Isolation of a nanomolar scFv inhibiting the endopeptidase activity of botulinum toxin A, by single-round panning of an immune phage-displayed library of macaque origin

Abstract

BackgroundBotulinum neurotoxin A (BoNT/A), mainly represented by subtype A1, is the most toxic substance known. It causes naturally-occurring food poisoning, and is among the biological agents at the highest risk of being weaponized. Several antibodies neutralizing BoNT/A by targeting its heavy chain (BoNT/A-H) have been isolated in the past. For the first time however, an IgG (4LCA) recently isolated by hybridoma technology and targeting the BoNT/A light chain (BoNT/A-L), was shown to inhibit BoNT/A endopeptidase activity and protect in vivo against BoNT/A. In the present study, a phage-displayed library was constructed from a macaque (Macaca fascicularis) hyper-immunized with BoNTA/L in order to isolate scFvs inhibiting BoNT/A endopeptidase activity for clinical use.ResultsDiversity of the scFvs constituting the library was limited due to the frequent presence, within the genes intended to be part of the library, of restriction sites utilized for its construction. After screening with several rounds of increasing stringency, as is usual with phage technology, the library got overwhelmed by phagemids encoding incomplete scFvs. The screening was successfully re-performed with a single round of high stringency. In particular, one of the isolated scFvs, 2H8, bound BoNT/A1 with a 3.3 nM affinity and effectively inhibited BoNT/A1 endopeptidase activity. The sequence encoding 2H8 was 88% identical to human germline genes and its average G-score was -0.72, quantifying the high human-like quality of 2H8.ConclusionsThe presence of restrictions sites within many of the sequences that were to be part of the library did not prevent the isolation of an scFv, 2H8, by an adapted panning strategy. ScFv 2H8 inhibited toxin endopeptidase activity in vitro and possessed human-like quality required for clinical development. More generally, the construction and screening of phage-displayed libraries built from hyper-immunized non-human primates is an efficient solution to isolate antibody fragments with therapeutic potential.

Development of neutralizing scFv-Fc against botulinum neurotoxin A light chain from a macaque immune library

“…In contrast, antibodies inhibiting BoNT/A light chain endopeptidase activity have rarely been described. Only one human (4LCA), one llama (Aa1) and one macaque (2H8) antibody with such inhibitory properties have been described to date 16 , 25 , 36 . Such antibodies may also be protective through other mechanisms in vivo, such as toxin clearance via the recruitment of immune effectors 37 …”

“…The diversity of a previous anti-BoNT/A1-L library was limited by the frequent presence of Nco I and Hind III restriction sites within the genes encoding the scFv. Nevertheless, one scFv (2H8) inhibiting the endopeptidase activity of BoNT/A in vitro was isolated 25 . In this study, we designed a new vector for macaque antibody-phage display and constructed a new immune library, using Sfi I rather than Nco I restriction sites.…”

Monoclonal Antibodies and Antibody Like Fragments Derived from Immunised Phage Display Libraries

“…BoNT/A which is the most potent among seven serotypes exerts its toxicity by the cleavage of SNAP-25 (synaptosomal-associated protein), mediated by its light chain (BoNT/A-L). This proteolysis causes blockage of nerve impulses and causes flaccid paralysis, including that of respiratory muscles resulting in death [16]. Immunisation of macaques (Macaca fascicularis) and construction of hyper immune phage display library resulted in the isolation of nanomolar sensitivity antibody fragments to the light chain fragment of BoNT/A that are capable of inhibiting BoNT/A endopeptidase activity in vitro.…”

Generation of Recombinant Antibodies Against Toxins and Viruses by Phage Display for Diagnostics and Therapy

“…So far, antibodyphage display was successfully used for antibody selection against a panel of toxins classified as category A agents, such as from Clostridium botulinum (botulism) [16,25,53,79] and Bacillus anthracis (anthrax) [97] and also against different category B agents, such as staphylococcal enterotoxin B [66,110] and ricin toxin from Ricinus communis [6,96]. An example for a high-risk microorganism that produces the most toxic substances known with the highest risk of potential use as bioweapons is the Gram-positive, anaerobic, spore-forming bacterium Clostridium botulinum and other Clostridium subspecies.…”

“…Here, antibody phage display provides a technology to generate toxin-neutralizing antibodies against each serotype. For instance, a macaque immune library was used to isolate neutralizing scFv with nm affinities against the light chain of BoNT/A [16,79], but also antibodies against the heavy chain or other relevant serotypes of BoNT are of therapeutical interest. Phage display technology was also used for isolation of single domain antibodies (VHH) after immunization of a llama with a cocktail of seven BoNT toxoids (A-F) [25].…”