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From Epitope Mapping to Autoimmune, Infectious Disease and Vaccine Research

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From Epitope Mapping to Autoimmune, Infectious Disease and Vaccine Research Epitope Mapping Kinase Substrate Optimization Biomarker Discovery Peptide Drug Development Laser-Printed Peptide Microarrays From Epitope Mapping to Autoimmune, Infectious Disease and Vaccine Research Corporate Presentation 11/2016 Company Overview History Scientific Background • based in Heidelberg, Germany • long-time research experience • privately held, independent company • proven track record (e.g. Science) • product launch in 2011 • broad patent coverage • ISO 9001 certified quality • 2010 Innovation Award of German Industry Products & Services Why PEPperPRINT? • PEPperCHIP® Peptide Microarrays • technological leadership with laser printing • PEPperMAP® Analysis Services • most flexible peptide microarray platform • accessories, software, synthetic peptides • recommended by 85% of our customers • serum biomarker discovery • excellent technical and scientific support → working with 9 of top 10 pharma companies Microarray Platform Technology Benefits • very low material consumption • high spot density / high content (1,000 peptides/cm2) • digital printing flexibility, fast production times • high peptide quality with routine double couplings Bayer et al., Science 2007, Stadler et al., Angew Chem Int Ed Engl. 2008 Competitive Advantage Standard Pre-synthesis of peptides Direct synthesis of peptides on chip pay per peptide high flexibility Spotting of peptides on chip Standard peptide array PEPperCHIP® Peptide Microarray number of different peptides resolution epitope mapping LOW HIGH customization economic → up to tens of thousands of peptide ELISA each microarray Peptide Microarray Toolkit PEPperCHIP® Peptide Microarray Platform Discovery Format Standard Format Mapping Format Multiplexed Format >60,000 Peptides Features >8,900 Peptide Features e.g. 5 x 1,200 Peptide Features 16 x 200 Peptide Features PEPperCHIP® Peptide Microarrays PEPperMAP® Services • PEPperCHIP® Staining Kit • Epitope Mapping, Epitope Analysis • PEPperCHIP® Incubation Trays • Serum Antibody Profiling • PepSlide® Analyzer • Antigen, Epitope & Peptide Discovery PEPperCHIP® Applications Antibody Analysis Vaccine Development Allergy Research Autoimmune Research Infectious Diseases Cancer Research Antibody Analysis PEPperMAP® Epitope Mapping • linear and conformational epitope mapping service with max. epitope resolution • including a custom peptide microarray with single or multiple antigens PEPperMAP® Epitope Substitution Scans • in-depth analysis of epitopes for the identification of essential and variable amino acids • ideal solution for specificity and/or cross-reactivity analyses of monoclonal antibodies PEPperMAP® Antibody Cross-Reactivity Analysis • identification of unknown antigens and epitopes with the Human Epitome Microarray • cross-reactivity profiling of antibodies PEPperMAP® Epitope Mapping Antigen Sequence Overlapping Peptides Secondary Antibody Target Antibody Peptide Microarray Microarray Scan (Double Spots) Monoclonal Sample Polyclonal Sample Epitope = Consensus Motif Data Analysis PEPperMAP® Epitope Mapping Length: 15 AA Overlap: 14 AA Maximum Epitope Resolution with Maximal Peptide Overlap Standard Length: 15 AA Overlap: 11 AA PEPperMAP® Epitope Mapping Standard Length 15 AA, Overlap 14 AA Length 15 AA, Overlap 11 AA • clear separation of two adjacent epitopes YES • easy determination of epitope length NO • exact identification of conserved core motifs PEPperMAP® Epitope Mapping Standard Length 15 AA, Overlap 14 AA Length 15 AA, Overlap 11 AA Samples: • monoclonal or polyclonal antibodies • synthetic or purified antibodies, hybridoma supernatant • plasma or serum samples • species: human, mouse, rabbit, goat, chicken, pig, bovine… • isotypes: total Ig, IgG, IgM, IgE, IgY, IgA… • clear separation of two adjacent epitopes YES • easy determination of epitope length NO • exact identification of conserved core motifs Conformational Epitope Mapping • model: therapeutic mAb Rituximab directed against the extracellular domain of human CD20 • data basis: X-ray Analysis of a co-crystal of Rituximab Fab fragment and CD20 peptide (163- 187)1 • observation: conformational epitope due to disulfide bond formation • outcome: 15 amino acid loop NIYNCEPANPSEKNSPSTQYCYSIQ covers the epitope of Rituximab Du et al., J.Biol.Chem. 2007; 282, 15073-15080 Conformational Epitope Mapping • goal: epitope mapping of Rituximab (Rituxan®), a chimeric monoclonal anti-CD20 antibody • microarray: CD20 as linear and cyclic constrained overlapping peptides (7 aa, 10 aa and 13 aa) • sample: monoclonal antibody Rituximab • outcome: high resolution data with EPANPSEK as Rituximab epitope 13 aa 10 aa 7 aa PEPperMAP® Epitope Substitution Scan • goal: in-depth epitope analysis to determine conserved and variable amino acids • microarray setup: full substitution scan of looped peptide NIYNCEPANPSEK with all 20 amino acids • data analysis: antibody binding relative to the wild type epitope at 100% • outcome: detailed information about essential, conserved and variable amino acids Microarray Scan Heatmap PEPperMAP® Epitope Substitution Scan • goal: in-depth epitope analysis to determine conserved and variable amino acids • microarray setup: full substitution scan of looped peptide NIYNCEPANPSEK with all 20 amino acids • data analysis: antibody binding relative to the wild type epitope at 100% • outcome: detailed information about essential, conserved and variable amino acids Applications: Microarray Scan • epitope mapping and analysis of therapeutic antibodies • cross-reactivity analysis with response matrix • correlation of response matrices with point mutations • patient stratification • specificity testing of diagnostic antibodies Heatmap Antibody Cross-Reactivity Analysis Human Monoclonal Antibody anti-c-Myc (chi9E10), 2 µg/ml The Human Epitome Microarray Human Epitome Microarray • covers all human epitopes of the Immune Epitope Database • cross-reactivity analysis with 28,895 • 8,504 epitopes of infectious diseases human database epitopes • 3,808 epitopes of autoimmune diseases • case study with human mAb anti-c- Myc (chi9E10) • 4,661 epitopes of the most common vaccines • few but clear peptide hits (red) with • 199 cancer epitopes, 3,451 epitopes of allergens 2 µg/ml antibody • 28,895 different peptides of 2,542 proteins and 468 organisms • cross-reactions against epitopes of nucleoprotein, erythrocyte membrane-associated giant protein antigen 332 and dihydrolipoamide S- acetyltransferase Antibody Cross-Reactivity Analysis Human Monoclonal Antibody anti-c-Myc (chi9E10), 2 µg/ml Human Epitome Microarray • cross-reactivity analysis with 28,895 human database epitopes • case study with human mAb anti-c- Myc (chi9E10) • few but clear peptide hits (red) with 2 µg/ml antibody • cross-reactions against epitopes of nucleoprotein, erythrocyte membrane-associated giant protein antigen 332 and dihydrolipoamide S- acetyltransferase Antibody Cross-Reactivity Analysis Peptide anti-c-Myc (chi9E10), 2 µg/ml Epitope ID Source Molecule Accession Source Molecule Name Source Organism ID Source Organism Name LGITAEDARLVSEIAMH 7.987,5 98874 127900 Nucleoprotein 11235 Measles virus strain Edmonston EEVVGEEKLVSEEIVT 2.535,0 11927 13508497 Erythrocyte Membrane-Associated Giant Protein Antigen 332 5833 Plasmodium Falciparum EVQEEGYLAKILVPE 1.960,5 125443 119587578 Dihydrolipoamide S-Acetyltransferase (E2 Component Of Pyruvate 9606DehydrogenaseHomo Complex), Sapiens Isoform Cra_A LAKILVPEGTRDVP 1.559,0 125468 119587578 Dihydrolipoamide S-Acetyltransferase (E2 Component Of Pyruvate 9606DehydrogenaseHomo Complex), Sapiens Isoform Cra_A TAEDARLVSEIAMHTTE 1.050,5 98874 127900 Nucleoprotein 11235 Measles virus strain Edmonston TYGRKLHLYSHPIILGF 1.007,5 190568 4323200 Polymerase 10407 Hepatitis B virus ALVAEGIEAIVFRTL 923,0 178535 15608176 Truncated Is1560 Transposase 1773 Mycobacterium tuberculosis NGFLDVFTSFGGLVAE 748,0 6520 75352214 Outer Surface Protein Vlse 224326 Borrelia Burgdorferi B31 FTSFGGLVAEAFGF 639,0 18069 75352214 Outer Surface Protein Vlse 224326 Borrelia Burgdorferi B31 QYLVGERTVLAGQCYI 620,0 133331 113118 Muscarinic Acetylcholine Receptor M1 9606 Homo Sapiens AQTQSLVYPF 593,0 78152 162797 Beta-Casein Precursor 9913 Bos Taurus VVSYVNTNVGLKFRQLL 547,5 53394 164509170 Precore Protein 10418 Hepatitis B virus subtype ayw VLNPWDQVKR 545,5 95970 115654 Alpha-S2-Casein Precursor 9913 Bos Taurus WLSLLVPFV 496,0 72794 15211906 Large Surface Antigen 10407 Hepatitis B virus GRSPRRRTPSPRRR 495,0 55713 16930336 Core Protein 10407 Hepatitis B virus GLSPTVWLSV 485,0 21139 128168864 Envelope Protein 10407 Hepatitis B virus PSPRRRRSQSPRRRR 475,5 55713 16930336 Core Protein 10407 Hepatitis B virus WSEGEGAVFYRVDLHFI 468,0 119822 62094 110 Kd Polyprotein Precursor 11041 Rubella virus GAAGTAAQAAVVRFQ 443,0 21976 15611010 10 Kda Culture Filtrate Antigen Esxb 1773 Mycobacterium tuberculosis PWATLVAES 418,0 140582 KYGGTEIKYNGEEYLI 414,0 34437 116200 10 Kda Chaperonin 1773 Mycobacterium tuberculosis RKLHLYSHPIILGFRKI 412,0 190568 4323200 Polymerase 10407 Hepatitis B virus SSLRGF 408,0 156460 MININIFMRESSRSFL 399,0 11711 RRRSQSPRRRR 396,5 55713 16930336 Core Protein 10407 Hepatitis B virus HVYLDTVVLLGALAN 383,0 25123 15609123 Probable Conserved Integral Membrane Protein 83332 Mycobacterium tuberculosis H37Rv RIRRSILPYGDSMDRI 377,0 125882 146345399 Collagen Alpha-1(Xvii) Chain 9606 Homo Sapiens WWARRRRRWRRWKRR 360,0 127897 5441235 Orf1 68887 Torque Teno Virus FPGGGQIVGGVYVLPRR 357,0 150483 974351 Core Protein 356114 Hepatitis C Virus Genotype 3 LVAEEDER 352,5 7979 SRC279960 Genome Polyprotein 11103 Hepatitis
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