Impact of IgG subclass on molecular properties of monoclonal antibodies

Abstract

Immunoglobulin G-based monoclonal antibodies (mAbs) have become a dominant class of biotherapeutics in recent decades. Approved antibodies are mainly of the subclasses IgG1, IgG2, and IgG4, as well as their derivatives. Over the decades, the selection of IgG subclass has frequently been based on the needs of Fc gamma receptor engagement and effector functions for the desired mechanism of action, while the effect on drug product developability has been less thoroughly characterized. One of the major reasons is the lack of systematic understanding of the impact of IgG subclass on the molecular properties. Several efforts have been made recently to compare molecular property differences among these IgG subclasses, but the conclusions from these studies are sometimes obscured by the interference from variable regions. To further establish mechanistic understandings, we conducted a systematic study by grafting three independent variable regions onto human IgG1, an IgG1 variant, IgG2, and an IgG4 variant constant domains and evaluating the impact of subclass and variable regions on their molecular properties. Structural and computational analysis revealed specific molecular features that potentially account for the differential behavior of the IgG subclasses observed experimentally. Our data indicate that IgG subclass plays a significant role on molecular properties, either through direct effects or via the interplay with the variable region, the IgG1 mAbs tend to have higher solubility than either IgG2 or IgG4 mAbs in a common pH 6 buffer matrix, and solution behavior relies heavily on the charge status of the antibody at the desirable pH.

Pharmacokinetic Developability and Disposition Profiles of Bispecific Antibodies: A Case Study with Two Molecules

“…The isoelectric points were measured or calculated as described previously [ 20 ]. The pI of each the Fab and scFv was calculated from the primary sequences using algorithm in Molecular Operating Environment (MOE2019) (Chemical Computing Group, Montreal, Canada) and outlined by Sillero and colleagues [ 21 ].…”

Factors affecting IgG4-mediated complement activation

“…IgG4 is believed to have mainly neutralizing and anti-inflammatory functions due to limited complement fixation and crosslinking, therefore it has been exploited for the development of therapeutic antibodies that are intended to block their target, often including a stabilizing S228P mutation to minimize in vivo Fab arm exchange. Currently IgG4 antibodies account for approximately 13% of all therapeutic monoclonal antibodies approved or in development ( 75 ). Two examples are the humanized kappa IgG4 antibodies pembrolizumab and nivolumab, which are both programmed death receptor-1 (PD1)-blocking antibodies ( 76 , 77 ).…”

Constant domain polymorphisms influence monoclonal antibody stability and dynamics

“…Embracing this naturally occurring sequence variation, particularly within the IgG3 subclass, could aid targeted mAb therapies (Rispens et al 2014; Richardson et al 2019; Stapleton et al 2011) and potentially unlock mAbs that exploit unique functional capabilities (Tay et al 2016). Despite the presence of allelic mutations distal to the CH1‐VH interface (Figure 2b), further studies using variable domains from clinical antibodies beyond trastuzumab will improve our understanding of variant influence on stability, especially since several reports indicate possible variable domain influence on constant region function (Tang et al 2021).…”