Allosteric Effects between the Antibody Constant and Variable Regions: A Study of IgA Fc Mutations on Antigen Binding

Abstract

Therapeutics antibodies have increasingly shifted the paradigm of disease treatments, from small molecules to biologics, especially in cancer therapy. Despite the increasing number of antibody candidates, much remains unknown about the antibody and how its various regions interact. In fact, the constant region can govern effects that might be useful in reducing the unwanted consequences resulted from systemic circulation. For this reason, apart from the commonly used IgG isotypes, IgA antibodies are promising therapeutics drugs, given its localized mucosal effects. While the antibody Fc effector cell activity has been well explored, recent research has shown evidences that the constant region of the antibody can also influence antigen binding, challenging the conventional idea of region-specific antibody functions. To further investigate this, we analyzed the IgA antibody constant and its allosteric effects onto the antigen binding regions, using recombinant Pertuzumab IgA1 and IgA2 variants. We found mutations in the C-region to reduce Her2 binding, and our computational structural analysis showed that such allosteric communications were highly dependent on the antibody hinge, providing the evidence to consider antibodies as a whole protein rather than a sum of functional regions.

Considerations of Antibody Geometric Constraints on NK Cell Antibody Dependent Cellular Cytotoxicity

“…Moving toward the Fc end of the antibody, there are several studies that indicate that perturbations to the Fc region can affect binding to antigen, for example with Pertuzumab (as an IgA1 and IgA2) and Her2 binding ( 256 ). Similarly, many other studies have shown that using identical variable regions with different constant regions (i.e., isotype or subclass switching) leads to variable antigen binding as well ( 257 ).…”

Essentially Leading Antibody Production: An Investigation of Amino Acids, Myeloma, and Natural V-Region Signal Peptides in Producing Pertuzumab and Trastuzumab Variants

“…In all experiments, the recombinant wild-type Pertuzumab (POK PG1) or Trastuzumab (HOK HG1) was used (shown in the first column) for normalization. The production rate of the variants utilizing the ‘IgE’ SP ( 23 ) was used as the reference for the production rate.…”

“…limited understanding to the utilization of the huge antibody SP repertoires. With recent studies demonstrating cross-talks of antibody elements, where the constant regions ( 22 , 23 ), variable regions and their pairings ( 24 , 25 ) affect antibody production and function, there is increasing evidence that antibodies ought to be investigated holistically ( 26 ), especially when gearing towards better therapeutics ( 27 ). Through the inclusion of antibody SP in the analysis, deeper insights into antibody V-region pairing effects in transient recombinant production ( 24 ) can be further holistically considered.…”

The structural basis for distinct binding avidity of Pertuzumab and Trastuzumab IgM towards HER2

“…Allosteric modulation has been an area of interest in molecular immunology (Janda et al, 2016;Lua et al, 2019;Su et al, 2017;Yang et al, 2017). Previous experimental and computational studies of IgG and IgA have suggested that the Fc and Fab regions of an antibody might communicate with one another Phua et al, 2019); for example, point mutations on the Fc region may reduce binding affinity, whilst antigen binding to the Fab region changes the conformation of the Fc domain to promote binding to its receptor (Lua et al, 2019;Oda et al, 2003;Schlessinger et al, 1975;Su et al, 2018). Allostery between the different subunits in a multimeric IgM, however, remain elusive.…”

“…A molecular-level understanding of how such distinct binding avidities arise for the same antigen is of importance for future design of therapeutic antibodies and epitope selection. Given that within the Pertuzumab and Trastuzumab models we have previously showed that the antibody-antigen interactions can be drastically affected by small changes in the antibody light chain (Su et al, 2017), antibody hinge (Su et al, 2018), V-region pairing , and VH families (Lua et al, 2019), it may be necessary to study the whole IgM molecule using a holistic approach, as previously proposed .…”