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

Chia-Yu Fan^1,*, Chuan-Chuan Huang^1,*, Wei-Chun Chiu^*, Chun-Chieh Lai, Gunn-Guang Liou, Hsiu-Chuan Li^* and Min-Yuan Chou^2,*

^* Biomedical Engineering Research Laboratories, Industrial Technology Research Institute, Taiwan; and

Division of Molecular and Genomic Medicine, National Health Research Institutes, Taiwan

²Correspondence: Biomedical Engineering Research Laboratories, Industrial Technology Research Institute, Bldg. 53, No 195, Sec. 4, Chung Hsing Rd., Chutung, Hsinchu 310, Taiwan, Republic of China. E-mail: minyuanc{at}itri.org.tw

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.

Key Words: phage-display • single-chain antibody • EGFR • CD3