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Evaluation of recombinant monoclonal antibody SVmab1 binding to Na V1.7 target sequences and block of human Na V1.7 currents

Identification of small and large molecule pain therapeutics that target the genetically validated voltage-gated sodium channel Na V1.7 is a challenging endeavor under vigorous pursuit. The monoclonal antibody SVmab1 was recently published to bind the Na V1.7 DII voltage sensor domain and block human Na V1.7 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 V1.7 DII voltage sensor domain via ELISA, and does not bind Na V1.7 in live HEK293, U-2 OS, and CHO-K1 cells via FACS. Whole cell manual patch clamp electrophysiology protocols interrogating diverse Na V1.7 gating states in HEK293 cells, revealed that recombinant SVmab1 does not block Na V1.7 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 V1.7 target sequences or specifically inhibit Na V1.7 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 ion 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 functions as 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 opening 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 ). 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.
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-Na V 1.7. After DNA sequence confirmation, pHTBV1.1-Na V 1.7 was used in a transposition reaction to generate recombinant full-length baculoviral genomic DNA carrying Na V 1.7, with transcription driven by the immediate early promoter from cytomegalovirus (Bac-to-Bac; Thermo Fisher Scientific). Transfection into Sf9 insect cells (Expression Systems) using FuGENE HD (Roche) allowed production of replication competent baculovirus, pseudotyped with VSV-G protein. The resulting transfection supernatant (P0 virus) was amplified twice, titered by endpoint dilution, as measured by gp64 expression (Expression Systems), and used in cell based assays.

Stable cell lines
Human Na V 1.7 HEK293 stably transfected cells were purchased from Eurofins Pharma Bioanalytics Services US, Inc., and human Na V 1.7 CHO-K1 stably transfected, inducible cells were purchased from Chantest.
The peptide was reconstituted to 10 mg/ml in DMSO and maleimide-activated BSA was made up to 10 mg/ml in dH 2 O. The BSA-conjugate was prepared by mixing 100ug of maleimide-activated BSA in 200uL PBS, 100ug synthetic peptide and 5mM TCEP (Thermo Fisher Scientific, #PI-77720), and the reaction was incubated at room temperature overnight. BSA-conjugated synthetic peptide (VELFLADVEG) was coated at 1µg/ml on a Costar 384well medium binding plate (#3702) using 40µL/well, in 1X PBS and incubated at 37 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 control mouse 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).

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 capillaries (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 a train of depolarizations 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 to 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    rSVmab1 was evaluated for functional 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=0.05 comparing 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; Dataset 6) demonstrated 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 the DII voltage sensor paddle region.  showing sequential addition of 0.3% bovine serum albumin (BSA) control, 100 nM rSVmab1 (after 20min incubation), and 500nM tetrodotoxin (TTX) on Na V 1.7 currents in the same HEK293 cell. (B) Summary of normalized Na V 1.7 currents. rSVmab1 did not block Na V 1.7 currents, whereas 500nM TTX robustly blocked Na V 1.7 currents. Data are mean ± SEM (n=3/group). (C) Voltage protocol used, where channels were held at -120 mV in the closed/resting state.
Both 500nM rSVmab1 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 (Figure 9; Dataset 7; p>0.05 for all group comparisons). In all these studies, antibodies were incubated on cells for 20min with 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 voltage 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 inactivation, in which 20% of Na V 1.7 channels are unavailable for opening and 80% of Na V 1.7 channels are closed/resting, 500nM rSVmab1 and isotype control antibody decreased currents similarly around 30% after 15min of antibody treatment (p>0.05 for BSA, IgG1, and rSVmab1 comparisons), whereas TTX robustly blocked currents within seconds of application ( Figure 10; Dataset 8). When cells were evaluated using a protocol that promotes transition of Na V 1.7 into a slow inactivated state, by maintaining cells at a resting potential of -20mV during antibody addition and between voltage measurements, 500nM rSVmab1 and isotype control IgG1 Ab both decreased currents ~35% after 15 min, whereas TTX again robustly blocked currents (Figure 11, P1 tonic measurements; Dataset 9; p>0.05 for BSA, IgG1, and rSVmab1 group comparisons). Layering on a 5 Hz use-dependent protocol with 150msec depolarizing pulses following induction of slow inactivation resulted in current reduction by ~65% for rSVmab1 and isotype control IgG1 groups after 15min of antibody treatment (Figure 11, P26 use measurements; Dataset 9; p<0.01 for BSA compared to IgG1, p<0.05 for BSA compared to rSVmab1, p>0.05 for IgG1 compared to rSV-mab1). In these experiments, effects of rSVmab1 were similar to those of the isotype control IgG1 antibody.

Conclusion
At the concentrations tested, recombinant monoclonal antibody SVmab1, generated from published sequence information 8 , did not  Exemplary raw traces at pulse 1 (P1) and pulse 100 (P100) following control or 20min of incubation with (E) 0.3% BSA, (F) 500nM IgG, and (G) 500nM rSVmab1. Summary of normalized currents at P1 (tonic block) and P100 (use-dependent block) following 20 min incubation with (H) 0.3% BSA, (I) 500 nM IgG, and (J) 500nM rSVmab1. Data are mean ± SEM (n=3-5/group).  bind to the following target sources: Na V 1.7 peptide VELFLAD-VEG, Na V 1.7 DII voltage sensor protein, and Na V 1.7 expressing mammalian cells (HEK293, CHO-K1, U-2 OS). Recombinant SVmab1 also did not specifically block Na V 1.7 currents in HEK293 cells, as assessed by whole cell manual patch clamp electrophysiology when channels were closed/resting, inactivated, or cycled through states to expose the voltage sensor paddle region using a train of depolarizing stimuli. Reductions in Na V 1.7 current were comparable when using an isotype control IgG1 or recombinant SVmab1 at 500nM. It is unknown why both isotype control IgG1 and recombinant SVmab1 produced current reductions larger than BSA vehicle control in some voltage protocols. In the absence of positive binding data or specific Na V 1.7 block, our results indicate that recombinant SVmab1 is not a robust large molecule Na V 1.7 antagonist. It should be noted that Lee et al. 6 utilized SVmab1 purified from a hybridoma, whereas the studies reported here employed recombinant SVmab1 purified from HEK293 6E cells. Differences in heavy and/or light chain antibody sequences from these sources could account for the observed differences in Na V 1.7 binding and block. In addition, it is conceivable that differences in Na V 1.7 glycosylation or beta subunit expression in HEK293 cells could impact epitope accessibility to SVmab1 in cell-based experiments; beta subunits have been reported to partially mask interactions between peptide toxins and Na V 1.2 11,12 . Other groups evaluating SVmab1 are encouraged to share their findings on Na V 1.7 binding and block to inform the research community on the utility of this reagent.

Data availability
Open Science Framework: Dataset: Evaluation of recombinant monoclonal antibody SVmab1 binding to Na V 1.7 target sequences and block of human Na V 1.7 currents, doi 10.17605/osf.io/4jbz7 13 . Na 1.7, and therefore does not represent a valid tool for the exploration of Na 1.7 biology.
These findings raise the important question as to why results with the recombinant form of the antibody differ so drastically from published results with SVmab purified from hybridoma. One possibility, alluded to by Liu , is that the published sequence is different from the mAb purified from the hybridoma. If the et al authors have access to the original hybridoma-derived SVmab, it would be interesting to see side-by-side studies with the two forms of the antibody (alternatively, it would be interesting to hear from Lee ., if et al they already have these comparative data at hand). Although unlikely, another possibility suggested by Liu , is that differences exist in Na 1.7 glycosylation or beta subunit expression in the HEK293 cell et al lines utilized in the two studies. Since Lee ., demonstrated that SVmab could block sodium currents et al (presumably Na 1.7) in small DRG neurons, it might be informative to know whether the recombinant SVmab was similarly able to block these native Na 1.7-mediated currents.
The original findings by Lee , indicated that mAbs directed to the DII VSD might represent a viable et al strategy for inhibiting Na 1.7 function. Unfortunately the rSVmab described in the present study is not a useful tool for further examination of this strategy. Interestingly however, Liu , show that the "positive et al. control" mAb from Abmart, binds with high affinity to the peptide immunogen and the purified Na 1.7 DII voltage sensor domain. It would be very interesting to know if this control mAb could inhibit Na 1.7 function in patch-clamp studies.
Minor points: rSVmab was tested at 200nM in the peptide ELISA, 13nM in the DII VSD ELISA, 33nM in the FACs assays and up to 500nM in the patch clamp studies. Why was the rSVmab tested at different concentrations in each study and why was binding not assessed at concentrations used in the functional studies?
Why was no capacitance or series resistance compensation applied in the patch clamp studies? Please provide an estimate of typical series resistance values and associated voltage-errors.
Please provide more information on "MABN41". Does this mAb recognize the DII VSD or a different extracellular epitope?
The recombinantly produced rSVmab was flash frozen following purification and prior to testing. Although many proteins tolerate freezing and thawing, including antibodies, there are some that do not, and this is a relatively minor variable worth considering.
We have read this submission. We believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.
No competing interests were disclosed.