SEEDbodies: fusion proteins based on strand-exchange engineered domain (SEED) CH3 heterodimers in an Fc analogue platform for asymmetric binders or immunofusions and bispecific antibodies

• Published in: Protein engineering, design and selection Vol. 23; no. 4; pp. 195 - 202
• Main Authors: Davis, Jonathan H., Aperlo, Christel, Li, Yue, Kurosawa, Emmi, Lan, Yan, Lo, Kin-Ming, Huston, James S.
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 01.04.2010
• Subjects: Amino Acid Sequence; Antibodies, Bispecific - chemistry; Antibodies, Bispecific - genetics; Antibodies, Bispecific - metabolism; Antibodies, Monoclonal - genetics; Antibodies, Monoclonal - metabolism; Antibody Specificity; CH3 domain; Dimerization; heterodimeric Fc analogue; Immunoglobulin Fc Fragments - chemistry; Immunoglobulin Fc Fragments - genetics; Immunoglobulin Fc Fragments - metabolism; Immunoglobulin G - chemistry; Immunoglobulin G - genetics; Immunoglobulin G - metabolism; Models, Molecular; Molecular Sequence Data; mono-Fab SEEDbody; Protein Engineering - methods; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; SEEDbody mono-IL2 fusion protein; Sequence Alignment; β-strand-exchange engineered domain; SEEDbody mono-IL2 fusion protein; β-strand-exchange engineered domain; C; heterodimeric Fc analogue; mono-Fab SEEDbody; 3 domain
• ISSN: 1741-0126; 1741-0134
• DOI: 10.1093/protein/gzp094

Bispecific antibodies and asymmetric Fc fusion proteins offer opportunities for important advances in therapeutics. Bivalent IgG depends upon in vivo dimerization of its heavy chains, mediated by homodimeric association of its CH3 domains. We have developed a heterodimeric Fc platform that supports the design of bispecific and asymmetric fusion proteins by devising strand-exchange engineered domain (SEED) CH3 heterodimers. These derivatives of human IgG and IgA CH3 domains create complementary human SEED CH3 heterodimers that are composed of alternating segments of human IgA and IgG CH3 sequences. The resulting pair of SEED CH3 domains preferentially associates to form heterodimers when expressed in mammalian cells. SEEDbody (Sb) fusion proteins consist of [IgG1 hinge]-CH2-[SEED CH3], that may be genetically linked to one or more fusion partners. This investigation reports on the generation of mono-Fab-Sb and Sb-IL2 monocytokine as models. They were expressed at high levels in NS/0 cells, purified on recombinant protein A resin and were well-behaved in solution. When administered intravenously to mice, Sb pharmacokinetics exhibited the long serum half-life extensions typical of comparable Fc-containing immunofusion and IgG1 controls.