Phage selections on whole cells ensures relevant presentation of membrane proteins in their native context. However, selections may be complicated by low target density, high background of irrelevant antigens, and structural features that limit accessibility of binding phage. A case study presents cell-based phage selections of two membrane proteins, which resulted in a highly successful antibody discovery effort and one that did not. Factors influencing discovery will be discussed.

Developing antibody agonists targeting G-protein-coupled receptors (GPCRs) remains a big challenge. Here we report the co-crystal structure of human apelin receptor (APJ) with a potent orthosteric single-domain antibody antagonist, JN241, and the structure-guided conversion of JN241 into a full agonist, JN241-9, to human APJ. Modeling and molecular dynamics simulation shed light on JN241-9-stimulated receptor activation, providing structural insights for discovering agonistic antibodies against class A GPCRs.

G protein-coupled receptors (GPCRs) represent a major therapeutic target class as they play a key role in many (patho)physiological processes. Besides small molecules, GPCRs can be effectively targeted by biologics. In particular, antibody fragments from camelid-derived heavy chain-only antibodies (nanobodies/VHHs) are attractive tools in detecting, stabilizing, modulating and therapeutically targeting GPCRs. The small size and molecular structure of nanobodies allow extracellular and intracellular modulation of GPCR function. Besides modulating GPCR activity as monovalent or multivalent constructs, nanobodies can also be functionalized for imaging and therapy. Examples of nanobodies targeting human and viral chemokine receptors will be provided. Moreover, GPCR-binding nanobodies have been instrumental in obtaining crystal structures of GPCRs, facilitating structure-based drug discovery. Thus, nanobodies are an important class of biologics targeting GPCRs. 

Synthetic antibody (sAB)-based fiducial marks were generated by phage display to G-protein receptor kinase 1 and the two G-protein families: Gi and Gs for structure determination of GPCR-signaling complexes by single particle cryo-EM. Several cross-reactive sABs were identified which can be used across all subclasses in a “plug and play” fashion. EM data indicated that these sABs provide effective single and dual fiducials for multiple GPCR signaling complexes.

Despite considerable interest in antibody-based therapeutics targeting GPCRs, few reported antibodies functionally modulate GPCR signaling. We developed a methodology to discover modulatory camelid antibody fragments (nanobodies) for GPCRs. Using this strategy, we identified nanobody antagonists for the angiotensin II type I receptor (AT1R), which regulates cardiovascular homeostasis. Our nanobody performs similarly to clinically used angiotensin receptor blockers (ARBs) and provides a strategy for targeting AT1R when ARBs are contraindicated.

Human cell microarray screening enables the discovery of specific primary cell surface receptors (i.e., druggable targets) as well as uncovering off-targets for a variety of biotherapeutic molecules, including peptides, antibodies and proteins, CAR T and other cell therapies. Case studies will focus on the assessment of off-target liabilities at an early stage and highlight the increasing role of the technology as an adjunct to, or replacement for, tissue cross reactivity studies prior to IND submissions.

The voltage-gated sodium channel Nav1.7 is critical for amplifying pain signals. Selective blockers of Nav1.7 could provide analgesia with minimal side effects. One source of Nav1.7 drug discovery starting points are spider venoms that block voltage-gated sodium channels. This talk will discuss engineering efforts on huwentoxin-IV and protoxin-II, alterations in them that produced favorable changes in Nav1.7 potency and/or selectivity, and their ability to pharmacologically recapitulate the Nav1.7-null phenotype.

Voltage-gated sodium channels enable translocation of sodium ions across cell membranes, playing crucial roles in electrical signaling in different tissues. Sodium channel mutations give rise to neurological and cardiovascular diseases; hence they are important targets for pharmaceutical drugs. Identifying existing drugs approved for use in humans and repurposing them for treatment of diseases may provide a rapid way forward for new therapeutic applications.

We have identified various agonists of TRPA1 channel, and determined their ligand-binding and gating mechanism through biophysical and structural studies. Different from electrophilic agonists, non-covalent agonists activate TRPA1 without desensitization or tachyphylaxis, and evoke pain responses with different kinetics and sensitivity to antagonist treatment. Our study provides a pathway for the discovery of drugs tailored to specific disease conditions.

Genetic data provides a rationale for targeting SCN9a, a voltage-gated sodium channel, as a step in developing novel therapeutics for severe and chronic pain. Q-State has designed antisense oligonucleotides (ASOs) targeting SCN9a and other pain-related voltage-gated sodium channels. The ASOs were characterized using qPCR and an all-optical electrophysiology (Optopatch) screening platform to measure functional effects in recombinant cells expressing SCN9a, primary sensory neurons and iPS-derived sensory cells.  

G protein-coupled receptors (GPCRs) and ion channels represent some of the most important membrane protein drug target classes across a wide range of therapeutic areas. An update on antibody-based therapeutics in the GPCR/ion channel pipeline will be provided outlining the breadth and diversity of the target landscape, progress in preclinical and clinical development, including examples that highlight the successes and challenges faced with these target classes.

γ-aminobutyric acid receptors (GABAARs) are Cl- -preferring ligand-gated ion channels that mediate fast (phasic) synaptic inhibition and tonic (sustained) inhibition in the adult brain. They are important drug targets for benzodiazepines, intravenous anesthetics, and neurosteroids. I will present recent work involving the development of large data-rich 'omic' technologies and bioinformatic analyses with the aim of discovering novel targets for neurological diseases.

Transporter proteins control the movement of nutrients and other molecules across the cell membrane and are targets for diseases including diabetes and cancer. The structural complexity of transporters including SLC2A4 (GLUT4) pose a barrier in discovering MAbs for research and therapeutics. We leveraged our MPS Antibody Discovery platform to discover the first functional, conformation-dependent MAbs against SLC2A4. These MAbs selectively bind state-specific active forms of the transporter, providing new molecular tools for therapeutic discovery.

Resistance to antimalarials such as chloroquine (CQ) and piperaquine (PPQ) has been associated with distinct sets of point mutations in the P. falciparum chloroquine resistance transporter PfCRT, a 49 kDa integral transmembrane protein localized in the digestive vacuole of the pathogenic parasite. We present the 3.2 Å structure of a PfCRT isoform from CQ-resistant, PPQ-sensitive South American 7G8 parasites, using a combination of single-particle cryo-electron microscopy and fragment antigen-binding technology.