Anti-C. Diff Toxin B Recombinant Antibody (Bezlotoxumab) (CAT#: TAB-725)

Figure 1 Tissue distribution of actoxumab-bezlotoxumab in hamsters.

(A) Levels of actoxumab-bezlotoxumab in homogenized whole intestinal tissues from healthy (white bars; n ϭ 4) and C. difficile -infected (black bars; n ϭ 3) hamsters, harvested 2 days after challenge. Values shown are means Ϯ SD. No significant differences in the levels of actoxumab-bezlotoxumab exist when comparing healthy to C. difficile -infected hamsters across all tissues. hIgG, human IgG; Duod, duodenum; Jej, jejunum; Ile, ileum; Col, colon; Cec, cecum. (B) Levels of actoxumab-bezlotoxumab in the lumenal contents of various intestinal segments from healthy (white bars; n ϭ 4) and C. difficile -infected (black bars; n ϭ 3) hamsters, harvested 2 days after challenge. Values shown are means Ϯ SD. ء, P Ͻ 0.01 (compared to results for corresponding lumenal contents in healthy hamsters). Representative experiments from 2 independent experiments are shown. (C) Localization of actoxumab-bezlotoxumab in the cecum wall of vehicle-injected healthy hamsters ("Control"), healthy hamsters injected with actoxumab-bezlotoxumab ("Healthy"), and infected hamsters injected with actoxumab-bezlotoxumab ("CDI") (for additional pictures, see Fig. S1 in the supplemental material), as determined by immunohistochemistry, harvested 2 days after challenge. Insets show representative epithelial layers of healthy and CDI hamsters at higher magnification. LP, lamina propria; E, epithelium; L, lumen; S, submucosa. Representative panels from at least 3 independent experiments are shown.

Zhang, Z., Chen, X., Hernandez, L. D., Lipari, P., Flattery, A., Chen, S. C.,... & Kelly, C. P. (2015). Toxin-mediated paracellular transport of antitoxin antibodies facilitates protection against Clostridium difficile infection. Infection and immunity, 83(1), 405-416.

Figure 2 Role of FcRn and other Fc receptors in protection against CDI.

(A) Kaplan-Meier survival curves of mice infected with C. difficile and treated with vehicle (dashed blue line) or with 250 ug actoxumab alone (dotted blue line), bezlotoxumab alone (thin blue line), or both antibodies together (thick blue line). (B) Survival curves of wild-type (dark blue lines) and FcRn knockout (light-blue lines) mice infected with C. difficile in the primary infection mode and treated with vehicle (dashed lines) or 250 ug actoxumab-bezlotoxumab (solid lines). (C) As in panel B but in the recurrent infection mode. (D) Survival curves of Syrian hamsters infected with C. difficile and treated with vehicle (black line), human IgG (red line), 50 mg/kg actoxumab-bezlotoxumab (blue line), or 50 mg/kg actoxumab-bezlotoxumab with human IgG (purple line).

Zhang, Z., Chen, X., Hernandez, L. D., Lipari, P., Flattery, A., Chen, S. C.,... & Kelly, C. P. (2015). Toxin-mediated paracellular transport of antitoxin antibodies facilitates protection against Clostridium difficile infection. Infection and immunity, 83(1), 405-416.

Figure 3 Effects of apical toxin on TER and neutralization by actoxumab-bezlotoxumab in Caco cells.

(A) Time-dependent effects on TER of TcdA added to the apical chamber (circles, 0.1 ng/ml; squares, 0.3 ng/ml; triangles, 1 ng/ml; inverted triangles, 3 ng/ml; diamonds, 10 ng/ml TcdA). B) Time-dependent effects on TER of TcdB added to the apical chamber (circles, 1 ng/ml; squares, 3 ng/ml; triangles, 10 ng/ml; inverted triangles, 30 ng/ml; diamonds, 100 ng/ml TcdB). Dashed box highlights timepoint shown in F). C) Same as A), but in the presence of 100 ng/ml actoxumab in the basolateral chamber. D) Same as B), but in the presence of 100 g/ml bezlotoxumab in the basolateral chamber. E) Concentration-dependent effects of TcdA on TER measured 24h after addition of toxin to the apical chamber in the absence (circles) or presence of 100 ng/ml actoxumab added to the apical chamber (squares) or to the basolateral chamber (triangles), or in the presence of 100 ng/ml F(ab')2 fragments of actoxumab added to the basolateral chamber (inverted triangles).

Zhang, Z., Chen, X., Hernandez, L. D., Lipari, P., Flattery, A., Chen, S. C.,... & Kelly, C. P. (2015). Toxin-mediated paracellular transport of antitoxin antibodies facilitates protection against Clostridium difficile infection. Infection and immunity, 83(1), 405-416.

Figure 4 Transport of actoxumab and bezlotoxumab from the basolateral to the apical side of Caco monolayers.

(A) Concentrations of actoxumab in the apical chamber at various times after addition of increasing concentrations of TcdA (as indicated, in ng/ml) to the apical chamber. (B) Concentrations of bezlotoxumab in the apical chamber at various times after addition of increasing concentrations of TcdB (as indicated, in ng/ml) to the apical chamber. ء, P Ͻ 0.05; ءء, P Ͻ 0.001 (compared to results for all other toxin concentrations at the same time point); (C) Time-dependent accumulation of actoxumab (circles and triangles) or F(ab = ) 2 fragments thereof (squares and diamonds) in the apical chamber after addition of buffer (circles and squares) or 64 ng/ml TcdA (triangles and diamonds) to the apical chamber. (D) Time-dependent accumulation of bezlotoxumab (circles and triangles) or F(ab = ) 2 fragments thereof (squares and diamonds) in the apical chamber after addition of buffer (circles and squares) or 256 ng/ml TcdB (triangles and diamonds) to the apical chamber.

Zhang, Z., Chen, X., Hernandez, L. D., Lipari, P., Flattery, A., Chen, S. C.,... & Kelly, C. P. (2015). Toxin-mediated paracellular transport of antitoxin antibodies facilitates protection against Clostridium difficile infection. Infection and immunity, 83(1), 405-416.

Figure 5 Toxin-induced transepithelial toxin neutralization and transport of basolateral antibodies in MDCK cells.

(A) Time-dependent effects on TER of TcdA added to the apical chamber (circles, 1 ng/ml TcdA; squares, 4 ng/ml; triangles, 16 ng/ml; inverted triangles, 64 ng/ml; diamonds, 256 ng/ml). (B) Like panel A but with 100 ug/ml actoxumab added to the basolateral chamber. (C) Concentrations of actoxumab (green bars) or bezlotoxumab (blue bars) in the apical chamber 42 h after addition of the antibodies individually to the basolateral chamber and 24 h after addition of various concentrations ( x axis) of TcdA to the apical chamber.(D) Like panel C but with TcdB added to the apical chamber. In all cases, antibodies were added to the basolateral chamber 18 h prior to addition of toxin. Values are means Ϯ SD from 4 independent experiments.

Zhang, Z., Chen, X., Hernandez, L. D., Lipari, P., Flattery, A., Chen, S. C.,... & Kelly, C. P. (2015). Toxin-mediated paracellular transport of antitoxin antibodies facilitates protection against Clostridium difficile infection. Infection and immunity, 83(1), 405-416.

Figure 6 Actoxumab-bezlotoxumab protects against systemic and intestinal toxin challenge in mice.

(A) Survival curve of CD1 mice treated with vehicle (blue line) or with 0.1 (light-pink line), 1 (pink line), and 10 (red line) mg/kg actoxumab-bezlotoxumab and systemically challenged 1 h later with TcdA and TcdB (25 ng each). Survival of mice was monitored over time. (B) Fluid accumulation in ileal loops after inoculation with vehicle (Veh) or C. difficile toxins (2.5 μg each; Tx) in mice treated with actoxumab-bezlotoxumab (A/B) or PBS, calculated as the ratio of loop weight (in mg) to loop length (in cm). (C) Histology sections from cecal wall of ileal loops taken from each group for panel B. (D to F) Histological analysis of ileal loop sections taken from each group in panel B. Damage score (D), average number of neutrophils (E), and average number of apoptotic cells (F) were assessed based on criteria listed in Materials and Methods.

Yang, Z., Ramsey, J., Hamza, T., Zhang, Y., Li, S., Yfantis, H. G.,... & Davis, N. M. (2015). Mechanisms of protection against Clostridium difficile infection by the monoclonal antitoxin antibodies actoxumab and bezlotoxumab. Infection and immunity, 83(2), 822-831.

Figure 7 Actoxumab-bezlotoxumab protects against damage and inflammation in primary CDI.

(A) Intestines of mice treated with either PBS or 10 mg/kg actoxumab-bezlotoxumab (WT) collected 48 h following spore challenge. Pictures are representative of three animals per group. (B) Hematoxylin-eosin-stained histology sections from the ceca of spore-challenged mice treated with PBS or with 10 mg/kg WT or N297Q mutant (N297Q) actoxumab-bezlotoxumab, collected 48 h following spore challenge. (C to E) Histological analysis of ileal loop sections taken from each group in panel B. Damage score (C), average number of neutrophils (D), and average number of apoptotic cells (E) were assessed based on criteria listed in Materials and Methods. *, P < 0.05, compared to the PBS group, as assessed by one-way ANOVA with Dunnett's posttest. (F) Toxin-dependent cell rounding in sera of mice treated with PBS or with WT or N297Q mutant actoxumab-bezlotoxumab. Cell rounding in Vero cells incubated with serum samples from mice on days 1 (red circles), 2 (green circles), 3 (pink triangles), 4 (blue inverted triangles), and 6 (brown diamonds) following spore challenge was assessed as described in Materials and Methods.

Yang, Z., Ramsey, J., Hamza, T., Zhang, Y., Li, S., Yfantis, H. G.,... & Davis, N. M. (2015). Mechanisms of protection against Clostridium difficile infection by the monoclonal antitoxin antibodies actoxumab and bezlotoxumab. Infection and immunity, 83(2), 822-831.

Figure 8 Methyl Cholate Selectively Stabilizes TcdB and Inhibits Autoprocessing and Receptor Binding.

(A) Structure of methyl cholate identified in the primary screen.(B) (Top) Dose titration of methyl cholate on IMR-90 cells treated with either TcdA (red) or TcdB (blue). (Bottom) Dose titration of cholic acid on IMR-90 cells treatedwith either TcdA (red) or TcdB (blue).(C) Temperature-dependent fluorescence measurements of TcdB melting temperature (Tm) in the presence of increasing concentrations of methyl cholate (blacksquares) or cholic acid (gray open circles).(D) Western blot assay using Mab102 to detect TcdB glucosyltransferase activity. The Rac1 band detected in the absence of GTD loses reactivity with Mab102following toxin-induced glucosylation.(E) Inhibition of autoactivation of full-length toxin (top) or a truncated construct of GTD-CPD (bottom). (F) Binding of TcdB to Vero cells. Vero cells were incubated with TcdB (100 ng/ml) in the absence or presence of bezlotoxumab (1.4 mM), methyl cholate (40 mM), orcholic acid (40 mM), as indicated. Binding of TcdB was assessed by preparing membrane fractions and immunoblotting against TcdB with bezlotoxumab or acadherin-specific antibody (loading control).

Tam, J., Beilhartz, G. L., Auger, A., Gupta, P., Therien, A. G., & Melnyk, R. A. (2015). Small molecule inhibitors of Clostridium difficile toxin B-induced cellular damage. Chemistry & biology, 22(2), 175-185.

Figure 9 Cell surface binding of TcdA and TcdB as measured by Western blot.

Western blots of cell membranes isolated from Vero cells following incubation with (A) TcdA or (B) TcdB, in the presence of vehicle, actoxumab, or bezlotoxumab (200 µg/ml), as indicated. The top blots in each panel show TcdA and TcdB, while the bottom blots show cadherin, used as a loading control.

Cox, M. A., Hernandez, L. D., Gupta, P., Zhang, Z., Racine, F., & Therien, A. G. (2017). Assays for Measuring C. difficile Toxin Activity and Inhibition in Mammalian Cells. In Clostridium Difficile-A Comprehensive Overview. IntechOpen.

Anti-C. difficile toxin B Therapeutic Antibody (bezlotoxumab) (TAB-725) in ELISA

ELISA analysis of TAB-725 was performed by coating with Recombinant C. difficile Toxin B/TcdB Protein, CF.