Evaluation of recombinant monoclonal antibody SVmab1 binding to Na_V1.7 target sequences and block of human Na_V1.7 currents

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 publication⁸. 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 sequences⁸. 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% Tryptone. 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) equilibrated with 20mM 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 River PTS Assay; 1.0-0.01 EU/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 peptide and D2S domain binding ELISAs, was generated against the DII voltage sensor peptide sequence VELFLADVEG 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-phase 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_V1.7 DII voltage sensor domain

DNA encoding human Na_V1.7 amino acids 709–857 (GenScript; 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 histidine affinity tag [D2S(709-857)-His₆] in the pFastBac vector (Thermo Fisher Scientific), and a recombinant baculovirus was generated (Bac-to-Bac; Thermo Fisher Scientific). In total, 12L of Sf9 insect cells (3 × 10⁶ 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 fluorescence size exclusion chromatography (Trp FSEC) was used to monitor the oligomerization state of D2S(709-857)-His₆ during purification. Trp FSEC was performed on a Superose 6 10/300 GL column (GE Healthcare Life Sciences) equilibrated with DDM buffer, using an Agilent HPLC system equipped with a fluorescence detector (272 nm excitation/327 nm emission). Absorbance at 280nm was used to determine the protein concentration of purified D2S(709-857)-His₆. N-terminal amino acid sequencing confirmed the identity of purified D2S(709-857)-His₆. The DDM soluble fraction was incubated with 10ml Talon Superflow resin (Clontech) for 14–16h on a rocker. The resin was collected into an XK 16 column (GE Healthcare Life Sciences) and washed with stepwise increases in imidazole concentration (10 c.v., 5mM; 10 c.v., 7.5mM; 5 c.v., 15mM; and 2 c.v., 25mM) in DDM buffer until the A280nm reached a stable minimum. Talon-bound protein was eluted with 200mM imidazole in DDM buffer. Fractions containing D2S(709-857)-His₆ were pooled, concentrated in Ultracel-30K MWCO ultrafiltration units (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)-His₆ was confirmed by Trp FSEC⁹. Monodisperse, micellar D2S(709-857)-His₆ 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)-His₆ 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)-His₆ was collected, aliquoted, and frozen at -80°C.

Generation of Na_V1.7 BacMam

A recombinant BacMam baculovirus expressing human Na_V1.7 was constructed as follows. A full-length cDNA clone of human Na_V1.7 was obtained from Origene (pCMV6-XL4-Na_V1.7) 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_V1.7 was used in an LR Gateway reaction with pHTBV1.1 to produce pHTBV1.1-Na_V1.7. After DNA sequence confirmation, pHTBV1.1-Na_V1.7 was used in a transposition reaction to generate recombinant full-length baculoviral genomic DNA carrying Na_V1.7, 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_V1.7 HEK293 stably transfected cells were purchased from Eurofins Pharma Bioanalytics Services US, Inc., and human Na_V1.7 CHO-K1 stably transfected, inducible cells were purchased from Chantest.

HEK293 complete media contained D-MEM/F-12 (1X) with 10% fetal bovine serum (FBS; US origin), 1x non-essential amino acids (NEAA; 10mM, 100X), 1x penicillin-streptomycin-glutamine (100X), and 400ug/ml Geneticin® Selective Antibiotic (all Invitrogen; #11330-033, #16000-044, 11140-050, 10378–016 and 10131-027, respectively).

CHO-K1 complete media contained F12 HAM (1X; Sigma-Aldrich, #N6658) with 10% FBS (US origin; Sigma-Aldrich, #F2442), 1x L-glutamine (Sigma-Aldrich, #G7513), 0.4mg/ml Zeocin (Invitrogen, #46-0509), and 0.01mg/ml blasticidin (Gibco, #A11139-03). CHO-K1 stable cells were seeded at 8×6¹⁰ cells in 20ml media with 1ug/ml tetracycline (Sigma-Aldrich, #T7660) and 100uM sodium butyrate (Sigma-Aldrich, #303410) in a T-175 flask and incubated 18–24hr prior to FACS analysis.

BacMam transduction

U-2 OS cells (ATCC; #HTB-96; RRID: CVCL_0042), cultured to 80% confluency, were rinsed with Ca and Mg-free DPBS (Gibco, #14190-144) and dissociated with Cell Dissociation Buffer (enzyme-free; Gibco, #13151-014) for 8–10 minutes in a 37°C incubator. Following addition of 5.0ml of complete growth medium, cells were dislodged with gentle pipetting, pelleted, and resuspended to 3×6¹⁰ cells/5ml growth medium. Cells and human Na_V1.7 BacMam virus added at 200 MOI were combined in a T-75 flask and incubated 18-24hr prior to FACS analysis.

U-2 OS complete media contains McCoy’s 5A with 10% FBS, 1x NEAA, 1x L-glutamine (200mM, 100X) and 1x penicillin-streptomycin (10,000U/ml, 100X) (all Gibco; #16600-082, #10099-141, #11140-050, #25030-081 and #15140-122, respectively).

Peptide binding ELISAs

The synthetic peptide VELFLADVEG (Abmart) was conjugated to maleimide-activated bovine serum albumin (BSA; Thermo Fisher Scientific, #PI-77116) through an N-terminal cysteine. The peptide was reconstituted to 10 mg/ml in DMSO and maleimide-activated BSA was made up to 10 mg/ml in dH₂O. 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 384-well medium binding plate (#3702) using 40μL/well, in 1X PBS and incubated at 37°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 rSVmab1 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).

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°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 VELFLADVEG 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_V1.7 stably transfected HEK293 cells, human Na_V1.7 stably transfected, inducible CHO-K1 cells, human Na_V1.7 BacMam 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_V1.7 antibody¹⁰) 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_V1.7 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 MultiClamp 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_V1.7 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_V1.7 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.