# Patent

## Title
Tumor antigens BFA4 and BCY1 for prevention and / or treatment of cancer

## 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.

## Background
RELATED APPLICATIONS
This application claims priority to U.S. Ser. Nos. 60/394,346 filed Jul. 3, 2002; 60/394,503 filed Jul. 9, 2002; 60/411,833 filed Sep. 18, 2002; and, 60/445,342 filed Feb. 6, 2003, all of which being hereby incorporated by reference.


FIELD OF THE INVENTION
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.
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 (1999) identified several genes differentially expressed in invasive and metastatic carcinoma cells with combined use of laser capture microdissection and cDNA microarrays. Several delivery systems like DNA or viruses could be used for therapeutic vaccination against human cancers (Bonnet et al, 2000) and can elicit immune responses and also break immune tolerance against TAAs. Tumour cells can be rendered more immunogenic by inserting transgenes encoding T cell co-stimulatory molecules such as B7.1 or cytokines such as IFN-γ, IL2, or GM-CSF, among others. Co-expression of a TAA and a cytokine or a co-stimulatory molecule can develop effective therapeutic vaccine (Hodge et al, 95, Bronte et al, 1995, Chamberlain et 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.: 1 or 3 or has the amino acid sequence of SEQ ID NO.: 2 or 4. 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. BFA4 cDNA sequence.
FIG. 2. BFA4 amino acid sequence.
FIG. 3. BCY1 nucleotide (A-C) and amino acid (D) sequences.



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 cellular response (i.e., increased T cells). An antigen that produces an immune response may alternatively be referred to as being immunogenic or as an immunogen. In describing the present invention, a TA may be referred to as an “immunogenic target”.
TA includes both tumor-associated antigens (TAAs) and tumor-specific antigens (TSAs), where a cancerous cell is the source of the antigen. A TAA is an antigen that is expressed on the surface of a tumor cell in higher amounts than is observed on 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 to whom the TA has been administered. 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. No. 6,235,525; CN 1319611), BAGE family antigens (Boel et al., Immunity, 2:167-175 (1995)), GAGE family antigens (i.e., GAGE-1,2; Van den Eynde et al., J. Exp. Med., 182:689-698 (1995); U.S. Pat. No. 6,013,765), RAGE family antigens (i.e., RAGE-1; Gaugler et at., Immunogenetics, 44:323-330 (1996); U.S. Pat. No. 5,939,526), N-acetylglucosaminyltransferase-V (Guilloux et at., J. Exp. Med., 183:1173-1183 (1996)), 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 at., Int. J Cancer, 56:40-45 (1994)), BCR-abl (Bocchia et al., Blood, 85:2680-2684 (1995)), p53 (Theobald et al., Proc. Natl. Acad. Sci. USA, 92:11993-11997 (1995)), p185 HER2/neu (erb-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-BR-85, NY-BR-87, NY-BR-96 (Scanlan, M. Serologic and Bioinformatic Approaches to the Identification of Human Tumor Antigens, in Cancer Vaccines 2000, Cancer Research Institute, New York, N.Y.), BFA4 (SEQ ID NOS.: 26 and 27), or BCY1 (SEQ ID NOS.: 28 and 29), including “wild-type” (i.e., normally encoded by the genome, naturally-occurring), modified, and mutated versions as well as other fragments and derivatives thereof. Any of these TAs may be utilized alone or in combination with one another in a co-immunization protocol.
In certain cases, it may be beneficial to co-immunize patients with both TA and other antigens, such as angiogenesis-associated antigens (“AA”). An AA is an immunogenic molecule (i.e., peptide, polypeptide) associated with cells involved in the induction and/or continued development of blood vessels. For example, an AA may be expressed on an endothelial cell (“EC”), which is a primary structural component of blood vessels. Where the cancer is cancer, it is preferred that that the AA be found within or near blood vessels that supply a 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; Bemardini, et al. J. Urol., 2001, 166(4): 1275-9; Stames, et al. J. Thorac. Cardiovasc. Surg., 2001, 122(3): 518-23; Dias, et al. Blood, 2002, 99: 2179-2184), the VEGF receptor (i.e., VEGF-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. Oncol., 2001, 19(2):305-10), k-ras (Zhang, et al. Cancer Res., 2001, 61(16):6050-4), Wnt (Zhang, et al. Cancer Res., 2001, 61(16):6050-4), cyclin-dependent kinases (CDKs; Drug Resist. Updat. 2000, 3(2):83-88), microtubules (Timar, et al. 2001. Path. Oncol. Res., 7(2): 85-94), heat shock proteins (i.e., HSP90 (Timar, supra)), heparin-binding factors (i.e., heparinase; Gohji, et al. Int. J. Cancer, 2001, 95(5):295-301), synthases (i.e., ATP synthase, thymidilate synthase), collagen receptors, integrins (i.e., ανβ3, ανβ5, α1β1, α2β1, α5β1), or surface proteolglycan NG2, 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-N6-methyladenosine, aziridinyl-cytosine, pseudoisocytosine, 5-(carboxyhydroxylmethyl) uracil, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxy-methylaminomethyluracil, dihydrouracil, inosine, N6-iso-pentenyladenine, 1-methyladenine, 1-methylpseudouracil, 1-methylguanine, 1-methylinosine, 2,2-dimethyl-guanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine, 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 be linked to all or a portion of a polynucleotide to which the nucleic acid molecule is linked in nature; (3) is operably linked to a polynucleotide which it is not linked to in nature; and/or, (4) does not occur in nature as part of a larger polynucleotide sequence. Preferably, the isolated nucleic acid molecule of the present invention is substantially free from any other contaminating nucleic acid molecule(s) or other contaminants that are found in its natural environment that would interfere with its use in polypeptide production or its therapeutic, diagnostic, prophylactic or research use. As used herein, the term “naturally occurring” or “native” or “naturally found” when used in connection with biological materials such as nucleic acid molecules, polypeptides, host cells, and the like, refers to materials which are found in nature and are not manipulated by man. Similarly, “non-naturally occurring” or “non-native” as used herein refers to a material that is not found in nature or that has been structurally modified or synthesized by man.
The identity of two or more nucleic acid or polypeptide molecules is determined by comparing the sequences. As known in the art, “identity” means the degree of sequence relatedness between nucleic acid molecules or polypeptides as determined by the match between the units making up the molecules (i.e., nucleotides or amino acid residues). Identity measures the percent of identical matches between the smaller of two or more sequences with gap alignments (if any) addressed by a particular mathematical model or computer program (i.e., an algorithm). Identity between nucleic acid sequences may also be determined by the ability of the related sequence to hybridize to the nucleic acid sequence or isolated nucleic acid molecule. In defining such sequences, the term “highly stringent conditions” and “moderately stringent conditions” refer to procedures that permit hybridization of nucleic acid strands whose sequences are complementary, and to exclude hybridization of significantly mismatched nucleic acids. Examples of “highly stringent conditions” for hybridization and washing are 0.015 M sodium chloride, 0.0015 M sodium citrate at 65-68° C. or 0.015 M sodium chloride, 0.0015 M sodium citrate, and 50% formamide at 42° C. (see, for example, Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory, 1989); Anderson et al., Nucleic Acid Hybridisation: A Practical Approach Ch. 4 (IRL Press Limited)). The term “moderately stringent conditions” refers to conditions under which a DNA duplex with a greater degree of base pair mismatching than could occur under “highly stringent conditions” is able to form. Exemplary moderately stringent conditions are 0.015 M sodium chloride, 0.0015 M sodium citrate at 50-65° C. or 0.015 M sodium chloride, 0.0015 M sodium citrate, and 20% formamide at 37-50° C. By way of example, moderately stringent conditions of 50° C. in 0.015 M sodium ion will allow about a 21% mismatch. During hybridization, other agents may be included in the hybridization and washing buffers for the purpose of reducing non-specific and/or background hybridization. Examples are 0.1% bovine serum albumin, 0.1% polyvinyl-pyrrolidone, 0.1% sodium pyrophosphate, 0.1% sodium dodecylsulfate, 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 preferred embodiments of the present invention, vectors are used to transfer a nucleic acid sequence encoding a polypeptide to a cell. A vector is any molecule used to transfer a nucleic acid sequence to a host cell. In certain cases, an expression vector is utilized. An expression vector is a nucleic acid molecule that is suitable for transformation of a host cell and contains nucleic acid sequences that direct and/or control the expression of the transferred nucleic acid sequences. Expression includes, but is not limited to, processes such as transcription, translation, and splicing, if introns are present. Expression vectors typically comprise one or more flanking sequences operably linked to a heterologous nucleic acid sequence encoding a polypeptide. Flanking sequences may be homologous (i.e., from the same species and/or strain as the host cell), heterologous (i.e., from a species other than the host cell species or strain), hybrid (i.e., a combination of flanking sequences from more than one source), or synthetic, for example.
A flanking sequence is preferably capable of effecting the replication, transcription and/or translation of the coding sequence and is operably linked to a coding sequence. As used herein, the term operably linked refers to a linkage of polynucleotide elements in a functional relationship. For instance, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence. However, a flanking sequence need not necessarily be contiguous with the coding sequence, so long as it functions correctly. Thus, for example, intervening untranslated yet transcribed sequences can be present between a promoter sequence and the coding sequence and the promoter sequence may still be considered operably linked to the coding sequence. Similarly, an enhancer sequence may be located upstream or downstream from the coding sequence and affect transcription of the sequence.
In certain embodiments, it is preferred that the flanking sequence is a trascriptional regulatory region that drives high-level gene expression in the target cell. The transcriptional regulatory region may comprise, for example, a promoter, enhancer, silencer, repressor element, or combinations thereof. The transcriptional regulatory region may be either constitutive, tissue-specific, cell-type specific (i.e., the region is drives higher levels of transcription in a one type of tissue or cell as compared to another), or regulatable (i.e., responsive to interaction with a compound such as tetracycline). The source of a transcriptional regulatory region may be any prokaryotic or eukaryotic organism, any vertebrate or invertebrate organism, or any plant, provided that the flanking sequence functions in a cell by causing transcription of a nucleic acid within that cell. A wide variety of transcriptional regulatory regions may be utilized in practicing the present invention.
Suitable transcriptional regulatory regions include the CMV promoter (i.e., the CMV-immediate early promoter); promoters from eukaryotic genes (i.e., the estrogen-inducible chicken ovalbumin gene, the interferon genes, the gluco-corticoid-inducible tyrosine aminotransferase gene, and the thymidine kinase gene); and the major early and late adenovirus gene promoters; the SV40 early promoter region (Bemoist and Chambon, 1981, Nature 290:304-10); the promoter contained in the 3′ long terminal repeat (LTR) of Rous sarcoma 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. U.S.A., 75:3727-31); or the tac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. US.A., 80:21-25). Tissue- and/or cell-type specific transcriptional control regions include, for example, the elastase I gene control region which is active in pancreatic acinar cells (Swift et al., 1984, Cell 38:639-46; Ornitz et al., 1986, Cold Spring Harbor Symp. Quant. Biol. 50:399-409 (1986); MacDonald, 1987, Hepatology 7:425-515); the insulin gene control region which is active in pancreatic beta cells (Hanahan, 1985, Nature 315:115-22); the immunoglobulin gene control region which is active in lymphoid cells (Grosschedl et al., 1984, Cell 38:647-58; 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 polypeptide” is linked in nature; (3) is operably linked (by covalent or noncovalent interaction) to a polypeptide with which it is not linked in nature; or, (4) does not occur in nature. Preferably, the isolated polypeptide is substantially free from any other contaminating polypeptides or other contaminants that are found in its natural environment that would interfere with its therapeutic, diagnostic, prophylactic or research use.
Immunogenic target polypeptides may be mature polypeptides, as defined herein, and may or may not have an amino terminal methionine residue, depending on the method by which they are prepared. Further contemplated are related polypeptides such as, for example, fragments, variants (i.e., allelic, splice), orthologs, homologues, and derivatives, for example, that possess at least one characteristic or activity (i.e., activity, antigenicity) of the immunogenic target. Also related are peptides, which refers to a series of contiguous amino acid residues having a sequence corresponding to at least a portion of the polypeptide from which its sequence is derived. In preferred embodiments, the peptide comprises about 5-10 amino acids, 10-15 amino acids, 15-20 amino acids, 20-30 amino acids, or 30-50 amino acids. In a more preferred embodiment, a peptide comprises 9-12 amino acids, suitable for presentation upon Class I MHC molecules, for example.
A fragment of a nucleic acid or polypeptide comprises a truncation of the sequence (i.e., nucleic acid or polypeptide) at the amino terminus (with or without a leader sequence) and/or the carboxy terminus. Fragments may also include variants (i.e., allelic, splice), orthologs, homologues, and other variants having one or more amino acid additions or substitutions or internal deletions as compared to the parental sequence. In preferred embodiments, truncations and/or deletions comprise about 10 amino acids, 20 amino acids, 30 amino acids, 40 amino acids, 50 amino acids, or more. The polypeptide fragments so produced will comprise about 10 amino acids, 25 amino acids, 30 amino acids, 40 amino acids, 50 amino acids, 60 amino acids, 70 amino acids, or more. Such polypeptide fragments may optionally comprise an amino terminal methionine residue. It will be appreciated that such fragments can be used, for example, to generate antibodies or cellular immune responses to immunogenic target polypeptides.
A variant is a sequence having one or more sequence substitutions, deletions, and/or additions as compared to the subject sequence. Variants may be naturally occurring or artificially constructed. Such variants may be prepared from the corresponding nucleic acid molecules. In preferred embodiments, the variants have from 1 to 3, or from 1 to 5, or from 1 to 10, or from 1 to 15, or from 1 to 20, or from 1 to 25, or from 1 to 30, or from 1 to 40, or from 1 to 50, or more than 50 amino acid substitutions, insertions, additions and/or deletions.
An allelic variant is one of several possible naturally-occurring alternate forms of a gene occupying a given locus on a chromosome of an organism or a population of organisms. A splice variant is a polypeptide generated from one of several RNA transcript resulting from splicing of a primary transcript. An ortholog is a similar nucleic acid or polypeptide sequence from another species. For example, the mouse and human versions of an immunogenic target polypeptide may be considered orthologs of each other. A derivative of a sequence is one that is derived from a parental sequence those sequences having 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 particlar, 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 tranduction or transcytosis sequences, include sequences derived from HIV tat (see Kim et al. 1997 J. Immunol. 159:1666), Drosophila antennapedia (see Schutze-Redelmeier et al. 1996 J. Immunol. 157:650), or human period-1 protein (hPER1; in particular, SRRHHCRSKAKRSRHH).
In addition, the polypeptide or variant thereof may be fused to a homologous polypeptide to form a homodimer or to a heterologous polypeptide to form a heterodimer. Heterologous peptides and polypeptides include, but are not limited to: an epitope to allow for the detection and/or isolation of a fusion polypeptide; a transmembrane receptor protein or a portion thereof, such as an extracellular domain or a transmembrane and intracellular domain; a ligand or a portion thereof which binds to a transmembrane receptor protein; an enzyme or portion thereof which is catalytically active; a polypeptide or peptide which promotes oligomerization, such as a leucine zipper domain; a polypeptide or peptide which increases stability, such as an immunoglobulin constant region; and a polypeptide which has a therapeutic activity different from the polypeptide or variant thereof.
In certain embodiments, it may be advantageous to combine a nucleic acid sequence encoding an immunogenic target, polypeptide, or derivative thereof with one or more co-stimulatory component(s) such as cell surface proteins, cytokines or chemokines in a composition of the present invention. The co-stimulatory component may be included in the composition as a polypeptide or as a nucleic acid encoding the polypeptide, for example. Suitable co-stimulatory molecules include, for instance, polypeptides that bind members of the CD28 family (i.e., CD28, ICOS; Hutloff, et al. Nature 1999, 397: 263-265; Peach, et al. J Exp Med 1994, 180: 2049-2058) such as the CD28 binding polypeptides B7.1 (CD80; Schwartz, 1992; Chen et al, 1992; Ellis, et al. J. Immunol., 156(8): 2700-9) and B7.2 (CD86; Ellis, et al. J. Immunol., 156(8): 2700-9); polypeptides which bind members of the integrin family (i.e., LFA-1 (CD11a/CD18); 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: 462469); polypeptides which bind heat stable antigen (HSA or CD24; Zhou, et al. Eur J Immunol 1997, 27: 2524-2528); polypeptides which bind to members of the TNF receptor (TNFR) family (i.e., 4-1BB (CD137; Vinay, et al. Semin Immunol 1998, 10: 481-489), OX40 (CD134; Weinberg, et al. Semin Immunol 1998, 10: 471-480; Higgins, et al. J Immunol 1999, 162: 486-493), and CD27 (Lens, et al. Semin Immunol 1998, 10: 491-499)) such as 4-1BBL (4-1BB ligand; Vinay, et al. Semin Immunol 1998, 10: 481-48; DeBenedette, et al. J Immunol 1997, 158: 551-559), TNFR associated factor-1 (TRAF-1; 4-1BB ligand; Saoulli, et al. J Exp Med 1998, 187: 1849-1862, Arch, et al. Mol Cell Biol 1998, 18: 558-565), TRAF-2 (4-1BB and OX40 ligand; Saoulli, et al. J Exp Med 1998, 187: 1849-1862; Oshima, et al. Int Immunol 1998, 10: 517-526, Kawamata, et al. J Biol Chem 1998, 273: 5808-5814), TRAF-3 (4-1BB and OX40 ligand; Arch, et al. Mol Cell Biol 1998, 18: 558-565; Jang, et al. Biochem Biophys Res Commun 1998, 242: 613-620; Kawamata S, et al. J Biol Chem 1998, 273: 5808-5814), OX40L (OX40 ligand; Gramaglia, et al. J Immunol 1998, 161: 6510-6517), TRAF-5 (OX40 ligand; Arch, et al. Mol Cell Biol 1998, 18: 558-565; Kawamata, et al. J Biol Chem 1998, 273: 5808-5814), and CD70 (CD27 ligand; Couderc, et al. Cancer Gene Ther., 5(3): 163-75). 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. J. 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 (Ahlers, et al. J. Immunol., 158: 3947-3958 (1997); Iwasaki, et al. J. Immunol. 158: 4591-4601 (1997)), IL-12+GM-CSF+TNF-α (Ahlers, et al. Int Immunol. 13: 897-908 (2001)), CD80+IL-12 (Fruend, et al. Int. J Cancer, 85: 508-517 (2000); Rao, et al. supra), and CD86+GM-CSF+IL-12 (Iwasaki, supra). One of skill in the art would be aware of additional combinations useful in carrying out the present invention.In addition, the skilled artisan would be aware of additional reagents or methods that may be used to modulate such mechanisms. These reagents and methods, as well as others known by those of skill in the art, may be utilized in practicing the present invention.
Additional strategies for improving the efficiency of nucleic acid-based immunization may also be used including, for example, the use of self-replicating viral replicons (Caley, et al. 1999. Vaccine, 17: 3124-2135; Dubensky, et al. 2000. Mol. Med. 6: 723-732; Leitner, et al. 2000. Cancer Res. 60: 51-55), codon optimization (Liu, et al. 2000. Mol. Ther., 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, supra; Cho, et al. J. Immunol. 168(10):4907-13), sequences for targeting of the endocytic or ubiquitin-processing pathways (Thomson, et al. 1998. J. Virol. 72: 2246-2252; Velders, et al. 2001. J. Immunol. 166: 5366-5373), Marek's disease virus type 1 VP22 sequences (J. Virol. 76(6):2676-82, 2002), prime-boost regimens (Gurunathan, supra; Sullivan, et al. 2000. Nature, 408: 605-609; Hanke, et al. 1998. Vaccine, 16: 439-445; Amara, et al. 2001. Science, 292: 69-74), and the use of mucosal delivery vectors such as Salmonella (Darji, et al. 1997. Cell, 91: 765-775; Woo, et al. 2001. Vaccine, 19: 2945-2954). Other methods are known in the art, some of which are described below.
Chemotherapeutic agents, radiation, anti-angiogenic compounds, or other agents may also be utilized in treating and/or preventing cancer using immunogenic targets (Sebti, et al. Oncogene 2000 Dec. 27;19(56):6566-73). For example, in treating metastatic breast cancer, useful chemotherapeutic agents include cyclophosphamide, 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 utlized for various reasons. A majority of breast cancer patients have estrogen-receptor positive (ER+) tumors and in these patients, endocrine therapy (i.e., tamoxifen) is preferred over chemotherapy. For such patients, tamoxifen or, as a second line therapy, progestins (medroxyprogesterone acetate or megestrol acetate) are preferred. Aromatase inhibitors (i.e., aminoglutethimide and analogs thereof such as letrozole) decrease the availability of estrogen needed to maintain 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 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, vinblastine, taxol, ketoconazole, thalidomide, dolestatin, combrestatin A, rapamycin (Guba, et al. 2002, Nature Med., 8: 128-135), CEP-7055 (available from Cephalon, Inc.), flavone acetic acid, Bay 12-9566 (Bayer Corp.), AG3340 (Agouron, Inc.), CGS 27023A (Novartis), tetracylcine derivatives (i.e., COL-3 (Collagenix, Inc.)), Neovastat (Aeterna), BMS-275291 (Bristol-Myers Squibb), low dose 5-FU, low dose methotrexate (MTX), irsofladine, radicicol, cyclosporine, captopril, celecoxib, D45152-sulphated polysaccharide, cationic protein (Protamine), cationic peptide-VEGF, Suramin (polysulphonated napthyl urea), compounds that interfere with the function or production of VEGF (i.e., SU5416 or SU6668 (Sugen), PTK787/ZK22584 (Novartis)), Distamycin A, Angiozyme (ribozyme), isoflavinoids, staurosporine derivatives, genistein, EMD121974 (Merck KcgaA), tyrphostins, isoquinolones, retinoic acid, carboxyamidotriazole, TNP-470, octreotide, 2-methoxyestradiol, aminosterols (i.e., squalamine), glutathione analogues (i.e., N-acteyl-L-cysteine), combretastatin A-4 (Oxigene), Eph receptor blocking agents (Nature, 414:933-938, 2001), Rh-Angiostatin, Rh-Endostatin (WO 01/93897), cyclic-RGD peptide, accutin-disintegrin, benzodiazepenes, humanized anti-avb3 Ab, Rh-PAI-2, amiloride, p-amidobenzamidine, anti-uPA ab, anti-uPAR Ab, L-phanylalanin-N-methylamides (i.e., Batimistat, Marimastat), AG3340, and minocycline. Many other suitable agents are known in the art and would suffice in practicing the present invention.
The present invention may also be utilized in combination with “non-traditional” methods of treating cancer. For example, it has recently been demonstrated that administration of certain anaerobic bacteria may assist in slowing 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 T2, 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., injection) or by implantation of a “producer cell line” in proximity to the target cell population (Culver, K., et al., 1994, Hum. Gene Ther., 5 (3): 343-79; Culver, K., et al., Cold Spring Harb. Symp. Quant. Biol., 59: 685-90); Oldfield, E., 1993, Hum. Gene Ther., 4 (1): 39-69). The producer cell line is engineered to produce a viral vector and releases viral particles in the vicinity of the target cell. A portion of the released viral particles contact the target cells and infect those cells, thus delivering a nucleic acid of the present invention to the target cell. Following infection of the target cell, expression of the nucleic acid of the vector occurs.
Adenoviral vectors have proven especially useful for gene transfer into eukaryotic cells (Rosenfeld, M., et al., 1991, Science, 252 (5004): 431-4; Crystal, R., et al., 1994, 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 vivo have included intratracheal instillation (Rosenfeld, M., et al., 1992, Cell, 68 (1): 143-55) injection into muscle (Quantin, B., et al., 1992, Proc. Natl. Acad. Sci. U.S.A., 89 (7): 2581-4), peripheral intravenous injection (Herz, J., and Gerard, R., 1993, Proc. Natl. Acad. Sci. U.S.A., 90 (7): 2812-6) and stereotactic inoculation to brain (Le Gal La Salle, G., et al., 1993, Science, 259 (5097): 988-90), among others.
Adeno-associated 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 yet another attractive vector system, especially for use in the nervous system because of its neurotropic property (Geller, A., et al., 1991, Trends Neurosci., 14 (10): 428-32; Glorioso, et al., 1995, Mol. Biotechnol., 4 (1): 87-99; Glorioso, et al., 1995, Annu. Rev. Microbiol., 49: 675-710).
Poxvirus is another useful expression vector (Smith, et al. 1983, Gene, 25 (1): 21-8; Moss, et al, 1992, Biotechnology, 20: 345-62; Moss, et al, 1992, Curr. Top. Microbiol. Immunol., 158: 25-38; Moss, et al. 1991. Science, 252: 1662-1667). Poxviruses shown to be useful include vaccinia, NYVAC, avipox, fowlpox, canarypox, ALVAC, and ALVAC(2), among others.
NYVAC (vP866) was derived from the Copenhagen vaccine strain of vaccinia 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 (14L). 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 American Type Culture Collection (ATCC), P.O. Box 1549, 10801 University Boulevard, Manassas, Va 20110-2209. USA under the terms of the Budapest Treaty, as accession numbers VR-2559, VR-2558, VR-2557, VR-2556, ATCC-97913, ATCC-97912, and ATCC-97914, respectively, all deposits made on Mar. 6, 1996.
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), P.O. Box 1549, 10801 University Boulevard, Manassas, Va. 20110-2209, USA, as ATCC accession number VR-2547, on November 14, 1996.
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 American Type Culture Collection (ATCC). P.O. Box 1549, 10801 University Boulevard, Manassas, Va. 20110-2209, USA, as accession number 2553, on Feb. 6, 1997.
“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 (BlueBachl, 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., TOPOTM 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, CaPO4 precipitation, 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
(Aggerbeck and Heron, 1995)



(“alum 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 oligonucleotides (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 (Ott 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); Lonberg et al., Nature 368 856-859 (1994); Morrison, Nature 368 812-13 (1994); Fishwild et al., Nature Biotechnology 14, 845-51 (1996); Neuberger, Nature Biotechnology 14, 826 (1996); Lonberg and Huszar, Intern. Rev. Immunol. 13 65-93 (1995); as well as U.S. Pat. Nos. 4,816,567; 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and, 5,661,016). The antibodies or derivatives therefrom may also be conjugated to therapeutic moieties such as cytotoxic drugs or toxins, or active fragments thereof such as diptheria A chain, exotoxin A chain, ricin A chain, abrin A chain, curcin, crotin, phenomycin, enomycin, among others. Cytotoxic agents may also include radiochemicals. Antibodies and their derivatives may be incorporated into compositions of the invention for use in vitro or in vivo.
Nucleic acids, proteins, or derivatives thereof representing an immunogenic target may be used in assays to determine the presence of a disease state in a patient, to predict prognosis, or to determine the effectiveness of a chemotherapeutic or other treatment regimen. Expression profiles, performed as is known in the art, may be used to determine the relative level of expression of the immunogenic target. The level of expression may then be correlated with base levels to determine whether a particular disease is present within the patient, the patient's prognosis, or whether a particular treatment regimen is effective. For example, if the patient is being treated with a particular chemotherapeutic regimen, an decreased level of expression of an immunogenic target in the patient's tissues (i.e., in peripheral blood) may indicate the regimen is decreasing the cancer load in that host. Similarly, if the level of expresssion is increasing, another therapeutic modality may need to be utilized. In one embodiment, nucleic acid probes corresponding to a nucleic acid encoding an immunogenic target may be attached to a biochip, as is known in the art, for the detection and quantification of expression in the host.
It is also possible to use nucleic acids, proteins, derivatives therefrom, or antibodies thereto as reagents in drug screening assays. The reagents may be used to ascertain the effect of a drug candidate on the expression of the immunogenic target in a cell line, or a cell or tissue of a patient. The expression profiling technique may be combined with high throughput screening techniques to allow rapid identification of useful compounds and monitor the effectiveness of treatment with a drug candidate (see, for example, Zlokarnik, et al., Science 279, 84-8 (1998)). 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 1×106 infectious particles per dose. Thus, the dosage regimen may vary widely, but can be determined routinely using standard methods.
A prime-boost regimen may also be utilized (WO 01/30382 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
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. 1 and the deduced amino acid sequence is shown in FIG. 2.
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:










CLP 2589
MVRKKNPPLRNVASEGEGQILE
BFA4 (1-22)



 


CLP 2590
SPKATEETGQAQSGQANCQGLS
BFA4 (157-178)


 


CLP 2591
VAKPSEKNSNKSIPALQSSDSG
BFA4 (371-392)


 


CLP 2592
NHLQGSDGQQSVKESKEHSCTK
BFA4 (649-670)


 


CLP 2593
NGEQIIRRRTRKRLNPEALQAE
BFA4 (940-961)


 


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:










BFA4 (1-22)
KLH-MVRKKNPPLRNVASEGEG
(CLP-2589)




QILE


 


BFA4 (157-178)
KLH-SPKATEETGQAQSGQANC
(CLP-2590)



QGLS


 


BFA4 (371-392)
KLH-VAKPSEKNSNKSIPALQS
(CLP-2591)



SDSG


 


BFA4 (649-670)
KLH-NHLQGSDGQQSVKESKEH
(CLP-2592)



SCTK


 


BFA4 (940-961)
KLH-NGEQIIRRRTRKRLNPEA
(CLP-2593)



LQAE


 


BFA4 (1178-1200)
KLH-ANGASKEKTKAPPNVKNE
(CLP-2594)



GPLNV






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:
5′ CGGGATCCACCATGGTCCGGAAAAAGAACCCC 3′(BamHI for DNA3.1, MP76)
Primer 7723: BFA4-BamHI/R1 (3′ end reverse 4 kb):
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 BFA4 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/microliter) 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 JB-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 TRPSI 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 pMP76 (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 EcoRl 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 EcoRl 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 polyclonal sera raised against BFA4 peptides. The following constructs were used to study expression of BFA4 gene: pcDNA 3.1 zeoR/BFA4, pMP76/BFA4, pcDNA 3.1 zeor/BFA4/Myc tag and pcDNA 3.1 zeoR/BFA4/MycHis tag. BFA4 expression plasmids (5 μg and 10 μg) were co-transfected with pGL3 Luciferase (1 □g) (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 CHOKI cell extracts (pCDNA3.1 /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 zeoR/BFA4 (2.5 μg and 20 μg), and pcDNA 3.1 zeoR/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 zeoR/BFA4, and pcDNA 3.1 zeor/BFA4/MycHis tag) were expanded to T75 cm2 flasks in the presence of Zeocin at 1 mg/ml. Five clones from each pool (pcDNA 3.1 zeoR/BFA4, and pcDNA 3.1 zeoR/BFA4/MycHis tag) were expanded to T75 cm2 flasks 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 BamHI-Xho-1 fragment (1.5 Kbp) fragment encoding N-terminal 54kDa BFDA4 from pCDNA3.1/BFA4 was cloned into pGEX4T1-6His (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. Therapeutic Anti-BFA4 Immune Responses
1. 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




DESIGNATION
SEQUENCE
IN PROTEIN



















CLP-2421
MVRKKNPPL
BFA4
(1-9)i′′



 


CLP-2422
KKNPPLRNV
BFA4
(4-12)i′′


 


CLP-2423
VASEGEGQI
BFA4
(12-20)i′′


 


CLP-2424
QILEPIGTE
BFA4
(19-27)i′′


 


CLP-2425
RNMLAFSFP
BFA4
(108-116)i′′


 


CLP-2426
NMLAFSFPA
BFA4
(109-117)i′′


 


CLP-2427
MLAFSFPAA
BFA4
(110-118)i′′


 


CLP-2428
FSFPAAGGV
BFA4
(113-121)i′′


 


CLP-2429
AAGGVCEPL
BFA4
(117-125)i′′


 


CLP-2430
SGQANCQGL
BFA4
(170-178)i′′


 


CLP-2431
ANCQGLSPV
BFA4
(172-180)i′′


 


CLP-2432
GLSPVSVAS
BFA4
(176-184)i′′


 


CLP-2433
SVASKNPQV
BFA4
(181-189)i′′


 


CLP-2434
RLNKSKTDL
BFA4
(196-204)i′′


 


CLP-2435
NDNPDPAPL
BFA4
(207-215)i′′


 


CLP-2436
DPAPLSPEL
BFA4
(211-219)i′′


 


CLP-2437
ELQDFKCNI
BFA4
(218-216)i′′


 


CLP-2438
GLHNRTRQD
BFA4
(249-257)i′′


 


CLP-2439
ELDSKILAL
BFA4
(259-267)i′′


 


CLP-2440
KILALHNMV
BFA4
(263-271)i′′


 


CLP-2441
ALHNMVQFS
BFA4
(266-284)i′′


 


CLP-2442
VNRSVFSGV
BFA4
(282-290)i′′


 


CLP-2443
FSGVLQDIN
BFA4
(287-295)i′′


 


CLP-2444
DINSSRPVL
BFA4
(293-301)i′′


 


CLP-2445
VLLNGTYDV
BFA4
(300-308)i′′


 


CLP-2446
FCNFTYMGN
BFA4
(337-345)i′′


 


CLP-2447
YMGNSSTEL
BFA4
(342-350)i′′


 


CLP-2448
FLQTHPNKI
BFA4
(354-362)i′′


 


CLP-2449
KASLPSSEV
BFA4
(363-371)i′′


 


CLP-2450
DLGKWQDKI
BFA4
(393-401)i′′


 


CLP-2451
VKAGDDTPV
BFA4
(403-411)i′′


 


CLP-2452
FSCESSSSL
BFA4
(441-449)i′′


 


CLP-2453
KLLEHYGKQ
BFA4
(450-458)i′′


 


CLP-2454
GLNPELNDK
BFA4
(466-474)i′′


 


CLP-2455
GSVINQNDL
BFA4
(478-486)i′′


 


CLP-2456
SVINQNDLA
BFA4
(479-487)i′′


 


CLP-2457
FCDFRYSKS
BFA4
(527-535)i′′


 


CLP-2458
SHGPDVIVV
BFA4
(535-543)i′′


 


CLP-2459
PLLRHYQQL
BFA4
(545-553)i′′


 


CLP-2460
GLCSPEKHL
BFA4
(570-578)i′′


 


CLP-2461
HLGEITYPF
BFA4
(577-585)i′′


 


CLP-2462
LGEITYPFA
BFA4
(578-586)i′′


 


CLP-2463
HCALLLLHL
BFA4
(594-602)i′′


 


CLP-2464
ALLLLHLSP
BFA4
(596-604)i′′


 


CLP-2465
LLLLHLSPG
BFA4
(597-605)i′′


 


CLP-2466
LLLHLSPGA
BFA4
(598-606)i′′


 


CLP-2467
LLHLSPGAA
BFA4
(599-607)i′′


 


CLP-2468
FTTPDVDVL
BFA4
(621-629)i′′


 


CLP-2469
TTPDVDVLL
BFA4
(622-630)i′′


 


CLP-2470
VLLFHYESV
BFA4
(628-636)i′′


 


CLP-2471
FITQVEEEI
BFA4
(673-681)i′′


 


CLP-2472
FTAADTQSL
BFA4
(699-707)i′′


 


CLP-2473
SLLEHFNTV
BFA4
(706-714)i′′


 


CLP-2474
STIKEEPKI
BFA4
(734-742)i′′


 


CLP-2475
KIDFRVYNL
BFA4
(741-749)i′′


 


CLP-2476
NLLTPDSKM
BFA4
(748-756)i′′


 


CLP-2479
VTWRGADIL
BFA4
(792-800)i′′


 


CLP-2480
ILRGSPSYT
BFA4
(799-807)i′′


 


CLP-2481
YTQASLGLL
BFA4
(806-814)i′′


 


CLP-2482
ASLGLLTPV
BFA4
(809-817)i′′


 


CLP-2483
GLLTPVSGT
BFA4
(812-820)i′′


 


CLP-2484
GTQEQTKTL
BFA4
(819-827)i′′


 


CLP-2485
KTLRDSPNV
BFA4
(825-833)i′′


 


CLP-2486
HLARPIYGL
BFA4
(837-845)i′′


 


CLP-2487
PIYGLAVET
BFA4
(841-849)i′′


 


CLP-2488
LAVETKGFL
BFA4
(845-853)i′′


 


CLP-2489
FLQGAPAGG
BFA4
(852-860)i′′


 


CLP-2490
AGGEKSGAL
BFA4
(858-866)i′′


 


CLP-2491
GALPQQYPA
BFA4
(864-872)i′′


 


CLP-2492
ALPQQYPAS
BFA4
(865-873)i′′


 


CLP-2493
FCANCLTTK
BFA4
(895-903)i′′


 


CLP-2494
ANGGYVCNA
BFA4
(911-919)i′′


 


CLP-2495
NACGLYQKL
BFA4
(918-926)i′′


 


CLP-2496
GLYQKLHST
BFA4
(921-929)i′′


 


CLP-2497
KLHSTPRPL
BFA4
(925-933)i′′


 


CLP-2498
STPRPLNII
BFA4
(928-936)i′′


 


CLP-2499
RLNPEALQA
BFA4
(952-960)i′′


 


CLP-2500
VLVSQTLDI
BFA4
(1020-1028)i′′


 


CLP-2501
DIHKRMQPL
BFA4
(1027-1035)i′′


 


CLP-2502
RMQPLHIQI
BFA4
(1031-1039)i′′


 


CLP-2503
YPLFGLPFV
BFA4
(1092-1100)i′′


 


CLP-2504
GLPFVHNDF
BFA4
(1096-1104)i′′


 


CLP-2505
FVHNDFQSE
BFA4
(1099-1107)i′′


 


CLP-2506
SVPGNPHYL
BFA4
(1120-1128)i′′


 


CLP-2507
GNPHYLSHV
BFA4
(1123-1131)i′′


 


CLP-2508
HYLSHVPGL
BFA4
(1126-1134)i′′


 


CLP-2509
YVPYPTFNL
BFA4
(1141-1149)i′′


 


CLP-2510
FNLPPHFSA
BFA4
(1147-1155)i′′


 


CLP-2511
NLPPHFSAV
BFA4
(1148-1156)i′′


 


CLP-2512
SAVGSDNDI
BFA4
(1154-1162)i′′


 


CLP-2513
KNEGPLNVV
BFA4
(1192-1200)i′′


 


CLP-2514
TKCVHCGIV
BFA4
(1215-1223)i′′


 


CLP-2515
CVHCGIVFL
BFA4
(1217-1225)i′′


 


CLP-2516
CGIVFLDEV
BFA4
(1220-1228)i′′


 


CLP-2517
FLDEVMYAL
BFA4
(1224-1232)i′′


 


CLP-2518
VMYALHMSC
BFA4
(1228-1236)i′′


 


CLP-2519
FQCSICQHL
BFA4
(1243-1251)i′′


 


CLP-2520
GLHRNNAQV
BFA4
(1265-1273)i′′









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







1
CLP-2421
MVRKKNPPL



 



CLP-2422
KKNPPLRNV


 



CLP-2423
VASEGEGQI


 



CLP-2424
QILEPIGTE


 



CLP-2425
RNMLAFSFP


 



CLP-2426
NMLAFSFPA


 



CLP-2427
MLAFSFPAA


 



CLP-2428
FSFPAAGGV


 



CLP-2429
AAGGVCEPL


 



CLP-2430
SGQANCQGL


 


2
CLP-2431
ANCQGLSPV


 



CLP-2432
GLSPVSVAS


 



CLP-2433
SVASKNPQV


 



CLP-2434
RLNKSKTDL


 



CLP-2435
NDNPDPAPL


 



CLP-2436
DPAPLSPEL


 



CLP-2437
ELQDFKCNI


 



CLP-2438
GLHNRTRQD


 



CLP-2439
ELDSKILAL


 



CLP-2440
KILALHNMV


 


3
CLP-2441
ALHNMVQFS


 



CLP-2442
VNRSVFSGV


 



CLP-2443
FSGVLQDIN


 



CLP-2444
DINSSRPVL


 



CLP-2445
VLLNGTYDV


 



CLP-2446
FCNFTYMGN


 



CLP-2447
YMGNSSTEL


 



CLP-2448
FLQTHPNKI


 



CLP-2449
KASLPSSEV


 



CLP-2450
DLGKWQDKI


 


4
CLP-2451
VKAGDDTPV


 



CLP-2452
FSCESSSSL


 



CLP-2453
KLLEHYGKQ


 



CLP-2454
GLNPELNDK


 



CLP-2455
GSVINQNDL


 



CLP-2456
SVINQNDLA


 



CLP-2457
FCDFRYSKS


 



CLP-2458
SHGPDVIVV


 



CLP-2459
PLLRHYQQL


 



CLP-2460
GLCSPEKHL


 


5
CLP-2461
HLGEITYPF


 



CLP-2462
LGEITYPFA


 



CLP-2463
HCALLLLHL


 



CLP-2464
ALLLLHLSP


 



CLP-2465
LLLLHLSPG


 



CLP-2466
LLLHLSPGA


 



CLP-2467
LLHLSPGAA


 



CLP-2468
FTTPDVDVL


 



CLP-2469
TTPDVDVLL


 



CLP-2470
VLLFHYESV


 


6
CLP-2471
FITQVEEEI


 



CLP-2472
FTAADTQSL


 



CLP-2473
SLLEHFNTV


 



CLP-2474
STIKEEPKI


 



CLP-2475
KIDFRVYNL


 



CLP-2476
NLLTPDSKM


 



CLP-2477
KMGEPVSES


 



CLP-2478
GLKEKVWTE


 



CLP-2479
VTWRGADIL


 



CLP-2480
ILRGSPSYT


 


7
CLP-2481
YTQASLGLL


 



CLP-2482
ASLGLLTPV


 



CLP-2483
GLLTPVSGT


 



CLP-2484
GTQEQTKTL


 



CLP-2485
KTLRDSPNV


 



CLP-2486
HLARPIYGL


 



CLP-2487
PIYGLAVET


 



CLP-2488
LAVETKGFL


 



CLP-2489
FLQGAPAGG


 



CLP-2490
AGGEKSGAL


 


8
CLP-2491
GALPQQYPA


 



CLP-2492
ALPQQYPAS


 



CLP-2493
FCANCLTTK


 



CLP-2494
ANGGYVCNA


 



CLP-2495
NACGLYQKL


 



CLP-2496
GLYQKLHST


 



CLP-2497
KLHSTPRPL


 



CLP-2498
STPRPLNII


 



CLP-2499
RLNPEALQA


 



CLP-2500
VLVSQTLDI


 


9
CLP-2501
DIHKRMQPL


 



CLP-2502
RMQPLHIQI


 



CLP-2503
YPLFGLPFV


 



CLP-2504
GLPFVHNDF


 



CLP-2505
FVHNDFQSE


 



CLP-2506
SVPGNPHYL


 



CLP-2507
GNPHYLSHV


 



CLP-2508
HYLSHVPGL


 



CLP-2509
YVPYPTFNL


 



CLP-2510
FNLPPHFSA


 


10
CLP-2511
NLPPHFSAV


 



CLP-2512
SAVGSDNDI


 



CLP-2513
KNEGPLNVV


 



CLP-2514
TKCVHCGIV


 



CLP-2515
CVHCGIVFL


 



CLP-2516
CGIVFLDEV


 



CLP-2517
FLDEVMYAL


 



CLP-2518
VMYALHMSC


 



CLP-2519
FQCSICQHL


 



CLP-2520
GLHRNNAQV









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 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 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 de-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 CTL 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

















CLP 2425
RNMLAFSFP
CLP 2425
RNMLAFSFP



CLP 2426
NMLAFSFPA
CLP 2426
NMLAFSFPA



CLP 2427
MLAFSFPAA
CLP 2427
MLAFSFPAA



CLP 2461
HLGEITYPF



CLP 2468
FTTPDVDVL
CLP 2468
FTTPDVDVL



CLP 2470
VLLFHYESV
CLP 2470
VLLFHYYESV



CLP 2474
KIDFRVYNL



CLP 2482
ASLGLLTPV
CLP 2482
ASLGLLTPV



CLP 2486
HLARPIYGL
CLP 2486
HLARPIYGL



CLP 2495
NACGLYQKL
CLP 2495
NACGLYQKL



CLP 2497
KLHSTPRPL



CLP 2499
RLNPEALQA
CLP 2499
RLNPEALQA



CLP 2503
YPLFGLPFV



CLP 2509
YVPYPTFNL
CLP 2509
YVPYPTFNL



CLP 2511
NLPPHFSAV



CLP 2518
VMYALHMSC



CLP 2520
GLHRNNAQV
CLP 2520
GLHRNNAQV










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 1-2×107 pfu 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 2
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 I partial DNA sequence and are designed to clone BCY I 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:
5′ CAGTACGGATCCACCATGGCCGAGCTGCGCCTGA




AGGGC 3′


 


9617SXC:
5′ CCACGAGGATCCTTAGGAGAATATTCGGATGGC



TTGCG 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 (FIG. 3). The following primers were utilized in sequencing the clones:











9620MC:
5′ TAATACGACTCACTATAGGG 3′




 



9621MC:
5′ TAGAAGGCACAGTCGAGG 3′



 



9618MC:
5′ GAAAACGACTTCCTGGCGGGGAG 3′



 



9619MC:
5′ GCTCACCCAGGCGTGGGGCCTC 3′






DNA sequencing of all six clones indicated a consensus sequence, as shown in FIGS. 3A, and B, C, and D 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. 3D.
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




Designation
Sequence
in Protein









*CLP-2599
VPVPTSEHV
  2




 



*CLP-2602
PTSEHVAEI
  5



 



*CLP-2609
EIVGRQCKI
 12



 



*CLP-2616
KIKALRAKT
 19



 



*CLP-2618
KALRAKTNT
 21



 



*CLP-2619
ALRAKTNTY
 22



 



*CLP-2620
LRAKTNTYI
 23



 



*CLP-2624
TNTYIKTPV
 27



 



*CLP-2627
YIKTPVRGE
 30



 



*CLP-2630
TPVRGEEPV
 33



 



*CLP-2633
RGEEPVFMV
 36



 



*CLP-2640
MVTGRREDV
 43



 



 CLP-2641
VTGRREDVA
 44



 



*CLP-2643
GRREDVATA
 46



 



 CLP-2647
DVATARREI
 50



 



 CLP-2648
VATARREII
 51



 



*CLP-2650
TARREIISA
 53



 



*CLP-2651
ARREIISAA
 54



 



*CLP-2655
IISAAEHFS
 58



 



*CLP-2656
ISAAEHFSM
 59



 



 CLP-2657
SAAEHFSMI
 60



 



*CLP-2659
AEHFSMIRA
 62



 



*CLP-2663
SMIRASRNK
 66



 



 CLP-2666
RASRNKSGA
 69



 



*CLP-2670
NKSGAAFGV
 73



 



*CLP-2673
GAAFGVAPA
 76



 



*CLP-2674
AAFGVAPAL
 77



 



*CLP-2677
GVAPALPGQ
 80



 



*CLP-2678
VAPALPGQV
 81



 



*CLP-2680
PALPGQVTI
 83



 



*CLP-2681
ALPGQVTIR
 84



 



*CLP-2682
LPGQVTIRV
 85



 



 CLP-2684
GQVTIRVRV
 87



 



*CLP-2689
RVRVPYRVV
 92



 



*CLP-2691
RVPYRVVGL
 94



 



*CLP-2692
VPYRVVGLV
 95



 



*CLP-2695
RVVGLVVGP
 98



 



*CLP-2698
GLVVGPKGA
101



 



*CLP-2699
LVVGPKGAT
102



 



*CLP-2700
VVGPKGATI
103



 



*CLP-2710
RIQQQTNTY
113



 



*CLP-2711
IQQQTNTYI
114



 



*CLP-2712
QQQTNTYII
115



 



*CLP-2713
QQTNTYIIT
116



 



*CLP-2718
YIITPSRDR
121



 



 CLP-2721
TPSRDRDPV
124



 



 CLP-2724
RDRDPVFEI
127



 



 CLP-2731
EITGAPGNV
134



 



 CLP-2734
GAPGNVERA
137



 



 CLP-2738
NVERAREEI
141



 



 CLP-2744
EEIETHIAV
147



 



 CLP-2746
IETHIAVRT
149



 



 CLP-2749
HIAVRTGKI
152



 



 CLP-2750
IAVRTGKIL
153



 



 CLP-2756
KILEYNNEN
159



 



 CLP-2760
YNNENDFLA
163



 



 CLP-2762
NENDFLAGS
165



 



 CLP-2766
FLAGSPDAA
169



 



 CLP-2767
LAGSPDAAI
170



 



 CLP-2774
AIDSRYSDA
177



 



 CLP-2777
SRYSDAWRV
180



 



 CLP-2785
VHQPGCKPL
188



 



 CLP-2793
LSTFRQNSL
196



 



 CLP-2801
LGCIGECGV
204



 



 CLP-2807
CGVDSGFEA
210



 



 CLP-2812
GFEAPRLDV
215



 



 CLP-2817
RLDVYYGVA
220



 



 CLP-2819
DVYYGVAET
222



 



 CLP-2823
GVAETSPPL
226



 



 CLP-2825
AETSPPLWA
228



 



 CLP-2830
PLWAGQENA
233



 



 CLP-2833
AGQENATPT
236



 



 CLP-2835
QENATPTSV
238



 



 CLP-2843
VLFSSASSS
246



 



 CLP-2857
KARAGPPGA
260



 



 CLP-2869
PATSAGPEL
272



 



 CLP-2870
ATSAGPELA
273



 



 CLP-2872
SAGPELAGL
275



 



 CLP-2879
GLPRRPPGE
282



 



 CLP-2887
EPLQGFSKL
290



 



 CLP-2892
FSKLGGGGL
295



 



 CLP-2894
KLGGGGLRS
297



 



 CLP-2899
GLRSPGGGR
302



 



 CLP-2909
CMVCFESEV
312



 



 CLP-2910
MVCFESEVT
313



 



 CLP-2911
VCFESEVTA
314



 



 CLP-2913
FESEVTAAL
316



 



 CLP-2916
EVTAALVPC
319



 



 CLP-2917
VTAALVPCG
320



 



 CLP-2920
ALVPCGHNL
323



 



 CLP-2921
LVPCGHNLF
324



 



 CLP-2922
VPCGHNLFC
325



 



 CLP-2927
NLFCMECAV
330



 



 CLP-2929
FCMECAVRI
332



 



 CLP-2933
CAVRICERT
336



 



 CLP-2936
RICERTDPE
339



 



 CLP-2940
RTDPECPVC
343



 



 CLP-2945
CPVCHITAT
348



 



 CLP-2947
VCHITATQA
350



 



 CLP-2950
ITATQAIRI
353

















TABLE IX







shows the groups of peptides used for immunological testing:









Peptide
Peptide
Peptide


Group
Number
Sequence












1
CLP 2887
EPLQGFSKL



CLP 2916
EVTAALVPC



CLP 2945
CPVCHITAT



CLP 2673
KIKALRAKT



CLP 2699
IISAAEHFS



CLP 2616
RASRNKSGA



CLP 2655
GAAFGVAPA



CLP 2731
LVVGPKGAT



CLP 2734
EITGAPGNV



CLP 2666
GAPGNVERA


2
CLP 2724
ALRAKTNTY



CLP 2689
VATARREII



CLP 2648
PALPGQVTI



CLP 2680
ALPGQVTIR



CLP 2619
RVRVPYRVV



CLP 2681
RDRDPVFEI



CLP 2689
RVRVPYRVV



CLP 2947
HIAVRTGKI



CLP 2762
NENDFLAGS



CLP 2933
CAVRICERT



CLP 2749
VCHITATQA


3
CLP 2647
GRREDVATA



CLP 2677
DVATARREI



CLP 2643
TARREIISA



CLP 2785
GVAPALPGQ



CLP 2917
RVVGLVVGP



CLP 2695
VHQPGCKPL



CLP 2650
PATSAGPEL



CLP 2869
VTAALVPCG


4
CLP 2812
VPVPTSEHV



CLP 2892
ARREIISAA



CLP 2738
RIQQQTNTY



CLP 2651
NVERAREEI



CLP 2870
GFEAPRLDV



CLP 2899
ATSAGPELA



CLP 2710
FSKLGGGGL



CLP 2599
GLRSPGGGR


5
CLP 2609
PTSEHVAEI



CLP 2602
EIVGRQCKI



CLP 2641
LRAKTNTYI



CLP 2620
VTGRREDVA



CLP 2940
SMIRASRNK



CLP 2921
CMVCFESEV



CLP 2936
LVPCGHNLF



CLP 2663
NLFCMECAV



CLP 2927
RICERTDPE



CLP 2909
RTDPECPVC


6
CLP 2766
MVTGRREDV



CLP 2711
GLVVGPKGA



CLP 2913
IQQQTNTYI



CLP 2823
FLAGSPDAA



CLP 2640
GVAETSPPL



CLP 2698
FESEVTAAL



CLP 2929
FCMECAVRI


7
CLP 2760
KALRAKTNT



CLP 2633
RGEEPVFMV



CLP 2700
SAAEHFSMI



CLP 2835
AAFGVAPAL



CLP 2618
VVGPKGATI



CLP 2657
YNNENDFLA



CLP 2674
LGCIGECGV



CLP 2911
QENATPTSV



CLP 2801
VCFESEVTA


8
CLP 2807
TNTYIKTPV



CLP 2872
NKSGAAFGV



CLP 2670
QQTNTYIIT



CLP 2756
KILEYNNEN



CLP 2825
CGVDSGFEA



CLP 2843
AETSPPLWA



CLP 2713
PLWAGQENA



CLP 2624
VLFSSASSS



CLP 2830
SAGPELAGL


9
CLP 2712
ISAAEHFSM



CLP 2744
QQQTNTYII



CLP 2774
EEIETHIAV



CLP 2819
IETHIAVRT



CLP 2656
LAGSPDAAI



CLP 2922
AIDSRYSDA



CLP 2746
DVYYGVAET



CLP 2767
VPCGHNLFC



CLP 2950
ITATQAIRI


10
CLP 2793
TPVRGEEPV



CLP 2777
AEHFSMIRA



CLP 2910
VAPALPGQV



CLP 2721
TPSRDRDPV



CLP 2630
IAVRTGKIL



CLP 2659
SRYSDAWRV



CLP 2678
LSTFRQNSL



CLP 2750
RLDVYYGVA



CLP 2833
AGQENATPT



CLP 2817
MVCFESEVT









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 BCY1 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. Many of these single peptides also induced CTL activity killing peptide-loaded human T2 lymphoma cell targets. Table IX lists these peptides.
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. A poxvirus expression vector for expressing the BFA4 protein in a cell, the vector comprising the nucleic acid sequence of SEQ ID NO.: 1 and at least one nucleic sequence encoding an angiogenesis-associated antigen, wherein administration of the vector to a mammal induces a T-cell response against the BFA4 protein.

2. The expression vector of claim 1 further comprising at least one additional tumor-associated antigen.

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

4. The expression vector of claim 3 wherein the poxvirus is selected from the group consisting of NYVAC, ALVAC, and ALVAC(2).

5. The expression vector of claim 1 wherein the poxvirus is selected from the group consisting of vaccinia, NYVAC, avipox, canarypox, ALVAC, ALVAC(2), fowlpox, and TROVAC.

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

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

8. The expression vector of claim 7 wherein the poxvirus is selected from the group consisting of vaccinia, NYVAC, avipox, canarypox, ALVAC, ALVAC(2), fowlpox, and TROVAC.

9. The poxvirus of claim 8 wherein the poxvirus is selected from the group consisting of NYVAC, ALVAC, and ALVAC(2).

10. A composition comprising the poxvirus expression vector of claim 1 and a pharmaceutically acceptable carrier.

11. The composition of claim 10 wherein the poxvirus is selected from the group consisting of vaccinia, NYVAC, avipox, canarypox, ALVAC, ALVAC(2), fowlpox, and TROVAC.

12. The composition of claim 11 wherein the poxvirus is selected from the group consisting of NYVAC, ALVAC, and ALVAC(2).

13. The expression vector of claim 2 wherein the at least one additional tumor-associated antigen has the amino acid sequence of SEQ ID NO:4.

14. The expression vector of claim 2 further comprising at least one nucleic acid sequence encoding a co-stimulatory component.

15. A composition comprising the poxvirus expression vector of claim 2 and a pharmaceutically acceptable carrier.

16. The composition of claim 15 wherein the poxvirus is selected from the group consisting of vaccinia, NYVAC, avipox, canarypox, ALVAC, ALVAC(2), fowlpox, and TROVAC.

17. The composition of claim 16 wherein the poxvirus is selected from the group consisting of NYVAC, ALVAC, and ALVAC(2).

18. A poxvirus expression vector comprising a nucleic acid sequence encoding a BFA4 protein having the amino acid sequence of SEQ ID NO:2 and at least one nucleic sequence encoding an angiogenesis-associated antigen, wherein administration of the vector to a mammal induces a T-cell response against the BFA4 protein.

19. The expression vector of claim 18 further comprising a nucleic acid encoding at least one additional tumor-associated antigen.

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

21. The expression vector of claim 20 wherein the poxvirus is selected from the group consisting of NYVAC, ALVAC, and ALVAC(2).

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

23. The expression vector of claim 22 wherein the poxvirus is selected from the group consisting of NYVAC, ALVAC, and ALVAC(2).

24. The expression vector of claim 18 further comprising at least one nucleic acid sequence encoding a co-stimulatory component.

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

26. The poxvirus of claim 25 wherein the poxvirus is selected from the group consisting of NYVAC, ALVAC, and ALVAC(2).

27. A composition comprising an expression vector of claim 19 and a pharmaceutically acceptable carrier.

28. A composition comprising an expression vector of claim 18 and a pharmaceutically acceptable carrier.

29. The expression vector of claim 7, 24, or 14 wherein the co-stimulatory component is B7.1.

30. The expression vector of claim 7, 24, or 14 wherein the co-stimulatory component is TRICOM comprising B7.1, LFA-3 and ICAM-1.

31. The expression vector of claim 1 or 18 wherein the angiogenesis-associated antigen is selected from the group consisting of vascular endothelial growth factor, vascular endothelial growth factor receptor EPH receptors, epidermal growth factor receptor, basic fibroblast growth factor, platelet-derived cell growth factor, platelet-derived endothelial cell growth factor, a transforming growth factor, endoglin, an Id proteins, uPA, uPAR, a matrix metalloproteinases, thrombospondin, and surface proteolglycan NG2.

32. The expression vector claim 2 or 19 wherein the at least one additional tumor-associated antigen is selected from the group consisting of gp100, TRP-1, NY-ESO-1, a MAGE antigen, MAGE-1, MAGE-3, carcinoembryonic antigen, MUC-1, and NY-BR-1.