TY - JOUR
T1 - Intramolecular trimerization, a novel strategy for making multispecific antibodies with controlled orientation of the antigen binding domains
AU - Alvarez-Cienfuegos, Ana
AU - Nuñez-Prado, Natalia
AU - Compte, Marta
AU - Cuesta, Angel M.
AU - Blanco-Toribio, Ana
AU - Harwood, Seandean Lykke
AU - Villate, Maider
AU - Merino, Nekane
AU - Bonet, Jaume
AU - Navarro, Rocio
AU - Muñoz-Briones, Clara
AU - Sørensen, Karen Marie Juul
AU - Mølgaard, Kasper
AU - Oliva, Baldo
AU - Sanz, Laura
AU - Blanco, Francisco J.
AU - Alvarez-Vallina, Luis
PY - 2016/6/27
Y1 - 2016/6/27
N2 - 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 (V HHs) 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 V HH-TIE XVIII trimerbodies were trimeric in solution and exhibited excellent antigen binding capacity. Furthermore, by connecting with two additional glycine-serine-based linkers three V HH-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 V HH-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 V HH-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 V HH-based trimerbodies are therefore ideal candidates for future applications in various therapeutic areas.
AB - 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 (V HHs) 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 V HH-TIE XVIII trimerbodies were trimeric in solution and exhibited excellent antigen binding capacity. Furthermore, by connecting with two additional glycine-serine-based linkers three V HH-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 V HH-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 V HH-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 V HH-based trimerbodies are therefore ideal candidates for future applications in various therapeutic areas.
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U2 - 10.1038/srep28643
DO - 10.1038/srep28643
M3 - Article
C2 - 27345490
AN - SCOPUS:84976628869
SN - 2045-2322
VL - 6
JO - Scientific reports
JF - Scientific reports
M1 - 28643
ER -