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中国生物工程杂志

China Biotechnology
China Biotechnology
China Biotechnology  2024, Vol. 44 Issue (2/3): 59-68    DOI: 10.13523/j.cb.2307036
    
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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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     
Received: 27 July 2023      Published: 03 April 2024
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Shijie LI
Weiyan DAI
Xuelian WANG
Chang LIU
Yaoji LIANG
Zhonghu BAI
Yongqi CHEN
Cite this article:

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.

URL:

https://manu60.magtech.com.cn/biotech/10.13523/j.cb.2307036     OR     https://manu60.magtech.com.cn/biotech/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
Table 1 Screening data from an alpaca natural phage display library
Fig.1 Human IL-5 binding assay for recombinant nanobodies Molar concentration (nmol/L) was calculated by the website(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
Table 2 EC50 of the binding assay of the recombinant nanobodies to human IL-5
Fig.2 Cynomolgus IL-5 binding assay for recombinant nanobodies
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
Table 3 EC50 of the binding assay of the recombinant nanobodies to cynomolgus IL-5
Fig.3 ELISA blocking assay of recombinant nanobodies
Fig.4 FACS blocking assay of AIL-A96-Fc
Fig.5 Cell proliferation inhibition assay of AIL-A96-Fc
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