title: Humanization of a phosphothreonine peptide-specific chicken antibody by combinatorial library optimization of the phosphoepitope-binding motif.
abstract: Detection of protein phosphorylation at a specific residue has been achieved by using antibodies, which have usually been raised by animal immunization. However, there have been no reports of the humanization of phosphospecific non-human antibodies. Here, we report the humanization of a chicken pT231 antibody specific to a tau protein-derived peptide carrying the phosphorylated threonine at residue 231 (pT231 peptide) as a model for better understanding the phosphoepitope recognition mechanism. In the chicken antibody, the phosphate group of the pT231-peptide antigen is exclusively recognized by complementarity determining region 2 of the heavy chain variable domain (VH-CDR2). Simple grafting of six CDRs of the chicken antibody into a homologous human framework (FR) template resulted in the complete loss of pT231-peptide binding. Using a yeast surface-displayed combinatorial library with permutations of 11 FR residues potentially affecting CDR loop conformations, we identified 5 critical FR residues. The back mutation of these residues to the corresponding chicken residues completely recovered the pT231-peptide binding affinity and specificity of the humanized antibody. Importantly, the back mutation of the FR 76 residue of VH (H76) (Asn to Ser) was critical in preserving the pT231-binding motif conformation via allosteric regulation of ArgH71, which closely interacts with ThrH52 and SerH52a residues on VH-CDR2 to induce the unique phosphate-binding bowl-like conformation. Our humanization approach of CDR grafting plus permutations of FR residues by combinatorial library screening can be applied to other animal antibodies containing unique binding motifs on CDRs specific to posttranslationally modified epitopes.
authors.affiliation: Department of Molecular Science and Technology, Ajou University, Suwon 443-749, Republic of Korea.
authors.forename: Du-San
authors.initials: DS
authors.lastname: Baek
authors.affiliation: Department of Molecular Science and Technology, Ajou University, Suwon 443-749, Republic of Korea. Electronic address: kimys@ajou.ac.kr.
authors.forename: Yong-Sung
authors.initials: YS
authors.lastname: Kim
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D061067:Antibodies, Monoclonal, Humanized / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D000915:Antibody Affinity; D002645:Chickens; D015723:Gene Library; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010769:Phosphothreonine / Q000737:chemistry / Q000276:immunology*; D015202:Protein Engineering; D016875:tau Proteins / Q000737:chemistry / Q000276:immunology*
chemical_list: D061067:Antibodies, Monoclonal, Humanized; D007135:Immunoglobulin Variable Region; D016875:tau Proteins; D010769:Phosphothreonine
keywords: Antibody engineering; Chicken antibody; Humanization; Phosphospecific antibody; Phosphothreonine-binding motif