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

Alvarez-Cienfuegos, Ana; Nuñez-Prado, Natalia; Compte, Marta; Cuesta, Angel M.; Blanco-Toribio, Ana; Harwood, Seandean Lykke; Villate, Maider; Merino, Nekane; Bonet Martínez, Jaume, 1982-; Navarro, Rocio; Muñoz-Briones, Clara; Sørensen, Karen Marie Juul; Mølgaard, Kasper; Oliva Miguel, Baldomero; Sanz, Laura; Blanco, Francisco; Alvarez-Vallina, Luis

doi:http://dx.doi.org/10.1038/srep28643

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

Citation

Alvarez-Cienfuegos A, Nuñez-Prado N, Compte M, Cuesta AM, Blanco-Toribio A, Harwood SL et al. Intramolecular trimerization, a novel strategy for making multispecific antibodies with controlled orientation of the antigen binding domains. Scientific Reports. 2016;6:28643. DOI: 10.1038/srep28643

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 (TIEXVIII) we produced monospecific trimerbodies that were efficiently secreted as soluble functional proteins by mammalian cells. The purified VHH-TIEXVIII trimerbodies were trimeric in solution and exhibited excellent antigen binding capacity. Furthermore, by connecting with two additional glycine-serine-based linkers three VHH-TIEXVIII 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.