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

Abstract

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.

Tribody: Robust Self-Assembled Trimeric Targeting Ligands with High Stability and Significantly Improved Target-Binding Strength

“…Compared with other trimeric scaffolds that have been reported, 8–10,21–22 this tribody approach possesses several key advantages. First, this trimerization domain is derived from a highly conserved extracellular protein that is abundant in both mouse and human.…”

“…Different scaffolds that allow for enhanced avidity have been reported. 1,2,5–7 These scaffolds include the bacteriophage T4 foldon domain, collagen like peptide (Gly-Pro-Pro) 10 , NC1 domain of collagen XV and XVIII domain, and GCN leucine zipper domain for trimers, 1,2,8,9,10 streptavidin and transcription factor p53 for tetramers, 11 the B-subunit of bacterial verotoxin and cartilage oligomeric matrix protein (COMP) for pentamers, 12,13 and recently the hyperthermophilic Sm protein for heptamers. 6 However, most of these scaffolds are derived from non-human proteins and have limited clinical application due to immunogenicity.…”

Multimerization of Peptide Mimotopes for Blocking of Factor VIII Neutralizing Antibodies

“…The IC 50 values of homo-and heteromultimers were significantly decreased relative to the IC 50 values of synthetic peptides, presumably for similar reasons as discussed for the observed EC 50 values. In addition to multimerization via C4bp a , other methods for multimerization have been described, including the use of streptavidin/neutravidin, a collagen-like polypeptide [20] or a different peptide linker. The latter approach was also used to combine different specificities in a single molecule with increased receptor binding.…”

Peptide‐Based Inhibitors of Enzymes

“…The triple-helical structure allows for interaction with both the active site and exosites [62]. Triple-helical conformation is also less susceptible to general proteolysis than peptides and other folded proteins [63,64]. In order to create the desired phosphinate transition state analogs, our laboratory prepared protected Fmoc-phosphinodipeptides [57,[65][66][67]].…”