RESUMO
Bispecific antibodies present unique opportunities in terms of new applications for engineered antibodies. However, designing ideal bispecific antibodies remains a challenge. Here we describe a novel bispecific antibody model in which five single domain antibodies (sdAbs) are fused via a linker sequence to the N-terminus of the verotoxin B (VTB) subunit, a pentamerization domain, and five sdAbs are fused via a linker sequence to the VTB C-terminus. Fifteen such decavalent bispecific molecules, termed decabodies, were constructed and characterized for the purpose of identifying an optimal decabody design. One of the fifteen molecules existed in a non-aggregated decavalent form. In conjunction with the isolation of sdAbs with the desired specificities from non-immune phage display libraries, the decabody strategy provides a means of generating high avidity bispecific antibody reagents, with good physical properties, relatively quickly.
Assuntos
Anticorpos Biespecíficos/biossíntese , Proteínas Recombinantes de Fusão/imunologia , Toxinas Shiga/genética , Sequência de Aminoácidos , Animais , Anticorpos Biespecíficos/química , Anticorpos Biespecíficos/imunologia , Afinidade de Anticorpos/imunologia , Reações Antígeno-Anticorpo/imunologia , Camelídeos Americanos , Cromatografia em Gel , Vetores Genéticos/genética , Modelos Moleculares , Dados de Sequência Molecular , Peso Molecular , Proteína Relacionada ao Hormônio Paratireóideo/imunologia , Fragmentos de Peptídeos/imunologia , Subunidades Proteicas/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Ressonância de Plasmônio de SuperfícieRESUMO
We describe a novel type of molecule in which single-domain antibodies (sdAbs) isolated from a nai;ve llama single domain antibody library are linked to an oligomerization domain to generate high-avidity, antigen-binding reagents. An sdAb is fused to the B-subunit of Escherichia coli verotoxin, or shiga-like toxin, which self-assembles to form a homopentamer and results in simultaneous sdAb pentamerization and introduction of avidity. Molecular modeling indicated that this fusion protein (PDB: 1OJF), termed pentabody, has structural flexibility for binding to surface-presented antigen. In the instance of an sdAb specific for a peptide antigen, pentamerization resulted in a dramatic increase in functional affinity for immobilized antigen. The pentabody was expressed in high yield in E.coli in a non-aggregated state, and exhibited excellent thermostability and protease resistance. This technology provides a relatively rapid means of generating novel antigen-binding molecules that bind strongly to immobilized antigen. It is expected that pentavalent sdAbs will have general applicability in proteomics, immunochemical staining, cancer diagnosis and other applications in which antigens are presented multivalently.
