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

Abstract

Human IgG2 antibodies may exist in at least three distinct structural isomers due to disulfide shuffling within the upper hinge region. Antibody interactions with Fc gamma receptors and the complement component C1q contribute to immune effector functions. These interactions could be impacted by the accessibility and structure of the hinge region. To examine the role structural isomers may have on effector functions, a series of cysteine to serine mutations were made on a human IgG2 backbone. We observed structural homogeneity with these mutants and mapped the locations of their disulfide bonds. Importantly, there was no observed difference in binding to any of the Fc gamma receptors or C1q between the mutants and the wild-type IgG2. However, differences were seen in the apparent binding affinity of these antibodies that were dependent on the selection of the secondary detection antibody used.

Disulfide Scrambling in IgG2 Monoclonal Antibodies: Insights from Molecular Dynamics Simulations

“…Hence, methods and strategies have been developed to eliminate or mitigate disulfide heterogeneity of human IgG2 mAbs (18,19,36). The main approach is to mutate the Cys residues involved in disulfide scrambling.…”

Complementary MS Methods Assist Conformational Characterization of Antibodies with Altered S–S Bonding Networks

“…The native ESI spray tip was custom-made by using a pulling machine (Sutter Instrument Co., Novato, CA, USA), as described elsewhere [40]. The protocols of cloning, expression, and purification for human IgG2 antibody (anti-CD44 monoclonal antibody) were reported previously [16]. Those protocols were adopted without modification for preparing the mAb samples used in these experiments.…”

“…Depending on redox environment, these isomers interconvert, possibly enabling IgG2 antibodies to regulate function [15]. To understand the role of isomers, Lightle et al [16] constructed cysteine-to-serine substitutions on the IgG2 anti-CD44 antibody. The single and double substitutions force the antibody into a single isoform that displays no significant binding differences to the CD44 extracellular domain [16].…”

“…To understand the role of isomers, Lightle et al [16] constructed cysteine-to-serine substitutions on the IgG2 anti-CD44 antibody. The single and double substitutions force the antibody into a single isoform that displays no significant binding differences to the CD44 extracellular domain [16]. A large difference occurs, however, in the binding of a secondary antibody to the hinge region of the IgG2, suggesting that disulfide isomers have structural alterations in the hinge region.…”

Global Shape and Ligand Binding Efficiency of the HIV-1-neutralizing Antibodies Differ from Those of Antibodies That Cannot Neutralize HIV-1

“…Portion-To analyze the binding affinity of IgG1 mAbs to C1q, ELISA was done following a previously published protocol with slight modification (25). The IgG1 mAbs were diluted to a concentration of 10 nM in PBS, and each mAb was coated in an individual well of a 96-well Maxisorp Immunoplate (Nunc, Rochester, NY) incubated at 4°C overnight.…”