Production of multivalent protein binders using a self-trimerizing collagen-like peptide scaffold

• Published in: The FASEB journal 2008-11, Vol.22 (11), p.3795-3804
• Main Authors: Fan, Chia-Yu, Huang, Chuan-Chuan, Chiu, Wei-Chun, Lai, Chun-Chieh, Liou, Gunn-Guang, Li, Hsiu-Chuan, Chou, Min-Yuan
• Format: Article
• Language: English
• Subjects: Animals; CD3; Cell Line, Tumor; Collagen - chemistry; Collagen - genetics; Collagen - metabolism; EGFR; Humans; Immunoglobulin Variable Region - chemistry; Immunoglobulin Variable Region - genetics; Immunoglobulin Variable Region - metabolism; Mice; Peptides - chemistry; Peptides - genetics; Peptides - metabolism; phage‐display; Protein Binding - genetics; Protein Structure, Quaternary - genetics; Receptor, Epidermal Growth Factor - chemistry; Receptor, Epidermal Growth Factor - genetics; Receptor, Epidermal Growth Factor - metabolism; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; single‐chain antibody
• ISSN: 0892-6638; 1530-6860
• DOI: 10.1096/fj.08-111484

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_scFv-Col bound to the extracellular domain of epidermal growth factor receptor with a binding strength ~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.