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  Page-4, an x-linked gage-like gene expressed in normal and neoplastic prostate, testis and uterus, and uses thereforUSPTO Application #: 20070248972Title: Page-4, an x-linked gage-like gene expressed in normal and neoplastic prostate, testis and uterus, and uses therefor Abstract: PAGE-4 is a gene preferentially expressed in normal male and female reproductive tissues, prostate, testis, fallopian tube, uterus and placenta, as well as in prostate cancer, testicular cancer and uterine cancer. This expression pattern makes it a target for diagnosis and for vaccine based therapy of neoplasms of prostate, testis and uterus. The invention provides immunogenic compositions comprising PAGE-4 protein or immunogenic peptides thereof, methods of inhibiting the growth of malignant cells expressing PAGE-4, and methods of inducing an enhanced immune response to PAGE-4-expressing cancers. (end of abstract) Agent: Klarquist Sparkman, LLP - Portland, OR, US Inventors: Ira H. Pastan, Ulrich Brinkmann, George Vasmatzis, Byungkook Lee USPTO Applicaton #: 20070248972 - Class: 435006000 (USPTO) Related Patent Categories: Chemistry: Molecular Biology And Microbiology, Measuring Or Testing Process Involving Enzymes Or Micro-organisms; Composition Or Test Strip Therefore; Processes Of Forming Such Composition Or Test Strip, Involving Nucleic Acid The Patent Description & Claims data below is from USPTO Patent Application 20070248972. Brief Patent Description - Full Patent Description - Patent Application Claims
CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application is a continuation of U.S. patent application Ser. No. 09/763,393 filed on Jul. 30, 2001, which is incorporated herein by reference. U.S. patent application Ser. No. 09/763,393 is a .sctn. 371 U.S. national stage of PCT Application No. PCT/US99/20046, filed Aug. 31, 1999, which was published in English under PCT Article 21(2), which in turn claims the benefit of U.S. Provisional Application 60/098,993 filed Sep. 1, 1998, which is incorporated herein by reference. BACKGROUND OF THE INVENTION [0002] ESTs are sequences derived from randomly selected clones from various cDNA libraries (Adams, M. D. et al. Science 252:1651-1656 (1991); Adams, M. D. et al. Nature 377:3-174 (1995); Adams, M. D. et al., Nature 355:632-634 (1992); Emmert-Buck et al., Science 274:998-1101 (1996); Krizman, D. B. et al., Cancer Res. 56:5380-5383 (1996); Strausberg, R. L. et al., Nat. Genet. 16:415-516 (1997)). Each cDNA clone is generated from a transcript, and the frequency and distribution of the many different transcripts in any given tissue depends on the tissue specific activity of the genes. The translation of transcript frequency and distribution into frequency and distribution of EST sequences depends not only on the specificity and magnitude of mRNA expression, but also on other factors such as mRNA stability and clonability of these EST sequences. Therefore, a specificity or frequency analysis of ESTs only provides a guide for the prediction of expression patterns. Nevertheless, ESTs provide a valuable source of information that may be utilized to predict the expression patterns of specific genes in different tissues. [0003] The recently developed NCl Cancer Genome Anatomy Project (CGAP) uses microdissection and laser-capture techniques to generate defined and tissue/tumor specific EST libraries (Emmert-Buck et al., Science 274:998-1101 (1996); Krizman, D. B. et al., Cancer Res. 56:5380-5383 (1996); Strausberg, R. L. et al., Nat. Genet. 16:415-516 (1997)). CGAP has already accumulated a vast number of tissue-specific sequences and the CGAP sequence data base is rapidly growing with the continuous addition of sequences from different tissues and tumor types. There are many ways by which the EST sequence data can be processed to cluster, sort and filter the cDNA sequences, in order to identify genes that are specifically expressed in certain tissues. Database "mining" for cDNAs that are preferentially or exclusively expressed in defined tissues, or in malignant/neoplastic tissues provides lists of potential target genes for cancer therapy (Emmert-Buck et al., Science 274:998-1101 (1996); Krizman, D. B. et al., Cancer Res. 56:5380-5383 (1996); Strausberg, R. L. et al., Nat. Genet. 16:415-516 (1997); Vasmatzis, G. et al., Proc. Natl. Acad. Sci., USA 95:300-304 (1998)). Although in many cases these "candidate genes", which appear tissue specific in database analyses, cannot be confirmed in their specificity by experimental techniques (e.g. Northern blots or PCR), a reasonable number of candidate genes remain for which the predicted and desired expression pattern can be experimentally confirmed (Vasmatzis, G. et al., Proc. Natl. Acad. Sci., USA 95:300-304 (1998); He, W. W. et al., Genomics 43:69-77 (1997)). These specifically expressed genes are of interest because of their functions in cell or tumor biology and may also be directly used as markers for cancer diagnosis and as the basis for a variety of methods of therapy. [0004] One method of therapy is to use the gene product as a vaccine to enhance the patient's immune response to the cancer. T cells play an important role in tumor regression in most murine tumor models. Tumor infiltrating lymphocytes ("TIL") that recognize unique cancer antigens can be isolated from many murine tumors. The adoptive transfer of these TIL plus interleukin-2 can mediate the regression of established lung and liver metastases (Rosenberg, S., et al., Science 233:1318-1321 (1986). In addition, the secretion of IFN-.gamma by injected TIL significantly correlates with in vivo regression of murine tumors suggesting activation of T-cells by the tumor antigens. (Barth, R., et al., J. Exp. Med. 173:647-658 (1991)). In humans, the ability of tumor TIL to mediate the regression of metastatic cancer in 35 to 40% of melanoma patients when adoptively transferred into patients with metastatic melanoma attests to the clinical importance of the antigens recognized (Rosenberg, S., et al., N Engl J Med 319:1676-1680 (1988); Rosenberg S., J. Clin. Oncol. 10:180-199 (1992)). [0005] T cell receptors on CD8+ T cells recognize a complex consisting of an antigenic peptide (9-10 amino acids for Human Leukocyte Antigen ("HLA")-A2), .beta.-2 microglobulin, and class I major histocompatibility complex ("MHC") heavy chain (HLA-A, B, C, in humans). Peptides generated by digestion of endogenously synthesized proteins are transported into the endoplasmic reticulum, bound to class I MHC heavy chain and .beta.-2 microglobulin, and finally expressed in the cell surface in the groove of the class I MHC molecule. Therefore, T cells can detect molecules that originate from proteins inside cells, in contrast to antibodies that detect molecules expressed on the cell surface. Antigens recognized by T cells thus may be particularly useful for inhibiting the progression of cancer. [0006] Strong evidence that an immune response to cancer exists in humans have been provided by, for example, the existence of lymphocytes within melanoma deposits. These lymphocytes, when isolated, are capable of recognizing specific tumor antigens on autologous and allogeneic melanomas in an MHC restricted fashion. (Kawakami, Y., et al., J. Immunol. 148:638-643 (1992); Hom, S., et al., J. Immunother. 13:18-30 (1993)). TIL from patients with metastatic melanoma recognize shared antigens including melanocyte-melanoma lineage specific tissue antigens in vitro (Kawakami, Y., et al., J. Immunother. 14:88-93 (1993); Anichini, A. et al., J. Exp. Med. 177:989-998 (1993)). [0007] Although several tumor associated antigens ("TAA") have been found for melanoma, there is a need to identify tissue specific genes whose expression is associated with cancers of other tissues. SUMMARY OF THE INVENTION [0008] This invention provides a new class of proteins which are preferentially expressed by cells of reproductive tissues, including, but not limited to, the prostate gland, testis, uterus, fallopian tubes, and placenta. The proteins are found in both normal and cancerous reproductive tissues. These proteins share some homology with the GAGE and MAGE family of proteins. [0009] In particular, the invention provides the PAGE-4 protein. The PAGE-4 protein and immunogenic peptides thereof can be used as immunogenic compositions to raise cytotoxic T lymphocyte responses against cells expressing PAGE-4 in vitro or in vivo. Such cells include cancers of the prostate, testis, and uterus. Nucleic acids encoding the protein or an immunogenic peptide thereof can also as immunogenic compositions. [0010] In addition to the uses as immunogenic compositions, the present invention also provides for methods of detecting the presence of the PAGE-4 protein in cells in cell samples or body tissues. The detection can be performed by detecting the protein, typically by using antibodies. Detection of the presence of the protein can also be accomplished by detecting nucleic acids that encode the proteins. Conveniently, this can be done by using detectable probes complementary to all or a portion of sequences encoding PAGE-4. The presence of PAGE-4 in tissues not related to reproduction could be indicative of the spread of cancerous reproductive tissue. [0011] In addition to diagnostic and vaccine uses, PAGE-4 protein and the nucleic acids encoding it can be used in therapeutic applications. PAGE-4 protein can be used to raise antibodies which can be used not only in the diagnostic assays described above, but also as the targeting moiety of immunoconjugates. The toxic moiety of the immunoconjugates can include, but is not limited to, toxins such as ricin, abrin, diphtheria toxin and subunits thereof, as well as botulinum toxins A through F, and Pseudomonas exotoxin (PE). DESCRIPTION OF THE DRAWINGS [0012] FIG. 1: Similarity of PAGE-4, GAGE and MAGE: (A) The predicted PAGE-4 reading frame (SEQ ID NO: 1) is derived from the full length PAGE-4 EST clone nh32c06. The GAGE and MAGE sequences are from SW: GGE1 (SEQ ID NO: 2), GGE2 (SEQ ID NO: 3), GGE3 (SEQ ID NO: 4), GGE4 (SEQ ID NO: 5), GGE5 (SEQ ID NO: 6), GGE6 (SEQ ID NO: 7), MAG5 (SEQ ID NO: 8) and MAG8_HUMAN (SEQ ID NO: 9). Note that the "MAGE-alignment" matches amino acids that occur in MAGE5 and/or MAGE8, which are similar to PAGE-4 and/or GAGE1-6; the homologies between single members of the MAGE and PAGE and GAGE protein families are weaker. (B) Alignment of PAGE-1 (SEQ ID NO: 10) with other PAGEs. PAGE-2 (SEQ ID NO: 11) was translated from the EST ai61a04 EST-cluster and PAGE-3 from om29f08. PAGE-3 (SEQ ID NO: 12) was translated from one single EST and it is possible that the truncated amino terminus results from a sequence artifact (the homology extends further to the N-terminus in another reading frame). Several other so far undefined EST clusters were found that have homology to PAGE as well as to GAGE. These clusters do not have the striking similarities that the other GAGE family members have to each other, but they are also not significantly more similar to PAGE than to GAGE. Representatives of some of these cDNA clusters are the ESTS yd88e11 (fetal liver/spleen), yw86a06 (placenta) and yi21h01 (placenta). [0013] FIG. 2: Hybridization analysis of PAGE-4 expression: (A) A MTN Dot blot (left) and Northern blots (middle to right) were probed with a 140 bp .sup.32P labeled PAGE-4 probe under very stringent hybridization conditions (50% formamide, 55.degree. C.). Specific PAGE-4 signals were observed in prostate, testis, placenta and uterus, but not in other tissues (Table 1 legend lists the analyzed tissues). Because the hybridization probe had some similarity with another PAGE-4-like EST cluster that is expressed in testis (PAGE-2 represented by the EST zv62h08, Table 2), we additionally used a probe with minimal homology to zv62h08 to confirm that the signal in testis corresponds to the expression of the authentic PAGE-4. (B) Blots containing 20 .mu.g/lane total RNA from normal or malignant ovary (right), fallopian tube (middle) and uterus (left) were hybridized under stringent conditions. PAGE-4 is expressed in fallopian tube, uterus and uterine cancer, but not in ovary and ovarian cancer. [0014] FIG. 3: Relation of the sequences of MAGE, GAGE, PAGE and other so far uncharacterized EST clusters: The GCG program "PILEUP" was used to compare the multiple protein sequences of the GAGE and PAGE protein family. The dendrogram shows that PAGE proteins are a separate group of proteins that are less related to GAGE proteins. [0015] FIG. 4: RT-PCR analysis of PAGE-4 expression: Ethidium bromide stained 2.5% agarose gel; PAGE-4 cDNA was amplified with 5' and 3'-end specific PAGE primers (40 cycles 94.degree. C.-58.degree. C.-72.degree. C., 1 min each). [0016] FIG. 5: Nucleotide sequence encoding PAGE-4 (SEQ ID NO: 13): The open reading frame is in bold type and underlined. DETAILED DESCRIPTION I. Introduction [0017] Here we describe the identification of an X-linked gene that is expressed in normal and malignant male and female reproductive tissues. This gene, PAGE-4 (which we originally called PAGE-1 and have now renumbered to be consistent with other findings), is homologous to a family of MAGE/GAGE like proteins and is expressed in normal prostate, testis, uterus, fallopian tube and placenta, as well as in prostate, testicular and uterine cancers. [0018] Prostate, testicular, and uterine cancers, are usually treated, in part, by the surgical removal of the affected organ. The metastases may not, however, be susceptible to surgical removal, or they may be too small to be readily detected. Enhancing the patient's immune response to the cancer, and particularly enhancing the response of cytotoxic T lymphocytes ("CTLs") to the cancer, can aid in slowing or stopping the progress of the disease. PAGE-4 and immunogenic peptides thereof can be used as a vaccine to enhance a patient's immune response against PAGE-4-expressing cancers. Vaccines of DNA encoding the protein or an immunogenic portion thereof can also be used. The vaccines can be administered with adjuvants which help induce a CTL response to the antigen. Since healthy people have tissues which express PAGE-4, it is contemplated that the vaccines of the invention would be used as therapeutic vaccines, rather than as prophylactic vaccines. The identification of this new gene therefore arms researchers and clinicians with a new weapon with which to attack prostate cancer, testicular cancer, and other disorders in which death of the specific tissues might be beneficial to the patient. Continue reading... Full patent description for Page-4, an x-linked gage-like gene expressed in normal and neoplastic prostate, testis and uterus, and uses therefor Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Page-4, an x-linked gage-like gene expressed in normal and neoplastic prostate, testis and uterus, and uses therefor patent application. ###  How KEYWORD MONITOR works... a FREE service from FreshPatents1. Sign up (takes 30 seconds). 2. Fill in the keywords to be monitored. 3. Each week you receive an email with patent applications related to your keywords. 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