Development of a bispecific antibody tetramerized through hetero-associating peptides.
The specific assembly of self-associating peptides can be useful in building a functional antibody complex from small antibody fragments. We have focused on the exceedingly specific heterotetrameric assembly of Lin-2 and Lin-7 (L27) domains, which work as protein-protein interaction modules in many scaffold proteins. Here, we describe a novel method for constructing a highly functional antibody based on the hetero-association of L27 domains. In this study, we used a bacterial expression system to produce a bispecific antibody that was heterotetramerized through L27 domains and that targeted both epidermal growth factor receptor (EGFR) and Fcγ receptor III (FcγRIII or CD16). Gel electrophoresis, mass spectrometry and gel filtration analyses revealed that the constructed recombinant antibody was a disulfide-linked heterotetramer. The tetramerized antibody bound to EGFR and CD16 simultaneously, according to results from flow cytometry and surface plasmon resonance spectroscopy, respectively. Furthermore, we demonstrated that the bispecific antibody showed cytotoxic activity against EGFR-expressing tumor cells by using CD16-positive lymphocytes as effectors, and its cytotoxicity was comparable to that of a commercial therapeutic antibody. Taken together, the results show that our method has high potential for the cost-efficient production of highly active therapeutic antibodies.
Department of Applied Chemistry and Bioengineering, Graduate School of Engineering, Osaka City University, Japan.
Tomohiro
T
Osaki
Department of Applied Chemistry and Bioengineering, Graduate School of Engineering, Osaka City University, Japan.
Shingo
S
Fujisawa
Department of Applied Chemistry and Bioengineering, Graduate School of Engineering, Osaka City University, Japan.
Masahiro
M
Kitaguchi
Department of Applied Chemistry and Bioengineering, Graduate School of Engineering, Osaka City University, Japan.
Masaya
M
Kitamura
Department of Applied Chemistry and Bioengineering, Graduate School of Engineering, Osaka City University, Japan.
Takeshi
T
Nakanishi
D018033:Antibodies, Bispecific / Q000737:chemistry* / Q000276:immunology; D002478:Cells, Cultured; D003602:Cytotoxicity, Immunologic; D066246:ErbB Receptors / Q000276:immunology*; D006801:Humans; D007694:Killer Cells, Natural / Q000276:immunology; D055503:Protein Multimerization; D017452:Receptors, IgG / Q000276:immunology*; D020349:Surface Plasmon Resonance
D018033:Antibodies, Bispecific; D017452:Receptors, IgG; D066246:ErbB Receptors
CD16; bispecific antibody; cancer therapy; epidermal growth factor receptor; self-associating peptide