Evaluation of recombinant monoclonal antibody SVmab1 binding to Na 1.7 target sequences and block of human Na 1.7

Identification of small and large molecule pain therapeutics that target the genetically validated voltage-gated sodium channel Na 1.7 is a challenging endeavor under vigorous pursuit. The monoclonal antibody SVmab1 was recently published to bind the Na 1.7 DII voltage sensor domain and block human Na 1.7 sodium currents in heterologous cells. We produced purified SVmab1 protein based on publically available sequence information, and evaluated its activity in a battery of binding and functional assays. Herein, we report that our recombinant SVmAb1 does not bind peptide immunogen or purified Na 1.7 DII voltage sensor domain via ELISA, and does not bind Na 1.7 in live HEK293, U-2 OS, and CHO-K1 cells via FACS. Whole cell manual patch clamp electrophysiology protocols interrogating diverse Na 1.7 gating states in HEK293 cells, revealed that recombinant SVmab1 does not block Na 1.7 currents to an extent greater than observed with an isotype matched control antibody. Collectively, our results show that recombinant SVmab1 monoclonal antibody does not bind Na 1.7 target sequences or specifically inhibit Na 1.7 current. V V


Tseng 
Epste
n Lf 

University-Purdue University Indianapolis
USA


Amgen Inc
Neuroscience, Thousand OaksUSA


Amgen British Columbia
BurnabyCanada


Molecular Engineering
Amgen Inc
CambridgeUSA


Discovery Attribute Sciences
Amgen Inc
Thousand OaksUSA


V V 1 2 3 2 2 4 2 2 2 2 1 1 2 3 4

Open Peer Review , Genentech, Inc USA
25 Nov 2016, :2764ADFDA3A72C3B69F22E2DA02777D2ACA010.12688/f1000research.9918.1Invited Referees version 1
Identification of small and large molecule pain th rapeutics that target the genetically validated voltage-gated sodium channel Na 1.7 is a challenging endeavor under vigorous pursuit.The monoclonal antibody SVmab1 was recently published to bind the Na 1.7 DII voltage sensor domain and block human Na 1.7 sodium currents in heterologous cells.We produced purified SVmab1 protein based on publically available sequence information, and evaluated its activity in a battery of binding and functional assays.Herein, we report that our recombinant SVmAb1 does not bind peptide immunogen or purified Na 1.7 DII voltage sensor domain via ELISA, and does not bind Na 1.7 in live HEK293, U-2 OS, and CHO-K1 cells via FACS.Whole cell manual patch clamp electrophysiology protocols interrogating diverse Na 1.7 gating states in HEK293 cells, revealed that recombinant SVmab1 does not block Na 1.7 currents to an extent greater than observed with an isotype matched control antibody.Collectively, our results show that recombinant SVmab1 monoclonal antibody does not bind Na 1.7 target sequences or specifically inhibit Na 1.7 current.

Introduction

Ion channels are attractive drug targets and small molecule therapeutic drugs to this protein family generate worldwide sales of approximately $12 billion 1 .Despite this attraction and the demonstrated involvement of i n channel antibodies in diverse autoimmune diseases 2 , no antibody-based ion channel therapeutic has progressed to the clinic, due to challenges in developing both optimal immunogens and robust screening processes to identify channel modulators 3 .

The genetically validated pain target Na V 1.7 f

ctions a
a voltagegated sodium channel expressed in nociceptive neurons in the peripheral nervous system 4 .Na V 1.7 is comprised of four domains (DI-DIV), each containing six transmembrane (TMD) helices, in which TMD helices S1-S4 contain the voltage sensor region and TMD helices S5-S6 contain the pore region.Upon membrane depolarization, the voltage sensor domains, in particular the voltage sensor paddle comprised of S3, the S3-S4 loop, and S4, move outward resulting in pore opening, influx of sodium into the cell, and action potential firing 5 .Recently, Lee et al. described a monoclonal antibody SVmab1 targeted to a peptide loop between DII S3-4 in the voltage sensor paddle region, which bound a Na V 1.7 DII voltage-sensor domain protein by ELISA and blocked Na V 1.7 function by electrophysiology 6 .In particular, SVmab1, purified from a hybridoma, was reported to block human Na V 1.7 currents in a use-dependent manner, in which repeated channel ope ing events uncovered the epitope for antibody binding in the paddle region, akin to antibody blockade of potassium channels 6,7 .The antigen used to generate SVmab1 was peptide VELFLADVEG, located in the DII paddle region and the sequence of this antibody was previously reported 8 .

We generated recombinant SVmab1 (rSVmab1) protein based on the publically available sequence information and evaluated its ability to bind peptide VELFLADVEG, purified DII voltage sensor domain protein, and cells expressing Na V 1.7, as well as block Na V 1.7 sodium currents in heterologous cells.


Methods


Cloning, expression, and purification of rSVmab1 and control antibodies

The amino acid sequences for the heavy and light chains of rSVmab1 were obtained from Table 2 of a publication 8 .The variable region heavy chain sequence corresponds to SEQ ID NO 4 and the variable region light chain sequence corresponds to SEQ ID NO 8 of this publication.Synthetic, human codon-optimized, reverse translated DNA was generated by Genewiz, and subcloned into pTT5 expression vectors (National Research Council Canada), containing murine IgG1 heavy chain or kappa light chain constant regions.The coding regions from the resulting constructs were confirmed by sequencing to match the published sequences 8 .Plasmids were purified (Endofree Quanta Mega Kit; MDI Healthcare Services India) and re-confirmed by both sequencing and diagnostic restriction digest prior to transfection.Heavy and light chain DNA constructs for rSVmab1 were transiently co-transfected into 1.6L of HEK293 6E cells in an Erlenmeyer shake flask.Cells were grown in Freestyle F17 media supplemented with 4mM L-glutamine, 0.1% pluronic acid and 1x antibiotic solution (Freestyle F17: Invitrogen, #12338-026; L-glutamine: Himedia, #TC243-1Kg; Antibiotic-Antimycotic: Invitrogen, #15140-062; Pluronic F-68; Invitrogen, #24040032; Tryptone N1: TekniScience Inc, #19553).Transfections were performed using polyethylenimine (PEI; Polysciences, #23967), at a DNA-PEI MAX ratio of 1:2.88.At 24 hours post-transfection, the cells were supplemented with 0.5% Try tone.Cells were harvested after 5 days of culture and the supernatant was used for antibody purification.Conditioned media was clarified and used for affinity chromatography using a MabSelect SuRe column (GE Healthcare Life Sciences, #17-5199-01).Fractions containing antibody were pooled and further purified by ion exchange chromatography using SP-Sepharose Fast Flow resin (GE Healthcare).Protein purification and integrity were monitored throughout by SDS-PAGE using 4-12% Bis-Tris gels (Invitrogen, #NP0322), MES SDS Running Buffer (20X; Invitrogen, #NP0002), LDS sample buffer (Invitrogen, #NP0007) and stained with Simply Blue Safe (Invitrogen, #LC6065).Purified antibody was buffer exchanged via dialysis into 10mM sodium acetate (pH5.2),containing 9% sucrose and concentrated (30kD Amicon Ultra centrifugal filter unit; Millipore, #UFC801096).The concentration of the purified antibody was determined by the A280 method on a Nanodrop 2000c (Thermo Fisher Scientific).The final antibody sample was verified by analytical size exclusion chromatography-high performance liquid chromatography (SEC-HPLC) using a YMC-Pack Diol-200, 300 × 8 mm column (YMC Co. Ltd., ID: 0830002871 P/No.DL20S05-3008WT)

uilibrated with 20
M sodium phosphate, 400mM sodium chloride, at a pH 7.2, maintaining a flow rate of 0.75ml/min.Finally, the rSVmab1 preparation was assayed for endotoxin levels using the Kinetic Endotoxin Assay (Charles Rive PTS Assay; 1.0-0.01EU/ml Sensitivity PTS Cartridge, #PTS2001F) and flash frozen in liquid nitrogen.The isotype-matched control antibody used for electrophysiology studies was a recombinant murine IgG1/kappa monoclonal derived from an unrelated immunization campaign.The positive control mouse monoclonal antibody, used for p ptide and D2S domain binding ELISAs, was generated against the DII voltage sensor peptide sequence VEL-FLADVEG by Abmart, which corresponds to the exact sequence used to generate SVmab1.


Mass spectrometry

Mass analysis of non-reduced rSVmab1 was performed on an Agilent TOF 6230 Mass Spectrometer coupled with an Agilent 1260 Infinity HPLC system.HPLC Mobile phases A and B were 0.1% trifluoroacetic acid (TFA) and 90% n-propanol/0.1%TFA, respectively.The reverse-ph se column was an Agilent Zorbax 300SB-C8, 3.5µm 2.1 × 50mm column (#865750-906), heated to 75°C.A 20µg aliquot of rSVmab1 was injected into the system.The sample was chromatographed at 0.2 ml/min with an 11 min gradient as follows: 20%B for 1 min; 20-70%B over 8 min; 70-100%B over 1 min; held at 100%B for 1 min.Mass spectrometer ionization and transmission settings were set as follows: Vcap, 5900V; fragmenter voltage, 460V; nebulizer gas, 25 psig; skimmer voltage, 95V; Oct RF Vpp voltage, 800V; and drying gas, 13 l/min.Purification of human Na V 1.7 DII vo tage sensor domain DNA encoding human Na V 1.7 amino acids 709-857 (Gen-Script; derived from sequence NM_002977.3;https://www.ncbi.nlm.nih.gov/nuccore/NM_002977.3; NCBI Nucleotide RRID: SCR_004860) was cloned N-terminal to a 6x histi

ne affinity tag [D2S(70
-857)-His 6 ] in the pFastBac vector (Thermo Fisher Scientific), and a recombinant baculovirus was generated (Bacto-Bac; Thermo Fisher Scientific).In total, 12L of Sf9 insect c lls (3 × 10 6 cell/ml; Expression Systems) were infected with 5% (v/v) virus, incubated at 27°C for 48 h in spinner flasks, harvested by centrifugation and stored at -80°C until use.The remainder of the purification was conducted at 4°C.The frozen cell pellet (175 g wet weight) was resuspended in lysis buffer [25 mM Tris-HCl (pH 7.4), 200 mM NaCl (TBS), containing 1% v/v protease inhibitor cocktail (Sigma-Aldrich, Inc., #P8340)], stirred until thawed and disrupted by passing the suspension through a high pressure homogenizer at 10,000 psi (Microfluidizer M110EHI; Microfluidics, Corp.).The crude lysate was centrifuged at 10,000 × g for 15 min and the resulting supernatant collected and centrifuged at 100,000 × g for 1.5 h in a 70 Ti rotor.The supernatant was decanted and the 100,000 × g pellet was collected, resuspended in lysis buffer and homogenized prior to solubilization.N-dodecyl-β-D-maltoside (DDM; Anatrace, Inc.) was added to the resuspended membranes to a final concentration of 40 mM, incubated for 1h on a rocker, followed by centrifugation at 100,000 × g to pellet insoluble material.The DDM soluble fraction (100ml) was decanted and used for purification.Preparative chromatography steps were performed on an AKTA Purifier (GE Lifesciences, Inc.) in TBS containing 1 mM DDM, unless noted.SDS-PAGE with Coomassie Blue staining was used to monitor purification.

Analytical tryptophan fluorescen

its (Millipore Corp.
Inc.) and chromatographed on a Superdex 200 10/300 column (GE Lifesciences, Inc.) to remove contaminating proteins and imidazole.The monodispersity of fractions containing D2S(709-857)-His 6 was confirmed by Trp FSEC 9 .Monodisperse, micellar D2S(709-857)-His 6 migrates at an apparent MW of 70kDa, which is similar in size to DDM micelles.Thus, the detergent concentrates during ultrafiltration and cannot be separated well using size exclusion chromatography (SEC), necessitating another Talon affinity step.SEC fractions containing monodisperse D2S(709-857)-His 6 were pooled, and incubated with 0.5ml Talon resin for 2h.The resin was collected in a 2ml gravity column, washed, and protein was eluted with 200mM imidazole in DDM buffer.The eluate was loaded into a 0.5-3ml 10K MWCO Slide-a-Lyzer cassette (Thermo Fisher Scientific) and imidazole was removed by dialysis against DDM buffer.The dialyzed D2S(709-857)-His 6 was collected, aliquoted, and frozen at -80°C.


Generation of Na V 1.7 BacMam

A recombinant BacMam baculovirus expressing human Na V 1.7 was constructed as follows.A full-length cDNA clone of human Na V 1.7 was obtained from Origene (pCMV6-XL4-Na V 1.7) and codon optimized using synthetic DNAs (Thermo Fisher Scientific) to produce a cDNA that was stable during DNA propagation in E. coli strain HB101.The resulting cDNA was cloned into pENTR-D-Topo (Thermo Fisher Scientific) and the sequence was confirmed.pENTR-D-Topo-Na V 1.7 was used in an LR Gateway reaction with pHTBV1.1 to produce pHTBV1.1-NaV 1.7.After DNA se

o C for 1hr.The plate
was washed three times with 90µL/well 1X PBS using a Biotek plate washer (ELx 405), blocked with 1% milk/1X PBS (90µl/well), and incubated at room temperature for 30 min.Blocking buffer was aspirated and rSV-mab1 or positive

ntrol mo
se monoclonal antibody against the DII sensor peptide VELFLADVEG was titrated from 200nM using 40µL/well in 1X PBS/1% milk and incubated at room temperature for 1hr.Plates were washed three times with 90µL/well 1X PBS.Polyclonal goat anti-mouse Fc HRP (Jackson ImmunoResearch Labs, #115-035-164; RRID: AB_2338510) was added at 100ng/mL in 1X PBS/1% milk (40µL/well) and incubated at room temperature for 1hr.Plates were washed an additional four times and the HRP signal was detected with 1-Step TMB (40µL/well; Neogenm #308177) for 30min followed by quenching with 1N hydrochloric acid (40µL/well).Plates were read at OD450 (Thermo Multiskan Ascent).


Soluble DIIS binding ELISAs

Purified DIIS was coated at 2µg/ml on a 96-well NiNTA plate pre-blocked by the manufacturer with bovine serum albumin (Thermo Fisher Scientific, #15442), (50µL/well), in 1X PBS/2mM n-dodecyl-β-D-maltoside (DDM) detergent (Calbiochem, 324355), and then incubated at 37 o C for 1hr.Plates were washed twice with 200µL/well of 1X PBS/2mM DDM.rSVmab1 or positive control mouse monoclonal antibody against the DII sensor peptide VEL-FLADVEG was titrated 1:2 from 13nM in 1% milk/1X PBS/2mM DDM (50µL/well) and then incubated at room temperature for 1hr.Following two washes with 200µL/well of 1X PBS/2mM DDM, polyclonal goat anti-mouse Fc HRP (Jackson ImmunoResearch Labs, #115-035-164; RRID: AB_2338510) was added at 400ng/ mL in 1% milk/1X PBS/2mM DDM (50µL/well), and incubated at room temperature for 1hr.Plates were washed an additional four times and the HRP signal was detected with 1-step TMB (50µL/ well), for 30min followed by quenching with 1N hydrochloric acid (50µL/well).Plates were read at OD450 (Thermo Multiskan Ascent).


FACS binding assays

Human Na V 1.7 stably transfected HEK293 cells, human Na V 1.7 stably transfected, inducible CHO-K1 cells, human Na V 1.7 Bac-Mam transduced U-2 OS and parental cells were treated with non-enzymatic dissociation buffer (Sigma-Aldrich, #C5914) to remove cells from the flask prior to FACS assays.In 96-well V-bottom plates (Costar, #3897), 50,000 cells/well were incubated with 33nM rSVmab1 or isotype control (R&D Systems, #MAB002; RRID: AB_357344; monoclonal mouse IgG1 isotype control) or positive control antibodies (Millipore, #MABN41; RRID: AB_10808664; monoclonal mouse anti-human Na V 1.7 antibody 10 ) in 50ul of FACS buffer (1X PBS+2% FBS; PBS: Hyclone, #SH30256.02;FBS: Sigma-Aldrich, #F2442, 500mL), and then incubated at 4°C for 1hr.Cells were isolated by centrifugation at 2500 RPM (664xg) for 2 min, the supernatant was removed and the cells were washed twice with 200ul/well FACS buffer.Cells were resuspended in 50ul (5ug/ml) polyclonal goat-anti-mouse IgG Fc Alexa 647 (Jackson ImmunoResearch Labs, #115-605-071; RRID: AB_2338909) and 2.5ug/ml 7-aminoactinomycin D (7AAD; Sigma, #A9400) and incubated at 4°C for 15min.Cells were then washed once, resuspended in 50ul FACS buffer and read on a Becton Dickenson Accuri Flow Cytometer using the Intellicyt Hypercyt Autosampler.Single cells were gated and geometric means (GeoMean) of 7AAD-negative cells were analyzed using the Intellicyte Forecyt 3.1 software (Intellicyt; http://intellicyt.com/products/software/).A minimum of 350 live cell events were collected per well.


Manual patch clamp electrophysiology

Human Na V 1.7 stably transfected HEK293 cells, plated on glass coverslips (Warner Instruments, CS-8R, #64-0701) for 18-28 hr before recording, were voltage clamped using the whole cell patch clamp configuration at room temperature (21-24°C), using a Mul-tiClamp 700B amplifier and DIGIDATA 1322A with pCLAMP 10.2 software (Molecular Devices; https://www.moleculardevices.com/systems/conventional-patch-clamp/pclamp-10-software; RRID: SCR_011323).Pipettes, pulled from borosilicate glass capil aries (World Precision Instruments), had resistances between 1.5 and 2.0MΩ.Whole cell capacitance was uncompensated and leak subtraction was not used.Currents were digitized at 50kHz and filtered (4-pole Bessel) at 10kHz using pClamp10.2.Cells were positioned directly in front of a micropipette connected to a solution exchange manifold for antibody perfusion.The external solution consisted of 140mM NaCl, 5.0mM KCl, 2.0mM CaCl2, 1.0mM MgCl2, 10mM HEPES, and 11mM glucose, with a pH 7.4 by NaOH.The internal solution consisted of 62.5mM CsCl, 75mM CsF, 2.5mM MgCl2, 5mM EGTA, and 10mM HEPES, with a pH 7.25 by CsOH.To record from closed/resting channels, cells were held at -120mV and pulsed to -10mV for 30msec at 0.1Hz.To record from partially inactivated channels, cells were held at -120mV initially and then switched to a voltage that yielded 20% channel inactivation.30msec pulses to -10 mV were delivered every 10 sec, and peak inward currents were recorded before and after antibody addition.To record from slow inactivated Na V 1.7 channels (P1) and following a train of depolarizing stimuli (P26), cells were voltage clamped to -110 mV for 3 sec and sodium currents were elicited by a train of 26 depolarizations of 150msec duration to -10 mV at a frequency of 5Hz.Cells were then clamped to -20mV while 500 nM rSVmab1, isotype-matched murine IgG1/kappa monoclonal antibody derived from an unrelated immunization campaign or 0.3% BSA control was added.At the 5 and 15 minute time points post-antibody addition, cells were reclamped to -110 mV for 3sec and put through the same 26 pulse voltage protocol as above.Peak inward current during the 1 st (slow inactivated) or 26 th (use-dependent) pulse to -10 mV in the presence of antibody was divided by the peak inward current evoked by the 1 st or 26 th pulse to -10 mV in the absence of antibody to determine percent inhibition.A separate use-dependent protocol was also employed that replicated conditions used by Lee et al. 6 , where cells were held at -120mV and sodium currents were elicited by

train of d
polarizations of 30msec duration to -10mV at a frequency of 10Hz.All testing solutions had 0.3% BSA (Sigma-Aldrich, #A2058) to prevent non-specific adhesion of proteins t tubing and recording chamber components, and solutions were perfused over cells at 1ml/min.The pore blocker tetrodotoxin (TTX; 500 nM; Alomone Labs, #T-550) was added at the end of experiments as a positive control for robust Na V 1.7 inhibition.Data were analyzed with pCLAMP and all figures were plotted using Origin Pro8 (OriginLab Corp).


Statistical analysis

Electrophysiology data are presented as mean ± SEM, and statistical significance was determined using two-tailed, paired or unpaired Student's t-test with Origin Pro 8 software, with p<0.05 denoting statistical significance.


Results

Recombinant SVmab1 (rSVmab1) was purified from transiently transfected HEK293 6E cells and analyzed by SDS-PAGE (Figure 1A) and SEC-HPLC (Figure 1B).rSVmab1 migrated at an observed molecular weight of ~150kDa in non-reducing SDS-PAGE, comprised distinct and appropriately sized heavy chain and light chain bands in reducing SDS-PAGE, and eluted as a single sharp peak in SEC-HPLC.Collectively, these findings are consistent with the production of an intact antibody.Mass spectrometry analysis of non-reduced rSVmab1 revealed the major peak mass to be 147,938Da, which closely matched the theoretical mass of 147,936Da for an agalactosylated/fucosylated bi-antennary glycoprotein (Figure 2).rSVmab1 binding to antigenic peptide was evaluated in an ELISA assay using peptide VELFLADVEG conjugated to BSA via an N-terminal cysteine residue.At 200nM rSVmab1, no peptide binding was observed, whereas binding of a positive control monoclonal antibody generated against this exact same peptide sequence was detected at a concentration as low as 2nM (Figure 3; Dataset 1).Next, purified DII voltage sensor domain protein, housing the SVmab1 epitope, was prepared as a detergent micelle in DDM and tested for rSVmab1 binding in an ELISA assay.At 13nM rSVmab1, no DIIS binding was observed, whereas binding of the positive control antibody, described above, was detected    at concentrations <1nM (Figure 4; Dataset 2).F

ally, FACS was use
to assess rSVmab1 binding to HEK293, CHO-K1, and U-2 OS cells expressing human Na V 1.7 protein.At 33nM rSVmab1, no cell binding was observed, whereas binding of a positive control Na V 1.7 Ab was detected in all three cell lines (Figure 5; Dataset 3).rSVmab1 was evaluated for functi nal inhibition of human Na V 1.7 currents in HEK293 cells using whole cell manual patch clamp electrophysiology.Protocols that mimic conditions reported by Lee et al. 6 , as well as protocols that interrogate diverse Na V 1.7 gating states, were employed.Na V channels exist in resting/closed states where the pore is shut, open states where sodium ions can permeate the pore, and one or more inactivated states where channels are recalcitrant to opening 5 .When 100nM rSVmab1 was applied to cells which were voltage clamped to a holding potential of -120mV with a 0.1Hz stimulation frequency, where Na V 1.7 channels are in the closed/resting state, no reduction of sodium current was detected following 20min of antibody treatment (Figure 6; Dataset 4; p>0.05 comparing BSA control to rSVmab1).Notably, the pore blocker tetrodotoxin (TTX) robustly inhibited currents under these conditions.For comparison, 100 nM SVmab1 was reported to block closed/resting Na V 1.7 by ~40% at 0.1Hz (Figure 3D of the study by Lee et al. 6 ) Increasing the concentration of rSVmab1 to 500nM for 20min resulted in reductions of Na V 1.7 current by 40% compared to reductions of 20% with an IgG1 isotype control (p=0.05comparing rSVmab1 to IgG1 isotype control).rSVmab1 and IgG1 isotype control both yielded significantly larger current reductions compared to a BSA vehicle control group (Figure 7; Dataset 5; p<0.01 for BSA compared to IgG1 isotype control and p<0.01 for BSA compared to rSVmab1).Conductance-voltage relationships (Figure 7; Dataset 5) and steady-state fast inactivation curves (Figure 8; D taset 6) demon trated that rSVmab1 did not affect Na V 1.7 gating properties.rSVmab1 was next evaluated in a use-dependent protocol using a 10Hz train of depolarizing stimuli (as per Lee et al. 6 ) to repeatedly cycle Na V 1.7 through open and inactive conformations in order to expose the SVmab1 epitope in th DII voltage sensor paddle region.Both 500nM rSVma 1 and an isotype control IgG1 antibody reduced tonic Na V 1.7 current 30-35% in the first pulse of the train with nominal evidence of use-dependent block in later pulses of the train (Fig re 9; Dataset 7; p>0.05 for all group comparisons).In all these studies, antibodies were incubated on cells for 20min w th constant perfusion to accommodate a potentially slow on-rate.For comparison, 100nM SVmab1 was reported to block Na V 1.7 current over 80% within 10sec (Figure 3C of the study by Lee et al. 6 ), using this 10Hz protocol.rSVmab1 was further evaluated using v ltage protocols that place Na V 1.7 channels in various inactivated states.When cells were voltage clamped at a potential that yielded 20% Na V 1.7 nactivation, in which 20% of Na V 1.7 ch