JP2008289472A - Composition and method for treating and diagnosing breast cancer - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a composition and a method for detecting and treating breast cancer. <P>SOLUTION: A nucleotide sequence which is preferentially expressed in breast tumor tissue, a polypeptide encoded by the nucleotide sequence, and a vaccine and a pharmaceutical composition each containing such a compound, are provided. The compound is usable for preventing and treating breast cancer. The polypeptide is also usable for the production of antibodies, which are useful for diagnosing and monitoring the progression of breast cancer in a relevant patient. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates generally to breast cancer detection and therapy. The present invention more particularly relates to nucleotide sequences that are preferentially expressed in breast tumor tissue and polypeptides encoded by such nucleotide sequences. Nucleotide sequences and polypeptides can be used in vaccines and pharmaceutical compositions for the prevention and treatment of breast cancer. The polypeptides can also be used for the production of compounds, such as antibodies, that are useful for the diagnosis and monitoring of progression of breast cancer in patients.

  Breast cancer is a significant health problem for women in the United States and around the world. Although progress has been made in disease detection and treatment, breast cancer is still the second leading cause of cancer-related death in women, affecting more than 180,000 women in the United States each year. For North American women, there is currently one in eight cases of lifetime breast cancer development.

  Vaccines or other universally successful methods for the prevention or treatment of breast cancer are not currently available. Disease management is currently a combination of aggressive treatment that can include early diagnosis (by routine breast screening procedures) and one or more different treatments (eg, surgery, radiation therapy, chemotherapy, and hormone therapy). It depends. A series of treatments for a particular breast cancer are often selected based on various prognostic parameters, including analysis of specific tumor markers. See, for example, Porter-Jordan and Lippman, Breast Cancer 8: 73-100 (1994). However, the use of established markers often results in difficult interpretation and the high mortality seen in breast cancer patients indicates that improvements in the treatment, diagnosis, and prevention of the disease are needed.

  Accordingly, there is a need in the art for improved methods for the treatment and diagnosis of breast cancer. The present invention fulfills these needs and further provides other related advantages.

Briefly stated, the present invention provides compositions and methods for the diagnosis and treatment of breast cancer. In one aspect, an isolated DNA molecule is provided, which is
(a) a nucleotide sequence that is preferentially expressed in breast cancer tissue compared to normal tissue;
(b) one or more than 20% (preferably not more than 5%) of the nucleotide positions so that the antigenic and / or immunogenic properties of the polypeptide encoded by the nucleotide sequence are retained. A variant of such a sequence comprising nucleotide substitutions, deletions, insertions and / or modifications; or (c) a nucleotide sequence encoding an epitope of a polypeptide encoded by at least one of the above sequences . In one embodiment, the isolated DNA molecule comprises the human endogenous retroviral sequence set forth in SEQ ID NO: 1. In other embodiments, the isolated DNA molecules are SEQ ID NOs: 3-26, 28-77, 142, 143, 146-152, 154-166, 168-176, 178-192, 194-198, 200- 204,206,207,209-214,216,218,219,221-240,243-245,247,250,251,253,255,257-266,268,269,271-273,275,276, It includes the nucleotide sequence set forth in any one of 278, 280, 281, 284, 288, and 291-297.

  In a related embodiment, the isolated DNA molecule encodes an epitope of a polypeptide, wherein the polypeptide is encoded by the following nucleotide sequence: (a) SEQ ID NO: 1, 3-26, 28 -77, 142, 143, 146-152, 154-166, 168-176, 178-192, 194-198, 200-204, 206, 207, 209-214, 216, 218, 219, 221-240, 243 ~ 245, 247, 250, 251, 253, 255, 257-266, 268, 269, 271-273, 275, 276, 278, 280, 281, 284, 288, and 291-297 A nucleotide sequence that hybridizes to the sequence under stringent conditions; and (b) SEQ ID NOs: 1, 3-26, 28-77, 142, 143, 146-152, 154-166, 168-176, 178-192, 194-198, 200-204, 206, 207, 209-214, 216, 218, 219, 221-240, 243-245, 247, 250, 251, 253, 255, 257-266, 268, 269, 271- 273, 275, 276, 278, 280, 281, 284, 288, and 291-2 A nucleotide sequence that is at least 80% identical to the sequence shown in any one of 97; and wherein the RNA corresponding to said nucleotide sequence is expressed at a higher level in human breast mass tissue than in normal breast tissue.

  In another embodiment, the present invention provides an isolated DNA molecule that encodes an epitope of a polypeptide. This polypeptide is encoded by: (a) a nucleotide sequence transcribed from the sequence of SEQ ID NO: 141; or (b) retains the antigenic and / or immunogenic properties of the polypeptide encoded by the nucleotide sequence. A variant of the above nucleotide sequence comprising one or more nucleotide substitutions, deletions, insertions and / or modifications not exceeding 20% of the nucleotide positions. Isolated DNA and RNA molecules comprising nucleotide sequences complementary to the above DNA molecules are also provided.

  In a related aspect, the present invention provides a recombinant expression vector comprising the DNA molecule described above and a host cell transformed or transfected with such an expression vector.

  In further aspects, polypeptides comprising amino acid sequences encoded by the DNA molecules described above and monoclonal antibodies that bind to such polypeptides are provided.

  In yet another aspect, a method is provided for determining the presence of breast cancer in a patient. In one embodiment, the method includes detecting the above-described polypeptide in a biological sample. In another embodiment, the method includes detecting an RNA molecule encoding the above-described polypeptide in a biological sample. In yet another embodiment, the method comprises (a) intradermal injection of the above polypeptide into a patient; and (b) detecting an immune response in the patient's skin and thereby determining the presence of the patient's breast cancer. Detecting. In a further embodiment, the present invention provides a method for determining the presence of breast cancer in a patient as described above, wherein said polypeptide is SEQ ID NO: 78-86, 144, 145, 153, 167, 177, 193, 199, 205, 208, 215, 217, 220, 241, 242, 246, 248, 249, 252, 256, 267, 270, 274, 277, 279, 282, 283, 285-287, 289, 290, And a nucleotide sequence selected from the group consisting of sequences that hybridize to them under stringent conditions.

In a related aspect, diagnostic kits useful for breast cancer determination are provided. This diagnostic kit generally comprises one or more of the above monoclonal antibodies, or SEQ ID NOs: 78-86, 144, 145, 153, 167, 177, 193, 199, 205, 208, 215, 217, 220, 241, 242 and 246, 248, 249, 252, 256, 267, 270, 274, 277, 279, 282, 283, 285 to 287, 289, 290 according to a nucleotide sequence selected from the group consisting of One or more monoclonal antibodies that bind to the encoded polypeptide, as well as a detection reagent.

  In a related aspect, the diagnostic kit comprises a first polymerase chain reaction primer and a second polymerase chain reaction primer, at least one of these primers being specific for the RNA molecule described herein. In one embodiment, at least one of these primers is at least about 10 contiguous nucleotides of the above RNA molecule, or SEQ ID NOs: 78-86, 144, 145, 153, 167, 177, 193, 199, 205. 208, 215, 217, 220, 241, 242, 246, 248, 249, 252, 256, 267, 270, 274, 277, 279, 282, 283, 285-287, 289, and 290 RNA molecules encoding a polypeptide encoded by a nucleotide sequence to be encoded.

  In another related aspect, the diagnostic kit comprises at least one oligonucleotide probe, which probe is specific for the DNA molecules described herein. In one embodiment, the probe comprises at least about 15 contiguous nucleotides of the above DNA molecule, or SEQ ID NOs: 78-86, 144, 145, 153, 167, 177, 193, 199, 205, 208, 215. 217, 220, 241, 242, 246, 248, 249, 252, 256, 267, 270, 274, 277, 279, 282, 283, 285-287, 289, and 290 including.

  In another related aspect, the invention provides a method for monitoring the progression of breast cancer in a patient. In one embodiment, the method comprises the following steps: (a) detecting the amount of the above-described polypeptide in the biological sample at a first time point; (b) at a subsequent time point, step (a) And (c) comparing the amount of polypeptide detected in steps (a) and (b), thereby monitoring the progression of breast cancer in the patient. In another embodiment, the method comprises the steps of: (a) detecting the amount of RNA molecule encoding the above polypeptide in a biological sample at a first time point; (b) at a subsequent time point (a And (c) comparing the amount of RNA molecules detected in step (a) and step (b), thereby monitoring the progression of breast cancer in the patient. In yet another embodiment, the invention provides a method for monitoring the progression of breast cancer in a patient as described above, wherein the polypeptide is SEQ ID NO: 78-86, 144, 145, 153, 167, 177, 193, 199, 205, 208, 215, 217, 220, 241, 242, 246, 248, 249, 252, 256, 267, 270, 274, 277, 279, 282, 283, 285-287, It is encoded by a nucleotide sequence selected from the group consisting of 289, 290, and sequences that hybridize to them under stringent conditions.

  In still other aspects, pharmaceutical compositions comprising the above polypeptides in combination with a physiologically acceptable carrier, and vaccines comprising the above polypeptides in combination with an immune response enhancer or adjuvant are provided. In yet another aspect, the invention provides SEQ ID NOs: 78-86, 144, 145, 153, 167, 177, 193, 199, 205, 208, 215, 217, 220, 241, 242 and 246, 248, 249 , 252, 256, 267, 270, 274, 277, 279, 282, 283, 285-287, 289, 290, and a nucleotide sequence selected from the group consisting of sequences that hybridize under stringent conditions Pharmaceutical compositions and vaccines comprising the encoded polypeptide are provided.

  In a related aspect, the invention provides a method for inhibiting the development of breast cancer in a patient, the method comprising administering to the patient a pharmaceutical composition or vaccine as described above.

  These and other aspects of the invention will be apparent upon reference to the following detailed description and attached drawings. From this, all references disclosed herein are incorporated by reference in their entirety as if each was individually incorporated.

As noted above, the present invention generally relates to compositions and methods for the diagnosis, monitoring, and treatment of breast cancer. The compositions described herein include polypeptides, nucleic acid sequences, and antibodies. The polypeptides of the invention generally comprise at least a portion of a protein that is expressed at a higher level in human breast mass tissue than in normal breast tissue (ie, the level of RNA encoding the polypeptide is increased in lung tumor tissue). At least twice as high). Such polypeptides are referred to herein as breast mass-specific polypeptides, and the cDNA molecules that encode such polypeptides are termed breast mass-specific cDNAs. The nucleic acid sequences of the invention generally include DNA or RNA sequences that encode all or part of the above-described polypeptides or that are complementary to such sequences. The antibody is generally an immune system protein, or a fragment thereof, that can bind to a portion of the polypeptide described above.
Antibodies include production of monoclonal antibodies via transfection of antibody genes into a suitable bacterial or mammalian cell host as described herein or to allow the production of recombinant antibodies. Can be produced by cell culture techniques.

  Polypeptides within the scope of the present invention include polypeptides (and epitopes thereof) encoded by human endogenous retroviral sequences, such as the sequence designated B18Ag1 (FIG. 5 and SEQ ID NO: 1). It is not limited to. Polypeptides encoded by other sequences within the retroviral genome including B18Ag1 (SEQ ID NO: 141) are also within the scope of the invention. Such sequences include, but are not limited to, the sequences shown in SEQ ID NO: 3 to SEQ ID NO: 10. B18Ag1 is homologous to the gag p30 gene of the endogenous human retroviral element S71 as described by Werner et al., Virology 174: 225-238 (1990), and with about 30 other retroviral gag genes. Also shows homology. As discussed in more detail below, the present invention also includes a number of additional breast mass-specific polypeptides (eg, SEQ ID NOs: 11-26, 28-77, 142, 143, 146-152, 154-166, 168-176, 178-192, 194-198, 200-204, 206, 207, 209-214, 216, 218, 219, 221-240, 243-245, 247, 250, 251, 253, 255, 257- 266, 268, 269, 271-273, 275, 276, 278, 280, 281, 284, 288, and the polypeptide encoded by the nucleotide sequence shown in 291-297). As used herein, the term “polypeptide” encompasses an amino acid chain of any length, including a full-length protein comprising the sequences set forth herein. A polypeptide comprising an epitope of a protein comprising a sequence described herein can consist entirely of that epitope or can comprise additional sequences. Additional sequences can be derived from native proteins or can be heterologous, and such sequences can (but need not have) immunogenic or antigenic properties.

  An “epitope” as used herein is a portion of a polypeptide that is recognized (ie, specifically bound) by a B cell and / or T cell surface antigen receptor. Epitopes are generally used using well-known techniques (eg, techniques summarized in Paul, Fundamental Immunology, 3rd edition, 243-247 (Raven Press, 1993) and references cited therein). Can be identified. Such techniques include screening polypeptides from native polypeptides for the ability to react with antigen-specific antisera and / or T cell lines or clones. Polypeptide epitopes are those moieties that react with such antisera and / or T cells at a level similar to that of full-length polypeptides (eg, in ELISA and / or T cell reactivity assays). Such screening can generally be performed using methods well known to those skilled in the art (eg, the method described in Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988). B cell and T cell epitopes can also be estimated via computer analysis. Polypeptides comprising an epitope of a polypeptide that is preferentially expressed in tumor tissue (with or without additional amino acid sequence) are within the scope of the invention.

  The compositions and methods of the invention also include variants of the above-described polypeptides and nucleic acid sequences that encode such polypeptides. As used herein, a polypeptide “variant” is a polypeptide that differs from the native polypeptide in substitutions and / or modifications such that the antigenicity and / or immunogenic properties of the polypeptide are retained. Such variants generally modify one of the above polypeptide sequences and evaluate the reactivity of the modified polypeptide with antisera and / or T cells as described above. Can be identified. Nucleic acid variants can include one or more substitutions, deletions, insertions and / or modifications such that the antigenicity and / or immunogenic properties of the encoded polypeptide are retained. One preferred variant of the polypeptides described herein is a variant that contains nucleotide substitutions, deletions, insertions and / or modifications at no more than 20% nucleotide positions.

  Preferably, the variant contains conservative substitutions. A “conservative substitution” is one in which an amino acid is replaced with another amino acid having similar properties, so that one skilled in the art of peptide chemistry will be substantially aware of the secondary structure and hydropathic properties of the polypeptide. Predict that it will not change. In general, the following groups of amino acids represent conservative changes: (1) ala, pro, gly, glu, asp, gln, asn, ser, thr; (2) cys, ser, tyr, thr; (3 ) val, ile, leu, met, ala, phe; (4) lys, arg, his; and (5) phe, tyr, trp, his.

  Variants can also (or alternatively) be modified, for example, by deletion or addition of amino acids that have minimal effect on the immunogenic or antigenic properties, secondary structure and hydropathic properties of the polypeptide. . For example, the polypeptide can be linked to a signal (or leader) sequence at the N-terminus of the protein that directs protein movement simultaneously with or after translation. Polypeptides can also be attached to linkers or other sequences for ease of synthesis, purification or identification of the polypeptide (eg, poly-His) or to enhance binding of the polypeptide to a solid support. Can be done. For example, the polypeptide can be bound to an immunoglobulin Fc region.

In general, nucleotide sequences encoding all or part of the polypeptides described herein can be prepared using any of a number of techniques. For example, cDNA molecules encoding such polypeptides can be cloned based on breast tumor specific expression of the corresponding mRNA using differential display PCR. This technique compares an amplification product from an RNA template prepared from normal tissue with an amplification product from an RNA template prepared from breast mass tissue. cDNA can be prepared by reverse transcription of RNA using (dT) ₁₂ AG primers. Following amplification of the cDNA with random primers, the band corresponding to the amplification product specific for tumor RNA was excised from the silver stained gel and the appropriate vector (eg, T-vector, Novagen, Madison, WI). ) Can be subcloned into. Nucleotide sequences encoding all or part of a breast mass-specific polypeptide disclosed herein are amplified from cDNA prepared as described above using random primers set forth in SEQ ID NOs: 87-125. obtain.

  Alternatively, a gene encoding a polypeptide (or portion thereof) described herein can be amplified from human genomic DNA or from breast mass cDNA via the polymerase chain reaction. For this approach, a B18Ag1 sequence specific primer can be designed based on the sequence provided in SEQ ID NO: 1 and can be purchased or synthesized. One suitable primer pair for amplification from breast tumor cDNA is (5'ATG GCT ATT TTC GGG GGC TGA CA) (SEQ ID NO: 126), and (5'CCG GTA TCT CCT CGT GGG TAT T) (sequence No. 127). The amplified portion of B18Ag1 is then ligated to human genomic DNA using well-known techniques (eg, those described in Sambrook et al., Molecular Cloning: A Laoratory Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor, NY (1989)). It can be used to isolate a full-length gene from a rally or from a breast mass cDNA library. Other sequences in the retroviral genome of which B18Ag1 is a part can be similarly prepared by screening a human genomic library using a B18Ag1 specific sequence as a probe. The nucleotide set forth in SEQ ID NO: 141 from the retroviral genome, which is then translated into protein, is suitable for cloning the corresponding cDNA, guessing the open reading frame, and the vector containing the viral promoter (eg, T7). It can be determined by cloning of cDNA. The resulting construct can be used in a translation reaction using techniques known to those skilled in the art to identify the nucleotide sequence that yields the expressed protein. Similarly, primers specific for the remaining breast mass-specific polypeptides described herein are also based on the nucleotide sequences provided in SEQ ID NO: 11-SEQ ID NO: 86, and SEQ ID NO: 142-SEQ ID NO: 297. Can be designed.

  The recombinant polypeptide encoded by the above DNA sequence can be readily prepared from the DNA sequence. For example, supernatant from a suitable host / vector system that secretes the recombinant protein or polypeptide into the culture medium can be first concentrated using a commercially available filter. Following concentration, the concentrate can be applied to a suitable purification matrix, such as an affinity matrix or ion exchange resin. Finally, one or more reverse phase HPLC steps can be used for further purification of the recombinant polypeptide.

  In general, any of a variety of expression vectors known to those of skill in the art can be used to express a recombinant polypeptide of the invention. Expression can be achieved in any appropriate host cell that has been transformed or transfected with an expression vector comprising a DNA molecule encoding the recombinant polypeptide. Suitable host cells include prokaryotic cells, yeast cells, and higher eukaryotic cells. Preferably, the host cell used is E. coli. coli, yeast, or a mammalian cell line (eg, COS or CHO).

  Such techniques can also be used to prepare polypeptides comprising epitopes or variants of native proteins. For example, native polypeptide variants can generally be prepared using standard mutagenesis techniques (eg, oligonucleotide-directed site-directed mutagenesis), and sections of the DNA sequence can be It can be removed to allow preparation of the peptide. Portions and other variants having less than about 100 amino acids, and generally less than about 50 amino acids, can also be made by synthetic means using techniques well known to those skilled in the art. For example, such polypeptides can be synthesized using any commercially available solid phase technique (eg, the Merrifield solid phase synthesis method, in which amino acids are added sequentially to an extending amino acid chain. Merrifield, J Am.Chem.Soc.85: 2149-2146 (1963) Equipment for automated synthesis of polypeptides is commercially available from suppliers such as Perkin Elmer / Applied BioSystems Division, Foster City, CA. And can be operated according to the manufacturer's instructions.

  In a specific embodiment, the polypeptide of the invention comprises SEQ ID NO: 1, 3-26, 28-77, 142, 143, 146-152, 154-166, 168-176, 178-192, 194-198, 200-204, 206, 207, 209-214, 216, 218, 219, 221-240, 243-245, 247, 250, 251, 253, 255, 257-266, 268, 269, 271-273, 275, Amino acid sequence encoded by a DNA molecule having the sequence shown in any one of 276, 278, 280, 281, 284, 288, and 291-297, one or more nucleotides at no more than 20% nucleotide positions It includes variants of such polypeptides encoded by DNA molecules containing substitutions, deletions, insertions, and / or modifications, as well as epitopes of such polypeptides. Polypeptides within the scope of the present invention can also be obtained under stringent conditions SEQ ID NOs: 1, 3-26, 28-77, 142, 143, 146-152, 154-166, 168-176, 178-192, 194. ~ 198,200 ~ 204,206,207,209 ~ 214,216,218,219,221 ~ 240,243 ~ 245,247,250,251,253,255,257 ~ 266,268,269,271 ~ 273 275, 276, 278, 280, 281, 284, 288, and a polypeptide encoded by a DNA sequence that hybridizes to a DNA molecule having the sequence shown in any one of 291 to 297 (and its epitope) Including. Here, the DNA sequence is at least 80% identical over the entire sequence to the sequence shown, and where the RNA corresponding to the nucleotide sequence is expressed at higher levels in human breast mass tissue than in normal breast tissue. The As used herein, “stringent conditions” include pre-washing in a solution of 6 × SSC, 0.2% SDS; 6 × SSC, 0.2% SDS, overnight hybridization at 65 ° C .; 2 times of washing in 1 × SSC, 0.1% SDS at 65 ° C. for 30 minutes each and 2 times washing in 0.2 × SSC, 0.1% SDS at 65 ° C. for 30 minutes each. DNA molecules according to the present invention include molecules encoding any of the above polypeptides.

  In another aspect of the invention, antibodies are provided. Such antibodies can be prepared by any of a variety of techniques known to those skilled in the art. See, for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. In one such technique, an immunogen comprising a polypeptide is first injected into a wide variety of any mammal (eg, mouse, rat, rabbit, sheep, or goat). In this step, the polypeptides of the invention can serve as unmodified immunogens. Alternatively, particularly for relatively short polypeptides, a superior immune response can be elicited when the polypeptide is conjugated to a carrier protein (eg, bovine serum albumin or keyhole limpet hemocyanin). The immunogen is preferably injected into the animal host according to a predetermined schedule that includes one or more booster immunizations, and the animals are bled regularly. Polyclonal antibodies specific for the polypeptide can then be purified from such antisera, for example, by affinity chromatography using the polypeptide bound to a suitable solid support.

  Monoclonal antibodies specific for the antigenic polypeptide of interest are described, for example, in Kohler and Milstein, Eur. J. Immunol. 6: 511-519 (1976) as well as improved methods thereof. Briefly, these methods involve the preparation of immortal cell lines that can produce antibodies with the desired specificity (ie, reactivity with the polypeptide of interest). Such cell lines can be produced, for example, from spleen cells obtained from animals immunized as described above. The spleen cells are then immortalized by, for example, fusion with a myeloma cell fusion partner (preferably a myeloma cell fusion partner that is syngeneic with the immunized animal). Various fusion techniques can be used. For example, spleen cells and myeloma cells can be combined with a nonionic surfactant for several minutes and then seeded at low density on selective media that supports hybrid cell growth but does not support myeloma cell growth. . A preferred selection technique uses HAT (hypoxanthine, aminopterin, thymidine) selection. Hybrid colonies are usually observed after a sufficient period of about 1-2 weeks. A single colony is selected and the culture supernatant is tested for binding activity against the polypeptide. Hybridomas with high reactivity and specificity are preferred.

  Monoclonal antibodies can be isolated from the supernatant of growing hybridoma colonies. In addition, various techniques (eg, injection of hybridoma cell lines into the peritoneal cavity of a suitable vertebrate host such as a mouse) can be used to enhance the yield. The monoclonal antibody can then be recovered from ascites or blood. Contaminants can be removed from the antibody by conventional techniques such as chromatography, gel filtration, sedimentation, and extraction. The polypeptides of the present invention can be used, for example, in a purification process in an affinity chromatography step.

  For example, the antibody can be used in a method for detecting breast cancer in a patient. Such methods include using antibodies to detect the presence or absence of a breast mass-specific polypeptide described herein in an appropriate biological sample. Suitable biological samples for use herein include tumor or normal tissue biopsies obtained from patients, mastectomy, blood, lymph node, serum, or urine samples, or other tissues, or homogenates or Including their extracts.

  There are a variety of assay formats known to those of skill in the art for the use of antibodies to detect polypeptide markers in a sample. See, for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. For example, the assay can be performed in a Western blot format. Here, a protein preparation from a biological sample is subjected to gel electrophoresis, transferred to a suitable membrane and reacted with an antibody. The presence of the antibody on the membrane can then be detected using a suitable detection reagent as described below.

  In another embodiment, the assay involves the use of an antibody immobilized on a solid support to bind the polypeptide and remove it from the rest of the sample. The bound polypeptide can then be detected using a secondary antibody or reagent having a reporter group. Alternatively, competition assays can be used in which the polypeptide is labeled with a reporter group and allowed to bind to the immobilized antibody after incubation of the antibody with the sample. The extent to which the sample components inhibit binding of labeled polypeptide to the antibody indicates the reactivity of the sample with the immobilized antibody and, as a result, indicates the concentration of the polypeptide in the sample.

The solid support can be any material known to those of skill in the art to which antibodies can be attached.
For example, the solid support can be a test well of a microtiter plate or a nitrocellulose filter, or other suitable membrane. Alternatively, the support can be a bead or disk (eg, glass, glass fiber, latex or a plastic material (eg, polystyrene or polyvinyl chloride)). The support can also be a magnetic particle or fiber optic sensor (eg, disclosed in US Pat. No. 5,359,681).

  The antibody can be immobilized on a solid support using a variety of techniques known in the art and described in detail in the patent and scientific literature. In the context of the present invention, the term “immobilization” can be a non-covalent association (eg adsorption) and covalent attachment (direct linkage between an antigen and a functional group on a support or by a cross-linking agent. Both). Fixation by adsorption to a well or membrane of a microtiter plate is preferred. In such cases, adsorption can be accomplished by contacting the antibody in a suitable buffer with the support for a suitable time. The contact time varies with temperature, but is typically between about 1 hour and 1 day. In general, contacting a well of a plastic microtiter plate (eg, polystyrene or polyvinyl chloride) with an amount of antibody in the range of about 10 ng to about 1 μg, and preferably about 100-200 ng, results in an appropriate amount of poly Sufficient to immobilize the peptide.

  Covalent attachment of the antibody to the solid support generally also involves first combining the support with a bifunctional reagent that reacts with both the support and a functional group (eg, a hydroxyl or amino group) on the antibody. It can be achieved by reacting. For example, the antibody can be covalently attached to a support having a suitable polymer coat using benzoquinone or by condensation of an aldehyde group on the support with an amine and active hydrogen on a binding partner (eg, Pierce). (See Immunotechnology Catalog and Handbook (1991) A12-A13).

  In certain embodiments, the assay for detection of the polypeptide in the sample is a two-antibody sandwich assay. This assay involves first contacting an antibody immobilized on a solid support (usually a well of a microtiter plate) and a biological sample so that the polypeptide in the sample binds to the immobilized antibody. Can be performed. The unbound sample is then removed from the immobilized polypeptide-antibody complex and a secondary antibody (containing a reporter group) that can bind to a different site on the polypeptide is added. The amount of secondary antibody that remains bound to the solid support is then determined using a method appropriate for the particular reporter group.

More particularly, once the antibody is immobilized on the support as described above, the remaining protein binding sites on the support are typically blocked. Any suitable blocking agent (eg, bovine serum albumin or Tween 20 ^™ (Sigma Chemical Co., St. Louis, MO)) is known to those skilled in the art. The immobilized antibody is then incubated with the sample and the polypeptide is allowed to bind to the antibody. The sample can be diluted with a suitable diluent (eg, phosphate buffered saline (PBS)) prior to incubation. In general, a suitable contact time (ie, incubation time) is a time sufficient to detect the presence of the polypeptide in a sample obtained from an individual having breast cancer. Preferably, the contact time is sufficient to achieve a level of binding that is at least 95% of the level of binding achieved at equilibrium between the bound and unbound polypeptides. One skilled in the art understands that the time required to achieve equilibrium is readily determined by assaying the level of binding that occurs over a period of time. At room temperature, an incubation time of about 30 minutes is generally sufficient.

Unbound sample can then be removed by washing the solid support with a suitable buffer (eg, PBS containing 0.1% Tween 20 ^™ ). A secondary antibody having a reporter group can then be added to the solid support. Preferred reporter groups include enzymes (eg, horseradish peroxidase), substrates, cofactors, inhibitors, dyes, radionuclides, luminescent groups, fluorescent groups, and biotin. Binding of the antibody to the reporter group can be accomplished using standard methods known to those skilled in the art.

  The secondary antibody is then incubated with the immobilized antibody-polypeptide complex for a time sufficient to detect the bound polypeptide. The appropriate length of time can generally be determined by assaying the level of binding that occurs over a given time. The unbound secondary antibody is then removed and the bound secondary antibody is detected using a reporter group. The method used to detect the reporter group depends on the nature of the reporter group. For radioactive groups, scintillation counting or autoradiographic methods are generally appropriate. Spectroscopy can be used to detect dyes, luminescent groups and fluorescent groups. Biotin can be detected using avidin coupled to different reporter groups (usually radioactive or fluorescent groups or enzymes). Enzyme reporter groups can generally be detected by addition of a substrate (generally for a specific time) followed by spectroscopic or other analysis of the reaction product.

  To determine the presence or absence of breast cancer, the signal detected from the reporter group that remains bound to the solid support generally results in a predetermined cut-off value established from non-tumor tissue. Compared with the corresponding signal. In one preferred embodiment, the cutoff value is the average signal obtained when the immobilized antibody is incubated with a sample from a patient who does not have breast cancer. In general, a sample that produces a signal that exceeds 3 standard deviations of the predetermined cut-off value can be considered positive for breast cancer. In another preferred embodiment, the cutoff value is determined using a Receiver Operator Curve according to the method of Sackett et al., Clinical Epidemiology: A Basic Science for Clinical Medicine, pages 106-7, (Little Brown and Co, 1985). Briefly, in this embodiment, the cutoff value is the true positive rate (ie sensitivity) and false positive rate (100% specificity) corresponding to each possible cutoff value for diagnostic test results. Can be determined by plotting pairs of The cutoff value closest to the upper left corner of the plot (i.e., the value surrounding the largest area) is the most accurate cutoff value, and the sample that produces a signal higher than the cutoff value determined by this method is It can be considered positive. Alternatively, the cutoff value can be shifted to the left along the plot to minimize the false positive rate or to the right to minimize the false negative rate. In general, a sample that produces a signal that is higher than the cut-off value determined by this method is considered positive for breast cancer.

  In related embodiments, the assay is performed in a flow-through test or strip test format. Here, the antibody is immobilized on a membrane (for example, nitrocellulose). In the flow-through test, the polypeptide in the sample binds to the immobilized antibody as the sample passes through the membrane. The labeled secondary antibody then binds to the antibody-polypeptide complex as the solution containing the secondary antibody flows through the membrane. Detection of bound secondary antibody can then be performed as described above. In the strip test format, one end of the membrane to which the antibody is bound is immersed in a solution containing the sample. The sample moves along the membrane through the area containing the secondary antibody and into the area of the immobilized antibody. The concentration of secondary antibody in the range of fixed antibodies indicates the presence of breast cancer. Typically, the concentration of secondary antibody at this site produces a pattern (eg, line) that can be read visually. The absence of such a pattern indicates a negative result. In general, the amount of antibody immobilized on the membrane is visible when the biological sample contains a sufficient level of polypeptide to produce a positive signal in the two-antibody sandwich assay of the format discussed above. Is selected so as to produce a pattern that can be discriminated automatically. Preferably, the amount of antibody immobilized on the membrane ranges from about 25 ng to about 1 μg, and more preferably from about 50 ng to about 1 μg. Such a test can typically be performed using a very small amount of biological sample.

  The presence or absence of breast cancer in a patient also causes the breast mass-specific polypeptide described herein in a biological sample (eg, a patient-derived biopsy, mastectomy, and / or blood sample). The level of encoding mRNA can be determined by evaluating against a predetermined cutoff value. Such assessment can be accomplished using any of a variety of methods known to those skilled in the art, such as in situ hybridization and amplification by polymerase chain reaction.

  For example, the polymerase chain reaction can be used to amplify sequences from cDNA prepared from RNA isolated from one of the above biological samples. Sequence specific primers for use in such amplification can be designed and purchased based on the sequence provided in any one of SEQ ID NO: 1, SEQ ID NO: 11-86, and SEQ ID NO: 142-297. Or it can be synthesized. For the B18Ag1 described herein, one suitable primer pair is B18Ag1-2 (5′ATG GCT ATT TTC GGG GGC TGA CA) (SEQ ID NO: 126) and B18Ag1-3 (5′CCG GTA TCT CCT CGT GGG TAT T) (SEQ ID NO: 127). The PCR reaction products can then be separated by gel electrophoresis and visualized according to methods well known to those skilled in the art. Amplification is typically performed in a sample obtained from a matched tissue pair (tumor tissue and non-tumor tissue from the same individual) or an unmatched tissue pair (tumor tissue and non-tumor tissue from a different individual). . The amplification reaction is preferably performed in several cDNA dilutions over two orders of magnitude. In some dilutions of the tumor sample, an increase in expression of more than 2-fold compared to a non-tumor sample of the same dilution is considered positive.

  As used herein, the term “primer / probe specific to a DNA / RNA molecule” refers to at least about 80% identity, preferably at least about 90%, to the DNA / RNA molecule in question. And more preferably oligonucleotide sequences having at least about 95% identity. Primers and / or probes that can be usefully used in the diagnostic methods of the present invention preferably have at least about 10-40 nucleotides. In a preferred embodiment, the polymerase chain reaction primer comprises at least about 10 consecutive DNA / RNA molecule nucleotides encoding one of the polypeptides disclosed herein. Preferably, the oligonucleotide probe used in the diagnostic methods of the invention comprises an oligonucleotide of at least about 15 consecutive DNA / RNA molecules encoding one of the polypeptides disclosed herein. Techniques for both PCR-based assays and in situ hybridization assays are well known in the art.

  Traditional RT-PCR protocols using agarose and ethidium bromide staining are important for defining gene specificity, but time and effort (ie, construction of saturation and / or titer curves) to quantify this This protocol does not lend itself to diagnostic kit development because of its need and sample throughput. This problem is overcome by the development of procedures such as real-time RT-PCR that allow the assay to be performed in one tube, which in turn can be modified for use in a 96 well plate format. Equipment for performing such methodologies is available from Perkin Elmer / Applied Biosystems Division. Alternatively, other high-throughput assays using a combination of labeled probes (eg, digoxigenin) and labeled (eg, enzyme, fluorescent, radioactive) antibodies against such probes are also used in the development of 96-well plate assays. obtain.

  In yet another method for determining the presence or absence of breast cancer in a patient, one or more of the described breast mass-specific polypeptides can be used in a skin test. As used herein, a “skin test” is any assay performed directly in a patient where a delayed type hypersensitivity (DTH) response (eg, swelling, reddening, or dermatitis) has occurred. Measured after intradermal injection of one or more of the above polypeptides. Such injection can be accomplished using any suitable device (eg, a tuberculin syringe or a 1 mL syringe) sufficient to contact the polypeptide to the patient's skin cells. Preferably, this response is measured at least 48 hours after injection, more preferably 48-72 hours.

  A DTH response is a cell-mediated immune response, which is greater in patients that have been previously exposed to a test antigen (ie, an immunogenic portion of the polypeptide used, or a variant thereof). This response can be measured visually using a ruler. In general, a response greater than about 0.5 cm in diameter, preferably greater than about 5.0 cm in diameter, is a positive response and is indicative of breast cancer.

The breast mass-specific polypeptide described herein is preferably at least one polypeptide and a physiologically acceptable carrier (eg, water, saline, alcohol) for use in skin testing. Or a buffer). Such compositions typically comprise one or more of the above polypeptides in a volume of 0.1 mL in an amount ranging from about 1 μg to 100 μg, preferably from about 10 μg to 50 μg. Preferably, the carrier used in such pharmaceutical compositions is a saline solution containing a suitable preservative (eg, phenol and / or Tween 80 ^™ ).

  In other aspects of the invention, progression and / or response to the treatment of breast cancer is performed by performing any of the above assays over a period of time and the level of response (ie, the amount of polypeptide or mRNA detected, or skin In the case of a test, it can be monitored by assessing changes in the degree of immune response detected. For example, the assay can be performed every month to every other month for a period of 1-2 years. In general, breast cancer is progressing in patients whose level of response increases over time. In contrast, breast cancer is not progressing if the detected signal remains constant or decreases over time.

  In a further aspect of the invention, the compounds described herein can be used for immunotherapy of breast cancer. In these aspects, the compound, which can be a polypeptide, antibody, or nucleic acid molecule, is preferably incorporated into a pharmaceutical composition or vaccine. A pharmaceutical composition includes one or more such compositions and a physiologically acceptable carrier. A vaccine can include one or more polypeptides and an immune response enhancer, such as an adjuvant or liposome in which the compound is incorporated. The pharmaceutical compositions and vaccines can further include delivery systems such as biodegradable microspheres, which are disclosed, for example, in US Pat. Nos. 4,897,268 and 5,075,109. Pharmaceutical compositions and vaccines within the scope of the present invention may also include other compounds, including one or more separate polypeptides.

  Alternatively, the vaccine may include DNA encoding one or more of the above polypeptides, such that the polypeptide is generated in situ. In such a vaccine, the DNA can be present in any of a variety of delivery systems known to those skilled in the art, including nucleic acid expression systems, bacterial and viral expression systems. Appropriate nucleic acid expression systems contain the necessary DNA sequences for expression in the patient (eg, appropriate promoters and termination signals). Bacterial delivery systems involve the administration of a bacterium (eg, Bacillus-Calmette-Guerrin) that expresses an immunogenic portion of the polypeptide on its cell surface. In preferred embodiments, the DNA can be introduced using a viral expression system (eg, vaccinia or other poxviruses, retroviruses, or adenoviruses), which is non-pathogenic (incomplete), replication competent. The use of various viruses. Techniques for incorporating DNA into such expression systems are well known to those skilled in the art. DNA can also be “naked” as described, for example, in Ulmer et al., Science 259: 1745-1749 (1993) and reviewed by Cohen, Science 259: 1691-1692 (1993). Naked DNA uptake can be increased by coating the DNA onto biodegradable beads, which are efficiently transported into the cell.

  Although any suitable carrier known to those skilled in the art can be used in the pharmaceutical compositions of the invention, the type of carrier will vary depending on the mode of administration. For parenteral administration (eg, subcutaneous injection), the carrier preferably comprises water, saline, alcohol, lipids, waxes, or buffers. For oral administration, any of the above carriers or a solid carrier such as mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, glucose, sucrose, and magnesium carbonate can be used. Biodegradable microspheres (eg, polylactate, polyglycolate) can also be used as carriers for the pharmaceutical compositions of the present invention.

  Any of a variety of adjuvants can be used to non-specifically enhance the immune response in the vaccines of the invention. Most adjuvants contain substances designed to protect the antigen from rapid metabolism (eg, aluminum hydroxide or mineral oil) and stimulators of the immune response (eg, proteins from lipid A, Bortadella pertussis or Mycobacterium tuberculosis) . Suitable adjuvants include, for example, Freund's Incomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit, MI), Merck Adjuvant 65 (Merck and Company, Inc., Rahway, NJ), alum, biodegradable microspheres, monophosphoryl lipid A , As well as quilA. Cytokines such as GM-CSF or interleukin-2, -7, or -12 can also be used as adjuvants.

  The pharmaceutical compositions and vaccines described above can be used, for example, for the treatment of breast cancer in patients. “Patient” as used herein refers to any warm-blooded animal, preferably a human. The patient may or may not suffer from breast cancer. Thus, the pharmaceutical compositions and vaccines described above can be used to prevent the development of breast cancer or to treat patients suffering from breast cancer. To prevent breast cancer development, a pharmaceutical composition or vaccine comprising one or more polypeptides described herein can be administered to a patient. Alternatively, naked DNA encoding the polypeptide or a plasmid or viral vector can be administered. To treat a patient with breast cancer, the pharmaceutical composition or vaccine is a nucleotide sequence that is complementary to one or more polypeptides, antibodies, or DNA encoding the polypeptides described herein. (Eg, antisense RNA or antisense deoxyribonucleotide oligonucleotide).

  The route and frequency of administration and dosage will vary from individual to individual. In general, pharmaceutical compositions and vaccines can be administered by injection (eg, intradermal, intramuscular, intravenous, or subcutaneous), intranasally (eg, by inhalation), or orally. A dose between 1 and 10 can be administered for 52 weeks. Preferably, 6 doses are administered at monthly intervals, and booster vaccine administration can thereafter be given periodically. Alternative protocols may be appropriate for individual patients. An appropriate dose is that amount of a compound that, when administered as described above, can promote an anti-tumor immune response. Such a response can be monitored by measuring anti-tumor antibodies in the patient or by vaccine-dependent generation of cytolytic effector cells that can kill the patient's tumor cells in vitro. Such vaccines also provide immune responses that lead to improved clinical outcomes (eg, more frequent remissions, complete or partial or longer disease-free survival) compared to unvaccinated patients Should be able to be induced in a vaccinated patient. In general, for pharmaceutical compositions and vaccines comprising one or more polypeptides, the amount of each polypeptide present in a dose ranges from about 100 μg to 5 mg. Appropriate dose sizes vary with the size of the patient, but typically range from about 0.1 mL to about 5 mL.

  The following examples are provided for purposes of illustration and not limitation.

Example 1
Preparation of Breast Mass Specific cDNA Using Differential Display RT-PCR This example illustrates the preparation of a cDNA molecule encoding a breast mass specific polypeptide using differential display screening.

A. Preparation of B18Ag1 cDNA and Characterization of mRNA Expression Tissue samples were prepared from breast masses and normal tissues of patients with breast cancer (as confirmed by pathology after removal of the tissue from the patient). Extract the normal RNA and Hai瘍RNA from the sample, and mRNA was isolated and converted into cDNA using 3 'primers that were ligated (dT) ₁₂ AG (SEQ ID NO: 130). Differential display PCR was then performed using a randomly selected primer (CTTCAACCTC) (SEQ ID NO: 103). Amplification conditions were standard buffer containing 1.5 mM MgCl ₂ , 20 pmol primer, 500 pmol dNTP, and 1 unit Taq DNA polymerase (Perkin-Elmer, Branchburg, NJ). Forty cycles of amplification were performed using denaturation at 94 ° C. for 30 seconds, annealing at 42 ° C. for 1 minute, and extension at 72 ° C. for 30 seconds. An RNA fingerprint containing 76 amplification products was obtained. The breast fingerprint tissue RNA fingerprint was more than 98% identical to the normal breast tissue RNA fingerprint, but the band was repeatedly observed to be specific for the tumor RNA fingerprint pattern . This band was excised from the silver stained gel, subcloned into T vector (Novagen, Madison, Wis.) And sequenced.

  The sequence of the cDNA (referred to as B18Ag1) is provided in SEQ ID NO: 1. By searching the GENBANK and EMBL databases, the first cloned B18Ag1 fragment is 77% identical to the endogenous human retrovirus element S71, which is a truncated retrovirus element homologous to the simian sarcoma virus (SSV) It became clear that. S71 contains an incomplete gag gene, part of the pol gene, and an LTR-like structure at the 3 ′ end (see Werner et al., Virology 174: 225-238 (1990)). B18Ag1 is also 64% identical to SSV in the region corresponding to the P30 (gag) locus. B18Ag1 contains three separate and incomplete reading frames that cover a region that shares significant homology to a wide variety of gag proteins of retroviruses that infect mammals. Furthermore, the homology to S71 extends not only within the gag gene, but also over several kb sequences including the LTR.

  B18Ag1-specific PCR primers were synthesized using computer analysis guidelines. RT-PCR amplification (94 ° C., 30 seconds; 60 ° C. → 42 ° C., 30 seconds; 72 ° C., 30 seconds 40 cycles) shows that B18Ag1 is present at relatively high levels in the breast mass tissue of the patient Confirmed to represent mRNA sequence. Primers used in amplification were B18Ag1-1 (CTG CCT GAG CCA CAA ATG) (SEQ ID NO: 128) and B18Ag1-4 (CCG GAG GAG GAA GCT AGA GGA ATA) (SEQ ID NO: 129) at 3.5 mM magnesium concentration and pH 8.5. ), And B18Ag1-2 (ATG GCT ATT TTC GGG GCC TGA CA) (SEQ ID NO: 126) and B18Ag1-3 (CCG GTA TCT CCT CGT GGG TAT T) (SEQ ID NO: 127) at 2 mM magnesium, pH 9.5 . The same experiment showed very low to nonexistent levels of expression in the patient's normal breast tissue (see FIG. 1). Then, using RT-PCR experiments, B18Ag1 mRNA is present in 9 other breast mass samples (from Brazilian and American patients) but not in normal breast tissue corresponding to each cancer patient Or present at very low levels. RT-PCR analysis also showed that B18Ag1 transcripts were absent in various normal tissues (including lymph nodes, myocardium, and liver) and at relatively low levels in PBMC and lung tissues . The presence of B18Ag1 mRNA in breast tumor samples and its absence from normal breast tissue has been confirmed by Northern blot analysis as shown in FIG.

  Differential expression of B18Ag1 in breast mass tissue was also confirmed by RNase protection assay. FIG. 3 shows the level of B18Ag1 mRNA in various tissue types determined in four different RNase protection assays. Lanes 1-12 represent various normal breast tissue samples, lanes 13-25 represent various breast mass samples; lanes 26-27 represent normal prostate samples; lanes 28-29 represent prostate tumor samples Lanes 30-32 represent colon tumor samples; Lane 33 represents normal aorta; Lane 34 represents normal small intestine; Lane 35 represents normal skin; Lane 36 represents normal lymph nodes Lane 37 represents a normal ovary; Lane 38 represents a normal liver; Lane 39 represents a normal skeletal muscle; Lane 40 represents a first normal stomach sample; Lane 41 represents a second Lanes represent normal lungs; lanes 43 represent normal kidneys; and lanes 44 represent normal pancreas. Comparison between experiments was facilitated by including a large amount of known β-actin message positive control RNA in each assay and normalizing the results of different assays with respect to this positive control.

  RT-PCR and Southern blot analysis indicated that the B18Ag1 locus was present as a single copy of an endogenous retroviral element in human genomic DNA. An approximately 12-18 kb genomic clone was isolated using the original B18Ag1 sequence as a probe. Four additional subclones were also isolated by XbaI digestion. Additional retroviral sequences obtained from the ends of the XbaI digests (located as shown in FIG. 4) of these clones are shown as SEQ ID NO: 3 to SEQ ID NO: 10, where SEQ ID NO: 3 is shown in FIG. , SEQ ID NO: 4 indicates the position of the sequence indicated as 11 to 29, SEQ ID NO: 5 indicates the position of the sequence indicated as 3, SEQ ID NO: 6 indicates 6 , SEQ ID NO: 7 indicates the position of the sequence indicated as 12, SEQ ID NO: 8 indicates the position of the sequence indicated as 13, SEQ ID NO: 9 indicates as 14 And SEQ ID NO: 10 indicates the position of the sequence labeled 11-22.

  Subsequent studies demonstrated that the 12-18 kb genomic clone contained approximately 7.75 kb of retroviral elements, as shown in FIGS. 5A and 5B. The sequence of this retroviral element is shown in SEQ ID NO: 141. The numbered line at the top of FIG. 5A represents the sense strand sequence of the retroviral genomic clone. The box below this line indicates the location of the selected restriction site. The arrows indicate the different overlapping clones used to sequence retroviral elements. The direction of the arrow indicates whether a single passage subclone sequence corresponds to either the sense or antisense strand. FIG. 5B is a schematic diagram of a retroviral element containing B18Ag1, showing the organization of viral genes within the element. An open box corresponds to a putative reading frame starting with methionine found throughout the element. Each of the six possible reading frames is shown as shown on the left of the box, and frames 1-3 correspond to those found on the sense strand.

  Using the cDNA of SEQ ID NO: 1 as a probe, a longer cDNA containing a slight nucleotide difference (less than 1%) was obtained compared to the genomic sequence shown in SEQ ID NO: 141 (SEQ ID NO: 227).

B. Preparation of cDNA molecules encoding other breast mass-specific polypeptides As described above, normal RNA and tumor RNA were prepared, and mRNA was isolated and using a 3 'primer linked to (dT) ₁₂ AG Converted into cDNA. Differential display PCR was then performed using randomly selected primers (SEQ ID NOs: 87-125). Amplification conditions were the same as above, and a band that was observed to be specific for the tumor RNA fingerprint pattern was excised from the silver stained gel and either T vector (Novagen, Madison, WI) or pCRII vector (Invitrogen, San) Diego, CA) and subcloned and sequenced. The sequences were provided in SEQ ID NO: 11 to SEQ ID NO: 86. Of the 79 sequences isolated, 67 were found to be novel (SEQ ID NOs: 11-26 and 28-77) (see also FIGS. 6-20).

  The extended DNA sequence (SEQ ID NO: 290) for the B15Ag1 antigen (originally identified partial sequence provided in SEQ ID NO: 27) was obtained in further studies. As described above, comparison of the sequence in Genebank with the sequence of SEQ ID NO: 290 showed homology to the known human β-A actin gene.

  Subsequent studies identified additional 146 sequences (SEQ ID NOs 142-289), of which 115 appear to be novel (SEQ ID NOs 142, 143, 146-152, 154-166, 168-176, 178-192, 194-198, 200-204, 206, 207, 209-214, 216, 218, 219, 221-240, 243-245, 247, 250, 251, 253, 255, 257-266, 268, 269, 271- 273, 275, 276, 278, 280, 281, 284, 288 and 291). To our knowledge, no previously identified sequence has ever been shown to be expressed at higher levels in human breast mass tissue than in normal breast tissue.

  In further studies, six different splice forms of B11Ag1 antigen were isolated, each different splice form containing a slightly different version of the B11Ag1 coding frame. Splice binding sequences define individual exons that constitute various splice forms in various patterns and arrangements. The primers were designed to test the expression pattern of each exon using RT-PCR as described below. Each exon showed the same expression pattern as the original B11Ag1 clone, and expression was found to be breast mass, prostate and testis specific. The determined cDNA sequences for the exons encoding the isolated protein are provided in SEQ ID NOs: 292-297, respectively.

Example 2
Preparation of B18Ag1 DNA from Human Genomic DNA This example illustrates the preparation of B18Ag1 DNA by amplification from human genomic DNA.

  B18Ag1 DNA can be prepared from 250 ng human genomic DNA using 20 pmol of B18Ag1 specific primer, 500 pmol of dNTP, and 1 unit of Taq DNA polymerase (Perkin Elmer, Branchburg, NJ) with the following amplification parameters: Denaturation at 94 ° C for 30 seconds, touchdown annealing in 2 ° C increments every 2 cycles from 60 ° C to 42 ° C for 30 seconds, and extension at 72 ° C for 30 seconds. The last increment (42 ° C annealing temperature) should be cycled 25 times. Primers were selected using computer analysis. The synthesized primers were B18Ag1-1, B18Ag1-2, B18Ag1-3, and B18Ag1-4. Primer pairs that can be used are 1 + 3, 1 + 4, 2 + 3, and 2 + 4.

  After gel electrophoresis, the band corresponding to B18Ag1 DNA can be excised and cloned into an appropriate vector.

Example 3
Preparation of B18Ag1 DNA from Breast Mass cDNA This example illustrates the preparation of B18Ag1 DNA by amplification from human breast mass cDNA.

First strand cDNA was purified from 500 ng poly A + RNA, 200 pmol primer (T) ₁₂ AG (ie TTT TTT TTT TTT AG) (SEQ ID NO: 130), 1 × first strand reverse transcriptase buffer in a final volume of 30 μl. Human breast mass in a reaction mixture containing solution, 6.7 mM DTT, 500 mmol dNTP, and 1 unit of AMV or MMLV reverse transcriptase (eg, from any supplier such as Gibco-BRL (Grand Island, NY)) Synthesize from RNA prepared from tissue. After first strand synthesis, the cDNA is diluted approximately 25-fold and 1 μl is used for amplification as described in Example 2. Some primer pairs can generate a heterogeneous population of transcripts, but primers B18Ag1-2 (5'ATG GCT ATT TTC GGG GGC TGA CA) (SEQ ID NO: 126) and B18Ag1-3 (5'CCG GTA TCT CCT CGT GGG TAT T) (SEQ ID NO: 127) yields a single 151 bp gain byproduct.

Example 4
Identification of B18 and T cell epitopes of B18Ag1 This example illustrates the identification of the B18Ag1 epitope.

  B18Ag1 sequences can be screened using various computer algorithms. To determine B cell epitopes, Hopp, Prog. Clin. Biol. Res. 172B: 367-77 (1985), or alternatively Cease et al. Exp. Med. 164: 1779-84 (1986) or Spouge et al. Immunol. 138: 204-12 (1987) can be used to screen sequences for hydrophobicity and hydrophilicity values. Further class II MHC (antibody or B cell) epitopes can be obtained from programs such as AMPHI (eg Margalit et al., J. Immunol. 138: 2213 (1987)) or Rothbard and Taylor (eg EMBO J. 7:93 (1988)). )).

  Once peptides (15-20 amino acids long) are identified using these techniques, individual peptides are available from manufacturers such as automated peptide synthesizers (Perkin Elumar Applied Biosystems Division, Foster City, CA). ) And Merrifield synthesis. After synthesis, the peptide can be used to screen serum collected from either normal or breast cancer patients to determine whether the breast cancer patient carries antibodies reactive with the peptide. The presence of such antibodies in breast cancer patients confirms the immunogenicity of the particular B cell epitope in question. Peptides can also be tested for their ability to generate serological or humoral immunity in an animal (mouse, rat, rabbit, chimpanzee, etc.) after immunization in vivo. Generation of such peptide-specific antisera after immunization further confirms the immunogenicity of the particular B cell epitope in question.

  To identify T cell epitopes, the B18Ag1 sequence can bind to an HLA class I MHC molecule (see, eg, Rammensee et al., Immunogenetics 41: 178-228 (1995)) 8-10 amino acids within the B18Ag1 sequence. Different computer algorithms useful in identifying motifs can be screened. After synthesis, such peptides can be tested for their ability to bind to class I MHC using standard binding assays (eg, Sette et al., J. Immunol. 153: 5586-92 (1994)) And more importantly, for example, Bakker et al., Cancer Res. 55: 5330-34 (1995); Visseren et al., J. MoI. Immunol. 154: 3991-98 (1995); Kawakami et al. Immunol. 154: 3961-68 (1995); and Kast et al. Immnunol. 152: 3904-12 (1994) can be used to test for its ability to generate antigen-reactive cytotoxic T cells following in vitro stimulation of patients or normal peripheral mononuclear cells. The successful in vitro generation of T cells that can kill autologous (carrying the same class I MHC molecule) tumor cells after in vitro peptide stimulation further confirms the immunogenicity of the B18Ag1 antigen. In addition, such peptides can be used to generate murine peptides and B18Ag1-reactive cytotoxic T cells following in vivo immunization in mice transgenic for the expression of certain human MHC class I haplotypes. (Vitiello et al., J. Exp. Med. 173: l007-15 (1991)).

A representative list of putative B18Ag1 B cell and T cell epitopes (analyzed according to putative HLA class I MHC binding antigens) is shown below:
Putative Th motif (B cell epitope) (SEQ ID NOs: 131-133)
Putative HLA A2.1 motif (T cell epitope) (SEQ ID NOs: 134-140)

Example 5
Characterization of the breast mass genes discovered by differential display PCR The specificity and sensitivity of the breast mass genes discovered by differential display PCR were determined using RT-PCR. This procedure allowed a rapid assessment of breast mass gene mRNA expression semi-quantitatively without using large amounts of RNA. Using gene-specific primers, various tissues (8 breast masses, 5 normal breasts, 2 prostate tumors, 2 rectal tumors, 1 lung tumor, and 14 other normal adult human tissues (normal prostate, MRNA levels in (including rectum, kidney, liver, lung, ovary, pancreas, skeletal muscle, skin, stomach, and testis) were examined.

  To confirm the semi-quantitative nature of RT-PCR, β-actin was used as an internal standard for each of the tissues tested. Serial dilutions of first strand cDNA were prepared and RT-PCR assays using β-actin specific primers were performed. The dilution that allowed β-actin template linear range amplification, which was sensitive enough to reflect the initial copy number differences, was then selected. Using this condition, β-actin levels were determined for each reverse transcription reaction from each tissue. DNA contamination was minimized by DNase treatment and by guaranteeing a negative result when using first strand cDNA prepared without the addition of reverse transcriptase.

Using gene specific primers, mRNA expression levels were determined in various tissues. To date, 38 genes have been successfully tested by RT-PCR, five of which (B15AG-1, B31GA1b, B38GA2a, B11A1a and B18AG1a) show good specificity and sensitivity to breast masses . Figures 21A and 21B show the results for three of these genes: B15AG-1 (SEQ ID NO: 27), B31GA1b (SEQ ID NO: 148) and B38GA2a (SEQ ID NO: 157). Table I summarizes the expression levels of all genes tested in normal breast tissue and breast mass, and also in other tissues.

  From the foregoing, it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without departing from the spirit and scope of the invention.

Separation by gel electrophoresis, obtained from differential display PCR products (lanes 1 and 2), obtained from cDNA prepared from normal breast tissue, and cDNA prepared from breast mass tissue from the same patient The differential display PCR product (lanes 3 and 4) is shown. The arrow indicates the band corresponding to B18Ag1. FIG. 6 is a Northern blot comparing B18Ag1 mRNA levels in breast mass tissue (lane 1) with levels in normal breast tissue. FIG. 5 shows the level of B18Ag1 mRNA in breast mass tissue compared to B18Ag1 mRNA in various normal and non-breast mass tissues determined by RNase protection assay. FIG. 5 is a genomic clone map showing the location of additional retroviral sequences obtained from the ends of XbaI restriction digests (provided in SEQ ID NO: 3 to SEQ ID NO: 10) compared to B18Ag1. Figures 5A and B show the sequencing strategy, genomic organization, and putative open reading frame for retroviral elements containing B18Ag1. The nucleotide sequence of a representative breast mass-specific cDNA B18Ag1 is shown. The nucleotide sequence of a representative breast mass-specific cDNA B17Ag1 is shown. The nucleotide sequence of a representative breast mass-specific cDNA B17Ag2 is shown. The nucleotide sequence of a representative breast mass-specific cDNA B13Ag2a is shown. The nucleotide sequence of a representative breast mass-specific cDNA B13Ag1b is shown. The nucleotide sequence of a representative breast mass-specific cDNA B13Ag1a is shown. The nucleotide sequence of a representative breast mass-specific cDNA B11Ag1 is shown. The nucleotide sequence of a representative breast mass-specific cDNA B3CA3c is shown. The nucleotide sequence of a representative breast mass-specific cDNA B9CG1 is shown. The nucleotide sequence of a representative breast mass-specific cDNA B9CG3 is shown. The nucleotide sequence of a representative breast mass-specific cDNA B2CA2 is shown. The nucleotide sequence of a representative breast mass-specific cDNA B3CA1 is shown. The nucleotide sequence of a representative breast mass-specific cDNA B3CA2 is shown. The nucleotide sequence of a representative breast mass-specific cDNA B3CA3 is shown. The nucleotide sequence of a representative breast mass-specific cDNA B4CA1 is shown. Breast tumor tissues (lanes 1-8) and normal breast tissues (lanes 9-13), and H ₂ 0 shows RT-PCR analysis of breast tumor genes in (lane 14). Prostate tumor (lane 1, 2), colon tumor (lane 3), lung tumor (lane 4), normal prostate (lane 5), normal colon (lane 6), normal kidney (lane 7), normal liver (lane 8) Normal lung (lane 9), normal ovary (lanes 10 and 18), normal pancreas (lanes 11 and 12), normal skeletal muscle (lane 13), normal skin (lane 14), normal stomach (lane 15), normal testis (Lane 16), normal small intestine (lane 17), HBL-100 (lane 19), MCF-12A (lane 20), breast mass (lanes 21-23), H ₂ O (lane 24), and colon lung tumor ( Shown is RT-PCR analysis of the breast mass gene in lane 25).

Claims (49)

An isolated DNA molecule comprising: (a) SEQ ID NO: 1, 3 to 26, 28 to 77, 142, 143, 146 to 152, 154 to 166, 168 to 176, 178 to 192, 194 to 198 , 200-204, 206, 207, 209-214, 216, 218, 219, 221-240, 243-245, 247, 250, 251, 253, 255, 257-266, 268, 269, 271-273, 275 276, 278, 280, 281, 284, 288, and a nucleotide sequence selected from the group consisting of 291-297;
(b) substitution, deletion, insertion of one or more nucleotides at no more than 20% nucleotide positions so that the antigenic and / or immunogenic properties of the polypeptide encoded by the nucleotide sequence are retained. And / or variants of the nucleotide sequence comprising modifications; or
(c) SEQ ID NOs: 1, 3-26, 28-77, 142, 143, 146-152, 154-166, 168-176, 178-192, 194-198, 200-204, 206, 207, 209-214 216, 218, 219, 221-240, 243-245, 247, 250, 251-253, 255, 257-266, 268, 269, 271-273, 275, 276, 278, 280, 281, 284, 288 And a nucleotide sequence encoding an epitope of a polypeptide encoded by at least one sequence selected from the group consisting of 291-297,
A DNA molecule comprising An isolated DNA molecule encoding an epitope of a polypeptide, wherein the polypeptide has the following nucleotide sequence:
(a) SEQ ID NOs: 1, 3-26, 28-77, 142, 143, 146-152, 154-166, 168-176, 178-192, 194-198, 200-204, 206, 207, 209-214 216, 218, 219, 221-240, 243-245, 247, 250, 251-253, 255, 257-266, 268, 269, 271-273, 275, 276, 278, 280, 281, 284, 288 And a nucleotide sequence that hybridizes under stringent conditions to a sequence selected from the group consisting of 291-297; and
(b) SEQ ID NOs: 1, 3-26, 28-77, 142, 143, 146-152, 154-166, 168-176, 178-192, 194-198, 200-204, 206, 207, 209-214 216, 218, 219, 221-240, 243-245, 247, 250, 251-253, 255, 257-266, 268, 269, 271-273, 275, 276, 278, 280, 281, 284, 288 And a nucleotide sequence that is at least 80% identical to a sequence selected from the group consisting of 291-297;
A DNA molecule encoded by An isolated DNA molecule encoding an epitope of a polypeptide, wherein the polypeptide is:
(a) a nucleotide sequence transcribed from the sequence of SEQ ID NO: 141; or
(b) substitution, deletion, insertion of one or more nucleotides at no more than 20% nucleotide positions so that the antigenic and / or immunogenic properties of the polypeptide encoded by the nucleotide sequence are retained. And / or a DNA molecule encoded by a variant of the nucleotide sequence, including modifications.   An isolated DNA or RNA molecule comprising a nucleotide sequence complementary to the DNA molecule of any one of claims 1-3.   A recombinant expression vector comprising the DNA molecule according to any one of claims 1 to 3.   A host cell transformed or transfected with the expression vector of claim 5.   A polypeptide comprising an amino acid sequence encoded by the DNA molecule of any one of claims 1-3.   8. The polypeptide of claim 7, wherein the polypeptide is SEQ ID NO: 1, 3-26, 28-77, 142, 143, 146-152, 154-166, 168-176, 178- 192,194-198,200-204,206,207,209-214,216,218,219,221-240,243-245,247,250,251,253,255,257-266,268,269, A polypeptide comprising an epitope of an amino acid sequence encoded by at least one nucleotide sequence selected from the group consisting of 271-273, 275, 276, 278, 280, 281, 284, 288, and 291-297.   A monoclonal antibody that binds to the polypeptide of claim 7.   A method for determining the presence of breast cancer in a patient comprising detecting at least one polypeptide according to claim 7 in a biological sample and thereby determining the presence of breast cancer in said patient. A method comprising the steps.   A method for determining the presence of breast cancer in a patient comprising the steps of SEQ ID NOs: 78-86, 144, 145, 153, 167, 177, 193, 199, 205, 208, 215, 217, in a biological sample. A nucleotide sequence selected from the group consisting of 220, 241, 242, 246, 248, 249, 252, 256, 267, 270, 274, 277, 279, 282, 283, 285-287, 289, and 290, and a string Detecting at least one polypeptide encoded by a sequence that hybridizes to them under gentle conditions.   12. A method according to claim 10 or 11, wherein the biological sample is part of a breast mass.   11. The method of claim 10, wherein the detecting step comprises contacting the biological sample with the monoclonal antibody of claim 9.   12. The method of claim 11, wherein the step of detecting comprises the biological sample and SEQ ID NOs: 78-86, 144, 145, 153, 167, 177, 193, 199, 205, 208, Nucleotides selected from the group consisting of 215, 217, 220, 241, 242, 246, 248, 249, 252, 256, 267, 270, 274, 277, 279, 282, 283, 285-287, 289, and 290 Contacting the sequence with a monoclonal antibody that binds to a polypeptide encoded by the sequence and a sequence that hybridizes to it under stringent conditions.   A method for detecting the presence of breast cancer in a patient comprising detecting an RNA molecule encoding at least one polypeptide of claim 7 in a biological sample, and thereby breast cancer in said patient Determining the presence of.   A method for determining the presence of breast cancer in a patient comprising the steps of SEQ ID NOs: 78-86, 144, 145, 153, 167, 177, 193, 199, 205, 208, 215, 217, in a biological sample. 220, 241, 242, 246, 248, 249, 252, 256, 267, 270, 274, 277, 279, 282, 283, 285-287, 289, 290, and hybridizes to these under stringent conditions A method comprising detecting at least one RNA molecule encoding a polypeptide encoded by a nucleotide sequence selected from the group consisting of sequences, and thereby determining the presence of breast cancer in the patient.   17. A method according to claim 15 or 16, wherein the biological sample is part of a breast mass. 16. The method of claim 15, wherein the detecting step is:
(a) preparing cDNA from RNA molecules in said biological sample; and
(b) a method comprising specifically amplifying a cDNA molecule capable of encoding at least a portion of the polypeptide of claim 7 and thereby determining the presence of breast cancer in said patient. 17. The method of claim 16, wherein the detecting step comprises:
(a) preparing cDNA from RNA molecules in said biological sample; and
(b) SEQ ID NOs: 78-86, 144, 145, 153, 167, 177, 193, 199, 205, 208, 215, 217, 220, 241, 242, 246, 248, 249, 252, 256, 267, 270 274, 277, 279, 282, 283, 285-287, 289, 290, and at least one polypeptide encoded by a nucleotide sequence selected from the group consisting of sequences that hybridize under stringent conditions Specifically amplifying a cDNA molecule capable of encoding a portion; and thereby determining the presence of breast cancer in the patient. A method for monitoring breast cancer progression in a patient, comprising:
(a) detecting an amount of at least one polypeptide according to claim 7 in a biological sample at an initial time point;
(b) repeating step (a) at subsequent time points; and (c) comparing the amount of polypeptide detected in steps (a) and (b), and thereby monitoring the progression of breast cancer in the patient The process of
Including the method. A method for monitoring breast cancer progression in a patient, comprising:
(a) detecting an amount of at least one polypeptide in a biological sample at an initial time point, the polypeptide comprising SEQ ID NOs: 78-86, 144, 145, 153, 167, 177, 193 , 199, 205, 208, 215, 217, 220, 241, 242, 246, 248, 249, 252, 256, 267, 270, 274, 277, 279, 282, 283, 285-287, 289, 290, and Encoded by a nucleotide sequence selected from the group consisting of sequences that hybridize to them under stringent conditions;
(b) repeating step (a) at subsequent times; and
(c) comparing the amount of polypeptide detected in steps (a) and (b), thereby monitoring the progression of breast cancer in the patient;
Including the method.   22. A method according to claim 20 or 21, wherein the biological sample is part of a breast mass.   21. The method of claim 20, wherein the detecting step comprises contacting a portion of the biological sample with the monoclonal antibody of claim 9.   24. The method of claim 21, wherein the detecting step comprises the biological sample and SEQ ID NOs: 78-86, 144, 145, 153, 167, 177, 193, 199, 205, 208, 215, 217, 220, 241, 242, 246, 248, 249, 252, 256, 267, 270, 274, 277, 279, 282, 283, 285-287, 289, 290, and hybridizes to these under stringent conditions Contacting with a monoclonal antibody that binds to a polypeptide encoded by a nucleotide sequence selected from the group consisting of soybean sequences. 21. The method of claim 20, wherein the polypeptide is SEQ ID NO: 1, 3-26, 28-77, 142, 143, 146-152, 154-166, 168-176, 178-192, 194-198, 200-204, 206, 207, 209-214, 216, 218, 219, 221-240, 243-245, 247, 250, 251, 253, 255, 257-266, 268, 269, 271- 273, 275, 276, 278, 280, 281, 284, 288, and an epitope of an amino acid sequence encoded by at least one nucleotide sequence selected from the group consisting of 291-297. A method for monitoring the progression of breast cancer in a patient comprising:
(a) detecting in an initial time the amount of at least one RNA molecule encoding a polypeptide according to claim 7 in a biological sample;
(b) repeating step (a) at subsequent times; and
(c) comparing the amount of RNA molecules detected in steps (a) and (b), thereby monitoring the progression of breast cancer in the patient;
Including the method. 27. The method of claim 26, wherein the detecting step comprises:
(a) preparing cDNA from RNA molecules in said biological sample; and
(b) a step of specifically amplifying a cDNA molecule capable of encoding at least a part of the polypeptide of claim 7;
Including the method. A method for monitoring breast cancer progression in a patient, comprising:
(a) detecting an amount of at least one RNA molecule in a biological sample at an initial time point, the RNA molecule comprising SEQ ID NOs: 78-86, 144, 145, 153, 167, 177, 193, 199, 205, 208, 215, 217, 220, 241, 242, 246, 248, 249, 252, 256, 267, 270, 274, 277, 279, 282, 283, 285-287, 289, 290, And encoding a polypeptide encoded by a nucleotide sequence selected from the group consisting of sequences that hybridize to them under stringent conditions;
(b) repeating step (a) at subsequent times; and
(c) comparing the amount of RNA molecules detected in steps (a) and (b), thereby monitoring the progression of breast cancer in the patient;
Including the method.   A pharmaceutical composition comprising the polypeptide of claim 7 and a physiologically acceptable carrier.   A pharmaceutical composition for inhibiting the development of breast cancer, wherein the pharmaceutical composition comprises a polypeptide and a physiologically acceptable carrier, the polypeptide comprising SEQ ID NOs: 78-86, 144, 145, 153, 167, 177, 193, 199, 205, 208, 215, 217, 220, 241, 242, 246, 248, 249, 252, 256, 267, 270, 274, 277, 279, 282, 283, 285- A pharmaceutical composition encoded by a nucleotide sequence selected from the group consisting of 287, 289, 290, and sequences that hybridize to them under stringent conditions.   A vaccine comprising the polypeptide according to claim 7 and an immune response enhancer.   A vaccine comprising the DNA molecule according to any one of claims 1-3.   A vaccine comprising a recombinant expression vector comprising the DNA molecule of any one of claims 1-3.   A vaccine for inhibiting the development of breast cancer, the vaccine comprising a polypeptide and an immune response enhancer, the polypeptide comprising SEQ ID NOs: 78-86, 144, 145, 153, 167, 177, 193, 199 205, 208, 215, 217, 220, 241, 242, 246, 248, 249, 252, 256, 267, 270, 274, 277, 279, 282, 283, 285-287, 289, 290 and stringent A vaccine encoded by a nucleotide sequence selected from the group consisting of sequences that hybridize to them under conditions.   31. A pharmaceutical composition according to any one of claims 29 to 30, for use in the manufacture of a medicament for inhibiting the development of breast cancer in a patient comprising the step of administering to the patient.   35. A vaccine according to any one of claims 31 to 34 for use in the manufacture of a medicament for inhibiting the development of breast cancer in a patient. Diagnostic kit with the following:
(a) one or more monoclonal antibodies according to claim 9; and
(b) A kit containing a detection reagent. Diagnostic kit with the following:
(a) SEQ ID NOs: 78-86, 144, 145, 153, 167, 177, 193, 199, 205, 208, 215, 217, 220, 241, 242, 246, 248, 249, 252, 256, 267, 270 274, 277, 279, 282, 283, 285-287, 289, 290, one or more monoclonal antibodies that bind to a polypeptide encoded by a nucleotide sequence selected from the group consisting of:
(b) A kit containing a detection reagent.   The kit according to any one of claims 37 and 38, wherein the monoclonal antibody is immobilized on a solid support.   A diagnostic kit comprising two polymerase chain reaction primers, wherein at least one primer is specific for the RNA molecule of claim 4.   41. The diagnostic kit of claim 40, wherein the at least one polymerase chain reaction primer comprises at least about 10 contiguous nucleotides of the RNA molecule of claim 4.   A diagnostic kit comprising two polymerase chain reaction primers, wherein at least one primer is SEQ ID NO: 78-86, 144, 145, 153, 167, 177, 193, 199, 205, 208, 215, 217, 220, Poly encoded by a nucleotide sequence selected from the group consisting of 241, 242, 246, 248, 249, 252, 256, 267, 270, 274, 277, 279, 282, 283, 285-287, 289, and 290 A diagnostic kit that is specific for an RNA molecule encoding a peptide.   At least one polymerase chain reaction primer is SEQ ID NO: 78-86, 144, 145, 153, 167, 177, 193, 199, 205, 208, 215, 217, 220, 241, 242, 246, 248, 249, 252 , 256, 267, 270, 274, 277, 279, 282, 283, 285-287, 289, and 290, at least about 10 RNA molecules encoding a polypeptide encoded by a nucleotide sequence selected from the group consisting of 43. The diagnostic kit of claim 42, comprising continuous nucleotides.   A diagnostic kit comprising at least one oligonucleotide probe, wherein the oligonucleotide probe comprises at least about 15 contiguous nucleotides of the DNA molecule of claim 4.   A diagnostic kit comprising at least one oligonucleotide probe, wherein the oligonucleotide probe comprises SEQ ID NOs: 78-86, 144, 145, 153, 167, 177, 193, 199, 205, 208, 215, 217, 220, At least about 15 of the DNA sequence selected from the group consisting of 241, 242, 246, 248, 249, 252, 256, 267, 270, 274, 277, 279, 282, 283, 285-287, 289, and 290 A diagnostic kit comprising sequential nucleotides.   A diagnostic kit comprising at least one oligonucleotide probe specific for the DNA molecule of claim 4.   47. The kit of claim 46, wherein the oligonucleotide probe comprises at least about 15 contiguous nucleotides of the DNA molecule of claim 4.   SEQ ID NOs: 78-86, 144, 145, 153, 167, 177, 193, 199, 205, 208, 215, 217, 220, 241, 242, 246, 248, 249, 252, 256, 267, 270, 274, A diagnostic kit comprising at least one oligonucleotide probe specific for a DNA sequence selected from the group consisting of 277, 279, 282, 283, 285-287, 289, and 290.   The oligonucleotide probes are SEQ ID NOs: 78-86, 144, 145, 153, 167, 177, 193, 199, 205, 208, 215, 217, 220, 241, 242, 246, 248, 249, 252, 256, 49. The kit of claim 48, comprising at least about 15 contiguous nucleotides of a DNA sequence selected from the group consisting of 267, 270, 274, 277, 279, 282, 283, 285-287, 289, and 290.