CN1878566B - Multi-antigen vectors of 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

Be used for melanomatous multi-antigen vectors

Invention field

The present invention relates to be used to prevent and/or treat the multi-antigen vectors of cancer.Especially, the present invention relates to be used to treat and/or prevent melanomatous multi-antigen vectors.

Background of invention

Technological at several kinds like high-density micro-array, SEREX, immunohistochemistry (IHC), RT-PCR, in situ hybridization (ISH) and laser capture microscopy (laser capturemicroscopy) (Rosenberg, Immunity, 1999; Sgroi et al, 1999, Schenaet al; 1995, Offringa et al, 2000) help under; Owing to, contain the existing huge increase of development of the cancer vaccine of tumor associated antigen (TAAs) recent years based on the huge advance made of primary tumo(u)r and Normocellular express spectra evaluation molecule.TAAs is can be specific by tumor cells expression or the antigen of cross expressing and to one or more tumors, and for example CEA antigen is expressed in colorectum, breast and pulmonary carcinoma.Sgroi et al (1999) adopts uniting of laser capture microdissection and cDNA microarray to use several genes of having identified differential expression in property invaded and metastatic carcinoma cell.Several kinds of delivery systems resemble DNA or virus and can be used for the therapeutic inoculation (Bonnet et al, 2000) of anti-human cancer and can cause immunoreation and cut off the immunologic tolerance to TAAs.Through inserting the for example transgenic of IFN-γ, IL2 or GM-CSF etc. of encode T cell costimulatory molecules such as B7.1 or cytokine, tumor cell can become and have higher immunogenicity.The coexpression of TAA and cytokine or costimulatory molecules can be developed efficacious therapy vaccine (Hodge et al, 95, Bronte et al, 1995, Chamberlain etal, 1996).

This area need be to immune response stimulating with prevention or treatment cancer useful reagent and method.The invention provides this reagent and the method that overcome many difficulties that others attempts to treat in the cancer and met with.

Summary of the invention

The invention provides and be used for using to prevent and/or treat the multi-antigen vectors of cancer to the patient.Especially, the multi-antigen vectors one or more tumor antigens (" TA ") of encoding.The multi-antigen vectors immunologic stimulant of can also encoding gives like costimulatory molecules and/or with adjuvant.

The accompanying drawing summary

Fig. 1. the figure of plasmid pALVAC.Tricom (#33) and pT1132.

Fig. 2. the DNA sequence of plasmid pALVAC.Tricom (#33) (SEQ ID NOS.1 and 2).

Fig. 3. the DNA sequence of plasmid pT1132 (SEQ ID NOS.3 and 4).

Fig. 4. the figure of plasmid pT3217.

Fig. 5. the DNA sequence of plasmid pT3217 (SEQ ID NOS.5 and 6).

Fig. 6. the NY-ESO-1 of example (SEQ ID NO.:7), TRP-2 (SEQ ID NO.:8), gp100 (SEQ ID NO.:9), gp100M (SEQ ID NO.:10), MART-1 (SEQ ID NO.:11), MAGE-1 (SEQ ID NO.:12), MAGE-3 (SEQ ID NO.:13), B7.1 (SEQ ID NO.:14), LFA-3 (SEQ ID NO.:15) and the proteic aminoacid sequence of ICAM-1 (SEQ ID NO.:16).

Detailed Description Of The Invention

The invention provides treating and/or preventing cancer useful reagent and method.All lists of references of quoting among the application are incorporated herein by reference.

In one embodiment, what the present invention relates to anti-one or more tumor antigens (" TA ") immunoreactively induces or strengthens, to prevent and/or treat cancer.In certain embodiments, one or more TAs can be combined.In preferred embodiments, give the antigenic nucleic acid carrier of codes for tumor or for example peptide or polypeptide form tumor antigen originally after one's death, the expression of TA in host cell causes immunoreation.

" antigen " in this use is in giving antigenic host, to produce immunoreactive molecule (like polypeptide) or its part.Immunoreation can comprise with the bonded production of antibodies of antigenic at least one epi-position and/or to the generation of the cell immune response of the cell of expressing this antigenic epi-position.This reaction can be present immunoreactive enhancing, through for example causing that production of antibodies that antibody produce to increase, increases with the antigen affinity increases or cell immune response increases (being immunocompetence T cell quantity or active increasing).Produce immunoreactive antigen and can be considered to have immunogenic or immunogen.When description was of the present invention, TA can be called as " immunogenicity target ".The invention provides the expression vector of in the host, expressing one or more immunogenicity targets.

Term TA comprises tumor associated antigen (TAAs) and tumor specific antigen (TSAs), and wherein cancerous cell is antigenic source.TAA be on the tumor cell surface to be higher than antigen that observed scale reaches on the normal cell or the antigen of in the fetal development process, on normal cell, expressing.TSA is to tumor cell antigen unique and that on normal cell, do not express.TA further comprises TAAs or TSAs, its antigenicity fragment and keeps their antigenic modified forms.

According to expression pattern, function or the genetic origin of TAs, they typically are divided into five types: carcinoma of testis (CT) antigen (being MAGE, NY-ESO-1); Melanocyte differentiation antigen (being MelanA/MART-1, tryrosinase, gp100); Sudden change antigen (being MUM-1, p53, CDK-4); Cross expression " self " antigen (being HER-2/neu, p53); And virus antigen (being HPV, EBV).Be the purpose of embodiment of the present invention, suitable TA is any TA that induces or strengthen the anti tumor immune response among the host who gives TA.Suitable TAs comprises for example various gp100 (Coxet al., Science, 264:716-719 (1994); United States Patent (USP) 6,500,919 B1 and WO01/30847, the 162nd residue is Val, is also referred to as " gp100M "; United States Patent (USP) 6,537,560B1, the 162nd residue is Phe), MART-1/Melan A (Kawakami etal., J.Exp.Med., 180:347-352 (1994); United States Patent (USP) 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; United States Patent (USP) 5,831,016 and 6,083,783), tryrosinase (Wolfel et al., Eur.J Immunol., 24:759-764 (1994); WO 200175117; WO 200175016; WO200175007), NY-ESO-1 (WO98/14464; WO99/18206; GenBank accession number P78358; United States Patent (USP) 5,804,381), melanoma Dan Baijutang (Hellstrom et al., J.Immunol., 130:1467-1472 (1983)), MAGE family antigen (are MAGE-1,2,3,4,6,12,51; Van der Bruggen et al., Science, 254:1643-1647 (1991); United States Patent (USP) 6,235,525; CN 1319611), BAGE family antigen (Boel et al., Immunity, 2:167-175 (1995)), GAGE family antigen (be GAGE-1,2; Van den Eynde etal., J.Exp.Med., 182:689-698 (1995); United States Patent (USP) 6,013,765), the RAGE family antigen (is RAGE-1; Gaugler et at., Immunogenetics, 44:323-330 (1996); United States Patent (USP) 5,939,526), N-acetylglucosaminyl transferase-V (Guilloux etat.; J.Exp.Med., 183:1173-1183 (1996)), p15 (Robbins et al., J.Immunol.154:5944-5950 (1995)), white (the Robbins et al. of beta-catenin; J.Exp.Med., 183:1185-1192 (1996)), MUM-1 (Coulie et al Proc.Natl.Acad.Sci.USA, 92:7976-7980 (1995)), cell cycle protein dependent kinase-4 (CDK4) (Wolfel et al.; Science; 269:1281-1284 (1995)), p21-ras (Fossum et at., 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-Bl; Fisk et.al., J.Exp.Med., 181:2109-2117 (1995)), EGF-R ELISA (EGFR) (Harris et al.; Breast Cancer Res.Treat; 29:1-2 (1994)), carcinoembryonic antigen (CEA) (Kwong et al., J Natl.Cancer Inst., 85:982-990 (1995) United States Patent (USP) 5; 756,103; 5,274,087; 5,571,710; 6,071,716; 5,698,530; 6,045,802; EP 263933; EP 346710 and EP784483); The relevant sudden change of cancer mucin (is the MUC-1 gene outcome; Jerome et al., J.Immunol., 151:1654-1662 (1993)); The EBNA gene outcome of EBV (is EBNA-1; Rickinson et al., Cancer Surveys, 13:53-80 (1992)); Human papillomavirus's E7, E6 albumen (Ressing et al., J.Immunol.154:5934-5943 (1995)); PSA (PSA; Xue et al., The Prostate, 30:73-78 (1997)); PSMA (PSMA; Israeli etal., Cancer Res., 54:1807-1811 (1994)); Idiotype epi-position or antigen, for example immunoglobulin idiotype or TXi Baoshouti idiotype (Chen et al., J.Immunol., 153:4775-4787 (1994)); KSA (United States Patent (USP) 5; 348; 887), kinesin 2 (Dietz, et al.Biochem Biophys Res Commun 2000 Sep 7:275 (3): 731-8), anti-the die factor (Toomey, the et al.Br J Biomed Sci 2001 of transferring of HIP-55, TGF β-1; 58 (3): 177-83), oncoprotein D52 (Bryne J.A.et al.; Genomics, 35:523-532 (1996)), HIFT, NY-BR-1 (WO 01/47959), NY-BR-62, NY-BR-75, NY-BR-85, NY-BR-87, NY-BR-96 (Scanlan, M.Serologic andBioinformatic Approaches to the Identification of Human TumorAntigens; In Cancer Vaccines 2000; Cancer Research Institute, NewYork, NY); Comprise " wild type " (promptly by genome normal encoding, naturally occurring), its modification and mutant form, and other fragment and derivant.Any of these TAs can use separately or in immunization protocol altogether one unite use with another.

Preferred TAs is useful to the immunoreation of inducing the melanoma cell.Term " melanoma " for example includes but not limited to melanoma, metastatic melanoma, by melanocyte or the relevant cell-derived melanoma of nevus of melanocyte, melanotic cancer, melanocyte epithelioma, malignant melanoma, melanoma in situ, shallow spreading property of table melanoma, nodular melanoma, lentigo maligna melanoma, acra lentigo melanoma, the property invaded melanoma and family atypia nevus and melanoma (FAM-M) syndrome.Usually, melanoma improper organization's expression, gene expression change or carcinogen lopsided by for example chromosome, that move back the disorder of shape property g and D, mitogen, ultraviolet radiation (UV), viral infection, gene causes.

In some cases, maybe be useful with TA with other antigen such as the common immune patients of angiogenesis related antigen (" AA ").AA is and relates at blood vessel is induced and/or developmental again cell is relevant immunogenic molecules (being peptide, polypeptide).For example, AA can go up at endotheliocyte (" EC ") and express, and endotheliocyte is the primary structure composition of blood vessel.Under the situation of cancer, preferably in the blood vessel of tumor is provided or near find AA.Preferred patient's anti-AA immunity produces anti-AA immunoreation, prevents whereby and/or suppresses near the tumor or the inner angiogenesis that takes place.The AAs of example comprises that for example VEGF (is 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), vegf receptor (is VEGF-R, flk-1/KDR; Starnes, et al.J.Thorac.Cardiovasc.Surg, 2001,122 (3): 518-23), the EPH receptor (is EPHA2; Gerety, et al.1999, Cell, 4:403-414), EGF-R ELISA (is EGFR; Ciardeillo, et al.Clin.Cancer Res., 2001,7 (10): 2958-70), basic fibroblast growth factor (is bFGF; Davidson, et al.Clin.Exp.Metastasis2000,18 (6): 501-7; Poon, et al.Am J.Surg., 2001,182 (3): 298-304), platelet-derived cell growth factor be PDGF-B), platelet-derived ECGF (PD-ECGF; Hong, et al.J.Mol.Med., 2001,8 (2): 141-8), transforming growth factor (that is TGF-α; Hong, et al.J.Mol.Med., 2001; 8 (2): 141-8), endoglin (Balza, et al.Int.J.Cancer, 2001; 94:579-585), Id albumen (Benezra; R.Trends Cardiovasc.Med., 2001,11 (6): 237-41), protease such as uPA, uPAR and matrix metalloproteinase (MMP-2, MMP-9; Djonov, et al.J.Pathol., 2001; 195 (2): 147-55), nitricoxide synthase (Am.J.Ophthalmol., 2001,132 (4): 551-6), aminopeptidase (Rouslhati; E.NatureCancer, 2:84-90,2002), thrombospondin (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), Wnt (Zhang; Et al.Cancer Res., 2001,61 (16): 6050-4), cell cycle protein dependent kinase (CDKs; Drug Resist.Updat.2000,3 (2): 83-88), microtubule (Timar, et al.2001.Path.Oncol.Res., 7 (2): 85-94), heat shock protein (being HSP90 (Timar, above-mentioned)), heparin binding growth factor (be heparinase; Gohji; Et al.Int.J.Cancer; 2001,95 (5): 295-301), synthase (being atp synthase, thymidilate synthase), collagen receptor, integrin (being α v β 3, α v β 5, α 1 β 1, α 2 β 1, α 5 β 1), surface protein polysaccharide NG2, AAC2-1 or AAC2-2 etc., comprising " wild type " (promptly by the genome normal encoding, naturally occurring); Its modification, mutant form, and other fragment and derivant.In embodiment of the present invention, any in these targets maybe be all suitable, separately combine with one another or with other reagent associating.

Nucleic acid molecules can comprise nucleotide sequence or its fragment or the derivant of one or more immunogenicity targets of encoding, or is made up of them, like the sequence that contains in the DNA insert in the ATCC preservation thing.Term " nucleotide sequence " or " nucleic acid molecules " are meant DNA or RNA sequence.This term comprises the molecule that any known base analogue by DNA and RNA forms; Such as but not limited to 4-acetylcytosine, 8-hydroxy-n 6-methyladenosine, aziridinyl-cytosine, false iso-cytosine, 5-(carboxyl hydroxymethyl) uracil, 5-fluorouracil, 5-bromouracil, 5-carboxyl methylamino methyl-2-thiouracil, 5-carboxyl-methylamino methyluracil, dihydrouracil, inosine, N6-different-pentenyl adenine, 1-methyladenine, 1-methyl pseudouracil, 1-methyl guanine, 1-methylinosine, 2; 2-dimethyl-guanine, 2-methyladenine, 2-methyl guanine, 3-methylcystein, 5-methylcytosine, N6-methyladenine, 7-methyl guanine, 5-methylamino methyluracil, 5-methoxyl group amino-methyl-2-thiouracil, β-D-mannosylqueosine, 5-methoxycarbonyl-methyluracil, 5-methoxyuracil, 2-methyl sulfo--N6-isopentenyl gland purine, uracil-5-glycolic methyl ester, uracil-5-glycolic, oxybutoxosine, pseudouracil, queosine, 2-sulfur cytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, methyl uracil, N-uracil-5-glycolic methyl ester, uracil-5-glycolic, pseudouracil, queosine, 2-sulfur cytosine and 2,6-diaminopurine etc.

Isolated nucleic acid molecule is (1) when TNA separates from derived cell, the nucleic acid molecules that is separated with the albumen, lipid, carbohydrate or other material that therewith find under about at least 50% the natural situation; (2) not with natural situation under the nucleic acid molecules of all or part of connection of the polynucleotide that this nucleic acid molecules was connected; (3) with natural situation under the polynucleotide that the are not attached thereto nucleic acid molecules that can be operatively connected; The nucleic acid molecules that the part of the polynucleotide sequence that (4) conduct is not bigger under the natural situation exists.Preferably, isolated nucleic acid molecule of the present invention be substantially free of any other contaminated nucleic acid molecule or in its natural environment, find will disturb the purposes of said nucleic acid molecules in polypeptide preparation or other pollutant of its treatment, diagnosis, prevention or research purposes.Term " naturally occurring (naturally occuring) " or " natural (native) " or " natural discovery " in this use; When with relevant use such as biomaterial such as nucleic acid molecules, polypeptide, host cell, be meant find under the nature situation with not by the material of people's operation.Similarly, be meant in " non-natural exists " or " non-natural " of this use and be not the material of finding under the nature situation or carried out structural modification or synthetic material by people.

The homogeneity of two or more nucleic acid or aminoacid sequence (identity) is confirmed through comparative sequences.As known in the art, " homogeneity " is the relevant degree of sequence between the nucleic acid confirmed through the pairing between the unit that forms this molecule (being nucleotide or amino acid residue) or the aminoacid sequence.Identical paired percentage ratio between the less sequence of two or more sequences of homogeneity mensuration, wherein breach comparison (if any) obtains through specific mathematical pattern or computer program (being algorithm).Homogeneity between the nucleotide sequence can also be confirmed through the ability that nucleotide sequence is hybridized each other.In limiting hybridizing method, term " height stringent condition " and " medium stringent condition " are meant and allow the complementary nucleic acid chains hybridization of sequence, and get rid of the condition of the hybridization of remarkable mispairing nucleic acid.Instance for " height stringent condition " of hybridization and washing is at 65-68 ℃, 0.015M sodium chloride, 0.0015M sodium citrate or at 42 ℃, 0.015M sodium chloride, 0.0015M sodium citrate and 50% Methanamide.(referring to for example Sambrook, Fritsch & Maniatis, Molecular Cloning:A Laboratory Manual (2nd ed., Cold SpringHarbor Laboratory, 1989); Anderson et al., N ucleic AcidHybridisation:A Practical Approach Ch.4 (IRL Press Limited)).Term " medium stringent condition " is meant the condition that can form than at " height stringent condition " the dna double spiral that contingent base-pair mismatch degree is bigger down.The medium stringent condition of example is at 50-65 ℃, 0.015M sodium chloride, 0.0015M sodium citrate or at 37-50 ℃, 0.015M sodium chloride, 0.0015M sodium citrate and 20% Methanamide.For instance, at 50 ℃, the medium stringent condition in the 0.015M sodium ion will allow about 21% mispairing.In the crossover process,, can comprise other reagent in hybridization and the lavation buffer solution in order to reduce the purpose of non-specific and/or background hybridization.Instance is 0.1% bovine serum albumin, 0.1% polyvinylpyrrolidone, 0.1% tetrasodium pyrophosphate, 0.1% sodium lauryl sulphate, NaDodSO ₄, (SDS), ficoll, Denhardt ' s solution, supersound process salmon sperm DNA (or another non-complementary DNA) and dextran sulfate, although can also use other suitable reagent.The concentration of these additives and type can change, and do not influence the stringency of hybridization conditions basically.Hybrid experiment carries out at pH6.8-7.4 usually; Yet under typical ionic strength conditions, hybridization speed is almost irrelevant with pH.

In a preferred embodiment of the invention, carrier is used for the nucleotide sequence of coding immunogenicity target is transferred to cell.Carrier is any molecule that is used for nucleotide sequence is transferred to host cell.In some cases, utilize expression vector.Expression vector is to be suitable for the conversion of host cell and to contain guidance and/or the nucleic acid molecules of the nucleotide sequence of the expression of control institute transfer nucleic acid sequence.Expression includes but not limited to various procedures, as transcribes, translates, and montage, if there is intron.Expression vector typically comprises one or more flanking sequences that the heterologous nucleic acid sequence with coded polypeptide can be operatively connected.Flanking sequence can be for example homologous (promptly from species identical with host cell and/or bacterial strain), allogenic (promptly from the species outside host cell species or the bacterial strain), the hybrid combination of flanking sequence of source more than (promptly from), or synthetic.

Preferred flanking sequence can be realized duplicating, transcribing and/or translating of (effect) coded sequence and can be operatively connected with coded sequence.Can be operatively connected at the term of this use and to be meant of the connection of polynucleotide element with functional relationship.For example, promoter or enhancer and coded sequence can be operatively connected, if it influences transcribing of coded sequence.Yet flanking sequence need not be close to coded sequence, as long as its correct performance function.Therefore, for example, insertion by translation but do not may reside between promoter sequence and the coded sequence by the sequence of being transcribed, promoter sequence still can be considered to can be operatively connected with coded sequence.Similarly, enhancer sequence can be positioned at the coded sequence upper reaches or downstream and influence transcribing of this sequence.

In certain embodiments, preferred flanking sequence is the transcriptional regulatory district that drives high-level gene expression in the target cell.The transcriptional regulatory district can comprise for example promoter, enhancer, silencer, resistance and meet construction element (repressor element) or its combination.The transcriptional regulatory district can be composing type, tissue-specific, cell type-specific (promptly compare with another kind tissue or cell type, higher level that this zone drives in a kind of tissue or cell type is transcribed) or adjustable (promptly to the responding property of interaction of chemical compound such as tetracycline).The source in transcriptional regulatory district can be any protokaryon or eukaryote, any vertebrates or invertebrates or any plant, condition be this flanking sequence through causing intracellular nucleic acid to transcribe in cell functionating.In embodiment of the present invention, can utilize transcriptional regulatory miscellaneous district.

Suitable transcriptional regulatory district comprises CMV promoter (being upright (immediateearly) promoter early of CMV); Promoter from eukaryotic gene (being estrogen-induced type chicken ovalbumin gene, interferon gene, glucocorticoid inducible type TAT gene and thymidine kinase gene); With adenoviral gene promoter in main early stage and late period; SV40 early promoter district (Bernoist and Chambon, 1981, Nature 290:304-10); The promoter (Yamamoto, et al., 1980, Cell 22:787-97) that contains among 3 ' long terminal repetition (LTR) of rous sarcoma virus (RSV); Herpes simplex virus thymidine kinase (HSV-TK) promoter (Wagner et al., 1981, Proc.Natl.Acad.Sci.U.S.A.78:1444-45); The adjusting sequence of metallothionein gene (Brinster et al., 1982, Nature 296:39-42); Prokaryotic expression carrier such as beta-lactamase promoter (Villa-Kamaroff et al., 1978, Proc.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 specificity transcripting controling area comprise for example activated elastoser 1 gene-controlled area (Swift et al., 1984, Cell 38:639-46 in pancreatic acinar cell; Omitz et al., 1986, Cold SpringHarbor Symp.Quant.Biol.50:399-409 (1986); MacDonald, 1987, Hepatology 7:425-515); Activated insulin gene control zone in pancreatic beta cell (Hanahan, 1985, Nature 315:115-22); Activated immunoglobulin gene control zone (Grosschedl et al., 1984, Cell 38:647-58 in lymphoid cell; Adameset al., 1985, Nature 318:533-38; Alexander et al., 1987, Mol.Cell.Biol., 7:1436-44); Mice mastoncus virus control zone (Leder et al., 1986, Cell 45:485-95) in testis, mammary gland, lymph appearance and mastocyte; Albumin gene control zone in the liver (Pinkert et al., 1987, Genes and Devel.1:268-76); α in the liver-feto-protein gene-controlled area (Krumlauf et al., 1985, Mol.Cell.Biol., 5:1639-48; Hammer et al., 1987, Science 235:53-58); Alpha1-antitrypsin gene-controlled area in the liver (Kelsey et al., 1987, Genes and Devel.1:161-71); Beta-globin gene-controlled area in the myeloid cell (Mogram et al., 1985, Nature 315:338-40; Kollias et al., 1986, Cell 46:89-94); MBP gene-controlled area in the brain in the oligodendroglia (Readhead et al., 1987, Cell 48:703-12); Myosin light chain 2 gene-controlled areas in the skeletal muscle (Sani, 1985, Nature 314:283-86); Tryrosinase promoter (Hart, I.Semin Oncol 1996 Feb in gonadotropin releasing hormone gene-controlled area in the hypothalamus (Mason et al., 1986, Science 234:1372-78) and the melanoma cells; 23 (1): 154-8; Siders, et al.Cancer Gene Ther 1998 Sep-Oct; 5 (5): 281-91) etc.Can also use at some chemical compound or condition activatory inducible promoter (referring to for example WO00/10612) in the presence of light, heat, radiation, tetracycline or the heat shock protein for example.Other suitable promoter is known in the art.

As stated, enhancer also can be suitable flanking sequence.Enhancer is the cis acting element that acts on the DNA that promoter transcribes with increase, the about 10-300bp of normal length.Enhancer typically is that direction and position are not dependent, 5 of controlled encoding sequence ' with 3 ' all identified enhancer.Known several enhancer sequence (being globin, elastoser, albumin, α-feto albumen and insulin) that can obtain from mammalian genes.Similarly, SV40 enhancer, the sub-enhancer of cytomegalovirus early promoter, polyoma enhancer and adenovirus enhancer are useful to the promoter in eukaryote sequence.Though enhancer can be in 5 of nucleic acid coding sequence ' or 3 ' the position montage go into carrier, typically it be positioned at 5 of promoter ' the site.Other suitable enhancer is known in the art, and will be applicable to the present invention.

When preparation reagent of the present invention, cell maybe be by transfection or conversion.Transfection is that phalangeal cell externally comes or the picked-up of exogenous DNA, and when exogenous DNA has been imported into cell membrane when inboard, cell is by transfection.A lot of rotaring dyeing technologies be well known (be Graham etal., 1973, Virology 52:456; Sambrook et al., Molecular Cloning, ALaboratoty Manual. (Cold Spring Harbor Laboratories, 1989); Daviset al., Basic Methods in Molecular Biology, (Elsevier, 19S6); With Chuet al., 1981, Gene 13:197).This technology can be used for one or more exogenous DNA are partly imported proper host cell.

In certain embodiments, the transfection of preferred cell causes those transformations.When the characteristic of cell changed, cell was transformed, and when it was contained novel nucleic acids by modification, cell was transformed (being transformed).After the transfection, the nucleic acid of transfection can through physics be integrated into cell chromosome and with the reorganization of the nucleic acid of cell, can be used as free of short duration the keeping of type element and be not replicated, or it is independently duplicated to can be used as plasmid.When nucleic acid duplicated with the division of cell, cell was by stable conversion.

Expression vector of the present invention also provides the segmental expression of immunogenicity target.Fragment can be included in the sequence that amino terminal (being with or without targeting sequencing) and/or carboxyl terminal block.Fragment also can comprise variant (promptly homotopic, montage), straight to congener, congener with compare other variant with one or more aminoacid addition or displacement or inner disappearance with parental array.In preferred embodiments, truncate and/or disappearance comprise about 1-5 aminoacid, a 5-10 aminoacid, a 10-20 aminoacid, a 20-30 aminoacid, a 30-40 aminoacid, a 40-50 aminoacid or more.This polypeptide fragment can be chosen wantonly and comprise the amino terminal methionine residues.This fragment should be understood and antibody or cell immune response can be used for for example producing to the immunogenicity target.

Variant is to compare the sequence that has one or more sequence displacements, disappearance and/or add with subject nucleotide sequence.Variant can be naturally occurring or artificial constructed.Can prepare this variant from the corresponding nucleic acids molecule.In preferred embodiments, variant has 1 to 3, or 1 to 5, or 1 to 10, or 1 to 15,1 to 20, or 1 to 25,1 to 30, or 1 to 40, or 1 to 50, or 50 above amino acid replacements, insertion, interpolation and/or disappearances.

Allelic variant be occupy sequence several of given locus on biology or the biotic population chromosome one of maybe naturally occurring replacement forms.Splicing variants is the polypeptide that produces from one of several rna transcription things that produced by the primary transcript montage.Straight is similar nucleotide sequence or peptide sequence from another species to congener.For example, mice and people's immunogenicity target can be considered to directly to congener each other.The sequence derivant is those sequences with displacement, interpolation, disappearance or chemical modification variation that obtained by parental array.Variant also can comprise fusion rotein, and it is meant that one or more first sequences (like peptide) are in the amino of at least one other sequence (like heterologous peptides) or the fusion of carboxyl terminal.

" similarity " is the notion relevant with homogeneity, the measurement of dependency comprises the identical pairing and the pairing of conservative substitution except similarity is meant.If two peptide sequences for example have 10/20 identical aminoacid, and remaining all is non-conservative substitution, and homogeneity and similarity percentage ratio will all be 50% so.If in same instance, also have on five positions conservative substitution is arranged, homogeneity percentage ratio remains 50% so, but similarity percentage ratio will be 75% (15/20).Therefore, having under the situation of conservative substitution, the similarity percentage ratio between two polypeptide will be higher than the homogeneity percentage ratio between those two polypeptide.

Displacement can be that guard or nonconservative or its any combination.The conserved amino acid of peptide sequence is modified (with the corresponding modification of coding nucleotide) and can be produced and have the function that is similar to parent's polypeptide and the polypeptide of chemical feature.For example; " conservative amino acid replacement " can comprise with non-natural residue displacement natural amino acid residue; So that size, polarity, electric charge, hydrophobicity or hydrophilic to the amino acid residue of that position have seldom or not influence, do not cause that especially immunogenicity reduces.Table I shows suitable conservative amino acid replacement.

Table I

Original residue	The displacement of example	Preferred displacement
			Ala	Val，Len，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, nor-leucine	Leu
			Leu	Nor-leucine, Ile, Val, Met, Ala, Phe	Ile
Lys	Arg, 1,4 diaminourea-butanoic 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, nor-leucine	Leu

Those skilled in the art can use and know the suitable variant that technology is confirmed the immunogenicity target.Do not destroy the appropriate area of biological activity (be MHC combine, immunogenicity) in order to identify can changing of this molecule, those skilled in the art can targeting those be considered to the unessential zone of the sort of activity.For example, during from same species or from the similar active immne originality of having of other species target, those skilled in the art can compare amino acid sequence of polypeptide and this similar polypeptide when known.Through carrying out this analysis, people can identify the residue and the part of conservative molecule.The change in the zone not conservative with respect to this similar immunogenicity target that should understand this molecule is with littler biological activity and/or the structure that influences polypeptide possibly unfriendly.Similarly, be known in conjunction with the required residue of MHC, and can be modified to improve combination.Yet, as a rule, cause that the modification that combines reduction with MHC is with inappropriate.Those skilled in the art also will know, even in conservative relatively zone, people also can use chemically the similarly naturally occurring residue of amino acid replacement, keep active simultaneously.Therefore, even also can stand conservative amino acid replacement, and not destroy the biological activity of immunogenicity target or can influence the structure of immunogenicity target sharply for biological activity or for the zone that weight of structure is wanted.

Other preferred polypeptide variant comprises the glycosylation variant, and wherein the quantity at glycosylation position and/or type are compared with the subject amino acid sequence and be changed.In one embodiment, polypeptide variants comprises the N linked glycosylation position than the more or less quantity of subject amino acid sequence.The characteristics at N linked glycosylation position (N-linked glycosylation site) are sequence A sn-X-Ser or Asn-X-Thr, and the amino acid residue that wherein is expressed as X can be any amino acid residue except that proline.The radical amino acid replacement that produces this sequence provides and has added the potential new position that N joins sugar chain.Perhaps, eliminate the N couplet sugar chain that this PERMUTATION OF SEQUENCES will be removed existence.The rearrangement that also provides N to join sugar chain, wherein one or more N linked glycosylation positions (typically being naturally occurring position) are eliminated and produce one or more new N and join the position.For influencing the O linked glycosylation of polypeptide, people will modify serine and/or threonine residues.

Preferred variant in addition comprises the cysteine variant, wherein compares with theme (subject) aminoacid sequence group, and one or more cysteine residues are deleted or by another amino acid replacement (like serine).When peptide or polypeptide must be folded into biology during activated conformation again, after insoluble inclusion bodies separating, the cysteine variant is useful.The cysteine variant has than native protein cysteine residues still less usually, typically has even number so that the interaction that not paired cysteine causes reduces to minimum.

In other embodiments, peptide or polypeptide can be connected with the one or more fusion fragments that help peptide purification.Can merge at the amino terminal or the carboxyl terminal of theme polypeptide variants.Can there be the direct fusion of joint or adapter molecule maybe can merge through joint or adapter molecule.Joint or adapter molecule can be one or more amino acid residues, typically about 20 to about 50 amino acid residues.Joint or adapter molecule also can be designed with the cleavage site of DNA restriction endonuclease or protease to allow to merge the separation of part.Should understand fused polypeptide and in a single day be fabricated, just can come derivatization according to method described here.Suitable fusion fragment comprises melts combine domain (like the polyhistidyl fragment); Immune globulin binding structural domain (is an A albumen; G albumen; The T cell; The B cell; Fc receptor or complement protein antibodies domain); Sugar binding structural domain (like the maltose binding structural domain) and/or " tag " domain (are at least a portion of alpha-galactosidase; Streptococcus labelling peptide (a strep tagpeptide); T7 labelling peptide; The FLAG peptide or use to combine other domain that chemical compound such as the monoclonal antibody of this domain can purification) etc.This labelling (tag) typically merges with peptide or polypeptide, and can be as the instrument from host cell affinity purification polypeptide of interest sequence after expressing.Affinity purification can be accomplished as the column chromatography of affinity substrate through for example using anti-tag antibody.Optional, in all sorts of ways subsequently as using some peptidase to cut, from the polypeptide of interest sequence of purification, remove this labelling.Be described below, for example also can and be total between stimulation component such as chemotactic factor CXC10 (IP-10), CCL7 (MCP-3) or the CCL5 (RANTES) and merge at TA.

Merge motif (motif) and can the enhances immunogenicity target process the transportation of compartment such as endoplasmic reticulum to MHC.Being called as these sequences that the transduction or the anuria during pregnancy gulp down sequence comprises and derives from HIVtat (referring to Kim et al.1997 J.Immunol.159:1666), Drosophila antennapedia (referring to Schutze-Redelmeier et al.1996 J.Immunol.157:650) or people 1 phase (period-1) albumen (hPER1; Especially the sequence of (SEQ ID NO.:17) SRRHHCRSKAKRSRHH).

In addition, polypeptide or its variant can merge the formation homodimer or merge the formation heterodimer with heterologous peptides or polypeptide with homeopeptide or polypeptide.Heterologous peptides and polypeptide include but not limited to allow to detect and/or separate the epi-position of fused polypeptide; Transmembrane receptor protein or its part such as ectodomain or stride film and born of the same parents' intracellular domain; With the bonded part of transmembrane receptor protein or its part; Enzyme or its part with catalytic activity; Promote polypeptide or the peptide such as the leucine zipper motif of oligomerization; Increase polypeptide or the peptide such as the constant region for immunoglobulin of stability; Peptide or polypeptide with the therapeutic activity that is different from this peptide or polypeptide; And/or its variant.

In certain embodiments, in the present composition, the nucleotide sequence of coding immunogenicity target and the combination of one or more stimulation component altogether maybe be useful, like cell surface protein, cytokine or chemotactic factor.Altogether stimulation component can be for example be included in the compositions as polypeptide or as the nucleic acid of this polypeptide of coding.Suitable costimulatory molecules comprise for example combine the CD28 family member (be CD28, ICOS; Hutloff, et al.Nature 1999,397:263-265; Peach, et al.J Exp Med 1994, polypeptide 180:2049-2058) combines polypeptide B7.1 (CD80 like CD28; Schwarz, 1992; Chen et al, 1992; Ellis, et al.J.Immunol., 156 (8): 2700-9), B7.2 (CD86; Ellis, et al.J.Immunol., 156 (8): 2700-9) with its mutant/variant (WO 00/66162); Integrin binding family member's polypeptide (is LFA-1 (CD 11a/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), comprises ICAM family member (being ICAM-1 ,-2 or-3); In conjunction with CD2 family member's polypeptide (be CD2, signal lymphocyte activation molecule (CDw150 or " SLAM "; Aversa, et al.J Immunol 1997,158:4036-4044)) like CD58 (LFA-3; The CD2 part; Davis, et al.Immunol Today 1996,17:177-187) or the SLAM part (Sayos, et al.Nature 1998,395:462-469); Polypeptide (HSA or CD24 in conjunction with heat stable antigen; Zhou, et al.Eur J Immunol 1997,27:2524-2528); Polypeptide in conjunction with TNF receptor (TNFR) family member (is 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)) like 4-1BBL (4-1BB part; Vinay, et al.Semin Immunol 1998,10:481-48; DeBenedette, et al.J Immunol 1997,158:551-559), TNFR correlation factor-1 (TRAF-1; The 4-1BB part; 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 part; Saoulli, et al.J Exp Med 1998,187:1849-1862; Oshima, et al.Int Immunol 1998,10:517-526, Kawamata, et al.J BiolChem 1998,273:5808-5814), TRAF-3 (4-1BB and OX40 part; Arch, et al.Mol Cen Biol 1998,18:558-565; Jang, et al.Biochem BiophysRes Commun 1998,242:613-620; Kawamata S, et al.J Biol Chem1998,273:5808-5814), OX40L (OX40 part; Gramaglia, et al.JImmunol 1998,161:6510-6517), TRAF-5 (OX40 part; Arch, et al.Mol Cell Biol 1998,18:558-565; Kawamata, et al.J Biol Chem 1998,273:5808-5814) and CD70 (CD27 part; Couderc, et al.Cancer GeneTher., 5 (3): 163-75).CD 154 (CD40 part or " CD40L "; Gurunathan, et al.J.Immunol., 1998,161:4563-4571; Sine, et al.Hum.Gene Ther., 2001,12:1091-1102) maybe be also suitable.

One or more cytokines also possibly be suitable common stimulation component or " adjuvants ", the polypeptide that contains as the present composition or by the nucleic acid coding that contains (Parmiani, et al.Immunol Lett 2000 Sep 15; 74 (1): 41-4; Berzofsky, et al.NatureImmunol.1:209-219).Suitable cytokine comprises for example interleukin-2 (IL-2) (Rosenberg, et al.Nature Med.4:321-327 (1998)), IL-4, IL-7, IL-12 (summary, 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. 2001.127 (12): 718-726), GM-CSF (CSF (Disis; Et al.Blood, 88:202-210 (1996)), tumor necrosis factor-alpha (TNF-α) or interferon such as IFN-α or IFN-γ.Other cytokine also possibly be suitable for embodiment of the present invention, as known in the art.

Also can use chemotactic factor, with polypeptide or nucleic acid form.Shown the fusion rotein inducing antitumor immunity that comprises CXCL10 (IP-10) and CCL7 (MCP-3) that merges with the tumor autoantigen (Biragyn, et al.Nature Biotech.1999,17:253-258).Chemotactic factor CCL3 (MIP-1 α) and CCL5 (RANTES) (Boyer, et al.Vaccine, 1999,17. (Supp.2): S53-S64) maybe be also useful in embodiment of the present invention.Other suitable chemotactic factor is known in the art.

Inhibition also known in the art or negativity are regulated immunologic mechanism and can be blocked, and cause enhanced immunoreation.For example, (Shrikant, et al.Immunity, 1996,14:145-155 have been shown with anti-CTLA-4; Sutmuller, et al.J.Exp.Med., 2001,194:823-832), anti-CD25 (Sutmuller; Above-mentioned), anti-CD4 (Matsui, et al.J.Immunol., 1999; 163:184-193), fusion rotein IL13Ra2-Fc (Terabe, et al.NatureImmunol., 2000; 1:515-520) and combination (promptly anti-CTLA-4 and anti-CD25, Sutmuller, above-mentioned) treatment raise anti tumor immune response and will be suitable for embodiment of the present invention.Especially this treatment also can be united with one or more immunogenicity targets of the present invention.

Any in these components can be united use separately or with other reagent.For example, the combination that has shown CD80, ICAM-1 and LFA-3 (" TRICOM ") can strengthen antitumor immune reaction (Hodge, et al.Cancer Res.59:5800-5807 (1999).Other effectively makes up and comprises 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-α (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. is above-mentioned) and CD86+GM-CSF+IL-12 (Iwasaki, above-mentioned).Those skilled in the art will know the useful other combination of embodiment of the present invention.In addition, those skilled in the art will know the other reagent or the method that can be used for regulating this mechanism.In embodiment of the present invention, can use these reagent and method, and other reagent well known by persons skilled in the art and method.

Also can use the other strategy of raising, comprise and for example use self replication virus replication (Caley, et al.1999.Vaccine, 17:3124-2135 based on the efficient of the immunity of nucleic acid; Dubensky, et al.2000.Mol.Med.6:723-732; Leitner, et al.2000. Cancer Res.60:51-55), codon optimized (Liu, et al.2000.Mol.Ther., 1:497-500; Dubensky, above-mentioned; Huang; Et al.2001.J.Virol.75:4947-4951), electroporation (Widera, et al.2000.J.Immunol.164:4635-3640), CpG stimulate (Gurunathan, the et al.Ann.Rev.Immunol. of mixing of motif in the body; 2000,18:927-974; Leitner, above-mentioned; Cho, et al.J.Immunol.168 (10): 4907-13), sequence (Thomson, the et al.1998.J.Virol.72:2246-2252 of targeting endocytosis or ubiquitin processing approach; Velders, et al.2001.J.Immunol.166:5366-5373), the sick viral 1 type VP22 sequence (J.Virol.76 (6): 2676-82,2002) of Marek, excite-strengthened scheme (prime-boost regimens) (Gurunathan, above-mentioned; Sullivan, et a.2000.Nature, 408:605-609; Hanke, et al.1998.Vaccine, 16:439-445; Amara, et al.2001.Science is 292:69-74) with use mucosal delivery carrier such as Salmonella (Darji, et al.1997.Cell, 91:765-775; Woo, et al.2001.Vaccine, 19:2945-2954).Other method is known in the art, and some of them are described below.

Also can utilize chemotherapeutics, ray, anti-angiogenic compounds or other reagent (Sebti, et al.Oncogene 2000 Dec27 in the cancer using the immunogenicity target to treat and/or prevent; 19 (56): 6566-73).For example, in the treatment metastatic melanoma, suitable chemotherapy regimen can comprise BELD (bleomycin, vindesine, CCNU, 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, D actinomycin D; Hochster, et al.Cancer TreatmentReports, 69:39-42) or POC (procarbazine, vincristine, lomustine; Carmo-Pereira, et al.1984.Cancer Treatment Reports, 68:1211-4) etc.Also can use other suitable chemotherapy regimen.

Many angiogenesis inhibitor reagent be known in the art and will be suitable for using jointly with immunogenicity target vaccine and/or chemotherapy regimen (referring to for example Timar, et al.2001.PathologyOncol.Res., 7 (2): 85-94).This reagent for example comprises that physiology reagent such as somatomedin (are ANG-2; NK1; 2; 4 (HGF) transform growth because of in β (TGF-β)), cytokine (be interferon such as IFN-α ,-β ,-γ, platelet factor 4 (PF-4), PR-39), protease (AT-III, collagen XVIII fragment (Endostatin), HmwKallikrein-d5 fibrinolysin fragment (Angiostatin), thrombinogen-F1-2, the TSP-1 that promptly cut), protease inhibitor (is the tissue depressant such as the TIMP-1 ,-2 or-3 of metalloproteases; Maspin; PAI such as PAI-1; Pigment epidermal derived factors (PEDF)), Tumstatin (through ILEX, Inc. can obtain), antibody product (are collagen binding antibody HUIV26, HUI77, XL313; Anti-VEGF; Anti-alpha 2 integrin (being Vitaxin, (Lxsys))) and glycosidase (be heparinase-I ,-III).Known or be considered to have " chemical reagent " of angiogenesis inhibitor potentiality or the physiology reagent of modifying; Comprise for example vinblastine, taxol, ketoconazole, Sa Li polyamines, dolestatin, combrestatin A, rapamycin (Guba; Et al.2002; Nature Med.; 8:128-135), CEP-7055 (can be from Cephalon; Inc. acquisition), flavone acetic acid, Bay 12-9566 (Bayer Corp.), AG3340 (Agouron; Inc.), CGS 27023A (Novartis), tetracylcine derivant (are COL-3 (Collagenix; Inc.)), chemical compound (being SU5416 or SU6668 (Sugen), PTK787/ZK22584 (Novartis)), Telomycin A, Angiozyme (ribozyme), isoflavinoids, staurosporine derivatives, genistein, EMD121974 (Merck KcgaA), tyrphostins, isoquinolones, tretinoin, carboxyamidotriazole, TNP-470, Sandostatin LAR Depot, 2-methoxyestradiol, aminosterols (being squalamine), glutathione analogs (being N-acetyl-L-cysteine), combretastatin A-4 (Oxigene), the Eph receptor blocking agent (Nature of Neovastat (Aeterna), BMS-275291 (Bristol-Myers Squibb), low dosage 5-FU, low dosage methotrexate (MTX), irsofladine, radicicol, cyclosporine, captopril, celecoxib, D45152 sulfated polysaccharides, cationic protein (protamine), cationic peptide VEGF, suramin (polysulphonated napthyl urea), interference VEGF function or output; 414:933-938; 2001), Rh-Angiostatin, Rh-Endostatin (WO 01/93897), ring-type RGD peptide, accutin-disintegrin, benzodiazepine , the anti-avb3 Ab of humanization, Rh-PAI-2, amiloride, p-amidobenzamidine, anti-uPA ab, anti-uPAR Ab, L-phanylalanin-N-methylamides (are Batimistat, Marimastat), AG3340 and minocycline.Many other suitable reagent are known in the art, and will be enough to put into practice the present invention.

The present invention also can unite use with " non-traditional " method of treatment cancer.For example, proved that recently giving some anaerobe can help to slow down tumor growth.In a research; Nuo Shi clostridium (Clostridium novyi) is modified removes the toxin gene that carries on the phage episome (episome) and is given the mice (Dang with colorectum tumor; Et al.P.N.A.S.USA, 98 (26): 15155-15160,2001).With chemotherapy combined, show that this treatment causes neoplasm necrosis in animal.Reagent of describing among the application and method can be united with this Therapeutic Method.

The nucleic acid of coding immunogenicity target can give the patient through any in several kinds of technology capable of using.The various viral vector that successfully have been used for nucleic acid is introduced the host comprise retrovirus, adenovirus, adeno-associated virus (AAV) (AAV), herpesvirus and poxvirus etc.This area is understood many in the art this viral vector and can be utilized.Carrier of the present invention can use the extensive available standard reorganization technique construction of those skilled in the art.In common molecular biology list of references, can find this technology, like Molecular Cloning:A LaboratoryManual (Sambrook, et al.; 1989, Cold Spring Harbor LaboratoryPress), 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).

Preferred retroviral vector is slow virus (lentivirus) derivant and Mus and the retroviral derivant of birds.Suitable retroviral vector instance comprises for example Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV), molluscum contagiosum adenoma virus (MuMTV), SIV, BIV, HIV and rous sarcoma virus (RSV).A lot of retroviral vectors can merge (incorporate) a plurality of exogenous nucleic acid sequences.Because recombinant retrovirus is defective, therefore in order to produce the infectiousness carrier granular, they need be assisted.This auxiliary can providing through the for example auxiliary cell line of encoding hiv reverse transcriptase structural gene.Suitable auxiliary cell line comprises Ψ 2, PA317 and PA12 etc.The vector virus particle that uses this cell line to produce then can be used for infected tissue's cell line, like the NIH3T3 cell to produce a large amount of chimeric retrovirus particles.Retroviral vector can give (Culver, K., et al., 1994, Hum.Gene Ther., 5 (3): 343-79 through traditional method (i.e. injection) or through the implantation near target cell crowd " production cell line "; Culver, K., et al., Cold Spring Harb.Symp.Quant.Biol., 59:685-90); Oldfield, E., 1993, Hum.Gene Ther., 4 (1): 39-69).Production cell line (producer cell line) is transformed to produce viral vector and near releasing virus granule target cell.The virion contact target cell that a part discharges also infects those cells, thus with delivery of nucleic acids of the present invention to target cell.After target cell infected, the expression of vector nucleic acid took place.

Proved that adenovirus vector is for gene transfer being gone into eukaryotic cell (Rosenfeld, M., etal., 1991, Science, 252 (5004): 431-4; Crystal, R., etal., 1994, Nat.Genet., 8 (1): 42-51), the research (Levrero of eukaryotic gene expression; M., et al., 1991, Gene, 101 (2): 195-202), vaccine development (Graham; F.and Prevec, L., 1992, Biotechnology, 20:363-90) with animal model in (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) particularly useful.The experimental approach of different tissues in the reorganization Ad donor is comprised intratracheal instillation (Rosenfeld, M., et al., 1992, Cell; 68 (1): 143-55), intramuscular injection (Quantin, B., et al., 1992, Proc.Natl.Acad.Sci.U.S.A.; 89 (7): 2581-4), injection (Herz, J., and Gerard, R. in the peripheral vein; 1993, Proc.Natl.Acad.Sci.U.S.A., 90 (7): 2812-6) inoculate (stereotactic inoculation) to brain (LeGal La Salle, G. with the entity that becomes; Et al., 1993, Science, 259 (5097): 988-90) etc.

Adeno-associated virus (AAV) (AAV) demonstrates high-level infectiousness, host range and be integrated into specificity (Hermonat, P., et al., 1984, Proc.Natl.Acad.Sci.U.S.A., 81 (20): 6466-70) of host cell gene group widely.And herpes simplex virus type 1 (HSV-1) is another attracting carrier system, especially owing to its neurotropism can be used for nervous system (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).

Poxvirus is another kind of 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).Prove that useful poxvirus comprises cowpox, NYVAC, fowl pox (avipox), bird pox (fowlpox), canary pox (canarypox), ALVAC and ALVAC (2) etc.

NYVAC (vP866) stems from Copenhagen vaccine strain of vaccinia virus, through deleting six nonessential regions (referring to for example United States Patent (USP) 5,364,773 and 5,494,807) of the known or potential virulence factor of genomic coding.The missing gene seat is also transform as the receptor gene seat that inserts exogenous gene.The disappearance zone is: thymidine kinase gene (TK; J2R); Hemorrhage district (u; B13R+B14R); The A type is forgiven tagma (ATI; A26L); Hemagglutination plain gene (HA; A56R); Host range gene district (C7L-K1L) and big subunit, ribonucleotide reductase (I4L).NYVAC is the vaccinia strain of genetic modification, is 18 ORFs generations through the specificity disappearance coding gene outcome relevant with toxicity and host range.Proved that NYVAC can be used for expressing TAs (referring to for example United States Patent (USP) 6,265,189).NYVAC (vP866), vP994, vCP205, vCP1433, placZH6H4Lreverse, pMPC6H6K3E3 and pC3H6FHVB also are deposited in ATCC according to the condition of budapest treaty, and preserving number is respectively VR-2559, VR-2558, VR-2557, VR-2556, ATCC-97913, ATCC-97912 and ATCC-97914.

Recombinant virus (being ALVAC-1 and ALVAC-2) based on ALVAC also is suitable for embodiment of the present invention (referring to for example United States Patent (USP) 5,756,103).ALVAC (2) is identical with ALVAC (1), except ALVAC (2) genome comprises cowpox E3L and K3L gene (United States Patent (USP) 6,130,066 under the control of cowpox promoter; Beattie et al., 1995a, 1995b, 1991; Chang et al., 1992; Davies et al., 1993).Proved that ALVAC (1) and ALVAC (2) can be used for expressing exogenous DNA array, like TAs (Tartaglia et al., 1993 a, b; United States Patent (USP) 5,833,975).ALVAC is deposited in American type culture collection (ATCC) according to the condition of budapest treaty, 10801 University Boulevard, Manassas, Va.20110-2209, the U.S., ATCC preserving number VR-2547.

Another kind of useful poxvirus vector is TROVAC.TROVAC is meant the bird pox of attenuation, is to derive from by the plaque clone and separate thing of permission to the FP-1 vaccine strain of the avipoxvirus of the little chicken inoculation of 1 age in days.TROVAC is deposited in ATCC according to the condition of budapest treaty equally, preserving number 2553.

" non-virus " plasmid vector possibly also be suitable for embodiment of the present invention.Preferred plasmid vector is compatible with antibacterial, insecticide and/or mammalian host cell.This carrier for example comprise PCR-II, pCR3 and pcDNA3.1 (Invitrogen, San Diego, CA), pBSII (Stratagene, La Jolla; CA), pET15 (Novagen, Madison, WI), pGEX (PharmaciaBiotech, Piscataway; NJ), pEGFP-N2 (Clontech, Palo Alto, CA), pETL (BlueBacII; Invitrogen), pDSR-α (PCT publication number WO 90/14363) and pFastBacDual (Gibco-BRL, Grand Island, NY) and The plasmid derivative thing (high copy number is based on the phasmid of COLEl, Stratagene CloningSystems, and La Jolla, CA), be the PCR cloned plasmids of clone Taq amplification PCR products design (TOPO for example ^TMTA

Kit,

Plasmidderivatives, Invitrogen, Carlsbad, CA).Bacteria carrier also can use with the present invention.These carriers comprise that for example shigella (Shigella), Salmonella (Salmonella), vibrio cholera (Vibrio cholerae), lactobacillus (Lactobacillus), Bacille calmette guerin (BCG) and streptococcus (Streptococcus) are (referring to for example WO88/6626; WO90/0594; WO 91/13157; WO 92/1796 and WO92/21376).Many other non-virus particle expression vectors and system are known in the art and can use with the present invention.

Suitable delivery of nucleic acids technology comprises DNA-part complex, adenovirus-part-dna complex, dna direct injection, CaPO ₄The sedimentation method, gene gun technology, electroporation and colloidal dispersion system etc.Colloidal dispersion system comprises polymer composite, nanometer wafer, microsphere, pearl and comprises oil in water emulsion, micelle, mixed micelle and liposome based on the system of lipid.The preferred colloidal dispersion of the present invention is liposome, and it is effective synthetic membrane vesicle as vehicle in external and the body.RNA, DNA and complete virion can be encapsulated in aqueous interior and with have biology activity form be delivered to cell (Fraley, R., et al., 1981, Trends Biochem.Sci., 6:77).The composition of liposome is phospholipid normally, the combination of particularly high phase transition temperature phospholipid, common and especially cholesterol combination of steroid.Also can use other phospholipid or other lipid.The physical property of liposome depends on the existence of pH, ionic strength and bivalent cation.The lipid instance useful to the preparation of liposome comprises the phosphatidyl chemical compound, like phosphatidyl glycerol, phosphatidylcholine, Phosphatidylserine, PHOSPHATIDYL ETHANOLAMINE, sphingolipid, cerebroside and ganglioside.Useful especially is the diacyl phosphatidyl glycerol, and wherein lipid part contains 14-18 carbon atom, a particularly 16-18 carbon atom and is saturated.Illustrative phospholipid comprises lecithin phatidylcholine, dipalmitoyl phosphatidyl choline and DSPC.

The immunogenicity target also can be united to giving the booster immunization reaction with one or more adjuvants.Following Table II has shown the adjuvant of example:

Table II

The type of immunological adjuvant

Compositions of the present invention to host's administration can be used any completion the in the various technology well known by persons skilled in the art.Can give patient's medical reagent with preparation according to the conventional method processing compositions of pharmacy industry, comprise people and other mammal (i.e. " pharmaceutical composition ").Preferred pharmaceutical compositions is with the dosage unit form preparation of the for example DNA, vector particles, polypeptide or the peptide that contain a specified rate.Can change to a great extent for people or other mammiferous suitable daily dose, depend on patient's the state of an illness and other factors, but can use conventional method to confirm equally.

Pharmaceutical composition can be oral, in the parenteral, suction spraying, rectum, tuberosity or topical administration, can accept the form of the dosage unit preparations of carrier, adjuvant and excipient to contain conventional medicine.Be meant the term " drug acceptable carrier " of this use or " physiology can be accepted carrier " and be suitable for accomplishing or strengthening one or more preparation materials that nucleic acid, polypeptide or peptide are sent as pharmaceutical composition." pharmaceutical composition " is to comprise the nucleic acid of treating effective dose or the compositions of polypeptide.Each is meant the amount that is used to induce or strengthen the nucleic acid or the polypeptide of effective immune response term " effective dose " and " treatment effective dose ".Preferred compositions of the present invention provides inducing of anti tumor immune response among the host or strengthens, and this immunoreation protection host avoids the development of tumor and/or allows the host to eliminate the tumor that exists from health.

For oral administration, pharmaceutical composition can be any form in several kinds of forms, comprises for example capsule, tablet, suspending agent or liquid etc.Liquid can be used as the compositions with suitable carrier and gives through injection, and said carrier comprises saline, glucose or water.In the term parenteral of this use comprises subcutaneous, intravenous, intramuscular, breastbone, transfusion or intraperitoneal administration.The drug suppository that is used for rectally can prepare through medicine is mixed with suitable nonirritant excipient, excipient such as cupu oil and Polyethylene Glycol, and they are solid-state under ordinary temp, but are liquid under rectal temperature.

Based on various factors, comprise disease type, age, body weight, sex, patient's the state of an illness, being in a bad way property, route of administration and employed specific compound with the dosage of compositions of the present invention immunity host or treatment disorder or disease.For example, poxvirus vector can be used as every dose and comprises 1 * 10 ⁶The compositions of individual infective granule gives.Therefore, dosage regimen can change to a great extent, and standard method is conventional to be confirmed but can use.

Also can use to excite-strengthened scheme (WO01/30382 A1), wherein the target immunogen gives with a kind of form in exciting step for the first time, is subsequently to strengthen step, and wherein the target immunogen gives with another kind of form.Excite with strengthen step in the immunogenic form of target be different.For example, if exciting step is used nucleic acid, strengthening step can give with polypeptide.Similarly, under the situation of the recombinant virus (being ALVAC) that the exciting step use is a type, strengthen step and can use another kind of type virus (being NYVAC).Verifiedly excite-strengthen medication to induce powerful immunoreation.Various forms of combinations are suitable for embodiment of the present invention.

Although compositions of the present invention can be used as unique active agents and gives, they also can use with one or more other compositionss or reagent (being other immunogenicity target, costimulatory molecules, adjuvant) combination.When combination gave, each composition can be formulated as component separately, and while or different time give, or made up each component with the form of single compositions.

Injection like aseptic injection aqueous or oily suspending agent, can use suitable dispersant or wetting agent and suspending agent to prepare according to known method.Injection also can be to be in nontoxic parenteral can accept aseptic parenteral solution or suspending agent in diluent or the solvent.Operable suitable carrier and solvent are water, ringer's solution and isotonic sodium chlorrde solution etc.For example, viral vector such as poxvirus can prepare in 0.4%NaCl.In addition, aseptic fixedness oils is used as solvent or suspension media usually.For this purpose, can use the fixed oil of any gentleness, comprise synthetic monoglyceride or diglyceride.In addition, fatty acid such as oleic acid are applied in the preparation of injection.

For topical, the suitable local dose of compositions can give for one to four time with every day, and preferred every day two or three times.Also at interval a couple of days gives application dose not during these days to this dosage.Suitable compositions can comprise 0.001% to 10%w/w, for example, the preparation of 1% to 2% weight, although it can comprise as many as 10%w/w, preferably 5%w/w, more preferably 0.1% to 1% preparation at the most.The preparation that is suitable for topical comprises the drop that is suitable for fluid or the semifluid preparation (for example liniment, washing liquid, ointment, emulsifiable paste or paste) through dermal osmosis and is suitable for giving eyes, ear or nose.

Pharmaceutical composition also can be prepared into solid-state form (comprising granule, powder or suppository).Pharmaceutical composition can stand conventional pharmaceutical operations as sterilizing and/or can containing conventional adjuvant, like antiseptic, stabilizing agent, wetting agent, emulsifying agent, buffer or the like.The solid dosage forms of oral administration can comprise capsule, tablet, pill, powder and granule.In this solid dosage forms, reactive compound can mix with at least a inert diluent such as sucrose, lactose or starch.This dosage form also can comprise the other material except that inert diluent, and as in ordinary production, this material is lubricant such as magnesium stearate for example.Under the situation of capsule, tablet and pill, dosage form also can comprise buffer agent.Tablet and pill can be prepared as in addition has enteric coating.The fluid dosage form of oral administration can comprise contain the normally used inert diluent in this area for example the medicine of water can accept Emulsion, solution, suspending agent, syrup and elixir.These compositionss also can comprise adjuvant such as wetting agent, sweeting agent, correctives and aromatic.

Any form during the pharmaceutical composition that comprises nucleic acid or polypeptide of the present invention can take several forms and can be through any the giving in several kinds of approach.In preferred embodiments, said composition is given through parenteral route (Intradermal, intramuscular or subcutaneous) and is given induction of immunity reaction in the host.Perhaps, said composition can directly be applied in lymph node (in the tuberosity) or the tumor mass (being administration in the tumor).For example, dosage can be 0,7 and 14 day subcutaneous giving.The appropriate method that use comprises the compositions immunity of TAs is known in the art; As for p53 (Hollstein et al.; 1991), p21-ras (Almoguera et al., 1988), HER-2 (Fendly et al., 1990), melanoma associated antigen (MAGE-1; MAGE-2) (van derBruggen et al., 1991), p97 (Hu et al., 1988), melanoma associated antigen E (WO99/30737) and carcinoembryonic antigen (CEA) (Kantor et al., 1993; Fishbein et al., 1992; Kaufman et al., 1991) etc.

The preferred embodiment that can give (administratable) compositions comprises the fluid preparation that for example contains nucleic acid or polypeptide, like suspending agent, syrup or elixir.Optimizing injection comprises nucleic acid or the polypeptide that for example is suitable for parenteral, subcutaneous, Intradermal, intramuscular or intravenous administration, like aseptic suspending agent or Emulsion.For example, recombinant poxvirus can mix with suitable carrier, diluent or excipient such as sterilized water, normal saline, glucose etc.Compositions also can provide with lyophilized form, is used in for example isotonic aqueous solution, brine buffer solution reconstruct.In addition, compositions can give or give in proper order with other antitumor agent, antitumor agent or anticarcinogen and/or with the reagent that reduces or alleviate the illeffects of antitumor agent, antitumor agent or anticarcinogen jointly.

The test kit that comprises the present composition also is provided.This test kit can comprise the separately container that contains suitable carriers, diluent or excipient.The reagent that this test kit can also comprise other anticarcinogen, antitumor agent or antitumor agent and/or reduction or alleviate the illeffects of antitumor agent, antitumor agent or anticarcinogen is used for common or the order administration.In addition, this test kit can comprise the description about mixing or composition and/or administration.

To obtain better understanding from the following example that provides with way of illustration to the present invention and lot of advantages thereof.

Embodiment

Embodiment 1

The structure of the former construct vT416 of multi-resistance

Use standard technique construction of expression vector vT416 (ALVAC-NY-ESO-1/Trp-2-LFA-3/ICAM-1/B7.1-E3L/K3L) in the ALVAC carrier.The DNA sequence of coding NY-ESO-1, Trp-2, LFA-3, ICAM-1, B7.1, vvE3L and vvK3L is inserted in the range gene seat in the ALVAC genome.DNA sequence (Chen et al.1997 PNAS 94:1914) and the TRP-2 (Wang et al.1996 J.Exp.Med.184:2207) of coding NY-ESO-1 are inserted in the C5 locus.Coding LFA-3 (Wallner; Et al. (1987) J.Exp.Med.166:923-932), ICAM-1 (Staunton; Et al. (1988) Cell 52:925-933) and the DNA sequence of B7.1 (Chen, et al. (1992) Cell 71:1093-1102) be inserted in the C3 locus.LFA-3, ICAM-1 and B7.1 form the expression cassette that is called TRICOM.The DNA sequence of coding vvE3L (Chang, et al.1992.Proc.Natl.Acad.Sci.U.S.A89:4825-4829) and vvK3L (Beattie, et al.1991.Virology 183:419-422) is inserted in the C6 locus.The promoter of using is following:

Table III

DNA sequence	Promoter
		E3L	Cowpox E3L
K3L	Cowpox H6
		LFA-3	Cowpox 30K
ICAM-1	Cowpox I3
		B7.1	sE/L
NY-ESO-1	Cowpox H6
		TRF-2	sE/L

Promoter sE/L is by Chakrabarti, and et al. (BioTechniques 23:1094-1097,1997) describes.Show the donor plasmid of use below:

Table IV

Plasmid	Size (bp)	Carrier	Antibiotics resistance gene
				pMPC6H6K3E3	-	pBS-SK	Amp
pALVAC，Tricom(C3)#33	10,470	pBS-SK	Amp
				pT1132	11,154	pBS-SK	Amp

The DNA sequence of NY-ESO-1 and TRP-2 is inserted among the ALVAC donor plasmid pT1132.This donor plasmid uses with pALVAC Tricom (C3) #33 then, so that produce to express the ALVAC-TRICOM recombinant of these genes with standard technique.Plasmid pALVAC Tricom (C3) #33 and pT1132 are shown in Fig. 1.The DNA sequence of pALVAC Tricom (C3) #33 and pT1132 is respectively shown in Fig. 2 and 3.

Embodiment 2

The structure of the former construct vT419 of multi-resistance

Use standard technique construction of expression vector vT419 (ALVAC-gp100M/Mart-1/Mage-1,3 mini-genes-LFA-3/ICAM-1/B7.1-E3L/K3L) in the ALVAC carrier.Coding gp100M/MART-1/MAGE-1, the DNA sequence of 3 mini-genes, LFA-3, ICAM-1, B7.1, vvE3L and vvK3L is inserted in the range gene seat in the ALVAC genome.Gp100M/MART-1/MAGE-1,3 mini-genes are inserted in the C5 locus.Coding LFA-3 (Wallner; Et al. (1987) J.Exp.Med.166:923-932), ICAM-1 (Staunton; Et al. (1988) Cell 52:925-933) and the DNA sequence of B7.1 (Chen, et al. (1992) Cell 71:1093-1102) be inserted in the C3 locus.LFA-3, ICAM-1 and B7.1 form the expression cassette that is called as TRICOM.The DNA sequence of coding vvE3L (Chang, et al.1992.Proc.Natl.Acad.Sci.U.S.A 89:4825-4829) and vvK3L (Beattie, et al.1991.Virology 183:419-422) is inserted in the C6 locus.The promoter of using is following:

Table V

Gene	Promoter
		E3L	Cowpox E3L
K3L	Cowpox H6
		LFA-3	Cowpox 30K
ICAM-1	Cowpox I3
		B7.1	sE/L
gp100(M)	Cowpox H6
		Mart-1	Cowpox 42K

Promoter sE/L is by Chakrabarti, and et al. (BioTechniques 23:1094-1097,1997) describes.Show the donor plasmid of use below:

Table VI

Plasmid	Size (bp)	Carrier	Antibiotics resistance gene
				?PMPC6H6K3E3	-	pBS-SK	Amp
?pALVAC，Tricom(C3)#33	10,470	pBS-SK	Amp
				?pT3217	11,465	pBS-SK	Amp

Gap100 (M), Mart-1 and Mage-1,3 mini-genes are inserted among the ALVAC C5 donor plasmid pT3217.This donor plasmid uses with pALVAC Tricom (C3) #33 then, so that produce to express the ALVAC-TRICOM recombinant of these genes with standard technique.This donor plasmid inserts in the C5 site.PALVAC.Tricom (C3) #33 is shown in Fig. 1 and Fig. 2.The pT3217 plasmid is shown in Fig. 4.The DNA sequence of pT3217 is shown in Fig. 5.

Embodiment 3

The immunologic evaluation of multi-antigen vectors

First zooperal result shows when vaccine gives with the injection of opening in twice minute, three in four antigen (Mart1, NY-ESO-1 and gap100) is tended to have higher immunoreation.Yet these differences do not have significance on statistics.In detail, (ALVAC (2)-gp100M/MART-1/MAGE-1/3 mini-gene/TRICOM) (ALVAC (2)-TRP-2/NY-ESO-1/TRICOM) gives and subcutaneous immune HLA-A2/k a position combination or as separating to inject with vT416 with vT419 ^bTransgenic mice (5/group).With the immune control mice of parent ALVAC (2).Mouse inoculation three times (with the interval in three weeks), and in the end strengthen three weeks of back, with IFN-g ELISPOT and CTL test, stimulate again with peptide is external before, the t cell responses in every mice is analyzed.Compare with control animal, demonstrate the ELISPOT reaction of the anti-MART-1 that significance is arranged on the statistics with the mice of multi-antigen vectors (at 2 positions) inoculation.IFN-gamma reaction to gp100M and NY-ESO-1 also can detect, although because the variability of reaction is few with the quantity of the culture of being tested, these are reflected at does not have significance on the statistics.In all groups (comprising control animal) of test, the antigenic ELISPOT reaction of anti-TRP-2 raises, and is general because the TRP-2 peptide (180-188) and the H-2k of leading A2 restriction ^bCross reaction, and, therefore on statistics, do not have remarkable meaning in the fact that can in inmature mice, induce low affinity t cell responses behind the In vitro culture.What is interesting is, be usually less than the mice of separately accepting every kind of virus with the reaction of the ELISPOT in the mice of the mixture of vT416 and vT419 injection, although these differences do not have statistical significance.The CTL data are negative basically, and except an intensive anti-gp100 reaction and one MIN (marginal) anti-MART-1 reaction, the two all takes place in the mice with vT416 and vT419 inoculation (two positions).Generally speaking, these results provide excitation people's data, and it has confirmed that multi-antigen vectors can produce anti-MART-1 reaction, and prompting also can be induced anti-gp100 and anti-NY-ESO-1 reaction.

Used the former ALVAC recombinant of melanoma multi-resistance to accomplish two other clinical preceding zooscopies.In these experiments, ((ALVAC (2)-TRP-2/NY-ESO-1/TRICOM) is a position combination or separate to inject and give and subcutaneous immune HLA-A2/K for ALVAC (2)-gp100M/MART-1/MAGE-1/3 mini-gene/TRICOM) and vT416 with vT419 ^bTransgenic mice (5/group).With the immune control mice of parent ALVAC (2).After the inoculation, after the external stimulation again of peptide, estimate the t cell responses in each mice with IFN-γ ELISPOT test.Different with the former experiment of former multi-resistance, the former experiment of said multi-resistance provides excitation people's immunogenicity data, and these two nearest researchs have produced uncertain data, because the high background response in the contrast immune animal.Therefore, think that generally speaking the result is uncertain.

First studies the result who produces for the immunogenicity of the former construct of confirmation multi-resistance with for repetition, has accomplished another clinical preceding zooscopy.((ALVAC (2)-TRP-2/NY-ESO-1/TRICOM) separately injection gives and subcutaneous immune HLA-A2/K for ALVAC (2)-gp100M/MART-1/MAGE-1/3 mini-gene/TRICOM) and vT416 with vT419 ^bTransgenic mice (10/group).With the immune control mice of parent ALVAC (2).Can detect the ELISPOT reaction of significance on the statistics of anti-gp100, Mart-1 and TRP-2, and detect some reactions of anti-NY-ESO-1, it is in the edge that statistics goes up significance.

Although described the present invention, be to be understood that those skilled in the art will expect variants and modifications according to preferred embodiment.Therefore, intention is that all this equivalent modifications within the claimed invention scope are included in the accompanying claims covering.

Claims (4)

1. be used for preparing the expression vector that is used to treat or prevent melanomatous pharmaceutical composition, at least two kinds of tumor antigens that wherein said expression vector comprises ALVAC (2), TRICOM and is selected from NY-ESO-1, TRP-2, gp100, gp100M, MART-1, MAGE-1 and MAGE-3.

2. being used for treatment or preventing melanomatous pharmaceutical composition of expression vector that comprises claim 1.

3. the pharmaceutical composition of the claim 2 that is suitable for using through injection.

4. pharmaceutical composition as claimed in claim 2, it comprises two kinds of carriers, and one of them carrier is ALVAC (2)-gp100M/MART-1/MAGE-1/3 mini-gene/TRICOM, and another carrier is ALVAC (2)-TRP-2/NY-ESO-1/TRICOM.

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