title: Novel selection method for engineered antibodies using the mechanism of Fv fragment stabilization in the presence of antigen.
abstract: Although the heavy and light chain domains of some antibody variable region fragments (Fvs) readily dissociate under physiological conditions, the Fvs are stable in the presence of antigen. This 'antigen-driven Fv stabilization mechanism' was applied to the selection of clones with specificity toward target antigens. The results can be summarized as follows. (i) Some of the residues in the heavy chain complementarity determining region 2 (HCDR2) of anti-hen egg white lysozyme (HEL) monoclonal antibody HyHEL10 heavy chain variable region (VH) were randomized. (ii) The randomized VH fragments of HyHEL10 were displayed on a filamentous bacteriophage and mixed with the target antigen, before being applied to a light chain variable region (VL) which was immobilized on microtiter plates and subjected to selection by panning. (iii) After four rounds of panning, four clones that showed significant binding to human lysozyme (hL), which HyHEL10 recognized poorly, were selected from the HCDR2 library. (iv) The soluble Fv fragments selected were expressed in Escherichia coli, purified, and subjected to an inhibition assay of lysozyme enzymatic activities and an isothermal titration calorimetry. These Fv fragments had increased affinity toward hL, and thermodynamic analysis suggested that the reduced entropy loss due to binding by the replacement of residues in HCDR2 resulted in the higher hL binding activity.
authors.affiliation: Department of Biochemistry and Engineering, Graduate School of Engineering, Tohoku University, Sendai, Japan.
authors.forename: K
authors.initials: K
authors.lastname: Tsumoto
authors.forename: Y
authors.initials: Y
authors.lastname: Nishimiya
authors.forename: N
authors.initials: N
authors.lastname: Kasai
authors.forename: H
authors.initials: H
authors.lastname: Ueda
authors.forename: T
authors.initials: T
authors.lastname: Nagamune
authors.forename: K
authors.initials: K
authors.lastname: Ogasahara
authors.forename: K
authors.initials: K
authors.lastname: Yutani
authors.forename: K
authors.initials: K
authors.lastname: Tokuhisa
authors.forename: M
authors.initials: M
authors.lastname: Matsushima
authors.forename: I
authors.initials: I
authors.lastname: Kumagai
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000276:immunology / Q000494:pharmacology; D000941:Antigens / Q000276:immunology*; D002151:Calorimetry; D002645:Chickens; D004355:Drug Stability; D004591:Electrophoresis, Polyacrylamide Gel; D015723:Gene Library; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry / Q000235:genetics* / Q000276:immunology*; D007128:Immunoglobulin Fragments / Q000737:chemistry / Q000235:genetics* / Q000276:immunology*; D007147:Immunoglobulin Light Chains / Q000235:genetics / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000276:immunology*; D008969:Molecular Sequence Data; D009113:Muramidase / Q000037:antagonists & inhibitors / Q000276:immunology; D016296:Mutagenesis; D015202:Protein Engineering; D013816:Thermodynamics
chemical_list: D000911:Antibodies, Monoclonal; D000941:Antigens; D007141:Immunoglobulin Fc Fragments; D007128:Immunoglobulin Fragments; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; C055966:immunoglobulin Fv; D009113:Muramidase