Target Discovery and Validation Reviews and Protocols

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Tumor Antigen Discovery 321 development of clonal heterogeneity in most solid tumors. To circumvent the selection and outgrowth of antigen-negative tumor cell clones that have a proliferation advantage under the selection pressure of antigen specific immunotherapy, polyvalent immunotherapy approaches targeting several tumor antigens expressed at the same time by an individual tumor need to be developed. To design such polyvalent immunotherapy strategies, it will be necessary to dissect the antigenic profile of an individual tumor. 2. Serological Analysis of Recombinant Tumor cDNA Expression Libraries The development of the new cloning technique SEREX with autologous serum (26) to identify tumor antigens based on spontaneous antibody responses in cancer patients made it possible to analyze tumor systems that typically do not grow in cell culture. Another advantage is that SEREX does not rely on tumor-specific T-cell lines (27). It was shown for several SEREX-defined antigens, e.g., the cancer-testis (CT) antigen NY-ESO-1, that spontaneous CD8 T-cell responses correlate with a spontaneous humoral immune response in patients with antigen-positive tumors. The repertoire of antibody-defined tumor antigens correlates tightly with the CD4 T-cell antigen repertoire of an individual patient. These antigens can potentially be recognized by CD8 T-cells (21,28). SEREX has been used to identify new tumor-associated antigens in different tumor types, which led to the identification of HOM-MEL-40, a gene identical to the synovial sarcoma/X breakpoint 2 gene (SSX2) involved in the t(x:18) translocation in synovial sarcoma (29), as well as other CT antigens, including NY-ESO-1 (30), CT7/MAGE-C1 (31), SCP-1 (32), cTAGE-1 (33), OY-TES-1 (34), HOM-TES-85 (35), CAGE (36), cTAGE (33), and recently NY-SAR-35 (37). We were successful using SEREX to identify new differentiation antigens, such as RAB38 (38) and NY-BR-1 (39), as well as overexpressed antigens, such as tumor protein D52, NY-BR-62, and NY-BR-85. SEREX represents a very useful technique to define the individual CD4 T-cell tumor antigen repertoire. Today, more than 1200 antigens have been identified by SEREX and are deposited in the SEREX database of the Ludwig Institute for Cancer Research (http:// www. licr.org/SEREX.html). 3. Critical Steps in Antigen Validation Several steps of analysis are necessary for SEREX-defined antigens to represent potential target antigens for active immunotherapy strategies in cancer patients. In the first step, a careful expression analysis for each individual antigen has to be performed by comparing the cDNA sequence to expressed sequence tag (EST) databases. Antigens with restricted expression by database analysis are being tested by reverse transcription (RT)-polymerase chain reaction by using a panel of normal tissue and tumor tissue cDNAs to confirm the restricted mRNA expression pattern.
322 Jäger Antigens that are tumor- or tissue-restricted expressed undergo a serological analysis to define the frequency of spontaneous humoral immune responses in sera derived from cancer patients as well as healthy individuals. A limited number of sera can be tested using the phage-plaque assay, for larger scale serology, the recombinant protein has to be produced and tested in Western blot or ELISA assays. Antigens with tumor- or tissue-specific expression and detectable humoral immune responses in tumor patients are being further analyzed for T-cell recognition. We are following an approach called “reverse immunology” where we test the protein sequence of the respective antigen for potential MHC class I binding sequences (http://www2.licr.org/, CancerImmunomeDB, SYFPEITHI database of MHC ligands, and peptide motifs at http://www.bmiheidelberg.com/ syfpeithi/). We focus on HLA-A2 epitopes because HLA-A2 represents the most frequent MHC class I allele in Western European populations. Peptide-specific CTLs can be generated upon in vitro stimulation, and recognition of target cells expressing the antigenic protein will be documented. CD8 T-cell reactivity to the identified epitope can be analyzed in peripheral blood and tumor lesions of a large series of HLA-A2–positive patients with antigen-positive tumors. Tumor antigens that fall into the categories of CT antigens or differentiation antigens, which represent target antigens for CD8 T-cells, can be used to immunize HLA-A2–positive patients with antigen-positive tumors. The peptides that are recognized by CD8 T-cells will be used for intradermal injection. The readout includes delayed-type hypersensitivity reaction, induction of peptide-specific CD8 T-cells (ELISPOT and Multimer), and clinical development of the tumor disease. 4. A New Breast Cancer Differentiation Antigen, NY-BR-1 In a recent autologous SEREX screening of a breast cancer cDNA expression library, we identified a previously unknown antigen designated as NY-BR-1. About half of the clones identified in the screening were derived from NY-BR-1, whereas the other clones were derived from distinct known and unknown genes. Comparison with EST databases showed only some cDNA sequences matching the NY-BR-1 cDNA. Interestingly, some sequences were derived from testis; one sequence was derived from normal breast. All other antigens identified in this screening matched to cDNA sequences derived from a wide array of normal tissues, and they were not further analyzed. Following a combined approach of rapid amplification of cDNA ends-PCR and database analysis for overlapping cDNA sequences to complete the 5′ cDNA sequence, we were able to clone the full-length NY-BR-1 cDNA, which is approx 6 kilobases, coding for a protein of approx 160 kDA. RT-PCR in a normal tissue
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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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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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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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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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Fig. 2. (Continued) the right ident
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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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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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12 The HUVEC/Matrigel Assay An In V
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Page 332 and 333: 15 Potential Target Antigens for Im
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Page 340 and 341: 16 Identification of Tumor Antigens
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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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