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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): 14-24    DOI: 10.13523/j.cb.2305033
研究报告     
具有人鼠交叉结合活性的抗IL-1R3抗体的筛选与鉴定
秦慧1,2,郁心蕊2,3,刘娇1,2,古丽赛娜·恰尔谢2,4,崔靖敏2,4,王茜2,杜鹏2,*(),周春阳1,*()
1 川北医学院药学院 川北医学院药物研究所 南充 637000
2 军事科学院军事医学研究院生物工程研究所 北京 100071
3 海南医学院基础医学与生命科学学院 海口 571158
4 沈阳药科大学医疗器械学院 本溪 117004
Screening and Identification of Antibodies with Cross-binding Activity to Human and Mouse IL-1R3
QIN Hui1,2,YU Xinrui2,3,LIU Jiao1,2,Gulisaina Qiaerxie2,4,CUI Jingmin2,4,WANG Xi2,DU Peng2,*(),ZHOU Chunyang1,*()
1 School of Pharmacy, North Sichuan Medical College, Institute of Pharmaceutical Research,North Sichuan Medical College, Nanchong 637000, China
2 Institute of Biotechnology, Academy of Military Medical Science, Academy of Military Science, Beijing 100071, China
3 School of Basic Medical Sciences and Life Sciences, Hainan Medical University, Haikou 571158, China
4 School of Medical Instrumentation, Shenyang Pharmaceutical University, Benxi 117004, China
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摘要：

目的: IL-1受体辅助蛋白(IL-1R3)是炎症调控的潜在新靶点。筛选获得具有人鼠交叉结合活性的抗IL-1R3抗体,为炎症干预机制探究与新药开发奠定基础。方法: 基于对人源和鼠源IL-1R3氨基酸序列与结构的比对,采用交替包被人源和鼠源IL-1R3的策略对噬菌体抗体库进行筛选;将获得的抗体可变区基因克隆到真核表达载体,制备抗体样品;在分子和细胞水平对候选抗体的结合活性与功能活性进行评价;采用重新配对抗体轻链与重链的策略获得新抗体,尝试改善其特性。结果: 筛选获得5个具有人鼠交叉结合活性的抗IL-1R3抗体,其中4个对人源和鼠源IL-1R3的亲和力相当,均在10-7 mol/L水平;2个溶解度较好的抗体在细胞水平上具有阻断活性;重新配对候选抗体的轻链与重链,获得1个溶解度明显提升且结合活性有所改善的新抗体。结论: 优选获得2个具有人鼠交叉结合活性的抗IL-1R3抗体AET1907和4H6L,为后续深入探索奠定了物质基础。
关键词： IL-1R3;  抗体;  噬菌体展示;  抗体库;  交叉结合活性;  溶解度    
Abstract:

Objective: Interleukin-1 receptor accessory protein (IL-1R3) is a potential new target for inflammation regulation. The aim of this study is to obtain anti-IL-1R3 antibodies with cross-binding activity to human and mouse IL-1R3 and to lay the foundation for the new drug development and pharmacodynamic mechanism research of a novel inflammation intervention strategy. Methods: Based on the sequence and structure alignment of human and mouse IL-1R3, a phage-displayed human single-chain (scFv) antibody library was challenged by alternately coating immune-tubes with human or mouse IL-1R3. The variable region genes of the resulting antibodies were cloned into eukaryotic expression vectors to prepare antibodies. The binding activities of the candidate antibodies were determined by ELISA and SPR and their functional activities were evaluated by cell assay. A strategy of re-pairing the light and heavy chains of these antibodies was then followed to obtain new antibodies with improved properties. Results: Five antibodies with cross-binding activity to human and mouse IL-1R3 were identified, four of which showed comparable affinity to both human and mouse IL-1R3, approximately 10-7 mol/L. Two candidate antibodies with favorable solubility effectively blocked IL-1R3-mediated activities in the ex vivo functional assay. A new antibody with re-paired light and heavy chains showed improved binding activity and apparently increased solubility. Conclusions: By introducing antibodies AET1907 and 4H6L with comparable human-mouse IL-1R3 cross-binding activity, this study has laid the groundwork for further investigation.
Key words: IL-1R3    Antibody    Phage-displayed    Antibody library    Cross-binding activity    Solubility
收稿日期: 2023-05-25 出版日期: 2024-04-03
ZTFLH:  Q342+.4  
通讯作者: *电子信箱:zhouchunyang@nsmc.edu.cn; dudedu@sina.com   
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秦慧
郁心蕊
刘娇
古丽赛娜·恰尔谢
崔靖敏
王茜
杜鹏
周春阳

引用本文:

秦慧, 郁心蕊, 刘娇, 古丽赛娜·恰尔谢, 崔靖敏, 王茜, 杜鹏, 周春阳. 具有人鼠交叉结合活性的抗IL-1R3抗体的筛选与鉴定[J]. 中国生物工程杂志, 2024, 44(2/3): 14-24.

QIN Hui, YU Xinrui, LIU Jiao, Gulisaina Qiaerxie, CUI Jingmin, WANG Xi, DU Peng, ZHOU Chunyang. Screening and Identification of Antibodies with Cross-binding Activity to Human and Mouse IL-1R3. China Biotechnology, 2024, 44(2/3): 14-24.

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https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2305033    或    https://manu60.magtech.com.cn/biotech/CN/Y2024/V44/I2/3/14

图1  人源IL-1R3(huIL-1R3)和鼠源IL-1R3(moIL-1R3)的序列与结构比对 A:人源和鼠源IL-1R3序列比对结果,氨基酸相似性通过背景颜色深浅标示 B:人源和鼠源IL-1R3胞外域D2结构域(图A中120~215位氨基酸)结构比对结果,该结构域涵盖了几乎全部配体结合区域
筛选轮次 1 2-1 2-2 3
包被 人源IL-1R3,20 mg 人源IL-1R3,3.0 mg 鼠源IL-1R3,3.0 mg 人源IL-1R3,1.0 mg
投入量/pfu 2 × 109 6 × 1010 1.2 × 1011 8 × 1011
产出量/pfu 2.0 × 104 3.6 × 105 3.0 × 104 5 400
产出阳性率/% 58 15.63 34
特异性阳性克隆数量 12 18 7
表1  抗IL-1R3单链抗体筛选过程
图2  筛选产出单克隆鉴定与富集抗体特异性鉴定 A:重组人源IL-1R3-His与阴性对照抗原BSA和KDR-His并行包被进行ELISA单克隆鉴定 B:ELISA检测特异性。人源和鼠源IL-1R3-His的包被量均为50 ng/孔,其他抗原包被量为100 ng/孔;抗体浓度为10 μg/mL
图3  抗体与人源IL-1R3(A)和鼠源IL-1R3(B)的结合活性 人源或鼠源IL-1R3按100 ng/孔包被于96孔板,ELISA检测梯度稀释的抗体
图4  SPR检测抗体亲和力的代表性传感图 抗体固定于检测芯片表面,梯度浓度的人源或鼠源IL-1R3(37.5~600 nmol/L)分别流经芯片通道进行检测。选择1∶1结合模式进行结果分析,获得拟合曲线(图中黑色细线),计算平衡解离常数(KD)。* 该数值通过稳态测定(1∶1结合)拟合计算获得
抗体 抗原 ka /[104mol/(L·s)] kd /(10-3s-1) KD /(10-7mol/L) χ2 (RU2)
AET1903 huIL-1R3 1.76 8.44 4.79 6.72
moIL-1R3 1.63 4.76 2.93 3.25
AET1904 huIL-1R3 8.68 8.81 1.02 5.72
moIL-1R3 4.11 10.12 2.47 2.89
AET1906 huIL-1R3 4.01 8.89 2.22 6.83
moIL-1R3 3.15 10.9 3.48 0.82
AET1907 huIL-1R3 26.7 42.6 1.60 19.50
moIL-1R3 - - 2.87* 0.84
AET1912 huIL-1R3 4.61 11.60 2.52 9.91
moIL-1R3 - - 16.6* 1.77
表2  抗体与人源IL-1R3和鼠源IL-1R3相互作用的动力学常数
图5  抗体可有效阻断IL-1R3介导的功能活性 A、B:AET1907(A)和AET1912(B)阻断人IL-1β促进A549细胞分泌IL-6结果 C、D:AET1907(C)和AET1912(D)阻断人IL-36α促进HaCaT细胞分泌IL-8结果。数据处理使用GraphPad Prism软件,以平均值±标准差呈现在图中。*** P<0.001, ** P<0.01, * P<0.05(t检验)
图6  抗体轻链与重链可变区序列比对(A)及4H6L亲和力测定结果(B) A:氨基酸相似性通过背景颜色深浅标示 B:重新配对抗体4H6L浓度依次为600 nmol/L、300 nmol/L、150 nmol/L、75 nmol/L和37.5 nmol/L。KD由1∶1结合模式的拟合曲线计算得到
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