Recombinant production of GPCRs is challenging due to their complex structure and low stability outside of the lipid bilayer. Selection of the appropriate model membrane system and protein engineering strategy can address some of these challenges. Antigen formats, including virus-like-particles or polymer extracted membranes, must be carefully considered within the application context. In this talk, I will discuss GPCR antigen generation and purification, supporting antibody discovery and characterization efforts.

Membrane proteins are challenging drug targets (GPCRs, ion channels, transporters,) and are notoriously difficult to work with. Our proprietary nano-membrane platform technology stabilises these important membrane proteins. We will present novel data on Salipro-CXCRs complexes, as well as case studies on other membrane protein types, to illustrate how the Salipro platform enables the development of next generation therapeutic (e.g. via SPR, phage display, B cell sorting and cryoEM.)

Generating monoclonal antibodies (mAbs) directed towards G protein-coupled receptors (GPCRs) poses many challenges, including antigen design, expression, and purification. In this presentation, I will share our experience using a stabilized, purified NK1 receptor as a soluble antigen in an in vitro selection campaign. Through this approach, we successfully identified mAbs that interact with the target: both on cells, and in surface plasmon resonance experiments.

Integral membrane proteins continue to be a hurdle for antibody therapeutic discovery. By combining a yeast-based, single B cell platform with humanized transgenic murine diversities, we have developed a high-throughput methodology for the discovery of membrane-obligate-specific antibodies. In the absence of soluble recombinant antigen, this platform enables the discovery of large, clonally-diverse panels of high-affinity, target-specific antibodies.

The FK506-binding protein 51 (FKBP51) is a known target for stress and metabolic disorders. The high structural similarity between FKBP homologs represents a hurdle to designing selective ligands. We generated a yeast display library expressing chicken-derived scFvs and screened it via FACS to identify conformation-locking scFvs that stabilize the transient binding pocket of FKBP51. Conformation-locking scFvs may facilitate drug discovery of ligands selective to the FKBP51 transient binding pocket.

• What are the challenges of working with in vitro and in vivo systems for membrane protein discovery?
• How can these approaches be effectively leveraged to identify antibodies targeting membrane proteins?
• What approaches are able to overcome typical hurdles that prevent efficient antibody affinity optimization against membrane protein targets?
• What technologies can be used to target specific epitopes on membrane proteins?​

• Affinity and Kinetics: Useful approaches for characterizing antibody binding
• Epitopes: Different techniques for epitope mapping; binning versus mapping; why do epitopes matter?
• Specificity: Understanding off-target binding
• Cell Function: Integrating functional assays into antibody discovery and development​

Structurally complex membrane targets such as GPCRs, ion channels, and transporters are acknowledged as challenging targets for monoclonal antibody discovery. Strategies for discovery of functional antibodies to these target classes are emerging. This presentation will provide an overview of these emerging strategies and provide a number of case studies, including a new anti-GPCR antibody case study, highlighting the discovery and optimization of antibodies against GPCRs and ion channels.

Using computational methods that score physical features of protein structures or apply artificial intelligence approaches, homologs and de novo proteins are screened in silico to replace human signaling proteins and their receptors. Predicted HLA-II epitopes that pose immunogenicity risks are removed through targeted mutations, without damaging structure or activity. The proteins have a range of affinities, specificities, and signaling properties, which are tuned through experimental deep mutagenesis and affinity optimization.

G protein coupled receptors (GPCRs) have a central role in many physiological processes, but are still underexplored (in many cases due to the absence of specific ligands.) Existing clinical stage GPCR drugs often result in treatment-limiting side effects, and could be replaced by therapeutic antibodies. We will show the unique potential of ConfoBody stabilized GPCRs to facilitate parallel discovery of highly-potent and selective (ant)agonistic biologics to peptide GPCRs.

The GPCR superfamily contains a subset of approximately 50 receptors, whose natural ligands are small proteins. While these receptors are valuable drug targets linked to serious diseases, many remain undrugged because they present challenges to standard drug discovery approaches. This presentation describes Orion's novel solution to target small protein GPCRs: engineering the natural small protein ligand to discover potent analogs with enhanced binding properties and user-defined signaling activity.

This talk will describe the discovery and biological characterisation of anti-GPCR mAbs. Antibodies against the target were obtained from a hybridoma discovery campaign. Clones selected for high cell binding potency and specificity to the target were engineered to enhance Fc effector function. We then identified Fc-enhanced clones displaying potent ADCC activity, in cells overexpressing and in cells endogenously expressing the target, as well as in primary target cells.