US20130011422A1

US20130011422A1 - Tumor Antigens for the Prevention and/or Treatment of Cancer

Info

Publication number: US20130011422A1
Application number: US13/485,668
Authority: US
Inventors: Neil Berinstein; Scott Gallichan; Corey Lovitt; Mark Parrington; Laszlo Radvanyi; Devender Singh-Sandhu
Original assignee: Individual
Current assignee: Individual
Priority date: 2003-05-16
Filing date: 2012-05-31
Publication date: 2013-01-10
Also published as: CN102964426B; WO2004104039A2; EP1631585A1; US20100278848A1; US8207314B2; JP5503630B2; CN1910198B; JP2011067207A; CN1910198A; CA2527640A1; JP5363446B2; CA2527640C; JP2008518583A; CN102964426A; WO2004104039A8; JP2012110328A

Abstract

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

Description

RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 10/557,066 filed Jul. 30, 2007, which was filed under 35 U.S.C. §371, and claims priority to International Application No. PCT/US2004/015202 filed May 15, 2004, which claims priority to Ser. Nos. 60/471,119 filed May 16, 2003 and 60/471,193 filed May 16, 2003.

FIELD OF THE INVENTION

BACKGROUND OF THE INVENTION

There has been tremendous increase in last few years in the development of cancer vaccines with Tumour-associated antigens (TAAs) due to the great advances in identification of molecules based on the expression profiling on primary tumours and normal cells with the help of several techniques such as high density microarray, SEREX, immunohistochemistry (IHC), RT-PCR, in-situ hybridization (ISH) and laser capture microscopy (Rosenberg, Immunity, 1999; Sgroi et al, 1999, Schena et al, 1995, Offringa et al, 2000). The TAAs are antigens expressed or over-expressed by tumour cells and could be specific to one or several tumours for example CEA antigen is expressed in colorectal, breast and lung cancers. Sgroi et al (1.999) identified several genes differentially expressed in invasive and metastatic carcinoma cells with combined use of laser capture microdissection and cDNA microarrays. Several delivery systems like DNA or viruses could be used for therapeutic vaccination against human cancers (Bonnet et al, 2000) and can elicit immune responses and also break immune tolerance against TAAs. Tumour cells can be rendered more immunogenic by inserting transgenes encoding T cell co-stimulatory molecules such as B7.1 or cytokines such as IFN-γ, IL2, or GM-CSF, among others. Co-expression of a TAA and a cytokine or a co-stimulatory molecule has also been shown to be useful in developing effective therapeutic vaccines (Hodge et al, 95, Bronte et al, 1995, Chamberlain et al, 1996).
There is a need in the art for reagents and methodologies useful in stimulating an immune response to prevent or treat cancers. The present invention provides such reagents and methodologies which overcome many of the difficulties encountered by others in attempting to treat cancer.

SUMMARY OF THE INVENTION

The present invention provides an immunogenic target for administration to a patient to prevent and/or treat cancer. In particular, the immunogenic target is a tumor antigen (“TA”) and or an angiogenesis-associated antigen (“AA”). In one embodiment, the immunogenic target is encoded by SEQ ID NO.: 34 or SEQ ID NO.: 36 or has the amino acid sequence of SEQ ID NO.: or SEQ ID NO.: 37. In certain embodiments, the TA and/or AA are administered to a patient as a nucleic acid contained within a plasmid or other delivery vector, such as a recombinant virus. The TA and/or AA may also be administered in combination with an immune stimulator, such as a co-stimulatory molecule or adjuvant.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. A, B. Nucleotide sequences of AAC2-1 and AAC2-2. C. Alignment of predicted amino acid sequence of AAC2-1 and AAC2-2. Missing nucleotides or amino acids are indicated by a “*”. Differences between sequences are underlined.

FIG. 2. Human lymphocytes differentiate into effector cells secreting IFN-γ in response to peptides derived from the AAC2-2 protein. T cells were stimulated with the groups of peptides shown n in Table III (groups 1-9). After three rounds of stimulation, the lymphocytes were analyzed for peptide-specific IFN-γ production by ELISPOT. The graph in the inset shows that activated cells stimulated by peptide Group #6 are capable of antigen-specific CTL activity killing peptide loaded T2 target cells. Peptide EC5 elicits dominant activity in inducing both CTL activity and IFN-γ secretion.

FIG. 3. Murine T cells from HLA-A2-Kb transgenic mice recognize and secrete IFN-γ in response to DNA immunization with a human AAC2-2-encoding DNA plasmid. Spleen cells from pEF6-hAAC2-2-immunized mice were re-stimulated with the different groups of peptides. After six days, the cells were harvested and tested for IFN-γ secretion in response to each respective peptide group or a control HLA-A2-binding 9-mer HIV peptide. ELISPOT plates were incubated over-night and developed. Each group responded with high levels of IFN-γ production (over 250 spots) in response to PMA and ionomycin used as a positive control. One of the highly reactive peptides groups (group 6) is also recognized by human lymphocytes from the HLA-A-0201⁺donors tested so far.

FIG. 4. DNA vaccination with a gene encoding human AAC2-2 completely abrogates the growth of implanted B16F10 melanoma cells. This effect is not due to a non-specific immune response as shown by the inability of plasmid encoding flu-NP protein and the human flk1 (VEGFR-2) to prevent tumor growth.

FIG. 5. Survival of mice after implantation of B16F10 melanoma cells into C57BL/6 mice showing the ability of DNA vaccination with a human AAC2-2 vector to completely protect against the effects of rumor growth. This protective effect is antigen-specific and can not be elicited through vaccination with other genes.

FIG. 6. T lymphocytes from C57BL/6 mice exhibit effector cell activity and secrete IFN-γ in response to peptides of human AAC2-2 following DNA vaccination with the pEF6-hAAC2-2 expression plasmid. These peptides can exhibit cross-reactivity on B6 MHC class I. The peptides in group 1 and group 5 induce strong reactivity by C57BL/6 T cells.

FIG. 7. BFA4 cDNA sequence.

FIG. 8. BFA4 amino acid sequence.

FIG. 9. BCY1 nucleotide (A) and amino acid (B) sequences.

FIG. 10. Immune response against specific BCY1 peptides.

FIG. 11. BFA5 cDNA sequence.

FIG. 12. BFA5 amino acid sequence.

FIGS. 13A, B and C. Immune response against BFA5-derived peptides.

FIG. 14. BCZ4 cDNA (A) and amino acid (B) sequences.

FIG. 15. Immune response against BCZ4-derived peptides (A: BCZ4 ELISPOT; B: BCZ4 Peptide Deconvolution; C: CTL response).

FIG. 16. BFY3 cDNA (A) and amino acid (B) sequences.

FIG. 17A-E. Immune response against BFY3-derived peptides.

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.
As used herein, an “antigen” is a molecule such as a polypeptide or a portion thereof that produces an immune response in a host to whom the antigen has been administered. The immune response may include the production of antibodies that bind to at least one epitope of the antigen and/or the generation of a cellular immune response against cells expressing an epitope of the antigen. The response may be an enhancement of a current immune response by, for example, causing increased antibody production, production of antibodies with increased affinity for the antigen, or an increased or more effective cellular response (i.e., increased T cells or T cells with higher anti-tumor activity). An antigen that produces an immune response may alternatively be referred to as being immunogenic or as an immunogen. In describing the present invention, a TA may be referred to as an “immunogenic target”.
TA includes both tumor-associated antigens (TAAs) and tumor-specific antigens (TSAs), where a cancerous cell is the source of the antigen. A TAA is an antigen that is expressed on the surface of a tumor cell in higher amounts than is observed on normal cells or an antigen that is expressed on normal cells during fetal development. A TSA is an antigen that is unique to tumor cells and is not expressed on normal cells. TA further includes TAAs or TSAs, antigenic fragments thereof, and modified versions that retain their antigenicity.
TAs are typically classified into five categories according to their expression pattern, function, or genetic origin: cancer-testis (CT) antigens (i.e., MAGE, NY-ESO-1); melanocyte differentiation antigens (i.e., Melan A/MART-1, tyrosinase, gp100); mutational antigens (i.e., MUM-1, p53, CDK-4); overexpressed ‘self’ antigens (i.e., HER-2/neu, p53); and, viral antigens (i.e., HPV, EBV). For the purposes of practicing the present invention, a suitable TA is any TA that induces or enhances an anti-tumor immune response in a host in whom the TA is expressed. Suitable TAs include, for example, gp100 (Cox et al., Science, 264:716-719 (1994)), MART-1/Melan A (Kawakami et al., J. Exp. Med., 180:347-352 (1994)), gp75 (TRP-1) (Wang et al., J. Exp. Med., 186:1131-1140 (1996)), tyrosinase (Wolfel et al., Eur. J. Immunol., 24:759-764 (1994); WO 200175117; WO 200175016; WO 200175007), NY-ESO-1 (WO 98/14464; WO 99/18206), melanoma proteoglycan (Hellstrom et al., J. Immunol. 130:1467-1472 (1983)), MAGE family antigens (i.e., MAGE-1, 2, 3, 4, 6, 12, 51; Van der Bruggen et al., Science, 254:1643-1647 (1991); U.S. Pat. Nos. 6,235,525; CN 1319611), BAGE family antigens (Boel et al., Immunity, 2:167-175 (1995)), GAGE family antigens (i.e., GAGE-1,2; Van den Eynde et al., J. Exp. Med., 182:689-698 (1995); U.S. Pat. No. 6,013,765), RAGE family antigens (i.e., RAGE-1; Gaugler et al., Immunogenetics, 44:323-330 (1996); U.S. Pat. No. 5,939,526), N-acetylglucosaminyltransferase-V (Guilloux et al., J. Exp. Med., 183:1173-1183 (1996)), p15 (Robbins et al., J. Immunol. 154:5944-5950 (1995)), 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 al., Int. J. Cancer, 56:40-45 (1994)), BCR-abl (Bocchia et al., Blood, 85:2680-2684 (1995)), p53 (Theobald et al., Proc. Natl. Acad. Sci. USA, 92:11993-11997 (1995)), p185 HER2/neu (erb-B1; Fisk et al., J. Exp. Med., 181:2109-2117 (1995)), epidermal growth factor receptor (EGFR) (Harris et al., Breast Cancer Res. Treat, 29:1-2 (1994)), carcinoembryonic antigens (CEA) (Kwong et al., J. Natl. Cancer Inst., 85:982-990 (1995) U.S. Pat. Nos. 5,756,103; 5,274,087; 5,571,710; 6,071,716; 5,698,530; 6,045,802; EP 263933; EP 346710; and, EP 784483); carcinoma-associated mutated mucins (i.e., MUC-1 gene products; Jerome et al., J. Immunol. 151:1654-1662 (1993)); EBNA gene products of EBV (i.e., EBNA-1; Rickinson et al., Cancer Surveys, 13:53-80 (1992)); E7, E6 proteins of human papillomavirus (Ressing et al., J. Immunol, 154:5934-5943 (1995)); prostate specific antigen (PSA; Xue et al., The Prostate, 30:73-78 (1997)); prostate specific membrane antigen (PSMA; Israeli, et al., Cancer Res., 54:1807-1811 (1994)); idiotypic epitopes or antigens, for example, immunoglobulin idiotypes or T cell receptor idiotypes (Chen et al., J. Immunol., 153:4775-4787 (1994)); KSA (U.S. Pat. No. 5,348,887), kinesin 2 (Dietz, et al. Biochem Biophys Res Commun 2000 Sep. 7; 275(3):731-8), HIP-55, 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-B R-85, NY-BR-87, NY-BR-96 (Scanlan, M. Serologic and Bioinformatic Approaches to the Identification of Human Tumor Antigens, in Cancer Vaccines 2000, Cancer Research Institute, New York, N.Y.), BFA4 (SEQ ID NOS.: 23 and 24), BCY1 (SEQ ID NOS.: 25 and 26), BFA5 (SEQ ID NOS.: 27 and 28), BCZ4 (SEQ ID NOS.: 29 and 30), and BFY3 (SEQ ID NOS. 31 and 32), 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. For treatment of cancer, it is preferred that that the AA be found within or near blood vessels that supply a tumor. Immunization of a patient against an AA preferably results in an anti-AA immune response whereby angiogenic processes that occur near or within tumors are prevented and/or inhibited.
Exemplary AAs include, for example, vascular endothelial growth factor (i.e., VEGF; Bernardini, et al. J. Urol., 2001, 166(4): 1275-9; Starnes, et al. J. Thorac. Cardiovasc. Surg., 2001, 122(3): 518-23; Dias, et al. Blood, 2002, 99: 2179-2184), the VEGF receptor (i.e., VEGF-R, 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-α; 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. Oncot., 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, α1β1, α2β1, α5β1), the surface proteolglycan NG2, AAC2-1 (SEQ ID NO.:1), or AAC2-2 (SEQ ID NO.:2), among others, including “wild-type” (i.e., normally encoded by the genome, naturally-occurring), modified, mutated versions as well as other fragments and derivatives thereof. Any of these targets may be suitable in practicing the present invention, either alone or in combination with one another or with other agents.
In certain embodiments, a nucleic acid molecule encoding an immunogenic target is utilized. The nucleic acid molecule may comprise or consist of a nucleotide sequence encoding one or more immunogenic targets, or fragments or derivatives thereof, such as that contained in a DNA insert in an ATCC Deposit. The term “nucleic acid sequence” or “nucleic acid molecule” refers to a DNA or RNA sequence. The term encompasses molecules formed from any of the known base analogs of DNA and RNA such as, but not limited to 4-acetylcytosine, 8-hydroxy-N-6-methyladenosine, aziridinyl-cytosine, pseudoisocytosine, 5-(carboxyhydroxylmethyl)uracil, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxy-methylaminomethyluracil, dihydrouracil, inosine, N6-iso-pentenyladenine, 1-methyladenine, 1-methylpseudouracil, 1-methylguanine, 1-methylinosine, 2,2-dimethyl-guanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyamino-methyl-2-thiouracil, beta-D-mannosylqueosine, 5′-methoxycarbonyl-methyluracil, 5-methoxyuracil, 2-methylthio-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 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 to 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 without manipulation by man. Similarly, “non-naturally occurring” or “non-native” as used herein refers to a material that is not found in nature or that has been structurally modified or synthesized by man.
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, NaDodSO₄, (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 certain embodiments of the present invention, vectors are used to transfer a nucleic acid sequence encoding a polypeptide to a cell. A vector is any molecule used to transfer a nucleic acid sequence to a host cell. In certain cases, an expression vector is utilized. An expression vector is a nucleic acid molecule that is suitable for transformation of a host cell and contains nucleic acid sequences that direct and/or control the expression of the transferred nucleic acid sequences. Expression includes, but is not limited to, processes such as transcription, translation, and splicing, if introns are present. Expression vectors typically comprise one or more flanking sequences operably linked to a heterologous nucleic acid sequence encoding a polypeptide. Flanking sequences may be homologous (i.e., from the same species and/or strain as the host cell), heterologous (i.e., from a species other than the host cell species or strain), hybrid (i.e., a combination of flanking sequences from more than one source), or synthetic, for example.
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 transcriptional regulatory region that drives high-level gene expression in the target cell. The transcriptional regulatory region may comprise, for example, a promoter, enhancer, silencer, repressor element or combinations thereof. The transcriptional regulatory region may be either constitutive, tissue-specific, cell-type specific (i.e., the region is drives higher levels of transcription in a one type of tissue or cell as compared to another), or regulatable (i.e., responsive to interaction with a compound). The source of a transcriptional regulatory region may be any prokaryotic or eukaryotic organism, any vertebrate or invertebrate organism, or any plant, provided that the flanking sequence functions in a cell by causing transcription of a nucleic acid within that cell. A wide variety of transcriptional regulatory regions may be utilized in practicing the present invention.
Suitable transcriptional regulatory regions include, for example, the CMV promoter (i.e., the CMV-immediate early promoter); promoters from eukaryotic genes (i.e., the estrogen-inducible chicken ovalbumin gene, the interferon genes, the gluco-corticoid-inducible tyrosine aminotransferase gene, and the thymidine kinase gene); and the major early and late adenovirus gene promoters; the SV40 early promoter region (Bernoist and Chambon, 1981, Nature 290:304-10); the promoter contained in the 3′ long terminal repeat (LTR) of Rous sarcoma virus (RSV) (Yamamoto, et al., 1980, Cell 22:787-97); the herpes simplex virus thymidine kinase (HSV-TK) promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. U.S.A. 78:1444-45); the regulatory sequences of the metallothionine gene (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. USA., 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 7:425-51.5); the insulin gene control region which is active in pancreatic beta cells (Hanahan, 1985, Nature 315:115-22); the immunoglobulin gene control region which is active in lymphoid cells (Grosschedl et al., 1984, Cell 38:647-58; Adames et al., 1985, Nature 318:533-38; Alexander et al., 1987, Mol. Cell. Biol., 7:1436-44); the mouse mammary tumor virus control region in testicular, breast, lymphoid and mast cells (Leder et al., 1986, Cell 45:485-95); the albumin gene control region in liver (Pinkert et al., 1987, Genes and Devel. 1:268-76); the alpha-feto-protein gene control region in liver (Krumlauf et al., 1985, Mol. Cell. Biol., 5:1639-48; Hammer et al., 1987, Science 235:53-58); the alpha 1-antitrypsin gene control region in liver (Kelsey et al., 1987, Genes and Devel. 1:161-71); the beta-globin gene control region in myeloid cells (Mogram et al., 1985, Nature 315:338-40; Kollias et al., 1986, Cell 46:89-94); the myelin basic protein gene control region in oligodendrocyte cells in the brain (Readhead et al., 1987, Cell 48:703-12); the myosin light chain-2 gene control region in skeletal muscle (Sani, 1985, Nature 314:283-86); the gonadotropic releasing hormone gene control region in the hypothalamus (Mason et al, 1986, Science 234:1372-78), and the tyrosinase promoter in melanoma cells (Hart, I. Semin Oncol 1996 February; 23(1):154-8; Siders, et al. Cancer Gene Ther 1998 September-October; 5(5):281-91), among others. Inducible promoters that are activated in the presence of a certain compound or condition such as light, heat, radiation, tetracycline, or heat shock proteins, for example, may also be utilized (see, for example, WO 00/10612). Other suitable promoters are known in the art.
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 polynucleotides, lipids, carbohydrates, or other materials with which it is naturally found when isolated from the source cell: (2) is not linked (by covalent or noncovalent interaction) to all or a portion of a polypeptide to which the “isolated poly peptide” 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 substitutions, additions, deletions, or chemically modified variants.
Variants may also include fusion proteins, which refers to the fusion of one or more first sequences (such as a peptide) at the amino or carboxy terminus of at least one other sequence (such as a heterologous peptide).
“Similarity” is a concept related to identity, except that similarity refers to a measure of relatedness which includes both identical matches and conservative substitution matches. If two polypeptide sequences have, for example, 10/20 identical amino acids, and the remainder are all non-conservative substitutions, then the percent identity and similarity would both be 50%. If in the same example, there are five more positions where there are conservative substitutions, then the percent identity remains 50%, but the percent similarity would be 75% ( 15/20). Therefore, in cases where there are conservative substitutions, the percent similarity between two polypeptides will be higher than the percent identity between those two polypeptides.
Substitutions may be conservative, or non-conservative, or any combination thereof. Conservative amino acid modifications to the sequence of a polypeptide (and the corresponding modifications to the encoding nucleotides) may produce polypeptides having functional and chemical characteristics similar to those of a parental polypeptide. For example, a “conservative amino acid substitution” may involve a substitution of a native amino acid residue with a non-native residue such that there is little or no effect on the size, polarity, charge, hydrophobicity, or hydrophilicity of the amino acid residue at that position and, in particular, does not result in decreased immunogenicity. Suitable conservative amino acid substitutions are shown in Table I.

TABLE I

Original		Preferred
Residues	Exemplary Substitutions	Substitutions

Ala	Val, Leu, Ile	Val
Arg	Lys, Gln, Asn	Lys
Asn	Gln	Gln
Asp	Glu	Glu
Cys	Ser, Ala	Ser
Gln	Asn	Asn
Glu	Asp	Asp
Gly	Pro, Ala	Ala
His	Asn, Gln, Lys, Arg	Arg
Ile	Leu, Val, Met, Ala, Phe, Norleucine	Leu
Leu	Norleucine, Ile, Val, Met, Ala, Phe	Ile
Lys	Arg, 1,4 Diamino-butyric Acid, Gln, Asn	Arg
Met	Leu, Phe, Ile	Leu
Phe	Leu, Val, Ile, Ala, Tyr	Leu
Pro	Ala	Gly
Ser	Thr, Ala, Cys	Thr
Thr	Ser	Ser
Trp	Tyr, Phe	Tyr
Tyr	Trp, Phe, Thr, Ser	Phe
Val	Ile, Met, Leu, Phe, Ala, Norleucine	Leu

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. For example, when similar polypeptides with similar activities from the same species or from other species are known, one skilled in the art may compare the amino acid sequence of a polypeptide to such similar polypeptides. By performing such analyses, one can identify residues and portions of the molecules that are conserved among similar polypeptides. It will be appreciated that changes in areas of the molecule that are not conserved relative to such similar polypeptides would be less likely to adversely affect the biological activity and/or structure of a polypeptide. Similarly, the residues required for binding to MHC are known, and may be modified to improve binding. However, modifications resulting in decreased binding to MHC will not be appropriate in most situations. One skilled in the art would also know that, even in relatively conserved regions, one may substitute chemically similar amino acids for the naturally occurring residues while retaining activity. Therefore, even areas that may be important for biological activity or for structure may be subject to conservative amino acid substitutions without destroying the biological activity or without adversely affecting the polypeptide structure.
Other preferred polypeptide variants include glycosylation variants wherein the number and/or type of glycosylation sites have been altered compared to the subject amino acid sequence. In one embodiment, polypeptide variants comprise a greater or a lesser number of N-linked glycosylation sites than the subject amino acid sequence. An N-linked glycosylation site is characterized by the sequence Asn-X-Ser or Asn-X-Thr, wherein the amino acid residue designated as X may be any amino acid residue except proline. The substitution of amino acid residues to create this sequence provides a potential new site for the addition of an N-linked carbohydrate chain. Alternatively, substitutions that eliminate this sequence will remove an existing N-linked carbohydrate chain. Also provided is a rearrangement of N-linked carbohydrate chains wherein one or more N-linked glycosylation sites (typically those that are naturally occurring) are eliminated and one or more new N-linked sites are created. To affect O-linked glycosylation of a polypeptide, one would modify serine and/or threonine residues.
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. These sequences, referred to as transduction or transcytosis sequences, include sequences derived from HIV tat (see Kim et al. 1997 J. Immunol. 159:1666), Drosophila antennapedia (see Schutze-Redelmeier et al. 1996 J. Immunol. 157:650), or human period-1 protein (hPER1; in particular, SRRHHCRSKAKRSHH (SEQ ID NO: 42)).
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-stimulator 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 (CD11a/CD8); Sedwick, et al. J Immunol 1999, 162: 1367-1375; Wülfing, et al. Science 1998, 282: 2266-2269; Lub, et al. Immunol Today 1995, 16: 479-483) including members of the ICAM family (i.e., ICAM-1, -2 or -3); polypeptides which bind CD2 family members (i.e., CD2, signalling lymphocyte activation molecule (CDw150 or “SLAM”; Aversa, et al. J Immunol 1997, 158: 4036-4044)) such as CD58 (LFA-3; CD2 ligand; Davis, et al. Immunol Today 1996, 17: 177-187) or SLAM ligands (Sayos, et al. Nature 1998, 395: 462-469); polypeptides which bind heat stable antigen (HSA or CD24; Zhou, et al. Eur J. Immunol 1997, 27: 2524-2528); polypeptides which bind to members of the TNF receptor (TNFR) family (i.e., 4-IBB (CD137; Vinay, et al. Semin Immunol 1998, 10: 481-489), OX40 (CD-134; 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-IBBL (4-IBB 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-IBB ligand; Saoulli, et al. J Exp Med 1998, 187: 1849-1862, Arch, et al. Mol Cell Biol 1998, 18: 558-565), TRAF-2 (4-IBB and OX40 ligand; Saoulli, et al. J Exp Med 1998, 187: 1849-1862: Oshima, et al. Int Immunol 1.998, 10: 517-526, Kawamata, et al. J Biol Chem 1.998, 273: 5808-5814), TRAF-3 (4-IBB and OX40 ligand; Arch, et al. Mol Cell Biol 1998, 18: 558-565; Jang, et al. Biochem Biophys Res Commun 1998, 242: 613-620; Kawamata S, et al. J Biol Chem 1998, 273: 5808-5814), OX40L (OX40 ligand; Gramaglia, et al. J Immunol 1998, 161: 6510-6517), TRAF-5 (OX40 ligand; Arch, et al. Mol Cell Biol 1998, 18: 558-565; Kawamata, et al. J Biol Chem 1998, 273: 5808-5814), and CD70 (CD27 ligand; Couderc, et al. Cancer Gene Ther., 5(3): 163-75). CD154 (CD40 ligand or “CD40L”; Gurunathan, et al. J. Immunol. 1998, 161: 4563-4571; Sine, et al. Hum. Gene Ther., 2001, 12: 1091-1102) may also be suitable.
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. T. Gene Med. 2000 July-August; 2(4):243-9; Rao, et al. J. Immunol. 156: 3357-3365 (1996)), IL-15 (Xin, et al. Vaccine, 17:858-866, 1999), IL-16 (Cruikshank, et al. J. Leuk Biol. 67(6): 757-66, 2000), IL-18 (J. Cancer Res. Clin. Oncol. 2001. 127(12): 718-726), GM-CSF (CSF (Disis, et al. Blood, 88: 202-210 (1996)), tumor necrosis factor-alpha (TNF-α), or interferons such as IFN-α or 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 tumor self-antigen have been shown to induce anti-tumor immunity (Biragyn, et al. Nature Biotech. 1999, 17: 253-258). The chemokines CCL3 (MIP-1α) 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 (Alers, 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 alt 1999, Vaccine, 17: 3124-2135; Dubensky, et al. 2000. Mol. Med. 6: 723-732; Leitner, et al. 2000. Cancer Res. 60: 51-55), codon optimization (Liu et al 2000. Mol Ther., 1: 497-500; Dubensky, supra; Huang, et al 2001. J. Virol. 75: 4947-4951), in vivo electroporation (Widera, et al. 2000. J. Immunol. 164: 4635-3640), incorporation of CpG stimulatory motifs (Gurunathan, et al. Ann. Rev. Immunol., 2000, 18: 927-974; Leitner, stpra; Cho, et al J. Immunol. 168(10):4907-13), sequences for targeting of the endocytic or tubiquitin-processing pathways (Thomson, et al. 1998, J. Virol. 72: 2246-2252; Velders, et al. 2001. J. Immunol. 166: 5366-5373), Marek's disease virus type 1 VP22 sequences (J. Virol. 76(61):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 alt Oncogene 2000 Dec. 27; 19(56):6566-73). For example, in treating metastatic breast cancer, useful chemotherapeutic agents include cyclophosphamide, doxorubicin, paclitaxel, docetaxel, navelbine, capecitabine, and mitomycin C, among others. Combination chemotherapeutic regimens have also proven effective including cyclophosphamide+methotrexate+5-fluorouracil; cyclophosphamide+doxorubicin-5-fluorouracil; or, cyclophosphamide+doxorubicin, for example. Other compounds such as prednisone, a taxane, navelbine, mitomycin C, or vinblastine have been utilized for various reasons. A majority of breast cancer patients have estrogen-receptor positive (ER+) tumors and in these patients, endocrine therapy (i.e., tamoxifen) is preferred over chemotherapy. For such patients, tamoxifen or, as a second line therapy, progestins (medroxyprogesterone acetate or megestrol acetate) are preferred. Aromatase inhibitors (i.e., aminoglutethimide and analogs thereof such as letrozole) decrease the availability of estrogen needed to maintain tumor growth and may be used as second or third line endocrine therapy in certain patients.
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 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-III, collagen XVIII fragment (Endostatin)), HmwKallikrein-d5 plasmin fragment (Angiostatin), prothrombin-F1-2, TSP-1), protease inhibitors (i.e., tissue inhibitor of metalloproteases such as TIMP-1, -2, or -3; maspin; plasminogen activator-inhibitors such as PAI-1; pigment epithelium derived factor (PEDF)), Tumstatin (available through ILEX, Inc.), antibody products (i.e., the collagen-binding antibodies HUIV26, HU177, XL313; anti-VEGF; anti-integrin (i.e., Vitaxin, (Lxsys))), and glycosidases (i.e., heparinase-I, -III). “Chemical” or modified physiological agents known or believed to have anti-angiogenic potential include, for example, viablastine, 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-methyl amides (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 tumor growth. In one study, Clostridium novyi was modified to eliminate a toxin gene carried on a phage episome and administered to mice with colorectal tumors (Dang, et al. P.N.A.S. USA, 98(26): 15155-15160, 2001). In combination with chemotherapy, the treatment was shown to cause tumor necrosis in the animals. The reagents and methodologies described in this application may be combined with such treatment methodologies.
Nucleic acids encoding immunogenic targets may be administered to patients by any of several available techniques. Various viral vectors that have been successfully utilized for introducing a nucleic acid to a host include retrovirus, adenovirus, adeno-associated virus (AAV), herpes virus, and poxvirus, among others. It is understood in the art that many such viral vectors are available in the art. The vectors of the present invention may be constructed using standard recombinant techniques widely available to one skilled in the art. Such techniques may be found in common molecular biology references such as Molecular Cloning: A Laboratory Manual (Sambrook, et al., 1989, Cold Spring Harbor Laboratory Press), Gene Expression Technology (Methods in Enzymology, Vol. 185, edited by D. Goeddel, 1991. Academic Press, San Diego, Calif.), and PCR Protocols: A Guide to Methods and Applications (Innis, et al. 1990. Academic Press, San Diego, Calif.).
Preferred retroviral vectors are derivatives of lentivirus as well as derivatives of murine or avian retroviruses. Examples of suitable retroviral vectors include, for example, Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), SIV, BIV, HIV and Rous Sarcoma Virus (RSV). A number of retroviral vectors can incorporate multiple exogenous nucleic acid sequences. As recombinant retroviruses are defective, they require assistance in order to produce infectious vector particles. This assistance can be provided by, for example, helper cell lines encoding retrovirus structural genes. 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 NIH 3T3 cells, to produce large quantities of chimeric retroviral virions. Retroviral vectors may be administered by traditional methods (i.e., infection) 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, Nat. 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 viro 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 virus (AAV) demonstrates high-level infectivity, broad host range and specificity in integrating into the host cell genome (Hermonat, P., et al., 1984, Proc. Natl. Acad. Sci. U.S.A., 81 (20): 6466-70). And Herpes Simplex Virus type-1 (HSV-1) is vet 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 virus by deleting six nonessential regions of the genome encoding known or potential virulence factors (see, for example, U.S. Pat. Nos. 5,364,773 and 5,494,807). The deletion loci were also engineered as recipient loci for the insertion of foreign genes. The deleted regions are: thymidine kinase gene (TK; J2R); hemorrhagic region (u; B13R+B14R); A type inclusion body region (ATI; A26L); hemagglutinin gene (HA; A56R); host range gene region (C7L-K1L); and, large subunit, ribonucleotide reductase (I4L). NYVAC is a genetically engineered vaccinia virus strain that was generated by the specific deletion of eighteen open reading frames encoding gene products associated with virulence and host range. NYVAC has been show to be useful for expressing TAs (see, for example, U.S. Pat. No. 6,265,189). NYVAC (vP866), vP994, vCP205, vCP1433, placZH6H4Lreverse, pMPC6H6K3E3 and pC3H6FHVB were also deposited with the ATCC under the terms of the Budapest Treaty, accession numbers VR-2559, VR-2558, VR-2557, VR-2556, ATCC-97913, ATCC-97912, and ATCC-97914, respectively.
ALVAC-based recombinant viruses (i.e., ALVAC-1 and ALVAC-2) are also suitable for use in practicing the present invention (see, for example, U.S. Pat. No. 5,756,103). ALVAC(2) is identical to ALVAC(1) except that ALVAC(2) genome comprises the vaccinia E3L and K3L genes under the control of vaccinia promoters (U.S. Pat. No. 6,130,066; Beattie et al., 1995a, 1995b, 1991; Chang et al., 1992; Davies et al., 1993). Both ALVAC(1) and ALVAC(2) have been demonstrated to be useful in expressing foreign DNA sequences, such as TAs (Tartaglia et 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 poxvirus vector is TROVAC. TROVAC refers to an attenuated fowlpox that was a plaque-cloned isolate derived from the FP-1 vaccine strain of fowlpoxvirus which is licensed for vaccination of 1 day old chicks. TROVAC was likewise deposited under the terms of the Budapest Treaty with the ATCC, accession number 2553.
“Non-viral” plasmid vectors may also be suitable in practicing the present invention. Preferred plasmid vectors are compatible with bacterial, insect, and/or mammalian host cells. Such vectors include, for example, PCR-II, pCR3, and pcDNA3.1 (Invitrogen, San Diego, Calif.), pBSII (Stratagene, La Jolla, Calif.), pET15 (Novagen, Madison, Wis.), pGEX (Pharmacia Biotech, Piscataway, N.J.), pEGFP-N2 (Clontech. Palo Alto, Calif.), pETL (BlueBacII, Invitrogen), pDSR-alpha (PCT pub. No. WO 90/14363) and pFastBacDual (Gibco-BRL, Grand Island, N.Y.) as well as Bluescript® plasmid derivatives (a high copy, number COLE1-based phagemid, Stratagene Cloning Systems, La Jolla, Calif.), PCR cloning plasmids designed for cloning Taq-amplified PCR products (e.g., TOPO™ TA Cloning® kit, PCR2.1® plasmid derivatives, invitrogen, Carlsbad, Calif.). Bacterial vectors may also be used with the current invention. These vectors include, for example, Shigella, Salmonella, Vibrio cholerae, Lactobacillus, Bacille calmette guérin (BCG) and Streptococcus (see for example, WO 88/6626; WO 90/0594; WO 91/13157; WO 92/1796; and WO 92/21376). Many other non-viral plasmid expression vectors and systems are known in the art and could be used with the current invention.
Suitable nucleic acid delivery techniques include DNA-ligand complexes, adenovirus-ligand-DNA complexes, direct injection of DNA, CaPO₄precipitation, gene gun techniques, electroporation, and colloidal dispersion systems, among others. Colloidal dispersion systems include macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. The preferred colloidal system of this invention is a liposome, which are artificial membrane vesicles useful as delivery vehicles in vitro and in vivo, RNA, DNA and intact virions can be encapsulated within the aqueous interior and be delivered to cells in a biologically active form (Fraley, R., et al., 1981, Trends Biochem. Sci., 6: 77). The composition of the liposome is usually a combination of phospholipids, particularly high-phase-transition-temperature phospholipids, usually in combination with steroids, especially cholesterol. Other phospholipids or other lipids may also be used. The physical characteristics of liposomes depend on pH, ionic strength, and the presence of divalent cations. Examples of lipids useful in liposome production include phosphatidyl compounds, such as phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides. Particularly useful are diacylphosphatidylglycerols, where the lipid moiety contains from 14-18 carbon atoms, particularly from 16-18 carbon atoms, and is saturated. Illustrative phospholipids include egg phosphatidylcholine, dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine.
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

Type of
Adjuvant	General Examples	Specific Examples/References

Gel-type	Aluminum hydroxide/phosphate (“alum	(Aggerbeck and Heron, 1995)
	adjuvants”)
	Calcium phosphate	(Relyveld, 1986)
Microbial	Muramyl dipeptide (MDP)	(Chedid et al., 1986)
	Bacterial exotoxins	Cholera toxin (CT), E. coli labile toxin
		(LT)(Freytag and Clements, 1999)
	Endotoxin-based adjuvants	Monophosphoryl lipid A (MPL)
		(Ulrich and Myers, 1995)
	Other bacterial	CpG oligomicleotides (Corral and
		Petray, 2000), BCG sequences (Krieg, et
		al, Nature, 374: 576), tetanus toxoid
		(Rice, et al, J. Immunol., 2001, 167:
		1558-1565)
Particulate	Biodegradable	(Gupta et al., 1998)
	Polymer microspheres
	Immunostimulatory complexes	(Morein and Bengtsson, 1999)
	(ISCOMs)
	Liposomes	(Wassef et al., 1994)
Oil-emulsion	Freund's incomplete adjuvant	(Jensen et al., 1998)
and	Microfluidized emulsions	MF59 (Otl et al., 1995)
surfactant-		SAF (Allison and Byars, 1992)
based		(Allison, 1999)
adjuvants	Saponins	QS-21 (Kensil, 1996)
Synthetic	Muramyl peptide derivatives	Murabutide (Lederer, 1986)
		Threony-MDP (Allison, 1997)
	Nonionic block copolymers	L121 (Allison, 1999)
	Polyphosphazene (PCPP)	(Payne et al., 1995)
	Synthetic polynucleotides	Poly A:U Poly I:C (Johnson, 1994)
	Thalidomide derivatives	CC-4047/ACTIMID (J. Immunol.,
		168(10): 4914-9)

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 Laboratory, Manual, Portable Protocol No. 1, 1998; Kohler and Milstein, Nature, 256:495 (1975)); Jones et al. Nature, 321:522-525 (1986); Riechmann et al. Nature, 332:323-329 (1988); Presta (Curr. Op. Struct. Biol., 2:593-596 (1992); 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); Boerner et al., J. Immunol., 147(1):86-95 (1991); Marks et al., Bio/Technology 10, 779-783 (1992); Lornberg 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, a decreased level of expression of an immunogenic target in the patient's tissues (i.e., in peripheral blood) may indicate the regimen is decreasing the cancer load in that host. Similarly, if the level of expression is increasing, another therapeutic modality may need to be utilized. In one embodiment, nucleic acid probes corresponding to a nucleic acid encoding an immunogenic target may be attached to a biochip, as is known in the art, for the detection and quantification of expression in the host.
It is also possible to use nucleic acids, proteins, derivatives therefrom, or antibodies thereto as reagents in drug screening assays. The reagents may be used to ascertain the effect of a drug candidate on the expression of the immunogenic target in a cell line, or a cell or tissue of a patient. The expression profiling technique may be combined with high throughput screening techniques to allow rapid identification of useful compounds and monitor the effectiveness of treatment with a drug candidate (see, for example, Zlokarnik, et al., Science 279, 84-8 (1998)). Drug candidates may be chemical compounds, nucleic acids, proteins, antibodies, or derivatives therefrom, whether naturally occurring or synthetically derived. Drug candidates thus identified may be utilized, among other uses, as pharmaceutical compositions for administration to patients or for use in further screening assays.
Administration of a composition of the present invention to a host may be accomplished using any of a variety of techniques known to those of skill in the art. The composition(s) may be processed in accordance with conventional methods of pharmacy to produce medicinal agents for administration to patients, including humans and other mammals (i.e., a “pharmaceutical composition”). The pharmaceutical composition is preferably made in the form of a dosage unit containing a given, amount of DNA, viral vector particles, polypeptide or peptide, for example. A suitable daily dose for a human or other mammal may vary widely depending on the condition of the patient and other factors, but, once again, can be determined using routine methods.
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 drug with a suitable non-irritating excipient such as cocoa butter and polyethylene glycols that are solid at ordinary temperatures but liquid at the rectal temperature.
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 1×10⁶infectious 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 (see, for example, WO 01/30382 A1) in which the targeted immunogen is initially administered in a priming step in one form followed by a boosting step in which the targeted immunogen is administered in another form. The form of the targeted immunogen in the priming and boosting steps are different. For instance, if the priming step utilized a nucleic acid, the boost may be administered as a peptide. Similarly, where a priming step utilized one type of recombinant virus (i.e., ALVAC), the boost step may utilize another type of virus (i.e., NYVAC). This prime-boost method of administration has been shown to induce strong immunological responses.
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 injectable aqueous or oleaginous suspensions, may be formulated according to known methods using suitable dispersing or wetting agents and suspending agents. The injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent. Suitable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution, among others. For instance, a viral vector such as a poxvirus may be prepared in 0.4% 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 formulation, although it may comprise as much as 10% w/w, but preferably not more than 5% w/w, and more preferably from 0.1% to 1% of the formulation. Formulations suitable for topical administration include liquid or semi-liquid preparations suitable for penetration through the skin (e.g., liniments, lotions, ointments, creams, or pastes) and drops suitable for administration to the eye, ear, or nose.
The pharmaceutical compositions may also be prepared in a solid form (including granules, powders or suppositories). The pharmaceutical compositions may be subjected to conventional pharmaceutical operations such as sterilization and/or may contain conventional adjuvants, such as preservatives, stabilizers, wetting agents, emulsifiers, buffers etc. Solid dosage forms for oral administration may include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active compound may be admixed with at least one inert diluent such as sucrose, lactose, or starch. Such dosage forms may also comprise, as in normal practice, additional substances other than inert diluents, e.g., lubricating agents such as magnesium stearate. In the case of capsules, tablets, and pills, the dosage forms may also comprise buffering agents. Tablets and pills can additionally be prepared with enteric coatings. Liquid dosage forms for oral administration may include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs containing inert diluents commonly used in the art, such as water. Such compositions may also comprise adjuvants, such as wetting sweetening, flavoring, and perfuming agents.
Pharmaceutical compositions comprising a nucleic acid or polypeptide of the present invention may take any of several forms and may be administered by any of several routes. In preferred embodiments, the compositions are administered via a parenteral route (intradermal, intramuscular or subcutaneous) to induce an immune response in the host. Alternatively, the composition may be administered directly into a lymph node (intranodal) or tumor mass (i.e., intratumoral administration). For example, the dose could be administered subcutaneously at days 0, 7, and 14. Suitable methods for immunization using compositions comprising TAs are known in the art, as shown for p53 (Hollstein et al., 1991), p21-ras (Almoguera et al., 1988), HER-2 (Fendly et al., 1990), the melanoma-associated antigens (MAGE-1; MAGE-2) (van der Bruggen et al, 1991), p97 (Hu et al., 1988), melanoma-associated antigen E (WO 99/30737) and carcinoembryonic antigen (CEA) (Kantor et al., 1993; Fishbein et al, 1992; Kaufman et al., 1991), among others.
Preferred embodiments of administratable compositions include, for example, nucleic acids or polypeptides in liquid preparations such as suspensions, syrups, or elixirs. Preferred injectable preparations include, for example, nucleic acids or polypeptides suitable for parental, subcutaneous, intradermal, intramuscular or intravenous administration such as sterile suspensions or emulsions. For example, a recombinant poxvirus may be in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose or the like. The composition may also be provided in lyophilized form for reconstituting, for instance, in isotonic aqueous, saline buffer. In addition, the compositions can be co-administered or sequentially administered with other antineoplastic, anti-tumor or anti-cancer agents and/or with agents which reduce or alleviate ill effects of antineoplastic, anti-tumor or anti-cancer agents.
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

Example 1

AAC2 Tumor Associated Antigen

A version of the AAC2 coding sequence (AAC2-1) was provided by a collaborator and found to have high sequence similarity to a murine bcl-6-associated zinc finger protein (“BAZF”). Based on this sequence information, PCR primers were designed as shown below:

	(forward primer; SEQ ID NO.: 6)
	CACCATGGGT TCCCCCGCCG CCCCGGA

	(reverse primer; SEQ ID NO.: 7)
	CTAGGGCCCC CCGAGAATGT GGTAGTGCAC TTT

RNA was isolated from confluent HUVEC (BioWhittacker; Cat. No. CC2517, Lot No. IF0141) cultures using Trizol™ as indicated by the manufacturer (Life Technologies, Inc., Cat. No. 15596). High fidelity RT-PCR was then performed using the forward and reverse primers (24 cycles at 94 degrees, 2 min.; 94 degrees, 30 see; 56.8 degrees, 30 sec; 68 degrees, 1 min 40 sec; cycle 25 is 68 degrees, 7 min) resulting in the isolation of a 1,447 base pair cDNA. The cDNA was cloned into the pEF6-TOPO eukaryotic expression plasmid and termed “pEF6-hAAC2-2”. The cDNA pEF6-hAAC2-2 was sequenced using four primers and aligned to the sequence of AAC2-1 and murine BAZF (FIG. 1). As shown therein, AAC2-2 is missing the serine residue (S) found at position 245 in AAC2-1. Secondly, a stretch of 17 amino acids at positions 298 to 316 (SEFFSCQNCEAVAGCSS) of AAC2-2 showed only 11.8% sequence identity with amino acids 298-316 of AAC2-1 (FIG. 1). Interestingly, the stretch of 17 amino acids between positions 298 and 316 is 100% identical with murine BAZF suggesting that this may be critical for transcription factor function along with the long stretch of serines (zinc finger). AAC2-2 was then cloned into the pcDNA3.1-zeo eukaryotic expression plasmid (“pcDNA3.1-hAAC2-2”).

Example 2

Human T-Cell Reactivity Against AAC-2 Peptides

Using the AAC2-2 amino acid sequence, a library of 9-mer peptides predicted to bind to HLA-A-0201 was constructed (Table III; “N” indicates the sequence is not found within the mouse homolog, while “Y” indicates the sequence is found within the mouse homolog). Twenty-three of the peptides were dissolved in DMSO at 10 mg/ml (Table IV) and used in human PBMC cultures to test for their ability to elicit CD8 and CD4 αβ T-cell responses in vitro.

TABLE III

Predicted HLA-A-0201-binding nonamer peptides
of human AAC2-2

		Position in	SEQ ID
Designation	Sequence	Protein	NO.

CLP-2954	RLSPTAATV	AAC2(256-264)	44

CLP-2955	SIFRGRAGV	AAC2(65-73)	45

CLP-2956	DVLGNLNEL	AAC2(23-31)	46

CLP-2957	GVGVDVLSL	AAC2(72-80)	47

CLP-2958	LLTSQAQDT	AAC2(277-285)	48

CLP-2959	VLNSQASQA	AAC2(201-209)	49

CLP-2960	VQFKCGAPA	AAC2(264-272)	50

CLP-2961	GQPCPQARL	AAC2(219-227)	51

CLP-2962	GAHRGLDSL	AAC2(312-320)	52

CLP-2963	GAPASTPYL	AAC2(269-277)	53

CLP-2964	VVQACHRFI	AAC2(123-131)	54

CLP-2965	PLGISLRPL	AAC2(137-145)	55

CLP-2966	PLRAHKAVL	AAC2(48-56)	56

CLP-2967	FVQVAHLRA	AAC2(394-402)	57

CLP-2968	APLLDFMYT	AAC2(90-98)	58

CLP-2969	RAGVGVDVL	AAC2(70-78)	59

CLP-2970	CETCGSRFV	AAC2(387-395)	60

CLP-2971	ATAPAVLAA	AAC2(106-114)	61

CLP-2972	SRFVQVAHL	AAC2(392-400)	62

CLP-2973	CNWKKYKYI	AAC2(192-200)	63

CLP-2974	SPAAPEGAL	AAC2(3-11)	64

			—

EC-1	ALGYVREFT	AAC2(10-18)	65

EC-3	RLRGILTDV	AAC2(32-40)	66

EC-4	GILTDVTLL	AAC2(35-43)	67

EC-5	ILTDVTLLV	AAC2(36-44)	68

EC-6	TLLVGGQPL	AAC2(41-49)	69

EC-9	FMYTSRLRL	AAC2(95-103)	70

EC-10	RLSPATAPA	AAC2(102-110)	71

EC-11	AVLAAATYL	AAC2(110-118)	72

EC-12	ATYLQMEHV	AAC2(115-123)	73

EC-13	LQMEHVVQA	AAC2(118-126)	74

EC-21	QVAHLRAHV	AAC2(390-398)	75

EC-22	HLQTLKSHV	AAC2(418-426)	76

EC-24	VVQACHRFI	AAC2(123-131)	77

Using GM-CSF and IL-4, dendritic cells (DC) were generated from peripheral blood monocytes of blood donors expressing HLA-A-0201. DC were pulsed with the different pools of 9-mer AAC2-2 peptides shown in Table IV.

TABLE IV

AAC2-2 Peptide Groups

1	CLP 2954	RLSPTAATV (SEQ ID NO.: 44)	AAC2(256-264)
	CLP 2956	DVLGNLNEL (SEQ ID NO.: 45)	AAC2(23-31)
	CLP 2957	GVGVDVLSL (SEQ ID NO.: 46)	AAC2(72-80)

2	CLP 2959	VLNSQASQA (SEQ ID NO.: 49)	AAC2(201-209)
	CLP 2960	VQFKCGAPA (SEQ ID NO.: 50)	AAC2(264-272)
	CLP 2963	GAPASTPYL (SEQ ID NO.: 53)	AAC2(269-277)

3	CLP 2964	VVQACHRFI (SEQ ID NO.: 54)	AAC2(123-131)
	CLP 2968	APLLDFMYT (SEQ ID NO.: 58)	AAC2(90-98)

4	CLP 2971	ATAPAVLAA (SEQ ID NO.: 61)	AAC2(106-114)
	CLP 2973	CNWKKYKYI (SEQ ID NO.: 63)	AAC2(192-200)

5	EC 1	ALGYVREFT (SEQ ID NO.: 65)	AAC2(10-18)
	EC 3	RLRGILTDV (SEQ ID NO.: 66)	AAC2(32-40)
	EC 3	GILTDVTLL (SEQ ID NO.: 67)	AAC2(35-43)

6	EC 5	ILTDVTLLV (SEQ ID NO.: 68)	AAC2(36-44)
	EC 6	ILLVGGQPL (SEQ ID NO.: 69)	AAC2(41-49)
	EC 9	FMYTSRLRL (SEQ ID NO.: 70)	AAC2(95-103)

7	EC 10	RLSPATAPA (SEQ ID NO.: 71)	AAC2(102-110)
	EC 11	AVLAAATYL (SEQ ID NO.: 72)	AAC2(110-118)
	EC 12	ATYLQMEHV (SEQ ID NO.: 73)	AAC2(115-123)

8	EC 13	LQMEHVVQA (SEQ ID NO.: 74)	AAC2(118-126)
	EC 21	QVAHLRAHV (SEQ ID NO.: 75)	AAC2(390-398)

9	EC 22	HLQTLKSHV (SEQ ID NO.: 76)	AAC2(418-426)
	EC 24	VVQACHRFI (SEQ ID NO.: 77)	AAC2(123-131)

These DC were used to stimulate autologous T-cell-enriched PBMC preparations. The T cells were re-stimulated with autologous PBMC and then re-stimulated with CD40-ligand-activated autologous B cells. After the third and fourth round of stimulation with each peptide pool. ELISPOT analysis for IFN-γ production indicated that the T cells responded most strongly to one of the pools of AAC2-2 peptides (peptide group 6; FIG. 2A). Peptide group 6 includes the following peptides: ILTDVTLLV (aa 36-44), TLLVGGQPL (aa 41-49), and FMYTSRLRL (aa 95-103). Flow cytometric analysis (FACS) showed that the lymphocytes from this peptide-specific line consisted of >50% CD8 T cells with a memory (CD45RO⁺) phenotype. Very few cells (<:2%) were stained with anti-CD56 antibodies, indicating that the observed IFN-γ production was not due to NK cell activity.
Analysis of CTL activity from this peptide pool-specific T-cell line also demonstrated that the activated T cells were capable of killing peptide-loaded TAP-deficient T2 cells in an HLA-A-0201-restricted fashion (FIG. 2B). This analysis also revealed that ILTDVTLLV was a dominant peptide that stimulated the majority of the peptide-specific CTL activity. Thus, it was determined that AAC2-2 peptides are immunogenic in the human immune system.

Example 3

Immunogenicity of AAC2-2 In Vivo

Using DNA immunization into HLA-A2-Kb transgenic mice, it was found that the AAC2-2 protein is processed into immunogenic peptides and can elicit an HLA-A-0201-restricted T-cell response in vivo. Mice were immunized on day 1 by injection with pEF6-hAAC2-2 and boosted with the same plasmid at day 21 Lymphocytes were harvested from immunized mice 21 days after boosting and re-stimulated in vitro with the different groups of AAC2-2 peptides shown in Table IV. Peptide-specific effector T-cell function towards these peptides was found using IFN-γ ELISPOT analysis (FIG. 3). It was found that the same pool of peptides (group 6) previously shown to be strongly immunogenic in human PBMC cultures also elicited significant reactivity by cells after DNA vaccination (FIG. 3). Thus, the AAC2 gene product administered as a DNA-based vaccine is immunogenic in vivo and elicits a strong cell-mediated immune response characterized by the activation of CTL activity.

Example 4

Therapeutic AAC2-2 Vaccine

Therapeutic vaccination against the AAC2-2 gene product using the pEF6-hAAC2-2 DNA vaccine was found to completely block the growth of a solid tumor. Groups of eight C57BJ6 mice were subcutaneously challenged with 10⁴B16F10 melanoma cells, a vigorous and relatively non-immunogenic tumor cell line. The mice were then immunized at weekly intervals starting at 6 days after tumor challenge. Control mice (eight per group) treated either with a plasmid encoding the flu-NP protein or saline alone all developed large tumors. In contrast, all the mice (8/8) immunized with pIF6-hA-AC2-2 had no detectable tumor over a 50-day period (FIG. 4). All mice remained tumor-free through 80 days (data not shown). FIG. 5 plots the survival of mice treated with the different DNA vectors shown after melanoma implantation showing again the complete effectiveness of AAC2-2 vaccination in protecting mice against melanoma growth. No adverse health effects have been observed as a result of immunization with the human AAC2-2 gene-encoding vector (immunized mice were as active as control mice and showed no weight loss).
As shown in FIGS. 4 and 5, vaccination with a plasmid encoding the human VEGFR-2 (pBLAST-hflk1) did not protect tumor-challenged mice. In fact, the tumors grew even more rapidly in these mice. Analysis of sera from mice vaccinated with the pBLAST-hflk1 plasmid by ELISA found that IgG against the VEGFR-2 protein is induced in significant titres (data not shown). These results suggest that an antibody-based immune response directed against VEGFR-2 may not be not effective in preventing angiogenesis and solid tumor growth.
Inhibition of melanoma solid tumor growth in C57BL/6 mice immunized with pEF6-hAAC2-2 correlates with an immune response against the protein (FIG. 6). Immunization of CS7BL/6 mice was performed as described above. Spleen cells from immunized mice were re-stimulated with the same peptide pools used in experiments with HLA-A2-Kb transgenic mice (Table III). A significant number of peptides cross-react on C57BL/6 class I MHC (Kb and Db molecules). Two pools of peptides in particular (group 1 and group 5) were found to elicit strong effector cell activity in the IFN-□ ELISPOT assays (FIG. 6). All of the peptides in these groups are also identical to the corresponding sequence in the murine BAZF protein. These results strongly suggest that immunization with the human AAC2-2 activates an immune response against its murine orthologue BAZF in mice and can inhibit tumor angiogenesis as a result.

Example 5

BFA4 Tumor Antigen

The BFA4 sequence was found to be the “trichorhinophalangeal syndrome 1” (TRPS-1) gene (Genebank ID #6684533; Momeniet et al, Nature Genetics, 24(1), 71-74, 2000), a known transcription factor with no function attributed previously in any form of cancer. The BFA4 cDNA sequence is shown in FIG. 7 (SEQ ID NO.: 28) and the deduced amino acid sequence is shown in FIG. 8 (SEQ ID NO.: 29)

A. BFA4 Peptides and Polyclonal Antisera

For monitoring purposes, rabbit anti-BFA4 polyclonal antibodies were generated. Six peptides (22-mers) were designed and synthesized to elicit antibody response to BFA4, as shown below:
(SEQ ID NO.: 78)

CLP 2589 MVRKKNPPLRNVASEGEGQILE BFA4 (1-22)

(SEQ ID NO.: 79)

CLP 2590 SPKATEETGQAQSGQANCQGLS BFA4 (157-178)

(SEQ ID NO.: 80)

CLP 2591 VAKPSEKNSNKSIPALQSSDSG BFA4 (371-392)

(SEQ ID NO.: 81)

CLP 2592 NHLQGSDGQQSVKESKEHSCTK BFA4 (649-670)

(SEQ ID NO.: 82)

CLP 2593 NGEQIIRRRTRKRLNPEALQAE BFA4 (940-961)

(SEQ ID NO.: 83)

CLP 2594 ANGASKEKTKAPPNVKNEGPLNV BFA4 (1178-1199)
Rabbits were immunized with the peptides, serum was isolated, and the following antibody titers were observed:


Rabbit #	Peptide	Titer (Bleed 2)	Titer (Final Bleed)

1, 2	CLP2589	800000, 1600000	2560000, 2560000
3, 4	CLP2590	12800, 6400	40000, 40000
5, 6	CLP2591	400000, 400000	320000, 320000
7, 8	CLP2592	25600, 12800	80000, 40000
9, 10	CLP2593	3200000, 51200	2560000, 160000
11, 12	CLP2594	409600, 409600	320000, 320000

These peptides were also modified by coupling with KLH peptides to enhance immune responses as shown below:

(CLP-2589; SEQ ID NO.: 78)

	BFA4(1-22)	KLH-MVRKKNPPLRNVASEGEGQILE

(CLP-2590; SEQ ID NO.: 79)

	BFA4(157-178)	KLH-SPKATEETGQAQSGQANCQGLS

(CLP-2591; SEQ ID NO.: 80)

	BFA4(371-392)	KLH-VAKPSEKNSNKSIPALQSSDSG

(CLP-2592; SEQ ID NO.: 81)

	BFA4(649-670)	KLH-NHLQGSDGQQSVKESKEHSCTK

(CLP-2593; SEQ ID NO.: 82)

	BFA4(940-961)	KLH-NGEQIIRRRTRKRLNPEALQAE

(CLP-2594; SEQ ID NO.: 83)

BFA4(1178-1200)

KLH-ANGADKEKTKAPPNVKNEGPLNV

The pcDNA3.2BFA4 (3.6 mg) was also used for DNA immunization to generate polyclonal sera in chickens.

B. Cloning of BFA4

Complete cDNA sequence for BFA4 is ˜10 kb and gene is expressed in BT474 ductal carcinoma cells. Primers 7717 (forward primer) and 7723 (reverse primer) were designed to amplify full-length BFA4 gene by amplification of 4 kb, 7 kb or 10 kb products by RT-PCR.

	Primer 7717:
	BFA4-BamH1/F1 (5′ end forward) with Kozak:
	(SEQ ID NO.: 84)
	5′ CGGGATCCACCATGGTCCGGAAAAAGAACCCC 3′
	(BamHI for DNA3.1, MP76)

	Primer 7723:
	BFA4-BamHI/R1 (3′ end reverse 4 kb):
	(SEQ ID NO.: 85)
	5′ CGGGATCCCTCTTTAGGTTTTCCATTTTTTTCCAC 3′
	(BamHI for DNA3.1, MP76)

Ten mg of total RNA isolated and frozen in different batches from BT-474 cells using Trizol as indicated by the manufacturer (Gibco BRL) was used in RT-PCR to amplify the BF-A4 gene. RT-PCR conditions were optimized using Taq Platinum High Fidelity enzyme, OPC (Oligo Purification Cartridge; Applied Biosystems) purified primers and purified total. RNA/polyA mRNA (BT 474 cells). Optimization resulted in a 4.0 kb fragment as a single band.
To re-amplify the BFA4 sequence, mRNA was treated with DNase per manufacturers' instructions (Gibco BRL). The 4 kb DNA was reamplified using PCR using primers 7717 and 7723 primers (10 pmole/microlitre) and Taq Platinum High Fidelity polymerase (GIBCO BRL) enzyme. Thermocycler conditions for both sets of reactions were as under: 94° C. (2 min), followed by 30 cycles of 94° C. (30 sec), 52° C. (30 sec), 67° C. (4 min) and 67° C. (5 min) and finally 40° C. for 10 min. Three BFA4 clones were identified after pCR2.1/TOPO-TA cloning.
Several mutations were identified during analysis of the BFA4 sequence. To correct these sequences, the BamHI/XhoI fragment (5′) of the BFA4 gene from clone JB-3552-1-2 (pCR2.1/TOPO/BFA4) was exchanged with the XhoI/BamHI fragment (3′) of the BFA4 gene from clone JB-3552-1-4 (pCR2.1/TOPO/BFA4). This recombined fragment was then ligated into pMCS5 BamHI/CAP. Clone JB-3624-1-5 was generated and found to contain the correct sequence.
Nucleotide 344 of the isolated BFA4 clone was different from the reported sequence (C in BFA4, T in TRPS-1). The change resulted in a phe to ser amino acid change. To change this sequence to the reported sequence, the EcoRI/BglII fragment (5′) of the BFA4 gene from clone JIB-3552-1-2 (pCR2.1/TOPO/BFA4) was subcloned into pUC8:2 to generate clone JB-3631-2. This clone was used as a template for Quickchange (Stratagene) mutagenesis to change amino acid 115 of the BFA4 protein from a serine to a phenylalanine as in the TRPS1 protein. The selected clone was JB-3648-2-3. Mutagenesis was also repeated with pMCS5 BFA4 (BT474) as a template for Quickchange (Stratagene) mutagenesis to change amino acid 115 of the BFA4 protein from a serine to a phenylalanine as in the TRPS1 protein. Several clones were found to be correct by DNA sequencing and one of the clones (JB-3685-1-18) was used for further subcloning.
JB-3685-1-18 was then used to subclone the BFA4 coding sequence into the BamHI sites of four different expression vectors: 1) the poxviral (NYVAC) vector pSD554VC (COPAK/H6; JB-3707-1-7); 2) pcDNA3.1/Zeo (+) (JB-3707-3-2); 3) pCAMycHis (JB-3707-5-1); and, 4) Semiliki Forest virus alphaviral replicon vector pMP176 (JB-3735-1-23). The BFA4 coding sequence within JB-3707-1-7, JB-3707-5-1, and JB-3735-1-23 was confirmed by DNA sequencing.
A stop codon was introduced near the end of the cloned sequence in the pcDNA3.1/Zeo/BFA4 construct (JB-3707-3-2). A unique EcoRI site was opened and filled in to introduce a stop codon in-frame with BFA4 coding sequence. Several putative clones were identified by the loss of EcoRI site, however three clones (JB-3756-1-2; JB-3756-3-1; and JB-3756-4-1) were sequenced. All three were found to be correct in the area of the fill-in. Clone JB-3756-3-1 identified as having the correct sequence and orientation.
Myc and myc/his tags (Evans et al, 1985) were introduced using oligonucleotides, which were annealed and ligated into the pcDNA3.1/Zeo/BFA4 construct (JB-3707-3-2) at the EcoRI/EcoRV sites. Several clones were obtained for these constructs. Three clones having the correct sequences and orientations were obtained: 1) PcDNA3.1/Zeo/BFA4/myc-tag (JB-3773-1-2); 2) PcDNA3.1/Zeo/BFA4/mychis-tag (JB-3773-2-1); and, 3) PcDNA3.1 Zeo/BFA4/mychis-tag (JB-3773-2-2).

C. Expression of BFA4

1. Expression from Poxviral Vectors
The pSD554VC (COPAK/H6; JB-3707-1-7) vector was used to generate NYVAC-BFA4 virus. In vitro recombination was performed with plasmid COPAK/H6/BFA4 and NYVAC in RK13/CEF cells. NYVAC-BFA4 (vP2033-NYVAC-RK13) was generated and amplified to P3 level after completion of three enrichments with final stock concentrations of 1.12×10⁹/ml (10 ml), Vero cells were infected with NYVAC-BFA4 at an M.O.I. of 0.5 pfu/cell. Lysates and media were harvested 24 h post-infection to confirm expression of BFA4 protein. One-twentieth of the concentrated media and 1/40 of the lysate were loaded onto a western blot and incubated with rabbit antisera against the BFA4 peptides CLP 2589, 2591, 2598 and 2594 (see above for peptide sequences and preparation of anti-BFA4 antisera). An approximate 120 kD band was detected in both the lysate and the concentrated media of NYVAC-BFA4-infected Vero cells which was not evident in either Vero control cells (“mock-infected”), Vero cells infected with the parental NYVAC virus, or concentrated media.
2. Expression from pcDNA3.1-Based Vectors
Transient transfection studies were performed to verify expression of BFA4 from the pcDNA-based vectors and to analyze quality of polyconal sera raised against BFA4 peptides. The following constructs were used to study expression of BFA4 gene: pcDNA 3.1 zeo^R/BFA4, pMP76/BFA4, pcDNA 3.1 zeo^R/BFA4/Myc tag and pcDNA 3.1 zeo^R/BFA4/MycHis tag. BFA4 expression plasmids (5 μg and 10 μg) were co-transfected with pGL3 Luciferase (1 μg) (Promega) with the Gene porter reagent (Gene Therapy Systems) as the transfection reagent. At 48 h post-transfection, whole cell extract was prepared by scraping cells in cell lysis reagent (200 μl) and 1 cycle of freeze-thaw (−20° C. freeze, 37° C. thaw). Transfection efficiency was quantitated by analyzing expression of the luciferase reporter gene by measuring Relative Luciferase Units (RLU) in duplicate. Similar RLU values were obtained in the samples co-transfected with luciferase construct in the presence and absence of BFA4 expression vectors. There was no significant difference observed in toxicity or RLU values with differential amount (5 μg and 10 μg) of BFA4 expression vectors. Preliminary western blot analysis using alkaline phosphatase system with the CHOK1 cell extracts (pcDNA3/zeo/BFA4/MycHisTag) and an anti-BFA4 polyclonal antisera, revealed a band at approximately 120 kDa band in extracts of BFA4 vector-transfected cells.
A stable transfection study was initiated to obtain stable clones of BFA4 expressing COS A2 cells. These cells are useful for in vitro stimulation assays. pcDNA 3.1 zeo^R/BFA4 (2.5 μg and 20 μg), and pcDNA 3.1 zeo^R/BFA4/MycHis tag (2.5 μg) were used to study expression of BFA4). pGL3 Luciferase (2.5 μg) was used as a control vector to monitor transfection efficiency. The Gene porter reagent was used to facilitate transfection of DNA vectors. After 48 h post-transfection, whole cell extract were prepared by scraping cells in the cell lysis reagent (200 μl) and 1 cycle of freeze-thaw at −20° C./37° C. for first experiment. Transfected cells obtained from the second experiment were trypsinized, frozen stock established and cells were plated in increasing concentrations of Zeocin (0, 250, 500, 750 and 1000 μg/ml). Non-transfected CosA2cells survived at 60-80% confluency for three weeks at 100 μg/ml (Zeocin) and 10% confluency at 250 μg/ml (Zeocin). However, after three weeks, at higher drug concentration (500-1000 μg/ml), live cells were not observed in the plates containing non-transfected cells and high Zeocin concentration (500-1000 μg/ml).
Several Zeocin-resistant clones growing in differential drug concentrations (Zeocin-250, 500, 750 and 1000 μg/ml) were picked from 10 cm plates after three weeks. These clones were further expanded in a 3.5 cm plate(s) in the presence of Zeocin at 500, 750 and 1000 μg/ml. Frozen lots of these clones were prepared and several clones from each pool (pcDNA 3.1 zeo^R/BFA4, and pcDNA 3.1 zeo^R/BFA4/MycHis tag) were expanded to T75 cm²flasks in the presence of Zeocin at 1 mg/ml. Five clones from each pool (pcDNA 3.1 zeo^R/BFA4, and pcDNA 3.1 zeo^R/BFA4/MycHis tag) were expanded to T75 cm²flasks in the presence of Zeocin at 1 mg/ml. Cells are maintained under Zeocin drug (1 mg/ml) selection, Six clones were used in BFA4 peptide-pulsed target experiment, and two clones were found to express BFA4 at a moderate level by immunological assays. The non-adherent cell lines K562A2 and EL4A2 were also transfected with these vectors to generate stable cell lines.

3. Prokaryotic Expression Vector

The BamH1-Xho-1 fragment (1.5 Kbp) fragment encoding N-terminal 54 kDa BFDA4 from pcDNA3.1/BFA4 was cloned into pGEX4T1-6H is (Veritas) plasmid. This vector contains the tac promoter followed by the N-terminal glutathione S-transferase (GST ˜26 kDa) and a hexahistidine tag to C terminus of the GST fusion protein.
The BFA4-N54 expression plasmid was transformed into BL21 cells and grown at 25° C. in antibiotic selection medium (2 L culture) to an OD (600 nm) and thereafter induced with 1 mM IPTG. GST-BFA4-N54 was found to be soluble protein. Clarified extract of the soluble fraction was adsorbed batchwise to glutathione-Sepharose 4B and eluted with 10 mM reduced glutathione, Fractions were analyzed after estimation of protein concentration and TCA precipitation. Specific polypeptide of Mr=85 kDa in the eluate was confirmed by SDS-PAGE. The recombinant protein was purified by gluathione-Sepharose was absorbed on a NiNTA column for further purification. The bound protein was eluted with 0.25M imidazole. The protein was dialyzed versus TBS containing 40% Glycerol, resulting in 4.5 mg GST-BFA4-N54-6 His (N terminus BFA4 protein) protein. Expression of BFA4 was confirmed using the rabbit anti-BFA4 polyclonal antibody by western blot.

D. Anti-BFA4 Immune Responses

I. BFA4 Peptides

In addition to genetic immunization vectors for BFA4, immunological reagents for BFA4 have been generated. A library of 100 nonamer peptides spanning the BFA4 gene product was synthesized. The peptides were chosen based on their potential ability to bind to HLA-A*0201. Table V lists 100 nonamer peptide epitopes for HLA-A*0201 from the BFA4 protein tested (see below):


PEPTIDE		POSITION IN
DESIGNATION	SEQUENCE	PROTEIN	SEQ ID.

CLP-2421	MVRKKNPPL	BFA4 (1-9)	131

CLP-2422	KKNPPLRNV	BFA4 (4-12)	132

CLP-2423	VASEGEGQI	BFA4 (12-20)	133

CLP-2424	QILEPIGTE	BFA4 (19-27)	134

CLP-2425	RNMLAFSFP	BFA4 (108-116)	135

CLP-2426	NMLAFSFPA	BFA4 (109-117)	136

CLP-2427	MLAFSFPAA	BFA4 (110-118)	137

CLP-2428	FSFPAAGGV	BFA4 (113-121)	138

CLP-2429	AAGGVCEPL	BFA4 (117-125)	139

CLP-2430	SGQANCQGL	BFA4 (170-178)	140

CLP-2431	ANCQGLSPV	BFA4 (172-180)	588

CLP-2432	GLSPVSVAS	BFA4 (176-184)	141

CLP-2433	SVASKNPQV	BFA4 (181-189)	142

CLP-2434	RLNKSKTDL	BFA4 (196-204)	143

CLP-2435	NDNPDPAPL	BFA4 (207-215)	144

CLP-2436	DPAPLSPEL	BFA4 (211-219)	145

CLP-2437	ELQDFKONI	BFA4 (218-216)	146

CLP-2438	GLHNRTRQD	BFA4 (249-257)	147

CLP-2439	ELDSKILAL	BFA4 (259-267)	148

CLP-2440	KILALHNMV	BFA4 (263-271)	149

CLP-2441	ALHNMVQFS	BFA4 (266-284)	150

CLP-2442	VNRSVFSGV	BFA4 (282-290)	151

CLP-2443	FSGVLQDIN	BFA4 (287-295)	152

CLP-2444	DINSSRPVL	BFA4 (293-301)	153

CLP-2445	VLLNGTYDV	BFA4 (300-308)	154

CLP-2446	FCNFTYMGN	BFA4 (337-345)	155

CLP-2447	YMGNSSTEL	BFA4 (342-350)	156

CLP-2448	FLQTHPNKI	BFA4 (354-362)	157

CLP-2449	KASLPSSEV	BFA4 (363-371)	158

CLP-2450	DLGKWQDKI	BFA4 (393-401)	159

CLP-2451	VKAGDDTPV	BFA4 (403-411)	160

CLP-2452	FSCESSSSL	BFA4 (441-449)	161

CLP-2453	KLLEHYGKQ	BFA4 (450-458)	162

CLP-2454	GLNPELNDK	BFA4 (466-474)	163

CLP-2455	GSVINQNDL	BFA4 (478-486)	164

CLP-2456	SVINQNDLA	BFA4 (479-487)	165

CLP-2457	FCDFRYSKS	BFA4 (527-535)	166

CLP-2458	SHGPDVIVV	BFA4 (535-543)	167

CLP-2459	PLLRHYQQL	BFA4 (545-553)	168

CLP-2460	GLCSPEKHL	BFA4 (570-578)	169

CLP-2461	HLGEITYPF	BFA4 (577-585)	170

CLP-2462	LGEITYPFA	BFA4 (578-586)	171

CLP-2463	HCALLLLHL	BFA4 (594-602)	172

CLP-2464	ALLLLHLSP	BFA4 (596-604)	173

CLP-2465	LLLLHLSPG	9FA4 (597-605)	174

CLP-2466	LLLHLSPGA	BFA4 (598-606)	175

CLP-2467	LLHLSPGAA	BFA4 (599-607)	176

CLP-2468	FTTPDVDVL	BFA4 (621-629)	177

CLP-2469	TTPDVDVLL	BFA4 (622-830)	178

CLP-2470	VLLFHYESV	BFA4 (628-636)	179

CLP-2471	FITQVEEEI	BFA4 (673-681)	180

CLP-2472	FTAADTQSL	BFA4 (699-707)	181

CLP-2473	SLLEHFNTV	BFA4 (706-714)	182

CLP-2474	STIKEEPKI	BFA4 (734-742)	86

CLP-2475	KIDFRVYNL	BFA4 (741-749)	87

CLP-2476	NLLTPDSKM	BFA4 (748-756)	88

CLP-2479	V1WRGADIL	BFA4 (792-800)	89

CLP-2480	ILRGSPSYT	BFA4 (799-807)	90

CLP-2481	YTQASLGLL	BFA4 (806-814)	91

CLP-2482	ASLGLLTPV	BFA4 (809-817)	92

CLP-2483	GLLTPVSGT	BFA4 (812-820)	93

CLP-2484	GTQEQTKTL	BFA4 (819-827)	94

CLP-2485	KTLRDSPNV	BFA4 (825-833)	95

CLP-2486	HLARPIYGL	BFA4 (837-845)	96

CLP-2487	PIYGLAVET	BFA4 (841-849)	97

CLP-2488	LAVETKGFL	BFA4 (845-853)	98

CLP-2489	FLQGAPAGG	BFA4 (852-860)	99

CLP-2490	AGGEKSGAL	BFA4 (858-866)	100

CLP-2491	GALPQQYPA	BFA4 (864-872)	101

CLP-2492	ALPQQYPAS	BFA4 (865-873)	102

CLP-2493	FCANCLTTK	BFA4 (895-903)	103

CLP-2494	ANGGYVCNA	BFA4 (911-919)	104

CLP-2495	NACGLYQKL	BFA4 (918-926)	105

CLP-2496	GLYQKLHST	BFA4 (921-929)	106

CLP-2497	KLHSTPRPL	BFA4 (925-933)	107

CLP-2498	STPRPLNII	BFA4 (928-936)	108

CLP-2499	RLNPEALQA	BFA4 (962-960)	109

CLP-2500	VLVSQTLDI	BFA4 (1020-1028)	110

CLP-2501	DIHKRIMPL	BFA4 (1027-1035)	111

CLP-2502	RMQPLHIQI	BFA4 (1031-1039)	112

CLP-2503	YPLFGLPFV	BFA4 (1092-1100)	113

CLP-2504	GLPFVHNDF	BFA4 (1096-1104)	114

CLP-2505	FVHNDFQSE	BFA4 (1099-1107)	115

CLP-2506	SVPGNPHYL	BFA4 (1120-1128)	116

CLP-2507	GNPHYLSHV	BFA4 (1123-1131)	117

CLP-2508	HYLSHVPGL	BFA4 (1126-1134)	118

CLP-2509	YVPYPTENL	BFA4 (1141-1149)	119

CLP-2510	FNLPPHFSA	BFA4 (1147-1155)	120

CLP-2511	NLPPHFSAV	BFA4 (1148-1156)	121

CLP-2512	SAVGSDNDI	BFA4 (1154-1162)	122

CLP-2513	KNEGPLNVV	BFA4 (1192-1200)	123

CLP-2514	TKCVHCGIV	BFA4 (1215-1223)	124

CLP-2515	CVHCGIVFL	BFA4 (1217-1225)	125

CLP-2516	CGtVFLDEV	BFA4 (1220-1228)	126

CLP-2517	FLDEVMYAL	BFA4 (1224-1232)	127

CLP-2518	VMYALHMSC	BFA4 (1228-1236)	128

CLP-2519	FQCSICOHL	BFA4 (1243-1251)	129

CLP-2520	GLHRNNAQV	BFA4 (1265-1273)	130

The peptide library was pooled into separate groups containing 7-10 different peptides for immunological testing as shown in Table VI (see below). In addition to a peptide library spanning BFA4, a recombinant protein spanning the N-terminal 300 amino acids (positions 1-300) has been synthesized and purified from E. coli.


PEPTIDE	PEPTIDE
GROUP	NUMBER	SEQUENCE	SEQ ID

1	CLP-2421	MVRKKNPPL	331
	CLP-2422	KKNPPLRNV	132
	CLP-2423	VASEGEGQI	133
	CLP-2424	QILEPIGTE	134
	CLP-2425	RNMLAFSFP	135
	CLP-2426	NMLAFSFPA	136
	CLP-2427	MLAFSFPAA	137
	CLP-2428	FSFPAAGGV	138
	CLP-2429	AAGGVCEPL	139
	CLP-2430	SGQANCQGL	140

2	CLP-2431	ANCQGLSPV	588
	CLP-2432	GLSPVSVAS	141
	CLP-2433	SVASKNPQV	142
	CLP-2434	RLNKSKTDL	143
	CLP-2435	NDNPDPAPL	144
	CLP-2436	DPAPLSPEL	145
	CLP-2437	ELQDFKCNI	146
	CLP-2438	GLHNRTRQD	147
	CLP-2439	ELDSKILAL	148
	CLP-2440	KtLALHNMV	149

3	CLP-2441	ALHNMVQFS	150
	CLP-2442	VNRSVFSGV	151
	CLP-2443	FSGVLODIN	152
	CLP-2444	DINSSRPVL	153
	CLP-2445	VLLNGTYDV	154
	CLP-2446	FCNFTYMGN	155
	CLP-2447	KASLPSSEV	156
	CLP-2448	FLOTHPNKI	157
	CLP-2449	KASLPSSEV	158
	CLP-2450	DLGKWQDKI	159

4	CLP-2451	VKAGDDTPV	160
	CLP-2452	FSCESSSSL	161
	CLP-2453	KLLEHYGKQ	162
	CLP-2454	GLNPELNDK	183
	CLP-2455	GSVINQNDL	164
	CLP-2456	SVINQNDLA	165
	CLP-2457	FCDFRYSKS	166
	CLP-2458	SHGPDVIVV	167
	CLP-2459	PLLRHYQQL	168
	CLP-2460	GLCSPEKHL	169

5	CLP-2461	HLGEITYPF	170
	CLP-2462	LGEITYPFA	171
	CLP-2463	HCALLLLHL	172
	CLP-2464	ALLLLHLSP	173
	CLP-2465	LLLLHLSPG	174
	CLP-2466	LLLHLSPGA	175
	CLP-2467	LLHLSPGAA	176
	CLP-2468	FTTPDVDVL	177
	CLP-2469	TTPOVDVLL	178
	CLP-2470	VLLFHYESV	179

6	CLP-2471	FITQVEEEI	180
	CLP-2472	FTAADTQSL	181
	CLP-2473	SLLEHFNTV	182
	CLP-2474	STIKEEPKI	86
	CLP-2475	KIDFRVYNL	87
	CLP-2476	NLLTPDSKM	88
	CLP-2477	KMGEPVSES	589
	CLP-2478	FLKEKVWTE	590
	CLP-2479	VTWRGADIL	89
	CLP-2460	ILRGSPSYT	90

7	CLP-2481	YTQASLGLL	91
	CLP-2482	ASLGLLTPV	92
	CLP-2483	GLLTPVSGT	93
	CLP-2484	GTQEQTKTL	94
	CLP-2485	KTLRDSPNV	95
	CLP-2486	HLARPIYGL	96
	CLP-2487	PIYGLAVET	97
	CLP-2488	LAVETKGFL	98
	CLP-2489	FLQGAPAGG	99
	CLP-2490	AGGEKSGAL	100

8	CLP-2491	GALPQQYPA	101
	CLP-2492	ALPQQYPAS	102
	CLP-2493	FCANCLTTK	103
	CLP-2494	ANGGYVCNA	104
	CLP-2495	NACGLYQKL	105
	CLP-2496	GLYQKLHST	106
	CLP-2497	KLHSTPRPL	107
	CLP-2498	STPRPLNII	108
	CLP-2499	RLNPEALQA	109
	CLP-2500	VLVSQTLDI	110

9	CLP-2501	DIHKRMQPL	111
	CLP-2502	RMQPLHIQI	112
	CLP-2503	YPLFGLPFV	113
	CLP-2504	GLPFVHNDF	114
	CLP-2505	FVHNDFQSE	115
	CLP-2506	SVPGNPHYL	116
	CLP-2507	GNPHYLSHV	117
	CLP-2508	HYLSHVPGL	118
	CLP-2509	YVPYPTFNL	119
	CLP-2510	FNLPPHFSA	120

10	CLP-2511	NLPPHFSAV	121
	CLP-2512	SAVGSDNDI	122
	CLP-2513	KNEGPLNVV	123
	CLP-2514	TKCVHCGIV	124
	CLP-2515	CVHCGIVFL	125
	CLP-2516	CGIVFLDEV	126
	CLP-2517	FLDEVMYAL	127
	CLP-2518	VMYALHMSC	128
	CLP-2519	FQCSICQHL	129
	CLP-2520	GLHRNNAQV	130

2. Immune Reactivity of BFA4 Peptides and Generation of Human Effector T Cells:

The BFA4 peptides were grouped into different pools of 7-10 peptides for immunological testing. Dissolved peptide pools were pulsed onto autologous HLA-A*0201 dendritic cells and used to activate autologous T-cell-enriched PBMC preparations. Activated T cells from each peptide-pool-stimulated culture were re-stimulated another 3 to 5 times using CD40L-activated autoloous B-cells. IFN-γ ELISPOT analysis and assays for CTL killing of peptide-pulsed target cells was performed to demonstrate the immunogenicity of these epitopes from BFA4.
Human T cells demonstrated effector cell activity against a number of pools of peptides from the BFA4 protein, as shown by their ability to secrete IFN-γ in ELISPOT assays. These experiments were repeated after different rounds of APC stimulation resulting in the same reactive peptide groups. Peptide groups 1, 2, 4, 5, 6, 7, 8, 9, and 10 were found to be immunoreactive in these assays. Subsequently, these reactive peptide groups were dc-convoluted in additional IFN-γ ELISPOT assays in which single peptides from each group were tested separately. The individual peptides from BFA4 peptide groups 1, 5 6, 7, 8, 9, and 10 in ELISPOT assays. This analysis revealed a number of individual strongly reactive peptides from the BFA4 protein recognized by human T cells. It was also observed that many of these single peptides also induced C TL activity killing peptide-loaded human T2 lymphoma cell targets. These peptides are listed in Table VII:

TABLE VII

List of highly immunoreactive peptides from BFA4

Strong IFN-γ Killing	Strong CTL Killing	SEQ ID

CLP 2425	RNMLAFSFP	CLP 2425	RNMLAFSFP	135

CLP 2426	NMLAFSFPA	CLP 2426	NMLAFSFPA	136

CLP 2427	MLAFSFPAA	CLP 2427	MLAFSFPAA	137

CLP 2461	HLGEITYPF			170

CLP 2468	FTTPDVDVL	CLP 2468	FTTPDVDVL	177

CLP 2470	VLLFHYESV	CLP 2470	VLLFHYYESV	179

CLP 2474	KIDFRVYNL			86

CLP 2482	ASLGLLTPV	CLP 2482	ASLGLLTPV	92

CLP 2486	HLARPIYGL	CLP 2486	HLARPIYGL	96

CLP 2495	NACGLYQKL	CLP 2495	NACGLYQKL	105

CLP 2497	KLHSTPRPL			107

CLP 2499	RLNPEALQA	CLP 2499	RLNPEALQA	109

CLP 2503	YPLFGLPEV			113

CLP 2509	YVPYPTFNL	CLP 2509	YVPYPTFNL	119

CLP 2511	NLPPHFSAV			121

CLP 2518	VMYALHMSC			128

CLP 2520	GLHRNNAQV	CLP 2520	GLHRNNAQV	130

D. Immune Responses Against BFA4 after Immunization In Vivo:

The pcDNA3.1/Zeo-BFA4 plasmid was used to immunize transgenic mice expressing a hybrid HLA-A*0201 α1α2 domain fused to a murine Kb α3 domain in C57BL/6 mice (A2-Kb mice). IFN-γ ELISPOT analysis using the groups of pooled peptides after DNA immunization and removal of activated spleen cells revealed a number of reactive BFA4 peptide groups. Some of these groups (especially group 7 and 8) also reacted strongly in human T-cell cultures suggesting that overlapping groups of peptides are recognized by human T cells and are naturally processed and presented on HLA-A2 after vaccination.
Vaccination experiments were also performed with the NYVAC-BFA4 and the MP76-18-BFA4 vectors in A2-Kb mice. Mice were immunized subcutaneously with 10-20 μg of MP-76-18-BFA4 and 1-2×10⁷pfu vP2033 (NYVAC-BFA4) and boosted 28 days later with the same amounts of each vector. Re-stimulation of spleen cells from the immunized mice with the pools of BFA4 peptides revealed induction of IFN-γ production in response to BFA4 peptide groups 2, 3, 4, 5, 7, 9, and 10 in ELISPOT assays. Thus, the BFA4 gene encoded in a CMV promoter driven eukaryotic plasmid, NYVAC, or a Semliki replicase-based DNA plasmid, were all capable of inducing T-cell responses against the BFA4 protein in vivo.

Example 6

BCY1 Tumor Antigen
The BCY1 gene was detected as a partial open reading frame (ORF) homologous to a nematode gene called “posterior-expressed maternal gene-3” (PEM-3) playing a role in posterior to anterior patterning in Caenorhabtidis elegans embryos. No previous involvement of this gene in cancer has been documented.

A. BCY1 and Amino Acid DNA Sequences

A partial DNA sequence was originally determined for BCY1. Primers, 9616SXC and 9617SXC, are derived from the BCY 1 partial DNA sequence and are designed to clone BCY 1 by RT-PCR from Calu 6 total RNA. The primers were designed such that the PCR product has BamHI sites at both ends and an ATG start codon and a Kozak sequence at the 5′ end, as shown below:

	9616SXC:
	(SEQ ID NO.: 183)
	5′ CAGTACGGATCCACCATGGCCGAGCTGCGCCTGAAGGGC 3′

	9617SXC:
	(SEQ ID NO.: 184)
	5′ CCACGAGGATCCTTAGGAGAATATTCGGATGGCTTGCG 3′

The 1.2 Kb expected amplicon was obtained using ThermoScript RT-PCR System (Invitrogen) under optimized conditions. The PCR products from three separate RT-PCR's were digested with BamHI and respectively inserted in pcDNA3.1/Zeo(+). The resulting clones were MC50A6, MC50A8 and MC50A19 from the first RT-PCR; MC54.21 from the second RT-PCR and MC55.29; and, MC55.32 from the third RT-PCR. The following primers were utilized in sequencing the clones:

	9620MC:
	(SEQ ID NO.: 185)
	5′ TAATACGACTCACTATAGGG 3′

	9621MC:
	(SEQ ID NO.: 186)
	5′ TAGAAGGCACAGTCGAGG 3′

	9618MC:
	(SEQ ID NO.: 187)
	5′ GAAAACGACTTCCTGGCGGGGAG 3′

	9619MC:
	(SEQ ID NO.: 188)

5′ GCTCACCCAGGCGTGGGGCCTC 3′

DNA sequencing of all six clones indicated a consensus sequence (SEQ ID NO.: 30), as shown in FIGS. 9A and 9B, having the following differences from the original partial BCY1 sequence: a C to G substitution at position 1031 resulting in an amino acid change of Ala to Gly; a GC deletion at position 1032-1034 resulting in a Thr deletion; and, an A to G substitution at position 1177 resulting in an amino acid change of Thr to Ala. Clones MC50A8 and MC55.29 are identical to the consensus sequence. The amino acid sequence of BCY1 is shown in FIG. 98B and (SEQ ID NO.: 31).

B. Immunological Reagents for BCY1 Breast Cancer Antigen:

A library of 100 nonamer peptides spanning the BCY1 gene product was synthesized. The peptides were chosen based on their potential ability to bind to HLA-A*0201. Table VIII lists 100 nonamer peptide epitopes for HLA-A*0201 from the BCY1 protein tested (see below):

TABLE VIII

Peptide		Position in
Designation	Sequence	Protein	SEQ ID

*CLP-2599

VPVPTSEHV

2

189

*CLP-2602	PTSEHVAEI	5	190

*CLP-2609	EIVGRQCKI	12	191

*CLP-2616	KIKALRAKT	19	192

*CLP-2618	KALRAKTNT	21	193

*CLP-2619	ALRAKTNTY	22	194

*CLP-2620	LRAKTNTYI	23	195

*CLP-2624	INTYIKTPV	27	196

*CLP-2627	YIKTPVRGE	30	197

*CLP-2630	TPVRGEEPV	33	198

*CLP-2633	RGEEPVFMV	36	199

*CLP-2640	MVTGRREDV	43	200

CLP-2641	VTGRREDVA	44	201

*CLP-2643	GRREDVATA	46	202

CLP-2647	DVATARREI	50	203

CLP-2648	VATARREII	51	204

*CLP-2650	TARREIISA	53	205

*CLP-2651	ARREIISAA	54	206

*CLP-2655	IISAAEHFS	58	207

*CLP-2656	ISAAEHFSM	59	208

CLP-2657	SAAEHFSMI	60	209

*CLP-2659	AEHFSMIRA	62	210

*CLP-2663	SMIRASRNK	66	211

CLP-2666	RASRNKSGA	69	212

*CLP-2670	NKSGAAFGV	73	213

*CLP-2673	GAAFGVAPA	76	214

*CLP-2674	AAFGVAPAL	77	215

*CEP- 2677	GVAPALPGQ	80	216

*CLP-2678	VAPALPGQV	81	217

*CLP-2680	PALPGOVTI	83	218

*CLP-2681	ALPGOVTIR	84	219

*CLP-2682	LPGQVTIRV	85	220

CLP-2684	GQVTIRVRV	87	221

*CLP-2689	RVRVPYRVV	92	222

*CLP-2691	RVPYRVVGL	94	223

*CLP-2692	VPYRVVGLV	95	224

*CLP-2695	RVVGLVVGP	98	225

*CLP-2698	GLVVGPKGA	101	226

*CLP-2699	LVVGPKGAT	102	227

*CLP-2700	VVGPKGATI	103	228

*CLP-2710	RIQQQTNTY	113	229

*CLP-2711	IQQQTNTYI	114	230

*CLP-2712	QQQTNTYII	115	231

*CLP-2713	QQTNTYIIT	116	232

*CLP-2718	YIITPSRDR	121	233

CLP-2721	TPSRDRDPV	124	234

CLP-2724	RDRDPVFEI	127	235

CLP-2731	EITGAPGNV	134	236

CLP-2734	GAPGNVERA	137	237

CLP-2738	NVERAREEI	141	238

CLP-2744	EEIETHIAV	147	239

CLP-2746	IETHIAVRT	149	240

CLP-2749	HEAVRTGKI	152	241

CLP-2750	IAVRTGKIL	153	242

CLP-2756	KILEYNNEN	159	243

CLP-2760	YNNENDFLA	163	244

CLP-2762	NENDFLAGS	165	245

CLP-2766	FLAGSPDAA	169	246

CLP-2767	LAGSPDAAI	170	247

CLP-2774	AIDSRYSDA	177	248

CLP-2777	SRYSDAWRV	180	249

CLP-2785	VHQPGCKPL	188	250

CLP-2793	LSTFRQNSL	196	251

CLP-2801	LGCIGECGV	204	252

CLP-2807	CGVDSGFEA	210	253

CLP-2812	GFEAPRLDV	215	254

CLP-2817	RLDVYYGVA	220	255

CLP-2819	DVYYGVAET	222	256

CLP-2823	GVAETSPPL	226	257

CLP-2825	AETSPPLWA	228	258

CLP-2830	PLWAGQENA	233	259

CLP-2833	AGQENATPT	236	260

CLP-2835	QENATPTSV	238	261

CLP-2843	VLFSSASSS	246	262

CLP-2857	KARAGPPGA	260	263

CLP-2869	PATSAGPEL	272	264

CLP-2870	ATSAGPELA	273	265

CLP-2872	SAGPELAGL	275	266

CLP-2879	GLPRRPPGE	282	267

CLP-2887	EPLQGFSKL	290	268

CLP-2892	FSKLGGGGL	295	269

CLP-2894	KLGGGGLRS	297	270

CLP-2899	GLRSPGGGR	302	271

CLP-2909	CMVCFESEV	312	272

CLP-2910	MVCFESEVT	313	273

CLP-2911	VCFESEVTA	314	274

CLP-2913	FESEVTAAL	316	275

CLP-2916	EVTAALVPC	319	276

CLP-2917	VTAALVPCG	320	277

CLP-2920	ALVPCGHNL	323	278

CLP-2921	LVPCGHNLF	324	279

CLP-2922	VPCGHNLFC	325	280

CLP-2927	NLFCMECAV	330	281

CLP-2929	FCMECAVRI	332	282

CLP-2933	CAVRICERT	336	283

CLP-2936	RICERTDPE	339	284

CLP-2940	RTDPECPVC	343	285

CLP-2945	CPVCHITAT	348	286

CLP-2947	VCHITATQA	350	287

CLP-2950	ITATQAIRI	353	288

Table IX shows the groups of peptides used for immunological testing:


Peptide	Peptide
Group	Sequence	SEQ ID

1	EPLQFGSKL	268
	EVTAALVPC	276
	CPVSHITAT	286
	KIKALRAKT	192
	IISAAEHFS	207
	RASRNKSGA	192
	GAAFGVAPA	207
	LVVGPKGAT	227
	EITGAPGNV	236
	GAPGNVERA	237

2	ALRAKTNTY	192
	VATARREII	204
	PALPGQVTI	218
	ALPGQVTIR	219
	RVTVPYRVV	222
	RDRDPVFEI	127
	RVRVPYRVV	222
	HIAVRTGKI	241
	NENDFLAGS	245
	CAVRICERT	283
	VCHITATQA	287

3	GRREDVATA	202
	DVATARREI	203
	TARREIISA	205
	GVAPALPGQ	216
	RVVGLVVGP	225
	VHQPGCKPL	250
	PATSAGPEL	264
	VTAALVPCG	277

4	VPVPTSEHV	189
	ARREIISAA	206
	RIQQQTNTY	229
	NVERAREEI	238
	GFEAPRLDV	254
	ATSAGPELA	265
	FSKLGGGGL	269
	GLRSPGGGR	271

5	PTSEHVAEI	190
	EIVGRQCKI	191
	LRAKTNTYI	195
	VTGRREDVA	201
	SMIRASRNK	211
	CMVCFESEV	272
	LVPCGHNLF	279
	NLFCMECAV	281
	RICERTDPE	284
	RTDPECPVC	285

6	MVTGRREDV	200
	GLVVGPKGA	226
	IQQQTNTYI	230
	FLAGSPDAA	246
	GVAETSPPL	257
	FESEVTAAL	275
	FCMECAVRI	282

7	KALRAKTNT	193
	RGEEPVFMV	199
	SAAEHFSMI	209
	AAFGVAPAL	215
	VVGPKGATI	228
	YNNENDFLA	244
	LGCIGECGV	252
	QENATPTSV	261
	VCFESEVTA	274

8	TNTYIKTPV	196
	NKSGAAFGV	213
	QQTNTYIIT	232
	KILEYNNEN	243
	CGVDSGFEA	253
	AETSPPLWA	258
	PLWAGQENA	259
	VLFSSASSS	262
	SAFPELAGL	266

9	ISAAEHFSM	208
	QQQTNTYII	231
	EEIETHIAV	239
	IETHIAVRT	240
	LAGSPDAAI	247
	AIDSRYSDA	248
	DVYYGVAET	256
	VPCGHNLFC	280
	ITATQAIRI	288

10	TPVRGEEPV	198
	AEHFSMIRA	210
	VAPALPGQV	217
	TPSRDRDPV	234
	IAVRTGKIL	242
	SRYSDAWRV	249
	LSTFRQNSL	251
	RLDVYYGVA	255
	AGQENATPT	260
	MVCFESEVT	273

C. Immune Reactivity of BCY1 Peptides and Generation of Human Effector T Cells

The library of 100 peptides from BCY1 was separated into 10 groups of 7-10 peptides for immunological testing. Dissolved peptide pools were pulsed onto autologous HLA-A*0201 dendritic cells and used to activate autologous T-cell-enriched PBMC preparations. Activated T cells from each peptide-pool-stimulated culture were re-stimulated another 3 to 5 times using CD40L-activated autologous B-cells. IFN-γ ELISPOT analysis and assays for CTL killing of peptide-pulsed target cells was performed to demonstrate the immunogenicity of these epitopes from BCY1.
Human T cells demonstrated effector cell activity against a number of pools of peptides from the BCY 1 protein, as shown by their ability to secrete IFN-γ in ELISPOT assays. These experiments were repeated after different rounds of APC stimulation resulting in the same reactive peptide groups. Peptide groups 1, 2, 3, 4, 5, 6, and 7 were found to be immunoreactive in these assays. Subsequently, these reactive peptide groups were de-convoluted in additional IFN-γ ELISPOT assays in which single peptides from each group were tested separately. This analysis revealed a number of individual strongly reactive peptides from the BCY1 protein recognized by human T cells (FIG. 10). Many of these single peptides also induced CTL activity killing peptide-loaded human T2 lymphoma cell targets. Table IX lists these peptides.

Example 7

BFA5/NYBR-1 Breast Cancer Antigen

A. Identification of BFA5

Microarray profiling analysis indicated that BFA5 was expressed at low to high levels in 41 out of 54 breast tumor biopsy samples (76%) and at high levels in 31 out of 54 breast tumors (57%), as compared to a panel of 52 normal, non-tumor tissues. In situ hybridization (ISH) was performed using a series of BFA5 DNA probes and confirmed the microarray with at least 61% of the tumors showing fairly strong signals. Further bioinformatics assessment confirmed the results of these gene expression analysis results.
Sequence analysis of the BFA5 nucleotide sequence revealed a high degree of similarity to two unidentified human genes: KIAA1074 (GenBank Accession No. XM_—159732); and, KIAA0565 (GenBank Accession No. AB011137) isolated from a number of fetal and adult brain cDNA clones (Kikuno, et al. The complete sequences of 100 new cDNA clones from brain which code for large proteins in vitro. DNA Res. 6: 1.97-205). These genes were found to contain putative Zn finger regions and a nuclear localization sequence. BFA5 was suggested by others to be a potential breast cancer antigen (Jager, et al. 2001. Identification of a tissue-specific putative transcription factor in breast tissue by serological screening of a breast cancer library. Cancer Res. 61: 2055-2061 and WO 01/47959). In each of these publications, the nucleotide sequence BFA5 was designated NYBR-1 (“New York Breast Cancer-1”; GenBank Accession Nos. AF269087 (nucleotide) and AAK27325 (amino acid). For the purposes of this application, the sequence is referred to as BFA-5, the terms BFA-5 and NYBR-1 are interchangeable.
As shown previously by Jager, et al. and described in WO 01/47959, supra, BFA5 is specifically expressed in mammary gland, being expressed in 12/19 breast tumors analyzed. The structure of the BFA5/NYBR-1 gene has revealed that it encodes a 150-160 kD nuclear transcription factor with a bZIP site (DNA-binding domain followed by a leucine zipper motif). The gene also contains 5 tandem ankyrin repeats implying a role in protein-protein interactions. These ankyrin repeats may play a role in homo-dimerization of the protein. The BFA5 cDNA sequence is shown in FIG. 11 and SEQ ID NO.: 32. The BFA5 amino acid sequence is shown in FIG. 12 and SEQ ID NO.: 33.

B. Immunoreactivity of BFA5

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

A library of 100 peptides from the BFA5/NYBR-11 coding sequence that are predicted to be medium to high binders to HLA-A*0201 were designed using Rammensee and Parker algorithms. The library was sub-divided into 10 pools of ten peptides (see Table XI), and each pool was used to activate 10 different T cell cultures after pulsing peptides on to mature autologous dendritic cells. Two experiments were performed with the library of BFA5/NYBR-1 peptides demonstrating immunoreactivity in HLA-A*0201 human T cells, as described below.

TABLE X

Peptide	CLP
Group	Number	Sequence	SEQ ID

BFA5	2983	LMDMQTFKA	289
Group 1	2984	KVISPTKAL	290
	2985	SIPTKALEL	291
	2986	LELKNEQTL	292
	2987	TVSQKDVCL	293
	2988	SVPNKALEL	294
	2989	CETVSQKDV	295
	2990	KINGKLEES	296
	2991	SLVEKTPDE	297
	2991	SLCETVSQK	298

BFA5	2993	EIDKINGKL	299
Group 2	2994	MLLQQNVDV	300
	2995	NMWLQQQLV	301
	2996	FLVDRKCQL	302
	2997	YLLHENCML	303
	2998	SLFESSAKI	304
	2999	KITIDIHFL	305
	3000	QLQSKNMWL	306
	3001	SLDQKLFQL	307
	3002	FLLIKNANA	308

BFA5	3003	KILDTVHSC	309
Group 3	3004	SLSKILDTV	310
	3005	ILIDSGADI	311
	3006	KVMEINREV	312
	3007	KLLSHGAVI	313
	3009	AVYSETLSV	314
	3010	KMNVDVSST	315
	3011	ILSVVAKLL	316
	3012	VLIAENTML	317

BFA5	3013	KLSKNHQNT	318
Group 4	3014	SLTPLLLSI	319
	3015	SQYSGQLKV	320
	3016	KELEVKQQL	321
	3017	QTMEYIRKL	322
	3018	AMLKLEIAT	323
	3019	VLHQPLSEA	324
	3020	GLLKATCGM	325
	3021	GLLKANCGM	326
	3022	QQLEQALRI	327

BFA5	3023	CMLKKEIAM	328
Group 5	3024	EQMKKKFCV	329
	3025	IQDIELKSV	330
	3026	SVPNKAFEL	331
	3027	SIYQKVMEI	332
	3028	NLNYAGDAL	333
	3029	AVQDHDQIV	334
	3030	LIAENTMLT	335
	3031	FELKNEQTL	336

BFA5	3033	FESSQKIQV	337
Group 6	3034	GVTAEHYAV	338
	3035	RVTSNKTKV	339
	3036	TVSQKDVCV	340
	3037	KSQEPAFHI	341
	3038	KVLIAENTM	342
	3039	MLKLEIATL	343
	3040	EILSVVAKL	344
	3041	MLKKETAML	345
	3042	LLEKENEEI	346

BFA5	3043	ALRIQDIEL	347
Group 7	3044	KIREELGRI	348
	3045	TLKLKEESL	349
	3046	ILNEKIREE	350
	3047	VLKKKLSEA	351
	3048	GTSDKIQCL	352
	3049	GADINLVDV	353
	3050	ELCSVRLTL	354
	3051	SVESNLNQV	355
	3052	SLKINLNYA	356

BFA5	3053	KTPDEAASL	357
Group 8	3054	ATCGMKVSI	358
	3055	LSHGAVIEV	359
	3056	ETAMLKLEI	360
	3057	AELQMTLKL	361
	3058	VFAADICGV	362
	3060	PAIEMQNSV	363
	3061	EIFNYNNHL	364
	3062	ILKEKNAEL	365

BFA5	3063	QLVHAHKKA	366
Group 9	3065	NIQDAQKRT	367
	3066	NLVDVYGNM	368
	3067	KCTALMLAV	369
	3068	KTQCLEKAT	370
	3069	KIAWEKKET	371
	3070	IAWEKKEDT	372
	3071	VGMLLQQNV	373
	3072	VKTGCVARV	374

BFA5	3074	ALHYAVYSE	375
Group 10	3075	QMKKKFCVL	376
	3076	ALQCHQEAC	377
	3077	SEQIVEFLL	378
	3078	AVIEVHNKA	379
	3079	AVTCGFHHI	380
	3080	ACLQRKMNV	381
	3081	ALVEGTSDK	382

ELISPOT analysis was performed on human T-cell cultures activated through four rounds of stimulation with each pool of BFA5 peptides. In FIG. 13A, the numbers under the X-axis indicate the number of each peptide pool (1-10). Reactivity against a CMV pp65 peptide and a Flu matrix peptide were used as positive controls for T-cell activation in the experiments. Each experiment was performed with PBMC and dendritic cells from a single HLA-A*0201⁺ donor designated as “AP10”. The results show that, although BFA4 is markedly reactive with high ELISPOT counts per 100,000 cells in the assay, BFA5 is even more reactive with 9/10 pools demonstrating ELISPOT reactivity. Similar results were obtained for both BFA4 and BFA5/NYBR-1 with a different HLA-A*0201. The bars reach a maximum at 600 spots because beyond that the ELISPOT reader does not give accurate counts. Cultures having a reading of 600 spots have more than this number of spots.
A large number of the BFA5 peptide pools of are reactive as shown by the high levels of lFN-γ production (FIG. 13A). Each reactive peptide pool was then separated into individual peptides and analyzed for immunogenicity using ELISPOT analysis to isolate single reactive BFA5 peptides. As shown in FIG. 13B, BFA5 is highly immunogenic with several reactive single peptides than that of BFA4. Similar results were obtained in two independent PBMC culture experiments.
In addition to ELISPOT analysis, human T cells activated by BFA5 peptides were assayed to determine their ability to function as CTL. The cells were activated using peptide-pulsed dendritic cells followed by CD40 ligand-activated B cells (5 rounds of stimulation). The experiment shown was performed with isolated PBMC from HLA-A*0201⁺donor AP31. Isolated T cells were tested in ⁵¹Cr-release assays using peptide-loaded T2 cells. The % specific lysis at a 10:1, 5:1, and 1:1 T-cell to target ratio is shown for T2 cells pulsed with either pools of BFA5/NYBR-1 peptides or with individual peptides. The graph shows CTL activity induced against targets loaded with a c non-specific HLA-A*0201-binding HIV peptide (control) followed by the CTL activity against the peptide pool (Pool 1 etc.) and then the activity induced by individual peptides from the respective pool to the right. A high level of cytotoxicity was observed for some peptides at a 1:1 E:T ratio. CTL activity (percent specific lysis) induced by the control HIV peptide was generally <10%. Similar results were obtained with another PBMC donor expressing HLA-A*0201 (AP10). FIG. 13C shows that a large number of BFA5 peptides trigger T cell-mediated cytotoxicity of BFA5 peptide-loaded target cells. Table XI lists those peptides having immunogenic properties. Five peptides (LMDMQTFKA, ILIDSGADI, ILSVVAKLL, SQYSGQLKV, and ELCSVRLTL) were found to induce both IFN-γ secretion and CTL activity in T cells from both donors.

TABLE XI

Immunoreactive peptides from BFA5

BFA5 peptides eliciting
high IFN-γ release	BFA5 peptides inducing
(>200 spots/100,000 cells)	CTL lysis of pulsed cells

Donor AP10	Donor AP31	Donor AP10	Donor AP31

LMDMQTFKA	LMDMQTFKA	LMDMQTFKA	LMDMQTFKA

KVSIPTKAL			KVSIPTKAL

SIPTKALEL			SIPTKALEL

TVSQKDVCL

SVPNKALEL

YLLHENCML	YLLHENCML	YLLHENCML

QLQSKNMWL	QLQSKNMWL		QLQSKNMWL

SLSKILDTV	SLSKILDTV		SLSKILDTV

ILIDSGADI	ILIDSGADI	ILIDSGADI	ILIDSGADI

KVMEINREV

AVYSEILSV

ILSVVAKLL	ILSVVAKLL	ILSVVAKLL	ILSVVAKLL

SLTPLLLSI	SLTPLLLSI		SLTPLLLSI

SQYSGQLKV	SQYSGQLKV	SQYSGQLKV	SQYSGQLKV

QIMEYIRKL	QIMEYIRKL		QIMEYIRKL

SVPNKAFEL

NLNYAGDAL	NLNYAGDAL

	GVTAEHYAV

	KSQEPAFHI

MLKLEIATL	MLKLEIATL		MLKLEIATL

	MLKKEIAML

ALRIQDIEL

	VLKKKLSEA

ELCSVRLTL	ELCSVRLTL	ELCSVRLTL	ELCSVRLTL

SLKINLNYA	SLKINLNYA		SLKINLNYA

ATCGMKVSI		ATCGMKVSI

AELQMTLKL		AELQMTLKL	AELQMTLKL

	VFAADICGV

ILKEKNAEL	ILKEKNAEL

NLVDVYGNM		NLVDVYGNM

KCTALMLAV

C. Immunological Reagents

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

(CLP-2988; SEQ ID NO.: 383)

	BFA5(1-23)	KLH-MTKRKKTINLNIQDAQKRTALHW

(CLP-2978; SEQ ID NO.: 384)

	BFA5(312-334)	KLH-TSEKFTWPAKGRPRKIAWEKKED

(CLP-2979; SEQ ID NO.: 385)

	BFA5(612-634)	KLH-DEILPSESKQKDYEENSWDTESL

(CLP-2980; SEQ ID NO.: 386)

	BFA5(972-994)	KLH-RLTLNQEEEKRRNADILNEKIRE

(CLP-2981; SEQ ID NO.: 387)

	BFA5(1117-1139)	KLH-AENTMLTSKLKEKQDKEILEAEI

(CLP-2982; SEQ ID NO.: 388)

BFA5(1319-1341)

KLH-NYNNHLKNRIYQYEKEKAETENS

Prebleed samples from rabbits were processed and stored at −20° C. Rabbits were immunized as follows: 1) the peptides were administered as an emulsion with Freund's Complete Adjuvant (FCA); and, 2) two weeks later, the peptides were coupled with Keyhole-Limpet Hemocyanin (KLH)-coupled and administered as an emulsion with Freund's Incomplete Adjuvant FIA. The following results were observed:

TABLE XII

		IgG titer × 10⁵(after
	IgG titer × 10⁵(after	second
	first Immunization	Immunization
Peptide/protein	Rb1/Rb2)	Rb1/Rb2)

CLP 2977	25/6	256/64
CLP 2978	25/25	64/256
CLP 2979	12/25	256/512
CLP 2980	25/12	1024/128
CLP 2981	8/4	256/64
CLP 2982	2/2	64/32

Prebleed Sample Results Exhibited IgG Titers <100 for all Samples.

To assess the quality of the polyclonal antisera, western blots were performed using sera against BFA5. Sera were separately screened against cell extracts obtained from the BT474, MDMB453, MCF-7, Calu-6, and CosA2 cells. The approximate expected MW_rof BFA5 protein is 153 kDa. A 220 kD band was observed in the BT474 extract with CLP2980 antibody but not in the MDMB453 cell extracts however a ˜130 kD band was present in the MDMB453 extract. Both bands were found to be consistent with the polyclonal antibosera tested in this analysis, Neither of these bands is present in the negative control. Thus, it can be concluded that the polyclonal antisera are specific for BFA5.

Example 8

BCZ4 Tumor Antigen

A. BCZ4 Sequence

The BCZ4 sequence was detected as an over-expressed sequence in breast cancer samples. The nucleotide sequence and deduced amino acid sequence of BCZ4 are shown in FIG. 14, SEQ ID NO. 34 (BCZ4 cDNA), and SEQ ID NO. 35 (BCZ4 amino acid sequence).

B. Immunological Reagents for BCZ4 Breast Cancer Antigen:

A library of 100 nonamer peptides spanning the BCZ4 gene product was synthesized. The peptides were chosen based on their potential ability to bind to HLA-A*0201. Table XIII lists 100 nonamer peptide epitopes for HLA-A*0201 from the BCZ4 protein tested (see below):

TABLE XIII

Peptide	CLP
Group	Number	Sequence	SEQ ID

BCZ4	3220	LDLETLTDI	389
Group 1	3221	DILQHQIRA	390
	3222	ILQHQIRAV	391
	3223	AVPFENLNI	392
	3224	NLNIHCGDA	393
	3225	AMDLGLEAI	394
	3226	GLEAIFDQV	395
	3227	LEAIFDQVV	396
	3228	WCLQVNHLL	397
	3229	QVNHLLYWA	398

BCZ4	3230	VNHLLYWAL	399
Group 2	3231	HLLYWALTT	400
	3232	LLYWALTTI	401
	3233	ALTTIGFET	402
	3234	LTTIGFETT	403
	3235	TTIGGETTM	404
	3236	TIGFETTML	405
	3237	TMLGGYVYS	406
	3238	MLGGYVYST	407
	3239	YSTGMIHLL	408

BCZ4	3240	STGMIHLLL	409
Group 3	3241	GMIHLLLQV	410
	3242	MIHLLLQVT	411
	3243	LLLQVTIDG	412
	3244	GTIDGRNYI	413
	3245	TIDGRNYIV	414
	3246	YIVDAGFGR	415
	3247	RSYQMWQPL	416
	3248	YQMWQPLEL	417
	3249	QMWQPLELI	418

BCZ4	3250	ISGKDQPQV	419
Group 4	3251	KDQPQVPCV	420
	3252	PQVPCVFRL	421
	3253	QVPCVFRLT	422
	3254	RLTEENGFW	423
	3255	TEENGFWYL	424
	3256	NFGWYLDQI	425
	3257	DQIRREQYI	426
	3258	YIPNEEFLH	427
	3259	YSFTLKPRT	428

BCZ4	3260	RTIEDFESM	429
Group 5	3261	YLQTSPSSV	430
	3262	QTSPSSVFT	431
	3263	SVFTSKSFC	432
	3264	FTSKSFCSL	433
	3265	CSLQTPDGV	434
	3266	LQTPDGVHC	435
	3267	QTPDGVHCL	436
	3268	TPDGVHCLV	437
	3269	GVHCLVGFT	438

BCZ4	3270	CLVGFTLTH	439
Group 6	3271	TLTHRRFNY	440
	3272	FNYKDNTDL	441
	3273	NTDLIEFKT	442
	3274	TDLIEFKTL	443
	3275	LSEEEIEKV	444
	3276	KVLKNIFNI	445
	3277	LKNIFNISL	446
	3278	NISLQRKLV	447
	3279	KHGDRFFTI	448

BCZ4	3280	DIEAYLERI	449
Group 7	3281	YLERIGYKK	450
	3282	RNKLDLETL	451
	3283	NKLDLETLT	452
	3284	KLDLETLTD	453
	3285	DLETLTDIL	454
	3286	TLTDILQHQ	455
	3287	LTDILQHQI	456
	3288	QIRAVPFEN	457
	3289	IRAVPFENL	458

BCZ4	3290	IHCGDAMDL	459
Group 8	3291	HCGDAMDLG	460
	3292	DLGLEAIFD	461
	3293	AIFDQVVRR	462
	3294	GWCLQVNHL	463
	3295	LQVNHLLYW	464
	3296	GGYVYSTPA	465
	3297	YCYSTPAKK	466
	3298	STPAKKYST	467
	3299	IHLLLQVTI	468

BCZ4	3300	HLLLQVTID	469
Group 9	3301	LLQVTIDGR	470
	3302	YLDQIRREQ	471
	3303	QYIPNEEFL	472
	3304	FLHSDLLED	473
	3305	DLLEDSKYR	474
	3306	YRKIYSFTL	475
	3307	KIYSFTLKP	476
	3308	TLKPRTIED	477
	3309	VHCLVGFTL	478

BCZ4	3310	LTHRRFNYK	479
Group 10	3311	DLIEFKTLS	480
	3312	LIEFKTLSE	481
	3313	FKTLSEEEI	482
	3314	TLSEEEIEK	483
	3315	EIEKVLKNI	484
	3316	FNISLQRKL	485
	3317	SLQRKLVPK	486
	3318	KLVPKHGDR	487
	3319	PKHGDRFFT	488

C. Immune reactivity of BCZ4 Peptides and Generation of Human Effector T Cells

Human PBMC from an HLA-A2.1 positive donor designated AP10 were activated with autologous dendritic cells pulsed with different pools of 9-mer peptides from the BCZ4 antigen (see Table XIII for list). The activated T cells were re-stimulated after 12 days with activated autologous CD40-ligand-activated B cells pulsed with the same respective peptide pools for another 8 to 10 days. This secondary activation was repeated more time for a total of 3 stimulations. The activated T cells were isolated after the 3^rdstimulation and subjected to ELISPOT analysis for human IFN-γ production against their respective BCZ4 peptide pools as shown (FIG. 15A). In FIG. 15A, the blue bars show reactivity against the BCZ4 peptide pools and the red bars are for an HLA-A2.1-binding HIV peptide as a negative control. Positive control HLA-A2.1-binding recall antigen peptides for CMV and flu were as used as positive control in the experiment. Standard deviations are indicated.
The experiment was repeated on activated T cells after an additional round of peptide stimulation with the similar results.
The peptide pools were deconvoluted using IFN-γ ELISPOT assays (FIG. 15B), Human T cells from donor AP10 were stimulated with the different pools of BCZ4 peptides shown in Table XIII. Stimulation was performed as described earlier for the other antigens described. After 4 and 5 rounds of stimulation, T cells were harvested and subjected to ELISPOT analysis for IFN-γ production with each individual peptide in each pool. The bars shown represent individual peptide reactivity for each specific pool. Table XIII identifies each of the reactive peptides. This experiment was repeated with similar results following another round of stimulation of AP10 donor T cells.
In addition to ELISPOT analysis, human T cells activated by BCZ4 peptides were assayed to determine their ability to function as CTL. The cells were activated using peptide-pulsed dendritic cells followed by CD40 ligand-activated B cells (5 rounds of stimulation). The experiment shown was performed with isolated PBMC from HLA-A*0201⁺ donor AP31. Isolated T cells were tested in ⁵¹Cr-release assays using peptide-loaded T2 cells. The % specific lysis at a 10:1 T-cell to target ratio is shown for T2 cells pulsed with individual BCZ4 peptides. A high level of cytotoxicity was observed for some peptides (FIG. 15C). CTL activity (percent specific lysis) induced by the control HIV peptide was generally <10%. Similar results were obtained with another PBMC donor expressing HLA-A*0201 (AP10).
Table XIV lists the reactivity of the individual peptides:

TABLE XIV

	Peptides	Peptides eliciting
	eliciting	CTL activity
	strong IFN-γ	(peptide	SEQ
	ELISPOT activity	pulsed targets)	ID

CLP 3222	ILQHQIRAV	ILQHQIRAV	391

CLP 3225	AMDLGLEAI		394

CLP 3226	GLEAIFDQV	GLEAIFDQV	395

CLP 3227	LEAIFDQVV		396

CLP 3229	QVNHLLYWA		398

CLP 3231	HLLYWALTT		400

CLP 3232	LLYWALTTI	LLYWALTTI	401

CLP 3235	TTIGFETTM		404

CLP 3237	TMLGGYVYS		406

CLP 3239	YSTGMIHLL		408

CLP 3240	STGMIHLLL		409

CLP 3248	YQMWQPLEL	YQMEQPLEL	417

CLP 3260	RTIEDFESM		429

CLP 3261	YLQTSPSSV	YLQTSPSSV	430

CLP 3266	LQTPDGVHC		435

CLP 3267	QTPDGVHCL		436

CLP 3268	TPDGVHCLV		437

CLP 3269	GVHCLVGFT		438

CLP 3271	TLTHRRFNY		440

CLP 3277	LKNIFNISL		446

CLP 3288	QIRAVPFEN		457

CLP 3289	IRAVPFENL		458

CLP 3294	GWCLQVNHL		463

CLP 3298	STPAKKYST		467

CLP 3299	IHLLLQVTI	IHLLLQVTI	468

CLP 3301	LLQVTIDGR		470

CLP 3306	YRKIYSFTL		475

CLP 3307	KIYSFTLKP		476

CLP 3308	TLKPRTIED		477

CLP 3309	VHCLVGFTL		478

CLP 3317	SLQRKLVPK		486

CLP CC19	PKRGDRFFT		488

D. BCZ4 Expression Vectors

BCZ4 was PCR amplified using plasmid called pSporty/BCZ4 as the template using Platinum Taq (Invitrogen). Amplification conditions were as follows: 1) 94° C. 2 minutes; 2) 35 cycles of 94° C., 30 seconds, 53° C. 30 seconds, 67° C. 2.5 minutes; and, 3) 67° C. 7 minutes. PCR primers were designed to include EcoRI restriction sites and directly flank the ORF (i.e., no extraneous sequence). Primer sequences were as follows: AS032F (forward primer) 5′ GGAATTCAACATGGACATTGAAGCATATCTTAAGAATTG 3′ (SEQ II NO.:591), AS034R (reverse primer) 5′ GGAATTCCTGGTGAGCTGGATGACAAATAGACAAAGATTG 3′ (SEQ ID NO.: 592). A Kozak sequence was also included in the forward primer. pcDNA3.1/Zeo(+) was cut with EcoRI and treated with CIP to prevent self-ligation. The BCZ4 amplicon was then ligated into EcoRI digested pcDNA3Zeo(+). Sequencing produced one clone (AS-579-5) which matched the expected BCZ4 sequence. BCZ4 protein was then expressed from this expression vector using standard techniques.

Example 9

BFY3 Tumor Antigen

A. BFY3 Sequence

The BFY3 sequence was detected as an over-expressed sequence in breast cancer samples. RT-PCR amplification of BFY3 w/EcoRI ends from HTB131 total RNA with AS007F (forward primer) 5′ GGAATTCACCATGCTTTGGAAATTGACGGAT 3′ (SEQ ID NO—: 593) and AS010R (reverse primer) 5′ GGAATTCCTCACTTTCTGTGCT TCTC CTCTTTGTCA 3′ (SEQ ID NO.: 594) was performed. PCR product was digested with EcoRI and cloned into EcoRI digested and CIP treated pcDNA3.1/Zeo(+) vector by ligation. Several positive clones were identified by restriction digestion and sequence results of AS-391-2 match expected BFY3 sequence. The nucleotide sequence and deduced amino acid sequence of BFY3 are shown in FIG. 16, SEQ ID NO. 36 (BFY3 cDNA), and SEQ ID NO. 37 (BFY3 amino acid sequence).

B. Immunological Reagents for BFY3 Breast Cancer Antigen

A library of 0.100 nonamer peptides spanning the BFY3 gene product was synthesized. The peptides were chosen based on their potential ability to bind to HLA-A*0201. Table XV lists 100 nonamer peptide epitopes for HLA-A*0201 from the BFY3 protein tested (see below):

TABLE XV

Peptide	CLP
Group	Number	Sequence	SEQ ID

BFY3	3320	MLWKLTDNI	489
Group 1	3321	KLTDNIKYE	490
	3322	GTSNGTARL	491
	3323	NGTARLPQL	492
	3324	ARLPQLGTV	493
	3325	GTVGQSPYT	494
	3326	SPYTSAPPL	495
	3327	FQPPYFPPP	496
	3328	YFPPPTQPI	497
	3329	QSQDPYSHV	498

BFY3	3330	SHVNDPYSL	499
Group 2	3331	SLNPLHAQP	500
	3332	RQSQESGLL	501
	3333	GLLHTHRGL	502
	3334	GLPHQLSGL	503
	3335	GLDPRRDYR	504
	3336	DLLHGPHAL	505
	3337	LLHGPHALS	506
	3338	ALSSGLGDL	507
	3339	SSGLGDLSI	508

BFY3	3340	GLGDLSIHS	509
Group 3	3341	LGDLSIHSL	510
	3342	SIHSLPHAI	511
	3343	SLPHAIEEV	512
	3344	HAIEEVPHV	513
	3345	GINIPDQTV	514
	3346	QTVIKKGPV	515
	3347	VIKKGPVSL	516
	3348	SLSKSNSNA	517
	3349	SNSNAVSAI	518

BFY3	3350	AIPINKDNL	519
Group 4	3351	NLFGGVVNP	520
	3352	FGGVVNPNE	521
	3353	GGVVNPNEV	522
	3355	NPNEVFCSV	523
	3356	CSVPGRLSL	524
	3357	SVPGRLSLL	525
	3358	SLLSSTSKY	526
	3359	LLSSTSKYK	527

BFY3	3360	LSSTSKYKV	528
Group 5	3361	STSKYKVTV	529
	3362	KYKVTVAEV	530
	3363	YKVTVAEVQ	531
	3364	TVAEVQRRL	532
	3365	RLSPPECLN	533
	3366	LNASLLGGV	534
	3367	NASLLGGVL	535
	3368	SLLGGVLRR	536
	3369	LLGGVLRRA	537

BFY3	3370	VLRRAKSKN	538
Group 6	3371	SLREKLDKI	539
	3372	KLDKIGLNL	540
	3373	KIGLNLPAG	541
	3374	GLNLPAGRR	542
	3375	NLPAGRRKA	543
	3376	AGRRKAANV	544
	3377	RKAANVTLL	545
	3378	KAANVTLLT	546
	3379	ANVTLLTSL	547

BFY3	3380	NVTLLTSLV	548
Group 7	3381	TLLTSLVEG	549
	3382	LLTSLVEGE	550
	3383	TSLVEGEAV	551
	3384	SLVEGEAVH	552
	3385	LVEGEAVHL	553
	3386	VEGEAVHLA	554
	3387	HLARDFGYV	555
	3388	YVCETEFPA	556
	3389	CETEFPAKA	557

BFY3	3390	AKAVAEFLN	558
Group 8	3391	AVAEFLNRQ	559
	3392	FLNRQHSDP	560
	3393	QVTRKNMLL	561
	3394	NMLLATKQI	562
	3395	MLLATKQIC	563
	3396	LLATKQICK	564
	3397	QICKEFTDL	565
	3398	ICKEFTDLL	566
	3399	LLAQDRSPL	567

BFY3	3400	ILEPGIQSC	568
Group 9	3401	LEPGIQSCL	569
	3402	QSCLTHFNL	570
	3403	SCLTHFNLI	571
	3404	NLISHGFGS	572
	3405	LISHGFGSP	573
	3406	ISHGFGSPA	574
	3407	SHGFGSPAV	575
	3408	FGSPAVCAA	576
	3409	GSPAVCAAV	577

BFY3	3410	AVCAAVTAL	578
Group 10	3411	AVTALQNYL	579
	3412	VTALQNYLT	580
	3413	ALQNYLTEA	581
	3414	LQNYLTEAL	582
	3415	YLTEALKAM	583
	3416	LKAMDKMYL	584
	3417	AMDKMYLSN	585
	3418	KMYLSNNPN	586
	3419	YLSNNPNSH	587

Human PBMC from an HLA-A2.1 positive donor designated AP31 were activated with autologous dendritic cells pulsed with different pools of 9-mer peptides from the BFY3 antigen (see Table 1 for list). The activated T cells were re-stimulated after 12 days with activated autologous CD40-ligand-activated B cells pulsed with the same respective peptide pools for another 8 to 10 days. This secondary activation was repeated 2 more time for a total of 4 stimulations. The activated T cells were isolated after the 4^thstimulation and subjected to ELISPOT analysis for human IFN-γ production against their respective BFY3 peptide pools as shown. The blue bars show reactivity against the BFY3 peptide pools and the red bars are for an HLA-A2.1-binding HIV peptide as a negative control. Standard deviations are indicated. The experiment was repeated 2 times on activated T cells from different rounds of peptide stimulation with the similar results (FIG. 17A).
The BFY3 peptide pools were deconvoluted and studied in IFN-γ ELISPOT assays, Human T cells from donor AP10 were stimulated with the different pools of BFY3 peptides shown in Table XV. Stimulation was performed as described earlier for the other antigens described. After 4 rounds of stimulation, the T cells from each culture were harvested and subjected to ELISPOT analysis for IFN-γ production with each individual peptide in each pool. FIG. 17B illustrates individual peptide reactivity for each specific pool.
In addition to ELISPOT analysis, human T cells activated by BFY3 peptides were assayed for reactivity. Ten pools of peptides consisting of ten peptides per pool used to generate CTL. These 10 groups of effectors used to kill targets pulsed with corresponding peptide pools. Peptides from pools 1, 3, 5, 6, and 7 found to be recognized, indicating that peptides in those pools are capable of generating CTL (FIG. 17C). From these ten pools, peptides 3344, 3320, 3378, 2272, and 3387 were strongly recognized by CTL (FIG. 17D). “Moderately recognized” peptides include 3369, 3355, and 336218D (FIG. 17D). CosA2 cells transfected with BFY3 were killed by CTL generated from pools 1 and 3 indicating that processed and presented epitopes from these pools are immunologically relevant (FIG. 17E). The peptides responsible for this cytotoxicity are 3320 and 3344. Table XVI summarizes the properties of the BFY3 peptides.

TABLE XVI

Summary of Immunoreactive BFY3 Nonamer Peptides

	Peptides	Peptides eliciting
	eliciting	CTL activity
	strong IFN-γ	(peptide	SEQ
	ELISPOT activity	pulsed targets)	ID

CLP 3320	MLWKLTDNI	MLWKLTDNI	489

CLP 3343	SLPHAIEEV		512

CLP 3344	HAIEEVPHV	HAIEEVPHV	513

CLP 3351	NLFGGVVNP		520

CLP 3362	KYKVTVAEV	KYKVTVAEV	530

CLP 3366	LNASLLGGV		534

CLP 3369	LLGGVLRRA	LLGGVLRRA	537

CLP 3372	KLDKIGLNL	KLDKIGLNL	540

CLP 3378	KAANVTLLT	KAANVTLLT	546

CLP 3380	NVTLLTSLV		548

CLP 3387	HLARDFGYV	HLARDFGYV	555

CLP 3403	SCLTHFNLI		571

CLP 3407	SHGFGSPAV		575

CLP 3415	YLTEALKAM		583

C. BFY3 Expression Vectors

To construct a BFY3 expression vector, RT-PCT amplification of BFY3 w/EcoRI ends from HTB131 total RNA with AS007F (forward primer) 5′ GGAATTCACCATGCTTTGGAAATTGACGGAT 3′ (SEQ ID NO.: 595) and AS010R (reverse primer) 5′ GGAATTCCTCACTTTCTGTGCTTCTCCTCTTTGTCA 3′ (SEQ ID NO.: 596) was performed. PCR was performed using standard techniques. The amplified product was digested with EcoRI and cloned into CIP treated pcDNA3.1/Zeo(+) vector by ligation using standard techniques. Several positive clones were identified by restriction digestion and sequenced. Sequencing indicated that the sequence of clone AS-391-2 matched the expected BFY3 sequence, BFY3 protein was then expressed from the BFY3 expression vector using standard techniques.

Example 10

Expression Vectors Encoding Multiple Tumor Antigens

In certain instances, it may be desirable to construct expression vectors encoding multiple tumor antigens. It has been determined that certain combinations of antigens, when combined into a single expression vector, encompasses the expression profiles of many patients in a single vector. For instance, one study of breast cancer samples from different patients indicated that the combination of BFA4 and BFA5 covered expression profiles of 74% of the samples; the combination of BCY1 and BFA5 covered 65% of the samples; the combination of BCZ4 and BFA5 covered 69% of the samples; the combination of BFY3 and BFA5 covered 67% of the samples; the combination of BCY1, BFA4 and BFA5 covered 78% of the samples; the combination of BCZ4, BFA4 and BFA5 covered 81% of the samples; and, the combination of BFY3, BFA4, and BFA5 covered 74% of the samples. Accordingly, a multi-antigen expression construct may be built such that the most common expression profiles among breast cancer patients may be addressed using a single vector. Such a multiantigen expression vector is constructed using standard cloning techniques positioning nucleic acids encoding each of the tumor antigen sequences in proximity to a promoter or other transcriptional regulatory sequence. The expression vector may be engineered such that each nucleotide sequence encoding a tumor antigen is operably linked to a specific promoter, or the tumor antigens may collectively be operably linked to a single promoter and expressed as a single expression unit. Where a single expression unit is constructed, nucleotide sequences useful in separating the tumor antigen sequences following expression may be inserted between the tumor antigen sequences. Sequences useful for include IRES sequences, nucleotide sequences encoding amino acid sequences corresponding to protease cleavage sites, and the like. Suitable vectors for constructing such multiantigen expression vectors include, for example, poxviruses such as vaccinia, avipox, ALVAC and NYVAC.
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

1. An expression vector comprising the nucleic acid sequence as illustrated in SEQ ID NO.: 29 or SEQ ID NO.: 31; a nucleic acid sequence encoding the amino acid sequence illustrated in SEQ ID NO.: 30 or SEQ ID NO.: 32; or a fragment thereof.

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

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

4. The expression vector of claim 3 wherein the viral vector is a poxvirus selected from the group consisting of vaccinia, NYVAC, avipox, canarypox, ALVAC, ALVAC(2), fowlpox, 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. The expression vector of claim 1 further comprising at least one additional tumor-associated anti gen.

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

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

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

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

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

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

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

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

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

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

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

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

19. The expression vector of claim 17 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 poxvirus of claim 18 wherein the viral vector is a poxvirus selected from the group consisting of NYVAC, ALVAC, and ALVAC(2).

21. The expression vector of claim 1, 6, 11 or 16 further comprising at least one nucleic acid sequence encoding a co-stimulatory component.

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

23. The expression vector of claim 23 wherein the viral vector is selected from the group consisting of poxvirus, adenovirus, retrovirus, herpesvirus, and adeno-associated virus.

24. The expression vector of claim 24 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 poxvirus of claim 18 wherein the viral vector is a poxvirus selected from the group consisting of NYVAC, ALVAC, and ALVAC(2).

26. A composition comprising an expression vector in a pharmaceutically acceptable carrier, said vector comprising the nucleic acid sequence shown in SEQ ID NO.: 29 or SEQ ID NO.: 31; a nucleic acid sequence encoding the amino acid sequence illustrated in SEQ ID NO.: 30 or SEQ ID NO.: 32; or a fragment thereof.

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

28. The expression vector of claim 27 wherein the viral vector is selected from the group consisting of poxvirus, adenovirus, retrovirus, herpesvirus, and adeno-associated virus.

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

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

31. A method for preventing or treating cancer comprising administering to a host an expression vector comprising the nucleic acid sequence illustrated in SEQ ID NO.: 29 or SEQ ID NO.: 31; a nucleic acid sequence encoding the amino acid sequence illustrated in SEQ ID NO.: 30 or SEQ ID NO.: 32; or a fragment thereof.

32. The expression vector of claim 31 wherein the vector is a plasmid or a viral vector.

33. The expression vector of claim 32 wherein the viral vector is selected from the group consisting of poxvirus, adenovirus, retrovirus, herpesvirus, and adeno-associated virus.

34. The expression vector of claim 33 wherein the viral vector is a poxvirus selected from the group consisting of vaccinia, NYVAC, avipox, canarypox, ALVAC, ALVAC(2), fowlpox, and TROVAC.

35. The poxvirus of claim 34 wherein the viral vector is a poxvirus selected from the group consisting of NYVAC, ALVAC, and ALVAC(2).

36. An isolated peptide derived from BFY3 as shown in Table XV or XVI.

37. A method for immunizing a host against the tumor antigen BFY3 comprising administering to the patient a peptide shown in Table XV or XVI, either alone or in combination with another agent, where the individual components of the combination are administered simultaneously or separately from one another.

38. An isolated peptide derived from BFY3 as shown in Table XV or XVI.

39. A method for immunizing a host against the tumor antigen BFY3 comprising administering to the patient a peptide shown in Table XV or XVI, either alone or in combination with another agent, where the individual components of the combination are administered simultaneously or separately from one another.

40. An isolated peptide derived from BCZ4 as shown in Table XIII or XVI.

41. A method for immunizing a host against the tumor antigen BCZ4 comprising administering to the patient a peptide shown in Table XIII or XIV, either alone or in combination with another agent; where the individual components of the combination are administered simultaneously or separately from one another.

42. An isolated peptide derived from BCZ4 as shown in Table XIII or XVI.

43. A method for immunizing a host against the tumor antigen BCZ4 comprising administering to the patient a peptide shown in Table XIII or XVI, either alone or in combination with another agent, where the individual components of the combination are administered simultaneously or separately from one another.

44. A expression vector for expression of multiple tumor antigens or fragments thereof, the expression vector comprising at least two nucleic acid sequences encoding at least two different tumor antigens or fragments thereof, the tumor antigens being selected from the group consisting of BFA4, BCY1, BFA5, BCZ4, and BFY3.

45. A expression vector for expression of multiple tumor antigens or fragments thereof, the expression vector comprising at least two nucleic acid sequences encoding at least two different tumor antigens or fragments thereof, the nucleic acid sequences being selected from the group consisting of SEQ ID NO.: 23, SEQ ID NO.: 25, SEQ ID NO.: 27, SEQ ID NO.: 29, and SEQ ID NO.: 31.

46. The expression vector of claim 44 or 45 wherein the vector is a plasmid or a viral vector.

47. The expression vector of claim 46 wherein the viral vector is selected from the group consisting of poxvirus, adenovirus, retrovirus, herpesvirus, and adeno-associated virus.

48. The expression vector of claim 47 wherein the viral vector is a poxvirus selected from the group consisting of vaccinia, NYVAC, avipox, canarypox, ALVAC, ALVAC(2), fowlpox, and TROVAC.

49. The expression vector of claim 48 wherein the viral vector is a poxvirus selected from the group consisting of NYVAC, ALVAC, and ALVAC(2).

50. The expression vector of any one of claims 44 to 49 further comprising at least one nucleic acid sequence encoding a co-stimulatory component.