MXPA06002477A - Multi-antigen vectors for melanoma - Google Patents

Abstract

The present invention relates to peptides, polypeptides, and nucleic acids and the use of the peptide, polypeptide or nucleic acid in preventing and / or treating cancer. In particular, the invention relates to peptides and nucleic acid sequences encoding such peptides for use in diagnosing, treating, or preventing melanoma.

Description

MULTI-ANTIGENIC VECTORS FOR MELANOMA FIELD OF THE INVENTION The present invention relates to multi i-antigen vectors for use in the prevention and / or treatment of cancer. In particular, the invention relates to multi-ion vectors for use in the treatment and / or prevention of melanomas.

BACKGROUND OF THE INVENTION There has been an extraordinary increase in recent years in the development of cancer vaccines with antigens associated with tumors (TAAs) due to the great advances in the identification of molecules based on the expression profiles on primary tumors and normal cells with the aid of various techniques such as, for example, high density micro-array, SEREX, immunohistochemistry (IHC), RT-PCR, in-situ hybridization (ISH) , for its acronym in English) and laser capture microscopy (Rosenberg, Immunity, 1999, Sgroi et al, 1999, Schena et al, 1995, Offringa et al, 2000). TAAs are antigens expressed or overexpressed by cancers. The present invention provides the reagents and methodologies that overcome many of the difficulties encountered by others in attempting to treat cancer.

BRIEF DESCRIPTION OF THE INVENTION The present invention provides uiti-antigen vectors for administration to a patient for preventing and / or treating cancer. In particular, the multi-antigenic vector encodes one or more tumor antigens (XXTA "). The uiti-antigenic vectors can also encode an immuno stimulator such as, for example, a co-stimulatory molecule and / or be administered with an adjuvant. .

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1. Schematic of pALVAC plasmids. Trico (# 33) and pT1132. Figure 2. DNA sequence of plasmid pALVAC. Tricom (# 33). Figure 3. DNA sequence of plasmid pT1132. Figure 4. Schematic of plasmid? T3217.

Figure 5. DNA sequence of plasmid pT3217. Figure 6. Amino acid sequences of the proteins NY-ESO-1, TRP-2, gplOO, gplOOM, MART-1, MAGE-1, MAGE-3, B7.1, LFA-3, and ICAM-1.

DETAILED DESCRIPTION The present invention provides useful reagents and methodologies for the treatment and / or prevention of cancer. All references cited in this application are incorporated by reference. In one embodiment, the present invention relates to the induction or enhancement of an immune response against one or more tumor antigens ("TA") for the prevention and / or treatment of cancer. In certain modalities, one or more of the TA may be combined. In preferred embodiments, the immune response is the result of the expression of a TA in a host cell after 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. In the sense in which it is used herein, an "antigen" is a molecule (such as, for example, 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 that express an epitope of the antigen. The response may be an improvement of a general immune response, for example, by causing increased production of antibodies, production of antibodies with increased affinity for the antigen, or an increase in cellular immune response (i.e., increased number or activity of the immunoreactive T lymphocytes). An antigen that produces an immune response alternatively can be referred to as immunogenic or immunogenic. In the description of the present invention, a TA can be referred to as an "immunogenic target". The present invention provides expression vectors that express one or more immunogenic targets in a host. The term TA includes both tumor-associated antigens (TAAs) and tumor-specific antigens (TSAs), where a cancer cell is the source of the antigen. A TAA is an antigen that is expressed on the surface of a tumor cell in greater quantities than those observed in normal cells or an antigen that is expressed in normal cells during fetal development. A TSA is an antigen that is unique to tumor cells and is not expressed in normal cells. The TA also includes the TAA or TSA, the antigenic fragments thereof, and the modified versions that retain their antigenicity. TAs are typically classified into five categories according to their pattern of expression, function, or genetic origin: antigens (ie, MAGE, NY-ESO-1) of testicular cancer (CT, for its acronym in English); antigens for melanocyte differentiation (ie, Melan A / MART-1, tyrosinase, gplOO); mutational antigens (ie, MUM-1, p53, CDK-4); ? auto 'over-expressed antigens (ie, HER-2 / neu, p53); and, viral antigens (ie, HPV, EBV). In order to practice the present invention, a suitable TA is any TA that induces or reinforces an anti-tumor immune response in a host to whom the TA has been administered. Suitable TAs include, for example, gplOO species (Cox et al., Sci en ce, 264: 716-719 (1994), U.S. Patent No. 6,500, 919 Bl and WO 01/30847 with Val. at residue 162, also referred to as "gplOOM", U.S. Patent No. 6,537,560 Bl with Phe at residue 162), MART-1 / Melan A (Kawaka i et al., J. Exp. Med., 180: 347-352 (1994), U.S. Patent No. 5,874,560), gp75 (TRP-1) (Wang et al., J. Exp. Med., 186: 1131-1140 (1996)), TRP -2 (Wang et al., 1996 J. Exp. Med. 184: 2207, U.S. Patent Nos. 5,831,016 and 6,083,783), tyrosinase (Wolfel et al., Eur. J. Immunol., 24: 759-764. (1994), WO 200175117, WO 200175016, W0200175007), NY-ESO-1 (W098 / 14464, WO99 / 18206, Accession No. GenBank P78358, U.S. Patent No. 5,804, 381), melanoma proteoglycan ( Hellstrom et al., J. Immunol., 130: 1467-1472 (1983)), antigens of the MAGE family (ie, MAGE-1, 2, 3, 4, 6, 12, 51; Vander Bruggen et al. al., Science, 254: 1643-1647 (1991); U.S. Patent Nos. 6,235,525; CN 1319611), the antigens of the BAGE family (Boel et al., Immunity, 2: 167-175 (1995)), the antigens of the GAGE family (ie, GAGE-1,2, Van den Eynde et al. , J. Exp. Med., 182: 689-698 (1995), U.S. Patent No. 6,013,765), antigens of the RAGE family (ie, RAGE-1, Gaugler et al., Immunogenetics, 44 : 323-330 (1996), U.S. Patent No. 5,939,526), N-acetylglucosaminyltransferase-V (Guilloux et al., J. Exp. Med., 183: 1173-1183 (1996)), pl5 ( Robbins et al., J. Immunol. 154: 5944-5950 (1995)), β-catenin (Robbins et al., J. Exp. Med., 183: 1185-1192 (1996)), MUM-1 (Coulie et al., Proc. Nati, Acad. Sci. USA, 92: 7976-7980 (1995)), cyclin-dependent kinase-4 (CDK4) (Wolfel et al., Science, 269: 1281-1284 (1995)) , p21-ras (Fossu et al., Int. J. Cancer, 56: 40-45 (1994)), BCR-abl (Bocchia et al., Blood, 85: 2680-2684 (1995)), p53 (Theobald et al., Proc. Nati, Acad. Sci. USA, 92: 11993-11997 (1995)), p85 HER2 / neu (erb-Bl; Fisk et al. ., J. Exp. Med., 181: 2109-2117 (1995)), the epidermal growth factor receptor (EGFR) (Harris et al., Breast Cancer Res. Treat, 29: 1-2 (1994)), carcinoembryonic antigens (CEA) (Kwong et al., J. Nati. Cancer Inst., 85: 982-990 (1995) U.S. Patent Nos. 5,756,103; 5,274,087; 5,571,710; 6,071,716; 5,698,530; 6,045,802; EP 263933; EP 346710; EP784483); mutated mucins associated with carcinoma (ie, products of the MUC-1 gene; Jerome et al., J. Immunol., 151: 1654-1662 (1993)); the EBNA EBNA gene products (ie, EBNA-1; Rickinson et al., Cancer Surveys, 13: 53-80 (1992)); the E7, E6 proteins of human papilloma virus (Ressing et al., J. Imm un ol, 154: 5934-5943 (1995)); the prostate-specific antigen (PSA; Xue et al., Th e Pros ta t e, 30: 73-78 (1997)); prostate-specific membranous antigen (PSMA; Israeli, et al., Cán cer Res. 54: 1807-1811 (1994)); epitopes or idiotypic antigens, for example, immunoglobulin idiotypes or idiotypes of the T lymphocyte receptor (Chen et al., J. Imm un ol., 153: 4775-4787 (1994)); KSA (U.S. Patent No. 5,348,887), kinesin 2 (Dietz, et al., Biochem Biophys Res Common 2000 Sep 7; 275 (3): 731-8), HIP-55, antiapoptotic factor TGFβ-1 ( Toomey, et al., Br J Biomed Sci 2001; 58 (3): 177-83), tumor protein D52 (Bryne JA, 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 Bioformatic Approaches to the Identification of Human Tumor Antigens, in Cán cer Vaccin is 2000, Cancer Research Institute, New Cork, NY), including "Wild type" (ie, normally encoded by the genome, which occurs in nature), modified and mutated versions, as well as also, other fragments and derivatives thereof. Any of 10 these TA can be used alone or in combination with each other in a co-immunization protocol. Preferred TAs are useful for inducing an immune response against melanoma cells. The term "melanoma" includes, but is not limited to: melanomas, metastatic melanomas, melanomas derived from its melanocytes or nevus cells related to melanocytes, melanocarcinomas, melanoepitheliomas, melanosarcomas, melanoma in itself, superficial spread melanoma, nodular melanoma, malignant lentigo melanoma , acral lentiginous melanoma, invasive melanoma and molar syndrome and familial atypical melanoma (FAM-M), for example, In general, melanomas are the result of chromosomal abnormalities, degenerative growth and developmental disorder, mitogenic agents, ultraviolet radiation (UV ), viral infections, expression of a gene in a suitable tissue, alterations in the expression of a gene or carcinogenic agents, for example. In certain cases, it may be convenient to coinmunize patients with both TA and other antigens, such as, for example, angiogenesis-associated antigens ("AA"). An AA is an immunogenic molecule (i.e., a peptide, a polypeptide) 11 associated with cells involved in the induction and / or continuous development of blood vessels. For example, an AA can be expressed in an endothelial cell ("CE") that is a primary structural component of blood vessels. When the disease is cancer, it is preferred that the AA be within or near the blood vessels that facilitate a tumor. Immunization of a patient against an AA preferably results in an anti-AA immune response with which the angiogenic processes occurring near or within the tumors are prevented and / or inhibited. Exemplary AAs include, for example, vascular endothelial growth factor (ie, 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 (ie, VEGF-R, flk-1 / KDR; Starnes, et al., J. Thorac, Cardiovasc. Surg, 2001, 122 (3): 518-23), EPH receptors (ie, EPHA2, Gerety, et al., 1999, Cell, 4: 403-414), receptor of epidermal growth factor (ie, 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): 12 501-7; Poon, et al. Am J. Surg., 2001, 182 (3): 298-304), platelet-derived cell growth factor (ie, PDGF-B), platelet-derived endothelial cell growth factor (PD-ECGF; Hong, et al. J. Mol. Med., 2001, 8 (2) -141-8), transforming growth factors (ie, TGF-a; Hong, et al., J. Mol. Med., 2001, 8 ( 2): 141-8), endoglin (Balza, et al., In J. Ca n cer, 2001, 94: 579-585), Id proteins (Benezra, R., Trends Cardiovasc. Med., 2001, 11 (6): 237-41), proteases such as, for example, uPA, uPAR, and matrix metalloproteinases (MMP-2, MMP-9; Djonov, et al., J. Pa. Ol., 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), thrombospondin ( that is, TSP-1, TSP-2, Alvarez, et al., Gynecol Oncol., 2001, 82 (2): 273-8, Seki, et al., Int. J. Oncol., 2001, 19 (2). : 305-10), k-ras (Zhang, et al, Cancer Res., 2001, 61 (16): 6050-4), Wn t (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. Pa th. On col. R es., 7 (2): 85-94), heat shock proteins (ie, HSP90 (Timar, upra heparin binding factors (ie heparinase, Gohji, et 13 to the. Int. J. Cancer, 2001, 95 (5): 295-301), syntans (ie, ATP synthase, thymidylate synthase), collagen receptors, integrins (ie, auß3, auß5, lßl, 2ßl, a5ßl), the surface proteolglycan NG2, AAC2-1, or AAC2-2, among others, including the "wild type" versions (ie, normally encoded by the genome, which occurs in nature), modified, mutated, as well as, other fragments and derivatives thereof. Any of these targets may be suitable for practicing the present invention, either alone or in combination with each other or with other agents. The nucleic acid molecule may comprise or may consist of a nucleotide sequence encoding one or more uninogenic targets, or fragments or derivatives thereof, such as, for example, same contents contained in a DNA insert in an ATCC Repository. . 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 DNA and RNA base analogues such as for example, but not limited to: -acet-il-cytosine, -hydroxy-N-6-methyladenosine, aziridinyl-cytosine, pseudoisocytosine, 5- (carboxyhydroxylmethyl) uracil, 5-fluorouracil, 5-bromouracil, 5-carboxymethylsinomethyl-2-thiouryl cyl, 5-carboxymethylenimethyl luracil, dihydrodracil, inosine, N6-iso-pentenyladenine, 1-methyladenine, -methylpseudouracil, l-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methoguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine, 7-methylguanine, 5-met ilesinomet iluracil, 5-methoxyamino-methyl-2-t iouracil, beta-D-mannosylqueosine, 5'-methoxycarbonyl-methyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methyl ester, uracil acid -5-oxyacetic acid, oxibut oxosin, pseudouracil, kerosine, 2-t iocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methylacilyl, N-uracil-5-oxyacetyl acid methyl ester , uracil-5-oxyacetic acid, pseudouracil, kerosine, 2-thiocytoxine, and 2,6-diaminpurine, among others. An isolated nucleic acid molecule is one that: (1) is separated from at least about 50 percent proteins, lipids, carbohydrates, or other materials with which they are found in nature when the total nucleic acid is isolated from source cells; (2) it is not linked to the whole or a 15 a portion of a polynucleotide to which the nucleic acid molecule is linked in nature; (3) is operably linked to a polynucleotide that is not linked in nature; and / or, (4) does not occur in nature as part of a larger sequence of polynucleotides. Preferably, the isolated nucleic acid molecule of the present invention is virtually free of any other contaminating nucleic acid molecules or other contaminants that are found in its natural environment that could interfere with its use in the production of polypeptides or their therapeutic use, diagnosis, prophylactic or research. As used herein, the term "occurs in nature" or "naturally occurring" or "naturally occurring" when used in conjunction with biological materials such as, for example, nucleic acid molecules, polypeptides, cells hosts, and the like, refers to materials found in nature and not manipulated by man. Similarly, "not occurring in nature" or "unnatural," in the sense in which it is used herein, refers to a material that is not found in nature or in the wild. that has been modified or synthesized structurally by man. The identity of two or more nucleic acid sequences or amino acids is determined by comparing the sequences. As is known in the art, "identity" means the degree of sequence relationship between the nucleic acid or amino acid sequences as determined by the match between the units that make up the molecules (ie, nucleotides or amino acid residues). Identity measures the percentage of identical matches between the lesser of two or more sequences with separation alignments (if any) directed by a mathematical model or particular computer program (ie, an algorithm). The identity between the nucleic acid sequences can also be determined by the ability of the nucleic acid sequences to hybridize with each other. To define the hybridization process, the term "fairly severe conditions" and "moderately severe conditions" refers to conditions that allow the hybridization of the nucleic acid strands whose sequences are complementary, and to exclude the hybridization of nucleic acids that do not match significantly. The examples of 17"quite severe conditions" for hybridization and washing are 0.015 M sodium chloride, 0.0015 M sodium citrate at 65-68 ° C or 0.015 M sodium chloride, 0.0015 M sodium citrate, and 50% formamide at 42 ° C. . (see, for example, Sambrook, Fritsch &Maniatis, Mol ecul ar Cl oning: A Labora t ory Manual (2nd ed., Col Spring Harbor Laboratory, 1989), Anderson et al., Nu cl ei c Aci d Hybri di sa tí on: A Pra cti cal Approa ch Ch. 4 (IRL Press Limited)). The term "moderately severe conditions" refers to the conditions under which a duplex DNA with a higher degree of mismatch of base pairs is capable of being formed under "fairly severe conditions". Slightly severe conditions of example are 0.015 M sodium chloride, 0.0015 M C sodium citrate at 50-65 ° C or 0.015 M sodium chloride, 0.0015 M sodium citrate, and 20% formamide at 37-50 ° C. As an example, the slightly severe conditions of 50 ° C in the 0.015 M sodium ion will approximately allow for a mismatch of 21%. During hybridization, other agents can be included in the hybridization and wash buffer in order to reduce 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, NaDodS04, (SDS), Denhardt's solution, salmon sperm DNA subjected to ultrasound ( or other non-complementary DNA), and dextran sulfate, although other suitable agents may also be used. The concentration and types of these additives can be changed without substantially affecting the severity of the hybridization conditions. Hybridization experiments are normally carried out at pH 6.8-7.4; however, at typical ionic strength conditions, the hybridization rate is almost independent of pH. In the preferred embodiments of the present invention, vectors are used to transfer a nucleic acid sequence encoding an immunogenic target 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 used. 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. The 19 Expression includes, but is not limited to, processes such as, for example, transcription, translation, and splicing, if introns are present. Expression vectors typically comprise one or more flanking sequences functionally linked to a heterologous nucleic acid sequence encoding a polypeptide. The flanking sequences can be homologous (ie, of the same species and / or strain as the host cell), heterologous (ie, from a species other than the host cell species or strain), hybrid (i.e., a combination of flanking sequences from more than a source), or synthetic, for example. A flanking sequence is preferably capable of carrying out the replication, transcription and / or translation of the coding sequence and is functionally linked to a coding sequence. In the sense in which it is used herein, the term functionally linked refers to a union of polynucleotide elements in a functional relationship. For example, a promoter or enhancer is functionally linked to a coding sequence if it affects the transcription of the coding sequence. However, a flanking sequence does not necessarily have to be contiguous with the coding sequence, as long as it works correctly. In this way, for example, the intercalation of untranslated sequences still transcribed may be present between a promoter sequence and the coding sequence and the promoter sequence can still be considered functionally linked to the coding sequence. Similarly, an enhancer sequence can be located in the 5 'or 3' direction of the coding sequence and affect the transcription of the sequence. In certain embodiments, it is preferred that the flanking sequence be 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, lentifier, repressor, or combinations thereof. The transcriptional regulatory region can be either constitutive, tissue-specific, cell-type specific (i.e., the region is driven to higher levels of transcription in one type of tissue or cell as compared to another), or dimmable (i.e. sensitive to interaction with a compound such as, for example, tetracycline). The source of a 21 The transcriptional regulatory region can be any prokaryotic or eukaryotic organism, any vertebrate or invertebrate organism, or any plant, with the proviso that the flanking sequence functions in a cell causing the transcription of a nucleic acid within that cell. For the practice of the present invention a wide variety of transcriptional regulatory regions can be used. Suitable transcriptional regulatory regions include the CMV promoter (ie, the immediate anterior promoter of CMV); promoters from eukaryotic genes (ie, the estrogen-inducible chicken ovalbumin gene, the interferon genes, the gluco-corticoid-inducible tyrosine-aminotransferase gene, and the thymidine kinase gene); and the early and late adenovirus gene promoters; the previous promoter region SV40 (Bernoist and Chambón, 1981, Na t ure 290: 304-10); the promoter contained in the 3 'long terminal repeat (LTR) of Rous sarcoma virus (RSV) (Yamamoto, et al., 1980, Cel l 22: 787-97); the thymidine kinase promoter of herpes simplex virus (HSV-TK) (Wagner et al., 1981, Proc.Na.i.Ac d.Sci.U.S.A. 78: 1444-45); the regulatory sequences of the 22 gene metallothionine (Brinster et al., 1982, Nature 296: 39-42); prokaryotic expression vectors such as, for example, the beta-lactamase promoter (Villa-Ka aroff et al., 1978, Proc.Nat.Acid.Sci.U.S.A., 75: 3727-31); or the tac promoter (DeBoer et al., 1983, Proc. Nati, Acad. Sci. U.S.A., 80: 21-25). The tissue and / or cell-specific transcriptional regulatory regions include, for example, the controlling region of the elastase I gene that is active in pancreatic acinar cells (Swift et al., 1984, Cell 38: 639-46; Ornitz et al. , 1983, Cold Spring Harbor Symp. Quant. Biol. 50: 399-409 (1986); MacDonald, 1987, Hepatology 7: 425-514); the controlling region of the insulin gene that is active in pancreatic beta cells (Hanahan, 1985, Nature 315: 115-22); the controlling region of the immunoglobulin gene that 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 controlling region of the mouse mammary tumor virus in testicular, breast, lymphoid and mammary cells (Leder et al., 1986, Cell 45: 485-95); the controlling region of the albumin gene in the liver (Pinker et al., 1987, Gener and Devel., 1: 268-76); the region 23 controller of the alpha-fetus-protein gene in the liver (Krumlanf et al., 1985, Mol.Cel. l Bi. bi., 5: 1639-48, Hammer et al., 1987, Sci in ce 235: 53-58); the controlling region of the alpha I-antitrypsin gene in the liver (Kelsey et al., 1987, Gen es and Devel. 1: 161-71); the controlling region of the beta-globin gene in myeloid cells (Mogram et al., 1985, Na t ure 315: 338-49, Kollias et al., 1986, Cell 46: 89-94); the controlling region of the myelin basic protein gene in oligodendrocytic cells in the brain (Readhead et al., 1987, Cell 48: 703-12); the gene controlling region of two myosin light chains in skeletal muscle (Sani, 1985, Na t ure 314: 283-86); the controlling region of the gonadotropic releasing hormone gene in the hypothelase (Mason et al., 1986, Sci in ce 234: 1372-78), and the tyrosinase promoter in melanoma cells (Hart, I. Semen Oncol 1996 Feb; 23 (1): 154-8, Siders, et al Cancer Gene Ther 1998 Sep-Oct; 5 (5): 281-91), among others. Inducible promoters that are activated in the presence of a certain compound or condition such as, for example, light, heat, radiation, tetracycline, or heat shock proteins, for example, can also be used (see, for example, WO 24). 00/10612). Other suitable promoters are known in the art. As described above, the intifiers can also be suitable flanking sequences. Intensifiers are cis-interpreting elements of DNA, usually about 10-300 base pairs in length, which act on the promoter to increase transcription. Intensifiers are typically independent of orientation and position, which have been identified both 5 'and 3' for the controlled coding sequences. Various available binding sequences of mammalian genes are known (ie, globin, elastase, albumin, alpha-fetus-protein and insulin). Similarly, the SV40 enhancer, the cytomegalovirus enhancer anterior promoter, the polyoma enhancer, and the adenovirus enhancers are useful with the eukaryotic promoter sequences. While an enhancer can be spliced into the vector at a 5 'or 3' position for the nucleic acid encoding the sequence, it is typically located at a 5 'site from the promoter. Other enhancers are known in the art suitable, and could be applied in the present invention. While the reagents of the present invention are being prepared, it may be necessary for the cells to be transfected or transformed. Transfection refers to the absorption of foreign or exogenous DNA by a cell, and a cell has been transfected when the exogenous DNA has been introduced into the cell membrane. Several transfection techniques are known in this field (ie, Graham et al., 1973, Vi rol ogy 52: 456, Sambrook et al., Molecule r Cl on ing, A Labora t ory Man ua l (Cold Spring Harbor Laboratories, 1989), Davis et al., Ba si c Methods in Mol e cula r Bi olgy, (Elsevier, 1986), and Chu et al., 1981, Gen e 13: 197). These techniques can be used to introduce one or more exogenous DNA entities into suitable host cells. In certain embodiments, it is preferred that the transfection of a cell results in the transformation of that cell. A cell is transformed when there is a change in a characteristic of Cell 1, which is transformed when it has been modified to contain a new nucleic acid. After transfection, the acid 26 Transfected nucleic acid can be recombined with that of the cell by physically integrating it into a Cel l chromosome, it can be transiently maintained as an episomal element without replicating, or it can be replicated independently as a plasmid. A cell is stably transformed when the nucleic acid replicates with the division of the cell. The expression vectors of the present invention also provide expression of immunogenic target fragments. The fragments may include sequences truncated at the amino terminus (with or without a leader sequence) and / or the term carboxy. Fragments may also include variants (ie, allelic, splicing), orthologs, homologs, and other variants having one or more amino acid additions or substitutions or internal deletions as compared to the parent sequence. In preferred embodiments, the truncations and / or deletions comprise about 1-5 amino acids, 5-10 amino acids, 10-20 amino acids, 20-30 amino acids, 30-40 amino acids, 40-50 amino acids, or more. These polypeptide fragments may optionally comprise an amino terminal methionine residue. It will be appreciated that these fragments can be used, for example, to generate antibodies or cellular immune responses to immunogenic targets. A variant is a sequence having one or more substitutions, deletions, and / or additions of the sequence in comparison with the exposed sequence. Variants can occur in nature or artificially. These variants can be prepared from the corresponding nucleic acid molecules. In the preferred modalities, the variants have from 1 to 3, or from 1 to 5, or from 1 to 10, or from 1 to 15, or from 1 to 20, or from 1 to 25, or from 1 to 30, or from 1 to 40 , or from 1 to 50, or more than 50 amino acid substitutions, insertions, additions and / or deletions. An allelic variant is one of several possible alternate forms that occur in the nature of a sequence that occupies a particular locus on a chromosome of an organism or a population of organisms. A splice variant is a polypeptide generated from one of several RNA transcripts that result from splicing a primary transcript. An ortholog is a similar nucleic acid or polypeptide sequence of another species. For example, mouse and human versions of an immunogenic target can be considered 28 orthographs to each other. A derivative of a sequence is one that is derived from a parental sequence of those sequences that have substitutions, additions, deletions, or chemically modified variants. Variants may also include fusion proteins, which refers to the fusion of one or more of first sequences (such as, for example, a peptide) to the amino or carboxy terminus of at least one other sequence (such as, for example, a heterologous peptide). "Similarity" is a concept related to identity, except that similarity refers to a measure of relationship that includes both identical matches and matches of conservative substitution. If two polypeptide sequences, for example, have 10/20 identical amino acids, and the rest are all non-conservative substitutions, then the percent identity and the similarity could both be 50%. If in the same example, there are five additional positions when there are conservative substitutions, then the percentage identity is still 50%, but the percentage similarity would be 75% (15/20). Therefore, in cases where there are conservative substitutions, the percentage similarity between two polypeptides will be greater than that of the percentage identity between those same two polypeptides. The substitutions may be conservative, or non-conservative, or any combination thereof. Conservative modifications of amino acids to the sequence of a polypeptide (and corresponding modifications to the coding nucleotides) can produce polypeptides having the functional and chemical characteristics similar to those of a parent polypeptide. For example, a "conservative amino acid substitution" may involve a substitution of a natural amino acid residue with a non-natural residue such that there is little or no effect on the size, polarity, charge, hydrophobicity, or hydrophilicity of the amino acid residue in that position and, in particular, does not result in decreased immunogenicity. In Table I conservative substitutions of suitable amino acids are shown.

TABLE I An expert will be able to determine the suitable variants of an immunogenic target using well-known techniques. For the identification of suitable areas of the molecule that can be changed without destroying the biological activity (i.e., binding, MHC immunogenicity), one skilled in the art can designate the areas that he does not think are important for that activity. For example, when immunogenic targets with similar activities of the same species or of another species are known, someone with 31 experience in the art can compare the amino acid sequence of a polypeptide for those similar polypeptides. By performing these analyzes, you can identify the residues and portions of the molecules that are conserved. It will be appreciated that changes in the areas of the molecule that are not conserved relative to these similar immunogenic targets might be less likely to adversely affect the activity and / or biological structure of a polypeptide. Similarly, the residues required for binding to MHC are known, and can be modified to improve binding. However, modifications that result from decreased binding to MHC will not be adequate in most situations. One skilled in the art would also know that, even in relatively conserved regions, similar amino acids can be chemically substituted for residues that occur in nature 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 structure of the immunogenic target. 32 Other preferred polypeptide variants include glycosylation variants wherein the number and / or type of glycosylation sites have been altered in comparison to the subject amino acid sequence. In one embodiment, the polypeptide variants comprise a greater or 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 can be any amino acid residue except proline. Substitution of the 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 suppress an existing N-linked carbohydrate chain. A restructuring of N-linked carbohydrate chains is also provided wherein one or more N-linked glycosylation sites (typically the same ones that occur in nature) are removed and one or more new N-linked sites are created. To affect the O-linked glycosylation of a polypeptide, could modify the serine and / or threonine residues. 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 set of subject amino acid sequences. Cysteine variants are useful when peptides or polypeptides must be replicated in a biologically active conformation such as, for example, after isolation of insoluble inclusion bodies. Cysteine variants generally have less cysteine residues than the natural protein, and typically have an equal number to minimize the interactions that result from unpaired cysteines. In other embodiments, the peptides or polypeptides can be attached to one or more fusion segments that aid in the purification of the polypeptides. Fusions can be made, either at the amino terminus or at the carboxy terminus of the subject polypeptide variant. The fusions can be directed without any binding or adapter molecule or can be made through a binding or adapter molecule. One molecule 34 Binder or adapter can be one or more amino acid residues, typically between about 20 and 50 amino acid residues. A linker or adapter molecule can also be designed with a cleavage site for a restriction endonuclease with DNA or for a protease to allow separation of the fused entities. It will be appreciated that once constructed, the fusion polypeptides can be derived 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 lymphocytes, B lymphocytes, Fc receptor , or binding domains with complementary protein antibodies), sugar binding domains (e.g., a maltose binding domain), and / or a "tag" domain (i.e., at least a portion of a-galactosidase, a strep tag peptide, a T7 tag peptide, a FLAG peptide, or other domains that can be purified using the domain binding compounds, such as, for example, monoclonal antibodies). This label is typically fused to the peptide or polypeptide and at the time of Expression can serve as a means for affinity purification of the polypeptide sequence of interest of the host cell. For example, affinity purification can be carried out by column chromatography using antibodies against the label as an affinity matrix. Optionally, the tag can be subsequently removed from the purified sequence of the polypeptide of interest by various means such as for example, using certain peptidases for cleavage. As will be described below, fusions can also be made between a TA and one of the co-stimulatory components such as, for example, the chemokines CXC10 (IP-10), CCL7 (MCP-3), or CCL5 (RANTES), for example. A fusion motif can improve the transport of an immunogenic target to an MHC processing compartment, such as, for example, the endoplasmic reticulum. These sequences, referred to as transduction and translational sequences, include sequences derived from HIV tat (see, Kim et al., 1997 J. Immunol., 159: 1666), Dros oph il to antennapedia (see, Schutze-Redelmeier et al. 1996 J. Immunol. 157: 650), or 36 human protein of period-1 (hPERl, in particular, SRRHHCRSKAKRSRHH). In addition, the polypeptide or variant thereof can be fused to a homologous peptide or polypeptide to form a homodimer or a heterologous peptide or polypeptide to form a heterodimer. Heterologous peptides and polypeptides include, but are not limited to, an epitope to allow the detection and / or isolation of a fusion polypeptide; a transmembrane receptor protein or a portion thereof, such as, for example, an extracellular domain or a transmembrane and intracellular domain; a ligand or a portion thereof that binds to a transmembrane receptor protein; an enzyme or portion thereof that is catalytically active; a polypeptide or peptide that stimulates oligomerization, such as, for example, a leucine closure domain; a polypeptide or peptide that increases stability, such as, for example, an immunoglobulin constant region; a peptide or polypeptide having a therapeutic activity different from the peptide or polypeptide; and / or variants thereof. In certain embodiments, it may be advantageous to combine a nucleic acid sequence encoding the for an immunogenic target with one or more co-stimulatory components such as, for example, cell surface proteins, cytokines or chemokines in a composition of the present invention. The co-stimulator component can be included in the composition as a polypeptide or as a nucleic acid encoding the polypeptide, for example. Suitable co-stimulatory molecules include, for example, polypeptides that bind members of the CD28 family (ie, CD28, ICOS; Hutloff, et al., Na ture 1999, 397: 263-265; Peach, et al. J Exp Med 1994, 180: 2049-2058) such as, for example, B7.1 polypeptides of binding with CD28 (CD80, Schwarz, 1992, Chen et al, 1992, Ellis, et al., J. Imm un ol., 156 (8): 2700-9), B7.2 (CD86, Ellis, et al., J. Imm un ol., 156 (8): 2700-9), and mutants / variants thereof (WO 00/66162); polypeptides that bind members of the integrin family (ie, LFA-1) (CDlla / CDl8); Sedwick, et al. J Imm un ol 1999, 162: 1367-1375; Wülfing, et al. Sci en ce 1998, 282: 2266-2269; Lub, et al. Imm ol Today 1995, 16: 479-483) including members of the ICAM family (ie, ICAM-1, -2 or -3); polypeptides that bind the members of the CD2 family (ie, CD2, signaling the activation molecule with lymphocytes (CDwl50 or "SLAM"; 38 Aversa, et al. J Immunol 1997, 158: 4036-4044)) such as for example CD58 (LFA-3; ligand CD2; Davis, et al., Immunol Today 1996, 17: 177-187) or SLAM ligands (Sayos, et al., Nature 1998, 395: 462-69); polypeptides that bind the heat-stable antigen (HSA or CD24; Zhou, et al., Eur J Immunol 1997, 27: 2524-2528); polypeptides that bind to the members of the TNF receptor family (TNFR) (ie, 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 for example , 4-1BBL (ligand 4-1BB; Vinay, et al., Semin Immunol 1998, 10: 481-48; DeBenedette, et al., J Immunol 1997, 158: 551-559), factor-1 associated with TNFR (TRAF- 1; ligand 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 ligand 0X40; Saoulli, et al J Exp Med 1998, 187: 18 9-1862; Oshima, et al., Immunol 1998, 10: 517-526, Kawamata, et al., J Biol Chem 1998, 273: 5808-581 ), TRAF-3 (4-1BB and the ligand OX40; Arch, et al., Mol Cell Biol 1998, 18: 558-565; Jang, et al., Biochem Biophys Res Commun 1998, 242: 613-620; Kawamata S, et 39 to the. J Biol Chem 1998, 273: 5808-5814), OX40L (ligand OX40, Gramaglia, et al., J Immunol 1998, 161: 6510-6517), TRAF-5 (ligand OX40; 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), ligand CD154 (ligand CD40 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. One or more cytokines can also be suitable co-substituents or "adjuvants" either as polypeptides or can be encoded by the nucleic acids contained within the compositions of the present invention (Parmiani, et al., Immunol Lett 2000 Sep 15; 74 (l): 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 Jul-Aug; 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 40 127 (12): 718-726), GM-CSF (CSF (Disis, et al., Bl or od, 88: 202-210 (1996)), tumor necrosis factor-alpha (TNF-a), or interferons such as for example, IFN- or INF-. Other cytokines may also be suitable for practicing the present invention, as is known in the art Chemokines, in any form of polypeptide or nucleic acid may also be used.

It has been shown that fusion proteins comprising CXCL10 (IP-10) and CCL7 (MCP-3) fused to a tumor autoantigen induce anti-tumor immunity (Biragyn, et al., 1999, 17: 253-258).

The chemokines CCL3 (MlP-la) and CCL5 (RANTES) (Boyer, et al., Vac cin, 1999, 17. (Supp.2): S53-S64) can also be used for the practice of the present invention. Other suitable chemokines are known in the art. It is also known in the art that suppressive or negative regulatory immunological mechanisms can be blocked, resulting in improved immunological responses. For example, treatment with anti-CTLA-4 (Shrikant, et al., Imm uni ty, 1996, 14: 145-155; Sutmuller, et al., J. Exp. Med., 2001, 194: 823-832), anti-CD25 (Sutmuller, s upra), anti-CD4 (Matsui, et al., J. Imm un ol., 1999, 41 163: 184-193), the IL13Ra2-Fc fusion protein (Terabe, et al., Na ture Immun ol., 2000, 1: 515-520), and combinations thereof (ie, anti-CTLA-4). and anti-CD25, Sutmuller, supra) has been shown to over-regulate anti-tumor immune responses and could be suitable for the practice of the present invention. These treatments, among others, may also be combined with one or more immunogenic targets of the present invention. Any of these components can be used alone or in combination with other agents. For example, it has been shown that a combination of CD80, ICAM-1 and LFA-3 ("TRICOM") can enhance anti-cancer immune responses (Hodge, et al., Cán cer Re s. 59: 5800-5807 (1999 Other effective combinations include, for example, IL-12 + GM-CSF (Ahlers, et al., J. Imm un ol., 158: 3947-3958 (1997); Iwasaki, et al., J. Imm un ol. 158: 4591-4601 (1997)), IL-12 + GM-CSF + TNF-α (Ahlers, et al., Inm.unm.Un.13: 897-908 (2001)), CD80 + IL-12 ( Fruend, et al., In., J. Cán cer, 85: 508-517 (2000), Rao, et al., Upra), and CD86 + GM-CSF + IL-12 (Iwasaki, supra). in the art one could realize additional combinations useful for carrying out the present invention. realize reagents or additional methods that can be used to modulate these mechanisms. These reagents and methods, as well as others known to those skilled in the art, can be used for the practice of the present invention. Additional strategies can also be used to improve the efficacy of nucleic acid-based immunization including, for example, the use of viral replicons for self-replication (Caley, et al., 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. : 4947-4951), in vivo electroporation (Widera, et al., 2000. J. Immunol., 164: 4635-3640), incorporation of the CpG stimulator motifs (Gurunn, et al., Ann. Rev. Immunol., 2000, 18 : 927-974; Leitner, supra; Cho, et al., J. Immunol., 168 (10): 4907-13), the sequences for the designation of endocytic trajectories- or processing with ubiquitin (Thomson, et al., 1998. J. Virol. 72: 2246-2252; Velders, et al. 2001. J. Immunol. 166: 5366-5373), the VP22 sequences type 1 of the disease virus 43 de Marek (J. Virol. 76 (6): 2676-82, 2002), main reinforcement regimes (Gurunn, upra, Sullivan, et al 2000. Na t ure, 408: 605-609; Hanke, et al. 1998. Vaccine e, 16: 439-445; Amara, et al., 2001. Sci en ce, 292: 69-74), and the use of vectors for mucosal delivery such as, for example, Sa lm on el la ( Darji, et al., 1997. Cel l, 91: 765-775; Woo, et al. 2001. Vaction e, 19: 2945-2954). Other methods are known in the art, some of which will be described later. Chemotherapeutic agents, radiation, anti-angiogenic compounds, or other agents can also be used in treatment and / or prevention of cancer using immunogenic targets (Sebti, et al., Oncogene 2000 Dec 27; 19 (56): 6566-73). For example, in the treatment of metastatic melanomas, suitable chemotherapeutic regimens may include BELD (bleomycin, vindesine, lomustine, and deacarbazine; Young, et al., 1985. Cancer, 55: 1879-81), BOLD (bleomycin, vincristine, lomustine , Dacarbazine; Seigler, et al., 1980. Cancer, 46: 2346-8); DD (dacarbazine, actinomycin, Hochster, et al., Cancer Treatment Reports, 69: 39-42), or POC (procarbazine, vincristine, lomustine, Carmo-Pereira, et al., 1984. Cancer Treatment Reports, 68: 1211-4) 44 among others. Other suitable chemotherapeutic regimens may also be used. Many anti-angiogenic agents are known in the art and could be suitable for co-administration with designated immunogenic vaccines and / or chemotherapeutic regimens (see, for example, Timar, et al., 2001. Pa tholgy On On. Re s. 7 (2): 85-94). These agents include, for example, physiological agents such as, for example, growth factors (ie, ANG-2, NK1, 2.4 (HGF), transforming growth factor-beta (TGF-β)), cytokines ( that is, interferons such as, for example, IFN-oc, -ß, -?, platelet factor 4 (PF-4), PR-39), proteases (ie, cleaved AT-III, collagen fragment XVIII ( Endostatin)), the plasmin fragment of HmwKalli krein-d5 (Angiost atina), prothrombin-Fl-2, TSP-1), protease inhibitors (i.e., inhibitor of metalloprotease tissues such as, for example, TIMP-1, -2, or -3; maspina, plasminogen activator inhibitors such as, for example, PAI-1, pigment epithelium-derived factor (PEDF)), Tumstatin (available through ILEX, Inc.), antibody products (ie, collagen binding antibodies HUIV26, HUI77, 45 XL313; and anti-VEGF; anti-integrin (ie, Vitaxin, (Lxsys))), and glycosidases (ie, heparinase-I, -III). It is known or believed that "chemical" or modified physiological agents have anti-angiogenic potential and include, for example, vinblastine, taxol, ketoconazole, thalidomide, dolestatin, combrestatin A, rapamycin (Guba, et al., 2002, Na t ure Med., 8: 128-135), CEP-7055 (available from Cephalon, Inc.), flavone acetic acid, Bay 12-9566 (Bayer Corp.), AG3340 (Agouron, Inc.), CGS 27023A (Novartis), tetracycline derivatives (i.e., COL-3 (Collagenix , Inc.)), Neovastat (Aeterna), BMS-275291 (Bristol-Myers Squibb), low dose of 5-FU, low dose of methotrexate (MTX), irsofladin, radicicol, cyclosporine, captopril, celecoxib, polysaccharide D45152- sulphated , cationic protein (Protamine), ionic cat peptide-VEGF, Suramine (naphthyl urea polysulfonated), compounds that interfere with the function or production of VEGF (ie, SU5416 or SU6668 (Sugen), PTK787 / ZK22584 (Novartis)), Distamícin A , Angiozyme (ribozyme), isoflavonoids, staurosporine derivatives, genistein, EMD121974 (Merck KcgaA), tyrphostins, isoquinolones, retinoic acid, carboxyazidot riazole, TNP-470, octreotide, 46 2-methoxyestradiol, aminosterols (ie, squalamine), glutathione analogue (ie, N-acetyl-L-cis theine), combretastatin A-4 (Oxigene), Eph receptor blocking agents (Na t ure, 414: 933 -938, 2001), Rh-Angiostat ina, Rh-Endost atina (WO 01/93897), cyclic peptide-RGD, acutin-disint egrina, benzodia zepenos, anti-avb3 humanized Ab, Rh-PAI-2, amiloride, p -amidobenzamidine, anti-uPA ab, anti-uPAR Ab, L-fanilalanin-N-methylamides (ie, Batimistat, Marimastate), AG3340, and Minocycline. Many other suitable agents are known in the art and could suffice for the practice of the present invention. The present invention can also be used in combination with "non-traditional" cancer treatment methods. For example, it has recently been shown that the administration of certain anaerobic bacteria can help to slow tumor growth. In one study, Cl or tri di um novyi was modified to remove a toxin gene carried in a phage epitome and administered to mice with colorectal tumors (Dang, et al., P.N.A.S. USA, 98 (26 ): 15155-15160, 2001). In combination with chemotherapy, it was shown that the treatment causes tumor necrosis in 47 animals. The reagents and methodologies described in this application can be combined with these treatment methodologies. Nucleic acids encoding immunogenic targets can be administered to patients by any of a variety of available techniques. Several viral vectors have been successfully used to introduce a nucleic acid to a host including retroviruses, adenovirus, adeno-associated virus (AAV), herpes virus, and poxvirus, among others. It is understood in the art that many of these viral vectors are available in the art. The vectors of the present invention can be constructed using standard recombinant techniques generally available to one of skill in the art. These techniques can be found in common molecular biology references such as, for example, Mol ecul ar Cl on i ng: A Labora t ory Man ua l (Sambrook, et al., 1989, Cold Spring Harbor Laboratory), Gene Expression Technology (Methods in Enzymology, Vol. 185, edited by D. Goeddel, 1991. Academic Press, San Diego, CA), and PCR ProtocolS: A Guide to Methods and Applications (Innis, et al., 1990. Academic Press, San Diego, CA ). 48 Preferred retroviral vectors are lentivirus derivatives as well as derivatives of murine or avian retroviruses. Examples of suitable retroviral vectors include, for example, Molonay murine leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), SIV, BIV, HIV and Rous sarcoma virus. (RSV). Several of the retroviral vectors can incorporate multiple exogenous nucleic acid sequences. When the recombinant retroviruses are defective, they require help to produce infectious vector particles. This help can be provided, for example, by helper cell lines that code for retrovirus structural genes. Suitable helper cell lines include γ2, PA317 and PA12, among others. The vector virions produced using these cell lines can then be used to infect a tissue cell line, such as, for example, NIH3T3 cells, to produce large amounts of chimeric retroviral virions. Retroviral vectors can be administered by traditional methods (ie injection) or by implantation of a "cell line" 49 producer "in proximity to the white cell population (Culver, K., et al., 1994, Hum Gene Ther., 5 (3): 343-79; Culver, K., et al., Cold Spring Harb. Symp. Wuant, Biol., 59: 685-90), Oldfield, E., 1993, Hum. Gene 'Ther., 4 (l): 39-69.) The producer cell line is engineered to produce a viral vector and releasing viral particles in the vicinity of the target cell A portion of the viral particles released are contacted with the target cells and infect those cells, thus supplying a nucleic acid of the present invention to the target cell. of the target cell, expression of the vector's nucleic acid is shown, It has been demonstrated that adenoviral vectors are especially useful for gene transfer in eukaryotic cells (Rosenfeld, M., et al., 1991, Science, 252 (5004 ): 431-4; Crystal, R., et al., 1994, Nat. Genet., 8 (1): 42-51), the expression of the study eukaryotic gene (Le Vrero, M., et al., 1991, Gene, 101 (2): 195-202), the development of vaccines (Graham, F., and Prevec, L., 1992, Biotechnology, 20: 363-90), and in animal models (Stratford-Perricaudet, L., et al. ., 1992, Bone Marrow Trassplant., 9 (Suppl 1): 151-2; fifty Rich, D., et al., 1993, Hum. Gen Ther. , 4 (4): 461-76). Experimental routes to administer recombinant Ad to different tissues in vivo have included intratracheal instillation (Rosenfeld, M., et al., 1992, Cell, 68 (1): 143-55) injection into the muscle (Quantin, B., et al. , 1992, Proc. Nati, Acad. Sci. USA, 89 (7): 2581-4), peripheral intravenous injection (Herz, J., and Gerard, R., 1993, Proc. Nati. Acad. Sci. USA , 90 (7): 2812-6) and inoculation is erectotactic in the head (Le Gal La Salle, G., et al., 1993, Science, 259 (5097): 988-90), among others. The adeno-associated virus (AAV) shows a high level of infectivity, a wide range of hosts and integration specificity in the genome of the host cell (Hermonat, P., et al., 1984, Proc. Nati. Acad. Sci USA, 81 (20): 6466-70). And the Type 1 Herpes Simplex Virus (HSV-1) still another attractive vector system, especially for use in the nervous system due to its neurotropic property (Geller, et al., 1991, Trains 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). Poxvirus is another useful expression vector (Smith, et al., 1983, Gene, 25 (l): 21-8; Moss, et al, 51 1992, Bi or techn olgy, 20: 345-62; Mos s, et al, 1992, Curr. Top My crobi ol. Im an ol. , 158: 25-38; Mos s, et al. 1991. Sci en ce, 252: 1662-1667). It is shown that poxviruses are useful for inclusion in vaccines, NYVAC, fowlpox, poultrypox, smallpox, ALVAC, and ALVAC (2), among others. NYVAC (vP866) was derived from the vaccinia virus vaccine Copenhagen strain by removing six non-essential regions of the genome that encodes known or potential virulence factors (see, for example, U.S. Patent Nos. 5,364,773 and 5,494,807). The deletion sites were also engineered as the recipient sites for the extraneous gene insertion. The suppressed regions are: the thymidine kinase gene (TK); J2R); the hemorrhagic region (u; B13R + B14R); the inclusion body region type A (ATI; A26L); the hemagglutinin gene (HA, A56R); the gene region with host range (C7L-K1L); and, the ribonucleotide reductase, with a large subunit, (I4L). NYVAC is a genetically engineered vaccinia virus strain that was generated by specifically suppressing eighteen open reading frames that encode the 52 gene products associated with virulence and the host range. It has been shown that NYVAC will be useful for expressing TAs (see, for example, U.S. Patent No. 6,265,189). NYVAC (vP866), vP994, VCP205, VCP1433, placZH6H4L reverse, pMPC6H6K3E3 and pC3H6FHVB were also deposited with the ATCC in accordance with the terms of the Budapest Treaty, accession numbers VR-2559, VR-2558, VR-2557, VR-2556 , ATCC-97913, ATCC-97912, and ATCC-97914, respectively. ALVAC-based recombinant viruses (ie, ALVAC-1 and ALVAC-2) are also suitable for use in the practice of the present invention (see, for example, U.S. Patent No. 5,756,103). ALVAC (2) is identical to ALVAC (1) except that the ALVAC genome (2) comprises the vaccinia genes E3L and K3L under the control of the vaccinia promoters (U.S. Patent 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 shown that they are useful for expressing foreign DNA sequences, such as for example TAs (Tartaglia et al., 1993 a, b, U.S. Patent No. 5,833,975). ALVAC was deposited in accordance with the conditions of 53 Treaty of Budapest with the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, Va. 20110-2209, USA, access number ATCC VR-2547. Another useful poxvirus vector is TROVAC.

TROVAC refers to an attenuated poultry pox which was an isolated cloned in plaques derived from the FP-1 vaccine strain of poultry pox virus which was authorized for vaccination of chicks 1 day of age. TROVAC was also deposited in accordance with the conditions of the Budapest Treaty with accession number ATCC 2553. The "non-viral" plasmid vectors may also be suitable for the practice of the present invention. Preferred plasmid vectors are compatible with bacterial, insect, and / or mammalian host cells. These vectors include, for example, PCR-11, pCR3, and pcDNA3.1 (Invitrogen, San Diego, CA), pBSII (Stratagene, La Jolla, CA), pET15 (Novagen, Madíson, Wl), pGEX (Pharmacia Bíotech, Piscataway, NJ), pEGFP-N2 (Clontech, Palo Alto, CA), pETL (BlueBacII, Invitrogen), pDSR-alpha (PCT Pub. No. WO 90/14363) and pFastBacDual (Gibco-BRL, Grand Island, NY), as well as, the 54 derivatives of the Bluescript® plasmid (a phagemid based on C0LE1 with large number of copies, Stratagene Cloning Systems, La Jolla, CA), plasmids for PCR cloning designed to clone the PCR products amplified by Taq (for example, plasmid derivatives) TOPOMR cloning® kit, PCR2.1®, Invitrogen, Carlsbad, CA). Bacterial vectors can also be used with the current invention. For example, these vectors include Shi gel la, Sa lmonel la, Vibri or chol era e, La ctoba ci ll us, Ba lle lle came t te guérin (BCG), and Strept oco ccus (see, for example, W088 / 6626, WO90 / 0594, WO 91/13157, WO 92/1796, and W092 / 21376). Many other vectors and expression systems of the non-viral plasmid could be used in the art and could be used with the present invention. Suitable nucleic acid delivery techniques include complexes with the DNA ligand, adenovirus-ligand-DNA complexes, direct DNA injection, CaP04 precipitation, gene acceleration techniques, electroporation, and colloidal dispersion systems, among others. Colloidal dispersion systems include complexes of macromolecules, nanocapsules, microspheres, beads, and lipid-based systems among which are they include oil-in-water emulsions, micelles, mixed micelles, and liposomes. The preferred colloidal system of this invention is a liposome, which are artificial membranous vesicles useful as vehicles for in vitro and in vivo delivery. RNA, DNA and intact virions can be encapsulated in the aqueous interior and can be delivered to cells in a biologically active form (Fraley, R, et al., 1981, Trencas Bi or chem. Sci. , 6: 77). The composition of the liposome is usually a combination of phospholipids, in particular the phospholipids of high phase transition temperature, usually in combination with steroids, especially cholesterol. Other phospholipids or other lipids can also be used. The physical characteristics of liposomes depend on pH, ionic strength, and the presence of divalent cations. Examples of lipids useful in the production of liposomes include phosphatidyl compounds, such as for example, phosphatidylglycerol, phosphatidylcholine, phosphatidyl-serine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides. Diacylphosphat idylglycerols are particularly useful, where the lipid entity contains 14-18 56 carbon atoms, in particular 16-18 carbon atoms, and saturates. Illustrative phospholipids include egg phosphatidylcholine, dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine. An immunogenic target can also be administered in combination with one or more adjuvants to enhance the immune response. In the following Table II the adjuvants of example are shown: TABLE II TYPES OF ADJUVANT S INMUNOLOGY S TYPE OF GENERAL EXAMINES EXAMPLE SPECIFIC ADJUVANT / REFERENCES Type gel Hydroxide / phosphate (Aggerbeck and Heron, 1995) aluminum ("Alum adjuvants") Calcium phosphate (Relyveld, 1986) Microbial Diphidoid murayl (MDP) (Chedid et al., 1986) Bacterial exotoxins Cholera toxin (CT), labile toxin from E. coli (LT) (Freytag and Clementes, 1999) Adjuvants based on lipid A of monophosphoryl endotoxin (MPL) (Ulrico and Myers, 1995) Other bacteria CpG oligonucleotides (Corral and Petray, 2000), BCG sequences (Krieg, et al., Naure, 374: 576), tetanus toxoid (Rice, et al., J. Immunol ., 2001, 167: 1558-1565) 57 The administration of a composition of the present invention to a host can be carried out using any of a variety of techniques known to those with experience in this field. The compositions can be processed according to conventional pharmacy methods to produce medicinal agents for administration to patients, including humans and other mammals (ie, a "pharmaceutical composition"). The pharmaceutical composition preferably occurs in the form of a unit of 58 dosage containing a certain amount of DNA, viral vector particles, polypeptide or peptide, for example. A suitable daily dose for a human or other mammal can vary, depending largely on the patient's condition and other factors, although, once again, it can be determined using routine methods. The pharmaceutical composition can be administered orally, parentally, by inhalation, rectal, intranodal spray, or topically in dosage unit formulations containing pharmaceutically acceptable and conventional carriers, adjuvants, and vehicles. The term "pharmaceutically acceptable carrier" or "physiologically acceptable carrier" in the sense in which it is used herein refers to one or more suitable formulation materials for carrying out 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 refers to the amount of a nucleic acid or polypeptide used to induce or improve an effective immune response. It is preferred that the compositions of the present invention be provided for the induction or amelioration of an anti-tumor immune response in a host that protects the host from the development of a tumor and / or allows the host to eliminate an existing tumor from the body. For oral administration, the pharmaceutical composition may be any of a variety of forms including, for example, a capsule, a tablet, a suspension, or liquid, among others. The liquids can be administered by injection as a composition with suitable carriers including saline, glucose, or water. The term parenteral, in the sense that is 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, for example, cocoa butter and polyethylene glycols that are solid at normal temperatures but liquid at the rectal temperature. 60 The dosage regimen for immunizing a host or otherwise for treating a disorder or disease with a composition of this invention is based on a variety of factors, including the type of disease, age, weight, sex, the patient's medical condition, the severity of the condition, the route of administration, and the particular compound used. For example, a poxviral vector can be administered as a composition comprising 1 x 106 infectious particles per dose. In this way, the dosage regimen can vary widely, although it can be determined using standard routine methods. A main reinforcement regime (WO 01/30382 Al) in which the target immunogen is initially administered in a priming step in a form followed by a booster step in which the target immunogen is administered in another form can also be used. . The shape of the white immunogen in the priming and reinforcement steps is different. For example, if the priming step used a nucleic acid, the reinforcement can be administered as a peptide. Similarly, when a priming step, a type of recombinant virus (i.e., ALVAC) is used, the booster step can use another type of virus (i.e., NYVAC). It has been shown that this method of administration of main reinforcement induces strong immunological responses. Various combinations of shapes are suitable for the practice of the present invention. 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-estimulatory 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 an individual composition. Injectable preparations such as, for example, sterile injectable aqueous or oleaginous suspensions, can be formulated, according to known methods using suitable dispersing or wetting agents and suspending agents. The injectable preparation can also be a sterile injectable solution or suspension in a diluent or non-toxic parenterally acceptable solvent. Suitable vehicles and solvents that can be used are water, Ringer's solution, and isotonic sodium chloride solution, among others. For example, a viral vector such as, for example, a poxvirus can be prepared in 0.4% NaCl. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any insipid, fixed oil, including synthetic mono or diglycerides, can be employed. In addition, fatty acids such as for example oleic acid find use in the preparation of injectable solutions. For topical administration, a suitable topical dose of a composition of one to four, and preferably two or three times a day, may be administered. The dose can also be administered with intermediate days during which it is not 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 can 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 preparations 63 or semi-liquid suitable for penetration through the skin (for example, liniments, lotions, ointments, creams, or pastes) and drops suitable for ocular, in the ear, or nose administration. The pharmaceutical compositions can also be prepared in a solid form (including granules, powders or suppositories). The pharmaceutical compositions can be subjected to conventional pharmaceutical operations such as for example, sterilization and / or may contain conventional adjuvants, such as, for example, preservatives, stabilizers, wetting agents, emulsifiers, buffers, etc. Solid dosage forms for oral administration may include capsules, tablets, pills, powders, and granules. In these solid dosage forms, the active compound can be mixed with at least one inert diluent such as, for example, sucrose, lactose, or starch. These dosage forms can also comprise, as in normal practice, additional substances other than inert diluents, for example, lubricating agents such as, for example, magnesium stearate. In the case of capsules, tablets, and pills, the dosage forms may also comprise agents that can be used in the preparation of the drug. shock absorbers. Tablets and pills can be additionally 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, for example, water. These compositions may also comprise adjuvants, such as, for example, wetting agents, sweeteners, flavors, and flavors. Pharmaceutical compositions comprising a nucleic acid or polypeptide of the present invention can take any of various forms and can 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 can be administered directly into a lymph node (intranodal) or tumor mass (i.e., int ratumoral administration). For example, the dose could be administered subcutaneously on days 0, 7, and 14. Appropriate methods are known in the art. immunization using the compositions comprising the TA, as shown for p53 (Hollstein et al., 1991), p21-ras (Almoguera et al., 1988), HER-2 (Fendly et al., 1990), antigens associated with melanoma (MAGE-1; MAGE-2) (van der Bruggen et al., 1991), p97 (Hu et al., 1988), E antigen associated with melanoma (WO 99/30737) and carcinoembryonic antigen (CEA) (Kantor et al., 1993; Fishbein et al., 1992; Kaufman et al., 1991), among others. Preferred embodiments of administrable compositions include, for example, nucleic acids or polypeptides in liquid preparations such as for example 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, for example, sterile suspensions or emulsions. For example, a recombinant poxvirus may be in combination with a suitable carrier, diluent, or excipient such as, for example, sterile water, physiological saline, glucose or the like. The composition can also be provided in lyophilized form 66 for reconstitution, for example, in aqueous, isotonic saline solution buffer. In addition, the compositions can be co-administered or sequentially administered with other anti-neoplastic, anti-tumor or anti-cancer agents and / or agents that reduce or alleviate the disease effects of anti-neoplastic, antitumor agents or anti-cancer. A kit comprising a composition of the present invention is also provided. The equipment may include a separate container containing a suitable carrier, diluent or excipient. The kit may also include an additional anti-cancer, anti-tumor or anti-neoplastic agent, and / or an agent that reduces or alleviates the disease effects of antineoplastic, anti-tumor or anti-cancer agents for co-administration or sequential administration. Additionally, the equipment may include instructions for mixing or combining the ingredients and / or administration. A better understanding of the present invention and many of its advantages will be obtained from the following examples, provided by way of illustration. 67 EXAMPLES EXAMPLE 1 Con s tru ct ura tion of the m ult ti -an ti gen i ct io n vT41 expression vector vT416 (ALVAC-NY-ESO- l / Trp-2-LFA-31ICAM-l / B7.1-E3L / K3L) was constructed in the ALVAC vector using standard techniques. The DNA sequences encoding NY-ESO-1, Trp-2, LFA-3, ICAM-1, B7.1, vvE3L and vvK3L were inserted at various sites within the ALVAC genome. The DNA sequences coding for NY-ESO-1 (Chen et al., 1997 PNAS 94: 1914) and TRP-2 (Wang et al., 1996).

J. Exp. Med. 184: 2207) were inserted into the C5 site.

The DNA sequences that code for LFA-3 (Wallner, et al (1987) J. Exp. Med. 166: 923-932), ICAM-1 (Staunton, et al. (1988) Cell 52: 925-933) and B7.1 (Chen, et al. . (1992) Cell 71: 1093-1102) were inserted into the C3 site. LFA-3, ICAM-1 and B7.1 form an expression cassette known as TRICOM. The DNA sequences encoding vvE3L (Chang, et al., 1992, Proc. Nati, Acad. Sci. USA 89: 4825-4829) and vvK3L (Beattie, et al., 1991. Virology 183: 419-422) were inserted. at the C6 site. The promoters were used as follows: 68 TABLE III The sE / L promoter is described by Chakrabarti, et al. (BioTechniques 23: 1094-1097, 1997). The donor plasmids used are shown below: TABLE IV DNA sequences NY-ESO-1 and TRP-2 were inserted into the donor plasmid pT1132 of ALVAC. This donor plasmid was then used with pALVAC. ricom (C3) # 33 to generate the recombinant ALVAC-TRICOM that expresses these genes using standard techniques. Plasmids pALVAC. Tricom (C3) # 33 and pT1132 are shown in Figure 1. The 69 pALVAC DNA sequences. Tricom (C3) # 33 and pT1132 are shown in Figures 2 and 3, respectively.

Example 2 Constructing the multilayered structure vT419 The expression vector vT419 (ALVAC-gplOOM / Mart-1 / Mage-1,3 minigen-LFA-3 / ICAM-1 / B7.1 -E3L / K3L) was constructed in the ALVAC vector using standard techniques. The DNA sequences coding for gpl 00M / MART-1 / MAGE-1, 3 minigen, LFA-3, ICAM-1, B7.1, vvE3L and vvK3L were inserted at various sites within the ALVAC genome. The gpl 00M / MART-1 / MAGE-1, 3 minigenes were inserted into the C5 site. The DNA sequences encoding LFA-3 (Wallner, et al (1987) J. Exp. Med. 166: 923-932), ICAM-1 (Staunton, et al. (1988) Cell 52: 925-933 ) and B7.1 (Chen, et al. (1992) Cell 71: 1093-1102) were inserted into the C3 site. LFA-3, ICAM-1 and B7.1 form an expression cassette known as TRICOM. The DNA sequences encoding wE3L (Chang, et al., 1992. Proc. Nati, Acad. Sci. USA 89: 4825-4829) and vvK3L (Beattie, et al., 1991. Virology 183: 419-422) were inserted. at the C6 site. The promoters were used as follows: 70 TABLE V The sE / L promoter is described by Chakrabarti, et al. (BioTechniques 23: 1094-1097, 1997). The donor plasmids used are shown below: TABLE VI gplOO (M), Mart-1 and the Mage-1,3 minigene were inserted into the donor plasmid pT3217 of ALVAC C5. This donor plasmid was then used with pALVAC. Tricom (C3) # 33 to generate the ALVAC-TRICOM recombinant that expresses these genes using standard techniques. This donor plasmid was inserted into the C5 site. pALVAC. Tricom (C3) # 33 is shown in 71 Figures 1 and 2. Plasmid pT3217 is shown in Figure 4. The DNA sequence of pT3217 is shown in Figure 5.

EXAMPLE 3 Immunological performance of the animal vectors The results of the first animal experiment indicated a tendency towards immunological responses greater than three (Marti, NY-ESO-1 and gplOO) of the four antigens when the vaccine was administered as two injections separately. However, these differences are not statistically significant. In detail, transgenic HLA-A2 / Kb mice (5 / group) were immunized subcutaneously with vT419 (ALVAC (2) -gpl 00M / MART-1 / minigen MAGE-1/3 / TRICOM) and vT416 (ALVAC (2) -TRP-2 / NY-ESO- 1 / TRICOM) either combined in a site or administered as separate injections. The control mice were immunized with parental ALVAC (2). Mice were vaccinated three times (at three-week intervals), and three weeks after the last responses to booster T lymphocytes were analyzed in individual mice by IFN-g ELISPOT and CTL analysis after re-stimulation in vi t ro with the 72 peptide. Compared to the control animals, mice vaccinated with multigenic vectors (at 2 sites) exhibited statistically significant responses to ELISPOT against MART-1. The response of IFN-gamma to gplOOM and NY-ESO-1 was also they could detect, although these responses were not statistically significant due to the variability of responses and the small number of crops tested. ELISPOT responses against TRP-2 antigen were elevated in all tested groups (including control animals), presumably due to the fact that the TRP-2 peptide A2-restrid dominant (180-188) cross-reacts with H-2Kb and can induce low avidity of T lymphocyte responses in mice that have not received prior treatment after in vitro culture, and therefore were not statistically significant. Interestingly, ELISPOT responses in mice injected with a combination of vT416 and vT419 were generally lower in mice that received each virus separately, although these differences did not reach statistical significance. The CTL data were quite negative, except for a strong anti-gplOO response and a marginal anti-MART-1 response, the 73 two were presented in mice vaccinated with vT416 and vT419 (two sites). In general, these results provided encouraging data that establish that multi-antigenic vectors can generate responses against MART-1, and suggest that anti-gplOO and anti-NY-ESO-1 responses can also be induced. Two additional pre-clinical animal studies have been completed using the ALVAC mult i-antigenic melanoma recombinants. In these experiments, the transgenic HLA ~ A2 / Kb mice (5 / group) were immunized subcutaneously with vT419 (ALVAC (2) -gplOOM / MART-l / minigen MAGE-1/3 / TRICOM) and VT416 (ALVAC (2) -TRP-2 / NY-ESO-1 / TRICOM) either combined at one site or administered as separate injections. The control mice were immunized with parental ALVAC (2). After vaccination, T lymphocyte responses in individual mice were assessed by IFN-gamma ELISPOT analysis after re-stimulation with the peptide. Unlike the previous multi-antigenic experiment, which provided the encouraging immunogenicity data, the two most recent studies generated inconclusive data, due to the high background responses in the control animals. immunized. Therefore, the results were generally judged as inconclusive. To confirm the immunogenicity of the multi-antigenic structures, and to repeat the results of the first study, another pre-clinical animal study was completed. Transgenic HLA-A2 / Kb mice (10 / group) were immunized subcutaneously with vT419 (ALVAC (2) -gplO OM / MART-1 / minigen MAGE-1/3 / TRICOM) and vT416 (ALVAC (2) -TRP- 2 / NY-ESO-1 / TRICOM) were administered as separate injections. The control mice were immunized with parental ALVAC (2). Statistically significant ELISPOT responses could be detected against gplOO, MART-1 and TRP-2, and some responses were detected against NY-ESO-1, which were at the limit of being statistically significant. While the present invention has been described in terms of the preferred embodiments, it should be understood that variations and modifications will be presented to those skilled in the art. Therefore, it is intended that the appended claims cover all of these equivalent variations as they fall within the scope of the invention as claimed.

Claims (19)

1. tumor cells and could be specific for one or several tumors, for example the CEA antigen is expressed in colorectal, breast and pulmonary cancers. Sgroi et al (1999) differentially identified diverse genes expressed in invasive and metastatic carcinoma cells with combined use of laser capture microdissection and cDNA micro-arrays. Various delivery systems similar to DNA or virus could be used for therapeutic vaccination against human cancers (Bounet et al, 2000) and can produce immunological responses and also the interruption of immunological tolerance against TAA. Tumor cells can be made more immunogenic by inserting transgenes encoding T-lymphocyte co-stimulating molecules such as, for example, B7.1 or cytokines, such as, for example, IFN- ?, IL2, or GM-CSF, among others The co-expression of a TAA and a cytokine or co-mimickers molecule can develop an effective therapeutic vaccine (Hodge et al, 95, Bronte et al, 1995, Chamberlain et al, 1996). There is a need in the art for reagents and methodologies useful for stimulating an immune response to prevent or treat CLAIMS 1. An expression vector for the coexpression of at least two immunogenic targets, wherein the immunogenic targets are selected from the group consisting of NY -ESO-1, TRP-2, gplOO, gplOOM, a MART antigen, MART-1, an MAGE antigen, MAGE-1, and MAGE-3.
2. 2. The expression vector according to claim 1 wherein the vector is a plasmid or a viral vector.
3. 3. The expression vector according to claim 2 wherein the viral vector is selected from the group consisting of poxvirus, adenovirus, retrovirus, herpesvirus, and adeno-associated virus.
4. 4. The expression vector according to claim 3 wherein the viral vector is a poxvirus selected from the group consisting of vaccinia, NYVAC, fowl pox, Canary pox, ALVAC, ALVAC (2), poultry pox, and TROVAC.
5. 5. The expression vector according to claim 4 wherein the viral vector is a poxvirus selected from the group consisting of NYVAC, ALVAC, and ALVAC (2).
6. 6. The expression vector according to claim 1 further comprising at least one nucleic sequence encoding an angiogenesis-associated antigen.
7. 7. The expression vector according to claim 6 wherein the vector is a plasmid or a viral vector.
8. 8. The expression vector according to claim 7 wherein the viral vector is selected from the group consisting of poxvirus, adenovirus, retrovirus, herpesvirus, and adeno-associated virus.
9. 9. The expression vector according to claim 8 wherein the viral vector is a poxvirus selected from the group consisting of vaccinia, NYVAC, fowl pox, Canary pox, ALVAC, ALVAC (2), poultry pox, and TROVAC.
10. 10. The expression vector according to claim 9 wherein the viral vector is a poxvirus selected from the group consisting of NYVAC, ALVAC, and ALVAC (2).
11. 11. The expression vector according to claim 1 or 6 further comprising at least one nucleic acid sequence encoding a co-stimulator component.
12. 12. The expression vector according to claim 11 wherein the vector is a plasmid or a viral vector.
13. 13. The expression vector according to claim 12 wherein the viral vector is selected from the group consisting of poxvirus, adenovirus, retrovirus, herpesvirus, and adeno-associated virus.
14. 14. The expression vector according to claim 13 wherein the viral vector is a poxvirus selected from the group consisting of vaccinia, NYVAC, fowl pox, Canary pox, ALVAC, ALVAC (2), poultry pox, and TROVAC.
15. 15. The expression vector according to claim 14 wherein the viral vector is a poxvirus selected from the group consisting of NYVAC, ALVAC, and ALVAC (2).
16. 16. The expression vector according to any of claims 11-15 wherein the co-esting component is human B7.1.
17. 17. A composition comprising an expression vector according to any of claims 1-16 in a pharmaceutically acceptable carrier.
18. 18. A method for preventing or treating cancer comprising administering to an host an expression vector according to any of claims 1-16.
19. 19. A method for preventing or treating cancer, comprising administering to a host a composition according to claim 17. SUMMARY OF THE INVENTION The present invention relates to peptides, polypeptides, and nucleic acids and the use of the peptide, polypeptide or nucleic acid for the prevention and / or treatment of cancer. In particular, the invention relates to peptides and nucleic acid sequences encoding the peptides for use in the diagnosis, treatment, or prevention of melanoma. FIGURE 2 DNA sequence of pALVAC.Tricom (C3) # 33 1 GGAAATTGTA AACGTTAATA TTTTQTTA &A ATTCGCGTTA AATTTT? GTT. CCTT? ÑACAT TTGCAATTA? AAAACñATTT TññGCGCAAr TT ñAAACAA • 5 51. AÑATCAGCTC ATTTTTTTAAC CAATAGGCCG AAATCGGCAA AATCCC? TAT TTTAG? CGAG TAAAAAATTG GTTA? CCGGC TTTAGCCGTT TT GG AAT 101 AAATCAAAAG AATAGACCGA GA2AGGGTTG AGTGTTGCTC CAGTTTGGAA TIGAGTTTTC TTATCGGGCT CTATCCCAAC TCACAACAAG GTCAAACCTT 151 CAAGAGTCCA CTATTSAAGA ACGTGGACTC CAACGTCAA & GGGCGAASAA 10 -. 10 - GTLCTCASGI GA? AATTTCT TGCACCTGAG GTTGCAGTTT CCCGC? TTTT 201 CCGTCTATCA GGGCGATGGC CC CTACGTG AACCATCACC CTAATCAAG? GGCAGATAGT CCCGCTACCG GGTGATGCAC TTGGTAGTGG GATTAGTTCA 251 'TTTTTGGGGT CGAGGTGCCG TAAAGCACTA AATCGGAACC CTAAAGGGAG AAAAACCCCA GCTCCÑCGGC ATTTCGTGAT TTAGCCTTGG GATTTCCCTC 15 '301 CCCCCGATTT A A CriGAC GGGGAAAGCC GGCGftACG? G GCfiAG & aAGG GGGGGCTAAA TC? CGñACTG CCCCTTTCGG CCGCTTGCAC CGCTCT? TCC -351 AAGGGAAGA AGCGAAAGGA GCGGGCGCTA GGGCGCTGGC SAGTGTAGCG . ' TTCCCTTCTT TCGCTTTCCT CGCCCGCGAT CCGGCGACCG TTCACASCGC . 401 GTCACGCTGC GCGTMCCAC CACACCCGCC GCGCTTAA? G CGCCGCTACA '20' CAGTGCGACG CGO &TTGGTG GTGTGGGCGG CGCGAATTAC GCGGCGATGT 451 GGGCGCG? CG CGCCATTCGC CATTCAGGCT GCGCAACTGT TGGGAAGGGC CCCC3CGCAGC GCGGTAAGCG GTAAGTCCGA CGCGTTGACA ACCCTTCCCG '501 GATCGGTGCG GGCCrCTTCG CTAISACGCC AGCTGGCGAA ÁGGGGGATGT CTAGCCACGC .CCGGAGAAGC GATAATGCGG 'JJCGACCGCTT TCCCCCTACA 25 .551 GCTGCAAGGC GATIAAGT? G GGTAACGCCA GGGTTTTCCC AGTC &CGACG CGACGTTCCG CTAATTCAAC CCATTGCGGT CCCAAAAGGG TCAGTGCTGC 601 TTGTAAAACG * ACGSCCAGTG AATTGTHATA CGACTCACTA TAGGGCGAAT AACATTTTGC TGCCGGTCAC T? AACATTAT GCTGAGTGAT ATCCCGCTTA 651. TGGGTACCGC GGCCGCGTCG ACATGCAT? G T? AGT? CTGI AGATCAGTAA 30 ACCCA? GGCG CCGGCGCAGC GGTACGTAAC AATCAAGACA TCTAGTCATT Left branch 701 CGTATAGCAT ACGAG? AIAA TTATCGTAGG -TAG? AGGTAT -CCTAAAA? AA. GCA? A? CGTA TGCTCATATT AATAGCATCC ATCATCCATA GGATTTTATT 35 Left branch 753, ATCTGATACA Gft.! CAA £ &ACT ?? GTAAATCA ATTCAGCAAT- TTCSCTATTA '• TAGACTATGE CTATTATTGA AACATTTAGT TAAGTCGTTA AAGAGATAAT 4'0 • Left branch 801 TCATGATAAT GATTAATACA CAGCGTGTCG TTATTTTTTG TTACGATAGT AGTftCTATTA CTAATTATGT GTCGCACAGC AATAAAAAAC AATGCTATCA Left rationing 45 851 ATTTCTAAAG TAAAGAGCAG GAATCCCIAG TATAATAGAA ÁTAATCCATA TAAAGATTTC ATTTCTCGTC CTTAGGGflTC ATATTATCTT TATTAGGTAT Branch left. .901 'TGAAAAATAT- AGTAATGTAC AT TTTCT TO TGTTAACATA TTTATAGGTA • 50 ACT? TTTATA TCATTACATG TAIAAAGATT ACAATTGTAT TiflATATCCAT' • Left branch 551, AATCCAGGAA GGGTAATTTT TACATATCTA TATACGCTTA .TTACAGTTA? TTAGGTCCTT CCCATTAAAA ATGTATAGAT ATATGCGAAT AATGTCAATA 2/53