title: Fab-scFv fusion protein: an efficient approach to production of bispecific antibody fragments.
abstract: The clinical development of bispecific antibodies (BsAb) as therapeutics has been hampered by the difficulty in preparing the materials in sufficient quantity and quality by traditional methods. Here, we describe an efficient approach for the production of a novel bispecific antibody fragment by genetically fusing a single-chain Fv (scFv) to the C-terminus of either the light chain or the heavy chain of a Fab fragment of different antigen-binding specificity. The bispecific Fab-scFv fragments were expressed in a single Escherichia coli host and purified to homogeneity by a one-step affinity chromatography. Two different versions of the bispecific Fab-scFv fragments were constructed using two antibodies directed against the two tyrosine kinase receptors of vascular endothelial growth factor. These bispecific antibody fragments not only retained the antigen-binding capacity of each of the parent antibodies, but also are capable of binding to both targets simultaneously as demonstrated by a cross-linking ELISA. Further, the bispecific antibodies were comparable to their parent antibodies in their potency in blocking ligand binding to the receptors and in inhibiting ligand-induced biological activities. This design for BsAb fragments should be applicable to any pair of antigen specificities.
authors.affiliation: Department of Antibody Technology, ImClone Systems Incorporated, 180 Varick Street, New York, NY 10014, USA.
authors.forename: Dan
authors.initials: D
authors.lastname: Lu
authors.forename: Xenia
authors.initials: X
authors.lastname: Jimenez
authors.forename: Haifan
authors.initials: H
authors.lastname: Zhang
authors.forename: Peter
authors.initials: P
authors.lastname: Bohlen
authors.forename: Larry
authors.initials: L
authors.lastname: Witte
authors.forename: Zhenping
authors.initials: Z
authors.lastname: Zhu
mesh_terms: D000818:Animals; D018033:Antibodies, Bispecific / Q000096:biosynthesis* / Q000235:genetics; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D002465:Cell Movement / Q000187:drug effects; D015195:Drug Design; D016228:Endothelial Growth Factors / Q000037:antagonists & inhibitors / Q000276:immunology / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000096:biosynthesis / Q000235:genetics; D007128:Immunoglobulin Fragments / Q000096:biosynthesis* / Q000235:genetics; D007700:Kinetics; D008222:Lymphokines / Q000037:antagonists & inhibitors / Q000276:immunology / Q000494:pharmacology; D051379:Mice; D000072483:Placenta Growth Factor; D011257:Pregnancy Proteins / Q000037:antagonists & inhibitors / Q000276:immunology / Q000494:pharmacology; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D014407:Tumor Cells, Cultured; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors
chemical_list: D018033:Antibodies, Bispecific; D016228:Endothelial Growth Factors; D007140:Immunoglobulin Fab Fragments; D007128:Immunoglobulin Fragments; D008222:Lymphokines; C000608036:PGF protein, human; C000608037:Pgf protein, mouse; D011257:Pregnancy Proteins; D011993:Recombinant Fusion Proteins; D042461:Vascular Endothelial Growth Factor A; D042442:Vascular Endothelial Growth Factors; C055966:immunoglobulin Fv; D000072483:Placenta Growth Factor