Impact of methionine oxidation in human IgG1 Fc on serum half-life of monoclonal antibodies

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Abstract

IgG monoclonal antibodies (mAbs) consist of two Fab fragments and one Fc fragment. The Fab fragments contain the variable regions and are responsible for drug specificity (via antigen binding); the Fc fragment contains constant regions and is responsible for effector functions (via interactions with Fcγ receptors) and extended serum half-life (via interaction with the neonatal Fc receptor, FcRn). There are two conserved methionine (Met) residues located in the FcRn binding site of the Fc fragment. It has been shown previously that oxidation of these two Met residues decreases the binding affinity to FcRn. We have further evaluated the impact of Met oxidation on serum half-lives of two humanized IgG1 mAbs in transgenic mice with human FcRn. Variable oxidation levels were obtained by several procedures: exposure to an oxidizing agent, accumulation during extended refrigerated storage, or chromatographic separation. Our results show that Met oxidation can result in a significant reduction of the serum circulation half-life and the magnitude of the change correlates well with the extent of Met oxidation and changes in FcRn binding affinities. The relatively low levels of Met oxidation accumulated during 3 years of refrigerated storage had minimal impact on FcRn binding and no detectable impact on the serum half-life.

Introduction

The enormous success that mAbs have as therapeutic agents can largely be attributed to their high specificity to targets and prolonged circulation time in the human body. The increased circulation time of IgGs compared to other serum proteins is due to their pH-dependent interaction with the neonatal Fc receptor (FcRn) (Ghetie and Ward, 2000). Like all other circulating serum proteins, IgGs are subject to non-specific pinocytosis by vascular endothelia cells and bone marrow-derived cells (Roopenian and Akilesh, 2007). Following pinocytosis, IgGs bind to FcRn with high affinity in the slightly acidic environment of endosomes, which protects them from lysosomal degradation. The IgG-FcRn complex is recycled to the cell surface where IgG can be released back into circulation due to low-affinity binding to FcRn at neutral pH. Though not without exceptions, there are many reports showing a correlation between the binding affinity to FcRn at pH 6.0 and in vivo half-life of mAbs (Zalevsky et al., 2010 and references within).

The molecular details of the IgG-FcRn interaction have been extensively studied and involve residues located at the CH2/CH3 interface in the Fc fragment. The IgG sequences involved in the FcRn binding are highly conserved among IgG isotypes and contain residues in the positions Met 252-Pro 257, Thr 307-Gln 311 and His 433-Tyr 436 (EU numbering) (Ghetie and Ward, 2000 and references within). The mapping of residues involved in the IgG-FcRn interaction has guided the development of engineered Fc variants with greater affinity for FcRn at pH 6.0 while maintaining low-affinity binding at neutral pH; these Fc variants obtained by rational design were demonstrated to have not only improved half-life, but also enhanced therapeutic efficacy as well (Zalevsky et al., 2010).

The affinity of IgGs for FcRn can be affected by post-translational modification of the residues in the binding site. The post-translational modifications may occur during production and/or storage of mAbs. In particular, the Fc fragment of IgG1 molecules contains two conserved Met residues at positions 252 and 428 (Jefferis and Lefranc, 2009) that are subject to oxidation. Met 252, which is part of the CH2 domain, and Met 428, which is part of the CH3 domain, are very close in the folded structure at the interface of the CH2/CH3 domains, where FcRn binds. There are many reports in the literature describing oxidation of Met residues in IgG molecules as a result of exposure to oxidizing agents (Keck, 1996), light (Qi et al., 2009, Liu et al., 2009), or simply during storage (Lam et al., 1997, Rao and Kroon, 1993). Some IgG1 alleles have an additional Met at position 358 (Jefferis and Lefranc, 2009), though oxidation of Met 358 is less likely to have an impact on FcRn binding because it is located at the CH3/CH3 interface, at significant distance from the FcRn binding site. We and others have also shown that Met 358 is oxidized at a slower rate than Met 252 or Met 428 (Bertolotti-Ciarlet et al., 2009, Shen et al., 1996); therefore, oxidation of Met 358 will not be evaluated in the current study.

The impact of Met oxidation on IgG structure has been extensively studied. Met oxidation induces subtle conformational changes that can be revealed by CD and DSC (Wang et al., 2010), but these subtle changes do not alter the susceptibility to protease cleavage (Liu et al., 2008b). The influence of Met oxidation on aglycosylated Fc fragment structure and stability was studied in detail by NMR and other biophysical and biochemical techniques (Liu et al., 2008a). The thermal stability of the CH2 domain depends on the extent of oxidation of both Met 252 and Met 428 and was confirmed by site-specific Met mutants (Liu et al., 2008a). More recently, Houde and colab. (Houde et al., 2010) have used Hydrogen/Deuterium exchange mass spectrometry to map the regions in IgG1 structure that undergo structural changes upon Met oxidation. Their results suggest that Met oxidation causes detectable changes in the backbone for residues 243–247, a region that contains a small α-helix and a loop, but has no influence on the backbone encompassing Met 428 because that region is stabilized by a rigid anti-parallel β-sheet structure (Houde et al., 2010).

When exposed to an oxidizing agent, Met 252 undergoes oxidation at a rate about twice that for Met 428 (Bertolotti-Ciarlet et al., 2009, Liu et al., 2008a), and the oxidation rates are not significantly affected by the presence or absence of the glycans (Liu et al., 2008b). The oxidation rate depends on the formulation composition and can be reduced by anti-oxidants (Lam et al., 1997) and/or controlling the oxygen content in the head space (S. Wang, personal communication). In comparison to other chemical degradation pathways, oxidation has a small activation energy (Thirumangalathu et al., 2007), so it may be the dominant cause of degradation under refrigerated conditions (Rao and Kroon, 1993).

The manner in which these structural changes induced by Met oxidation at the CH2/CH3 interface affect the function of mAbs is a topic of extreme interest and is extensively studied. It was reported that Met oxidation reduces the binding affinity of IgGs to Protein A and Protein G (Gaza-Bulseco et al., 2008, Pan et al., 2009) and has little or no effect on the binding affinity to Fcγ receptors (Bertolotti-Ciarlet et al., 2009). Of critical importance is the finding that the binding of both IgG1 (Bertolotti-Ciarlet et al., 2009) and IgG2 (Pan et al., 2009) to FcRn at pH 6.0 is reduced upon Met oxidation. It is therefore important to further understand the impact of Met oxidation on the serum circulation half-life for mAbs as a result of altered FcRn binding. In this report, we describe the results of an investigation involving the in vivo effect in human FcRn transgenic mice using two different humanized IgG1 mAbs with different levels of oxidation. The human FcRn transgenic mice have been shown to represent a valuable surrogate system to study half-life of human IgGs (Petkova et al., 2006, Zalevsky et al., 2010). The material used for in vivo studies represented a wide range of oxidation; extensively oxidized material was obtained by prolonged exposure to an oxidizing agent (extreme condition) or by chromatographic separation; mildly oxidized material was obtained by short exposure to an oxidizing agent or by extended refrigerated storage (to mimic “real-life” conditions). The in vivo observations were correlated with in vitro results regarding the impact of oxidation on FcRn binding.

Section snippets

Sample preparation

The two humanized IgG1 molecules used in this study have been described in a previous paper (Bertolotti-Ciarlet et al., 2009). Antibody 1, hereafter referred to as AbM, was produced by Merck Sharp and Dohme Corp.; Antibody 2, hereafter referred to as AbH, is Herceptin® and is commercially available. The two humanized IgG1 molecules have identical Fc sequences but different Fab sequences (they target different antigens). Both antibodies are designed to bind receptors expressed at the surface of

Characterization of the oxidation levels in samples following long-term storage

Oxidized variants of two IgG1 mAbs can be obtained using different approaches. We have reported levels of Met oxidation and corresponding impact on FcRn binding for AbM and AbH following incubation with oxidation reagent (Bertolotti-Ciarlet et al., 2009). Here, AbM was allowed to undergo oxidation under long-term refrigeration (3 years), which is the typical storage condition for mAbs with pharmaceutical applications. A sample stored frozen at −40 °C served as a control. The extent of oxidation

Discussion

There has been a tremendous effort in the past years to modulate the function of the Fc fragment of mAbs by rational design (Zalevsky et al., 2010). Earlier experiments (Kamei et al., 2005) have demonstrated that mutation of Met 252 along with other residues in the CH2 domain can significantly decrease or increase the affinity for FcRn; the FcA2 mutant (M252L/T256F) results in a 5-fold increase in affinity, while the FcA7 mutant (M252A/R255A/T256K/T250Q) represents a nearly 50-fold decrease in

Acknowledgements

We thank Dr. Michael Washabaugh for supporting this study and Mrs. Lisa McCormick for editing suggestions.

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