A Bispecific Monoclonal Antibody Targeting Psl and PcrV Enhances Neutrophil-Mediated Killing of Pseudomonas aeruginosa in Patients with Bronchiectasis.

Rationale: Pseudomonas aeruginosa infection is associated with worse outcomes in bronchiectasis. Impaired neutrophil antimicrobial responses contribute to bacterial persistence. Gremubamab is a bivalent, bispecific monoclonal antibody targeting Psl exopolysaccharide and the type 3 secretion system component PcrV. Objectives: This study evaluated the efficacy of gremubamab to enhance killing of P. aeruginosa by neutrophils from patients with bronchiectasis and to prevent P. aeruginosa-associated cytotoxicity. Methods: P. aeruginosa isolates from a global bronchiectasis cohort (n = 100) underwent whole-genome sequencing to determine target prevalence. Functional activity of gremubamab against selected isolates was tested in vitro and in vivo. Patients with bronchiectasis (n = 11) and control subjects (n = 10) were enrolled, and the effect of gremubamab in peripheral blood neutrophil opsonophagocytic killing (OPK) assays against P. aeruginosa was evaluated. Serum antibody titers to Psl and PcrV were determined (n = 30; 19 chronic P. aeruginosa infection, 11 no known P. aeruginosa infection), as was the effect of gremubamab treatment in OPK and anti-cytotoxic activity assays. Measurements and Main Results: Psl and PcrV were conserved in isolates from chronically infected patients with bronchiectasis. Seventy-three of 100 isolates had a full psl locus, and 99 of 100 contained the pcrV gene, with 20 distinct full-length PcrV protein subtypes identified. PcrV subtypes were successfully bound by gremubamab and the monoclonal antibody-mediated potent protective activity against tested isolates. Gremubamab increased bronchiectasis patient neutrophil-mediated OPK (+34.6 ± 8.1%) and phagocytosis (+70.0 ± 48.8%), similar to effects observed in neutrophils from control subjects (OPK, +30.1 ± 7.6%). No evidence of competition between gremubamab and endogenous antibodies was found, with protection against P. aeruginosa-induced cytotoxicity and enhanced OPK demonstrated with and without addition of patient serum. Conclusions: Gremubamab enhanced bronchiectasis patient neutrophil phagocytosis and killing of P. aeruginosa and reduced virulence.

Characterization of mAbs against Klebsiella pneumoniae type 3 fimbriae isolated in a target-independent phage display campaign.

We used phage display, antibody engineering, and high-throughput assays to identify antibody-accessible targets of Klebsiella pneumoniae. We report the discovery of monoclonal antibodies (mAbs) binding to type 3 fimbrial proteins, including MrkA. We found that anti-MrkA mAbs were cross-reactive to a diverse panel of K. pneumoniae clinical isolates, representing different O-serotypes. mAbs binding to MrkA have previously been described and have been shown to provide prophylactic protection, although only modest protection when dosed therapeutically in vivo in a murine lung infection model. Here, we used a combination of binding and opsonophagocytic killing studies using a high-content imaging platform to provide a possible explanation for the modest therapeutic efficacy in vivo reported in that model. Our work shows that expression of K. pneumoniae type 3 fimbriae in in vitro culture is not homogenous within a bacterial population. Instead, sub-populations of bacteria that do, and do not, express type 3 fimbriae exist. In a high-content opsonophagocytic killing assay, we showed that MrkA-targeting antibodies initially promote killing by macrophages; however, over time, this effect is diminished. We hypothesize the reason for this is that bacteria not expressing MrkA can evade opsonophagocytosis. Our data support the fact that MrkA is a conserved, immunodominant protein that is antibody accessible on the surface of K. pneumoniae and suggest that additional studies should evaluate the potential of using anti-MrkA antibodies in different stages of K. pneumoniae infection (different sites in the body) as well as against K. pneumoniae biofilms in the body during infection and associated with medical devices.IMPORTANCEThere is an unmet, urgent need for the development of novel antimicrobial therapies for the treatment of Klebsiella pneumoniae infections. We describe the use of phage display, antibody engineering, and high-throughput assays to identify antibody-accessible targets of K. pneumoniae. We discovered monoclonal antibodies (mAbs) binding to the type 3 fimbrial protein MrkA. The anti-MrkA mAbs were found to be highly cross-reactive, binding to all K. pneumoniae strains tested from a diverse panel of clinical isolates, and were active in an opsonophagocytic killing assay at pM concentrations. MrkA is important for biofilm formation; thus, our data support further exploration of the use of anti-MrkA antibodies for preventing and/or controlling K. pneumoniae in biofilms and during infection.