WO2000055629A2 - Nouvelles techniques permettant de traiter et de diagnostiquer le cancer du sein, compositions et techniques de criblage pour modulateurs de cancer du sein - Google Patents

Nouvelles techniques permettant de traiter et de diagnostiquer le cancer du sein, compositions et techniques de criblage pour modulateurs de cancer du sein Download PDF

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Publication number
WO2000055629A2
WO2000055629A2 PCT/US2000/006952 US0006952W WO0055629A2 WO 2000055629 A2 WO2000055629 A2 WO 2000055629A2 US 0006952 W US0006952 W US 0006952W WO 0055629 A2 WO0055629 A2 WO 0055629A2
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WIPO (PCT)
Prior art keywords
breast cancer
protein
bch1
expression
antibody
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PCT/US2000/006952
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English (en)
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WO2000055629A3 (fr
Inventor
David Mack
Kurt C. Gish
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Eos Biotechnology, Inc.
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Publication date
Application filed by Eos Biotechnology, Inc. filed Critical Eos Biotechnology, Inc.
Priority to JP2000605210A priority Critical patent/JP2004537710A/ja
Priority to CA002368152A priority patent/CA2368152A1/fr
Priority to MXPA01009367A priority patent/MXPA01009367A/es
Priority to EP00916419A priority patent/EP1159619A2/fr
Priority to AU37527/00A priority patent/AU3752700A/en
Priority to US09/642,034 priority patent/US6762020B1/en
Publication of WO2000055629A2 publication Critical patent/WO2000055629A2/fr
Priority to US09/747,371 priority patent/US6750013B2/en
Publication of WO2000055629A3 publication Critical patent/WO2000055629A3/fr
Priority to US10/769,612 priority patent/US20040141983A1/en
Priority to US12/796,244 priority patent/US8313745B2/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4748—Tumour specific antigens; Tumour rejection antigen precursors [TRAP], e.g. MAGE
    • 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
    • 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

Definitions

  • the invention relates to the identification of expression profiles and the nucleic acids involved in breast cancer, and to the use of such expression profiles and nucleic acids in diagnosis, prognosis and treatment of breast cancer
  • the invention further relates to methods for identifying and using candidate agents and/or targets which modulate breast cancer
  • the present invention provides methods for screening for compositions which modulate breast cancer Also provided herein are methods of inhibiting proliferation of a cell, preferably a breast cancer cell Methods of treatment of cancer, as well as compositions, are also provided herein
  • 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 are genes which are differentially expressed in cancer cells, preferably breast cancer cells, compared to other cells
  • Preferred embodiments of expression profile genes used in the methods herein include but are not limited to the group consisting of BCR3, BCQ8, BCQ5, BCH1 , BCN1 , BCN2, BCN5, BC02, BCX2, BCJ7, BCY3, BCX3, BCA2 and BCR2, fragments of the proteins of this group are also preferred
  • a nucleic acid is selected from Figures 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , or 12
  • Preferred nucleic acids are in Figure 10, more preferably Figure 1 1 , and most preferably in Figure 12
  • 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
  • the method comprising combining the BCMP and a candidate bioactive agent, and determining the binding of the candidate agent to the BCMP
  • the BCMP is a protein or fragment thereof selected from the group consisting of BCR3, BCQ8, BCQ5, BCH1 , BCN1 , BCN2, BCN5, BC02, BCX2, BCJ7, BCY3, BCX3, BCA2 and BCR2
  • the protein is encoded by a nucleic acid selected from Figures 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , or 12 Preferred nucleic acids are in Figure 10, more preferably Figure 11 , and most preferably in Figure 12
  • the method comprises combining the BCMP and a candidate bioactive agent, and determining the effect of the candidate agent on the bioactivity of the BCMP
  • the BCMP is a protein or fragment thereof selected from the group consisting of BCR3, BCQ8, BCQ5, BCH1 , BCN1 , BCN2, BCN5, BC02, BCX2, BCJ7, BCY3, BCX3, BCA2 and BCR2
  • the protein is encoded by a nucleic acid selected from Figures 1 , 2, 3, 4, 5, 6, 7,
  • Preferred nucleic acids are in Figure 10, more preferably Figure 11 , and most preferably in Figure 12
  • Also provided is a method of evaluating the effect of a candidate breast cancer drug comprising administering the drug to a transgenic animal expressing or over-expressing the BCMP, or an animal lacking the BCMP, for example as a result of a gene knockout
  • a method of evaluating the effect of a candidate 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 biochip comprising a nucleic acid segment which encodes a breast cancer protein, preferably selected from the group consisting of BCR3, BCQ8, BCQ5, BCH1 , BCN1 , BCN2, BCN5, BC02, BCX2, BCJ7, BCY3, BCX3, BCA2 and BCR2, or a fragment thereof, wherein the biochip comprises fewer than 1000 nucleic acid probes Preferably at least two nucleic acid segments are included in another embodiment, the nucleic acid selected from Figures 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , or 12 Preferred nucleic acids are in Figure 10 more preferably Figure 11 , and most preferably in Figure 12
  • a method of diagnosing a disorder associated with breast cancer comprises determining the expression of a gene which encodes a breast cancer protein preferably selected from the group consisting of BCR3, BCQ8, BCQ5, BCH1 , BCN1 , BCN2, BCN5, BC02, BCX2, BCJ7, BCY3, BCX3, BCA2 and BCR2 or a fragment thereof in a first tissue type of a first individual, and comparing the distribution to the expression of the gene from a second normal tissue type from the first individual or a second unaffected individual
  • the protein is encoded by a nucleic acid selected from Figures 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , or 12 Preferred nucleic acids are in Figure 10, more preferably Figure 11 , and most preferably in Figure 12
  • a difference in the expression indicates that the first individual has a disorder associated with breast cancer
  • the present invention provides an antibody which specifically binds to a breast cancer protein, preferably selected from the group consisting of BCR3, BCQ8, BCQ5, BCH1 , BCN1 , BCN2, BCN5, BC02, BCX2, BCJ7, BCY3, BCX3, BCA2 and BCR2, or a fragment thereof
  • the protein is encoded by a nucleic acid selected from Figures 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , or 12 Preferred nucleic acids are in Figure 10, more preferably Figure 11 , and most preferably in Figure 12
  • the fragment of BCH1 is selected from BCH1p1 and BCH1p2
  • 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 a breast cancer protein (BCMP) or a fragment thereof and an antibody which binds to said BCMP or fragment thereof comprises combining a BCMP or fragment thereof, a candidate bioactive agent and an antibody which binds to said BCMP or fragment thereof
  • the method further includes determining the binding of said BCMP 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 breast cancer
  • a method for inhibiting breast cancer comprises administering to a cell a composition comprising an antibody to a breast modulating protein, preferably selected from the group consisting of BCR3, BCQ8, BCQ5, BCH1 , BCN1 , BCN2, BCN5, BC02, BCX2, BCJ7, BCY3, BCX3, BCA2 and BCR2, or a fragment thereof
  • the protein is encoded by a nucleic acid selected from Figures 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , or 12
  • Preferred nucleic acids are in Figure 10, more preferably Figure 11 , and most preferably in Figure 12
  • the method can be performed in vitro or in vivo, preferably in vivo to an individual
  • the method of inhibiting breast cancer is provided to an individual with cancer
  • methods of inhibiting breast cancer can be performed by administering an inhibitor of breast cancer protein activity, including an antisense molecules, and preferably small molecules
  • a method provided herein comprises administering to an individual a composition comprising a breast modulating protein, preferably selected from the group consisting of BCR3, BCQ8, BCQ5, BCH1 , BCN1 , BCN2, BCN5, BC02, BCX2, BCJ7, BCY3, BCX3, BCA2 and BCR2, or a fragment thereof
  • the protein is encoded by a nucleic acid selected from Figures 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , or 12 Preferred nucleic acids are in Figure 10, more preferably Figure 11 , and most preferably in Figure 12
  • said composition comprises a nucleic acid comprising a sequence encoding a breast modulating protein, preferably selected from the group consisting of BCR3, BCQ8, BCQ5, BCH1 , BCN1 , BCN2, BCN5, BC02, BCX2, BCJ7, BCY3, BCX3, BCA2 and BCR2, or a fragment
  • compositions capable of eliciting an immune response in an individual
  • a composition provided herein comprises a breast modulating protein, preferably selected from the group consisting of BCR3, BCQ8, BCQ5, BCH1 , BCN1 , BCN2, BCN5, BC02, BCX2, BCJ7, BCY3, BCX3, BCA2 and BCR2, or a fragment thereof, and a pharmaceutically acceptable carrier
  • the protein is encoded by a nucleic acid selected from Figures 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , or 12 Preferred nucleic acids are in Figure 10, more preferably Figure 11 , and most preferably in Figure 12
  • said composition comprises a nucleic acid comprising a sequence encoding a breast modulating protein, preferably selected from the group consisting of BCR3, BCQ8, BCQ5, BCH1 , BCN1 , BCN2, BCN5, BC02, BCX2, BCJ7, BCY3,
  • nucleic acid is selected from Figures 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , or 12 Preferred nucleic acids are in Figure 10, more preferably Figure 11 , and most preferably in Figure 12
  • a method of neutralizing the effect of a breast cancer protein preferably selected from the group consisting of BCR3, BCQ8, BCQ5, BCH1 , BCN1 , BCN2, BCN5, BC02, BCX2, BCJ7, BCY3, BCX3,
  • the method comprises contacting an agent specific for said protein with said protein in an amount sufficient to effect neutralization
  • the protein is encoded by a nucleic acid selected from Figures
  • nucleic acids are in Figure 10, more preferably Figure
  • a method of treating an individual for breast cancer comprises administering to said individual an inhibitor of BCH1
  • the individual is non-responsive to an anti-estrogen and is positive for estrogen receptor
  • the method further comprises administering an anti-estrogen to said individual
  • Also provided herein is method for determining the prognosis of an individual with breast cancer comprising determining the level of BCH1 in a sample, wherein a high level of BCH1 indicates a poor prognosis Moreover, in yet another aspect of the invention, a method is provided for determining whether an individual with breast cancer will be non-responsive to anti-estrogen therapies comprising determining the level of BCH1 wherein a high level of BCH1 indicates that an individual will be non- responsive Preferably, the methods herein are used to identify persons who are tamoxifen resistant
  • Figure 1 provides the Accession numbers for genes, including expression sequence tags, (incorporated in their entirety here and throughout the application where Accession numbers are provided), downregulated in tumor tissue compared to normal breast tissue
  • Figure 2 provides the Accession numbers for genes, including expression sequence tags, downregulated in tumor tissue compared to normal breast tissue
  • Figure 3 provides the Accession numbers for genes, including expression sequence tags, downregulated in tumor tissue compared to normal breast tissue
  • Figure 4 provides the Accession numbers for genes, including expression sequence tags, upregulated in tumor tissue compared to normal breast tissue
  • Figure 5 provides the Accession numbers for genes, including expression sequence tags, upregulated in tumor tissue compared to normal breast tissue
  • Figure 6 provides the Accession numbers for genes, including expression sequence tags, upregulated in tumor tissue compared to normal breast tissue
  • Figure 7 provides the Accession numbers for genes, including expression sequence tags, upregulated in tumor tissue compared to normal breast tissue Open reading frames in the sequences have been characterized as having a signal sequence (SS), a transmembrane domain (TM) or other
  • Figure 8 provides the Accession numbers for genes, including expression sequence tags, upregulated in tumor tissue compared to normal breast tissue Specifically, one column shows the ratio of expression of the indicated gene in breast tumor tissue compared to other body tissues, and another column shows the ratio of expression of the indicated gene in breast tumor tissue compared to normal breast tissue
  • Figure 9 depicts the Accession numbers for genes, including expression sequence tags, upregulated in tumor tissue compared to normal breast tissue
  • Figure 10 depicts a preferred group of 1007 Accession numbers for genes, including expression sequence tags, upregulated in tumor tissue compared to normal breast tissue
  • Figure 11 depicts a preferred group of 123 Accession numbers for genes, including expression sequence tags, upregulated in tumor tissue compared to normal breast tissue
  • Figure 12 depicts a preferred group of 10 Accession numbers for genes, including expression sequence tags, upregulated in tumor tissue compared to normal breast tissue
  • Figure 13 shows an embodiment of a nucleic acid (mRNA) which includes a sequence which encodes a breast cancer protein provided herein, BCR3 The start and stop codons are underlined
  • Figure 14 shows an embodiment of an open reading frame of a nucleic acid encoding BCR3, wherein the start and stop codons are underlined
  • Figure 15 shows an embodiment of an ammo acid sequence of BCR3
  • the signal peptide is underlined and the transmembrane domain is shaded
  • a soluble form of BCR3 is provided wherein the signal peptide is deleted or preferably naturally cleaved, and the transmembrane domain is deleted, inactivated, or BCR3 is truncated to exclude the transmembrane domain
  • Figure 16 shows the ammo acid sequence of BCR3p1 and BCR3p2
  • Figure 17 shows the relative amount of expression of BCR3 in various samples of breast cancer tissue (dark bars) and many normal tissue types (light bars)
  • Figure 18 shows an embodiment of a nucleic acid (mRNA) which includes a sequence which encodes a breast cancer protein provided herein, BCQ8 The start and stop codon are underlined
  • Figure 19 shows an embodiment of an open reading frame of a nucleic acid encoding BCQ8, wherein the start and stop codons are underlined
  • Figure 20 shows an embodiment of an ammo acid sequence of BCQ8
  • the signal peptide is underlined twice and the transmembrane domain is underlined
  • a soluble form of BCQ8 is provided wherein the signal peptide is deleted, and the transmembrane domain is deleted, inactivated, or BCQ8 is truncated on either end as desired, to exclude the transmembrane domain
  • Figure 21 shows the ammo acid sequence of BCQ8p1 and BCQ8p2
  • Figure 22 shows the relative amount of expression of BCQ8 in various samples of breast cancer tissue (dark bars) and many normal tissue types (light bars)
  • Figure 23 shows an embodiment of a nucleic acid (mRNA) which includes a sequence which encodes a differentially expressed protein provided herein, human BCQ5
  • Figure 24 shows an embodiment of an open reading frame of a nucleic acid encoding human BCQ5
  • Figure 25 shows embodiments of ammo acid sequences of BCQ5 by providing an alignment wherein human is above mouse which is above rat
  • Figure 26 shows the ammo acid sequence of BCQ5p1 , BCQ5p2 and BCQ5p3
  • Figure 27 shows the relative amount of expression of BCQ5 in various samples of breast cancer tissue, colorectal cancer tissue, angiogenesis models wherein tubes are formed from endothe al cells, and normal tissue types
  • Figure 28 shows an embodiment of a nucleic acid (mRNA) which includes a sequence which encodes a differentially expressed protein provided herein, mouse BCQ5
  • Figure 29 shows an embodiment of an open reading frame of a nucleic acid encoding mouse BCQ5
  • Figure 30 shows an embodiment of a nucleic acid (partial mRNA) which includes a sequence which encodes a differentially expressed protein provided herein, rat BCQ5
  • Figure 31 shows an embodiment of a partial open reading frame of a nucleic acid encoding rat BCQ5
  • Figure 32 shows an embodiment of a nucleic acid (mRNA) which includes a sequence which encodes a breast cancer protein provided herein, BCH1 Start and stop codons are underlined
  • mRNA nucleic acid
  • Figure 33 shows an embodiment of an open reading frame of a nucleic acid encoding BCH1 , wherein start and stop codons are underlined
  • Figure 34 shows an embodiment of an ammo acid sequence of BCH1
  • isolated BCH1 excludes the signal sequence, ammo acids 1-19
  • Figure 35 shows the ammo acid sequence of BCH1 p1 and BCH1 p2 (below)
  • Figure 36 shows the relative amount of expression of BCH1 in various samples of breast cancer tissue
  • Figure 37 shows a graph correlating expression of estrogen receptor (ER) (vertical bar) and BCH1 (horizontal bar) with localization to the nucleus (diamonds) or the cytoplasm (squares)
  • ER estrogen receptor
  • BCH1 horizontal bar
  • Figures 38A and 38B show breast carcinoma tissue specimens having high levels of BCH1 expression as indicated by ant ⁇ -BCH1 antibodies
  • Figures 39A and 39B show samples taken from the tissues shown in Figures 38A and 38B respectively, wherein anti-estrogen receptor antibodies indicate that estrogen receptor is localized exclusively to the cytoplasm
  • Figures 40A and 40B show breast carcinoma tissue specimens having low levels of BCH1 expression as indicated by ant ⁇ -BCH1 antibodies
  • Figures 41 A and 41 B show samples taken from the tissues shown in Figures 40A and 40B respectively, wherein anti-estrogen receptor antibodies indicate that estrogen receptor in low BCH1 expression tissue does not correlate with estrogen receptor localization
  • Figure 42 shows an embodiment of a nucleic acid (mRNA) which includes a sequence which encodes a breast cancer protein provided herein, BCN1 Start and stop codons are shaded, and Accession number AA419622 sequence is underlined
  • mRNA nucleic acid
  • Figure 43 shows an embodiment of an ammo acid sequence of BCN1 A putative transmembrane domain is predicted to be at least at approximately positions 201-217 and 67-83
  • the protein may be a type Ilia membrane protein and may have additional transmembrane domains
  • Figure 44 shows the ammo acid sequence of BCN1 p1 and BCN1 p2
  • Figure 45 shows the relative amount of expression of BCN1 in various samples of breast cancer tissue
  • Figures 46A-46D show an embodiment of a nucleic acid (mRNA) which includes a sequence which encodes a breast cancer protein provided herein, BCN2 Start and stop codons are shaded, and Accession number AA428090 sequence is underlined
  • Figure 47 shows an embodiment of an ammo acid sequence of BCN2
  • Figure 48 shows the relative amount of expression of BCN2 in various samples of breast cancer tissue (dark bars), colon cancer tissue (light bars) and many normal tissue types (medium light dotted bars)
  • Figures 49A-49B show an embodiment of a nucleic acid (mRNA) which includes a sequence which encodes a breast cancer protein provided herein, BCN5 Start and stop codons are shaded, and
  • Figure 50 shows an embodiment of an ammo acid sequence of BCN5 A putative signal sequence is shaded and preferred sequence is underlined
  • Figure 51 shows the relative amount of expression of BCN5 in various samples of breast cancer tissue (dark bars), colon cancer tissue (light bars) and many normal tissue types (medium light dotted bars)
  • Figure 52 shows an embodiment of a nucleic acid (mRNA) which includes a sequence which encodes a breast cancer protein provided herein, BC02 Start and stop codons are underlined
  • mRNA nucleic acid
  • Figure 53 shows an embodiment of an open reading frame of a nucleic acid encoding BC02
  • Figure 54 shows an embodiment of an am o acid sequence of BC02
  • Figure 55 shows an alignment of ammo acids for human BC02 above mouse BC02
  • Figure 56 shows the relative amount of expression of BC02 in various samples of breast cancer tissue (dark bars) and many normal tissue types (light bars)
  • Figure 57 shows an embodiment of a nucleic acid (mRNA) which includes a sequence which encodes a breast cancer protein provided herein, BCX2 Start and stop codons are underlined
  • mRNA nucleic acid
  • Figure 58 shows an embodiment of an open reading frame of a nucleic acid encoding BCX2
  • Figure 59 shows an embodiment of an ammo acid sequence of BCX2
  • Figure 60 shows the relative amount of expression of BCX2 in va ⁇ ous samples of breast cancer tissue (dark bars) and many normal tissue types (light bars)
  • Figure 61 shows the relative amount of expression of BCX2 in various samples of colon cancer tissue (light bars), non-cancer cell lines (dark bars) and many cell lines (medium light bars)
  • Figure 62 shows an embodiment of a nucleic acid (mRNA) which includes a sequence which encodes a breast cancer protein provided herein, BCX3 Start and stop codons are underlined
  • mRNA nucleic acid
  • Figure 63 shows an embodiment of an open reading frame of a nucleic acid encoding BCX3
  • Figure 64 shows an embodiment of an ammo acid sequence of BCX3
  • Figure 65 shows the relative amount of expression of BCX3 in various samples of breast cancer tissue (dark bars) and many normal tissue types (light bars)
  • Figure 66 shows an embodiment of a nucleic acid (mRNA) which includes a sequence which encodes a breast cancer protein provided herein, BCA2 Start and stop codons are shaded, and Accession number D12485 sequence is underlined
  • mRNA nucleic acid
  • Figure 67 shows an embodiment of an ammo acid sequence of BCA2
  • Figure 68 shows an embodiment of a nucleic acid (mRNA) which includes a sequence which encodes a breast cancer protein provided herein, BCR2 Start and stop codons are shaded, and Accession number AA609773 sequence is shown underlined
  • mRNA nucleic acid
  • Figure 69 shows an embodiment of an ammo acid sequence of BCR2
  • Figure 70 shows the relative amount of expression of BCR2 in various samples of breast cancer tissue
  • Figure 71 shows the relative amount of expression of BCR2 in various samples of normal tissue types (light bars)
  • Figure 72 shows an embodiment of a nucleic acid which includes a sequence which encodes a breast cancer protein provided herein, BCJ7
  • Figure 73 shows an embodiment of a nucleic acid which includes a sequence which encodes a breast cancer protein provided herein, BCY3
  • the present invention provides novel methods for diagnosis and prognosis evaluation for breast cancer, as well as methods for screening for compositions which modulate breast cancer
  • 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 That is, normal tissue may be distinguished from breast cancer tissue, and within breast 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
  • breast cancer sequences include those that are up-regulated (i e expressed at a higher level) in breast cancer, as well as those that are down-regulated (i e expressed at a lower level) in breast cancer
  • the breast cancer sequences are from humans, however, as will be appreciated by those in the art, breast cancer sequences from other organisms may be useful in animal models of disease and drug evaluation, thus, other 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)
  • rodents rats, mice, hamsters, guinea pigs, etc
  • primates farm animals
  • breast cancer sequences can include both nucleic acid and am o acid sequences
  • the 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
  • 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 breast cancer protein from one organism in a different organism or host cell Alternatively, the protein may be made at a significantly higher concentration than is normally seen, through the use of an inducible promoter or high
  • the breast cancer sequences are nucleic acids
  • 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 breast cancer sequences can be generated In the broadest sense, then, by "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), Sp ⁇ nzl et al , Eur J
  • 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
  • PNA backbones are substantially non-ionic under neutral conditions, in contrast to the highly charged phosphodiester backbone of naturally occurring nucleic acids.
  • Tm melting temperature
  • RNA typically exhibit a 2-4°C drop in Tm for an internal mismatch
  • 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 As will be appreciated by those in the art, the depiction of a single strand ("Watson") 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 deoxy ⁇ bo- and ⁇ bo-nucleotides, and any combination of bases, including uracil, adenine, thymine, cytosme, guanine, inosine, xanth e hypoxanthine, isocytosme, isoguanme, etc
  • the term "nucleoside” includes nucleotides and nucleoside and nucleotide analogs, and modified nucleosides such as ammo modified nucle
  • the breast 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 Affymet ⁇ x 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, brain, liver, breast, kidney, muscle, prostate, small intestine, large intestine, spleen, bone, and placenta
  • those genes identified during the breast 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
  • breast cancer sequences are those that are up-regulated in breast cancer, that is, the expression of these genes is higher in breast carcinoma as compared to normal breast tissue
  • Up-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
  • GenBank GenBank sequence database
  • 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 were found to be expressed in a limited relative amount or not at all in heart, brain, lung, liver, kidney, prostate, small intestine, testes, fibroblasts and spleen
  • breast cancer sequences are those that are down-regulated in breast cancer, that is, the expression of these genes is lower in 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
  • Breast cancer proteins of the present invention may be classified as secreted proteins, transmembrane proteins or mtracellular proteins
  • the breast cancer protein is an mtracellular protein
  • 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 deregulated cellular processes
  • many intracellular proteins have enzymatic activity such as protein kmase 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
  • the breast 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 Frequently the intracellular domain of transmembrane proteins serves both roles
  • certain receptor tyrosine kmases have both protein kmase activity and SH2 domains
  • autophosphorylation of tyrosmes 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 kmases, certain cytokine receptors, receptor guanylyl cyclases and receptor se ⁇ ne/threonine protein kmases contain a single transmembrane domain
  • transmembrane domains include approximately 20 consecutive hydrophobic ammo acids that may be followed by charged ammo acids Therefore, upon analysis of the ammo acid sequence of a particular protein, the localization and number of transmembrane domains within the protein may be predicted
  • transmembrane proteins are diverse, however, conserved motifs are found repeatedly among various extracellular domains conserveed structure and/or functions have been ascribed to different extracellular motifs
  • Immunoglobulin-like domains are highly conserved Mucm-like domains may be involved in cell adhesion and leucine- ⁇ ch repeats participate in protein-protein interactions It is understood that the transmembrane domains may be removed to create soluble proteins herein
  • 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 cytokmes, 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 glycosylphosphatidyl ositol (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
  • GPI glycosylphosphatidyl ositol
  • the breast 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 autoc ⁇ ne manner (acting on the cell that secreted the factor), a paracnne 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
  • Breast 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
  • a breast cancer sequence is initially identified by substantial nucleic acid and/or ammo acid sequence homology to the breast cancer sequences outlined herein Such homology can be based upon the overall nucleic acid or ammo acid sequence, and is generally determined as outlined below, using either homology programs or hybridization conditions
  • a nucleic acid is a "breast cancer nucleic acid” if the overall homology of the nucleic acid sequence to the nucleic acid sequences encoding the ammo 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 Biool 48 443 (1970), by the search for similarity method of Pearson & Lipman, PNAS USA 85 2444 (1988), by computerized implementations of these algorithms (GAP,
  • sequences which are used to determine sequence identity or similarity are selected from the sequences set forth in the figures In one embodiment the sequences utilized herein are those set forth in the figures In another embodiment, the sequences are naturally occurring allelic variants of the sequences set forth in the figures In another embodiment, the sequences are sequence variants as further described herein
  • PILEUP 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 35 351-360 (1987), the method is similar to that described by Higgms & Sharp CABIOS 5 151-153 (1989)
  • 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 % ammo acid sequence identity value is determined by the number of matching identical residues divided by the total number of residue
  • 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 the se
  • 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
  • sequences which contain either more or fewer nucleosides than those of the figures it is understood that 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 proteins identified in the figures, or their complements are considered a breast cancer sequence
  • High stringency conditions are known in the art, see for example Maniatis et al , Molecular
  • 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 Ausubel, supra, and Tijssen, supra
  • breast 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 breast 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 breast cancer nucleic acid Once the breast cancer nucleic acid is identified, it can be cloned and, if necessary, its constituent parts recombined to form the entire breast cancer nucleic acid Once isolated from its natural source, e g , contained within a plasmid or other vector or excised therefrom as a linear nucleic acid segment, the recombinant breast cancer nucleic acid can be further-used as a probe to identify and isolate other breast cancer nucleic acids, for example additional coding regions It can also be used as a
  • nucleic acid probes to the breast 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 breast cancer nucleic acids that include coding regions of breast cancer proteins can be put into expression vectors for the expression of breast cancer proteins, again either for screening purposes or for administration to a patient
  • nucleic acid probes to breast cancer nucleic acids are made
  • the nucleic acid probes attached to the biochip are designed to be substantially complementary to the breast 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
  • 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
  • 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, streptavidm to the support and the non- covalent binding of the biotinylated probe to the streptavidm
  • 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
  • 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
  • substrates 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 ), polysaccha ⁇ des, 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 copendmg application entitled A Reusable Low Fluorescent Plastic Biochip, U S
  • 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, ammo groups, carboxy groups, oxo groups and thiol groups, with ammo groups being particularly preferred
  • the probes can be attached using functional groups on the probes
  • nucleic acids containing ammo groups can be attached to surfaces comprising ammo 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 streptavidm, resulting in attachment
  • the oligonucleotides may be synthesized on the surface, as is known in the art
  • photoactivation techniques utilizing photopolyme ⁇ zation 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
  • breast cancer nucleic acids encoding breast cancer proteins are used to make a variety of expression vectors to express breast 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 breast 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 ⁇ bosome 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 preprotem 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
  • a ⁇ bosome 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
  • enhancers do not have to be contiguous Linking is accomplished by gation at convenient restriction sites If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice
  • the transcriptional and translational regulatory nucleic acid will generally be appropriate to the
  • 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
  • the breast cancer proteins of the present invention are produced by culturing a host cell transformed with an expression vector containing nucleic acid encoding a breast cancer protein, under the appropriate conditions to induce or cause expression of the breast cancer protein
  • the conditions appropriate for breast 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
  • the use of constitutive promoters in the expression vector will require optimizing the growth and proliferation of the host cell, 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, archaebacte ⁇ a, fungi, and insect and animal cells, including mammalian cells Of particular interest are Drosophila melangaster cells, Saccharomyces cerevisiae and other yeasts, E coli, Bacill
  • the breast 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
  • breast cancer proteins are expressed in bacterial systems Bacterial expression systems are well known in the art Promoters from bacte ⁇ ophage may also be used and are known in the art In addition, synthetic promoters and hybrid promoters are also useful, for example, the tac promoter is a hybrid of the trp and lac promoter sequences Furthermore, 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 ⁇ bosome binding site is desirable
  • the expression vector may also include a signal peptide sequence that provides for secretion of the breast cancer protein in bacteria The protein is either secreted into the growth media (gram-positive bacteria) or into the pe ⁇ plasmic 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
  • breast 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
  • breast 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 guillerimondn and P pastons, Schizosaccharomyces pombe, and Yarrowia lipolytica
  • the breast cancer protein may also be made as a fusion protein, using techniques well known in the art Thus, for example, for the creation of monoclonal antibodies, if the desired epitope is small, the breast cancer protein may be fused to a carrier protein to form an immunogen Alternatively, the breast cancer protein may be made as a fusion protein to increase expression, or for other reasons For example, when the breast cancer protein is a breast cancer peptide, the nucleic acid encoding the peptide may be linked to other nucleic acid for expression purposes
  • the breast 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 breast 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, 1 C, 32 P, 35 S, or 125 l, a fluorescent or chemiluminescent compound, such as fluorescein isothiocyanate, rhodamme, or luciferin, or an enzyme, such as alkaline phosphatase, beta- galactosidase or horseradish peroxid
  • a breast 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 ammo acid sequence, or by comparing it to known sequences to search for homology to provide a frame, assuming the breast 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 Advanced BLAST parameters
  • 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
  • ammo acid variants of the naturally occurring sequences are also included within one embodiment of breast cancer proteins.
  • the variants are preferably greater than about
  • 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
  • breast cancer proteins of the present invention may be shorter or longer than the wild type am o acid sequences
  • included within the definition of breast cancer proteins are portions or fragments of the wild type sequences herein
  • the breast 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 breast cancer proteins are derivative or variant breast cancer proteins as compared to the wild-type sequence That is, as outlined more fully below, the derivative breast cancer peptide will contain at least one ammo acid substitution, deletion or insertion, with ammo acid substitutions being particularly preferred The am o acid substitution, insertion or deletion may occur at any residue within the breast cancer peptide
  • ammo acid sequence variants are also included in an embodiment of breast cancer proteins of the present invention.
  • ammo acid sequence variants fall into one or more of three classes substitutional, msertional or deletional variants
  • These variants ordinarily are prepared by site specific mutagenesis of nucleotides in the DNA encoding the breast 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
  • variant breast cancer protein fragments having up to about 100-150 residues may be prepared by in vitro synthesis using established techniques
  • Ammo 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 breast cancer protein ammo 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 breast 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 breast cancer protein activities
  • Ammo acid substitutions are typically of single residues, insertions usually will be on the order of from about 1 to 20 am o 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 ammo 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 breast cancer protein are desired, substitutions are generally made in accordance with the following chart
  • substitutions 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
  • 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, (b) a cysteine or prolme is substituted for (or by) any other residue, (c) a residue having an electropositive side chain, e g lysyl, argmyl, or histidyl, is substituted for (or
  • 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 breast cancer proteins as needed Alternatively, the variant may be designed such that the biological activity of the breast cancer protein is altered For example, glycosylation sites may be altered or removed
  • Covalent modifications of breast cancer polypeptides are included within the scope of this invention
  • One type of covalent modification includes reacting targeted ammo acid residues of a breast cancer polypeptide with an organic de ⁇ vatizing agent that is capable of reacting with selected side chains or the N-or C-termmal residues of a breast cancer polypeptide
  • De ⁇ vatization with bifunctional agents is useful, for instance, for crosslinkmg breast cancer to a water-insoluble support matrix or surface for use in the method for purifying anti-breast cancer antibodies or screening assays, as is more fully described below
  • Commonly used crosslinkmg agents include, e g , 1 ,1-b ⁇ s(d ⁇ azoacetyl)-2- phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with 4-az ⁇ dosal ⁇ cyl ⁇ c acid, homobifunctional imidoesters, including disuccmimidyl esters such as 3,3'
  • Another type of covalent modification of the breast 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 breast cancer polypeptide, and/or adding one or more glycosylation sites that are not present in the native sequence breast cancer polypeptide
  • Addition of glycosylation sites to breast cancer polypeptides may be accomplished by altering the ammo acid sequence thereof
  • the alteration may be made, for example, by the addition of, or substitution by, one or more se ⁇ ne or threonine residues to the native sequence breast cancer polypeptide (for O-hnked glycosylation sites)
  • the breast cancer ammo acid sequence may optionally be altered through changes at the DNA level, particularly by mutating the DNA encoding the breast cancer polypeptide at preselected bases such that codons are generated that will translate into the desired ammo acids
  • Another means of increasing the number of carbohydrate moieties on the breast 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 Aplm and W ⁇ ston, breast cancer
  • Removal of carbohydrate moieties present on the breast cancer polypeptide may be accomplished chemically or enzymatically or by mutational substitution of codons encoding for ammo acid residues that serve as targets for glycosylation
  • Chemical deglycosylation techniques are known in the art and described, for instance, by Hakimuddm, 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 breast cancer comprises linking the breast cancer polypeptide to one of a variety of nonprotemaceous 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
  • breast cancer polypeptides of the present invention may also be modified in a way to form chimeric molecules comprising a breast cancer polypeptide fused to another, heterologous polypeptide or ammo acid sequence
  • a chimeric molecule comprises a fusion of a breast 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 ammo-or carboxyl-terminus of the breast cancer polypeptide
  • the presence of such epitope-tagged forms of a breast cancer polypeptide can be detected using an antibody against the tag polypeptide
  • provision of the epitope tag enables the breast 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 breast cancer polypeptide with an immunoglobulm or a particular region of an immunoglobulm For a bivalent form of
  • 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)], tubulm epitope peptide [Skinner et al , J Biol Chem , 266 15163-15166 (1991)], and the T7 gene 10 protein peptide tag [Lutz-Freyermuth et al , Proc Natl Acad Sci USA, 87 6393-6397 (1990)]
  • breast cancer protein in one embodiment are other breast cancer proteins of the breast cancer family, and breast 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 breast cancer proteins from humans or other organisms
  • particularly useful probe and/or PCR primer sequences include the unique areas of the breast 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
  • breast 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
  • breast cancer proteins may also be identified as being encoded by breast cancer nucleic acids
  • breast cancer proteins are encoded by nucleic acids that will hybridize to the sequences of the sequence listings, or their complements, as outlined herein
  • the breast cancer protein when the breast cancer protein is to be used to generate antibodies, for example for immunotherapy, the breast 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 Thus, in most instances, antibodies made to a smaller breast cancer protein will be able to bind to the full length protein
  • the epitope is unique, that is, antibodies generated to a unique epitope show little or no cross-reactivity
  • the epitope is selected from BCH1p1 , BCH1 p2, BCR3p1 , BCR3p2, BCQ8p1 , BCQ8p2, BCQ5p1 , BCQ5p2, BCN1p1 , BCN1p2, wherein BCH1 p1 and BCH1p2 are preferred In one embodiment the epitope or fragment of BCH1p1
  • 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 or those 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 Typically, the immunizing agent and/or adjuvant will be injected in the mammal by multiple subcutaneous or intrapentoneal injections
  • the immunizing agent may include the BCH1 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 Examples of such immunogemc proteins include but are not limited to keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsm inhibitor Examples of 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 undu
  • the antibodies may, alternatively, be monoclonal antibodies
  • Monoclonal antibodies may be prepared using hybridoma methods, such as those described by Kohler and Milstem, 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 wfro
  • the immunizing agent will typically include the BCH1 polypeptide or fragment thereof or a fusion protein thereof Generally, either peripheral blood lymphocytes ("PBLs”) 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
  • PBLs peripheral blood lymphocytes
  • spleen cells or lymph node cells are used if non-human mammalian sources are desired
  • the lymphocytes are then fused with an immortal
  • the antibodies are bispe ⁇ fic 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 breast cancer protein 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 breast cancer are capable of reducing or eliminating the biological function of breast cancer, as is described below That is, the addition of anti-breast cancer antibodies (either polyclonal or preferably monoclonal) to breast cancer (or cells containing breast cancer) may reduce or eliminate the breast 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 breast cancer proteins are humanized antibodies
  • Humanized forms of non-human (e g , murine) antibodies are chimeric molecules of immunoglobulins, immunoglobulm 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 immunoglobulm
  • 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
  • donor antibody such as mouse, rat or rabbit having the desired specificity, affinity and capacity
  • Fv framework residues of the human immunoglobulm 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
  • a humanized antibody has one or more ammo acid residues introduced into it from a source which is non-human
  • 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 , J 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 Cancer Therapy, Alan R Liss, p 77 (1985) and Boerner et al , J Immunol , 147(1 ) 86-95 (1991 )1 Similarly, human antibodies can be made by introducing of human immunoglobulm loci into transgenic animals, e g , mice in which the endogenous immunoglobulm 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
  • immunotherapy is meant treatment of breast cancer with an antibody raised against breast 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 breast 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 breast cancer protein
  • the breast cancer protein to which antibodies are raised is a transmembrane protein
  • antibodies used for treatment bind the extracellular domain of the breast 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 breast cancer protein
  • the antibody may be a competitive, non-competitive or uncompetitive inhibitor of protein binding to the extracellular domain of the breast cancer protein
  • the antibody is also an antagonist of the breast cancer protein
  • the antibody prevents activation of the transmembrane breast 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, vmblastme, actmomycin D,
  • the antibody is conjugated to a therapeutic moiety
  • the therapeutic moiety is a small molecule that modulates the activity of the breast cancer protein
  • the therapeutic moiety modulates the activity of molecules associated with or in close proximity to the breast cancer protein
  • the therapeutic moiety may inhibit enzymatic activity such as protease or protein kmase activity associated with breast 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 breast 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 dipthe ⁇ a A chain, exotoxin A chain, ⁇ cin A chain, ab ⁇ n A chain, curcin, crotin, phenomycm, enomycm and the like Cytotoxic agents also include radiochemicals
  • the breast cancer antibodies of the invention specifically bind to breast 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
  • the breast cancer protein is purified or isolated after expression
  • Breast 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 chromatofocusmg
  • the breast cancer protein may be purified using a standard anti-breast cancer antibody column Ultrafiltration and diafiltration techniques, in conjunction with protein concentration, are also useful For general guidance in suitable purification techniques, see Scopes, R , Protein Purification, Sp ⁇ nger-Verlag, NY (1982) The degree of purification necessary will vary depending on the use of the breast cancer protein In some instances no purification will be necessary
  • breast 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 breast cancer phenotype, that is, the expression levels of genes in normal breast tissue and in breast cancer tissue (and in some cases, for varying seventies of breast cancer that relate to prognosis, as outlined below) are evaluated to provide expression profiles
  • An expression profile of a particular cell state or point of development is essentially a "fingerprint" of the 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 of the 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 breast cancer tissue
  • differential expression refers to both qualitative as well as quantitative differences in the genes' temporal and/or cellular expression patterns within and among the cells
  • a differentially expressed gene can qualitatively have its expression altered, including an activation or mactivation, in, for example, normal versus breast cancer tissue That is, genes may be turned on or turned off in a particular state, relative to another state
  • 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 Alternatively, 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 Affymet ⁇ x GeneChipTM expression
  • 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 breast 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
  • mass spectroscopy assays i e those identified as being important in a breast cancer phenotype
  • 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 breast cancer nucleic acid probes are attached to biochips as outlined herein for the detection and quantification of breast cancer sequences in a particular cell
  • the assays are further described below in the example
  • nucleic acids encoding the breast cancer protein are detected
  • DNA or RNA encoding the breast cancer protein may be detected
  • methods wherein the mRNA encoding a breast cancer protein is detected The presence of mRNA in a sample is an indication that the breast 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 permeabihzed cells or tissue samples are contacted with a detect
  • any of the three classes of proteins as described herein secreted, transmembrane or intracellular proteins are used in diagnostic assays
  • the breast cancer proteins, antibodies, nucleic acids, modified proteins and cells containing breast 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
  • breast cancer proteins including intracellular, transmembrane or secreted proteins, find use as markers of breast cancer Detection of these proteins in putative breast cancer tissue or patients allows for a determination or diagnosis of breast cancer
  • antibodies are used to detect breast 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 isoelect ⁇ c focusing gels and the like) Following separation of proteins, the breast cancer protein is detected by immunoblotting with antibodies raised against the breast cancer protein Methods of immunoblotting are well known to those of ordinary skill in the art
  • antibodies to the breast cancer protein find use in in situ imaging techniques
  • cells are contacted with from one to many antibodies to the breast cancer prote ⁇ n(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 breast cancer prote ⁇ n(s) contains a detectable label
  • each one of multiple primary antibodies contains a distinct and detectable label
  • 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 breast cancer from blood samples
  • certain breast cancer proteins are secreted/circulating molecules Blood samples, therefore, are useful as samples to be probed or tested for the presence of secreted breast cancer proteins
  • Antibodies can be used to detect the breast 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 breast cancer nucleic acid probes to tissue arrays is done For example, arrays of tissue samples, including breast cancer tissue and/or normal tissue, are made In situ hybridization as is known in the art can then be done
  • the breast cancer proteins, antibodies, nucleic acids, modified proteins and cells containing breast cancer sequences are used in prognosis assays
  • gene expression profiles can be generated that correlate to breast 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 breast cancer probes are attached to biochips for the detection and quantification of breast 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 breast cancer proteins, antibodies, nucleic acids, modified proteins and cells containing breast 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), Held, 1996 #69
  • the breast cancer proteins, antibodies, nucleic acids, modified proteins and cells containing the native or modified breast cancer proteins are used in screening assays That is, the present invention provides novel methods for screening for compositions which modulate the breast 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"
  • 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 Having identified the differentially expressed genes herein, a variety of 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 breast 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
  • 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 breast 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 breast cancer nucleic acid probes are attached to biochips as outlined herein for the detection and quantification of breast cancer sequences in a particular cell
  • the assays are further described below
  • a candidate bioactive agent is added to the cells prior to analysis Moreover, screens are provided to identify a candidate bioactive agent which modulates breast cancer, modulates breast cancer proteins, binds to a breast cancer protein, or interferes between the binding of a breast cancer protein and an antibody
  • the term “candidate bioactive agent” or “drug candidate” or grammatical equivalents as used herein describes any molecule, e g , protein, oligopeptide, small organic molecule, polysaccha ⁇ de, polynucleotide, etc , to be tested for bioactive agents that are capable of directly or indirectly altering either the breast cancer phenotype or the expression of a breast 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 breast cancer phenotype, for example to a normal breast tissue fingerprint
  • the candidate agent preferably suppresses a severe breast cancer phenotype
  • a plurality of assay mixtures are run in parallel with different agent concentrations to obtain a differential response to the various concentrations Typically, one of these concentrations serves as a negative control, i e , at zero concentration or below the level of detection
  • 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 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 amme, 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, saccha ⁇ des, fatty acids, steroids, pu ⁇ nes, pyrimidmes, 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, este ⁇ fication, amidification to produce structural analogs
  • the candidate bioactive agents are proteins
  • protein herein is meant at least two covalently attached am o acids, which includes proteins, polypeptides, ohgopeptides and peptides
  • the protein may be made up of naturally occurring ammo acids and peptide bonds, or synthetic peptidomimetic structures
  • “ammo acid”, or “peptide residue”, as used herein means both naturally occurring and synthetic am o acids
  • homo-phenylalanme, citrullme and noreleuc e are considered ammo acids for the purposes of the invention
  • Ammo acid also includes imino acid residues such as prolme and hydroxyprolme
  • the side chains may be in either the (R) or the (S) configuration
  • the ammo acids are in the (S) or L-configu ration If non-naturally occurring side chains are used, non-ammo 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 protemaceous cellular extracts may be used In this way libraries of procaryotic and eucaryotic proteins may be made for screening in the methods of the invention
  • the candidate bioactive agents are peptides of from about 5 to about 30 ammo acids, with from about 5 to about 20 ammo 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 ammo acids, respectively Since generally these random peptides (or nucleic acids, discussed below) are chemically synthesized, they may incorporate any nucleotide or ammo 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 protemaceous 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 ammo acid residues are randomized within a defined class, for example, of hydrophobic ammo acids, hydrophilic residues, ste ⁇ cally biased (either small or large) residues, towards the creation of nucleic acid binding domains, the creation of cysteines, for cross-linking, prolmes for SH-3 domains, se ⁇ nes, threonmes, tyrosmes or histidmes for phosphorylation sites, etc , or to pu ⁇ nes, 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 Generally, the nucleic acids are labeled with biotm-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 streptavidm
  • the streptavidm is labeled as described above, thereby, providing a detectable signal for the bound target sequence
  • unbound labeled streptavidm 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
  • 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 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 breast cancer phenotype Specifically, there are several types of screens that can be run 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 breast cancer similar to the expression profile of normal breast tissue is expected to result in a suppression of the breast cancer phenotype Thus, in this embodiment, 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 differentially expressed gene
  • 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 breast 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 After identifying a candidate agent based upon its ability to suppress a breast cancer expression pattern leading to a normal expression pattern, or modulate a single breast 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 breast cancer tissue reveals genes that are not expressed in normal tissue or breast 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 breast cancer genes or proteins In particular these sequences and the proteins they encode find use in marking or identifying agent treated cells In addition, antibodies can be raised against the agent induced proteins and used to target novel therapeutics to the treated breast cancer tissue sample
  • a candidate agent is administered to a population of breast cancer cells, that thus has an associated breast cancer expression profile
  • 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 protemaceous candidate agent i e a peptide
  • a viral construct such as a retroviral construct
  • 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
  • breast cancer tissue may be screened for agents that reduce or suppress the breast cancer phenotype
  • a change in at least one gene of the expression profile indicates that the agent has an effect on breast cancer activity
  • screens may be done on individual genes and gene products (proteins) That is, having identified a particular differentially expressed 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 differentially expressed genes are sometimes referred to herein as "breast cancer proteins", “breast cancer modulating proteins” "BCP” or a "BCMP”
  • BCMP is termed BCH1
  • BCH1 can be identified as described for identifying breast cancer proteins herein
  • BCH1 is depicted in Figure 34
  • the BCMP may be a fragment, or alternatively, be the full length protein to the fragment shown herein
  • the BCMP is a fragment of approximately 14 to 24 amino acids long More preferably the fragment is a soluble fragment
  • the breast cancer protein or nucleic acid encoding a breast cancer protein may be selected from any sequence provided in the figures including those wherein the accession numbers are provided Preferred sequences are in Figure 10, more preferably Figure 11 and most preferably Figure 12 Preferred sequences are also selected from the group consisting of BCH1 , BCA2, BCJ7, BCN1 , BCN5, BC02, BCQ5, BCR2, BCR3, BCQ8, BCN2, BCX3, BCX2 and BCY3 Most preferably, the sequence is selected from the group consisting of BCH1 , BCA2, BCJ7, BCN1 , BCN5, BC02, BCQ5, BCR2, BCX2 and BCY3 It is understood that any protein can also be selected from any subset of this group of proteins, for example, BCH1 can be selected from the whole group, or from the subset of BCH1 and BCN1
  • 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 differentially expressed proteins, and then these agents may be used in assays that evaluate the ability of the candidate agent to modulate differentially expressed 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 differentially expressed 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 breast cancer proteins can be used in the assays
  • the methods comprise combining a breast cancer protein and a candidate bioactive agent, and determining the binding of the candidate agent to the breast cancer protein
  • Preferred embodiments utilize the human breast cancer protein, although other mammalian proteins may also be used, for example for the development of animal models of human disease
  • variant or derivative breast cancer proteins may be used
  • the breast 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 )
  • 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
  • suitable insoluble supports include microtiter plates, arrays, membranes and beads These are typically made of glass, plastic (e g , polystyrene), polysaccha ⁇ des, nylon or nitrocellulose, teflonTM, etc 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 P
  • the breast 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 breast 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
  • 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 candidate bioactive agent is labelled, and binding determined directly For example, this may be done by attaching all or a portion of the breast cancer protein to a solid support, adding a labelled candidate agent (for example a fluorescent label), washing off excess reagent, and determining whether the label is present on the solid support Various blocking and washing steps may be utilized as is known in the art
  • 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 streptavidm, digoxm and antidigoxm 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 Alternatively, 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 breast cancer), such as an antibody, peptide, binding partner, ligand, etc
  • the target molecule i e breast 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 breast cancer protein and thus is capable of binding to, and potentially modulating, the activity of the breast 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 breast cancer protein with a higher affinity
  • the presence of the label on the support, coupled with a lack of competitor binding may indicate that the candidate agent is capable of binding to the breast cancer protein
  • the methods comprise differential screening to identity bioactive agents that are capable of modulating the activity of the breast cancer proteins
  • the methods comprise combining a breast cancer protein and a competitor in a first sample
  • a second sample comprises a candidate bioactive agent, a breast cancer protein and a competitor
  • the binding of the 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 breast 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 breast cancer protein
  • a preferred embodiment utilizes differential screening to identify drug candidates that bind to the native breast cancer protein, but cannot bind to modified breast cancer proteins
  • the structure of the breast cancer protein may be modeled, and used in rational drug design to synthesize agents that interact with that site
  • Drug candidates that affect breast 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
  • 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
  • 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 breast cancer proteins comprise the steps of adding a candidate bioactive agent to a sample of breast cancer proteins, as above, and determining an alteration in the biological activity of breast cancer proteins "Modulating the activity of breast 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 breast 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 breast cancer proteins
  • the methods comprise combining a breast cancer sample and a candidate bioactive agent, and evaluating the effect on breast cancer activity
  • breast cancer activity or grammatical equivalents herein is meant one of the breast cancer's biological activities, including, but not limited to, cell division, preferably in breast tissue, cell proliferation, tumor growth, transformation of cells
  • breast cancer activity include activation of breast cancer protein, BCH1 , for example, or a substrate thereof by the breast cancer protein
  • An inhibitor of breast cancer activity is the inhibition of any one or more breast cancer activities
  • the activity of the breast cancer protein is increased, in another preferred embodiment, the activity of the breast 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 breast cancer protein
  • the methods comprise adding a candidate bioactive agent, as defined above, to a cell comprising breast cancer proteins
  • Preferred cell types include almost any cell
  • the cells contain a recombinant nucleic acid that encodes a breast 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, cytokmes, growth factors, action potentials, pharmacological agents including chemotherapeutics, radiation, carcmogenics, or other cells (i e cell-cell contacts)
  • physiological signals for example hormones, antibodies, peptides, antigens, cytokmes, growth factors, action potentials, pharmacological agents including chemotherapeutics, radiation, carcmogenics, or other cells (i e cell-cell contacts)
  • the determinations are determined at different stages of the cell cycle process In this way, bioactive agents are identified Compounds with pharmacological activity are able to enhance or interfere with the activity of the breast cancer protein
  • breast cancer protein activity includes at least one of the following breast cancer activity, binding to a breast cancer protein, activation of a breast cancer protein or activation of substrates of a breast cancer protein by a breast cancer protein
  • a method of inhibiting breast cancer cell division comprises administration of a breast cancer inhibitor
  • a method of inhibiting tumor growth comprises administration of a breast cancer inhibitor
  • methods of treating cells or individuals with cancer comprises administration of a breast cancer inhibitor
  • a breast cancer inhibitor is an antibody as discussed above
  • the breast 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 breast cancer molecules
  • a preferred antisense molecule is for BCH1 or for a ligand or activator thereof
  • Antisense or sense oligonucleotides, according to the present invention 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, Stem and Cohen (Cancer Res 48 2659, 1988) and van der Krol et al (BioTechniques 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 cytokmes, or other ligands that bind to cell surface receptors
  • conjugation of the 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- hpid 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
  • 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, intrape ⁇ toneally, mtravascularly, 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 invention provides methods for identifying cells containing variant breast cancer genes comprising determining all or part of the sequence of at least one endogeneous breast cancer genes in a cell
  • this may be done using any number of sequencing techniques
  • the invention provides methods of identifying the breast cancer genotype of an individual comprising determining all or part of the sequence of at least one breast 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 of the same tissue
  • the method may include comparing the sequence of the sequenced breast cancer gene to a known breast cancer gene, i e a wild-type gene
  • the sequence of all or part of the breast cancer gene can then be compared to the sequence of a known breast cancer gene to determine if any differences exist This can be done using any number of known homology programs, such as Bestfit,
  • the breast cancer genes are used as probes to determine the number of copies of the breast cancer gene in the genome
  • breast cancer genes are used as probed to determine the chromosomal localization of the breast 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 breast cancer gene loci
  • the methods comprise administering to a cell an anti-breast cancer antibody that reduces or eliminates the biological activity of an endogenous breast cancer protein
  • the methods comprise administering to a cell or organism a recombinant nucleic acid encoding a breast cancer protein
  • this may be accomplished in any number of ways
  • the activity of the breast cancer gene is increased by increasing the amount of breast cancer in the cell, for example by overexpressmg the endogenous breast cancer or by administering a gene encoding the breast cancer 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 Alternatively, for example when the breast cancer
  • EHR enhanced homologous recombination
  • the breast cancer proteins of the present invention may be used to generate polyclonal and monoclonal antibodies to breast cancer proteins, which are useful as described herein Similarly, the breast cancer proteins can be coupled, using standard technology, to affinity chromatography columns These columns may then be used to purify breast cancer antibodies In a preferred embodiment, the antibodies are generated to epitopes unique to a breast cancer protein, that is, the antibodies show little or no cross-reactivity to other proteins These antibodies find use in a number of applications For example, the breast cancer antibodies may be coupled to standard affinity chromatography columns and used to purify breast cancer proteins The antibodies may also be used as blocking polypeptides, as outlined above, since they will specifically bind to the breast cancer protein
  • a therapeutically effective dose of a breast 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 ascertamable by one skilled in the art using known techniques As is known in the art, adjustments for protein 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 ascertamable 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 breast 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, mtranasally, transdermally, intrape ⁇ toneally, intramuscularly, intrapulmonary, vagmally, rectally, or mtraocularly
  • the breast cancer proteins and modulators may be directly applied as a solution or spray
  • compositions of the present invention comprise a breast 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, succmic acid, fuma ⁇ c acid, tarta ⁇ c acid, citric acid, benzoic acid, cinnamic acid, mandehc acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid
  • compositions may also include one or more of the following carrier proteins such as serum albumin, buffers, fillers such as microcrystallme cellulose, lactose, corn and other starches, binding agents, sweeteners and other flavoring agents, coloring agents, and polyethylene glycol Additives are well known in the art, and are used in a variety of formulations
  • breast cancer proteins and modulators are administered as therapeutic agents, and can be formulated as outlined above
  • breast cancer genes can be administered in gene therapy applications, as is known in the art
  • These breast 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
  • breast cancer genes are administered as DNA vaccines, either single genes or combinations of breast cancer genes Naked DNA vaccines are generally known in the art Brower, Nature Biotechnology, 16 1304-1305 (1998)
  • breast 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 breast cancer gene or portion of a breast cancer gene under the control of a promoter for expression in a breast cancer patient
  • the breast cancer gene used for DNA vaccines can encode full-length breast cancer proteins, but more preferably encodes portions of the breast cancer proteins including peptides derived from the breast cancer protein
  • a patient is immunized with a DNA vaccine comprising a plurality of nucleotide sequences derived from a breast 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 breast cancer proteins
  • the DNA vaccines include a gene encoding an adjuvant molecule with the
  • DNA vaccine Such adjuvant molecules include cytokmes that increase the immunogenic response to the breast 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
  • breast cancer genes find use in generating animal models of breast cancer
  • gene therapy technology wherein antisense RNA directed to the breast cancer gene will also dimmish or repress expression of the gene
  • An animal generated as such serves as an animal model of breast 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 breast cancer protein
  • tissue-specific expression or knockout of the breast cancer protein may be necessary
  • breast cancer protein is overexpressed in breast cancer
  • transgenic animals can be generated that overexpress the breast 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 of the expression level of the transgene
  • Animals generated by such methods find use as animal models of breast cancer and are additionally useful in screening for bioactive molecules to treat breast cancer
  • ER expression is an important clinical prognostic marker for breast cancer Because signaling through ER to activate most estrogen-responsive genes is believed to require translocation of activated ER to the nucleus, high expression of BCH1 is predicted to correlate with functionally- negative ER in ER+ tumors Since ER- correlates with poor prognosis, BCH1 may also be a valuable prognostic marker, as it may also correlate with poor prognosis, or to no-responsiveness to anti- estrogen therapies (e g , tamoxifen), since approximately 50% of the ER+ patients do not respond to tamoxifen Tamoxifen works against the effects of estrogen on these cells It is often called an "anti- estrogen " As a treatment for breast cancer, the drug slows or stops the growth of cancer cells that are already present in the body As adjuvant therapy, tamoxifen has been shown to help prevent the original breast cancer from returning and also prevent the development of new cancers in the opposite breast
  • BCH1 may have a casual role in ER translocation to the nucleus, by either directly or indirectly preventing nuclear translocation, thereby eliminating or altering responsiveness of the cell to estrogens or their analogs

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Abstract

L'invention concerne des techniques utilisées pour diagnostiquer et pronostiquer le cancer du sein. L'invention concerne également des techniques pouvant être utilisées pour cribler des agents bioactifs candidats, permettant de moduler le cancer du sein. L'invention concerne, en outre, des techniques et des cibles moléculaires (gènes et leurs produits) permettant d'intervenir thérapeutiquement dans le cancer du sein et dans d'autres cancers.
PCT/US2000/006952 1999-03-15 2000-03-15 Nouvelles techniques permettant de traiter et de diagnostiquer le cancer du sein, compositions et techniques de criblage pour modulateurs de cancer du sein WO2000055629A2 (fr)

Priority Applications (9)

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JP2000605210A JP2004537710A (ja) 1999-03-15 2000-03-15 乳癌の診断及び治療の新規方法、組成物及び乳癌調節剤用の選別方法
CA002368152A CA2368152A1 (fr) 1999-03-15 2000-03-15 Nouvelles techniques permettant de traiter et de diagnostiquer le cancer du sein, compositions et techniques de criblage pour modulateurs de cancer du sein
MXPA01009367A MXPA01009367A (es) 1999-03-15 2000-03-15 Nuevos metodos de diagnostico y tratamiento de cancer mamario, composiciones y metodo para seleccionar moduladores de cancer mamario.
EP00916419A EP1159619A2 (fr) 1999-03-15 2000-03-15 Nouvelles techniques permettant de traiter et de diagnostiquer le cancer du sein, compositions et techniques de criblage pour modulateurs de cancer du sein
AU37527/00A AU3752700A (en) 1999-03-15 2000-03-15 Novel methods of diagnosing and treating breast cancer, compositions, and methods of screening for breast cancer modulators
US09/642,034 US6762020B1 (en) 1999-03-15 2000-08-18 Methods of diagnosing breast cancer
US09/747,371 US6750013B2 (en) 1999-12-02 2000-12-21 Methods for detection and diagnosing of breast cancer
US10/769,612 US20040141983A1 (en) 1999-03-15 2004-01-29 Compositions against cancer antigen LIV-1 and uses thereof
US12/796,244 US8313745B2 (en) 1999-03-15 2010-06-08 Compositions against cancer antigen LIV-1 and uses thereof

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US26886599A 1999-03-15 1999-03-15
US09/268,865 1999-03-15
US43987899A 1999-11-12 1999-11-12
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US09/440,370 1999-11-12
US09/439,878 1999-11-12
US44049399A 1999-11-15 1999-11-15
US09/440,493 1999-11-15
US44067799A 1999-11-16 1999-11-16
US44067699A 1999-11-16 1999-11-16
US09/440,677 1999-11-16
US09/440,676 1999-11-16
US45081099A 1999-11-29 1999-11-29
US09/450,810 1999-11-29
US45313799A 1999-12-02 1999-12-02
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