Rapid single B cell antibody discovery using nanopens and structured light.

Accelerated development of monoclonal antibody (mAb) tool reagents is an essential requirement for the successful advancement of therapeutic antibodies in today's fast-paced and competitive drug development marketplace. Here, we describe a direct, flexible, and rapid nanofluidic optoelectronic single B lymphocyte antibody screening technique (NanOBlast) applied to the generation of anti-idiotypic reagent antibodies. Selectively enriched, antigen-experienced murine antibody secreting cells (ASCs) were harvested from spleen and lymph nodes. Subsequently, secreted mAbs from individually isolated, single ASCs were screened directly using a novel, integrated, high-content culture, and assay platform capable of manipulating living cells within microfluidic chip nanopens using structured light. Single-cell polymerase chain reaction-based molecular recovery on select anti-idiotypic ASCs followed by recombinant IgG expression and enzyme-linked immunosorbent assay (ELISA) characterization resulted in the recovery and identification of a diverse and high-affinity panel of anti-idiotypic reagent mAbs. Combinatorial ELISA screening identified both capture and detection mAbs, and enabled the development of a sensitive and highly specific ligand binding assay capable of quantifying free therapeutic IgG molecules directly from human patient serum, thereby facilitating important drug development decision-making. The ASC import, screening, and export discovery workflow on the chip was completed within 5 h, while the overall discovery workflow from immunization to recombinantly expressed IgG was completed in under 60 days.

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.

Evaluation of recombinant monoclonal antibody SVmab1 binding to Na V1.7 target sequences and block of human Na V1.7 currents.

Identification of small and large molecule pain therapeutics that target the genetically validated voltage-gated sodium channel Na V1.7 is a challenging endeavor under vigorous pursuit. The monoclonal antibody SVmab1 was recently published to bind the Na V1.7 DII voltage sensor domain and block human Na V1.7 sodium currents in heterologous cells. We produced purified SVmab1 protein based on publically available sequence information, and evaluated its activity in a battery of binding and functional assays. Herein, we report that our recombinant SVmAb1 does not bind peptide immunogen or purified Na V1.7 DII voltage sensor domain via ELISA, and does not bind Na V1.7 in live HEK293, U-2 OS, and CHO-K1 cells via FACS. Whole cell manual patch clamp electrophysiology protocols interrogating diverse Na V1.7 gating states in HEK293 cells, revealed that recombinant SVmab1 does not block Na V1.7 currents to an extent greater than observed with an isotype matched control antibody. Collectively, our results show that recombinant SVmab1 monoclonal antibody does not bind Na V1.7 target sequences or specifically inhibit Na V1.7 current.

Discovery and bio-optimization of human antibody therapeutics using the XenoMouse® transgenic mouse platform.

Since the late 1990s, the use of transgenic animal platforms has transformed the discovery of fully human therapeutic monoclonal antibodies. The first approved therapy derived from a transgenic platform--the epidermal growth factor receptor antagonist panitumumab to treat advanced colorectal cancer--was developed using XenoMouse(®) technology. Since its approval in 2006, the science of discovering and developing therapeutic monoclonal antibodies derived from the XenoMouse(®) platform has advanced considerably. The emerging array of antibody therapeutics developed using transgenic technologies is expected to include antibodies and antibody fragments with novel mechanisms of action and extreme potencies. In addition to these impressive functional properties, these antibodies will be designed to have superior biophysical properties that enable highly efficient large-scale manufacturing methods. Achieving these new heights in antibody drug discovery will ultimately bring better medicines to patients. Here, we review best practices for the discovery and bio-optimization of monoclonal antibodies that fit functional design goals and meet high manufacturing standards.