Precision Epitope Mapping

Defining linear, conformational and discontinuous epitopes: accurate, fast and cost-effective

Exact definition of antibody epitopes is important for various reasons, such as:

  • Selection and characterization of antibodies, in particular where epitope similarity or dissimilarity issues are involved;
  • Intellectual Property (IP) purposes: evaluating the possibilities to establish IP rights (patenting, ‘freedom to operate’ assessment);
  • Regulatory filings:  FDA¹ and EMA² guidelines require specific binding site information to be included in regulatory dossiers for novel antibodies

The surface of a protein interacting with an antibody is not always a simple string of amino acids, but often consists of a complex of loops and folds determined by the interaction between side chains of the residues.

Epitopes can be therefore classified in three types:

  • Linear epitopes: defined by the primary amino acid sequence of a particular region of a protein. The epitope can usually be mimicked with linear peptides.
  • Conformational epitopes: defined not only by a primary amino acid sequence, but also by its spatial conformation. Appropriate epitope mimicry usually requires constrained peptides.
  • Discontinuous epitopes: the epitope consists of non-adjacent parts of the protein sequence, which are brought together in 3D structure by constraints.

The vast majority of therapeutic antibodies have a conformational or discontinuous epitope. Therefore Pepscan, as the inventor of epitope mapping with linear peptides, perfected its technology through the addition of CLIPS technology to enable addressing the 3D spatial conformation of epitopes. This has resulted in CLIPS Precision Epitope Mapping, a unique, high resolution mapping technology using 3D-structured peptides to characterize discontinuous and conformational epitopes with high accuracy. Many leading companies therefore apply our Precision Epitope Mapping to their promising therapeutic antibodies, several of which received FDA breakthrough status or accelerated approval, such as daratumumab (JNJ), ofatumumab (GSK/Novartis) and obinutuzumab (Roche).

Precision Epitope Mapping is characterized by a number of unique features and benefits:

Broadest applicability: the only technology to identify any type of epitope
Linear epitopes,
Conformational epitopes,
Discontinuous epitopes, and
Complex epitopes involving multimeric protein complexes

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

Highest success rate
Unmatched sensitivity through high peptide density

Structurally and functionally customized arrays
Including PTM’s, cyclizations, helices, β-sheets, etc.

CLIPS Precision Epitope Mapping uses arrays with large, surface-immobilized libraries of conformationally CLIPS-constrained peptides derived from the target protein. The binding of the antibody to each peptide construct of the entire library is determined. This affinity information is used in iterative screens to define the sequence and conformation of epitopes in detail. For more details see the Technology section.

A unique advantage of Precision Epitope Mapping resides in the fact that the arrays of 3D-structured peptides are re-usable and allow multiple screenings. Thus for panels of therapeutic antibody candidates (up to 100’s) the precise structured epitope data can be obtained for each individual antibody (Comparative Mapping). This makes Precision Epitope Mapping the technology of choice for comparative mapping and therefore ideally suited for characterization and selection of therapeutic antibody candidates for a/o IP purposes (patenting, assessing freedom to operate). Numerous recent patent filings using Pepscan’s Precision Epitope Mapping data testify to its importance for IP purposes:

Acticor – WO2017021539Canimguide – 20160311854Numab – WO/2016/184570
Janssen – US20170051069Calypso – WO/2016/001275R peptide – WO/2016/137950
Lundbeck – US20170015739Chiome – US20160333110R peptide – WO/2016/137947
NRC Canada: US20170015748Fraunhofer – WO2016165729 C2N – WO/2015/200806
Regeneron – WO2017027316Genmab – US20160237161Medimmune – WO/2015/169811
Aduro – WO/2016/110587GSK – 20160333086Medimmune – WO20150376285
Alector – WO2016201388Mass Gen Hosp – WO/2016/187068Netris – WO/2015/104360
Amphivena – US20160002333 McMaster – US2016034375UMCU – WO/2015/088348
Bayer – US20160319029Minomic – US20160349263Dyax – US8822653/2014

Pepscan’s Precision Epitope Mapping offers a comprehensive epitope mapping services package, addressing all needs occurring in antibody characterization or vaccine development. Work with the experts in a project that is tailored to your needs!

Case example: Revealing the discontinuous epitope of human anti-SIRPα

Anti SIRPα is a monoclonal antibody that specifically recognizes human Signal Regulatory Protein Alpha (SIRPα) and has poor or no cross-reactivity with variants of human SIRPβ. SIRPα is a regulatory membrane glycoprotein from SIRP family expressed mainly by myeloid cells and also by stem cells or neurons. SIRP α acts as inhibitory receptor and interacts with a broadly expressed transmembrane protein CD47.

Because the epitope of the anti-SIRPα antibody could not be mapped with linear peptides, Pepscan applied CLIPS 3D Structured Epitope Mapping. Based on a matrix design, a comprehensive library of combinatorial peptides based on the sequence of SIRPα by introducing structural constraints in arrayed peptides was synthesized. Heatmap analyses showed that only peptide chimeras containing both peptide stretches YYAVKERKGSPDDVE and GRELIYNQKEGHEPR strongly bind to the anti-SIRPα antibody and thus represent the core epitope (Figure). Linear peptides or simple looped peptides are not bound by anti-SIRPα antibody (data not shown).


Identification of the discontinuous epitope for anti-SIRPα using double looped matrix CLIPS peptides derived from the sequences of each individual domain of human SIRPα (2wng.pdb). Individual domains are correspondingly labeled. Only mimics containing residues YYAVKERKGSPDDVE (red) and GRELIYNQKEGHEPR (blue) located within the first lg-domain were bound by mAb4546. On the right is the vizualization of the identified stretches onto 3D structure of SIRPα, where the partial epitope candidates are colored respectively.

Other illustrative case examples, such as the elucidation of the discontinuous epitope of daratumumab (Darzalex, JNJ) or the helical (conformational) epitope mapping of a Flu virus antibody can be found in the case example section.

Refs: 1. FDA: February 27, 1997: Docket No. 94D-0259 (document UCM153182)
2. EMA: effective since July 1, 2009: EMEA/CHMP/BWP/157653/2007

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)

Epitope fine mapping: revealing fine details of anti-HIV-1 F425-B4e8

Having a detailed understanding of the binding specificity of an antibody surely is appealing from the perspective of basic science, but also to secure IP or ensure freedom to operate.  Epitope fine mapping defined the fine features of an anti-HIV-1 antibody and provided a unique insight into the breadth of HIV strains that can be recognized by this antibody.

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FDA breakthrough therapy designation for two Pepscan-mapped antibodies

Two antibodies for which Pepscan was asked to perform the epitope mapping, recently received breakthrough therapy designation by the FDA. It concerns Roche/Genentech’s anti-CD20 mAb obinutuzumab and Genmab/Johnson & Johnson’s anti-CD38 mAb daratumumab. We are proud to have contributed to these projects and congratulate our clients with their success.

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Revealing the discontinuous epitope of human anti-SIRPα in full detail

The epitope of an anti-SIRPα antibody, which cannot be mapped either with linear peptides or with structural mimics, was successfully elucidated via CLIPS Precision Epitope Mapping using a comprehensive library of combinatorial CLIPS peptides based on the sequence of SIRPα.

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Determining the discontinuous epitope of the anti-CD38 antibody daratumumab

CD38 is a 46kDa trans-membrane glycoprotein highly expressed in hematological malignancies. A two-stage CLIPS Precision Epitope Mapping strategy precisely determined the discontinuous epitope for daratumumab on the membrane-bound CD38.

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Conformational Epitope Mapping of a Flu Virus Antibody

FI6 is a monoclonal antibody that potentially neutralizes all influenza viruses. To reveal the molecular basis of this pan-influenza cross-reactivite antibody, we fine-mapped the conformational epitope recognized by FI6. (Corti et al, Science 2011).

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The molecular basis for distinction between GA-101 and Rituximab

Rituximab, a monoclonal antibody targeting CD20, has improved the treatment of malignant lymphomas. Therapeutic CD20 antibodies are classified as either type I or II based on different mechanisms of killing malignant B cells. To reveal the molecular basis of this distinction, we fine-mapped the epitopes recognized by Rituximab (Type I) and novel GA101 (Type II).

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Conformational Protein-Protein Interaction Mapping of etanercept (Enbrel®)

The CLIPS Epitope Mapping technology has been shown an effective tool for mapping of conformational and discontinuous epitopes of therapeutic antibodies. This case study demonstrates that the technology can also be used to identify the interaction site between hormones and cell-bound receptors. Etanercept (Enbrel®) is a chimeric TNFa receptor that is used to treat autoimmune disease by interfering with TNFa (tumor necrosis factor). The interaction between Etanercept and the TNFa trimer was studied in full detail through the CLIPS Mapping technology.

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Epitope Fingerprinting of large sets of disease-related polyclonal sera

Pepscan’s CLIPS Epitope Mapping not only suitable for precision mapping of one single antibody , but also for detailed epitope fingerprinting analysis of large sets of antibodies or polyclonal sera. This case report demonstrates the application in profiling the epitope landscapes of large numbers of sera from diseased and healthy origin to identify epitopes linked to protection.

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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


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.