Isolation of a human-like antibody fragment (scFv) that neutralizes ricin biological activity

Abstract

Background: Ricin is a lethal toxin that inhibits protein synthesis. It is easily extracted from a ubiquitously grown plant, Ricinus communis, and thus readily available for use as a bioweapon (BW). Anti-ricin antibodies provide the only known therapeutic against ricin intoxication.

Results: In this study, after immunizing a non-human primate (Macaca fascicularis) with the ricin chain A (RTA), a phage-displayed immune library was built (2 x 108 clones), that included the lambda light chain fragment. The library was screened against ricin, and specific binders were sequenced and further analyzed. The best clone, 43RCA, was isolated using a new, stringent neutralization test. 43RCA had a high, picomolar affinity (41 pM) and neutralized ricin efficiently (IC50 = 23 +/- 3 ng/ml, corresponding to a [scFv]/[ricin] molar ratio of 4). The neutralization capacity of 43RCA compared favourably with that of polyclonal anti-deglycosylated A chain (anti-dgRCA) IgGs, obtained from hyperimmune mouse serum, which were more efficient than any monoclonal at our disposal. The 43RCA sequence is very similar to that for human IgG germline genes, with 162 of 180 identical amino acids for the VH and VL (90% sequence identity).

Conclusion: Results of the characterization studies, and the high degree of identity with human germline genes, altogether make this anti-ricin scFv, or an IgG derived from it, a likely candidate for use in humans to minimize effects caused by ricin intoxication.

Figure 1

Ricin neutralization in cell-based assays. Ricin neutralization capacity of scFv 43RCA, calculated as (signal in average test wells minus signal in 4 no-toxin control wells)/(signal in 4 toxin-only control wells minus signal in 4 no-toxin control wells) and expressed as a function of 43RCA concentration ("ng/ml"). 43RCA IC₅₀ = 23 ± 3 ng/ml

Figure 2

Ricin neutralization in cell-free assays. Neutralization of ricin biological activity by 43RCA (diamond, solid line), anti-dgRCA-A IgG (square, dotted line), or non-specific murine Ig (triangle, solid line). Dilutions of the antibodies were mixed with whole ricin and incubated with ricin (4.0 nM) before adding to the translation assay. Luminescence was measured after 90 min at 37°C. Values were calculated as the % control [(CPS treated sample/CPS untreated, no ricin control) × 100]. Results represent the average ± S.D. for three individual experiments.

Figure 3

Sensorgram of 43RCA. 43RCA affinity was measured at 40.9 pM against ricin, utilizing 4 h elution times; K_on = 3.34 10⁵ M ^-1 S ^-1 and K_off = 1.36 10^-5 s^-1. The ricin concentrations utilized for the measurement are indicated in the legend box; the solution at 32 nM was tested twice (noted (1) and (2) in the box) and the 256 nM concentration was utilized for the long term elution (noted (LT) in the box).

Figure 4

Grouping of the 19 non redundant scFvs, in the form of phylogenetic tree. The tree root (extreme left) indicates that the scFvs are grouped in two clusters, A (scFvs 48, 11, 34, 9, 47, 13, 30, 37, 7, 15) on the upper part, and B (scFvs 8, 1, 23, 4, 43, 35, 44, 38, 31) on the lower part of the figure.

Figure 5

IMGT Collier de Perles graphical 2D representation of 43RCA (Figure 5a: VH fragment, Figure 5b: light chain). IMGT Colliers de Perles representations are displayed according to the IMGT unique numbering. Black squares indicate differences from the human genes most similar to 43RCA, and 43RCA. 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 neighbouring FR-IMGT. Hatched circles correspond to missing positions according to the IMGT unique numbering. 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.