AV<inf>H</inf>H that neutralizes the zinc metalloproteinase activity of botulinum neurotoxin type A

Abstract

The current treatment of botulism is to administer animal-derived antitoxin, which frequently causes severe adverse reactions in the recipients. In this study, a heavy chain antibody fragment (VH/VHH) phage display library was constructed by amplification of the immunoglobulin genes of a nonimmune camel, Camelus dromedarius, using primers specific to human VH gene segments. A recombinant light chain of type A botulinumtoxin, BoTxA/LC, with zinc endoprotease activity was used in phage bio-panning to select phage clones displaying BoTxA/LC-bound VH/VHH. Soluble VH/VHHwere produced and purified from 10 VH/VHH phagemid-transformed E. coli clones. Complementary determining regions (CDRs) and immunoglobulin frameworks (FRs) of the 10 camel VH/VHH-deduced amino acid sequences were determined. FR2 sequences of two clones showed a hallmark of camel V HH, i.e. (F/Y)42E49R50(G/F) 52. The remaining eight clones had an FR2 amino acid tetrad of conventional VH, i.e. V42G49L50W52. VHH of one clone (VHH17) neutralized the SNAP25 hydrolytic activity of BoTxA/LC, whereas mouse polyclonal anti-BoTxA/LC did not have such activity. Mimotope sequences of VHH17 matched with the 194-206 amino acid residues of BoTxA/LC, which are located near the S′1 subsite of the catalytic cleft of the enzyme. Molecular docking revealed that CDR3 of the VHH17 bound to epitope in the toxin enzymatic cleft. Therefore, the BoTxA/LC neutralization by the VHH17 should be due to the V HH insertion into the enzymatic cleft of the toxin, which is usually inaccessible to a conventional antibody molecule. This antibody fragment warrants further development as a therapeutic agent for botulism. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.