Design and evaluation of a diabody to improve protection against a potent scorpion neurotoxin

Abstract

Diabodies are recombinant, dimeric, antibody-based molecules composed of two non-covalently associated single-chain antibody fragments that bind to an antigen in a divalent manner. In an attempt to develop more effective therapeutic molecules against scorpion venoms, we designed a diabody derived from monoclonal antibody 9C2, which neutralizes the toxicity of scorpion neurotoxin AahI in mammals. The recombinant diabody produced in the periplasm of Escherichia coli was purified to homogeneity in a single step by protein L-agarose affinity chromatography. It was functional, and possessed a high binding affinity to AahI (8 x 10(-11) M). The bivalence of the diabody was confirmed by size-exclusion chromatography, isoelectrofocussing and electron microscopic observations. Finally, the diabody showed high thermal stability in serum and demonstrated protective activity when injected intraperitoneally in mice experimentally envenomed with toxin AahI. In conclusion, the diabody format gives the 9C2 molecule advantageous properties that are particularly important for potential clinical applications in the treatment of envenomations.

Antibody Fragments and Their Purification by Protein L Affinity Chromatography

“…While Ab-fragments are typically monovalent, they can be combined (pre-or post-initial processing) into multivalent entities, such as diabodies, triabodies, and tetrabodies [15,27,73,80,81]. Modification in valency may lead to recruitment or modification of effector functions [11] which may be important in different treatment regimens.…”

“…Such affinity resins have been used in the noncommercial purification of scFv's [31,41,80,89,105,106], Fab's [91,[106][107][108], and single-domain antibodies [52,91,98,103,108,109].…”

Diabody Mixture Providing Full Protection against Experimental Scorpion Envenoming with Crude Androctonus australis Venom

“…Transformed bacteria were grown at 37°C to A 600 nm ϭ 1.2 in 2ϫ YT medium (Sigma) containing 0.05 g/liter ampicillin (Euromedex). Isopropyl ␤-Dthiogalactoside (Euromedex) was then added to yield a final concentration of 0.1 and 0.84 mM for pSW1-Db4C1op and pSW1-Db9C2, respectively, and growth was then continued at 16°C for 18 h. The bacterial cells were harvested by centrifuging at 4°C (5000 ϫ g, 20 min), and periplasmic extracts were prepared as described previously (29). All the soluble periplasmic proteins were extensively dialyzed against phosphate-buffered saline (PBS), pH 7.4, and finally centrifuged (10,000 ϫ g, 4°C, 30 min).…”

“…The fragment was cloned into pMA (AmpR) using the SacI and KpnI cloning sites (Invitrogen). The synthetic gene was then subcloned in-frame with the leader sequence pelB into the expression vector pSW1 using PstI and XhoI restriction enzymes, as described previously (29). Finally, the plasmid pSW1-4C1op was used as a template to create the diabody4C1op gene (Db4C1op) in a PCR to modify the flanking regions of the gene encoding the 4C1op VL domain with the primers VLF4C1, to delete the MRC-OX74 sequence, and Link5R4C1, to generate subsequently a short rigid (Gly 4 -Ser) intramolecular linker.…”

“…Recombinant anti-AahI antibody fragment derived from hybridoma 9C2 ((scFv 5 9C2) 2 (also known as diabody Db9C2)) was produced and purified as described previously (29).…”