WO2002016939A2 - Procedes de diagnostic du cancer, compositions et procedes de depistage de modulateurs du cancer - Google Patents

Procedes de diagnostic du cancer, compositions et procedes de depistage de modulateurs du cancer Download PDF

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WO2002016939A2
WO2002016939A2 PCT/US2001/025997 US0125997W WO0216939A2 WO 2002016939 A2 WO2002016939 A2 WO 2002016939A2 US 0125997 W US0125997 W US 0125997W WO 0216939 A2 WO0216939 A2 WO 0216939A2
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prostate
breast
ovarian
bcr4
cancer
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PCT/US2001/025997
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English (en)
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WO2002016939A3 (fr
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David Mack
Kurt C. Gish
Keith E. Wilson
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Eos Biotechnology, Inc.
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Priority claimed from US09/642,034 external-priority patent/US6762020B1/en
Application filed by Eos Biotechnology, Inc. filed Critical Eos Biotechnology, Inc.
Priority to AU2001283463A priority Critical patent/AU2001283463A1/en
Publication of WO2002016939A2 publication Critical patent/WO2002016939A2/fr
Publication of WO2002016939A3 publication Critical patent/WO2002016939A3/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/0004—Screening or testing of compounds for diagnosis of disorders, assessment of conditions, e.g. renal clearance, gastric emptying, testing for diabetes, allergy, rheuma, pancreas functions
    • A61K49/0008—Screening agents using (non-human) animal models or transgenic animal models or chimeric hosts, e.g. Alzheimer disease animal model, transgenic model for heart failure
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/0002—General or multifunctional contrast agents, e.g. chelated agents
    • 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
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3015—Breast
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3069—Reproductive system, e.g. ovaria, uterus, testes, prostate
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57415—Specifically defined cancers of breast
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57434—Specifically defined cancers of prostate
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/5748—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving oncogenic 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
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • 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
    • C12Q2600/136—Screening for pharmacological compounds
    • 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 invention relates to the identification of expression profiles and the nucleic acids involved in prostate cancer, breast cancer, ovarian cancer and/or bladder cancer and to the use of such expression profiles and nucleic acids in diagnosis and prognosis of such cancers.
  • the invention further relates to methods for identifying and using candidate agents and/or targets which modulate these forms of cancer.
  • Antigens suitable for immunotherapeutic strategies should be highly expressed in cancer tissues and ideally not expressed in normal adult tissues. Expression in tissues that are dispensable for life, however, may be tolerated. Examples of such antigens include Her2/neu and the B-cell antigen CD20. Humanized monoclonal antibodies directed to Her2/neu (Herceptin) are currently in use for the treatment of metastatic breast cancer (Ross and Fletcher, 1998, Stem Cells 16:413-428).
  • anti-CD20 monoclonal antibodies are used to effectively treat non-Hodgekin's lymphoma (Maloney et al., 1997, Blood 90:2188-2195; Leget and Czuczman, 1998, Curr. Opin. Oncol. 10:548-551).
  • Prostate cancer is the most commonly diagnosed internal malignancy and second most common cause of cancer death in men in the U.S., resulting in approximately 40,000 deaths each year (Landis et al., CA Cancer J. Clin. 48:6-29 (1998); Greenlee et al., CA Cancer J. Clin. 50(1):7-13 (2000)), and incidence of prostate cancer has been increasing rapidly over the past 20 years in many parts of the world (Nakata et al., Int. J. Urol. 7(7):254-257 (2000); Majeed et al., BJU Int. 85(9):1058-1062 (2000)). It develops as the result of a pathologic transformation of normal prostate cells. In tumorigenesis, the cancer cell undergoes initiation, proliferation and loss of contact inhibition, culminating in invasion of surrounding tissue and, ultimately, metastasis.
  • PSA prostate-specific antigen
  • Treatments such as surgery (prostatectomy) , radiation therapy, and cryotherapy are potentially curative when the cancer remains localized to the prostate. Therefore, early detection of prostate cancer is important for a positive prognosis for treatment.
  • Systemic treatment for metastatic prostate cancer is limited to hormone therapy and chemotherapy.
  • Chemical or surgical castration has been the primary treatment for symptomatic metastatic prostate cancer for over 50 years. This testicular androgen deprivation therapy usually results in stabilization or regression of the disease (in 80% of patients), but progression of metastatic prostate cancer eventually develops (Panvichian et al., Cancer Control 3(6):493-500 (1996)).
  • Metastatic disease is currently considered incurable, and the primary goals of treatment are to prolong survival and improve quality of life (Rago, Cancer Control 5(6):513-521 (1998)).
  • PSMA prostate-specific membrane antigen
  • PSCA prostate stem cell antigen
  • PSMA serpentine transmembrane epithelial antigen of the prostate
  • STAP serpentine transmembrane epithelial antigen of the prostate
  • PSMA is a type II transmembrane hydrolase with significant homology to a rat neuropeptidase (Carter et al., 1996, Proc. Natl. Acad. Sci. USA 93:749-753).
  • Antibodies directed towards PSMA are currently being used to detect metastasized prostate cancer as the Prostascint Scan (Sodee et al., 1996, Clin. Nucl. Med.
  • PSCA is a member of the Thy-1/Ly-6 family of glycosylphosphatidylinositol-linked plasma membrane proteins (Reiter et al., 1998, Proc. Natl. Acad. Sci. USA 95:1735-1740). Immunohistochemical data shows that PSCA is up-regulated in the majority of prostate cancer epithelia and is also detected in bone metastasis (Gu et al., 2000, Oncogene 19:1288-1296).
  • STEAP is a multi-transmembrane prostate-specific protein that may function as a channel or transporter protein (Hubert et al., 1999, Proc. Natl. Acad.
  • Breast cancer is also a significant cancer in Western populations. It develops as the result of a pathologic transformation of normal breast epithelium to an invasive cancer. There have been a number of recently characterized genetic alterations that have been implicated in breast cancer. However, there is a need to identify all of the genetic alterations involved in the development of breast cancer.
  • Ovarian cancer and bladder cancer are also significant diseases Western populations, both in terms of incidence and mortality. In both of these cancers, a major problem has been that diagnosis is not made until late stages of the disease in a majority of cases. As with most cancers, late diagnosis presents a considerably less favorable prognosis for effective treatment and recovery.
  • the LIV-1 protein has been implicated in breast cancer, and study of the protein structure suggest that it is a transmembrane protein with a zinc-binding domain which may be involved in zinc transport (Taylor, IUBMB Life 49(4):249-253 (2000)). However, this protein has not been associated with any other disease state.
  • methods that can be used in diagnosis and prognosis of prostate cancer and/or breast cancer Further provided are methods that can be used to screen candidate bioactive agents for the ability to modulate prostate cancer and/or breast cancer. Additionally, provided herein are molecular targets for therapeutic intervention in prostate and breast cancer, as well as other cancers.
  • a method of screening drug candidates comprises providing a cell that expresses an expression profile gene or fragments thereof.
  • Preferred embodiments of the expression profile gene as described herein include the sequence comprising BCR4 or a fragment thereof.
  • the method further includes adding a drug candidate to the cell and determining the effect of the drug candidate on the expression of the expression profile gene.
  • the method of screening drug candidates includes comparing the level of expression in the absence of the drug candidate to the level of expression in the presence of the drug candidate, wherein the concentration of the drug candidate can vary when present, and wherein the comparison can occur after addition or removal of the drug candidate.
  • the cell expresses at least two expression profile genes. The profile genes may show an increase or decrease.
  • Also provided herein is a method of screening for a bioactive agent capable of binding to a prostate cancer modulating protein (PCMP) and/or breast cancer modulating protein (BCMP) or a fragment thereof, the method comprising combining the PCMP and/or BCMP or fragment thereof and a candidate bioactive agent, and determining the binding of the candidate agent to the PCMP and/or BCMP or fragment thereof.
  • PCMP prostate cancer modulating protein
  • BCMP breast cancer modulating protein
  • the PCMP and/or BCMP is BCR4.
  • the method comprises combining the PCMP and/or BCMP or fragment thereof and a candidate bioactive agent, and determining the effect of the candidate agent on the bioactivity of the PCMP and/or BCMP or the fragment thereof.
  • the PCMP and/or BCMP is BCR4
  • Also provided herein is a method of evaluating the effect of a candidate prostate cancer and/or breast cancer drug comprising administering the drug to a transgenic animal expressing or over-expressing a PCMP and/or BCMP or a fragment thereof, or an animal lacking a PCMP and/or BCMP for example as a result of a gene knockout.
  • the PCMP and/or BCMP is BCR4.
  • a method of evaluating the effect of a candidate prostate cancer and/or breast cancer drug comprising administering the drug to a patient and removing a cell sample from the patient. The expression profile of the cell is then determined. This method may further comprise comparing the expression profile to an expression profile of a healthy individual.
  • a method of diagnosing prostate cancer and/or breast cancer comprises determining the expression of a gene which encodes BCR4 or a fragment thereof in a first tissue type of a first individual, and comparing this to the expression of the gene from a second unaffected individual. A difference in the expression indicates that the first individual has prostate cancer or breast cancer.
  • the present invention provides an antibody which specifically binds to BCR4, or a fragment thereof.
  • the antibody is a monoclonal antibody.
  • the antibody can be a fragment of an antibody such as a single stranded antibody as further described herein, or can be conjugated to another molecule.
  • the antibody is a humanized antibody.
  • a method for screening for a bioactive agent capable of interfering with the binding of BCR4 or a fragment thereof and an antibody which binds to said BCR4 or fragment thereof comprises combining
  • the method further includes determining the binding of said BCR4 or fragment thereof and said antibody. Wherein there is a change in binding, an agent is identified as an interfering agent.
  • the interfering agent can be an agonist or an antagonist.
  • the antibody as well as the agent inhibits prostate cancer and/or breast cancer.
  • a method for inhibiting the activity of a prostate cancer and/or breast cancer modulating protein comprises binding an inhibitor to the protein.
  • the protein is BCR4.
  • the invention provides a method for neutralizing the effect of a prostate cancer and/or breast cancer modulating protein. The method comprises contacting an agent specific for the protein with the protein in an amount sufficient to effect neutralization.
  • the protein is BCR4.
  • a method for treating or inhibiting prostate cancer and/or breast cancer comprises administering to a cell a composition comprising an antibody to BCR4 or a fragment thereof.
  • the antibody is conjugated to a therapeutic moiety.
  • therapeutic moieties include a cytotoxic agent and a radioisotope.
  • the method can be performed in vitro or in vivo, preferably in vivo to an individual.
  • the method of inhibiting prostate cancer and/or breast cancer is provided to an individual with such cancer.
  • a BCR4 inhibitor is an antisense molecule to a nucleic acid encoding BCR4.
  • biochip comprising a nucleic acid segment which encodes
  • the biochip comprises fewer than 1000 nucleic acid probes.
  • a method provided herein comprises administering to an individual a composition comprising BCR4 or a fragment thereof.
  • said composition comprises a nucleic acid comprising a sequence encoding BCR4 or a fragment thereof.
  • compositions capable of eliciting an immune response in an individual.
  • a composition provided herein comprises BCR4 or a fragment thereof and a pharmaceutically acceptable carrier.
  • said composition comprises a nucleic acid comprising a sequence encoding BCR4 or a fragment thereof and a pharmaceutically acceptable carrier.
  • BCR4 nucleic acids, amino acids and antibodies find similar utility in methods and compositions for the diagnosis, determination of prognosis and treatment of ovarian cancer and bladder cancer as described above for breast cancer and prostate cancer.
  • Figures 1A and 1B show an embodiment of a nucleic acid (mRNA) which includes a sequence which encodes a prostate/breast/ovarian/bladder cancer protein provided herein, human BCR4.
  • the start (ATG and stop (TAG) codons are underlined, defining an open reading frame.
  • the BCR4 sequence differs from the published sequence of accession number
  • Figure 2 shows an embodiment of an amino acid sequence of human BCR4. A signal sequence is shown in bold, and putative transmembrane sequences are underlined.
  • Figure 3 shows an embodiment of a nucleic acid (mRNA) which includes a sequence which encodes a prostate/breast/ovarian/bladder cancer protein provided herein, mouse BCR4.
  • mRNA nucleic acid
  • Figure 4 shows an embodiment of an amino acid sequence of mouse BCR4.
  • Figure 5 shows an alignment of the human and mouse BCR4 amino acid sequences. Identical amino acid residues are indicated with an "*" below the aligned sequences.
  • Figures 6-8B show the relative amounts of expression of BCR4 in several different prostate cancer tissue samples (Figure 6), several different breast cancer tissue samples ( Figure 7) and various normal tissue types ( Figures 8A and 8B). These data reveal that BCR4 is overexpressed or upregulated in both prostate cancer tissue and breast cancer tissue, as compared with normal tissues.
  • FIG. 9 shows the relative expression of BCR4 in several different cell lines.
  • BRC4 is highly expressed in the LNCP cell line, which is from a prostate tumor, and the MCF7 cell line, which is from a breast tumor.
  • LNCP cell line which is from a prostate tumor
  • MCF7 cell line which is from a breast tumor.
  • Such expression allows for the use of these cells lines to produce animal models of prostate and breast cancer, which may be used, for example, to test antibodies directed to BCR4 in vivo.
  • Figures 10 and 11 show the relative amounts of expression of BCR4 in several different ovarian cancer tissue samples ( Figure 10) and several different bladder cancer tissue samples ( Figure 11). These data reveal that BCR4 is overexpressed or upregulated in both ovarian cancer tissue and bladder cancer tissue, as compared with normal tissues.
  • the present invention provides novel methods for diagnosis and prognosis evaluation for prostate cancer, breast cancer, ovarian cancer and/or bladder cancer as well as methods for screening for compositions which modulate such cancer and compositions which bind to modulators of these cancers.
  • the expression levels of genes are determined in different patient samples for which either diagnosis or prognosis information is desired, to provide expression profiles.
  • An expression profile of a particular sample is essentially a "fingerprint" of the state of the sample; while two states may have any particular gene similarly expressed, the evaluation of a number of genes simultaneously allows the generation of a gene expression profile that is unique to the state of the cell.
  • normal tissue may be distinguished from cancer tissue, and within similar types of cancer tissue, different prognosis states (good or poor long term survival prospects, for example) may be determined.
  • prognosis states good or poor long term survival prospects, for example
  • By comparing expression profiles of cancer tissue in different states information regarding which genes are important (including both up- and down-regulation of genes) in each of these states is obtained.
  • the identification of sequences that are differentially expressed in prostate cancer, breast cancer, ovarian cancer and/or bladder cancer tissue versus normal tissue, as well as differential expression resulting in different prognostic outcomes, allows the use of this information in a number of ways. For example, the evaluation of a particular treatment regime may be evaluated: does a chemotherapeutic drug act to improve the long-term prognosis in a particular patient.
  • diagnosis may be done or confirmed by comparing patient samples with the known expression profiles.
  • these gene expression profiles allow screening of drug candidates with an eye to mimicking or altering a particular expression profile; for example, screening can be done for drugs that suppress the specific cancer expression profile or convert a poor prognosis profile to a better prognosis profile. This may be done by making biochips comprising sets of the relevant cancer genes, which can then be used in these screens. These methods can also be done on the protein basis; that is, protein expression levels of the specific cancer proteins can be evaluated for diagnostic and prognostic purposes or to screen candidate agents.
  • the nucleic acid sequences associated with a specific cancer can be administered for gene therapy purposes, including the administration of antisense nucleic acids, or cancer proteins (including antibodies and other modulators thereof) administered as therapeutic drugs.
  • the present invention provides nucleic acid and protein sequences that are differentially expressed in prostate cancer, breast cancer, ovarian cancer and/or bladder cancer when compared to normal tissue.
  • the differentially expressed sequences provided herein are termed "prostate cancer sequences” or “breast cancer sequences” or “ovarian cancer sequences” or “bladder cancer sequences” or “prostate/breast/ovarian/bladder” (read: prostate, breast, ovarian and/or bladder) cancer sequences" or grammatical equivalents thereof.
  • prostate cancer sequences or "breast cancer sequences” or “ovarian cancer sequences” or “bladder cancer sequences” or “prostate/breast/ovarian/bladder” (read: prostate, breast, ovarian and/or bladder) cancer sequences” or grammatical equivalents thereof.
  • the designation of a sequence as being associated with a specific cancer type neither necessarily includes or excludes its association with any other of the specific cancer types discussed herein.
  • prostate cancer sequences include those that are up-regulated (i.e. expressed at a higher level) in prostate cancer, as well as those that are down-regulated (i.e. expressed at a lower level) in prostate cancer.
  • breast cancer sequences, ovarian cancer sequences and bladder cancer sequences include those that are up-regulated (i.e. expressed at a higher level) in breast cancer, ovarian cancer and bladder cancer, respectively, as well as those that are down-regulated (i.e. expressed at a lower level) in breast cancer, ovarian cancer and bladder cancer.
  • the prostate/breast/ovarian/bladder cancer sequences are from humans; however, as will be appreciated by those in the art, prostate/breast cancer sequences from other organisms may be useful in animal models of disease and drug evaluation; thus, other prostate/breast cancer sequences are provided, from vertebrates, including mammals, including rodents (rats, mice, hamsters, guinea pigs, etc.), primates, farm animals (including sheep, goats, pigs, cows, horses, etc). Prostate/breast/ovarian/bladder cancer sequences from other organisms may be obtained using the techniques outlined below.
  • the prostate/breast/ovarian/bladder cancer sequences are those of nucleic acids encoding BCR4 or fragments thereof.
  • the sequence is that depicted in figure 1 , or a fragment thereof.
  • the prostate/breast/ovarian/bladder cancer sequences encode a protein having the amino acid sequence depicted in figure 2, or a fragment thereof.
  • BCR4 is a human LIV-1 protein.
  • Prostate/breast/ovarian/bladder cancer sequences can include both nucleic acid and amino acid sequences.
  • the prostate/breast cancer sequences are recombinant nucleic acids.
  • recombinant nucleic acid herein is meant nucleic acid, originally formed in vitro, in general, by the manipulation of nucleic acid by polymerases and endonucleases, in a form not normally found in nature.
  • an isolated nucleic acid, in a linear form, or an expression vector formed in vitro by ligating DNA molecules that are not normally joined, are both considered recombinant for the purposes of this invention.
  • nucleic acid once a recombinant nucleic acid is made and reintroduced into a host cell or organism, it will replicate non-recombinantly, i.e. using the in vivo cellular machinery of the host cell rather than in vitro manipulations; however, such nucleic acids, once produced recombinantly, although subsequently replicated non-recombinantly, are still considered recombinant for the purposes of the invention.
  • a "recombinant protein” is a protein made using recombinant techniques, i.e. through the expression of a recombinant nucleic acid as depicted above.
  • a recombinant protein is distinguished from naturally occurring protein by at least one or more characteristics.
  • the protein may be isolated or purified away from some or all of the proteins and compounds with which it is normally associated in its wild type host, and thus may be substantially pure.
  • an isolated protein is unaccompanied by at least some of the material with which it is normally associated in its natural state, preferably constituting at least about 0.5%, more preferably at least about 5% by weight of the total protein in a given sample.
  • a substantially pure protein comprises at least about 75% by weight of the total protein, with at least about 80% being preferred, and at least about 90% being particularly preferred.
  • the definition includes the production of a protein from one organism in a different organism or host cell.
  • the protein may be made at a significantly higher concentration than is normally seen, through the use of an inducible promoter or high expression promoter, such that the protein is made at increased concentration levels.
  • the protein may be in a form not normally found in nature, as in the addition of an epitope tag or amino acid substitutions, insertions and deletions, as discussed below.
  • the prostate/breast/ovarian/bladder cancer sequences are nucleic acids.
  • prostate/breast cancer sequences are useful in a variety of applications, including diagnostic applications, which will detect naturally occurring nucleic acids, as well as screening applications; for example, biochips comprising nucleic acid probes to the prostate/breast cancer sequences can be generated.
  • diagnostic applications which will detect naturally occurring nucleic acids, as well as screening applications; for example, biochips comprising nucleic acid probes to the prostate/breast cancer sequences can be generated.
  • nucleic acid or oligonucleotide or grammatical equivalents herein means at least two nucleotides covalently linked together.
  • a nucleic acid of the present invention will generally contain phosphodiester bonds, although in some cases, as outlined below, nucleic acid analogs are included that may have alternate backbones, comprising, for example, phosphoramidate (Beaucage et al., Tetrahedron 49(10):1925 (1993) and references therein; Letsinger, J. Org. Chem. 35:3800 (1970); Sblul et al., Eur. J. Biochem. 81:579 (1977); Letsinger et al., Nucl. Acids Res.
  • nucleic acid analogs may find use in the present invention.
  • mixtures of naturally occurring nucleic acids and analogs can be made; alternatively, mixtures of different nucleic acid analogs, and mixtures of naturally occurring nucleic acids and analogs may be made.
  • PNA peptide nucleic acids
  • These backbones are substantially non-ionic under neutral conditions, in contrast to the highly charged phosphodiester backbone of naturally occurring nucleic acids. This results in two advantages.
  • the PNA backbone exhibits improved hybridization kinetics. PNAs have larger changes in the melting temperature (Tm) for mismatched versus perfectly matched basepairs. DNA and RNA typically exhibit a 2-4°C drop in Tm for an internal mismatch. With the non-ionic PNA backbone, the drop is closer to 7-9°C.
  • Tm melting temperature
  • RNA typically exhibit a 2-4°C drop in Tm for an internal mismatch.
  • the non-ionic PNA backbone the drop is closer to 7-9°C.
  • hybridization of the bases attached to these backbones is relatively insensitive to salt concentration.
  • PNAs are not degraded by cellular enzymes, and thus can be more stable.
  • the nucleic acids may be single stranded or double stranded, as specified, or contain portions of both double stranded or single stranded sequence.
  • the depiction of a single strand also defines the sequence of the other strand (“Crick"); thus the sequences described herein also includes the complement of the sequence.
  • the nucleic acid may be DNA, both genomic and cDNA, RNA or a hybrid, where the nucleic acid contains any combination of deoxyribo- and ribo-nucleotides, and any combination of bases, including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine, isoguanine, etc.
  • nucleoside includes nucleotides and nucleoside and nucleotide analogs, and modified nucleosides such as amino modified nucleosides.
  • nucleoside includes non-naturally occurring analog structures. Thus for example the individual units of a peptide nucleic acid, each containing a base, are referred to herein as a nucleoside.
  • a prostate/breast/ovarian/bladder cancer sequence can be initially identified by substantial nucleic acid and/or amino acid sequence homology to the sequences disclosed herein. Such homology can be based upon the overall nucleic acid or amino acid sequence, and is generally determined as outlined below, using either homology programs or hybridization conditions.
  • the prostate/breast/ovarian/bladder cancer sequences of the invention can be identified as follows. Samples of normal and tumor tissue are applied to biochips comprising nucleic acid probes. The samples are first microdissected, if applicable, and treated as is know in the art for the preparation of mRNA. Suitable biochips are commercially available, for example from Affymetrix. Gene expression profiles as described herein are generated, and the data analyzed.
  • the genes showing changes in expression as between normal and disease states are compared to genes expressed in other normal tissues, including, but not limited to lung, heart, bladder, brain, liver, breast, kidney, muscle, ovary, prostate, small intestine, large intestine, spleen, bone, and placenta.
  • those genes identified during the prostate/breast/ovarian/bladder cancer screen that are expressed in any significant amount in other tissues are removed from the profile, although in some embodiments, this is not necessary. That is, when screening for drugs, it is preferable that the target be disease specific, to minimize possible side effects.
  • prostate/breast/ovarian/bladder cancer sequences are those that are up-regulated in the specified cancer; that is, for example, the expression of these genes is higher in prostate carcinoma as compared to normal prostate tissue and/or the expression is higher in breast carcinoma as compared to normal breast tissue.
  • Up-regulation means at least about a 50% increase, preferably a two-fold change, more preferably at least about a three fold change, with at least about five-fold or higher being preferred. All accession numbers herein are for the GenBank sequence database and the sequences of the accession numbers are hereby expressly incorporated by reference.
  • GenBank is known in the art, see, e.g., Benson, DA, et al., Nucleic Acids Research 26:1-7 (1998) and http://www.ncbi.nlm.nih.gov/.
  • these genes are generally found to be expressed in a limited amount or not at all in bladder, bone marrow, brain, colon, fibroblasts, heart, kidney, liver, lung, muscle, pancreas, prostate, skin, small intestine, spleen, stomach and testes.
  • BCR4 is up-regulated in prostate cancer, breast cancer, ovarian cancer and. or bladder cancer.
  • prostate/breast/ovarian/bladder cancer sequences are those that are down-regulated in the specific cancer; that is, the expression of these genes is lower in, for example, prostate carcinoma as compared to normal prostate tissue and/or breast carcinoma as compared to normal breast tissue.
  • Down-regulation as used herein means at least about a two-fold change, preferably at least about a three fold change, with at least about five-fold or higher being preferred.
  • prostate/breast/ovarian/bladder cancer proteins of the present invention may be classified as secreted proteins, transmembrane proteins or intracellular proteins.
  • the prostate/breast/ovarian/bladder cancer protein is an intracellular protein.
  • Intracellular proteins may be found in the cytoplasm and/or in the nucleus. Intracellular proteins are involved in all aspects of cellular function and replication (including, for example, signaling pathways); aberrant expression of such proteins results in unregulated or disregulated cellular processes. For example, many intracellular proteins have enzymatic activity such as protein kinase activity, protein phosphatase activity, protease activity, nucleotide cyclase activity, polymerase activity and the like. Intracellular proteins also serve as docking proteins that are involved in organizing complexes of proteins, or targeting proteins to various subcellular localizations, and are involved in maintaining the structural integrity of organelles.
  • Src-homology-2 (SH2) domains bind tyrosine-phosphorylated targets in a sequence dependent manner.
  • PTB domains which are distinct from SH2 domains, also bind tyrosine phosphorylated targets.
  • SH3 domains bind to proline-rich targets.
  • PH domains, tetratricopeptide repeats and WD domains have been shown to mediate protein-protein interactions.
  • these motifs can be identified on the basis of primary sequence; thus, an analysis of the sequence of proteins may provide insight into both the enzymatic potential of the molecule and/or molecules with which the protein may associate.
  • the prostate/breast/ovarian/bladder cancer sequences are transmembrane proteins.
  • Transmembrane proteins are molecules that span the phospholipid bilayer of a cell. They may have an intracellular domain, an extracellular domain, or both.
  • the intracellular domains of such proteins may have a number of functions including those already described for intracellular proteins.
  • the intracellular domain may have enzymatic activity and/or may serve as a binding site for additional proteins.
  • the intracellular domain of transmembrane proteins serves both roles.
  • certain receptor tyrosine kinases have both protein kinase activity and SH2 domains.
  • autophosphorylation of tyrosines on the receptor molecule itself creates binding sites for additional SH2 domain containing proteins.
  • Transmembrane proteins may contain from one to many transmembrane domains.
  • receptor tyrosine kinases certain cytokine receptors, receptor guanylyl cyclases and receptor serine/threonine protein kinases contain a single transmembrane domain.
  • various other proteins including channels and adenylyl cyclases contain numerous transmembrane domains.
  • Many important cell surface receptors are classified as "seven transmembrane domain" proteins, as they contain 7 membrane spanning regions.
  • transmembrane protein receptors include, but are not limited to insulin receptor, insulin-like growth factor receptor, human growth hormone receptor, glucose transporters, transferrin receptor, epidermal growth factor receptor, low density lipoprotein receptor, epidermal growth factor receptor, leptin receptor, interleukin receptors, e.g. IL-1 receptor, IL-2 receptor, etc.
  • Other transmembrane proteins serve to bind and/or transport ions across membranes.
  • Characteristics of transmembrane domains include approximately 20 consecutive hydrophobic amino acids that may be followed by charged amino acids. Therefore, upon analysis of the amino acid sequence of a particular protein, the localization and number of transmembrane domains within the protein may be predicted.
  • extracellular domains are involved in binding to other molecules.
  • extracellular domains are receptors.
  • Factors that bind the receptor domain include circulating ligands, which may be peptides, proteins, or small molecules such as adenosine and the like.
  • growth factors such as EGF, FGF and PDGF are circulating growth factors that bind to their cognate receptors to initiate a variety of cellular responses.
  • Other factors include cytokines, mitogenic factors, neurotrophic factors and the like.
  • Extracellular domains also bind to cell-associated molecules. In this respect, they mediate cell-cell interactions.
  • Cell- associated ligands can be tethered to the cell for example via a glycosylphosphatidylinositol (GPI) anchor, or may themselves be transmembrane proteins.
  • Extracellular domains also associate with the extracellular matrix and contribute to the maintenance of the cell structure.
  • prostate/breast/ovarian/bladder cancer proteins that are transmembrane are particularly preferred in the present invention as they are good targets for immunotherapeutics, as are described herein.
  • transmembrane proteins can be also useful in imaging modalities.
  • BCR4 is a transmembrane protein.
  • BCR4 is localized to the plasma membrane.
  • transmembrane protein can be made soluble by removing transmembrane sequences, for example through recombinant methods.
  • transmembrane proteins that have been made soluble can be made to be secreted through recombinant means by adding an appropriate signal sequence.
  • the prostate/breast/ovarian/bladder cancer proteins are secreted proteins; the secretion of which can be either constitutive or regulated. These proteins have a signal peptide or signal sequence that targets the molecule to the secretory pathway.
  • Secreted proteins are involved in numerous physiological events; by virtue of their circulating nature, they serve to transmit signals to various other cell types.
  • the secreted protein may function in an autocrine manner (acting on the cell that secreted the factor), a paracrine manner (acting on cells in close proximity to the cell that secreted the factor) or an endocrine manner (acting on cells at a distance).
  • secreted molecules find use in modulating or altering numerous aspects of physiology.
  • Prostate/breast/ovarian/bladder cancer proteins that are secreted proteins are particularly preferred in the present invention as they serve as good targets for diagnostic markers, for example for blood tests.
  • BCR4 has been modified to be a secreted protein.
  • a prostate/breast/ovarian/bladder cancer sequence is initially identified by substantial nucleic acid and/or amino acid sequence homology to the prostate/breast cancer sequences outlined herein. Such homology can be based upon the overall nucleic acid or amino acid sequence, and is generally determined as outlined below, using either homology programs or hybridization conditions.
  • a nucleic acid is a "prostate/breast/ovarian/bladder cancer nucleic acid” if the overall homology of the nucleic acid sequence to the nucleic acid sequences encoding the amino acid sequences of the figures is preferably greater than about 75%, more preferably greater than about 80%, even more preferably greater than about 85% and most preferably greater than 90%. In some embodiments the homology will be as high as about 93 to 95 or 98%. Homology in this context means sequence similarity or identity, with identity being preferred. A preferred comparison for homology purposes is to compare the sequence containing sequencing errors to the correct sequence.
  • This homology will be determined using standard techniques known in the art, including, but not limited to, the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biobl. 48:443 (1970), by the search for similarity method of Pearson & Lipman, PNAS USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wl), the Best Fit sequence program described by Devereux et al., Nucl. Acid Res. 12:387-395 (1984), preferably using the default settings, or by inspection.
  • sequences which are used to determine sequence identity or similarity are selected from the sequences set forth in the figures, preferably that shown in Figure 1 and fragments thereof.
  • sequences utilized herein are those set forth in the figures.
  • sequences are naturally occurring allelic variants of the sequences set forth in the figures.
  • sequences are sequence variants as further described herein.
  • PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments. It can also plot a tree showing the clustering relationships used to create the alignment. PILEUP uses a simplification of the progressive alignment method of Feng & Doolittle, J. Mol. Evol.
  • Useful PILEUP parameters including a default gap weight of 3.00, a default gap length weight of 0.10, and weighted end gaps.
  • BLAST BLAST algorithm
  • WU-BLAST-2 WU-BLAST-2 uses several search parameters, most of which are set to the default values.
  • the HSP S and HSP S2 parameters are dynamic values and are established by the program itself depending upon the composition of the particular sequence and composition of the particular database against which the sequence of interest is being searched; however, the values may be adjusted to increase sensitivity.
  • a % amino acid sequence identity value is determined by the number of matching identical residues divided by the total number of residues of the "longer" sequence in the aligned region.
  • the "longer" sequence is the one having the most actual residues in the aligned region (gaps introduced by WU-Blast-2 to maximize the alignment score are ignored).
  • percent (%) nucleic acid sequence identity is defined as the percentage of nucleotide residues in a candidate sequence that are identical with the nucleotide residues of Figure 1.
  • a preferred method utilizes the BLASTN module of WU-BLAST-2 set to the default parameters, with overlap span and overlap fraction set to 1 and 0.125, respectively.
  • the alignment may include the introduction of gaps in the sequences to be aligned.
  • the percentage of homology will be determined based on the number of homologous nucleosides in relation to the total number of nucleosides.
  • homology of sequences shorter than those of the sequences identified herein and as discussed below will be determined using the number of nucleosides in the shorter sequence.
  • the nucleic acid homology is determined through hybridization studies.
  • nucleic acids which hybridize under high stringency to the nucleic acid sequences which encode the peptides identified in the figures, or their complements are considered a prostate/breast/ovarian/bladder cancer sequence.
  • High stringency conditions are known in the art; see for example Maniatis et al., Molecular Cloning: A Laboratory Manual, 2d Edition, 1989, and Short Protocols in Molecular Biology, ed. Ausubel, et al., both of which are hereby incorporated by reference. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures.
  • Tm thermal melting point
  • Stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short probes (e.g. 10 to 50 nucleotides) and at least about 60°C for long probes (e.g. greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.
  • less stringent hybridization conditions are used; for example, moderate or low stringency conditions may be used, as are known in the art; see Maniatis and
  • prostate/breast/ovarian/bladder cancer nucleic acid sequences of the invention are fragments of larger genes, i.e. they are nucleic acid segments. "Genes" in this context includes coding regions, non-coding regions, and mixtures of coding and non-coding regions. Accordingly, as will be appreciated by those in the art, using the sequences provided herein, additional sequences of the prostate/breast/ovarian/bladder cancer genes can be obtained, using techniques well known in the art for cloning either longer sequences or the full length sequences; see Maniatis et al., and Ausubel, et al., supra, hereby expressly incorporated by reference.
  • the prostate/breast/ovarian/bladder cancer nucleic acid Once the prostate/breast/ovarian/bladder cancer nucleic acid is identified, it can be cloned and, if necessary, its constituent parts recombined to form the entire prostate/breast/ovarian/bladder cancer nucleic acid.
  • the recombinant prostate/breast/ovarian/bladder cancer nucleic acid can be further- used as a probe to identify and isolate other prostate/breast/ovarian/bladder cancer nucleic acids, for example additional coding regions. It can also be used as a "precursor" nucleic acid to make modified or variant prostate cancer nucleic acids and proteins.
  • the prostate/breast/ovarian/bladder cancer nucleic acids of the present invention are used in several ways.
  • nucleic acid probes to the prostate/breast/ovarian/bladder cancer nucleic acids are made and attached to biochips to be used in screening and diagnostic methods, as outlined below, or for administration, for example for gene therapy and/or antisense applications.
  • the prostate/breast/ovarian/bladder cancer nucleic acids that include coding regions of prostate/breast/ovarian/bladder cancer proteins can be put into expression vectors for the expression of prostate/breast/ovarian/bladder cancer proteins, again either for screening purposes or for administration to a patient.
  • nucleic acid probes to prostate/breast/ovarian/bladder cancer nucleic acids are made.
  • the nucleic acid probes attached to the biochip are designed to be substantially complementary to the prostate/breast/ovarian/bladder cancer nucleic acids, i.e. the target sequence (either the target sequence of the sample or to other probe sequences, for example in sandwich assays), such that hybridization of the target sequence and the probes of the present invention occurs.
  • this complementarity need not be perfect; there may be any number of base pair mismatches which will interfere with hybridization between the target sequence and the single stranded nucleic acids of the present invention. However, if the number of mutations is so great that no hybridization can occur under even the least stringent of hybridization conditions, the sequence is not a complementary target sequence.
  • substantially complementary herein is meant that the probes are sufficiently complementary to the target sequences to hybridize under normal reaction conditions, particularly high stringency conditions, as outlined herein.
  • a nucleic acid probe js generally single stranded but can be partially single and partially double stranded.
  • the strandedness of the probe is dictated by the structure, composition, and properties of the target sequence.
  • the nucleic acid probes range from about 8 to about 100 bases long, with from about 10 to about 80 bases being preferred, and from about 30 to about 50 bases being particularly preferred. That is, generally whole genes are not used. In some embodiments, much longer nucleic acids can be used, up to hundreds of bases.
  • more than one probe per sequence is used, with either overlapping probes or probes to different sections of the target being used. That is, two, three, four or more probes, with three being preferred, are used to build in a redundancy for a particular target.
  • the probes can be overlapping (i.e. have some sequence in common), or separate.
  • nucleic acids can be attached or immobilized to a solid support in a wide variety of ways.
  • immobilized and grammatical equivalents herein is meant the association or binding between the nucleic acid probe and the solid support is sufficient to be stable under the conditions of binding, washing, analysis, and removal as outlined below.
  • the binding can be covalent or non-covalent.
  • non-covalent binding and grammatical equivalents herein is meant one or more of either electrostatic, hydrophilic, and hydrophobic interactions. Included in non-covalent binding is the covalent attachment of a molecule, such as, streptavidin to the support and the non-covalent binding of the biotinylated probe to the streptavidin.
  • covalent binding and grammatical equivalents herein is meant that the two moieties, the solid support and the probe, are attached by at least one bond, including sigma bonds, pi bonds and coordination bonds. Covalent bonds can be formed directly between the probe and the solid support or can be formed by a cross linker or by inclusion of a specific reactive group on either the solid support or the probe or both molecules. Immobilization may also involve a combination of covalent and non-covalent interactions.
  • the probes are attached to the biochip in a wide variety of ways, as will be appreciated by those in the art.
  • the nucleic acids can either be synthesized first, with subsequent attachment to the biochip, or can be directly synthesized on the biochip.
  • the biochip comprises a suitable solid substrate.
  • substrate or “solid support” or other grammatical equivalents herein is meant any material that can be modified to contain discrete individual sites appropriate for the attachment or association of the nucleic acid probes and is amenable to at least one detection method.
  • the number of possible substrates are very large, and include, but are not limited to, glass and modified or functionalized glass, plastics (including acrylics, polystyrene and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethanes, TeflonJ, etc.), polysaccharides, nylon or nitrocellulose, resins, silica or silica-based materials including silicon and modified silicon, carbon, metals, inorganic glasses, plastics, etc.
  • the substrates allow optical detection and do not appreciably fluorescese.
  • a preferred substrate is described in copending application entitled Reusable Low Fluorescent Plastic Biochip filed March 15, 1999, herein incorporated by reference in its entirety.
  • the substrate is planar, although as will be appreciated by those in the art, other configurations of substrates may be used as well.
  • the probes may be placed on the inside surface of a tube, for flow-through sample analysis to minimize sample volume.
  • the substrate may be flexible, such as a flexible foam, including closed cell foams made of particular plastics.
  • the surface of the biochip and the probe may be derivatized with chemical functional groups for subsequent attachment of the two.
  • the biochip is derivatized with a chemical functional group including, but not limited to, amino groups, carboxy groups, oxo groups and thiol groups, with amino groups being particularly preferred.
  • the probes can be attached using functional groups on the probes.
  • nucleic acids containing amino groups can be attached to surfaces comprising amino groups, for example using linkers as are known in the art; for example, homo-or hetero-bifunctional linkers as are well known (see 1994 Pierce Chemical Company catalog, technical section on cross-linkers, pages 155-200, incorporated herein by reference).
  • additional linkers such as alkyl groups (including substituted and heteroalkyl groups) may be used.
  • the oligonucleotides are synthesized as is known in the art, and then attached to the surface of the solid support. As will be appreciated by those skilled in the art, either the 5' or 3' terminus may be attached to the solid support, or attachment may be via an internal nucleoside.
  • the immobilization to the solid support may be very strong, yet non-covalent.
  • biotinylated oligonucleotides can be made, which bind to surfaces covalently coated with streptavidin, resulting in attachment.
  • the oligonucleotides may be synthesized on the surface, as is known in the art.
  • photoactivation techniques utilizing photopolymerization compounds and techniques are used.
  • the nucleic acids can be synthesized in situ, using well known photolithographic techniques, such as those described in WO 95/25116; WO 95/35505; U.S. Patent Nos. 5,700,637 and 5,445,934; and references cited within, all of which are expressly incorporated by reference; these methods of attachment form the basis of the Affimetrix GeneChipTM technology.
  • prostate/breast/ovarian/bladder cancer nucleic acids encoding prostate/breast/ovarian/bladder cancer proteins are used to make a variety of expression vectors to express prostate/breast/ovarian/bladder cancer proteins which can then be used in screening assays, as described below.
  • the expression vectors may be either self-replicating extrachromosomal vectors or vectors which integrate into a host genome.
  • these expression vectors include transcriptional and translational regulatory nucleic acid operably linked to the nucleic acid encoding the prostate/breast/ovarian/bladder cancer protein.
  • control sequences refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism.
  • the control sequences that are suitable for prokaryotes include a promoter, optionally an operator sequence, and a ribosome binding site.
  • Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.
  • Nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence.
  • DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide;
  • a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or
  • a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation.
  • "operably linked” means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous.
  • the transcriptional and translational regulatory nucleic acid will generally be appropriate to the host cell used to express the prostate/breast/ovarian/bladder cancer protein; for example, transcriptional and translational regulatory nucleic acid sequences from Bacillus are preferably used to express the prostate/breast/ovarian/bladder cancer protein in
  • the transcriptional and translational regulatory sequences may include, but are not limited to, promoter sequences, ribosomal binding sites, transcriptional start and stop sequences, translational start and stop sequences, and enhancer or activator sequences.
  • the regulatory sequences include a promoter and transcriptional start and stop sequences.
  • Promoter sequences encode either constitutive or inducible promoters.
  • the promoters may be either naturally occurring promoters or hybrid promoters.
  • Hybrid promoters which combine elements of more than one promoter, are also known in the art, and are useful in the present invention.
  • the expression vector may comprise additional elements.
  • the expression vector may have two replication systems, thus allowing it to be maintained in two organisms, for example in mammalian or insect cells for expression and in a procaryotic host for cloning and amplification.
  • the expression vector contains at least one sequence homologous to the host cell genome, and preferably two homologous sequences which flank the expression construct.
  • the integrating vector may be directed to a specific locus in the host cell by selecting the appropriate homologous sequence for inclusion in the vector. Constructs for integrating vectors are well known in the art.
  • the expression vector contains a selectable marker gene to allow the selection of transformed host cells.
  • Selection genes are well known in the art and will vary with the host cell used.
  • the prostate/breast/ovarian/bladder cancer proteins of the present invention are produced by culturing a host cell transformed with an expression vector containing nucleic acid encoding a prostate/breast/ovarian/bladder cancer protein, under the appropriate conditions to induce or cause expression of the prostate/breast/ovarian/bladder cancer protein.
  • the conditions appropriate for prostate/breast/ovarian/bladder cancer protein expression will vary with the choice of the expression vector and the host cell, and will be easily ascertained by one skilled in the art through routine experimentation.
  • constitutive promoters in the expression vector will require optimizing the growth and proliferation of the host eel!, while the use of an inducible promoter requires the appropriate growth conditions for induction.
  • the timing of the harvest is important.
  • the baculoviral systems used in insect cell expression are lytic viruses, and thus harvest time selection can be crucial for product yield.
  • Appropriate host cells include yeast, bacteria, archaebacteria, fungi, and insect and animal cells, including mammalian cells. Of particular interest are Drosophila melangaster cells, Saccharomyces cerevisiae and other yeasts, E. coli, Bacillus subiilis, Sf9 cells, C129 cells, 293 cells, Neurospora, BHK, CHO, COS, HeLa cells, THP1 cell line (a macrophage cell line) and human cells and cell lines.
  • the prostate/breast/ovarian/bladder cancer proteins are expressed in mammalian cells.
  • Mammalian expression systems are also known in the art, and include retroviral systems.
  • a preferred expression vector system is a retroviral vector system such as is generally described in PCT/US97/01019 and PCT/US97/01048, both of which are hereby expressly incorporated by reference.
  • mammalian promoters are the promoters from mammalian viral genes, since the viral genes are often highly expressed and have a broad host range. Examples include the SV40 early promoter, mouse mammary tumor virus LTR promoter, adenovirus major late promoter, herpes simplex virus promoter, and the CMV promoter.
  • transcription termination and polyadenylation sequences recognized by mammalian cells are regulatory regions located 3' to the translation stop codon and thus, together with the promoter elements, flank the coding sequence.
  • transcription terminator and polyadenlytion signals include those derived form SV40.
  • prostate/breast/ovarian/bladder cancer proteins are expressed in bacterial systems.
  • Bacterial expression systems are well known in the art. Promoters from bacteriophage may also be used and are known in the art.
  • synthetic promoters and hybrid promoters are also useful; for example, the tac promoter is a hybrid of the trp and lac promoter sequences.
  • a bacterial promoter can include naturally occurring promoters of non-bacterial origin that have the ability to bind bacterial RNA polymerase and initiate transcription. In addition to a functioning promoter sequence, an efficient ribosome binding site is desirable.
  • the expression vector may also include a signal peptide sequence that provides for secretion of the prostate/breast/ovarian/bladder cancer protein in bacteria.
  • the protein is either secreted into the growth media (gram-positive bacteria) or into the periplasmic space, located between the inner and outer membrane of the cell (gram-negative bacteria).
  • the bacterial expression vector may also include a selectable marker gene to allow for the selection of bacterial strains that have been transformed. Suitable selection genes include genes which render the bacteria resistant to drugs such as ampicillin, chloramphenicol, erythromycin, kanamycin, neomycin and tetracycline. Selectable markers also include biosynthetic genes, such as those in the histidine, tryptophan and leucine biosynthetic pathways. These components are assembled into expression vectors.
  • Expression vectors for bacteria are well known in the art, and include vectors for Bacillus subtilis, E. coli, Streptococcus cremoris, and Streptococcus lividans, among others.
  • the bacterial expression vectors are transformed into bacterial host cells using techniques well known in the art, such as calcium chloride treatment, electroporation, and others.
  • prostate/breast/ovarian/bladder cancer proteins are produced in insect cells.
  • Expression vectors for the transformation of insect cells, and in particular, baculovirus- based expression vectors, are well known in the art.
  • prostate/breast/ovarian/bladder cancer protein is produced in yeast cells.
  • yeast expression systems are well known in the art, and include expression vectors for Saccharomyces cerevisiae, Candida albicans and C. maltosa, Hansenula polymorpha, Kluyveromyces fragilis and K. lactis, Pichia guillerimondii and P. pastoris, Schizosaccharomyces pombe, and Yarrow/a lipolytica.
  • the prostate/breast/ovarian/bladder cancer protein may also be made as a fusion protein, using techniques well known in the art.
  • the prostate/breast/ovarian/bladder cancer protein may be fused to a carrier protein to form an immunogen.
  • the prostate/breast/ovarian/bladder cancer protein may be made as a fusion protein to increase expression, or for other reasons.
  • the nucleic acid encoding the peptide may be linked to other nucleic acid for expression purposes.
  • the prostate/breast/ovarian/bladder cancer nucleic acids, proteins and antibodies of the invention are labeled.
  • labeled herein is meant that a compound has at least one element, isotope or chemical compound attached to enable the detection of the compound.
  • labels fall into three classes: a) isotopic labels, which may be radioactive or heavy isotopes; b) immune labels, which may be antibodies or antigens; and c) colored or fluorescent dyes.
  • the labels may be incorporated into the prostate/breast/ovarian/bladder cancer nucleic acids, proteins and antibodies at any position.
  • the label should be capable of producing, either directly or indirectly, a detectable signal.
  • the detectable moiety may be a radioisotope, such as 3 H, 14 C, 32 P, 35 S, or 125 l, a fluorescent or chemiluminescent compound, such as fluorescein isothiocyanate, rhodamine, or luciferin, or an enzyme, such as alkaline phosphatase, beta-galactosidase or horseradish peroxidase.
  • a radioisotope such as 3 H, 14 C, 32 P, 35 S, or 125 l
  • a fluorescent or chemiluminescent compound such as fluorescein isothiocyanate, rhodamine, or luciferin
  • an enzyme such as alkaline phosphatase, beta-galactosidase or horseradish peroxidase.
  • Any method known in the art for conjugating the antibody to the label may be employed, including those methods described by Hunter et al., Nature. 144:945 (1962); David et al
  • the present invention also provides prostate/breast/ovarian/bladder cancer protein sequences.
  • a prostate/breast/ovarian/bladder cancer protein of the present invention may be identified in several ways. "Protein” in this sense includes proteins, polypeptides, and peptides.
  • the nucleic acid sequences of the invention can be used to generate protein sequences. There are a variety of ways to do this, including cloning the entire gene and verifying its frame and amino acid sequence, or by comparing it to known sequences to search for homology to provide a frame, assuming the prostate/breast/ovarian/bladder cancer protein has homology to some protein in the database being used.
  • the nucleic acid sequences are input into a program that will search all three frames for homology. This is done in a preferred embodiment using the following NCBI
  • the program is blastx or blastn.
  • the database is nr.
  • the input data is as "Sequence in FASTA format”.
  • the organism list is “none”.
  • the “expect” is 10; the filter is default.
  • the “descriptions” is 500, the “alignments” is 500, and the “alignment view” is pairwise.
  • the "Query Genetic Codes” is standard (1).
  • the matrix is BLOSUM62; gap existence cost is 11 , per residue gap cost is 1 ; and the lambda ratio is .85 default. This results in the generation of a putative protein sequence.
  • amino acid variants of the naturally occurring sequences are preferably greater than about 75% homologous to the wild-type sequence, more preferably greater than about 80%, even more preferably greater than about 85% and most preferably greater than 90%.
  • the homology will be as high as about 93 to 95 or 98%.
  • nucleic acids homology in this context means sequence similarity or identity, with identity being preferred. This homology will be determined using standard techniques known in the art as are outlined above for the nucleic acid homologies.
  • Prostate/breast/ovarian/bladder cancer proteins of the present invention may be shorter or longer than the wild type amino acid sequences.
  • included within the definition of prostate/breast/ovarian/bladder cancer proteins are portions or fragments of the wild type sequences, herein.
  • the prostate/breast/ovarian/bladder cancer nucleic acids of the invention may be used to obtain additional coding regions, and thus additional protein sequence, using techniques known in the art.
  • the prostate/breast/ovarian/bladder cancer proteins are derivative or variant prostate/breast/ovarian/bladder cancer proteins as compared to the wild-type sequence. That is, as outlined more fully below, the derivative prostate/breast/ovarian/bladder cancer peptide will contain at least one amino acid substitution, deletion or insertion, with amino acid substitutions being particularly preferred. The amino acid substitution, insertion or deletion may occur at any residue within the prostate/breast/ovarian/bladder cancer peptide.
  • variants are also included in an embodiment of prostate/breast/ovarian/bladder cancer proteins of the present invention.
  • These variants fall into one or more of three classes: substitutional, insertional or deletional variants.
  • These variants ordinarily are prepared by site specific mutagenesis of nucleotides in the DNA encoding the prostate/breast/ovarian/bladder cancer protein, using cassette or PCR mutagenesis or other techniques well known in the art, to produce DNA encoding the variant, and thereafter expressing the DNA in recombinant cell culture as outlined above.
  • variant prostate/breast/ovarian/bladder cancer protein fragments having up to about 100- 50 residues may be prepared by in vitro synthesis using established techniques.
  • Amino acid sequence variants are characterized by the predetermined nature of the variation, a feature that sets them apart from naturally occurring allelic or interspecies variation of the prostate/breast/ovarian/bladder cancer protein amino acid sequence.
  • the variants typically exhibit the same qualitative biological activity as the naturally occurring analogue, although variants can also be selected which have modified characteristics as will be more fully outlined below.
  • the mutation per se need not be predetermined.
  • random mutagenesis may be conducted at the target codon or region and the expressed prostate/breast/ovarian/bladder cancer variants screened for the optimal combination of desired activity.
  • Techniques for making substitution mutations at predetermined sites in DNA having a known sequence are well known, for example, M13 primer mutagenesis and PCR mutagenesis. Screening of the mutants is done using assays of prostate/breast/ovarian/bladder cancer protein activities.
  • Amino acid substitutions are typically of single residues; insertions usually will be on the order of from about 1 to 20 amino acids, although considerably larger insertions may be tolerated. Deletions range from about 1 to about 20 residues, although in some cases deletions may be much larger.
  • substitutions deletions, insertions or any combination thereof may be used to arrive at a final derivative. Generally these changes are done on a few amino acids to minimize the alteration of the molecule. However, larger changes may be tolerated in certain circumstances. When small alterations in the characteristics of the prostate/breast/ovarian/bladder cancer protein are desired, substitutions are generally made in accordance with the following chart:
  • substitutions that are less conservative than those shown in Chart I.
  • substitutions may be made which more significantly affect: the structure of the polypeptide backbone in the area of the alteration, for example the alpha-helical or beta-sheet structure; the charge or hydrophobicity of the molecule at the target site; or the bulk of the side chain.
  • the substitutions which in general are expected to produce the greatest changes in the polypeptide's properties are those in which (a) a hydrophilic residue, e.g. seryl or threonyl is substituted for (or by) a hydrophobic residue, e.g.
  • leucyl isoleucyl, phenylalanyl, valyl or alanyl
  • a cysteine or proline is substituted for (or by) any other residue
  • a residue having an electropositive side chain e.g. lysyl, arginyl, or histidyl
  • an electronegative residue e.g. glutamyl or aspartyl
  • a residue having a bulky side chain e.g. phenylalanine, is substituted for (or by) one not having a side chain, e.g. glycine.
  • variants typically exhibit the same qualitative biological activity and will elicit the same immune response as the naturally-occurring analogue, although variants also are selected to modify the characteristics of the prostate/breast/ovarian/bladder cancer proteins as needed.
  • the variant may be designed such that the biological activity of the prostate/breast/ovarian/bladder cancer protein is altered. For example, glycosylation sites may be altered or removed.
  • Covalent modifications of prostate/breast/ovarian/bladder cancer polypeptides are included within the scope of this invention.
  • One type of covalent modification includes reacting targeted amino acid residues of a prostate/breast/ovarian/bladder cancer polypeptide with an organic derivatizing agent that is capable of reacting with selected side chains or the N-or C-terminal residues of a prostate/breast/ovarian/bladder cancer polypeptide.
  • Derivatization with bifunctional agents is useful, for instance, for crosslinking prostate/breast/ovarian/bladder cancer polypeptides to a water-insoluble support matrix or surface for use in the method for purifying anti-prostate cancer and/or anti-breast cancer antibodies or screening assays, as is more fully described below.
  • Commonly used crosslinking agents include, e.g., 1,1-bis(diazo- acetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with
  • 4-azidosalicylic acid homobifunctional imidoesters, including disuccinimidyl esters such as 3,3'-dithiobis(succinimidylpropionate), bifunctional maleimides such as bis-N-maleimido-1 ,8- octane and agents such as methyl-3-[(p-azidophenyl)dithio]propioimidate.
  • Another type of covalent modification of the prostate/breast/ovarian/bladder cancer polypeptide included within the scope of this invention comprises altering the native glycosylation pattern of the polypeptide.
  • "Altering the native glycosylation pattern" is intended for purposes herein to mean deleting one or more carbohydrate moieties found in native sequence prostate/breast/ovarian/bladder cancer polypeptide, and/or adding one or more glycosylation sites that are not present in the native sequence prostate/breast/ovarian/bladder cancer polypeptide.
  • Addition of glycosylation sites to prostate/breast/ovarian/bladder cancer polypeptides may be accomplished by altering the amino acid sequence thereof.
  • the alteration may be made, for example, by the addition of, or substitution by, one or more serine or threonine residues to the native sequence prostate/breast/ovarian/bladder cancer polypeptide (for O-linked glycosylation sites).
  • the prostate/breast/ovarian/bladder cancer amino acid sequence may optionally be altered through changes at the DNA level, particularly by mutating the DNA encoding the prostate/breast/ovarian/bladder cancer polypeptide at preselected bases such that codons are generated that will translate into the desired amino acids.
  • Another means of increasing the number of carbohydrate moieties on the prostate/breast/ovarian/bladder cancer polypeptide is by chemical or enzymatic coupling of glycosides to the polypeptide. Such methods are described in the art, e.g., in WO 87/05330 published 11 September 1987, and in Aplin and Wriston, Crit. Rev. Biochem., pp. 259-306 (1981).
  • Removal of carbohydrate moieties present on the prostate/breast/ovarian/bladder cancer polypeptide may be accomplished chemically or enzymatically or by mutational substitution of codons encoding for amino acid residues that serve as targets for glycosylation.
  • Chemical deglycosylation techniques are known in the art and described, for instance, by Hakimuddin, et al., Arch. Biochem. Biophys., 259:52 (1987) and by Edge et al., Anal. Biochem., 118:131 (1981).
  • Enzymatic cleavage of carbohydrate moieties on polypeptides can be achieved by the use of a variety of endo-and exo-glycosidases as described by Thotakura et al., Meth. Enzymol., 138:350 (1987).
  • Another type of covalent modification of prostate/breast/ovarian/bladder cancer protein comprises linking the prostate/breast/ovarian/bladder cancer polypeptide to one of a variety of nonproteinaceous polymers, e.g., polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, in the manner set forth in U.S. Patent Nos. 4,640,835; 4,496,689; 4,301 ,144; 4,670,417; 4,791 ,192 or 4,179,337.
  • nonproteinaceous polymers e.g., polyethylene glycol, polypropylene glycol, or polyoxyalkylenes
  • Prostate/breast/ovarian/bladder cancer polypeptides of the present invention may also be modified in a way to form chimeric molecules comprising a prostate/breast/ovarian/bladder cancer polypeptide fused to another, heterologous polypeptide or amino acid sequence.
  • such a chimeric molecule comprises a fusion of a prostate/breast/ovarian/bladder cancer polypeptide with a tag polypeptide which provides an epitope to which an anti-tag antibody can selectively bind.
  • the epitope tag is generally placed at the arriino-or carboxyl-terminus of the prostate/breast/ovarian/bladder cancer polypeptide.
  • the presence of such epitope-tagged forms of a prostate/breast/ovarian/bladder cancer polypeptide can be detected using an antibody against the tag polypeptide.
  • provision of the epitope tag enables the prostate/breast/ovarian/bladder cancer polypeptide to be readily purified by affinity purification using an anti-tag antibody or another type of affinity matrix that binds to the epitope tag.
  • the chimeric molecule may comprise a fusion of a prostate/breast/ovarian/bladder cancer polypeptide with an immunoglobulin or a particular region of an immunoglobulin. For a bivalent form of the chimeric molecule, such a fusion could be to the Fc region of an IgG molecule.
  • tag polypeptides and their respective antibodies are well known in the art. Examples include poly-histidine (poly-his) or poly-histidine-glycine (poly-his-gly) tags; the flu HA tag polypeptide and its antibody 12CA5 [Field et al., Mol. Cell.
  • tag polypeptides include the Flag-peptide [Hopp et al., BioTechnology, 6:1204-1210 (1988)]; the KT3 epitope peptide [Martin et al., Science, 255:192-194 (1992)]; tubulin epitope peptide
  • prostate/breast/ovarian/bladder cancer protein in one embodiment are other prostate/breast/ovarian/bladder cancer proteins of the specific cancer family, and prostate/breast/ovarian/bladder cancer proteins from other organisms, which are cloned and expressed as outlined below.
  • probe or degenerate polymerase chain reaction (PCR) primer sequences may be used to find other related prostate/breast/ovarian/bladder cancer proteins from humans or other organisms.
  • particularly useful probe and/or PCR primer sequences include the unique areas of the prostate/breast/ovarian/bladder cancer nucleic acid sequence.
  • preferred PCR primers are from about 15 to about 35 nucleotides in length, with from about 20 to about 30 being preferred, and may contain inosine as needed.
  • the conditions for the PCR reaction are well known in the art.
  • prostate/breast/ovarian/bladder cancer proteins can be made that are longer than those depicted in the figures, for example, by the elucidation of additional sequences, the addition of epitope or purification tags, the addition of other fusion sequences, etc.
  • Prostate/breast/ovarian/bladder cancer proteins may also be identified as being encoded by prostate/breast/ovarian/bladder cancer nucleic acids.
  • prostate/breast/ovarian/bladder cancer proteins are encoded by nucleic acids that will hybridize to the sequences ofthe sequence listings, or their complements, as outlined herein.
  • the prostate/breast/ovarian/bladder cancer protein when the prostate/breast/ovarian/bladder cancer protein is to be used to generate antibodies, for example for immunotherapy, the prostate/breast/ovarian/bladder cancer protein should share at least one epitope or determinant with the full length protein.
  • epitope or “determinant” herein is meant a portion of a protein which will generate and/or bind an antibody or T-cell receptor in the context of MHC.
  • the epitope is unique; that is, antibodies generated to a unique epitope show little or no cross-reactivity.
  • the term "antibody” includes antibody fragments, as are known in the art, including Fab, Fab 2 , single chain antibodies (Fv for example), chimeric antibodies, etc., either produced by the modification of whole antibodies orthose synthesized de novo using recombinant DNA technologies.
  • Polyclonal antibodies can be raised in a mammal, for example, by one or more injections of an immunizing agent and, if desired, an adjuvant.
  • the immunizing agent and/or adjuvant will be injected in the mammal by multiple subcutaneous or intraperitoneal injections.
  • the immunizing agent may include the BCR4 or fragment thereof or a fusion protein thereof. It may be useful to conjugate the immunizing agent to a protein known to be immunogenic in the mammal being immunized.
  • immunogenic proteins include but are not limited to keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor.
  • adjuvants which may be employed include Freund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate).
  • the immunization protocol may be selected by one skilled in the art without undue experimentation.
  • the antibodies may, alternatively, be monoclonal antibodies.
  • Monoclonal antibodies may be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975).
  • a hybridoma method a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent.
  • the lymphocytes may be immunized in vitro.
  • the immunizing agent will typically include the BCR4 polypeptide or fragment thereof or a fusion protein thereof.
  • peripheral blood lymphocytes are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired.
  • the lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell [Goding, Monoclonal Antibodies: Principles and Practice. Academic Press, (1986) pp.59-103].
  • Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin. Usually, rat or mouse myeloma cell lines are employed.
  • the hybridoma cells may be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells.
  • a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells.
  • the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine ("HAT medium"), which substances preventthe growth of HGPRT-deficient cells.
  • the antibodies are bispecific antibodies.
  • Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens.
  • one of the binding specificities is for the BCR4 or a fragment thereof, the other one is for any other antigen, and preferably for a cell-surface protein or receptor or receptor subunit, preferably one that is tumor specific.
  • the antibodies to prostate/breast/ovarian/bladder cancer are capable of reducing or eliminating the biological function of prostate/breast/ovarian/bladder cancer proteins, as is described below. That is, the addition of anti-prostate/breast/ovarian/bladder cancer antibodies (either polyclonal or preferably monoclonal) to prostate/breast/ovarian/bladder cancer (or cells containing such cancer) may reduce or eliminate the prostate/breast/ovarian/bladder cancer activity. Generally, at least a 25% decrease in activity is preferred, with at least about 50% being particularly preferred and about a 95-100% decrease being especially preferred.
  • the antibodies to the prostate/breast/ovarian/bladder cancer proteins are humanized antibodies.
  • Humanized forms of non-human (e.g., murine) antibodies are chimeric molecules of immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab') 2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin.
  • Humanized antibodies include human immunoglobulins (recipient antibody) in which residues form a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity.
  • CDR complementary determining region
  • Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence.
  • the humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin [Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.. 2:593-596 (1992)].
  • Fc immunoglobulin constant region
  • a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as import residues, which are typically taken from an import variable domain. Humanization can be essentially performed following the method of Winter and co-workers [Jones et al., Nature, 321 :522-525 (1986); Riechmann et al.. Nature.332:323-327 (1988); Verhoeyen et al.. Science, 239:1534-1536
  • humanized antibodies are chimeric antibodies (U.S. Patent No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species.
  • humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.
  • Human antibodies can also be produced using various techniques known in the art, including phage display libraries [Hoogenboom and Winter, J. Mol. Biol.. 227:381 (1991 ); Marks et al., L Mol. Biol.. 222:581 (1991)].
  • the techniques of Cole et al. and Boerner et al. are also available for the preparation of human monoclonal antibodies (Cole et al., Monoclonal Antibodies and
  • human antibodies can be made by introducing of human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Patent Nos.
  • immunotherapy is meant treatment of prostate/breast/ovarian/bladder cancer with an antibody raised against prostate/breast/ovarian/bladder cancer proteins.
  • immunotherapy can be passive or active. Passive immunotherapy as defined herein is the passive transfer of antibody to a recipient (patient). Active immunization is the induction of antibody and/or T-cell responses in a recipient (patient). Induction of an immune response is the result of providing the recipient with an antigen to which antibodies are raised.
  • the antigen may be provided by injecting a polypeptide against which antibodies are desired to be raised into a recipient, or contacting the recipient with a nucleic acid capable of expressing the antigen and under conditions for expression of the antigen.
  • the prostate/breast/ovarian/bladder cancer proteins against which antibodies are raised are secreted proteins as described above.
  • antibodies used for treatment bind and prevent the secreted protein from binding to its receptor, thereby inactivating the secreted prostate/breast/ovarian/bladder cancer protein.
  • the prostate/breast/ovarian/bladder cancer protein to which antibodies are raised is a transmembrane protein.
  • antibodies used for treatment bind the extracellular domain of the prostate/breast/ovarian/bladder cancer protein and prevent it from binding to other proteins, such as circulating ligands or cell-associated molecules.
  • the antibody may cause down-regulation of the transmembrane prostate/breast/ovarian/bladder cancer protein.
  • the antibody may be a competitive, non-competitive or uncompetitive inhibitor of protein binding to the extracellular domain of the prostate/breast/ovarian/bladder cancer protein.
  • the antibody is also an antagonist ofthe prostate/breast/ovarian/bladder cancer protein. Further, the antibody prevents activation of the transmembrane prostate/breast/ovarian/bladder cancer protein. In one aspect, when the antibody prevents the binding of other molecules to the prostate/breast/ovarian/bladder cancer protein, the antibody prevents growth of the cell.
  • the antibody also sensitizes the cell to cytotoxic agents, including, but not limited to TNF- ⁇ , TNF- ⁇ , IL-1 , INF- ⁇ and IL-2, or chemotherapeutic agents including 5FU, vinblastine, actinomycin D, cisplatin, methotrexate, and the like.
  • the antibody belongs to a sub-type that activates serum complement when complexed with the transmembrane protein thereby mediating cytotoxicity.
  • prostate/breast/ovarian/bladder cancer is treated by administering to a patient antibodies directed against the transmembrane prostate/breast/ovarian/bladder cancerprotein.
  • the antibody is conjugated to a therapeutic moiety.
  • the therapeutic moiety is a small molecule that modulates the activity of the prostate/breast/ovarian/bladder cancer protein.
  • the therapeutic moiety modulates the activity of molecules associated with or in close proximity to the prostate/breast/ovarian/bladder cancer protein.
  • the therapeutic moiety may inhibit enzymatic activity such as protease or protein kinase activity associated with prostate/breast/ovarian/bladder cancer.
  • the therapeutic moiety may also be a cytotoxic agent.
  • targeting the cytotoxic agent to tumor tissue or cells results in a reduction in the number of afflicted cells, thereby reducing symptoms associated with prostate/breast/ovarian/bladder cancer.
  • Cytotoxic agents are numerous and varied and include, but are not limited to, cytotoxic drugs or toxins or active fragments of such toxins. Suitable toxins and their corresponding fragments include diptheria A chain, exotoxin A chain, ricin A chain, abrin A chain, curcin, crotin, phenomycin, enomycin and the like.
  • Cytotoxic agents also include radiochemicals made by conjugating radioisotopes to antibodies raised against prostate/breast/ovarian/bladder cancer proteins, or binding of a radionuclide to a chelating agent that has been covalently attached to the antibody.
  • Targeting the therapeutic moiety to transmembrane prostate/breast/ovarian/bladder cancer proteins not only serves to increase the local concentration of therapeutic moiety in the cancer afflicted area, but also serves to reduce deleterious side effects that may be associated with the therapeutic moiety.
  • the PC protein against which the antibodies are raised is an intracellular protein.
  • the antibody may be conjugated to a protein which facilitates entry into the cell.
  • the antibody enters the cell by endocytosis.
  • a nucleic acid encoding the antibody is administered to the individual or cell.
  • an antibody thereto contains a signal for that target localization, i.e., a nuclear localization signal.
  • the prostate/breast/ovarian/bladder cancer antibodies of the invention specifically bind to t prostate/breast/ovarian/bladder cancer proteins.
  • specifically bind herein is meant that the antibodies bind to the protein with a binding constant in the range of at least 10 "4 - 10 "6 M “1 , with a preferred range being 10 "7 - 10 "9 M “1 .
  • the prostate/breast/ovarian/bladder cancer protein is purified or isolated after expression.
  • Prostate/breast/ovarian/bladder cancer proteins may be isolated or purified in a variety of ways known to those skilled in the art depending on what other components are present in the sample. Standard purification methods include electrophoretic, molecular, immunological and chromatographic techniques, including ion exchange, hydrophobic, affinity, and reverse-phase HPLC chromatography, and chromatofocusing.
  • the prostate/breast/ovarian/bladder cancer protein may be purified using a standard anti-prostate cancer and/or anti-breast cancer antibody column. Ultrafiltration and diafiltration techniques, in conjunction with protein concentration, are also useful.
  • prostate/breast/ovarian/bladder cancer proteins and nucleic acids are useful in a number of applications.
  • the expression levels of genes are determined for different cellular states in the prostate/breast/ovarian/bladder cancer phenotype; that is, for example, the expression levels of genes in normal prostate tissue and in prostate cancer tissue (and in some cases, for varying severities of prostate cancer that relate to prognosis, as outlined below) are evaluated to provide expression profiles.
  • the expression levels of genes in normal breast tissue and in breast cancer tissue are evaluated to provide expression profiles.
  • An expression profile of a particular cell state or point of development is essentially a "fingerprint" ofthe state; while two states may have any particular gene similarly expressed, the evaluation of a number of genes simultaneously allows the generation of a gene expression profile that is unique to the state ofthe cell.
  • information regarding which genes are important (including both up- and down-regulation of genes) in each of these states is obtained. Then, diagnosis may be done or confirmed: does tissue from a particular patient have the gene expression profile of normal or cancer tissue.
  • a prostate/breast/ovarian/bladder cancer gene can qualitatively have its expression altered, including an activation or inactivation, in, for example, normal versus cancer tissue. That is, genes may be turned on or turned off in a particular state, relative to another state. As is apparent to the skilled artisan, any comparison of two or more states can be made. Such a qualitatively regulated gene will exhibit an expression pattern within a state or cell type which is detectable by standard techniques in one such state or cell type, but is not detectable in both.
  • the determination is quantitative in that expression is increased or decreased; that is, the expression of the gene is either upregulated, resulting in an increased amount of transcript, or downregulated, resulting in a decreased amount of transcript.
  • the degree to which expression differs need only be large enough to quantify via standard characterization techniques as outlined below, such as by use of Affymetrix GeneChipTM expression arrays, Lockhart, Nature Biotechnology, 14:1675-1680 (1996), hereby expressly incorporated by reference.
  • Other techniques include, but are not limited to, quantitative reverse transcriptase PCR, Northern analysis and RNase protection.
  • the change in expression i.e. upregulation or downregulation
  • this may be done by evaluation at either the gene transcript, or the protein level; that is, the amount of gene expression may be monitored using nucleic acid probes to the DNA or RNA equivalent of the gene transcript, and the quantification of gene expression levels, or, alternatively, the final gene product itself (protein) can be monitored, for example through the use of antibodies to the prostate/breast/ovarian/bladder cancer protein and standard immunoassays (ELISAs.e tc.) or other techniques, including mass spectroscopy assays, 2D gel electrophoresis assays, etc.
  • ELISAs.e tc. standard immunoassays.
  • the proteins corresponding to prostate/breast/ovarian/bladder cancer genes i.e. those identified as being important in a cancer phenotype, can be evaluated in a cancer diagnostic test.
  • gene expression monitoring is done and a number of genes, i.e. an expression profile, is monitored simultaneously, although multiple protein expression monitoring can be done as well. Similarly, these assays may be done on an individual basis as well.
  • the prostate/breast/ovarian/bladder cancer nucleic acid probes are attached to biochips as outlined herein for the detection and quantification of prostate/breast/ovarian/bladder cancer sequences in a particular cell.
  • the assays are further described below in the example.
  • nucleic acids encoding the prostate/breast/ovarian/bladder cancer protein are detected.
  • DNA or RNA encoding the prostate/breast/ovarian/bladder cancer protein may be detected, of particular interest are methods wherein the mRNA encoding a prostate/breast/ovarian/bladder cancer protein is detected.
  • the presence of mRNA in a sample is an indication that the prostate/breast/ovarian/bladder cancer gene has been transcribed to form the mRNA, and suggests that the protein is expressed.
  • Probes to detect the mRNA can be any nucleotide/deoxynucleotide probe that is complementary to and base pairs with the mRNA and includes but is not limited to oligonucleotides, cDNA or RNA. Probes also should contain a detectable label, as defined herein.
  • the mRNA is detected after immobilizing the nucleic acid to be examined on a solid support such as nylon membranes and hybridizing the probe with the sample. Following washing to remove the non-specifically bound probe, the label is detected.
  • detection of the mRNA is performed in situ. In this method permeabilized cells or tissue samples are contacted with a detectably labeled nucleic acid probe for sufficient time to allow the probe to hybridize with the target mRNA.
  • RNA probe that is complementary to the mRNA encoding a prostate/breast/ovarian/bladder cancer protein is detected by binding the digoxygenin with an anti- digoxygenin secondary antibody and developed with nitro blue tetrazolium and 5-bromo-4-chloro-3-indoyl phosphate.
  • any of the three chipses of proteins as described herein are used in diagnostic assays.
  • the prostate/breast/ovarian/bladder cancer proteins, antibodies, nucleic acids, modified proteins and cells containing prostate/breast/ovarian/bladder cancer sequences are used in diagnostic assays. This can be done on an individual gene or corresponding polypeptide level.
  • the expression profiles are used, preferably in conjunction with high throughput screening techniques to allow monitoring for expression profile genes and/or corresponding polypeptides.
  • prostate/breast/ovarian/bladder cancer proteins including intracellular, transmembrane or secreted proteins, find use as markers of one or more of the specified cancers. Detection of these proteins in putative cancer tissue of patients allows for a determination ordiagnosis of prostate/breast/ovarian/bladder cancer. Numerous methods known to those of ordinary skill in the art find use in detecting prostate/breast/ovarian/bladder cancer.
  • antibodies are used to detect prostate/breast/ovarian/bladder cancer proteins.
  • a preferred method separates proteins from a sample or patient by electrophoresis on a gel (typically a denaturing and reducing protein gel, but may be any other type of gel including isoelectric focusing gels and the like).
  • the prostate/breast/ovarian/bladder cancer protein is detected by immunoblotting with antibodies raised against the prostate/breast/ovarian/bladder cancer protein. Methods of immunoblotting are well known to those of ordinary skill in the art.
  • antibodies to the prostate/breast/ovarian/bladder cancer protein find use in in situ imaging techniques.
  • cells are contacted with from one to many antibodies to the prostate/breast/ovarian/bladder cancer protein(s). Following washing to remove non-specific antibody binding, the presence of the antibody or antibodies is detected.
  • the antibody is detected by incubating with a secondary antibody that contains a detectable label.
  • the primary antibody to the prostate/breast/ovarian/bladder cancer protein(s) contains a detectable label.
  • each one of multiple primary antibodies contains a distinct and detectable label. This method finds particular use in simultaneous screening for a pluralilty of prostate/breast/ovarian/bladder cancer proteins. As will be appreciated by one of ordinary skill in the art, numerous other histological imaging techniques are useful in the invention.
  • the label is detected in a fluorometer which has the ability to detect and distinguish emissions of different wavelengths.
  • a fluorescence activated cell sorter FACS
  • FACS fluorescence activated cell sorter
  • antibodies find use in diagnosing prostate/breast/ovarian/bladder cancer from blood samples.
  • certain prostate/breast/ovarian/bladder cancer proteins are secreted/circulating molecules. Blood samples, therefore, are useful as samples to be probed or tested for the presence of secreted prostate/breast/ovarian/bladder cancer proteins.
  • Antibodies can be used to detect the cancer by any of the previously described immunoassay techniques including ELISA, immunoblotting (Western blotting), immunoprecipitation, BIACORE technology and the like, as will be appreciated by one of ordinary skill in the art.
  • in situ hybridization of labeled prostate/breast/ovarian/bladder cancer nucleic acid probes to tissue arrays is done.
  • arrays of tissue samples, including prostate/breast/ovarian/bladder cancer tissue and/or normal tissue are made.
  • In situ hybridization as is known in the art can then be done.
  • the prostate/breast/ovarian/bladder cancer proteins, antibodies, nucleic acids, modified proteins and cells containing prostate/breast/ovarian/bladder cancer sequences are used in prognosis assays.
  • gene expression profiles can be generated that correlate to cancer severity, in terms of long term prognosis. Again, this may be done on either a protein or gene level, with the use of genes being preferred.
  • the prostate/breast/ovarian/bladder cancer probes are attached to biochips for the detection and quantification of prostate/breast/ovarian/bladder cancer sequences in a tissue or patient. The assays proceed as outlined for diagnosis.
  • any of the three classes of proteins as described herein are used in drug screening assays.
  • the prostate/breast/ovarian/bladder cancer proteins, antibodies, nucleic acids, modified proteins and cells containing prostate/breast/ovarian/bladder cancer sequences are used in drug screening assays or by evaluating the effect of drug candidates on a "gene expression profile" or expression profile of polypeptides.
  • the expression profiles are used, preferably in conjunction with high throughput screening techniques to allow monitoring for expression profile genes after treatment with a candidate agent, Zlokarnik, et al., Science 279, 84-8 (1998), Heid, 1996 #69.
  • the prostate/breast/ovarian/bladder cancer proteins, antibodies, nucleic acids, modified proteins and cells containing the native or modified prostate/breast/ovarian/bladder cancer proteins are used in screening assays. That is, the present invention provides novel methods for screening for compositions which modulate the cancer phenotype. As above, this can be done on an individual gene level or by evaluating the effect of drug candidates on a "gene expression profile". In a preferred embodiment, the expression profiles are used, preferably in conjunction with high throughput screening techniques to allow monitoring for expression profile genes after treatment with a candidate agent, see Zlokarnik, supra.
  • assays may be executed.
  • assays may be run on an individual gene or protein level. That is, having identified a particular gene as up regulated in prostate/breast/ovarian/bladder cancer, candidate bioactive agents may be screened to modulate this gene's response; preferably to down regulate the gene, although in some circumstances to up regulate the gene.
  • “Modulation” thus includes both an increase and a decrease in gene expression. The preferred amount of modulation will depend on the original change of the gene expression in normal versus tumor tissue, with changes of at least 10%, preferably 50%, more preferably 100-300%, and in some embodiments 300-1000% or greater. Thus, if a gene exhibits a 4 fold increase in tumor compared to normal tissue, a decrease of about four fold is desired; a 10 fold decrease in tumor compared to normal tissue gives a 10 fold increase in expression for a candidate agent is desired.
  • this may be done by evaluation at either the gene or the protein level; that is, the amount of gene expression may be monitored using nucleic acid probes and the quantification of gene expression levels, or, alternatively, the gene product itself can be monitored, for example through the use of antibodies to the prostate/breast/ovarian/bladder cancer protein and standard immunoassays.
  • gene expression monitoring is done and a number of genes, i.e. an expression profile, is monitored simultaneously, although multiple protein expression monitoring can be done as well.
  • the prostate/breast/ovarian/bladder cancer nucleic acid probes are attached to biochips as outlined herein for the detection and quantification of prostate/breast/ovarian/bladder cancer sequences in a particular cell.
  • the assays are further described below.
  • a candidate bioactive agent is added to the cells prior to analysis.
  • screens are provided to identify a candidate bioactive agent which modulates prostate/breast/ovarian/bladder cancer, modulates prostate/breast/ovarian/bladder cancer proteins, binds to a prostate/breast/ovarian/bladder cancer protein, or interferes between the binding of a prostate/breast/ovarian/bladder cancer protein and an antibody.
  • bioactive agent or “drug candidate” or grammatical equivalents as used herein describes any molecule, e.g., protein, oligopeptide, small organic molecule, polysaccharide, polynucleotide, etc. , to be tested for bioactive agents that are capable of directly or indirectly altering the cancer phenotype or the expression of a prostate/breast/ovarian/bladder cancer sequence, including both nucleic acid sequences and protein sequences.
  • the bioactive agents modulate the expression profiles, or expression profile nucleic acids or proteins provided herein.
  • the candidate agent suppresses a prostate/breast/ovarian/bladder cancer phenotype, for example to a normal fingerprint ofthe same tissue type.
  • the candidate agent preferably suppresses a severe cancer phenotype.
  • a plurality of assay mixtures are run in parallel with different agent concentrations to obtain a differential response to the various concentrations.
  • one of these concentrations serves as a negative control, i.e., at zero concentration or below the level of detection.
  • a candidate agent will neutralize the effect of a CRC protein.
  • neutralize is meant that activity of a protein is either inhibited or counter acted against so as to have substantially no effect on a cell.
  • Candidate agents encompass numerous chemical classes, though typically they are organic molecules, preferably small organic compounds having a molecular weight of more than 100 and less than about 2,500 daltons (D). Preferred small molecules are less than 2000, or less than 1500 or less than 1000 or less than 500 D.
  • Candidate agents comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups.
  • the candidate agents often comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups.
  • Candidate agents are also found among biomolecules including peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof. Particularly preferred are peptides.
  • Candidate agents are obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means. Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification to produce structural analogs.
  • the candidate bioactive agents are proteins.
  • protein herein is meant at least two covalently attached amino acids, which includes proteins, polypeptides, oligopeptides and peptides.
  • the protein may be made up of naturally occurring amino acids and peptide bonds, or synthetic peptidomimetic structures.
  • amino acid or “peptide residue”, as used herein means both naturally occurring and synthetic amino acids. For example, homo- phenylalanine, citrulline and noreleucine are considered amino acids for the purposes of the invention.
  • Amino acid also includes imino acid residues such as proline and hydroxyproline.
  • the side chains may be in either the (R) or the (S) configuration. In the preferred embodiment, the amino acids are in the (S) or L-configuration. If non-naturally occurring side chains are used, non-amino acid substituents may be used, for example to prevent or retard in vivo degradations.
  • the candidate bioactive agents are naturally occurring proteins or fragments of naturally occurring proteins.
  • cellular extracts containing proteins, or random or directed digests of proteinaceous cellular extracts may be used.
  • libraries of procaryotic and eucaryotic proteins may be made for screening in the methods of the invention.
  • Particularly preferred in this embodiment are libraries of bacterial, fungal, viral, and mammalian proteins, with the latter being preferred, and human proteins being especially preferred.
  • the candidate bioactive agents are peptides of from about 5 to about 30 amino acids, with from about 5 to about 20 amino acids being preferred, and from about 7 to about 15 being particularly preferred.
  • the peptides may be digests of naturally occurring proteins as is outlined above, random peptides, or "biased” random peptides.
  • randomized or grammatical equivalents herein is meant that each nucleic acid and peptide consists of essentially random nucleotides and amino acids, respectively. Since generally these random peptides (or nucleic acids, discussed below) are chemically synthesized, they may incorporate any nucleotide or amino acid at any position.
  • the synthetic process can be designed to generate randomized proteins or nucleic acids, to allow the formation of all or most of the possible combinations over the length of the sequence, thus forming a library of randomized candidate bioactive proteinaceous agents.
  • the library is fully randomized, with no sequence preferences or constants at any position.
  • the library is biased. That is, some positions within the sequence are either held constant, or are selected from a limited number of possibilities.
  • the nucleotides or amino acid residues are randomized within a defined class, for example, of hydrophobic amino acids, hydrophilic residues, sterically biased (either small or large) residues, towards the creation of nucleic acid binding domains, the creation of cysteines, for cross-linking, prolines for SH-3 domains, serines, threonines, tyrosines or histidines for phosphorylation sites, etc., or to purines, etc.
  • the candidate bioactive agents are nucleic acids, as defined above.
  • nucleic acid candidate bioactive agents may be naturally occurring nucleic acids, random nucleic acids, or "biased" random nucleic acids.
  • digests of procaryotic or eucaryotic genomes may be used as is outlined above for proteins.
  • the candidate bioactive agents are organic chemical moieties, a wide variety of which are available in the literature.
  • the sample containing the target sequences to be analyzed is added to the biochip.
  • the target sequence is prepared using known techniques.
  • the sample may be treated to lyse the cells, using known lysis buffers, electroporation, etc., with purification and/or amplification such as PCR occurring as needed, as will be appreciated by those in the art.
  • an in vitro transcription with labels covalently attached to the nucleosides is done.
  • nucleic acids are labeled with biotin-FITC or PE, or with cy3 or cy5.
  • the target sequence is labeled with, for example, a fluorescent, a chemiluminescent, a chemical, or a radioactive signal, to provide a means of detecting the target sequence's specific binding to a probe.
  • the label also can be an enzyme, such as, alkaline phosphatase or horseradish peroxidase, which when provided with an appropriate substrate produces a product that can be detected.
  • the label can be a labeled compound or small molecule, such as an enzyme inhibitor, that binds but is not catalyzed or altered by the enzyme.
  • the label also can be a moiety or compound, such as, an epitope tag or biotin which specifically binds to streptavidin.
  • the streptavidin is labeled as described above, thereby, providing a detectable signal for the bound target sequence.
  • unbound labeled streptavidin is removed prior to analysis.
  • these assays can be direct hybridization assays or can comprise "sandwich assays", which include the use of multiple probes, as is generally outlined in U.S. Patent Nos. 5,681 ,702, 5,597,909, 5,545,730, 5,594,117, 5,591 ,584, 5,571 ,670, 5,580,731, 5,571 ,670, 5,591 ,584, 5,624,802, 5,635,352, 5,594,118, 5,359,100, 5,124,246 and
  • the target nucleic acid is prepared as outlined above, and then added to the biochip comprising a plurality of nucleic acid probes, under conditions that allow the formation of a hybridization complex.
  • hybridization conditions may be used in the present invention, including high, moderate and low stringency conditions as outlined above.
  • the assays are generally run under stringency conditions which allows formation of the label probe hybridization complex only in the presence of target.
  • Stringency can be controlled by altering a step parameter that is a thermodynamic variable, including, but not limited to, temperature, formamide concentration, salt concentration, chaotropic salt concentration pH, organic solvent concentration, etc.
  • reaction may be accomplished in a variety of ways, as will be appreciated by those in the art. Components of the reaction may be added simultaneously, or sequentially, in any order, with preferred embodiments outlined below.
  • the reaction may include a variety of other reagents may be included in the assays. These include reagents like salts, buffers, neutral proteins, e.g. albumin, detergents, etc which may be used to facilitate optimal hybridization and detection, and/or reduce non-specific or background interactions. Also reagents that otherwise improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, anti-microbial agents, etc., may be used, depending on the sample preparation methods and purity of the target.
  • the data is analyzed to determine the expression levels, and changes in expression levels as between states, of individual genes, forming a gene expression profile.
  • the screens are done to identify drugs or bioactive agents that modulate the prostate/breast/ovarian/bladder cancer phenotype.
  • a preferred embodiment is in the screening of candidate agents that can induce or suppress a particular expression profile, thus preferably generating the associated phenotype. That is, candidate agents that can mimic or produce an expression profile in prostate/breast/ovarian/bladder cancer similar to the expression profile of normal tissue of the same type is expected to result in a suppression of the cancer phenotype.
  • mimicking an expression profile, or changing one profile to another is the goal.
  • screens can be run to alter the expression of the genes individually. That is, screening for modulation of regulation of expression of a single gene can be done; that is, rather than try to mimic all or part of an expression profile, screening for regulation of individual genes can be done. Thus, for example, particularly in the case of target genes whose presence or absence is unique between two states, screening is done for modulators of the target gene expression.
  • screening is done to alter the biological function of the expression product of the prostate/breast/ovarian/bladder cancer gene. Again, having identified the importance of a gene in a particular state, screening for agents that bind and/or modulate the biological activity of the gene product can be run as is more fully outlined below.
  • screening of candidate agents that modulate the prostate/breast/ovarian/bladder cancer phenotype either at the gene expression level or the protein level can be done.
  • screens can be done for novel genes that are induced in response to a candidate agent.
  • a candidate agent After identifying a candidate agent based upon its ability to suppress a prostate/breast/ovarian/bladder cancer expression pattern leading to a normal expression pattern, or modulate a single prostate/breast/ovarian/bladder cancer gene expression profile so as to mimic the expression of the gene from normal tissue, a screen as described above can be performed to identify genes that are specifically modulated in response to the agent. Comparing expression profiles between normal tissue and agent treated prostate/breast/ovarian/bladder cancer tissue reveals genes that are not expressed in normal tissue or cancer tissue, but are expressed in agent treated tissue.
  • agent specific sequences can be identified and used by any of the methods described herein for prostate/breast/ovarian/bladder cancer genes or proteins.
  • sequences and the proteins they encode find use in marking or identifying agent treated cells.
  • antibodies can be raised against the agent induced proteins and used to target novel therapeutics to the treated prostate/breast/ovarian/bladder cancer tissue sample.
  • a candidate agent is administered to a population of prostate cancer or breast cancer cells, that thus has an associated prostate cancer or breast cancer expression profile, respectively.
  • administration or “contacting” herein is meant that the candidate agent is added to the cells in such a manner as to allow the agent to act upon the cell, whether by uptake and intracellular action, or by action at the cell surface.
  • nucleic acid encoding a proteinaceous candidate agent i.e. a peptide
  • the cells can be washed if desired and are allowed to incubate under preferably physiological conditions for some period of time.
  • the cells are then harvested and a new gene expression profile is generated, as outlined herein.
  • prostate/breast/ovarian/bladder cancertissue may be screened for agents that reduce or suppress the cancer phenotype.
  • a change in at least one gene of the expression profile indicates that the agent has an effect on cancer activity.
  • screens may be done on individual genes and gene products (proteins). That is, having identified a particular prostate/breast/ovarian/bladder cancer gene as important in a particular state, screening of modulators of either the expression of the gene or the gene product itself can be done.
  • the gene products of prostate/breast/ovarian/bladder cancer genes are sometimes referred to herein as "prostate cancer proteins” or “prostate cancer modulating proteins” or "PCMP” and/or "breast cancer proteins” or “breast cancer modulating proteins” or “BCMP”. Additionally, “modulator” and “modulating” proteins are sometimes used interchangeably herein.
  • the prostate/breast/ovarian/bladder cancer protein is termed BCR4.
  • BCR4 sequences can be identified as described herein for prostate/breast/ovarian/bladder cancer sequences.
  • a BCR4 protein sequence is as depicted in Figure 2.
  • the prostate/breast/ovarian/bladder cancer protein may be a fragment, or alternatively, be the full length protein to the fragment shown herein.
  • the prostate/breast/ovarian/bladder cancer protein is a fragment.
  • the amino acid sequence which is used to determine sequence identity or similarity is that depicted in figure 2.
  • sequences are naturally occurring allelic variants of a protein having the sequence depicted in figure 2.
  • sequences are homologs or orthologs of the human BRC4 sequence, such as the murine BCR4 sequence shown in Figure 4.
  • sequences are sequence variants as further described herein.
  • the prostate/breast/ovarian/bladder cancer protein is a fragment of approximately 14 to 24 amino acids long. More preferably the fragment is a soluble fragment.
  • the fragment includes a non-transmembrane region.
  • the fragment has an N-terminal Cys to aid in solubility.
  • the c-terminus of the fragment is kept as a free acid and the n-terminus is a free amine to aid in coupling, i.e., to cysteine.
  • a BCR4 fragment has at least one BCR4 bioactivity as defined below.
  • prostate/breast/ovarian/bladder cancer proteins are conjugated to an immunogenic agent as discussed herein . In one embodiment the prostate/breast/ovarian/bladder cancer protein is conjugated to BSA.
  • screening for modulators of expression of specific genes can be done. This will be done as outlined above, but in general the expression of only one or a few genes are evaluated.
  • screens are designed to first find candidate agents that can bind to prostate/breast/ovarian/bladder cancer proteins, and then these agents may be used in assays that evaluate the ability of the candidate agent to modulate prostate/breast/ovarian/bladder cancer activity.
  • binding assays and activity assays there are a number of different assays which may be run; binding assays and activity assays.
  • binding assays are done.
  • purified or isolated gene product is used; that is, the gene products of one or more prostate/breast/ovarian/bladder cancer nucleic acids are made. In general, this is done as is known in the art.
  • antibodies are generated to the protein gene products, and standard immunoassays are run to determine the amount of protein present.
  • cells comprising the prostate/breast/ovarian/bladder cancer proteins can be used in the assays.
  • the methods comprise combining a prostate/breast/ovarian/bladder cancer protein and a candidate bioactive agent, and determining the binding of the candidate agent to the prostate/breast/ovarian/bladder cancer protein.
  • Preferred embodiments utilize the human prostate/breast/ovarian/bladder cancer protein, although other mammalian proteins may also be used, for example for the development of animal models of human disease.
  • variant or derivative prostate/breast/ovarian/bladder cancer proteins may be used.
  • the prostate/breast/ovarian/bladder cancer protein or the candidate agent is non-diffusably bound to an insoluble support having isolated sample receiving areas (e.g. a microtiter plate, an array, etc.).
  • isolated sample receiving areas e.g. a microtiter plate, an array, etc.
  • soluble assays known in the art may be performed.
  • the insoluble supports may be made of any composition to which the compositions can be bound, is readily separated from soluble material, and is otherwise compatible with the overall method of screening.
  • the surface of such supports may be solid or porous and of any convenient shape. Examples of suitable insoluble supports include microtiter plates, arrays, membranes and beads.
  • Microtiter plates and arrays are especially convenient because a large number of assays can be carried out simultaneously, using small amounts of reagents and samples.
  • the particular manner of binding of the composition is not crucial so long as it is compatible with the reagents and overall methods of the invention, maintains the activity of the composition and is nondiffusable.
  • Preferred methods of binding include the use of antibodies (which do not sterically block either the ligand binding site or activation sequence when the protein is bound to the support), direct binding to "sticky" or ionic supports, chemical crosslinking, the synthesis of the protein or agent on the surface, etc. Following binding of the protein or agent, excess unbound material is removed by washing. The sample receiving areas may then be blocked through incubation with bovine serum albumin (BSA), casein or other innocuous protein or other moiety.
  • BSA bovine serum albumin
  • the prostate/breast/ovarian/bladder cancer protein is bound to the support, and a candidate bioactive agent is added to the assay.
  • the candidate agent is bound to the support and the prostate/breast/ovarian/bladder cancer protein is added.
  • Novel binding agents include specific antibodies, non-natural binding agents identified in screens of chemical libraries, peptide analogs, etc. Of particular interest are screening assays for agents that have a low toxicity for human cells. A wide variety of assays may be used for this purpose, including labeled in vitro protein-protein binding assays, electrophoretic mobility shift assays, immunoassays for protein binding, functional assays (phosphorylation assays, etc.) and the like.
  • the determination of the binding of the candidate bioactive agent to the prostate/breast/ovarian/bladder cancer protein may be done in a number of ways.
  • the candidate bioactive agent is labeled, and binding determined directly. For example, this may be done by attaching all or a portion of the prostate/breast/ovarian/bladder cancer protein to a solid support, adding a labeled candidate agent (for example a fluorescent label), washing off excess reagent, and determining whether the label is present on the solid support.
  • a labeled candidate agent for example a fluorescent label
  • washing off excess reagent for example a fluorescent label
  • label herein is meant that the compound is either directly or indirectly labeled with a label which provides a detectable signal, e.g. radioisotope, fluorescers, enzyme, antibodies, particles such as magnetic particles, chemiluminescers, or specific binding molecules, etc.
  • Specific binding molecules include pairs, such as biotin and streptavidin, digoxin and antidigoxin etc.
  • the complementary member would normally be labeled with a molecule which provides for detection, in accordance with known procedures, as outlined above.
  • the label can directly or indirectly provide a detectable signal.
  • the proteins may be labeled at tyrosine positions using 125 l, or with fluorophores.
  • more than one component may be labeled with different labels; using 125 l for the proteins, for example, and a fluorophor for the candidate agents.
  • the binding of the candidate bioactive agent is determined through the use of competitive binding assays.
  • the competitor is a binding moiety known to bind to the target molecule (i.e. prostate cancer), such as an antibody, peptide, binding partner, ligand, etc.
  • the target molecule i.e. prostate cancer
  • the candidate bioactive agent is labeled.
  • Either the candidate bioactive agent, or the competitor, or both, is added first to the protein for a time sufficient to allow binding, if present.
  • Incubations may be performed at any temperature which facilitates optimal activity, typically between 4 and 40°C. Incubation periods are selected for optimum activity, but may also be optimized to facilitate rapid high through put screening. Typically between 0.1 and 1 hour will be sufficient. Excess reagent is generally removed or washed away. The second component is then added, and the presence or absence of the labeled component is followed, to indicate binding.
  • the competitor is added first, followed by the candidate bioactive agent.
  • Displacement of the competitor is an indication that the candidate bioactive agent is binding to the prostate/breast/ovarian/bladder cancer protein and thus is capable of binding to, and potentially modulating, the activity of the prostate/breast/ovarian/bladder cancer protein.
  • either component can be labeled.
  • the presence of label in the wash solution indicates displacement by the agent.
  • the candidate bioactive agent is labeled, the presence of the label on the support indicates displacement.
  • the candidate bioactive agent is added first, with incubation and washing, followed by the competitor.
  • the absence of binding by the competitor may indicate that the bioactive agent is bound to the prostate/breast/ovarian/bladder cancer protein with a higher affinity.
  • the candidate bioactive agent is labeled, the presence ofthe label on the support, coupled with a lack of competitor binding, may indicate that the candidate agent is capable of binding to the prostate/breast/ovarian/bladder cancer protein.
  • the methods comprise differential screening to identity bioactive agents that are capable of modulating the activity of the prostate/breast/ovarian/bladder cancer proteins.
  • the methods comprise combining a prostate/breast/ovarian/bladder cancer protein and a competitor in a first sample.
  • a second sample comprises a candidate bioactive agent, a prostate/breast/ovarian/bladder cancer protein and a competitor.
  • the binding ofthe competitor is determined for both samples, and a change, or difference in binding between the two samples indicates the presence of an agent capable of binding to the prostate/breast/ovarian/bladder cancer protein and potentially modulating its activity. That is, if the binding of the competitor is different in the second sample relative to the first sample, the agent is capable of binding to the prostate/breast/ovarian/bladder cancer protein.
  • a preferred embodiment utilizes differential screening to identify drug candidates that bind to the native prostate/breast/ovarian/bladder cancer protein, but cannot bind to modified prostate/breast/ovarian/bladder cancer proteins.
  • the structure of the prostate/breast/ovarian/bladder cancer protein may be modeled, and used in rational drug design to synthesize agents that interact with that site.
  • Drug candidates that affect prostate cancer bioactivity are also identified by screening drugs for the ability to either enhance or reduce the activity of the protein.
  • Positive controls and negative controls may be used in the assays.
  • Preferably all control and test samples are performed in at least triplicate to obtain statistically significant results. Incubation of all samples is for a time sufficient for the binding of the agent to the protein. Following incubation, all samples are washed free of non-specifically bound material and the amount of bound, generally labeled agent determined. For example, where a radiolabel is employed, the samples may be counted in a scintillation counter to determine the amount of bound compound.
  • reagents may be included in the screening assays. These include reagents like salts, neutral proteins, e.g. albumin, detergents, etc which may be used to facilitate optimal protein-protein binding and/or reduce non-specific or background interactions. Also reagents that otherwise improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, anti-microbial agents, etc., may be used. The mixture of components may be added in any order that provides for the requisite binding.
  • methods for screening for a bioactive agent capable of modulating the activity of prostate/breast/ovarian/bladder cancer proteins comprise the steps of adding a candidate bioactive agent to a sample of prostate/breast/ovarian/bladder cancer proteins, as above, and determining an alteration in the biological activity of prostate/breast/ovarian/bladder cancer proteins.
  • “Modulating the activity" of prostate/breast/ovarian/bladder cancer includes an increase in activity, a decrease in activity, or a change in the type or kind of activity present.
  • the candidate agent should both bind to prostate/breast/ovarian/bladder cancer proteins (although this may not be necessary), and alter its biological or biochemical activity as defined herein.
  • the methods include both in vitro screening methods, as are generally outlined above, and in vivo screening of cells for alterations in the presence, distribution, activity or amount of prostate/breast/ovarian/bladder cancer proteins.
  • the methods comprise combining a prostate cancer sample and a candidate bioactive agent, and evaluating the effect on prostate cancer activity.
  • prostate cancer activity and/or “breast cancer activity” or grammatical equivalents herein is meant at least one of the cancer's biological activities, including, but not limited to, cell division, preferably in prostate or breast tissue, cell proliferation, tumor growth, and transformation of cells.
  • prostate/breast/ovarian/bladder cancer activity includes activation of BCR4 or a substrate thereof by BCR4.
  • An inhibitor of prostate/breast/ovarian/bladder cancer activity is an agent which inhibits any one or more prostate/breast/ovarian/bladder cancer activities.
  • the activity of the prostate/breast/ovarian/bladder cancer protein is increased; in another preferred embodiment, the activity of the prostate/breast/ovarian/bladder cancer protein is decreased.
  • bioactive agents that are antagonists are preferred in some embodiments, and bioactive agents that are agonists may be preferred in other embodiments.
  • the invention provides methods for screening for bioactive agents capable of modulating the activity of a prostate/breast/ovarian/bladder cancer protein.
  • the methods comprise adding a candidate bioactive agent, as defined above, to a cell comprising prostate/breast/ovarian/bladder cancer proteins.
  • Preferred cell types include almost any cell.
  • the cells contain a recombinant nucleic acid that encodes a prostate/breast/ovarian/bladder cancer protein.
  • a library of candidate agents are tested on a plurality of cells.
  • the assays are evaluated in the presence or absence or previous or subsequent exposure of physiological signals, for example hormones, antibodies, peptides, antigens, cytokines, growth factors, action potentials, pharmacological agents including chemotherapeutics, radiation, carcinogenics, or other cells (i.e. cell-cell contacts).
  • physiological signals for example hormones, antibodies, peptides, antigens, cytokines, growth factors, action potentials, pharmacological agents including chemotherapeutics, radiation, carcinogenics, or other cells (i.e. cell-cell contacts).
  • the determinations are determined at different stages of the cell cycle process.
  • prostate cancer protein activity includes at least one of the following: prostate cancer activity, zinc binding, zinc transport, binding to BCR4, activation of BCR4 or activation of substrates of BCR4 by BCR4.
  • breast cancer protein activity or “breast cancer protein bioactivity”
  • ovarian cancer protein activity or “ovarian cancer protein bioactivity”
  • bladedder cancer protein activity or “bladder cancer protein bioactivity” and grammatical equivalents thereof as used herein includes at least one ofthe following: breast cancer activity, ovarian cancer activity and bladdercancer activity, respectively, as well as zinc binding, zinc transport, binding to BCR4, activation of BCR4 or activation of substrates of BCR4 by BCR4.
  • An inhibitor of BCR4 inhibits at least one of BCR4's bioactivities.
  • a method of inhibiting prostate cancer or breast cancer cell division comprises administration of a prostate cancer or breast cancer inhibitor, respectively.
  • a method of inhibiting ovarian cancer or bladder cancer cell division is provided.
  • the method comprises administration of a ovarian cancer or bladder cancer inhibitor, respectively.
  • a method of inhibiting prostate or breast tumor growth is provided.
  • the method comprises administration of a prostate cancer or breast cancer inhibitor, respectively.
  • a method of inhibiting ovarian or bladder tumor growth is provided.
  • the method comprises administration of a ovarian cancer or bladder cancer inhibitor, respectively.
  • the inhibitor is an inhibitor of BCR4.
  • methods of treating cells or individuals with prostate cancer or breast cancer comprise administration of a prostate cancer or breast cancer inhibitor, respectively.
  • methods of treating cells or individuals with ovarian cancer or bladder cancer are provided.
  • the method comprises administration of a ovarian cancer or bladder cancer inhibitor, respectively.
  • the inhibitor is an inhibitor of BCR4.
  • a prostate/breast/ovarian/bladder cancer inhibitor is an antibody as discussed above.
  • the prostate/breast/ovarian/bladder cancer inhibitor is an antisense molecule.
  • Antisense molecules as used herein include antisense or sense oligonucleotides comprising a singe-stranded nucleic acid sequence (either RNA or DNA) capable of binding to target mRNA (sense) or DNA (antisense) sequences for prostate/breast/ovarian/bladder cancer molecules.
  • a preferred antisense molecule is for BCR4 or for a ligand or activator thereof.
  • Antisense or sense oligonucleotides comprise a fragment generally at least about 14 nucleotides, preferably from about 14 to 30 nucleotides.
  • the ability to derive an antisense or a sense oligonucleotide, based upon a cDNA sequence encoding a given protein is described in, for example, Stein and Cohen (Cancer Res. 48:2659, 1988) and van der Krol et al. (BioTechnioues 6:958, 1988).
  • Antisense molecules may be introduced into a cell containing the target nucleotide sequence by formation of a conjugate with a ligand binding molecule, as described in WO 91/04753.
  • Suitable ligand binding molecules include, but are not limited to, cell surface receptors, growth factors, other cytokines, or other ligands that bind to cell surface receptors.
  • conjugation ofthe ligand binding molecule does not substantially interfere with the ability of the ligand binding molecule to bind to its corresponding molecule or receptor, or block entry of the sense or antisense oligonucleotide or its conjugated version into the cell.
  • a sense or an antisense oligonucleotide may be introduced into a cell containing the target nucleic acid sequence by formation of an oligonucleotide-lipid complex, as described in WO 90/10448. It is understood that the use of antisense molecules or knock out and knock in models may also be used in screening assays as discussed above, in addition to methods of treatment.
  • the compounds having the desired pharmacological activity may be administered in a physiologically acceptable carrier to a host, as previously described.
  • the agents may be administered in a variety of ways, orally, parenterally e.g., subcutaneously, intraperitoneally, intravascularly, etc. Depending upon the manner of introduction, the compounds may be formulated in a variety of ways.
  • the concentration of therapeutically active compound in the formulation may vary from about 0.1-100 wt.%.
  • the agents may be administered alone or in combination with other treatments, i.e., radiation.
  • compositions can be prepared in various forms, such as granules, tablets, pills, suppositories, capsules, suspensions, salves, lotions and the like.
  • Pharmaceutical grade organic or inorganic carriers and/or diluents suitable for oral and topical use can be used to make up compositions containing the therapeutically-active compounds.
  • Diluents known to the art include aqueous media, vegetable and animal oils and fats. Stabilizing agents, wetting and emulsifying agents, salts for varying the osmotic pressure or buffers for securing an adequate pH value, and skin penetration enhancers can be used as auxiliary agents.
  • the various prostate/breast/ovarian/bladder cancer sequences are important in prostate/breast/ovarian/bladder cancer. Accordingly, disorders based on mutant or variant prostate/breast/ovarian/bladder cancer genes may be determined.
  • the invention provides methods for identifying cells containing variant prostate/breast/ovarian/bladder cancer genes comprising determining all or part ofthe sequence of at least one endogeneous prostate/breast/ovarian/bladder cancer gene in a cell. As will be appreciated by those in the art, this may be done using any number of sequencing techniques.
  • the invention provides methods of identifying the cancer genotype of an individual comprising determining all or part of the sequence of at least one prostate/breast/ovarian/bladder cancer gene of the individual. This is generally done in at least one tissue of the individual, and may include the evaluation of a number of tissues or different samples ofthe same tissue. The method may include comparing the sequence ofthe sequenced gene to a known gene, i.e. a wild-type gene.
  • the sequence of all or part of the prostate/breast/ovarian/bladder cancer gene can then be compared to the sequence of a known prostate/breast/ovarian/bladder cancer gene to determine if any differences exist. This can be done using any number of known homology programs, such as Bestfit, etc.
  • the presence of a difference in the sequence between the prostate/breast/ovarian/bladder cancer gene of the patient and the known prostate/breast/ovarian/bladder cancer gene is indicative of a disease state or a propensity for a disease state, as outlined herein.
  • the prostate/breast/ovarian/bladder cancer genes are used as probes to determine the number of copies of the prostate/breast/ovarian/bladder cancer gene in the genome.
  • prostate/breast/ovarian/bladder cancer genes are used as probed to determine the chromosomal localization ofthe prostate/breast/ovarian/bladder cancer genes.
  • Information such as chromosomal localization finds use in providing a diagnosis or prognosis in particular when chromosomal abnormalities such as translocations, and the like are identified in prostate/breast/ovarian/bladder cancer gene loci.
  • methods of modulating prostate/breast/ovarian/bladder cancer in cells or organisms comprise administering to a cell an antibody that reduces or eliminates the biological activity of an endogenous prostate/breast/ovarian/bladder cancer protein.
  • the methods comprise administering to a cell or organism a recombinant nucleic acid encoding a prostate/breast/ovarian/bladder cancer protein. As will be appreciated by those in the art, this may be accomplished in any number of ways.
  • the activity of the cancer gene is increased by increasing the amount in the cell, for example by overexpressing the endogenous protein or by administering a gene encoding the sequence, using known gene-therapy techniques, for example.
  • the gene therapy techniques include the incorporation of the exogenous gene using enhanced homologous recombination (EHR), for example as described in PCT/US93/03868, hereby incorporated by reference in its entirety.
  • EHR enhanced homologous recombination
  • the activity of the endogeneous gene is decreased, for example by the administration of an inhibitor of prostate or breast cancer, such as an antisense nucleic acid.
  • the prostate/breast/ovarian/bladder cancer proteins ofthe present invention may be used to generate polyclonal and monoclonal antibodies to such proteins, which are useful as described herein.
  • the prostate/breast/ovarian/bladder cancer proteins can be coupled, using standard technology, to affinity chromatography columns. These columns may then be used to purify prostate/breast/ovarian/bladder cancer antibodies.
  • the antibodies are generated to epitopes unique to a prostate/breast/ovarian/bladder cancer protein; that is, the antibodies show little or no cross-reactivity to other proteins. These antibodies find use in a number of applications.
  • the prostate/breast/ovarian/bladder cancer antibodies may be coupled to standard affinity chromatography columns and used to purify prostate/breast/ovarian/bladder cancer proteins.
  • the antibodies may also be used as blocking polypeptides, as outlined above, since they will specifically bind to the prostate/breast/ovarian/bladder cancer protein.
  • a therapeutically effective dose of a prostate/breast/ovarian/bladder cancer or modulator thereof is administered to a patient.
  • therapeutically effective dose herein is meant a dose that produces the effects for which it is administered. The exact dose will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques. As is known in the art, adjustments for degradation, systemic versus localized delivery, and rate of new protease synthesis, as well as the age, body weight, general health, sex, diet, time of administration, drug interaction and the severity of the condition may be necessary, and will be ascertainable with routine experimentation by those skilled in the art.
  • a "patient” for the purposes of the present invention includes both humans and other animals, particularly mammals, and organisms. Thus the methods are applicable to both human therapy and veterinary applications.
  • the patient is a mammal, and in the most preferred embodiment the patient is human.
  • the administration of the prostate/breast/ovarian/bladder cancer proteins and modulators of the present invention can be done in a variety of ways as discussed above, including, but not limited to, orally, subcutaneously, intravenously, intranasally, transdermally, intraperitoneally, intramuscularly, intrapulmonary, vaginally, rectally, or intraocularly.
  • the prostate/breast/ovarian/bladder cancer proteins and modulators may be directly applied as a solution or spray.
  • compositions of the present invention comprise a prostate/breast/ovarian/bladder cancer protein in a form suitable for administration to a patient.
  • the pharmaceutical compositions are in a water soluble form, such as being present as pharmaceutically acceptable salts, which is meant to include both acid and base addition salts.
  • “Pharmaceutically acceptable acid addition salt” refers to those salts that retain the biological effectiveness of the free bases and that are not biologically or otherwise undesirable, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and the like.
  • inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like
  • organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic acid,
  • “Pharmaceutically acceptable base addition salts” include those derived from inorganic bases such as sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Particularly preferred are the ammonium, potassium, sodium, calcium, and magnesium salts.
  • Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, and ethanolamine.
  • compositions may also include one or more of the following: carrier proteins such as serum albumin; buffers; fillers such as microcrystalline cellulose, lactose, corn and other starches; binding agents; sweeteners and other flavoring agents; coloring agents; and polyethylene glycol.
  • carrier proteins such as serum albumin
  • buffers such as buffers
  • fillers such as microcrystalline cellulose, lactose, corn and other starches
  • binding agents such as microcrystalline cellulose, lactose, corn
  • prostate/breast/ovarian/bladder cancer proteins and modulators are administered as therapeutic agents, and can be formulated as outlined above.
  • prostate/breast/ovarian/bladder cancer genes (including both the full-length sequence, partial sequences, or regulatory sequences ofthe coding regions) can be administered in gene therapy applications, as is known in the art.
  • These prostate/breast/ovarian/bladder cancer genes can include antisense applications, either as gene therapy (i.e. for incorporation into the genome) or as antisense compositions, as will be appreciated by those in the art.
  • prostate/breast/ovarian/bladder cancer genes are administered as DNA vaccines, either single genes or combinations of prostate/breast/ovarian/bladder cancer genes. Naked DNA vaccines are generally known in the art. Brower, Nature Biotechnology, 16:1304-1305 (1998).
  • prostate/breast/ovarian/bladder cancer genes of the present invention are used as DNA vaccines.
  • Methods for the use of genes as DNA vaccines are well known to one of ordinary skill in the art, and include placing a prostate cancer or breast cancer gene or portion of such a gene under the control of a promoter for expression in a patient with prostate cancer or breast cancer, respectively.
  • the prostate/breast/ovarian/bladder cancer gene used for DNA vaccines can encode full-length proteins, but more preferably encodes portions of the prostate/breast/ovarian/bladder cancer proteins including peptides derived from the protein.
  • a patient is immunized with a DNA vaccine comprising a plurality of nucleotide sequences derived from a prostate/breast/ovarian/bladder cancer gene.
  • a DNA vaccine comprising a plurality of nucleotide sequences derived from a prostate/breast/ovarian/bladder cancer gene.
  • expression of the polypeptide encoded by the DNA vaccine, cytotoxic T-cells, helper T-cells and antibodies are induced which recognize and destroy or eliminate cells expressing prostate cancer or breast cancer proteins.
  • the DNA vaccines include a gene encoding an adjuvant molecule with the DNA vaccine.
  • adjuvant molecules include cytokines that increase the immunogenic response to the prostate/breast/ovarian/bladder cancer polypeptide encoded by the DNA vaccine. Additional or alternative adjuvants are known to those of ordinary skill in the art and find use in the invention.
  • prostate/breast/ovarian/bladder cancer genes find use in generating animal models of prostate cancer or breast cancer.
  • gene therapy technology wherein antisense RNA directed to the cancer gene will also diminish or repress expression of the gene.
  • An animal generated as such serves as an animal model of prostate/breast/ovarian/bladder cancer that finds use in screening bioactive drug candidates.
  • gene knockout technology for example as a result of homologous recombination with an appropriate gene targeting vector, will result in the absence of the prostate/breast/ovarian/bladder cancer protein.
  • tissue-specific expression or knockout of the prostate/breast/ovarian/bladder cancer protein may be necessary.
  • the prostate/breast/ovarian/bladder cancer protein is overexpressed in prostate cancer or breast cancer or both.
  • transgenic animals can be generated that overexpress the prostate/breast/ovarian/bladder cancer protein.
  • promoters of various strengths can be employed to express the transgene.
  • the number of copies of the integrated transgene can be determined and compared for a determination ofthe expression level ofthe transgene. Animals generated by such methods find use as animal models of prostate cancer or breast cancer and are additionally useful in screening for bioactive molecules to treat disorders related to the prostate/breast/ovarian/bladder cancer protein.
  • RNA is purified from cells or tissue
  • double-stranded QDNA is prepared from the RNA
  • the cDNA is purified
  • the cDNA is then labeled with biotin during an in vitro transcription (IVT) reaction
  • the cRNA prepared in the IVT reaction is purified, fragmented, and hybridized to an oligonucleotide array.
  • tissue homogenizer Polytron PT3100 fitted with probe 9100072, Kinematica
  • Sterilize by running the homogenizer in ethanol, and then run the homogenizer in at least 3 mL of TRIzol reagent (Life Technology/GibcoBRL).
  • tissue weight Homogenize tissue samples in 1 mL of TRIzol per 50 mg of tissue. If cells derived from experimental model systems are used as the source of RNA, use 1 mL of TRIzol per 5-10 x 106 cells. Homogenize tissue or cells thoroughly.
  • the probe After each sample homogenization run the probe in at least 3 mL fresh TRIzol, and then add this TRIzol back to the homogenized sample. Wash the probe with at least 50 mL fresh RNase-free water before proceeding to the next sample.
  • centrifuge sample in a microfuge at 12 OOOg for 10 min at 4°C (microfuge tubes) or in a Sorvall centrifuge (Sorvall Centrifuge RT7 Plus) at 4000 RPM for 60 min at 4°C (15 mL conical tubes).
  • RNA quantification and quality control Resuspend RNA pellet in 50 uL RNase-free water. Vortex. Incubate at 65°C for 10 minutes, vortex for 3 seconds to resuspend pellet, and spin briefly to collect sample in the bottom of the microcentrifuge tube. RNA quantification and quality control
  • RNA sample 1 uL of RNA sample to quantify RNA in a spectrometer.
  • the ratio of the optical density readings at 260 and 280 nm should be between 1.4 and 2.0 OD.
  • Use 250-500 ng of RNA sample to run on a 1 % agarose electrophoretic gel to check integrity of 28S, 18S and 5S RNAs. Smearing of the RNA should be minimal and not biased toward RNAs of lower molecular weight.
  • RNA purification no more than 100 ug of RNA on an individual RNeasy column (Qiagen).
  • manufacturer's instructions for RNA purification Adjust the sample to a volume of 100 uL with RNase-free water. Add 350 uL Buffer RLT and then 250 uL ethanol to the sample. Mix gently by pipetting and then apply sample to the RNeasy column. Centrifuge in a microcentrifuge for 15 seconds at 10 000 RPM.
  • PolyA ⁇ RNA can be purified from total RNA if desired using the Oligotex mRNA Purification System (Qiagen) by following the manufacturer's instructions. Before proceeding with cDNA synthesis the polyA ⁇ RNA must be ethanol precipitated and resuspended as the Oligotex procedure leaves a reagent in the polyA+RNA which inhibits downstream reactions.
  • Oligotex mRNA Purification System Qiagen
  • RNA RNA at 70°C for 2 minutes to disloge RNA that is adhering to the plastic tube. Vortex, spin briefly in microcentrifuge, and then keep RNA at room temperature until aliquot is taken.
  • T7-(dT) 24 primer (100 pmol/uL) 1 uL (2 ug/uL)
  • Total volume of second strand reaction mix per sample is 130 u L. Add mix to first strand cDNA synthesis sample.
  • T7 10XATP (75 mM) 2 uL T7 10XGTP (75 mM) 2 uL T7 10XCTP (75 mM) 1.5 uL T7 10XUTP (75 mM) 1.5 uL Bio-11-UTP (10 mM) 3.75 uL (Boehringer Mannheim or Enzo Diagnostics)
  • RNeasy columns Qiagen
  • RNA purification using RNeasy Kit RNA purification using RNeasy Kit
  • BioC control cRNA (Affymetrix) 5 pM
  • BioD control cRNA (Affymetrix) 25 pM
  • CRE control cRNA 100 pM Herring sperm DNA (10 mg/mL) 3 uL
  • Bovine serum albumin (50 mg/mL) 3 uL
  • RNAs from two different sources on the same oligonucleotide array for example, RNA prepared from tumor tissue versus RNA prepared from normal tissue.
  • the starting material for this method is IVT product prepared as described in Example 1 , above.
  • the cRNA is reverse transcribed in the presence of either Cy3 (sample 1 ) or Cy5 (sample 2) conjugated dUTP.
  • the RNA is degraded and the samples are purified to recover the Cy3 and Cy5 dUTP.
  • the differentially labelled samples are combined and the cDNA is further purified to remove fragments less than 100 bp in length.
  • the sample is then fragmented and hybridized to oligonucleotide arrays. Labeling of cRNA
  • RNase-free water 15 u L Reverse transcription is performed on the IVT product by adding the following reagents from the Superscript Choice System for cDNA Synthesis kit (GibcoBRL) to the IVT-random hexamer mixture.
  • cDNA is purified using the Qiaquick PCR Purification Kit (Qiagen), following the manufacturer's directions.
  • Buffer PB 5 volumes Apply sample to Qiaquick column. Spin at 10 OOOg in a microcentrifuge for 10 minutes Discard flow through and add 750 uL Buffer PB to column. Centrifuge at 10 OOOg for 1 minute. Discard flow through. Spin at maximum speed for 1 minute to dry column.
  • Second wash Wash slides for 5 minutes at room temperature in Buffer 2
  • oligonucleotide microarrays were interrogated with cRNAs derived from multiple tissues. More specifically, biotinylated-cRNAs were generated by in vitro transcription reactions (IVTs) from primary prostate, breast, ovarian and bladder tumors and samples from adult tissues and organs including: adrenal gland, aorta, aortic valve, artery, bladder, bone marrow, brain, breast, colonic epithelium, cervix, colon, diaphragm, esophagus, gallbladder, heart, kidney, liver, lung, lymph node, muscle, omentum, pancreas, prostate, rectum, salivary gland, skin, small intestine (incl.
  • IVTTs in vitro transcription reactions
  • Hybridization of cDNA to the oligonucleotide microarrays was measured by average fluorescence intensity (Al), which is directly proportional to the expression level ofthe gene. To specifically calculate the overexpression of any gene in a specific cancer, the following calculations were made:
  • the lowest value was set at 10 units for the purpose of calculating cancer.normal tissue expression ratios.
  • the expression ratio of each gene was calculated to be the 90 th percentile of breast cancer expression divided by the 85 th percentile of normal adult tissue expression.
  • the 90 th percentile is mathematically equal to the median ofthe top 20% of samples; likewise, the 85 th percentile corresponds to the median of the top 30%.
  • the genes were sorted by descending ratio.
  • BCR4 human BCR4 in several different cancer tissue samples and samples of various normal tissues was determined as described above. A nucleic acid having the sequence as shown in accession number AA242758 was used as a probe on the biochip. The relative amounts of BCR4 expressed in the various samples are shown in Figures 6-8B, 10 and 11. BCR4 was overexpressed or upregulated in cancer tissue, breast cancer tissue, ovarian cancer tissue and bladder cancer tissue as compared with normal tissues. Human BCR4 is found on chromosome 18 at cytoband 18q12.
  • BCR4 The cDNA identified for BCR4 encodes a 3461 residue mRNA (Fig. 1 ). Putative start (ATG) and stop (TAG) codons are underlined in Figure 1 , defining an open reading frame (ORF) which encodes a 755 amino acid protein (Fig. 2). The BCR4 protein appears to have an N-terminal signal sequence (bold). Putative transmembrane domains are underlined in Figure 2.
  • Animal models of prostate cancer or breast cancer are provided by grafting cells of a prostate cancer cell line or breast cancer cell line, respectively, within an experimental animal. Model tumors may then develop. The animals are administered antibodies to BCR4, or a fragment thereof, and development the tumors is monitored as compared with untreated model tumor- bearing animals. Alternatively, antibodies to BCR4, or a fragment thereof, which comprise a therapeutic moiety are administered and tumor development is monitored as above. Differential tumor size in antibody treated animals as compared with untreated animals is indicative of the efficacy of the antibodies for treatment of prostate cancer or breast cancer.

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Abstract

L'invention concerne des procédé utiles pour diagnostiquer et pronostiquer le cancer de la prostate, du sein, des ovaires et/ou de la vésicule. L'invention concerne également des procédés pouvant servir à dépister des agents candidats réactifs pour la capacité à moduler le cancer de la prostate, du sein, des ovaires et/ou de la vésicule. Enfin, l'invention concerne des procédés et des cibles moléculaires (gènes et leurs produits) pour l'intervention thérapeutique dans des cas de cancer de la prostate, du sein et d'autres cancers.
PCT/US2001/025997 2000-08-18 2001-08-20 Procedes de diagnostic du cancer, compositions et procedes de depistage de modulateurs du cancer WO2002016939A2 (fr)

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003081239A2 (fr) * 2002-03-25 2003-10-02 Theryte Ltd Traitement du cancer
US7285382B2 (en) * 2000-01-25 2007-10-23 Genentech, Inc. Compositions and methods for treatment of cancer
US8455200B2 (en) 2009-10-15 2013-06-04 Traxxsson, Llc Measurement of PKA for cancer detection
EP3211421A1 (fr) 2010-09-09 2017-08-30 Traxxsson, LLC Combinaison de procédés de diagnostic du cancer chez un patient
EP3394084B1 (fr) * 2015-12-22 2021-04-07 Immatics Biotechnologies GmbH Peptides et combinaison de peptides à utiliser en immunothérapie contre le cancer du sein et d'autres cancers
US11065314B2 (en) 2015-12-22 2021-07-20 Immatics Biotechnologies Gmbh Peptides and combination of peptides for use in immunotherapy against breast cancer and other cancers

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
No further relevant documents disclosed *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7285382B2 (en) * 2000-01-25 2007-10-23 Genentech, Inc. Compositions and methods for treatment of cancer
US7691566B2 (en) 2000-01-25 2010-04-06 Genentech, Inc. Compositions and methods for treatment of cancer
WO2003081239A2 (fr) * 2002-03-25 2003-10-02 Theryte Ltd Traitement du cancer
WO2003081239A3 (fr) * 2002-03-25 2004-09-16 Theryte Ltd Traitement du cancer
US8455200B2 (en) 2009-10-15 2013-06-04 Traxxsson, Llc Measurement of PKA for cancer detection
EP3211421A1 (fr) 2010-09-09 2017-08-30 Traxxsson, LLC Combinaison de procédés de diagnostic du cancer chez un patient
EP3394084B1 (fr) * 2015-12-22 2021-04-07 Immatics Biotechnologies GmbH Peptides et combinaison de peptides à utiliser en immunothérapie contre le cancer du sein et d'autres cancers
US11065314B2 (en) 2015-12-22 2021-07-20 Immatics Biotechnologies Gmbh Peptides and combination of peptides for use in immunotherapy against breast cancer and other cancers

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