ProImmune REVEAL & ProVE Rapid Epitope Discovery System and Cellular Analysis Services

Transcription

1 ProImmune REVEAL & ProVE Rapid Epitope Discovery System and Cellular Analysis Services Overview of ProImmune REVEAL & ProVE Rapid Epitope Discovery System 2-4 Applications 4 Service in Detail 5-9 Module 1: Custom Peptide Library Synthesis Module 2: MHC-Peptide Binding Assay Module 3: Synthesis of ProVE MHC Class I Pentamers Module 4: Rate Assays Technical Report Case Studies ProImmune REVEAL & ProVE Rapid Epitope Discovery System used to identify novel T cell epitopes in HIV-1 (Imperial College, London, UK) 2. Class II ProImmune REVEAL Binding assay helps characterize epitopes in Graves disease (Universitat Autònoma de Barcelona, Spain) 3. Identification of new epitopes in Type 1 Diabetes (INRA/ENV de Nantes, France) 4. Novel epitope detection in phase II trial for colorectal cancer (Oxford BioMedica, Oxford, UK) Cellular Analysis Services ELISpot Assays Intracellular Cytokine Staining Detection of Antigen-Specific T Cells with ProVE or Pro5 MHC Pentamers in Flow Cytometry CFSE T Cell Proliferation Assays HLA Tissue Typing of Donors Integration of the ProImmune REVEAL & ProVE System with Cellular Analysis Services 25 Page 1 of 25

2 ProImmune REVEAL & ProVE Rapid Epitope Discovery System ProImmune REVEAL & ProVE is a novel technology for analyzing proteins for potential T cell epitopes in only a few weeks. The process saves weeks or months when compared with existing labor-intensive and expensive methods for epitope discovery and reduces risk in the development stages of novel bio-therapeutic molecules. The technology is widely applicable across many disease areas including all areas of cancer and infectious diseases. New CD4 + and CD8 + T cell epitopes identified with the help of this technology can be used as core building blocks for vaccine development or as targets for new immunotherapy. Summary of the Process CINGVCWTV INGVCWTVY NGVCWTVYH MHC Class I Select peptides Select peptides MHC Class II MAPRTLVLLLSGALA APRTLVLLLSGALAL PRTLVLLLSGALALT MHC-peptide binding assay MHC-peptide binding assay A*0201_# 34 % Assembled Rate assays for kinetic analysis Rate assays for kinetic analysis Time (h) Use ProVE MHC Class I Pentamers to confirm epitopes by flow cytometry Functional cellular assays, such as ELISpot, with well defined hits Figure 1 Class I ProImmune REVEAL & ProVE combines the powerful in vitro MHC-peptide binding and rate assays with the rapid and cost effective synthesis of ProVE MHC Class I Pentamers, ready for conclusive confirmation of epitopes on patient samples. For Class II HLA alleles, the MHC-peptide binding and rate assays are available for rapid prediction and analysis of CD4 + T cell antigens in any protein sequence. This may be followed by epitope validation using functional cellular assays, such as ELISpot. ProImmune REVEAL & ProVE is the only fully integrated, commercially available epitope discovery technology. The service saves time and money when compared with traditional epitope discovery methods and other commercial products. ProImmune s custom service delivers fast and accurate results, taking away the laborintensive epitope discovery process, removing uncertainty from confounding results, and allowing the customer to focus on the core objectives of the research project. Page 2 of 25

3 Background and Overview of Technology The development of improved vaccines to prevent or treat conditions that have failed to respond to previous treatments and prevention approaches is one of the fastest growing areas in pharmaceutical development. Many diseases that represent major unmet medical need today, including HIV, viral hepatitis, malaria, tuberculosis, and most forms of cancer, represent important targets for improved vaccination. In order to design optimized vaccination systems it is critical to identify which antigens or epitopes can be recognized and targeted by the immune system in these conditions. Knowledge of such epitopes can be used to monitor and improve our understanding of the immunological effects of vaccination throughout the product development process. Using algorithms to predict peptide binding to MHC molecules does not always reveal which epitopes are the most naturally immunogenic and thus the most appropriate for inclusion in vaccines. Functional in vitro assays, such as ELISpot and intracellular cytokine staining, which test whether pools of peptides can induce T cells to produce cytokines, are better predictors of this, as are functional cellular assays, like the T2 assay that monitors the ability of a peptide to stabilize an MHC class I molecule. However, these approaches usually require several rounds of screening and are thus labor intensive. Even using several of these approaches together it is difficult to assess the true MHC restriction of the epitope involved in generating the immune response. Carrying out exhaustive screening of peptide libraries using these functional assays with samples from infected patients is also strongly limited by the quantity of sample that can be obtained from each patient. Restricted sample numbers and the heterogeneity of patients tissue types can also confound results. ProImmune s Rapid Epitope Discovery System provides a solution to these problems and significantly accelerates the epitope discovery process, while reducing time variances in product development programmes. It combines the powerful in vitro MHC-peptide binding and rate assays, with the synthesis of ProVE MHC Class I Pentamers for conclusive confirmation of epitopes on clinical cell samples. This predictable step-by-step approach allows the narrowing down of many candidate epitopes to identify those that actually cause relevant immune responses, in a matter of weeks rather than months. The technology is widely applicable across many disease areas including all areas of cancer and infectious disease, and is ideally suited to accelerate research programmes with particular time pressures, such as vaccine development for emerging diseases. Rapid The MHC-peptide binding assay determines the MHC restriction in just a few weeks. For example, the binding assays for 94 peptides plus 2 controls can be carried out in only five working days and may be carried out with several alleles in parallel. ProVE MHC Class I Pentamers can be produced in a further five working days for peptides generating positive results in the binding assay, providing a tool for conclusive validation of new T cell epitopes by flow cytometry. Informative On- and off-rate binding assays for class I and class II alleles can be carried out for specific peptides of interest to improve understanding of their kinetics. Peptides with rapid on-rates and longer off-rates are more likely to be good T cell epitopes. Affordable ProImmune REVEAL & ProVE has been developed as a modular system in order to offer rapid epitope discovery at an affordable price. When compared with existing labor-intensive methods and other commercial products the technology saves time and money. Page 3 of 25

4 Convenient The assays are carried out in a controlled environment to ensure maximum accuracy and reproducibility. There is no need for assay set up by the researcher, or purchase of reagents or software for analysis of results. Furthermore, epitope validation can be outsourced using ProImmune s Cellular Analysis Services. ELISpot assays, Pentamer flow cytometry testing, and even tissue typing are offered, removing the need for the researcher to set up and maintain these assays in the laboratory. Flexible Any starting set of peptide sequences may be used, as long as the length matches the restriction of the alleles to be investigated. The customer may select any number or all of the modules that make up the ProImmune REVEAL & ProVE service. On- and off-rate binding assays may be ordered at the same time, or later if required. Improved Intellectual Property Position Identification of the individual epitopes of a therapeutic protein allows separation of the intellectual property (IP) of that protein from the IP around the method of delivery or its therapeutic use. During vaccine development, defining IP for specific epitopes increases the options for future changes in product design. If the IP for the specific antigens in a protein of interest is not secured, competitive organizations may have the opportunity to develop an obstructive patent position. Applications of the ProImmune REVEAL & ProVE Discovery System T Cell Epitope Mapping Rapid, flexible and comprehensive CD4 + and CD8 + T cell epitope mapping to confirm minimal epitopes and accurate MHC restrictions. Start from an unmapped protein sequence, overlapping peptides, pre-scored sequences identified with algorithms, or sequences pre-screened by ELISpot or intracellular cytokine staining. Improve Vaccine Design and Efficiency Optimize epitopes delivered in novel vaccines and eliminate irrelevant sequences to increase vaccine safety. Optimize Clinical Trial Design Following biomarker identification, use the specific MHC Pentamer to monitor antigen-specific T cells in your target patient group. Define Specific Model Antigens Identification of the epitopes most likely to play a key role in the process under investigation Investigate the effect of post-translational modifications Peptides with modifications, e.g. citrullination and oxidation, can be incorporated into REVEAL assays to determine the effect on MHC-peptide binding and stability. Page 4 of 25

5 The ProImmune REVEAL & ProVE Rapid Epitope Discovery System in Detail ProImmune's Rapid Epitope Discovery System is a customized, modular process for rapidly determining T cell epitopes from a set of candidate peptides. ProImmune REVEAL & ProVE is designed to be highly time and cost efficient and hence significantly reduce the overall effort required in discovering new T cell epitopes. Module 1: Custom Peptide Synthesis Any starting set of peptide sequences can be screened using the MHC-peptide binding assay, providing the length of these matches the restriction of the alleles to be investigated. Candidate sequences may be generated initially as an overlapping peptide library from a known protein. Alternatively, epitopes can be chosen from a more limited selection of known sequences that have been pre-screened using an algorithm of choice to identify those that are most likely to bind to MHC molecules. The peptides are provided as a Prospector PEPscreen : Custom Peptide Library. Peptides 6-20 amino acids in length are synthesized in 0.5-2mg quantities with high average purity. Quality control by MALDI-TOF Mass Spectrometry is carried out on 100% of samples and the library is ready in just working days. On request, a second set of peptides can be synthesized for use in functional cellular assays, such as ELISpot. ProImmune s technical advisors can assist with the design of a suitable peptide library for the protein of interest. Module 2: MHC-Peptide Binding Assay The high-throughput binding assay determines the ability of each candidate peptide to bind to one or more MHC class I or class II allele and stabilize the MHC-peptide complex (figure 2). By comparing the binding to both a positive control peptide and an intermediate control peptide (one that has intermediate affinity to the MHC allele of interest), the most likely immunogenic peptides in a protein sequence can be identified. Detection is based on the presence or absence of the native conformation of the MHC-peptide complex. Unlike traditional functional assay approaches, the binding assay determines the MHC restriction of peptides at the outset. Figure 2: Illustration of the binding of a peptide (red) to the MHC class I protein (blue) and beta-2 microglobulin (grey); when the individual components come together in the correct conformation a positive signal is generated via a labeled antibody (pink). Each peptide is given a score relative to the positive control peptide. In addition results for an intermediate control peptide are shown for comparison. The intermediate control is a known T cell epitope, or in the case of certain class II alleles, a known epitope for the beta-chain in the allele alpha and beta pair. The score is reported quantitatively as a percentage of the signal generated by the test peptide versus the positive control peptide. Page 5 of 25

6 The customer may select any or all of the peptides that pass the MHC-peptide binding assay for further rate or stability assays. Additionally, for class I alleles any peptides may be selected for ProVE Pentamer synthesis, allowing confirmation of epitopes on clinical or mouse cell samples by flow cytometry. The assay results can be used to identify those epitopes that could play a key role as targets in the development of novel immunotherapies. Table of alleles available for MHC-peptide binding and rate assays MHC Class I MHC Class II Human Mouse Rhesus Macaque Human Mouse * A*01:01 B*07:02 H-2Db Mamu A*01 DR1 DRB1*01:01 H-2 IAb A*02:01 B*08:01 H-2Dd Mamu A*02 DR2 DRB1*15:01 H-2 IAd A*03:01 B*14:02 H-2Kb DR3 DRB1*03:01 A*11:01 B*15:01 H-2Kd DR4 DRB1*04:01 A*24:02 B*27:05 H-2Ld DR5 DRB1*11:01 A*29:02 B*35:01 DR7 DRB1*07:01 B*40:01 * MHC-peptide binding assay only available for mouse class II alleles. Class II alleles available for HLA-peptide binding and Quick Check stability assays Name Allele Name Allele Name Allele R01 DRA1*01:01 + DRB1*01:01 R32 DRA1*01:01 + DRB1*15:02 P01 DPA1*01:03 + DPB1*01:01 R02 DRA1*01:01 + DRB1*15:01 R33 DRA1*01:01 + DRB1*15:03 P02 DPA1*01:03 + DPB1*02:01 R03 DRA1*01:01 + DRB1*03:01 R34 DRA1*01:01 + DRB1*16:01 P03 DPA1*01:03 + DPB1*03:01 R04 DRA1*01:01 + DRB1*04:01 R35 DRA1*01:01 + DRB1*16:02 P04 DPA1*01:03 + DPB1*04:01 R05 DRA1*01:01 + DRB1*11:01 R36 DRA1*01:01 + DRB3*02:02 P05 DPA1*01:03 + DPB1*04:02 R06 DRA1*01:01 + DRB1*1301 R37 DRA1*01:01 + DRB3*03:01 P06 DPA1*01:03 + DPB1*05:01 R07 DRA1*01:01 + DRB1*07:01 R38 DRA1*01;01 + DRB5*01:01 P14 DPA1*02:01 + DPB1*01:01 R08 DRA1*01:01 + DRB1*01:02 Q01 DQA1*01:01 + DQB1*05:01 P15 DPA1*02:01 + DPB1*02:01 R11 DRA1*01:01 + DRB1*04:02 Q02 DQA1*05:01 + DQB1*02:01 P16 DPA1*02:01 + DPB1*03:01 R13 DRA1*01:01 + DRB1*04;04 Q03 DQA1*01:02 + DQB1*05:02 P17 DPA1*02:01 + DPB1*04:01 R14 DRA1*01:01 + DRB1*04:05 Q06 DQA1*01:02 + DQB1*06:02 P18 DPA1*02:01 + DPB1*04:02 R15 DRA1*01:01 + DRB1*04:07 Q08 DQA1*03:01 + DQB1*03:02 P19 DPA1*02:01 + DPB1*05:01 R16 DRA1*01:01 + DRB1*04:08 Q09 DQA1*01:02 + DQB1*06:04 P20 DPA1*02:01 + DPB1*06:01 R19 DRA1*01:01 + DRB1*08:04 Q10 DQA1*05;01 + DQB1*03:01 P21 DPA1*02:01 + DPB1*09:01 R20 DRA1*01:01 + DRB1*09:01 Q11 DQA1*02:01 + DQB1*02:02 P22 DPA1*02:01 + DPB1*11:01 R21 DRA1*01:01 + DRB1*10:01 Q12 DQA1*03:01 + DQB1*03:01 P23 DPA1*02:01 + DPB1*13:01 R22 DRA1*01:01 + DRB1*11:02 Q15 DQA1*02:01 + DQB1*03:03 P24 DPA1*02:01 + DPB1*14:01 R23 DRA1*01:01 + DRB1*11:03 Q16 DQA1*03:03 + DQB1*03:03 P25 DPA1*02:01 + DPB1*15:01 R24 DRA1*01:01 + DRB1*11:04 P26 DPA1*02:01 + DPB1*17:01 Page 6 of 25

7 Module 3: ProVE Pentamer Synthesis Following the identification of candidate epitopes using the MHC-peptide binding assay for class I alleles, ProImmune can generate ProVE MHC Class I Pentamers with specificity for these peptides. ProVE Pentamers are suitable for use in flow cytometry, enabling the customer to identify and quantify the antigen-specific CD8 + T cells and thus attain conclusive validation of new T cell epitopes. With this system the testing of precious patient sample is shifted to the final validation step at the end of the discovery process, ensuring that the use of these samples is optimized. MHC-peptide complex Coiled-coil domain Pro5 Fluorotag or Biotag Figure 3: ProVE Pentamers are generated using a rapid, high-throughput, parallel synthesis method. They comprise five MHCpeptide complexes assembled through a coiled-coil domain. Due to their planar configuration, all five MHC-peptide complexes in the Pentamer are available for binding to complementary T cell receptors. ProVE Pentamers are supplied unlabeled and at a minimum quantity of 20 tests (one test labels 1-2 x 10 6 cells). A Pro5 Fluorotag or Biotag is supplied (R-PE, APC or biotin) for two-layer fluorescent staining. Each Pentamer is QC tested using ELISA and is guaranteed for 3 months when stored at 4 o C. Table of alleles available for ProVE MHC Class I Pentamer synthesis Human Mouse Macaque A*01:01 B*07:02 H-2Db Mamu A*01 A*02:01 B*08:01 H-2Dd Mamu A*02 A*03:01 B*14:02 H-2Kb A*11:01 B*15:01 H-2Kd A*24:02 B*27:05 H-2Ld A*29:02 B*35:01 B*40:01 Module 4: Rate Assays and Stability Assays Rate assays assess the on- and off-rates for peptide binding to MHC class I and class II molecules, and by indicating how long individual epitopes could be presented to T cells they can help to identify which candidate epitopes will be appropriate for use in vaccine development. If cell samples used for final epitope validation are limited in supply, rate assay data can also be used to prioritize downstream work on the most promising candidate peptides. Rate assays can be particularly important for intermediate affinity peptides, which may exhibit slower on-rate when binding to a particular MHC allele than high affinity peptides. If a slow off-rate is also seen this indicates that once the peptide is bound it could be presented for a considerable period of time, and it would thus merit further study as a potential epitope There are three options available for Module 4: (1) Quick Check Off Rate Assay, (2) Complete Rate Assay, and (3) Quick Check Stability Assay Page 7 of 25

8 Quick Check Off-Rate Assay, available for class I alleles The Quick Check option for module 4 must be ordered in conjunction with module 3, ProVE MHC Class I Pentamers. The off-rates for the MHC-peptide complexes are measured at 0 h, 2 h and 24 h, at 37 C. Results are supplied graphi cally and in terms of t 1/2 (h) values. The Quick Check off-rate assay provides information relating to the likelihood of a peptide being presented long enough for it to be a good T cell epitope. The results of this analysis can be combined with the results from the MHC-peptide binding assay, which typically reflect the onrate properties of a peptide more strongly than the stability of assembled complexes. The results are less comprehensive than the Complete Rate Assay but the cost of the Quick Check assay is significantly less. The results can be used as a cost effective way to prioritize which of the ProVE MHC Pentamers should be used on cellular samples first. This enables the user to optimize the use of cell samples and the time needed for testing. Complete Rate Assay, available for all class I and selected class II alleles The Complete Rate Assay may be ordered at any point in the ProImmune REVEAL & ProVE process. The on- and off-rates of peptides that have passed the MHC-binding assay are measured at six time points over 48 hours (on-rate) and 24 hours (off-rate). The Complete Rate Assay module is ideal for studying the peptide presentation in depth, and results can be analyzed alongside those from other quantitative assays, such as functional assays and staining with Pentamers. DR1_#84 DR1_Intermediate Control % Assembled % Assembled Time (h) Time (h) Figure 4: The on-rate of peptide number 84 (from a library of peptides used in ProImmune s study to search for novel class II epitopes from the human GAD65 protein) is measured in comparison with the on-rate of the intermediate control peptide. DR1_#84 DR1_Intermediate Control % Denaturation % Denaturation Time 37 C (h) Time 37 C (h) Figure 5: Comparison of the off-rate for Peptide 84 and the intermediate control peptide. Page 8 of 25

9 Quick Check Stability Assay, available for all class II alleles The Quick Check Stability Assay is available for any of the DR, DQ and DP class II alleles, and must be ordered at the same time as the MHC-peptide binding assay. The stability assay measures the amount of peptide bound to the allele at time zero and time 24 hours, at 37 C, and gives an indication of the stability of the MHC-peptide complex. Each peptide is given a stability index, allowing comparison of the relative stability of the different peptide epitopes. The MHC-peptide binding assay reflects the on-rate properties of a peptide more strongly than the stability of the assembled complex. However, when the outcomes of the stability assay and the binding assay are combined, the subsequent data provide more complete information as to whether the peptide could be presented long enough for it to be a good T cell epitope. This index provides a method for comparing the results of different peptides across the allele being measured. The results can be used as a cost effective way to prioritize which of the peptides could merit further study in functional cellular assays and enables the user to optimize the use of cell samples and the time needed for testing. Technical Report The results are presented by module in a detailed technical report. Shortly after delivery of the report, ProImmune s technical team contact the recipient to review the results. Module 1: Custom Peptide Library Synthesis All peptides that passed QC and went on for use in the MHC-peptide binding assay are presented in a table. Module 2: MHC-Peptide Binding assay Each peptide is given a score relative to the positive control peptide. The score is reported as a percentage of the signal generated by the test peptides or the intermediate control peptide versus the positive control peptide. Results are tabulated and presented graphically. A threshold percentage is assigned to offer an indication of those peptides that could be considered for further investigation. Module 3: ProVE MHC Pentamer Synthesis All ProVE Pentamers with successful synthesis are tabulated, with the number of tests and quantity required per test for staining in flow cytometry. Module 4: Rate Assays and Stability Assays Rate assay results are tabulated as t 1/2 (h) values. Peptides are given a Kinetic Score based on the results of on- and off-rates; a higher score indicates a better candidate T cell epitope. Graphs for the on- and off-rates are also shown. Where both MHC-peptide binding and rate assays have been ordered, the R score is defined for each peptide. This incorporates the rate assay score with the MHC-peptide binding assay score; the higher the R score, the better the epitope. Scores from the assays are offered as a general guideline; peptide epitopes should be validated using antigen-specific T cell detection with MHC Class I Pentamers and using functional cellular assays. The stability assay results are presented as tabulated peptide Stability Index data and in graphical format for each allele investigated. In cases where peptides have been screened with the binding, rate or stability assays against more than one class II DR allele (for alleles DR1-DR7), the results are also presented in a Pan-DR table and graph, showing a standardized score. This allows comparison of the results for each peptide and indicates the potential impact of that peptide as an epitope across all the DR alleles investigated. Page 9 of 25

10 Case Study 1 ProImmune REVEAL & ProVE Rapid Epitope Discovery System used to identify novel T cell epitopes in HIV-1 Westrop et al. used the ProImmune REVEAL MHC-peptide binding assay to screen 9-mer peptide sequences from the HIV-1 Gag protein against the HLA-B*35:01 allele. A previously unknown epitope was identified, which may be involved in an immune response in B*35:01- positive individuals. This is of particular importance as B*35:01 is an allele associated with a rapid disease progression in HIV-1 + patients. ProImmune synthesized forty-four 9-mer HIV-1 Gag peptides as a PEPscreen Custom Peptide Library, which were then assessed using the MHC-peptide binding assay. The binding affinity of the peptides was compared to a pass/fail control peptide known to have marginal affinity for the B*35:01 MHC complex. Peptides with a conformational signal >80% of the pass/fail control signal were considered to be potential epitopes (figure 6). Twelve HIV- 1 Gag peptides fulfilled this criterion, and these peptides were further analyzed using the Quick Check Off Rate Assay. The rates of dissociation of the peptide-mhc complexes were measured at 0, 2 and 24 hours and the half-lives of the complexes were calculated to give an indication of their overall stability. Two peptides (YPLTSLRSL and HPVHAGPIA) were found to form a stable complex with B*35:01, indicating that these may be potential novel epitopes. Figure 6. Results of the MHC-peptide binding assay for controls and the 12 peptides that were considered potential epitopes; binding signal is shown as a percentage relative to the pass/fail control; peptide 8, HPVHAGPIA (red) was further validated by Pro5 Pentamer staining Pentamer R-PE Westrop et al. then carried out Pro5 MHC Class I Pentamer staining, which showed a welldefined Pentamer + /CD8 + population for the B*35:01/HPVHAGPIA epitope (figure 7). Further validation with ELISpot assays confirmed a high IFN-gamma response to the HPVHAGPIA peptide. Figure 7. Pro5 Pentamer staining of live 0.0% 0.03% 0.01% 0.39% lymphocytes gated on CD3 + cells; the left plot shows staining with an allele mismatched negative control Pentamer, the right plot shows staining with the HA9 (B*35:01/HPVHAGPIA) Pentamer. 0.39% of CD3 + live lymphocytes are CD8 + /HA Pentamer + with a background stain of 0.03%. CD8 FITC This study highlights the value of the ProImmune REVEAL & ProVE technology as a superior tool for identifying potentially immunogenic epitopes in a cell free environment. Validation of the identified epitopes can be addressed with Pro5 Pentamer staining and other functional assays, such as ELISpot. Westrop S.J. et al. (2009) Novel approach to recognition of predicted HIV-1 Gag B*3501-restricted CD8 T-cell epitopes by HLA-B*35:01 + patients: Confirmation by quantitative ELISpot analyses and characterisation using multimers. J Immunol Methods 341: Page 10 of 25

11 Case Study 2 Class II ProImmune REVEAL HLA-peptide binding assay helps characterize peptides in Graves' disease To date there have been very few studies into HLA-DR epitopes that use human autoimmune tissue, mainly due to low availability of such tissues, low HLA-class II expression and heterozygosity of samples. Despite these constraints, a study by Muixí et al. identified seven different HLA-DR-associated peptides belonging to thyroglobulin, a known target autoantigen in autoimmune thyroid diseases. The HLA-peptide binding assay helped to overcome the experimental limitations, as the assay is cell free and peptide sequences can be screened against a wide array of alleles. The assay was used to assess the binding of potential thyroglobulin epitopes to HLA alleles DRB1*15:01 and DRB1*03:01. HLA class II-peptide complexes were first purified from ex vivo thyroid samples of patients with Graves disease and the peptide sequences determined by mass spectrometry. Of the 150 peptides isolated with DR molecules, 7 thyroglobulin-specific peptides were identified. To verify the binding affinity to HLA-DRB1*15:01 and HLA-DRB1*03:01, the peptides were analyzed using the class II MHC-peptide binding assay. The results showed that thyroglobulin ( ) binds to DRB1*03:01 (see figure 8), which is in agreement with results from the ProPred binding analysis software. Also in support of these findings, other recent studies have demonstrated the pathogenicity of this epitope using a DR3 transgenic mouse model of experimental autoimmune thyroiditis. % relative to intermediate control DRB1*1501 DRB1* ve +ve Peptide ID Figure 8: Results of the HLA-peptide binding assay. Thyroglobulin peptides (1-9) were tested alongside other peptides that had been theoretically assigned to one DR allele (10-13). Binding data are expressed as the percentage binding compared with a control peptide. Peptide 9 was confirmed as a medium-binder to DR3. The alleles assigned to peptides 10, 11 and 15 were also confirmed. Copyright 2008 The American Association of Immunologists, Inc. Muixí et al. (2008) Thyroglobulin Peptides Associate In Vivo to HLA-DR in Autoimmune Thyroid Glands. J. Immunol. (2008) 181: Page 11 of 25

12 Case Study 3 Identification of new epitopes in type I diabetes (T1D) CD8 + T cells have been shown to have a major role in pancreatic beta cell injury leading to T1D. Defining pancreatic beta cell autoantigenic epitopes is essential for the design of peptide-based immunotherapy in T1D. However, exhaustive screening of peptide libraries with human PBMC from T1D patients is strongly limited by the quantity of blood that can be obtained from T1D patients and the low frequency of autoreactive CD8 + T1D cells in human peripheral blood. A study by Blancou et al used a combination of epitope identification methods to find novel T cell epitopes in the proteins GAD65 and IA-2, including pre-screening of candidate peptides with the algorithm SYFPEITHI, DNA and peptide immunization of HLA transgenic HHDII mice, ELISpot and T2 killing assays of human and murine PBMC, and ProImmune s REVEAL MHC-peptide binding assay. Strongly immunodominant responses against two GAD peptides, GAD and GAD , and one IA-2 peptide, IA were found. The results of the study show that binding scores from the MHC-peptide binding assay correlate well with the responses observed by ELISpot in immunized HHDII mice. Significantly the three most important new epitopes identified in >25% of T1D patients were all predicted by the ProImmune REVEAL MHCbinding assay with scores similar to the positive control peptide used in the assay. Blancou, P. et al. (2007) Immunization of HLA class I transgenic mice identifies autoantigenic epitopes eliciting dominant responses in type 1 diabetes patients. J. Immunol. 178: Case Study 4 Novel epitope detection in phase II trial for colorectal cancer Scientists at Oxford BioMedica used ProImmune s PEPscreen peptides and ProVE MHC Class I Pentamers to detect novel T cell epitopes in patient samples following TroVax administration. Using ProVE Pentamers they confirmed the MHC restriction of the T cell epitopes identified and demonstrated that the T cells were truly antigen-specific. TroVax consists of a recombinant vaccinia virus (MVA) encoding the tumor-associated antigen 5T4, which is rarely detected on normal tissues but is expressed at high levels on a broad range of solid tumors. The presence of the 5T4 antigen correlates with poor prognosis. In Oxford BioMedica s phase I/II trial for colorectal cancer, 94% of patients responded to the TroVax antigen. A positive correlation was found between the magnitude of the immune response and time to disease progression. Based on this information, it was of great importance for Oxford BioMedica to analyze the 5T4-specific T cell response induced by TroVax in further detail. In particular, researchers wanted to understand the magnitude, specificity and phenotype of these immune responses. Two subsequent phase II studies in metastatic colorectal cancer reiterated the high frequency of responses observed in the earlier phase I/II. In the immunological analysis of these studies, Oxford BioMedica used a ProImmune PEPscreen 9mer peptide library for IFNgamma ELISpot assays to begin the validation process for 5T4 epitopes implicated in earlier MHC-peptide binding studies. This identified patients who showed no detectable 5T4-specific cellular responses prior to treatment, but who mounted very strong responses following TroVax vaccination (Figure 9). However, the IFN gamma ELISpot assay indicates the frequency of cells that secrete IFN gamma following peptide stimulation, but does not reveal their phenotype. Page 12 of 25

13 Baseline (<100,000) Post-TroVax vaccination Figure 9: Patients received a total of 6 intramuscular TroVax vaccinations. Blood samples were taken prior to the initial vaccination and 2 weeks after completion of chemotherapy. A 96-well culture plate was coated with an anti-ifn gamma antibody (1-D1K). 200,000 PBMC were plated per well and incubated overnight at 37 C with 5ug/ml peptide. Cells were removed and the plate washed prior to addition of a biotinylated anti-ifn gamma detection antibody (7-B6-1). Upon addition of streptavidin-alp, followed by a precipitating substrate for ALP, spots developed and were counted. The figure shows results for a single patient. Prior to treatment, an average of one spot could be detected per 200,000 cells. However, following a combination of chemotherapy and TroVax treatment, an average of 93 spots per 200,000 could be detected, indicating that a strong response to 5T4-specific peptide had been mounted. In order to confirm the nature of the immune responses in patients that had received TroVax, Oxford BioMedica wished to identify the presence of increased antigen-specific T cell populations in these patients. They turned again to ProImmune for the rapid synthesis of a ProVE Pentamer Library. ProImmune supplied ten 5T4-specific ProVE Pentamers that were used to validate the responses detected by IFN gamma ELISpot and to confirm the MHC restriction of the 5T4 T cell epitopes (Figure 10). The precursor frequencies detected using the ProVE Pentamers were approximately 2-fold greater than those detected using the IFN gamma ELISpot assay against the same peptide antigen, demonstrating a good correlation between these two assays. (A) Positive Pentamer Post-vaccination (B) Positive Pentamer Pre-vaccination (C) Negative Pentamer Figure 10: Patient blood samples were taken prior to the initial vaccination and 2 weeks after completion of chemotherapy. For flow cytometry staining, 2 x 10 6 PBMC were incubated with 1 test (0.5ug) ProVE Pentamer for 10 minutes at room temperature, followed by 1 test R-PElabeled Pro5 Fluorotag and 1 test FITC-labeled anti-cd8 antibody (clone RPA-T8) for 20 minutes at 4ºC. Samples were analyzed by flow cytometry and 500,000 live events collected. Results are shown for the same patient as in figure 9. A clear population of 5T4-specific CD8 + T cells was detected in the sample taken after completion of chemotherapy using an A*02:01- restricted ProVE Pentamer (A, 0.22% of live gate). Such antigen-specific cells were not present prior to vaccination with TroVax (B). No antigen-specific cells were detected at either time-point when an A*01:01-restricted ProVE Pentamer (negative Pentamer) was used for staining (C). Administration of TroVax vaccine clearly elicits potent cellular immune responses in the patient studied, demonstrated by the expansion of antigen-specific T lymphocytes that recognize specific epitopes of 5T4. ProVE MHC class I Pentamers provide a powerful means to elucidate a detailed profile of cellular immune responses in patients undergoing immunotherapy. As this study shows, CTL responses to single epitopes can be determined clearly, confirming the applicability of ProVE Pentamers in the clinical development of new immunotherapies. Page 13 of 25

14 Cellular Analysis Services Are you facing major obstacles to running cell mediated immunity assays in-house? Protocol optimization, lack of experienced operators and the cost and time needed to set up and maintain assays are common limitations to obtaining fast, reproducible results for your research study, or preclinical or clinical trial. ProImmune s Cellular Analysis Services offer you optimized, rapid and affordable cellular assays to GLP/GCP standards. Our service is designed to help you accelerate your immune monitoring or epitope discovery projects, by saving you the cost and effort of setting up and maintaining these assay platforms in your own laboratory. Services are based on a choice of standard assay formats in order to maintain affordability. ELISpot assays Intracellular Cytokine Staining Antigen-specific CD8 + T cell detection with Pro5 Pentamers in flow cytometry NKT cell detection with CD1d tetramers in flow cytometry HLA Tissue typing of donors Isolation and cryopreservation of PBMC from fresh, whole blood Dendritic cell (DC)-T cell assays with whole proteins Naïve CFSE T cell proliferation assays with peptides To make life easier, outsource your cellular assays to ProImmune. We have developed a straightforward, step-wise approach that allows us to understand your requirements quickly and that sets out clearly what you can expect from us at each stage. The steps include assay design, cell preparation and shipping, HLA tissue typing, assay fulfilment, preparation and delivery of the final report and post-project consultation with a technical expert. As a consequence you can maximize the amount of work outsourced, with minimum inconvenience. Free up time for core research and project planning Remove the cost and effort of setting up assays in your laboratory Optimized protocols to minimize assay variation Rapid turnaround of projects Order individually or as part of a more complete epitope discovery project Outsource to our experienced team Page 14 of 25

15 Assay Specifications and Applications ELISpot Intracellular cytokine staining Flow Cytometry with Pro5 Pentamers Flow Cytometry with CD1d Tetramers Peptide CFSE T Cell Proliferation DC-T Cell Proliferation HLA Tissue Typing Assay Specifications ProImmune supplied donor cells Customer sourced samples * Whole blood accepted for cryopreservation Frozen cells accepted Genomic DNA accepted Analysis performed on frozen cells Applications Assessment of CD4 + T Cells Assessment of CD8 + T Cells Immune monitoring Cell surface phenotyping Epitope mapping Antigen-specific CD8 + T cell detection Natural Killer T (NKT) cell detection Proliferation of T cells Batch release assessment Whole protein antigenicity assessment HLA tissue typing of donor samples * Inquire Page 15 of 25

16 Sample Management All cellular analysis services are carried out at ProImmune s qualified facilities. ProImmune has substantial experience in the shipping of customer cell samples worldwide. We handle the shipping process seamlessly from your facility through our own pre-qualified, specialized sample shipping providers. Alternatively, you can use your own shipper under our instruction. Samples can be provided fresh or cryopreserved. Fresh samples must be cryopreserved for storage before the assays are performed. We recommend that all fresh samples be processed within 24 hours of collection from the donor. We can supply validated cryopreservation protocols, which help ensure high viability of the samples once they are thawed at our laboratories under optimal conditions. Alternatively, we can process your samples for you. Clinical trial samples can be received under GCP conditions. All assays are carried out on cryopreserved samples. While cryopreservation reduces cell viability, we have extensively validated that this loss can be limited to only 15-20% by following best practise methods. By taking this controllable effect into account, an accurate and valid picture of the original sample can be formed. For each project we will discuss any special requirements for sample handling with you in detail in advance. When shipping, we ensure that all required customs and export regulation information is on hand, and in the unlikely event of any delay in the package clearing customs, we and our shippers ensure that the appropriate coolant is topped up to maintain sample temperature. Our streamlined service makes outsourcing to ProImmune rapid and affordable compared to other commercial providers. The flow diagram below gives an overview of the processes involved and the specific assays currently offered. Overview of ProImmune s Cellular Analysis Services Figure 11: Overview of ProImmune s cellular analysis services. Page 16 of 25

17 ELISpot Assay Service When set up correctly, the Enzyme-Linked ImmunoSpot (ELISpot) assay is a highly reproducible and sensitive cellular assay, particularly suited to high-throughput analysis of antigen-specific CD4 + and CD8 + T cell immune responses. It can allow detection of a secreted cytokine at the single cell level, as low as 1 cell in 100,000. For these reasons, ELISpot is now a widely adopted assay standard for monitoring T cell immune responses and validating new T cell epitopes. Carrying out ELISpot assays in-house, particularly with inexperienced staff, can require significant effort and can be time consuming with variable results. You can now save time and resources, and minimize risk by relying on the expertise of ProImmune s experienced team, who perform these assays routinely using optimized protocols. We have developed a streamlined service that makes outsourcing ELISpot to ProImmune rapid and affordable compared to other commercial providers. Our assays are based on simple standardized formats, which follow a step-by-step process. IFN gamma, IL-2 and Granzyme B to GLP or GCP quality standard Other cytokines available on request Detection of CD8 + and CD4 + T cell responses on frozen unmodified PBMC Detection of CD4 + T cell responses on frozen PBMC depleted of CD8 + T cells after thawing HLA tissue typing of donor samples if required Project turnaround in 6-8 weeks, including peptide synthesis and shipment of cells (varies according to exact requirements) Assay report and data CD, including images from the automated ELISpot reader Project follow-up consultation with technical specialist ELISpot assay validation report available on request Applications Clinical immune monitoring Epitope discovery Determine functionality of preclinical and clinical samples ELISpot Assay Validation To be confident that your ELISpot assay is consistently performing well, validation is essential. ProImmune offers assays to GLP- or GCP-accredited standards. We have carried out validation testing to confirm reproducibility between assays and between operators. A sample of our data is shown below. Figure 12: Low inter-assay variability in ProImmune ELISpot Assays: normalized data from two users across six IFN gamma ELISpot assays performed over three days. The data show the consistency of the response for positive control and two test peptides. Page 17 of 25

18 Intracellular Cytokine Staining Intracellular cytokine staining by flow cytometry is a powerful technique that allows the multiparametric analysis of individual cells in a mixed population. The procedure relies upon the stimulation of T cells in the presence of an inhibitor of protein transport, in order to retain the cytokines inside the cell. Cells are first stimulated with antigen, followed by staining with antibodies specific for extracellular epitopes, such as CD4 and CD8. Intracellular cytokine staining follows fixation and membrane permeabilization. The frequency of cells that produce a particular cytokine is measured using fluorescent antibodies. ProImmune offers rapid and reliable flow cytometric detection of IFN gamma, IL-2 and TNF alpha cytokine production in CD4 + and CD8 + T cells through its experienced applications team. Successful intracellular cytokine staining requires the design of suitably controlled experiments and a well-maintained facility. By outsourcing your intracellular cytokine staining experiments to ProImmune, you take advantage of our technical proficiency and save time by passing collection and analysis of data to our experts. Multicolor analysis is possible through the use of our 4-color or 8-color flow cytometry instruments with 96-well high-throughput capability. Intracellular cytokine staining can be used as a monitoring tool to measure the immune response to known antigens, and for epitope discovery, to identify and validate novel T cell epitopes. Flow cytometric detection of intracellular cytokines allows for simultaneous detailed phenotyping and gating of cells, to select the specific live lymphocyte population. Figure 13: Example staining for an intracellular cytokine staining experiment. Our service is based on a standardized format, which follows a step-by-step process. IFN gamma, IL-2 and TNF alpha; other cytokines on request HLA tissue typing of donor samples if required Project turnaround from just 3 weeks, including peptide synthesis and shipment of cells (varies according to exact requirements) Assay report and data DVD, including raw data.fcs files. Project follow-up consultation with technical specialist Applications Determine functionality of samples Measure immunomodulatory effects of test compounds or biologics Epitope discovery Antigen-specific functional readout when combined with Pro5 Pentamer Immune subset phenotyping, e.g. Th17, T regs Page 18 of 25

19 Flow Cytometry Testing with Pro5 MHC Class I Pentamers and CD1d Tetramers ProImmune offers a flexible flow cytometry testing service for measuring antigen-specific CD8 + T cell responses using Pro5 MHC Class I Pentamers, and NKT cells using CD1d tetramers. MHC Pentamers bind directly to T cell receptors of a single specificity, determined by the MHC allele and peptide combination, and by use of flow cytometry can detect antigen-specific T cell populations as rare as 0.02% of lymphocytes. Staining cells with MHC Pentamers provides a quantitative analysis of lymphocytes that express a specific T cell receptor and in conjunction with other functional assays, such as ELISpot, gives complementary information about an immune response. For increased depth of information, MHC Pentamer staining can be combined with a wide range of phenotyping antibodies. ProImmune is the only commercial source worldwide for fluorescently labeled human and mouse CD1d tetramers. The tetramers are available pre-loaded with alpha-galactosyl Ceramide* (α-galcer) or empty for loading with ligand of choice. Each tetramer also has a relevant negative control. The negative controls are mock-loaded with carrier only (no ligand) and will not bind to NKT cells. Tetrameric CD1d-lipid complexes bind to receptors on NKT cells of a particular specificity (as determined by the lipid ligand used), allowing identification and enumeration of antigen-specific CD1d-restricted NKT cells by flow cytometry. Additional co-staining for intracellular cytokines such as IFN gamma or IL-2, and/or surface markers e.g. CD69 can yield functional data for the antigen-specific subset. * The alpha GalCer molecule and derivatives thereof are covered by US Patent No.5,936,076, which is owned by Kirin Pharma. The alpha GalCer molecule is licensed to Funakoshi Co. Ltd. for research use worldwide. Even for researchers experienced in flow cytometry techniques, cell preparation, staining and analysis all require careful planning and execution. By outsourcing flow cytometry testing to ProImmune, you immediately benefit from many years experience from the team that has developed MHC Pentamer technology. Protocols are carried out to optimized procedures to give reliable and consistent results. Our affordable, streamlined service is based on simple standardized formats. Pro5 MHC Class I Pentamer staining of antigen-specific T cells CD1d tetramer staining of NKT cells Additional phenotyping antibodies on request HLA tissue typing of donor samples if required Project turnaround from 1 week (varies according to exact requirements) Assay report and data DVD, including raw data.fcs files. Project follow-up consultation with technical specialist Applications Immune monitoring Epitope validation Page 19 of 25

20 Example Flow Cytometry Testing with Human CD1d Tetramers 1.31% 0.03% αgalcer loaded Human CD1d APC Negative control Human CD1d APC CD3 CD3 Figure 14: 1 x 10 6 PBMC were incubated with 1 test (0.5 µl) APC labeled, α GalCer loaded human CD1d tetramer (left plot), or 1 test (0.5 µl) APC labeled, negative control human CD1d tetramer (right plot) for 30 minutes at 4 o C. Following a wash step the cells were incubated at 4 o C for 20 minutes with anti-cd3 FITC and anti-cd19 PE in 50 µl total volume. Following two further washes the cells were acquired and analyzed by flow cytometry. Non-specific staining was eliminated from the plot by gating on CD19 negative cells before plotting CD3 vs. CD1d tetramer. Page 20 of 25

21 T Cell Proliferation Assays The T cell proliferation assay has been developed to identify the presence or absence of potential T cell epitopes and is useful in preclinical screening of novel peptides or proteins. The assays can address issues of relative antigenicity between structurally similar molecules; for example, they can help to distinguish between different candidates, or can give an indication of the success of protein engineering strategies in deimmunization of a potential immunotherapeutic. ProImmune offers two types of T cell proliferation assay: naïve primary T cell assay for peptide epitope screening and dendritic cell (DC)-T cell assay for whole protein antigenicity assessment. Cells are labeled with the fluorescent dye 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE). Cells that proliferate in response to antigen show a reduction in CFSE fluorescence intensity, which is measured directly by flow cytometry. This technology enables us to determine accurately the percentage of proliferating CD4 + cells, offers detailed phenotyping of T cell responses, and is more sensitive than traditional assays based on radioactive thymidine incorporation. Naïve Primary T cell Assay for Peptide Epitope Screening This assay is used to identify epitope sequences that can elicit helper T cell proliferation and therefore potentially cause a helper T cell immune response. The assays are particularly suited to measure the stimulation of naïve T cells. As an example, in order to assess the potential for a peptide to be antigenic in a wider population group, the assays can be carried out on samples provided by ProImmune from healthy donors, who are presumed to be naïve to the antigens encountered. The donor group would typically include up to 50 individuals, chosen to reflect a broad HLA background. Percentage Antigenicity split by responding donors PPD KLH HA peptide Percentage Antigenicity TT peptide Figure 15: Graph showing the proportion of responding donors out of a total of 41 donors screened against 4 controls and 35 peptides. Each colored segment represents a different donor. A positive response in more than two independent donor samples is considered indicative of a potential T cell epitope. Depending on the size of the study, (20, 40 or 50 donors), this sets a threshold of 10%, 5% or 4% respectively. In this study, a threshold of 4.9% antigenicity was set. Thus, peptides 3, 5, 26, 32 and 33 can be considered as potential T cell epitopes. Applications Immune monitoring Validate or confirm epitopes in samples Page 21 of 25

22 DC-T Cell Assays for Whole Protein Screening The dendritic cell (DC)-T cell assay has wide-ranging applications. It allows for an overall comparison of the T cell driven antigenicity of any number of drug candidates at a pre-clinical stage. Crucially, it can also be used for assessing the impact on antigenicity of factors other than protein sequence. Such differences may include a comparison of biosimilars, protein modifications, degradation products, chemical entities given in combination therapies, and other parameters related to manufacturing processes, excipients, drug formulation and stability. Additionally, in some cases it may not be possible to use the antigen to stimulate PBMC directly, particularly if the antigen involved modifies the function of responding T cells. To avoid such assay interference, antigens can be presented using dendritic cells, allowing the relative antigenicity of different leads to be compared directly. Donor PBMC are used as a source of monocytes that are cultured in defined media to generate immature dendritic cells. Dendritic cells are loaded with test antigen (whole protein), and are then induced into a more mature phenotype by further culture in defined media. CD8 + T cell-depleted donor PBMC from the same donor sample are labeled with CFSE then cultured with the antigen-primed DC. Each DC-T cell culture includes a set of untreated control wells. The assay also incorporates reference antigen controls, comprising two potent whole protein antigens. Figure 16: In a study at ProImmune, the DC-T cell proliferation assay was used to compare the relative antigenicity of a number of whole protein antibody drugs. Example staining data from DC-T cell proliferation assay: CFSE-labeled T cells incubated with (1) DC not cocultured with antigen, (2) DC previously co-cultured with whole protein antigen (Remicade, a chimeric antibody), (3) DC previously co-cultured with control protein, Tuberculin PPD. When evaluating antigenicity, both the strength and frequency of a response should be considered. The strength of positive donor cell responses is determined by taking an average of the percentage stimulation above background obtained across accepted donors for each drug. A Response Index is determined by multiplying this value for strength with the frequency of the donor cell responses, and this index is more representative of the level of antigenicity than methods of analysis that rely on the frequency of response alone. Page 22 of 25