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  • Intramolecular trimerization, a novel strategy for making multispecific antibodies with controlled orientation of the antigen binding domains.

Intramolecular trimerization, a novel strategy for making multispecific antibodies with controlled orientation of the antigen binding domains.

Scientific reports (2016-06-28)
Ana Alvarez-Cienfuegos, Natalia Nuñez-Prado, Marta Compte, Angel M Cuesta, Ana Blanco-Toribio, Seandean Lykke Harwood, Maider Villate, Nekane Merino, Jaume Bonet, Rocio Navarro, Clara Muñoz-Briones, Karen Marie Juul Sørensen, Kasper Mølgaard, Baldo Oliva, Laura Sanz, Francisco J Blanco, Luis Alvarez-Vallina
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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Human Serum, from human male AB plasma, USA origin, sterile-filtered
Sigma-Aldrich
Anti-Mouse IgG (Fc specific)–Peroxidase antibody produced in goat, affinity isolated antibody
Sigma-Aldrich
Anti-Rabbit IgG (whole molecule)–Peroxidase antibody produced in goat, IgG fraction of antiserum, buffered aqueous solution
Sigma-Aldrich
Carcinoembryonic Antigen, Human Colon Adenocarcinoma Cell Line, Carcinoembryonic Antigen, Human Colon Adenocarcinoma Cell Line, is a native CEA from human colon adenocarcinoma cells.