com   
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李诗洁
代维燕
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白仲虎
陈永奇

引用本文:

李诗洁, 代维燕, 王雪莲, 刘畅, 梁耀极, 白仲虎, 陈永奇. 抗IL-5纳米抗体筛选及活性检测*[J]. 中国生物工程杂志, 2024, 44(2/3): 59-68.

LI Shijie, DAI Weiyan, WANG Xuelian, LIU Chang, LIANG Yaoji, BAI Zhonghu, CHEN Yongqi. Anti-IL-5 Nanobody Screening and Activity Detection. China Biotechnology, 2024, 44(2/3): 59-68.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2307036    或    https://manu60.magtech.com.cn/biotech/CN/Y2024/V44/I2/3/59

编号 筛选抗原 抗原浓度 负筛 噬菌体加入 噬菌体加入的
滴度/cfu		 筛选后洗脱的
滴度/cfu		 空白
/cfu		 筛选后洗脱
的滴度
/噬菌体加入
的滴度		 筛选后洗
脱的滴度
/空白
1-1 IL-5-His 100 μg/mL N/A 羊驼天然库 2.20×1013 1.40×108 N/A 0.01 N/A
1-2 IL-5-His-bio 300 nmol/L N/A 羊驼天然库 3.30×1013 3.60×108 N/A 0.01 N/A
2-1 IL-5-His 30 μg/mL N/A 1-1 1.80×1012 1.00×107 1.12×107 0.01 0.89
2-2 IL-5-His-bio 100 nmol/L N/A 1-1 9.00×1011 4.20×106 4.80×107 0 0.09
2-3 IL-5-Fc 30 μg/mL BCMA-Fc 1-1 1.20×1012 3.20×107 1.12×107 0.03 2.86
2-4 IL-5-Fc-bio 100 nmol/L BCMA-Fc 1-1 5.00×1011 4.80×106 4.80×107 0.01 0.1
2-5 IL-5-His 30 μg/mL N/A 1-2 1.40×1012 6.40×106 1.12×107 0 0.57
2-6 IL-5-His-bio 100 nmol/L N/A 1-2 6.50×1011 1.80×107 4.80×107 0.03 0.38
2-7 IL-5-Fc-bio 100 nmol/L BCMA-Fc 1-2 6.00×1011 2.10×107 4.80×107 0.04 0.44
2-8 IL-5-Fc 30 μg/mL BCMA-Fc 1-2 1.00×1012 1.12×107 1.12×107 0.01 1
3-1 IL-5-His 10 μg/mL N/A 2-1 4.60×1011 2.40×106 6.00×106 0.01 0.4
3-2 IL-5-His 10 μg/mL N/A 2-2 2.40×1011 3.20×106 6.00×106 0.01 0.53
3-3 IL-5-His 10 μg/mL N/A 2-3 3.60×1011 5.20×106 6.00×106 0.01 0.87
3-4 IL-5-His 10 μg/mL N/A 2-4 5.00×1011 8.00×106 6.00×106 0.02 1.33
3-5 IL-5-His-bio 30 nmol/L N/A 2-5 2.40×1011 4.40×107 9.20×108 0.18 0.05
3-6 IL-5-His-bio 30 nmol/L N/A 2-6 1.70×1011 8.00×108 9.20×108 4.71 0.87
3-7 IL-5-His-bio 30 nmol/L N/A 2-7 1.15×1011 1.72×108 9.20×108 1.5 0.19
3-8 IL-5-His-bio 30 nmol/L N/A 2-8 1.35×1011 5.60×107 9.20×108 0.41 0.06
表1  羊驼天然库海选数据
图1  重组纳米抗体与人IL-5结合检测 摩尔浓度(nmol/L)参考摩尔浓度计算器进行计算(https://www.selleck.cn/molaritycalculator.jsp)
样品名称 EC50 样品名称 EC50 样品名称 EC50 样品名称 EC50
Mepolizumab 0.482 5 Mepolizumab 0.431 6 Mepolizumab 0.511 Mepolizumab 0.449 8
Reslizumab 0.396 4 Reslizumab 0.384 5 Reslizumab 0.264 4 Reslizumab 0.305 7
AIL-A3-Fc 16.9 AIL-A30-Fc N/A AIL-A75-Fc N/A AIL-A188-Fc 14.5
AIL-A103-Fc N/A AIL-A303-Fc 约1 778 AIL-A76-Fc 0.808 8 AIL-A19-Fc N/A
AIL-A107-Fc N/A AIL-A318-Fc N/A AIL-A86-Fc N/A AIL-A191-Fc N/A
AIL-A1-Fc 10.76 AIL-A38-Fc N/A AIL-A96-Fc 0.940 9 AIL-A2-Fc 13.21
AIL-A12-Fc 0.101 8 AIL-A39-Fc N/A AIL-B108-Fc N/A AIL-A23-Fc 0.959 8
AIL-A14-Fc 0.248 1 AIL-A4-Fc N/A AIL-B124-Fc 4.92 AIL-A233-Fc 117.8
AIL-A163-Fc N/A AIL-A5-Fc N/A AIL-B127-Fc 0.243 6
AIL-A178-Fc 23.84 AIL-A6-Fc N/A AIL-B90-Fc 0.623 5
表2  重组纳米抗体与人IL-5结合检测的EC50
图2  重组纳米抗体与猴IL-5结合检测
样品名称 EC50 样品名称 EC50 样品名称 EC50 样品名称 EC50
Mepolizumab 0.565 3 Mepolizumab 0.484 9 Mepolizumab 0.460 6 Mepolizumab 0.613 3
Reslizumab 0.378 6 Reslizumab 0.195 8 Reslizumab 0.304 6 Reslizumab 0.273 5
AIL-A3-Fc 25.2 AIL-A30-Fc N/A AIL-A75-Fc N/A AIL-A188-Fc 18.26
AIL-A103-Fc N/A AIL-A303-Fc 约431.6 AIL-A76-Fc 0.575 1 AIL-A19-Fc 144.3
AIL-A107-Fc N/A AIL-A318-Fc N/A AIL-A86-Fc N/A AIL-A191-Fc N/A
AIL-A1-Fc 16.16 AIL-A38-Fc N/A AIL-A96-Fc 0.654 4 AIL-A2-Fc 24.6
AIL-A12-Fc 0.089 39 AIL-A39-Fc N/A AIL-B108-Fc N/A AIL-A23-Fc 0.515 4
AIL-A14-Fc 0.391 7 AIL-A4-Fc N/A AIL-B124-Fc 2.7 AIL-A233-Fc 约152 7
AIL-A163-Fc N/A AIL-A5-Fc N/A AIL-B127-Fc 0.090 9
AIL-A178-Fc 15.28 AIL-A6-Fc N/A AIL-B90-Fc 0.868 4
表3  重组纳米抗体与猴IL-5结合检测的EC50
图3  重组纳米抗体ELISA阻断检测
图4  AIL-A96-Fc抗体FACS阻断检测
图5  AIL-A96-Fc抗体细胞增殖抑制检测
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