Understanding the Significance and Implications of Antibody Numbering and Antigen-Binding Surface/Residue Definition

Abstract

Monoclonal antibodies are playing an increasing role in both human and animal health. Different strategies of protein and chemical engineering, including humanization techniques of non-human antibodies were applied successfully to optimize clinical performances of antibodies. Despite the emergence of techniques allowing the development of fully human antibodies such as transgenic Xeno-mice, antibody humanization remains a standard procedure for therapeutic antibodies. An important prerequisite for antibody humanization requires standardized numbering methods to define precisely complementary determining regions (CDR), frameworks and residues from the light and heavy chains that affect the binding affinity and/or specificity of the antibody-antigen interaction. The recently generated deep-sequencing data and the increasing number of solved three-dimensional structures of antibodies from human and non-human origins have led to the emergence of numerous databases. However, these different databases use different numbering conventions and CDR definitions. In addition, the large fluctuation of the variable chain lengths, especially in CDR3 of heavy chains (CDRH3), hardly complicates the comparison and analysis of antibody sequences and the identification of the antigen binding residues. This review compares and discusses the different numbering schemes and “CDR” definition that were established up to date. Furthermore, it summarizes concepts and strategies used for numbering residues of antibodies and CDR residues identification. Finally, it discusses the importance of specific sets of residues in the binding affinity and/or specificity of immunoglobulins.

A compact vocabulary of paratope-epitope interactions enables predictability of antibody-antigen binding

“…Abhinandan and Martin further refined the Chothia numbering scheme by making corrections, not only in the CDRs but also in the FRs. Here, we used the Martin numbering scheme to annotate the FRs and CDRs of antibodies as it was found to be suitable for structural and antibody engineering (Dondelinger et al, 2018) and it is the most recent of the presently available numbering schemes. Supplementary Table S1 summarizes the position of FR and CDR regions and the position of insertions according to the Martin numbering scheme.…”

“…While the percentage of interacting residues found in the CDR3 was consistent between heavy and light chains, only half as many interacting residues mapped to CDR-H1 (14.8%) as compared to CDR-L1 (32.5%), whereas the reverse was true for CDR-H2 (36.1%) and CDR-L2 (13.9%). Since we used the Martin numbering scheme for CDR and FR annotation (see Methods), which mostly excludes germline gene residues from the CDR3, the above numbers demonstrate that germline-gene residues surrounding the CDR3 (FR3, FR4) contribute relatively little to antibody-interaction and that CDR3 paratope-epitope interaction is essentially non-germline gene residue driven (Abhinandan and Martin, 2008;Dondelinger et al, 2018). Finally, we found that paratope interacting residues correlate positively with the sites of (inferred) somatic hypermutation (SHM) (Spearman (Pearson) correlation: 0.31-0.52 (0.44-0.58), Suppl Fig.…”

Loxoscelism: Advances and Challenges in the Design of Antibody Fragments with Therapeutic Potential

“…The most commonly employed method for humanization consists in grafting murine CDRs into whole human framework regions. Still, it has been demonstrated that some murine framework residues, denoted as vernier zone residues, are able to interfere in the CDR loops conformation thus affecting antibody binding affinity [38,41]. Although many methods have been reported for humanization, the immunogenicity of therapeutic antibodies is still a discussed matter [42,43].…”

Conformational changes in a Vernier zone region: Implications for antibody dual specificity

“…The main differences between all antibody variable domains are content, structure, and conformations of CDR loops. Non‐CDR framework regions are mostly conserved and have a high degree of structural conservation forming a core β‐sheet structure 28 . Although several studies point out that non‐CDR regions are important for biophysical properties 29 and humanization, 30,31 their roles on affinity/specificity are underestimated 29,32 .…”

“…Back mutations on the Vernier zone can provide the desired canonical structure of CDRs to obtain restored binding affinity 36 . However, there is no study investigating the relationship between Vernier zone residues and antibody specificity 28 . Although Vernier zone residues are mostly engineered for humanization efforts to regain/improve affinity, it can be hypothesized that features of Vernier zone residues might also affect many antibody properties such as binding specificity.…”