Compositions and methods for the therapy and diagnosis of ovarian cancer

Compositions and methods for the therapy and diagnosis of cancer, such as ovarian cancer, are disclosed. Compositions may comprise one or more ovarian tumor proteins, immunogenic portions thereof, or polynucleotides that encode such portions. Alternatively, a therapeutic composition may comprise an antigen presenting cell that expresses an ovarian tumor protein, or a T cell that is specific for cells expressing such a protein. Such compositions may be used, for example, for the prevention and treatment of diseases such as ovarian cancer. Diagnostic methods based on detecting an ovarian tumor protein, or mRNA encoding such a protein, in a sample are also provided.

EXAMPLE 1 
 Identification of Ovarian Tumor Protein cDNAs This Example illustrates the identification of cDNA molecules encoding ovarian tumor proteins. The polymerase chain reaction (PCR)-Select™ cDNA subtraction methodology of Clontech (Palo Alto, Calif.) was utilized to create libraries enriched for cDNAs that encode ovary tissue-specific antigens, ovary metastatic tumor-expressed antigens and primary ovary tumor-expressed antigens. See U.S. Pat. Nos. 5,565,340 and 5,759,822, incorporated herein by reference, for a description of the (PCR)-Select™ cDNA subtraction methodology. This methodology permits the selective amplification of differentially expressed genes such as those expressed in one cell type but poorly expressed, or absent altogether, in another cell type. PolyA mRNA was prepared from (a) a pool of four ovary omentum metastases and (b) four ovary primary tumors (including an endometroid adenocarcinoma, a germ cell tumor, a papillary serous adenocarcinoma and a clear cell carcinoma). The resulting transcripts were independently subtracted with a set of transcripts from normal brain, pancreas, bone marrow, lung, heart, kidney, liver, and trachea. Following mRNA isolation, cDNA was synthesized and hybridization and PCR amplification reactions were performed in accordance with Clontech's user manual with the following modifications: (1) cDNA was digested with a mixture of restriction enzymes including MscI, PvuII, StuI and DraI instead of the enzyme, RsaI, recommended by Clontech and (2) the ratio of driver to tester cDNA in the hybridization steps was increased to 1:76 in order to increase the stringency of the subtraction. While these are the tumor and normal cell types and (PCR)-Select™ cDNA subtraction reaction conditions used to achieve the polynucleotides disclosed herein, it will be apparent to one skilled in the art that each of these parameters may be modified as required for the particular application contemplated. Thus, for example, the artisan will recognize that the choice of restriction enzymes for cDNA digestion and the ratios of tester and driver for the hybridization steps may be varied in order to influence the efficiency and complexity of the resulting set of subtracted sequences. Using the PCR-Select cDNA Subtraction methodology, three ovary tumor PCR-subtracted libraries were constructed by ligating cDNAs into the pCR2.1-TOPO plasmid vector (Invitrogen; Carlsbad, Calif.). One library was constructed using a pool of four ovary metastatic omentum “tester” mRNAs (OvMS Library; Ovarian Metastatic Subtraction Library) and two libraries were constructed using a pool of primary ovary tumor “tester” mRNAs, including an endometroid adenocarcinoma, a germ cell tumor, a papillary serous adenocarcioma, and a clear cell carcinoma (PrOvS Library; Primary Ovarian Tumor Subtraction Library). The PROvS1 and PROvS2 libraries were constructed from different PCR amplified pools from the same initial subtraction. Eight normal tissues were represented in the “driver” mRNA pool, including brain, pancreas, bone marrow, lung, heart, kidney, liver and trachea. To analyze the efficiency of the subtraction, housekeeping genes such as actin and GAPDH were PCR amplified from dilutions of subtracted as well as unsubtracted PCR samples. The complexity and redundancy of each library was characterized by sequencing 96 clones from each of the PCR subtraction libraries (OvMS, PROvS1 and PROvS2). These analyses revealed that the libraries were enriched for genes overexpressed in ovary tissue and ovary tumor samples. Individual library members were isolated and the cDNA inserts were amplified by PCR. The inserts were purified and subjected to automated sequencing. Disclosed herein in SEQ ID NOs:1-1412 and 1509-1730 are the sequences of 1,634 cDNA clones isolated from the ovarian PCR-subtracted libraries. 
 EXAMPLE 2 
 Synthesis of Ovarian Tumor Specific Polypeptides This Example illustrates a methodology for the preparation of ovarian tumor specific polypeptides according to the present invention. Polypeptides may be synthesized on a Perkin Elmer/Applied Biosystems Division 430A peptide synthesizer using FMOC chemistry with HPTU (O-Benzotriazole-N,N,N′,N′-tetramethyluronium hexafluorophosphate) activation. A Gly-Cys-Gly sequence may be attached to the amino terminus of the peptide to provide a method of conjugation, binding to an immobilized surface, or labeling of the peptide. Cleavage of the peptides from the solid support may be carried out using the following cleavage mixture: trifluoroacetic acid:ethanedithiol:thioanisole:water:phenol (40:1:2:2:3). After cleaving for 2 hours, the peptides may be precipitated in cold methyl-t-butyl-ether. The peptide pellets may then be dissolved in water containing 0.1% trifluoroacetic acid (TFA) and lyophilized prior to purification by C18 reverse phase HPLC. A gradient of 0%-60% acetonitrile (containing 0. 1% TFA) in water (containing 0. 1% TFA) may be used to elute the peptides. Following lyophilization of the pure fractions, the peptides may be characterized using electrospray or other types of mass spectrometry and by amino acid analysis. 
 EXAMPLE 3 
 Analysis of Ovarian Tumor cDNA Expression Using Microarray Technology This example describes microarray expression analysis of ovary tumor-and tissue-specific cDNAs. Sequences disclosed herein were found to be overexpressed in specific tumor tissues as determined by microarray analysis. Using this approach, cDNA sequences are PCR amplified and their mRNA expression profiles in tumor and normal tissues are examined using cDNA microarray technology essentially as described (Schena, M. et al., 1995 Science 270:467-70). In brief, the clones are arrayed onto glass slides as multiple replicas, with each location corresponding to a unique cDNA clone (as many as 5500 clones can be arrayed on a single slide, or chip). Each chip is hybridized with a pair of cDNA probes that are fluorescence-labeled with Cy3 and Cy5, respectively. Typically, 1 &mgr;g of polyA &plus; RNA is used to generate each cDNA probe. After hybridization, the chips are scanned and the fluorescence intensity recorded for both Cy3 and Cy5 channels. There are multiple built-in quality control steps. First, the probe quality is generally monitored using a panel of ubiquitously expressed genes. Secondly, the control plate also can include yeast DNA fragments of which complementary RNA may be spiked into the probe synthesis for measuring the quality of the probe and the sensitivity of the analysis. Currently, the technology offers a sensitivity of about 1 in 100,000 copies of mRNA. Finally, the reproducibility of this technology can be ensured by including duplicated control cDNA elements at different locations. The clones described herein were randomly picked from an ovarian metastatic PCR subtracted library (OvMS library; also called OPs library) and a primary ovarian tumor PCR-subtraction library (PrOvS library; also called OTs library) described in Example 1. A total of 2016 clones (1056 from OPs and 960 from OTs) were arrayed. cDNA inserts for arraying were amplified by PCR using vector specific primers. The resulting PCR products were sequenced in one direction and the trimmed sequences disclosed in Example 1 (SEQ ID NO:1-1412 and 1509-1730). The arrays were probed with 35 probe pairs (normal tissues and ovary tumor and normal-specific probes). Analysis consists of determining the ratio of the mean or median hybridization signal for a particular element (cDNA) using two sets of probes. The ratio is a reflection of the over- or under-expression of the element (cDNA) within the probe population. Probe groups were set up to identify elements (cDNAs) with high differential expression in probe group&num;1: Analysis &num;1 compared ovary tumor (probe group &num;1) and non-ovary normal tissue (probe group &num;2); Analysis &num;2 compared normal and tumor ovary (probe group &num;1) and normal essential tissues (probe group &num;2); Analysis &num;3 used the same probe groups as analysis &num;2 with the addition of probe pairs that had to be repeated due to initial failure. A threshold (fold-overexpression in probe group &num;1) was set at 3.0 (analysis &num;1) or 2.5 (analysis &num;2 and &num;3). This threshold was set based on experience to identify elements with overexpression that could be reproducibly detected. Elements identified by these analyses are shown in Tables 2-4. Elements within each analysis were ranked on mean signal 2 and mean signal 1: Mean Signal 2<0.1 identifies elements with potentially very low background; Mean Signal 2 0.1-0.2 identifies elements with potentially some background; Mean signal 2>2.0 identifies elements with potentially higher background. Tabulation of analysis &num;2 only shows elements that are not identified in analysis &num;1. Analysis &num;3 only shows elements that are not identified by either analysis &num;1 or &num;2. The elements identified were compared to sequences in publically available databases. Those sequences showing some degree of similarity are detailed in Tables 2-4. Sequences of elements identified by this analysis are disclosed in SEQ ID NO:1413-1508. 2 TABLE 2 Analysis &num;1: High Differential Expression in Group 1 Ovary Tumor cy3>Normal Tissue cy5 Threshold: 3 SEQ Clone ID NO Identifier &num; Ratio Mean Signal 1 Mean Signal 2 EST/GenBank Annotation 1413 33482 4.34 0.366 0.084 Novel &equals; O772P 1414 33380 3.54 0.315 0.089 Novel &equals; O772P 1415 33498 3.31 0.313 0.095 Novel &equals; O772P 807 33161 3.63 0.273 0.075 Retinoic acid-binding protein II (CRABP-II) 1416 33855 3.04 0.27 0.089 Glucose regulated protein, GRP78 1417 33872 4.83 0.241 0.05 Novel &equals; O772P 1418 34863 4.01 0.226 0.056 Wnt inhibitory factor-1 (WIF-1) 1419 34558 3.09 0.224 0.073 Zinc finger protein HZF1. Genomic clone, chromosome 19 1420 34582 3.52 0.217 0.062 TAR RNA loop binding protein (TRP-185) 43 34409 3.25 0.204 0.063 Fibroblast growth factor inducible gene 14 (FTN14) 1421 34598 3.01 0.198 0.066 Genomic clone, chromosome 20p11.21-11.23 &equals; O8E 1422 34517 7.11 0.169 0.024 Mitotic checkpoint kinase Bub 1 (BUB 1) 1423 34093 3.37 0.165 0.049 Novel 1424 33345 4.06 0.775 0.191 cDNA clone, KIAA0762 1425 34445 3.58 0.709 0.198 Ribophorin I 382 34008 5 0.681 0.136 Genomic clone, chromosome 20 1426 34946 3.59 0.678 0.189 Wnt inhibitory factor-1 (WIF-1) 1427 34751 4.74 0.608 0.128 Putative Integral membrane transporter protein (LC27) 1428 33813 4.69 0.592 0.126 Ribosomal protein L3 1429 34122 4.73 0.587 0.124 Ribophorin I 1430 34162 3.18 0.466 0.147 Keratin 19 1431 33858 3.08 0.379 0.123 Fibrillarin (Hfib 1), casein kinase II beta subunit 1432 34861 3.14 0.363 0.116 Keratin 19 1433 34237 4.08 13.106 3.211 1434 34496 4.23 5.957 1.408 1435 33673 4.22 6.908 1.638 Elongation factor 1-alpha (EF-1) 1436 34058 3.31 5.396 1.629 Elongation factor 1-alpha 1 1437 34143 3.5 3.477 0.995 GAPDH 1438 33603 3.39 1.861 0.548 Ribosomal Protein S3 1439 33742 3.74 1.644 0.439 Ribosomal protein S3 1440 33555 3.81 1.581 0.415 Collagen alpha-2 type I (COL1A2) 1441 33656 5.13 1.569 0.306 Collagen alpha-1 type I (COL1A1) 1442 33562 3.19 1.536 0.481 Collagen alpha-2 type I(COL1A2) 1443 34501 3.61 1.456 0.403 hmRNP A1 1444 33302 3.33 1.454 0.437 Ribosomal Protein S3 1445 34276 3.35 1.429 0.427 eIF-4AII 1446 33967 3.96 1.369 0.346 Fibronectin 1447 33220 3.97 1.326 0.334 Collagen alpha-1 type I (COL1A1) 1448 34006 3.26 1.284 0.394 Carcinoma associated antigen GA733-2/KSA 1449 34088 4.89 1.277 0.261 Putative integral membrane transporter protein (LC27) 1450 33720 3.87 1.277 0.33 Collagen alpha-1 type I (COL1A1) 1451 34801 4.3 1.267 0.295 eIF-4A 1452 33033 4.71 1.174 0.249 Keratin 8 1453 34310 3.02 1.114 0.369 Genomic clone, BAC from 7p15 1454 33592 3.53 1.004 0.285 Collagen alpha-1 type III (COL3A1) 632 33055 4.31 0.955 0.222 cDNA clone DKFZp564N1116 1455 34279 3.78 0.939 0.248 hnRNP A1 1456 34541 3.65 0.937 0.257 hnRNP A1 1457 34380 3.66 0.867 0.237 eIF-4AI 1458 34675 3.63 0.775 0.214 Ribosomal protein L5 1459 33291 3.14 0.698 0.222 Collagen alpha-1 type III (COL3A1) 1460 34694 3.2 0.679 0.212 Genomic clone, Xq13; X inactivation transcript (XIST) 3 TABLE 3 Analysis &num;2: High Differential Expression in Group 1 Normal and Tumor Ovary > Normal Essential Tissues Threshold: 2.5 SEQ Clone ID NO Identifier &num; Ratio Mean Signal 1 Mean Signal 2 EST/GenBank Annotation 1461 33660 5.05 0.491 0.097 1-218 is novel; 218-305 is vector&quest; 1462 33277 2.93 0.284 0.097 Partial unknown mRNA from drug- resistant melanoma cells 1463 33376 3.19 0.253 0.079 Novel &equals; O772P 1464 33462 2.7 0.238 0.088 Novel &equals; O772P 1465 32951 2.78 0.234 0.084 Keratin 19 1466 34405 2.63 0.232 0.088 c-kit proto-oncogene (stem cell growth factor receptor) 1467 34305 2.57 0.217 0.085 Wnt inhibitory factor-1 (WIF-1) 1468 33508 2.68 0.203 0.076 Insulin-like growth factor II (IGF-2) exon 7 with additional ORF 1212 33750 2.68 0.187 0.07 DEK putative oncogene (leukemia); 1-387 bp; 387&plus; &equals; vector 1469 34873 3.02 0.165 0.055 Apurinic/apyrimidinic endonuclease (HAP1); REDOX factor 1377 33885 2.83 0.143 0.051 Genomic clones, HLA class I region&quest;Bases after 101 are vector&quest; 1470 34542 2.62 0.125 0.048 Estrogen receptor (ESR1) 1471 33644 3.01 0.557 0.185 Novel &equals; O772P 1472 34344 2.62 0.505 0.193 HER2/c-erb-B-2 (tyrosine kinase receptor) &equals; Her 2 neu 1473 33414 2.66 0.494 0.186 Collagen alpha-2 type I (COL&excl;A2) 1474 34298 2.78 0.488 0.175 Keratin 19 1475 33624 2.72 0.442 0.163 Jerky (mouse) homolog-like (JRKL); nucl. reg., role in epilepsy&quest; 1476 34047 3.38 0.406 0.12 Fibroblast growth factor inducible gene 14 (FIN14) 1477 33173 2.84 0.343 0.121 Fibronectin (FN1) 1478 34607 2.59 0.34 0.131 HER2/c-erb-B-2 (tyrosine kinase receptor) &equals; Her 2 neu 1479 33404 2.65 0.32 0.121 Novel &equals; O772P 1480 33069 2.8 7.953 2.841 1481 33847 3.42 2.541 0.743 1482 34527 2.51 2.089 0.833 1483 33427 2.68 1.999 0.746 1484 33035 3.31 1.799 0.543 1485 34079 2.57 1.562 0.608 1486 33680 2.53 1.477 0.584 1487 33463 2.87 1.207 0.42 1488 33207 2.87 1.053 0.367 1489 34256 2.63 0.911 0.347 1490 33581 2.81 0.86 0.306 1491 33634 2.71 0.794 0.293 1492 34539 2.85 0.637 0.224 1493 32961 2.98 0.626 0.21 1494 33790 2.58 0.621 0.24 1495 33284 2.58 0.617 0.239 1496 33183 2.64 0.589 0.223 1497 33388 2.5 0.557 0.223 4 TABLE 4 Analysis &num;3: High Differential Expression in Group 1 N and T Ovary (re-probe) > Normal Essential Tissues (re-probe) Threshold: 2.5 SEQ Clone ID NO Identifier &num; Ratio Mean Signal 1 Mean Signal 2 EST/GenBank Annotation 1498 33090 2.53 0.247 0.098 Genomic clone, chromosome 20p12.3-13 1499 33239 2.73 0.52 0.191 Genomic clone, chromosome 6; (breast cancer antigen discovery) 886 34943 2.52 0.317 0.126 Wnt inhibitory factor-1 (WIF-1) 1500 33654 2.51 6.272 2.499 Collagen alpha-1 type 1 (COL1A1) 1501 33999 2.52 2.285 0.908 Thymosin beta-4; interferon- inducible Based on the microarray profile and database search results, several cDNAs identified above were further analyzed by Real Time PCR. Clone 0990P (SEQ ID NO:1502; Clone Identifier &num;:33055/61229) showed overexpression in most tumors and some expression in bone marrow and brain. Clone 0989P (SEQ ID NO:1506; Clone Identifier &num;33161/59657) was overexpressed in most tumors (n&equals;13) and showed some expression in normal breast, ureter, and oesophagus. From the foregoing it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims.