Immuno Tools for> Biomarker Discovery

> Seroscreening

> B- and T-cell Epitope Identification and Validation

> T-cell Assays

> Vaccine Development

> Antibody Signature Profiling

Innovative Peptide Solutions

Imm

uno

Tool

s

Imm

uno

Tool

s

HistoryJPT Peptide Technologies is a service provider based in Berlin, Germany and has achieved worldwide credibility for its commitment to rigorous quality standards and a reputation for developing and implementing innovative peptide based services and research tools for immunological and proteomic applications.

Together with its US-subsidiary, based in Acton near Boston, Massachusetts, JPT is serving a worldwide customer base in the pharmaceutical and biotechnology industries as well as researchers in universities, governmental and non-profit organizations.

Technology & ApplicationOver the past decade, JPT has developed a portfolio of proprietary technologies and developed a series of unique products and services which support research efforts in proteomics and in all developmental phases of novel vaccines against infectious and autoimmune diseases as well as against allergies and cancer.

In addition, JPT’s technologies find application in areas where peptide screening is needed. Typical examples are: screening for proteoptypic peptides, enzymatic activities (proteases, kinases, phosphatases, methyltransferases), or systematic optimization of peptide lead structures.

Quality AssuranceJPT is DIN EN ISO 9001:2008 certified and GCLP audited.

JPT’s proprietary key technologies are:

SPOT

High throughput peptide synthesisand screening technology forpeptides & peptidomimetics forT-cell epitope and peptide leaddiscovery and characterization on proteome-wide levels.

PepStar™

Proprietary technology platformfor humoral immune responses for biomarker discovery, seroscreening and immune monitoring.

PepMix™

Defined antigen spanning peptide pools to stimulate CD4+ and CD8+ T-cell responses.

PepTrack™

Peptide libraries of individual peptides optimized for T-cell assays.

SpikeTides™

Small scale light and heavily labeled peptides for proteomics.

1

Imm

uno

Tool

sTa

ble

of c

onte

ntsCellular immune responses

02 / PepTrack™ Peptide Libraries Individual peptides for specific requirements of T-cell assays

06 / PepMix™ Peptide Pools Antigen spanning or mixed epitope pools for immune monitoring,

vaccine efficacy testing & adoptive immunotherapy

08 / Custom Peptides ISO 9001:2008 certified synthesis for provision of peptides

fulfilling highest quality requirements

10 / Micro-Scale Peptides The most inexpensive source of small scale peptides on the market

Humoral immune responses 12 / PepStar™ Customized high density peptide microarrays for seromarker

discovery and immune monitoring

14 / PepSpots™ Customized cellulose bound peptide arrays for epitope mapping

16 / Epitope Discovery & Antibody Profiling Service Incubation and data evaluation service using peptide arrays with

your serum, lysate, antibody or enzyme

18 / BioTides™ High throughput assembly of biotinylated peptides for ELISA

development, microarray generation and more

19 / Bioinformatics & Cheminformatics Comprehensive bio- & cheminformatic services supporting peptide

library and array design for biomarker discovery, validation and optimization programs

20 / RepliTope™ Predefined peptide microarrays for mapping of immunodominant

regions

Our PepTrack™ options Research, Discovery and Trial Track are delivered in 96-tube racks for individual tube handling.

2

Imm

uno

Tool

sC

ellu

lar

imm

une

resp

onse

s PepTrack™ Peptide Libraries

Custom peptide libraries optimized for performance in T-cell assays such as ELISPOT, CFC, etc.

Applications for PepTrack™

• T-cell epitope discovery • Peptide vaccine development • T-cell epitope mapping and validation • Immune monitoring • Optimization and validation of T-cell assays • Vaccine efficacy testing • Cell therapy approaches • Biological screening • Identification of proteotypic peptides

Benefits of PepTrack™

• Peptide specifications tailored for cell based assay applications

• Best pricing in industry starts at 7 $/€ per peptide • All production performed in a regulated environ-

ment according to DIN EN ISO 9001:2008 & GCLP standards; Audits welcome!

• Peptide protocols designed to avoid toxic contaminants

• Special synthesis technology inhibits side products causing false positive T-cell responses

• Provision of freeze dried aliquots for enhanced stability, validated pooling & vialing available

• Proven track record for applications in clinical studies

Contact us at peptide@jpt.com and find the perfect peptide library specifications for your application with the help of our experts!

3

Imm

uno

Tool

sC

ellu

lar

imm

une

resp

onse

s

What is PepTrack™?

JPT Peptide Technologies is the leading provider of peptide libraries for T-cell assays and pro-teomic applications worldwide. Together with its partners in the vaccine industry, governmen-tal and non-profit organisations, JPT defined specific requirements for peptides to be used for T-cell assays in the various phases of vaccine development. These specifications were translated to a product line called PepTrack™ Peptide Libraries offering tailor made solutions for all peptide needs in vaccine development. Using its regulated environment (DIN EN ISO 9001:2008, GCLP) JPT produces more individual peptides on an annual basis than any other company worldwide.

PepTrack™ Options

Fast Track

Synthesized by JPT’s proprietary SPOT synthesis method, Fast Track Peptide Libraries represent the most inexpensive source of peptides worldwide. The ability to produce tens-of-thousands of pep-tides within weeks enables T-cell epitope discovery, biological screening and identification of pro-teotypic peptides at proteome-wide levels.

Research TrackResearch Track Peptide Libraries are designed for applications in T-cell assays requiring large num-bers of fully analyzed peptides in quantities of 1-5mg. All peptides are fully analyzed for identity and purity by LC-MS (no MALDI-MS!). In addition, JPT’s peptide synthesis protocols are optimized to avoid contaminations and failure sequences which cause either false positive T-cell responses or toxic inhibition.

Purity QA/QC* Length Scale Delivery Price/peptide

Unpurified - no purity guarantee 5% LC-MS 7-15aa 50 - 250nmol 2 weeks from 7 $/€

* additional QC available upon request Delivery Format: 96-well plate format

Purity QA/QC Length Scale* Delivery Price/peptide

Unpurified - no purity guarantee LC-MS 7-15aa 1-5mg 2 weeks from 25 $/€

main product= target peptide LC-MS 7-15aa 1-5mg 2 weeks please inquire

* for larger amounts please inquireDelivery Format: 96-array tube format

4

Imm

uno

Tool

sC

ellu

lar

imm

une

resp

onse

s Discovery Track

Discovery Track Peptide Libraries have defined purities for applications requiring similar and specified purities of all peptides (i.e. immune monitoring). Each peptide has a guaranteed mini-mum specification. All peptides will be QC’d by LC-MS (not MALDI-MS!).

Trial Track Trial Track Peptide Libraries were designed to provide a reliable and competitive source of pep-tides and peptide pools for application as monitoring tools in clinical vaccination trials. All peptides have guaranteed purities according to specifications.

Purity QA/QC Length Scale* Delivery Price/peptide

> 70 % LC-MS 7-15aa 1-5mg 3 weeks from 45 $/€

* for larger amounts please inquireDelivery Format: 96-array tube format

Purity QA/QC* Length Scale** Delivery Price/peptide

> 80 % LC-MS 7-15 aa > 1 mg 3 weeks from 69 $/€

> 90 % LC-MS 7-15 aa > 1 mg 3 weeks please inquire

> 95 % LC-MS 7-15 aa > 1 mg 4 weeks please inquire

> 97 % LC-MS 7-15 aa > 1 mg 5 weeks please inquire

* full analytical coverage including amino acids analysis, capillary electrophoresis, peptide

content determination, determination of residual solvents etc. is available upon request ** for larger amounts please inquireDelivery Format: 96-array tube format

5

Imm

uno

Tool

sC

ellu

lar

imm

une

resp

onse

s

Selected References> “Strong Human Endogenous Retrovirus-Specific

T cell Responses Are Associated with Control of HIV-1 in Chronic Infection”

SenGupta et al., J. Virol. (2011)

> “Functional Characterization of Aquaporin-4 Specific T Cells: Towards a Model for Neuromyelitis Optica”

Reddy Kalluri et al., PLoS ONE (2011)

> “Telomerase and HER-2/neu as Targets of Genetic Cancer Vaccines in Dogs”

Peruzzi et al., Vaccine 28 (2010)

> “Phase I Safety and Immunogenicity Evaluation of MVA-CMDR, a Multigenic, Recombinant Modified Vaccinia Ankara-HIV-1 Vaccine Candidate”

Currier et al., PLoS ONE (2010)

> “Structural Basis for the Immunogenic Properties of the Meningococcal Vaccine Candidate LP2086”

Mascioni et al., The Journal of Biological Chemistry (2009)

> “Comprehensive Analysis of the α-Fetoprotein-Specific CD8+ T Cell Responses in Patients with Hepatocellular Carcinoma”

Thimme et al., Hepatology (2008)

> ”Identification and Functional Validation of MHC Class I Epitopes in the Tumor-Associated Antigen 5T4”

Shingler et al., International Immunology (2008)

> ”Acquisition of Direct Antiviral Effector Functions by CMV-Specific CD4+T Lymphocytes with Cellular Maturation”

Casazza et al., J. Exp. Med. (2006)

“I have been collaborating with JPT for several years. Their unique peptide library technologies and knowledge of specific peptide requirements for T-cell assays made them a valuable, long-term partner in our efforts to develop novel adoptive immunotherapy approaches to prevent and treat viral infections post-transplant as well as for the treatment of virus and non virus-associated malignancies. Furthermore, their reagents allow us to precisely monitor and track specific T-cell populations post-infusion.”Ann Leen, PhD, Baylor College of Medicine, Center for Cell and Gene Therapy, Houston, TX, USA

6

Imm

uno

Tool

s

Validated pooling and aliquoting is performed in an environment regulated by ISO 9001:2008 and GCLP.

The 19 PepMixes™ of our new HCMV PepMix™ Collection are delivered in individual tubes (2 aliquots each) in a microtube rack.

Applications for PepMix™

• Immunostimulation of T-lymphocytes • Monitoring of immune status during

diseases & therapeutic interventions• Assessment of vaccine efficacy

What is a PepMix™?Antigenic protein sequences will be divided into overlapping peptides, chemically synthesized and analyzed to comply with the requirements of T-cell assays. Pooling is performed using validated proto-cols ensuring the presence of all peptides in the respective pools. Our fully automated aliquotation service yields freeze dried pool aliquots minimizing deterioration of peptides that easily occurs in DMSO stock solutions.

> Peptide scan format: length 15 amino acids with overlap 11 amino acids

> Pool aliquots: 25 tests per vial (25 µg / peptide)

Benefits of PepMix™

• Simultaneous detection of CD4+ and CD8+ responses in a single sample

• Similar T-cell responses compared to whole protein antigens

• Longer shelf life and batch to batch reproducibility • Improved responses in stored blood and PBM cells

compared to whole protein antigens

Selection of available PepMixes™

Positive & negative control pools: CEF, CEFT & EF Pools, HCMV (pp65, IE-1...),

Human Actin

Infectious Diseases: EBV (EBNAs, BLZF1...), BKV (VP1, largeT...),

Adenovirus, HPV, HIV, Influenza and many more

Tumor Associated Antigens: Melanoma, prostate & breast cancer, glioma, gastric

cancer, testis/ovary cancer, Wilms tumor and others Cel

lula

r im

mun

e re

spon

ses

Sub-Pool IVMarkerPeptide

Sub-Pool I Sub-Pool II Sub-Pool IIIMarkerPeptide

MarkerPeptide

MarkerPeptide

Individual Peptides

PepMix™

Antigen spanning or mixed epitope pools for in vitro stimulation of CD4+ and CD8+ T-cell responses. Both pre-made and customized peptide pools are available.

Please inquire for details on customized PepMixes™ via email to peptide@jpt.com!

NEW: HCMV PepMix™ Collection

All important 19 antigens of HCMV combined in one rack for reliable immune response profiling in transplant patients.

Have a look at the complete and up-to-date list of over 100 PepMixes™ under shop.jpt.com!

All important 19 antigens of HCMV combined in one rack for

NEW: HCMV PepMix™ Collection

All important 19 antigens of HCMV combined in one rack for

7

Cel

lula

r im

mun

e re

spon

ses

PepMix™ peptide pools will be provided as freeze dried aliquots for highest shelf-stability and with full QC/QA documentation and application protocol.

Selected References> “Innate and Adaptive Immune Responses Both

Contribute to Pathological CD4 T Cell Activation in HIV-1 Infected Ugandans”

Eller et al., PLoS ONE (2011)

> “Correlating Cellular and Molecular Signatures of Mucosal Immunity that Distinguish HIV Controllers from Non-Controllers”

Loke et al., Blood (2011)

> “A Whole Blood Monokine-based Reporter Assay Provides a Sensitive and Robust Measurement of the Antigen-Specific T Cell Response”

Chakera et al., Journal of Translational Medicine (2011)

> “Live and Inactivated Influenza Vaccines Induce Similar Humoral Responses, but Only Live Vaccines Induce Diverse T-Cell Responses in Young Children”

Hoft et al., The Journal of Infectious Disease (2011)

> “Prophylactic Transfer of BCR-ABL–, PR1-, and WT1-Reactive Donor T Cells after T cell–Depleted Allogeneic Hematopoietic Cell Transplantation in Patients with Chronic Myeloid Leukemia”

Bornhäuser et al., Blood (2011)

> “Induction of Complete and Molecular Remissions in Acute Myeloid Leukemia by Wilms’ Tumor 1 Antigen-Targeted Dendritic Cell Vaccination”

Tendeloo et al., PNAS (2010)

“The main focus of my research group at the Charité in Berlin is the development of novel immunotherapeutic approaches against cancer and infectious diseases.For reliable monitoring of tumor and virus specific T-cell responses we have a permanent need for peptides and pep-tide pools that are produced in a regula-ted environment for application in a clini-cal environment. JPT has been a long term and dedicated partner in this regard which continuously works on improving its peptide based services.”Carmen Scheibenbogen, Professor, Charité Berlin, Medical Immunology, Germany

Imm

uno

Tool

s

Freeze dried peptides will be delivered with full analytical coverage.

Automated synthesis allows a largevariety of scales and chemistries.

Imm

uno

Tool

sC

ellu

lar

imm

une

resp

onse

s

8

Custom Peptides

The exceptional quality and reliability of this service has been appreciated by customers worldwide for many years. JPT is the premier provider of custom peptides and related services.

Options and Specialties

• Fluorogenic and chromogenic peptides • Internally quenched peptides

(Abz/nitroTyr, EDANS/DABCYL, MCA/DNP) guaranteed without flourescent impurities

• Immunogenic peptides (MAPs, palmitinylation, Pam3Cys labeling, etc.)

• Phospho-peptides and peptidomimetics (amide bond isosteres, non-natural amino acids, etc.)

• Non-commercial building blocks available • Labeling

(non-radioactive isotopes, chromophores, etc.) • Site-directed conjugations with KLH, BSA,

ovalbumin or other carriers • Cyclic peptides

(disulfide bridges, lactams, thioether-bridges, etc.) • Long peptides (> 70 amino acids)• Scales ranging from 1 mg to several grams

Need other modifications or specifications? We will give our best to make it happen! Contact us at peptide@jpt.com!

Benefits of JPT´s Custom Peptides

• Proprietary synthesis technologies warrant fastest turnaround & most competitive pricing

• Reliable and stringent QC/QA• ISO 9001:2008 and GCLP compliance• Rapid order processing • Large variety of chemistry protocols• Fully automated pooling, aliquoting and vialing• Solubility & stability testing• Personal consultation with experienced scientists • Highest purities available (> 95 %, > 97 %)• Full range of analyses including LC-MS (trap and/or

quad), MALDI-MS, HPLC, AAA, NMR, CE, as well as peptide content determination to confirm the identity and demonstrate the high quality of our peptides

• Substantial, long-standing expertise in providing custom peptides

• Highly skilled and committed scientific staff

Selected References> “Functional Gap Junctions Accumulate at

the Immunological Synapse and Contribute to T Cell Activation”

Mendoza-Naranjo et al., J. Immunol. (2011)

> “The Immunodominant CD8 T Cell Response to the Human Cytomegalovirus Tegument Phosphoprotein pp65495–503 Epitope Critically Depends on CD4 T Cell Help in Vaccinated HLA-A*0201 Transgenic Mice”

Reiser et al., J. Immunol. (2011)

> “A Whole Blood Monokine-based Reporter Assay Provides a Sensitive and Robust Measurement of the Antigen-Specific T Cell Response”

Chakera et al., Journal of Translational Medicine (2011)

Cel

lula

r im

mun

e re

spon

ses

Quality Assurance

• JPT´s entire peptide production, purification and analysis procedures are backed by astringent DIN ISO 9001:2008 certified Quality Management System.

• All quality relevant processes are well documented and regulated according to a comprehensive SOP system.

• All peptide production is performed at JPT´s headquarters in Berlin, Germany under continuous quality measures.

9

Imm

uno

Tool

s

“Our research relies heavily on developing robust high-throughput screens with fluorescent peptides. We have found that JPT’s are the best on the market because the signal-to-noise ratio is very high, providing the sensitivity we need for the screens. Their peptides always perform well. In addition, the knowledge, wonderful customer support, and fast turnaround time provided by JPT have been invaluable in helping us develop the best peptides for our assays.”Carla Koehler, Professor, UCLA, Chemistry & Biochemistry, Los Angeles, CA

10

Imm

uno

Tool

sC

ellu

lar

imm

une

resp

onse

s Micro-Scale Peptides

Ultra-high-throughput, small scale synthesis generating your:

> Antigen spanning peptide library

> Proteotypic peptide sets

> Random peptide libraries

> Peptide biomarker library

> Peptide substrate collection

> Peptide scans covering entire pathogenic proteomes

Applications for Micro-Scale Peptides

• Screening for proteotypic peptides • Validation and optimization of SRM assays • Systematic biomarker screening • T-cell epitope discovery • Optimization of peptide lead structures • Generation of peptide pools for immune

monitoring • Screening assays for biological activity • GPCR deorphanization

What are Micro-Scale Peptides?

Our SPOT technology is the fastest and most eco-nomic peptide source on the market. Annually more than 3 million peptides are synthesized. The result-ing singular peptides are provided as lyophilized materials in microtiter plates.

> Amounts of 50-250 nmol per peptide> Peptide length up to 20 aa> Ready-to-use peptides in 96- or 384-well plates

Benefits of Micro-Scale Peptides

• Highly parallel synthesis approach (30 000 peptides/ week)

• Pricing: from 7 $/€ / peptide • Full analysis (MALDI, LC-MS) available• Non natural and isotopically labeled amino

acids possible• Purified peptides available• Peptide pools available upon request

Your Micro-Scale Peptide Sets will be delivered freeze dried in microtiter plates.

Documentation includes QC / QA report and plate alignment.

Recent Cooperation Projects> “Libraries of thousands of Micro-Scale Peptides

for high throughput screening against undisclosed drug targets”

Bayer AG, Germany

> “Over 45 000 SpikeTides™ used for the creation of a high-resolution fragmentation database of 150 000 protein-specific fragments (human SRMAtlas)”

Institute for Systems Biology, USA

> “More than 50 000 peptides for systematic discovery of T-cell epitopes to accelerate the development of vaccines against tuberculosis”

Oregon Health and Science University, USA

Selected References> “Screening of Predicted Secreted Antigens from

Mycobacterium bovis Identifies Potential Novel Differential Diagnostic Reagents“

Jones et al., Clin. Vaccine Immunol. (2010)

> “Extensive Major Histocompatibility Complex Class I Binding Promiscuity for Mycobacterium Tuberculosis TB10.4 Peptides and Immune Dominance of Human Leucocyte Antigen (HLA)-B*0702 and HLA-B*0801 Alleles in TB10.4 CD8+ T-cell Responses”

Axelsson-Robertson et al., Immunology (2009)

“Our group at OHSU has purchased more than 50 000 peptides over the past few years from JPT to support our proteome wide TB epitope discovery program. We are impressed by the turnaround times and highly competitive pricing structure for such large numbers of peptides and are very satisfied with the responsive and professional service.”Gwendolyn Swarbrick, PhD, Oregon Health & Science University, Portland, USA

Micro-Scale Peptides represent the most economic custom peptide source in the market.

11

Imm

uno

Tool

sC

ellu

lar

imm

une

resp

onse

s

12

Imm

uno

Tool

sH

umor

al im

mun

e re

spon

ses PepStar™

Your customized high content microarray could contain

> Peptide scans through antigens or whole genomes

> Huge random peptide libraries

> Tumor associated antigen scans

> Annotated phosphorylation sites

> Annotated cleavage sites

Applications for PepStar™

• Peptide biomarker discovery• Seromarker identification & validation• Mutiplexed immune monitoring in clinical trials• Elucidation of antigen and epitope spreading

during disease progression and therapeutic intervention

• Mapping of immunodominant regions in antigens

• QC / QA of therapeutic biologics • Vaccine target identification• Identification & optimization of enzyme

substrates

What are PepStar™ peptide microarrays?

Large numbers (up to 21 000) of peptides are N-termi-nally attached to glass slides by directed and chemo-selective immobilization. Patented high-throughput synthesis of peptides results in high-content peptide arrays. Yield of synthesis is sufficient to generate hundreds of identical slides. Incubation can be per-formed with proteins and patient samples. Read-out is achieved by fluorescence using validated protocols and commercial equipment.

Benefits of PepStar™

• Cost effective provision of hundreds of identical microarrays from a single synthesis batch

• Directed immobilization of purified peptides • Flexible co-immobilization of controls• Chemical synthesis and analysis warrant batch-

to-batch reproducibility • High shelf stability • High assay sensitivity• Defined posttranslational modifications

are possible• Commercial incubation and read-out equipment

can be applied • Low consumption of patient materials and

proteins • Incubation and read-out protocols available

We will be happy to discuss the design of your peptide microarray. Contact our support team at peptide@jpt.com!

Your PepStar™ microarrays are delivered with detailed QC / QA documentation and application protocol.

State-of-the-art printing devices support high accuracy and batch-to-batch reproducibility.

scale unit: mm

Lot #

Subarray 1 Subarray 2 Subarray 3 K1

Controls

K2K3

Peptides

13

Imm

uno

Tool

sH

umor

al im

mun

e re

spon

ses

Selected References> “Polyclonal B Cell Responses to Conserved

Neutralization Epitopes in a Subset of HIV-1-Infected Individuals“

Tomaras et al., J. Virol. (2011)

> “Epitope Mapping of Atlantic Salmon Major Allergen by Peptide Microarray Immunoassay”

Perez-Gordo et al., Int Arch Allergy Immunol (2011)

> “Epitope Mapping of Antibodies to VlsE Protein of Borrelia Burgdorferi in Post-Lyme Disease Syndrome”

Chandra, et al., Clin. Immunol. (2011)

> “Impact of Valency of a Glycoprotein B Specific Monoclonal Antibody on Neutralization of Herpes Simplex Virus”

Krawczyk et al., J. Virol. (2011)

> “The Protein Interaction Network Mediated by Human SH3 Domains”

Carducci et al., Biotechnol Adv (2011)

> “Computational Analysis of High-density Peptide Microarray Data with Application from Systemic Sclerosis to Multiple Sclerosis”

Hecker et al., Autoimmunity Reviews (2011)

> “Mapping Mouse IL-13 Binding Regions Using Structure Modeling, Molecular Docking, and High-Density Peptide Microarray Analysis”

Madala et al., Proteins (2010)

> “Vaccination with ALVAC and AIDSVAX to prevent HIV-1 infection in Thailand“

Supachai et al., N. Engl. J. Med (2009)

“One focus of our group is to decipher the nature of immune responses by identification of biomarkers and indicators of immune protection. With the support of JPT‘s high content peptide microarray platform, we created a peptide chip which contains 22 000 individual peptides. This enabled the visualization of the B-cell „signature“ in individuals with TB-infection vs. non-infected individuals. In our hands, JPT‘s peptide microarrays turned out to be very robust tools to identify novel peptide based biomarkers in the context of novel diagnostics and vaccine target identification.”Prof. Markus Maeurer, Karolinska Institute, Solna, Sweden

Above: Standard Layout of a PepStar™ microarray. The whole set of peptides and controls is printed in three copies (one in each subarray). Other formats are available upon request.

Layout of one subarray. Each peptide is displayed once per subarray, each control (e.g. full length antigens, antibodies or other proteins) is printed in three copies per subarray (in total 9 copies on each PepStar™ peptide microarray).

14

Imm

uno

Tool

sH

umor

al im

mun

e re

spon

ses PepSpots™

Your customized cellulose bound peptide library could display

> Peptide scans through antigens

> Random peptides

> Positional scans

> Alanine & D-amino acid scans

> Truncation libraries

Applications for PepSpots™

• Antibody epitope mapping & characterization• Characterization of protein contact sites between - Receptor-ligand - Enzyme-substrate - Protein modules in signal transduction - Recognition sequences for heat shock proteins• Systematic optimization of peptide lead structures

What are PepSpots™ ?

Peptides (up to 20 mers) are synthesized directly on a cellulose membrane. They are C-terminally attached via a flexible linker. The membrane can be used directly for incubation with antibodies or other pro-teins and binding events read-out via chemilumines-cence. Membranes will be delivered with a detailed application protocol.

Benefits of the PepSpots™ peptidemembranes

• Incubation by standard equipment• Detection using routine ELISA protocols• Rapid, economical, and flexible synthesis

of any set of peptides • Hydrophilic cellulose membranes minimize

unspecific interactions • Detection of low affinity interactions• Incubation protocols available

Above: Automated synthesis of PepSpots™ membrane.

Left: Your PepSpots™ package includes: PepSpots™ membrane, data CD and application protocol.

15

Imm

uno

Tool

sH

umor

al im

mun

e re

spon

ses

Selected References> “HSP70 Natively and Specifically Associates

with an N-terminal Dermcidin-derived Peptide That Contains an HLA-A*03 Antigenic Epitope”

Stocki et al., J. Biol. Chem. (2011)

> “Biological Effects and Use of PrPSc- and PrP-Specific Antibodies Generated by Immunizing with Purified Full Length Native Mouse Prions”

Petsch et al., J. Virol. (2011)

> “Binding of the Human Complement Regulators CFHR1 and Factor H by Streptococcal Collagen-like Protein 1 (Scl1) via Their Conserved C Termini Allows Control of the Complement Cascade at Multiple Levels”

Reuter et al., The Journal of biological Chemistry (2010)

> “Identification of Potential HLA Class I and Class II Epitope Precursors Associated with Heat Shock Protein 70 (HSPA)”

Stocki et al., Cell Stress Chaperones (2010)

> “A Monoclonal Antibody Against Mutated Nucleophosmin 1 for the Molecular Diagnosis of Acute Myeloid Leukemias”

Gruszka et al., Blood (2010)

> “N-terminal Acetylation of Cellular Proteins Creates Specific Degradation Signals”

Hwang HS et al., Science (2010)

“My group at the University Clinics in Düsseldorf works on the investigation of pro-tein misassembly, misfolding or misprocessing in brain diseases. Our research has an ongoing need for peptide related tools that allow systematic elucidation of such protein pathologies. JPT’s PepSpots™ arrays and JPT’s ability to reproducibly assem-ble even challenging and modified peptides contributed to a noticeable degree to the success of our work.”Prof. Dr. med. Carsten Korth, Institute for Neuropathology, University of Düsseldorf, Germany

For a typical order we synthesize overlapping peptides of your protein of interest on cellulose membranes. Resulting PepSpots™ membranes can be incubated with your sample and binding region detected by chemiluminescence read-out.

Antigen of interest Overlapping peptides PepSpots™ membrane Result of incubation Binding region

16

Imm

uno

Tool

sH

umor

al im

mun

e re

spon

ses

Applications for our profiling service

• Identification of seromarkers for cancer, infectious & autoimmune diseases and allergies

• Peptide biomarker validation • Multiplexed immune monitoring in clinical trials• Elucidation of antibody signatures in vaccination

studies• Monitoring of humoral immune responses • Identification of novel vaccine targets • Substrate identification and specificity testing

Benefits of our profiling service

• Take advantage of our proprietary peptide microarray platform

• Well established assay conditions for seroscreening, protein-protein interactions and enzyme profiling

• All sample handling and profiling in environment regulated by DIN ISO 9001:2008 and GCLP

• Flexible co-immobilization of controls• Strong bioinformatic support for array design

and data interpretation

What services are provided?Send us a short outline of your project and we will:

> Suggest the appropriate array platform to be used

> Provide bioinformatic support for peptide and microarray design

> Provide project proposal & quotation> Perform synthesis of peptides and generate

peptide arrays > Incubate peptide arrays with your protein or

patient sample and perform control experiments> Evaluate and interprete data - Assign signals to peptide sequences - Provide documentation of results> Provide comprehensive and confidential report

Epitope Discovery & Antibody Profiling Service

JPT offers a comprehensive profiling and screening service using its peptide array platforms PepSpot™ and PepStar™ with your antibody / protein preparations or patient samples. JPT will provide comprehensive profiling reports with all results and detailed data analysis.

Discuss your project directly with our scientists. Contact us at peptide@jpt.com!

Let us incubate our PepStar™ peptide microarrays with your antibody, protein or patient serum.

Schematic representation of a PepStar™ peptide microarray displaying 3 identical subarrays.

Fluorescence image of a single block within subarray after incubation with human serum and fluorescently labeled secondary antibody.

Results from incubations with different patient sera. Heatmap of signal intensities for peptides scanning the sequence of a single protein (y-axis) incubated with different patient sera (x-axis).

Scatterplot of signal intensities of different subarrays.

Data analysis

PAT A1

PAT A2

PAT B1

PAT B2

PAT C1

PAT C2

PAT D1

PAT D2

PAT E1

PAT E2

PAT F1

PAT F2

PAT G1

PAT G2

PAT H1

PAT H2

PAT I1

PAT I2

PAT K1

PAT K2

PAT L1

PAT L2

Check of data quality

Microarray Layout and Data Analysis

Section of one subarray

Incubation

17

Imm

uno

Tool

sH

umor

al im

mun

e re

spon

ses

“Carolus’ technology enabled the development of therapeutic peptides and antibodies that target specific interactions between macrophage migration inhibiting factor and its chemokine receptors as well as RANTES and platelet factor-4. JPT's PepStar™ peptide microarray platform as well as its full profiling service and data interpretation capabilities have been a reliable and robust approach to elucidate the molecular details of these protein-protein interactions.”Joshua Schultz, PhD, Carolus Therapeutics Inc., San Diego, USA

load beads for affinity

purifications

immobilize onto microtiter plates

add enzyme print microarray

18

Applications for BioTides™

• Identification and optimization of kinase-, phosphatase-, acetyltransferase- and histone deacetylase-substrates via standard screening systems (AlphaScreen, FlashPlates, SPA-Beads, Luminex and many more)

• Mapping of protein/protein interaction sites • Peptide ELISA assays• Production of peptide microarrays • Loading of columns for affinity chromatography

What are BioTides™?BioTides™ are custom synthesized inexpensive sets of small scale biotinylated peptides. Thousands of BioTides™ are available within days.

> Amounts of 50 - 250 nmol per peptide> Peptide length up to 20 aa > Ready-to-use soluble peptides in 96- or 384-well

plates delivered freeze dried

Benefits of BioTides™

• Thousands of unpurified biotinylated peptides for screening and peptide array production

• Unmatched turnaround times (10 000 peptides per week!)

• Delivery in ready-to-use microtiter plates • Lowest price in the industry due to patented

technology (0.99 $/€ per amino acid residue)• Complete QC (LC-MS, MALDI etc.) and aliquotation

service available

Selected References> “GP4 of Porcine Reproductive and Respiratory

Syndrome Virus Contains a Neutralizing Epitope that is Susceptible to Immuno-Selection in Vitro”

Costers et al., Arch. Virol. (2010)

> “Screening of Predicted Secreted Antigens from Mycobacterium bovis Reveals the Immunodominance of the ESAT-6 Protein Family”

Jones et al., Infect. Immun. (2010)

Your BioTides™ will be delivered in 96-well plates with detailed documentation and QC / QA report on a CD-ROM.

Use of BioTides™ for binding and enzymatic assays.

BioTides™

Biotinylated peptides for your biomedical assays using streptavidin coated beads, membranes, glass slides or microtiter plates.

Imm

uno

Tool

sH

umor

al im

mun

e re

spon

ses

Applications for our bioinformatics service

• Design of peptide content for high-density peptide microarrays and complex peptide libraries

• Generation of homology models and use of available structural information for peptide selection

• Optimization of hit and lead structures (affinity, physicochemical properties, ADME)

• Prediction and modeling of data• Scaffold design for native-like presentation

of peptides• Interpretation, management and integration

of data

Benefits of our bioinformatics service

• Long term track record on the discovery and development of peptides in drug discovery and diagnostic development

• State of the art prediction, data interpretation and data mining algorithms and software paired with chemical and biological know-how

• Expertise available as a fee-for-service or in collaborative partnerships

Service Specifications

• Detailed discussion of your project and definition of a suitable strategy based on scientific feasibility and experience

• Receive project proposal on how our bio- and cheminformatic expertise can support your project

• Obtain detailed and comprehensive service reports

Selected References> “Probing the Epitope Signatures of IgG Antibodies in

Human Serum from Patients with Autoimmune Disease”

Lorenz et al., Methods Mol. Biol. (2009)

> “Novel Small Molecule Bradykinin B2 Receptor Antagonists”

Gibson et al., J. Med. Chem. (2009)

> “Effect of Thioxopeptide Bonds on Alpha-Helix Structure and Stability”

Reimer et al., J. Am. Chem. Soc. (2008)

JPT has a long standing and focused expertise to escort biomedical screening and peptide lead optimization programs with its comprehensive bioinformatic and cheminformatic capabilities.We offer this unique know-how and expertise as part of our high content peptide microarray and library services or in R&D collaborations focusing on peptide hit discovery and optimization.

Bioinformatics & Cheminformatics

19

Use of homology model for the selection of cytosolic loop peptides for a GPCR (MSH).

X-ray loop structure (left) and model (right) stabilized by scaffold.

Imm

uno

Tool

sH

umor

al im

mun

e re

spon

ses

Discuss your project directly with our computer scientists. Contact us at peptide@jpt.com!

20

Imm

uno

Tool

sH

umor

al im

mun

e re

spon

ses

RepliTope™ microarrays are delivered together with application protocol, gal-file and QC/QA report.

JPT’s printing tools for high-content microarray generation.

Applications for RepliTope™

• Efficient profiling of antibody collections directed against the same antigen

• Identification of biomarkers for infectious and autoimmune diseases, allergy and cancer

• Monitoring humoral immune responses using sera in preclinical and clinical trials

• Characterization of potential therapeutic antibodies

• Elucidation of protein-protein and enzyme -substrate interactions in general

• In vitro study of anti-antibody / neutralizing antibody action

• Development of phosphorylation dependent antibodies

• Monitoring of side effects of protein drugs during clinical trials

What is RepliTope™?Catalog Peptide Microarrays that contain peptide scans through a large number of relevant antigens or random peptide libraries. Every peptide is present 3 times on each microarray to ensure reproducibility of results.

Benefits of RepliTope™

• Ready-to-use RepliTope™ peptide microarrays through hundreds of relevant antigens available within days

• Compared to protein arrays: high shelf stability, high batch-to-batch reproducibility, regio-specific immobilization of peptides

• Optimized surfaces and built-in quality controls • Fully compatible with standard ELISA protocols • Flexible and economical access to many

identical peptide microarrays• Reliable epitope mapping using minimal

amounts of antibody• All in one service available (incubation, data

analysis and a detailed report)

RepliTope™

RepliTopes™ are premade peptide microarrays displaying peptide scans (15 mers, overlapping by 11 aa) through annotated proteins or random peptide libraries on glass slides. Read-out is performed via fluorescence. Every peptide is present in 3 copies on each microarray to ensure reproducibility of results.

21

Selection of available RepliTopes™

BKV (VP1, largeT...), EBV (EBNAs, BMRF1), HIV, HCMV (pp65, IE1...), influenza, tumor associated antigens (mela-noma, prostate and breast cancer...) and many more

NEW: RepliTope™ Antigen Collections

All important antigens of a given pathogen on one microarray saving time and consumables!BKV, EBV, HCMV, TB, Vaccinia, Random peptide libraries and many more

Have a look at the complete and up-to-date list of more than 100 catalog microarrays under shop.jpt.com!

Selected References> “Epitope Mapping Using Randomly Generated

Peptide Libraries” Bongartz et al., Methods in Molecular Biology,

Epitope Mapping Protocols (2009)

> “Validation of Peptide Epitope Microarray Experiments and Extraction of Quality Data”

Nahtman et al., J. Immunol. Methods (2007)

> “Prion Recognition Elements Govern Nucleation, Strain Specificity and Species Barriers”

Tessier & Lindquist, Nature (2007)

“Over the past five years my research team has been active in the develop-ment of species-specific peptide arrays. Our ability to advance this technology into new research areas has been dependent on working with the highest quality of arrays. In this regard, JPT’s commitment to excel-lence has been a critical foundation for the success of our research program. I have been consistently impressed with their customer service, technical expertise and professiona-lism. When it comes to peptide work there are no other options for our group…JPT is the best.”Scott Napper, PhD, Vaccine and Infectious Disease Organization (VIDO), University of Saskatchewan, Canada

All important antigens of a given pathogen on one

Imm

uno

Tool

sIm

mun

o To

ols

Imm

uno

Tool

sH

umor

al im

mun

e re

spon

ses

> Peptide design for T-cell assays

Recommended peptide length:

• 9 or 10 mers for stimulation of CD8+ cells only• 15 mers for stimulation of both, CD4+ & CD8+cells,

in parallel• Overlapping peptides with an offset of 4 amino

acids or less• References:

- Kern et al., Human Immunol. (2004)- Maecker et al., JIM (2001)

Recommended peptide purity:

• Crude peptides often give false positive T-cell responses

• Even small contaminations (below 1%) can provoke false T-cell responses

> Hits should always be validated using highly purified peptides

• References: - Burgers et al., RCMS (2007)- Mannering et al., Vaccine (2003)- Cox et al., Clin. & Vaccine Immun. (2008)

Recommended pool size:

• Pools with many peptides trigger lower responses > Peptide pools should contain 150 peptides

at most• Reference:

- McElrath et al., (JID 2003)

> Peptide content & net weight

• Peptides contain 10-40% of non-peptidic components (water, counter-ions, etc.)

• Peptide content = the total percentage of peptide within a synthetic sample

• Peptide content is determined by amino acid analysis

• Net weight= peptide amount (mg) considering the peptide content

• Net weight is calculated acc. to measured peptide content

> How to dissolve peptides

Solubility of peptides is hard to predict as it depends on various parameters (ratio of charged to uncharged/hydrophobic residues, pH value, primary and secon-dary structure...). Therefore, there is no ideal solvent for all peptides. If an identical solvent for many peptides (e.g. of a library) is needed, JPT recommends to use a small volume of organic solvent (DMSO is preferred) first, followed by stepwise dilution with the appropri-ate buffer. However, to find the optimal solvent for one or several peptides, it may be necessary to use a series of increasingly powerful solvents until the peptide dis-solves. Please proceed according to the workflow shown on the next page.

Hydrophobic (non-polar) Ala, Ile, Leu, Met, Phe, Pro, Trp, Val

Hydrophilic (polar & uncharged)

Asn, Cys, Gly, Gln, Ser, Thr, Tyr

Charged (acidic) Asp, Glu, free C-terminus

Charged (basic) Arg, His, Lys, free N-terminus

The 20 natural amino acids sorted acc. to their hydrophobicity.

Application Recommended Specification

High-throughput discovery (Epitopes, biomarkers...)

Unpurified (Main product = target peptide, optional “capping“ to avoid dele-tion sequences)

Epitope mapping Guaranteed purity: 70% or 80%

Epitope validationControl peptides

Guaranteed purity: 90% or 95%, purified by HPLC

Immune monitoringClinical trials

Guaranteed purity: >80%, >90%, >95% purified by HPLC

Note: JPT uses optimized synthesis protocols to minimize contaminants even in unpurified peptides!

< Turn the page for more technical support.

Tech

nica

l Sup

port

Peptide Parameters Influencing T-cell AssaysIm

mun

o To

ols

Hydrophobic (non-polar) Ala, Ile, Leu, Met, Phe, Pro, Trp, Val

Hydrophilic (polar & uncharged)

Asn, Cys, Gly, Gln, Ser, Thr, Tyr

Charged (acidic) Asp, Glu, free C-terminus

Charged (basic) Arg, His, Lys, free N-terminus

The 20 natural amino acids sorted acc. to their hydrophobicity.Te

chni

cal S

uppo

rt> Peptide stability

Chemical stability of peptides depends on amino acid composition and sequence. Peptides are generally more stable if lyophilized. Main degradation problems are:• Hydrolysis of Asp-Pro, Asp-Gly and Asp-Ser bonds• Deamidation at Asn-Gly or Gln-Gly (Q-G) bonds• Oxidation at Cys or Met• Pyroglutamic acid formation at N-terminal Gln

(avoided by N-terminal acetylation)• References:

- Landon, Methods in Enzymology (1977)- Stephenson and Clarke, J. Biol. Chem. (1989)- Robinson et al., J. Amer. Chem. Soc. (1973)- Scothcler and Robinson, Analytical Biochem

(1974) - Powel, Formulation and Delivery of Proteins

and Peptides (1994)

> How to store peptides

JPT recommends to store all peptides in frozen and freeze dried aliquots!

Storing freeze dried peptides:• Store in dry, cool and dark place• If possible store lyophilized not in solution• Short term storage (for several days) at room

temperature is fine• Long term storage at -20°C, or even better at -80°C• Before opening the vial, allow it to adjust to

room temperature• Reseal vial (under dry inert gas if possible)

Note: JPT offers provision of small ready-to-use, freeze dried aliquots and stability testing services!

Storing dissolved peptides:• Storage at -80°C or in liquid nitrogen• Avoid freeze-thaw cycles (instead freeze small

aliquots)• Store in glass or chemically inert plastic vials

(e.g. polypropylene)• Dissolve in sterile buffers• Use within a few days

Note: DMSO, DMF and NMP are toxic and DMSO leads to faster oxidation of cysteine containing peptides!

Workflow to find solvents for pep-tides with different physicochemi-cal properties.

For further questions contact our customer support team at peptide@jpt.com!

Peptide Parameters Influencing

T-cell Assays

add water or buffer stepwise until final concentration is

reached

insoluble

still insoluble

still insoluble

sonification

sonificationsonification

sterile distilled water / sterile acetic acid (0.1%)

examine amino acid composition

hydrophobic peptide (or little/no charge)

basic peptides: 10% acetic acid or TFA

acidic peptides: aqueous ammonia or 10% ammonium bicarbonate

remove solvents by lyophilization

dissolve in small amount of 100% organic solvent (DMSO or

DMF, NMP, isopropanol)

Imm

uno

Tool

s

Issu

e D

ate:

Nov

embe

r 201

1

We take pride in our competent service and swift response.Please do not hesitate to contact us for further information.We also very much welcome your feedback and comments.

Volmerstraße 5 (UTZ)12489 BerlinGermany

P. O. Box 2619Acton, MA 01720USA

USA/Canada T 1-888-578-2660F 1-888-578-2666

European Head Office T +49-30 - 6392-7878F +49-30 - 6392-7888

JPT Peptide Technologieswww.jpt.com | peptide@ jpt.com