Di-diabody: a novel tetravalent bispecific antibody molecule by design.
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.
Lu
Dan
D
Department of Antibody Technology, ImClone Systems Incorporated, New York, NY 10014, USA.
Jimenez
Xenia
X
Zhang
Haifan
H
Atkins
Amanda
A
Brennan
Laura
L
Balderes
Paul
P
Bohlen
Peter
P
Witte
Larry
L
Zhu
Zhenping
Z
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
D043925:Angiogenesis Inducing Agents; D018033:Antibodies, Bispecific; D000941:Antigens; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A