Anti-P. aeruginosa PcrV Recombinant Antibody (MAb 166) (MRO-156MZ)

Fab 1A8 (50 nM) was mixed with various concentrations of either MAb 166 or MAb 3.7.5, and residual binding of Fab 1A8 to GST-PcrV was detected using anti-human kappa-HRP conjugate.

An Engineered Human Antibody Fab Fragment Specific for Pseudomonas aeruginosa PcrV Antigen Has Potent Antibacterial Activity

X63 cells were incubated with PA103 (MOI = 10) for 3 h in the presence of various concentrations of Fab 1A8, MAb 166, or control Fab with no binding to PcrV or other P. aeruginosa proteins. Percent cytotoxicity was determined by PI staining and flow cytometry. Data are from three independent assays. Mean IC50 values ± SE are shown. A t test analysis showed no significant difference between MAb 166 IgG and Fab 1A.

An Engineered Human Antibody Fab Fragment Specific for Pseudomonas aeruginosa PcrV Antigen Has Potent Antibacterial Activity

MAb 166 and Fab fragments were coinstilled with 1.5 × 106 bacteria via the intratracheal route (five mice per group, with four mice for MAb 166 Fab groups). The control was a nonspecific Fab with no binding to PcrV or any P. aeruginosa protein. Mice were treated with antibody doses of 10 μg (A), 5 μg (B), 2.5 μg (C) 1.25 μg (*, P = 0.01 for Fab 1A8 versus MAb 166 Fab) (D), 0.625 μg (*, P = 0.002 for 1A8 versus MAb 166 Fab) (E), 0.3125 μg (F), 0.16 μg (G), and 0.08 μg (H). P values for differences between treatment groups were determined by a Mantel-Cox log-rank test.

An Engineered Human Antibody Fab Fragment Specific for Pseudomonas aeruginosa PcrV Antigen Has Potent Antibacterial Activity

Rectal temperatures are shown for 48 h or until mortality (means ± SE). Antibody doses were 10 μg. *, P < 0.05 for differences in body temperature between control mice and mice treated with MAb 166 IgG, MAb 166 Fab, or human Fab 1A8 at 10-μg or 5-μg doses (unpaired t test).

An Engineered Human Antibody Fab Fragment Specific for Pseudomonas aeruginosa PcrV Antigen Has Potent Antibacterial Activity

Full-length (aa 1–294), different parts (aa 1–46, 1–76, 1–134, 1–172, and 139–294), and internal deletions (aa 1–75 and 173–294) of pcrV were subcloned for expression as glutathione S–transferase (GST)–tagged fusion proteins in the vector pGEX-2TK. The restriction site used for the initial construct is indicated, and the size of the expressed protein (in kDa) is indicated in parentheses. Bacterial lysates were prepared after induction and were analyzed for antibody binding by Western blot analysis. An immunoblot gel from an assay using rabbit (Rab) anti-PcrV as the primary antibody is shown (top). The data indicate that the clones are expressed and in frame with the predicted protein. Also shown is a gel from the same assay, except that MAb 166 was used as the primary antibody (bottom). MAb 166 bound to full-length clones and a carboxyl-terminal expression clone but not to amino-terminal clones or an internal deletion protein.

Generation and Characterization of a Protective Monoclonal Antibody to Pseudomonas aeruginosa PcrV