Membrane Antibody Therapeutic Approach to Ion Channelopathies

What is channelopathy?


Ions channels are specific transmembrane proteins - encoded by more than 500 genes in human body - whose role is the selective passage of ions across the cell membrane following electrochemical gradients.


When a malfunction of this receptor occurs, either an abnormal gain or a loss of function, the resulting disorders are described as channelopathies.


Since ion channels are present throughout the entire human body, a channelopathy implies a wide variety of consequences including central nervous system (migraine syndromes, epilepsy, ataxia), cardiac arrhythmia diseases (long QT syndrome, short QT syndrome, Brugada syndrome), pain, kidney, pancreas, skeletal muscle (myotonic dystrophy) and bone diseases (osteoporosis).


These particular pathologies related to membrane receptor dysfunction have two distinct forms.


The first form is innate channelopathy. Hereditary malfunctions of the transmembrane protein result from genetic mutations in the subunits of an ion channel or in the regulatory proteins linked to it.


The second is acquired channelopathy. Ion channel abnormalities develop independently of the genetic background, following exposure to drugs, toxic products or as a consequence of peripheral nerve injury due to accident or specific medical conditions like autoimmune diseases (diabetes, lupus, Guillain-Barre syndrome, Sjogren's syndrome...).


Inherited channelopathies are individually rare. For instance, the prevalence of the most common form of congenital long-QT syndrome, Romano-Ward syndrome (RWS), is close to 1/2,500 live births, when the prevalence of Brugada syndrome is estimated to be around 5/10,000 live births and myotonic dystrophy type 1 (DM1) is the most common muscle disease with a prevalence of 5.5/100,000.


Acquired channelopathies are considerably more common.


It is important to remember that channelopathies are governed by two fundamental concepts. On the one hand, different mutations in the same gene can cause different diseases, this is called phenotypic heterogeneity. On the other hand, mutations in different genes can cause the same apparent disease phenotype, which is called genetic heterogeneity. As the genotype-phenotype correlation cannot be easily defined, an accurate diagnosis for many channelopathies remains difficult and therapeutic treatment is not facilitated.


Therapeutic approaches to ion channel diseases: can channelopathies be cured?


By greatly simplifying, we could classify ion channels as the following, depending on the primary factors that lead to channel opening and closing:

  • Voltage-gated potassium channels, like hERG, Kv1.1, Kv1.4, and Kv1.5
  • Voltage-gated sodium channels like Nav1.2, Nav1.5, and Nav1.7
  • Voltage-gated calcium channels CCDV like Cav1.1, Cav1.2, Cav1.3, Cav1.4, Cav2.1, Cav2.2, Cav2.3, Cav3.1, Cav3.2 and Cav3.3
  • Ligand-gated channels like GABAA, P2X, KATP, SKCa, BKCa, and nAChR
  • Transient receptor potential channels (TRP channels) including TRPA, TRPC, TRPM, TRPML, TRPN, TRPP, TRPS, TRPV, TRPVL, TRPY
  • Mechanosensitive or stretch-activated potassium channels

It is proven that small molecule drugs can modulate these transmembrane receptors. Before 1997, many of these chemical compounds have had great success in the treatment of hypertension, arrhythmia, and pain or used as anaesthetics. As a result, over 6,5 billion dollars in yearly sales were generated by calcium channel antagonists.


On the other hand, some have had major setbacks as certain analgesics to treat chronic pain, such as aryl sulfonamides directed against the voltage-dependent sodium channel Nav1.7.


One revealed major issue that has adversely affected successful translational from preclinical to clinics of small molecules is their drug-like properties like pharmacokinetics (PK), absorption, distribution, metabolism, and excretion (ADME) and toxicology.


Another issue remains the lack of specificity of small molecules for a dedicated ion channel. In fact, several chemical structures are not able to discriminate among ion channel isoforms - for example, the non-functional variant of P2X7 (nfP2X7), the neo-natal splice variant of Nav1.5 (nNav1.5), or isoforms of Kv11.1B that are up-regulated in certain tumors - hence at risk of causing unwanted side effects.


A judicious alternative to chemical molecules and small peptides with short half-life is the monoclonal antibody where high levels of specificity would be expected to mitigate off-target effects, and generate safer and more potent classes of drugs.


So why aren't there more therapeutic antibodies on the market targeting ion channels?


Therapeutic antibodies & channelopathies : about the difficulty in generating antibodies against ion channels


Although the idea of generating therapeutic monoclonal antibodies that block or agonize ion channels, which are membrane proteins, seems to be self-evident, its implementation is more complicated than expected.


Difficult access of antibodies to the most relevant but hidden epitopes of membrane targets


First, like all transmembrane receptors, ion channels are largely embedded in the lipid cell membrane bilayer, leaving only small epitopic regions, their extracellular loops being even smaller than those of GPCRs, which are already known to be difficult to access.


Highly conserved sequences of transmembrane ortholog proteins within different species


Secondly, the sequence homology of inter-species ion channels is at least 70% when the epitopic region can be even 100% conserved! This means that traditional antigens injected to classical mammals through common immunization routes may not allow a breakthrough of immunotolerance to generate robust immunogenicity.


Problems of targeting conformational, discontinuous, and complex epitopes of receptors


Thirdly, the epitope is not necessarily linear which adds complexity to the antigenic design, the targeting of a conformational or discontinuous area, and the screening step to ensure correct recognition of the native protein.


Challenges in membrane protein expression, purification and characterization


Fourth difficulty, transmembrane receptors are generally weakly expressed on healthy cells, overexpressed on diseased tissues, difficult to purify which causes major problems for the access to starting material for immunization and screening campaign.


Complexity of identifying rare antigen-engaged subclustered B cell populations


Another difficulty is that obtaining a binder antibody is no guarantee of obtaining an antibody with therapeutic activating, blocking or depleting effects. The B lymphocyte subpopulations of interest are rare, difficult to access and embedded in numerous plasma cells generating off-target antibodies. If the number of ion channel binders is low, the amount of effector antibodies is infinitesimal.


Considerable lack of tools available for the valorization of antibodies targeting transmembrane receptors


Finally, as if all this were not complicated enough, many ion channels are still poorly described or are orphaned for their ligand. Not knowing the ligand, or being the company that will generate a first-in-class monoclonal antibody means that no tools will be available to do the work of characterizing the antibody and proving its efficacy. Not only does the biotech take the technical risk of a failure to generate the antibody, but also the financial risk of its proof of concept. As far as I know, and I speak only for SynAbs here, this is the critical bottleneck.


How am I going to create the cell line that will verify that my antibody actually activates the signaling? How do I verify that my antibody blocks and competes with the receptor's natural ligand if I don't know what ligand? What in-vivo proof-of-concept animal model will I be able to use if nothing is described in the literature and I'm the only one (well I think...but statistically there is bound to be someone else at world’s end who has the exact same belief, not ?) to work on it?


So many questions that will keep you up at night.