Multimechanistic Monoclonal Antibodies (MAbs) Targeting Staphylococcus aureus Alpha-Toxin and Clumping Factor A: Activity and Efficacy Comparisons of a MAb Combination and an Engineered Bispecific Antibody Approach

Abstract

Secreted alpha-toxin and surface-localized clumping factor A (ClfA) are key virulence determinants in Staphylococcus aureus bloodstream infections. We previously demonstrated that prophylaxis with a multimechanistic monoclonal antibody (MAb) combination against alpha-toxin (MEDI4893*) and ClfA (11H10) provided greater strain coverage and improved efficacy in an S. aureus lethal bacteremia model. Subsequently, 11H10 was found to exhibit reduced affinity and impaired inhibition of fibrinogen binding to ClfA002 expressed by members of a predominant hospital-associated methicillin-resistant S. aureus (MRSA) clone, ST5. Consequently, we identified another anti-ClfA MAb (SAR114) from human tonsillar B cells with >100-fold increased affinity for three prominent ClfA variants, including ClfA002, and potent inhibition of bacterial agglutination by 112 diverse clinical isolates. We next constructed bispecific Abs (BiSAbs) comprised of 11H10 or SAR114 as IgG scaffolds and grafted anti-alpha-toxin (MEDI4893*) single-chain variable fragment to the amino or carboxy terminus of the anti-ClfA heavy chains. Although the BiSAbs exhibited in vitro potencies similar to those of the parental MAbs, only 11H10-BiSAb, but not SAR114-BiSAb, showed protective activity in murine infection models comparable to the respective MAb combination. In vivo activity with SAR114-BiSAb was observed in infection models with S. aureus lacking ClfA. Our data suggest that high-affinity binding to ClfA sequesters the SAR114-BiSAb to the bacterial surface, thereby reducing both alpha-toxin neutralization and protection in vivo These results indicate that a MAb combination targeting ClfA and alpha-toxin is more promising for future development than the corresponding BiSAb.

Keywords: Staphylococcus aureus; alpha toxin; clumping factor A; monoclonal antibodies.

FIG 1

SAR114 MAb broad reactivity against the three main ClfA genotypes in vitro. Inhibition of fibrinogen binding to the three main ClfA genotypes was measured in the presence of serially diluted (from 666 μM to 2.55 μM) anti-ClfA MAb 11H10 (A) or SAR114 (B). Data are representative of three independent experiments. (C) Agglutination of S. aureus clinical isolates in the presence of human plasma. The graph shows the minimum concentration of 11H10 (■) and SAR114 (■) required to inhibit bacterial agglutination. Data are representative of two independent experiments. c-IgG, used as a negative control, did not show any inhibition at 200 μg/ml.

FIG 2

SAR114 and 11H10 share an overlapping epitope on ClfA001 genotype. SAR114 and 11H10 compete for binding to the ClfA001 genotype. (A) ELISA competition binding. Serial dilution (1:2 or 1:600) of SAR114 (■) or 11H10 (■) was added to ClfA-coated plates, followed by addition of biotinylated 11H10 (1:600). Percent competition was calculated as 100 × (OD_{MAb + 11H10biot}/OD_11H10biot). Data represent the mean values ± standard deviations (SD). (B) Octet binding. SAR114 (•) or 11H10 (■), diluted at 5 μg/ml, was captured on an APS biosensor and blocked with BSA, followed by addition of ClfA001 (2.5 μg/ml), and then a second MAb diluted at 5 μg/ml was added. Real-time measurement of the binding to the biosensors (in nanometers) was registered.

FIG 3

ClfA-alpha-toxin bispecific antibodies compared to IgG1. (A) Bispecific constructs using anti-ClfA MAb as a scaffold. scFv of anti-alpha-toxin MAb MEDI4893* were linked via a 10-amino-acid linker (GGGGx2) to the ClfA MAb heavy-chain N terminus (B) or heavy-chain C terminus (C).

FIG 4

In vitro characterization of anti-ClfA SAR114 or 11H10/MEDI4893* BiSAbs. (A) BiS₂ and BiS₃ activities compared to that of MEDI4893* in an alpha-toxin-mediated rabbit RBC hemolytic assay. Serial dilutions of BiS₂, BiS₃, and MEDI4893* were incubated with purified alpha-toxin and rabbit RBC. Hemolysis was measured by the amount of hemoglobin released in the supernatant. Percent hemolysis inhibition was calculated as 100 × [100 − (OD_{alpha-toxin+MAb}/OD_{alpha-toxin alone})]. Data are representative of three independent experiments. (B) Anti-ClfA MAb SAR114 or 11H10 in an immobilized fibrinogen binding assay. ClfA binding to fibrinogen was measured in the presence of serial dilutions (200 to 0.5 μM) of BiS2, BiS3, and SAR114 or a nonspecific c-IgG MAb. Data represent the mean values and standard deviations from three separate experiments. Percent inhibition binding was calculated as 100 × [100 − (OD_ClfA+MAb/OD_{ClfA alone})].

FIG 5

Efficacy for anti-ClfA MAb/MEDI4893* BiSAbs in i.v. bacteremia. BALB/c mice (n = 10) were passively immunized i.p. with SAR114/MEDI4893* BiS₂, BiS₃, or an SAR114-MEDI4893* combination at the indicated concentrations and were i.v. infected 24 h later with the LD₉₀ of SF8300 (6e7 CFU) (A) and 3049057 (5e7 CFU) (B). Protective efficacy for 11H10-MEDI4893* BiS₂, BiS₃, or 11H10-MEDI4893* MAbs was evaluated against SF8300 (C) or 3049057 (D) challenge. Survival was monitored for 2 weeks. Results were analyzed with a log rank (Mantel-Cox) test. Statistical analysis of results compared to those for c-IgG were considered statistically different at a P value of <0.05 and are indicated with an asterisk. Data are representative of three independent experiments.

FIG 6

ClfA sequesters SAR114/MEDI4893* BiSAb in i.n. lethal pneumonia. C57/B6 mice (n = 10) were passively immunized i.p. with BiS₂, BiS₃, MEDI4893*, or the SAR114-MEDI4893* MAb combination at the indicated concentrations and i.n. infected 24 h later with 1.5e6 CFU of SF8300 (A) or the SF8300 ΔclfA isogenic mutant (B). Survival was monitored for 6 days. Results were analyzed with a log rank (Mantel-Cox) test. Results compared to those for c-IgG were considered statistically different at a P value of <0.05. Data are representative of three independent experiments.