Anti-P. aeruginosa exopolysaccharide Psl Recombinant Antibody (Cam-003) (MRO-160MZ) (CAT#: MRO-160MZ)

Figure 1 Functional activity screening of antibodies derived from phage scFv patient libraries.

In vitro functional screens included OPK assays and cell attachment assays using the lung epithelial cell line A549. R347, an isotype-matched human mAb that does not bind P. aeruginosa antigens, was used as a negative control.

Identification of broadly protective human antibodies to Pseudomonas aeruginosa exopolysaccharide Psl by phenotypic screening

Figure 2 Identification of the P. aeruginosa Psl exopolysaccharide as the target of antibodies derived from phenotypic screening.

Reactivity of antibodies was determined by whole-cell ELISA on plates coated with indicated P. aeruginosa strains: mAb (Genway Biosciences) specific to a P. aeruginosa outer membrane protein was used as a positive control.

Identification of broadly protective human antibodies to Pseudomonas aeruginosa exopolysaccharide Psl by phenotypic screening

Figure 3 Identification of the P. aeruginosa Psl exopolysaccharide as the target of antibodies derived from phenotypic screening.

FACS binding analysis of Cam-003 to PAO1 and PAO1ΔpslA. Cam-003 is indicated by a green line; an isotype matched non–P. aeruginosa–specific human IgG1 antibody was used as a negative control and is indicated by the blue line. Washed cells were stained with BacLight to differentiate live from dead cells. Staining with the secondary antibody alone plus BacLight was used as an additional control.

Identification of broadly protective human antibodies to Pseudomonas aeruginosa exopolysaccharide Psl by phenotypic screening

Figure 4 Anti-Psl binding to P. aeruginosa passaged in vivo.

To test if Psl expression is maintained in vivo, BALB/c mice were injected intraperitoneally with P. aeruginosa isolates, followed by harvesting of bacteria by peritoneal lavage 4 h after infection. The presence of Psl was analyzed with a control antibody and Cam-003 by FACS. For the positive control, Cam-003 was assayed for binding to strains grown in vitro to log-phase from an overnight culture (∼5 × 108/ml).

Identification of broadly protective human antibodies to Pseudomonas aeruginosa exopolysaccharide Psl by phenotypic screening

Figure 5 Psl mAb Cam-003 protects mice against lethal challenge in a P. aeruginosa acute pneumonia model.

Animals were monitored for survival between 72 and 120 h after infection. In all experiments, PBS and R347 served as negative controls. A PBS control was not tested in this experiment because previous results indicated no difference in survival versus mice treated with R347. In its place, we used challenge with PAO1ΔpslA as an additional control. Results are represented as Kaplan-Meier survival curves; differences in survival were calculated by the log-rank test for Cam-003 versus R347.

Identification of broadly protective human antibodies to Pseudomonas aeruginosa exopolysaccharide Psl by phenotypic screening

Figure 6 Transcytosis of Cam-003 peptide fusions in pIgR-transfected MDCK cells.

At 24 hours, antibodies in the top chamber (apical) were quantified by ELISA. Cam-003 levels were similar in all 3 cell lines, whereas Cam-003–QRN and Cam-003–KLKL, as well as human dimeric IgA, had increased accumulation in the apical chamber.

Enhancing IgG distribution to lung mucosal tissue improves protective effect of anti–Pseudomonas aeruginosa antibodies

Figure 1 Anti-Human P. aeruginosa exopolysaccharide Psl Antibody in SDS-PAGE

SDS-PAGE analysis of MRO-160MZ In non-reduced (lane 1) and β-mercaptoethanol-reduced (lane 2) conditions.

Figure 2 Anti-Human P. aeruginosa exopolysaccharide Psl Antibody in HPLC

The purity of MRO-160MZ was greater than 95% as determined by SEC-HPLC.