Construction of IgG4 Fc variants and their serum half-lives.

Xun GUO; Huijun XIE; Yuan ZHANG

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Construction of IgG4 Fc variants and their serum half-lives.

Author: Xun GUO ¹ ; Huijun XIE ¹ ; Yuan ZHANG ¹
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1. School of Biomedical Sciences and Engineering, South China University of Technology, Guangzhou 510006, Guangdong, China.
Publication Type:Journal Article
Keywords: Fc variant; IgG4; antibody; fusion protein; half-life; point mutation
MeSH: Immunoglobulin G/blood*; Immunoglobulin Fc Fragments/biosynthesis*; Half-Life; Animals; Escherichia coli/metabolism*; Humans; Recombinant Fusion Proteins/biosynthesis*; Recombinant Proteins/biosynthesis*; Mice
From: Chinese Journal of Biotechnology 2025;41(8):3143-3154
CountryChina
Language:Chinese
Abstract: In this study, we constructed a series of recombinant Fc variants of immunoglobulin G4 (IgG4), screened the fragment crystallizable (Fc) variants with significantly prolonged serum half-lives, and analyzed the relationship between mutation site and half-life, aiming to provide a theoretical basis for the development of IgG4 antibodies and Fc fusion protein-based drugs. Nine gene sites were selected for mutation, and different mutation sites were combined. The variant expression plasmids pET24b-Fc were constructed by molecular cloning and point mutation. The plasmids were transformed into Escherichia coli BL21(DE3) for the expression of different recombinant proteins of Fc. Fc2 and Fc3 variants had slightly lower recombinant protein yields, and the expression of other variants was not affected. The toxicity of different Fc variants was determined by cell counting kit-8 (CCK-8) and calcein acetoxymethyl ester/ propidium iodide (calcein AM/PI) in vitro and enzyme-linked immuno sorbent assay (ELISA) in vivo. The results showed that the recombinant Fc variants had good biocompatibility and safety. Finally, the Fc variants were labeled with fluorescent markers, and the effects of different mutations on their serum half-lives were investigated by in vivo experiments. The Fc5 variant with prolonged serum half-life was successfully screened out, which provided a theoretical and practical basis for the optimal design of IgG4 subtype antibody and Fc fusion protein drugs.