Discovery of Functional Antibodies Targeting Ion Channels

http://jbx.sagepub.com/Journal of Biomolecular Screening

http://jbx.sagepub.com/content/early/2014/12/03/1087057114560698The online version of this article can be found at:

DOI: 10.1177/1087057114560698

published online 3 December 2014J Biomol ScreenTrevor C. I. Wilkinson, Matthew J. Gardener and Wendy A. Williams

Discovery of Functional Antibodies Targeting Ion Channels

Published by:

http://www.sagepublications.com

On behalf of:

Journal of Biomolecular Screening

can be found at:Journal of Biomolecular ScreeningAdditional services and information for

http://jbx.sagepub.com/cgi/alertsEmail Alerts:

http://jbx.sagepub.com/subscriptionsSubscriptions:

http://www.sagepub.com/journalsReprints.navReprints:

http://www.sagepub.com/journalsPermissions.navPermissions:

What is This?

- Dec 3, 2014OnlineFirst Version of Record >>

at TEXAS SOUTHERN UNIVERSITY on December 9, 2014jbx.sagepub.comDownloaded from at TEXAS SOUTHERN UNIVERSITY on December 9, 2014jbx.sagepub.comDownloaded from

http://jbx.sagepub.com/

http://jbx.sagepub.com/content/early/2014/12/03/1087057114560698

http://www.sagepublications.com

http://slas.org

http://jbx.sagepub.com/cgi/alerts

http://jbx.sagepub.com/subscriptions

http://www.sagepub.com/journalsReprints.nav

http://www.sagepub.com/journalsPermissions.nav

http://jbx.sagepub.com/content/early/2014/12/03/1087057114560698.full.pdf

http://online.sagepub.com/site/sphelp/vorhelp.xhtml



Journal of Biomolecular Screening 1 –14© 2014 Society for LaboratoryAutomation and ScreeningDOI: 10.1177/1087057114560698jbx.sagepub.com

Review Article

Introduction

Ion channels form a large family of polytopic membrane proteins that function by allowing passage of ions across cellular membranes. They are present in all cell types, and they play critical roles in biological processes and disease as a consequence of their modulation of functions such as electrical excitability, secretion, cell migration, and gene transcription. Ion channels are widely regarded as attractive targets to exploit by therapeutic drugs and comprise the third best-selling group of prescribed drugs with estimated worldwide sales of $12 billion.1 Despite this success in the use of ion channel modulators, only a few of the estimated 400 annotated ion channel genes predicted in the human genome have been targeted to date.2,3 The majority of the drugs that have been marketed or are under development are small chemical entities with the most notable exceptions being the peptide toxin, ziconotide, and the proanthocyani-din oligomer, crofelemer.4,5 Alternative modalities of target-ing ion channels comprise the use of biological toxins derived from venomous species and the use of biologics such as antibodies.6,7 Despite the attractiveness of targeting ion channels with antibodies due to their high specificity, no antibody-based molecule has been developed to date that has progressed to clinical use. In this article, we will review the current state of ion channel biologics drug discovery

with a particular focus on antibodies. We also highlight the challenges in isolating functional antibodies to these targets and how these challenges are being addressed.

Overview of Ion Channels as Drug Targets

Ion channels are complex polytopic transmembrane proteins that control the transfer of ions across biological membranes via a gated pore. The family of ion channels is involved in a range of diverse biological processes, which include nerve and muscle excitability, sensory and cognitive function, sig-nal transduction, blood pressure regulation, and cell prolifer-ation.2 This wide range of activities and the modulation of ion channel function have translated into links to a variety of pathophysiologies that include cardiac and neurological

560698 JBXXXX10.1177/1087057114560698Journal of Biomolecular ScreeningWilkinson et al.research-article2014

1Antibody Discovery and Protein Engineering, MedImmune, Cambridge, UK

Received Jul 31, 2014, and in revised form Oct 21, 2014. Accepted for publication Oct 22, 2014.

Corresponding Author:Trevor C. I. Wilkinson, MedImmune, Milstein Building, Granta Park, Cambridge, CB21 6GH, United Kingdom. Email: [email protected]

Discovery of Functional Antibodies Targeting Ion Channels

Trevor C. I. Wilkinson1, Matthew J. Gardener1, and Wendy A. Williams1

AbstractIon channels play critical roles in physiology and disease by modulation of cellular functions such as electrical excitability, secretion, cell migration, and gene transcription. Ion channels represent an important target class for drug discovery that has been largely addressed, to date, using small-molecule approaches. A significant opportunity exists to target these channels with antibodies and alternative formats of biologics. Antibodies display high specificity and affinity for their target antigen, and they have the potential to target ion channels very selectively. Nevertheless, isolating antibodies to this target class is challenging due to the difficulties in expression and purification of ion channels in a format suitable for antibody drug discovery in addition to the complexity of screening for function.

In this article, we will review the current state of ion channel biologics discovery and the progress that has been made. We will also highlight the challenges in isolating functional antibodies to these targets and how these challenges may be addressed. Finally, we also illustrate successful approaches to isolating functional monoclonal antibodies targeting ion channels by way of a number of case studies drawn from recent publications.

Keywordsion channels, monoclonal antibodies, screening, antigens

at TEXAS SOUTHERN UNIVERSITY on December 9, 2014jbx.sagepub.comDownloaded from


2 Journal of Biomolecular Screening

disorders, perception of pain, kidney failure, blindness, and oncogenesis.8–15 A large number of channelopathies have also been described in which rare genetic variants give rise to disease that is linked to point or nonsense mutations in ion channels.16 In addition, a number of autoimmune disorders exist in which antibodies are directed to ion channels leading to adverse clinical effects. Examples of such autoimmune disorders include Lambert–Eaton myasthenic syndrome and Isaacs’ syndrome in which autoantibodies target voltage-gated calcium channels and voltage-gated potassium chan-nels, respectively.17–20 Given the importance of ion channels in modulation of cellular function and their role in disease, it is clear that ion channels are recognized as important thera-peutic targets.

Analysis of the human genome shows that several hun-dred genes encode ion channels. The ion channel superfam-ily is classified broadly into two types (voltage-gated or ligand-gated) depending on the factors that control opening and closing of the ion channel pore.21,22 Examples of modu-lation of both channel types by small molecules have been described. This can be exemplified by the action of lido-caine on voltage-gated sodium channels, the action of diaz-epam on the ligand-gated γ-aminobutyric acid (GABA) class A channel, and the action of sulphonylureas such as glipizide on the KATP channel.2

Ion channel drug development throughout the past 50 years has focused on the discovery of small-molecule thera-peutics. The discovery of these drugs ranges from opportu-nistic observations made during in vivo testing of compounds to the use of high-throughput compound screen-ing.2,23 These efforts have given rise to approximately 20 marketed compounds targeting a very limited number of ion channels.2 Although the pace of new drug approvals against this target class slowed in the past decade, two new drugs targeting ion channels have been approved for clinical use in 2012. These new drugs are Ivacaftor (Kalydeco), which targets the cystic fibrosis transmembrane regulator (CFTR) chloride channel, and crofelemer (Fulyzaq), which inhibits both CFTR and the calcium-activated chloride channel TMEM16A.5,24,25 These new drug approvals provide renewed confirmation of the value of ion channel targets and a significant stimulus to the field of ion channel drug discovery.

Given the importance of this target class, a significant opportunity exists to target ion channels with biologics such as antibodies or antibody fragments. Antibodies demon-strate exquisite specificity against their targets, and their affinity and potency against a target can be further opti-mized by protein engineering.26 In addition, the effector function and pharmacokinetics of antibodies can be modu-lated to provide further control of their mode and duration of action.27,28 To date, however, there has been limited prog-ress in identifying antibodies with the appropriate proper-ties to modulate ion channel function in a pharmacologically

relevant way. To some extent, this reflects the challenges in identifying functional antibodies against this complex and diverse target class.

Targeting Ion Channels with Antibody Therapeutics

Antibodies and related molecules represent the fastest-growing class of therapeutic drugs.29 In 2012, 34 approved monoclonal antibodies were described in a compendium of marketed therapeutic antibodies.30 As a result, antibody therapeutics are a well-established drug class, and they ben-efit from a high success rate from their first time in human studies through to regulatory approval. Several features of antibodies make them attractive as potential therapeutics for ion channels that address some of the barriers to small-molecule drug discovery such as difficulties in achieving specificity to highly homologous ion channel subtypes. Antibodies typically bind to their target antigens with very high specificity allowing them to be used for targeted thera-pies. Antibodies can also be generated that have high antigen-binding affinities in the nanomolar to subpicomolar range, and options exist for optimizing antibody affinity via pro-tein-engineering strategies targeting the antibody para-tope.27,31 This affinity maturation typically leads to large reductions in the dissociation rate of the antibody and may also lead to increased biological potency against a target.27 These features of antibodies reside in the variable-domain regions of antibodies.

In addition to the variable domains of antibodies mediat-ing binding to target antigens, features of the antibody con-stant domains affect the effector function of antibodies (e.g., antibody-dependent cellular cytoxicity) and also the pharmacokinetics exhibited with antibodies typically hav-ing a long plasma half-life (several weeks) allowing for reduced frequency of administration.28 These properties of the constant region are also amenable to protein engineer-ing, and it is possible to “tune” the properties of antibodies to increase or decrease effector functions such as antibody-dependent cellular cytotoxicity and enhance the half-life of antibodies to affect dosing frequency and duration of action. One other feature of antibodies is that their distribution is peripherally restricted, which lends itself to targeting ion channels in which the functional effect needs to be limited to the periphery to avoid CNS side effects. In contrast, tech-nologies are emerging by which antibodies can be modified and targeted to cross the blood–brain barrier, which holds the promise of being able to devise strategies to target ion channels in the CNS.32–34

A variety of alternative antibody formats also exist and add to the options for targeting ion channels. Although not specifically the focus of this review, scFv’s, Fab frag-ments,35,36 nanobodies,37,38 and a variety of alternative non-antibody scaffolds39,40 have been described that all have



Wilkinson et al. 3

potential to target ion channels. Alternative non-antibody scaffolds and nanobodies are smaller in size than intact immunoglobulin G (IgG) molecules, and these smaller binding protein formats may be useful to target small epit-opes and binding pockets on ion channels that may not be accessible to full IgGs. These formats also allow multiple different epitopes to be targeted by linking two or more binding proteins together as a fusion protein. This strategy has been described by Ablynx in conference proceedings (Collaborations in Ion Channel Drug Discovery, Cambridge, UK, 2014) in which variable domains of heavy-chain-only camelid antibodies (nanobodies) have been isolated that show function against the Kv1.3 ion channel when com-bined in a multivalent format.41

Challenges in Identifying Functional Antibodies to Ion Channels

Although small-molecule approaches have been successful in delivering marketed therapeutic drugs targeting ion chan-nels, targeting ion channels with functional antibodies offers significant opportunities. Generating antibodies against these integral membrane proteins does, however, pose some challenges. Ion channels comprise a very diverse collection of structural and functional types with selected examples shown schematically in Figure 1. Ligand-gated

ion channels feature large extracellular domains that have defined ligand-binding pockets to allow for activation of the channel following ligand binding.42,43 In principle, these large extracellular domains are accessible for antibody binding, and examples of antibodies targeting these ion channels have been described.44 Identifying antibodies that target voltage-gated ion channels may be more challenging particularly because the extracellular loops of these chan-nels are typically small and therefore less accessible to anti-bodies. In addition to these structural considerations, a number of general features of ion channels linked to the difficulty of expressing and manipulating ion channels and the complexities of functional screening also add complex-ity to the isolation and characterization of antibodies. These challenges are described in the following sections.

Antigen and Immunogen Formats

Identification of functional antibodies directed to specific targets fundamentally relies on the selection of a suitable antigen and its presentation to elicit antibody generation.45,46 The aim of immunization methodologies is to present an antigen to the host in a form that stimulates a strong and specific immune response. To isolate antibodies from tech-nologies such as phage display, the aim is to present a pure antigen to allow selection of a diverse panel of binding and

Figure 1. Illustration of the diversity of transmembrane topologies of voltage-gated and ligand-gated ion channels. Representations of three types of ion channel to demonstrate their diverse structures and the relative differences in extracellular loops and domains that could be targeted by antibodies. Shown here are two ligand-gated ion channels [AMPA receptor (AMPAR) and P2X receptor] and a voltage-gated calcium channel (VGCC). Structures were generated by PyMOL software using information in the Protein Data Bank [AMPAR 3KG2,106 P2X receptor 3I5D,42 VGCC 4MTO,107 and immunoglobulin G (IgG) 1IGT108]. The ligand-gated ion channels both have large extracellular domains that bind agonist ligands. The voltage-gated ion channels typically have four homologous domains that are arranged around a central pore. Voltage-gated ion channels have relatively small extracellular loops in comparison to the ligand-gated ion channels represented here. A structure of an IgG antibody is shown approximately to scale.




functional antibodies. Although the precise format of the antigen will affect the selection and identification of bind-ing antibodies, the production of antigens for soluble pro-teins such as cytokines, growth factors, protein subdomains, and globular extracellular domains of receptors is relatively straightforward.47 A plethora of methods exist for the expression and preparation of these antigens in a pure, well-characterized, and correctly folded state.

By contrast, methods for the expression and purification of ion channel antigens face a number of challenges that are linked to the complex topology of ion channels and the inti-mate association with the membrane lipid bilayer. In the fol-lowing sections, we will highlight some of the challenges and strategies that may be used to overcome the challenges.

Synthetic peptides represent the simplest antigen format that can be used for both immunizations and selection-based antibody discovery strategies.48 Typically, peptides require conjugation to a carrier protein such as keyhole lim-pet hemocyanin or bovine serum albumin prior to use as an immunogen. Peptides may be used as surrogates for pro-teins and result in a highly specific targeted epitope antigen. Peptides may be used in a linear format, or more complex peptides may be synthesized when chemical methods are used to constrain the conformation of single or multiple peptides as defined loops. Examples of the use of chemi-cally constrained, more native-like peptides exist for gener-ating antibodies targeting G-protein coupled receptors (GPCRs). GPCRs exhibit similar issues to ion channels with respect to the challenges faced in isolating functional antibodies. Biotinylated cyclic peptides have been used to isolate functional scFv antibodies to CCR5 by phage dis-play methods.49,50 Functional antibodies to CXCR7 have also been reported to have been raised using CLiPS (chemi-cal linkage of peptides to scaffolds) peptide technology that is marketed by Pepscan.51 Generation of functional antibod-ies to GPCRs using constrained peptides suggests that this type of method could be extended to ion channels. The abil-ity to modify peptides by chemical methods to introduce moieties such as biotin facilitates phage display panning methods and detection methods by allowing capture onto beads or solid-phase surfaces coated with streptavidin.48

A particularly successful strategy for obtaining poly-clonal antibodies to ion channels has been the use of pep-tides derived from the E3 pore loop region of a variety of channels.52 Several channel classes, including voltage-gated ion channels (with Na, K, and Ca selectivities) and the tran-sient receptor potential (TRP) channels, exhibit a common structural theme of six transmembrane spanning helices (S1–S6) per subunit with intracellular amino and carboxyl termini and three extracellular loops (E1–E3). The E3 loop is implicated in the ion selectivity filter for ion channels, and because it is typically relatively long, it lends itself to being an accessible epitope for antibodies (Fig. 2). Using this loop as an immunogen, antibodies have been generated

to Nav1.5, Cav2.1/2, Kv1.2, Kv3.1, Eag1, TRPC1, TRPC5, TRPM3, and TRPV1.53–55 It is noteworthy that the majority of antibodies generated using E3 loop antigens have been polyclonal antibodies, but they have nevertheless shown functionality in blocking channel function, albeit in a partial manner. In one case (TRPV1), both polyclonal and mono-clonal antibodies were isolated with only the polyclonal antibodies showing activity.55 Recent studies, however, have demonstrated that monoclonal antibodies targeting the E3 loop can be effective antagonists. A functional monoclo-nal antibody against Eag1 has been generated by using the E3 loop strategy, and this example is exemplified as one of the case studies in this review.54 More recently, Miller et al.109 explored the feasibility of blocking the transient receptor channel TRPM8 by evaluating the functional effects of commercially available polyclonal and monoclo-nal antibodies raised against E3 loop peptides. In this study, a polyclonal antibody (ACC-049; Alomone Laboratories, Jerusalem, Israel) and a monoclonal antibody (MBS609041; My Biosource, San Diego, CA) were reported to completely inhibit ligand activation of human TRPM8. Although the authors stated that the potencies of the antibodies were not sufficient for therapeutic consideration, this study supports the feasibility of generating monoclonal antibodies target-ing the pore loop that block channel activation.

As an alternative to using peptides as immunogens, whole cells expressing ion channels of interest can be used for immunizations and in vitro display technologies.44 The use of cells in this way is affected by some general challenges raised

S1 S2 S3 S4 S5 S6

+

+

E3 loop

Figure 2. Schematic representation of the functional unit of a voltage-gated ion channel. Transmembrane folding topology of a single domain of the voltage-gated Nav or Kv ion channel, illustrating the position of the E3 extracellular loop. Sodium channels are single polypeptides in which four repeated domains, each with six transmembrane helices, are arrayed around a single ion-conducting pore. Potassium channels with a similar six-transmembrane topology contain four identical subunits that assemble to form a pore. A similar topology is exhibited by transient receptor potential channels such as TRPV1.



Wilkinson et al. 5

by expressing ion channels. Ion channels are integral mem-brane proteins and are often found in low abundance. Family members within specific ion channel classes often exhibit high sequence identities. Overexpression of ion channels can be difficult to achieve because methods for monitoring or selecting for high expression are compromised by the lack of effective tool antibodies to check for expression and the difficulty of assaying for channel function with relatively low-throughput electrophysiology methods. High-level expression of ion channels can also be difficult because the overexpression can sometimes result in toxicity to cells, pre-cluding the generation of stable cell lines.56 It is possible to address some of these concerns by using methods that enhance detection of ion channels or that result in transient, regulated expression of the ion channels. To improve detec-tion of ion channels, a wide range of strategies have been used to introduce epitope tags into ion channel sequences to facilitate detection using methods such as flow cytometry. Epitope tags including, but not limited to, Flag, hemaggluti-nin (HA), V5 epitope, 6-His, and c-myc tag have been used to facilitate ion channel detection.57 Green fluorescent pro-tein (GFP) has also gained significant utility in tagging ion channels to monitor expression in cells and to facilitate ion channel purification.58–60 The use of epitope tags has been comprehensively reviewed by Maue.57 Controlling the tim-ing of gene expression is another method that can be used to allow overexpression and reduce the issues associated with channels that cause toxicity. In this approach, the ion channel gene of interest is placed in a vector that allows for inducible promoter expression by administration of agents such as tet-racycline.61 A number of commercial products are available for controlling gene expression in mammalian cells that include the T-Rex, Tet-On 3G, and Cumate inducible sys-tems.61–63 Finally, it is also possible to prepare significant quantities of cells by transient transfection, which may also assist high-level expression. This can be done using polyca-tion or lipid-based transfection reagents or by the use of sys-tems such as the Maxcyte electroporation system that allow electroporation of gene expression vectors into large batches of cells.64

In addition to considering methods to achieve overex-pression of ion channels, the cell host chosen for expression also represents a significant factor. Ion channels may be expressed in a variety of cell types (e.g., HEK293, CHO, and U2OS). In each case, subsequent screening on cells for isolated antibodies will require both the parental host cell and the overexpressing cell line. When using host cells expressing ion channels as immunogens, an immune response will also be mounted to other proteins in the host cell. To maximize the opportunity for a specific antibody response to the target of interest, human target genes can be overexpressed on a host cell that is syngeneic to the immu-nization host strain (e.g., BALB/C mouse).44 In this case, a significantly reduced background immune response would

be expected with an improved response to the target of interest and increased yield of monoclonal antibodies against the target protein.

If overexpression of ion channel genes can be achieved in cells, another potential option is to generate virus-like parti-cles (VLPs) as an immunogen.65,66 In this approach, cells overexpressing the channel are transfected with vectors encoding a viral protein such as Gag, which causes budding of the cell membrane with the release of VLPs. These small particles are often enriched for the gene of interest, compared with the host cell, and can be used to support both immuniza-tion and phage display antibody isolation methods.67 Recent conference reports indicate that this approach has been suc-cessful in the isolation of antibodies against ion channels with functional antibodies to P2X3 reported by Integral Molecular and Crystal Biosciences.67

Purified, correctly folded, and functional protein pro-vides one of the best and most versatile of antigens for gen-erating functional antibodies. Purified protein can be used to support immunization strategies as well as support isola-tion of antibodies by in vitro display methods. In the case of ion channels, significant challenges are posed by the inte-gral membrane location of the channels and the requirement for extraction and purification of the proteins in detergents. Solubilization of membrane proteins by detergents typically requires screening of multiple factors to extract functional protein and can often result in protein instability, unfolding, and aggregation that are all detrimental to production of quality antigen. It is clear that progress is being made in the purification of ion channel proteins as demonstrated by the increasing number of crystal structures of these integral membrane proteins, but success still requires a diverse array of methods to achieve well-behaved, correctly folded, sta-ble protein.42,68 Methods to achieve this include using spe-cies homologues of human proteins and engineered proteins that effectively “screen” for proteins with amino acid sequences that have well-defined behavior in detergents. Examples of this include the use of bacterial homologues of potassium channels68 and species variants of P2X chan-nels.42 Although this demonstrates that ion channel purifica-tion is feasible, it does not solve the issue that human proteins are required to support therapeutic antibody dis-covery. Other features of detergent extraction that may affect antibody isolation include the fact that longer-chain detergents typically required for stabilization of membrane proteins may mask critical epitopes. Purified proteins will also have both intracellular and extracellular loops avail-able, which may result in antibodies being isolated to intra-cellular epitopes.

One other approach that may improve our ability to purify ion channels is the use of mutagenesis to create sta-bilized ion channel proteins as has been demonstrated in the case of GPCRs and transporters.69–71 The use of a stabilized receptor protein for functional antibody generation has been




validated for the β1-adrenergic receptor.72 This approach appears feasible for ion channels, but in some cases (e.g., mammalian voltage-gated sodium channels), the large size of the protein makes the sequence space that needs to be explored challenging.

Given the challenges described in expression and purifi-cation of these complex antigens, an alternative strategy for antibody isolation involves DNA immunization.73 In this approach, expression plasmids encoding a particular gene of interest are used for immunization, and DNA is delivered by injection or via a gene gun into a suitable host.73,74 This method would typically require that a check is made prior to immunization that the plasmid supports gene expression, and there is still a requirement to have purified antigen, high-expressing cell lines, or functional assays for the ion channel of interest to allow effective screening for binders and functional antibodies. Improved methods for DNA immunization have been described recently, leading to suc-cessful antibody isolation against a membrane transporter, which is a target with similar challenges to ion channels. This report described the use of hydrodynamic tail vein injection of DNA encoding the transporter gene in conjunc-tion with immune modulators.75 This method shows great promise for complex membrane proteins such as ion chan-nels. In a further extension of this DNA immunization strat-egy, it is possible to also use viruses encoding the ion channel as immunogens. This particular approach has shown some success using adenovirus, although it is not entirely clear in the published work which of several strate-gies led to the final functional monoclonal antibody.76 Examples of strategies that have been used to isolate func-tional monoclonal antibodies described in the public domain are described in the case studies in this review.

Challenges Facing Ion Channel Biologics Screening

The field of ion channel screening for biologics is still in its infancy with a limited number of successful campaigns resulting in examples of functional antibodies against ion channels.77 In general, identifying antibodies against ion channels can be achieved through a variety of approaches, and the range of commercial antibodies is increasing rap-idly, highlighting the feasibility of identifying binding non-functional and polyclonal antibodies to ion channels.78 Identification of functionally active antagonist or agonist antibodies is more challenging and requires the use of screening assays that add additional complexity along with the requirement for investment in technology and expertise. Historically, there is a high attrition between identifying binding antibodies and those exhibiting functional activity as evidenced by the limited number of functional antibodies reported in Table 1. To reduce this attrition, many antibody screening campaigns implement functional assays as a first

screen, and this can be successful for cell surface targets such as GPCRs, in which difficulties such as antibody expression, media tolerance, and antibody potency can be overcome.79

Outputs from phage display selection using naïve anti-body libraries typically require expression of scFv in Escherichia coli before screening that leads to variable anti-body concentration in bacterial lysates or periplasmic extracts.35 Buffers used in the isolation of these samples may affect the ability to assay ion channel activity due to nonspecific interference by buffer components, ionic con-centration, and osmolarity, which are key factors in regulat-ing ion channel function and cell volume. To avoid these challenges, direct binding assays are often implemented as a first-line screen to identify antibodies specific for the ion channel of interest, reducing the number taken forward for functional screening. Initial screens using formats such as ELISA (using immobilized peptides or purified proteins) can provide a high concentration of immobilized antigen and facilitate the identification of binding antibodies as reported by Lee at al.80 and Gómez-Varela et al.54 The con-formational changes that can occur during immobilization are unknown, however, and this could lead to a bias in the epitopes being presented that potentially affects the diver-sity of the antibody panel selected and hence their func-tional characteristics. Other options for initial screening by ELISA include immobilizing liposome vesicles containing the protein of interest, which may overcome some of the conformational changes seen when immobilizing directly onto a plastic substrate.81

Ideally, cell-based binding assays can be implemented in which the ion channel is more likely to be in a native, active conformation due to its location in the plasma membrane. This format requires cell lines that either stably express the target of interest or can be induced to express the target fol-lowing the activation of an inducible promoter.82 Typically, it has been challenging to achieve high copy numbers of ion channels because overexpression of the channel can be det-rimental to cell health, possibly as a result of compromised membrane integrity. Poor cell viability in a screening assay will increase identification of false positives due to nonspe-cific binding and uptake of fluorophore labels into the trans-fected cells. Therefore, the ion channel expression level needs to be optimized to maintain cell health and to allow detection of relatively low-affinity clones in phage libraries. The benefit, however, of cell-binding assays is that they are normally more tolerant to sample media and can provide significant sensitivity.83 Once antibodies have been identi-fied by this process, they need to be tested for function in a relevant assay. Due to the issues mentioned above regarding assay interference, the antibodies are purified and prepared in a buffer compatible with downstream functional assays.

Patch clamp electrophysiology remains the gold standard for measurement of the effects of molecules on ion channel



Wilkinson et al. 7

function. The classic manual patch clamp (see Neher and Sakmann84 and Neher et al.85) has too low a throughput for meaningful drug discovery efforts, and whereas there are non–patch clamp high-throughput screening assay technolo-gies such as fluorescent imaging plate reader assays, which use either membrane potential or voltage-sensitive dyes, these do not measure the effects of modulators directly on the ion channel. There are limited options to screen ion channels in other assay formats, and the development of reporter assays, internalization assays, and downstream readouts that incorporate signal amplification is still lacking for ion

channel targets. Small-molecule screening has historically required the testing of extensive chemical libraries in excess of 1×106 compounds, and it has necessitated higher-through-put screening approaches. Biologics screening, specifically the identification of monoclonal antibodies that modulate ion channels, uses far smaller subsets of molecules because the original libraries are essentially screened by phage display followed by standard binding assays, resulting in greatly reduced numbers of screening samples. This allows the direct screening of all binding monoclonal antibodies with auto-mated patch clamp platforms described below.

Table 1. Key Information on Published Functional Monoclonal Antibodies That Form the Basis of the Case Studies Described in This Review.

Target Channel Type ReferenceMethod of Antibody

Generation ImmunogenScreening Methods

Number of Functional Antibodies Reported

P2X7 Ligand-gated Buell et al. (1998)44

Mouse immunization and hybridoma generation

Cells expressing P2X7

Cell-binding assays, whole-cell patch clamp, and recognition of native P2X7

1

Eag-1 Voltage- gated Gómez-Varela et al. (2007)54


Fusion protein containing two domains: residues 374 to 452, including the E3 segment, and residues 872 to 932, including the tetramerizing coiled-coil

ELISA and SPR, and whole-cell patch clamp

1

Orai1 Calcium release-activated channel

Lin et al. (2013)95 Mouse immunization and hybridoma generation

Cells overexpressing Orai1

Cell-binding assays, store-operated calcium influx, and NFAT-dependent luciferase activity

3

Orai1 Calcium release-activated channel

Cox et al. (2013)96


Peptide corresponding to second extracellular loop fused with BSA

ELISA and cell-binding assays, calcium flux, Orai1 internalization, and T-cell proliferation

1

TrpA1 Transient receptor potential channel

Lee et al. (2014)76


Cells, plasmid and recombinant adenoviral vector expressing TRPA1

Cell-binding and radioactive calcium uptake assay

2

Nav1.7 Voltage- gated Lee JH et al. (2014)80


Peptide corresponding to S3–S4 loop of domain II

ELISA using purified sensor domain protein and whole-cell patch clamp

1

BSA, bovine serum albumin; ELISA, enzyme-linked immunosorbent assay; NFAT, nuclear factor of activated T cells; SPR, surface plasmon resonance.




There are multiple automated patch clamp systems avail-able, ranging from full 384-well screening platforms down to simplified manual patch clamp equipment, and these have been recently reviewed (see Dunlop et al.86 and Trevedi et al.87). Screening for functional antibodies against ion chan-nels has a unique set of issues to overcome, including buffer tolerance (typically overcome by purification) and incubation times required to see an effect. Monoclonal antibodies likely exert a modulatory effect via allosteric mechanisms on ligand-gated ion channels due to their size and lack of access to the ligand-binding site as well as via inhibition of ion channel domain movement on activation. To address these issues, the ability to perform baseline readings followed by prolonged incubation times before a test stimulus is applied as a prerequi-site for screening. This requirement moves antibody screen-ing toward lower-throughput systems such as QPatch and PatchXpress, which offer more control over the experimental protocols than higher-throughput systems such as the Ionworks Quattro. The introduction of higher-throughput automated patch clamp platforms such as the Ionworks Barracuda (Molecular Devices), SyncroPatch 384PE (Nanion), and Qube (Sophion) allows more detailed 384-well patch clamp assays to be performed. The Ionworks Barracuda has been validated for screening of focused small-molecule compound libraries (in the range of 20,000–100,000 compounds) against voltage-gated calcium channels.88 Such studies show there is a signifi-cant opportunity to use these automated patch clamp platforms for biologics screening, and time will tell if they are suitable for screening and identification of functional antibodies directed against ion channels.

As the number of successful campaigns increases, so will our knowledge regarding the best technologies to use and the most sensitive assays to implement for each type of ion chan-nel. Until that time, the assays used for biologics screening for ion channel targets will require a mixture of assays from stan-dard antibody isolation methods to any cell surface target (reviewed by Cariuk et al.89) and the use of assays previously developed for screening large small-molecule libraries (reviewed by McManus96 and Falconer and Smith91). This, however, can lead to assumptions regarding how antibodies may modulate ion channels, and it could lead to screening cam-paigns that are biased to certain mechanisms or, worse still, assays not sensitive enough to identify any antibody modula-tors. Nevertheless, the case studies described in the following section demonstrate that progress is being made in addressing screening methods to identify functional antibodies.

Case Studies of Isolation of Functional Monoclonal Antibodies to Ion Channels

Despite the challenges outlined in previous sections to iso-late antibodies against ion channel targets, there have been some significant advances in the past decade. In this

section, we describe a number of case studies taken from published literature that highlight advances in the field. Table 1 summarizes key information from these publica-tions, and more detailed descriptions follow below.

P2X7

P2X receptors are ligand-gated ion channels that are acti-vated by extracellular adenosine triphosphate (ATP). Activation results in opening of a cation channel that allows entry of calcium and depolarization of the cell membrane.92 P2X7 receptors have been implicated in the immune response with P2X7 upregulation on macrophages and microglial cells following lipopolysaccharide or interferon-γ stimulation.

Buell et al.44 successfully isolated a functional monoclo-nal antibody directed to P2X7. This was achieved by immu-nization of mice with a mouse myeloma cell line (XS63) stably transfected with human P2X7. Following hybridoma generation, screening for binding antibodies was performed by flow cytometry using XS63 cells expressing P2X7 and a parental control XS63 cell line to check for specificity of binding to P2X7. The specificity of the binding was also confirmed by assessing binding to HEK293 cells express-ing P2X1, P2X4, and P2X7. The monoclonal IgG2b anti-body isolated was specific for P2X7 in these assays. The antibody was also shown to bind endogenous P2X7 on human monocytes and macrophages. Further studies dem-onstrated that the antibody was functional and was shown to inhibit ATP-stimulated release of interleukin 1β (IL-1β) from THP-1 cells. Interestingly, the monoclonal antibody was able to immunoprecipitate P2X7 from XS63 cells over-expressing the receptor, whereas the antibody was nonfunc-tional in western blotting. This suggests that the antibody recognizes a conformational epitope.

Eag-1

Potassium channels are increasingly thought to play an important role in cellular processes such as excitability, cell cycle progression, and cellular proliferation. Human Eag 1 is a voltage-gated potassium channel modulated throughout the cell cycle, and it is believed to be involved in tumori-genesis.13 Observations linking high expression levels of Eag1 in tumor tissues have led to the suggestion that Eag1 is involved in progression of malignant disease and may represent a valid target for therapeutic intervention.

The high sequence homology between voltage-gated potassium channel subtypes presents a significant hurdle in the development of selective inhibitors. In the case of Eag1, the closest family member is Eag2, which has 73% sequence identity with Eag1. Specificity is particularly important for Eag because it is related to the human ether-à-go-go related channel (hERG), whose inhibition is linked to severe and



Wilkinson et al. 9

dangerous adverse cardiac events.93 Given this background, monoclonal antibodies targeting the Eag1 channel offer the potential for highly specific binding.

Gómez-Varela et al.54 isolated a monoclonal antibody to Eag1 by immunizing mice with a fusion protein composed of the linker between the S5–S6 transmembrane segments (E3 loop, residues 374–452) fused to the C-terminal tetra-merization domain of the channel (residues 872–932). This strategy was performed to mimic the native tetrameric state of the potassium channels. Following immunization and hybridoma generation, screening was performed using a sequence of steps. First, an ELISA was used to confirm binding of antibodies to the recombinant antigen followed by surface plasmon resonance analysis to confirm those antibodies binding to the E3 loop and those binding selec-tively to Eag1 over Eag2. Function was then confirmed by testing the effect of the antibodies on Eag1 currents in Xenopus oocytes. This gave rise to a single monoclonal antibody named mAb56.

Further analysis of mAb56 showed that it recognized a linear epitope (GSGSGKWEG), and the antibody was able to bind to HEK293 and CHO cells expressing Eag1. This bind-ing could be inhibited by co-incubation of the synthetic pep-tide epitope. The antibody was also able to inhibit Eag1 currents in HEK cells expressing the channel by ~40%, and mAb56 did not inhibit hERG currents demonstrating selec-tivity. The authors also demonstrated that mAb56 was able to inhibit anchorage-independent cancer cell growth in vitro.

Orai1

Calcium release-activated calcium channels are activated in response to the depletion of intracellular calcium stores in the endoplasmic reticulum (ER). The channel has two compo-nents: stromal interaction molecule 1 (STIM1), which acts as a calcium store sensor in the ER, and Orai1, which is a pore-forming protein subunit located in the plasma membrane.94 These two proteins are responsible for store-operated cal-cium influx, which is responsible for refilling intracellular calcium stores following cell activation involving release of calcium as a second messenger. STIM1 acts as a calcium sen-sor detecting reduced calcium concentration in the ER, which subsequently activates Orai1 in the plasma membrane result-ing in calcium influx. Orai1 and STIM1 are present in T-cells, and the calcium entry following antigen stimulation of T-cells is critical for NFAT-mediated T-cell activation. Human muta-tions in STIM1 and Orai1 that affect protein expression or function give rise to a hereditary form of severe combined immunodeficiency syndrome.94 Autoreactive T-cells play a key role in autoimmune diseases such as rheumatoid arthritis, and this observation highlights Orai1 as a potential target for therapeutics that inhibit autoimmune disease and chronic inflammation as well as for potential therapeutics for use in organ transplantation. Although small-molecule antagonists

have been developed and studied, none have advanced to therapeutic use. This resulted in Lin et al.95 and Cox et al.96 pursuing strategies to develop monoclonal antibodies as alternative antagonists of Orai1 function.

Lin et al.95 obtained fully human antibodies targeted to Orai1 by immunization of the Xenomouse with U2OS cells overexpressing human Orai1. The Xenomouse is a trans-genic system in which human immunoglobulin loci are introduced into the germ line of mice.97 One hundred and two fully human antibodies were isolated that showed bind-ing to CHO-AM1 cells stably overexpressing both human Orai1 and a YFP-tagged STIM1. From these antibodies, 11 unique monoclonal antibodies were expressed and purified for more detailed study. Antibody 2C1.1 was studied in some detail and was shown to bind to Orai1 expressed in HEK293 cells and also to endogenously expressed Orai1 in Jurkat cells by flow cytometry. A total of four antibodies were described (2C1.1, 2D2.1, 2B7.1, and 5F7.1), which were all shown to bind Orai1 with high affinity by the KinExA instrument with dissociation constants (Kd) of ~20–100 pMolar. The antibodies isolated were unable to bind to rat and mouse Orai1, and the authors used this infor-mation to suggest putative epitope sites for the antibodies. Extracellular loop 2 (ECL2) shows only 60% identity between human and rat/mouse Orai1 species variants, sug-gesting this may be the epitope location. Human/Mouse chimeric proteins were constructed in which the ECL2 of human Orai1 was replaced with the mouse ECL2 and a reverse construct was made with Mouse Orai1 containing the human Orai1 ECL2. Binding studies on cells overex-pressing the chimeras showed that the monoclonal antibod-ies were unable to bind to the Human Orai1 containing mouse ECL2 and that antibodies did bind to Mouse Orai1 containing the human ECL2. These results confirmed ECL2 as the binding epitope for the Orai1 antibodies. Further studies based on site-directed mutagenesis further defined the epitope to the region of amino acid residues 210–217, with some subtle differences in the precise epitope targeted by the four antibodies.

Subsequent studies demonstrated that the antibodies showed inhibition of Orai1 function. This was demonstrated by dose-dependent inhibition of cytokine release from thapsigargin-treated whole blood (2D2.1, 2C1.1, and 2B7.1) and dose-dependent inhibition by the 2C1.1 antibody of IL-17, IL-4, and IL-10 release from phorbol myristate ace-tate and ionomycin-stimulated whole blood. Antibody 2C1.1 was also shown to inhibit Orai1 current measured by PatchXpress recordings in HEK293 cells stably expressing Orai1 and STIM1. It is noteworthy that the antibody only caused partial inhibition of currents in this experimental format.

Cox et al.96 adopted a more direct strategy to target the extracellular loops of Orai1 by using peptides spanning the first and second extracellular loops to immunize mice.




Although both peptides gave rise to immune responses as detected by peptide ELISA, only ECL2 gave immune titers that recognized native Orai1 expressed on cells. A single Orai1 monoclonal antibody (10F8) was identified following hybridoma generation and was characterized in more detail. 10F8 was shown to bind to Orai1 overexpressed in a Ba/F3 host cell but did not bind to Ba/F3 cells expressing the related family members Orai2 and Orai3. The antibody was also able to bind to endogenous Orai1 expressed in Jurkat cells, and this was confirmed by reduced binding of the antibody to cells in which the Orai1 gene was knocked down using short hairpin RNA interference.

Further studies with the 10F8 monoclonal antibody showed that thapsigargin-induced calcium flux was specifi-cally inhibited, confirming the antibody was functional. Interestingly, the authors also demonstrated that antibody-mediated internalization of Orai1 contributes to the func-tional inhibition observed and 10F8 induced internalization of Orai1 in primary T-cells. The antibody was also shown to inhibit T-cell proliferation and inhibited IL-2 and interferon (IFN)-γ production in both αCD3/αCD28-stimulated peripheral-blood mononuclear cells from healthy donors and stimulated synovial-fluid mononuclear cells obtained from rheumatoid arthritis patients. These studies were extended to demonstrate that antibody 10F8 was efficacious in vivo in a human T-cell-mediated graft-versus-host dis-ease mouse model (GvHD). This was demonstrated by a significant delay in time to and incidence of GvHD mea-sured by weight loss, reduction in CD4+ and CD8+ T-cell numbers, and reduced IFN-γ plasma levels compared to iso-type controls.

These two studies both indicate the importance of ECL2 as a target epitope to generate functional monoclonal antibodies and illustrate that these antibodies can be successfully obtained either by strategies using whole-cell immunizations or via direct use of ECL2-derived peptides. An important feature of both studies was the ability to screen for binding to native Orai1 on cells stably expressing Orai1. A common theme that also emerges from the studies is that the monoclonal antibodies obtained only recognized the human Orai1 with no observed cross-reactivity to rodent Orai1. This illustrates an important consideration for the drug discovery process because the lack of cross-reactivity will affect the ability to conduct experimen-tal in vivo studies for efficacy. In this case, it is known that the human Orai1 ECL2 has relatively poor sequence identity to rodent Orai1 ECL2 (60%), and this makes obtaining cross-species recognition with a monoclonal antibody challenging.

TRPA1

The transient receptor potential ankyrin 1 (TRPA1) channel is a nonselective cation channel that has been implicated in pathophysiologies that include asthma, cough, itch, and inflammatory pain.98 Agonists of the channel include mol-ecules such as mustard oil and allicin, which cause pain and

pain-related behavior. TRPA1 antagonists are therefore being pursued as potential therapeutics.

Lee et al.76 recently described the isolation of antagonist monoclonal antibodies that inhibit activation of TRPA1 by a variety of stimulants including chemical irritants, cold, and osmotic effects. The strategy used to isolate antibodies involved immunization of mouse strains with cells, plasmids, and adenoviruses expressing TRPA1 followed by generation of hybridomas and screening of conditioned media from hybridoma lines to identify mAbs showing selective bind-ing to U2OS cells overexpressing TRPA1. The TRPA1-overexpressing cells were isolated by inserting a FLAG tag into the predicted extracellular loop 1 of TRPA1, which enabled fluorescent cell sorting to be used to enrich for high-expressing cells. These cells were also checked to confirm that the insertion of the epitope tag did not affect ion channel activ-ity detected by 45Ca influx. Sixteen antibodies were identified that showed greater than twofold specific binding to TRPA1 U2OS cells compared to parental control cells. Purified anti-bodies were then tested for binding to a second cell line (CHO) expressing TRPA1 and were also tested for their ability to inhibit allyl isothiocyanate (AITC)-stimulated 45Ca uptake. Two hybridoma lines (2B10 and 2D1) were identified that showed greater than 50% inhibition of AITC-induced TRPA1 activation, and the antibodies were subcloned to confirm monoclonality. These antibodies were further tested and shown to cause concentration-related inhibition of AITC-induced 45Ca uptake, cold-induced 45Ca uptake, and osmotic pressure–induced 45Ca uptake. Although detailed epitope mapping was not done, the authors suggested that the antibodies were target-ing ECL3 (which is part of the pore) based on considerations of activity against both human and rat TRPA1 and sequence identities in the loop 3 region.

This successful isolation of functional antibodies can be attributed to the immunogenicity of the pore loop sequence and the very effective screening strategy using direct bind-ing assays to cells (with the added benefit of a positive con-trol due to the FLAG tag) and subsequent activity assays. Interestingly, the authors do state that the potency of the antibodies is too weak to consider for therapeutic applica-tions, and it is also noteworthy that the antibodies are unable to completely block the channel unlike a small-molecule inhibitor, AMG9090.

Nav 1.7

Voltage-gated sodium channels (Nav) are responsible for action potential initiation and propagation in excitable cells with nine highly homologous subtypes encoded by the human genome.99,100 The pore-forming unit of Nav channels consists of a single polypeptide with four non-identical but homolo-gous repeat domains (domains I–IV). Each repeat consists of six transmembrane helices (S1–S6) with the first four helices (S1–S4) acting as a voltage sensor domain (VSD) and the S5–S6 segments assembling together as a tetramer in the



Wilkinson et al. 11

membrane to form the ion-conducting pore.103 In the VSD, S4 contains arginine residues that sense membrane potential changes and together with S3 form a unit known as the volt-age sensor paddle, which moves in response to membrane potential changes and is coupled to pore opening and closing. This voltage sensor paddle has been the target of a number of natural peptide toxins isolated from venomous species of spi-ders and scorpions that cause potent inhibition of Nav chan-nels.104,105 Each subtype is implicated in various physiological and disease processes.

Nav1.7 has generated particular interest because loss-of-function mutations in SCN9A, the gene encoding Nav1.7 in humans, lead to congenital inability to sense pain without affecting other sensory pathways.99,101 There are also gain-of-function mutations in SCN9A that lead to episodic pain dis-orders such as erythromelalgia and paroxysmal extreme pain disorder.102 Together, these observations support Nav1.7 as a potential analgesic target for a variety of pain disorders.

In 2014, Lee at al.80 described the first monoclonal antibody targeting Nav1.7. This seminal paper describes the use of an elegantly simple strategy to isolate the functional antibody. Isolation of a monoclonal antibody against the channel was achieved by immunizing mice with a peptide (VELFLADVEG) that corresponds to the loop between helices S3 and S4 in domain II. Thus, the antibody response was directed to the voltage sensor paddle of the channel. The hybridomas derived from the mice were screened to identify antibodies that bind to the recombinant domain II VSD. A single functional IgG1 (SVmab1) was obtained by this approach. A negative control monoclonal antibody (CTmab) was raised by targeting the peptide sequence, HHPMTEEFKN, in the S1–S2 loop. The S1–S2 loop does not move in response to changes in mem-brane potential and is therefore not likely to be involved in channel gating. It is also interesting to note that the peptide sequence used as antigen to obtain SVmab1 is part of the rec-ognition site for two inhibitory scorpion toxins (Huwentoxin IV104 and Hainantoxin III105). This highlights the importance of this amino acid sequence region for functional activity and as a potential functional antibody epitope.

SVmab1 and CTmab were tested in patch clamp experi-ments on HEK293 cells transiently expressing human Nav1.7. SVmab1 significantly reduced the sodium currents (75% inhibition at 100 nM), whereas CTmab had no effect on the currents at 1 µM. The specificity of S3–S4 loop tar-geting was confirmed by demonstrating that the inhibitory effects of SVmab1 on the sodium currents was eliminated in the presence of the peptide that was used as the antigen. Further characterization of SVmab1 demonstrated that it was Nav1.7-specific and did not inhibit other sodium chan-nel subtypes with the exception of Nav1.6, in which it caused a partial inhibition of currents. This latter observa-tion is expected because the S3–S4 loop of Nav1.6 contains a peptide sequence that is 80% identical to the antigen. Following the demonstration of Nav 1.7 inhibition, the authors used the monoclonal antibody in a variety of in vivo

models of pain and itch and were able to demonstrate sup-pression of inflammatory and neuropathic pain and inhibi-tion of acute and chronic itch.

Discussion and Perspectives

Ion channels are widely regarded as important therapeutic targets that to date have been targeted mainly by small mol-ecules. Antibodies represent a significant and growing class of successful therapeutic drugs and are well established as effective treatment options for diseases such as cancer, inflammatory disease, and autoimmune disorders. A signifi-cant literature has demonstrated that antibodies can be used as experimental tools to study ion channel function, but to date no antibodies suitable for therapeutic intervention have been developed. Nevertheless, antibodies represent a sig-nificant opportunity by virtue of their specific properties, which include exquisite specificity, options for tailoring their affinity and potency, and the ability to modulate their effector function and pharmacokinetic properties by engi-neering of the Fc region. It is also possible now to consider modifying antibodies to favor delivery of molecules across the blood–brain barrier to enable targeting of not only peripheral ion channels but also those located in the CNS.

Although no therapeutic ion channel antibodies are described to date, there are a number of new studies report-ing functional monoclonal antibodies that give rise to some optimism that effective drugs are on the horizon. Challenges remain, however, and issues relating to effective antigen generation, expression of ion channels, and screening meth-ods suggest that antibody screening campaigns will require a portfolio of techniques to isolate effective functional anti-bodies. It is also noteworthy that in several recently reported cases of functional antibodies, relatively few functional antibodies were isolated from immunization campaigns and only partial inhibition of ion channel activity was achieved. This further confirms the challenging nature of the task and suggests that the epitopes targeted by the antibodies were suboptimal. Many reports to date also describe mouse monoclonal antibodies, and this raises a further requirement for humanization and possibly further optimization of bind-ing properties before clinical use can be considered.

In summary, the feasibility of identifying functional antibodies to ion channels has taken significant steps for-ward in the past decade, and advances in the field of ion channel biochemistry and structural understanding coupled with advances in antibody discovery and screening tech-nologies strongly suggest that effective functional antibod-ies may be developed with significant opportunities for therapeutic treatment of diseases covering a wide spectrum of pathophysiologies.

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.




Funding

The authors received support through their employment at MedImmune for the research, authorship, and/or publication of this article.

References

1. Wickenden, A.; Priest, B.; Erdemli, G. Ion Channel Drug Discovery: Challenges and Future Directions. Future Med. Chem. 2012, 4, 661–679.

2. Bagal, S. K.; Brown, A. D.; Cox, P. J.; et al. Ion Channels as Therapeutic Targets: A Drug Discovery Perspective. J. Med. Chem. 2013, 56, 593–624.

3. Overington, J. P.; Al-Lazikani, B.; Hopkins, A. L. How Many Drug Targets Are There? Nat. Rev. Drug. Disc. 2006, 5, 993–996.

4. McGivern, J. G. Ziconotide: A Review of Its Pharmacology and Use in the Treatment of Pain. Neuropsych. Treat. Dis. 2007, 3, 69–85.

5. Tradtrantip, L.; Namkung, W.; Verkman, A. S. Crofelemer, An Antisecretory Antidiarrheal Proanthocyanidin Oligomer Extracted from Croton lechleri, Targets Two Distinct Intestinal Chloride Channels. Mol. Pharmacol. 2010, 77, 69–78.

6. Bosmans, F.; Swartz, K. J. Targeting Voltage Sensors in Sodium Channels with Spider Toxins. Trends Pharmacol. Sci. 2010, 31, 175–182.

7. Beck, A.; Wurch, T.; Bailly, C.; et al. Strategies and Challenges for the Next Generation of Therapeutic Antibodies. Nat. Rev. Immunol. 2010, 10, 345–352.

8. Pederson, S. F.; Stock, C. Ion Channels and Transporters in Cancer: Pathophysiology, Regulation and Clinical Potential. Cancer Res. 2013, 73, 1658–1661.

9. Imbrici, P.; Camerino, D. C.; Tricarico, D. Major Channels Involved in Neuropsychiatric Disorders and Therapeutic Perspectives. Frontiers Genet. 2013, 4, 1–17.

10. Srivastava, R.; Aslam, M.; Kalluri, S. R.; et al. Potassium Channel KIR4.1 as an Immune Target for Multiple Sclerosis. New Eng. J. Med. 2012, 367, 115–123.

11. Huber, S. M. Oncochannels. Cell Calcium. 2013, 53, 241–255.

12. Wemmie, J. A.; Taugher, R. J.; Kreple, C. J. Acid-Sensing Ion Channels in Pain and Disease. Nature Rev. Neurosci. 2013, 14, 461–471.

13. Wulff, H.; Castle, N. A.; Pardo, L. A. Voltage-Gated Potassium Channels as Therapeutic Targets. Nat. Rev. Drug. Disc. 2009, 8, 982–1001.

14. Perret, D.; Luo, Z. D. Targeting Voltage-Gated Calcium Channels for Neuropathic Pain Management. Neurotherapeutics. 2009, 6, 679–692.

15. Mathie, A. Ion Channels as Novel Therapeutic Targets in the Treatment of Pain. J. Pharm. Pharmacol. 2010, 62, 1089–1095.

16. Bennett, D. L. H.; Woods, C. G. Painful and Painless Channelopathies. Lancet Neurol. 2014, 13, 587–599.

17. Lambert, E. H.; Eaton, L. M.; Rooke, E. D. Defect of Neuromuscular Conduction Associated with Malignant Neoplasm. Am. J. Physiol. 1956, 187, 612–613.

18. Meriney, S. D.; Hulsizer, S. C.; Lennon, V. A.; et al. Lambert-Eaton Myasthenic Syndrome Immunoglobulins React with

Multiple Types of Calcium Channels in Small-Cell Lung Carcinoma. Ann. Neurol. 1996, 40, 739–749

19. Newsom-Davis, J.; Buckley, C.; Clover, L.; et al. Autoimmune Disorders of Neuronal Potassium Channels. Ann. N.Y. Acad. Sci. 2003, 998, 202–210.

20. Rana, S. S.; Ramanathan, R. S.; Small, G.; et al. Paraneoplastic Isaacs’ Syndrome: A Case Series and Review of the Literature. J. Clin. Neuromusc. Dis. 2012, 13, 228–233.

21. Yu, F. K.; Yarov-Yarovoy, V.; Gutman, G. A.; et al. Overview of Molecular Relationships in the Voltage-Gated Ion Channel Superfamily. Pharmacol. Rev. 2005, 57, 387–395.

22. Li, S.; Gosling, M.; Poll, C. T.; et al. Therapeutic Scope of Modulation of Non-Voltage Gated Cation Channels. Drug Disc. Today. 2005, 10, 129–137.

23. Kaczorowski, G. J.; McManus, O. B.; Priest, B. T.; et al. Ion Channels as Drug Targets: The Next GPCR’s. J. Gen. Physiol. 2008, 131, 399–405.

24. Eckford, P. D. W.; Li, C.; Ramjeesingh, M.; et al. Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Potentiator VX-770 (Ivacaftor) Opens the Defective Channel Gate of Mutant CFTR in a Phosphorylation-Dependent but ATP-Independent Manner. J. Biol. Chem. 2012, 287, 36639–36649.

25. Sermet-Gaudelus, I. Ivacaftor Treatment in Patients with Cystic Fibrosis and the G551D-CFTR Mutation. Eur. Respir. Rev. 2013, 22, 66–71.

26. Dimitrov, D. Therapeutic Proteins. In Therapeutic Proteins: Methods and Protocols, Methods in Molecular Biology; Voynov, V., Caravella, J. A., Eds.; Humana Press: Totowa, NJ, 2012, pp 1–26.

27. Carter, P. J. Potent Antibody Therapeutics by Design. Nat. Rev. Immunol. 2006, 6, 343–357.

28. Hogarth, P. M.; Pietersz, G. A. Fc Receptor-Targeted Therapies for the Treatment of Inflammation, Cancer and Beyond. Nat. Rev. Drug. Disc. 2012, 11, 311–331.

29. Nelson, A. L.; Dhimolea, E.; Reichart, J. M. Development Trends for Human Monoclonal Antibody Therapeutics. Nat. Rev. Drug. Disc. 2010, 9, 767–774.

30. Reichart, J. M. Marketed Therapeutic Antibodies Compendium. mAbs. 2012, 4, 413–415.

31. Groves, M. A. T.; Amanuel, L.; Campbell, J. I.; et al. Antibody VH and VL Recombination Using Phage and Ribosome Display Technologies Reveals Distinct Structural Routes to Affinity Improvements with VH-VL Interface Residues Providing Important Structural Diversity. mAbs. 2014, 6, 236–245.

32. Atwal, J. K.; Chen, Y.; Chiu, C.; et al. A Therapeutic Antibody Targeting BACE1 Inhibits Amyloid-β Production in Vivo. Science Trans. Med. 2011, 3, 84ra43, 1–12.

33. Yu, Y. J.; Zhang, Y.; Kenrick, M.; et al. Boosting Brain Uptake of a Therapeutic Antibody by Reducing its Affinity for a Transcytosis Target. Science Trans. Med. 2011, 3, 84ra44, 1–8.

34. Niewoehner, J.; Bohrmann, B.; Collin, L.; et al. Increased Brain Penetration and Potency of a Therapeutic Antibody Using a Monovalent Molecular Shuttle. Neuron. 2014, 81, 49–60.

35. Vaughan, T. J.; Williams, A. J.; Pritchard, K.; et al. Human Antibodies with Sub-Nanomolar Affinities Isolated from a Large Non-Immunised Phage Display Library. Nature Biotech. 1996, 14, 309–314.



Wilkinson et al. 13

36. Schwimmer, L. J.; Huang, B.; Giang, H.; et al. Discovery of Diverse and Functional Antibodies from Large Human Repertoire Antibody Libraries. J. Immunol. Meth. 2013, 391, 60–71.

37. Muyldermans, S. Nanobodies: Natural Single-Domain Antibodies. Ann. Rev. Biochem. 2013, 82, 775–797.

38. Mujić-Delić, A.; de Wit, R. H.; Verkaar, F.; et al. GPCR-Targeting Nanobodies: Attractive Research Tools, Diagnostics, and Therapeutics. Trends Pharm. Sci. 2014, 35, 247–255.

39. Skerra, A. Alternative Non-Antibody Scaffolds for Molecular Recognition. Curr. Opin. Biotech. 2007, 18, 295–304.

40. Gebauer, M.; Skerra, A. Engineered Protein Scaffolds as Next-Generation Antibody Therapeutics. Curr. Opin. Chem. Biol. 2009, 13, 245–255.

41. Ablynx nv. Characterization of Anti-Kv1.3 Nanobodies and Activity in Inflammatory Model Systems. http://www.ablynx .com (accessed Jun 26, 2014).

42. Kawate, T.; Michel, J. C.; Birdsong, W. T.; et al. Crystal Structure of the ATP Gated P2X(4) Ion Channel in the Closed State. Nature. 2009, 460, 592–598.

43. Mayer, M. L. Emerging Models of Glutamate Receptor Ion Channel Structure and Function. Structure. 2011, 19, 1370–1380.

44. Buell, G.; Chessell, I. P.; Michel, A. D.; et al. Blockade of P2X7 Receptor Function with a Monoclonal Antibody. Blood. 1998, 92, 3251–3528.

45. Ebersbach, H.; Proetzel, G.; Zhang, C. Antigen Presentation for the Generation of Binding Molecules. Meth. Mol Biol. 2012, 901, 1–10.

46. Chiarelli, P.; Fazio, V. M. Mouse Monoclonal Antibodies in Biological Research: Strategies for High-Throughput Production. Biotechnol. Lett. 2008, 30, 1303–1310.

47. Ebersbach, H.; Geisse, S. Antigen Generation and Display in Therapeutic Antibody Drug Discovery—a Neglected but Critical Player. Biotechnol. J. 2012, 7, 1–11.

48. Chames, P.; Hoogenboom, H. R.; Henderikx, P. Selection of Antibodies against Biotinylated Peptides. Meth. Mol. Biol. 2002, 178, 147–157.

49. Sommerfelt, M. A. Circular CCR5 Peptide Conjugates and Uses Thereof (WO2008074895). Exp. Opin. Ther. Patents. 2009, 19, 1323–1328

50. Zhang, Y.; Pool, C.; Sadler, K.; et al. Selection of Active scFv to G-Protein-Coupled Receptor CCR5 Using Surface Antigen-Mimicking Peptides. Biochemistry. 2004, 43, 12575–12584.

51. Pepscan. Antibody Discovery to G-Protein Coupled Receptors. http://www.pepscan.com (accessed Jun 14, 2014).

52. Naylor, J.; Beech, D. J. Extracellular Ion Channel Inhibitor Antibodies. Open Drug Disc. J. 2009, 1, 36–42.

53. Xu, S-Z.; Zeng, F.; Lei, M.; et al. Generation of Functional Ion-Channel Tools by E3 Targeting. Nat. Biotech. 2005, 23, 1289–1293.

54. Gómez-Varela, D.; Zwick-Wallasch, E.; Knötgen, H.; et al. Monoclonal Antibody Blockade of the Human Eag1 Potassium Channel Function Exerts Antitumor Activity. Cancer Res. 2007, 67, 7343–7349.

55. Klionsky, L.; Tamir, R.; Holzinger, B.; et al. A Polyclonal Antibody to the Prepore Loop of Transient Receptor Potential Vanilloid Type 1 Blocks Channel Activation. J. Pharm. Exp. Ther. 2006, 319, 192–198.

56. Clare, J. J. Functional Expression of Ion Channels in Mammalian Systems. In Expression and Analysis of Recombinant Ion Channels; Clare, J. J., Tresize, D. J., Eds.; Wiley-VCH: Weinheim, Germany, 2006.

57. Maue, R. A. Understanding Ion Channel Biology Suing Epitope Tags: Progress, Pitfalls and Promise. J. Cell. Physiol. 2007, 213, 618–625.

58. Kawate, T.; Gouaux, E. Fluorescence-Detection Size-Exclusion Chromatography for Precrystallization Screening of Integral Membrane Proteins. Structure. 2006, 14, 673–681.

59. Agharkar, A.; Rzadkowolski, J.; McBroom, M.; et al. Detergent Screening of the Human Voltage-Gated Proton Channel Using Fluorescence-Detection Size-Exclusion Chromatography. Prot. Sci. 2014, 23, 1136–1147.

60. Stojilkovic, S. S.; Yan, Z.; Obsil, T.; et al. Structural Insights into the Function of P2X4: An ATP Gated Cation Channel of Neuroendocrine Cells. Cell. Mol. Neurobiol. 2010, 30, 1251–1258.

61. Clontech. Tet-on 3G Tetracycline Inducible Expression System. http://www.clontech.com (accessed Jun 27, 2014).

62. Corin, K.; Baaske, P.; Geissler, S.; et al. Structure and Function Analyses of the Purified GPCR Human Vomeronasal Type 1 Receptor 1. Scientific Reports. 2011, 172, 1–6.

63. Gaillet, B.; Gilbert, R.; Broussau, S.; et al. High-Level Recombinant Protein Production in CHO Cells Using Lentiviral Vectors and the Cumate Gene-Switch. Biotechnol. Bioeng. 2010, 106, 203–215.

64. Johansson, T.; Norris, T.; Peilot-Sjögren, H. Yellow Fluorescent Protein-Based Assay to Measure GABA

A Channel Activation and Allosteric Modulation in CHO-K1 Cells. PLoS One. 2013, 8, e59429.

65. Willis, S.; Davidoff, C.; Schilling, J.; et al. Virus-Like Particles as Quantitative Probes of Membrane Protein Interactions. Biochemistry. 2008, 47, 6988–6990.

66. Sulli, C.; Banik, S. S. R.; Schilling, J.; et al. Detection of Proton Movement Directly across Viral Membranes to Identify Novel Influenza Virus M2 Inhibitors. J. Virol. 2013, 87, 10679–10686.

67. Integral Molecular. http://www.integralmolecular.com (accessed Nov 6, 2014).

68. Doyle, D. A.; Cabral, J. M.; Pfuetzner, R. A.; et al. The Structure of the Potassium Channel: Molecular Basis of K+ Conduction and Selectivity. Science. 1998, 280, 69–77.

69. Robertson, N.; Jazayeri, A.; Errey, J.; et al. The Properties of Thermostabilised G Protein-Coupled Receptors (StaRs) and Their Use in Drug Discovery. Neuropharmacology. 2011, 60, 36–44.

70. Magnani, F.; Shibata, Y.; Serano-Vega, M. J.; et al. Co-Evolving Stability and Conformational Homogeneity of the Human Adenosine A2a Receptor. Proc. Natl. Acad. Sci. 2008, 105, 10744–10749.

71. Abdul-Hussein, S.; Andrell, J.; Tate, C. G. Thermostabilisation of the Serotonin Transporter in a Cocaine-Bound Conformation. J. Mol. Biol. 2013, 425, 2198–2207.

72. Hutchings, C. J.; Cseke, G.; Osborne, G.; et al. Monoclonal Anti-β1-Adrenergic Receptor Antibodies Activate G Protein Signalling in the Absence of β-Arrestin Recruitment. mAbs. 2014, 6, 246–261.

73. Chowdhury, P. S. DNA Immunization as a Means to Generate Antibodies to Proteins. Methods. Mol. Biol. 2003, 207, 57–62.




74. Chowdhury, P. S.; Gallo, M.; Pastan, I. Generation of High Titer Antisera in Rabbits by DNA Immunization. J. Immunol. Methods. 2001, 249, 147–154.

75. Hazen, M.; Bhakta, S.; Vij, R.; et al. An Improved and Robust DNA Immunization Method to Develop Antibodies against Extra-Cellular Loops of Multi-Transmembrane Proteins. mAbs. 2014, 6, 95–107.

76. Lee, K. J.; Wang, W.; Padaki, R.; et al. Mouse Monoclonal Antibodies to Transient Receptor Potential Ankyrin 1 Act as Antagonists of Multiple Modes of Channel Activation. J. Pharm. Exp. Ther. 2014, 350, 223–231.

77. Sin, H.; Li, M. 2013. Antibody Therapeutics Targeting Ion Channels: Are We There Yet? Acta Pharmacologica Sinica. 2013, 34, 199–204.

78. Brass, D.; Grably, M. R.; Bronstein-Sitton, N.; et al. Using Antibodies against P2Y and P2X Receptors in Purinergic Signaling Research. Purinergic Sig. 2012, 8 (Suppl. 1), 61–79.

79. Smith, A. J.; Hancock, M. K.; Bi, K.; et al. Feasibility of Implementing Cell-Based Pathway Reporter Assays in Early High-Throughput Screening Assay Cascades for Antibody Drug Discovery. J. Biomol. Screening, 2012, 17, 713–726.

80. Lee, J-H.; Park, C-K.; Chen, G.; et al. A Monoclonal Antibody That Targets a Nav1.7 Channel Voltage Sensor for Pain and Itch Relief. Cell. 2014, 157, 1–12.

81. Sohma, Y.; Fujita, R.; Katoh, S.; et al. Recognition of Liposome-Bound Antigens by Antipeptide Antibody. Appl. Biochem. Biotech. 1993, 38, 179–188.

82. Yao, F.; Theopold, C.; Hoeller, D.; et al. Highly Efficient Regulation of Gene Expression by Tetracycline in a Replication-Defective Herpes Simplex Viral Vector. Mol. Ther. 2006, 13, 1133–1141.

83. Lee, R.; Tran, M.; Nocerini, M.; et al. 2008. A High-Throughput Hybridoma Selection Method Using Fluorometric Microvolume Assay Technology. J. Biomol. Screening, 2008, 13, 210–217.

84. Neher, E.; Sakmann, B. Single-Channel Currents Recorded from Membrane of Denervated Frog Muscle Fibres. Nature, 1976, 260, 799–802.

85. Neher, E.; Sakmann, B.; Steinbach, J. H. The Extracellular Patch Clamp: A Method for Resolving Currents through Individual Open Channels in Biological Membranes. Pflug Archiv Eur. J. Physiol. 1978, 375 (2), 219–228.

86. Dunlop, J.; Bowlby, M.; Peri, R.; et al. High-Throughput Electrophysiology: An Emerging Paradigm for Ion-Channel Screening and Physiology. Nat. Rev. Drug Disc. 2008, 7, 358–368.

87. Trivedi, T.; Liu, J.; Liu, R; et al. Advances in Functional Assays for High-Throughput Screening of Ion Channels Targets. Expert Opin. Drug Disc., 2010, 5 (10), 995–1006.

88. Kuryshev, Y. A.; Brown, A. M.; Duzic, E.; et al. Evaluating State Dependence and Subtype Selectivity of Calcium Channel Modulators in Automated Electrophysiology Assays. Assay Drug Dev Tech. 2014, 12, 110–119.

89. Cariuk, P.; Gardener, M. J.; Vaughan, T. J. Evolution of Biologics Screening Technologies. Pharmaceuticals .2013, 6, 681–688.

90. McManus, O. B. HTS Assays for Developing the Molecular Pharmacology of Ion Channels. Curr. Opin. Pharmacol. 2014, 15, 91–96.

91. Falconer, M.; Smith, F.; Surah-Narwal, S.; et al. High-Throughput Screening for Ion Channel Modulators. J. Biomol. Screen. 2002, 7, 460–465.

92. North, R. A.; Jarvis, M. F. P2X Receptors as Drug Targets. Mol. Pharmacol. 2013, 83, 759–769.

93. Roy, M-L.; Dumaine, R.; Brown, A.M. HERG, a Primary Ventricular Target of the Nonsedating Antihistamine Terfanidine. Circulation. 1996, 94, 817–823.

94. Srikanth, S.; Gwack, Y. Orai1, STIM1 and Their Associating Partners. J. Physiol. 2012, 590, 4169–4177.

95. Lin, F-F.; Elliott, R.; Colombero, A.; et al. Generation and Characterization of Fully Human Monoclonal Antibodies against Human Orai1 for Autoimmune Disease. J. Pharm. Exp. Ther. 2013, 345, 225–238.

96. Cox, J. H.; Hussell, S.; Sondergaard, H.; et al. Antibody-Mediated Targeting of the Orai1 Calcium Channel Inhibits T Cell Function. Plos One. 2013, 8, 1–11.

97. Jakobovits, A.; Amado, R. G.; Yang, X.; et al. From Xenomouse Technology to Panitimumab, the First Fully Human Antibody Product from Transgenic Mice. Nat. Biotech. 2007, 10, 1134–1143.

98. Moran, M. M.; McAlexander, M. A.; Bíró, T.; et al. Transient Receptor Potential Channels as Therapeutic Targets. Nat. Rev. Drug Disc. 2011, 10, 601–620.

99. Dib-Haji, S. D.; Yang, Y.; Black, J. A.; et al. The Nav1.7

Sodium Channel: From Molecule to Man. Nat. Rev. Neurosci. 2013, 14, 49–62.

100. Catterall, W. A. Structure and Function of Voltage-Gated Sodium Channels at Atomic Resolution. Exp. Physiol. 2014, 99, 35–51.

101. Clare, J. J. Targeting Voltage-Gated Sodium Channels for Pain Therapy. Expert Opin. Investig. Drugs. 2010, 19, 45–62.

102. Wu, M-T.; Huang, P-Y.; Yen, C-T.; et al. A Novel SCN9A Mutation Responsible for Primary Erythromelalgia and Is Resistant to the Treatment of Sodium Channel Blockers. Plos One. 2013, 8, e55212.

103. Catterall, W. A. Ion Channel Voltage Sensors: Structure, Function and Pathophysiology. Neuron. 2010, 67, 915–928.

104. Minassian, N. A.; Gibbs, A.; Shih, A. Y.; et al. Analysis of the Structural and Molecular Basis of Voltage-Sensitive Sodium Channel Inhibition by the Spider Toxin Huwentoxin-IV (µ-TRTX-Hh2a). J. Biol. Chem. 2013, 288, 22707–22720.

105. Liu, Z.; Cai, T.; Zhu, Q.; et al. Structure and Function of Hainantoxin-III, a Selective Antagonist of Neuronal Tetrodotoxin-Sensitive Voltage Gated Sodium Channels Isolated from the Chinese Bird Spider Ornithoctonus hain-ana. J. Biol. Chem. 2013, 288, 20392–20403.

106. Sobolevsky, A. I.; Rosconi, M. P.; Gouaux, E. C. X-Ray Structure, Symmetry and Mechanism of an AMPA-Subtype Glutamate Receptor. Nature. 2009, 462, 745–756.

107. Tang, L.; Gamel El-Din, T. M.; Payendah, J.; et al. Structural Basis for Ca2+ Selectivity of a Voltage-Gated Calcium Channel. Nature. 2014, 505, 56–61.

108. Harris, L. J.; Larson, S. B.; Hasel, K. W.; et al. Refined Structure of an Intact IgG2a Monoclonal Antibody. Biochemistry. 1997, 36, 1581–1597.

109. Miller, S.; Rao, S.; Wang, W.; et al. Antibodies to the Extracellular Pore Loop of TRPM8 Act as Antagonists of Channel Activation. Plos One. 2014, 9, e107151.



Documents

Discovery of Functional Antibodies Targeting Ion Channels