ZA200602771B - Multi-antigen vectors for melanoma - Google Patents

Description

Multi-Antigen Vectors for Melanoma oC

FIELD OF THE INVENTION

The present invention relates to multi-antigen vectors for use in preventing and / or treating cancer. In particular, the invention relates to multi-antigen vectors for use in treating and/or preventing melanoma. hE

BACKGROUND OF THE INVENTION

There has been tremendous increase in last few years in the development of cancer vaccines with tumour-associated antigens (TAAs) due to the great advances in identification of molecules based on the expression profiling on primary tumours and normal cells with the help of several techniques such as high density microarray, SEREX, immunohistochemistry (IHC),

RT-PCR, in-situ hybridization (ISH) and laser capture microscopy (Rosenberg, Immunity, 1999;

Sgroi et al, 1999, Schena et al, 1995, Offringa et al, 2000). The TAAs are antigens expressed or over-expressed by tumour cells and could be specific to one or several tumours for example CEA antigen is expressed in colorectal, broast and Jung cancers. Sgroi et al (1999) identified several genes differentially expressed in invasive and metastatic carcinoma cells with combined use of laser capture microdissection and cDNA microarrays. Several delivery systems like DNA or viruses could be used for therapeutic vaccination against human cancers (Bonnet et al, 2000) and can elicit immune responses and also break immune tolerance against TAAs. Tumour cells can be rendered more immunogenic by inserting transgenes encoding T cell co-stimulatory molecules such as B7.1 or cytokines such as IFN-y, IL2, or GM-CSF, among others. Co- expression of a TAA and a cytokine or a co-stimulatory molecule can develop effective therapeutic vaccine (Hodge et al, 95, Bronte et al, 1995, Chamberlain et a, 1996). Ce 55 . Thereisaneed in the art for reagents and methodologies useful in stimulating an immune fesponse to prevent or treat cancers. The present invention provides: such reagents and methodologies that overcome many of the difficulties encountered by others in attempting to

SUMMARY OF THE INVENTION

The present invention provides multi-antigen vectors for administration to a patient 0 prevent and / or treat cancer. In particular, the multi-antigen vector encodes one or more tumor antigens (“TA”). The multi-antigen vector may also encode an immune stimulator such as a co- stimulatory molecule and/or be administered with an adjuvant.

Figure 1. Schematic of plasmids pALVAC.Tricom(#33) and pT1132.

Figure 2. DNA sequence of plasmid pALVAC.Tricom(#33).

Figure 3. DNA sequence of plasmid pT1132.. "Figure 4. Schematic of plasmid pT3217. . ‘Figure 5. DNA sequence of plasmid pT3217 .

Figure 6. Amino acid sequences of exemplary NY-ESO-1, TRP-2, gp100, gp100M, MART- - 1, MAGE-1, MAGE-3, B7.1, LFA-3, and ICAM-1 proteins. oo : DETAILED DESCRIPTION

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

In one embodiment, the present invention relates to the induction or enhancement of an 50 immune response against one or more tumor antigens (“TA”) to prevent and / or treat cancer. In certain embodiments, one or more TAs may be combined. In preferred embodiments, the mmune response results from expression of a TA in a host cell following administration of a mucleic acid vector encoding the tumor antigen or the tumor antigen itself in the form of a peptide or polypeptide, for example. : 235 As used herein, an “antigen” is a molecule (such as a polypeptide) of a portion thereof that produces an immune response in a host to whom the antigen has been administered. The -pxphune response may include the production of antibodies that bind to at least one epitope of the antigen and / or the generation of a cellular immune response against cells expressing an epitope of the antigen. The response may be an enhancement of a current immune response by, for "30 example, causing increased antibody production, production of antibodies with increased affinity for the antigen, or an increase in the cellular immune response (i.e., increased number or activity

Co WO 2005/026370 PCT/US2004/028751 of immunoreactive T cells). An antigen that produces an immune response may alternatively be referred to as being immunogenic or as an immunogen. In describing the present invention, 8

TA may be referred to as an «jmmunogenic target”. The present invention provide expression vectors for expressing in a host one or more immunogenic targets. SE 5 . ~~ The term TA includes both tumor-associated antigens (TAAs) and. tumor-specific oC antigens (TSAs), where a cancerous cell is the source of the antigen. ATAA isan antigen that is expressed on the surface of a tumor cell in higher amounts than is observed on normal cellsoran antigen that is expressed on normal cells during fetal development. A TSA is an antigen that is unique to tumor cells and is not expressed on normal cells. TA further includes TAAs or TSAs, . 10 - antigenic fragments thereof, and modified versions that retain their antigenicity. co

TAs are typically classified into five categories according to their expression pattern, function, or genetic origin: cancer-testis (CT) antigens (i.e, MAGE, NY-ESO-1); melanocyte differentiation antigens (i.e, Melan A/MART-1, tyrosinase, gp100); mutational antigens (ie, -

MUM-1, p53, CDK-4); overexpressed ‘self antigens (i. HER-2/neu, p53); and, viral antigens - 15 (ie. HPV, EBV). For the purposes of practicing the present invention, a suitable TA is any TA that induces or enhances an anti-tumor immune response in a host to whom the TA has been administered. Suitable TAs include, for example, species of gp100 (Cox et al., Science, 264:716- 719 (1994); US. Pat. No. 6,500,919 Bl and WO 01/30847 with Val at residue 162, also referred to as “gpl00M”; U.S. Pat. No. 6,537,560 Bl with Phe at residue 162), MART-1/Melan A (Kawakami et al, J. Exp. Med., 180:347-352 (1994); U.S. Pat. No. 5,874,560), gp75 (TRP-1) (Wang ct al. J. Exp. Med, 186:1131-1140 (1996), TRP-2 (Wang ct al. 1996 J. Exp. Med. 184:2207; U.S. Pat. Nos. 5,831,016 and 6,083,783), tyrosinase (Wolfel et al, Eur. J. Immunol., 24:759-764 (1994); WO 200175117; WO 200175016; WO 200175007), NY-ESO-1 (WO 0814464; WO 99/18206; GenBank Accession No. P78358; US. Pet. No. 5,804,381), melaioma »s proteoglycan (Hellstrom et al, J. Immunol., 130:1467-1472 (1983)), MAGE family antigens (ic, MAGE-1, 2,3,4,6,12, 51; Van der Bruggen et al., Science, 254:1643-1647 (1991); US. Pat.

Nos. 6,235,525; CN 1319611), BAGE family antigens (Boel et al., Immunity, 2:167-175 (1995),

GAGE family antigens (ie, GAGE-1,2; Van den Eynde ct al, J. Exp. Med., 182:689-698 (1995); U.S. Pat. No. 6,013,765), RAGE family antigens (ie. RAGE-1; Gaugler et sat. 3 Immunogenetics, 44:323-330 (1996); U.S. Pat. No. 5,939,526), N-acetylglucosaminyltransferase-

V (Guilloux ct at, J. Exp. Med., 183:1173-1183 (1996), p15 (Robbins et al, J. immuno. i» : 3

© WO 2005/026370 PCT/US2004/028751 154:5044-5050 (1995), B-catenin (Robbins ct al, J. Exp- Med. 183:1185-1192 (1996); MUM-1 (Coulie et al., Proc. Natl. Acad. Sci. USA, 92:7976-7980 (1995)), cyclin dependent kinase-4 (CDK4) (Wolfel et al., Science, 269:1281-1284 (1995)), p21-ras (Fossum et at., Int. J. Cancer, 56:40-45 (1994)), BCR-abl (Bocchia et al., Blood, 85:2680-2684 (1995), p53 (Theobald et al, s Proc. Natl. Acad. Sci. USA, 92:11993-11997 (1995), p185 HERZ/nen (erb-B1; Fisk etal, J

Exp. Med., 181:2109-2117 (1995), epidermal growth factor receptor (EGFR) (Harris et als.

Breast. Cancer Res. Treat, 29:1-2 (1994), carcinoembryonic antigens (CEA) (Kwong et al, J.

Natl. Cancer Inst, 85:982-990 (1995) U.S. Pat. Nos. 5,756,103; 5,274,087; 5,571,110; 6,071,716; 5,698,530; 6,045,802; EP 263933; EP 346710; and, EP 784483); carcinoma- lo associated mutated mucins (ie., MUC-T gene products; Jerome et al., J. Immunol, 151:1654- 1662 (1993)); EBNA gene products of EBV (ie, EBNA-1; Rickinson et al, Cancer Surveys, 13:53-80 (1992)); E7, B6 proteins of human papillomavirus (Ressing et al. J. Immunol, 154:5034-5943 (1995); prostate specific antigen (PSA; Xue et al, The Prostate, 30:13-18 (1997)); prostate specific membrane antigen (PSMA; Israeli, et al, Cancer Res., 54:1807-1811 (1994); idiotypic epitopes or antigens, for example, immunoglobulin idiotypes or T cell receptor idiotypes (Chen et al, J. Immunol., 153:4775-4787 (1994)); KSA (U.S. Patent No. 5,348,887), \inesin 2 (Dietz, et al. Biochem Biophys Res Commun 2000 Sep 7:275(3):731-8), HIP-55,

TGFp-1 anti-apoptotic factor (Toomey, et al. BrJ Biomed Sci 2001;58(3):177-83), tumor protein

P52 (Bryne J.A., et al, Genomics, 35:523-532 (1996)), HIFT, NY-BR-1 (WO 01/47959), NY- 220 "BR-62, NY-BR-75, NY-BR-85, NY-BR-87, NY-BR-96 (Scanlan, M. ‘Serologic and

Bioinformatic Approaches to the Identification of Human Tumor Antigens, in Cancer Vaccines “2000, Cancer Research Institute, New York, NY), including «wild-type” (i.¢., normally encoded by the genome, naturally-occurting), modified, and mutated versions as well as other fragments and derivatives thereof. Any of these TAs may be utilized alone or in combination with one another in a co-immunization protocol. : Preferred TAs are useful for inducing an immune response against melanoma cells, The . tem nmelanoma” includes but is not limited to melanomas, metastatic melanomas, melanomas derived from either melanocytes or melanocyte related nevus cells, melanocarcinomas, melanoepitheliomas, melanosarcomas, melanoma in situ, superficial spreading ‘melanoma, nodular melanoma, lentigo maligna melanoma, acral lentiginous melanoma, invasive melanoma ‘and familial atypical mole and melanoma (AMM) syndrome, for example. In general,

melanomas result from chromosomal abnormalities, degenerative growth and development disorders, mitogenic agents, ultraviolet radiation (UV), viral infections, inappropriate tissue expression of a gene, alterations in expression of a gene or carcinogenic agents, for example. : In certain cases, it may be beneficial to co-immunize patients with both TA and other antigens, such as angiogenesis-associated antigens (“AA”). An AA isan immunogenic molecule (i.e., peptide, polypeptide) associated with cells involved in the induction and / or continued development of blood vessels. For example, an AA may be expressed on an endothelial cell (“EC”), whichis a primary structural component of blood vessels. Where the cancer is cancer, it is preferred that that the AA be found within or near blood vessels that supply a tumor.

Immunization of a patient against an AA preferably results in an anti-AA immune response : whereby angiogenic processes that occur near or within tumors are prevented and / or inhibited. . Exemplary AAs include, for example, vascular endothelial growth factor (ie. VEGF;

Bernardini, ct al. J. Urol, 2001, 1664): 1275-9; Stames, et al. J. Thorac. Cardiovasc Surg, 2001, 122(3): 518-23; Dias, et al. Blood, 2002, 99: 2179-2184), the VEGF receptor (i.e, VEGF- sR fik-I/KDR; Stames, ot al. J. Thorac. Cardiovasc. Surg., 2001, 122(3): 518-23), BPH receptors (ie., EPHA2; Gerety, ot al. 1999, Cell, 4: 403-414), epidermal growth factor receptor (i.c.,

EGFR; Ciardeillo, et al. Clin. Cancer Res., 2001, 7(10): 2958-70), basic fibroblast growth factor (0 bFGF; Davidson, et al. Clin. Exp. Metastasis 2000,18(6): 501-7; Poon, et al. Am J. Surg, © 2001, 182(3):298-304), platelet-derived cell growth factor (i.e, PDGF-B), platelet-derived 2 endothelial cell growth factor (PD-ECGF; Hong, et al. J. Mol. Med., 2001, 8(2):141-8), wansforming growth factors (i.e., TGF-a; Hong, et al. J. Mol. Med., 2001, 8(2):141-8), endoglin ‘Balzi et al. Int. J. Cancer, 2001, 94: 579-585), 1d proteins (Benezra, R. Trends Cardiovasc.

Med., 2001, 11(6):23741), proteases such as uPA, uPAR, and matrix metalloproteinases (MMP- : 2, MMP-9; Djonov, et al. J. Pathol, 2001, 195(2):147-55), nitric oxide synthase (Am. 1.

Ophthalmol, 2001, 132(4):551-6), aminopeptidase (Rouslhati, E. Nature Cancer, 2: 84-90, © 2002), thrombospondins (i.e., TSP-1, TSP-2; Alvarez, et al. Gynecol. Oncol., 2001, 82(2):273-8;

Seki, ct al. Int J. Oncol, 2001, 19(2):305-10), k-ras (Zbavg, et al. Cancer Res, 2001, .61(16):6050-4), Wnt (Zhang, et al. Cancer Res., 2001, 61(16):6050-4), cyclin-dependent kinases (CDKs; Drug Resist. Updat. 2000, 3(2):83-88), microtubules (Tima, et al. 2001. Path. Oncol. "40 Res, 7(2): 85-94), heat shock proteins (i.c., HSP9O (Timr, supra)), heparin-binding factors (i. heparinase; Gohji, et al. Int. J. Cancer, 2001, 95(5):295-301), synthases (i.e., ATP synthase,

hymidilate synthase), collagen receptors, integrins (i.e, avp3, aps, alpl, a2Bl, spl), the surface proteolglycan NG2, AAC2-1, or AAC2-2, among others, including wild-type” (ier normally encoded by the genome, naturally-occurring), modified, mutated versions as well as other fragments and derivatives thereof. Any of these targets may be suitable in practicing the present invention, either alone or in combination with one another or with other agents.

The nucleic acid molecule may comprise or consist of a nucleotide sequence encoding one or more immunogenic targets, or fragments or derivatives thereof, such as that contained ina.

DNA insert in an ATCC Deposit. The term “nucleic acid sequence” or “nucleic acid molecule” refers to a DNA or RNA sequence. The term encompasses molecules formed from any of the known base analogs of DNA and RNA such as, but not limited to 4-acetylcytosine, 8-hydroxy-

N6-methyladenosine, aziridinyl-cytosine, pseudoisocytosine, 5-(catboxyhydroxylmethy?) uracil, sfnorouracil, S-bromouracil, S-carboxymethylaminomethyl-2-thiouracil, S.carboxy- methylaminomethyluraci, dihydrouracil, inosine, N6-iso-pentenyladenine, ]-methyladenine, 1- methylpseudouracil, 1-methylguanine, 1-methylinosine, 2,2-dimethyl-guanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine, 7-methylguanine, 5- mefhylaminomethyluraci, 5.methoxyamino-methyl-2-thiouracil, beta-D-mannosylqueosine, 5 - methoxycarbonyl-methyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5- - oxyacetic acid methylester, uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, ‘queosine, 2- thiocytosine, 5-methyl-2-thiouracil, 2-thiouraci, = 4-thiourecil 5-methyluracil, N-uracils- oxyacetic acid methylester, uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, and 2,6-diaminopurine, among others. : RE

An isolated nucleic acid molecule is one that: (1) is separated froin at least about 50 percent ‘of proteins, lipids, carbohydrates, or other materials with which it is naturally found when total nucleic acid is isolated from the source cells; (2) is not be linked to all or a portion of ‘25 a polynucleotide to which the nucleic acid molecule is linked in nature; (3) is operably linked to . a polynucleotide which it is not linked to in nature; and / or, (4) does not occur in nature as part of a larger polynucleotide sequence. Preferably, the isolated nucleic acid molecule of the present . jnivention is substantially free from any other contaminating nucleic acid molecule(s) or other contaminants that are found in its naturai environment that would interfere with its use in 30, polypeptide production or its therapeutic, diagnostic, prophylactic or research use. As used herein, the term “naturally occurring” or “native” or “naturally found” when used in connection with biological materials such as nucleic acid molecules, polypeptides, host cells, and the like, refers to materials which are found in nature and are not manipulated by man. Similarly, “non- naturally occurring” or “non-native” as used herein refers to a material that is not found in nature or that has been structurally modified or synthesized by man.

The identity of two or more aucleic acid or amino acid sequences is determined by comparing the sequences. As known in the art, “identity” means the degree of sequence relatedness between nucleic acid or amino acid sequences as determined by the match between the units making up the molecules (ie. nucleotides or amino acid residues). Identity measures r the percent of identical matches between the smaller of two or more ‘sequences with gap alignments (if any) addressed by a particular mathematical model or computer program (ie, an algorithm). dentity between nucleic acid sequences may also be determined by the ability of the ~ nucleic acid sequences to hybridize to one another. In defining the process of hybridization, the term “highly stringent conditions” and “moderately stringent conditions” refer to conditions that permit hybridization of nucleic acid strands whose sequences are complementary, and to exclude 1s hybridization of significantly ismatched nucleic acids. Examples of “highly stringent - conditions” for hybridization and washing are 0.015 M sodium chloride, 0.0015 M sodium "citrate at 65-68°C or 0.015 M sodium chloride, 0.0015 M sodium citrate, and 50% formamide at

R 42°C. (see, for example, Sambrook, Fritsch '& Maniatis, Molecular Cloning: A Laboratory "Manual (nd od., Cold Spring Harbor Laboratory, 1989); Anderson ef al, Nucleic Acid

Hybridisation: A Practical Approach Ch. 4 (IRL Press Limited). The term “moderately stringent conditions” refers to conditions under which a DNA duplex with a greater degree of base pair mismatching than could occur under “highly stringent conditions” is able to form.

Exemplary moderately stringent conditions are 0.015 M sodium chloride, 0.0015 M sodium : citrate at 50-65°C or 0.015 M sodium chloride, 0.0015 M sodium citrate, and 20% formamide at 37-50°C. By way of example, moderately stringent conditions of 50°C in 0.015 M sodium ion will allow about a 21% mismatch. During hybridization, other agents may be included in the hybridization and washing buffers for the purpose of reducing non-specific and/or background "hybridization. Examples are 0.1% bovine serum albumin, 0.1% polyvinyl-pyrrolidone, 0.1% sodium pyrophosphate, 0.1% sodium dodecylsulfate, NaDodSOs, (SDS), ficoll, Dénhardt’s solution, sonicated salmon sperm DNA. (or another non-complementary DNA), and dextran sulfate, although other suitable agents can also be used. The concentration and types of these additives can be changed ‘without substantially affecting the stringency of the hybridization conditions. Hybridization experiments are usually carried out at pH 6.8-7.4; however, at typical jonic strength conditions, the rate of hybridization is nearly independent of pH. - In 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 t0 transfer a nucleic acid sequence to a host cell. In certain cases, an expression vector is utilized.

An expression vector is a nucleic acid molecule that is suitable for transformation of a host cell and contains mucleic acid sequences that direct and / or contro} the expression of the transferred nucleic acid sequences. Expression includes, but is not limited to, processes such as . 10 - transcription, translation, and splicing, if introns are present. Expression vectors typically . comprise one or more flanking sequences operably linked to a heterologous mucleic acid sequence encoding a polypeptide. Flanking sequences maybe homologous (i.e., from the same * species and / or strain as the host cell), heterologous (i.e., from a species other than the host cell species or strain), hybrid (i.e, 8 combination of flanking sequences from more than one Source), or synthetic, for example. | | So : A flanking sequence is preferably capable of effecting the replication, transcription and / or translation of the coding sequence and is operably linked to a coding sequence. As used herein, the term operably linked refers to 8 linkage of polynucleotide elements in a functional - relationship. For instance, a promoter or enhancer is operably linked to a coding sequence if it 50 affects the transcription of the coding sequence. However, a flanking sequence need not necessarily be contiguous with the coding sequence, so long as it functions correctly. Thus, for example, intervening untranslated yet transcribed sequences can be present between a promoter sequence ‘and thie coding sequence and the promoter sequence may still be considered operably linked to the coding sequence. Similarly, an enhancer sequence may be located upstream or downstream from the coding sequence and affect transcription of the sequence.

In certain embodiments, it is preferred that the flanking sequence is a transcriptional regulatory region that drives high-level gene expression in the target cell. The transcriptional : regulatory region may comprise, for example, a promoter, enhancer, silencer, repressor clement, or combinations thereof. The transcriptional regulatory region may be either constitutive, tissue- - specific, cell-type specific (i.e., the region is drives higher levels of transcription in a one type of tissue or cell as compared to another), or regulatable (i.e., responsive to interaction with a compound such as tetracycline). The source of a transcriptional regulatory region may be any prokaryotic or eukaryotic organism, any vertebrate or invertebrate organism, or any plant, "provided that the flanking sequence functions in a cell by causing transcription of a nucleic acid oo within that cell. A wide variety of transcriptional regulatory regions may be utilized in practicing 5s the present invention. "Suitable transcriptional regulatory regions include the CMV promoter (i.e., the CMV- immediate early promoter); promoters from eukaryotic genes (i.e, the estrogen-inducible chicken ovalbumin gene, the interferon genes, the gluco-corticoid-inducible tyrosine : aminotransferase gene, and the thymidine kinase gene); and the major early and late adenovirus fo gene promoters; the SV40 early promoter rogion (Bernoist and Chambon, 1981, Nature 290:304- ~ 10); the promoter contained in the 3° long terminal repeat (LTR) of Rous sarcoma virus RSY) ©. © - (Yamamoto, et al., 1980, Cell 22:787-97); the herpes simplex virus thymidine kinase (HSV-TK) promoter (Wagner ef al, 1981, Proc. Natl. Acad. Sci. USA 78:1444-45); the regulatory - sequences of the metallothionine gene (Brinster et al, 1982, Nature 296:39-42); prokaryotic - expression vectors such as the beta-lactamase promoter \% illa-Kamaroff et al., 1978, Proc. Natl. oo Acad. Sci. USA, 75:3727-31); or the tac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci.

USA, 80:21-25). Tissue- and / or cell-type specific transcriptional control regions include, for ’ example, the elastase 1 gene control region which is active in pancreatic acinar cells (Swift et al., © 1984, Cell 38:639-46; Omitz et al, 1986, Cold Spring Harbor Symp. Quant. Biol. 50:399-409 (1986); MacDonald, 1987, Hepatology 7:425-515); the insulin gene control region which is active in pancreatic beta cells (Hanahan, 1985, Nature 315:1 15-22); the immunoglobulin gene. control region which is active in lymphoid cells (Grossched! et al., 1984, Cell 38:647-58; oo Adames et al., 1985, Nature 318:533-38; Alexander et al., 1987, Mol. Cell. Biol, 7:1436-44); the : mouse mammary tumor virus control region in testicular, breast, lymphoid and mast cells (Leder et al., 1986, Cell 45:485-95); the albumin gene control region in liver (Pinkert et al., 1987, Genes and Devel. 1:268-76); the alpha-feto-protein gene control region in liver (Krumlauf et al., 1985,

Mol. Cell. Biol., 5:1639-48; Hammer et al., 1987, Science 235:53-58); the alpha 1-antitrypsin gene control region in liver (Kelsey et al., 1987, Genes and Devel. 1:161-71); the beta-globin gene control region in myeloid cells (Mogram et al., 1985, Nature 315:338-40; Kollias et al, "3 1986, Cell 46:89-94); the myelin basic protein gene control region in oligodendrocyte cells in the brain (Readhead et al., 1987, Cell 48:703-12); the myosin light chain-2 gene control region in skeletal muscle (Sani, 1985, Nature 314:283-86); the gonadotropic releasing hormone gene control région in the hypothalamus (Mason et al, 1986, Science 234:1372-78), and the . tyrosinase promoter in melanoma cells (Hart, I. Semin 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 light, heat, radiation, : tetracycline, or heat shock proteins, for example, may also be utilized (see, for example, wO 00/10612). Other suitable promoters are known in the art.

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

Co and insulin). Similarly, the SV40 enhancer, the cytomegalovirus early promoter enhancer, the polyoma enhancer, and adenovirus enhancers are useful with eukaryotic promoter sequences. - While an enhancer may be spliced into the vector at a position: 5’ or 3’ 10 nucleic acid coding x sequence, it is typically located at a site 5° from the promoter. Other suitable enhancers are own in the art, and would be applicable to the present invention. oo ‘While preparing reagents of the present invention, cells may need to be transfected or transformed. Transfection refers to the uptake of foreign or €XOgEnOus DNA by a cell, and a cell 2 has been transfected when the exogenous DNA has been introduced inside the cell membrane. A umber of transfection techniques are well known in the art (i.e., Grabam et al., 1973, Virology © 53.456; Sambrook et al, Molecular Cloning, A Laboratory Manual (Cold Spring Harbor

Laboratories, 1989); Davis et al., Basic Methods in Molecular Biology (Elsevier, 1986); and Chu

E et al., 1981, Gene 13:197). Such techniques can be used to introduce one or more EX0gENOUS

DNA moieties into suitable host cells. | oo _- In certain embodiments, it is preferred that transfection of a cell results in transformation "of that cell. A cell is transformed when there is a change in a characteristic of the cell, being © reniforined when it has been modified to contain a pew mueleic acid. Following transfection, “the transfected nucleic acid may recombine with that of the cell by physically integrating into a "30 chromosome of the cell, may be maintained transiently as an episomal element without being : 10 . oo replicated, or may replicate independently as a plasmid. A cell is stably transformed when the nucleic acid is replicated with the division of the cell.

The expression vectors of the present invention also provide for expression of fragments of immunogenic targets. Fragments may include sequences truncated at the amino terminus (with or without a leader sequence) and / or the carboxy terminus. Fragments may also include variants (i.e., allelic, splice), orthologs, homologues, and other variants having one or more amino acid additions or substitutions or internal deletions as compared to the parental sequence.

In preferred embodiments, truncations and/or deletions comprise about 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. Such polypeptide fragments may optionally comprise an amino terminal methionine residue. It will be appreciated that such fragments can be used, for example, to generate antibodies or cellular immune responses to immunogenic targets. :

A variant is a sequence having one or more sequence substitutions, deletions, and/or additions as compared to the subject sequence. Variants may be naturally occurring or artificially constructed. Such variants may be prepared from the corresponding nucleic acid : molecules. In preferred embodiments, the variants have from 1 to 3, or from 1 to 5, or from 1 to 10, or from i to 15, or from 1 to 20, or from I 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 naturally-occurring alternate forms of a sequence occupying a given locus on a chromosome of an organism or a population of organisms. A splice variant is a polypeptide generated from one of several RNA . transcript resulting from splicing of a primary transcript. An ortholog is a similar nucleic acid or polypeptide sequence from another species. For example, the mouse and human versions of an immunogenic target may be considered orthologs of each other. A derivative of a sequence is 25s one that is derived from a parental sequence those sequences having substitutions, additions, deletions, or chemically modified variants. Variants may also include fusion proteins, which refers to the fusion of one or more first sequences (such as a peptide) at the amino or carboxy terminus of at least one other sequence (such as a heterologous peptide). ". “Similarity” is a concept related to identity, except that similarity refers to a measure of relatedness which includes both identical matches and conservative substitution matches. If two polypeptide sequences have, for example, 10/20 identical amino acids, and the remainder are all non-conservative substitutions, then the percent identity and similarity would both be 50%. If in the same example, there are five more positions where there are conservative substitutions, then the percent identity remains 50%, but the percent similarity would be 75% (15/20). Therefore, in : cases where there are conservative substitutions, the percent similarity between two polypeptides ~~ s will be higher than the percent identity between those two polypeptides. :

EE Substitutions may be conservative, or non-conservative, or any combination thereof.

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

ETRE CL | Tablel

Residues : Substitutions —Giy | __ ProAla | Ala oo im Cnlwaz | Aw

Oe | Leu, Val, Met, Ala, Phe, Norleucine TT Lew

Sl Notleucive, lle, Val, Met, Ala, Phe | Te | Co

Lys |Arg, 1,4 Diamino-butyric Acid, Gln, Asn Arg

Fhe TewPhele | Le oo [Phe | Leu, Val, Ile, Ala, T - [Tyr | "Tip, Phe; Thr, Ser | Phe

Tic, Met, Low, Phe, Ala, Norleucine | Leu 12 oo

_ A skilled artisan will be able to determine suitable variants of an immunogenic target using well-known techniques. For identifying suitable areas of the molecule that may be changed without destroying biological activity (i.e, MHC binding, immunogenicity), one skilled in the art may target areas not believed to be important for that activity. - For example, when immunogenic targets with similar activities from the same species or from other species are known, one skilled in the art may compare the amino acid sequence of a polypeptide to'such similar polypeptides. By performing such analyses, one can identify residues and portions of the molecules that are conserved. It will be appreciated that changes in areas of the molecule that

SE (2 are not conserved relative to such similar immunogenic targets would be less likely to adversely © affect the biological activity and/or structure of a polypeptide. Similarly, the residues Tequired for binding to MHC are known, and may be modified to improve binding. - However, modifications resulting in decreased binding to MHC will not-be appropriate in most situations.

One skilled in the art would also know that, even in relatively conserved regions, one may substitute chemically similar amino acids for the naturally occurring residues while retaining activity. Therefore, even arcas 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.

Other preferred polypeptide variants include glycosylation variants wherein the mumber 50 - andlor type of glycosylation sites have been altered compared tothe subject amino acid sequence. In one embodiment, polypeptide variants comprise a greater or a lesser number of N- ‘Jiked glycosylation sites than the subject amino acid sequence. An N-linked glycosylation site . - is characterized by the sequence Asn-X-Ser or Asn-X-Thr, wherein the amino acid residue designated as X may be any amino acid residue except proline. The substitution of amino acid residues to create this sequence provides a potential new site for the addition of an N-linked _ carbohydrate chain. Altematively, substitutions that eliminate this sequence will remove an existing N-linked carbohydrate chain. Also provided is a rearrangement of N-linked carbohydrate chains wherein one or more N-linked glycosylation sites (typically those that are naturally occurring) are eliminated and one or more new N-linked sites are created. To.affect O- linked glycosylation of a polypeptide, one would modify serine and / or threonine residues. : | B Co oo WO 2005/026370 PCT/US2004/028751

Additional preferred variants include cysteine variants, wherein one or more cysteine residues are deleted or substituted with another amino acid (e.g., serine) as compared to the subject amino acid sequence set. Cysteine variants are useful when peptides or polypeptides must be refolded into a biologically active conformation such as after the isolation of insoluble inclusion bodies. Cysteine variants generally have fewer cysteine residues than the native protein, and typically have an even number to minimize interactions resulting from unpaired cysteines. Co : : * In other embodiments, the peptides or polypeptides may be attached to one or more fusion segments that assist in purification of the polypeptides. Fusions can be made either at the = amino terminus or at the carboxy terminus of the subject polypeptide variant thereof. Fusions . may be direct with no linker or adapter molecule or may be through alinker or adapter molecule.

A linker or adapter molecule may be one or more amino acid residues, typically from about 20 to about 50 amino acid residues. A linker or adapter molecule may also be designed with a cleavage site for a DNA restriction endonuclease or for a protease to allow for the separation of the fused moieties. It will be appreciated that once constructed; the fusion polypeptides can be

IE derivatized according to the methods described herein. Suitable fusion segments incfude, among others, metal binding domains (¢.g., a poly-histidine segment), immunoglobulin binding domains

Co Ge. Protein A, Protein G, T cell, B cell, Fc receptor, or complement protein antibody-binding © domains), sugar binding domsins (e.g., a maltose binding domsin), and/or a "tag" domain (i.c., 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 compounds that. bind to the domain, such as monoclonal.

Co antibodies). This tag is typically fused to the peptide or polypeptide and upon expression may _ serve 83 a means for affinity purification of the sequence of interest polypeptide from the host oC cell: - Affinity purification can be accomplished, for example, by column chromatography using "3s aiitibodies against the tag as an affinity matrix. Optionally, the tag can subsequently be removed from the purified sequence of interest polypeptide by various means such as using certain peptidases for cleavage. As described below, fusions may also be made between a TA and a co- stimulatory components such as the chemokines CXC10 (IP-10), CCL7 (MCP-3), or CCL3 (RANTES), for example. oo | A fusion motif may enhance transport of an immunogenic target to an MHC processing compartment, such as the endoplasmic reticulum, These sequences, referred to as tranduction or transcytosis sequences, include sequences derived from HIV tat (see Kim et al. 1997 J. Immunol. 159:1666), Drosophila antennapedia (see Schutze-Redelmeier et al. 1996 J. Immunol. 157.650), or human period-1 protein (hPERL1; in particular, SRRHHCRSKAKRSRHH). - In addition, the polypeptide or variant thereof may be fused to a homologous peptide or 5s polypeptide to form a homodimer or to a heterologous peptide or polypeptide to form a heterodimer. Heterologous peptides and polypeptides include, but are not limited to an epitope to allow for the detection and/or isolation of a fusion polypeptide; a transmembrane receptor protein or a portion thereof, such as an extracellular domain or a transmembrane and intracellular domain; a ligand or a portion thereof which binds to a transmembrane receptor protein; an enzyme or portion thereof which is catalytically active; a polypeptide or peptide which promotes oo oligomerization, such as a- leucine zipper domain; a polypeptide or peptide which increases stability, such as an immunoglobulin constant region; a peptide or polypeptide: which has a therapeutic activity different from the peptide or polypeptide; and/or variants thereof. SL

Co In certain embodiments, it may be advantageous to combine a nucleic acid sequence encoding. an immunogenic target with one or more co-stimulatory component(s) such as cell surface proteins, cytokines or chemokines in a composition of the present invention. The co- stimulatory component may be included in the composition as a polypeptide or as a nucleic acid encoding the polypeptide, for example. Suitable co-stimulatory molecules include; for instance, polypeptides that bind members of the CD28 family (i.e., CD28, 1COS; ‘Hutloff, et al. Nature 1999, 397: 263-265; Peach, et ol. J Exp Med 1994, 180: 2049-2058) such as the CD28 binding polypeptides B7.1 (CD80; Schwartz, 1992; Chen et al; 1992; Ellis, et al. J. Immunol., 156(8): 2700-9), B7.2 (CD86; Ellis, et al. J. Immunol, 156(8): 2700-9), and’ mutants / variants thereof (WO 00/66162); polypeptides ‘which bind members of the integrin family (i.e, LFA-1 (CD11a/

CD18); Sedwick, et al. J Immunol 1999, 162; 1367-1375; Wlfing, et al. Science 1998, 28%: 2266-2269; Lub, et al. Immunol Today 1995, 16: 479-483) including members of the ICAM family (ie, ICAM-1, -2 or -3); polypeptides which bind CD? family members (ie, CD2,

Ggnalling lymphocyte activation molecule (CDW1S0. or “SLAM”; Aversa, et al. iJ Immunol 1997, 158: 4036-4044)) such as CD58 (LFA-3; CD2 ligand; Davis, et al. Immunol oo

Today 1996, 17: 177-187) or SLAM ligands (Sayos, et al. Nature 1998, 395: 462-469); polypeptides which bind heat stable antigen (HSA or CD24; Zhou, et al. Eur J Immunol 1997, 27: 2524-2528); polypeptides which bind to members of the TNF receptor (TNFR) family. Ge,

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 1995, 162: 486-493), and

CD27 (Lens, et al. Semin Immunol 1998, 10: 491-499) such as 4-1BBL (4-1BB ligand; Vinay, otal Semin Irmunol 1998, 10: 481-48; DeBencdette, ct al. J Immunol 1997, 158: 551-559), s TNFR associated factor-1 (TRAF-1; 4-1BB ligand; Saculli, et al. J Exp Med 1998, 187: 1849— 1862, Arch, et al. Mol Cell Biol 1998, 18: 558-565), TRAF-2 (4-1BB and 0X40 ligand; Saoulli, et al. J Exp Med 1998, 187: 1849-1862; Oshima, et al. Int Immunol 1998, 10: 517-526,. "Kawamata, et al. J Biol Chem 1998, 273: 5808-5814), TRAE-3 (4-1BB and OX40 ligand; Arch, : et al. . Mol Cell Biol 1998, 18: 558-565; Jang, et al. Biochem Biophys Res Commun 1998, 242: 613-620; Kawamata S, et al. J Biol Chem 1998, 273: 5808-5814), OX40L (0X40 ligand; Gramaglia, et al. J Immunol 1998, 161: 6510-6517), TRAF-5 (0X40 ligand; Arch, et al. Mol -

Cell Biol 1998, 18: 558-565; Kawamata, et al. J Biol Chem 1998, 273: 5808-5814), and CD70 © (CD27 ligand; Couderc, et al. Cancer Gene Ther., 5(3): 163-75). CD154 (CD40 ligand or © «CDA40L": Gurunathan, et al. J. Immunol., 1998, 161: 4563-4571; Sine, ct al. Hum. Gene Ther. 2001, 12: 1091-1102) may also be suitable. 0 CL : . One or more cytokines may also be suitable co-stimulatory components or “adjuvants”, either as polypeptides or being encoded by nucleic acids contained within the compositions of the present invention (Parmiani, et al. Immunol Lett 2000 Sep 15; 74(1): 41-4; Berzofsky, etal.

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, ct al. J. Gene Med. 2000 Jul-Aug;2(4):243-9; Rao, ct al. J. Immunol. 16 3357- 3365 (1996), IL-15 (Xin, et al. Vaccine, 17:858-866, 1999), I-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-a), or 2s interferons such as IFN-a or INF-y, Other cytokines may also be suitsble for practicing the . present invention, as is known in the art -

Chemokines may also be utilized, in either polypeptide or nucleic acid form. Fusion proteins comprising CXCL10 (I-10) and CCL7 (MCP-3) fosed to a tumor self-antigen have been shown to induce anti-tumor immunity (Biragyn, et al. Nature Biotech. 1999, 17; 253-258). ‘The chemokines CCL3 (MIP-1a) and CCLS (RANTES) (Boyer, et al: Vaccine, 1999, 17 (Supp.

2): $53-S64) may also be of use in practicing the present invention. Other suitable chemokines are known in the art.

It is also known in the art that suppressive or negative regulatory immune mechanisms may be blocked, resulting in enhanced immune responses; For instance, treatment with anti- . CTLA-4 (Shrikant, et al. Immunity, 1996, 14: 145-155; Sutmuller, et al. J. Exp. Med., 2001, 194: 823-832), anti-CD25 (Sutmuller, supra), anti-CD4 (Matsui, et al. J. Immunol, 1999, 163: 184-193), the fusion protein I1.13Ra2-Fc (Terabe, et al. Nature Immunol., 2000, 1: 515-520), and combinations thereof (i.e., anti-CTLA-4 and anti-CD?25, Sutmuller, supra) have been shown to upregulate anti-tumor immune responses and would be suitable in practicing the present invention. Such treatments, among others, may also be combined with the one or more immunogenic targets of the present invention. _ To

Any of these components may be used alone or in combination with other agents. For instance, it has been shown that a combination of CD80, ICAM-1 and LFA-3 (“TRICOM”) may potentiate anti-cancer immune responses (Hodge, et al. Cancer Res. 59: 5800-5807 (1999). «Other effective combinations include, for example, IL-12 + GM-CSF (Ablers, ct al. J. Immunol., © 158: 3947-3958 (1997); Iwasaki, et al. J. Immunol, 158: 4591-4601 (1997), IL-12 + GM-CSF +

TNF-a (Ablers, et al. Int. Immunol. ‘13: 897-908 (2001)), CD80 + IL-12 (Fruend, et al. Int. J. . Cancer, 85: 508-517 (2000); Rao, et al. supra), and CD86 + GM-CSF + [1-12 (Iwasaki, supra). * One of skill in the art would be aware of additional combinations useful in carrying out the present invention. In addition, the skilled artisan would be aware of additional reagents or riethods that may be used to modulate such mechanisms. ‘These reagents and methods, as well as others known by those of skill in the art, may be utilized in practicing the present invention. : Additional strategies for improving the efficiency of nucleic acid-based immunization : may also be used including, for example, the use of self-replicating viral replicons (Caley, et al. 1999. Vaccine, 17: 3124-2135; Dubensky, et al. 2000. Mol. Med. 6: 723-732; Leitner, et al. 2000. Cancer Res. 60: 51-55), codon optimization (Liu, et al. - 2000. Mol. Ther., 1: 497-500;

Dubensky, supra; Huang, et al. 2001. J. Virol. 75: 4947-4951), in vivo electroporation (Widera, et al. 2000. J. Immunol. 164: 4635-3640), incorporation of CpG stimulatory motifs (Gurumsthan, et al. Ann. Rev. Immumol., 2000, 18: 927-974; Leitner, supra; Cho, ¢t al. J.

Immunol. 168(10):4907-13), sequences for targeting of the endocytic or ubiquitin-processing pathways (Thomson, et al. 1998. J. Virol 72; 22462252 Velders, et al. 2001. J. Immunol.

166: 5366-5373), Marek's disease virus type 1 VP22 sequences (J. Virol. 76(6):2676-82, 2002), prime-boost regimens (Gurunathan, supra; Sullivan, et al. 2000. Nature, 408: 605-609; Hanke, et al. 1998. Vaccine, 16: 439-445; Amara, et al. 2001. Science, 292: 69-74), and the use of mucosal delivery vectors such as Salmonella (Dasji, etal. 1997. Cell, 91:765-715; Woo, etal. s 2001. Vaccine, 19: 2945-2954). Other methods are known in the art, some of which are described below. | | oo . Chemotherapeutic agents, radiation, anti-angiogenic compounds, or other agents may . also be utilized in treating and / or preventing cancer using immunogenic targets (Sebti, et al.

Oncogene 2000 Dec 27;19(56):6566-73). For example, in treating metastatic melanoma, 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: 1080. 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) among others. Other suitable chemotherapeutic regimens may also be utilized. IE © Many anti-angiogenic agents are known in the art and would be suitable for co- administration with the immunogenic target vaccines and/or chemotherapeutic regimens (see, for . example, Tima, et al. 2001. Pathology Oncol. Res., 7(2): 85-94). Such agents include, for © example, physiological agents such as growth factors (ie, ANG-2, NKi24 (HGF), transforming growth factor beta (TGF-B)), cytokines (i.e., interferons such as IFN-a, -B, -1, platelet factor 4 (PF-4), PR-39), proteases (i.e. cleaved AT-IN, collagen XVIII fragment (Endostatin)), HmwKallikrein-dS plasmin fragment (Angiostatin), prothrombin-F1.2, TSP-1), proteasé inhibitors (i.e., tissue inhibitor of metalloproteases such as TIMP-1, -2, or —3; maspin; plasminogen activator-inhibitors such as PAI-1; pigment epithelium derived factor (PEDF)), »s Tumstatin (availble through ILEX, Inc), antibody products (i.e. the collagen-binding antibodies HUIV26, HUI77, XL313; anti-VEGF; anti-integrin. (ie., Vitaxin, {Lxsys))), and - glycosidases (i.c., heparinase-], -IIT). “Chemical” or modified physiological agents known ‘or ‘believed to have anti-angiogenic potential include, for example, vinblastine, taxol, ketoconazole, hs thalidomide, dolestatin, combrestatin A, rapamycin {Guba, et al. 2002, Nature Med., 8: 128- 3 135), CEP-7055 (available from Cephalon, Inc.), flavone acetic acid, Bay 12-9566 (Bayer

Corp), AG3340 (Agouron, Inc), CGS 270234 (Novartis), tetracylcine derivatives (i.e., COL-3

(Collagenix, Inc.)), Neovastat (Aeterna), BMS-275291 (Bristol-Myers Squibb), low dose 5-FU, low dose methotrexate (MTX), -irsofladine, radicicol, cyclosporine, "captopril, celecoxib, ". D45152-sulphated polysaccharide, cationic protein (Protamine), cationic peptide-VEGF, Suramin (polysulphonated napthyl ures), compounds that interfere with the function or production of VEGF (ie, SU5416 or SU6668 (Sugen), PTK787/ZK22584 (Novartis), : Distamycin A, Angiozyme (ribozyme), isoflavinoids, staurosporine derivatives, genistein,

EMD121974 (Merck KcgaA), tyrphostins, isoquinolones, retinoic acid, carboxyamidotriazole,

TNP-470, octreotide, 2: methoxyestradiol, aminosterols (i.e., squalamine), glutathione analogues

G.e., N-acteyl-L-cysteine), combretastatin A-4 (Oxigene), Eph receptor blocking agents (Nature, 414:933-938, 2001), Rh-Angiostatin, Rh-Endostatin (WO 01/93897), cyclic-RGD peptide; " accutin-disintegrin, benzodiazepenes,” humanized anti-avb3 Ab, Rh-PAI-2, amiloride, p- "* amidobenzamidine, anti-uPA ab, anti-uPAR Ab, L-phanylalanin-N-methylamides (i.e.

Batimistat, Marimastat), AG3340, and minocycline. Many other suitable agents are known in the art and would suffice in practicing the present invention. is The present invention may also be utilized in combination with “non-traditional” - methods of treating cancer. For example, it has recently been demonstrated that administration of certain anaerobic bacteria may assist in slowing tumor growth. In one study, Clostridium : novyi was modified to eliminate a toxin gene carried on a phage episome and administered to - mice with colorectal tumors (Dang, et al. P.NAS. USA, 98(26): 15155-15160, 2001). In 50 combination with chemotherapy, the treatment was shown to cause tumor necrosis in the - animals. The reagents and methodologies described in this application may be combined with such treatment methodologies. : : aE Nucleic acids encoding immunogenic targets may be administered to patients by any of ~~ several available techniques. Various viral vectors that have been successfully utilized for 5 introducing a nucleic acid to a host include retrovirus, adenovirus, adeno-associated virus © (AAV), berpes virus, and poxvirus, among others. It is understood in the art that many such viral vectors are available in the art. The vectors of the present invention may be constructed using standard recombinant techniques widely available to one skilled in the art. Such techniques may be found in common molecular biology references such as Molecular Cloning: A Laboratory "30 Manual (Sambrook, et al, 1989, Cold Spring Harbor Laboratory Press), Gene Expression . Technology (Methods in Enzymology, Vol. 185, edited by D. Goeddel, 1991. Academic Press,

San Diego, CA), and PCR Protocols: A Guide to Methods and Applications (Tnnis, et al. 1990.

Academic Press, San Diego, CA)- ~~ =

Preferred retroviral vectors are derivatives of lentivirus as well as derivatives of murine or avian retroviruses. Examples of suitable fetroviral vectors include, for example, Moloney murine Jeukemis virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV), murine mammary : qumor virus (MuMTV), SIV, BIV, HIV and Rous Sarcoma Virus (RSV). A number of retroviral vectors can incorporate multiple exogenous nucleic acid sequences. As recombinant retroviruses are defective, they require assistance in order to produce infectious vector particles. - This assistance can be provided by, for example, helper cell lines encoding retrovirus structural genes. lo - Suitable helper cell lines include ¥2, PA317 and PA12, among others. The vector virions produced ‘using such cell lines may then be used to infect a tissue cell line, such as NIH 313 cells, to produce large quantities of chimeric retroviral virions. Retroviral vectors may be administered by traditional methods (i.e, injection) or by implantation of a “producer cell line” in proximity to the target cell population (Culver, K., et al, 1994, Hum. Gene Ther., 5 (3): 343- 15° 79: Culver, K., et al, Cold Spring Harb. Symp. Quant. Biol, 59: 685-90); Oldfield, E., 1993,

Hum. Gene Ther., 4 (1): 39-69). The producer cell line is engineered to produce a viral vector and releases viral particles in the vicinity of the target cell. A portion of the released viral : particles contact the target cells and infect those cells, thus delivering a nucleic acid of the

Co present invention fo the target cell. Following infection of the target cell, expression of the “nucleic acid of the vector occurs. oo . Co

Adenoviral vectors have proven especially useful for gene transfer inta eukaryotic cells (Rosenfeld, M., et al,, 1991, Science, 252 (5004): 4314; Crystal, R., et al. 1994, Nat, Genet., 8 (1): 42-51), the study eukaryotic gene expression (Levrero, M., etal., 1991, Gene, 101 (2): 195- 202), vaccine development (Graham, F. and Prevec, L., 1992, Biotechnology, 20: 363-90), and in 5s animal models (Stratford-Perricaudet, L., et al., 1992, Bone Marrow Transplant., 9 (Suppl. 1): .151.2 ; Rich, D,, et al., 1993, Hum. Gene Ther, 4 (4): 461-16). Experimental routes for administrating recombinant Ad to different tissues in vivo have inchided intratracheal instillation (Rosenfeld, M., et al, 1992, Cell, 68 (1): 143-55) injection into muscle (Quantin, B., et al., 1992,

Proc. Natl. dcad, Sci. USA., 89 (7): 2581-4), peripheral intravenous injection (Herz, J., and

Gerard, R., 1993, Proc. Nat. Acad, Sci. USA, 90 (7): 2812-6) and stereotactic inoculation to brain (Le Gal La Salle, G., et al, 1993, Science, 259 (5097): 988-90), among others. iE

Co WO 2005/026370 PCT/US2004/028751 = Adeno-associated virus (AAV) demonstrates high-level infectivity, broad host range and . specificity in integrating into the host cell genome (Hermonat, P., et al, 1984, Proc. Natl. Acad. ©. Sci. USA, 81 (20): 6466-70). And Herpes Simplex Virus type-1 (HSV-1) is yet another attractive vector system, especially for use in the nervous system ‘because of its neurotropic 5" property (Geller, A., et al, 1991, Trends Neurosci, 14 (10): 428-32; Glorioso, et al., 1995, Mol.

Biotechnol. 4 (1): 87-99; Glorioso, et al., 1995, Annu. Rev. Microbiol., 49: 675-710). ° Poxvirus is another useful expression vector (Smith, et al. 1983, Gene, 25 (1): 21-8;

Moss, et al, 1992, Biotechnology, 20: 345-62; Moss, et al, 1992, Curr. Top. Microbiol. Immunol. : 158: 25-38: Moss, et al. 1991. Science, 252: 1662-1667)... Poxviruses shown to be useful include vaccinia, NYVAC, avipox, fowlpox,. canarypox, ALVAC, and ALVAC(2), among "others. - oo © NYVAC (vP866) wes derived from the Copenhagen vaccine strain of vaccinia virus by deleting six nonessential regions of the genome encoding known or potential virulence factors © (see; for example, U.S. Pat. Nos. 5,364,773 and 5,494,807). The deletion loci were also engineered as recipient loci for the insertion of foreign genes. The deleted regions are: "thymidine kinase gene (TK; J2R); hemorrhagic region (1; B13R+B14R); A type inclusion body oo region (ATI; A26L); hemagglutinin gene (HA; A56R); host range gene region (C7TL-KI1L); and, large subunit, ribonucleotide reductase (14L). NYVAC is a genetically engineered vaccinia virus © gtrain that was generated by the specific deletion of eighteen open reading frames encoding gene % products associated with virulence and host range. NYVAC has been show to be useful for expressing TAs (see, for example, U.S. Pat. No. 6,265,189). NYVAC (vP866), vP994, vCP205, © yCP1433, placZH6HA4Lreverse, pMPCSH6K3E3 and pC3HEFHVB were also deposited with the © ATCC under the terms of the Budapest Treaty, accession numbers VR-2559, VR-2558, VR- ©. 2557,VR-2556, ATCC-97913, ATCC-97912, and ATCC-97914, respectively.

ALVAC-based recombinant viruses (i.c., ALVAC-1 and ALVAC-2) are also suitable for © use in practicing the present invention (see, for example, U.S. Pat. No. 5,756,103). ALVAC(2) is identical to ALVAC(1) except that ALVAC(2) genome comprises the vaccinia E3L and K3L "genes under the control of vaccinia promoters (U.S. Pat. No. 6,130,066; Beatti ef al, 19952, 11995b, 1991; Chang et al, 1992; Davies et al, 1993). Both ALVAC(1) and ALVAC(2) have "30 been demonstrated to be useful in expressing foreign DNA sequences, such as TAs (Tartaglia et al, 1993 ab; U.S. Pat. No. 5,833,975). ALVAC was deposited under the terms of the Budapest 21 :

Treaty with the American Type Culture Collection (ATCC), 10801 University Boulevard, © Manassas, Va. 20110-2209, USA, ATCC accession number VR-2547. ©" Another useful poxvirus vector is TROVAC. TROVAC refers to an attenuated fowlpox that was a plaque-cloned isolate derived from the FP-1.vaccine strain of fowlpoxvirus which is s licensed for vaccination of 1 day old chicks. TROVAC was likewise deposited under the terms oo of the Budapest Treaty with the ATCC, accession number 2553. : * «“Non-viral” plasmid vectors may also be suitable in practicing the present invention.

Preferred plasmid vectors are compatible with bacterial, insect, and / or mammalian host cells. : Such vectors inchude, for example, PCR-II, pCR3, and pcDNA3.1 (Invitrogen, San Diego, CA), : 10. pBSII (Stratagene, La Jolla, CA), pET1S (Novagen, Madison, WD, pGEX (Pharmacia Biotech,

Piscataway, NJ), pEGFP-N2 (Clontech, Palo Alto, CA), pETL (BlueBacll, Invitroger), pDSR- alpha (PCT pub. No. WO 90/14363) and pFastBacDual (Gibco-BRL, Grand Island, NY) as well as Bluescript® plasmid derivatives (a high copy number COLE1-based phagemid, Stratagene

Cloning Systems, La Jolla, CA), PCR cloning plasmids designed for cloning Taq-amplified PCR ~~ © 4s products (e.g., TOPO™ TA cloning® kit, PCR2.1® plasmid derivatives, Invitrogen, Carlsbad, oo CA). Bacterial vectors may also be used with the current invention. These vectors include, for example, Shigella, Salmonella, Vibrio cholerae, Lactobacillus, Bacille calmette guérin (BCG), - and Streptococcus (see for example, WO 88/6626; WO 90/0594; WO 91/13157; WO 92/1796; . and WO 92/21376). Many other non-viral plasmid expression vectors and systems are known in the art and could be used with the current invention. -

Suitable nucleic acid delivery techniques include DNA-ligand complexes, adenovirus- = ligand-DNA complexes, direct injection of DNA, CaPO, precipitation, gene gun techniques, oo “electroporation, and: colloidal dispersion systems, among others. Colloidal dispersion systems “include macromolecule complexes, nanocapsules, microspheres, beads, and tipid-based systems : “including oil-in-water emulsions, micelles, mixed micelles, and liposomes. The preferred colloidal system of this invention is a liposome, which are artificial membrane vesicles useful as delivery vehicles in vitro and in vivo. RNA, DNA and intact virions can be encapsulated within + the aqueous interior and be delivered to cells in a biologically active form (Fraley, R,etal, oo. 198], Trends Biochem. Sci., 6: 77). The composition of the liposome is usually a combination of phospholipids, particularly high-phase-transition-teinperature phospholipids, usually in oo | py combination with steroids, especially cholesterol. Other phospholipids or other lipids may also be used. The physical characteristics of liposomes depend on pH, jonic strength, and the presence of divalent cations. Examples of lipids useful in liposome production include phosphatidyl ~~ compounds, such as phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides.

Particularly useful are diacylphosphatidylglycerols, where the lipid moiety contains from 14-18 carbon atoms, particularly from 16-18 carbon atoms, and-is saturated. Dlustrative phospholipids include egg phosphatidylcholine, dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine. oo

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

Table I B

Types of Immunologic Adjuvants RE oo

EE | aruree| setcruminims

Adjuvant General Examples Specific Examples/References adjuvants

Microbial © ag and Clements, 1999 —— [ep oo ich and Myers, 199 . Other bacterial CpG oligonucleotides (Corral and oo . : Petray, 2000), BCG sequences (Krieg, ct al. Nature, 374:576), tetanus toxoid

KE (Rice, etal. J. Immumol. , 2001, 167: J ) . - , { 1558-1565 .

Ni } Polymer microspheres : ni

SCOMs

Oil-cmulsion and Microfluidized emulsions : we based - Allison, 1999 adjuvants Spm [QS2l(Kensi}, 196) =

Threony-MDP (Allison, 199 1 23 .

_ 168(10):4914-9 ~ Administrafion of a composition of the present invention to a host may be accomplished "using any of a variety of techniques known to those of skill in the art. The compositions) may be processed in accordance with conventional methods of pharmacy to produce medicinal agents for administration to- patients, including humans and other mammals (i.e., 8 “sharmaceutical composition”). The pharmaceutical composition is preferably made in the form of a dosage unit : containing a given amount of DNA, viral vector particles, polypeptide or peptide, for example. A oo guitable daily dose for a human or other mammal may vary widely depending onthe condition of © ‘the patient and other factors, but, once again, can be determined using routine methods. -

The pharmaceutical composition may be administered orally, parentally, by inhalation spray, rectally, intranodally, or topically in dosage unit formulations containing conventional ~ - pharmaceutically acceptable camiers, adjuvants, and vehicles. The term “pharmaceutically acceptable carrier” or “physiologically acceptable carrier” as used herein refers to one or more oo formulation materials suitable for accomplishing or enhancing the delivery of a nucleic acid, polypeptide, or peptide as a pharmaceutical composition. A “pharmaceutical composition” is a a. composition comprising a therapeutically effective amount of a nucleic acid or polypeptide. The terms “effective amount” and “therapeutically effective amount” each refer to the amount of a . nucleic acid or polypeptide used to induce. or enhance an effective immune response. [tis preferred that compositions of the present invention provide for the induction or enhancement of 20. an anti-tumor immune response in a host which protects the host from the development of a : amar and / or allows the host to eliminate an existing tumor from the body. oo .~. ... For oral administration, the pharmaceutical composition may be of any of several forms including, for example, a capsule, a tablet, a suspension, or liquid, among others. Liquids may "be administered by injection as a composition with suitable carriers including saline, dextrose, or +25 water. The term parenteral as used herein includes subcutaneous, intravenous, intramuscular, intrasternal, infusion; or intraperitoneal administration. Suppositories for rectal administration of the drug can be prepared by mixing the drug with a auitable non-irritating excipient Such as . © cheoa butter and polyethylene glycols that are solid at ordinary temperatures but liquid at the rectal temperature. :

Co 24 : : ee

The dosage regimen for immunizing a host or otherwise treating a. disorder or a disease with a composition of this invention is based on a variety of factors, including the type of disease, the age, weight, sex, medical condition of the patient, the severity of the condition, the route of administration, and the particular compound employed. For example, 8 poxviral vector may be administered as a composition comprising 1 x 10° infectious particles per dose. Thus, the dosage regimen may vary widely, but can be determined routinely using standard methods.

A prime-boost regimen may also be utilized (WO 01/30382 Al) in which the targeted. immunogen is initially administered in a priming step in one form followed by a boosting step in which the targeted immunogen is administered in another form. The form of the targeted - immunogen in the priming and boosting steps are different. For instance, if the priming step utilized a nucleic acid, the boost may be administered as a peptide. Similarly, Where a priming step utilized one type of recombinant virus (ie. ALVAC), the boost step may utilize another type of virus (ie, NYVAC). This prime-boost method of administration has been shown to induce strong immunological responses. Various combinations of forms are suitable in practicing the present invention. | IE

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

Injectable preparations, such as sterile injectable aqueous or oleaginous suspensions, may be formulated according to known methods using suitable dispersing or wetting agents and suspending agents. The injectable preparation may also be a sterile injectable solution or . 25 suspension in a non-toxic parenterally acceptable diluent or solvent. Suitable vehicles and . solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution, among others. For instance, a viral vector such as a poxvirus may be prepared in 0.4%

NaCl. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending’ oo medium. For this purpose, any biand fixed oil may be employed, including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.

For topical administration, a suitable topical dose of a composition may be administered one to four, and preferably two or three times daily. The dose may also be administered with intervening days during which no does is applied. Suitable compositions may comprise from 0.001% to 10% w/w, for example, from 1% to 2% by weight of the formulation, although it may comprise as much as 10% w/w, but preferably not more than 5% w/w, and more preferably from oo 0.1% to 1% of the formulation. Formulations suitable for topical administration include liquid or semi-liquid preparations suitable for penetration through the skin (e.g., liniments, lotions, ointments, creams, or pastes) and drops suitable for administration to the eye, ear, Or nose. : The pharmaceutical compositions may also be prepared in a solid form (including granules, powders or. suppositories). The pharmaceutical compositions may be subjected to oe conventional pharmaceutical operations such as sterilization and/or may contain conventional a “adjuvants, such as preservatives, stabilizers, wetting agents, emulsifiers, buffers etc. Solid dosage forms for oral administration may include capsules, tablets, pills, powders, and granules. oo - In such solid dosage forms, the active compound may be admixed with at least one inert diluent such as SUCTOSE, lactose, or starch. Such dosage forms may also comprise, as in normal practice, . additional substances other than inert diluents, e.g., lubricating agents such as’ magnesium stearate. In the case of capsules, tablets, and pills, the dosage forms may also comprise buffering agents. Tablets and pills can additionally be prepared with enteric coatings. Liquid dosage + forms for oral. administration may include pharmaceutically acceptable emulsions, solutions, % suspensions, syiups, and elixirs containing inert diluents commonly used in the art, such as water. Such compositions may also comprise adjuvants, such as wetting sweetening, flavoring, - and perfuming agents. : :

Ce RE Pharmaceutical compositions comprising a nucleic acid or polypeptide of the present - invention may take any of several forms and may be administered by any of several routes. In preferred embodiments, the compositions are administered via a parenteral route (intradermal, . - jptramuscular or subcutaneous) to induce an immune response in the host. Alternatively, the composition may be administered directly into a lymph node (intranodal) or tumor mass-(i.e., intratumoral administration). For example, the dose could be administered subcutaneously at days 0, 7, and 14. Suitable methods for immunization using compositions comprising TAs are “46 known in the art, as shown for ps3 (Hollstein et al, 1991), p21-ras (Almoguera et al., 1988),

HER-2 (Fendly et al., 1990), the melanome-associated antigens (MAGE-1; MAGE-2) (van der

: Bruggen etal,, 1991), p97 (Hu et al, 1988), melanoma-associated antigen E.(WO 99130737) and carcinoembryonic antigen (CBA) (Kantor et al., 1993; Fishbein et al, 1992; Kaufman et al. 1991), among others.

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

Co agents which reduce or alleviate ill effects of antineoplastic, anti-tumor or anti-cancer agents. oo A kit comprising a composition of the present invention is also provided. The kit can include a separate container containing a suitable carrier, diluent or excipient. The kit can also include an additional anti-cancer, anti-tumor or antineoplastic agent and/or an agent that reduces or alleviates ill effects of antineoplastic, anti-tumor or anti-cancer agents for co- or sequential- administration. Additionally, the kit can include instructions for mixing or combining ingredients and/or administration. . oo Bh 0 oo . oo

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

Example 1 :

Construction of the Multi-Antigen Construct vI416 + .. The expression .vector VvT416 (ALVAC-NY-ESO-1/Trp-2-LFA-3/ICAM-1/B7.1-

E3L/K3L) was constructed in the ALVAC vector using standard techniques. DNA sequences’ oo encoding NY-ESO-1, Trp-2, LFA-3, ICAM-1, B7.1, vwE3L and vwK3L were inserted into various loci within the ALVAC genome. DNA sequences encoding 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 CS locus. DNA sequences encoding LFA-3 (Wallner, et al. (1987) 1. 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 locus. LFA-3, ICAM-1 and B7.1 form an expression cassette known as TRICOM. DNA sequences encoding VE3L (Chang, et al. 1952. Proc. Netl. 3

Acad. Sci. U. S. A 89:4825-4829) and vK3L (Beattie, et al. 1991. Virology 183:419-422) were inserted into the C6 locus. Promoters were utilized as follows: _ © TableIll

Cw. -

Promoter SE/L is described by Chakrabarti, et al. (BioTechniques 23: 1094-1097, 1997). oo "The donor plasmids utilized are shown below: oo ce SE 5 : :

TablelV : : , Plasmid | size(p) | Vector | Antibiotic } Resitance Gene | . : ~~ pMPCSH6K3E3 _ | pBSSK | Amp.

FAVA eon(C PI | 1040 | pBSSK | Amp [pris | 11,154 | pBSSK | Amp ) NY-ESO-1 and TRP-2 DNA sequences were inserted into the ALVAC donor plasmid pT1132. This donor plasmid was then used with pALVAC.Tricom(C3) #33 to generate the

ALVAC-TRICOM recombinant expressing these genes using standard techniques. The plasmids pALVAC.Tricom(C3) #33 and pT1132 are shown in Figure 1. - The DNA sequences of pALVAC.Tricom(C3) #33 and pT1132 are shown in Figures 2 and 3, respectively.

© WO 2005/026370 PCT/US2004/028751

Example 2

Construction of the Multi-Antigen Construct vI419

The expression vector vT419 (ALVAC-gp100M/Mart-1/ Mage-1,3 minigene-LFA- 3/ICAM-1/B7.1-B3L/K3L) was constructed in the ALVAC vector using standard techniques.

DNA sequences encoding the gpl 00M/MART-1/MAGE-1,3 minigene, LFA-3, ICAM-1, B7.1, vvE3L and vvK3L were inserted into various loci within the ALVAC genome. The gp100M/

MART-1/MAGE-1,3 minigene was inserted into the C5 locus. 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 locus. "10 LFA-3, ICAM-1 and B7.1 form an expression cassette known as TRICOM. DNA sequences encoding VvE3L (Chang, et al. 1992. Proc. Natl. Acad. Sci. U. S. A 89:4825-4829) and vwK3L + - (Beattie, et al. 1991. Virology 183:419-422) were inserted into the C6 locus. Promoters were utilized as follows: : 5s : TableV

DE A ERY wr | vecmefis vo Ee — a a

Promoter sF/L is described by Chakrabarti, et al. (BioTechniques 23: 1094-1097, 1997).

The donor plasmids utilized are shown below:

Table VI oo

Plasmid | Size (bp) Vector Antibiotic oo Resitance Gene _ >ALVAC.Tricom(C3) #33 10470 | pBSSK | Amp gp100(M), Mart-1 and Mage-1,3 minigene were inserted into the ALVAC C5 donor © + plasmid pT3217. This donor plasmid was then used with pALVAC. Tricom(C3) #33 to generate : the ALVAC-TRICOM recombinant expressing these genes using standard techniques. This donor plasmid inserts into the C5 site. pALVAC.Tricom(C3) #33 is shown in Figures 1'and2. s The pT3217 plasmid is shown in Figure 4. The DNA sequence of pT3217 is shown in Figure.

B Immunological Assessment of Multi-Antigen Vectors :

The results of the first animal experiment indicated a trend toward higher immunological - responses to three (Mart 1, NY-ESO-1 and gp100) of the four antigens when the vaccine was given as two separate injections. However, thesé differences were not statistically significant. In detail, HLA-A2/K® transgenic mice (S/group) were immunized subcutaneously with vT419 (ALVAC(2)-gp! 00M/MART-1/MAGE-1/3 minigene/TRICOM) and vI416 (ALVAC(2}TRP- 2/NY-ESO-1/TRICOM) either combined at one site or given as separate injections. Control mice is were immunized with parental ALVAC(2). Mice were vaccinated three times (at three week intervals), and three weeks after the last boost T cell responses in individual mice were analyzed by IFN-g ELISPOT and CTL assays following in vitro restimulation with peptide. Compared to contro} animals, mice vaccinated with the multi-antigen vectors (at 2 sites) exhibited statistically significant ELISPOT responses against MART-1. The IFN-gamma response to gpl00M and NY- ‘20 ESO-1 were also detectable, although these responses were not statistically significant due to response variability and the small number of cultures tested. ELISPOT responses. against the

TRP-2 antigen were elevated in all groups tested (including control animals), presumably due to the fact that the dominant A2-restricted TRP-2 peptide (180-188) cross-reacts with H-2K® and can induce low avidity T cell responses in naive mice following in vitro, culture, and: were 55 therefore not statistically significant. Interestingly, ELISPOT responses in mice injected withan admixture of vI416 and vT419 were generally lower them in mice receiving each virus separately, although these differences did not achieve statistical significance. The CTL data were largely negative, except for one strong anti-gp100 response and one marginal anti-MART-1 response, both of which occurred in mice vaccinated with vT416 and VT419 {two sites). Overall, : 30 these results provided encouraging data that establish that the multi-antigen vectors can generate

EE Co responses against MART-1, and suggest that anti-gp100 and anti-NY-ESO-1 responses can also Co © beinduced. = . Two additional pre-clinical animal studies have been completed using the melanoma multi-antigen ALVAC recombinants. In these experiments, HLA-A2/K® transgenic’ mice 's (5/group) were immunized subcutaneously with vI419 (ALVAC(2)-gp100M/MART-1/MAGE- 1/3 minigene/TRICOM) and vT416 (ALVAC(2)-TRP-2/NY-ESO-1/TRICOM) either combined at one site or given as separate injections. Control mice were immunized with parental . ALVAC(2). After vaccination, the T cell responses in individual mice were assessed by IFN- . gamma ELISPOT assay following in vitro restimulation with peptide. Unlike the previous multi- antigen experiment, which provided encouraging immunogenicity data, the two most recent studies generated inconclusive data, due to high background responses in control immunized ." animals. Therefore, overall the results were deemed as inconclusive. : o To confirm the immunogenicity of the multi-antigen constructs, and to repeat results "from the first study, another preclinical animal study has ‘been completed.” HLA-A2ZK® = - transgenic mice’ (10/group) were immunized subcutaneously with VI419 (ALVACE)- .- gpl 0OM/MART-1/MAGE-1/3 minigene/TRICOM) and vT416 (ALVAC(2)-TRP-2/NY-ESO- "1/TRICOM) given as separate injections. Control mice were immunized with parental

Co ALVAC(2). Statistically significant ELISPOT responses were detectable against gp100, Mart-1 : and TRP-2, and some responses were detected against NY-ESO-1, which were at the border of being statistically significant. : I - © While the present invention has been-described in terms of the preferred embodiments, it ©. is understood that variations and modifications will occur to those skilled in the art. Therefore, it

SS intended that the appended claims cover all such equivalent variations that come within the scope of the invention as claimed. Ll nN ps

Claims (19)

What is claimed is: Co

1. An expression vector for co-expressing at least two immunogenic targets, wherein said immunogenic targets are selected from the group consisting of NY-ESO-1, TRP-2, gp100, ¢p100M, a MART antigen, MART-1, a MAGE antigen, MAGE-1, and MAGE-3.’ Co

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

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

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

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

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

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

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

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

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

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

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

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

14. The expression vector of claim 13 wherein the viral vector is a poxvirus selected: from the 36 group consisting of vaccinia, NYVAC, avipox, canarypox, ALVAC, ALVAC(2), fowlpox, © and TROVAC. | SE SE 32 . } ,

: © WO 2005/026370 PCT/US2004/028751

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

16. The expression vector of any one claims 11-15 wherein the co-stimulatory component is human B7.1. 0

11.A composition comprising an expression vector of any one of claims 1-16 in a pharmaceutically acceptable carrier.

18. A method for preventing or treating cancer comprising administering to a host an expression vector of any one of claims 1-16.

19. A method for preventing or treating cancer comprising administering to a host a composition _ ofclaim 17. | -