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

Thibaut Pelat,Marie-Paule Lefranc,Philippe Thullier,Michael Hust,Stefan Dübel,Martha Hale

doi:10.1186/1472-6750-9-60

Abstract

BackgroundRicin 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.ResultsIn this study, after immunizing a non-human primate (Macaca fascicularis) with the ricin chain A (RTA), a phage-displayed immune library was built (2 × 108 clones), that included the λ 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).ConclusionResults 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.

Highlights

Ricin is a lethal toxin that inhibits protein synthesis
Three days after the fifth boost, amplification was detected with 7 of the 9 pairs of primers utilized for amplifying DNA encoding the VH fragment, and with all (7) primer pairs utilized for VLκ
The most diverse DNA was obtained on days 7 and 10, when all pairs of primers strongly amplified DNA that declined on the 14th day (6) pairs positive for VH and for VLκ)

Summary

Introduction

Ricin is a lethal toxin that inhibits protein synthesis. It is extracted from a ubiquitously grown plant, Ricinus communis, and readily available for use as a bioweapon (BW). A 60 to 65 kDa glycoprotein derived from beans of the castor plant (Ricinus communis), is a lectin and member of the A-B toxin family. The B-chain carries the lectin function and binds to specific sugar residues of the target cell surface, allowing ricin to be internalized by endocytosis [1]. The A-chain (RCA-A) has RNA N-glycosidase activity, removing a highly conserved adenine residue in the sarcin/ricin loop of 28S rRNA. The RNA depurination in the ribosome inhibits docking of elongation factor 2, and prevents attachment of amino acids to the polypeptide chain. Ricin is on the second priority list of the CDC and is regarded as a high risk for being utilised as a bioweapon

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