Preparation of a Fully Human Anti-PD-L1 Disulfide-stabilized Diabody with High Affinity and Stability

doi:10.13523/j.cb.2302020

China Biotechnology

2023, Vol. 43

Issue (6): 20-30 DOI: 10.13523/j.cb.2302020

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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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)

Received: 10 February 2023 Published: 04 July 2023

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	GUO Rao-qing
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	GU Jiang-tao
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Cite this article:

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.

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

Table1 Enrichment effect of PD-L1 extracellular segment protein as antigen on Fab phage antibody

Fig.1 Selection and soluble expression of anti-PD-L1 antibody from naive Fab phage library (a) Identification of positive phage Fab antibodies against PD-L1 by phage-ELISA. F1-F8: Positive phage antibody clones; BSA was irrelevant control; VCSM13 was negative control (n=3/well) (b) Colony PCR analysis of positive phage antibodies (c) SDS-PAGE analysis of Fab antibody expression. NC: Negative control; F2-F4, F6-F8: Positive Fab antibody clones; Lane 1, 5, 9, 13, 17, 21: Reductive treatment of pre-induction supernatant; Lane 2, 6, 10, 14, 18, 22: Reductive treatment of post-induction supernatant; Lane 3, 7, 11, 15, 19, 23: Non-reductive treatment of post-induction supernatant; Lane 4, 8, 12, 16, 20, 24: Reductive treatment of post-induction precipitation (d) The antigen-binding activities of anti-PD-L1 Fab antibodies by ELISA (e) SDS-PAGE analysis of the purified Fab antibody in reducing condition. The Fab antibody was clone F6

Table 2 Sequence analysis of the variable region of the light chain

Table 3 Sequence analysis of variable region of heavy chain

Fig.2 Construction and screening of phage antibody mutation library (a) The CDR3 region base sites of light-chain and heavy-chain. Single underlined were mutation hotspots (b) Relative affinity assay of phage mutant antibodies against PD-L1. Mut was the mutant strain (c) Identification of positive mutant clones against PD-L1 by phage-ELISA. Mut was the mutant strain

Table 4 The input and output titers of phage antibody mutation library by four rounds of screening

Fig.3 Sequence analysis of positive phage antibody mutant strains (a) Sequence alignment of the VL domains of F6 antibody with its corresponding variants isolated from the mutant library (b) Sequence alignment of the VH domains of F6 antibody with its corresponding variants isolated from the mutant library A dash (-) denoted residues identical to those found in wild-type F6 antibody

Fig.4 The expression, purification and binding activity studies of ds-Diabody against PD-L1 in Pichia pastoris (a) Selection of positive clones by PCR amplification (b) The SDS-PAGE assay of the purified ds-Diabody in reducing condition (c) The Western blot assays of ds-Diabody expression. Number 1-17: Different yeast recombinant transformants; NC: Negative control (d) The antigen combined activity of ds-Diabody yeast expression supernatant against PD-L1 by ELISA. Number 1-17: Different yeast recombinant transformants; NC: Negative control (n=3/well) (e) The antigen-binding activity of the purified ds-Diabody and Fab antibody against PD-L1. PBS was negative control (n=3/well) (f) The thermal stability of the ds-Diabody and Fab antibody against PD-L1 under 37℃ incubation (n=3/well)


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