CLIPSᵀᴹ conformational and discontinuous mapping
Precise and cost-effective definition of conformational and discontinuous epitopes
Given that the vast majority of therapeutic antibodies have a conformational or discontinuous epitope (Figure 1), Pepscan has developed a unique platform that can characterize such epitopes with high accuracy. At Pepscan, we address the 3D spatial conformation of epitopes by using our in-house developed and proprietary CLIPSTM (Chemical Linkage of Peptides onto Scaffolds) technology to map epitopes contained within looped, beta sheet or helical structures with constrained peptides (Figure 2).
Finding full epitopes using CLIPSTM discontinuous epitope mapping platform
A discontinuous epitope consists of non-adjacent parts of the protein sequence that form a specific 3D conformation in a protein tertiary structure. CLIPSTM discontinuous epitope mapping applies a combinatorial matrix design (Figure 3), in which sequences from the target protein are screened against each other in a bicyclic loop construct. The target protein is converted into an extensive library of conformationally constrained mimics that has sequences which are not adjacent in the primary sequence brought together by a CLIPSTM scaffold. This library of CLIPS-based tertiary structure mimics is then synthesized on a solid support using high-throughput microarray synthesis technology. If an antibody binds to a discontinuous epitope, peptide constructs containing these parts should bind at a much higher affinity than their single-loop counterparts.
Bioinformatic statistics-based analysis of the combined binding data is used to define the sequence and conformation of epitopes in detail. CLIPSTM discontinuous epitope mapping also enables detection of discontinuous epitopes involving dimeric or multimeric protein complexes, simply by introducing screening of one sequence in one loop against the sequence of a second protein in the second loop.
Case report 1: CLIPSTM precision discontinuous mapping identifies additional epitope details
The majority of antibodies have a discontinuous epitope, unless they were raised against single peptides. Mapping an anti-anthrax antibody using linear and CLIPSTM conformational mapping showed only a single part of the epitope. Supplemental mapping using matrix-style scanning of the antigen sequence through a bicyclic loop revealed additional binding sites that were directly adjacent to the dominant part of the epitope. While the dominant part was found in the linear and conformational screen, often such details will determine whether an antibody is patent-protected or if there is freedom to operate.
Case report 2: CLIPSTM precision epitope mapping differentiates anti-CD20 monoclonal antibodies
CLIPSTM precision epitope mapping revealed distinctly different binding sites on CD20 for two antibody drugs and demonstrated that one of the drugs uses a unique discontinuous binding site. This information was crucial to confirm the freedom to operate and for the antibody patent filings. It also contributed to the understanding of the mechanism of action and offered a valuable opportunity for marketing differentiation.