Abstract
IgG subclass-specific differences in biological function and in vitro stability are often referred to variations in the conformational flexibility, while this flexibility has rarely been characterized. Here, small-angle X-ray scattering data from IgG1, IgG2 and IgG4 antibodies, which were designed with identical variable regions, were thoroughly analysed by the ensemble optimization method. The extended analysis of the optimized ensembles through shape clustering reveals distinct subclass-specific conformational preferences, which provide new insights for understanding the variations in physical/chemical stability and biological function of therapeutic antibodies. Importantly, the way that specific differences in the linker region correlate with the solution structure of intact antibodies is revealed, thereby visualizing future potential for the rational design of antibodies with designated physicochemical properties and tailored effector functions. In addition, this advanced computational approach is applicable to other flexible multi-domain systems and extends the potential for investigating flexibility in solutions of macromolecules by small-angle X-ray scattering.
Highlights
Immunoglobulin G (IgG) antibodies are the dominant antibodies in the human immune system and the IgG monoclonal antibodies are a major class of biopharmaceuticals with high antigen specificity and long half-lives
There are four human IgG subclasses, namely IgG1, IgG2, IgG3 and IgG4, which have been commonly described as flexible adaptor molecules with dual function, i.e. antigen binding and effector function
Human IgG subclasses differ doi:10.1107/S205225251402209X 9 research papers predominantly in their hinge-region sequence, length and disulfide bond structure (Liu & May, 2012), so solution conformations may differ depending on the nature of the antibody hinge region
Summary
Immunoglobulin G (IgG) antibodies are the dominant antibodies in the human immune system and the IgG monoclonal antibodies (mAbs) are a major class of biopharmaceuticals with high antigen specificity and long half-lives. There are four human IgG subclasses, namely IgG1, IgG2, IgG3 and IgG4, which have been commonly described as flexible adaptor molecules with dual function, i.e. antigen binding (via the variable regions) and effector function (via the constant regions). The primary sequence in the constant regions is more than 90% identical, the IgG subclasses exhibit different effector functions, including complement activation and antibody-dependent cell-mediated cytotoxicity (Bruhns et al, 2009; Bruggemann et al, 1987). IgG subclasses with identical variable regions exhibit different functional affinity to their antigens and differ in their ranking orders amongst different binding studies (Hubbard et al, 2013; McCloskey et al, 1996; Morelock et al, 1994). In-depth characterization of the solution structure of intact antibodies under physiological and formulationrelevant conditions could facilitate antibody engineering and isotype selection in the development of antibody therapeutics
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