De novo design of antibody complementarity determining regions binding a FLAG tetra-peptide

Kevin C Entzminger,Costas D Maranas,Robert J Pantazes,Ahlam N Qerqez,Andrew D Ellington,Jeong-Min Hyun,Athena C Patterson-Orazem,Raquel L Lieberman,Jennifer A Maynard,Zach P Frye,Randall A Hughes

doi:10.1038/s41598-017-10737-9

Abstract

Computational antibody engineering efforts to date have focused on improving binding affinities or biophysical characteristics. De novo design of antibodies binding specific epitopes could greatly accelerate discovery of therapeutics as compared to conventional immunization or synthetic library selection strategies. Here, we employed de novo complementarity determining region (CDR) design to engineer targeted antibody–antigen interactions using previously described in silico methods. CDRs predicted to bind the minimal FLAG peptide (Asp–Tyr–Lys–Asp) were grafted onto a single-chain variable fragment (scFv) acceptor framework. Fifty scFvs comprised of designed heavy and light or just heavy chain CDRs were synthesized and screened for peptide binding by phage ELISA. Roughly half of the designs resulted in detectable scFv expression. Four antibodies, designed entirely in silico, bound the minimal FLAG sequence with high specificity and sensitivity. When reformatted as soluble antigen-binding fragments (Fab), these clones expressed well, were predominantly monomeric and retained peptide specificity. In both formats, the antibodies bind the peptide only when present at the amino-terminus of a carrier protein and even conservative peptide amino acid substitutions resulted in a complete loss of binding. These results support in silico CDR design of antibody specificity as an emerging antibody engineering strategy.

Highlights

Antibodies are one of the most important protein classes, widely used in commercial diagnostics and therapeutics that garnered global sales of $75 billion in 20131
Optimal CDR (OptCDR) starts with a database of canonical structure backbones for each complementarity determining region (CDR) derived from known antibody structures
Given a position of the antigen, for each canonical structure, OptCDR calculates a geometric score where CDR backbone atoms close to the antigen are rewarded as having the potential to contribute to binding but van der Waals clashes are penalized

Summary

Introduction

Antibodies are one of the most important protein classes, widely used in commercial diagnostics and therapeutics that garnered global sales of $75 billion in 20131. A computational method, Optimal CDR (OptCDR) for de novo design of antibody binding interfaces complementary to specific three-dimensional epitopes was recently reported[14] This approach is unique in that it first selects canonical CDR backbone loop structures, decorates them with specific amino acid side chains, using energy minimization to refine the structure and maximize predicted interactions with the target epitope. We used this approach to design CDRs binding the minimal FLAG peptide (sequence: DYKD)[15]. These results support de novo CDR design to target specific epitopes as a viable engineering approach

Methods

Results

Conclusion