WO2005035724A2 - Cancer-linked genes as targets for chemotherapy - Google Patents

Cancer-linked genes as targets for chemotherapy Download PDF

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WO2005035724A2
WO2005035724A2 PCT/US2004/033072 US2004033072W WO2005035724A2 WO 2005035724 A2 WO2005035724 A2 WO 2005035724A2 US 2004033072 W US2004033072 W US 2004033072W WO 2005035724 A2 WO2005035724 A2 WO 2005035724A2
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gene
cancer
expression
agent
activity
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PCT/US2004/033072
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WO2005035724A3 (en
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Colyn B. Cain
Steven K. Horrigan
Jeffrey W. Strovel
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Avalon Pharmaceuticals, Inc
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Publication of WO2005035724A3 publication Critical patent/WO2005035724A3/en
Priority to US11/599,845 priority Critical patent/US20080025981A1/en

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    • 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
    • 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

Definitions

  • the present invention relates to methods of identifying cancer-target genes and screening such cancer-target 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 the design of small organic compounds and other molecules, and in the diagnosis of cancer.
  • 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.
  • cancer-target genes and encoded polypeptides, have been identified. Such genes are 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.
  • cancer-target genes genes related to, or linked to, cancer, or otherwise involved in the cancer initiating and facilitating process and referred to as cancer-target genes, as well as polypeptides encoded by such genes.
  • such genes are those corresponding to KIAA1274, NEK6, PAK2, PAK4, STK38L, ACP1 , ARHC, CDC6, CDK7, CDKN3, CRK7, DUSP16, FIGNL1, GUK1, ITPR2, KCNK1 , KCNK5, PRO2000, RFC2 and RIPK2 and which encode polypeptides.
  • 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 one of the genes designated KIAA1274, NEK6, PAK2, PAK4, STK38L,
  • the present invention also relates to a method for treating cancer comprising contacting a cancerous cell with an agent having activity against an expression product encoded by one or more of 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 encoded by one of KIAA1274, NEK6, PAK2, PAK4, STK38L, ACP1, ARHC, CDC6, CDK7, CDKN3; CRK7, DUSP16, FIGNL1, GUK1 , ITPR2, KCNK1 , KCNK5, PRO2000, RFC2 and RIPK2.
  • druggable or “druggable domain” refers to a gene that encodes a protein domain known to be modulated by chemical compounds.
  • 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.
  • portion refers 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.
  • 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 genes of KIAA1274, NEK6, PAK2, PAK4, STK38L, ACP1 , ARHC, CDC6, CDK7, CDKN3, CRK7, DUSP16, FIGNL1, GUK1 , ITPR2, KCNK1 , KCNK5, PRO2000, RFC2 and RIPK2.
  • the term “correspond” means that the gene has the same nucleotide sequence as a gene disclosed herein or that it encodes substantially the same RNA as would be encoded by the disclosed gene, the term “substantially” meaning at least 90% identical as defined elsewhere herein and includes splice variants thereof.
  • Percent identity 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.
  • 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.
  • protein domain refers to a discrete portion of a single polypeptide chain with its own function. The combination of domains in a single protein determines its overall function. Protein domains can act as independent units, to the extent that they can be excised from the chain, and still be shown to fold correctly, and often still exhibit biological activity. Another property of domains is that they are regions which are usually conserved during recombination events. This means that along a protein sequence, the domains will tend to be fairly well conserved, and conversely, the interdomain regions will be more divergent
  • the term "conservative amino acid substitution” are defined herein as exchanges within one of the following five groups: I. Small aliphatic, nonpolar residues: Ala, Gly; II. Negatively charged residues: Asp, Glu III. Positively charged residues: His, Arg, Lys IV. Large, aliphatic, nonpolar residues: Met, Leu, He, Val, Cys V. Aromatic residues: Phe, Tyr, Trp, Pro VI. Polar residues Ser, Thr VII. Amides Asn, Gin SEQUENCE LISTING ON CD-ROM ONLY
  • sequences disclosed herein as SEQ ID NO: 1-501 in the sequence listing are contained on compact disc (CD-ROM) only (denoted as Filename: Avalon 213 (1 ,965 kB), 4 copies of which appear on discs denoted Copy 1 , Copy 2, Copy 3 and CRF, and which discs were generated on 6 October 2004), which accompanies this application and the contents of said CD-ROMs are hereby incorporated by reference in their entirety.
  • CD-ROM compact disc
  • the present invention relates to processes for identifying and/or utilizing cancer-target genes, and expression products of such genes, as targets for chemotherapeutic agents, especially anti-cancer agents.
  • Genes 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 are disclosed herein or that are identified by methods disclosed herein. These include genes having structural and/or functional similarity to the genes disclosed herein and include genes that are substantially identical to said genes.
  • genes are at least about 90% identical, preferably 95% identical, most preferably at least about 98% identical and especially where such gene is a gene of KIAA1274, NEK6, PAK2, PAK4, STK38L, ACP1 , ARHC, CDC6, CDK7, CDKN3, CRK7, DUSP16, FIGNL1, GUK1 , ITPR2, KCNK1 , KCNK5, PRO2000, RFC2 and RIPK2.
  • the genes disclosed herein according to the invention were identified within an amplified chromosomal region in a cancer cell line(s) and exhibit RNA over-expression in the cell line(s) and clinical tumor tissues by Affymetrix microarray analysis. Each disclosed gene contains sequences that encode a protein domain previously described as being modulated by chemical compounds.
  • Each such gene was identified in a cancer cell line(s) for which high- resolution comparative genomic hybridization (CGH) data and Affymetrix U133 chip expression data were generated. Each meets the following criteria:
  • RNA expression level of at least 1.5 fold or higher in tumor tissue samples compared to corresponding normal tissue samples in a genetic database with Gene Logic GX2000 database being a non-limiting example.
  • the gene encodes a protein domain known to be modulated by chemical compounds (i.e., a "druggable" domain).
  • the genes identified herein represent a subset of all genes in these classes.
  • the present invention relates to nucleotide sequences and derived polypeptides having the following characteristics:
  • Affymetrix fragment 231887_s_at Affymetrix ID: 262356
  • Druggable domain phosphatase Arbitrary Gene Name: NEK6
  • Gencarta No. T11445 (Gene 12) Cytogenetic location: 9q33.3 Affymetrix fragment: 223158_s_at Affymetrix ID: 253651 Cell lines involved: Colo205 Druggable domain: kinase
  • NIMA-related kinases are mammalian serine/threonine protein kinases structurally related to Aspergillus NIMA (never in mitosis, gene A), which play essential roles in mitotic signaling.
  • Affymetrix fragment 208875_s_at Affymetrix ID: 239505
  • Druggable domain kinase
  • PAK p21 -activated kinases
  • the PAK proteins are a family of serine/threonine kinases that serve as targets for the small GTP binding proteins, CDC42 and RAC1 , and have been implicated in a wide range of biological activities.
  • the p21 activated kinases are critical effectors that link Rho GTPases to cytoskeleton reorganization and nuclear signaling.
  • the PAK proteins are a family of serine/threonine kinases that serve as targets for the small GTP binding proteins, CDC42 and RAC1 , and have been implicated in a wide range of biological activities.
  • Affymetrix fragment 212565_at
  • Affymetrix fragment 201629_s_at Affymetrix ID: 232293
  • Druggable domain phosphatase
  • the product of this gene belongs to the phosphotyrosine protein phosphatase family of proteins. It functions as an acid phosphatase and a protein tyrosine phosphatase by hydrolyzing protein tyrosine phosphate to protein tyrosine and orthophosphate.
  • This gene is genetically polymorphic, and three common alleles segregating at the corresponding locus give rise to six phenotypes. Each allele appears to encode at least two electrophoretically different isozymes, Bf and Bs, which are produced in allele-specific ratios. Three transcript variants encoding distinct isoforms have been identified for this gene (Bryson et al., Genomics 1995 Nov 20;30(2): 133-40).
  • Affymetrix fragment 229484_at Affymetrix ID: 259953
  • Druggable domain phosphatase ARHC (UniGene Hs.179735) sits next to the gene for hypothetical protein MGC19531.
  • the two sequences are in close proximity and they are annotated as the same gene in GenCarta, but they are listed as two distinct genes in UCSC Goldenpath.
  • the Affymetrix fragment maps within the sequence for hypothetical protein MGC19531 , but we are inferring that this fragment is detecting expression for ARHC.
  • ARHC encodes a ras-related GTP binding protein of the rho subfamily, member C (RhoC) that regulates remodeling of the actin cytoskeleton during cell morphogenesis and motility.
  • RhoC ras-related GTP binding protein of the rho subfamily, member C
  • Up regulation of RhoC through increased expression of ARHC has been reported in breast, ovarian and pancreatic cancer as well as melanoma and has been associated with progression to a metastatic phenotype in each cancer type (van Golen et al., Cancer Res. 2000 Oct 15;60(20):5832-8, Horiuchi A et al. Lab Invest. 2003 83(6):861-70, Suwa et al. Br J Cancer. 1998 77(1):147-52, Clark et al., Nature, 2000 406(6795):532-5).
  • CDC6 is expressed selectively in proliferating but not quiescent mammalian cells, both in culture and within tissues in intact animals.
  • transcription of mammalian CDC6 is regulated by E2F proteins as revealed by a functional analysis of the promoter and by the ability of exogenously expressed E2F proteins to stimulate endogenous CDC6.
  • Immunodepletion of CDC6 protein by microinjection of anti-CDC6 antibody blocked initiation of DNA replication in a human tumor cell line. The authors concluded that expression of human CDC6 is regulated in response to mitogenic signals through transcriptional control mechanisms involving E2F proteins, and that CDC6 protein is required for initiation of DNA replication in mammalian cells.
  • Cyclin-dependent kinase 7 (M015 homolog, Xenopus laevis)
  • Druggable protein domain kinase
  • CDK7 The protein encoded by CDK7 is a member of the cyclin-dependent protein kinase (CDK) family, which are known to be important regulators of cell cycle progression.
  • CDK cyclin-dependent protein kinase
  • This protein forms a trimeric complex with cyclin H and MAT1 , which functions as a Cdk-activating kinase (CAK) (Fisher and Morgan, Cell 78:713-724,1994). It is an essential component of the transcription factor IIH (TFIIH) that is involved in transcription initiation and DNA repair (Shiekhattar et al., Nature 374: 283-287, 1995). This protein is thought to serve as a direct link between the regulation of transcription and the cell cycle.
  • TFIIH transcription factor IIH
  • CDKN3 Description: cyclin-dependent kinase inhibitor 3 (CDK2-associated dual specificity phosphatase)
  • the protein encoded by this gene is a human dual specificity protein phosphatase that was identified as a cyclin-dependent kinase inhibitor, and has been shown to interact with and dephosphorylate CDK2 kinase and thus prevent the activation of CDK2 kinase.
  • the gene has been reported to be deleted, mutated, or overexpressed in several kinds of cancers.
  • CDKN3 an overexpressed gene in breast and prostate cancer by using a phosphatase domain-specific differential-display PCR strategy. They report in normal cells, CDKN3 protein is primarily found in the perinuclear region, but in tumor cells, a significant portion of the protein is found in the cytoplasm. Blocking CDKN3 expression by antisense in a tetracycline-regulatable system resulted in a reduced population of S-phase cells and reduced Cdk2 kinase activity.
  • CDKN3 transcripts found aberrant CDKN3 transcripts in hepatocellular tumors and showed mutant proteins were defective in interacting with Cdk2.
  • Affymetrix fragment 225697_at
  • CrkRS CDC2-related kinase 7
  • RS arginine/serine-rich domain
  • CrkRS is a 1490 amino acid protein where the protein kinase domain is 89% identical to CHED protein kinase.
  • CrkRS has extensive proline-rich regions that match the consensus for SH3 and WW domain binding sites and RS domain that is predominantly found in splicing factors. The authors describe CrkRS as a novel, conserved link between the transcription and splicing machinery of a cell.
  • Druggable domain phosphatase
  • MAPK Mitogen-activated protein kinase
  • MKPs Mitogen-activated protein kinase
  • DUSP16 is a dual specificity phosphatase that functions as a MAPK phosphatase, also known as MKP7. Masuda et al.
  • MAPK7 behaves as a nuclear shuttle for c-Jun terminal kinase (JNK) group of MAPKs as well as a phosphatase.
  • JNK c-Jun terminal kinase
  • Druggable domain AAA ATPase
  • Affymetrix fragment 200075_s_at Affymetrix ID: 231232
  • Druggable domain kinase Guanylate kinase catalyzes the phosphorylation of either GMP to GDP or dGMP to dGDP and is an essential enzyme in nucleotide metabolism pathways.
  • GUK2 and GUK3 are isoforms, GUK2 and GUK3, determined by different loci. Brady et al. [J Biol Chem. 1996 12;271 (28): 16734-40] stated that the guanylate kinases are targets for cancer chemotherapy and are inhibited by the drug 6-thioguanine. They report a model of the tertiary structure designed to be used in the development of chemotherapy drugs.
  • Affymetrix fragment 211360_s_at
  • KCNK1 potassium channel, subfamily K, member 1
  • Druggable domain potassium channel
  • This gene encodes one of the members of the superfamily of potassium channel proteins containing two pore-forming P domains and 4 transmembrane segments. Potassium channels are functionally important to a large number of cellular processes including maintenance of the action potential, muscle contraction, hormone secretion, osmotic regulation and ion flow.
  • This gene encodes one of the members of the superfamily of potassium channel proteins containing two pore-forming P domains.
  • MDA_MB231 MDA_MB436, MDA_MB453, SKBR3, T47D, Colo201 , HCT116, SW620, HT29, HCC827, NCI-H23, NCI-H460
  • Druggable domain AAA ATPase
  • AAA ATPases Associated with diverse cellular Activities
  • AAA family proteins often perform chaperone-like functions that assist in the assembly, operation, or disassembly of protein complexes. The exact function of the PRO2000 protein is unknown.
  • RFC proliferating cell nuclear antigen
  • RFC replication factor C
  • Affymetrix fragment 209545_s_at Affymetrix ID: 240173
  • Druggable domain kinase
  • the methods of the invention utilize these genes, designated as
  • genes disclosed herein may be used in any of the methods of the invention and modulation, as used herein, may include modulation of the gene, such as an increase or decrease in transcription or translation, and may include differences in the amount and/or the rate of production of RNA and/or polypeptide. Such modulation may affect any of the transcripts disclosed for the genes of the invention or any of the encoded polypeptides, as identified in Table 6.
  • Antibodies useful in the invention would include those specific for any of the polypeptides encoded by these genes, especially any polypeptide whose sequences are provided in Figure 1 , as identified in Table 6. A brief summary of these genes identified by their respective GenBank Accession Nos. is provided in Table 1 .
  • D26350 241911 inositol 1,4,5-triphosphate receptor, type 2 Hs.406293 253470 neurotrophic tyrosine kinase, receptor, type 1
  • Table 2 describes the location of the cancer target genes of the present invention while Table 3 describes primers used to locate these genes. An additional set of primers is provided in Table 5 while additional gene data is provided in Table 4.
  • nucleotides and polypeptides, as gene products, used in the methods 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.
  • a fragment, derivative or analog of a polypeptide encoded by one of KIAA1274, NEK6, PAK2, PAK4, STK38L, ACP1 , ARHC, CDC6, CDK7, CDKN3, CRK7, DUSP16, FIGNL1, GUK1 , ITPR2, KCNK1 , KCNK5, PRO2000, RFC2 and RIPK2 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
  • genes and gene products useful in practicing the methods of the present invention may likewise be obtained in an isolated or purified form.
  • 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.
  • 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.
  • Expression products of the genes disclosed herein for use in the methods of the invention may be in an isolated form.
  • the present invention relates to a method for identifying a cancer-target gene, comprising: a) identifying a gene that is at least 5 fold over-expressed in a cancer cell line and that maps to a chromosomal region with a CGH ratio of at least 1.25; b) determining an RNA expression level of said gene of at least 1.5 fold in a tumor tissue compared to corresponding normal tissue in a genetic database, c. determining that said gene encodes a protein domain known to be modulated, or shown to be modulated, by chemical compounds wherein a gene that meets the criteria of a, b and c is considered to be a cancer-target gene, thereby identifying a cancer-target gene.
  • the present invention also relates to a set of cancer-target genes identified using such methods. The genes disclosed herein form such a set. In addition, subsets of such sets are specifically contemplated by the invention.
  • the present invention relates to a method for identifying an agent that modulates the activity of a cancer-target gene comprising: (a) contacting a test compound with a cell containing a polynucleotide that corresponds to a gene that has the properties of a, b and c of claim 1 and under conditions promoting the expression of said gene, and (b) determining a difference in expression of said gene relative to when said test compound is not present wherein said difference indicates gene modulating activity, thereby identifying an agent that modulates the activity of a cancer- related gene.
  • said gene was first identified as a cancer target gene using one or more of the methods of the invention.
  • the gene is a gene selected from the group consisting of KIAA1274, NEK6, PAK2, PAK4, STK38L, ACP1, ARHC, CDC6, CDK7, CDKN3, CRK7, DUSP16, FIGNL1, GUK1 , ITPR2, KCNK1 , KCNK5, PRO2000, RFC2 and RIPK2.
  • Table 6 where they are described in terms of a consensus sequence along with identified polynucleotide transcripts and polypeptides.
  • expression may be determined by determining transcription (to form RNA), as by measuring the rate or amount of RNA formed, or translation (to form protein), such as where antibodies may be used to determine the amount of polypeptide or protein formed from the gene in question or where the activity of such protein is determined, such as where the protein is an enzyme and the amount of enzyme activity can be determined.
  • the cell is a cancer cell and the determined difference in expression is a decrease in expression.
  • the cell is a recombinant cell, such as one comprising a gene as disclosed herein, and the difference in expression is a decrease in expression.
  • the present invention also relates to a method for identifying an anti- neoplastic agent comprising contacting a cell exhibiting neoplastic activity with a compound first identified as a cancer target gene modulator using one of the methods of the invention and detecting a decrease in said neoplastic activity after said contacting compared to when said contacting does not occur.
  • the neoplastic activity is accelerated cellular replication.
  • the decrease in neoplastic activity results from the death of the cell.
  • the compound is one that modulates, preferably inhibits, a gene disclosed herein, most preferably a gene identified in Table 6.
  • the present invention also relates to a method for identifying an anti- neoplastic agent comprising administering to an animal exhibiting a cancerous condition an effective amount of a cancer target gene modulating agent by a method of the invention and detecting a decrease in said cancerous condition.
  • the compound is one that modulates, preferably inhibits, a gene disclosed herein, most preferably a gene identified in Table 6.
  • 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 or cancer-target gene sequence such as that of KIAA1274, NEK6, PAK2, PAK4, STK38L, ACP1 , ARHC, CDC6, CDK7, CDKN3, CRK7, DUSP16, FIGNL1, GUK1 , ITPR2, KCNK1 , KCNK5, PRO2000, RFC2 and RIPK2
  • a cancer-linked or cancer-target gene sequence such as that of KIAA1274, NEK6, PAK2, PAK4, STK38L, ACP1 , ARHC, CDC6, CDK7, CDKN3, CRK7, DUSP16, FIGNL1, GUK1 , ITPR2, KCNK1 , KCNK5, PRO2000, RFC2 and RIPK2
  • 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.
  • genes have been identified that are expressed at a level in cancer cells that is different from the expression level in non-cancer cells.
  • the identified genes are expressed at higher levels in cancer cells than in normal cells.
  • genes useful in the methods of the invention can include fully operational 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.
  • a selected set of said genes are expressed in the reference cell, including the gene(s) 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.
  • cancer-related genes or cancer-target 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 of KIAA1274, NEK6, PAK2, PAK4, STK38L, ACP1 , ARHC, CDC6, CDK7, CDKN3, CRK7, DUSP16, FIGNL1, GUK1, ITPR2, KCNK1, KCNK5, PRO2000, RFC2 and RIPK2.
  • the genes disclosed herein may be genomic in nature and thus represent an actual gene as found in nature, 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.
  • mRNA messenger RNA
  • the genes disclosed herein may represent less than the full genomic nucleotide 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 of genes 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 as corresponding to genes disclosed herein 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).
  • a cDNA for example, a sequence as disclosed herein.
  • the sequences of genes 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" the genes of KIAA1274,
  • RNA processed or unprocessed, including naturally occurring splice variants and alleles
  • ACP1, ARHC, CDC6, CDK7, CDKN3, CRK7, DUSP16 FIGNL1, GUK1, ITPR2, KCNK1 , KCNK5, PRO2000, RFC2 and RIPK2 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 encoding the same proteins as any of these genes 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.
  • genes will also encode the same or similar polypeptide sequence as the genes KIAA1274, NEK6, PAK2, PAK4, STK38L, ACP1 , ARHC, CDC6, CDK7, CDKN3, CRK7, DUSP16, FIGNL1, GUK1 , ITPR2, KCNK1 , KCNK5,
  • amino acid sequences may be within the scope of the present invention where they react with the same antibodies that react with polypeptides encoded by genes disclosed herein, preferably KIAA1274, NEK6, PAK2, PAK4, STK38L, ACP1 , ARHC, CDC6, CDK7, CDKN3, CRK7, DUSP16, FIGNL1, GUK1 , ITPR2, KCNK1 , KCNK5, PRO2000, RFC2 and RIPK2.
  • the present invention also relates to methods of assaying potential antitumor agents based on their modulation of the expression of the disclosed genes according to the invention and methods for diagnosing cancerous, or potentially cancerous, conditions as a result of the patterns of expression of the a gene disclosed herein as well as related genes 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 genes 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 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 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.
  • Changes in gene copy number may be determined by determining a change in expression of messenger RNA encoded by a particular gene, 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, especially that of KIAA1274, NEK6, PAK2, PAK4, STK38L, ACP1 , ARHC, CDC6, CDK7, CDKN3, CRK7, DUSP16, FIGNL1, GUK1 , ITPR2, KCNK1 , KCNK5, PRO2000, RFC2 and RIPK2.
  • 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.
  • a gene corresponding to one of KIAA1274, NEK6, PAK2, PAK4, STK38L, ACP1 , ARHC, CDC6, CDK7, CDKN3, CRK7, DUSP16, FIGNL1, GUK1 , ITPR2, KCNK1, KCNK5, PRO2000, RFC2 and RIPK2, 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 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 any of KIAA1274, NEK6, PAK2,
  • the present invention further relates to a method for identifying an agent that modulates the activity of a cancer-target polypeptide comprising: (a) contacting a test compound with a cell expressing a polypeptide encoded by a polynucleotide corresponding to a gene having the properties of a, b and c disclosed above for identifying a cancer-target gene and under conditions promoting the expression of said polypeptide; and (b) determining a difference in expression of said polypeptide relative to when said test compound is not present wherein said difference indicates cancer-target polypeptide modulating activity, thereby identifying a cancer-target polypeptide modulating agent.
  • the present invention further relates to a method for identifying an agent that modulates the activity of a cancer-target polypeptide comprising: (a) contacting a test compound with a polypeptide encoded by a polynucleotide corresponding to a gene having the properties of a, b and c of claim 1 and under conditions promoting the activity of said polypeptide; and (b) determining a difference in activity of said polypeptide relative to when said test compound is not present wherein said difference indicates cancer-target polypeptide modulating activity, thereby identifying a cancer-target polypeptide modulating agent.
  • a preferred embodiment utilizes a gene selected from KIAA1274, NEK6, PAK2, PAK4, STK38L, ACP1 , ARHC, CDC6, CDK7, CDKN3, CRK7, DUSP16, FIGNL1, GUK1 , ITPR2, KCNK1 , KCNK5, PRO2000, RFC2 and RIPK2.
  • 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 of KIAA1274, NEK6, PAK2, PAK4, STK38L, ACP1 , ARHC, CDC6, CDK7, CDKN3, CRK7, DUSP16, FIGNL1, GUK1 , ITPR2, KCNK1 , KCNK5, PRO2000, RFC2 and RIPK2, including genes 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 useful in the methods of the invention 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.
  • mRNA messenger RNA
  • 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 methods 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 relates to a method for treating cancer comprising contacting a cancerous cell with an effective amount of an agent that can reduce the activity of a cancer-target gene (i.e., a gene having the properties of a, b and c disclosed herein for identifying a cancer-target gene).
  • a cancer-target gene i.e., a gene having the properties of a, b and c disclosed herein for identifying a cancer-target gene.
  • said gene is one of KIAA1274, NEK6, PAK2, PAK4, STK38L, ACP1 , ARHC, CDC6, CDK7, CDKN3, CRK7, DUSP16, FIGNL1, GUK1 , /7Pf?2, KCNK1 , KCNK5, PRO2000, RFC2 and RIPK2.
  • Such embodiments include use of any of the agents having activity in one or more of the screening methods disclosed herein, most preferably wherein the agent was first identified as having such activity using one or more of said methods.
  • the cancerous cell is contacted in vivo.
  • the agent has affinity for an expression product of said gene, such as where the agent is an antibody, preferably one disclosed according to the present invention.
  • 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 /n wt ' ro 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.
  • 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, H 1 , H2, H3) for the respective light (L) and heavy (H) chains.
  • the accepted CDR regions have been described by Kabat et al, J. ⁇ /o/. Cftem. 252:6609-661 6 ( 1 977). The numbering scheme is shown in the figures, where the CDRs are underlined and the numbers follow the Kabat scheme.
  • antibody polypeptides contain constant amino acids
  • variable regions 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 encoded by a gene identified by the methods of the invention, preferably one of KIAA1274, NEK6, PAK2, PAK4, STK38L, ACP1 , ARHC, CDC6, CDK7, CDKN3, CRK7, DUSP16, FIGNL1, GUK1 , ITPR2, KCNK1 , KCNK5, PRO2000, RFC2 and RIPK2.
  • Such antibodies may commonly be in the form of a composition, especially a pharmaceutical composition.
  • the present invention contemplates an antibody that binds to a polypeptide encoded by a cancer-target gene (i.e., a gene having the properties of a, b and c disclosed above for identifying a cancer-target gene).
  • a cancer-target gene i.e., a gene having the properties of a, b and c disclosed above for identifying a cancer-target gene.
  • the polypeptide or protein is encoded by one or more of genes KIAA1274, NEK6, PAK2, PAK4, STK38L, ACP1, ARHC, CDC6, CDK7, CDKN3, CRK7, DUSP16, FIGNL1, GUK1 , ITPR2, KCNK1 , KCNK5, PRO2000, RFC2 and RIPK2.
  • this may include a naturally occurring antibody, such as polyclonal antibodies, or more preferably a monoclonal antibody, or a recombinant antibody or a partly or wholly synthetic antibody.
  • the antibody further comprises a cytotoxic agent, such as an apoptotic agent.
  • 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.
  • 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 contemplated by the invention preferably those encoded by one or more of KIAA1274, NEK6, PAK2, PAK4, STK38L, ACP1 , ARHC, CDC6, CDK7, CDKN3, CRK7, DUSP16, FIGNL1, GUK1 , ITPR2, KCNK1 , KCNK5, PRO2000, RFC2 and RIPK2, 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.
  • CTLs cytotoxic T lymphocytes
  • 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 encoded by one or more of KIAA1274, NEK6, PAK2, PAK4, STK38L, ACP1 , ARHC, CDC6, CDK7, CDKN3, CRK7, DUSP16, FIGNL1, GUK1 , ITPR2, KCNK1 , KCNK5, PRO2000, RFC2 and RIPK2, 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.
  • the present invention also relates to a process that comprises a method for producing a product, such as by generating test data to facilitate identification of such 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.
  • a method for producing a product such as by generating test data to facilitate identification of such 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 one of KIAA1274, NEK6, PAK2, PAK4, STK38L, ACP1 , ARHC, CDC6, CDK7, CDKN3, CRK7, DUSP16, FIGNL1, GUK1 ,
  • KCNK1 KCNK5
  • PRO2000 PRO2000
  • RFC2 RFC2
  • RIPK2 RIPK2
  • genes that may be over-expressed in malignant cancer may be over-expressed in malignant cancer.
  • 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.
  • genes 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.
  • genes 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.
  • a gene corresponding to a polynucleotide disclosed herein when in normal tissues, may indicate a predisposition towards development of colon cancer.
  • the encoded polypeptide might then present a potentially useful cell surface target for therapeutic molecules such as cytolytic antibodies, or antibodies attached to cytotoxic, or cytolytic, agents.
  • the present invention specifically contemplates use of antibodies against polypeptides encoded by the genes disclosed herein, whereby said antibodies are conjugated to one or more cytotoxic agents so that the antibodies serve to target the conjugated immunotoxins to a region of cancerous activity, such as a solid tumor.
  • cytotoxic agents lack of selectivity has presented a drawback to their use as therapeutic agents in the treatment of malignancies.
  • the class of two-chain toxins consisting of a binding subunit (or B-chain) linked to a toxic subunit (A-chain) are extremely cytotoxic.
  • agents as ricin, a protein isolated from castor beans, kills cells at very low concentrations (even less than 10 "11 M) by inactivating ribosomes in said cells (see, for example, Lord et al., Ricin: structure, mode of action, and some current applications. Faseb J, 8; 201-208 (1994), and Blattler et al., Realizing the full potential of immunotoxins. Cancer Cells, 1. 50-55 (1989)).
  • A-chains of protein toxins that functionally resemble ricin A-chain are only weakly cytotoxic for intact cells (in the concentration range of 10 "7 to 10 "6 M), they are very potent cytotoxic agents inside the cells.
  • a single molecule of the A-subunit of diphtheria toxin can kill a cell once inside (see: Yamaizumi et al., One molecule of diphtheria toxin fragment A introduced into a cell can kill the cell. Cell, 15: 245-250, 1978).
  • the present invention solves this selectivity problem by using antibodies specific for antigens present on cancer cells to target the cytotoxins to said cells.
  • use of antibodies decreases toxicity because the antibodies are non-toxic until they reach the tumor and, because the cytotoxin is bound to the antibody, it is presented with less opportunity to cause damage to non-targeted tissues.
  • ADCC antibody-dependent cellular cytotoxic response
  • a number of recombinant immunotoxins for example, consisting of Fv regions of cancer specific antibodies fused to truncated bacterial toxins are well known (see, for example, Smyth et al., Specific targeting of chlorambucil to tumors with the use of monoclonal antibodies, J. Natl. Cancer Inst, 76(3):503-510 (1986); Cho et al., Single-chain Fv/folate conjugates mediate efficient lysis of folate-receptor-positive tumor cells, Bioconjug. Chem., 8(3):338-346 (1997)).
  • these may contain, for example, a truncated version of Pseudomonas exotoxin as a toxic moiety but the toxin is modified in such a manner that by itself it does not bind to normal human cells, but it retains all other functions of cytotoxicity.
  • recombinant antibody fragments target the modified toxin to cancer cells which are killed, such as by direct inhibition of protein synthesis, or by concomitant induction of apoptosis. Cells that are not recognized by the antibody fragment, because they do not carry the cancer antigen, are not affected. Good activity and specificity has been observed for many recombinant immunotoxins in in vitro assays using cultured cancer cells as well as in animal tumor models.
  • cytotoxic agents specifically contemplated for use as immunoconjugates according to the present invention are Calicheamicin, a highly toxic enediyne antibiotic isolated from M/cromonospora ecfi/nospora ssp. Ca//c/?e/7s/s, and which binds to the minor groove of DNA to induce double strand breaks and cell death (see: Lee et al., Calicheamicins, a novel family of antitumor antibiotics. 1. Chemistry and partial structure of calichemicin gi..
  • calicheamicin gamma 11 an antitumor antibiotic that cleaves double-stranded DNA site specifically, Science, 240: 1198-1201 (1988)).
  • useful derivatives of the calicheamicins include mylotarg and 138H11-Cam ⁇ .
  • Mylotarg is an immunoconjugate of a humanized anti-CD33 antibody (CD33 being found in leukemic cells of most patients with acute myeloid leukemia) and N-acetyl gamma colicheamicin dimethyl hydrazide, the latter of which is readily coupled to an antibody of the present invention (in place of the anti- CD33 but which can also be humanized by substitution of human framework regions into the antibody during production as described elsewhere herein) to form an immunoconjugate of the invention, (see: Hamann et al. Gemtuzumab Ozogamicin, A Potent and Selective Anti-CD33 Antibody-Calicheamicin Conjugate for Treatment of Acute Myeloid Leukemia, Bioconjug. Chem.
  • 138H11 is an anti- ⁇ -glutamyl transferase antibody coupled to theta calicheamicin through a disulfide linkage and found useful in vitro against cultured renal cell carcinoma cells, (see: Knoll et al., Targeted therapy of experimental renal cell carcinoma with a novel conjugate of monoclonal antibody 138H11 and calicheamicin ⁇ Cancer Res, 60: 6089-6094 (2000) The same linkage may be utilized to link this cytotoxic agent to an antibody of the present invention, thereby forming a targeting structure for colon cancer cells.
  • DC1 a disulfide-containing analog of adozelesin, that kills cells by binding to the minor groove of DNA, followed by alkylation of adenine bases.
  • Adozelesin is a structural analog of CC-1065, an anti-tumor antibiotic isolated from microbial fermentation of Streptomyces zelensis, and is about 1 ,000 fold more toxic to cultured cell lines that other DNA interacting agents, such as cis-platin and doxorubicin. This agent is readily linked to antibodies through the disulfide bond of adozelesin.
  • Maytansine a highly cytotoxic microtubular inhibitor isolated from the shrub Maytenus serrata found to have little value in human clinical trials, is much more effective in its derivatized form, denoted DM1 , containing a disulfide bond to facilitate linkage to antibodies, is up to 10-fold more cytotoxic (see: Chari et al., Immunoconjugates containing novel maytansinoids: promising anticancer drugs, Cancer Res, 52: 127-131 (1992)).
  • a preferred embodiment of the present invention includes where the cytotoxic agent is a calicheamicin, a maytansinoid, an adozelesin, DC1 , a cytotoxic protein, a taxol, a taxotere, or a taxoid.
  • the cytotoxic agent is a calicheamicin, a maytansinoid, an adozelesin, DC1 , a cytotoxic protein, a taxol, a taxotere, or a taxoid.
  • the calicheamicin is calicheamicin ⁇ i 1 , N-acetyl gamma calicheamicin dimethyl hydrazide or calicheamicin ⁇ i 1
  • the maytansinoid is DM1
  • the cytotoxic protein is ricin, abrin, gelonin, pseudomonas exotoxin or diphtheria toxin
  • the taxol is paclitaxel
  • the taxotere is docetaxel.
  • cytotoxic proteins include a number of different classes, such as those that inhibit protein synthesis: ribosome-inactivating proteins of plant origin, such as ricin, abrin, gelonin, and a number of others, and bacterial toxins such as pseudomonas exotoxin and diphtheria toxin.
  • ribosome-inactivating proteins of plant origin such as ricin, abrin, gelonin, and a number of others
  • bacterial toxins such as pseudomonas exotoxin and diphtheria toxin.
  • Another useful class is the one including taxol, taxotere, and taxoids.
  • paclitaxel taxol
  • its analog docetaxel taxotere
  • derivatives thereof paclitaxel
  • the first two are clinical drugs used in treating a number of tumors while the taxoids act to induce cell death by inhibiting the de-polymerization of tubulin.
  • Such agents are readily linked to antibodies through disulfide bonds without disadvantageous effects on binding specificity.
  • a truncated Pseudomonas exotoxin was fused to an anti-CD22 variable fragment and used successfully to treat patients with chemotherapy-resistant hairy-cell leukemia, (see: Kreitman et al., Efficacy of the anti-CD22 recombinant immunotoxin BL22 in chemotherapy-resistant hairy-cell leukemia, N Engl J Med, 345: 241-247 (2001))
  • the cancer-linked peptides of the present invention offer the opportunity to prepare antibodies, recombinant or otherwise, against the appropriate antigens to target solid tumors, preferably those of malignancies of colon tissue, using the same or similar cytotoxic conjugates.
  • the immunoconjugates formed using the antibodies prepared against the cancer-linked antigens disclosed herein can be formed by any type of chemical coupling.
  • the cytotoxic agent of choice, along with the immunoglobulin can be coupled by any type of chemical linkage, covalent or non-covalent, including electrostatic linkage, to form the immunoconjugates of the present invention.
  • the antitumor agents of the present invention represent a class of pro-drugs that are relatively non-toxic when first administered to an animal (due mostly to the stability of the immunoconjugate), such as a human patient, but which are targeted by the conjugated immunoglobulin to a cancer cell where they then exhibit good toxicity.
  • the tumor-related, associated, or linked, antigens preferably those presented herein, serve as targets for the antibodies (monoclonal, recombinant, and the like) specific for said antigens.
  • the end result is the release of active cytotoxic agent inside the cell after binding of the immunoglobulin portion of the immunoconjugate.
  • the present invention provides highly useful cancer-associated antigens for generation of antibodies for linkage to a number of different cytotoxic agents which are already known to have some in vitro toxicity and possess chemical groups available for linkage to antibodies.
  • 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.
  • SW480 cells are grown to a density of 10 5 cells/cm 2 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.
  • Table 6 contains a listing of the genes (numbered 1 to 20) along with their Gencarta names (or accessions). Each gene is represented as a consensus sequence followed by predicted mRNA transcripts and then predicted polypeptides. All of the sequences, with additional information, are presented in Figure 1.
  • the present invention also relates to an isolated cancer target gene wherein said gene is a gene identified in Table 6.
  • the present invention encompasses isolated genes identified herein as KIAA1274, NEK6, PAK2, PAK4, STK38L, ACP1 , ARHC, CDC6, CDK7, CDKN3, CRK7, DUSP16, FIGNL1, GUK1 , ITPR2, KCNK1 , KCNK5, PRO2000, RFC2 and RIPK2 and uses of these genes, whether isolated or not, in any of the methods of the invention.

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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 priority of U.S. Provisional Application 60/509,515, filed 8 October 2003, the disclosure of which is hereby incorporated by reference in its entirety.
FIELD OF THE INVENTION
The present invention relates to methods of identifying cancer-target genes and screening such cancer-target 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 the design of small organic compounds and other molecules, and in the diagnosis of cancer.
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, cancer-target genes, and encoded polypeptides, have been identified. Such genes are 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 referred to as cancer-target genes, as well as polypeptides encoded by such genes.
In a particular embodiment, such genes are those corresponding to KIAA1274, NEK6, PAK2, PAK4, STK38L, ACP1 , ARHC, CDC6, CDK7, CDKN3, CRK7, DUSP16, FIGNL1, GUK1, ITPR2, KCNK1 , KCNK5, PRO2000, RFC2 and RIPK2 and which encode polypeptides.
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 one of the genes designated KIAA1274, NEK6, PAK2, PAK4, STK38L,
ACP1 , ARHC, CDC6, CDK7, CDKN3, CRK7, DUSP16, FIGNL1, GUK1, ITPR2, KCNK1 , KCNK5, PRO2000, RFC2 and RIPK2 of genes substantially identical to said genes and/or encode the same or similar polypeptide 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, or target 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 method for treating cancer comprising contacting a cancerous cell with an agent having activity against an expression product encoded by one or more of 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 encoded by one of KIAA1274, NEK6, PAK2, PAK4, STK38L, ACP1, ARHC, CDC6, CDK7, CDKN3; CRK7, DUSP16, FIGNL1, GUK1 , ITPR2, KCNK1 , KCNK5, PRO2000, RFC2 and RIPK2. DEFINITIONS
As used herein and except as noted otherwise, all terms are defined as given below.
The term "druggable" or "druggable domain" refers to a gene that encodes a protein domain known to be modulated by chemical compounds.
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. 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.
As used herein, the term "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 genes of KIAA1274, NEK6, PAK2, PAK4, STK38L, ACP1 , ARHC, CDC6, CDK7, CDKN3, CRK7, DUSP16, FIGNL1, GUK1 , ITPR2, KCNK1 , KCNK5, PRO2000, RFC2 and RIPK2.
As used herein, the term "correspond" means that the gene has the same nucleotide sequence as a gene disclosed herein or that it encodes substantially the same RNA as would be encoded by the disclosed gene, the term "substantially" meaning at least 90% identical as defined elsewhere herein and includes splice variants thereof.
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.
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 "protein domain" refers to a discrete portion of a single polypeptide chain with its own function. The combination of domains in a single protein determines its overall function. Protein domains can act as independent units, to the extent that they can be excised from the chain, and still be shown to fold correctly, and often still exhibit biological activity. Another property of domains is that they are regions which are usually conserved during recombination events. This means that along a protein sequence, the domains will tend to be fairly well conserved, and conversely, the interdomain regions will be more divergent
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 residues: Ala, Gly; II. Negatively charged residues: Asp, Glu III. Positively charged residues: His, Arg, Lys IV. Large, aliphatic, nonpolar residues: Met, Leu, He, Val, Cys V. Aromatic residues: Phe, Tyr, Trp, Pro VI. Polar residues Ser, Thr VII. Amides Asn, Gin SEQUENCE LISTING ON CD-ROM ONLY
The sequences disclosed herein as SEQ ID NO: 1-501 in the sequence listing are contained on compact disc (CD-ROM) only (denoted as Filename: Avalon 213 (1 ,965 kB), 4 copies of which appear on discs denoted Copy 1 , Copy 2, Copy 3 and CRF, and which discs were generated on 6 October 2004), which accompanies this application and the contents of said CD-ROMs are hereby incorporated by reference in their entirety. These sequence numbers also appear in Table 6 where all sequences are referred to as consecutive Sequence ID Nos. for reference.
DETAILED SUMMARY OF THE INVENTION
The present invention relates to processes for identifying and/or utilizing cancer-target genes, and expression products of such genes, as targets for chemotherapeutic agents, especially anti-cancer agents. Genes 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 are disclosed herein or that are identified by methods disclosed herein. These include genes having structural and/or functional similarity to the genes disclosed herein and include genes that are substantially identical to said genes. In terms of nucleotide sequence, such genes are at least about 90% identical, preferably 95% identical, most preferably at least about 98% identical and especially where such gene is a gene of KIAA1274, NEK6, PAK2, PAK4, STK38L, ACP1 , ARHC, CDC6, CDK7, CDKN3, CRK7, DUSP16, FIGNL1, GUK1 , ITPR2, KCNK1 , KCNK5, PRO2000, RFC2 and RIPK2. The genes disclosed herein according to the invention were identified within an amplified chromosomal region in a cancer cell line(s) and exhibit RNA over-expression in the cell line(s) and clinical tumor tissues by Affymetrix microarray analysis. Each disclosed gene contains sequences that encode a protein domain previously described as being modulated by chemical compounds.
Each such gene was identified in a cancer cell line(s) for which high- resolution comparative genomic hybridization (CGH) data and Affymetrix U133 chip expression data were generated. Each meets the following criteria:
1) At least 5 fold over-expressed in a cancer cell line(s) and mapped to a chromosomal region with a CGH ratio of 1.25 or above.
2) RNA expression level of at least 1.5 fold or higher in tumor tissue samples compared to corresponding normal tissue samples in a genetic database (with Gene Logic GX2000 database being a non-limiting example).
3) The gene encodes a protein domain known to be modulated by chemical compounds (i.e., a "druggable" domain). The genes identified herein represent a subset of all genes in these classes.
In accordance with the foregoing, the present invention relates to nucleotide sequences and derived polypeptides having the following characteristics:
Arbitrary Gene Name: KIAA1274
Description: KIAA protein (similar to mouse paladin) UniGene: Hs.300646
Accession: AB033100
Gencarta No. AA553584 (Gene 2)
Cytogenetic location: 10q22.1
Affymetrix fragment: 231887_s_at Affymetrix ID: 262356
Cell lines involved: SW620
Druggable domain: phosphatase Arbitrary Gene Name: NEK6
Description: NIMA (never in mitosis gene a)-related kinase 6 UniGene: Hs.9625 Accession: BE616825
Gencarta No. T11445 (Gene 12) Cytogenetic location: 9q33.3 Affymetrix fragment: 223158_s_at Affymetrix ID: 253651 Cell lines involved: Colo205 Druggable domain: kinase
NIMA-related kinases (NEKs) are mammalian serine/threonine protein kinases structurally related to Aspergillus NIMA (never in mitosis, gene A), which play essential roles in mitotic signaling.
Arbitrary Gene Name: PAK2
Description: p21 -activated kinase 2 UniGene: Hs.56974
Accession: BF796470
Gencarta No. Z26993 (Gene 17)
Cytogenetic location: 3q29
Affymetrix fragment: 208875_s_at Affymetrix ID: 239505
Cell lines involved: HCC1954, HCC202, HCC70, MDA_MB453, T47D
Druggable domain: kinase
The p21 -activated kinases (PAK) are critical effectors that link Rho GTPases to cytoskeleton reorganization and nuclear signaling. The PAK proteins are a family of serine/threonine kinases that serve as targets for the small GTP binding proteins, CDC42 and RAC1 , and have been implicated in a wide range of biological activities.
Arbitrary Gene Name: PAK4
Description: p21 -activated kinase 4
UniGene: Hs.20447
Accession: AF005046 Gencarta No. R09837 (Gene 8)
Cytogenetic location: 19q13.2
Affymetrix fragment: 33814_at
Affymetrix ID: 107335
Cell lines involved: HCC202, MDA MB468 Druggable domain: kinase
The p21 activated kinases (PAK) are critical effectors that link Rho GTPases to cytoskeleton reorganization and nuclear signaling. The PAK proteins are a family of serine/threonine kinases that serve as targets for the small GTP binding proteins, CDC42 and RAC1 , and have been implicated in a wide range of biological activities.
Arbitrary Gene Name: STK38L
Description: serine/threonine kinase 38 like
UniGene: Hs.184523
Accession: AW779556 Gencarta No. R14324 (Gene 9)
Cytogenetic location: 12p11.23
Affymetrix fragment: 212565_at
Affymetrix ID: 243092
Cell lines involved: HCC827, BEN Druggable domain: kinase
Arbitrary Gene Name: ACP1
Description: acid phosphatase 1, soluble UniGene: Hs.75393
Accession: BE872974
Gencarta No. HUMAAPA (Gene 6)
Cytogenetic location: 2p25.3
Affymetrix fragment: 201629_s_at Affymetrix ID: 232293
Cell lines involved: BEN, NCI-H460, NCI-H522, MCF7, MDA-MB436, MDA-
MB468
Druggable domain: phosphatase
The product of this gene belongs to the phosphotyrosine protein phosphatase family of proteins. It functions as an acid phosphatase and a protein tyrosine phosphatase by hydrolyzing protein tyrosine phosphate to protein tyrosine and orthophosphate. This gene is genetically polymorphic, and three common alleles segregating at the corresponding locus give rise to six phenotypes. Each allele appears to encode at least two electrophoretically different isozymes, Bf and Bs, which are produced in allele-specific ratios. Three transcript variants encoding distinct isoforms have been identified for this gene (Bryson et al., Genomics 1995 Nov 20;30(2): 133-40).
Arbitrary Gene Name: ARHC
Description: ras homolog gene family, member C (hypothetical protein
MGC19531) UniGene: Hs.446391
Accession: AW117553
Gencarta No. AA383349 (Gene 1)
Cytogenetic location: 1p13.2
Affymetrix fragment: 229484_at Affymetrix ID: 259953
Cell lines involved: HCC202, MDA_MB468
Druggable domain: phosphatase ARHC (UniGene Hs.179735) sits next to the gene for hypothetical protein MGC19531. The two sequences are in close proximity and they are annotated as the same gene in GenCarta, but they are listed as two distinct genes in UCSC Goldenpath. The Affymetrix fragment maps within the sequence for hypothetical protein MGC19531 , but we are inferring that this fragment is detecting expression for ARHC.
ARHC encodes a ras-related GTP binding protein of the rho subfamily, member C (RhoC) that regulates remodeling of the actin cytoskeleton during cell morphogenesis and motility. Up regulation of RhoC through increased expression of ARHC has been reported in breast, ovarian and pancreatic cancer as well as melanoma and has been associated with progression to a metastatic phenotype in each cancer type (van Golen et al., Cancer Res. 2000 Oct 15;60(20):5832-8, Horiuchi A et al. Lab Invest. 2003 83(6):861-70, Suwa et al. Br J Cancer. 1998 77(1):147-52, Clark et al., Nature, 2000 406(6795):532-5).
Arbitrary Gene Name: CDC6 Description: CDC6 cell division cycle 6 homolog (S. cerivisiae) UniGene: Hs.69563 Accession: U77949 Gencarta No. T83032 (Gene 16) Cytogenetic location: 17q21.3 Affymetrix fragment: 203967_at Affymetrix ID: 234629 Cell lines involved: NCI-H522, NCI-H23 Druggable domain: AAA ATPase
Yan et al. [Proc Nat Acad Sci 94:142-147 (1998)] showed that CDC6 is expressed selectively in proliferating but not quiescent mammalian cells, both in culture and within tissues in intact animals. During the transition from a growth-arrested to a proliferative state, transcription of mammalian CDC6 is regulated by E2F proteins as revealed by a functional analysis of the promoter and by the ability of exogenously expressed E2F proteins to stimulate endogenous CDC6. Immunodepletion of CDC6 protein by microinjection of anti-CDC6 antibody blocked initiation of DNA replication in a human tumor cell line. The authors concluded that expression of human CDC6 is regulated in response to mitogenic signals through transcriptional control mechanisms involving E2F proteins, and that CDC6 protein is required for initiation of DNA replication in mammalian cells.
Arbitrary Gene Name: CDK7
Description: Cyclin-dependent kinase 7 (M015 homolog, Xenopus laevis)
UniGene: Hs.184298 Accession: X77743
Gencarta No. F02366 (Gene 4)
Cytogenetic location: 5q13.2
Affymetrix fragment: 211297_s_at
Affymetrix ID: 241855 Cell lines involved: SW620
Druggable protein domain: kinase
The protein encoded by CDK7 is a member of the cyclin-dependent protein kinase (CDK) family, which are known to be important regulators of cell cycle progression. This protein forms a trimeric complex with cyclin H and MAT1 , which functions as a Cdk-activating kinase (CAK) (Fisher and Morgan, Cell 78:713-724,1994). It is an essential component of the transcription factor IIH (TFIIH) that is involved in transcription initiation and DNA repair (Shiekhattar et al., Nature 374: 283-287, 1995). This protein is thought to serve as a direct link between the regulation of transcription and the cell cycle.
Arbitrary Gene Name: CDKN3 Description: cyclin-dependent kinase inhibitor 3 (CDK2-associated dual specificity phosphatase)
UniGene: Hs.84113
Accession: AF213033
Gencarta No. HUMPTPB (Gene 7) Cytogenetic location: 14q22.2
Affymetrix fragment: 209714_s_at
Affymetrix ID: 240337
Cell lines involved: NCI-H460, HCC827, NCI-H23, HCC202, MCF7, MDA-
MB453, T47D Druggable domain: phosphatase
The protein encoded by this gene is a human dual specificity protein phosphatase that was identified as a cyclin-dependent kinase inhibitor, and has been shown to interact with and dephosphorylate CDK2 kinase and thus prevent the activation of CDK2 kinase. The gene has been reported to be deleted, mutated, or overexpressed in several kinds of cancers.
Lee et al. [Mol Cell Biol. 2000 Mar;20(5): 1723-32} identified the CDKN3 as an overexpressed gene in breast and prostate cancer by using a phosphatase domain-specific differential-display PCR strategy. They report in normal cells, CDKN3 protein is primarily found in the perinuclear region, but in tumor cells, a significant portion of the protein is found in the cytoplasm. Blocking CDKN3 expression by antisense in a tetracycline-regulatable system resulted in a reduced population of S-phase cells and reduced Cdk2 kinase activity. Furthermore, lowering CDKN3 expression led to inhibition of the transformed phenotype, with reduced anchorage-independent growth and tumorigenic potential in athymic nude mice. They suggest that therapeutic intervention might be aimed at repression of CDKN3 gene overexpression in human breast and prostate cancer.
Yeh et al. [Cancer Res. 2000 Sep 1 ;60(17):4697-4700] analyzed CDKN3 mRNA in hepatocellular carcinoma by reverse transcription-PCR (RT-
PCR), followed by cloning and sequencing. They found aberrant CDKN3 transcripts in hepatocellular tumors and showed mutant proteins were defective in interacting with Cdk2.
Arbitrary Gene Name: CRK7
Description: CDC2-related protein kinase 7
UniGene: Hs.278346
Accession: AI651265 Gencarta No. T60764 (Gene 14)
Cytogenetic location: 17q12
Affymetrix fragment: 225697_at
Affymetrix ID: 256169
Cell lines involved: HCC1954, HCC202, SKBR3 Druggable domain: kinase
Ko et al. [Journal of Cell Science, 114,2591-2603 (2001)] isolated and characterized CrkRS, CDC2-related kinase 7, as a novel human protein with an arginine/serine-rich (RS) domain that is most closely related to the cyclin- dependent kinase family. They report CrkRS is a 1490 amino acid protein where the protein kinase domain is 89% identical to CHED protein kinase. CrkRS has extensive proline-rich regions that match the consensus for SH3 and WW domain binding sites and RS domain that is predominantly found in splicing factors. The authors describe CrkRS as a novel, conserved link between the transcription and splicing machinery of a cell.
Arbitrary Gene Name: DUSP16 Description: dual specificity phosphatase 16
UniGene: Hs.20281
Accession: AB051487
Gencarta No. T23935 (Gene 13)
Cytogenetic location: 12p13.2 Affymetrix fragment: 224832_at Affymetrix ID: 255305
Cell lines involved: HCC827
Druggable domain: phosphatase
Mitogen-activated protein kinase (MAPK) phosphatases (MKPs) negatively regulate MAPK activity. DUSP16 is a dual specificity phosphatase that functions as a MAPK phosphatase, also known as MKP7. Masuda et al.
[J Biol Chem. 2001 276(42):39002-11] showed that MAPK7 behaves as a nuclear shuttle for c-Jun terminal kinase (JNK) group of MAPKs as well as a phosphatase.
Arbitrary Gene Name: FIGNL1 Description: fidgetin-like 1
UniGene: Hs.137516
Accession: AA805691
Gencarta No. H61320 (Gene 5)
Cytogenetic location: 7p12.2 Affymetrix fragment: 222843_at
Affymetrix ID: 253337
Cell lines involved: SW620, BEN, HCC827
Druggable domain: AAA ATPase
Arbitrary Gene Name: GUK1
Description: guanylate kinase 1 UniGene: Hs.3764
Accession: BC006249
Gencarta No. T08090 (Gene 11)
Cytogenetic location: 1q42.13
Affymetrix fragment: 200075_s_at Affymetrix ID: 231232
Cell lines involved: HCC202, MDA_MB436, MDA_MB453, MDA_MB468
Druggable domain: kinase Guanylate kinase catalyzes the phosphorylation of either GMP to GDP or dGMP to dGDP and is an essential enzyme in nucleotide metabolism pathways. There are several isoforms, GUK2 and GUK3, determined by different loci. Brady et al. [J Biol Chem. 1996 12;271 (28): 16734-40] stated that the guanylate kinases are targets for cancer chemotherapy and are inhibited by the drug 6-thioguanine. They report a model of the tertiary structure designed to be used in the development of chemotherapy drugs.
Arbitrary Gene Name: ITPR2
Description: inositol 1,4,5-triphosphate receptor, type 2
UniGene
Accession: D26350 Gencarta No. Z38709 (Gene 18)
Cytogenetic location: 12p11.23
Affymetrix fragment: 211360_s_at
Affymetrix ID: 241911
Cell lines involved: BEN, HCC827 Druggable domain: Ion transport
Arbitrary Gene Name: KCNK1 Description: potassium channel, subfamily K, member 1
UniGene: Hs.79351
Accession: U90065
Gencarta No. Z39663 (Gene 19)
Cytogenetic location: 1q42.2 Affymetrix fragment: 204678_s_at
Affymetrix ID: 235340
Cell lines involved: HCC202, HCC70, MDA_MB436, MDA_MB453,
MDA_MB468
Druggable domain: potassium channel
This gene encodes one of the members of the superfamily of potassium channel proteins containing two pore-forming P domains and 4 transmembrane segments. Potassium channels are functionally important to a large number of cellular processes including maintenance of the action potential, muscle contraction, hormone secretion, osmotic regulation and ion flow.
Arbitrary Gene Name: KCNK5
Description: potassium channel, subfamily K, member 5
UniGene: Hs.127007 Accession: AI678413 Gencarta No. R25184 (Gene 10) Cytogenetic location: 6p21.2 Affymetrix fragment: 69854_at Affymetrix ID: 153971
Cell lines involved: Colo201, Colo205 Druggable domain: K+ channel
This gene encodes one of the members of the superfamily of potassium channel proteins containing two pore-forming P domains.
Arbitrary Gene Name: PRO2000
Description: Hypothetical protein MGC5254
UniGene: Hs.222088
Accession: AI925583
Gencarta No. Z44462 (Gene 20) Cytogenetic location: 8q24.13
Affymetrix fragment: 222740_at
Affymetrix ID: 253234
Cell lines involved: BT549, HCC1954, HCC202, HCC70, Hs578t, MCF7,
MDA_MB231 , MDA_MB436, MDA_MB453, SKBR3, T47D, Colo201 , HCT116, SW620, HT29, HCC827, NCI-H23, NCI-H460
Druggable domain: AAA ATPase
A large family of ATPases has been described, whose key feature is that they share a conserved region of about 220 amino acids that contains an ATP-binding site. The protein encoded by PR02000 contains two AAA (ATPases Associated with diverse cellular Activities) domains as well as a bromodomain. AAA family proteins often perform chaperone-like functions that assist in the assembly, operation, or disassembly of protein complexes. The exact function of the PRO2000 protein is unknown.
Fellenberg et al. [Int J Cancer 105(5);636-643 (2003)] report PRO2000 is up-regulated >2 fold in osteosarcoma cell line (Saos-2) following treatment with cisplatin, methotrexate and doxorubicin. Arbitrary Gene Name: RFC2 Description: replication factor C (activator 1) 2, 40kDa UniGene: Hs.139226 Accession: M87338 Gencarta No. T62520 (Gene 15) Cytogenetic location: 7q11.23 Affymetrix fragment: 1053_at Affymetrix ID: 113880
Cell lines involved: HCC827, NCI_H23, NCI_H522 Druggable domain: AAA ATPase
The elongation of primed DNA templates by DNA polymerase delta and epsilon requires the action of the accessory proteins proliferating cell nuclear antigen (PCNA) and replication factor C (RFC). RFC, also called activator 1 , is a protein complex consisting of five distinct subunits of 145, 40, 38, 37, and 36.5 kD. This gene encodes the 40 kD subunit, which has been shown to be responsible for binding ATP. Alternatively spliced transcript variants encoding distinct isoforms have been described.
Arbitrary Gene Name: RIPK2
Description: receptor-interacting serine-threonine kinase 2 UniGene: Hs.103755
Accession: AF064824
Gencarta No. D61791 (Gene 3)
Cytogenetic location: 8q21.3
Affymetrix fragment: 209545_s_at Affymetrix ID: 240173
Cell lines involved: HCT116
Druggable domain: kinase The methods of the invention utilize these genes, designated as
KIAA1274, NEK6, PAK2, PAK4, STK38L, ACP1 , ARHC, CDC6, CDK7, CDKN3, CRK7, DUSP16, FIGNL1, GUK1 , ITPR2, KCNK1 , KCNK5, PRO2000, RFC2 and RIPK2. The genes disclosed herein may be used in any of the methods of the invention and modulation, as used herein, may include modulation of the gene, such as an increase or decrease in transcription or translation, and may include differences in the amount and/or the rate of production of RNA and/or polypeptide. Such modulation may affect any of the transcripts disclosed for the genes of the invention or any of the encoded polypeptides, as identified in Table 6. Antibodies useful in the invention would include those specific for any of the polypeptides encoded by these genes, especially any polypeptide whose sequences are provided in Figure 1 , as identified in Table 6. A brief summary of these genes identified by their respective GenBank Accession Nos. is provided in Table 1 .
Table 1. Brief summary of cancer target genes.
Accession unigene affy Description
AI651265 Hs.278346 256169 CDC2-related protein kinase 7
U77949 Hs.69563 234629 CDC6 cell division cycle 6 homolog (S. cerevisiae)
X77743 Hs.184298 241855 cyclin-dependent kinase 7 (M015 homolog, Xenopus laevis, cdk-activating kinase)
AA805691 Hs.137516 253337 fidgetin-like 1
AI925583 Hs.222088 253234 hypothetical protein MGC5254
D26350 241911 inositol 1,4,5-triphosphate receptor, type 2 Hs.406293 253470 neurotrophic tyrosine kinase, receptor, type 1
BE616825 Hs.9625 253651 NIMA (never in mitosis gene a)-related kinase 6
BF796470 Hs.56974 239505 p21 (CDKNIA)-activated kinase 2
AF005046 Hs.20447 107335 p21(CDKN1A)-activated kinase 4
U90065 Hs.79351 235340 potassium channel, subfamily K, member 1
AF064824 Hs.103755 240173 receptor-interacting serine-threonine kinase 2
M87338 Hs.139226 113180 replication factor C (activator 1) 2, 40kDa
AW779556 Hs.184523 243092 serine/threonine kinase 38 like
AI678413 Hs.127007 153971 potassium channel, subfamily K, member 5
AF213033 Hs.84113 240337 cyclin-dependent kinase inhibitor 3 (CDK2- associated dual specificity phosphatase)
BE872974 Hs.75393 232293 acid phosphatase 1 , soluble
AW117553 Hs.446391 259953 hypothetical protein MGC19531 (ras homolog gene family, member C)
BC006249 Hs.3764 231232 guanylate kinase 1
AB033100 Hs.300646 262356 KIAA protein (similar to mouse paladin)
AB051487 Hs.20281 255305 dual specificity phosphatase 16 Table 2 describes the location of the cancer target genes of the present invention while Table 3 describes primers used to locate these genes. An additional set of primers is provided in Table 5 while additional gene data is provided in Table 4.
The nucleotides and polypeptides, as gene products, used in the methods 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.
Table 2. Chromosome Location of Cancer Target Genes
Accession Chromosome band Description indication Primer AI651265 chr17 q12 kinase PR3869 U77949 chr17 q21.2 AAA ATPase breast PR3870 X77743 chr5 q13.2 kinase ovary PR3871 AA805691 chr7 p12.2 AAA ATPase lung PR3872 AI925583 chr8 q24.13 AAA ATPase PR3873 D26350 chr12 P11.23 ion transport PR3874 chrl q21.3 tk melanoma PR3875 and LN mets BE616825 chr9 q33.3 kinase sarcoma PR3876 BF796470 chr3 q29 kinase ovary PR3877 AF005046 chrl 9 q13.2 kinase PR3878 U90065 chrl q42.2 K+ channel pancreas PR3879 AF064824 chr8 q21.3 kinase ovary PR3880 M87338 chr7 q11.23 AAA ATPase PR3881 AW779556 chr12 p11.23 kinase pancreas PR3882 AI678413 chr6 p21.2 K+ channel PR3883 AF213033 chr14 q22.2 PR3884 BE872974 chr2 p25.3 PR3885 AW117553 chrl p13.2 Phosphatase breast PR3886 BC006249 chrl q42.13 PR3887 AB033100 chr10 q22.1 PR3888 AB051487 chr12 p13.2 PR3889 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 a polypeptide encoded by one of KIAA1274, NEK6, PAK2, PAK4, STK38L, ACP1 , ARHC, CDC6, CDK7, CDKN3, CRK7, DUSP16, FIGNL1, GUK1 , ITPR2, KCNK1 , KCNK5, PRO2000, RFC2 and RIPK2 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 genes and gene products useful in practicing the methods 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. Table 3. Primers used to Identify Genes
Accession Left primer 1 Right primer 1 AI651265 TCTTGGGCATCTCAACG CACATAAAGCAGGTTGTAGAACG U77949 TATTCAGCTGGCATTTAGAGAGC ACACTTGCAAGCACATTGGC X77743 TGGACAACATTTTACTACTGAGGG TAAGTTTTCCCTAATGCATTTTCA
AA805691 TTAGTCCTGATAAAAATTAAAAACC AGAAAGTGCCTTCCTGATG AI925583 TAAACCATTTGGGATGGCAT TGGAACAAGCTGTTAACACCC D26350 CTCTGAGGACATTCCCGTTAGAA CAGGTGTTTCAAGGAAGAGGAAA ATGACATGGGGCTTGC AACACCTGAGGGGGCTT
BE616825 TCTTCATGAATTCTAAGTAACTC GTCACTGACTTCATGACA
BF796470 TAACAAGCGATTCTAAACCACC ATGGATGCAAATTCTTTAAGCA
AF005046 AACTAACTCGAGGCAGGGGT CTGCCCTTATTGGGGGAC U90065 TTCCCCTTATTTTATTGTAGCAA GGTTTATGTGTACTGGTTTGCA
AF064824 AAATGGGGACAGGAAGCC GCTTAATTGCCCTACAAAGGG M87338 CAACAAACACTGCAAGGCTT TCTCCATCCTGGGGAAAAA
AW779556 TGCCACCAAAACATTTTTGA ATGTGAGGGGATATTGCTGC AI678413 AACTACTACACACAGAAGCTGC AAGCCAGCTTCAGATGTATAT
AF213033 CCATGTCTGAAATGTCAGTTCTC AAAACTTTAGGAATATCTGCACATG
BE872974 TCAGAGGCAAAGTGGTTCAG AATCAGTCGTTGGCACCTTC
AW117553 TCTTGACACATACGAAGCC GTAGAAGCAGAGTCCCTGG
BC006249 AGGCTTGCTGTCTGTTCTCG TTTATTAGGATGTCAGCCCTGG
AB033100 CTTCTCCTCAGTCTCAAACCCAA ATCCATCTCTCTGACAGTGCTGA
AB051487 ATCCCATTTTAAACAATTCTTTGA GCTGAACCACCAGGAACCT
Table 4. Further Description of Cancer Target Genes
Accession UniSTS Gencarta Name AI651265 PR3890 CRK7 SHGC-58832 T60764 U77949 PR3891 CDC6 RH70424 T83032 X77743 PR3892 CDK7 SHGC-149358 F02366 AA805691 PR3893 FIGNL1 RH 103568 H61320 AI925583 PR3894 PRO2000 RH80934 Z44462 D26350 PR3895 ITPR2 SHGC-106565 Z38709 PR3896 SHGC-69193 BE616825 PR3897 NEK6 RH62928 T11445 BF796470 PR3898 PAK2 SHGC-35416 Z26993 AF005046 PR3899 PAK4 RH39107 R09837 U90065 PR3900 KCNK1 55164 Z39663 AF064824 RIPK2 D61791 M87338 PR3901 RFC2 47404 T62520 AW779556 PR3902 STK38L 182659 R14324 AI678413 PR3903 KCNK5 83108 R25184 AF213033 PR3904 CDKN3 24341 HUMPTPB BE872974 PR3905 ACP1 91295 HUMAAPA AW117553 PR3906 ARHC RH49960 AA383349 BC006249 PR3907 GUK1 38548 T08090 AB033100 PR3908 KIAA1274 148013 AA553584 AB051487 PR3909 DUSP16 85676 T23935
Table 5. Additional Primers for Cancer Target Genes
Accession Left primer 2 Right primer 2 AI651265 GTGGGGCCCAATAACTCAAA TTTTGAATCTGGCCTTGCCT U77949 TTATGACCCCAACGCC AAGCAAGTCCACATGGAG X77743 CAGAGGTTCCCTCTTAAAAATTCA AAAGTGAAGTATTGGCTGGGC AA805691 CCATCCATGGAATCCTAGACA TTATCCTACCACTTTGCGGG AI925583 AAGAGTTGGCCAAACTTCAACTATT TGTCATGTCCGCCTAATTGA D26350 CAAAGCCTCAAGACCTTTTTCAA AAGGTACCAGCTAAACCTCTTTGC GAGAAAGGGAGGGATCGTTC TGTGAGGGGCTATGCTGG BE616825 TTCCACTTTATCCCTTTACAACA GGCTTATGCTAACAGGAGACTTG BF796470 TCACTGCTGTGGCCTCATAC TCAGTCCACAATTCCTTCTGG AF005046 GGGGGACGCTGTCATTCAC TTCCCAGTACCGCAGAGCC U90065 GGTCCTCTACTTCCACAT GCTCTCTGAAI I I I I GATT AF064824 M87338 GCAGAGACTTCACTGACTGAC TGACCTCAGGTGATCCACCTG AW779556 TTTAGCAAAACTTGGAGCTGGAG AAAACCATTCTCTACTAACTACCCCC AI678413 TTTTGCAAGGCAACTGAGG GATACGGCAGCCTCTACTGC AF213033 CACATGGCCTAGTAGTTTGG GTTCCAACTGCTTAGATCAGC BE872974 TGAACAAAGAGCTGGGCTTT ACTGAGGCAGGTTCGTGC AW117553 AGCCTGTAGCCTTTATCCATG CTTCTGGCTCACAGGAAAATG BC006249 CTGCTCTTTACCTGGGGTTG GAGCCACAGAGGAGTGAAGG AB033100 ACATGTGCCCTACACACAC AGCTGTCACATAAATAGAACCC AB051487 ATCAGACATTCTCAAGTTTCACACA GGACCATGGCCAAGAGAAG
Expression products of the genes disclosed herein for use in the methods of the invention may be in an isolated form.
Methods of producing vectors comprising genes disclosed herein, or recombinant cells expressing such genes, 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 one aspect, the present invention relates to a method for identifying a cancer-target gene, comprising: a) identifying a gene that is at least 5 fold over-expressed in a cancer cell line and that maps to a chromosomal region with a CGH ratio of at least 1.25; b) determining an RNA expression level of said gene of at least 1.5 fold in a tumor tissue compared to corresponding normal tissue in a genetic database, c. determining that said gene encodes a protein domain known to be modulated, or shown to be modulated, by chemical compounds wherein a gene that meets the criteria of a, b and c is considered to be a cancer-target gene, thereby identifying a cancer-target gene. The present invention also relates to a set of cancer-target genes identified using such methods. The genes disclosed herein form such a set. In addition, subsets of such sets are specifically contemplated by the invention.
In another aspect, the present invention relates to a method for identifying an agent that modulates the activity of a cancer-target gene comprising: (a) contacting a test compound with a cell containing a polynucleotide that corresponds to a gene that has the properties of a, b and c of claim 1 and under conditions promoting the expression of said gene, and (b) determining a difference in expression of said gene relative to when said test compound is not present wherein said difference indicates gene modulating activity, thereby identifying an agent that modulates the activity of a cancer- related gene.
In a preferred embodiment, said gene was first identified as a cancer target gene using one or more of the methods of the invention. In another preferred embodiment, the gene is a gene selected from the group consisting of KIAA1274, NEK6, PAK2, PAK4, STK38L, ACP1, ARHC, CDC6, CDK7, CDKN3, CRK7, DUSP16, FIGNL1, GUK1 , ITPR2, KCNK1 , KCNK5, PRO2000, RFC2 and RIPK2. These 20 genes are contained in Table 6 where they are described in terms of a consensus sequence along with identified polynucleotide transcripts and polypeptides.
In the methods of the invention, expression may be determined by determining transcription (to form RNA), as by measuring the rate or amount of RNA formed, or translation (to form protein), such as where antibodies may be used to determine the amount of polypeptide or protein formed from the gene in question or where the activity of such protein is determined, such as where the protein is an enzyme and the amount of enzyme activity can be determined.
In one preferred embodiment, the cell is a cancer cell and the determined difference in expression is a decrease in expression. In another embodiment, the cell is a recombinant cell, such as one comprising a gene as disclosed herein, and the difference in expression is a decrease in expression.
The present invention also relates to a method for identifying an anti- neoplastic agent comprising contacting a cell exhibiting neoplastic activity with a compound first identified as a cancer target gene modulator using one of the methods of the invention and detecting a decrease in said neoplastic activity after said contacting compared to when said contacting does not occur. In preferred embodiments, the neoplastic activity is accelerated cellular replication. In another preferred embodiment, the decrease in neoplastic activity results from the death of the cell. In a preferred embodiment, the compound is one that modulates, preferably inhibits, a gene disclosed herein, most preferably a gene identified in Table 6. The present invention also relates to a method for identifying an anti- neoplastic agent comprising administering to an animal exhibiting a cancerous condition an effective amount of a cancer target gene modulating agent by a method of the invention and detecting a decrease in said cancerous condition. In a preferred embodiment, the compound is one that modulates, preferably inhibits, a gene disclosed herein, most preferably a gene identified in Table 6.
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 are thus disclosed herein methods for using a cancer-linked or cancer-target gene sequence (such as that of KIAA1274, NEK6, PAK2, PAK4, STK38L, ACP1 , ARHC, CDC6, CDK7, CDKN3, CRK7, DUSP16, FIGNL1, GUK1 , ITPR2, KCNK1 , KCNK5, PRO2000, RFC2 and RIPK2) 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 are 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 genes useful in the methods of the invention can include fully operational 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.
In a preferred embodiment a selected set of said genes are expressed in the reference cell, including the gene(s) 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.
The genes identified by the present disclosure are considered "cancer- related" genes, or cancer-target" 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 of KIAA1274, NEK6, PAK2, PAK4, STK38L, ACP1 , ARHC, CDC6, CDK7, CDKN3, CRK7, DUSP16, FIGNL1, GUK1, ITPR2, KCNK1, KCNK5, PRO2000, RFC2 and RIPK2. The genes disclosed herein may be genomic in nature and thus represent an actual gene as found in nature, 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 genes disclosed herein may represent less than the full genomic nucleotide 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 of genes 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 as corresponding to genes disclosed herein 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 of genes 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" the genes of KIAA1274,
NEK6, PAK2, PAK4, STK38L, ACP1, ARHC, CDC6, CDK7, CDKN3, CRK7, DUSP16, FIGNL1, GUK1, ITPR2, KCNK1 , KCNK5, PRO2000, RFC2 and RIPK2 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 genes of KIAA1274, NEK6, PAK2, PAK4, STK38L, ACP1 , ARHC, CDC6, CDK7, CDKN3, CRK7, DUSP16, FIGNL1, GUK1 , ITPR2, KCNK1 , KCNK5, PRO2000, RFC2 or RIPK2, 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, genes encoding the same proteins as any of these genes, 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 genes will also encode the same or similar polypeptide sequence as the genes KIAA1274, NEK6, PAK2, PAK4, STK38L, ACP1 , ARHC, CDC6, CDK7, CDKN3, CRK7, DUSP16, FIGNL1, GUK1 , ITPR2, KCNK1 , KCNK5,
PRO2000, RFC2 and RIPK2 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 encoded by genes disclosed herein, preferably KIAA1274, NEK6, PAK2, PAK4, STK38L, ACP1 , ARHC, CDC6, CDK7, CDKN3, CRK7, DUSP16, FIGNL1, GUK1 , ITPR2, KCNK1 , KCNK5, PRO2000, RFC2 and RIPK2.
The present invention also relates to methods of assaying potential antitumor agents based on their modulation of the expression of the disclosed genes according to the invention and methods for diagnosing cancerous, or potentially cancerous, conditions as a result of the patterns of expression of the a gene disclosed herein as well as related genes 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 genes 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, corresponding to any of the genes disclosed herein, 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 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.
Changes in gene copy number may be determined by determining a change in expression of messenger RNA encoded by a particular gene, 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, especially that of KIAA1274, NEK6, PAK2, PAK4, STK38L, ACP1 , ARHC, CDC6, CDK7, CDKN3, CRK7, DUSP16, FIGNL1, GUK1 , ITPR2, KCNK1 , KCNK5, PRO2000, RFC2 and RIPK2. 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 one of KIAA1274, NEK6, PAK2, PAK4, STK38L, ACP1 , ARHC, CDC6, CDK7, CDKN3, CRK7, DUSP16, FIGNL1, GUK1 , ITPR2, KCNK1, KCNK5, PRO2000, RFC2 and RIPK2, 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 genes, 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 any of KIAA1274, NEK6, PAK2,
PAK4, STK38L, ACP1 , ARHC, CDC6, CDK7, CDKN3, CRK7, DUSP16,
FIGNL1, GUK1 , ITPR2, KCNK1 , KCNK5, PRO2000, RFC2 and RIPK2.
Thus, the present invention further relates to a method for identifying an agent that modulates the activity of a cancer-target polypeptide comprising: (a) contacting a test compound with a cell expressing a polypeptide encoded by a polynucleotide corresponding to a gene having the properties of a, b and c disclosed above for identifying a cancer-target gene and under conditions promoting the expression of said polypeptide; and (b) determining a difference in expression of said polypeptide relative to when said test compound is not present wherein said difference indicates cancer-target polypeptide modulating activity, thereby identifying a cancer-target polypeptide modulating agent. The present invention further relates to a method for identifying an agent that modulates the activity of a cancer-target polypeptide comprising: (a) contacting a test compound with a polypeptide encoded by a polynucleotide corresponding to a gene having the properties of a, b and c of claim 1 and under conditions promoting the activity of said polypeptide; and (b) determining a difference in activity of said polypeptide relative to when said test compound is not present wherein said difference indicates cancer-target polypeptide modulating activity, thereby identifying a cancer-target polypeptide modulating agent. In any of these methods, a preferred embodiment utilizes a gene selected from KIAA1274, NEK6, PAK2, PAK4, STK38L, ACP1 , ARHC, CDC6, CDK7, CDKN3, CRK7, DUSP16, FIGNL1, GUK1 , ITPR2, KCNK1 , KCNK5, PRO2000, RFC2 and RIPK2. 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 of KIAA1274, NEK6, PAK2, PAK4, STK38L, ACP1 , ARHC, CDC6, CDK7, CDKN3, CRK7, DUSP16, FIGNL1, GUK1 , ITPR2, KCNK1 , KCNK5, PRO2000, RFC2 and RIPK2, including genes 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 useful in the methods of the invention 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.
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 methods 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.
Thus, the present invention relates to a method for treating cancer comprising contacting a cancerous cell with an effective amount of an agent that can reduce the activity of a cancer-target gene (i.e., a gene having the properties of a, b and c disclosed herein for identifying a cancer-target gene). In a preferred embodiment, said gene is one of KIAA1274, NEK6, PAK2, PAK4, STK38L, ACP1 , ARHC, CDC6, CDK7, CDKN3, CRK7, DUSP16, FIGNL1, GUK1 , /7Pf?2, KCNK1 , KCNK5, PRO2000, RFC2 and RIPK2.
Such embodiments include use of any of the agents having activity in one or more of the screening methods disclosed herein, most preferably wherein the agent was first identified as having such activity using one or more of said methods. In a preferred embodiment, the cancerous cell is contacted in vivo.
In an additional preferred embodiment, the agent has affinity for an expression product of said gene, such as where the agent is an antibody, preferably one disclosed according to the present invention.
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 /n wt'ro 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, /n v/tro or /n Wvo, 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, H 1 , H2, H3) for the respective light (L) and heavy (H) chains. The accepted CDR regions have been described by Kabat et al, J. β/o/. Cftem. 252:6609-661 6 ( 1 977). 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 encoded by a gene identified by the methods of the invention, preferably one of KIAA1274, NEK6, PAK2, PAK4, STK38L, ACP1 , ARHC, CDC6, CDK7, CDKN3, CRK7, DUSP16, FIGNL1, GUK1 , ITPR2, KCNK1 , KCNK5, PRO2000, RFC2 and RIPK2. Such antibodies may commonly be in the form of a composition, especially a pharmaceutical composition.
Thus, the present invention contemplates an antibody that binds to a polypeptide encoded by a cancer-target gene (i.e., a gene having the properties of a, b and c disclosed above for identifying a cancer-target gene). In a preferred embodiment, the polypeptide or protein is encoded by one or more of genes KIAA1274, NEK6, PAK2, PAK4, STK38L, ACP1, ARHC, CDC6, CDK7, CDKN3, CRK7, DUSP16, FIGNL1, GUK1 , ITPR2, KCNK1 , KCNK5, PRO2000, RFC2 and RIPK2. In specific embodiments, this may include a naturally occurring antibody, such as polyclonal antibodies, or more preferably a monoclonal antibody, or a recombinant antibody or a partly or wholly synthetic antibody. In additional preferred embodiments, the antibody further comprises a cytotoxic agent, such as an apoptotic agent. 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 a gene 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 contemplated by the invention, preferably those encoded by one or more of KIAA1274, NEK6, PAK2, PAK4, STK38L, ACP1 , ARHC, CDC6, CDK7, CDKN3, CRK7, DUSP16, FIGNL1, GUK1 , ITPR2, KCNK1 , KCNK5, PRO2000, RFC2 and RIPK2, 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 encoded by one or more of KIAA1274, NEK6, PAK2, PAK4, STK38L, ACP1 , ARHC, CDC6, CDK7, CDKN3, CRK7, DUSP16, FIGNL1, GUK1 , ITPR2, KCNK1 , KCNK5, PRO2000, RFC2 and RIPK2, 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, such as by generating test data to facilitate identification of such 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 one of KIAA1274, NEK6, PAK2, PAK4, STK38L, ACP1 , ARHC, CDC6, CDK7, CDKN3, CRK7, DUSP16, FIGNL1, GUK1 ,
ITPR2, KCNK1 , KCNK5, PRO2000, RFC2 and RIPK2 and represent genes that may be over-expressed in malignant cancer. 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 genes 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 genes 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.
Expression of a gene corresponding to a polynucleotide disclosed herein, when in normal tissues, may indicate a predisposition towards development of colon cancer. The encoded polypeptide might then present a potentially useful cell surface target for therapeutic molecules such as cytolytic antibodies, or antibodies attached to cytotoxic, or cytolytic, agents. The present invention specifically contemplates use of antibodies against polypeptides encoded by the genes disclosed herein, whereby said antibodies are conjugated to one or more cytotoxic agents so that the antibodies serve to target the conjugated immunotoxins to a region of cancerous activity, such as a solid tumor. For many known cytotoxic agents, lack of selectivity has presented a drawback to their use as therapeutic agents in the treatment of malignancies. For example, the class of two-chain toxins, consisting of a binding subunit (or B-chain) linked to a toxic subunit (A-chain) are extremely cytotoxic. Thus, such agents as ricin, a protein isolated from castor beans, kills cells at very low concentrations (even less than 10"11 M) by inactivating ribosomes in said cells (see, for example, Lord et al., Ricin: structure, mode of action, and some current applications. Faseb J, 8; 201-208 (1994), and Blattler et al., Realizing the full potential of immunotoxins. Cancer Cells, 1. 50-55 (1989)). While isolated A-chains of protein toxins that functionally resemble ricin A-chain are only weakly cytotoxic for intact cells (in the concentration range of 10"7 to 10"6 M), they are very potent cytotoxic agents inside the cells. Thus, a single molecule of the A-subunit of diphtheria toxin can kill a cell once inside (see: Yamaizumi et al., One molecule of diphtheria toxin fragment A introduced into a cell can kill the cell. Cell, 15: 245-250, 1978).
The present invention solves this selectivity problem by using antibodies specific for antigens present on cancer cells to target the cytotoxins to said cells. In addition, use of antibodies decreases toxicity because the antibodies are non-toxic until they reach the tumor and, because the cytotoxin is bound to the antibody, it is presented with less opportunity to cause damage to non-targeted tissues.
In addition, use of such antibodies alone can provide therapeutic effects on the tumor through the antibody-dependent cellular cytotoxic response (ADCC) and complement-mediated cell lysis mechanisms. A number of recombinant immunotoxins (for example, consisting of Fv regions of cancer specific antibodies fused to truncated bacterial toxins) are well known (see, for example, Smyth et al., Specific targeting of chlorambucil to tumors with the use of monoclonal antibodies, J. Natl. Cancer Inst, 76(3):503-510 (1986); Cho et al., Single-chain Fv/folate conjugates mediate efficient lysis of folate-receptor-positive tumor cells, Bioconjug. Chem., 8(3):338-346 (1997)). As noted in the literature, these may contain, for example, a truncated version of Pseudomonas exotoxin as a toxic moiety but the toxin is modified in such a manner that by itself it does not bind to normal human cells, but it retains all other functions of cytotoxicity. Here, recombinant antibody fragments target the modified toxin to cancer cells which are killed, such as by direct inhibition of protein synthesis, or by concomitant induction of apoptosis. Cells that are not recognized by the antibody fragment, because they do not carry the cancer antigen, are not affected. Good activity and specificity has been observed for many recombinant immunotoxins in in vitro assays using cultured cancer cells as well as in animal tumor models. Ongoing clinical trials provide examples where the promising pre-clinical data correlate with successful results in experimental cancer therapy, (see, for example, Brinkmann U., Recombinant antibody fragments and immunotoxin fusions for cancer therapy, In Vivo (2000) 14:21-27).
While the safety of employing immunoconjugates in humans has been established, in vivo therapeutic results have been less impressive. Because clinical use of mouse MAbs in humans is limited by the development of a foreign anti-globulin immune response by the human host, genetically engineered chimeric human-mouse MAbs have been developed by replacing the mouse Fc region with the human constant region. In other cases, the mouse antibodies have been "humanized" by replacing the framework regions of variable domains of rodent antibodies by their human equivalents. Such humanized and engineered antibodies can even be structurally arranged to have specificities and effector functions determined by design and which characteristics do not appear in nature. The development of bispecific antibodies, having different binding ends so that more than one antigenic site can be bound, have proven useful in targeting cancer cells. Thus, such antibody specificity has been improved by chemical coupling to various agents such as bacterial or plant toxins, radionuclides or cytotoxic drugs and other agents, (see, for example, Bodey, B. et al). Genetically engineered monoclonal antibodies for direct anti-neoplastic treatment and cancer cell specific delivery of chemotherapeutic agents. Curr PΛam? Dβs (2000) Feb;6(3):261 -76). See also, Garnett, M. C, Targeted drug conjugates: principles and progress. Adv. Drug Deliv. Rev. (2001 Dec 17) 53(2): 171 -216; Brinkmann et al., Recombinant immunotoxins for cancer therapy. Expert Opin Biol Ther. (2001) 1(4):693-702.
Among the cytotoxic agents specifically contemplated for use as immunoconjugates according to the present invention are Calicheamicin, a highly toxic enediyne antibiotic isolated from M/cromonospora ecfi/nospora ssp. Ca//c/?e/7s/s, and which binds to the minor groove of DNA to induce double strand breaks and cell death (see: Lee et al., Calicheamicins, a novel family of antitumor antibiotics. 1. Chemistry and partial structure of calichemicin gi.. J Am Chem Soc, 109: 3464-3466 (1987); Zein et al., Calicheamicin gamma 11: an antitumor antibiotic that cleaves double-stranded DNA site specifically, Science, 240: 1198-1201 (1988)). Useful derivatives of the calicheamicins include mylotarg and 138H11-Camθ. Mylotarg is an immunoconjugate of a humanized anti-CD33 antibody (CD33 being found in leukemic cells of most patients with acute myeloid leukemia) and N-acetyl gamma colicheamicin dimethyl hydrazide, the latter of which is readily coupled to an antibody of the present invention (in place of the anti- CD33 but which can also be humanized by substitution of human framework regions into the antibody during production as described elsewhere herein) to form an immunoconjugate of the invention, (see: Hamann et al. Gemtuzumab Ozogamicin, A Potent and Selective Anti-CD33 Antibody-Calicheamicin Conjugate for Treatment of Acute Myeloid Leukemia, Bioconjug. Chem. 13, 47-58 (2002)) For use with 138H11-Camθ, 138H11 is an anti-γ-glutamyl transferase antibody coupled to theta calicheamicin through a disulfide linkage and found useful in vitro against cultured renal cell carcinoma cells, (see: Knoll et al., Targeted therapy of experimental renal cell carcinoma with a novel conjugate of monoclonal antibody 138H11 and calicheamicin θ Cancer Res, 60: 6089-6094 (2000) The same linkage may be utilized to link this cytotoxic agent to an antibody of the present invention, thereby forming a targeting structure for colon cancer cells.
Also useful in forming the immunoconjugates of the invention is DC1 , a disulfide-containing analog of adozelesin, that kills cells by binding to the minor groove of DNA, followed by alkylation of adenine bases. Adozelesin is a structural analog of CC-1065, an anti-tumor antibiotic isolated from microbial fermentation of Streptomyces zelensis, and is about 1 ,000 fold more toxic to cultured cell lines that other DNA interacting agents, such as cis-platin and doxorubicin. This agent is readily linked to antibodies through the disulfide bond of adozelesin. (see: Chari et al., Enhancement of the selectivity and antitumor efficacy of a CC-1065 analogue through immunoconjugate formation, Cancer Res, 55: 4079-4084 (1995)). Maytansine, a highly cytotoxic microtubular inhibitor isolated from the shrub Maytenus serrata found to have little value in human clinical trials, is much more effective in its derivatized form, denoted DM1 , containing a disulfide bond to facilitate linkage to antibodies, is up to 10-fold more cytotoxic (see: Chari et al., Immunoconjugates containing novel maytansinoids: promising anticancer drugs, Cancer Res, 52: 127-131 (1992)). These same in vitro studies showed that up to four DM1 molecules could be linked to a single immunoglobulin without destroying the binding affinity. Such conjugates have been used against breast cancer antigens, such as the neu/HER2/erbB-2 antigen, (see: Goldmacher et al., Immunogen, Inc., (2002) in press); also see Liu, C. et al., Eradication of large colon tumor xenografts by targeted delivery of maytansinoids, Proc. Natl. Acad. Sci. USA, 93, 8618-8623 (1996)). For example, Liu et al. (1996) describes formation of an immunoconjugate of the maytansinoid cytotoxin DM1 and C242 antibody, a murine lgG1 immunoglobulin, available from Pharmacia and which has affinity for a mucin- like glycoprotein variably expressed by human colorectal cancers. The latter immunoconjugate was prepared according to Chari et al., Cancer Res., 52:127-131 (1992) and was found to be highly cytotoxic against cultured colon cancer cells as well as showing anti-tumor effects in vivo in mice bearing subcutaneous COLO 205 human colon tumor xenografts using doses well below the maximum tolerated dose. In accordance with the foregoing, a preferred embodiment of the present invention includes where the cytotoxic agent is a calicheamicin, a maytansinoid, an adozelesin, DC1 , a cytotoxic protein, a taxol, a taxotere, or a taxoid. In especially preferred embodiments, the calicheamicin is calicheamicin γi1, N-acetyl gamma calicheamicin dimethyl hydrazide or calicheamicin θi1, the maytansinoid is DM1 , the cytotoxic protein is ricin, abrin, gelonin, pseudomonas exotoxin or diphtheria toxin, the taxol is paclitaxel, and the taxotere is docetaxel.
In addition, there are a variety of protein toxins (cytotoxic proteins), which include a number of different classes, such as those that inhibit protein synthesis: ribosome-inactivating proteins of plant origin, such as ricin, abrin, gelonin, and a number of others, and bacterial toxins such as pseudomonas exotoxin and diphtheria toxin. Another useful class is the one including taxol, taxotere, and taxoids.
Specific examples include paclitaxel (taxol), its analog docetaxel (taxotere), and derivatives thereof. The first two are clinical drugs used in treating a number of tumors while the taxoids act to induce cell death by inhibiting the de-polymerization of tubulin. Such agents are readily linked to antibodies through disulfide bonds without disadvantageous effects on binding specificity. In one instance, a truncated Pseudomonas exotoxin was fused to an anti-CD22 variable fragment and used successfully to treat patients with chemotherapy-resistant hairy-cell leukemia, (see: Kreitman et al., Efficacy of the anti-CD22 recombinant immunotoxin BL22 in chemotherapy-resistant hairy-cell leukemia, N Engl J Med, 345: 241-247 (2001)) Conversely, the cancer-linked peptides of the present invention offer the opportunity to prepare antibodies, recombinant or otherwise, against the appropriate antigens to target solid tumors, preferably those of malignancies of colon tissue, using the same or similar cytotoxic conjugates. Thus, many of the previously used immunoconjugates have been formed using antibodies against general antigenic sites linked to cancers whereas the antibodies formed using the peptides disclosed herein are more specific and target the antibody-cytotoxic agent to a particular tissue or organ, thus further reducing toxicity and other undesirable side effects.
In addition, the immunoconjugates formed using the antibodies prepared against the cancer-linked antigens disclosed herein can be formed by any type of chemical coupling. Thus, the cytotoxic agent of choice, along with the immunoglobulin, can be coupled by any type of chemical linkage, covalent or non-covalent, including electrostatic linkage, to form the immunoconjugates of the present invention.
When used as immunoconjugates, the antitumor agents of the present invention represent a class of pro-drugs that are relatively non-toxic when first administered to an animal (due mostly to the stability of the immunoconjugate), such as a human patient, but which are targeted by the conjugated immunoglobulin to a cancer cell where they then exhibit good toxicity. The tumor-related, associated, or linked, antigens, preferably those presented herein, serve as targets for the antibodies (monoclonal, recombinant, and the like) specific for said antigens. The end result is the release of active cytotoxic agent inside the cell after binding of the immunoglobulin portion of the immunoconjugate. The cited references describe a number of useful procedures for the chemical linkage of cytotoxic agents to immunoglobulins and the disclosures of all such references cited herein are hereby incorporated by reference in their entirety. For other reviews see Ghetie et al., Immunotoxins in the therapy of cancer: from bench to clinic, Pharmacol Ther, 63: 209-234 (1994), Pietersz et al. The use of monoclonal antibody immunoconjugates in cancer therapy, Adv Exp Med Biol, 353:169-179 (1994), and Pietersz, G. A. The linkage of cytotoxic drugs to monoclonal antibodies for the treatment of cancer, Bioconjug Chem, 1 : 89-95 (1990).
Thus, the present invention provides highly useful cancer-associated antigens for generation of antibodies for linkage to a number of different cytotoxic agents which are already known to have some in vitro toxicity and possess chemical groups available for linkage to antibodies.
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. EXAMPLE
SW480 cells are grown to a density of 105 cells/cm2 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.
Table 6 below contains a listing of the genes (numbered 1 to 20) along with their Gencarta names (or accessions). Each gene is represented as a consensus sequence followed by predicted mRNA transcripts and then predicted polypeptides. All of the sequences, with additional information, are presented in Figure 1.
The present invention also relates to an isolated cancer target gene wherein said gene is a gene identified in Table 6. Thus, the present invention encompasses isolated genes identified herein as KIAA1274, NEK6, PAK2, PAK4, STK38L, ACP1 , ARHC, CDC6, CDK7, CDKN3, CRK7, DUSP16, FIGNL1, GUK1 , ITPR2, KCNK1 , KCNK5, PRO2000, RFC2 and RIPK2 and uses of these genes, whether isolated or not, in any of the methods of the invention.
Table 6. Sequence Identification Numbers for genes, transcripts and polypeptides*
Figure imgf000057_0001
*Accession numbers in Table 6 are for the Gencarta database.

Claims

WHAT IS CLAIMED IS:
1. A method for identifying a cancer-target gene, comprising: a) identifying a gene that is at least 5 fold over-expressed in a cancer cell line and that maps to a chromosomal region with a CGH ratio of at least
1.25; b) determining an RNA expression level of said gene of at least 1.5 fold in a tumor tissue compared to corresponding normal tissue in a genetic database, and c) determining that said gene encodes a protein domain that is modulated by chemical compounds, wherein a gene that meets the criteria of steps a, b and c is considered to be a cancer-target gene, thereby identifying a cancer-target gene.
2. A set of cancer-target genes identified by the method of claim 1.
3. A method for identifying an agent that modulates the activity of a cancer-target gene comprising: (a) contacting a test compound with a cell that expresses a polynucleotide that corresponds to a gene that has the properties of a, b and c of claim 1 and under conditions supporting said expression; and (b) determining a difference in expression of said gene relative to when said test compound is not present wherein said difference indicates gene modulating activity, thereby identifying said test compound as an agent that modulates the activity of said cancer-related gene.
4. The method of claim 3 wherein said gene was first identified as a cancer target gene using the method of claim 1 or 2.
5. The method of claim 4 wherein said gene is a gene selected from the group consisting of KIAA1274, NEK6, PAK2, PAK4, STK38L, ACP1, ARHC, CDC6, CDK7, CDKN3, CRK7, DUSP16, FIGNL1, GUK1 , ITPR2, KCNK1 , KCNK5, PRO2000, RFC2 and RIPK2.
6. The method of claim 3 wherein said expression is transcription to form RNA.
7. The method of claim 3 wherein said expression is translation to form protein.
8. The method of claim 4 wherein the cell is a cancer cell and the determined difference in expression is a decrease in expression.
9. The method of claim 4 wherein the cell is a recombinant cell and the difference in expression is a decrease in expression.
10. A method for identifying an anti-neoplastic agent comprising contacting a cell exhibiting neoplastic activity with a compound first identified as a cancer target gene modulator using the method of claim 3 and detecting a decrease in said neoplastic activity after said contacting compared to when said contacting does not occur.
11. The process of claim 10 wherein said neoplastic activity is accelerated cellular replication.
12. The process of claim 10 wherein said decrease in neoplastic activity results from the death of the cell.
13. A method for identifying an anti-neoplastic agent comprising contacting a cell exhibiting neoplastic activity with a compound that modulates expression of at least one of genes 1 to 20 of Table 6 and detecting a decrease in said neoplastic activity after said contacting compared to when said contacting does not occur.
14. The process of claim 13 wherein said neoplastic activity is accelerated cellular replication.
15. The process of claim 13 wherein said decrease in neoplastic activity results from the death of the cell.
16. A method for identifying an anti-neoplastic agent comprising administering to an animal exhibiting a cancerous condition an effective amount of an agent that modulates expression of at least one of genes 1 to 20 of Table 6 and detecting a decrease in said cancerous condition.
17. A method for identifying an anti-neoplastic agent comprising administering to an animal exhibiting a cancerous condition an effective amount of a cancer target gene modulating agent by the method of claim 3 and detecting a decrease in said cancerous condition.
18. A method 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 meets the criteria of a, b and c of claim 1.
19. The method of claim 14 wherein said gene is a gene selected from the group consisting of KIAA1274, NEK6, PAK2, PAK4, STK38L, ACP1 ,
ARHC, CDC6, CDK7, CDKN3, CRK7, DUSP16, FIGNL1, GUK1 , ITPR2, KCNK1 , KCNK5, PRO2000, RFC2 and RIPK2.
20. A method for identifying an agent that modulates the activity of a cancer-target polypeptide comprising: (a) contacting a test compound with a cell expressing a polypeptide encoded by a polynucleotide corresponding to a gene having the properties of a, b and c of claim 1 and under conditions promoting the expression of said polypeptide; and (b) determining a difference in expression of said polypeptide relative to when said test compound is not present wherein said difference indicates cancer-target polypeptide modulating activity, thereby identifying a cancer-target polypeptide modulating agent.
21. The method of claim 20 wherein said polypeptide is encoded by a gene selected from the group consisting of KIAA1274, NEK6, PAK2, PAK4, STK38L, ACP1 , ARHC, CDC6, CDK7, CDKN3, CRK7, DUSP16, FIGNL1, GUK1 , ITPR2, KCNK1 , KCNK5, PRO2000, RFC2 and RIPK2.
22. A method for identifying an agent that modulates the activity of a cancer-target polypeptide comprising: (a) contacting a test compound with a polypeptide encoded by a polynucleotide corresponding to a gene having the properties of a, b and c of claim 1 and under conditions promoting the activity of said polypeptide; and (b) determining a difference in activity of said polypeptide relative to when said test compound is not present wherein said difference indicates cancer-target polypeptide modulating activity, thereby identifying a cancer-target polypeptide modulating agent.
23. The method of claim 22 wherein said polypeptide is encoded by a gene selected from the group consisting of KIAA1274, NEK6, PAK2, PAK4, STK38L, ACP1 , ARHC, CDC6, CDK7, CDKN3, CRK7, DUSP16, FIGNL1, GUK1 , ITPR2, KCNK1 , KCNK5, PRO2000, RFC2 and RIPK2.
24. An antibody that binds to a polypeptide encoded by a gene having the properties of a, b and c of claim 1.
25. The antibody of claim 24 wherein said gene is a gene selected from the group consisting of KIAA1274, NEK6, PAK2, PAK4, STK38L, ACP1, ARHC, CDC6, CDK7, CDKN3, CRK7, DUSP16, FIGNL1, GUK1 , ITPR2, KCNK1 , KCNK5, PRO2000, RFC2 and RIPK2.
26. The antibody of claim 24 wherein said antibody is a monoclonal antibody.
27. The antibody of claim 24 wherein said antibody is a recombinant antibody.
28. The antibody of claim 24 wherein said antibody is a synthetic antibody.
29. The antibody of claim 24 wherein said antibody further comprises a cytotoxic agent.
30. The antibody of claim 29 wherein said cytotoxic agent is an apoptotic agent.
31. A method for treating cancer comprising contacting a cancerous cell with an effective amount of an agent that can reduce the activity of a gene having the properties of a, b and c of claim 1.
32. The method of claim 31 wherein said agent having activity in the method of claim 3.
33. The method of claim 31 wherein said agent was first identified as having such activity using the method of claim 3.
34. The method of claim 31 wherein said agent having activity in the method of claim 16.
35. The method of claim 31 wherein said agent was first identified as having such activity using the method of claim 16.
36. The method of claim 31 wherein said agent having activity in the method of claim 22.
37. The method of claim 31 wherein said agent was first identified as having such activity using the method of claim 22.
38. The method of claim 31 wherein said gene is a gene selected from the group consisting of KIAA1274, NEK6, PAK2, PAK4, STK38L, ACP1 , ARHC, CDC6, CDK7, CDKN3, CRK7, DUSP16, FIGNL1, GUK1 , ITPR2, KCNK1 , KCNK5, PRO2000, RFC2 and RIPK2.
39. The method of claim 31 wherein said cancerous cell is contacted in vivo.
40. The method of claim 31 wherein said agent has affinity for an expression product of said gene.
41. The method of claim 40 wherein said agent is an antibody of claim 24 - 30.
42. A cancer target gene wherein said gene is a gene identified in Table 6.
43. A method for producing test data with respect to the gene modulating activity of a compound comprising: (a) contacting a compound with a cell containing a polynucleotide comprising a nucleotide sequence corresponding to a gene whose expression is increased in a cancerous cell over that in a non-cancerous cell and under conditions wherein said polynucleotide is being expressed, (b) determining a change in expression of polynucleotides as a result of said contacting, and (c) producing test data with respect to the gene modulating activity of said compound based on a decrease in the expression of the determined gene whose expression is otherwise increased in a cancerous cell over that in a non-cancerous cell indicating gene modulating activity.
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