Tumor Antigens BFA5 for 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 claims priority to U.S. Provisional Application No. 60/462,945 filed Apr. 15, 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 to 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, Offring a 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. (1999) 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, Bronze 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.: 5 or has the amino acid sequence of SEQ ID NO.: 6. 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 additional tumor antigens (i.e., SEQ ID NOS.: 1-4) and/or an immune stimulator, such as a co-stimulatory molecule or adjuvant.

BRIEF DESCRIPTION OF THE DRAWINGS

[0006] FIG. 1. BFA4 cDNA sequence.

[0007] FIG. 2. BFA4 amino acid sequence.

[0008] FIG. 3. BCY1 nucleotide (A) and amino acid (B) sequences.

[0009] FIG. 4. BFA5 cDNA sequence.

[0010] FIG. 5. BFA5 amino acid sequence.

DETAILED DESCRIPTION

[0011] The present invention provides reagents and methodologies useful for treating and/or preventing cancer. All references cited within this application are incorporated by reference.

[0012] 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.

[0013] 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".

[0014] 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.

[0015] 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-BR-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.: 1 and 2), BCY1 (SEQ ID NOS.: 3 and 4), and BFA5 (SEQ ID NOS.: 5 and 6) 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.

[0016] 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.

[0017] 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, fik-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 to al. Int. J. Oncol., 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..nu..beta.3, .alpha..nu..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.

[0018] 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-N6-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-N-6-isopentenyladenine, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouraoil, 4-thiouracil, 5-methyluracil, N-uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, and 2,6-diaminopurine, among others.

[0019] 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 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 is 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.

[0020] 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.

[0021] 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.

[0022] 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.

[0023] 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.

[0024] 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 (Brirster 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. U.S.A., 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; Omitz et al., 1986, Cold Spring Harbor Symp. Quant. Biol. 50:399-409 (1986); MacDonald, 1987, Hepatology 7:425-515); 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.

[0025] As described above, enhancers may also be suitable flanking sequences. Enhancers are cis-acting elements of DNA, usually about 10-300 by 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). 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.

[0026] 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.

[0027] 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.

[0028] 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 polypeptide" 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.

[0029] 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.

[0030] 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.

[0031] 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 Ito 40, or from 1 to 50, or more than 50 amino acid substitutions, insertions, additions and/or deletions.

[0032] 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. 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).

[0033] "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, 1020 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.

[0034] 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

[0035] 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 to 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.

[0036] 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.

[0037] 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.

[0038] 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 to 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.

[0039] 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 tranduction 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, SRRHHCRSKAKRSRHH).

[0040] 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.

[0041] 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 nucleic acid sequences encoding one or more co-stimulatory 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); mutated and derivative B7 molecules (WO 00/66162); polypeptides which bind members of the integrin family (i.e., LFA-1 (CD11a/CD18); 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., 4-1BB (CD137; Vinay, et al. Semin Immunol 1998, 10: 481-489), OX40 (CD134; 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 4-1BBL (4-1BB 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; 4-1BB Saoulli, et al. J Exp Med 1998, 187: 1849-1862, Arch, et al. Mol Cell Biol 1998, 18: 558-565), TRAF-2 (4-1BB and OX40 ligand; Saoulli, et al. J Exp Med 1998, 187: 1849-1862; Oshima, et al. Int Immunol 1998, 10: 517-526, Kawamata, et al. J Biol Chem 1998, 273: 5808-5814), TRAF-3 (4-1BB 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.

[0042] 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. J. 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.

[0043] 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.

[0044] 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.

[0045] 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 (Ahlers; 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.

[0046] 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 al. b 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, supra; Cho, et al. J. Immunol. 168(10):4907-13), sequences for targeting of the endocytic or ubiquitin-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(6):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.

[0047] Chemotherapeutic agents, radiation, anti-angiogenic compounds, or other agents may also be utilized in treating and/or preventing cancer using immunogenic targets (Sebti, et al. 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.

[0048] 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 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.

[0049] 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 (HOF), 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, HUI77, 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, vinblastine, 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-methylamides (i.e., Batimistat, Marimastat), AG3340, and minocycline. Many other suitable agents are known in the art and would suffice in practicing the present invention.

[0050] 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.

[0051] 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.).

[0052] 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, BJV, 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., injection) 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.

[0053] 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 (I): 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 vivo have included intratracheal instillation (Rosenfeld, M., et al., 1992, Cell, 68 (1): 143-55) injection into muscle (Quentin, 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.

[0054] 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 yet 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).

[0055] 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.

[0056] 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.

[0057] 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 to 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.

[0058] 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.

[0059] "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.), pETI 5 (Novagen, Madison, Wis.), pGEX (Pharmacia Biotech, Piscataway, N.J.), pEGFP-N2 (Clontech, Palo Alto, Calif.), pETL (BlueBacll, 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 COLE I-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.

[0060] 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.

[0061] 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 Specific Examples/ Adjuvant General Examples References Gel-type Aluminum hydroxide/ (Aggerbeck and Heron, phosphate ("alum adjuvants") 1995) 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 oligonucleotides (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 (Morein and Bengtsson, complexes (ISCOMs) 1999) Liposomes (Wassef et al., 1994) Oil-emulsion Freund's incomplete adjuvant (Jensen et al., 1998) and Microfluidized emulsions MF59 (Ott et al., 1995) surfactant- SAF (Allison and Byars, based 1992) (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)

[0062] The immunogenic targets of the present invention may also be used to generate antibodies for use in screening assays or for immunotherapy, which are another aspect of the present invention. 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.

[0063] 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); Verboeyen 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); Lonberg 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.

[0064] 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 is 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.

[0065] 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.

[0066] 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.

[0067] 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.

[0068] 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.

[0069] 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 poxyiral 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.

[0070] 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.

[0071] 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.

[0072] 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% to 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.

[0073] 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.

[0074] 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.

[0075] 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.

[0076] 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.

[0077] 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.

[0078] 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

BFA5 Breast Cancer Antigen

A. Identification of BFA5

[0079] 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.

[0080] 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: 197-205). 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).

[0081] 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. 4 and SEQ ID NO.: S. The BFA5 amino acid sequence is shown in FIG. 5 and SEQ ID NO.: 6.

B. Immunoreactivity of BFA5

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

[0082] A library of 100 peptides from the BFA5/NYBR-1 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 (Table III), 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-00003 TABLE III BFA5 Peptide Pools Peptide Group CLP number Sequence BFA5 2983 LMOMQTFKA Group 1 2984 KVSIPTKAL 2985 SIPTKALEL 2986 LELKNEQTL 2987 TVSQKDVCL 2988 SVPNKALEL 2989 CETVSQKDV 2990 KINGKLEES 2991 SLVEKTPDE 2992 SLCETVSQK BFA5 2993 ESDKINGKL Group 2 2994 MLLQQNVDV 2995 NMWLQQQLV 2996 FLVDRKCQL 2997 YLLHENCML 2998 SLFESSAKI 2999 KITIDIHFL 3000 QLQSKNMWL 3001 SLDQKLFQL 3002 FLLIKNANA BFA5 3003 KILDTVHSC Group 3 3004 SLSKILDTV 3005 ILIDSGADI 3006 KVMEINREV 3007 KLLSHGAVI 3009 AVYSEILSV 3010 KMNVDVSST 3011 ILSVVAKLL 3012 VLIAENTML BFA5 3013 KLSKNHQNT Group 4 3014 SLTPLLLSI 3015 SQYSGQLKV 3016 KELEVKQQL 3017 QIMEYIRKL 3018 AMLKLEIAT 3019 VLHQPLSEA 3020 GLLKATCGM 3021 GLLKATCGM 3022 QQLEQALRI BFA5 3023 CMLKKEIAM Group 5 3024 EQMKKKFCV 3025 IQDIELKSV 3026 SVPNKAFEL 3027 SIYQKVMEI 3028 NLNYAGDAL 3029 AVQDHDQIV BFA5 3033 FESSAKIOV Group 6 3034 GVTAEHYAV 3035 RVTSNKTKV 3036 TVSQKDVCV 3037 KSOEPAPHI 3038 KVLIAENTM 3039 MLKLEIATL 3040 EILSVVAKL 3041 MLKKEIAML 3042 LLKEKNEEI BFA5 3043 ALRIQDIEL Group 7 3044 KIREELGRI 3045 TLKLKEESL 3046 ILNEKIREE 3047 VLKKKLSEA 3048 GTSOKIQCL 3049 GADINLVDV 3050 ELCSVRLTL 3051 SVESNLNQV 3052 SLKINLNYA BFA5 3053 KTPOEAASL Group 8 3054 ATCGMKVSI 3055 LSHGAVIEV 3056 EIAMLKLEI 3057 AELQMTLKL 3058 VFAADICGV 3060 PAIEMQNSV 3061 EIFNYNNHL 3062 ILKERMAEL BFA5 3063 QLVHAHKKA Group 9 3065 NIQDAQKRT 3066 NLVDVYGNM 3067 KCTALMLAV 3068 KIQCLEKAT 3069 KIAWEKKET 3070 IAWEKKEDT 3071 VGMLLQQNV 3072 VKTGCVARV BFA5 3074 ALHYAVYSE Group 10 3075 QMKKKFCVL 3076 ALQCHQEAC 3077 SEQIVEFLL 3078 AVIEVHNKA 3079 AVTCGFHHI 3080 ACLQRKMNV 3081 SLVEGTSDK

[0083] ELISPOT analysis was performed on human T-cell cultures activated through four rounds of stimulation with each pool of BFA5 peptides. 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.

[0084] A large number of the BFA5 peptide pools of are reactive as shown by the high levels of IFN-.gamma. production. Each reactive peptide pool was then separated into individual peptides and analyzed for immunogencity using ELISPOT analysis to isolate single reactive BFA5 peptides. BFA5 is highly immunogenic with several reactive single peptides than that of BFA4. Similar results were obtained in two independent PBMC culture experiments.

[0085] 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 peptide's. 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 HTV peptide was generally <10%. Similar results were obtained with another PBMC donor expressing HLA-A*0201 (AP10). A large number of BFA5 peptides trigger T cell-mediated cytotoxicity of BFA5 peptide-loaded target cells. Table IV lists those peptides having immunogenic properties. Five peptides (LMDMQTFKA, ILIDSGADI, 1LSVVAICLL, SQYSGQLKV, and ELCSVRLTL) were found to induce both IFN-.gamma. secretion and CTL activity in T cells from both donors.

TABLE-US-00004 TABLE IV Immunoreactive peptides from BFA5 BFA5 peptides eliciting BFA5 peptides high IFN-.gamma. release inducing CTL lysis (>200 spots/100,000 cells) of pulsed cells Donor AP10 Donor AP31 Donor AP10 Donor AP31 LMDMQTFKA LMDMQTFKA LMDMQTFKA LMOMQTFKA 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

[0086] Polyclonal antisera were generated against the following series of 22- to 23-mer peptides of BFA5:

TABLE-US-00005 BFA5(1-23) KLH-MTKRKKTINLNIQDAQKRTALHW (CLP-2977) BFA5(312-334) KLH-TSEKFTWPAKGRPRKIAWEKKED (CLP-2978) BFA5(612-634) KLH-DEILPSESKQKDYEENSWDTESL (CLP-2979) BFA5(972-994) KLH-RLTLNQEEEKRRNADILNEKIRE (CLP-2980) BFA5(1117-1139) KLH-AENTMLTSKLKEKQDKEILEAEI (CLP-2981) BFA5(1319-1341) KLH-NYNNHLKNRIYQYEKEKAETENS (CLP-2982)

[0087] 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-00006 TABLE V IgG titer .times. 10.sup.5 (after IgG titer .times. 10.sup.5 (after first Immunization second 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.

[0088] 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, 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.

[0089] 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

10513846DNAHomo sapiens 1atggtccgga 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 384621281PRTHomo sapiens 2Met Val Arg Lys Lys Asn Pro Pro Leu Arg Asn Val Ala Ser Glu Gly1 5 10 15Glu Gly Gln Ile Leu Glu Pro Ile Gly Thr Glu Ser Lys Val Ser Gly 20 25 30Lys Asn Lys Glu Phe Ser Ala Asp Gln Met Ser Glu Asn Thr Asp Gln 35 40 45Ser Asp Ala Ala Glu Leu Asn His Lys Glu Glu His Ser Leu His Val 50 55 60Gln Asp Pro Ser Ser Ser Ser Lys Lys Asp Leu Lys Ser Ala Val Leu65 70 75 80Ser Glu Lys Ala Gly Phe Asn Tyr Glu Ser Pro Ser Lys Gly Gly Asn 85 90 95Phe Pro Ser Phe Pro His Asp Glu Val Thr Asp Arg Asn Met Leu Ala 100 105 110Phe Ser Phe Pro Ala Ala Gly Gly Val Cys Glu Pro Leu Lys Ser Pro 115 120 125Gln Arg Ala Glu Ala Asp Asp Pro Gln Asp Met Ala Cys Thr Pro Ser 130 135 140Gly Asp Ser Leu Glu Thr Lys Glu Asp Gln Lys Met Ser Pro Lys Ala145 150 155 160Thr Glu Glu Thr Gly Gln Ala Gln Ser Gly Gln Ala Asn Cys Gln Gly 165 170 175Leu Ser Pro Val Ser Val Ala Ser Lys Asn Pro Gln Val Pro Ser Asp 180 185 190Gly Gly Val Arg Leu Asn Lys Ser Lys Thr Asp Leu Leu Val Asn Asp 195 200 205Asn Pro Asp Pro Ala Pro Leu Ser Pro Glu Leu Gln Asp Phe Lys Cys 210 215 220Asn Ile Cys Gly Tyr Gly Tyr Tyr Gly Asn Asp Pro Thr Asp Leu Ile225 230 235 240Lys His Phe Arg Lys Tyr His Leu Gly Leu His Asn Arg Thr Arg Gln 245 250 255Asp Ala Glu Leu Asp Ser Lys Ile Leu Ala Leu His Asn Met Val Gln 260 265 270Phe Ser His Ser Lys Asp Phe Gln Lys Val Asn Arg Ser Val Phe Ser 275 280 285Gly Val Leu Gln Asp Ile Asn Ser Ser Arg Pro Val Leu Leu Asn Gly 290 295 300Thr Tyr Asp Val Gln Val Thr Ser Gly Gly Thr Phe Ile Gly Ile Gly305 310 315 320Arg Lys Thr Pro Asp Cys Gln Gly Asn Thr Lys Tyr Phe Arg Cys Lys 325 330 335Phe Cys Asn Phe Thr Tyr Met Gly Asn Ser Ser Thr Glu Leu Glu Gln 340 345 350His Phe Leu Gln Thr His Pro Asn Lys Ile Lys Ala Ser Leu Pro Ser 355 360 365Ser Glu Val Ala Lys Pro Ser Glu Lys Asn Ser Asn Lys Ser Ile Pro 370 375 380Ala Leu Gln Ser Ser Asp Ser Gly Asp Leu Gly Lys Trp Gln Asp Lys385 390 395 400Ile Thr Val Lys Ala Gly Asp Asp Thr Pro Val Gly Tyr Ser Val Pro 405 410 415Ile Lys Pro Leu Asp Ser Ser Arg Gln Asn Gly Thr Glu Ala Thr Ser 420 425 430Tyr Tyr Trp Cys Lys Phe Cys Ser Phe Ser Cys Glu Ser Ser Ser Ser 435 440 445Leu Lys Leu Leu Glu His Tyr Gly Lys Gln His Gly Ala Val Gln Ser 450 455 460Gly Gly Leu Asn Pro Glu Leu Asn Asp Lys Leu Ser Arg Gly Ser Val465 470 475 480Ile Asn Gln Asn Asp Leu Ala Lys Ser Ser Glu Gly Glu Thr Met Thr 485 490 495Lys Thr Asp Lys Ser Ser Ser Gly Ala Lys Lys Lys Asp Phe Ser Ser 500 505 510Lys Gly Ala Glu Asp Asn Met Val Thr Ser Tyr Asn Cys Gln Phe Cys 515 520 525Asp Phe Arg Tyr Ser Lys Ser His Gly Pro Asp Val Ile Val Val Gly 530 535 540Pro Leu Leu Arg His Tyr Gln Gln Leu His Asn Ile His Lys Cys Thr545 550 555 560Ile Lys His Cys Pro Phe Cys Pro Arg Gly Leu Cys Ser Pro Glu Lys 565 570 575His Leu Gly Glu Ile Thr Tyr Pro Phe Ala Cys Arg Lys Ser Asn Cys 580 585 590Ser His Cys Ala Leu Leu Leu Leu His Leu Ser Pro Gly Ala Ala Gly 595 600 605Ser Ser Arg Val Lys His Gln Cys His Gln Cys Ser Phe Thr Thr Pro 610 615 620Asp Val Asp Val Leu Leu Phe His Tyr Glu Ser Val His Glu Ser Gln625 630 635 640Ala Ser Asp Val Lys Gln Glu Ala Asn His Leu Gln Gly Ser Asp Gly 645 650 655Gln Gln Ser Val Lys Glu Ser Lys Glu His Ser Cys Thr Lys Cys Asp 660 665 670Phe Ile Thr Gln Val Glu Glu Glu Ile Ser Arg His Tyr Arg Arg Ala 675 680 685His Ser Cys Tyr Lys Cys Arg Gln Cys Ser Phe Thr Ala Ala Asp Thr 690 695 700Gln Ser Leu Leu Glu His Phe Asn Thr Val His Cys Gln Glu Gln Asp705 710 715 720Ile Thr Thr Ala Asn Gly Glu Glu Asp Gly His Ala Ile Ser Thr Ile 725 730 735Lys Glu Glu Pro Lys Ile Asp Phe Arg Val Tyr Asn Leu Leu Thr Pro 740 745 750Asp Ser Lys Met Gly Glu Pro Val Ser Glu Ser Val Val Lys Arg Glu 755 760 765Lys Leu Glu Glu Lys Asp Gly Leu Lys Glu Lys Val Trp Thr Glu Ser 770 775 780Ser Ser Asp Asp Leu Arg Asn Val Thr Trp Arg Gly Ala Asp Ile Leu785 790 795 800Arg Gly Ser Pro Ser Tyr Thr Gln Ala Ser Leu Gly Leu Leu Thr Pro 805 810 815Val Ser Gly Thr Gln Glu Gln Thr Lys Thr Leu Arg Asp Ser Pro Asn 820 825 830Val Glu Ala Ala His Leu Ala Arg Pro Ile Tyr Gly Leu Ala Val Glu 835 840 845Thr Lys Gly Phe Leu Gln Gly Ala Pro Ala Gly Gly Glu Lys Ser Gly 850 855 860Ala Leu Pro Gln Gln Tyr Pro Ala Ser Gly Glu Asn Lys Ser Lys Asp865 870 875 880Glu Ser Gln Ser Leu Leu Arg Arg Arg Arg Gly Ser Gly Val Phe Cys 885 890 895Ala Asn Cys Leu Thr Thr Lys Thr Ser Leu Trp Arg Lys Asn Ala Asn 900 905 910Gly Gly Tyr Val Cys Asn Ala Cys Gly Leu Tyr Gln Lys Leu His Ser 915 920 925Thr Pro Arg Pro Leu Asn Ile Ile Lys Gln Asn Asn Gly Glu Gln Ile 930 935 940Ile Arg Arg Arg Thr Arg Lys Arg Leu Asn Pro Glu Ala Leu Gln Ala945 950 955 960Glu Gln Leu Asn Lys Gln Gln Arg Gly Ser Asn Glu Glu Gln Val Asn 965 970 975Gly Ser Pro Leu Glu Arg Arg Ser Glu Asp His Leu Thr Glu Ser His 980 985 990Gln Arg Glu Ile Pro Leu Pro Ser Leu Ser Lys Tyr Glu Ala Gln Gly 995 1000 1005Ser Leu Thr Lys Ser His Ser Ala Gln Gln Pro Val Leu Val Ser 1010 1015 1020Gln Thr Leu Asp Ile His Lys Arg Met Gln Pro Leu His Ile Gln 1025 1030 1035Ile Lys Ser Pro Gln Glu Ser Thr Gly Asp Pro Gly Asn Ser Ser 1040 1045 1050Ser Val Ser Glu Gly Lys Gly Ser Ser Glu Arg Gly Ser Pro Ile 1055 1060 1065Glu Lys Tyr Met Arg Pro Ala Lys His Pro Asn Tyr Ser Pro Pro 1070 1075 1080Gly Ser Pro Ile Glu Lys Tyr Gln Tyr Pro Leu Phe Gly Leu Pro 1085 1090 1095Phe Val His Asn Asp Phe Gln Ser Glu Ala Asp Trp Leu Arg Phe 1100 1105 1110Trp Ser Lys Tyr Lys Leu Ser Val Pro Gly Asn Pro His Tyr Leu 1115 1120 1125Ser His Val Pro Gly Leu Pro Asn Pro Cys Gln Asn Tyr Val Pro 1130 1135 1140Tyr Pro Thr Phe Asn Leu Pro Pro His Phe Ser Ala Val Gly Ser 1145 1150 1155Asp Asn Asp Ile Pro Leu Asp Leu Ala Ile Lys His Ser Arg Pro 1160 1165 1170Gly Pro Thr Ala Asn Gly Ala Ser Lys Glu Lys Thr Lys Ala Pro 1175 1180 1185Pro Asn Val Lys Asn Glu Gly Pro Leu Asn Val Val Lys Thr Glu 1190 1195 1200Lys Val Asp Arg Ser Thr Gln Asp Glu Leu Ser Thr Lys Cys Val 1205 1210 1215His Cys Gly Ile Val Phe Leu Asp Glu Val Met Tyr Ala Leu His 1220 1225 1230Met Ser Cys His Gly Asp Ser Gly Pro Phe Gln Cys Ser Ile Cys 1235 1240 1245Gln His Leu Cys Thr Asp Lys Tyr Asp Phe Thr Thr His Ile Gln 1250 1255 1260Arg Gly Leu His Arg Asn Asn Ala Gln Val Glu Lys Asn Gly Lys 1265 1270 1275Pro Lys Glu 128031203DNAHomo sapiens 3atggccgagc 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 12034400PRTHomo sapiens 4Met Ala Glu Leu Arg Leu Lys Gly Ser Ser Asn Thr Thr Glu Cys Val1 5 10 15Pro Val Pro Thr Ser Glu His Val Ala Glu Ile Val Gly Arg Gln Gly 20 25 30Cys Lys Ile Lys Ala Leu Arg Ala Lys Thr Asn Thr Tyr Ile Lys Thr 35 40 45Pro Val Arg Gly Glu Glu Pro Val Phe Met Val Thr Gly Arg Arg Glu 50 55 60Asp Val Ala Thr Ala Arg Arg Glu Ile Ile Ser Ala Ala Glu His Phe65 70 75 80Ser Met Ile Arg Ala Ser Arg Asn Lys Ser Gly Ala Ala Phe Gly Val 85 90 95Ala Pro Ala Leu Pro Gly Gln Val Thr Ile Arg Val Arg Val Pro Tyr 100 105 110Arg Val Val Gly Leu Val Val Gly Pro Lys Gly Ala Thr Ile Lys Arg 115 120 125Ile Gln Gln Gln Thr Asn Thr Tyr Ile Ile Thr Pro Ser Arg Asp Arg 130 135 140Asp Pro Val Phe Glu Ile Thr Gly Ala Pro Gly Asn Val Glu Arg Ala145 150 155 160Arg Glu Glu Ile Glu Thr His Ile Ala Val Arg Thr Gly Lys Ile Leu 165 170 175Glu Tyr Asn Asn Glu Asn Asp Phe Leu Ala Gly Ser Pro Asp Ala Ala 180 185 190Ile Asp Ser Arg Tyr Ser Asp Ala Trp Arg Val His Gln Pro Gly Cys 195 200 205Lys Pro Leu Ser Thr Phe Arg Gln Asn Ser Leu Gly Cys Ile Gly Glu 210 215 220Cys Gly Val Asp Ser Gly Phe Glu Ala Pro Arg Leu Gly Glu Gln Gly225 230 235 240Gly Asp Phe Gly Tyr Gly Gly Tyr Leu Phe Pro Gly Tyr Gly Val Gly 245 250 255Lys Gln Asp Val Tyr Tyr Gly Val Ala Glu Thr Ser Pro Pro Leu Trp 260 265 270Ala Gly Gln Glu Asn Ala Thr Pro Thr Ser Val Leu Phe Ser Ser Ala 275 280 285Ser Ser Ser Ser Ser Ser Ser Ala Lys Ala Arg Ala Gly Pro Pro Gly 290 295 300Ala His Arg Ser Pro Ala Thr Ser Ala Gly Pro Glu Leu Ala Gly Leu305 310 315 320Pro Arg Arg Pro Pro Gly Glu Pro Leu Gln Gly Phe Ser Lys Leu Gly

325 330 335Gly Gly Gly Leu Arg Ser Pro Gly Gly Gly Arg Asp Cys Met Val Cys 340 345 350Phe Glu Ser Glu Val Thr Ala Ala Leu Val Pro Cys Gly His Asn Leu 355 360 365Phe Cys Met Glu Cys Ala Val Arg Ile Cys Glu Arg Thr Asp Pro Glu 370 375 380Cys Pro Val Cys His Ile Thr Ala Ala Gln Ala Ile Arg Ile Phe Ser385 390 395 40054026DNAHomo sapiens 5atgacaaaga 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 402661341PRTHomo sapiens 6Met Thr Lys Arg Lys Lys Thr Ile Asn Leu Asn Ile Gln Asp Ala Gln1 5 10 15Lys Arg Thr Ala Leu His Trp Ala Cys Val Asn Gly His Glu Glu Val 20 25 30Val Thr Phe Leu Val Asp Arg Lys Cys Gln Leu Asp Val Leu Asp Gly 35 40 45Glu His Arg Thr Pro Leu Met Lys Ala Leu Gln Cys His Gln Glu Ala 50 55 60Cys Ala Asn Ile Leu Ile Asp Ser Gly Ala Asp Ile Asn Leu Val Asp65 70 75 80Val Tyr Gly Asn Met Ala Leu His Tyr Ala Val Tyr Ser Glu Ile Leu 85 90 95Ser Val Val Ala Lys Leu Leu Ser His Gly Ala Val Ile Glu Val His 100 105 110Asn Lys Ala Ser Leu Thr Pro Leu Leu Leu Ser Ile Thr Lys Arg Ser 115 120 125Glu Gln Ile Val Glu Phe Leu Leu Ile Lys Asn Ala Asn Ala Asn Ala 130 135 140Val Asn Lys Tyr Lys Cys Thr Ala Leu Met Leu Ala Val Cys His Gly145 150 155 160Ser Ser Glu Ile Val Gly Met Leu Leu Gln Gln Asn Val Asp Val Phe 165 170 175Ala Ala Asp Ile Cys Gly Val Thr Ala Glu His Tyr Ala Val Thr Cys 180 185 190Gly Phe His His Ile His Glu Gln Ile Met Glu Tyr Ile Arg Lys Leu 195 200 205Ser Lys Asn His Gln Asn Thr Asn Pro Glu Gly Thr Ser Ala Gly Thr 210 215 220Pro Asp Glu Ala Ala Pro Leu Ala Glu Arg Thr Pro Asp Thr Ala Glu225 230 235 240Ser Leu Val Glu Lys Thr Pro Asp Glu Ala Ala Pro Leu Val Glu Arg 245 250 255Thr Pro Asp Thr Ala Glu Ser Leu Val Glu Lys Thr Pro Asp Glu Ala 260 265 270Ala Ser Leu Val Glu Gly Thr Ser Asp Lys Ile Gln Cys Leu Glu Lys 275 280 285Ala Thr Ser Gly Lys Phe Glu Gln Ser Ala Glu Glu Thr Pro Arg Glu 290 295 300Ile Thr Ser Pro Ala Lys Glu Thr Ser Glu Lys Phe Thr Trp Pro Ala305 310 315 320Lys Gly Arg Pro Arg Lys Ile Ala Trp Glu Lys Lys Glu Asp Thr Pro 325 330 335Arg Glu Ile Met Ser Pro Ala Lys Glu Thr Ser Glu Lys Phe Thr Trp 340 345 350Ala Ala Lys Gly Arg Pro Arg Lys Ile Ala Trp Glu Lys Lys Glu Thr 355 360 365Pro Val Lys Thr Gly Cys Val Ala Arg Val Thr Ser Asn Lys Thr Lys 370 375 380Val Leu Glu Lys Gly Arg Ser Lys Met Ile Ala Cys Pro Thr Lys Glu385 390 395 400Ser Ser Thr Lys Ala Ser Ala Asn Asp Gln Arg Phe Pro Ser Glu Ser 405 410 415Lys Gln Glu Glu Asp Glu Glu Tyr Ser Cys Asp Ser Arg Ser Leu Phe 420 425 430Glu Ser Ser Ala Lys Ile Gln Val Cys Ile Pro Glu Ser Ile Tyr Gln 435 440 445Lys Val Met Glu Ile Asn Arg Glu Val Glu Glu Pro Pro Lys Lys Pro 450 455 460Ser Ala Phe Lys Pro Ala Ile Glu Met Gln Asn Ser Val Pro Asn Lys465 470 475 480Ala Phe Glu Leu Lys Asn Glu Gln Thr Leu Arg Ala Asp Pro Met Phe 485 490 495Pro Pro Glu Ser Lys Gln Lys Asp Tyr Glu Glu Asn Ser Trp Asp Ser 500 505 510Glu Ser Leu Cys Glu Thr Val Ser Gln Lys Asp Val Cys Leu Pro Lys 515 520 525Ala Thr His Gln Lys Glu Ile Asp Lys Ile Asn Gly Lys Leu Glu Glu 530 535 540Ser Pro Asn Lys Asp Gly Leu Leu Lys Ala Thr Cys Gly Met Lys Val545 550 555 560Ser Ile Pro Thr Lys Ala Leu Glu Leu Lys Asp Met Gln Thr Phe Lys 565 570 575Ala Glu Pro Pro Gly Lys Pro Ser Ala Phe Glu Pro Ala Thr Glu Met 580 585 590Gln Lys Ser Val Pro Asn Lys Ala Leu Glu Leu Lys Asn Glu Gln Thr 595 600 605Trp Arg Ala Asp Glu Ile Leu Pro Ser Glu Ser Lys Gln Lys Asp Tyr 610 615 620Glu Glu Asn Ser Trp Asp Thr Glu Ser Leu Cys Glu Thr Val Ser Gln625 630 635 640Lys Asp Val Cys Leu Pro Lys Ala Ala His Gln Lys Glu Ile Asp Lys 645 650 655Ile Asn Gly Lys Leu Glu Gly Ser Pro Val Lys Asp Gly Leu Leu Lys 660 665 670Ala Asn Cys Gly Met Lys Val Ser Ile Pro Thr Lys Ala Leu Glu Leu 675 680 685Met Asp Met Gln Thr Phe Lys Ala Glu Pro Pro Glu Lys Pro Ser Ala 690 695 700Phe Glu Pro Ala Ile Glu Met Gln Lys Ser Val Pro Asn Lys Ala Leu705 710 715 720Glu Leu Lys Asn Glu Gln Thr Leu Arg Ala Asp Glu Ile Leu Pro Ser 725 730 735Glu Ser Lys Gln Lys Asp Tyr Glu Glu Ser Ser Trp Asp Ser Glu Ser 740 745 750Leu Cys Glu Thr Val Ser Gln Lys Asp Val Cys Leu Pro Lys Ala Thr 755 760 765His Gln Lys Glu Ile Asp Lys Ile Asn Gly Lys Leu Glu Glu Ser Pro 770 775 780Asp Asn Asp Gly Phe Leu Lys Ala Pro Cys Arg Met Lys Val Ser Ile785 790 795 800Pro Thr Lys Ala Leu Glu Leu Met Asp Met Gln Thr Phe Lys Ala Glu 805 810 815Pro Pro Glu Lys Pro Ser Ala Phe Glu Pro Ala Ile Glu Met Gln Lys 820 825 830Ser Val Pro Asn Lys Ala Leu Glu Leu Lys Asn Glu Gln Thr Leu Arg 835 840 845Ala Asp Gln Met Phe Pro Ser Glu Ser Lys Gln Lys Lys Val Glu Glu 850 855 860Asn Ser Trp Asp Ser Glu Ser Leu Arg Glu Thr Val Ser Gln Lys Asp865 870 875 880Val Cys Val Pro Lys Ala Thr His Gln Lys Glu Met Asp Lys Ile Ser 885 890 895Gly Lys Leu Glu Asp Ser Thr Ser Leu Ser Lys Ile Leu Asp Thr Val 900 905 910His Ser Cys Glu Arg Ala Arg Glu Leu Gln Lys Asp His Cys Glu Gln 915 920 925Arg Thr Gly Lys Met Glu Gln Met Lys Lys Lys Phe Cys Val Leu Lys 930 935 940Lys Lys Leu Ser Glu Ala Lys Glu Ile Lys Ser Gln Leu Glu Asn Gln945 950 955 960Lys Val Lys Trp Glu Gln Glu Leu Cys Ser Val Arg Leu Thr Leu Asn 965 970 975Gln Glu Glu Glu Lys Arg Arg Asn Ala Asp Ile Leu Asn Glu Lys Ile 980 985 990Arg Glu Glu Leu Gly Arg Ile Glu Glu Gln His Arg Lys Glu Leu Glu 995 1000 1005Val Lys Gln Gln Leu Glu Gln Ala Leu Arg Ile Gln Asp Ile Glu 1010 1015 1020Leu Lys Ser Val Glu Ser Asn Leu Asn Gln Val Ser His Thr His 1025 1030 1035Glu Asn Glu Asn Tyr Leu Leu His Glu Asn Cys Met Leu Lys Lys 1040 1045 1050Glu Ile Ala Met Leu Lys Leu Glu Ile Ala Thr Leu Lys His Gln 1055 1060 1065Tyr Gln Glu Lys Glu Asn Lys Tyr Phe Glu Asp Ile Lys Ile Leu 1070 1075 1080Lys Glu Lys Asn Ala Glu Leu Gln Met Thr Leu Lys Leu Lys Glu 1085 1090 1095Glu Ser Leu Thr Lys Arg Ala Ser Gln Tyr Ser Gly Gln Leu Lys 1100 1105 1110Val Leu Ile Ala Glu Asn Thr Met Leu Thr Ser Lys Leu Lys Glu 1115 1120 1125Lys Gln Asp Lys Glu Ile Leu Glu Ala Glu Ile Glu Ser His His 1130 1135 1140Pro Arg Leu Ala Ser Ala Val Gln Asp His Asp Gln Ile Val Thr 1145 1150 1155Ser Arg Lys Ser Gln Glu Pro Ala Phe His Ile Ala Gly Asp Ala 1160 1165 1170Cys Leu Gln Arg Lys Met Asn Val Asp Val Ser Ser Thr Ile Tyr 1175 1180 1185Asn Asn Glu Val Leu His Gln Pro Leu Ser Glu Ala Gln Arg Lys 1190 1195 1200Ser Lys Ser Leu Lys Ile Asn Leu Asn Tyr Ala Gly Asp Ala Leu 1205 1210 1215Arg Glu Asn Thr Leu Val Ser Glu His Ala Gln Arg Asp Gln Arg 1220 1225 1230Glu Thr Gln Cys Gln Met Lys Glu Ala Glu His Met Tyr Gln Asn 1235 1240 1245Glu Gln Asp Asn Val Asn Lys His Thr Glu Gln Gln Glu Ser Leu 1250 1255 1260Asp Gln Lys Leu Phe Gln Leu Gln Ser Lys Asn Met Trp Leu Gln 1265 1270 1275Gln Gln Leu Val His Ala His Lys Lys Ala Asp Asn Lys Ser Lys 1280 1285 1290Ile Thr Ile Asp Ile His Phe Leu Glu Arg Lys Met Gln His His 1295 1300 1305Leu Leu Lys Glu Lys Asn Glu Glu Ile Phe Asn Tyr Asn Asn His 1310 1315 1320Leu Lys Asn Arg Ile Tyr Gln Tyr Glu Lys Glu Lys Ala Glu Thr 1325 1330 1335Glu Asn Ser 134079PRTHomo sapiens 7Leu Met Asp Met Gln Thr Phe Lys Ala1 589PRTHomo sapiens 8Lys Val Ser Ile Pro Thr Lys Ala Leu1 599PRTHomo sapiens 9Ser Ile Pro Thr Lys Ala Leu Glu Leu1 5109PRTHomo sapiens 10Leu Glu Leu Lys Asn Glu Gln Thr Leu1 5119PRTHomo sapiens 11Thr Val Ser Gln Lys Asp Val Cys Leu1 5129PRTHomo sapiens 12Ser Val Pro Asn Lys Ala Leu Glu Leu1 5139PRTHomo sapiens 13Cys Glu Thr Val Ser Gln Lys Asp Val1 5149PRTHomo sapiens 14Lys Ile Asn Gly Lys Leu Glu Glu Ser1 5159PRTHomo sapiens 15Ser Leu Val Glu Lys Thr Pro Asp Glu1 5169PRTHomo sapiens 16Ser Leu Cys Glu Thr Val Ser Gln Lys1 5179PRTHomo sapiens 17Glu Ile Asp Lys Ile Asn Gly Lys Leu1 5189PRTHomo sapiens 18Met Leu Leu Gln Gln Asn Val Asp Val1 5199PRTHomo sapiens 19Asn Met Trp Leu Gln Gln Gln Leu Val1 5209PRTHomo sapiens 20Phe Leu Val Asp Arg Lys Cys Gln Leu1 5219PRTHomo sapiens 21Tyr Leu Leu His Glu Asn Cys Met Leu1 5229PRTHomo sapiens 22Ser Leu Phe Glu Ser Ser Ala Lys Ile1 5239PRTHomo sapiens 23Lys Ile Thr Ile Asp Ile His Phe Leu1 5249PRTHomo sapiens 24Gln Leu Gln Ser Lys Asn Met Trp Leu1 5259PRTHomo sapiens 25Ser Leu Asp Gln Lys Leu Phe Gln Leu1 5269PRTHomo sapiens 26Phe Leu Leu Ile Lys Asn Ala Asn Ala1 5279PRTHomo sapiens 27Lys Ile Leu Asp Thr Val His Ser Cys1 5289PRTHomo sapiens 28Ser Leu Ser Lys Ile Leu Asp Thr Val1 5299PRTHomo sapiens 29Ile Leu Ile Asp Ser Gly Ala Asp Ile1 5309PRTHomo sapiens 30Lys Val Met Glu Ile Asn Arg Glu Val1 5319PRTHomo sapiens 31Lys Leu Leu Ser His Gly Ala Val Ile1 5329PRTHomo sapiens 32Ala Val Tyr Ser Glu Ile Leu Ser Val1 5339PRTHomo sapiens 33Lys Met Asn Val Asp Val Ser Ser Thr1 5349PRTHomo sapiens 34Ile Leu Ser Val Val Ala Lys Leu Leu1 5359PRTHomo sapiens 35Val Leu Ile Ala Glu Asn Thr Met Leu1 5369PRTHomo sapiens 36Lys Leu Ser Lys Asn His Gln Asn Thr1 5379PRTHomo sapiens 37Ser Leu Thr Pro Leu Leu Leu Ser Ile1 5389PRTHomo sapiens 38Ser Gln Tyr Ser Gly Gln Leu Lys Val1 5399PRTHomo sapiens 39Lys Glu Leu Glu Val Lys Gln Gln Leu1 5409PRTHomo sapiens 40Gln Ile Met Glu Tyr Ile Arg Lys Leu1 5419PRTHomo sapiens 41Ala Met Leu Lys Leu Glu Ile Ala Thr1 5429PRTHomo sapiens 42Val Leu His Gln Pro Leu Ser Glu Ala1 5439PRTHomo sapiens 43Gly Leu Leu Lys Ala Thr Cys Gly Met1 5449PRTHomo sapiens 44Gly Leu Leu Lys Ala Asn Cys Gly Met1 5459PRTHomo sapiens 45Gln Gln Leu Glu Gln Ala Leu

Arg Ile1 5469PRTHomo sapiens 46Cys Met Leu Lys Lys Glu Ile Ala Met1 5479PRTHomo sapiens 47Glu Gln Met Lys Lys Lys Phe Cys Val1 5489PRTHomo sapiens 48Ile Gln Lys Ile Glu Leu Lys Ser Val1 5499PRTHomo sapiens 49Ser Val Pro Asn Lys Ala Phe Glu Leu1 5509PRTHomo sapiens 50Ser Ile Tyr Gln Lys Val Met Glu Ile1 5519PRTHomo sapiens 51Asn Leu Asn Tyr Ala Gly Asp Ala Leu1 5529PRTHomo sapiens 52Ala Val Gln Asp His Asp Gln Ile Val1 5539PRTHomo sapiens 53Phe Glu Ser Ser Ala Lys Ile Gln Val1 5549PRTHomo sapiens 54Gly Val Thr Ala Glu His Tyr Ala Val1 5559PRTHomo sapiens 55Arg Val Thr Ser Asn Lys Thr Lys Val1 5569PRTHomo sapiens 56Thr Val Ser Gln Lys Asp Val Cys Val1 5579PRTHomo sapiens 57Lys Ser Gln Glu Pro Ala Phe His Ile1 5589PRTHomo sapiens 58Lys Val Leu Ile Ala Glu Asn Thr Met1 5599PRTHomo sapiens 59Met Leu Lys Leu Glu Ile Ala Thr Leu1 5609PRTHomo sapiens 60Glu Ile Leu Ser Val Val Ala Lys Leu1 5619PRTHomo sapiens 61Met Leu Lys Lys Glu Ile Ala Met Leu1 5629PRTHomo sapiens 62Leu Leu Lys Glu Lys Asn Glu Glu Ile1 5639PRTHomo sapiens 63Ala Leu Arg Ile Gln Asp Ile Glu Leu1 5649PRTHomo sapiens 64Lys Ile Arg Glu Glu Leu Gly Arg Ile1 5659PRTHomo sapiens 65Thr Leu Lys Leu Lys Glu Glu Ser Leu1 5669PRTHomo sapiens 66Ile Leu Asn Glu Lys Ile Arg Glu Glu1 5679PRTHomo sapiens 67Val Leu Lys Lys Lys Leu Ser Glu Ala1 5689PRTHomo sapiens 68Gly Thr Ser Asp Lys Ile Gln Cys Leu1 5699PRTHomo sapiens 69Gly Ala Asp Ile Asn Leu Val Asp Val1 5709PRTHomo sapiens 70Glu Leu Cys Ser Val Arg Leu Thr Leu1 5719PRTHomo sapiens 71Ser Val Glu Ser Asn Leu Asn Gln Val1 5729PRTHomo sapiens 72Ser Leu Lys Ile Asn Leu Asn Tyr Ala1 5739PRTHomo sapiens 73Lys Thr Pro Asp Glu Ala Ala Ser Leu1 5749PRTHomo sapiens 74Ala Thr Cys Gly Met Lys Val Ser Ile1 5759PRTHomo sapiens 75Leu Ser His Gly Ala Val Ile Glu Val1 5769PRTHomo sapiens 76Glu Ile Ala Met Leu Lys Leu Glu Ile1 5779PRTHomo sapiens 77Ala Glu Leu Gln Met Thr Leu Lys Leu1 5789PRTHomo sapiens 78Val Phe Ala Ala Asp Ile Cys Gly Val1 5799PRTHomo sapiens 79Pro Ala Ile Glu Met Gln Asn Ser Val1 5809PRTHomo sapiens 80Glu Ile Phe Asn Tyr Asn Asn His Leu1 5819PRTHomo sapiens 81Ile Leu Lys Glu Lys Asn Ala Glu Leu1 5829PRTHomo sapiens 82Gln Leu Val His Ala His Lys Lys Ala1 5839PRTHomo sapiens 83Asn Ile Gln Asp Ala Gln Lys Arg Thr1 5849PRTHomo sapiens 84Asn Leu Val Asp Val Tyr Gly Asn Met1 5859PRTHomo sapiens 85Lys Cys Thr Ala Leu Met Leu Ala Val1 5869PRTHomo sapiens 86Lys Ile Gln Cys Leu Glu Lys Ala Thr1 5879PRTHomo sapiens 87Lys Ile Ala Trp Glu Lys Lys Glu Thr1 5889PRTHomo sapiens 88Ile Ala Trp Glu Lys Lys Glu Asp Thr1 5899PRTHomo sapiens 373Val Gly Met Leu Leu Gln Gln Asn Val1 5909PRTHomo sapiens 90Val Lys Thr Gly Cys Val Ala Arg Val1 5919PRTHomo sapiens 91Ala Leu His Tyr Ala Val Tyr Ser Glu1 5929PRTHomo sapiens 92Gln Met Lys Lys Lys Phe Cys Val Leu1 5939PRTHomo sapiens 93Ala Leu Gln Cys His Gln Glu Ala Cys1 5949PRTHomo sapiens 94Ser Glu Gln Ile Val Glu Phe Leu Leu1 5959PRTHomo sapiens 95Ala Val Ile Glu Val His Asn Lys Ala1 5969PRTHomo sapiens 96Ala Val Thr Cys Gly Phe His His Ile1 5979PRTHomo sapiens 97Ala Cys Leu Gln Arg Lys Met Asn Val1 5989PRTHomo sapiens 98Ser Leu Val Glu Gly Thr Ser Asp Lys1 59923PRTHomo sapiens 99Met Thr Lys Arg Lys Lys Thr Ile Asn Leu Asn Ile Gln Asp Ala Gln1 5 10 15Lys Arg Thr Ala Leu His Trp 2010023PRTHomo sapiens 100Thr Ser Glu Lys Phe Thr Trp Pro Ala Lys Gly Arg Pro Arg Lys Ile1 5 10 15Ala Trp Glu Lys Lys Glu Asp 2010123PRTHomo sapiens 101Asp Glu Ile Leu Pro Ser Glu Ser Lys Gln Lys Asp Tyr Glu Glu Asn1 5 10 15Ser Trp Asp Thr Glu Ser Leu 2010223PRTHomo sapiens 102Arg Leu Thr Leu Asn Gln Glu Glu Glu Lys Arg Arg Asn Ala Asp Ile1 5 10 15Leu Asn Glu Lys Ile Arg Glu 2010323PRTHomo sapiens 103Ala Glu Asn Thr Met Leu Thr Ser Lys Leu Lys Glu Lys Gln Asp Lys1 5 10 15Glu Ile Leu Glu Ala Glu Ile 2010423PRTHomo sapiens 104Asn Tyr Asn Asn His Leu Lys Asn Arg Ile Tyr Gln Tyr Glu Lys Glu1 5 10 15Lys Ala Glu Thr Glu Asn Ser 2010516PRTHomo sapiens 105Ser Arg Arg His His Cys Arg Ser Lys Ala Lys Arg Ser Arg His His1 5 10 15

Claims

1-63. (canceled)

64. An expression vector comprising the nucleic acid sequence as illustrated in SEQ ID NO.: 5 or a fragment thereof.

65. The expression vector of claim 64 wherein the vector is a plasmid or a viral vector.

66. The expression vector of claim 65 wherein the viral vector is selected from the group consisting of poxvirus, adenovirus, retrovirus, herpesvirus, and adeno-associated virus.

67. The expression vector of claim 66 wherein the viral vector is a poxvirus selected from the group consisting of vaccinia, NYVAC, avipox, canarypox, ALVAC, ALVAC(2), fowlpox, and TROVAC.

68. The expression vector of claim 66 wherein the viral vector is a poxvirus selected from the group consisting of NYVAC, ALVAC, and ALVAC(2).

69. The expression vector of claim 1 further comprising at least one additional tumor-associated antigen.

70. An isolated peptide derived from BFA5 as shown in Table X or XI.

71. A method for immunizing a host against the tumor antigen BFA5 comprising administering to the patient a peptide of claim 74, either alone or in combination with another agent, where the individual components of the combination are administered simultaneously or separately from one another.

72. An expression vector comprising a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO.: 6 or a fragment thereof.

73. A composition comprising an expression vector of claim 1 and a pharmaceutically acceptable carrier.

74. A composition comprising an expression vector of claim 76 and a pharmaceutically acceptable carrier.

75. An antibody having the ability to bind the amino acid sequence of SEQ ID NO.: 6 or a fragment thereof.

76. The antibody of claim 79 wherein the fragment is selected from the group consisting of SEQ ID NO.:7, SEQ ID NO.:8, SEQ ID NO.:9, SEQ ID NO.:11, SEQ ID NO.:12, SEQ ID NO.:21, SEQ ID NO.:24, SEQ ID NO.:29, SEQ ID NO.:30, SEQ ID NO.:32, SEQ ID NO.:34, SEQ ID NO.:37, SEQ ID NO.:38, SEQ ID NO.:40, SEQ ID NO.:49, SEQ ID NO.:51, SEQ ID NO.:54, SEQ ID NO.:57, SEQ ID NO.:59, SEQ ID NO.:61, SEQ ID NO.:63, SEQ ID NO.:67, SEQ ID NO.:70, SEQ ID NO.:72, SEQ ID NO.:74, SEQ ID NO.:77, SEQ ID NO.:78, SEQ ID NO.:81, SEQ ID NO.:84, and SEQ ID NO.:85.

77. A composition comprising an antibody of claim 79 and a pharmaceutically acceptable carrier.

78. A composition comprising an antibody of claim 80 and a pharmaceutically acceptable carrier.

79. An isolated peptide selected from the group consisting of SEQ ID NO.:8, SEQ ID NO.:9, SEQ ID NO.:11, and SEQ ID NO.:12.

80. A composition comprising an isolated peptide of claim 79 and a pharmaceutically acceptable carrier.

81. A method for inducing an immune response against the tumor antigen BFA5 (SEQ ID NO.: 6) in a host comprising administering to the host the isolated peptide of claim 79.

82. A method for inducing an immune response against the tumor antigen BFA5 (SEQ ID NO.: 6) in a host comprising administering to the host the composition of claim 79.