WO1997002280A1 - Breast specific genes and proteins - Google Patents

Breast specific genes and proteins Download PDF

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Publication number
WO1997002280A1
WO1997002280A1 PCT/US1995/008295 US9508295W WO9702280A1 WO 1997002280 A1 WO1997002280 A1 WO 1997002280A1 US 9508295 W US9508295 W US 9508295W WO 9702280 A1 WO9702280 A1 WO 9702280A1
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WIPO (PCT)
Prior art keywords
polypeptide
dna
breast
gene
polynucleotide
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PCT/US1995/008295
Other languages
French (fr)
Inventor
Hongjun Ji
Craig A. Rosen
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Human Genome Sciences, Inc.
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Publication date
Application filed by Human Genome Sciences, Inc. filed Critical Human Genome Sciences, Inc.
Priority to JP9505093A priority Critical patent/JPH11509093A/en
Priority to AU30929/95A priority patent/AU3092995A/en
Priority to CA002225824A priority patent/CA2225824A1/en
Priority to PCT/US1995/008295 priority patent/WO1997002280A1/en
Priority to EP95926616A priority patent/EP0851869A4/en
Publication of WO1997002280A1 publication Critical patent/WO1997002280A1/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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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

  • This invention relates to newly identified polynucleotides, polypeptides encoded by such polynucleotides, and the use of such polynucleotides and polypeptides for detecting disorders of the breast, particularly the presence of breast cancer and breast cancer metastases.
  • the present invention further relates to inhibiting the production and function of the polypeptides of the present invention.
  • the twenty breast specific genes of the present invention are sometimes hereinafter referred to as "BSG1", “BSG2" etc.
  • the mammary gland is subject to a variety of disorders that should be readily detectable. Detection may be accomplished by inspection which usually consists of palpation. Unfortunately, so few periodic self-examinations are made that many breast masses are discovered only by accidental palpation. Aspiration of suspected cysts with a fine-gauge needle is another fairly common diagnostic practice. Mammography or xeroradiography (soft-tissue x-ray) of the breast of yet another. A biopsy of a lesion or suspected area is an extreme method of diagnostic test.
  • Fibroadenomas is the most common benign breast tumor. As a pathological entity, it ranks third behind cystic disease and carcinoma, respectively. These tumors are seen most frequently in young people and are usually readily recognized because they feel encapsulated. Fibrocystic disease, a benign condition, is the most common disease of the female breast, occurring in about 20% bf pre- menopausal women. Lipomas of the breast are also common and they are benign in nature. Carcinoma of the breast is the most common malignant condition among women and carries with it the highest fatality rate of all cancers affecting this sex. At some during her life, one of every 15 women in the USA will develop cancer of the breast.
  • nucleic acid probes comprising nucleic acid molecules of sufficient length to specifically hybridize to the RNA transcribed from the human breast specific genes of the present invention or to DNA corresponding to such RNA.
  • a method of and products for diagnosing breast cancer formation and breast cancer metastases by detecting the presence of RNA transcribed from the human breast specific genes of the present invention or DNA corresponding to such RNA in a sample deprived from a host.
  • a method of and products for diagnosing breast cancer formation and breast cancer metastases by detecting an altered level of a polypeptide corresponding to the breast specific genes of the present invention in a sample derived from a host, whereby an elevated level of the polypeptide indicates a breast cancer diagnosis.
  • isolated polynucleotides encoding human breast specific polypeptides, including mRNAs, DNAs, cDNAs, genomic DNAs, as well as antisense analogs and biologically active and diagnostically or therapeutically useful fragments thereof.
  • human breast specific genes which include polynucleotides as set forth in the sequence listing.
  • novel polypeptides encoded by the polynucleotides as well as biologically active and diagnostically or therapeutically useful fragments, analogs and derivatives thereof.
  • a process for producing such polypeptides by recombinant techniques comprising culturing recombinant prokaryotic and/or eukaryotic host cells, containing a polynucleotide of the present invention, under conditions promoting expression of said proteins and subsequent recovery of said proteins.
  • antibodies specific to such polypeptides which may be employed to detect breast cancer cells or breast cancer metastasis.
  • processes for using one or more of the polypeptides of the present invention to treat breast cancer and for using the polypeptides to screen for compounds which interact with the polypeptides, for example, compounds which inhibit or activate the polypeptides of the present invention are provided.
  • a screen for detecting compounds which inhibit activation of one or more of the polynucleotides and/or polypeptides of the present invention which may be used to therapeutically, for example, in the treatment of breast cancer.
  • Figure 1 is a full length cDNA sequence of breast specific gene 1 of the present invention.
  • Figure 2 is a partial cDNA sequence and the corresponding deduced amino acid sequence of breast specific gene 2 of the present invention.
  • Figure 3 is a partial cDNA sequence and deduced amino acid sequence of breast specific gene 3 of the invention.
  • Figure 4 is a partial cDNA sequence and the corresponding deduced amino acid sequence of breast specific Figure 5 is a partial cDNA sequence of breast specific gene 5 of the present invention.
  • Figure 6 is a partial cDNA and deduced amino acid sequence of breast specific gene 6 of the present invention.
  • Figure 7 is a partial cDNA sequence of breast specific gene 7 of the present invention.
  • Figure 8 is a partial cDNA sequence of breast specific gene 8 of the present invention.
  • Figure 9 is a partial cDNA sequence of breast specific gene 9 of the present invention.
  • Figure 10 is a partial cDNA sequence of breast specific gene 10 of the pre ⁇ ent invention.
  • Figure 11 is a partial cDNA sequence of breast specific gene 11 of the present invention.
  • Figure 12 i ⁇ a partial cDNA sequence of breast specific gene 12 of the present invention.
  • Figure 13 i ⁇ a partial cDNA sequence of breast specific gene 13 of the present invention.
  • Figure 14 is a partial cDNA sequence of breast specific gene 14 of the present invention.
  • Figure 15 is a partial cDNA sequence of breast specific gene 15 of the present invention.
  • Figure 16 is a partial cDNA sequence of breast specific gene 16 of the present invention.
  • Figure 17 i ⁇ a partial cDNA ⁇ equence of breast specific gene 17 of the present invention.
  • Figure 18 is a partial cDNA sequence of breast specific gene 18 of the present invention.
  • Figure 19 i ⁇ a partial cDNA ⁇ equence of brea ⁇ t ⁇ pecific gene 19 of the pre ⁇ ent invention.
  • breast specific gene means that ⁇ uch gene is primarily expres ⁇ ed in ti ⁇ ue ⁇ derived from the brea ⁇ t, and ⁇ uch gene ⁇ may be expre ⁇ ed in cell ⁇ derived from tissue ⁇ other than from the breast. However, the expression of such genes is significantly higher in tissues derived from the breast than from non-breast tis ⁇ ue ⁇ .
  • a ⁇ pect of the present invention there is provided a polynucleotide which encodes the mature polypeptides having the deduced amino acid sequence of Figure 1 (SEQ ID NO:l) and fragments, analogues and derivatives thereof.
  • a polynucleotide which encodes the same mature polypeptide a ⁇ a human gene having a coding portion which contains a polynucleotide which is at least 90% identical (preferably at least 95% identical and most preferably at least 97% or 100% identical) to one of the polynucleotides of Figures 2-20 (SEQ ID NO:2-20) , as well as fragments thereof.
  • a polynucleotide which encodes for the same mature polypeptide as a human gene whose coding portion includes a polynucleotide which is at least 90% identical to (preferably at least 95% identical to and most preferably at least 97% or 100% identical) to one of the polynucleotides included in ATCC Deposit No. 97175 deposited June 2, 1995.
  • a polynucleotide probe which hybridizes to mRNA (or the corre ⁇ ponding cDNA) which is transcribed from the coding portion of a human gene which coding portion include ⁇ a DNA sequence which is at least 90% identical to (preferably at least 95% identical to) and most preferably at least 97% or 100% identical) to one of the polynucleotide sequences of Figures 1-20 (SEQ ID NO:1-20) .
  • the present invention further relate ⁇ to a mature polypeptide encoded by a coding portion of a human gene which coding portion include ⁇ a DNA sequence which i ⁇ at le ⁇ t 90% identical to (preferably at least 95% identical to and more preferably 97% or 100% identical to) one of the polynucleotides of Figures 2-20 (SEQ ID NO:2-20) , as well as analogues, derivatives and fragment ⁇ of such polypeptides.
  • the present invention also relates to one of the mature polypeptides of Figure 1 (SEQ ID N0:1) and fragments, analogues and derivatives of such polypeptides.
  • the present invention further relates to the same mature polypeptide encoded by a human gene whose coding portion include ⁇ DNA which i ⁇ at least 90% identical to (preferably at least 95% identical to and more preferably at least 97% or 100% identical to) one of the polynucleotides included in ATCC Deposit No. 97175 depo ⁇ ited June 2, 1995.
  • nucleic acids which encode for the mature polypeptides having the deduced amino acid sequence of Figure 1 (SEQ ID N0:1) or fragments, analogues or derivatives thereof.
  • the polynucleotides of the present invention may be in the form of RNA or in the form of DNA, which DNA includes cDNA, genomic DNA, and synthetic DNA.
  • the DNA may be double- stranded or single-stranded, and if single stranded may be the coding strand or non-coding (anti-sense) strand.
  • the coding sequence which encodes the mature polypeptide may include DNA identical to Figures 1-20 (SEQ ID NO:1-20) or that of the deposited clone or may be a different coding sequence which coding sequence, as a result of the redundancy or degeneracy of the genetic code, encodes the same mature polypeptide as the coding sequence of a gene which coding sequence includes the DNA of Figures 1-20 (SEQ ID NO:1-20) or the deposited cDNA.
  • the polynucleotide which encodes a mature polypeptide of the present invention may include, but is not limited to: only the coding sequence for the mature polypeptide; the coding sequence for the mature polypeptide and additional coding sequence such a ⁇ a leader or secretory sequence or a proprotein sequence; the coding sequence for the mature polypeptide (and optionally additional coding sequence) and non-coding sequence, such as introns or non-coding sequence 5' and/or 3' of the coding sequence for the mature polypeptide.
  • polynucleotide encoding a polypeptide encompasses a polynucleotide which includes only coding sequence for the polypeptide as well as a polynucleotide which include ⁇ additional coding and/or non-coding ⁇ equence.
  • the present invention further relates to variants of the hereinabove described polynucleotides which encode fragments, analogs and derivatives of a mature polypeptide of the present invention.
  • the variant of the polynucleotide may be a naturally occurring allelic variant of the polynucleotide or a non-naturally occurring variant of the polynucleotide.
  • the present invention includes polynucleotide ⁇ encoding the ⁇ ame mature polypeptide a ⁇ hereinabove described as well as variants of such polynucleotides which variants encode a fragment, derivative or analog of a polypeptide of the invention.
  • Such nucleotide variants include deletion variants, sub ⁇ titution variants and addition or insertion variants.
  • the polynucleotides of the invention may have a coding sequence which is a naturally occurring allelic variant of the human gene who ⁇ e coding ⁇ equence include ⁇ DNA as shown in Figures 1-20 (SEQ ID NO:1-20) or of the coding sequence of the DNA in the deposited clone.
  • an allelic variant is an alternate form of a polynucleotide sequence which may have a sub ⁇ titution, deletion or addition of one or more nucleotide ⁇ , which doe ⁇ not substantially alter the function of the encoded polypeptide.
  • the present invention also includes polynucleotides, wherein the coding sequence for the mature polypeptide may be fused in the same reading frame to a polynucleotide sequence which aids in expre ⁇ sion and secretion of a polypeptide from a host cell, for example, a leader ⁇ equence which functions as a secretory sequence for controlling transport of a polypeptide from the cell.
  • the polypeptide having a leader sequence is a preprotein and may have the leader sequence cleaved by the host cell to form the mature form of the polypeptide.
  • the polynucleotides may also encode a proprotein which is the mature protein plus additional 5' amino acid residues.
  • a mature protein having a prosequence is a proprotein and is an inactive form of the protein. Once the prosequence i ⁇ cleaved an active mature protein remains.
  • the polynucleotide of the pre ⁇ ent invention may encode a mature protein, or a protein having a pro ⁇ equence or a protein having both a pre ⁇ equence and a pre ⁇ equence (leader sequence) .
  • the polynucleotides of the present invention may also have the coding sequence fused in frame to a marker sequence which allows for purification of the polypeptide of the present invention.
  • the marker sequence may be a hexa- histidine tag supplied by a pQE-9 vector to provide for purification of the mature polypeptide fused to the marker in the case of a bacterial host, or, for example, the marker sequence may be a hemagglutinin (HA) tag when a mammalian host, e.g. COS-7 cells, is used.
  • the HA tag correspond ⁇ to an epitope derived from the influenza hemagglutinin protein (Wil ⁇ on, I., et al., Cell, 37:767 (1984)).
  • the present invention further relates to polynucleotides which hybridize to the hereinabove-described polynucleotides if there is at least 70%, preferably at least 90%, and more preferably at least 95% identity between the sequence ⁇ .
  • the pre ⁇ ent invention particularly relate ⁇ to polynucleotide ⁇ which hybridize under ⁇ tringent conditions to the hereinabove-described polynucleotides.
  • stringent conditions means hybridization will occur only if there is at least 95% and preferably at least 97% identity between the sequences.
  • polypeptide ⁇ which retain ⁇ ub ⁇ tantially the ⁇ ame biological function or activity a ⁇ the mature polypeptide of the present invention encoded by a coding sequence which includes the DNA of Figures 1-20 (SEQ ID NO:l-20) or the deposited cDNA(s) .
  • the polynucleotide may have at least 10 or 20 ba ⁇ es, preferably at least 30 base ⁇ , and more preferably at least 50 bases which hybridize to a polynucleotide of the pre ⁇ ent invention and which ha ⁇ an identity thereto, a ⁇ hereinabove de ⁇ cribed, and which may or may not retain activity.
  • such polynucleotides may be employed as probes for polynucleotides, for example, for recovery of the polynucleotide or as a diagnostic probe or as a PCR primer.
  • the present invention is directed to polynucleotides having at least a 70% identity, preferably at least 90% and more preferably at least 95% identity to a polynucleotide which encodes the mature polypeptide encoded by a human gene which includes the DNA of one of Figures 1-20 (SEQ ID NO:1-20) as well as fragments thereof, which fragments have at least 30 bases and preferably at least 50 bases and to polypeptides encoded by such polynucleotide ⁇ .
  • the partial ⁇ equence ⁇ are ⁇ pecific tag ⁇ for me ⁇ senger RNA molecules.
  • the complete sequence of that mes ⁇ enger RNA, in the form of cDNA can be determined u ⁇ ing the partial ⁇ equence as a probe to identify a cDNA clone corresponding to a full-length transcript, followed by sequencing of that clone.
  • the partial cDNA clone can also be used as a probe to identify a genomic clone or clones that contain the complete gene including regulatory and promoter regions, exons, and introns.
  • the partial sequences of Figures 2-20 may be used to identify the corresponding full length gene from which they were derived.
  • the partial sequences can be nick-translated or end-labelled with 3 P u ⁇ ing polynucleotide kina ⁇ e using labelling methods known to those with skill in the art (Basic Methods in Molecular Biology, L.G. Davi ⁇ , M.D. Dibner, and J.F. Battey, ed. , El ⁇ evier Press, NY, 1986).
  • a lambda library prepared from human breast tissue can be directly screened with the labelled sequences of interest or the library can be converted en masse to pBluescript (Stratagene Cloning System ⁇ , La Jolla, CA 92037) to facilitate bacterial breasty screening.
  • pBluescript Stratagene Cloning System ⁇ , La Jolla, CA 92037
  • Both method ⁇ are well known in the art. Briefly, filter ⁇ with bacterial colonies containing the library in pBluescript or bacterial lawns containing lambda plaques are denatured and the DNA is fixed to the filters. The filters are hybridized with the labelled probe using hybridization conditions described by Davis et al., supra.
  • the partial sequences, cloned into lambda or pBluescript, can be used as positive controls to asses ⁇ background binding and to adjust the hybridization and washing stringencies necessary for accurate clone identification.
  • the resulting autoradiograms are compared to duplicate plates of colonies or plaques; each exposed spot correspond ⁇ to a positive breasty or plaque.
  • the colonies or plaques are selected, expanded and the DNA i ⁇ i ⁇ olated from the colonies for further analysi ⁇ and ⁇ equencing.
  • Po ⁇ itive cDNA clones are analyzed to determine the amount of additional sequence they contain using PCR with one primer from the partial ⁇ equence and the other primer from the vector.
  • Clones with a larger vector-insert PCR product than the original partial sequence are analyzed by restriction digestion and DNA sequencing to determine whether they contain an insert of the same size or similar as the mRNA size determined from Northern blot Analysi ⁇ . Once one or more overlapping cDNA clones are identified, the complete sequence of the clones can be determined.
  • the preferred method is to use exonuclease III digestion (McCombie, W.R, Kirknes ⁇ , E., Fleming, J.T.
  • the DNA sequences (as well as the corresponding RNA sequences) also include sequences which are or contain a DNA sequence identical to one contained in and isolatable from ATCC Deposit No. 97175, deposited June 2, 1995, and fragments or portions of the isolated DNA sequences (and corresponding RNA sequence ⁇ ) , a ⁇ well a ⁇ DNA (RNA) sequences encoding the ⁇ ame polypeptide.
  • depo ⁇ it( ⁇ ) referred to herein will be maintained under the term ⁇ of the Budapest Treaty on the International Recognition of the Deposit of Micro-organisms for purposes of Patent Procedure. These depo ⁇ it ⁇ are provided merely a ⁇ convenience to tho ⁇ e of ⁇ kill in the art and are not an admi ⁇ ion that a deposit is required under 35 U.S.C. ⁇ 112.
  • the sequence of the polynucleotides contained in the deposited materials, as well as the amino acid sequence of the polypeptides encoded thereby, are incorporated herein by reference and are controlling in the event of any conflict with any description of sequence ⁇ herein.
  • a licen ⁇ e may be required to make, u ⁇ e or ⁇ ell the depo ⁇ ited material ⁇ , and no ⁇ uch licen ⁇ e i ⁇ hereby granted.
  • the present invention further relates to polynucleotides which have at least 10 base ⁇ , preferably at least 20 base ⁇ , and may have 30 or more ba ⁇ e ⁇ , which polynucleotide ⁇ are hybridizable to and have at least a 70% identity to RNA (and DNA which correspond ⁇ to ⁇ uch RNA) tran ⁇ cribed from a human gene whose coding portion includes DNA as hereinabove described.
  • the polynucleotide sequences which hybridize as described above may be u ⁇ ed to hybridize to and detect the expression of the human genes to which they correspond for use in diagnostic assays as hereinafter described.
  • diagnostic assays for detecting micrometastases of breast cancer in a host. While applicant does not wish to limit the reasoning of the present invention to any specific scientific theory, it is believed that the presence of active transcription of a breast specific gene of the present invention in cells of the host, other than those derived from the breast, is indicative of breast cancer metasta ⁇ es. This is true because, while the breast specific genes are found in all cells of the body, their transcription to mRNA, cDNA and expression products is primarily limited to the breast in non-diseased individuals.
  • breast cancer cells migrate from the cancer to other cell ⁇ , ⁇ uch that the ⁇ e other cell ⁇ are now actively tran ⁇ cribing and expre ⁇ ing a breast specific gene at a greater level than is normally found in non-disea ⁇ ed individual ⁇ , i.e., tran ⁇ cription is higher than found in non- breast tis ⁇ ue ⁇ in healthy individual ⁇ . It i ⁇ the detection of this enhanced transcription or enhanced protein expression in cells, other than tho ⁇ e derived from the brea ⁇ t, which i ⁇ indicative of meta ⁇ ta ⁇ es of breast cancer.
  • an RNA sequence in a sample derived from a tis ⁇ ue other than the breast is detected by hybridization to a probe.
  • the sample contains a nucleic acid or a mixture of nucleic acids, at least one of which is su ⁇ pected of containing a human brea ⁇ t ⁇ pecific gene or fragment thereof of the present invention which is transcribed and expressed in such tissue.
  • RNA is isolated from the cells.
  • a sample may be obtained from cells derived from tis ⁇ ue other than from the breast including but not limited to blood, urine, saliva, tissue biopsy and autopsy material.
  • the use of such methods for detecting enhanced transcription to mRNA from a human breast specific gene of the present invention or fragment thereof in a sample obtained from cell ⁇ derived from other than the breast is well within the scope of those skilled in the art from the teachings herein.
  • the isolation of mRNA comprises isolating total cellular RNA by disrupting a cell and performing differential centrifugation. Once the total RNA is isolated, mRNA is isolated by making use of the adenine nucleotide residues known to those skilled in the art as a poly(A) tail found on virtually every eukaryotic mRNA molecule at the 3' end thereof. Oligonucleotide ⁇ compo ⁇ ed of only deoxythymidine [oligo(dT)] are linked to cellulo ⁇ e and the oligo(dT) - cellulo ⁇ e packed into ⁇ mall columns.
  • the mRNA molecules bind to the oligo(dT) by the poly(A)tails while the rest of the RNA flows through the column. The bound mRNAs are then eluted from the column and collected.
  • One example of detecting isolated mRNA transcribed from a breast specific gene of the present invention comprises screening the collected mRNAs with the gene specific oligonucleotide probes, as hereinabove described.
  • probes can be and are preferably labeled with an analytically detectable reagent to facilitate identification of the probe.
  • useful reagents include but are not limited to radioactivity, fluorescent dyes or enzymes capable of catalyzing the f ⁇ mation of a detectable product.
  • An example of detecting a polynucleotide complementary to the mRNA ⁇ equence (cDNA) utilizes the polymerase chain reaction (PCR) in conjunction with reverse transcriptase. PCR is a very powerful method for the specific amplification of DNA or RNA stretches (Saiki et al . , Nature, 234:163-166 (1986)).
  • nucleic acid probe technology to bring up nucleic acid sequence ⁇ pre ⁇ ent in low copy numbers to a detectable level.
  • Numerou ⁇ diagno ⁇ tic and ⁇ cientific application ⁇ of thi ⁇ method have been described by H.A. Erlich (ed.) in PCR Technology- Principles and Applications for DNA Amplification, Stockton Pre ⁇ , USA, 1989, and by M.A. Inis (ed.) in PCR Protocols, Academic Pre ⁇ , San Diego, USA, 1990.
  • RT-PCR i ⁇ a combination of PCR with the reverse transcriptase enzyme.
  • Reverse transcriptase is an enzyme which produce ⁇ cDNA molecule ⁇ from corre ⁇ ponding mRNA molecule ⁇ .
  • Thi ⁇ i ⁇ important ⁇ ince PCR amplifie ⁇ nucleic acid molecule ⁇ , particularly DNA, and thi ⁇ DNA may be produced from the mRNA isolated from a sample derived from the ho ⁇ t.
  • a ⁇ pecific example of an RT-PCR diagno ⁇ tic assay involves removing a sample from a tis ⁇ ue of a ho ⁇ t. Such a ⁇ ample will be from a ti ⁇ ue, other than the brea ⁇ t, for example, blood. Therefore, an example of ⁇ uch a diagno ⁇ tic assay comprises whole blood gradient isolation of nucleated cells, total RNA extraction, RT-PCR of total RNA and agarose gel electrophore ⁇ is of PCR products.
  • the PCR products comprise cDNA complementary to RNA transcribed from one or more breast specific genes of the present invention or fragment ⁇ thereof.
  • a blood sample i ⁇ obtained and the whole blood i ⁇ combined with an equal volume of phosphate buffered saline, centrifuged and the lymphocyte and granulocyte layer is carefully aspirated and rediluted in phosphate buffered saline and centrifuged again. The supernate is discarded and the pellet containing nucleated cells is used for RNA extraction using the RNazole B method as described by the manufacturer (Tel-Test Inc. , Friendswood, TX) .
  • Oligonucleotide primers and probes are prepared with high specificity to the DNA sequences of the present invention.
  • the probes are at least 10 base pairs in length, preferably at least 30 base pairs in length and most preferably at least 50 base pair ⁇ in length or more.
  • the rever ⁇ e tran ⁇ criptase reaction and PCR amplification are performed sequentially without interruption. Taq polymerase is used during PCR and the PCR products are concentrated and the entire sample is run on a Tris-borate-EDTA agarose gel containing ethidium bromide.
  • a method of diagnosing a disorder of the breast for example breast cancer, by determining altered levels of the breast specific polypeptides of the present invention in a biological sample, derived from tissue other than from the breast. Elevated levels of the breast specific polypeptides of the present invention, indicates active transcription and expre ⁇ ion of the corre ⁇ ponding breast specific gene product.
  • Assay ⁇ used to detect levels of a breast specific gene polypeptide in a sample derived from a host are well-known to those skilled in the art and include radioimmunoas ⁇ ays, competitive-binding assays, Western blot analysis, ELISA as ⁇ ay ⁇ and " ⁇ andwich" assays.
  • a biological sample may include, but is not limited to, tissue extracts, cell sample ⁇ or biological fluid ⁇ , however, in accordance with the pre ⁇ ent invention, a biological ⁇ ample ⁇ pecifically doe ⁇ not include ti ⁇ ue or cells of the breast.
  • An ELISA assay (Coligan, et al., Current Protocols in Immunology. 1(2), Chapter 6, 1991) initially comprises preparing an antibody specific to a breast specific polypeptide of the present invention, preferably a monoclonal antibody.
  • a reporter antibody i ⁇ prepared against the monoclonal antibody.
  • a detectable reagent such as a ⁇ radioactivity, fluorescence or, in this example, a horseradish peroxidase enzyme.
  • a sample is removed from a host and incubated on a solid ⁇ upport, e.g., a poly ⁇ tyrene di ⁇ h, that bind ⁇ the protein ⁇ in the sample.
  • any free protein binding site ⁇ on the dish are then covered by incubating with a non-specific protein, such as BSA.
  • a non-specific protein such as BSA.
  • the monoclonal antibody is incubated in the dish during which time the monoclonal antibodie ⁇ attach to the brea ⁇ t ⁇ pecific polypeptide attached to the polystyrene dish. All unbound monoclonal antibody is washed out with buffer.
  • Peroxidase sub ⁇ trate ⁇ are then added to the di ⁇ h and the amount of color developed in a given time period i ⁇ a mea ⁇ urement of the amount of the brea ⁇ t specific polypeptide present in a given volume of patient sample when compared against a standard curve.
  • a competition assay may be employed where antibodies specific to a brea ⁇ t specific polypeptide are attached to a solid support. The breast specific polypeptide is then labeled and the labeled polypeptide a sample derived from the host are pa ⁇ ed over the solid support and the amount of label detected, for example, by liquid scintillation chromatography, can be correlated to a quantity of the breast ⁇ pecific polypeptide in the ⁇ ample.
  • a " ⁇ andwich” assay is similar to an ELISA as ⁇ ay.
  • a " ⁇ andwich” a ⁇ ay brea ⁇ t ⁇ pecific polypeptides are pas ⁇ ed over a ⁇ olid ⁇ upport and bind to antibody attached to the ⁇ olid support.
  • a second antibody is then bound to the breast specific polypeptide.
  • a third antibody which is labeled and is specific to the ⁇ econd antibody, i ⁇ then passed over the solid support and binds to the ⁇ econd antibody and an amount can then be quantified.
  • labeled antibodies to a breast specific polypeptide are used.
  • the target molecule if it is present, is immobilized and incubated with a labeled antibody.
  • the labeled antibody binds to the immobilized target molecule. After washing to remove the unbound molecules, the sample is assayed for the presence of the label.
  • immobilized target molecule is incubated with an unlabeled antibody.
  • the target molecule-labeled antibody complex if present, is then bound to a second, labeled antibody that is specific for the unlabeled antibody.
  • the sample is washed and assayed for the presence of the label.
  • Such antibodies specific to breast specific gene proteins for example, anti-idiotypic antibodies, can be used to detect breast cancer cells by being labeled and described above and binding tightly to the breast cancer cells, and, therefore, detect their presence.
  • the antibodies may also be used to target breast cancer cell ⁇ , for example, in a method of homing interaction agent ⁇ which, when contacting breast cancer cell ⁇ , destroy them. This is true ⁇ ince the antibodies are specific for breast specific genes which are primarily expressed in breast cancer, and a linking of the interaction agent to the antibody would cause the interaction agent to be carried directly to the breast.
  • Antibodies of thi ⁇ type may al ⁇ o be u ⁇ ed to do in vivo imaging, for example, by labeling the antibodies to facilitate scanning of the breast.
  • One method for imaging comprises contacting any cancer cells of the brea ⁇ t to be imaged with an anti-brea ⁇ t ⁇ pecific gene protein antibody labeled with a detectable marker. The method i ⁇ performed under conditions such that the labeled antibody binds to the breast s ecific gene proteins.
  • the antibodies interact with the breast, for example, breast cancer cells, and fluoresce upon such contact such that imaging and visibility of the breast is enhanced to allow a determination of the diseased or non-disea ⁇ ed ⁇ tate of the breast.
  • labeled antibodies will vary depending upon the application. However, the choice of marker is readily determinable to one skilled in the art.
  • These labeled antibodies may be used in immunoassays as well as in histological applications to detect the presence of the proteins.
  • the labeled antibodies may be polyclonal or monoclonal.
  • thi ⁇ phenomenon may have important clinical implications since the method of treating a localized, as opposed to a metastasized, tumor is entirely different.
  • breast specific gene 1 i ⁇ a full-length gene.
  • Brea ⁇ t ⁇ pecific gene 1 i ⁇ 79% identical and 83% similar to human Alzheimer disea ⁇ e amyloid gene.
  • Breast specific gene 2 is 30% identical and 48% similar to human hydroxyindole-o-methyltransfera ⁇ e gene.
  • Brea ⁇ t specific gene 3 i ⁇ 58% identical and 62% ⁇ imilar to human 06-methylguanine-DNA methyltransferase gene.
  • Breast specific gene 4 is 34% identical and 65% similar to the mouse pl20 gene.
  • Breast specific gene 5 is 78% identical and 89% similar to human p70 ribosomal S6 kinase alpha-II gene.
  • Breast specific gene 6 is 77% identical and 79% similar to the human transcription factor NFATp gene.
  • the brea ⁇ t ⁇ pecific gene ⁇ of the present invention are putative molecular markers in the diagnosis of breast cancer formation, and breast cancer metastases.
  • Table 1 the presence of the breast specific genes when tested in normal breast, breast cancer, embryo and other cancer libraries, the breast specific gene ⁇ of the present invention were found to be most prevalent in the breast cancer library, indicating that the genes of the present invention may be employed for detecting breast cancer, as discussed previously.
  • the table also indicates a putative identification, based on homology, of BSGl through BSG6 to known genes.
  • the as ⁇ ay ⁇ described above may also be used to test whether bone marrow preserved before chemotherapy i ⁇ contaminated with micrometasta ⁇ e ⁇ of a breast cancer cell.
  • blood cells from the bone marrow are isolated and treated as described above, this method allows one to determine whether preserved bone marrow is still suitable for tran ⁇ plantation after chemotherapy.
  • the pre ⁇ ent invention further relates to mature polypeptides, for example the BSGl polypeptide, as well a ⁇ fragment ⁇ , analog ⁇ and derivatives of such polypeptide.
  • fragment when referring to the polypeptides encoded by the genes of the invention means a polypeptide which retains essentially the ⁇ ame biological function or activity as such polypeptide.
  • an analog includes a proprotein which can be activated by cleavage of the proprotein portion to produce an active mature polypeptide.
  • polypeptides of the present invention may be recombinant polypeptides, natural polypeptides or ⁇ ynthetic polypeptides, preferably recombinant polypeptides.
  • the fragment, derivative or analog of the polypeptides encoded by the genes of the invention may be (i) one in which one or more of the amino acid residue ⁇ are ⁇ ub ⁇ tituted with a con ⁇ erved or non-con ⁇ erved amino acid re ⁇ idue (preferably a con ⁇ erved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) one in which one or more of the amino acid re ⁇ idue ⁇ includes a substituent group, or (iii) one in which the polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol) , or (iv) one in which the additional amino acids are fused to the polypeptide, such as a leader or secretory sequence or a sequence which is employed for purification of the mature polypeptide or a proprotein sequence.
  • Such fragments, derivative ⁇ and analog ⁇ are deemed to be within the scope of
  • polypeptides and polynucleotides of the present invention are preferably provided in an isolated form, and preferably are purified to homogeneity.
  • isolated means that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring) .
  • a naturally- occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide, separated from ⁇ ome or all of the coexi ⁇ ting materials in the natural sy ⁇ tem, i ⁇ i ⁇ olated.
  • Such polynucleotides could be part of a vector and/or such polynucleotides or polypeptides could be part of a composition, and still be isolated in that such vector or composition is not part of its natural environment.
  • polypeptides of the present invention include the polypeptides of Figure 1 (SEQ ID NO:l) (in particular the mature polypeptides) as well as polypeptides which have at least 70% similarity (preferably at lea ⁇ t a 70% identity) to the polypeptides of Figure 1 (SEQ ID NO:l) and more preferably at least a 90% similarity (more preferably at least a 90% identity) to the polypeptides of Figures 8 and 9 (SEQ ID NO:8 and 9) and still more preferably at least a 95% similarity (still more preferably at least 95% identity) to the polypeptides of Figure 1 (SEQ ID N0:1) and al ⁇ o include portion ⁇ of ⁇ uch polypeptides with such portion of the polypeptide generally containing at least 30 amino acids and more preferably at least 50 amino acids.
  • similarity between two polypeptides is determined by comparing the amino acid sequence and it ⁇ con ⁇ erved amino acid substitutes of one polypeptide to the sequence of a second polypeptide.
  • Fragments or portions of the polypeptides of the present invention may be employed for producing the corresponding full-length polypeptide by peptide synthesis; therefore, the fragments may be employed as intermediates for producing the full-length polypeptide ⁇ . Fragments or portions of the polynucleotides of the present invention may be used to synthesize full-length polynucleotide ⁇ of the pre ⁇ ent invention.
  • the present invention also relates to vectors which include polynucleotides of the present invention, host cells which are genetically engineered with vectors of the invention and the production of polypeptides of the invention by recombinant techniques.
  • Host cells are genetically engineered (transduced or transformed or transfected) with the vectors of this invention which may be, for example, a cloning vector or an expression vector.
  • the vector may be, for example, in the form of a plasmid, a viral particle, a phage, etc.
  • the engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, ⁇ electing tran ⁇ formant ⁇ or amplifying the breast specific genes.
  • the culture conditions such as temperature, pH and the like, are those previously used with the host cell selected for expres ⁇ ion, and will be apparent to those of ordinarily skill in the art.
  • the polynucleotides of the present invention may be employed for producing polypeptides by recombinant technique ⁇ .
  • the polynucleotide may be included in any one of a variety of expression vectors for expressing a polypeptide.
  • Such vectors include chromosomal, nonchromosomal and synthetic DNA sequence ⁇ , e.g., derivative ⁇ of SV40; bacterial pla ⁇ mid ⁇ ; phage DNA; baculoviru ⁇ ; yea ⁇ t pla ⁇ mid ⁇ ; vectors derived from combinations of plasmids and phage DNA, viral DNA such a ⁇ vaccinia, adenovirus, fowl pox viru ⁇ , and pseudorabies.
  • any other vector may be used as long as it is replicable and viable in the host.
  • the appropriate DNA sequence may be inserted into the vector by a variety of procedures.
  • the DNA sequence is inserted into an appropriate restriction endonuclease ⁇ ite( ⁇ ) by procedures known in the art. Such procedures and others are deemed to be within the scope of tho ⁇ e ⁇ killed in the art.
  • the DNA ⁇ equence in the expre ⁇ ion vector i ⁇ operatively linked to an appropriate expre ⁇ sion control sequence( ⁇ ) (promoter) to direct mRNA ⁇ ynthe ⁇ i ⁇ .
  • an appropriate expre ⁇ sion control sequence( ⁇ ) promoter
  • a ⁇ repre ⁇ entative examples of such promoters there may be mentioned: LTR or SV40 promoter, the E. coli. lac or trp. the phage lambda P L promoter and other promoters known to control expres ⁇ ion of gene ⁇ in prokaryotic or eukaryotic cell ⁇ or their viru ⁇ e ⁇ .
  • the expression vector also contains a ribosome binding ⁇ ite for tran ⁇ lation initiation and a tran ⁇ cription terminator.
  • the vector may al ⁇ o include appropriate ⁇ equences for amplifying expres ⁇ ion.
  • the expression vectors preferably contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or such as tetracycline or ampicillin resistance in E. coli.
  • the vector containing the appropriate DNA sequence as hereinabove described, as well as an appropriate promoter or control sequence, may be employed to transform an appropriate host to permit the host to express the protein.
  • appropriate hosts there may be mentioned: bacterial cells, such as E. coli. Streptomyces, Salmonella typhimurium; fungal cells, such as yeast; in ⁇ ect cell ⁇ such as Drosophila S2 and Spodoptera Sf9; animal cell ⁇ ⁇ uch a ⁇ CHO, COS or Bowes melanoma; adenoviru ⁇ e ⁇ ; plant cell ⁇ , etc.
  • the ⁇ election of an appropriate ho ⁇ t is deemed to be within the scope of those skilled in the art from the teachings herein.
  • the present invention also includes recombinant constructs comprising one or more of the sequences as broadly described above.
  • the constructs comprise a vector, such as a plasmid or viral vector, into which a ⁇ equence of the invention ha ⁇ been inserted, in a forward or reverse orientation.
  • the construct further comprises regulatory sequence ⁇ , including, for example, a promoter, operably linked to the sequence.
  • regulatory sequence ⁇ including, for example, a promoter, operably linked to the sequence.
  • Bacterial pQE70, pQE60, pQE-9 (Qiagen) , pBS, pDIO, phagescript, psiXl74, pblue ⁇ cript SK, pBSKS, pNH8A, pNH16a, pNH18A, pNH46A (Stratagene) ; ptrc99a, pKK223- 3, pKK233-3, pDR540, pRIT5 (Pharmacia).
  • Eukaryotic pWLNEO, PSV2CAT, pOG44, pXTl, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia) .
  • any other plasmid or vector may be used as long as they are replicable and viable in the host.
  • Promoter regions can be ⁇ elected from any desired gene using CAT (chloramphenicol transferase) vectors or other vectors with selectable markers.
  • Two appropriate vector ⁇ are pKK232-8 and pCM7.
  • Particular named bacterial promoter ⁇ include lacl, lacZ, T3, T7, gpt, lambda P R , P L and trp.
  • Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retroviru ⁇ , and mou ⁇ e metallothionein-I. Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art.
  • the present invention relates to host cells containing the above-described constructs.
  • the host cell can be a higher eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell.
  • Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE- Dextran mediated tran ⁇ fection, or eiectroporation (Davis, L. , Dibner, M. , Battey, I., Basic Methods in Molecular Biology, (1986)) .
  • the constructs in host cell ⁇ can be u ⁇ ed in a conventional manner to produce the gene product encoded by the recombinant sequence.
  • the polypeptides of the invention can be synthetically produced by conventional peptide synthe ⁇ izer ⁇ .
  • Protein ⁇ can be expre ⁇ ed in mammalian cells, yeast, bacteria, or other cells under the control of appropriate promoters.
  • Cell-free translation sy ⁇ tems can also be employed to produce ⁇ uch proteins using RNAs derived from the DNA constructs of the present invention.
  • Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described by Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989), the disclosure of which is hereby incorporated by reference.
  • Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp that act on a promoter to increase its transcription. Examples including the SV40 enhancer on the lat ⁇ e side of the replication origin bp 100 to 270, a cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.
  • recombinant expression vectors will include origins of replication and selectable markers permitting transformation of the host cell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiae TRPl gene, and a promoter derived from a highly-expressed gene to direct transcription of a downstream structural sequence.
  • promoters can be derived from operons encoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK) , ⁇ -factor, acid phosphata ⁇ e, or heat shock proteins, among others.
  • the heterologous structural sequence is as ⁇ embled in appropriate pha ⁇ e with translation initiation and termination sequences.
  • the heterologous sequence can encode a fusion protein including an N-terminal identification peptide imparting desired characteristics, e.g., stabilization or simplified purification of expres ⁇ ed recombinant product.
  • U ⁇ eful expression vectors for bacterial use are constructed by inserting a structural DNA sequence encoding a desired protein together with suitable translation initiation and termination signal ⁇ in operable reading frame with a functional promoter.
  • the vector will comprise one or more phenotypic selectable marker ⁇ and an origin of replication to en ⁇ ure maintenance of the vector and to, if desirable, provide amplification within the host.
  • Suitable prokaryotic hosts for transformation include E. coli. Bacillus subtilis. Salmonella typhimurium and various specie ⁇ within the genera Pseudomonas, Streptomyces, and Staphylococcus, although others may also be employed as a matter of choice.
  • u ⁇ eful expression vectors for bacterial use can comprise a selectable marker and bacterial origin of replication derived from commercially available plasmids comprising genetic elements of the well known cloning vector pBR322 (ATCC 37017) .
  • cloning vector pBR322 ATCC 37017
  • Such commercial vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEMl (Promega Biotec, Madison, WI, USA) .
  • pBR322 "backbone" section ⁇ are combined with an appropriate promoter and the structural sequence to be expressed.
  • the selected promoter is induced by appropriate means (e.g., temperature shift or chemical induction) and cells are cultured for an additional period.
  • Cell ⁇ are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.
  • Microbial cells employed in expres ⁇ ion of protein ⁇ can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents, such methods are well know to tho ⁇ e ⁇ killed in the art.
  • Variou ⁇ mammalian cell culture ⁇ y ⁇ tem ⁇ can also be employed to expres ⁇ recombinant protein.
  • Example ⁇ of mammalian expre ⁇ ion system ⁇ include the COS-7 line ⁇ of monkey kidney fibroblasts, described by Gluzman, Cell, 23:175 (1981) , and other cell lines capable of expressing a compatible vector, for example, the C127, 3T3, CHO, HeLa and BHK cell lines.
  • Mammalian expres ⁇ ion vector ⁇ will comprise an origin of replication, a suitable promoter and enhancer, and also any neces ⁇ ary ribosome binding ⁇ ite ⁇ , polyadenylation ⁇ ite, ⁇ plice donor and acceptor ⁇ ites, transcriptional termination sequence ⁇ , and 5' flanking nontranscribed sequences.
  • DNA sequence ⁇ derived from the SV40 splice, and polyadenylation site ⁇ may be u ⁇ ed to provide the required nontran ⁇ cribed genetic element ⁇ .
  • the brea ⁇ t ⁇ pecific gene polypeptide ⁇ can be recovered and purified from recombinant cell culture ⁇ by method ⁇ including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Protein refolding steps can be used, as necessary, in completing configuration of the mature protein. Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps.
  • HPLC high performance liquid chromatography
  • the polynucleotides of the present invention may have the coding sequence fused in frame to a marker sequence which allows for purification of the polypeptide of the present invention.
  • a marker sequence is a hexahistidine tag which may be supplied by a vector, preferably a pQE-9 vector, which provides for purification of the polypeptide fused to the marker in the case of a bacterial host, or, for example, the marker sequence may be a hemagglutinin (HA) tag when a mammalian host, e.g. COS-7 cells, i ⁇ used.
  • the HA tag correspond ⁇ to an epitope derived from the influenza hemagglutinin protein (Wilson, I., et al., Cell, 37:767 (1984)) .
  • polypeptides of the present invention may be a naturally purified product, or a product of chemical synthetic procedures, or produced by recombinant techniques from a prokaryotic or eukaryotic host (for example, by bacterial, yeast, higher plant, insect and mammalian cells in culture) .
  • a prokaryotic or eukaryotic host for example, by bacterial, yeast, higher plant, insect and mammalian cells in culture
  • the polypeptides of the present invention may be glycosylated or may be non-glycosylated.
  • Polypeptides of the invention may also include an initial methionine amino acid residue.
  • BSGl, and other breast specific genes, and the protein product thereof may be employed for early detection of breast cancer ⁇ ince they are over-expres ⁇ ed in the breast cancer state.
  • ⁇ ay ⁇ which may be u ⁇ ed to screen for therapeutics to inhibit the action of the breast specific genes or breast ⁇ pecific protein ⁇ of the pre ⁇ ent invention.
  • the present invention discloses methods for selecting a therapeutic which forms a complex with breast specific gene proteins with sufficient affinity to prevent their biological action.
  • the method ⁇ include various assays, including competitive assays where the proteins are immobilized to a support, and are contacted with a natural substrate and a labeled therapeutic either ⁇ imultaneously or in either consecutive order, and determining whether the therapeutic effectively competes with the natural substrate in a manner sufficient to prevent binding of the protein to its sub ⁇ trate.
  • the substrate is immobilized to a support, and is contacted with both a labeled breast specific polypeptide and a therapeutic (or unlabeled proteins and a labeled therapeutic) , and it is determined whether the amount of the breast specific polypeptide bound to the substrate is reduced in comparison to the as ⁇ ay without the therapeutic added.
  • the brea ⁇ t specific polypeptide may be labeled with antibodies.
  • Potential therapeutic compounds include antibodies and anti-idiotypic antibodie ⁇ a ⁇ de ⁇ cribed above, or in ⁇ ome cases, an oligonucleotide, which binds to the polypeptide.
  • Anti ⁇ en ⁇ e technology can be u ⁇ ed to control gene expre ⁇ ion through triple-helix formation or antisense DNA or RNA, both of which methods are based on binding of a polynucleotide to DNA or RNA.
  • a DNA oligonucleotide is designed to be complementary to a region of the gene involved in transcription (triple helix -see Lee et al., Nucl. Acids Res., 6:3073 (1979); Cooney et al, Science, 241:456 (1988); and Dervan et al., Science, 251: 1360 (1991)), thereby preventing transcription and the production of a breast specific polynucleotide.
  • the antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks tran ⁇ lation of the mRNA molecule into the breast specific genes polypeptide (antisen ⁇ e - Okano, J.
  • oligonucleotides described above can also be delivered to cells ⁇ uch that the antisense RNA or DNA may be expres ⁇ ed in vivo to inhibit production of the breast specific polypeptides.
  • Another example is a ⁇ mall molecule which bind ⁇ to and occupies the active site of the brea ⁇ t ⁇ pecific polypeptide thereby making the active ⁇ ite inacce ⁇ ible to ⁇ ub ⁇ trate ⁇ uch that normal biological activity is prevented.
  • small molecules include but are not limited to ⁇ mall peptide ⁇ or peptide-like molecules.
  • These compounds may be employed to treat breast cancer, since they interact with the function of breast specific polypeptides in a manner sufficient to inhibit natural function which i ⁇ nece ⁇ ary for the viability of breast cancer cells. Thi ⁇ i ⁇ true since the BSGs and their protein product ⁇ are primarily expres ⁇ ed in brea ⁇ t cancer ti ⁇ ue ⁇ and are, therefore, ⁇ u ⁇ pected of being critical to the formation of this state.
  • the compounds may be employed in a composition with a pharmaceutically acceptable carrier, e.g., as hereinafter described.
  • a pharmaceutically acceptable carrier e.g., as hereinafter described.
  • the compounds of the present invention may be employed in combination with a ⁇ uitable pharmaceutical carrier.
  • Such compo ⁇ itions compri ⁇ e a therapeutically effective amount of the polypeptide, and a pharmaceutically acceptable carrier or excipient.
  • a carrier includes but is not limited to saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the formulation should suit the mode of administration.
  • the invention also provides a pharmaceutical pack or kit compri ⁇ ing one or more containers filled with one or more of the ingredients of the pharmaceutical composition ⁇ of the invention.
  • a pharmaceutical pack or kit compri ⁇ ing one or more containers filled with one or more of the ingredients of the pharmaceutical composition ⁇ of the invention.
  • Associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
  • the pharmaceutical compositions may be employed in conjunction with other therapeutic compounds.
  • the pharmaceutical compositions may be administered in a convenient manner such as by the oral, topical, intravenous, intraperitoneal, intramuscular, subcutaneou ⁇ , intrana ⁇ al, intra-anal or intradermal routes.
  • the pharmaceutical compositions are administered in an amount which is effective for treating and/or prophylaxis of the specific indication. In general, they are administered in an amount of at least about 10 ⁇ g/kg body weight and in most ca ⁇ es they will be administered in an amount not in exces ⁇ of about 8 mg/Kg body weight per day. In mo ⁇ t ca ⁇ e ⁇ , the do ⁇ age i ⁇ from about 10 ⁇ g/kg to about l mg/kg body weight daily, taking into account the route ⁇ of admini ⁇ tration, ⁇ ymptom ⁇ , etc.
  • brea ⁇ t specific genes and compounds which are polypeptides may also be employed in accordance with the present invention by expre ⁇ ion of ⁇ uch polypeptide ⁇ in vivo, which i ⁇ often referred to a ⁇ "gene therapy.”
  • cells from a patient may be engineered with a polynucleotide (DNA or RNA) encoding a polypeptide ex vivo, with the engineered cells then being provided to a patient to be treated with the polypeptide.
  • DNA or RNA polynucleotide
  • cells may be engineered by procedures known in the art by use of a retroviral particle containing RNA encoding a polypeptide of the present invention.
  • cells may be engineered in vivo for expression of a polypeptide in vivo by, for example, procedures known in the art.
  • a producer cell for producing a retroviral particle containing RNA encoding a polypeptide of the present invention may be administered to a patient for engineering cells in vivo and expression of the polypeptide in vivo.
  • the expression vehicle for engineering cells may be other than a retrovirus, for example, an adenovirus which may be used to engineer cells in vivo after combination with a suitable delivery vehicle.
  • Retroviruses from which the retroviral plasmid vectors hereinabove mentioned may be derived include, but are not limited to, Moloney Murine Leukemia Virus, spleen necrosi ⁇ viru ⁇ , retroviru ⁇ es such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis viru ⁇ , gibbon ape leukemia virus, human immunodeficiency virus, adenovirus, Myeloproliferative Sarcoma Virus, and mammary tumor virus.
  • the retroviral plasmid vector is derived from Moloney Murine Leukemia Virus.
  • the vector includes one or more promoter ⁇ .
  • Suitable promoters which may be employed include, but are not limited to, the retroviral LTR; the SV40 promoter; and the human cytomegalovirus (CMV) promoter described in Miller, et al., Biotechniques. Vol. 7, No. 9, 980-990 (1989) , or any other promoter (e.g., cellular promoters such as eukaryotic cellular promoters including, but not limited to, the histone, pol III, and ⁇ -actin promoter ⁇ ) .
  • CMV cytomegalovirus
  • viral promoter ⁇ which may be employed include, but are not limited to, adenovirus promoters, thymidine kinase (TK) promoters, and B19 parvovirus promoters. The selection of a suitable promoter will be apparent to those skilled in the art from the teachings contained herein.
  • Suitable promoters which may be employed include, but are not limited to, adenoviral promoters, such as the adenoviral major late promoter; or heterologous promoters, such as the cytomegalovirus (CMV) promoter; the respiratory syncytial virus (RSV) promoter; inducible promoters, such as the MMT promoter, the metallothionein promoter; heat shock promoters; the albumin promoter; the ApoAI promoter; human globin promoters,- viral thymidine kinase promoters, such as the Herpes Simplex thymidine kinase promoter; retroviral LTRs (including the modified retroviral LTRs hereinabove de ⁇ cribed) ; the ⁇ -actin promoter; and human growth hormone promoter ⁇ .
  • the promoter al ⁇ o may be the native promoter which control ⁇ the
  • the retroviral pla ⁇ mid vector is employed to transduce packaging cell lines to form producer cell lines.
  • packaging cells which may be transfected include, but are not limited to, the PE501, PA317, ⁇ -2 , ⁇ -AM, PA12, T19-14X, VT-19-17-H2, ⁇ CRE, ⁇ CRIP, GP+E-86, GP+envAml2, and DAN cell lines as described in Miller, Human Gene Therapy. Vol. 1, pgs. 5-14 (1990), which is incorporated herein by reference in its entirety.
  • the vector may transduce the packaging cells through any means known in the art. Such means include, but are not limited to, eiectroporation, the use of liposomes, and CaP0 4 precipitation.
  • the retroviral plasmid vector may be encapsulated into a liposome, or coupled to a lipid, and then administered to a ho ⁇ t.
  • the producer cell line generate ⁇ infectiou ⁇ retroviral vector particle ⁇ which include the nucleic acid sequence(s) encoding the polypeptides.
  • retroviral vector particles then may be employed, to transduce eukaryotic cells, either in vitro or in vivo.
  • the transduced eukaryotic cells will express the nucleic acid sequence(s) encoding the polypeptide.
  • Eukaryotic cells which may be transduced include, but are not limited to, embryonic ⁇ tem cells, embryonic carcinoma cells, as well as hematopoietic stem cells, hepatocytes, fibroblasts, myoblast ⁇ , keratinocytes, endothelial cells, and bronchial epithelial cells.
  • This invention is also related to the use of a breast specific genes of the present invention as a diagnostic. For example, some disease ⁇ re ⁇ ult from inherited defective genes.
  • the breast specific gene ⁇ , CSG7 and CSG10 for example, have been found to have a reduced expre ⁇ ion in brea ⁇ t cancer cells as compared to that in normal cells. Further, the remaining breast specific genes of the present invention are overexpressed in breast cancer. Accordingly, a mutation in these genes allows a detection of breast di ⁇ orders, for example, breast cancer.
  • a mutation in a breast specific gene of the present invention at the DNA level may be detected by a variety of techniques.
  • Nucleic acids used for diagnosis may be obtained from a patient's cells, other than from the breast, such as from blood, urine, saliva, tis ⁇ ue biop ⁇ y and autop ⁇ y material.
  • the genomic DNA may be u ⁇ ed directly for detection or may be amplified enzymatically by u ⁇ ing PCR (Saiki, et al., Nature, 324:163- 166 (1986)) prior to analy ⁇ i ⁇ .
  • RNA or cDNA may al ⁇ o be u ⁇ ed for the ⁇ ame purpose.
  • PCR primers complementary to the nucleic acid of the instant invention can be used to identify and analyze mutations in a breast specific polynucleotide of the present invention. For example, deletions and insertions can be detected by a change in size of the amplified product in comparison to the normal genotype. Point mutations can be identified by hybridizing amplified DNA to radiolabelled breast specific RNA or, alternatively, radiolabelled antisense DNA sequences.
  • SSCP single-strand conformation polymorphism
  • Sequence difference ⁇ between the reference gene and "mutant ⁇ ” may be revealed by the direct DNA sequencing method.
  • cloned DNA ⁇ egment ⁇ may be used as probes to detect specific DNA ⁇ egment ⁇ .
  • the sensitivity of this method is greatly enhanced when combined with PCR.
  • a sequencing primer is used with double-stranded PCR product or a single-stranded template molecule generated by a modified PCR.
  • the sequence determination is performed by conventional procedures with -radiolabeled nucleotides or by automatic sequencing procedures with fluorescent-tag ⁇ .
  • DNA sequence differences may be achieved by detection of alteration in electrophoretic mobility of DNA fragments and gels with or without denaturing agents. Small sequence deletions and insertions can be visualized by high-resolution gel electrophoresis. DNA fragment ⁇ of different sequences may be di ⁇ tingui ⁇ hed on denaturing formamide gradient gel ⁇ in which the mobilities of different DNA fragments are retarded in the gel at different position ⁇ according to their specific melting or partial melting temperatures (see, e.g., Myers, et al., Science. 230:1242 (1985)). In addition, sequence alterations, in particular small deletions, may be detected as changes in the migration pattern of DNA.
  • Sequence changes at specific locations may also be revealed by nuclease protection as ⁇ ay ⁇ , such as Rnase and Si protection or the chemical cleavage method (e.g., Cotton, et al., PNAS. USA. 85:4397-4401 (1985)).
  • the detection of the specific DNA sequence may be achieved by methods such as hybridization, RNase protection, chemical cleavage, direct DNA sequencing, or the use of restriction enzymes (e.g., Restriction Fragment Length Polymorphisms (RFLP)) and Southern blotting.
  • restriction enzymes e.g., Restriction Fragment Length Polymorphisms (RFLP)
  • the sequence ⁇ of the present invention are also valuable for chromosome identification.
  • the ⁇ equence i ⁇ specifically targeted to and can hybridize with a particular location on an individual human chromosome.
  • Few chromosome marking reagents based on actual sequence data (repeat polymorphisms) are presently available for marking chromosomal location.
  • the mapping of DNA ⁇ to chromo ⁇ ome ⁇ according to the pre ⁇ ent invention i ⁇ an important fir ⁇ t ⁇ tep in correlating tho ⁇ e ⁇ equence ⁇ with gene ⁇ a ⁇ ociated with di ⁇ ease.
  • sequences can be mapped to chromosome ⁇ by preparing PCR primer ⁇ (preferably 15-25 bp) from the cDNA. Computer analysi ⁇ of the 3' untranslated region is used to rapidly select primers that do not span more than one exon in the genomic DNA, thus complicating the amplification process. These primers are then used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the primer will yield an amplified fragment.
  • mapping of ⁇ omatic cell hybrid ⁇ i ⁇ a rapid procedure for a ⁇ signing a particular DNA to a particular chromosome.
  • sublocalization can be achieved with panels of fragments from specific chromosome ⁇ or pools of large genomic clones in an analogous manner.
  • Other mapping strategies that can similarly be used to map to it ⁇ chromosome include in si tu hybridization, prescreening with labeled flow-sorted chromosome ⁇ and pre ⁇ election by hybridization to con ⁇ truct chromosome specific-cDNA libraries.
  • Fluorescence in situ hybridization of a cDNA clone to a metaphase chromosomal spread can be used to provide a precise chromo ⁇ omal location in one step.
  • This technique can be used with cDNA as short as 50 or 60 bases.
  • Verma et al. Human Chromosome ⁇ : a Manual of Ba ⁇ ic Technique ⁇ , Pergamon Press, New York (1988) .
  • a cDNA precisely localized to a chromosomal region associated with the disease, ⁇ ould be one of between 50 and 500 potential causative genes. (Thi ⁇ as ⁇ ume ⁇ 1 megabase mapping resolution and one gene per 20 kb) .
  • polypeptides, their fragments or other derivatives, or analogs thereof, or cells expres ⁇ ing them can be u ⁇ ed as an immunogen to produce antibodies thereto.
  • These antibodies can be, for example, polyclonal or monoclonal antibodies.
  • the present invention also includes chimeric, single chain, and humanized antibodies, as well as Fab fragments, or the product of an Fab expres ⁇ ion library. Variou ⁇ procedure ⁇ known in the art may be used for the production of such antibodies and fragments.
  • Antibodies generated against the polypeptides corre ⁇ ponding to a ⁇ equence of the pre ⁇ ent invention can be obtained by direct injection of the polypeptides into an animal or by administering the polypeptides to an animal, preferably a nonhuman. The antibody so obtained will then bind the polypeptides itself. In this manner, even a ⁇ equence encoding only a fragment of the polypeptide ⁇ can be used to generate antibodies binding the whole native polypeptide ⁇ . Such antibodies can then be used to isolate the polypeptide from tissue expressing that polypeptide.
  • any technique which provide ⁇ antibodie ⁇ produced by continuou ⁇ cell line culture ⁇ can be used.
  • Examples include the hybridoma technique (Kohler and Milstein, 1975, Nature, 256:495-497), the trioma technique, the human B-cell hybridoma technique (Kozbor et al., 1983, Immunology Today 4:72), and the EBV- hybridoma technique to produce human monoclonal antibodies (Cole, et al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).
  • Antibodies of this type may also be used to do in vivo imaging, for example, by labeling the antibodies to facilitate scanning of the pelvic area and the breast.
  • One method for imaging compri ⁇ e ⁇ contacting any cancer cells of the breast to be imaged with an anti-breast specific protein- antibody labeled with a detectable marker. The method is performed under condition ⁇ such that the labeled antibody binds to the breast specific polypeptides.
  • the antibodies interact with the brea ⁇ t, for example, breast cancer cell ⁇ , and fluore ⁇ ce upon contact such that imaging and visibility of the breast are enhanced to allow a determination of the diseased or non-disea ⁇ ed ⁇ tate of the brea ⁇ t.
  • Plasmids are designated by a lower case p preceded and/or followed by capital letters and/or numbers.
  • the starting plasmid ⁇ herein are either commercially available, publicly available on an unre ⁇ tricted ba ⁇ is, or can be constructed from available plasmid ⁇ in accord with published procedures.
  • equivalent plasmid ⁇ to those described are known in the art and will be apparent to the ordinarily ⁇ killed artisan.
  • “Digestion” of DNA refers to catalytic cleavage of the DNA with a re ⁇ triction enzyme that acts only at certain sequences in the DNA.
  • the various restriction enzymes used herein are commercially available and their reaction conditions, cofactors and other requirements were used as would be known to the ordinarily skilled artisan.
  • For analytical purposes typically 1 ⁇ g of plasmid or DNA fragment is used with about 2 units of enzyme in about 20 ⁇ l of buffer solution.
  • For the purpose of isolating DNA fragments for plasmid construction typically 5 to 50 ⁇ g of DNA are digested with 20 to 250 units of enzyme in a larger volume. Appropriate buffers and sub ⁇ trate amount ⁇ for particular restriction enzymes are specified by the manufacturer. Incubation times of about 1 hour at 37"C are ordinarily used, but may vary in accordance with the supplier's instructions. After digestion the reaction is electrophoresed directly on a polyacrylamide gel to isolate the desired fragment.
  • Oligonucleotides refers to either a single stranded polydeoxynucleotide or two complementary polydeoxynucleotide strands which may be chemically synthe ⁇ ized. Such synthetic oligonucleotides have no 5' phosphate and thus will not ligate to another oligonucleotide without adding a phosphate with an ATP in the presence of a kinase. A synthetic oligonucleotide will ligate to a fragment that has not been dephosphorylated.
  • Ligase refers to the proces ⁇ of forming phosphodiester bonds between two double stranded nucleic acid fragment ⁇ (Maniatis, T., et al., Id., p. 146). Unless otherwise provided, ligation may be accomplished using known buffers and condition ⁇ with 10 unit ⁇ of T4 DNA ligase ("ligase”) per 0.5 ⁇ g of approximately equimolar amount ⁇ of the DNA fragment ⁇ to be ligated.
  • ligase T4 DNA ligase
  • the lymphocyte and granulocyte layer (approximately 5 ml) is carefully aspirated and rediluted up to 50 ml with phosphate-buffered saline in a 50- ml tube, which is centrifuged again at 1800 X g for 20 min. at 5°C. The ⁇ upernatant is discarded and the pellet containing nucleated cells is used for RNA extraction using the RNazole B method a ⁇ de ⁇ cribed by the manufacturer (Tel- Te ⁇ t Inc., Friend ⁇ wood, TX) .
  • a probe is designed with an identity to at least a portion of the mRNA sequence transcribed from a human gene whose coding portion includes a DNA sequence of Figures 1-20 (SEQ ID NO:1-20) .
  • This probe is mixed with the extracted RNA and the mixed DNA and RNA are precipitated with ethanol -70°C for 15 minutes) .
  • the pellet is resuspended in hybridization buffer and di ⁇ solved.
  • the tubes containing the mixture are incubated in a 72°C water bath for 10-15 mins. to denature the DNA.
  • the tubes are rapidly transferred to a water bath at the desired hybridization temperature. Hybridization temperature depend ⁇ on the G + C content of the DNA.
  • Hybridization i ⁇ done for 3 hr ⁇ . 0.3 ml of nuclea ⁇ e-Sl buffer i ⁇ added and mixed well. 50 ⁇ l of 4.0 M ammonium acetate and 0.1 M EDTA i ⁇ added to stop the reaction. The mixture i ⁇ extracted with phenol/chloroform and 20 ⁇ g of carrier tRNA is added and precipitation is done with an equal volume of isopropanol. The precipitate is dissolved in 40 ⁇ l of TE (pH 7.4) and run on an alkaline agarose gel. Following electrophoresis, the RNA is microsequenced to confirm the nucleotide sequence. (See Favaloro, J. et al., Methods Enzymol., 65:718 (1980) for a more detailed review).
  • Two oligonucleotide primers are employed to amplify the sequence isolated by the above methods.
  • the 5' primer is 20 nucleotides long and the 3' primer is a complimentary ⁇ equence for the 3' end of the i ⁇ olated mRNA.
  • the primer ⁇ are cu ⁇ tom de ⁇ igned according to the i ⁇ olated mRNA.
  • the rever ⁇ e tran ⁇ cripta ⁇ e reaction and PCR amplification are performed ⁇ equentially without interruption in a Perkin Elmer 9600 PCR machine (Emeryville, CA) .
  • Four hundred ng total RNA in 20 ⁇ l diethylpyrocarbonate-treated water are placed in a 65°C water bath for 5 min.
  • PCR conditions are as follows: cycle 1 i ⁇ 42°C for 15 min then 97°C for 15 ⁇ (1 cycle) ; cycle 2 i ⁇ 95°C for 1 min. 60°C for 1 min, and 72°C for 30 ⁇ (15 cycles) ; cycle 3 is 95°C for 1 min. 60°C for 1 min., and 72°C for 1 min. (10 cycles); cycle 4 is 95°C for 1 min., 60°C for 1 min., and 72°C for 2 min. (8 cycles) ; cycle 5 is 72°C for 15 min. (1 cycle) ,- and the final cycle is a 4°C hold until sample is taken out of the machine.
  • RNA in the pellet may be quantified in numerous way ⁇ , for example, it may be weighed.
  • Verification of the nucleotide ⁇ equence of the PCR products is done by microsequencing.
  • the PCR product is purified with a Qiagen PCR Product Purification Kit (Qiagen, Chatsworth, CA) as described by the manufacturer.
  • Qiagen Quality of Service
  • One ⁇ g of the PCR product undergoes PCR sequencing by using the Taq DyeDeoxy Terminator Cycle sequencing kit in a Perkin-Elmer 9600 PCR machine as described by Applied Biosy ⁇ tem ⁇ (Fo ⁇ ter, CA) .
  • the sequenced product is purified using Centri-Sep columns (Princeton Separations, Adelphia, NJ) as described by the company. This product is then analyzed with an ABI model 373A DNA sequencing sy ⁇ tem (Applied Bio ⁇ ystems) integrated with a Macintosh Uci computer.
  • the DNA sequence encoding a polypeptide of the present invention for this example BSGl, ATCC # 97175, i ⁇ initially amplified u ⁇ ing PCR oligonucleotide primer ⁇ corresponding to the 5' sequences of the protein and the vector sequences 3' to the protein. Additional nucleotides corresponding to the DNA sequence are added to the 5' and 3' sequence ⁇ respectively.
  • the 5' oligonucleotide primer has the sequence 5' GCC ⁇ CC ⁇ TGGATGTTTTCAAG 3' (SEQ ID NO:21) and contains an Ncol restriction enzyme site followed by 15 nucleotides of coding sequence starting from the initial aminp acid of the processed protein.
  • the 3' sequence 5' GCGCAGATCTGTCT CCCCCACTCTGGGC 3' (SEQ ID NO:22) and contains a complementary ⁇ equence to a Bglll restriction enzyme ⁇ ite and i ⁇ followed by 18 nucleotides of the nucleic acid sequence encoding the protein.
  • the restriction enzyme sites correspond to the restriction enzyme site ⁇ on a bacterial expre ⁇ ion vector, pQE-60 (Qiagen, Inc. Chatsworth, CA) .
  • pQE-60 encodes antibiotic resistance (Amp r ) , a bacterial origin of replication (ori) , an IPTG-regulatable promoter operator (P/O) , a ribosome binding site (RBS) , a 6-His tag and re ⁇ triction enzyme sites.
  • pQE-60 is then digested with Ncol and Bglll.
  • the amplified sequence ⁇ are ligated into pQE-60 and in ⁇ erted in frame with the ⁇ equence encoding for the hi ⁇ tidine tag and the RBS.
  • the ligation mixture is then used to transform an E. coli strain M15/rep 4 (Qiagen) by the procedure described in Sambrook, J.
  • M15/rep4 contains multiple copies of the plasmid pREP4, which expresse ⁇ the lacl repressor and also confers kanamycin resistance (Kan r ) .
  • Transformants are identified by their ability to grow on LB plates and ampicillin/kanamycin re ⁇ i ⁇ tant colonie ⁇ are ⁇ elected. Plasmid DNA is isolated and confirmed by restriction analysis.
  • Clones containing the desired constructs are grown overnight (O/N) in liquid culture in LB media ⁇ upplemented with both Amp (100 ug/ml) and Kan (25 ug/ml) .
  • the O/N culture is used to inoculate a large culture at a ratio of 1:100 to 1:250.
  • the cells are grown to an optical density 600 (O.D. 600 ) of between 0.4 and 0.6.
  • IPTG Isopropyl-B-D- thiogalacto pyrano ⁇ ide
  • IPTG induces by inactivating the lacl repressor, clearing the P/O leading to increased gene expression.
  • Cells are grown an extra 3 to 4 hour ⁇ .
  • Cell ⁇ are then harve ⁇ ted by centrifugation.
  • the cell pellet i ⁇ ⁇ olubilized in the chaotropic agent 6 Molar Guanidine HCl.
  • ⁇ olubilized protein i ⁇ purified from this solution by chromatography on a Nickel-Chelate column under conditions that allow for tight binding by proteins containing the 6-His tag (Hochuli, E. et al., J. Chromatography 411:177-184 (1984)).
  • BSGl protein (>90% pure) is eluted from the column in 6 molar guanidine HCl pH 5.0 and for the purpose of renaturation adjusted to 3 molar guanidine HCl, lOOmM sodium phosphate, 10 mmolar giutathione (reduced) and 2 mmolar giutathione (oxidized) . After incubation in this solution for 12 hours the protein is dialyzed to 10 mmolar sodium phosphate.
  • the protein purified in this manner may be used as an epitope to raise monoclonal antibodies specific to such protein.
  • the monoclonal antibodies generated against the polypeptide the isolated protein can be obtained by direct injection of the polypeptides into an animal or by administering the polypeptides to an animal. The antibodies so obtained will then bind to the protein itself. Such antibodies can then be used to isolate the protein from tis ⁇ ue expressing that polypeptide by the use of an, for example, ELISA assay.
  • RNA i ⁇ prepared from tissue ⁇ by the guanidinium-phenol method as previously described (P. Chomczynski and N. Sacchi, Anal. Biochem., 162: 156-159 (1987)) using RNAzol (Cinna-Biotecx) . An additional ethanol precipitation of the RNA is included. Poly A mRNA is isolated from the total RNA using oligo dT-coated latex beads (Qiagen) . Two rounds of poly A ⁇ election are performed to ensure better separation from non-polyadenylated material when sufficient quantities of total RNA are available.
  • the mRNA selected on the oligo dT is used for the synthesi ⁇ of cDNA by a modification of the method of Gobbler and Hoffman (Gobbler, U. and B.J. Hoffman, 1983, Gene, 25.:263) .
  • the first strand synthesis is performed using either Moloney murine ⁇ arcoma viru ⁇ rever ⁇ e tran ⁇ criptase (Stratagene) or Superscript II (RNase H minus Moloney murine rever ⁇ e tran ⁇ cripta ⁇ e, Gibco-BRL) .
  • the nucleotide mix used in the synthesis contains methylated dCTP to prevent restriction within the cDNA sequence.
  • E. coli polymerase Klenow fragment is u ⁇ ed and [ 32 P] -dATP i ⁇ incorporated a ⁇ a tracer of nucleotide incorporation.
  • the cDNA i ⁇ made blunt ended u ⁇ ing either T4 DNA polymerase or Klenow fragment.
  • Eco RI adapters are added to the cDNA and the cDNA is restricted with Xho I.
  • the cDNA is ⁇ ize fractionated over a Sephacryl S-500 column (Pharmacia) to remove exce ⁇ linker ⁇ and cDNAs under approximately 500 base pairs.
  • the cDNA is cloned unidirectionally into the Eco RI-Xho I sites of either pBluescript II phagemid or lambda Uni-zap XR (Stratagene) .
  • the plasmids are electroporated into E.coli SURE competent cell ⁇ (Stratagene) .
  • E.coli SURE competent cell ⁇ (Stratagene)
  • the cDNA i ⁇ cloned into Uni-Zap XR it i ⁇ packaged u ⁇ ing the Gigipack II packaging extract (Stratagene) .
  • the packaged phage i ⁇ used to infect SURE cells and amplified.
  • the pBlue ⁇ cript phagemid containing the cDNA in ⁇ erts are excised from the lambda Zap phage using the helper phage ExAs ⁇ i ⁇ t (Stratagene) .
  • the rescued phagemid is plated on SOLR E.coli cells (Stratagene) .
  • Template DNA for sequencing is prepared by 1) a boiling method or 2) PCR amplification.
  • the boiling method is a modification of the method of Holmes and Quigley (Holmes, D.S. and M. Quigley, 1981, Anal. Biochem., 114:193) .
  • Colonies from either cDNA cloned into Bluescript II or re ⁇ cued Blue ⁇ cript phagemid are grown in an enriched bacterial media overnight. 400 ⁇ l of cells are centrifuged and resu ⁇ pended in STET (O.lM NaCl, lOmM TRIS Ph 8.0, 1.0 mM EDTA and 5% Triton X-100) including lysozyme (80 ⁇ g/ml) and RNase A (4 ⁇ g/ml) .
  • Cells are boiled for 40 second ⁇ and centrifuged for 10 minute ⁇ . The supernatant is removed and the DNA is precipitated with PEG/NaCl and washed with 70% ethanol (2x) . Templates are resuspended in water at approximately 250 ng/ ⁇ l.
  • the primer ⁇ et cho ⁇ en for amplification of the template ⁇ is outside of primer sites chosen for sequencing of the templates.
  • the primers used are 5'-ATGCTTCCGGCTCGTATG-3' (SEQ ID NO:23) which is 5' of the M13 reverse sequence in pBluescript and 5'-GGGTTTTCCCAGTCACGAC-3' (SEQ ID NO:24) which is 3' Of the M13 forward primer in pBluescript.
  • any primer ⁇ which corre ⁇ pond to the sequence flanking the M13 forward and reverse sequence ⁇ can be u ⁇ ed.
  • Perkin-Elmer 9600 thermocycler ⁇ are u ⁇ ed for amplification of the templates with the following cycler conditions: 5 min at 94°C (1 cycle) ; (20 sec at 94°C) ; 20 sec at 55°C (l min at 72°C) (30 cycles) ; 7 min at 72°C (1 cycle) .
  • the PCR templates are precipitated using PEG/NaCl and washed three times with 70% ethanol. The templates are resuspended in water.
  • Example 4 Isolation of a Selected Clone From Brea ⁇ t Ti ⁇ ue Two approaches are u ⁇ ed to i ⁇ olate a particular clone from a cDNA library prepared from human brea ⁇ t ti ⁇ ue.
  • a clone is isolated directly by screening the library using an oligonucleotide probe.
  • a specific oligonucleotide with 30-40 nucleotides is synthesized using an Applied Biosy ⁇ tem ⁇ DNA synthesizer according to one of the partial sequences described in this application.
  • the oligonucleotide is labeled with 32 P- -ATP u ⁇ ing T4 polynucleotide kinase and purified according to the standard protocol (Maniatis et al. , Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring, NY, 1982) .
  • the Lambda cDNA library is plated on 1.5% agar plate to a density of 20,000-50,000 pfu/150 mm plate. These plates are screened using Nylon membranes according to the standard phage screening protocol (Stratagene, 1993) . Specifically, the Nylon membrane with denatured and fixed phage DNA is prehybridized in 6 x SSC, 20 mM NaH 2 P0 4 , 0.4% SDS, 5 x Denhardt's 500 ⁇ g/ml denatured, sonicated salmon sperm DNA; and 6 x SSC, 0.1% SDS.
  • the membrane is hybridized with hybridization buffer 6 x SSC, 20 mM NaH 2 P0 4 , 0.4% SDS, 500 ⁇ g/ml denatured, sonicated salmon sperm DNA with 1 x IO 6 cpm/ml 32 P-probe overnight at 42°C.
  • the membrane is washed at 45-50°C with washing buffer 6 x SSC, 0.1% SDS for 20-30 minute ⁇ dried and exposed to Kodak X-ray film overnight.
  • Positive clones are isolated and purified by secondary and tertiary screening. The purified clone sequenced to verify its identity to the partial sequence described in this application.
  • An alternative approach to screen the cDNA library prepared from human breast tissue is to prepare a DNA probe corresponding to the entire partial sequence.
  • a probe two oligonucleotide primers of 17-20 nucleotides derived from both end ⁇ of the partial ⁇ equence reported are ⁇ ynthe ⁇ ized and purified. These two oligonucleotides are used to amplify the probe using the cDNA library template.
  • the DNA template is prepared from the phage lysate of the cDNA library according to the ⁇ tandard phage DNA preparation protocol (Maniatis et al.) .
  • the polymerase chain reaction i ⁇ carried out in 25 ⁇ l reaction mixture with 0.5 ⁇ g of the above cDNA template.
  • the reaction mixture is 1.5-5 mM MgCl 2 , 0.01% (w/v) gelatin, 20 ⁇ M each of dATP, dCTP, dGTP, dTTP, 25 pmol of each primer and 0.25 Unit of Taq polymerase.
  • Thirty five cycles of PCR (denaturation at 94°C for 1 min; annealing at 55°C for 1 min; elongation at 72°C for 1 min) are performed with the Perkin-Elmer Cetus automated thermal cycler.
  • the amplified product is analyzed by agarose gel electrophoresis and the DNA band with expected molecular weight is excised and purified.
  • the PCR product is verified to be the probe by subcloning and sequencing the DNA product.
  • the probe is labeled with the Multiprime DNA Labelling System (Amersham) at a ⁇ pecific activity ⁇ 1 x IO 9 dmp/ ⁇ g. This probe is u ⁇ ed to ⁇ creen the lambda cDNA library according to Stratagene' ⁇ protocol.
  • Hybridization i ⁇ carried out with 5X TEN 920XTEN:0.3M Tri ⁇ -HCl pH 8.0, 0.02M EDTA and 3MNaCl) , 5X Denhardt' ⁇ , 0.5% ⁇ odium pyropho ⁇ phate, 0.1% SDS, 0.2 mg/ml heat denatured salmon sperm DNA and l x IO 6 cpm/ml of [ 32 P] - labeled probe at 55°C for 12 hour ⁇ .
  • the filters are washed in 0.5X TEN at room temperature for 20-30 min., then at 55°C for 15 min.
  • the filters are dried and autoradiographed at - 70°C using Kodak XAR-5 film.
  • the positive clones are purified by secondary and tertiary ⁇ creening. The ⁇ equence of the i ⁇ olated clone are verified by DNA sequencing.
  • Selected human DNA from the partial ⁇ equence clone (the cDNA clone that wa ⁇ ⁇ equenced to give the partial ⁇ equence) is purified e.g. , by endonuclease dige ⁇ tion using Eco-Rl, gel electrophoresi ⁇ , and i ⁇ olation of the clone by removal from low melting agarose gel.
  • the isolated insert DNA is radiolabeled e.g., with 32 P labels, preferably by nick tran ⁇ lation or random primer labeling.
  • the labeled in ⁇ ert i ⁇ u ⁇ ed as a probe to screen a lambda phage cDNA library or a plasmid cDNA library.
  • Colonies containing clones related to the probe cDNA are identified and purified by known purification methods.
  • the end ⁇ of the newly purified clone ⁇ are nucleotide sequenced to identify full length sequence ⁇ .
  • Complete sequencing of full length clones i ⁇ then performed by Exonuclea ⁇ e III digestion or primer walking.
  • Northern blots of the mRNA from various tis ⁇ ues using at least part of the depo ⁇ ited clone from which the partial sequence is obtained as a probe can optionally be performed to check the size of the mRNA against that of the purported full length CDNA.
  • the following procedures 2 and 3 can be used to obtain full length genes or full length coding portions of genes where a clone isolated from the deposited clone mixture doe ⁇ not contain a full length ⁇ equence.
  • a library derived from human brea ⁇ t ti ⁇ ue or from the depo ⁇ ited clone mixture i ⁇ also applicable to obtaining full length sequence ⁇ from clone ⁇ obtained from ⁇ ource ⁇ other than the depo ⁇ ited mixture by u ⁇ e of the partial ⁇ equences of the present invention.
  • Partial cDNA clone ⁇ can be made full-length by utilizing the rapid amplification of cDNA end ⁇ (RACE) procedure described in Frohman, M.A. , Dush, M.K. and Martin, G.R. (1988) Proc. Nat'l. Acad. Sci. USA, 85:8998-9002.
  • RACE rapid amplification of cDNA end ⁇
  • a cDNA clone mis ⁇ ing either the 5' or 3' end can be recon ⁇ tructed to include the ab ⁇ ent ba ⁇ e pair ⁇ extending to the tran ⁇ lational ⁇ tart or ⁇ top codon, re ⁇ pectively. In most case ⁇ , cDNA ⁇ are mi ⁇ sing the start of translation therefor. The following briefly describes a modification of this original 5' RACE procedure.
  • RNA is rever ⁇ e transcribed with Superscript II (Gibco/BRL) and an antisense or complementary primer specific to the cDNA sequence.
  • the primer is removed from the reaction with a Microcon Concentrator (Amicon) .
  • the first-strand cDNA is then tailed with dATP and terminal deoxynucleotide transferase (Gibco/BRL) . Thu ⁇ , an anchor sequence is produced which is needed for PCR amplification.
  • the second strand is synthesized from the dA-tail in PCR buffer, Taq DNA polymerase (Perkin-Elmer Cetus) , an oligo-dT primer containing three adjacent restriction sites (Xhol.
  • This double-stranded cDNA is PCR amplified for 40 cycles with the same primers as well as a nested cDNA- ⁇ pecific anti ⁇ en ⁇ e primer.
  • the PCR product ⁇ are ⁇ ize- ⁇ eparated on an ethidium bromide-agarose gel and the region of gel containing cDNA products the predicted size of missing protein-coding DNA is removed.
  • cDNA is purified from the agarose with the Magic PCR Prep kit (Promega) , restriction digested with Xhol or Sail, and ligated to a plasmid such as pBluescript SKII (Stratagene) at Shol and EcoRV site ⁇ .
  • kit ⁇ Several quality-controlled kit ⁇ are available for purchase. Similar reagents and methods to those above are supplied in kit form from Gibco/BRL. A second kit is available from Clontech which i ⁇ a modification of a related technique, SLIC ( ⁇ ingle- ⁇ tranded ligation to ⁇ ingle- ⁇ tranded cDNA) developed by Dumas et al. (Dumas, J.B., Edwards, M. , Delort, J. and Mallet, Jr., 1991, Nucleic Acids Res., 19:5227-5232) .
  • SLIC ⁇ ingle- ⁇ tranded ligation to ⁇ ingle- ⁇ tranded cDNA
  • RNA is alkaline hydrolyzed after reverse transcription and RNA liga ⁇ e is used to join a re ⁇ triction site-containing anchor primer to the first- ⁇ trand cDNA. This obviates the necessity for the dA-tailing reaction which results in a polyT stretch that is difficult to ⁇ equence past.
  • An alternative to generating 5' cDNA from RNA is to use cDNA library double- ⁇ tranded DNA.
  • An asymmetric PCR- amplified antisen ⁇ e cDNA strand is ⁇ ynthe ⁇ ized with an anti ⁇ ense cDNA- ⁇ pecific primer and a plasmid-anchored primer. These primers are removed and a symmetric PCR reaction is performed with a nested cDNA-specific antisense primer and the plasmid-anchored primer.
  • a gene of interest is identified, several methods are available for the identification of the 5' or 3' portions of the gene which may not be present in the original deposited clone. These methods include but are not limited to filter probing, clone enrichment using specific probes and protocol ⁇ ⁇ imilar and identical to 5' and 3' RACE. While the full length gene may be pre ⁇ ent in a library and can be identified by probing, a u ⁇ eful method for generating the 5' end i ⁇ to u ⁇ e the exi ⁇ ting ⁇ equence information from the original partial ⁇ equence to generate the mis ⁇ ing information. A method similar to 5' RACE is available for generating the mis ⁇ ing 5' end of a de ⁇ ired full-length gene.
  • RNA oligonucleotide is ligated to the 5' ends of a population of RNA presumably containing full-length gene RNA transcript and a primer ⁇ et containing a primer specific to the ligated RNA oligonucleotide.
  • a primer specific to a known sequence (EST) of the gene of interest is u ⁇ ed to PCR amplify the 5' portion of the de ⁇ ired full length gene which may then be ⁇ equenced and used to generate the full length gene.
  • RNA isolation starts with total RNA isolated from the desired source, poly A RNA may be used but is not a prerequisite for this procedure.
  • the RNA preparation may then be treated with phosphatase if necessary to eliminate 5' phosphate groups on degraded or damaged RNA which may interfere with the later RNA ligase step.
  • the phosphatase if used is then inactivated and the RNA is treated with tobacco acid pyrophosphatase in order to remove the cap structure present at the 5' ends of messenger RNAs.
  • Thi ⁇ reaction leaves a 5' phosphate group at the 5' end of the cap-cleaved RNA which can then be ligated to an RNA oligonucleotide using T4 RNA ligase.
  • This modified RNA preparation can then be used as a template for first strand cDNA synthe ⁇ is using a gene-specific oligonucleotide.
  • the first stand synthesis reaction can then be used as a template for PCR amplification of the de ⁇ ired 5' end u ⁇ ing a primer specific to the ligated RNA oligonucleotide and a primer specific to the known sequence (EST) of the gene of interest.
  • the resultant product is then sequenced and analyzed to confirm that the 5' end sequence belongs to the partial sequence.
  • the 5' primer has the sequence 5' AAAGGATCCCCCGCCATCATGG ATGTTTTCAAGAAG 3' (SEQ ID NO:25) and contains a BamHl restriction enzyme site (in bold) followed by 8 nucleotides resembling an efficient signal for the initiation of translation in eukaryotic cells (Kozak, M. , J. Mol. Biol., 196:947-950 (1987) of the BSGl gene (the initiation codon for translation "ATG" is underlined) .
  • the 3' primer has the sequence 5' AAATCTAGACTAGTCTCCCCC ACTCTG 3' (SEQ ID NO:26) and contain ⁇ the cleavage ⁇ ite for the restriction endonuclease Xbal and 21 nucleotides complementary to the 3' sequence of the BSGl gene.
  • the amplified sequences were isolated from a 1% agarose gel using a commercially available kit ("Geneclean, " BIO 101 Inc., La Jolla, Ca.) .
  • the fragment wa ⁇ then digested with the endonucleases BamHl and Xbal and then purified again on a 1% agarose gel. This fragment is designated F2.
  • the vector pA2 (modification of pVL941 vector, discussed below) is used for the expression of the BSGl protein using the baculovirus expression sy ⁇ tem (for review see: Summers, M.D. and Smith, G.E. 1987, A manual of methods for baculoviru ⁇ vectors and insect cell culture procedures, Texas Agricultural Experimental Station Bulletin No. 1555) .
  • This expression vector contains the strong polyhedrin promoter of the Autographa californica nuclear polyhedrosi ⁇ viru ⁇ (AcMNPV) followed by the recognition sites for the restriction endonucleases BamHl and Xbal.
  • the polyadenylation site of the simian virus (SV)40 is used for efficient polyadenylation.
  • the beta-galactosidase gene from E.coli is inserted in the same orientation as the polyhedrin promoter followed by the polyadenylation signal of the polyhedrin gene.
  • the polyhedrin ⁇ equences are flanked at both ⁇ ide ⁇ by viral ⁇ equence ⁇ for the cell-mediated homologous recombination of co-transfected wild-type viral DNA.
  • Many other baculovirus vectors could be used in place of pA2 such as pRGl, pAc373, pVL941 and pAcIMl (Luckow, V.A. and Summers, M.D., Virology, 170:31-39) .
  • the plasmid was dige ⁇ ted with the re ⁇ triction enzyme ⁇ BamHl and Xbal and depho ⁇ phorylated u ⁇ ing calf inte ⁇ tinal pho ⁇ phata ⁇ e by procedures known in the art.
  • the DNA was then i ⁇ olated from a 1% agarose gel using the commercially available kit ("Geneclean" BIO 101 Inc., La Jolla, Ca.) .
  • This vector DNA is designated V2.
  • Fragment F2 and the dephosphorylated plasmid pA2 were ligated with T4 DNA ligase.
  • E.coli HB101 cells were then transformed and bacteria identified that contained the plasmid (pBacBSGl) with the BSGl gene using the enzymes BamHl and Xbal. The sequence of the cloned fragment was confirmed by DNA sequencing.
  • the plate was rocked back and forth to mix the newly added solution.
  • the plate wa ⁇ then incubated for 5 hours at 27°C.
  • the transfection solution was removed from the plate and 1 ml of Grace's insect medium supplemented with 10% fetal calf serum was added.
  • the plate was put back into an incubator and cultivation continued at 27°C for four days.
  • plaque assay An agarose gel with "Blue Gal” (Life Technologies Inc., Gaithersburg) wa ⁇ used which allows an ea ⁇ y i ⁇ olation of blue ⁇ tained plaque ⁇ . (A detailed description of a "plaque assay” can also be found in the u ⁇ er's guide for insect cell culture and baculovirology distributed by Life Technologies Inc., Gaithersburg, page 9- 10) .
  • the virus was added to the cells and blue ⁇ tained plaque ⁇ were picked with the tip of an Eppendorf pipette.
  • the agar containing the recombinant viru ⁇ e ⁇ was then resuspended in an Eppendorf tube containing 200 ⁇ l of Grace's medium.
  • the agar was removed by a brief centrifugation and the supernatant containing the recombinant baculovirus was used to infect Sf9 cells seeded in 35 mm dishes. Four days later the supernatants of these culture dishes were harvested and then ⁇ tored at 4°C.
  • Sf9 cell ⁇ were grown in Grace's medium ⁇ upplemented with 10% heat-inactivated FBS.
  • the cells were infected with the recombinant baculovirus V-BSG1 at a multiplicity of infection
  • plasmid, BSGl HA is derived from a vector pcDNAI/Amp (Invitrogen) containing: 1) SV40 origin of replication, 2) ampicillin resistance gene, 3) E.coli replication origin, 4) CMV promoter followed by a polylinker region, an SV40 intron and polyadenylation site.
  • the HA tag corresponds to an epitope derived from the influenza hemagglutinin protein as previously described (I. Wilson, H.
  • HA tag to the target protein allows easy detection of the recombinant protein with an antibody that recognizes the HA epitope.
  • the plasmid construction strategy is described as follows:
  • the DNA sequence encoding BSGl, ATCC # 97175 was constructed by PCR using two primers: the 5' primer AAAGGA TCCCCCGCCIATCATCGATGTTTTCAAGAAG 3' (SEQ ID NO:27) contain ⁇ a BamHl ⁇ ite followed by 18 nucleotide ⁇ of BSGl coding sequence starting from the initiation codon; the 3' sequence AAATC TAGACTAAAGCGTAGTC ⁇ 3GGACX ⁇ rr ⁇ , ATGGGTACTCCTOGGGT 3' (SEQ ID NO:28) contains complementary sequences to an Xbal site, translation stop codon, HA tag and the la ⁇ t 18 nucleotide ⁇ of the BamHl coding sequence (not including the stop codon) .
  • the PCR product contain ⁇ an BamHl ⁇ ite, BSGl coding sequence followed by HA tag fused in frame, a translation termination stop codon next to the HA tag, and an Xbal site.
  • the PCR amplified DNA fragment and the vector, pcDNAI/Amp were digested with BamHl and Xbal restriction enzyme and ligated.
  • the ligation mixture was transformed into E. coli strain SURE (available from Stratagene Cloning Systems, 11099 North Torrey Pines Road, La Jolla, CA 92037) the transformed culture was plated on ampicillin media plates and re ⁇ i ⁇ tant colonie ⁇ were ⁇ elected.
  • COS cells were transfected with the expres ⁇ ion vector by DEAE-DEXTRAN method (J. Sambrook, E. Fritsch, T. Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Laboratory Pres ⁇ , (1989)).
  • the expre ⁇ ion of the BSG HA protein was detected by radiolabelling and immunoprecipitation method (E. Harlow, D. Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Pres ⁇ , (1988)).
  • Cell ⁇ were labelled for 8 hour ⁇ with 35 S-cysteine two days post transfection.

Abstract

Human breast specific gene polypeptides and DNA (RNA) encoding such polypeptides and a procedure for producing such polypeptides by recombinant techniques is disclosed. Also disclosed are methods for utilizing such polynucleotides or polypeptides as a diagnostic marker for breast cancer and as an agent to determine if breast cancer has metastasized. Also disclosed are antibodies specific to the breast specific gene polypeptides which may be used to target cancer cells and be used as part of a breast cancer vaccine. Methods of screening for antagonists for the polypeptide and therapeutic uses thereof are also disclosed.

Description

BREAST SPECIFIC GENES AND PROTEINS
This invention relates to newly identified polynucleotides, polypeptides encoded by such polynucleotides, and the use of such polynucleotides and polypeptides for detecting disorders of the breast, particularly the presence of breast cancer and breast cancer metastases. The present invention further relates to inhibiting the production and function of the polypeptides of the present invention. The twenty breast specific genes of the present invention are sometimes hereinafter referred to as "BSG1", "BSG2" etc.
The mammary gland is subject to a variety of disorders that should be readily detectable. Detection may be accomplished by inspection which usually consists of palpation. Unfortunately, so few periodic self-examinations are made that many breast masses are discovered only by accidental palpation. Aspiration of suspected cysts with a fine-gauge needle is another fairly common diagnostic practice. Mammography or xeroradiography (soft-tissue x-ray) of the breast of yet another. A biopsy of a lesion or suspected area is an extreme method of diagnostic test.
There are many types of tumors and cysts which affect the mammary gland. Fibroadenomas is the most common benign breast tumor. As a pathological entity, it ranks third behind cystic disease and carcinoma, respectively. These tumors are seen most frequently in young people and are usually readily recognized because they feel encapsulated. Fibrocystic disease, a benign condition, is the most common disease of the female breast, occurring in about 20% bf pre- menopausal women. Lipomas of the breast are also common and they are benign in nature. Carcinoma of the breast is the most common malignant condition among women and carries with it the highest fatality rate of all cancers affecting this sex. At some during her life, one of every 15 women in the USA will develop cancer of the breast. Its reported annual incidence is 70 per 100,000 females in the population in 1947, rising to 72.5 in 1969 for whites, and rising from 47.8 to 60.1 for blacks. The annual mortality rate from 1930 to the present has remained fairly constant, at approximately 23 per 100,000 female population. Breast cancer is rare in men, but when it does occur, it usually not recognized until late, and thus the results of treatment are poor. In women, carcinoma of the breast is rarely seen before age 30 and the incidence rises rapidly after menopause. For this reason, post-menopausal breast masses should be considered cancer until proved otherwise.
In accordance with an aεpect of the present invention, there are provided nucleic acid probes comprising nucleic acid molecules of sufficient length to specifically hybridize to the RNA transcribed from the human breast specific genes of the present invention or to DNA corresponding to such RNA.
In accordance with another aspect of the present invention there is provided a method of and products for diagnosing breast cancer formation and breast cancer metastases by detecting the presence of RNA transcribed from the human breast specific genes of the present invention or DNA corresponding to such RNA in a sample deprived from a host. In accordance with yet another aspect of the present invention, there is provided a method of and products for diagnosing breast cancer formation and breast cancer metastases by detecting an altered level of a polypeptide corresponding to the breast specific genes of the present invention in a sample derived from a host, whereby an elevated level of the polypeptide indicates a breast cancer diagnosis.
In accordance with another aspect of the present invention, there are provided isolated polynucleotides encoding human breast specific polypeptides, including mRNAs, DNAs, cDNAs, genomic DNAs, as well as antisense analogs and biologically active and diagnostically or therapeutically useful fragments thereof.
In accordance with still another aspect of the present invention there are provided human breast specific genes which include polynucleotides as set forth in the sequence listing.
In accordance with a further aspect of the present invention, there are provided novel polypeptides encoded by the polynucleotides, as well as biologically active and diagnostically or therapeutically useful fragments, analogs and derivatives thereof.
In accordance with yet a further aspect of the present invention, there is provided a process for producing such polypeptides by recombinant techniques comprising culturing recombinant prokaryotic and/or eukaryotic host cells, containing a polynucleotide of the present invention, under conditions promoting expression of said proteins and subsequent recovery of said proteins.
In accordance with yet a further aspect of the present invention, there are provided antibodies specific to such polypeptides, which may be employed to detect breast cancer cells or breast cancer metastasis. In accordance with another aspect of the present invention, there are provided processes for using one or more of the polypeptides of the present invention to treat breast cancer and for using the polypeptides to screen for compounds which interact with the polypeptides, for example, compounds which inhibit or activate the polypeptides of the present invention.
In accordance with yet another aspect of the present invention, there is provided a screen for detecting compounds which inhibit activation of one or more of the polynucleotides and/or polypeptides of the present invention which may be used to therapeutically, for example, in the treatment of breast cancer.
In accordance with yet a further aspect of the present invention, there are provided processes for utilizing such polypeptides, or polynucleotides encoding such polypeptideε, for in vitro purposes related to scientific research, synthesis of DNA and manufacture of DNA vectors.
These and other aspects of the present invention should be apparent to those skilled in the art from the teachings herein.
The following drawings are illustrative of embodiments of the invention and are not meant to limit the scope of the invention as encompassed by the claims.
Figure 1 is a full length cDNA sequence of breast specific gene 1 of the present invention.
Figure 2 is a partial cDNA sequence and the corresponding deduced amino acid sequence of breast specific gene 2 of the present invention.
Figure 3 is a partial cDNA sequence and deduced amino acid sequence of breast specific gene 3 of the invention.
Figure 4 is a partial cDNA sequence and the corresponding deduced amino acid sequence of breast specific Figure 5 is a partial cDNA sequence of breast specific gene 5 of the present invention.
Figure 6 is a partial cDNA and deduced amino acid sequence of breast specific gene 6 of the present invention.
Figure 7 is a partial cDNA sequence of breast specific gene 7 of the present invention.
Figure 8 is a partial cDNA sequence of breast specific gene 8 of the present invention.
Figure 9 is a partial cDNA sequence of breast specific gene 9 of the present invention.
Figure 10 is a partial cDNA sequence of breast specific gene 10 of the preεent invention.
Figure 11 is a partial cDNA sequence of breast specific gene 11 of the present invention.
Figure 12 iε a partial cDNA sequence of breast specific gene 12 of the present invention.
Figure 13 iε a partial cDNA sequence of breast specific gene 13 of the present invention.
Figure 14 is a partial cDNA sequence of breast specific gene 14 of the present invention.
Figure 15 is a partial cDNA sequence of breast specific gene 15 of the present invention.
Figure 16 is a partial cDNA sequence of breast specific gene 16 of the present invention.
Figure 17 iε a partial cDNA εequence of breast specific gene 17 of the present invention.
Figure 18 is a partial cDNA sequence of breast specific gene 18 of the present invention.
Figure 19 iε a partial cDNA εequence of breaεt εpecific gene 19 of the preεent invention.
Figure 20 iε a partial cDNA εequence of breast specific gene 20 of the present invention.
The term "breast specific gene" means that εuch gene is primarily expresεed in tiεεueε derived from the breaεt, and εuch geneε may be expreεεed in cellε derived from tissueε other than from the breast. However, the expression of such genes is significantly higher in tissues derived from the breast than from non-breast tisεueε.
In accordance with one aεpect of the present invention there is provided a polynucleotide which encodes the mature polypeptides having the deduced amino acid sequence of Figure 1 (SEQ ID NO:l) and fragments, analogues and derivatives thereof.
In accordance with a further aspect of the present invention there is provided a polynucleotide which encodes the same mature polypeptide aε a human gene having a coding portion which contains a polynucleotide which is at least 90% identical (preferably at least 95% identical and most preferably at least 97% or 100% identical) to one of the polynucleotides of Figures 2-20 (SEQ ID NO:2-20) , as well as fragments thereof.
In accordance with still another aspect of the present invention there is provided a polynucleotide which encodes for the same mature polypeptide as a human gene whose coding portion includes a polynucleotide which is at least 90% identical to (preferably at least 95% identical to and most preferably at least 97% or 100% identical) to one of the polynucleotides included in ATCC Deposit No. 97175 deposited June 2, 1995.
In accordance with yet another aspect of the present invention, there is provided a polynucleotide probe which hybridizes to mRNA (or the correεponding cDNA) which is transcribed from the coding portion of a human gene which coding portion includeε a DNA sequence which is at least 90% identical to (preferably at least 95% identical to) and most preferably at least 97% or 100% identical) to one of the polynucleotide sequences of Figures 1-20 (SEQ ID NO:1-20) .
The present invention further relateε to a mature polypeptide encoded by a coding portion of a human gene which coding portion includeε a DNA sequence which iε at leεt 90% identical to (preferably at least 95% identical to and more preferably 97% or 100% identical to) one of the polynucleotides of Figures 2-20 (SEQ ID NO:2-20) , as well as analogues, derivatives and fragmentε of such polypeptides.
The present invention also relates to one of the mature polypeptides of Figure 1 (SEQ ID N0:1) and fragments, analogues and derivatives of such polypeptides.
The present invention further relates to the same mature polypeptide encoded by a human gene whose coding portion includeε DNA which iε at least 90% identical to (preferably at least 95% identical to and more preferably at least 97% or 100% identical to) one of the polynucleotides included in ATCC Deposit No. 97175 depoεited June 2, 1995.
In accordance with an aspect of the present invention, there are provided isolated nucleic acids (polynucleotides) which encode for the mature polypeptides having the deduced amino acid sequence of Figure 1 (SEQ ID N0:1) or fragments, analogues or derivatives thereof.
The polynucleotides of the present invention may be in the form of RNA or in the form of DNA, which DNA includes cDNA, genomic DNA, and synthetic DNA. The DNA may be double- stranded or single-stranded, and if single stranded may be the coding strand or non-coding (anti-sense) strand. The coding sequence which encodes the mature polypeptide may include DNA identical to Figures 1-20 (SEQ ID NO:1-20) or that of the deposited clone or may be a different coding sequence which coding sequence, as a result of the redundancy or degeneracy of the genetic code, encodes the same mature polypeptide as the coding sequence of a gene which coding sequence includes the DNA of Figures 1-20 (SEQ ID NO:1-20) or the deposited cDNA.
The polynucleotide which encodes a mature polypeptide of the present invention may include, but is not limited to: only the coding sequence for the mature polypeptide; the coding sequence for the mature polypeptide and additional coding sequence such aε a leader or secretory sequence or a proprotein sequence; the coding sequence for the mature polypeptide (and optionally additional coding sequence) and non-coding sequence, such as introns or non-coding sequence 5' and/or 3' of the coding sequence for the mature polypeptide.
Thus, the term "polynucleotide encoding a polypeptide" encompasses a polynucleotide which includes only coding sequence for the polypeptide as well as a polynucleotide which includeε additional coding and/or non-coding εequence.
The present invention further relates to variants of the hereinabove described polynucleotides which encode fragments, analogs and derivatives of a mature polypeptide of the present invention. The variant of the polynucleotide may be a naturally occurring allelic variant of the polynucleotide or a non-naturally occurring variant of the polynucleotide.
Thus, the present invention includes polynucleotideε encoding the εame mature polypeptide aε hereinabove described as well as variants of such polynucleotides which variants encode a fragment, derivative or analog of a polypeptide of the invention. Such nucleotide variants include deletion variants, subεtitution variants and addition or insertion variants.
The polynucleotides of the invention may have a coding sequence which is a naturally occurring allelic variant of the human gene whoεe coding εequence includeε DNA as shown in Figures 1-20 (SEQ ID NO:1-20) or of the coding sequence of the DNA in the deposited clone. As known in the art, an allelic variant is an alternate form of a polynucleotide sequence which may have a subεtitution, deletion or addition of one or more nucleotideε, which doeε not substantially alter the function of the encoded polypeptide.
The present invention also includes polynucleotides, wherein the coding sequence for the mature polypeptide may be fused in the same reading frame to a polynucleotide sequence which aids in expreεsion and secretion of a polypeptide from a host cell, for example, a leader εequence which functions as a secretory sequence for controlling transport of a polypeptide from the cell. The polypeptide having a leader sequence is a preprotein and may have the leader sequence cleaved by the host cell to form the mature form of the polypeptide. The polynucleotides may also encode a proprotein which is the mature protein plus additional 5' amino acid residues. A mature protein having a prosequence is a proprotein and is an inactive form of the protein. Once the prosequence iε cleaved an active mature protein remains.
Thus, for example, the polynucleotide of the preεent invention may encode a mature protein, or a protein having a proεequence or a protein having both a preεequence and a preεequence (leader sequence) .
The polynucleotides of the present invention may also have the coding sequence fused in frame to a marker sequence which allows for purification of the polypeptide of the present invention. The marker sequence may be a hexa- histidine tag supplied by a pQE-9 vector to provide for purification of the mature polypeptide fused to the marker in the case of a bacterial host, or, for example, the marker sequence may be a hemagglutinin (HA) tag when a mammalian host, e.g. COS-7 cells, is used. The HA tag correspondε to an epitope derived from the influenza hemagglutinin protein (Wilεon, I., et al., Cell, 37:767 (1984)).
The present invention further relates to polynucleotides which hybridize to the hereinabove-described polynucleotides if there is at least 70%, preferably at least 90%, and more preferably at least 95% identity between the sequenceε. The preεent invention particularly relateε to polynucleotideε which hybridize under εtringent conditions to the hereinabove-described polynucleotides. As herein used, the term "stringent conditions" means hybridization will occur only if there is at least 95% and preferably at least 97% identity between the sequences. The polynucleotides which hybridize to the hereinabove described polynucleotides in a preferred embodiment encode polypeptideε which retain εubεtantially the εame biological function or activity aε the mature polypeptide of the present invention encoded by a coding sequence which includes the DNA of Figures 1-20 (SEQ ID NO:l-20) or the deposited cDNA(s) .
Alternatively, the polynucleotide may have at least 10 or 20 baεes, preferably at least 30 baseε, and more preferably at least 50 bases which hybridize to a polynucleotide of the preεent invention and which haε an identity thereto, aε hereinabove deεcribed, and which may or may not retain activity. For example, such polynucleotides may be employed as probes for polynucleotides, for example, for recovery of the polynucleotide or as a diagnostic probe or as a PCR primer.
Thus, the present invention is directed to polynucleotides having at least a 70% identity, preferably at least 90% and more preferably at least 95% identity to a polynucleotide which encodes the mature polypeptide encoded by a human gene which includes the DNA of one of Figures 1-20 (SEQ ID NO:1-20) as well as fragments thereof, which fragments have at least 30 bases and preferably at least 50 bases and to polypeptides encoded by such polynucleotideε.
The partial εequenceε are εpecific tagε for meεsenger RNA molecules. The complete sequence of that mesεenger RNA, in the form of cDNA, can be determined uεing the partial εequence as a probe to identify a cDNA clone corresponding to a full-length transcript, followed by sequencing of that clone. The partial cDNA clone can also be used as a probe to identify a genomic clone or clones that contain the complete gene including regulatory and promoter regions, exons, and introns.
The partial sequences of Figures 2-20 (SEQ ID NO:2-20) may be used to identify the corresponding full length gene from which they were derived. The partial sequences can be nick-translated or end-labelled with 3P uεing polynucleotide kinaεe using labelling methods known to those with skill in the art (Basic Methods in Molecular Biology, L.G. Daviε, M.D. Dibner, and J.F. Battey, ed. , Elεevier Press, NY, 1986). A lambda library prepared from human breast tissue can be directly screened with the labelled sequences of interest or the library can be converted en masse to pBluescript (Stratagene Cloning Systemε, La Jolla, CA 92037) to facilitate bacterial breasty screening. Regarding pBluescript, see Sambrook et al., Molecular Cloning-A Laboratory Manual, Cold Spring Harbor Laboratory Presε (1989), pg. 1.20. Both methodε are well known in the art. Briefly, filterε with bacterial colonies containing the library in pBluescript or bacterial lawns containing lambda plaques are denatured and the DNA is fixed to the filters. The filters are hybridized with the labelled probe using hybridization conditions described by Davis et al., supra. The partial sequences, cloned into lambda or pBluescript, can be used as positive controls to assesε background binding and to adjust the hybridization and washing stringencies necessary for accurate clone identification. The resulting autoradiograms are compared to duplicate plates of colonies or plaques; each exposed spot correspondε to a positive breasty or plaque. The colonies or plaques are selected, expanded and the DNA iε iεolated from the colonies for further analysiε and εequencing.
Poεitive cDNA clones are analyzed to determine the amount of additional sequence they contain using PCR with one primer from the partial εequence and the other primer from the vector. Clones with a larger vector-insert PCR product than the original partial sequence are analyzed by restriction digestion and DNA sequencing to determine whether they contain an insert of the same size or similar as the mRNA size determined from Northern blot Analysiε. Once one or more overlapping cDNA clones are identified, the complete sequence of the clones can be determined. The preferred method is to use exonuclease III digestion (McCombie, W.R, Kirknesε, E., Fleming, J.T. , Kerlavage, A.R., Iovannisci, D.M. , and Martin-Gallardo, R. , Methods, 3_:33-40, 1991) . A series of deletion clones are generated, each of which is sequenced. The resulting overlapping sequences are assembled into a single contiguous sequence of high redundancy (usually three to five overlapping sequences at each nucleotide position) , reεulting in a highly accurate fina1 sequence.
The DNA sequences (as well as the corresponding RNA sequences) also include sequences which are or contain a DNA sequence identical to one contained in and isolatable from ATCC Deposit No. 97175, deposited June 2, 1995, and fragments or portions of the isolated DNA sequences (and corresponding RNA sequenceε) , aε well aε DNA (RNA) sequences encoding the εame polypeptide.
The depoεit(ε) referred to herein will be maintained under the termε of the Budapest Treaty on the International Recognition of the Deposit of Micro-organisms for purposes of Patent Procedure. These depoεitε are provided merely aε convenience to thoεe of εkill in the art and are not an admiεεion that a deposit is required under 35 U.S.C. §112. The sequence of the polynucleotides contained in the deposited materials, as well as the amino acid sequence of the polypeptides encoded thereby, are incorporated herein by reference and are controlling in the event of any conflict with any description of sequenceε herein. A licenεe may be required to make, uεe or εell the depoεited materialε, and no εuch licenεe iε hereby granted.
The present invention further relates to polynucleotides which have at least 10 baseε, preferably at least 20 baseε, and may have 30 or more baεeε, which polynucleotideε are hybridizable to and have at least a 70% identity to RNA (and DNA which correspondε to εuch RNA) tranεcribed from a human gene whose coding portion includes DNA as hereinabove described.
Thuε, the polynucleotide sequences which hybridize as described above may be uεed to hybridize to and detect the expression of the human genes to which they correspond for use in diagnostic assays as hereinafter described.
In accordance with still another aspect of the present invention there are provided diagnostic assays for detecting micrometastases of breast cancer in a host. While applicant does not wish to limit the reasoning of the present invention to any specific scientific theory, it is believed that the presence of active transcription of a breast specific gene of the present invention in cells of the host, other than those derived from the breast, is indicative of breast cancer metastaεes. This is true because, while the breast specific genes are found in all cells of the body, their transcription to mRNA, cDNA and expression products is primarily limited to the breast in non-diseased individuals. However, if breast cancer iε preεent, breast cancer cells migrate from the cancer to other cellε, εuch that theεe other cellε are now actively tranεcribing and expreεεing a breast specific gene at a greater level than is normally found in non-diseaεed individualε, i.e., tranεcription is higher than found in non- breast tisεueε in healthy individualε. It iε the detection of this enhanced transcription or enhanced protein expression in cells, other than thoεe derived from the breaεt, which iε indicative of metaεtaεes of breast cancer.
In one example of such a diagnoεtic assay, an RNA sequence in a sample derived from a tisεue other than the breast is detected by hybridization to a probe. The sample contains a nucleic acid or a mixture of nucleic acids, at least one of which is suεpected of containing a human breaεt εpecific gene or fragment thereof of the present invention which is transcribed and expressed in such tissue. Thus, for example, in a form of an assay for determining the presence of a specific RNA in cells, initially RNA is isolated from the cells.
A sample may be obtained from cells derived from tisεue other than from the breast including but not limited to blood, urine, saliva, tissue biopsy and autopsy material. The use of such methods for detecting enhanced transcription to mRNA from a human breast specific gene of the present invention or fragment thereof in a sample obtained from cellε derived from other than the breast is well within the scope of those skilled in the art from the teachings herein.
The isolation of mRNA comprises isolating total cellular RNA by disrupting a cell and performing differential centrifugation. Once the total RNA is isolated, mRNA is isolated by making use of the adenine nucleotide residues known to those skilled in the art as a poly(A) tail found on virtually every eukaryotic mRNA molecule at the 3' end thereof. Oligonucleotideε compoεed of only deoxythymidine [oligo(dT)] are linked to celluloεe and the oligo(dT) - celluloεe packed into εmall columns. When a preparation of total cellular RNA is passed through such a column, the mRNA molecules bind to the oligo(dT) by the poly(A)tails while the rest of the RNA flows through the column. The bound mRNAs are then eluted from the column and collected.
One example of detecting isolated mRNA transcribed from a breast specific gene of the present invention comprises screening the collected mRNAs with the gene specific oligonucleotide probes, as hereinabove described.
It is also appreciated that such probes can be and are preferably labeled with an analytically detectable reagent to facilitate identification of the probe. Useful reagents include but are not limited to radioactivity, fluorescent dyes or enzymes capable of catalyzing the fα mation of a detectable product. An example of detecting a polynucleotide complementary to the mRNA εequence (cDNA) utilizes the polymerase chain reaction (PCR) in conjunction with reverse transcriptase. PCR is a very powerful method for the specific amplification of DNA or RNA stretches (Saiki et al . , Nature, 234:163-166 (1986)). One application of this technology is in nucleic acid probe technology to bring up nucleic acid sequenceε preεent in low copy numbers to a detectable level. Numerouε diagnoεtic and εcientific applicationε of thiε method have been described by H.A. Erlich (ed.) in PCR Technology- Principles and Applications for DNA Amplification, Stockton Preεε, USA, 1989, and by M.A. Inis (ed.) in PCR Protocols, Academic Preεε, San Diego, USA, 1990.
RT-PCR iε a combination of PCR with the reverse transcriptase enzyme. Reverse transcriptase is an enzyme which produceε cDNA moleculeε from correεponding mRNA moleculeε. Thiε iε important εince PCR amplifieε nucleic acid moleculeε, particularly DNA, and thiε DNA may be produced from the mRNA isolated from a sample derived from the hoεt.
A εpecific example of an RT-PCR diagnoεtic assay involves removing a sample from a tisεue of a hoεt. Such a εample will be from a tiεεue, other than the breaεt, for example, blood. Therefore, an example of εuch a diagnoεtic assay comprises whole blood gradient isolation of nucleated cells, total RNA extraction, RT-PCR of total RNA and agarose gel electrophoreεis of PCR products. The PCR products comprise cDNA complementary to RNA transcribed from one or more breast specific genes of the present invention or fragmentε thereof. More particularly, a blood sample iε obtained and the whole blood iε combined with an equal volume of phosphate buffered saline, centrifuged and the lymphocyte and granulocyte layer is carefully aspirated and rediluted in phosphate buffered saline and centrifuged again. The supernate is discarded and the pellet containing nucleated cells is used for RNA extraction using the RNazole B method as described by the manufacturer (Tel-Test Inc. , Friendswood, TX) .
Oligonucleotide primers and probes are prepared with high specificity to the DNA sequences of the present invention. The probes are at least 10 base pairs in length, preferably at least 30 base pairs in length and most preferably at least 50 base pairε in length or more. The reverεe tranεcriptase reaction and PCR amplification are performed sequentially without interruption. Taq polymerase is used during PCR and the PCR products are concentrated and the entire sample is run on a Tris-borate-EDTA agarose gel containing ethidium bromide.
In accordance with another aspect of the present invention, there is provided a method of diagnosing a disorder of the breast, for example breast cancer, by determining altered levels of the breast specific polypeptides of the present invention in a biological sample, derived from tissue other than from the breast. Elevated levels of the breast specific polypeptides of the present invention, indicates active transcription and expreεεion of the correεponding breast specific gene product. Assayε used to detect levels of a breast specific gene polypeptide in a sample derived from a host are well-known to those skilled in the art and include radioimmunoasεays, competitive-binding assays, Western blot analysis, ELISA asεayε and "εandwich" assays. A biological sample may include, but is not limited to, tissue extracts, cell sampleε or biological fluidε, however, in accordance with the preεent invention, a biological εample εpecifically doeε not include tiεεue or cells of the breast.
An ELISA assay (Coligan, et al., Current Protocols in Immunology. 1(2), Chapter 6, 1991) initially comprises preparing an antibody specific to a breast specific polypeptide of the present invention, preferably a monoclonal antibody. In addition, a reporter antibody iε prepared against the monoclonal antibody. To the reporter antibody is attached a detectable reagent such aε radioactivity, fluorescence or, in this example, a horseradish peroxidase enzyme. A sample is removed from a host and incubated on a solid εupport, e.g., a polyεtyrene diεh, that bindε the proteinε in the sample. Any free protein binding siteε on the dish are then covered by incubating with a non-specific protein, such as BSA. Next, the monoclonal antibody is incubated in the dish during which time the monoclonal antibodieε attach to the breaεt εpecific polypeptide attached to the polystyrene dish. All unbound monoclonal antibody is washed out with buffer. The reporter antibody linked to horseradiεh peroxidaεe iε now placed in the diεh reεulting in binding of the reporter antibody to any monoclonal antibody bound to the breaεt specific gene polypeptide. Unattached reporter antibody is then washed out. Peroxidase subεtrateε are then added to the diεh and the amount of color developed in a given time period iε a meaεurement of the amount of the breaεt specific polypeptide present in a given volume of patient sample when compared against a standard curve.
A competition assay may be employed where antibodies specific to a breaεt specific polypeptide are attached to a solid support. The breast specific polypeptide is then labeled and the labeled polypeptide a sample derived from the host are paεεed over the solid support and the amount of label detected, for example, by liquid scintillation chromatography, can be correlated to a quantity of the breast εpecific polypeptide in the εample.
A "εandwich" assay is similar to an ELISA asεay. In a "εandwich" aεεay, breaεt εpecific polypeptides are pasεed over a εolid εupport and bind to antibody attached to the εolid support. A second antibody is then bound to the breast specific polypeptide. A third antibody which is labeled and is specific to the εecond antibody, iε then passed over the solid support and binds to the εecond antibody and an amount can then be quantified.
In alternative methods, labeled antibodies to a breast specific polypeptide are used. In a one-step assay, the target molecule, if it is present, is immobilized and incubated with a labeled antibody. The labeled antibody binds to the immobilized target molecule. After washing to remove the unbound molecules, the sample is assayed for the presence of the label. In a two-step assay, immobilized target molecule is incubated with an unlabeled antibody. The target molecule-labeled antibody complex, if present, is then bound to a second, labeled antibody that is specific for the unlabeled antibody. The sample is washed and assayed for the presence of the label.
Such antibodies specific to breast specific gene proteins, for example, anti-idiotypic antibodies, can be used to detect breast cancer cells by being labeled and described above and binding tightly to the breast cancer cells, and, therefore, detect their presence.
The antibodies may also be used to target breast cancer cellε, for example, in a method of homing interaction agentε which, when contacting breast cancer cellε, destroy them. This is true εince the antibodies are specific for breast specific genes which are primarily expressed in breast cancer, and a linking of the interaction agent to the antibody would cause the interaction agent to be carried directly to the breast.
Antibodies of thiε type may alεo be uεed to do in vivo imaging, for example, by labeling the antibodies to facilitate scanning of the breast. One method for imaging comprises contacting any cancer cells of the breaεt to be imaged with an anti-breaεt εpecific gene protein antibody labeled with a detectable marker. The method iε performed under conditions such that the labeled antibody binds to the breast s ecific gene proteins. in a specific example, the antibodies interact with the breast, for example, breast cancer cells, and fluoresce upon such contact such that imaging and visibility of the breast is enhanced to allow a determination of the diseased or non-diseaεed εtate of the breast.
The choice of marker used to label the antibodies will vary depending upon the application. However, the choice of marker is readily determinable to one skilled in the art. These labeled antibodies may be used in immunoassays as well as in histological applications to detect the presence of the proteins. The labeled antibodies may be polyclonal or monoclonal.
The presence of active transcription, which is greater than that normally found, of the breast specific genes in cells other than from the breast, by the presence of an altered level of mRNA, cDNA or expression products is an important indication of the presence of a breaεt cancer which haε metaεtasized, since breast cancer cells are migrating from the breast into the general circulation. Accordingly, thiε phenomenon may have important clinical implications since the method of treating a localized, as opposed to a metastasized, tumor is entirely different.
Of the 20 breast specific genes disclosed, only breast specific gene 1 iε a full-length gene. Breaεt εpecific gene 1 iε 79% identical and 83% similar to human Alzheimer diseaεe amyloid gene. Breast specific gene 2 is 30% identical and 48% similar to human hydroxyindole-o-methyltransferaεe gene. Breaεt specific gene 3 iε 58% identical and 62% εimilar to human 06-methylguanine-DNA methyltransferase gene. Breast specific gene 4 is 34% identical and 65% similar to the mouse pl20 gene. Breast specific gene 5 is 78% identical and 89% similar to human p70 ribosomal S6 kinase alpha-II gene. Breast specific gene 6 is 77% identical and 79% similar to the human transcription factor NFATp gene. As stated previously, the breaεt εpecific geneε of the present invention are putative molecular markers in the diagnosis of breast cancer formation, and breast cancer metastases. As shown in the following Table 1, the presence of the breast specific genes when tested in normal breast, breast cancer, embryo and other cancer libraries, the breast specific geneε of the present invention were found to be most prevalent in the breast cancer library, indicating that the genes of the present invention may be employed for detecting breast cancer, as discussed previously. The table also indicates a putative identification, based on homology, of BSGl through BSG6 to known genes.
Table 1
Figure imgf000022_0001
BSG14 2
BSG15 3
BSG16 1 1 1
BSGl7 2 1
BSGl8 2
BSGl9 1 1
BSG20 2
The asεayε described above may also be used to test whether bone marrow preserved before chemotherapy iε contaminated with micrometastaεeε of a breast cancer cell. In the asεay, blood cells from the bone marrow are isolated and treated as described above, this method allows one to determine whether preserved bone marrow is still suitable for tranεplantation after chemotherapy.
The preεent invention further relates to mature polypeptides, for example the BSGl polypeptide, as well aε fragmentε, analogε and derivatives of such polypeptide.
The terms "fragment," "derivative" and "analog" when referring to the polypeptides encoded by the genes of the invention means a polypeptide which retains essentially the εame biological function or activity as such polypeptide. Thus, an analog includes a proprotein which can be activated by cleavage of the proprotein portion to produce an active mature polypeptide.
The polypeptides of the present invention may be recombinant polypeptides, natural polypeptides or εynthetic polypeptides, preferably recombinant polypeptides.
The fragment, derivative or analog of the polypeptides encoded by the genes of the invention may be (i) one in which one or more of the amino acid residueε are εubεtituted with a conεerved or non-conεerved amino acid reεidue (preferably a conεerved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) one in which one or more of the amino acid reεidueε includes a substituent group, or (iii) one in which the polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol) , or (iv) one in which the additional amino acids are fused to the polypeptide, such as a leader or secretory sequence or a sequence which is employed for purification of the mature polypeptide or a proprotein sequence. Such fragments, derivativeε and analogε are deemed to be within the scope of those skilled in the art from the teachings herein.
The polypeptides and polynucleotides of the present invention are preferably provided in an isolated form, and preferably are purified to homogeneity.
The term "isolated" means that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring) . For example, a naturally- occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide, separated from εome or all of the coexiεting materials in the natural syεtem, iε iεolated. Such polynucleotides could be part of a vector and/or such polynucleotides or polypeptides could be part of a composition, and still be isolated in that such vector or composition is not part of its natural environment.
The polypeptides of the present invention include the polypeptides of Figure 1 (SEQ ID NO:l) (in particular the mature polypeptides) as well as polypeptides which have at least 70% similarity (preferably at leaεt a 70% identity) to the polypeptides of Figure 1 (SEQ ID NO:l) and more preferably at least a 90% similarity (more preferably at least a 90% identity) to the polypeptides of Figures 8 and 9 (SEQ ID NO:8 and 9) and still more preferably at least a 95% similarity (still more preferably at least 95% identity) to the polypeptides of Figure 1 (SEQ ID N0:1) and alεo include portionε of εuch polypeptides with such portion of the polypeptide generally containing at least 30 amino acids and more preferably at least 50 amino acids.
As known in the art "similarity" between two polypeptides is determined by comparing the amino acid sequence and itε conεerved amino acid substitutes of one polypeptide to the sequence of a second polypeptide.
Fragments or portions of the polypeptides of the present invention may be employed for producing the corresponding full-length polypeptide by peptide synthesis; therefore, the fragments may be employed as intermediates for producing the full-length polypeptideε. Fragments or portions of the polynucleotides of the present invention may be used to synthesize full-length polynucleotideε of the preεent invention.
The present invention also relates to vectors which include polynucleotides of the present invention, host cells which are genetically engineered with vectors of the invention and the production of polypeptides of the invention by recombinant techniques.
Host cells are genetically engineered (transduced or transformed or transfected) with the vectors of this invention which may be, for example, a cloning vector or an expression vector. The vector may be, for example, in the form of a plasmid, a viral particle, a phage, etc. The engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, εelecting tranεformantε or amplifying the breast specific genes. The culture conditions, such as temperature, pH and the like, are those previously used with the host cell selected for expresεion, and will be apparent to those of ordinarily skill in the art.
The polynucleotides of the present invention may be employed for producing polypeptides by recombinant techniqueε. Thus, for example, the polynucleotide may be included in any one of a variety of expression vectors for expressing a polypeptide. Such vectors include chromosomal, nonchromosomal and synthetic DNA sequenceε, e.g., derivativeε of SV40; bacterial plaεmidε; phage DNA; baculoviruε; yeaεt plaεmidε; vectors derived from combinations of plasmids and phage DNA, viral DNA such aε vaccinia, adenovirus, fowl pox viruε, and pseudorabies. However, any other vector may be used as long as it is replicable and viable in the host.
The appropriate DNA sequence may be inserted into the vector by a variety of procedures. In general, the DNA sequence is inserted into an appropriate restriction endonuclease εite(ε) by procedures known in the art. Such procedures and others are deemed to be within the scope of thoεe εkilled in the art.
The DNA εequence in the expreεεion vector iε operatively linked to an appropriate expreεsion control sequence(ε) (promoter) to direct mRNA εyntheεiε. Aε repreεentative examples of such promoters, there may be mentioned: LTR or SV40 promoter, the E. coli. lac or trp. the phage lambda PL promoter and other promoters known to control expresεion of geneε in prokaryotic or eukaryotic cellε or their viruεeε. The expression vector also contains a ribosome binding εite for tranεlation initiation and a tranεcription terminator. The vector may alεo include appropriate εequences for amplifying expresεion.
In addition, the expression vectors preferably contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or such as tetracycline or ampicillin resistance in E. coli.
The vector containing the appropriate DNA sequence as hereinabove described, as well as an appropriate promoter or control sequence, may be employed to transform an appropriate host to permit the host to express the protein. As representative examples of appropriate hosts, there may be mentioned: bacterial cells, such as E. coli. Streptomyces, Salmonella typhimurium; fungal cells, such as yeast; inεect cellε such as Drosophila S2 and Spodoptera Sf9; animal cellε εuch aε CHO, COS or Bowes melanoma; adenoviruεeε; plant cellε, etc. The εelection of an appropriate hoεt is deemed to be within the scope of those skilled in the art from the teachings herein.
More particularly, the present invention also includes recombinant constructs comprising one or more of the sequences as broadly described above. The constructs comprise a vector, such as a plasmid or viral vector, into which a εequence of the invention haε been inserted, in a forward or reverse orientation. In a preferred aspect of this embodiment, the construct further comprises regulatory sequenceε, including, for example, a promoter, operably linked to the sequence. Large numbers of suitable vectors and promoters are known to those of skill in the art, and are commercially available. The following vectors are provided by way of example. Bacterial: pQE70, pQE60, pQE-9 (Qiagen) , pBS, pDIO, phagescript, psiXl74, pblueεcript SK, pBSKS, pNH8A, pNH16a, pNH18A, pNH46A (Stratagene) ; ptrc99a, pKK223- 3, pKK233-3, pDR540, pRIT5 (Pharmacia). Eukaryotic: pWLNEO, PSV2CAT, pOG44, pXTl, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia) . However, any other plasmid or vector may be used as long as they are replicable and viable in the host.
Promoter regions can be εelected from any desired gene using CAT (chloramphenicol transferase) vectors or other vectors with selectable markers. Two appropriate vectorε are pKK232-8 and pCM7. Particular named bacterial promoterε include lacl, lacZ, T3, T7, gpt, lambda PR, PL and trp. Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retroviruε, and mouεe metallothionein-I. Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art.
In a further embodiment, the present invention relates to host cells containing the above-described constructs. The host cell can be a higher eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell. Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE- Dextran mediated tranεfection, or eiectroporation (Davis, L. , Dibner, M. , Battey, I., Basic Methods in Molecular Biology, (1986)) .
The constructs in host cellε can be uεed in a conventional manner to produce the gene product encoded by the recombinant sequence. Alternatively, the polypeptides of the invention can be synthetically produced by conventional peptide syntheεizerε.
Proteinε can be expreεεed in mammalian cells, yeast, bacteria, or other cells under the control of appropriate promoters. Cell-free translation syεtems can also be employed to produce εuch proteins using RNAs derived from the DNA constructs of the present invention. Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described by Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989), the disclosure of which is hereby incorporated by reference.
Transcription of the DNA encoding the polypeptides of the present invention by higher eukaryotes is increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp that act on a promoter to increase its transcription. Examples including the SV40 enhancer on the lat^e side of the replication origin bp 100 to 270, a cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.
Generally, recombinant expression vectors will include origins of replication and selectable markers permitting transformation of the host cell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiae TRPl gene, and a promoter derived from a highly-expressed gene to direct transcription of a downstream structural sequence. Such promoters can be derived from operons encoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK) , α-factor, acid phosphataεe, or heat shock proteins, among others. The heterologous structural sequence is asεembled in appropriate phaεe with translation initiation and termination sequences. Optionally, the heterologous sequence can encode a fusion protein including an N-terminal identification peptide imparting desired characteristics, e.g., stabilization or simplified purification of expresεed recombinant product.
Uεeful expression vectors for bacterial use are constructed by inserting a structural DNA sequence encoding a desired protein together with suitable translation initiation and termination signalε in operable reading frame with a functional promoter. The vector will comprise one or more phenotypic selectable markerε and an origin of replication to enεure maintenance of the vector and to, if desirable, provide amplification within the host. Suitable prokaryotic hosts for transformation include E. coli. Bacillus subtilis. Salmonella typhimurium and various specieε within the genera Pseudomonas, Streptomyces, and Staphylococcus, although others may also be employed as a matter of choice.
As a representative but nonlimiting example, uεeful expression vectors for bacterial use can comprise a selectable marker and bacterial origin of replication derived from commercially available plasmids comprising genetic elements of the well known cloning vector pBR322 (ATCC 37017) . Such commercial vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEMl (Promega Biotec, Madison, WI, USA) . These pBR322 "backbone" sectionε are combined with an appropriate promoter and the structural sequence to be expressed.
Following transformation of a suitable host strain and growth of the host strain to an appropriate cell density, the selected promoter is induced by appropriate means (e.g., temperature shift or chemical induction) and cells are cultured for an additional period.
Cellε are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.
Microbial cells employed in expresεion of proteinε can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents, such methods are well know to thoεe εkilled in the art.
Variouε mammalian cell culture εyεtemε can also be employed to expresε recombinant protein. Exampleε of mammalian expreεεion systemε include the COS-7 lineε of monkey kidney fibroblasts, described by Gluzman, Cell, 23:175 (1981) , and other cell lines capable of expressing a compatible vector, for example, the C127, 3T3, CHO, HeLa and BHK cell lines. Mammalian expresεion vectorε will comprise an origin of replication, a suitable promoter and enhancer, and also any necesεary ribosome binding εiteε, polyadenylation εite, εplice donor and acceptor εites, transcriptional termination sequenceε, and 5' flanking nontranscribed sequences. DNA sequenceε derived from the SV40 splice, and polyadenylation siteε may be uεed to provide the required nontranεcribed genetic elementε.
The breaεt εpecific gene polypeptideε can be recovered and purified from recombinant cell cultureε by methodε including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Protein refolding steps can be used, as necessary, in completing configuration of the mature protein. Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps.
The polynucleotides of the present invention may have the coding sequence fused in frame to a marker sequence which allows for purification of the polypeptide of the present invention. An example of a marker sequence is a hexahistidine tag which may be supplied by a vector, preferably a pQE-9 vector, which provides for purification of the polypeptide fused to the marker in the case of a bacterial host, or, for example, the marker sequence may be a hemagglutinin (HA) tag when a mammalian host, e.g. COS-7 cells, iε used. The HA tag correspondε to an epitope derived from the influenza hemagglutinin protein (Wilson, I., et al., Cell, 37:767 (1984)) .
The polypeptides of the present invention may be a naturally purified product, or a product of chemical synthetic procedures, or produced by recombinant techniques from a prokaryotic or eukaryotic host (for example, by bacterial, yeast, higher plant, insect and mammalian cells in culture) . Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or may be non-glycosylated. Polypeptides of the invention may also include an initial methionine amino acid residue.
BSGl, and other breast specific genes, and the protein product thereof may be employed for early detection of breast cancer εince they are over-expresεed in the breast cancer state. In accordance with another aspect of the present invention there are provided asεayε which may be uεed to screen for therapeutics to inhibit the action of the breast specific genes or breast εpecific proteinε of the preεent invention. The present invention discloses methods for selecting a therapeutic which forms a complex with breast specific gene proteins with sufficient affinity to prevent their biological action. The methodε include various assays, including competitive assays where the proteins are immobilized to a support, and are contacted with a natural substrate and a labeled therapeutic either εimultaneously or in either consecutive order, and determining whether the therapeutic effectively competes with the natural substrate in a manner sufficient to prevent binding of the protein to its subεtrate.
In another embodiment, the substrate is immobilized to a support, and is contacted with both a labeled breast specific polypeptide and a therapeutic (or unlabeled proteins and a labeled therapeutic) , and it is determined whether the amount of the breast specific polypeptide bound to the substrate is reduced in comparison to the asεay without the therapeutic added. The breaεt specific polypeptide may be labeled with antibodies.
Potential therapeutic compounds include antibodies and anti-idiotypic antibodieε aε deεcribed above, or in εome cases, an oligonucleotide, which binds to the polypeptide.
Another example is an antisenεe conεtruct prepared uεing antiεenεe technology, which iε directed to a breaεt specific polynucleotide to prevent transcription. Antiεenεe technology can be uεed to control gene expreεεion through triple-helix formation or antisense DNA or RNA, both of which methods are based on binding of a polynucleotide to DNA or RNA. For example, the 5' coding portion of the polynucleotide sequence, which encodes for the mature polypeptides of the preεent invention, iε used to design an antisense RNA oligonucleotide of from about 10 to 40 baεe pairε in length. A DNA oligonucleotide is designed to be complementary to a region of the gene involved in transcription (triple helix -see Lee et al., Nucl. Acids Res., 6:3073 (1979); Cooney et al, Science, 241:456 (1988); and Dervan et al., Science, 251: 1360 (1991)), thereby preventing transcription and the production of a breast specific polynucleotide. The antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks tranεlation of the mRNA molecule into the breast specific genes polypeptide (antisenεe - Okano, J. Neurochem., 56:560 (1991); Oligodeoxynucleotideε aε Antisense Inhibitorε of Gene Expreεεion, CRC Press, Boca Raton, FL (1988)) . The oligonucleotides described above can also be delivered to cells εuch that the antisense RNA or DNA may be expresεed in vivo to inhibit production of the breast specific polypeptides.
Another example is a εmall molecule which bindε to and occupies the active site of the breaεt εpecific polypeptide thereby making the active εite inacceεεible to εubεtrate εuch that normal biological activity is prevented. Examples of small molecules include but are not limited to εmall peptideε or peptide-like molecules.
These compounds may be employed to treat breast cancer, since they interact with the function of breast specific polypeptides in a manner sufficient to inhibit natural function which iε neceεεary for the viability of breast cancer cells. Thiε iε true since the BSGs and their protein productε are primarily expresεed in breaεt cancer tiεεueε and are, therefore, εuεpected of being critical to the formation of this state.
The compounds may be employed in a composition with a pharmaceutically acceptable carrier, e.g., as hereinafter described. The compounds of the present invention may be employed in combination with a εuitable pharmaceutical carrier. Such compoεitions compriεe a therapeutically effective amount of the polypeptide, and a pharmaceutically acceptable carrier or excipient. Such a carrier includes but is not limited to saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The formulation should suit the mode of administration.
The invention also provides a pharmaceutical pack or kit compriεing one or more containers filled with one or more of the ingredients of the pharmaceutical compositionε of the invention. Associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration. In addition, the pharmaceutical compositions may be employed in conjunction with other therapeutic compounds.
The pharmaceutical compositions may be administered in a convenient manner such as by the oral, topical, intravenous, intraperitoneal, intramuscular, subcutaneouε, intranaεal, intra-anal or intradermal routes. The pharmaceutical compositions are administered in an amount which is effective for treating and/or prophylaxis of the specific indication. In general, they are administered in an amount of at least about 10 μg/kg body weight and in most caεes they will be administered in an amount not in excesε of about 8 mg/Kg body weight per day. In moεt caεeε, the doεage iε from about 10 μg/kg to about l mg/kg body weight daily, taking into account the routeε of adminiεtration, εymptomε, etc.
The breaεt specific genes and compounds which are polypeptides may also be employed in accordance with the present invention by expreεεion of εuch polypeptideε in vivo, which iε often referred to aε "gene therapy." Thus, for example, cells from a patient may be engineered with a polynucleotide (DNA or RNA) encoding a polypeptide ex vivo, with the engineered cells then being provided to a patient to be treated with the polypeptide. Such methods are well-known in the art. For example, cells may be engineered by procedures known in the art by use of a retroviral particle containing RNA encoding a polypeptide of the present invention.
Similarly, cells may be engineered in vivo for expression of a polypeptide in vivo by, for example, procedures known in the art. As known in the art, a producer cell for producing a retroviral particle containing RNA encoding a polypeptide of the present invention may be administered to a patient for engineering cells in vivo and expression of the polypeptide in vivo. These and other methods for administering a polypeptide of the present invention by such method should be apparent to those skilled in the art from the teachings of the present invention. For example, the expression vehicle for engineering cells may be other than a retrovirus, for example, an adenovirus which may be used to engineer cells in vivo after combination with a suitable delivery vehicle.
Retroviruses from which the retroviral plasmid vectors hereinabove mentioned may be derived include, but are not limited to, Moloney Murine Leukemia Virus, spleen necrosiε viruε, retroviruεes such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis viruε, gibbon ape leukemia virus, human immunodeficiency virus, adenovirus, Myeloproliferative Sarcoma Virus, and mammary tumor virus. In one embodiment, the retroviral plasmid vector is derived from Moloney Murine Leukemia Virus.
The vector includes one or more promoterε. Suitable promoters which may be employed include, but are not limited to, the retroviral LTR; the SV40 promoter; and the human cytomegalovirus (CMV) promoter described in Miller, et al., Biotechniques. Vol. 7, No. 9, 980-990 (1989) , or any other promoter (e.g., cellular promoters such as eukaryotic cellular promoters including, but not limited to, the histone, pol III, and β-actin promoterε) . Other viral promoterε which may be employed include, but are not limited to, adenovirus promoters, thymidine kinase (TK) promoters, and B19 parvovirus promoters. The selection of a suitable promoter will be apparent to those skilled in the art from the teachings contained herein.
The nucleic acid sequence encoding the polypeptide of the present invention is under the control of a suitable promoter. Suitable promoters which may be employed include, but are not limited to, adenoviral promoters, such as the adenoviral major late promoter; or heterologous promoters, such as the cytomegalovirus (CMV) promoter; the respiratory syncytial virus (RSV) promoter; inducible promoters, such as the MMT promoter, the metallothionein promoter; heat shock promoters; the albumin promoter; the ApoAI promoter; human globin promoters,- viral thymidine kinase promoters, such as the Herpes Simplex thymidine kinase promoter; retroviral LTRs (including the modified retroviral LTRs hereinabove deεcribed) ; the β-actin promoter; and human growth hormone promoterε. The promoter alεo may be the native promoter which controlε the geneε encoding the polypeptideε.
The retroviral plaεmid vector is employed to transduce packaging cell lines to form producer cell lines. Examples of packaging cells which may be transfected include, but are not limited to, the PE501, PA317, ψ-2 , φ-AM, PA12, T19-14X, VT-19-17-H2, ψCRE, ψCRIP, GP+E-86, GP+envAml2, and DAN cell lines as described in Miller, Human Gene Therapy. Vol. 1, pgs. 5-14 (1990), which is incorporated herein by reference in its entirety. The vector may transduce the packaging cells through any means known in the art. Such means include, but are not limited to, eiectroporation, the use of liposomes, and CaP04 precipitation. In one alternative, the retroviral plasmid vector may be encapsulated into a liposome, or coupled to a lipid, and then administered to a hoεt.
The producer cell line generateε infectiouε retroviral vector particleε which include the nucleic acid sequence(s) encoding the polypeptides. Such retroviral vector particles then may be employed, to transduce eukaryotic cells, either in vitro or in vivo. The transduced eukaryotic cells will express the nucleic acid sequence(s) encoding the polypeptide. Eukaryotic cells which may be transduced include, but are not limited to, embryonic εtem cells, embryonic carcinoma cells, as well as hematopoietic stem cells, hepatocytes, fibroblasts, myoblastε, keratinocytes, endothelial cells, and bronchial epithelial cells.
This invention is also related to the use of a breast specific genes of the present invention as a diagnostic. For example, some diseaseε reεult from inherited defective genes. The breast specific geneε, CSG7 and CSG10, for example, have been found to have a reduced expreεεion in breaεt cancer cells as compared to that in normal cells. Further, the remaining breast specific genes of the present invention are overexpressed in breast cancer. Accordingly, a mutation in these genes allows a detection of breast diεorders, for example, breast cancer. A mutation in a breast specific gene of the present invention at the DNA level may be detected by a variety of techniques. Nucleic acids used for diagnosis (genomic DNA, mRNA, etc.) may be obtained from a patient's cells, other than from the breast, such as from blood, urine, saliva, tisεue biopεy and autopεy material. The genomic DNA may be uεed directly for detection or may be amplified enzymatically by uεing PCR (Saiki, et al., Nature, 324:163- 166 (1986)) prior to analyεiε. RNA or cDNA may alεo be uεed for the εame purpose. As an example, PCR primers complementary to the nucleic acid of the instant invention can be used to identify and analyze mutations in a breast specific polynucleotide of the present invention. For example, deletions and insertions can be detected by a change in size of the amplified product in comparison to the normal genotype. Point mutations can be identified by hybridizing amplified DNA to radiolabelled breast specific RNA or, alternatively, radiolabelled antisense DNA sequences.
Another well-established method for screening for mutations in particular segmentε of DNA after PCR amplification is single-strand conformation polymorphism (SSCP) analysis. PCR productε are prepared for SSCP by ten cycles of reamplification to incorporate 32P-dCTP, digested with an appropriate restriction enzyme to generate 200-300 bp fragments, and denatured by heating to 85°C for 5 min. and then plunged into ice. Electrophoresis iε then carried out in a nondenaturing gel (5% glycerol, 5% acrylamide) (Glavac, D. and Dean, M. , Human Mutation, 2:404-414 (1993)) .
Sequence differenceε between the reference gene and "mutantε" may be revealed by the direct DNA sequencing method. In addition, cloned DNA εegmentε may be used as probes to detect specific DNA εegmentε. The sensitivity of this method is greatly enhanced when combined with PCR. For example, a sequencing primer is used with double-stranded PCR product or a single-stranded template molecule generated by a modified PCR. The sequence determination is performed by conventional procedures with -radiolabeled nucleotides or by automatic sequencing procedures with fluorescent-tagε.
Genetic teεting baεed on DNA sequence differences may be achieved by detection of alteration in electrophoretic mobility of DNA fragments and gels with or without denaturing agents. Small sequence deletions and insertions can be visualized by high-resolution gel electrophoresis. DNA fragmentε of different sequences may be diεtinguiεhed on denaturing formamide gradient gelε in which the mobilities of different DNA fragments are retarded in the gel at different positionε according to their specific melting or partial melting temperatures (see, e.g., Myers, et al., Science. 230:1242 (1985)). In addition, sequence alterations, in particular small deletions, may be detected as changes in the migration pattern of DNA.
Sequence changes at specific locations may also be revealed by nuclease protection asεayε, such as Rnase and Si protection or the chemical cleavage method (e.g., Cotton, et al., PNAS. USA. 85:4397-4401 (1985)).
Thus, the detection of the specific DNA sequence may be achieved by methods such as hybridization, RNase protection, chemical cleavage, direct DNA sequencing, or the use of restriction enzymes (e.g., Restriction Fragment Length Polymorphisms (RFLP)) and Southern blotting.
The sequenceε of the present invention are also valuable for chromosome identification. The εequence iε specifically targeted to and can hybridize with a particular location on an individual human chromosome. Moreover, there is a current need for identifying particular sites on the chromosome. Few chromosome marking reagents based on actual sequence data (repeat polymorphisms) are presently available for marking chromosomal location. The mapping of DNAε to chromoεomeε according to the preεent invention iε an important firεt εtep in correlating thoεe εequenceε with geneε aεεociated with diεease.
Briefly, sequences can be mapped to chromosomeε by preparing PCR primerε (preferably 15-25 bp) from the cDNA. Computer analysiε of the 3' untranslated region is used to rapidly select primers that do not span more than one exon in the genomic DNA, thus complicating the amplification process. These primers are then used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the primer will yield an amplified fragment.
PCR mapping of εomatic cell hybridε iε a rapid procedure for aεsigning a particular DNA to a particular chromosome. Using the present invention with the same oligonucleotide primers, sublocalization can be achieved with panels of fragments from specific chromosomeε or pools of large genomic clones in an analogous manner. Other mapping strategies that can similarly be used to map to itε chromosome include in si tu hybridization, prescreening with labeled flow-sorted chromosomeε and preεelection by hybridization to conεtruct chromosome specific-cDNA libraries.
Fluorescence in situ hybridization (FISH) of a cDNA clone to a metaphase chromosomal spread can be used to provide a precise chromoεomal location in one step. This technique can be used with cDNA as short as 50 or 60 bases. For a review of this technique, see Verma et al., Human Chromosomeε: a Manual of Baεic Techniqueε, Pergamon Press, New York (1988) .
Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. Such data are found, for example, in V. McKusick, Mendelian Inheritance in Man (available on line through Johnε Hopkins University Welch Medical Library) . The relationship between genes and diseases that have been mapped to the same chromosomal region are then identified through linkage analysis (coinheritance of physically adjacent genes) .
Next, it is necessary to determine the differences in the cDNA or genomic εequence between affected and unaffected individualε. If a mutation is observed in some or all of the affected individuals but not in any normal individuals, then the mutation is likely to be the causative agent of the disease.
With current resolution of physical mapping and genetic mapping techniques, a cDNA precisely localized to a chromosomal region associated with the disease,^ould be one of between 50 and 500 potential causative genes. (Thiε asεumeε 1 megabase mapping resolution and one gene per 20 kb) .
The polypeptides, their fragments or other derivatives, or analogs thereof, or cells expresεing them can be uεed as an immunogen to produce antibodies thereto. These antibodies can be, for example, polyclonal or monoclonal antibodies. The present invention also includes chimeric, single chain, and humanized antibodies, as well as Fab fragments, or the product of an Fab expresεion library. Variouε procedureε known in the art may be used for the production of such antibodies and fragments.
Antibodies generated against the polypeptides correεponding to a εequence of the preεent invention can be obtained by direct injection of the polypeptides into an animal or by administering the polypeptides to an animal, preferably a nonhuman. The antibody so obtained will then bind the polypeptides itself. In this manner, even a εequence encoding only a fragment of the polypeptideε can be used to generate antibodies binding the whole native polypeptideε. Such antibodies can then be used to isolate the polypeptide from tissue expressing that polypeptide.
For preparation of monoclonal antibodies, any technique which provideε antibodieε produced by continuouε cell line cultureε can be used. Examples include the hybridoma technique (Kohler and Milstein, 1975, Nature, 256:495-497), the trioma technique, the human B-cell hybridoma technique (Kozbor et al., 1983, Immunology Today 4:72), and the EBV- hybridoma technique to produce human monoclonal antibodies (Cole, et al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).
Techniques described for the production of εingle chain antibodies (U.S. Patent 4,946,778) can be adapted to produce single chain antibodies to immunogenic polypeptide products of this invention. Transgenic mice may also be uεed to generate antibodieε. The antibodies may also be employed to target breast cancer cellε, for example, in a method of homing interaction agentε which, when contacting breaεt cancer cells, destroy them. This is true since the antibodies are specific for the breaεt specific polypeptides of the preεent invention. A linking of the interaction agent to the antibody would cause the interaction agent to be carried directly to the breast.
Antibodies of this type may also be used to do in vivo imaging, for example, by labeling the antibodies to facilitate scanning of the pelvic area and the breast. One method for imaging compriεeε contacting any cancer cells of the breast to be imaged with an anti-breast specific protein- antibody labeled with a detectable marker. The method is performed under conditionε such that the labeled antibody binds to the breast specific polypeptides. In a specific example, the antibodies interact with the breaεt, for example, breast cancer cellε, and fluoreεce upon contact such that imaging and visibility of the breast are enhanced to allow a determination of the diseased or non-diseaεed εtate of the breaεt.
The preεent invention will be further deεcribed with reference to the following exampleε; however, it iε to be underεtood that the present invention is not limited to such examples. All parts or amounts, unless otherwise specified, are by weight.
In order to facilitate understanding of the following examples certain frequently occurring methods and/or terms will be described.
"Plasmids" are designated by a lower case p preceded and/or followed by capital letters and/or numbers. The starting plasmidε herein are either commercially available, publicly available on an unreεtricted baεis, or can be constructed from available plasmidε in accord with publiεhed procedures. In addition, equivalent plasmidε to those described are known in the art and will be apparent to the ordinarily εkilled artisan.
"Digestion" of DNA refers to catalytic cleavage of the DNA with a reεtriction enzyme that acts only at certain sequences in the DNA. The various restriction enzymes used herein are commercially available and their reaction conditions, cofactors and other requirements were used as would be known to the ordinarily skilled artisan. For analytical purposes, typically 1 μg of plasmid or DNA fragment is used with about 2 units of enzyme in about 20 μl of buffer solution. For the purpose of isolating DNA fragments for plasmid construction, typically 5 to 50 μg of DNA are digested with 20 to 250 units of enzyme in a larger volume. Appropriate buffers and subεtrate amountε for particular restriction enzymes are specified by the manufacturer. Incubation times of about 1 hour at 37"C are ordinarily used, but may vary in accordance with the supplier's instructions. After digestion the reaction is electrophoresed directly on a polyacrylamide gel to isolate the desired fragment.
Size separation of the cleaved fragments is performed using 1 percent TAE agarose gel described by Sambrook, et al., "Molecular Cloning: A Laboratory Manual" Cold Spring Laboratory Press, (1989) .
"Oligonucleotides" refers to either a single stranded polydeoxynucleotide or two complementary polydeoxynucleotide strands which may be chemically syntheεized. Such synthetic oligonucleotides have no 5' phosphate and thus will not ligate to another oligonucleotide without adding a phosphate with an ATP in the presence of a kinase. A synthetic oligonucleotide will ligate to a fragment that has not been dephosphorylated.
"Ligation" refers to the procesε of forming phosphodiester bonds between two double stranded nucleic acid fragmentε (Maniatis, T., et al., Id., p. 146). Unless otherwise provided, ligation may be accomplished using known buffers and conditionε with 10 unitε of T4 DNA ligase ("ligase") per 0.5 μg of approximately equimolar amountε of the DNA fragmentε to be ligated.
Unless otherwise stated, transformation was performed as described in the method of Graham, F. and Van der Eb, A., Virology, 52:456-457 (1973).
Example 1 Determination of Transcription of a breast specific gene
To assess the presence or absence of active tranεcription of a breast specific gene RNA, approximately 6 ml of venous blood is obtained with a standard venipuncture technique using heparinized tubes. Whole blood is mixed with an equal volume of phosphate buffered saline, which is then layered over 8 ml of Ficoll (Pharmacia, Uppsala, Sweden) in a 15-ml polystyrene tube. The gradient is centrifuged at 1800 X g for 20 min at 5°C. The lymphocyte and granulocyte layer (approximately 5 ml) is carefully aspirated and rediluted up to 50 ml with phosphate-buffered saline in a 50- ml tube, which is centrifuged again at 1800 X g for 20 min. at 5°C. The εupernatant is discarded and the pellet containing nucleated cells is used for RNA extraction using the RNazole B method aε deεcribed by the manufacturer (Tel- Teεt Inc., Friendεwood, TX) .
To determine the quantity of mRNA from the gene of interest, a probe is designed with an identity to at least a portion of the mRNA sequence transcribed from a human gene whose coding portion includes a DNA sequence of Figures 1-20 (SEQ ID NO:1-20) . This probe is mixed with the extracted RNA and the mixed DNA and RNA are precipitated with ethanol -70°C for 15 minutes) . The pellet is resuspended in hybridization buffer and diεsolved. The tubes containing the mixture are incubated in a 72°C water bath for 10-15 mins. to denature the DNA. The tubes are rapidly transferred to a water bath at the desired hybridization temperature. Hybridization temperature dependε on the G + C content of the DNA. Hybridization iε done for 3 hrε. 0.3 ml of nucleaεe-Sl buffer iε added and mixed well. 50 μl of 4.0 M ammonium acetate and 0.1 M EDTA iε added to stop the reaction. The mixture iε extracted with phenol/chloroform and 20 μg of carrier tRNA is added and precipitation is done with an equal volume of isopropanol. The precipitate is dissolved in 40 μl of TE (pH 7.4) and run on an alkaline agarose gel. Following electrophoresis, the RNA is microsequenced to confirm the nucleotide sequence. (See Favaloro, J. et al., Methods Enzymol., 65:718 (1980) for a more detailed review).
Two oligonucleotide primers are employed to amplify the sequence isolated by the above methods. The 5' primer is 20 nucleotides long and the 3' primer is a complimentary εequence for the 3' end of the iεolated mRNA. The primerε are cuεtom deεigned according to the iεolated mRNA. The reverεe tranεcriptaεe reaction and PCR amplification are performed εequentially without interruption in a Perkin Elmer 9600 PCR machine (Emeryville, CA) . Four hundred ng total RNA in 20 μl diethylpyrocarbonate-treated water are placed in a 65°C water bath for 5 min. and then quickly chilled on ice immediately prior to the addition of PCR reagentε. The 50-μl total PCR volume conεiεted of 2.5 unitε Taq polymerase (Perkin-Elmer) . 2 units avian myeloblastoεiε virus reverse transcriptase (Boehringer Mannheim, Indianapolis, IN); 200 μM each of dCTP, dATP, dGTP and dTTP (Perkin Elmer) ; 18 pM each primer, 10 mM Tris-HCl; 50 mM KCI; and 2 mM MgCl2 (Perkin Elmer). PCR conditions are as follows: cycle 1 iε 42°C for 15 min then 97°C for 15 ε (1 cycle) ; cycle 2 iε 95°C for 1 min. 60°C for 1 min, and 72°C for 30 ε (15 cycles) ; cycle 3 is 95°C for 1 min. 60°C for 1 min., and 72°C for 1 min. (10 cycles); cycle 4 is 95°C for 1 min., 60°C for 1 min., and 72°C for 2 min. (8 cycles) ; cycle 5 is 72°C for 15 min. (1 cycle) ,- and the final cycle is a 4°C hold until sample is taken out of the machine. The 50-μl PCR products are concentrated down to 10 μl with vacuum centrifugation, and a sample iε then run on a thin 1.2 % Triε-borate-EDTA agarose gel containing ethidium bromide. A band of expected size would indicate that this gene is present in the tissue assayed. The amount of RNA in the pellet may be quantified in numerous wayε, for example, it may be weighed.
Verification of the nucleotide εequence of the PCR products is done by microsequencing. The PCR product is purified with a Qiagen PCR Product Purification Kit (Qiagen, Chatsworth, CA) as described by the manufacturer. One μg of the PCR product undergoes PCR sequencing by using the Taq DyeDeoxy Terminator Cycle sequencing kit in a Perkin-Elmer 9600 PCR machine as described by Applied Biosyεtemε (Foεter, CA) . The sequenced product is purified using Centri-Sep columns (Princeton Separations, Adelphia, NJ) as described by the company. This product is then analyzed with an ABI model 373A DNA sequencing syεtem (Applied Bioεystems) integrated with a Macintosh Uci computer.
Example 2 Bacterial Expreεsion and Purification of the BSG Proteins and Use For Preparing a Monoclonal Antibody
The DNA sequence encoding a polypeptide of the present invention, for this example BSGl, ATCC # 97175, iε initially amplified uεing PCR oligonucleotide primerε corresponding to the 5' sequences of the protein and the vector sequences 3' to the protein. Additional nucleotides corresponding to the DNA sequence are added to the 5' and 3' sequenceε respectively. The 5' oligonucleotide primer has the sequence 5' GCCΑCCΑTGGATGTTTTCAAG 3' (SEQ ID NO:21) and contains an Ncol restriction enzyme site followed by 15 nucleotides of coding sequence starting from the initial aminp acid of the processed protein. The 3' sequence 5' GCGCAGATCTGTCT CCCCCACTCTGGGC 3' (SEQ ID NO:22) and contains a complementary εequence to a Bglll restriction enzyme εite and iε followed by 18 nucleotides of the nucleic acid sequence encoding the protein. The restriction enzyme sites correspond to the restriction enzyme siteε on a bacterial expreεεion vector, pQE-60 (Qiagen, Inc. Chatsworth, CA) . pQE-60 encodes antibiotic resistance (Ampr) , a bacterial origin of replication (ori) , an IPTG-regulatable promoter operator (P/O) , a ribosome binding site (RBS) , a 6-His tag and reεtriction enzyme sites. pQE-60 is then digested with Ncol and Bglll. The amplified sequenceε are ligated into pQE-60 and inεerted in frame with the εequence encoding for the hiεtidine tag and the RBS. The ligation mixture is then used to transform an E. coli strain M15/rep 4 (Qiagen) by the procedure described in Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, Cold Spring Laboratory Press, (1989) . M15/rep4 contains multiple copies of the plasmid pREP4, which expresseε the lacl repressor and also confers kanamycin resistance (Kanr) . Transformants are identified by their ability to grow on LB plates and ampicillin/kanamycin reεiεtant colonieε are εelected. Plasmid DNA is isolated and confirmed by restriction analysis.
Clones containing the desired constructs are grown overnight (O/N) in liquid culture in LB media εupplemented with both Amp (100 ug/ml) and Kan (25 ug/ml) . The O/N culture is used to inoculate a large culture at a ratio of 1:100 to 1:250. The cells are grown to an optical density 600 (O.D.600) of between 0.4 and 0.6. IPTG ("Isopropyl-B-D- thiogalacto pyranoεide") iε then added to a final concentration of 1 mM. IPTG induces by inactivating the lacl repressor, clearing the P/O leading to increased gene expression. Cells are grown an extra 3 to 4 hourε. Cellε are then harveεted by centrifugation. The cell pellet iε εolubilized in the chaotropic agent 6 Molar Guanidine HCl. After clarification, εolubilized protein iε purified from this solution by chromatography on a Nickel-Chelate column under conditions that allow for tight binding by proteins containing the 6-His tag (Hochuli, E. et al., J. Chromatography 411:177-184 (1984)). BSGl protein (>90% pure) is eluted from the column in 6 molar guanidine HCl pH 5.0 and for the purpose of renaturation adjusted to 3 molar guanidine HCl, lOOmM sodium phosphate, 10 mmolar giutathione (reduced) and 2 mmolar giutathione (oxidized) . After incubation in this solution for 12 hours the protein is dialyzed to 10 mmolar sodium phosphate.
The protein purified in this manner may be used as an epitope to raise monoclonal antibodies specific to such protein. The monoclonal antibodies generated against the polypeptide the isolated protein can be obtained by direct injection of the polypeptides into an animal or by administering the polypeptides to an animal. The antibodies so obtained will then bind to the protein itself. Such antibodies can then be used to isolate the protein from tisεue expressing that polypeptide by the use of an, for example, ELISA assay.
Example 3 Preparation of cDNA Libraries from Breaεt Tiεsue
Total cellular RNA iε prepared from tissueε by the guanidinium-phenol method as previously described (P. Chomczynski and N. Sacchi, Anal. Biochem., 162: 156-159 (1987)) using RNAzol (Cinna-Biotecx) . An additional ethanol precipitation of the RNA is included. Poly A mRNA is isolated from the total RNA using oligo dT-coated latex beads (Qiagen) . Two rounds of poly A εelection are performed to ensure better separation from non-polyadenylated material when sufficient quantities of total RNA are available.
The mRNA selected on the oligo dT is used for the synthesiε of cDNA by a modification of the method of Gobbler and Hoffman (Gobbler, U. and B.J. Hoffman, 1983, Gene, 25.:263) . The first strand synthesis is performed using either Moloney murine εarcoma viruε reverεe tranεcriptase (Stratagene) or Superscript II (RNase H minus Moloney murine reverεe tranεcriptaεe, Gibco-BRL) . Firεt strand syntheεiε iε primed uεing a primer/linker containing an Xho I reεtriction site. The nucleotide mix used in the synthesis contains methylated dCTP to prevent restriction within the cDNA sequence. For second-strand synthesis E. coli polymerase Klenow fragment is uεed and [32P] -dATP iε incorporated aε a tracer of nucleotide incorporation.
Following 2nd εtrand synthesis, the cDNA iε made blunt ended uεing either T4 DNA polymerase or Klenow fragment. Eco RI adapters are added to the cDNA and the cDNA is restricted with Xho I. The cDNA is εize fractionated over a Sephacryl S-500 column (Pharmacia) to remove exceεε linkerε and cDNAs under approximately 500 base pairs.
The cDNA is cloned unidirectionally into the Eco RI-Xho I sites of either pBluescript II phagemid or lambda Uni-zap XR (Stratagene) . In the case of cloning into pBluescript II, the plasmids are electroporated into E.coli SURE competent cellε (Stratagene) . When the cDNA iε cloned into Uni-Zap XR it iε packaged uεing the Gigipack II packaging extract (Stratagene) . The packaged phage iε used to infect SURE cells and amplified. The pBlueεcript phagemid containing the cDNA inεerts are excised from the lambda Zap phage using the helper phage ExAsεiεt (Stratagene) . The rescued phagemid is plated on SOLR E.coli cells (Stratagene) . Preparation of Sequencing Templates
, Template DNA for sequencing is prepared by 1) a boiling method or 2) PCR amplification.
The boiling method is a modification of the method of Holmes and Quigley (Holmes, D.S. and M. Quigley, 1981, Anal. Biochem., 114:193) . Colonies from either cDNA cloned into Bluescript II or reεcued Blueεcript phagemid are grown in an enriched bacterial media overnight. 400 μl of cells are centrifuged and resuεpended in STET (O.lM NaCl, lOmM TRIS Ph 8.0, 1.0 mM EDTA and 5% Triton X-100) including lysozyme (80 μg/ml) and RNase A (4 μg/ml) . Cells are boiled for 40 secondε and centrifuged for 10 minuteε. The supernatant is removed and the DNA is precipitated with PEG/NaCl and washed with 70% ethanol (2x) . Templates are resuspended in water at approximately 250 ng/μl.
Preparation of templates by PCR iε a modification of the method of Rosenthal et al. (Rosenthal, et al., Nucleic Acids Res., 1993, 21:173-174). Colonies containing cDNA cloned into pBluescript II or rescued pBluescript phagemid are grown overnight in LB containing ampicillin in a 96 well tisεue culture plate. Two μl of the cultureε are uεed aε template in a PCR reaction (Saiki, RK, et al., Science, 239:487-493. 1988; and Saiki, RK, et al., Science, 23_0:1350-1354, 1985) uεing a tricine buffer εyεtem (Ponce and Micol., Nucleic Acids Res., 1992, 2_0:1992.) and 200 μM dNTPs. The primer εet choεen for amplification of the templateε is outside of primer sites chosen for sequencing of the templates. The primers used are 5'-ATGCTTCCGGCTCGTATG-3' (SEQ ID NO:23) which is 5' of the M13 reverse sequence in pBluescript and 5'-GGGTTTTCCCAGTCACGAC-3' (SEQ ID NO:24) which is 3' Of the M13 forward primer in pBluescript. Any primerε which correεpond to the sequence flanking the M13 forward and reverse sequenceε can be uεed. Perkin-Elmer 9600 thermocyclerε are uεed for amplification of the templates with the following cycler conditions: 5 min at 94°C (1 cycle) ; (20 sec at 94°C) ; 20 sec at 55°C (l min at 72°C) (30 cycles) ; 7 min at 72°C (1 cycle) . Following amplification the PCR templates are precipitated using PEG/NaCl and washed three times with 70% ethanol. The templates are resuspended in water.
Example 4 Isolation of a Selected Clone From Breaεt Tiεεue Two approaches are uεed to iεolate a particular clone from a cDNA library prepared from human breaεt tiεεue.
In the first, a clone is isolated directly by screening the library using an oligonucleotide probe. To isolate a particular clone, a specific oligonucleotide with 30-40 nucleotides is synthesized using an Applied Biosyεtemε DNA synthesizer according to one of the partial sequences described in this application. The oligonucleotide is labeled with 32P- -ATP uεing T4 polynucleotide kinase and purified according to the standard protocol (Maniatis et al. , Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring, NY, 1982) . The Lambda cDNA library is plated on 1.5% agar plate to a density of 20,000-50,000 pfu/150 mm plate. These plates are screened using Nylon membranes according to the standard phage screening protocol (Stratagene, 1993) . Specifically, the Nylon membrane with denatured and fixed phage DNA is prehybridized in 6 x SSC, 20 mM NaH2P04, 0.4% SDS, 5 x Denhardt's 500 μg/ml denatured, sonicated salmon sperm DNA; and 6 x SSC, 0.1% SDS. After one hour of prehybridization, the membrane is hybridized with hybridization buffer 6 x SSC, 20 mM NaH2P04, 0.4% SDS, 500 μg/ml denatured, sonicated salmon sperm DNA with 1 x IO6 cpm/ml 32P-probe overnight at 42°C. The membrane is washed at 45-50°C with washing buffer 6 x SSC, 0.1% SDS for 20-30 minuteε dried and exposed to Kodak X-ray film overnight. Positive clones are isolated and purified by secondary and tertiary screening. The purified clone sequenced to verify its identity to the partial sequence described in this application.
An alternative approach to screen the cDNA library prepared from human breast tissue is to prepare a DNA probe corresponding to the entire partial sequence. To prepare a probe, two oligonucleotide primers of 17-20 nucleotides derived from both endε of the partial εequence reported are εyntheεized and purified. These two oligonucleotides are used to amplify the probe using the cDNA library template. The DNA template is prepared from the phage lysate of the cDNA library according to the εtandard phage DNA preparation protocol (Maniatis et al.) . The polymerase chain reaction iε carried out in 25 μl reaction mixture with 0.5 μg of the above cDNA template. The reaction mixture is 1.5-5 mM MgCl2, 0.01% (w/v) gelatin, 20 μM each of dATP, dCTP, dGTP, dTTP, 25 pmol of each primer and 0.25 Unit of Taq polymerase. Thirty five cycles of PCR (denaturation at 94°C for 1 min; annealing at 55°C for 1 min; elongation at 72°C for 1 min) are performed with the Perkin-Elmer Cetus automated thermal cycler. The amplified product is analyzed by agarose gel electrophoresis and the DNA band with expected molecular weight is excised and purified. The PCR product is verified to be the probe by subcloning and sequencing the DNA product. The probe is labeled with the Multiprime DNA Labelling System (Amersham) at a εpecific activity < 1 x IO9 dmp/μg. This probe is uεed to εcreen the lambda cDNA library according to Stratagene'ε protocol. Hybridization iε carried out with 5X TEN 920XTEN:0.3M Triε-HCl pH 8.0, 0.02M EDTA and 3MNaCl) , 5X Denhardt'ε, 0.5% εodium pyrophoεphate, 0.1% SDS, 0.2 mg/ml heat denatured salmon sperm DNA and l x IO6 cpm/ml of [32P] - labeled probe at 55°C for 12 hourε. The filters are washed in 0.5X TEN at room temperature for 20-30 min., then at 55°C for 15 min. The filters are dried and autoradiographed at - 70°C using Kodak XAR-5 film. The positive clones are purified by secondary and tertiary εcreening. The εequence of the iεolated clone are verified by DNA sequencing.
General procedures for obtaining complete sequences from partial εequences deεcribed herein are εummarized aε followε,- Procedure 1
Selected human DNA from the partial εequence clone (the cDNA clone that waε εequenced to give the partial εequence) is purified e.g. , by endonuclease digeεtion using Eco-Rl, gel electrophoresiε, and iεolation of the clone by removal from low melting agarose gel. The isolated insert DNA, is radiolabeled e.g., with 32P labels, preferably by nick tranεlation or random primer labeling. The labeled inεert iε uεed as a probe to screen a lambda phage cDNA library or a plasmid cDNA library. Colonies containing clones related to the probe cDNA are identified and purified by known purification methods. The endε of the newly purified cloneε are nucleotide sequenced to identify full length sequenceε. Complete sequencing of full length clones iε then performed by Exonucleaεe III digestion or primer walking. Northern blots of the mRNA from various tisεues using at least part of the depoεited clone from which the partial sequence is obtained as a probe can optionally be performed to check the size of the mRNA against that of the purported full length CDNA.
The following procedures 2 and 3 can be used to obtain full length genes or full length coding portions of genes where a clone isolated from the deposited clone mixture doeε not contain a full length εequence. A library derived from human breaεt tiεεue or from the depoεited clone mixture iε also applicable to obtaining full length sequenceε from cloneε obtained from εourceε other than the depoεited mixture by uεe of the partial εequences of the present invention.
Procedure 2
RACE Protocol For Recovery of Full-Length Genes
Partial cDNA cloneε can be made full-length by utilizing the rapid amplification of cDNA endε (RACE) procedure described in Frohman, M.A. , Dush, M.K. and Martin, G.R. (1988) Proc. Nat'l. Acad. Sci. USA, 85:8998-9002. A cDNA clone misεing either the 5' or 3' end can be reconεtructed to include the abεent baεe pairε extending to the tranεlational εtart or εtop codon, reεpectively. In most caseε, cDNAε are miεsing the start of translation therefor. The following briefly describes a modification of this original 5' RACE procedure. Poly A+ or total RNA is reverεe transcribed with Superscript II (Gibco/BRL) and an antisense or complementary primer specific to the cDNA sequence. The primer is removed from the reaction with a Microcon Concentrator (Amicon) . The first-strand cDNA is then tailed with dATP and terminal deoxynucleotide transferase (Gibco/BRL) . Thuε, an anchor sequence is produced which is needed for PCR amplification. The second strand is synthesized from the dA-tail in PCR buffer, Taq DNA polymerase (Perkin-Elmer Cetus) , an oligo-dT primer containing three adjacent restriction sites (Xhol. Sail and Clal) at the 5' end and a primer containing just these restriction sites. This double-stranded cDNA is PCR amplified for 40 cycles with the same primers as well as a nested cDNA-εpecific antiεenεe primer. The PCR productε are εize-εeparated on an ethidium bromide-agarose gel and the region of gel containing cDNA products the predicted size of missing protein-coding DNA is removed. cDNA is purified from the agarose with the Magic PCR Prep kit (Promega) , restriction digested with Xhol or Sail, and ligated to a plasmid such as pBluescript SKII (Stratagene) at Shol and EcoRV siteε. Thiε DNA iε tranεformed into bacteria and the plasmid clones sequenced to identify the correct protein- coding insertε. Correct 5' endε are confirmed by comparing thiε εequence with the putatively identified homologue and overlap with the partial cDNA clone.
Several quality-controlled kitε are available for purchase. Similar reagents and methods to those above are supplied in kit form from Gibco/BRL. A second kit is available from Clontech which iε a modification of a related technique, SLIC (εingle-εtranded ligation to εingle-εtranded cDNA) developed by Dumas et al. (Dumas, J.B., Edwards, M. , Delort, J. and Mallet, Jr., 1991, Nucleic Acids Res., 19:5227-5232) . The major differences in procedure are that the RNA is alkaline hydrolyzed after reverse transcription and RNA ligaεe is used to join a reεtriction site-containing anchor primer to the first-εtrand cDNA. This obviates the necessity for the dA-tailing reaction which results in a polyT stretch that is difficult to εequence past.
An alternative to generating 5' cDNA from RNA is to use cDNA library double-εtranded DNA. An asymmetric PCR- amplified antisenεe cDNA strand is εyntheεized with an antiεense cDNA-εpecific primer and a plasmid-anchored primer. These primers are removed and a symmetric PCR reaction is performed with a nested cDNA-specific antisense primer and the plasmid-anchored primer.
Procedure 3
RNA Ligase Protocol For Generating The 5' End Sequences To
Obtain Full Length Genes
Once a gene of interest is identified, several methods are available for the identification of the 5' or 3' portions of the gene which may not be present in the original deposited clone. These methods include but are not limited to filter probing, clone enrichment using specific probes and protocolε εimilar and identical to 5' and 3' RACE. While the full length gene may be preεent in a library and can be identified by probing, a uεeful method for generating the 5' end iε to uεe the exiεting εequence information from the original partial εequence to generate the misεing information. A method similar to 5' RACE is available for generating the misεing 5' end of a deεired full-length gene. (This method was published by Fromont-Racine et al, Nucleic Acids Res., 21(7) :1683-1684 (1993). Briefly, a specific RNA oligonucleotide is ligated to the 5' ends of a population of RNA presumably containing full-length gene RNA transcript and a primer εet containing a primer specific to the ligated RNA oligonucleotide. A primer specific to a known sequence (EST) of the gene of interest is uεed to PCR amplify the 5' portion of the deεired full length gene which may then be εequenced and used to generate the full length gene. This method starts with total RNA isolated from the desired source, poly A RNA may be used but is not a prerequisite for this procedure. The RNA preparation may then be treated with phosphatase if necessary to eliminate 5' phosphate groups on degraded or damaged RNA which may interfere with the later RNA ligase step. The phosphatase if used is then inactivated and the RNA is treated with tobacco acid pyrophosphatase in order to remove the cap structure present at the 5' ends of messenger RNAs. Thiε reaction leaves a 5' phosphate group at the 5' end of the cap-cleaved RNA which can then be ligated to an RNA oligonucleotide using T4 RNA ligase. This modified RNA preparation can then be used as a template for first strand cDNA syntheεis using a gene-specific oligonucleotide. The first stand synthesis reaction can then be used as a template for PCR amplification of the deεired 5' end uεing a primer specific to the ligated RNA oligonucleotide and a primer specific to the known sequence (EST) of the gene of interest. The resultant product is then sequenced and analyzed to confirm that the 5' end sequence belongs to the partial sequence.
Example 5 Cloning and expreεεion of BSGl uεing the baculoviruε expreεεion εvεtem
The DNA εequence encoding the full length BSGl protein, ATCC # 97175, waε amplified uεing PCR oligonucleotide primerε correεponding to the 5' and 3' εequences of the gene:
The 5' primer has the sequence 5' AAAGGATCCCCCGCCATCATGG ATGTTTTCAAGAAG 3' (SEQ ID NO:25) and contains a BamHl restriction enzyme site (in bold) followed by 8 nucleotides resembling an efficient signal for the initiation of translation in eukaryotic cells (Kozak, M. , J. Mol. Biol., 196:947-950 (1987) of the BSGl gene (the initiation codon for translation "ATG" is underlined) .
The 3' primer has the sequence 5' AAATCTAGACTAGTCTCCCCC ACTCTG 3' (SEQ ID NO:26) and containε the cleavage εite for the restriction endonuclease Xbal and 21 nucleotides complementary to the 3' sequence of the BSGl gene. The amplified sequences were isolated from a 1% agarose gel using a commercially available kit ("Geneclean, " BIO 101 Inc., La Jolla, Ca.) . The fragment waε then digested with the endonucleases BamHl and Xbal and then purified again on a 1% agarose gel. This fragment is designated F2.
The vector pA2 (modification of pVL941 vector, discussed below) is used for the expression of the BSGl protein using the baculovirus expression syεtem (for review see: Summers, M.D. and Smith, G.E. 1987, A manual of methods for baculoviruε vectors and insect cell culture procedures, Texas Agricultural Experimental Station Bulletin No. 1555) . This expression vector contains the strong polyhedrin promoter of the Autographa californica nuclear polyhedrosiε viruε (AcMNPV) followed by the recognition sites for the restriction endonucleases BamHl and Xbal. The polyadenylation site of the simian virus (SV)40 is used for efficient polyadenylation. For an eaεy selection of recombinant virus the beta-galactosidase gene from E.coli is inserted in the same orientation as the polyhedrin promoter followed by the polyadenylation signal of the polyhedrin gene. The polyhedrin εequences are flanked at both εideε by viral εequenceε for the cell-mediated homologous recombination of co-transfected wild-type viral DNA. Many other baculovirus vectors could be used in place of pA2 such as pRGl, pAc373, pVL941 and pAcIMl (Luckow, V.A. and Summers, M.D., Virology, 170:31-39) .
The plasmid was digeεted with the reεtriction enzymeε BamHl and Xbal and dephoεphorylated uεing calf inteεtinal phoεphataεe by procedures known in the art. The DNA was then iεolated from a 1% agarose gel using the commercially available kit ("Geneclean" BIO 101 Inc., La Jolla, Ca.) . This vector DNA is designated V2. Fragment F2 and the dephosphorylated plasmid pA2 were ligated with T4 DNA ligase. E.coli HB101 cells were then transformed and bacteria identified that contained the plasmid (pBacBSGl) with the BSGl gene using the enzymes BamHl and Xbal. The sequence of the cloned fragment was confirmed by DNA sequencing.
5 μg of the plasmid pBacBSGl was co-transfected with l.0 μg of a commercially available linearized baculoviruε
("BaculoGold™ baculovirus DNA", Pharmingen, San Diego, CA.) uεing the lipofection method (Feigner et al. Proc. Natl.
Acad. Sci. USA, 84:7413-7417 (1987)). lμg of BaculoGold™ virus DNA and 5 μg of the plasmid pBacBSGl were mixed in a sterile well of a microtiter plate containing 50 μl of serum free Grace's medium (Life Technologies Inc., Gaithersburg, MD) . Afterwards 10 μl Lipofectin plus 90 μl Grace's medium were added, mixed and incubated for 15 minutes at room temperature. Then the transfection mixture was added drop-wise to the Sf9 inεect cellε (ATCC CRL 1711) seeded in a 35 mm tisεue culture plate with 1 ml Grace's medium without serum. The plate was rocked back and forth to mix the newly added solution. The plate waε then incubated for 5 hours at 27°C. After 5 hours the transfection solution was removed from the plate and 1 ml of Grace's insect medium supplemented with 10% fetal calf serum was added. The plate was put back into an incubator and cultivation continued at 27°C for four days.
After four days the supernatant was collected and a plaque aεεay performed similar as described by Summerε and Smith (εupra) . As a modification an agarose gel with "Blue Gal" (Life Technologies Inc., Gaithersburg) waε used which allows an eaεy iεolation of blue εtained plaqueε. (A detailed description of a "plaque assay" can also be found in the uεer's guide for insect cell culture and baculovirology distributed by Life Technologies Inc., Gaithersburg, page 9- 10) . Four days after the serial dilution, the virus was added to the cells and blue εtained plaqueε were picked with the tip of an Eppendorf pipette. The agar containing the recombinant viruεeε was then resuspended in an Eppendorf tube containing 200 μl of Grace's medium. The agar was removed by a brief centrifugation and the supernatant containing the recombinant baculovirus was used to infect Sf9 cells seeded in 35 mm dishes. Four days later the supernatants of these culture dishes were harvested and then εtored at 4°C.
Sf9 cellε were grown in Grace's medium εupplemented with 10% heat-inactivated FBS. The cells were infected with the recombinant baculovirus V-BSG1 at a multiplicity of infection
(MOI) of 2. Six hours later the medium was removed and replaced with SF900 II medium minus methionine and cysteine
(Life Technologies Inc., Gaithersburg) . 42 hours later 5 μCi of 35S-methionine and 5 μCi 35S cysteine (Amersham) were added. The cells were further incubated for 16 hours before they were harvested by centrifugation and the labelled proteins viεualized by SDS-PAGE and autoradiography.
Example 6 Expression of Recombinant BSGl in COS cellε
The expression of plasmid, BSGl HA is derived from a vector pcDNAI/Amp (Invitrogen) containing: 1) SV40 origin of replication, 2) ampicillin resistance gene, 3) E.coli replication origin, 4) CMV promoter followed by a polylinker region, an SV40 intron and polyadenylation site. A DNA fragment encoding the entire precursor and a HA tag fused in frame to itε 3' end waε cloned into the polylinker region of the vector, therefore, the recombinant protein expreεεion iε directed under the CMV promoter. The HA tag corresponds to an epitope derived from the influenza hemagglutinin protein as previously described (I. Wilson, H. Niman, R. Heighten, A Cherenson, M. Connolly, and R. Lerner, 1984, Cell 37:767, (1984)) . The infusion of HA tag to the target protein allows easy detection of the recombinant protein with an antibody that recognizes the HA epitope.
The plasmid construction strategy is described as follows:
The DNA sequence encoding BSGl, ATCC # 97175, was constructed by PCR using two primers: the 5' primer AAAGGA TCCCCCGCCIATCATCGATGTTTTCAAGAAG 3' (SEQ ID NO:27) containε a BamHl εite followed by 18 nucleotideε of BSGl coding sequence starting from the initiation codon; the 3' sequence AAATC TAGACTAAAGCGTAGTCπτ3GGACXΞrrσπ,ATGGGTACTCCTOGGGT 3' (SEQ ID NO:28) contains complementary sequences to an Xbal site, translation stop codon, HA tag and the laεt 18 nucleotideε of the BamHl coding sequence (not including the stop codon) . Therefore, the PCR product containε an BamHl εite, BSGl coding sequence followed by HA tag fused in frame, a translation termination stop codon next to the HA tag, and an Xbal site. The PCR amplified DNA fragment and the vector, pcDNAI/Amp, were digested with BamHl and Xbal restriction enzyme and ligated. The ligation mixture was transformed into E. coli strain SURE (available from Stratagene Cloning Systems, 11099 North Torrey Pines Road, La Jolla, CA 92037) the transformed culture was plated on ampicillin media plates and reεiεtant colonieε were εelected. Plasmid DNA waε isolated from transformantε and examined by reεtriction analysis for the presence of the correct fragment. For expression of the recombinant BSG protein, COS cells were transfected with the expresεion vector by DEAE-DEXTRAN method (J. Sambrook, E. Fritsch, T. Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Laboratory Presε, (1989)). The expreεεion of the BSG HA protein was detected by radiolabelling and immunoprecipitation method (E. Harlow, D. Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Presε, (1988)). Cellε were labelled for 8 hourε with 35S-cysteine two days post transfection. Culture media was then collected and cells were lysed with detergent (RIPA buffer (150 mM NaCl, 1% NP-40, 0.1% SDS, 1% NP-40, 0.5% DOC, 50mM Tris, pH 7.5) (Wilson, I. et al., Id. 37:767 (1984)) . Both cell lysate and culture media were precipitated with an HA specific monoclonal antibody. Proteins precipitated were analyzed on 15% SDS-PAGE gels.
Numerous modifications and variations of the preεent invention are poεsible in light of the above teachings and, therefore, within the εcope of the appended claimε, the invention may be practiced otherwise than as particularly described.

Claims

WHAT IS CLAIMED IS:
1. An isolated polynucleotide comprising a member selected from the group conεiεting of
(a) a polynucleotide encoding the εame polypeptide aε the polynucleotide of Figure 1 (SEQ ID NO:l) ;
(b) a polynucleotide encoding the same mature polypeptide as a human gene having a coding portion which includes DNA having at least a 90% identity to the DNA of one of Figures 2-20 (SEQ ID NO:2-20) ;
(c) a polynucleotide which hybridizes to the polynucleotide of (a) and which haε at leaεt a 70% identity thereto; and
(d) a polynucleotide encoding the same mature polypeptide as a human gene having a coding portion which includes DNA having at least a 90% identity to a DNA included in the deposited clone.
2. The polynucleotide of Claim 1 wherein the human gene includes DNA contained in the deposited clone.
3. The polynucleotide of Claim 1 wherein the member is a polynucleotide encoding the εame polypeptide aε the polynucleotide of Figure 1 (SEQ ID NO:l) .
4. A vector containing the polynucleotide of claim 1.
5. A hoεt cell tranεformed or tranεfected with the vector of Claim 4.
6. A process for producing cells capable of expressing a polypeptide comprising genetically engineering cells with the vector of Claim 4.
7. A procesε for producing a polypeptide comprising: expressing from the host cell of Claim 5 the polypeptide encoded by said polynucleotide.
8. A polypeptide comprising a member selected from the group consisting of: (i) a polypeptide encoded by a human gene, said human gene having a coding portion whose DNA has at least a 90% identity to the DNA of one of Figures 2-20 (SEQ ID NO:2-20) ; (ii) a polypeptide having the deduced amino acid εequence aε set forth in Figure 1 (SEQ ID NO:l) and fragments, analogs and derivatives thereof; and (iii) a polypeptide encoded by the human gene whose coding region includes a DNA having at least a 90% identity to the DNA contained in the deposited clone and fragments, analogs and derivatives of said polypeptide.
9. The polypeptide of Claim 8 wherein the polypeptide has the deduced amino acid sequence as set forth in Figure 1 (SEQ ID NO:l) .
10. An antibody against the polypeptide of claim 8.
11. A compound which inhibitε activation of the polypeptide of claim 8.
12. A method for the treatment of a patient having need to inhibit a breast specific gene protein comprising: adminiεtering to the patient a therapeutically effective amount of the compound of Claim 11.
13. The method of claim 12 wherein the compound is a polypeptide and the therapeutically effective amount of the compound is administered by providing to the patient DNA encoding said polypeptide and expressing said polypeptide in vivo.
14. A method for the treatment of a patient having need of a breast specific gene protein comprising: administering to the patient a therapeutically effective amount of the polypeptide of claim 8.
15. A process for diagnosing a disorder of the breast in a host comprising: determining transcription of a human gene in a sample derived from non-breast tissue of a host, said gene having a coding portion which includeε DNA having at leaεt 90% identity to DNA selected from the group consisting of the DNA of Figures 1-20 (SEQ ID NO:1-20), whereby said transcription indicates a disorder of the breaεt in the hoεt.
16. The proceεε of claim 15 wherein tranεcription is determined by detecting the presence of an altered level of RNA transcribed from said human gene.
17. The process of claim 15 wherein transcription is determined by detecting the presence of an altered level of DNA complementary to the RNA tranεcribed from εaid human gene.
18. The process of claim 15 wherein transcription is determined by detecting the presence of an altered level of an expression product of said human gene.
19. A process for determining a disorder of a breast in a host compriεing: contacting an antibody specific to a BSG antigen or an epitopic portion thereof, to a fluid sample derived from a host; determining the presence of an altered level of a BSG gene product in εaid εample.
20. A process for identifying antagonistε to the polypeptide of claim 8 compriεing: contacting said polypeptide with a natural substrate and a labeled compound to be screened either simultaneously or in either consecutive order,- and determining whether the therapeutic effectively competes with the natural substrate in a manner sufficient to prevent binding of the protein to its substrate.
PCT/US1995/008295 1995-06-30 1995-06-30 Breast specific genes and proteins WO1997002280A1 (en)

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WO1998033915A1 (en) * 1997-02-03 1998-08-06 Human Genome Sciences, Inc. Breast cancer specific gene 1
WO1998059049A1 (en) * 1997-06-20 1998-12-30 Abbott Laboratories Reagents and methods useful for detecting diseases of the breast
WO1999007740A2 (en) * 1997-08-06 1999-02-18 Zymogenetics, Inc. Lipocalin homologs
EP0908727A1 (en) * 1997-09-19 1999-04-14 Neuropa Limited Synuclein-based assay and synuclein protein
WO1999025877A1 (en) * 1997-11-18 1999-05-27 Abbott Laboratories Reagents and methods useful for detecting diseases of the breast
WO1999033869A2 (en) * 1997-12-24 1999-07-08 Corixa Corporation Compounds for immunotherapy and diagnosis of breast cancer and methods for their use
WO1999034017A1 (en) * 1997-12-26 1999-07-08 Abbott Laboratories Reagents and methods useful for detecting diseases of the breast
WO2000061756A2 (en) * 1999-04-09 2000-10-19 Corixa Corporation Compounds for immunotherapy and diagnosis of breast cancer and methods for their use
US6379951B1 (en) 1997-12-24 2002-04-30 Corixa Corporation Compounds for immunotherapy of breast cancer and methods for their use
US6432707B1 (en) 1997-12-24 2002-08-13 Corixa Corporation Compositions and methods for the therapy and diagnosis of breast cancer
US6468758B1 (en) 1998-09-23 2002-10-22 Corixa Corporation Compositions and methods for ovarian cancer therapy and diagnosis
US7563880B2 (en) 1999-11-30 2009-07-21 Corixa Corporation Compositions and methods for the therapy and diagnosis of breast cancer
US7598226B2 (en) 1998-12-28 2009-10-06 Corixa Corporation Compositions and methods for the therapy and diagnosis of breast cancer
US7855271B2 (en) 1998-12-28 2010-12-21 Corixa Corporation Compositions and methods for the therapy and diagnosis of breast cancer

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Cited By (19)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998033915A1 (en) * 1997-02-03 1998-08-06 Human Genome Sciences, Inc. Breast cancer specific gene 1
WO1998059049A1 (en) * 1997-06-20 1998-12-30 Abbott Laboratories Reagents and methods useful for detecting diseases of the breast
WO1999007740A2 (en) * 1997-08-06 1999-02-18 Zymogenetics, Inc. Lipocalin homologs
WO1999007740A3 (en) * 1997-08-06 1999-04-29 Zymogenetics Inc Lipocalin homologs
EP0908727A1 (en) * 1997-09-19 1999-04-14 Neuropa Limited Synuclein-based assay and synuclein protein
WO1999025877A1 (en) * 1997-11-18 1999-05-27 Abbott Laboratories Reagents and methods useful for detecting diseases of the breast
WO1999033869A3 (en) * 1997-12-24 1999-12-23 Corixa Corp Compounds for immunotherapy and diagnosis of breast cancer and methods for their use
WO1999033869A2 (en) * 1997-12-24 1999-07-08 Corixa Corporation Compounds for immunotherapy and diagnosis of breast cancer and methods for their use
US6379951B1 (en) 1997-12-24 2002-04-30 Corixa Corporation Compounds for immunotherapy of breast cancer and methods for their use
US6410507B1 (en) 1997-12-24 2002-06-25 Corixa Corporation Compounds for immunotherapy and diagnosis of breast cancer and methods for their use
US6432707B1 (en) 1997-12-24 2002-08-13 Corixa Corporation Compositions and methods for the therapy and diagnosis of breast cancer
WO1999034017A1 (en) * 1997-12-26 1999-07-08 Abbott Laboratories Reagents and methods useful for detecting diseases of the breast
US6468758B1 (en) 1998-09-23 2002-10-22 Corixa Corporation Compositions and methods for ovarian cancer therapy and diagnosis
US7598226B2 (en) 1998-12-28 2009-10-06 Corixa Corporation Compositions and methods for the therapy and diagnosis of breast cancer
US7855271B2 (en) 1998-12-28 2010-12-21 Corixa Corporation Compositions and methods for the therapy and diagnosis of breast cancer
US8182823B2 (en) 1998-12-28 2012-05-22 Corixa Corporation Compositions and methods for the therapy and diagnosis of breast cancer
WO2000061756A2 (en) * 1999-04-09 2000-10-19 Corixa Corporation Compounds for immunotherapy and diagnosis of breast cancer and methods for their use
WO2000061756A3 (en) * 1999-04-09 2001-02-08 Corixa Corp Compounds for immunotherapy and diagnosis of breast cancer and methods for their use
US7563880B2 (en) 1999-11-30 2009-07-21 Corixa Corporation Compositions and methods for the therapy and diagnosis of breast cancer

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AU3092995A (en) 1997-02-05
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CA2225824A1 (en) 1997-01-23

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