High-affinity, human antibody-like antibody fragment (single-chain variable fragment) neutralizing the lethal factor (LF) of Bacillus anthracis by inhibiting protective antigen-LF complex formation

Abstract

The anthrax lethal toxin (LT) consists of two subunits, the protective antigen (PA) and the lethal factor (LF), and is essential for anthrax pathogenesis. Several recombinant antibodies directed against PA and intended for medical use have been obtained, but none against LF, despite the recommendations of anthrax experts. Here we describe an anti-LF single-chain variable fragment (scFv) that originated from an immunized macaque (Macaca fascicularis) and was obtained by phage display. Panning of the library of 1.8 x 10(8) clones allowed the isolation of 2LF, a high-affinity (equilibrium dissociation constant, 1.02 nM) scFv, which is highly neutralizing in the standardized in vitro assay (50% inhibitory concentration, 1.20 +/- 0.06 nM) and in an in vivo assay. The scFv neutralizes anthrax LT by inhibiting the formation of the LF-PA complex. The genes encoding 2LF are very similar to those of human immunoglobulin germ line genes, sharing substantial (84.2%) identity with their most similar, germinally encoded counterparts; this feature favors medical applications. These results, and others formerly published, demonstrate that our approach can generate antibody fragments suitable for prophylaxis and therapeutics.

FIG. 1.

Neutralization capacity of 2LF. LT neutralization was calculated as [(signal in average test wells) − (signal in four no-toxin control wells)]/[(signal in four toxin-only control wells) − (signal in four no-toxin control wells)] and expressed as a function of 2LF concentration (nM). When not visible, the error bars fall within the symbol itself.

FIG. 2.

Gel mobility shift assay. Lane A, LF; lane B, PA₆₃ plus LF, showing the LT (upper band) and LF in excess (lower band); lane C, LF plus 2LF, showing only the complexes; lane D, LF plus PA₆₃ plus 2LF, showing only the LF-2LF complexes and no LT. Control lanes with PA only or scFv only showed no band; PA probably formed aggregates and did not enter the gel, and 2LF migrated with the migration front (data not shown).

FIG. 3.

IMGT “collier de perle” graphical 2-dimensional representation of 2LF. (A) Fd fragment; (B) light chain. IMGT “collier de perle” representations are displayed according to IMGT unique numbering (29, 30). Dots indicate differences between 2LF and the human genes most similar to 2LF. Hydrophobic amino acids (those with positive hydropathy index values, i.e., I, V, L, F, C, M, and A) and tryptophan (W) are shown in blue circles. All proline (P) residues are shown in yellow circles. The complementarity-determining region (CDR-IMGT) sequences are delimited by amino acids shown in squares (anchor positions), which belong to the neighboring FR (FR-IMGT). Gray circles correspond to missing positions according to IMGT unique numbering. Colors of circle outlines indicate regions: in the V_H domain, red for CDR1-IMGT, orange for CDR2-IMGT, and purple for CDR3-IMGT; in the V_κ domain, blue for CDR1-IMGT, green for CDR2-IMGT, and turquoise for CDR3-IMGT.