Structure of Fab408 and homology models of Fab403 and Fab410.

<p><b>Left</b>: Overall views of (<b>A</b>) Fab408, with a light blue L chain and yellow H chain; (<b>B</b>) Fab403, with a violet L chain and orange H chain; (<b>C</b>) Fab410, with a dark blue L chain and salmon H chain, displayed with their N-terminal variable region on top and C-terminal constant region at bottom. CDR-L1, CDR-L2, CDR-L3 are displayed in blue, light green, dark green and CDR-H1, CDR-H2, CDR-H3 in red, orange, purple. The extended CDR-H3 in Fab408 is clearly visible. <b>Center</b>: Close-up views of the combining sites, displayed as molecular surfaces with the CDRs colored as on the left panels and labeled. Differences in the sizes of the CDRs and the shapes of the combining surfaces (pocket in Fab408 <i>versus</i> extended surfaces in Fab403 and Fab410) are evident. <b>Right</b>: Distribution of the electrostatic potentials mapped on the Fab molecular surfaces at -3 kT/e (red) to +3 kT/e (blue) (same orientation as on the central panels). Note the electronegative combining site in Fab408 <i>versus</i> the electropositive combining sites in Fab403 (centered around the L-chain CDRs) and Fab410 (centered around the H-chain CDRs).</p

Sequences and numbering of the three anti-EeAChE Fabs.

<p>The light (L, top) and heavy (H, bottom) chains are displayed. The residue numbering and secondary structure elements displayed above the alignment are those of Fab408. Conserved residues are shown on a <i>black</i> background and non-conserved residues on a <i>white</i> background. The CDRs, defined according to <a href="http://www.bioinf.org.uk/abs/" target="_blank"><u>http://www.bioinf.org.uk/abs/</u></a> (cf. Figure S1 in File S1), are highlighted as grey boxes and labeled.</p

BoNT/A1, BoNT/A2, and BoNT/A3 protection by the murine and chimeric TA12 mAbs.

<p>MAb protection activity was determined using the mouse protection assay with 5 estimated MLD₅₀/ml of BoNT/A1, BoNT/A2 or BoNT/A3 and serial dilutions of mAbs. 5 estimated MLD₅₀/mouse were incubated for 30 min at room temperature with 2,500 to 0.25 ng of Mab and the mixture (0.5 ml) was injected intraperitoneally in each mouse of a group of 8 to 10 mice. Results are expressed as the number of surviving mice versus the total number of mice. ND, not done.</p

Physical and functional characterization of the purified Fabs (typical experiments).

<p>(<b>A</b>) MALDI-TOF mass spectrometry profiles, showing both the di-charged and mono-charged entities. Note the satisfactory homogeneity in mass of the latter. (<b>B</b>) Electrophoresis patterns obtained by SDS-PAGE <i>in </i><i>non-reducing </i><i>conditions</i> (12.5% PhastGel, left) and native-PAGE (7.5% PhastGel, center) with migration from the cathode (top) toward the anode (bottom), and by isoelectric focusing (pI 3-9 PhastGel, right). The three Fabs are more homogenous in mass than in charge, a feature that likely result from variations in the C-terminus generated by papaine cleavage of the CH chain; yet, all isoforms bind EeAChE equally, as verified by a native-PAGE mobility shift assay (not shown). The neutral average pI of Fab408 and cationic average pIs of Fab403 and Fab410 are evident. (<b>C</b>) Inhibition of native EeAChE (closed symbols, full lines) and N-deglycosylated EeAChE (open symbols, dashed lines) by the three Fabs. The higher residual activity recorded at saturating concentration of Fab408, compared with Fab403 and Fab410, is evident. Removal of the six N-linked glycan chains of EeAChE results in 6.5-fold higher affinity for Fab403 but unaltered residual activity at saturating Fab403 concentration. Data points correspond to the average values of duplicates or mean values of triplicates. Non-linear fitting of the data points used a sigmoidal equation. Mean values of the mass, pI, IC50 and residual activity of each Fab are reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077226#pone-0077226-t001" target="_blank">Table 1</a>.</p

Production of cTA12 in <i>Sf9</i> cells by infection of a recombinant baculovirus at different multiplicities of infection (MOI).

<p>1.35×10⁷ cells seeded in T75 flasks were infected at different MOI (•: 0.04; ▪: 0.12; <b>▴</b>0.4; ⧫1.2) with the recombinant baculovirus stock. cTA12 containing supernatants were harvested at different times and antibody was quantified by enzyme immunoassay.</p

Pharmacokinetic analysis of cTA12 and mTA12 in mice.

<p>50 µg of purified antibody was intraperitoneally injected into Swiss mice (n = 3 or 4). Mice were sacrificed at different times to calculate plasma concentrations of mAb (• mTA12; cTA12) using a competitive immunoassay. Data were analysed and fitted with WinNonLin professional software (Pharsight®).</p

Western blot analysis of cTA12 produced in insect cells and mammalian cells.

<p>Samples of TA12 were denatured at 95°C in Laemmli buffer and electrophoresed in 13% SDS-PAGE; in non-reducing conditions (A), lane 1: cTA12 purified from ascitic fluids (clone 16G7) and lane 2: cTA12 purified from Sf9 supernatant; and in reducing conditions (B) Lane 1: Sf9 supernatant, lane 2: cTA12 purified from Sf9 supernatant, lane 3: cTA12 purified from ascitic fluids (clone 16G7), lane 4: unpurified ascitic fluids (clone 16G7). After transfer, membranes were incubated with a polyclonal rabbit anti-human antibody, detected with HRP-conjugated anti-rabbit IgG and revealed using chemiluminescence.</p

Neutralization potency of cTA12 using the <i>in vivo</i> co-injection protocol.

<p>The neutralizing activity was determined using the mouse protection assay with 5 estimated MLD₅₀/mouse of BoN/A1. BoNT/A1 (0.5 ml) and serial dilutions of cTA12 (0.5 ml) were injected separately into each mouse of a group of 8–10. Results are expressed as surviving mice versus the total number of mice.</p

Productivity of different <i>SP2/0-Ag14</i> clones secreting cTA12.

<p>6-well plates were seeded with 2.5×10⁵ cells/well in 3 ml of medium culture. The productivity of 12 clones obtained after stable transfection and limiting dilution cloning was tested +12A1; ¤12A2; ▴13E1; <b>X</b> 13E4; ⧫13E5; •16C2; □16C4; ▵16G1; ▾16G2; ◊16G3; ○16G6; ▪16G7). Supernatants were harvested every 24 h to measure cell density and cTA12 concentration (by enzyme immunoassay).</p

Quantification of functional cTA12 recognising native BoNT/A1 by a sandwich immunoassay.

<p>Native BoNT/A1 from culture supernatant (100 µl at 300 LD100) was incubated in 96-well microtitre plates coated with different dilutions of purified TA12 (from 0.05 to 1.5 µg/ml). TA13-labeled AChE was used as the tracer antibody, and absorbance was measured at 414 nm after incubation with AChE substrate. cTA12 purified from ascitic fluids and cTA12 purified from Sf9 supernatant were compared using mTA12 as reference (□ purified mTA12; ▪ cTA12 purified from ascitic fluids; ▪ cTA12 purified from S<i>f9</i> supernatant).</p