The neutrophil dynamic mass redistribution assay as a medium throughput primary cell screening assay.

In a typical GPCR drug discovery campaign, an in vitro primary functional screening assay is often established in a recombinant system over-expressing the target of interest which offers advantages with respect to overall throughput and robustness of compound testing. Subsequently, compounds are then progressed into more physiologically relevant but lower throughput ex vivo primary cell assays and finally in vivo studies. Here we describe a dynamic mass redistribution (DMR) assay which has been developed in a format suitable to support medium throughput drug screening in primary human neutrophils. Neutrophils are known to express both CXCR1 and CXCR2 chemokine receptors that are thought to play significant roles in various inflammatory disorders and cancer. Using multiple relevant chemokine ligands and a range of selective and non-selective small and large molecule antagonists that block CXCR1 and CXCR2 responses, we demonstrate distinct pharmacological profiles in neutrophil DMR from those observed in recombinant assays but predictive of activity in neutrophil chemotaxis and CD11b upregulation, a validated target engagement marker previously utilized in clinical studies of CXCR2 antagonists. The primary human neutrophil DMR cell system is highly reproducible, robust, and less prone to donor variability observed in CD11b and chemotaxis assays and thus provides a unique, more physiologically relevant, and higher throughput assay to support drug discovery and translation to early clinical trials. Significance Statement Neutrophil dynamic mass redistribution assays provide a higher throughput screening assay to profile compounds in primary cells earlier in the screening cascade enabling a higher level of confidence in progressing the development of compounds towards the clinic. This is particularly important for chemokine receptors where redundancy contributes to a lack of correlation between recombinant screening assays and primary cells, with the co-expression of related receptors confounding results.

Discovery and design of G protein-coupled receptor targeting antibodies.

INTRODUCTION: G protein-coupled receptors (GPCRs) are the target of one-third of all approved drugs; however, these drugs only target about one-eighth of the human repertoire of GPCRs. GPCRs regulate a diverse range of critical physiological processes including organ development, cardiovascular function, mood, cognition, multicellularity, cellular motility, immune responses and sensation of light, taste, and odor. However, many GPCRs are expressed poorly, and a significant proportion have unknown ligands and unclear signaling pathways. AREAS COVERED: GPCRs are better suited to be targeted by monoclonal antibodies (mAbs) because of the challenges encountered in small-molecule discoveries such as druggability, selectivity, and distribution. mAbs have better drug-like properties in these respects. Herein, the authors review previously discovered functional mAbs that target GPCRs that are in the clinic and/or in development. They also review the biophysical considerations that make GPCRs so challenging to work with but also provide opportunities for biologic druggability. EXPERT OPINION: GPCRs are proven targets of small molecules yet remain an under-represented target of biologics. We believe that antibody drugs that target GPCRs have the potential to unlock new therapeutic avenues and also uncover previously unappreciated receptor biology, particularly when harnessing next-generation biologic modalities.

A review of antibody-based therapeutics targeting G protein-coupled receptors: an update.

INTRODUCTION: G protein-coupled receptors (GPCRs) play key roles in many biological functions and are linked to many diseases across all therapeutic areas. As such, GPCRs represent a significant opportunity for antibody-based therapeutics. AREAS COVERED: The structure of the major GPCR families is summarized in the context of choice of antigen source employed in the drug discovery process and receptor biology considerations which may impact on targeting strategies. An overview of the therapeutic GPCR-antibody target landscape and the diversity of current therapeutic programs is provided along with summary case studies for marketed antibody drugs or those in advanced clinical studies. Antibodies in early clinical studies and the emergence of next-generation modalities are also highlighted. EXPERT OPINION: The GPCR-antibody pipeline has progressed significantly with a number of technical developments enabling the successful resolution of some of the challenges previously encountered and this has contributed to the growing interest in antibody-based therapeutics addressing this target class.

Mini-review: antibody therapeutics targeting G protein-coupled receptors and ion channels.

Antibodies are now well established as therapeutics with many additional advantages over small molecules and peptides relative to their selectivity, bioavailability, half-life and effector function. Major classes of membrane-associated protein targets include G protein-coupled receptors (GPCRs) and ion channels that are linked to a wide range of disease indications across all therapeutic areas. This mini-review summarizes the antibody target landscape for both GPCRs and ion channels as well as current progress in the respective research and development pipelines with some example case studies highlighted from clinical studies, including those being evaluated for the treatment of symptoms in COVID-19 infection.

Ion channels as therapeutic antibody targets.

It is now well established that antibodies have numerous potential benefits when developed as therapeutics. Here, we evaluate the technical challenges of raising antibodies to membrane-spanning proteins together with enabling technologies that may facilitate the discovery of antibody therapeutics to ion channels. Additionally, we discuss the potential targeting opportunities in the anti-ion channel antibody landscape, along with a number of case studies where functional antibodies that target ion channels have been reported. Antibodies currently in development and progressing towards the clinic are highlighted.

A monoclonal antibody raised against a thermo-stabilised ß1-adrenoceptor interacts with extracellular loop 2 and acts as a negative allosteric modulator of a sub-set of ß1-adrenoceptors expressed in stable cell lines.

Recent interest has focused on antibodies that can discriminate between different receptor conformations. Here we have characterised the effect of a monoclonal antibody (mAb3), raised against a purified thermo-stabilised turkey ß1-adrenoceptor (ß1AR-m23 StaR), on ß1-ARs expressed in CHO-K1 or HEK 293 cells. Immunohistochemical and radioligand-binding studies demonstrated that mAb3 was able to bind to ECL2 of the tß1-AR, but not its human homologue. Specific binding of mAb3 to tß1-AR was inhibited by a peptide based on the turkey, but not the human, ECL2 sequence. Studies with [3H]-CGP 12177 demonstrated that mAb3 prevented the binding of orthosteric ligands to a subset (circa 40%) of turkey ß1-receptors expressed in both CHO K1 and HEK 293 cells. MAb3 significantly reduced the maximum specific binding capacity of [3H]-CGP-12177 without influencing its binding affinity. Substitution of ECL2 of tß1-AR with its human equivalent, or mutation of residues D186S, P187D, Q188E prevented the inhibition of [3H]-CGP 12177 binding by mAb3. MAb3 also elicited a negative allosteric effect on agonist-stimulated cAMP responses. The identity of the subset of turkey ß1-adrenoceptors influenced by mAb3 remains to be established but mAb3 should become an important tool to investigate the nature of ß1-AR conformational states and oligomeric complexes.

Opportunities for therapeutic antibodies directed at G-protein-coupled receptors.

G-protein-coupled receptors (GPCRs) are activated by a diverse range of ligands, from large proteins and proteases to small peptides, metabolites, neurotransmitters and ions. They are expressed on all cells in the body and have key roles in physiology and homeostasis. As such, GPCRs are one of the most important target classes for therapeutic drug discovery. The development of drugs targeting GPCRs has therapeutic value across a wide range of diseases, including cancer, immune and inflammatory disorders as well as neurological and metabolic diseases. The progress made by targeting GPCRs with antibody-based therapeutics, as well as technical hurdles to overcome, are presented and discussed in this Review. Antibody therapeutics targeting C-C chemokine receptor type 4 (CCR4), CCR5 and calcitonin gene-related peptide (CGRP) are used as illustrative clinical case studies.

Monoclonal anti-ß1-adrenergic receptor antibodies activate G protein signaling in the absence of ß-arrestin recruitment.

Thermostabilized G protein-coupled receptors used as antigens for in vivo immunization have resulted in the generation of functional agonistic anti-ß1-adrenergic (ß1AR) receptor monoclonal antibodies (mAbs). The focus of this study was to examine the pharmacology of these antibodies to evaluate their mechanistic activity at ß1AR. Immunization with the ß1AR stabilized receptor yielded five stable hybridoma clones, four of which expressed functional IgG, as determined in cell-based assays used to evaluate cAMP stimulation. The antibodies bind diverse epitopes associated with low nanomolar agonist activity at ß1AR, and they appeared to show some degree of biased signaling as they were inactive in an assay measuring signaling through ß-arrestin. In vitro characterization also verified different antibody receptor interactions reflecting the different epitopes on the extracellular surface of ß1AR to which the mAbs bind. The anti-ß1AR mAbs only demonstrated agonist activity when in dimeric antibody format, but not as the monomeric Fab format, suggesting that agonist activation may be mediated through promoting receptor dimerization. Finally, we have also shown that at least one of these antibodies exhibits in vivo functional activity at a therapeutically-relevant dose producing an increase in heart rate consistent with ß1AR agonism.

Therapeutic antibodies directed at G protein-coupled receptors.

G protein-coupled receptors (GPCRs) are one of the most important classes of targets for small molecule drug discovery, but many current GPCRs of interest are proving intractable to small molecule discovery and may be better approached with bio-therapeutics. GPCRs are implicated in a wide variety of diseases where antibody therapeutics are currently used. These include inflammatory diseases such as rheumatoid arthritis and Crohn disease, as well as metabolic disease and cancer. Raising antibodies to GPCRs has been difficult due to problems in obtaining suitable antigen because GPCRs are often expressed at low levels in cells and are very unstable when purified. A number of new developments in over-expressing receptors, as well as formulating stable pure protein, are contributing to the growing interest in targeting GPCRs with antibodies. This review discusses the opportunities for targeting GPCRs with antibodies using these approaches and describes the therapeutic antibodies that are currently in clinical development.