WO2003095642A2 - Polyepitopes and mini-genes for cancer treatment - Google Patents

Abstract

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

Description

POLYEPITOPES AND MINI-GENES FOR CANCER TREATMENT

RELATED APPLICATIONS

This application claims priority to U.S. 60/378,416 filed May 7, 2002.

FIELD OF THE INVENTION

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

BACKGROUND OF THE INVENTION

One goal of most current vaccination strategies is to optimize the elicitation of T cell responses against defined class I-restricted epitopes (Raychaudhuri, et al. Nature Biotech, 1998, 16: 1025-1031; Moingeon, J. Biotech., 2002, in press). Importantly, strategies are needed to elicit balanced immune responses against multiple T cell epitopes, both immunodominant and cryptic, presented at the same time to the immune system. In this regard, epitope-based strategies might offer a means to boost responses against weak or cryptic epitopes, some of which may be important to protective or immunotherapeutic responses (Thomson, et al. Proc. Natl. Acad. Sci. (USA) 1995; 92:5845-5849; Thomson, et al. J. Immunol. 1996;157:822-826). The approaches which have been evaluated as of today include the expression of nonamer peptide sequences as single epitope minigenes (Anton, et al. J. Immunol. 1997; 158: 2535-2542), as well as the expression of linked epitopes in a string of beads configuration, such that a single polypeptide is produced containing multiple epitopes (Thomson, supra; Anton, supra; An, et al. J. Virol 1997;71:2292-2302; Mateo, et al. J. Immunol. 1999;163:4058-4063; Ishioka, et al. J. Immunol. 1999;162:3915-3925; Elliott, et al. Vaccine 1999;17:2009-2019). An advantage of the minigene approach is that further proteasomal processing is not required for association with MHC class I molecules. However, in order to immunize against a variety of epitopes, many constructs would be required. Thus, the advantage of the polyepitope approach is that a single constmct could potentially immunize against many epitopes. However, a successfull immunization with polyepitope-based vaccines requires that each component epitope is successfully processed by the proteasome and that cryptic epitopes are recognized by the immune system even in the presence of immunodominant epitopes.

Inducing such a balanced immune response against multiple T cell epitopes is desirable in the development of cancer vaccines to fully exploit the diversity of the human T cell repertoire, in its capacity to recognize tumor-associated antigens (TAAs). One approach to optimize the immunogenicity of cryptic epitopes is to modify anchor residues in order to enhance the association with MHC class I. By altering the class I anchor residues in epitopes from the melanoma TAA gplOO from non-optimal to optimal amino acids, both higher affinity association with class I and an enhanced ability to stimulate T cells were observed. Further, immunization of cancer patients with such modified epitopes resulted in readily detectable clinical responses in at least some patients, indicating that some of these T cells could recognize native gp 100 expressed by tumor cells.

There is a need in the art for reagents and methodologies useful in stimulating an immune response to prevent or treat cancers. The present invention provides such reagents and methodologies which overcome many of the difficulties encountered by others in attempting to treat cancers such as cancer. To this end, an epitope-based strategy has been utilized to elicit optimized and well-balanced T cell responses against a range of HLA-A2.1.1 restricted epitopes from the g lOO and Melan A/MART- 1 molecules. These tumor- associated molecules could be used as targets for a melanoma vaccine, for example. To facilitate the induction of T cell responses, the epitopes may be delivered using a recombinant DNA vector such as a plasmid or virus.

SUMMARY OF THE INVENTION

The present invention provides an immunogenic target for administration to a patient to prevent and / or treat cancer. In one embodiment, the immunogenic target is a tumor antigen ("TA") expressed from a recombinant DNA molecule comprising multiple epitopes or one or more mini-genes derived from a TA-encoding nucleic acid sequence. In certain embodiments, the recombinant DNA is a plasmid or other delivery vector, such as a recombinant virus. The TA and / or AA may also be administered in combination with an immune stimulator, such as a co-stimulatory component or adjuvant. BRIEF DESCRIPTION OF THE DRAWINGS

Figures 1A-C. Lysis by TIL lines of J82 target cells infected with ALVAC-polyepitope recombinants. Panel A, MART-l^27"35-specifιc lysis by TIL 620; Panel B, gplOO^154"162- specific lysis by TIL 1200; Panel C, gpl00 ^09_217-specific lysis by TIL 620. The X axis of each panel indicates J82 cells modified by pulsing with epitope peptides (MART27 pep =

MART-1^27"35, gpl00-154 pep = gplOO^154"162, gpl00-209 pep = gplOO^209"217, gpl00-280 pep = gplOO^280"288) or infected with ALVAC recombinants expressing full length TAAs (AL-

MART-1, AL-gplOO), polyepitope constructs (AL-PE01, AL-PE02, AL-PE03), or single epitope constructs (AL-MART-27, AL-gpl00-154, AL-gpl00-209, AL-gp 100-280). Data shown was obtained at an E:T ratio of 50, and lytic activity was determined in triplicates

(with standard deviation < 5%) after 4 hours of incubation. As a negative control, lysis of impulsed or uninfected J82 cells by TIL lines was always < 5%. Discrimination of MART^27" and gplOO ^" killing by TIL 620 was performed by parallel assays in the presence and absence of cold target J82 cells infected with ALVAC-MART-27.

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 immune response against one or more tumor antigens ("TA") to prevent and / or treat cancer. In certain embodiments, one or more TAs may be combined. In preferred embodiments, the immune response results from expression of a TA in a host cell following administration of a nucleic acid vector encoding the tumor antigen or the tumor antigen itself in the form of a peptide or polypeptide, for example. In other preferred embodiments, the TA is administered as part of a "polyepitope" in which multiple Tas or immunogenic portions thereof are encoded on a single recombinant DNA molecule. In still other preferred embodiments, the TA is administered as a "mini-gene" in which a minimal immunogenic portion of the TA is encoded by the recombinant DNA molecule.

As used herein, an "antigen" is a molecule (such as a polypeptide) or a portion thereof that produces an immune response in a host to whom the antigen has been administered. The immune response may include the production of antibodies that bind to at least one epitope of the antigen and / or the generation of a cellular immune response against cells expressing an epitope of the antigen. The response may be an enhancement of a current immune response by, for example, causing increased antibody production, production of antibodies with increased affinity for the antigen, or an increased cellular response (i.e., increased 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, a TA may be referred to as an "immunogenic target".

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

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, gplOO); mutational antigens (i.e., MUM-1, p53, CDK-4); overexpressed 'self antigens (i.e., HER-2/neu, p53); and, viral antigens (i.e., HPV, EBV). For the purposes of practicing the present invention, a suitable TA is any TA that induces or enhances an anti-tumor immune response in a host to whom the TA has been administered. Suitable TAs include, for example, gplOO (Cox et al., Science, 264:716-719 (1994)), MART-1/Melan A (Kawakami et al., J. Exp. Med., 180:347-352 (1994)), gp75 (TRP-1) (Wang et al, J. Exp. Med., 186:1131-1140 (1996)), tyrosinase (Wolfel et al., Eur. J. Immunol, 24:759-764 (1994); WO 200175117; WO 200175016; WO 200175007), NY-ESO-1 (WO 98/14464; WO 99/18206), melanoma proteoglycan (Hellstrom et al., J. Immunol, 130:1467-1472 (1983)), MAGE family antigens (i.e., MAGE-1, 2,3,4,6,12, 51; Van der Bruggen et al., Science, 254:1643-1647 (1991); U.S. Pat. Nos. 6,235,525; CN 1319611), BAGE family antigens (Boel et al., Immunity, 2:167-175 (1995)), GAGE family antigens (i.e., GAGE-1,2; Van den Eynde et al., J. Exp. Med., 182:689-698 (1995); U.S. Pat. No. 6,013,765), RAGE family antigens (i.e., RAGE-1; Gaugler et at., Immunogenetics, 44:323-330 (1996); U.S. Pat. No. 5,939,526), N- acetylglucosaminyltransferase-V (Guilloux et at., J. Exp. Med., 183:1173-1183 (1996)), pl5 (Robbins et al., J. Immunol. 154:5944-5950 (1995)), β-catenin (Robbins et al., J. Exp. Med., 183:1185-1192 (1996)), MUM-1 (Coulie et al., Proc. 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, Proc. Natl. Acad. Sci. USA, 92:11993-11997 (1995)), pl85 HER2/neu (erb-Bl; Fisk et al., J. Exp. Med., 181:2109-2117 (1995)), epidermal growth factor receptor (EGFR) (Harris et al., Breast Cancer Res. Treat, 29:1-2 (1994)), carcinoembryonic antigens (CEA) (Kwong et al., J. Natl. Cancer Inst., 85:982-990 (1995) U.S. Pat. Nos. 5,756,103; 5,274,087; 5,571,710; 6,071,716; 5,698,530; 6,045,802; EP 263933; EP 346710; and, EP 784483; CAP(6D), CAP(6D)-1,2); carcinoma-associated mutated mucins (i.e., MUC-1 gene products; Jerome et al., J. Immunol, 151:1654-1662 (1993)); EBNA gene products of EBV (i.e., EBNA-1; Rickinson et al., Cancer Surveys, 13:53-80 (1992)); E7, E6 proteins of human papillomavirus (Ressing et al., J. Immunol, 154:5934-5943 (1995)); prostate specific antigen (PSA; Xue et al., The Prostate, 30:73-78 (1997)); prostate specific membrane antigen (PSMA; Israeli, et al., Cancer Res., 54:1807- 1811 (1994)); idiotypic epitopes or antigens, for example, immunoglobulin idiotypes or T cell receptor idiotypes (Chen et al., J. Immunol, 153:4775-4787 (1994)); KSA (U.S. Patent No. 5,348,887), kinesin 2 (Dietz, et al. Biochem Biophys Res Commun 2000 Sep 7;275(3):731- 8), HIP-55, TGFβ-1 anti-apoptotic factor (Toomey, et al. Br J Biomed Sci 2001;58(3):177- 83), tumor protein D52 (Bryne J.A., et al., Genomics, 35:523-532 (1996)), H1FT, NY-BR-1 (WO 01/47959), NY-BR-62, NY-BR-75, NY-BR-85, NY-BR-87, NY-BR-96 (Scanlan, M. Serologic and Bioinformatic Approaches to the Identification of Human Tumor Antigens, in Cancer Vaccines 2000, Cancer Research Institute, New York, NY), AAC2-1, or AAC2-2, including "wild-type" (i.e., normally encoded by the genome, naturally-occurring), modified, and mutated versions as well as other fragments and derivatives thereof. Any of these TAs may be utilized alone or in combination with one another in a co-immunization protocol.

In certain cases, it may be beneficial to co-immunize patients with both TA and other antigens, such as angiogenesis-associated antigens ("AA"). An AA is an immunogenic molecule (i.e., peptide, polypeptide) associated with cells involved in the induction and / or continued development of blood vessels. For example, an AA may be expressed on an endothelial cell ("EC"), which is a primary structural component of blood vessels. Where the cancer is cancer, it is preferred that that the AA be found within or near blood vessels that supply a tamor. 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 (i.e., VEGF; Bemardini, et al. J. UroL, 2001, 166(4): 1275-9; Starnes, et al. J. Thorac. Cardiovasc. Surg., 2001, 122(3): 518-23; Dias, et al. Blood, 2002, 99: 2179-2184), the VEGF receptor (i.e., VEGF-R, flk-1/KDR; Starnes, et al. J. Thorac. Cardiovasc. Surg., 2001, .122(3): 518-23), EPH receptors (i.e., EPHA2; Gerety, et al. 1999, Cell, 4: 403-414), epidermal growth factor receptor (i.e., EGFR; Ciardeillo, et al. Clin. Cancer Res., 2001, 7(10): 2958-70), basic fibroblast growth factor (i.e., bFGF; Davidson, et al. Clin. Exp. Metastasis 2000,18(6): 501-7; Poon, et al. Am J. Surg., 2001, 182(3):298-304), platelet-derived cell growth factor (i.e., PDGF-B), platelet-derived endothelial cell growth factor (PD-ECGF; Hong, et al. J. Mol. Med., 2001, 8(2):141-8), transforming growth factors (i.e., TGF- ; Hong, et al. J. Mol. Med., 2001, 8(2):141-8), endoglin (Balza, et al. Int. J. Cancer, 2001, 94: 579-585), Id proteins (Benezra, R. Trends Cardiovasc. Med., 2001, 11(6):237-41), proteases such as uPA, uPAR, and matrix metalloproteinases (MMP-2, MMP-9; Djonov, et al. J. Pathol., 2001, 195(2): 147- 55), nitric oxide synthase (Am. J. Ophthalmol., 2001, 132(4):551-6), aminopeptidase (Rouslhati, E. Nature Cancer, 2: 84-90, 2002), thrombospondins (i.e., TSP-1, TSP-2; Alvarez, et al. Gynecol. Oncol., 2001, 82(2):273-8; Seki, et al. Int. J. Oncol., 2001, 19(2):305-10), k- ras (Zhang, et al. Cancer Res., 2001, 61(16):6050-4), Wnt (Zhang, et al. Cancer Res., 2001, 61(16):6050-4), cyclin-dependent kinases (CDKs; Drug Resist. Updat. 2000, 3(2):83-88), microtubules (Timar, et al. 2001. Path. Oncol. Res., 7(2): 85-94), heat shock proteins (i.e., HSP90 (Timar, supra)), heparin-binding factors (i.e., heparinase; Gohji, et al. Int. J. Cancer, 2001, 95(5):295-301), synthases (i.e., ATP synthase, thymidilate synthase), collagen receptors, integrins (i.e., αυβ3, αυβ5, αlβl, α2βl, α5βl), the surface proteolglycan NG2, AAC2-1, or AAC2-2, among others, including "wild-type" (i.e., normally encoded by the genome, naturally-occurring), modified, mutated versions as well as other fragments and derivatives thereof. Any of these targets may be suitable in practicing the present invention, either alone or in combination with one another or with other agents and may be also be referred to as "immunogenic targets".

Polyepitopic preparations may also be utilized to generate an immune response. For example, particular immunogenic regions of a TA or AA may be combined and co- administered to a patient. The immunogenic regions may be combined either as peptides, polypeptides or nucleic acid sequences. The nucleic acid sequences may be combined into a single or multiple recombinant plasmids or viral vectors and administered, for instance. In one embodiment, nucleic acid sequences encoding immunogenic regions of Melan-A/MART- 1 or gplOO are incorporated into a recombinant DNA molecule. Exemplary immunogenic regions of TAs include, for example, MART-1^27"35, gplOO^154"162, gplOO^209"217, gplOO^280"288, gpl00-209-2M, and gplOO-280-9V. Spacers such as nucleic acid sequences encoding the amino acid sequence AAA or NKRK may be optionally included to separate the immunogenic regions from one another. The immunogenic regions may also be utilized separately as "mini-genes".

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

An isolated nucleic acid molecule is one that: (1) is separated from at least about 50 percent of proteins, lipids, carbohydrates, or other materials with which it is naturally found when total nucleic acid is isolated from the source cells; (2) is not be linked to all or a portion of a polynucleotide to which the nucleic acid molecule is linked in nature; (3) is operably linked to a polynucleotide which it is not linked to in nature; and / or, (4) does not occur in nature as part of a larger polynucleotide sequence. Preferably, the isolated nucleic acid molecule of the present invention is substantially free from any other contaminating nucleic acid molecule(s) or other contaminants that are found in its natural environment that would interfere with its use in polypeptide production or its therapeutic, diagnostic, prophylactic or research use. As used herein, the term "naturally occurring" or "native" or "naturally found" when used in connection 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 nucleic acid or polypeptide molecules is determined by comparing the sequences. As known in the art, "identity" means the degree of sequence relatedness between nucleic acid molecules or polypeptides as determined by the match between the units making up the molecules (i.e., nucleotides or amino acid residues). Identity measures the percent of identical matches between the smaller of two or more sequences with gap alignments (if any) addressed by a particular mathematical model or computer program (i.e., an algorithm). Identity between nucleic acid sequences may also be determined by the ability of the related sequence to hybridize to the nucleic acid sequence or isolated nucleic acid molecule. In defining such sequences, the term "highly stringent conditions" and "moderately stringent conditions" refer to procedures that permit hybridization of nucleic acid strands whose sequences are complementary, and to exclude hybridization of significantly mismatched nucleic acids. Examples of "highly stringent conditions" for hybridization and washing are 0.015 M sodium chloride, 0.0015 M sodium citrate at 65-68°C or 0.015 M sodium chloride, 0.0015 M sodium citrate, and 50% formamide at 42°C. (see, for example, Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory, 1989); Anderson et al., Nucleic Acid Hybridisation: A Practical Approach Ch. 4 (IRL Press Limited)). The term "moderately stringent conditions" refers to conditions under which a DNA duplex with a greater degree of base pair mismatching than could occur under "highly stringent conditions" is able to form. Exemplary moderately stringent conditions are 0.015 M sodium chloride, 0.0015 M sodium citrate at 50-65°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, NaDodSO , (SDS), ficoll, Denhardt's solution, sonicated salmon sperm DNA (or another non-complementary DNA), and dextran sulfate, although other suitable agents can also be used. The concentration and types of these additives can be changed without substantially affecting the stringency of the hybridization conditions. Hybridization experiments are usually carried out at pH 6.8-7.4; however, at typical ionic strength conditions, the rate of hybridization is nearly independent of pH. In preferred embodiments of the present invention, vectors are used to transfer a nucleic acid sequence encoding a polypeptide to a cell. A vector is any molecule used to transfer a nucleic acid sequence to a host cell. In certain cases, an expression vector is utilized. An expression vector is a nucleic acid molecule that is suitable for transformation of a host cell and contains nucleic acid sequences that direct and / or control the expression of the transferred nucleic acid sequences. Expression includes, but is not limited to, processes such as transcription, translation, and splicing, if introns are present. Expression vectors typically comprise one or more flanking sequences operably linked to a heterologous nucleic acid sequence encoding a polypeptide. Flanking sequences may be homologous (i.e., from the same species and / or strain as the host cell), heterologous (i.e., from a species other than the host cell species or strain), hybrid (i.e., a combination of flanking sequences from more than one source), or synthetic, for example.

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

In certain embodiments, it is preferred that the flanking sequence is a trascriptional regulatory region that drives high-level gene expression in the target cell. The transcriptional regulatory region may comprise, for example, a promoter, enhancer, silencer, repressor element, or combinations thereof. The transcriptional regulatory region may be either constitutive, tissue-specific, cell-type specific (i.e., the region is drives higher levels of transcription in a one type of tissue or cell as compared to another), or regulatable (i.e., responsive to interaction with a compound 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 within that cell. A wide variety of transcriptional regulatory regions may be utilized in practicing 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 adeno virus gene promoters; the SV40 early promoter region (Bemoist and Chambon, 1981, Nature 290:304-10); the promoter contained in the 3' long terminal repeat (LTR) of Rous sarcoma vims (RSV) (Yamamoto, et al, 1980, Cell 22:787-97); the herpes simplex vims thymidine kinase (HSV-TK) promoter (Wagner et al, 1981, Proc. Natl. Acad. Sci. U.S.A. 78:1444-45); the regulatory sequences of the metallothionine gene (Brinster et al, 1982, Nature 296:39-42); prokaryotic expression vectors such as the beta-lactamase promoter (Villa-Kamaroff et al, 1978, Proc. Natl. Acad. Sci. U.S.A., 75:3727-31); or the tac promoter (DeBoer et al, 1983, Proc. Natl. Acad. Sci. U.S.A., 80:21-25). Tissue- and / or cell-type specific transcriptional control regions include, for example, the elastase I gene control region which is active in pancreatic acinar cells (Swift et al, 1984, Cell 38:639-46; Ornitz et al, 1986, Cold Spring Harbor Symp. Quant. Biol 50:399-409 (1986); MacDonald, 1987, Hepatology 7:425-515); the insulin gene control region which is active in pancreatic beta cells (Hanahan, 1985, Nature 315:115-22); the immunoglobulin gene control region which is active in lymphoid cells (Grosschedl et al, 1984, Cell 38:647-58; Ada es et al, 1985, Nature 318:533-38; Alexander et al, 1987, Mol. Cell. Biol, 7:1436-44); the mouse mammary tamor virus control region in testicular, breast, lymphoid and mast cells (Leder et al, 1986, Cell 45:485-95); the albumin gene control region in liver (Pinkert et al, 1987, Genes and Devel. 1 :268-76); the alpha-feto-protein gene control region in liver (Krumlauf et al, 1985, Mol. Cell. Biol, 5:1639-48; Hammer et al, 1987, Science 235:53-58); the alpha 1- antitrypsin gene control region in liver (Kelsey et al, 1987, Genes and Devel. 1:161-71); the beta-globin gene control region in myeloid cells (Mogram et al, 1985, Nature 315:338-40; Kollias et al, 1986, Cell 46:89-94); the myelin basic protein gene control region in oligodendrocyte cells in the brain (Readhead et al, 1987, Cell 48:703-12); the myosin light chain-2 gene control region in skeletal muscle (Sani, 1985, Nature 314:283-86); the gonadotropic releasing hormone gene control region in the hypothalamus (Mason et al, 1986, Science 234:1372-78), and the tyrosinase promoter in melanoma cells (Hart, I. Semin Oncol 1996 Feb;23(l): 154-8; Siders, et al. Cancer Gene Ther 1998 Sep-Oct;5(5):281-91), among others. 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, elastase, albumin, alpha- feto-protein and insulin). Similarly, the SV40 enhancer, the cytomegalovirus early promoter enhancer, the polyoma enhancer, and adenovirus enhancers are useful with eukaryotic promoter sequences. While an enhancer may be spliced into the vector at a position 5' or 3' to nucleic acid coding sequence, it is typically located at a site 5' from the promoter. Other suitable enhancers are known in the art, and would be applicable to the present invention. While preparing reagents of the present invention, cells may need to be transfected or transformed. Transfection refers to the uptake of foreign or exogenous DNA by a cell, and a cell has been transfected when the exogenous DNA has been introduced inside the cell membrane. A number of transfection techniques are well known in the art (i.e., Graham et al, 1973, Virology 52:456; Sambrook et al, Molecular Cloning, A Laboratory Manual (Cold Spring Harbor Laboratories, 1989); Davis et al, Basic Methods in Molecular Biology (Elsevier, 1986); and Chu et al, 1981, Gene 13: 197). Such techniques can be used to introduce one or more exogenous DNA moieties into suitable host cells.

In certain embodiments, it is preferred that transfection of a cell results in transformation of that cell. A cell is transformed when there is a change in a characteristic of the cell, being transformed when it has been modified to contain a new nucleic acid. Following transfection, the transfected nucleic acid may recombine with that of the cell by physically integrating into a chromosome of the cell, may be maintained transiently as an episomal element without being replicated, or may replicate independently as a plasmid. A cell is stably transformed when the nucleic acid is replicated with the division of the cell. The present invention further provides isolated immunogenic targets in polypeptide form. A polypeptide is considered isolated where it: (1) has been separated from at least about 50 percent of polymicleotides, lipids, carbohydrates, or other materials with which it is naturally found when isolated from the source cell; (2) is not linked (by covalent or noncovalent interaction) to all or a portion of a polypeptide to which the "isolated polypeptide" is linked in nature; (3) is operably linked (by covalent or noncovalent interaction) to a polypeptide with which it is not linked in nature; or, (4) does not occur in nature. Preferably, the isolated polypeptide is substantially free from any other contaminating polypeptides or other contaminants that are found in its natural environment that would interfere with its therapeutic, diagnostic, prophylactic or research use.

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

A fragment of a nucleic acid or polypeptide comprises a truncation of the sequence (i.e., nucleic acid or polypeptide) at the amino terminus (with or without a leader sequence) and / or the carboxy terminus. Fragments may also include variants (i.e., allelic, splice), orthologs, homologues, and other variants having one or more amino acid additions or substitutions or internal deletions as compared to the parental sequence. In preferred embodiments, truncations and or deletions comprise about 10 amino acids, 20 amino acids, 30 amino acids, 40 amino acids, 50 amino acids, or more. The polypeptide fragments so produced will comprise about 10 amino acids, 25 amino acids, 30 amino acids, 40 amino acids, 50 amino acids, 60 amino acids, 70 amino acids, or more. Such polypeptide fragments may optionally comprise an amino terminal methionine residue. It will be appreciated that such fragments can be used, for example, to generate antibodies or cellular immune responses to immunogenic target polypeptides. A variant is a sequence having one or more sequence substitutions, deletions, and/or additions as compared to the subject sequence. Variants may be naturally occurring or artificially constructed. Such variants may be prepared from the corresponding nucleic acid molecules. In preferred embodiments, the variants have from 1 to 3, or from 1 to 5, or from 1 to 10, or from 1 to 15, or from 1 to 20, or from 1 to 25, or from 1 to 30, or from 1 to 40, or from 1 to 50, or more than 50 amino acid substitutions, insertions, additions and/or deletions. An allelic variant is one of several possible naturally-occurring alternate forms of a gene occupying a given locus on a chromosome of an organism or a population of organisms. A splice variant is a polypeptide generated from one of several RNA transcript resulting from splicing of a primary transcript. An ortholog is a similar nucleic acid or polypeptide sequence from another species. For example, the mouse and human versions of an immunogenic target polypeptide may be considered orthologs of each other. A derivative of a sequence is one that is derived from a parental sequence those sequences having substitations, 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 will be higher than the percent identity between those two polypeptides.

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

A skilled artisan will be able to determine suitable variants of polypeptide using well- known techniques. For identifying suitable areas of the molecule that may be changed without destroying biological activity (i.e., MHC binding, immunogenicity), one skilled in the art may target areas not believed to be important for that activity. Increased binding to MHC class I may be accomplished by, for example, reducing the off-rate by modifying anchor residues, or introducing a tyrosine residue in position PI (Parkhurst, et al. J. Immunol 1996. 157:2539-2548; Tourdot, et al. Eur. J. Immunol. 2000; 30: 3411-3421; Valmori, et al. J. Immunol. 1998. 160:1750-1758). Other possible T cell epitope enhancement strategies focus on amino-acid residues which are recognized by the T cell receptor complex (Zaremba, et al. Cancer Res. 1997;57:4570-4577). When performing such epitope modifications, in any case it becomes critical to confirm that the point mutation introduced does not preclude processing and presentation of the T cell epitope (Ossendorp, et al. Immunity 1996; 5: 115- 124). Further, when similar polypeptides with similar activities from the. same species or from other species are known, one skilled in the art may compare the amino acid sequence of a polypeptide to such similar polypeptides. By performing such analyses, one can identify residues and portions of the molecules that are conserved among similar polypeptides. It will be appreciated that changes in areas of the molecule that are not conserved relative to such similar polypeptides would be less likely to adversely affect the biological activity and/or structure of a polypeptide. One skilled in the art would also know that, even in relatively conserved regions, one may substitute chemically similar amino acids for the naturally occurring residues while retaining activity. Therefore, even areas that may be important for biological activity or for structure may be subject to conservative amino acid substitations without destroying the biological activity or without adversely affecting the polypeptide structure.

Other preferred polypeptide variants include glycosylation variants wherein the number and/or type of glycosylation sites have been altered compared to the subject amino acid sequence. In one embodiment, polypeptide variants comprise a greater or a lesser number of N-linked glycosylation sites than the subject amino acid sequence. An N-linked glycosylation site is characterized by the sequence Asn-X-Ser or Asn-X-Thr, wherein the amino acid residue designated as X may be any amino acid residue except proline. The substitution of amino acid residues to create this sequence provides a potential new site for the addition of an N-linked carbohydrate chain. Alternatively, substitations 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.

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

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

A fusion motif may enhance transport of an immunogenic target to an MHC processing compartment, such as the endoplasmic reticulum, may also be included. 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 (hPERl; in particular, SRRHHCRSKAKRSRHH). In addition, the polypeptide or variant thereof may be fused to a homologous polypeptide to form a homodimer or to a heterologous polypeptide to form a heterodimer. Heterologous peptides and polypeptides include, but are not limited to: an epitope to allow for the detection and/or isolation of a fusion polypeptide; a transmembrane receptor protein or a portion thereof, such as an extracellular domain or a transmembrane and intracellular domain; a ligand or a portion thereof which binds to a transmembrane receptor protein; an enzyme or portion thereof which is catalytically active; a polypeptide or peptide which promotes oligomerization, such as a leucine zipper domain; a polypeptide or peptide which increases stability, such as an immunoglobulin constant region; and a polypeptide which has a therapeutic activity different from the polypeptide or variant thereof.

In certain embodiments, it may be advantageous to combine a nucleic acid sequence encoding an immunogenic target, polypeptide, or derivative thereof with one or more co- stimulatory component(s) such as cell surface proteins, cytokines or chemokines in a composition of the present invention. The co-stimulatory component may be included in the composition as a polypeptide or as a nucleic acid encoding the polypeptide, for example. Suitable co-stimulatory molecules include, for instance, polypeptides that bind members of the CD28 family (i.e., CD28, ICOS; Hutloff, et al. Nature 1999, 397: 263-265; Peach, et al. J Exp Med 1994, 180: 2049-2058) such as the CD28 binding polypeptides B7.1 (CD80; Schwartz, 1992; Chen et al, 1992; Ellis, et al. J. Immunol, 156(8): 2700-9) and B7.2 (CD86; Ellis, et al. J. Immunol, 156(8): 2700-9); polypeptides which bind members of the integrin family (i.e., LFA-1 (GDI la / CD18); Sedwick, et al. J Immunol 1999, 162: 1367-1375; Wύlfmg, et al. Science 1998, 282: 2266-2269; Lub, et al. Immunol Today 1995, 16: 479- 483) including members of the ICAM family (i.e., ICAM-1, -2 or -3); polypeptides which bind CD2 family members (i.e., CD2, signalling lymphocyte activation molecule (CDwl50 or "SLAM"; Aversa, et al.

J Immunol 1997, 158: 4036-4044)) such as CD58 (LFA-3; CD2 ligand; Davis, et al. Immunol Today 1996, 17: 177-187) or SLAM ligands (Sayos, et al. Nature 1998, 395: 462- 469); polypeptides which bind heat stable antigen (HSA or CD24; Zhou, et al. Eur J Immunol 1997, 27: 2524-2528); polypeptides which bind to members of the TNF receptor (TNFR) family (i.e., 4-1BB (CD137; Vinay, et al. Semin Immunol 1998, 10: 481-489), OX40 (CD134; Weinberg, et al. Semin Immunol 1998, 10: 471-480; Higgins, et al. J Immunol 1999, 162: 486-493), and CD27 (Lens, et al. Semin Immunol 1998, 10: 491-499)) such as 4-1BBL (4-1BB ligand; Vinay, et al. Semin Immunol 1998, 10: 481-48; DeBenedette, et al. J Immunol 1997, 158: 551-559), TNFR associated factor-1 (TRAF-1; 4- 1BB ligand; Saoulli, et al. J Exp Med 1998, 187: 1849-1862, Arch, et al. Mol Cell Biol 1998, 18: 558-565), TRAF-2 (4-1BB and OX40 ligand; Saoulli, et al. J Exp Med 1998, 187: 1849-1862; Oshima, et al. Int Immunol 1998, 10: 517-526, Kawamata, et al. J Biol Chem 1998, 273: 5808-5814), TRAF-3 (4-1BB and OX40 ligand; Arch, et al. Mol Cell Biol 1998, 18: 558-565; Jang, et al. Biochem Biophys Res Commun 1998, 242: 613-620; Kawamata S, et al. J Biol Chem 1998, 273: 5808-5814), OX40L (OX40 ligand; Gramaglia, et al. J Immunol 1998, 161: 6510-6517), TRAF-5 (OX40 ligand; Arch, et al. Mol Cell Biol 1998, 18: 558-565; Kawamata, et al. J Biol Chem 1998, 273: 5808-5814), and CD70 (CD27 ligand; Couderc, et al. Cancer Gene Ther., 5(3): 163-75). CD 154 (CD40 ligand or "CD40L"; Gurunathan, et al. J Immunol, 1998, 161: 4563-4571; Sine, et al. Hum. Gene Ther. , 2001 , 12 : 1091 - 1102) may also be suitable. One or more cytokines may also be suitable co-stimulatory components or

"adjuvants", either as polypeptides or being encoded by nucleic acids contained within the compositions of the present invention (Parmiani, et al. Immunol Lett 2000 Sep 15; 74(1): 41- 4; Berzofsky, et al. Nature Immunol. 1: 209-219). Suitable cytokines include, for example, interleukin-2 (IL-2) (Rosenberg, et al. Nature Med. 4: 321-327 (1998)), IL-4, IL-7, IL-12 (reviewed by Pardoll, 1992; Harries, et al. J. Gene Med. 2000 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 interferons (i.e., IFN-α, INF-γ). Other cytokines may also be suitable for practicing the present invention, as is known in the art.

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

It is also known in the art that suppressive or negative regulatory immune mechanisms may be blocked, resulting in enhanced immune responses. For instance, treatment with anti-CTLA-4 (Shrikant, et al. Immunity, 1996, 14: 145-155; Sutmuller, et al. J. Exp. Med., 2001, 194: 823-832), anti-CD25 (Sutmuller, supra), anti-CD4 (Matsui, et al. J. Immunol, 1999, 163: 184-193), the fusion protein IL13Ra2-Fc (Terabe, et al. Nature Immunol, 2000, 1: 515-520), and combinations thereof (i.e., anti-CTLA-4 and anti-CD25, Sutmuller, supra) have been shown to upregulate anti-tumor immune responses and would be suitable in practicing the present invention. Any of these components may be used alone or in combination with other agents. For instance, it has been shown that a combination of CD80, ICAM-1 and LFA-3'("TRICOM") may potentiate anti-cancer immune responses (Hodge, et al. Cancer Res. 59: 5800-5807 (1999). Other effective combinations include, for example, IL-12 + GM-CSF (Ahlers, et al. J. Immunol, 158: 3947-3958 (1997); Iwasaki, et al. J. Immunol. 158: 4591-4601 (1997)), IL- 12 + GM-CSF + TNF-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. supra), and CD86 + GM-CSF + IL-12 (Iwasaki, supra). One of skill in the art would be aware of additional combinations useful in carrying out the present invention .In addition, the skilled artisan would be aware of additional reagents or methods that may be used to modulate such mechanisms. These reagents and methods, as well as others known by those of skill in the art, may be utilized in practicing the present invention.

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., I: 491- 500; Dubensky, supra; Huang, et al. 2001. J. Virol. 75: 4947-4951), in vivo electroporation (Widera, et al. 2000. J. Immunol 164: 4635-3640), incorporation of CpG stimulatory motifs (Gurunathan, et al. Ann. Rev. Immunol, 2000, 18: 927-974; Leitner, supra), sequences for targeting of the endocytic or ubiquitin-processing pathways (Thomson, et al. 1998. J. Virol. 72: 2246-2252; Velders, et al. 2001. J. Immunol 166: 5366-5373), Marek's disease vims type 1 VP22 sequences (J. Virol. 76(6):2676-82, 2002), prime-boost regimens (Gurunathan, supra; Sullivan, et al. 2000. Nature, 408: 605-609; Hanke, et al. 1998. Vaccine, 16: 439- 445; Amara, et al. 2001. Science, 292: 69-74), and the use of mucosal delivery vectors such as Salmonella (Darji, et al. 1997. Cell, 91: 765-775; Woo, et al. 2001. Vaccine, 19: 2945- 2954). Other methods are known in the art, some of which are described below.

Chemotherapeutic agents, radiation, anti-angiogenic compounds, or other agents may also be utilized in treating and / or preventing cancer using immunogenic targets (Sebti, et al. Oncogene 2000 Dec 27;19(56):6566-73). For example, in treating metastatic breast cancer, useful chemotherapeutic agents include cyclophosphamide, doxombicin, paclitaxel, docetaxel, navelbine, capecitabine, and mitomycin C, among others. Combination chemotherapeutic regimens have also proven effective including cyclophosphamide + methotrexate + 5-fluorouracil; cyclophosphamide + doxombicin + 5-fluorouracil; or, cyclophosphamide + doxombicin, for example. Other compounds such as prednisone, a taxane, navelbine, mitomycin C, or vinblastine have been utlized for various reasons. A majority of breast cancer patients have estrogen-receptor positive (ER+) tumors and in these patients, endocrine therapy (i.e., tamoxifen) is preferred over chemotherapy. For such patients, tamoxifen or, as a second line therapy, progestins (medroxyprogesterone acetate or megestrol acetate) are preferred. Aromatase inhibitors (i.e., aminoglutethimide and analogs thereof such as letrozole) decrease the availability of estrogen needed to maintain tamor growth and may be used as second or third line endocrine therapy in certain patients. Other cancers may require different chemotherapeutic regimens. For example, metastatic colorectal cancer is typically treated with Camptosar (irinotecan or CPT-11), 5- fluorouracil or leucovorin, alone or in combination with one another. Proteinase and integrin inhibitors such as as the MMP inhibitors marimastate (British Biotech), COL-3 (Collagenex), Neovastat (Aeterna), AG3340 (Agouron), BMS-275291 (Bristol Myers Squibb), CGS 27023A (Novartis) or the integrin inhibitors Vitaxin (Medimmune), or MED1522 (Merck KgaA) may also be suitable for use. As such, immunological targeting of immunogenic targets associated with colorectal cancer could be performed in combination with a treatment using those chemotherapeutic agents. Similarly, chemotherapeutic agents used to treat other types of cancers are well-known in the art and may be combined with the immunogenic targets described herein.

Many anti-angiogenic agents are known in the art and would be suitable for co- administration with the immunogenic target vaccines (see, for example, Timar, et al. 2001. Pathology Oncol. Res., 7(2): 85-94). Such agents include, for example, physiological agents such as growth factors (i.e., ANG-2, NK1,2,4 (HGF), transforming growth factor beta (TGF- β)), cytokines (i.e., interferons such as IFN-α, -β, -γ, platelet factor 4 (PF-4), PR-39), proteases (i.e., cleaved AT-IH, collagen XViπ fragment (Endostatin)), HmwKallikrein-d5 plasmin fragment (Angiostatin), prothrombin-Fl-2, TSP-1), protease inhibitors (i.e., tissue inhibitor of metalloproteases such as TIMP-1, -2, or -3; maspin; plasminogen activator- inhibitors such as PAI-1; pigment epithelium derived factor (PEDF)), Tumstatin (available through ILEX, Inc.), antibody products (i.e., the collagen-binding antibodies HUIV26, HUI77, XL313; anti-VEGF; anti-integrin (i.e., Vitaxin, (Lxsys))), and glycosidases (i.e., heρarinase-I, -III). "Chemical" or modified physiological agents known or believed to have anti-angiogenic potential include, for example, vinblastine, taxol, ketoconazole, thalidomide, dolestatin, combrestatin A, rapamycin (Guba, et al. 2002, Nature Med., 8: 128-135), CEP- 7055 (available from Cephalon, Inc.), flavone acetic acid, Bay 12-9566 (Bayer Corp.), AG3340 (Agouron, Inc.), CGS 27023A (Novartis), tetracylcine derivatives (i.e., COL-3 (Collagenix, Inc.)), Neovastat (Aeterna), BMS-275291 (Bristol-Myers Squibb), low dose 5- FU, low dose methotrexate (MTX), irsofladine, radicicol, cyclosporine, captopril, celecoxib, D45152-sulphated polysaccharide, cationic protein (Protamine), cationic peptide-VEGF, Suramin (polysulphonated napthyl urea), compounds that interfere with the function or production of VEGF (i.e., SU5416 or SU6668 (Sugen), PTK787/ZK22584 (Novartis)), Distamycin A, Angiozyme (ribozyme), isoflavinoids, staurosporine derivatives, genistein, EMD121974 (Merck KcgaA), tyrphostins, isoquinolones, retinoic acid, carboxyamidotriazole, TNP-470, octreotide, 2-methoxyestradiol, aminosterols (i.e., squalamine), glutathione analogues (i.e., N-acteyl-L-cysteine), combretastatin A-4 (Oxigene), Eph receptor blocking agents (Nature, 414:933-938, 2001), Rh-Angiostatin, Rh-Endostatin (WO 01/93897), cyclic-RGD peptide, accutin-disintegrin, benzodiazepenes, humanized anti- avb3 Ab, Rh-PAI-2, amiloride, p-amidobenzamidine, anti-uPA ab, anti-uPAR Ab, L- phanylalanin-N-methylamides (i.e., Batimistat, Marimastat), AG3340, and minocycline. Many other suitable agents are known in the art and would suffice in practicing the present invention.

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 tamor growth. In one study, Clostridium novyi was modified to eliminate a toxin gene carried on a phage episome and administered to mice with colorectal tumors (Dang, et al. P.N.A.S. USA, 98(26): 15155- 15160, 2001). In combination with chemotherapy, the treatment was shown to cause tamor necrosis in the animals. The reagents and methodologies described in this application may be combined with such treatment methodologies.

Nucleic acids encoding immunogenic targets may be administered to patients by any of several available techniques. Various viral vectors that have been successfully utilized for introducing a nucleic acid to a host include retrovirus, adenovirus, adeno-associated virus (AAV), herpes virus, and poxvims, among others. It is understood in the art that many such viral vectors are available in the art. The vectors of the present invention may be constructed using standard recombinant techniques widely available to one skilled in the art. Such techniques may be found in common molecular biology references such as Molecular Cloning: A Laboratory Manual (Sambrook, et al., 1989, Cold Spring Harbor Laboratory Press), Gene Expression Technology (Methods in Enzymology, Vol. 185, edited by D. Goeddel, 1991. Academic Press, San Diego, CA), and PCR Protocols: A Guide to Methods and Applications (Innis, et al. 1990. Academic Press, San Diego, CA). Preferred retroviral vectors are derivatives of lentivirus as well as derivatives of murine or avian retrovirases. Examples of suitable retroviral vectors include, for example, Moloney murine leukemia vims (MoMuLV), Harvey murine sarcoma vims (HaMuSV), murine mammary tumor vims (MuMTV), SIV, BIN, HIV and Rous Sarcoma Virus (RSV). A number of retroviral vectors can incorporate multiple exogenous nucleic acid sequences. As recombinant retroviruses are defective, they require assistance in order to produce infectious vector particles. This assistance can be provided by, for example, helper cell lines encoding retrovirus structural genes. Suitable helper cell lines include Ψ2, 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 ΝIH 3T3 cells, to produce large quantities of chimeric retroviral virions. Retroviral vectors may be administered by traditional methods (i.e., injection) or by implantation of a "producer cell line" in proximity to the target cell population (Culver, K., et al., 1994, Hum. Gene Ther., 5 (3): 343-79; Culver, K., et al, Cold Spring Harb. Symp. Quant. Biol, 59: 685-90); Oldfield, E., 1993, Hum. Gene Ther., 4 (1): 39-69). The producer cell line is engineered to produce a viral vector and releases viral particles in the vicinity of the target cell. A portion of the released viral particles contact the target cells and infect those cells, thus delivering a nucleic acid of the present invention to the target cell. Following infection of the target cell, expression of the nucleic acid of the vector occurs.

Adenoviral vectors have proven especially useful for gene transfer into eukaryotic cells (Rosenfeld, M., et al, 1991, Science, 252 (5004): 431-4; Crystal, R., et al, 1994, Not. Genet., 8 (1): 42-51), the study eukaryotic gene expression (Levrero, M., et al, 1991, Gene, 101 (2): 195-202), vaccine development (Graham, F. and Prevec, L., 1992, Biotechnology, 20: 363-90), and in animal models (Stratford-Perricaudet, L., et al, 1992, Bone Marrow Transplant., 9 (Suppl. 1): 151-2 ; Rich, D., et al, 1993, Hum. Gene Ther., 4 (4): 461-76). Experimental routes for administrating recombinant Ad to different tissues in vivo have included intratracheal instillation (Rosenfeld, M., et al, 1992, Cell, 68 (1): 143-55) injection into muscle (Quantin, B., et al, 1992, Proc. Natl Acad. Sci. U.S.A., 89 (7): 2581-4), peripheral intravenous injection (Herz, J., and Gerard, R., 1993, Proc. Natl. Acad. Sci. U.S.A., 90 (7): 2812-6) and stereotactic inoculation to brain (Le Gal La Salle, G., et al, 1993, Science, 259 (5097): 988-90), among others.

Adeno-associated vims (AAV) demonstrates high-level infectivity, broad host range and specificity in integrating into the host cell genome (Hermonat, P., et al., 1984, Proc. Natl Acad. Sci. U.S.A., 81 (20): 6466-70). And Herpes Simplex Vims type-1 (HSV-1) is yet another attractive vector system, especially for use in the nervous system because of its neurotropic property (Geller, A., et al, 1991, Trends Neurosci, 14 (10): 428-32; Glorioso, et al, 1995, Mol. Biotechnol, 4 (1): 87-99; Glorioso, et al, 1995, Annu. Rev. Microbiol, 49: 675-710). 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. NYVAC (vP866) was derived from the Copenhagen vaccine strain of vaccinia vims by deleting six nonessential regions of the genome encoding known or potential vimlence factors (see, for example, U.S. Pat. Nos. 5,364,773 and 5,494,807). The deletion loci were also engineered as recipient loci for the insertion of foreign genes. The deleted regions are: thymidine kinase gene (TK; J2R); hemorrhagic region (u; B13R+B14R); A type inclusion body region (ATI; A26L); hemagglutinin gene (HA; A56R); host range gene region (C7L- K1L); and, large subunit, ribonucleotide reductase (I4L). NYVAC is a genetically engineered vaccinia vims strain that was generated by the specific deletion of eighteen open reading frames encoding gene products associated with vimlence and host range. NYVAC has been show to be useful for expressing TAs (see, for example, U.S. Pat. No. 6,265,189). NYVAC (vP866), vP994, vCP205, vCP1433, placZH6H4Lreverse, pMPC6H6K3E3 and pC3H6FHVB were also deposited with the ATCC under the terms of the Budapest Treaty, accession numbers VR-2559, VR-2558, VR-2557, VR-2556, ATCC-97913, ATCC-97912, and ATCC-97914, respectively.

ALVAC-based recombinant viruses (i.e., ALVAC-1 and ALVAC-2) are also suitable for use in practicing the present invention (see, for example, U.S. Pat. No. 5,756,103; Perkus, et al. J. Tissue Cult. Meth. 1993;15:72-81; Perkus, et al. . J. Leukocyte Biol. 1995;58:1-13; Bonnet, et al. Immunol Let. 2000;74:11-25; Tartaglia, et al. Vaccine 2001; 19: 2571-2575). ALVAC(2) is identical to ALVAC(l) except that ALVAC(2) genome comprises the vaccinia E3L and K3L genes under the control of vaccinia promoters (U.S. Pat. No. 6,130,066; Beattie et al., 1995a, 1995b, 1991; Chang et al., 1992; Davies et al, 1993). Both ALVAC(l) and ALVAC(2) have been demonstrated to be useful in expressing foreign DNA sequences, such as TAs (Tartaglia et al., 1993 a,b; U.S. Pat. No. 5,833,975). ALVAC was deposited under the terms of the Budapest Treaty with the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, Va. 20110-2209, USA, ATCC accession number VR-2547.

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

"Non-viral" plasmid vectors may also be suitable in practicing the present invention.

Preferred plasmid vectors are compatible with bacterial, insect, and / or mammalian host cells. Such vectors include, for example, PCR-II, pCR3, and pcDNA3.1 (Invitrogen, San Diego, CA), pBSII (Stratagene, La Jolla, CA), ρET15 (Novagen, Madison, WI), pGEX

(Pharmacia Biotech, Piscataway, NJ), pEGFP-N2 (Clontech, Palo Alto, CA), pETL

(BlueBacII, Invitrogen), pDSR-alpha (PCT pub. No. WO 90/14363) and pFastBacDual

© (Gibco-BRL, Grand Island, NY) as well as Bluescript plasmid derivatives (a high copy number COLE 1 -based phagemid, Stratagene Cloning Systems, La Jolla, CA), PCR cloning plasmids designed for cloning Taq-amplified PCR products (e.g., TOPO™ TA cloning kit,

® PCR2.1 plasmid derivatives, Invitrogen, Carlsbad, CA). Bacterial vectors may also be used with the current invention. These vectors include, for example, Shigella, Salmonella, Vibrio cholerae, Lactobacillus, Bacille calmette guerin (BCG), and Streptococcus (see for example,

WO 88/6626; WO 90/0594; WO 91/13157; WO 92/1796; and WO 92/21376). Many other non-viral plasmid expression vectors and systems are known in the art and could be used with the current invention.

Suitable nucleic acid delivery techniques include DNA-ligand complexes, adenovirus- ligand-DNA complexes, direct injection of DNA, CaPO₄ precipitation, gene gun techniques, electroporation, and colloidal dispersion systems, among others. Colloidal dispersion systems include, for example, macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. The preferred colloidal system of this invention is a liposome, which are artificial membrane vesicles useful as delivery vehicles in vitro and in vivo. RNA, DNA and intact virions can be encapsulated within the aqueous interior and be delivered to cells in a biologically active form (Fraley, R., et al, 1981, Trends Biochem. Sci., 6: 11). The composition of the liposome is usually a combination of phospholipids, particularly high- phase-transition-temperature phospholipids, usually in combination with steroids, especially cholesterol. The physical characteristics of liposomes depend on pH, ionic strength, and the presence of divalent cations.

Other lipids or phospholipids may also be used. Examples of lipids useful in liposome production include phosphatidyl compounds, such as phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides. Particularly useful are diacylphosphatidylglycerols, where the lipid moiety contains from 14-18 carbon atoms, particularly from 16-18 carbon atoms, and is saturated. Illustrative phospholipids include egg phosphatidylcholine, dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine.

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

Table II Types of Immunologic Adjuvants

The immunogenic targets of the present invention may also be used to generate antibodies for use in screening assays or for immunotherapy. Other uses would be apparent to one of skill in the art. The term "antibody" includes antibody fragments, as are known in the art, including Fab, Fab₂, single chain antibodies (Fv for example), humanized antibodies, chimeric antibodies, human antibodies, produced by several methods as are known in the art. Methods of preparing and utilizing various types of antibodies are well-known to those of skill in the art and would be suitable in practicing the present invention (see, for example, Harlow, et al. Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988; Harlow, et al. Using Antibodies: A Laboratoiγ Manual, Portable Protocol No. 1, 1998; Kohler and Milstein, Nature, 256:495 (1975)); Jones et al. Nature, 321:522-525 (1986); Riechmann et al. Nature, 332:323-329 (1988); Presta (Curr. Op. Struct. Biol., 2:593-596 (1992); Verhoeyen et al. (Science, 239:1534-1536 (1988); Hoogenboom et al., J. Mol. Biol, 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991); Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boemer et al., J. Immunol., 147(l):86-95 (1991); Marks et al., Bio/Technology 10, 779-783 (1992); Lonberg et al., Nature 368 856-859 (1994); Morrison, Nature 368 812-13 (1994); Fishwild et al., Nature Biotechnology 14, 845-51 (1996); Neuberger, Nature Biotechnology 14, 826 (1996); Lonberg and Huszar, Intern. Rev. Immunol. 13 65-93 (1995); as well as U.S. Pat. Nos. 4,816,567; 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and, 5,661,016). The antibodies or derivatives therefrom may also be conjugated to therapeutic moieties such as cytotoxic drugs or toxins, or active fragments thereof such as diptheria A chain, exotoxin A chain, ricin A chain, abrin A chain, curcin, crotin, phenomycin, enomycin, among others. Cytotoxic agents may also include radiochemicals. Antibodies and their derivatives may be incorporated into compositions of the invention for use in vitro or in vivo.

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

It is also possible to use nucleic acids, proteins, derivatives therefrom, or antibodies thereto as reagents in drug screening assays. The reagents may be used to ascertain the effect of a drug candidate on the expression of the immunogenic target in a cell line, or a cell or tissue of a patient. The expression profiling technique may be combined with high throughput screening techniques to allow rapid identification of useful compounds and monitor the effectiveness of treatment with a drug candidate (see, for example, Zlokarnik, et al., Science 279, 84-8 (1998)). Drag candidates may be chemical compounds, nucleic acids, proteins, antibodies, or derivatives therefrom, whether naturally occurring or synthetically derived. Drag candidates thus identified may be utilized, among other uses, as pharmaceutical compositions for administration to patients or for use in further screening assays. Administration of a composition of the present invention to a host may be accomplished using any of a variety of techniques known to those of skill in the art. The compositiθn(s) may be processed in accordance with conventional methods of pharmacy to produce medicinal agents for administration to patients, including humans and other mammals (i.e., a "pharmaceutical composition"). The pharmaceutical composition is preferably made in the form of a dosage unit containing a given amount of DNA, viral vector particles, polypeptide or peptide, for example. A suitable daily dose for a human or other mammal may vary widely depending on the condition of the patient and other factors, but, once again, can be determined using routine methods.

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

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

The dosage regimen for immunizing a host or otherwise treating a disorder or a disease with a composition of this invention is based on a variety of factors, including the type of disease, the age, weight, sex, medical condition of the patient, the severity of the condition, the route of administration, and the particular compound employed. For example, a poxviral vector may be administered as a composition comprising 1 x 10⁶ 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 (i.e., ALVAC), the boost step may utilize another type of viras (i.e., NYNAC). This prime-boost method of administration has been shown to induce strong immunological responses.

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

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 fomiulation, although it may comprise as much as 10% w/w, but preferably not more than 5% w/w, and more preferably from 0.1% to 1% of the formulation. Formulations suitable for topical administration include liquid or semi-liquid preparations suitable for penetration through the skin (e.g., liniments, lotions, ointments, creams, or pastes) and drops suitable for administration to the eye, ear, or nose. The pharmaceutical compositions may also be prepared in a solid form (including granules, powders or suppositories). The phannaceutical compositions may be subjected to conventional pharmaceutical operations such as sterilization and/or may contain conventional adjuvants, such as preservatives, stabilizers, wetting agents, emulsifiers, buffers etc. Solid dosage forms for oral administration may include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active compound may be admixed with at least one inert diluent such as sucrose, lactose, or starch. Such dosage forms may also comprise, as in normal practice, additional substances other than inert diluents, e.g., lubricating agents such as magnesium stearate. In the case of capsules, tablets, and pills, the dosage forms may also comprise buffering agents. Tablets and pills can additionally be prepared with enteric coatings. Liquid dosage forms for oral administration may include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs containing inert diluents commonly used in the art, such as water. Such compositions may also comprise adjuvants, such as wetting sweetening, flavoring, and perfuming agents. Pharmaceutical compositions comprising a nucleic acid or polypeptide of the present invention may take any of several forms and may be administered by any of several routes. In preferred embodiments, the compositions are administered via a parenteral route (intradermal, intramuscular or subcutaneous) to induce an immune response in the host. Alternatively, the composition may be administered directly into a lymph node (intranodal) or tamor mass (i.e., intratamoral administration). For example, the dose could be administered subcutaneously at days 0, 7, and 14. Suitable methods for immunization using compositions comprising TAs are known in the art, as shown for p53 (Hollstein et al., 1991), p21-ras (Almoguera et al., 1988), HER-2 (Fendly et al., 1990), the melanoma-associated antigens (MAGE-1; MAGE-2) (van der Braggen et al., 1991), ρ97 (Hu et al., 1988), and carcinoembryonic antigen (CEA) (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 poxvims may be in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose or the like. The composition may also be provided in lyophilized form for reconstituting, for instance, in isotonic aqueous, saline buffer. In addition, the compositions can be co- administered or sequentially administered with other antineoplastic, anti-tumor or anti-cancer agents and/or with agents which reduce or alleviate ill effects of antineoplastic, anti-tumor or anti-cancer agents.

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.

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

Generation of ALVAC recombinants

ALVAC recombinants expressing various single epitope, polyepitope, or full length gene constructs were generated by in vitro recombination using standard methods as previously described (Perkus, et al. Methodology of Using Vaccinia Viras to Express Foreign Genes in Tissue Culture. J. Tissue Cult. Meth. 1993;15:72-81; Perkus, et al. Poxvirus-based vaccine candidates for cancer, AIDS, and infectious diseases. J. Leukocyte Biol. 1995;58:1- 13). Expression cassettes consisting of single or polyepitope gene constructs were generated by overlap-extension PCR with synthetic oligonucleotides, and placed under the control of the vaccinia H6 promoter by use of standard gene cloning techniques. A summary of the recombinants and constructs evaluated in this study are listed in Table III. These constructs are based on HLA-A2.1 -restricted epitopes identified in Melan A/MART-1 (MART-1^27"35) and gplOO (gplOO^154"162, gplOO^209"217, gplOO^280"288). The MARTI ^27'35 epitope is an immunodominant epitope. gplOO^154"162 represents also an HLA -A2 restricted epitope with high affinity for MHC class I and high immunogenicity (Salgaller, et al. Can. Res. 1996; 56:4749-4757). A basic polyepitope construct was derived, called PE01, consisting of the MART-1^27-35, gplOO^154-162, gplOO^209"217, and gplOO^280"288 epitopes with no spacer amino acids flanking the epitopes.

Table πi

Cell lines

The EBV-transformed human B-LCL line BN was derived from an HLA-A2.1- positive donor. The HLA-A2.1 -positive human bladder carcinoma line J82 was obtained from ATCC. TIL lines 620 (specific for the HLA-A2.1 -restricted epitopes MART-1^27"35 and gplOO^209"217), 1200 (specific for HLA-A2.1 -restricted epitope gplOO^154"162), were provided by Dr. U. Kawakami (NCI, USA) and were maintained in AIM V medium supplemented with 10% human AB serum and 6000 U/ml IL-2 [25].

Cytotoxicity assays with TIL lines The processing and presentation of epitopes expressed by gene constructs in ALVAC was assessed by evaluating the ability of TIL lines to lyse J82 target cells infected with appropriate ALVAC recombinants. J82 cells were infected with ALVAC recombinants for 18 hours using a multiplicity of infection of 5. These targets were subsequently incubated with TIL lines at various effector to target (E:T) ratios in a standard four hour ⁵¹Cr release assay for cytotoxicity. Controls included in each experiment were unmodified J82 cells, J82 cells pulsed with the appropriate Melan A/MART-1 or gplOO epitope peptides at 100 μg peptide/ 10⁶ cells, and J82 cells infected with parental ALVAC viras. For the TIL line 620, which exhibits multiple epitope specificities, lytic assays were performed in the presence of unlabelled J82 cells infected with an ALVAC recombinant expressing the MART-1^27"35 epitope. These cold targets were included in the assays at a ratio of 30:1 cokhradio-labelled target. Results obtained in parallel assays which lacked the cold targets allowed the discrimination of lysis directed against each of the epitope specificities.

Immunization and CTL evaluation of HHD mice Transgenic HLA-A2.1 HHD mice have been previously described (Pascolo, et al. J.

Exp. Med. 1997; 12: 2043-2051; Firat, et al. Eur. J. Immunol. 1999; 29:3112-3121).- These mice express a transgene consisting of the αl and α2 domains of the human HLA-A2.1 class I molecule linked to the α3, transmembrane, and cytoplasmic domains of the mouse class I molecule H- 2D . This hybrid molecule is linked via the αl domain to human β₂-microglobulin, and its hybrid nature facilitates further interaction with mouse CD8 on T cells. This strain was generated on the H-2D^b double knockout genetic background, and thus the only class I molecule expressed by HHD mice is the HLA-A2.1/H-2D^b hybrid transgene. This strain provides a good model to evaluate the capacity of various immunogens to elicit CTL responses restricted by the HLA-A2.1 molecule. Immunization experiments and read-outs of CTL activity were performed as described elsewhere. Briefly, groups of six mice were immunized with 10^s pfu of appropriate ALVAC recombinants through the intraperitoneal route. Three weeks after immunization, splenocytes were harvested and restimulated in vitro with peptide-pulsed LPS blasts. After six days in culture, the lytic activity was assessed in a standard ⁵¹Cr-release assay, using RMAS-HHD cells pulsed with appropriate epitope peptides as targets.

Processing and presentation of polyepitope constructs or minigenes The processing and presentation of polyepitope constructs or minigenes expressed by

ALVAC was assessed by evaluating the ability of ALVAC-infected HLA-A2.1+ bladder carcinoma cells (J82) to stimulate the lytic activity of epitope-specific TIL lines. ALVAC recombinants expressing the various polyepitope configurations were compared in this manner to recombinants expressing both full length Melan A/MART-1 and gplOO, or alternatively single epitope minigenes. Peptide-pulsed J82 cells and non-pulsed J82 cells were used as positive and negative controls, respectively. As confirmed from the specific lysis by TIL 620, MART-1^27"35 is processed and presented from each of the three tested polypepitopes (Figure 1). The levels of lysis were similar with targets expressing each of the constructs. Good responses are also stimulated by cells expressing the Melan A/MART-1^27"35 minigene (Figure 1A). As shown in Figure IB, cells expressing the ALVAC gplOO^154"162 single epitope minigene stimulated strong cytotoxic responses from the specific TIL lines. Associated results obtained with TIL 620 in the presence of cold target cells expressing the

97 oc 9f)0 917

Melan A/MART-1 ^" epitope indicate that the gp 100 ^" epitope is very poorly processed and presented from either ALVAC expressing full length gplOO or from the three polypepitope constracts, as evidenced following infection of J82 cells (Figure IC).

90Q 17

Interestingly, cells expressing the gplOO ^" single epitope minigene did stimulate cytotoxic responses, suggesting that for this epitope, expression and processing as a minigene were better than when relying upon the full length construct. Other constracts containing potential spacer amino acid sequences flanking the component epitopes, either AAA (PE03) or NKRK (PE02) were also produced (Table III), in order to test the hypothesis that flanking residues can facilitate processing and presentation of the T cell epitope [28-30]. In contrast with some of the studies, but in agreement with others [3,4], we did not see any impact of the spacer in

9RO 9RR these experiments. In the absence of specific Tils against the gp 100 ^" epitope, we were not able to evaluate the processing and presentation of this epitope from these constructs. Altogether, reliable processing and presentation were observed for all assessable epitopes, albeit at different levels. Although good Melan A/MART-1^27"35 stimulation was seen in general, for the gplOO^209"217 and gplOO^154"162 epitopes, minigenes were found to be significantly better for expressing the corresponding epitope and stimulating the TIL lines than full length antigens. These results are in agreement with previous studies.

Immunization of HHD mice Immunogenicity studies were subsequently performed in vivo, using HLA-A2.1 transgenic mice as a model. In a first set of experiments, we compared ALVAC-polyepitopes and ALVAC minigenes with respect to their capacity to elicit CTLs in HHD mice. Based on the observation that the MART-1^27"35 epitope was not immunogenic in such mice, we focused on T cell responses against gplOO epitopes (Table IV). In a first series of experiments, HHD mice were immunized with ALVAC constructs expressing either the tall length gplOO gene, polyepitopes, or single minigenes.

A first observation was that the polyepitope constracts do not elicit broad CTL responses against most of the component epitopes (Table IV). One exception was PEO2, which elicits some weak responses against gplOO^{154"1 5}, in 2 out of 6 mice. Noteworthy, these results are in agreement with our first set of experiments, showing weak, albeit detectable, expression of some of the epitopes when relying upon polyepitopes. These results were also confirmed by a lack of stimulation of gplOO specific CTLs in vitro from human peripheral blood mononuclear cells derived from HLA-A2.1 donors (not shown). Altogether, these results confirm the difficulty to elicit broad and balanced immune responses against multiple epitopes when relying on polyepitopes. Interestingly, in contrast, immunization with ALVAC-based single epitope constructs was found to be effective, excepted for the gplOO^280" 980 9KR epitope. As stated above, we cannot rule out that the gplOO ^" was not expressed optimally by the ALVAC recombinant. Here again, these immunogenicity data correlates with our expression data shown in Figure 1. For comparison, the full length gplOO recombinant is effective in inducing CTLs against gplOO ^{5 "}' , but not against any of the other cryptic HLA-A2.1 restricted epitopes (Table IV).

Table IV

Polyepitope enhancement approaches

A second series of constructs was generated to create immunogens in which immunostimulatory properties of weak immunogens could be 'enhanced'. To this aim, we evaluated epitopes that had been modified to increase their affinity for class I HLA molecules. Given the relatively good immunogenicity of the gplOO^154"162 epitope, we elected to focus on improving the immunogenicity of the gplOO^209"217 and gplOO^280"288 epitopes. Two ALVAC recombinants were made, which are listed in Table II. These include a construct expressing tall length gplOO that has been modified to contain the 209-2M and 280-9 V amino acid modifications (gplOOM), and a polyepitope constmct expressing single epitopes

(ie gplOO 154-162 gp lOO ,2"0™9-2M and gplOO 280-9V under separate promoters (Table III). These constructs were used to immunize HHD mice, as shown in Table V, no CTLs specific for gplOO were induced by ALVAC alone, used as a control. When using ALVAC expressing full-length gplOOM (with modified epitopes) CTLs against all three epitopes were detected. The modified polyepitopic constmct (154/2M/9V) was also found to elicit strong CTL responses, and potentially at a level higher than the full length immunogen: with the full length gplOOM, one or two mice did not respond for each epitope, whereas all mice (6 out of 6) receiving the modified polyepitope mounted CTLs against each single epitope (Table V). Importantly, the protocol used to detect CTL responses in HHD mice detects only CTLs which cross-react with natural unmodified gplOO epitopes. This confirms that enhanced immunogens with improved affinity for MHC Class I molecules have a better immunogenicity, and can elicit CTLs recognizing native gplOO T cell epitopes (i.e., as they are expressed by tumor cells).

Table V

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

Claims

CLAIMS What is claimed is:

1. A recombinant expression vector comprising a nucleic acid sequence encoding at least one polyepitope selected from the group consisting of MART-1 - gplOO ^" - gplOO^209"217 - gplOO^280"288; MART-1^27"35 -NKRK-gpl00^154",o -NKRK-gpl00^209"217-

NKRK- gplOO^280"288; and, MART-1^27"35 -AAA- gplOO^154"162 -AAA- gplOO^209"217 - AAA- gplOO^280"288.

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 poxvims, adenoviras, retrovirus, herpesvirus, and adeno-associated viras.

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

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

6. A recombinant expression vector comprising a nucleic acid sequence encoding at least one minigene selected from the group consisting of MART-1^27"35; gplOO^154"162; gplOO^{209" 217}; gplOO^280"288; gpl00-209-2M; and, gpl00-280-9V.

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

8. The expression vector of claim 2 wherein the viral vector is selected from the group consisting of poxvirus, adenoviras, retrovirus, herpesvirus, and adeno-associated viras.

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

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

11. A composition comprising an expression vector comprising a nucleic acid sequence encoding at least one polyepitope selected from the group consisting of MART-1^27"35 - gplOO^154"162 - gplOO^209"217 - gplOO^280"288; MART-1^27"35 -NKRK-gpl00^154"162-NKRK- gpl00^209"217-NKRK- gplOO^280"288; and, MART-1^27"35 -AAA- gplOO^154"162 -AAA- gpl00^209"2I7 -AAA- gpl00²⁸⁰-²⁸⁸.

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

13. The composition of claim 12 wherein the viral vector is selected from the group consisting of poxvims, adenoviras, retrovirus, herpesvirus, and adeno-associated viras.

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

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

1 . A method for preventing or treating cancer comprising administering to a host an expression vector comprising a nucleic acid sequence encoding at least one polyepitope selected from the group consisting of MART-1^27"35 - gplOO^154"162 - gplOO^209"217 - gplOO^280"288; MART-1^27"35 -NKRK-g l00^154"162-NKRK-gpl00^209"217-NKRK- gplOO^{280" 288}; and, MART-1^27"35 -AAA- gplOO^154"162 -AAA- gplOO^209"217 -AAA- gplOO^280"288.

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

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

19. The method of claim 18 wherein the viral vector is a poxvirus selected from the group consisting of vaccinia, NYVAC, avipox, canarypox, ALVAC, ALVAC(2), fowlpox, and TROVAC.

20. The method of claim 19 wherein wherein the viral vector is a poxvirus selected from the group consisting of NYVAC, ALVAC, and ALVAC(2).

21. A composition comprising an expression vector comprising a nucleic acid sequence encoding at least one minigene selected from the group consisting of MART-1 97 ^" ^S ; gplOO^154"162; gplOO^209"217; gplOO^280"288; gpl00-209-2M; and, gpl00-280-9V.

22. The composition of claim 21 wherein the expression vector is a plasmid or a viral vector.

23. The composition of claim 22 wherein the viral vector is selected from the group consisting of poxvims, adenoviras, retro vims, herpesvirus, and adeno-associated vims.

24. The composition of claim 23 wherein the viral vector is a poxvirus selected from the group consisting of vaccinia, NYVAC, avipox, canarypox, ALVAC, ALVAC(2), fowlpox, and TROVAC.

25. The composition of claim 24 wherein the viral vector is a poxvirus selected from the group consisting of NYVAC, ALVAC, and ALVAC(2).

26. A method for preventing or treating cancer comprising administering to a host an expression vector comprising a nucleic acid sequence encoding at least one minigene selected from the group consisting of MART-1^27"35; gplOO^154"162; gplOO^209"217; gplOO^{280" 288}; gpl00-209-2M; and, gpl00-280-9V.

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

28. The method of claim 27 wherein the viral vector is selected from the group consisting of poxvirus, adenoviras, retrovirus, herpesvirus, and adeno-associated viras.

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

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