The botulinum neurotoxins (BoNTs) produced by Clostridium botulinum are the most poisonous protein substances known. The neutralizing antibodies against botulinum neurotoxin can effectively prevent and cure the toxicosis. Using purified Hc fragments of botulinum neurotoxin serotype A (BoNT/A-Hc) as antigen, 2 specific neutralizing antibodies mapping different epitopes were selected from a fully synthetic human antibody library. The 2 antibodies can effectively inhibit the binding between BoNT/A-Hc and differentiated PC-12 cells in vitro, and the neutralization was evaluated in vivo. Although no single mAb completely protected mice from toxin, they both could prolong time to death when challenged with 20 LD 50s (50% lethal doses) of BoNT/A. When used together, the mAbs completely neutralized 1000 LD50s/mg Ab, suggesting their high neutralizing potency in vivo. The results would lead to further production of neutralizing antibody drugs against BoNT/A. It also proved that it was a quick method to obtain human therapeutic antibodies by selecting from the fully synthetic human antibody phage display library. (Journal of Biomolecular Screening 2009:991-998)
Arnon SS, Schechter R., Inglesby TV, Henderson DA, Bartlett JG, Ascher MS, et al: Botulinum toxin as a biological weapon: medical and public health management. JAMA2001;285:1059-1070.
2.
Amersdorfer P., Wong C., Chen S., Smith T., Deshpande S., Sheridan R., et al: Molecular characterization of murine humoral immuneresponse to botulinum neurotoxin type A binding domain as assessed by using phage antibody libraries. Infect Immun1997;65:3743-3752.
3.
Sheets MD, Amersdorfer P., Finnern R., Sargent P., Lindquist E., Schier R., et al: Efficient construction of large non-immune phage antibody library: the production of high affinity human single-chain antibodies to protein antigens . Proc Natl Acad Sci USA1998;95:6157-6162.
4.
de Haard HJ, van Neer N., Reurs A., Hufton SE, Roovers RC, Henderikx P., et al: A large non-immunized human Fab fragment phage library that permits rapid isolation and kinetic analysis of high affinity antibodies. J Bio Chi1999;274:18218-18230.
5.
Griffiths AD , Williams SC, Hartley O., Tomlinson IM, Waterhouse P., Crosby WL, et al: Isolation of high affinity human antibodies directly from large synthetic repertoires . EMBO J1994;13:3245-3260.
6.
Knappik A., Ge L., Honegger A., Pack P., Fischer M., Wellnhofer G., et al: Fully synthetic human combinatorial antibody libraries (HuCAL) based on modular consensus frameworks and CDRs randomized with trinucleotides . J Mol Biol2000;296:57-86.
7.
Volkel T., Muller R., Kontermann REIsolation of endothelial cell-specific human antibodies from a novel fully synthetic scFv library. Bioch Biophy Res Commun2004;317:515-521.
8.
Silacci M., Brack S., Schirru G., Mårlind J., Ettorre A., Merlo A., et al: Design, construction, and characterization of a large synthetic human antibody phage display library. Proteomics2005;5:2340-2350.
9.
Lacy DB, Tepp W., Cohen AC, DasGupta BR, Stevens RC: Crystal structure of botulinum neurotoxin type A and implications for toxicity . Nat Struct Biol1998;5:898-902.
10.
Tavallaie M. , Chenal A., Gillet D., Pereira Y., Manich M., Gibert M., et al: Interaction between the two subdomains of the C-terminal part of the botulinum neurotoxin A is essential for the generation of protective antibodies. FEBS Lett2004;572:299-306.
11.
Yu YZ, Sun ZW, Wang S., Yu WY: High-level expression of the Hc domain of Clostridium botulinum neurotoxin serotype A in Escherichia coli and its immunogenicity as an antigen. Sheng Wu Gong Cheng Xue Bao2007;23:812-817.
12.
Tomlinson IM , Cox JP, Gherardi E., Lesk AM, Chothia C.: The structural repertoire of the human Vκ domain. EMBO J1995;14:4628-4638.
13.
Williams SC , Frippiat JP, Tomlinson IM, Ignatovich O., Lefranc MP, Winter G.: Sequence and evolution of the human germline Vλ repertoire. J Mol Biol1996;264:220-232.
14.
Shirai H., Kidera A., Nakamura H.: Structural classification of CDR-H3 in antibodies. FEBS Lett1996;399:1-8.
15.
Oliva B., Bates PA, Querol E., Avilés FX, Sternberg MJ: Automated classification of antibody complementarity determining region 3 of the heavy chain(H3) loops into canonical forms and its application to protein structure prediction. J Mol Biol1998;279:1193-1210.
16.
Shone CC, Melling J.: Inhibition of calcium-dependent release of noradrenaline from PC-12 cells by botulinum type-A neurotoxin. Eur J Biochem1992;207:1009-1016.
17.
Burnett JC , Ruthel G., Stegmann CM, Panchal RG, Nguyen TL, Hermone AR, et al: Inhibition of metalloprotease botulinum serotype A from a pseudo-peptide binding mode to a small molecule that is active in primary neurons. J Biol Chem2007 ;282:5004-5014.
18.
Blommaert A. , Turcaud S., Anne C., Roques BP: Small tripeptide surrogates with low nanomolar affinity as potent inhibitors of the botulinum neurotoxin B metallo-proteolytic activity. Bioorg Med Chem2004;12:3055-3062.
19.
de Paiva A. , Ashton AC, Foran P., Schiavo G., Montecucco C., Dolly JO: Botulinum A like type B and tetanus toxins fulfils criteria for being a zinc-dependent protease. J Neurochem1993;61:2338-2341.
20.
Marks JD: Deciphering antibody properties that lead to potent botulinum neurotoxin neutralization . Mov Disord2004;19(suppl 8):S101-S108.
21.
Mowry MC, Meagher M., Smith L., Marks J., Subramanian AP: Production and purification of a chimeric monoclonal antibody against botulinum neurotoxin serotype A. Protein Expr Purif2004;37:399-408.
22.
Mah DC, Hu WG, Pon JK, Masri SA, Fulton RE, Monette PL, et al: Recombinant anti-botulinum neurotoxin a single-chain variable fragment antibody generated using a phage display system. Hybrid Hybridomics2003;22:277-283.
23.
Pless DD, Torres ER, Reinke EK, Bavari S.: High-affinity, protective antibodies to the binding domain of botulinum neurotoxin type A. Infect Immun2001;69:570-574.
24.
Amersdorfer P., Marks JD: Phage libraries for generation of anti-botulinum scfv antibodies. Methods Mol Biol2000;145:219-240.
25.
Lee MS, Lee JC, Choi CY, Chung J.: Production and characterization of monoclonal antibody to botulinum neurotoxin type B light chain by phage display. Hybridoma ( Larchmt) 2008;27:18-24.
26.
Nowakowski A., Wang C., Powers DB, Amersdorfer P., Smith TJ, Montgomery VA, et al: Potent neutralization of botulinum neurotoxin by recombinant oligoclonal antibody . Proc Natl Acad Sci USA2002;99: 11346-11350.
27.
Amersdorfer P., Wong C., Smith T., Chen S., Deshpande S., Sheridan R., et al: Genetic and immunological comparison of anti-botulinum type A antibodies from immune and non-immune human phage libraries. Vaccine2002;20:1640-1648.
28.
Hoogenboom HR, Winter G.: By-passing immunization: human antibodies from synthetic repertoires of germline VH gene segments rearranged in vitro . J Mol Biol1992;227:381-388.
29.
Montecucco C.: How do tetanus and botulinum toxins bind to neuronal membranes?Trends Biochem Sci1986;11:315-317.
