5Y56

Fc mutant (K392D/K409D/D399K)

Summary for 5Y56

• Entry DOI: 10.2210/pdb5y56/pdb
• Descriptor: Immunoglobulin gamma-1 heavy chain, beta-D-galactopyranose-(1-4)-2-acetamido-2-deoxy-alpha-D-glucopyranose-(1-2)-alpha-D-mannopyranose-(1-6)-[2-acetamido-2-deoxy-beta-D-glucopyranose-(1-2)-beta-D-mannopyranose-(1-3)]beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-[beta-L-fucopyranose-(1-6)]2-acetamido-2-deoxy-beta-D-glucopyranose, beta-D-galactopyranose-(1-4)-2-acetamido-2-deoxy-alpha-D-glucopyranose-(1-2)-alpha-D-mannopyranose-(1-6)-[beta-D-mannopyranose-(1-3)]beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose, ... (4 entities in total)
• Functional Keywords: igg fc, homodimer, triple mutantion (k392d/k409d/d399k), immune system
• Biological source: Homo sapiens (Human)
• Cellular location: Secreted : P0DOX5
• Total number of polymer chains: 2
• Total formula weight: 50082.76

Authors

Ye, S.,Xu, T.,Yu, J.,Wang, X.,Xu, T.,Jin, Q.,Duan, J.,Wu, J.,Wu, H. (deposition date: 2017-08-07, release date: 2017-09-13, Last modification date: 2023-11-22)

Primary citation

Yu, J.,Wang, X.,Xu, T.,Jin, Q.,Duan, J.,Wu, J.,Wu, H.,Xu, T.,Ye, S.
A rational approach to enhancing antibody Fc homodimer formation for robust production of antibody mixture in a single cell line
J. Biol. Chem., 292:17885-17896, 2017

PubMed Abstract: 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.

PubMed: 28878018
DOI: 10.1074/jbc.M116.771188

Experimental method

X-RAY DIFFRACTION (2.653 Å)