MXPA00002810A - Mammaglobin, a secreted mammary-specific breast cancer protein - Google Patents

Abstract

A purified and isolated DNA sequence and the encoded mammary-specific secreted protein, mammaglobin, are disclosed. Also disclosed are methods for detecting breast cancer based upon the overexpression and secretion of mammaglobin by breast cancer cells. The methods detect and/or quantitate the presence of mammaglobin or the mRNA encoding mammaglobin. Immunotherapy-based methods for treating a breast cancer patient with a mammaglobin-expressing tumor are also disclosed. The methods involve using mammaglobin antigens to induce a humoral and/or cell-mediated immune response against the tumor.

Description

MAMAGLOBINA. A SECRETED PROTEIN OF SPECIFIC MAMMARY GLAND CANCER RELATED REQUESTS This application is a continuation that forms part of document PCT / US96 / 08235, filed on May 31, 1996, which is a continuation that forms part of the application of E.U.A. No. 08 / 455,896, filed May 31, 1995.

BACKGROUND OF THE INVENTION FIELD OF THE INVENTION This invention relates generally to the field of the pathogenesis of breast cancer and, more particularly, to a cDNA sequence and mammary gland-specific encoded protein for use in the detection and treatment of breast cancer.

DESCRIPTION OF THE RELATED TECHNIQUE Breast cancer is one of the most common and potentially lethal cancers.

Although early diagnosis and treatment can reduce morbidity and mortality related to the disease, it has been estimated that the value of the positive prognosis of mammography is only about 25% (Hall et al., N Engl J Med 327: 319 -328,1992, citation incorporated herein by reference). Therefore, it would be convenient to have means to detect cancer before cancer can be detected using mammography, and a genetic or biochemical marker could be able to provide such means to complement and increase the prognostic value of mammography (Hayes, Hematol Oncol Clin N Am 8: 485, 1994, citation incorporated herein by reference). The development of breast cancer is accompanied by a number of genetic changes (for a review, see Porter-Jordan, Hematol Oncol Clin N Am 8:73, 1994, citation incorporated herein by reference). Such changes include obvious chromosomal alterations and loss of genetic markers (Devilee et al, Biochim Biophys Acta 7798: 1 13,1994, Callahan et al, J Cell Biochem Suppl 77: 167, 1993, citation incorporated herein by reference). It has also been shown that the progression of breast neoplasia results in qualitative and quantitative changes in the expression of previously identified genes encoding growth factors and their receptors (Zajchowski et al., Cancer Res 48: 7041, 1988, citation incorporated herein by reference), structural proteins (Trask et al., Proc Nati Acad Sci 87: 2319, 1990, citation incorporated herein by reference), second messenger proteins (Ohuchi et al., Cancer Res 26: 251 1, 1986, citation incorporated herein by reference) and transcription factors (Harris, Adv Cancer Res 59:69, 1992, citation incorporated herein by reference). These changes in gene expression could potentially form the basis for the development of a marker for breast cancer, although the precise role of these gene changes in the pathogenesis of breast carcinoma in patient biopsies is not well understood. In addition to providing a genetic or biochemical marker for breast cancer to detect the disease early, it would also be convenient to have a tumor marker that could provide an estimate of the prognosis, means to select and evaluate the therapy and means to direct the latter. Although numerous tissue markers have been identified, none of them is sufficiently sensitive or tumor specific to be ideally adapted for diagnosis or for selection of the general population (Id.). Thus, there is a continuing need for a cancer marker. breast such as a gene together with its expressed protein that can be used to specifically and selectively identify the appearance and pathogenic development of breast cancer in a patient, and that can be used in tumor-specific immunotherapy. Using a modified differential display polymerase chain reaction technique to isolate differentially expressed sequence tags of mammary carcinoma, several sequence fragments that were uniquely expressed in neoplastic mammary epithelial tissue were isolated, compared to normal tissue controls (Watson and Fleming , Cancer Res 54: 4598-4602, 1994, citation incorporated herein by reference). The discovery of one of these sequence tags identified as DEST002 has led to the discovery and isolation of the novel full-length cDNA and the encoded protein now identified as mamaglobin. The cDNA and the protein are new.

BRIEF DESCRIPTION OF THE INVENTION Therefore, in summary, the present invention is directed to the identification of novel genes whose expression is increased in breast cancer, and to the isolation of cDNA molecules from messenger RNA molecules of these genes: Accordingly, applicants have succeeded in discovering a novel cDNA and mammary gland-specific secretory protein, mammaglobin. The cDNA is in purified and isolated form and has a nucleotide sequence identified as SEQ ID NO: 1, and the encoded protein, mamaglobin, is in purified and isolated form, and has an amino acid sequence identified as SEQ ID NO: 2. Mammaglobin is overexpressed in 27% of primary stage I breast cancer tumors. This suggests that dysmagulation of the mamaglobin gene occurs early and often in breast cancer. The discovery of mammaglobin and its cDNA, therefore, provides the basis for the development of novel methods and compositions for detection and treatment in humans and other mammals. Therefore, the present invention is directed to novel methods for detecting the presence of breast neoplasia cells in a sample. In one embodiment, cDNA encoding mamaglobin or a derivative of said cDNA is used to detect the presence of mammaglobin messenger RNA in a sample. The method comprises the steps of: (a) providing a polynucleotide containing a nucleotide sequence having the sequence SEQ ID NO: 1 or a derivative thereof, (b) incubating the nucleotide sequence with the sample under conditions in the which sequence can hybridize with messenger RNA from breast neoplasia cells, and (c) detect the existence of a DNA-RNA hybridization complex. Another aspect of the present invention provides a device for detecting the presence of breast neoplasia cells in a sample by hybridization. The kit comprises a polynucleotide containing a nucleotide sequence having the sequence of SEQ ID NO: 1 or a derivative thereof packaged in a container. In another embodiment of the present invention, the expression of the mammaglobin in a sample is determined by detecting the presence of cDNA that is reverse transcribed from the mammaglobin messenger RNA in the sample. The method comprises the steps of: (a) producing a cDNA coding for mamaglobin from messenger RNA using the reverse transcription method in a sample obtained from a patient, (b) providing two primers for the chain reaction method of polymerase comprising oligomers that flank or that are within the cDNA encoding mamaglobin; and (c) amplifying the cDNA encoding mamaglobin by the polymerase chain reaction method. The two primers have nucleotide sequences comprising SEQ ID NO: 3 and SEQ ID NO: 4. Another embodiment of the present invention provides a kit for detecting the presence of breast neoplasia cells in a sample by the chain reaction of polymerase The kit comprises two primers for the polymerase chain reaction method, which comprises oligomers that flank or are within a cDNA sequence encoding mamaglobin packaged in a container. The two primers have nucleotide sequences comprising SEQ ID NO: 3 and SEQ ID NO: 4. In another embodiment of the present invention, the presence of the mammaglobin protein expressed by a tumor cell is detected in a sample using antibodies specific for the protein mamaglobin. The specific antibodies can be polyclonal or monoclonal antibodies. The invention is also directed to novel compositions and methods for detecting neoplastic breast disease using mamaglobin antigens capable of inducing an immune response mediated by antibodies and / or cell mediated, ie, through T cell activities, against a tumor which expresses mammaglobin. An embodiment of a composition in accordance with The invention comprises a B-cell antigen of mammaglobin capable of activating mammaglobin-specific B cells. The B cell antigen comprises a B-cell epitope specific to mamaglobin and an epitope T, O determinant, recognized by T helper cells. In another embodiment, the mamaglobin antigen is an antigen of magelobulin Te cells recognized by mammaglobin-specific cytotoxic T lymphocytes which comprise an epitope of Te cells, and a binding site, or agrétope, for an MHC class I molecule. Yet another embodiment of a composition according to the invention comprises B cell antigens and Te cells. Methods for treating a patient with a mammaglobin-expressing tumor include adoptive immunotherapy, which comprises stimulation ex vivo with a mamaglobin antigen of mamaglobin-specific lymphocytes isolated from the patient, and subsequent administration of the lymphocytes activated to the patient, as well as as the in vivo stimulation of an anti-mamaglobin immune response, comprising administering to the patient a vaccine comprising a mamaglobin antigen. Among the various advantages that have been found can be achieved by the present invention, the provision of a nucleotide sequence and an encoded sequence of amino acids that can function as markers of breast cancer cells can therefore be emphasized; the provision of methods for the early detection of the presence of breast cancer cells; the provision of means to detect breast cancer that can complement mammography and increase the prognostic value; the provision of methods that can provide an estimate of the forecast; the provision of markers for directing therapy; and the provision of compositions to stimulate a cellular and humoral immune response against the tumor.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 illustrates the strategy used to isolate full-length mamaglobin cDNA, including the polymerase chain reaction (PCR) technique for rapid amplification of cDNA ends (RACE), and subsequent subcloning in vectors pGEM7Z and pCEV27. Figure 2 illustrates the human cDNA sequence of SEQ ID NO: 1 (nucleotides recited above), and the amino acid sequence of the mammary gland-specific encoded protein, mammaglobin (SEQ ID NO: 2) (amino acids listed below) , the solid bar indicating the 403 bp fragment (SEQ ID NO: 5) isolated by the PCR method for RACE, and the clear bar indicating the 206 bp DEST002 sequence (SEQ ID NO: 6); Figure 3 illustrates the amino acid sequence of the mammary gland specific protein, mamaglobin (hMAM), (SEQ ID NO: 2), comparatively with the C3 subunit of the steroidal binding prostatic protein (rPSC3) (SEQ ID NO: 7), and 10 kD protein of human Clara cells (hCC10) (SEQ ID NO: 8) with identities marked by bold letters and double lines, and structurally similar amino acids marked by simple lines; Figure 4 illustrates (A) Northern biot analysis of the hybridization of the human cDNA sequence encoding the mammary gland specific protein, mammaglobin (hMAM), with messenger RNA expressed by breast, normal breast and breast neoplasm tissues. other adult tissues, and (B) the analysis of tissue samples amplified by RT / PCR of breast cancer tissues, normal breast and other adult tissues; Figure 5 illustrates the translation of the mammary gland-specific cDNA sequence into an in vitro rabbit reticulocyte lysate test system. Figure 6 illustrates Northern biot hybridization with the cDNA encoding mamaglobin, showing the detection of messenger RNA in tumor 2410, in tumors of three of eight other patients (shown in bold), and to a lesser degree, in normal tissue of breast (shown in italics), and comparing in two cases (the four bands on the right) the expression of the mammaglobin messenger RNA in tumor tissue and normal tissue compared in patients; Figure 7 illustrates Western biot analysis using polyclonal antibody for the C-terminus of mamaglobin (SEQ ID NO: 14) of the conditioned medium (S) and of the cell lysate (C) of breast tumor cells MDA-MB-415 in absence (-) and presence (+) of the immunization peptide showing the detection of the precursor and secreted forms of the mamaglobin protein in the cell medium and the cell lysate, respectively; Figure 8 illustrates the Western biot analysis using polyclonal anti-mamaglobin antibody from the conditioned medium (S) and the cell lysate (C) of MDA-MB-415 breast tumor cells developed in the absence (-) and presence (+ ) of tunicamycin, which blocks glycosylation, showing the lack of detectable mamaglobin protein in the lysate or cell medium in which N-linked glycosylation is inhibited; Figure 9 illustrates the Western biot analysis of lysates of human breast tumor cell cells showing detection of the precursor mamaglobin protein using the polyclonal anti-mamaglobin antibody and goat anti-rabbit antibody visualized by enzyme-linked chemiluminescence; Figure 10 illustrates Western biot analysis using the polyclonal anti-mammaglobin antibody from human breast fluid secretions during pregnaand postpartum showing the detection of the secreted mammaglobin protein in the proliferating mammary gland; Figure 11A illustrates in color a paraffin-fixed section of breast cancer cells of a patient specimen stained immunohistochemically using the polyclonal anti-mammaglobin antibody and goat anti-rabbit antibody labeled with horseradish peroxidase and DAB as a substrate showing a brown staining of cells expressing the mamaglobin protein; and Figure 11B illustrates in black and white a paraffin-fixed section of breast cancer cells of a patient specimen stained immunohistochemically using the polyclonal anti-mammaglobin antibody and goat anti-rabbit antibody labeled with horseradish peroxidase and DAB as a substrate where the brown staining of the cells expressing the mamaglobin protein is indicated.

DESCRIPTION OF THE PREFERRED MODALITIES One aspect of the present invention is based on the identification and sequencing of the cDNA identified as SEQ ID NO: 1, which codes for a mammary gland specific secretory protein, mammaglobin, identified by SEQ ID NO: 2 (Fig. 2). As described below, the full length mamaglobin cDNA was isolated starting from tumor cell messenger RNA which was reverse transcribed, amplified using the PCR technique, and subcloned into expression vectors. In addition, the protein, mamaglobin, encoded by the cDNA, was identified and characterized. Using the anonymous sequence tag previously designated DEST002, it was shown that the corresponding gene product, which was hitherto unknown, but in the present one identified as mamaglobin, is particularly abundant in the breast cancer tumor cell line MDA-MB -415 To isolate the full-length mamaglobin cDNA, the messenger RNA was reverse transcribed from this cell line, and cloned using the RACE PCR technique (Edwards et al., Nucleic Acids Research 19: 5227-32, 1991). , citation incorporated herein by reference). This technique is based on the ligation strategy of the single chain oligodeoxyribonucleotide to the 3 'end of the single chain cDNA. The method by which the mamaglobin cDNA was isolated is depicted schematically in Figure 1. The cDNA sequence of 503 bp of total length (SEQ ID NO: 1) was deduced from the sequence information obtained from the 403 bp fragment (SEQ ID NO: 5) (Fig. 2) isolated by this technique, together with the sequence information previously obtained from the corresponding DEST sequence (DEST002, SEQ ID NO: 6) (Figure 2) in the authors' previous study (Watson and Fleming, cited above). Within the 503 bp cDNA is an open reading frame of 279 bp, which codes for a polypeptide of 93 amino acids and predicted molecular mass of 10.5 kD (SEQ ID NO: 2) (Figure 2). The initial methionine of this open reading frame is within an almost perfect Kozak consensus sequence (Kozak, Cell 22: 7-8, 1980, citation incorporated herein by reference). The 60 bp towards the 5 'end of this sequence does not contain other methionines or translation stops within the framework. The 3 'untranslated cDNA sequence constitutes 163 bp, and contains a polyadenylation signal, AATAAA, 12 bp towards the 5' end of the initiation site of the original DEST002 sequence. These data indicate that the full-length mamaglobin cDNA has been isolated. The first 19 residues of the encoded polypeptide predict a hydrophobic sequence of signal peptides, and residues 53 to 55 and 68 to 70 are N-linked consensus glycosylation sites, indicating that the mammaglobin is a secreted glycoprotein. The search for DNA sequences similar to the mamaglobin cDNA sequence in the gene bank using the BLAST algorithm (Benson et al., Nucí Acid Res 27: 2963-2965, 1993; Altschul et al, J Mol Biol 275: 403 -410, 1990, citation incorporated herein by reference), did not allow to identify obvious homologies of the DNA sequence. In this way, it is thought that the mamaglobin cDNA is a novel DNA sequence, hitherto unknown. The search for other polypeptides for sequences related to the mammaglobin revealed a homology of amino acid sequences between the mammaglobin and other polypeptides. Mammaglobin exhibited 42% amino acid identity (58% including conservative substitutions) with the C3 subunit of the steroid-binding prostatic protein (prostatein) (rPSC3) (Figure 3) (SEQ ID NO: 7). The rat steroid-binding prostatic protein is a major secretory protein in the rat ventral prostate consisting of a tetrameric protein composed of two different dimeric subunits: C3 / C1 and C3 / C2 (Parker et al., Ann NY Acad Sci 438: 1 15-124; Parker et al., J Steroid Biochem 20: 67-71, 1984, citations incorporated herein by reference). The C1, C2 and C3 genes code for secretory proteins of approximately 6 kD, and it is thought that they have arisen from gene duplication, but while the C1 and C2 genes show strong homology to each other, they are much less similar to the C3 gene. Accordingly, mammaglobin does not show sequence homology with the C1 or C2 proteins. As noted above, the steroid-binding prostatic protein (prostatein) is the main secretory protein in the ventral prostate of rat, and its expression is regulated by androgenic steroids (Parker et al, Ann NY Acad Sci 438: 115-24, 1984; Parker et al, J Steroid Biochem 20: 67-71, 1984, citations incorporated herein by reference). It has been reported that another protein, the human estramustine binding protein (hEMBP), is expressed in the human prostate, human breast cancer and human malignant melanoma (Bjork et al, Cancer Res 42: 1935-1942, 1982 Bjork et al, Anticancer Res 77: 1173-82, 1991, citations incorporated herein by reference). The human estramustine binding protein is immunochemically similar to the rat estramustine binding protein, which has been postulated is identical to the rat steroid binding protein, prostatein. As noted above, the amino acid sequence of the mamaglobin exhibited 42% amino acid identity and 58% homology including conservative substitutions with the C3 subunit of prostatein. In this way, it is possible that the mammaglobin could be in some way related to hEMBP. However, while prostatein and hEMBP are detected in the prostate gland, the messenger RNA of mamaglobin is completely missing in this tissue. Therefore, mammaglobin is neither the same protein nor a subunit of hEMBP and, in addition, the sequence of hEMBP has not been determined, so it is not known if there is even any similarity of the mammaglobin with any fragment or subunit of hEMBP. Although recent reports have shown that the rPSC3 promoter fused to the SV40 T antigen produces prosthetic and mammary carcinomas in transgenic mice (Maroulakou et al, Proc Nat Acad Sci US 97: 11236-1 1240, 1994, Sandmoller et al, Oncogene 9: 2805- 2815, 1994, citations incorporated herein by reference), the actual biological function of this protein is unknown. Furthermore, notwithstanding the presumed relationship of the prostatic steroid-binding protein with the human EMBP, no human polypeptide or human gene corresponding to rPSC3 has been identified. In this way, the mammaglobin and the cDNA coding for it represent novel sequences hitherto unknown. Using manual alignment with other sequences that had less significant BLAST scores with mamaglobin and rPSC3 protein sequences, other homologies were identified with the 10kD protein of human Clara cells (hCD10) (SEQ ID NO: 8) (Peri et al, J Clin Invest 92: 2099-2109, 1993, citation incorporated herein by reference) (Fig. 3) and, in addition, with mouse and rabbit uteroglobin proteins (Miele et al., Endocrine Rev 8: 474-90 , 1987, Cato and Beato, Anticancer Res 5: 65-72, 1985; Miele et al., J Endocrinol Invest 77: 679-692, 1994, citations incorporated herein by reference). These homologies, depending on the species, had 26% identity or 40%, including conservative substitutions. In particular, a number of amino acids was perfectly conserved among all proteins, including Cys-3 and Cys-69, which are known to play a role in the formation of disulfide bridges between the subunits of uteroglobin (see below). These homologies suggest that mammaglobin is a novel member of a small family of proteins that are secreted by epithelial cells (Miele et al, 1994, cited above). The hCC10 gene is the human homolog of rabbit and mouse uteroglobin genes (Peri et al, J Clin Invest 92: 2099-2109, 1993, citation incorporated herein by reference). Uteroglobin was originally characterized as a secretory protein in the rabbit uterus, but has since been found in other epithelial organs, including lung, breast and prostate. Unlike rat prostatein, uteroglobin is a homodimeric protein coupled by two disulfide bonds in the conserved residues Cys-2 and Cys-69 (Miele et al, 1994, cited above). Although transcription of the uteroglobin gene is regulated by steroid hormones, the ability of the protein itself to bind to progesterone or other steroid hormones is debatable and again, the actual biological function of this protein is unknown (Miele et al., 1994, cited above).

The expression of mammaglobin is restricted to the mammary gland. This contrasts with the observation that rPSC3 is expressed in the rat ventral prostate (Parker et al., Ann NY Acad Sci 438: 115-1124, 1984), and the expression of hCC10 / uteroglobin in numerous tissues including lung, uterus , prostate and breast (Miele et al., 1987, cited above, Cato and Beato, cited above, Miele et al., 1994, cited above). Due to the sequence homology between the mammaglobin and these proteins, the specific expression pattern in tissues was determined. The 500 bp mamaglobin messenger RNA was easily detected in tumor specimen 2410 (the tissue from which this original sequence tag was isolated), and to a much lesser extent in normal human breast tissue (Figure 4A). The mammaglobin messenger RNA could not be detected in the immortalized breast epithelial cell line B5-589. Mammaglobin expression also could not be detected in the uterus and human lung, two uteroglobin expression sites. Amplification was performed using mammaglobin messenger RNA detected by RT / PCR in tumor specimen 2410 and normal breast tissue, but not in other tissues, including tissues that normally express rPSC3 and uteroglobin (lung, uterus, prostate), tissues steroidogenic and hormone-responsive (ovary, testis, placenta), and other secretory epithelial organs (colon) (Figure 4B). Therefore, the expression of the mammaglobin messenger RNA is relatively specific for breast tissue.

Based on the studies in this report, mammaglobin is a relatively specific protein of the mammary gland. Two other genes known to be overexpressed in breast carcinoma are erb-B and cyclin D (Jardines et al, Pathobiology 67: 268-282, 1994, Keyomars and Pardee, Proc Nat Acad Sci US 90: 1 1 12- 116, 1993, citations incorporated herein by reference). Unlike overexpression of erb-B or cyclin D, overexpression of mammaglobin may reflect a more specific alteration of the mammary epithelial cell, rather than an overall increased growth of mitotic or potential velocity. As such, the occurrence of dysregulation of the mamaglobin gene may have a more specific relevance to the therapeutic vulnerability or clinical course of a tumor. Mammaglobin expression may not be detected in normal lymph nodes or peripheral lymphocytes at the level of sensitivity produced by a one-step RT / PCR test. This suggests that the analysis of mamaglobin transcripts in peripheral lymph nodes may be useful for detecting hidden metastases of breast cancer, as has been suggested for other specific epithelial genes (Schoenfeld et al., Cancer Res 54: 2986-90, citation incorporated herein by reference). To demonstrate that the mamaglobin cDNA encoded a translatable protein, the cDNA clone was used in an in vitro translation test. Figure 5 shows the protein product of a rabbit reticulocyte lysate programmed with the mammaglobin cDNA. A protein of approximately 6 kD is generated using the mamaglobin cDNA. The apparent molecular weight is lower than that predicted from the conceptual translation of the open reading frame, but this finding is also commonly observed with rabbit and human uteroglobin translation products. Although overexpression of mamaglobin RNA was detected in a tumor specimen (ie, 2410), it was not clear at what frequency this overexpression is observed in other breast carcinomas. A panel of 15 primary stage I mammary carcinomas of different histological types was then examined by Northern biot hybridization with the mamaglobin cDNA probe. Due to the potential variability in expression due to environmental influences (for example, the patient's hormonal status), we also sought to compare tumor specimens directly with samples of normal breast tissues compared in patients, although this was not possible in many cases. As shown in Figure 6, the 500 bp mamaglobin messenger RNA was detected again in normal breast tissue and the tumor specimen 2410. Mammaglobin was also detected in three other tumors, two of which showed little or no expression in normal tissue compared in patients (BO15 and BO22). 4 of the 15 tumors (27%) examined overexpressed mamaglobin messenger RNA. These data suggest that overexpression of mammaglobin is not unique to an individual tumor specimen and is, in fact, relatively frequent among primary breast tumors. In addition, the fact that all the tumors examined were stage I suggests that this dysregulation occurs relatively early in the progression of breast neoplasia. Since applicants believe that mammaglobin is a secreted protein, its presence would be expected to be detectable in sera from patients whose tumors overexpress this gene product. As such, it is likely that mammaglobin is clinically as useful as prostate-specific antigen (PSA) and other solid tumor markers for the management of breast cancer patients (Tumor markers in diagnostic pathology, Clin Lab Med 70: 1- 250, 1990, citation incorporated herein by reference). The frequency of mammaglobin was determined as a tumor marker in the general population of breast cancer tumors, examining the expression of mammaglobin in several primary breast carcinomas. Although the number of specimens examined in this study was small, 27% of the tumors evaluated overexpressed mammaglobin messenger RNA. This percentage is comparable to the frequency of other genetic alterations such as the amplification of erb-B and the mutation of p53 (Slamon et al., Sci 244: 707-712, 1989; Thor et al. J Nat'l Cancer Inst 84: 845-855, 1992, citations incorporated herein by reference). In addition, because the analysis has been restricted to stage I tumors, overexpression of mammaglobin would actually be more frequent than any other genetic alteration reported in this subgroup of tumors (Alllerd et al., J Nat'l Cancer Inst 85: 200 -206, 1993, citation incorporated herein by reference). The identification of mammaglobin as a marker of breast cancer provides the basis for another aspect of the present invention, which includes methods for detecting the presence of breast cancer in a patient. The term "detection" as used herein in the context of detection of neoplastic breast disease, has the purpose of being an aspect that encompasses the determination of the presence of breast cancer in a patient, distinguishing breast cancer from other diseases, estimate the prognosis in terms of the probable outcome of the disease and the prospects of recovery, monitoring of the disease state or recurrence thereof, determination of a preferred therapeutic regimen for the patient, and assignment of antitumor therapy. One method for detecting breast cancer comprises hybridizing a polynucleotide with the messenger RNA of breast neoplasia cells. The polynucleotide comprises SEQ ID NO: 1 or a derivative thereof. A derivative of a nucleotide sequence means that the derived nucleotide sequence is substantially identical to the sequence from which it is derived, since the derived nucleotide sequence has sufficient sequence complementarity with the sequence from which it is derived, to hybridize with the messenger RNA of breast neoplasm cells under the same conditions of astringency as the sequence from which it is derived, and hybridizes with the messenger RNA of the breast neoplasia cells. The derived nucleotide sequence is not necessarily physically derived from the nucleotide sequence, but can be generated in any form that includes, for example, chemical synthesis, DNA replication, reverse transcription or transcription. To detect the presence of messenger RNA encoding mamaglobin in a breast cancer detection system, a sample is obtained from a patient. The sample may be a tissue biopsy sample or a sample of blood, plasma, serum, or the like. The sample can be treated to extract the nucleic acids contained therein. The nucleic acid resulting from the sample is subjected to gel electrophoresis or other size separation techniques. The detection involves contacting the nucleic acids, and in particular the messenger RNA of the sample with a DNA sequence that serves as a probe to form hybrid duplexes. The term "probe" refers to a structure formed of a polynucleotide which forms a hybrid structure with a target sequence, due to the complementarity of the sequence of the probe with a sequence in the target region. The detection of the resulting duplex is usually achieved by the use of labeled probes. Alternatively, the probe may not be labeled, but may be detectable by specific binding to a ligand which is labeled, either directly or indirectly. Suitable labels and methods for labeling probes and ligands are known in the art, and include, for example, radioactive labels which can be incorporated by known methods (eg nick translation or kinase treatment), biotin, fluorescent groups, groups chemiluminescents (e.g., dioxetanes, particularly dioxetanes triggered), enzymes, antibodies, and the like. When the cDNA encoding mamaglobin or a derivative thereof is used as a probe, high stringency conditions can be used to prevent the occurrence of false positives. When using sequences derived from mammaglobin, less astringent conditions can be used. The stringency of hybridization is determined by a number of factors during hybridization and during the washing procedure, including temperature, ionic strength, time and concentration of formamide. These factors are described, for example, in Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd ed., 1989). To increase the sensitivity of the detection of magelobin messenger RNA in a sample, the reverse transcription / polymerase chain reaction (RT / PCR) technique can be used to amplify the cDNA transcribed from the messenger RNA encoding mamaglobin. The RT / PCR method is well known in the art (for example, see Watson and Fleming, cited above). The RT / PCR method can be carried out in the following manner. Total cellular RNA is isolated, for example, by the standard guanidium isothiocyanate method, and the total RNA is reverse transcribed. The reverse transcription method involves the synthesis of DNA on an RNA template using a reverse transcriptase enzyme, and a 3 'end promoter. Typically, the initiator contains an oligo (dT) sequence. The cDNA produced in this way is then amplified using the PCR method and specific primers of mamaglobin (Belyavsky et al., Nucí Acid Res 17: 2919-2932, 1989; Krug and Berger, Methods in Enzymology, Academic Press, N.Y., Vol. 152, pp. 316-325, 1987, citations incorporated herein by reference). The polymerase chain reaction method is carried out using two oligonucleotide primers which are complementary to the two flanking reactions of the DNA segment to be amplified. The primers towards the 5 'end and towards the 3' end are typically 20 to 30 base pairs in length, and hybridize with the flanking regions for replication of the nucleotide sequence. Initiators that are substantially complementary to the cDNA chain to be amplified are selected. Therefore, the primers do not need to reflect the exact sequence of the template, but must be sufficiently complementary to hybridize selectively with the chain that is being amplified. Polymerization from the primers is catalyzed by a DNA polymerase in the presence of deoxynucleotide triphosphates or nucleotide analogs to produce double-stranded DNA molecules. The double chains are then separated by any denaturing method including physical, chemical or enzymatic methods. Typically, the physical denaturing method which includes heating the nucleic acid is used, typically at temperatures of about 80 ° C to 105 ° C for times ranging from about 1 to 10 minutes. The procedure is repeated according to the desired number of cycles. After amplification, the PCR product is then detected by staining with ethidium bromide (Sambrook et al., 1989, cited above). In another embodiment of the present invention, the mamaglobin cDNA sequence or derivative thereof can be used to characterize any alteration of the mamaglobin gene (i.e., gene rearrangement, gene amplification or gene deletion) in a specimen of a patient suffering from breast cancer. This provides a method by which specimens or samples of patients, which do not contain intact messenger RNA, can be examined even for changes in the structure of the genes. In one application of this technique, the mamaglobin cDNA sequence or derivative thereof is hybridized to the genomic DNA of the patient which has been isolated from the tumor, normal tissue or lymphocytes of a patient, and digested with one or more endonucleases from restriction. Using the Southern biot protocol, which is well known in the art, this test determines whether a patient or a breast tumor of the patient has a mamaglobia gene, which was deleted, rearranged or amplified. The detection of these changes can then provide important information useful for predicting the prognosis and for managing the patient. In a second application of this technique, one or more pairs of oligonucleotide primers based on the mamaglobin cDNA sequence or derivative thereof, could be used in the polymerase chain reaction to amplify segments of the mamaglobin gene from the sample of a patient. Analysis of the resulting PCR products indicates whether a particular segment of the mamaglobin gene is deleted or rearranged. This information is useful for the prognosis and management of the patient. Another method for detecting breast cancer comprises detecting the presence of the precursor and / or the secreted forms of the mamaglobin polypeptide in a sample obtained from a patient. Any method known in the art can be used to detect proteins. Such methods include, but are not limited to, immunodiffusion, immunoelectrophoresis, immunochemical methods, linker-ligand tests, immunohistochemical techniques, agglutination and complementary tests (e.g., see Basic and Clinical Immunology, Sites and Terr, eds., Appleton & Lange, Norwalk, Conn. Pp 217-262, 1991, citation incorporated herein by reference). Preferred are ligand-ligand immunoassay methods which include reacting antibodies with an epitope or mammaglobin epitopes, and competitively displacing a labeled mamaglobin polypeptide or derivative thereof.

As used herein, the term "mammaglobin polypeptide" encompasses naturally occurring mammaglobin, including non-glycosylated and glycosylated precursor forms and the secreted glycosylated form, derivatives and fragments thereof. By the term "occurring naturally" is meant a polypeptide which can be isolated from a source in nature, for example, from healthy and / or diseased organisms, and which has not been intentionally modified by man. A "mamaglobin derivative" refers to polypeptides which are formed of a segment of at least 10 amino acids and which has substantial identity with a naturally occurring portion of mammaglobin. The segment having substantial identity is preferably at least about 20 amino acids, more preferably at least about 50 amino acids, and most preferably at least about 75 amino acids. Two polypeptides have substantial identity when after optimal alignment by sequence alignment programs such as BLAST, they share at least 80% sequence identity, preferably at least 95% sequence identity, more preferably at least 95% sequence identity, most preferably at least 99% sequence identity. Preferably, the positions of the residues that are not identical differ in conservative amino acid substitutions. Conservative amino acid substitutions refer to the exchange capacity of residues having similar side chains.

For example, a group of amino acids having aliphatic side chains is that of glycine, alanine, valine, leucine and isoleucine; a group of amino acids that have aliphatic hydroxyl side chains is that of serine and threonine; a group of amino acids having side chains containing amide is that of asparagine and glutamine; a group of amino acids that have aromatic side chains is phenylalanine, tyrosine and tryptophan; a group of amino acids that have basic side chains is lysine, arginine and histidine; and a group of amino acids that have side chains containing sulfur is that of cysteine and methionine. Preferred conservative amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine and asparagine-glutamine. A polypeptide derived from mamaglobin will preferably cross-react with an anti-mamaglobin antibody, monoclonal or polyclonal, which is specific for the naturally occurring mamaglobin, or fragments thereof. As used herein, the terms "fragment" and "peptide" refer to a mamaglobin polypeptide having an amino acid sequence identical to the deduced amino acid sequence of a full-length mamaglobin cDNA (e.g., SEQ ID. NO: 1), or derivative thereof, but having an amino-terminal and / or carboxy-terminal deletion. Typically, the mamaglobin fragments or peptides are at least 3 amino acids in length. Preferably, a mamaglobin fragment or peptide is at least 6 amino acid residues in length, more preferably approximately 12 amino acid residues in length, even more preferably approximately 25 amino acid residues in length, and most preferably 50 residues in amino acids. amino acids or more. Numerous competitive and non-competitive protein binding immunoassays are well known in the art. The antibodies used in such tests may be unlabelled, for example, as used in agglutination tests, or marked for use in a wide variety of test methods. The labels that may be used include radionuclides, enzymes, fluorescent molecules, chemiluminescent molecules, cofactors or enzyme substrates, enzyme inhibitors, particles, dyes, and the like, for use in radioimmunoassay (RIA)enzyme immunoassays, for example, enzyme-linked immunosorbent assay (ELISA), immunoassays of fluorescent molecules, and the like. Polyclonal or monoclonal antibodies can be obtained for a mamaglobin polypeptide comprising a B cell epitope, for use in immunoassays by any of a number of methods well known in the art. As used herein, the term "B-cell epitope" refers to an antigenic determinant of a mamaglobin polypeptide. A B-cell epitope could comprise 3 amino acids in a spatial conformation which is unique to the epitope. In general, a B cell epitope consists of at least 5 of said amino acids. Methods for determining the spatial conformation of amino acids are well known in the art and include, for example, X-ray crystallography and two-dimensional nuclear magnetic resonance. A method for preparing antibodies for a protein is the selection and preparation of an amino acid sequence of a whole protein or part thereof, chemically synthesizing the sequence and injecting it into a suitable animal, usually a rabbit or a mouse. Methods for the preparation of a mamaglobin polypeptide include, but are not limited to, chemical synthesis, recombinant DNA techniques or isolation of biological samples. The chemical synthesis of a peptide comprising an epitope can be carried out, for example, by the classical Merrifeld method of solid-phase peptide synthesis (Merrifeld, J. Am. Chem Soc 85: 2149, 1693, citation incorporated in present as reference) or the FMOC strategy based on a rapid, automated and multiple peptide synthesis system (DuPont Company, Wilmington, DE) (Caprino and Han, J. Org Chem 37: 3404, 1972, citation incorporated herein by reference). Polyclonal antibodies can be prepared by immunizing rabbits by injecting antigens into the popliteal lymph nodes, followed by subsequent boosts at two week intervals with peritoneal injection of the antigen. The animals are bled and the sera tested against purified mamaglobin protein, usually by test ELISA Monoclonal antibodies can be prepared following the method of Milstein and Kohler by fusing splenocytes from immunized mice with continuously replicating tumor cells, such as myeloma or lymphoma cells (Milstein and Kohler Nature 256: 495-497, 1975; Gulfre and Milstein, Methods in Enzymology: Immunochemical Techniques 73: 1-46, Langone and Banatis eds., Academic Press, 1981, citations incorporated herein by reference). The hybridoma cells thus formed are then cloned by limiting dilution methods, and the supernatants tested for antibody production by ELISA or RIA tests. In this manner, monoclonal or polyclonal antibodies prepared for mammaglobin can be used to isolate and purify precursor and secreted forms of mamaglobin from cells expressing same. For example, as shown below, a polyclonal antibody generated against the predicted C-terminal 16 amino acids of the mamaglobin cDNA (Glu-Val-Phe-Met-GIn-Leu-lle-Tyr-Asp-Ser-Ser-Leu Cys-Asp-Leu-Phe, SEQ ID NO: 14) binds to the precursor and secreted forms of mamaglobin, as well as to the mamaglobin that has been synthesized in an in vitro translation system. Isolation of the mamaglobin using an anti-mamaglobin antibody can be achieved using procedures well known in the art, such as affinity chromatography. The unique ability of antibodies to specifically recognize and bind target antigens expressed by a tumor cell provides an alternative for the treatment of cancer (for a review, see LoBuglio and Saleh, Am J Med Sci 304: 214-224, 1992; Bagshawe Adv Pharmacol 24: 99-121, 1993, citations incorporated herein by reference). Thus, another aspect of the present invention provides a method to prevent the onset of breast cancer, as well as the treatment thereof in a mammal, based on the use of antibodies to mammaglobin, which has been found to be overexpressed by breast cancer cells. Antibodies specific for mamaglobin, either polyclonal or monoclonal, are produced by methods well known in the art. For example, murine or human monoclonal antibodies can be produced by hybridoma technology. Alternatively, mamaglobin, or an immunologically active derivative or fragment thereof, or an anti-idiotypic antibody, or fragment thereof, can be administered to an animal to induce the production of antibodies by B cells capable of recognizing cells. that express the mamaglobina. The antibodies or fragments thereof produced in this manner are labeled with one or more oncolytic substances such as radionuclides, toxins or cytotoxic drugs, and administered to a patient suspected of having breast cancer. The binding of the labeled antibody to the mammaglobin that is being overexpressed by the breast cancer cells will cause the death of the cancer cell.

Any of a variety of oncolytic substances known in the art can be used to produce said labeled antibodies. For example, immunotoxins can be obtained by coupling toxins from plants and bacteria to antibodies. Such toxins include, for example, ricin, diphtheria toxin and Pseudomonas exotoxin A. Drug-antibody conjugates can also be obtained in which the chemotherapeutic agents are linked to the antibody. Suitable chemotherapeutic agents for such use include, for example, tomoxifene, doxorubicin, methotrexate, chlorambucil, Vinca alkaloids, and mitomycin. In addition, radioimmunoconjugates can be obtained in which a radionuclide is stably bound to the antibody. Suitable radionuclides for obtaining radioimmunoconjugates include, for example, β-emitters such as 131l, 188Re, 186Re, 67Cu, 90Y and 47Sc.; transmitters such as 211At, 212B¡ and 212Pb; probe electron emitters such as 125l and 77Br; and fissile nuclides such as 10B. The finding that a significant percentage of breast tumors express mammaglobin is the basis of another aspect of the invention, which consists in the activation of B-cell and / or T-cell lymphocytes specific for mammaglobin with antigens from mamaglobin Accordingly, the invention provides antigens of B cells and antigens of mammaglobin Tc cells; vaccines comprising at least one B-cell antigen of mammaglobin and / or at least one T-cell antigen of mammaglobin for inducing immune responses mediated by cells and / or antibodies against tumors expressing mammaglobin, and methods for the treatment of a patient suffering from breast cancer with a tumor that expresses mammaglobin. A method according to the invention comprises administering to the patient mamaglobin-specific activated lymphocytes. Another method comprises administering a specific mammaglobin vaccine to the patient. As used herein, "mamaglobin antigen" includes naturally occurring mamaglobin polypeptides, derivatives and fragments thereof, which contain an epitope of B cells or Tc cells, recognized by B cells or Tc cells specific to mamaglobin. A mamaglobin B-cell antigen comprises a mammaglobin-specific B-cell epitope and an epitope of TH cells. The term "B cell epitope" refers to any antigen, hapten, epitope or antigenic determinant which is recognized by anti-mammaglobin immunoglobulin receptors in B cells, and is capable of inducing the production of antibodies with the appropriate help of cells TH when administered to an animal. The B cell epitope comprises an amino acid sequence of at least 4 amino acids. Preferably, the B cell epitope is between at least 6 and 25 amino acids in length, and more preferably is between about 15 and 22 amino acids in length. The amino acid sequence of the B cell epitope can be identical or substantially identical to a continuous sequence of amino acids in a naturally occurring mamaglobin fragment. Alternatively, the amino acid sequence of a B cell epitope is identical to, or substantially identical to, a discontinuous amino acid sequence that represents an assembled topographic determinant of mamaglobin. The term "TH cell epitope" refers to any antigenic determinant recognized by helper T cells by association with MHC class II molecules. The activation of the T helper cells induces the differentiation of resting B cells from mammaglobin in higher affinity cells that secrete IgG, that is, induces a secondary antibody response. It is known in the art to prepare and use immunogenic peptides containing B and TH cell determinants to produce higher titres of specific B cells producing antibodies by the intervention of helper T cells; see, for example, Cheronis et al., U.S. Patent. No. 5,573,916; Denton et al., Cancer Letters 70: 143-150 (1993); Borras-Cuesta et al., Eur. J. Immunol. 17, 1213-1215 (1987); and Good et al., Science 235: 1059-1062 (1987), citations which are incorporated herein by reference. The TH cell epitope can comprise a mamaglobin amino acid sequence or a heterologous protein. For example, Dentón et.al. describe the induction of antibody responses to mucins, which are complex glycoproteins expressed in secretory epithelia and are related to breast and other carcinomas in mice immunized with a synthetic peptide containing a B cell determinant region from the MUC mucin nucleus -1 linked to sequence 1 1 1-120 of influenza A / X-31 haemagglutinin, a known helper T-cell determinant. The TH cell epitope comprises an amino acid sequence of about 6 to about 20 amino acid residues, preferably between 8 residues and 18 residues approximately, very preferably still between 9 residues and 15 residues. An antigen of Mamaglobin Te cells comprises a Tc cell epitope and an MHC class I agrétope. The term "Te cell epitope" means any antigen, epitope or antigenic determinant which is recognized by mammaglobin-specific T cells when presented by a MHC class I molecule on the surface of a cell that presents antigens. The term "agrétope MHC class I" refers to any amino acid sequence recognized by an MHC class I molecule that allows the mamaglobin antigen to be presented to a mamaglobin-specific Te cell by the MHC class I molecule in a cell that presents antigens (APC). The Te cell epitope and the MHC class I agrétope are contained in an amino acid sequence between about 6 to about 11 amino acids, which is identical or substantially identical to the amino acid sequence of a mamaglobin fragment as presented in nature. Preferably, the sequence is 8 or 9 amino acids in length. Methods for identifying B cell and Tc epitopes for a protein antigen are known in the art. For example, the ability of mammaglobin-specific Tc cells or isolated mammaglobin-specific B cells to respond to overlapping synthetic peptides spanning secreted mammaglobin can be determined using normal immunobiology techniques. Those peptides identified as antigenic can then be modified in one or several amino acids at a time to optimize their ability to stimulate mammaglobin-specific B or T cells. B-cell epitopes can also be mapped using commercially available epitope mapping equipment that includes the selection of random peptides linked at the C-terminus for polyethylene multipirin supports, e.g., Cambridge Research Biochemicals. Alternatively, the predicted amino acid sequence of mamaglobin can be searched for sequences that conform to known binding motifs of MHC class I or MHC class II molecules. See for example, Hill et.al, Nature 360: 434 (1992), Pamer et.al, Nature 360: 852 (1992) and Hammer et.al, J. Exp. Med. 176: 1007 (1992), and Falk. et.al, Nature 357: 290-296 (991), each of which is incorporated herein by reference. For example, antigenic peptides that can be recognized by breast tumor-specific CTLs can be identified by searching for the amino acid sequence of mamaglobin for peptides linked to HLA-A2-, as described by Peoples et.al, Proc. Nati Acad. Sci. 92: 432-436 (1995), which is incorporated herein by reference. The selection of HLA-A2 as the molecule presenting the antigen is appropriate where the patient expresses HLA-A2 (approximately 50% of Caucasians express HLA-A2). The predicted HLA-A2 binding peptides can be synthesized and evaluated for antigenicity by loading the synthetic peptides into the T2 cell line, a fusion product of human B cells / T cells containing a defect in the presentation of the antigen, so that the HLA-A2 molecules on the surface of T2 cells can be loaded effectively with exogenous HLA-A2 binding peptides (Henderson, et.al, Science 255: 1264-1266 (1992) incorporated herein by reference). A normal cytotoxicity test is then performed comprising the incubation of T2 cells loaded with peptides with breast-specific CTLs derived from tumor infiltration lymphocytes (TlLs) isolated from a breast tumor expressing mammaglobin, for example, see Peoples et.al, pages 432-433 and Toso et.al, Cancer Reseach 56: 16-20 (1996), incorporated herein by reference. Antigenic mamaglobin peptides containing epitopes of Te cells can also be identified by endogenous peptides that are eluted with acid presented by HLA class I molecules on the tumor cell surface. (See, for example, Peoples et.al, supra, p. 433). The eluted peptides can be separated by any number of techniques known in the art, including HPLC fractionation. The different peptide fractions are loaded into T2 cells and the loaded T2 cells are incubated with breast tumor-specific CTLs to determine which peptides are recognized by the CTLs using normal immunobiology techniques. One use of a mamaglobin antigen in accordance with the invention is in adoptive immunotherapy. This therapy encompasses in vitro activation and expansion by a mamaglobin antigen of anti-mamaglobin antibody producing B cells and / or mammaglobin-specific cytotoxic T lymphocytes (CTLs) isolated from a patient with a tumor expressing mamaglobin. The method can also be practiced with a composition comprising B cell antigens and mamaglobin Tc cells. The activated lymphocytes are then reintroduced into the patient for adoptive immunotherapy. A mamaglobin antigen according to the invention is also useful as a component of a mammaglobin-specific vaccine. The vaccine includes an immunogenically stimulating amount of a mamaglobin antigen. As used herein, an "immunostimulatory amount" refers to that amount of antigen that is capable of stimulating the desired immune response in the recipient for the improvement or treatment of breast cancer. This amount can be determined empirically by normal procedures already known to those skilled in the art without undue experimentation. The antigen can be provided in any number of vaccine formulations that are designed to induce the desired type of immune response, for example, mediated by antibodies and / or cells. Such formulations are known in the art. See, for example, A. Lanzavecchia, Science 260: 937-944 (1993) and patent of E.U.A. No. 5,585,103 to Raychandhuri, each of which is incorporated herein by reference. Examples of vaccine formulations used to stimulate immune responses include pharmaceutically acceptable adjuvants, such as aluminum salts; emulsions of squalene or squalane and muramyl dipeptide; liposomes; and a composition comprising a stabilizing detergent, a micelle-forming agent and a biodegradable and biocompatible oil (Raychandhuri, supra). A specific mammaglobin vaccine can also include a carrier cell loaded with a mamaglobin antigen. Preferably, the carrier cell is prepared from cells displaying autologous professional antigens (APCs), such as macrophages, dendritic cells or activated B or T lymphocytes. See, for example, Lanzavecchia, supra, p. 937. Professional APCs express a B7 ligand, which binds CD28 or CTLA4 on T cells to deliver a non-specific antigen-co-stimulatory signal known as signal 2 that prevents anergy or inactivity of T cells. Therefore, the vaccine also it may include interleukin-2 or another costimulatory signal to counteract the induction of anergy. (Lanzavecchia, supra, page 938). Another formulation of a mammaglobin-specific vaccine comprises a recombinant vector that contains a nucleotide sequence that codes for expression for a mamaglobin antigen. The use of infectious agents to stimulate cytotoxic T lymphocytes is known in the art. (Raychaudhuri, supra). Chimeric vectors have been described using vaccinia viruses, poliovirus, adeno- and retro-virus, as well as bacteria, such as listeria and BCG. For example, a canarypox virus vector, ALVAC, has been shown to induce strong cellular immune responses against heterologous encoded gene products (Taylor et.al, Virology 787: 321-328 (1991), incorporated herein by reference). reference). In addition, an ALVAC recombinant expressing the human melanoma rejection antigen MZ2-E encoded by the MAGE-1 gene is able to stimulate in vitro MAGE-1 CTL activities in a TIL population derived from a breast tumor expressing MAGE-1 MRNA (Toso et.al, supra). In another scope described in the patent of E.U.A. No. 5593972 to Weiner et.al (incorporated herein by reference), a recombinant expression vector encoding an antigen of an immunogenic protein to be identified is administered directly to an individual, either in vivo, for example, a muscle cells, or the cells of an ex vivo individual together with an agent that facilitates the delivery of DNA into cells. Those skilled in the art can easily determine how to formulate a suitable vaccine to achieve the desired immune response. For example, to induce the production of anti-mamaglobin antibodies in vivo, a mammaglobin-specific vaccine comprises at least one B-cell antigen of mammaglobin comprising a B-cell epitope and an epitope of TH cells. The TH cell epitope is preferably paired with the appropriate MHC class II haplotype of the intended recipient of the vaccine. Alternatively, a TH cell epitope known to be universally recognized by humans regardless of the HLA type, such as the "universal" T cell epitope of tetanus toxoid (Panina-Bordignon et al., Eur. J. Immunol, may be used. 79: 2237 (1989), incorporated herein by reference). Preferably, the vaccine comprises a plurality of mamaglobin B cell antigens with TH epitopes recognized by MHC class II molecules of different HLA types. Another embodiment of a mammaglobin-specific vaccine induces a cell-mediated response and comprises at least one Tc antigen of mammaglobin capable of activating the Tc cells specific for mammaglobin. Preferably, the vaccine comprises several antigens of Te- cells. A specific mammaglobin vaccine can also be formulated to induce cell-mediated responses and antibodies. This embodiment comprises Te cell antigens and mamaglobin B cells. A patient with a tumor expressing mammaglobin can be treated by administering to the patient an immunostimulatory amount of a mammaglobin-specific vaccine according to the present invention. The administration of the vaccine can be by any known or normal technique. These include, but are not limited to intravenous, intraperitoneal, intramuscular, subcutaneous or intramammary injection. Preferred embodiments of the invention are described in the following examples. Other embodiments within the scope of the claims in the present invention will become apparent to those skilled in the art upon consideration of the specification or practice of the invention as described herein. The specification, together with the examples, is intended to be considered as exemplary only, since the scope and spirit of the invention are indicated by the claims subsequent to the examples. In the following examples, the cell lines were obtained from the American Type Culture Collection and cultured in minimal Dulbecco's essential medium, supplemented with 10% fetal calf serum. Tissue biopsy specimens were obtained from the Human Cooperative Tissue Network (LiVoIsi et al, Cancer 77: 1391-1394, 1993, which is incorporated herein by reference).

EXAMPLE 1 This example illustrates the isolation of mamaglobin cDNA. The total cellular RNA of the MDA-MB415 cell line was isolated using the normal guanidinium isothiocyanate method. (Belyavsky et.al, supra). This RNA was used in the PCR procedure for RACE that it uses in the Amplifinder (Clonetech) device and that follows the manufacturer's protocol. The first strand synthesis of cDNA was performed in a normal reaction containing 1 μg of RNA, 10 μM of specific mammaglobin primer D2R (5'-ATA AGA AAG AGA TGG GG3 ') (SEQ ID NO: 4), 4μl of 5X pH regulator RT (250 mM TrisCI pH8.3, 375mM Kcl, 15mM MgCl2) 2 μl of DTT at 100 mM, 1 μl of dNTPs at 10 mM and 200 units of Superscrip ™ ll reverse transcriptase (Gibco / BRL) at a reaction volume of 20 μl. The reaction was continued for 1 hour at 45 ° C and was concluded by incubation at 95 ° C for 5 minutes. The RNA was hydrolyzed with 400 μM NaOH at 65 ° C for 30 minutes and neutralized with 400 μM acetic acid. The reaction mixture was then added to 3 volumes of 6M Nal and 10 μl of treated glass waits. The beads were washed 3 times with 80% EtOH and the nucleic acid was eluted from the beads in 45 μl of water. Then, the nucleic acid was precipitated and resuspended in 10 μl of water. The first purified strand of cDNA was ligated to the anchor oligonucleotide provided by the manufacturer (SEQ ID NO: 9, 5'-CAC GAA TTC ACT ATC GAT TCT GGA ACC TTC AGA GG-3 '), using T4 RNA ligase at 27 ° for 20 hours. One-tenth of a ligation reaction was used for PCR amplification in a 50 μl reaction containing an anchor initiator at 1 μM from the manufacturer (SEQ ID NO: 10, 5'-CTG GTT CGG CCC ACC TCT GAA GGT TCC AGA ATC GAT AG-3 '), 1μM of specific primer of mamaglobin D2Rb (SEQ ID NO: 11, 5'-AAT CCG TAG TTG GTT TCT CAC C-3'), 200 μM dNTPs, 5 units of DNA Vent ™ polymerase and 1X polymerase pH regulator (10mM Kcl, 20mM TrisCI, 10mM (NH4) 2SO4, 2mM MgSO4, 0.1% Triton X-100). The reaction was incubated at 94 ° for 2 minutes and then at 94 ° for 45 seconds, 50 ° for 1 minute and 72 ° for 90 seconds for a total of 40 times. The two nested oligonucleotides specific for mamaglobin towards the 3 'end were D2R (SEQ ID NO: 4) and D2Rb (SEQ ID NO: 1 1). A mamaglobin-specific control oligonucleotide towards the 5 'end was also used, according to the manufacturer's recommendations D2F (5'-CTT GCC AGA CCT TTG GC-3') (SEQ ID NO: 12). All amplifications by PCR were performed with Vent DNA polymerase (New England Biolabs). The amplified RACE product was digested with EcoRI and ligated into the EcoRI and Smal sites of the plasmid vector pGEM7Z (Promega, Madison, Wl). All sequences were performed using the Taq DNA polymerase thermal cycle sequencing kit, according to the manufacturer's protocol (Promega). Briefly, the method used is as follows. Ten pmol of sequence specific oligonucleotide were labeled at the end with 10 pmoles of 33P-? ATP (3,000 Ci / mmol and 10 mCi / ml) using T4 polynucleotide kinase in 10 μl of a reaction for 30 minutes at 37 ° C. A polymerization reaction containing 100 ng of plasmid template, 1.5 pmol of labeled sequence initiator, and 5 units of sequence-grade Taq polymerase was created in 17 μl of the sequence pH regulator provided by the manufacturer. This reaction was divided into aliquots to a set of four reaction tubes containing the deoxynucleotide mixture provided by the manufacturer, and dideoxy-A, C, G or T. The set of four tubes was incubated at 95 ° C for 2 minutes and then at 94 ° C for 45 seconds, 45 ° C for 30 seconds and 72 ° C for 1 minute for 30 times. After completing the reactions, 3 μl of 80% bromophenol blue / formamide dye was added to each tube. The samples were heated at 70 ° C for 2 minutes and loaded onto a 6% acrylamide / 7.5M urea sequence gel and spun for 2-4 hours and constant power of 60W. The gel was dried and then exposed to Kodak XAR5 X-ray film for 2 to 24 hours. In this way, the obtained sequence was a fragment of 403 bp (SEQ ID NO: 5) as illustrated in figure 2, solid bar. In previous works, the dial sequence of DEST002 was isolated (Watson and Fleming, supra). This sequence was a fragment of 206 bp (SEQ ID NO: 6), as it appears in figure 2, open bar. The combination of information from these two sequences allowed the 503 bp total length of mammaglobin cDNA to be reduced (FIG 2).

EXAMPLE 2 This example demonstrates that the expression of mammaglobin is restricted to mammary gland tumor cells and, to a lesser degree, normal mammary gland cells. Total cellular RNA samples were isolated using the guanidinium isothiocyanate method and treated with RNase-free DNase (Promega). For analysis by RT / PCR, 1 μg of indicated total RNA was transcribed in reverse with oligo dT2? (SEQ ID NO: 13) and Superscript II reverse transcriptase (Gibco / BRL) in accordance with the manufacturer's protocol. Two hundred ng of oligo dT2? (SEQ ID NO: 13) and 1 μg total RNA were incubated at 65 ° C for 5 minutes in a volume of 10 μl. The sample was cooled in ice and 4μl of 5X RT pH buffer (250mM TrisClpH8.3, 375mM Kcl, 15mM MgCl2), 2μl of DTT at 100mM, 1μl of dNTPs at 10mM and 200 units were added. of Superscript ™ reverse transcriptase! I (Gibco / BRL). The reaction was continued for 1 hour at 45 ° C and concluded by incubation at 95 ° C for 5 minutes. One tenth of each RT reaction was subjected to PCR analysis using the D2R-specific mammaglobin primers (5'ATA AGA AG AGG TGT GG-3 ') (SEQ ID NO: 4) and d2102 (5'-CAG CGG CTT CCT TGA TCC TTG-3 ') (SEQ ID NO: 3) and normal reaction conditions for 40 cycles at 94 ° x 30 sec / 55 ° x 1 min / 72 ° x 1 min. For Northern analysis, 20 μg of total RNA was analyzed, as already described (Watson and Fleming, supra) using the full length mamaglobin cDNA probe. The integrity and equal loading of each RNA sample was examined by staining with ethidium bromide. As shown in Figure 4A, the 500 bp mammaglobin message is easily detected in the tumor specimen 2410 (tissue from which this original DEST was isolated) and to a much lesser degree in normal human breast tissue, but not in the immortalized epithelial cell of breast line B5-589, or in human lung, placenta, uterus and ovary (FIG 4A). After amplification using RT / PCR analysis, the expression of mamaglobin was not yet detected in 15 tissues evaluated (Figure 4B). Message detection of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Figure 4B) and EGF receptor message (data not illustrated) in these reactions demonstrated that the absence of expression was not due to degraded RNA or other trivial explanations. In this way, the expression of mamaglobin mRNA is relatively specific for breast tissue.

EXAMPLE 3 This example demonstrates that the mamaglobin cDNA encodes a translatable nucleotide sequence that results in an appropriately predicted molecular mass protein product. The in vitro translations were performed using the TNT ™ rabbit reticulocyte translation kit with T7 RNA polymerase (Promega) and 35S-methionine (> 100 Ci / mmol, 10 mCi / ml, Amersham) in accordance with the manufacturer's protocol . To 25 μl of TNT ™ rabbit reticulocyte lysate was added 2 μl of the reaction pH buffer prepared by the manufacturer, T7 RNA polymerase, 20 μM of amino acid mixture minus methionine, 40 μ of Ci35S-methionine (1000 Ci / mmol). and 10 mCi / ml), 40 units of ribonuclease inhibitor, 1 μg of mammaglobin / plasmid pGEM7 and enough DEPC treated with water to create a final reaction volume of 50 μl. This reaction was incubated at 30 ° C for 60 minutes. 5 μl of this reaction was removed in 20 μl of SDS gel buffer, boiled for 2 minutes, and loaded on an SDS-polyacrylamide gel at 17.5%. The rabbit reticulocyte lysate programmed with mamaglobin cDNA produced a 6kD protein, whereas that which was programmed without cDNA did not result in any protein product.

EXAMPLE 4 This example illustrates the prevalence of overexpression of mammaglobin in primary breast carcinoma. To determine the frequency of overexpression of mammaglobin in breast carcinomas, a panel of 15 stage 1 primary breast carcinomas of different histological types was examined using Northern biot hybridization with the mammaglobin cDNA probe. Samples of normal breast tissues compared in patients were also compared in tissues from two patients (Figure 6). 500 mb mamaglobin mRNA was detected in normal breast tissue and 2410 tumor in three other tumors, two of which showed little or no expression in normal tissue compared in patients (BO15 v. BO16; B022 v. B023) (FIG 6). In all, 4 of 15 (27%) of tumors examined overexpressed mamaglobin mRNA. These data indicate that overexpression of mammaglobin is not unique to a single tumor specimen, and in fact is relatively common among primary breast tumors. In addition, the fact that all tumors examined were stage 1 suggests that this dysregulation occurs relatively early in the progression of breast neoplasia.

EXAMPLE 5 The following example illustrates the detection of the mammaglobin protein using a polyclonal antimamaglobin antibody. The anti-mamaglobin polyclonal antibody was prepared by coupling a peptide corresponding to the predicted C-terminal 16 amino acids from mamaglobin cDNA (Glu-Val-Phe-Met-GIn-Leu-lle-Tyr-Asp-Ser-Ser- Leu-Cys-Asp-Leu-Phe, SEQ ID NO: 14) for Lapa Hemocyanin, and which was injected into rabbits with Freund's adjuvant. The inoculated rabbits were boosted at 3 week intervals, and at week 12, the rabbits were bled and the sera were analyzed for their ability to detect mamaglobin in a serum-free conditioned medium from cultures of the tumor cell lines. breast MDA-MB-415 and MCF-7. MDA-MB-415 have previously been identified as a cell line overexpressing the message of mamaglobin and MCF-7 has been identified as a cell line that produces non-detectable mamaglobin mRNA.

Conditioned media were grown from a 24-hour culture, and were resolved in 12% SDS acrylamide gel under reducing conditions (ie, the sample was boiled in pH regulator containing dithiothreitol (DTT) and 2- mercaptoethanol (BME) to reduce disulfide bonds), subjected to biot on a Nytran filter and analyzed by normal Western biot protocols using the antibody described previously to the C-terminal peptide as the primary antibody in this test. After the primary antibody binding, the biot was washed, and the secondary antibody (goat anti-rabbit) was added. The mamaglobin-antibody complexes were visualized by enzyme-linked chemiluminescence (ECL Western Blotting Detecting Reagent, Amersham, Arlington Heights, IL). The anti-mamaglobin polyclonal antibody detected a band with an apparent molecular weight of approximately 21 kD in the conditioned media of the MDA-MB-415 cell culture (data not shown). No bands were detected in the conditioned medium of MCF-7 cell culture (data not illustrated). In this way, according to the mRNA data, the MDA-MB-415 cells secrete mamaglobin protein, but the MCF-7 cells do not. The apparent molecular weight of the secreted mammaglobin in culture media MDA-MB-415 is greater than the molecular weight of 10.5 kDa calculated from the predicted amino acid sequence of SEQ ID NO: 2. Since almost all secreted proteins are When glycosylated, the cytosol of MDA-MB-415 cells was analyzed with the polyclonal anti-mamaglobin antibody to see if any forms of secreted mammaglobin precursors could be detected. The MDA-MG-415 cells were cultured for 24 hours in serum free media, the culture media were harvested, centrifuged and the resulting supernatant was collected. The adjoining cells were washed with phosphate buffered saline (PBS) and used with 1X pH regulator for Laemmii samples (2% SDS, 10% glycerol, DTT at 100 mM, Tris at 60 mM, pH 6.8, 0.001% bromophenol blue dye). The lysis mixture was boiled for 5 minutes and then centrifuged at 10,000 g for 5 minutes to pellet the cell debris. The cell lysate was transferred to a new tube and used to perform the Western biot analysis, as described below. The culture supernatant and the cell lysate were run on a 12% SDS acrylamide gel under reducing conditions (i.e., the samples boiled in pH buffer containing DTT and BME) and subjected to biot on a membrane PVDF using normal techniques. The biot was probed with the polyclonal antibody to the C-terminal peptide in the presence and absence of the competition peptide used to generate the antibody. The visualization of mamaglobin-antibody complexes was as described above. As can be seen in figure 7, in the absence of competition peptide (-), the conditioned media (S) have the 21 kD band representative of the secreted mamaglobin protein. The cell lysate (C) showed a prominent band at approximately 14 kD, and several bands of higher molecular weight, including one at approximately 21 kD. When the Western biot is performed in the presence of the competition peptide (+), the secreted form and the intracellular forms of mamaglobin are not visualized, indicating that these proteins contain the peptide to which the antibody was synthesized. The 14 kD band detected only in the cell lysate probably represents a precursor, or unprocessed, form of mamaglobin. Since the predicted amino acid sequence for mamaglobin has the consensus N-glycosylation site, Asn-X-Thr, located at residues 53-55 and residues 68-70 of SEQ ID NO: 2, the 21 kD form secreted and observed probably represents some glycosylated form of the protein. This hypothesis was tested by culturing cells MDA-MB-415 in the presence and absence of tunicamycin, a drug that blocks N-linked glycosylation of eukaryotic proteins. Tunicamycin was added to one or two identical cultures at 1 ug / ml and both cultures were incubated overnight for more hours. The culture media and the cell lysate from the treated and control cultures were prepared and analyzed by Western biot analysis, as described above. As shown in FIG. 8, the culture media (S) treated with detectable levels of tunicamycin (+) deficiency of secreted mamaglobin, suggesting that the secreted mamaglobin is glycosylated. Surprisingly, the cytosol form of mamaglobin cells (14 kD) was also not detectable in lysates of MDA-MB-415 cells treated with tunicamycin (right-most band). It is thought that the blocking of early glycosylation events with tunicamycin causes instability and degradation of mamaglobin precursor forms, which explains the lack of 14 kD protein detectable in the cytosol of cells treated with tunicamycin. The polyclonal antibody to the C-terminal peptide of mammaglobin has also detected the 14 kDa precursor form of mamaglobin in cell lysates from specimens of primary human breast tumors. As seen in Figure 9, the precursor form of mammaglobin is present in tumor specimen B023, but is not detectable in a normal breast tissue sample from the same patient (B022). Interestingly, some tumor samples expressing the mammaglobin transcript (ie, 087R, 014, 75A and 2410) do not contain detectable levels of mamaglobin protein, as tested by Western biot analysis. A consistent hypothesis with these data is that the expression of mamaglobin is regulated differentially in the levels of transcription and translation, and that this differential regulation is determined at the stage of tumor development. The polyclonal anti-mamaglobin antibody has also been used to search for secreted mammaglobin in breast secretions from mammary proliferation glands. Colostrum or mature milk fluid (500 ul samples) was collected by manual expression of a pregnant woman during the first and third trimesters, at birth, and on day 3, 14 and 21 after delivery. Samples were diluted with an equal volume of 2X pH buffer for laemmii samples (4% SDS, 20% glycerol, DTT at 200 mM, Tris at 120 mM, pH 6.8, 0.002% bromophenol blue dye). The diluted samples were boiled for 5 minutes and then centrifuged at 10,000 g for 5 minutes at 4 ° C to transform pellets of cell debris. The denatured samples were transferred to a new tube and stored at -20 ° C before the Western biot analysis, as described above. As shown in Figure 10, the antibody detected the secreted mammaglobin 21 kD in breast secretions that were taken as samples during pregnancy, a period of high proliferation of breast epithelial cells. However, at the beginning of lactation, a stage of breast epithelial differentiation, mamaglobin levels decreased significantly by 3 days after delivery and was no longer observed in 14 days after delivery. These results indicate that the secreted mammaglobin is related to proliferating breast epithelial cells, an observation consistent with the detection of mammaglobin secreted in human breast cancer. The reaction with the antibody to the mamaglobin peptide has also been shown for breast tumor cells by immunohistochemical staining of paraffin-fixed sections of a patient specimen with breast cancer (Figure 11). Immunohistochemical staining was performed using the antibody to the mamaglobin peptide as the primary antibody and then the mamaglobin-antibody complex was detected using the labeled anti-rabbit antibody with horseradish peroxidase and 3,3 'diamino-benzene-tetrahydrochloride ( DAB) as a substrate. The cells expressing the mamaglobin protein showed a brown stain. From these results, it is believed that the mamaglobin protein is synthesized as a precursor protein, and post-translational modifications, such as N-linked glycosylation, increase its apparent molecular weight prior to secretion; that the stability of mamaglobin precursor forms depends on the N-linked glycosylation: and that the mamaglobin protein is secreted by proliferating breast tumor cells. The detection of a mamaglobin protein is applicable in cancer diagnostics using the mammaglobin protein as an indicator of breast tumor, in the recurrent evaluation of breast tumor, in the monitoring of autologous stem cell / bone marrow transplants to contaminate the cells of tumor, and in the indication as a target of breast tumor cells for therapeutic intervention by complexes mediated by antibodies. A purified and isolated mamaglobin polypeptide is useful for accelerating antibodies against breast tumors and in the development of other tumor-specific immunotherapy regimens. In view of the foregoing, it will be noted that the various advantages of the invention are achieved, and other convenient results are obtained. Since various changes could be made to the above methods and compositions without departing from the scope of the invention, it is intended that all of the material contained in the above description and illustrated in the accompanying drawings will be construed as illustrative and not in a limiting sense .

LIST OF SEQUENCES (1) GENERAL INFORMATION: (i) APPLICANT: UNIVERSITY OF WASHINGTON (I) TITLE OF THE INVENTION: MAMAGLOBIN, A SECRETED PROTEIN OF SPECIFIC BREAST CANCER OF MAMMARY GLAND (iii) NUMBER OF SEQUENCES: 14 (iv) ADDRESS FOR CORRESPONDENCE: (A) RECIPIENT: HOWELL & HAFERKAMP, L.C. (B) STREET: 7733 FORSYTH BOULEVARD, SUITE 1400 (C) CITY: ST. LOUIS (D) STATE: MISSOURI (E) COUNTRY: UNITED STATES OF NORTH AMERICA (F) POSTAL CODE: 63105-1817 (v) LEGIBLE COMPUTER FORM: (A) TYPE OF MEDIA: Flexible Disk (B) COMPUTER: Compatible with PC IBM (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) PROGRAMS: Patentln Relay # 1.0, Version # 1.25 (vi) COMMON DATA OF THE APPLICATION: (A) REQUEST NUMBER: (B) DATE OF SUBMISSION: ( C) CLASSIFICATION: (viii) INFORMATION OF THE EMPLOYEE / AGENT: (A) NAME: HENDERSON, MELODIE W. (B) REGISTRY NUMBER: 37,848 (C) REFERENCE / CASE NUMBER: 6029-6443 (¡x) TELECOMMUNICATION INFORMATION: ( A) TELEPHONE: (314) 727-5188 (B) TELEFAX: (314) 727-6092 (2) INFORMATION FOR SEQ ID NO: 1: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 503 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear ( ii) TYPE OF MOLECULE: cDNA for mRNA (iii) HYPOTHETIC: No (iv) ANTI-SENSE: No (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 1: GACAGCGGCT TCCTTGATCC TTGCCACCCG CGACTGAACA CCGACAGCAG CAGCCTCACC 60 ATGAAGTTGC TGATGGTCCT CATGCTGGCG GCCCTCTCCC AGCACTGCTA CGCAGGCTCT 120 GGCTGCCCCT TATTGGAGAA TGTGATTTCC .AAGACAATCA ATCCACAAGT GTCTAAGACT 180 GAATACAAAG AACTTCTTCA AGAGTTCATA GACGACAATG CCACTACAAA TGCCATAGAT 240 GAATTGAAGG AATGTTTTCT TAACCAAACG GATGAAACTC TGAGCAATGT TGAGGTGTTT 300 ATGCAATTAA TATATGACAG CAGTCTTTGT GATTTATTTT AACTTTCTGC AAGACCTTTG 360 GCTCACAGAA CTGCAGGGTA TGGTGAGAAA CCAACTACGG ATTGCTGCAA ACCACACCTT 420 'J- -.- 1 -1 -.-. -l-. Lri --- AC .. ". -.... G - T.1-.Í - A 1 - oí i uru-ii -.-.-. - b ^ lp.r.l 4S0 TTTATTTTAA TAAATTGATG GCA 503 (2) INFORMATION FOR SEQ ID NO: 2: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 93 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (i) TYPE OF MOLECULE: protein (iii) HYPOTHETIC: No (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 2: Met Lvs Leu Leu; t Vax .-- eu .-. Et _- Ala Leu Ser Gln His Cys 15 Tyr Wing Gly Ser Gly Cys Pro Leu Leu Glu Asn Val lie Ser Lys Thr 20 25 30 lie Asn Pro Gln / al Ser Lys Tr.r Glu Tyr Lys Glu Leu Leu Gln Glc 35 40 45 Phe lie Asp Asp Asn Ala Thr Thr Asn Ala He Asp Glu Leu Lys Gla 50 55 60 Cys Phe Leu Asn Gln Thr Asp Clu Thr L ~ u Ser Asn Val Glu Val Phe 65 70 75 80 Met Gln Leu He Tyr Aso Ser S; : r Leu C.S Asp Leu Phe 65 90 (2) INFORMATION FOR SEQ ID NO: 3: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear ( ii) TYPE OF MOLECULE: cDNA (iii) HYPOTHETIC: No (iv) ANTI-SUIT: No (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 3: CAGCGGCTTC CTTGATCCTT G 21 (2) INFORMATION FOR SEQ ID NO: 4: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear ( I) TYPE OF MOLECULE: cDNA (iii) HYPOTHETIC: No (iv) ANTI-SUIT: No (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 4: ATAAGAAAGA GAAGGTGTGG 20 (2) INFORMATION FOR SEQ ID NO: 5: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 403 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear ( ii) TYPE OF MOLECULE: cDNA for mRNA (iii) HYPOTHETIC: No (iv) ANTI-SENSE: No (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 5: GACAGCGGCT TCCTTGATCC TTGCCACCCG CGACTGAACA CCGACAGCAG CAGCCTCACC 60 ATGAAGTTGC TGATGGTCCT CATGCTGGCG GCCCTCTCCC AGCACTGCTA CGCAGGCTCT 120 GGCTGCCCCT TATTGGAGAA TGTGATTTCC AAGACAATCA ATCCACAAGT GTCTAAGACT 180 GAATACAAAG AACTTCTTCA AGAGTTCATA GACGACAATG CCACTACAAA TGCCATAGAT 240 GAATTGAAGG AAT3TTTTCT TAACCAAACG GATGAAACTC TGAGCAATGT TGAGGTGTTT 300 ATGCAATTAA TATATGACAG CAGTCTTTGT GATTTATTTT AACTTTCTGC AAGACCTTTG 360 GCTCACAGAA CTGCAGGGTA TGGTGAGAAA CCAACTACGG ATT 403 (2) INFORMATION FOR SEQ ID NO: 6: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 206 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear ( ii) TYPE OF MOLECULE: cDNA for mRNA (iii) HYPOTHETIC: No (iv) ANTI-SENSE: No (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 6: TTTATGCAAT TAATATATGA CAGCAGTCTT TGTGATTTAT TTTAACTTTC TGCAAGACCT 60 TTGGCTCACA GAACTGCAGG GTATGGTGAG AAACCAACTA CGGATTGCTG CAAACCACAC 120 CTTCTCTTTC TTATGTCTTT TTACTACAAA CTACAAGACA ATTGTTGAAA CCTGCTATAC 180 ATGTTTA.TTT TAATAAATTG ATGGCA 206 (2) INFORMATION FOR SEQ ID NO: 7: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 95 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (iii) HYPOTHETIC: No (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 7: Met Lys Leu Vai Phe Leu Phe Leu Leu Vai Thr lie Pro laugh Cys Cys 5 10 15 Tyr Wing Ser Giy Ser Gly Cys Ser lie Leu Asp Glu Val He Arg Gly 20 25 30 Thr lie Asn Ser Thr Val Thr Leu His Asp Tyr Met Lys Leu Val Lys 35 40 45 Pro Tyr Vai Gin Asp His Phe Thr Glu Lys Wing Val Lys Gln Phe Lys 50 55 60; in: -.- s Phe _ = aso JA? D Lvs Thr Leu GH Val Gly Val "80 Met Met Glu .-. The He Pne A.sn Ser Giu Ser Cys Gln Gln Pro Ser 85 90 S5 (2) INFORMATION FOR SEQ ID NO: 8: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 91 amino acids (B) TYPE: amino acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (i) TYPE OF MOLECULE: protein (iii) HYPOTHETIC: No (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 8: Met Lys Leu Aia Val Thr Leu Thr Leu Vai Thr Leu Ala Leu Cys Cys 1 5 10 15 Being Be Wing Being Wing Glu He Cys Pro Being Phe Gln Arg Val He Glu 20 25 30 Thr Leu Leu Met Asp Thr Pro Being Ser Tyr Glu Wing Wing Met Glu Leu 35 40 45 Phe Ser Pro Asp Gln Asp Met Arg Giu Wing Gly Aia Gin Leu Lys Lys 50 55 '60 Leu Val Asp Thr Leu Pro Gln Lys Pro Arg Glu Ser He He Lys Leu 65 70 75 80 Met Glu Lys He Wing Gln Ser Ser Leu Cys Asn 85 90 (2) INFORMATION FOR SEQ ID NO: 9: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 35 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear ( ii) TYPE OF MOLECULE: cDNA (iii) HYPOTHETIC: No (iv) ANTI-SUIT: No (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 9: CACGAATTCA CTATCGATTC TGGAACCTTC AGAGG 35 (2) INFORMATION FOR SEQ ID NO: 10: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 38 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: simple (D) ) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (iii) HYPOTHETIC: No (iv) ANTI-SENSE: No (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 10: C G '? S (2) INFORMATION FOR SEQ ID NO: 1 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (iii) HYPOTHETIC: No (iv) ANTI-SENSE: No (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 1 1: AATCCGTAGT TGGTTTCTCA CC 22 (2) INFORMATION FOR SEQ ID NO: 12: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear ( ii) TYPE OF MOLECULE: cDNA (iii) HYPOTHETIC: No (iv) ANTI-SUIT: No (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 12: -. , (2) INFORMATION FOR SEQ ID NO: 13: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear ( ii) TYPE OF MOLECULE: cDNA for mRNA (iii) HYPOTHETIC: No (iv) ANTI-SENSE: No (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 13: twenty-one (2) INFORMATION FOR SEQ ID NO: 14: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 16 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (iii) HYPOTHETICAL: No (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 14: Glu Val Phe Met Gln -eu Tyr Asp Being Ser Leu Cys Asp Leu Phe 10 15

Claims (20)

NOVELTY OF THE INVENTION CLAIMS

1. A mammaglobin-specific vaccine comprising an immunogenically effective amount of at least one mamaglobin antigen comprising at least six contiguous amino acids of SEQ ID NO: 2.

2. The vaccine according to claim 1, further characterized in that said mamaglobin antigen is carried in a carrier cell.

3. The vaccine according to claim 1, further characterized in that it comprises a pharmaceutically acceptable adjuvant.

4. The vaccine according to claim 1, further characterized comprising a mixture of antigens of B cells of mamaglobin and antigens of Tc cells of mamaglobin.

5. A mammaglobin-specific vaccine comprising a recombinant vector that includes a nucleotide sequence that codes for the expression of an antigen of B cells of mammaglobin and / or a antigen of Mamaglobin-T cells.

6. The use of a specific mammaglobin vaccine comprising an immunogenically effective amount of a mamaglobin antigen to treat a patient with a tumor that expresses mamaglobin.

7. The use of said vaccine as claimed in claim 6, wherein an antigen is carried in a carrier cell.

8. The use of said vaccine as claimed in claim 5, wherein said vaccine also includes a pharmaceutically acceptable adjuvant.

9. The use of said vaccine as claimed in claim 5, wherein said mamaglobin antigen comprises an amino acid sequence that is encoded for expression by a nucleotide sequence in a recombinant vector.

10. A method for the treatment of a patient with a breast tumor expressing mammaglobin comprising tumor-isolating mamaglobin-specific lymphocytes, which activate the lymphocytes with at least one mamaglobin antigen, and administration of the activated lymphocytes to the patient.

1 1. An isolated and purified polypeptide comprising at least one mamaglobin antigen that includes at least six contiguous amino acids of SEQ ID NO: 2, further characterized in that said mamaglobin antigen is recognized by B cells and / or Te cells. specific for secreted mamaglobin polypeptide as it occurs in nature, which is glycosylated and consists of amino acids 20-93 of SEQ ID NO: 2.

12. The isolated and purified polypeptide according to claim 1, further characterized in that the mamaglobin antigen comprises at least 12 contiguous amino acids of SEQ ID NO: 2.

13. The polypeptide isolated and purified according to claim 12, further characterized in that the mamaglobin antigen comprises at least 25 contiguous amino acids of SEQ ID NO: 2.

14. The polypeptide isolated and purified according to claim 13, further characterized in that the mamaglobin antigen is glycosylated and consists of amino acids 20-93 of SEQ ID NO: 2.

15. The polypeptide isolated and purified according to claim 13, further characterized in that the mamaglobin antigen is glycosylated and consists of SEQ ID NO: 2.

16. The polypeptide isolated and purified according to claim 1, further characterized in that the mamaglobin antigen is a B-cell antigen of mammaglobin that induces in vitro activation and expansion of B cells of a patient with breast cancer.

17. The polypeptide isolated and purified according to claim 16, further characterized in that the B-cell antigen of mamaglobin includes a TH cell epitope of a heterologous protein.

18. The polypeptide isolated and purified according to claim 11, further characterized in that the mamaglobin antigen is an antigen of Mamaglobin Te cells that induces the in vitro activation and expansion of Te cells from a patient with breast cancer.

19. - The polypeptide isolated and purified according to claim 18, further characterized in that the antigen of mamaglobin Te cells comprises 8 or 9 contiguous amino acids of SEQ ID NO: 2.

20. The polypeptide isolated and purified according to claim 11, further characterized in that it comprises a B-cell antigen from mammaglobin and a magenlobin-T cell antigen.