Production Of Multivalent Protein Binders Using A Self-Trimerizing Collagen-Like Peptide Scaffold

A class of multivalent protein binders was designed to overcome the limitations of low-affinity therapeutic antibodies. These binders, termed "collabodies," use a triplex-forming collagen-like peptide to drive the trimerization of a heterologous target-binding domain. Different forms of collabody, consisting of the human single-chain variable fragment ( scFv) fused to either the N or C terminus of the collagen-like peptide scaffold (Gly-Pro-Pro) 10, were stably expressed as soluble secretory proteins in mammalian cells. The collabody consisting of scFv fused to the N terminus of collagen scaffold is present as a homotrimer, whereas it exhibited a mixture of trimer and interchain disulfide-bonded hexamer when cysteine residues were introduced and flanked the scaffold. The collagenous motif in collabody is prolyl-hydroxylated, with remarkable thermal and serum stabilities. The collabody erb_scFvCol bound to the extracellular domain of epidermal growth factor receptor with a binding strength similar to 20- and 1000-fold stronger than the bivalent and monovalent counterparts, respectively. The trimeric collagen scaffold does not compromise the functionality of the binding moieties of parental immunoglobulin G (IgG); therefore, it could be applied to fuse other protein molecules to acquire significantly improved targeting-binding strengths.-Fan, C.-Y., Huang, C.-C., Chiu, W.-C., Lai, C.-C., Liou, G.-G., Li, H.-C., and Chou, M.-Y. Production of multivalent protein binders using a self-trimerizing collagen-like peptide scaffold. FASEB J. 22, 3795-3804 (2008)