Understanding differences between synthetic and natural antibodies can help improve antibody engineering

Abstract

Synthetic libraries are a major source of human-like antibody (Ab) drug leads. To assess the similarity between natural Abs and the products of these libraries, we compared large sets of natural and synthetic Abs using "CDRs Analyzer," a tool we introduce for structural analysis of Ab-antigen (Ag) interactions. Natural Abs, we found, recognize their Ags by combining multiple complementarity-determining regions (CDRs) to create an integrated interface. Synthetic Abs, however, rely dominantly, sometimes even exclusively on CDRH3. The increased contribution of CDRH3 to Ag binding in synthetic Abs comes with a substantial decrease in the involvement of CDRH2 and CDRH1. Furthermore, in natural Abs CDRs specialize in specific types of non-covalent interactions with the Ag. CDRH1 accounts for a significant portion of the cation-pi interactions; CDRH2 is the major source of salt-bridges and CDRH3 accounts for most hydrogen bonds. In synthetic Abs this specialization is lost, and CDRH3 becomes the main sources of all types of contacts. The reliance of synthetic Abs on CDRH3 reduces the complexity of their interaction with the Ag: More Ag residues contact only one CDR and fewer contact 3 CDRs or more. We suggest that the focus of engineering attempts on CDRH3 results in libraries enriched with variants that are not natural-like. This may affect not only Ag binding, but also Ab expression, stability and selectivity. Our findings can help guide library design, creating libraries that can bind more epitopes and Abs that better mimic the natural antigenic interactions.

Keywords: Antigen binding site; antibody–antigen interactions; human-like antibodies; libraries; synthetic.

Figure 1.

The binding contribution score of CDRs in natural and synthetic Abs –Binding contribution score (varies between 4 to 16) of each CDR in each Ab-Ag complex was calculated using the “CDRs Analyzer.” On the Y axis are the average scores of a given CDR across all of the natural (white bars) or synthetic (gray bar) Ab-Ag complexes. Error bars represent standard error.

Figure 2.

Distribution of H-bonds, salt-bridges and cation-pi across the CDRs- Percentage of salt-bridges, H-bonds and cation-pi interactions (top to bottom) that occur in each CDR in natural Abs and synthetic Abs. Labels are composed of CDR name (e.g., H1,L2) and the percentage of the specific interaction that are from residues in this CDR .

Figure 3.

Complexity of Ab-Ag interactions in terms of independent and integrated epitope residues – The average percentage of independent residues in the epitope (Ag residues contacting only one CDR), are shown for natural and synthetic Abs (A) and for each CDR of the 2 groups (B). The average percentage of integrated residues in the epitope (Ag residues contacting at least 3 CDRs) are shown for natural Ab and synthetic Abs (C) and for each CDR of the 2 groups (D). An Integrated residue in the epitope was attributed to a certain CDR if the residue contacts that CDR and at least 2 other CDRs. Error Bars represent standard error.

Figure 4.

Natural and synthetic Ab-Ag interactions - The complex between hemagglutinin (HA) and the natural 2D1 Ab (PDBID: 3LZF) (A) and the complex between membrane-type serine protease 1 (MT-SP1) and the synthetic E2 Abs (PDBID: 3BN9) (B) are shown. Ags are presented in a gray surface view. Abs are presented in gray ribbon for non CDRs residues. CDRs are in ribbon representation colored according to their binding contribution score, from low contribution (blue) to high contribution (red). Images were generated using Discovery Studio Visualizer. Each Ab-Ag complex is accompanied by a table, detailing the calculated parameters, the binding contribution score and the percent of independent and integrated epitope residues.