Discovery of the Migraine Prevention Therapeutic Aimovig (Erenumab), the First FDA-Approved Antibody against a G-Protein-Coupled Receptor.

In 2018, the United States Food and Drug Administration (FDA) approved Aimovig (erenumab) for the prevention of migraine. Erenumab is the first FDA approved antibody therapeutic against a G-protein-coupled receptor, the canonical receptor of calcitonin gene related peptide (CGRP-R). A novel, epitope-focused antigen was created to reconstruct the extracellular domains of the CGRP-R in a stable conformation. Successful inoculation of XenoMouse animals and careful screening yielded multiple candidate molecules for high potency and exquisite selectivity toward the CGRP-R over related receptors. These efforts led to the discovery of erenumab which has demonstrated the desired efficacy and safety profiles in multiple clinical studies for the prevention of migraine. The innovation developed in the discovery of erenumab furthers the ability to target G-coupled protein receptors using antibody approaches.

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.

High-throughput screening of hybridoma supernatants using multiplexed fluorescent cell barcoding on live cells.

With current available assay formats using either immobilized protein (ELISA, enzyme-linked immunosorbent assay) or immunostaining of fixed cells for primary monoclonal antibody (mAb) screening, researchers often fail to identify and characterize antibodies that recognize the native conformation of cell-surface antigens. Therefore, screening using live cells has become an integral and important step contributing to the successful identification of therapeutic antibody candidates. Thus the need for developing high-throughput screening (HTS) technologies using live cells has become a major priority for therapeutic mAb discovery and development. We have developed a novel technique called Multiplexed Fluorescent Cell Barcoding (MFCB), a flow cytometry-based method based upon the Fluorescent Cell Barcoding (FCB) technique and the Luminex fluorescent bead array system, but is applicable to high-through mAb screens on live cells. Using this technique in our system, we can simultaneously identify or characterize the antibody-antigen binding of up to nine unique fluorescent labeled cell populations in the time that it would normally take to process a single population. This has significantly reduced the amount of time needed for the identification of potential lead candidates. This new technology enables investigators to conduct large-scale primary hybridoma screens using flow cytometry. This in turn has allowed us to screen antibodies more efficiently than before and streamline identification and characterization of lead molecules.