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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  2023, Vol. 43 Issue (6): 20-30    DOI: 10.13523/j.cb.2302020
技术与方法     
具有高亲和力和稳定性的人源性抗PD-L1二硫键稳定Diabody的制备*
郭娆晴,黄嘉雯,张利刚,赵文丽,辜江涛,邓宁**()
暨南大学生命科学技术学院生物系 广东省抗体药物与免疫检测工程技术研究中心 广州 510632
Preparation of a Fully Human Anti-PD-L1 Disulfide-stabilized Diabody with High Affinity and Stability
GUO Rao-qing,HUANG Jia-wen,ZHANG Li-gang,ZHAO Wen-li,GU Jiang-tao, DENG-Ning**()
Guangdong Province Engineering Research Center for Antibody Drug and Immunoassay, Department of Biology, College of Life Science and Technology, Jinan University, Guangzhou 510632, China
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摘要：

目的:对天然噬菌体抗体库进行筛选并对抗体进行体外亲和力成熟,获得高亲和力人源性抗PD-L1抗体,然后对该抗体进行二硫键稳定改造,获得具有高亲和力和稳定性的人源性抗PD-L1的二硫键稳定Diabody。方法:首先以PD-L1重组蛋白为抗原在天然噬菌体Fab抗体库中筛选Fab抗体,其次分析结合能力较好的抗PD-L1的Fab抗体可变区基因中的热点,通过对轻链、重链CDR3区的7处热点随机突变构建噬菌体抗体突变库,从中筛选出亲和力得到提高的抗体。最后在抗体骨架区引入两个二硫键,构建二硫键稳定的抗PD-L1的ds-Diabody,并在毕赤酵母GS115中进行表达。结果:该方法筛选获得了6株特异性抗PD-L1噬菌体Fab抗体,对结合能力较好的其中一株抗体CDR3区的热点进行随机突变,成功构建库容为1.14×108 CFU/mL的噬菌体抗体突变库,并从中筛选出亲和力提高约6倍的噬菌体抗体突变株。对该抗体骨架区进行二硫键引入,成功构建与表达二硫键稳定的ds-Diabody。结论:构建的ds-Diabody比Fab抗体与PD-L1结合亲和力高、稳定性好,为药物开发、肿瘤治疗等研究PD-1/PD-L1途径提供有力依据。
关键词： PD-L1;  噬菌体抗体库;  抗体亲和力成熟;  二硫键稳定双链抗体    
Abstract:

Objective: To isolate a fully human anti-PD-L1 antibody from a naïve phage-display human Fab library and increase its affinity through in vitro antibody affinity maturation, and then modify the antibody with disulfide bond stability to obtain a humanized anti-PD-L1 disulfide-stabilized diabody with high affinity and stability. Methods: First, the PD-L1 recombinant protein was used as the antigen in the naïve phage Fab antibody library to screen Fab antibodies. Then the hot spots in the variable region gene of Fab antibody with good binding ability against PD-L1 were analyzed, and the phage antibody mutation library was constructed by randomly mutating 7 hot spots in the light chain and heavy chain CDR3 region, from which antibodies with improved affinity were screened. Finally, two disulfide bonds were introduced in the antibody backbone region to construct disulfide-stabilized anti-PD-L1 ds-Diabody, which was expressed in GS115. Results: Six specific anti-PD-L1 phage Fab antibodies were screened and obtained, the CDR3 region hot spot of one of the antibodies with better binding ability was randomly mutated to successfully construct a phage antibody mutant library with a library capacity of 1.14×108 CFU/mL, and phage antibody mutant strains with increased affinity of about 6 times were screened. The disulfide bond was introduced to the backbone region of the antibody, and the ds-Diabody with stable disulfide bond was successfully constructed and expressed. Conclusion: The ds-Diabody has higher binding affinity and better stability than Fab antibody to PD-L1, which provides a strong basis for drug development, tumor treatment and other research on PD-1/PD-L1 pathway.

Key words: PD-L1    Phage antibody library    Antibody affinity maturation    Disulfide-stabilized diabody (ds-Diabody)
收稿日期: 2023-02-10 出版日期: 2023-07-04
ZTFLH:  Q819  
基金资助: * 国家自然科学基金(81972705)
通讯作者: **电子信箱:tdengn@jnu.edu.cn   
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郭娆晴
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辜江涛
邓宁

引用本文:

郭娆晴, 黄嘉雯, 张利刚, 赵文丽, 辜江涛, 邓宁. 具有高亲和力和稳定性的人源性抗PD-L1二硫键稳定Diabody的制备*[J]. 中国生物工程杂志, 2023, 43(6): 20-30.

GUO Rao-qing, HUANG Jia-wen, ZHANG Li-gang, ZHAO Wen-li, GU Jiang-tao, DENG-Ning. Preparation of a Fully Human Anti-PD-L1 Disulfide-stabilized Diabody with High Affinity and Stability. China Biotechnology, 2023, 43(6): 20-30.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2302020    或    https://manu60.magtech.com.cn/biotech/CN/Y2023/V43/I6/20

轮数 PD-L1/(μg/μL) 输入滴度/(CFU/mL) 输出滴度/(CFU/mL) 收获率 倍数
1 50 1.6×1013 1.14×106 7.13×10-8 1
2 25 7.36×1012 1.99×106 2.7×10-7 4
3 12.5 1.84×1012 8.54×106 4.64×10-6 65
4 6.25 4.24×1013 6.84×107 1.61×10-5 226
表1  以PD-L1胞外段蛋白为抗原对Fab噬菌体抗体库的富集效应
图1  天然噬菌体Fab抗体库中抗PD-L1抗体的筛选与可溶性表达
Fab 克隆 VL 种系基因 同源性/% CDR1 CDR2 CDR3
F2 Homsap IGKV3-11*01 95.51 QSVSNY DAS QQRHSWPLT
F3 Homsap IGLV1-40*02 95.14 SSNIGAGSD ANT QSYDSSLSGWV
F4 Homsap IGKV1-39*01 95.96 QRINSY TTS QQSFSNPYT
F6 Homsap IGLV3-19*01 99.62 SLRSYY GKN NSRDSSGNWV
F7 Homsap IGKV4-1*01 89.73 QNILYSSKNKNY WAS CSYAGSSSFV
F8 Homsap IGKV1-33*01 96.69 QDISNY DAS QQSGA
表2  轻链基因可变区序列分析
Fab 克隆 VH 种系基因 同源性/% CDR1 CDR2 CDR3
F2 Homsap IGHV1-8*01 F 97.57 GYTFTSYD MNPNSGNT ARGTPYYDFWSGYYSPHYYYYYYMDV
F3 Homsap IGHV4-39*07 F 95.17 GGSISSSNYY MSYSGSS ASTRSYYANWFDP
F4 Homsap IGHV3-7*05 F 94.10 GFPFSSYW IKPDGSDT ARGTSWNIFDS
F6 Homsap IGHV3-7*01 F 85.07 GGSISSYY IKQDESTK ARVWGTSGWAGFDY
F7 Homsap IGHV3-9*01 F 98.94 GFTFDDYA ISWNSGSI AKDIRYSSSWGPFDY
F8 Homsap IGHV3-23*03 F 90.97 GFSGSTFE IDSGGSSR VKGGWLDY
表3  重链基因可变区序列分析
图2  噬菌体抗体突变文库的构建与筛选
轮数 PD-L1/(μg/μL) 输入滴度/(CFU/mL) 输出滴度/(CFU/mL) 收获率 倍数
1 40 1.45×1013 1.82×106 1.26×10-7 1
2 20 3.83×1012 4.27×106 1.11×10-6 9
3 10 2.03×1012 8.82×106 4.34×10-6 34
4 5 1.36×1012 3.04×107 2.24×10-5 178
表4  噬菌体抗体突变文库4轮筛选的输入和输出滴度
图3  噬菌体抗体突变株阳性的序列分析
图4  抗PD-L1的ds-Diabody在毕赤酵母的表达、纯化和结合活性研究
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