Novel chimpanzee/human monoclonal antibodies that neutralize anthrax lethal factor, and evidence for possible synergy with anti-protective antigen antibody

Abstract

Three chimpanzee Fabs reactive with lethal factor (LF) of anthrax toxin were isolated and converted into complete monoclonal antibodies (MAbs) with human gamma1 heavy-chain constant regions. In a macrophage toxicity assay, two of the MAbs, LF10E and LF11H, neutralized lethal toxin (LT), a complex of LF and anthrax protective antigen (PA). LF10E has the highest reported affinity for a neutralizing MAb against LF (dissociation constant of 0.69 nM). This antibody also efficiently neutralized LT in vitro, with a 50% effective concentration (EC(50)) of 0.1 nM, and provided 100% protection of rats against toxin challenge with a 0.5 submolar ratio relative to LT. LF11H, on the other hand, had a slightly lower binding affinity to LF (dissociation constant of 7.4 nM) and poor neutralization of LT in vitro (EC(50) of 400 nM) and offered complete protection in vivo only at an equimolar or higher ratio to toxin. Despite this, LF11H, but not LF10E, provided robust synergistic protection when combined with MAb W1, which neutralizes PA. Epitope mapping and binding assays indicated that both LF10E and LF11H recognize domain I of LF (amino acids 1 to 254). Although domain I is responsible for binding to PA, neither MAb prevented LF from binding to activated PA. Although two unique MAbs could protect against anthrax when used alone, even more efficient and broader protection should be gained by combining them with anti-PA MAbs.

FIG. 1.

Alignment of the deduced amino acid sequences of the variable domains of the heavy (A) and kappa (B) chains of anti-LF clones. Substitutions relative to LF9D are shown as single amino acid letters. Identical residues are indicated by dashes. The absence of corresponding residues relative to the longest sequence is indicated by asterisks. Complementarity-determining regions (CDR1, CDR2, and CDR3) and framework regions (1, 2, 3, and 4) are indicated above the sequence alignments.

FIG. 2.

ELISA titration of anti-LF MAbs. Recombinant LF was used to coat ELISA plates. Wells were then incubated with various dilutions of LF9D, LF10E, and LF11H IgGs, and the bound IgGs were detected by the addition of peroxidase-conjugated anti-human Fc antibody followed by tetramethylbenzidine substrate. OD₄₅₀, optical density at 450 nm.

FIG. 3.

LF10E and LF11H neutralize LT toxicity. LT (100 ng/ml) was incubated with serial dilutions of each antibody (1 h, 37°C) prior to treatment of RAW264.7 macrophage cells with LT-MAb mixtures for 4 h. Cell viability was assessed by 3-(4,5-dimethylthiazo-2-yl)-2,5-diphenyltetrazolium bromide staining and is presented as a percentage of the value for untreated controls.

FIG. 4.

LF10E and LF11H bind to LF amino acids 1 to 254 and prevent LT-mediated MEK cleavage in cells but do not prevent LF binding to cleaved PA. Purified toxin proteins LF, PA, and EF and fusion proteins FP59 and FP119 were loaded on SDS-polyacrylamide gels (2 ng/well for panel A and 10 ng/well for panel B) and probed with LF10E (A) or LF11H (B) by Western blotting. LT (1 μg/ml) was preincubated with MAbs (100 μg/ml) for 1 h prior to addition of LT or LT-MAb mixtures to CHO cells (C) or RAW 264.7 cells (D). Toxins were then allowed to bind to cells for 1 h prior to Western blotting as described in Materials and Methods. No treatment (NT) was used as a negative control.