Discovery of functional monoclonal antibodies targeting G-protein-coupled receptors and ion channels.

The development of recombinant antibody therapeutics is a significant area of growth in the pharmaceutical industry with almost 50 approved monoclonal antibodies on the market in the US and Europe. Despite this growth, however, certain classes of important molecular targets have remained intractable to therapeutic antibodies due to complexity of the target molecules. These complex target molecules include G-protein-coupled receptors and ion channels which represent a large potential target class for therapeutic intervention with monoclonal antibodies. Although these targets have typically been addressed by small molecule approaches, the exquisite specificity of antibodies provides a significant opportunity to provide selective modulation of these target proteins. Given this opportunity, substantial effort has been applied to address the technical challenges of targeting these complex membrane proteins with monoclonal antibodies. In this review recent progress made in the strategies for discovery of functional monoclonal antibodies for these challenging membrane protein targets is addressed.

Screening Strategies for the Discovery of Ion Channel Monoclonal Antibodies.

Ion channels play crucial roles in physiology by modulation of cellular functions that include electrical excitability, secretion, cell migration, and gene transcription. They are an important target class for drug discovery and have historically been targeted using small molecule approaches. A significant opportunity exists to target these channels with antibodies and alternative forms of biologics. Antibodies display high specificity, selectivity, and affinity for their target antigen, thus having the potential to target ion channels very precisely. Nonetheless, isolating antibodies to ion channels is challenging due to the difficulties in expression and purification of ion channels in a format suitable for antibody drug discovery and due to the complexities of screening for function. In this overview, we focus on an array of screening methods, ranging from direct antibody binding screens to complex electrophysiological assays, and describe how these assays can be used to identify functional monoclonal antibodies. We also provide some insights into specific considerations which are required to enable these screens to be used for antibody drug discovery. © 2018 by John Wiley & Sons, Inc.

Development of therapeutic antibodies to G protein-coupled receptors and ion channels: Opportunities, challenges and their therapeutic potential in respiratory diseases.

The development of recombinant antibody therapeutics continues to be a significant area of growth in the pharmaceutical industry with almost 50 approved monoclonal antibodies on the market in the US and Europe. Therapeutic drug targets such as soluble cytokines, growth factors and single transmembrane spanning receptors have been successfully targeted by recombinant monoclonal antibodies and the development of new product candidates continues. Despite this growth, however, certain classes of important disease targets have remained intractable to therapeutic antibodies due to the complexity of the target molecules. These complex target molecules include G protein-coupled receptors and ion channels which represent a large target class for therapeutic intervention with monoclonal antibodies. Although these targets have typically been addressed by small molecule approaches, the exquisite specificity of antibodies provides a significant opportunity to provide selective modulation of these important regulators of cell function. Given this opportunity, a significant effort has been applied to address the challenges of targeting these complex molecules and a number of targets are linked to the pathophysiology of respiratory diseases. In this review, we provide a summary of the importance of GPCRs and ion channels involved in respiratory disease and discuss advantages offered by antibodies as therapeutics at these targets. We highlight some recent GPCRs and ion channels linked to respiratory disease mechanisms and describe in detail recent progress made in the strategies for discovery of functional antibodies against challenging membrane protein targets such as GPCRs and ion channels.

Overcoming the Refractory Expression of Secreted Recombinant Proteins in Mammalian Cells through Modification of the Signal Peptide and Adjacent Amino Acids.

The expression and subsequent purification of mammalian recombinant proteins is of critical importance to many areas of biological science. To maintain the appropriate tertiary structure and post-translational modifications of such proteins, transient mammalian expression systems are often adopted. The successful utilisation of these systems is, however, not always forthcoming and some recombinant proteins prove refractory to expression in mammalian hosts. In this study we focussed on the role of different N-terminal signal peptides and residues immediately downstream, in influencing the level of secreted recombinant protein obtained from suspension HEK293 cells. Using secreted alkaline phosphatase (SEAP) as a model protein, we identified that the +1/+2 downstream residues flanking a heterologous signal peptide significantly affect secreted levels. By incorporating these findings we conducted a comparison of different signal peptide sequences and identified the most productive as secrecon, a computationally-designed sequence. Importantly, in the context of the secrecon signal peptide and SEAP, we also demonstrated a clear preference for specific amino acid residues at the +1 position (e.g. alanine), and a detrimental effect of others (cysteine, proline, tyrosine and glutamine). When proteins that naturally contain these "undesirable" residues at the +1 position were expressed with their native signal peptide, the heterologous secrecon signal peptide, or secrecon with an additional alanine at the +1 or +1 and +2 position, the level of expression differed significantly and in an unpredictable manner. For each protein, however, at least one of the panel of signal peptide/adjacent amino acid combinations enabled successful recombinant expression. In this study, we highlight the important interplay between a signal peptide and its adjacent amino acids in enabling protein expression, and we describe a strategy that could enable recombinant proteins that have so far proved refractory to expression in HEK293 cells, to be produced in sufficient quantities to answer important biological questions.

Discovery of functional antibodies targeting ion channels.

Ion channels play critical roles in physiology and disease by modulation of cellular functions such as electrical excitability, secretion, cell migration, and gene transcription. Ion channels represent an important target class for drug discovery that has been largely addressed, to date, using small-molecule approaches. A significant opportunity exists to target these channels with antibodies and alternative formats of biologics. Antibodies display high specificity and affinity for their target antigen, and they have the potential to target ion channels very selectively. Nevertheless, isolating antibodies to this target class is challenging due to the difficulties in expression and purification of ion channels in a format suitable for antibody drug discovery in addition to the complexity of screening for function. In this article, we will review the current state of ion channel biologics discovery and the progress that has been made. We will also highlight the challenges in isolating functional antibodies to these targets and how these challenges may be addressed. Finally, we also illustrate successful approaches to isolating functional monoclonal antibodies targeting ion channels by way of a number of case studies drawn from recent publications.

Generation of potent mouse monoclonal antibodies to self-proteins using T-cell epitope "tags".

Immunization of mice or rats with a "non-self" protein is a commonly used method to obtain monoclonal antibodies, and relies on the immune system's ability to recognize the immunogen as foreign. Immunization of an antigen with 100% identity to the endogenous protein, however, will not elicit a robust immune response. To develop antibodies to mouse proteins, we focused on the potential for breaking such immune tolerance by genetically fusing two independent T-cell epitope-containing sequences (from tetanus toxin (TT) and diphtheria toxin fragment A (DTA)) to a mouse protein, mouse ST2 (mST2). Wild-type CD1 mice were immunized with three mST2 tagged proteins (Fc, TT and DTA) and the specific serum response was determined. Only in mice immunized with the T-cell epitope-containing antigens were specific mST2 serum responses detected; hybridomas generated from these mice secreted highly sequence-diverse IgGs that were capable of binding mST2 and inhibiting the interaction of mST2 with its ligand, mouse interleukin (IL)-33 (mIL-33). Of the hundreds of antibodies profiled, we identified five potent antibodies that were able to inhibit IL-33 induced IL-6 release in a mast cell assay; notably one such antibody was sufficiently potent to suppress IL-5 release and eosinophilia infiltration in an Alternaria alternata challenge mouse model of asthma. This study demonstrated, for the first time, that T-cell epitope-containing tags have the ability to break tolerance in wild-type mice to 100% conserved proteins, and it provides a compelling argument for the broader use of this approach to generate antibodies against any mouse protein or conserved ortholog.