Characterization of a humanized monoclonal antibody recognizing clumping factor A expressed by Staphylococcus aureus

Abstract

We report the humanization and characterization of monoclonal antibody (MAb) T1-2 or tefibazumab, a monoclonal antibody that recognizes clumping factor A expressed on the surface of Staphylococcus aureus. We demonstrate that the binding kinetics of MAb T1-2 is indistinguishable compared to that of its murine parent. Furthermore, MAb T1-2 is shown to enhance the opsonophagocytic uptake of ClfA-coated latex beads, protect against an intravenous challenge in a prophylactic model of rabbit infective endocarditis, and enhance the efficacy of vancomycin therapy in a therapeutic model of established infective endocarditis.

FIG. 1.

Opsonization of ClfA-coated fluorescent beads. PMNs were incubated with unopsonized beads, complement-opsonized beads, T1-2 plus complement-opsonized beads, and nonspecific human IgG1 complement-opsonized beads. The PP represents the mean beads per cell multiplied by the percent fluorescent PMNs, as determined via flow cytometric analysis. C′, complement.

FIG. 2.

Serum levels of MAbs and blood or target tissue densities in the prophylactic rabbit model of IE (harvested 72 h postinfection). (A) Blood levels of T1-2 (at 10 [n = 7] and 30 [n = 9] mg/kg) were assessed at 48, 72, and 96 h post-antibody treatment. (B) Number of MRSA recovered from the blood at 96 h in prophylactically treated rabbits with MAb T1-2 (10 or 30 mg/kg) versus that in animals receiving IVIG. Vegetation bacterial densities (C), as well as infection in other target organs, such as the kidney (D), were also quantified 96 h after MAb treatment. In panels B, C, and D, data are represented as the bacterial density in each animal, with the horizontal line indicating the median. Detection limits: blood, 1.0 CFU/ml; vegetations, 9.0 CFU/g average; kidneys, 4.0 CFU/g average. Analysis of the infection rates in each rabbit treatment group was carried out using the Fisher exact test analysis with GraphPad Prism for Windows (version 3.02). The staphylococcal densities in target tissues (log₁₀ CFU/g tissue) for each group were also compared using Kruskal-Wallis analysis of variance, with Dunn's multiple comparison post-test analysis, and P values of <0.05 were considered statistically significant.

FIG. 3.

Serum levels of MAbs and target tissue densities associated with MRSA challenge in the therapeutic rabbit model of IE. Bacterial challenge was at 0 h, 1× MAb therapy was at 24 h, 2× MAb therapy was at 96 h, and harvest of tissues was at 120 h postinfection. Control, no treatment (n = 8); vancomycin (Vanco.), (twice daily) alone at 7.5 mg/kg (n = 9); and vancomycin (twice daily) at 7.5 mg/kg plus MAb T1-2 at 30 mg/kg (n = 9). Animals given a second dose of MAb T1-2 received it 72 h after the initial dose (96 h postchallenge, designated by the arrow in panel A). (A) Blood levels of MAb T1-2 post-i.v. infusion. (B) Vegetation bacterial densities. (C) Kidney bacterial densities. (D) Spleen bacterial densities. All values were from sacrifices performed at 96 h after vancomycin ± MAb T1-2 (1× or 2×) treatment (120 h postinfection). In panels B, C, and D, data are represented as the bacterial density observed in tissues of individual animals with the horizontal line indicating the median. Detection limits: vegetations, 54 CFU/g average; kidneys, 9.5 CFU/g average; spleen, 7.3 CFU/g average. Statistical analysis was performed as described for Fig. 2.