R&D Collaboration. R & D Collaborations

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1 R&D Collaboration R & D Collaborations > Biomarker Discovery > Immune Monitoring > Vaccine Development > Antibody Signature Profiling > Functional & Targeted Proteomics > Drug Discovery & Optimization Innovative Peptide Solutions

2 Immuno Tools JPT s proprietary key technologies are: Why JPT Use our proprietary synthesis and screening technologies Take advantage of JPT's integrated R&D team for organic & medicinal chemistry, bioinformatics, structural biology, data management and assay development Rely on our solid track record and ISO 9001 certification Benefit from flexible business arrangements for collaborative projects Technologies & Applications Over the past decade, JPT has developed a portfolio of proprietary technologies and 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 all 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. SPOT High throughput peptide synthesis and screening technology for peptides & peptidomimetics for T-cell epitope and peptide lead discovery and characterization on proteome-wide levels. PepStar Proprietary technology platform for 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 Light and stable isotope labeled peptides for proteomics. Quality Assurance JPT is DIN EN ISO 9001:2008 certified and GCLP audited.

3 R & D Collaboration 02 / About JPT 03 / Projects & Partners 04 / Immune Monitoring & Biomarker Discovery 05 / Humoral Immune Response 06 / Cellular Immune Response 07 / Case Studies 09 / Proteomics 10 / Discovery & Targeted Proteomics 11 / Functional Proteomics 12 / Case Studies Table of Contents 13 / Drug Discovery & Optimization 14 / Peptide Discovery 14 / Hit to Lead / Qualification 14 / Peptide Optimization 15 / Case Studies 17 / Technologies & Know How 17 / Bio-, Cheminformatics & Modeling 18 / Assays & Screening 20 / Chemistry and Technologies 1

4 2R About JPT & D Collaboration About JPT JPT Peptide Technologies is an experienced partner for peptide based R&D collaborations. Located in Berlin, Germany, JPT has achieved world wide credibility for its commitment to rigorous quality standards and a reputation for developing and implementing innovative peptide based services and research tools for various applications. JPT serves a broad clientele in the pharmaceutical and biotechnology industries as well as researchers in universities and other non-profit organizations. JPT offers its services in flexible business arrangements Fee-for-service projects FTE based and milestone driven collaboration projects Shared risk, partnered development projects SME qualification for participation on publicly granted R&D projects Company History JPT Peptide Technologies GmbH was established in 2004 as a subsidiary of Jerini AG, a spin-off of Charité in Berlin, focused on the discovery and development of novel peptide-based drugs. In 2008, JPT was ac quired to become a part of BioNTech AG, Mainz (Germany), a company that develops novel immune therapy and diagnostic approaches for various cancers. JPT is your preferred partner for peptide related projects because of its > Proprietary ultra high-throughput synthesis and screening basis > Integrated in-house R &D team for organic and medicinal chemistry, bioinformatics, structural biology, data management and assay development > Solid track record of successful R &D projects > ISO 9001:2008 and GCLP regulated quality assurance system > Professional project management and communication > Flexible business arrangements for collaborative projects

5 R & D Collaboration Recent Projects & Partners Here we present our extensive but not complete list of recent projects and project partners. Drug Discovery Substrate identification and optimization for se veral orphan enzymes for screening assays Design and production of screening libraries of peptides and peptidomimetics Identification and optimization of agonistic and antagonistic ligands Identification and optimization of affinity ligands Identification and optimization of stabilizing peptides as excipients Development of surrogate bioassays for antigenfree detection of therapeutic proteins Immunology Identification of serological predictive, prognostic and surrogate biomarkers Seromarker identification as immune correlates for protection Proteome wide identification of T-cell epitopes for pathogens Companion diagnostics for vaccination studies in cancer and infectious diseases Design of comprehensive peptide libraries for companion diagnostics and treatment Proteomics Development of next generation standards for targeted proteomics Characterization of specificity profiles for pro teins and protein families Design and synthesis of peptide libraries for proteome wide detection and quantification of proteins Development of molecular probes for the multiplexed detection and quantification of proteins from biological samples Discovery of proteotypic peptide markers for functional proteomics Identification of epigenetic states of proteins and characterization of enzymatic alterations of epige netic states Partners include > Bayer Pharma AG > BioNTech AG > Cancer Immunotherapy Trials Network (CITN) > Charité Universitätsmedizin Berlin > Duke University > ETH Zürich > Harvard Medical School > Hoffmann La-Roche > Immudex > Institute for Systems Biology, Seattle > International AIDS Vaccine Initiative (IAVI) > National Institute for Biological Standards and Control (NIBSC) > Oregon Health and Science University > Zellnet and various undisclosed partners About JPT Left: JPT's team of skilled staff and knowledgeable scientists 3

6 4R Immune Monitoring & Biomarker Discovery & D Collaboration Immune Monitoring & Biomarker Discovery Over the last decade, JPT has developed several unique peptide technologies to enable efficient and proteome spanning B- and T-cell epitope-based biomarker discovery. Several successful projects have lead to new diagnostic and stratification markers in indications such as cancer, infections and autoimmune diseases and allergies. Furthermore, we developed tailored profiling workflows for humoral and cellular immune responses that can be correlated to clinical data. JPT s com prehensive serological assay platform has been applied in collaborations devel oping epitope resolved immune monitoring tools and molecular correlates for protection in various indications. Correspondingly, JPT s proprietary overlapping peptide pool platform finds widespread application in the development of clinical immune monitoring tools and novel diagnostic applications focusing on cellular immune responses. Benefits Multiplexed identification of epitopes from antigens or entire proteomes State of the art infrastructure (cleanroom, BSL2, organic chemistry labs) & bioinformatic capabilities Solid track record Chemical synthesis methods address sequence diversity and post-translational modifications Selected References > Nonreplicating vaccines can protect african green monkeys from the memphis 37 strain of respiratory syncytial virus Eyles et al., JID (2013) > Serum autoantibodies to myelin peptides distinguish acute disseminated encephalomyelitis from relapsing remitting multiple sclerosis Van Haren et al., Multiple Sclerosis Journal (2013) > Neutralization of porcine endogenous retrovirus by antibodies against the characterization of humoral and cellular immune responses elicited by a recombinant adenovirus serotype 26 HIV-1 Env vaccine in healthy adults (IPCAVD 001) Barouch et al., JID (2013)

7 R & D Collaboration Humoral Immune Response JPT offers a comprehensive workflow to detect circulating antibodies in patient samples for the development of diagnostic and stratification biomarkers in indications such as infectious and autoimmune diseases as well as cancer. Full length proteins as capture reagents allow the identification and quantification of antibodies directed against entire antigens. In contrast, peptides as capture reagents yield an additional dimension of information. An overlapping peptide scan through an antigen, antigen family or entire proteome allows the detailed mapping of linear epitopes for antibodies along the antigen sequence. Thus, different epitope patterns, epitope spreading, as well as changes of B-cell immune responses against linear epitopes within any antigen can be monitored in detail and correlated to disease progression, therapeutic intervention or any other clinical evidence. The investigation of samples at different time points and with secondary detection anti - bodies of different isotype specificity allows the characterization of time dependency and involvement of different phases of the humoral immune response. In addition, immobilised synthetic peptide libraries allow representation of sequence diversity and defined posttranslational modifications. JPT s seroprofiling platform consists of three synergistic but independent modules as follows: 1) PepStar High Content Peptide Microarrays for multiplexed biomarker discovery 2) PepStar Multiwell Peptide Microarray for biomarker verification 3) Peptide ELISA for validation of detected biomarkers The three modules can be applied sequentially in milestone-driven biomarker discovery programs or individually in projects targeting the development of robust humoral immune monitoring tools in indications such as cancer, infectious and autoimmune diseases as well as allergies. Library Content Sample Throughput High Content Peptide Microarray > 5000 peptides ~ 100 samples Multiwell Peptide Microarray ~200 peptides ~1000 samples Peptide ELISA flexible design Multiplexed Epitope Discovery is carried out for candidate proteins. Thousands of peptides can be screened in parallel using high content peptide microarrays. Hits are confirmed by Selective Antigen Profiling with multiple serum samples. Applying multiwell peptide microarrays, thousands of samples can be tested. Validation of identified biomarkers by flexible and robust peptide ELISA. Results are transferred towards diagnostic assays. Immune Monitoring & Biomarker Discovery 5

8 6R Immune Monitoring & Biomarker Discovery & D Collaboration Cellular Immune Response JPT offers its expertise in discovery projects for the definition of immunodominant epi topes from various antigens or proteomes and for the development of tailored immune monitoring tools. Our peptide pool technique takes advantage of synthetic overlapping peptides that span entire antigen sequences and has been reported as an excellent tool for the antigen specific stimulation of CD4 + and CD8 + T-cell responses in vitro. As a consequence, JPT's PepMix peptide pools have been used as independent readout systems to monitor clinical vaccination and immunotherapy trials and to reactivate polyclonal CD4 + and CD8 + T-cells specific for a spectrum of viral antigens for stem cell recipients in an FDA approved trial. For discovery projects, JPT developed a unique and economic workflow for proteome spanning T-cell epitope discovery using well established and commonly used T-cell assays such as ELISpot. For development of robust immune monitoring tools, our peptide library platform provides access to high quality peptides designed for performance in cellular assays accompanying clinical trials. Virus, cancer cell or other Antigen sequences Overlapping peptide scan through antigen sequences Pooling (approx. 100 peptides / pool) Aliquoting T-cell assays lead to identi fication of immunodominant peptides Optional deconvolution and therapeutic options Pooling 317 peptides / 4 pools Pooling 86 peptides / 1 pool Pooling 119 peptides / 2 pools

9 Case Studies R & D Collaboration Antibody response to HIV gp120 is critical for protective activity in HIV-vaccination RV144-study RV144 Thai trial is the first successful HIV vaccination study Correlate analysis looking for multiple parameters uses JPT's peptide microarrays for the mapping of humoral immune response Microarray analysis shows that specific antibody reactivities in V2 loop correlate with a lower risk of HIV infection > The thai phase III HIV type 1 vaccine trial (RV144) regimen induces antibodies that target conserved regions within the V2 loop of gp120 Karasavvas et al., AIDS Res Hum Retroviruses (2012) > A computational framework for the analysis of peptide microarray antibody binding data with application to HIV vaccine profiling Imholte et al., J Immunol Methods (2013) > Analysis of V2 antibody responses induced in vaccinees in the ALVAC/AIDSVAX HIV-1 vaccine efficacy trial Zolla-Pazner et al., PLoS One (2013) > Peptide array mapping of env-specific IgG responses in HIV-1 vaccine efficacy trials and chronically infected individuals Gottardo et al., PLoS One (2013) Identification of immunodominant T-cell antigens in Mycobacterium bovis for vaccine design 119 antigens of M. bovis were produced as peptide pools representing each antigen in overlapping peptides and screened for IFN-γ response in blood from 23 M. bovis-positive cattle 59% of the tested antigens induced positive responses Proteins of the ESAT-6 family are immunodominant Epitope mapping allows for the identification of the most frequently recognized peptides > Screening of predicted secreted antigens from mycobacterium bovis reveals the immunodominance of the ESAT-6 protein family Jones et al., Infect Immun (2010) Immune Monitoring & Biomarker Discovery Identification of target antigens and immunodominant T-cell epitopes for vaccine development or biomarker discovery and validation. 7

10 8R & D Collaboration Immune Monitoring & Biomarker Discovery Case Studies Dose finding for an experimental HIV vaccine in a phase I clinical trial Patient samples from IPCAVD 001, the first-inhuman evaluation of a prototype Ad26 vectorbased vaccine expressing clade A HIV-1 Env (Ad26.ENVA.01) were evaluated on peptide microarrays for antibody reactivities Dose dependent expansion of the magnitude, breadth and epitopic diversity was measured A critical dose for the stimulation of a Env-V2 specific B-cell immune response could be defined > Characterization of humoral and cellular immune responses elicited by a recombinant adenovirus serotype 26 HIV-1 Env vaccine in healthy adults (IPCAVD 001) Barouch et al., JID (2013) Antigen-specific binding antibody profiles. Sera were tested on peptide arrays to monitor linear antibody responses to peptides spanning Env from clades M, A, B, C, D, CRF02, and CRF01. Mean signal intensity (in week 28 with baselines subtracted) from all subjects in each group is depicted. Black asterisks denote peptides with significantly elevated mean signal intensity as compared to placebos (p<0.05, Wilcoxon Mann-Whitney tests), and red asterisks denote trends (p<0.10). Mean Signal Intensivity week 28 - pre vaccination Peptide Number

11 R & D Collaboration Proteomics JPT is providing the Proteomics community with a multitude of innovative research tools. Besides high-throughput chemical and analytical approaches to accelerate the discovery of novel biomarkers from biological samples, JPT has developed a new peptide standard technique for absolute quantification of target proteins in clinical and targeted proteomics. Furthermore, JPT has a decade-long track record providing services and project expertise in the area of functional proteomics ranging from elucidation of protein-protein interactions and enzymatic activities to epigenetic events. Proteomics Benefits Parallel identification and quantification of hundreds of proteins Simultaneous determination of post-translational modifications Cost efficient multiplexed assay Reliable high-throughput technique High sensitivity Selected References > Seminal plasma as a source of prostate cancer peptide biomarker candidates for detection of indolent and advanced disease Neuhaus et al., PLOS ONE (2013) > Multiple reaction monitoring of multiple low-abundance transcription factors in whole lung cancer cell lysates Kim et al., Journal of the Proteome Research (2013) > Automatic generation of predictive dynamic models reveals nuclear phosphorylation as the key Msn2 control mechanism Sunnåker et al., Sci Signal (2013) > N-glycoprotein SRMAtlas: a resource of massspectrometric assays for n-glycosites enabling consistent and multiplexed protein quantification for clinical applications Hüttenhain et al., Mol Cell Proteomics (2013) > Occurrence and detection of phosphopeptide isomers in large-scale phosphoproteomics experiments Courcelles et al., Journal of Proteome Research (2012) > Using irt, a normalized retention time for more targeted measurement of peptides Reiter et al., Proteomics (2012) > Automated workflow for large-scale selected reaction monitoring experiments Malmström et al., Journal of Proteome Research (2012) > Peptides quantification by liquid chromatography with matrix-assisted laser desorption/ Ionization and selected reaction monitoring detection Lesur et al., Journal of Proteome Research (2012) 9

12 R & D Collaboration Proteomics Discovery & Targeted Proteomics Two main approaches for mass spectrometry based proteomics exist, discovery or shotgun proteomics and targeted proteomics. Discovery or shotgun proteomics usually aims at the identification of large sets of proteins in complex samples. In targeted proteomics high-selectivity quantitative assays for lower numbers of proteins are developed. The ultimate goal of targeted proteomics is to specifically address defined biological questions. The significance of targeted proteomics is exemplified by the fact that it was selected as Nature Method of the Year Multiple reaction monitoring (MRM), which is one of the most frequently used targeted proteomics approaches, utilizes a triple quadrupole mass spectrometer to detect precursor and production pairs, furnishing assays for the detection and quantitation of multiple proteins in biological samples. JPT has been providing the proteomics community with tools for targeted proteomics for years. The needs of researchers for inexpensive, heavily labeled or unlabeled and absolutely quantified peptides triggered the development of the SpikeTides product family, which finds widespread application in many laboratories. In addition, JPT offers complete proteomic studies in collaboration with its partners. Please feel free to discuss all aspects of your project with our experts. Our Bioinformatics expertise allows for intelligent peptide library design that, in combination with out technologies, helps to solve complex problems in target discovery and validation as well as biomedical research. Impact of JPT's technologies on the proteomics workflow. Discovery Proteomics Targeted Proteomics Trypsin Trypsin Protein(s) of interest in complex sample Proteotypic peptides Proteotypic peptides Shotgun experiment (MS spectra of as many proteins as possible) SpikeTides or SpikeTides L for SRM/MRM assay setup SRM/MRM assay setup MS conditions for identification of characteristic proteotypic peptides Identification of characteristic proteotypic peptides with sequence information from databases 10 High-throughput identification of proteins SpikeTides Fragmentation information SpikeTides L for relative quantification SpikeTides TQL for absolute quantification Identification and relative/ absolute quantification of protein(s) of interest SRM/MRM Experiment (Selected fragmentation reactions monitored)

13 R & D Collaboration Functional Proteomics Functional proteomics studies the biological function of proteins or protein groups and classes on a proteome-wide level. The data are the basis for the elucidation of signaling networks and therefore to understand cell function. Functional proteomics includes the characterization of enzyme activities as well as protein-protein interactions and the functional state of proteins. Combining our technologies and expertise, we are able to map changes in signaling pathways as a consequence of cell-treatment or changes in environmental conditions. This can either be done by the large scale measurement of enzymatic activities such as kinase, phosphatase, protease or of other protein modifying enzymes. In addition, valuable information about the epigenetic state of proteins can be collected. Proteomics Additionally, we have developed innovative ways to evaluate and present complex data sets that is indispensable to the optimal exploitation of experimental results. Characterization of signal transduction networks. Proteome wide screening for substrates of Sirtuin class enzymes by peptide microarrays and structural characterization of enzyme-substrate complexes. For further details see page 12. Substrate Specificity Profiles Complex Structure 35.8 % SIRT 1 depleted enriched 35.8 % 33.0 % SIRT 2 depleted enriched 33.0 % 26.6 % SIRT 5 depleted enriched 26.6 % 11

14 R & D Collaboration Case Studies Proteomics Characterization of PRMT5-MEP50 histone methyltransferase in dependence of post-translational substrate modifications Methylation of arginine and lysine residues is part of the epigenetic code in histones PRMT5-MEP50 is known to catalyze the methylation of Arg-residues A complex peptide library displayed on peptide microarrays was used to characterize the histone methyltransferase PRMT5-MEP50 Results show that PRMT5-MEP50 modifies Arg3 in the N-terminal tail of H2A and H4 This activity is modulated by substrate PTMs: phosphorylation of neighbouring Ser inhibits activity, acetylation of C-terminal Lys residues enhances activity > Structure of the arginine methyltransferase PRMT5- MEP50 reveals a mechanism for substrate specificity Ho et al., PLoS One (2013) PRMT5-MEP50 histone methyltransferase activity is modulated by substrate PTMs. High-density histone peptide arrays incubated with PRMT5-MEP50. Data from H4 peptides are shown, each row represents a discrete peptide. The left panel shows individual modifications present on each peptide, the histogram on the right panel shows the relative activity on each peptide. The signal on the unmodified 1-20 peptide is indicated in blue. Inhibition by Ser1 phosphorylation is indicated in red. Deciphering substrate specificity of human sirtuins The human class of sirtuins consists of 7 members, only a limited number of substrates is known The 7 human sirtuins were tested for enzymatic activity on a library of 6802 human acetylation sites immobilized on peptide microarrays Substrates were identified and substrate specificity profiles could be generated (see figure on p. 11) These results help to understand the role of single sirtuins in signaling networks > An acetylome peptide microarray reveals specificities and deacetylation substrates for all human sirtuin isoforms Rauh et al., Nat Commun (2013) 12

15 R & D Collaboration Drug Discovery & Optimization JPT Peptide Technologies applies its portfolio of proprietary peptide technologies and bioinformatic and medicinal chemistry know-how to manage R &D projects for contract research and R &D collaborations in peptide discovery, hit-to-lead qualification and peptide optimization. Our experience in drug discovery and optimization finds application in peptide based drug discovery and vaccine projects, in the discovery of peptidic additives for the cosmetic and nutritional industry, in the development of affinity ligands for purification of biomolecules, as well as in content determination for novel immune diagnostics. Peptide Discovery Random/ Knowledge-based Libraries Example Projects Identification and optimization of agonistic and antagonistic ligands Enzyme substrate identification and optimization for screening assay development Design and production of screening libraries of peptides and peptidomimetics Identification and optimization of affinity ligands Identification and optimization of stabilizing peptides as excipients Development of surrogate bioassays for antigenfree detection of therapeutic proteins Hit to Lead Qualification SAR Analysis Prioritization Selected References Peptide Optimization Iterative Activity/ PhysChem/ADME Optimization > High density peptide microarrays for proteomewide fingerprinting of kinase activities in cell lysates Thiele et al., Methods Mol. Biol. (2010) > Novel small molecule bradykinin B1 receptor antagonists. Part 1-3 Schnatbaum et al., Bioorg. Med. Chem. Lett. (2010) > Small molecule bradykinin B2 receptor modulators Gibson, Schnatbaum et al., WO 2010/ (2010) > TFPI Inhibitors and methods of use Reimer et al., WO 2010/ (2010) Drug Discovery & Optimization > Small molecule antagonists of the bradykinin B1 receptor Reimer et al., WO 2009/ (2009) > Peptide arrays for enzyme profiling Zerweck et al., Methods Mol. Biol. (2009) 13

16 R & D Collaboration Drug Discovery & Optimization Peptide Discovery Over the past decade, JPT has run several successful projects for the identification of peptide hits. Among them were specific substrates for orphan proteases, kinases, phosphatases and acetyltransferases. The key to most of these screening projects was the application of JPT's proprietary PepSpot or PepStar technologies. These respective libraries were designed by either knowledge based methods or from randomized sequences. In collaboration with Bayer AG, Germany, high content peptide and peptidomimetics libraries were created for screening of undisclosed targets. Overall, several million compounds were selected, synthesized and screened. Hit-to-lead / Qualification At JPT, we pay particular attention to the thorough qualification of hits. For this, hits are resynthesized, purified and characterized in multiple assays that are relevant to the target parameters/properties. Along with SAR analyses, profiling results enable a clear prioritization and selection of the most promising leads for optimization. JPT has been engaged in several hit qualification/hit-to-lead projects, where hits for kinases, proteases, transferases, isomerases and phosphatases were transferred into qualified leads. The projects were done in-house or during perennial collaborations with industry partners. The hit qualification process at JPT is supported by an established network of service providers which addresses basically all properties relevant to drug discovery and other applications. Peptide Optimization Peptide optimization projects at JPT are conducted with purified compounds that are synthesized on polymeric resins or in solution. If required, proprietary high content technologies such as SPOT and PepStar support the optimization work. The process is highly iterative, taking into account all relevant optimization parameters. In this res pect, we emphasize the evaluation and consider late-stage goals (e.g. PK/ Tox properties) from very early in the optimization process (to allow early fall-back if necessary). During optimization, the lead peptides are usually converted to smaller and less peptide-like compounds (i.e. by incorporating unnatural amino acids, small molecule building blocks or steric constraints). Lead optimization projects are not limited to the area of drug discovery. Our expertise is employed in many other areas of biomedical research as well as the nutritional and cosmetic industry. Exem plarily, JPT s scientists have a profound experience in the development of specific peptidic affinity ligands for the purification of therapeutic proteins. 14

17 Case Studies R & D Collaboration SAR characterization and optimization of a 13-meric cyclic antigen for anti-myc antibody The key residues of a cyclic peptide for binding to an anti-myc antibody were to be identified A substitutional analysis of the myc tag sequence (EQKLISEEDL, incorporated into a 13-meric cyclic peptide) was performed using more than 170 cyclic peptides on microarray slides. All peptides were screened for binding to an anti-c-myc antibody. One peptide was also inserted into a proteolytically stable microprotein to enhance pharmacokinetic properties Key residues and tolerable substitutions were identified. Cyclization was shown to be beneficial for binding affinity Drug Discovery & Optimization Substrate specificity analysis of a 13-meric cyclic antigen for antimyc antibody Left: Cyclic myc peptide with highlighted key residues ESIL. The Cys-Cys bridge is marked in yellow. Right: Results of a substitutional analysis of this cyclic myc peptide. Red colour indicates strong specific binding, light yellow indicates less or no binding. The results for the WT peptideare highlighted by the black boxes. 15

18 R & D Collaboration Case Studies Drug Discovery & Optimization Optimization of a 21-mer hit to a 9-mer peptide Various cycles of substitutional anlyses, truncation and deletion analyses were used to optimize a peptide in terms of molecular weight, binding characteristics and physicochemical properties The 21-mer hit peptide with two internal disulphide bonds was optimized towards a nonapeptide with retained biological activity and superior physicochemical properties Left: The figures depict the results of substitutional analyses of the initial peptides, which where used as a basis for convertion into a smaller and more stable peptide. Red indicates strong binding light yellow or white indicate less or no binding. Black brackets show positions of disulfide bonds. Below: Table showing various properties measured for intermediate compounds at different stages of peptide optimization. 16

19 Technologies & Know How R & D Collaboration Technologies & Know How Bio-, Cheminformatics & Molecular Modeling JPT Peptide Technologies has long standing expertise in biomedical screening and peptide lead optimization programs with its comprehensive bioinformatic and cheminformatic capabilities. We offer this unique know-how as part of our high content peptide microarray and custom peptide synthesis service or in R &D collaborations focusing on seromarker profiling, peptide hit discovery and optimization. Service Specifications In depth discussions of your project and definition of a suitable strategy based on scientific feasibility and experience Detailed project proposal on how our bioinformatic and cheminformatic expertise can support your project Obtain detailed and comprehensive service reports Selected References > Design and synthesis of a new class of selective integrin α5β1 antagonists Stragies et al., J. of Medicinal Chemistry (2007) Benefits Long term track record on the discovery and deve lopment 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 > Translating peptides into small molecules Hummel et al., Mol. BioSyst. (2006) X-ray loop structure (left) and model (right) stabilized by scaffold. Use of homology model for the selection of cytosolic loop peptides for a GPCR (MSH). 17

20 R & D Collaboration 18Technologies & Know How Assays & Screening JPT's experienced scientists will develop the optimal peptide screening strategy for your pro ject. Assays are performed in an S2 laboratory environment using automated incubation stations assuring robust and reproducible performance. Applications Biomarker discovery for cancer, infectious & autoimmune disease and allergy Seroprofiling and antibody epitope mapping Development of immune diagnostic and patient stratification tools Clinical monitoring and companion diagnostics Elucidation of antigen and epitope spreading during disease progression and therapeutic intervention Detection of immunodominant regions in antigens and of novel vaccine targets Identification of substrates for orphan enzymes and enzymatic assays Characterization of protein-protein interactions Investigation of posttranslational modifications and epigenetics Antigen sequence Overlapping peptides PepStar Microarray Selected References > Characterization of humoral and cellular immune responses elicited by a recombinant adenovirus serotype 26 HIV-1 Env vaccine in healthy adults Barouch et al., J Infect Dis (2013) > Peptide array mapping of env-specific IgG responses in HIV-1 vaccine efficacy trials and chronically infected individuals Gottardo et al., PLoS One (2013) > Serum autoantibodies to myelin peptides distinguish acute disseminated encephalomyelitis from relapsing - remitting multiple sclerosis Haren et al., Multiple Sclerosis J (2013) > A sequence in subdomain 2 of DBL1a of plasmodium falciparum erythrocyte membrane protein 1 induces strain transcending antibodies Blonqvist et al., PLoS One (2013) Blood serum / plasma Labeled secondary antibody Principle of antibody epitope mapping by overlapping peptides. The antigen sequence is displayed by a set of overlapping peptides. Antibodies from patient sample bind to epitopes presented by the peptides and are subsequently detected by a labeled secondary antibody. Using this information, epitopes recognized by circulating antibodies can be determined. Binding region Antigen of interest

21 R & D Collaboration Benefits Proprietary technologies for multiplexed identi fication and quantification of epitopes for multiple antigens or entire proteomes Addressing of natural sequence diversity and posttranslational modifications Cost efficient and modular workflow allows efficient and tailored planning of projects (see figure below). 1) Multiplexed epitope discovery enabling screening of thousands of peptides in parallel using high content peptide microarrays. 2) Selective antigen profiling for systematic screening of large numbers of test samples using multiwell peptide microarrays. 3) Marker validation and transfer of results towards diagnostic assays using peptide ELISA Technologies & Know How Minimal consumption of patient material and proteins Low background combined with high sensitivity Robust assay protocols ensuring reproducible results Technologies defining JPT's biomarker discovery platform. High Content and Multiwell Microarrays are two manifestations of our unique PepStar technology. These formats allow efficient screening of a high number of peptides and samples, respectively. The robust and straightforward Peptide ELISA is applied, as an alter native technology, for the validation and development of diagnostic tests. Peptide Microarray Peptide Microarray Peptide ELISA High Content: 6912 peptides in 3 copies/sample Multiwell: 192 peptides in 3 copies/21 samples 1-96 peptides/samples A B C D E F G H > Multiplexed Epitope Discovery > Selective Antigen Profiling > Marker Validation 19

22 20Technologies & Know How R & D Collaboration Chemistry and Technologies Peptide Synthesis Extraordinarily high capacity and quality in automated synthesis, analysis and purification. Applications and Specifications Applied in all kinds of peptide based projects Wide range of peptide modifications From 1 mg to several grams Analyses: LC-MS MALDI-MS, HPLC, optionally AAA, NMR, CE, peptide content, solubility, stability testing, bacterial endotoxin and sterility analysis High-troughput SPOT Synthesis JPT Peptide Technologies has the unique ability to produce peptide libraries made of up to 1 million single, individual peptides with rapid turnaround at unmatched pricing. Applications and Specifications Screening projects for bioactive peptides, T-cell epitopes, peptidic ligands, substrates, proteotypic peptides, biomarkers etc. Systematic optimization of peptidic lead structures Assembly of peptide pools for immune monitoring Scale: nmol / compound High Content PepStar Microarrays JPT Peptide Technologies applies its unique PepStar peptide microarray platform to generate high content peptide microarrays on glass slides. Applications and Specifications Antibody epitope mapping Seromarker discovery Development of immune diagnostics Peptide optimization Screening for protein-protein interactions Characterization of enzymes and enzyme substrate identification/optimization (e.g. kinases, acetyl transferases, proteases) Up to 7000 peptides/slide in triplicates >1000 slides from one synthesis HT-Purification by chemo-selective immobilization of target peptides Benefits Substantial, long-standing expertise Exceptional quality and reliability for even complex or unusual peptide sequences Appreciated by customers worldwide for many years DIN ISO 9001 certification Stringent quality control Benefits Highly parallel synthesis approach ( peptides or mimetics / week) Quick turnaround (2 weeks) Low cost (from 7 /$ per peptide) Benefits Screening of tens of thousands of peptides in parallel Only smallest sample volumes needed (sera, blood, purified biologics ) Economical production of large batches of identical microarrays Peptides in triplicates guarantee high reliability Low background combined with high sensitivity Incubation manually or automated Flexible read-out via fluorescence, radioactive labeling or silver staining

23 Technologies & Know How R&D Collaboration Proteotypic SpikeTides Peptides SpikeTides are small-scale, inexpensive, heavily labeled or unlabeled and/ or absolutely quantified peptides for single reaction monitoring (SRM) and multiple reaction monitoring (MRM). Applications Optimization and validation of SRM/MRM assays Relative and absolute quantification of proteins Selection of best proteotypic peptides showing high mass spectrometry signal response Development of kits for the quantification of entire proteomic pathways Medicinal Chemistry Long-standing experience from discovery to preclinical development projects backed by very high synthesis capacities. Applications Stepwise transformation of peptides into more druglike molecules Example: Identification and optimization of specific substrates for orphan proteases, kinases, phosphatases and acetyltransferases Example: Development of specific affinity ligands for the purification of therapeutic proteins Benefits Thousands of economic SpikeTides for MRM assays Unmatched turnaround times ( peptides per week!) Fully compatible with downstream analyses like MS and HPLC SpikeTides quantification significantly more cost effective than AAA Benefits Experienced project team (from discovery to preclinical development) Long standing record in peptide and small molecule chemistry High capacity automated synthesis, analytics and purification Solid and solution phase capabilities Extensive building block library 21

24 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. JPT Peptide Technologies European Head Office T F Volmerstraße Berlin Germany USA/Canada T F P. O. Box 2619 Acton, MA USA Issue Date: October 2013