title: Intramolecular trimerization, a novel strategy for making multispecific antibodies with controlled orientation of the antigen binding domains.
abstract: Here, we describe a new strategy that allows the rapid and efficient engineering of mono and multispecific trivalent antibodies. By fusing single-domain antibodies from camelid heavy-chain-only immunoglobulins (VHHs) to the N-terminus of a human collagen XVIII trimerization domain (TIE(XVIII)) we produced monospecific trimerbodies that were efficiently secreted as soluble functional proteins by mammalian cells. The purified VHH-TIE(XVIII) trimerbodies were trimeric in solution and exhibited excellent antigen binding capacity. Furthermore, by connecting with two additional glycine-serine-based linkers three VHH-TIE(XVIII) modules on a single polypeptide chain, we present an approach for the rational design of multispecific tandem trimerbodies with defined stoichiometry and controlled orientation. Using this technology we report here the construction and characterization of a tandem VHH-based trimerbody capable of simultaneously binding to three different antigens: carcinoembryonic antigen (CEA), epidermal growth factor receptor (EGFR) and green fluorescence protein (GFP). Multispecific tandem VHH-based trimerbodies were well expressed in mammalian cells, had good biophysical properties and were capable of simultaneously binding their targeted antigens. Importantly, these antibodies were very effective in inhibiting the proliferation of human epidermoid carcinoma A431 cells. Multispecific VHH-based trimerbodies are therefore ideal candidates for future applications in various therapeutic areas.
authors.affiliation: Department of Antibody Engineering, Leadartis SL, Ferraz 3, 28008 Madrid, Spain.
authors.forename: Ana
authors.initials: A
authors.lastname: Alvarez-Cienfuegos
authors.affiliation: Molecular Immunology Unit, Hospital Universitario Puerta de Hierro Majadahonda, Manuel de Falla 1, 28222 Madrid, Spain.
authors.forename: Natalia
authors.initials: N
authors.lastname: Nuñez-Prado
authors.affiliation: Department of Antibody Engineering, Leadartis SL, Ferraz 3, 28008 Madrid, Spain.
authors.forename: Marta
authors.initials: M
authors.lastname: Compte
authors.affiliation: Molecular Immunology Unit, Hospital Universitario Puerta de Hierro Majadahonda, Manuel de Falla 1, 28222 Madrid, Spain.
authors.forename: Angel M
authors.initials: AM
authors.lastname: Cuesta
authors.affiliation: Department of Antibody Engineering, Leadartis SL, Ferraz 3, 28008 Madrid, Spain.
authors.forename: Ana
authors.initials: A
authors.lastname: Blanco-Toribio
authors.affiliation: Department of Engineering, Aarhus University, Gustav Wieds Vej 10, 8000 C Aarhus, Denmark.
authors.forename: Seandean Lykke
authors.initials: SL
authors.lastname: Harwood
authors.affiliation: Structural Biology Unit, CIC bioGUNE, Parque Tecnológico de Bizkaia, 48160 Derio, Spain.
authors.forename: Maider
authors.initials: M
authors.lastname: Villate
authors.affiliation: Structural Biology Unit, CIC bioGUNE, Parque Tecnológico de Bizkaia, 48160 Derio, Spain.
authors.forename: Nekane
authors.initials: N
authors.lastname: Merino
authors.affiliation: Structural Bioinformatics Laboratory, Biomedical Informatics Research Unit, Parc de Recerca Biomèdica de Barcelona, 08003 Barcelona, Spain.
authors.forename: Jaume
authors.initials: J
authors.lastname: Bonet
authors.affiliation: Molecular Immunology Unit, Hospital Universitario Puerta de Hierro Majadahonda, Manuel de Falla 1, 28222 Madrid, Spain.
authors.forename: Rocio
authors.initials: R
authors.lastname: Navarro
authors.affiliation: Molecular Immunology Unit, Hospital Universitario Puerta de Hierro Majadahonda, Manuel de Falla 1, 28222 Madrid, Spain.
authors.forename: Clara
authors.initials: C
authors.lastname: Muñoz-Briones
authors.affiliation: Molecular Immunology Unit, Hospital Universitario Puerta de Hierro Majadahonda, Manuel de Falla 1, 28222 Madrid, Spain.|Department of Engineering, Aarhus University, Gustav Wieds Vej 10, 8000 C Aarhus, Denmark.
authors.forename: Karen Marie Juul
authors.initials: KM
authors.lastname: Sørensen
authors.affiliation: Department of Engineering, Aarhus University, Gustav Wieds Vej 10, 8000 C Aarhus, Denmark.
authors.forename: Kasper
authors.initials: K
authors.lastname: Mølgaard
authors.affiliation: Structural Bioinformatics Laboratory, Biomedical Informatics Research Unit, Parc de Recerca Biomèdica de Barcelona, 08003 Barcelona, Spain.
authors.forename: Baldo
authors.initials: B
authors.lastname: Oliva
authors.affiliation: Molecular Immunology Unit, Hospital Universitario Puerta de Hierro Majadahonda, Manuel de Falla 1, 28222 Madrid, Spain.
authors.forename: Laura
authors.initials: L
authors.lastname: Sanz
authors.affiliation: Structural Biology Unit, CIC bioGUNE, Parque Tecnológico de Bizkaia, 48160 Derio, Spain.|IKERBASQUE, Basque Foundation for Science, 48013 Bilbao, Spain.
authors.forename: Francisco J
authors.initials: FJ
authors.lastname: Blanco
authors.affiliation: Department of Engineering, Aarhus University, Gustav Wieds Vej 10, 8000 C Aarhus, Denmark.
authors.forename: Luis
authors.initials: L
authors.lastname: Alvarez-Vallina
mesh_terms: D000818:Animals; D018033:Antibodies, Bispecific / Q000737:chemistry / Q000235:genetics; D002161:Camelids, New World; D006801:Humans; D051379:Mice; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics; D057127:Single-Chain Antibodies / Q000737:chemistry / Q000235:genetics
chemical_list: D018033:Antibodies, Bispecific; D011993:Recombinant Fusion Proteins; D057127:Single-Chain Antibodies