title: Di-diabody: a novel tetravalent bispecific antibody molecule by design.
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. In recent years, a variety of recombinant methods have been developed for efficient production of BsAb, both as antibody fragments and as full-length IgG-like molecules. These recombinant antibody molecules possess dual antigen-binding capability with, in most cases, monovalency to each of their target antigens. Here, we describe an efficient approach for the production of a novel tetravalent BsAb, with two antigen-binding sites to each of its target antigens, by genetically fusing a bispecific/divalent diabody to, via the hinge region, the N-terminus of the CH(3) domain of an IgG. The novel BsAb, which we termed "di-diabody", represents a tetravalent diabody dimer resulting from dimerization between the hinge region and the CH(3) domains. A di-diabody was constructed using two antibodies directed against the two tyrosine kinase receptors of vascular endothelial growth factor, expressed both in a single Escherichia coli host and in mammalian cells, and purified to homogeneity by a one-step affinity chromatography. Compared to the bispecific/divalent diabody, the tetravalent di-diabody binds more efficiently to both of its target antigens and is more efficacious in blocking ligand binding to the receptors. The di-diabody retained good antigen-binding activity after incubation at 37 degrees C in mouse serum for 72 h, demonstrating good product stability. Finally, expression of the di-diabody in mammalian cells yielded higher level of production and better antibody activity. This design and expression for BsAb fragments should be applicable to any pair of antigen specificities.
authors.affiliation: Department of Antibody Technology, ImClone Systems Incorporated, 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: Amanda
authors.initials: A
authors.lastname: Atkins
authors.forename: Laura
authors.initials: L
authors.lastname: Brennan
authors.forename: Paul
authors.initials: P
authors.lastname: Balderes
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: D043925:Angiogenesis Inducing Agents / Q000378:metabolism; D018033:Antibodies, Bispecific / Q000276:immunology*; D000918:Antibody Specificity / Q000276:immunology; D000941:Antigens / Q000276:immunology; D002465:Cell Movement / Q000276:immunology / Q000502:physiology; D003001:Cloning, Molecular; D004730:Endothelium, Vascular / Q000276:immunology; D006801:Humans; D007700:Kinetics; D007938:Leukemia / Q000276:immunology / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods*; D042461:Vascular Endothelial Growth Factor A
chemical_list: D043925:Angiogenesis Inducing Agents; D018033:Antibodies, Bispecific; D000941:Antigens; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A