Tracking The Clinical And Preclinical Progress In Targeting Membrane Proteins

June 24, 2019 | G protein-coupled receptors (GPCRs), ion channels, and transporters represent some of the most important drug target classes across a wide range of diseases. According to Catherine Hutchings, progress in targeting these membrane proteins requires a recognition of not only what works, but also what doesn't.

Using over 20 years of industry expertise, Hutchings provides independent scientific and strategic consultancy to biotechnology companies with particular focus on GPCRs, ion channels, immune-oncology, platform positioning and target/product evaluation.

On behalf of Bio-IT World, Kent Simmons, Senior Conference Director at Cambridge Healthtech Institute (CHI), spoke with Hutchings about her work tracking the progress in developing biotherapeutics against GPCR and ion channel targets, the successes and failures of targeting membrane proteins, and what technologies in the R&D space have the potential to be game changers.

Editor's note: Simmons is producing the Antibodies Against Membrane Protein Targets program as part of the upcoming Discovery on Target (DOT) conference in Boston, September 16-19. Hutchings will be speaking on the program. Their conversation has been edited for length and clarity.

Bio-IT World: Over the last decade, you've published several pivotal survey papers that track the progress by industry and academia in developing biotherapeutics against GPCR  and ion channel targets (2010 GPCR, 2017 GPCR, 2019 ion channel). What are your observations on the developments that have occurred in the membrane protein space between the first big paper in 2010 and the 2017 paper?

Catherine Hutchings: In parallel to the strong outlook emerging in the global antibody pipeline in general, there has been a growing interest in targeting GPCRs with antibodies with increasing investment of resources and technologies into the development of these as therapeutics. We have seen several programs progress clinically, with notably two approvals (US and EU) for mogamulizumab (CCR4) and erenumab (CGRP Receptor).

In addition, next-generation antibody therapeutic programmes, such as ADCs, bispecifics, alternative biological scaffolds, are also adding to the diversity of modalities being pursued to enhance the potential value of this target class.

Antibodies directed at GPCRs have considerable potential in the diagnosis, prognosis, and treatment of cancers either by directly inhibiting pathways driving tumour proliferation and metastasis, or by modulating GPCRs in the TME to target the immune system, including challenging GPCR targets as evidenced by the emerging interest of adhesion GPCRs as oncology tar­gets.

Intelligently designed antigen formats (e.g., stabilization, formulation into liposomes, nanodiscs, etc.), alternative expression systems coupled with advances in purification methodology, a deeper understanding of receptor biology and signalling, as well as the industry-wide dramatic increase in the implementation of automation for sampling/assay throughput/assay miniaturisation, have all contributed to the rise in the number of GPCR-targeting antibody programs in the R&D pipeline. It is increasingly common to identify functional GPCR-targeting antibodies by applying a combination of antigen formats with antibody platform capabilities tailored to the GPCR target in question and applying key learnings to overcome the technical challenges encountered with this target class.

And then more recently, I believe you co-authored a detailed survey of the ion channel space. What were some of the findings from that study?

Key learnings garnered from GPCR-antibody drug discovery are transferable to ion channels given that they are multi-spanning membrane proteins with restricted extracellular surfaces.

As with GPCRs, there is a wide range of physiological processes involving ion channels. The ion channel antibody space is reminiscent of how the GPCR antibody field looked 10 years ago, and while it is still in its infancy, the rate of success as measured by the number of programs progressing to preclinical development is encouraging. Currently, there is only one ion channel targeting antibody (BIL010t which is a polyclonal) in early clinical development (Phase 1; Biosceptre) for the treatment of basal cell carcinoma and has the potential to become a first-in-class therapy.

However, the ion channel drug target class still remains significantly under-exploited as antibody drug targets, primarily due to specific challenges such as attaining sufficient levels of cellular expression that are not toxic to the cell, the ability to purify sufficient protein in a biologically relevant conformation for antibody discovery purposes, as well screening cascade approaches, such as the identification of high throughput methodologies that can be used for patch-clamping if other functional assays are not relevant/available.

Nevertheless, advances have been made in generating crystal structures and, more recently, improvements in methodology have enhanced the number of cryo-electron microscopy structures being generated for ion channels. These advances, coupled with the deepening knowledge of ion channel gating and target biology validation, now provide an informed base on which to progress and streamline antibody-based approaches in targeting ion channels to treat a variety of diseases.

What do you think is working and what is not in efforts to target membrane proteins?

This is still a work in progress and it has been much easier to identify what has worked versus what hasn't, as folks are reluctant to share failure experiences and there is no journal of experiments that didn't work! I think both success and failure are multi-factorial, with some similarities shared in the technical challenges identified from GPCR-antibody discovery.

One key challenge is that ion channel extracellular loops are very short and therefore will not contain many epitopes or have limited accessibility. In addition, loops can be highly conserved in some ion channel families at the primary amino acid sequence level, and thus lack sufficient immunogenicity to generate robust antibody responses in mammalian hosts. However, evaluation of the utility of different/smaller antibody formats, such as nanobodies and supplementation of immunogen with adjuvants to enhance the immune response, has yielded some success.

Even in cases where the extracellular domains are large, the proteins themselves are sometimes either poorly expressed or difficult to purify from conventional platforms used for recombinant protein production. This, in turn, can limit the starting material available for large-scale immunization and screening campaigns, which is the first critical factor for success.

As with GPCRs, there is a requirement to maintain the purified ion channel in physiological context – particularly if a discontinuous epitope or conformational epitope needs to be targeted, so this is most likely why peptides as antigens have not yielded success. In addition to complexity, immunogenicity, and mechanistic properties, the size of the extracellular domains where antibodies are expected to bind can vary considerably. Some will be easier to target than others (e.g., Na_v versus P2X).

The experience thus far of workers in the field suggest that current methodologies yield therapeutically valuable antibodies that are typically rare and difficult to identify in any given discovery program. Therefore, efforts have been undertaken to increase a specific immune response against the target ion channel and/or deeply mine an immune repertoire in an effort to capture as many potential hits as possible. As with GPCRs, I think the discovery approaches will need to be tailored on a case-by-case basis.

However, we know there is precedence for the immunogenicity of ion channels as evidenced by the existence of autoantibodies (many of which are thought to be functional) to a range of ion channel families, so we should persevere with inventive approaches. For example, using alternative expression hosts such as Tetrahymena, the design of chimeric ion channels as antigen to enhance expression and maintain the correct conformation of extracellular loops, as well as engineering exquisite function into target antibody binders, as exemplified by Iontas' KnotBodies, where target binding is usually mediated by the HCDR3 and function introduced by the introduction of toxin peptide sequences (based on knottins) into one of the other CDRs.

Looking toward the future, are you seeing any technologies coming along in the R&D space that have the potential to be game changers in this space?

Disruptive technologies, such as AI and robotics, are mooted to improve our understanding of biology and subsequently revolutionize the drug discovery process, as is being pioneered by the Rosalind Franklin Institute in the UK.  In the more immediate term, the advances made with cryo-EM application to therapeutically relevant ion channels will be illuminating from an antibody targeting/available epitopes perspective, whether this is a co-structure with a peptide or antibody/antibody fragment. The strategy that is used to undertake such structural studies may also enable significant advances. For example, the Science publication (DOI: 10.1126/science.aaw2493) authored by Shen et al. earlier this year concerning Na_v1.7 revealed the two co-structures were achieved by complexing with both auxiliary subunits and two combinations of pore blockers and gating modifier toxins (GMTs), tetrodotoxin with protoxin-II and saxitoxin with huwentoxin-IV, providing overall resolutions of 3.2 angstroms.

Another game changer for all would be the ability to identify diverse, functional, conformationally specific/inducing antibodies as early as possible in the screening cascade using cost-effective methods that ideally could be applied to any antibody platform. This would be particularly enabling for identifying agonist mechanisms of action. Progress towards this approach is being explored. For example, AbTLAS is developing an innovative strategy for complex targets including GPCRs and ion channels using FAST (Functional Antibody Screening Technology), which employs HTS of a spatially addressed Fv antibody library with cells expressing target of interest. Similarly, an encapsulated assay for B cells to evaluate binding and/or signalling activation would enable early identification from immunization campaigns. Crystal Bioscience (acquired by Ligand Pharmaceuticals) made progress towards this goal with their GEM assay. However, the read-out needs to be manually evaluated by eye rather than being fully automated. Cost and accessibility are particularly relevant for many small companies or academic researchers endeavouring to expedite their discovery pipeline or validate the biology of an emerging target without the requirement for ultra-specialized instrumentation or expensive assay components that will consume all their funding.