Preclinical in vitro and in vivo characterization of the fully human monoclonal IgM antibody KBPA101 specific for Pseudomonas aeruginosa serotype IATS-O11

Abstract

Pseudomonas aeruginosa infection in ventilator-associated pneumonia is a serious and often life-threatening complication in intensive care unit patients, and new treatment options are needed. We used B-cell-enriched peripheral blood lymphocytes from a volunteer immunized with a P. aeruginosa O-polysaccharide-toxin A conjugate vaccine to generate human hybridoma cell lines producing monoclonal antibodies specific for individual P. aeruginosa lipopolysaccharide serotypes. The fully human monoclonal antibody secreted by one of these lines, KBPA101, is an IgM/kappa antibody that binds P. aeruginosa of International Antigenic Typing System (IATS) serotype O11 with high avidity (5.81 x 10(7) M(-1) +/- 2.8 x 10(7) M(-1)) without cross-reacting with other serotypes. KBPA101 specifically opsonized the P. aeruginosa of IATS O11 serotype and mediated complement-dependent phagocytosis in vitro by the human monocyte-like cell line HL-60 at a very low concentration (half-maximal phagocytosis at 0.16 ng/ml). In vivo evaluation of KBPA101 demonstrated a dose-response relationship for protection against systemic infections in a murine burn wound sepsis model, where 70 to 100% of animals were protected against lethal challenges with P. aeruginosa at doses as low as 5 microg/animal. Furthermore, a high efficacy of KBPA101 in protection from local respiratory infections in an acute lung infection model in mice was demonstrated. Preclinical toxicology evaluation on human tissue, in rabbits, and in mice did not indicate any toxicity of KBPA101. Based on these preclinical findings, the first human clinical trials have been initiated.

FIG. 1.

KBPA101 specifically and exclusively reacts with clinical isolates of P. aeruginosa serotype O11. (A) The binding of KBPA101 to 20 clinical P. aeruginosa isolates of serotype O11 (serotype confirmed by agglutination and PCR) was tested by whole-cell ELISA. Reference strain FT-2 was used as a positive control. (B) The specificity of KBPA101 for O11 was confirmed by the absence of binding to clinical P. aeruginosa isolates of serotypes other than O11. Various murine MAbs with specificity for serotypes O1, O16, O3, O6, or O10 were used as a positive control.

FIG. 2.

KBPA101 specifically mediates complement-dependent opsonophagocytosis of P. aeruginosa serotype O11 in vitro. FITC-conjugated P. aeruginosa (reference strain FT2, ATCC 27131 [A]; clinical isolate 2310.55 [B]) were incubated with KBPA101, together with baby rabbit complement (BRS, ▴) and the phagocytic cell line (HL-60). The amount of phagocytosis was measured by flow cytometry. KBPA101 mediated phagocytosis in a dose-dependent manner, showing half-maximal phagocytosis at a concentration of 0.1 ng/ml for reference strain FT2, as well as the clinical isolate 2310.55. No opsonophagocytosis was observed in the absence of active complement (heat-inactivated complement, HBRS, ▪). (C) Cross-sectional confocal microscopy of HL-60 cells after phagocytosis of opsonized FITC-conjugated P. aeruginosa strain FT2. On the right side and at the bottom, overlays of 10 sections are shown. (D) KBPA101 mediated complement-dependent killing of P. aeruginosa serotype O11. Bacteria were incubated in the presence or absence of rabbit complement with increasing concentrations of KBPA101 for 90 min. Surviving bacteria were plated on agarose plates and incubated overnight at 37°C. Percent survival rates are plotted against MAb concentrations.

FIG. 3.

KBPA101 protects mice from systemic infection with P. aeruginosa serotype O11 in a murine burn wound model. Various doses of KBPA101 were given to NMRI mice (n = 10/dose/experiment) prior to challenge with a lethal dose of live bacteria. Upon inflicting a burn wound to animals under deep anesthesia, P. aeruginosa serotype O11 (clinical isolate 2310.55) was injected subcutaneously (s.c.) under the burn wound. The experiment was repeated four times (experiments 1 through 4). The average survival rates 3 days after challenge are shown from the four independent experiments. A linear regression was calculated from the mean values. Control animals receiving no antibody had survival rates of <10%, whereas KBPA101-treated animals had increasing survival rates in a dose-dependent manner. No mortality was observed in mice treated equally but injected s.c. with PBS instead of live bacteria (data not shown).

FIG. 4.

Treatment with KBPA101 extends the survival of mice with systemic infection with P. aeruginosa serotype O11 in a murine burn wound model. Upon inflicting a burn wound to animals under deep anesthesia, a lethal dose of live P. aeruginosa serotype O11 (clinical isolate 2310.55) was injected s.c. under the burn wound. Control animals were treated with the highest dose of KBPA101 immediately after challenge, whereas experimental animals were treated 4 h postchallenge with a single i.v. injection of various doses of KBPA101. A significant survival benefit was observed for treated animals compared to untreated animals. No dose response was seen for treatment effect. Significant survival rates compared to control group are marked with an asterisk (*, P < 0.001 [log-rank test]).

FIG. 5.

KBPA101 protects mice from local lung infection with P. aeruginosa serotype O11 in an acute lung infection model. KBPA101 or an equivalent volume of PBS was injected i.v. into BALB/c mice 2 h before challenge with 3.5 × 10⁷ CFU of P. aeruginosa serotype O11 (clinical isolate 2310.55). Groups of 10 mice were sacrificed 6, 12, 24, and 48 h after intratracheal challenge, and the bacterial load was determined in homogenates of the lungs (A) and spleens (B). Time points with significant differences between treated and untreated controls are marked with an asterisk (*, P < 0.05 [Mann-Whitney test]). (C) Pathological evaluation, wherein the macroscopic lung pathology was expressed as the lung index of macroscopic pathology (LIMP) as described previously (the LIMP is the ratio of the lung area with pathological changes divided by the area of the whole lung). Statistical significant differences are marked with an asterisk (*, P < 0.05 [Mann-Whitney test]).