title: Neutralizing human monoclonal antibodies binding multiple serotypes of botulinum neurotoxin.
abstract: Botulism, a disease of humans characterized by prolonged paralysis, is caused by botulinum neurotoxins (BoNTs), the most poisonous substances known. There are seven serotypes of BoNT (A-G) which differ from each other by 34-64% at the amino acid level. Each serotype is uniquely recognized by polyclonal antibodies, which originally were used to classify serotypes. To determine if there existed monoclonal antibodies (mAbs) capable of binding two or more serotypes, we evaluated the ability of 35 yeast-displayed single-chain variable fragment antibodies generated from vaccinated humans or mice for their ability to bind multiple BoNT serotypes. Two such clonally related human mAbs (1B18 and 4E17) were identified that bound BoNT serotype A (BoNT/A) and B or BoNT/A, B, E and F, respectively, with high affinity. Using molecular evolution techniques, it proved possible to both increase affinity and maintain cross-serotype reactivity for the 4E17 mAb. Both 1B18 and 4E17 bound to a relatively conserved epitope at the tip of the BoNT translocation domain. Immunoglobulin G constructed from affinity matured variants of 1B18 and 4E17 were evaluated for their ability to neutralize BoNT/B and E, respectively, in vivo. Both antibodies potently neutralized BoNT in vivo demonstrating that this epitope is functionally important in the intoxication pathway. Such cross-serotype binding and neutralizing mAbs should simplify the development of antibody-based BoNT diagnostics and therapeutics.
authors.affiliation: Department of Anesthesia and Pharmaceutical Chemistry, University of California-San Francisco, Rm 3C-38, San Francisco General Hospital, 1001 Potrero Ave., San Francisco, CA 94110, USA.
authors.forename: C
authors.initials: C
authors.lastname: Garcia-Rodriguez
authors.forename: I N
authors.initials: IN
authors.lastname: Geren
authors.forename: J
authors.initials: J
authors.lastname: Lou
authors.forename: F
authors.initials: F
authors.lastname: Conrad
authors.forename: C
authors.initials: C
authors.lastname: Forsyth
authors.forename: W
authors.initials: W
authors.lastname: Wen
authors.forename: S
authors.initials: S
authors.lastname: Chakraborti
authors.forename: H
authors.initials: H
authors.lastname: Zao
authors.forename: G
authors.initials: G
authors.lastname: Manzanarez
authors.forename: T J
authors.initials: TJ
authors.lastname: Smith
authors.forename: J
authors.initials: J
authors.lastname: Brown
authors.forename: W H
authors.initials: WH
authors.lastname: Tepp
authors.forename: N
authors.initials: N
authors.lastname: Liu
authors.forename: S
authors.initials: S
authors.lastname: Wijesuriya
authors.forename: M T
authors.initials: MT
authors.lastname: Tomic
authors.forename: E A
authors.initials: EA
authors.lastname: Johnson
authors.forename: L A
authors.initials: LA
authors.lastname: Smith
authors.forename: J D
authors.initials: JD
authors.lastname: Marks
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D057134:Antibodies, Neutralizing / Q000737:chemistry / Q000276:immunology*; D000915:Antibody Affinity; D000918:Antibody Specificity; D001905:Botulinum Toxins / Q000737:chemistry / Q000276:immunology*; D016466:CHO Cells; D017124:Conserved Sequence; D006224:Cricetinae; D003412:Cricetulus; D003429:Cross Reactions; D019020:Directed Molecular Evolution; D018604:Epitope Mapping; D000939:Epitopes / Q000276:immunology; D005260:Female; D006801:Humans; D007074:Immunoglobulin G / Q000276:immunology; D051379:Mice; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D057127:Single-Chain Antibodies / Q000235:genetics / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D057134:Antibodies, Neutralizing; D000939:Epitopes; D007074:Immunoglobulin G; D057127:Single-Chain Antibodies; D001905:Botulinum Toxins