Evaluation of recombinant monoclonal antibody SVmab1 binding to NaV1.7 target sequences and block of human NaV1.7 currents

Figure 1.. Analysis of rSVmab1. ( A) SDS-PAGE of 0.5 and 5.0 ug non-reduced and reduced rSVmab1. ( B) Size exclusion chromatography-high performance liquid chromatography elution profile of rSVmab1. The main peak comprised 97.7% of the area.

Figure 2.. rSVmab1 evaluation by mass spectrometry. The major glycoform on non-reduced rSVmab1 is G0F (agalactosylated/fucosylated bi-antennary glycan) with a calculated mass of 147,938Da. Glycosylation of each heavy chain is denoted (G0F) 2. Additional peaks not matching the theoretical mass of 147,936Da are extended glycoforms of the intact molecule and correspond to addition of galactoses (G1F = +1 galactose; G2F= +2 galactose) or aglyco = no glycan.

Figure 3.. rSVmab1 does not bind to human Na V1.7 DII voltage sensor domain S3-S4 peptide. Peptide ELISA of increasing concentrations of rSVmab1 (blue circles) or positive control antibody (red squares) binding to the BSA-conjugated peptide VELFLADVEG. Absorbance values after subtraction of non-specific binding to uncoated plates represent means ± standard deviation of the mean of at least two independent experiments.

Figure 4.. rSVmab1 does not bind to the soluble DII voltage sensor domain from human Na V1.7. ELISA analysis of increasing concentrations of rSVmab1 (blue circles) or positive control antibody (red squares) binding to purified, soluble Na V1.7 DII voltage sensor domain. Absorbance values after subtraction of non-specific binding to uncoated plates represent means ± standard deviation of the mean of at least two independent experiments.

Figure 5.. rSVmab1 does not bind to cell lines expressing human Na V1.7 by FACS. FACS histograms of rSVmab1, positive control Na V1.7 antibody (Millipore, #MABN41), and an isotype control (R&D, #MAB002) (all at 33nM) binding to cell lines stably or transiently expressing human Na V1.7, or their respective parental cell lines.

Figure 6.. Effect of rSVmAb1 on human Na V1.7 channels in the resting/closed state when tested at 100 nM. ( A) Exemplary raw traces showing sequential addition of 0.3% bovine serum albumin (BSA) control, 100 nM rSVmab1 (after 20min incubation), and 500nM tetrodotoxin (TTX) on Na V1.7 currents in the same HEK293 cell. ( B) Summary of normalized Na V1.7 currents. rSVmab1 did not block Na V1.7 currents, whereas 500nM TTX robustly blocked Na V1.7 currents. Data are mean ± SEM (n=3/group). ( C) Voltage protocol used, where channels were held at -120 mV in the closed/resting state.

Figure 7.. Effect of rSVmab1 on human Na V1.7 channels in the resting/closed state when tested at 500nM. ( A– F) Traces and I-V curves following control or 20min incubation with ( A and D) 0.3% bovine serum albumin (BSA), ( B and E) 500nM IgG, and ( C and F) 500 nM rSVmab1. ( G– I) Conductance-voltage relationships following control or 20 min incubation with ( G) 0.3% BSA, ( H) 500 nM IgG, and ( I) 500 nM rSVmab1. ( J) Voltage protocol used for panels A– F. ( K) Summary of normalized peak Na V1.7 currents from cells incubated with 0.3% BSA, 500 nM IgG, or 500 nM rSVmab1 (after 20 min incubation) followed by 500nM tetrodotoxin (TTX), which blocked nearly all current. Data are mean ± SEM (n=5–8/group). ** p<0.01 for BSA compared to IgG and BSA compared to rSVmab1 at 20 min.

Figure 8.. Effect of rSVmab1 on human Na V1.7 fast inactivation. Steady state fast inactivation curves following control or 20min incubation with ( A) 0.3% bovine serum albumin (BSA), ( B) 500nM IgG, and ( C) 500nM rSVmab1. Data are mean ± SEM (n=4–5/group). ( D) Voltage protocol used for panels A– C.

Figure 9.. Effect of rSVmab1 on human Na V1.7 channels following a 10Hz use-dependent protocol. Normalized current over 100 pulses at 10Hz following control or 20min incubation with ( A) 0.3% bovine serum albumin (BSA), ( B) 500nM IgG, and ( C) 500nM rSVmab1. ( D) Voltage protocol used for panels A– C. 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).

Figure 10.. Effect of rSVmab1 on partially inactivated human Na V1.7 channels. Exemplary raw traces following control or 15min incubation with ( A) 0.3% bovine serum albumin (BSA), ( B) 500 nM IgG, and ( C) 500nM rSVmab1. ( D) Voltage-protocol used for panels A– C. Exemplary time courses following incubation with ( E) 0.3% BSA, ( F) 500nM IgG, and ( G) 500nM rSVmab1. ( H) Voltage protocol employed for panels E– G, where cells were held at a voltage yielding 20% channel inactivation during antibody addition. Summary of normalized currents following 15 min incubation with ( I) 0.3% BSA, ( J) 500nM IgG, and ( K) 500nM rSVmab1. Data are mean ±SEM (n=3–5/group).

Figure 11.. Effect of rSVmab1 on slow inactivated human Na V1.7 channels followed by a 5Hz use-dependent protocol. Exemplary raw traces following 5min or 15min incubation with ( A) 0.3% bovine serum albumin BSA, ( B) 500nM IgG, and ( C) 500nM rSVmab1. P1 = first pulse (tonic block); P26 = 26 th pulse (use-dependent block). ( D) Voltage protocol used for panels A– C. Cells were clamped to -20mV during addition of antibodies and between voltage measurements. Summary of normalized currents following 5 min or 15 min incubation with 0.3% ( E) BSA, ( F) 500nM IgG, and ( G) 500nM rSVmab1. Data are mean ± SEM (n=4–5/group). ** p<0.01 for BSA compared to IgG (15 min, P26); * p<0.05 for BSA compared to rSVmab1 (15 min, P26).