Epitope Fine Mapping

Fine mapping determines the role of each individual residue within the epitope, and inventorizes the tolerance of the paratope towards variants of the optimal binder. Even when antibodies target the same or an overlapping epitope, the sensitivity to replacements on certain positions within the epitope may differ and point towards functional differences between these antibodies. This allows selecting most promising candidates depending on how strict target selectivity requirements for a given antibody should be.

Full epitope substitution scanning covers the stepwise substitution of all amino acid positions of an identified epitope with all 20 proteinogenic (canonical) amino acids. In certain cases, non-canonical or specifically modified residues are available per request.

Fine mapping through Epitope Substitution Scanning enables:

  • the identification of essential and variable amino acid positions within an epitope
  • epitope comparison on single residue level
  • detailed cross-reactivity analysis
  • proving candidate biosimilar antibodies similar to the original for regulatory purposes

Full substitution analysis using all proteogenic as well as non-proteogenic and/or specifically modified residues allows establishing tolerance of paratope to all substitutions in the corresponding epitope. In specific cases also substitutions which result in increased binding may be of interest. This sort of information provides a fresh glance onto the binding thermodynamics determined by epitope-paratope fit, be it perfect or almost perfect. It also allows developing superior peptide binders as potential inhibitors of protein activity (binding competitors, enzyme inhibitors and etc) and is therefore frequently applied by Pepscan for peptide lead optimization.

Epitope length scanning is complimentary to the full substitution analysis and employs a comprehensive library of epitope candidates of all lengths that is tested with antibody and allows identification of the “perfect fit” epitope candidate based on the binding intensity.

Case example: Fine mapping of the linear epitope of B4e8

The F425-B4e8 is one of few broadly neutralizing anti-HIV monoclonal antibodies. The antibody recognizes the highly flexible V3 variable loop region on the gp120 subunit of HIV-1 virus. To date it is known that V3 loop mediates coreceptor binding thereby assisting in the entry of HIV-1 virus. Deep understanding of recognition mechanisms of broadly neutralizing antibodies would not only extend our knowledge on HIV-1 entry, but also may inspire new immunogen designs for vaccine development. In this study HiSense linear mapping together with subsequent full replacement analysis were used to examine the fine features of the F425-B4e8 epitope.

Core epitope sequence 310HIGPRAFY318 was identified using a library of all overlapping linear peptides designed based on the sequence of gp160. Using these results the sequence of top binder 306RKRIHIGPGRAFYT319 was used to generate a comprehesive series of epitope mutants, where each position of the epitope is replaced by all other proteogenic amino acids (schematic on top right). Binding of F425-B4e8 to each epitope permutation was recorded and compared to that of the native sequence (bottom right). Results of the experiment show that 311IGPGRAF317 are essential for the binding as most if not all (R315) diminish the antibody binding. Most of replacements of I309 significantly decrease the binding, but do not fully abolish it.

These pepscan results are in agreement 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.

HiSense linear re-usable peptide arrays for epitope mappingare functionalized with a propritary polymeric hydrogel, which allows achieving high peptide density and at the same time working with all types of biological samples.

Fine mapping

Initially, the linear epitope for antibody F425-B4e8 was mapped using a HiSense linear epitope approach. Binding profile is shown on the top left panel. To reveal fine features of the antibody binding a full replacement analysis was used, as outlined in the top right panel. In short, such analysis allows identification of essential for the binding residues and those, which provide context for the binding. On the bottom right panel is a letterplot that shows how mutation of positions 311IGPGRAF317 diminishes antibody binding, while replacements of other residues are mostly tolerated. Agreement of the results obtained from Pepscan analysis and X-ray crystallography is visualized using 3D coordinate file 2qsc.pdb on the bottom left panel.

Pepscan’s exclusive expertise in linear, conformational and discontinuous epitopes allows identification residues that are crucial for binding as well as those that provide the proper structural context.

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.