Design Strategy to Create Antibody Mimetics Harbouring Immobilised Complementarity Determining Region Peptides for Practical Use

Tetsuya Kadonosono,Shinae Kizaka-Kondoh,Tadashi Shiozawa,Wanaporn Yimchuen,Takahiro Kuchimaru,Kyra See,Akash Patil,Yumi Ota,Yu Goto,Maika Kitazawa,Tadaomi Furuta

doi:10.1038/s41598-020-57713-4

Abstract

Monoclonal antibodies (mAbs) are attractive therapeutics for treating a wide range of human disorders, and bind to the antigen through their complementarity-determining regions (CDRs). Small stable proteins containing structurally retained CDRs are promising alternatives to mAbs. In this report, we present a method to create such proteins, named fluctuation-regulated affinity proteins (FLAPs). Thirteen graft acceptor (GA) sites that efficiently immobilise the grafted peptide structure were initially selected from six small protein scaffolds by computational identification. Five CDR peptides extracted by binding energy calculations from mAbs against breast cancer marker human epithelial growth factor receptor type 2 (HER2) were then grafted to the selected scaffolds. The combination of five CDR peptides and 13 GA sites in six scaffolds revealed that three of the 65 combinations showed specific binding to HER2 with dissociation constants (KD) of 270–350 nM in biolayer interferometry and 24–65 nM in ELISA. The FLAPs specifically detected HER2-overexpressing cancer cells. Thus, the present strategy is a promising and practical method for developing small antibody mimetics.

Highlights

The antigen-binding surface of Monoclonal antibodies (mAbs) is often composed of six complementarity-determining regions (CDRs), spread across the heavy chain and light chain variable domains
Our fast and easy method for developing fluctuation-regulated affinity proteins (FLAPs) includes computational methods for selecting short peptides from the CDR that help with binding to the target and for determining the graft acceptor (GA) sites, which are crucial for proper immobilisation of the grafted short peptide structure, in small protein scaffolds
We aimed to develop a strategy to computationally design small antibody mimetics, named FLAPs, by selecting adequate small protein scaffolds and extracting target-binding sequences from mAb drugs (Fig. 1)

Summary

Introduction

The antigen-binding surface of mAbs is often composed of six complementarity-determining regions (CDRs), spread across the heavy chain and light chain variable domains. Small antibody mimetics targeting lysozyme have been generated by grafting CDRs from a single domain antibody to non-Ig scaffolds, including neocarzinostatin[12], ubiquitin[13] and Affitins[14], mimicking the CDR conformations from the parental antibody These small antibody mimetics have the same CDRs; they showed various affinities toward lysozyme (KD = 0.5–230 μM), indicating that scaffolds affect the affinity of grafted CDRs. when designing antibody mimetics for practical use, it is extremely important both to select appropriate non-Ig scaffolds and to determine the proper peptide sequences in CDRs. In this report, we aimed to develop a clinically applicable small antibody mimetic termed fluctuation-regulated affinity proteins (FLAPs). Among the 65 anti-HER2 FLAP candidates created, three bind to the same epitope as the parental mAb, and their KD values are within a clinically relevant range that binds to high HER2-expressing cells but reduces on-target off-tumour binding risk, demonstrating that our strategy is a promising method to develop small antibody mimetics that could contribute to molecular-targeted therapy and diagnosis

Objectives

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Results

Conclusion