GI value of the humanized variants of the different humanized variants of SEM120-IIIC1 and A1HC38.

<p>GI value of the humanized variants of the different humanized variants of SEM120-IIIC1 and A1HC38.</p

GI value of the humanized variants of the different humanized variants of BLC7 and B2-7.

<p>GI value of the humanized variants of the different humanized variants of BLC7 and B2-7.</p

Antigen ELISA of the humanized variants of SEM120-IIIC1 against recombinant light chain of BoNT/A1.

<p>Binding of the germline-humanized anti-BoNT/A1 antibodies (hu1-hu16SEM120-IIIC1) as scFv-Fc (each 1 μg) was tested on 100 ng recombinant BoNT/A1 light chain.</p

Affinity measurement of the humanized SEM120-IIIC1 variants as scFv-Fc against holotoxin BoNT/A1 (no affinities for hu9-hu16, no reactivity).

<p>Affinity measurement of the humanized SEM120-IIIC1 variants as scFv-Fc against holotoxin BoNT/A1 (no affinities for hu9-hu16, no reactivity).</p

Sequence of the macaque framework regions and those coded by the most similar human germline genes.

<p>Based on the physiochemical classes of the amino acids (AA), differences in the framework regions are classified into very similar AA (green), similar AA (blue), dissimilar AA (orange) and very dissimilar AA (red).</p

3D structure of the humanized VH variants.

<p>A) Comparison hu₁VH (red) vs. SEM120-IIIC1 (black). B) Comparison hu₁VH/V21>L (blue) vs. SEM120-IIIC1 (black). C) Comparison hu₂VH/V21>L (green) vs. SEM120-IIIC1 (black). D) Comparison hu₃VH/V21>L (orange) vs. SEM120-IIIC1 (black).</p

Protection capacities of IgGs. <i>In vivo</i> mouse paralysis assay was performed to determine protection capacities of IgGs targeting BoNT/A (5A) or BoNT/B (5B) light and heavy chains.

<p>A) Pure BoNT/A1 (0.4 LD₅₀ per dose, 1.74 pg) was pre-mixed with either hu8SEM120-IIIC1 (light chain) or hu8A1HC38 (heavy chain) at 100 μg, 50 μg, 10 μg, 5 μg or 1 μg of antibody per dose. When tested in combination IgGs (hu8SEM120-IIIC1 and hu8A1HC38 50) were pre-mixed with the same concentration of BoNT/A1 and 50 μg, 10 μg, 2.5 μg, 1.0 μg, 0.25 μg or 0.1 μg of each antibody per dose (100 μg, 20 μg, 5.0 μg, 2.0 μg, 0.5 μg or 0.2 μg total IgG). B) Complex BoNT/B2 (0.2 LD₅₀ per dose) was pre-mixed with either hu8BLC3 (light chain) or hu8B2-7 (heavy chain) at 100 μg, 10 μg, 1 μg or 0.1 μg per dose. When tested in combination IgGs (hu8BLC3 and hu8B2-7) were pre-mixed with the same concentration of BoNT/B2 and 50 μg or 0.25 μg of each antibody per dose (100 μg or 0.5 μg total IgG). In both studies mixtures of toxin and antibody were left for 30 min at room temperature before 0.1 mL was injected subcutaneously into left inguinocrural region of female MF1 strain of mice (n = 4). Animals were scored at 48 hr post injection. Results are expressed as mean score of 4 mice ± SEM. Positive control group of mice received a single injection of either BoNT/A1 (0.4 LD₅₀ per dose) or BoNT/B2 (0.2 LD₅₀ per dose). In each study negative control comprised of one group of mice (n = 4) injected with the highest concentration of IgG mixtures (50 μg each) without toxin (data not shown).</p

Protection capacities of IgGs in <i>in vivo</i> mouse assay.

<p>Neutralization potencies of hu8BLC3 and hu8B2-7, tested individually or in combination, against 5 MLD₅₀ of BoNT/B2 in complex form. The preparations of antibodies and toxin (0.5 mL) were injected intraperitoneally into Swiss mice (20–22 g). The results are expressed as the number of the surviving mice of the total number of mice injected.</p

Human germline genes most similar to the genes encoding the four anti-BoNT antibodies and the corresponding GI value.

<p>Human germline genes most similar to the genes encoding the four anti-BoNT antibodies and the corresponding GI value.</p