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Tesi etd-05172008-180530

Thesis type
Tesi di dottorato di ricerca
Selection of catalytic recombinant antibodies with acetylcholinesterase-like activity
Settore scientifico disciplinare
Corso di studi
Relatore Prof. Podestà, Adriano
Parole chiave
  • catalytic recombinant antibodies
Data inizio appello
Data di rilascio
Riassunto analitico
Irreversible inhibition of acetylcholinesterase, due to accidental exposure, suicide or homicide, is responsible of hundreds of thousands deaths, every year, all around the world.
The enzyme is targeted also by the chemical weapons known as nerve agents, extremely toxic and very rapid poisons that killed thousands of unarmed people in recent years.
The inability of inhibited AchE to hydrolyse acetylcholine causes transmitter accumulation in synapses of both the peripheral and central nervous systems and in neuromuscular junctions. This results in generalised over-stimulation of cholinergic receptors, followed by breakdown of ganglionic and neuromuscular transmission. While atropine and other anticholinergic drugs competitively counteract muscarinic symptoms, they do not restore the impaired neuromuscular transmission and are ineffective in preventing respiratory muscles failure. On the other hand, oximes, such as HI-6 (2-hydroxyiminomethyl-4’-carbamoyl-piridinium-1’-methylether dichloride monohydrate), pralidoxime, obidoxime and others, can reverse the complex of acetylcholinesterase and organophosphate through a radical oxygen group and restore acetylcholinesterase activity, but they are rather ineffective against certain nerve agents, e.g. soman and cyclosarin and fully ineffective against the aged phosphorylated enzyme.

We attempted an alternative strategy for the management of acethylcholinesterase poisoning, i.e. the production of a catalytic recombinant antibody with specific esterase activity, a small size and poor immunogenicity.
The Tomlinson I + J libraries, obtained from MRC geneservice (Cambridge, UK) were used for selection. The human ScFv repertoire of > 100 million sequences was recovered from the TG1 libraries with the helper phage KM13 and the E. Coli strain HB2151 were infected for expression of soluble antibody fragments, while avoiding any panning procedure. Transformed HB2151 cells were then grown in an agar selective medium, containing ampicillin and IPTG for induction, and a specific selective agent, whose identity is maintained confidential.
Under the culture conditions in use, isolated colonies became detectable after a few days incubation in aerobiosis at 37 °C. Cholinesterase activity against acetylthiocholine was detectable in all supernatants of the liquid cultures and an active His-tagged molecule was purified by affinity against nichel ions.
The protein is an unconjugated polypeptide that catalyses the acetylthiocholine hydrolysis according to a Michaelis-Menten kinetics with Km and kcat values of 7.34 mM and 17 molecules/sec, respectively.

The catalytic ScFv is resistant to paraoxon inhibition even at 10 fold molar excess for an extended period of time. Paraoxon (O,O-diethyl O-p-nitrophenyl phosphate) is the neurotoxic metabolite of the insecticide parathion (O,O-diethyl O-p-nitrophenyl phosphorothioate).
We can state that the ScFv we isolated is fully resistant to its toxic effects.
In conclusion, the original strategy we set up to attempt introducing a new protein tool in management of acute cholinergic syndromes was successful. Further experiments are in progress to test its in vivo activity and to set up the conditions for its clinical usage as a protein therapeutics.