Epitope Mapping Kinase Substrate Optimization Biomarker Discovery Peptide Drug Development

Laser-Printed Peptide Microarrays From Mapping to Autoimmune, Infectious Disease and 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 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 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 for the identification of essential and variable amino acids

• ideal solution for specificity and/or cross-reactivity analyses of monoclonal

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 • 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 C Virus DVKFPGGGQI 332,0 10636 130461 Genome Polyprotein 11103 Hepatitis C Virus GPSVFLF 331,0 107421 494350 Chain H, Three-Dimensional Structure Of A Human Immunoglobulin9606 With A Hinge HomoDeletion Sapiens AIAEYERSAAVLVRYPF 329,5 193035 564602885 Peptidylarginine Deiminase 837 Porphyromonas Gingivalis NPGLLRFLPQLSERL 324,0 179310 15609704 Hypothetical Protein 1773 Mycobacterium tuberculosis FLPSDFFPSV 311,5 79531 116946 Capsid Protein 10418 Hepatitis B virus Subtype Ayw ELGGKPALVPDRQVLYQ 311,0 20145 81992797 Genome Polyprotein 31647 Hepatitis C Virus Subtype 1B VFCVQP 311,0 68440 130458 Genome Polyprotein 31647 Hepatitis C Virus Subtype 1B QSLSFDSSNPEYFDGYW 309,0 176618 226694183 Integrin Alpha-Iib 9606 Homo Sapiens AQLLTEFAI 308,0 4002 57117045 Ppe Family Protein 83332 Mycobacterium tuberculosis H37Rv CLLCAYSIEF 301,5 7713 124757 Ovomucoid Precursor 9031 Gallus Gallus

• cross-reactivity profile over 28,895 known human epitopes with database annotations • identification of conserved and variable amino acid positions by MEME motif discovery PEPperCHIP® Applications

Antibody Analysis Vaccine Development Allergy Research

Autoimmune Research Infectious Diseases Cancer Research Autoimmune Research

PEPperCHIP® Autoimmune Epitope Microarray 3.0

• covering 4,287 linear autoimmune epitopes of the Immune Epitope Database

• including 286 citrullinated peptides and the corresponding arginine controls

PEPperCHIP® Cyclic Citrullinated Peptide Microarray

• covering 577 different cyclic peptides of the Immune Epitope Database and literature

• including 337 different cyclic citrullinated peptides and the corresponding arginine controls

PEPperCHIP® Human Epitome Microarray

• covering all linear human B-cell epitopes of the Immune Epitope Database

• most comprehensive human epitope microarray with 28,895 different peptides Autoimmune Epitope Microarray 3.0

• goal: identification of IgG and IgM autoantibody profiles in patient serum • microarray setup: Autoimmune Epitope Microarray 3.0 with 4,287 linear autoimmune epitopes • sample: serum of 56 year old female diabetes mellitus type 1 patient (1:500, 3 µl sample need) • outcome: IgG response against IA-2, IgG and IgM responses against Sjögren’s syndrome epitopes

Protein tyrosine phosphatase Human poliovirus 3 strain Sabin Human coxsackievirus A9 Human herpesvirus 4 Human coxsackievirus A9

Human herpesvirus 4

Calreticulin precursor

Integrin beta-3 precursor Small nuclear ribonucleoprotein Sm D1/ Ribosomal protein P-JL5 Human herpesvirus 4 Histone H1b High mobility group protein B1 The Human Epitome Microarray

Human Serum 110116, 1:250, IgG (red) vs. IgM (green)

High-Throughput Screening of Serum Antibodies

• corresponding to 28,895 peptide ELISA at once Human Serum 110116 • epitome-wide antibody screening • very strong IgG responses • isotype-specific antibody profiling (e.g. IgG vs. IgM) • strong IgM responses • case study with clinical serum 110116 of SLE patient • top hits against EBV and polio, herpes simplex

• Ro ribonucleoprotein as top autoantigen (Sjögren, SLE)

• various IgM responses against calreticulin (SLE, coeliac disease) The Human Epitome Microarray

Human Serum 110116, 1:250, IgG (red) vs. IgM (green)

Human Serum 110116

• very strong IgG responses

• strong IgM responses

• top hits against EBV and polio, herpes simplex

• Ro ribonucleoprotein as top autoantigen (Sjögren, SLE)

• various IgM responses against calreticulin (SLE, coeliac disease) Autoantibody Epitope Mapping

• goal: high resolution epitope mapping of autoantibodies in systemic sclerosis sera • microarray setup: CENPA and KDM6B translated into 1,831 overlapping peptides • samples: three systemic sclerosis patient sera vs. one control serum • outcome: fingerprint of antibody responses of patient sera; common epitopes for ELISA tests Autoantibody Epitope Mapping

• goal: high resolution epitope mapping of autoantibodies in systemic sclerosis sera • microarray setup: CENPA and KDM6B translated into 1,831 overlapping peptides • samples: three systemic sclerosis patient sera vs. one control serum • outcome: fingerprint of antibody responses of patient sera; common epitopes for ELISA tests

Application Note “High Resolution PEPperMAP® Epitope Mapping of Autoimmune Sera” available as download on our website Autoantibody Epitope Mapping

Autoantibody Epitope Mapping

→ 8,700 epitopes of human and viral antigens and random peptides Cyclic Citrullinated Peptide Microarray

• goal: identification of IgG and IgM autoantibody profiles in rheumatoid arthritis patient serum • microarray setup: 337 different cyclic citrullinated peptides including the arginine variants as controls • sample: rheumatoid arthritis patient and control sera (1:100, 2.5 µl sample need) • outcome: IgG and IgM response profiles with common and differential ACPA responses

Apolipoprotein E Fibrinogen alpha chain Vimentin

78 kDa glucose-regulated protein

Profilaggrin PEPperCHIP® SeroRA Library

• application: patient stratification, serum-based IVD and CDx development for rheumatoid arthritis

• setup: >100,000 linear and cyclic constrained peptides with citrulline and homocitrulline variants

• antigens: epitope-wide coverage of standard autoantigens like Vimentin, Fillagrin, Enolase or Fibrinogen, new autoantigens (BiP, α-1B-Glycoprotein) and pathogen-derived antigens (EBNA-1)

Standard RA Antigens Other RA Antigens Pathogen-Based RA Antigens

Vimentin α-1B-Glycoprotein EBNA-1

Filaggrin Tryptase Nuclear protein EBNA-2

Enolase Aggrecan DnaJ (E.coli)

Fibrinogen Clusterin EBV Envelope glycoprotein B

Actin 78 kDa glucose-regulated protein Immunoreactive 23 kDa antigen PG3 Collagen alpha-1(II) chain Glucose-6-phosphate Receptor antigen A

Histone 2A Apolipoprotein E Minor fimbrium tip subunit MfA4

Ro ribonucleoprotein BiP Peptidylarginine deiminase

Rheumatoid factor RF Myeloid cell nuclear antigen 19 kDa Lipoprotein Antigen Myelin basic protein Biglycan Hemolysin transporter protein HpmB

Standard Peptides Citrullinated Peptides Homocitrullinated Peptides

→ >100,000 peptides of human and pathogen origin PEPperCHIP® SeroRA Library

• application: patient stratification, serum-based IVD and CDx development for rheumatoid arthritis

• setup: >100,000 linear and cyclic constrained peptides with citrulline and homocitrulline variants

• antigens: epitope-wide coverage of standard autoantigens like Vimentin, Fillagrin, Enolase or Fibrinogen, new autoantigens (BiP, α-1B-Glycoprotein) and pathogen-derived antigens (EBNA-1)

Biggest RA peptide library based on hundreds of antigens

First comprehensive library with homocitrulline

Unlimited flexibility in terms of peptide content

Higher epitope specificity than protein-based assays

Closes gap for anti-CCP, anti-MCV and RF negative RA PEPperCHIP® SeroRA Library

Microarray Epitope Hit Selection for Cohort Statistical Data Validation and Transfer to Set-Up Discovery 2nd Microarray Screening Analysis Optimization ELISA/Luminex •custom or •discovery chip •focused peptide •assays with •data quantifi- •selection of top •generation of standard content format for hit library based on hundreds of cation and peptides for loaded MTPs for identification top hits samples formatting validation ELISA/Luminex •any peptide or beads protein list •up to 30,000 •screening format •single or dual •statistical •optimization of peptides in for higher sample isotype read-out microarray data peptides by •assay develop- •selection by duplicate per numbers analysis substitution ment for final test PEPperPRINT microarray scans format

Capacities:

• IVD and CDx discovery and development from peptide library design to test assay format

• proven track record, projects with 9 of the top 10 pharma companies

• test development based on virtually any peptide microarray content and disease area

• on request: partner companies for test verification and validation according to ISO 13485 PEPperCHIP® SeroRA Library

Summary

• SeroRA Library for the identification of prognostic RA peptides

• >100,000 peptides from hundreds of autoantigens and pathogen antigens

• including linear and cyclic constrained peptides

• including citrullinated and homocitrullinated peptide variants

• world’s biggest library for the analysis of antibody responses in RA patients

• implementation of custom or new antigens in few weeks

• new marker or marker combinations with higher sensitivity than standard tests

• potential to close the gap with seronegative RA patients

SeroRA Library Availability

• discovery and development of prognostic marker peptides for CDx and IVD

• from discovery to test development; optional validation and verification

• exclusive or non-exclusive set-up PEPperCHIP® Applications

Antibody Analysis Vaccine Development Allergy Research

Autoimmune Research Infectious Diseases Cancer Research Infectious Disease Research

PEPperCHIP® Infectious Disease Epitope Microarray

• covering 3,857 linear infectious disease epitopes of the Immune Epitope Database

• epitopes of 192 different pathogens including bacterial, fungal, parasitic and viral pathogens

PEPperCHIP® Human Epitome Microarray

• covering all linear human B-cell epitopes of the Immune Epitope Database

• most comprehensive human epitope microarray with 28,895 different peptides

Various PEPperCHIP® Standard Peptide Microarrays

• PEPperCHIP® Dengue Virus Proteome Microarrays

• PEPperCHIP® MERS-CoV Proteome Microarray

• PEPperCHIP® Influenza Virus H1N1 Proteome Microarray

• PEPperCHIP® Oncovirus Epitope Microarray and many more… Infectious Disease Epitope Microarray

• goal: screening of IgG and IgA antibody responses in patient serum against 196 pathogens • microarray setup: Infectious Disease Epitope Microarray with 4,347 linear database epitopes • sample: classified serum of patient with Helicobacter pylori infection (1:250, 6 µl sample need) • outcome: clear IgA response against H. pylori, IgG and IgA responses against E. coli and HSV

Human Coxsackievirus/Poliovirus Human Poliovirus 3 Strain Sabin

Enterovirus C

gondii

gondii

Herpes Simplex Virus 1

crassiceps

Escherichia coli Escherichia

Toxoplasma

Toxoplasma Taenia

Helicobacter pylori Escherichia coli Virus Proteome Microarrays

• goal: identification of West Nile virus epitopes in infected patients • microarray setup: West Nile virus genome polyprotein translated into 3,438 overlapping peptides • samples: various human sera at dilutions of 1:1000 (1 µl sample need) • outcome: two main WNV epitopes in serine protease NS3 and RNA-directed polymerase NS5 Zika Virus Proteome Microarrays

• goal: identification of Zika virus epitopes in patient sera, differentiation between patient groups • microarray setup: Zika virus genome polyprotein translated into 2,744 overlapping peptides • samples: Zika patient sera from Brazil (pregnancy, microcephaly, neurozika, dengue negative sera) • outcome: common IgG epitopes in dengue negative, pregnancy and microcephaly cases

Zika infection during Microcephaly, blood was Zika+/Dengue-, acute Neurozika, acute conditions, pregnancy taken right after birth conditions, > 10 days 6-15 days

→ representative microarray scans of all patient groups Zika Virus Proteome Microarrays

• goal: identification of Zika virus epitopes in patient sera, differentiation between patient groups • microarray setup: Zika virus genome polyprotein translated into 2,744 overlapping peptides • samples: Zika patient sera from Brazil (pregnancy, microcephaly, neurozika, dengue negative sera) • outcome: common IgG epitopes in dengue negative, pregnancy and microcephaly cases

Zika infection during OtherMicrocephaly, Virus Proteome blood was MicroarraysZika+/Dengue -, acute Neurozika, acute conditions, pregnancy taken right after birth conditions, > 10 days 6-15 days • Vaccinia Virus, SARS-CoV, HCV

• MERS-CoV, HCV, H5N1, Yellow Fever

• Foot and Mouth disease, Tick-Borne Encephalitis

• any many more including custom proteomes

→ representative microarray scans of all patient groups PEPperCHIP Flavivirus Immune Profiling

• application: identification of neutralizing and prognostic antibodies for a specific flavivirus, diseases state (acute vs. hemorrhagic fever, primary vs. secondary infections etc.) and isotype (IgG, IgM)

• setup: proteome-wide high resolution epitope mappings of most important flaviviruses

• virus proteomes: dengue 1-4, Zika, yellow fever, West Nile, Chikungunya, tick-borne encephalitis...

Most comprehensive and flexible flavivirus epitope library

Proteome-wide epitope coverage

Higher specificity than protein-based tests

Isotype-specific fingerprint analysis

Identification of prognostic and protective antibodies PEPperCHIP Flavivirus Immune Profiling

Microarray Epitope Hit Selection for Cohort Statistical Data Validation and Transfer to Set-Up Discovery 2nd Microarray Screening Analysis Optimization ELISA/Luminex •custom or •discovery chip •focused peptide •assays with •data quantifi- •selection of top •generation of standard content format for hit library based on hundreds of cation and peptides for loaded MTPs for identification top hits samples formatting validation ELISA/Luminex •any peptide or beads protein list •up to 30,000 •screening format •single or dual •statistical •optimization of peptides in for higher sample isotype read-out microarray data peptides by •assay develop- •selection by duplicate per numbers analysis substitution ment for final test PEPperPRINT microarray scans format

Capacities:

• IVD and CDx discovery and development from peptide library design to test assay format

• proven track record, projects with 9 of the top 10 pharma companies

• test development based on virtually any peptide microarray content and disease area

• on request: partner companies for test verification and validation according to ISO 13485 PEPperCHIP Flavivirus Immune Profiling

Summary

• Flavivirus Immune Profiling Library with proteome-wide epitope coverage

• including dengue 1-4, Zika, yellow fever, West Nile, Chikungunya etc.

• enables the fingerprint analysis of antibody responses in patient sera

• identify prognostic and protective antibodies

• differentiation between various disease states (primary and secondary infections, mild symptoms vs. hemorrhagic fever)

• implementation of new viruses or strains in 4 weeks

• higher specificity than protein-based assays due to thorough epitope coverage

Flavivirus Immune Profiling Library Availability

• discovery and development of prognostic marker peptides for CDx and IVD

• from discovery to test development; optional validation and verification

• discovery and development of protective antibodies for new vaccines PEPperCHIP® Applications

Antibody Analysis Vaccine Development Allergy Research

Autoimmune Research Infectious Diseases Cancer Research Cancer Research

PEPperCHIP® Oncovirus Epitope Microarray

• covering 3,653 linear B-cell epitopes of human oncoviruses

• including epitopes of hepatitis B and C, human herpesviruses, human papillomavirus etc.

PEPperCHIP® Tumor Antigen Microarray

• covering 22 selected tumor antigens as overlapping peptides (p53, NY-ESO-1 etc.)

• multiplexed and isotype-specific epitope mappings against general tumor antigens

PEPperCHIP® Melanoma Microarray

• covering 21 melanoma-associated antigens as overlapping peptides (e.g. MAGEA1, 2, 3)

• multiplexed and isotype-specific epitope mappings against melanoma antigens

PEPperCHIP® Custom Peptide Microarrays

• covering any epitope or antigen collection with linear or cyclic constrained peptides

• isotype-specific screening of antibody responses against linear or conformational epitopes Tumor Antigen Microarray

• goal: identification of IgG responses in tumor patient serum on the epitope level • microarray setup: Tumor Antigen Microarray with 22 tumor antigens as overlapping peptides • sample: clinical serum of tumor patient (1:250, 6 µl sample need) • outcome: main IgG response against various epitopes of protein SSX2 and cancer/testis antigen 1

QGNDLDNDP (Protein SSX2)

STGDADGPG (Cancer/testis antigen 1) MYLPGAEVPEPAA TTARELPIPEPEG (Transcription factor SOX-2) (Melanocyte protein PMEL) Breast Cancer Epitope Discovery

• goal: identification of breast cancer epitopes in patient sera for cohort screening in clinical trial • microarray layout: multiplexed epitope mapping of 61 breast cancer antigens (35,672 peptides) • samples: breast cancer patient sera of clinical trial on viral stimulation of immune responses • outcome: multiple IgG epitopes in all patient sera with common responses for cohort screening Custom Peptide Microarrays PEPperCHIP® Applications

Antibody Analysis Vaccine Development Allergy Research

Autoimmune Research Infectious Diseases Cancer Research Vaccine Development

PEPperMAP® Immune Monitoring

• linear and conformational epitope mapping of immunogens with max. epitope resolution

• fingerprint analysis of B-cell responses at various time points before and after immunization

PEPperMAP® HLA Class II Screening

• linear mapping of HLA class II binding peptides for peptide vaccine design

• identification of HLA II DR specific epitopes with stable recombinant HLA proteins

Peptide Pools for T-Cell Stimulation

• unpurified & purified custom peptides with varying QC levels from 6 - 25 amino acids

• peptide pools with hundreds of peptides from 2-10 µmol scale in few weeks PEPperCHIP® Immune Monitoring

• goal: in-depth analysis of immune responses upon immunization or vaccination • microarray: antigen translated into overlapping peptides with maximum peptide overlap • samples: pre-immune and immune sera (mice, chicken, human) of different time points • outcome: time-resolved B-cell responses with detailed epitope information (ELISA: yes/no)

Pre-Immune Serum

Second Immunization First Immunization PEPperCHIP® Immune Monitoring

• goal: in-depth analysis of immune responses upon immunization or vaccination • microarray: antigen translated into overlapping peptides with maximum peptide overlap • samples: pre-immune and immune sera (mice, chicken, human) of different time points • outcome: time-resolved B-cell responses with detailed epitope information (ELISA: yes/no)

Applications:Pre-Immune Serum

• analysis of vaccination strategies

• adjuvant testing

• analysis of anti-drug antibodies

• monitoring of vaccination trials

• compliance markers for animal vaccines (DIVA) Second Immunization First Immunization PEPperMAP® HLA Class II Screening

• goal: identification of HLA class II binding peptides for peptide vaccine design • microarray: target protein translated into overlapping peptides • sample: stable recombinant HLA protein to be provided by customer • assay: screening with multiple HLA II alleles to yield binding data of HLA II DR specific epitopes

Target Protein Overlapping Peptides Peptide Microarray

Data Analysis HLA Class II Screening HLA II

anti- HLA II Ab Peptide Pools for T-Cell Stimulation

• unpurified & purified custom peptides with varying QC levels (MS, HPLC) • from 6 - 25 amino acids in 2-10 µmol scale • T-cell stimulation: 8 - 15 amino acids each peptide with free amino and carboxyl termini • individual peptides and peptide pools, optional modifications like acetylation, phosphorylation etc.

General Capacities • 8 peptide synthesizers, 4 RP-HPLC units (analytical & preparative) • more than 20 RP-HPLC columns • Bruker Ultraflex MALDI-TOF

Delivery Times • set of 384 peptides, < 2µmol scale (research grade): 6 weeks • set of 192 peptides, 2-10 µmol scale (research grade): 4-5 weeks • for higher peptide purities (>80% purity), delivery times may vary

→ about 100 customers all over the world Identification of Neutralizing Abs

• Malaria problem: over 400,000 deaths per year, no efficient vaccine available yet • goal: identification of neutralizing antibodies for the development of Malaria vaccines (MalVa GmbH) • microarray: ~3,800 Malaria peptides from selected antigens and vaccines, ~1,900 random peptides • samples: sera from Malaria infected and immune patients, control sera

12 top vaccine Malaria Malaria candidates as 1,116 Control (infected) (immune) overlapping peptides

Bioinformatical • Strongest signals in high selection of 1,912 Pf immunity patients proteome peptides 12 top vaccine • Differences of infected and candidates1,912 random as 1,116 Pf overlappingpeptides selected peptides immune sera important 12 top vaccine fromcandidates the Pf proteome as 1,116 Bioinformatical • Clear indications for overlappingselection of peptides1,912 Pf proteome peptides protective antibodies in Bioinformatical immune group selection1,912 random of 1,912 Pf Pf proteomepeptides selectedpeptides • General approach from the Pf proteome 1,912 random Pf applicable to any pathogen peptides selected from the Pf proteome PEPperCHIP® Applications

Antibody Analysis Vaccine Development Allergy Research

Autoimmune Research Infectious Diseases Cancer Research Allergy Research

PEPperCHIP® Allergen Epitope Microarray

• covering 4,022 linear autoimmune epitopes of the Immune Epitope Database

• epitopes of 223 different allergens of 90 organisms with a broad disease coverage

Custom PEPperCHIP® Peptide Microarray

• covering e.g. selected allergens as overlapping peptides for IgE epitope mapping

• high resolution IgE epitope data from single allergens to multiplexed analyses

PEPperCHIP® Human Epitome Microarray

• covering all linear human B-cell epitopes of the Immune Epitope Database

• most comprehensive human epitope microarray with 28,895 different peptides Allergen Epitope Microarray

• goal: identification of IgG and IgE antibody responses against 223 different allergens at once • microarray setup: Allergen Epitope Microarray with 4,022 linear allergen epitopes of the IEDB • sample: classified serum of patient with multiple allergic responses • outcome: clear IgG and IgE responses against various allergens, thorough ELISA validation

Allergen Pen N 18 Penicillium chrysogenum Allergen Ara H 1, Clone P41B Precursor

Arachis hypogaea

B 5 B Alkaline Serine Protease

Penicillium chrysogenum

Hev

Precursor

japonica

Small Rubber Particle Protein Alpha S1 Casein Hevea brasiliensis brasiliensis Bos taurus Polygalacturonase Precursor

Cryptomeria japonica

Polygalacturonase Cryptomeria

Major Latex Latex Major Allergen Hevea Cashew Nut IgE Epitope Mapping

• goal: IgE epitope mapping from human patient sera vs. control serum • microarray setup: custom layout, cashew nut allergen Ana o 3 translated into overlapping peptides • sample: human patient sera with known cashew nut allergy vs. classified control serum • outcome: polyclonal IgE responses of patient sera, no response in control serum Custom Peptide Microarrays PEPperCHIP® Applications

Antibody Analysis Vaccine Development Allergy Research

Autoimmune Research Infectious Diseases Cancer Research Summary

Why PEPperPRINT?

• access to most flexible peptide microarray platform with any content

• from up to 30,000 different peptides per microarray to prototype test format

• linear and cyclic constrained peptides

• incorporation of peptides with posttranslational modifications

• high peptide quality and chip reproducibility , ISO 9001-certified quality

• custom and adjusted glass slide coatings for best signal-to-noise ratios

• extensive knowhow from peptide selection to bioinformatics

• custom and scientific reporting instead of machine-made reports

• thorough scientific support and fast response times More Information

Tutorial Video & Webinar Application Notes

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