Reconstitution of an Anti‐HER2 Antibody Paratope by Grafting Dual CDR‐Derived Peptides onto a Small Protein Scaffold

Abstract

Target-binding small proteins are promising alternatives to conventional monoclonal antibodies (mAbs), offering advantages in terms of tissue penetration and manufacturing costs. Recently, a design strategy to create small proteins called fluctuation-regulated affinity proteins (FLAPs) consisting of a structurally immobilized peptide from the complementarity-determining region (CDR) loops of mAbs (CDR-derived peptide) and a protein scaffold was developed. Because mAb paratopes are usually composed of multiple CDRs, FLAPs with multiple binding peptides may have an enhanced target-binding capability. Here, a strategy to create FLAPs bearing dual CDR-derived peptides (D-FLAPs) using the anti-human epithelial growth factor receptor type 2 (HER2) mAb trastuzumab as a basis is developed. Computationally selected CDR-derived peptides are first grafted onto two adjacent loops of the fibronectin type III domain (FN3) scaffold, yielding 80 D-FLAP candidates. After computational screening based on their similarity to the parental mAb with regard to the conformation of paratope residues, two candidates are selected. After further evaluation with ELISA, one D-FLAP with HYTTPP and GDGFYA peptides from CDR-L3 and CDR-H3 of the parental mAb, respectively, is found to bind HER2 with a dissociation constant of 58 nm. This method applies to various mAb drugs and allows the rational design of small protein alternatives.

Mutational and biophysical robustness in a prestabilized monobody

“…Furthermore, there is growing evidence that monobody scaffolds can take on valuable loop sequences and their resulting affinity to clinical targets (56), such as in the transfer of anti-HER2 CDRs (57) or the anchoring of peptides (58). However, it is unclear how much of the previously published antibody CDR loop sequences can be transferred to monobody domains, especially if it is a complicated process that involves iterative redesign (55).…”

Nanomaterials for Biomedical Applications

“…Also, the preparation of nanostructures through self‐assembly and engineering of natural substances such as peptides, magnetosomes, and exosomes is introduced, [ 10–12 ] In these reports, the successful fabrication of nanovesicles with better affinity and stability for cancer cell targets is suggested with magnetic reactivity or tissue‐regeneration potential. Another example is the use of the physalis mottle virus nanoparticles for intelligent delivery of an anti‐cancer drug.…”

A reengineered common chain cytokine augments CD8+ T cell–dependent immunotherapy

“…As OMCP binds to NKG2D as a single domain, it provides advantages over antibody-mediated delivery, where dimerization of heavy and light chains must occur for optimal target binding. In addition, OMCP's small size facilitates tissue penetration over larger antibodies (57). Furthermore, monomeric OMCP binding does not result in NKG2D costimulation (Supplemental Figure 1A), a phenomenon that might occur with antibody-mediated clustering of receptors.…”