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 n m . This method applies to various mAb drugs and allows the rational design of small protein alternatives.