CA2232239A1 - Mammaglobin, antibodies thereto, and their use for detecting diseases of the breast - Google Patents

Abstract

A set of contiguous and partially overlapping RNA sequences and polypeptides encoded thereby, designated as mammaglobin and transcribed from breast tissue, is described. These sequences are useful for the detecting, diagnosing, staging, monitoring, prognosticating, preventing or treating, or determining the predisposition of an individual to diseases and conditions of the breast such as breast cancer. Also provided are antibodies which specifically bind to mammaglobin-encoded polypeptide or protein, and agonists or inhibitors which prevent action of the tissue-specific mammaglobin polypeptide, which molecules are useful for the therapeutic treatment of breast diseases, tumors or metastases.

Description

W O 98/077~3 PCTrUS97114666 MAM MAGLOBnN,ANTIBOD~ T~E~ETO,AND T~E~ US~ FOR D~lk~l~G DISEASES OF
THE f3REAST

Bacl~ground of the Invention This invention relates generally to detecting diseases of the breast using a member of the uteroglobin family of proteins termed m:~mm~lobin. Furthermore, the invention also relates to reagents and methods for detecting diseases of thebreast. More particularly, the present invention relates to reagents such as m~mm~globin-specific polynucleotide sequcnces and the polypeptidc sequences encoded thereby, as well as methods which utilize these sequences. The polynucleotide and polypeptide sequences are use~ul for detecting, diagnosing, staging, monitoring, prognosticating, preventing or treating, or determining predisposition to diseases or conditions of the breast such as breast cancer.
Breast cancer is the most common form of cancer occurring in females in the US. The incidence of breast cancers in the United States is projected to be 180,200 cases diagnosed and 43,900 breast cancer-related deaths to occur during 1997 (American Cancer Society statistics). Worldwide, the incidence of breast cancer has increased from 700,000 in 198~ to about 900,00û in 1990.
.N. Hortobagyi et al., CA CancerJ. Clin. 45: 199-226 (1995).
Procedures used for detecting, diagnosing, staging, monitoring, prognosticating, preventing or treating, or determining predisposition to diseases or conditions of the breast, such as breast cancer, are of critical importance to the outcome of the patient. For example, patients diagnosed with early breast cancerhave greater than a 90% five-year relative survival rate as compared to a survival rate of about 20% for patients diagnosed with distantly metastasized breast cancers.
(American Cancer Society statistics). Currently, the best initial indicators of early breast cancer are physical e~ in~tion of the breast and mammography. J.R. Harriset al. In: ~n~er: Principles and Practice of Oncology. Fourth Edition. pp. 1264-1332, Philadelphia, PA: J/B. Lippincott Co. (1993). Mammography may detect a breast tumor before it can be detected by physical examination, but it has limitations.
For example, the predictive value of mammography depends on the observer's skill W 098/077~3 PCT~US97/14666 and the quality of the mammogram. In addition, 80 to 93% of suspicious mammograms are false positives, and 10 to 15% of women with breast cancer have false negative mammograms. C.J. Wright et al., L,ance~ 346: 29-32 (1995). New diagnostic methods which are more sensitive and specific for detecting early breast 5 cancer are clearly needed.
Breast cancer patients are closely monitored following initial therapy and during adjuvant therapy to determine response to therapy, and to detect persistent or recurrent disease, or early distant metastasis. Current diagnostic proceduresfor monitoring breast cancer includc mammography, bone scan, chest 10 radiographs, liver function tests and tcsts for serum markers. Thc serum tumor markers most comrnonly used for monitoring patients are carcinoembryonic antigen (CEA) and CA 15-3. 1 imi~ ions of CE~ include absence of elevated serum levels in about 40% of women with metastatic disease. In addition, CEA
elevation during adjuvant therapy may not be related to recurrence but to other 15 factors that are not clinically important. CA 15-3 can also be negative in a significant number of patients with progressive disease and, therefore, fail to predict metastasis. Both CEA and CA 15-3 can be elevated in nonmalignant, benign conditions giving rise to false positive results. Therefore, it would be clinically beneficial to find a breast associated marker which is more sensitive20 and specific in detecting cancer recurrence. J. R. Harris, et al., ~. M. K.
Schwartz, Ln: Cancer: Principles and Practice of Oncolo~v. Vol. 1, Fourth Edition, pp. 531 - 542, Philadelphia, PA: J/B. Lippincott Co. (1993).
Another important step in m~n~ging breast cancer is to detcrmine the stage of the patient's disease because stage determination has potential 25 prognostic value and provides criteria for designing optimal therapy. Currently, pathological staging of breast cancer is preferable over clinical staging becausc the former gives a more accurate prognosis. J. R. Harris, et al., supra. On the other hand, clinical staging would be preferred were it at least as accurate as pathological staging because it does not depend on an invasive procedure to 30 obtain tissue for pathological evaluation. Staging of breast cancer could be improved by detecting new markers in serum or urine which could differentiate between different stages of invasion. Such markers could be mRNA or protein markers expressed by cells originating from the primary tumor in the breast but residing in blood, bone marrow or Iymph nodes and could serve as sensitive 35 indicators for metastasis to these distal organs. For example, specific protein W O 98/07753 PCTrUS97/14666 antigens and mRNA, associated witll breast epithelial cells, have been detected by immunohistochemical techniques and RT-PCR, respectively, in bone marrow, Iymph nodes and blood of breast cancer patients suggesting metastasis.
K. Pantel, et al., Onkologie 18: 394-401 (1995).
Such procedures also could include assays based upon the appcarance of various disease markers in test samples such as blood, plasma, serum or urine obtained by minim~lly invasive procedures which are detectable by immunological methods. These procedures would provide information to aid the physician in m~n~ing the patient with disease of the breast, at low cost to the patient. Markers such as prostate specific antigen (PSA) and human chorionic gonadotropin (hCG) exist and are used clinically for screening patients for prostate cancer and testicular cancer, respectively. For example, PSA normally is secreted by the prostate at high levels into the seminal fluid, but is present in very low levels in the blood of men with normal prostates. Elevated levels of PSA protein in serum are used in the early detection of prostate cancer or disease in asymptomatic men. See, for example, G.E. Hanks, et al., In: Cancer: Principles and Practice of Oncology, Vol. 1, Fourth Edition, pp. 1073-1113, Philadelphia, PA: J.B. Lippincott Co. (1993); M. K.
Schwartz, et al., In: Cancer: Principles and Practice of Oncology, Vol. 1, Fourth Edition, pp. 531-542, Philadelphia, PA: J.B. Lippincott Co. (1993). Likewise, the management of breast diseases could bc improved by the use of new markers normally expressed in the breast but found in elevated amounts in an inappropriate body compartment as a result of the disease of the breast.
Further, new markers which could predict the biologic behavior of early breast cancers would also be of significant value. Early breast cancers that threaten or will threaten the life of the patient are more clinically important than those that do not or will not be a threat. G.E. Hanks, supra. Such markers are needed to predict which patients with histologically negative Iymph nodes will experience recurrence of cancer and also to predict which cases of ductal carcinoma in situ will develop into invasive breast carcinoma. More accurate prognostic markers would allow theclinician to accurately identify early cancers localized to the breast which will progress and met~ct~i7~ if not treated aggressively. Additionally, the absence of a marker for an aggressive cancer in the patient could spare the patient expensive and non-beneficial treatment. J. R. ~arris et al., supra. E. R. Frykberg et al., C~ancer 74: 350-36 I (1994).

W O 98/07753 PCT~US97/14666 Attempts have been made to discover improved tumor markers for breast cancer by first identifying differentially expressed cellular components in breast tumor tissue compared to normal breast tissue. Two-dimensional polyacrylamide gel electrophoresis has been used to characterize quantitative and qualitative differences in polypeptide composition. P.J. Wirth, Electrophoresis 10:543-554 (1989). The sensitivity of this techni~ue, however, is limited by the degree of protein resolution of the two electrophoretic steps and by the detection step which depends on staining protein in gels. About 1000 polypeptides could be compared between tissues. P.~. Wirth, supra. Also, polypeptide instability wil} generate artifacts in the two-dimensional pattern. Subtractivc hybridization has been used to screen for differences in gene expression at the mRNA level. The NM23 gene was found to be differentially expressed between tumor cells of varying metastatic potential. P.S. Steeg et al., J. Natl. Cancer Inst. 80: 200-204 (1988). This technique has lirnitations in detecting ml~NA species in tissues present in low abundance, and screening for mRNA is very laborious. A more sensitive and less technically dem:~n~ling method for identifying differentially expressed genes isdifferential display. P. Liang et al., Cancer Res. 52:6966-6g68 ( l 992). This method involves the rcverse transcription of cellular mRNAs to cDNAs followed byPCR amplification of a cDNA subpopulation. Comparison of amplified cDNA
subpopulations between normal and tumor breast tissues allows identification of mRNA species that are differentially expressed. This technique has greater sensitivity than subtractive hybridization for detecting mRNAs of low abundance.Several genes have been shown to be differentially expresscd between normal and tumor breast tissues by differential display. P. Liang, et al., supra.; M.A. Watson et al., Cancer Res. 54:4598-4602 (1994); R.A. Jensen et al., PNAS USA 91:9257-9261 (1994). An example of a gene found differentially expressed in breast tumorversus normal breast tissue by differential display is the mammaglobin gene, which is part of the uteroglobin family of proteins. Mammaglobin cDNA was only detected in breast tissue and was elevated in some breast tumors compared to itslevels in normal breast tissues. M.A. Watson et al., Cancer Res. 56:860-865 (1996).
It would be advantageous, therefore, to provide spccific methods and rea~gents useful for detecting, diagnosing, staging, monitoring, prognosticating, preventing or treating, or determining predisposition to diseases or conditions of the breast. Such methods would include assaying a test sample for products of a W O 98/07753 PCT~US97/14666 gene which are overexpressed in diseases and conditions associated with the breast, including cancer. Such methods may also include assaying a test sample for products of a gene which have been altered by the disease or condition associated with the breast, including cancer. Such methods may further include assaying a test sample for products of a gene whose distribution among the various tissues and compartments of the body have been altered by a breast-associated disease or condition including cancer. Such methods would comprise making cDNA from mRNA in the test sample, amplifying, when necessary, portions of the cDNA corresponding to thc gene or a fragment thereof, and detecting the cDNA product as an indication of the presence of the disease or condition including cancer or detecting translation products of the mRNAs comprising gene sequences as an indication of the presence of the disease.
Useful reagents include polynucleotide(s), or fragment(s) thereof which may be used in diagnostic methods such as reverse transcriptase-polymerase chain reaction (~T-PCR), PCR, or hybridization assays of mRNA extracted from biopsied tissue, blood or other test samples; or proteins which are the translation products of such mRNAs; or antibodies directed against these proteins. Such assays would include methods for assaying a sample for product(s) of the gene and detecting the product(s) as an indication of disease of the breast. Drug treatment or gene therapy for diseases and conditions of the breast including cancer can be based on thesc identirled gene se~uences or their expressed proteins, and efficacy of any particular therapy can be monitored. Furthermore, it would be advantageous to have available alternative, non-surgical diagnostic methods capable of detecting early stage breast disease such as cancer.
Summary of the Invention The present invention provides a method of detecting a target m~mm~globin polynucleotide in a test sample which compriscs contacting the test sample with at least one m:~rnm~globin-specific polynucleotide and detecting the presence of the ~ 30 target m~mm~globin polynucleotide in the test sample. The m~mm~lobin-specific polynucleotide has at least 50% identity with a polynucleotide selected from the~ group consisting of SE~UENCE ID NO 1, SEQUENCE ID NO 2, and fragments or complements thereof. Also, the m~mm~globin-spccific polynucleotide may be attached to a solid phase prior to a performing the method.

The present invention also provides a method for detecting m~mm:~globin rnRNA in a test sample, which comprises performing reverse transcription (RT) with at least one primer in order to produce cDNA, amplifying the cDNA so obtained using m~mm~lobin oligonucleotides as sense and antisense primers to obtain m~rnm~globin amplicon, and detecting the presence of the m~mm~globin amplicon as an indication of the presence of mammaglobin mRNA in the test sample, wherein the m~rnm~globin oligonucleotides havc at least 50% identity to a sequence selected from the group consisting of SEQUENCE ID NO I, SEQUENCE
ID NO 2, and fragments or complements thereof. Amplification can be performed 10 by the polymerase chain reaction. Also, the test sample can be reacted with a solid phase prior to performing the method, prior to amplification or prior to detection.
This reaction can be a direct or an indireci reaction. Furthcr, the detection step can comprise utilizing a detectable label capable of generating a measurable signal. The detectable label can bc attached to a solid phase.
The present invention further provides a method of detecting a target m~mm~globin polynucleotide in a test sample suspected of containing target mammaglobin polynucleotides, which compri~es (a) contacting the test sample withat least one r~mm~globin oligonucleotide as a sense primer and at least one m:~mm~globin oligonucleotide as an anti-sense primer, and amplifying same to 20 obtain a first stage reaction product; (b) contacting the first stage rcaction product with at least one other m~mm~globin oligonucleotidc to obtain a second stage reaction product, with the proviso that the other mammaglobin oligonucleotidc islocated 3' to the m~mm~globin oligonucleotides utilized in step (a) and is complemcntaly to the first stage reaction product; and (c) detecting the second stage 25 reaction product as an indication of the presence of a target mammaglobin polynucleotide in the test sample. The mammaglobin oligonucleotides selected as reagents in the method have at least 50% identity to a sequence selccted from thc group consisting of SEQU~3NCE ID NO 1, SEQUENCE ID NO 2, and fragments or complements thereof. Amplification may be performed by the polymerase chain 30 reaction. The tes~ sample can be reacted either directly or indirectly with a solid phase prior to pcrforming the method, or prior to ampli~lcation, or prior to detection.
The detection step also comprises ntili~.ing a detectable label capable of generating a measurable signal; further, the detectable label can be attached to a solid phase. Test kits useful for detecting a target m~rnm~globin polynucleotide in a test sample arc 35 also provided which comprise a container containing at least one m~mm~globin W O 9XJ'~7/~ PCTAJS97/14666 specific polynucleotide sclected from the group consisting of SEQUENCEIDNOl, SEQUENCEIDNO2, and fragments or complements thereof. These test kits further comprise containers with tools useful for collecting test samples (such as, for example, blood, urine, saliva and stool). Such tools include lancets and absorbent paper or cloth for collccting and stabilizing blood; swabs for collecting and stabilizing saliva; and cups for collecting and stabilizing urine or stool samples.
Collection materials such as, papcrs, cloths, swabs, cups and the like, may optionally be treated to avoid denaturation or irreversible adsorption o~ the sample.
The collection materials also may be treated with or contain preservatives, stabilizers or antimicro~ial agents to help maintain the integrity of the specimens. Specific primers useful in these methods and test kits include a primer selected from thegroup consisting of SEQUENCEIDNO3,SEQUENCEIDNO4,SEQUENCEID
NOS, and SEQUENCE ~ NO6.
The present invention provides a purified polynucleotide or fragment thereof derived from a m~mm~globin gene. The puri~led polynucleotide is capable of selectively hybridizing to the nucleic acid of the mammaglobin gene, or a complement thereof. The polynucleotide has at least 50% identity to a polynucleotide selected from the group consisting of SEQUENCE TD NOl, SEQUENCEIDNO2, and fragments or complements thereof. Further, the purified polynucleotide can be produced by recombinant and/or synthe~ic techniques. The purifled recombinant polynucleotide can be contained within a recombinant vector.
The invention further comprises a host cell transfected with said vector.
The present invention further provides a recombinant expression system comprising a nucleic acid sequence that includes an open reading frame derived from m~mm~globin. The nucleic acid sequence has at least 50% identity with a sequencese}ected from the group consisting of SEQUENCEIDNOI,SEQUENCEIDNO

2, and fragments or complements thereof. The nucleic acid sequence is opcrably linked to a control sequence compatible with a desired host. Also provided is a cell transfected with this recombinant expression system.
The present invention also provides polypeptides encoded by mammaglobin.
The polypeptidcs can be produced by recombinant technology, provided in purifiedform, or produced by synthetic techniques. The polypeptides comprise amino acid sequences which have at least 50% identity to an amino acid sequence selected from the group consisting of SEQUENCEIDNOS 17-26, and fragments thereof.

W O 98/077~3 PCTrUS97/14666 Also provided is an antibody which specifically binds to at least one m,.mm:~lobin epitope. The antibody can be a polyclonal or monoclonal antibody.
The epitope is derive~ from an amino acid sequence selected from the group consisting of SEQUEi:NCE ID NOS 17-26, and fragments thereof. Assay kits for 5 deterrnining the presence of m~mm~globin antigen or anti-m~mm~.~lobin antibody in a test sample are also included. In one embodiment, the assay kits comprise a container con~inin~ at least one m~7mm~7globin polypeptide having at least 50%
identity to an amino acid sequence selected from the group consisting of SEQUENCE ID NOS 17-26, and fragments thereof. Purther, the test kit can 10 comprisc a container with tools useful for collecting test samples (such as blood, urine, saliva and stool). Such tools include lancets and absorbent paper or cloth for collecting and stabilizing blood; swabs for collecting and stabilizing saliva; and cups for collecting and stabilizing urine or stool samples. Collection materials such as, papers, cloths, swabs, cups and the like, may optionally be treated to avoid 15 denaturation or irreversible adsorption of the sample. These collection materials also may be treated with or contain preservatives, stabilizers or antimicrobial agents to help m,.int~in the integrity of the specimens. Also, the polypeptide can be attachcd to a solid phase.
Another assay kit for determining the presence of mammaglobin antigen or 20 anti-m,.mm~lobin antibody in a test sample comprises a container containing an antibody which specifically binds to a mammaglobin antigen, wherein the m,.mm~globin antigen comprises at least one mammaglobin epitope. The m,.mm~EIobin antigen has at least about 60% se~uence similarity to a sequence of a m~rnm,.globin antigen selected from the group consisting of SEQUENCE ID NOS
25 17-26, and fragments thereof. These test kits can further comprise containers with tools useful for collecting test samples (such as blood, urine, saliva and stool).
Such tools include lancets and absorbent paper or cloth for collecting and stabilizing blood; swabs for collecting and stabilizing saliva; cups for collecting and stabilizing urine or stool samples. Collection materials, papers, cloths, swabs, cups and the 30 like, may optionally bc treated to avoid denaturation or irreversible adsorption of the sample. These collection materials also may be treated with, or contain, preservatives, stabilizers or antimicrobial agents to help maintain the integrity of the specimens. The antibody can be attached to a solid phase.
A method for producing a polypeptide which contains at least one epitope of 35 m~mm .globin is provided, which method comprises incubating host cells W O 98/07753 PCTrUS97/14666 transfected with an expression vector. This vector comprises a polynucleotide sequence encoding a polypeptide, wherein the polypeptide comprises an amino acidsequence having at least 50% identity to a m~rnm~globin amino acid sequence selected from the group consisting of SEQUENCE ID NOS 17-26, and fragments 5 thereof.
A method for detecting m~mm~globin antigen in a test sample suspected of containing m~r m~globin antigen also is provided. The method comprises contacting the test sample with an antibody or fragment thereof which specifically binds to at least one epitope of a m~mrn~globin antigen, for a time and under 10 conditions sufficient for the formation of antibody/antigen complexes; and detecting the presence of such complexes con~ining the antibody as an indication of the presence of a m~mmz~globin antigen in the test sample. The antibody can be attached to a solid phase and be either a monoclonal or polyclonal antibody. Furthermore,the antibody specifically binds to at least one mammaglobin antigen selected from the 15 group consisting of SEQUENCE ID NOS 17-26, and fragments thereof.
Another method is provided which detects antibodies which specifically bind to a m~rnm~globin antigen in a test sample suspected of containing thesc antibodies.
The method comprises contacting the test sample with a polypeptide which contains at least one m~mm~globin epitope, wherein the mammaglobin epitope comprises an 20 amino acid sequence having at least 50% identity with an amino acid sequence encoded by a m~mm~globin polynucleotide, or a fragment thereof. Contacting is carricd out for a time and under conditions suf~lcient to allow antigen/antibodycomplexes to form. The method further entails detecting complexes which contain the polypeptide. The polypeptide can be attached to a solid phase. Further, the 25 polypeptide can be a recombinant protein or a synthetic peptide having at least 50%
identity to an amino acid sequence selected from the group consisting of SEQUENCE ID NOS 17-26, and fragments thereof.
The present invention provides a cell transfected with a m~mmzlglobin nucleic acid sequence that encodes at least one epitope of a m~rnm~globin antigen, or 30 fragment thereof. The nucleic acid sequence is selected from the group consisting of SEQUENCE ID NO 1, SEQUENCE ID NO 2, and fragments or complements thereof.
A method for producing antibodies to mammaglobin antigen also is provided, which method comprises administering to an individual an isolated 35 irnmunogenic polypeptide or fragment thereof, wherein the isolated immunogenic W O 98t077~3 PCTAUS97/14666 polypeptidc comprises at least one mammaglobin epitope in an amount sufficient to produce an immune response. The isolated, immunogenic polypeptide comprises an amino acid sequence selected from the group consisting of SEQUENCE ID NOS
17-26, and fragments thereof.
Another method for producing antibodies which specifically bind to mammaglobin antigen is disclosed, which method comprises ~lmini~ ring to a m~mm~l a plasrnid comprising a nucleic acid sequence which encodes at least one mammaglobin epitope derived from an amino acid sequence selected from the group consisting of SEQUENCE ID NOS 17-26, and fragmcnts thereof.
Also provided is a composition of matter that comprises a m~rnm~globin polynucleotide of at least about 10-12 nucleotides having at least 50% identity to a polynucleotide selected from the group consisting of SEQUENOE ID NO l, SEQUENCE ID NO 2, and fragments or complements thereof. The mammaglobin polynucleotide encodes an amino acid sequence having at least onc mammaglobin epitope. Another composition of matter provided by the prcsent invention comprises a polypeptide with at least one mzlmm:~globin epitope of about 8-10 amino acids.The polypeptide comprises an amino acid sequence having at least 50% identity to an amino acid sequence selected from the group consisting of S~QUENCE ID NOS
17-26, and fragments thereoi. Also provided is a gene or fragment thereof codingfor a mammaglobin polypeptide which has at least 50% identity to SEQUENCE ID
NO 17, and a gene or a fragment thereof comprising DNA having at least 50%
identity to SEQUE~NCE ID NO I or SEQUENCE ID NO 2.
Brief Description of the Drawin~s FIGURE IA represents a scan of an ethidium bromide stained agarose gel of RNA from various tissue extracts; FIGURE IB dcpicts a northern blot of RNA from various tissue extracts using a mammaglobin radiolabelled probe;
FIGURE 2 represents a scan of a stained agarose gcl of mammaglobin-specific primed PCR amplification products;
FIaURE 3 is a representation of a film of a western blot of different tissue protein extracts using MAM. I antiserum.
l~etailed l~escription of the Invention The present invention provides a gene or a fragment thereof which codes for a m~mm~g10bin polypeptide having at least about 50% identity to SEQUENCE3 ID

W 098/07753 PCTrUS97/14666 NO 17. The present invention further encomp~ces a m~mm~globin gene or a fragment thereof comprising DNA which has at least about 50% identity to ~ SEQUE~CE ID NO I or SEQUENCE ID NO 2.
The present invention provides methods for assaying a test sample for 5 products of a breast tissue gene designated as mammaglobin, which comprises making cDNA from mRNA in the test sample, and detecting the cDNA as an indication of the presence of the breast tissue gene m:~mm~globin. The method may include an amplification step, wherein one or more portions of the mRNA from mammaglobin corresponding to the gene or fragments thereof, is amplified.
lO Methods also are provided for assaying for the translation products of mammaglobin. Test samples which may be assayed by the methods provided herein include tissues, cells, body fluids and secretions. The present invention also provides reagents such as oligonucleotide primers and polypeptides which are useful in performing these methods.
Portions of the nucleic acid sequences disclosed herein are useful as primers for the reverse transcription of RNA or for the amplification of cDNA; or as probes to determine the presence of certain mRNA sequences in test samples. Also disclosed are nucleic acid sequences which permit the production of encoded polypeptide sequences which are useful as standards or reagents in diagnostic 20 immunoassays, as targets for pharm~reutical screening assays and/or as components or as target sites for various therapies. Monoclonal and polyclonal antibodies directed against at least one epitope contained within these polypeptide sequences are useful as delivery agents for therapeutic agents as well as for diagnostic tests and for screening for diseases or conditions associatcd with m~mm~globin, especially breast 25 cancer. Isolation of sequences of other portions of the gene of interest can be accomplished utilizing probes or PCR primers derived from these nucleic acid sequences. This allows additional probes of the mRNA or cDNA of interest to be established, as well as corresponding encoded polypeptide sequences. These additional molecules are useful in detecting, diagnosing, staging, monitoring, 30 prognosticating, preventing or treating, or determining the predisposition to, diseases and conditions of the breast such as breast cancer, characterized by m~rnm~globin, as disclosed herein.
Techniques for determining amino acid sequence "similarity" are well-known in the art. In general, "similarity" means the exact amino acid to amino acid 35 comparison of two or more polypeptides at the appropriate place, where amino acids W O 98/07753 PCT~US97/14666 are id~ntic~l or possess similar chemical and/or physical properties such as charge or hydropho~icity. A so-termed "percent similarity" then can be de~ lined between the compared polypeptide sequences. Techniques for determining nucleic acid and amino acid sequence identity also are well known in the art and include determining S the nucleotide sequence of the mRNA for that gene (usually via a cDNA
intermcdiate) and deterrnining the amino acid sequence encoded thereby, and comparing this to a second amino acid sequence. In general, "identity" refers to an exact nucleotide to nucleotide or amino acid to amino acid correspondence of twopolynucleotides or polypeptide sequences, respectively. Two or more 10 polynucleotide sequences can be compared by determining their "pcrcent identity."
Two or more arnino acid sequences likewise can be compared by deterrnining their"percent identity." The programs available in the Wisconsin Sequence Analysis Package, Version 8 (available from C~enetics Computer Group, Madison, WI), for example, the GAP program, are capable of calculating both the identity between two 15 polynucleotides and the identity and similarity between two polypeptide sequences, respectively. Other programs for calculating identity or similarity between sequences are known in the art.
The compositions and methods described herein will enable the identification of certain markers as indicative of a breast tissue disease or condition; the 20 information obtained therefrom will aid in the detecting, diagnosing, staging, monitoring, prognosticating, preventing or treating, or determining diseases or conditions associated with mammaglobin, especially breast cancer. Test methods include, for example, probe assays which utilize the sequence(s) provided hereinand which also may utilize nucleic acid amplification methods such as the 25 polymerase chain reaction (PCR), the ligase chain reaction (LCR), and hybridization. In addition, the nucleotide sec~uences provided herein contain open reading frarncs from which an immunogenic epitope may be found. This epitope is believed to be unique to the disease state or condition associated with mammaglobin.
It also is thought that the polynucleotides or polypeptides and protein encoded by 30 the m~mm~globin gene are useful as a marker. This marker is eithcr elevated in disease such as breast canccr, altered in disease such as breast cancer, or present as a normal protein but appearing in an inappropriate body compartment. The uniqueness of the epitope may be determined by (i) its immunological reactivity and specificity with antibodies directed against protcins and po';ypeptides encoded by the 35 rn~mm:~globin gene, and (ii) its nonreactivity with any other tissue markers.

W O 98/07753 PCT~US97tl4666 - Methods for determining immllnnlogical reactivity are well-known and include but are not limited to, for example, radi~ .lllulloassay (RIA~, enzyme-linked ~ immunosorbent assay (ELISA), hemagglll~in~ion (HA), fluorescence polarization immunoassay (FPIA), chemiluminescent immunoassay (CLIA) and others. Several 5 examples of suitable methods are described herein.
Unless otherwise stated, the following terms shall have the following me:~ningc A polynucleotide "derived from" or "specific for" a designated sequence refers to a polynucleotide sequence which comprises a contiguous sequence of 10 approximately at least about 6 nucleotides, preferably at least about 8 nucleotides, more preferably at least about 10-12 nucleotides, and even more preferably at least about 15-20 nucleotides corresponding, i.e., identical or complementary to, a region of the designated nucleotide sequence. The sequence may be complementary or identical to a sequence which is unique to a particular polynucleotide sequence as 15 determined by techniques known in the art. Comparisons to sequences in databanks, for example, can be used as a method to determine the uniqucness of adesignated sequence. Regions from which sequences may be derived, include but are not limited to, regions encoding specific epitopes, as well as non-tr~n.cl:l~ed and/or non-transcribed regions.
The derived polynucleotide will not necessarily be derived physically from the nucleotide sequence of interest under study, but may be generated in any manner, including but not limited to chemical synthesis, replication, reverse transcription or transcription, which is based on the information provided by the sequence of bases in the region(s) from which the polynucleotide is derived. As such, it may represent either a sense or an antisense orientation of the original polynucleotide. In addition, combinations of regions corresponding to that of the designated sequence may be modified in ways known in the art to be consistent with the intended use.
A "fragment" of a specified polynuclcotide refers to a polynucleotide sequence which comprises a contiguous sequence of approximately at least about 6nucleotides, preferably at least about 8 nucleotides, more preferably at least about 10-12 nucleotides, and even more preferably at least about 15-20 nucleotides corresponding, i.e., identical or complementary to, a region of the specified nucleotide sequence.

CA 02232239 l998-04-20 W O 98/07753 PCTrUS97/14666 The term "primer" denotes a specific oligonucleotide sequence which is complementary to a target nucleotide sequence and used to hybridize to the target nucleotide sequence. A primer serves as an initiation point for nucleotide polymerization catalyzed by either DNA polymerase, RNA polymerase or revcrse 5 transcriptase.
The term "probe" denotes a defined nucleic acid segment (or nucleotide analog segment, e.g., PNA as defined hereinbelow) which can be used to identify a specific polynucleotide present in samples bearing the complementary sequence.
"Encoded by" refers to a nucleic acid sequence which codes for a 10 polypeptide sequence, wherein the polypeptide sequence or a portion thereof contains an amino acid sec~uence of at least 3 to 5 amino acids, more preferably at least 8 to 10 amino acids, and even more preferably at least 15 to 20 amino acids from a polypeptide encoded by the nucleic acid sequencc. Also encompassed are polypeptide sequences which are immunologically identifiable with a polypeptide 15 encoded by the sequence. Thus, a "polypeptide," "protein," or "amino acid"
sequence has at least about 50% identity, preferably about 60% identity, more preferably about 75-85% identity, and most preferably ~bout 90-95% or more identity to a m~mm~globin amino acid sequence. ~urther, the m~mm~3~lobin "polypeptide," "protein," or "amino acid" sequence may have at least about 60%
20 similarity, preferably at least about 75% similarity, more preferably about 85%
similarity, and most preferably about 95% or more similarity to a polypeptide orarnino acid sequence of mammaglobin. This amino acid sequence can be selected from the group consisting of SEQUENCE ID NOS 17-26, and fragments thereof.
A "recombinant polypeptide," ~'recombinant protein," or "a polypeptide 25 produced by recombinant techniques," which terms may be used interchangeably herein, describes a polypeptide which by virtue of its origin or manipulation is not associated with all or a portion of the polypeptide with which it is associated in nature and/or is linked to a polypeptide other than that to which it is linked in nature.
A recombinant or encoded polypeptide or protein is not necessarily translated from a 30 designated nucleic acid sequence. It also may be generated in any manner, includin~
chemical synthesis or expression of a recombinant expression system.
The term "synthetic peptide" as used herein means a polymeric form of amino acids of any length, which may be chemically synthesized by methods well-known to the routineer. These synthetic peptides are useful in various applications.

The term "polynucleotide" as used herein means a polymeric form of nuclcotides of any length, either ribonucleotides or deoxyribonucleotides. This term ~ refers only to the primary structure of the molecule. Thus, the term includes double-and single-stranded DNA, as well as double- and single-stranded RNA. It also includes modifications, such as methylation or capping and unmodified forms of the polynucleotide. The terms "polynucleotide," "oligomer," "oligonucleotide," and "oligo" are used interchangeably herein.
"A sequence corresponding to a cDNA" means that the sequence contains a polynucleotide sequence that is identical or complementary to a sequence in the de.sign~t~ (l DNA. The degree (or "p.~rcent") of identity or complementarity to the cDNA will be approximately 50% or greater, preferably at least about 70% or greater, and more preferably at least about 90% or greater. The sequence that corresponds to the identified c~NA will be at least about S0 nucleotides in length, preferably at least about 60 nucleotides in length, and more preferably at least about 70 nucleotides in length. The correspondence between thc gene or gene fragment of interest and the cDNA can be determined by methods known in the art and include,for example, a direct comparison of the sequenced material with the cDNAs described, or hybridization and digestion with single strand nucleases, followed by size determination of the digested fragments.
"Purificd polynucleotide" refers to a polynucleotide of interest or fragment thereof which is essentially free, e.g., contains less than about 50%, prcferably less than about 70%, and more preferably lcss than about 90%, of the protein with which the polynucleotide is naturally associated. Techni~ues for purifying polynucleotides of interest are well-known in the art and include, for example, disruption of the cell containing the polynucleotide with a chaotropic agent and separation of the polynucleotide(s) and proteins by ion-exchange chromatography, affinity chromatography and sedimentation according to density.
"Purificd polypeptide" or "purified protein" means a polypeptide of interest or fragment thereof which is essentially free of, e.g., contains less than about 50%, preferably less than about 70%, and more preferably less than about 90%, cellular components with which the polypeptide of interest is naturally associated. Methods for purifying polypeptides of interest are known in the art.
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 prcsent in a living W O 98/07753 rCT~US97/14666 animal is not isolated, but the same polynucleotide or ~NA or polypeptide, which is separated from some or all of the coexisting materials in the natural system, isisolated. Such polynucleotide could be part of a vector and/or such polynucleotide or polypeptide could be part of a composition, and still bc isolated in that the vector 5 or composition is not part of its natural environment.
"Polypeptide" and "protein" are used interchangeably herein and indicate at least one molecular chain of amino acids linked through covalent and/or non-covalent bonds. The terms do not refer to a specific length of the product. Thuspcptides, oligopeptides and proteins are included within the definition of 10 polypeptide. The terms include post-translational modi~lcations of the polypeptidc, for example, glycosylations, acetylations, phosphorylations and the like. In addition, protein fragments, analogs, mutated or variant proteins, fusion proteins and the like are included within the meaning of polypeptide.
A "fragment" of a specified polypeptide refers to an amino acid sequence which comprises at least about 3-5 amino acids, more preferably at least about 8-10 amino acids, and even more preferably at least about 15-20 amino acids derived from the specified polypeptide.
"Recombinant host cells," "host cells?" "cells," ''cell lines," "ccll cultures,"and other such terms denoting microorganisms or higher eukaryotic cell lines 20 cultured as unicellular entities refer to cells which can be, or have been, used as recipients for recombinant vectors or other transferred DNA, and include the original progeny of the original cell which has been transfected.
As used herein "replicon" means any genetic element, such as a plasmid, a chromosome or a virus, that behaves as an autonomous unit of polynucleotide 25 replication within a cell.
A "vector" is a replicon in which another polynucleotide segment is attached, such as to bring about the replication and/or expression of the attached segment.
The term "control sequence" refers to a polynucleotide sequence which is necessary to effect thc expression of a coding sequence to which it is ligated. The 30 nature of such control sequences differs depending upon the host organism. Inprokaryotes, such control sequences generally include a promoter, a ribosomal binding site and terminators; in eukaryotes, such control sequences generally include promoters, terminators and, in some instances, enhancers. The term "control sequence" thus is intended to include at a minimum all components whose presence W O 98/077~3 PCT~US97/14666 is necessary for expression, and also may include additional components whose presence is advantageous, for example, leader sequences.
~ "Operably linked" refers to a situation wherein the components described are in a relalionship permitting them to function in their intended manner. Thus, for example, a control sequence "operably linked" to a coding sequence is ligated insuch a manner that expression of the coding sequence is achieved under conditions compatible with the control sequence.
The term "open reading frame" or "ORF" refers to a region of a polynucleotide sequence which encodes a polypeptide. This region may represent aportion of a coding sequence or a total coding sequence.
A "coding sequence" is a polynucleotide sequence which is transcribed into mRNA and tr~ncl~cl into a polypeptide when placed under the control of applop~iate regulatory sequences. The boundaries of the coding sequence are determined by a translation start codon at the ~' -terminus and a translation stop codon at the 3' -terminus. A coding sequence can include, but is not limited to,mRNA, cDNA and recombinant polynucleotide sequences.
The term "immunologically identifiable with/as" refers to the presence of epitope(s) and polypeptide(s) which also are present in and are unique to the designated polypeptide(s). Immunological identity may be determined by antibody binding and/or competition in binding. These techniques are known to the routineer and also are described herein. The uniqueness of an epitope also can be determined by computer searches of known data banks, such as C~enBank, for the polynucleotide sequence which encodes the epitope and ~y amino acid sequence comparisons with other known proteins.
As used herein, "epitope" means an antigenic determinant of a polypeptide or protein. Conceivably, an epitope can comprise three amino acids in a spatial conformation which is unique to the epitope. Generally, an epitope consists of at least five such amino acids and more usually, it consists of at least eight to ten amino acids. ~ethods of examining spatial conformation are known in the art and include, for example, x-ray crystallography and two-dimensional nuclear magnetic resonance.
A "conformational epitope" is an epitope that is comprised of specific juxtaposition of amino acids in an immunologically recognizable structure, such amino acids being present on the same polypeptide in a contiguous or non-contiguous order or present on different polypeptides.

W O 98/07753 PCTrUS97/14666 A polypeptide is "immunologically reactive" with an antibody when it binds to an antibody due to antibody recognition of a specific epitope contained within the polypeptide. Immunological reactivity may be detertnincd by antibody binding, more particularly, by the kinetics of antibody binding, and/or by competition inbinding using as competitor(s) a known polypeptide(s) containing an epitope against which the antibody is directed. The methods for determining whether a polypeptide is immunologically reactive with an antibody are known in thc art.
As uscd herein, the term "immunogenic polypeptide containing an epitope of interest" means naturally occurring polypeptides of interest or fragments thereof, as well as polypeptides prepared by other means, for example, by chemical synthesisor the expression of the polypeptide in a recombinant organism.
The term "transfection" refers to the introduction of an exogenous polynucleotide into a prokaryotic or eucaryotic host cell, irrespective of the method used for the introduction. The term "transfection" refers to both stablc and transient introduction of the polynucleotide, and encompasses direct uptal~e of polynucleotides, transformation, transduction, and f-mating. Once introduced into the host cell, the exogenous polynucleotide may be maintained as a non-integrated replicon, for example, a plasmid, or alternatively, may be integrated into the host genome.
"Treatment" refers to prophylaxis and/or therapy.
- - The term "individual" as used herein refers to vertebrates, palticularly membcrs of the m:~mm~ n species and includes, but is not limited to, domestic animals, sports ~nirn~l~, primates and humans; more particularly the term refers to humans.
The term '~sense strand" or "plus strand" (or ''+") as used herein denotes a nucleic acid that contains the sequence that encodes the polypeptide. The term "antisense strand" or "minus strand" (or "-") denotes a nucleic acid that contains a sequence that is complementary to that of the "plus" strand.
The term "test sample" refers to a component of an individual's body which is the source of the analyte (such as, antibodies of interest or antigens of interest).
These components are well known in the art. A test sample is typically anything suspected of containing a target sequence. Test samples can be prepared using methodologies well known in the art such as by obtaining a specimen from an individual and, if necessary, disrupting any cells contained thereby to release target nucleic acids. These test samples include biological samplcs which can be tested by W O 98/07753 PCT~US97/14666 ~ the methods of the present invention described herein and include human and animal body fluids such as whole blood, serum, plasma, cerebrospinal fluid, sputum, bronchial washing, bronchial aspirates, urine, lymph fluids and various externalsecretions of the respiratory, intestinal and genitourinary tracts, tears, saliva, milk, 5 white blood cells, myelomas and the like; biological fluids such as cell culture supcrnatants; tissue specimens which may be fixed; and cell specimens which may be fixed.
"Purified product" refers to a prcparation of the product which has been isolated from the cellular constituents with which the product is normally associated 10 and from other types of cells which may be present in the sample of interest."PNA" denotes a "peptide nucleic acid analog" which may be utilized in a procedure such as an assay described herein to determine the presence of a target.
"MA" denotes a "morpholino analog" which may be utilized in a procedure such as an assay described herein to determine the presence of a targct. See, for example, U.S. Patent No. 5,378,841. PNAs are neutrally charged moieties which can be dirccted against RNA targets or DNA. PNA probes used in assays in place of, for example, the DNA probes of the present invention, offer advantages not achievable when DNA probes are used. These advantages include manufacturability, large scale labeling, reproducibility, stability, insensitivity to changes in ionic strength and 20 resistance to enzymatic degradation which is present in methods utilizing DNA or E~NA. These PNAs can be labeled with ("attached to") such signal generating compounds as fluorescein, radionucleotides, chemiluminescent compounds and the like. PNAs or other nucleic acid analogs such as MAs thus can be used in assay methods in place of DNA or RNA. Although assays are described herein utilizing 25 DNA probes, it is within the scope of the routineer that PNAs or MAs can be substituted for RNA or DNA with appl-3pl iate changes if and as needed in assay reagents.
"Analyte," as used herein, is the substance to be detected which may be present in the test sample. The analyte can be any substance for which there exists a 30 natura]ly occurring specific binding member (such as, an antibody), or for which a specific binding member can be prepared. Thus, an analyte is a substance that can bind to one or more specific binding members in an assay. "Analyte" also includes any antigenic substances, haptens, antibodies and combinations thereof. As a member of a specific binding pair, the analyte can be detected by means of naturally 35 occurring specific binding partners (pairs) such as the use of intrinsic factor protein W O 98/07753 PCTnUS97/14666 as a member of a specific binding pair for the determination of Vitamin B 12, the use of folate-binding protein to determine folic acid, or the use of a lectin as a member of a specific binding pair for the determination of a carbohydrate. The analyte caninclude a protein, a polypeptide, an amino acid, a nucleotide target and the like.
"Diseases of the breast" or "breast disease," or "condition of the breast" as used herein, refer to any disease or condition of the breast including, but not limited to, atypical hyperplasia, fibroadenoma, cystic breast disease, and cancer.
"Breast cancer," as used herein, refers to any malignant disease of the breast inc}uding, but not limited to, ductal carcinoma in situ, lobular carcinoma in situ, 10 infiltrating ductal carcinoma, me~ ry carcinoma, tubular carcinoma, mucinous carcinoma, infiltrating lobular carcinoma, infiltrating comedocarcinoma and infl~mm~tory carcinoma.
An "Expressed Sequence Tag" or "EST" refers to thc partial sequence of a cDNA insert which has been made by reverse transcription of mRNA extracted from 15 a tissue followed by insertion into a vector.
A "transcript image" refers to a tablc or list giving the quantitative distribution of ESTs in a library and represents the genes active in the tissuc from which the library was madc.
The present invention provides assays which utilizc spccific binding 20 members. A "specific binding member," as used herein, is a member of a specific binding pair. That is, two different molecules where one of thc molecules, through chemical or physical means, specifically binds to the second moleculc. Thereforc, in addition to antigen and antibody specific binding pairs of common immunoassays, other specific binding pairs can include biotin and avidin, carbohydratcs and Icctins, 25 complementary nucleotide sequences, effector and receptor molecules, cofactors and enzymes, enzyme inhibitors, and enzymes and the likc. Furthermorc, specific binding pairs can include members that are analogs of the original specific binding members, for example, an analyte-analog. Immunoreactive specific binding members include antigens, antigen fragments, antibodies and antibody fragments, 30 both monoclonal and polyclonal and complexes thercof, including those formcd by recombinant DNA molecules.
The term "hapten," as used herein~ refers to a partial antigen or non-protcin binding member which is capable of binding to an antibody, but which is not capable of eliciting antibody formation unlcss coupled to a carricr protein.

W O 98/07753 PCT~US97/14666 A "capture reagent," as used herein, refers to an unlabeled specific binding member which is specific either for the analyte as in a sandwich assay, for the ~ indicator reagent or analyte as in a competitive assay, or for an ancillary specific binding member, which itself is specific for the analyte, as in an indirect assay. The 5 capture reagent can be directly or indirectly bound to a solid phase material before the performance of the assay or during the performance of the assay, thereby enabling the separation of immobilized complexes from the test sample.
"Specific binding mernber" as used herein means a member of a specific binding pair. That is, two different molecules where one of the molecules through 10 chemical or physical means specif~cally binds to the second molecule.
The "indicator reagent" comprises a "signal-generating compound" ("label") which is capable of generating and generates a measurable signal detectable by external means, conjugated ("attached") to a specific binding member. In addition to being an antibody member of a specific binding pair, the indicator reagent also can 15 be a member of any specific binding pair, including either hapten-anti-haptensystems such as biotin or anti-biotin, avidin or biotin, a carbohydrate or a lectin, a complementary nucleotide sequence, an effector or a receptor molecule, an enzymecofactor and an enzyme, an enzyme inhibitor or an enzyme and the like. An immunoreactive specific binding member can be an antibody, an antigen, or an 20 antibody/antigen complex that is capable of binding either to the polypeptide of interest as in a sandwich assay, to the capture reagent as in a competitive assay, or to the ancillary specific binding member as in an indirect assay. When describing probes and probe assays, the term "reporter molecule" may be used. A reporter molecule comprises a signal generating compound as described hereinabove 25 conjugated to a specific binding member of a specific binding pair, such as carbazole or ~ n~ne.
The various "signal-generating compounds" (labels) contemplated include chromagens, catalysts such as enzymes, luminescent compounds such as fluoresceinand rhodamine, chemiluminescent compounds such as dioxetanes, acridiniums, 30 phenanthridiniums and luminol, radioactive elements and direct visual labels.Examples of enzymes include alkaline phosphatase, horseradish peroxidase, beta-galactosidase and the like. The selection of a particular label is not critical, but it must be capable of producing a signal either by itself or in conjunction with one or more additional substances.

W O 98/07753 rCT~US97/14666 "Solid phases" ("solid supports") are known to those in the art and include the walls of wells of a reaction tray, test tubes, polystyrene beads, magnetic or non-magnetic beads, nitrocellulose strips, membranes, microparticles such as latex particles, sheep (or other animal) red blood cells and Duracytes~ (red blood cells "fixed" by pyruvic aldehyde and formaldehyde, available from Abbott Laboratories, Abbott Park, IL) and others. The "solid phase" is not critical and can be selected by one skilled in the art. Thus, latex particles, microparticles, magnetic or non-magnetic beads, membranes, plastic tubes, walls of microtiter wells, glass or silicon chips, sheep (or other suitable animal's) red blood cells and Duracytes~ are allsuitable examples. Suitable methods for immobilizing peptides on solid phases include ionic, hydrophobic, covalent interactions and the like. A "solid phase," as used herein, refers to any material which is insoluble, or can be made insoluble by a subsequent reaction. The solid phase can be chosen for its intrinsic ability to attract and immobilize the capture reagent. Alternatively, the solid phase can retain anadditional receptor which has the ability to attract and immobilize the capture reagent The additional receptor can include a charged substance that is oppositely charged with respect to the capture reagent itself or to a charged substance conjugated to the capture reagent. As yet another alternative, the receptor molecule can be any specific binding member which is immobilized upon (attached to) the solid phase and whichhas the ability to irnmobilize the capture reagent through a specific binding reaction.
The receptor molecule enables the indirect binding of the capture reagent to a solid phase material before the performance of the assay or during the performance of the assay. The solid phase thus can be a plastic, derivatizcd plastic, magnetic or non-magnetic metal, glass or silicon surface of a test tube, microtiter well, sheet, bead, microparticle, chip, sheep ~or other suitable animal's) red blood cells, Duracytes~
and other configurations known to those of ordinary skill in the ar~.
It is contemplated and within the scope of the present invention that the solid phase also can comprise any suitable porous material with sufficient porosity toallow access by detection antibodies and a suitable surface affinity to bind antigens.
Microporous structures generally are ~l~;rell~d, but materials with a gel structure in the hydrated state may be used as well. Such useful solid supports include, but are not limited to, nitrocellulose and nylon. It is contemplated that such porous solid supports described herein preferably are in the form of sheets of thickness fromabout 0.01 to 0.5 mm, preferably about 0.1 mm. The pore size may vary within wide limits and preferably is from about 0.025 to 15 microns, especially from about W O 98/07753 PCTrUS97/14666 0.15 to 15 microns. The surface of such supports may be activated by chemical processes which cause covalent linkage of the antigen or antibody to the support.
The irreversible binding of the antigen or antibody is obtained, however, in general, by adsorption on the porous material by poorly understood hydrophobic forces.
Other suitable solid supports are known in the art.
Reagents.
The present invention provides reagents such as polynucleotide sequences derived from a breast tissue of interest and dcsignated as m~ m~globin, polypeptides encoded thereby and antibodies specific for these polypeptides. Thcpresent invention also provides reagents such as oligonucleotide fragments derived from the disclosed polynucleotides and nucleic acid sequences complementary to these polynucleotides. The polynucleotides, polypeptides, or antibodies of the present invention may be used to provide information leading to the detecting, diagnosing, staging, monitoring, prognosticating, preventing or treating of, or determining the predisposition to, diseases and conditions of the breast such ascancer. The sequences disclosed herein represent unique polynucleotides which can be used in assays or for producing a specific profile of gene transcription activity.
Such assays are disclosed in European Patent Number 0373203B I and Intemational Publication No. WO 95/11995.
Selected m:~mm~globin-derived polynucleotides can be used in the methods described herein for the detection of normal or altered gene expression. Such methods may employ m~mm~globin polynucleotides or oligonucleotides, fragments or derivatives thereof, or nucleic acid sequences complementary thereto.
The polynucleotides disclosed herein, their complementary sequences, or fragments of either, can be used in assays to detect, amplify or ~uantify genes,nucleic acids, cDNAs or mRNAs relating to breast tissue disease and conditions associated therewith. They also can bc used to identify an entire or partial coding region of a m~mm~globin polypeptide. They further can be provided in individual containers in the form of a kit for assays, or provided as individual compositions. If provided in a kit for assays, other suitable reagents such as buffers, conjugates and the like may be included.
The polynucleotide may be in the form of RNA or DNA. Polynucleotides in thc form of DNA, cDNA, genomic DNA, nucleic acid analogs and synthetic DNA
are within the scope of the present invention. The DNA may be double-stranded orsingle-stranded, and if single stranded, may be the coding (sense) strand or non-W O 9~ 7/~ PCTrUS97/14666 coding (anti-sense) strand. The coding sequence which encodes the polypeptide may be identical to the coding sequence provided herein 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 polypeptide as the DNA provided herein.
This polynucleotide may include only the coding sequence for the polypeptide, or the coding sequence for the polypeptide and an additional codingsequence such as a leader or secretory sequence or a proprotein sequence, or thecoding sequence for the polypeptide (and optionally an additional coding sequence) and non-coding sequence, such as a non-coding sequence 5' and/or 3' of the coding sequence for the polypeptide.
In addition, the invention includes variant polynucleotides containing modifications such as polynucleotide deletions, substitutions or additions; and any polypeptide modification resulting from the variant polynucleotide sequence. A
polynucleotide of the present invention also may have a coding sequence which is a l S naturally occurring allelic variant of the coding sequence provided herein.
In addition, the coding sequence for the polypeptide may be fuscd in the same reading frame to a polynucleotide sequence which aids in expression and secretion of a polypeptide from a host cell, for example, a leader sequence which functions as a secretory sequence for controlling transport of a polypeptide from the cell. The polypeptide having a leader sequence is a prepro~n and may have the leader sequence cleaved by the host cell to forrn the polypeptide. The polynucleotides may also encode for a proprotcin which is the protein plus additional 5' amino acid residues. A protein having a prosequence is a proprotein and may, in some cases, be an inactive form of the protein. Once the prosequence is cleaved, an active protein remains. Thus, the polynucleotide of the present invention may encode for a protein, or for a protein having a prosequence or for a protein having both a presequence (leader sequence) and a prosequence.
The polynucleotides of the present invention may also have the coding sequence fused in frame to a marker sequencc 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 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 m~rnm~ n host, e.g. a COS-7 cell line, is used. The HA tag corresponds to an epitope derived from the influenza hemagglutinin protein. See, for example, I. Wilson, et al., Cell 37:767 (1984).

It is contemp}ated that polynucleotides will be considered to hybridize to the sequences provided herein if there is at least 50%, preferably at least 70%, and more preferably at least 90% identity between the polynucleotide and the sequence.
The present invention also provides an antibody produced by using a 5 purified m~mm~globin polypeptide of which at least a portion of the polypeptide is encoded by a m~mm:~globin polynucleotide selected from the polynucleotides provided herein. These antibodies may be used in the methods provided herein forthe detection of m~rnm~lobin antigen in test samplcs. The presence of m~n~m~globin antigen in the test samples is indicative of the presence of a breast 10 disease or condition. The antibody also may be used for therapeutic purposes, for example~ in neutralizing the activity of mammaglobin polypeptide in conditions associated with altered or abnormal expression.
The present invention further relates to a m~mm:lglobin polypeptide which has the df~ e-l amino acid sequence as provided herein, as well as fragments, 15 analogs and derivatives of such polypeptide. The polypeptide of the present invention may be a recombinant polypeptide, a natural purified polypeptide or a synthetic polypeptide. The fragment, derivative or analog of the mammaglobin polypeptide may be one in which one or more of the amino acid residues is substituted with a conserved or non-conserved amino acid residue (preferably a 20 conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code; or it may be one in which one or more of the amino acid residues includes a substituent group; or ii may be 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 it may be one in 25 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 polypeptide or a proprotein sequence. Such fragments, derivatives and analogs are within the scope of the present invention. The polypeptides and polynucleotides of the present invention are provided preferably in an isolated form and preferably30 purified.
Thus, a polypeptide of the present invention may have an amino acid sequence that is identical to that of the naturally occurring polypeptide or that is different by minor variations due to one or more amino acid substitutions. The variation may be a "conservative change" typically in the range of about 1 to 5 amino 35 acids, wherein the substituted amino acid has similar structural or chemical W O 98/07753 PCT~US97/14666 properties, e.g., replacement of leucine with isoleucine or threonine with serine. In contrast, variations may include nonconservative changes, e.g., replacement of aglycine with a tryptophan. Similar minor variations may also include amino acid deletions or insertions, or both. Guidance in deterrnining which and how many amino acid residues may be substituted, inserted or deleted without changing biological or imrnunological activity may be found using computer programs well l~nown in the art, for example, DNASTAR software (DNASTAR Inc., Madison WI).
Probes constructed according to the polynuc}eotide sequences of the present 10 invention can be used in various assay methods to provide various types of analysis.
For example, such probes can be used in fluorescent in situ hybridization (FISH~technology to perform chromosomal analysis, and used to identify cancer-specificstructural alterations in the chromosomes, such as deletions or translocations that are visible from chromosome spreads or detectable using PCR-generated and/or allele 15 specific oligonucleotides probes, allele specific amplification or by direct sequencing. Probes also can be labeled with radioisotopes, dircctly- or indirectly-detectable haptens, or fluorescent molecules, and utilized for in situ hybridization studies to evaluate the mRNA expression of the gene comprising the polynucleotidc in tissue specimens or cells.
This invention also provides teachings as to the production of the polynucleotides and polypeptides provided herein.
SEQUENCE ID NO 1 and SEQUENCE ID NO 2 present the nucleotide sequence of the m~mm~globin gene. SEQUENCE ID NO 3, SEQUENCE ID NO
4, SEQUENCE ID NO 5, and SEQUENCE ID NO 6, present probcs useful in 25 performing probe assays while SEQI JENCE ID NOS 17-26 present polypeptidcs useful for perforrning immunoassays, in purification and as immunogens for antibodies specific to m~mm~lobin.

Probe Assays The sequences provided herein may be used to produce probes which can be used in assays for the detection of nucleic acids in test samples. The probes may be designed from conserved nucleotide regions of the polynucleotides of interest orfrom non-conserved nucleotide regions of the polynuclcotide of interest. The design of such probes for optimization in assays is within the skill of the routineer.
35 Generally, nucleic acid probes are developed from non-conserved or uniquc regions W O 981~7/-.~ PCTAJS97/14666 when maximum specificity is desired, and nucleic acid probes are developed from conserved regions when assaying for nucleotide regions that are closely related to, for example, different members of a multi-gene family or in related species likemouse and man.
The polymerase chain reaction (PCR) is a technique for amplifying a desired nucleic acid sequence (target) contained in a nucleic acid or mixture thereof. In PCR, a pair of primers are employed in excess to hybridize to the complementary strands of the target nucleic acid. The primers are each extended by a polymerase using the target nucleic acid as a template. The extension products become target sequences themselves, following dissociation from the original target strand. New primers then are hybridized and extended by a polymerase, and the cycle is repeated to geometrically increase the number of target sequence molecules. PCR is disclosed in U.S. Patents 4,683,1g5 and 4,683,202.
The Ligase Chain Reaction (LCR) is an alternate method for nucleic acid amplification. In LCR, probe pairs are used which include two primary (first andsecond) and two secondary (third and fourth) probes, all of which are employed in molar excess to target. The first probe hybridizes to a first segment of the target strand, and the second probe hybridizes to a second segmcnt of the target strand, the first and second segments being contiguous so that the primary probes abut one another in 5' phosphate-3' hydroxyl relationship, and so that a ligase can covalently fuse or ligate the two probes into a fused product. In addition, a third (secondary) probe can hybridize to a portion of the first probe and a fourth (secondary) probe can hybridize to a portion of the second probe in a similar abutting fashion. Of course, if the target is initially double stranded, the secondary probes also will hybridize to the target complement in the first instance. Once the ligated strand of primary probes is separated from the target strand, it will hybridize with the third and fourth probes which can be ligated to form a complcmentary, secondary ligated product. It is important to realize that the ligated products are functionally equivalent to either the target or its complement. By repeated cycles of hybridization and ligation, amplification of the target sequence is achieved. This technique is described more completely in EP-A- 320 308 to K. Backman published June 16, 1989 and EP-A-439 182 to K. Backman et al., published July 31, 1991.
For amplification of mRNAs, it is within the scope of the present invention to reverse transcribe mRNA into cDNA followed by polymcrase chain reaction (RT-PCR); or, to use a single enzyme for both stcps as described in U.S. Patent No.

WO 98/077!!;3 PCT/US97/14666 ~,322,770; or reverse transcribe mRNA into cDNA followed by asymmetric gap ligase chain reaceion (RT-AGLCR) as described by R.L. Marshall et al., P(~R
Methods and Applications 4: 80-84 (1994).
Other known amplification methods which can be utilized herein include but are not limited to the so-called "NASBA" or "3SR" technique described by J.(~.
Guatelli et al., PNA~ USA 87:1874-1878 (1990) and also described by J.
Compton, Nature 350 (No. 6313):91-92 (1991); Q-beta amplification as described in published European Patent Application (EPA) No. 4544610; strand displacement amplification ~as described in G.T. Walkeret al., Clin. Chem. 42:9-13 tl996]) and 10 E~uropean Patent Application No. 684315; and target mediated amplification, as described in International Publication No. WO 93/22461.
Detection of m~mm~lobin may be accomplished using any suitable detection method, including those detection methods which are currently well known in the art, as well as detection strategies which may evolve later. See, for example, 15 Caskey et al., U.S. Patent No. 5,582,g89, Gelfand ct al., U.S. Patent No.
5,210,015. Examples of such detection methods include target amplification methods as well as signal amplification technologies. An example of presently known detection methods would include the nucleic acid amplification technologies referred to as PCR, LCR, NASBA, SDA, RCR and TMA. See, for example, 20 Caskey et al., U.S. Patent No. 5,582,989, Gelfand et al., U.S. Patent No.
5,210,015. Detection may also be accomplished using signal amplification such asthat disclosed in Snitman et al., U.S. Patent No. 5~273,882. While the amplification of target or signal is preferred at present, it is contemplated and within the scope of the present invention that ultrasensitive detection methods which do not 25 require amplification can be utilized herein.
Detection, both amplified and non-amplified, may be (combined) carried out using a variety of heterogeneous and homogeneous detection formats. Examples of heterogeneous detection formats are disclosed in Snitman et al., U.S. Patent No.5,273,882, Albarella et al in EP-84114441.9, Urdea et al., U.S. Patent No.
30 5,124,246, Ullman et al., U.S. Patent No. 5,185,243 and Kourilsky et al., U.S.
Patent No. 4,581,333. Examples of homogeneous detection formats are discloscd in, Caskey et al., U.S. Patent No. 5,582,989, Gelfand et al., U.S. Patent No.
5,210,015. Also contemplated and within the scope of the present invention is the use of multiple probes in the hybridization assay, which use improves sensitivity W O 98/~7753 PCT~US97/14666 and amplification of the m~rnm~globin signal. See, for example, Caskey et al., U.S. Patent No. 5,582,989, Gelfand et al., U.S. Patent No. 5,210,Q15.
In one embodiment, the present invention generally comprises the steps of contacting a test sample suspected of containing a target polynucleotide sequence S with amplification reaction reagents CC~ isillg an amplification primer, and a detection probe that can hybridize with an internal region of the amplicon sequences.
Probes and primers employed according to the method provided herein are labeled with capture and detection labels, wherein probes are labeled with one type of label and primers are labeled with another type of label. Additionally, the primers and probes are selected such that thc probe sequence has a lower melt temperature than the primer sequences. The amplification reagents, detection reagents and test sample are placed under amplification conditions whereby, in the presence of target sequence, copies of the target sequence (an amplicon) are produced. In the usualcase, the amplicon is double stranded because primers are provided to amplify a target sequence and its complementary strand. The double stranded amplicon then is thermally denatured to produce single stranded amplicon members. Upon formation of the single stranded amplicon members, the mixture is cooled to allow the formation of complexes between the probes and single stranded amplicon members.
As the single stranded amplicon se~uences and probe sequences are cooled, the probe sequences preferentially bind the single stranded amplicon members. This finding is counterintuitive given that the probe sequences generally are selected to be shorter than the primer sequences and therefore have a lower melt temperature than the primers. Accordingly, the melt temperature of the amplicon produced by the primers should also have a higher melt temperature than the probes. Thus, as themixture cools, the re-formation of the double stranded amplicon would be expected.
As previously stated, however, this is not the case. The probes are found to preferentially bind the single stranded amplicon members. Moreover, this preference of probe/single stranded amplicon binding exists even when the primersequences are added in excess of the probes.
After the probe/single stranded amplicon member hybrids are formed, they are detected. Standard heterogeneous assay formats are suitable for detecting the ~ hybrids using the detection labels and capture labels present on the primers and probes. The hybrids can be bound to a solid phase reagent by virtue of the capture label and detected by virtue of the detection label. In cases where the detection label is directly detectable, the presence of the hybrids on the solid phase can be detected W O 98/07753 P~llUS97/14666 -3~

by causing the label to produce a detectable signal, if n~cess~ry, and <~et~cting the signal. In cases where the label is not directly detectable, the captured hybrids can be contacted with a conjugate, which generally comprises a binding member attached to a directly detectable label. The conjugate becomes bound to the complexes andS the conjugates presence on the complexes can be detected with the directly detectable label. Thus, the presence of the hybrids on the solid phase reagent can be deterrnined. Those skilled in the art will recognize that wash steps may be employed to wash away unhybridized amplicon or probe as well as unbound conjugate.
Although the target sequence is described as single stranded, it also is l 0 contemplated to include the case where the target sequence is actually double stranded but is merely separated from its complement prior to hybridization with the amplification primer sequences. In the case where PCR is employed in this method, the ends of the target sequences are usually known. In cases where LCR or a modification thereof is employed in the preferred method, the entire targct sequence 15 is usually known. Typically, the target sequence is a nucleic acid sequence such as, for example, RNA or DNA.
The method provided herein can be used in well-known amplification reactions that include thermal cycle reaction mixtures, particularly in PCR and gap LCR (GLCR). Amplification reactions typically employ primers to repeatedly 20 generate copies of a target nucleic acid sequence, which target sequence is usually a small region of a much larger nucleic acid sequence. Primers ar~ thcmselves nucleic acid sequences that are complementary to regions of a target sequence. Under amplification conditions, these primers hybridize or bind to the complcmentary regions of the target sequence. Copies of the target sequence typically are generatcd 25 by the process of primer extension and/or ligation which utilizes enzymes with polymerase or ligase activity, separately or in combination, to add nucleotides to the hybridized primers and~or ligate adjacent probe pairs. The nuclcotides that are added to the primers or probes, as monomers or preformed oligomers, are also complementary to the target sequence. Once the primers or probes have been 30 sufficiently extended and/or ligated, they are separated from the target sequence, for example, by heating the reaction mixture to a "melt temperature" which is one inwhich complementary nucleic acid strands dissociate. Thus, a sequence complementary to the target sequence is formed.
A new amplification cycle then can take place to further amplify the number 35 of target sequences by separating any double stranded scquences, allowing primers W O 98107753 PCTrUS97114666 - or probes to hybridize to their respective targets, extending and/or ligating the hybridized primers or probes and re-separating. The complementary sequences thatare generated by amplification cycles can serve as templates for primer extension or fil}ing the gap of two probes to further amplify the number of target sequences.5 Typically, a reaction mixture is cycled between 20 and 100 times, more typically, a reaction mixture is cycled between 25 and 50 times. The numbers of cycles can bedetermined by the routineer. ~n this manner, multiple copies of the target sequence and its complementary sequence are produced. Thus, primers initiate amplification of the target sequence when it is present under amplification conditions.
Generally, two primers which are complementary to a portion of a target strand and its complement are employed in PCR. For LCR, four probes, two of which are complementary to a target sequence and two of which are similarly complementary to the target's complement, are generally employed. In addition tothe primer sets and enzymes previously mentioned, a nucleic acid amplification 15 reaction mixture may also comprise other rcagents which are well known and include but are not limited to: enzyme cofactors such as manganese; magnesium;
salts; nicotinamide adenine dinucleotide (NAD); and deoxynucleotide triphosphates (dNTPs) such, as for example, deoxyadenine triphosphate, deoxyguanine triphosphate, deoxycytosine triphosphate and deoxythymine triphosphate.
While the amplification primers initiate amplification of the target sequence, the detection (or hybridization) probe is not involved in amplification. Detection probes are generally nucleic acid sequences or uncharged nucleic acid analogs such as, for example, peptide nucleic acids which are disclosed in International Publication No. WO 92/20702; morpholino analogs which are described in U.S.
25 Patents Nos 5,185,444, 5,034,506 and 5,142,047; and the like. Depending upon the type of label carried by the probe, the probe is employed to capture or detect the amplicon generated by the amplification reaction. The probe is not involved in amplification of the target sequence and therefore may have to be rendercd "non-extendible" in that additional dNTPs cannot be added to the probe. In and of 30 themselves, analogs usually are non-extendible and nucleic acid probes can berendered non-extendible by modifying the 3' end of the probe such that the hydroxyl group is no longer capable of participating in elongation. For example, the 3' end of the probe can be functionalized with the capture or detection label to thereby consume or othcrwise block the hydroxyl group. Alternatively, the 3' hydroxyl 35 group simply can be cleaved, replaced or modified. U.S. Patent Application Serial W O 98/07753 PCT~US97/14666 No. 07/049,061 filed April 19, 1993 describes modi~lcations which can be used torender a probe non-extendible.
The ratio of primers to probes is not important Thus, either the probes or primers can be added to the reaction mixture in excess whereby the concentration of 5 one would be greater than the concentration of the other. Alternatively, primers and probes can be employed in equivalent concentrations Preferably, however, the primers are added to the reaction mixture in excess of the probes. Thus, primer to probe ratios of, for example, 5: 1 and 20: 1 are preferred While the length of the primers and probes can vary, the probe sequences are 10 selected such that they have a lower melt temperature than the primer sequences.
Hence, the primer sequences are generally longer than the probe sequences.
Typically, the primer sequences are in the range of between 20 and 50 nucleotides long, more typically in the range of between 20 and 30 nucleotides long. The typical probe is in the range of between 10 and 25 nucleotides long.
Various methods for synthesizing primers and probes are well known in the art. Similarly, methods for attaching labels to primers or probes are also well known in the art. For example, it is a matter of routine to synthesize desired nucleic acid primers or probes using conventional nucleotide phosphoramidite chemistry and instruments available from Applied Biosystems, Inc., (Foster City, C~), Dupont (Wilmington, DE), or Milligen (Bedford MA). Many methods have been described for labeling oligonucleotides such as the primers or probes of the present invention.
Enzo Biochemical (New York, NY) and Clontech (Palo Alto, CA) both have described and commercialized probe labeling techniques. For example, a primary amine can be attached to a 3' oligo terminus using 3'-Amine-ON CPGTM (Clontech, Palo Alto, CA). Similarly, a primary amine can be attached to a S' oligo terminus using Aminomodifier II~ (Clontech). The amines can be reacted to various haptensusing conventional activation and linking chemistries. In addition, copending applications U.S. Serial Nos. 625,566, fîled December 11, 19g0 and 630,908, filed December 20, 1990, teach methods for labeling probes at their 5' and 3' termini,respectively. International Publication Nos WO 92/l0505, published 25 June 1992,and WO 92/11388, published 9 July 1992, teach methods for labeling probes at their S' and 3' ends, respectively. According to one known method for labeling an oligonucleotide, a label-phosphoramidite reagent is prepared and used to add thelabel to the oligonucleotide during its synthesis. See, for example, N.T. Thuong et al., Tet. Letters 29(46):5905-5908 (1988); or J.S. Cohen et al., published U.S.

CA 02232239 l998-04-20 W O 98/07753 PCTrUS97/14666 Patent Application 07/246,688 (NTIS ORDER No. PAT-APPL~7-246,688) (1989).
Preferably, probes are labeled at their 3' and 5' ends.
A capture label is attached to the primers or probes and can be a specific binding member which forms a binding pair with the solid phase reagent's specific binding member. It will be understood that the primer or probe itself may serve as the capture label. For example, in the case where a solid phase reagent's binding member is a nucleic acid sequence, it may be selected such that it binds a complementary portion of the primer or probe to thereby immobilize the primer orprobe to the solid phase. In cases where the probe itself serves as the binding 10 member, those skilled in the art will recognize that the probe will contain a sequence or "tail" that is not complementary to the single stranded amplicon members. In the case where the primer itself serves as the capture label, at least a portion of the primer will be free to hybridize with a nucleic acid on a solid phase because the probe is selected such that it is not fully complementary to the primer sequence.
Generally, probe/single strandcd amplicon member complexes can bc detected using techniques commonly employed to perform heterogeneous immunoassays. Preferably, in this embodiment, detection is performed according to the protocols used by the commercially available Abbott LCx~ instr~ mentation (Abbott Laboratories, Abbott Park, IL).
The primers and probes disclosed herein are useful in typical PCR assays, wherein the test sample is contacted with a pair of primcrs, amplification is performed, the hybridization probe is added, and detection is performed.
Another method provided by the present invention comprises contacting a test sample with a plurality of polynucleotides, wherein at least one polynucleotide is 25 a m~rnm~globin molccule as described herein, hybridizing the test samplc with the plurality of polynucleotides and detecting hybridization complexes. Hybridization complexes are identified and quantitated to compile a profile which is indicative of breast tissue disease, such as breast cancer. Expressed RNA sequences may further be detected by reverse transcription and amplification of the DNA product by 30 procedures well-known in the art, including polymerase chain reaction (PCR).
LCR primers are provided herein. They include SEQUENCE ID NO 3, SEQUENCE ID NO 4, SEQUENCE ID NO 5, and SEQUENCE ID NO 6.

W O 98/07753 PCT~US97114666 Dru~ Screening and (;~ene Therapy.
The present invention also encompasses the use of gene therapy methods for the introduction of anti-sense m~mm~globin dcrived molecules, such as polynucleotides or oligonucleotides of the present invention, into patients withconditions associated with abnormal expression of polynucleotides related to a breast tissue disease or condition especially breast cancer. These molecules, includingantisense RNA and DNA fragments and ribozymes, are designed to inhibit the translation of mammaglobin-rnRNA, and may be used therapeutically in the treatment of conditions associated with altered or abnormal expression of a 10 m~mm~globin polynucleotide.
Alternatively, the oligonucleotides described above can be delivered to cells by procedures known in the art such that the anti-sense RNA or DNA may be expressed in vivo to inhibit production of a m~mm:~globin polypeptide in the manner described above. Antisense constructs to a mammaglobin polynucleotide, therefore, 15 reverse the action of m~mm~globin transcripts and may be used for treating breast tissue disease conditions, such as breast cancer. These antisense constructs mayalso be used to treat tumor metastases.
The present invention also provides ? method of screening a plurality of compounds for specific binding to mammaglobin polypeptide(s), or any fragment 20 thereof, to identify at least one compound which specifically binds the mammaglobin polypeptide. Such a method comprises the steps of providing at least one compound; combining the mammaglobin polypeptide with each compound under suitable conditions for a time sufficient to allow binding; and detecting the m~Tnm:~globin polypeptide binding to each compound.
The polypeptide or peptide fragment empioyed in such a test may either be free in solution, affixed to a solid support, borne on a cell surface or locatedintracellularly. One method of drug sc-eening utilizes eukaryotic or prokaryotic host cells which are stably transfected with recombinant nucleic acids which can express the polypeptide or peptide fragment. Drugs may be screened against such 30 transfected cells in competitive binding assays. For example, the formation of complexes between a polypeptide and the agent being tested can be measured in either viable or fixed cells.
The present invention thus provides methods of screening for drugs or any other agent which can be used to treat diseases associated with m~mm~globin.
35 These methods comprise contacting the drug with a polypeptide or fragment thereof W O 981~7753 PCT~US97/14666 and assaying for either the presence of a complex between the agent and the polypeptide, or for the presence of a complex between the polypeptide and the cell.
In competitive binding assays, the polypeptide typically is labeled. After suitable incubation, free ~or uncomplexed) polypeptide or fragment thereof is separated from that present in bound form, and the amount of free or uncomplexed label is used as a measure of the ability of the particular drug to bind to the polypeptide or to interfere with the polypeptide/cell complex.
The present invention also encompasses the use of competitive drug screening assays in which neutralizing antibodies capable of binding polypeptide10 specifically compete with a test drug for binding to the polypeptide or fragment thereof. In this manner, the antibodies can be used to detect the presence of any polypeptide in the test sample which shares one or more antigenic determinants with a m:~mm~globin polypeptide as provided herein.
Another techni~lue for drug screening provides high throughput screening for 15 compounds having suitable binding affinity to at least one polypeptide of m~mm~globin disclosed herein. Briefly, large numbers of different small peptide test compounds are synthesized on a solid phase, such as plastic pins or some other surface. The peptide test compounds are reacted with polypeptide and washed.
Polypeptide thus bound to the solid phase is detected by methods well-known in the 20 art. Purified polypeptide can also be coated directly onto plates for use in the drug screening techniques described herein. In addition, non-neutralizing antibodies can be used to capture the polypeptide and immobilize it on the solid support. See, for example, EP 84/03564, published on September 13, 1984.
The goal of rational drug design is to produce structural analogs of 25 biologically active polypeptides of interest or of the small molecules including agonists, antagonists, or inhibitors with which they interact. Such structural analogs can be used to design drugs which are more active or stable forms of the polypeptide or which enhance or il~t~lrele with the function of a polypeptide in vivo. J.
Hodgson, Bio/Technolo~y 9: 19-21 ( 1991).
For example, in one approach, the three-dimensional structure of a polypeptide, or of a polypeptide-inhibitor complex, is determined by x-ray crystallography, by computer modeling or, most typically, by a combination of the two approaches. Both the shapc and charges of the polypeptide must be ascertained to elucidate the structure and to determine active site(s) of the molecule. Lcss often, 35 useful information regarding the structure of a polypeptide may be gained by W O 98/077S3 PCT~US97114666 modeling based on the structure of homologous proteins. In both cases, relevant structural information is used to design analogous polypeptide-like molecules or to identify efficient inhibitors Useful exarnples of rational drug design may include molecules which have improved activity or stability as shown by S. Braxton et al., Biochemistry 31:7796-7801 (1992), or which act as inhibitors, agonists, or antagonists of native peptides as shown by S.B.P. Athauda et al., J. Biochem. (Tokyo) 113 (6):742-746 (1993).
It also is possible to isolate a target-specific antibody selected by an assay as described hereinabove, and then to determine its crystal structure. In principle, this 10 approach yields a pharmacophore upon which subsequent drug design can be based.
It further is possible to bypass protein crystallography altogether by generating anti-idiotypic antibodies ("anti-ids") to a functional, pharmacologically active antibody.
As a mirror image of a mirror image, the binding site of the anti-id is an analog of the original receptor. The anti-id then can be used to identify and isolate peptides 15 from banks of chemically or biologically produced peptides. The isolated peptides then can act as the pharmacophore (that is, a prototype pharmaceutical drug).
A sufficient amount of a recombinant polypeptide of the present invention may be made available to perform analytical studies such as X-ray crystallography.
In addition, knowledge of the polypeptide amino acid sequcnce which is derivable20 from the nucleic acid sequence provided herein will provide guidance to thoseemploying computer modeling techniques in place of, or in addition to, x-ray crystallography .
Antibodies specific to a m~mm:~globin polypeptide (e.g., anti-mammaglobin antibodies~ further may be used to inhibit the biological action of the polypeptide by 25 binding to the polypeptide. In this manner, the antibodies may be used in therapy, for example, to treat breast tissue diseases including breast cancer and its metastases.
Further, such antibodies can detect the presence or absence of a m~mm~l~lobin polypeptide in a test sample and, therefore, are useful as diagnostic markers for the diagnosis of a breast tissue disease or condition especially breast 30 cancer. Such antibodies may also function as a diagnostic marker for breast tissue disease conditions such as breast cancer. The present invention also is directed to antagonists and inhibitors of the polypeptides of the present invention. The antagonists and inhibitors are those which inhibit or elirninate the function of the polypeptide. Thus, for example, an antagonist may bind to a polypeptide of the 35 present invention and inhibit or elimin~tf~ its function. The antagonist, for example, W O 98/07753 PCT~US97/14666 could be an antibody against the polypeptide which elimin~t~s the activity of a m~mm~globin polypeptide by binding a m~mm~globin polypeptide, or in some cases the antagonist may be an oligonucleotide. Examples of small rnolecule inhibitorsinclude, but are not limited to, small peptides or peptide-like molecules.
The antagonists and inhibitors may be employed as a composition with a pharm~reutically acceptable carrier including, but not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol and combinations thereof. ~rlmini~tration of m~mm:~lobin polypeptide inhibitors is preferably systemic. The present invention also provides an antibody which inhibits the action of such a polypeptide.
Antisense technology can be used to reduce gene expression through triple-helix forrnation 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 ofthe polynucleotide sequence, which encodes for the polypeptide of the present invention, is used to design an antisense RNA oligonucleotide of from 10 to 40 base pairs in length. A DNA oligonucleotide is designed to be complemcntary to a region of the gene involved in transcription, thereby preventing transcription and the production of the m~mm~globin polypeptide. For triple helix, sce, for example, Lee et al., Nuc. Acids Res. 6:3073 (1979); Cooney et al., Science 241:456 (1988); and Dervan et al., Science 251:1360(1991) The antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks translation of a mRNA molecule into the m:3mm~lobin polypeptide. For antisense, see, for example, Okano, J. Neurochem.
56:560 (1991); and "Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression", CRC Press, Boca Raton, Fla. (1988). Antisense oligonuclcotides act with greater efficacy when modified to contain artificial internucleotide linkages w-hich render the molecule resistant to nucleolytic cleavage. Such artificial internucleotide linkages include, but are not limited to, methylphosphonate, phosphorothiolate and phosphoroamydate internucleotide linkages.
Recombinant Technolo~y.
The present invention provides host cells and expression vectors comprising m~mm:~globin polynucleotides of the present invention and methods for the production of the polypeptide(s) they encode. Such methods comprise culturing the host cells under conditions suitable for the expression of the m~mm~globin polynucleotide and recovering the m~mm~globin polypcptide from the ccll culture.The present invention also provides vectors which include m~mm~globin polynucleotides of the present invention, host cclls which are genetically engineered W O 98/07753 PCT~US97/14666 with vectors of the present invention and thc production of polypeptides of the present invention by recombinant techniques.
Host cells are genetically engineered (transfected, tr~n~ ced or transformed) with the vectors of this invention which may be cloning vectors or expression vectors. The vector may be 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, selecting transfected cells, or amplifyingm~mm~globin gene(s). The culture conditions, such as temperature, pH and the like, are those previously used with the host cell selccted for expression, and will be 10 apparent to the ordinarily skilled artisan.
The polynucleotides of the present invention may be employed for producing a polypeptide by recombinant techniques. Thus, the polynucleotide sequence may be included in any one of a variety of expression vehicles, in particular vectors or plasmids for expressing a polypeptide. Such vectors include chromosomal, 15 nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40; bacterial plasrnids; phage DNA; yeast plasmids; vectors derived from combinations of plasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox virus and pseudorabies. However, any other plasmid or vector may be uscd so long as itis replicable and viable in the host.
The ~upliate DNA sequence may be inserted into the vector by a variety of procedures. In general, the DNA sequence is inserted into appropriate restriction endonuclease sites by procedures known in the art. Such procedures and others are deemed to be within the scope of thosc skilled in the art. The DNA sequence in the expression vector is operatively linked to an appropriate expression control 25 sequence(s) (promoter) to direct mRNA synthesis. Representative examples of such promoters include, but are not limited to, the LTR or the SV40 promoter, the E. coli lac or trp, the phage lambda P sub L promoter and other promoters known to control expression of genes in prokaryotic or eukaryotic cells or their viruses. The expression vector also contains a ribosome binding site for translation initiation and 30 a transcription terminator. The vector may also include appropriate sequences for amplifying expression. In addition~ the expression vectors preferably contain a gene to provide a phenotypic trait for selection of transfected host cells such as dihydrofolate rcductase or neomycin resistance for cukaryotic cell culture, or such as tetracycline or ampicillin resistance in E. coli.

W O 98/077'.3 PCT~US97/14666 .

The vector cont~ining the a~lupl iate DNA sequence as hereinabove described, as well as an ~L~lupliate promoter or control sequence, may be emp'ioyed to transfect an ~p~ .l iate host to permit the host to express the protein. As representative examples of al)l)ru~l iate hosts, there may be mentioned: bacterial cells, such as E coli, Salmonella typhimurium: Streptomyces ~; fungal cells, such as yeast; insect cells such as Drosophila and Sf9; animal cells such as CHO, COS or Bowes melanoma; plant cells, etc. The selection of an a~ Iiate host is deemed tobe within the scope of those skilled in the art from the teachings provided herein.
More particularly, thc 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 sequence of the invention has been inserted, in a forward or reverse orientation. In a preferred aspect of this embodiment, the construct further comprises regulatory sequences, inc}uding, 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 ofexample. Bacterial: pINCY (Incyte Pharmaceuticals Inc., Palo Alto, CA), pSPORT1 (Life Technologies, Gaithersburg, MD), pQE70, pQE60, pQE-9 (Qiagen) pBs, phagescript, psiX174, pBluescript SK, pBsKS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene); pTrc99A, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia); Eukaryotic: pVVLneo, pSV2cat, pOG44, pXT 1, pSG ~Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia). However, any other plasmid or vector may be used as long as it is replicable and viable in the host.
Plasmid pINCY is generally identical to the plasmid pSPORT1 (available from Life Technologies, Gaithersburg, MD) with the exception that it has two modifications in the polylinker (multiple cloning site). These modifications are ( I ) it lacks a Hindm restriction site and (2) its EcoRI restriction site lies at a different location. pINCY is created from pSPORTI by cleaving pSPORTI with both Hindm and EcoRI and replacing the excised fragment of the polylinker with synthetic DNA fragments (SEQUENCE ID NO 7 and SEQUENCE ID NO 8). This replacement may be made in any manner known to those of ordinary skill in the art.
For example, the two nucleotide sequences, SEQUENCE ID NO 7 and SEOUENCE ID NO 8, may be generated synthetically with 5' terminal phosphates, mixed together, and then ligated under standard conditions for performing staggered end ligations into the pSPORTI plasmid cut with HindIII and EcoRI. Suitable host W O 98/07753 PCTrUS97/14666 ~0-cells (such as E. ç~ DH5~x cells) then are trans~ected with the ligated DNA and recombinant clones are selected for ampicillin resistance. Plasmid DNA then is prepared from individual clones and subjected to restriction enzyme analysis or DNA
sequencing in order to confirm the presence of insert sequences in the proper 5 oricntation. Other cloning strategies known to the ordinary artisan also may be employed.
Promoter regions can be selected from any desired gene using CAT
(chloramphenicol transferase) vectors or other vectors with sclectable markers. Two a~ Opl iate vectors are pKK232-8 and pCM7. Particular named bacterial promotcrs 10 include lacI, lacZ, T3, SP6, T7, gpt, lambda P sub R, P sub L and trp. Eukaryotic promoters include cytomegalovirus (CM~) immediate early, herpes simplcx virus (HSV) thymidine kinase, early and late SV40, LTRs from retroviruses and mouse metallothionein-I. Selection of the ap~ )pl iate vector and promoter is well within the Icvel of ordinary skill in the art.
In a further embodiment, the present invention provides host cells containing the above-described construct. The host cell can be a higher eukaryotic cell~ such as a mamm~ n 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 thc construct into the host cell can be effected by calcium phosphate transfection, DEAE-Dextran mediated 20 transfection, or electroporation (L. Davis et al., "Basic Methods in Molecular Biology", 2nd cdition, Appleton and Lang, Paramount Publishing, East Norwalk, CT (1994)).
The constructs in host cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence. Alternatively, the 25 polypeptides of the invention can be synthetically produced by conventional peptide synthesizers.
Recombinant proteins can be exprcssed in m~mm~ n cells, yeast, bacteria, or other cells, under the control of ~ l iate promotcrs. Cell-free translation systems can also be employed to produce such proteins using RNAs derived from 30 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 Clonin~: A I~aboratorv Manual, Second Edition, (Cold Spring Harbor, N.Y., 1989).
Transcription of a DNA encoding the polypcptide(s) of the present invention 35 by highcr eukaryotcs is increased by inserting an enhancer sequence into the vector.

F.nh~n~ers are cis-acting elements of DNA, usually about from 10 to 300 bp, that act on a promoter to increase its transcription. Examples include the SV40 enhancer on - the late side of the replication origin (bp 100 to 270), a cytomegalovirus early promoter enhancer, a 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 transfection of the host cell, e.g., the ampicillin resistance gene of E. coli and S . cerevisiae TRP I 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), alpha factor, acid phosphatase, or heat shock proteins, among others. The heterologous structural sequence is assembled in appr~,p,iate phase with translation initiation and termination sequences, and preferably, a leader sequence capable of directing secretion of tr~n~l~t~-l protein into the periplasmic space or extracellular medium. 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 expressed recombinant product.
Useful expression vectors for bacterial use are constructed by inserting a structural DNA sequence encoding a desired protein together with suitable translation initiation and termination signals in operable reading phase with a functional promotcr. The vector will comprise onc or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and to, if desirable, provide amplification within the host. Suitable prokaryotic hosts fortransfection include E. coli. Bacillus subtilis. Salmonella typhimurium and various species within the genera Pseudomonas. Streptomvces and Staphylococcus.
although, others may also be employed as a routine matter of choice.
Useful expression vectors for bacterial use comprise a selectable marker and bacterial origin of rep]ication derived from plasmids comprising genetic elements of the well-known cloning vector pBR322 (ATCC 37017). Other vectors include but are not limited to PKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM 1 (Promega Biotec, Madison, WI). Thesc pBR322 "backbone" sections are combined with an apL)lupl iate promoter and the structural sequence to be expressed.
Following transfection of a suitable host and growth of the host to an apl.,upliate cell density, the selected promoter is derepressed by ~L~p,opliate means W O g8J'~ PCTnUS97/14666 (e.g., temperature shift or chernical induction), and cells are cultured for an additional period. Cells are typically harvested by centrifugation, disrupted byphysical or chernical means, and the resulting crude extract retained for further purification. Microbial cells employed in expression of proteins can be disrupted by 5 any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell Iysing agents; such methods are well-known to the ordinary artisan.
Various m:~mm~lian cell culture systems can also be employed to express recombinant protein. Examples of m~mm~ n expression systems include the COS-7 lines of monkey kidney fibroblasts described by Gluzman, Cell 23:175 (1981), and other cell lines capable of expressing a compatible vector, such as the C127, HEK-293, 3T3, CHO, HeLa and BHK cell lines. Mammalian expression vectors will comprise an origin of replication, a suitable promoter and enhancer and also any necessary ribosome binding sites, polyadenylation sites, splice donor and acceptor sites, transcriptional termination sequences and 5' flanking nontranscribed sequences. DNA sequences derived from the SV40 viral genome, for example, SV40 origin, early promoter, enhancer, splice, and polyadenylation sites may be used to provide the required nontranscribed genetic elements. Representative, useful vectors include p~c/CMV and pcDNA3 (available from Invitrogen, San Diego, CA).
Mamm:~globin polypeptides are recovered and purified from recombinant cell cultures by known methods including affinity chromatography, ammonium sulfatc or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, hydroxyapatite chromatography or lectin chromatography. It is preferred to have ~5 low concentrations (approximately 0.1-5 mM) of calcium ion present during purification (Price, et al., J. Biol. Chem. 244:917 (1969)). Protein refolding steps can be used, as necessary, in completing configuration of the polypeptide. Finally, high performance liquid chromatography (HPLC:~) can be employed for final purification steps.
Thus, polypeptides of the present invention may be naturally purified products expressed from a high expressing cell line, 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 m, rnm~ n cells in culture~. Depcnding upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated W O ~X~7/5~ PC~US97/14666 with m:~mmz~ n or other eukaryotic carbohydrates or may be non-glycosylated.
The polypeptides of the invention may also include an initial methionine amino acid residue.
The starting plasmids can be constructed from available plasmids in accord 5 with published, known procedures. In addition, equivalent plasmids to those described are known in the art and will be a~l,al~;nt to the ordinarily skilled artisan.
The following is the general procedure for the isolation and analysis of cDNA clones. In a particular embodiment disclosed herein, m~mm,.globin mRNA
was isolated from human breast tissue and used to generate the cDNA library.
The nucleotide sequence of m,.mm~globin may contain an entire open reading frame with or without associated regulatory sequences for a particular gene, or they may encode only a portion of the gene of interest. This is attributed to the fact that many genes are several hundred, and sometimes several thousand, bases in length and, with current technology, cannot be cloncd in their entirety because of 15 vector limitations, incomplete reverse transcription of the first strand, or incomplete replication of the second strand. Contiguous, secondary clones containing additional nucleotide sequences may be obtained using a variety of methods knownto those of skill in the art.
Methods for DNA sequencing are well known in the art. Conventional 20 enzymatic methods employ DNA polymerase, Klenow fragment, Sequenase (US
Biochemical Corp, Cleveland, OH) or Taq polymerase to extend DNA chains from an oligonucleotide primer annealed to the DNA template of interest. Methods havebeen developed for the use of both single-stranded and double-stranded templates.
The chain termination reaction products may be electrophoresed on 25 urea/polyacrylarnide gels and detected either by autoradiography (for radionucleotide labeled precursors) or by fluorescence (for fluorescent-labeled precursors). Recent improvements in mechz-ni7e.d reaction preparation, sequencing and analysis using the fluorescent detection method have permitted expansion in the number of sequencesthat can be determined per day using machines such as the Applied Biosystems 37730 DNA Sequencers (Applied Biosystems, Foster City, CA).
The reading frame of the nucleotide sequence can be ascertained by several types of analyses. First, reading framcs contained within the coding sequence can be analyzed for the presence of start codon ATG and stop codons TGA, TAA or TAG. Typically, one reading frame will continue throughout the major portion of a 35 cDNA sequence while other reading frames tend to contain numerous stop codons.

In such cases, reading frame determination is straightforward. In other more difficult cases, further analysis is recluired.
Algorithms have been created to analyze the occurrence of individual nucleotide bases at each putative codon triplet. See, for example J.W. Fickett, Nuc AcidsRes 10:5303 (1982). Coding DNA for particular organisms (bacteria, plants and animals) tends to contain certain nucleotides within certain triplet periodicities, such as a significant preference for pyrimidines in the third codon position. These preferences have been incorporated into widely available software which can be used to determine coding potential (and frame) of a given stretch of DNA. The algorithm-derived inforrnation combined with start/stop codon information can be used to determine proper frame with a high degree of certainty. This, in turn, readily permits cloning of the sequence in the correct reading frame into appl~iate expression vectors.
The nucleic acid sequences disclosed herein may be joined to a variety of other polynucleotide sequences and vectors of interest by means of well-established recombinant DNA techniques. See J. Sambrook et al., ~E_- Vectors of interest include cloning vectors, such as plasmids, cosmids, phage derivatives, phagemids, as well as sequencing, replication and expression vectors, and the like. In general, such vectors contain an origin of replication functional in at least one organism, convenient restriction endonuclease digestion sites and selectable markers appropriate for particular host cells. The vectors can be transferred by a variety of means known to those of skill in the art into suitable host cells which thcn produce the desired DNA, RNA or polypeptides.
Occasionally, sequencing or random reverse transcription errors will mask the presence of the appfopliate open reading frame or regulatory element. In such cases, it is possible to determine the correct reading frame by attempting to express the polypeptidc and determining the amino acid sequence by standard peptide mapping and sequencing techniques. See, F.M. Ausubel, et al., Current Protocols in Molecular Biolo~y. John Wiley & Sons~ New York, NY ( 1989). Additionally, the actual reading frame of a given nucleotide sequence may be determined by transfection of host cells with vectors containing all threc potential reading frames.
Only those cells with the nucleotide sequence in the correct reading frame will produce a peptide of the predicted length.
The nucleotide sequences provided herein have been prepared by current, state-of-the-art, automated methods and as such may contain unidentified W O 98/077~3 PCTr~S97/14666 ~5-nucleotides. These will not present a problem to those skilled in the art who wish to practice the invention. Several methods employing standard recombinant ~ techniques, described in J. Sambrook (~) or periodic updates thereof, may be used to complete the missing sequence information. The same techniques used for 5 obtaining a full length sequence, as described herein, may be used to obtain nucleotide sequences.
Exp}ession of a particular cDNA may be accomplished by subcloning the cDNA into an ap~.opliate expression vector and transfecting this vector into an ay~lo~liate expression host. The cloning vector used for the generation of the breast 10 tissue cDNA library can be used for transcribing mRNA of a particular cDNA and contains a promoter for beta-galactosidase, an amino-terminal met and thc subsequent seven amino acid residues of beta-galactosidase. lmmt~ t~ly followingthese eight residues is an engineered bacteriophage promoter, useful for artificial priming and transcription, as well as a number of unique restriction sites, including 15 EcoRl, for cloning. The vector can be transfected into an applop,iate host strain of E. co]i.
Induction of the isolated bacterial strain with isopropylthiogalactosidc ~IPTG) using standard methods will produce a fusion protein which contains the first seven residues of beta-galactosidase, about 15 residucs of linker and the peptide 20 encoded within the cDNA. Since cDNA clone inscrts are generated by an essentially random process, there is one chance in three that the included cDNA will lie in the correct frame for proper translation. If the cDNA is not in the proper reading frame, the correct frame can be obtained by deletion or insertion of an ~pl~p,iate number of bases by well known methods including in vitro mutagenesis, digestion with 25 exonuclease III or mung bean nuclease, or oligonucleotide linker inclusion.
The cDNA can be shuttled into other vectors known to be useful for expression of protein in specific hosts. Oligonucleotide primers containing cloning sites and segments of DNA, sufficient to hybridize to stretches at both ends of the target cDNA, can be synthe~i7erl chemically by standard methods. These primers 30 can then be used to amplify the desired gene segments by PCR. The resulting new gene segments can be digested with appropriate restriction enzymes under standard conditions and isolated by gel electrophoresis. Alternately, similar gene segments can be produced by digestion of the cDNA with appropriate restriction enzymcs and filling in the m;~ing gene segments with chemically synthesized oligonucleotides.

W O 98/077~3 PCTrUS97/14666 Segments of the coding sequence from more than one gene can be ligated together and cloned in app~ riate vectors to optimize expression of recombinant sequence.Suitable expression hosts for such chimeric molecules include but are not limited to, m~mm~ n cells such as Chinese Hamster Ovary (CHO) and human 5 embryonic kidney (HEK) 293 cells, insect cells such as Sf9 cells, yeast cells such as Saccharomyces cerevisiae and bacteria such as E~. coli. For each of these cell systems, a useful expression vector may also include an origin of rep~ication toallow propagation in bacteria and a selectable marker, such as the beta-lactamase antibiotic resistance gene, to allow selection in bacteria. In addition, the vectors may 10 include a second selectable marker such as the neomycin phosphotransferase gene to allow selection in transfected eukaryotic host cells. Vectors for use in eukaryotic expression hosts may require the addition of 3' poly A tail if the sequence of interest lacks poly A.
Additionally, the vector may contain promoters or enhancers which increase 15 gene expression. Such promoters are host specific and include, but are not limited to, MMTV, SV40, or metallothionine promoters for CHO cells; trp, lac, tac or T7 promoters for bacterial hosts; or alpha factor, alcohol oxidase or PGH promotcrs for yeast. Adenoviral vectors with or without transcription enhancers, such as the rous sarcoma virus (RSV) enhancer, may be used to drivc protein expression in 20 m~mm:~lian cell lines. Once homogeneous cultures of recombinant cells are obtained, large quantities of recombinantly produced protein can be recovered from the conditioned medium and analyzed using chromatographic methods well known in the art. An alternative method for the production of large amounts of secreted protein involves the transfection of m~mm~ n embryos and the recovery of thc 2~ recombinant protein from milk produced by transgenic cows, goats, sheep, etc.Polypeptides and closely related molecules may be expressed recombinantly in such a way as to facilitate protein purification. One approach involves expression of a chimeric protein which includes one or more additional polypeptide domains not naturally present on human polypeptides. Such purification-facilitating domains 30 include, but are not limited to, metal-chelating peptides such as histidine-tryptophan domains that allow purification on irnmobilized metals, protein A domains that allow purification on immobilized immunoglobulin and the domain utilized in the F~AGS
extension/affinity purification system (Immunex Corp, Seattle, WA). The inclusion of a cleavable linker secluence such as Factor XA or enterokinase from Invitrogen W O 98/077~3 PCT~US97/14666 (San Diego, CA) between the polypeptide sequence and the purification domain maybe usefu} for recovering the polypeptide.

Immunoassays.
M~mm~lobin polypeptides, including fragments, derivatives, and analogs thereof, or cells expressing such polypeptides, can be utilized in a variety of assays, many of which are described herein, for the detection of antibodies to m~rnm~globin. They also can be used as immunogens to produce antibodics. These antibodies can be, for example, polyclonal or monoclonal antibodies, chimeric, 10 single chain and hllm~ni7e~i antibodies, as well as Fab fragments, or the product of an Fab expression library. Various procedures known in the art may be used for the production of such antibodies and fr~gm~ n~c.
For example, antibodies generated against a polypeptide comprising a sequence of the present invention can be obtained by direct injection of the 15 polypeptide into an animal or by administering the polypeptide to an animal such as a mouse, rabbit, goat or human. A mouse, rabbit or goat is preferred. The polypeptide is selected from the group consisting of SEQUENCE ID NOS 17-26, and fragments thereof. The antibody so obtained then will bind the polypeptide itself. In this manner, even a sequence encoding only a fragment of the polypeptide 20 can be used to generate antibodies that bind the native polypeptide. Such antibodies then can be used to isolate the polypeptide from tcst samples such as tissue suspected of containing that polypeptide. For preparation of monoclonal antibodies, any technique which provides antibodies produced by continuous cell line cultures can be used. Examples include the hybridoma technique as described by Kohler and25 Milstein, Nature 256:495-497 (1975), the trioma technique, the human B-cell hybridoma technique as describcd by Kozbor et al., Immun. Today 4:72 (1983) and the EBV-hybridoma technique to produce human monoclonal antibodies as described by Cole et al., in Monoclonal Antibodies and Cancer Therapy. Alan R.
Liss, Inc, New York, NY, pp. 77-96 (1985). Techniques described for the 30 production of single chain antibodies can be adapted to produce single chain antibodies to immunogenic polypeptide products of this invention. See, for example, U.S. Patent No. 4,946,778.
Various assay formats may utilize the antibodies of the present invention, including "sandwich" immunoassays and probe assays. For example, the antibodies 35 of the present invention, or fragments thereof, can be cmployed in various assay WO 98/077~3 PCT~US97/1~666 systems to determine the presence, if any, of m~mm~lobin antigen in a test sample.
For example, in a ~Irst assay format, a polyclonal or monoclonal antibody or fragment thereof, or a combination of these antibodies, which has been coated on a solid phase, is contacted with a test sample, to forrn a first mixture. This first 5 mixture is incubated for a time and under conditions sufficient to form antigen/antibody complexes. Then, an indicator reagent comprising a monoclonal or a polyclonal antibody or a fragment thereof, or a combination of these antibodies, to which a signal generating compound has been attached, is contacted with the antigen/antibody complexes to form a second mixture. This second mixture then is10 incubated for a time and under conditions sufficient to form antibody/antigen/antibody complexes. The presence of m:~rnm:~globin antigen in thc test sample and captured on the solid phase, if any, is determined by detecting the measurable signal generated by the signal generating c~ und. The amount of m~mm~globin antigen present in the test sample is proportional to the signal 1 5 generated.
In an alternative assay format, a mixture is formed by contacting: ( I ) a polyclonal antibody, monoclonal antibody, or fragment thereof, which specifically binds to m~mm~globin antigen, or a combination of such antibodies bound to a solid ~iUI~)Ol L; (2~ the test sample; and (3) an indicator reagent comprising a monoclonal 20 antibody, polyclonal antibody, or fragment thereof, which specifically binds to a different m~mm~globin antigen (or a combination of these antibodies) to which a signal generating compound is attached. This mixture is incubated for a time andunder conditions sufficient to form antibody/antigen/antibody complexes. The presence, if any, of m~mm~globin antigen present in the test sample and captured on 25 the solid phase is determined by detecting the measurable signal generated by the signal generating compound. The amount of mammaglobin antigen present in the test sample is proportional to the signal generated.
In another assay format, one or a combination of at least two monoclonal antibodies of the invention can be employed as a competitive probe for the detection 30 of antibodies to m~mm~globin antigen. For example, m~mm~globin polypeptides such as the recombinant antigens disclosed herein, either alone or in combination, are coated on a solid phase. A test sample suspected of cont~ining antibody to m~mm~globin antigen then is incubated with an indicator reagent comprising a signal generating compound and at least one monoclonal antibody of the invention for a 35 time and under conditions sufficient to form antigen/antibody complexes of either the W 098/07753 PCT~US97/14666 ~9 ~ test sample and indicator reagent bound to the solid phase or the indicator reagent bound to the solid phase. The reduction in binding of the monoclonal antibody tothe solid phase can be qll~ntit~tively measured.
In yet another detection method, each of the monoclonal or polyclonal 5 antibodies of the present invention can be employed in the detection of m~mm~globin antigens in tissue sections, as well as in cells, by immunohistochemical analysis. Cytochemical analysis wherein these antibodies arelabeled directly (with, for example, fluoresccin, colloidal gold, horseradish peroxidase, alkaline phosphatase, etc.) or are labeled by using secondary labeled 10 anti-species antibodies (with various labels as exemplified herein) to track the histopathology of disease also are within the scope of the present invention.
In addition, these monoclonal antibodies can be bound to matrices similar to CNBr-activated Sepharose and used for the affinity purification of specific m~rnm~globin polypeptides from cell cultures or biological tissues such as to purify 15 recombinant and native m~mm~globin proteins.
The monoclonal antibodies of the invention also can be used for the generation of chimeric antibodies for therapeutic use, or other similar applications.
The monoclonal antibodies or fragments thereof can be provided individually to detect m~mm~globin antigens. Combinations of the monoclonal antibodies (and 20 fragments thereof) provided herein also may be used together as components in a mixture or "cocktail" of at least one m~mm:~globin antibody of the invention, along with antibodies which specifically bind to other mzlmm~globin regions, each antibody having dirrel~nt binding specificities. Thus, this cocktail can include the monoclonal antibodies of the invention which are directed to m~mm~globin 2~ polypeptides disclosed herein and other monoclonal antibodies specific to other antigenic determinants of m~mm~globin antigens or other related proteins.
The polyclonal antibody or fragment thereof which can be used in the assay formats should specifically bind to a m~rnm~giobin polypeptide or other miqmm~globin polypeptides additionally used in the assay. The polyclonal antibody 30 used preferably is of m~mm~ n origin such as, human, goat, rabbit or sheep polyclonal antibody which binds m~mm:~globin polypeptide. Most preferably, the polyclonal antibody is of rabbit origin. The polyclonal antibodies used in the assays can be used either alone or as a cocktail of polyclonal antibodies. Since the cocktails used in the assay formats are comprised of either monoclonal antibodies or 35 polyclonal antibodies having different binding specificity to m~mm~globin WO ~ .7/~ PCT/USg7/14666 polypeptides, they are useful for the dto~cting, diagnosing, staging, monitoring, prognosticating, preventing or treating, or determinin~ the predisposition to, diseases and conditions of the breast such as breast cancer.
It is contemplated and within the scope of the present invention that 5 m~mm~globin antigen may be ~ t.-.ct~hle in assays by use of a recombinant antigen as well as by use of a synthetic peptide or purified peptide, which peptide comprises an amino acid sequence of m~mm~globin. The amino acid sequence of such a poJypeptide is selected from the group consisting of SEQU~NCE ID NOS 17-26, and fragments thereof. It also is within the scope of the present invention that10 different synthetic, recombinant or purifled peptides, identifying different epitopes of m~mm~globin, can be used in combination in an assay for the detecting, diagnosing, staging, monitoring, prognosticating, preventing or treating, or determining the predisposition to diseases and conditions of the breast such as breast cancer. In this case, all of these peptides can be coated onto one solid phase; or cach 15 separate peptide may be coated onto separate solid phases, such as microparticles, and then combined to form a mixture of peptides which can be later used in assays.
Furthermore, it is contemplated that multiple peptides which define epitopes from different antigens may be used for the detection, diagnosis, staging, monitoring, prognosis, prevention or treatment of, or determining the predisposition to, diseascs 20 and conditions of the breast, such as breast cancer. Peptidcs coated on solid phases or labeled with detectable labels are then allowed to compete with those present in a patient sample (if any) for a limited amount of antibody. A reduction in binding of the synthetic, recombinant, or purified peptides to the antibody ~or antibodies) is an indication of the presence of m~mm~globin antigen in the patient sample. The 25 presence of m~mmslglobin antigen indicates the presence of breast tissue disease, especially breast cancer, in the patient. Variations of assay formats are known to those of ordinary skill in the art and many are discussed herein below.
In another assay format, the presence of anti-m~mm~;lobin antibody and/or m~mm~globin antigen can be detected in a .cimlllt~neous assay, as follows. A test 30 sample is simultaneously contacted with a capture reagent of a first analyte, wherein said capture reagent comprises a first binding member specific for a first analyte attached to a solid phase and a capture reagent ~or a second analyte, wherein said capture reagent comprises a first binding member for a second analyte attached to a second solid phase, to thereby form a mixture. This mixture is incubated for a time 35 and under conditions sufficient to form capture reagent/first analyte and capture W O 98/07753 PCT~US97/14666 reagent/second analyte complexes. These so-formed complexes then are contacted with an indicator reagent comprising a member of a binding pair specific for the first ~ analyte labeled with a signal generating compound and an incli(~tor reagent Colllplisil~g a member of a binding pair specific for the second analyte labeled with a signal generating compound to form a second mixture. This second mixture is incubated for a time and under conditions sufficient to form capture reagent/first analyte/indicator rcagent complexes and capture reagent/second analyte/indicatorreagent complexes. The presence of one or more analytes is determined by detecting a signal generated in connection with the complexes formed on either or both solid 10 phases as an indication of the presence of one or more analytes in the test sample. In this assay format, recombinant antigens derived from the expression systems disclosed herein may be lltili7e(1, as well as monoclonal antibodies produced from the proteins derived from the expression systems as disclosed herein. For example, in this assay system, m~mm~globin antigen can be the first analyte. Such assay systems are described in greater detail in EP Publication No. 0473065.
In yet other assay formats, the polypeptides disclosed herein may be utilized to detect the presence of antibody against mammaglobin antigen in test samples. For example, a test sampie is incubated with a solid phase to which at least one polypeptide such as a recombinant protein or synthetic peptide has been attached.
The polypeptide is selected from the group consisting of SEQUENCE ID NOS 17-26, and fragments thereof. These are rcacted for a time and under conditions sufflcient to form antigen/antibody complexes. Following incubation, the antigen/antibody complex is detected. Indicator reagents may be used to facilitate detection, depending upon the assay system chosen. In another assay format, a test sample is contacted with a solid phase to which a recombinant protein produced as described hercin is attached, and also is contacted with a monoclonal or polyclonal antibody specific for the protein, which preferably has been labeled with an indicator reagent. After incubation for a time and under conditions sufficient for antibody/antigen complexcs to form, the solid phase is separated from the free phase, and the labcl is detected in either the solid or free phase as an indication of the presence of antibody against n~rnm~globin antigen. Other assay formats utilizingthe recombinant antigens disclosed herein are contemplated. Thesc include contacting a test sample with a solid phase to which at least one antigen from a first source has been attached, incubating the solid phase and test sample for a time and under conditions sufficient to form antigen/antibody complexes, and then contacting W O 98/077~3 PCT~US97/14666 the solid phase with a labe1ed antigen, which antigen is derived from a second source different from the first source. For example, a recombinant protein derived from a first source such as E. coli is used as a capture antigen on a solid phase, a test sample is added to the so-prepared solid phase, and following standard incubation 5 and washing steps as deemed or required, a recombinant protein derived from a different source (i.e., non-E. coli) is utilized as a part of an indicator reagent which subsequently is detected. Likewise, combinations of a recombinant antigen on a solid phase and synthetic peptide in the indicator phase also are possible. Any assay format which utilizes an antigen specific for mammaglobin produced or derived from 10 a first source as the capture antigen and an antigen specific for m~ntm~globin from a different second source is contemplated. Thus, various combinations of recombinant antigens, as well as the use of synthetic peptides, purified proteins and the likc, are within the scope of this invention. Assays such as this and others are described in U.S. Patent No. 5,254,458.
Other embodiments which utilize various other solid phases also are contemplated and are within the scope of this invention. For example, ion capture procedures for immobilizing an immobilizable reaction complex with a negatively charged polymer (described in EP publication 0326100 and EP publication No.
0406473), can be employed according to the present invention to effect a fast solution-phase immunochemical reaction. An immobilizable immune complex is separated from the rest of the reaction mixture by ionic interactions between the negatively charged poly-anion/irnmune complex and the previously treated, positively charged porous matrix and detected by using various signal generatingsystems previously described, including those described in chemiluminescent signal measurements as described in EPO Publication No. 0 273,115.
Also, the methods of the present invention can be adapted for use in systems which utilize microparticle technology including automated and semi-automated systems wherein the solid phase comprises a microparticle (magnetic or non-magnetic). Such systems include those described in, for example, published EPO
applications Nos. EP 0 425 633 and EP 0 424 634, respectively.
The use of scanning probe microscopy (SPM) for immunoassays also is a technology to which the monoclonal antibodies of the present invention are easily adaptable. In scanning probe microscopy, particularly in atomic force microscopy, the capture phase, for example, at least one of the monoclonal antihodies of theinvention, is adhered to a solid phase and a scanning probe microscopc is utilized to ~ detect antigen/antibody complexes which may be present on the surface of the solid phase. The use of sc7~nning tunneling microscopy eliminates the need for labels which normally must be utilized in many immunoassay systems to detect antigen/antibody complexes. The use of SPM to monitor specific binding reactions5 can occur in many ways. In one embodiment, one member of a specific binding partner (analyte specific substance which is the monoclonal antibody of the invention) is attached to a surface suitable for scanning. The attachment of theanalyte specific substance may be by adsorption to a test piece which comprises a solid phase of a plastic or metal surface, following methods known to those of 10 ordinary skill in the art. Or, covalent attachment of a specific binding partner (analyte specific substance) to a test piece which test piece comprises a solid phase of derivatized plastic, metal, silicon, or glass may be utilized. Covalent attachment methods are known to those skilled in the art and include a variety of means to irreversibly link specific binding partners to the test piece. If the test piece is silicon 15 or glass, the surface must be activated prior to attaching the specific binding partner.
Also, polyelectrolyte interactions may be used to immobilize a specific binding partner on a surface of a test piece by using techniques and chemistries. The preferred method of attachment is by covalent means. Following attachment of a specific binding member, the surface may be further treated with materials such as 20 serum, proteins, or other blocking agents to minimize non-specific binding. The surface also may be scanned either at the site of manuf~cture or point of use to verify its suitability for assay purposes. The scanning process is not anticipated to alter the specific binding ~ )e~Lies of the test piece.
While the present invention discloses the preference for the use of solid 25 phases, it is contemplated that the reagents such as antibodies, proteins and peptides of the present invention can be utilized in non-solid phase assay systems. Theseassay systems are known to those skilled in the art, and are considered to be within the scope of the present invention.
It is contemplated that the reagent employed for the assay can be provided in 30 the form of a test kit with one or more containers such as vials or bottles, with each container containing a separate reagent such as a probe, primer, monoclonal antibody or a cocktail of monoclonal antibodies, or a polypeptide (e.g.
recombinantly, synthetically produced or purified) employed in the assay. The polypeptide is selected from the group consisting of SEQUENCE ID NOS 17-26, 35 and fragments thereof. Other components such as buffers, controls and the like, W O 98/07753 PCT~US97/1~666 known to those of ordinary skill in art, may be included in such test kits. It also is contemplated to provide test kits which have means for collecting test samples comprising accessible body fluids, e.g., blood, urine, saliva and stool. Such tools useful for col]ection ~'collection materials") include lancets and absorbent paper or 5 cloth for collecting and stabilizing blood; swabs for collecting and stabilizing saliva;
cups for collecting and stabilizing urine or stool samples. C~ollection matcrials, papers, clotlls, swabs, cups and the like, may optionally be treated to avoid denaturation or irreversible adsorption of the sample. The collection materials also may be treated with or contain preservatives, stabilizers or antimicrobial agents to 10 help maintain the integrity of thc specimens. Test kits designed for the collection, stabilization and preservation of test specimens obtained by surgery or needle biopsy are also useful. It is contemplated that all kits may bc configured in two components which can be provided separately; one component for collection and transport of the specimen and the other component for the analysis of the specimen. The collection 15 component, for example, can be provided to the open market user while the components for analysis can be provided to others such as laboratory personnel for determination of the presence, absence or amount of analyte. Further, kits for the collection, stabilization and preservation of test spccimens may be configured for use by untrained personnel and may be availabie in the open market for use at home with 20 subsequent transportation to a laboratory for analysis of the test sample.
The present invention will now be described by way of examples, which are meant to illustrate, but not to limit, the scope of the present invention.
E~AMPLES
25 Example 1: Identification of Breast Tissue Library Marnm~globin Gene-Specific Clones A. Library Comparison of ExFressed Sequence Ta,,s ~ESTs) or Transcript Images. Partial sequences of cDNA clone inserts, so-called "expressed sequcnce tags" (ESTs), were derived from cDNA libraries made from breast tumor tissues, 30 breast non-tumor tissues and nurnerous other tissues, both tumor and non-tumor and entered into a database (L~SEQTM database, available from Incyte Pharmaceuticals, Palo Alto, CA) as gene transcript imagcs. See International Publication No. WO 95/20681. (A transcript image is a listing of the number of EST's for each of the represented genes in a given tissue library. ESTs sharing 35 regions of mutual sequence ovcrlap are classified into clusters. A cluster is assigned W O 98/07753 PCT~US97/14666 - a clone number from a representative 5' EST. Often, a cluster of interest can be extended by cull,pa. ing its consensus sequence with sequences of other EST's which did not meet the criteria for automated clustering. The :~lignm~nt of all available clusters and single ESTs represent a contig from which a consensus 5 sequence is derived.) The transcript images then were evaluated to identify EST
clusters that were representative primarily of the breast tissue libraries. These target clusters then were ranked according to their abundance (occurrence of ES'r members) in the target libraries and their absence from background libraries. Higher abundance clusters with low background occurrence were given higher study 10 priority. A cluster of over 70 clones was identified which was over-expressed in two breast tumor libraries, but was present at 5-fold or more lower levels in 6 of 7 non-tumor breast tissue libraries.
B. Electronic Northern. Clones from the cluster were assembled into a contig and a consensus sequence was generated. The consensus sequence was 15 reviewed in the Ll~SEQ~M program to deterrnine its occurrence in other non-breast tissue cDNA libraries using an "electronic northern" application of the LIFESEQTM
software package. Default settings were usec}. It was found that each sequence occurred only in the breast tissue libraries previously identified above.
C. BLAST. The consensus sequence was compared with other sequences 20 in LIFESEQTM database using the BLAST function (Altschul, S. F. et al., J. Mol.
Biol. ~, 403-410, 1990). The consensus sequence was identified as l~eing identical with that of m~mm~globin, a protein in the uteroglobin family of proteins.
M. A. Watson et al., Cancer Research 56, 860-865 (1996).

Lxample 2: Sequencing of Mammaglobin EST-Specific Clones The DNA sequence for clone 899895 (SEQUENCE ID NO 2) which comprises the 5'-most EST of the m~mm~globin gene contig was determined using dideoxy termination sequencing with dye terminators following known methods (F.
Sanger et al., PNAS U.S.A. 74:5463 (1977)~.
Because the pSPORT1 vector (Life Technologies, Gaithersburg, MD) contains universal priming sites just adjacent to the 3' and 5' ligation junctions of the inserts, approximately 300 bases of the insert were sequenced in both directionsusing universal primers, SEQUENCE ID NO 9 and SEQUENCE ID NO 10 ( New England Biolabs, Beverly, MA and Applied Biosystems Inc, Foster City, CA, respectively). The sequencing reactions were run on a polyacrylamide denaturing - - .

W O 98/077S3 PCT~US97/14666 gel, and the sequences were determined by an Applied Biosystems 377 Sequencer (available from Applied Biosystems, Foster City, CA) or other sequencing apparatus. Additional sequencing primers were designed, MAM.~I and MAM.R I
(SEt2UENCE ~D NO 11 and S~3QUENCE ID NO 12, respectively) from the 3'-5 most end each of the sequences already determined from the initial and subsequentsequencing reactions, and were used in sequential fashion to obtain the sequence of the entire cloned insert in both directions, as previously described.
E3xample 3: Nucleic Acid Preparation A. RN~ E~xtraction from Tissue. Total RNA was isolated ~rom solid breast tissues or cells and from non-breast tissues. Various methods were utilized, including but not limited to the lithium chloride/urea technique, known and described in the art (Kato et al., J. Virol. 61:2182-2191, [19~7]), UltraspecTM (Biotecx Laboratories, Inc., Houston Texas), and TRIzolTM (Life Technologies, Inc., Gaithersburg, MD).
For northern blot analysis, the tissue was placed in a sterile conical tube on ice and 10-15 volumes of 3 M LiCI, 6 M urea, 5 mM EDTA, 0.1 M ,B-mercaptoethanol, 50 mM Tris-HCl (pH 7.5) were added. The tissue was homogenized with a Polytron~ homogenizer (Brinkman Instruments, Inc., Westbury, NY) for 30-50 sec on ice. The solution was transferred to a 15 ml plastic centrifuge tube and placed overnight at -20~C. The tube was centrifuged for 90 min at 9,000 x g at 0-4~C, and the supernatant w~s immediately decanted. Then, 10 mlof 3 M LiCI were added, the tuhe was vortexed for 5 scc and centrifuged for 45 min at 11,000 x g at 0-4~C. Dccanting, resuspension in LiCI, and ccntrifugation wererepeated. The final pellet was air dried and resuspended in 2 ml of I mM ~DTA, 0.5% SDS, 10 mM Tris (p~ 7.5). Then, 20 ,ul of Proteinase K (20 mg/ml~ were added, and the solution was incubated for 30 min at 37~C with occasional mixin~.One-tenth volume (0.22-0.25 ml) of 3 M NaCI was addcd, and the solution was vortexed before transfer into another tube which contained 2 ml of phenol/chloroform/isoamyl alcohol (PCI). The tube was vortexed for 1-3 sec and centrifuged for 20 min at 3,000 x g at 10~C. The PCI extraction was repeated twice more, followed by two similar extractions with chloroform/isoamyl alcohol. The final aqueous solution was transferred to a pre-chilled 15 ml corcx glass tube containing 6 ml of 100% absolute ethanol, the tube was covered with parafilm andplaced at -20~C overnight. The tube was centrifuged for 30 min at 10,000 x g at 0-W O 98/07753 PCTr~S97/14666 4~C, and the ethanol supernatant was decanted imm~ t~ly. The RNA pellet was washed four times with 10 ml of 75% ice-cold ethanol, followed each time by centrifugation at 10,000 x g for 10 min. The final pellet was air dried for 15 min at room tempcrature. The RNA was suspended in 0.5 ml of 10 mM Tris (pH 7.6), 1 5 mM EDTA, and its concentration was determined spectrophotometrically. RNA
samples were aliquoted and stored at -70~C as ethanol precipitates.
The quality of the RNA was determined by agarose gel electrophoresis (see Lxample 5) and staining with 0.5 ,ug/ml ethidium bromide for one hour. RNA
samples that did not contain intact 28S/18S rRNAs were excludcd from the study.
Alternatively, for RT-PCR analysis, 1 ml of Ultraspec RNA reagent was added to 120 mg of pulverized tissue in a 2.0 ml polypropylene microfuge tube, homogenized with a Polytron~ homogenizer (Brinkman Instruments, Inc., Westbury, NY) for 50 sec and left on ice for 5 min. Then, 0.2 ml of chloroform was added to each sample, followed by vortexing for 15 sec. The sample was left in ice for another 5 min, followed by centrifugation at 12,000 x g for 15 min at 4~C. The upper layer was collected and transferred to another RNase-free ~.0 ml microfugetube. An equal volume of isopropanol was added to each sample, and the solution was placed on ice for 10 min. The sample was centrifuged at 12,000 x g for 10 min at 4~C, and the supernatant was discarded. The remaining pellet was washed twice20 with cold 75% ethanol, resuspended by vortexing, and the resuspended material was then re-pelleted by centrifugation at 7500 x g for 5 min at 4~C. Finally, the RNA
pellet was dried in a speedvac for at least 5 min and reconstituted in RNase free water.
B. RNA Extraction from Blood Mononuclear Cells. Mononuclear cells are 25 isolated from blood sarnples from patients by centrifugation using Ficoll-Hypaque as follows. A 10 ml volume of whole blood is mixed with an equal volume of RPMI
Medium (Life Technologies, Gaithersburg, MD). This mixture is then underlayed with ~0 ml of Ficoll-Hypaque (Pharmacia, Piscataway, NJ) and centrifuged for 30 minutes at 200 x g. The buffy coat containing the mononuclear cells is removed, 30 diluted to 50 ml with Dulbecco's PBS (Life Technologies, Gaithersburg, MD) and the mixture centrifuged for 10 minutes at 200 x g. After two washcs, the resulting pellet is resuspended in Dulbecco's PBS to a final volume of I ml.
RNA is prepared from the isolated mononuclear cells as described by N.
Kato et al., supra. Briefly, the pelleted mononuclear cells are brought to a final of 1 W O 98/077~3 PCTAUS97/14666 ml volume and then are resuspended in 250 ~LL of PBS and mixed with 2.5 ml of 3M LiCl, 6M urea, 5rnM EDTA, 0.1 M 2-mercaptoethanol, 50mM Tris-HCl (pH
7.5). The resulting mixture is homogenized and incubated at -20~C overnight. Thehomogenate is spun at 8,000 RPM in a Beclcman J2-21M rotor for 90 minutes at 0-4~C. Thc pellet is resuspended in 10 ml 3M LiCI by vortexing and then spun at 10,000 RPM in a Beckrnan J2-21M rotor centrifuge for 45 minutes at 0-4~C. The resuspending and pelleting steps then are repeated. The pellet is resuspended in 2 rnl of I mM EDTA, 0.5% SDS, 10 mM Tris (pH 7.5) and 400,ug Proteinase K with vortexing and then it is incubated at 37~C for 30 minutes with .ch~king. One tenth volume of 3M NaCl then is added and the vortexed mixture. Proteins are removed by two cycles of extraction with phenol/ chloroform/ isoamyl alcohol followed byone extraction with chloroformt isoamyl alcohol. RNA is precipitated by thc addition of 6 ml of ethanol followed by overnight incubation at -20~C. After theprecipitated RNA is collected by centrifugation, the pellet is washed 4 times in 75%
ethanol. The pelleted RNA is then dissolved in 1 mM EDTA, 1 OmM T~is-HCI (pH
7.5).
Non-breast tissues are used as negativc controls. The mRNA can be further purified from total RNA by using commercially available kits such as oligo dT
cellulose spin columns (RediColTM from Pharmaeia, Uppsala, Sweden~ for the isolation of poly-adenylated RNA. Total or mRNA can be dissolved in Iysis buffer(SM guanidine thiocyanate, 0. lM EDTA, pH 7.0) for analysis in the ribonuclease protcction assay.
C. RNA Extraction from polysomes. Tissue is minced in saline at 4~C and mixed with 2.5 volumes of 0.8 M sucrose in a TK,50M (150 mM KCl, 5 mM
MgCl~, 50 mM Tris-HCl, pH 7.43 solution containing 6 mM 2-mercaptoethanol.
The tissue is homogenized in a Teflon-glass Potter homogenizer with fivc strokes at 100-200 rpm followed by six strokes in a Dounce homogenizer, as described by B.
Mechler, Methods in lE~nzvmology 152:241-248 (1987). The homogenate then is centrifuged at 12,000 x g for 15 min at 4~C to sediment the nuclei. The polysomes are isolated by mixing 2 ml of the supernatant with 6 ml of 2.5 M sucrose in TKI50M
and layering ehis mixture over 4 ml of 2.5 M sucrose in TK,50M in a 38 ml polyallomer tube. Two additional sucrose TKI50M solutions are successively layered onto the extract fraction; a first layer of 13 ml 2.05 M sucrose followed by a second layer of 6 ml of 1.3 M sucrose. The polysomes are isolated by centrifuging the gradient at 90,000 x g for 5 h at 4~C. The fraction then is taken from the 1.3 M

W O 98t077S3 PCTrUS97/14666 sucrose/2.05 M sucrose interface with a siliconized pasteur pipette and diluted in an equal volume of TE (10 mM Tris-HCI, pH 7.4, 1 mM EDTA). An equal volume of 90~C SDS buffer (1% SDS, 200 mM NaCI, 20 mM Tris-HCI, pH 7.4) is added and the solution is incllh~terl in a boiling water bath for 2 min. Proteins next are digested with a Proteinase-K digestion (50 mg/ml) for 15 min at 37~C. The mRNA is purified with 3 equal volumes of phenol-chloroform extractions followed by precipitation with 0.1 volume of 2 M sodium acetate (pH 5.2) and 2 volumes of 100% ethanol at -20~C overnight. The precipitated RNA is recovered by centrifugation at 12,000 x g for 10 min at 4~C. Thc RNA is dried and resuspended10 in TE (pH 7.4) or distilled water. The resuspendcd RNA then can be used in a slot blot or dot blot hybridization assay to check for the presence of mammaglobin mRNA (see Example 6).
The quality of nucleic acid and proteins is dependent on the method of preparation used. Each sample may require a different preparation technique to 15 maximize isolation efficiency of the target molecule. These preparation techniques are within the skill of the ordinary artisan.
~xample 4: Ribonuclease Protection Assay A. Synthesis of Labeled Complementary RNA (cRNA) Hybridization Probe 20 and Unlabeled Sense Strand. A pSPORTI plasmid containing the m:~mm~globin gene cDNA sequence insert (clone 8998g5), flanked by opposed SP6 and T7 polymerase promoters, was purified using Qiagen Plasmid Purification Kit (Qiagen, Chatsworth, CA). Then, 10 ,ug of the plasmid were linearized by cutting with 10 U
Dde I restriction enzyme for 1 h at 37~C. The linearized plasmid was purified using 25 QIAprep kits (Qiagen, Chatsworth, CA) and used for the synthcsis of antisensctranscript labeled with 6.3 ,uM (alpha32P) UTP (Amersham Life Scicnces, Inc.
Arlington Heights, IL) from the SP6 promoter using the Riboprobea~ in vitro Transcription System (Promega Corporation, Madison, WI), as described by the supplier's instructions. To gencrate the sense strand, 10 ,ug of the purified plasmid 30 were cut with restriction enzymes 10U Xba l and 10 U Not I, and transcribed as above from the T7 promoter. Both sense and antisense strands were isolated by spin column chromatography. Unlabeled sense strand was quantitated by UV
absorption at 260 nm.
B. Hybridization of Labeled Probe to Target. Frozen tissue was pulverized 35 to powder under liquid nitrogen and 100-500 mg were dissolved in I ml of Iysis CA 02232239 l998-04-20 W O 98/07753 PCTrUS97/1~666 buffer as available as a component of the DirectProtect~M Lysate RNase Protection kit (Ambion, Inc., Austin, TX). Further dissolution was achieved using a tissue homogenizer. In addition, a dilution series of a known amount of sense strand inmouse liver Iysate was made for use as a positive control. Finally, 45 ~1 of 5 solubilized tissue or diluted sense strand was mixed directly with I xlOs cpm of radioactively labeled probe in S ,ul of Iysis buffer. Hybridization was allowed to proceed overnight at 37~C.
C. RNase Digestion. R~A that was not hybridized to probe was removed from the reaction as per the Direct Protect~ protocol using a solution of RNase A
and RNase T1 for 30 min at 37~C, followed by removal of RNase by Proteinase-K
digestion in the presence of sodium sarcosy3. Hybridized fragments protected from digestion were then prccipitated by the addition of an equal volume of isopropanol and placed at -70~C for 3 h. The precipitates were collected by centrifugation at 12,000 x g for 20 min.
p. Fr;~ment Analysis. The precipitates were dissolved in denaturing gel loading dye (80% formamide, 10 mM EDTA (pH 8.0), I mg/ml xylene cyanol, I
mg/ml bromophenol blue), heat denatured, and electrophorescd in 65~o polyacrylamide TBE, 8 M urea denaturing gels. The gels were imaged and analyzed 20 using the STORMTM storage phosphor autoradiography system ~Molecular Dynamics, Sunnyvale, CA). Quantitation of protected fragment bands, expressed infemtograms (fg), was achieved by comparing the peak areas obtained from the testsamples to those from thc known dilutions of the positive control sense strand (sec Section B, supra). In addition, the concentration of DNA in the Iysate was assayed 25 to estimate the numbcr of cells in the test sample Iysates. The results are expresscd in molecules of m~mm~globin RNA/cell and as an image rating score (Table 1).
High level expression of mRNA corresponding to a sequence sclected from the group consisting of SEQUENCE ID NO 1. SEQUENCE ID NO 2, and fragments or complements thereof, indicated the presence of mammaglobin mRNA(s), 30 suggesting a diagnosis of a breast tissue disease or condition, such as breast canccr.

-W O 98/07753 PCTrUS97/14666 Table 1 Tissue ID Number m~rnm~lobin Score*
RNA/cell Norrnal BreastC157 37 2+
C007G ' 4 2+
C0'7~ ~ ~
C0 6~ ~ +
C1~5~ 0.3 +
Malignant Breast C01 ~J >41 3+
C02 G 0.5 +
C01 '.G 0 C03 R >76 3+

~iormal Lung C005R 0 Malignant LungC037G 0 ~ormal Colon C027G 0 *Samples with no ~IP.t~ ct~hle protected fragment were scored "-"; samples with detectable protected fragment, the fg values of which were within the std curve,were scored "+"; samples with detectable protected fragment, the fg values of which were 2 to 10 fold above the std curve, were scored "2+"; samples with detectableprotected fragment, the fg values of which were 10 fold or more above the std curve, were scored "3+"

Example 5: Northern Blottin~
The northern blot technique was used to idcntify a specific size RNA species in a complex population of RNA using agarose gel electrophoresis and nucleic acid hybridization. Briefly, 5-10,ug of total RNA (see Example 3) was incubated in 15,ul of a solution containing 40 mM morphilinopropanesulfonic acid (MOPS) (pH
7.0), 10 mM sodium acetate, 1 mM EDTA, 2.2 M formaldehyde, 50% v/v formamide for 15 min at 65~C. The denatured RNA was mixed with 2 ,ul of loading buffer (50% glycerol, 1 mM EDTA, 0.4% bromophenol blue, 0.4% xylene cyanol) and loaded into a denaturing 1.0% agarose gel containing 40 mM MOPS (pH 7.0), 10 rnM sodium acetate, 1 mM EDTA and 2.2 M formaldehyde. The gel was electrophoresed at 60 V for 1.5 h and rinsed in RNAse free water. RNA was transferred from the gel onto nylon membranes (Brightstar-Plus, Ambion, Inc., Austin, TX) for 1.5 hours using the downward alkaline capillary transfer method (Chomczynski, Anal. Biochem. 201:134-139, 1992). The filterwas rinsed with lX
SSC, and RNA was crosslinked to the filter using a Stratalinker (Stratagene, Inc., W O 98/07753 PCTrUS97/14666 La Jolla, CA) on the autocro~.~linking mode and dried for 15 min. The membrane was then placed into a hybridization tube cont:~ining 20 ml of preheated prehybridization solution (5X SSC, 50% formamide, SX Denhardt's solution, 100 ,ug/ml denatured salmon sperm DNA) and incubated in a 42~C hybridization oven for 5 at least 3 hr. While the blot was prehybridizing, a 32P-labeled random-primed probc was generated using the m:~mm~globin insert fragment (obtained by digesting clone 899895 with XbaI and NotI) using Random Primer DNA Labeling System (Life Technologies, lnc., Gaithersburg, MD) according to the manufacturer's instructions. Half of the probe was boiled for 10 min, quick chillcd on ice and 10 added to the hybridization tube. Hybridization was carried out at 42~C for at least 12 hr. The hybridization solution was discarded and the filter was washed in 30 ml of 3X SSC, 0.1% SDS at 42~C for 15 min, followed by 30 ml of 3X SSC, 0.1% SDS
at 42~C for 15 min. The filter was wrapped in saran wrap, exposed to Kodak XAR-Omat film for 8-96 hr, and the film was developed for analysis.
~esults of the analysis of RNA quality using an ethidium bromide stained agarose gel and the corresponding northern blot using mammaglobin probe hybridized to RNAs from breast tissues and non-breast tissues are shown in Figures lA & B, respectively. The positions of RNA size standards (in kb) are shown to the left of each panel. The mammaglobin prol~e hybridized to an RNA band at 1.3 Kb only in a breast sample in lane 1 but not to RNAs in the breast sample in ianc 3 or the seven non-breast samples in lanes 4-10 (colon, colon, lung, lung, ovary, prostate, and spleen, respectively) (Fig. IB). I,ane 2 is blank.
Example 6: Pot Blot/Slot Blot Dot and slot blot assays are quick methods to evaluate the presence of a specific nucleic acid sequence in a complex mix of nucleic acid. To perform suchassays, up to 50 ~Lg of RNA is mixed in 50 ~ul of 50% formamide, 7%
formaldehyde, lX SSC, incubated 15 min at 68~C, and then cooled on icc. Then, 100 ~1 of 20X SSC is added to the RNA mixture and loaded under vacuum onto a manifold apparatus that has a prepared nitrocellulose or nylon membrane. The membrane is soaked in water, 20X SSC for 1 hour, placed on two sheets of 20X
SSC prewet Whatman #3 filter paper, and loaded into a slot blot or dot blot vacuum manifold apparatus. The slot blot is analyzed with probes prepared and labeled as described in Example 4, supra. Detection of m~NA corresponding to a sequence selected from the group consisting of SEQUENCE ID NO 1, SEQUENCE ID NO

W O 98/07753 PCT~US97/14666 2, and fragments or complements thereof, is an indication of the presence of m~mm~lobin, suggesting a diagnosis of a breast tissue disease or condition, suchas breast cancer.
Other methods and buffers which can be utilized in the methods described in S Examples 5 and 6, but not specifically detailed herein, are known in the art and are described in J. Sambrook et al., supra .

~xamplc 7: In Situ Hybridization This method is useful to directly detect specific target nucleic acid sequences 10 in cells using detectable nucleic acid hybridization probes.
Tissues are prepared with cross-linking fixative agents such as paraformaldehyde or glutaraldchyde for maximum cellular RNA retention. See, L.
Angereret al., Methods in Cell Biol. 35:37-71 (1991). Briefly, the tissue is placed in greater than S volumes of 1 % glutaraldehyde in 50 mM sodium phosphate, p~
7.5 at 4~C for 30 min. The solution is changed with fresh glutaraldehyde solution (1 % glutaraldehyde in 50mM sodium phosphate, pE~ 7.5) for a further 30 min fixing. The fixing solution should have an osmola~ity of approximately 0.375~o NaCI. The tissue is washed once in isotonic NaCI to remove the phosphate.
The fixed tissues then are embedded in paraffin as follows. The tissue is dehydrated though a series of ethanol concentrations for 15 min each: 50% (twice), 70% ~twice), 85%, 90% and then 100% (twice). Next, the tissue is soaked in two changes of xylene for 20 min each at room temperature. The tissue is then soaked in two changes of a 1: 1 mixture of xylene and paraffin for 20 min each at 60~C; and then in three final changes of paraffin for 15 min each.
Next, the tissue next is cut in 5 ,um sections using a standard microtome and placed on a slide previously treated with a tissue adhesive such as 3-aminopropyltriethoxysilane.
Paraffin is removed from the tissue by two 10 min xylene soaks and rehydrated in a series of ethanol concentrations: 99% twice, 95%, ~5%, 70%, 50%,30%, and then distilled water twice. The sections are pre-treated with 0.2 M HClfor 10 min and permeabilized with 2,ug/ml Proteinase-K at 37~C for 15 min.
Labeled riboprobes transcribed from the m~mm~globin gene plasmid (see Example 4~ are hybridized to the prepared tissue sections and incubated overnight at 56~C in 3X standard saline extract and 50% formamide. Excess probe is removed by washing in 2X standard saline citrate and 50% formamide followed by digestion W O 981077~3 PCTAUS97114666 with 100 ,ug/ml RNase A at 37~(~ for 30 min. Fluoresccncc probe is vi.c~ rl by illumination with ultraviolet (UV) light under a microscope. Fluorescence in thecytoplasm is indicative of m~mm~globin mRNA. Alternatively, the sections can be vi~ i7e~1 by autoradiography.
Example 8: Reverse Transcription PCR
~. Qne Step RT-PCR Assay. Target-specific primers are designed to detect the above-described target sequenccs by reverse transcription PCR using methods known in the art. One step RT-PCR is a sequential procedure that performs both RT
10 and PCR in a single reaction mixture. The procedure is performed in a 200 ,ulreaction mixture containing 50 n~M (N,N,-bis~2-Hydroxyethyl]glycine), pH 8.15, 81.7 mM KOAc, 33.33 n3M KOH, 0.01 mg/ml bovine serum albumin, 0.1 mM
ethylene diaminetetraacetic acid, 0.02 mg/ml NaN3, 8% w/v glycerol, 150 ,uM eachof dNTP, 0.25 ~M each primer, 5U rTth polymerase, 3.25 mM Mn(OAc)2 and 5 ,u3 15 of target RNA (see Example 3). Since RNA and the rTth polymerase enzyme are unstable in the presence of Mn(OAc) 2, the Mn(OAc)2 should bc added just before target addition. Optimal conditions for cDNA synthesis and thermal cycling readily can be determined by those skilled in the art. The reaction is incubated in a Perkin-Elmer Thermal Cycler 480. Optimal conditions for cDNA synthesis and thcrmal 20 cycling can readily be determined by those skilled in the art. Conditions which rnay be found useful include cDNA synthesis at 60~-70~C for 15-45 min and 30-45 amplification cycles at 94~C, I min; 55~-70~C, I min; 72~C, 2 min. One step RT-PCR also may be perrormed by using a dual enzyme procedure with Taq polymerase and a reversc transcriptase enzyme, such as MMLV or AMV RT enzymes.
1~. Traditional RT-P(~R. A traditional two-step RT-PCR reaction was performed, as described by K.Q. Hu et al., Virology 181:721-726 (1991~. Briefly,0.5 ,ug of extracted mRNA (see Exarnple 3) was reverse transcribcd in a 20 ,ul reaction mixture containing lX PCR II buffer (Perkin-Elmer), 5 mM MgC12, 1 mM
dNTP, 20 U RNasin, 2.5 ~uM random hexamers, and 50 U MMLV (Moloney 30 murine leukemia virus) reverse transcriptase (RT). Reverse transcription was performed at room temperature for 10 min, 42~C for 30 min in a PE~80 thermal cycler, followed by further incubation at ~5~C for 5 min to inactivate the RT. PCR
was performcd using 2 ,ul of the cDNA reaction in a final PCR reaction volume of50 ~I containing 10 mM Tris-HCl (pH 8.3), 50 mM KCI, 2 rnM MgCl2, 200 ~M
35 dNTP, 0.5 ,uM of each sense and antisense primer, SEQUENCE ID NO 13 and W O 98/07753 PCT~US97/14666 SEQUENCE ID NO 14, respectively, and 2.5 U of Taq polymerase. The reaction was incubated in an MJ Research Model PTC-200 as follows: 40 cycles of amplification (g4~C, 20 sec; 58~C, 30 sec; 72~C, 30 sec); a final extension (72~C, 10 min); and a soak at 4~C.
C. PCR Fragment Analysis. The corrcct products were verifled by size determination using gel electrophoresis with a SYBR~ Green I fluorescent intercalator (Molecular Probes, Eugene, OR) and imaged using a STORM im"ging system (Fig. 2). Figure 2 shows a DNA band at 231 bases which is indicative of am~rnm~globin -specific PCR product, in both cancerous (lanes 1-5) and normal (lanes 6- 10) breast tissues, and not in any lung (lanes 12- 16) or colon (lanes 17-21) tissues. Detection of a product comprising a sequence selected from the group consisting of SEQUENCE ID NO 1, SEQUENCE ID NO 2, and fragments or complements thereof, indicated the presence of m~mm~lobin mRNA(s), suggesting a diagnosis of a breast tissue disease or condition, such as breast cancer.
~xamplc 9: OH-PCR
A. Probe selection and Labeling. Target-specific primers and probcs are designed to detect the above-described target se~uences by oligonucleotide hybridization PCR. International Publication Nos WO 92/10505, published 25 June 1992, and WO 92/11388, published 9 July 1992, teach methods for labeling oligonucleotides at their 5' and 3' ends, respectively. According to one known method for labeling an oligonucleotide, a label-phosphoramidite reagent is prepared and used to add the label to the oligonucleotide during its synthesis. For example, see N. T. Thuong et al., Tet. Letters 29(46):5905-5908 (198~',); or J. S. Cohen et al., published U.S. Patent Application 07/246,688 (NTIS ORDER No. PAT-APPL-7-246,688) (1989~. Preferably, probes are labeled at their 3' end to prevent participation in PCR and the formation of undesired extension products. For one step OH-PCR, the probe should have a TM at least 15~C below the TM of the primers. The primers and probes are utilized as specific binding members, with or - 30 without detectable labels, using standard phosphoramidite chemistry and/or post-synthetic labeling methods which are well-known to one skilled in the art.
B. One Step Oligo Hybridization PCR. OH-PCR is performed on a 200 ,ul reaction containing 50 mM (N,N,-bis[2-Hydroxyethyl]glycine), pH 8.15, 81.7 mM
KOAc, 33.33 mM KOH, 0.01 mg/ml bovine serum albumin, 0.1 mM ethylene diaminetetraacetic acid, 0.02 mg/ml NaN3, 8% w/v glycerol, 150 ,uM each of dNTP, WO ~7/~3 PCTrUS97/14666 0.25 ~M each primer, 3.75 nM probe, 5U rTth polymerase, 3.25 mM Mn(OAc)2 and 5 ,ul blood equivalents of target (see Example 3). Since RNA and the rTth polymerase enzyme are unstable in the presence of Mn(OAc)2, the Mn(OAc)2 should be added just before target addition. The reaction is incubated in a Perkin-Elmer Therrnal Cycler 480. Optimal conditions for cDNA synthesis and therrnal cycling can be readily determined by those skilled in the art. Conditions which may be found useful include cDNA synthesis (60~C, 30 min), 30-45 amplification cycles (94~C, 40 sec; 55-70~C, 60 sec), oligo-hybridization (97~C, 5 min; 15~C, 5 min;
15~C soak). The correct reaction product contains at least one of the strands of the PCR product and an internally hybridized probe.
C. OH-PCR Product ~nalysis. Ampli~led reaction products are detected on an LCx(~) analyzer system (available from Abbott Laboratories, Abbott Park, IL).Briefly, the correct reaction product is captured by an antibody labeled microparticle at a capturable site on cither the PCR product strand or the hybridization probe, and the complex is detected by binding of a rl~t~ct~hle antibody conjugate to either a detectable site on the probe or the PCR strand. Only a complex containing a PCR
strand hybridized with the internal probe is detectable. The detection of this connplex then is indicative of the presence of m~mm~lobin mRNA, suggesting a diagnosis ofa breast disease or condition, such as breast cancer.
Many other detection formats exist which can be used and/or modified by those skilled in the art to detect the presence of amplified or non-amplified m~mm~globin-derived nucleic acid sequences including, but not limited to, ligasechain reaction (LCR, Abbott Laboratories, Abbott Park, IL); Q-beta replicase (Gene-TrakTM, Naperville, Illinois), branched chain reaction (Chiron, 13meryville, CA) and strand displacement assays (Becton Dickinson, Research Triangle Park, NC).
Example 10: Synthetic Peptide Production Synthetic peptides, MAM. 1 -MAM.9 (SEQUENCE ID NOS 18-26, respectively) were prepared based upon the predicted amino acid sequence of the open reading frame of m~Tnm~globin (SEQUENCE ID NO 17) ~see Example 1). All peptides were synthesized on a Symphony Peptide Synthesizer (available from Rainin Instrument Co, Emeryville California), using FMOC chemistry, standard cycles and in-situ HBTIJ activation. Cleavage and deprotection conditions were as follows: a volume of 2.5 ml of cleavage reagent (77.5% v/v trifluoroacetic acid,15% v/v ethanedithiol, 2.5% v/v water, 5% v/v thioanisole, 1-2% w/v phenol) was W O 98/07753 PCT~US97/14666 added to the resin, and ~gif~ at room temperature for 2-4 hours. The filtrate was then removed and the peptide was precipitated from the cleavage reagent with cold diethyl ether. Each peptide was then filtered, purified via reverse-phase preparative HPLC using a water/acetonitrile/0.1 % TFA gradient, and Iyophilized. The product5 was confirmed by mass spectrometry (data not shown).
The purified peptides were conjugated to Keyhole Limpet Hemocyanin with glutaraldehyde, mixed with adjuvant, and injected into rabbits (see Example 14).
Example 11 a: Expression of Protein in a Cell Line I Jsin~ Plasmid 577 A. Construction of a M~mm:~lobin Expression Plasmid. Plasmid 577, described in U.S. patent application Serial No. 08/478,073, filed June 7, 199~, has becn constructed for the expression of secreted antigens in a permanent cell line.
This plasmid contains the following DNA segments: (a) a 2.3 Kb fragment of pBR322 containing bacterial beta~ t~m~e and origin of DNA replication; (b) a 1.815 Kb cassette directing expression of a neomycin resistance gene under control of HSV-I thymidine kinase promoter and poly-A addition signals; (c) a I .9 Kb cassette directing expression of a dihydrofolate reductase genc under the control of an SV40 promoter and poly-A addition signals; (d) a 3.5 Kb cassette directing expression of a rabbit immunoglobulin heavy chain signal sequence fused to a modified hepatitis C
20 virus (HCV) E2 protein under the control of the Simian Virus 40 T-Ag promoter and transcription enhancer, the hepatitis B virus surface antigen (HBsAg) enhancer Ifollowed by a fragment of Herpes Simplex Virus- I (HSV- I ) genome providing poly-A addition signals; and (e) a residual 0.7 Kb fragment of Simian Virus 40 genome late region of no function in this plasmid. All of thc segments of the vector 25 were assembled by standard methods known to those skilled in the art of molecular biology.
Plasmids for the expression of secrctable mammaglobin proteins are constructed by replacing the hepatitis C virus E2 protein coding sequence in plasmid 577 with that of a mammaglobin polynucleotide sequence selected from the group 30 consisting of SEQUENCE ID NO l, SEQUENCE ID NO 2, and fragments or complements thereof, as follows. Digestion of plasmid 577 with XbaI releases the~ hepatitis C virus E2 gene fragment. The resulting plasmid backbone allows insertion of the m~mm~globin cDNA insert downstream of the rabbit immunoglobulin heavy chain signal sequence which directs the expressed proteins 35 into thc secretory pathway of the cell. The mammaglobin cDNA fragment is W O 98/07753 PCTnJS97/14666 generated by PCR using standard procedures. Encoded in the sense PCR primer sequence is an XbaI site, immediately followed by a 12 nucleotide se~uence that encodes the amino acid sequence Ser-Asn-Glu-Leu ~"SNEL") to promote signal protease processing, efficient secretion and final product stability in culture fluids.
5 Immediately following this 12 nucleotide sequence the primer contains nucleotides complementaly to template sequences encoding amino acids of the mammaglobin gene . The antisense primer incorporates a sequence encoding the following eightamino acids just before the stop codons: Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (SEQUENCE ID NO 27). Within this sequence is incorporated a recognition site to 10 aid in analysis and purification of the m~mm~globin protein product. A recognition site (termed "FLAG") that is recognized by a commercially available monoclonal antibody designated anti-FLAG M2 (Eastman Kodak, Co., Ncw Haven, CT) can be utilized, as well as other comparable sequences and their corresponding antibodies.
For example, PCR is performed using GeneAmp~ reagents obtained from Perkin-15 Elmer-Cetus, as directed by the supplier's instructions. PCR primers are uscd at a final concentration of 0.5 ~M. PCR is performed on the mammaglobin plasmid templatc in a 100 111 reaction for 35 cycles (94~C, 30 seconds; 55~C,30 seconds;72~C, 90 seconds) followed by an extension cycle of 72~C for 10 min.
B. Transfection of Vihydrofolate ~cductase Deficicnt Chinesc Hamster 20 Ovary Cells. The plasmid described supra is transfected into C~O/dhfr~ cells - ~DXB-I I I, Uriacio, et al., PN~S 77:4451-446G (1980)). These cclls are availablc from the A.T.C.C., 12301 Parklawn Drive, Rockville, MD 20852, undcr Accession No. CRL 9096. Transfection is carried out using thc cationic liposome-mcdiated procedure described by P. L. Felgneret al., PNAS 84:7413-7417 (1987).
25 Particularly, CHO/dhfr- cclls are cultured in Ham's F- 12 media supplemented with 10% fetal calf serum, L-~ t~rnine. ~ I mM) and f~eshly seeded into a flask at a density of 5 - 8 x 105cells per flask. The cells are grown to a confluency of betwecn 60 and 80% for transfection. Twenty micrograms (20,ug) of plasmid DNA is added to 1.5 ml of Opti-MEM I medium and 100 ~1 of Lipofectin Rcagent (Gibco-BRL;
30 Grand Island, NY) are added to a second 1.5 ml portion of Opti-MEM I media. Thc two solutions are mixed and incubated at room tempcrature for 20 min. After the culture medium is removed from the cells, the cells are rinsed 3 times with 5 ml of Opti-~EM I medium. The Opti-MEM I-Lipofection-plasmid DNA solution then is overlaid onto the cells. The cells are incubated for 3 h at 37~C, after which time the W O 98/077S3 PCT~US97/14666 Opti-MEM I-Lipofectin-D~A solution is replaced with culture medium for an additional 24 h prior to selection.
C. Selection and Amplification. One day after transfection, cells are passaged 1:3 and incubated with dhfr/G418 selection medium (hereafter, "F-12 5 minus medium G"). Selection medium is Ham's F-12 with L-glutamine and without hypoxanthine, thymidine and glycine (JRH Biosciences, Lenexa, Kansas) and 300 ,ug per ml G418 (Gibco-BRL; Grand Island, NY). Media volume-to-surface area ratios of 5 ml per 25 cm2 are maintained. After approximately two weeks, DHFR/G418 cells are expanded to allow passage and continuous maintenance in ~-10 12 minus medium G.
Amplification of each of the transfected m~rnm:~globin cDNA sequences isachieved by stepwise selection of DHFR+, G418+ cells with methotrexate (reviewed by R. Schimke, Cell 37:705-713 [1984]). Cells are incubated with F-12 minus medium G containing 150 nM methotrexate (MTX) (Sigma, St. Louis, MO) for 15 approximately two weeks until resistant colonies appear. Further gene amplification is achieved by selection of 150 nM adapted cells with 5 ,uM MT~.
D. Antigen Production. F-12 minus medium G supplemented with 5 ~lM
MTX is overlaid onto just confluent monolayers for 12 to 24 h at 37~C in 5% C02.The growth medium is removed and the cells are rinsed 3 times with Dulbecco's 20 phosphate buffered saline (PBS) (with calcium and magnesium) (Gibco-BRL;
Grand Island, NY) to remove the remaining media/serum which may be present.
Cells then are incubated with VAS custom medium (VAS custom formulation with L-glutamine with HEPES without phenol red, available from JRH Bioscience;
Lenexa, KS, product number 52-08678P), for 1 h at 37~C in 5% CO2. Cells then 25 are overlaid with VAS for production at 5 ml per T flask. Medium is removed after seven days of incubation, retained, and then frozen to await purification with harvests 2, 3 and 4. The monolayers are overlaid with VAS for 3 more seven day harvests.
E. Analysis of Breast Tissue Gene Mammaglobin Anti~en Expression.
30 Aliquots of VAS supernatants from the cells expressing the mammaglobin protein construct are analyzed, either by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) using standard methods and reagents known in the art (Laemmli discontinuous gels), or by mass spectrometry.
F. Purification. Purification of the mammaglobin protein containing the 35 FLAG sequence is performed by immunoaffinity chromatography using an affinity W O 98/07753 PCT~US97/14666 matrix comprising anti-FLAG M2 monoclonal antibody covalently attached to agarose by hydrazide linkage (Eastman Kodak Co., New Haven, CT). Prior to affinity purification, protein in pooled VAS medium harvests from roller bottles is exchanged into 50 mM Tris-HCI (pH 7.5), 150 mM NaCl buffer using a Sephadex 5 G-25 (Pharmacia Biotech Inc., Uppsala, Swedcn) column. Protein in this buffer is applied to the anti-FI~AG M2 antibody affinity column. Non-binding protein is eluted by washing the column with 50 mM Tris-HCl (pH 7.5), 150 mM NaCI
buffer. Bound protein is eluted using an excess of FLAG peptide in 50 mM Tris-HCI (pH 7.5), 150 mM NaCl. The excess FLAG peptide can be removed from the 10 purified m~mm~globin protein by gel electrophoresis or HPLC.
Although plasmid 577 is utilized in this examplc, it is known to those skilled in the art that other comparable expression systems, such as CMV, can be utilized herein with ~l~pliate modifications in reagent and/or techniques and are within the skill of the ordinary artisan.
The largest cloned insert cont~ining the coding region of the m~rnm~;lobin gene is thcn sub-cloned into either (i) a eukaryotic expression vector which maycontain, for example, a cytomegalovirus (CMV) promoter and/or protein fusible sequences which aid in protein expression and detection, or (ii) a bacterial expression vector containing a superoxide-dismutase ~SOD) and CMP-KDO
synthetase (CKS) or other protein fusion gene for expression of the protein sequence. Methods and vectors which are useful for the production of polypeptides which contain fusion sequences of SOD are described in EPO 0196056, published October 1, 1986, and those containing fusion sequences of CKS are described in EPO Publication No. 0331961, published September 13, 1989. This so-purified protein can be used in a variety of techniques, including but not limited to animal immunization studies, solid phase immunoassays, etc.
Examl~le 1 lb: Expression of Protein in a Cell Line Using pcDNA3. l/Myc-His ~. Construction of a Mamm:~lobin Expression Plasmid. Plasmid pcDNA3.1/Myc-His (Cat.# V855-20, Invitrogen, Carlsbad, C~A) has been constructed, in the past, for the expression of secreted antigens by most mammalian cell lines. Expressed protein inserts are fused to a myc-his peptidc tag. The myc-his tag (SEQUENCE ID NO 28) comprises a c-myc oncoprotein epitope and a polyhistidine sequence which are useful for the puri~lcation of an expressed fusion W O g8/07753 PCTAUS97/14666 protein by using either anti-myc or anti-his affinity columns, or metalloproteinbinding colum~s.
A plasmid for the expression of secretable m~mm:~globin protein was constructed by inserting a mz~mm~globin polynucleotide sequence from clone 899895 into the pcDNA3. 1/Myc-His vector. Prior to construction of a m~mm~globin expression plasmid, the m~mm~globin cDNA sequence was first cloned into a pCR~-Blunt vector. The m~mm~globin cDNA fragment was generated by PCR performed using Stratagene~ reagents obtained from Stratagene, as directed by the supplier's instructions. PCR primers are used at a final concentration of 0.5 ,uM. PCR using S U of pfu polymerase (Stratagene, La Jolla, CA) is performed on the m~mm~lobin plasmid template (see Example 2) in a 50 ,ul reaction for 30 cycles (94~C, I min; 65~C, 1.5 min; 72~C, 3 min) followed by an extension cycle of 72~Cfor 8 min. (The sense PCR primer sequence, SEQUENCE ID NO 15, comprises nucleotides which are identical to the pSPORT vector directly upstream of the m~mm~Elobin gene insert. The antisense primer, SEQUENCE ID NO 16, incorporated a 5' Not I restriction sequence and a sequence complementary to the 3' end of the m~mm~globin cDNA insertiust upstream of the 3'-most, in-frame stop codon.) Five microliters (5 ,ul) of the resulting blunted-ended PCR product wereligated into 25 ng of linearized pCR~3-Blunt vector (Invitrogen, Carlsbad, CA) interrupting the lethal ccdB gene of the vector. The resulting ligated vector was transformed into TOP10 E. coli (Invitrogen, Carlsbad, CA) using a One ShotTM
transformation kit (Invitrogen, Carlsbad, CA) following supplier's directions. The transformed cells were grown on LB-Kan (50 ,ug/ml kanamycin) selection plates at37~C. Only cells containing a plasmid with an interrupted ccdB gene grew after transformation (Grant, S.G.N., PNAS 87:4645-4649 (l990)). Transformed colonies were picked and grown up in 3 ml of LB-Kan broth at 37~C. Plasmid DNA
was isolated by using a QIAprep6~ (Qiagen Inc., Santa Clarita, CA) procedure, asdirected by the supplier's instructions. The DNA was digested with EcoRI and NotI
restriction enzymes to release the m~rnmzlglobin insert fragment. The fragment was electrophoresed on 1% Seakem~D LE agarose (FMC, Rockland, ME)/0.5 ,ug/ml ethidium bromide/TE gel, visualized by UV illumination, excised and purified using QIAquickTM (Qiagen Inc., Santa Clarita, CA) procedures, as directed by the supplier's instructions.
The pcDNA3. 1/Myc-His plasmid DNA was linearized by digestion with EcoRI and NotI, sites present in the polylinker region of the plasmid DNA. The W O ~J'~/a~ PCTrUS971~4666 m~mm~lobin purified fragment, supra, was ligated with the resu1ting plasmid DNA
backbone downstream from a CMV promoter, and transformed into DH5 alpha~M
cells (GibcoBRL Gaithersburg, Md), as directed by the supplier's instructions.
Brie~ly, 10 ng of pcDNA3.1/Myc-His containing the m~mm~lobin insert were added to 50,ul of competent DH5 alpha cells, and the contents were mixed gently.The mixture was incubated on ice for 30 min, heat shocked for 20 sec at 37~C, and placed on ice for an additional 2 min. Upon addition of 0.95 ml of LB medium, thc mixture was incubated for I h at 37~C while shaking at 225 rpm. The transformed cells then were plated onto 100 mm LB/Amp (50,ug/ml ampicillin) plates and grown10 at 37~C. Colonies were picked and grown in 3 ml of LB/ampicillin broth. Plasmid DN~ was purified using a QIAprep kit. The presence of the insert was confirmcd using restriction enzyme digestion and gel analysis. (J. Sambrook et al., supra.) B. Transfection of ~uman Embryonic Kidnev Cell 293 Cells. The m~rnm~lobin expression plasmid described in section A, supra, was retransformed 15 into DH5 alpha cells, plated onto LB/ampicillin agar, and grown up in 10 ml of LB/arnpicillin broth, as described hereinabove. The plasmid was purified using aQIAfilterT~ maxi kit (Qiagen, Chatsworth, CA) and transfected into HEK293 cells (F.L. Graham et al., J. Gen. Vir. 36:59-72 [1977~). These cells are available from the A.T.C.C., 12301 Parklawn Drive, Rockville, MD 20852, under Accession No.
20 CRL 1573. Transfection was carried out using the cationic lipofectaminc-mediated procedure described by P. Hawley-Nelson et al., Focus 15.73 (1993). HEK293 cells were cultured in 10 ml DMEM media supplcmented with 10% fetal bovine serum (FBS), L-gl~lt~mine (2 mM) and freshly seeded into 100 mm culture plates at a density of 9 x 106 cells per plate . The cells were grown at 37 ~C to a confluency 25 of between 70% and 80% for transfection. Eight micrograms (8 ,ug) of plasmid DNA were added to 800,ul of Opti-MEM I~ medium (Gibco-BRL, Grand Island, NY), and 48-96 ,ul of LipofectamineTM Reagent (Gibco-BRL, Grand Island, NY) werc added to a second 800,ul portion of DMEM serum-free medium. The two solutions were mixed and incubated at room temperature for 15-30 min. After the 30 culture medium was removed from the cells, the cells were washed once with 10 ml of serum-free DMEM. The Opti-MEM I-Lipofectamine-plasmid DNA solution was diluted with 6.4 ml of serum-free DMEM and then overlaid onto lhe cells. The cells were incnh~t~.rl for 5 h at 37~C, after which time, an additional 8 ml of DM~M with ~0% FBS were added. After 18-24 h, the old medium was aspirated, and the cells W O ~8~'~//5~ PCTrUS97/14666 were overlaid with 5 ml of fresh DMEM with 5% FBS. Supernatants and cell extracts were analyzed for m~mm~lobin gene activity 72 h after transfection.
C. Analysis of Breast Tissue Gene Mammaglobin Anti~en Expression. The culture supernatant, supra, is transferred to cryotubes and stored on ice. H~K293 cells are harvested by washing twice with 10 ml of cold Dulbecco's PBS and Iysing by addition of 1.5 ml of CAT lysis buffer (Boehringer Mannheim, Indianapolis, IN), followed by incubation for 30 min at room temperature. Lysate is transferred to 1.7 ml polypropylene microfuge tubes and centrifuged at 1000 x g for 10 min.
The supernatant is transferred to new cryotubes and stored on ice. Aliquots of supernatants from the cells and the lysate of the cells expressing the m~mm~globin protein construct are analyzed for the presence of m~mm~globin recombinant protein. The aiiquots can be run on SDS-polyacrylamide gel electrophoresis (SDS-PAGE) using standard methods and reagents known in the art. (J. Sambrook et al.,~) These gels can then be blotted onto a solid medium such as nitrocellulose, nytran, etc., and the m~rnm~globin protein band can be visualized using western blotting techniques with anti-myc epitope or anti-histidine monoclonal antibodies (Invitrogen, Carlsbad, CA) or anti-m~mm~globin polyclonal serum (see Example 14). Alternatively, the expressed m:~mm:~lobin recombinant protein can be analyzed by mass spectrometry (see Example 12~.
C)A Purification. Purification of the mammaglobin recombinantprotein containing the myc-his sequence is performed using the Xpress~ affinity chromatography system (Invitrogen, Carlsbad, CA) containing a nickel-charged agarose resin which specifically binds polyhistidine residues. Supernatants from 10 x 100 mm plates, prepared as described supra, are pooled and passed over the nickel-charged column. Non-binding protein is eluted by washing the column with 50 mM Tris-HCI (pH 7.5)/150 mM NaCl buffer, leaving only the myc-his fusion proteins. Bound m~mm~globin recombinant protein then is eluted from the column using either an excess of imidazole or histidine, or a low pH buffer. Alternatively, the recombinant protein can also be purified by binding at the myc-his sequence to an affinity column consisting of either anti-myc or anti-histidine monoclonal antibodies conjugated through a hydrazide or other linkage to an agarose resin and eluting with an excess of myc peptide or histidine, respectively.
The purified recombinant protein can then be covalently cross-linked to a solid phase, such as N-hydroxysuccinimide-activated sepharose columns (Pharmacia Biotech, Piscataway, NJ), as directed by supplier's instructions. These columns cont~inin~ covalently linked m~mm~globin recombinant protein, can then be used to purify anti-m~mm~lobin antibodies from rabbit or mouse sera (see Examples 13 and 143.
E. Coatin~ Microtiter Plates with Marnnl~lobin E~xpressedProte;n~.
Supernatant from a 100 mm plate, as described supra, is diluted in an a~pl-~pliate volume of PBS. Then, 100 ,ul of the resulting mixture is placed into each well of a Reacti-BindTM metal chelate microtiter plate (Pierce, Rockford, IL), incubated at room temperature while shaking, and followed by three washes with 200 ,ul each of PBS with 0.05% Tween~ 20. The prepared microtiter plate can then be used to 10 screen polyclonal antisera for the presence of mammaglobin antibodies (see Example 17).
Although pcDNA3.1/Myc-His is utilized in this example, it is known to those skilled in the art that other comparable expression systems can be utilized herein with ~r~liate modifications in reagent and/or techniques and are within the 15 skill of one of ordiDary skill in the art. The largest cloned insert containing the coding region of the mammaglobin gene is sub-cloned into either (i) a eukaryoticexpression vector which may contain, for cxample, a cytomegalovirus (CMV) promoter and/or protein fusible sequences which aid in protein expression and detection, or (ii) a bacterial expression vector containing a superoxide-dismutase 20 (SOD) and CMP-KDO synthetase (CKS) or other protein fusion gene for expression of the protein sequence. Methods and vectors which are useful for the production of polypeptides which contain fusion sequences of ~OD are dcscribed in published EPO application No. EP 0 196 056, published Octobcr 1, 1986, and vectors containing fusion sequences of CKS are describcd in published EPO application No.
25 EP 0 331 961, published September 13, 1989. The purified protein can be used in a variety of techniques, including but not limited to, animal immllni7~tion studies, solid phase immunoassays, etc.
~xarnple 12: Chemical Analysis of Breast Tissue Proteins A. Analysis of Tryptic Peptide Fragments Using M~. Sera from patients with breast disease such as breast cancer, sera from patients with no breast disease, extracts of breast tissues or cells from patients with breast disease such as breast canccr, extracts of breast tissues or cells from patients with no breast disease, and extracts of tissues or cells from other non-diseased or diseased organs of patients arc 35 run on a polyacrylamide gel using standard procedures and stained with Coomassie -W O 98/07753 PCT~US97/14666 Blue. Sections of the gel suspected of containing the unknown polypeptide are excised and subjected to an in-gel reduction, ~eet~m~ tion and tryptic digestion. P.
Jeno et al., Anal. Bio. 224:451-455 (1995) and J. Rosenfeld et al., Anal. Bio.
203:173-179 (1992). The gel sections are washed with 100 mM NH4HC03 and acetonitrile. The shrunken gel pieces are swollen in digestion buffer (50 mM
NH4HCO3, 5 rnM CaCl2 and 12.5 ~g/ml trypsin) at 4~C for 45 rnin. The supernatant is aspirated and replaced with 5 to 10,ul of digestion buffer without trypsin and allowed to incubate overnight at 37~C. Peptides are extracted with 3changes of 5% formic acid and acetonitrile and evaporated to dryness. The peptides are adsorbed to approximately 0.1,ul of POROS R2 sorbent (Perseptive Biosystems, Fr~mingh~m, Massachusetts) trapped in the tip of a drawn gas chromatography capillary tube by dissolving them in 10 ~LI of 5% formic acid andpassing it through the capillary. The adsorbed pcptides are washed with water and elutcd with 5% formic acid in 60% methanol. The eluant is passed directly into the spraying capillary of an API III mass spectrometer (Perkin-Elmer Sciex, Thornhill, Ontario, Canada) for analysis by nano-electrospray mass spectrometry. M. Wilm etal., Int. J. Mass Spectrom. Ion Process 136: 167- 180 (1994) and M. Wilm et al.,Anal. Chem. 66: 1-8 (1994). The masses of the tryptic peptides are determined from the mass spectrum obtained off the first quadrupole. Masses corresponding to predicted peptides can be further analyzed in MS/MS mode to give the amino acid se~uence of the peptide.
B. Peptide Fra~ment Analysis Using LC/MS. The presence of polypeptides predicted from mRNA sequences found in hyperplastic disease tissues also can be confirmed using liquid chromatography/tandem mass spectrometry (LC/MS/MS).
D. Hess et al., ME~THODS. A Companion to Methods in Enzymolo~y 6:227-238 (1994). The serum specimen or tumor extract from the patient is denatured with SDS and reduced with dithiothreitol (1.5 mg/ml) for 30 min at 90~C followed by alkylation with iodo~r.et~mi(le (4 mg/ml) for 15 min at 25~C. Following acrylamide electrophoresis, the polypcptides are electroblotted to a cationic membrane and stained with Coomassie Blue. Following staining, the membranes are washed and sections thought to contain the unknown polypeptides are cut out and dissected into small pieces. The membranes are placed in 500 ~LI microcentrifuge tubes and immersed in 10 to 20 ~l of proteolytic digestion buffer (100 mM Tris-HCI, pH 8.2, containing 0.1 M NaCI, 10% acetonitrilc, 2 mM CaCI~ and 5 ,ug/ml trypsin) (Sigma~
St. Louis, M~). After 15 h at 37~C~ 3 111 of saturated urea and I ,ul of 100 ~ug/ml W O ~ //a~ PCTAUS97114666 trypsin are added and incubated for an additional 5 h at 37~C. The digestion mixture is acidified with 3 ,ul of 10% trifluoroacetic acid and centrifuged to separate supernatant from membrane. The supernatant is injected directly onto a microbore, reverse phase HPLC column and eluted with a linear gradient of acetonitrile in 5 0.05% trifluoroacetic acid. The eluate is fed directly into an electrospray mass spectrometcr, after passing though a stream splittcr if necessary to adjust the volume of material. The data is analyzed following the procedures set forth in Example 12, Scction A.
F.xample 13: Gene Trnm~ llni7~fion Protocol A. In Vivo Antigen Fxpression. Gene immunization circumvents protein purification steps by directly expressing an an~igen in vivo after inoculation of thc appropriate expression vector. Also, production of antigen by this method may allow correct protein folding and glycosylation since the protein is produced in15 m~mrn~ n tissue. The method utilizes insertion of the gene sequence into a plasmid which contains a CMV promoter, expansion and purification of the plasmid and injection of the plasmid DNA into the muscle tissue of an animal. Preferred animals include mice and rabbits. See, for example, H. Davis et al., Human Molecular Genetics 2:1847-~851 (l9g3). After one or two booster immllni7~tions, the animal20 can then be bled, ascites fluid collected, or the animal's spleen can be harvested for production of hybridomas.
B. Plasmid Preparation and Purification. Mammaglobin cDNA insert was released from the m~mm~;lobin vector described in Example I Ib by digestion withEcoRI and NotI restriction en~:ymes. The digested plasmid fragments were 25 electrophoresed on a 1% Seakem KE agaroselO.5 ~g/ml ethidium bromide/TE gel and the bands were vj.cu~li7~.-l by UV illumination. The insert fragment was excised from the gel and purified using the QIAquick procedurc, describcd supra. The fragment was ligated into an EcoRI/NotI digested pcDNA3. I vector (Invitrogen, Carlsbad, CA) and transformed into DH5 alpha cells as described supra. The 30 plasmid DNA was purified from the bacterial Iysate using a QIAprep column. All these techni~ues are familiar to one of ordinary skill in the art of molecular biology.
C. Tmmllni7~fion Protocol. Anesthetized animals are immnni7Pcl intramuscularly with 0.1-100 !lg of the purified plasmid diluted in PBS or otherDNA uptake enhancers (Cardiotoxin, 25% sucrose). See, for example, H. Davis et al., Human Gene Therapy 4:733-740 (1993); and P. W. Wolff et al., Biotechni~ues 1 1:474-485 (1991). One to two booster injections are given at monthly intervals.
D. Testing and Use of Antiserum. Animals are b5ed and the resultant sera tested for antibody using peptides synthesized from the known gene sequence (see5 Example 16~ using techniques known in the art, such as western blotting or EIAtechniques. Antisera produced by this method can then be used to detect the presence of the antigen in a patient's tissue or cell extract or in a patient's serum by ELISA or Western blotting techniques, such as those described in Examples 15 through 18.
;Example 14: Production of Antibodies Against M~mm~lobin A. Production of Polyclonal Antisera. Antiserum against m~rnmz~globin was prepared by injecting rabbits with peptides whose scquences were derived from that of the predicted amino acid sequence of the m~mm~globin gene sequence 15 (SEQUENCE ID NO 1 and SEQUENCE ID NO 2). The synthesis of peptides (SEQUENCE ID NOS 18-26) was described in Example 10. Peptides used as immunogen were either conjugated to a carrier, keyhole limpet hemocyanin (KLH) (SEQUENCE ID NO 18, SEQUENCE ID NO 20, and SEQUENCE ID NO 21), prepared as described hereinbelow, or unconjugated (i.e., not conjugated to a carrier 20 such as KLH) (SEQUENCE ID NO 19).
1. Peptide Conjugation. Peptide was conjugated to maleimide - activated keyhole limpet hemocyanin (KLH, commercially available as Imject~, available from Pierce Chemical Company, Rockford, IL). Imject~ contains about 250 moles of reactive maleimide groups per mole of hemocyanin. The activated 25 KL~ was dissolved in phosphate buffered saline (PBS, pH 8.4) at a concentration of about 7.7 mg/ml. The peptide was conjugated through cysteines occurring in the peptide sec~uence, or to a cysteine previously added to the synthesized peptide in order to provide a point of attachment. The peptide was dissolved in dimethyl sulfoxide (DMSO, Sigma Chemical Company, St. Louis, MO) and reacted with the 30 activated KLH at a mole ratio of about 1.5 moles of peptide per mole of reactive maleimide attached to the KLH. A procedure for the conjugation of peptides (SEQUENCE ID NO 18, SEQUENCE ID NO 20, and SEQUENCE ID NO 21) is provided hereinbelow. It is known to the ordinary artisan that the amounts, times and conditions of such a procedure can be varied to optimize peptide conjugation.

W O 98/07753 PCT~US97/14666 The conjugation reaction described hereinbelow was based on obtaining 3 mg of KLH peptide conjugate ("conjugated peptide"), which contains about 0.77 ,umoles of reactive maleimide groups. This quantity of peptide ConJugate usuallywas adequate for one primary injection and four booster injections for production of polyclonal antisera in a rabbit. Briefly, each pcptide (SEQUENCE ID NO 18, SEQUENCE ID NO 20, and S~QUENCE ID NO 21 ) was dissolved in DMSO at a concentration of 1.16 ~umoles/l 00 ,ul of DMSO. One hundred microliters ( 100 ,ul) of the DMSO solution were added to 380 ,ul of the activaled KLH solution preE)ared as described hereinabove, and 20 ~1 of PBS (pH 8.4) was added to bring the volume to 500 ,ul. The reaction was incubated overnight at room temperature withstirring. The extent of reaction was deterrnined by measuring the amount of unreacted thiol in the reaction mixture. The difference between the starting concentration of thiol and the final concentration was assumed to be the concentration of peptide which has coupled to the activated KLH. The amount of remaining thiol was measured using Ellman's reagent (5,5'-dithiobis(2-nitrobenzoic acid), Pierce Chemical Company, Rockford, lL). Cysteine standards were madc at a concentration of 0, 0.1, 0.5, 2, 5 and 20 mM by dissolving 35 mg of cysteine HCl (Pierce Chemical Company, Rockford, IL) in 10 ml of PBS (pH 7.2) and diluting the stock solution to the desired concentration(s). The photomctric determination of the concentration of thiol was accomplished by placing 200 ,ul of PBS (pH 8.4) in each well of an Immulon 2~ microwell plate (Dynex Technologies, Chantilly, VA).
Next, 10 ~1 of standard or reaction mixture was added to each well. Finally, 20 ~11 of Ellman's reagent at a concentration of 1 mg/ml in PBS (pH 8.4) was added to each well. The wells were incubated for 10 minutes at room temperature, and the absorbance of all wells was read at 415 nm with a microplate reader (such as theBioRad Model 3550, BioRad, Richrnond, CA). The absorbance of the standards was used to construct a standard curve and the thiol concentration of the reaction mixture was determined from the standard curve. A decrease in the concentration of free thiol was indicative of a successful conjugation reaction. In addition, c~lc~ ion of free thiol in the peptide solution, prior to addition of the maleimide activated KLH and upon completion of the reaction, allowed deterrnination of thesubstitution ratio of moles of peptidc/mole of KLH for each peptide x KLH
conjugate. In all cases, the reaction went to completion, and there were approxirnately 250 peptides/KLH molecule for each of the peptide conjugates preparcd. Any unreacted peptide was removed by dialysis against PBS (pH 7.2) at W O 98/07753 . PCT~US97/14666 room t~ elatu1~ for 6 hours. The conjugate was stored at 2-8~C if it was to be used immediately; otherwise, it was stored at -20~~ or colder.
2. Animal Tmmllni7:~tion. Female white New 7~ nd rabbits weighing 2 kg or more were used for raising polyclonal antiserum. Generally, one5 animal was immllni7~? per unconjugated or conjugated peptide (MAM. l-MAM.4, prepared as described hereinabove). One week prior to the first immnni7~tion, 5 to l0 ml of blood were obtained from the animal to serve as a non-imm11ne prebleed sample.
Each of the unconjugated or conjugated peptides, MAM. l -MAM.4, was l 0 used to prepare the primary immunogen by emulsifying 0.5 ml of the peptide at a concentration of 2 mg/ml in PBS (pH 7.2) with 0.5 ml of complete Freund's adjuvant (CFA) (Difco, Detroit, MI). The immunogen was injected into several sites of the animal via subcutaneous, intraperitoneal, and/or intramuscular routes of ~lmini~tration. Four weeks following the primary immllni7:~tion7 a booster 15 irnmuni7~tion was ~Amini.ctered. The immunogen used for the booster immllni7~tion dose was prepared by emulsifying 0.5 ml of the same unconjugated or conjugated peptide used for the primary immunogen, except that the peptide now was diluted to I mg/ml with 0.5 ml of incomplete Freund's adjuvant (IFA) (Difco, Detroit, MI).
Again, the booster dose was ~rimini~tered into several sites and utilized 20 subcutaneous, intraperitoneal and intramuscular types of injections. The animal was bled (5 ml) two weeks after the booster imm-lni7~tion and the serum was tested for immunoreactivity to the peptide, as dcscribed below. The booster and bleed schedule was repeated at 4 week intervals until an adequate titer was obtained. The titer or concentration of antiserum was determined by microtiter EIA as described in 25 Example 17, below using the corresponding unconjugated peptide as the antigencoated on the solid phase, except in the case of anti-MAM.2 antisera. For anti-MAM.2 antisera testing, the microtiter plates were coated with MAM.3 peptide which contained three additional amino acids on the N-terminal end of the peptide compared to the MAM.2 peptide. An antibody titer of 1:500 or greater was 30 considered an adequate titer for further use and study.

B. Production of Monoclonal Antibody.
1. Tmmnni7~tion Protocol. Mice are imm1lni7.D~l using immunogens prepared as described hereinabove, except that the amount of the unconjugated or35 conjugated peptide for monoclonal antibody production in mice is one-tenth the CA 02232239 l99X-04-20 W O ~8~'~7/-.~ PCTAUS97/14666 amount used to produce polyclonal antisera in rabbits. Thus, the primary immunogcn consists of 100 ,ug of unconjugated or conjugated peptide in 0.1 ml ofCFA emulsion; while the immunogen used for booster immllni7zltions consists of 50 ,ug of unconjugated or conjugated peptide in 0.1 ml of IFA. Hybridomas for thc 5 generation of monoclonal antibodies are prepared and screened using standard techniques. The methods used for monoclonal antibody development follow procedures known in the art such as those detailed in Kohler and Milstein, Nature 256:494 (1975) and reviewed in J.G.R. Hurrel, ed., Monoclonal Hybridoma Antibodies Techni~ues and Applications. CRC Press, Inc., Boca Raton, FL
10 (1982). Another method of monoclonal antibody development which is based on the Kohler and Milstein method is that of L.T. Mimms et al., Virolo~y 176:604-619 (1990).
The immllni7~fion regimen (per mouse) consists of a primary immuni7~tion with additional booster immllni7,.tions. The primary immunogen used for the 15 primary immllni7~tion consists of 100 ~g of unconjugated or conjugated peptide in 50 ,ul of PBS (pH 7.2) previously emulsified in 50 ,ul of CFA. Booster immuni7~tions performed at approximately two weeks and four weeks post primary immllni7:~tion consist of 50 ~Lg of unconjugated or conjugated peptide in ~0 ,ul of PBS (pH 7.2) emulsified with 50 ,ul IFA. A total of lO0 ,u] of this immunogen is20 inoculated intraperitoneally and subcutaneously into each mouse. Individual mice are screened for immune response by microtiter plate enzyme immunoassay (EIA) asdescribed in Example 17 approximately four weeks after the third immuni7~tion.
Mice are inoculated either intravenously, intrasplenically or intraperitoncally with 50 ~g of unconjugated or con~ugated peptide in PBS (pH 7.2) approximately fifteen 25 weeks after the third immllni7~fion..
Three days after this intravenous boost, splenocytes are fused with, for example, Sp2/0-Agl4 myeloma cells (Milstein Laboratories, England) using the polyethylene glycol (PEG) method. The fusions are cultured in Iscove's Modified Dulbecco's Medium ~IMDM) containing 10% fetal calf serum (FCS), plus 1%
30 hypoxanthine, aminopterin and thymidine (HAT). Bulk culturcs are screened by microtiter plate EIA following the protocol in Example 17. Clones reactive with the peptide used an immunogcn and non-reactive with other peptides (i.e., peptides of m~mm~Elobin not used as the immunogen) are selected for final expansion. Clones thus selected are expanded, aliquoted and frozen in IMDM containing 10% FCS and 35 10% dimethyl-sulfoxide.

W O 98/07753 rCTrUS97/14666 ~ 2. Production of Ascites Fluid Cont~ining Monoclonal Antibodies.
Frozen hybridoma cells prepared as described hereinabove are thawed and placed into expansion culture. Viable hybridoma cells are inoculated inl.~liluneally into Pristane treated mice. Ascitic fluid is removed from the mice, pooled, filtered through a 0.2 ,u filter and subjected to an immunoglobulin class G (IgG) analysis to determine the volume of the Protein A column required for the purification.
3. Purification of Monoclonal Antibodies From Ascites Fluid.
Briefly, filtered and thawed ascites fluid is mixed with an equal volume of Protein A
sepharose binding buffer (1.5 M glycine, 3.0 M NaCI, pH 8.9) and refiltered 10 through a 0.2 ,u filter. The volume of the Protein A column is determined by the quantity of IgG present in the ascites fluid. The eluate then is dialyzed against PBS
(pH 7.2) overnight at 2-8~C. The dialyzed monoclonal antibody is sterile filtered and dispensed in aliquots. The immunoreactivity of the purified monoclonal antibody is confirrned by determining its ability to specifically bind to the peptide 15 used as the immunogen by use of the EIA microtiter plate assay procedure of Example 17. The specificity of the purified monoclonal antibody is confirmed by determining its lack of binding to irrelevant peptides such as peptides of m~mm~globin not used as the immunogen. The purified anti-m:~m~globin monoclonal thus prepared and characterized is placed at either 2-8~C for short term 20 storage or at -80~C for long term storage.
4. Further Characterization of Monoclonal Antibody. The isotype and subtype of the monoclonal antibody produced as described hereinabove can be determined using commercially available kits ~available from Amersham. Inc., Arlington Heights, IL). Stability testing also can be performed on the monoclonal 25 antibody by placing an aliquot of the monoclonal antibody in continuous storage at 2-8~C and assaying optical density (OD) readings throughout the course of a given period of time.
C. Use of Recombinant Proteins as Immunogens. It is within the scope of the present invention that recombinant proteins made as described herein can be 30 utilized as immunogens in the production of polyclonal and monoclonal antibodies, with corresponding changes in reagents and techniques known to those skilled in the art.

W O 98/07753 PCT~US97/14666 E~ le 15: Purification of Serum Antibodies Which Sl~ecifically F~ind to M~mm:~lobin Peptides Immune sera, obtained as described hereinabove in Examples 13 and/or 14, is affinity purified using irnmobilized synthetic peptides prepared as described in Example 10, or recombinant proteins prepared as described in Example 11. An IgG
fraction of the antiserum is obtained by passing the diluted, crude antiserum over a Protein A column (Affi-Gel protein A, Bio-Rad, Hercules, CA~. Elution with a buffer (Binding Buffer, supplied by the manufacturer) removes substantially all proteins that are not immunoglobulins. Elution with 0.1M buffered glycine (p~ 3)gives an immunoglobulin ~ Lion that is substantially free of albumin and other serum proteins.
Immunoaffinity chromatography is performed to obtain a preparation with a higher fraction of specific antigen-binding antibody. The peptide used to raise the antiserum is immobilized on a chromatography resin, and the specific antibodies directed against its epitopes are adsorbed to the resin. After washing away non-binding components, the specific antibodies are eluted with 0.1 M glycine buffer(pH 2.3). Antibody fractions are im mt~ tely neutralized with 1.0 M Tris buffer (pH 8.0) to preserve immunoreactivity. The chromatography resin chosen depends on the reactive groups present in the peptide. If the peptide has an amino group, a resin such as Affi-Gel 10 or Affi-Gel 15 is used (Bio-Rad, Hercules, CA). If coupling through a carboxy group on the peptide is desired, Affi-Gel 102 can be used (Bio-Rad, Hercules, CA). If the peptide has a free sulfhydryl group, an organomercurial resin such as Affi-~el 501 (Bio-Rad, Hercules, CA) or SulkfoLinkTM (Pierce, Rockford, IL) can be used. The amount of peptide immobilized on the resin can be determined using Nano Orange TM (Molecular Probes, Eugene, OR).
Alternatively, spleens can be harvested and used in the production of hybridomas to produce monoclonal antibodies following routine methods known in the art as described hereinabove.

Fxample 16: Western Blotting of Tissue Samples Protein extracts were prepared by homogenizing tissue samples in 0.1M
Tris-HCI (pH 7.5), 15% (w/v) glycerol, 0.2mM EDTA, 1.0 mM 1,4-dithiothreitol, W O 9~/07753 PCT~US97/14666 - 10 ~Lglml l~up~ and 1.0 mM phenylmethylsulfonylfluoride (Kain et al., Biotechniques,17:982 (1994)). Following homogenization, the homogenates were centrifuged at 4~C for 5 mimltes to :iepald~; supernate from debris. For proteinqll~ntit~tion, 3-10 ,uL of supernate was added to 1.5 ml of bicinchoninic acid reagent 5 (Sigma, St. Louis, MO), and the resulting absorbance at 562 nm was measured.
For SDS-PA(~F,~:~mples were adjusted to desired protein concentration with Tricine ~uffer (Novex,San Diego,CA). They were then mixed with an equal volume of 2X Tricine sample buffer ~Novex, San Diego,CA). and heated for 5 ~ o~s at 100~C in a thermal cycler. Samples were then applied to a Novex 10-20% Precast 10 Tricine Gel for electrophoresis. Following electrophoresis samples were transferred from the gels to nitrocellulose membranes in Novex Tris Glycine Transfer buffer.Membranes were then probed with specific anti-peptide antibodies using the reagents and procedures provided in the Western Lights Plus or Western Lights (Tropix, Bedford, MA) chemiluminesence detection kits. Chemiluminesent bands were 15 vicll~li7.o-1 by exposing the developed membranes to Hyperfilm ECL (Amersham, Arlington Heights, IL).
Figure 3 shows the results of the western blot performed on a panel of tissue protein extracts (CloneTech, Palo Alto, CA) using MAM.1 antiserum (see Example 14). Each lane of Fig. 3 ~ples~ ; a different tissue protein extract (1, prostate CA;
2, prostate BPE~; 3, prostate BPH; 4, prostate BPH; 5, breast CA; 6, breast normal;
7, breast normal; 8, breast CA; g, breast CA; 10, breast CA; 11, breast CA; 12, breast CA; 13, breast CA; 14, markers). Two clusters of bands at 21 kD and 12 kD(see arrows), as determined by protein size markers (lane 14), were detected in 2 of 9 breast cancer tissue protein extracts (lanes 8 and 13) and not in any other of the tissue protein extracts. The multiple bands are most likely due to post-translational modifications of the ll....~ globin protein.
Competition experiments were carried out in an analogous manner as above, with the following exception; the primary antibodies (anti-peptide polyclonal antisera) were pre-inc~lh~t~ for 30 minutes at room temperature with varying 30 concentrations of peptide immunogen prior to exposure to the nitrocellulose filter.
Development of the Western was continued as above. Antibody binding to the bandsat 21 kDa and 12 kDa were inhibited at a concentration of 100 nM peptide MAM. 1.After vi~l,.li7~tion of the bands on film, the bands can also be vis~ li7t -1 directly on the membranes by the addition and development of a chromogenic 35 substrate such as 5-bromo-4-chloro-3-indolyl phosphate (BCIP). This chromogenic W O ~ 7la~ PCT~US97/14666 solution contains 0.016% BCIP in a solution cont~ining 100 mM NaCI, 5 mM
MgC12 and 100 mM Tris-HCI, pH 9.5. The filter is incubated in the solution at room temperature until the bands develop to the desired intensity. Molecular mass determination is made based upon the mobility of pre-stained molecular weight S standards (Novex, San Diego, CA) or biotinylated molecular weight standards (Tropix, Bedford, MA).

Ex,.rnple 17: EIA Microtiter Plate Assay The immunoreactivity of ~ antiserum obtaincd from rabbits as described in Example 14 was determined by means of a microtiter plate EIA (Table 2). BrielCly, synthetic peptides prepared as described in Fxample 10 were dissol~ed in carbonate buffer (50 mM, pH 9.63 to a final concentration of 2 mg/ml. Next, 100 ~ul of thcpeptide or protein solution was placed in each well of an Immulon 2'19 microtiter plate (Dynex Technologies, Chantilly, VA). The plate was incubated overnight at room telllL)eldLule and then washed four times with deionized water. The wells were blocked by adding 125 ,ul of Superblock~9 (Pierce Chemical Company, Rockford, IL) to each wcll and then imml-rlj~tf~ly discarding the solution. This blocking procedure was performed three times. Antiserum obtained ~rom immllni~ l rabbits prepared as previously described was diluted in a protcin blocking agent (e.g., a 3%
Superblock'!D solution) in PBS containing 0.05% Tween-20~ (monolaurate polyoxyethylene ether) (Sigma Chemical Company, St. Louis, MO) and 0.05%
sodium azide at dilutions of 1:500, 1:2500, 1:12,500, 1:62,500 and 1:312,500 andplaced in each well of the coated microtiter plate. Thc wells thcn were incubated for three hours at room t~ pt;l~tuLe. E~ach well was washed four times with deionized water. One hundred microliters (100 ,ul) of ~lk~line phosphatase-conjugated goatanti-rabbit IgG (Southern Biotech, Birmingham, AL), diluted 1:2000 in 3%
Superblock~' solution in phosphate buffered saline containing 0.05% Tween 20a3 and 0.05% sodium azide, was added to each well. The wells were incubated for two hours at room temperature. Next, each well was washed four times with deionized watcr. One hundred microliters ( 100 ,ul) of paranitrophenyl phosphate substrate(Kirkegaard and Perry Laboratories, Gaithersburg, MD) then was added to each well. The wells were incubated for thirty minutes at room temperature. The absorbance at 405 nm was read in each well. Positive reactions were identified by an increase in absorbance at ~05 nm in the test well above that absorbance given by a non-irnmune serum (negative control). A positive reaction was indicative of the W O 98/07753 PCT~US97/14666 ~5 ~ presence of cl~tect~hle anti-m~rnm~lobin antibodies. Titers of the anti-peptide antisera were calculated from the previously described dilutions of antisera anddefined as the calculated dilution where A4OSnm=0.5 OD.

Table 2 Titer of polyclonal antibodies produced against Mammaglobin.
Peptide Peptide Titer ImmunogenConjugated?
MAM. I yes/KLH 56,000 MAM.2 no 44,000 MAM.4 yes/KLH 500 MAM.5 yes/KLH 42,000 Example 18: Coatin~ of Solid Phase Particles A. Coating of Microparticles with Antibodie.s Which Specifically Bind to mammaglobin Antigen. Affinity purified antibodies which specifically bind to 15 m~mm~globin protein (see Example 15) are coated onto microparticles of polystyrene, carboxylated polystyrene, polymethylacrylate or similar particles having a radius in the range of about 0.1 to 20 ,um. Microparticles may be either passively or actively coated. One coating method comprises coating EDAC ( 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (Aldrich Chemical Co., 20 Milwaukee, WI) activated carboxylated latex microparticles with antibodies which specifically bind to m:~mm~globin protein, as follows. Briefly, a final 0.375% solid suspension of resin washed carboxylated latex microparticles (available from Bangs Laboratories, Carmel, IN or Serodyn, Indianapolis, IN) are mixed in a solution containing 50 mM MES buffer, pH 4.0 and 150 mg/l of affinity purified anti-25 m~mm:~g~obin antibody (see Example 14) for 15 min in an a~plu~liate container.EDAC coupling agent is added to a final concentration of 5.5 ~g/ml to the mixture and mixed for 2.5 h at room temperature.
The microparticles then are washed with 8 volumes of a Tween 20~/sodium phosphate wash buffer (pH 7.2) by tangential flow filtration using a 0.2 ,um 30 Microgon Filtration module. Washed microparticles are stored in an ~plupliate W O 98/07753 PCTnUS97/14666 buffer which usually contains a dilute surfactant and irrelevant protein as a blocking agent, until needed.
B. Coatin~ of 1/4 Inch Beads. Antibodies which specifically bind to mammaglobin-antigen also may be coated on the surface of 1/4 inch polystyrene beads by routine methods known in the art (Snitman et al., US Patent 5,273,882) and used in competitive binding or EIA sandwich assays.
Polystyrene beads hrst are cleaned by ultrasonicating them for about 15 seconds in 10 mM NaHCO3 buffer at pH 8Ø The beads then are washed in deionizcd water until all fines are removed. Beads then are immersed in an antibody 10 solution in 10 mM carbonate buffer, p~ 8 to 9.5. The antibody solution can be as dilute as I ,ug/ml in the casc of high af~lnity monoclonal antibodies or as concentrated as about 500 ~g/ml for polyclonal antibodies which have not been affinity purified. Beads are coated for at least 12 llours at room temperature, and then they are washcd with deionized water. Beads may be air dried or stored wet (in 15 PBS, pH 7.4). They also may be overcoated with protcin stabilizers (such as sucrose) or protein blocking agents used as non-specihc binding blockers (such as irrelevant proteins, Carnation skim milk, Superblock~, or the like).
Fx~ ple 19: Microparticle l~nzyme Immunoassay ~M~IA) 2û m~mm~globin antigens are detccted in patient test samples by performing a standard antigen competition EIA or antibody sandwich EIA and utili~ing a solid phase such as microparticles (MEIA). The assay can be performcd on an automated analyzer such as the IMx~ Analyzer (Abbott Laboratories, Abbott Park, IL).
A. Antibody Sandwich EIA. Briefly, samples suspected of containing 25 m:~mm~globin antigen are incubated in the presence of anti-m~mm~globin antibody-coated microparticles (prepared as described in l~xample 17) in order to form antigcn/antibody complexes. The microparticles then are washed and an indicator reagent comprising an antibody conjugated to a signal gencrating compound (i.e.,enzymes such as alkaline phosphatase or horseradish peroxide) is added to the 30 antigen/antibody complexes or the microparticles and incubated. The microparticles are washed and the bound antibody/antigen/antibody complexes are detected by adding a substrate (e.g., 4-methyl umbelliferyl phosphate (MUP), or OPD/peroxide, respectively), that reacts with the signal generating compound to generate a measurable signal. An elevated signal in the test sample, compared to the signal35 generated by a negative control, detects the presence of m:~rnm~globin antigen. The W O 98/077~3 PCTrUS97/14666 ~ presence of m~mm~globin antigen in the test sample is indicative of a diagnosis of a breast disease or condition, such as breast cancer.
B. (~ompetitive Bindin~ Assay. The competitive binding assay uses a peptide or protein that generates a measurable signal when the labeled peptide is 5 contacted with an anti-peptide antibody coated microparticle. This assay can be performed on the IMx~ Analyzer (available from Abbott Laboratories, Abbott Park,IL). The labeled peptide is added to the m~rnm~globin antibody-coated microparticles (prepared as described in Example 17) in thc presence of a test sample suspected of containing m~mm~globin antigen, and incubated for a time and under 10 conditions sufficient to form labeled m~mm:~globin peptide (or labeled protein) /
bound antibody complexes and/or patient mammaglobin antigen / bound antibody complexes. The m~mm:~globin antigen in the test sample competes with the labeledm~mm~globin peptide (or m,lmm~globin protein) for binding sites on the microparticlc. m~mm~globin antigen in the test sample results in a lowered binding 15 of labeled peptide and antibody coated microparticles in the assay since antigen in the test sample and the m:~rnm~globin peptide or mammaglobin protein compete for antibody binding sites. A lowered signal (comparcd to a control) indicates thc presence of m:~mm~globin antigen in the test sample. The presence of m~mm~globinantigen suggests the diagnosis of a breast disease or condition, such as breast 2() cancer.
The mammaglobin polynucleotides and the proteins encoded thereby which are provided and discussed hereinabove are useful as markers of breast tissue disease, especially breast cancer. Tests based upon the appearance of this marker in a test sample such as blood, plasma or serum can provide low cost, non-invasive,2~ diagnostic information to aid the physician to make a diagnosis of cancer, to help select a therapy protocol, or to monitor the success of a chosen therapy. This marker may appear in readily accessible body fluids such as blood, urine or stool as antigens derived from the diseased tissue which are detectable by immunological methods. This marker may be elevated in a disease state, altered in a disease state, 30 or be a normal protein of the breast which appears in an inappropriate body compartment.

WO 98107753 PCTrUS97/14666 SEQUENCE LISTING
(l) GENERAL INFORMATION
(i) APPLICANT: Billing-Medel, Patricia A.
Cohen, Maurice Colpitts, Tracey L.
Friedman, Paula N.
Go~don, Julian Granados, Edward N.
Hodges, Steven C.
Klass, Michael R.
Kratochvil, Jon D.
Roberts-Rapp, Lisa Russell, John C.
Stroupe, Steven D.
(ii) TITLE OF THE l~V~llON: REAGENTS AND METHODS USEFUL
FOR DETECTING DISEASES OF THE BREAST
(iii) NUMBER OF SE~N~S: 2B
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Abbott Laboratories (B) STREET: l00 Abbott Park Road (C.~ CITY: Abbott Park (D~ STATE: IL
(E~ COUNTRY: USA
(Fl ZIP: 60064-3500 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Diskette (B) COMPUTER: IBM Compatible (C) OPERATING SYSTEM: DOS
(D) SOFTWARE: FastSEQ for Windows Version 2.0 (vi) CURRENT APPLICATION DATA:
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(A) APPLICATION NUMBER:
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(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Becker, Cheryl L
(B) REGISTRATION NUMBER: 35,441 (C) REFERENCE/DOCKET NUMBER: 5g73.US.Pl (ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 847/935-1729 (B) TELEFAX: 847/938-2623 (C) TELEX:

(2) INFORMATION FOR SEQ ID NO:l:

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GAATTGAAGG AA~ CT TAACCAAACG GATGA~ACTC TGAGCAATGT TGAGGTGTTT 300 ATGCAATTAA TATATGACAG CAGTCTTTGT GATTTATTTT AA~lll~lGC AAGACCTTTG 360 CTCTTTCTTA 'l'~'l'~'l"l"l"l"l'A CTACAAACTA CAAGACAATT GTTGAAACCT GCTATACATG 480TTTATTTTAA TAAATTGATG GCA 503 (2) INFORMATION FOR SEQ ID NO:2:
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GCCACTACAA ATGCCATAGA TGAATTGAAG GAATGTTTTC TTA~CCAAAC GGATGAAACT 300 TAA~lll~lG CAAGACCTTT GGCTCACAGA ACTGCAGGGT ATGGTGAGAA ACCAGCTACG 420 GATTGCTGCA AACCACACCT TCT~~ l~ll ATGTCTTTTT ACTACAAACT ACAAGACAAT 480 TGTTGAAACC TGCTATACAT GTTTATTTTA ATAAATTGAT GGCAAAAAA~ AP}a~AA~ 540 A~AAAAGG 548 (2) INFORMATION FOR SEQ ID NO:3:
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TATGAACTCT TGAAGAAGTT ~lll~lAT 28 (2) INFORMATION FOR SEQ ID NO:5:
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AGTTTCATCC ~'l"l"l'G~'l"l'A 19 (2) INFORMATION FOR SEQ ID NO:12:
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GCGGCCGCCA AATA~ATCAC AAAGACTGC 29 (2) INFORMATION FOR SEQ ID NO:17:
(i) SEQUENCE CHARACTERISTICS:
(A'~ LENGTH: 93 amino acids (B TYPE: amino acid (CI STR~Nn~n~S single (D,~ TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:
Met Lys Leu Leu Met Val Leu Met Leu Ala Ala Leu Ser Gln His Cys ~ Tyr Ala Gly Ser Gly Cy9 Pro Leu Leu Glu Asn Val Ile Ser Lys Thr Ile Asn Pro Gln Val Ser Lys Thr Glu Tyr Lys Glu Leu Leu Gln Glu Phe Ile Asp Asp Asn Ala Thr Thr Asn Ala Ile Asp Glu Leu Lys Glu Cys Phe Leu Asn Gln Thr Asp Glu Thr Leu Ser Asn Val Glu Val Phe Met Gln Leu Ile Tyr Asp Ser Ser Leu Cys Asp Leu Phe (2) INFORMATION FOR SEQ ID NO:18:
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Pro Gln Val Ser Lys Thr Glu Tyr Lys Glu Leu Leu Gln Glu Phe Ile Asp Asp Asn Ala Thr Thr Asn Ala Ile Asp Glu Leu Lys Glu Cys Phe Leu (2) INFORMATION FOR SEQ ID NO:19:
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Leu Asn Gln Thr Asp Glu Thr Leu Ser Asn Val Glu Val Phe Met Gln Leu Ile Tyr Asp Ser Ser Leu Cys Asp Leu Phe (2) INFORMATION FOR SEQ ID NO:20:
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Glu Cys Phe Leu Asn Gln Thr Asp Glu Thr Leu Ser Asn Val Glu Val Phe Met Gln Leu Ile Tyr Asp Ser Ser Leu Cy9 Asp Leu Phe (2) INFORMATION FOR SEQ ID NO:21:
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(A) LENGTH: 20 amino acids (B) TYPE: amino acid (C) STRANn~n~S: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:
Glu Leu Leu Gln Glu Phe Ile Asp Asp Asn Ala Thr Thr Asn Ala Ile Asp Glu Leu Lys (2) INFORMATION FOR SEQ ID NO:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids (B) TYPE: amino acid (C) STR~Nn~nM~s single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:
Ser Gly Cy6 Pro Leu Leu Glu Asn Val Ile Ser Lys (2) INFORMATION FOR SEQ ID NO:23:

W O 98/07753 PCT~US97/14666 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTE: 15 amino acids (B) TYPE: amino acid (C) STRANv~vNhSS: 9 ingle ~D) TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi) ~N~ DESCRIPTION: SEQ ID NO:23:
Thr Ile Asn Pro Gln Val Ser Lys Thr Glu Tyr Lys Glu Leu Cys (2) INFORMATION FOR SEQ ID NO:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 amino acids (B) TYPE: amino acid (C) STRANv~vN~SS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:
Glu Cys Phe Leu Asn Gln Thr Asp Glu Thr Leu Ser Asn Val Glu Val (2) INFORMATION FOR SEQ ID NO:25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids (B) TYPE: amino acid (C) STR~N~ S: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:
Phe Met Gln Leu Ile Tyr Asp Ser Ser Leu Cys Asp Leu Phe (2) INFORMATION FOR SEQ ID NO:26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 amino acids (B) TYPE: amino acid (C) STRPNI~ )N~ : single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:
Glu Leu Leu Gln Glu Phe Ile Asp Asp Asn Ala Thr Thr Asn Ala Ile Asp Glu Leu Lys Glu Cys (2) INFORMATION FOR SEQ ID NO:27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acid8 (B) TYPE: amino acid CA 02232239 l998-04-20 WO 98/07753 PCTrUS97/14666 (C) STR~N~ liN~ S: ~ingle (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:
ARP Tyr Ly~ Asp Asp Asp Asp Ly~

(2) INFORMATION FOR SEQ ID NO:28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 amino acids (B) TYPE: amino acid (C) STR~MD~nM~S: ~ingle (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:
Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Asn Met His Thr Glu His His His His His His

Claims (8)

We Claim:

1. An antibody which specifically binds to at least one mammaglobin epitope, wherein the mammaglobin epitope is derived from an amino acid sequence having at least 50% identity to an amino acid sequence selected from the group consisting of SEQUENCE ID NOS 17-26, and fragments thereof.

2. An assay kit for determining the presence of mammaglobin antigen in a test sample, comprising a container containing an antibody according to claim 1.

3. The assay kit of claim 2, wherein said antibody is attached to a solid phase.

4. The assay kit of claim 2, further comprising a container with tools useful for collection of said sample, wherein the tools are selected from the group consisting of lancets, absorbent paper, cloth, swabs and cups.

5 . A method for detecting mammaglobin antigen in a test sample suspected of containing said mammaglobin antigen, comprising:
(a) contacting the test sample with an antibody according to claim 1, or fragment thereof, wherein said contacting is carried out for a time and under conditions sufficient for the formation of antibody/antigen complexes; and (b) detecting said complexes.

6. The method of claim 5, wherein said antibody is attached to a solid phase.

7 . A method for producing antibodies which specifically bind to a mammaglobin antigen, comprising administering to an individual an isolated immunogenic polypeptide or fragment thereof in an amount sufficient to elicit animmune response, wherein said immunogenic polypeptide comprises at least one mammaglobin epitope and has at least 50% identity to a sequence selected from the group consisting of SEQUENCE ID NOS 17-26, and fragments thereof.

8. A method for producing antibodies which specifically bind to a mammaglobin antigen, comprising administering to a mammal a plasmid comprising a sequence which encodes at least one mammaglobin epitope derived from a polypeptide having an amino acid sequence selected from the group consisting of SEQUENCE ID NOS 17-26, and fragments thereof.