Fine mapping

Define the epitope to single residue level

Fine mapping determines the role of each individual residue within the epitope and investigates the tolerance of an antibody’s binding to variants of the optimal binder. Initial peptide mapping studies using overlapping linear, conformational, or discontinuous mapping may identify stretches of residues of 3-8 amino acids, depending on the mode of binding. Within these stretches, not all residues will be involved in the binding.

Our fine mapping platform

Full single amino acid replacement scanning involves the stepwise substitution of all amino acid positions of an identified epitope with all 20 proteinogenic (natural) amino acids. In certain cases, non-natural or specifically modified residues are available upon request. Substitutions which result in increased binding may also be of interest. This sort of information can provide insights into the thermodynamics of epitope-paratope binding. For protein-protein (peptide) interaction, fine mapping enables the development of superior peptide binders as potential inhibitors of protein activity (binding competitors, enzyme inhibitors, etc.) and is therefore frequently applied by Pepscan for peptide lead optimization.

Epitope length scanning complements full substitution analysis and employs a comprehensive library of epitope candidates of all lengths. These candidates are tested with the antibody to identify the best fit based on binding intensity.

Case report 1: Fine mapping of the linear epitope of anti-HIV monoclonal antibodies (F425-B4e8)

The F425-B4e8 is one of a few broadly neutralizing anti-HIV monoclonal antibodies. The antibody recognizes the highly flexible V3 variable loop region on the gp120 subunit of the HIV-1 virus. A deeper understanding of recognition mechanisms of broadly neutralizing antibodies would not only extend our knowledge of HIV-1 entry, but also may inspire new immunogen designs for vaccine development. In this study, linear mapping and subsequent full replacement analysis were used to examine the fine features of the F425-B4e8 epitope.

The top binding sequence 306RKRIHIGPGRAFYT319 was identified in a library of fully overlapping linear peptides based on the sequence of gp160. A full single amino replacement analysis, in which each position of the epitope was substituted with all of the other natural amino acids, was then conducted. Binding of F425-B4e8 to each epitope permutation was recorded and compared to that of the native sequence (Figure 1, bottom right). The experiment revealed that within motif 311IGPGRAF317, all amino acids are essential for the binding; most substitutions diminished the antibody binding. Most of the replacements of I309 significantly decreased the binding, but did not fully abolish it.

These Pepscan results are in line with those obtained by X-ray crystallography (adapted from Bell et al., J Mol Biol. 2008, 2QSC.pdb; bottom left), but allow even more detailed determination of the paratope tolerance.

Fine mapping

Figure 1. Initially, the linear epitope for antibody F425-B4e8 was mapped using a linear epitope mapping approach. The binding profile is shown at top left. To reveal the fine features of the antibody binding, a full replacement analysis was used, as outlined at top right. In short, such analysis enables the identification of binding-essential residues and provides context for the binding. At bottom right is a letter plot that shows how the mutation of positions ₃₁₁IGPGRAF₃₁₇ diminishes antibody binding, while replacements of other residues are mostly tolerated. Agreement of the results obtained from Pepscan’s analysis and X-ray crystallography is visualized using 3D coordinate file 2qsc.pdb at bottom left.