Charge-mediated influence of the antibody variable domain on FcRn-dependent pharmacokinetics

• Published in: Proceedings of the National Academy of Sciences - PNAS 2015-05, Vol.112 (19), p.5997-6002
• Main Authors: Schoch, Angela, Kettenberger, Hubert, Mundigl, Olaf, Winter, Gerhard, Engert, Julia, Heinrich, Julia, Emrich, Thomas
• Format: Article
• Language: English
• Subjects: Animals; Antibodies - chemistry; Antibodies, Monoclonal - chemistry; Antibodies, Monoclonal, Humanized - chemistry; Antigen-Antibody Reactions; beta 2-Microglobulin - chemistry; Biological Sciences; Chromatography; Chromatography, Affinity; Female; Histocompatibility Antigens Class I - chemistry; Homeostasis; Human Umbilical Vein Endothelial Cells; Humans; Hydrogen-Ion Concentration; Immunoglobulin Fc Fragments - chemistry; Immunoglobulin G - chemistry; Kinetics; Mice; Mice, Transgenic; Microscopy, Confocal; Molecular Dynamics Simulation; Pharmacology; Protein Binding; Protein Conformation; Protein Engineering; Protein Multimerization; Receptors, Fc - chemistry; Simulation; Static Electricity; Surface Plasmon Resonance; Ustekinumab; Variables
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.1408766112

Here, we investigated the influence of the variable fragment (Fv) of IgG antibodies on the binding to the neonatal Fc receptor (FcRn) as well as on FcRn-dependent pharmacokinetics (PK). FcRn plays a key role in IgG homeostasis, and specific manipulation in the crystallizable fragment (Fc) is known to affect FcRn-dependent PK. Although the influence of the antigen-binding fragment (Fab) on FcRn interactions has been reported, the underlying mechanism is hitherto only poorly understood. Therefore, we analyzed the two IgG1 antibodies, briakinumab and ustekinumab, that have similar Fc parts but different terminal half-lives in human and systematically engineered variants of them with cross-over exchanges and varied charge distribution. Using FcRn affinity chromatography, molecular dynamics simulation, and in vivo PK studies in human FcRn transgenic mice, we provide evidence that the charge distribution on the Fv domain is involved in excessive FcRn binding. This excessive binding prevents efficient FcRn–IgG dissociation at physiological pH, thereby reducing FcRn-dependent terminal half-lives. Furthermore, we observed a linear correlation between FcRn column retention times of the antibody variants and the terminal half-lives in vivo. Taken together, our study contributes to a better understanding of the FcRn–IgG interaction, and it could also provide profound potential in FcRn-dependent antibody engineering of the variable Fab region. Significance Therapeutic antibodies of the immunoglobulin G (IgG) isotype show a pharmacokinetic (PK) profile that is strongly mediated by the interaction with the neonatal Fc receptor (FcRn). Therefore, modulating the FcRn–IgG interaction allows altering PK characteristics of therapeutic antibodies. So far, engineering the crystallizable fragment (Fc) is known to affect PK, and, although the influence of the antigen binding fragment (Fab) on FcRn interactions has been reported, the underlying mechanism remains unknown. Here, we demonstrate that the charge distribution in the distal variable fragment (Fv) of IgGs is involved in excessive binding to the FcRn, thereby reducing FcRn-dependent terminal half-lives in vivo. These findings contribute to a better understanding of the FcRn–IgG interaction.