Highly Enhanced Cytotoxicity of a Dimeric Bispecific Diabody, the hEx3 Tetrabody

• Published in: The Journal of biological chemistry 2010-07, Vol.285 (27), p.20844-20849
• Main Authors: Asano, Ryutaro, Ikoma, Keiko, Sone, Yukiko, Kawaguchi, Hiroko, Taki, Shintaro, Hayashi, Hiroki, Nakanishi, Takeshi, Umetsu, Mitsuo, Katayose, Yu, Unno, Michiaki, Kudo, Toshio, Kumagai, Izumi
• Format: Article
• Language: English
• Subjects: Antibodies; Antibodies, Bispecific - genetics; Antibodies, Bispecific - therapeutic use; Antibodies, Bispecific - toxicity; Anticancer Drug; Antigens - immunology; Bispecific Diabody; Bispecific Tetrabody; Cancer Immunotherapy; Cancer Therapy; CD3 Complex - immunology; Cell Division - drug effects; Chromatography, Gel; Dimerization; ErbB Receptors - immunology; Humans; Killer Cells, Lymphokine-Activated - drug effects; Killer Cells, Lymphokine-Activated - immunology; Kinetics; Light; Neoplasms - drug therapy; Neoplasms - immunology; Neoplasms - pathology; Protein Folding; Protein Structure and Folding; Protein-Protein Interactions; Recombinant Proteins - therapeutic use; Scattering, Radiation; ScFv Multimer; Small Recombinant Antibody; Software; Surface Plasmon Resonance; Thermodynamics; Tyrosine Protein Kinase (Tyrosine Kinase)
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M110.120444

We previously reported the utility for cancer immunotherapy of a humanized bispecific diabody (hEx3) that targets epidermal growth factor receptor and CD3. Here, we used dynamic and static light scattering measurements to show that the multimer fraction observed in hEx3 in solution is a monodisperse tetramer. The multimerization into tetramers increased the inhibition of cancer cell growth by the hEx3 diabody. Furthermore, 1:2 stoichiometric binding for both antigens was observed in a thermodynamic analysis, indicating that the tetramer has bivalent binding activity for each target, and the structure may be in a circular configuration, as is the case for the single-chain Fv tetrabody. In addition to enhanced cytotoxicity, the functional affinity and stability of the hEx3 tetrabody were superior to those of the hEx3 diabody. The increase in molecular weight is also expected to improve the pharmacokinetics of the bispecific diabody, making the hEx3 tetrabody attractive as a therapeutic antibody fragment for cancer immunotherapy.