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.
Highlights
Enhances functional affinity, decreases dissociation rates and improves biodistribution[5]
Under reducing conditions, demonstrated that αGFPN7 and αEGFRN7 trimerbodies were single-chain-type molecules with a migration pattern consistent with the molecular weights calculated from their amino acid sequences (22.4 and 27.1 kDa, respectively) (Fig. 2C)
We studied whether the binding sites of the purified ttαGFP-αCEA(1:2) and ttαGFP-αEGFR-αCEA trimerbodies can bind concurrently to their cognate antigens
Summary
Enhances functional affinity, decreases dissociation rates and improves biodistribution[5]. Engineered homotrimeric antibodies have been obtained by fusing scFv fragments with collagen-derived trimerization (TIE) domains, composed of the N-terminal trimerization region of collagen XVIII NC1 or collagen XV NC1 flanked by flexible peptide linkers[11]. Using this technology we have generated monospecific trivalent trimerbodies (N-trimerbodies or C-trimerbodies; 110 kDa) and monospecific or bispecific hexavalent trimerbodies (N/C-trimerbodies; 190 kDa). Recombinant VHH-based trimerbodies were efficiently secreted as soluble proteins by transfected human HEK-293 cells and were able to recognize their cognate antigen/s with high affinity and specificity. As this new antibody format can target two or more antigens it might have therapeutic potential in multiples diseases, simultaneously inhibiting different pathways involved in their etiopathogenesis as a means to avoid the appearance of resistance
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