SignificanceTherapeutic 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.

The success of recombinant monoclonal immunoglobulins (IgG) is rooted in their ability to target distinct antigens with high affinity combined with an extraordinarily long serum half-life, typically around 3 weeks. The pharmacokinetics of IgGs is intimately linked to the recycling mechanism of the neonatal Fc receptor (FcRn). For long serum half-life of therapeutic IgGs, the highly pH-dependent interaction with FcRn needs to be balanced to allow efficient FcRn binding and release at slightly acidic pH and physiological pH, respectively. Some IgGs, like the antibody briakinumab has an unusually short half-life of ϳ8 days. Here we dissect the molecular origins of excessive FcRn binding in therapeutic IgGs using a combination of hydrogen/deuterium exchange mass spectrometry and FcRn affinity chromatography. We provide experimental evidence for a two- (7-12) and the molecular mechanism is not clear. Limited structural information is available for the complex of full-length IgG and FcRn and much of our knowledge of the binding interface comes from X-ray crystal structures of only the Fc region bound to FcRn (3,4).We recently applied a method based on hydrogen/deuterium exchange mass spectrometry (HDX-MS) to detect local structural conformation and dynamics of IgG and FcRn upon interaction (13). HDX-MS monitors the isotopic exchange of hydrogen and deuterium of proteins in solution and reports on the protection of amide hydrogens from intra-and intermolecular hydrogen bonds and to a lesser extent solvent accessibility (14, 15). Our studies (13) revealed structural stabilization of the Fab regions upon FcRn interaction, which could be caused by a conformational link between the Fc and Fab regions or by direct interaction with FcRn.Here, we perform HDX-MS analyses of a unique set of antibodies with different FcRn binding characteristics to dissect the conformational origins of the involvement of Fab in FcRn binding. The antibodies ustekinumab (Stelara) and briakinumab (Ozespa) targeting the p40 subunit of IL-12 and From the ‡Department

In pharmacokinetic studies, intravenous (i.v.) administration of antibodies to mice is usually done via the lateral tail vein. This approach can cause stress to the mice and has a high rate of failure because it is challenging to perform correctly. Administration via the retro-orbital venous sinus has been suggested as a good alternative to tail vein i.v. administration of antibodies. Evidence is still needed, however, to determine whether the route of administration has an effect on the absorption or the pharmacokinetic activity of the injected antibody. The authors compared serum concentrations and pharmacokinetic parameters of a therapeutic antibody administered via tail vein injection or via retro-orbital injection. The findings suggest that there is no difference in the absorption or pharmacokinetic activity of therapeutic antibodies when administered via the lateral tail vein versus the retro-orbital venous sinus.