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_scFv-Col bound to the extracellular domain of epidermal growth factor receptor with a binding strength approximately 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
Chia-Yu
CY
Biomedical Engineering Research Laboratories, Industrial Technology Research Institute, Bldg. 53, No 195, Sec. 4, Chung Hsing Rd., Chutung, Hsinchu 310, Taiwan, Republic of China.
Huang
Chuan-Chuan
CC
Chiu
Wei-Chun
WC
Lai
Chun-Chieh
CC
Liou
Gunn-Guang
GG
Li
Hsiu-Chuan
HC
Chou
Min-Yuan
MY
D000818:Animals; D045744:Cell Line, Tumor; D003094:Collagen / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D066246:ErbB Receptors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D051379:Mice; D010455:Peptides / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D020836:Protein Structure, Quaternary / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D007135:Immunoglobulin Variable Region; D010455:Peptides; D011993:Recombinant Fusion Proteins; D003094:Collagen; C512478:EGFR protein, human; D066246:ErbB Receptors