Structural and Functional Characterization of Disulfide Isoforms of the Human IgG2 Subclass

Abstract

we have identified that the human IgG2 subclass exists as an ensemble of distinct isoforms, designated IgG2-A, -B, and -A/B, which differ by the disulfide connectivity at the hinge region. In this report, we studied the structural and functional properties of the IgG2 disulfide isoforms and compared them to IgG1. Human monoclonal IgG1 and IgG2 antibodies were designed with identical antigen binding regions, specific to interleukin-1 cell surface receptor type 1. In vitro biological activity measurements showed an increased activity of the IgG1 relative to the IgG2 in blocking interleukin-1␤ ligand from binding to the receptor, suggesting that some of the IgG2 isoforms had lower activity. Under reduction-oxidation conditions, the IgG2 disulfide isoforms converted to IgG2-A when 1 M guanidine was used, whereas IgG2-B was enriched in the absence of guanidine. The relative potency of the antibodies in cell-based assays was: IgG1 > IgG2-A > IgG2 Ͼ Ͼ IgG2-B. This difference correlated with an increased hydrodynamic radius of IgG2-A relative to IgG2-B, as shown by biophysical characterization. The enrichment of disulfide isoforms and activity studies were extended to additional IgG2 monoclonal antibodies with various antigen targets. All IgG2 antibodies displayed the same disulfide conversion, but only a subset showed activity differences between IgG2-A and IgG2-B. Additionally, the distribution of isoforms was influenced by the light chain type, with IgG2 composed mostly of IgG2-A. Based on crystal structure analysis, we propose that IgG2 disulfide exchange is caused by the close proximity of several cysteine residues at the hinge and the reactivity of tandem cysteines within the hinge. Furthermore, the IgG2 isoforms were shown to interconvert in whole blood or a "bloodlike" environment, thereby suggesting that the in vivo activity of human IgG2 may be dependent on the distribution of isoforms.

Rapid, automated characterization of disulfide bond scrambling and IgG2 isoform determination

“…For example, the LC212-HC129 bond was detected only in the A and A/B forms using this method, which is consistent with previous reports about the LC-HC connectivity in these isoforms. 11,12 Most native intra-chain and inter-chain disulfide bonds (12 total for an IgG2) were detected in all isoforms. However, the intra-chain HC22-HC96 could not be detected in neither the A nor the B isoforms, suggesting possible trypsin missed cleavage at this site or low ionization of this particular DSB peptide.…”

“…11–13 The inter-chain disulfides of the A isoform are configured in the classical IgG arrangement, where the LC-HC disulfide is found in the same antigen-binding fragment (Fab) region and the four hinge cysteines are bonded in a parallel fashion with the same residue of the opposite HC. The A isoform is furthermore subcategorized as the A1 and A2 forms, which have identical disulfide configurations but the A2 form has one hinge disulfide that is resistant to reduction.…”

“…15,17,19,27–30 Several mass spectrometry (MS)/MS approaches for characterizing disulfide linkages have been described using collision-induced dissociation (CID), electron-transfer dissociation (ETD), or electron-capture dissociation (ECD). 11–13,29,31–35 These approaches often require multiple analysis, comparison of reduced and non-reduced peptide maps, interpretation of complex data sets, or large sample consumption. Electrospray ionization MS/MS characterization of IgG2 hinge disulfides can also be difficult due to the large size of hinge peptides (5 – 10 kDa, depending on disulfide configuration and number of missed cleavages) that generate low-intensity, multiply charged peak clusters that complicate the deconvolution of the MS/MS spectra.…”

“…Electrospray ionization MS/MS characterization of IgG2 hinge disulfides can also be difficult due to the large size of hinge peptides (5 – 10 kDa, depending on disulfide configuration and number of missed cleavages) that generate low-intensity, multiply charged peak clusters that complicate the deconvolution of the MS/MS spectra. 11,12,32 Quantification of IgG2 disulfide isoforms requires a separate reverse-phase chromatography or charge distribution analysis of the intact protein. 11,12 The advantage of using matrix-assisted laser desorption/ionization (MALDI)-MS/MS for IgG2 disulfide-bonded (DSB) peptide analysis is the predominance of singly-charged ions in the MALDI spectra, which considerably simplifies data analysis and renders it compatible with both semi-quantitative output and automation from a single LC-MS/MS analysis.…”

“…11,12,32 Quantification of IgG2 disulfide isoforms requires a separate reverse-phase chromatography or charge distribution analysis of the intact protein. 11,12 The advantage of using matrix-assisted laser desorption/ionization (MALDI)-MS/MS for IgG2 disulfide-bonded (DSB) peptide analysis is the predominance of singly-charged ions in the MALDI spectra, which considerably simplifies data analysis and renders it compatible with both semi-quantitative output and automation from a single LC-MS/MS analysis. 36–38 …”