CLIPS Discontinuous Epitope Mapping

Precisely defines discontinuous and conformational epitopes

Given that the vast majority of therapeutic antibodies have a conformational or discontinuous epitope, Pepscan has developed a unique, high resolution mapping technology to characterize such epitopes with high accuracy. Many leading companies apply our CLIPS Precision Epitope Mapping to their most promising antibodies, several of which recently received FDA breakthrough status.

As a discontinuous epitope consists of non-adjacent parts of the protein sequence, CLIPS Discontinuous Epitope Mapping applies a combinatorial matrix design and different CLIPS scaffolds to convert the target protein into an extensive library of conformationally constrained mimics that has sequences which are not adjacent in the primary sequence brought together by a CLIPS scaffold. This library of CLIPS-based tertiary structure mimics is then synthesized on a solid support, using high-throughput microarray synthesis technology. The binding of the antibody to each peptide construct of the entire library is determined. Designed constructs containing both parts of the interaction site in the correct orientation are bound with the highest affinity by the test antibody, which is detected and quantified. Constructs representing the incomplete epitope bind the antibody with much lower affinity, whereas constructs not containing (parts of) the epitope are not bound by the antibody at all.  Bioinformatic statistics-based analysis of the combined binding data is used to define the sequence and conformation of epitopes in detail. CLIPS Precision Epitope Mapping allows detecting of conformational, discontinuous, and complex epitopes involving dimeric or multimeric protein complexes. For further details see the Technology section.

Strong binding of antibodies to CLIPS peptide libraries

The target protein containing a discontinuous conformational epitope (left cartoon) is converted into a library of linear peptides as well as CLIPS constructs via a combinatorial matrix design. Peptides are synthesized on a proprietary minicard and chemically converted into spatially defined CLIPS constructs (right). Binding of antibodies is quantified using an automated ELISA-type read-out. Constructs representing both parts of the discontinuous epitope in the correct orientation best binds the antibody.

CLIPS Precision Epitope Mapping offers unrivalled single residue resolution and can determine the role of individual amino acids to the binding of an antibody (Fine Mapping). Because the arrays can be washed and re-used multiple times, it is the only method for comparative mapping of a series of samples.

Case example: CLIPS Precision Epitope Mapping differentiates Ofatumumab from Rituximab

CLIPS Precision Epitope Mapping revealed distinctly different binding sites for rituximab and ofatumumab on CD20 and demonstrated that ofatumumab uses a unique discontinuous binding site. This information was crucial in assuring freedom to operate and for patent filings for ofatumumab. It also contributed to the understanding of the mechanism of action and offered a valuable opportunity for marketing differentiation.

Distinctly different binding sites for Rituximab and Ofatumumab identified with Precision Epitope Mapping

Precision Epitope Mapping revealed distinctly different binding sites for rituximab and ofatumumab on CD20 and demonstrated that ofatumumab uses a unique discontinuous binding site.

The only technology for all types of epitopes:
Linear, conformational and discontinuous

Re-usable arrays for multiple screenings:
Comparative mapping and epitope fingerprinting of up to 100’s of samples

Highest sensitivity through high peptide density:
Also effective for weakly binding antibodies

Structurally & functionally customized peptide arrays:
Include post-translational modifications, cyclizations, helices, β-sheets

Applicable to all kinds of samples:
Mabs, antibody-like scaffolds and polyclonal sera

Applicable to all kinds of target proteins:
Soluble as well as membrane integrated proteins, viral capsids

Unrivalled single residue resolution:
Solid support for patent claims and Freedom to Operate assessments

Reliable, fast and cost-effective:
No-crystallization required, multiple screenings on one array

Full service with minimal material consumption:
20 μg antibody or 20 μl of serum + target protein sequence (Uniprot/FASTA)

Precision Epitope Mapping:

Using its extensive expertise in peptide synthesis and vaccine development Pepscan has developed the Precision Epitope Mapping platform to profile all types of epitopes for big panels of biological samples (antibodies and antibody fragments, purified proteins and sera). Applying its thorough expertise in structured peptides, Pepscan generated various strategies in addressing linear, conformational and discontinuous epitopes via fully customized library designs and bio-informatical data analysis tools.

Linear vs Conformational Epitope Mapping

Linear Epitope Mapping

The concept of mapping linear epitopes using libraries of overlapping synthetic peptides was for pioneered by Pepscan founders Geysen and Meloen. Since then this technology was widely applied by many companies and research groups for various projects. As the inventor of the technology Pepscan has long standing expertise in addressing linear epitopes by directly synthesizing libraries of linear peptides on a solid support covered with a proprietary hydrogel formulation, which allows working with biomolecules and can be easily regenerated for profiling big sample sets. To generate a library of linear mimics, the correct amino acid sequence of the immunogen (or target protein) is split in overlapping fragments in silico, which are then synthesized on a solid support. Once the linear array is synthesized, binding of a test antibody to such library is quantified and compared via an ELISA. When the epitope sequence is present in linear peptides, the antibody avidly binds this set of peptides (as schematically shown below).

Precision Epitope Mapping Technology with linear overlapping peptides

The target linear sequence is converted into a library of all overlapping linear peptides directly synthesized on a proprietary solid support called “mini-card”. Binding of antibodies is quantified using an automated ELISA-type read-out. Constructs containing the right amino acid sequence in the correct conformation best bind the antibody.

However, the majority of biomolecules of therapeutic interest recognize conformational or discontinuous epitopes, which cannot be reliably (if at all) addressed by means of linear epitope mapping. For many antibodies, the primary sequence of amino acids is not sufficient for binding and additional 3D structure features are needed. This is why Pepscan perfected its platform to enable systematic mapping of conformational and discontinuous epitopes.

CLIPS Epitope Mapping

One example is creating simple secondary structure mimics by applying different CLIPS scaffolds allowing to thermodynamically favour a limited series of peptide conformations. In such a manner CLIPS peptide library can mimic secondary structure elements, such as loops, α-helixes and β-strands. A schematic representation of this approach is drawn in the figure below, where all three secondary structure elements present in the target’s 3D structure are mimicked using various CLIPS chemistry strategies.

Conformational Epitope Mapping

The target protein contains α-helixes, β-sheets separated by loops is converted into different conformational libraries using a CLIPS scaffold. Peptides are synthesized on a proprietary minicard and chemically converted into spatially defined CLIPS constructs (right). Binding of antibodies is quantified using an automated ELISA-type read-out. Constructs containing the right amino acid sequence in the correct conformation best bind the antibody.

It is also possible to create a large combinatorial library of CLIPS based tertiary structure mimics. Using a combinatorial matrix design and different CLIPS scaffolds, 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 on a CLIPS scaffold. This library of CLIPS-based tertiary structure mimics is then synthesized on a solid support, using high-throughput microarray synthesis technology.
Subsequently, the binding of the antibody to each construct of the entire library is determined, using an automated ELISA-type read-out. This identifies those CLIPS-constructs that best mimic the interaction site of interest. A schematic representation of the approach is presented in the figure below. Designed constructs containing both parts of the interaction site in the correct orientation are bound with the highest affinity by the test antibody, which is detected and quantified. Constructs representing theincomplete epitope bind the antibody with much lower affinity, whereas constructs not containing (parts of) the epitope are not bound by the antibody at all. Bioinformatic statistics-based analysis of the combined binding data is used to define the sequence and conformation of epitopes in detail. CLIPS Precision Epitope Mapping also allows detecting of conformational, discontinuous, and complex epitopes involving dimeric or multimeric protein complexes.

Strong binding of conformational peptide to epitope

The target protein containing a discontinuous conformational epitope (left cartoon) is converted into a library of linear peptides as well as CLIPS constructs via a combinatorial matrix design. Peptides are synthesized on a proprietary minicard and chemically converted into spatially defined CLIPS constructs (right). Binding of antibodies is quantified using an automated ELISA-type read-out. Constructs representing both parts of the discontinuous epitope in the correct orientation best binds the antibody.

Making surface-bound conformationally constrained peptide libraries

The Precision Epitope Mapping is based on Pepscan’s proprietary platform for making microarrays containing large libraries of surface-immobilized linear, secondary and tertiary structure CLIPS-based epitope mimics. Using high-throughput parallel microarray synthesis technology, a full library of linear, conformational and discontinuous epitope mimics, is synthesized on a proprietary surface with a polymeric graft optimized for low non-specific binding and high peptide construct loading resulting in high sensitivity of the Precision Epitope Mapping technology. Via Pepscan’s patented CLIPS technology these peptides are structurally fixed into defined three-dimensional structures. This enables mimicking even the most complex binding sites. The CLIPS technology is now routinely used to create peptide libraries of single- or double- looped structures, as well as sheet- and helix-like folds.

CLIPS technology; from linear peptide to T3 double loop peptide

Using the CLIPS technology, peptides derived from native proteins are transformed into CLIPS constructs with a range of structures. From left to right: linear, single mP2 loops, stabilized beta sheet, alpha helix, and T3 double loop.

Precision Epitope Mapping: how does it work?

Using its extensive expertise in peptide synthesis and vaccine development Pepscan has developed the Precision Epitope Mapping platform to profile all types of epitopes for big panels of biological samples (antibodies and antibody fragments, purified proteins and sera). Applying its thorough expertise in structured peptides, Pepscan generated various strategies in addressing linear, conformational and discontinuous epitopes via fully customized library designs and bio-informatical data analysis tools.

The concept of mapping linear epitopes using libraries of overlapping synthetic peptides was for pioneered by Pepscan founders Geysen and Meloen. Since then this technology was widely applied by many companies and research groups for various projects. As the inventor of the technology Pepscan has long standing expertise in addressing linear epitopes by directly synthesizing libraries of linear peptides on a solid support covered with a proprietary hydrogel formulation, which allows working with biomolecules and can be easily regenerated for profiling big sample sets. To generate a library of linear mimics, the correct amino acid sequence of the immunogen (or target protein) is split in overlapping fragments in silico, which are then synthesized on a solid support. Once the linear array is synthesized, binding of a test antibody to such library is quantified and compared via an ELISA. When the epitope sequence is present in linear peptides, the antibody avidly binds this set of peptides (as schematically shown below).

Linear concept

The target linear sequence is converted into a library of all overlapping linear peptides directly synthesized on a proprietary solid support called “mini-card”. Binding of antibodies is quantified using an automated ELISA-type read-out. Constructs containing right amino acid sequence in the correct conformation best bind the antibody.

However, the majority of biomolecules of therapeutic interest recognize conformational or discontinuous epitopes, which cannot be reliably (if at all) addressed by means of linear epitope mapping. For many antibodies the primary sequence of amino acids is not sufficient for binding and additional 3D structure features are needed. This is why Pepscan perfected its platform to enable systematic mapping of conformational and discontinuous epitopes.

One example is creating simple secondary structure mimics by applying different CLIPS scaffolds allowing to thermodynamically favour a limited series of peptide conformations. In such a manner CLIPS peptide libraries can mimic secondary structure elements, such as loops, α-helixes and β-strands. A schematic representation of this approach is drawn in the figure below, where all three secondary structure elements present in the target’s 3D structure are mimicked using various CLIPS chemistry strategies.

Conformational concept

The target protein contains α-helixes, β-sheets separated by loops is converted into different conformational libraries using a CLIPS scaffold. Peptides are synthesized on a proprietary minicard and chemically converted into spatially defined CLIPS constructs (right). Binding of antibodies is quantified using an automated ELISA-type read-out. Constructs containing the right amino acid sequence in the correct conformation best bind the antibody.

It is also possible to create a large combinatorial library of CLIPS based tertiary structure mimics. Using a combinatorial matrix design and different CLIPS scaffolds, 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 on a CLIPS scaffold. This library of CLIPS-based tertiary structure mimics is then synthesized on a solid support, using high-throughput microarray synthesis technology.
Subsequently the binding of the antibody to each construct of the entire library is determined, using an automated ELISA-type read-out. This identifies those CLIPS-constructs that best mimic the interaction site of interest. A schematic representation of the approach is presented in the figure below. Designed constructs containing both parts of the interaction site in the correct orientation are bound with the highest affinity by the test antibody, which is detected and quantified. Constructs representing theincomplete epitope bind the antibody with much lower affinity, whereas constructs not containing (parts of) the epitope are not bound by the antibody at all. Bioinformatic statistics-based analysis of the combined binding data is used to define the sequence and conformation of epitopes in detail. CLIPS Precision Epitope Mapping also allows detecting of conformational, discontinuous, and complex epitopes involving dimeric or multimeric protein complexes.

Discontinuous concept

The target protein containing a discontinuous conformational epitope (left cartoon) is converted into a library of linear peptides as well as CLIPS constructs via a combinatorial matrix design. Peptides are synthesized on a proprietary minicard and chemically converted into spatially defined CLIPS constructs (right). Binding of antibodies is quantified using an automated ELISA-type read-out. Constructs representing both parts of the discontinuous epitope in the correct orientation best binds the antibody.

Making surface-bound conformationally constrained peptide libraries

The Precision Epitope Mapping is based on Pepscan’s proprietary platform for making microarrays containing large libraries of surface-immobilized linear, secondary and tertiary structure CLIPS-based epitope mimics.
Using high-throughput parallel microarray synthesis technology, a full library of linear, conformational and discontinuous epitope mimics, is synthesized on a proprietary surface with a polymeric graft optimized for low non-specific binding and high peptide construct loading resulting in high sensitivity of the Precision Epitope Mapping technology. Via Pepscan’s patented CLIPS technology these peptides are structurally fixed into defined three-dimensional structures. This enables mimicking even the most complex binding sites.
The CLIPS technology is now routinely used to create peptide libraries of single- or double- looped structures, as well as sheet- and helix-like folds.

All mimics

Using the CLIPS technology, peptides derived from native proteins are transformed into CLIPS constructs with a range of structures. From left to right: linear, single mP2 loops, stabilized beta sheet, alpha helix, and T3 double loop.

Peptide synthesisFMOC chemistry. Maximum peptide length over 40 residues. All amino acids including D-amino acids and non-natural amino acids.
Capacity50.000 peptides per run with custom high-througput parallel synthesis robots.
Peptide library formatProprietary ‘Minicard’ format with solid phase-bound peptide constructs in 455 microwells. Surface chemistry: proprietary polymeric graft optimized for low non-specific binding and high peptide construct loading.
Combinatorial library complexityMatrix analysis e.g. 50 x 50 = 2.500 double loop T3 CLIPS™. All matrix combinations within 40-mers possible. All overlapping single loops usually 15 – 20-mers. All overlapping peptides of a protein usually 15 – 20-mers. Full positional scan libraries of all epitopes.
Spatial construct complexitySingle loops on T2 CLIPS.
Double loop combinations on T3 or 2 x T2 CLIPS
Sheet-like T2 CLIPS. Helix-like T2 CLIPS.
All loop structures with 2-6 cysteines and 1 or 2 CLIPS.
Peptide library reusabilityAt least 20 times but up to 100 depending on the samples. Library storage and re-use up to years.
Binding detectionBinding of the antibodies to the CLIPS peptides is determined in an ELISA. The resulting color in each well is quantified with a CDD camera.
Binding detection sensitivityOptimized for epitope mapping of even low affinity binding antibodies (down to Kd=10-3M)
Required material and information20 μg antibody or 20 μl polyclonal serum. Sequence of target protein in FASTA format or UniProt ID.
Project run-through timePriority 1.5 months. Standard up to 3 months.
ReportingBinding values of all peptides are quantified and stored in the PepLab™ database. A full report is provided including details on binding and specificity for each residue optimized for registration regulatory and/or IP purposes. Full support is offered for IP generation and publishing.

Pepscan provides Precision Epitope Mapping as a complete end-to-end service. Supported by the expertise of our scientific team this allows each customer to fully benefit from all options the Precision Epitope Mapping technology offers.

A project usually starts with a discussion with the customer to define the project objectives. Before a project is discussed in detail, upon request, Pepscan is willing to sign a Confidentiality Disclosure Agreement (CDA) to assure absolute confidentiality.

Customer data input and sample requirements

  • Sequence of target protein in FASTA format or UniProt ID.
  • Minimal material consumption: 20 μg antibody or 20 μl polyclonal serum.

Tailor made project proposal

  • Full description of project study proposal
  • Timelines and budget

Design and synthesis of target-derived peptide arrays on re-usable "Minicards"

  • Design based on protein sequence (linear, single-loops, double loops)
  • Tailored additions to library based on 3D structure or customer input
  • Potential to address Post Translational Modifications

Screening of samples

  • On each re-usable Minicard up to 100 antibodies or sera can be screened
  • Data quantification
  • Storage in PepLab database

Extensive analysis of data

  • Matrix scan / Box plots / Heat maps
  • 3D imaging and modeling of protein
  • Mapping of epitopes on protein
  • Amino acid residue replacement analysis
  • Fine Mapping of epitopes on protein

Deliverable: a detailed report

  • Study design
  • Protocols
  • Experimental details
  • Results

Timelines

The timeline of a project starts with the design of the peptide arrays based on the sequence of target protein provided by the customer. Depending on the size of the target protein and the complexity of the project the synthesis of target-derived peptide arrays usually takes around 4 – 6 weeks. We usually ask to ship the sample(s) within this period. The duration of the screening depends on the number of samples to be tested, followed by 1-2 weeks for data analysis, evaluation and preparation of the report. In most cases a project is completed within 3 months, although more time may be needed for large series of samples.