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 2 Correspondence: 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@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
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Production of multivalent protein binders using a self-trimerizing collagen-like peptide scaffold
Fan, Chia-Yu; Huang, Chuan-Chuan; Chiu, Wei-Chun; Lai, Chun-Chieh; Liou, Gunn-Guang; Li, Hsiu-Chuan; Chou, Min-Yuan
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, Volume 22 (11): 3795
Fed of American Socs for Experimental Biology – Nov 1, 2008
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