CLIPSᵀᴹ conformational and discontinuous epitope mapping

Figure 1: Conformational epitope (left) and discontinuous epitope (right), as highlighted in red in the ribbon diagrams of ubiquitin. To indicate this part in the sequence, it is also highlighted in the horizontal bars, which represents the sequences from N- to C-terminus.

Precise and cost-effective definition of conformational epitopes 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).

Figure 2. Linearly generated peptides are cyclized via insertion of cysteines at specific locations in the peptide chain. A chemical linker is added, which is then CLIPSed into a constrained peptide. Illustrated here is the formation of a looped peptide, and an example showing that an antibody recognizing a loop in a structure of follicle stimulating hormone (FSH) can be recognized only if the CLIPS reaction is applied.

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.

Figure 3. The target protein containing a discontinuous conformational epitope (left) is converted into a library of linear peptides as well as CLIPSTM constructs via a combinatorial matrix design. Peptides are synthesized on a proprietary minicard and chemically converted into spatially defined CLIPS constructs (right). Antibody binding is quantified using an automated ELISA-type readout. Constructs representing both parts of the discontinuous epitope in the correct orientation are best for binding to the antibody.

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.

Figure 4. Discontinuous epitope mapping revealed additional parts of the epitope for an anti-anthrax antibody. While the linear (green) and conformational (blue) mapping did reveal a single part of the epitope (left), our matrix discontinuous scan revealed other parts of the epitope that contribute to the binding, as well (right). The higher intensity peptides are highlighted in red.

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.


Figure 5. Discontinuous epitope mapping revealed distinctly different binding sites on CD20 for two antibody drugs and demonstrated a clear and unique discontinuous binding site for one of the antibody drugs, confirming the freedom to operate.

We use cookies to ensure that we give you the best experience on our website. If you would like to, you can change your cookie settings in your browser.
For more information check our privacy policy
Find out more
Accept All Cookies
By continuing to browse or by clicking "Accept All Cookies" you agree to the storing of first and third-party cookies on your device to enhance site navigation, analyze site usage, and assist in our marketing efforts.
Cookie Policy
Cookie Settings
Accept All Cookies