title: Development of Cys38 knock-out and humanized version of NbAahII10 nanobody with improved neutralization of AahII scorpion toxin.
abstract: During scorpion envenoming, highly toxic small polypeptides of the venom diffuse rapidly within the victim, causing serious medical problems. Nanobodies (Nbs), the recombinant single-domain antigen-binding fragments of camel-specific heavy-chain only antibodies, offer special advantages in therapy over classic antibody fragments due to their robustness and smaller size, matching the size of the scorpion toxins. Recently, a potent AahII scorpion toxin-neutralizing Nb was identified. However, this NbAahII10 contains a single Cys in its first antigen-binding loop, leading to Nb dimerization upon prolonged storage. In this work, we first investigate the efficacy of NbAahII10 variants in which this Cys was substituted by Ala, Ser or Thr. Second, the NbAahII10 Cys/Ser mutant displaying the best functional properties is subsequently humanized. It is demonstrated that the maximally humanized version of NbAahII10 Cys/Ser maintains its high affinity for the antigen without conceding much on expression yield and stability. More importantly, its neutralizing capacity is preserved as all mice survive injections of seven LD(50) and 50% of mice survived nine LD(50) of the scorpion toxin. Thus, this humanized Nb is the best candidate to develop a therapy in human against the most toxic venom compound of one of the most dangerous scorpions.
authors.affiliation: Laboratoire des Venins et Toxines, Institut Pasteur de Tunis, 13 Place Pasteur, BP-74, 1002 Tunis, Tunisia.
authors.forename: Rahma
authors.initials: R
authors.lastname: Ben Abderrazek
authors.forename: Cécile
authors.initials: C
authors.lastname: Vincke
authors.forename: Issam
authors.initials: I
authors.lastname: Hmila
authors.forename: Dirk
authors.initials: D
authors.lastname: Saerens
authors.forename: Naima
authors.initials: N
authors.lastname: Abidi
authors.forename: Mohamed
authors.initials: M
authors.lastname: El Ayeb
authors.forename: Serge
authors.initials: S
authors.lastname: Muyldermans
authors.forename: Balkiss
authors.initials: B
authors.lastname: Bouhaouala-Zahar
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D061067:Antibodies, Monoclonal, Humanized / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D057134:Antibodies, Neutralizing / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D000915:Antibody Affinity; D002162:Camelus; D002850:Chromatography, Gel; D003545:Cysteine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D055785:Gene Knockdown Techniques; D006801:Humans; D007128:Immunoglobulin Fragments / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D007928:Lethal Dose 50; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009500:Neutralization Tests; D055503:Protein Multimerization; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D012604:Scorpion Venoms / Q000276:immunology* / Q000378:metabolism
chemical_list: D061067:Antibodies, Monoclonal, Humanized; D057134:Antibodies, Neutralizing; D007128:Immunoglobulin Fragments; D007143:Immunoglobulin Heavy Chains; D011994:Recombinant Proteins; D012604:Scorpion Venoms; C053969:scorpion toxin II, Androctonus; D003545:Cysteine