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WO2000052165A2 - Compositions et methodes de therapie et de diagnostic du cancer du sein - Google Patents

Compositions et methodes de therapie et de diagnostic du cancer du sein Download PDF

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Publication number
WO2000052165A2
WO2000052165A2 PCT/US2000/005431 US0005431W WO0052165A2 WO 2000052165 A2 WO2000052165 A2 WO 2000052165A2 US 0005431 W US0005431 W US 0005431W WO 0052165 A2 WO0052165 A2 WO 0052165A2
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Prior art keywords
breast tumor
antigen
polynucleotide
tumor antigen
polynucleotides
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PCT/US2000/005431
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English (en)
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WO2000052165A3 (fr
Inventor
Michael J. Lodes
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Corixa Corporation
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Priority to AU33912/00A priority Critical patent/AU3391200A/en
Publication of WO2000052165A2 publication Critical patent/WO2000052165A2/fr
Publication of WO2000052165A3 publication Critical patent/WO2000052165A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/82—Translation products from oncogenes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/515—Animal cells
    • A61K2039/5156—Animal cells expressing foreign proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present invention relates generally to therapy and diagnosis of breast cancer.
  • the invention is more specifically related to polypeptides comprising at least a portion of a breast tumor antigen, and to polynucleotides encoding such polypeptides.
  • polypeptides and polynucleotides may be used in vaccines and pharmaceutical compositions for treatment of breast cancer, and for the diagnosis and monitoring of such cancer.
  • breast cancer is a significant health problem for women in the United States and throughout the world. Although advances have been made in detection and treatment of the disease, breast cancer remains the second leading cause of cancer- related deaths in women, affecting more than 180,000 women in the United States each year. For women in North America, the life-time odds of getting breast cancer are now one in eight.
  • this invention provides compositions and methods for the therapy of cancer, such as breast cancer.
  • the present invention provides polypeptides comprising an immunogenic portion of a breast tumor antigen, or a variant thereof that differs in one or more substitutions, deletions, additions and/or insertions such that the ability of the variant to react with antigen-specific antisera is not substantially diminished.
  • the polypeptide comprises a sequence that is encoded by a polynucleotide sequence selected from the group consisting of sequences recited in Figures 1A-1L (SEQ ID NOs: 1-35), Figures 2A-2I (SEQ ID NOs: 36-57) and SEQ ID NOs:90-186, and complements of such polynucleotides.
  • the present invention further provides polynucleotides that encode a polypeptide as described above or a portion thereof, expression vectors comprising such polynucleotides and host cells transformed or transfected with such expression vectors.
  • the present invention provides pharmaceutical compositions and vaccines.
  • a pharmaceutical composition comprises: (a) a polypeptide comprising at least an immunogenic portion of a breast tumor antigen, or a variant thereof that differs in one or more substitutions, deletions, additions and/or insertions such that the ability of the variant to react with antigen- specific antisera is not substantially diminished, wherein the breast tumor antigen comprises an amino acid sequence that is encoded by a polynucleotide sequence J
  • a vaccine comprises: (a) a polypeptide comprising at least an immunogenic portion of a breast tumor antigen, or a variant thereof that differs in one or more substitutions, deletions, additions and/or insertions such that the ability of the variant to react with antigen-specific antisera is not substantially diminished, wherein the breast tumor antigen comprises an amino acid sequence that is encoded by a polynucleotide sequence selected from the group consisting of: (i) polynucleotides recited in Figures 1A-1L (SEQ ID NOs: 1-35), Figures 2A-2I (SEQ ID NOs: 36-57) and SEQ ID NOs:90-186; and (ii) complements of the foregoing polynucleotides; and (b) a non-specific immune response enhancer.
  • compositions provided herein comprise: (a) a polynucleotide encoding at least 15 amino acid residues of a breast tumor antigen, or a variant thereof that differs in one or more substitutions, deletions, additions and/or insertions such that the ability of the variant to react with antigen- specific antisera is not substantially diminished, wherein the breast tumor antigen comprises an amino acid sequence that is encoded by a polynucleotide sequence selected from the group consisting of: (i) polynucleotides recited in Figures 1A-1L (SEQ ID NOs: 1-35), Figures 2A-2I (SEQ ID NOs: 36-57) and SEQ ID NOs:90-186; and (ii) complements of the foregoing polynucleotides; and (b) a physiologically acceptable carrier.
  • the present invention provides vaccines comprising: (a) a polynucleotide encoding at least 15 amino acid residues of a breast tumor antigen, or a variant thereof that differs in one or more substitutions, deletions, additions and/or insertions such that the ability of the variant to react with antigen- specific antisera is not substantially diminished, wherein the breast tumor antigen comprises an amino acid sequence that is encoded by a polynucleotide sequence selected from the group consisting of: (i) polynucleotides recited in Figures 1A-1L (SEQ ID NOs: 1-35), Figures 2A-2I (SEQ ID NOs: 36-57) and SEQ ID NOs:90-186; and (ii) complements of the foregoing polynucleotides; and (b) a non-specific immune response enhancer.
  • a polynucleotide encoding at least 15 amino acid residues of a breast tumor antigen, or a variant thereof that differs in one
  • the present invention further provides pharmaceutical compositions that comprise: (a) an antibody or antigen-binding fragment thereof that specifically binds to a breast tumor antigen, wherein the breast tumor antigen comprises an amino acid sequence that is encoded by a polynucleotide sequence selected from the group consisting of: (i) polynucleotides recited in Figures 1A-1L (SEQ ID NOs: 1-35), Figures 2A-2I (SEQ ID NOs: 36-57) and SEQ ID NOs:90-186; and (ii) complements of the foregoing polynucleotides; and (b) a physiologically acceptable carrier.
  • a polynucleotide sequence selected from the group consisting of: (i) polynucleotides recited in Figures 1A-1L (SEQ ID NOs: 1-35), Figures 2A-2I (SEQ ID NOs: 36-57) and SEQ ID NOs:90-186; and (ii) complements of the foregoing poly
  • the present invention provides pharmaceutical compositions comprising: (a) an antigen presenting cell that expresses a polypeptide comprising at least an immunogenic portion of a breast tumor antigen, or a variant thereof that differs in one or more substitutions, deletions, additions and/or insertions such that the ability of the variant to react with antigen-specific antisera is not substantially diminished, wherein the breast tumor antigen comprises an amino acid sequence that is encoded by a polynucleotide sequence selected from the group consisting of: (i) polynucleotides recited in Figures 1A-1L (SEQ ID NOs: 1-35), Figures 2A-2I (SEQ ID NOs: 36-57) and SEQ ID NOs:90-186; and (ii) complements of the foregoing polynucleotides; and (b) a pharmaceutically acceptable carrier or excipient.
  • Antigen presenting cells include dendritic cells and macrophages.
  • vaccines comprising: (a) an antigen presenting cell that expresses a polypeptide comprising at least an immunogenic portion of a breast tumor antigen, or a variant thereof that differs in one or more substitutions, deletions, additions and/or insertions such that the ability of the variant to react with antigen-specific antisera is not substantially diminished, wherein the breast tumor antigen comprises an amino acid sequence that is encoded by a polynucleotide sequence selected from the group consisting of: (i) polynucleotides recited in Figures 1A-1L (SEQ ID NOs: 1-35), Figures 2A-2I (SEQ ID NOs: 36-57) and SEQ ID NOs:90-186; and (ii) complements of the foregoing polynucleotides; and (b) a non-specific immune response enhancer.
  • a polynucleotide sequence selected from the group consisting of: (i) polynucleotides recited in Figures 1A
  • the present invention further provides, in other aspects, fusion proteins that comprise at least one polypeptide as described above, as well as polynucleotides encoding such fusion proteins.
  • compositions comprising a fusion protein or polynucleotide encoding a fusion protein in combination with a physiologically acceptable carrier are provided.
  • Vaccines are further provided, within other aspects, comprising a fusion protein or polynucleotide encoding a fusion protein in combination with a non-specific immune response enhancer.
  • the present invention provides methods for inhibiting the development of a cancer in a patient, comprising administering to a patient a pharmaceutical composition or vaccine as recited above.
  • the present invention further provides, within other aspects, methods for removing tumor cells from a biological sample, comprising contacting a biological sample with T cells that specifically react with a breast tumor antigen, wherein the breast tumor antigen comprises an amino acid sequence that is encoded by a polynucleotide sequence selected from the group consisting of: (i) polynucleotides recited in Figures 1A-1L (SEQ ID NOs: 1-35), Figures 2A-2I (SEQ ID NOs: 36-57) and SEQ ID NOs:90-186; and (ii) complements of the foregoing polynucleotides, wherein the step of contacting is performed under conditions and for a time sufficient to permit the removal of cells expressing the antigen from the sample.
  • methods for inhibiting the development of breast cancer in a patient, comprising administering to a patient a biological sample treated as described above.
  • Methods are further provided, within other aspects, for stimulating and/or expanding T cells specific for a breast tumor antigen, comprising contacting T cells with one or more of: (i) a polypeptide comprising at least an immunogenic portion of a breast tumor antigen, or a variant thereof that differs in one or more substitutions, deletions, additions and/or insertions such that the ability of the variant to react with antigen-specific antisera is not substantially diminished, wherein the breast tumor antigen comprises an amino acid sequence that is encoded by a polynucleotide sequence selected from the group consisting of: polynucleotides recited in Figures 1A-1L (SEQ ID NOs: 1-35), Figures 2A-2I (SEQ ID NOs: 36-57) and SEQ ID NOs:90-186; and complements of the foregoing polynucleotides; (ii) a polynucleotide encoding such a polypeptide; and/or (iii) an antigen presenting
  • the present invention provides methods for inhibiting the development of breast cancer in a patient, comprising administering to a patient an effective amount of a T cell population as described above.
  • the present invention further provides methods for inhibiting the development of breast cancer in a patient, comprising the steps of: (a) incubating CD4 + and/or CD8+ T cells isolated from a patient with one or more of: (i) a polypeptide comprising at least an immunogenic portion of an breast tumor antigen or a variant thereof that differs in one or more substitutions, deletions, additions and/or insertions such that the ability of the variant to react with antigen-specific antisera is not substantially diminished, wherein the breast tumor antigen comprises an amino acid sequence that is encoded by a polynucleotide sequence selected from the group consisting of: polynucleotides recited in Figures 1A-1L (SEQ ID NOs: 1-35), Figures 2A-2I (SEQ ID NOs: 36-57) and SEQ ID NOs:90-186; and complements of such polynucleotides; (ii) a polynucleotide encoding such a polypeptide; or (ii
  • the present provides methods for determining the presence or absence of a cancer in a patient, comprising (a) contacting a biological sample obtained from a patient with a binding agent that binds to a polypeptide as recited above; (b) detecting in the sample an amount of polypeptide that binds to the binding agent; and (c) comparing the amount of polypeptide with a predetermined cutoff value, and therefrom determining the presence or absence of a cancer in the patient.
  • the binding agent is an antibody, more preferably a monoclonal antibody.
  • the cancer may be breast cancer.
  • the present invention also provides, within other aspects, methods for monitoring the progression of a cancer in a patient.
  • Such methods comprise the steps of: (a) contacting a biological sample obtained from a patient at a first point in time with a binding agent that binds to a polypeptide as recited above; (b) detecting in the sample an amount of polypeptide that binds to the binding agent; (c) repeating steps (a) and (b) using a biological sample obtained from the patient at a subsequent point in time; and (d) comparing the amount of polypeptide detected in step (c) with the amount detected in step (b) and therefrom monitoring the progression of the cancer in the patient.
  • the present invention further provides, within other aspects, methods for determining the presence or absence of a cancer in a patient, comprising the steps of: (a) contacting a biological sample obtained from a patient with an oligonucleotide that hybridizes to a polynucleotide that encodes a breast tumor antigen, wherein the antigen comprises an amino acid sequence that is encoded by a polynucleotide sequence selected from the group consisting of: (i) polynucleotides recited in Figure 1 or Figure 2; and (ii) complements of the foregoing polynucleotides; (b) detecting in the sample a level of a polynucleotide that hybridizes to the oligonucleotide; and (c) comparing the level of polynucleotide that hybridizes to the oligonucleotide with a predetermined cut- off value, and therefrom determining the presence or absence of a cancer in the patient.
  • the amount of mRNA is detected via polymerase chain reaction using, for example, at least one oligonucleotide primer that hybridizes to a polynucleotide that encodes a polypeptide as recited above, or a complement of such a polynucleotide.
  • the amount of mRNA is detected using a hybridization technique, employing an oligonucleotide probe that hybridizes to a polynucleotide that encodes a polypeptide as recited above, or a complement of such a polynucleotide.
  • methods for monitoring the progression of a cancer in a patient comprising the steps of: (a) contacting a biological sample obtained from a patient with an oligonucleotide that hybridizes to a polynucleotide that encodes a breast tumor antigen, wherein the antigen comprises an amino acid sequence that is encoded by a polynucleotide sequence selected from the group consisting of: (i) polynucleotides recited in Figures 1A-1L (SEQ ID NOs: 1-35), Figures 2A-2I (SEQ ID NOs: 36-57) and SEQ ID NOs:90-186; and (ii) complements of the foregoing polynucleotides; (b) detecting in the sample an amount of a polynucleotide that hybridizes to the oligonucleotide; (c) repeating steps (a) and (b) using a biological sample obtained from the patient at a subsequent point in time; and (d)
  • the present invention provides antibodies, such as monoclonal antibodies, that bind to a polypeptide as described above, as well as diagnostic kits comprising such antibodies. Diagnostic kits comprising one or more oligonucleotide probes or primers as described above are also provided.
  • Figures 1A-1L depict partial sequences of representative polynucleotides encoding breast tumor antigens (SEQ ID NOs: 1-35), and the predicted amino acid sequences of the encoded polypeptides (SEQ ID NOs: 58-77).
  • Figures 2A-2I depict partial sequences of representative polynucleotides encoding further breast tumor antigens (SEQ ID NOs: 36-57). and the predicted amino acid sequences of the encoded polypeptides (SEQ ID NOs: 78-89). DETAILED DESCRIPTION OF THE INVENTION
  • compositions and methods for the therapy and diagnosis of cancer such as breast cancer.
  • the compositions described herein may include immunogenic polypeptides, nucleic acid sequences encoding such polypeptides, binding agents such as antibodies that bind to a polypeptide, antigen presenting cells (APCs) that express such a polypeptide and/or immune system cells (e.g., T cells).
  • APCs antigen presenting cells
  • Polypeptides of the present invention generally comprise at least an immunogenic portion of a breast tumor antigen or a variant thereof.
  • breast tumor antigen is a protein that is expressed by breast tumor cells (preferably human cells) and that reacts detectably (within an immunoassay, such as an ELISA or Western blot) with antisera of a patient afflicted with breast cancer.
  • Nucleic acid sequences of the subject invention generally comprise a DNA or RNA sequence that encodes all or a portion of such a polypeptide, or that is complementary to such a sequence.
  • Antibodies are generally immune system proteins, or antigen-binding fragments thereof, that are capable of binding to a portion of a polypeptide as described above.
  • Antigen presenting cells include dendritic cells and macrophages that express a polypeptide as described above.
  • T cells that may be employed within such compositions are generally T cells that are specific for a polypeptide as described above.
  • the present invention is based on the discovery of previously unknown human breast tumor antigens. Partial sequences of polynucleotides encoding specific breast tumor antigens are provided in Figures 1A-1L (SEQ ID NOs: 1-35), Figures 2A- 21 (SEQ ID NOs: 36-57) and SEQ ID NOs:90-186. Certain extended sequences are also provided in SEQ ID NOs: 187- 192.
  • Any polynucleotide that encodes a breast tumor antigen or a portion or other variant thereof as described herein is encompassed by the present invention.
  • Preferred polynucleotides comprise at least 15 consecutive nucleotides. and preferably at least 30 consecutive nucleotides, that encode a portion of a breast tumor antigen.
  • a polynucleotide encodes an immunogenic portion of a breast tumor antigen.
  • Polynucleotides complementary to any such sequences are also encompassed by the present invention.
  • Polynucleotides may be single-stranded (coding or antisense) or double-stranded, and may be DNA (genomic, cDNA or synthetic) or RNA molecules. Additional coding or non-coding sequences may, but need not, be present within a polynucleotide of the present invention, and a polynucleotide may, but need not, be linked to other molecules and/or support materials.
  • Polynucleotides may comprise a native sequence (i.e., an endogenous sequence that encodes a breast tumor antigen or a portion thereof) or may comprise a variant of such a sequence.
  • Polynucleotide variants may contain one or more substitutions, additions, deletions and/or insertions such that the immunogenicity of the encoded polypeptide is not diminished, relative to a native breast tumor antigen.
  • the effect on the immunogenicity of the encoded polypeptide may generally be assessed as described herein.
  • Variants preferably exhibit at least about 70% identity, more preferably at least about 80% identity and most preferably at least about 90% identity to a polynucleotide sequence that encodes a native breast tumor antigen or a portion thereof.
  • the percent identity may be readily determined by comparing sequences using computer algorithms well known to those of ordinary skill in the art, such as Megalign, using default parameters. Certain variants are substantially homologous to a native gene, or a portion or complement thereof. Such polynucleotide variants are capable of hybridizing under moderately stringent conditions to a naturally occurring DNA sequence encoding a native breast tumor antigen (or a complementary sequence).
  • Suitable moderately stringent conditions include prewashing in a solution of 5 X SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0); hybridizing at 50°C-65°C, 5 X SSC, overnight; followed by washing twice at 65°C for 20 minutes with each of 2X, 0.5X and 0.2X SSC containing 0.1 % SDS.
  • Polynucleotides may be prepared using any of a variety of techniques. For example, a polynucleotide may be identified, as described in more detail below, by screening a breast tumor cDNA expression library with sera of patients with breast cancer. Alternatively, polypeptides may be amplified from cDNA prepared from breast tumor cells. Such polynucleotides may be amplified via polymerase chain reaction (PCR). For this approach, sequence-specific primers may be designed based on the sequences provided herein, and may be purchased or synthesized. An amplified portion may be used to isolate a full length gene from a suitable library (e.g., a breast tumor cDNA library) using well known techniques.
  • a suitable library e.g., a breast tumor cDNA library
  • a library (cDNA or genomic) is screened using one or more polynucleotide probes or primers suitable for amplification.
  • a library is size-selected to include larger molecules. Random primed libraries may also be preferred for identifying 5' and upstream regions of genes. Genomic libraries are preferred for obtaining introns and extending 5' sequences.
  • a partial sequence may be labeled (e.g., by nick-translation or end-labeling with 32 P) using well known techniques.
  • a bacterial or bacteriophage library is then screened by hybridizing filters containing denatured bacterial colonies (or lawns containing phage plaques) with the labeled probe (see Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor, NY, 1989). Hybridizing colonies or plaques are selected and expanded, and the DNA is isolated for further analysis.
  • cDNA clones may be analyzed to determine the amount of additional sequence by, for example, PCR using a primer from the partial sequence and a primer from the vector.
  • Restriction maps and partial sequences may be generated to identify one or more overlapping clones.
  • the complete sequence may then be determined using standard techniques, which may involve generating a series of deletion clones.
  • the resulting overlapping sequences are then assembled into a single contiguous sequence.
  • a full length cDNA molecule can be generated by ligating suitable fragments, using well known techniques.
  • Primers are preferably 22-30 nucleotides in length, have a GC content of at least 50% and anneal to the target sequence at temperatures of about 68°C to 72°C.
  • the amplified region may be sequenced as described above, and overlapping sequences assembled into a contiguous sequence.
  • One such amplification technique is inverse PCR (see Triglia et al., Nucl.
  • the fragment is then circularized by intramolecular ligation and used as a template for PCR with divergent primers derived from the known region.
  • sequences adjacent to a partial sequence may be retrieved by amplification with a primer to a linker sequence and a primer specific to a known region.
  • the amplified sequences are typically subjected to a second round of amplification with the same linker primer and a second primer specific to the known region.
  • a variation on this procedure, which employs two primers that initiate extension in opposite directions from the known sequence, is described in WO 96/38591.
  • RACE Rapid amplification of cDNA ends
  • This technique involves the use of an internal primer and an external primer, which hybridizes to a polyA region or vector sequence, to identify sequences that are 5' and 3' of a known sequence. Additional techniques include capture PCR (Lagerstrom et al., PCR Methods Applic. 7:1 1 1-19, 1991) and walking PCR (Parker et al, Nucl. Acids. Res. 79:3055-60, 1991). Other methods employing amplification may also be employed to obtain a full length cDNA sequence.
  • EST expressed sequence tag
  • FIGS 1A-1L Certain nucleic acid sequences of cDNA molecules encoding portions of breast tumor antigens are provided in Figures 1A-1L (SEQ ID NOs: 1-35), Figures 2A- 21 (SEQ ID NOs: 36-57) and SEQ ID NOs:90-186. These polynucleotides were isolated by serological screening of a breast tumor cDNA expression library. The library was prepared from unamplified cDNA derived from a pool of three human breast tumors grown in a SCID mouse, in the vector pScreen. Sera from two human patients with breast cancer were pooled for the screen.
  • polynucleotides recited herein, as well as full length polynucleotides comprising such sequences, other portions of such full length polynucleotides, and sequences complementary to all or a portion of such full length molecules, are specifically encompassed by the present invention.
  • Polynucleotide variants may generally be prepared by any method known in the art, including chemical synthesis by, for example, solid phase phosphoramidite chemical synthesis. Modifications in a polynucleotide sequence may also be introduced using standard mutagenesis techniques, such as oligonucleotide- directed site-specific mutagenesis (see Adelman et al., DNA 2:183, 1983). Alternatively, RNA molecules may be generated by in vitro or in vivo transcription of DNA sequences encoding a breast tumor antigen, or portion thereof, provided that the DNA is incorporated into a vector with a suitable RNA polymerase promoter (such as T7 or SP6). Certain portions may be used to prepare an encoded polypeptide, as described herein.
  • a suitable RNA polymerase promoter such as T7 or SP6
  • a portion may be administered to a patient such that the encoded polypeptide is generated in vivo (e.g., by transfecting antigen-presenting cells such as dendritic cells with a cDNA construct encoding a breast tumor antigen polypeptide, and administering the transfected cells to the patient).
  • antigen-presenting cells such as dendritic cells
  • a cDNA construct encoding a breast tumor antigen polypeptide
  • a portion of a sequence complementary to a coding sequence may also be used as a probe or to modulate gene expression.
  • cDNA constructs that can be transcribed into antisense RNA may also be introduced into cells of tissues to facilitate the production of antisense RNA.
  • An antisense polynucleotide may be used, as described herein, to inhibit expression of a breast tumor antigen.
  • Antisense technology can be used to control gene expression through triple- helix formation, which compromises the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors or regulatory molecules (see Gee et al., In Huber and Carr, Molecular and Immunologic Approaches. Futura Publishing Co.
  • an antisense molecule may be designed to hybridize with a control region of a gene (e.g., promoter, enhancer or transcription initiation site), and block transcription of the gene; or to block translation by inhibiting binding of a transcript to ribosomes.
  • a control region of a gene e.g., promoter, enhancer or transcription initiation site
  • a portion of a coding sequence or a complementary sequence may also be designed as a probe or primer to detect gene expression.
  • Probes may be labeled by a variety of reporter groups, such as radionuclides and enzymes, and are preferably at least 10 nucleotides in length, more preferably at least 20 nucleotides in length and still more preferably at least 30 nucleotides in length.
  • Primers as noted above, are preferably 22-30 nucleotides in length. Any polynucleotide may be further modified to increase stability in vivo.
  • flanking sequences at the 5' and/or 3' ends Possible modifications include, but are not limited to, the addition of flanking sequences at the 5' and/or 3' ends; the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase linkages in the backbone; and/or the inclusion of nontraditional bases such as inosine, queosine and wybutosine, as well as acetyl- methyl-, thio- and other modified forms of adenine, cytidine, guanine, thymine and uridine.
  • Nucleotide sequences as described herein may be joined to a variety of other nucleotide sequences using established recombinant DNA techniques.
  • a polynucleotide may be cloned into any of a variety of cloning vectors, including plasmids, phagemids, lambda phage derivatives and cosmids.
  • Vectors of particular interest include expression vectors, replication vectors, probe generation vectors and sequencing vectors.
  • a vector will contain an origin of replication functional in at least one organism, convenient restriction endonuclease sites and one or more selectable markers. Other elements will depend upon the desired use, and will be apparent to those of ordinary skill in the art.
  • polynucleotides may be formulated so as to permit entry into a cell of a mammal, and expression therein. Such formulations are particularly useful for therapeutic purposes, as described below.
  • a polynucleotide may be incorporated into a viral vector such as, but not limited to, adenovirus, adeno-associated virus, retrovirus, or vaccinia or other pox virus (e.g., avian pox virus). Techniques for incorporating DNA into such vectors are well known to those of ordinary skill in the art.
  • a retroviral vector may additionally transfer or incorporate a gene for a selectable marker (to aid in the identification or selection of transduced cells) and/or a targeting moiety, such as a gene that encodes a ligand for a receptor on a specific target cell, to render the vector target specific. Targeting may also be accomplished using an antibody, by methods known to those of ordinary skill in the art.
  • Other formulations for therapeutic purposes include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.
  • a preferred colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (i.e., an artificial membrane vesicle). The preparation and use of such systems is well known in the art.
  • polypeptides may comprise at least an immunogenic portion of a breast tumor antigen or a variant thereof, as described herein.
  • a breast tumor antigen is a protein that is expressed by breast tumor cells and that reacts detectably within an immunoassay (such as an ELISA) with antisera from a patient with breast cancer.
  • Polypeptides as described herein may be of any length. Additional sequences derived from the native protein and/or heterologous sequences may be present, and such sequences may (but need not) possess further immunogenic or antigenic properties.
  • immunogenic portion is a portion of an antigen that is recognized (i.e., specifically bound) by a B-cell and/or T-cell surface antigen receptor.
  • immunogenic portions generally comprise at least 5 amino acid residues, more preferably at least 10, and still more preferably at least 20 amino acid residues of a breast tumor antigen or a variant thereof.
  • Preferred immunogenic portions are encoded by sequences recited in Figures 1A-1L (SEQ ID NOs: 1-35), Figures 2A-2I (SEQ ID NOs: 36-57) and SEQ ID NOs:90-186, or the complements thereof.
  • immunogenic portions may generally be identified using well known techniques, such as those summarized in Paul, Fundamental Immunology, 3rd ed., 243-247 (Raven Press, 1993) and references cited therein. Such techniques include screening polypeptides for the ability to react with antigen-specific antibodies, antisera and/or T-cell lines or clones.
  • antisera and antibodies are "antigen-specific” if they specifically bind to an antigen (i.e., they react with the antigen in an ELISA or other immunoassay, and do not react detectably with unrelated proteins).
  • antisera and antibodies may be prepared as described herein, and using well known techniques.
  • An immunogenic portion of a native breast carcinoma antigen is a portion that reacts with such antisera and/or T-cells at a level that is not substantially less than the reactivity of the full length polypeptide (e.g., in an ELISA and/or T-cell reactivity assay). Such immunogenic portions may react within such assays at a level that is similar to or greater than the reactivity of the full length polypeptide.
  • Such screens may generally be performed using methods well known to those of ordinary skill in the art, such as those described in Hariow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988.
  • a polypeptide may be immobilized on a solid support and contacted with patient sera to allow binding of antibodies within the sera to the immobilized polypeptide. Unbound sera may then be removed and bound antibodies detected using, for example, 125 I-labeled Protein A.
  • a composition may comprise a variant of a native breast tumor antigen.
  • a polypeptide "variant,” as used herein, is a polypeptide that differs from a native breast tumor antigen in one or more substitutions, deletions, additions and/or insertions, such that the immunogenicity of the polypeptide is not substantially diminished.
  • the ability of a variant to react with antigen-specific antisera may be enhanced or unchanged, relative to the native antigen, or may be diminished by less than 50%, and preferably less than 20%. relative to the native antigen.
  • Such variants may generally be identified by modifying one of the above polypeptide sequences and evaluating the reactivity of the modified polypeptide with antigen-specific antibodies or antisera as described herein.
  • a variant contains conservative substitutions.
  • a "conservative substitution” is one in which an amino acid is substituted for another amino acid that has similar properties, such that one skilled in the art of peptide chemistry would expect the secondary structure and hydropathic nature of the polypeptide to be substantially unchanged.
  • Amino acid substitutions may generally be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or the amphipathic nature of the residues.
  • negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; and serine, threonine, phenylalanine and tyrosine.
  • amino acids that may represent conservative changes include: (1) ala, pro, gly, glu, asp, gin, 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.
  • a variant may also, or alternatively, contain nonconservative changes.
  • Variants may also (or alternatively) be modified by, for example, the deletion or addition of amino acids that have minimal influence on the immunogenicity, secondary structure and hydropathic nature of the polypeptide.
  • polypeptides may comprise a signal (or leader) sequence at the N-terminal end of the protein which co-translationally or post- translationally directs transfer of the protein.
  • the polypeptide may also be conjugated to a linker or other sequence for ease of synthesis, purification or identification of the polypeptide (e.g., poly-His), or to enhance binding of the polypeptide to a solid support.
  • a polypeptide may be conjugated to an immunoglobulin Fc region.
  • Polypeptides may be prepared using any of a variety of well known techniques. Recombinant polypeptides encoded by DNA sequences as described above may be readily prepared from the DNA sequences using any of a variety of expression vectors known to those of ordinary skill in the art.
  • Expression may be achieved in any appropriate host cell that has been transformed or transfected with an expression vector containing a DNA molecule that encodes a recombinant polypeptide.
  • Suitable host cells include prokaryotes, yeast and higher eukaryotic cells.
  • the host cells employed are E. coli, yeast or a mammalian cell line such as COS or CHO.
  • Supematants from suitable host/vector systems which secrete recombinant protein or polypeptide into culture media may be first concentrated using a commercially available filter. Following concentration, the concentrate may be applied to a suitable purification matrix such as an affinity matrix or an ion exchange resin. Finally, one or more reverse phase HPLC steps can be employed to further purify a recombinant polypeptide.
  • Portions and other variants having fewer than about 100 amino acids, and generally fewer than about 50 amino acids, may also be generated by synthetic means, using techniques well known to those of ordinary skill in the art.
  • such polypeptides may be synthesized using any of the commercially available solid- phase techniques, such as the Merrifield solid-phase synthesis method, where amino acids are sequentially added to a growing amino acid chain. See Merrifield, J. Am. Chem. Soc. 55:2149-2146, 1963.
  • Equipment for automated synthesis of polypeptides is commercially available from suppliers such as Applied BioSystems, Inc. (Foster City, CA), and may be operated according to the manufacturer's instructions.
  • a polypeptide may be a fusion protein that comprises multiple polypeptides as described herein, or that comprises one polypeptide as described herein and a known tumor antigen, such as a breast tumor antigen, or a variant of such an antigen. Fusion proteins may generally be prepared using standard techniques. For example, a fusion protein may be prepared recombinantly. Briefly, DNA sequences encoding the polypeptide components may be assembled separately, and ligated into an appropriate expression vector. The 3' end of the DNA sequence encoding one polypeptide component is ligated. with or without a peptide linker, to the 5' end of a DNA sequence encoding the second polypeptide component so that the reading frames of the sequences are in phase. This permits translation into a single fusion protein that retains the biological activity of both component polypeptides.
  • a peptide linker sequence may be employed to separate the first and the second polypeptide components by a distance sufficient to ensure that each polypeptide folds into its secondary and tertiary structures.
  • Such a peptide linker sequence is incorporated into the fusion protein using standard techniques well known in the art.
  • Suitable peptide linker sequences may be chosen based on the following factors: (1) their ability to adopt a flexible extended conformation; (2) their inability to adopt a secondary structure that could interact with functional epitopes on the first and second polypeptides; and (3) the lack of hydrophobic or charged residues that might react with the polypeptide functional epitopes.
  • Preferred peptide linker sequences contain Gly, Asn and Ser residues.
  • linker sequences which may be usefully employed as linkers include those disclosed in Maratea et al., Gene 40:39-46, 1985; Murphy et al., Proc. Natl. Acad. Sci. USA 53:8258-8262, 1986; U.S. Patent No. 4,935,233 and U.S. Patent No. 4,751,180.
  • the linker sequence may generally be from 1 to about 50 amino acids in length. Linker sequences are not required when the first and second polypeptides have non-essential N-terminal amino acid regions that can be used to separate the functional domains and prevent steric interference.
  • the ligated DNA sequences are operably linked to suitable transcriptional or translational regulatory elements.
  • the regulatory elements responsible for expression of DNA are located only 5' to the DNA sequence encoding the first polypeptides.
  • stop codons required to end translation and transcription termination signals are only present 3' to the DNA sequence encoding the second polypeptide.
  • Fusion proteins are also provided that comprise a polypeptide of the present invention together with an unrelated immunogenic protein.
  • the immunogenic protein is capable of eliciting a recall response.
  • examples of such proteins include tetanus, tuberculosis and hepatitis proteins (see, for example, Stoute et al. NeM' Engl J. Med., 536:86-91 , 1997).
  • polypeptides including fusion proteins and polynucleotides as described herein are isolated.
  • An "isolated" polypeptide or polynucleotide is one that is removed from its original environment.
  • a naturally-occu ⁇ ing protein is isolated if it is separated from some or all of the coexisting materials in the natural system.
  • polypeptides are at least about 90% pure, more preferably at least about 95% pure and most preferably at least about 99% pure.
  • a polynucleotide is considered to be isolated if, for example, it is cloned into a vector that is not a part of the natural environment.
  • the present invention further provides agents, such as antibodies and antigen-binding fragments thereof, that specifically bind to a breast tumor antigen.
  • an antibody, or antigen-binding fragment thereof is said to "specifically bind" to a breast tumor antigen if it reacts at a detectable level (within, for example, an ELISA) with a breast tumor antigen, and does not react detectably with unrelated proteins under similar conditions.
  • binding refers to a noncovalent association between two separate molecules such that a "complex" is formed. The ability to bind may be evaluated by, for example, determining a binding constant for the formation of the complex.
  • the binding constant is the value obtained when the concentration of the complex is divided by the product of the component concentrations.
  • two compounds are said to "bind,” in the context of the present invention, when the binding constant for complex formation exceeds about 10 3 L/mol.
  • the binding constant maybe determined using methods well known in the art.
  • Binding agents may be further capable of differentiating between patients with and without a cancer, such as breast cancer, using the representative assays provided herein.
  • a cancer such as breast cancer
  • antibodies or other binding agents that bind to a breast tumor antigen will generate a signal indicating the presence of a cancer in at least about 20% of patients with the disease, and will generate a negative signal indicating the absence of the disease in at least about 90% of individuals without the cancer.
  • biological samples e.g., blood, sera, urine and/or tumor biopsies
  • tissue samples e.g., blood, sera, urine and/or tumor biopsies
  • each binding agent should satisfy the above criteria; however, those of ordinary skill in the art will recognize that binding agents may be used in combination to improve sensitivity.
  • a binding agent may be a ribosome, with or without a peptide component, and RNA molecule or a polypeptide.
  • a binding agent is an antibody or an antigen-binding fragment thereof.
  • Antibodies may be prepared by any of a variety of techniques known to those of ordinary skill in the art. See, e.g., Hariow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. In general, antibodies can be produced by cell culture techniques, including the generation of monoclonal antibodies as described herein, or via transfection of antibody genes into suitable bacterial or mammalian cell hosts, in order to allow for the production of recombinant antibodies.
  • an immunogen comprising the polypeptide is initially injected into any of a wide variety of mammals (e.g., mice, rats, rabbits, sheep or goats).
  • the polypeptides of this invention may serve as the immunogen without modification.
  • a superior immune response may be elicited if the polypeptide is joined to a carrier protein, such as bovine serum albumin or keyhole limpet hemocyanin.
  • the immunogen is injected into the animal host, preferably according to a predetermined schedule incorporating one or more booster immunizations, and the animals are bled periodically.
  • Monoclonal antibodies specific for the antigenic polypeptide of interest may be prepared, for example, using the technique of Kohler and Milstein, Ewr. J. Immunol. 6:511-519, 1976, and improvements thereto. Briefly, these methods involve the preparation of immortal cell lines capable of producing antibodies having the desired specificity (i.e., reactivity with the polypeptide of interest). Such cell lines may be produced, for example, from spleen cells obtained from an animal immunized as described above.
  • the spleen cells are then immortalized by, for example, fusion with a myeloma cell fusion partner, preferably one that is syngeneic with the immunized animal.
  • a myeloma cell fusion partner preferably one that is syngeneic with the immunized animal.
  • a variety of fusion techniques may be employed.
  • the spleen cells and myeloma cells may be combined with a nonionic detergent for a few minutes and then plated at low density on a selective medium that supports the growth of hybrid cells, but not myeloma cells.
  • a preferred selection technique uses HAT (hypoxanthine, aminopterin, thymidine) selection. After a sufficient time, usually about 1 to 2 weeks, colonies of hybrids are observed. Single colonies are selected and their culture supematants tested for binding activity against the polypeptide. Hybridomas having high reactivity and specificity are preferred.
  • Monoclonal antibodies may be isolated from the supematants of growing hybridoma colonies.
  • various techniques may be employed to enhance the yield, such as injection of the hybridoma cell line into the peritoneal cavity of a suitable vertebrate host, such as a mouse.
  • Monoclonal antibodies may then be harvested from the ascites fluid or the blood. Contaminants may be removed from the antibodies by conventional techniques, such as chromatography, gel filtration, precipitation, and extraction.
  • the polypeptides of this invention may be used in the purification process in, for example, an affinity chromatography step.
  • the use of antigen-binding fragments of antibodies may be preferred. Such fragments include Fab fragments, which may be prepared using standard techniques.
  • immunoglobulins may be purified from rabbit serum by affinity chromatography on Protein A bead columns (Hariow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988) and digested by papain to yield Fab and Fc fragments.
  • the Fab and Fc fragments may be separated by affinity chromatography on protein A bead columns.
  • Monoclonal antibodies of the present invention may be coupled to one or more therapeutic agents.
  • Suitable agents in this regard include radionuclides.
  • Preferred radionuclides include 90 Y, 123 I, 125 I, ,3, I, ,86 Re, 188 Re, 21 , At, and 212 Bi.
  • Prefe ⁇ ed drugs include methotrexate, and pyrimidine and purine analogs.
  • Prefe ⁇ ed differentiation inducers include phorbol esters and butyric acid.
  • Prefe ⁇ ed toxins include ricin, abrin, diptheria toxin, cholera toxin, gelonin. Pseudomonas exotoxin, Shigella toxin, and pokeweed antiviral protein.
  • a therapeutic agent may be coupled (e.g., covalently bonded) to a suitable monoclonal antibody either directly or indirectly (e.g., via a linker group).
  • a direct reaction between an agent and an antibody is possible when each possesses a substituent capable of reacting with the other.
  • a nucleophilic group such as an amino or sulfhydryl group
  • on one may be capable of reacting with a carbonyl- containing group, such as an anhydride or an acid halide, or with an alkyl group containing a good leaving group (e.g. , a halide) on the other.
  • a linker group can function as a spacer to distance an antibody from an agent in order to avoid interference with binding capabilities.
  • a linker group can also serve to increase the chemical reactivity of a substituent on an agent or an antibody, and thus increase the coupling efficiency. An increase in chemical reactivity may also facilitate the use of agents, or functional groups on agents, which otherwise would not be possible. It will be evident to those skilled in the art that a variety of bifunctional or polyfunctional reagents, both homo- and hetero-functional (such as those described in the catalog of the Pierce Chemical Co., Rockford, IL), may be employed as the linker group.
  • Coupling may be effected, for example, through amino groups, carboxyl groups, sulfhydryl groups or oxidized carbohydrate residues.
  • a linker group which is cleavable during or upon intemalization into a cell.
  • a number of different cleavable linker groups have been described.
  • the mechanisms for the intracellular release of an agent from these linker groups include cleavage by reduction of a disulfide bond (e.g., U.S. Patent No.
  • multiple molecules of an agent are coupled to one antibody molecule.
  • more than one type of agent may be coupled to one antibody.
  • immunoconjugates with more than one agent may be prepared in a variety of ways.
  • more than one agent may be coupled directly to an antibody molecule, or linkers which provide multiple sites for attachment can be used.
  • linkers which provide multiple sites for attachment can be used.
  • a ca ⁇ ier can be used.
  • a ca ⁇ ier may bear the agents in a variety of ways, including covalent bonding either directly or via a linker group.
  • Suitable ca ⁇ iers include proteins such as albumins (e.g., U.S. Patent No. 4,507,234, to Kato et al.), peptides and polysaccharides such as aminodextran (e.g., U.S. Patent No. 4,699,784, to Shih et al.).
  • a ca ⁇ ier may also bear an agent by noncovalent bonding or by encapsulation, such as within a liposome vesicle (e.g., U.S. Patent Nos. 4,429,008 and 4,873,088).
  • Ca ⁇ iers specific for radionuclide agents include radiohalogenated small molecules and chelating compounds.
  • U.S. Patent No. 4,735,792 discloses representative radiohalogenated small molecules and their synthesis.
  • a radionuclide chelate may be formed from chelating compounds that include those containing nitrogen and sulfur atoms as the donor atoms for binding the metal, or metal oxide, radionuclide.
  • U.S. Patent No. 4,673,562 to Davison et al. discloses representative chelating compounds and their synthesis.
  • routes of administration for the antibodies and immunoconjugates may be used. Typically, administration will be intravenous, intramuscular, subcutaneous or in the bed of a resected tumor. It will be evident that the precise dose of the antibody/immunoconjugate will vary depending upon the antibody used, the antigen density on the tumor, and the rate of clearance of the antibody.
  • Immunotherapeutic compositions may also, or alternatively, comprise T cells specific for a breast tumor protein.
  • T cells may generally be prepared in vitro or ex vivo, using standard procedures.
  • T cells may be present within (or isolated from) bone ma ⁇ ow, peripheral blood or a fraction of bone ma ⁇ ow or peripheral blood of a mammal, such as a patient, using a commercially available cell separation system, such as the CEPRATETM system, available from CellPro Inc., Bothell WA (see also U.S. Patent No. 5,240,856; U.S. Patent No. 5,215,926; WO 89/06280; WO 91/161 16 and WO 92/07243).
  • T cells may be derived from related or unrelated humans, non-human animals, cell lines or cultures.
  • T cells may be stimulated with a breast tumor polypeptide, polynucleotide encoding a breast tumor polypeptide and/or an antigen presenting cell (APC) that expresses such a polypeptide.
  • APC antigen presenting cell
  • Such stimulation is performed under conditions and for a time sufficient to permit the generation of T cells that are specific for the polypeptide.
  • a breast tumor polypeptide or polynucleotide is present within a delivery vehicle, such as a microsphere, to facilitate the generation of specific T cells.
  • T cells are considered to be specific for a breast tumor antigen polypeptide if the T cells kill target cells coated with a breast tumor antigen polypeptide or expressing a gene encoding such a polypeptide.
  • T cell specificity may be evaluated using any of a variety of standard techniques. For example, within a chromium release assay or proliferation assay, a stimulation index of more than two fold increase in lysis and/or proliferation, compared to negative controls, indicates T cell specificity. Such assays may be performed, for example, as described in Chen et al.. Cancer Res. 54: 1065-1070, 1994. Alternatively, detection of the proliferation of T cells may be accomplished by a variety of known techniques.
  • T cell proliferation can be detected by measuring an increased rate of DNA synthesis (e.g., by pulse-labeling cultures of T cells with tritiated thymidine and measuring the amount of tritiated thymidine incorporated into DNA).
  • a breast tumor antigen polypeptide 200 ng/ml - 100 ⁇ g/ml, preferably 100 ng/ml - 25 ⁇ g/ml
  • contact with a breast tumor antigen polypeptide 200 ng/ml - 100 ⁇ g/ml, preferably 100 ng/ml - 25 ⁇ g/ml
  • contact as described above for 2-3 hours should result in activation of the T cells, as measured using standard cytokine assays in which a two fold increase in the level of cytokine release (e.g., TNF or IFN- ⁇ ) is indicative of T cell activation (see Coligan et al., Cu ⁇ ent Protocols in Immunology, vol.
  • T cells that have been activated in response to a breast tumor antigen polypeptide, polynucleotide or polypeptide-expressing APC may be CD4" and/or CD8 + .
  • Breast tumor antigen-specific T cells may be expanded using standard techniques. Within prefe ⁇ ed embodiments, the T cells are derived from a patient or a related or unrelated donor and are administered to the patient following stimulation and expansion.
  • CD4 + or CD8 + T cells that proliferate in response to a breast tumor antigen polypeptide, polynucleotide or APC can be expanded in number either in vitro or in vivo. Proliferation of such T cells in vitro may be accomplished in a variety of ways.
  • the T cells can be re-exposed to a breast tumor antigen, with or without the addition of T cell growth factors, such as interleukin-2, and/or stimulator cells that synthesize a breast tumor antigen polypeptide.
  • T cell growth factors such as interleukin-2
  • stimulator cells that synthesize a breast tumor antigen polypeptide.
  • one or more T cells that proliferate in the presence of a breast tumor antigen can be expanded in number by cloning. Methods for cloning cells are well known in the art, and include limiting dilution.
  • polypeptides, polynucleotides and/or binding agents may be incorporated into pharmaceutical compositions or vaccines.
  • Pha ⁇ naceutical compositions comprise one or more such compounds and a physiologically acceptable ca ⁇ ier.
  • Vaccines may comprise one or more such compounds and a non-specific immune response enhancer.
  • a non-specific immune response enhancer may be any substance that enhances an immune response to an exogenous antigen. Examples of non-specific immune response enhancers include adjuvants, biodegradable microspheres (e.g., polylactic galactide) and liposomes (into which the compound is incorporated; see e.g., Fullerton, U.S. Patent No. 4.235,877).
  • compositions and vaccines within the scope of the present invention may also contain other compounds, which may be biologically active or inactive.
  • one or more immunogenic portions of other tumor antigens may be present, either incorporated into a fusion polypeptide or as a separate compound within the composition or vaccine.
  • a pharmaceutical composition or vaccine may contain DNA encoding one or more of the polypeptides as described above, such that the polypeptide is generated in situ.
  • the DNA may be present within any of a variety of delivery systems known to those of ordinary skill in the art, including nucleic acid expression systems, bacteria and viral expression systems. Numerous gene delivery techniques are well known in the art, such as those described by Rolland, Crit. Rev. Therap. Drug Carrier Systems -75:143-198, 1998, and references cited therein. Appropriate nucleic acid expression systems contain the necessary DNA sequences for expression in the patient (such as a suitable promoter and terminating signal).
  • Bacterial delivery systems involve the administration of a bacterium (such as Bacillus-Calmette- Guerri ) that expresses an immunogenic portion of the polypeptide on its cell surface.
  • the DNA may be introduced using a viral expression system (e.g., vaccinia or other pox virus, retrovirus, or adenovirus), which may involve the use of a non-pathogenic (defective), replication competent virus.
  • a viral expression system e.g., vaccinia or other pox virus, retrovirus, or adenovirus
  • Suitable systems are disclosed, for example, in Fisher-Hoch et al., Proc. Natl. Acad. Sci. USA 5(5:317-321, 1989; Flexner et al., Ann. N. Y. Acad. Sci.
  • compositions of the present invention may be formulated for any appropriate manner of administration, including for example, topical, oral, nasal, intravenous, intracranial, intraperitoneal. subcutaneous or intramuscular administration.
  • the ca ⁇ ier preferably comprises water, saline, alcohol, a fat, a wax or a buffer.
  • any of the above ca ⁇ iers or a solid ca ⁇ ier such as mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, glucose, sucrose, and magnesium carbonate, may be employed.
  • Biodegradable microspheres e.g., polylactate polyglycolate
  • Suitable biodegradable microspheres are disclosed, for example, in U.S. Patent Nos. 4,897,268 and 5,075,109.
  • compositions may also comprise buffers (e.g., neutral buffered saline or phosphate buffered saline), carbohydrates (e.g., glucose, mannose, sucrose or dextrans), mannitol, proteins, polypeptides or amino acids such as glycine. antioxidants, chelating agents such as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide) and/or preservatives.
  • buffers e.g., neutral buffered saline or phosphate buffered saline
  • carbohydrates e.g., glucose, mannose, sucrose or dextrans
  • mannitol proteins
  • proteins polypeptides or amino acids
  • proteins e.glycine
  • antioxidants e.g., g., g., glycine
  • chelating agents such as EDTA or glutathione
  • adjuvants e.g., aluminum hydroxide
  • preservatives e.
  • an adjuvant may be included.
  • Most adjuvants contain a substance designed to protect the antigen from rapid catabolism, such as aluminum hydroxide or mineral oil, and a stimulator of immune responses, such as lipid A, Bortadella pertussis or Mycobacterium tuberculosis derived proteins.
  • Suitable adjuvants are commercially available as, 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 and quil A.
  • Cytokines such as GM-CSF or interleukin-2, -7, or -12, may also be used as adjuvants.
  • the adjuvant composition is preferably designed to induce an immune response predominantly of the Thl type.
  • High levels of Thl -type cytokines e.g., IFN- ⁇ , IL-2 and IL-12
  • Th2-type cytokines e.g., IL-4, IL-5, IL-6, IL-10 and TNF- ⁇
  • a patient will support an immune response that includes Thl- and Th2-type responses.
  • Thl -type cytokines will increase to a greater extent than the level of Th2-type cytokines.
  • the levels of these cytokines may be readily assessed using standard assays. For a review of the families of cytokines, see Mosmann and Coffman, Ann. Rev. Immunol. 7: 145-173, 1989.
  • Prefe ⁇ ed adjuvants for use in eliciting a predominantly Thl -type response include, for example, a combination of monophosphoryl lipid A, preferably 3- de-O-acylated monophosphoryl lipid A (3D-MPL), together with an aluminum salt.
  • MPL adjuvants are available from Ribi ImmunoChem Research Inc. (Hamilton, MT; see US Patent Nos. 4,436,727; 4,877,61 1 ; 4,866,034 and 4,912,094).
  • AS-2 SmithKline Beecham
  • CpG-containing oligonucleotides in which the CpG dinucleotide is unmethylated also induce a predominantly Thl response.
  • oligonucleotides are well known and are described, for example, in WO 96/02555.
  • Another prefe ⁇ ed adjuvant is a saponin, preferably QS21, which may be used alone or in combination with other adjuvants.
  • QS21 a monophosphoryl lipid A and saponin derivative, such as the combination of QS21 and 3D-MPL as described in WO 94/00153, or a less reactogenic composition where the QS21 is quenched with cholesterol, as described in WO 96/33739.
  • Other prefe ⁇ ed formulations comprises an oil-in-water emulsion and tocopherol.
  • a particularly potent adjuvant formulation involving QS21, 3D-MPL and tocopherol in an oil-in-water emulsion is described in WO 95/17210.
  • Any vaccine provided herein may be prepared using well known methods that result in a combination of antigen, immune response enhancer and a suitable ca ⁇ ier or excipient.
  • compositions described herein may be administered as part of a sustained release formulation (i.e., a formulation such as a capsule or sponge that effects a slow release of compound following administration).
  • a sustained release formulation i.e., a formulation such as a capsule or sponge that effects a slow release of compound following administration.
  • Such formulations may generally be prepared using well known technology and administered by, for example, oral, rectal or subcutaneous implantation, or by implantation at the desired target site.
  • Sustained-release formulations may contain a polypeptide, polynucleotide or antibody dispersed in a ca ⁇ ier matrix and/or contained within a reservoir su ⁇ ounded by a rate controlling membrane.
  • Ca ⁇ iers for use within such formulations are biocompatible, and may also be biodegradable; preferably the formulation provides a relatively constant level of active component release.
  • the amount of active compound contained within a sustained release formulation depends upon the site of implantation, the rate and expected duration of release and the nature of the condition to be treated or prevented.
  • Delivery vehicles include antigen presenting cells, such as dendritic cells and macrophages. Such cells may be transfected with a polynucleotide encoding a breast tumor antigen (or portion or other variant thereof) such that the breast tumor antigen polypeptide is expressed on the cell surface. Such transfection may take place ex vivo, and a composition or vaccine comprising such transfected cells may then be used for therapeutic purposes, as described herein.
  • a gene delivery vehicle that targets a dendritic or other antigen presenting cell may be administered to a patient, resulting in transfection that occurs in vivo.
  • In vivo and ex vivo transfection of dendritic cells may generally be performed using any methods known in the art, such as those described in WO 97/24447, or the gene gun approach described by Mahvi et al., Immunology and cell Biology 75:456-460, 1997.
  • compositions described herein may be used for immunotherapy of cancer, such as breast cancer.
  • pharmaceutical compositions and vaccines are typically administered to a patient.
  • a patient refers to any warm-blooded animal, preferably a human.
  • a patient may or may not be afflicted with cancer.
  • the above pharmaceutical compositions and vaccines may be used to prevent the development of a cancer or to treat a patient afflicted with a cancer.
  • a patient is afflicted with breast cancer.
  • Such cancer may be diagnosed using criteria generally accepted in the art, including the presence of a malignant tumor.
  • Pharmaceutical compositions and vaccines may be administered either prior to or following surgical removal of primary tumors and/or treatment such as administration of radiotherapy or conventional chemotherapeutic drugs.
  • immunotherapy may be active immunotherapy, in which treatment relies on the in vivo stimulation of the endogenous host immune system to react against tumors with the administration of immuno response-modifying agents (such as tumor vaccines, bacterial adjuvants and/or cytokines).
  • immuno response-modifying agents such as tumor vaccines, bacterial adjuvants and/or cytokines.
  • immunotherapy may be passive immunotherapy, in which treatment involves the delivery of agents with established tumor-immune reactivity (such as effector cells or antibodies) that can directly or indirectly mediate antitumor effects and does not necessarily depend on an intact host immune system.
  • agents with established tumor-immune reactivity such as effector cells or antibodies
  • effector cells include T lymphocytes (such as CD8 + cytotoxic T lymphocytes and CD4 + T-helper tumor-infiltrating lymphocytes), killer cells (such as Natural Killer cells and lymphokine-activated killer cells), B cells and antigen-presenting cells (such as dendritic cells and macrophages) expressing a polypeptide provided herein.
  • T cell receptors and antibody receptors specific for the polypeptides recited herein may be cloned, expressed and transfe ⁇ ed into other vectors or effector cells for adoptive immunotherapy.
  • the polypeptides provided herein may also be used to generate antibodies or anti-idiotypic antibodies (as described above and in U.S. Patent No. 4,918,164) for passive immunotherapy.
  • Effector cells may generally be obtained in sufficient quantities for adoptive immunotherapy by growth in vitro, as described herein.
  • Culture conditions for expanding single antigen-specific effector cells to several billion in number with retention of antigen recognition in vivo are well known in the art.
  • Such in vitro culture conditions typically use intermittent stimulation with antigen, often in the presence of cytokines (such as IL-2) and non-dividing feeder cells.
  • cytokines such as IL-2
  • immunoreactive polypeptides as provided herein may be used to rapidly expand antigen-specific T cell cultures in order to generate a sufficient number of cells for immunotherapy.
  • antigen-presenting cells such as dendritic, macrophage or B cells
  • antigen-presenting cells may be pulsed with immunoreactive polypeptides or transfected with one or more polynucleotides using standard techniques well known in the art.
  • antigen-presenting cells can be transfected with a polynucleotide having a promoter appropriate for increasing expression in a recombinant virus or other expression system.
  • Cultured effector cells for use in therapy must be able to grow and distribute widely, and to survive long term in vivo.
  • the polypeptides provided herein may also be used to generate and/or isolate tumor-reactive T cells, which can then be administered to a patient.
  • antigen-specific T cell lines may be generated by in vivo immunization with short peptides co ⁇ esponding to immunogenic portions of the disclosed polypeptides.
  • the resulting antigen-specific CD8 * CTL clones may be isolated from the patient, expanded using standard tissue culture techniques and returned to the patient.
  • Polypeptides may also be used for ex vivo treatment of a cancer, such as breast cancer.
  • cells of the immune system such as T cells
  • T cells may be isolated from the peripheral blood of a patient, using a commercially available cell separation system, such as CellPro Incorporated's (Bothell, WA) CEPRATETM system (see U.S. Patent No. 5,240,856; U.S. Patent No. 5,215,926; WO 89/06280; WO 91/161 16 and WO 92/07243).
  • the separated cells are stimulated with one or more of the immunoreactive polypeptides contained within a delivery vehicle, such as a microsphere, to provide antigen-specific T cells.
  • the population of tumor antigen- specific T cells is them expanded using standard techniques and the cells may be administered back to the patient as described, for example, by Chang et al., Crit. Rev. Oncol. Hematol. 22:213, 1996.
  • syngeneic or autologous dendritic cells may be pulsed with peptides co ⁇ esponding to at least an immunogenic portion of a polypeptide disclosed herein.
  • the resulting antigen-specific dendritic cells may either be transfe ⁇ ed into a patient or employed to stimulate T cells to provide antigen-specific T cells which may, in turn, be administered to a patient.
  • the use of peptide-pulsed dendritic cells to generate antigen-specific T cells and the subsequent use of such antigen-specific T cells to eradicate tumors in a murine model has been demonstrated by Cheever et al., Immunological Reviews 157: 77, 1997.
  • a vector expressing a polypeptide recited herein may be introduced into antigen presenting cells taken from a patient and clonally propagated ex vivo for transplant back into the same patient.
  • Transfected cells may be reintroduced into the patient using any means known in the art, preferably in sterile form by intravenous, intracavitary, intraperitoneal or intratumor administration.
  • compositions and vaccines may be administered by injection (e.g., intracutaneous, intramuscular, intravenous or subcutaneous), intranasally (e.g., by aspiration) or orally.
  • injection e.g., intracutaneous, intramuscular, intravenous or subcutaneous
  • intranasally e.g., by aspiration
  • between 1 and 10 doses may be administered over a 52 week period.
  • 6 doses are administered, at intervals of 1 month, and booster vaccinations may be given periodically thereafter.
  • Alternate protocols may be appropriate for individual patients.
  • a suitable dose is an amount of a compound that, when administered as described above, is capable of promoting an anti-tumor immune response, and is at least 10-50% above the basal (i.e., untreated) level..
  • Such response can be monitored by measuring the anti-tumor antibodies in a patient or by vaccine- dependent generation of cytolytic effector cells capable of killing the patient's tumor cells in vitro.
  • Such vaccines should also be capable of causing an immune response that leads to an improved clinical outcome (e.g., more frequent remissions, complete or partial or longer disease-free survival) in vaccinated patients as compared to non- vaccinated patients.
  • the amount of each polypeptide present in a dose ranges from about 100 ⁇ g to 5 mg per kg of host. Suitable dose sizes will vary with the size of the patient, but will typically range from about 0.1 mL to about 5 mL.
  • an appropriate dosage and treatment regimen provides the active compound(s) in an amount sufficient to provide therapeutic and/or prophylactic benefit.
  • a response can be monitored by establishing an improved clinical outcome (e.g., more frequent remissions, complete or partial, or longer disease-free survival) in treated patients as compared to non-treated patients.
  • Increases in preexisting immune responses to a breast tumor antigen generally co ⁇ elate with an improved clinical outcome.
  • Such immune responses may generally be evaluated using standard proliferation, cytotoxicity or cytokine assays, which may be performed using samples obtained from a patient before and after treatment.
  • a cancer may be detected in a patient based on the presence of one or more breast tumor antigens and/or polynucleotides encoding such antigens in a biological sample obtained from the patient.
  • such antigens may be used as markers to indicate the presence or absence of a cancer such as breast cancer.
  • such antigens may be useful for the detection of other cancers.
  • the binding agents provided herein generally permit detection of the level of antigen that binds to the agent in the biological sample.
  • Polynucleotide primers and probes may be used to detect the level of mRNA encoding an antigen, which is also indicative of the presence or absence of a cancer.
  • the presence or absence of a cancer in a patient may be determined by (a) contacting a biological sample obtained from a patient with a binding agent; (b) detecting in the sample a level of polypeptide that binds to the binding agent; and (c) comparing the level of polypeptide with a predetermined cut-off value.
  • the assay involves the use of binding agent immobilized on a solid support to bind to and remove the polypeptide from the remainder of the sample.
  • the bound polypeptide may then be detected using a detection reagent that contains a reporter group and specifically binds to the binding agent/polypeptide complex.
  • detection reagents may comprise, for example, a binding agent that specifically binds to the polypeptide or an antibody or other agent that specifically binds to the binding agent, such as an anti-immunoglobulin, protein G, protein A or a lectin.
  • a competitive assay may be utilized, in which a polypeptide is labeled with a reporter group and allowed to bind to the immobilized binding agent after incubation of the binding agent with the sample.
  • the extent to which components of the sample inhibit the binding of the labeled polypeptide to the binding agent is indicative of the reactivity of the sample with the immobilized binding agent.
  • Suitable polypeptides for use within such assays include full length breast tumor antigens and portions thereof to which the binding agent binds, as described above.
  • the solid support may be any material known to those of ordinary skill in the art to which the antigen may be attached.
  • the solid support may be a test well in a microtiter plate or a nitrocellulose or other suitable membrane.
  • the support may be a bead or disc, such as glass, fiberglass, latex or a plastic material such as polystyrene or polyvinylchloride.
  • the support may also be a magnetic particle or a fiber optic sensor, such as those disclosed, for example, in U.S. Patent No. 5,359,681.
  • the binding agent may be immobilized on the solid support using a variety of techniques known to those of skill in the art, which are amply described in the patent and scientific literature.
  • immobilization refers to both noncovalent association, such as adsorption, and covalent attachment (which may be a direct linkage between the antigen and functional groups on the support or may be a linkage by way of a cross-linking agent). Immobilization by adsorption to a well in a microtiter plate or to a membrane is prefe ⁇ ed. In such cases, adsorption may be achieved by contacting the binding agent, in a suitable buffer, with the solid support for a suitable amount of time. The contact time varies with temperature, but is typically between about 1 hour and about 1 day.
  • contacting a well of a plastic microtiter plate (such as polystyrene or polyvinylchloride) with an amount of binding agent ranging from about 10 ng to about 10 ⁇ g, and preferably about 100 ng to about 1 ⁇ g, is sufficient to immobilize an adequate amount of binding agent.
  • a plastic microtiter plate such as polystyrene or polyvinylchloride
  • Covalent attachment of binding agent to a solid support may generally be achieved by first reacting the support with a bifunctional reagent that will react with both the support and a functional group, such as a hydroxyl or amino group, on the binding agent.
  • a bifunctional reagent that will react with both the support and a functional group, such as a hydroxyl or amino group, on the binding agent.
  • the binding agent may be covalently attached to supports having an appropriate polymer coating using benzoquinone or by condensation of an aldehyde group on the support with an amine and an active hydrogen on the binding partner (see, e.g., Pierce Immunotechnology Catalog and Handbook, 1991, at A12-A13).
  • the assay is a two-antibody sandwich assay.
  • This assay may be performed by first contacting an antibody that has been immobilized on a solid support, commonly the well of a microtiter plate, with the sample, such that polypeptides within the sample are allowed to bind to the immobilized antibody. Unbound sample is then removed from the immobilized polypeptide-antibody complexes and a detection reagent (preferably a second antibody capable of binding to a different site on the polypeptide) containing a reporter group is added. The amount of detection reagent that remains bound to the solid support is then determined using a method appropriate for the specific reporter group.
  • a detection reagent preferably a second antibody capable of binding to a different site on the polypeptide
  • the immobilized antibody is then incubated with the sample, and polypeptide is allowed to bind to the antibody.
  • the sample may be diluted with a suitable diluent, such as phosphate-buffered saline (PBS) prior to incubation.
  • PBS phosphate-buffered saline
  • an appropriate contact time is a period of time that is sufficient to detect the presence of polypeptide within a sample obtained from an individual with breast cancer.
  • the contact time is sufficient to achieve a level of binding that is at least about 95% of that achieved at equilibrium between bound and unbound polypeptide.
  • a level of binding that is at least about 95% of that achieved at equilibrium between bound and unbound polypeptide.
  • the time necessary to achieve equilibrium may be 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 may then be removed by washing the solid support with an appropriate buffer, such as PBS containing 0.1% Tween 20TM.
  • the second antibody which contains a reporter group, may then be added to the solid support.
  • Prefe ⁇ ed reporter groups include those groups recited above.
  • the detection reagent is then incubated with the immobilized antibody- polypeptide complex for an amount of time sufficient to detect the bound polypeptide.
  • An appropriate amount of time may generally be determined by assaying the level of binding that occurs over a period of time.
  • Unbound detection reagent is then removed and bound detection reagent is detected using the reporter group.
  • the method employed for detecting the reporter group depends upon the nature of the reporter group. For radioactive groups, scintillation counting or autoradiographic methods are generally appropriate. Spectroscopic methods may be used to detect dyes, luminescent groups and fluorescent groups. Biotin may be detected using avidin, coupled to a different reporter group (commonly a radioactive or fluorescent group or an enzyme). Enzyme reporter groups may generally be detected by the addition of substrate (generally for a specific period of time), followed by spectroscopic or other analysis of the reaction products.
  • the signal detected from the reporter group that remains bound to the solid support is generally compared to a signal that co ⁇ esponds to a predetermined cut-off value.
  • the cut-off value for the detection of breast cancer is the average mean signal obtained when the immobilized antibody is incubated with samples from patients without breast cancer.
  • a sample generating a signal that is three standard deviations above the predetermined cut-off value is considered positive for breast cancer.
  • the cut-off value is determined using a Receiver Operator Curve, according to the method of Sackett et al., Clinical Epidemiology: A Basic Science for Clinical Medicine, Little Brown and Co., 1985, p. 106-7. Briefly, in this embodiment, the cut-off value may be determined from a plot of pairs of true positive rates (i.e., sensitivity) and false positive rates (100%-specificity) that co ⁇ espond to each possible cut-off value for the diagnostic test result. The cut-off value on the plot that is the closest to the upper left-hand comer (i.e..).
  • a sample generating a signal that is higher than the cut-off value determined by this method may be considered positive.
  • the cut-off value may be shifted to the left along the plot, to minimize the false positive rate, or to the right, to minimize the false negative rate.
  • a sample generating a signal that is higher than the cut-off value determined by this method is considered positive for breast cancer.
  • the assay is performed in a flow-through or strip test format, wherein the binding agent is immobilized on a membrane, such as nitrocellulose.
  • a membrane such as nitrocellulose.
  • polypeptides within the sample bind to the immobilized binding agent as the sample passes through the membrane.
  • a second, labeled binding agent then binds to the binding agent-polypeptide complex as a solution containing the second binding agent flows through the membrane.
  • the detection of bound second binding agent may then be performed as described above.
  • the strip test format one end of the membrane to which binding agent is bound is immersed in a solution containing the sample. The sample migrates along the membrane through a region containing second binding agent and to the area of immobilized binding agent.
  • Concentration of second binding agent at the area of immobilized antibody indicates the presence of breast cancer.
  • concentration of second binding agent at that site generates a pattern, such as a line, that can be read visually. The absence of such a pattern indicates a negative result.
  • the amount of binding agent immobilized on the membrane is selected to generate a visually discernible pattern when the biological sample contains a level of polypeptide that would be sufficient to generate a positive signal in the two-antibody sandwich assay, in the format discussed above.
  • Prefe ⁇ ed binding agents for use in such assays are antibodies and antigen-binding fragments thereof.
  • the amount of antibody immobilized on the membrane ranges from about 25 ng to about l ⁇ g, and more preferably from about 50 ng to about 500 ng. Such tests can typically be performed with a very small amount of biological sample.
  • the above polypeptides may be used as markers for the progression of cancer, such as breast cancer.
  • assays as described above for the diagnosis of a cancer may be performed over time, and the change in the level of reactive polypeptide(s) evaluated.
  • the assays may be performed every 24-72 hours for a period of 6 months to 1 year, and thereafter performed as needed.
  • a cancer is progressing in those patients in whom the level of polypeptide detected by the binding agent increases over time. In contrast, the cancer is not progressing when the level of reactive polypeptide either remains constant or decreases with time.
  • a cancer may also, or alternatively, be detected based on the level of mRNA encoding a breast tumor antigen in a biological sample.
  • At least two oligonucleotide primers may be employed in a polymerase chain reaction (PCR) based assay to amplify a portion of a breast tumor antigen cDNA derived from a biological sample, wherein at least one of the oligonucleotide primers is specific for (i.e., hybridizes to) a polynucleotide encoding the breast tumor antigen.
  • PCR polymerase chain reaction
  • the amplified cDNA is then separated and detected using techniques well known in the art, such as gel electrophoresis.
  • oligonucleotide probes that specifically hybridize to a polynucleotide encoding a breast tumor antigen may be used in a hybridization assay to detect the presence of polynucleotide encoding the antigen in a biological sample.
  • oligonucleotide primers and probes should comprise an oligonucleotide sequence that has at least about 60%, preferably at least about 75% and more preferably at least about 90%, identity to a portion of a polynucleotide encoding a breast tumor antigen that is at least 10 nucleotides, and preferably at least 20 nucleotides, in length.
  • Oligonucleotide primers and/or probes which may be usefully employed in the diagnostic methods described herein preferably are at least 10-40 nucleotides in length.
  • the oligonucleotide primers comprise at least 10 contiguous nucleotides, more preferably at least 15 contiguous nucleotides, of a DNA molecule having a sequence recited in any one of Figures 1A-1L (SEQ ID NOs: 1-35), Figures 2A-2I (SEQ ID NOs: 36-57) or SEQ ID NOs:90-186.
  • Methods for both PCR based assays and hybridization assays are well known in the art (see, for example, Mullis et al., Cold Spring Harbor Symp. Quant. BioL, 51:263, 1987; Erlich ed., RC.7? Technology, Stockton Press, NY, 1989).
  • RNA is extracted from a sample tissue and is reverse transcribed to produce cDNA molecules.
  • PCR amplification using at least one specific primer generates a cDNA molecule, which may be separated and visualized using, for example, gel electrophoresis.
  • Amplification may be performed on samples obtained from biological samples taken from a test patient and an individual who is not afflicted with breast cancer.
  • the amplification reaction may be performed on several dilutions of cDNA spanning two orders of magnitude. A two-fold or greater increase in expression in several dilutions of the test patient sample as compared to the same dilutions of the non-cancerous sample is typically considered positive.
  • Certain in vivo diagnostic assays may be performed directly on a tumor.
  • One such assay involves contacting tumor cells with a binding agent.
  • the bound binding agent may then be detected directly or indirectly via a reporter group.
  • binding agents may also be used in histological applications.
  • polynucleotide probes may be used within such applications.
  • multiple breast tumor antigen markers may be assayed within a given sample. It will be apparent that binding agents specific for different antigens provided herein may be combined within a single assay. Further, multiple primers or probes may be used concu ⁇ ently. The selection of antigen markers may be based on routine experiments to determine combinations that results in optimal sensitivity. In addition, or alternatively, assays for antigens provided herein may be combined with assays for other known tumor antigens.
  • kits for use within any of the above diagnostic methods.
  • Such kits typically comprise two or more components necessary for performing a diagnostic assay.
  • Components may be compounds, reagents, containers and/or equipment.
  • one container within a kit may contain a monoclonal antibody or fragment thereof that specifically binds to a breast tumor antigen.
  • Such antibodies or fragments may be provided attached to a support material, as described above.
  • One or more additional containers may enclose elements, such as reagents or buffers, to be used in the assay.
  • Such kits may also, or alternatively, contain a detection reagent as described above that contains a reporter group suitable for direct or indirect detection of antibody binding.
  • kits may be designed to detect the level of mRNA encoding a breast tumor antigen in a biological sample.
  • kits generally comprise at least one oligonucleotide probe or primer, as described above, that hybridizes to a polynucleotide encoding a breast tumor antigen.
  • Such an oligonucleotide may be used, for example, within a PCR or hybridization assay. Additional components that may be present within such kits include a second oligonucleotide and/or a diagnostic reagent or container to facilitate the detection of a polynucleotide encoding a breast tumor antigen.
  • This Example illustrates the identification of cDNA molecules encoding breast tumor antigens.
  • BR1-1 1 Novel (SEQ ID NO:46; predicted amino acid sequence SEQ ID NO:85); similar to chicken inner centromere protein

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Abstract

L'invention concerne des compositions et des méthodes de thérapie et de diagnostic du cancer, tel que le cancer du sein. Les compositions peuvent contenir un ou plusieurs antigènes de tumeur du sein, des parties immunogènes de ceux-ci ou des polynucléotides qui codent pour ces parties. Dans un autre mode de réalisation, une composition thérapeutique peut contenir une cellule présentant un antigène qui exprime un antigène de tumeur du sein, ou une cellule T spécifique de cellules exprimant un antigène de tumeur du sein. Ces compositions peuvent être utilisées, par exemple, pour prévenir et traiter des maladies telles que le cancer du sein. L'invention concerne également des méthodes diagnostiques basées sur la détection d'un antigène de tumeur du sein dans un échantillon.
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