Impact of antibody subclass and disulfide isoform differences on the biological activity of CD200R and ßklotho agonist antibodies.

Agonism of cell surface receptors by monoclonal antibodies is dependent not only on its ability to bind the target, but also to deliver a biological signal through receptors to the cell. Immunoglobulin G2 antibodies (IgG2s) are made up of a mixture of distinct isoforms (IgG2-A, -B and A/B), which differ by the disulfide connectivity at the hinge region. When evaluating panels of agonistic antibodies against CD200 receptor (CD200R) or ßklotho receptor (ßklotho), we noticed striking activity differences of IgG1 or IgG2 antibodies with the same variable domains. For the CD200R antibody, the IgG2 antibody demonstrated higher activity than the IgG1 or IgG4 antibody. More significantly, for ßklotho, agonist antibodies with higher biological activity as either IgG2 or IgG1 were identified. In both cases, ion exchange chromatography was able to isolate the bioactivity to the IgG2-B isoform from the IgG2 parental mixture. The subclass-related increase in agonist activity was not correlated with antibody aggregation or binding affinity, but was driven by enhanced avidity for the CD200R antibody. These results add to the growing body of evidence that show that conformational differences in the antibody hinge region can have a dramatic impact on the antibody activity and must be considered when screening and engineering therapeutic antibody candidates. The results also demonstrate that the IgG1 (IgG2-A like) or the IgG2-B form may provide the most active form of agonist antibodies for different antibodies and targets.

Flow Cytometry-Based Epitope Binning Using Competitive Binding Profiles for the Characterization of Monoclonal Antibodies against Cellular and Soluble Protein Targets.

A key step in the therapeutic antibody drug discovery process is early identification of diverse candidate molecules. Information comparing antibody binding epitopes can be used to classify antibodies within a large panel, guiding rational lead molecule selection. We describe a novel epitope binning method utilizing high-throughput flow cytometry (HTFC) that leverages cellular barcoding or spectrally distinct beads to multiplex samples to characterize antibodies raised against cell membrane receptor or soluble protein targets. With no requirement for sample purification or direct labeling, the method is suited for early characterization of antibody candidates. This method generates competitive binding profiles of each antibody against a defined set of known or unknown reference antibodies for binding to epitopes of an antigen. Antibodies with closely related competitive binding profiles indicate similar epitopes and are classified in the same bin. These large, high-throughput, multiplexed experiments can yield epitope bins or clusters for the entire antibody panel, from which a conceptual map of the epitope space for each antibody can be created. Combining this valuable epitope information with other data, such as functional activity, sequence, and selectivity of binding to orthologs and paralogs, enables us to advance the best epitope-diverse candidates for further development.