Understanding the impacts of dual methionine oxidations in complementarity-determining regions on the structure of monoclonal antibodies

Abstract

Methionine oxidation can substantially alter the structure and functionality of monoclonal antibodies (mAbs), especially when it occurs in the complementarity-determining regions (CDRs). It is imperative to fully understand the effects of methionine oxidation because these modifications can affect the binding affinity, stability, and immunogenicity of mAbs. Moreover, the presence of multiple methionines in close proximity within the amino acid sequence increases the complexity of accurate characterization, and sophisticated analytical methods are required to detect these modifications. In this study, we used hydrogen deuterium exchange mass spectrometry (HDX-MS) and homology modeling to investigate the effects of dual methionine oxidations (heavy chain (HC) Met111 and Met115) within a single CDR on the structure of a mAb. Our findings reveal that the solvent-accessible methionine (HC Met111) is more prone to oxidation, but such a modification does not result in conformational changes in the mAb. In contrast, the methionine (HC Met115) at the V_H-V_L interface, when subjected to different oxidative stresses, can undergo oxidation with selective stereochemistry. This can lead to predominant formation of either the S- or R-form of methionine sulfoxide diastereomer, each of which can induce distinct local conformational changes. A mechanism is proposed to elucidate these observations in this particular antibody. Furthermore, binding assays confirm that both CDR methionine oxidations do not compromise antigen binding, which alleviates concerns about potential loss of therapeutic efficacy.

Keywords: Antibody higher order structure; Antigen binding; Complementarity determining region; HDX-MS; Methionine oxidation; Stereochemistry; homology modeling.

Figure 1.

Extracted ion chromatograms (XIC) of tryptic peptide GGPV¹¹¹M[O]NYYYYYG¹¹⁵M[O]DWGQGTTVTVSSASTK³⁺ in (a) mAb1-oxi-2 (stress under native condition) and mAb1-oxi-3 (stress under denaturing condition). (b) mAb1-oxi-2 digests with MsrA treatment at T0, 7 hours and 24 hours. (c) mAb1-oxi-3 digests with MsrA treatment at T0, 7 hours and 24 hours. (d) Proposed reduction pathways for mAb1 samples with oxidations at both HC Met111 and HC Met115 when treated with methionine sulfoxide reductase a (MsrA), in which S-[O] at either site was reduced while R-[O] still survived, resulting in multiple XIC peaks for the tryptic peptide (as shown in table S2 and figure S4).

Figure 2.

HDX-MS kinetics plots of peptide sequences (a) HC 108-111.2, (b) HC 112.1-116, (c) LC 103-124, (d) LC 53-67, (e) LC 19-24. HDX of oxidized peptide is compared with the native peptide in specific samples, as denoted in the figure legend. The means and error bars (standard deviations) are based on triplicated experiments. (f) Structural homology of the fab domain of mAb1-ds. HC Met111, HC Met115 and peptide sequences of (a)-(e) are mapped on the homology model.

Figure 3.

(a) Homology structures and interactions of HC Met115, Met115-S-sulfoxide and Met115-R-sulfoxide (left to right). Related residues are shown as sticks and numbered. The bond angle and length of hydrogen bonding are indicated. Surface structures of V_H-V_L interface from mAb1 containing (b) unmodified HC Met115 (c) HC Met115-S-sulfoxide, and (d) HC Met115-R-sulfoxide, with hydrophobic patches in yellow, negatively charged oxygens of glutamate and aspartate in red and positively charged nitrogens of arginine and lysine in blue. peptides exhibit significant difference in deuterium uptake due to HC Met115 oxidation were labeled and represented with cartoon structures.

Figure 4.

(a) Proposed mechanism for the formation of HC Met115-r/s-sulfoxide diastereomers under different stress conditions. HDX-MS kinetics plots of peptide sequences (b) HC 108-111.2 and (c) HC 112.1-116 with unfolding and refolding treatment.

Figure 5.

HDX-MS kinetics plots of peptide sequences prior and post antibody-antigen binding (a) HC 108–111.2 from HC CDR3 of antibody, (b) HC 112.1–116 from HC CDR3 of antibody, (c) peptide 32–47 from epitope of antigen, (d) peptide 335–347 from epitope of antigen. Traces with solid and empty symbols represent unbound and bound states, respectively.