A rational approach to enhancing antibody Fc homodimer formation for robust production of antibody mixture in a single cell line

• Published in: The Journal of biological chemistry Vol. 292; no. 43; pp. 17885 - 17896
• Main Authors: Yu, Jie, Wang, Xiaoxiao, Xu, Tao, Jin, Qiuheng, Duan, Jinyuan, Wu, Jie, Wu, Haiyan, Xu, Ting, Ye, Sheng
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 27-10-2017; American Society for Biochemistry and Molecular Biology
• Subjects: antibody engineering; crystal structure; epidermal growth factor receptor (EGFR); HEK293 Cells; Humans; Immunoglobulin Fc Fragments - chemistry; Immunoglobulin Fc Fragments - genetics; Immunoglobulin Fc Fragments - metabolism; monoclonal antibody; Protein Domains; protein engineering; Protein Multimerization; Protein Structure and Folding; structure based protein engineering; antibody engineering; crystal structure; protein engineering; monoclonal antibody; epidermal growth factor receptor (EGFR); structure based protein engineering
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M116.771188

Combinations of different antibodies have been shown to be more effective for managing certain diseases than monotherapy. Co-expression of the antibody mixture in a single cell line is key to reducing complexity during antibody development and manufacturing. However, co-transfection of multiple light and heavy chains into cells often leads to production of mismatched, heterodimeric by-products that are inactive, making the development of co-expression systems that robustly and efficiently produce highly active antibody mixtures a high priority. In this study, we modified the CH3 domain interface of the antibody fragment crystallizable (Fc) region by changing several charge pairs to create electrostatic interactions favoring Fc homodimer formation and disfavoring Fc heterodimer formation. When co-expressed, these modified antibodies with altered charge polarity across the Fc dimer interface preferentially formed homodimers that fully preserved the functions of each component, rather than inactive heterodimers whose formation was reduced because of rationally designed repulsive interactions. We designed eight different combinations and experimentally screened the best one, which enabled us to produce a binary antibody mixture against the EGF receptor with a minimal heterodimer contaminant. We further determined the crystal structure of a triple-mutated Fc variant in the best combination, and we elucidated the molecular interactions favoring Fc homodimer over heterodimer formation, which provided a structural basis for further optimization. The approach presented here demonstrates the feasibility of rational antibody modification for efficient and consistent production of monoclonal antibody mixtures in a single cell line and thus broadens our options for manufacturing more effective antibody-based therapeutic agents.