Impact of Molecular Processing in the Hinge Region of Therapeutic IgG4 Antibodies on Disposition Profiles in Cynomolgus Monkeys
Open Access
- 22 October 2009
- journal article
- Published by American Society for Pharmacology & Experimental Therapeutics (ASPET) in Drug Metabolism and Disposition
- Vol. 38 (1), 84-91
- https://doi.org/10.1124/dmd.109.029751
Abstract
The IgG4 isotype antibody is a potential candidate for immunotherapy when reduced effector functions are desirable. However, antigen binding fragment (Fab) arm exchange leads to functional monovalency with potentially reduced therapeutic efficacy. Mutagenesis studies suggested that the CH3 domain and not the core hinge is dominantly involved in in vivo molecular processing. This work investigated whether stabilization of the core hinge of a therapeutic IgG4 antibody by mutation of Ser228 to Pro (S228P) would be sufficient to prevent in vivo Fab arm exchange. In vitro experiments evaluated the influence of different levels of oxidation-reduction conditions in buffer and serum on Fab arm exchange (swapping) of wild-type (WT) IgG4 and IgG1 and of IgG4 S228P, which included a sterically neutral second mutation (Leu235 replaced by Glu). The objective of single-dose pharmacokinetic experiments in cynomolgus monkeys was to determine whether the mutation reduced IgG4 swapping in vivo. The results indicated that S228P mutation did not completely prevent Fab arm exchange in vitro in buffer under reducing conditions relative to IgG4 WT. The immunoassay findings were confirmed by mass spectrometry measurements. Results of the in vivo studies suggested that the therapeutic IgG4 WT antibody exchanged Fab arms with endogenous cynomolgus monkey IgG4, resulting in bispecific IgG4 antibodies with monovalency for the therapeutic target. In contrast, serum from cynomolgus monkeys dosed with the IgG4 mutant was virtually free of swapped IgG4. In conclusion, the results indicated that IgG4 swapping in vivo was markedly attenuated by S228P mutation.Keywords
This publication has 12 references indexed in Scilit:
- Therapeutic IgG4 antibodies engage in Fab-arm exchange with endogenous human IgG4 in vivoNature Biotechnology, 2009
- Monoclonal antibodies as innovative therapeutics.Current Pharmaceutical Biotechnology, 2008
- Considerations for the development of therapeutic monoclonal antibodiesCurrent Opinion in Immunology, 2008
- Human Monoclonal Antibodies from Transgenic MiceHandbook of Experimental Pharmacology, 2008
- Isotype selection in antibody engineeringNature Biotechnology, 2007
- Antibody therapeutics:Expert Opinion on Biological Therapy, 2007
- Potent antibody therapeutics by designNature Reviews Immunology, 2006
- IgG4 breaking the rulesImmunology, 2002
- Redox state of glutathione in human plasmaFree Radical Biology & Medicine, 2000
- Normal human immunoglobulin G4 is bispecific: it has two different antigen‐combining sitesImmunology, 1999