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16 Identification of Tumor Antigens by Using Proteomics François Le Naour Summary The recent progress of proteomics has opened new avenues for tumor-associated antigen discovery. Here, I describe a two-dimensional (2D), gel-based Western blot approach for screening and identification of proteins eliciting a humoral response in cancer. Sera from patients are used in 2D Western blot experiments for screening of autoantibodies, and the immunoreactive target proteins are subsequently identified by mass spectrometry. Applied to several types of cancer, this proteomic-based approach has revealed a high frequency of autoantibodies in sera from cancer patients and has led to the identification of novel tumor antigens. Relevant examples are described. Key Words: Autoantibodies; mass spectrometry; proteomics; two-dimensional (2D) Western blot; tumor antigens. 1. Introduction Cancer immunology has a long history since it has been worded more than a century ago that tumor cells could be considered as “nonself” (1). This basic concept, suggesting that the immune system could be able to recognize tumor cells and then destroy them, has been strengthened by the observation of spontaneous tumor regression in some patients with different types of cancer. To develop immunotherapeutic intervention, also called immunotherapy or cancer vaccine, in cancer patients, the field of cancer immunology has been dominated by the search of tumor-specific and tumor-associated antigens (TAAs) as well as by the characterization of the immune cells involved in tumor recognition by the autologous host. It has now been demonstrated that cancer may elicit autologous immune response involving CD8+ cytotoxic T-lymphocytes (CTLs), CD4+ T-helper cells as well as antibodies (2–4). Tumor rejection is critically From: Methods in Molecular Biology, vol. 360, Target Discovery and Validation Reviews and Protocols Volume I, Emerging Strategies for Targets and Biomarket Discovery Edited by: M. Sioud © Humana Press Inc., Totowa, NJ 327
328 Le Naour dependent on CD8+ cytotoxic T-lymphocytes. Thus, attention has been focused on tumor-specific CD8+ T-cells. Since the cloning of MAGE-1, the first reported antigen to encode a human tumor antigen recognized by autologous CTLs (5), an array of autoimmunogenic tumor antigens have been identified (6). Although the characterization of tumor antigen recognized by cytotoxic T-cells is crucial, a limitation stems from this approach requiring the establishment of autologous target cells in culture and the isolation of stable lines of autologous CTLs that recognize antigens. In addition, some epithelial cell types are poorly susceptible to CTLs in vitro. In addition, the B-cell response (antibodies) also has been demonstrated in cancer patients. Recently, a method called serological analysis of recombinant tumor cDNA expression libraries (SEREX) has been used for the identification of tumor antigens (7). SEREX is based on screening of autoantibodies in sera from patients with cancer against an expression library made with mRNAs from the autologous tumor (see Chapter 15, Volume 1). By applying this strategy to human tumors of different origin, an unexpected frequency of tumor antigens that elicit specific immune responses in the autologous host has been demonstrated (7–10). However, SEREX is limited by the necessity to construct expression libraries, and the analysis is usually restricted to one or a few patients (7–11). The recent progress of proteomics, allowing large-scale analysis of proteins within a single experiment, has opened new avenues for TAA discovery. This chapter focuses the utility of proteomics for identification of tumor-associated antigens with emphasis on proteins eliciting humoral response in cancer patients. 2. Proteomics: The State of the Art Proteomics designates the study of the proteome. It involves a variety of approaches combining high-resolution separation techniques with identification methods such as mass spectrometry. For many years, two-dimensional (2D) polyacrylamide gel electrophoresis (PAGE) has been the primary technique for proteomic-based biomarker discovery. Thus, 2D electrophoresis (2DE) allows the separation of the proteins according to their charge in a first dimension where they stop their migration after reaching their isoelectric point (pI) and then in a second dimension according to their molecular mass. The proteins can be visualized by different staining, such as Coomassie blue or silver, and digitalized. 2DE is a highly resolutive technique leading to the visualization of at least 1000 protein spots in one experiment. Therefore, 2DE allows comparative analysis of protein expression levels and the visualization of various protein isoforms. The identification of the proteins can be performed after enzymatic digestion of the proteins in-gel followed by analysis of the resulting peptides by mass spectrometry. This process can be performed by mass fingerprinting, usually using matrix-assisted laser desorption ionization/time of flight mass spectrometry or
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METHODS IN MOLECULAR BIOLOGY 360 T
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M E T H O D S I N M O L E C U L A R
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© 2007 Humana Press Inc. 999 River
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vi Preface get. Approaches to targe
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viii Contents 10 Transgenic Animal
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x Contributors SAHOHIME MATSUMOTO
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xii Contents of Volume 2 12 Validat
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2 Sioud disease pathogenesis and pe
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4 Sioud A more fruitful approach fo
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6 Sioud both transient and permanen
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8 Sioud serum biomarkers. This syne
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10 Sioud Fig. 2. Schematic of targe
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12 Sioud 13. Magin, T. M. (1998) Le
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Harnessing the Human Genome 15 The
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Harnessing the Human Genome 17 Fig.
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Harnessing the Human Genome 23 The
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Harnessing the Human Genome 27 repr
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Harnessing the Human Genome 29 14.
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Harnessing the Human Genome 31 45.
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34 Imoto et al. (4,5), and Bayesian
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36 Imoto et al. 2. Materials Two ty
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38 Imoto et al. Fig. 3. Graphical v
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40 Imoto et al. Fig. 5. Result of t
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42 Imoto et al. p(D |G) = ∫ p(D,
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44 Imoto et al. β k (k = 1,…,q j
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46 Imoto et al. Fig. 7. Partial res
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48 Imoto et al. Fig. 8. Strategy to
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50 Imoto et al. Fig. 9. (continued)
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52 Imoto et al. Table 3 List of Roo
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54 Imoto et al. 5. De Hoon, M. J. L
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56 Imoto et al. 39. Kamimura, T., S
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58 Beaty et al. cytotoxic drugs and
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60 Beaty et al. Fig. 1. Principles
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62 Beaty et al. oligonucleotide mic
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64 Beaty et al. 3. The 12% polyacry
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78 Beaty et al. using a protein ass
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82 Beaty et al. Fig. 2. Preparation
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84 Beaty et al. search in. A widely
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86 Beaty et al. 3. Kern, S. E., Hru
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88 Beaty et al. 34. Peters, D. G.,
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5 Molecular Classification of Breas
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Molecular Profiling of Breast Cance
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6 Discovery of Differentially Expre
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Gene Target Discovery 117 In the fi
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Gene Target Discovery 119 sequences
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Gene Target Discovery 121 There is
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Gene Target Discovery 123 digestion
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Gene Target Discovery 125 Fig. 1. P
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Gene Target Discovery 127 5. Sargen
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Gene Target Discovery 129 41. Berry
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132 Matsumoto, Miyagishi, and Taira
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144 Fig. 1. The ribozyme-expression
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148 Sano and Taira ribozymes may cl
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150 Fig. 3. A system for the identi
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152 Sano and Taira 4. Notes 1. We r
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9 Production of siRNA- and cDNA-Tra
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Transfected Cell Arrays 157 Fig. 1.
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Transfected Cell Arrays 159 2. The
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164 Houdebine Fig. 1. Different met
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166 Houdebine concentrations become
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170 Houdebine integrate into the me
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174 Houdebine as insulators. The co
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178 Houdebine systems. Moreover, st
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204 Vijayaraj, Söhl, and Magin 1.
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12 The HUVEC/Matrigel Assay An In V
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256 Skovseth, Küchler, and Haralds
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Page 332 and 333: 15 Potential Target Antigens for Im
Page 334 and 335: Tumor Antigen Discovery 321 develop
Page 336 and 337: Tumor Antigen Discovery 323 panel b
Page 338 and 339: Tumor Antigen Discovery 325 16. Joc
Page 342 and 343: Immunoproteomics 329 by “shot gun
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Page 350 and 351: Protein Arrays 337 Table 1 Classifi
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Page 354 and 355: Protein Arrays 341 combinatorial sc
Page 356 and 357: Protein Arrays 343 The ideal proteo
Page 358 and 359: Protein Arrays 345 18. Cekaite, H.
Page 360 and 361: Protein Arrays 347 49. Yuko Kawahas
Page 362 and 363: Index 349 Index A Acetyl-CoA carbox
Page 364 and 365: Index 351 conditional by using FRT
Page 366 and 367: Index 353 Recombinant tumor antigen
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