EP0918788A1 - Compositions brca1 et procedes de diagnostic et de traitement du cancer du sein - Google Patents

Compositions brca1 et procedes de diagnostic et de traitement du cancer du sein

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Publication number
EP0918788A1
EP0918788A1 EP97933344A EP97933344A EP0918788A1 EP 0918788 A1 EP0918788 A1 EP 0918788A1 EP 97933344 A EP97933344 A EP 97933344A EP 97933344 A EP97933344 A EP 97933344A EP 0918788 A1 EP0918788 A1 EP 0918788A1
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EP
European Patent Office
Prior art keywords
brcal
protein
peptide
cell
antibody
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP97933344A
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German (de)
English (en)
Other versions
EP0918788A4 (fr
Inventor
Wen-Hwa Lee
Yumay Chen
Chi-Fen Chen
Phang-Lang Chen
Andrew A. Farmer
Diane C. Jones
D. Craig Allred
C. Kent Osborne
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University of Texas System
Original Assignee
University of Texas System
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Publication date
Application filed by University of Texas System filed Critical University of Texas System
Publication of EP0918788A1 publication Critical patent/EP0918788A1/fr
Publication of EP0918788A4 publication Critical patent/EP0918788A4/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/04—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates generally to the field of molecular biology. More particularly, certain embodiments concern methods and compositions comprising BRCAl compositions and methods for the diagnosis and treatment of breast cancer.
  • breast cancer is the most common fatal malignancy affecting females in developed countries.
  • the etiology of breast cancer involves a complex interplay of genetic, hormonal, and dietary factors that are superimposed on the physiological status of the host.
  • Extensive genetic analysis of breast tumors has identified several alterations in gene expression associated with the disease.
  • At the molecular level in addition to frequently observed gene amplification (Escot et al, 1986, Lidereau et al, 1988, Slamon et al, 1987, van de Vijver et al, 1987, Varley et al, 1988), breast tumor development is thought to be the consequence of unmasking one or more recessive genes by mutation.
  • BRCAl may be inactivated only in familial breast cancers (Boyd, 1995; Castilla et al, 1994), and only a subset of these, since other breast cancer genes, including BRCA2 on chromosome 13ql2-13 (Wooster et al, 1994; 1995), have been mapped to different genetic loci.
  • the pathogenesis of cancer is a multistep process, and alternative pathways can eventually lead to the same or similar consequences.
  • BRCAl complementary DNA encodes a 1863-amino acid protein whose predicted structure includes two zinc finger domains near the NH 2 -terminus and an acidic COOH-terminal domain, leading to speculation that the BRCAl protein is a transcription factor (Miki et al, 1994; Vogelstein and Kinzler, 1994).
  • BRCAl the gene on human chromosome 17q21 responsible for almost 50% of inherited breast cancer, remains an enigma. While mutations in BRCAl have been clearly linked to inherited breast and ovarian cancer, no sporadic breast cancers, and only 10% of sporadic ovarian cancers have been found to harbor BRCAl mutations (Futreal et al, 1994; Hosking et al, 1995; Merajver et al, 1995). Rather, it has been suggested that BRCAl is functionally inactivated by mislocation from its normal location within the nucleus to the cytoplasm in spontaneous cancers (Chen et al, 1995). The defect responsible is presumably in a protein required for the translocation of BRCAl to the nucleus, since tagged. exogenous wild-type BRCAl is similarly mislocated in breast cancer cell lines (Che et al, 1996).
  • BRCAl is a transcription factor, based on the presence of a RING finger motif close to the N-terminus and a C-terminal segment rich in acidic residues (Miki et al, 1994). This would be consistent with the reported nuclear localization of BRCAl.
  • BRCAl is a transcription factor
  • n situ hybridization data have suggested that BRCAl may play a critical role in cellular growth and differentiation, since BRCAl mRNA appears to be generally expressed throughout developing mouse embryos, with particularly high activity seen to correlate with tissues undergoing rapid proliferation and differentiation (Lane et al, 1995; Marquis et al, 1995).
  • BRCAl While two groups have found BRCAl to be a 220 kDa nuclear protein (Chen et al, 1995; Scully et al., 1996), others using similar antibodies have suggested BRCAl to be a 190 kDa secreted protein (Gudas et al, 1995; Jensen et al, 1996).
  • BRCAl located on chromosome 17q21 , is broadly believed to be responsible for about 50% of familial breast and ovarian cancers. Based on the presence of a zinc finger motif and an acidic activation domain, it has been speculated that BRCAl is a transcription factor (Miki et al, 1994). However, to date, no gene activation or repressor function has been documented. BRCAl may play a role in cellular growth and differentiation since its mRNA is widely expressed in developing embryos, being especially high in tissues where cells are rapidly proliferating and differentiating (Lane et al, 1995; Marquis et al, 1995).
  • BRCAl As a tumor suppressor gene, it is paradoxical that mutations in BRCAl are clearly linked to inherited breast and ovarian cancers, but are rarely found in sporadic tumors (Miki et al, 1994; Futreal et al, 1994; Hosking et al, 1995).
  • BRCAl may be functionally inactivated by mislocation from the nuclear to cytoplasmic compartments in sporadic breast cancer cells (Chen et al , 1995; 1996).
  • the problem is either in nuclear transport, retention or cytoplasmic confinement since epitope-tagged exogenous wild-type BRCAl protein is also cytoplasmic in at least two lines of breast cancer cells (Chen et al, 1996).
  • BRCAl has a molecular mass of approximately 220 kDa, presumably it is actively translocated from the cytoplasm to the nucleus by direct interactions with the nuclear localization signal receptor or by indirect interactions with other NLS-containing proteins (Hicks and Rikhel, 1995; Dingwall and Laskey, 1991).
  • the direct import of karyophilic proteins through the nuclear pore complex requires energy (Newmeyer and Forbes, 1988; Richardson et al, 1988) and a nuclear localization sequence (NLS) located in the transport substrate (Dingwall et al, 1982; alderon et al, 1984) to which a cytosolic receptor complex, importin- ⁇ and importin- ⁇ , binds (Gorlich et al, 1994; 1995).
  • a GTP-binding protein, RAN mediates the energy-dependent translocation of the substrate-receptor complex through the nuclear pore complex (Moore and Blobel, 1993). After translocation, importin- ⁇ dissociates from the complex in the vicinity of the inner aspect of the nuclear envelope while importin- ⁇ accompanies the substrate to its sites of function (Gorlich et al, 1995).
  • BRCAl is a secreted protein (Jensen et al, 1996). Since the subcellular location of proteins is a fundamental aspect of their function, it is important to solidify the data regarding the location of BRCAl in normal and cancer cells. More importantly, the subcellular compartmentation of BRCAl is also a critical issue with regard to its role in breast tumorigenesis.
  • the present invention overcomes one or more of these and other drawbacks inherent in the prior art by providing novel compositions and methods for their use in the diagnosis and treatment of breast cancer.
  • the invention provides a method of localizing a BRCAl protein or peptide in a cell.
  • the method generally involves contacting the cell with a labeled antibody that specifically binds to a BRCAl or BRCAl -associated protein or peptide, under conditions effective to allow the formation of immune complexes; and determining the location of the immune complexes in the cell. WT ⁇ en such complexes are localized in the cytoplasm of the cell, there is an indication of metastasis or primary cancer of the cell.
  • Such information is useful in the early detecting and screening for cancers, and in particular, breast and ovarian cancers, which the inventors have shown to be correlated with cytoplasmic subcellular localization of BRCAl protein.
  • the cells are human, and in particular, a breast or an ovarian cell.
  • a further object of the invention is a method of identifying a breast or ovarian cancer cell in a sample.
  • the method generally involves obtaining an ovarian or breast tumor cell suspected of being cancerous and determining the subcellular location of a BRCAl protein or peptide in the tumor cell.
  • subcellular localization of the BRCAl protein or peptide to the cytoplasm of the cell has been demonstrated by the inventors to be indicative of the presence of cancerl
  • the method generally involves identifying in the cell a cytoplasmically- localized BRCAl or BRCAl -associated protein or peptide, wherein the presence of the protein or peptide in the cytoplasm is indicative of susceptibility of the cell to cancer.
  • the invention provides nucleic acid sequences encoding a BRCAl -associated protein (BAP).
  • BAP BRCAl -associated protein
  • a BAP gene means a nucleic acid sequence encoding a
  • a preferred nucleic acid sequence encoding a BAP gene is a nucleotide sequence which encodes the amino acid sequence of SEQ ID NO: l . It is expected that the gene encoding BAP may vary in nucleic acid sequence from sample to sample, but that the variation in nucleic acid sequence will not preclude hybridization between sequences encoding BAP of each sample under strict hybridization conditions.
  • a strain variant of BAP means any polypeptide encoded, in whole or in part, by a nucleic acid sequence which hybridizes under strict hybridization conditions to a nucleic acid sequence which encodes the amino acid sequence of SEQ ID NO: l .
  • a BAP is also understood to mean a polypeptide that is immunologically reactive with antibodies generated against the BAP protein of SEQ ID NO:l .
  • BRCAl is understood to mean a polypeptide that is capable of eliciting antibodies that are immunologically reactive with BRCAl and BRCAl -like gene products
  • BAP is understood to mean a polypeptide that is capable of eliciting antibodies that are immunologically reactive with a BAP encoded by a nucleic acid sequence which encodes the amino acid sequence of SEQ ID NO: 1.
  • an active fragment of BAP includes B APs which are modified by conventional techniques, e.g., by addition, deletion, or substitution, but which active fragment exhibits substantially the same structure and function as BAP as described herein, antigenicity according to conventional methods.
  • the present invention concerns DNA segments, that can be isolated from virtually any bacterial source, that are free from total genomic DNA and that encode proteins having BAP-like activity. DNA segments encoding BAP-like species may prove to encode proteins, polypeptides, subunits, functional domains, and the like.
  • DNA segment refers to a DNA molecule that has been isolated free of total genomic DNA of a particular species. Therefore, a DNA segment encoding BAP refers to a DNA segment that contains BAP coding sequences yet is isolated away from, or purified free from, total genomic DNA of the species from which the DNA segment is obtained. Included within the term “DNA segment”, are DNA segments and smaller fragments of such segments, and also recombinant vectors, including, for example, plasmids, cosmids, phagemids, phage, viruses, and the like.
  • a DNA segment comprising an isolated or purified BAP gene refers to a DNA segment including BAP coding sequences and, in certain aspects, regulatory sequences, isolated substantially away from other naturally occurring genes or protein encoding sequences.
  • the term "gene” is used for simplicity to refer to a functional protein, polypeptide or peptide encoding unit.
  • this functional term includes both genomic sequences, extra-genomic and plasmid-encoded sequences and smaller engineered gene segments that express, or may be adapted to express, proteins, polypeptides or peptides. Such segments may be naturally isolated, or modified synthetically by the hand of man.
  • isolated substantially away from other coding sequences means that the gene of interest, in this case, a gene encoding BAP, forms the significant part of the coding region of the DNA segment, and that the DNA segment does not contain large portions of naturally-occurring coding DNA, such as large chromosomal fragments or other functional genes or polypeptide coding regions. Of course, this refers to the DNA segment as originally isolated, and does not exclude genes or coding regions later added to the segment by the hand of man.
  • amino acid and nucleic acid sequences may include additional residues, such as additional N- or C-terminal amino acids or 5' or 3' sequences, and yet still be essentially as set forth in one of the sequences disclosed herein, so long as the sequence meets the criteria set forth above, including the maintenance of biological protein activity where protein expression is concerned.
  • the addition of terminal sequences particularly applies to nucleic acid sequences that may, for example, include various non-coding sequences flanking either of the 5' or 3' portions of the coding region or may include various upstream or downstream regulatory or structural genes.
  • nucleic acid sequences that are “complementary” are those that are capable of base-pairing according to the standard Watson-Crick complementarity rules.
  • complementary sequences means nucleic acid sequences that are substantially complementary, as may be assessed by the same nucleotide comparison set forth above, or as defined as being capable of hybridizing to a nucleic acid segment which encodes the amino acid sequence of SEQ ID NO:l, under relatively stringent conditions such as those described herein.
  • nucleic acid segments of the present invention may be combined with other DNA sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their overall length may vary considerably. It is therefore contemplated that a nucleic acid fragment of almost any length may be employed, with the total length preferably being limited by the ease of preparation and use in the intended recombinant DNA protocol.
  • nucleic acid fragments may be prepared that include a short contiguous stretch identical to or complementary to a nucleic acid sequence which encodes the amino acid sequence of SEQ ID NO:l, such as about 14 nucleotides, and that are up to about 10,000 or about 5,000 base pairs in length, with segments of about 3,000 being preferred in certain cases.
  • DNA segments with total lengths of about 2,000, about 1,000, about 500, about 200, about 100 and about 50 base pairs in length (including all intermediate lengths) are also contemplated to be useful. It will be readily understood that "intermediate lengths", in these contexts, means any length between the quoted ranges, such as 14, 15, 16, 17, 18, 19, 20, etc.; 21, 22, 23.
  • Recombinant vectors and isolated DNA segments may therefore variously include the BAP coding regions themselves, coding regions bearing selected alterations or modifications in the basic coding region, or they may encode larger polypeptides that nevertheless include BAP coding regions or may encode biologically functional equivalent proteins or peptides that have variant amino acids sequences.
  • fusion proteins and peptides e.g., where the BAP coding regions are aligned within the same expression unit with other proteins or peptides having desired functions, such as for purification or immunodetection purposes (e g-, proteins that may be purified by affinity chromatography and enzyme label coding regions, respectively).
  • Recombinant vectors form further aspects of the present invention.
  • Particularly useful vectors are contemplated to be those vectors in which the coding portion of the DNA segment, whether encoding a full length protein or smaller peptide, is positioned under the control of a promoter.
  • the promoter may be in the form of the promoter that is naturally associated with a BAP gene, as may be obtained by isolating the 5' non-coding sequences located upstream of the coding segment, for example, using recombinant cloning and/or PCRTM technology, in connection with the compositions disclosed herein.
  • a recombinant or heterologous promoter is intended to refer to a promoter that is not normally associated with a BAP gene in its natural environment.
  • Such promoters may include BAP promoters normally associated with other genes, and/or promoters isolated from any bacterial, viral, eukaryotic, or mammalian cell.
  • BAP promoters normally associated with other genes
  • promoter and cell type combinations for protein expression is generally known to those of skill in the art of molecular biology, for example, see Sambrook et al, 1989.
  • the promoters employed may be constitutive, or inducible, and can be used under the appropriate conditions to direct high level expression of the introduced DNA segment, such as is advantageous in the large-scale production of recombinant proteins or peptides.
  • Prokaryotic expression of nucleic acid segments of the present invention may be performed using methods known to those of skill in the art, and will likely comprise expression vectors and promoter sequences such as those provided by tac, trp, lac, lacUV ⁇ or T7.
  • BRCAl -like proteins is desired in eukaryotic cells, a number of expression systems are available and known to those of skill in the art.
  • An exemplary eukaryotic promoter system contemplated for use in high-level expression is the Pichia expression vector system (Pharmacia LKB Biotechnology).
  • Pichia expression vector system Pharmacia LKB Biotechnology
  • BRCAl and/or related peptides it is contemplated that longer DNA segments will most often be used, with DNA segments encoding the entire BAP or BRCAl or functional domains, epitopes, ligand binding domains, subunits, etc. being most preferred.
  • DNA segments that encode peptide antigens from about 15 to about 100 amino acids in length, or more preferably, from about 15 to about 50 amino acids in length are contemplated to be particularly useful.
  • the BAP gene and DNA segments may also be used in connection with somatic expression in an animal or in the creation of a transgenic animal. Again, in such embodiments, the use of a recombinant vector that directs the expression of the full length or active BAP protein is particularly contemplated. Expression of a BAP transgene in animals is particularly contemplated to be useful in the production of anti- BAP antibodies for use in passive immunization methods and treatment of particular breast cancers. 2.2 RECOMBINANT EXPRESSION OF BAP AND BRCAl
  • engineered or "recombinant” cell is intended to refer to a cell into which a recombinant gene, such as a gene encoding a BAP or BRCAl has been introduced. Therefore, engineered cells are distinguishable from naturally occurring cells which do not contain a recombinantly introduced gene. Engineered cells are thus cells having a gene or genes introduced through the hand of man.
  • Recombinantly introduced genes will either be in the form of a single structural gene, an entire genomic clone comprising a structural gene and flanking DNA, or an operon or other functional nucleic acid segment which may also include genes positioned either upstream and/or downstream of the promoter, regulatory elements, or structural gene itself, or even genes not naturally associated with the particular structural gene of interest.
  • constitutive eukaryotic promoters include viral promoters such as the cytomegalovirus (CMV) promoter, the Rous sarcoma long-terminal repeat (LTR) sequence, or the SV40 early gene promoter. The use of these constitutive promoters will ensure a high, constant level of expression of the introduced genes.
  • CMV cytomegalovirus
  • LTR Rous sarcoma long-terminal repeat
  • the level of expression from the introduced genes of interest can vary in different clones, or genes isolated from different strains or bacteria.
  • the level of expression of a particular recombinant gene can be chosen by evaluating different clones derived from each transfection experiment; once that line is chosen, the constitutive promoter ensures that the desired level of expression is permanently maintained. It may also be possible to use promoters that are specific for cell type used for engineering, such as the insulin promoter in insulinoma cell lines, or the prolactin or growth hormone promoters in anterior pituitary cell lines.
  • the recombinant GST-BRCAl ⁇ 5g/II gene fusion disclosed herein was deposited with the American Type Culture Collection in E. coli DH5 ⁇ TMF' under the terms of the Budapest Treaty and was assigned the following accession number: ATCC 98100.
  • the present invention concerns immunodetection methods and associated kits. It is contemplated that the proteins or peptides of the invention may be employed to detect antibodies having reactivity therewith, or, alternatively, antibodies prepared in accordance with the present invention, may be employed to detect BAP or BRCAl peptides.
  • the kits may also be used in antigen or antibody purification, as appropriate.
  • the preferred immunodetection methods will include first obtaining a sample suspected of containing a BAP or BRCAl -reactive antibody, such as a biological sample from a patient, and contacting the sample with a first BAP or BRCAl peptide under conditions effective to allow the formation of an immunocomplex (primary immune complex). One then detects the presence of any primary immunocomplexes that are formed.
  • Detection of primary immune complexes is generally based upon the detection of a label or marker, such as a radioactive, fluorescent, biological or enzymatic label, with enzyme tags such as alkaline phosphatase, urease, horseradish peroxidase and glucose oxidase being suitable.
  • a label or marker such as a radioactive, fluorescent, biological or enzymatic label
  • enzyme tags such as alkaline phosphatase, urease, horseradish peroxidase and glucose oxidase being suitable.
  • the particular antigen employed may itself be linked to a detectable label, wherein one would then simply detect this label, thereby allowing the amount of bound antigen present in the composition to be determined.
  • the primary immune complexes may be detected by means of a second binding ligand that is linked to a detectable label and that has binding affinity for the first protein or peptide.
  • the second binding ligand is itself often an antibody, which may thus be termed a "secondary" antibody.
  • the primary immune complexes are contacted with the labeled, secondary binding ligand, or antibody, under conditions effective and for a period of time sufficient to allow the formation of secondary immune complexes.
  • the secondary immune complexes are then generally washed to remove any non-specifically bound labeled secondary antibodies and the remaining bound label is then detected.
  • sample suspected of containing the antibodies of interest may be employed.
  • exemplary samples include clinical samples obtained from a patient such as blood or serum samples, cerebrospinal, synovial, or bronchoalveolar fluid, ear swabs, sputum samples, middle ear fluid or even perhaps urine samples may be employed.
  • clinical samples may have application to non-clinical samples, such as in the titering of antibody samples, in the selection of hybridomas, and the like.
  • the clinical samples may be from veterinary sources and may include such domestic animals as cattle, sheep, and goats. Samples from feline, canine, and equine sources may also be used in accordance with the methods described herein.
  • kits in accordance with the present invention contemplates the preparation of kits that may be employed to detect the presence of BAP- or BRCAl -specific antibodies in a sample.
  • kits in accordance with the present invention will include a suitable protein or peptide together with an immunodetection reagent, and a means for containing the protein or peptide and reagent.
  • the immunodetection reagent will typically comprise a label associated with a BAP or BRCAl peptide, or associated with a secondary binding ligand.
  • exemplary ligands might include a secondary antibody directed against the first BAP or BRCAl peptide or antibody, or a biotin or avidin (or streptavidin) ligand having an associated label.
  • Detectable labels linked to antibodies that have binding affinity for a human antibody are also contemplated, e.g., for protocols where the first reagent is a BAP or BRCAl peptide that is used to bind to a reactive antibody from a human sample.
  • the kits may contain antigen or antibody-label conjugates either in fully conjugated form, in the form of intermediates, or as separate moieties to be conjugated by the user of the kit.
  • the container means will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which the antigen may be placed, and preferably suitably allocated.
  • the kit will also generally contain a second vial or other container into which this ligand or antibody may be placed.
  • the kits of the present invention will also typically include a means for containing the vials in close confinement for commercial sale, such as, e.g., injection or blow-molded plastic containers into which the desired vials are retained.
  • BRCAl or a BRCAl -associated protein may be desirable to administer BRCAl or a BRCAl -associated protein to the human or animal subject in a pharmaceutically acceptable composition comprising an immunologically effective amount of BRCAl or a BRCAl -associated protein mixed with other excipients, carriers, or diluents which may improve or otherwise alter stimulation of B cell and/or T cell responses, or immunologically inert salts, organic acids and bases, carbohydrates, and the like, which promote stability of such mixtures.
  • Immunostimulatory excipients may include salts of aluminum (often referred to as Alums), simple or complex fatty acids and sterol compounds, physiologically acceptable oils, polymeric carbohydrates, chemically or genetically modified protein toxins, and various particulate or emulsified combinations thereof.
  • Alums aluminum
  • BRCAl, BRCAl -derived peptides, or one or more BAPs may be formulated within these mixtures, or each variant if more than one are present, would be expected to comprise about 0.0001 to 1.0 milligrams, or more preferably about 0.001 to 0.1 milligrams, or even more preferably less than 0.1 milligrams per dose.
  • Attenuated organisms may be engineered to express recombinant BRCAl gene products or a BRCAl -associated protein and themselves be delivery vehicles for the invention.
  • Particularly preferred are attenuated bacterial species such as Mycobacterium, and in particular M. bovis, M. smegmatis, or BCG.
  • pox-, polio-, adeno-, or other viruses, and bacteria such as Salmonella, or Shigella, species may also be used in conjunction with the methods and compositions disclosed herein.
  • the naked DNA technology has been shown to be suitable for protection against infectious organisms.
  • DNA segments could be used in a variety of forms including naked DNA and plasmid DNA, and may administered to the subject in a variety of ways including parenteral, mucosal, and so-called microprojectile-based "gene-gun” inoculations.
  • the use of BRCAl or BAP gene nucleic acid compositions of the present invention in such immunization techniques is thus proposed to be useful in the formulation of antibodies directed against such proteins.
  • an optimal dosing schedule of a vaccination regimen may include as many as five to six, but preferably three to five, or even more preferably one to three administrations of the immunizing entity given at intervals of as few as two to four weeks, to as long as five to ten years, or occasionally at even longer intervals.
  • Particular aspects of the invention concern the use of plasmid vectors for the cloning and expression of recombinant peptides, and particular peptide epitopes comprising either native, or site-specifically mutated BRCAl or BRCAl -associated protein epitopes.
  • the generation of recombinant vectors, transformation of host cells, and expression of recombinant proteins is well-known to those of skill in the art.
  • Prokaryotic hosts are preferred for expression of the peptide compositions of the present invention.
  • An example of a preferred prokaryotic host is E. coli, and in particular, E. coli strains ATCC69791, BL21(D ⁇ 3), JM101, XLl-Blue ® , RR1, LE392, B, P 1776 (ATCC No.
  • plasmid vectors containing replicon and control sequences which are derived from species compatible with the host cell are used in connection with these hosts.
  • the vector ordinarily carries a replication site, as well as marking sequences which are capable of providing phenotypic selection in transformed cells. For example, E.
  • coli may be typically transformed using vectors such as pBR322, or any of its derivatives (Bolivar et al, 1977).
  • pBR322 contains genes for ampicillin and tetracycline resistance and thus provides easy means for identifying transformed cells.
  • pBR322 its derivatives, or other microbial plasmids or bacteriophage may also contain, or be modified to contain, promoters which can be used by the microbial organism for expression of endogenous proteins.
  • phage vectors containing replicon and control sequences that are compatible with the host microorganism can be used as transforming vectors in connection with these hosts.
  • bacteriophage such as ⁇ GEMTM-l l may be utilized in making a recombinant vector which can be used to transform susceptible host cells such as E. coli LE392.
  • Those promoters most commonly used in recombinant DNA construction include the ⁇ -lactamase (penicillinase) and lactose promoter systems (Chang et al. 1978; Itakura et al, 1977; Goeddel et al, 1979) or the tryptophan (trp) promoter system (Goeddel et al, 1980).
  • eukaryotic microbes such as yeast cultures may also be used in conjunction with the methods disclosed herein.
  • Saccharomyces cerevisiae, or common bakers' yeast is the most commonly used among eukaryotic microorganisms, although a number of other species may also be employed for such eukaryotic expression systems.
  • the plasmid YRp7 for example, is commonly used (Stinchcomb et al, 1979; Kingsman et al, 1979; Tschemper et al, 1980).
  • This plasmid already contains the trpL gene which provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example ATCC No. 44076 or PEP4-1 (Jones, 1977).
  • the presence of the trpL lesion as a characteristic of the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan.
  • Suitable promoting sequences in yeast vectors include the promoters for 3- phosphoglycerate kinase (Hitzeman et al, 1980) or other glycolytic enzymes (Hess et al, 1968; Holland et al, 1978), such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase.
  • 3- phosphoglycerate kinase Hitzeman et al, 1980
  • other glycolytic enzymes Hess et al, 1968; Holland et al, 1978
  • enolase glyceraldehyde-3-phosphate dehydrogenas
  • the termination sequences associated with these genes are also ligated into the expression vector 3' of the sequence desired to be expressed to provide polyadenylation of the mRNA and termination.
  • Other promoters which have the additional advantage of transcription controlled by growth conditions are the promoter region for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, and the aforementioned glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization.
  • Any plasmid vector containing a yeast-compatible promoter, an origin of replication, and termination sequences is suitable.
  • cultures of cells derived from multicellular organisms may also be used as hosts in the routine practice of the disclosed methods.
  • any such cell culture is workable, whether from vertebrate or invertebrate culture.
  • interest has been greatest in vertebrate cells, and propagation of vertebrate cells in culture (tissue culture) has become a routine procedure in recent years.
  • useful host cell lines are VERO and HeLa cells, Chinese hamster ovary (CHO) cell lines, and WI 38, BHK, COS-7, 293 and MDCK cell lines.
  • Expression vectors for such cells ordinarily include (if necessary) an origin of replication, a promoter located in front of the gene to be expressed, along with any necessary ribosome binding sites, RNA splice sites, polyadenylation site, and transcriptional terminator sequences.
  • control functions on the expression vectors are often provided by viral material.
  • promoters are derived from polyoma, Adenovirus 2, and most frequently Simian Virus 40 (SV40).
  • SV40 Simian Virus 40
  • the early and late promoters of SV40 virus are particularly useful because both are obtained easily from the virus as a fragment which also contains the SV40 viral origin of replication (Fiers et al, 1978). Smaller or larger SV40 fragments may also be used, provided there is included the approximately 250 bp sequence extending from the H dIII site toward the BgH site located in the viral origin of replication.
  • the origin of replication may be provided either by construction of the vector to include an exogenous origin, such as may be derived from SV40 or other viral (e.g., Polyoma, Adeno, VSV, BPV) source, or may be provided by the host cell chromosomal replication mechanism. If the vector is integrated into the host cell chromosome, the latter is often sufficient.
  • polypeptides may be present in quantities below the detection limits of the Coomassie brilliant blue staining procedure usually employed in the analysis of SDS/PAGE gels, or that their presence may be masked by an inactive polypeptide of similar M r .
  • other detection techniques may be employed advantageously in the visualization of particular polypeptides of interest.
  • Immunologically-based techniques such as Western blotting using enzymatically-, radiolabel-, or fluorescently-tagged antibodies described herein are considered to be of particular use in this regard.
  • the peptides of the present invention may be detected by using antibodies of the present invention in combination with secondary antibodies having affinity for such primary antibodies.
  • This secondary antibody may be enzymatically- or radiolabeled, or alternatively, fluorescently-, or colloidal gold-tagged.
  • FIG. 1A A schematic showing the 3 overlapping cDNA clones used to construct a full-length BRCAl cDNA. The 3 regions of the BRCAl cDNA used to make
  • GST fusion-proteins are also outlined.
  • FIG. IB Identification of BRCAl as a 220-kDa protein in human cells.
  • FIG. 2A Comparison of the mobility of in vitro translated BRCAl with that from HBL100 cells.
  • Lanes 1, 2 BRCAl from HBL100 cells (1 x 10 ? /lane) precipitated with anti-BRCAl .
  • Lanes 3, 4, and 5 in vitro translated BRCAl ( 1 /20th total product) immunoprecipitated with each of the three antisera.
  • FIG. 2B Comparison of the mobility of recombinant, baculovirus-derived
  • Lanes 1 , 2 HBL100 cells (0.5 x 10 6 /lane). Lane
  • Lanes 4-7 infected SF9 cells. Lanes 1 and 4: immunoprecipitated with preimmune serum. Lanes 2, 3, and 5: immunoprecipitated with anti-BRCAl . Lane 6: immunoprecipitated with anti-BRCAl Bgl. Lane 7: immunoprecipitated with anti-BRCAlN. Immunoprecipitates were detected by western blotting and probing with anti-BRCAl monoclonal MAb 6B4.
  • FIG. 3A BRCAl expression and phosphorylation is cell cycle dependent. Extracts from synchronized T24 cells were aliquoted, separated by SDS-PAGE, western
  • Top Panel 1 x 10 cells/lane probed for BRCAl using
  • Middle Panel 1 x 10 cells/lane probed for pl l ORB using MAb
  • Lane 1 1D7 Lower Panel: 5 x 10 cells/lane probed for p84 with anti-N5-3.
  • FIG. 3B BRCAl expression and phosphorylation is cell cycle dependent.
  • FIG. 3C BRCAl expression and phosphorylation is cell cycle dependent. FACS analysis of the synchronized cells at the time points assayed showing the % distribution of cells in the various stages of the cell cycle.
  • FIG. 3D BRCAl expression and phosphorylation is cell cycle dependent. Immunofluorescence staining for BRCAl during cell cycle progression. Panels a, c, e, g, i, k: DAPI staining for DNA. Panels b, d, f, h, j.
  • FIG. 4 Phosphorylation of BRCAl by various cyclin-dependent kinases.
  • Extracts from HBL100 cells were precipitated with various anti-cyclin/cyclin-dependent kinase antibodies, as shown. Precipitates were incubated in kinase buffer in the presence 32 of [ ⁇ - P]ATP, and then washed and dissociated. The resultant supernatants were reprecipitated using anti-BRCAl, separated by SDS-PAGE, and the gels dried and autoradiographed .
  • FIG. 5A Identification of BRCAl . Diploid human breast epithelial cells (HBLIOO, about 1 x 10 7 cells per lane) were incubated with 35 S-methionine (lanes 1 to 6)
  • FIG. 5B Detection of full-length BRCAl in normal breast epithelial cells and breast cancer cell lines.
  • Established cell lines were obtained from American Type Culture Tissue Collection. Malignant cells from pleural effusions, immediately after being withdrawn from patients, were washed in 50:50 Ham's F-12-Dulbecco's modified Eagle's medium (DMEM) and frozen in liquid nitrogen without passage, in the same medium plus 50% fetal calf serum (FCS) and 10% dimethyl sulfoxide. Before fixation for immunostaining the cells were washed, then plated for 12 hours in Ham's F-12-DMEM plus 10% FCS. Viable cells were cytospun onto glass cover slips where they were fixed as described for established cell lines.
  • DMEM Ham's F-12-Dulbecco's modified Eagle's medium
  • FCS fetal calf serum
  • Viable cells were cytospun onto glass cover slips where they were fixed as described for established cell lines.
  • Lane 1 Human breast cell lines (5 x 10 cells per lane) were labeled with [ PJphosphoric acid.
  • Lane 1 HBLIOO lysate immunoprecipitated by preimmune mouse serum.
  • Cell lysates in lanes 2 to 1 1 immunoprecipitated by anti-BRCAl lane 2, T47D; lane 3, MCF7; lane 4, MB468; lane 5, MB175-7; lane 6, MB-361; lane 7 MB-231; lane 8, MB-435S; lane 9, MB415; lane 10, HS578T; and lane 1 1 , HBLIOO.
  • Sections 5- ⁇ m-thick from randomly selected, formalin-fixed, paraffin-embedded, breast cancer biopsies in the inventors' tumor bank were immunostained by a modification of the avidin-biotin-horseradish peroxidase complex (ABC) method (Hsu et al, 1981).
  • Anti-BRCAl was used at 1 :100 dilution. Both cases of invasive breast cancer showing no cytoplasmic or nuclear immunostaining for BRCAl did show positive immunostaining for the nuclear proliferation antigen MiBl .
  • FIG. 5C. Full-length BRCAl is expressed in tumor cell lines derived from tissues other than breast.
  • rhabdomyosarcoma lane 7, HCT116 (colon carcinoma); lane, SW620 (colon carcinoma); lane 9, C41 1 (cervical carcinoma); lane 10, MS751 (cervical carcinoma); lane 1 1, SAOS-2 (osteosarcoma); and lane 12, U2OS (osteosarcoma).
  • FIG. 6A Localization of BRCAl in normal and breast cancer cells. Fractionation of HBLIOO cells. Cells (1.5 x 10 ) were labeled with 35 S-methionine; 5 x
  • T, lane 1 10 6 cells were left unfractionated (total or T, lane 1) and the remainder were separated into nuclear (N, lane 2), cytoplasmic (C, lane 3), and membrane (M, lane 4) fractions (Chen et al, 1995; Abrams et al, 1982).
  • pl lO 1 * served as a marker for nuclear distribution and GST for cytoplasmic distribution.
  • Small aliquots were incubated with GST beads, separated by SDS-PAGE, and stained with Coomassie Brilliant Blue to visualize the expected 26-kDa glutathione-S-transferase (GST) band.
  • FIG. 6B Localization of BRCAl in normal and breast cancer cells.
  • DAPI 4,6-diamidino-2-phenolindole propidium iodide
  • FIG. 6C Localization of BRCAl in normal and breast cancer cells. Detection of BRCAl in the nuclei of cell lines derived from tissues other than breast, (i, k, m, o) DAPI staining; (j, 1, n, p) BRCAl staining, (i and j) DU145 (prostate carcinoma) cells; (k and 1) RAT2 fibroblasts; (m and n) T24 (TCC bladder) cells; (o and p) CV1 (monkey kidney epithelial) cells.
  • FIG. 6D Localization of BRCAl in normal and breast cancer cells.
  • Cytoplasmic localization of BRCAl in breast cancer cells (a) through (h) breast cancer line T47D; (k and 1) breast cancer line MCF7; (m and n) cells from primary malignant effusion #22550; (o and p) cells from primary effusion #23159.
  • FIG. 7 A Primary breast cancer sections stained for BRCAl by the immunoperoxidase method. Sections 5- ⁇ m-thick from randomly selected, formalin-fixed, paraffin-embedded, breast cancer biopsies in the inventors' tumor bank were immunostained by a modification of the avidin-biotin-horseradish peroxidase complex (ABC) method (Hsu et al, 1981). AntiBRCAl was used at 1 : 100 dilution.
  • ABSC avidin-biotin-horseradish peroxidase complex
  • FIG. 7B Primary breast cancer sections stained for BRCAl by the immunoperoxidase method. Sections 5- ⁇ m-thick from randomly selected, formalin-fixed, paraffin-embedded, breast cancer biopsies in the inventors' tumor bank were immunostained by a modification of the avidin-biotin-horseradish peroxidase complex (ABC) method (Hsu et al, 1981). Anti-BRCAl was used at 1 :100 dilution. Both cases of invasive breast cancer showing no cytoplasmic or nuclear immunostaining for BRCAl did show positive immunostaining for the nuclear proliferation antigen MiBl . BRCAl localized only to cytoplasm.
  • ABSC avidin-biotin-horseradish peroxidase complex
  • FIG. 7C Primary breast cancer sections stained for BRCA 1 by the immunoperoxidase method. Sections 5- ⁇ m-thick from randomly selected, formalin-fixed, paraffin-embedded, breast cancer biopsies in the inventors' tumor bank were immunostained by a modification of the avidin-biotin-horseradish peroxidase complex (ABC) method (Hsu et al, 1981). Anti-BRCAl was used at 1 : 100 dilution.
  • ABSC avidin-biotin-horseradish peroxidase complex
  • FIG. 8 Shown are NLS Deletion-Mutant Constructs. Schematic showing the positions and sequences of the three putative NLS motifs in BRCAl together with the respective changes made in each by PCR-based mutagenesis. As shown, these constructs have been cloned, in-frame with the FLAG epitope, into the pCEP4 vector, which directs high-level expression of inserted cDNAs under the control of the CMV major-late promoter.
  • FIG. 9 Identification of a fraAw-activation domain in BRCA 1 using a yeast "one-hybrid" system.
  • the various fragments of BRCAl shown were fused in- frame to the GAL4 DNA-binding domain, expressed in the yeast vector pAS.
  • These constructs were then transfected into the yeast strain Y153, which harbors a GAL4 responsive b-galactosidase reporter gene b-galactosidase activity was determined either qualitatively by streaking transformants onto plates and doing a colony lift assay, or quantitatively by CPRG assay. These assays have been described by the inventors previously (Durfee , et al, 1993).
  • FIG. 10 Schematic showing the regions of BRCAl used as bait in the yeast two-hybrid screen. The putative Zn-finger, NLS motifs, and /raws-activation domain of
  • BRCAl are depicted on a schematic for the BRCAl cDNA. Below this are shown the positions of the two regions used as bait in the yeast two-hybrid screen. These regions were cloned in-frame with the Gal4 DNA-binding domain of the yeast expression vector pAS. Numbers above the bars represent positions of amino acids within the sequence.
  • kits comprising, in suitable container means, a BAP or BRCAl composition of the present invention in a pharmaceutically acceptable formulation represent another aspect of the invention.
  • the BAP or BRCAl composition may be native BAP or BRCAl, truncated BAP or BRCAl, site-specifically mutated BAP or
  • BRCAl, or BAP- or BRCAl -encoded peptide epitopes or alternatively antibodies which bind native BAP or BRCAl, truncated BAP or BRCAl , site-specifically mutated BAP or BRCAl, or BAP- or BRCAl -encoded peptide epitopes.
  • the BAP or BRCAl composition may be nucleic acid segments encoding native BAP or BRCAl, truncated BAP or BRCAl, site-specifically mutated BAP or BRCAl , or BAP- or
  • Such nucleic acid segments may be DNA or RNA, and may be either native, recombinant, or mutagenized nucleic acid segments.
  • kits may comprise a single container means that contains the BAP or BRCAl composition.
  • the container means may, if desired, contain a pharmaceutically acceptable sterile excipient, having associated with it, the BRCAl or BAP composition and, optionally, a detectable label or imaging agent.
  • the formulation may be in the form of a gelatinous composition, e.g., a coIlagenous-BRCAl or BAP composition, or may even be in a more fluid form that nonetheless forms a gel-like composition upon administration to the body.
  • the container means may itself be a syringe, pipette, or other such like apparatus, from which the BRCAl or BAP composition may be applied to a particular site.
  • the single container means may contain a dry, or lyophilized, mixture of a BRCAl or BAP composition, which may or may not require pre-wetting before use.
  • kits of the invention may comprise distinct container means for each component.
  • one container would contain the BAP or BRCAl composition, either as a sterile DNA solution or in a lyophilized form, and the other container would include the matrix, which may or may not itself be pre-wetted with a sterile solution, or be in a gelatinous, liquid or other syringeable form.
  • kits may also comprise a second or third container means for containing a sterile, pharmaceutically acceptable buffer, diluent or solvent.
  • a sterile, pharmaceutically acceptable buffer, diluent or solvent Such a solution may be required to formulate the BAP or BRCAl component into a more suitable form for application to the body, e.g., as a topical preparation, or alternatively, in oral, parenteral, or intravenous forms.
  • all components of a kit could be supplied in a dry form (lyophilized), which would allow for "wetting" upon contact with body fluids.
  • the presence of any type of pharmaceutically acceptable buffer or solvent is not a requirement for the kits of the invention.
  • the kits may also comprise a second or third container means for containing a pharmaceutically acceptable detectable imaging agent or composition.
  • the container means will generally be a container such as a vial, test tube, flask, bottle, syringe or other container means, into which the components of the kit may placed.
  • the matrix and gene components may also be aliquoted into smaller containers, should this be desired.
  • the kits of the present invention may also include a means for containing the individual containers in close confinement for commercial sale, such as, e.g., injection or blow-molded plastic containers into which the desired vials or syringes are retained. Irrespective of the number of containers, the kits of the invention may also comprise, or be packaged with, an instrument for assisting with the placement of the ultimate matrix-gene composition within the body of an animal. Such an instrument may be a syringe, pipette, forceps, or any such medically approved delivery vehicle.
  • Affinity chromatography is generally based on the recognition of a protein by a substance such as a ligand or an antibody.
  • the column material may be synthesized by covalently coupling a binding molecule, such as an activated dye, for example to an insoluble matrix.
  • the column material is then allowed to adsorb the desired substance from solution. Next, the conditions are changed to those under which binding does not occur and the substrate is eluted.
  • the requirements for successful affinity chromatography are:
  • BRCAl or alternatively, peptide epitopes derived from either BAP or BRCAl, covalently-coupled to a suitable matrix such as e.g., Sepharose CL6B or CL4B.
  • a suitable matrix such as Sepharose CL6B or CL4B.
  • This matrix binds the antibodies of the present invention directly and allows their separation by elution with an appropriate gradient such as salt, GuHCl, pH, or urea.
  • Another preferred embodiment of the present invention is an affinity chromatography method for the purification of BAP, BRCAl, or related peptide epitopes from solution.
  • the matrix binds the amino acid compositions of the present invention directly, and allows their separation by elution with a suitable buffer as described above.
  • nucleic acid segments disclosed herein will be used to transfect appropriate host cells.
  • Technology for introduction of DNA into cells is well-known to those of skill in the art.
  • Four general methods for delivering a nucleic segment into cells have been described: ( 1 ) chemical methods (Graham and Van der Eb, 1973);
  • the inventors contemplate the use of liposomes and/or nanocapsules for the introduction of particular peptides or nucleic acid segments into host cells. Such formulations may be preferred for the introduction of pharmaceutically- acceptable formulations of the nucleic acids, peptides, and/or antibodies disclosed herein.
  • liposomes The formation and use of liposomes is generally known to those of skill in the art (see for example, Couvreur et al, 1977 which describes the use of liposomes and nanocapsules in the targeted antibiotic therapy of intracellular bacterial infections and diseases). Recently, liposomes were developed with improved serum stability and circulation half- times (Gabizon and Papahadjopoulos, 1988; Allen and Choun, 1987).
  • Nanocapsules can generally entrap compounds in a stable and reproducible way (Henry-Michelland et al, 1987). To avoid side effects due to intracellular polymeric overloading, such ultrafine particles (sized around 0.1 ⁇ m) should be designed using polymers able to be degraded in vivo. Biodegradable polyalkyl-cyanoacrylate nanoparticles that meet these requirements are contemplated for use in the present invention, and such particles may be are easily made, as described (Couvreur et al, 1977; 1988).
  • Liposomes are formed from phospholipids that are dispersed in an aqueous medium and spontaneously form multilamellar concentric bilayer vesicles (also termed multilamellar vesicles (MLVs).
  • MLVs generally have diameters of from 25 nm to 4 ⁇ m.
  • Sonication of MLVs results in the formation of small unilamellar vesicles (SUVs) with diameters in the range of 200 to 500 A, containing an aqueous solution in the core.
  • SUVs small unilamellar vesicles
  • Phospholipids can form a variety of structures other than liposomes when dispersed in water, depending on the molar ratio of lipid to water. At low ratios the liposome is the preferred structure.
  • the physical characteristics of liposomes depend on pH, ionic strength and the presence of divalent cations. Liposomes can show low permeability to ionic and polar substances, but at elevated temperatures undergo a phase transition which markedly alters their permeability. The phase transition involves a change from a closely packed, ordered structure, known as the gel state, to a loosely packed, less-ordered structure, known as the fluid state. This occurs at a characteristic phase-transition temperature and results in an increase in permeability to ions, sugars and drugs.
  • Liposomes interact with cells via four different mechanisms: Endocytosis by phagocytic cells of the reticuloendothelial system such as macrophages and neutrophils; adsorption to the cell surface, either by nonspecific weak hydrophobic or electrostatic forces, or by specific interactions with cell-surface components; fusion with the plasma cell membrane by insertion of the lipid bilayer of the liposome into the plasma membrane, with simultaneous release of liposomal contents into the cytoplasm; and by transfer of liposomal lipids to cellular or subcellular membranes, or vice versa, without any association of the liposome contents. It often is difficult to determine which mechanism is operative and more than one may operate at the same time.
  • the present invention contemplates an antibody that is immunoreactive with a polypeptide of the invention.
  • one of the uses for BRCAl and BRCAl -derived epitopic peptides or BAP and BAP-derived epitopic peptides according to the present invention is to generate antibodies.
  • Reference to antibodies throughout the specification includes whole polyclonal and monoclonal antibodies (mAbs), and parts thereof, either alone or conjugated with other moieties.
  • Antibody parts include Fab and F(ab) 2 fragments and single chain antibodies.
  • the antibodies may be made in vivo in suitable laboratory animals or in vitro using recombinant DNA techniques.
  • an antibody is a polyclonal antibody.
  • a polyclonal antibody is prepared by immunizing an animal with an immunogen comprising a polypeptide of the present invention and collecting antisera from that immunized animal.
  • an immunogen comprising a polypeptide of the present invention
  • a wide range of animal species can be used for the production of antisera.
  • an animal used for production of anti-antisera is a rabbit, a mouse, a rat, a hamster or a guinea pig.
  • Antibodies both polyclonal and monoclonal, specific for BRCAl and BRCAl -derived epitopes, or alternatively, BAP and BAP-derived epitopes, may be prepared using conventional immunization techniques, as will be generally known to those of skill in the art.
  • a composition containing antigenic epitopes of the particular BRCA Is and BAPs disclosed herein can be used to immunize one or more experimental animals, such as a rabbit or mouse, which will then proceed to produce specific antibodies against BAP or BRCAl peptides.
  • Polyclonal antisera may be obtained, after allowing time for antibody generation, simply by bleeding the animal and preparing serum samples from the whole blood.
  • the amount of immunogen composition used in the production of polyclonal antibodies varies upon the nature of the immunogen, as well as the animal used for immunization.
  • routes can be used to administer the immunogen (subcutaneous, intramuscular, intradermal, intravenous and intraperitoneal).
  • the production of polyclonal antibodies may be monitored by sampling blood of the immunized animal at various points following immunization. A second, booster injection, also may be given. The process of boosting and titering is repeated until a suitable titer is achieved.
  • the immunized animal can be bled and the serum isolated and stored, and/or the animal can be used to generate mAbs (below).
  • mAbs a polyclonal sera that is relatively homogenous with respect to the specificity of the antibodies therein.
  • polyclonal antisera is derived from a variety of different "clones,” i.e., B-cells of different lineage.
  • mAbs by contrast, are defined as coming from antibody-producing cells with a common B-cell ancestor, hence their "mono" clonality.
  • polyclonal antisera When peptides are used as antigens to raise polyclonal sera, one would expect considerably less variation in the clonal nature of the sera than if a whole antigen were employed. Unfortunately, if incomplete fragments of an epitope are presented, the peptide may very well assume multiple (and probably non-native) conformations. As a result, even short peptides can produce polyclonal antisera with relatively plural specificities and, unfortunately, an antisera that does not react or reacts poorly with the native molecule. Polyclonal antisera according to present invention is produced against peptides that are predicted to comprise whole, intact epitopes. It is believed that these epitopes are.
  • the present invention provides for polyclonal antisera where the clonality, i.e., the percentage of clone reacting with the same molecular determinant, is at least 80%. Even higher clonality - 90%, 95% or greater - is contemplated.
  • the clonality i.e., the percentage of clone reacting with the same molecular determinant, is at least 80%. Even higher clonality - 90%, 95% or greater - is contemplated.
  • an experimental animal often preferably a mouse, with a BRCAl -containing composition.
  • the spleen or lymph cells can then be fused with cell lines, such as human or mouse myeloma strains, to produce antibody-secreting hybridomas. These hybridomas may be isolated to obtain individual clones which can then be screened for production of antibody to the desired peptide.
  • Hybridomas which produce mAbs to the selected antigens are identified using standard techniques, such as ELISA and Western blot methods.
  • Hybridoma clones can then be cultured in liquid media and the culture supernatants purified to provide the BAP- or BRCAl -specific mAbs.
  • the mAbs of the present invention will also find useful application in immunochemical procedures, such as ELISA and Western blot methods, as well as other procedures such as immunoprecipitation, immunocytological methods, etc. which may utilize antibodies specific to BAPs or BRCAl .
  • BAP or BRCAl antibodies may be used in immunoabsorbent protocols to purify native or recombinant BAPs, BRCAl or BAP- or BRCAl -derived peptide species or synthetic or natural variants thereof.
  • the antibodies disclosed herein may be employed in antibody cloning protocols to obtain cDNAs or genes encoding BAPs or BRCAl s from other species or organisms, or to identify proteins having significant homology to BAP or BRCAl .
  • Anti-BRCAl or anti-BAP antibodies will also be useful in immunolocalization studies to analyze the distribution of BRCAl or BAP protein under different physiological conditions.
  • a particularly useful application of such antibodies is in purifying native or recombinant BAPs or BRCAl, for example, using an antibody affinity column. The operation of all such immunological techniques will be known to those of skill in the art in light of the present disclosure.
  • Recombinant clones expressing the BAP or BRCAl nucleic acid segments may be used to prepare purified recombinant BRCAl (rBRCAl), purified rBRCAl -derived peptide antigens or, alternatively, purified recombinant BAP (rBAP), purified rBAP- derived peptide antigens, as well as mutant or variant recombinant protein species in significant quantities.
  • the selected antigens, and variants thereof, are proposed to have significant utility in diagnosing and treating breast cancers.
  • rBAPs, rBRCAls, peptide variants thereof, and/or antibodies against such rBAPs or rBRCAls may also be used in immunoassays to detect localization of BAP or BRCAl in vivo or as vaccines or immunotherapeutics to treat breast cancers.
  • the present invention allows the ready preparation of so-called "second generation" molecules having modified or simplified protein structures. Second generation proteins will typically share one or more properties in common with the full-length antigen, such as a particular antigenic/immunogenic epitopic core sequence. Epitopic sequences can be provided on relatively short molecules prepared from knowledge of the peptide, or encoding DNA sequence information.
  • Such variant molecules may not only be derived from selected immunogenic/ antigenic regions of the protein structure, but may additionally, or alternatively, include one or more functionally equivalent amino acids selected on the basis of similarities or even differences with respect to the natural sequence. 4.7 ANTIBODY COMPOSITIONS AND FORMULATIONS THEREOF
  • a polyclonal antibody is prepared by immunizing an animal with an immunogenic composition in accordance with the present invention and collecting antisera from that immunized animal.
  • an immunogenic composition in accordance with the present invention
  • a wide range of animal species can be used for the production of antisera.
  • the animal used for production of anti-antisera is a rabbit, a mouse, a rat, a hamster, a guinea pig or a goat. Because of the relatively large blood volume of rabbits, a rabbit is a preferred choice for production of polyclonal antibodies.
  • a given composition may vary in its immunogenicity. It is often necessary therefore to boost the host immune system, as may be achieved by coupling a peptide or polypeptide immunogen to a carrier.
  • exemplary and preferred carriers are keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albumins such as ovalbumin, mouse serum albumin or rabbit serum albumin can also be used as carriers.
  • KLH keyhole limpet hemocyanin
  • BSA bovine serum albumin
  • Other albumins such as ovalbumin, mouse serum albumin or rabbit serum albumin can also be used as carriers.
  • Means for conjugating a polypeptide to a carrier protein are well known in the art and include glutaraldehyde, m-maleimidobenzoyl-/V-hydroxysuccinimide ester, carbodiimide and bis-biazotized benzidine.
  • mAbs may be readily prepared through use of well-known techniques, such as those exemplified in U. S. Patent 4,196,265, inco ⁇ orated herein by reference.
  • this technique involves immunizing a suitable animal with a selected immunogen composition, e.g., a purified or partially purified protein, polypeptide or peptide.
  • the immunizing composition is administered in a manner effective to stimulate antibody producing cells.
  • Rodents such as mice and rats are preferred animals, however, the use of rabbit, sheep or frog cells is also possible.
  • the use of rats may provide certain advantages (Goding, 1986), but mice are preferred, with the B ALB/c mouse being most preferred as this is most routinely used and generally gives a higher percentage of stable fusions.
  • somatic cells with the potential for producing antibodies, specifically B-lymphocytes (B-cells), are selected for use in the mAb generating protocol.
  • B-cells B-lymphocytes
  • These cells may be obtained from biopsied spleens, tonsils or lymph nodes, or from a peripheral blood sample. Spleen cells and peripheral blood cells are preferred, the former because they are a rich source of antibody-producing cells that are in the dividing plasmablast stage, and the latter because peripheral blood is easily accessible.
  • a panel of animals will have been immunized and the spleen of animal with the highest antibody titer will be removed and the spleen lymphocytes obtained by homogenizing the spleen with a syringe.
  • 7 ft contains approximately about 5 x 10 to about 2 x 10 lymphocytes.
  • the antibody-producing B lymphocytes from the immunized animal are then fused with cells of an immortal myeloma cell, generally one of the same species as the animal that was immunized.
  • Myeloma cell lines suited for use in hybridoma-producing fusion procedures preferably are non-antibody-producing, have high fusion efficiency, and enzyme deficiencies that render them incapable of growing in certain selective media which support the growth of only the desired fused cells (hybridomas). Any one of a number of myeloma cells may be used, as are known to those of skill in the art (Goding, 1986; Campbell, 1984).
  • the immunized animal is a mouse
  • rats one may use R210.RCY3, Y3-Ag 1.2.3, IR983F and 4B210; and U-266, GM1500-GRG2, LICR-LON-HMy2 and UC729-6 are all useful in connection with human cell fusions.
  • NS-1 myeloma cell line also termed P3-NS-l-Ag4-l
  • P3-NS-l-Ag4-l NS-1 myeloma cell line
  • Another mouse myeloma cell line that may be used is the 8-azaguanine-resistant mouse murine myeloma
  • Methods for generating hybrids of antibody-producing spleen or lymph node cells and myeloma cells usually comprise mixing somatic cells with myeloma cells in a 2: 1 ratio, though the ratio may vary from about 20: 1 to about 1 :1 , respectively, in the presence of an agent or agents (chemical or electrical) that promote the fusion of cell membranes.
  • Fusion methods using Sendai virus have been described (Kohler and Milstein, 1975; 1976), and those using polyethylene glycol (PEG), such as 37% (v/v) PEG, by Gefter et al. (1977).
  • PEG polyethylene glycol
  • the use of electrically induced fusion methods is also appropriate (Goding, 1986).
  • Fusion procedures usually produce viable hybrids at low frequencies, about 1 x 10 " to about 1 x 10 ' . However, this does not pose a problem, as the viable, fused hybrids are differentiated from the parental, unfused cells (particularly the unfused myeloma cells that would normally continue to divide indefinitely) by culturing in a selective medium.
  • the selective medium is generally one that contains an agent that blocks the de novo synthesis of nucleotides in the tissue culture media.
  • Exemplary and preferred agents are aminopterin, methotrexate, and azaserine. Aminopterin and methotrexate block de novo synthesis of both purines and pyrimidines, whereas azaserine blocks only purine synthesis.
  • the media is supplemented with hypoxanthine and thymidine as a source of nucleotides (HAT medium).
  • HAT medium a source of nucleotides
  • azaserine the media is supplemented with hypoxanthine.
  • the preferred selection medium is HAT. Only cells capable of operating nucleotide salvage pathways are able to survive in HAT medium. The myeloma cells are defective in key enzymes of the salvage pathway, e.g., hypoxanthine phosphoribosyl transferase (HPRT), and they cannot survive. The B-cells can operate this pathway, but they have a limited life span in culture and generally die within about two weeks. Therefore, the only cells that can survive in the selective media are those hybrids formed from myeloma and B-cells.
  • HPRT hypoxanthine phosphoribosyl transferase
  • This culturing provides a population of hybridomas from which specific hybridomas are selected.
  • selection of hybridomas is performed by culturing the cells by single-clone dilution in microtiter plates, followed by testing the individual clonal supernatants (after about two to three weeks) for the desired reactivity.
  • the assay should be sensitive, simple and rapid, such as radioimmunoassays, enzyme immunoassays, cytotoxicity assays, plaque assays, dot immunobinding assays, and the like.
  • the selected hybridomas would then be serially diluted and cloned into individual antibody-producing cell lines, which clones can then be propagated indefinitely to provide mAbs.
  • the cell lines may be exploited for mAb production in two basic ways.
  • a sample of the hybridoma can be injected (often into the peritoneal cavity) into a histocompatible animal of the type that was used to provide the somatic and myeloma cells for the original fusion.
  • the injected animal develops tumors secreting the specific mAb produced by the fused cell hybrid.
  • the body fluids of the animal such as serum or ascites fluid, can then be tapped to provide mAbs in high concentration.
  • the individual cell lines could also be cultured in vitro, where the mAbs are naturally secreted into the culture medium from which they can be readily obtained in high concentrations.
  • mAbs produced by either means may be further purified, if desired, using filtration, centrifugation and various chromatographic methods such as HPLC or affinity chromatography.
  • native and synthetically-derived peptides and peptide epitopes of the invention will find utility as immunogens, e.g., in connection with vaccine development, or as antigens in immunoassays for the detection of reactive antibodies.
  • preferred immunoassays of the invention include the various types of enzyme linked immunosorbent assays (ELISAs), as are known to those of skill in the art.
  • ELISAs enzyme linked immunosorbent assays
  • BRCAl -derived proteins and peptides is not limited to such assays, and that other useful embodiments include RIAs and other non- enzyme linked antibody binding assays and procedures.
  • BRCAl, rBRCAl, or BAP or BRCAl -derived protein antigen sequences are immobilized onto a selected surface, preferably a surface exhibiting a protein affinity.
  • a selected surface preferably a surface exhibiting a protein affinity.
  • a nonspecific protein that is known to be antigenically neutral with regard to the test antisera, such as bovine serum albumin (BSA) or casein, onto the well.
  • BSA bovine serum albumin
  • the immobilizing surface is contacted with the antisera or clinical or biological extract to be tested in a manner conducive to immune complex (antigen/antibody) formation.
  • Such conditions preferably include diluting the antisera with diluents such as BSA, bovine gamma globulin (BGG) and phosphate buffered saline (PBS)/Tween . These added agents also tend to assist in the reduction of nonspecific background.
  • the layered antisera is then allowed to incubate for, e.g., from 2 to 4 hours, at temperatures preferably on the order of about 25° to about 27°C. Following incubation, the antisera-contacted surface is washed so as to remove non-immunocomplexed material.
  • a preferred washing procedure includes washing with a solution such as PBS/Tween , or borate buffer.
  • the occurrence and the amount of immunocomplex formation may be determined by subjecting the complex to a second antibody having specificity for the first.
  • the second antibody will preferably be an antibody having specificity for human antibodies.
  • the second antibody will preferably have an associated detectable label, such as an enzyme label, that will generate a signal, such as color development upon incubating with an appropriate chromogenic substrate.
  • a urease or peroxidase-conjugated anti -human IgG for a period of time and under conditions that favor the development of immunocomplex formation (e.g.. incubation for 2 hours at room temperature in a PBS-containing solution such as PBS-Tween ® ).
  • the amount of label is quantified by incubation with a chromogenic substrate such as urea and bromocresol purple or 2,2'-azino-di-(3- ethyl-benzthiazoline)-6-sulfonic acid (ABTS) and H 2 O 2 , in the case of peroxidase as the enzyme label. Quantitation is then achieved by measuring the degree of color generation, e.g.. using a visible spectrum spectrophotometer. ELISAs may be used in conjunction with the invention.
  • proteins or peptides incorporating antigenic sequences of the present invention are immobilized onto a selected surface, preferably a surface exhibiting a protein affinity such as the wells of a polystyrene microtiter plate. After washing to remove incompletely adsorbed material, it is desirable to bind or coat the assay plate wells with a nonspecific protein that is known to be antigenically neutral with regard to the test antisera such as bovine serum albumin (BSA), casein or solutions of powdered milk.
  • BSA bovine serum albumin
  • the anti-BRCAl and anti-BAP antibodies of the present invention are particularly useful for the isolation of BRCAl and BAP antigens by immunoprecipitation. Immunoprecipitation involves the separation of the target antigen component from a complex mixture, and is used to discriminate or isolate minute amounts of protein. In an alternative embodiment the antibodies of the present invention are useful for the close juxtaposition of two antigens. This is particularly useful for increasing the localized concentration of antigens, e.g., enzyme-substrate pairs.
  • compositions of the present invention will find great use in immunoblot or western blot analysis.
  • the anti-BRCAl and anti-BAP antibodies may be used as high- affinity primary reagents for the identification of proteins immobilized onto a solid support matrix, such as nitrocellulose, nylon or combinations thereof.
  • a solid support matrix such as nitrocellulose, nylon or combinations thereof.
  • immunoprecipitation followed by gel electrophoresis, these may be used as a single step reagent for use in detecting antigens against which secondary reagents used in the detection of the antigen cause an adverse background.
  • the antigens studied are immunoglobulins (precluding the use of immunoglobulins binding bacterial cell wall components), the antigens studied cross-react with the detecting agent, or they migrate at the same relative molecular weight as a cross-reacting signal.
  • lmmunologically-based detection methods in conjunction with Western blotting are considered to be of particular use in this regard.
  • the present invention contemplates vaccines for use in both active and passive immunization embodiments.
  • Immunogenic compositions proposed to be suitable for use as a vaccine may be prepared most readily directly from the novel immunogenic proteins and/or peptide epitopes described herein.
  • the antigenic material is extensively dialyzed to remove undesired small molecular weight molecules and/or lyophilized for more ready formulation into a desired vehicle.
  • vaccines that contain peptide sequences as active ingredients
  • U. S. Patents 4,608,251 ; 4,601,903; 4,599,231 ; 4,599,230; 4,596,792; and 4,578,770 all inco ⁇ orated herein by reference.
  • such vaccines are prepared as injectables, either as liquid solutions or suspensions, solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared.
  • the preparation may also be emulsified.
  • the active immunogenic ingredient is often mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredient.
  • Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof.
  • the vaccine may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, or adjuvants that enhance the effectiveness of the vaccines.
  • a composition comprising BAP, BRCAl or BRCAl -derived proteins and/or native or modified epitopic peptides therefrom could also be the basis for human vaccines.
  • the preparation of such compositions that are essentially free from endotoxin can be achieved by following the published methodology, for example, U. S. Patent 4,271.147 (inco ⁇ orated herein by reference) discloses methods for the preparation of Neisseria meningitidis membrane proteins for use in vaccines.
  • BAP, BRCAl, BRCAl -derived and BAP-derived epitope-based vaccines may be conventionally administered parenterally, by injection, for example, either subcutaneously or intramuscularly. Additional formulations that are suitable for other modes of administration include suppositories and, in some cases, oral formulations.
  • suppositories traditional binders and carriers may include, for example, polyalkalene glycols or triglycerides: such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably 1-2%.
  • Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain 10-95% of active ingredient, preferably 25-70%.
  • the proteins may be formulated into the vaccine as neutral or salt forms.
  • Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the peptide) and those that are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.
  • inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like.
  • Salts formed with the free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such
  • the vaccines may be administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective and immunogenic.
  • the quantity to be administered depends on the subject to be treated, including, e.g., the capacity of the individual's immune system to synthesize antibodies, and the degree of protection desired. Precise amounts of active ingredient required to be administered will be readily determinable by the skilled practitioner. However, suitable dosage ranges are of the order of several hundred micrograms active ingredient per vaccination. Suitable regimes for initial administration and booster shots are also variable, but are typified by an initial administration followed by subsequent inoculations or other administrations.
  • Any of the conventional methods for administration of a vaccine are applicable. These are believed to include oral application on a solid physiologically acceptable base or in a physiologically acceptable dispersion, parenterally, by injection or the like.
  • the dosage of the vaccine will depend on the route of administration and will vary according to the size of the host.
  • Various methods of achieving adjuvant effect for the vaccine includes use of agents such as aluminum hydroxide or phosphate (alum), commonly used as 0.05 to 0.1 percent solution in phosphate buffered saline, admixture with synthetic polymers of sugars (Carbopol ) used as 0.25% solution, aggregation of the protein in the vaccine by heat treatment with temperatures ranging between about 70° and about 101 °C for 30 second to 2 minute periods respectively. Aggregation by reactivating with pepsin treated F(ab) antibodies to albumin, mixture with bacterial ceils such as C.
  • agents such as aluminum hydroxide or phosphate (alum), commonly used as 0.05 to 0.1 percent solution in phosphate buffered saline, admixture with synthetic polymers of sugars (Carbopol ) used as 0.25% solution, aggregation of the protein in the vaccine by heat treatment with temperatures ranging between about 70° and about 101 °C for 30 second to 2 minute periods respectively. Aggregation by re
  • parvum or endotoxins or lipopolysaccharide components of gram-negative bacteria emulsion in physiologically acceptable oil vehicles such as mannide monooleate (Aracel-ATM) or emulsion with 20 percent solution of a perfluorocarbon (Fluosol-DATM) used as a block substitute may also be employed.
  • physiologically acceptable oil vehicles such as mannide monooleate (Aracel-ATM) or emulsion with 20 percent solution of a perfluorocarbon (Fluosol-DATM) used as a block substitute may also be employed.
  • the vaccine will be desirable to have multiple administrations of the vaccine, usually not exceeding six vaccinations, more usually not exceeding four vaccinations and preferably one or more, usually at least about three vaccinations.
  • the vaccinations will normally be at from two to twelve week intervals, more usually from three to five week intervals. Periodic boosters at intervals of 1-5 years, usually three years, will be desirable to maintain protective levels of the antibodies.
  • the course of the immunization may be followed by assays for antibodies for the supernatant antigens.
  • the assays may be performed by labeling with conventional labels, such as radionuclides, enzymes, fluorescers, and the like. These techniques are well known and may be found in a wide variety of patents, such as U. S. Patent Nos. 3,791,932; 4,174,384 and 3,949,064, as illustrative of these types of assays.
  • compositions disclosed herein may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or they may be enclosed in hard or soft shell gelatin capsule, or they may be compressed into tablets, or they may be inco ⁇ orated directly with the food of the diet.
  • the active compounds may be inco ⁇ orated with excipients and used in the form of ingestible tablets, buccal tables, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.
  • Such compositions and preparations should contain at least 0.1% of active compound.
  • the percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 2 to about 60% of the weight of the unit.
  • the amount of active compounds in such therapeutically useful compositions is such that a suitable dosage will be obtained.
  • the tablets, troches, pills, capsules and the like may also contain the following: a binder, as gum tragacanth, acacia, cornstarch, or gelatin; excipients, such as dicalcium phosphate; a disintegrating agent, such as corn starch, potato starch, alginic acid and the like; a lubricant, such as magnesium stearate; and a sweetening agent, such as sucrose, lactose or saccharin may be added or a flavoring agent, such as peppermint, oil of wintergreen, or cherry flavoring.
  • a binder as gum tragacanth, acacia, cornstarch, or gelatin
  • excipients such as dicalcium phosphate
  • a disintegrating agent such as corn starch, potato starch, alginic acid and the like
  • a lubricant such as magnesium stearate
  • a sweetening agent such as sucrose, lactose or saccharin may be added or a flavor
  • any material may be present as coatings or to otherwise modify the physical form of the dosage unit.
  • tablets, pills, or capsules may be coated with shellac, sugar or both.
  • a syrup of elixir may contain the active compounds sucrose as a sweetening agent methyl and propylparabens as preservatives, a dye and flavoring, such as cherry or orange flavor.
  • any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed.
  • the active compounds may be inco ⁇ orated into sustained-release preparation and formulations.
  • the active compounds may also be administered parenterally or intraperitoneally.
  • Solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose.
  • Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
  • the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • a coating such as lecithin
  • surfactants for example, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium bicarbonate, sodium sorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars or sodium chloride.
  • Prolonged abso ⁇ tion of the injectable compositions can be brought about by the use in the compositions of agents delaying abso ⁇ tion, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions are prepared by inco ⁇ orating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by inco ⁇ orating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum-drying and freeze- drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and abso ⁇ tion delaying agents and the like.
  • the use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated.
  • Supplementary active ingredients can also be inco ⁇ orated into the compositions.
  • the polypeptide may be inco ⁇ orated with excipients and used in the form of non-ingestible mouthwashes and dentifrices.
  • a mouthwash may be prepared inco ⁇ orating the active ingredient in the required amount in an appropriate solvent, such as a sodium borate solution (Dobell's Solution).
  • the active ingredient may be inco ⁇ orated into an antiseptic wash containing sodium borate. glycerin and potassium bicarbonate.
  • the active ingredient may also be dispersed in dentifrices, including: gels, pastes, powders and slurries.
  • the active ingredient may be added in a therapeutically effective amount to a paste dentifrice that may include water, binders, abrasives, flavoring agents, foaming agents, and humectants.
  • compositions that do not produce an allergic or similar untoward reaction when administered to a human.
  • pharmaceutically-acceptable refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a human.
  • aqueous composition that contains a protein as an active ingredient is well understood in the art.
  • injectables either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection can also be prepared.
  • the preparation can also be emulsified.
  • composition can be formulated in a neutral or salt form.
  • Pharmaceutically-acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic. tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine. histidine, procaine and the like. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.
  • the formulations are easily administered in a variety of dosage forms such as injectable solutions, drug release capsules and the like.
  • parenteral administration in an aqueous solution for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose.
  • aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration.
  • sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure.
  • one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences” 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biologies standards.
  • the present invention is also directed to protein or peptide compositions, free from total cells and other peptides, which comprise a purified protein or peptide which inco ⁇ orates an epitope that is immunologically cross-reactive with one or more of the antibodies of the present invention.
  • the term "inco ⁇ orating an epitope(s) that is immunologically cross-reactive with one or more anti-BRCAl antibodies” is intended to refer to a peptide or protein antigen which includes a primary, secondary or tertiary structure similar to an epitope located within a BAP or BRCAl polypeptide.
  • the level of similarity will generally be to such a degree that monoclonal or polyclonal antibodies directed against the BAP or BRCAl polypeptide will also bind to, react with, or otherwise recognize, the cross-reactive peptide or protein antigen.
  • BAP or BRCAl epitopes such as those derived from BAP or
  • BRCAl or --3/?C4-/-like gene products and/or their functional equivalents, suitable for use in vaccines is a relatively straightforward matter.
  • Hopp as taught in U.S. Patent 4,554,101 , inco ⁇ orated herein by reference, which teaches the identification and preparation of epitopes from amino acid sequences on the basis of hydrophilicity.
  • the methods described in several other papers, and software programs based thereon, can also be used to identify epitopic core sequences
  • amino acid sequence of these "epitopic core sequences" may then be readily inco ⁇ orated into peptides, either through the application of peptide synthesis or recombinant technology.
  • Preferred peptides for use in accordance with the present invention will generally be on the order of about 5 to about 25 amino acids in length, and more preferably about 8 to about 20 amino acids in length. It is proposed that shorter antigenic peptide sequences will provide advantages in certain circumstances, for example, in the preparation of vaccines or in immunologic detection assays. Exemplary advantages include the ease of preparation and purification, the relatively low cost and improved reproducibility of production, and advantageous biodistribution.
  • An epitopic core sequence is a relatively short stretch of amino acids that is "complementary" to, and therefore will bind, antigen binding sites on BAP or BRCAl epitope-specific antibodies. Additionally or alternatively, an epitopic core sequence is one that will elicit antibodies that are cross-reactive with antibodies directed against the peptide compositions of the present invention. It will be understood that in the context of the present disclosure, the term “complementary” refers to amino acids or peptides that exhibit an attractive force towards each other. Thus, certain epitope core sequences of the present invention may be operationally defined in terms of their ability to compete with or perhaps displace the binding of the desired protein antigen with the corresponding protein-directed antisera.
  • the size of the polypeptide antigen is not believed to be particularly crucial, so long as it is at least large enough to carry the identified core sequence or sequences.
  • the smallest useful core sequence expected by the present disclosure would generally be on the order of about 5 amino acids in length, with sequences on the order of 8 or 25 being more preferred.
  • this size will generally correspond to the smallest peptide antigens prepared in accordance with the invention.
  • the size of the antigen may be larger where desired, so long as it contains a basic epitopic core sequence.
  • epitopic core sequences are known to those of skill in the art, for example, as described in U.S. Patent 4,554,101 , inco ⁇ orated herein by reference, which teaches the identification and preparation of epitopes from amino acid sequences on the basis of hydrophilicity.
  • numerous computer programs are available for use in predicting antigenic portions of proteins (see e.g., Jameson and Wolf, 1988; Wolf et al , 1988).
  • Computerized peptide sequence analysis programs e.g., DNA Star® software, DNAStar, Inc., Madison, WI
  • the peptides provided by this invention are ideal targets for use as vaccines or immunoreagents for the detection of BAP, BRCAl and BAP- or BRCAl -encoding genes, or alternatively the detection of either BAP, BRCAl or BRCAl -like gene product(s).
  • particular advantages may be realized through the preparation of synthetic peptides that include epitopic/immunogenic core sequences. These epitopic core sequences may be identified as hydrophilic and/or mobile regions of the polypeptides or those that include a T cell motif. It is known in the art that such regions represent those that are most likely to promote B cell or T cell stimulation, and, hence, elicit specific antibody production.
  • a protein or peptide is immunologically cross-reactive with, or a biological functional equivalent of, one or more epitopes of the disclosed peptides is also a straightforward matter.
  • This can be readily determined using specific assays, e.g., of a single proposed epitopic sequence, or using more general screens, e.g., of a pool of randomly generated synthetic peptides or protein fragments.
  • the screening assays may be employed to identify either equivalent antigens or cross-reactive antibodies.
  • the principle is the same, i.e., based upon competition for binding sites between antibodies and antigens.
  • Suitable competition assays include protocols based upon immunohistochemical assays, ELISAs, RIAs, Western or dot blotting and the like.
  • one of the binding components generally the known element, such as the BRCAl -derived peptide, or a known antibody, will be labeled with a detectable label and the test components, that generally remain unlabeled, will be tested for their ability to reduce the amount of label that is bound to the corresponding reactive antibody or antigen.
  • BAP or BRCAl to conduct a competition study between a BAP or a BRCAl and any test antigen
  • a detectable label such as, e.g., biotin or an enzymatic, radioactive or fluorogenic label
  • the known antibody would be immobilized, e.g., by attaching to an ELISA plate.
  • the ability of the mixture to bind to the antibody would be determined by detecting the presence of the specifically bound label. This value would then be compared to a control value in which no potentially competing (test) antigen was included in the incubation.
  • the assay may be any one of a range of immunological assays based upon hybridization, and the reactive antigens would be detected by means of detecting their label, e.g., using streptavidin in the case of biotinylated antigens or by using a chromogenic substrate in connection with an enzymatic label or by simply detecting a radioactive or fluorescent label.
  • An antigen that binds to the same antibody as BRCAl, for example, will be able to effectively compete for binding to and thus will significantly reduce BRCAl binding, as evidenced by a reduction in the amount of label detected.
  • the reactivity of the labeled antigen, e.g., a BAP or BRCAl composition, in the absence of any test antigen would be the control high value.
  • the control low value would be obtained by incubating the labeled antigen with an excess of unlabeled BAP or BRCA1 antigen, when competition would occur and reduce binding.
  • a significant reduction in labeled antigen reactivity in the presence of a test antigen is indicative of a test antigen that is "cross-reactive", i.e., that has binding affinity for the same antibody.
  • "A significant reduction" in terms of the present application may be defined as a reproducible (i.e., consistently observed) reduction in binding.
  • peptidyl compounds described herein may be formulated to mimic the key portions of the peptide structure.
  • Such compounds which may be termed peptidomimetics, may be used in the same manner as the peptides of the invention and hence are also functional equivalents.
  • the generation of a structural functional equivalent may be achieved by the techniques of modeling and chemical design known to those of skill in the art. It will be understood that all such sterically similar constructs fall within the scope of the present invention.
  • Syntheses of epitopic sequences, or peptides which include an antigenic epitope within their sequence are readily achieved using conventional synthetic techniques such as the solid phase method (e.g., through the use of a commercially-available peptide synthesizer such as an Applied Biosystems Model 430A Peptide Synthesizer). Peptide antigens synthesized in this manner may then be aliquoted in predetermined amounts and stored in conventional manners, such as in aqueous solutions or, even more preferably, in a powder or lyophilized state pending use.
  • peptides may be readily stored in aqueous solutions for fairly long periods of time if desired, e.g., up to six months or more, in virtually any aqueous solution without appreciable degradation or loss of antigenic activity.
  • agents including buffers such as Tris or phosphate buffers to maintain a pH of about 7.0 to about 7.5.
  • agents which will inhibit microbial growth such as sodium azide or Merthiolate.
  • the peptides are stored in a lyophilized or powdered state, they may be stored virtually indefinitely, e.g. , in metered aliquots that may be rehydrated with a predetermined amount of water (preferably distilled) or buffer prior to use.
  • Site-specific mutagenesis is a technique useful in the preparation of individual peptides, or biologically functional equivalent proteins or peptides, through specific mutagenesis of the underlying DNA.
  • the technique well-known to those of skill in the art, further provides a ready ability to prepare and test sequence variants, for example, inco ⁇ orating one or more of the foregoing considerations, by introducing one or more nucleotide sequence changes into the DNA.
  • Site-specific mutagenesis allows the production of mutants through the use of specific oligonucleotide sequences which encode the DNA sequence of the desired mutation, as well as a sufficient number of adjacent nucleotides, to provide a primer sequence of sufficient size and sequence complexity to form a stable duplex on both sides of the deletion junction being traversed.
  • a primer of about 14 to about 25 nucleotides in length is preferred, with about
  • the technique of site-specific mutagenesis is well known in the art, as exemplified by various publications.
  • the technique typically employs a phage vector which exists in both a single stranded and double stranded form.
  • Typical vectors useful in site-directed mutagenesis include vectors such as the Ml 3 phage. These phage are readily commercially-available and their use is generally well-known to those skilled in the art.
  • Double-stranded plasmids are also routinely employed in site directed mutagenesis which eliminates the step of transferring the gene of interest from a plasmid to a phage.
  • site-directed mutagenesis in accordance herewith is performed by first obtaining a single-stranded vector or melting apart of two strands of a double-stranded vector which includes within its sequence a DNA sequence which encodes the desired peptide.
  • An oligonucleotide primer bearing the desired mutated sequence is prepared, generally synthetically. This primer is then annealed with the single-stranded vector, and subjected to DNA polymerizing enzymes such as E. coli polymerase I Klenow fragment, in order to complete the synthesis of the mutation-bearing strand.
  • DNA polymerizing enzymes such as E. coli polymerase I Klenow fragment
  • This heteroduplex vector is then used to transform appropriate cells, such as E. coli cells, and clones are selected which include recombinant vectors bearing the mutated sequence arrangement.
  • appropriate cells such as E. coli cells
  • clones are selected which include recombinant vectors bearing the mutated sequence arrangement.
  • sequence variants of the selected peptide-encoding DNA segments using site-directed mutagenesis is provided as a means of producing potentially useful species and is not meant to be limiting as there are other ways in which sequence variants of peptides and the DNA sequences encoding them may be obtained.
  • recombinant vectors encoding the desired peptide sequence may be treated with mutagenic agents, such as hydroxylamine, to obtain sequence variants.
  • the PCRTM-based strand overlap extension (SO ⁇ ) (Ho et al, 1989) for site- directed mutagenesis is particularly preferred for site-directed mutagenesis of the nucleic acid compositions of the present invention.
  • SO ⁇ strand overlap extension
  • the techniques of PCRTM are well-known to those of skill in the art, as described hereinabove.
  • the SO ⁇ procedure involves a two- step PCRTM protocol, in which a complementary pair of internal primers (B and C) are used to introduce the appropriate nucleotide changes into the wild-type sequence.
  • flanking PCRTM primer A (restriction site inco ⁇ orated into the oligo) and primer D (restriction site inco ⁇ orated into the oligo) are used in conjunction with primers B and C, respectively to generate PCRTM products AB and CD.
  • the PCRTM products are purified by agarose gel electrophoresis and the two overlapping PCRTM fragments AB and CD are combined with flanking primers A and D and used in a second PCRTM reaction.
  • the amplified PCRTM product is agarose gel purified, digested with the appropriate enzymes, ligated into an expression vector, and transformed into E.
  • Modification and changes may be made in the structure of the peptides of the present invention and DNA segments which encode them and still obtain a functional molecule that encodes a protein or peptide with desirable characteristics.
  • the following is a discussion based upon changing the amino acids of a protein to create an equivalent, or even an improved, second-generation molecule.
  • the amino acid changes may be achieved by changing the codons of the DNA sequence, according to Table 1.
  • amino acids may be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity with structures such as, for example, antigen-binding regions of antibodies or binding sites on substrate molecules. Since it is the interactive capacity and nature of a protein that defines that protein's biological functional activity, certain amino acid sequence substitutions can be made in a protein sequence, and, of course, its underlying DNA coding sequence, and nevertheless obtain a protein with like properties. It is thus contemplated by the inventors that various changes may be made in the peptide sequences of the disclosed compositions, or corresponding DNA sequences which encode said peptides without appreciable loss of their biological utility or activity.
  • the hydropathic index of amino acids may be considered.
  • the importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte and Doolittle, 1982, inco ⁇ orated herein by reference). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like.
  • Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics (Kyte and Doolittle, 1982).
  • hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 ⁇ 1 ) glutamate (+3.0 ⁇ 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0) threonine (-0.4); proline (-0.5 ⁇ 1 ); alanine (-0.5); histidine (-0.5); cysteine (-1.0) methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3) phenylalanine (-2.5); tryptophan (-3.4).
  • an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent protein.
  • substitution of amino acids whose hydrophilicity values are within +2 is preferred, those which are within ⁇ 1 are particularly preferred, and those within ⁇ 0.5 are even more particularly preferred.
  • amino acid substitutions are generally therefore based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like.
  • Exemplary substitutions which take various of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.
  • NUCLEAR TRANSPORT Nuclear transport is a multi-step process. Following synthesis in the cytoplasm, proteins that contain an active nuclear localization sequence (NLS) are transported to the nucleus, entering through pore complexes in the nuclear envelope (Silver, 1991). There are several critical steps in this process, involving multiple proteins. First, the NLS of the proteins to be imported must be recognized. Second, the proteins are brought to the nuclear pore complex. Third, the pore complex mediates selective entry of these proteins. In addition, there are several examples of proteins that are tethered in the cytoplasm by other proteins that release them for nuclear transport in response to specific signals.
  • NLS nuclear localization sequence
  • the NF-kB/IkB and the Hsp90/glucocorticoid receptor interactions are paradigms of such regulation (Sanchez et al, 1985; Ghosh and Baltimore, 1990).
  • the phosphorylation status of a protein may also affect its localization.
  • the yeast transcription factor PHO4 is prevented from translocating to the nucleus when it is phosphorylated by the PHO850-PHO85-cyclin-CDK complex (O'Neill et al, 1996).
  • the mislocation of BRCAl in advanced breast tumor cells may be due to failure in any one of these steps. However, it is unlikely that a major nuclear transport system is defective in these tumor cells, since they are viable. Most likely, subtle regulators, such as proteins required for modification of BRCAl in order to expose its NLS, or a protein that specifically recognizes the NLS of BRCAl , may fail to function properly.
  • CIP calf intestinal alkaline phosphatase
  • FACS fluorescence-activated cell sorting
  • EGF epidermal growth factor
  • SDS sodium dodecyl sulfate
  • BRCAl is a 220 kDa phosphoprotein in human cells, and that it is expressed and phosphorylated in a cell cycle dependent manner and is localized to the nuclei of normal cells.
  • a full-length BRCAl cDNA was obtained by using a PCR-generated fragment of exon 1 1 to probe a human cDNA library (Zhu, et al, 1995). Three overlapping clones were identified that together encoded the entire cDNA. Convenient restriction sites within these clones were then used to assemble a full-length clone (see FIG. 1A) in pBSK (Stratagene, La Jolla, CA).
  • This construct was co-transfected into SF9 cells (obtained from ATTC, Rockville, MD) together with BaculoGold® viral D ⁇ A (Pharmingen), according to the manufacturer's protocols. After 5 days, the culture medium was collected, and a plaque assay done, as per the manufacturer's protocols.
  • plaques were identified and picked.
  • Several plaque-purified viruses were then screened for BRCAl production by using nickel-affinity chromatography to purify expressed protein from infected SF9 cells. Purified BRCAl was detected either by Coomassie Brilliant Blue staining or by western blotting with MAb 6B4.
  • Mouse polyclonal antisera have been generated against human BRCAl using GST fusion-proteins encoding three different regions of the BRCAl protein as immunogens (depicted schematically in FIG. 1A). As shown in FIG. IB (lanes 2, 4, and 6), each of the three independent antisera immunoprecipitated a protein, running as a
  • BRCAl was generated. This was used to drive in vitro translation of the gene product, which was then immunoprecipitated with the three antisera. As shown in FIG. 2A all three antisera recognize a 220 kDa protein from the in vitro translation mixture that co- migrates with the lower band of the doublet detected in whole-cell lysates (FIG. 2A, lanes 1 , 3, 4, and 5). The upper band was shown to inco ⁇ orate radioactive phosphate, suggesting that it might be a phosphorylated form of BRCAl (Chen, et al, 1995).
  • FIG. 3 A depicts an IP/western for BRCAl using extracts made either at various times following release of T24 cells from density arrest, or from T24 cells arrested in M-phase using nocodazole (0.4 ⁇ g/ml for 8 hrs).
  • FIG. 3E The cell cycle distribution profile at each time-point, determined by FACS analysis, is presented in FIG. 3E.
  • the phosphorylation status of Rb in the same extracts was used as an additional indicator of cell cycle progression (FIG. 3B, middle panel), and staining for p84 (FIG. 3C, bottom panel) served to quantify loading.
  • BRCAl is readily detected in unsynchronized cells (lane 1), it is expressed at very low levels in early GI , such that it is undetectable by western analysis until 18 hrs post-release (lane 4). This corresponds to late GI , since the cells still have a 2N DNA content by FACS analysis, but Rb has already become phosphorylated (FIG. 3B, lane 4).
  • BRCAl is CELL-CYCLE DEPENDENT
  • FIG. 2A BRCAl is phosphorylated in vivo, resulting in the detection of both hypo- and hyper-phosphorylated species as a 220-kDa doublet by immunoprecipitation.
  • the dependence of this phosphorylation on cell cycle progression was investigated by pulse-labeling synchronized T24 cells with 32p ortho phosphate at various times following release from GO arrest, or following nocodazole arrest. Cell extracts were then immunoprecipitated with anti-BRCAl .
  • Antisera specific for BRCAl has been characterized and used to analyze BRCAl expression in normal cells. The results confirmed and extended previous observation that BRCAl is a 220 kDa nuclear phosphoprotein in normal cells. It was shown that the polyclonal antisera, raised against three different regions of the BRCAl protein, all specifically recognized a 220 kDa protein in whole-cell lysates. Immunostaining reconfirmed previous observations that BRCAl is a nuclear protein in normal cells (Chen et al, 1995).
  • both in vitro translated BRCAl and recombinant BRCAl expressed using the baculovirus system were shown to co-migrate with BRCAl from HBLIOO cells.
  • BRCAl migrates as a doublet, the upper band of the doublet being a phosphorylated form of BRCAl .
  • the 220 kDa size is fully consistent with the predicted molecular weight for full-length BRCAl , and is in agreement with previous data (Chen et al, 1995; Scully et al, 1996). Others have reported detecting a 190-kDa protein using antibodies raised against the same immunogen as Scully et al.
  • BRCAl is reported to undergo alternative splicing (Miki et al, 1994) and it is possible that the 190 kDa species is an alternatively spliced variant of BRCAl expressed in some cell types.
  • the same size protein was also detected in cells transfected with a retrovirus expressing a full-length BRCAl cDNA (Holt et al, 1996).
  • the same group of authors also described a baculovirus-derived. recombinant BRCAl with a molecular weight of 180 kDa (Jensen et al, 1996).
  • baculovirus-derived BRCAl was expressed as a 220 kDa protein that co-migrated with BRCAl from HBLIOO cells.
  • This 220 kDa protein could be detected by three separate criteria: nickel-affinity chromatography, immuno- precipitation with three independent BRCAl -specific antisera, and western blotting with a BRCAl -specific monoclonal antibody, make it very unlikely that this protein is not correctly synthesized.
  • the peptide antisera used in the other studies are not specific for BRCAl, but for some other protein such as the EGF receptor.
  • BRCAl plays a critical role in the regulation of cell growth and determination, at least in mice.
  • BRCAl nullizygous mice die at the egg cylinder stage of development (5-6 days p.c), suggesting a role for
  • BRCAl in early cell fate determination (Chia-Yang Liu, et al, 1996) Whether BRCAl has a similar role in humans is unclear.
  • a developmentally normal woman has been described carrying germline nonsense mutations in both alleles of BRCAl (Boyd et al, 1995). It is possible that in humans there is functional redundancy between BRCAl and another protein, or that different mutations have varying effects.
  • homozygous-null individuals are yet to be reported among the extensively studied Ashkenazi Jewish population that has a high incidence of breast and ovarian cancer due to a founder BRCAl mutation (Friedman et al, 1995).
  • BRCAl To characterize BRCAl, the inventors generated polyclonal antibodies to BRCAl (anti-BRCAl) by creating a glutathione-S-transferase (GST)-BRCAl fusion protein containing amino acids encoded by a 3' portion of BRCAl exon 1 1.
  • GST glutathione-S-transferase
  • GST glutathione-S-transferase
  • PCRTM was used to amplify two exon BRCAl fragments from WI 38 cell (human diploid lung) genomic DNA.
  • a fragment of -1.9 kb was amplified with two 27-nucleotide primers synthesized according to the published BRCAl sequence: BRCA9 [5'-TTGCAAACTGAAAGATCTGTAGAGAGT-3'J (SEQ ID NO:2), upstream of a Bglll site, and BRCA7 [5'-TTCCAAGCCCGTTCCTCTTTCTTCCAT-3'J, (SEQ ID NO:3) downstream of a BamHI site.
  • the amplified genomic DNA was then digested with Bglll and BamHI to create a 1.8-kb fragment from codons 762 to 1315.
  • This fragment was purified and subcloned into the GST expression vector pGEX-2T to create pGST-BRCAl .
  • GST-BRCAl-Bgl another 27-nucleotide primer, BRCA8 [5'-GATTTGAACACCACTGAGAAGCGTGCA-3'J (SEQ ID NO:4), beginning at codon 245, and primer BRCA9 were used to amplify a 3.2-kb fragment comprising almost all of exon 1 1.
  • This fragment was then digested with BgH ⁇ to create a 1.2-kb fragment from codons 341 to 748, which was subcloned into a modified pGEX-2T.
  • Each of the two fusion proteins was expressed in Escherichia coli and purified with glutathione-Sepharose beads for use as an antigen in mice. Serum from immunized mice was then preabsorbed on GST affinity columns. The serum raised against the first GST-BRCA1 protein was used in all studies illustrated in the figures. Preimmune serum was obtained from the same mice and used at the same dilution. Anti-BRCAl serum specifically immunoprecipitated a protein with a molecular mass of 220-kDa in HBLIOO human diploid breast epithelial cells metabolically labeled with 3 S-methionine (FIG. 5A). The protein migrated at approximately the size predicted from the 1863-amino acid sequence (Miki et al, 1994).
  • Immunoprecipitated proteins were boiled in a denaturing buffer [20 mM Tris-HCl (pH 7.4), 50 mM NaCl, 1% SDS, and 5 M dithiothreitol] for 5 min, diluted 10-fold with a lysis-50 buffer containing different detergents [20 mM TrisHCl (pH 7.4), 50 mM NaCl, 1% NP-40, and 1% deoxycholate], and reimmunoprecipitated by anti-BRCAl in the same buffer. This doubly immunoprecipitated protein was then washed with lysis-250 buffer before separation by SDS-polyacrylamide gel electrophoresis (SDS-PAGE).
  • SDS-PAGE SDS-polyacrylamide gel electrophoresis
  • C20 directed against an epitope near the COOH-terminus, and BRCAl- ⁇ g/, raised against a fusion protein with sequences encoded by the more 5' portion of exon 1 1, identified the same protein as the first antibody (FIG. 5A, lane 6).
  • Rabbit polyclonal antibody C20 raised against a synthetic peptide corresponding to amino acids 1843 to 1862 of BRCAl, was purchased from Santa Cruz Biotechnology, Inc.
  • GST glutathione-S-transferase
  • GST-BRCA l-Bgl For creation of a second plasmid, GST-BRCA l-Bgl, another 27-nucleotide primer, BRCA8 [5'-GATTTGAACACCACTGAGAAGCGTGCA-3'J (SEQ ID NO:4), beginning at codon 245, and primer BRCA9 were used to amplify a 3.2-kb fragment comprising almost all of exon 11. This fragment was then digested with Bglll to create a 1.2-kb fragment from codons 341 to 748, which was subcloned into a modified pGEX-2T. Each of the two fusion proteins was expressed in Escherichia coli and purified with glutathione-Sepharose beads for use as an antigen in mice.
  • Serum from immunized mice was then preabsorbed on GST affinity columns.
  • the serum raised against the first GST-BTAC1 protein was used in all studies illustrated in the figures.
  • Preimmune serum was obtained from the same mice and used at the same dilution. The same results were obtained when each step in the double immunoprecipitation was performed with a different polyclonal antibody.
  • These immunological results demonstrated that the 220-kDa protein is the BRCAl gene product.
  • the immunoprecipitate of lysate from HBLIOO cells labeled with [ PJphosphoric acid contained only a single, more slowly migrating species (lane 7) and thus showed that BRCAl is a phosphoprotein.
  • BRCAl is present not only in normal breast epithelial cells like the HBLI OO line, but in all breast cancer lines tested (FIG. 5B). It appears to be expressed largely intact in these cells, because the proteins identified by P labeling and immunoprecipitation with anti-BRCAl all migrated in the gel at -220 kDa. Thus BRCAl is not mutated by truncation in most breast cancer cell lines. In tumor lines derived from tissues other than breast, BRCAl appears to be more abundant than in breast cancer lines; it can be detected more easily in bladder, cervical, colon, and other cancers by labeling with 35 S-methionine (FIG. 5C).
  • HBLIOO cells into nuclear, cytoplasmic, and membrane components (Chen et al, 1994; Abrams et al, 1982). BRCAl was detected in normal cells mainly in nuclei (FIG. 6A). Furthermore, indirect immunostaining of intact cells, including HBLIOO, several other normal cell lines, and tumor cells derived from tissues other than breast or ovary also localized BRCAl to nuclei (FIG. 6B and FIG. 6C; Table 2). In contrast. BRCAl was detected mainly in the cytoplasm of almost all breast cancer cell lines tested (FIG. 6C; Table 2).
  • MDA-MB361 which was originally derived from a brain metastasis (Cailleau et al, 1978), contained two distinct populations of cells: A less abundant fraction of larger, more heterogeneous cells in which BRCAl localized to the nuclei, and a more abundant fraction of smaller, more homogeneous cells in which BRCAl localized to the cytoplasm. Similar results were obtained by cell fractionation in several of the same cell lines. These results suggest that BRCAl is located aberrantly in the cytoplasm of most breast and ovarian cancer cell lines.
  • BRCAl was also located primarily in the cytoplasm (FIG. 6D, panels n and p; Table 2).
  • Other tumor cells grown in suspension such as leukemia lines CEM, HL60, and Molt4
  • metastatic to pleura K562 and U937 stained mainly in nuclei (Table 2).
  • BRCAl staining was mainly cytoplasmic in 6 (12%), cytoplasmic and nuclear to a variable extent in 34 (68%), primarily nuclear in 10 (20%), and absent in 2(4%) (Table 2).
  • the BRCAl amino acid sequence does not have typical bipartite nuclear localization signals (NLSs) (Miki et al, 1994), but does contain at least two other putative NLSs (Boulikas, 1994).
  • NLSs nuclear localization signals
  • NKLKRKRRP SEQ ID NO:5
  • amino acids 419 to 427 SEQ ID NO:6
  • NRLRRKS SEQ ID NO:6
  • amino acids 609 to 615 are similar to sequences found in estrogen, progesterone, and other steroid hormone receptor molecules (Boulikas, 1994; Arriza et al, 1987; Danielsen et al, 1986; Green et al, 1986; Kastner et al, 1990; Laudet et al, 1991).
  • BRCAl may normally localize to the nucleus in a similar manner, by dissociation from proteins that anchor it in the cytoplasm, as a passenger with other nuclear proteins, or after modification to expose its own potential NLS. Similar transport mechanisms have been demonstrated for other transcription factors including SV40 large T antigen and c-Fos (Schneider et al, 1988; Roux et al, 1990; Moll et al, 1991).
  • the mutations of molecules involved in the pathway of BRCAl transport from its site of synthesis to sites of action in the nucleus may be alternative ways to inactivate the same crucial protein in many sporadic breast cancers.
  • KLS 6I ⁇ and 65I KLA were tagged with the flag epitope, expressed in human DU145 cells, and detected with the M2 monoclonal antibody.
  • the KLP mutant completely fails to localize in nuclei, whereas the KLS mutant is mostly cytoplasmic with occasional nuclear localization.
  • the KLN protein is always located in nuclei.
  • hSRPl ⁇ a component of the NLS receptor complex
  • BRCAl yeast two-hybrid screen using BRCAl as the bait.
  • the specificity of the interaction between BRCAl and importin- ⁇ was further demonstrated by showing that the 503 KRKRRP 508 (SEQ ID NO:7) and 606 PKKNRLRRKS 651 (SEQ ID NO:8) regions, but not 63 I KKKKYN 656 (SEQ ID NO:9), are critical for this interaction.
  • Human cell lines DU145 prostate cancer
  • T24 bladder cancer
  • T47D ZR75
  • MCF7 breast epithelial cells immortalized with SV40
  • CV1 monkey kidney cell line
  • Each 10-cm dish of cells grown to 60% confluency was transfected with 10 ⁇ g of plasmid DNA using the calcium phosphate method (Kingston, 1994).
  • the calcium phosphate precipitate was left in the culture medium for 6-8 h. At that time the medium was drained, and the cells were refed with fresh medium.
  • Plasmid BSK-BRCAla that contains a full-length BRCAl cDNA (Chen et al, 1996) was used as the template for PCRTM amplifications using each pair of internal and external primers.
  • the resulting DNA fragments were gel purified and cut with Aflll and Hindlll for the N-terminal cDNA fragments, and with Kpnl and H dlll for the C-terminal cDNA fragments.
  • the N- and C-terminal fragments were then used to replaced the Aflll/ Kpnl fragment in pBSK-BRCAla. Ligation of the Hindlll site at each of the NLS sites generated in-frame deletions and additions of CTT codons for leucine residues.
  • AfllllKpnl fragments from pBSK-BRCAl-KLP, KPS, and KLN] were then used to replace a similar fragment in the expression vector pCEP-FlagBRCAl (Chen et al, 1996) to generate pCEP-FlagBRCAl KLp , pCEP-FlagBRCAl KLS and
  • Cells were transfected with pCEP-FlagBRCAl KLp , pCEP-FlagBRCAl RLS or pCEP-FlagBRCAl KLN for expression of the epitope- (Flag, Kodak, IBI) tagged NLS mutated proteins and pCEP-FlagBRCAl for Flag -tagged wild-type BRCAl.
  • the cells were fixed and indirectly immunostained with the M2 Flag mAb (Kodak, IBI) using previously described procedures (Mancini et al, 1994).
  • the microscopic images were acquired using a Hammamatsu Color Chilled 3CCD camera attached to a Zeiss AxiophotTM fluorescence microscope.
  • the image files were digitally processed for presentation using Adobe Photoshop®.
  • the BRCAl proteins were immunoprecipitated as described (Chen et al, 1995) using mouse an ⁇ -BRCAl-Bg ⁇ antibodies (Chen et ⁇ l, 1995). After SDS/PAGE, epitope-tagged BRCAl protein was detected in Western blots using the anti-Flag M2 mAB (Kodak, IBI) and endogenous BRCAl was detected with the BRCAl-Bg antibodies (Chen et ⁇ l, 1995).
  • the identification of an activation domain in BRCAl was done by a yeast one-hybrid assay in Saccharomyces cerevisiae strain Y153, which contains a l ⁇ cZ reporter under the control of a promoter with GAL4-binding sites in the upstream activating sequence of GAL1 (UAS G ) (Durfee et ⁇ l, 1993).
  • ⁇ -Galactosidase activity was determined by colony color and quantitated using chlorophenyl red ⁇ -D-galactopyranoside in assays as described previously (Durfee et ⁇ l, 1993).
  • a cDNA library prepared from human B lymphocytes was screened as described previously (Durfee et ⁇ l, 1993).
  • the protein from pAS-BRCA3.5 (see FIG. 2) sewed as the "bait,” which consisted of amino acids 1-1142 of BRCAl fused to the GAL4
  • pAS-KLP, pAS-KLS, mad pAS-KLN were constructed by fusing BRCAl 142 cDNAs from pBSK-BRCAl-KLP, KLS, and KLN to the DNA-binding domain of GAL4 in pAS (Durfee et al, 1993). ⁇ -galactosidase activity was assayed as described above.
  • BRCAl nuclear transport of BRCAl clearly requires interactions with other cellular proteins.
  • the inventors elected to use the yeast two-hybrid method to identify and clone genes encoding BRCAl -interacting proteins. Since BRCAl has been proposed to be a transcription factor (Miki et al, 1994), it may therefore have transactivation activity.
  • hBRAP21 is identical to that of the nuclear localization signal receptor hSRPl ⁇ (Weis et al, 1995), also known as importin- ⁇ (Gorlich et al, 1994) or karyopherin- ⁇ (Radu et al, 1995). TABLE 3 SUMMARY OF CLONES ENCODING BRCAI-INTERACTING PROTEINS
  • Insert size is given in kilobase pairs.
  • pAS-BRCA3.5 with wild-type BRCAl amino acid sequence 1-1142 or the same region containing the mutated NLS sequences, KLP, KLS and KLN, cloned into the pAS expression vector (pAS-KLP, pASKLS, and pASKLN) were used.
  • a region of importin- ⁇ from amino acid 220 to 529, which is known to interact with BRCAl was translationally fused to the activation domain of GAL4 in pACT (Durfee et al, 1993).
  • expression of pAS-BRCA3.5 encoding wild-type BRCAl produced blue colonies and had a 100 fold increase in ⁇ -galactosidase activity over that of the negative control, untransfected Y153 cells. Consistent with the ability of Flag- ⁇ -?C4/ RLN to translocate into the nucleus, the assay of pAS-KLN also resulted in blue colonies and a 150 fold increase in ⁇ -galactosidase activity.
  • BRCAl CYTOPLASMIC LOCALIZATION IN BREAST CANCER CELLS previously, the inventors transfected an expression plasmid containing flag-tagged BRCAl into two breast cancer cell lines, T47D and MB468, and one immortalized non-breast ,epithelial cell line, HBLIOO.
  • the flag-tagged BRCAl protein was found in the cytoplasm of the T47D and MB468 cells and the nucleus of HBLIOO cells by iimmunostaining with anti-flag M2 monoclonal antibody.
  • the identification of two regions of charged, basic amino acids between 503 and 508 and between 606 and 615 that are both crucial for efficient nuclear transport of the BRCAl protein further supports this notion.
  • the distance between these two motifs is much greater than the 10 amino acids separating the bipartite sites of nucleoplasmin (Robbins et al, 1991).
  • the structure and function of the NLSs in BRCAl is similar to other nuclear proteins in which two NLSs are more widely spaced such as those in the polyoma large T antigen (Richardson et al, 1986), influenza A virus NSl protein (Greenspan et al, 1988), and adenovirus DNA-binding protein (Morin et al, 1989). While the inventors cannot rule out the possibility that other sequences are also required for translocation of BRCAl from the cytoplasm to the nucleus, the NLS at 503-508 is essential for this process.
  • BRCAl Using mouse polyclonal antibodies specific for the BRCAl protein, the inventors have consistently found BRCAl to be a 220-kDa nuclear protein that is aberrantly located in the cytoplasm of advanced breast cancer cells (Chen et al, 1995; Chen et al , 1996b). However, Scully et al. (Scully et al, 1996) reported that the 220-kDa BRCAl protein remains in the nucleus of some breast cancer cell lines. Although the precise reason for this discrepancy is unclear, one cannot exclude the possibility of less specific antibodies, potential immunostaining artifacts, or both.
  • BRCAl expressed in breast cancer cells remains in the cytoplasm. This result further suggests that its mislocation in breast cancer cells is not due to mutations of BRCAl itself. Rather, the aberrant localization seems to be the result of alterations in the cells, perhaps at the level of nuclear transport Of BRCAl .
  • the demonstration here that BRCAl interacts with the importin-a subunit of the nuclear transport receptor complex could be an important clue. However, if there is a problem with the importin- ⁇ subunit or the importin-substrate complex, why is it manifested in breast epithelial cells? Does this indicate an unsuspected specificity of importin- ⁇ for BRCAl? And, does the defect in the function of BRCAl reside in the cytoplasm or the nucleus?
  • Fragments of BRCAl that exclude any potential intrinsic /raw-activation activity are fused, in-frame, with the Gal4 DNA-binding domain. These are used to screen a cDNA library, fused to the Gal4 activation domain, using a yeast strain containing a Gal4 responsive b-galactosidase gene. The clones are analyzed based on the following criteria. First, whether they map to chromosomal regions known to undergo frequent LOH in breast cancer. Second, whether large scale deletions in the genes are detected by Southern analysis of DNA from tumor lines in which BRCAl is mislocated. And third, whether more subtle mutations in these genes can be detected in the tumor lines by RT-
  • PCRTM-based single-strand conformational polymo ⁇ hism (RT-PCRTM/SSCP) assay. Genes in which mutations are found are strong candidates for the gene responsible for translocation of BRCAl to the nucleus.
  • in vitro binding and in vivo co-immunoprecipitation assays are performed using wild-type BRCAl, NLS-deficient mutants, and deletion mutants encompassing other regions of BRCAl.
  • BRCAl localization in breast cancer cells it is necessary to clone full-length cDNAs.
  • antibodies are raised against the protein products to permit their detection. If mutations in the identified gene(s) are responsible for the mislocalization of BRCAl in the tumor lines, then these antibodies are also used in screening tumor samples.
  • the cDNAs identified are expressed in breast cancer cell lines under the control of a tetracycline-inducible promoter, so that expression is regulated by the presence of tetracycline in the culture medium. This avoids potentially toxic effects due to the expression of these proteins.
  • the effect of their expression on the localization of endogenous and/or exogenous wild-type BRCAl is monitored by immunofluorescence and cell fractionation assays.
  • BRCAl MISLOCALIZATION IN ADVANCED BREAST TUMOR CELLS Wild-type BRCAl may be expressed in normal cells and breast tumor lines.
  • This example is designed to confirm that a defective nuclear transport system is a common cause of the mislocation of BRCAl protein in advanced breast cancer cells.
  • the nuclear-matrix associated protein, p84(N5) is used as a control for nuclear transport function.
  • Immortalized human breast epithelial cells (HBLIOO), and CV1 cells can be used as controls for normal BRCAl localization.
  • Tagged BRCAl proteins with deletions in each of the three NLS motifs may be constructed and expressed in normal cells, so as to identify the motif required for transportation of BRCAl into the nucleus. If the three putative NLS motifs are not responsible for the transportation, a series of systematic deletion mutants can be generated and similarly tested to define the functional NLS motif.
  • the NLS mutants can be used as tools to screen the BRCA 1 -associated proteins identified in aim 2 to determine if any interact with the NLS.
  • the inventors have constructed 3 BRCAl deletion mutants, each specifically removing one of the putative NLS motifs (amino acids: 500-508, 606-614, and 650-655). These have been cloned, in-frame with the FLAG epitope, into the pCEP4 vector (FIG. 8).
  • the mutant proteins can be transiently expressed in normal cells, and their localization determined by indirect-immunofluorescence using the anti-FLAG antibody M2 (Kodak).
  • NLS motifs can promote BRCAl translocation to the nucleus.
  • double and triple deletion- mutants are also constructed to test this possibility. From previous experience in determining the NLS of the Rb-associated protein, mitosin (Zhu, et al, 1995), it is clear that the rules for determining functional NLS motifs are not absolute, and it may be necessary to broaden the spectrum of potential NLS candidate sequences. Should this be the case, a series of systematic BRCAl deletion mutants can be generated and tested for their ability to translocate to the nucleus. These studies are useful in identifying residues crucial for nuclear transport of BRCA 1.
  • the yeast two-hybrid system has been very successful in isolating a total of 25 Rb-associated proteins (Durfee et al, 1993).
  • An advantage of this method is that it is able to detect fairly weak interactions and is more sensitive than co-immunoprecipitation assays that depend on efficiency of metabolic labeling, affinity of the immunoprecipitating antibody, and the half-life of the complex assayed.
  • BRCAl is a transcription factor. This is based on the presence of a putative DNA-binding, Zn-finger domain in the N-terminal region of the protein, three putative NLS motifs, and a potential acidic /r ⁇ ra-activation domain towards the C-terminus.
  • the nature of the yeast two-hybrid assay requires that the bait protein does not contain intrinsic /n s-activation activity.
  • the inventors first screened a series of BRCAl deletion mutants to identify regions that contain such activity. As shown in FIG. 9, a region of about 600 amino acids towards the C-terminus contains strong /r ⁇ w-activation activity. Therefore, the inventors have used fragments of BRCAl not encoding this region as baits to identify associated proteins.
  • Two BRCAl expression plasmids have been constructed: pAS-BRCA2.5 (amino acids 303-1 142) and pAS-BRCA3.5 (amino acids 1-1 142) (FIG. 10). These create hybrid molecules between sequences for the DNA-binding domain of the yeast transcription factor Gal4 (amino acids 1-147; (Keegan et al, 1986)) and portions of the BRCAl protein. These constructs have been used to screen a cDNA library from human B-lymphocytes, which was cloned into a second expression plasmid containing sequences for the Gal4 activation domain II (amino acids 768-881 ; (Ma and Ptashne, 1987)).
  • BRCAl -associated proteins by co-immunoprecipitation provides a useful complement to the yeast two-hybrid screen described above.
  • This method has two significant advantages over the yeast two-hybrid system. First, it is not restricted by the presence of a potential /r ⁇ ra-activation domain in BRCAl . Thus, protein interactions with full-length BRCAl can be addressed. This is important, since although defects in proteins that interact with the NLS of BRCAl are prime suspects for the mislocation of BRCAl in advanced breast cancers, it is equally likely that proteins interacting with other regions of BRCAl are important in this process, perhaps by causing conformational changes in BRCAl to reveal an otherwise hidden NLS motif.
  • proteins that interact with other regions of BRCAl could be critical for the nuclear transport of BRCAl .
  • a protein that tethers BRCAl in the cytoplasm through phosphorylation similar to the action of PHO80-PHO85-CDK on PHO4 (O'Neill et al, 1996), or by directly masking the NLS, similar to the action of IkB on NF-kB (Ghosh and Baltimore, 1990), need not necessarily bind to the NLS of BRCAl .
  • the second advantage of this method is that it provides a more directed search for proteins involved in BRCAl transport than does the yeast two-hybrid screen.
  • At least five proteins with molecular weights of: 40, 41 , 49, 95, and 140 kDa are specifically co-immunoprecipitated with BRCAl in HBLIOO cells. Further analysis includes the following: First, proteins that co-immunoprecipitate with wild-type BRCAl in normal cells and breast tumor cells may be compared. This should identify interacting proteins that are potentially involved in BRCAl import and which may be defective in cancer cells. Second, differences in the co-immunoprecipitated proteins between the NLS mutants and wild-type BRCAl in normal cells are determined. This is done to identify proteins that specifically interact with the NLS of BRCAl . Taken together, these results permit the identification of proteins that are critical for BRCAl nuclear- import.
  • the inventors over- express the FLAG-tagged BRCAl protein in the cells by transfection.
  • the tetracycline-inducible system can be used.
  • the FLAG-tag provides a convenient handle to mark exogenous protein, since it is located at the N-terminus of the protein and is thus less likely to induce steric hindrance between BRCAl and any interacting proteins.
  • the high affinity monoclonal antibody against FLAG (M2, Kodak) is highly specific, thus reducing problems of contamination of the co-precipitate with unrelated, cross-reacting proteins.
  • the genes encoding the identified proteins may be cloned according to procedures previously established in the laboratory for cloning Rb-associated proteins (Qian et al, 1993). Briefly, sufficient protein are purified by co-immunoprecipitation to permit peptide sequencing to be done. A comparison of the obtained peptide sequences with the GenBank database reveals if the genes are novel and may provide additional clues as to their function. Novel genes are cloned by screening a cDNA library with degenerate oligonucleotides based on the peptide sequences obtained. The identity of the cloned genes and the co-immunoprecipitated proteins are confirmed in several ways.
  • This strain was constructed using conventional gene targeting techniques (Lee et al, 1992; Liu et al, 1996).
  • a targeting plasmid was constructed using an 8.0 kb Hindlll-BamHl DNA fragment containing Brcal [exons 9, 10, and part of 1 1J.
  • a single IoxP site derived from pGEM-30 (Gu et al, 1993) was inserted into a unique 5 ⁇ site within intron 9 using EcoRI-linkers.
  • a second set of-loxP sites in conjunction with a neomycin resistance cassette derived from P12-neo r [obtained from H. Gu, NH-I/NIAID] was then inserted into a unique Xhol site within intron 10.
  • the resulting plasmid was designated Brcal-loxP.
  • a pMC l-tk cassette was placed at the 5'end of the construct to generate the final targeting vector: Brcal-loxP ko.
  • the construct was linearized by BamHI and individually transfected into ⁇ S cells [an early passage of ⁇ 14-1 (Handyside et al, 1989)], as described previously (Lee et al, 1992; Liu et al, 1996). Colonies doubly resistant to G418 and FIAU were isolated from which the DNA was analyzed by Southern blotting to identify clones containing a site specific integration of the Brcal-floxP gene resulting from homologous recombination.
  • Neo cassette intron 10 could disrupt Brcal expression by- interfering with RNA processing.
  • targeted ES cells are transfected with pIC-Cre (Sternberg et al, 1981) to initiate the transient expression of Cre recombinase. Under these conditions, it is expected that Cre-mediated excision will only occur once in some cells (Gu et al, 1994). Since there are three possible recombination events, a screen of 100 clones subsequent to transfection is used to generate the desired recombinant that has deleted the Neo cassette but not exon 10.
  • ES cells with the desired placement of IoxP sites are independently injected into C57BL/6 blastocysts, which are implanted into the uteri of pseudo-pregnant F, [CBA X C57BL/6] female foster mice to generate chimeras.
  • Germline transmission are tested by back crossing chimeric male mice with C57BL/6 females. Because the IoxP sites are inserted into intron sequences, they are not expected to affect endogenous Brcal gene function (Gu et al, 1994).
  • Germline chimeras are used to establish Brcal-loxP heterozygous and homozygous transgenic lines.
  • transgenes driven by the WAP promoter can be activated cyclically during estrus in about 30% of the secretory alveolar structures
  • Homozygous transgenic lines with high expression of Cre recombinase are mated with homozygous Brcal-loxP mice. This initially generates obligate heterozygotes for both WPA-rtTA;Cre and Brcal-loxP. These F, heterozygotes are then be bred to generate an F 2 population in which 1 out of 16 mice is expected to be homozygous for both alleles. Breeding these animals generates obligate
  • WPA-rtTA;Cre/ Brcal -IoxP homozygotes in the F 3 generation that may then be used for the targeted-excision studies.
  • Doxycycline is given to female mice in their drinking water [or. alternatively, through sub cutaneous slow release pellets, Alternative Research of America] to induce expression of the Cre recombinase.
  • four time points are used for the administration of doxycycline; estrus, late pregnancy [days 15-18 ], and day 3 of lactation.
  • Previous studies have shown that the WAP promoter is capable of activating reporter gene expression at these time points.
  • the inventors expect rtTA expression to be induced at these times (Robinson et al, 1995). With doxycycline treatment, rtTA should activate expression of the Cre recombinase, which in turn will catalyze the targeted excision of Brcal exon 10.
  • Negative controls are female littermates that have not been treated with doxycycline. To confirm that Brcal exon 10 is deleted following induction of Cre, histological sections are made from the mammary glands of mice from each of the treatment time points. These are then examined for Brcal by standard immunohistochemical methods using BRCAl antibodies that specifically recognize the
  • microdissection followed by PCRTM analysis as described previously may be used to screen directly for genomic deletion of exon 10. Having confirmed that the excision event can be induced, groups of mice are followed for the development of mammary glands and the genesis of tumors, as outlined below. Subsequent to treatment of WPArtTA;Cre/.Brca- -/o P females with doxycycline, breast is prepared for examination as follows. In 4 month virgins either untreated or treated with doxycycline during estrus, the tissues [eight breasts per animalj are collected for analysis according to the schedule shown. In pregnant animals, doxycycline treatment is administered beginning on day 14 and tissues collected according to a prescribed schedule.
  • PCRTM is done on individual alveolar secretory units (Liu et al, 1996). Two breasts per animal serve for this analysis. If deletion of Brcal perturbs mammary gland development, then the inventors might expect that abnormalities of mammary epithelial cells will be revealed by histocytochemistry studies. Markers of differentiation commonly used are milk protein genes, specifically for the inventors' purposes [ ⁇ -casein and WDNM1 [early pregnancy], and whey acidic protein and ⁇ -lactalbumin [later near the end of gestation] (Robinson et al, 1995). Two breasts per animal serve as a source of tissue for either immunohistochemistry studies [antibodies can be obtained from L. Hennighausen, NIH-NIDDK], or in situ hybridization.
  • the dynamics of tumor development may be determined by sacrificing mice for histological examination of breasts over several time-points, from the initial deletion event to the time at which overt tumors are detectable. In this way, it should be possible to document tumor formation from its earliest stages until the last stages of malignancy, including metastasis. This approach was recently used successfully in documenting the dynamics of melanotrophic tumor formation in Rb + " mice (Nikitin and Lee, 1996).
  • the primary amino acid sequence of the hBRAP12 protein is shown in SEQ ID NO:l . Notably, it contains eight zinc fingers of the C2H2 class that are highly homologous to each other but are not homologous to any other proteins, including known zinc finger domains in the GenBank.
  • hB RAP 12 To determine the subcellular localization of hB RAP 12, a full-length cDNA was translationally fused to the green fluorescent protein. The subcellular localization of the fusion protein is nuclear in CV1 and HBLIOO cells.
  • hBRAP12 is a nuclear protein is consistent with an interaction with BRCAl .
  • In vitro interactions may be be confirmed by GST pull-down assays as previously applied for E2F-retinoblastoma protein interactions (Shan et al. 1992).
  • In vivo interactions between BRCAl and hBRAP12 is ascertained using reciprocal immunoprecipitation combined with Western blotting (Shan et al, 1992).
  • hB RAP 12 The interaction of hB RAP 12 with BRCAl may be assessed at two levels. First, their capacity to associate in vitro is determined by assaying the ability of GST- hBRAP12 bound to glutathione-agarose beads, to bind S-methionine labeled, in vitro translated BRCAl or baculo virus-made BRCAl (Chen et al, 1996b). A negative control is GST alone. The converse study using different fragments of GST-BRCA1 fusion proteins bound to glutathione beads (Chen et al, 1996b) and in vitro translated hBRAP12 is also done as previously described (Shan et al, 1992).
  • hBRAP12 and BRCAl are tested by co-immunoprecipitation assays of whole cell extracts (Shan et al, 1992) using antibodies generated against hBRAP12 and those available for BRCAl (Chen et al, 1995). Highly specific monoclonal antibodies for BRCAl raised against two different epitopes in exon 1 1 of BRCAl may also be used in in vivo and in vitro binding assays.
  • Mouse antisera against hBRAP12 has been generated using GST-hBRAP12 fusion proteins expressed in E. coli as previously described (Chen et al, 1995).
  • the hBRAP12 antiserum is preabsorbed using GST-glutathionine beads (Chen et al, 1995).
  • the co-immunoprecipitation assays are performed either using cells that co-express sufficient quantities of endogenous proteins, or using cells co-transfected with expression vectors for either tagged (flag) or untagged BRCAl and hBRAP12.
  • hB RAP 12 deletion mutants including the N- and C-terminus and zinc-finger domains may be generated and tested for binding to full-length BRCAl, as described above.
  • the inventors' initial hypothesis is that, in addition to DNA-binding, the zinc finger domain will also be important for the BRCAl interaction. In any event, these studies should be effective in determining the region of hBRAP12 required for binding to BRCAl .
  • deletion mutants of BRCAl may be assayed for their ability to interact with GST-hBRAP12 in an in vitro binding assay, as described above.
  • the RING-finger domain of BRCAl appears to be important for this interaction, and GST-fusions have been prepared for either the wild-type N-terminal region of BRCAl or the same region with a single point mutation (T to G substitution) which results in a Cys61 to Gly found in a familial breast cancer case (Johannsson, et al, 1996). This mutation disrupts the RING-finger domain of
  • hB RAP 12 is a DNA-binding transcription factor, it should recognize a specific DNA sequence. To identify this sequence, one may use the method of random sequence selection and PCRTM (Perkins et al, 1991 ; Blackwell and Weintraub, 1990) to define the consensus DNA binding site for BRCAl . Importantly, the identification of the cognate binding site for hBRAP12 permits functional testing of hBRAP12 activator function as described. The identification of the cognate binding site is also important for the identification of the downstream target genes for hBRAP12.
  • the cDNAs for these constructions were generated using standard PCRTM and cloning methodologies. After expression in E. coli, the bacterial lysates were incubated with glutathione agarose beads and washed extensively to remove non-specific binding proteins. After quantification of GST-hBRAP12, GST-hBRAP12-Z g and ⁇ Zn- hBRAP12 protein-binding, the respective beads were used to screen the random sequence DNA library.
  • hBRAP12 binding sites 10 ⁇ g of random oligonucleotides is incubated with beads bound with ten ⁇ g of GST- ⁇ Zn-hBRAP12 protein in a binding buffer containing ZnSO 4 and 100 ⁇ g/ml of tRNA as a non-specific competitor. The pu ⁇ ose of this first incubation is to remove DNA that binds non- specifically.
  • either GST-hBRAP or GST-hBRAP12Z 8 is incubated with the pre- bound random DNA library. After extensive washing with low salt buffer, the DNA is eluted with high salt buffer (1.0 M NaCl) and precipitated with ethanol in the presence of tRNA carrier.
  • high salt buffer 1.0 M NaCl
  • the recovered DNA will then be subjected to PCRTM amplification and the binding process repeated four times.
  • the DNA is cleaved with -B ⁇ mHI and Hindlll, purified on 15% acrylamide gels and ligated into pBSK vector (Stratagene). Random colonies are picked, and inipreparations of plasmid DNA sequenced according to standard procedures. From this sequence analysis, consensus binding sequences may be identified. The progress of the selection will be monitored using gel shift analysis (Shan et al, 1992). To do this, the PCRTM reaction is performed with a primer end-labeled with 3 p- ⁇ ATP using T4 polynucleotide kinase.
  • Specific binding by the consensus binding site is determined by gel shift analyses using specific and non-specific competitor oligonucleotides (Shan et al. 1992), and/or DNAasel footprinting (Cao et al, 1988) with wild-type hBRAP12 or with ⁇ Zn-hBRAP12 proteins.
  • An alternative to affinity purification is to use electrophoretic mobility shifts to isolate the DNA fragments (Kinzler and Vogelstein, 1989; Caubin et al, 1994) which is amplified by PCRTM and then selected and amplified four additional times. The resulting fragments are digested and ligated into pBSK, as described above.
  • N nucleus C: cytoplasm N/D: not done
  • a full length cDNA for hBRAP12 was isolated from a human fibroblast library using the partial cDNA from a yeast two-hybrid screen as the hybridization probe.
  • the 8 zinc fingers begin at residue 208 and end at 431.
  • the Cys and His residues in each finger are in bold.
  • hBRAP Primary Sequence SEQ ID NO:l
  • MIQAQESITLEDVAVDFT EEWQLLGAAQKDLYRDVMLENYSNLVAVGYQASK PDALFKLEQGEQPWTIEDGIHSGACSDIWKVDHVLERLQSESLVNRRKPCHEH DAFENIVHCSKSQFLLGQNHDIFDLRGKSLKSNLTLVNQSKGYEIKNSVEFTG NGDSFLHANHERLHTAIKFPASQKLISTKSQFISPKHQKTRKLEKHHVCSECG KAFIKKSWLTDHQVMHTGEKPHRCSLCEKAFSRKFMLTEHQRTHTGEKPYECP ECGKAFLKKSRLNIHQKTHTGEKPYICSECGKGFIQKGNLIVHQRIHTGEKPY ICNECGKGFIQKTCLIAHQRFHTGKTPFVCSECGKSCSQKSGLIKHQRIHTGE KPFECSECGKAFSTKQKLIVHQRTHT
  • Eglitis, M.A., and Anderson, W.F. "Retroviral vectors for introduction of genes into mammalian cells," Biotechniques 6(7):608-614, 1988.
  • Eglitis, M.A. Kantoff, P.W., Kohn, D.B., Karson, E., Moen, R.C., Lothrop, CD.,
  • CD34(3+) hematopoietic stem/progenitor cells from human umbilical cord blood J. Exp. Med. 178(6):2089-2096, 1993. Lundberg, C, Skoog, L., Cavenee, W.K., Nordenskjold, M., "Loss of heterozygosity in human ductal breast tumors indicates a recessive mutation," Proc. Natl. Acad.
  • Drash syndrome Cell, 61A31-AA3, 1991. Perkins, Fishel, Jenkins, Copeland, "Evi-1, a murine zinc finger proto-oncogene, encodes a sequence-specific DNA-binding protein," Molecular and Cellular Biology,
  • Shan, B., Lee, W.H. "Deregulated expression of E2F-1 induces S-phase entry and leads to apoptosis," Mol. Cell Biol, 14:8166-73, 1994.
  • Shan, B., Zhu, X., Chen, P.L., Durfee, T., Yang, Y vigorous Sharp, D., Lee, W.H. "Molecular cloning of cellular genes encoding retinoblastoma-associated proteins: identification of a gene with properties of the transcription factor E2F. " . Mol. Cell
  • compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the composition, methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims. Accordingly, the exclusive rights sought to be patented are as described in the claims below.

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Abstract

L'invention concerne des procédés et des compositions s'appliquant au diagnostic et au traitement du cancer du sein et autres cancers apparentés. L'invention concerne également des compositions et des procédés de détection du produit génique BRCA1 in vivo et in vitro, ainsi que des procédés de diagnostic de localisation aberrante de la protéine BRCA1 dans des cellules mettant en oeuvre des anticorps anti-BRCA1. L'invention concerne de plus des procédés d'identification des protéines associées à BRCA1 qui agissent dans la translocation correcte du produit génique BRCA1 vers le noyau cellulaire.
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WO1998005968A1 (fr) 1996-08-02 1998-02-12 The Wistar Institute Of Anatomy And Biology Proteine bap-1 associee a la brca1 et ses utilisations
WO2001025255A2 (fr) * 1999-10-07 2001-04-12 Baylor College Of Medicine Nouvelle proteine a doigt de zinc
US20050065333A1 (en) * 2001-04-27 2005-03-24 Arun Seth Breast cancer-associated genes and uses thereof
KR100468466B1 (ko) * 2001-08-25 2005-01-27 주식회사 랩 지노믹스 유방암 및 난소암 진단에 유용한 ｂｒｃａ１, ｂｒｃａ２유전자 돌연변이 및 이를 이용한 검색 방법
JP4628674B2 (ja) * 2001-09-12 2011-02-09 ザ ウォルター アンド エリザ ホール インスティテュートオブ メディカル リサーチ 診断および治療の方法ならびにそのために有用な作用物質
CN111253483B (zh) * 2020-03-02 2021-07-30 江苏莱森生物科技研究院有限公司 一种抗brca1单克隆抗体及其用途

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995019369A1 (fr) * 1994-01-14 1995-07-20 Vanderbilt University Procede de detection et de traitement du cancer du sein
US5654155A (en) * 1996-02-12 1997-08-05 Oncormed, Inc. Consensus sequence of the human BRCA1 gene
US5756294A (en) * 1995-09-25 1998-05-26 Oncormed, Inc. Susceptibility mutation for breast and ovarian cancer

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995019369A1 (fr) * 1994-01-14 1995-07-20 Vanderbilt University Procede de detection et de traitement du cancer du sein
US5756294A (en) * 1995-09-25 1998-05-26 Oncormed, Inc. Susceptibility mutation for breast and ovarian cancer
US5654155A (en) * 1996-02-12 1997-08-05 Oncormed, Inc. Consensus sequence of the human BRCA1 gene

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
BRODY L C ET AL: "Construction of a transcription map surrounding the BRCA1 locus of human chromosome 17;MTO120 Homo sapiens genomic clone MTO120" EMBL NUCLEOTIDE SEQUENCE, XX, XX, 31 December 1994 (1994-12-31), XP002134210 *
BROWN M A ET AL: "PHYSICAL MAPPING, CLONING, AND IDENTIFICATION OF GENES WITHIN A 500-KB REGION CONTAINING BRCA1" PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA, NATIONAL ACADEMY OF SCIENCE. WASHINGTON, US, vol. 92, 1 May 1995 (1995-05-01), pages 4362-4366, XP002017873 ISSN: 0027-8424 *
HAKEM RAZQALLAH ET AL: "The tumor suppressor gene Brca1 is required for embryonic cellular proliferation in the mouse." CELL, vol. 85, no. 7, 1996, pages 1009-1023, XP002174576 ISSN: 0092-8674 *
See also references of WO9801460A1 *
SZABO CSILLA I ET AL: "Human, canine and murine BRCA1 genes: Sequence comparison among species." HUMAN MOLECULAR GENETICS, vol. 5, no. 9, 1996, pages 1289-1298, XP002174577 ISSN: 0964-6906 *

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