WO2002031198A2 - Cancer-linked genes as targets for chemotherapy - Google Patents

Cancer-linked genes as targets for chemotherapy Download PDF

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Publication number
WO2002031198A2
WO2002031198A2 PCT/US2001/031607 US0131607W WO0231198A2 WO 2002031198 A2 WO2002031198 A2 WO 2002031198A2 US 0131607 W US0131607 W US 0131607W WO 0231198 A2 WO0231198 A2 WO 0231198A2
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gene
expression
sequence
seq
cancer
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PCT/US2001/031607
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French (fr)
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WO2002031198A3 (en
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Paul E. Young
Stephen Horrigan
Zoe Weaver
Gregory A. Endress
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Avalon Pharmaceuticals
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Priority to CA 2425643 priority Critical patent/CA2425643A1/en
Priority to US10/399,225 priority patent/US20040072192A1/en
Priority to EP20010981443 priority patent/EP1399584A2/en
Priority to JP2002534562A priority patent/JP2004533206A/en
Priority to AU2002213084A priority patent/AU2002213084A1/en
Publication of WO2002031198A2 publication Critical patent/WO2002031198A2/en
Publication of WO2002031198A3 publication Critical patent/WO2002031198A3/en

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    • 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
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • 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
    • G01N33/57484—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
    • 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
    • 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
    • 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
    • C12Q2600/136—Screening for pharmacological 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
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

  • the present invention relates to methods of screening cancer-linked genes and expression products for involvement in the cancer initiation and facilitation process and the use of such genes for screening potential anti- cancer agents, including small organic compounds and other molecules.
  • Cancer-linked genes are valuable in that they indicate genetic differences between cancer cells and normal cells, such as where a gene is expressed in a cancer cell but not in a non-cancer cell, or where said gene is over-expressed or expressed at a higher level in a cancer as opposed to normal or non-cancer cell.
  • the expression of such a gene in a normal cell but not in a cancer cell, especially of the same type of tissue can indicate important functions in the cancerous process. For example, screening assays for novel drugs are based on the response of model cell based systems in vitro to treatment with specific compounds.
  • cytokines cytokines
  • alterations in cell surface markers activation of specific enzymes
  • alterations in ion flux and/or pH Some such screens rely on specific genes, such as oncogenes (or gene mutations).
  • a cancer-linked gene has been identified and its putative amino acid sequence worked out.
  • Such gene is useful in the diagnosing of cancer, the screening of anticancer agents and the treatment of cancer using such agents, especially in that these genes encode polypeptides that can act as markers, such as cell surface markers, thereby providing ready targets for anti-tumor agents such as antibodies, preferably antibodies complexed to cytotoxic agents, including apoptotic agents. .
  • such genes are those corresponding to the sequences of SEQ ID NO: 1 , 3, 5, 7, 9, 11 , 13, 15, 17 and 19 and which encode polypeptides, including those comprising a sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20.
  • genes whose expression is changed in cancerous, as compared to non-cancerous cells, from a specific tissue, for example, lung, where the gene would include a polynucleotide corresponding to the nucleotide sequence of SEQ ID NO: 1 or sequences that are substantially identical to said sequence and/or encode the polypeptide with amino acid sequence of SEQ ID NO: 2 or a polypeptide differing therefrom by conservative amino acid substitutions.. It is another object of the present invention to provide methods of using such characteristic genes as a basis for assaying the potential ability of selected chemical agents to modulate upward or downward the expression of said cancer characteristic, or related, genes.
  • the present invention also relates to a process for treating cancer comprising contacting a cancerous cell with an agent having activity against an expression product encoded by the genes, which process may be conducted either ex vivo or in vivo and which product is disclosed herein.
  • agents may comprise an antibody or other molecule or portion that is specific for said expression product.
  • the polypeptide product of such genes is a polypeptide as disclosed herein, such as SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20.
  • the present invention relates to processes for utilizing a nucleotide sequence for a cancer-linked gene (SEQ ID NO: 1 , 3, 5, 7, 9, 11 , 13, 15, 17 and 19) and the derived amino acid sequence (SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20) as targets for chemotherapeutic agents, especially anti-cancer agents.
  • Characteristic gene sequences whose expression, or non-expression, or change in expression, are indicative of the cancerous or non-cancerous status of a given cell and whose expression is changed in cancerous, as compared to non-cancerous cells, from a specific tissue, are genes that include the nucleotide sequences disclosed herein or sequences that are substantially identical to said sequence, at least about 90% identical, preferably 95% identical, most preferably at least about 98% identical and especially where such gene has the sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17 and 19. Such sequences have been searched within the GenBank database, with the following results.
  • the deposited information represented a prediction of coding sequence deduced from a cDNA clone of unknown function. In accordance with the present invention, this message was up-regulated by at least 3-fold in lung cancer versus normal lung tissue.
  • a search against the Prosite database reveals a domain with 100% similarity to the bromodomain 2 sequence, which is contained in a number of transcription factors.
  • a key role for bromodomain proteins in maintaining normal proliferation is indicated by the implication of several bromodomain proteins in cancer, with four of these identified at translocation breakpoints.
  • TLRs 1-5 Five of the human Toll-like receptors (called TLRs 1-5) may be direct homologs of the corresponding fly molecule and may constitute an important component of innate immunity in humans. Expression analysis shows that this gene is specifically expressed by B-lymphocytes.
  • Genbank Accession Number: AB015631 A nucleotide sequence with Genbank Accession Number: AB015631 with the following specific characteristics: Genbank Accession Number: AB015631; UniGene Cluster: Hs.8752; Locus Link ID: 10330; Cluster Name: Transmembrane protein 4; Sequence Information: 814 bp mRNA (cDNA is SEQ ID NO: 5 with encoded polypeptide as SEQ ID NO: 6) and Chromosomal Location: 12.
  • the UniGene cluster is composed of over 22 sequences derived from a number of tissues. Strongest levels of expression in normal tissues were detected in skeletal muscle.
  • the UniGene cluster contains over 150 EST sequences from a variety of tissue sources.
  • the top BLAST score was to protein kinase nekl , which contains an N-terminal protein kinase domain with about 42% identity to the catalytic domain of NIMA, a protein kinase that controls initiation of mitosis in Aspergillus nidulans.
  • both Nek1 and NIMA have a long, basic C- terminal extension and are therefore similar in overall structure.
  • the UniGene cluster contained over 215 EST sequences from a variety of tissue sources.
  • Two of these p21 GTPase-regulated kinases, the myotonic dystrophy protein kinase-related Cdc42-binding kinases (MRCKalpha and beta), have been demonstrated to phosphorylate nonmuscle myosin light chain, a prerequisite for the activation of actin-myosin contractility.
  • a BLAST search showed A portion of SEQ ID NO: 10 to have about 49% identity to human myotonic dystrophy kinase.
  • the UniGene cluster is composed of 64 sequences derived from a number of tissues. PCTAIRE-3 was expressed in colon adenocarcinomas tested and was expressed at a lower level or not at all in normal colon tissue samples tested. A nucleotide sequence with Genbank Accession Number: NM_006293 with the following specific characteristics: UniGene Cluster: Hs.301 ; Locus Link ID: 7301 ; Cluster Name: TYR03 protein tyrosine kinase; Sequence Information: 4364 bp mRNA (SEQ ID NO: 13 with derived amino acid sequence SEQ ID NO: 14) with Chromosomal Location: 15q15.1-q21.1. The UniGene cluster is composed of over 45 sequences derived from a number of tissues.
  • SEQ ID NO: 14 displays appreciable homology to a variety of receptor tyrosine kinases. For example, a portion of SEQ ID NO: 14 has at least about 43% identity to AXL receptor tyrosine kinase.
  • Over-expression of axl cDNA in NIH 3T3 cells induces neoplastic transformation with the concomitant appearance of a 140 kD axl tyrosine-phosphorylated protein.
  • Locus Link ID 6300; Cluster Name: Mitogen-activated protein kinase 12; Sequence Information: 1457 bp mRNA (SEQ ID NO: 15 with derived amino acid sequence of SEQ ID NO: 16) with Chromosomal Location: 22q13.33.
  • the UniGene cluster is composed of over 22 sequences derived from a number of tissues. The strongest levels of expression in normal tissues were detected in skeletal muscle. This sequence displays appreciable homology to a variety of mitogen-activated protein kinases, for example, with human mitogen-activated protein kinase p38delta.
  • the p38 mitogen-activated protein kinases (MAPK) play a crucial role in stress and inflammatory responses and are also involved in activation of the human immunodeficiency virus gene expression.
  • the UniGene cluster is composed of over 9 sequences, all of which are derived from parathyroid.
  • GenBank database shows an exact match to AF153819 (Homo sapiens inwardly-rectifying potassium channel Kir2.1). This match is entirely confined to the 3' untranslated region of the GenBank entry.
  • the translation product of thyrocarcin and the Kir2.1 gene are identical; however, we cannot formally rule out the possibility that thyrocarcin is a completely different gene that shares some splicing with Kir2.1.
  • SEQ ID NO: 17 shows the nucleotide sequence for Kir2.1 and SEQ ID NO: 21 shows EST cluster identified from expression analysis that is specific for thyroid adenocarcinoma.
  • the derived amino acid sequence from SEQ ID NO: 17 is shown as SEQ ID NO: 18.
  • the sequence of the EST cluster displays no obvious homology to known proteins. However, for the case where the EST cluster is simply the 3'-untranslated region of Kir2.1, this gene is an inwardly- rectifying potassium channel.
  • a nucleotide sequence with Genbank Accession Number: AA133334, representing a Sox2-like HMG-box Oncogenically Expressed Sequence with the following specific characteristics: UniGene Cluster: Hs.129911; Locus Link ID: none; Sequence Information: 1050 bp mRNA (bp base pair, SEQ ID NO: 19), 264 bp ORF and 88 amino acids (SEQ ID NO: 20) with Chromosomal Location: unknown.
  • SEQ ID NO: 19 is present as an EST (Expressed Sequence Tag) in the Gene Logic database. It has been elongated to 1050 bp by overlapping contigs in the public databases.
  • the unigene cluster indicates widespread expression and it was found that the message is upregulated by at least 3-fold in lung cancer versus normal lung tissue.
  • This sequence significant homolgy with Ovis aries SOX-2 gene, and to a slightly lesser extent the murine SOX-2.
  • the Sox gene family consists of a large number of embryonically expressed genes related via the possession of a 79-amino-acid DNA-binding domain known as the HMG box. These genes are transcription factors likely to be involved in the regulation of gene expression.
  • nucleotides and polypeptides, as gene products, used in the processes of the present invention may comprise a recombinant polynucleotide or polypeptide, a natural polynucleotide or polypeptide, or a synthetic polynucleotide or polypeptide, preferably a recombinant polynucleotide or polypeptide. Fragments of such polynucleotides and polypeptides as are disclosed herein may also be useful in practicing the processes of the present invention.
  • a fragment, derivative or analog of the polypeptide may be (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) one in which one or more of the amino acid residues includes a substituent group, or (iii) one in which the mature polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol), or (iv) one in which the additional amino acids are fused to the mature polypeptide, such as a leader or secretory sequence or a sequence which is employed for purification of the mature polypeptide or a proprotein sequence.
  • Such fragments, derivatives and analogs are deemed to be within the scope of those skilled in the art from the teachings herein.
  • the present invention relates to an isolated polynucleotide comprising a polynucleotide at least 65% identical to the polynucleotide of SEQ ID NO: 3, or its complement.
  • said isolated polynucleotide comprises a polynucleotide that has sequence identity of at least 80%, preferably at least about 90%, most preferably at least about 95%, especially at least about 98% and most especially is identical to the sequence of SEQ ID NO: 3.
  • An isolated polynucleotide of the invention may also include the complement of any of the foregoing.
  • the present invention relates to an isolated polypeptide, including a purified polypeptide, comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 4.
  • said isolated polypeptide comprises an amino acid sequence having sequence identity of at least 95%, preferably at least about 98%, and especially is identical to, the sequence of SEQ ID NO: 4.
  • the present invention also includes isolated active fragments of such polypeptides where said fragments retain the biological activity of the polypeptide or where such active fragments are useful as specific targets for cancer treatment, prevention or diagnosis.
  • polypeptides and polypeptides useful in practicing the processes of the present invention may likewise be obtained in an isolated or purified form.
  • polypeptide disclosed herein as being useful in practicing the processes of the invention include different types of proteins in terms of function so that, as recited elsewhere herein, some are enzymes, some are transcription factors and other may be cell surface receptors. Precisely how such cancer- linked proteins are used in the processes of the invention may thus differ depending on the function and cellular location of the protein and therefore modification, or optimization, of the methods disclosed herein may be desirable in light of said differences.
  • a cell-surface receptor is an excellent target for cytotoxic antibodies whereas a transcription factor or enzyme is a useful target for a small organic compound with anti-neoplastic activity.
  • isolated means that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring). It could also be produced recombinantly and subsequently purified.
  • a naturally-occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated.
  • Such polynucleotides for example, those prepared recombinantly, could be part of a vector and/or such polynucleotides or polypeptides could be part of a composition, and still be isolated in that such vector or composition is not part of its natural environment.
  • such isolated, or purified, polypeptide is useful in generating antibodies for practicing the invention, or where said antibody is attached to a cytotoxic or cytolytic agent, such as an apoptotic agent.
  • GenBank submissions can readily be utilized by those skilled in the art to prepare the corresponding full-length polypeptide by peptide synthesis. The same is true for either the polynucleotides or polypeptides disclosed herein for use in the methods of the invention.
  • portion when used in relation to polypeptides, refer to a continuous sequence of residues, such as amino acid residues, which sequence forms a subset of a larger sequence.
  • the oligopeptides resulting from such treatment would represent portions, segments or fragments of the starting polypeptide.
  • such terms refer to the products produced by treatment of said polynucleotides with any of the common endonucleases.
  • the present invention further relates to a vector comprising any of the polynucleotides disclosed herein and to a recombinant cell comprising such vectors, or such polynucleotides or expressing the polypeptides disclosed herein, especially the polypeptide whose amino acid sequence is the sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20.
  • the present invention relates to a process for identifying an agent that modulates the activity of a cancer-related gene comprising: (a) contacting a compound with a cell containing a gene that corresponds to a polynucleotide having a sequence selected from the group consisting of SEQ ID NO: 1 , 3, 5, 7, 9, 11 , 13, 15, 17 and 19 and under conditions promoting the expression of said gene; and (b) detecting a difference in expression of said gene relative to when said compound is not present thereby identifying an agent that modulates the activity of a cancer- related gene.
  • the genes useful for the invention comprise genes that correspond to polynucleotides having a sequence selected from SEQ ID NO: 1 , 3, 5, 7, 9, 11, 13, 15, 17 and 19, or may comprise the sequence of any of the polynucleotides disclosed herein (where the latter are cDNA sequences).
  • corresponding genes refers to genes that encode an RNA that is at least 90% identical, preferably at least 95% identical, most preferably at least 98% identical, and especially identical, to an RNA encoded by one of the nucleotide sequences disclosed herein (i.e., SEQ ID NO: 1 , 3, 5, 7, 9, 11 , 13, 15, 17 and 19).
  • Such genes will also encode the same polypeptide sequence as any of the sequences disclosed herein, preferably SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20, but may include differences in such amino acid sequences where such differences are limited to conservative amino acid substitutions, such as where the same overall three dimensional structure, and thus the same antigenic character, is maintained.
  • amino acid sequences may be within the scope of the present invention where they react with the same antibodies that react with polypeptides comprising the sequences of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 as disclosed herein.
  • model cellular systems using cell lines, primary cells, or tissue samples are maintained in growth medium and may be treated with compounds that may be at a single concentration or at a range of concentrations.
  • cellular RNAs are isolated from the treated cells, primary cells or tumors, which RNAs are indicative of expression of selected genes. The cellular RNA is then divided and subjected to analysis that detects the presence and/or quantity of specific
  • RNA transcripts which transcripts may then be amplified for detection purposes using standard methodologies, such as, for example, reverse transcriptase polymerase chain reaction (RT-PCR), etc.
  • RT-PCR reverse transcriptase polymerase chain reaction
  • the presence or absence, or levels, of specific RNA transcripts are determined from these measurements and a metric derived for the type and degree of response of the sample to the treated compound compared to control samples.
  • a cancer-linked gene sequence (SEQ ID NO: 1 , 3, 5, 7, 9, 11 , 13, 15, 17 and 19) whose expression is, or can be, as a result of the methods of the present invention, linked to, or used to characterize, the cancerous, or non-cancerous, status of the cells, or tissues, to be tested.
  • the processes of the present invention identify novel anti-neoplastic agents based on their alteration of expression of the polynucleotide sequence disclosed herein in specific model systems.
  • the methods of the invention may therefore be used with a variety of cell lines or with primary samples from tumors maintained in vitro under suitable culture conditions for varying periods of time, or in situ in suitable animal models. More particularly, genes have been identified that is expressed at a level in cancer cells that is different from the expression level in non-cancer cells. In one instance, the identified genes are expressed at higher levels in cancer cells than in normal cells.
  • polynucleotides of the invention can include fully operation genes with attendant control or regulatory sequences or merely a polynucleotide sequence encoding the corresponding polypeptide or an active fragment or analog thereof.
  • said gene modulation is downward modulation, so that, as a result of exposure to the chemical agent to be tested, one or more genes of the cancerous cell will be expressed at a lower level (or not expressed at all) when exposed to the agent as compared to the expression when not exposed to the agent.
  • the gene encoding the polypeptide of SEQ ID NO: 2 is expressed at a higher level in cells of lung cancer than in normal lung cells.
  • a selected set of said genes are expressed in the reference cell, including the gene(s) sequences identified for use according to the present invention, but are not expressed in the cell to be tested as a result of the exposure of the cell to be tested to the chemical agent.
  • said chemical agent causes the gene, or genes, of the tested cell to be expressed at a lower level than the same genes of the reference, this is indicative of downward modulation and indicates that the chemical agent to be tested has anti-neoplastic activity.
  • Sequences encoding the same proteins as any of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20, regardless of the percent identity of such sequences, are also specifically contemplated by any of the methods of the present invention that rely on any or all of said sequences, regardless of how they are otherwise described or limited. Thus, any such sequences are available for use in carrying out any of the methods disclosed according to the invention. Such sequences also include any open reading frames, as defined herein, present within the sequence of SEQ ID NO: 1.
  • genes identified by the present disclosure are considered "cancer- related" genes, as this term is used herein, and include genes expressed at higher levels (due, for example, to elevated rates of expression, elevated extent of expression or increased copy number) in cancer cells relative to expression of these genes in normal (i.e., non-cancerous) cells where said cancerous state or status of test cells or tissues has been determined by methods known in the art, such as by reverse transcriptase polymerase chain reaction (RT-PCR) as described in the Example below.
  • RT-PCR reverse transcriptase polymerase chain reaction
  • this relates to the genes whose sequences correspond to the sequences of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17 and 19.
  • the term “correspond” means that the gene has the indicated nucleotide sequence or that it encodes substantially the same RNA as would be encoded by the indicated sequence, the term “substantially” meaning about at least 90% identical as defined elsewhere herein and includes splice variants thereof.
  • sequences disclosed herein may be genomic in nature and thus represent the sequence of an actual gene, such as a human gene, or may be a cDNA sequence derived from a messenger RNA (mRNA) and thus represent contiguous exonic sequences derived from a corresponding genomic sequence or they may be wholly synthetic in origin for purposes of practicing the processes of the invention. Because of the processing that may take place in transforming the initial RNA transcript into the final mRNA, the sequences disclosed herein may represent less than the full genomic sequence. They may also represent sequences derived from ribosomal and transfer RNAs.
  • mRNA messenger RNA
  • genes present in the cell (and representing the genomic sequences) and the sequences disclosed herein, which are mostly cDNA sequences, may be identical or may be such that the cDNAs contain less than the full genomic sequence.
  • Such genes and cDNA sequences are still considered corresponding sequences because they both encode similar RNA sequences.
  • a gene that encodes an RNA transcript, which is then processed into a shorter mRNA is deemed to encode both such RNAs and therefore encodes an RNA complementary to (using the usual Watson-Crick complementarity rules), or that would otherwise be encoded by, a cDNA (for example, a sequence as disclosed herein).
  • sequences disclosed herein correspond to genes contained in the cancerous or normal cells used to determine relative levels of expression because they represent the same sequences or are complementary to RNAs encoded by these genes.
  • genes also include different alleles and splice variants that may occur in the cells used in the processes of the invention.
  • genes of the invention "correspond to" a polynucleotide having a sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17 and 19 if the gene encodes an RNA (processed or unprocessed, including naturally occurring splice variants and alleles) that is at least 90% identical, preferably at least 95% identical, most preferably at least 98% identical to, and especially identical to, an RNA that would be encoded by, or be complementary to, such as by hybridization with, a polynucleotide having the indicated sequence.
  • RNA processed or unprocessed, including naturally occurring splice variants and alleles
  • genes including sequences at least 90% identical to a sequence selected from SEQ ID NO: 1 , 3, 5, 7, 9, 11, 13, 15, 17 and 19, preferably at least about 95% identical to such a sequence, more preferably at least about 98% identical to such sequence and most preferably comprising such sequence are specifically contemplated by all of the processes of the present invention as being genes that correspond to these sequences.
  • sequences encoding the same proteins as any of these sequences, regardless of the percent identity of such sequences are also specifically contemplated by any of the methods of the present invention that rely on any or all of said sequences, regardless of how they are otherwise described or limited. Thus, any such sequences are available for use in carrying out any of the methods disclosed according to the invention.
  • sequences also include any open reading frames, as defined herein, present within any of the sequences of SEQ ID NO: 1 , 3, 5, 7, 9, 11 , 13, 15, 17 and 19.
  • percent identity or “percent identical,” when referring to a sequence, means that a sequence is compared to a claimed or described sequence after alignment of the sequence to be compared (the "Compared Sequence") with the described or claimed sequence (the “Reference Sequence”). The Percent Identity is then determined according to the following formula:
  • C is the number of differences between the Reference Sequence and the Compared Sequence over the length of alignment between the Reference Sequence and the Compared Sequence wherein (i) each base or amino acid in the Reference Sequence that does not have a corresponding aligned base or amino acid in the Compared Sequence and (ii) each gap in the Reference Sequence and (iii) each aligned base or amino acid in the Reference Sequence that is different from an aligned base or amino acid in the Compared Sequence, constitutes a difference; and R is the number of bases or amino acids in the Reference Sequence over the length of the alignment with the Compared Sequence with any gap created in the Reference Sequence also being counted as a base or amino acid.
  • the Compared Sequence has the specified minimum percent identity to the Reference Sequence even though alignments may exist in which the hereinabove calculated Percent Identity is less than the specified Percent Identity.
  • DNA segment refers to a DNA polymer, in the form of a separate fragment or as a component of a larger DNA construct, which has been derived from DNA isolated at least once in substantially pure form, i.e., free of contaminating endogenous materials and in a quantity or concentration enabling identification, manipulation, and recovery of the segment and its component nucleotide sequences by standard biochemical methods, for example, using a cloning vector.
  • segments are provided in the form of an open reading frame uninterrupted by internal nontranslated sequences, or introns, which are typically present in eukaryotic genes. Sequences of non- translated DNA may be present downstream from the open reading frame, where the same do not interfere with manipulation or expression of the coding regions.
  • coding region refers to that portion of a gene which either naturally or normally codes for the expression product of that gene in its natural genomic environment, i.e., the region coding in vivo for the native expression product of the gene.
  • the coding region can be from a normal, mutated or altered gene, or can even be from a DNA sequence, or gene, wholly synthesized in the laboratory using methods well known to those of skill in the art of DNA synthesis.
  • nucleotide sequence refers to a heteropolymer of deoxyribonucleotides.
  • DNA segments encoding the proteins provided by this invention are assembled from cDNA fragments and short oligonucleotide linkers, or from a series of oligonucleotides, to provide a synthetic gene which is capable of being expressed in a recombinant transcriptional unit comprising regulatory elements derived from a microbial or viral operon.
  • expression product means that polypeptide or protein that is the natural translation product of the gene and any nucleic acid sequence coding equivalents resulting from genetic code degeneracy and thus coding for the same amino acid(s).
  • active fragment when referring to a coding sequence, means a portion comprising less than the complete coding region whose expression product retains essentially the same biological function or activity as the expression product of the complete coding region.
  • primer means a short nucleic acid sequence that is paired with one strand of DNA and provides a free 3'-OH end at which a DNA polymerase starts synthesis of a deoxyribonucleotide chain.
  • promoter means a region of DNA involved in binding of RNA polymerase to initiate transcription.
  • enhancer refers to a region of DNA that, when present and active, has the effect of increasing expression of a different DNA sequence that is being expressed, thereby increasing the amount of expression product formed from said different DNA sequence.
  • ORF open reading frame
  • reference to a DNA sequence includes both single stranded and double stranded DNA.
  • specific sequence unless the context indicates otherwise, refers to the single strand DNA of such sequence, the duplex of such sequence with its complement (double stranded
  • the present invention also relates to methods of assaying potential antitumor agents based on their modulation of the expression of the gene sequence according to the invention and methods for diagnosing cancerous, or potentially cancerous, conditions as a result of the patterns of expression of the gene sequence disclosed herein as well as related gene sequence based on common expression or regulation of such genes.
  • relative antineoplastic activity may be ascertained by the extent to which a given chemical agent modulates the expression of genes present in a cancerous cell.
  • a first chemical agent that modulates the expression of a gene associated with the cancerous state i.e., a gene that includes one of the sequences of the invention as disclosed herein and present in cancerous cells
  • a second chemical agent tested by the assays of the invention is thereby deemed to have higher, or more desirable, or more advantageous, anti-neoplastic activity than said second chemical agent.
  • RNA expression is commonly assayed using RNA expression as an indicator.
  • RNA expression is commonly assayed using RNA expression as an indicator.
  • messenger RNA messenger RNA
  • gene expression is determined by the relative expression of the RNAs encoded by such genes.
  • RNA may be isolated from samples in a variety of ways, including lysis and denaturation with a phenolic solution containing a chaotropic agent (e.g., triazol) followed by isopropanol precipitation, ethanol wash, and resuspension in aqueous solution; or lysis and denaturation followed by isolation on solid support, such as a Qiagen resin and reconstitution in aqueous solution; or lysis and denaturation in non-phenolic, aqueous solutions followed by enzymatic conversion of RNA to DNA template copies.
  • a chaotropic agent e.g., triazol
  • steady state RNA expression levels for the genes, and sets of genes, disclosed herein will have been obtained. It is the steady state level of such expression that is affected by potential anti-neoplastic agents as determined herein. Such steady state levels of expression are easily determined by any methods that are sensitive, specific and accurate.
  • Such methods include, but are in no way limited to, real time quantitative polymerase chain reaction (PCR), for example, using a Perkin-Elmer 7700 sequence detection system with gene specific primer probe combinations as designed using any of several commercially available software packages, such as Primer Express software., solid support based hybridization array technology using appropriate internal controls for quantitation, including filter, bead, or microchip based arrays, solid support based hybridization arrays using, for example, chemiluminescent, fluorescent, or electrochemical reaction based detection systems.
  • PCR polymerase chain reaction
  • solid support based hybridization array technology using appropriate internal controls for quantitation, including filter, bead, or microchip based arrays
  • solid support based hybridization arrays using, for example, chemiluminescent, fluorescent, or electrochemical reaction based detection systems.
  • the gene patterns indicative of a cancerous state need not be characteristic of every cell found to be cancerous.
  • the methods disclosed herein are useful for detecting the presence of a cancerous condition within a tissue where less
  • a set of selected genes comprising sequences corresponding to the sequence of SEQ ID NO: 1, may be found, using appropriate probes, either DNA or RNA, to be present in as little as 60% of cells derived from a sample of tumorous, or malignant, tissue while being absent from as much as 60% of cells derived from corresponding non- cancerous, or otherwise normal, tissue (and thus being present in as much as 40% of such normal tissue cells).
  • such gene pattern is found to be present in at least 50% of cells drawn from a cancerous tissue, such as the lung cancer disclosed herein.
  • such gene pattern is found to be present in at least 100% of cells drawn from a cancerous tissue and absent from at least 100% of a corresponding normal, non-cancerous, tissue sample, although the latter embodiment may represent a rare occurrence.
  • the present invention relates to a process for determining the cancerous status of a test cell, comprising determining expression in said test cell of a gene sequence as disclosed herein and then comparing said expression to expression of said at least one gene in at least one cell known to be non-cancerous whereby a difference in said expression indicates that said cell is cancerous.
  • said change in expression is a change in copy number, including either an increase or decrease in copy number.
  • said change in gene copy number may be determined by determining a change in expression of messenger RNA encoded by said gene sequence.
  • Changes in gene copy number may be determined by determining a change in expression of messenger RNA encoded by a particular gene sequence, especially that of Such change in gene copy number may be determined by determining a change in expression of messenger RNA encoded by a particular gene sequence, especially that of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17 and 19.
  • said gene may be a cancer initiating gene, a cancer facilitating gene, or a cancer suppressing gene.
  • a cancer facilitating gene is a gene that, while not directly initiating or suppressing tumor formation or growth, said gene acts, such as through the actions of its expression product, to direct, enhance, or otherwise facilitate the progress of the cancerous condition, including where such gene acts against genes, or gene expression products, that would otherwise have the effect of decreasing tumor formation and/or growth.
  • the presence or absence of expression of a gene corresponding to a sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17 and 19 may be indicative of a cancerous status for a given cell
  • the mere presence or absence of such a gene may not alone be sufficient to achieve a malignant condition and thus the level of expression of such gene pattern may also be a significant factor in determining the attainment of a cancerous state.
  • the level of expression as determined by any of the methods disclosed herein, all of which are well known in the art, may differ between the cancerous versus the non-cancerous cells.
  • the level of expression of the polypeptides disclosed herein is also a measure of gene expression, such as polypeptides having sequence identical, or similar to any polypeptide encoded by the sequence of SEQ ID NO: 1, 3, 5,
  • the present invention further relates to a process for determining the cancerous status of a cell to be tested, comprising determining the level of expression in said cell of at least one gene that includes one of the nucleotide sequences selected from the sequences of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17 and 19, including sequences substantially identical to said sequences, or characteristic fragments thereof, or the complements of any of the foregoing and then comparing said expression to that of a cell known to be non-cancerous whereby the difference in said expression indicates that said cell to be tested is cancerous.
  • gene expression for a gene that includes as a portion thereof one of the sequences of SEQ ID NO: 1 , 3, 5, 7, 9, 11, 13, 15, 17 and 19, is preferably determined by use of a probe that is a fragment of such nucleotide sequence
  • the probe may be formed from a different portion of the gene.
  • Expression of the gene may be determined by use of a nucleotide probe that hybridizes to messenger RNA (mRNA) transcribed from a portion of the gene other than the specific nucleotide sequence disclosed herein.
  • genes there are a variety of different contexts in which genes have been evaluated as being involved in the cancerous process.
  • some genes may be oncogenes and encode proteins that are directly involved in the cancerous process and thereby promote the occurrence of cancer in an animal.
  • other genes may serve to suppress the cancerous state in a given cell or cell type and thereby work against a cancerous condition forming in an animal.
  • Other genes may simply be involved either directly or indirectly in the cancerous process or condition and may serve in an ancillary capacity with respect to the cancerous state. All such types of genes are deemed with those to be determined in accordance with the invention as disclosed herein.
  • the gene determined by said process of the invention may be an oncogene, or the gene determined by said process may be a cancer facilitating gene, the latter including a gene that directly or indirectly affects the cancerous process, either in the promotion of a cancerous condition or in facilitating the progress of cancerous growth or otherwise modulating the growth of cancer cells, either in vivo or ex vivo.
  • the gene determined by said process may be a cancer suppressor gene, which gene works either directly or indirectly to suppress the initiation or progress of a cancerous condition.
  • Such genes may work indirectly where their expression alters the activity of some other gene or gene expression product that is itself directly involved in initiating or facilitating the progress of a cancerous condition.
  • a gene that encodes a polypeptide, either wild or mutant in type, which polypeptide acts to suppress of tumor suppressor gene, or its expression product will thereby act indirectly to promote tumor growth.
  • the process of the present invention includes cancer modulating agents that are themselves either polypeptides, or small chemical entities, that affect the cancerous process, including initiation, suppression or facilitation of tumor growth, either in vivo or ex vivo.
  • Said cancer modulating agent may have the effect of increasing gene expression or said cancer modulating agent may have the effect of decreasing gene expression as such terms have been described herein.
  • the present invention also relates to a process for treating cancer comprising contacting a cancerous cell with an agent having activity against an expression product encoded by a gene sequence as disclosed herein, such as the sequence of SEQ ID NO: 1 , 3, 5, 7, 9, 11 , 13, 15, 17 and 19.
  • the proteins encoded by the genes disclosed herein due to their expression, or elevated expression, in cancer cells represent highly useful therapeutic targets for "targeted therapies” utilizing such affinity structures as, for example, antibodies coupled to some cytotoxic agent.
  • targeted therapies utilizing such affinity structures as, for example, antibodies coupled to some cytotoxic agent.
  • such approaches include the use of so-called suicide "bullets" against intracellular proteins
  • Such antibodies can be produced by either cloning the gene sequences encoding the polypeptide chains of said antibodies or by direct synthesis of said polypeptide chains, with in vitro assembly of the synthesized chains to form active tetrameric (H 2 L 2 ) structures with affinity for specific epitopes and antigenic determinants. This has permitted the ready production of antibodies having sequences characteristic of neutralizing antibodies from different species and sources.
  • all antibodies have a similar overall 3 dimensional structure.
  • This structure is often given as H 2 L 2 and refers to the fact that antibodies commonly comprise 2 light (L) amino acid chains and 2 heavy (H) amino acid chains. Both chains have regions capable of interacting with a structurally complementary antigenic target. The regions interacting with the target are referred to as "variable” or "V" regions and are characterized by differences in amino acid sequence from antibodies of different antigenic specificity.
  • variable regions of either H or L chains contains the amino acid sequences capable of specifically binding to antigenic targets. Within these sequences are smaller sequences dubbed “hypervariable” because of their extreme variability between antibodies of differing specificity. Such hypervariable regions are also referred to as “complementarity determining regions” or “CDR” regions. These CDR regions account for the basic specificity of the antibody for a particular antigenic determinant structure.
  • the CDRs represent non-contiguous stretches of amino acids within the variable regions but, regardless of species, the positional locations of these critical amino acid sequences within the variable heavy and light chain regions have been found to have similar locations within the amino acid sequences of the variable chains.
  • the variable heavy and light chains of all antibodies each have 3 CDR regions, each noncontiguous with the others (termed L1 , L2, L3, H1 , H2, H3) for the respective light (L) and heavy (H) chains.
  • the accepted CDR regions have been described by Kabat et al, J. Biol. Chem. 252:6609-6616 (1977). The numbering scheme is shown in the figures, where the CDRs are underlined and the numbers follow the Kabat scheme.
  • antibody polypeptides contain constant (i.e., highly conserved) and variable regions, and, within the latter, there are the CDRs and the so-called "framework regions” made up of amino acid sequences within the variable region of the heavy or light chain but outside the CDRs.
  • the antibodies disclosed according to the invention may also be wholly synthetic, wherein the polypeptide chains of the antibodies are synthesized and, possibly, optimized for binding to the polypeptides disclosed herein as being receptors.
  • Such antibodies may be chimeric or humanized antibodies and may be fully tetrameric in structure, or may be dimeric and comprise only a single heavy and a single light chain.
  • Such antibodies may also include fragments, such as Fab and F(ab 2 )' fragments, capable of reacting with and binding to any of the polypeptides disclosed herein as being receptors.
  • the present invention relates to immunoglobulins, or antibodies, as described herein, that react with, especially where they are specific for, the polypeptides having amino acid sequences as disclosed herein, preferably those having an amino acid sequence of one of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20.
  • Such antibodies may commonly be in the form of a composition, especially a pharmaceutical composition.
  • compositions useful herein also contain a pharmaceutically acceptable carrier, including any suitable diluent or excipient, which includes any pharmaceutical agent that does not itself induce the production of antibodies harmful to the individual receiving the composition, and which may be administered without undue toxicity.
  • a pharmaceutically acceptable carrier including any suitable diluent or excipient, which includes any pharmaceutical agent that does not itself induce the production of antibodies harmful to the individual receiving the composition, and which may be administered without undue toxicity.
  • Pharmaceutically acceptable carriers include, but are not limited to, liquids such as water, saline, glycerol and ethanol, and the like, including carriers useful in forming sprays for nasal and other respiratory tract delivery or for delivery to the ophthalmic system.
  • liquids such as water, saline, glycerol and ethanol, and the like, including carriers useful in forming sprays for nasal and other respiratory tract delivery or for delivery to the ophthalmic system.
  • the process of the present invention includes embodiments of the above-recited processes wherein the cancer cell is contacted in vivo as well as ex vivo, preferably wherein said agent comprises a portion, or is part of an overall molecular structure, having affinity for said expression product.
  • said portion having affinity for said expression product is an antibody, especially where said expression product is a polypeptide or oligopeptide or comprises an oligopeptide portion, or comprises a polypeptide.
  • Such an agent can therefore be a single molecular structure, comprising both affinity portion and anti-cancer activity portions, wherein said portions are derived from separate molecules, or molecular structures, possessing such activity when separated and wherein such agent has been formed by combining said portions into one larger molecular structure, such as where said portions are combined into the form of an adduct.
  • Said anti- cancer and affinity portions may be joined covalently, such as in the form of a single polypeptide, or polypeptide-like, structure or may be joined non- covalently, such as by hydrophobic or electrostatic interactions, such structures having been formed by means well known in the chemical arts.
  • the anti-cancer and affinity portions may be formed from separate domains of a single molecule that exhibits, as part of the same chemical structure, more than one activity wherein one of the activities is against cancer cells, or tumor formation or growth, and the other activity is affinity for an expression product produced by expression of genes related to the cancerous process or condition.
  • a chemical agent such as a protein or other polypeptide
  • an agent such as an antibody
  • an agent having affinity for an expression product of a cancerous cell, such as a polypeptide or protein encoded by a gene related to the cancerous process, especially a gene as disclosed herein according to the present invention.
  • binding of said agent to said expression product will have the effect of negating said tumor promoting activity.
  • said agent is an apoptosis-inducing agent that induces cell suicide, thereby killing the cancer cell and halting tumor growth.
  • genes within the cancer cell that are regulated in a manner similar to that of the genes disclosed herein and thus change their expression in a coordinated way in response to chemical compounds represent genes that are located within a common metabolic, signaling, physiological, or functional pathway so that by analyzing and identifying such commonly regulated groups of genes (groups that include the gene, or similar sequences, disclosed according to the invention, one can (a) assign known genes and novel genes to specific pathways and (b) identify specific functions and functional roles for novel genes that are grouped into pathways with genes for which their functions are already characterized or described.
  • the processes disclosed according to the present invention at once provide a novel means of assigning function to genes, i.e.
  • Such chemical compounds may have therapeutic relevance to a variety of diseases outside of cancer as well, in cases where such diseases are known or are demonstrated to involve the specific cellular pathway that is affected.
  • polypeptides disclosed herein preferably those of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20, also find use as vaccines in that, where the polypeptide represents a surface protein present on a cancer cell, such polypeptide may be administered to an animal, especially a human being, for purposes of activating cytotoxic T lymphocytes (CTLs) that will be specific for, and act to lyze, cancer cells in said animal. Where used as vaccines, such polypeptides are present in the form of a pharmaceutical composition.
  • the present invention may also employ polypeptides that have the same, or similar, immunogenic character as the polypeptides of SEQ ID NO: 2, 4, 6, 8,
  • the present invention also relates to a process that comprises a method for producing a product comprising identifying an agent according to one of the disclosed processes for identifying such an agent (i.e., the therapeutic agents identified according to the assay procedures disclosed herein) wherein said product is the data collected with respect to said agent as a result of said identification process, or assay, and wherein said data is sufficient to convey the chemical character and/or structure and/or properties of said agent.
  • the present invention specifically contemplates a situation whereby a user of an assay of the invention may use the assay to screen for compounds having the desired enzyme modulating activity and, having identified the compound, then conveys that information (i.e., information as to structure, dosage, etc) to another user who then utilizes the information to reproduce the agent and administer it for therapeutic or research purposes according to the invention.
  • information i.e., information as to structure, dosage, etc
  • the user of the assay may screen a number of test compounds without knowing the structure or identity of the compounds (such as where a number of code numbers are used the first user is simply given samples labeled with said code numbers) and, after performing the screening process, using one or more assay processes of the present invention, then imparts to a second user (user 2), verbally or in writing or some equivalent fashion, sufficient information to identify the compounds having a particular modulating activity (for example, the code number with the corresponding results).
  • This transmission of information from user 1 to user 2 is specifically contemplated by the present invention.
  • genes useful in the methods of the invention disclosed herein are genes corresponding to a polynucleotide having the sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17 or 19 and represent genes that may be over- expressed in malignant cancer, such as is a gene corresponding to SEQ ID NO: 1 for lung, the latter being expressed at least three fold higher in lung cancer samples as compared to normal lung tissues.
  • a gene corresponding to SEQ ID NO: 1 for lung for lung, the latter being expressed at least three fold higher in lung cancer samples as compared to normal lung tissues.
  • not all cancer cells may express this gene a substantial expression thereof in a substantial number of such cells is sufficient to warrant a determination of a cancerous, or potentially cancerous, condition.
  • polynucleotide sequences disclosed according to the present invention are expressed in cancer compared to normal tissue samples or may be expressed at a higher level in cancer as compared to normal tissues. Further, such polynucleotide, or gene, sequence expression in normal tissues may correlate with individuals having a family history of cancer.
  • Such gene sequences may play a direct role in cancer progression, such as in cancer initiation or cancer cell proliferation/survival.
  • one or more genes encoding the same polypeptide as one or more of the sequences disclosed herein represent novel individual gene targets for screening and discovery of small molecules that inhibit enzyme or other cellular functions, e.g. kinase inhibitors. Such molecules represent valuable therapeutics for cancer.
  • small molecules or agents, such as small organic molecules, that down-regulate the expression of these genes in cancer would represent valuable anti-cancer therapeutics.
  • Expression of the gene in normal tissues may indicate a predisposition towards development of lung cancer.
  • the encoded polypeptide might represent a potentially useful cell surface target for therapeutic molecules such as cytolytic antibodies, or antibodies attached to cytotoxic, or cytolytic, agents. .
  • any reference to particular buffers, media, reagents, cells, culture conditions and the like are not intended to be limiting, but are to be read so as to include all related materials that one of ordinary skill in the art would recognize as being of interest or value in the particular context in which that discussion is presented. For example, it is often possible to substitute one buffer system or culture medium for another and still achieve similar, if not identical, results. Those of skill in the art will have sufficient knowledge of such systems and methodologies so as to be able, without undue experimentation, to make such substitutions as will optimally serve their purposes in using the methods and procedures disclosed herein. The present invention will now be further described by way of the following non-limiting example.
  • SW480 cells are grown to a density of 10 cells/cm in Leibovitz's L-15 medium supplemented with 2 mM L-glutamine (90%) and 10% fetal bovine serum. The cells are collected after treatment with 0.25% trypsin, 0.02% EDTA at 37°C for 2 to 5 minutes. The trypsinized cells are then diluted with 30 ml growth medium and plated at a density of 50,000 cells per well in a 96 well plate (200 ⁇ l/well). The following day, cells are treated with either compound buffer alone, or compound buffer containing a chemical agent to be tested, for 24 hours.
  • RNA is quantitated and 10 ng of sample in 1 ⁇ l are added to 24 ⁇ l of Taqman reaction mix containing 1X PCR buffer, RNAsin, reverse transcriptase, nucleoside triphosphates, amplitaq gold, tween 20, glycerol, bovine serum albumin (BSA) and specific PCR primers and probes for a reference gene (18S RNA) and a test gene (Gene X). Reverse transcription is then carried out at 48°C for 30 minutes.
  • the sample is then applied to a Perlin Elmer 7700 sequence detector and heat denatured for 10 minutes at 95°C. Amplification is performed through 40 cycles using 15 seconds annealing at 60°C followed by a 60 second extension at 72°C and 30 second denaturation at 95°C. Data files are then captured and the data analyzed with the appropriate baseline windows and thresholds. The quantitative difference between the target and reference gene is then calculated and a relative expression value determined for all of the samples used. This procedure is then repeated for other genes functionally related to the gene as disclosed herein and the level of function, or expression, noted.
  • the relative expression ratios for each pair of genes is determined (i.e., a ratio of expression is determined for each target gene versus each of the other genes for which expression is measured, where each gene's absolute expression is determined relative to the reference gene for each compound, or chemical agent, to be screened).
  • the samples are then scored and ranked according to the degree of alteration of the expression profile in the treated samples relative to the control.
  • the overall expression of the particular gene relative to the controls, as modulated by one chemical agent relative to another, is also ascertained. Chemical agents having the most effect on a given gene, or set of genes, are considered the most anti- neoplastic.

Abstract

Cancer-linked gene sequences, and derived amino acid sequences, are disclosed along with processes for assaying potential antitumor agents based on their modulation of the expression of these cancer-linked genes. Also disclosed are antibodies that react with the disclosed polypeptides and methods of diagnosing and treating cancer using the gene sequences. A novel gene and polypeptide are also disclosed.

Description

CANCER-LINKED GENES AS TARGETS FOR CHEMOTHERAPY
This application claims the benefit of U.S. provisional application Serial No. 60/239,294, filed 11 October 2000; 60/239,297, filed 11 October 2000
60/239,605, filed 11 October 2000 60/239,802, filed 12 October 2000 60/239,805, filed 12 October 2000 60/239,806, filed 12 October 2000
60/240,622, filed 16 October 2000 60/241,682, filed 19 October 2000
60/241,723, filed 19 October 2000; and 60/244,932, filed 31 October 2000, the disclosures of which are hereby incorporated by reference in their entirety.
FIELD OF THE INVENTION The present invention relates to methods of screening cancer-linked genes and expression products for involvement in the cancer initiation and facilitation process and the use of such genes for screening potential anti- cancer agents, including small organic compounds and other molecules.
BACKGROUND OF THE INVENTION
Cancer-linked genes are valuable in that they indicate genetic differences between cancer cells and normal cells, such as where a gene is expressed in a cancer cell but not in a non-cancer cell, or where said gene is over-expressed or expressed at a higher level in a cancer as opposed to normal or non-cancer cell. In addition, the expression of such a gene in a normal cell but not in a cancer cell, especially of the same type of tissue, can indicate important functions in the cancerous process. For example, screening assays for novel drugs are based on the response of model cell based systems in vitro to treatment with specific compounds. Various measures of cellular response have been utilized, including the release of cytokines, alterations in cell surface markers, activation of specific enzymes, as well as alterations in ion flux and/or pH. Some such screens rely on specific genes, such as oncogenes (or gene mutations). In accordance with the present invention, a cancer-linked gene has been identified and its putative amino acid sequence worked out. Such gene is useful in the diagnosing of cancer, the screening of anticancer agents and the treatment of cancer using such agents, especially in that these genes encode polypeptides that can act as markers, such as cell surface markers, thereby providing ready targets for anti-tumor agents such as antibodies, preferably antibodies complexed to cytotoxic agents, including apoptotic agents. .
BRIEF SUMMARY OF THE INVENTION
In accordance with the present invention, there is provided herein a set of genes related to, or linked to, cancer, or otherwise involved in the cancer initiating and facilitating process and the derived amino acid sequences thereof.
In a particular embodiment, such genes are those corresponding to the sequences of SEQ ID NO: 1 , 3, 5, 7, 9, 11 , 13, 15, 17 and 19 and which encode polypeptides, including those comprising a sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20.
More particularly, such genes whose expression is changed in cancerous, as compared to non-cancerous cells, from a specific tissue, for example, lung, where the gene would include a polynucleotide corresponding to the nucleotide sequence of SEQ ID NO: 1 or sequences that are substantially identical to said sequence and/or encode the polypeptide with amino acid sequence of SEQ ID NO: 2 or a polypeptide differing therefrom by conservative amino acid substitutions.. It is another object of the present invention to provide methods of using such characteristic genes as a basis for assaying the potential ability of selected chemical agents to modulate upward or downward the expression of said cancer characteristic, or related, genes.
It is a further object of the present invention to provide methods of detecting the expression, or non-expression, or amount of expression, of said characteristic gene, or portions thereof, as a means of determining the cancerous, or non-cancerous, status (or potential cancerous status) of selected cells as grown in culture or as maintained in situ.
It is a still further object of the present invention to provide methods for treating cancerous conditions utilizing selected chemical agents as determined from their ability to modulate (i.e., increase or decrease) the characteristic gene, or its protein product.
The present invention also relates to a process for treating cancer comprising contacting a cancerous cell with an agent having activity against an expression product encoded by the genes, which process may be conducted either ex vivo or in vivo and which product is disclosed herein. Such agents may comprise an antibody or other molecule or portion that is specific for said expression product. In a preferred embodiment, the polypeptide product of such genes is a polypeptide as disclosed herein, such as SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20.
DETAILED SUMMARY OF THE INVENTION
The present invention relates to processes for utilizing a nucleotide sequence for a cancer-linked gene (SEQ ID NO: 1 , 3, 5, 7, 9, 11 , 13, 15, 17 and 19) and the derived amino acid sequence (SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20) as targets for chemotherapeutic agents, especially anti-cancer agents. Characteristic gene sequences whose expression, or non-expression, or change in expression, are indicative of the cancerous or non-cancerous status of a given cell and whose expression is changed in cancerous, as compared to non-cancerous cells, from a specific tissue, are genes that include the nucleotide sequences disclosed herein or sequences that are substantially identical to said sequence, at least about 90% identical, preferably 95% identical, most preferably at least about 98% identical and especially where such gene has the sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17 and 19. Such sequences have been searched within the GenBank database, with the following results.
The present invention relates to nucleotide sequences and derived polypeptides having the following characteristics:
A nucleotide sequence with Genbank Accession Numbers: NM_014109 and AA235448, representing an amplified bromodomain- containing protein in cancer that was identified with the following specific characteristics: UniGene Cluster: Hs. 46677; Locus Link ID: none; Sequence Information: 1934 bp mRNA (cDNA is SEQ ID NO: 1) 1086 bp ORF and 362 amino acids (SEQ ID NO: 2) with Chromosomal Location: 8q24 (based on alignment to mapped human genomic sequence). The deposited information represented a prediction of coding sequence deduced from a cDNA clone of unknown function. In accordance with the present invention, this message was up-regulated by at least 3-fold in lung cancer versus normal lung tissue. A search against the Prosite database reveals a domain with 100% similarity to the bromodomain 2 sequence, which is contained in a number of transcription factors. A key role for bromodomain proteins in maintaining normal proliferation is indicated by the implication of several bromodomain proteins in cancer, with four of these identified at translocation breakpoints.
A novel gene identified based upon EST sequences present within dbEST. This novel gene represents a novel member of the family of Toll-like receptors and a portion of the polypeptide derived therefrom has at least 39% sequence identity to human toll-like receptor 1. Five of the human Toll-like receptors (called TLRs 1-5) may be direct homologs of the corresponding fly molecule and may constitute an important component of innate immunity in humans. Expression analysis shows that this gene is specifically expressed by B-lymphocytes. Characteristics were: Genbank Accession Number for sample EST in cluster: AA648836; UniGene Cluster: Hs.89206; Locus Link ID: 10330; Cluster Name: ESTs, Weakly similar to TLR6 [H.sapiens]; Sequence Information: 1274 bp mRNA (cDNA is SEQ ID NO: 3 with encoded polypeptide SEQ ID NO: 4. The UniGene cluster is composed of 10 sequences, all derived from tonsil. Microarray expression analysis indicates specific expression in B-lymphocytes.
A nucleotide sequence with Genbank Accession Number: AB015631 with the following specific characteristics: Genbank Accession Number: AB015631; UniGene Cluster: Hs.8752; Locus Link ID: 10330; Cluster Name: Transmembrane protein 4; Sequence Information: 814 bp mRNA (cDNA is SEQ ID NO: 5 with encoded polypeptide as SEQ ID NO: 6) and Chromosomal Location: 12. The UniGene cluster is composed of over 22 sequences derived from a number of tissues. Strongest levels of expression in normal tissues were detected in skeletal muscle.
A nucleotide sequence with Genbank Accession Number: NM_014397, AB026289 and with the following specific characteristics: UniGene Cluster: Hs.9625; Locus Link ID: 27073; Public Cluster Name: SID6-1512, putative serine-threonine protein kinase; Sequence Information: 1597 bp mRNA (SEQ ID NO: 7), 921 bp ORF and 307 amino acids (and SEQ ID NO: 8) with Chromosomal Location: 9q33. From the record in Genbank the complete mRNA and protein sequences are obtained. SID6-1512 shares sequence similarity with murine NEK1, a kinase involved in cell cycle regulation. The UniGene cluster contains over 150 EST sequences from a variety of tissue sources. The top BLAST score was to protein kinase nekl , which contains an N-terminal protein kinase domain with about 42% identity to the catalytic domain of NIMA, a protein kinase that controls initiation of mitosis in Aspergillus nidulans. In addition, both Nek1 and NIMA have a long, basic C- terminal extension and are therefore similar in overall structure.
A nucleotide sequence with Genbank Accession Number: NM_006035, AF 28625 and with the following specific characteristics: UniGene Cluster: Hs.12908; Locus Link ID: 9578; Cluster Name: CDC42-binding protein kinase beta (DMPK-like; RCKbeta); Sequence Information: 6780 bp mRNA (SEQ ID NO: 9), 5136 bp ORF and 1711 amino acids (SEQ ID NO: 10) with Chromosomal Location: 14q32.3. The UniGene cluster contained over 215 EST sequences from a variety of tissue sources. The p21 GTPases, Rho and Cdc42, regulate numerous cellular functions by binding to members of a serine/threonine protein kinase subfamily. These functions include the remodeling of the cell cytoskeleton that is a feature of cell growth and differentiation. Two of these p21 GTPase-regulated kinases, the myotonic dystrophy protein kinase-related Cdc42-binding kinases (MRCKalpha and beta), have been demonstrated to phosphorylate nonmuscle myosin light chain, a prerequisite for the activation of actin-myosin contractility. A BLAST search showed A portion of SEQ ID NO: 10 to have about 49% identity to human myotonic dystrophy kinase.
A nucleotide sequence with Genbank Accession Number: NM_002654 with the following specific characteristics: UniGene Cluster: Hs.198281; Locus Link ID: 5315; Cluster Name: Pyruvate kinase, muscle; Sequence Information: 2287 bp mRNA (SEQ ID NO: 11 with derived amino acid sequence SEQ ID NO: 12)) with Chromosomal Location: 15q22. This gene in a member of a small sub-family within the cdk family of protein kinases. PCTIARE-3 appears to play a role in signal transduction in terminally differentiated cells. The cloning of the human and murine PCTAIRE-3 genes have been described but no other information is available in the scientific literature. The UniGene cluster is composed of 64 sequences derived from a number of tissues. PCTAIRE-3 was expressed in colon adenocarcinomas tested and was expressed at a lower level or not at all in normal colon tissue samples tested. A nucleotide sequence with Genbank Accession Number: NM_006293 with the following specific characteristics: UniGene Cluster: Hs.301 ; Locus Link ID: 7301 ; Cluster Name: TYR03 protein tyrosine kinase; Sequence Information: 4364 bp mRNA (SEQ ID NO: 13 with derived amino acid sequence SEQ ID NO: 14) with Chromosomal Location: 15q15.1-q21.1. The UniGene cluster is composed of over 45 sequences derived from a number of tissues. Strongest levels of expression in normal tissues detected in brain. SEQ ID NO: 14 displays appreciable homology to a variety of receptor tyrosine kinases. For example, a portion of SEQ ID NO: 14 has at least about 43% identity to AXL receptor tyrosine kinase. Over-expression of axl cDNA in NIH 3T3 cells induces neoplastic transformation with the concomitant appearance of a 140 kD axl tyrosine-phosphorylated protein.
A nucleotide sequence with Genbank Accession Number: NM_002969 with the following specific characteristics: UniGene Cluster: Hs.55039;
Locus Link ID: 6300; Cluster Name: Mitogen-activated protein kinase 12; Sequence Information: 1457 bp mRNA (SEQ ID NO: 15 with derived amino acid sequence of SEQ ID NO: 16) with Chromosomal Location: 22q13.33. The UniGene cluster is composed of over 22 sequences derived from a number of tissues. The strongest levels of expression in normal tissues were detected in skeletal muscle. This sequence displays appreciable homology to a variety of mitogen-activated protein kinases, for example, with human mitogen-activated protein kinase p38delta. The p38 mitogen-activated protein kinases (MAPK) play a crucial role in stress and inflammatory responses and are also involved in activation of the human immunodeficiency virus gene expression.
A nucleotide sequence with Genbank Accession Number: W31344 with the following specific characteristics: UniGene Cluster: Hs.55444; Locus Link ID: unknown; Cluster Name: ESTs; Chromosomal Location: unknown. The UniGene cluster is composed of over 9 sequences, all of which are derived from parathyroid. The GenBank database shows an exact match to AF153819 (Homo sapiens inwardly-rectifying potassium channel Kir2.1). This match is entirely confined to the 3' untranslated region of the GenBank entry. The translation product of thyrocarcin and the Kir2.1 gene are identical; however, we cannot formally rule out the possibility that thyrocarcin is a completely different gene that shares some splicing with Kir2.1. SEQ ID NO: 17 shows the nucleotide sequence for Kir2.1 and SEQ ID NO: 21 shows EST cluster identified from expression analysis that is specific for thyroid adenocarcinoma. The derived amino acid sequence from SEQ ID NO: 17 is shown as SEQ ID NO: 18. The sequence of the EST cluster displays no obvious homology to known proteins. However, for the case where the EST cluster is simply the 3'-untranslated region of Kir2.1, this gene is an inwardly- rectifying potassium channel.
A nucleotide sequence with Genbank Accession Number: AA133334, representing a Sox2-like HMG-box Oncogenically Expressed Sequence with the following specific characteristics: UniGene Cluster: Hs.129911; Locus Link ID: none; Sequence Information: 1050 bp mRNA (bp = base pair, SEQ ID NO: 19), 264 bp ORF and 88 amino acids (SEQ ID NO: 20) with Chromosomal Location: unknown. SEQ ID NO: 19 is present as an EST (Expressed Sequence Tag) in the Gene Logic database. It has been elongated to 1050 bp by overlapping contigs in the public databases. The unigene cluster indicates widespread expression and it was found that the message is upregulated by at least 3-fold in lung cancer versus normal lung tissue. This sequence significant homolgy with Ovis aries SOX-2 gene, and to a slightly lesser extent the murine SOX-2. The Sox gene family consists of a large number of embryonically expressed genes related via the possession of a 79-amino-acid DNA-binding domain known as the HMG box. These genes are transcription factors likely to be involved in the regulation of gene expression.
The nucleotides and polypeptides, as gene products, used in the processes of the present invention may comprise a recombinant polynucleotide or polypeptide, a natural polynucleotide or polypeptide, or a synthetic polynucleotide or polypeptide, preferably a recombinant polynucleotide or polypeptide. Fragments of such polynucleotides and polypeptides as are disclosed herein may also be useful in practicing the processes of the present invention. For example, a fragment, derivative or analog of the polypeptide (SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20) may be (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) one in which one or more of the amino acid residues includes a substituent group, or (iii) one in which the mature polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol), or (iv) one in which the additional amino acids are fused to the mature polypeptide, such as a leader or secretory sequence or a sequence which is employed for purification of the mature polypeptide or a proprotein sequence. Such fragments, derivatives and analogs are deemed to be within the scope of those skilled in the art from the teachings herein.
In one aspect, the present invention relates to an isolated polynucleotide comprising a polynucleotide at least 65% identical to the polynucleotide of SEQ ID NO: 3, or its complement. In preferred embodiments, said isolated polynucleotide comprises a polynucleotide that has sequence identity of at least 80%, preferably at least about 90%, most preferably at least about 95%, especially at least about 98% and most especially is identical to the sequence of SEQ ID NO: 3. An isolated polynucleotide of the invention may also include the complement of any of the foregoing.
In another aspect, the present invention relates to an isolated polypeptide, including a purified polypeptide, comprising an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 4. In preferred embodiments, said isolated polypeptide comprises an amino acid sequence having sequence identity of at least 95%, preferably at least about 98%, and especially is identical to, the sequence of SEQ ID NO: 4. The present invention also includes isolated active fragments of such polypeptides where said fragments retain the biological activity of the polypeptide or where such active fragments are useful as specific targets for cancer treatment, prevention or diagnosis.
The polynucleotides and polypeptides useful in practicing the processes of the present invention may likewise be obtained in an isolated or purified form. In addition, the polypeptide disclosed herein as being useful in practicing the processes of the invention include different types of proteins in terms of function so that, as recited elsewhere herein, some are enzymes, some are transcription factors and other may be cell surface receptors. Precisely how such cancer- linked proteins are used in the processes of the invention may thus differ depending on the function and cellular location of the protein and therefore modification, or optimization, of the methods disclosed herein may be desirable in light of said differences. For example, a cell-surface receptor is an excellent target for cytotoxic antibodies whereas a transcription factor or enzyme is a useful target for a small organic compound with anti-neoplastic activity.
As used herein, the term "isolated" means that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring). It could also be produced recombinantly and subsequently purified. For example, a naturally-occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated. Such polynucleotides, for example, those prepared recombinantly, could be part of a vector and/or such polynucleotides or polypeptides could be part of a composition, and still be isolated in that such vector or composition is not part of its natural environment. In one embodiment of the present invention, such isolated, or purified, polypeptide is useful in generating antibodies for practicing the invention, or where said antibody is attached to a cytotoxic or cytolytic agent, such as an apoptotic agent.
As known in the art "similarity" between two polypeptides is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one polypeptide to the sequence of a second polypeptide. The sequence information disclosed herein, as derived from the
GenBank submissions, can readily be utilized by those skilled in the art to prepare the corresponding full-length polypeptide by peptide synthesis. The same is true for either the polynucleotides or polypeptides disclosed herein for use in the methods of the invention.
As used herein, the terms "portion," "segment," and "fragment," when used in relation to polypeptides, refer to a continuous sequence of residues, such as amino acid residues, which sequence forms a subset of a larger sequence. For example, if a polypeptide were subjected to treatment with any of the common endopeptidases, such as trypsin or chymotrypsin, the oligopeptides resulting from such treatment would represent portions, segments or fragments of the starting polypeptide. When used in relation to a polynucleotides, such terms refer to the products produced by treatment of said polynucleotides with any of the common endonucleases.
The present invention further relates to a vector comprising any of the polynucleotides disclosed herein and to a recombinant cell comprising such vectors, or such polynucleotides or expressing the polypeptides disclosed herein, especially the polypeptide whose amino acid sequence is the sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20.
Methods of producing such cells and vectors are well known to those skilled in the molecular biology art. See, for example, Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989), Wu et al, Methods in Gene Biotechnology (CRC Press, New York, NY, 1997), and Recombinant Gene Expression Protocols, in Methods in Molecular Biology, Vol. 62, (Tuan, ed., Humana Press, Totowa, NJ, 1997), the disclosures of which are hereby incorporated by reference.
In another aspect, the present invention relates to a process for identifying an agent that modulates the activity of a cancer-related gene comprising: (a) contacting a compound with a cell containing a gene that corresponds to a polynucleotide having a sequence selected from the group consisting of SEQ ID NO: 1 , 3, 5, 7, 9, 11 , 13, 15, 17 and 19 and under conditions promoting the expression of said gene; and (b) detecting a difference in expression of said gene relative to when said compound is not present thereby identifying an agent that modulates the activity of a cancer- related gene.
In specific embodiments of the present invention, the genes useful for the invention comprise genes that correspond to polynucleotides having a sequence selected from SEQ ID NO: 1 , 3, 5, 7, 9, 11, 13, 15, 17 and 19, or may comprise the sequence of any of the polynucleotides disclosed herein (where the latter are cDNA sequences). As used herein, "corresponding genes" refers to genes that encode an RNA that is at least 90% identical, preferably at least 95% identical, most preferably at least 98% identical, and especially identical, to an RNA encoded by one of the nucleotide sequences disclosed herein (i.e., SEQ ID NO: 1 , 3, 5, 7, 9, 11 , 13, 15, 17 and 19). Such genes will also encode the same polypeptide sequence as any of the sequences disclosed herein, preferably SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20, but may include differences in such amino acid sequences where such differences are limited to conservative amino acid substitutions, such as where the same overall three dimensional structure, and thus the same antigenic character, is maintained. Thus, amino acid sequences may be within the scope of the present invention where they react with the same antibodies that react with polypeptides comprising the sequences of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 as disclosed herein.
As used herein, the term "conservative amino acid substitution" are defined herein as exchanges within one of the following five groups:
I. Small aliphatic, nonpolar or slightly polar residues:
Ala, Ser, Thr, Pro, Gly;
II. Polar, negatively charged residues and their amides: Asp, Asn, Glu, Gin;
III. Polar, positively charged residues:
His, Arg, Lys;
IV. Large, aliphatic, nonpolar residues: Met Leu, He, Val, Cys
V. Large, aromatic residues:
Phe, Tyr, Trp
In accordance with the present invention, model cellular systems using cell lines, primary cells, or tissue samples are maintained in growth medium and may be treated with compounds that may be at a single concentration or at a range of concentrations. At specific times after treatment, cellular RNAs are isolated from the treated cells, primary cells or tumors, which RNAs are indicative of expression of selected genes. The cellular RNA is then divided and subjected to analysis that detects the presence and/or quantity of specific
RNA transcripts, which transcripts may then be amplified for detection purposes using standard methodologies, such as, for example, reverse transcriptase polymerase chain reaction (RT-PCR), etc. The presence or absence, or levels, of specific RNA transcripts are determined from these measurements and a metric derived for the type and degree of response of the sample to the treated compound compared to control samples.
In accordance with the foregoing, there is thus disclosed herein processes for using a cancer-linked gene sequence (SEQ ID NO: 1 , 3, 5, 7, 9, 11 , 13, 15, 17 and 19) whose expression is, or can be, as a result of the methods of the present invention, linked to, or used to characterize, the cancerous, or non-cancerous, status of the cells, or tissues, to be tested. Thus, the processes of the present invention identify novel anti-neoplastic agents based on their alteration of expression of the polynucleotide sequence disclosed herein in specific model systems. The methods of the invention may therefore be used with a variety of cell lines or with primary samples from tumors maintained in vitro under suitable culture conditions for varying periods of time, or in situ in suitable animal models. More particularly, genes have been identified that is expressed at a level in cancer cells that is different from the expression level in non-cancer cells. In one instance, the identified genes are expressed at higher levels in cancer cells than in normal cells.
The polynucleotides of the invention can include fully operation genes with attendant control or regulatory sequences or merely a polynucleotide sequence encoding the corresponding polypeptide or an active fragment or analog thereof.
In one embodiment of the present invention, said gene modulation is downward modulation, so that, as a result of exposure to the chemical agent to be tested, one or more genes of the cancerous cell will be expressed at a lower level (or not expressed at all) when exposed to the agent as compared to the expression when not exposed to the agent. For example, the gene encoding the polypeptide of SEQ ID NO: 2 is expressed at a higher level in cells of lung cancer than in normal lung cells.
In a preferred embodiment a selected set of said genes are expressed in the reference cell, including the gene(s) sequences identified for use according to the present invention, but are not expressed in the cell to be tested as a result of the exposure of the cell to be tested to the chemical agent. Thus, where said chemical agent causes the gene, or genes, of the tested cell to be expressed at a lower level than the same genes of the reference, this is indicative of downward modulation and indicates that the chemical agent to be tested has anti-neoplastic activity.
Sequences encoding the same proteins as any of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20, regardless of the percent identity of such sequences, are also specifically contemplated by any of the methods of the present invention that rely on any or all of said sequences, regardless of how they are otherwise described or limited. Thus, any such sequences are available for use in carrying out any of the methods disclosed according to the invention. Such sequences also include any open reading frames, as defined herein, present within the sequence of SEQ ID NO: 1.
The genes identified by the present disclosure are considered "cancer- related" genes, as this term is used herein, and include genes expressed at higher levels (due, for example, to elevated rates of expression, elevated extent of expression or increased copy number) in cancer cells relative to expression of these genes in normal (i.e., non-cancerous) cells where said cancerous state or status of test cells or tissues has been determined by methods known in the art, such as by reverse transcriptase polymerase chain reaction (RT-PCR) as described in the Example below. In specific embodiments, this relates to the genes whose sequences correspond to the sequences of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17 and 19. As used herein, the term "correspond" means that the gene has the indicated nucleotide sequence or that it encodes substantially the same RNA as would be encoded by the indicated sequence, the term "substantially" meaning about at least 90% identical as defined elsewhere herein and includes splice variants thereof.
The sequences disclosed herein may be genomic in nature and thus represent the sequence of an actual gene, such as a human gene, or may be a cDNA sequence derived from a messenger RNA (mRNA) and thus represent contiguous exonic sequences derived from a corresponding genomic sequence or they may be wholly synthetic in origin for purposes of practicing the processes of the invention. Because of the processing that may take place in transforming the initial RNA transcript into the final mRNA, the sequences disclosed herein may represent less than the full genomic sequence. They may also represent sequences derived from ribosomal and transfer RNAs. Consequently, the genes present in the cell (and representing the genomic sequences) and the sequences disclosed herein, which are mostly cDNA sequences, may be identical or may be such that the cDNAs contain less than the full genomic sequence. Such genes and cDNA sequences are still considered corresponding sequences because they both encode similar RNA sequences. Thus, by way of non-limiting example only, a gene that encodes an RNA transcript, which is then processed into a shorter mRNA, is deemed to encode both such RNAs and therefore encodes an RNA complementary to (using the usual Watson-Crick complementarity rules), or that would otherwise be encoded by, a cDNA (for example, a sequence as disclosed herein). Thus, the sequences disclosed herein correspond to genes contained in the cancerous or normal cells used to determine relative levels of expression because they represent the same sequences or are complementary to RNAs encoded by these genes. Such genes also include different alleles and splice variants that may occur in the cells used in the processes of the invention.
The genes of the invention "correspond to" a polynucleotide having a sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17 and 19 if the gene encodes an RNA (processed or unprocessed, including naturally occurring splice variants and alleles) that is at least 90% identical, preferably at least 95% identical, most preferably at least 98% identical to, and especially identical to, an RNA that would be encoded by, or be complementary to, such as by hybridization with, a polynucleotide having the indicated sequence. In addition, genes including sequences at least 90% identical to a sequence selected from SEQ ID NO: 1 , 3, 5, 7, 9, 11, 13, 15, 17 and 19, preferably at least about 95% identical to such a sequence, more preferably at least about 98% identical to such sequence and most preferably comprising such sequence are specifically contemplated by all of the processes of the present invention as being genes that correspond to these sequences. In addition, sequences encoding the same proteins as any of these sequences, regardless of the percent identity of such sequences, are also specifically contemplated by any of the methods of the present invention that rely on any or all of said sequences, regardless of how they are otherwise described or limited. Thus, any such sequences are available for use in carrying out any of the methods disclosed according to the invention. Such sequences also include any open reading frames, as defined herein, present within any of the sequences of SEQ ID NO: 1 , 3, 5, 7, 9, 11 , 13, 15, 17 and 19. Further in accordance with the present invention, the term "percent identity" or "percent identical," when referring to a sequence, means that a sequence is compared to a claimed or described sequence after alignment of the sequence to be compared (the "Compared Sequence") with the described or claimed sequence (the "Reference Sequence"). The Percent Identity is then determined according to the following formula:
Percent Identity = 100 [1-(C/R)]
wherein C is the number of differences between the Reference Sequence and the Compared Sequence over the length of alignment between the Reference Sequence and the Compared Sequence wherein (i) each base or amino acid in the Reference Sequence that does not have a corresponding aligned base or amino acid in the Compared Sequence and (ii) each gap in the Reference Sequence and (iii) each aligned base or amino acid in the Reference Sequence that is different from an aligned base or amino acid in the Compared Sequence, constitutes a difference; and R is the number of bases or amino acids in the Reference Sequence over the length of the alignment with the Compared Sequence with any gap created in the Reference Sequence also being counted as a base or amino acid.
If an alignment exists between the Compared Sequence and the Reference Sequence for which the percent identity as calculated above is about equal to or greater than a specified minimum Percent Identity then the Compared Sequence has the specified minimum percent identity to the Reference Sequence even though alignments may exist in which the hereinabove calculated Percent Identity is less than the specified Percent Identity.
As used herein and except as noted otherwise, all terms are defined as given below.
In accordance with the present invention, the term "DNA segment" or "DNA sequence" refers to a DNA polymer, in the form of a separate fragment or as a component of a larger DNA construct, which has been derived from DNA isolated at least once in substantially pure form, i.e., free of contaminating endogenous materials and in a quantity or concentration enabling identification, manipulation, and recovery of the segment and its component nucleotide sequences by standard biochemical methods, for example, using a cloning vector. Such segments are provided in the form of an open reading frame uninterrupted by internal nontranslated sequences, or introns, which are typically present in eukaryotic genes. Sequences of non- translated DNA may be present downstream from the open reading frame, where the same do not interfere with manipulation or expression of the coding regions.
The term "coding region" refers to that portion of a gene which either naturally or normally codes for the expression product of that gene in its natural genomic environment, i.e., the region coding in vivo for the native expression product of the gene. The coding region can be from a normal, mutated or altered gene, or can even be from a DNA sequence, or gene, wholly synthesized in the laboratory using methods well known to those of skill in the art of DNA synthesis.
In accordance with the present invention, the term "nucleotide sequence" refers to a heteropolymer of deoxyribonucleotides. Generally, DNA segments encoding the proteins provided by this invention are assembled from cDNA fragments and short oligonucleotide linkers, or from a series of oligonucleotides, to provide a synthetic gene which is capable of being expressed in a recombinant transcriptional unit comprising regulatory elements derived from a microbial or viral operon.
The term "expression product" means that polypeptide or protein that is the natural translation product of the gene and any nucleic acid sequence coding equivalents resulting from genetic code degeneracy and thus coding for the same amino acid(s).
The term "active fragment," when referring to a coding sequence, means a portion comprising less than the complete coding region whose expression product retains essentially the same biological function or activity as the expression product of the complete coding region.
The term "primer" means a short nucleic acid sequence that is paired with one strand of DNA and provides a free 3'-OH end at which a DNA polymerase starts synthesis of a deoxyribonucleotide chain.
The term "promoter" means a region of DNA involved in binding of RNA polymerase to initiate transcription. The term "enhancer" refers to a region of DNA that, when present and active, has the effect of increasing expression of a different DNA sequence that is being expressed, thereby increasing the amount of expression product formed from said different DNA sequence.
The term "open reading frame (ORF)" means a series of triplets coding for amino acids without any termination codons and is a sequence (potentially) translatable into protein.
As used herein, reference to a DNA sequence includes both single stranded and double stranded DNA. Thus, the specific sequence, unless the context indicates otherwise, refers to the single strand DNA of such sequence, the duplex of such sequence with its complement (double stranded
DNA) and the complement of such sequence.
The present invention also relates to methods of assaying potential antitumor agents based on their modulation of the expression of the gene sequence according to the invention and methods for diagnosing cancerous, or potentially cancerous, conditions as a result of the patterns of expression of the gene sequence disclosed herein as well as related gene sequence based on common expression or regulation of such genes.
In carrying out the foregoing assays, relative antineoplastic activity may be ascertained by the extent to which a given chemical agent modulates the expression of genes present in a cancerous cell. Thus, a first chemical agent that modulates the expression of a gene associated with the cancerous state (i.e., a gene that includes one of the sequences of the invention as disclosed herein and present in cancerous cells) to a larger degree than a second chemical agent tested by the assays of the invention is thereby deemed to have higher, or more desirable, or more advantageous, anti-neoplastic activity than said second chemical agent.
The gene expression to be measured is commonly assayed using RNA expression as an indicator. Thus, the greater the level of RNA (messenger RNA) detected the higher the level of expression of the corresponding gene. Thus, gene expression, either absolute or relative, is determined by the relative expression of the RNAs encoded by such genes.
RNA may be isolated from samples in a variety of ways, including lysis and denaturation with a phenolic solution containing a chaotropic agent (e.g., triazol) followed by isopropanol precipitation, ethanol wash, and resuspension in aqueous solution; or lysis and denaturation followed by isolation on solid support, such as a Qiagen resin and reconstitution in aqueous solution; or lysis and denaturation in non-phenolic, aqueous solutions followed by enzymatic conversion of RNA to DNA template copies.
Normally, prior to applying the processes of the invention, steady state RNA expression levels for the genes, and sets of genes, disclosed herein will have been obtained. It is the steady state level of such expression that is affected by potential anti-neoplastic agents as determined herein. Such steady state levels of expression are easily determined by any methods that are sensitive, specific and accurate. Such methods include, but are in no way limited to, real time quantitative polymerase chain reaction (PCR), for example, using a Perkin-Elmer 7700 sequence detection system with gene specific primer probe combinations as designed using any of several commercially available software packages, such as Primer Express software., solid support based hybridization array technology using appropriate internal controls for quantitation, including filter, bead, or microchip based arrays, solid support based hybridization arrays using, for example, chemiluminescent, fluorescent, or electrochemical reaction based detection systems. The gene patterns indicative of a cancerous state need not be characteristic of every cell found to be cancerous. Thus, the methods disclosed herein are useful for detecting the presence of a cancerous condition within a tissue where less than all cells exhibit the complete pattern. Thus, for example, a set of selected genes, comprising sequences corresponding to the sequence of SEQ ID NO: 1, may be found, using appropriate probes, either DNA or RNA, to be present in as little as 60% of cells derived from a sample of tumorous, or malignant, tissue while being absent from as much as 60% of cells derived from corresponding non- cancerous, or otherwise normal, tissue (and thus being present in as much as 40% of such normal tissue cells). In a preferred embodiment, such gene pattern is found to be present in at least 50% of cells drawn from a cancerous tissue, such as the lung cancer disclosed herein. In an additional embodiment, such gene pattern is found to be present in at least 100% of cells drawn from a cancerous tissue and absent from at least 100% of a corresponding normal, non-cancerous, tissue sample, although the latter embodiment may represent a rare occurrence.
In another aspect the present invention relates to a process for determining the cancerous status of a test cell, comprising determining expression in said test cell of a gene sequence as disclosed herein and then comparing said expression to expression of said at least one gene in at least one cell known to be non-cancerous whereby a difference in said expression indicates that said cell is cancerous.
In one embodiment, said change in expression is a change in copy number, including either an increase or decrease in copy number. In accordance with the present invention, said change in gene copy number may be determined by determining a change in expression of messenger RNA encoded by said gene sequence.
Changes in gene copy number may be determined by determining a change in expression of messenger RNA encoded by a particular gene sequence, especially that of Such change in gene copy number may be determined by determining a change in expression of messenger RNA encoded by a particular gene sequence, especially that of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17 and 19. Also in accordance with the present invention, said gene may be a cancer initiating gene, a cancer facilitating gene, or a cancer suppressing gene. In carrying out the methods of the present invention, a cancer facilitating gene is a gene that, while not directly initiating or suppressing tumor formation or growth, said gene acts, such as through the actions of its expression product, to direct, enhance, or otherwise facilitate the progress of the cancerous condition, including where such gene acts against genes, or gene expression products, that would otherwise have the effect of decreasing tumor formation and/or growth.
Although the presence or absence of expression of a gene corresponding to a sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17 and 19 may be indicative of a cancerous status for a given cell, the mere presence or absence of such a gene may not alone be sufficient to achieve a malignant condition and thus the level of expression of such gene pattern may also be a significant factor in determining the attainment of a cancerous state. Thus, while a pattern of genes may be present in both cancerous and non- cancerous cells, the level of expression, as determined by any of the methods disclosed herein, all of which are well known in the art, may differ between the cancerous versus the non-cancerous cells. Thus, it becomes essential to also determine the level of expression of a gene such as that disclosed herein, including substantially similar sequences and sequences comprising said sequence, as a separate means of diagnosing the presence of a cancerous status for a given cell, groups of cells, or tissues, either in culture or in situ.
The level of expression of the polypeptides disclosed herein is also a measure of gene expression, such as polypeptides having sequence identical, or similar to any polypeptide encoded by the sequence of SEQ ID NO: 1, 3, 5,
7, 9, 11, 13, 15, 17 and 19, such as the polypeptide whose amino acid sequence is the sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20. In accordance with the foregoing, the present invention further relates to a process for determining the cancerous status of a cell to be tested, comprising determining the level of expression in said cell of at least one gene that includes one of the nucleotide sequences selected from the sequences of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17 and 19, including sequences substantially identical to said sequences, or characteristic fragments thereof, or the complements of any of the foregoing and then comparing said expression to that of a cell known to be non-cancerous whereby the difference in said expression indicates that said cell to be tested is cancerous.
In accordance with the invention, although gene expression for a gene that includes as a portion thereof one of the sequences of SEQ ID NO: 1 , 3, 5, 7, 9, 11, 13, 15, 17 and 19, is preferably determined by use of a probe that is a fragment of such nucleotide sequence, it is to be understood that the probe may be formed from a different portion of the gene. Expression of the gene may be determined by use of a nucleotide probe that hybridizes to messenger RNA (mRNA) transcribed from a portion of the gene other than the specific nucleotide sequence disclosed herein. ,
It should be noted that there are a variety of different contexts in which genes have been evaluated as being involved in the cancerous process. Thus, some genes may be oncogenes and encode proteins that are directly involved in the cancerous process and thereby promote the occurrence of cancer in an animal. In addition, other genes may serve to suppress the cancerous state in a given cell or cell type and thereby work against a cancerous condition forming in an animal. Other genes may simply be involved either directly or indirectly in the cancerous process or condition and may serve in an ancillary capacity with respect to the cancerous state. All such types of genes are deemed with those to be determined in accordance with the invention as disclosed herein. Thus, the gene determined by said process of the invention may be an oncogene, or the gene determined by said process may be a cancer facilitating gene, the latter including a gene that directly or indirectly affects the cancerous process, either in the promotion of a cancerous condition or in facilitating the progress of cancerous growth or otherwise modulating the growth of cancer cells, either in vivo or ex vivo. In addition, the gene determined by said process may be a cancer suppressor gene, which gene works either directly or indirectly to suppress the initiation or progress of a cancerous condition. Such genes may work indirectly where their expression alters the activity of some other gene or gene expression product that is itself directly involved in initiating or facilitating the progress of a cancerous condition. For example, a gene that encodes a polypeptide, either wild or mutant in type, which polypeptide acts to suppress of tumor suppressor gene, or its expression product, will thereby act indirectly to promote tumor growth.
In accordance with the foregoing, the process of the present invention includes cancer modulating agents that are themselves either polypeptides, or small chemical entities, that affect the cancerous process, including initiation, suppression or facilitation of tumor growth, either in vivo or ex vivo. Said cancer modulating agent may have the effect of increasing gene expression or said cancer modulating agent may have the effect of decreasing gene expression as such terms have been described herein.
In keeping with the disclosure herein, the present invention also relates to a process for treating cancer comprising contacting a cancerous cell with an agent having activity against an expression product encoded by a gene sequence as disclosed herein, such as the sequence of SEQ ID NO: 1 , 3, 5, 7, 9, 11 , 13, 15, 17 and 19.
The proteins encoded by the genes disclosed herein due to their expression, or elevated expression, in cancer cells, represent highly useful therapeutic targets for "targeted therapies" utilizing such affinity structures as, for example, antibodies coupled to some cytotoxic agent. In such methodology, it is advantageous that nothing need be known about the endogenous ligands or binding partners for such cell surface molecules. Rather, an antibody or equivalent molecule that can specifically recognize the cell surface molecule (which could include an artificial peptide, a surrogate ligand, and the like) that is coupled to some agent that can induce cell death or a block in cell cycling offers therapeutic promise against these proteins. Thus, such approaches include the use of so-called suicide "bullets" against intracellular proteins
With the advent of methods of molecular biology and recombinant technology, it is now possible to produce antibody molecules by recombinant means and thereby generate gene sequences that code for specific amino acid sequences found in the polypeptide structure of the antibodies. Such antibodies can be produced by either cloning the gene sequences encoding the polypeptide chains of said antibodies or by direct synthesis of said polypeptide chains, with in vitro assembly of the synthesized chains to form active tetrameric (H2L2) structures with affinity for specific epitopes and antigenic determinants. This has permitted the ready production of antibodies having sequences characteristic of neutralizing antibodies from different species and sources.
Regardless of the source of the antibodies, or how they are recombinantly constructed, or how they are synthesized, in vitro or in vivo, using transgenic animals, such as cows, goats and sheep, using large cell cultures of laboratory or commercial size, in bioreactors or by direct chemical synthesis employing no living organisms at any stage of the process, all antibodies have a similar overall 3 dimensional structure. This structure is often given as H2L2 and refers to the fact that antibodies commonly comprise 2 light (L) amino acid chains and 2 heavy (H) amino acid chains. Both chains have regions capable of interacting with a structurally complementary antigenic target. The regions interacting with the target are referred to as "variable" or "V" regions and are characterized by differences in amino acid sequence from antibodies of different antigenic specificity.
The variable regions of either H or L chains contains the amino acid sequences capable of specifically binding to antigenic targets. Within these sequences are smaller sequences dubbed "hypervariable" because of their extreme variability between antibodies of differing specificity. Such hypervariable regions are also referred to as "complementarity determining regions" or "CDR" regions. These CDR regions account for the basic specificity of the antibody for a particular antigenic determinant structure.
The CDRs represent non-contiguous stretches of amino acids within the variable regions but, regardless of species, the positional locations of these critical amino acid sequences within the variable heavy and light chain regions have been found to have similar locations within the amino acid sequences of the variable chains. The variable heavy and light chains of all antibodies each have 3 CDR regions, each noncontiguous with the others (termed L1 , L2, L3, H1 , H2, H3) for the respective light (L) and heavy (H) chains. The accepted CDR regions have been described by Kabat et al, J. Biol. Chem. 252:6609-6616 (1977). The numbering scheme is shown in the figures, where the CDRs are underlined and the numbers follow the Kabat scheme.
In all mammalian species, antibody polypeptides contain constant (i.e., highly conserved) and variable regions, and, within the latter, there are the CDRs and the so-called "framework regions" made up of amino acid sequences within the variable region of the heavy or light chain but outside the CDRs.
The antibodies disclosed according to the invention may also be wholly synthetic, wherein the polypeptide chains of the antibodies are synthesized and, possibly, optimized for binding to the polypeptides disclosed herein as being receptors. Such antibodies may be chimeric or humanized antibodies and may be fully tetrameric in structure, or may be dimeric and comprise only a single heavy and a single light chain. Such antibodies may also include fragments, such as Fab and F(ab2)' fragments, capable of reacting with and binding to any of the polypeptides disclosed herein as being receptors. In one aspect, the present invention relates to immunoglobulins, or antibodies, as described herein, that react with, especially where they are specific for, the polypeptides having amino acid sequences as disclosed herein, preferably those having an amino acid sequence of one of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20. Such antibodies may commonly be in the form of a composition, especially a pharmaceutical composition.
The pharmaceutical compositions useful herein also contain a pharmaceutically acceptable carrier, including any suitable diluent or excipient, which includes any pharmaceutical agent that does not itself induce the production of antibodies harmful to the individual receiving the composition, and which may be administered without undue toxicity.
Pharmaceutically acceptable carriers include, but are not limited to, liquids such as water, saline, glycerol and ethanol, and the like, including carriers useful in forming sprays for nasal and other respiratory tract delivery or for delivery to the ophthalmic system. A thorough discussion of pharmaceutically acceptable carriers, diluents, and other excipients is presented in REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Pub. Co., N.J. current edition).
The process of the present invention includes embodiments of the above-recited processes wherein the cancer cell is contacted in vivo as well as ex vivo, preferably wherein said agent comprises a portion, or is part of an overall molecular structure, having affinity for said expression product. In one such embodiment, said portion having affinity for said expression product is an antibody, especially where said expression product is a polypeptide or oligopeptide or comprises an oligopeptide portion, or comprises a polypeptide.
Such an agent can therefore be a single molecular structure, comprising both affinity portion and anti-cancer activity portions, wherein said portions are derived from separate molecules, or molecular structures, possessing such activity when separated and wherein such agent has been formed by combining said portions into one larger molecular structure, such as where said portions are combined into the form of an adduct. Said anti- cancer and affinity portions may be joined covalently, such as in the form of a single polypeptide, or polypeptide-like, structure or may be joined non- covalently, such as by hydrophobic or electrostatic interactions, such structures having been formed by means well known in the chemical arts. Alternatively, the anti-cancer and affinity portions may be formed from separate domains of a single molecule that exhibits, as part of the same chemical structure, more than one activity wherein one of the activities is against cancer cells, or tumor formation or growth, and the other activity is affinity for an expression product produced by expression of genes related to the cancerous process or condition.
In one embodiment of the present invention, a chemical agent, such as a protein or other polypeptide, is joined to an agent, such as an antibody, having affinity for an expression product of a cancerous cell, such as a polypeptide or protein encoded by a gene related to the cancerous process, especially a gene as disclosed herein according to the present invention. Thus, where the presence of said expression product is essential to tumor initiation and/or growth, binding of said agent to said expression product will have the effect of negating said tumor promoting activity. In one such embodiment, said agent is an apoptosis-inducing agent that induces cell suicide, thereby killing the cancer cell and halting tumor growth..
Other genes within the cancer cell that are regulated in a manner similar to that of the genes disclosed herein and thus change their expression in a coordinated way in response to chemical compounds represent genes that are located within a common metabolic, signaling, physiological, or functional pathway so that by analyzing and identifying such commonly regulated groups of genes (groups that include the gene, or similar sequences, disclosed according to the invention, one can (a) assign known genes and novel genes to specific pathways and (b) identify specific functions and functional roles for novel genes that are grouped into pathways with genes for which their functions are already characterized or described. For example, one might identify a group of 10 genes, at least one of which is the gene as disclosed herein, that change expression in a coordinated fashion and for which the function of one, such as the polypeptide encoded by the sequence disclosed herein, is known then the other genes are thereby implicated in a similar function or pathway and may thus play a role in the cancer-initiating or cancer-facilitating process. In the same way, if a gene were found in normal cells but not in cancer cells, or happens to be expressed at a higher level in normal as opposed to cancer cells, then a similar conclusion may be drawn as to its involvement in cancer, or other diseases. Therefore, the processes disclosed according to the present invention at once provide a novel means of assigning function to genes, i.e. a novel method of functional genomics, and a means for identifying chemical compounds that have potential therapeutic effects on specific cellular pathways. Such chemical compounds may have therapeutic relevance to a variety of diseases outside of cancer as well, in cases where such diseases are known or are demonstrated to involve the specific cellular pathway that is affected.
The polypeptides disclosed herein, preferably those of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20, also find use as vaccines in that, where the polypeptide represents a surface protein present on a cancer cell, such polypeptide may be administered to an animal, especially a human being, for purposes of activating cytotoxic T lymphocytes (CTLs) that will be specific for, and act to lyze, cancer cells in said animal. Where used as vaccines, such polypeptides are present in the form of a pharmaceutical composition. The present invention may also employ polypeptides that have the same, or similar, immunogenic character as the polypeptides of SEQ ID NO: 2, 4, 6, 8,
10, 12, 14, 16, 18 and 20 and thereby elicit the same, or similar, immunogenic response after administration to an animal, such as an animal at risk of developing cancer, or afflicted therewith. Thus, the polypeptides disclosed according to the invention will commonly find use as immunogenic compositions. The present invention also relates to a process that comprises a method for producing a product comprising identifying an agent according to one of the disclosed processes for identifying such an agent (i.e., the therapeutic agents identified according to the assay procedures disclosed herein) wherein said product is the data collected with respect to said agent as a result of said identification process, or assay, and wherein said data is sufficient to convey the chemical character and/or structure and/or properties of said agent. For example, the present invention specifically contemplates a situation whereby a user of an assay of the invention may use the assay to screen for compounds having the desired enzyme modulating activity and, having identified the compound, then conveys that information (i.e., information as to structure, dosage, etc) to another user who then utilizes the information to reproduce the agent and administer it for therapeutic or research purposes according to the invention. For example, the user of the assay (user 1) may screen a number of test compounds without knowing the structure or identity of the compounds (such as where a number of code numbers are used the first user is simply given samples labeled with said code numbers) and, after performing the screening process, using one or more assay processes of the present invention, then imparts to a second user (user 2), verbally or in writing or some equivalent fashion, sufficient information to identify the compounds having a particular modulating activity (for example, the code number with the corresponding results). This transmission of information from user 1 to user 2 is specifically contemplated by the present invention.
The genes useful in the methods of the invention disclosed herein are genes corresponding to a polynucleotide having the sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17 or 19 and represent genes that may be over- expressed in malignant cancer, such as is a gene corresponding to SEQ ID NO: 1 for lung, the latter being expressed at least three fold higher in lung cancer samples as compared to normal lung tissues. In addition, in any given sample, not all cancer cells may express this gene a substantial expression thereof in a substantial number of such cells is sufficient to warrant a determination of a cancerous, or potentially cancerous, condition. Thus, the polynucleotide sequences disclosed according to the present invention are expressed in cancer compared to normal tissue samples or may be expressed at a higher level in cancer as compared to normal tissues. Further, such polynucleotide, or gene, sequence expression in normal tissues may correlate with individuals having a family history of cancer.
Such gene sequences may play a direct role in cancer progression, such as in cancer initiation or cancer cell proliferation/survival. For example, one or more genes encoding the same polypeptide as one or more of the sequences disclosed herein represent novel individual gene targets for screening and discovery of small molecules that inhibit enzyme or other cellular functions, e.g. kinase inhibitors. Such molecules represent valuable therapeutics for cancer. In addition, small molecules or agents, such as small organic molecules, that down-regulate the expression of these genes in cancer would represent valuable anti-cancer therapeutics. Expression of the gene in normal tissues may indicate a predisposition towards development of lung cancer. The encoded polypeptide might represent a potentially useful cell surface target for therapeutic molecules such as cytolytic antibodies, or antibodies attached to cytotoxic, or cytolytic, agents. .
It should be cautioned that, in carrying out the procedures of the present invention as disclosed herein, any reference to particular buffers, media, reagents, cells, culture conditions and the like are not intended to be limiting, but are to be read so as to include all related materials that one of ordinary skill in the art would recognize as being of interest or value in the particular context in which that discussion is presented. For example, it is often possible to substitute one buffer system or culture medium for another and still achieve similar, if not identical, results. Those of skill in the art will have sufficient knowledge of such systems and methodologies so as to be able, without undue experimentation, to make such substitutions as will optimally serve their purposes in using the methods and procedures disclosed herein. The present invention will now be further described by way of the following non-limiting example. In applying the disclosure of the example, it should be kept clearly in mind that other and different embodiments of the methods disclosed according to the present invention will no doubt suggest themselves to those of skill in the relevant art. The following example shows how a potential anti-neoplastic agent may be identified using one or more of the genes disclosed herein.
EXAMPLE
SW480 cells are grown to a density of 10 cells/cm in Leibovitz's L-15 medium supplemented with 2 mM L-glutamine (90%) and 10% fetal bovine serum. The cells are collected after treatment with 0.25% trypsin, 0.02% EDTA at 37°C for 2 to 5 minutes. The trypsinized cells are then diluted with 30 ml growth medium and plated at a density of 50,000 cells per well in a 96 well plate (200 μl/well). The following day, cells are treated with either compound buffer alone, or compound buffer containing a chemical agent to be tested, for 24 hours. The media is then removed, the cells lysed and the RNA recovered using the RNAeasy reagents and protocol obtained from Qiagen. RNA is quantitated and 10 ng of sample in 1 μl are added to 24 μl of Taqman reaction mix containing 1X PCR buffer, RNAsin, reverse transcriptase, nucleoside triphosphates, amplitaq gold, tween 20, glycerol, bovine serum albumin (BSA) and specific PCR primers and probes for a reference gene (18S RNA) and a test gene (Gene X). Reverse transcription is then carried out at 48°C for 30 minutes. The sample is then applied to a Perlin Elmer 7700 sequence detector and heat denatured for 10 minutes at 95°C. Amplification is performed through 40 cycles using 15 seconds annealing at 60°C followed by a 60 second extension at 72°C and 30 second denaturation at 95°C. Data files are then captured and the data analyzed with the appropriate baseline windows and thresholds. The quantitative difference between the target and reference gene is then calculated and a relative expression value determined for all of the samples used. This procedure is then repeated for other genes functionally related to the gene as disclosed herein and the level of function, or expression, noted. The relative expression ratios for each pair of genes is determined (i.e., a ratio of expression is determined for each target gene versus each of the other genes for which expression is measured, where each gene's absolute expression is determined relative to the reference gene for each compound, or chemical agent, to be screened). The samples are then scored and ranked according to the degree of alteration of the expression profile in the treated samples relative to the control. The overall expression of the particular gene relative to the controls, as modulated by one chemical agent relative to another, is also ascertained. Chemical agents having the most effect on a given gene, or set of genes, are considered the most anti- neoplastic.

Claims

WHAT IS CLAIMED IS:
1. A process for identifying an agent that modulates the activity of a cancer-related gene comprising: (a) contacting a compound with a cell containing a gene that corresponds to a polynucleotide having a sequence selected from the group consisting of SEQ ID NO: 1 , 3, 5, 7, 9, 11 , 13, 15, 17 and 19 and under conditions promoting the expression of said gene; and
(b) detecting a difference in expression of said gene relative to when said compound is not present thereby identifying an agent that modulates the activity of a cancer- related gene.
2. The process of claim 1 wherein said gene has a sequence selected from the group consisting of SEQ ID NO: SEQ ID NO: 1 , 3, 5, 7, 9, 11 , 13, 15,
17 and 19.
3. The process of claim 1 wherein the cell is a cancer cell and the difference in expression is a decrease in expression.
4. The process of claim 2 wherein the cell is a cancer cell and the difference in expression is a decrease in expression.
5. A process for identifying an anti-neoplastic agent comprising contacting a cell exhibiting neoplastic activity with a compound first identified as a cancer related gene modulator using a process of one of claims 1 - 4 and detecting a decrease in said neoplastic activity after said contacting compared to when said contacting does not occur.
6. The process of claim 5 wherein said neoplastic activity is accelerated cellular replication.
7. The process of claim 5 wherein said decrease in neoplastic activity results from the death of the cell.
8. A process for identifying an anti-neoplastic agent comprising administering to an animal exhibiting a cancer condition an effective amount of an agent first identified according to a process of one of claims 1-7 and detecting a decrease in said cancerous condition.
9. A process for determining the cancerous status of a cell, comprising determining an increase in the level of expression in said cell of at least one gene that corresponds to a polynucleotide having a sequence selected from the group consisting of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17 and 19 wherein an elevated expression relative to a known non-cancerous cell indicates a cancerous state or potentially cancerous state.
10. An antibody that reacts with a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20.
11. The antibody of claim 10 wherein said antibody is a monoclonal antibody.
12. The antibody of claim 10 wherein said antibody is a recombinant antibody.
13. The antibody of claim 10 wherein said antibody is a synthetic antibody.
14. The antibody of claim 10 wherein said antibody further comprises a cytotoxic agent.
15. The antibody of claim 14 wherein said cytotoxic agent is an apoptotic agent.
16. A process for treating cancer comprising contacting a cancerous cell with an agent having activity against an expression product encoded by a gene sequence selected from the group consisting of SEQ ID NO: 1 , 3, 5, 7, 9, 11, 13, 15, 17 and 19.
17. The process of claim 16 wherein said cancerous cell is contacted in vivo.
18. The process of claim 16 wherein said agent has affinity for said expression product.
19. The process of claim 18 wherein said agent is an antibody of claim
10 - 15.
20. An immunogenic composition comprising a polypeptide comprising an amino acid sequence with at least 90% identity to a sequence selected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 and wherein and amino acid difference results only from conservative amino acid substitutions.
21. The immunogenic composition of claim 20 wherein said percent identity is at least 95%.
22. The immunogenic composition of claim 20 wherein said percent identity is at least 98%.
23. The immunogenic composition of claim 20 wherein said polypeptide has the sequence of a member selected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20.
24. A process for treating cancer in an animal afflicted therewith comprising administering to said animal an amount of an immunogenic composition of claim 20-23 sufficient to elicit the production of cytotoxic T lymphocytes specific for said immunogenic composition.
25. The process of claim 24 wherein said animal is a human being.
26. A process for treating a cancerous condition in an animal afflicted therewith comprising administering to said animal a therapeutically effective amount of an agent first identified as having anti-neoplastic activity using the process of claim 8.
27. A process for protecting an animal against cancer comprising administering to an animal at risk of developing cancer a therapeutically effective amount of an agent first identified as having anti-neoplastic activity using the process of claim 8.
28. A method for producing a product comprising identifying an agent according to the process of claim 1 - 8 wherein said product is the data collected with respect to said agent as a result of said process and wherein said data is sufficient to convey the chemical structure and/or properties of said agent.
29. An isolated polynucleotide comprising a polynucleotide having at least 95% sequence identity to a member selected from the group consisting of SEQ ID NO: 3 or the complement thereof.
30. The isolated polynucleotide of claim 29 wherein said polynucleotide comprises the sequence of SEQ ID NO: 3.
31. An isolated polynucleotide comprising a polynucleotide selected from the group consisting of: (a) a polynucleotide encoding the amino acid sequence of SEQ ID NO:
4, and
(b) the complement of (a).
32. An isolated polynucleotide comprising an amino acid sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 4 and wherein any difference in sequence identity results only from conservative amino acid substitutions.
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