Engineering of a recombinant Fab from a neutralizing IgG directed against scorpion neurotoxin AahI, and functional evaluation versus other antibody fragments Nicolas Aubrey a,d , Julien Muzard a , Jean Christophe Peter c , Herve ´ Rochat b , Max Goyffon a , Christiane Devaux b , Philippe Billiald a,d, * a Muse ´um National d’Histoire Naturelle, USM 0505—Lerai, 57 rue Cuvier, F75231 Paris cedex 05, France b CNRS UMR 6560, Faculte ´ de Me ´decine Nord, Bd Dramard, F13916 Marseille cedex 20, France c CNRS UPR 9021, Immunologie et Chimie the ´rapeutique, IBMC, 15 rue Descartes, F67084 Strasbourg, France d UMR Universite ´-INRA Immunologie parasitaire et vaccinologie, Faculte ´ de Pharmacie, 31 Avenue Monge, F37200 Tours cedex, France Received 21 August 2003; revised 15 October 2003; accepted 25 November 2003 Abstract Antibody-based therapy is the only specific treatment for scorpion envenomation. However, there are still major drawbacks associated with its use; mainly because antivenoms are still prepared from immune equine serum raised against crude venoms, whereas only a limited number of neurotoxins are responsible for the lethality of the venom. Using a murine hybridoma that secretes a well-characterized neutralizing IgG directed to neurotoxins AahI and AahIII from the venom of the scorpion Androctonus australis, we constructed a recombinant Fab (rFab) fragment, which was produced and purified from transformed bacteria. It recognized toxin AahI with a high affinity ðK D ¼ 8:2 £ 10 211 MÞ equivalent to the homologous pFab prepared by papain digestion of whole IgG. Although the AahI-neutralizing capacity of protein L-purified rFab was low compared to other recombinant antibody formats (scFv and diabody) investigated in parallel, the antibody engineering approach presented here provides an innovative way to synthesize novel toxin-neutralizing molecules. It may serve as a strategy for designing a new generation of antivenoms. q 2003 Elsevier Ltd. All rights reserved. Keywords: Antivenom; Androctonus australis; Antibody engineering; Scorpion; Toxin; Immunotherapy 1. Introduction Human poisoning from scorpion stings is a serious threat in many subtropical and tropical countries. Several hundred deaths were previously reported each year in Tunisia, but more recently, there have been an average of 40,000 scorpion stings a year, with only about 20–40 deaths, mostly in children. This low mortality rate has been attributed to intensive-care treatment, but also to the widespread use of antivenoms, which is the standard treatment in many countries (Krifi et al., 1999; Possani, 2000). However, no major improvements in antivenom manufacture have been made for a long time, and these medicines are still made of equine polyclonal antibody fragments, usually directed against the crude venom, even though the number of clinically important toxic components of the venom is very limited (Chippaux and Goyffon, 1998). As a result, the administration of antivenom to human subjects is sometimes associated with major drawbacks. The low specific neutralizing antibody content of antivenoms makes it necessary to inject a high and potentially toxic dose, with the risk of life-threatening anaphylactic shock and of late serum sickness (Bond, 1992; Sofer et al., 1994). In addition, Fab or F(ab) 0 2 , which are the only active 0041-0101/$ - see front matter q 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.toxicon.2003.11.024 Toxicon 43 (2004) 233–241 www.elsevier.com/locate/toxicon * Corresponding author. Tel.: þ33-140-793-155; fax: þ 33-140- 793-441. E-mail address: billiald@univ-tours.fr (P. Billiald).