title: Design and Generation of Humanized Single-chain Fv Derived from Mouse Hybridoma for Potential Targeting Application.
abstract: Single-chain variable antibody fragments (scFvs) are attractive candidates for targeted immunotherapy in several human diseases. In this study, a concise humanization strategy combined with an optimized production method for humanizing scFvs was successfully employed. Two antibody clones, one directed against the hemagglutinin of H5N1 influenza virus, the other against EpCAM, a cancer biomarker, were used to demonstrate the validity of the method. Heavy chain (VH) and light chain (VL) variable regions of immunoglobulin genes from mouse hybridoma cells were sequenced and subjected to the construction of mouse scFv 3-D structure. Based on in silico modeling, the humanized version of the scFv was designed via complementarity-determining region (CDR) grafting with the retention of mouse framework region (FR) residues identified by primary sequence analysis. Root-mean-square deviation (RMSD) value between mouse and humanized scFv structures was calculated to evaluate the preservation of CDR conformation. Mouse and humanized scFv genes were then constructed and expressed in Escherichia coli. Using this method, we successfully generated humanized scFvs that retained the targeting activity of their respective mouse scFv counterparts. In addition, the humanized scFvs were engineered with a C-terminal cysteine residue (hscFv-C) for site-directed conjugation for use in future targeting applications. The hscFv-C expression was extensively optimized to improve protein production yield. The protocol yielded a 20-fold increase in production of hscFv-Cs in E. coli periplasm. The strategy described in this study may be applicable in the humanization of other antibodies derived from mouse hybridoma.
authors.affiliation: 1 Research Division, Faculty of Medicine Siriraj Hospital, Mahidol University , Bangkok, Thailand .
authors.forename: Kannika
authors.initials: K
authors.lastname: Khantasup
authors.affiliation: 2 Graduate Program in Immunology, Faculty of Medicine Siriraj Hospital, Mahidol University , Bangkok, Thailand .|4 National Nanotechnology Center , National Science and Technology Development Agency, Pathumthani, Thailand .
authors.forename: Warangkana
authors.initials: W
authors.lastname: Chantima
authors.affiliation: 5 Department of Chemistry, Faculty of Science, Kasetsart University , Bangkok, Thailand .
authors.forename: Chak
authors.initials: C
authors.lastname: Sangma
authors.affiliation: 6 Biotechnology Program, School of Bioresources and Technology, King Mongkut's University of Technology , Bangkok, Thailand .
authors.forename: Kanokwan
authors.initials: K
authors.lastname: Poomputsa
authors.affiliation: 3 Department of Immunology, Faculty of Medicine Siriraj Hospital, Mahidol University , Bangkok, Thailand .|4 National Nanotechnology Center , National Science and Technology Development Agency, Pathumthani, Thailand .
authors.forename: Tararaj
authors.initials: T
authors.lastname: Dharakul
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D061067:Antibodies, Monoclonal, Humanized / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D000941:Antigens / Q000032:analysis* / Q000235:genetics / Q000276:immunology; D000951:Antigens, Neoplasm / Q000032:analysis / Q000235:genetics / Q000276:immunology; D015815:Cell Adhesion Molecules / Q000032:analysis / Q000235:genetics / Q000276:immunology; D003001:Cloning, Molecular; D022801:Complementarity Determining Regions / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D003198:Computer Simulation; D000071858:Epithelial Cell Adhesion Molecule; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000032:analysis / Q000235:genetics / Q000276:immunology; D006801:Humans; D006825:Hybridomas / Q000737:chemistry / Q000276:immunology; D007143:Immunoglobulin Heavy Chains / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D007147:Immunoglobulin Light Chains / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D017422:Sequence Analysis, DNA; D057127:Single-Chain Antibodies / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D040681:Structural Homology, Protein
chemical_list: D061067:Antibodies, Monoclonal, Humanized; D000941:Antigens; D000951:Antigens, Neoplasm; D015815:Cell Adhesion Molecules; D022801:Complementarity Determining Regions; C499379:EPCAM protein, human; D000071858:Epithelial Cell Adhesion Molecule; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D011994:Recombinant Proteins; D057127:Single-Chain Antibodies; C483468:hemagglutinin, human influenza A virus