Tumor Antigens for the Prevention and/or Treatment of Cancer

Abstract

The present invention relates to a nucleic acid encoding a polypeptide and the use of the nucleic acid or polypeptide in preventing and/or treating cancer. In particular, the invention relates to improved vectors for the insertion and expression of foreign genes encoding tumor antigens for use in immunotherapeutic treatment of cancer.

Description

RELATED APPLICATIONS

[0001] This application is a continuation of U.S. Ser. No. 10/557,066 filed Jul. 30, 2007, which was filed under 35 U.S.C. .sctn.371, and claims priority to International Application No. PCT/US2004/015202 filed May 15, 2004, which claims priority to Ser. Nos. 60/471,119 filed May 16, 2003 and 60/471,193 filed May 16, 2003.

FIELD OF THE INVENTION

[0002] The present invention relates to a nucleic acid encoding a polypeptide and the use of the nucleic acid or polypeptide in preventing and/or treating cancer. In particular, the invention relates to improved vectors for the insertion and expression of foreign genes encoding tumor antigens for use in immunotherapeutic treatment of cancer.

BACKGROUND OF THE INVENTION

[0003] There has been tremendous increase in last few years in the development of cancer vaccines with Tumour-associated antigens (TAAs) due to the great advances in identification of molecules based on the expression profiling on primary tumours and normal cells with the help of several techniques such as high density microarray, SEREX, immunohistochemistry (IHC), RT-PCR, in-situ hybridization (ISH) and laser capture microscopy (Rosenberg, Immunity, 1999; Sgroi et al, 1999, Schena et al, 1995, Offringa et al, 2000). The TAAs are antigens expressed or over-expressed by tumour cells and could be specific to one or several tumours for example CEA antigen is expressed in colorectal, breast and lung cancers. Sgroi et al (1.999) identified several genes differentially expressed in invasive and metastatic carcinoma cells with combined use of laser capture microdissection and cDNA microarrays. Several delivery systems like DNA or viruses could be used for therapeutic vaccination against human cancers (Bonnet et al, 2000) and can elicit immune responses and also break immune tolerance against TAAs. Tumour cells can be rendered more immunogenic by inserting transgenes encoding T cell co-stimulatory molecules such as B7.1 or cytokines such as IFN-.gamma., IL2, or GM-CSF, among others. Co-expression of a TAA and a cytokine or a co-stimulatory molecule has also been shown to be useful in developing effective therapeutic vaccines (Hodge et al, 95, Bronte et al, 1995, Chamberlain et al, 1996).

[0004] There is a need in the art for reagents and methodologies useful in stimulating an immune response to prevent or treat cancers. The present invention provides such reagents and methodologies which overcome many of the difficulties encountered by others in attempting to treat cancer.

SUMMARY OF THE INVENTION

[0005] The present invention provides an immunogenic target for administration to a patient to prevent and/or treat cancer. In particular, the immunogenic target is a tumor antigen ("TA") and or an angiogenesis-associated antigen ("AA"). In one embodiment, the immunogenic target is encoded by SEQ ID NO.: 34 or SEQ ID NO.: 36 or has the amino acid sequence of SEQ ID NO.: or SEQ ID NO.: 37. In certain embodiments, the TA and/or AA are administered to a patient as a nucleic acid contained within a plasmid or other delivery vector, such as a recombinant virus. The TA and/or AA may also be administered in combination with an immune stimulator, such as a co-stimulatory molecule or adjuvant.

BRIEF DESCRIPTION OF THE DRAWINGS

[0006] FIG. 1. A, B. Nucleotide sequences of AAC2-1 and AAC2-2. C. Alignment of predicted amino acid sequence of AAC2-1 and AAC2-2. Missing nucleotides or amino acids are indicated by a "*". Differences between sequences are underlined.

[0007] FIG. 2. Human lymphocytes differentiate into effector cells secreting IFN-.gamma. in response to peptides derived from the AAC2-2 protein. T cells were stimulated with the groups of peptides shown n in Table III (groups 1-9). After three rounds of stimulation, the lymphocytes were analyzed for peptide-specific IFN-.gamma. production by ELISPOT. The graph in the inset shows that activated cells stimulated by peptide Group #6 are capable of antigen-specific CTL activity killing peptide loaded T2 target cells. Peptide EC5 elicits dominant activity in inducing both CTL activity and IFN-.gamma. secretion.

[0008] FIG. 3. Murine T cells from HLA-A2-Kb transgenic mice recognize and secrete IFN-.gamma. in response to DNA immunization with a human AAC2-2-encoding DNA plasmid. Spleen cells from pEF6-hAAC2-2-immunized mice were re-stimulated with the different groups of peptides. After six days, the cells were harvested and tested for IFN-.gamma. secretion in response to each respective peptide group or a control HLA-A2-binding 9-mer HIV peptide. ELISPOT plates were incubated over-night and developed. Each group responded with high levels of IFN-.gamma. production (over 250 spots) in response to PMA and ionomycin used as a positive control. One of the highly reactive peptides groups (group 6) is also recognized by human lymphocytes from the HLA-A-0201.sup.+ donors tested so far.

[0009] FIG. 4. DNA vaccination with a gene encoding human AAC2-2 completely abrogates the growth of implanted B16F10 melanoma cells. This effect is not due to a non-specific immune response as shown by the inability of plasmid encoding flu-NP protein and the human flk1 (VEGFR-2) to prevent tumor growth.

[0010] FIG. 5. Survival of mice after implantation of B16F10 melanoma cells into C57BL/6 mice showing the ability of DNA vaccination with a human AAC2-2 vector to completely protect against the effects of rumor growth. This protective effect is antigen-specific and can not be elicited through vaccination with other genes.

[0011] FIG. 6. T lymphocytes from C57BL/6 mice exhibit effector cell activity and secrete IFN-.gamma. in response to peptides of human AAC2-2 following DNA vaccination with the pEF6-hAAC2-2 expression plasmid. These peptides can exhibit cross-reactivity on B6 MHC class I. The peptides in group 1 and group 5 induce strong reactivity by C57BL/6 T cells.

[0012] FIG. 7. BFA4 cDNA sequence.

[0013] FIG. 8. BFA4 amino acid sequence.

[0014] FIG. 9. BCY1 nucleotide (A) and amino acid (B) sequences.

[0015] FIG. 10. Immune response against specific BCY1 peptides.

[0016] FIG. 11. BFA5 cDNA sequence.

[0017] FIG. 12. BFA5 amino acid sequence.

[0018] FIGS. 13A, B and C. Immune response against BFA5-derived peptides.

[0019] FIG. 14. BCZ4 cDNA (A) and amino acid (B) sequences.

[0020] FIG. 15. Immune response against BCZ4-derived peptides (A: BCZ4 ELISPOT; B: BCZ4 Peptide Deconvolution; C: CTL response).

[0021] FIG. 16. BFY3 cDNA (A) and amino acid (B) sequences.

[0022] FIG. 17A-E. Immune response against BFY3-derived peptides.

DETAILED DESCRIPTION

[0023] The present invention provides reagents and methodologies useful for treating and/or preventing cancer. All references cited within this application are incorporated by reference.

[0024] In one embodiment, the present invention relates to the induction or enhancement of an immune response against one or more tumor antigens ("TA") to prevent and/or treat cancer. In certain embodiments, one or more TAs may be combined. In preferred embodiments, the immune response results from expression of a TA in a host cell following administration of a nucleic acid vector encoding the tumor antigen or the tumor antigen itself in the form of a peptide or polypeptide, for example.

[0025] As used herein, an "antigen" is a molecule such as a polypeptide or a portion thereof that produces an immune response in a host to whom the antigen has been administered. The immune response may include the production of antibodies that bind to at least one epitope of the antigen and/or the generation of a cellular immune response against cells expressing an epitope of the antigen. The response may be an enhancement of a current immune response by, for example, causing increased antibody production, production of antibodies with increased affinity for the antigen, or an increased or more effective cellular response (i.e., increased T cells or T cells with higher anti-tumor activity). An antigen that produces an immune response may alternatively be referred to as being immunogenic or as an immunogen. In describing the present invention, a TA may be referred to as an "immunogenic target".

[0026] TA includes both tumor-associated antigens (TAAs) and tumor-specific antigens (TSAs), where a cancerous cell is the source of the antigen. A TAA is an antigen that is expressed on the surface of a tumor cell in higher amounts than is observed on normal cells or an antigen that is expressed on normal cells during fetal development. A TSA is an antigen that is unique to tumor cells and is not expressed on normal cells. TA further includes TAAs or TSAs, antigenic fragments thereof, and modified versions that retain their antigenicity.

[0027] TAs are typically classified into five categories according to their expression pattern, function, or genetic origin: cancer-testis (CT) antigens (i.e., MAGE, NY-ESO-1); melanocyte differentiation antigens (i.e., Melan A/MART-1, tyrosinase, gp100); mutational antigens (i.e., MUM-1, p53, CDK-4); overexpressed `self` antigens (i.e., HER-2/neu, p53); and, viral antigens (i.e., HPV, EBV). For the purposes of practicing the present invention, a suitable TA is any TA that induces or enhances an anti-tumor immune response in a host in whom the TA is expressed. Suitable TAs include, for example, gp100 (Cox et al., Science, 264:716-719 (1994)), MART-1/Melan A (Kawakami et al., J. Exp. Med., 180:347-352 (1994)), gp75 (TRP-1) (Wang et al., J. Exp. Med., 186:1131-1140 (1996)), tyrosinase (Wolfel et al., Eur. J. Immunol., 24:759-764 (1994); WO 200175117; WO 200175016; WO 200175007), NY-ESO-1 (WO 98/14464; WO 99/18206), melanoma proteoglycan (Hellstrom et al., J. Immunol. 130:1467-1472 (1983)), MAGE family antigens (i.e., MAGE-1, 2, 3, 4, 6, 12, 51; Van der Bruggen et al., Science, 254:1643-1647 (1991); U.S. Pat. Nos. 6,235,525; CN 1319611), BAGE family antigens (Boel et al., Immunity, 2:167-175 (1995)), GAGE family antigens (i.e., GAGE-1,2; Van den Eynde et al., J. Exp. Med., 182:689-698 (1995); U.S. Pat. No. 6,013,765), RAGE family antigens (i.e., RAGE-1; Gaugler et al., Immunogenetics, 44:323-330 (1996); U.S. Pat. No. 5,939,526), N-acetylglucosaminyltransferase-V (Guilloux et al., J. Exp. Med., 183:1173-1183 (1996)), p15 (Robbins et al., J. Immunol. 154:5944-5950 (1995)), .beta.-catenin (Robbins et al., J. Exp. Med., 183:1185-1192 (1996)), MUM-1 (Coulie et al. Proc. Natl. Acad. Sci. USA, 92:7976-7980 (1995)), cyclin dependent kinase-4 (CDK4) (Wolfel et al., Science, 269:1281-1284 (1995)), p21-ras (Fossum et al., Int. J. Cancer, 56:40-45 (1994)), BCR-abl (Bocchia et al., Blood, 85:2680-2684 (1995)), p53 (Theobald et al., Proc. Natl. Acad. Sci. USA, 92:11993-11997 (1995)), p185 HER2/neu (erb-B1; Fisk et al., J. Exp. Med., 181:2109-2117 (1995)), epidermal growth factor receptor (EGFR) (Harris et al., Breast Cancer Res. Treat, 29:1-2 (1994)), carcinoembryonic antigens (CEA) (Kwong et al., J. Natl. Cancer Inst., 85:982-990 (1995) U.S. Pat. Nos. 5,756,103; 5,274,087; 5,571,710; 6,071,716; 5,698,530; 6,045,802; EP 263933; EP 346710; and, EP 784483); carcinoma-associated mutated mucins (i.e., MUC-1 gene products; Jerome et al., J. Immunol. 151:1654-1662 (1993)); EBNA gene products of EBV (i.e., EBNA-1; Rickinson et al., Cancer Surveys, 13:53-80 (1992)); E7, E6 proteins of human papillomavirus (Ressing et al., J. Immunol, 154:5934-5943 (1995)); prostate specific antigen (PSA; Xue et al., The Prostate, 30:73-78 (1997)); prostate specific membrane antigen (PSMA; Israeli, et al., Cancer Res., 54:1807-1811 (1994)); idiotypic epitopes or antigens, for example, immunoglobulin idiotypes or T cell receptor idiotypes (Chen et al., J. Immunol., 153:4775-4787 (1994)); KSA (U.S. Pat. No. 5,348,887), kinesin 2 (Dietz, et al. Biochem Biophys Res Commun 2000 Sep. 7; 275(3):731-8), HIP-55, TGF.beta.-1 anti-apoptotic factor (Toomey, et al. Br J Biomed Sci 2001; 58(3): 177-83), tumor protein D52 (Bryne J. A., et al., Genomics, 35:523-532 (1996)), H1FT, NY-BR-1 (WO 01/47959), NY-BR-62, NY-BR-75, NY-B R-85, NY-BR-87, NY-BR-96 (Scanlan, M. Serologic and Bioinformatic Approaches to the Identification of Human Tumor Antigens, in Cancer Vaccines 2000, Cancer Research Institute, New York, N.Y.), BFA4 (SEQ ID NOS.: 23 and 24), BCY1 (SEQ ID NOS.: 25 and 26), BFA5 (SEQ ID NOS.: 27 and 28), BCZ4 (SEQ ID NOS.: 29 and 30), and BFY3 (SEQ ID NOS. 31 and 32), including "wild-type" (i.e., normally encoded by the genome, naturally-occurring), modified, and mutated versions as well, as other fragments and derivatives thereof. Any of these TAs may be utilized alone or in combination with one another in a co-immunization protocol.

[0028] In certain cases, it may be beneficial to co-immunize patients with both TA and other antigens, such as angiogenesis-associated antigens ("AA"). An AA is an immunogenic molecule (i.e., peptide, polypeptide) associated with cells involved in the induction and/or continued development of blood vessels. For example, an AA may be expressed on an endothelial cell ("EC"), which is a primary structural component of blood vessels. For treatment of cancer, it is preferred that that the AA be found within or near blood vessels that supply a tumor. Immunization of a patient against an AA preferably results in an anti-AA immune response whereby angiogenic processes that occur near or within tumors are prevented and/or inhibited.

[0029] Exemplary AAs include, for example, vascular endothelial growth factor (i.e., VEGF; Bernardini, et al. J. Urol., 2001, 166(4): 1275-9; Starnes, et al. J. Thorac. Cardiovasc. Surg., 2001, 122(3): 518-23; Dias, et al. Blood, 2002, 99: 2179-2184), the VEGF receptor (i.e., VEGF-R, flk-1/KDR; Starnes, et al. J. Thorac. Cardiovasc. Surg., 2001, 122(3): 518-23), EPH receptors (i.e., EPHA2; Gerety, et al. 1999, Cell, 4: 403-414), epidermal growth factor receptor (i.e., EGFR; Ciardeillo, et al. Clin. Cancer Res., 2001, 7(10): 2958-70), basic fibroblast growth factor (i.e., bFGF; Davidson, et al. Clin. Exp. Metastasis 2000, 18(6): 501-7; Poon, et al. Am J. Surg., 2001, 182(3):298-304), platelet-derived cell growth factor (i.e., PDGF-B), platelet-derived endothelial cell growth factor (PD-ECGF; Hong, et al. J. Mol. Med., 2001, 8(2):141-8), transforming growth factors (i.e., TGF-.alpha.; Hong, et al. J. Mol. Med., 2001, 8(2):141-8), endoglin (Balza, et al., Int. J. Cancer, 2001, 94: 579-585), Id proteins (Benezra, R. Trends Cardiovasc. Med., 2001, 11(6):237-41), proteases such as uPA, uPAR, and matrix metalloproteinases (MMP-2, MMP-9; Djonov, et al. J. Pathol., 2001, 195(2):147-55), nitric oxide synthase (Am. J. Ophthalmol., 2001, 132(4):551-6), aminopeptidase (Rouslhati, E. Nature Cancer, 2: 84-90, 2002), thrombospondins (i.e., TSP-1, TSP-2; Alvarez, et al. Gynecol. Oncol., 2001, 82(2):273-8; Seki, et al. Int. J. Oncot., 2001, 19(2):305-10), k-ras (Zhang, et al. Cancer Res., 2001, 61(16):6050-4), Wnt (Zhang, et al. Cancer Res., 2001, 61(16):6050-4), cyclin-dependent kinases (CDKs; Drug Resist. Updat. 2000, 3(2):83-88), microtubules (Timar, et al. 2001. Path. Oncol. Res., 7(2): 85-94), heat shock proteins (i.e., HSP90 (Timar, supra)), heparin-binding factors (i.e., heparinase; Gohji, et al. Int. J. Cancer, 2001, 95(5):295-301), synthases (i.e., ATP synthase, thymidilate synthase), collagen receptors, integrins (i.e., .alpha..upsilon..beta.3, .alpha..upsilon..beta.5, .alpha.1.beta.1, .alpha.2.beta.1, .alpha.5.beta.1), the surface proteolglycan NG2, AAC2-1 (SEQ ID NO.:1), or AAC2-2 (SEQ ID NO.:2), among others, including "wild-type" (i.e., normally encoded by the genome, naturally-occurring), modified, mutated versions as well as other fragments and derivatives thereof. Any of these targets may be suitable in practicing the present invention, either alone or in combination with one another or with other agents.

[0030] In certain embodiments, a nucleic acid molecule encoding an immunogenic target is utilized. The nucleic acid molecule may comprise or consist of a nucleotide sequence encoding one or more immunogenic targets, or fragments or derivatives thereof, such as that contained in a DNA insert in an ATCC Deposit. The term "nucleic acid sequence" or "nucleic acid molecule" refers to a DNA or RNA sequence. The term encompasses molecules formed from any of the known base analogs of DNA and RNA such as, but not limited to 4-acetylcytosine, 8-hydroxy-N-6-methyladenosine, aziridinyl-cytosine, pseudoisocytosine, 5-(carboxyhydroxylmethyl)uracil, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxy-methylaminomethyluracil, dihydrouracil, inosine, N6-iso-pentenyladenine, 1-methyladenine, 1-methylpseudouracil, 1-methylguanine, 1-methylinosine, 2,2-dimethyl-guanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyamino-methyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarbonyl-methyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, N-uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, and 2,6-diaminopurine, among others.

[0031] An isolated nucleic acid molecule is one that: (1) is separated from at least about 50 percent of proteins, lipids, carbohydrates, or other materials with which it is naturally found when total nucleic acid is isolated from the source cells; (2) is not linked to all or a portion of a polynucleotide to which the nucleic acid molecule is linked in nature; (3) is operably linked to a polynucleotide which it is not linked to in nature; and/or, (4) does not occur in nature as part of a larger polynucleotide sequence. Preferably, the isolated nucleic acid molecule of the present invention is to substantially free from any other contaminating nucleic acid molecule(s) or other contaminants that are found in its natural environment that would interfere with its use in polypeptide production or its therapeutic, diagnostic, prophylactic or research use. As used herein, the term "naturally occurring" or "native" or "naturally found" when used in connection with biological materials such as nucleic acid molecules, polypeptides, host cells, and the like, refers to materials which are found in nature without manipulation by man. Similarly, "non-naturally occurring" or "non-native" as used herein refers to a material that is not found in nature or that has been structurally modified or synthesized by man.

[0032] The identity of two or more nucleic acid or polypeptide molecules is determined by comparing the sequences. As known in the art, "identity" means the degree of sequence relatedness between nucleic acid molecules or polypeptides as determined by the match between the units making up the molecules (i.e., nucleotides or amino acid residues). Identity measures the percent of identical matches between the smaller of two or more sequences with gap alignments (if any) addressed by a particular mathematical model or computer program (i.e., an algorithm). Identity between nucleic acid sequences may also be determined by the ability of the related sequence to hybridize to the nucleic acid sequence or isolated nucleic acid molecule. In defining such sequences, the term "highly stringent conditions" and "moderately stringent conditions" refer to procedures that permit hybridization of nucleic acid strands whose sequences are complementary, and to exclude hybridization of significantly mismatched nucleic acids. Examples of "highly stringent conditions" for hybridization and washing are 0.015 M sodium chloride, 0.0015 M sodium citrate at 65-68.degree. C. or 0.015 M sodium chloride, 0.0015 M sodium citrate, and 50% formamide at 42.degree. C. (see, for example, Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory, 1989); Anderson et al., Nucleic Acid Hybridisation: A Practical Approach Ch. 4 (IRL Press Limited)). The term "moderately stringent conditions" refers to conditions under which a DNA duplex with a greater degree of base pair mismatching than could occur under "highly stringent conditions" is able to form. Exemplary moderately stringent conditions are 0.015 M sodium chloride, 0.0015 M sodium citrate at 50-65.degree. C. or 0.015 M sodium chloride, 0.0015 M sodium citrate, and 20% formamide at 37-50.degree. C. By way of example, moderately stringent conditions of 50.degree. C. in 0.015 M sodium ion will allow about a 21% mismatch. During hybridization, other agents may be included in the hybridization and washing buffers for the purpose of reducing non-specific and/or background hybridization. Examples are 0.1% bovine serum albumin, 0.1% polyvinyl-pyrrolidone, 0.1% sodium pyrophosphate, 0.1% sodium dodecylsulfate, NaDodSO.sub.4, (SDS), ficoll, Denhardt's solution, sonicated salmon sperm DNA (or another non-complementary DNA), and dextran sulfate, although other suitable agents can also be used. The concentration and types of these additives can be changed without substantially affecting the stringency of the hybridization conditions. Hybridization experiments are usually carried out at pH 6.8-7.4; however, at typical ionic strength conditions, the rate of hybridization is nearly independent of pH.

[0033] In certain embodiments of the present invention, vectors are used to transfer a nucleic acid sequence encoding a polypeptide to a cell. A vector is any molecule used to transfer a nucleic acid sequence to a host cell. In certain cases, an expression vector is utilized. An expression vector is a nucleic acid molecule that is suitable for transformation of a host cell and contains nucleic acid sequences that direct and/or control the expression of the transferred nucleic acid sequences. Expression includes, but is not limited to, processes such as transcription, translation, and splicing, if introns are present. Expression vectors typically comprise one or more flanking sequences operably linked to a heterologous nucleic acid sequence encoding a polypeptide. Flanking sequences may be homologous (i.e., from the same species and/or strain as the host cell), heterologous (i.e., from a species other than the host cell species or strain), hybrid (i.e., a combination of flanking sequences from more than one source), or synthetic, for example.

[0034] A flanking sequence is preferably capable of effecting the replication, transcription and/or translation of the coding sequence and is operably linked to a coding sequence. As used herein, the term operably linked refers to a linkage of polynucleotide elements in a functional relationship. For instance, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence. However, a flanking sequence need not necessarily be contiguous with the coding sequence, so long as it functions correctly. Thus, for example, intervening untranslated yet transcribed sequences can be present between a promoter sequence and the coding sequence and the promoter sequence may still be considered operably linked to the coding sequence. Similarly, an enhancer sequence may be located upstream or downstream from the coding sequence and affect transcription of the sequence.

[0035] In certain embodiments, it is preferred that the flanking sequence is a transcriptional regulatory region that drives high-level gene expression in the target cell. The transcriptional regulatory region may comprise, for example, a promoter, enhancer, silencer, repressor element or combinations thereof. The transcriptional regulatory region may be either constitutive, tissue-specific, cell-type specific (i.e., the region is drives higher levels of transcription in a one type of tissue or cell as compared to another), or regulatable (i.e., responsive to interaction with a compound). The source of a transcriptional regulatory region may be any prokaryotic or eukaryotic organism, any vertebrate or invertebrate organism, or any plant, provided that the flanking sequence functions in a cell by causing transcription of a nucleic acid within that cell. A wide variety of transcriptional regulatory regions may be utilized in practicing the present invention.

[0036] Suitable transcriptional regulatory regions include, for example, the CMV promoter (i.e., the CMV-immediate early promoter); promoters from eukaryotic genes (i.e., the estrogen-inducible chicken ovalbumin gene, the interferon genes, the gluco-corticoid-inducible tyrosine aminotransferase gene, and the thymidine kinase gene); and the major early and late adenovirus gene promoters; the SV40 early promoter region (Bernoist and Chambon, 1981, Nature 290:304-10); the promoter contained in the 3' long terminal repeat (LTR) of Rous sarcoma virus (RSV) (Yamamoto, et al., 1980, Cell 22:787-97); the herpes simplex virus thymidine kinase (HSV-TK) promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. U.S.A. 78:1444-45); the regulatory sequences of the metallothionine gene (Brinster et al., 1982, Nature 296:39-42); prokaryotic expression vectors such as the beta-lactamase promoter (Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. USA., 75:3727-31); or the tac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. U.S.A., 80:21-25). Tissue- and/or cell-type specific transcriptional control regions include, for example, the elastase I gene control region which is active in pancreatic acinar cells (Swift et al., 1984, Cell 38:639-46; Ornitz et al., 1986, Cold Spring Harbor Symp. Quant. Biol. 50:399-409 (1986); MacDonald, 1987, Hepatology 7:425-51.5); the insulin gene control region which is active in pancreatic beta cells (Hanahan, 1985, Nature 315:115-22); the immunoglobulin gene control region which is active in lymphoid cells (Grosschedl et al., 1984, Cell 38:647-58; Adames et al., 1985, Nature 318:533-38; Alexander et al., 1987, Mol. Cell. Biol., 7:1436-44); the mouse mammary tumor virus control region in testicular, breast, lymphoid and mast cells (Leder et al., 1986, Cell 45:485-95); the albumin gene control region in liver (Pinkert et al., 1987, Genes and Devel. 1:268-76); the alpha-feto-protein gene control region in liver (Krumlauf et al., 1985, Mol. Cell. Biol., 5:1639-48; Hammer et al., 1987, Science 235:53-58); the alpha 1-antitrypsin gene control region in liver (Kelsey et al., 1987, Genes and Devel. 1:161-71); the beta-globin gene control region in myeloid cells (Mogram et al., 1985, Nature 315:338-40; Kollias et al., 1986, Cell 46:89-94); the myelin basic protein gene control region in oligodendrocyte cells in the brain (Readhead et al., 1987, Cell 48:703-12); the myosin light chain-2 gene control region in skeletal muscle (Sani, 1985, Nature 314:283-86); the gonadotropic releasing hormone gene control region in the hypothalamus (Mason et al, 1986, Science 234:1372-78), and the tyrosinase promoter in melanoma cells (Hart, I. Semin Oncol 1996 February; 23(1):154-8; Siders, et al. Cancer Gene Ther 1998 September-October; 5(5):281-91), among others. Inducible promoters that are activated in the presence of a certain compound or condition such as light, heat, radiation, tetracycline, or heat shock proteins, for example, may also be utilized (see, for example, WO 00/10612). Other suitable promoters are known in the art.

[0037] As described above, enhancers may also be suitable flanking sequences. Enhancers are cis-acting elements of DNA, usually about 10-300 bp in length, that act on the promoter to increase transcription. Enhancers are typically orientation- and position-independent, having been identified both 5' and 3' to controlled coding sequences. Several enhancer sequences available from mammalian genes are known (i.e., globin, elastase, albumin, alpha-feto-protein and insulin).

[0038] Similarly, the SV40 enhancer, the cytomegalovirus early promoter enhancer, the polyoma enhancer, and adenovirus enhancers are useful with eukaryotic promoter sequences. While an enhancer may be spliced into the vector at a position 5' or 3' to nucleic acid coding sequence, it is typically located at a site 5' from the promoter. Other suitable enhancers are known in the art, and would be applicable to the present invention.

[0039] While preparing reagents of the present invention, cells may need to be transfected or transformed. Transfection refers to the uptake of foreign or exogenous DNA by a cell, and a cell has been transfected when the exogenous DNA has been introduced inside the cell membrane. A number of transfection techniques are well known in the art (i.e., Graham et al., 1973, Virology, 52:456; Sambrook et al., Molecular Cloning, A Laboratory Manual (Cold Spring Harbor Laboratories, 1989); Davis et al., Basic Methods in Molecular Biology (Elsevier, 1986); and Chu et al., 1981, Gene 13:197). Such techniques can be used to introduce one or more exogenous DNA moieties into suitable host cells.

[0040] In certain embodiments, it is preferred that transfection of a cell results in transformation of that cell. A cell is transformed when there is a change in a characteristic of the cell, being transformed when it has been modified to contain a new nucleic acid. Following transfection, the transfected nucleic acid may recombine with that of the cell by physically integrating into a chromosome of the cell, may be maintained transiently as an episomal element without being replicated, or may replicate independently as a plasmid. A cell is stably transformed when the nucleic acid is replicated with the division of the cell.

[0041] The present invention further provides isolated immunogenic targets in polypeptide form. A polypeptide is considered isolated where it: (1) has been separated from at least about 50 percent of polynucleotides, lipids, carbohydrates, or other materials with which it is naturally found when isolated from the source cell: (2) is not linked (by covalent or noncovalent interaction) to all or a portion of a polypeptide to which the "isolated poly peptide" is linked in nature; (3) is operably linked (by covalent or noncovalent interaction) to a polypeptide with which it is not linked in nature; or, (4) does not occur in nature. Preferably, the isolated polypeptide is substantially free from any other contaminating polypeptides or other contaminants that are found in its natural environment that would interfere with its therapeutic, diagnostic, prophylactic or research use.

[0042] Immunogenic target polypeptides may be mature polypeptides, as defined herein, and may or may not have an amino terminal methionine residue, depending on the method by which they are prepared. Further contemplated are related polypeptides such as, for example, fragments, variants (i.e., allelic, splice), orthologs, homologues, and derivatives, for example, that possess at least one characteristic or activity (i.e., activity, antigenicity) of the immunogenic target. Also related are peptides, which refers to a series of contiguous amino acid residues having a sequence corresponding to at least a portion of the polypeptide from which its sequence is derived. In preferred embodiments, the peptide comprises about 5-10 amino acids, 10-15 amino acids, 15-20 amino acids, 20-30 amino acids, or 30-50 amino acids. In a more preferred embodiment, a peptide comprises 9-12 amino acids, suitable for presentation upon Class I MHC molecules, for example.

[0043] A fragment of a nucleic acid or polypeptide comprises a truncation, of the sequence (i.e., nucleic acid or polypeptide) at the amino terminus (with or without a leader sequence) and/or the carboxy terminus. Fragments may also include variants (i.e., allelic, splice), orthologs, homologues, and other variants having one or more amino acid additions or substitutions or internal deletions as compared to the parental sequence. In preferred embodiments, truncations and/or deletions comprise about 10 amino acids, 20 amino acids, 30 amino acids, 40 amino acids, 50 amino acids, or more. The polypeptide fragments so produced will comprise about 10 amino acids, 25 amino acids, 30 amino acids, 40 amino acids, 50 amino acids, 60 amino acids, 70 amino acids, or more. Such polypeptide fragments may optionally comprise an amino terminal methionine residue, it will be appreciated that such fragments can be used, for example, to generate antibodies or cellular immune responses to immunogenic target polypeptides.

[0044] A variant is a sequence having one or more sequence substitutions, deletions, and/or additions as compared to the subject sequence. Variants may be naturally occurring or artificially constructed. Such variants may be prepared from the corresponding nucleic acid molecules. In preferred embodiments, the variants have from 1 to 3, or from 1 to 5, or from 1 to 10, or from 1 to 15, or from 1 to 20, or from 1 to 25, or from 1 to 30, or from 1 to 40, or from 1 to 50, or more than 50 amino acid substitutions, insertions, additions and/or deletions.

[0045] An allelic variant is one of several possible naturally-occurring alternate forms of a gene occupying a given locus on a chromosome of an organism or a population of organisms. A splice variant is a polypeptide generated from one of several RNA transcript resulting from splicing of a primary transcript. An ortholog is a similar nucleic acid or polypeptide sequence from another species. For example, the mouse and human versions of an immunogenic target polypeptide may be considered orthologs of each other. A derivative of a sequence is one that is derived from a parental sequence those sequences having substitutions, additions, deletions, or chemically modified variants.

[0046] Variants may also include fusion proteins, which refers to the fusion of one or more first sequences (such as a peptide) at the amino or carboxy terminus of at least one other sequence (such as a heterologous peptide).

[0047] "Similarity" is a concept related to identity, except that similarity refers to a measure of relatedness which includes both identical matches and conservative substitution matches. If two polypeptide sequences have, for example, 10/20 identical amino acids, and the remainder are all non-conservative substitutions, then the percent identity and similarity would both be 50%. If in the same example, there are five more positions where there are conservative substitutions, then the percent identity remains 50%, but the percent similarity would be 75% ( 15/20). Therefore, in cases where there are conservative substitutions, the percent similarity between two polypeptides will be higher than the percent identity between those two polypeptides.

[0048] Substitutions may be conservative, or non-conservative, or any combination thereof. Conservative amino acid modifications to the sequence of a polypeptide (and the corresponding modifications to the encoding nucleotides) may produce polypeptides having functional and chemical characteristics similar to those of a parental polypeptide. For example, a "conservative amino acid substitution" may involve a substitution of a native amino acid residue with a non-native residue such that there is little or no effect on the size, polarity, charge, hydrophobicity, or hydrophilicity of the amino acid residue at that position and, in particular, does not result in decreased immunogenicity. Suitable conservative amino acid substitutions are shown in Table I.

TABLE-US-00001 TABLE I Original Preferred Residues Exemplary Substitutions Substitutions Ala Val, Leu, Ile Val Arg Lys, Gln, Asn Lys Asn Gln Gln Asp Glu Glu Cys Ser, Ala Ser Gln Asn Asn Glu Asp Asp Gly Pro, Ala Ala His Asn, Gln, Lys, Arg Arg Ile Leu, Val, Met, Ala, Phe, Norleucine Leu Leu Norleucine, Ile, Val, Met, Ala, Phe Ile Lys Arg, 1,4 Diamino-butyric Acid, Gln, Asn Arg Met Leu, Phe, Ile Leu Phe Leu, Val, Ile, Ala, Tyr Leu Pro Ala Gly Ser Thr, Ala, Cys Thr Thr Ser Ser Trp Tyr, Phe Tyr Tyr Trp, Phe, Thr, Ser Phe Val Ile, Met, Leu, Phe, Ala, Norleucine Leu

[0049] A skilled artisan will be able to determine suitable variants of polypeptide using well-known techniques. For identifying suitable areas of the molecule that may be changed without destroying biological activity (i.e., MHC binding, immunogenicity), one skilled in the art may target areas not believed to be important for that activity. For example, when similar polypeptides with similar activities from the same species or from other species are known, one skilled in the art may compare the amino acid sequence of a polypeptide to such similar polypeptides. By performing such analyses, one can identify residues and portions of the molecules that are conserved among similar polypeptides. It will be appreciated that changes in areas of the molecule that are not conserved relative to such similar polypeptides would be less likely to adversely affect the biological activity and/or structure of a polypeptide. Similarly, the residues required for binding to MHC are known, and may be modified to improve binding. However, modifications resulting in decreased binding to MHC will not be appropriate in most situations. One skilled in the art would also know that, even in relatively conserved regions, one may substitute chemically similar amino acids for the naturally occurring residues while retaining activity. Therefore, even areas that may be important for biological activity or for structure may be subject to conservative amino acid substitutions without destroying the biological activity or without adversely affecting the polypeptide structure.

[0050] Other preferred polypeptide variants include glycosylation variants wherein the number and/or type of glycosylation sites have been altered compared to the subject amino acid sequence. In one embodiment, polypeptide variants comprise a greater or a lesser number of N-linked glycosylation sites than the subject amino acid sequence. An N-linked glycosylation site is characterized by the sequence Asn-X-Ser or Asn-X-Thr, wherein the amino acid residue designated as X may be any amino acid residue except proline. The substitution of amino acid residues to create this sequence provides a potential new site for the addition of an N-linked carbohydrate chain. Alternatively, substitutions that eliminate this sequence will remove an existing N-linked carbohydrate chain. Also provided is a rearrangement of N-linked carbohydrate chains wherein one or more N-linked glycosylation sites (typically those that are naturally occurring) are eliminated and one or more new N-linked sites are created. To affect O-linked glycosylation of a polypeptide, one would modify serine and/or threonine residues.

[0051] Additional preferred variants include cysteine variants, wherein one or more cysteine residues are deleted or substituted with another amino acid (e.g., serine) as compared to the subject amino acid sequence set. Cysteine variants are useful when polypeptides must be refolded into a biologically active conformation such as after the isolation of insoluble inclusion bodies, Cysteine variants generally have fewer cysteine residues than the native protein, and typically have an even number to minimize interactions resulting from unpaired cysteines.

[0052] In other embodiments, the isolated polypeptides of the current invention include fusion polypeptide segments that assist in purification of the polypeptides. Fusions can be made either at the amino terminus or at the carboxy terminus of the subject polypeptide variant thereof. Fusions may be direct with no linker or adapter molecule or may be through a linker or adapter molecule. A linker or adapter molecule may be one or more amino acid residues, typically from about 20 to about 50 amino acid residues. A linker or adapter molecule may also be designed with a cleavage site for a DNA restriction endonuclease or for a protease to allow for the separation of the fused moieties. It will be appreciated that once constructed, the fusion polypeptides can be derivatized according to the methods described herein. Suitable fusion segments include, among others, metal binding domains (e.g., a poly-histidine segment), immunoglobulin binding domains (i.e., Protein A, Protein G, T cell, B cell, Fc receptor, or complement protein antibody-binding domains), sugar binding domains (e.g., a maltose binding domain), and/or a "tag" domain (i.e., at least a portion of .alpha.-galactosidase, a strep tag peptide, a T7 tag peptide, a FLAG peptide, or other domains that can be purified using compounds that bind to the domain, such as monoclonal antibodies). This tag is typically fused to the polypeptide upon expression of the polypeptide, and can serve as a means for affinity purification of the sequence of interest polypeptide from the host cell. Affinity purification can be accomplished, for example, by column chromatography using antibodies against the tag as an affinity matrix. Optionally, the tag can subsequently be removed from the purified sequence of interest polypeptide by various means such as using certain peptidases for cleavage. As described below, fusions may also be made between a TA and a co-stimulatory components such as the chemokines CXC10 (IP-10), CCL7 (MCP-3), or CCL5 (RANTES), for example.

[0053] A fusion motif may enhance transport of an immunogenic target to an MHC processing compartment, such as the endoplasmic reticulum. These sequences, referred to as transduction or transcytosis sequences, include sequences derived from HIV tat (see Kim et al. 1997 J. Immunol. 159:1666), Drosophila antennapedia (see Schutze-Redelmeier et al. 1996 J. Immunol. 157:650), or human period-1 protein (hPER1; in particular, SRRHHCRSKAKRSHH (SEQ ID NO: 42)).

[0054] In addition, the polypeptide or variant thereof may be fused to a homologous polypeptide to form a homodimer or to a heterologous polypeptide to form a heterodimer. Heterologous peptides and polypeptides include, but are not limited to: an epitope to allow for the detection and/or isolation of a fusion polypeptide; a transmembrane receptor protein or a portion thereof, such as an extracellular domain or a transmembrane and intracellular domain; a ligand or a portion thereof which binds to a transmembrane receptor protein; an enzyme or portion thereof which is catalytically active; a polypeptide or peptide which promotes oligomerization, such as a leucine zipper domain; a polypeptide or peptide which increases stability, such as an immunoglobulin constant region; and a polypeptide which has a therapeutic activity different from the polypeptide or variant thereof.

[0055] In certain embodiments, it may be advantageous to combine a nucleic acid sequence encoding an immunogenic target, polypeptide, or derivative thereof with one or more co-stimulator component(s) such as cell surface proteins, cytokines or chemokines in a composition of the present invention. The co-stimulatory component may be included in the composition as a polypeptide or as a nucleic acid encoding the polypeptide, for example. Suitable co-stimulatory molecules include, for instance, polypeptides that bind members of the CD28 family (i.e., CD28, ICOS; Hutloff et al. Nature 1999, 397: 263-265: Peach, et al. J. Exp Med 1994, 180: 2049-2058) such as the CD28 binding polypeptides B7.1 (CD80; Schwartz, 1992; Chen et al. 1992; Ellis, et al., J. Immunol., 156(8): 2700-9) and B7.2 (CD86; Ellis, et al., J. Immunol., 156(8): 2700-9); polypeptides which bind members of the integrin family (i.e., LFA-1 (CD11a/CD8); Sedwick, et al. J Immunol 1999, 162: 1367-1375; Wulfing, et al. Science 1998, 282: 2266-2269; Lub, et al. Immunol Today 1995, 16: 479-483) including members of the ICAM family (i.e., ICAM-1, -2 or -3); polypeptides which bind CD2 family members (i.e., CD2, signalling lymphocyte activation molecule (CDw150 or "SLAM"; Aversa, et al. J Immunol 1997, 158: 4036-4044)) such as CD58 (LFA-3; CD2 ligand; Davis, et al. Immunol Today 1996, 17: 177-187) or SLAM ligands (Sayos, et al. Nature 1998, 395: 462-469); polypeptides which bind heat stable antigen (HSA or CD24; Zhou, et al. Eur J. Immunol 1997, 27: 2524-2528); polypeptides which bind to members of the TNF receptor (TNFR) family (i.e., 4-IBB (CD137; Vinay, et al. Semin Immunol 1998, 10: 481-489), OX40 (CD-134; Weinberg, et al. Semin Immunol 1998, 10: 471-480; Higgins, et al. J Immunol 1999, 162: 486-493), and CD27 (Lens, et al. Semin Immunol 1998, 10: 491-499)) such as 4-IBBL (4-IBB ligand; Vinay, et al. Semin Immunol 1998, 10: 481-48; DeBenedette et al. J Immunol 1997, 158: 551-559), TNFR associated factor-1 (TRAF-1; 4-IBB ligand; Saoulli, et al. J Exp Med 1998, 187: 1849-1862, Arch, et al. Mol Cell Biol 1998, 18: 558-565), TRAF-2 (4-IBB and OX40 ligand; Saoulli, et al. J Exp Med 1998, 187: 1849-1862: Oshima, et al. Int Immunol 1.998, 10: 517-526, Kawamata, et al. J Biol Chem 1.998, 273: 5808-5814), TRAF-3 (4-IBB and OX40 ligand; Arch, et al. Mol Cell Biol 1998, 18: 558-565; Jang, et al. Biochem Biophys Res Commun 1998, 242: 613-620; Kawamata S, et al. J Biol Chem 1998, 273: 5808-5814), OX40L (OX40 ligand; Gramaglia, et al. J Immunol 1998, 161: 6510-6517), TRAF-5 (OX40 ligand; Arch, et al. Mol Cell Biol 1998, 18: 558-565; Kawamata, et al. J Biol Chem 1998, 273: 5808-5814), and CD70 (CD27 ligand; Couderc, et al. Cancer Gene Ther., 5(3): 163-75). CD154 (CD40 ligand or "CD40L"; Gurunathan, et al. J. Immunol. 1998, 161: 4563-4571; Sine, et al. Hum. Gene Ther., 2001, 12: 1091-1102) may also be suitable.

[0056] One or more cytokines may also be suitable co-stimulatory components or "adjuvants", either as polypeptides or being encoded by nucleic acids contained within the compositions of the present invention (Parmiani, et al. Immunol Lett 2000 Sep. 15; 74(1): 41-4; Berzofsky, et al. Nature Immunol. 1: 209-219). Suitable cytokines include, for example, interleukin-2 (IL-2) (Rosenberg, et al. Nature Med. 4: 321-327 (1998)), IL-4, IL-7, IL-12 (reviewed by Pardoll, 1992; Harries, et al. T. Gene Med. 2000 July-August; 2(4):243-9; Rao, et al. J. Immunol. 156: 3357-3365 (1996)), IL-15 (Xin, et al. Vaccine, 17:858-866, 1999), IL-16 (Cruikshank, et al. J. Leuk Biol. 67(6): 757-66, 2000), IL-18 (J. Cancer Res. Clin. Oncol. 2001. 127(12): 718-726), GM-CSF (CSF (Disis, et al. Blood, 88: 202-210 (1996)), tumor necrosis factor-alpha (TNF-.alpha.), or interferons such as IFN-.alpha. or INF-.gamma.. Other cytokines may also be suitable for practicing the present invention, as is known in the art.

[0057] Chemokines may also be utilized. For example, fusion proteins comprising CXCL10 (IP-10) and CCL7 (MCP-3) fused to a tumor self-antigen have been shown to induce anti-tumor immunity (Biragyn, et al. Nature Biotech. 1999, 17: 253-258). The chemokines CCL3 (MIP-1.alpha.) and CCL5 (RANTES) (Boyer, et al. Vaccine, 1999, 17 (Supp. 2): S53-S64) may also be of use in practicing the present invention. Other suitable chemokines are known in the art.

[0058] It is also known in the art that suppressive or negative regulatory immune mechanisms may be blocked, resulting in enhanced immune responses. For instance, treatment with anti-CTLA-4 (Shrikant, et al. Immunity, 1996, 14: 145-155; Sutmuller, et al., J Exp. Med., 2001, 194: 823-832), anti-CD25 (Sutmuller, supra), anti-CD4 (Matsui, et al. J. Immunol., 1999, 163: 184-193), the fusion protein IL13Ra2-Fc (Terabe, et al. Nature Immunol., 2000, 1: 515-520), and combinations thereof (i.e., anti-CTLA-4 and anti-CD25, Sutmuller, supra) have been shown to upregulate anti-tumor immune responses and would be suitable in practicing the present invention.

[0059] Any of these components may be used alone or in combination with other agents. For instance, it has been shown that a combination of CD80, ICAM-1 and LFA-3 ("TRICOM") may potentiate anti-cancer immune responses (Hodge, et al. Cancer Res. 59: 5800-5807 (1999). Other effective combinations include, for example, IL-12+GM-CSF (Alers, et al. J. Immunol., 158: 3947-3958 (1997); Iwasaki, et al. J. Immunol. 158: 4591-4601 (1997)), IL-12-GM-CSF+TNF-.alpha. (Ahlers, et al. Int. Immunol. 13: 897-908 (2001)), CD80+IL-12 (Fruend, et al. Int. J. Cancer, 85: 508-517 (2000); Rao, et al. supra), and CD86+GM-CSF+IL-12 (Iwasaki, supra). One of skill in the art would be aware of additional combinations useful in carrying out the present invention. In addition, the skilled artisan would be aware of additional reagents or methods that may be used to modulate such mechanisms. These reagents and methods, as well as others known by those of skill in the art, may be utilized in practicing the present invention.

[0060] Additional strategies for improving the efficiency of nucleic acid-based immunization may also be used including, for example, the use of self-replicating viral replicons (Caley, et alt 1999, Vaccine, 17: 3124-2135; Dubensky, et al. 2000. Mol. Med. 6: 723-732; Leitner, et al. 2000. Cancer Res. 60: 51-55), codon optimization (Liu et al 2000. Mol Ther., 1: 497-500; Dubensky, supra; Huang, et al 2001. J. Virol. 75: 4947-4951), in vivo electroporation (Widera, et al. 2000. J. Immunol. 164: 4635-3640), incorporation of CpG stimulatory motifs (Gurunathan, et al. Ann. Rev. Immunol., 2000, 18: 927-974; Leitner, stpra; Cho, et al J. Immunol. 168(10):4907-13), sequences for targeting of the endocytic or tubiquitin-processing pathways (Thomson, et al. 1998, J. Virol. 72: 2246-2252; Velders, et al. 2001. J. Immunol. 166: 5366-5373), Marek's disease virus type 1 VP22 sequences (J. Virol. 76(61):2676-82, 2002), prime-boost regimens (Gurunathan, supra; Sullivan, et al. 2000. Nature, 408: 605-609; Hanke, et al. 1998. Vaccine, 16: 439-445; Amara, et al. 2001. Science, 292: 69-74), and the use of mucosal delivery vectors such as Salmonella (Darji, et al. 1997. Cell, 91: 765-775; Woo, et al. 2001. Vaccine, 19: 2945-2954). Other methods are known in the art, some of which are described below.

[0061] Chemotherapeutic agents, radiation, anti-angiogenic compounds, or other agents may also be utilized in treating and/or preventing cancer using immunogenic targets (Sebti, et alt Oncogene 2000 Dec. 27; 19(56):6566-73). For example, in treating metastatic breast cancer, useful chemotherapeutic agents include cyclophosphamide, doxorubicin, paclitaxel, docetaxel, navelbine, capecitabine, and mitomycin C, among others. Combination chemotherapeutic regimens have also proven effective including cyclophosphamide+methotrexate+5-fluorouracil; cyclophosphamide+doxorubicin-5-fluorouracil; or, cyclophosphamide+doxorubicin, for example. Other compounds such as prednisone, a taxane, navelbine, mitomycin C, or vinblastine have been utilized for various reasons. A majority of breast cancer patients have estrogen-receptor positive (ER+) tumors and in these patients, endocrine therapy (i.e., tamoxifen) is preferred over chemotherapy. For such patients, tamoxifen or, as a second line therapy, progestins (medroxyprogesterone acetate or megestrol acetate) are preferred. Aromatase inhibitors (i.e., aminoglutethimide and analogs thereof such as letrozole) decrease the availability of estrogen needed to maintain tumor growth and may be used as second or third line endocrine therapy in certain patients.

[0062] Other cancers may require different chemotherapeutic regimens. For example, metastatic colorectal cancer is typically treated with Camptosar (irinotecan or CPT-11), 5-fluorouracil or leucovorin, alone or in combination with one another. Proteinase and integrin inhibitors such as the MMP inhibitors marimastate (British Biotech), COL-3 (Collagenex), Neovastat (Aeterna), AG3340 (Agouron), BMS-275291 (Bristol Myers Squibb), CGS 27023A (Novartis) or the integrin inhibitors Vitaxin (Medimmune), or MED1522 (Merck KgaA) may also be suitable for use. As such, immunological targeting of immunogenic targets associated with colorectal cancer could be performed in combination with a treatment using those chemotherapeutic agents. Similarly, chemotherapeutic agents used to treat other types of cancers are well-known in the art and may be combined with the immunogenic targets described herein.

[0063] Many anti-angiogenic agents are known in the art and would be suitable for co-administration with the immunogenic target vaccines (see, for example, Timar, et al. 2001. Pathology Oncol. Res., 7(2): 85-94). Such agents include, for example, physiological agents such as growth factors (i.e., ANG-2, NK1,2,4 (HGF) transforming growth factor beta (TGF-.beta.)), cytokines (i.e., interferons such as IFN-.alpha., -.beta., -.gamma., platelet factor 4 (PF-4), PR-39), proteases (i.e., cleaved AT-III, collagen XVIII fragment (Endostatin)), HmwKallikrein-d5 plasmin fragment (Angiostatin), prothrombin-F1-2, TSP-1), protease inhibitors (i.e., tissue inhibitor of metalloproteases such as TIMP-1, -2, or -3; maspin; plasminogen activator-inhibitors such as PAI-1; pigment epithelium derived factor (PEDF)), Tumstatin (available through ILEX, Inc.), antibody products (i.e., the collagen-binding antibodies HUIV26, HU177, XL313; anti-VEGF; anti-integrin (i.e., Vitaxin, (Lxsys))), and glycosidases (i.e., heparinase-I, -III). "Chemical" or modified physiological agents known or believed to have anti-angiogenic potential include, for example, viablastine, taxol, ketoconazole, thalidomide, dolestatin, combrestatin A, rapamycin (Guba, et al. 2002, Nature Med., 8: 128-135), CEP-7055 (available from Cephalon, Inc.), flavone acetic acid, Bay 12-9566 (Bayer Corp.), AG3340 (Agouron, Inc.), CGS 27023A (Novartis), tetracylcine derivatives (i.e., COL-3 (Collagenix, Inc.)), Neovastat (Aeterna). BMS-275291 (Bristol-Myers Squibb), low dose 5-FU, low dose methotrexate (MTX), irsofladine, radicicol, cyclosporine, captopril, celecoxib, D45152-sulphated polysaccharide, cationic protein (Protamine), cationic peptide-VEGF, Suramin (polysulphonated napthyl urea), compounds that interfere with the function or production of VEGF (i.e., SU5416 or SU6668 (Sugen) PTK787/ZK22584 (Novartis)), Distamycin A, Angiozyme (ribozyme), isoflavinoids, staurosporine derivatives, genistein, EMD121974 (Merck KcgaA), tyrphostins, isoquinolones, retinoic acid, carboxyamidotriazole, TNP-470, octreotide, 2-methoxyestradiol, aminosterols (i.e., squalamine), glutathione analogues (i.e., N-acteyl-L-cysteine), combretastatin A-4 (Oxigene), Eph receptor blocking agents (Nature 414:933-938, 2001), Rh-Angiostatin. Rh-Endostatin (WO 01/93897), cyclic-RGD peptide, accutin-disintegrin, benzodiazepenes, humanized anti-avb3 Ab, Rh-PAI-2, amiloride, p-amidobenzamidine, anti-uPA ab, anti-uPAR Ab L-phanylalanin-N-methyl amides (i.e., Batimistat, Marimastat), AG3340, and minocycline. Many other suitable agents are known in the art and would suffice in practicing the present invention.

[0064] The present invention may also be utilized in combination with "non-traditional" methods of treating cancer. For example, it has recently been demonstrated that administration of certain anaerobic bacteria may assist in slowing tumor growth. In one study, Clostridium novyi was modified to eliminate a toxin gene carried on a phage episome and administered to mice with colorectal tumors (Dang, et al. P.N.A.S. USA, 98(26): 15155-15160, 2001). In combination with chemotherapy, the treatment was shown to cause tumor necrosis in the animals. The reagents and methodologies described in this application may be combined with such treatment methodologies.

[0065] Nucleic acids encoding immunogenic targets may be administered to patients by any of several available techniques. Various viral vectors that have been successfully utilized for introducing a nucleic acid to a host include retrovirus, adenovirus, adeno-associated virus (AAV), herpes virus, and poxvirus, among others. It is understood in the art that many such viral vectors are available in the art. The vectors of the present invention may be constructed using standard recombinant techniques widely available to one skilled in the art. Such techniques may be found in common molecular biology references such as Molecular Cloning: A Laboratory Manual (Sambrook, et al., 1989, Cold Spring Harbor Laboratory Press), Gene Expression Technology (Methods in Enzymology, Vol. 185, edited by D. Goeddel, 1991. Academic Press, San Diego, Calif.), and PCR Protocols: A Guide to Methods and Applications (Innis, et al. 1990. Academic Press, San Diego, Calif.).

[0066] Preferred retroviral vectors are derivatives of lentivirus as well as derivatives of murine or avian retroviruses. Examples of suitable retroviral vectors include, for example, Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), SIV, BIV, HIV and Rous Sarcoma Virus (RSV). A number of retroviral vectors can incorporate multiple exogenous nucleic acid sequences. As recombinant retroviruses are defective, they require assistance in order to produce infectious vector particles. This assistance can be provided by, for example, helper cell lines encoding retrovirus structural genes. Suitable helper cell lines include .PSI.2, PA317 and PA12, among others. The vector virions produced using such cell lines may then be used to infect a tissue cell line, such as NIH 3T3 cells, to produce large quantities of chimeric retroviral virions. Retroviral vectors may be administered by traditional methods (i.e., infection) or by implantation of a "producer cell line" in proximity to the target cell population (Culver, K., et al., 1994, Hum. Gene Ther., 5 (3): 343-79; Culver, K., et al, Cold Spring Harb. Symp. Quant. Biol., 59: 685-90); Oldfield, E., 1993, Hum. Gene Ther., 4 (1): 39-69). The producer cell line is engineered to produce a viral vector and releases viral particles in the vicinity of the target cell. A portion of the released viral particles contact the target cells and infect those cells, thus delivering a nucleic acid of the present invention to the target cell. Following infection of the target cell, expression of the nucleic acid of the vector occurs.

[0067] Adenoviral vectors have proven especially useful for gene transfer into eukaryotic cells (Rosenfeld, M., et al, 1991, Science, 252 (5004): 431-4; Crystal, R., et al., 1994, Nat. Genet., 8 (1): 42-51), the study eukaryotic gene expression (Levrero, M., et al., 1991, Gene, 101 (2): 195-202), vaccine development (Graham, F. and Prevec, L., 1992, Biotechnology, 20: 363-90), and in animal models (Stratford-Perricaudet, L., et al., 1992, Bone Marrow Transplant., 9 (Suppl. 1): 151-2; Rich, D., et al., 1993, Hum. Gene Ther., 4 (4): 461-76). Experimental routes for administrating recombinant Ad to different tissues in viro have included intratracheal instillation (Rosenfeld, M., et al., 1992, Cell, 68 (1): 143-55) injection into muscle (Quantin, B., et al., 1992, Proc. Natl. Acad Sci. U.S.A., 89 (7): 2581-4), peripheral intravenous injection (Herz, J., and Gerard, R., 1993, Proc. Natl. Acad. Sci. U.S.A., 90 (7): 2812-6) and stereotactic inoculation to brain (Le Gal La Salle, G., et al., 1993, Science, 259 (5097): 988-90), among others.

[0068] Adeno-associated virus (AAV) demonstrates high-level infectivity, broad host range and specificity in integrating into the host cell genome (Hermonat, P., et al., 1984, Proc. Natl. Acad. Sci. U.S.A., 81 (20): 6466-70). And Herpes Simplex Virus type-1 (HSV-1) is vet another attractive vector system, especially for use in the nervous system because of its neurotropic property (Geller, A., et al., 1991, Trends Neurosci., 14 (10): 428-32; Glorioso, et al., 1995, Mol. Biotechnol., 4 (1): 87-99; Glorioso, et al., 1995, Annu. Rev. Microbiol., 49: 675-710).

[0069] Poxvirus is another useful expression vector (Smith, et al. 1983, Gene, 25 (1): 21-8: Moss, et al, 1992, Biotechnology, 20: 345-62; Moss, et al, 1992, Curr. Top. Microbiol. Immunol., 158: 25-38: Moss, et al. 1991. Science, 252: 1662-1667). Poxviruses shown to be useful include vaccinia, NYVAC, avipox, fowlpox, canarypox, ALVAC, and ALVAC(2), among others.

[0070] NYVAC (vP866) was derived from the Copenhagen vaccine strain of vaccinia virus by deleting six nonessential regions of the genome encoding known or potential virulence factors (see, for example, U.S. Pat. Nos. 5,364,773 and 5,494,807). The deletion loci were also engineered as recipient loci for the insertion of foreign genes. The deleted regions are: thymidine kinase gene (TK; J2R); hemorrhagic region (u; B13R+B14R); A type inclusion body region (ATI; A26L); hemagglutinin gene (HA; A56R); host range gene region (C7L-K1L); and, large subunit, ribonucleotide reductase (I4L). NYVAC is a genetically engineered vaccinia virus strain that was generated by the specific deletion of eighteen open reading frames encoding gene products associated with virulence and host range. NYVAC has been show to be useful for expressing TAs (see, for example, U.S. Pat. No. 6,265,189). NYVAC (vP866), vP994, vCP205, vCP1433, placZH6H4Lreverse, pMPC6H6K3E3 and pC3H6FHVB were also deposited with the ATCC under the terms of the Budapest Treaty, accession numbers VR-2559, VR-2558, VR-2557, VR-2556, ATCC-97913, ATCC-97912, and ATCC-97914, respectively.

[0071] ALVAC-based recombinant viruses (i.e., ALVAC-1 and ALVAC-2) are also suitable for use in practicing the present invention (see, for example, U.S. Pat. No. 5,756,103). ALVAC(2) is identical to ALVAC(1) except that ALVAC(2) genome comprises the vaccinia E3L and K3L genes under the control of vaccinia promoters (U.S. Pat. No. 6,130,066; Beattie et al., 1995a, 1995b, 1991; Chang et al., 1992; Davies et al., 1993). Both ALVAC(1) and ALVAC(2) have been demonstrated to be useful in expressing foreign DNA sequences, such as TAs (Tartaglia et al., 1993 a,b; U.S. Pat. No. 5,833,975). ALVAC was deposited under the terms of the Budapest Treaty with the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, Va. 20110-2209, USA, ATCC accession number VR-2547.

[0072] Another useful poxvirus vector is TROVAC. TROVAC refers to an attenuated fowlpox that was a plaque-cloned isolate derived from the FP-1 vaccine strain of fowlpoxvirus which is licensed for vaccination of 1 day old chicks. TROVAC was likewise deposited under the terms of the Budapest Treaty with the ATCC, accession number 2553.

[0073] "Non-viral" plasmid vectors may also be suitable in practicing the present invention. Preferred plasmid vectors are compatible with bacterial, insect, and/or mammalian host cells. Such vectors include, for example, PCR-II, pCR3, and pcDNA3.1 (Invitrogen, San Diego, Calif.), pBSII (Stratagene, La Jolla, Calif.), pET15 (Novagen, Madison, Wis.), pGEX (Pharmacia Biotech, Piscataway, N.J.), pEGFP-N2 (Clontech. Palo Alto, Calif.), pETL (BlueBacII, Invitrogen), pDSR-alpha (PCT pub. No. WO 90/14363) and pFastBacDual (Gibco-BRL, Grand Island, N.Y.) as well as Bluescript.RTM. plasmid derivatives (a high copy, number COLE1-based phagemid, Stratagene Cloning Systems, La Jolla, Calif.), PCR cloning plasmids designed for cloning Taq-amplified PCR products (e.g., TOPO.TM. TA Cloning.RTM. kit, PCR2.1.RTM. plasmid derivatives, invitrogen, Carlsbad, Calif.). Bacterial vectors may also be used with the current invention. These vectors include, for example, Shigella, Salmonella, Vibrio cholerae, Lactobacillus, Bacille calmette guerin (BCG) and Streptococcus (see for example, WO 88/6626; WO 90/0594; WO 91/13157; WO 92/1796; and WO 92/21376). Many other non-viral plasmid expression vectors and systems are known in the art and could be used with the current invention.

[0074] Suitable nucleic acid delivery techniques include DNA-ligand complexes, adenovirus-ligand-DNA complexes, direct injection of DNA, CaPO.sub.4 precipitation, gene gun techniques, electroporation, and colloidal dispersion systems, among others. Colloidal dispersion systems include macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. The preferred colloidal system of this invention is a liposome, which are artificial membrane vesicles useful as delivery vehicles in vitro and in vivo, RNA, DNA and intact virions can be encapsulated within the aqueous interior and be delivered to cells in a biologically active form (Fraley, R., et al., 1981, Trends Biochem. Sci., 6: 77). The composition of the liposome is usually a combination of phospholipids, particularly high-phase-transition-temperature phospholipids, usually in combination with steroids, especially cholesterol. Other phospholipids or other lipids may also be used. The physical characteristics of liposomes depend on pH, ionic strength, and the presence of divalent cations. Examples of lipids useful in liposome production include phosphatidyl compounds, such as phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides. Particularly useful are diacylphosphatidylglycerols, where the lipid moiety contains from 14-18 carbon atoms, particularly from 16-18 carbon atoms, and is saturated. Illustrative phospholipids include egg phosphatidylcholine, dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine.

[0075] An immunogenic target may also be administered in combination with one or more adjuvants to boost the immune response. Exemplary adjuvants are shown in Table II below:

TABLE-US-00002 TABLE II Types of Immunologic Adjuvants Type of Adjuvant General Examples Specific Examples/References Gel-type Aluminum hydroxide/phosphate ("alum (Aggerbeck and Heron, 1995) adjuvants") Calcium phosphate (Relyveld, 1986) Microbial Muramyl dipeptide (MDP) (Chedid et al., 1986) Bacterial exotoxins Cholera toxin (CT), E. coli labile toxin (LT)(Freytag and Clements, 1999) Endotoxin-based adjuvants Monophosphoryl lipid A (MPL) (Ulrich and Myers, 1995) Other bacterial CpG oligomicleotides (Corral and Petray, 2000), BCG sequences (Krieg, et al, Nature, 374: 576), tetanus toxoid (Rice, et al, J. Immunol., 2001, 167: 1558-1565) Particulate Biodegradable (Gupta et al., 1998) Polymer microspheres Immunostimulatory complexes (Morein and Bengtsson, 1999) (ISCOMs) Liposomes (Wassef et al., 1994) Oil-emulsion Freund's incomplete adjuvant (Jensen et al., 1998) and Microfluidized emulsions MF59 (Otl et al., 1995) surfactant- SAF (Allison and Byars, 1992) based (Allison, 1999) adjuvants Saponins QS-21 (Kensil, 1996) Synthetic Muramyl peptide derivatives Murabutide (Lederer, 1986) Threony-MDP (Allison, 1997) Nonionic block copolymers L121 (Allison, 1999) Polyphosphazene (PCPP) (Payne et al., 1995) Synthetic polynucleotides Poly A:U Poly I:C (Johnson, 1994) Thalidomide derivatives CC-4047/ACTIMID (J. Immunol., 168(10): 4914-9)

[0076] The immunogenic targets of the present invention may also be used to generate antibodies for use in screening assays or for immunotherapy. Other uses would be apparent to one of skill in the art. The term "antibody" includes antibody fragments, as are known in the art, including Fab, Fab.sub.2, single chain antibodies (Fv for example), humanized antibodies, chimeric antibodies, human antibodies, produced by several methods as are known in the art. Methods of preparing and utilizing various types of antibodies are well-known to those of skill in the art and would be suitable in practicing the present invention (see, for example, Harlow, et al. Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988; Harlow, et al. Using Antibodies: A Laboratory, Manual, Portable Protocol No. 1, 1998; Kohler and Milstein, Nature, 256:495 (1975)); Jones et al. Nature, 321:522-525 (1986); Riechmann et al. Nature, 332:323-329 (1988); Presta (Curr. Op. Struct. Biol., 2:593-596 (1992); Verhoeyen et al. (Science, 239:1534-1536 (1988); Hoogenboom et al, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991); Cole et al, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J. Immunol., 147(1):86-95 (1991); Marks et al., Bio/Technology 10, 779-783 (1992); Lornberg et al., Nature 368 856-859 (1994); Morrison, Nature 368 812-13 (1994); Fishwild et al., Nature Biotechnology 14, 845-51 (1996); Neuberger, Nature Biotechnology 14, 826 (1996); Lonberg and Huszar, Intern. Rev. Immunol. 13 65-93 (1995); as well as U.S. Pat. Nos. 4,816,567; 5,545.807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and, 5,661,016). The antibodies or derivatives therefrom may also be conjugated to therapeutic moieties such as cytotoxic drugs or toxins, or active fragments thereof such as diptheria A chain, exotoxin A chain, ricin A chain, abrin A chain, curcin, crotin, phenomycin, enomycin, among others. Cytotoxic agents may also include radiochemicals. Antibodies and their derivatives may be incorporated into compositions of the invention for use in vitro or in vivo.

[0077] Nucleic acids, proteins, or derivatives thereof representing an immunogenic target may be used in assays to determine the presence of a disease state in a patient, to predict prognosis, or to determine the effectiveness of a chemotherapeutic or other treatment regimen. Expression profiles, performed as is known in the art, may be used to determine the relative level of expression of the immunogenic target. The level of expression may then be correlated with base levels to determine whether a particular disease is present within the patient, the patient's prognosis, or whether a particular treatment regimen is effective. For example, if the patient is being treated with a particular chemotherapeutic regimen, a decreased level of expression of an immunogenic target in the patient's tissues (i.e., in peripheral blood) may indicate the regimen is decreasing the cancer load in that host. Similarly, if the level of expression is increasing, another therapeutic modality may need to be utilized. In one embodiment, nucleic acid probes corresponding to a nucleic acid encoding an immunogenic target may be attached to a biochip, as is known in the art, for the detection and quantification of expression in the host.

[0078] It is also possible to use nucleic acids, proteins, derivatives therefrom, or antibodies thereto as reagents in drug screening assays. The reagents may be used to ascertain the effect of a drug candidate on the expression of the immunogenic target in a cell line, or a cell or tissue of a patient. The expression profiling technique may be combined with high throughput screening techniques to allow rapid identification of useful compounds and monitor the effectiveness of treatment with a drug candidate (see, for example, Zlokarnik, et al., Science 279, 84-8 (1998)). Drug candidates may be chemical compounds, nucleic acids, proteins, antibodies, or derivatives therefrom, whether naturally occurring or synthetically derived. Drug candidates thus identified may be utilized, among other uses, as pharmaceutical compositions for administration to patients or for use in further screening assays.

[0079] Administration of a composition of the present invention to a host may be accomplished using any of a variety of techniques known to those of skill in the art. The composition(s) may be processed in accordance with conventional methods of pharmacy to produce medicinal agents for administration to patients, including humans and other mammals (i.e., a "pharmaceutical composition"). The pharmaceutical composition is preferably made in the form of a dosage unit containing a given, amount of DNA, viral vector particles, polypeptide or peptide, for example. A suitable daily dose for a human or other mammal may vary widely depending on the condition of the patient and other factors, but, once again, can be determined using routine methods.

[0080] The pharmaceutical composition may be administered orally, parentally, by inhalation spray, rectally, intranodally, or topically in dosage unit formulations containing conventional pharmaceutically acceptable carriers, adjuvants, and vehicles. The term "pharmaceutically acceptable carrier" or "physiologically acceptable carrier" as used herein refers to one or more formulation materials suitable for accomplishing or enhancing the delivery of a nucleic acid, polypeptide, or peptide as a pharmaceutical composition. A "pharmaceutical composition" is a composition comprising a therapeutically effective amount of a nucleic acid or polypeptide. The terms "effective amount" and "therapeutically effective amount" each refer to the amount of a nucleic acid or polypeptide used to induce or enhance an effective immune response. It is preferred that compositions of the present invention provide for the induction or enhancement of an anti-tumor immune response in a host which protects the host from the development of a tumor and/or allows the host to eliminate an existing tumor from the body.

[0081] For oral administration, the pharmaceutical composition may be of any of several forms including, for example, a capsule, a tablet, a suspension, or liquid, among others. Liquids may be administered by injection as a composition with suitable carriers including saline, dextrose, or water. The term parenteral as used herein includes subcutaneous, intravenous, intramuscular, intrasternal, infusion, or intraperitoneal administration. Suppositories for rectal administration of the drug can be prepared by mixing the drug with a suitable non-irritating excipient such as cocoa butter and polyethylene glycols that are solid at ordinary temperatures but liquid at the rectal temperature.

[0082] The dosage regimen for immunizing a host or otherwise treating a disorder or a disease with a composition of this invention is based on a variety of factors, including the type of disease, the age, weight, sex, medical condition of the patient, the severity of the condition, the route of administration, and the particular compound employed. For example, a poxviral vector may be administered as a composition comprising 1.times.10.sup.6 infectious particles per dose. Thus, the dosage regimen may vary widely, but can be determined routinely using standard methods.

[0083] A prime-boost regimen may also be utilized (see, for example, WO 01/30382 A1) in which the targeted immunogen is initially administered in a priming step in one form followed by a boosting step in which the targeted immunogen is administered in another form. The form of the targeted immunogen in the priming and boosting steps are different. For instance, if the priming step utilized a nucleic acid, the boost may be administered as a peptide. Similarly, where a priming step utilized one type of recombinant virus (i.e., ALVAC), the boost step may utilize another type of virus (i.e., NYVAC). This prime-boost method of administration has been shown to induce strong immunological responses.

[0084] While the compositions of the invention can be administered as the sole active pharmaceutical agent, they can also be used in combination with one or more other compositions or agents (i.e., other immunogenic targets, co-stimulatory molecules, adjuvants). When administered as a combination, the individual components can be formulated as separate compositions administered at the same time or different times, or the components can be combined as a single composition.

[0085] Injectable preparations, such as sterile injectable aqueous or oleaginous suspensions, may be formulated according to known methods using suitable dispersing or wetting agents and suspending agents. The injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent. Suitable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution, among others. For instance, a viral vector such as a poxvirus may be prepared in 0.4% NaCl. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed, including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.

[0086] For topical administration, a suitable topical dose of a composition may be administered one to four, and preferably two or three times daily. The dose may also be administered with intervening days during which no does is applied. Suitable compositions may comprise from 0.001% to 10% w/w, for example, from 1% to 2% by weight of the formulation, although it may comprise as much as 10% w/w, but preferably not more than 5% w/w, and more preferably from 0.1% to 1% of the formulation. Formulations suitable for topical administration include liquid or semi-liquid preparations suitable for penetration through the skin (e.g., liniments, lotions, ointments, creams, or pastes) and drops suitable for administration to the eye, ear, or nose.

[0087] The pharmaceutical compositions may also be prepared in a solid form (including granules, powders or suppositories). The pharmaceutical compositions may be subjected to conventional pharmaceutical operations such as sterilization and/or may contain conventional adjuvants, such as preservatives, stabilizers, wetting agents, emulsifiers, buffers etc. Solid dosage forms for oral administration may include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active compound may be admixed with at least one inert diluent such as sucrose, lactose, or starch. Such dosage forms may also comprise, as in normal practice, additional substances other than inert diluents, e.g., lubricating agents such as magnesium stearate. In the case of capsules, tablets, and pills, the dosage forms may also comprise buffering agents. Tablets and pills can additionally be prepared with enteric coatings. Liquid dosage forms for oral administration may include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs containing inert diluents commonly used in the art, such as water. Such compositions may also comprise adjuvants, such as wetting sweetening, flavoring, and perfuming agents.

[0088] Pharmaceutical compositions comprising a nucleic acid or polypeptide of the present invention may take any of several forms and may be administered by any of several routes. In preferred embodiments, the compositions are administered via a parenteral route (intradermal, intramuscular or subcutaneous) to induce an immune response in the host. Alternatively, the composition may be administered directly into a lymph node (intranodal) or tumor mass (i.e., intratumoral administration). For example, the dose could be administered subcutaneously at days 0, 7, and 14. Suitable methods for immunization using compositions comprising TAs are known in the art, as shown for p53 (Hollstein et al., 1991), p21-ras (Almoguera et al., 1988), HER-2 (Fendly et al., 1990), the melanoma-associated antigens (MAGE-1; MAGE-2) (van der Bruggen et al, 1991), p97 (Hu et al., 1988), melanoma-associated antigen E (WO 99/30737) and carcinoembryonic antigen (CEA) (Kantor et al., 1993; Fishbein et al, 1992; Kaufman et al., 1991), among others.

[0089] Preferred embodiments of administratable compositions include, for example, nucleic acids or polypeptides in liquid preparations such as suspensions, syrups, or elixirs. Preferred injectable preparations include, for example, nucleic acids or polypeptides suitable for parental, subcutaneous, intradermal, intramuscular or intravenous administration such as sterile suspensions or emulsions. For example, a recombinant poxvirus may be in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose or the like. The composition may also be provided in lyophilized form for reconstituting, for instance, in isotonic aqueous, saline buffer. In addition, the compositions can be co-administered or sequentially administered with other antineoplastic, anti-tumor or anti-cancer agents and/or with agents which reduce or alleviate ill effects of antineoplastic, anti-tumor or anti-cancer agents.

[0090] A kit comprising a composition of the present invention is also provided. The kit can include a separate container containing a suitable carrier, diluent or excipient. The kit can also include an additional anti-cancer, anti-tumor or antineoplastic agent and/or an agent that reduces or alleviates ill effects of antineoplastic, anti-tumor or anti-cancer agents for co- or sequential-administration. Additionally, the kit can include instructions for mixing or combining ingredients and/or administration.

[0091] A better understanding of the present invention and of its many advantages will be had from the following examples, given by way of illustration.

EXAMPLES

Example 1

AAC2 Tumor Associated Antigen

[0092] A version of the AAC2 coding sequence (AAC2-1) was provided by a collaborator and found to have high sequence similarity to a murine bcl-6-associated zinc finger protein ("BAZF"). Based on this sequence information, PCR primers were designed as shown below:

TABLE-US-00003 (forward primer; SEQ ID NO.: 6) CACCATGGGT TCCCCCGCCG CCCCGGA (reverse primer; SEQ ID NO.: 7) CTAGGGCCCC CCGAGAATGT GGTAGTGCAC TTT

[0093] RNA was isolated from confluent HUVEC (BioWhittacker; Cat. No. CC2517, Lot No. IF0141) cultures using Trizol.TM. as indicated by the manufacturer (Life Technologies, Inc., Cat. No. 15596). High fidelity RT-PCR was then performed using the forward and reverse primers (24 cycles at 94 degrees, 2 min.; 94 degrees, 30 see; 56.8 degrees, 30 sec; 68 degrees, 1 min 40 sec; cycle 25 is 68 degrees, 7 min) resulting in the isolation of a 1,447 base pair cDNA. The cDNA was cloned into the pEF6-TOPO eukaryotic expression plasmid and termed "pEF6-hAAC2-2". The cDNA pEF6-hAAC2-2 was sequenced using four primers and aligned to the sequence of AAC2-1 and murine BAZF (FIG. 1). As shown therein, AAC2-2 is missing the serine residue (S) found at position 245 in AAC2-1. Secondly, a stretch of 17 amino acids at positions 298 to 316 (SEFFSCQNCEAVAGCSS) of AAC2-2 showed only 11.8% sequence identity with amino acids 298-316 of AAC2-1 (FIG. 1). Interestingly, the stretch of 17 amino acids between positions 298 and 316 is 100% identical with murine BAZF suggesting that this may be critical for transcription factor function along with the long stretch of serines (zinc finger). AAC2-2 was then cloned into the pcDNA3.1-zeo eukaryotic expression plasmid ("pcDNA3.1-hAAC2-2").

Example 2

Human T-Cell Reactivity Against AAC-2 Peptides

[0094] Using the AAC2-2 amino acid sequence, a library of 9-mer peptides predicted to bind to HLA-A-0201 was constructed (Table III; "N" indicates the sequence is not found within the mouse homolog, while "Y" indicates the sequence is found within the mouse homolog). Twenty-three of the peptides were dissolved in DMSO at 10 mg/ml (Table IV) and used in human PBMC cultures to test for their ability to elicit CD8 and CD4 .alpha..beta. T-cell responses in vitro.

TABLE-US-00004 TABLE III Predicted HLA-A-0201-binding nonamer peptides of human AAC2-2 Position in SEQ ID Designation Sequence Protein NO. CLP-2954 RLSPTAATV AAC2(256-264) 44 CLP-2955 SIFRGRAGV AAC2(65-73) 45 CLP-2956 DVLGNLNEL AAC2(23-31) 46 CLP-2957 GVGVDVLSL AAC2(72-80) 47 CLP-2958 LLTSQAQDT AAC2(277-285) 48 CLP-2959 VLNSQASQA AAC2(201-209) 49 CLP-2960 VQFKCGAPA AAC2(264-272) 50 CLP-2961 GQPCPQARL AAC2(219-227) 51 CLP-2962 GAHRGLDSL AAC2(312-320) 52 CLP-2963 GAPASTPYL AAC2(269-277) 53 CLP-2964 VVQACHRFI AAC2(123-131) 54 CLP-2965 PLGISLRPL AAC2(137-145) 55 CLP-2966 PLRAHKAVL AAC2(48-56) 56 CLP-2967 FVQVAHLRA AAC2(394-402) 57 CLP-2968 APLLDFMYT AAC2(90-98) 58 CLP-2969 RAGVGVDVL AAC2(70-78) 59 CLP-2970 CETCGSRFV AAC2(387-395) 60 CLP-2971 ATAPAVLAA AAC2(106-114) 61 CLP-2972 SRFVQVAHL AAC2(392-400) 62 CLP-2973 CNWKKYKYI AAC2(192-200) 63 CLP-2974 SPAAPEGAL AAC2(3-11) 64 -- EC-1 ALGYVREFT AAC2(10-18) 65 EC-3 RLRGILTDV AAC2(32-40) 66 EC-4 GILTDVTLL AAC2(35-43) 67 EC-5 ILTDVTLLV AAC2(36-44) 68 EC-6 TLLVGGQPL AAC2(41-49) 69 EC-9 FMYTSRLRL AAC2(95-103) 70 EC-10 RLSPATAPA AAC2(102-110) 71 EC-11 AVLAAATYL AAC2(110-118) 72 EC-12 ATYLQMEHV AAC2(115-123) 73 EC-13 LQMEHVVQA AAC2(118-126) 74 EC-21 QVAHLRAHV AAC2(390-398) 75 EC-22 HLQTLKSHV AAC2(418-426) 76 EC-24 VVQACHRFI AAC2(123-131) 77

[0095] Using GM-CSF and IL-4, dendritic cells (DC) were generated from peripheral blood monocytes of blood donors expressing HLA-A-0201. DC were pulsed with the different pools of 9-mer AAC2-2 peptides shown in Table IV.

TABLE-US-00005 TABLE IV AAC2-2 Peptide Groups 1 CLP 2954 RLSPTAATV (SEQ ID NO.: 44) AAC2(256-264) CLP 2956 DVLGNLNEL (SEQ ID NO.: 45) AAC2(23-31) CLP 2957 GVGVDVLSL (SEQ ID NO.: 46) AAC2(72-80) 2 CLP 2959 VLNSQASQA (SEQ ID NO.: 49) AAC2(201-209) CLP 2960 VQFKCGAPA (SEQ ID NO.: 50) AAC2(264-272) CLP 2963 GAPASTPYL (SEQ ID NO.: 53) AAC2(269-277) 3 CLP 2964 VVQACHRFI (SEQ ID NO.: 54) AAC2(123-131) CLP 2968 APLLDFMYT (SEQ ID NO.: 58) AAC2(90-98) 4 CLP 2971 ATAPAVLAA (SEQ ID NO.: 61) AAC2(106-114) CLP 2973 CNWKKYKYI (SEQ ID NO.: 63) AAC2(192-200) 5 EC 1 ALGYVREFT (SEQ ID NO.: 65) AAC2(10-18) EC 3 RLRGILTDV (SEQ ID NO.: 66) AAC2(32-40) EC 3 GILTDVTLL (SEQ ID NO.: 67) AAC2(35-43) 6 EC 5 ILTDVTLLV (SEQ ID NO.: 68) AAC2(36-44) EC 6 ILLVGGQPL (SEQ ID NO.: 69) AAC2(41-49) EC 9 FMYTSRLRL (SEQ ID NO.: 70) AAC2(95-103) 7 EC 10 RLSPATAPA (SEQ ID NO.: 71) AAC2(102-110) EC 11 AVLAAATYL (SEQ ID NO.: 72) AAC2(110-118) EC 12 ATYLQMEHV (SEQ ID NO.: 73) AAC2(115-123) 8 EC 13 LQMEHVVQA (SEQ ID NO.: 74) AAC2(118-126) EC 21 QVAHLRAHV (SEQ ID NO.: 75) AAC2(390-398) 9 EC 22 HLQTLKSHV (SEQ ID NO.: 76) AAC2(418-426) EC 24 VVQACHRFI (SEQ ID NO.: 77) AAC2(123-131)

[0096] These DC were used to stimulate autologous T-cell-enriched PBMC preparations. The T cells were re-stimulated with autologous PBMC and then re-stimulated with CD40-ligand-activated autologous B cells. After the third and fourth round of stimulation with each peptide pool. ELISPOT analysis for IFN-.gamma. production indicated that the T cells responded most strongly to one of the pools of AAC2-2 peptides (peptide group 6; FIG. 2A). Peptide group 6 includes the following peptides: ILTDVTLLV (aa 36-44), TLLVGGQPL (aa 41-49), and FMYTSRLRL (aa 95-103). Flow cytometric analysis (FACS) showed that the lymphocytes from this peptide-specific line consisted of >50% CD8 T cells with a memory (CD45RO.sup.+) phenotype. Very few cells (<:2%) were stained with anti-CD56 antibodies, indicating that the observed IFN-.gamma. production was not due to NK cell activity.

[0097] Analysis of CTL activity from this peptide pool-specific T-cell line also demonstrated that the activated T cells were capable of killing peptide-loaded TAP-deficient T2 cells in an HLA-A-0201-restricted fashion (FIG. 2B). This analysis also revealed that ILTDVTLLV was a dominant peptide that stimulated the majority of the peptide-specific CTL activity. Thus, it was determined that AAC2-2 peptides are immunogenic in the human immune system.

Example 3

Immunogenicity of AAC2-2 In Vivo

[0098] Using DNA immunization into HLA-A2-Kb transgenic mice, it was found that the AAC2-2 protein is processed into immunogenic peptides and can elicit an HLA-A-0201-restricted T-cell response in vivo. Mice were immunized on day 1 by injection with pEF6-hAAC2-2 and boosted with the same plasmid at day 21 Lymphocytes were harvested from immunized mice 21 days after boosting and re-stimulated in vitro with the different groups of AAC2-2 peptides shown in Table IV. Peptide-specific effector T-cell function towards these peptides was found using IFN-.gamma. ELISPOT analysis (FIG. 3). It was found that the same pool of peptides (group 6) previously shown to be strongly immunogenic in human PBMC cultures also elicited significant reactivity by cells after DNA vaccination (FIG. 3). Thus, the AAC2 gene product administered as a DNA-based vaccine is immunogenic in vivo and elicits a strong cell-mediated immune response characterized by the activation of CTL activity.

Example 4

Therapeutic AAC2-2 Vaccine

[0099] Therapeutic vaccination against the AAC2-2 gene product using the pEF6-hAAC2-2 DNA vaccine was found to completely block the growth of a solid tumor. Groups of eight C57BJ6 mice were subcutaneously challenged with 10.sup.4 B16F10 melanoma cells, a vigorous and relatively non-immunogenic tumor cell line. The mice were then immunized at weekly intervals starting at 6 days after tumor challenge. Control mice (eight per group) treated either with a plasmid encoding the flu-NP protein or saline alone all developed large tumors. In contrast, all the mice (8/8) immunized with pIF6-hA-AC2-2 had no detectable tumor over a 50-day period (FIG. 4). All mice remained tumor-free through 80 days (data not shown). FIG. 5 plots the survival of mice treated with the different DNA vectors shown after melanoma implantation showing again the complete effectiveness of AAC2-2 vaccination in protecting mice against melanoma growth. No adverse health effects have been observed as a result of immunization with the human AAC2-2 gene-encoding vector (immunized mice were as active as control mice and showed no weight loss).

[0100] As shown in FIGS. 4 and 5, vaccination with a plasmid encoding the human VEGFR-2 (pBLAST-hflk1) did not protect tumor-challenged mice. In fact, the tumors grew even more rapidly in these mice. Analysis of sera from mice vaccinated with the pBLAST-hflk1 plasmid by ELISA found that IgG against the VEGFR-2 protein is induced in significant titres (data not shown). These results suggest that an antibody-based immune response directed against VEGFR-2 may not be not effective in preventing angiogenesis and solid tumor growth.

[0101] Inhibition of melanoma solid tumor growth in C57BL/6 mice immunized with pEF6-hAAC2-2 correlates with an immune response against the protein (FIG. 6). Immunization of CS7BL/6 mice was performed as described above. Spleen cells from immunized mice were re-stimulated with the same peptide pools used in experiments with HLA-A2-Kb transgenic mice (Table III). A significant number of peptides cross-react on C57BL/6 class I MHC (Kb and Db molecules). Two pools of peptides in particular (group 1 and group 5) were found to elicit strong effector cell activity in the IFN-.quadrature. ELISPOT assays (FIG. 6). All of the peptides in these groups are also identical to the corresponding sequence in the murine BAZF protein. These results strongly suggest that immunization with the human AAC2-2 activates an immune response against its murine orthologue BAZF in mice and can inhibit tumor angiogenesis as a result.

Example 5

BFA4 Tumor Antigen

[0102] The BFA4 sequence was found to be the "trichorhinophalangeal syndrome 1" (TRPS-1) gene (Genebank ID #6684533; Momeniet et al, Nature Genetics, 24(1), 71-74, 2000), a known transcription factor with no function attributed previously in any form of cancer. The BFA4 cDNA sequence is shown in FIG. 7 (SEQ ID NO.: 28) and the deduced amino acid sequence is shown in FIG. 8 (SEQ ID NO.: 29)

A. BFA4 Peptides and Polyclonal Antisera

[0103] For monitoring purposes, rabbit anti-BFA4 polyclonal antibodies were generated. Six peptides (22-mers) were designed and synthesized to elicit antibody response to BFA4, as shown below:

TABLE-US-00006 (SEQ ID NO.: 78) CLP 2589 MVRKKNPPLRNVASEGEGQILE BFA4 (1-22) (SEQ ID NO.: 79) CLP 2590 SPKATEETGQAQSGQANCQGLS BFA4 (157-178) (SEQ ID NO.: 80) CLP 2591 VAKPSEKNSNKSIPALQSSDSG BFA4 (371-392) (SEQ ID NO.: 81) CLP 2592 NHLQGSDGQQSVKESKEHSCTK BFA4 (649-670) (SEQ ID NO.: 82) CLP 2593 NGEQIIRRRTRKRLNPEALQAE BFA4 (940-961) (SEQ ID NO.: 83) CLP 2594 ANGASKEKTKAPPNVKNEGPLNV BFA4 (1178-1199)

[0104] Rabbits were immunized with the peptides, serum was isolated, and the following antibody titers were observed:

TABLE-US-00007 Rabbit # Peptide Titer (Bleed 2) Titer (Final Bleed) 1, 2 CLP2589 800000, 1600000 2560000, 2560000 3, 4 CLP2590 12800, 6400 40000, 40000 5, 6 CLP2591 400000, 400000 320000, 320000 7, 8 CLP2592 25600, 12800 80000, 40000 9, 10 CLP2593 3200000, 51200 2560000, 160000 11, 12 CLP2594 409600, 409600 320000, 320000

[0105] These peptides were also modified by coupling with KLH peptides to enhance immune responses as shown below:

TABLE-US-00008 (CLP-2589; SEQ ID NO.: 78) BFA4(1-22) KLH-MVRKKNPPLRNVASEGEGQILE (CLP-2590; SEQ ID NO.: 79) BFA4(157-178) KLH-SPKATEETGQAQSGQANCQGLS (CLP-2591; SEQ ID NO.: 80) BFA4(371-392) KLH-VAKPSEKNSNKSIPALQSSDSG (CLP-2592; SEQ ID NO.: 81) BFA4(649-670) KLH-NHLQGSDGQQSVKESKEHSCTK (CLP-2593; SEQ ID NO.: 82) BFA4(940-961) KLH-NGEQIIRRRTRKRLNPEALQAE (CLP-2594; SEQ ID NO.: 83) BFA4(1178-1200) KLH-ANGADKEKTKAPPNVKNEGPLNV

[0106] The pcDNA3.2BFA4 (3.6 mg) was also used for DNA immunization to generate polyclonal sera in chickens.

B. Cloning of BFA4

[0107] Complete cDNA sequence for BFA4 is .about.10 kb and gene is expressed in BT474 ductal carcinoma cells. Primers 7717 (forward primer) and 7723 (reverse primer) were designed to amplify full-length BFA4 gene by amplification of 4 kb, 7 kb or 10 kb products by RT-PCR.

TABLE-US-00009 Primer 7717: BFA4-BamH1/F1 (5' end forward) with Kozak: (SEQ ID NO.: 84) 5' CGGGATCCACCATGGTCCGGAAAAAGAACCCC 3' (BamHI for DNA3.1, MP76) Primer 7723: BFA4-BamHI/R1 (3' end reverse 4 kb): (SEQ ID NO.: 85) 5' CGGGATCCCTCTTTAGGTTTTCCATTTTTTTCCAC 3' (BamHI for DNA3.1, MP76)

[0108] Ten mg of total RNA isolated and frozen in different batches from BT-474 cells using Trizol as indicated by the manufacturer (Gibco BRL) was used in RT-PCR to amplify the BF-A4 gene. RT-PCR conditions were optimized using Taq Platinum High Fidelity enzyme, OPC (Oligo Purification Cartridge; Applied Biosystems) purified primers and purified total. RNA/polyA mRNA (BT 474 cells). Optimization resulted in a 4.0 kb fragment as a single band.

[0109] To re-amplify the BFA4 sequence, mRNA was treated with DNase per manufacturers' instructions (Gibco BRL). The 4 kb DNA was reamplified using PCR using primers 7717 and 7723 primers (10 pmole/microlitre) and Taq Platinum High Fidelity polymerase (GIBCO BRL) enzyme. Thermocycler conditions for both sets of reactions were as under: 94.degree. C. (2 min), followed by 30 cycles of 94.degree. C. (30 sec), 52.degree. C. (30 sec), 67.degree. C. (4 min) and 67.degree. C. (5 min) and finally 40.degree. C. for 10 min. Three BFA4 clones were identified after pCR2.1/TOPO-TA cloning.

[0110] Several mutations were identified during analysis of the BFA4 sequence. To correct these sequences, the BamHI/XhoI fragment (5') of the BFA4 gene from clone JB-3552-1-2 (pCR2.1/TOPO/BFA4) was exchanged with the XhoI/BamHI fragment (3') of the BFA4 gene from clone JB-3552-1-4 (pCR2.1/TOPO/BFA4). This recombined fragment was then ligated into pMCS5 BamHI/CAP. Clone JB-3624-1-5 was generated and found to contain the correct sequence.

[0111] Nucleotide 344 of the isolated BFA4 clone was different from the reported sequence (C in BFA4, T in TRPS-1). The change resulted in a phe to ser amino acid change. To change this sequence to the reported sequence, the EcoRI/BglII fragment (5') of the BFA4 gene from clone JIB-3552-1-2 (pCR2.1/TOPO/BFA4) was subcloned into pUC8:2 to generate clone JB-3631-2. This clone was used as a template for Quickchange (Stratagene) mutagenesis to change amino acid 115 of the BFA4 protein from a serine to a phenylalanine as in the TRPS1 protein. The selected clone was JB-3648-2-3. Mutagenesis was also repeated with pMCS5 BFA4 (BT474) as a template for Quickchange (Stratagene) mutagenesis to change amino acid 115 of the BFA4 protein from a serine to a phenylalanine as in the TRPS1 protein. Several clones were found to be correct by DNA sequencing and one of the clones (JB-3685-1-18) was used for further subcloning.

[0112] JB-3685-1-18 was then used to subclone the BFA4 coding sequence into the BamHI sites of four different expression vectors: 1) the poxviral (NYVAC) vector pSD554VC (COPAK/H6; JB-3707-1-7); 2) pcDNA3.1/Zeo (+) (JB-3707-3-2); 3) pCAMycHis (JB-3707-5-1); and, 4) Semiliki Forest virus alphaviral replicon vector pMP176 (JB-3735-1-23). The BFA4 coding sequence within JB-3707-1-7, JB-3707-5-1, and JB-3735-1-23 was confirmed by DNA sequencing.

[0113] A stop codon was introduced near the end of the cloned sequence in the pcDNA3.1/Zeo/BFA4 construct (JB-3707-3-2). A unique EcoRI site was opened and filled in to introduce a stop codon in-frame with BFA4 coding sequence. Several putative clones were identified by the loss of EcoRI site, however three clones (JB-3756-1-2; JB-3756-3-1; and JB-3756-4-1) were sequenced. All three were found to be correct in the area of the fill-in. Clone JB-3756-3-1 identified as having the correct sequence and orientation.

[0114] Myc and myc/his tags (Evans et al, 1985) were introduced using oligonucleotides, which were annealed and ligated into the pcDNA3.1/Zeo/BFA4 construct (JB-3707-3-2) at the EcoRI/EcoRV sites. Several clones were obtained for these constructs. Three clones having the correct sequences and orientations were obtained: 1) PcDNA3.1/Zeo/BFA4/myc-tag (JB-3773-1-2); 2) PcDNA3.1/Zeo/BFA4/mychis-tag (JB-3773-2-1); and, 3) PcDNA3.1 Zeo/BFA4/mychis-tag (JB-3773-2-2).

C. Expression of BFA4

[0115] 1. Expression from Poxviral Vectors

[0116] The pSD554VC (COPAK/H6; JB-3707-1-7) vector was used to generate NYVAC-BFA4 virus. In vitro recombination was performed with plasmid COPAK/H6/BFA4 and NYVAC in RK13/CEF cells. NYVAC-BFA4 (vP2033-NYVAC-RK13) was generated and amplified to P3 level after completion of three enrichments with final stock concentrations of 1.12.times.10.sup.9/ml (10 ml), Vero cells were infected with NYVAC-BFA4 at an M.O.I. of 0.5 pfu/cell. Lysates and media were harvested 24 h post-infection to confirm expression of BFA4 protein. One-twentieth of the concentrated media and 1/40 of the lysate were loaded onto a western blot and incubated with rabbit antisera against the BFA4 peptides CLP 2589, 2591, 2598 and 2594 (see above for peptide sequences and preparation of anti-BFA4 antisera). An approximate 120 kD band was detected in both the lysate and the concentrated media of NYVAC-BFA4-infected Vero cells which was not evident in either Vero control cells ("mock-infected"), Vero cells infected with the parental NYVAC virus, or concentrated media.

2. Expression from pcDNA3.1-Based Vectors

[0117] Transient transfection studies were performed to verify expression of BFA4 from the pcDNA-based vectors and to analyze quality of polyconal sera raised against BFA4 peptides. The following constructs were used to study expression of BFA4 gene: pcDNA 3.1 zeo.sup.R/BFA4, pMP76/BFA4, pcDNA 3.1 zeo.sup.R/BFA4/Myc tag and pcDNA 3.1 zeo.sup.R/BFA4/MycHis tag. BFA4 expression plasmids (5 .mu.g and 10 .mu.g) were co-transfected with pGL3 Luciferase (1 .mu.g) (Promega) with the Gene porter reagent (Gene Therapy Systems) as the transfection reagent. At 48 h post-transfection, whole cell extract was prepared by scraping cells in cell lysis reagent (200 .mu.l) and 1 cycle of freeze-thaw (-20.degree. C. freeze, 37.degree. C. thaw). Transfection efficiency was quantitated by analyzing expression of the luciferase reporter gene by measuring Relative Luciferase Units (RLU) in duplicate. Similar RLU values were obtained in the samples co-transfected with luciferase construct in the presence and absence of BFA4 expression vectors. There was no significant difference observed in toxicity or RLU values with differential amount (5 .mu.g and 10 .mu.g) of BFA4 expression vectors. Preliminary western blot analysis using alkaline phosphatase system with the CHOK1 cell extracts (pcDNA3/zeo/BFA4/MycHisTag) and an anti-BFA4 polyclonal antisera, revealed a band at approximately 120 kDa band in extracts of BFA4 vector-transfected cells.

[0118] A stable transfection study was initiated to obtain stable clones of BFA4 expressing COS A2 cells. These cells are useful for in vitro stimulation assays. pcDNA 3.1 zeo.sup.R/BFA4 (2.5 .mu.g and 20 .mu.g), and pcDNA 3.1 zeo.sup.R/BFA4/MycHis tag (2.5 .mu.g) were used to study expression of BFA4). pGL3 Luciferase (2.5 .mu.g) was used as a control vector to monitor transfection efficiency. The Gene porter reagent was used to facilitate transfection of DNA vectors. After 48 h post-transfection, whole cell extract were prepared by scraping cells in the cell lysis reagent (200 .mu.l) and 1 cycle of freeze-thaw at -20.degree. C./37.degree. C. for first experiment. Transfected cells obtained from the second experiment were trypsinized, frozen stock established and cells were plated in increasing concentrations of Zeocin (0, 250, 500, 750 and 1000 .mu.g/ml). Non-transfected CosA2cells survived at 60-80% confluency for three weeks at 100 .mu.g/ml (Zeocin) and 10% confluency at 250 .mu.g/ml (Zeocin). However, after three weeks, at higher drug concentration (500-1000 .mu.g/ml), live cells were not observed in the plates containing non-transfected cells and high Zeocin concentration (500-1000 .mu.g/ml).

[0119] Several Zeocin-resistant clones growing in differential drug concentrations (Zeocin-250, 500, 750 and 1000 .mu.g/ml) were picked from 10 cm plates after three weeks. These clones were further expanded in a 3.5 cm plate(s) in the presence of Zeocin at 500, 750 and 1000 .mu.g/ml. Frozen lots of these clones were prepared and several clones from each pool (pcDNA 3.1 zeo.sup.R/BFA4, and pcDNA 3.1 zeo.sup.R/BFA4/MycHis tag) were expanded to T75 cm.sup.2 flasks in the presence of Zeocin at 1 mg/ml. Five clones from each pool (pcDNA 3.1 zeo.sup.R/BFA4, and pcDNA 3.1 zeo.sup.R/BFA4/MycHis tag) were expanded to T75 cm.sup.2 flasks in the presence of Zeocin at 1 mg/ml. Cells are maintained under Zeocin drug (1 mg/ml) selection, Six clones were used in BFA4 peptide-pulsed target experiment, and two clones were found to express BFA4 at a moderate level by immunological assays. The non-adherent cell lines K562A2 and EL4A2 were also transfected with these vectors to generate stable cell lines.

3. Prokaryotic Expression Vector

[0120] The BamH1-Xho-1 fragment (1.5 Kbp) fragment encoding N-terminal 54 kDa BFDA4 from pcDNA3.1/BFA4 was cloned into pGEX4T1-6H is (Veritas) plasmid. This vector contains the tac promoter followed by the N-terminal glutathione S-transferase (GST .about.26 kDa) and a hexahistidine tag to C terminus of the GST fusion protein.

[0121] The BFA4-N54 expression plasmid was transformed into BL21 cells and grown at 25.degree. C. in antibiotic selection medium (2 L culture) to an OD (600 nm) and thereafter induced with 1 mM IPTG. GST-BFA4-N54 was found to be soluble protein. Clarified extract of the soluble fraction was adsorbed batchwise to glutathione-Sepharose 4B and eluted with 10 mM reduced glutathione, Fractions were analyzed after estimation of protein concentration and TCA precipitation. Specific polypeptide of Mr=85 kDa in the eluate was confirmed by SDS-PAGE. The recombinant protein was purified by gluathione-Sepharose was absorbed on a NiNTA column for further purification. The bound protein was eluted with 0.25M imidazole. The protein was dialyzed versus TBS containing 40% Glycerol, resulting in 4.5 mg GST-BFA4-N54-6 His (N terminus BFA4 protein) protein. Expression of BFA4 was confirmed using the rabbit anti-BFA4 polyclonal antibody by western blot.

D. Anti-BFA4 Immune Responses

I. BFA4 Peptides

[0122] In addition to genetic immunization vectors for BFA4, immunological reagents for BFA4 have been generated. A library of 100 nonamer peptides spanning the BFA4 gene product was synthesized. The peptides were chosen based on their potential ability to bind to HLA-A*0201. Table V lists 100 nonamer peptide epitopes for HLA-A*0201 from the BFA4 protein tested (see below):

TABLE-US-00010 PEPTIDE POSITION IN DESIGNATION SEQUENCE PROTEIN SEQ ID. CLP-2421 MVRKKNPPL BFA4 (1-9) 131 CLP-2422 KKNPPLRNV BFA4 (4-12) 132 CLP-2423 VASEGEGQI BFA4 (12-20) 133 CLP-2424 QILEPIGTE BFA4 (19-27) 134 CLP-2425 RNMLAFSFP BFA4 (108-116) 135 CLP-2426 NMLAFSFPA BFA4 (109-117) 136 CLP-2427 MLAFSFPAA BFA4 (110-118) 137 CLP-2428 FSFPAAGGV BFA4 (113-121) 138 CLP-2429 AAGGVCEPL BFA4 (117-125) 139 CLP-2430 SGQANCQGL BFA4 (170-178) 140 CLP-2431 ANCQGLSPV BFA4 (172-180) 588 CLP-2432 GLSPVSVAS BFA4 (176-184) 141 CLP-2433 SVASKNPQV BFA4 (181-189) 142 CLP-2434 RLNKSKTDL BFA4 (196-204) 143 CLP-2435 NDNPDPAPL BFA4 (207-215) 144 CLP-2436 DPAPLSPEL BFA4 (211-219) 145 CLP-2437 ELQDFKONI BFA4 (218-216) 146 CLP-2438 GLHNRTRQD BFA4 (249-257) 147 CLP-2439 ELDSKILAL BFA4 (259-267) 148 CLP-2440 KILALHNMV BFA4 (263-271) 149 CLP-2441 ALHNMVQFS BFA4 (266-284) 150 CLP-2442 VNRSVFSGV BFA4 (282-290) 151 CLP-2443 FSGVLQDIN BFA4 (287-295) 152 CLP-2444 DINSSRPVL BFA4 (293-301) 153 CLP-2445 VLLNGTYDV BFA4 (300-308) 154 CLP-2446 FCNFTYMGN BFA4 (337-345) 155 CLP-2447 YMGNSSTEL BFA4 (342-350) 156 CLP-2448 FLQTHPNKI BFA4 (354-362) 157 CLP-2449 KASLPSSEV BFA4 (363-371) 158 CLP-2450 DLGKWQDKI BFA4 (393-401) 159 CLP-2451 VKAGDDTPV BFA4 (403-411) 160 CLP-2452 FSCESSSSL BFA4 (441-449) 161 CLP-2453 KLLEHYGKQ BFA4 (450-458) 162 CLP-2454 GLNPELNDK BFA4 (466-474) 163 CLP-2455 GSVINQNDL BFA4 (478-486) 164 CLP-2456 SVINQNDLA BFA4 (479-487) 165 CLP-2457 FCDFRYSKS BFA4 (527-535) 166 CLP-2458 SHGPDVIVV BFA4 (535-543) 167 CLP-2459 PLLRHYQQL BFA4 (545-553) 168 CLP-2460 GLCSPEKHL BFA4 (570-578) 169 CLP-2461 HLGEITYPF BFA4 (577-585) 170 CLP-2462 LGEITYPFA BFA4 (578-586) 171 CLP-2463 HCALLLLHL BFA4 (594-602) 172 CLP-2464 ALLLLHLSP BFA4 (596-604) 173 CLP-2465 LLLLHLSPG 9FA4 (597-605) 174 CLP-2466 LLLHLSPGA BFA4 (598-606) 175 CLP-2467 LLHLSPGAA BFA4 (599-607) 176 CLP-2468 FTTPDVDVL BFA4 (621-629) 177 CLP-2469 TTPDVDVLL BFA4 (622-830) 178 CLP-2470 VLLFHYESV BFA4 (628-636) 179 CLP-2471 FITQVEEEI BFA4 (673-681) 180 CLP-2472 FTAADTQSL BFA4 (699-707) 181 CLP-2473 SLLEHFNTV BFA4 (706-714) 182 CLP-2474 STIKEEPKI BFA4 (734-742) 86 CLP-2475 KIDFRVYNL BFA4 (741-749) 87 CLP-2476 NLLTPDSKM BFA4 (748-756) 88 CLP-2479 V1WRGADIL BFA4 (792-800) 89 CLP-2480 ILRGSPSYT BFA4 (799-807) 90 CLP-2481 YTQASLGLL BFA4 (806-814) 91 CLP-2482 ASLGLLTPV BFA4 (809-817) 92 CLP-2483 GLLTPVSGT BFA4 (812-820) 93 CLP-2484 GTQEQTKTL BFA4 (819-827) 94 CLP-2485 KTLRDSPNV BFA4 (825-833) 95 CLP-2486 HLARPIYGL BFA4 (837-845) 96 CLP-2487 PIYGLAVET BFA4 (841-849) 97 CLP-2488 LAVETKGFL BFA4 (845-853) 98 CLP-2489 FLQGAPAGG BFA4 (852-860) 99 CLP-2490 AGGEKSGAL BFA4 (858-866) 100 CLP-2491 GALPQQYPA BFA4 (864-872) 101 CLP-2492 ALPQQYPAS BFA4 (865-873) 102 CLP-2493 FCANCLTTK BFA4 (895-903) 103 CLP-2494 ANGGYVCNA BFA4 (911-919) 104 CLP-2495 NACGLYQKL BFA4 (918-926) 105 CLP-2496 GLYQKLHST BFA4 (921-929) 106 CLP-2497 KLHSTPRPL BFA4 (925-933) 107 CLP-2498 STPRPLNII BFA4 (928-936) 108 CLP-2499 RLNPEALQA BFA4 (962-960) 109 CLP-2500 VLVSQTLDI BFA4 (1020-1028) 110 CLP-2501 DIHKRIMPL BFA4 (1027-1035) 111 CLP-2502 RMQPLHIQI BFA4 (1031-1039) 112 CLP-2503 YPLFGLPFV BFA4 (1092-1100) 113 CLP-2504 GLPFVHNDF BFA4 (1096-1104) 114 CLP-2505 FVHNDFQSE BFA4 (1099-1107) 115 CLP-2506 SVPGNPHYL BFA4 (1120-1128) 116 CLP-2507 GNPHYLSHV BFA4 (1123-1131) 117 CLP-2508 HYLSHVPGL BFA4 (1126-1134) 118 CLP-2509 YVPYPTENL BFA4 (1141-1149) 119 CLP-2510 FNLPPHFSA BFA4 (1147-1155) 120 CLP-2511 NLPPHFSAV BFA4 (1148-1156) 121 CLP-2512 SAVGSDNDI BFA4 (1154-1162) 122 CLP-2513 KNEGPLNVV BFA4 (1192-1200) 123 CLP-2514 TKCVHCGIV BFA4 (1215-1223) 124 CLP-2515 CVHCGIVFL BFA4 (1217-1225) 125 CLP-2516 CGtVFLDEV BFA4 (1220-1228) 126 CLP-2517 FLDEVMYAL BFA4 (1224-1232) 127 CLP-2518 VMYALHMSC BFA4 (1228-1236) 128 CLP-2519 FQCSICOHL BFA4 (1243-1251) 129 CLP-2520 GLHRNNAQV BFA4 (1265-1273) 130

The peptide library was pooled into separate groups containing 7-10 different peptides for immunological testing as shown in Table VI (see below). In addition to a peptide library spanning BFA4, a recombinant protein spanning the N-terminal 300 amino acids (positions 1-300) has been synthesized and purified from E. coli.

TABLE-US-00011 PEPTIDE PEPTIDE GROUP NUMBER SEQUENCE SEQ ID 1 CLP-2421 MVRKKNPPL 331 CLP-2422 KKNPPLRNV 132 CLP-2423 VASEGEGQI 133 CLP-2424 QILEPIGTE 134 CLP-2425 RNMLAFSFP 135 CLP-2426 NMLAFSFPA 136 CLP-2427 MLAFSFPAA 137 CLP-2428 FSFPAAGGV 138 CLP-2429 AAGGVCEPL 139 CLP-2430 SGQANCQGL 140 2 CLP-2431 ANCQGLSPV 588 CLP-2432 GLSPVSVAS 141 CLP-2433 SVASKNPQV 142 CLP-2434 RLNKSKTDL 143 CLP-2435 NDNPDPAPL 144 CLP-2436 DPAPLSPEL 145 CLP-2437 ELQDFKCNI 146 CLP-2438 GLHNRTRQD 147 CLP-2439 ELDSKILAL 148 CLP-2440 KtLALHNMV 149 3 CLP-2441 ALHNMVQFS 150 CLP-2442 VNRSVFSGV 151 CLP-2443 FSGVLODIN 152 CLP-2444 DINSSRPVL 153 CLP-2445 VLLNGTYDV 154 CLP-2446 FCNFTYMGN 155 CLP-2447 KASLPSSEV 156 CLP-2448 FLOTHPNKI 157 CLP-2449 KASLPSSEV 158 CLP-2450 DLGKWQDKI 159 4 CLP-2451 VKAGDDTPV 160 CLP-2452 FSCESSSSL 161 CLP-2453 KLLEHYGKQ 162 CLP-2454 GLNPELNDK 183 CLP-2455 GSVINQNDL 164 CLP-2456 SVINQNDLA 165 CLP-2457 FCDFRYSKS 166 CLP-2458 SHGPDVIVV 167 CLP-2459 PLLRHYQQL 168 CLP-2460 GLCSPEKHL 169 5 CLP-2461 HLGEITYPF 170 CLP-2462 LGEITYPFA 171 CLP-2463 HCALLLLHL 172 CLP-2464 ALLLLHLSP 173 CLP-2465 LLLLHLSPG 174 CLP-2466 LLLHLSPGA 175 CLP-2467 LLHLSPGAA 176 CLP-2468 FTTPDVDVL 177 CLP-2469 TTPOVDVLL 178 CLP-2470 VLLFHYESV 179 6 CLP-2471 FITQVEEEI 180 CLP-2472 FTAADTQSL 181 CLP-2473 SLLEHFNTV 182 CLP-2474 STIKEEPKI 86 CLP-2475 KIDFRVYNL 87 CLP-2476 NLLTPDSKM 88 CLP-2477 KMGEPVSES 589 CLP-2478 FLKEKVWTE 590 CLP-2479 VTWRGADIL 89 CLP-2460 ILRGSPSYT 90 7 CLP-2481 YTQASLGLL 91 CLP-2482 ASLGLLTPV 92 CLP-2483 GLLTPVSGT 93 CLP-2484 GTQEQTKTL 94 CLP-2485 KTLRDSPNV 95 CLP-2486 HLARPIYGL 96 CLP-2487 PIYGLAVET 97 CLP-2488 LAVETKGFL 98 CLP-2489 FLQGAPAGG 99 CLP-2490 AGGEKSGAL 100 8 CLP-2491 GALPQQYPA 101 CLP-2492 ALPQQYPAS 102 CLP-2493 FCANCLTTK 103 CLP-2494 ANGGYVCNA 104 CLP-2495 NACGLYQKL 105 CLP-2496 GLYQKLHST 106 CLP-2497 KLHSTPRPL 107 CLP-2498 STPRPLNII 108 CLP-2499 RLNPEALQA 109 CLP-2500 VLVSQTLDI 110 9 CLP-2501 DIHKRMQPL 111 CLP-2502 RMQPLHIQI 112 CLP-2503 YPLFGLPFV 113 CLP-2504 GLPFVHNDF 114 CLP-2505 FVHNDFQSE 115 CLP-2506 SVPGNPHYL 116 CLP-2507 GNPHYLSHV 117 CLP-2508 HYLSHVPGL 118 CLP-2509 YVPYPTFNL 119 CLP-2510 FNLPPHFSA 120 10 CLP-2511 NLPPHFSAV 121 CLP-2512 SAVGSDNDI 122 CLP-2513 KNEGPLNVV 123 CLP-2514 TKCVHCGIV 124 CLP-2515 CVHCGIVFL 125 CLP-2516 CGIVFLDEV 126 CLP-2517 FLDEVMYAL 127 CLP-2518 VMYALHMSC 128 CLP-2519 FQCSICQHL 129 CLP-2520 GLHRNNAQV 130

2. Immune Reactivity of BFA4 Peptides and Generation of Human Effector T Cells:

[0123] The BFA4 peptides were grouped into different pools of 7-10 peptides for immunological testing. Dissolved peptide pools were pulsed onto autologous HLA-A*0201 dendritic cells and used to activate autologous T-cell-enriched PBMC preparations. Activated T cells from each peptide-pool-stimulated culture were re-stimulated another 3 to 5 times using CD40L-activated autoloous B-cells. IFN-.gamma. ELISPOT analysis and assays for CTL killing of peptide-pulsed target cells was performed to demonstrate the immunogenicity of these epitopes from BFA4.

[0124] Human T cells demonstrated effector cell activity against a number of pools of peptides from the BFA4 protein, as shown by their ability to secrete IFN-.gamma. in ELISPOT assays. These experiments were repeated after different rounds of APC stimulation resulting in the same reactive peptide groups. Peptide groups 1, 2, 4, 5, 6, 7, 8, 9, and 10 were found to be immunoreactive in these assays. Subsequently, these reactive peptide groups were dc-convoluted in additional IFN-.gamma. ELISPOT assays in which single peptides from each group were tested separately. The individual peptides from BFA4 peptide groups 1, 5 6, 7, 8, 9, and 10 in ELISPOT assays. This analysis revealed a number of individual strongly reactive peptides from the BFA4 protein recognized by human T cells. It was also observed that many of these single peptides also induced C TL activity killing peptide-loaded human T2 lymphoma cell targets. These peptides are listed in Table VII:

TABLE-US-00012 TABLE VII List of highly immunoreactive peptides from BFA4 Strong IFN-.gamma. Killing Strong CTL Killing SEQ ID CLP 2425 RNMLAFSFP CLP 2425 RNMLAFSFP 135 CLP 2426 NMLAFSFPA CLP 2426 NMLAFSFPA 136 CLP 2427 MLAFSFPAA CLP 2427 MLAFSFPAA 137 CLP 2461 HLGEITYPF 170 CLP 2468 FTTPDVDVL CLP 2468 FTTPDVDVL 177 CLP 2470 VLLFHYESV CLP 2470 VLLFHYYESV 179 CLP 2474 KIDFRVYNL 86 CLP 2482 ASLGLLTPV CLP 2482 ASLGLLTPV 92 CLP 2486 HLARPIYGL CLP 2486 HLARPIYGL 96 CLP 2495 NACGLYQKL CLP 2495 NACGLYQKL 105 CLP 2497 KLHSTPRPL 107 CLP 2499 RLNPEALQA CLP 2499 RLNPEALQA 109 CLP 2503 YPLFGLPEV 113 CLP 2509 YVPYPTFNL CLP 2509 YVPYPTFNL 119 CLP 2511 NLPPHFSAV 121 CLP 2518 VMYALHMSC 128 CLP 2520 GLHRNNAQV CLP 2520 GLHRNNAQV 130

D. Immune Responses Against BFA4 after Immunization In Vivo:

[0125] The pcDNA3.1/Zeo-BFA4 plasmid was used to immunize transgenic mice expressing a hybrid HLA-A*0201 .alpha.1.alpha.2 domain fused to a murine Kb .alpha.3 domain in C57BL/6 mice (A2-Kb mice). IFN-.gamma. ELISPOT analysis using the groups of pooled peptides after DNA immunization and removal of activated spleen cells revealed a number of reactive BFA4 peptide groups. Some of these groups (especially group 7 and 8) also reacted strongly in human T-cell cultures suggesting that overlapping groups of peptides are recognized by human T cells and are naturally processed and presented on HLA-A2 after vaccination.

[0126] Vaccination experiments were also performed with the NYVAC-BFA4 and the MP76-18-BFA4 vectors in A2-Kb mice. Mice were immunized subcutaneously with 10-20 .mu.g of MP-76-18-BFA4 and 1-2.times.10.sup.7 pfu vP2033 (NYVAC-BFA4) and boosted 28 days later with the same amounts of each vector. Re-stimulation of spleen cells from the immunized mice with the pools of BFA4 peptides revealed induction of IFN-.gamma. production in response to BFA4 peptide groups 2, 3, 4, 5, 7, 9, and 10 in ELISPOT assays. Thus, the BFA4 gene encoded in a CMV promoter driven eukaryotic plasmid, NYVAC, or a Semliki replicase-based DNA plasmid, were all capable of inducing T-cell responses against the BFA4 protein in vivo.

Example 6

[0127] BCY1 Tumor Antigen

[0128] The BCY1 gene was detected as a partial open reading frame (ORF) homologous to a nematode gene called "posterior-expressed maternal gene-3" (PEM-3) playing a role in posterior to anterior patterning in Caenorhabtidis elegans embryos. No previous involvement of this gene in cancer has been documented.

A. BCY1 and Amino Acid DNA Sequences

[0129] A partial DNA sequence was originally determined for BCY1. Primers, 9616SXC and 9617SXC, are derived from the BCY 1 partial DNA sequence and are designed to clone BCY 1 by RT-PCR from Calu 6 total RNA. The primers were designed such that the PCR product has BamHI sites at both ends and an ATG start codon and a Kozak sequence at the 5' end, as shown below:

TABLE-US-00013 9616SXC: (SEQ ID NO.: 183) 5' CAGTACGGATCCACCATGGCCGAGCTGCGCCTGAAGGGC 3' 9617SXC: (SEQ ID NO.: 184) 5' CCACGAGGATCCTTAGGAGAATATTCGGATGGCTTGCG 3'

[0130] The 1.2 Kb expected amplicon was obtained using ThermoScript RT-PCR System (Invitrogen) under optimized conditions. The PCR products from three separate RT-PCR's were digested with BamHI and respectively inserted in pcDNA3.1/Zeo(+). The resulting clones were MC50A6, MC50A8 and MC50A19 from the first RT-PCR; MC54.21 from the second RT-PCR and MC55.29; and, MC55.32 from the third RT-PCR. The following primers were utilized in sequencing the clones:

TABLE-US-00014 9620MC: (SEQ ID NO.: 185) 5' TAATACGACTCACTATAGGG 3' 9621MC: (SEQ ID NO.: 186) 5' TAGAAGGCACAGTCGAGG 3' 9618MC: (SEQ ID NO.: 187) 5' GAAAACGACTTCCTGGCGGGGAG 3' 9619MC: (SEQ ID NO.: 188) 5' GCTCACCCAGGCGTGGGGCCTC 3'

[0131] DNA sequencing of all six clones indicated a consensus sequence (SEQ ID NO.: 30), as shown in FIGS. 9A and 9B, having the following differences from the original partial BCY1 sequence: a C to G substitution at position 1031 resulting in an amino acid change of Ala to Gly; a GC deletion at position 1032-1034 resulting in a Thr deletion; and, an A to G substitution at position 1177 resulting in an amino acid change of Thr to Ala. Clones MC50A8 and MC55.29 are identical to the consensus sequence. The amino acid sequence of BCY1 is shown in FIG. 98B and (SEQ ID NO.: 31).

B. Immunological Reagents for BCY1 Breast Cancer Antigen:

[0132] A library of 100 nonamer peptides spanning the BCY1 gene product was synthesized. The peptides were chosen based on their potential ability to bind to HLA-A*0201. Table VIII lists 100 nonamer peptide epitopes for HLA-A*0201 from the BCY1 protein tested (see below):

TABLE-US-00015 TABLE VIII Peptide Position in Designation Sequence Protein SEQ ID *CLP-2599 VPVPTSEHV 2 189 *CLP-2602 PTSEHVAEI 5 190 *CLP-2609 EIVGRQCKI 12 191 *CLP-2616 KIKALRAKT 19 192 *CLP-2618 KALRAKTNT 21 193 *CLP-2619 ALRAKTNTY 22 194 *CLP-2620 LRAKTNTYI 23 195 *CLP-2624 INTYIKTPV 27 196 *CLP-2627 YIKTPVRGE 30 197 *CLP-2630 TPVRGEEPV 33 198 *CLP-2633 RGEEPVFMV 36 199 *CLP-2640 MVTGRREDV 43 200 CLP-2641 VTGRREDVA 44 201 *CLP-2643 GRREDVATA 46 202 CLP-2647 DVATARREI 50 203 CLP-2648 VATARREII 51 204 *CLP-2650 TARREIISA 53 205 *CLP-2651 ARREIISAA 54 206 *CLP-2655 IISAAEHFS 58 207 *CLP-2656 ISAAEHFSM 59 208 CLP-2657 SAAEHFSMI 60 209 *CLP-2659 AEHFSMIRA 62 210 *CLP-2663 SMIRASRNK 66 211 CLP-2666 RASRNKSGA 69 212 *CLP-2670 NKSGAAFGV 73 213 *CLP-2673 GAAFGVAPA 76 214 *CLP-2674 AAFGVAPAL 77 215 *CEP- 2677 GVAPALPGQ 80 216 *CLP-2678 VAPALPGQV 81 217 *CLP-2680 PALPGOVTI 83 218 *CLP-2681 ALPGOVTIR 84 219 *CLP-2682 LPGQVTIRV 85 220 CLP-2684 GQVTIRVRV 87 221 *CLP-2689 RVRVPYRVV 92 222 *CLP-2691 RVPYRVVGL 94 223 *CLP-2692 VPYRVVGLV 95 224 *CLP-2695 RVVGLVVGP 98 225 *CLP-2698 GLVVGPKGA 101 226 *CLP-2699 LVVGPKGAT 102 227 *CLP-2700 VVGPKGATI 103 228 *CLP-2710 RIQQQTNTY 113 229 *CLP-2711 IQQQTNTYI 114 230 *CLP-2712 QQQTNTYII 115 231 *CLP-2713 QQTNTYIIT 116 232 *CLP-2718 YIITPSRDR 121 233 CLP-2721 TPSRDRDPV 124 234 CLP-2724 RDRDPVFEI 127 235 CLP-2731 EITGAPGNV 134 236 CLP-2734 GAPGNVERA 137 237 CLP-2738 NVERAREEI 141 238 CLP-2744 EEIETHIAV 147 239 CLP-2746 IETHIAVRT 149 240 CLP-2749 HEAVRTGKI 152 241 CLP-2750 IAVRTGKIL 153 242 CLP-2756 KILEYNNEN 159 243 CLP-2760 YNNENDFLA 163 244 CLP-2762 NENDFLAGS 165 245 CLP-2766 FLAGSPDAA 169 246 CLP-2767 LAGSPDAAI 170 247 CLP-2774 AIDSRYSDA 177 248 CLP-2777 SRYSDAWRV 180 249 CLP-2785 VHQPGCKPL 188 250 CLP-2793 LSTFRQNSL 196 251 CLP-2801 LGCIGECGV 204 252 CLP-2807 CGVDSGFEA 210 253 CLP-2812 GFEAPRLDV 215 254 CLP-2817 RLDVYYGVA 220 255 CLP-2819 DVYYGVAET 222 256 CLP-2823 GVAETSPPL 226 257 CLP-2825 AETSPPLWA 228 258 CLP-2830 PLWAGQENA 233 259 CLP-2833 AGQENATPT 236 260 CLP-2835 QENATPTSV 238 261 CLP-2843 VLFSSASSS 246 262 CLP-2857 KARAGPPGA 260 263 CLP-2869 PATSAGPEL 272 264 CLP-2870 ATSAGPELA 273 265 CLP-2872 SAGPELAGL 275 266 CLP-2879 GLPRRPPGE 282 267 CLP-2887 EPLQGFSKL 290 268 CLP-2892 FSKLGGGGL 295 269 CLP-2894 KLGGGGLRS 297 270 CLP-2899 GLRSPGGGR 302 271 CLP-2909 CMVCFESEV 312 272 CLP-2910 MVCFESEVT 313 273 CLP-2911 VCFESEVTA 314 274 CLP-2913 FESEVTAAL 316 275 CLP-2916 EVTAALVPC 319 276 CLP-2917 VTAALVPCG 320 277 CLP-2920 ALVPCGHNL 323 278 CLP-2921 LVPCGHNLF 324 279 CLP-2922 VPCGHNLFC 325 280 CLP-2927 NLFCMECAV 330 281 CLP-2929 FCMECAVRI 332 282 CLP-2933 CAVRICERT 336 283 CLP-2936 RICERTDPE 339 284 CLP-2940 RTDPECPVC 343 285 CLP-2945 CPVCHITAT 348 286 CLP-2947 VCHITATQA 350 287 CLP-2950 ITATQAIRI 353 288

Table IX shows the groups of peptides used for immunological testing:

TABLE-US-00016 Peptide Peptide Group Sequence SEQ ID 1 EPLQFGSKL 268 EVTAALVPC 276 CPVSHITAT 286 KIKALRAKT 192 IISAAEHFS 207 RASRNKSGA 192 GAAFGVAPA 207 LVVGPKGAT 227 EITGAPGNV 236 GAPGNVERA 237 2 ALRAKTNTY 192 VATARREII 204 PALPGQVTI 218 ALPGQVTIR 219 RVTVPYRVV 222 RDRDPVFEI 127 RVRVPYRVV 222 HIAVRTGKI 241 NENDFLAGS 245 CAVRICERT 283 VCHITATQA 287 3 GRREDVATA 202 DVATARREI 203 TARREIISA 205 GVAPALPGQ 216 RVVGLVVGP 225 VHQPGCKPL 250 PATSAGPEL 264 VTAALVPCG 277 4 VPVPTSEHV 189 ARREIISAA 206 RIQQQTNTY 229 NVERAREEI 238 GFEAPRLDV 254 ATSAGPELA 265 FSKLGGGGL 269 GLRSPGGGR 271 5 PTSEHVAEI 190 EIVGRQCKI 191 LRAKTNTYI 195 VTGRREDVA 201 SMIRASRNK 211 CMVCFESEV 272 LVPCGHNLF 279 NLFCMECAV 281 RICERTDPE 284 RTDPECPVC 285 6 MVTGRREDV 200 GLVVGPKGA 226 IQQQTNTYI 230 FLAGSPDAA 246 GVAETSPPL 257 FESEVTAAL 275 FCMECAVRI 282 7 KALRAKTNT 193 RGEEPVFMV 199 SAAEHFSMI 209 AAFGVAPAL 215 VVGPKGATI 228 YNNENDFLA 244 LGCIGECGV 252 QENATPTSV 261 VCFESEVTA 274 8 TNTYIKTPV 196 NKSGAAFGV 213 QQTNTYIIT 232 KILEYNNEN 243 CGVDSGFEA 253 AETSPPLWA 258 PLWAGQENA 259 VLFSSASSS 262 SAFPELAGL 266 9 ISAAEHFSM 208 QQQTNTYII 231 EEIETHIAV 239 IETHIAVRT 240 LAGSPDAAI 247 AIDSRYSDA 248 DVYYGVAET 256 VPCGHNLFC 280 ITATQAIRI 288 10 TPVRGEEPV 198 AEHFSMIRA 210 VAPALPGQV 217 TPSRDRDPV 234 IAVRTGKIL 242 SRYSDAWRV 249 LSTFRQNSL 251 RLDVYYGVA 255 AGQENATPT 260 MVCFESEVT 273

C. Immune Reactivity of BCY1 Peptides and Generation of Human Effector T Cells

[0133] The library of 100 peptides from BCY1 was separated into 10 groups of 7-10 peptides for immunological testing. Dissolved peptide pools were pulsed onto autologous HLA-A*0201 dendritic cells and used to activate autologous T-cell-enriched PBMC preparations. Activated T cells from each peptide-pool-stimulated culture were re-stimulated another 3 to 5 times using CD40L-activated autologous B-cells. IFN-.gamma. ELISPOT analysis and assays for CTL killing of peptide-pulsed target cells was performed to demonstrate the immunogenicity of these epitopes from BCY1.

[0134] Human T cells demonstrated effector cell activity against a number of pools of peptides from the BCY 1 protein, as shown by their ability to secrete IFN-.gamma. in ELISPOT assays. These experiments were repeated after different rounds of APC stimulation resulting in the same reactive peptide groups. Peptide groups 1, 2, 3, 4, 5, 6, and 7 were found to be immunoreactive in these assays. Subsequently, these reactive peptide groups were de-convoluted in additional IFN-.gamma. ELISPOT assays in which single peptides from each group were tested separately. This analysis revealed a number of individual strongly reactive peptides from the BCY1 protein recognized by human T cells (FIG. 10). Many of these single peptides also induced CTL activity killing peptide-loaded human T2 lymphoma cell targets. Table IX lists these peptides.

Example 7

BFA5/NYBR-1 Breast Cancer Antigen

A. Identification of BFA5

[0135] Microarray profiling analysis indicated that BFA5 was expressed at low to high levels in 41 out of 54 breast tumor biopsy samples (76%) and at high levels in 31 out of 54 breast tumors (57%), as compared to a panel of 52 normal, non-tumor tissues. In situ hybridization (ISH) was performed using a series of BFA5 DNA probes and confirmed the microarray with at least 61% of the tumors showing fairly strong signals. Further bioinformatics assessment confirmed the results of these gene expression analysis results.

[0136] Sequence analysis of the BFA5 nucleotide sequence revealed a high degree of similarity to two unidentified human genes: KIAA1074 (GenBank Accession No. XM.sub.--159732); and, KIAA0565 (GenBank Accession No. AB011137) isolated from a number of fetal and adult brain cDNA clones (Kikuno, et al. The complete sequences of 100 new cDNA clones from brain which code for large proteins in vitro. DNA Res. 6: 1.97-205). These genes were found to contain putative Zn finger regions and a nuclear localization sequence. BFA5 was suggested by others to be a potential breast cancer antigen (Jager, et al. 2001. Identification of a tissue-specific putative transcription factor in breast tissue by serological screening of a breast cancer library. Cancer Res. 61: 2055-2061 and WO 01/47959). In each of these publications, the nucleotide sequence BFA5 was designated NYBR-1 ("New York Breast Cancer-1"; GenBank Accession Nos. AF269087 (nucleotide) and AAK27325 (amino acid). For the purposes of this application, the sequence is referred to as BFA-5, the terms BFA-5 and NYBR-1 are interchangeable.

[0137] As shown previously by Jager, et al. and described in WO 01/47959, supra, BFA5 is specifically expressed in mammary gland, being expressed in 12/19 breast tumors analyzed. The structure of the BFA5/NYBR-1 gene has revealed that it encodes a 150-160 kD nuclear transcription factor with a bZIP site (DNA-binding domain followed by a leucine zipper motif). The gene also contains 5 tandem ankyrin repeats implying a role in protein-protein interactions. These ankyrin repeats may play a role in homo-dimerization of the protein. The BFA5 cDNA sequence is shown in FIG. 11 and SEQ ID NO.: 32. The BFA5 amino acid sequence is shown in FIG. 12 and SEQ ID NO.: 33.

B. Immunoreactivity of BFA5

1. Activation of Human T Cells and IFN-.gamma. Secretion in ELISPOT

[0138] A library of 100 peptides from the BFA5/NYBR-11 coding sequence that are predicted to be medium to high binders to HLA-A*0201 were designed using Rammensee and Parker algorithms. The library was sub-divided into 10 pools of ten peptides (see Table XI), and each pool was used to activate 10 different T cell cultures after pulsing peptides on to mature autologous dendritic cells. Two experiments were performed with the library of BFA5/NYBR-1 peptides demonstrating immunoreactivity in HLA-A*0201 human T cells, as described below.

TABLE-US-00017 TABLE X Peptide CLP Group Number Sequence SEQ ID BFA5 2983 LMDMQTFKA 289 Group 1 2984 KVISPTKAL 290 2985 SIPTKALEL 291 2986 LELKNEQTL 292 2987 TVSQKDVCL 293 2988 SVPNKALEL 294 2989 CETVSQKDV 295 2990 KINGKLEES 296 2991 SLVEKTPDE 297 2991 SLCETVSQK 298 BFA5 2993 EIDKINGKL 299 Group 2 2994 MLLQQNVDV 300 2995 NMWLQQQLV 301 2996 FLVDRKCQL 302 2997 YLLHENCML 303 2998 SLFESSAKI 304 2999 KITIDIHFL 305 3000 QLQSKNMWL 306 3001 SLDQKLFQL 307 3002 FLLIKNANA 308 BFA5 3003 KILDTVHSC 309 Group 3 3004 SLSKILDTV 310 3005 ILIDSGADI 311 3006 KVMEINREV 312 3007 KLLSHGAVI 313 3009 AVYSETLSV 314 3010 KMNVDVSST 315 3011 ILSVVAKLL 316 3012 VLIAENTML 317 BFA5 3013 KLSKNHQNT 318 Group 4 3014 SLTPLLLSI 319 3015 SQYSGQLKV 320 3016 KELEVKQQL 321 3017 QTMEYIRKL 322 3018 AMLKLEIAT 323 3019 VLHQPLSEA 324 3020 GLLKATCGM 325 3021 GLLKANCGM 326 3022 QQLEQALRI 327 BFA5 3023 CMLKKEIAM 328 Group 5 3024 EQMKKKFCV 329 3025 IQDIELKSV 330 3026 SVPNKAFEL 331 3027 SIYQKVMEI 332 3028 NLNYAGDAL 333 3029 AVQDHDQIV 334 3030 LIAENTMLT 335 3031 FELKNEQTL 336 BFA5 3033 FESSQKIQV 337 Group 6 3034 GVTAEHYAV 338 3035 RVTSNKTKV 339 3036 TVSQKDVCV 340 3037 KSQEPAFHI 341 3038 KVLIAENTM 342 3039 MLKLEIATL 343 3040 EILSVVAKL 344 3041 MLKKETAML 345 3042 LLEKENEEI 346 BFA5 3043 ALRIQDIEL 347 Group 7 3044 KIREELGRI 348 3045 TLKLKEESL 349 3046 ILNEKIREE 350 3047 VLKKKLSEA 351 3048 GTSDKIQCL 352 3049 GADINLVDV 353 3050 ELCSVRLTL 354 3051 SVESNLNQV 355 3052 SLKINLNYA 356 BFA5 3053 KTPDEAASL 357 Group 8 3054 ATCGMKVSI 358 3055 LSHGAVIEV 359 3056 ETAMLKLEI 360 3057 AELQMTLKL 361 3058 VFAADICGV 362 3060 PAIEMQNSV 363 3061 EIFNYNNHL 364 3062 ILKEKNAEL 365 BFA5 3063 QLVHAHKKA 366 Group 9 3065 NIQDAQKRT 367 3066 NLVDVYGNM 368 3067 KCTALMLAV 369 3068 KTQCLEKAT 370 3069 KIAWEKKET 371 3070 IAWEKKEDT 372 3071 VGMLLQQNV 373 3072 VKTGCVARV 374 BFA5 3074 ALHYAVYSE 375 Group 10 3075 QMKKKFCVL 376 3076 ALQCHQEAC 377 3077 SEQIVEFLL 378 3078 AVIEVHNKA 379 3079 AVTCGFHHI 380 3080 ACLQRKMNV 381 3081 ALVEGTSDK 382

[0139] ELISPOT analysis was performed on human T-cell cultures activated through four rounds of stimulation with each pool of BFA5 peptides. In FIG. 13A, the numbers under the X-axis indicate the number of each peptide pool (1-10). Reactivity against a CMV pp65 peptide and a Flu matrix peptide were used as positive controls for T-cell activation in the experiments. Each experiment was performed with PBMC and dendritic cells from a single HLA-A*0201.sup.+ donor designated as "AP10". The results show that, although BFA4 is markedly reactive with high ELISPOT counts per 100,000 cells in the assay, BFA5 is even more reactive with 9/10 pools demonstrating ELISPOT reactivity. Similar results were obtained for both BFA4 and BFA5/NYBR-1 with a different HLA-A*0201. The bars reach a maximum at 600 spots because beyond that the ELISPOT reader does not give accurate counts. Cultures having a reading of 600 spots have more than this number of spots.

[0140] A large number of the BFA5 peptide pools of are reactive as shown by the high levels of lFN-.gamma. production (FIG. 13A). Each reactive peptide pool was then separated into individual peptides and analyzed for immunogenicity using ELISPOT analysis to isolate single reactive BFA5 peptides. As shown in FIG. 13B, BFA5 is highly immunogenic with several reactive single peptides than that of BFA4. Similar results were obtained in two independent PBMC culture experiments.

[0141] In addition to ELISPOT analysis, human T cells activated by BFA5 peptides were assayed to determine their ability to function as CTL. The cells were activated using peptide-pulsed dendritic cells followed by CD40 ligand-activated B cells (5 rounds of stimulation). The experiment shown was performed with isolated PBMC from HLA-A*0201.sup.+ donor AP31. Isolated T cells were tested in .sup.51Cr-release assays using peptide-loaded T2 cells. The % specific lysis at a 10:1, 5:1, and 1:1 T-cell to target ratio is shown for T2 cells pulsed with either pools of BFA5/NYBR-1 peptides or with individual peptides. The graph shows CTL activity induced against targets loaded with a c non-specific HLA-A*0201-binding HIV peptide (control) followed by the CTL activity against the peptide pool (Pool 1 etc.) and then the activity induced by individual peptides from the respective pool to the right. A high level of cytotoxicity was observed for some peptides at a 1:1 E:T ratio. CTL activity (percent specific lysis) induced by the control HIV peptide was generally <10%. Similar results were obtained with another PBMC donor expressing HLA-A*0201 (AP10). FIG. 13C shows that a large number of BFA5 peptides trigger T cell-mediated cytotoxicity of BFA5 peptide-loaded target cells. Table XI lists those peptides having immunogenic properties. Five peptides (LMDMQTFKA, ILIDSGADI, ILSVVAKLL, SQYSGQLKV, and ELCSVRLTL) were found to induce both IFN-.gamma. secretion and CTL activity in T cells from both donors.

TABLE-US-00018 TABLE XI Immunoreactive peptides from BFA5 BFA5 peptides eliciting high IFN-.gamma. release BFA5 peptides inducing (>200 spots/100,000 cells) CTL lysis of pulsed cells Donor AP10 Donor AP31 Donor AP10 Donor AP31 LMDMQTFKA LMDMQTFKA LMDMQTFKA LMDMQTFKA KVSIPTKAL KVSIPTKAL SIPTKALEL SIPTKALEL TVSQKDVCL SVPNKALEL YLLHENCML YLLHENCML YLLHENCML QLQSKNMWL QLQSKNMWL QLQSKNMWL SLSKILDTV SLSKILDTV SLSKILDTV ILIDSGADI ILIDSGADI ILIDSGADI ILIDSGADI KVMEINREV AVYSEILSV ILSVVAKLL ILSVVAKLL ILSVVAKLL ILSVVAKLL SLTPLLLSI SLTPLLLSI SLTPLLLSI SQYSGQLKV SQYSGQLKV SQYSGQLKV SQYSGQLKV QIMEYIRKL QIMEYIRKL QIMEYIRKL SVPNKAFEL NLNYAGDAL NLNYAGDAL GVTAEHYAV KSQEPAFHI MLKLEIATL MLKLEIATL MLKLEIATL MLKKEIAML ALRIQDIEL VLKKKLSEA ELCSVRLTL ELCSVRLTL ELCSVRLTL ELCSVRLTL SLKINLNYA SLKINLNYA SLKINLNYA ATCGMKVSI ATCGMKVSI AELQMTLKL AELQMTLKL AELQMTLKL VFAADICGV ILKEKNAEL ILKEKNAEL NLVDVYGNM NLVDVYGNM KCTALMLAV

C. Immunological Reagents

[0142] Polyclonal antisera were generated against the following series of 22- to 23-mer peptides of BFA5:

TABLE-US-00019 (CLP-2988; SEQ ID NO.: 383) BFA5(1-23) KLH-MTKRKKTINLNIQDAQKRTALHW (CLP-2978; SEQ ID NO.: 384) BFA5(312-334) KLH-TSEKFTWPAKGRPRKIAWEKKED (CLP-2979; SEQ ID NO.: 385) BFA5(612-634) KLH-DEILPSESKQKDYEENSWDTESL (CLP-2980; SEQ ID NO.: 386) BFA5(972-994) KLH-RLTLNQEEEKRRNADILNEKIRE (CLP-2981; SEQ ID NO.: 387) BFA5(1117-1139) KLH-AENTMLTSKLKEKQDKEILEAEI (CLP-2982; SEQ ID NO.: 388) BFA5(1319-1341) KLH-NYNNHLKNRIYQYEKEKAETENS

[0143] Prebleed samples from rabbits were processed and stored at -20.degree. C. Rabbits were immunized as follows: 1) the peptides were administered as an emulsion with Freund's Complete Adjuvant (FCA); and, 2) two weeks later, the peptides were coupled with Keyhole-Limpet Hemocyanin (KLH)-coupled and administered as an emulsion with Freund's Incomplete Adjuvant FIA. The following results were observed:

TABLE-US-00020 TABLE XII IgG titer .times. 10.sup.5 (after IgG titer .times. 10.sup.5 (after second first Immunization Immunization Peptide/protein Rb1/Rb2) Rb1/Rb2) CLP 2977 25/6 256/64 CLP 2978 25/25 64/256 CLP 2979 12/25 256/512 CLP 2980 25/12 1024/128 CLP 2981 8/4 256/64 CLP 2982 2/2 64/32

Prebleed Sample Results Exhibited IgG Titers <100 for all Samples.

[0144] To assess the quality of the polyclonal antisera, western blots were performed using sera against BFA5. Sera were separately screened against cell extracts obtained from the BT474, MDMB453, MCF-7, Calu-6, and CosA2 cells. The approximate expected MW.sub.r of BFA5 protein is 153 kDa. A 220 kD band was observed in the BT474 extract with CLP2980 antibody but not in the MDMB453 cell extracts however a .about.130 kD band was present in the MDMB453 extract. Both bands were found to be consistent with the polyclonal antibosera tested in this analysis, Neither of these bands is present in the negative control. Thus, it can be concluded that the polyclonal antisera are specific for BFA5.

Example 8

BCZ4 Tumor Antigen

A. BCZ4 Sequence

[0145] The BCZ4 sequence was detected as an over-expressed sequence in breast cancer samples. The nucleotide sequence and deduced amino acid sequence of BCZ4 are shown in FIG. 14, SEQ ID NO. 34 (BCZ4 cDNA), and SEQ ID NO. 35 (BCZ4 amino acid sequence).

B. Immunological Reagents for BCZ4 Breast Cancer Antigen:

[0146] A library of 100 nonamer peptides spanning the BCZ4 gene product was synthesized. The peptides were chosen based on their potential ability to bind to HLA-A*0201. Table XIII lists 100 nonamer peptide epitopes for HLA-A*0201 from the BCZ4 protein tested (see below):

TABLE-US-00021 TABLE XIII Peptide CLP Group Number Sequence SEQ ID BCZ4 3220 LDLETLTDI 389 Group 1 3221 DILQHQIRA 390 3222 ILQHQIRAV 391 3223 AVPFENLNI 392 3224 NLNIHCGDA 393 3225 AMDLGLEAI 394 3226 GLEAIFDQV 395 3227 LEAIFDQVV 396 3228 WCLQVNHLL 397 3229 QVNHLLYWA 398 BCZ4 3230 VNHLLYWAL 399 Group 2 3231 HLLYWALTT 400 3232 LLYWALTTI 401 3233 ALTTIGFET 402 3234 LTTIGFETT 403 3235 TTIGGETTM 404 3236 TIGFETTML 405 3237 TMLGGYVYS 406 3238 MLGGYVYST 407 3239 YSTGMIHLL 408 BCZ4 3240 STGMIHLLL 409 Group 3 3241 GMIHLLLQV 410 3242 MIHLLLQVT 411 3243 LLLQVTIDG 412 3244 GTIDGRNYI 413 3245 TIDGRNYIV 414 3246 YIVDAGFGR 415 3247 RSYQMWQPL 416 3248 YQMWQPLEL 417 3249 QMWQPLELI 418 BCZ4 3250 ISGKDQPQV 419 Group 4 3251 KDQPQVPCV 420 3252 PQVPCVFRL 421 3253 QVPCVFRLT 422 3254 RLTEENGFW 423 3255 TEENGFWYL 424 3256 NFGWYLDQI 425 3257 DQIRREQYI 426 3258 YIPNEEFLH 427 3259 YSFTLKPRT 428 BCZ4 3260 RTIEDFESM 429 Group 5 3261 YLQTSPSSV 430 3262 QTSPSSVFT 431 3263 SVFTSKSFC 432 3264 FTSKSFCSL 433 3265 CSLQTPDGV 434 3266 LQTPDGVHC 435 3267 QTPDGVHCL 436 3268 TPDGVHCLV 437 3269 GVHCLVGFT 438 BCZ4 3270 CLVGFTLTH 439 Group 6 3271 TLTHRRFNY 440 3272 FNYKDNTDL 441 3273 NTDLIEFKT 442 3274 TDLIEFKTL 443 3275 LSEEEIEKV 444 3276 KVLKNIFNI 445 3277 LKNIFNISL 446 3278 NISLQRKLV 447 3279 KHGDRFFTI 448 BCZ4 3280 DIEAYLERI 449 Group 7 3281 YLERIGYKK 450 3282 RNKLDLETL 451 3283 NKLDLETLT 452 3284 KLDLETLTD 453 3285 DLETLTDIL 454 3286 TLTDILQHQ 455 3287 LTDILQHQI 456 3288 QIRAVPFEN 457 3289 IRAVPFENL 458 BCZ4 3290 IHCGDAMDL 459 Group 8 3291 HCGDAMDLG 460 3292 DLGLEAIFD 461 3293 AIFDQVVRR 462 3294 GWCLQVNHL 463 3295 LQVNHLLYW 464 3296 GGYVYSTPA 465 3297 YCYSTPAKK 466 3298 STPAKKYST 467 3299 IHLLLQVTI 468 BCZ4 3300 HLLLQVTID 469 Group 9 3301 LLQVTIDGR 470 3302 YLDQIRREQ 471 3303 QYIPNEEFL 472 3304 FLHSDLLED 473 3305 DLLEDSKYR 474 3306 YRKIYSFTL 475 3307 KIYSFTLKP 476 3308 TLKPRTIED 477 3309 VHCLVGFTL 478 BCZ4 3310 LTHRRFNYK 479 Group 10 3311 DLIEFKTLS 480 3312 LIEFKTLSE 481 3313 FKTLSEEEI 482 3314 TLSEEEIEK 483 3315 EIEKVLKNI 484 3316 FNISLQRKL 485 3317 SLQRKLVPK 486 3318 KLVPKHGDR 487 3319 PKHGDRFFT 488

C. Immune reactivity of BCZ4 Peptides and Generation of Human Effector T Cells

[0147] Human PBMC from an HLA-A2.1 positive donor designated AP10 were activated with autologous dendritic cells pulsed with different pools of 9-mer peptides from the BCZ4 antigen (see Table XIII for list). The activated T cells were re-stimulated after 12 days with activated autologous CD40-ligand-activated B cells pulsed with the same respective peptide pools for another 8 to 10 days. This secondary activation was repeated more time for a total of 3 stimulations. The activated T cells were isolated after the 3.sup.rd stimulation and subjected to ELISPOT analysis for human IFN-.gamma. production against their respective BCZ4 peptide pools as shown (FIG. 15A). In FIG. 15A, the blue bars show reactivity against the BCZ4 peptide pools and the red bars are for an HLA-A2.1-binding HIV peptide as a negative control. Positive control HLA-A2.1-binding recall antigen peptides for CMV and flu were as used as positive control in the experiment. Standard deviations are indicated.

[0148] The experiment was repeated on activated T cells after an additional round of peptide stimulation with the similar results.

[0149] The peptide pools were deconvoluted using IFN-.gamma. ELISPOT assays (FIG. 15B), Human T cells from donor AP10 were stimulated with the different pools of BCZ4 peptides shown in Table XIII. Stimulation was performed as described earlier for the other antigens described. After 4 and 5 rounds of stimulation, T cells were harvested and subjected to ELISPOT analysis for IFN-.gamma. production with each individual peptide in each pool. The bars shown represent individual peptide reactivity for each specific pool. Table XIII identifies each of the reactive peptides. This experiment was repeated with similar results following another round of stimulation of AP10 donor T cells.

[0150] In addition to ELISPOT analysis, human T cells activated by BCZ4 peptides were assayed to determine their ability to function as CTL. The cells were activated using peptide-pulsed dendritic cells followed by CD40 ligand-activated B cells (5 rounds of stimulation). The experiment shown was performed with isolated PBMC from HLA-A*0201.sup.+ donor AP31. Isolated T cells were tested in .sup.51Cr-release assays using peptide-loaded T2 cells. The % specific lysis at a 10:1 T-cell to target ratio is shown for T2 cells pulsed with individual BCZ4 peptides. A high level of cytotoxicity was observed for some peptides (FIG. 15C). CTL activity (percent specific lysis) induced by the control HIV peptide was generally <10%. Similar results were obtained with another PBMC donor expressing HLA-A*0201 (AP10).

[0151] Table XIV lists the reactivity of the individual peptides:

TABLE-US-00022 TABLE XIV Peptides Peptides eliciting eliciting CTL activity strong IFN-.gamma. (peptide SEQ ELISPOT activity pulsed targets) ID CLP 3222 ILQHQIRAV ILQHQIRAV 391 CLP 3225 AMDLGLEAI 394 CLP 3226 GLEAIFDQV GLEAIFDQV 395 CLP 3227 LEAIFDQVV 396 CLP 3229 QVNHLLYWA 398 CLP 3231 HLLYWALTT 400 CLP 3232 LLYWALTTI LLYWALTTI 401 CLP 3235 TTIGFETTM 404 CLP 3237 TMLGGYVYS 406 CLP 3239 YSTGMIHLL 408 CLP 3240 STGMIHLLL 409 CLP 3248 YQMWQPLEL YQMEQPLEL 417 CLP 3260 RTIEDFESM 429 CLP 3261 YLQTSPSSV YLQTSPSSV 430 CLP 3266 LQTPDGVHC 435 CLP 3267 QTPDGVHCL 436 CLP 3268 TPDGVHCLV 437 CLP 3269 GVHCLVGFT 438 CLP 3271 TLTHRRFNY 440 CLP 3277 LKNIFNISL 446 CLP 3288 QIRAVPFEN 457 CLP 3289 IRAVPFENL 458 CLP 3294 GWCLQVNHL 463 CLP 3298 STPAKKYST 467 CLP 3299 IHLLLQVTI IHLLLQVTI 468 CLP 3301 LLQVTIDGR 470 CLP 3306 YRKIYSFTL 475 CLP 3307 KIYSFTLKP 476 CLP 3308 TLKPRTIED 477 CLP 3309 VHCLVGFTL 478 CLP 3317 SLQRKLVPK 486 CLP CC19 PKRGDRFFT 488

D. BCZ4 Expression Vectors

[0152] BCZ4 was PCR amplified using plasmid called pSporty/BCZ4 as the template using Platinum Taq (Invitrogen). Amplification conditions were as follows: 1) 94.degree. C. 2 minutes; 2) 35 cycles of 94.degree. C., 30 seconds, 53.degree. C. 30 seconds, 67.degree. C. 2.5 minutes; and, 3) 67.degree. C. 7 minutes. PCR primers were designed to include EcoRI restriction sites and directly flank the ORF (i.e., no extraneous sequence). Primer sequences were as follows: AS032F (forward primer) 5' GGAATTCAACATGGACATTGAAGCATATCTTAAGAATTG 3' (SEQ II NO.:591), AS034R (reverse primer) 5' GGAATTCCTGGTGAGCTGGATGACAAATAGACAAAGATTG 3' (SEQ ID NO.: 592). A Kozak sequence was also included in the forward primer. pcDNA3.1/Zeo(+) was cut with EcoRI and treated with CIP to prevent self-ligation. The BCZ4 amplicon was then ligated into EcoRI digested pcDNA3Zeo(+). Sequencing produced one clone (AS-579-5) which matched the expected BCZ4 sequence. BCZ4 protein was then expressed from this expression vector using standard techniques.

Example 9

BFY3 Tumor Antigen

A. BFY3 Sequence

[0153] The BFY3 sequence was detected as an over-expressed sequence in breast cancer samples. RT-PCR amplification of BFY3 w/EcoRI ends from HTB131 total RNA with AS007F (forward primer) 5' GGAATTCACCATGCTTTGGAAATTGACGGAT 3' (SEQ ID NO--: 593) and AS010R (reverse primer) 5' GGAATTCCTCACTTTCTGTGCT TCTC CTCTTTGTCA 3' (SEQ ID NO.: 594) was performed. PCR product was digested with EcoRI and cloned into EcoRI digested and CIP treated pcDNA3.1/Zeo(+) vector by ligation. Several positive clones were identified by restriction digestion and sequence results of AS-391-2 match expected BFY3 sequence. The nucleotide sequence and deduced amino acid sequence of BFY3 are shown in FIG. 16, SEQ ID NO. 36 (BFY3 cDNA), and SEQ ID NO. 37 (BFY3 amino acid sequence).

B. Immunological Reagents for BFY3 Breast Cancer Antigen

[0154] A library of 0.100 nonamer peptides spanning the BFY3 gene product was synthesized. The peptides were chosen based on their potential ability to bind to HLA-A*0201. Table XV lists 100 nonamer peptide epitopes for HLA-A*0201 from the BFY3 protein tested (see below):

TABLE-US-00023 TABLE XV Peptide CLP Group Number Sequence SEQ ID BFY3 3320 MLWKLTDNI 489 Group 1 3321 KLTDNIKYE 490 3322 GTSNGTARL 491 3323 NGTARLPQL 492 3324 ARLPQLGTV 493 3325 GTVGQSPYT 494 3326 SPYTSAPPL 495 3327 FQPPYFPPP 496 3328 YFPPPTQPI 497 3329 QSQDPYSHV 498 BFY3 3330 SHVNDPYSL 499 Group 2 3331 SLNPLHAQP 500 3332 RQSQESGLL 501 3333 GLLHTHRGL 502 3334 GLPHQLSGL 503 3335 GLDPRRDYR 504 3336 DLLHGPHAL 505 3337 LLHGPHALS 506 3338 ALSSGLGDL 507 3339 SSGLGDLSI 508 BFY3 3340 GLGDLSIHS 509 Group 3 3341 LGDLSIHSL 510 3342 SIHSLPHAI 511 3343 SLPHAIEEV 512 3344 HAIEEVPHV 513 3345 GINIPDQTV 514 3346 QTVIKKGPV 515 3347 VIKKGPVSL 516 3348 SLSKSNSNA 517 3349 SNSNAVSAI 518 BFY3 3350 AIPINKDNL 519 Group 4 3351 NLFGGVVNP 520 3352 FGGVVNPNE 521 3353 GGVVNPNEV 522 3355 NPNEVFCSV 523 3356 CSVPGRLSL 524 3357 SVPGRLSLL 525 3358 SLLSSTSKY 526 3359 LLSSTSKYK 527 BFY3 3360 LSSTSKYKV 528 Group 5 3361 STSKYKVTV 529 3362 KYKVTVAEV 530 3363 YKVTVAEVQ 531 3364 TVAEVQRRL 532 3365 RLSPPECLN 533 3366 LNASLLGGV 534 3367 NASLLGGVL 535 3368 SLLGGVLRR 536 3369 LLGGVLRRA 537 BFY3 3370 VLRRAKSKN 538 Group 6 3371 SLREKLDKI 539 3372 KLDKIGLNL 540 3373 KIGLNLPAG 541 3374 GLNLPAGRR 542 3375 NLPAGRRKA 543 3376 AGRRKAANV 544 3377 RKAANVTLL 545 3378 KAANVTLLT 546 3379 ANVTLLTSL 547 BFY3 3380 NVTLLTSLV 548 Group 7 3381 TLLTSLVEG 549 3382 LLTSLVEGE 550 3383 TSLVEGEAV 551 3384 SLVEGEAVH 552 3385 LVEGEAVHL 553 3386 VEGEAVHLA 554 3387 HLARDFGYV 555 3388 YVCETEFPA 556 3389 CETEFPAKA 557 BFY3 3390 AKAVAEFLN 558 Group 8 3391 AVAEFLNRQ 559 3392 FLNRQHSDP 560 3393 QVTRKNMLL 561 3394 NMLLATKQI 562 3395 MLLATKQIC 563 3396 LLATKQICK 564 3397 QICKEFTDL 565 3398 ICKEFTDLL 566 3399 LLAQDRSPL 567 BFY3 3400 ILEPGIQSC 568 Group 9 3401 LEPGIQSCL 569 3402 QSCLTHFNL 570 3403 SCLTHFNLI 571 3404 NLISHGFGS 572 3405 LISHGFGSP 573 3406 ISHGFGSPA 574 3407 SHGFGSPAV 575 3408 FGSPAVCAA 576 3409 GSPAVCAAV 577 BFY3 3410 AVCAAVTAL 578 Group 10 3411 AVTALQNYL 579 3412 VTALQNYLT 580 3413 ALQNYLTEA 581 3414 LQNYLTEAL 582 3415 YLTEALKAM 583 3416 LKAMDKMYL 584 3417 AMDKMYLSN 585 3418 KMYLSNNPN 586 3419 YLSNNPNSH 587

[0155] Human PBMC from an HLA-A2.1 positive donor designated AP31 were activated with autologous dendritic cells pulsed with different pools of 9-mer peptides from the BFY3 antigen (see Table 1 for list). The activated T cells were re-stimulated after 12 days with activated autologous CD40-ligand-activated B cells pulsed with the same respective peptide pools for another 8 to 10 days. This secondary activation was repeated 2 more time for a total of 4 stimulations. The activated T cells were isolated after the 4.sup.th stimulation and subjected to ELISPOT analysis for human IFN-.gamma. production against their respective BFY3 peptide pools as shown. The blue bars show reactivity against the BFY3 peptide pools and the red bars are for an HLA-A2.1-binding HIV peptide as a negative control. Standard deviations are indicated. The experiment was repeated 2 times on activated T cells from different rounds of peptide stimulation with the similar results (FIG. 17A).

[0156] The BFY3 peptide pools were deconvoluted and studied in IFN-.gamma. ELISPOT assays, Human T cells from donor AP10 were stimulated with the different pools of BFY3 peptides shown in Table XV. Stimulation was performed as described earlier for the other antigens described. After 4 rounds of stimulation, the T cells from each culture were harvested and subjected to ELISPOT analysis for IFN-.gamma. production with each individual peptide in each pool. FIG. 17B illustrates individual peptide reactivity for each specific pool.

[0157] In addition to ELISPOT analysis, human T cells activated by BFY3 peptides were assayed for reactivity. Ten pools of peptides consisting of ten peptides per pool used to generate CTL. These 10 groups of effectors used to kill targets pulsed with corresponding peptide pools. Peptides from pools 1, 3, 5, 6, and 7 found to be recognized, indicating that peptides in those pools are capable of generating CTL (FIG. 17C). From these ten pools, peptides 3344, 3320, 3378, 2272, and 3387 were strongly recognized by CTL (FIG. 17D). "Moderately recognized" peptides include 3369, 3355, and 336218D (FIG. 17D). CosA2 cells transfected with BFY3 were killed by CTL generated from pools 1 and 3 indicating that processed and presented epitopes from these pools are immunologically relevant (FIG. 17E). The peptides responsible for this cytotoxicity are 3320 and 3344. Table XVI summarizes the properties of the BFY3 peptides.

TABLE-US-00024 TABLE XVI Summary of Immunoreactive BFY3 Nonamer Peptides Peptides Peptides eliciting eliciting CTL activity strong IFN-.gamma. (peptide SEQ ELISPOT activity pulsed targets) ID CLP 3320 MLWKLTDNI MLWKLTDNI 489 CLP 3343 SLPHAIEEV 512 CLP 3344 HAIEEVPHV HAIEEVPHV 513 CLP 3351 NLFGGVVNP 520 CLP 3362 KYKVTVAEV KYKVTVAEV 530 CLP 3366 LNASLLGGV 534 CLP 3369 LLGGVLRRA LLGGVLRRA 537 CLP 3372 KLDKIGLNL KLDKIGLNL 540 CLP 3378 KAANVTLLT KAANVTLLT 546 CLP 3380 NVTLLTSLV 548 CLP 3387 HLARDFGYV HLARDFGYV 555 CLP 3403 SCLTHFNLI 571 CLP 3407 SHGFGSPAV 575 CLP 3415 YLTEALKAM 583

C. BFY3 Expression Vectors

[0158] To construct a BFY3 expression vector, RT-PCT amplification of BFY3 w/EcoRI ends from HTB131 total RNA with AS007F (forward primer) 5' GGAATTCACCATGCTTTGGAAATTGACGGAT 3' (SEQ ID NO.: 595) and AS010R (reverse primer) 5' GGAATTCCTCACTTTCTGTGCTTCTCCTCTTTGTCA 3' (SEQ ID NO.: 596) was performed. PCR was performed using standard techniques. The amplified product was digested with EcoRI and cloned into CIP treated pcDNA3.1/Zeo(+) vector by ligation using standard techniques. Several positive clones were identified by restriction digestion and sequenced. Sequencing indicated that the sequence of clone AS-391-2 matched the expected BFY3 sequence, BFY3 protein was then expressed from the BFY3 expression vector using standard techniques.

Example 10

Expression Vectors Encoding Multiple Tumor Antigens

[0159] In certain instances, it may be desirable to construct expression vectors encoding multiple tumor antigens. It has been determined that certain combinations of antigens, when combined into a single expression vector, encompasses the expression profiles of many patients in a single vector. For instance, one study of breast cancer samples from different patients indicated that the combination of BFA4 and BFA5 covered expression profiles of 74% of the samples; the combination of BCY1 and BFA5 covered 65% of the samples; the combination of BCZ4 and BFA5 covered 69% of the samples; the combination of BFY3 and BFA5 covered 67% of the samples; the combination of BCY1, BFA4 and BFA5 covered 78% of the samples; the combination of BCZ4, BFA4 and BFA5 covered 81% of the samples; and, the combination of BFY3, BFA4, and BFA5 covered 74% of the samples. Accordingly, a multi-antigen expression construct may be built such that the most common expression profiles among breast cancer patients may be addressed using a single vector. Such a multiantigen expression vector is constructed using standard cloning techniques positioning nucleic acids encoding each of the tumor antigen sequences in proximity to a promoter or other transcriptional regulatory sequence. The expression vector may be engineered such that each nucleotide sequence encoding a tumor antigen is operably linked to a specific promoter, or the tumor antigens may collectively be operably linked to a single promoter and expressed as a single expression unit. Where a single expression unit is constructed, nucleotide sequences useful in separating the tumor antigen sequences following expression may be inserted between the tumor antigen sequences. Sequences useful for include IRES sequences, nucleotide sequences encoding amino acid sequences corresponding to protease cleavage sites, and the like. Suitable vectors for constructing such multiantigen expression vectors include, for example, poxviruses such as vaccinia, avipox, ALVAC and NYVAC.

[0160] While the present invention has been described in terms of the preferred embodiments, it is understood that variations and modifications will occur to those skilled in the art. Therefore, it is intended that the appended claims cover all such equivalent variations that come within the scope of the invention as claimed.

Sequence CWU 1

1

59713562DNAHomo sapiensmisc_feature(1663)..(1663)n is A, T, G, or C 1agcaggaccg gggcctgtgt cgctatgggt tcccccgccg ccccggaggg agcgctgggc 60tacgtccgcg agttcactcg ccactcctcc gacgtgctgg gcaacctcaa cgagctgcgc 120ctgcgcggga tcctcactga cgtcacgctg ctggttggcg ggcaacccct cagagcacac 180aaggcagttc tcatcgcctg cagtggcttc ttctattcaa ttttccgggg ccgtgcggga 240gtcggggtgg acgtgctctc tctgcccggg ggtcccgaag cgagaggctt cgcccctcta 300ttggacttca tgtacacttc gcgcctgcgc ctctctccag ccactgcacc agcagtccta 360gcggccgcca cctatttgca gatggagcac gtggtccagg catgccaccg cttcatccag 420gccagctatg aacctctggg catctccctg cgccccctgg aagcagaacc cccaacaccc 480ccaacggccc ctccaccagg tagtcccagg cgctccgaag gacacccaga cccacctact 540gaatctcgaa gctgcagtca aggccccccc agtccagcca gccctgaccc caaggcctgc 600aactggaaaa agtacaagta catcgtgcta aactctcagg cctcccaagc agggagcctg 660gtcggggaga gaagttctgg tcaaccttgc ccccaagcca ggctccccag tggagacgag 720gcctccagca gcagcagcag cagcagcagc agcagcagtg aagaaggacc cattcctggt 780ccccagagca ggctctctcc aactgctgcc actgtgcagt tcaaatgtgg ggctccagcc 840agtaccccct acctcctcac atcccaggct caagacacct ctggatcacc ctctgaacgg 900gctcgtccac taccgggagt gaatttttca gctgccagaa ctgtgaggct gtggcagggt 960gctcatcggg ggctggactc cttggttcct ggggacgaag acaaacccta taagtgtcag 1020ctgtgccggt cttcgttccg ctacaagggc aaccttgcca gtcaccgtac agtgcacaca 1080ggggaaaagc cttaccactg ctcaatctgc ggagcccgtt ttaaccggcc agcaaacctg 1140aaaacgcaca gccgcatcca ttcgggagag aagccgtata agtgtgagac gtgcggctcg 1200cgctttgtac aggtggcaca tctgcgggcg cacgtgctga tccacaccgg ggagaagccc 1260tacccttgcc ctacctgcgg aacccgcttc cgccacctgc agaccctcaa gagccacgtt 1320cgcatccaca ccggagagaa gccttaccac tgcgacccct gtggcctgca tttccggcac 1380aagagtcaac tgcggctgca tctgcgccag aaacacggag ctgctaccaa caccaaagtg 1440cactaccaca ttctcggggg gccctagctg agcgcaggcc caggccccac ttgcttcctg 1500cgggtgggaa agctgcaggc ccaggccttg cttccctatc aggcttgggc ataggggtgt 1560gccaggccac tttggtatca gaaattgcca ccctcttaat ttctcactgg ggagagcagg 1620ggtggcagat cctggctaga tctgcctctg ttttgctggt canaccctct tccccacaag 1680ccagattgtt tctgaggaga gagctagcta ggggctggga aaggggagag attggagtcc 1740tggtctccct aagggaatag ccctccacct gtggccccca ttgcattcag tttatctgta 1800aaatataatt tattgaggcc tttgggtggc accggggcct tcattcgatt gcatttccca 1860ctcccctctt ccacaagtgt gattaaaagt gaccagaaac acagaaggtg agatcacagc 1920tctgctggca gagattacta gcccttggct ctctcgtttg gcttgggtat tttatattat 1980ttctgtcata acttttatct ttagaattgt tctttctcct gtttgtttgc ttgttagttt 2040gtttaaaatg gaaaaagggg ttctctgtgt tctgcccctg taattctagg tctggaacct 2100ttatttgttc tagggcagct ctgggaacat gcgggattgt ggaattgggt caggaaccct 2160ctctggtatt ctggatgttg taggttctct agcagtctag aaatggatac agacatttct 2220ctgttcttca agggtgatag gaaccattat gttgagccca aaatggaagt aataataaat 2280gcctcctgga ggctgtgggt gtgggggatt ctgtatctgg attccgtatc actccaactg 2340gaggctgtgg gtgtggggga ttctgtatct ggattccgta tcactccaag tggaggctgg 2400caggtttttc tgcaagatgg tccagaatct aaaatgtccc attaatctgg tcacttgggt 2460ttggctctgc tgtatccatc tatagtggta gagacccacc agggctcaag tggagtccat 2520catcctccca cgggggcctg ttcttagtac tgagttgatc gctccatggg ggagagatca 2580gacattcctt atcagagatg atgtgacctt ttctgactct gcccagtctc tatgaatgtt 2640atggcctagg gaagaatcat gaaactcttt agcttgatta gatggtaaac agtgttaacc 2700catcctttac tacagaggca tatgggtttg aatgttacct ggggttctct ctattgagtt 2760gagccccttc ttcctttagt gggttttgga catcttctgg caagtgtcca gatgccagaa 2820ccttcttttc ctctagaagg gatggtgctt ggtaacctta ccttttaaaa gctgggtctg 2880tgacctggtc ttcccatccc tgcattcctg tctggaacca gtgaatgcat tagaaccttc 2940cataggaaaa gaaaaggggc tgagttccat tctgggtttg ctgtagtttg gttgggatta 3000ttgttggcat tacagatgta aaagattgac tagcccatag gccaaaggcc tgttctagtt 3060gaccaagttt caagtaggat taagaggttg gttgaggggt gcagtttctg gtgtaggcca 3120ggtaggtaga aagtgaggaa cagggttgcc tcttggctgg gtggagtctc tgaaatgtta 3180gaagaagcgc tgaagccttg attgatagtt ctgccccttg ttgccctggg gcttatctga 3240ttatgggacg agggtagaaa gtaagaagca cttttgaatt tgtggggtag aacttcaaca 3300ataagtcagt tctagtggct gtcgcctggg gactagtgag aaagctactc ttctccctct 3360tccctctttc tccccatggc cccactgcag aattaaagaa ggaagaaggg aaggcggagg 3420agtctataag aaggaatcat gatttctatt tagcagattg gatgggcagg tggagaatgc 3480ctgggggtag aaatgttaga tcttgcaaca tcagatcctt ggaataaaga agcctctctg 3540cgcaaaaaaa aaaaaaaaaa aa 35622480PRTHomo sapiens 2Met Gly Ser Pro Ala Ala Pro Glu Gly Ala Leu Gly Tyr Val Arg Glu 1 5 10 15 Phe Thr Arg His Ser Ser Asp Val Leu Gly Asn Leu Asn Glu Leu Arg 20 25 30 Leu Arg Gly Ile Leu Thr Asp Val Thr Leu Leu Val Gly Gly Gln Pro 35 40 45 Leu Arg Ala His Lys Ala Val Leu Ile Ala Cys Ser Gly Phe Phe Tyr 50 55 60 Ser Ile Phe Arg Gly Arg Ala Gly Val Gly Val Asp Val Leu Ser Leu 65 70 75 80 Pro Gly Gly Pro Glu Ala Arg Gly Phe Ala Pro Leu Leu Asp Phe Met 85 90 95 Tyr Thr Ser Arg Leu Arg Leu Ser Pro Ala Thr Ala Pro Ala Val Leu 100 105 110 Ala Ala Ala Thr Tyr Leu Gln Met Glu His Val Val Gln Ala Cys His 115 120 125 Arg Phe Ile Gln Ala Ser Tyr Glu Pro Leu Gly Ile Ser Leu Arg Pro 130 135 140 Leu Glu Ala Glu Pro Pro Thr Pro Pro Thr Ala Pro Pro Pro Gly Ser 145 150 155 160 Pro Arg Arg Ser Glu Gly His Pro Asp Pro Pro Thr Glu Ser Arg Ser 165 170 175 Cys Ser Gln Gly Pro Pro Ser Pro Ala Ser Pro Asp Pro Lys Ala Cys 180 185 190 Asn Trp Lys Lys Tyr Lys Tyr Ile Val Leu Asn Ser Gln Ala Ser Gln 195 200 205 Ala Gly Ser Leu Val Gly Glu Arg Ser Ser Gly Gln Pro Cys Pro Gln 210 215 220 Ala Arg Leu Pro Ser Gly Asp Glu Ala Ser Ser Ser Ser Ser Ser Ser 225 230 235 240 Ser Ser Ser Ser Ser Glu Glu Gly Pro Ile Pro Gly Pro Gln Ser Arg 245 250 255 Leu Ser Pro Thr Ala Ala Thr Val Gln Phe Lys Cys Gly Ala Pro Ala 260 265 270 Ser Thr Pro Tyr Leu Leu Thr Ser Gln Ala Gln Asp Thr Ser Gly Ser 275 280 285 Pro Ser Glu Arg Ala Arg Pro Leu Pro Gly Val Asn Phe Ser Ala Ala 290 295 300 Arg Thr Val Arg Leu Trp Gln Gly Ala His Arg Gly Leu Asp Ser Leu 305 310 315 320 Val Pro Gly Asp Glu Asp Lys Pro Tyr Lys Cys Gln Leu Cys Arg Ser 325 330 335 Ser Phe Arg Tyr Lys Gly Asn Leu Ala Ser His Arg Thr Val His Thr 340 345 350 Gly Glu Lys Pro Tyr His Cys Ser Ile Cys Gly Ala Arg Phe Asn Arg 355 360 365 Pro Ala Asn Leu Lys Thr His Ser Arg Ile His Ser Gly Glu Lys Pro 370 375 380 Tyr Lys Cys Glu Thr Cys Gly Ser Arg Phe Val Gln Val Ala His Leu 385 390 395 400 Arg Ala His Val Leu Ile His Thr Gly Glu Lys Pro Tyr Pro Cys Pro 405 410 415 Thr Cys Gly Thr Arg Phe Arg His Leu Gln Thr Leu Lys Ser His Val 420 425 430 Arg Ile His Thr Gly Glu Lys Pro Tyr His Cys Asp Pro Cys Gly Leu 435 440 445 His Phe Arg His Lys Ser Gln Leu Arg Leu His Leu Arg Gln Lys His 450 455 460 Gly Ala Ala Thr Asn Thr Lys Val His Tyr His Ile Leu Gly Gly Pro 465 470 475 480 33676DNAHomo sapiens 3tctgcgtgtg ccggggctag gggctggaag tcctggctct agttgcacct cggaaggaaa 60aggcaaacag aggagggaag gcgtcttagg actgcctgga tccagagcac tttcctcggc 120ctctacaggc ctgtgtcgct atgggttccc ccgccgcccc ggagggagcg ctgggctacg 180tccgcgagtt cactcgccac tcctccgacg tgctgggcaa cctcaacgag ctgcgcctgc 240gcgggatcct cactgacgtc acgctgctgg ttggcgggca acccctcaga gcacacaagg 300cagttctcat cgcctgcagt ggcttcttct attcaatttt ccggggccgt gcgggagtcg 360gggtggacgt gctctctctg cccgggggtc ccgaagcgag aggcttcgcc cctctattgg 420acttcatgta cacttcgcgc ctgcgcctct ctccagccac tgcaccagca gtcctagcgg 480ccgccaccta tttgcagatg gagcacgtgg tccaggcatg ccaccgcttc atccaggcca 540gctatgaacc tctgggcatc tccctgcgcc ccctggaagc agaaccccca acacccccaa 600cggcccctcc accaggtagt cccaggcgct ccgaaggaca cccagaccca cctactgaat 660ctcgaagctg cagtcaaggc ccccccagtc cagccagccc tgaccccaag gcctgcaact 720ggaaaaagta caagtacatc gtgctaaact ctcaggcctc ccaagcaggg agcctggtcg 780gggagagaag ttctggtcaa ccttgccccc aagccaggct ccccagtgga gacgaggcct 840ccagcagcag cagcagcagc agcagcagca gtgaagaagg acccattcct ggtccccaga 900gcaggctctc tccaactgct gccactgtgc agttcaaatg tggggctcca gccagtaccc 960cctacctcct cacatcccag gctcaagaca cctctggatc accctctgaa cgggctcgtc 1020cactaccggg aagtgaattt ttcagctgcc agaactgtga ggctgtggca gggtgctcat 1080cggggctgga ctccttggtt cctggggacg aagacaaacc ctataagtgt cagctgtgcc 1140ggtcttcgtt ccgctacaag ggcaaccttg ccagtcatcg tacagtgcac acaggggaaa 1200agccttacca ctgctcaatc tgcggagccc gttttaaccg gccagcaaac ctgaaaacgc 1260acagccgcat ccattcggga gagaagccgt ataagtgtga gacgtgcggc tcgcgctttg 1320tacaggtggc acatctgcgg gcgcacgtgc tgatccacac cggggagaag ccctaccctt 1380gccctacctg cggaacccgc ttccgccacc tgcagaccct caagagccac gttcgcatcc 1440acaccggaga gaagccttac cactgcgacc cctgtggcct gcatttccgg cacaagagtc 1500aactgcggct gcatctgcgc cagaaacacg gagctgctac caacaccaaa gtgcactacc 1560acattctcgg ggggccctag ctgagcgcag gcccaggccc cacttgcttc ctgcgggtgg 1620gaaagctgca ggcccaggcc ttgcttccct atcaggcttg ggcatagggg tgtgccaggc 1680cactttggta tcagaaattg ccaccctctt aatttctcac tggggagagc aggggtggca 1740gatcctggct agatctgcct ctgttttgct ggtcaaaacc tcttccccac aagccagatt 1800gtttctgagg agagagctag ctaggggctg ggaaagggga gagattggag tcctggtctc 1860cctaagggaa tagccctcca cctgtggccc ccattgcatt cagtttatct gtaaatataa 1920tttattgagg cctttgggtg gcaccggggc cttcattcga ttgcatttcc cactcccctc 1980ttccacaagt gtgattaaaa gtgaccagaa acacagaagg tgagatcaca gctctgctgg 2040cagagattac tagcccttgg ctctctcgtt tggcttgggt attttatatt atttctgtca 2100taacttttat ctttagaatt gttctttctc ctgtttgttt gcttgttagt ttgtttaaaa 2160tggaaaaagg ggttctctgt gttctgcccc tgtaattcta ggtctggaac ctttatttgt 2220tctagggcag ctctgggaac atgcgggatt gtggaattgg gtcaggaacc ctctctggta 2280ttctggatgt tgtaggttct ctagcagtct agaaatggat acagacattt ctctgttctt 2340caagggtgat aggaaccatt atgttgagcc caaaatggaa gtaataataa atgcctcctg 2400gaggctgtgg gtgtggggga ttctgtatct ggattccgta tcactccaac tggaggctgt 2460gggtgtgggg gattctgtat ctggattccg tatcactcca agtggaggct ggcaggtttt 2520tctgcaagat ggtccagaat ctaaaatgtc ccattaatct ggtcacttgg gtttggctct 2580gctgtatcca tctatagtgg tagagaccca ccagggctca agtggagtcc atcatcctcc 2640cacgggggcc tgttcttagc actgagttga tcgctccatg ggggagagat cagacattcc 2700ttatcagaga tgatgtgacc ttttctgact ctgcccagtc tctatgaatg ttatggccta 2760gggaagaatc atgaaactct ttagcttgat tagatggtaa acagtgttaa cccatccttt 2820actacagagg catatgggtt tgaatgttac ctggggttct ctctattgag ttgagcccct 2880tcttccttta gtgggttttg gacatcttct ggcaagtgtc cagatgccag aaccttcttt 2940tcctctagaa gggatggtgc ttggtaacct taccttttaa aagctgggtc tgtgacctgg 3000tcttcccatc cctgcattcc tgtctggaac cagtgaatgc attagaacct tccataggaa 3060aagaaaaggg gctgagttcc attctgggtt tgctgtagtt tggttgggat tattgttggc 3120attacagatg taaaagattg actagcccat aggccaaagg cctgttctag ttgaccaagt 3180ttcaagtagg attaagaggt tggttgaggg gtgcagtttc tggtgtaggc caggtaggta 3240gaaagtgagg aacagggttg cctcttggct gggtggagtc tctgaaatgt tagaagaagc 3300gctgaagcct tgattgatag ttctgcccct tgttgccctg gggcttatct gattatggga 3360cgagggtaga aagtaagaag cacttttgaa tttgtggggt agaacttcaa caataagtca 3420gttctagtgg ctgtcgcctg gggactagtg agaaagctac tcttctccct cttccctctt 3480tctccccatg gccccactgc agaattaaag aaggaagaag ggaaggcgga ggagtctata 3540agaaggaatc atgatttcta tttagcagat tggatgggca ggtggagaat gcctgggggt 3600agaaatgtta gatcttgcaa catcagatcc ttggaataaa gaagcctctc tgygcwraaa 3660aaaaaaaaaa aaaaaa 367641440DNAHomo sapiens 4atgggttccc ccgccgcccc ggagggagcg ctgggctacg tccgcgagtt cactcgccac 60tcctccgacg tgctgggcaa cctcaacgag ctgcgcctgc gcgggatcct cactgacgtc 120acgctgctgg ttggcgggca acccctcaga gcacacaagg cagttctcat cgcctgcagt 180ggcttcttct attcaatttt ccggggccgt gcgggagtcg gggtggacgt gctctctctg 240cccgggggtc ccgaagcgag aggcttcgcc cctctattgg acttcatgta cacttcgcgc 300ctgcgcctct ctccagccac tgcaccagca gtcctagcgg ccgccaccta tttgcagatg 360gagcacgtgg tccaggcatg ccaccgcttc atccaggcca gctatgaacc tctgggcatc 420tccctgcgcc ccctggaagc agaaccccca acacccccaa cggcccctcc accaggtagt 480cccaggcgct ccgaaggaca cccagaccca cctactgaat ctcgaagctg cagtcaaggc 540ccccccagtc cagccagccc tgaccccaag gcctgcaact ggaaaaagta caagtacatc 600gtgctaaact ctcaggcctc ccaagcaggg agcctggtcg gggagagaag ttctggtcaa 660ccttgccccc aagccaggct ccccagtgga gacgaggcct ccagcagcag cagcagcagc 720agcagcagca gtgaagaagg acccattcct ggtccccaga gcaggctctc tccaactgct 780gccactgtgc agttcaaatg tggggctcca gccagtaccc cctacctcct cacatcccag 840gctcaagaca cctctggatc accctctgaa cgggctcgtc cactaccggg aagtgaattt 900ttcagctgcc agaactgtga ggctgtggca gggtgctcat cggggctgga ctccttggtt 960cctggggacg aagacaaacc ctataagtgt cagctgtgcc ggtcttcgtt ccgctacaag 1020ggcaaccttg ccagtcatcg tacagtgcac acaggggaaa agccttacca ctgctcaatc 1080tgcggagccc gttttaaccg gccagcaaac ctgaaaacgc acagccgcat ccattcggga 1140gagaagccgt ataagtgtga gacgtgcggc tcgcgctttg tacaggtggc acatctgcgg 1200gcgcacgtgc tgatccacac cggggagaag ccctaccctt gccctacctg cggaacccgc 1260ttccgccacc tgcagaccct caagagccac gttcgcatcc acaccggaga gaagccttac 1320cactgcgacc cctgtggcct gcatttccgg cacaagagtc aactgcggct gcatctgcgc 1380cagaaacacg gagctgctac caacaccaaa gtgcactacc acattctcgg ggggccctag 14405479PRTHomo sapiens 5Met Gly Ser Pro Ala Ala Pro Glu Gly Ala Leu Gly Tyr Val Arg Glu 1 5 10 15 Phe Thr Arg His Ser Ser Asp Val Leu Gly Asn Leu Asn Glu Leu Arg 20 25 30 Leu Arg Gly Ile Leu Thr Asp Val Thr Leu Leu Val Gly Gly Gln Pro 35 40 45 Leu Arg Ala His Lys Ala Val Leu Ile Ala Cys Ser Gly Phe Phe Tyr 50 55 60 Ser Ile Phe Arg Gly Arg Ala Gly Val Gly Val Asp Val Leu Ser Leu 65 70 75 80 Pro Gly Gly Pro Glu Ala Arg Gly Phe Ala Pro Leu Leu Asp Phe Met 85 90 95 Tyr Thr Ser Arg Leu Arg Leu Ser Pro Ala Thr Ala Pro Ala Val Leu 100 105 110 Ala Ala Ala Thr Tyr Leu Gln Met Glu His Val Val Gln Ala Cys His 115 120 125 Arg Phe Ile Gln Ala Ser Tyr Glu Pro Leu Gly Ile Ser Leu Arg Pro 130 135 140 Leu Glu Ala Glu Pro Pro Thr Pro Pro Thr Ala Pro Pro Pro Gly Ser 145 150 155 160 Pro Arg Arg Ser Glu Gly His Pro Asp Pro Pro Thr Glu Ser Arg Ser 165 170 175 Cys Ser Gln Gly Pro Pro Ser Pro Ala Ser Pro Asp Pro Lys Ala Cys 180 185 190 Asn Trp Lys Lys Tyr Lys Tyr Ile Val Leu Asn Ser Gln Ala Ser Gln 195 200 205 Ala Gly Ser Leu Val Gly Glu Arg Ser Ser Gly Gln Pro Cys Pro Gln 210 215 220 Ala Arg Leu Pro Ser Gly Asp Glu Ala Ser Ser Ser Ser Ser Ser Ser 225 230 235 240 Ser Ser Ser Ser Glu Glu Gly Pro Ile Pro Gly Pro Gln Ser Arg Leu 245 250 255 Ser Pro Thr Ala Ala Thr Val Gln Phe Lys Cys Gly Ala Pro Ala Ser 260 265 270 Thr Pro Tyr Leu Leu Thr Ser Gln Ala Gln Asp Thr Ser Gly Ser Pro 275 280 285 Ser Glu Arg Ala Arg Pro Leu Pro Gly Ser Glu Phe Phe Ser Cys Gln 290 295 300 Asn Cys Glu Ala Val Ala Gly Cys Ser Ser Gly Leu Asp Ser Leu Val 305 310 315 320 Pro Gly Asp Glu Asp Lys Pro Tyr Lys Cys Gln Leu Cys Arg Ser Ser 325 330 335 Phe Arg Tyr Lys Gly Asn Leu Ala Ser His Arg Thr Val His Thr Gly 340 345 350 Glu Lys Pro Tyr His Cys Ser Ile Cys Gly Ala Arg Phe Asn Arg Pro 355 360 365 Ala Asn Leu Lys Thr His Ser Arg Ile His Ser Gly Glu Lys Pro Tyr 370 375 380 Lys Cys Glu Thr Cys Gly Ser Arg Phe Val Gln Val Ala His Leu Arg 385 390 395 400 Ala His Val Leu Ile His Thr Gly Glu Lys Pro Tyr Pro Cys Pro Thr 405 410 415 Cys Gly Thr Arg Phe Arg His Leu Gln Thr Leu Lys Ser His Val Arg 420 425 430 Ile His Thr Gly Glu Lys Pro Tyr His Cys Asp Pro Cys Gly Leu His 435 440 445 Phe Arg His Lys Ser Gln Leu Arg Leu His Leu Arg Gln Lys His Gly 450 455 460

Ala Ala Thr Asn Thr Lys Val His Tyr His Ile Leu Gly Gly Pro 465 470 475 627DNAHomo sapiens 6caccatgggt tcccccgccg ccccgga 27733DNAHomo sapiens 7ctagggcccc ccgagaatgt ggtagtgcac ttt 33865DNAHomo sapiens 8atacccggaa ctccctaagc cttctattag ctccaataat agtaagcctg tcgaagacaa 60agatg 65970DNAHomo sapiens 9gcctgtgtcc cctagactcc aactcagcaa cggaaataga actctgaccc tgtttaacgt 60gaccaggaac 701070DNAHomo sapiens 10acgtgcttta cggacccgat gctcctacaa tcagccctct aaacacaagc tatagatcag 60gggaaaatct 701170DNAHomo sapiens 11acgttaaaca gggtcagagt tctatttccg ttgctgagtt ggagtctagg ggacacaggc 60agggactggt 701270DNAHomo sapiens 12ctgatctata gcttgtgttt agagggctga ttgtaggagc atcgggtccg taaagcacgt 60tgagaatcac 701363DNAHomo sapiens 13gatccactat tgttcacggt aatattggga atgaacagtt cctgggtgga ctgttggaaa 60gtg 631470DNAHomo sapiens 14gacacagcaa gctacaaatg cgaaacccaa aatccagtca gcgccaggag gtctgattca 60gtgattctca 701570DNAHomo sapiens 15tgaatcagac ctcctggcgc tgactggatt ttgggtttcg catttgtagc ttgctgtgtc 60gttcctggtc 701679DNAHomo sapiens 16gatcctacac gtgccaagct cacaatagcg acaccggact caaccgcaca accgtgacga 60cgattaccgt gtatgccga 791770DNAHomo sapiens 17catcctcaac tgggttagaa ttgttactag ttatgaatgg ttttggtggc tcggcataca 60cggtaatcgt 701880DNAHomo sapiens 18ttctaaccca gttgaggatg aggacgcagt tgcattaact tgtgagccag agattcaaaa 60taccacttat ttatggtggg 801980DNAHomo sapiens 19gtctaatgat aaccgcacat tgacactcct gtccgttact cgcaatgatg taggacctta 60tgagtgtggc attcagaatg 802080DNAHomo sapiens 20tttgtatggc ccagacgacc caactatatc tccatcatac acctactacc gtcccggcgt 60gaacttgagc ctttcttgcc 802180DNAHomo sapiens 21tgatggaaac attcagcagc atactcaaga gttatttata agcaacataa ctgagaagaa 60cagcggactc tatacttgcc 802280DNAHomo sapiens 22taaaacaata actgtttccg cggagctgcc caagccctcc atctccagca acaactccaa 60acccgtggag gacaaggatg 802380DNAHomo sapiens 23atgtgcggtt atcattagac aactgcaagc gtgggctaac cggcaaactt tggttattga 60cccaccataa ataagtggta 802480DNAHomo sapiens 24ggtcgtctgg gccatacaaa acattaagga taacagggtc ggagtgatca acggataatt 60cattctgaat gccacactca 802580DNAHomo sapiens 25gctgctgaat gtttccatca atcagccagg agtactgtgc aggggggttg gatgctgcat 60ggcaagaaag gctcaagttc 802680DNAHomo sapiens 26cggaaacagt tattgtttta actgtagtcc tgctgtgacc actggctgag ttattggcct 60ggcaagtata gagtccgctg 802747DNAHomo sapiens 27cctcaggttc acaggtgaag gccacagcat ccttgtcctc cacgggt 47283846DNAHomo sapiens 28atggtccgga aaaagaaccc ccctctgaga aacgttgcaa gtgaaggcga gggccagatc 60ctggagccta taggtacaga aagcaaggta tctggaaaga acaaagaatt ctctgcagat 120cagatgtcag aaaatacgga tcagagtgat gctgcagaac taaatcataa ggaggaacat 180agcttgcatg ttcaagatcc atcttctagc agtaagaagg acttgaaaag cgcagttctg 240agtgagaagg ctggcttcaa ttatgaaagc cccagtaagg gaggaaactt tccctccttt 300ccgcatgatg aggtgacaga cagaaatatg ttggctttct catttccagc tgctggggga 360gtctgtgagc ccttgaagtc tccgcaaaga gcagaggcag atgaccctca agatatggcc 420tgcaccccct caggggactc actggagaca aaggaagatc agaagatgtc accaaaggct 480acagaggaaa cagggcaagc acagagtggt caagccaatt gtcaaggttt gagcccagtt 540tcagtggcct caaaaaaccc acaagtgcct tcagatgggg gtgtaagact gaataaatcc 600aaaactgact tactggtgaa tgacaaccca gacccggcac ctctgtctcc agagcttcag 660gactttaaat gcaatatctg tggatatggt tactacggca acgaccccac agatctgatt 720aagcacttcc gaaagtatca cttaggactg cataaccgca ccaggcaaga tgctgagctg 780gacagcaaaa tcttggccct tcataacatg gtgcagttca gccattccaa agacttccag 840aaggtcaacc gttctgtgtt ttctggtgtg ctgcaggaca tcaattcttc aaggcctgtt 900ttactaaatg ggacctatga tgtgcaggtg acttcaggtg gaacattcat tggcattgga 960cggaaaacac cagattgcca agggaacacc aagtatttcc gctgtaaatt ctgcaatttc 1020acttatatgg gcaactcatc caccgaatta gaacaacatt ttcttcagac tcacccaaac 1080aaaataaaag cttctctccc ctcctctgag gttgcaaaac cttcagagaa aaactctaac 1140aagtccatcc ctgcacttca atccagtgat tctggagact tgggaaaatg gcaggacaag 1200ataacagtca aagcaggaga tgacactcct gttgggtact cagtgcccat aaagcccctc 1260gattcctcta gacaaaatgg tacagaggcc accagttact actggtgtaa attttgtagt 1320ttcagctgtg agtcatctag ctcacttaaa ctgctagaac attatggcaa gcagcacgga 1380gcagtgcagt caggcggcct taatccagag ttaaatgata agctttccag gggctctgtc 1440attaatcaga atgatctagc caaaagttca gaaggagaga caatgaccaa gacagacaag 1500agctcgagtg gggctaaaaa gaaggacttc tccagcaagg gagccgagga taatatggta 1560acgagctata attgtcagtt ctgtgacttc cgatattcca aaagccatgg ccctgatgta 1620attgtagtgg ggccacttct ccgtcattat caacagctcc ataacattca caagtgtacc 1680attaaacact gtccattctg tcccagagga ctttgcagcc cagaaaagca ccttggagaa 1740attacttatc cgtttgcttg tagaaaaagt aattgttccc actgtgcact cttgcttctg 1800cacttgtctc ctggggcggc tggaagctcg cgagtcaaac atcagtgcca tcagtgttca 1860ttcaccaccc ctgacgtaga tgtactcctc tttcactatg aaagtgtgca tgagtcccaa 1920gcatcggatg tcaaacaaga agcaaatcac ctgcaaggat cggatgggca gcagtctgtc 1980aaggaaagca aagaacactc atgtaccaaa tgtgatttta ttacccaagt ggaagaagag 2040atttcccgac actacaggag agcacacagc tgctacaaat gccgtcagtg cagttttaca 2100gctgccgata ctcagtcact actggagcac ttcaacactg ttcactgcca ggaacaggac 2160atcactacag ccaacggcga agaggacggt catgccatat ccaccatcaa agaggagccc 2220aaaattgact tcagggtcta caatctgcta actccagact ctaaaatggg agagccagtt 2280tctgagagtg tggtgaagag agagaagctg gaagagaagg acgggctcaa agagaaagtt 2340tggaccgaga gttccagtga tgaccttcgc aatgtgactt ggagaggggc agacatcctg 2400cgggggagtc cgtcatacac ccaagcaagc ctggggctgc tgacgcctgt gtctggcacc 2460caagagcaga caaagactct aagggatagt cccaatgtgg aggccgccca tctggcgcga 2520cctatttatg gcttggctgt ggaaaccaag ggattcctgc agggggcgcc agctggcgga 2580gagaagtctg gggccctccc ccagcagtat cctgcatcgg gagaaaacaa gtccaaggat 2640gaatcccagt ccctgttacg gaggcgtaga ggctccggtg ttttttgtgc caattgcctg 2700accacaaaga cctctctctg gcgaaagaat gcaaatggcg gatatgtatg caacgcgtgt 2760ggcctctacc agaagcttca ctcgactccc aggcctttaa acatcattaa acaaaacaac 2820ggtgagcaga ttattaggag gagaacaaga aagcgcctta acccagaggc acttcaggct 2880gagcagctca acaaacagca gaggggcagc aatgaggagc aagtcaatgg aagcccgtta 2940gagaggaggt cagaagatca tctaactgaa agtcaccaga gagaaattcc actccccagc 3000ctaagtaaat acgaagccca gggttcattg actaaaagcc attctgctca gcagccagtc 3060ctggtcagcc aaactctgga tattcacaaa aggatgcaac ctttgcacat tcagataaaa 3120agtcctcagg aaagtactgg agatccagga aatagttcat ccgtatctga agggaaagga 3180agttctgaga gaggcagtcc tatagaaaag tacatgagac ctgcgaaaca cccaaattat 3240tcaccaccag gcagccctat tgaaaagtac cagtacccac tttttggact tccctttgta 3300cataatgact tccagagtga agctgattgg ctgcggttct ggagtaaata taagctctcc 3360gttcctggga atccgcacta cttgagtcac gtgcctggcc taccaaatcc ttgccaaaac 3420tatgtgcctt atcccacctt caatctgcct cctcattttt cagctgttgg atcagacaat 3480gacattcctc tagatttggc gatcaagcat tccagacctg ggccaactgc aaacggtgcc 3540tccaaggaga aaacgaaggc accaccaaat gtaaaaaatg aaggtccctt gaatgtagta 3600aaaacagaga aagttgatag aagtactcaa gatgaacttt caacaaaatg tgtgcactgt 3660ggcattgtct ttctggatga agtgatgtat gctttgcata tgagttgcca tggtgacagt 3720ggacctttcc agtgcagcat atgccagcat ctttgcacgg acaaatatga cttcacaaca 3780catatccaga ggggcctgca taggaacaat gcacaagtgg aaaaaaatgg aaaacctaaa 3840gagtaa 3846291281PRTHomo sapiens 29Met Val Arg Lys Lys Asn Pro Pro Leu Arg Asn Val Ala Ser Glu Gly 1 5 10 15 Glu Gly Gln Ile Leu Glu Pro Ile Gly Thr Glu Ser Lys Val Ser Gly 20 25 30 Lys Asn Lys Glu Phe Ser Ala Asp Gln Met Ser Glu Asn Thr Asp Gln 35 40 45 Ser Asp Ala Ala Glu Leu Asn His Lys Glu Glu His Ser Leu His Val 50 55 60 Gln Asp Pro Ser Ser Ser Ser Lys Lys Asp Leu Lys Ser Ala Val Leu 65 70 75 80 Ser Glu Lys Ala Gly Phe Asn Tyr Glu Ser Pro Ser Lys Gly Gly Asn 85 90 95 Phe Pro Ser Phe Pro His Asp Glu Val Thr Asp Arg Asn Met Leu Ala 100 105 110 Phe Ser Phe Pro Ala Ala Gly Gly Val Cys Glu Pro Leu Lys Ser Pro 115 120 125 Gln Arg Ala Glu Ala Asp Asp Pro Gln Asp Met Ala Cys Thr Pro Ser 130 135 140 Gly Asp Ser Leu Glu Thr Lys Glu Asp Gln Lys Met Ser Pro Lys Ala 145 150 155 160 Thr Glu Glu Thr Gly Gln Ala Gln Ser Gly Gln Ala Asn Cys Gln Gly 165 170 175 Leu Ser Pro Val Ser Val Ala Ser Lys Asn Pro Gln Val Pro Ser Asp 180 185 190 Gly Gly Val Arg Leu Asn Lys Ser Lys Thr Asp Leu Leu Val Asn Asp 195 200 205 Asn Pro Asp Pro Ala Pro Leu Ser Pro Glu Leu Gln Asp Phe Lys Cys 210 215 220 Asn Ile Cys Gly Tyr Gly Tyr Tyr Gly Asn Asp Pro Thr Asp Leu Ile 225 230 235 240 Lys His Phe Arg Lys Tyr His Leu Gly Leu His Asn Arg Thr Arg Gln 245 250 255 Asp Ala Glu Leu Asp Ser Lys Ile Leu Ala Leu His Asn Met Val Gln 260 265 270 Phe Ser His Ser Lys Asp Phe Gln Lys Val Asn Arg Ser Val Phe Ser 275 280 285 Gly Val Leu Gln Asp Ile Asn Ser Ser Arg Pro Val Leu Leu Asn Gly 290 295 300 Thr Tyr Asp Val Gln Val Thr Ser Gly Gly Thr Phe Ile Gly Ile Gly 305 310 315 320 Arg Lys Thr Pro Asp Cys Gln Gly Asn Thr Lys Tyr Phe Arg Cys Lys 325 330 335 Phe Cys Asn Phe Thr Tyr Met Gly Asn Ser Ser Thr Glu Leu Glu Gln 340 345 350 His Phe Leu Gln Thr His Pro Asn Lys Ile Lys Ala Ser Leu Pro Ser 355 360 365 Ser Glu Val Ala Lys Pro Ser Glu Lys Asn Ser Asn Lys Ser Ile Pro 370 375 380 Ala Leu Gln Ser Ser Asp Ser Gly Asp Leu Gly Lys Trp Gln Asp Lys 385 390 395 400 Ile Thr Val Lys Ala Gly Asp Asp Thr Pro Val Gly Tyr Ser Val Pro 405 410 415 Ile Lys Pro Leu Asp Ser Ser Arg Gln Asn Gly Thr Glu Ala Thr Ser 420 425 430 Tyr Tyr Trp Cys Lys Phe Cys Ser Phe Ser Cys Glu Ser Ser Ser Ser 435 440 445 Leu Lys Leu Leu Glu His Tyr Gly Lys Gln His Gly Ala Val Gln Ser 450 455 460 Gly Gly Leu Asn Pro Glu Leu Asn Asp Lys Leu Ser Arg Gly Ser Val 465 470 475 480 Ile Asn Gln Asn Asp Leu Ala Lys Ser Ser Glu Gly Glu Thr Met Thr 485 490 495 Lys Thr Asp Lys Ser Ser Ser Gly Ala Lys Lys Lys Asp Phe Ser Ser 500 505 510 Lys Gly Ala Glu Asp Asn Met Val Thr Ser Tyr Asn Cys Gln Phe Cys 515 520 525 Asp Phe Arg Tyr Ser Lys Ser His Gly Pro Asp Val Ile Val Val Gly 530 535 540 Pro Leu Leu Arg His Tyr Gln Gln Leu His Asn Ile His Lys Cys Thr 545 550 555 560 Ile Lys His Cys Pro Phe Cys Pro Arg Gly Leu Cys Ser Pro Glu Lys 565 570 575 His Leu Gly Glu Ile Thr Tyr Pro Phe Ala Cys Arg Lys Ser Asn Cys 580 585 590 Ser His Cys Ala Leu Leu Leu Leu His Leu Ser Pro Gly Ala Ala Gly 595 600 605 Ser Ser Arg Val Lys His Gln Cys His Gln Cys Ser Phe Thr Thr Pro 610 615 620 Asp Val Asp Val Leu Leu Phe His Tyr Glu Ser Val His Glu Ser Gln 625 630 635 640 Ala Ser Asp Val Lys Gln Glu Ala Asn His Leu Gln Gly Ser Asp Gly 645 650 655 Gln Gln Ser Val Lys Glu Ser Lys Glu His Ser Cys Thr Lys Cys Asp 660 665 670 Phe Ile Thr Gln Val Glu Glu Glu Ile Ser Arg His Tyr Arg Arg Ala 675 680 685 His Ser Cys Tyr Lys Cys Arg Gln Cys Ser Phe Thr Ala Ala Asp Thr 690 695 700 Gln Ser Leu Leu Glu His Phe Asn Thr Val His Cys Gln Glu Gln Asp 705 710 715 720 Ile Thr Thr Ala Asn Gly Glu Glu Asp Gly His Ala Ile Ser Thr Ile 725 730 735 Lys Glu Glu Pro Lys Ile Asp Phe Arg Val Tyr Asn Leu Leu Thr Pro 740 745 750 Asp Ser Lys Met Gly Glu Pro Val Ser Glu Ser Val Val Lys Arg Glu 755 760 765 Lys Leu Glu Glu Lys Asp Gly Leu Lys Glu Lys Val Trp Thr Glu Ser 770 775 780 Ser Ser Asp Asp Leu Arg Asn Val Thr Trp Arg Gly Ala Asp Ile Leu 785 790 795 800 Arg Gly Ser Pro Ser Tyr Thr Gln Ala Ser Leu Gly Leu Leu Thr Pro 805 810 815 Val Ser Gly Thr Gln Glu Gln Thr Lys Thr Leu Arg Asp Ser Pro Asn 820 825 830 Val Glu Ala Ala His Leu Ala Arg Pro Ile Tyr Gly Leu Ala Val Glu 835 840 845 Thr Lys Gly Phe Leu Gln Gly Ala Pro Ala Gly Gly Glu Lys Ser Gly 850 855 860 Ala Leu Pro Gln Gln Tyr Pro Ala Ser Gly Glu Asn Lys Ser Lys Asp 865 870 875 880 Glu Ser Gln Ser Leu Leu Arg Arg Arg Arg Gly Ser Gly Val Phe Cys 885 890 895 Ala Asn Cys Leu Thr Thr Lys Thr Ser Leu Trp Arg Lys Asn Ala Asn 900 905 910 Gly Gly Tyr Val Cys Asn Ala Cys Gly Leu Tyr Gln Lys Leu His Ser 915 920 925 Thr Pro Arg Pro Leu Asn Ile Ile Lys Gln Asn Asn Gly Glu Gln Ile 930 935 940 Ile Arg Arg Arg Thr Arg Lys Arg Leu Asn Pro Glu Ala Leu Gln Ala 945 950 955 960 Glu Gln Leu Asn Lys Gln Gln Arg Gly Ser Asn Glu Glu Gln Val Asn 965 970 975 Gly Ser Pro Leu Glu Arg Arg Ser Glu Asp His Leu Thr Glu Ser His 980 985 990 Gln Arg Glu Ile Pro Leu Pro Ser Leu Ser Lys Tyr Glu Ala Gln Gly 995 1000 1005 Ser Leu Thr Lys Ser His Ser Ala Gln Gln Pro Val Leu Val Ser 1010 1015 1020 Gln Thr Leu Asp Ile His Lys Arg Met Gln Pro Leu His Ile Gln 1025 1030 1035 Ile Lys Ser Pro Gln Glu Ser Thr Gly Asp Pro Gly Asn Ser Ser 1040 1045 1050 Ser Val Ser Glu Gly Lys Gly Ser Ser Glu Arg Gly Ser Pro Ile 1055 1060 1065 Glu Lys Tyr Met Arg Pro Ala Lys His Pro Asn Tyr Ser Pro Pro 1070 1075 1080 Gly Ser Pro Ile Glu Lys Tyr Gln Tyr Pro Leu Phe Gly Leu Pro 1085 1090 1095 Phe Val His Asn Asp Phe Gln Ser Glu Ala Asp Trp Leu Arg Phe 1100 1105 1110 Trp Ser Lys Tyr Lys Leu Ser Val Pro Gly Asn Pro His Tyr Leu 1115 1120 1125 Ser His Val Pro Gly Leu Pro Asn Pro Cys Gln Asn Tyr Val Pro 1130 1135 1140 Tyr Pro Thr Phe Asn Leu Pro Pro His Phe Ser Ala Val Gly Ser 1145 1150 1155 Asp Asn Asp Ile Pro Leu Asp Leu Ala Ile Lys His Ser Arg Pro 1160 1165 1170 Gly Pro Thr Ala Asn Gly Ala Ser Lys Glu Lys Thr Lys Ala Pro 1175 1180 1185 Pro Asn Val Lys Asn Glu Gly Pro Leu Asn Val Val Lys Thr Glu 1190 1195 1200 Lys Val Asp Arg Ser Thr Gln Asp Glu Leu Ser Thr Lys Cys Val 1205 1210 1215 His Cys Gly Ile Val Phe Leu Asp Glu Val Met Tyr Ala Leu His 1220 1225 1230

Met Ser Cys His Gly Asp Ser Gly Pro Phe Gln Cys Ser Ile Cys 1235 1240 1245 Gln His Leu Cys Thr Asp Lys Tyr Asp Phe Thr Thr His Ile Gln 1250 1255 1260 Arg Gly Leu His Arg Asn Asn Ala Gln Val Glu Lys Asn Gly Lys 1265 1270 1275 Pro Lys Glu 1280 301203DNAHomo sapiens 30atggccgagc tgcgcctgaa gggcagcagc aacaccacgg agtgtgttcc cgtgcccacc 60tccgagcacg tggccgagat cgtgggcagg caaggctgca agattaaggc cttgagggcc 120aagaccaaca cctacatcaa gacaccggtg aggggcgagg aaccagtgtt catggtgaca 180gggcgacggg aggacgtggc cacagcccgg cgggaaatca tctcagcagc ggagcacttc 240tccatgatcc gtgcctcccg caacaagtca ggcgccgcct ttggtgtggc tcctgctctg 300cccggccagg tgaccatccg tgtgcgggtg ccctaccgcg tggtggggct ggtggtgggc 360cccaaagggg caaccatcaa gcgcatccag cagcaaacca acacatacat tatcacacca 420agccgtgacc gcgaccccgt gttcgagatc acgggtgccc caggcaacgt ggagcgtgcg 480cgcgaggaga tcgagacgca catcgcggtg cgcactggca agatcctcga gtacaacaat 540gaaaacgact tcctggcggg gagccccgac gcagcaatcg atagccgcta ctccgacgcc 600tggcgggtgc accagcccgg ctgcaagccc ctctccacct tccggcagaa cagcctgggc 660tgcatcggcg agtgcggagt ggactctggc tttgaggccc cacgcctggg tgagcagggc 720ggggactttg gctacggcgg gtacctcttt ccgggctatg gcgtgggcaa gcaggatgtg 780tactacggcg tggccgagac tagccccccg ctgtgggcgg gccaggagaa cgccacgccc 840acctccgtgc tcttctcctc tgcctcctcc tcctcctcct cttccgccaa ggcccgcgct 900gggcccccgg gcgcacaccg ctcccctgcc acttccgcgg gacccgagct ggccggactc 960ccgaggcgcc ccccgggaga gccgctccag ggcttctcta aacttggtgg gggcggcctg 1020cggagccccg gcggcgggcg ggattgcatg gtctgctttg agagcgaagt gactgccgcc 1080cttgtgccct gcggacacaa cctgttctgc atggagtgtg cagtacgcat ctgcgagagg 1140acggacccag agtgtcccgt ctgccacatc acagccgcgc aagccatccg aatattctcc 1200taa 120331400PRTHomo sapiens 31Met Ala Glu Leu Arg Leu Lys Gly Ser Ser Asn Thr Thr Glu Cys Val 1 5 10 15 Pro Val Pro Thr Ser Glu His Val Ala Glu Ile Val Gly Arg Gln Gly 20 25 30 Cys Lys Ile Lys Ala Leu Arg Ala Lys Thr Asn Thr Tyr Ile Lys Thr 35 40 45 Pro Val Arg Gly Glu Glu Pro Val Phe Met Val Thr Gly Arg Arg Glu 50 55 60 Asp Val Ala Thr Ala Arg Arg Glu Ile Ile Ser Ala Ala Glu His Phe 65 70 75 80 Ser Met Ile Arg Ala Ser Arg Asn Lys Ser Gly Ala Ala Phe Gly Val 85 90 95 Ala Pro Ala Leu Pro Gly Gln Val Thr Ile Arg Val Arg Val Pro Tyr 100 105 110 Arg Val Val Gly Leu Val Val Gly Pro Lys Gly Ala Thr Ile Lys Arg 115 120 125 Ile Gln Gln Gln Thr Asn Thr Tyr Ile Ile Thr Pro Ser Arg Asp Arg 130 135 140 Asp Pro Val Phe Glu Ile Thr Gly Ala Pro Gly Asn Val Glu Arg Ala 145 150 155 160 Arg Glu Glu Ile Glu Thr His Ile Ala Val Arg Thr Gly Lys Ile Leu 165 170 175 Glu Tyr Asn Asn Glu Asn Asp Phe Leu Ala Gly Ser Pro Asp Ala Ala 180 185 190 Ile Asp Ser Arg Tyr Ser Asp Ala Trp Arg Val His Gln Pro Gly Cys 195 200 205 Lys Pro Leu Ser Thr Phe Arg Gln Asn Ser Leu Gly Cys Ile Gly Glu 210 215 220 Cys Gly Val Asp Ser Gly Phe Glu Ala Pro Arg Leu Gly Glu Gln Gly 225 230 235 240 Gly Asp Phe Gly Tyr Gly Gly Tyr Leu Phe Pro Gly Tyr Gly Val Gly 245 250 255 Lys Gln Asp Val Tyr Tyr Gly Val Ala Glu Thr Ser Pro Pro Leu Trp 260 265 270 Ala Gly Gln Glu Asn Ala Thr Pro Thr Ser Val Leu Phe Ser Ser Ala 275 280 285 Ser Ser Ser Ser Ser Ser Ser Ala Lys Ala Arg Ala Gly Pro Pro Gly 290 295 300 Ala His Arg Ser Pro Ala Thr Ser Ala Gly Pro Glu Leu Ala Gly Leu 305 310 315 320 Pro Arg Arg Pro Pro Gly Glu Pro Leu Gln Gly Phe Ser Lys Leu Gly 325 330 335 Gly Gly Gly Leu Arg Ser Pro Gly Gly Gly Arg Asp Cys Met Val Cys 340 345 350 Phe Glu Ser Glu Val Thr Ala Ala Leu Val Pro Cys Gly His Asn Leu 355 360 365 Phe Cys Met Glu Cys Ala Val Arg Ile Cys Glu Arg Thr Asp Pro Glu 370 375 380 Cys Pro Val Cys His Ile Thr Ala Ala Gln Ala Ile Arg Ile Phe Ser 385 390 395 400 324026DNAHomo sapiens 32atgacaaaga ggaagaagac catcaacctt aatatacaag acgcccagaa gaggactgct 60ctacactggg cctgtgtcaa tggccatgag gaagtagtaa catttctggt agacagaaag 120tgccagcttg acgtccttga tggcgaacac aggacacctc tgatgaaggc tctacaatgc 180catcaggagg cttgtgcaaa tattctgata gattctggtg ccgatataaa tctcgtagat 240gtgtatggca acatggctct ccattatgct gtttatagtg agattttgtc agtggtggca 300aaactgctgt cccatggtgc agtcatcgaa gtgcacaaca aggctagcct cacaccactt 360ttactatcca taacgaaaag aagtgagcaa attgtggaat ttttgctgat aaaaaatgca 420aatgcgaatg cagttaataa gtataaatgc acagccctca tgcttgctgt atgtcatgga 480tcatcagaga tagttggcat gcttcttcag caaaatgttg acgtctttgc tgcagatata 540tgtggagtaa ctgcagaaca ttatgctgtt acttgtggat ttcatcacat tcatgaacaa 600attatggaat atatacgaaa attatctaaa aatcatcaaa ataccaatcc agaaggaaca 660tctgcaggaa cacctgatga ggctgcaccc ttggcggaaa gaacacctga cacagctgaa 720agcttggtgg aaaaaacacc tgatgaggct gcacccttgg tggaaagaac acctgacacg 780gctgaaagct tggtggaaaa aacacctgat gaggctgcat ccttggtgga gggaacatct 840gacaaaattc aatgtttgga gaaagcgaca tctggaaagt tcgaacagtc agcagaagaa 900acacctaggg aaattacgag tcctgcaaaa gaaacatctg agaaatttac gtggccagca 960aaaggaagac ctaggaagat cgcatgggag aaaaaagaag acacacctag ggaaattatg 1020agtcccgcaa aagaaacatc tgagaaattt acgtgggcag caaaaggaag acctaggaag 1080atcgcatggg agaaaaaaga aacacctgta aagactggat gcgtggcaag agtaacatct 1140aataaaacta aagttttgga aaaaggaaga tctaagatga ttgcatgtcc tacaaaagaa 1200tcatctacaa aagcaagtgc caatgatcag aggttcccat cagaatccaa acaagaggaa 1260gatgaagaat attcttgtga ttctcggagt ctctttgaga gttctgcaaa gattcaagtg 1320tgtatacctg agtctatata tcaaaaagta atggagataa atagagaagt agaagagcct 1380cctaagaagc catctgcctt caagcctgcc attgaaatgc aaaactctgt tccaaataaa 1440gcctttgaat tgaagaatga acaaacattg agagcagatc cgatgttccc accagaatcc 1500aaacaaaagg actatgaaga aaattcttgg gattctgaga gtctctgtga gactgtttca 1560cagaaggatg tgtgtttacc caaggctaca catcaaaaag aaatagataa aataaatgga 1620aaattagaag agtctcctaa taaagatggt cttctgaagg ctacctgcgg aatgaaagtt 1680tctattccaa ctaaagcctt agaattgaag gacatgcaaa ctttcaaagc ggagcctccg 1740gggaagccat ctgccttcga gcctgccact gaaatgcaaa agtctgtccc aaataaagcc 1800ttggaattga aaaatgaaca aacatggaga gcagatgaga tactcccatc agaatccaaa 1860caaaaggact atgaagaaaa ttcttgggat actgagagtc tctgtgagac tgtttcacag 1920aaggatgtgt gtttacccaa ggctgcgcat caaaaagaaa tagataaaat aaatggaaaa 1980ttagaagggt ctcctgttaa agatggtctt ctgaaggcta actgcggaat gaaagtttct 2040attccaacta aagccttaga attgatggac atgcaaactt tcaaagcaga gcctcccgag 2100aagccatctg ccttcgagcc tgccattgaa atgcaaaagt ctgttccaaa taaagccttg 2160gaattgaaga atgaacaaac attgagagca gatgagatac tcccatcaga atccaaacaa 2220aaggactatg aagaaagttc ttgggattct gagagtctct gtgagactgt ttcacagaag 2280gatgtgtgtt tacccaaggc tacacatcaa aaagaaatag ataaaataaa tggaaaatta 2340gaagagtctc ctgataatga tggttttctg aaggctccct gcagaatgaa agtttctatt 2400ccaactaaag ccttagaatt gatggacatg caaactttca aagcagagcc tcccgagaag 2460ccatctgcct tcgagcctgc cattgaaatg caaaagtctg ttccaaataa agccttggaa 2520ttgaagaatg aacaaacatt gagagcagat cagatgttcc cttcagaatc aaaacaaaag 2580aaggttgaag aaaattcttg ggattctgag agtctccgtg agactgtttc acagaaggat 2640gtgtgtgtac ccaaggctac acatcaaaaa gaaatggata aaataagtgg aaaattagaa 2700gattcaacta gcctatcaaa aatcttggat acagttcatt cttgtgaaag agcaagggaa 2760cttcaaaaag atcactgtga acaacgtaca ggaaaaatgg aacaaatgaa aaagaagttt 2820tgtgtactga aaaagaaact gtcagaagca aaagaaataa aatcacagtt agagaaccaa 2880aaagttaaat gggaacaaga gctctgcagt gtgagattga ctttaaacca agaagaagag 2940aagagaagaa atgccgatat attaaatgaa aaaattaggg aagaattagg aagaatcgaa 3000gagcagcata ggaaagagtt agaagtgaaa caacaacttg aacaggctct cagaatacaa 3060gatatagaat tgaagagtgt agaaagtaat ttgaatcagg tttctcacac tcatgaaaat 3120gaaaattatc tcttacatga aaattgcatg ttgaaaaagg aaattgccat gctaaaactg 3180gaaatagcca cactgaaaca ccaataccag gaaaaggaaa ataaatactt tgaggacatt 3240aagattttaa aagaaaagaa tgctgaactt cagatgaccc taaaactgaa agaggaatca 3300ttaactaaaa gggcatctca atatagtggg cagcttaaag ttctgatagc tgagaacaca 3360atgctcactt ctaaattgaa ggaaaaacaa gacaaagaaa tactagaggc agaaattgaa 3420tcacaccatc ctagactggc ttctgctgta caagaccatg atcaaattgt gacatcaaga 3480aaaagtcaag aacctgcttt ccacattgca ggagatgctt gtttgcaaag aaaaatgaat 3540gttgatgtga gtagtacgat atataacaat gaggtgctcc atcaaccact ttctgaagct 3600caaaggaaat ccaaaagcct aaaaattaat ctcaattatg caggagatgc tctaagagaa 3660aatacattgg tttcagaaca tgcacaaaga gaccaacgtg aaacacagtg tcaaatgaag 3720gaagctgaac acatgtatca aaacgaacaa gataatgtga acaaacacac tgaacagcag 3780gagtctctag atcagaaatt atttcaacta caaagcaaaa atatgtggct tcaacagcaa 3840ttagttcatg cacataagaa agctgacaac aaaagcaaga taacaattga tattcatttt 3900cttgagagga aaatgcaaca tcatctccta aaagagaaaa atgaggagat atttaattac 3960aataaccatt taaaaaaccg tatatatcaa tatgaaaaag agaaagcaga aacagaaaac 4020tcatga 4026331341PRTHomo sapiens 33Met Thr Lys Arg Lys Lys Thr Ile Asn Leu Asn Ile Gln Asp Ala Gln 1 5 10 15 Lys Arg Thr Ala Leu His Trp Ala Cys Val Asn Gly His Glu Glu Val 20 25 30 Val Thr Phe Leu Val Asp Arg Lys Cys Gln Leu Asp Val Leu Asp Gly 35 40 45 Glu His Arg Thr Pro Leu Met Lys Ala Leu Gln Cys His Gln Glu Ala 50 55 60 Cys Ala Asn Ile Leu Ile Asp Ser Gly Ala Asp Ile Asn Leu Val Asp 65 70 75 80 Val Tyr Gly Asn Met Ala Leu His Tyr Ala Val Tyr Ser Glu Ile Leu 85 90 95 Ser Val Val Ala Lys Leu Leu Ser His Gly Ala Val Ile Glu Val His 100 105 110 Asn Lys Ala Ser Leu Thr Pro Leu Leu Leu Ser Ile Thr Lys Arg Ser 115 120 125 Glu Gln Ile Val Glu Phe Leu Leu Ile Lys Asn Ala Asn Ala Asn Ala 130 135 140 Val Asn Lys Tyr Lys Cys Thr Ala Leu Met Leu Ala Val Cys His Gly 145 150 155 160 Ser Ser Glu Ile Val Gly Met Leu Leu Gln Gln Asn Val Asp Val Phe 165 170 175 Ala Ala Asp Ile Cys Gly Val Thr Ala Glu His Tyr Ala Val Thr Cys 180 185 190 Gly Phe His His Ile His Glu Gln Ile Met Glu Tyr Ile Arg Lys Leu 195 200 205 Ser Lys Asn His Gln Asn Thr Asn Pro Glu Gly Thr Ser Ala Gly Thr 210 215 220 Pro Asp Glu Ala Ala Pro Leu Ala Glu Arg Thr Pro Asp Thr Ala Glu 225 230 235 240 Ser Leu Val Glu Lys Thr Pro Asp Glu Ala Ala Pro Leu Val Glu Arg 245 250 255 Thr Pro Asp Thr Ala Glu Ser Leu Val Glu Lys Thr Pro Asp Glu Ala 260 265 270 Ala Ser Leu Val Glu Gly Thr Ser Asp Lys Ile Gln Cys Leu Glu Lys 275 280 285 Ala Thr Ser Gly Lys Phe Glu Gln Ser Ala Glu Glu Thr Pro Arg Glu 290 295 300 Ile Thr Ser Pro Ala Lys Glu Thr Ser Glu Lys Phe Thr Trp Pro Ala 305 310 315 320 Lys Gly Arg Pro Arg Lys Ile Ala Trp Glu Lys Lys Glu Asp Thr Pro 325 330 335 Arg Glu Ile Met Ser Pro Ala Lys Glu Thr Ser Glu Lys Phe Thr Trp 340 345 350 Ala Ala Lys Gly Arg Pro Arg Lys Ile Ala Trp Glu Lys Lys Glu Thr 355 360 365 Pro Val Lys Thr Gly Cys Val Ala Arg Val Thr Ser Asn Lys Thr Lys 370 375 380 Val Leu Glu Lys Gly Arg Ser Lys Met Ile Ala Cys Pro Thr Lys Glu 385 390 395 400 Ser Ser Thr Lys Ala Ser Ala Asn Asp Gln Arg Phe Pro Ser Glu Ser 405 410 415 Lys Gln Glu Glu Asp Glu Glu Tyr Ser Cys Asp Ser Arg Ser Leu Phe 420 425 430 Glu Ser Ser Ala Lys Ile Gln Val Cys Ile Pro Glu Ser Ile Tyr Gln 435 440 445 Lys Val Met Glu Ile Asn Arg Glu Val Glu Glu Pro Pro Lys Lys Pro 450 455 460 Ser Ala Phe Lys Pro Ala Ile Glu Met Gln Asn Ser Val Pro Asn Lys 465 470 475 480 Ala Phe Glu Leu Lys Asn Glu Gln Thr Leu Arg Ala Asp Pro Met Phe 485 490 495 Pro Pro Glu Ser Lys Gln Lys Asp Tyr Glu Glu Asn Ser Trp Asp Ser 500 505 510 Glu Ser Leu Cys Glu Thr Val Ser Gln Lys Asp Val Cys Leu Pro Lys 515 520 525 Ala Thr His Gln Lys Glu Ile Asp Lys Ile Asn Gly Lys Leu Glu Glu 530 535 540 Ser Pro Asn Lys Asp Gly Leu Leu Lys Ala Thr Cys Gly Met Lys Val 545 550 555 560 Ser Ile Pro Thr Lys Ala Leu Glu Leu Lys Asp Met Gln Thr Phe Lys 565 570 575 Ala Glu Pro Pro Gly Lys Pro Ser Ala Phe Glu Pro Ala Thr Glu Met 580 585 590 Gln Lys Ser Val Pro Asn Lys Ala Leu Glu Leu Lys Asn Glu Gln Thr 595 600 605 Trp Arg Ala Asp Glu Ile Leu Pro Ser Glu Ser Lys Gln Lys Asp Tyr 610 615 620 Glu Glu Asn Ser Trp Asp Thr Glu Ser Leu Cys Glu Thr Val Ser Gln 625 630 635 640 Lys Asp Val Cys Leu Pro Lys Ala Ala His Gln Lys Glu Ile Asp Lys 645 650 655 Ile Asn Gly Lys Leu Glu Gly Ser Pro Val Lys Asp Gly Leu Leu Lys 660 665 670 Ala Asn Cys Gly Met Lys Val Ser Ile Pro Thr Lys Ala Leu Glu Leu 675 680 685 Met Asp Met Gln Thr Phe Lys Ala Glu Pro Pro Glu Lys Pro Ser Ala 690 695 700 Phe Glu Pro Ala Ile Glu Met Gln Lys Ser Val Pro Asn Lys Ala Leu 705 710 715 720 Glu Leu Lys Asn Glu Gln Thr Leu Arg Ala Asp Glu Ile Leu Pro Ser 725 730 735 Glu Ser Lys Gln Lys Asp Tyr Glu Glu Ser Ser Trp Asp Ser Glu Ser 740 745 750 Leu Cys Glu Thr Val Ser Gln Lys Asp Val Cys Leu Pro Lys Ala Thr 755 760 765 His Gln Lys Glu Ile Asp Lys Ile Asn Gly Lys Leu Glu Glu Ser Pro 770 775 780 Asp Asn Asp Gly Phe Leu Lys Ala Pro Cys Arg Met Lys Val Ser Ile 785 790 795 800 Pro Thr Lys Ala Leu Glu Leu Met Asp Met Gln Thr Phe Lys Ala Glu 805 810 815 Pro Pro Glu Lys Pro Ser Ala Phe Glu Pro Ala Ile Glu Met Gln Lys 820 825 830 Ser Val Pro Asn Lys Ala Leu Glu Leu Lys Asn Glu Gln Thr Leu Arg 835 840 845 Ala Asp Gln Met Phe Pro Ser Glu Ser Lys Gln Lys Lys Val Glu Glu 850 855 860 Asn Ser Trp Asp Ser Glu Ser Leu Arg Glu Thr Val Ser Gln Lys Asp 865 870 875 880 Val Cys Val Pro Lys Ala Thr His Gln Lys Glu Met Asp Lys Ile Ser 885 890 895 Gly Lys Leu Glu Asp Ser Thr Ser Leu Ser Lys Ile Leu Asp Thr Val 900 905 910 His Ser Cys Glu Arg Ala Arg Glu Leu Gln Lys Asp His Cys Glu Gln 915 920 925 Arg Thr Gly Lys Met Glu Gln Met Lys Lys Lys Phe Cys Val Leu Lys 930 935 940 Lys Lys Leu Ser Glu Ala Lys Glu Ile Lys Ser Gln Leu Glu Asn Gln 945 950 955 960 Lys Val Lys Trp Glu Gln Glu Leu Cys Ser Val Arg Leu Thr Leu Asn 965 970 975 Gln Glu Glu Glu Lys Arg Arg Asn Ala Asp Ile Leu Asn Glu Lys Ile 980 985 990 Arg Glu Glu Leu Gly Arg Ile Glu Glu Gln His Arg Lys Glu Leu Glu 995 1000 1005 Val Lys Gln Gln Leu Glu Gln Ala Leu Arg Ile Gln Asp Ile Glu 1010 1015 1020

Leu Lys Ser Val Glu Ser Asn Leu Asn Gln Val Ser His Thr His 1025 1030 1035 Glu Asn Glu Asn Tyr Leu Leu His Glu Asn Cys Met Leu Lys Lys 1040 1045 1050 Glu Ile Ala Met Leu Lys Leu Glu Ile Ala Thr Leu Lys His Gln 1055 1060 1065 Tyr Gln Glu Lys Glu Asn Lys Tyr Phe Glu Asp Ile Lys Ile Leu 1070 1075 1080 Lys Glu Lys Asn Ala Glu Leu Gln Met Thr Leu Lys Leu Lys Glu 1085 1090 1095 Glu Ser Leu Thr Lys Arg Ala Ser Gln Tyr Ser Gly Gln Leu Lys 1100 1105 1110 Val Leu Ile Ala Glu Asn Thr Met Leu Thr Ser Lys Leu Lys Glu 1115 1120 1125 Lys Gln Asp Lys Glu Ile Leu Glu Ala Glu Ile Glu Ser His His 1130 1135 1140 Pro Arg Leu Ala Ser Ala Val Gln Asp His Asp Gln Ile Val Thr 1145 1150 1155 Ser Arg Lys Ser Gln Glu Pro Ala Phe His Ile Ala Gly Asp Ala 1160 1165 1170 Cys Leu Gln Arg Lys Met Asn Val Asp Val Ser Ser Thr Ile Tyr 1175 1180 1185 Asn Asn Glu Val Leu His Gln Pro Leu Ser Glu Ala Gln Arg Lys 1190 1195 1200 Ser Lys Ser Leu Lys Ile Asn Leu Asn Tyr Ala Gly Asp Ala Leu 1205 1210 1215 Arg Glu Asn Thr Leu Val Ser Glu His Ala Gln Arg Asp Gln Arg 1220 1225 1230 Glu Thr Gln Cys Gln Met Lys Glu Ala Glu His Met Tyr Gln Asn 1235 1240 1245 Glu Gln Asp Asn Val Asn Lys His Thr Glu Gln Gln Glu Ser Leu 1250 1255 1260 Asp Gln Lys Leu Phe Gln Leu Gln Ser Lys Asn Met Trp Leu Gln 1265 1270 1275 Gln Gln Leu Val His Ala His Lys Lys Ala Asp Asn Lys Ser Lys 1280 1285 1290 Ile Thr Ile Asp Ile His Phe Leu Glu Arg Lys Met Gln His His 1295 1300 1305 Leu Leu Lys Glu Lys Asn Glu Glu Ile Phe Asn Tyr Asn Asn His 1310 1315 1320 Leu Lys Asn Arg Ile Tyr Gln Tyr Glu Lys Glu Lys Ala Glu Thr 1325 1330 1335 Glu Asn Ser 1340 34873DNAHomo sapiens 34atggacattg aagcatatct tgaaagaatt ggctataaga agtctaggaa caaattggac 60ttggaaacat taactgacat tcttcaacac cagatccgag ctgttccctt tgagaacctt 120aacatccatt gtggggatgc catggactta ggcttagagg ccatttttga tcaagttgtg 180agaagaaatc ggggtggatg gtgtctccag gtcaatcatc ttctgtactg ggctctgacc 240actattggtt ttgagaccac gatgttggga gggtatgttt acagcactcc agccaaaaaa 300tacagcactg gcatgattca ccttctcctg caggtgacca ttgatggcag gaactacatt 360gtcgatgctg ggtttggacg ctcataccag atgtggcagc ctctggagtt aatttctggg 420aaggatcagc ctcaggtgcc ttgtgtcttc cgtttgacgg aagagaatgg attctggtat 480ctagaccaaa tcagaaggga acagtacatt ccaaatgaag aatttcttca ttctgatctc 540ctagaagaca gcaaataccg aaaaatctac tcctttactc ttaagcctcg aacaattgaa 600gattttgagt ctatgaatac atacctgcag acatctccat catctgtgtt tactagtaaa 660tcattttgtt ccttgcagac cccagatggg gttcactgtt tggtgggctt caccctcacc 720cataggagat tcaattataa ggacaataca gatctaatag agttcaagac tctgagtgag 780gaagaaatag aaaaagtgct gaaaaatata tttaatattt ccttgcagag aaagcttgtg 840cccaaacatg gtgatagatt ttttactatt tag 87335290PRTHomo sapiens 35Met Asp Ile Glu Ala Tyr Leu Glu Arg Ile Gly Tyr Lys Lys Ser Arg 1 5 10 15 Asn Lys Leu Asp Leu Glu Thr Leu Thr Asp Ile Leu Gln His Gln Ile 20 25 30 Arg Ala Val Pro Phe Glu Asn Leu Asn Ile His Cys Gly Asp Ala Met 35 40 45 Asp Leu Gly Leu Glu Ala Ile Phe Asp Gln Val Val Arg Arg Asn Arg 50 55 60 Gly Gly Trp Cys Leu Gln Val Asn His Leu Leu Tyr Trp Ala Leu Thr 65 70 75 80 Thr Ile Gly Phe Glu Thr Thr Met Leu Gly Gly Tyr Val Tyr Ser Thr 85 90 95 Pro Ala Lys Lys Tyr Ser Thr Gly Met Ile His Leu Leu Leu Gln Val 100 105 110 Thr Ile Asp Gly Arg Asn Tyr Ile Val Asp Ala Gly Phe Gly Arg Ser 115 120 125 Tyr Gln Met Trp Gln Pro Leu Glu Leu Ile Ser Gly Lys Asp Gln Pro 130 135 140 Gln Val Pro Cys Val Phe Arg Leu Thr Glu Glu Asn Gly Phe Trp Tyr 145 150 155 160 Leu Asp Gln Ile Arg Arg Glu Gln Tyr Ile Pro Asn Glu Glu Phe Leu 165 170 175 His Ser Asp Leu Leu Glu Asp Ser Lys Tyr Arg Lys Ile Tyr Ser Phe 180 185 190 Thr Leu Lys Pro Arg Thr Ile Glu Asp Phe Glu Ser Met Asn Thr Tyr 195 200 205 Leu Gln Thr Ser Pro Ser Ser Val Phe Thr Ser Lys Ser Phe Cys Ser 210 215 220 Leu Gln Thr Pro Asp Gly Val His Cys Leu Val Gly Phe Thr Leu Thr 225 230 235 240 His Arg Arg Phe Asn Tyr Lys Asp Asn Thr Asp Leu Ile Glu Phe Lys 245 250 255 Thr Leu Ser Glu Glu Glu Ile Glu Lys Val Leu Lys Asn Ile Phe Asn 260 265 270 Ile Ser Leu Gln Arg Lys Leu Val Pro Lys His Gly Asp Arg Phe Phe 275 280 285 Thr Ile 290 361314DNAHomo sapiens 36atgctttgga aattgacgga taatatcaag tacgaggact gcgaggaccg tcacgacggc 60accagcaacg ggacggcacg gttgccccag ctgggcactg taggtcaatc tccctacacg 120agcgccccgc cgctgtccca cacccccaat gccgacttcc agcccccata cttcccccca 180ccctaccagc ctatctaccc ccagtcgcaa gatccttact cccacgtcaa cgacccctac 240agcctgaacc ccctgcacgc ccagccgcag ccgcagcacc caggctggcc cggccagagg 300cagagccagg agtctgggct cctgcacacg caccgggggc tgcctcacca gctgtcgggc 360ctggatcctc gcagggacta caggcggcac gaggacctcc tgcacggccc acacgcgctc 420agctcaggac tcggagacct ctcgatccac tccttacctc acgccatcga ggaggtcccg 480catgtagaag acccgggtat taacatccca gatcaaactg taattaagaa aggccccgtg 540tccctgtcca agtccaacag caatgccgtc tccgccatcc ctattaacaa ggacaacctc 600ttcggcggcg tggtgaaccc caacgaagtc ttctgttcag ttccgggtcg cctctcgctc 660ctcagctcca cctcgaagta caaggtcacg gtggcggaag tgcagcggcg gctctcacca 720cccgagtgtc tcaacgcgtc gctgctgggc ggagtgctcc ggagggcgaa gtctaaaaat 780ggaggaagat ctttaagaga aaaactggac aaaataggat taaatctgcc tgcagggaga 840cgtaaagctg ccaacgttac cctgctcaca tcactagtag agggagaagc tgtccaccta 900gccagggact ttgggtacgt gtgcgaaacc gaatttcctg ccaaagcagt agctgaattt 960ctcaaccgac aacattccga tcccaatgag caagtgacaa gaaaaaacat gctcctggct 1020acaaaacaga tatgcaaaga gttcaccgac ctgctggctc aggaccgatc tcccctgggg 1080aactcacggc ccaaccccat cctggagccc ggcatccaga gctgcttgac ccacttcaac 1140ctcatctccc acggcttcgg cagccccgcg gtgtgtgccg cggtcacggc cctgcagaac 1200tatctcaccg aggccctcaa ggccatggac aaaatgtacc tcagcaacaa ccccaacagc 1260cacacggaca acaacgccaa aagcagtgac aaagaggaga agcacagaaa gtga 131437437PRTHomo sapiens 37Met Leu Trp Lys Leu Thr Asp Asn Ile Lys Tyr Glu Asp Cys Glu Asp 1 5 10 15 Arg His Asp Gly Thr Ser Asn Gly Thr Ala Arg Leu Pro Gln Leu Gly 20 25 30 Thr Val Gly Gln Ser Pro Tyr Thr Ser Ala Pro Pro Leu Ser His Thr 35 40 45 Pro Asn Ala Asp Phe Gln Pro Pro Tyr Phe Pro Pro Pro Tyr Gln Pro 50 55 60 Ile Tyr Pro Gln Ser Gln Asp Pro Tyr Ser His Val Asn Asp Pro Tyr 65 70 75 80 Ser Leu Asn Pro Leu His Ala Gln Pro Gln Pro Gln His Pro Gly Trp 85 90 95 Pro Gly Gln Arg Gln Ser Gln Glu Ser Gly Leu Leu His Thr His Arg 100 105 110 Gly Leu Pro His Gln Leu Ser Gly Leu Asp Pro Arg Arg Asp Tyr Arg 115 120 125 Arg His Glu Asp Leu Leu His Gly Pro His Ala Leu Ser Ser Gly Leu 130 135 140 Gly Asp Leu Ser Ile His Ser Leu Pro His Ala Ile Glu Glu Val Pro 145 150 155 160 His Val Glu Asp Pro Gly Ile Asn Ile Pro Asp Gln Thr Val Ile Lys 165 170 175 Lys Gly Pro Val Ser Leu Ser Lys Ser Asn Ser Asn Ala Val Ser Ala 180 185 190 Ile Pro Ile Asn Lys Asp Asn Leu Phe Gly Gly Val Val Asn Pro Asn 195 200 205 Glu Val Phe Cys Ser Val Pro Gly Arg Leu Ser Leu Leu Ser Ser Thr 210 215 220 Ser Lys Tyr Lys Val Thr Val Ala Glu Val Gln Arg Arg Leu Ser Pro 225 230 235 240 Pro Glu Cys Leu Asn Ala Ser Leu Leu Gly Gly Val Leu Arg Arg Ala 245 250 255 Lys Ser Lys Asn Gly Gly Arg Ser Leu Arg Glu Lys Leu Asp Lys Ile 260 265 270 Gly Leu Asn Leu Pro Ala Gly Arg Arg Lys Ala Ala Asn Val Thr Leu 275 280 285 Leu Thr Ser Leu Val Glu Gly Glu Ala Val His Leu Ala Arg Asp Phe 290 295 300 Gly Tyr Val Cys Glu Thr Glu Phe Pro Ala Lys Ala Val Ala Glu Phe 305 310 315 320 Leu Asn Arg Gln His Ser Asp Pro Asn Glu Gln Val Thr Arg Lys Asn 325 330 335 Met Leu Leu Ala Thr Lys Gln Ile Cys Lys Glu Phe Thr Asp Leu Leu 340 345 350 Ala Gln Asp Arg Ser Pro Leu Gly Asn Ser Arg Pro Asn Pro Ile Leu 355 360 365 Glu Pro Gly Ile Gln Ser Cys Leu Thr His Phe Asn Leu Ile Ser His 370 375 380 Gly Phe Gly Ser Pro Ala Val Cys Ala Ala Val Thr Ala Leu Gln Asn 385 390 395 400 Tyr Leu Thr Glu Ala Leu Lys Ala Met Asp Lys Met Tyr Leu Ser Asn 405 410 415 Asn Pro Asn Ser His Thr Asp Asn Asn Ala Lys Ser Ser Asp Lys Glu 420 425 430 Glu Lys His Arg Lys 435 3841DNAHomo sapiens 38ggaattcaac atggacattg aagcatatct tgaaagaatt g 413939DNAHomo sapiens 39ggaattcctg gtgagctgga tgacaaatag acaagattg 394031DNAHomo sapiens 40ggaattcacc atgctttgga aattgacgga t 314136DNAHomo sapiens 41ggaattcctc actttctgtg cttctcctct ttgtca 364217PRTHomo sapiens 42Ser Ser Arg Arg His His Cys Arg Ser Lys Ala Lys Arg Ser Arg His 1 5 10 15 His 4317PRTHomo sapiens 43Ser Glu Phe Phe Ser Cys Gln Asn Cys Glu Ala Val Ala Gly Cys Ser 1 5 10 15 Ser 449PRTHomo sapiens 44Arg Leu Ser Pro Thr Ala Ala Thr Val 1 5 459PRTHomo sapiens 45Ser Ile Phe Arg Phe Arg Ala Gly Val 1 5 469PRTHomo sapiens 46Asp Val Leu Gly Asn Leu Asn Glu Leu 1 5 479PRTHomo sapiens 47Gly Val Gly Val Asp Val Leu Ser Leu 1 5 489PRTHomo sapiens 48Leu Leu Thr Ser Gln Ala Gln Asp Thr 1 5 499PRTHomo sapiens 49Val Leu Asn Ser Gln Ala Ser Gln Ala 1 5 509PRTHomo sapiens 50Val Gln Phe Lys Cys Gly Ala Pro Ala 1 5 519PRTHomo sapiens 51Gly Gln Pro Cys Pro Gln Ala Arg Leu 1 5 529PRTHomo sapiens 52Gly Ala His Arg Gly Leu Asp Ser Leu 1 5 539PRTHomo sapiens 53Gly Ala Pro Ala Ser Thr Pro Tyr Leu 1 5 549PRTHomo sapiens 54Val Val Gln Ala Cys His Arg Phe Ile 1 5 559PRTHomo sapiens 55Pro Leu Gly Ile Ser Leu Arg Pro Leu 1 5 569PRTHomo sapiens 56Pro Leu Arg Ala His Lys Ala Val Leu 1 5 579PRTHomo sapiens 57Phe Val Gln Val Ala His Leu Arg Ala 1 5 589PRTHomo sapiens 58Ala Pro Leu Leu Asp Phe Met Tyr Thr 1 5 599PRTHomo sapiens 59Arg Ala Gly Val Gly Val Asp Val Leu 1 5 609PRTHomo sapiens 60Cys Glu Thr Cys Gly Ser Arg Phe Val 1 5 619PRTHomo sapiens 61Ala Thr Ala Pro Ala Val Leu Ala Ala 1 5 629PRTHomo sapiens 62Ser Arg Phe Val Gln Val Ala His Leu 1 5 639PRTHomo sapiens 63Cys Asn Trp Lys Lys Tyr Lys Tyr Ile 1 5 649PRTHomo sapiens 64Ser Pro Ala Ala Pro Glu Gly Ala Leu 1 5 659PRTHomo sapiens 65Ala Leu Gly Tyr Val Arg Glu Phe Thr 1 5 669PRTHomo sapiens 66Arg Leu Arg Gly Ile Leu Thr Asp Val 1 5 679PRTHomo sapiens 67Gly Ile Leu Thr Asp Val Thr Leu Leu 1 5 689PRTHomo sapiens 68Ile Leu Thr Asp Val Thr Leu Leu Val 1 5 699PRTHomo sapiens 69Thr Leu Leu Val Gly Gly Gln Pro Leu 1 5 709PRTHomo sapiens 70Phe Met Tyr Thr Ser Arg Leu Arg Leu 1 5 719PRTHomo sapiens 71Arg Leu Ser Pro Ala Thr Ala Pro Ala 1 5 729PRTHomo sapiens 72Ala Val Leu Ala Ala Ala Thr Tyr Leu 1 5 739PRTHomo sapiens 73Ala Thr Tyr Leu Gln Met Glu His Val 1 5 749PRTHomo sapiens 74Leu Gln Met Glu His Val Val Gln Ala 1 5 759PRTHomo sapiens 75Gln Val Ala His Leu Arg Ala His Val 1 5 769PRTHomo sapiens 76His Leu Gln Thr Leu Lys Ser His Val 1 5 779PRTHomo sapiens 77Val Val Gln Ala Cys His Arg Phe Ile 1 5 7822PRTHomo sapiens 78Met Val Arg Lys Lys Asn Pro Pro Leu Arg Asn Val Ala Ser Glu Gly 1 5 10 15 Glu Gly Gln Ile Leu Glu 20 7922PRTHomo sapiens 79Ser Pro Lys Ala Thr Glu Glu Thr Gly Gln Ala Gln Ser Gly Gln Ala 1 5 10 15 Asn Cys Gln Gly Leu Ser 20 8022PRTHomo sapiens 80Val Ala Lys Pro Ser Glu Lys Asn Ser Asn Lys Ser Ile Pro Ala Leu 1 5 10 15 Gln Ser Ser Asp Ser Gly 20 8122PRTHomo sapiens 81Asn His Leu Gln Gly Ser Asp Gly Gln Gln Ser Val Lys Glu Ser Lys 1 5 10 15 Glu His Ser Cys Thr Lys 20 8222PRTHomo sapiens 82Asn Gly Glu Gln Ile Ile Arg Arg Arg Thr Arg Lys Arg Leu Asn Pro 1 5 10 15 Glu Ala Leu Gln Ala Glu 20 8323PRTHomo sapiens 83Ala Asn Gly Ala Ser Lys Glu Lys Thr Lys Ala Pro Pro Asn Val Lys 1 5 10 15 Asn Glu Gly Pro Leu Asn Val 20 8431DNAHomo sapiens 84cggatccacc atggtccgga aaaagaaccc c 318535DNAHomo sapiens 85cgggatccct ctttaggttt tccatttttt tccac 35869PRTHomo sapiens 86Ser Thr Ile Lys Glu Glu Pro Lys Ile 1 5 879PRTHomo sapiens 87Lys Ile Asp Phe Arg Val Tyr Asn Leu 1 5 889PRTHomo sapiens 88Asn Leu Leu Thr Pro Asp Ser Lys Met 1 5 899PRTHomo sapiens 89Val Thr Trp Arg Gly Ala Asp Ile Leu 1 5 909PRTHomo sapiens 90Ile Leu Arg Gly Ser Pro Ser Tyr Thr 1 5 919PRTHomo sapiens 91Tyr Thr Gln Ala Ser Leu Gly Leu Leu 1 5 929PRTHomo sapiens 92Ala Ser Leu Gly Leu Leu Thr Pro Val 1 5 939PRTHomo sapiens 93Gly Leu Leu Thr Pro Val Ser Gly Thr 1 5 949PRTHomo sapiens 94Gly Thr Gln Glu Gln Thr Lys Thr Leu 1 5 959PRTHomo sapiens 95Lys Thr Leu Arg Asp Ser Pro Asn Val 1 5 969PRTHomo sapiens 96His Leu Ala Arg Pro Ile Tyr Gly Leu 1 5 979PRTHomo sapiens 97Pro Ile Tyr Gly Leu Ala Val Glu Thr 1 5 989PRTHomo sapiens 98Leu Ala Val Glu Thr Lys Gly Phe Leu 1 5 999PRTHomo sapiens 99Phe Leu Gln Gly Ala Pro Ala Gly Gly 1 5 1009PRTHomo sapiens 100Ala Gly Gly Glu Lys Ser Gly Ala Leu 1 5 1019PRTHomo sapiens 101Gly Ala Leu Pro Gln Gln Tyr Pro Ala 1 5 1029PRTHomo sapiens 102Ala Leu Pro Gln Gln Tyr Pro Ala Ser 1 5 1039PRTHomo sapiens 103Phe Cys Ala Asn Cys Leu Thr Thr Lys 1 5 1049PRTHomo sapiens 104Ala Asn Gly Gly Tyr Val Cys Asn Ala 1 5

1059PRTHomo sapiens 105Asn Ala Cys Gly Leu Tyr Gln Lys Leu 1 5 1069PRTHomo sapiens 106Gly Leu Tyr Gln Lys Leu His Ser Thr 1 5 1079PRTHomo sapiens 107Lys Leu His Ser Thr Pro Arg Pro Leu 1 5 1089PRTHomo sapiens 108Ser Thr Pro Arg Pro Leu Asn Ile Ile 1 5 1099PRTHomo sapiens 109Arg Leu Asn Pro Glu Ala Leu Gln Ala 1 5 1109PRTHomo sapiens 110Val Leu Val Ser Gln Thr Leu Asp Ile 1 5 1119PRTHomo sapiens 111Asp Ile His Lys Arg Met Gln Pro Leu 1 5 1129PRTHomo sapiens 112Arg Met Gln Pro Leu His Ile Gln Ile 1 5 1139PRTHomo sapiens 113Tyr Pro Leu Phe Gly Leu Pro Phe Val 1 5 1149PRTHomo sapiens 114Gly Pro Leu Phe Val His Asn Asp Phe 1 5 1159PRTHomo sapiens 115Phe Val His Asn Asp Phe Gln Ser Glu 1 5 1169PRTHomo sapiens 116Ser Val Pro Gly Asn Pro His Tyr Leu 1 5 1179PRTHomo sapiens 117Gly Asn Pro His Tyr Leu Ser His Val 1 5 1189PRTHomo sapiens 118His Tyr Leu Ser His Val Pro Gly Leu 1 5 1199PRTHomo sapiens 119Tyr Val Pro Tyr Pro Thr Phe Asn Leu 1 5 1209PRTHomo sapiens 120Phe Asn Leu Pro Pro His Phe Ser Ala 1 5 1219PRTHomo sapiens 121Asn Leu Pro Pro His Phe Ser Ala Val 1 5 1229PRTHomo sapiens 122Ser Ala Val Gly Ser Asp Asn Asp Ile 1 5 1239PRTHomo sapiens 123Lys Asn Glu Gly Pro Leu Asn Val Val 1 5 1249PRTHomo sapiens 124Thr Lys Cys Val His Cys Gly Ile Val 1 5 1259PRTHomo sapiens 125Cys Val His Cys Gly Ile Val Phe Leu 1 5 1269PRTHomo sapiens 126Cys Gly Ile Val Phe Leu Asp Glu Val 1 5 1279PRTHomo sapiens 127Phe Leu Asp Glu Val Met Tyr Ala Leu 1 5 1289PRTHomo sapiens 128Val Met Tyr Ala Leu His Met Ser Cys 1 5 1299PRTHomo sapiens 129Phe Gln Cys Ser Ile Cys Gln His Leu 1 5 1309PRTHomo sapiens 130Gly Leu His Arg Asn Asn Ala Gln Val 1 5 1319PRTHomo sapiens 131Met Val Arg Lys Lys Asn Pro Pro Leu 1 5 1329PRTHomo sapiens 132Lys Lys Asn Pro Pro Leu Arg Asn Val 1 5 1339PRTHomo sapiens 133Val Ala Ser Glu Gly Glu Gly Gln Ile 1 5 1349PRTHomo sapiens 134Gln Ile Leu Glu Pro Ile Gly Thr Glu 1 5 1359PRTHomo sapiens 135Arg Asn Met Leu Ala Phe Ser Phe Pro 1 5 1369PRTHomo sapiens 136Asn Met Leu Ala Phe Ser Phe Pro Ala 1 5 1379PRTHomo sapiens 137Met Leu Ala Phe Ser Phe Pro Ala Ala 1 5 1389PRTHomo sapiens 138Phe Ser Phe Pro Ala Ala Gly Gly Val 1 5 1399PRTHomo sapiens 139Ala Ala Gly Gly Val Cys Glu Pro Leu 1 5 1409PRTHomo sapiens 140Ser Gly Gln Ala Asn Cys Gln Gly Leu 1 5 1419PRTHomo sapiens 141Gly Leu Ser Pro Val Ser Val Ala Ser 1 5 1429PRTHomo sapiens 142Gly Leu Ser Pro Val Ser Val Ala Ser 1 5 1439PRTHomo sapiens 143Ser Val Ala Ser Lys Asn Pro Gln Val 1 5 1449PRTHomo sapiens 144Arg Leu Asn Lys Ser Lys Thr Asp Leu 1 5 1459PRTHomo sapiens 145Asn Asp Asn Pro Asp Pro Ala Pro Leu 1 5 1469PRTHomo sapiens 146Asp Pro Ala Pro Leu Ser Pro Glu Leu 1 5 1479PRTHomo sapiens 147Glu Leu Gln Asp Phe Lys Cys Asn Ile 1 5 1489PRTHomo sapiens 148Gly Leu His Asn Arg Thr Arg Gln Asp 1 5 1499PRTHomo sapiens 149Glu Leu Asp Ser Lys Ile Leu Ala Leu 1 5 1509PRTHomo sapiens 150Lys Ile Leu Ala Leu His Asn Met Val 1 5 1519PRTHomo sapiens 151Ala Leu His Asn Met Val Gln Phe Ser 1 5 1529PRTHomo sapiens 152Val Asn Arg Ser Val Phe Ser Gly Val 1 5 1539PRTHomo sapiens 153Phe Ser Gly Val Leu Gln Asp Ile Asn 1 5 1549PRTHomo sapiens 154Asp Ile Asn Ser Ser Arg Pro Val Leu 1 5 1559PRTHomo sapiens 155Val Leu Leu Asn Gly Thr Tyr Asp Val 1 5 1569PRTHomo sapiens 156Phe Cys Asn Phe Thr Tyr Met Gly Asn 1 5 1579PRTHomo sapiens 157Tyr Met Gly Asn Ser Ser Thr Glu Leu 1 5 1589PRTHomo sapiens 158Phe Leu Gln Thr His Pro Asn Lys Ile 1 5 1599PRTHomo sapiens 159Lys Ala Ser Leu Pro Ser Ser Glu Val 1 5 1609PRTHomo sapiens 160Asp Leu Gly Lys Trp Gln Asp Lys Ile 1 5 1619PRTHomo sapiens 161Val Lys Ala Gly Asp Asp Thr Pro Val 1 5 1629PRTHomo sapiens 162Phe Ser Cys Glu Ser Ser Ser Ser Leu 1 5 16310PRTHomo sapiens 163Lys Leu Leu Leu Glu His Tyr Gly Lys Gln 1 5 10 1649PRTHomo sapiens 164Gly Leu Asn Pro Glu Leu Asn Asp Lys 1 5 1659PRTHomo sapiens 165Gly Ser Val Ile Asn Gln Asn Asp Leu 1 5 1669PRTHomo sapiens 166Ser Val Ile Asn Gln Asn Asp Leu Ala 1 5 1679PRTHomo sapiens 167Phe Cys Asp Phe Arg Tyr Ser Lys Ser 1 5 1689PRTHomo sapiens 168Pro Leu Leu Arg His Tyr Gln Gln Leu 1 5 1699PRTHomo sapiens 169Gly Leu Cys Ser Pro Glu Lys His Leu 1 5 1709PRTHomo sapiens 170His Leu Gly Glu Ile Thr Tyr Pro Phe 1 5 1719PRTHomo sapiens 171Leu Gly Glu Ile Thr Tyr Pro Phe Ala 1 5 1729PRTHomo sapiens 172His Cys Ala Leu Leu Leu Leu His Leu 1 5 1739PRTHomo sapiens 173Ala Leu Leu Leu Leu His Leu Ser Pro 1 5 1749PRTHomo sapiens 174Leu Leu Leu Leu His Leu Ser Pro Gly 1 5 1759PRTHomo sapiens 175Leu Leu Leu His Leu Ser Pro Gly Ala 1 5 1769PRTHomo sapiens 176Leu Leu His Leu Ser Pro Gly Ala Ala 1 5 1779PRTHomo sapiens 177Phe Thr Thr Pro Asp Val Asp Val Leu 1 5 1789PRTHomo sapiens 178Thr Thr Pro Asp Val Asp Val Leu Leu 1 5 1799PRTHomo sapiens 179Val Leu Leu Phe His Tyr Glu Ser Val 1 5 1809PRTHomo sapiens 180Phe Ile Thr Gln Val Glu Glu Glu Ile 1 5 1819PRTHomo sapiens 181Phe Thr Ala Ala Asp Thr Gln Ser Leu 1 5 1829PRTHomo sapiens 182Ser Leu Leu Glu His Phe Asn Thr Val 1 5 18339DNAHomo sapiens 183cagtacggat ccaccatggc cgagctgcgc ctgaagggc 3918438DNAHomo sapiens 184ccacgaggat ccttaggaga atattcggat ggcttgcg 3818520DNAHomo sapiens 185taatacgact cactataggg 2018618DNAHomo sapiens 186tagaaggcac agtcgagg 1818723DNAHomo sapiens 187gaaaacgact tcctggcggg gag 2318822DNAHomo sapiens 188gctcacccag gcgtggggcc tc 221899PRTHomo sapiens 189Val Pro Val Pro Thr Ser Glu His Val 1 5 1909PRTHomo sapiens 190Pro Thr Ser Glu His Val Ala Glu Ile 1 5 1919PRTHomo sapiens 191Glu Ile Val Gly Arg Gln Cys Lys Ile 1 5 1929PRTHomo sapiens 192Lys Ile Lys Ala Leu Arg Ala Lys Thr 1 5 1939PRTHomo sapiens 193Lys Ala Leu Arg Ala Lys Thr Asn Thr 1 5 1949PRTHomo sapiens 194Ala Leu Arg Ala Lys Thr Asn Thr Tyr 1 5 1959PRTHomo sapiens 195Leu Arg Ala Lys Thr Asn Thr Tyr Ile 1 5 1969PRTHomo sapiens 196Thr Asn Thr Tyr Ile Lys Thr Pro Val 1 5 1979PRTHomo sapiens 197Tyr Ile Lys Thr Pro Val Arg Gly Glu 1 5 1989PRTHomo sapiens 198Thr Pro Val Arg Gly Glu Glu Pro Val 1 5 1999PRTHomo sapiens 199Arg Gly Glu Glu Pro Val Phe Met Val 1 5 2009PRTHomo sapiens 200Met Val Thr Gly Arg Arg Glu Asp Val 1 5 2019PRTHomo sapiens 201Val Thr Gly Arg Arg Glu Asp Val Ala 1 5 2029PRTHomo sapiens 202Gly Arg Arg Glu Asp Val Ala Thr Ala 1 5 2039PRTHomo sapiens 203Asp Val Ala Thr Ala Arg Arg Glu Ile 1 5 2049PRTHomo sapiens 204Val Ala Thr Ala Arg Arg Glu Ile Ile 1 5 2059PRTHomo sapiens 205Thr Ala Arg Arg Glu Ile Ile Ser Ala 1 5 2069PRTHomo sapiens 206Ala Arg Arg Glu Ile Ile Ser Ala Ala 1 5 2079PRTHomo sapiens 207Ile Ile Ser Ala Ala Glu His Phe Ser 1 5 2089PRTHomo sapiens 208Ile Ser Ala Ala Glu His Phe Ser Met 1 5 2099PRTHomo sapiens 209Ser Ala Ala Glu His Phe Ser Met Ile 1 5 2109PRTHomo sapiens 210Ala Glu His Phe Ser Met Ile Arg Ala 1 5 2119PRTHomo sapiens 211Ser Met Ile Arg Ala Ser Arg Asn Lys 1 5 2129PRTHomo sapiens 212Arg Ala Ser Arg Asn Lys Ser Gly Ala 1 5 2139PRTHomo sapiens 213Asn Lys Ser Gly Ala Ala Phe Gly Val 1 5 2149PRTHomo sapiens 214Gly Ala Ala Phe Gly Val Ala Pro Ala 1 5 2159PRTHomo sapiens 215Ala Ala Phe Gly Val Ala Pro Ala Leu 1 5 2169PRTHomo sapiens 216Gly Val Ala Pro Ala Leu Pro Gly Gln 1 5 2179PRTHomo sapiens 217Val Ala Pro Ala Leu Pro Gly Gln Val 1 5 2189PRTHomo sapiens 218Pro Ala Leu Pro Gly Gln Val Thr Ile 1 5 2199PRTHomo sapiens 219Ala Leu Pro Gly Gln Val Thr Ile Arg 1 5 2209PRTHomo sapiens 220Leu Pro Gly Gln Val Thr Ile Arg Val 1 5 2219PRTHomo sapiens 221Gly Gln Val Thr Ile Arg Val Arg Val 1 5 2229PRTHomo sapiens 222Arg Val Arg Val Pro Tyr Arg Val Val 1 5 2239PRTHomo sapiens 223Arg Val Pro Tyr Arg Val Val Gly Leu 1 5 2249PRTHomo sapiens 224Val Pro Tyr Arg Val Val Gly Leu Val 1 5 2259PRTHomo sapiens 225Arg Val Val Gly Leu Val Val Gly Pro 1 5 2269PRTHomo sapiens 226Gly Leu Val Val Gly Pro Lys Gly Ala 1 5 2279PRTHomo sapiens 227Leu Val Val Gly Pro Lys Gly Ala Thr 1 5 2289PRTHomo sapiens 228Val Val Gly Pro Lys Gly Ala Thr Ile 1 5 2299PRTHomo sapiens 229Arg Ile Gln Gln Gln Thr Asn Thr Tyr 1 5 2309PRTHomo sapiens 230Ile Gln Gln Gln Thr Asn Thr Tyr Ile 1 5 2319PRTHomo sapiens 231Gln Gln Gln Thr Asn Thr Tyr Ile Ile 1 5 2329PRTHomo sapiens 232Gln Gln Thr Asn Thr Tyr Ile Ile Thr 1 5 2339PRTHomo sapiens 233Tyr Ile Ile Thr Pro Ser Arg Asp Arg 1 5 2349PRTHomo sapiens 234Thr Pro Ser Arg Asp Arg Asp Pro Val 1 5 2359PRTHomo sapiens 235Arg Asp Arg Asp Pro Val Phe Glu Ile 1 5 2369PRTHomo sapiens 236Glu Ile Thr Gly Ala Pro Gly Asn Val 1 5 2379PRTHomo sapiens 237Gly Ala Pro Gly Asn Val Glu Arg Ala 1 5 2389PRTHomo sapiens 238Asn Val Glu Arg Ala Arg Glu Glu Ile 1 5 2399PRTHomo sapiens 239Glu Glu Ile Glu Thr His Ile Ala Val 1 5 2409PRTHomo sapiens 240Ile Glu Thr His Ile Ala Val Arg Thr 1 5 2419PRTHomo sapiens 241His Ile Ala Val Arg Thr Gly Lys Ile 1 5 2429PRTHomo sapiens 242Ile Ala Val Arg Thr Gly Lys Ile Leu 1 5 2439PRTHomo sapiens 243Lys Ile Leu Glu Tyr Asn Asn Glu Asn 1 5 2449PRTHomo sapiens 244Tyr Asn Asn Glu Asn Asp Phe Leu Ala 1 5 2459PRTHomo sapiens 245Asn Glu Asn Asp Phe Leu Ala Gly Ser 1 5 2469PRTHomo sapiens 246Phe Leu Ala Gly Ser Pro Asp Ala Ala 1 5 2479PRTHomo sapiens 247Leu Ala Gly Ser Pro Asp Ala Ala Ile 1 5 2489PRTHomo sapiens 248Ala Ile Asp Ser Arg Tyr Ser Asp Ala 1 5 2499PRTHomo sapiens 249Ser Arg Tyr Ser Asp Ala Trp Arg Val 1 5 2509PRTHomo sapiens 250Val His Gln Pro Gly Cys Lys Pro Leu 1 5 2519PRTHomo sapiens 251Leu Ser Thr Phe Arg Gln Asn Ser Leu 1 5 2529PRTHomo sapiens 252Leu Gly Cys Ile Gly Glu Cys Gly Val 1 5 2539PRTHomo sapiens 253Cys Gly Val Asp Ser Gly Phe Glu Ala 1 5 2549PRTHomo sapiens 254Gly Phe Glu Ala Pro Arg Leu Asp Val 1 5 2559PRTHomo sapiens 255Arg Leu Asp Val Tyr Tyr Gly Val Ala 1 5 2569PRTHomo sapiens 256Asp Val Tyr Tyr Gly Val Ala Glu Thr 1 5 2579PRTHomo sapiens 257Gly Val Ala Glu Thr Ser Pro Pro Leu 1 5 2589PRTHomo sapiens 258Ala Glu Thr Ser Pro Pro Leu Trp Ala 1 5 2599PRTHomo sapiens 259Pro Leu Trp Ala Gly Gln Glu Asn Ala 1 5 2609PRTHomo sapiens 260Gln Gly Gln Glu Asn Ala Thr Pro Thr 1 5 2619PRTHomo sapiens 261Gln Glu Asn Ala Thr Pro Thr Ser Val 1 5 2629PRTHomo sapiens 262Val Leu Phe Ser Ser Ala Ser Ser Ser 1 5 2639PRTHomo sapiens 263Lys Ala Arg Ala Gly Pro Pro Gly Ala 1 5 2649PRTHomo sapiens 264Pro Ala Thr Ser Ala Gly Pro Glu Leu 1 5 2659PRTHomo sapiens 265Ala Thr Ser Ala Gly Pro Glu Leu Ala 1 5 2669PRTHomo sapiens 266Ser Ala Gly Pro Glu Leu Ala Gly Leu 1 5 2679PRTHomo sapiens 267Gly Leu Pro Arg Arg Pro Pro Gly Glu 1 5 2689PRTHomo sapiens 268Glu Pro Leu Gln Phe Gly Ser Lys Leu 1 5 2699PRTHomo sapiens 269Phe Ser Lys Leu Gly Gly Gly Gly Leu 1 5 2709PRTHomo sapiens 270Lys Leu Gly Gly Gly Gly Leu Arg Ser 1 5 2719PRTHomo sapiens 271Gly Leu Arg Ser Pro Gly Gly Gly Arg 1 5 2729PRTHomo sapiens 272Cys Met Val Cys Phe Glu Ser Glu Val 1 5 2739PRTHomo sapiens 273Met Val Cys Phe Glu Ser Glu Val Thr 1 5 2749PRTHomo sapiens 274Val Cys Phe Glu Ser Glu Val Thr Ala 1 5 2759PRTHomo sapiens 275Phe Glu Ser Glu Val Thr Ala Ala Leu 1 5 2769PRTHomo sapiens 276Glu Val Thr Ala Ala Leu Val Pro Cys 1 5 2779PRTHomo sapiens 277Val Thr Ala Ala Leu Val Pro Cys Gly 1 5 2789PRTHomo sapiens 278Ala Leu Val Pro Cys Gly His Asn Leu 1 5 2799PRTHomo sapiens 279Leu Val Pro Cys Gly His Asn Leu Phe 1 5 2809PRTHomo sapiens 280Val Pro Cys Gly His Asn Leu Phe Cys 1 5 2819PRTHomo sapiens 281Asn Leu Phe Cys Met Glu Cys Ala Val 1 5 2829PRTHomo sapiens 282Phe Cys Met Glu Cys Ala Val Arg Ile 1 5 2839PRTHomo sapiens 283Cys Ala Val Arg Ile Cys Glu Arg Thr 1 5 2849PRTHomo sapiens 284Arg Ile Cys Glu Arg Thr Asp Pro Glu 1 5 2859PRTHomo sapiens 285Arg Thr Asp Pro Glu Cys Pro Val Cys 1 5 2869PRTHomo sapiens 286Cys Pro Val Cys His Ile Thr Ala Thr 1 5 2879PRTHomo sapiens 287Val Cys His Ile Thr Ala Thr Gln Ala 1 5 2889PRTHomo sapiens 288Ile Thr Ala Thr Gln Ala Ile Arg Ile 1 5 2899PRTHomo sapiens 289Leu Met Asp Met Gln Thr Phe Lys Ala 1 5 2909PRTHomo sapiens 290Lys Val Ser Ile Pro Thr Lys Ala Leu 1 5 2919PRTHomo sapiens 291Ser Ile Pro Thr Lys Ala Leu Glu Leu 1 5 2929PRTHomo sapiens 292Leu Glu Leu Lys Asn Glu Gln Thr Leu 1 5 2939PRTHomo sapiens 293Thr

Val Ser Gln Lys Asp Val Cys Leu 1 5 2949PRTHomo sapiens 294Ser Val Pro Asn Lys Ala Leu Glu Leu 1 5 2959PRTHomo sapiens 295Cys Glu Thr Val Ser Gln Lys Asp Val 1 5 2969PRTHomo sapiens 296Lys Ile Asn Gly Lys Leu Glu Glu Ser 1 5 2979PRTHomo sapiens 297Ser Leu Val Glu Lys Thr Pro Asp Glu 1 5 2989PRTHomo sapiens 298Ser Leu Cys Glu Thr Val Ser Gln Lys 1 5 2999PRTHomo sapiens 299Glu Ile Asp Lys Ile Asn Gly Lys Leu 1 5 3009PRTHomo sapiens 300Met Leu Leu Gln Gln Asn Val Asp Val 1 5 3019PRTHomo sapiens 301Asn Met Trp Leu Gln Gln Gln Leu Val 1 5 3029PRTHomo sapiens 302Phe Leu Val Asp Arg Lys Cys Gln Leu 1 5 3039PRTHomo sapiens 303Tyr Leu Leu His Glu Asn Cys Met Leu 1 5 3049PRTHomo sapiens 304Ser Leu Phe Glu Ser Ser Ala Lys Ile 1 5 3059PRTHomo sapiens 305Lys Ile Thr Ile Asp Ile His Phe Leu 1 5 3069PRTHomo sapiens 306Gln Leu Gln Ser Lys Asn Met Trp Leu 1 5 3079PRTHomo sapiens 307Ser Leu Asp Gln Lys Leu Phe Gln Leu 1 5 3089PRTHomo sapiens 308Phe Leu Leu Ile Lys Asn Ala Asn Ala 1 5 3099PRTHomo sapiens 309Lys Ile Leu Asp Thr Val His Ser Cys 1 5 3109PRTHomo sapiens 310Ser Leu Ser Lys Ile Leu Asp Thr Val 1 5 3119PRTHomo sapiens 311Ile Leu Ile Asp Ser Gly Ala Asp Ile 1 5 3129PRTHomo sapiens 312Lys Val Met Glu Ile Asn Arg Glu Val 1 5 3139PRTHomo sapiens 313Lys Leu Leu Ser His Gly Ala Val Ile 1 5 3149PRTHomo sapiens 314Ala Val Tyr Ser Glu Ile Leu Ser Val 1 5 3159PRTHomo sapiens 315Lys Met Asn Val Asp Val Ser Ser Thr 1 5 3169PRTHomo sapiens 316Ile Leu Ser Val Val Ala Lys Leu Leu 1 5 3179PRTHomo sapiens 317Val Leu Ile Ala Glu Asn Thr Met Leu 1 5 3189PRTHomo sapiens 318Lys Leu Ser Lys Asn His Gln Asn Thr 1 5 3199PRTHomo sapiens 319Ser Leu Thr Pro Leu Leu Leu Ser Ile 1 5 3209PRTHomo sapiens 320Ser Gln Tyr Ser Gly Gln Leu Lys Val 1 5 3219PRTHomo sapiens 321Lys Glu Leu Glu Val Lys Gln Gln Leu 1 5 3229PRTHomo sapiens 322Gln Ile Met Glu Tyr Ile Arg Lys Leu 1 5 3239PRTHomo sapiens 323Ala Met Leu Lys Leu Glu Ile Ala Thr 1 5 3249PRTHomo sapiens 324Val Leu His Gln Pro Leu Ser Glu Ala 1 5 3259PRTHomo sapiens 325Gly Leu Leu Lys Ala Thr Cys Gly Met 1 5 3269PRTHomo sapiens 326Gly Leu Leu Lys Ala Asn Cys Gly Met 1 5 3279PRTHomo sapiens 327Gln Gln Leu Glu Gln Ala Leu Arg Ile 1 5 3289PRTHomo sapiens 328Cys Met Leu Lys Lys Glu Ile Ala Met 1 5 3299PRTHomo sapiens 329Glu Gln Met Lys Lys Lys Phe Cys Val 1 5 3309PRTHomo sapiens 330Ile Gln Lys Ile Glu Leu Lys Ser Val 1 5 3319PRTHomo sapiens 331Ser Val Pro Asn Lys Ala Phe Glu Leu 1 5 3329PRTHomo sapiens 332Ser Ile Tyr Gln Lys Val Met Glu Ile 1 5 3339PRTHomo sapiens 333Asn Leu Asn Tyr Gln Gly Asp Ala Leu 1 5 3349PRTHomo sapiens 334Ala Val Gln Asp His Asp Gln Ile Val 1 5 3359PRTHomo sapiens 335Leu Ile Ala Glu Asn Thr Met Leu Thr 1 5 3369PRTHomo sapiens 336Phe Glu Leu Lys Asn Glu Gln Thr Leu 1 5 3379PRTHomo sapiens 337Phe Glu Ser Ser Ala Lys Ile Gln Val 1 5 3389PRTHomo sapiens 338Gly Val Thr Ala Glu His Tyr Ala Val 1 5 3399PRTHomo sapiens 339Arg Val Thr Ser Asn Lys Thr Lys Val 1 5 3409PRTHomo sapiens 340Thr Val Ser Gln Lys Asp Val Cys Val 1 5 3419PRTHomo sapiens 341Lys Ser Gln Glu Pro Ala Phe His Ile 1 5 3429PRTHomo sapiens 342Lys Asn Leu Ile Ala Glu Asn Thr Met 1 5 3439PRTHomo sapiens 343Met Leu Lys Leu Glu Ile Ala Thr Leu 1 5 3449PRTHomo sapiens 344Glu Ile Leu Ser Val Val Ala Lys Leu 1 5 3459PRTHomo sapiens 345Met Leu Lys Lys Glu Ile Ala Met Leu 1 5 3469PRTHomo sapiens 346Leu Leu Lys Glu Lys Asn Glu Glu Ile 1 5 3479PRTHomo sapiens 347Ala Leu Arg Ile Gln Asp Ile Glu Leu 1 5 3489PRTHomo sapiens 348Lys Ile Arg Glu Glu Leu Gly Arg Ile 1 5 3499PRTHomo sapiens 349Thr Leu Lys Leu Lys Glu Glu Ser Leu 1 5 3509PRTHomo sapiens 350Ile Leu Asn Glu Lys Ile Arg Glu Glu 1 5 3519PRTHomo sapiens 351Val Leu Lys Lys Lys Leu Ser Glu Ala 1 5 3529PRTHomo sapiens 352Gly Thr Ser Asp Lys Ile Gln Cys Leu 1 5 3539PRTHomo sapiens 353Gly Ala Asp Ile Asn Leu Val Asp Val 1 5 3549PRTHomo sapiens 354Glu Leu Cys Ser Val Arg Leu Thr Leu 1 5 3559PRTHomo sapiens 355Ser Val Glu Ser Asn Leu Asn Gln Val 1 5 3569PRTHomo sapiens 356Ser Leu Lys Ile Asn Leu Asn Tyr Ala 1 5 3579PRTHomo sapiens 357Lys Thr Pro Asp Glu Ala Ala Ser Leu 1 5 3589PRTHomo sapiens 358Ala Thr Cys Gly Met Lys Val Ser Ile 1 5 3599PRTHomo sapiens 359Leu Ser His Gly Ala Val Ile Glu Val 1 5 3609PRTHomo sapiens 360Glu Ile Ala Met Leu Lys Leu Glu Ile 1 5 3619PRTHomo sapiens 361Ala Glu Leu Gln Met Thr Leu Lys Leu 1 5 3629PRTHomo sapiens 362Val Phe Ala Ala Asp Ile Cys Gly Val 1 5 3639PRTHomo sapiens 363Pro Ala Ile Glu Met Gln Asn Ser Val 1 5 3649PRTHomo sapiens 364Glu Ile Phe Asn Tyr Asn Asn His Leu 1 5 3659PRTHomo sapiens 365Ile Leu Lys Glu Lys Asn Ala Glu Leu 1 5 3669PRTHomo sapiens 366Gln Leu Val His Ala His Lys Lys Ala 1 5 3679PRTHomo sapiens 367Asn Ile Gln Asp Ala Gln Lys Arg Thr 1 5 3689PRTHomo sapiens 368Asn Leu Val Asp Val Tyr Gly Asn Met 1 5 3699PRTHomo sapiens 369Lys Cys Thr Ala Leu Met Leu Ala Val 1 5 3709PRTHomo sapiens 370Lys Ile Gln Cys Leu Glu Lys Ala Thr 1 5 3719PRTHomo sapiens 371Lys Ile Ala Trp Glu Lys Lys Glu Thr 1 5 3729PRTHomo sapiens 372Ile Ala Trp Glu Lys Lys Glu Asp Thr 1 5 3739PRTHomo sapiens 373Val Gly Met Leu Leu Gln Gln Asn Val 1 5 3749PRTHomo sapiens 374Val Lys Thr Gly Cys Val Ala Arg Val 1 5 3759PRTHomo sapiens 375Ala Leu His Tyr Ala Val Tyr Ser Glu 1 5 3769PRTHomo sapiens 376Gln Met Lys Lys Lys Phe Cys Val Leu 1 5 3779PRTHomo sapiens 377Ala Leu Gln Cys His Gln Glu Ala Cys 1 5 3789PRTHomo sapiens 378Ser Glu Gln Ile Val Glu Phe Leu Leu 1 5 3799PRTHomo sapiens 379Ala Val Ile Glu Val His Asn Lys Ala 1 5 3809PRTHomo sapiens 380Ala Val Thr Cys Gly Phe His His Ile 1 5 3819PRTHomo sapiens 381Ala Cys Leu Gln Arg Lys Met Asn Val 1 5 3829PRTHomo sapiens 382Ser Leu Val Glu Gly Thr Ser Asp Lys 1 5 38323PRTHomo sapiens 383Met Thr Lys Arg Lys Lys Thr Ile Asn Leu Asn Ile Gln Asp Ala Gln 1 5 10 15 Lys Arg Thr Ala Leu His Trp 20 38423PRTHomo sapiens 384Thr Ser Glu Lys Phe Thr Trp Pro Ala Lys Gly Arg Pro Arg Lys Ile 1 5 10 15 Ala Trp Glu Lys Lys Glu Asp 20 38523PRTHomo sapiens 385Asp Glu Ile Leu Pro Ser Glu Ser Lys Gln Lys Asp Tyr Glu Glu Asn 1 5 10 15 Ser Trp Asp Thr Glu Ser Leu 20 38623PRTHomo sapiens 386Arg Leu Thr Leu Asn Gln Glu Glu Glu Lys Arg Arg Asn Ala Asp Ile 1 5 10 15 Leu Asn Glu Lys Ile Arg Glu 20 38723PRTHomo sapiens 387Ala Glu Asn Thr Met Leu Thr Ser Lys Leu Lys Glu Lys Gln Asp Lys 1 5 10 15 Glu Ile Leu Glu Ala Glu Ile 20 38823PRTHomo sapiens 388Asn Tyr Asn Asn His Leu Lys Asn Arg Ile Tyr Gln Tyr Glu Lys Glu 1 5 10 15 Lys Ala Glu Thr Glu Asn Ser 20 3899PRTHomo sapiens 389Leu Asp Leu Glu Thr Leu Thr Asp Ile 1 5 3909PRTHomo sapiens 390Asp Ile Leu Gln His Gln Ile Arg Ala 1 5 3919PRTHomo sapiens 391Ile Leu Gln His Gln Ile Arg Ala Val 1 5 3929PRTHomo sapiens 392Ala Val Pro Phe Glu Asn Leu Asn Ile 1 5 3939PRTHomo sapiens 393Asn Leu Asn Ile His Cys Gly Asp Ala 1 5 3949PRTHomo sapiens 394Ala Met Asp Leu Gly Leu Glu Ala Ile 1 5 3959PRTHomo sapiens 395Gly Leu Glu Ala Ile Phe Asp Gln Val 1 5 3969PRTHomo sapiens 396Leu Glu Ala Ile Phe Asp Gln Val Val 1 5 3979PRTHomo sapiens 397Trp Cys Leu Gln Val Asn His Leu Leu 1 5 3989PRTHomo sapiens 398Gln Val Asn His Leu Leu Tyr Trp Ala 1 5 3999PRTHomo sapiens 399Val Asn His Leu Leu Tyr Trp Ala Leu 1 5 4009PRTHomo sapiens 400His Leu Leu Tyr Trp Ala Leu Thr Thr 1 5 4019PRTHomo sapiens 401Leu Leu Tyr Trp Ala Leu Thr Thr Ile 1 5 4029PRTHomo sapiens 402Ala Leu Thr Thr Ile Gly Phe Glu Thr 1 5 4039PRTHomo sapiens 403Leu Thr Thr Ile Gly Phe Glu Thr Thr 1 5 4049PRTHomo sapiens 404Thr Thr Ile Gly Phe Glu Thr Thr Met 1 5 4059PRTHomo sapiens 405Thr Ile Gly Phe Glu Thr Thr Met Leu 1 5 4069PRTHomo sapiens 406Thr Met Leu Gly Gly Tyr Val Tyr Ser 1 5 4079PRTHomo sapiens 407Met Leu Gly Gly Tyr Val Tyr Ser Thr 1 5 4089PRTHomo sapiens 408Tyr Ser Thr Gly Met Ile His Leu Leu 1 5 4099PRTHomo sapiens 409Ser Thr Gly Met Ile His Leu Leu Leu 1 5 4109PRTHomo sapiens 410Gly Met Ile His Leu Leu Leu Gln Val 1 5 4119PRTHomo sapiens 411Met Ile His Leu Leu Leu Gln Val Thr 1 5 4129PRTHomo sapiens 412Leu Leu Leu Gln Val Thr Ile Asp Gly 1 5 4139PRTHomo sapiens 413Val Thr Ile Asp Gly Arg Asn Tyr Ile 1 5 4149PRTHomo sapiens 414Thr Ile Asp Gly Arg Asn Tyr Ile Val 1 5 4159PRTHomo sapiens 415Tyr Ile Val Asp Ala Gly Phe Gly Arg 1 5 4169PRTHomo sapiens 416Arg Ser Tyr Gln Met Trp Gln Pro Leu 1 5 4179PRTHomo sapiens 417Tyr Gln Met Trp Gln Pro Leu Glu Leu 1 5 4189PRTHomo sapiens 418Gln Met Trp Gln Pro Leu Glu Leu Ile 1 5 4199PRTHomo sapiens 419Ile Ser Gly Lys Asp Gln Pro Gln Val 1 5 4209PRTHomo sapiens 420Lys Asp Gln Pro Gln Val Pro Cys Val 1 5 4219PRTHomo sapiens 421Pro Gln Val Pro Cys Val Phe Arg Leu 1 5 4229PRTHomo sapiens 422Gln Val Pro Cys Val Phe Arg Leu Thr 1 5 4239PRTHomo sapiens 423Arg Leu Thr Glu Glu Asn Gly Phe Trp 1 5 4249PRTHomo sapiens 424Thr Glu Glu Asn Gly Phe Trp Tyr Leu 1 5 4259PRTHomo sapiens 425Asn Gly Phe Trp Tyr Leu Asp Gln Ile 1 5 4269PRTHomo sapiens 426Asp Gln Ile Arg Arg Glu Gln Tyr Ile 1 5 4279PRTHomo sapiens 427Tyr Ile Pro Asn Glu Glu Phe Leu His 1 5 4289PRTHomo sapiens 428Tyr Ser Phe Thr Leu Lys Pro Arg Thr 1 5 4299PRTHomo sapiens 429Arg Thr Ile Glu Asp Phe Glu Ser Met 1 5 4309PRTHomo sapiens 430Tyr Leu Gln Thr Ser Pro Ser Ser Val 1 5 4319PRTHomo sapiens 431Gln Thr Ser Pro Ser Ser Val Phe Thr 1 5 4329PRTHomo sapiens 432Ser Val Phe Thr Ser Lys Ser Phe Cys 1 5 4339PRTHomo sapiens 433Phe Thr Ser Lys Ser Phe Cys Ser Leu 1 5 4349PRTHomo sapiens 434Cys Ser Leu Gln Thr Pro Asp Gly Val 1 5 4359PRTHomo sapiens 435Leu Gln Thr Pro Asp Gly Val His Cys 1 5 4369PRTHomo sapiens 436Gln Thr Pro Asp Gly Val His Cys Leu 1 5 4379PRTHomo sapiens 437Thr Pro Asp Gly Val His Cys Leu Val 1 5 4389PRTHomo sapiens 438Gly Val His Cys Leu Val Gly Phe Thr 1 5 4399PRTHomo sapiens 439Cys Leu Val Gly Phe Thr Leu Thr His 1 5 4409PRTHomo sapiens 440Thr Leu Thr His Arg Arg Phe Asn Tyr 1 5 4419PRTHomo sapiens 441Phe Asn Tyr Lys Asp Asn Thr Asp Leu 1 5 4429PRTHomo sapiens 442Asn Thr Asp Leu Ile Glu Phe Lys Thr 1 5 4439PRTHomo sapiens 443Thr Lys Leu Ile Glu Phe Lys Thr Leu 1 5 4449PRTHomo sapiens 444Leu Ser Glu Glu Glu Ile Glu Lys Val 1 5 4459PRTHomo sapiens 445Lys Val Leu Lys Asn Ile Phe Asn Ile 1 5 4469PRTHomo sapiens 446Leu Lys Asn Ile Phe Asn Ile Ser Leu 1 5 4479PRTHomo sapiens 447Asn Ile Ser Leu Gln Arg Lys Leu Val 1 5 4489PRTHomo sapiens 448Lys His Gly Asp Arg Phe Phe Thr Ile 1 5 4499PRTHomo sapiens 449Asp Ile Glu Ala Tyr Leu Glu Arg Ile 1 5 4509PRTHomo sapiens 450Tyr Leu Glu Thr Ile Gly Tyr Lys Lys 1 5 4519PRTHomo sapiens 451Arg Asn Lys Leu Asp Leu Glu Thr Leu 1 5 4529PRTHomo sapiens 452Asn Lys Leu Asp Leu Glu Thr Leu Thr 1 5 4539PRTHomo sapiens 453Lys Leu Asp Leu Glu Thr Leu Thr Asp 1 5 4549PRTHomo sapiens 454Asp Leu Glu Thr Leu Thr Asp Ile Leu 1 5 4559PRTHomo sapiens 455Thr Leu Thr Asp Ile Leu Gln His Gln 1 5 4569PRTHomo sapiens 456Leu Thr Asp Ile Leu Gln His Gln Ile 1 5 4579PRTHomo sapiens 457Gln Ile Arg Ala Val Pro Phe Glu Asn 1 5 4589PRTHomo sapiens 458Ile Arg Ala Val Pro Phe Glu Asn Leu 1 5 4599PRTHomo sapiens 459Ile His Cys Gly Asp Ala Met Asp Leu 1 5 4609PRTHomo sapiens 460His Cys Gly Asp Ala Met Asp Leu Gly 1 5 4619PRTHomo sapiens 461Asp Leu Gly Leu Glu Ala Ile Phe Asp 1 5 4629PRTHomo sapiens 462Ala Ile Phe Asp Gln Val Val Arg Arg 1 5 4639PRTHomo sapiens 463Gly Trp Cys Leu Gln Val Asn His Leu 1 5 4649PRTHomo sapiens 464Leu Gln Val Asn His Leu Leu Tyr Trp 1 5 4659PRTHomo sapiens 465Gly Gly Tyr Val Tyr Ser Thr Pro Ala 1 5 4669PRTHomo sapiens 466Tyr Val Tyr Ser Thr Pro Ala Lys Lys 1 5 4679PRTHomo sapiens 467Ser Thr Pro Ala Lys Lys Tyr Ser Thr 1 5 4689PRTHomo sapiens 468Ile His Leu Leu Leu Gln Val Thr Ile 1 5 4699PRTHomo sapiens 469His Leu Leu Leu Gln Val Thr Ile Asp 1 5 4709PRTHomo sapiens 470Leu Leu Gln Val Thr Ile Asp Gly Arg 1 5 4719PRTHomo sapiens 471Tyr Leu Asp Gln Ile Arg Arg Glu Gln 1 5 4729PRTHomo sapiens 472Gln Tyr Ile Pro Asn Glu Glu Phe Leu 1 5 4739PRTHomo sapiens 473Phe Leu His Ser Asp Leu Leu Glu Asp 1 5 4749PRTHomo sapiens 474Asp Leu Leu Glu Asp Ser Lys

Tyr Arg 1 5 4759PRTHomo sapiens 475Tyr Arg Lys Ile Tyr Ser Phe Thr Leu 1 5 4769PRTHomo sapiens 476Lys Ile Tyr Ser Phe Thr Leu Lys Pro 1 5 4779PRTHomo sapiens 477Thr Leu Lys Pro Arg Thr Ile Glu Asp 1 5 4789PRTHomo sapiens 478Val His Cys Leu Val Gly Phe Thr Leu 1 5 4799PRTHomo sapiens 479Leu Thr His Arg Arg Phe Asn Tyr Lys 1 5 4809PRTHomo sapiens 480Asp Leu Ile Glu Phe Lys Thr Leu Ser 1 5 4819PRTHomo sapiens 481Leu Ile Glu Phe Lys Thr Leu Ser Glu 1 5 4829PRTHomo sapiens 482Phe Lys Thr Leu Ser Glu Glu Glu Ile 1 5 4839PRTHomo sapiens 483Thr Leu Ser Glu Glu Glu Ile Glu Lys 1 5 4849PRTHomo sapiens 484Glu Ile Glu Lys Val Leu Lys Asn Ile 1 5 4859PRTHomo sapiens 485Phe Asn Ile Ser Leu Gln Arg Lys Leu 1 5 4869PRTHomo sapiens 486Ser Leu Gln Arg Lys Leu Val Pro Lys 1 5 4879PRTHomo sapiens 487Lys Leu Val Pro Lys His Gly Asp Arg 1 5 4889PRTHomo sapiens 488Pro Lys His Gly Asp Arg Phe Phe Thr 1 5 4899PRTHomo sapiens 489Met Leu Trp Lys Leu Thr Asp Asn Ile 1 5 4909PRTHomo sapiens 490Lys Leu Thr Asp Asn Ile Lys Tyr Glu 1 5 4919PRTHomo sapiens 491Gly Thr Ser Asn Gly Thr Ala Arg Leu 1 5 4929PRTHomo sapiens 492Asn Gly Thr Ala Arg Leu Pro Gln Leu 1 5 4939PRTHomo sapiens 493Ala Arg Leu Pro Gln Leu Gly Thr Val 1 5 4949PRTHomo sapiens 494Gly Thr Val Gly Gln Ser Pro Tyr Thr 1 5 4959PRTHomo sapiens 495Ser Pro Tyr Thr Ser Ala Pro Pro Leu 1 5 4969PRTHomo sapiens 496Phe Gln Pro Pro Tyr Phe Pro Pro Pro 1 5 4979PRTHomo sapiens 497Tyr Phe Pro Pro Pro Tyr Gln Pro Ile 1 5 4989PRTHomo sapiens 498Gln Ser Gln Asp Pro Tyr Ser His Val 1 5 4999PRTHomo sapiens 499Ser His Val Asn Asp Pro Tyr Ser Leu 1 5 5009PRTHomo sapiens 500Ser Leu Asn Pro Leu His Ala Gln Pro 1 5 5019PRTHomo sapiens 501Arg Gln Ser Gln Glu Ser Gly Leu Leu 1 5 5029PRTHomo sapiens 502Gly Leu Leu His Thr His Arg Gly Leu 1 5 5039PRTHomo sapiens 503Gly Leu Pro His Gln Leu Ser Gly Leu 1 5 5049PRTHomo sapiens 504Gly Leu Asp Pro Arg Arg Asp Tyr Arg 1 5 5059PRTHomo sapiens 505Asp Leu Leu His Gly Pro His Ala Leu 1 5 5069PRTHomo sapiens 506Leu Leu His Gly Pro His Ala Leu Ser 1 5 5079PRTHomo sapiens 507Ala Leu Ser Ser Gly Leu Gly Asp Leu 1 5 5089PRTHomo sapiens 508Ser Ser Gly Leu Gly Asp Leu Ser Ile 1 5 5099PRTHomo sapiens 509Gly Leu Gly Asp Leu Ser Ile His Ser 1 5 5109PRTHomo sapiens 510Leu Gly Asp Leu Ser Ile His Ser Leu 1 5 5119PRTHomo sapiens 511Ser Ile His Ser Leu Pro His Ala Ile 1 5 5129PRTHomo sapiens 512Ser Leu Pro His Ala Ile Glu Glu Val 1 5 5139PRTHomo sapiens 513His Ala Ile Glu Glu Val Pro His Val 1 5 5149PRTHomo sapiens 514Gly Ile Asn Ile Pro Asp Gln Thr Val 1 5 5159PRTHomo sapiens 515Gln Thr Val Ile Lys Lys Gly Pro Val 1 5 5169PRTHomo sapiens 516Val Ile Lys Lys Gly Pro Val Ser Leu 1 5 5179PRTHomo sapiens 517Ser Leu Ser Lys Ser Asn Ser Asn Ala 1 5 5189PRTHomo sapiens 518Ser Asn Ser Asn Ala Val Ser Ala Ile 1 5 5199PRTHomo sapiens 519Ala Ile Pro Ile Asn Lys Asp Asn Leu 1 5 5209PRTHomo sapiens 520Asn Leu Phe Gly Gly Val Val Asn Pro 1 5 5219PRTHomo sapiens 521Phe Gly Gly Val Val Asn Pro Asn Glu 1 5 5229PRTHomo sapiens 522Gly Gly Val Val Asn Pro Asn Glu Val 1 5 5239PRTHomo sapiens 523Asn Pro Asn Glu Val Phe Cys Ser Val 1 5 5249PRTHomo sapiens 524Cys Ser Val Pro Gly Arg Leu Ser Leu 1 5 5259PRTHomo sapiens 525Ser Val Pro Gly Arg Leu Ser Leu Leu 1 5 5269PRTHomo sapiens 526Ser Leu Leu Ser Ser Thr Ser Lys Tyr 1 5 5279PRTHomo sapiens 527Leu Leu Ser Ser Thr Ser Lys Tyr Lys 1 5 5289PRTHomo sapiens 528Leu Ser Ser Thr Ser Lys Tyr Lys Val 1 5 5299PRTHomo sapiens 529Ser Thr Ser Lys Tyr Lys Val Thr Val 1 5 5309PRTHomo sapiens 530Lys Tyr Lys Val Thr Val Ala Glu Val 1 5 5319PRTHomo sapiens 531Tyr Lys Val Thr Val Ala Glu Val Gln 1 5 5329PRTHomo sapiens 532Thr Val Ala Glu Val Gln Arg Arg Leu 1 5 5339PRTHomo sapiens 533Arg Leu Ser Pro Pro Glu Cys Leu Asn 1 5 5349PRTHomo sapiens 534Leu Asn Ala Ser Leu Leu Gly Gly Val 1 5 5359PRTHomo sapiens 535Asn Ala Ser Leu Leu Gly Gly Val Leu 1 5 5369PRTHomo sapiens 536Ser Leu Leu Gly Gly Val Leu Arg Arg 1 5 5379PRTHomo sapiens 537Leu Leu Gly Gly Val Leu Arg Arg Ala 1 5 5389PRTHomo sapiens 538Val Leu Arg Arg Ala Lys Ser Lys Asn 1 5 5399PRTHomo sapiens 539Ser Leu Arg Glu Lys Leu Asp Lys Ile 1 5 5409PRTHomo sapiens 540Lys Leu Asp Lys Ile Gly Leu Asn Leu 1 5 5419PRTHomo sapiens 541Lys Ile Gly Leu Asn Leu Pro Ala Gly 1 5 5429PRTHomo sapiens 542Gly Leu Asn Leu Pro Ala Gly Arg Arg 1 5 5439PRTHomo sapiens 543Asn Leu Pro Ala Gly Arg Arg Lys Ala 1 5 5449PRTHomo sapiens 544Ala Gly Arg Arg Lys Ala Ala Asn Val 1 5 5459PRTHomo sapiens 545Arg Lys Ala Ala Asn Val Thr Leu Leu 1 5 5469PRTHomo sapiens 546Lys Ala Ala Asn Val Thr Leu Leu Thr 1 5 5479PRTHomo sapiens 547Ala Asn Val Thr Leu Leu Thr Ser Leu 1 5 5489PRTHomo sapiens 548Asn Val Thr Leu Leu Thr Ser Leu Val 1 5 5499PRTHomo sapiens 549Thr Leu Leu Thr Ser Leu Val Glu Gly 1 5 5509PRTHomo sapiens 550Leu Leu Thr Ser Leu Val Glu Gly Glu 1 5 5519PRTHomo sapiens 551Thr Ser Leu Val Glu Gly Glu Ala Val 1 5 5529PRTHomo sapiens 552Ser Leu Val Glu Gly Glu Ala Val His 1 5 5539PRTHomo sapiens 553Leu Val Glu Gly Glu Ala Val His Leu 1 5 5549PRTHomo sapiens 554Val Glu Gly Glu Ala Val His Leu Ala 1 5 5559PRTHomo sapiens 555His Leu Ala Arg Asp Phe Gly Tyr Val 1 5 5569PRTHomo sapiens 556Tyr Val Cys Glu Thr Glu Phe Pro Ala 1 5 5579PRTHomo sapiens 557Cys Glu Thr Glu Phe Pro Ala Lys Ala 1 5 5589PRTHomo sapiens 558Ala Lys Ala Val Ala Glu Phe Leu Asn 1 5 5599PRTHomo sapiens 559Ala Val Ala Glu Phe Leu Asn Arg Gln 1 5 5609PRTHomo sapiens 560Phe Leu Asn Arg Gln His Ser Asp Pro 1 5 5619PRTHomo sapiens 561Gln Val Thr Arg Lys Asn Met Leu Leu 1 5 5629PRTHomo sapiens 562Asn Met Leu Leu Ala Thr Lys Gln Ile 1 5 5639PRTHomo sapiens 563Met Leu Leu Ala Thr Lys Gln Ile Cys 1 5 5649PRTHomo sapiens 564Leu Leu Ala Thr Lys Gln Ile Cys Lys 1 5 5659PRTHomo sapiens 565Gln Ile Cys Lys Glu Phe Thr Asp Leu 1 5 5669PRTHomo sapiens 566Ile Cys Lys Glu Phe Thr Asp Leu Leu 1 5 5679PRTHomo sapiens 567Leu Leu Ala Gln Asp Arg Ser Pro Leu 1 5 5689PRTHomo sapiens 568Ile Leu Glu Pro Gly Ile Gln Ser Cys 1 5 5699PRTHomo sapiens 569Leu Glu Pro Gly Ile Gln Ser Cys Leu 1 5 5709PRTHomo sapiens 570Gln Ser Cys Leu Thr His Phe Asn Leu 1 5 5719PRTHomo sapiens 571Ser Cys Leu Thr His Phe Asn Leu Ile 1 5 5729PRTHomo sapiens 572Asn Leu Ile Ser His Gly Phe Gly Ser 1 5 5739PRTHomo sapiens 573Leu Ile Ser His Gly Phe Gly Ser Pro 1 5 5749PRTHomo sapiens 574Ile Ser His Gly Phe Gly Ser Pro Ala 1 5 5759PRTHomo sapiens 575Ser His Gly Phe Gly Ser Pro Ala Val 1 5 5769PRTHomo sapiens 576Phe Gly Ser Pro Ala Val Cys Ala Ala 1 5 5779PRTHomo sapiens 577Gly Ser Pro Ala Val Cys Ala Ala Val 1 5 5789PRTHomo sapiens 578Ala Val Cys Ala Ala Val Thr Ala Leu 1 5 5799PRTHomo sapiens 579Ala Val Thr Ala Leu Gln Asn Tyr Leu 1 5 5809PRTHomo sapiens 580Val Thr Ala Leu Gln Asn Tyr Leu Thr 1 5 5819PRTHomo sapiens 581Ala Leu Gln Asn Tyr Leu Thr Glu Ala 1 5 5829PRTHomo sapiens 582Leu Gln Asn Tyr Leu Thr Glu Ala Leu 1 5 5839PRTHomo sapiens 583Tyr Leu Thr Glu Ala Leu Lys Ala Met 1 5 5849PRTHomo sapiens 584Leu Lys Ala Met Asp Lys Met Tyr Leu 1 5 5859PRTHomo sapiens 585Ala Met Asp Lys Met Tyr Leu Ser Asn 1 5 5869PRTHomo sapiens 586Lys Met Tyr Leu Ser Asn Asn Pro Asn 1 5 5879PRTHomo sapiens 587Tyr Leu Ser Asn Asn Pro Asn Ser His 1 5 5889PRTHomo sapiens 588Ala Asn Cys Gln Gly Leu Ser Pro Val 1 5 5899PRTHomo sapiens 589Lys Met Gly Glu Pro Val Ser Glu Ser 1 5 5909PRTHomo sapiens 590Gly Leu Lys Glu Lys Val Trp Thr Glu 1 5 59141DNAHomo sapiens 591ggaattcaac atggacattg aagcatatct tgaaagaatt g 4159239DNAHomo sapiens 592ggaattcctg gtgagctgga tgacaaatag acaagattg 3959331DNAHomo sapiens 593ggaattcacc atgctttgga aattgacgga t 3159436DNAHomo sapiens 594ggaattcctc actttctgtg cttctcctct ttgtca 3659531DNAHomo sapiens 595ggaattcacc atgctttgga aattgacgga t 3159636DNAHomo sapiens 596ggaattcctc actttctgtg cttctcctct ttgtca 365971443DNAHomo sapiens 597atgggttccc ccgccgcccc ggagggagcg ctgggctacg tccgcgagtt cactcgccac 60tcctccgacg tgctgggcaa cctcaacgag ctgcgcctgc gcgggatcct cactgacgtc 120acgctgctgg ttggcgggca acccctcaga gcacacaagg cagttctcat cgcctgcagt 180ggcttcttct attcaatttt ccggggccgt gcgggagtcg gggtggacgt gctctctctg 240cccgggggtc ccgaagcgag aggcttcgcc cctctattgg acttcatgta cacttcgcgc 300ctgcgcctct ctccagccac tgcaccagca gtcctagcgg ccgccaccta tttgcagatg 360gagcacgtgg tccaggcatg ccaccgcttc atccaggcca gctatgaacc tctgggcatc 420tccctgcgcc ccctggaagc agaaccccca acacccccaa cggcccctcc accaggtagt 480cccaggcgct ccgaaggaca cccagaccca cctactgaat ctcgaagctg cagtcaaggc 540ccccccagtc cagccagccc tgaccccaag gcctgcaact ggaaaaagta caagtacatc 600gtgctaaact ctcaggcctc ccaagcaggg agcctggtcg gggagagaag ttctggtcaa 660ccttgccccc aagccaggct ccccagtgga gacgaggcct ccagcagcag cagcagcagc 720agcagcagca gcagtgaaga aggacccatt cctggtcccc agagcaggct ctctccaact 780gctgccactg tgcagttcaa atgtggggct ccagccagta ccccctacct cctcacatcc 840caggctcaag acacctctgg atcaccctct gaacgggctc gtccactacc gggagtgaat 900ttttcagctg ccagaactgt gaggctgtgg cagggtgctc atcgggggct ggactccttg 960gttcctgggg acgaagacaa accctataag tgtcagctgt gccggtcttc gttccgctac 1020aagggcaacc ttgccagtca tcgtacagtg cacacagggg aaaagcctta ccactgctca 1080atctgcggag cccgttttaa ccggccagca aacctgaaaa cgcacagccg catccattcg 1140ggagagaagc cgtataagtg tgagacgtgc ggctcgcgct ttgtacaggt ggcacatctg 1200cgggcgcacg tgctgatcca caccggggag aagccctacc cttgccctac ctgcggaacc 1260cgcttccgcc acctgcagac cctcaagagc cacgttcgca tccacaccgg agagaagcct 1320taccactgcg acccctgtgg cctgcatttc cggcacaaga gtcaactgcg gctgcatctg 1380cgccagaaac acggagctgc taccaacacc aaagtgcact accacattct cggggggccc 1440tag 1443

Claims

1. An expression vector comprising the nucleic acid sequence as illustrated in SEQ ID NO.: 29 or SEQ ID NO.: 31; a nucleic acid sequence encoding the amino acid sequence illustrated in SEQ ID NO.: 30 or SEQ ID NO.: 32; or a fragment thereof.

2. The expression vector of claim 1 wherein the vector is a plasmid or a viral vector.

3. The expression vector of claim 2 wherein the viral vector is selected from the group consisting of poxvirus, adenovirus, retrovirus, herpesvirus, and adeno-associated virus.

4. The expression vector of claim 3 wherein the viral vector is a poxvirus selected from the group consisting of vaccinia, NYVAC, avipox, canarypox, ALVAC, ALVAC(2), fowlpox, and TROVAC.

5. The expression vector of claim 4 wherein the viral vector is a poxvirus selected from the group consisting of NYVAC, ALVAC, and ALVAC(2).

6. The expression vector of claim 1 further comprising at least one additional tumor-associated anti gen.

7. The expression vector of claim 6 wherein the vector is a plasmid or a viral vector.

8. The expression vector of claim 7 wherein the viral vector is selected from the group consisting of poxvirus, adenovirus, retrovirus, herpesvirus, and adeno-associated virus.

9. The expression vector of claim 8 wherein the viral vector is a poxvirus selected from the group consisting of vaccinia, NYVAC, avipox, canarypox, ALVAC, ALVAC(2), fowlpox, and TROVAC.

10. The expression vector of claim 9 wherein the viral vector is a poxvirus selected from the group consisting of NYVAC, ALVAC, and ALVAC(2).

11. The expression vector of claim 1 further comprising at least one nucleic sequence encoding an angiogenesis-associated antigen.

12. The expression vector of claim 11 wherein the vector is a plasmid or a viral vector.

13. The expression vector of claim 12 wherein the viral vector is selected from the group consisting of poxvirus, adenovirus, retrovirus, herpesvirus, and adeno-associated virus.

14. The expression vector of claim 13 wherein the viral vector is a poxvirus selected from the group consisting of vaccinia, NYVAC, avipox, canarypox, ALVAC, ALVAC(2), fowlpox, and TROVAC.

15. The expression vector of claim 14 wherein the viral vector is a poxvirus selected from the group consisting of NYVAC, ALVAC, and ALVAC(2).

16. The expression vector of claim 6 further comprising at least one nucleic sequence encoding an angiogenesis-associated antigen.

17. The expression vector of claim 16 wherein the vector is a plasmid or a viral vector.

18. The expression vector of claim 17 wherein the viral vector is selected from the group consisting of poxvirus, adenovirus, retrovirus, herpesvirus, and adeno-associated virus.

19. The expression vector of claim 17 wherein the viral vector is a poxvirus selected from the group consisting of vaccinia, NYVAC, avipox, canarypox, ALVAC, ALVAC(2), fowlpox, and TROVAC.

20. The poxvirus of claim 18 wherein the viral vector is a poxvirus selected from the group consisting of NYVAC, ALVAC, and ALVAC(2).

21. The expression vector of claim 1, 6, 11 or 16 further comprising at least one nucleic acid sequence encoding a co-stimulatory component.

22. The expression vector of claim 22 wherein the vector is a plasmid or a viral vector.

23. The expression vector of claim 23 wherein the viral vector is selected from the group consisting of poxvirus, adenovirus, retrovirus, herpesvirus, and adeno-associated virus.

24. The expression vector of claim 24 wherein the viral vector is a poxvirus selected from the group consisting of vaccinia, NYVAC, avipox, canarypox, ALVAC, ALVAC(2), fowlpox, and TROVAC.

25. The poxvirus of claim 18 wherein the viral vector is a poxvirus selected from the group consisting of NYVAC, ALVAC, and ALVAC(2).

26. A composition comprising an expression vector in a pharmaceutically acceptable carrier, said vector comprising the nucleic acid sequence shown in SEQ ID NO.: 29 or SEQ ID NO.: 31; a nucleic acid sequence encoding the amino acid sequence illustrated in SEQ ID NO.: 30 or SEQ ID NO.: 32; or a fragment thereof.

27. The expression vector of claim 26 wherein the vector is a plasmid or a viral vector.

28. The expression vector of claim 27 wherein the viral vector is selected from the group consisting of poxvirus, adenovirus, retrovirus, herpesvirus, and adeno-associated virus.

29. The expression vector of claim 28 wherein the viral vector is a poxvirus selected from the group consisting of vaccinia, NYVAC, avipox, canarypox, ALVAC, ALVAC(2), fowlpox, and TROVAC.

30. The poxvirus of claim 29 wherein the viral vector is a poxvirus selected from the group consisting of NYVAC, ALVAC, and ALVAC(2).

31. A method for preventing or treating cancer comprising administering to a host an expression vector comprising the nucleic acid sequence illustrated in SEQ ID NO.: 29 or SEQ ID NO.: 31; a nucleic acid sequence encoding the amino acid sequence illustrated in SEQ ID NO.: 30 or SEQ ID NO.: 32; or a fragment thereof.

32. The expression vector of claim 31 wherein the vector is a plasmid or a viral vector.

33. The expression vector of claim 32 wherein the viral vector is selected from the group consisting of poxvirus, adenovirus, retrovirus, herpesvirus, and adeno-associated virus.

34. The expression vector of claim 33 wherein the viral vector is a poxvirus selected from the group consisting of vaccinia, NYVAC, avipox, canarypox, ALVAC, ALVAC(2), fowlpox, and TROVAC.

35. The poxvirus of claim 34 wherein the viral vector is a poxvirus selected from the group consisting of NYVAC, ALVAC, and ALVAC(2).

36. An isolated peptide derived from BFY3 as shown in Table XV or XVI.

37. A method for immunizing a host against the tumor antigen BFY3 comprising administering to the patient a peptide shown in Table XV or XVI, either alone or in combination with another agent, where the individual components of the combination are administered simultaneously or separately from one another.

38. An isolated peptide derived from BFY3 as shown in Table XV or XVI.

39. A method for immunizing a host against the tumor antigen BFY3 comprising administering to the patient a peptide shown in Table XV or XVI, either alone or in combination with another agent, where the individual components of the combination are administered simultaneously or separately from one another.

40. An isolated peptide derived from BCZ4 as shown in Table XIII or XVI.

41. A method for immunizing a host against the tumor antigen BCZ4 comprising administering to the patient a peptide shown in Table XIII or XIV, either alone or in combination with another agent; where the individual components of the combination are administered simultaneously or separately from one another.

42. An isolated peptide derived from BCZ4 as shown in Table XIII or XVI.

43. A method for immunizing a host against the tumor antigen BCZ4 comprising administering to the patient a peptide shown in Table XIII or XVI, either alone or in combination with another agent, where the individual components of the combination are administered simultaneously or separately from one another.

44. A expression vector for expression of multiple tumor antigens or fragments thereof, the expression vector comprising at least two nucleic acid sequences encoding at least two different tumor antigens or fragments thereof, the tumor antigens being selected from the group consisting of BFA4, BCY1, BFA5, BCZ4, and BFY3.

45. A expression vector for expression of multiple tumor antigens or fragments thereof, the expression vector comprising at least two nucleic acid sequences encoding at least two different tumor antigens or fragments thereof, the nucleic acid sequences being selected from the group consisting of SEQ ID NO.: 23, SEQ ID NO.: 25, SEQ ID NO.: 27, SEQ ID NO.: 29, and SEQ ID NO.: 31.

46. The expression vector of claim 44 or 45 wherein the vector is a plasmid or a viral vector.

47. The expression vector of claim 46 wherein the viral vector is selected from the group consisting of poxvirus, adenovirus, retrovirus, herpesvirus, and adeno-associated virus.

48. The expression vector of claim 47 wherein the viral vector is a poxvirus selected from the group consisting of vaccinia, NYVAC, avipox, canarypox, ALVAC, ALVAC(2), fowlpox, and TROVAC.

49. The expression vector of claim 48 wherein the viral vector is a poxvirus selected from the group consisting of NYVAC, ALVAC, and ALVAC(2).

50. The expression vector of any one of claims 44 to 49 further comprising at least one nucleic acid sequence encoding a co-stimulatory component.