WO2003062446A9 - Mrp9 et son utilisation pour la detection et le traitement du cancer - Google Patents

Mrp9 et son utilisation pour la detection et le traitement du cancer

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Publication number
WO2003062446A9
WO2003062446A9 PCT/US2003/001340 US0301340W WO03062446A9 WO 2003062446 A9 WO2003062446 A9 WO 2003062446A9 US 0301340 W US0301340 W US 0301340W WO 03062446 A9 WO03062446 A9 WO 03062446A9
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WIPO (PCT)
Prior art keywords
mrp9
seq
antibody
polypeptide
cancer
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PCT/US2003/001340
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English (en)
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WO2003062446A3 (fr
WO2003062446A2 (fr
Inventor
Ira H Pastan
Tapan K Bera
Byungkook Lee
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Us Gov Health & Human Serv
Ira H Pastan
Tapan K Bera
Byungkook Lee
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Application filed by Us Gov Health & Human Serv, Ira H Pastan, Tapan K Bera, Byungkook Lee filed Critical Us Gov Health & Human Serv
Priority to AU2003209265A priority Critical patent/AU2003209265A1/en
Publication of WO2003062446A2 publication Critical patent/WO2003062446A2/fr
Publication of WO2003062446A9 publication Critical patent/WO2003062446A9/fr
Publication of WO2003062446A3 publication Critical patent/WO2003062446A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4748—Tumour specific antigens; Tumour rejection antigen precursors [TRAP], e.g. MAGE
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3015—Breast
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • This application relates to the field of cancer, specifically to MRP9, an ATP binding cassette transporter, that is of use in detecting cancer, such as breast, testicular, or pancreatic cancer.
  • This application also relates to the immunotherapeutic agents directed to MRP9 in the treatment of cancer.
  • Breast cancer is the most common type of epithelial cancer among women in the United States. More than 180,000 women are diagnosed with breast cancer each year. About 1 in 8 women in the United States (approximately 12.8 percent) will develop breast cancer during her lifetime. At present there are no curative therapies available for breast cancer that has metastasized from its site of origination.
  • Immunotherapy is a potent new weapon against cancer that has been suggested to be useful in treating many types of cancer, including breast cancer. Immunotherapy involves evoking an immune response against cancer cells based on their production of target antigens. Immunotherapy based on cell-mediated immune responses involves generating a cell-mediated response to cells that produce particular antigenic determinants, while immunotherapy based on humoral immune responses involves generating specific antibodies to cells that produce particular antigenic determinants.
  • Cancer cells produce various proteins that can become the target of immunotherapy; antigenic determinants normally present on a specific cell type can also be immunogenic. For example, it has been shown that tumor infiltrating lymphocytes target and recognize antigenic determinants of the protein MART-1, produced by both normal melanocytes and malignant melanoma cells. Furthermore, active or passive immunotherapy directed against MART-1 or peptides of it that bind to MHC Class I molecules (epitopes of HLA A2, in particular) results in the destruction of melanoma cells as well as normal cells that produce MART-1 ( awakami et al., J. Immunol. 21:237, 1998). As disclosed herein, discovery of antigens expressed by the breast, pancreas and testes can similarly be used to design immunotherapy methods for pancreatic, testicular, and breast cancer.
  • MPR9 is expressed in cells of the breast, testes, and pancreas, and in cancer cells.
  • an antibody that specifically binds an antigenic epitope of an MRP9 polypeptide.
  • a method is provided for detecting cancer in subject. The method includes detection of MRP9 polypeptide in a sample from the subject. As disclosed herein, two transcripts are made from an endogenous MRP9 gene, a transcript of about 1.3 kb in length and a transcript of about 4.5 kb in length. Breast cells and cancer cells express the 4.5 kb variant of MRP9. In one embodiment, methods are provided for detecting cancer cells in a sample, by detecting mRNA encoding MRP9 of about 4.5 kb in length. In one specific, non- limiting example, the cancer cells are breast cancer cells.
  • immunotherapeutics are provided that are of use in the treatment of cancer.
  • Figs. 1A-1D are a set of schematic diagrams of the MRP9 cDNA and its variants.
  • Fig. 1A is a schematic diagram showing variants of MRP9 transcript and predicted open reading frames. The 4.5 kb transcript has 26 exons and the open reading frame starts from exon 1.
  • the MRP9 gene has 29 exons (GenBank Accession No. AY040220, herein incorporated by reference). However, the cDNA described herein isolated from testes has an ORF of only 930 amino acids. The major difference between GenBank Accession No.
  • AY040220 and the cDNA from the testes is that exons 5, 16, and 26 were not detected, and a 30 base pair sequence at the 5' end of exon 22 was not detected (Fig. IB).
  • the 1.8 kb transcript has seven exons and has the ORF of 233 amino acids.
  • the name and the location of the PCR primers used are shown by arrows and the location of the probes is shown by black rectangles.
  • Fig. 1C is a schematic diagram showing the probable topology of the MRP9 translated protein. Eight possible membrane spanning regions are numbered and the number of amino acids exposed to the outside of the cells are mentioned.
  • Fig. ID is a schematic diagram showing the design and sequence of the PCR primers used in Fig 4.
  • the sequence of the T418 primer (SEQ ID NO: 2), the T419 primer (SEQ LD NO: 3), a portion of the exon 21 sequence (SEQ ID NO: 4), and exon 22 (SEQ ID NO: 5) are shown.
  • Figs. 2A-2D are a set of digital images showing the tissue distribution of MRP9 mRNA expression.
  • Fig. 2A is a digital image showing DNA hybridization of a multiple tissue dot blot containing mRNA from 50 normal human cell types or tissues using a cDNA probe from the 3'end of the MRP9 transcript.
  • Fig. 2B is a digital image of RNA hybridization of the same blot used in A with 5' specific probe. A specific signal is detected only in testis (F8).
  • Fig. 2B is a digital image of RNA hybridization of the same blot used in A with 5' specific probe. A specific signal is detected only in testis (F8).
  • 2C is a digital image of PCR on cDNA from 24 different human tissues (Rapid Scan panel, Origene); expected size of the MRP9 PCR product is 400 bp using a 3' specific primer pair.
  • Lanes are: 1, Brain; 2, Heart; 3, Kidney; 4, Spleen; 5, Liver; 6, Colon; 7, Lung; 8, Small Intestine; 9, Muscle; 10, Stomach; 11, Testis; 12, Placenta; 13, Salivary gland; 14, Thyroid gland; 15, Adrenal gland; 16, Pancreas; 17, Ovary; 18, Uterus; 19, Prostate; 20, Skin; 21, Peripheral blood leukocytes; 22, Bone marrow; 23, Fetal brain; 24, Fetal Liver. Fig.
  • 2D is a digital image of a PCR using 5'- specific primer pair on cDNA from 24 different human tissues.
  • the expected size of the MRP9 PCR product is 400 bp.
  • PCR product is detected in testis (lane 11), normal breast (lane Br) and breast cancer cell lines (lane Be).
  • Figs. 3A-3D are a set of digital images of a Northern blot analyses showing expression and transcript sizes of MRP9 in different normal-tissues.
  • a PCR generated probe from the 3'end (Fig. 3A) and (Fig. 3B) and from the 5' end (Fig. 3C) of the MRP9 cDNA was used for hybridization.
  • Lanes are: Spleen, S; Thymus, Th; Prostate, Pr; Testis, Te; Ovary, Ov; Small intestine, In; Colon, C; Peripheral blood leukocyte, Pb; Heart, H; Brain, Bn; Placenta, PI; Lung, Lu; Liver, Li; Skeletal muscle, Sm; Kidney, K and Pancreas, Pn.
  • Figs. 4A-4B are a set of digital images of PCR analysis of MRP9 variant in different tissues.
  • Fig. 4 A is a digital image of an RT PCR analysis of testis and breast RNA using either T416/T399 or T417/T399 primer pair. Lanes 1 (T417/T399 ) and 2 (T416/T399) for breast; lane 3 (T417/T399) and 4 (T416/T399) for testis. Lane 5 is negative control and MW is molecular weight standard.
  • Fig. 4B is a digital image of an RT-PCR analysis of brain and testis RNA with T412, T413, T414 and T415 as 5' primer and T386 as 3' primer. Lanes 1 to 4 are for primers T412, T413, T414 and T415 respectively for brain; lanes 5 to 8 are for primers T412, T413, T414 and T415 respectively for testis. MW is molecular weight standard
  • Fig. 5 is a digital image of Rapid Scan PCR analysis on cDNAs from 12 different breast cancer specimens (lane 1 to 12).
  • Fig. 6 is a set of digital image of in situ hybridization experiments. Breast cancer tissue section stained with CD22 (Fig. 6A) and U6 probe (Fig. 6B) used as a negative control (CD22) and positive control (U6) respectively.
  • Fig. 6C is a digital image of a serial section of the same cancer tissue stained with a 5' specific MRP9 probe (MRP9-5'). A strong signal is found in the tumor cells.
  • Figs. 7A-7B are a set of digital images showing an analysis of the protein product encoded by 4.5 kb variant of MRP9.
  • Fig. 7A is a digital image of an analysis of the in vitro translated products of MRP9 cDNA: 4.5 kb variant of MRP9 cDNA was transcribed in vitro with T7 RNA polymerase and coupled translated with rabbit reticulocyte lysate in presence of 35S-methionine. The translated products were analyzed by SDS-PAGE and fluorography. Lane 1, luciferase cDNA as positive control; lane 2, no DNA; lane 3, MRP9 cDNA on anti-sense orientation; lane 4, MRP9 cDNA in sense orientation.
  • Fig. 1 is a digital image of an analysis of the in vitro translated products of MRP9 cDNA: 4.5 kb variant of MRP9 cDNA was transcribed in vitro with T7 RNA polymerase and coupled translated with rabbit reticulocyte lysate
  • FIG. 7B is a digital image of a Western blot analysis of anti-MRP9 peptide antisera.
  • a specific protein of molecular weight about 100 kDa is detected by anti-MRP9 IgG only in testis (Te) tissue extract.
  • the tissue extract from brain (Bn), heart (H), liver (Li), kidney (Ki) and prostate (Pr) showed no detectable signal.
  • SEQ ID NO: 1 is the amino acid sequence of MRP9.
  • SEQ ID NO: 2 is the nucleic acid sequence of the T418 primer.
  • SEQ ID NO: 3 is nucleic acid sequence of the T419 primer.
  • SEQ ID NO: 4 is the nucleic acid sequence of a portion of the MRP9 gene.
  • SEQ ID NO: 5 is the nucleic acid sequence of exon 22 of MRP9.
  • SEQ ID NO: 6 is the sequence of T399.
  • SEQ ID NO: 7 is the nucleic acid sequence of the T385 primer.
  • SEQ ID NO: 8 is the nucleic acid sequence of the T386 primer.
  • SEQ ID NO: 9 is the nucleic acid sequence of the T396 primer.
  • SEQ ID NO: 10 is the nucleic acid sequence of the T412 primer.
  • SEQ ID NO: 11 is the nucleic acid sequence of the T413 primer.
  • SEQ ID NO: 12 is the nucleic acid sequence of the T414 primer.
  • SEQ ID NO: 13 is the nucleic acid sequence of the T415 primer.
  • SEQ ID NO: 14 is the nucleic acid sequence of a nucleic acid encoding SEQ ID NO: l.
  • ABCC ATP binding cassette transporter
  • C ATP adenosine triphosphate
  • MRP9 Multidrug resistance protein 9 ORF: open reading frame TM: transmembrane
  • ABC Transporter A protein encoded by the ABC (ATP-binding cassette transporter) gene superfamily. This family of genes encodes active transporter proteins and constitutes a family of proteins that have been extremely well conserved through evolution, from bacteria to humans (Ames and Lecar, FASEB I., 6:2660-2666, 1992).
  • the ABC proteins are involved in extra- and intracellular membrane transport of various substrates, for example ions, amino acids, peptides, sugars, vitamins, or steroid hormones.
  • the prototype ABC protein binds ATP and uses the energy from ATP hydrolysis to drive the transport of various molecules across cell membranes.
  • the functional protein contains two ATP-binding domains (nucleotide binding fold, NBF) and two transmembrane (TM) domains.
  • the genes are typically organized as full transporters containing two of each domain, or half transporters with only one of each domain. Most full transporters are arranged in a TM-NBF-TM-NBF fashion (Dean et al., Curr Opin Genet, 5:79-785, 1995).
  • Abnormal A deviation from normal characteristics. "Normal" characteristics can be found in a control, a standard for a population, etc. For instance, where the abnormal condition is a tumor, such as a breast or a testicular tumor, sources of normal characteristics might include an individual who does not have the tumor, a population standard of individuals believed not to have a neoplastic disease, etc.
  • Amplification With regard to a nucleic acid molecule (e.g., a DNA or RNA molecule, amplification refers to use of a technique that increases the number of copies of a nucleic acid molecule in a specimen.
  • An example of amplification is the polymerase chain reaction, in which a biological sample collected from a subject is contacted with a pair of oligonucleotide primers, under conditions that allow for the hybridization of the primers to a nucleic acid template in the sample.
  • the primers are extended under suitable conditions, dissociated from the template, and then re-annealed, extended, and dissociated to amplify the number of copies of the nucleic acid.
  • the product of amplification may be characterized by electrophoresis, restriction endonuclease cleavage patterns, oligonucleotide hybridization or ligation, and/or nucleic acid sequencing using standard techniques.
  • Other examples of amplification include strand displacement amplification, as disclosed in U.S. Patent No. 5,744,311; transcription-free isothermal amplification, as disclosed in U.S. Patent No. 6,033,881; repair chain reaction amplification, as disclosed in WO 90/01069; ligase chain reaction amplification, as disclosed in EP-A-320 308; gap filling ligase chain reaction amplification, as disclosed in U.S. Patent No.
  • Antibody Immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that specifically binds (immunoreacts with) an antigen. Monoclonal and polyclonal antibodies are included.
  • a naturally occurring antibody e.g., IgG, IgM, IgD
  • IgG, IgM, IgD includes four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds.
  • H heavy
  • L light
  • binding fragments encompassed within the term antibody include (i) a Fab fragment consisting of the VL, NH, CL and CHI domains; (ii) an Fd fragment consisting of the VH and CHI domains; (iii) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (iv) a dAb fragment (Ward et al., Nature 341:544-546, 1989) which consists of a VH domain; (v) an isolated complimentarity determining region (CDR); and (vi) a F(ab') 2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region.
  • Immunoglobulins and certain variants thereof are known and many have been prepared in recombinant cell culture (e.g., see U.S. Patent No. 4,745,055; U.S. Patent No. 4,444,487; WO 88/03565; EP 256,654; EP 120,694; EP 125, 023; Faoulkner et al, Nature 298:286, 1982; Morrison, J. Immunol. 123:793, 1979; Morrison et al., Ann Rev. Immunol 2:239, 1984).
  • Animal Living multi-cellular vertebrate organisms, a category that includes, for example, mammals and birds.
  • mammal includes both human and non-human mammals.
  • subject includes both human and veterinary subjects.
  • Avidity The overall strength of interaction between two molecules, such as an antigen and an antibody. Avidity depends on both the affinity and the valency of interactions. Therefore, the avidity of a pentameric IgM antibody, with ten antigen binding sites, for a multivalent antigen may be much greater than the avidity of a dimeric IgG molecule for the same antigen.
  • Biological sample Any sample in which the presence of a protein and/or ongoing expression of a protein may be detected. Suitable biological samples include samples containing genomic DNA or RNA (including mRNA), obtained from body cells of a subject, such as those present in peripheral blood, urine, saliva, tissue biopsy, surgical specimen, amniocentesis samples and autopsy material.
  • Breast cancer A neoplastic condition of breast tissue that can be benign or malignant.
  • the most common type of breast cancer is ductal carcinoma.
  • Ductal carcinoma in situ is a non-invasive neoplastic condition of the ducts.
  • Lobular carcinoma is not an invasive disease but is an indicator that a carcinoma may develop.
  • Infiltrating (malignant) carcinoma of the breast can be divided into stages (I, IIA, IIB, ⁇ iA, mB, and IN).
  • Chemotherapeutic agents Any chemical agent with therapeutic usefulness in the treatment of diseases characterized by abnormal cell growth. Such diseases include tumors, neoplasms, and cancer as well as diseases characterized by hyperplastic growth such as psoriasis.
  • a chemotherapeutic agent is an agent of use in treating breast cancer.
  • a chemotherapeutic agent is a radioactive compound.
  • One of skill in the art can readily identify a chemotherapeutic agent of use (e.g. see Slapak and Kufe, Principles of Cancer Therapy, Chapter 86 in Harrison's Principles of Internal Medicine, 14th edition; Perry et al., Chemotherapy, Ch.
  • cD A complementary D ⁇ A: A piece of D ⁇ A lacking internal, non- coding segments (introns) and regulatory sequences that determine transcription. cD ⁇ A is synthesized in the laboratory by reverse transcription from messenger R ⁇ A extracted from cells.
  • Degenerate variant A polynucleotide encoding a MRP9 polypeptide that includes a sequence that is degenerate as a result of the genetic code. There are 20 natural amino acids, most of which are specified by more than one codon. Therefore, all degenerate nucleotide sequences are included in the invention as long as the amino acid sequence of the MRP9 polypeptide encoded by the nucleotide sequence is unchanged.
  • Epitope An antigenic determinant. These are particular chemical groups or peptide sequences on a molecule that are antigenic, i.e. that elicit a specific immune response. An antibody specifically binds a particular antigenic epitope on a polypeptide, such as an MRP9 polypeptide.
  • Expression Control Sequences Nucleic acid sequences that regulate the expression of a heterologous nucleic acid sequence to which it is operatively linked. Expression control sequences are operatively linked to a nucleic acid sequence when the expression control sequences control and regulate the transcription and, as appropriate, translation of the nucleic acid sequence.
  • expression control sequences can include appropriate promoters, enhancers, transcription terminators, a start codon (i.e., ATG) in front of a protein-encoding gene, splicing signal for introns, maintenance of the correct reading frame of that gene to permit proper translation of mRNA, and stop codons.
  • control sequences is intended to include, at a minimum, components whose presence can influence expression, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences.
  • Expression control sequences can include a promoter.
  • a promoter is a minimal sequence sufficient to direct transcription.
  • those promoter elements which are sufficient to render promoter- dependent gene expression controllable for cell-type specific, tissue-specific, or inducible by external signals or agents; such elements may be located in the 5 ' or 3' regions of the gene. Both constitutive and inducible promoters, are included (see e.g., Bitter et al., Methods in Enzymology 153:516-544, 1987).
  • inducible promoters such as pL of bacteriophage lambda , plac, ptrp, ptac (ptrp-lac hybrid promoter) and the like may be used.
  • promoters derived from the • genome of mammalian cells e.g., metallothionein promoter
  • mammalian viruses e.g., the retrovirus long terminal repeat; the adenovirus late promoter; the vaccinia virus 7.5K promoter
  • Promoters produced by recombinant DNA or synthetic techniques may also be used to provide for transcription of the nucleic acid sequences.
  • Host cells Cells in which a vector can be propagated and its DNA expressed.
  • the cell may be prokaryotic or eukaryotic.
  • the term also includes any progeny of the subject host cell. It is understood that all progeny may not be identical to the parental cell since there may be mutations that occur during replication. However, such progeny are included when the term "host cell” is used.
  • Immune response A response of a cell of the immune system, such as a B cell, T cell, or monocyte, to a stimulus. In one embodiment, the response is specific for a particular antigen (an "antigen-specific response"). In one embodiment, an immune response is a T cell response, such as a CD4+ response or a CD8+ response. In another embodiment, the response is a B cell response, and results in the production of specific antibodies.
  • Immunoconjugate A covalent linkage of an effector molecule to an antibody.
  • the effector molecule can be a chemotherapeutic agent or an immunotoxin.
  • toxins include, but are not limited to, abrin, ricin A, Pseudomonas exotoxin (PE, such as PE35, PE37, PE38, and PE40), diphtheria toxin (DT), botulinum toxin, or modified toxins thereof, or other toxic agents that directly or indirectly inhibit cell growth or kill cells.
  • PE and DT are highly toxic compounds that typically bring about death through liver toxicity.
  • PE and DT can be modified into a form for use as an immunotoxin by removing the native targeting component of the toxin (e.g., domain la of PE and the B chain of DT) and replacing it with a different targeting moiety, such as an antibody.
  • a "chimeric molecule” is a targeting moiety, such as a ligand or an antibody, conjugated (coupled) to an effector molecule.
  • conjugated or “linked” refers to making two polypeptides into one contiguous polypeptide molecule.
  • an antibody is joined to an effector molecule (EM).
  • an antibody joined to an effector molecule is further joined to a lipid or other molecule, such as a protein or peptide, to increase its half-life in the body.
  • the linkage can be either by chemical or recombinant means.
  • the linkage is chemical, wherein a reaction between the antibody moiety and the effector molecule has produced a covalent bond formed between the two molecules to form one molecule.
  • a peptide linker (short peptide sequence) can optionally be included between the antibody and the effector molecule.
  • Immunogenic peptide A peptide which comprises an allele-specific motif or other sequence such that the peptide will bind an MHC molecule and induce a cytotoxic T lymphocyte ("CTL") response, or a B cell response (e.g. antibody production) against the antigen from which the immunogenic peptide is derived.
  • CTL cytotoxic T lymphocyte
  • B cell response e.g. antibody production
  • immunogenic peptides are identified using sequence motifs or other methods, such as neural net or polynomial determinations, known in the art. Typically, algorithms are used to determine the "binding threshold" of peptides to select those with scores that give them a high probability of binding at a certain affinity and will be immunogenic.
  • the algorithms are based either on the effects on MHC binding of a particular amino acid at a particular position, the effects on antibody binding of a particular amino acid at a particular position, or the effects on binding of a particular substitution in a motif-containing peptide.
  • a conserved residue is one which appears in a significantly higher frequency than would be expected by random distribution at a particular position in a peptide.
  • a conserved residue is one where the MHC structure may provide a contact point with the immunogenic peptide.
  • Immunogenic composition A composition comprising a MRP9 polypeptide that induces a measurable CTL response against cells expressing MRP9 polypeptide, or induces a measurable B cell response (e.g. production of antibodies that specifically bind MRP9) against a MRP9 polypeptide. It further refers to isolated nucleic acids encoding a MRP9 polypeptide that can be used to express the MRP9 polypeptide (and thus be used to elicit an immune response against this polypeptide).
  • the immunogenic composition may consist of the isolated protein or peptide.
  • the immunogenic composition will typically comprise the protein or peptide in pharmaceutically acceptable carriers, and/or other agents. Any particular peptide, MRP9 polypeptide, or nucleic acid encoding the polypeptide, can be readily tested for its ability to induce a T cell or B cell response by art-recognized assays.
  • Isolated An "isolated" biological component (such as a nucleic acid or protein or organelle) has been substantially separated or purified away from other biological components in the cell of the organism in which the component naturally occurs, i.e., other chromosomal and extra-chromosomal DNA and RNA, proteins and organelles.
  • Nucleic acids and proteins that have been "isolated” include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids.
  • Label A detectable compound or composition that is conjugated directly or indirectly to another molecule to facilitate detection of that molecule. Specific, non- limiting examples of labels include fluorescent tags, enzymatic linkages, and radioactive isotopes.
  • Lymphocytes A type of white blood cell that is involved in the immune defenses of the body. There are two main types of lymphocytes: B cells and T cells. Mammal: This term includes both human and non-human mammals.
  • subject includes both human and veterinary subjects.
  • Monoclonal antibody An antibody produced by a single clone of B- lymphocytes. Monoclonal antibodies are produced by methods known to those of skill in the art, for instance by making hybrid antibody-forming cells from a fusion of myeloma cells with immune spleen cells.
  • MRP9 A polypeptide, also known as ABCC12, that binds ATP and functions in transportation of a molecule across a membrane.
  • MRP9 has a sequence as set forth as GenBank Accession No. XM_054602, AY040220, or NM_033226, all of which are herein incorporated by reference, and conservative variants thereof.
  • Oligonucleotide A linear polynucleotide sequence of up to about 100 nucleotide bases in length.
  • ORF Open reading frame
  • a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence.
  • a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.
  • operably linked DNA sequences are contiguous and, where necessary to join two protein-coding regions, in the same reading frame.
  • Peptide A chain of amino acids of between 3 and 30 amino acids in length. In one embodiment, a peptide is from about 10 to about 25 amino acids in length. In yet another embodiment, a peptide is from about 11 to about 20 amino acids in length. In yet another embodiment, a peptide is about 8 amino acids in length.
  • Peptide Modifications MRP9 polypeptides include synthetic embodiments of peptides described herein. In addition, analogues (non-peptide organic molecules), derivatives (chemically functionalized peptide molecules obtained starting with the disclosed peptide sequences) and variants (homologs) of these proteins can be utilized in the methods described herein. Each polypeptide of the invention is comprised of a sequence of amino acids, which may be either L- and/or D- amino acids, naturally occurring and otherwise.
  • Peptides may be modified by a variety of chemical techniques to produce derivatives having essentially the same activity as the unmodified peptides, and optionally having other desirable properties.
  • carboxylic acid groups of the protein may be provided in the form of a salt of a pharmaceutically-acceptable cation or esterified to form a C ⁇ -C 16 ester, or converted to an amide of formula NRiR wherein Ri and R 2 are each independently H or Ci-C 16 alkyl, or combined to form a heterocyclic ring, such as a 5- or 6- membered ring.
  • Amino groups of the peptide may be in the form of a pharmaceutically-acceptable acid addition salt, such as the HCl, HBr, acetic, benzoic, toluene sulfonic, maleic, tartaric and other organic salts, or may be modified to -C ⁇ 6 alkyl or dialkyl amino or further converted to an amide.
  • Hydroxyl groups of the peptide side chains may be converted to Ci- ⁇ alkoxy or to a C ⁇ -C 16 ester using well-recognized techniques.
  • Phenyl and phenolic rings of the peptide side chains may be substituted with one or more halogen atoms, such as fluorine, chlorine, bromine or iodine, or with Ci-C ⁇ 6 alkyl, C ⁇ -C 16 alkoxy, carboxylic acids and esters thereof, or amides of such carboxylic acids.
  • Methylene groups of the peptide side chains can be extended to homologous C 2 -C alkylenes.
  • Thiols can be protected with any one of a number of well-recognized protecting groups, such as acetamide groups.
  • Peptidomimetic and organomimetic embodiments are envisioned, whereby the three-dimensional arrangement of the chemical constituents of such peptido- and organomimetics mimic the three-dimensional arrangement of the peptide backbone and component amino acid side chains, resulting in such peptido- and organomimetics of a MRP9 polypeptide having measurable or enhanced ability to generate an immune response.
  • a pharmacophore is an idealized, three-dimensional definition of the structural requirements for biological activity.
  • Peptido- and organomimetics can be designed to fit each pharmacophore with current computer modeling software (using computer assisted drug design or CADD).
  • compositions and formulations suitable for pharmaceutical delivery of the fusion proteins herein disclosed are conventional. Remington's Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, PA, 15th Edition (1975), describes compositions and formulations suitable for pharmaceutical delivery of the fusion proteins herein disclosed.
  • parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle.
  • pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle.
  • physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like
  • solid compositions e.g., powder, pill, tablet, or capsule forms
  • conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate.
  • compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
  • non-toxic auxiliary substances such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
  • Polynucleotide refers to a polymeric form of nucleotide at least 10 bases in length.
  • a recombinant polynucleotide includes a polynucleotide that is not immediately contiguous with both of the coding sequences with which it is immediately contiguous (one on the 5' end and one on the 3' end) in the naturally occurring genome of the organism from which it is derived.
  • the term therefore includes, for example, a recombinant DNA which is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., a cDNA) independent of other sequences.
  • the nucleotides can be ribonucleotides, deoxyribonucleotides, or modified forms of either nucleotide.
  • the term includes single -and double -stranded forms of DNA.
  • Polypeptide Any chain of amino acids, regardless of length or post- translational modification (e.g., glycosylation or phosphorylation).
  • the polypeptide is MRP9 polypeptide.
  • a probe comprises an isolated nucleic acid attached to a detectable label or reporter molecule.
  • Primers are short nucleic acids, preferably DNA oligonucleotides 15 nucleotides or more in length. Primers may be annealed to a complementary target DNA strand by nucleic acid hybridization to form a hybrid between the primer and the target DNA strand, and then extended along the target DNA strand by a DNA polymerase enzyme. Primer pairs can be used for amplification of a nucleic acid sequence, e.g., by the polymerase chain reaction (PCR) or other nucleic-acid amplification methods known in the art.
  • PCR polymerase chain reaction
  • a promoter is an array of nucleic acid control sequences that directs transcription of a nucleic acid.
  • a promoter includes necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element.
  • a promoter also optionally includes distal enhancer or repressor elements which can be located as much as several thousand base pairs from the start site of transcription.
  • promoters include promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the retrovirus long terminal repeat; the adenovirus late promoter; the vaccinia virus 7.5K promoter) may be used. Promoters produced by recombinant DNA or synthetic techniques may also be used.
  • a polynucleotide can be inserted into an expression vector that contains a promoter sequence which facilitates the efficient transcription of the inserted genetic sequence of the host.
  • the expression vector typically contains an origin of replication, a promoter, as well as specific nucleic acid sequences that allow phenotypic selection of the transformed cells
  • the MRP9 polypeptides disclosed herein can be purified by any of the means known in the art. See, e.g., Guide to Protein Purification, ed. Academic Press, San Diego, 1990; and Scopes, Protein Purification: Principles and Practice, Springer Verlag, New York, 1982. Recombinant expression is one method for producing purified proteins. Substantial purification denotes purification from other proteins or cellular components. A substantially purified protein is at least 60%, 70%, 80%, 90%, 95% or 98% pure. Thus, in one specific, non-limiting example, a substantially purified protein is 90% free of other proteins or cellular components.
  • purified does not require absolute purity; rather, it is intended as a relative term.
  • a purified peptide preparation is one in which the peptide or protein is more enriched than the peptide or protein is in its natural environment within a cell.
  • a preparation is purified such that the protein or peptide represents at least 50% of the total peptide or protein content of the preparation.
  • Radioimmune assay a technique used to detect the presence of antigen-antibody binding using the measurement of radioactivity as the method of detection. Such techniques are well known in the art (See, for example, Raychaudhuri et al, J. Virol. 72(9): 7467-76, 1998).
  • Recombinant A recombinant nucleic acid is one that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques.
  • nucleic acid hybridization reactions the conditions used to achieve a particular level of stringency will vary, depending on the nature of the nucleic acids being hybridized. For example, the length, degree of complementarity, nucleotide sequence composition (e.g., GC v. AT content), and nucleic acid type (e.g., RNA v. DNA) of the hybridizing regions of the nucleic acids can be considered in selecting hybridization conditions. An additional consideration is whether one of the nucleic acids is immobilized, for example, on a filter.
  • a specific, non-limiting example of progressively higher stringency conditions is as follows: 2 x SSC/0.1% SDS at about room temperature (hybridization conditions); 0.2 x SSC/0.1% SDS at about room temperature (low stringency conditions); 0.2 x SSC/0.1% SDS at about 42° C (moderate stringency conditions); and 0.1 x SSC at about 68° C (high stringency conditions).
  • Hybridization conditions 2 x SSC/0.1% SDS at about room temperature
  • low stringency conditions low stringency conditions
  • 0.2 x SSC/0.1% SDS at about 42° C moderate stringency conditions
  • 0.1 x SSC at about 68° C high stringency conditions
  • Washing can be carried out using only one of these conditions, e.g., high stringency conditions, or each of the conditions can be used, e.g., for 10-15 minutes each, in the order listed above, repeating any or all of the steps listed.
  • optimal conditions will vary, depending on the particular hybridization reaction involved, and can be determined empirically.
  • Sequence identity The similarity between amino acid sequences is expressed in terms of the similarity between the sequences, otherwise referred to as sequence identity. Sequence identity is frequently measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences are. Homologues or variants of a MRP9 polypeptide will possess a relatively high degree of sequence identity when aligned using standard methods. Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith and Waterman, Adv. Appl. Math. 2:482, 1981; Needleman and Wunsch, J. Mol Biol 48:443, 1970; Pearson and Lipman, Proc. Natl Acad. Sci. U.S.A.
  • NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol Biol. 215:403, 1990) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, MD) and on the Internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. A description of how to determine sequence identity using this program is available on the NCBI website on the internet.
  • Homologues and variants of a MRP9 polypeptide are typically characterized by possession of at least 80%, for example at least 85%, sequence identity counted over the full length alignment with the amino acid sequence of MRP9 using the NCBI Blast 2.0, gapped blastp set to default parameters.
  • the Blast 2 sequences function is employed using the default BLOSUM62 matrix set to default parameters, (gap existence cost of 11, and a per residue gap cost of 1).
  • the alignment should be performed using the Blast 2 sequences function, employing the PAM30 matrix set to default parameters (open gap 9, extension gap 1 penalties). Proteins with even greater similarity to the reference sequences will show increasing percentage identities when assessed by this method, such as at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.
  • homologues and variants When less than the entire sequence is being compared for sequence identity, homologues and variants will typically possess at least 80% sequence identity over short windows of 10-20 amino acids, and may possess sequence identities of at least 85% or at least 90% or 95% depending on their similarity to the reference sequence. Methods for determining sequence identity over such short windows are available at the NCBI website on the internet. One of skill in the art will appreciate that these sequence identity ranges are provided for guidance only; it is entirely possible that strongly significant homologues could be obtained that fall outside of the ranges provided.
  • Specific binding agent An agent that binds substantially only to a defined target.
  • a MRP9 specific binding agent is an agent that binds substantially to an MRP9 polypeptide.
  • the specific binding agent is a monoclonal or polyclonal antibody that specifically binds MRP9.
  • Antibodies can be produced using molecular procedures described in a number of texts, including Harlow and Lane (Antibodies, A Laboratory Manual, CSHL, New York, 1988). The determination that a particular agent specifically binds to a target MRP9 polypeptide indicates that the antibody binds substantially only to the MRP9 polypeptide and not to unrelated polypeptides. A determination that an antibody specifically binds MRP9 can readily be made by using or adapting routine procedures.
  • One suitable in vitro assay makes use of the Western blotting procedure (described in many standard texts, including Harlow and Lane, Antibodies, A Laboratory Manual, CSHL, New York, 1988). Western blotting may be used to determine that a given protein binding agent, such as an anti-MRP9 antibody, specifically binds the specified MRP9 polypeptide.
  • Subject Living multi-cellular vertebrate organisms, a category that includes both human and veterinary subjects, including human and non-human mammals.
  • T Cell A white blood cell critical to the immune response.
  • T cells include, but are not limited to, CD4 + T cells and CD8 + T cells.
  • a CD4 + T lymphocyte is an immune cell that carries a marker on its surface known as "cluster of differentiation 4" (CD4). These cells, also known as helper T cells, help orchestrate the immune response, including antibody responses as well as killer T cell responses.
  • CD8 + T cells carry the "cluster of differentiation 8" (CD8) marker.
  • a CD8 T cells is a cytotoxic T lymphocytes, hi another embodiment, a CD8 cell is a suppressor T cell.
  • Therapeutically active polypeptide An agent, such as a MRP9 polypeptide that causes induction of an immune response, as measured by clinical response (for example increase in a population of immune cells, production of antibody that specifically binds MRP9, or measurable reduction of tumor burden).
  • Therapeutically active molecules can also be made from nucleic acids. Examples of a nucleic acid based therapeutically active molecule include a nucleic acid sequence that encodes a MRP9 polypeptide, wherein the nucleic acid sequence is operably linked to a control element such as a promoter.
  • Therapeutically active agents can also include organic or other chemical compounds that mimic the effects of MRP9.
  • the terms "therapeutically effective fragment of MRP9” or “therapeutically effective variant of MRP9” includes any fragment of MRP9, or variant of MRP9, that retains a function of MRP9, or retains an antigenic epitope of MRP9.
  • MRP9 is an amount used to generate an immune response, or to treat breast cancer in a subject.
  • Specific, non-limiting examples are the N-terminal half of MRP9 or the
  • transduced A transduced cell is a cell into which has been introduced a nucleic acid molecule by molecular biology techniques.
  • transduction encompasses all techniques by which a nucleic acid molecule might be introduced into such a cell, including transfection with viral vectors, transformation with plasmid vectors, and introduction of naked DNA by electroporation, lipofection, and particle gun acceleration.
  • a nucleic acid molecule as introduced into a host cell, thereby producing a transformed host cell.
  • a vector may include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication.
  • a vector may also include one or more selectable marker genes and other genetic elements known in the art.
  • MRP9 is a polypeptide, also known as ABCC12, that binds ATP and functions in transportation of a molecule across a membrane.
  • MRP9 has a sequence as set forth as GenBank Accession No. XM_054602, AY040220, or NM_033226, all of which are herein incorporated by reference, and conservative variants thereof.
  • the polypeptide sequence of MRP9 follows:
  • MVGEGPYLISDLDQRGRRRSFAERYDPSLKTMIPVRPCARLAP NPVDDAGLLSFATFSWLTPVMVKGYRQRLTVDTLPPLSTYDS SDTNAKRFRVLWDEEVARVGPEKASLSHVVWKFQRTRVLM DIVANILCII-MAAIGPTVLIHQILQQTERTSGKVWVGIGLCIALF , ATEFTKVFFWALAWAINYRTAIRLKVALSTLVFENLVSFKTLT fflSVGEMFMAKLNSAFRRSAILVTDKRVQTMNEFLTCIRLIKM YAWEKSFTNTIQDRRRERKLLEKAGFVQSGNSALAPIVSTIAI VLTLSCHII-IJiRKLTAPVAre
  • nucleic acid sequence that encodes SEQ ID NO: 1 is as follows:
  • MRP9 polypeptides also include a series of contiguous amino acid resides from an MRP9 polypeptide of between about 7 and about 30 amino acids in length, such as between about 8 and about 20 amino acids in length. These polypeptides can be immunogenic. Specific, non-limiting examples of an MRP polypeptide, include, but are not limited to, at least about 15 consecutive amino acids of SEQ ID NO: 1, such as about 8, about 10, or about 15 consecutive amino acids of SEQ ID NO: 1.
  • an MRP9 polypeptide is a peptide that has an amino acid sequence at least 80% homologous to SEQ ID NO: 1, such as 85%, 90%, 95%, or 98% homologous to SEQ ID NO: 1, wherein the polypeptide retains an activity of MRP0 (e.g. binds to an antibody that binds MRP9 or can transport molecules across a cell membrane).
  • an MRP9 polypeptide is a conservative variant of SEQ ID NO: 1, wherein an antibody that binds SEQ ID NO: 1 binds the conservative variant.
  • an MRP9 polypeptide is a conservative variant of SEQ ID NO: 1, wherein the polypeptide retains the ability to transport molecules across a cell membrane.
  • MRP9 polypeptide or a fragment or conservative variant thereof can be used to produce antibodies which are immunoreactive or bind to an epitope of MRP9.
  • representative MRP9 polypeptides include, for example, polypeptides with at least 80% identity to SEQ ID NO: 1, such as about 85%, 90%, or 95% identity with SEQ ID NO: 1.
  • MRP9 polypeptides also include fusion proteins including an MRP9 sequence, such as a polypeptide including a histidine tag (e.g. six histidine residues) or another marker polypeptide (e.g. myc, beta-galactosidase, or any other antigenic epitope).
  • MRP9 polypeptides also include conservative variants of MRP9.
  • a conservative variant does not include amino acid substitutions in a transmembrane region of MRP9.
  • a conservative variant does not include substitutions in the ABC signature domain.
  • a conservative variant does not include substitutions in either the transmembrane domains or the ABC signature domain.
  • the transmembrane (TM) regions of SEQ ID NO: 1 are listed below:
  • TM2 159 to 182 of SEQ ID NO: 1 VWVGIGLCIALFATEFTKVFFWAL
  • the ABC signature motif is: Amino acids 544-560 of SEQ ID NO: 1
  • MRP9 polypeptides also include immunogenic fragments of at least about 8 amino acids in length, such as about ten, about twelve, about fifteen, or about fifteen consecutive residues of MRP9. It should be noted that the amino acid residues of MRP9 exposed to the outside of cells is enumerated in Fig. IB. Thus, using the information provided herein, including the amino acid sequence of MRP9, an antibody can be generated that specifically binds to one or more of the regions of MRP9 exposed to the outside of cells. Thus, for example, an antibody can be produced that binds to one or more of
  • amino acid residues included in the fourteen amino acids located in the region between transmembrane regions 3 and 4 amino acid residues included in the 39 amino acids located in the region between transmembrane regions 5 and 6; and/or
  • antibodies can be produced that specifically bind amino acids at the amino terminal or the carboxy terminal ends of the transmembrane region.
  • antibodies can be produced that specifically bind amino acid residues exposed to the outside of the cell, in addition to amino acid residues located in one or more transmembrane region.
  • Polyclonal antibodies which consist essentially of pooled monoclonal antibodies with different epitopic specificities, as well as distinct monoclonal antibody preparations are included herein.
  • polyclonal antibodies The preparation of polyclonal antibodies is well known to those skilled in the art. See, for example, Green et al., "Production of Polyclonal Antisera, in: Immunochemical Protocols pages 1-5, Manson, ed., Humana Press 1992; Coligan et al., "Production of Polyclonal Antisera in Rabbits, Rats, Mice and Hamsters, in: Current Protocols in Immunology, section 2.4.1, 1992. Production of a polyclonal antibody that binds MRP9 is described in the Examples.
  • monoclonal antibodies can be obtained by injecting mice with a composition "comprising an antigen, verifying the presence of antibody production by removing a serum sample, removing the spleen to obtain B lymphocytes, fusing the B lymphocytes with myeloma cells to produce hybridomas, cloning the hybridomas, selecting positive clones that produce antibodies to the antigen, and isolating the antibodies from the hybridoma cultures.
  • Monoclonal antibodies can be isolated and purified from hybridoma cultures by a variety of well- established techniques. Such isolation techniques include affinity chromatography with Protein- A Sepharose, size-exclusion chromatography, and ion-exchange chromatography.
  • Multiplication in vitro may be carried out in suitable culture media such as Dulbecco's Modified Eagle Medium or RPMI 1640 medium, optionally supplemented by a mammalian serum such as fetal calf serum or trace elements and growth-sustaining supplements such as normal mouse peritoneal exudate cells, spleen cells, thymocytes or bone marrow macrophages.
  • suitable culture media such as Dulbecco's Modified Eagle Medium or RPMI 1640 medium
  • a mammalian serum such as fetal calf serum or trace elements
  • growth-sustaining supplements such as normal mouse peritoneal exudate cells, spleen cells, thymocytes or bone marrow macrophages.
  • Multiplication in vivo may be carried out by injecting cell clones into mammals histocompatible with the parent cells, e.g., syngeneic mice, to cause growth of antibody-producing tumors.
  • the animals are primed with a hydrocarbon, especially oils such as pristane (tetramethylpentadecane) prior to injection. After one to three weeks, the desired monoclonal antibody is recovered from the body fluid of the animal.
  • Antibodies can also be derived from subhuman primate antibody. General techniques for raising therapeutically useful antibodies in baboons can be found, for example, in WO 91/11465, 1991, and Losman et al., Int I. Cancer 46:310, 1990.
  • an antibody that specifically binds an MRP9 polypeptide can be derived from a humanized monoclonal antibody. Humanized monoclonal antibodies are produced by transferring mouse complementarity determining regions from heavy and light variable chains of the mouse immunoglobulin into a human variable domain, and then substituting human residues in the framework regions of the murine counterparts. The use of antibody components derived from humanized monoclonal antibodies obviates potential problems associated with the immunogenicity of murine constant regions.
  • Antibodies can be derived from human antibody fragments isolated from a combinatorial immunoglobulin library. See, for example, Barbas et al., in: Methods: a Companion to Methods in Enzymology, Vol. 2, page 119, 1991; Winter et al., Ann. Rev. Immunol. 12:433, 1994.
  • Cloning and expression vectors that are useful for producing a human immunoglobulin phage library can be obtained, for example, from STRATAGENE Cloning Systems (La Jolla, CA).
  • antibodies can be derived from a human monoclonal antibody.
  • Such antibodies are obtained from transgenic mice that have been "engineered” to produce specific human antibodies in response to antigenic challenge.
  • elements of the human heavy and light chain loci are introduced into strains of mice derived from embryonic stem cell lines that contain targeted disruptions of the endogenous heavy and light chain loci.
  • the transgenic mice can synthesize human antibodies specific for human antigens, and the mice can be used to produce human antibody-secreting hybridomas.
  • Methods for obtaining human antibodies from transgenic mice are described by Green et al., Nature Genet. 7:13, 1994; Lonberg et al, Nature 368:856, 1994; and Taylor et al., Int. Immunol. 6:579, 1994.
  • Antibodies include intact molecules as well as fragments thereof, such as
  • Fab the fragment which contains a monovalent antigen-binding fragment of an antibody molecule, can be produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain;
  • Fab' the fragment of an antibody molecule can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; two Fab' fragments are obtained per antibody molecule;
  • (Fab') 2 the fragment of the antibody that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction
  • F(ab') 2 is a dimer of two Fab' fragments held together by two disulfide bonds
  • Fv defined as a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains
  • Single chain antibody defined as a genetically engineered molecule containing the variable region of the light chain, the variable region of the heavy chain, linked by a suitable polypeptide linker as a genetically fused single chain molecule.
  • An epitope is any antigenic determinant on an antigen to which the paratope of an antibody binds.
  • Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics.
  • Antibody fragments can be prepared by proteolytic hydrolysis of the antibody or by expression in E. coli of DNA encoding the fragment.
  • Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods.
  • antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab') 2 .
  • This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab' monovalent fragments.
  • cleaving antibodies such as separation of heavy chains to form monovalent light-heavy chain fragments, further cleavage of fragments, or other enzymatic, chemical, or genetic techniques may also be used, so long as the fragments bind to the antigen that is recognized by the intact antibody.
  • Fv fragments comprise an association of V H and V L chains. This association may be noncovalent (Inbar et al., Proc. Nat'l Acad. Sci. U.S.A. 69:2659, 1972).
  • the variable chains can be linked by an intermolecular disulfide bond or cross-linked by chemicals such as glutaraldehyde. See, e.g., Sandhu, supra.
  • the Fv fragments comprise VH and V L chains connected by a peptide linker.
  • sFv single-chain antigen binding proteins
  • the structural gene is inserted into an expression vector, which is subsequently introduced into a host cell such as E. coli.
  • the recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains.
  • Methods for producing sFvs are known in the art (see Whitlow et al., Methods: a Companion to Methods in Enzymology, Vol. 2, page 97, 1991; Bird et al, Science 242:423, 1988; U.S. Patent No. 4,946,778; Pack et al., Bio/Technology 11:1271, 1993; and Sandhu, supra).
  • CDR peptides (“minimal recognition units") can be obtained by constructing genes encoding the CDR of an antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region from RNA of antibody-producing cells (Larrick et al., Methods: a Companion to Methods in Enzymology, Vol. 2, page 106, 1991). Antibodies can be prepared using an intact polypeptide or fragments containing small peptides of interest as the immunizing antigen.
  • the polypeptide or a peptide used to immunize an animal can be derived from substantially purified polypeptide produced in host cells, in vitro translated cDNA, or chemical synthesis which can be conjugated to a carrier protein, if desired.
  • a carrier protein e.g., a carrier protein, a carrier protein, if desired.
  • commonly used carriers which are chemically coupled to the peptide include keyhole limpet hemocyanin (KLH), thyroglobulin, bovine serum albumin (BSA), and tetanus toxoid.
  • KLH keyhole limpet hemocyanin
  • BSA bovine serum albumin
  • the coupled peptide is then used to immunize the animal (e.g., a mouse, a rat, or a rabbit).
  • Polyclonal or monoclonal antibodies can be further purified, for example, by binding to and elution from a matrix to which the polypeptide or a peptide to which the antibodies were raised is bound.
  • a matrix to which the polypeptide or a peptide to which the antibodies were raised is bound.
  • monoclonal antibodies See for example, Coligan et al., Unit 9, Current Protocols in Immunology, Wiley Interscience, 1991). It is also possible to use the anti-idiotype technology to produce monoclonal antibodies which mimic an epitope.
  • an anti-idiotypic monoclonal antibody made to a first monoclonal antibody will have a binding domain in the hypervariable region that is the "image" of the epitope bound by the first monoclonal antibody.
  • Effector molecules e.g., therapeutic, diagnostic, or detection moieties, can be linked to an antibody that specifically binds MRP9, using any number of means known to those of skill in the art. Both covalent and noncovalent attachment means may be used. The procedure for attaching an effector molecule to an antibody varies according to the chemical structure of the effector.
  • Polypeptides typically contain a variety of functional groups; e.g., carboxylic acid (COOH), free amine (-NH 2 ) or sulfhydryl (-SH) groups, which are available for reaction with a suitable functional group on an antibody to result in the binding of the effector molecule.
  • the antibody is derivatized to expose or attach additional reactive functional groups.
  • the derivatization may involve attachment of any of a number of linker molecules such as those available from Pierce Chemical Company, Rockford Illinois.
  • the linker can be any molecule used to join the antibody to the effector molecule.
  • the linker is capable of forming covalent bonds to both the antibody and to the effector molecule.
  • Suitable linkers are well known to those of skill in the art and include, but are not limited to, straight or branched-chain carbon linkers, heterocyclic carbon linkers, or peptide linkers. Where the antibody and the effector molecule are polypeptides, the linkers may be joined to the constituent amino acids through their side groups (e.g., through a disulfide linkage to cysteine) or to the alpha carbon amino and carboxyl groups of the terminal amino acids.
  • immunoconjugates will comprise linkages that are cleavable in the vicinity of the target site. Cleavage of the linker to release the effector molecule from the antibody may be prompted by enzymatic activity or conditions to which the immunoconjugate is subjected either inside the target cell or in the vicinity of the target site.
  • a linker which is cleavable under conditions present at the tumor site e.g. when exposed to tumor-associated enzymes or acidic pH
  • An MRP9 polypeptide can be administered to a subject in order to generate an immune response.
  • a therapeutically effective amount of an MRP9 polypeptide is administered to a subject to treat cancer, such as breast, pancreatic, or testicular cancer.
  • the MRP9 polypeptide can be administered by any means known to one of skill in the art (see Banga, A., Parenteral Controlled Delivery of Therapeutic Peptides and Proteins, in Therapeutic Peptides and Proteins, Technomic Publishing Co., Inc., Lancaster, PA, 1995) such as by intramuscular, subcutaneous, or intravenous injection, but even oral, nasal, or anal administration is contemplated. In one embodiment, administration is by subcutaneous or intramuscular injection. To extend the time during which the peptide or protein is available to stimulate a response, the peptide or protein can be provided as an implant, an oily injection, or as a particulate system.
  • the particulate system can be a microparticle, a microcapsule, a microsphere, a nanocapsule, or similar particle, (see, e.g., Banja, supra).
  • a particulate carrier based on a synthetic polymer has been shown to act as an adjuvant to enhance the immune response, in addition to providing a controlled release.
  • Aluminum salts may also be used as adjuvants to produce a humoral immune response.
  • a MRP9 polypeptide is administered in a manner to induce a humoral response.
  • an MRP9 polypeptide is administered in a manner to direct the immune response to a cellular response (that is, a CTL response), rather than a humoral (antibody) response.
  • a cellular response that is, a CTL response
  • humoral (antibody) response a cellular response
  • Lipids have been identified as agents capable of assisting in priming CTL in vivo against various antigens. For example, as described in U.S. Patent No.
  • palmitic acid residues can be attached to the alpha and epsilon amino groups of a lysine residue and then linked (e.g., via one or more linking residues, such as glycine, glycine-glycine, serine, serine-serine, or the like) to an immunogenic peptide.
  • the lipidated peptide can then be injected directly in a micellar form, incorporated in a liposome, or emulsified in an adjuvant.
  • coli lipoproteins such as tripalmitoyl-S- glycerylcysteinlyseryl-serine can be used to prime tumor specific CTL when covalently attached to an appropriate peptide (see, Deres et al., Nature 342:561, 1989). Further, as the induction of neutralizing antibodies can also be primed with the same molecule conjugated to a peptide which displays an appropriate epitope, the two compositions can be combined to elicit both humoral and cell-mediated responses where that is deemed desirable.
  • a MHC class Il-restricted T-helper epitope is added to the CTL antigenic peptide to induce T-helper cells to secrete cytokines in the microenvironment to activate CTL precursor cells.
  • the technique further involves adding short lipid molecules to retain the construct at the site of the injection for several days to localize the antigen at the site of the injection and enhance its proximity to dendritic cells or other "professional” antigen presenting cells over a period of time (see Chesnut et al., "Design and Testing of Peptide-Based Cytotoxic T-Cell-Mediated Immunotherapeutics to Treat Infectious Diseases and Cancer," in Powell et al., eds., Vaccine Design, the Subunit and Adjuvant Approach, Plenum Press, New York, 1995).
  • a pharmaceutical composition including a MRP9 polypeptide can be utilized to evoke an immune response.
  • the MRP9 polypeptide, or fragment thereof is mixed with an adjuvant containing two or more of a stabilizing detergent, a micelle-forming agent, and an oil.
  • a stabilizing detergent is any detergent that allows the components of the emulsion to remain as a stable emulsion.
  • Such detergents include polysorbate, 80 (TWEEN) (Sorbitan-mono-9-octadecenoate- poly(oxy-l,2-ethanediyl; manufactured by ICI Americas, Wilmington, DE), TWEEN 40TM, TWEEN 20TM, TWEEN 60TM, ZwittergentTM 3-12, TEEPOL HB7 TM, and SPAN 85 TM. These detergents are usually provided in an amount of approximately 0.05 to 0.5%, preferably at about 0.2%.
  • a micelle-forming agent is an agent which is able to stabilize the emulsion formed with the other components such that a micelle-like structure is formed. Such agents generally cause some irritation at the site of injection in order to recruit macrophages to enhance the cellular response.
  • agents examples include polymer surfactants described by BASF Wyandotte publications, e.g., Schmolka, J. Am. Oil. Chem. Soc. 54:110, 1977, and Hunter et al., J. Immunol 129:1244, 1981, PLURONIC TM L62LF, L101, and L64, PEG1000, and TETRONICTM 1501, 150R1, 701, 901, 1301, and 130R1.
  • the chemical structures of such agents are well known in the art.
  • the agent is chosen to have a hydrophile-lipophile balance (HLB) of between 0 and 2, as defined by Hunter and Bennett, J. Immun. 133:3167, 1984.
  • HLB hydrophile-lipophile balance
  • the agent can be provided in an effective amount, for example between 0.5 and 10%, most preferably in an amount between 1.25 and 5%.
  • the oil included in the composition is chosen to promote the retention of the antigen in oil-in-water emulsion, i.e., to provide a vehicle for the desired antigen, and preferably has a melting temperature of less than 65° C such that emulsion is formed either at room temperature (about 20° C to 25° C), or once the temperature of the emulsion is brought down to room temperature.
  • oils include squalene, Squalane, EICOSANE TM, tetratetracontane, glycerol, and peanut oil or other vegetable oils.
  • the oil is provided in an amount between 1 and 10%, most preferably between 2.5 and 5%.
  • the oil should be both biodegradable and biocompatible so that the body can break down the oil over time, and so that no adverse effects, such as granulomas, are evident upon use of the oil.
  • an adjuvant can be included in the composition.
  • the adjuvant is a mixture of a stabilizing detergent, a micelle-forming agent, and an oil available under the name Provax® (IDEC Pharmaceuticals, San Diego, CA).
  • a pharmaceutical composition including a nucleic acid encoding a MRP9 polypeptide or an immunogenic fragment thereof is utilized to induce an immune response.
  • a therapeutically effective amount of the MRP9 polynucleotide is administered to a subject in order to generate an immune response.
  • a therapeutically effective amount of the MRP9 polynucleotide is administered to a subject to treat cancer, such as breast, testicular, or pancreatic cancer.
  • nucleic acids are direct immunization with plasmid DNA, such as with a mammalian expression plasmid.
  • plasmid DNA such as with a mammalian expression plasmid.
  • the nucleotide sequence encoding MRP9, or an immunogenic peptide thereof can be placed under the control of a promoter to increase expression of the molecule.
  • the methods include liposomal delivery of the nucleic acids (or of the synthetic peptides themselves), and immune- stimulating constructs, or ISCOMSTM, negatively charged cage-like structures of 30- 40 nm in size formed spontaneously on mixing cholesterol and Quil ATM (saponin).
  • ISCOMSTM immune- stimulating constructs
  • Protective immunity has been generated in a variety of experimental models of infection, including toxoplasmosis and Epstein-Barr virus-induced tumors, using ISCOMSTM as the delivery vehicle for antigens (Mowat and Donachie, Immunol. Today 12:383, 1991).
  • Doses of antigen as low as 1 ⁇ g encapsulated in ISCOMSTM have been found to produce class I mediated CTL responses (Takahashi et al., Nature 344:873, 1990).
  • an MRP9 polypeptide or an immunogenic peptide thereof can also be expressed by attenuated viral hosts or vectors or bacterial vectors.
  • Recombinant vaccinia virus, adeno- associated virus (AAV), herpesvirus, retrovirus, or other viral vectors can be used to express the peptide or protein, thereby eliciting a CTL response.
  • vaccinia vectors and methods useful in immunization protocols are described in U.S. Patent No. 4,722,848.
  • BCG Bacillus Calmette Guerin
  • a nucleic acid encoding a MRP9 polypeptide or an immunogenic fragment thereof is introduced directly into cells.
  • the nucleic acid may be loaded onto gold microspheres by standard methods and introduced into the skin by a device such as Bio-Rad's HeliosTM Gene Gun.
  • the nucleic acids can be "naked," consisting of plasmids under control of a strong promoter.
  • the DNA is injected into muscle, although it can also be injected directly into other sites, including tissues in proximity to metastases. Dosages for injection are usually around 0.5 ⁇ g/kg to about 50 mg/kg, and typically are about 0.005 mg/kg to about 5 mg/kg (see, e.g., U.S. Patent No.
  • the cell growth inhibiting chimeric molecules including an antibody that specifically binds MRP9 linked to a toxin can be prepared in pharmaceutical compositions.
  • These cell growth inhibiting molecules can be administered by any method known to one of skill in the art.
  • the pharmaceutical compositions of this invention can be administered directly into the breast tissue. Metastases of breast cancer may be treated by intravenous administration or by localized delivery to the tissue surrounding the tumor.
  • compositions for administration will commonly comprise a solution of the cell growth inhibiting chimeric molecules dissolved in a pharmaceutically acceptable carrier, preferably an aqueous carrier.
  • a pharmaceutically acceptable carrier preferably an aqueous carrier.
  • aqueous carriers can be used, e.g., buffered saline and the like. These solutions are sterile and generally free of undesirable matter.
  • These compositions may be sterilized by conventional, well-known sterilization techniques.
  • the compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like.
  • concentration of cell growth inhibiting molecules in these formulations can vary widely, and will be selected primarily based on fluid volumes, viscosities, body weight and the like in accordance with the particular mode of administration selected and the patient's needs.
  • a pharmaceutical composition for intravenous administration such as an immunotoxin
  • compositions can be administered for therapeutic treatments.
  • compositions are administered to a patient suffering from a disease, such as cancer (for example breast cancer), in a therapeutically effective amount, which is an amount sufficient to cure or at least partially arrest the disease or a sign or symptom of the disease. Amounts effective for this use will depend upon the severity of the disease and the general state of the patient's health. An effective amount of the compound is that which provides either subjective relief of a symptom(s) or an objectively identifiable improvement as noted by the clinician or other qualified observer.
  • compositions are administered depending on the dosage and frequency as required and tolerated by the patient.
  • the dosage is administered once as a bolus, but in another embodiment it can be applied periodically, until a therapeutic result is achieved.
  • the dose is sufficient to treat or ameliorate symptoms or signs of disease without producing unacceptable toxicity to the patient.
  • Controlled release parenteral formulations of cell growth inhibiting chimeric molecules can be made as implants, oily injections, or as particulate systems.
  • Particulate systems include microspheres, microparticles, microcapsules, nanocapsules, nanospheres, and nanoparticles.
  • Microcapsules contain the therapeutic protein as a central core. In microspheres the therapeutic is dispersed throughout the particle. Particles, microspheres, and microcapsules smaller than about 1 ⁇ m are generally referred to as nanoparticles, nanospheres, and nanocapsules, respectively.
  • Capillaries have a diameter of approximately 5 ⁇ m so that only nanoparticles are administered intravenously. Microparticles are typically around 100 ⁇ m in diameter and are administered subcutaneously or intramuscularly (see Kreuter, J., Colloidal Drug Delivery Systems, J. -Kreuter, ed., Marcel Dekker, Inc., New York, NY, pp. 219-342, 1994; Tice & Tabibi, Treatise on Controlled Drug Delivery, A. Kydonieus, ed., Marcel Dekker, Inc. New York, NY, pp. 315-339, 1992).
  • Polymers can be used for ion-controlled release.
  • Various degradable and nondegradable polymeric matrices for use in controlled drug delivery are known in the art (Langer, R., Accounts Chem. Res. 26:537, 1993).
  • the block copolymer, polaxamer 407 exists as a viscous yet mobile liquid at low temperatures but forms a semisolid gel at body temperature. It has shown to be an effective vehicle for formulation and sustained delivery of recombinant interleukin-2 and urease (Johnston et al., Pharm. Res. 9:425, 1992); and Pec et al., I. Parent. Sci. Tech. 44(2):58, 1990).
  • hydroxyapatite has been used as a microcarrier for controlled release of proteins (Ijntema et al., Int. J. Pharm. 112:215, 1994).
  • liposomes are used for controlled release as well as drug targeting of the lipid-capsulated drug (Betageri et al., Liposome Drug Delivery Systems, Technomic Publishing Co., Inc., Lancaster, PA, 1993).
  • Numerous additional systems for controlled delivery of therapeutic proteins are known (e.g., U.S. Pat. No. 5,055,303, U.S. Patent No. 5,188,837, U.S. Patent No. 4,235,871, U.S. Patent No. 4,501,728, U.S. Patent No.
  • a method is provided herein for the detection of MRP9-expressing cells or ' tissue in a biological sample.
  • MRP9-expressing cells in a breast biopsy.
  • the detection of MRP9 expressing cells is used in staging a cancer.
  • expression of MRP9 can be used as a marker in the classification of a breast cancer, such as classifying a ductal carcinoma as stage I, HA, IDB, IIIA, IIIB, or IV.
  • the sample can be any sample that includes MRP9 polypeptide or a nucleic acid encoding MRP9 polypeptide.
  • samples include, but are not limited to, tissue from biopsies, autopsies, and pathology specimens.
  • Biological samples also include sections of tissues, such as frozen sections taken for histological purposes. Biological samples further include body fluids, such as blood, serum, or urine.
  • a biological sample is typically obtained from a mammal, such as a rat, mouse, cow, dog, guinea pig, rabbit, or primate. In one embodiment the primate is macaque, chimpanzee, or a human. In a further embodiment the subject has breast cancer, or is suspected of having breast cancer.
  • kits for detecting a MRP9 polypeptide of fragment thereof will typically comprise an antibody that specifically binds MRP9.
  • an antibody fragment such as an Fv fragment is included in the kit.
  • the antibody is preferably an scFv fragment.
  • the antibody is labeled (e.g. fluorescent, radioactive, or an enzymatic label).
  • a kit in one embodiment, includes instructional materials disclosing means of use of an antibody that specifically binds an MRP9 polypeptide or fragment thereof (e.g. for detection of MRP9 expressing cells in a sample).
  • the instructional materials may be written, in an electronic form (e.g. computer diskette or compact disk) or may be visual (e.g. video files).
  • the kits may also include additional components to facilitate the particular application for which the kit is designed.
  • the kit may additionally contain means of detecting a label (e.g. enzyme substrates for enzymatic labels, filter sets to detect fluorescent labels, appropriate secondary labels such as a secondary antibody, or the like).
  • the kits may additionally include buffers and other reagents routinely used for the practice of a particular method.
  • the diagnostic kit comprises an immunoassay.
  • the method of detecting a MRP9 polypeptide or fragment thereof in a biological sample generally comprises the steps of contacting the biological sample with an antibody which specifically reacts, under immunologically reactive conditions, to MRP9.
  • the antibody is allowed to specifically bind under immunologically reactive conditions to form an immune complex, and the presence of the immune complex (bound antibody) is detected directly or indirectly.
  • kits can be provided for detecting nucleic acids encoding MRP9 or a fragment thereof in a biological sample, such as a mRNA of about 4.5 kb in length encoding MRP9.
  • a sample from a subject can be tested to determine whether nucleic acids encoding MRP9 protein are present.
  • an amplification procedure is utilized to detect nucleic acids encoding MRP9.
  • a blotting procedure e.g. Northern Blot or Dot Blot
  • a kit can include probes or primers that specifically hybridize to nucleic acids encoding MRP9, such as that specifically detect or amplify a transcript of about 4.5 kb in length.
  • a kit provides a primer that amplifies nucleic acid encoding MRP9.
  • the amplification is performed by polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • the RNA can be reverse transcribed into DNA and then amplified by LCR, PCR, or other methods.
  • An exemplar protocol for conducting reverse transcription of RNA is taught in U.S. Patent No. 5,705,365. Selection of appropriate primers and PCR protocols are taught, for example, in Innis, M. et al., eds., PCR Protocols 1990 (Academic Press, San Diego CA).
  • the kit includes instructional materials disclosing means of use for the primer or probe.
  • the kits may also include additional components to facilitate the particular application for which the kit is designed.
  • the kits may additionally include buffers and other reagents routinely used for the practice of a particular method. Such kits and appropriate contents are well known to those of skill in the art.
  • RNA Hybridization was performed on multiple tissue Northern blots (MTN, Clontech, Palo Alto, CA) and a Human Multiple Tissue Expression Array
  • a Rapid Scan gene expression panel containing PCR-ready first-strand cDNA from 24 different tissues (Catalog Number HSCA-101; OriGene, Rockville, MD) was used as a template for PCR with a primer pair (T385 and T386) that should give a 400 bp fragment.
  • a human breast cancer Rapid Scan panel Catalog Number TSCE-101; OriGene, Rockville, MD
  • Rapid amplification of cDNA ends was performed on Marathon Ready brain and testis cDNA (Clontech, Palo Alto, CA). Gene specific primers T385 and T386 were used for the 3' and 5' RACE, respectively.
  • the PCR product was gel purified and cloned into the pCR2.1 TOPO vector (Invitrogen, Carlsbad, CA). The longest clones were identified by restriction digestion and sequenced using Perkin-Elmer's dRhodamine terminator sequencing kit (Perkin-Elmer Applied System, Warrington, UK).
  • a peptide of fourteen amino acids (amino acids 15 to 28) was synthesized, conjugated with KLH and injected into rabbits with complete Freund' s adjuvant for the first immunization, and with incomplete Freund' s adjuvant for subsequent immunizations. Sera were collected after the fourth and fifth immunizations and titrated by ELISA against the synthesized peptide. Total IgG was then purified with immobilized protein A (Pierce) matrix following the supplier's instructions. Western blot analysis
  • Filters were probed with 10 ig/ml of protein A purified anti-MRP9 antiserum or pre-immune sera and their respective signals were detected using a chemiluminescence Western blotting kit according to the manufacturer's instructions (Roche Molecular Biochemicals, Indianapolis, IN).
  • Biotinylated probes were prepared using 600 bp 5 '-end of MRP9 and U6 (250bp) cDNA cloned in pBluescript II (+) plasmid. Biotinylated pBluescript II (+) with a CD22 insert was used as a negative control. Probe labeling, hybridization and washing conditions were similar as described before (Olsson et al., 2001). Microscopic evaluation (brightfield) was performed using a Nikon Eclipse 800 microscope (Kumar and Collins, 1994).
  • MRP9 a member of ABC transporter super family is located in a genomic region of over 80.4 kb on chromosome 16ql2.1. Because some members of the ABC transporter super family (MDR1 and MDR2) are located near to each other, genes 100 kb upstream and downstream of the MRP8 gene were analyzed by the GeneScan gene prediction program to identify possible genes in this region. A gene was identified next to MRP8 that has sequence similarity with the ABCC subfamily. This gene was named MRP9 (Bera et al., Molec. Med. 7:509-516, 2001).
  • MRP9 is detected in different parts of brain (1A to IG; A2, D2, F2 and B3), testis (F8), and pancreas (B9).
  • a more sensitive PCR based analysis was used to validate tissue specific expression of MRP9.
  • a panel of cDNAs was used that were isolated from 24 different normal tissues. PCR reactions were performed with a primer pair (the same primer pair which is used to generate the probe for the dot blot ) designed from the 3' -DNA sequence of MRP9. As shown in Fig. 2B, a specific band of 400 bp is detected in brain (lane 12) testis (lane 2), ovary (lane 20) and in skeletal muscle (lane 4).
  • MRP9 gene is transcribed as a 5 kb transcript that encodes a 1359 amino acid open reading frame that is expressed in testis, prostate and ovary.
  • the cDNA that was isolated from testis has an ORF of only 930 amino acids.
  • the major difference between the sequence published by Tammur et al. (Gene 273(l):89-96, 2001) and the sequence obtained in these studies is that there is an extra 30 bp sequence in the sequence disclosed herein at the 5' end of the exon 20 (Fig. 1).
  • a stop codon TAG is incorporated in the cDNA sequence that was isolated in these studies. Consequently the size of the ORF becomes 930 amino acids.
  • this region of the cDNA was PCR cloned (Fig. 1) using primer pair T396 and T399 and testis and breast cDNA sources. More than ten clones were sequenced. Every clone that was analyzed had the 30 bp extra sequence at the 5' end of exon 20. To determine if the variant, which does not contain the extra 30 bp sequence, is expressed (if at all exists) in various tissues, a sensitive PCR based analysis was utilized. 5' primers were designed that were specific for each variant (T419 for the variant which contain the 30 bp extra sequence and T418 for the possible variant which does not contain the extra 30 bp; Fig. 1C).
  • the same 3' primer (T399) was used for PCR amplification using PCR ready cDNAs from testis and breast. As shown in Fig. 4A (lanes 2 and 4), a specific 300 bp PCR product was detected only when primers T419 and T399 were used. No detectable PCR product was observed when primers T418 and T399 were used. This result shows that in both testis and in breast the expressed MRP9 transcript contains the extra 30 bp sequence at the 5' end of the exon 20. Since the cDNAs used in this experiment were generated from pooled tissues from more than nine individuals, the presence of the extra 30 bp sequence is real and not due to isolated phenomena.
  • the dot blot and Rapid Scan PCR analysis shown in Fig. 2 indicates that MRP9 is highly expressed in brain.
  • the Northern analysis in Fig. 3 A shows that the transcript size of MRP9 in brain is about 1.3 kb, which is much smaller than the RNA detected in testis.
  • RACE-PCR was employed using the T385 primer for 3' RACE and the T386 p ⁇ mer for 5' RACE (Fig. 1). Marathon-ready cDNA from brain was used as a template.
  • the 5' RACE reaction gave a DNA fragment of 850 bp, and the product from the 3' RACE was about 1.1 kb in size.
  • a PCR analysis on brain and testis cDNA was performed using several 5' primers (T412, T413, T414 and T415, Fig 4A) and T386 as 3' primer (Fig 1 A).
  • T412, T413, T414 and T415, Fig 4A 5' primers
  • T386 3' primer
  • the expected sized PCR product was obtained with all four 5' primers with testis cDNA.
  • brain cDNA only the T415 primer (which is within exon 21) gave a PCR product of 800 bp in size.
  • the major PCR product for brain cDNA with the T415 and T386 primer pair contains an extra 79 bp from exon 23 A. There is a very weak band of about 700 bp in size that accounts for the transcripts without exon 23A (Fig. 4B, lane 4).
  • Example 5 Long Transcript of MRP9 is Specifically Expressed in Testis and Breast
  • a multi-tissue dot blot and Rapid Scan analysis was performed with a 5'- specific probe and a 5 '-specific primer pair respectively.
  • MRP9 is detected only in testis.
  • RT-PCR a specific band of 400 bp is also detected in testis (Fig. 2, lane 11) normal breast (lane 13), and breast cancer and in cDNA prepared from a pool of four breast cancer cell lines (lane 26). This band is not seen in 23 other tissues tested including heart, brain and lung.
  • RNA demonstrated expression of MRP9 in CRL1500, but not in the other breast cancer cell lines examined.
  • 5' specific probe was used in the multi-tissue Northern blot, a band of 4.5 kb in size was detected in testis (Fig 3C).- A 2.4 kb band was also identified in testis, which could be splice variant of the gene. No band was detected in any other tissues tested, which include ovary and brain.
  • MRP9 is expressed in different samples of normal breast and primary breast cancer
  • a RT-PCR analysis was carried out using a human breast cancer Rapid Scan panel, which contains cDNAs from 12 different normal breast and breast cancer specimens.
  • the expected 400 bp PCR product was detected in 9 out of 12 breast cancer samples. The signal was not detected in normal breast.
  • in situ hybridization was performed. These studies utilized a biotin labeled 5' specific MRP9 cDNA (nucleotide 1 to 604) probe.
  • the MRP9 cDNA has a predicted open reading frame of 930 amino acids with a calculated molecular weight of 95 kDa.
  • in vitro transcription and translation was performed using the rabbit reticulocyte lysate system. SDS-PAGE analysis and fluorography of the translated product showed a doublet about 100 kDa in size (Fig. 7A) probably due to different amount of glycosylation.
  • the size of the protein products agrees with the predicted open reading frame of the cDNA.
  • Polyclonal antibodies were produced in rabbits against a synthetic peptide (amino acid 15 to 28) of MRP9. Serum isolated from these animals specifically recognized MRP9.
  • MRP9 a new member of ABC transporter super family.
  • the experimental data disclosed herein demonstrates that MRP9 transcript is detected as different variants in different tissues.
  • the larger 4.5 kb transcript is expressed in breast cancer, and in testis and encodes a protein of 100 kDa molecular weight.
  • the smaller 1.3 kb transcript is expressed in brain, ovary and few other tissues tested.
  • the smaller transcript has an open reading frame of 234 amino acids.
  • the multidrug resistance/ ATP-binding cassette (MDR/ABC) superfamily of membrane transporters is one of the largest protein families and is involved in energy-dependent transport of a variety of substrates across the membrane.
  • ABC-A to -G subfamilies
  • MRP9 sequence like MRP8, is closely related to MRP5, with an overall 44% identity and 55% sequence similarity. Between MRP8 and MRP9 sequence, the overall sequence identity and similarity is 47% and 56%, respectively.
  • MRP9 has only one ATP- binding domain.
  • MRP9 is predicted to have four membrane-spanning regions in the first transmembrane domain and only three membrane-spanning region in the second transmembrane domain.
  • the longer (4.5 kb) transcript is specifically expressed in breast cancer, normal breast and testis.
  • In situ RNA analysis using a breast cancer tissue array also indicated that human cancer specimens express MRP9.
  • MRP9 is a membrane protein and it has very restricted expression in essential tissues, it is a target for immuno-based targeted therapy.
  • Example 10 Radioimmunoassay to Detect MRP9
  • the following example sets forth an exemplary protocol for a radioimmunoassay to detect the presence MRP9 in a sample.
  • a standard curve is established by mixing a fixed amount of labeled MRP9 ( ⁇ 0.2 ng at about 170 ⁇ dl ⁇ g) with different concentrations of unlabeled MRP9 (O.lng - 50ng) in 250 ⁇ l buffer (PBS with 0.25% bovine serum albumin) containing 1 ⁇ g of anti-MRP9 antibody. The samples are incubated at room temperature for 4 hours. Protein A sepharose beads are added and incubated for another hour. Finally the beads are collected by centrifugation and washed with buffer 3 times. The remaining bead pellet is measured for radioactivity in a gamma counter.
  • Juvenile female rhesus monkeys (Macaca mulatta), ages 1 to 2 years, are assigned to groups (e.g., three vaccination groups of four animals each, a low dose, a high dose and a control group).
  • a mastectomy is performed in one animal from each group to parallel two situations with regard to potential therapy in humans: (a) breast intact, with primary and/or metastatic disease; or (b) patients who have undergone mastectomy with cancer metastatic deposits. Animals are immunized 3 times over a two month period with a recombinant virus (e.g. a pox virus, see U.S. Patent No. 6,165,460).
  • a recombinant virus e.g. a pox virus, see U.S. Patent No. 6,165,460.
  • a dose of either lxlO 7 or lxlO 8 PFU of a recombinant pox virus encoding MRP9 is administered to 4 animals by skin scarification.
  • a control vector e.g. V-Wyeth, lxlO 8 PFU
  • V-Wyeth, lxlO 8 PFU is administered to a control group of animals.
  • ketamine Ketamine® HCl, 10 mg/kg I.M. sedated animals. Rectal temperatures and weights are recorded for each monkey on a weekly basis. The vaccination site is observed and erythema and swelling of the vaccination site are measured by caliper. Each animal is examined for regional lymphadenopathy, hepatomegaly, and splenomegaly. Any other gross abnormalities were also recorded.
  • Blood is obtained by venipuncture from the femoral vein of ketamine sedated animals before and after each immunization. A complete blood count, differential, hepatic and renal chemistry evaluation is performed on each monkey. Results are compared to normal primate values. Circulating levels of MRP9 before and after immunization are analyzed (e.g. by immunoassay or Northern blot).
  • anti- MRP9 antibody Prior to each immunization and 2 weeks following each immunization, anti- MRP9 antibody is quantified by ELISA.
  • Microtiter plates are coated with purified MRP9 (e.g. 100 ng/well,), ovalbumin (100 ng/well, Sigma), or lxlO 7 PFU/well UN- inactivated N-Wyeth in phosphate buffered saline (PBS). The plates are blocked (e.g. using 2% BSA in PBS), dried, and stored at -20° C. until used. The plates are incubated with serum (e.g. diluted 1:5), as well as a monoclonal antibody for MRP9 as a standard control, for 24 hours at 4° C.
  • serum e.g. diluted 1:5
  • Plates are washed several times (e.g. with PBS containing 1% BSA), and incubated with a commercially labeled antibody that specifically binds the anti-MRP9 monoclonal antibody.
  • An appropriate reagent system is used to visualize antibody binding.
  • the antibody is labeled with horseradish peroxidase (HRP), and detected by HRP substrate system (Kirkegaard & Perry Laboratories, Gaithersburg, MD) according to the manufacture's instructions.
  • HRP substrate system Kirkegaard & Perry Laboratories, Gaithersburg, MD
  • the absorbance of each well is read at 405 nm using a Bio-Tek EL310 microplate ELISA reader (Winooski, NT). Sera from each monkey is analyzed by ELISA for immunoreactivity to MRP9.
  • MRP9 specific T cell responses in monkeys immunized with MRP9 containing vector or control vector are also analyzed using a lymphoproliferative assays using peripheral blood mononuclear cells.
  • Breast cancer is known to metastasize to other areas of the body, such as lymph note, bone, and brain.
  • Antibodies to a MRP9 polypeptide can be used to detect breast cancer cells at locations other than the prostate.
  • a kit that provides an immunoassay that can be used to confirm that the cancer cells are of breast origin.
  • a biological sample of the metastasis is obtained.
  • the sample a biopsysample.
  • Non-specific immunoreactive sites on biological sample are blocked with a commercially available blocking agent, such as 10% bovine serum albumin in phosphate buffered saline (PBS), for thirty minutes at room temperature.
  • PBS phosphate buffered saline
  • the sample is then contacted with a mouse monoclonal antibody that specifically binds MRP9 for an incubation period sufficient to allow formation of an immune complex (e.g.
  • a fluorescent labeled (e.g. fluorescein isothiocyanate, FITC) goat anti-mouse antibody is diluted 1:100 in
  • a kit that detects the presence of a MRP9 transcript of about 4.5 kb in length.
  • a biological sample is obtained, and mRNA is purified from the sample.
  • a probe is then utilized that specifically detects a transcript of about 4.5 kb in length.
  • a PCR assay is utilized.
  • a Northern analysis or a dot blot analysis is utilized. Detection of a MRP9 transcript of about 4.5 kb in length indicates the metastasis is of breast origin.

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Abstract

La présente invention concerne le MRP9 susceptible d'expression dans des cellules cancéreuses. L'invention concerne également un anticorps se liant de façon spécifique à un épitope antigène d'un polypeptide MRP9. L'invention concerne également des procédés permettant de détecter des cellules cancéreuses par détection d'une ARN messager codant le MRP9, ou par détection d'un polypeptide MRP9. L'invention concerne enfin des thérapies immunitaires reposant sur le MRP9. Ces thérapies immunitaires sont indiquées dans le traitement du cancer.
PCT/US2003/001340 2002-01-17 2003-01-15 Mrp9 et son utilisation pour la detection et le traitement du cancer WO2003062446A2 (fr)

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CN115772219B (zh) * 2022-07-29 2023-11-14 生工生物工程（上海）股份有限公司 一种特异性结合mrp1蛋白的结合蛋白、试剂盒及其应用

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