Quantitative methods for developing Fc mutants with extended half-lives

• Published in: Biotechnology and bioengineering 2005-12, Vol.92 (6), p.748-760
• Main Authors: Kamei, Daniel T., Lao, Bert J., Ricci, Margaret Speed, Deshpande, Rohini, Xu, Han, Tidor, Bruce, Lauffenburger, Douglas A.
• Format: Article
• Language: English
• Subjects: Animals; Binding sites; Biotechnology; cell-level model; Half-Life; Histocompatibility Antigens Class I - chemistry; Histocompatibility Antigens Class I - metabolism; Humans; Immunoglobulin Fc Fragments - chemistry; Immunoglobulin Fc Fragments - genetics; Immunoglobulin Fc Fragments - metabolism; Immunoglobulin G - genetics; Immunoglobulin G - metabolism; Immunoglobulin G - therapeutic use; Mice; Models, Biological; Models, Molecular; molecular modeling; Mutagenesis, Site-Directed; Mutation; Protein Binding - physiology; Protein Structure, Quaternary; Protein Structure, Tertiary; Proteins; Receptors, Fc - chemistry; Receptors, Fc - metabolism; Sequence Homology, Amino Acid; Surface Plasmon Resonance; therapeutic half-lives; Toxicity; trafficking assay
• ISSN: 0006-3592; 1097-0290
• DOI: 10.1002/bit.20624

Fc mutants with increased binding affinity for the neonatal receptor, FcRn, exhibit increased half‐lives in vivo, and represent an attractive means for extending the half‐lives of therapeutic antibodies. The half‐lives of other therapeutic molecules (e.g., proteins) may also be extended by conjugating them to Fc fragments, thus decreasing the frequency of patient injections and allowing the administration of low and potentially nontoxic concentrations of the therapeutics. To investigate the possibility for further increasing the half‐life of Fc, a pair of quantitative methods is presented to complement combinatorial screening and in vivo testing. Specifically, a simple molecular modeling procedure was developed to predict relative Gibbs free energies of binding values (ΔΔGbind) between Fc and FcRn across different mutants and species. This procedure was found to reasonably reproduce experimental ΔΔGbind values from our experiments and the literature, and may be used as an initial screen to explore Fc sequence space more fully prior to experimental testing. In addition, a mathematical model of Fc trafficking was formulated and combined with a cell‐level pulse‐chase assay to obtain a quantitative recycling parameter in human T84 cells. This Fc recycling parameter was found to be correlated with binding affinity, but captures the pH dependent nature of the interaction between Fc and FcRn and may serve as an additional screen following combinatorial experiments. © 2005 Wiley Periodicals, Inc.