title: Affinity transfer to a human protein by CDR3 grafting of camelid VHH.
abstract: VHH is the binding domain of the IgG heavy chain. Some VHHs have an extremely long CDR3 that contributes to antigen binding. We studied the antigen binding ability of CDR3 by grafting a CDR3 from an antigen-binding VHH onto a nonbinding VHH. cAb-CA05-(1RI8), the CDR3-grafted VHH, had an antigen-binding ability. To find a human scaffold protein acceptable for VHH CDR3 grafting, we focused on the conserved structure of VHH, especially the N-terminal and C-terminal amino acid residues of the CDR3 loop and the Cys residue of CDR1. Human origin protein structures with the same orientation were searched in PDB and ubiquitin was selected. Ubi-(1RI8), the CDR3-grafted ubiquitin, had antigen-binding ability, though the affinity was relatively low compared to cAb-CA05-(1RI8). The thermodynamic parameters of Ubi-(1RI8) binding to HEWL were different from cAb-CA05-(1RI8). Hydrogen-deuterium exchange experiments showed decreased stability around the CDR3 grafting region of Ubi-(1RI8), which might explain the decreased antigen-binding ability and the differences in thermodynamic properties. We concluded that the orientation of the CDR3 sequence of Ubi-(1RI8) could not be reconstructed correctly.
authors: Kyowa Hakko Kirin Co., Ltd., Machida, Tokyo, Japan.
authors: Hidetoshi
authors: H
authors: Inoue
authors: Akiko
authors: A
authors: Iihara
authors: Hideo
authors: H
authors: Takahashi
authors: Ichio
authors: I
authors: Shimada
authors: Isao
authors: I
authors: Ishida
authors: Yoshitake
authors: Y
authors: Maeda
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002161:Camelids, New World / Q000276:immunology*; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry* / Q000235:genetics / Q000276:immunology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology*; D016415:Sequence Alignment; D025801:Ubiquitin / Q000737:chemistry* / Q000235:genetics / Q000276:immunology*
chemical_list: D007143:Immunoglobulin Heavy Chains; D011994:Recombinant Proteins; D025801:Ubiquitin