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

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.

AI842, AI843, AI844 and AI177 antibodies do not recognize a FLAG-tagged protein by immunofluorescence in D. discoideum cells

“…ABCD_AI177, ABCD_AI842, ABCD_AI843, and ABCD_AI844 antibodies (ABCD nomenclature, web.expasy.org/abcd/; Lima et al, 2019) were produced by the Geneva Antibody Facility (www.unige.ch/medecine/antibodies/) as mini-antibodies with the antigen-binding scFv fused to a human IgG1 Fc. The synthesized scFv sequences (GeneArt, Invitrogen) correspond to the sequences of the variable regions of the clones 2H8, EEh14.3, EEh13.6, andEEf15.4 (Sasaki et al, 2012, andEntzminger et al, 2017) joined by a peptide linker (GGGS) 3 . HEK293 suspension cells (growing in FreeStyle™ 293 Expression Medium, Gibco #12338) were transiently transfected with the vector coding for the scFv-Fc.…”

Mass spectrometry-basedde novosequencing of the anti-FLAG-M2 antibody using multiple proteases and a dual fragmentation scheme

“…There are four other monoclonal antibody sequences against the FLAG™ tag publicly available through the ABCD (AntiBodies Chemically Defined) database [34][35][36] . Comparison of the CDRs of anti-FLAG™-M2 with these additional four monoclonal antibodies reveals a few common motifs that may determine FLAG™-tag binding specificity (see Table S3).…”

OptMAVEn-2.0: De novo Design of Variable Antibody Regions against Targeted Antigen Epitopes

“…An integrated workflow would take advantage of the high affinities reached by phage display, as well as OptMAVEn-2.0′s speed (typically <24 h to design hundreds of variable domains) and abilities to minimize immunogenicity and target a specific epitope. Thus, we believe OptMAVEn-2.0 could enable the rapid design of candidate antibodies for experimental validation using only the antigen structure, unlike all other computational methods to our knowledge [16,17,18,19,55].…”