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WO1997028193A1 - Compositions et procedes utiles dans la detection et/ou le traitement d'etats cancereux - Google Patents

Compositions et procedes utiles dans la detection et/ou le traitement d'etats cancereux Download PDF

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Publication number
WO1997028193A1
WO1997028193A1 PCT/US1997/001586 US9701586W WO9728193A1 WO 1997028193 A1 WO1997028193 A1 WO 1997028193A1 US 9701586 W US9701586 W US 9701586W WO 9728193 A1 WO9728193 A1 WO 9728193A1
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Prior art keywords
mader
cells
gene
tissue sample
probe
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PCT/US1997/001586
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English (en)
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WO1997028193A9 (fr
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Judith P. Johnson
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Melcorp Diagnostics, Inc.
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Priority to AU21151/97A priority Critical patent/AU2115197A/en
Publication of WO1997028193A1 publication Critical patent/WO1997028193A1/fr
Publication of WO1997028193A9 publication Critical patent/WO1997028193A9/fr

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    • 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
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/82—Translation products from oncogenes
    • 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
    • G01N33/57407—Specifically defined cancers
    • G01N33/57415—Specifically defined cancers of breast
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/5743—Specifically defined cancers of skin, e.g. melanoma
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57496—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving intracellular compounds
    • 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
    • C12Q2600/112—Disease subtyping, staging or classification

Definitions

  • the present invention relates generally to diagnostic and therapeutic compositions and methods which target a melanoma associated delayed early response (MADER) gene and its expression products.
  • MADER delayed early response
  • the invention relates to the diagnostic use of anti-MADER molecules, and the use of MADER polynucleotides and polypeptides in the diagnosis and treatment of malignant melanomas and other cancerous conditions.
  • chromosomal alterations leading to changes in gene expression have implicated a number of molecules in the early events of tu origenesis (Vogelstein et al. (1993) Trends Genet . 9:138-141). Many of the implicated molecules appear to function in DNA repair and growth regulation, and have been found to be mutated or deleted in a variety of different carcinomas. For example, in human cutaneous melanoma, alterations in the expression of cell and matrix adhesion molecules accompanying tumor progression and metastasis development have been extensively described. See, e . g. , Albelda et al. (1990) Cancer Res . 5_0:6757-6764; Johnson et al. (1989) Proc. Natl . Acad .
  • the present inventors have discovered a novel nuclear protein that is associated with growth alterations in malignant melanomas and other cancerous conditions.
  • the subject protein is the "melanoma associated delayed early response" protein, and termed "MADER 11 herein.
  • MADER 11 melanoma associated delayed early response protein
  • Studies described below establish that MADER mRNA is rapidly induced in a wide range of cells following exposure to mitogens or growth factors. This induction requires protein synthesis indicating that MADER belongs to the set of growth factor dependent delayed early response genes that are induced during the GI stage of the cell cycle (Nathans et al. (1988) Cold Spring Harbor Symposia on Quantitative Biology LJ__f_I:883-900) .
  • the MADER gene is highly conserved, as cross-hybridizing DNA sequences have been observed in species as diverse as Rhesus and S . cerevisiae .
  • the predicted MADER expression product is approximately 55 kD, and is characterized as having two proline rich domains, 15 potential phosphorylation sites, a nuclear localization signal, and multiple S(T)PXX motifs that are characteristic of regulatory DNA binding proteins. Immunological studies presented below confirm that MADER is localized to the nucleus. These features suggest that MADER participates in growth regulation. Although Northern analyses indicate that low-level expression of MADER is ubiquitous among most types of cells, MADER expression is only rarely detectable in normal tissue.
  • the MADER gene and its expression product present novel targets for malignant melanoma, and other cancer diagnostics and therapeutics.
  • the present invention provides diagnostic and therapeutic compositions and methods which target the MADER gene and its expression product.
  • monoclonal antibody molecules capable of specifically binding to the approximately 55 kD MADER protein are provided.
  • the subject anti-MADER monoclonal antibodies are used herein in immunohistochemical methods of detecting malignant melanoma cells, or other cancerous cells, in a tissue sample. Particularly, methods are described wherein tissue samples suspected of containing cancerous cells, such as malignant melanoma cells, are exposed to anti-MADER monoclonal compositions, and the presence or absence of bound antibodies on the tissue sample is determined as an indication of the presence or absence of a cancerous condition, such as a melanoma malignancy. Visualization of positive binding events is effected by detectably labeling either the anti-MADER monoclonals (direct detection) , or by detecting secondary molecules capable of binding the anti-MADER molecules (indirect detection).
  • MADER polynucleotides are targeted in cytological analysis methods for detecting a cancerous condition, such as a melanoma malignancy, in a tissue sample. Particularly, methods are provided for detecting the presence of cells that over-express the MADER gene.
  • Detectably labeled oligonucleotide probes are provided which comprise a nucleotide sequence that is complementary to a region of mRNA transcribed from the MADER gene.
  • Mixed-phase hybridizations are conducted by incubating the subject probes with cell lysates (obtained from a selected tissue sample) under suitable conditions. A detection step is then carried out to detect the presence of any labeled probe on the substrate.
  • in- situ hybridizations can be carried out by incubating the subject probes with immobilized tissue sections (obtained from a selected tissue sample) under suitable conditions.
  • methods are provided for visualizing the presence or absence of a translocation of MADER in cellular genomes using fluorescence in situ hybridization.
  • MADER translocations are thought to give rise to over ⁇ expression of MADER in malignant melanoma cells and other cancerous cells; and, in this manner, these in situ hybridizations can be used to detect a cancerous condition, such as malignant melanoma, in a tissue sample.
  • compositions containing MADER immunogens are provided for use in immunizing a subject having a cancerous condition such as malignant melanoma.
  • the MADER immunogens can be obtained from either MADER polypeptides or polynucleotides.
  • MADER antisense molecules are used in tumor treatments. More particularly, antisense oligonucleotides capable of selectively binding to target MADER sequences are provided.
  • the antisense oligonucleotides generally comprise synthetic nucleic acid sequences that bind specifically and predictably to complementary regions of MADER mRNA, thereby inhibiting MADER protein biosynthesis.
  • Other related therapeutics include ribozymes that are capable of degrading MADER mRNAs.
  • Figure 1 depicts the nucleotide sequence (SEQ ID NO: ) of human MADER cDNA for the Drop9 variant.
  • the deduced amino acid sequence (SEQ ID NO: ) corresponding to the longest open reading frame
  • Figure 2 depicts the nucleotide sequence (SEQ ID NO: ) of human MADER cDNA for the Drops splice variant.
  • the deduced amino acid sequence (SEQ ID NO: ) is also shown.
  • the nucleotide sequence of the Drop ⁇ variant lacks 192 base pairs as compared to the Drop9 variant. These missing 192 base pairs are underlined and italicized in the Figure.
  • Figure 3 depicts the mRNA sequence (SEQ ID NO.: ) of yet another MADER variant.
  • MADER polypeptide encoded by yet a further splice variant is also shown in the figure.
  • Figure 4 depicts the results from a genomic Southern blot analysis of the MADER gene. Genomic DNA from EBV-transformed B cells of a normal individual was digested with the indicated restriction endonucleases. The positions of the molecular weight markers are indicated on the left of the gel.
  • Figure 5 depicts the hybridization of human cDNA with DNAs isolated from a variety of different species (Genomic Zoo blot) .
  • the gel contains 2?coRI digested DNA from human (lane 1) , Rhesus monkey (lane 2), rat (lane 3), mouse (lane 4), dog (lane 5), cow (lane 6), rabbit (lane 7), chicken (lane 8), S. cerevisiae (lane 9) .
  • Arrowheads in the Figure indicate the location of the weakly hybridizing bands in the rat (lane 3) and the chicken (lane 8) .
  • the blots were hybridized with [ 32 P]-labeled complete Mader cDNA.
  • Figure 6 depicts induction of Mader mRNA production in Mel JuSo cells by exposure to 20% serum (FCS) or phorbol 12-myristate 13-acetate (PMA) .
  • FCS serum
  • PMA phorbol 12-myristate 13-acetate
  • the cells were harvested at the indicated times following stimulation. 20 ⁇ g total RNA was used, and the blots were hybridized with [ 32 P]-labeled complete Mader cDNA, or with oligonucleotides detecting c-fos or GAPDH. All blots were analyzed by autoradiography and densitometry.
  • Figure 7 depicts induction of Mader mRNA production in lymphocytes stimulated with PHA. Freshly isolated PBMC were exposed to PHA for the indicated times. GAPDH was used as control for RNA loading. All blots were analyzed by autoradiography and densitometry.
  • Figure 8 depicts the effect of cycloheximide on the induction of Mader mRNA production in Mel JuSo cells.
  • the cells were grown to confluence and treated for 2 hours with 10 ng/ml PMA in the presence or absence of cycloheximide (CHX) as indicated in the Figure.
  • RNA was separated, blotted, and hybridized with probes detecting MADER, c-fos and GAPDH, respectively. The basal levels of the three mRNAs are shown in lane 1.
  • an "antigen” includes any substance that may be specifically bound by an antibody molecule.
  • An "immunogen” is a macromolecular antigen that is capable of initiating lymphocyte activation resulting in an antigen-specific immune response.
  • An immunogen therefore includes any molecule which contains one or more epitopes that will stimulate a host's immune system to initiate a secretory, humoral and/or cellular antigen-specific response.
  • antibody encompasses polyclonal and monoclonal antibody preparations, as well as preparations including hybrid antibodies, altered antibodies, F(ab ) 2 fragments, F(ab) fragments, Fv fragments, single domain antibodies, chimeric antibodies, humanized antibodies, and functional fragments thereof which exhibit immunological binding properties of the parent antibody molecule, alone, or linked to another polypeptide, or as a fusion protein.
  • monoclonal antibody refers to an antibody molecule derived from a single original lymphocyte. The term is not limited regarding the species or source of the antibody, nor is it intended to be limited by the manner in which it is made.
  • immunoglobulins encompasses whole immunoglobulins as well as fragments such as Fab, F(ab') 2 , Fv, and other fragments derived from a parent monoclonal antibody molecule which fragments exhibit immunological binding properties of the parent molecule.
  • immunological binding refers to non-covalent interactions of the type which occur between an immunoglobulin molecule and an antigen for which the immunoglobulin is specific.
  • the strength, or affinity of immunological binding interactions can be expressed in terms of the dissociation constant (K d ) of the interaction, wherein a smaller K d represents a greater affinity.
  • Immunological binding properties of selected polypeptides can be quantified using methods well known in the art.
  • One such method entails measuring the rates of antigen-binding site/antigen complex formation and dissociation, wherein those rates depend on the concentrations of the complex partners, the affinity of the interaction, and on geometric parameters that equally influence the rate in both directions.
  • K on an "on rate constant”
  • K off an “off rate constant”
  • K d The ratio of K off /K on enables cancellation of all parameters not related to affinity, and is thus equal to the dissociation constant K d . See, generall , Davies et al. (1990) Annual Rev . Biochem . 59:439-473.
  • polypeptide refers to a polymer of amino acids and does not refer to a specific length of the product; thus, peptides, oligopeptides, and proteins are included within the definition of polypeptide. This term also does not refer to, or exclude, post expression modifications of the polypeptide; for example, glycosylations, acetylations, phosphorylations and the like. Included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (e.g., unnatural amino acids) , polypeptides with substituted linkages, as well as other modifications known in the art, both naturally occurring and non-naturally occurring.
  • amino acid e.g., unnatural amino acids
  • a polypeptide or amino acid sequence "derived from" a designated nucleic acid sequence refers to a polypeptide having an amino acid sequence identical to that of a polypeptide encoded in the sequence, or a portion thereof wherein the portion consists of at least 3-5 amino acids, preferably at least 4-7 amino acids, more preferably at least 8-10 amino acids, and even more preferably at least 11-15 amino acids, or which is immunologically identifiable with a polypeptide encoded in the sequence.
  • This terminology also includes a polypeptide expressed from a designated nucleic acid sequence.
  • oligonucleotide and “polynucleotide” shall be generic to polydeoxy- ribonucleotides (containing 2-deoxy-D-ribose) , to polyribonucleotides (containing D-ribose) , to any other type of polynucleotide which is an N-glycoside of a purine or pyrimidine base, and to other polymers containing nonnucleotidic backbones, provided that the polymers contain nucleobases in a configuration which allows for base pairing and base stacking, such as is found in DNA and RNA.
  • polynucleotide and “oligonucleotide,” and these terms may be used interchangeably. These terms refer only to the primary structure of the molecule. Thus, these terms include double- and single-stranded DNA, as well as double- and single-stranded RNA and DNA:RNA hybrids, and also include known types of modifications, for example, labels which are known in the art.
  • purified and “isolated” is meant, when referring to a polypeptide or nucleotide sequence, that the indicated molecule is present in the substantial absence of other biological macromolecules of the same type.
  • purified as used herein preferably means at least 75% by weight, more preferably at least 85% by weight, more preferably still at least 95% by weight, and most preferably at least 98% by weight, of biological macromolecules of the same type are present.
  • "Homology” refers to the percent of identity between two polynucleotide or two polypeptide moieties. The correspondence between the sequence from one moiety to another can be determined by techniques known in the art. For example, homology can be determined by a direct comparison of the sequence information between two polypeptide molecules by aligning the sequence information and using readily available computer programs.
  • homology can be determined by hybridization of polynucleotides under conditions which form stable duplexes between homologous regions, followed by digestion with single- stranded-specific nuclease(s) , and size determination of the digested fragments.
  • Two DNA, or two polypeptide sequences are "substantially homologous" to each other when at least about 80%, preferably at least about 90%, and most preferably at least about 95% of the nucleotides or amino acids match over a defined length of the molecules, as determined using the methods above.
  • recombinant DNA molecule or “recombinant nucleic acid molecule” are used herein to refer to a polynucleotide of genomic, cDNA, semisynthetic, or synthetic origin which, by virtue of its origin or manipulation: (1) is not associated with all or a portion of a polynucleotide with which it is associated in nature, (2) is linked to a polynucleotide other than that to which it is linked in nature, or (3) does not occur in nature.
  • the term encompasses “synthetically derived” nucleic acid molecules.
  • cancer condition refers to any neoplastic disease characterized by the presence of cancerous cells (or tissue) .
  • Cancer cells unlike benign tumor cells, exhibit the properties of invasion and metastasis and are generally highly anaplastic.
  • melanomas e.g., carcinomas of the breast, prostate and lung
  • neuroblastomas e.g., lymphomas and leukemias.
  • a “malignant cell” refers to a cancerous cell which has undergone phenotypic transformation, such as, but not limited to, transformation by oncogenes, protooncogenes, TS mutations, or other such mechanisms.
  • phenotypic transformation such as, but not limited to, transformation by oncogenes, protooncogenes, TS mutations, or other such mechanisms.
  • Malignant cells are generally characterized by their capacity for unregulated growth, and have the properties of anaplasia, invasion and metastasis. Such cells have one or more phenotypic derangements which may be expressed as alterations in cellular membranes, in the expression levels of certain cellular proteins (e.g., enzymes involved in nucleic acid synthesis and metabolism) , or by the appearance of inappropriate gene products.
  • melanoma encompasses those tumors arising from the melanocytic system of the skin and other organs.
  • a "malignant melanoma” is a cutaneous malignant neoplasm of melanocytes, arising de novo or from a preexisting benign nevus or lentigo maligna. Malignant melanomas most often occur in the skin.
  • nucleic acid analyte refers to a single- or double-stranded nucleic acid molecule which contains a target nucleotide sequence.
  • target region or “target nucleotide sequence” refers to a probe binding region contained within the target molecule.
  • target sequence refers to a sequence with which a probe will form a stable hybrid under suitable hybridization conditions.
  • probe refers to a structure comprised of an oligonucleotide, as defined above, which contains a nucleic acid sequence complementary to a nucleic acid sequence present in another molecule of interest.
  • the oligonucleotide regions of probes may be composed of DNA, and/or RNA, and/or synthetic nucleotide analogues.
  • Two nucleotide sequences are “complementary” to one another when those molecules share base pair organization homology. "Complementary" nucleotide sequences will combine with specificity to form a stable duplex under appropriate hybridization conditions.
  • DNA sequences that are complementary can be identified using Southern blot hybridization under, for example, stringent conditions as defined for that particular system. Southern, E. (1975) J . Mol . Biol . 9_8:503. Defining appropriate hybridization conditions is within the skill of the art. See, e .g . , Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual , Second Edition (1989).
  • label and “detectable label” refer to a molecule capable of detection, including, but not limited to, radioactive isotopes, fluorescers, chemiluminescers, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, dyes, metal ions, ligands (e.g., biotin or haptens) and the like.
  • fluorescer refers to a substance or a portion thereof which is capable of exhibiting fluorescence in the detectable range.
  • cofactor is used broadly herein to include any molecular moiety which participates in an enzymatic reaction.
  • labels which may be used under the invention include fluorescein, rhodamine, dansyl, umbelliferone, Texas red, luminol, NADPH, ⁇ - ⁇ -galactosidase and horseradish peroxidase.
  • FIG. 1 Two potential translation start sites (ATG) , respectively occurring at positions 104 and 119 (where the first site is in a more favorable Kozak consensus sequence (Kozak, M. (1986) Cell 44 . :283-292) ) , are embedded within an open reading frame encoding a putative protein of 440 amino acids with a deduced molecular weight of approximately 55 kD.
  • a hydrophobicity analysis calculated by the method of Kyte and Doolittle indicates no evidence of a transmembrane region.
  • the 3' untranslated region is approximately 770 bp, and has one polyadenylation site (AATAAA) at position 2160. Located within the 3' region are three ATTTA repeats which have been underlined in Figure 1. ATTTA repeats have been implicated in rapid message turnover (Shaw et al. (1986) Cell 4_6: 659-667; Sachs, A.B. (1993) Cell 21:413-421) . A search of available data banks with the programs FASTA and Blast revealed no significant homology between MADER and known sequences. The above-described cDNA sequence for
  • MADER has been deposited with the EMBL data bank under the accession number X70991 HSDR0P9 (GenBank accession number X70991) , and is referred to herein as the "Drop9 variant.”
  • the nucleotide sequence (SEQ ID NO. : ) and predicted amino acid sequence (SEQ ID NO. : ) of a shorter splice variant of MADER are depicted in Figure 2.
  • the shorter splice variant is referred to herein as the "Drop ⁇ variant.”
  • Drops lacks 192 base pairs (which are italicized and underlined in Figure 2) from the Drop9 variant sequence, and is presumably a splice variant of the longer Drop9 sequence, lacking one exon.
  • Monoclonal antibodies produced against the Drop9 MADER variant also bind to the Drop ⁇ variant. These monoclonal antibody molecules are described below.
  • a longer version of MADER was obtained from human placental cDNA by PCR amplification using the following primers derived from the Drop9 variant sequence: GCCAACCTCCTTTCCTACTATG (SEQ ID NO.: ) and
  • GGCGAAGCTTCTGCCGGCTGGCCTCAGCCTC (SEQ ID NO. : ) .
  • the resulting fragment was combined with the 5' end of the human cDNA obtained by amplification of cDNA ends, and cloned into a cytomegalovirus-driven expression vector to yield a construct termed pCMVNAB2.
  • Svaren et al. (1996) Molec . Cell . Biol . 16:3545-3553.
  • a 1735 bp nucleotide sequence (mRNA) (SEQ ID NO. : )
  • a full-length, 525 residue amino acid sequence (SEQ ID NO. :
  • a 320 residue amino acid sequence (SEQ ID NO.: ) of yet a further MADER polypeptide is also shown in Figure 3.
  • This particular splice variant is approximately one-third the size of the Drop9 variant, and has been observed in human placental tissue.
  • Svaren et al. (1996) Molec . Cell . Biol . 16:3545-3553.
  • Analysis of DNA encoding this shorter splice variant has revealed that it has a deleted internal sequence, producing a frameshift that causes premature termination of translation. Termination results in the loss of approximately one-third of the full- length, 525 amino acid MADER molecule.
  • Analysis of human placental DNA has revealed that this alternatively spliced form of MADER is found in an approximately 1:1 ratio with the full-length molecule.
  • the shorter, 320 amino acid MADER polypeptide has an altered function, and fails to repress erg-1 activity. Svaren (1996), supra .
  • the putative 55 kD MADER protein is rich in proline residues which are concentrated in two regions, one near the N-terminus and one near the C-terminus.
  • the N-terminal region lies between amino acids 61-185, and contains a high proportion of glycine (23%) , and proline (20%) .
  • the second domain, occurring between amino acids 371 and 458, is rich in proline (20%) and leucine (16%). Both regions contain repeated S(T)PXX motifs (indicated by boxed residues in Figure 1) which are characteristic of gene regulatory DNA binding proteins (Suzuki, M. (1989) J. Mol. Biol . 207:61-84) .
  • the predicted MADER protein also contains a putative bipartite nuclear localization signal, occurring in the middle of the coding region (indicated by boxed residues and an overlying dotted line in Figure 1) .
  • a comparison between the MADER signal sequence and that of nucleoplasmin (Robbins et al. (1991) Cell 64.:615-623; Dingwell et al. (1991) TIBS 16_:478-481) reveals a strong sequence motif homology.
  • the putative MADER amino acid sequence includes three CK-2 sites within 30 residues of the nuclear localization signal. Flanking CK-2 phosphorylation sites have been found to be important in the regulation of nuclear localization for some proteins (Rihs et al.
  • MADER mRNA is detectable at low levels in a number of cell lines derived from various tumors.
  • the expression of MADER in such cell lines can be rapidly up-regulated in response to growth factors or mitogens. Such increased expression is transient, generally returning to basal levels within about six hours.
  • Expression of detectable levels of MADER protein in normal cell lines and frozen tissue is relatively rare.
  • strong uniform nuclear expression of MADER has been observed in all malignant melanoma lesions thus far examined. Since such expression has not been seen in normal epidermal melanocytes and benign melanocytic tumors (nevi) , it appears that malignant transformation of melanocytes is associated with over-expression of the MADER protein. Further, over ⁇ expression of the MADER protein may be associated with other malignant conditions.
  • the MADER protein is used in the practice of the invention to provide a malignant melanoma-associated marker molecule for immunohistochemical diagnosis of melanocytic lesions.
  • anti-MADER monoclonal antibodies are used in various diagnostic methods to detect melanoma malignancies in tissue samples.
  • Anti-MADER monoclonal antibodies are also used to detect other cancers, such a breast carcinomas, in tissue samples.
  • the MADER protein, and fragments or analogs thereof, also find use herein as immunogens for eliciting an immune response against cells which over-express MADER in an immunized subject.
  • Fluorescence in situ hybridization (FISH) localization of the MADER gene to human chromosomes has been carried out in order to gain further insight into the function of MADER.
  • FISH Fluorescence in situ hybridization
  • Region 12ql3-15 is also known as a preferential site for human papillomavirus integration in cervical carcinomas (Popescu et al. (1987) J. Virol . 6L:1682-1685; and Sastre-Garau et al. (1990) Cancer Genet . Cytogenet . 4_4:253-261) , and microcell fusion-mediated transfer of the particular region of chromosome 12 into a prostate cancer cell line can suppress tumorigenicity (Berube et al. (1994) Cancer Res . 54.:3077-3081) .
  • the MADER gene can thus be used as a target to detect translocation events in tumors, where translocation of the MADER gene gives rise to over ⁇ expression of MADER in malignant cells.
  • MADER RNAs are useful as targets in methods of determining the presence or absence of MADER over ⁇ expression in a tissue sample, where such over- expression is indicative of the presence of malignant cells, including malignant melanoma cells.
  • MADER polynucleotides also find use herein as targets in various therapeutic methods for treating malignant melanoma, and other cancers, in a subject.
  • Compositions including MADER DNA polynucleotide immunogens are used to elicit an immune response against cells which over-express MADER in an immunized subject.
  • MADER mRNAs are used as targets for antisense oligonucleotides in antisense therapies directed at inhibiting the expression of MADER in a treated subject.
  • MADER RNAs are also used as targets for ribozymes capable of degrading MADER mRNAs.
  • one embodiment of the present invention pertains to the production of monoclonal antibody molecules capable of specifically binding to the approximately 55 kD MADER protein.
  • Anti-MADER monoclonals can be readily produced by one skilled in the art using general hybridoma methodology.
  • immortal antibody-producing cell lines can be created by cell fusion or by other techniques such as direct transformation of B lymphocytes with oncogenic DNA or by transfection with Epstein-Barr virus. See, e .g . , M. Schreier et al . , Hybridoma Techniques (1980) ; Hammerling et al . , Monoclonal Antibodies and T-cell Hybridomas (1981); Kennett et al .
  • monoclonal antibodies can be readily produced using the method of Kohler and Milstein, Nature (1975) 256:495-497, or a modification thereof.
  • a suitable murine host animal is immunized with one or more MADER peptide antigens.
  • the peptide antigens can be linked to a carrier prior to immunization.
  • Suitable carriers are typically large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, lipid aggregates (such as oil droplets or liposomes) , and inactive virus particles.
  • the MADER antigen may be conjugated to a carrier molecule in order to enhance the immunogenicity thereof.
  • Dissociated spleen cells, or isolated B-lymphoblasts are then induced to fuse with myeloma cells to form hybridomas which are then cultured in a selective medium (e.g., hypoxanthine, aminopterin, thymidine medium, "HAT") .
  • a selective medium e.g., hypoxanthine, aminopterin, thymidine medium, "HAT"
  • Available murine myeloma lines such as those available from the American Type Culture Collection (ATCC, Rockville, MD) can be used in the fusions.
  • the resulting hybridomas are plated by limiting dilution, and assayed for the production of antibody molecules which bind specifically to the immunizing MADER antigen.
  • the selected monoclonal antibody-secreting hybridomas are then cultured, either in vitro (e . g . , in tissue culture bottles or hollow fiber reactors), or in vivo ( e . g . , as ascites in mice) .
  • Suitable MADER antigens used in the production of the above monoclonal antibodies include immunogenic peptide fragments that are at least about 5 amino acids in length, preferably 7-10 amino acids in length, and most preferably at least about 10 to 15 amino acids in length. There is no critical upper limit to the length of the fragment, which can comprise an entire MADER sequence, or alternatively one or more MADER peptides fused to a heterologous protein sequence.
  • MADER peptide antigens may be synthesized by protein synthesis techniques known to those of skill in the art. In general, these methods employ either solid or solution phase synthesis methods.
  • peptides can also be produced using recombinant techniques that are known in the art. For example, a DNA sequence encoding all or a portion of a MADER peptide can be synthesized using standard methods. See , e . g. , Edge (1981) Nature 292:756; Nambair et al.
  • the sequence can be derived from genomic or cDNA.
  • the DNA is cloned into an appropriate expression vector, either procaryotic or eucaryotic, using conventional methods. Suitable host cells are then transfected with the expression vector and cultured under conditions allowing for expression of the MADER peptides.
  • MADER peptide antigens can be produced by enzymatic or chemical cleavage of purified MADER protein. Such procedures are conventional and well-known in the art.
  • the anti-MADER monoclonal antibodies are available for use in immunohistochemical methods of detecting malignant melanoma cells, or other cancerous cells, present in a tissue sample.
  • Functional fragments of the anti-MADER antibody molecules will also find use with the present invention, and can be produced by cleaving a constant region from an antibody molecule using, e.g., pepsin, to produce F(ab') 2 fragments.
  • Fab fragments can be produced using known methods, e.g., by digestion of the monoclonal antibodies with papain.
  • tissue samples suspected of containing malignant melanoma cells, or other cancerous cells are then exposed to the anti-MADER monoclonal preparations, and the presence or absence of bound antibodies on the tissue sample is determined as an indication of the presence or absence of a malignancy.
  • the tissue samples generally comprise nodular or tumorous skin lesions that have been biopsied for histological analysis.
  • the samples are prepared for analysis using known techniques. For example, biopsy tissue samples may be reduced to single cell suspensions using techniques known in the art such as physical maceration, sonication, centrifugation or the like. Cell lysates are then obtained which are suitable for reaction with the monoclonal antibody molecules.
  • tissue sections are prepared using either freezing or paraffin sectioning methods.
  • Paraffin tissue sections e.g., 5 ⁇ m sections
  • Frozen tissue sections can be prepared by immediately deep freezing a 2-3 mm tissue slice (obtained from a fresh surgical biopsy) in OCT compound using the method of Ruiter, D.J., (1990) Curr. Opinion Oncol . 2.:377-387.
  • the tissue sections can then be fixed in acetone and placed on a slide.
  • the prepared slides are then incubated with a preparation containing one or more of the above-described anti-MADER monoclonal antibodies.
  • the primary anti-MADER monoclonal antibody in a direct procedure
  • a second or third antibody capable of recognizing the primary antibody in an indirect procedure
  • Suitable labels include, for example, enzymes, radioisotopes, fluorescers, chromophores, chemiluminescers, dyes, metal ions or ligands which provide for rapid and sensitive detection using techniques known in the art.
  • Particularly suitable labels include enzymes such as horse radish peroxidase or alkaline phosphatase, or complexes such as avidin-biotin.
  • a number of techniques to detect the presence of the label are known, e.g. , fluorometric, spectrophotometric, autoradiography, scintillation counting, and visual (e.g., colorimetric or chemiluminescence) techniques.
  • oligonucleotide probes comprising a nucleotide sequence having at least a 17 base region that is complementary to a region of mRNA transcribed from the MADER gene.
  • the oligonucleotide probes may be composed of DNA, RNA, and/or synthetic nucleotide analogues.
  • the nucleotide may be isolated from a suitable biological source using known methods such as by the chemical action of detergents, bases, acids, chaotropic salts or mixtures thereof. If desired, the average size of the nucleic acid sequence may be decreased by enzymatic, physical or chemical means, e.g., using restriction enzymes, sonication, chemical degradation and the like.
  • the oligonucleotide probes may be synthetically derived, using a combination of solid phase direct oligonucleotide synthesis chemistry and enzymatic ligation methods which are conventional in the art.
  • cDNA sequences of the MADER gene are known, and are depicted in Figure 1 (SEQ ID NO. : ) and Figure 2 (SEQ ID NO.: ).
  • a MADER mRNA sequence is also readily available (GenBank accession number
  • Synthetic sequences may be prepared under the invention using commercially available oligonucleotide synthesis devices such as those devices available from Applied Biosystems, Inc. (Foster City, CA) .
  • the oligonucleotide probes are labeled using a suitable detectable moiety.
  • a wide variety of methods of detectably labeling target oligonucleotides are known in the art. See, e . g . , Dunn et al. (1980) Methods Enzymol . .6J5:468-478; Palva et al. (1983) Journal Clin . Micro . 18 . :92-100; Ranki et al (1983) Gene 2_l:77-85; Polsky-Cynkin et al. (1985) Clin . Chem . 11:1438-1443; and U.S. Patent Nos. 4,486,539 and 4,563,419.
  • the labeled probes are used in mixed phase hybridization assays to detect the presence of MADER mRNAs in a selected tissue sample.
  • suitable mixed-phase hybridization techniques are well known in the art. Proceeding with the method, tissue samples suspected of containing cells which over-express the MADER gene are reduced to single cell suspensions using known techniques. The cell suspensions are treated, e.g., lysed, to release the cellular oligonucleotides including RNAs. Chemical lysing may be performed using dilute aqueous alkali, e.g., 0.1 to 1.0 M sodium hydroxide. The alkali serves to denature the RNAs.
  • RNA is extracted from the lysate and purified using methods such as density gradient centrifugation, ethanol precipitation, phenol extraction, or the like. See , e .
  • RNA may be digested using restriction endonucleases to provide smaller nucleotide segments.
  • the sample RNAs are immobilized to a solid support or substrate. Immobilization of oligonucleotides to a suitable substrate may be performed using conventional techniques. See, e . g. , Letsinger et al. (1975) Nucl . Acids Res . 2 :773-786; "Oligonucleotide Synthesis, a Practical Approach , " Gait, M.J. (ed.), Oxford, England: IRL Press (1984).
  • Commonly used solid supports include nitrocellulose or nylon, and methods of immobilizing nucleotides to such supports include transfer of selected sequences onto nitrocellulose filters or nylon membranes using Southern blot, colony and plaque blot, or dot and slot blot techniques.
  • the immobilized cellular RNA and the detectably labeled oligonucleotide probes are incubated under hybridizing conditions to provide an RNA-probe hybrid.
  • Hybridization generally takes from about 30 minutes to about 2 hours. The hybridization occurs at the highest rate at approximately 25°C below the temperature at which the nucleotide hybrid is 50% melted (the "T m ”) .
  • the T m for a particular hybridization pair will vary with the length and nature of the nucleotides and may be readily determined by those of ordinary skill in the art.
  • hybridizations are carried out in a buffered aqueous medium typically formulated with a salt buffer, detergents, nuclease inhibitors and chelating agents. Such formulations may be selected to preclude significant non-specific binding of nucleotides with the support surface. Depending on the nature of the particular oligonucleotide binding pair, various solvents may be added to the medium such as formamide, dimethylformamide and dimethylsulfoxide, and the stringency of the hybridization medium may be controlled by temperature, pH, salt concentration, solvent system, or the like. Defining appropriate hybridization conditions is within the skill of the art. See, e . g . , Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual , Second Edition (1989) .
  • a washing step is performed to provide an immobilized RNA-probe complex substantially free of unbound probe.
  • a detection step is then carried out to detect the presence of any labeled probe on the substrate. In this manner, a signal can be obtained which is indicative of the presence or absence of cells in the tissue sample which over-express the MADER gene.
  • the detection step is carried out under suitable conditions, such as in a detection solution formulated according to a particular detection means (e.g., where the label employed is an enzyme, the solution is formulated to include the selected enzyme substrate and any necessary reagents) .
  • in-situ hybridization is carried out to detect the presence of MADER mRNAs in a selected tissue sample.
  • tissue samples can be post-fixed, frozen, and then sectioned to provide 5 to 35 ⁇ m sections.
  • the sections can be mounted onto suitable substrates, such as polylysine- coated slides, and treated with proteinase K, aceticanhydride, and then dehydrated in graded alcohol.
  • suitable substrates such as polylysine- coated slides, and treated with proteinase K, aceticanhydride, and then dehydrated in graded alcohol.
  • the mounted sections can then be incubated under hybridizing conditions with detectably labeled oligonucleotide probes, prepared as described above, to provide an RNA-probe hybrid.
  • the hybridizations are carried out as above, the mounted washed, and then a detection step is performed to detect the presence of any labeled probe on the substrate.
  • a method for detecting a melanoma malignancy, or other cancerous condition, in a tissue sample which method generally comprises visualizing the presence or absence of a translocation of MADER in cellular genomes using fluorescence in situ hybridization (FISH) .
  • FISH fluorescence in situ hybridization
  • the method of the invention generally entails providing immobilized chromosomal target DNA that has been obtained from a tissue sample suspected of including cells having undergone a MADER translocation.
  • the target DNA is immobilized to a suitable substrate (e.g., fixed to a microscope slide) using oligonucleotide immobilization techniques such as those described above.
  • Oligonucleotide probes having a nucleotide sequence that is complementary to a MADER nucleotide sequence, are provided as described above.
  • the probes are generally synthetically derived using a combination of conventional solid phase direct oligonucleotide synthesis chemistry and enzymatic ligation methods. "Oligonucleotide Synthesis, a Practical Approach , " Gait, M.J. (ed.), Oxford, England: IRL Press (1984).
  • the oligonucleotide probes are preferably DNA fragments ranging from about 200 to 1000 bp in length which have been labeled with a fluorescent moiety. However, the length of the probe is not considered limiting in the present invention. Suitable fluorescent labels include, but are not limited to, fluorescein isothiocyanate (Fitc) , phycoerythrin (PE) , rhodamine and Texas red.
  • Both the labeled oligonucleotide probe and the immobilized target chromosomal DNA are denatured separately, then incubated together under hybridizing conditions to provide a target-probe hybrid. After any unbound probe is washed off, the bound probe is detected using standard fluorescent microscopy. In this manner, the position of the MADER gene sequence can be readily visualized to determine the presence or absence of MADER translocation events.
  • novel compositions containing MADER immunogens are provided for use in immunotherapeutic methods for treating a subject having malignant melanoma, or another cancerous condition.
  • MADER immunogens can be obtained from either MADER polypeptides or polynucleotides.
  • Polypeptide MADER immunogens can comprise a natural or synthetically derived full length protein, or one or more immunogenic fragments of a MADER polypeptide molecule.
  • Immunogenic MADER peptide fragments comprise a MADER amino acid sequence of at least about 6 residues or greater.
  • Polynucleotide MADER immunogens can comprise one or more DNA molecules containing polynucleotide sequences of at least about 18 to 30 bp that are substantially homologous to regions of the MADER gene.
  • MADER polynucleotide and polypeptide sequences of various lengths have been described herein, and are depicted in Figures 1-3.
  • the MADER immunogens are generally combined with a pharmaceutically acceptable vehicle to provide compositions for use in eliciting an immune response against cells over-expressing MADER in an immunized subject.
  • a pharmaceutically acceptable carrier or vehicle includes any and all solvents, dispersion media, antibacterial and antifungal agents that are substantially non-toxic to humans. The use of such media and agents for pharmaceutically active substances is well known in the art.
  • the compositions may be emulsified or the active ingredient encapsulated in liposome vehicles.
  • the MADER immunogen can be mixed with vehicles containing excipients which are pharmaceutically acceptable and compatible with the immunogen.
  • Suitable vehicles are, for example, water, saline, dextrose, glycerol, ethanol, or the like, and combinations thereof.
  • the vehicle may contain minor amounts of auxiliary substances such as wetting or emulsifying agents and/or pH buffering agents.
  • auxiliary substances such as wetting or emulsifying agents and/or pH buffering agents.
  • Actual methods of preparing such compositions are known, or will be apparent, to those skilled in the art. See, e . g. , Remington 's Pharmaceutical Sciences , Mack Publishing Company, Easton, Pennsylvania, 18th edition, 1990.
  • the compositions of the present invention will, in any event, contain a quantity of the MADER immunogen adequate to achieve the desired immunized state in the subject being treated.
  • suitable adjuvants may be incorporated into the compositions to enhance the immunogenicity of the compositions.
  • suitable adjuvants include organic molecules (e.g., muramyl dipeptide and tuftsin) , synthetic adjuvants (e.g. levamisole and isoprinosine) and other agents such as aliphatic nitrogenous bases (e.g., dimethyldioctadecylammonium bromide (DDA) and N,N-dioctadecyl-N,N-bis(2- hydroxyethyl)propanediamine (“avridine”) .
  • DDA dimethyldioctadecylammonium bromide
  • avridine N,N-dioctadecyl-N,N-bis(2- hydroxyethyl)propanediamine
  • Such agents act non-specifically to enhance the immunogenicity of a particular composition, thus reducing the quantity of antigen necessary in any given vaccine, as well as the frequency of injection.
  • the MADER immunogen compositions can be administered to a subject using parenteral (e.g., intravenous or intramuscular) injection, or by other suitable routes such as subcutaneous or intradermal injection, transdermal delivery, or like modes of administration. Effective dosages for the MADER immunogens in immunotherapies can be determined by routine experimentation, keeping in mind the objective of the treatment.
  • pharmaceutical forms of MADER immunogens suitable for injectable use include sterile aqueous solutions or dispersions.
  • Sterile injectable solutions can be prepared by incorporating the MADER immunogens of the invention into an appropriate solvent, such as sodium phosphate-buffered saline, followed by filter sterilization.
  • MADER proteins are coupled to approximately 10-35 ⁇ m biodegradable beads using known methods of attachment.
  • DeLuca et al. (1987) “Porous Biodegradable Microspheres for Parenteral Administration , " Topics in Pharmaceutical Sciences, Elsevier Science Publishers, B.V., Amsterdam. Immunization with such particulate antigens targets the proteins to the major histocompatibility complex class I pathway, thus eliciting a cytotoxic response.
  • MADER antisense molecules are used in tumor therapies.
  • a number of pharmaceuticals have been described in the art which are capable of binding specifically and predictably to certain nucleic acid target sequences in order to inhibit or modulate the expression of disease-causing genes. Neckers et al (1992) Crit . Rev . Oncogenesi ⁇ 2:175; Simons et al. (1992) Nature 359:67; Bayever et al. (1992) Antisense Res . Dev . 2 :109; Whitesell et al. (1991) Antisense
  • antisense oligonucleotides capable of selectively binding to target MADER sequences are provided herein for use in antisense therapeutics.
  • the antisense oligonucleotides are synthetic oligonucleotides that bind via Watson-Crick base pairing to complementary regions of MADER mRNA, thereby inhibiting MADER protein biosynthesis.
  • synthetic MADER antisense oligonucleotide molecules can be prepared using solid phase chemistry techniques in combination with the phosphoramidite method. See, e.g., Beaucage et al. (1992) Tetrahedron 48:2223.
  • any suitable synthetic method known in the art may be used herein to provide the subject antisense oligonucleotides.
  • the molecules range from about 10 to about 30 bases in length, wherein a 17-mer oligonucleotide sequence is most preferable since such a sequence is generally regarded to appear statistically only once in the human genome.
  • the antisense oligonucleotides must be sufficiently stable in serum and within target cells, must be capable of penetrating cell membranes and of forming stable complexes with their target MADER sequences under physiological conditions. Accordingly, a number of chemical modifications to the "natural" structure of the synthetic oligonucleotides need to be made such as, but not limited to, modifications or replacement of the phosphodiester backbone and base and sugar modifications. Such modifications are within the general skill of the art.
  • the MADER oligonucleotide antisense molecules are combined with suitable pharmaceutically acceptable vehicles to provide compositions for administration (e.g., via parenteral injection) to a subject. Improved cellular uptake of the antisense molecules can be achieved by lipophilic derivatization of the oligonucleotides (e.g., as lipophilic conjugates at the 5' terminus) , by conjugation to poly-L-lysine, or by packaging into antibody-targeted liposomes.
  • the subject compositions can preferably be injected directly into malignant melanoma tissue, or into other cancerous tissue.
  • a construct containing the MADER gene is also suitable for DNA immunizations using known techniques. See, e . g. , U.S. Patent No. 5,589,466 to Feigner et al.
  • the monoclonal antibodies 5B3 (IgGl) and 5H1 (IgGl) were produced against recombinant MADER protein as described above.
  • the monoclonal antibody W6/32 (IgG2a) directed against HLA class I molecules was obtained from the American Type Culture Collection (Rockville MD) .
  • Monoclonal antibody M701 (IgGl) directed against the CD45 leukocyte antigen was obtained from Dako (Glostrup, DK)
  • monoclonal antibody G7A5 (IgGl) directed against the melanoma-nevus associated proteoglycan (Garrigues et al. (1986) J . Cell . Biol . 102:1699-1710) was obtained from Immunotech (Marseille, FR) .
  • PBMC peripheral blood mononuclear cells
  • RNA was prepared from cells and tissues using a modified method of Okayama et al. (1987) Methods Enzymol . 154:3-28. Cesium trifluoroacetate (CsTFA, Pharmacia) was used for isolation and separation of RNA by isopycnic centrifugation. 20 ⁇ g total RNA were denatured in formaldehyde, separated on a 1.2 % agarose/formaldehyde gel, transferred to Hybond-N nylon membrane (Amersham, Braunschweig) , and UV cross-linked. Genomic DNA was digested with restriction endonucleases, electrophoresed, transferred to Hybond-N nylon membrane and hybridized as described for plasmid blots.
  • CsTFA Cesium trifluoroacetate
  • TTTCCAC—3' (SEQ ID NO.: ) (Dugaiczyk et al. (1985)
  • Biochem . 2J2:1605-1613 were end-labeled using terminal desoxynucleotidyl transferase (Gibco BRL) . Filters were hybridized and washed as previously described (Sers et al. (1993) Proc . Natl . Acad . Sci USA 90:8514-8518) at 65°C. Quantitation of autoradiograms was performed with an Elscript densitometer (Hirschmann, Unterhaching) .
  • Membranes were hybridized overnight at 65°C in 6x SSC/5x Denhardt's solution/0.5 % SDS/salmon sperm DNA (20 ⁇ g/ml) . The filters were washed at 65°C in 3x SSC /0.1% SDS, lx SSC/0.1% SDS, 0.3X SSC/0.1% SDS, and 0.Ix SSC/0.1% SDS (40 minutes each) .
  • Double-stranded DNA sequencing reaction was performed on the various clones in pUC18 using the dideoxy nucleotide chain termination method (Sanger et al. (1977) Proc . Natl . Acad . Sci . USA 24:5436-5467) with Sequenase enzyme (USB, Amersham) and with an AutoRead Sequencing Kit (Pharmacia, Uppsala) using oligonucleotides complementary to the vector and to the cDNA insert.
  • Genomic Southern blot analysis was performed with DNA from normal individuals and from a variety of tumor cell lines.
  • genomic DNA from EBV-transformed B cells obtained from a normal individual was digested with the restriction endonucleases BamHI and HindiII and gel electrophoresed. The positions of the molecular weight markers are indicated on the left of the gel.
  • a Genomic Zoo blot is shown containing EcoRI digested DNA from human (lane 1), Rhesus monkey (lane 2), rat (lane 3), mouse (lane 4), dog (lane 5), cow (lane 6) , rabbit (lane 7) , chicken (lane 8) , and S. cerevisiae (lane 9) .
  • Arrowheads in the Figure indicate the location of the weakly hybridizing bands in the rat (lane 3) and the chicken (lane 8).
  • the blots were hybridized with 32 P-labeled complete MADER cDNA.
  • the ability to up-regulate MADER expression in normal leukocytes stimulated with the PHA mitogen was ascertained as follows. Freshly isolated PBMC were exposed to PHA, and cells were harvested at 1, 2, 4, 6, and 10 hours after exposure. RNA isolation and Northern analysis were performed as described above. GADPH was used as a control for RNA loading. All blots were analyzed by autoradiography and densitometry. The results are depicted in Figure 7. As can be seen, MADER expression in normal leukocytes can also be up-regulated in response to mitogen exposure.
  • cycloheximide did not alter the basal level of MADER mRNA (lanes 3 and 4), but completely prevented its induction in response to PMA (lanes 6 and 7 as compared with lanes 9 and 10) .
  • cycloheximide treatment resulted in an increase in c-fos mRNA (lanes 3 and 4) and in a superinduction following exposure to phorbol ester (lanes 6 and 7) as previously described (Treisman, R. (1985) Cell 42:889- 902).
  • MADER gene expression is induced by mitogens and growth factors with mRNA levels reaching a maximum of approximately 10 fold by 2 hours and returning to basal levels by 6 hours. This up-regulation is completely blocked by pretreatment with cycloheximide, indicating that it requires de novo protein synthesis.
  • Genes that are expressed in the first hours following stimulation by growth factors or mitogens have been divided into two groups, the immediate early and the delayed early response genes (Pardee, A.B. (1989) Science 246:603- 608; Nathans et al. (1988) Cold Spring Harbor Symposia on Quantitative Biology LIII:883-900) .
  • the immediate early response genes include transcription factors such as c-fos (Greenberg et al.
  • MADER antigens were produced according to the new England Bio-Labs Protein Fusion and Purification System, using the pMAL-c2 expression vector. Particularly, a full length MADER cDNA clone of the Drop9 variant ( Figure 1, SEQ ID NO.: ) was cloned into the pMAL expression vector and expressed in bacteria as a maltose-binding protein fusion product. The fusion protein was isolated from bacterial extracts using an amylose separation column. The subject MADER cDNA clone encodes a fusion protein referred to as the "Drop9" fusion protein. Immunizations:
  • B-cells obtained from the spleens were induced to fuse with P3x53Ag8.653 myeloma cells to form hybridomas. After selection in HAT medium, the resulting hybridomas were plated by limiting dilution and assayed for the production of antibodies capable of binding to the MADER antigen.
  • a panel of frozen tissue sections from normal human tissue and human malignant melanomas were examined to determine the level of expression of the MADER 55 kD protein.
  • Cryocentrifuge preparations of cell lines or 5- ⁇ m frozen tissue sections were air dried, fixed for 10 minutes in acetone and incubated for 1 hour at room temperature with the monoclonal antibodies produced in Example 4 in the form of tissue culture supernatants or as purified antibodies at 20 ⁇ g/ml.
  • the slides were washed 3 times in phosphate buffered saline (PBS) and incubated for 1 hr with a peroxidase conjugated antiserum v. mouse immunoglobulin (Jackson ImmunoResearch Laboratories, West Grove PA).
  • PBS phosphate buffered saline
  • human malignant melanomas consistently showed strong nuclear staining.
  • the anti-MADER monoclonal antibody 3B3 also exhibited strong nuclear staining in the melanoma cell line Mel Wei.
  • the same regions were also stained with an antibody directed to the melanoma associated proteoglycan.
  • the majority of tumor cells examined in both metastases were found to express easily detectable levels of MADER, while the surrounding leukocytes were found to be negative.
  • cytoplasmic, but not nuclear determinants of MADER were stained with the 1F12 anti- MADER antibody on paraffin sections treated as detailed below. In these cases, melanomas of early progression, but not metastases were stained, thus allowing further differentiation of tumor stages.
  • tissue sections were treated with 0.25% trypsin (Sigma, St. Louis, Mo.) and 0.25% protease type 24 (Sigma) for 5 min.
  • the sections were then blocked with 0.2% BSA (Sigma A-3059) in PBS with sodium azide.
  • BSA Sigma A-3059
  • the first incubation was carried out with monoclonal antibody as culture supernatant (1:2), for 1 hour.
  • the sections were then carefully washed in PBS (the antibody was first drained off, and then slides were put into staining jars with PBS) ; incubation with PBS 4X (4 changes) , for 5 min each.
  • the designated deposits will be maintained for a period of thirty (30) years from the date of deposit, or for five (5) years after the last request for the deposit; or for the enforceable life of the U.S. patent, whichever is longer. Should a culture become nonviable or be inadvertently destroyed, or, in the case of plas id-containing strains, lose its plasmid, it will be replaced with a viable culture(s) of the same taxonomic description.

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Abstract

La présente invention concerne le diagnostic ainsi que les compositions et les procédés thérapeutiques ayant pour cible un gène a réaction précoce retardée associé au mélanome (MADER) et ses produits d'expression. Elle concerne en particulier la production, la caractérisation et l'utilisation d'anticorps monoclonaux capables de se lier spécifiquement à une protéine MADER d'environ 55 kD se trouvant en surexpression dans les mélanomes malins chez l'homme et dans d'autres tissus cancéreux humains. Ces anticorps sont capables de détecter une protéine MADER en surexpression dans des cellules cultivées ainsi que dans des segments congelés ou inclus en paraffine d'une matière de prélèvement biopsique humaine. La protéine MADER, des fragments ou des analogues de celle-ci, ou son gène présent dans un vecteur convenant à un vaccin ADN, sont utilisés en tant qu'immunogènes anticancéreux dans le traitement immunothérapeutique de mélanomes malins et d'autres états cancéreux. De même, on utilise des polynucléotides MADER dans divers procédés cytologiques pour détecter des cellules qui révèlent une surexpression de protéine MADER. Les ARN messagers de MADER servent de cibles dans les thérapies par antisens et ribozyme destinées à empêcher l'expression de la protéine MADER chez un sujet en traitement.
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EP1068530A4 (fr) * 1998-03-03 2003-07-30 Univ Emory Diagnostic du cancer de la prostate metastatique
US6824995B1 (en) 1998-03-03 2004-11-30 Emory University Diagnostic for metastatic prostate cancer
US7411037B2 (en) 1999-03-08 2008-08-12 Genentech, Inc. Polypeptides encoded by a nucleic acid underexpressed in melanoma
US7378501B2 (en) 1999-03-08 2008-05-27 Genentech, Inc. Secreted and transmembrane polypeptides and nucleic acids encoding the same
US7371814B2 (en) 1999-03-08 2008-05-13 Genentech, Inc. Secreted and transmembrane polypeptides and nucleic acids encoding the same
EP1623991A3 (fr) * 1999-09-01 2006-06-07 Genentech, Inc. Polypeptides sécrétés et transmembranaires ainsi que les acides nucléiques codant pour ceux-ci
EP1637541A2 (fr) * 1999-09-01 2006-03-22 Genentech, Inc. Polypeptides sécrétés et transmembranaires ainsi que les acides nucléiques codant pour ceux-ci
EP1623992A3 (fr) * 1999-09-01 2006-05-10 Genentech, Inc. Polypeptides sécrétés et transmembranaires ainsi que les acides nucléiques codant pour ceux-ci
EP1623991A2 (fr) * 1999-09-01 2006-02-08 Genentech, Inc. Polypeptides sécrétés et transmembranaires ainsi que les acides nucléiques codant pour ceux-ci
EP1637541A3 (fr) * 1999-09-01 2006-06-07 Genentech, Inc. Polypeptides sécrétés et transmembranaires ainsi que les acides nucléiques codant pour ceux-ci
EP1623992A2 (fr) * 1999-09-01 2006-02-08 Genentech, Inc. Polypeptides sécrétés et transmembranaires ainsi que les acides nucléiques codant pour ceux-ci
WO2002031198A3 (fr) * 2000-10-11 2003-12-31 Avalon Pharmaceuticals Genes lies au cancer utilises comme cibles pour la chimiotherapie
WO2002031198A2 (fr) * 2000-10-11 2002-04-18 Avalon Pharmaceuticals Genes lies au cancer utilises comme cibles pour la chimiotherapie
US7429241B2 (en) 2005-09-29 2008-09-30 Codman & Shurtleff, Inc. Dural graft and method of preparing the same

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