Selection of a macaque Fab with framework regions like those in humans, high affinity, and ability to neutralize the protective antigen (PA) of Bacillus anthracis by binding to the segment of PA between residues 686 and 694

Abstract

Human anthrax infection cannot always be treated successfully by antibiotics, as highlighted by recent bioterrorist attacks; thus, adjunct therapies are clearly needed for the future. There is a particular need to further develop adjunct therapies that can neutralize secreted toxins, such as antibodies directed towards the 83-kDa protective antigen (PA(83)). In the absence of human donors, we immunized a macaque (Macaca fascicularis) with PA(83) to obtain such antibodies suitable as an adjunct therapy for human anthrax infection. By using bone marrow as a template, we PCR amplified specific Fab-encoding genes and cloned them as an immune library (10(7) clones). We isolated a high-affinity (equilibrium dissociation constant [K(D)], 3.4 nM), highly neutralizing (50% inhibitory concentration, 5.6 +/- 0.13 nM) Fab (designated 35PA(83)) from this library by panning. Its epitope was localized by Pepscan analysis between residues 686 and 694 of PA(83) and is part of the region which directly interacts with the cell receptor. 35PA(83) may thus neutralize the anthrax toxin by competing directly for its receptor. The genes encoding 35PA(83) were similar to those of a human immunoglobulin germ line and were assigned to subgroups of human V, (D), or J genes by IMGT/V-QUEST analysis. The 35PA(83) framework regions were 92% identical to a representative allele of each subgroup. When compared to framework regions coded by related human germ line genes, only 2 of 74 (VH) or 75 (VK) analyzed amino acids of 35PA(83) have different chemical characteristics. A very high degree of identity with human framework regions makes 35PA(83) well suited for expression as a whole primatized immunoglobulin G and demonstrates the practicality of using macaque Fabs when immunized human plasma cell donors are not available.

FIG. 1.

Toxin neutralization assay. The assay was performed in triplicate as described in Materials and Methods.

FIG. 2.

Analysis of the 35PA₈₃ macaque Fab by surface plasmon resonance. Fab 35PA₈₃ binding responses at concentrations of 1.25 μM, 625 nM, 312 nM, 125 nM, 25 nM, and 0 nM. The black lines represent a global fit for a 1:1 interaction model.

FIG. 3.

IMGT Colliers de Perles of the VH and VK domains of the M. fascicularis Fab 35PA₈₃ on one layer (A) and on two layers (B). IMGT Colliers de Perles representations are displayed according to the IMGT unique numbering (23). Dots indicate differences from the human genes most similar to 35PA₈₃, and 35PA₈₃. The positions of hydrophobic amino acids (hydropathy index with positive value, i.e., I, V, L, F, C, M, A) and tryptophan (W) are shown in blue. All proline (P) residues are shown in yellow. The CDR-IMGT sequences are limited by amino acids shown in squares (anchor positions), which belong to the neighboring FR-IMGT. Hatched circles correspond to missing positions according to the IMGT unique numbering. Arrows indicate the direction of the beta strands (from IMGT Repertoire, http://imgt.cines.fr). For the VH domain, CDR1-IMGT is shown in red, CDR2-IMGT in orange, and CDR3-IMGT in purple. For the V-KAPPA domain, CDR1-IMGT is shown in blue, CDR2-IMGT in green, and CDR3-IMGT in turquoise (21).