Human IgG2 Antibody Disulfide Rearrangement in Vivo

Abstract

Proteins destined to circulate in the blood are first folded and assembled in the endoplasmic reticulum of secretory cells. For antibodies, like many other serum proteins, the folding and assembly steps involve the formation of disulfide bonds. Such bonds have been thought to be static features of proteins, stabilizing domains, and linking polypeptide chains, although some cases of extracellular disulfide bond cleavage have been noted. Recently, the human IgG2 antibody subclass was found to possess multiple structures differing in specific disulfide linkages. These structures are naturally occurring and can, in some cases, affect the activity of the antibody. Here we show that these IgG2 disulfide linkages interconvert while circulating in humans. Secretory cells initially produce primarily one form (IgG2-A), which is rapidly converted to a second form (IgG2-A/B) while circulating in the blood, followed by a slower conversion to a third form (IgG2-B). This work demonstrates that the disulfide structure of the IgG2 antibody is dynamic in vivo, on a time scale similar to that of the protein's lifetime. Thus, changes to the IgG2 disulfide structure provide a marker of the protein's age and may alter its activity over its lifetime.

Mutations within a human IgG2 antibody form distinct and homogeneous disulfide isomers but do not affect Fc gamma receptor or C1q binding

“…However, it has recently been reported that IgG2 antibody preparations are not homogeneous and instead consist of distinct isoforms. [19][20][21][22][23] The isomeric structures are due to disulfide shuffling between cysteine residues within the light (LC) and heavy chains (HC) with the upper hinge sequence. Three IgG2 isomers (A, B, and A/B) have been characterized and shown to be structurally and, in at least some instances, functionally distinct.…”

“…21 The canonical IgG2 structure, now referred to as IgG2-A, is present in low concentrations in purified antibody preparations and has been shown to convert to the A/B and B isomers in human serum. 22 In the IgG2-A isomer, the cysteine residue at or near the end of each LC pairs with the cysteine residue within CH1 of the HC and the four cysteine residues within the hinge region form four interchain disulfide bonds. In isomer IgG2-B, the C-terminal cysteine residues in both of the LC pair with either one of the upper two cysteine residues in the hinge.…”

Anticorps thérapeutiques et dérivés : une palette de structures pour une pléthore d’indications

“…Ces appariements sont sensibles à l'environnement redox in vitro et in vivo (par ex. au taux de glutathion réduit présent sur les hématies 6 ) [22]. Ils peuvent varier au cours du temps (compartiment cellulaire, âge de l'anticorps) et avoir un impact sur la fonctionnalité des anticorps.…”

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

“…Although the isoforms only differ in the arrangement of the inter-chain disulfide bonds, each disulfide pattern alters the higher-order structure of the mAb, thereby affecting thermal stability, binding efficiency, surface hydrophobicity, isoelectric point, and possibly influencing developability. 15–19 For example, A-isoform enriched panitumumab and denosumab demonstrated higher apparent affinity to the human IgG Fc receptor-like 5 (FCRL5) compared to the B isoform. 20 Disulfide isoform distribution may also affect the conjugation profile of cysteine-linked antibody-drug conjugates.…”

“…17,20,22–26 The flexible and solvent-accessible hinge region is particularly crucial, as it tends to be labile to reduction and oxidation that can lead to disulfide scrambling or trisulfide formation. 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.…”