Our T cell epitope test service offers you:

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1 Our T cell epitope test service offers you: high throughput peptide discovery fast lead-finding maximum of epitopes in a minimum of blood saving effort and costs on developing your own platforms custom-made advice by in-house scientific experts in-house cell therapy centre for fast translational research Sanquin Blood Supply Division Reagents reagents@sanquin.nl

2 Test Service for T cell Epitope and Biomarker Discovery

3 Division Reagents of Sanquin Blood Supply provides state of the art immunomonitoring services for MHC Class I T cell epitope discovery in the field of vaccine development, cellular immunotherapy and biomarker discovery and analysis. Our services make use of award-winning platforms and are designed to accelerate and improve your discovery phase, and your preclinical and clinical development phases by means of providing comprehensive and high quality techniques in quick response to your needs. To date, we have entered into several agreements with pharmaceutical companies and research organisations rendering our services for the development of innovative therapeutics. For detailed information, please contact us (see page 6 for contact information). Innovative technologies to rapidly detect immunogenic peptides, biomarkers and identify new T cell epitopes.

4 Sanquin Blood Supply, Division Reagents The division Reagents is part of Sanquin Blood Supply, an internationally reputed blood bank organisation with over 3,000 employees with its headquarters in Amsterdam, the Netherlands. Sanquin was established in 1998 through a merger between the Dutch blood banks and the Central Laboratory of the Netherlands Red Cross Blood Transfusion Service (CLB). Sanquin is responsible for blood supply in the Netherlands and advances fundamental and translational research in transfusion medicine. Benefiting from the Sanquin research environment and diagnostic laboratory settings, division Reagents has developed a broad range of laboratory reagents in the fields of immunohematology and immunology, including innovative products for diagnostic use and for fundamental and clinical research. These reagents are available world-wide through a network of distributors and OEM arrangements. Division Reagents is ISO 9001 and ISO certified. In 1999, Sanquin and the Netherlands Cancer Institute (NKI) started a collaboration to advance MHC Class I T cell epitope discovery. As a result of this collaboration an MHC multimer core facility at Sanquin s premises was launched that same year and since 2000 we offer class I PeliMers (original MHC tetramers), custom synthesis and peptides. In 2005 we introduced a novel, patent pending platform to simplify the discovery of new epitopes (UV induced peptide exchange technology), followed in 2008 by the introduction of another novel, patent pending platform allowing simultaneous antigen-specific detection of many different MHC Class I T cells in a single blood sample (combinatorial coding technology). World-wide successful scientific collaborations and publications in peer-reviewed international journals have demonstrated the validity of our MHC Class I T cell epitope discovery and immunomonitoring products and technologies. In response to an increasing demand we now offer these products and technologies as a service to pharmaceutical and biotechnology companies and research organisations for the development of new (immuno)therapeutics (including vaccines), cellular immunotherapy and biomarker discovery. 1

5 Fast screening of HLA-peptide combinations by UV-exchange Figure 1 The mechanism of UV induced peptide exchange How we can speed up your T cell epitope-biomarker discovery? Finding the right peptides for MHCmonomers is essential for identification of new T cell epitopes. In normal routine, the binding between MHC and peptides is tested for each peptide of interest, or predicted with a computer algorithm. These methods are time consuming and often indefinite, which is in fact waste of time and money. We can speed up this process for you by using a unique concept: fast screening of MHC-peptide combinations by UV induced peptide exchange. How does UV induced peptide exchange work? In our system peptides of interest binding to MHC Class I can be identified with a unique method that utilises an MHC Class I complex harbouring a UV labile peptide. By bringing this combination in contact with each peptide to be tested (in a 96 or 384-well plate or a multitude of this), we can screen these peptides on their actual binding capacities with the MHC class I molecule. In figure 1 the mechanism is explained. Have you ever speeded up T cell analysis with a factor 25? Stable MHC Class I complex with the UV cleavable peptide Result In this way a fast and efficient screening can be performed and within only a few hours we have the immunogenic peptides binding to the MHC-I molecule of choice for you, saving you time and money. Next step finding the right T cells Our UV induced peptide exchange protocol allows rapid production of large numbers of MHC tetramers. The suitable T cell epitope candidates which are found (even if this is a large number of candidates) can now easily be detected (figure 2). 2

6 UV 366 nm Peptide candidate Stable MHC class I complex Unstable MHC class I complex Positive in screening Multimerisation Negative screening Figure 2 Identification of MHC-I binding peptides. 4 assay controls peptides of interest 3 OD 414 nm High Medium Low Positive Negative UV only EBNA HIV MAGE PSMA FLU GP100 ELISA UV-exchange 3

7 A maximum of immunogenic epitopes tested with a minimum of patient blood! Finding the right T cells in a patient sample of only 8 ml? Finding T cells that recognise the immunogenic peptides is the goal in development of new vaccines or biomarker analysis in immunotherapy. In clinical trials blood of the patient is very valuable, thus doing more tests with less blood is a great step forward. This can be achieved with our new test format. For conventional testing methods you need 5 ml of patient blood to analyse one or a very limited number of T cell binding tetramers. Analysing 100 different tetramers would require a considerable volume of patient blood, which makes large studies troublesome. We developed a test method in which we are able to test 28 tetramers simultaneously in a sample volume of merely 8 ml patient blood. Result Offering you this solution, we are able to test up to 28 tetramers simultaneously in one small patient sample (figure 4). This technique leads to faster finding the right T cell population, using much less patient sample than conventional techniques. Your trials will be more efficient and cheaper. Figure 4 28 possible unique color combinations High Tech principle The method is based on two-dimensional coding of the same tetramer-peptide combination. In a flowcytometer we can detect the combination of colours that bind to a specific T cell population. In the schematic picture the sequence of actions is depicted (figure 3). Fluorochrome

8 Figure 3 Schematic illustration of Combinatorial Coding Multimerisation Green fluorochrome Multimerisation Red fluorochrome Two-dimensional coding Flow cytometry T cell Red Green 5

9 T cell epitope test service The most high tech offer in the market Division Reagents of Sanquin Blood Supply offers these high-tech techniques as a test service. Our facility is designed to accelerate your epitope discovery and immune monitoring, saving you the effort and costs of setting up these assay platforms in your own laboratory. Expertise within Sanquin Blood Supply All research within Sanquin is related to the many aspects of blood and blood transfusions, the Division Research employs around 300 scientists. A recently installed cell therapy centre can help bring your knowledge one step further. The facility is our partner in translational research, a state-of-the-art facility for preparation of cellular products for patient care and diagnostics. The Cell Analysis core facility is equipped with several flowcytometers and several wide field imaging microscopes. More information? Please contact us if you want more information regarding feasibility of your research project, pre-clinical or clinical study. Sanquin Blood Supply Division Reagents Amsterdam The Netherlands Tel: reagents@sanquin.nl Website: Scientific expertise Sanquin Blood Supply, Division Reagents, Amsterdam Product and assay development, Dr. W.J.E. van Esch. Dutch Cancer Institute, Division Immunology, Amsterdam Research and assay development, Prof. Dr. T. Schumacher. Dutch Cancer Institute, Division Cellular Biochemistry, Amsterdam Research and assay development, Dr. H. Oova. 6

10 Collaborations Dutch Cancer Institute, Div Immunology and Div Cellular Biochemistry, Amsterdam, The Netherlands Dutch Vaccine Institute, Research & Development: Dept Vaccine Research, Test Development and Formulation, Bilthoven, The Netherlands University of Duisburg-Essen, Institute for Virology, Essen, Germany Large pharmaceutical companies, World-wide Patents WO 2006/080837: Means and methods for breaking noncovalent binding interactions between molecules (NKI and Sanquin). PCT/EP2008/009356: Detecting antigen responsive cells in a sample (NKI and Sanquin). 7

11 Key publications: 1. Schulte, I et al. (2011) Characterization of CD8+ T cell response in acute and resolved hepatitis A virus infection. J Hepatol 54: Velthuis, JH et al. (2010) Simultaneous detection of circulating autoreactive CD8+ T cells specific for different islet cell-associated epitopes using combinatorial MHC-multimers. Diabetes 59: Hadrup SR and Schumacher TN. (2010) MHC-based detection of antigen-specific CD8+ T cell responses. Cancer Immunol Immunother 59: Toebes, M et al. (2009) Generation of peptide MHC class I monomers and multimers through ligand exchange. Curr Protoc Immunol: Chapter 18: Unit Hadrup, SR et al. (2009) High-throughput T cell epitope discovery through MHC peptide exchange. Methods Mol Biol 524: Hadrup, SR et al. (2009) Parallel detection of antigen-specific T cell responses by multidimensional encoding of MHC multimers. Nat Methods 6: Celie, PH et al. (2009) UV-induced ligand exchange in MHC class I protein crystals. J Am Chem Soc 131: Bakker, AH et al. (2008) Conditional MHC class I ligands and peptide exchange technology for the human MHC gene products HLA-A1, -A3, -A11, and -B7. Proc Natl Acad Sci USA 105: Grotenbreg, GM et al. (2008) Discovery of CD8+ T cell epitopes in Chlamydia trachomatis infection through use of caged class I MHC tetramers. Proc Natl Acad Sci USA 105: Gredmark-Russ, S et al. (2008) The CD8 T cell response against murine Gammaherpesvirus 68 is directed toward a broad repertoire of epitopes from both early and late antigens. J Virol 82: Frickel, EM et al. (2008) Parasite stage specific recognition of endogenous Toxoplasma gondii derived CD8+ T cell epitopes. J Infectious Diseases 198: Rodenko, B et al. (2006) Generation of peptide MHC class I complexes through UV-mediated ligand exchange. Nat Protocols 1: Toebes, M et al. (2006) Design and use of conditional MHC class I ligands. Nat Med 12:

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