High Resolution Conformational Epitope Mapping

Therapeutic antibodies often bind to conformational epitopes, which can hardly be mapped with linear peptides. Cyclic constrained peptides can overcome this limitation by mimicking looped epitope structures. PEPperMAP® Cyclic Peptide Microarrays combine the advantages of PEPperPRINT's unrivalled peptide sequence flexibility with on-array generation of cyclic constrained peptides.

 

By using antigen-specific PEPperCHIP® Cyclic Peptide Microarrays with cyclic constrained peptides, our PEPperMAP® Conformational Epitope Mapping enables the characterization of monoclonal antibodies with conformational epitopes in a uniquely high epitope resolution.

 

The detailed analysis of conserved and variable amino acids of a known conformational epitope by a Conformational PEPperMAP® Epitope Substitution Scan paves the way to in silico cross-reactivity analyses of monoclonal antibodies, the identification of non-responders and responders by correlation with sequencing data, or the differentiation between similar monoclonal antibodies or a standard antibody from its biosimilar for regulatory purposes.

 

 

Specifications of PEPperMAP® Conformational Epitope Mapping

High resolution PEPperMAP® Conformational Epitope Mapping is based on an antigen translated into 7, 10 and 13 aa cyclic constrained peptides with max. peptide overlaps of 6, 9 and 12 aa. Different peptide lengths are applied to mimic different loop sized and hence epitope conformations.

Conformational Epitope Mapping

PEPperCHIP® Peptide Microarrays covering the extracellular domain of CD20 translated into overlapping linear and constrained cyclic peptides. The first double-row corresponds to the 7 amino acid peptides, the second and the third double-row to the 10 and 13 amino acid peptides. The arrays were assayed with 100 µg/ml Rituximab followed by staining with anti-human IgG (red) and anti-Flag (green) antibodies. Linear peptides did not show any response against Rituximab, whereas the constrained cyclic peptides exhibited clear epitope stretches. Flag control peptides framing the peptide array showed the expected well-defined spot pattern.

 

    Microarray Content:    Single to multiple antigens translated into overlapping peptides
    Peptide Length:     7, 10 and 13 amino acids by default (adjustable)
    Peptide Overlap:     6, 9 and 12 amino acids by default (adjustable)
    Peptide Repeats:     Peptide double spots by default (adjustable)
    Peptide Cyclization:     Cystein disulfide bridging or thioether formation
   Control Peptides:     HA tags (YPYDVPDYAG) by default
    Microarray Example:     A 400 amino acid protein sequence will be translated into 3 x 400 = 1200 peptides in duplicate

 

 

Specifications of Conformational PEPperMAP® Epitope Substitution Scan

The Conformational PEPperMAP® Substitution Scan covers the stepwise substitution of all amino acid positions of a given constrained cyclic epitope or peptide with all 20 main amino acids and optionally additional non-natural amino acids. Conformational PEPperMAP® Epitope Substitution Scans enable:


  • the identification of conserved and variable amino acid positions of a conformational epitope
  • a detailed cross-reactivity analysis
  • the differentiation of similar monoclonal antibodies or biosimilars for regulatory purposes
  • the identification of responders and non-responders for therapeutic antibodies based on epitope sequencing data

Substitution Scan of Conformational Epitope

Conformational Substitution Scan microarray with the CD20 wild type peptide NIYNCEPANPSEK (left on top). Each amino acid position was gradually exchanged by the 20 L-amino acids from L-alanine (A) on left to L-tyrosine (Y) on right. Three single spot subarrays were printed next to each other and framed by Flag control peptides. The array was assayed with 100 µg/ml Rituximab followed by staining with anti-human IgG (red) and anti-Flag (green) antibodies. The heatmap displays the average signal intensities of the three subarrays (left on bottom). The amino acid plot reflects the intensity ratio of an epitope variant compared to the wild type epitope at 100% (right). The heatmap and the amino acid plot of the substitution scan of CD20 peptide 1NIYNCEPANPSEK13 highlight a conserved core motif 6EPANPSEK13 as well as a variable amino acid stretch 1NIYNC5 at the N-terminus of the wild type peptide. Moreover, the amino acid plot provides a very detailed insight on conserved and variable amino acids as well as tolerated amino acid exchanges.

 

    Microarray Content:     Gradual exchange of all amino acid positions with all amino acids
    Amino Acids:
    20 main amino acids by default, additional non-natural amino acids on demand
    Peptide Length:
    Up to 13 amino acids by default (adjustable)
    Motif Length for Scan:
    Up to 13 amino acids by default (adjustable)
    Peptide Repeats:     Peptide double spots by default (adjustable)
    Peptide Cyclization:
    Cystein disulfide bridging or thioether formation
    Control Peptides:
    HA tags (YPYDVPDYAG) by default
    Microarray Example:
    A 13 aa starting peptide will be translated into 13 x 20 = 260 peptides printed in duplicate
    Data Output:
    Detailed amino acid plots and substitution matrizes with conserved and variable amino acids

 

The detailed analysis of each individual amino acid of an epitope paves the way for a precise bioinformatic analysis of possible cross-reactions of a monoclonal antibody: Instead of a standard blast search of the conserved core motif, the consideration of tolerated epitope sequence variants clearly levels the significance of in silico cross-reactivity analyses of monoclonal antibodies. The relative intensity data of the amino acid plot even enables a semi-quantitative prediction of cross-reaction probabilities on a proteome-wide level. Furthermore, the detailed epitope data of the PEPperMAP® Substitution Scan helps to distinguish two similar monoclonal antibodies or a standard antibody from its biosimilar for regulatory purposes. Even more important is the differentiation between responders and non-responders for therapeutic antibodies based on sequencing data of the personalized epitope sequence.



Input Data & Sample Material

  • Protein sequence(s) in the FASTA format or with UniProt ID
  • 200 µl serum or 200 µg purified antibody

 

 

Further Information

 

 

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