Target Discovery and Validation Reviews and Protocols

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Tumor Antigen Discovery 323 panel by using NY-BR-1–specific primers showed NY-BR-1 mRNA to be exclusively expressed in normal breast, normal testis, and variable in normal prostate. In tumor tissues, NY-BR-1 is expressed in 70–80% of breast cancers, and 25% of prostate cancers; all other tumor types are NY-BR-1 negative (39). Based on the expression analysis, NY-BR-1 represents a new breast differentiation antigen. We produced a short carboxy-terminal recombinant protein for larger scale serological analysis and found humoral immune responses in approx 10% of patients with NY-BR-1–positive breast cancers. Importantly, all normal sera were antibody negative. To analyze T-cell responses against NY-BR-1, the entire NY-BR-1 protein sequence was screened for potential HLA-A2 binding motifs. Numerous potential binding peptides were synthesized, and CD8 T-cells derived from seropositive HLA-A2–positive breast cancer patients were stimulated once with each of the peptides and then tested in ELISPOT assay for peptide-specific recognition. We identified several peptides that were specifically recognized by CD8 T-cells. We could further demonstrate that those peptides represent naturally processed and presented NY-BR-1 epitopes. In summary, we (1) discovered a new tumor antigen by applying SEREX, (2) demonstrated a tumor-specific expression pattern, and (3) identified NY-BR-1–derived peptide epitopes that are naturally processed and presented to CD8 T-cells. A phase I clinical trial for HLA-A2–positive patients with advanced NY-BR-1–expressing breast cancer is planned. Vaccination with the two NY-BR-1–derived peptides will show whether potent antigen-specific CD8 T-cell responses can be induced. Clinical effects and potential side effects in normal breast tissue will be carefully analyzed. Vaccine strategies aiming at the induction of an integrated immune response (antigen-specific antibody, CD4, and CD8 cells) use longer peptide sequences or the entire protein. Such vaccine approaches been have shown to be efficient in early clinical trials in melanoma patients, and they are currently being tested in phase III trials in high-risk melanoma patients after resection of lymph node metastases. 5. Conclusions SEREX is one of several techniques that identify tumor-associated antigens that have potential use as target antigens for immunotherapy approaches. In the future, we aim at immunizing patients with a whole array of different tumor-associated antigens expressed by the individual tumor to induce integrated immune responses targeting a maximal percentage of cancer cells. References 1. Naito, Y., Saito, K., Shiiba, K., et al. (1998) CD8+ T cells infiltrated within cancer cell nests as a prognostic factor in human colorectal cancer. Cancer Res. 58, 3491–3494.
324 Jäger 2. Nakano, O., Sato, M., Naito, Y., et al. (2001) Proliferative activity of intratumoral CD8(+) T-lymphocytes as a prognostic factor in human renal cell carcinoma: clinicopathologic demonstration of antitumor immunity. Cancer Res. 61, 5132–5136. 3. Schumacher, K., Haensch, W., Roefzaad, C., and Schlag, P. M. (2001) Prognostic significance of activated CD8(+) T cell infiltrations within esophageal carcinomas. Cancer Res. 61, 3932–3936. 4. Eerola, A. K., Soini, Y., and Paakko, P. (2000) A high number of tumor-infiltrating lymphocytes are associated with a small tumor size, low tumor stage, and a favorable prognosis in operated small cell lung carcinoma. Clin. Cancer Res. 6, 1875–1881. 5. van der Bruggen, P., Traversari, C., and Chomez, P. (1991) A gene encoding an antigen recognized by cytolytic T lymphocytes on a human melanoma. Science 254, 1643–1647. 6. Boel, P., Wildmann, C., Sensi, M. L., et al. (1995) BAGE: a new gene encoding an antigen recognized on human melanomas by cytolytic T lymphocytes. Immunity 2, 167–175. 7. Van den Eynde, B., Peeters, O., De Backer, O., Gaugler, B., Lucas, S., and Boon, T. (1995) A new family of genes coding for an antigen recognized by autologous cytolytic T lymphocytes on a human melanoma. J. Exp. Med. 182, 689–698. 8. Boon, T. and van der Bruggen, P. (1996) Human tumor antigens recognized by T lymphocytes. J. Exp. Med. 183, 725–729. 9. Scanlan, M. J., Gure, A. O., Jungbluth, A. A., Old, L. J., and Chen, Y. T. (2002) Cancer/testis antigens: an expanding family of targets for cancer immunotherapy. Immunol. Rev. 188, 22–32. 10. Coulie, P. G., Brichard, V., Van Pel, A., et al. (1994) A new gene coding for a differentiation antigen recognized by autologous cytolytic T lymphocytes on HLA-A2 melanomas. J. Exp. Med. 180, 35–42. 11. Brichard, V., Van Pel, A., Wolfel, T., et al. (1993) The tyrosinase gene codes for an antigen recognized by autologous cytolytic T lymphocytes on HLA-A2 melanomas. J. Exp. Med. 178, 489–495. 12. Kawakami, Y., Eliyahu, S., Delgado, C. H., et al. (1994) Identification of a human melanoma antigen recognized by tumor- infiltrating lymphocytes associated with in vivo tumor rejection. Proc. Natl. Acad. Sci. USA 91, 6458–6462. 13. Wang, R. F., Robbins, P. F., Kawakami, Y., Kang, X. Q., and Rosenberg, S. A. (1995) Identification of a gene encoding a melanoma tumor antigen recognized by HLA-A31-restricted tumor-infiltrating lymphocytes. [published erratum appears in J. Exp. Med. (1995) Mar 1;181(3),1261]. J. Exp. Med. 181, 799–804. 14. Wolfel, T., Hauer, M., Schneider, J., et al. (1995) A p16INK4a-insensitive CDK4 mutant targeted by cytolytic T lymphocytes in a human melanoma. Science 269, 1281–1284. 15. Gnjatic, S., Cai, Z., Viguier, M., Chouaib, S., Guillet, J. G., and Choppin, J. (1998) Accumulation of the p53 protein allows recognition by human CTL of a wild-type p53 epitope presented by breast carcinomas and melanomas. J. Immunol. 160, 328–333.
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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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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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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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Transfected Cell Arrays 161 5. Simp
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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 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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