WO2006109086A2 - Procede - Google Patents

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Publication number
WO2006109086A2
WO2006109086A2 PCT/GB2006/001384 GB2006001384W WO2006109086A2 WO 2006109086 A2 WO2006109086 A2 WO 2006109086A2 GB 2006001384 W GB2006001384 W GB 2006001384W WO 2006109086 A2 WO2006109086 A2 WO 2006109086A2
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WIPO (PCT)
Prior art keywords
tumor
expression
mammal
genes
inhibitor
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PCT/GB2006/001384
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WO2006109086A3 (fr
Inventor
Lucienne Ronco
Kevin Webster
Lihua Yu
Xiaolin Zhang
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AstraZeneca UK Ltd
AstraZeneca AB
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AstraZeneca UK Ltd
AstraZeneca AB
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Publication of WO2006109086A2 publication Critical patent/WO2006109086A2/fr
Publication of WO2006109086A3 publication Critical patent/WO2006109086A3/fr
Anticipated expiration legal-status Critical
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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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/575—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57557—Immunoassay; Biospecific binding assay; Materials therefor for cancer of other specific parts of the body, e.g. brain
    • 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
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the present invention relates to predicting the sensitivity of tumors to a particular therapeutic agent.
  • the invention can be used to determine the suitability of cancer patients for particular treatments by measuring the levels of particular genes or their gene products in tumor tissue.
  • a biomarker is a characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacological responses to a therapeutic intervention, Atkinson et al, Clin. Pharmacol. Ther., 69: 89-95 (2001). It is believed that the development of new validated biomarkers will lead both to significant reductions in healthcare and drug development costs and to significant improvements in treatment for a wide variety of diseases and conditions. Human Eg5 has been identified to play a role in cancer.
  • Eg5 or KSP kinesin spindle protein
  • Eg5 inhibitors As the importance of using Eg5 inhibitors increases as an anti-cancer drug, it is important to be able identify patients who have tumors which will most likely benefit from therapeutic administration of an Eg5 inhibitor and spare those unlikely to benefit from the toxicity, cost and inconvenience of taking a pharmaceutical agent not expected to benefit an individual patient. Therefore there is a need for methods that can classify tumors as Eg5 inhibitor responders and methods to predict a patients' response to a drug based on the results of a test that indicates whether the patient is likely to respond to treatment or to be resistant to treatment. Summary of the Invention
  • the present invention is based on the discovery, in part, of methods of classifying if a tumor is sensitive to responding to an Eg5 inhibitor. By measuring the levels of particular genes or proteins in a tumor tissue it is possible to determine, prior to administration of the treatment, if the tumor will be responsive to the Eg5 inhibitor. Thus, the present invention can be used to predict the suitability of administering an Eg5 inhibitor to a mammal having cancer.
  • the mammal can be any mammal including a mouse, rat, dog, rabbit or human.
  • the invention provides a method of selecting a mammal having, or suspected of having, a tumor and determining if the subject should be treated with an Eg5 inhibitor drug.
  • the method includes providing a biological sample from a subject having cancer and testing the biological sample for expression of any one of the genes listed in Tables 1, 2 and 5, or their gene products, thereby to predict an increased likelihood of response to the Eg5 inhibitor drug.
  • the method includes testing the biological sample for at least 5, 10, 20, 30, or 40 of the genes listed in Tables 1-5.
  • the biological sample can be any tumor sample such as leukaemia, multiple myeloma, lymphoma, bile duct, bone, bladder, brain, CNS, glioblastoma, breast, colorectal, cervical, endometrial, gastric, head, neck, hepatic, lung, muscle, neuronal, oesophageal, ovarian, pancreatic, pleural membrane, peritoneal membrane, prostate, renal, skin, testicular, thyroid, uterine and vulval.
  • tumor sample such as leukaemia, multiple myeloma, lymphoma, bile duct, bone, bladder, brain, CNS, glioblastoma, breast, colorectal, cervical, endometrial, gastric, head, neck, hepatic, lung, muscle, neuronal, oesophageal, ovarian, pancreatic, pleural membrane, peritoneal membrane, prostate, renal, skin, testicular, thyroid, uter
  • Expression of the gene can be determined using any known method in the art such as determining its transcript level or determining for the level of production of its gene product, for example, the protein.
  • the invention includes a method of selecting a mammal having or suspected of having a tumor for treatment with an Eg5 inhibitor drug which includes testing a biological sample from the mammal for increased expression of any one of the genes listed in Table 3.
  • the invention includes a method of selecting a mammal having or suspected of having a tumor for treatment with an Eg5 inhibitor drug which comprises testing a biological sample from the mammal for decreased expression of any one of the genes listed in Table 4.
  • the invention includes a method of determining if a tumor is responsive to an Eg5 inhibitor including providing a tumor sample from a test mammal; and determining for the presence of a particular expression profile of two, three, four, five or more of the genes listed in Tables 1-5, or their gene products, whereby a particular expression profile is indicative that the tumor is responsive to treatment with an Eg5 inhibitor.
  • the invention further includes a kit for determining if a tumor is responsive for treatment with an Eg5 inhibitor.
  • the kit can include one or more probes or primers for detecting the expression level of one or more genes as listed in Tables 1-5.
  • the Eg5 inhibitor can be any known Eg5 inhibitor.
  • the Eg5 inhibitor can be any molecule such as a peptidomimetic, protein, peptide, nucleic acid, small molecule, an antibody or other drug candidate.
  • Known small molecule Eg5 inhibitors include those disclosed in patent applications WO200130768, WO03039460, WO0349527, WO03049678, WO03049679, WO03050064, WO03050122, WO0278639, WO-02079169, WO-02079149, WO-03099286, WO2004/078758 and WO-2004092147.
  • Other Eg5 inhibitors have been reported in the literature including Mayer et al., Science.
  • Eg5 inhibitors include antisense oligonucleotides as disclosed in WO-00107602 and WO-03030832. The inhibitors listed in the publications above are incorporated herein by reference. The methods of the invention can be used to determine if a mammal is responsive to
  • the response can include biological or cellular, or clinical response.
  • a cellular response includes growth inhibition of the cell. Methods to determine growth inhibition are known in the art, as described in Boyd et al, In Cytotoxic Anticancer Drugs: Models and Concepts for Drug Discovery and Development; 1992.
  • a clinical response can be defined by disease stabilization, tumor shrinkage, prolonged survival of the mammal, etc.
  • Figure 1 is a histogram of logl0(GI50)s for 60 tumor cell lines treated by an Eg5 inhibitor.
  • Figure 2 depicts two-way clustering diagram based on the gene expression pattern of 7095 genes inNCI60 cell lines and 40 tumor samples.
  • the present invention is particularly suitable for use in predicting the response of a mammal having a tumor to an Eg5 inhibitor drug.
  • the present invention provides one or more biomarkers which can be used to determine whether the tumor is likely to be responsive to the treatment of an Eg5 inhibitor.
  • a biomarker can be a gene or gene product whose pattern of expression, optionally together with other genes or gene products, can be used to determine whether the tumor is likely to respond to an Eg5 inhibitor.
  • the gene or gene product is up- or down-regulated as an indicator that the cell will be responsive to treatment with an Eg5 inhibitor compared to a control.
  • the biomarker(s) of the invention include one or more genes listed in Tables 1-5, or their gene products.
  • the present invention is based on the finding that the biomarkers listed in Table 1-5 are differentially expressed in Eg5 inhibitor sensitive and resistant tumor cells.
  • the expression profile can be a set of values representing mRNA levels of one or more genes listed in Table 1-5 or can include a set of values representing one or more protein or polypeptide encoded by the genes listed in Table 1-5 levels in a cell.
  • the expression levels of particular genes can be positively or negatively correlated with whether or not the tumor will respond to treatment with an Eg5 inhibitor (See Tables 3 and 4).
  • the tumor can be a non-solid tumor such as leukaemia, multiple myeloma or lymphoma, or can be a solid tumor, for example bile duct, bone, bladder, brain/CNS, breast, colorectal, cervical, endometrial, gastric, head and neck, hepatic, lung, muscle, neuronal, oesophageal, ovarian, pancreatic, pleural/peritoneal membranes, prostate, renal, skin, testicular, thyroid, uterine and vulval tumors. Detection of a biomarker
  • the method includes determining from a tumor sample of a test cancer mammal for an expression profile which is indicative that the tumor is responsive to Eg5 inhibitor treatment.
  • the present invention includes determining for the level of expression of the genes in Table 1, 2 or 5 in the test tumor sample.
  • the gene profile obtained is compared against controls, i.e., expression patterns, which is indicative that a tumor is responsive to Eg5 treatment.
  • the nucleic acid sequences of the biomarker s referred to in Table 1-5 are provided in Appendix A.
  • the gene sequences of each of the biomarkers listed in Tables 1-5 can be detected using agents that can be used to specifically detect the gene or other biological molecules relating to it, for example, RNA transcribed from the gene or polypeptides encoded by the gene.
  • Exemplary detection agents are nucleic acid probes, which hybridize to nucleic acids corresponding to the gene, and antibodies.
  • biomarkers listed in Tables 1-5 are intended to also include naturally occurring sequences including allelic variants and other family members.
  • the biomarkers of the invention also include sequences that are complementary to those listed sequences resulting from the degeneracy of the code and also sequences that are sufficiently homologous and sequences which hybridize under stringent conditions to the genes listed in Tables 1-5. Conditions for hybridization are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6.
  • a preferred, non-limiting example of highly stringent hybridization conditions are hybridization in 6 X sodium chloride/sodium citrate (SSC) at about 45 0 C, followed by one or more washes in 0.2 X SSC, 0.1% SDS at 50-65 0 C.
  • SSC sodium chloride/sodium citrate
  • a sufficient or minimum number of identical or equivalent e.g., an amino acid residue which has a similar side chain
  • amino acid residues or nucleotides to a second amino acid or nucleotide sequence such that the first and second amino acid or nucleotide sequences share common structural domains or motifs and/or a common functional activity.
  • amino acid or nucleotide sequences which share common structural domains have at least about 50% homology, preferably 60% homology, more preferably 70%- 80%, and even more preferably 90-95% homology across the amino acid sequences of the domains and contain at least one and preferably two structural domains or motifs, are defined herein as sufficiently homologous.
  • amino acid or nucleotide sequences which share at least 50%, preferably 60%, more preferably 70-80% or 90-95% homology and share a common functional activity are defined herein as sufficiently homologous.
  • the comparison of sequences and determination of percent homology between two sequences can be accomplished using a mathematical algorithim.
  • a preferred, non-limiting example of a mathematical algorithim utilized for the comparison of sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-68, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-77. Such an algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. MoI. Biol. 215:403-10.
  • Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Research 25(17):3389-3402.
  • the default parameters of the respective programs e.g., XBLAST and NBLAST
  • the invention provides a list of genes or gene products that can be used to produce an expression profile which is characteristic of the efficacy of an Eg5 compound in a tumor cell. Any method known in the art can be used to determine whether a tumor cell is responsive to treatment with an Eg5 inhibitor.
  • the method comprises determining mRNA and/or protein level of the biomarkers of a mammal, such as by Northern blot analysis, reverse transcription- polymerase chain reaction (RT-PCR), in situ hybridization, immunoprecipitation, Western blot hybridization, or immunohistochemistry.
  • RT-PCR reverse transcription- polymerase chain reaction
  • cells may be obtained from a subject and the levels of the biomarker's protein or mRNA level are determined and compared to a control.
  • the method comprises using a nucleic acid probe to determine whether a mammal is responsive to Eg5 inhibition.
  • the method includes: providing a nucleic acid probe comprising a nucleotide sequence, for example, at least
  • the methods of the invention include determining expression profiles with microarrays involves the following steps: (a) obtaining a mRNA sample from a subject and preparing labeled nucleic acids therefrom (the "target nucleic acids" or "targets");
  • the method of analyzing the results can include measuring the levels of at least one of the genes in Tables 1-5, wherein the difference compared to control values indicates that the mammal is responsive to Eg5 inhibition.
  • the control for comparison can be determined by one skilled in the art.
  • the control is determined by choosing a value that serves as a cut-off value.
  • the value can be a value that differenciates between those biological samples that are responsive to an Eg5 inhibitor and those that are unresponsiveness to the inhibitor.
  • the difference between the measured value in the test sample and the control value can be used to indicate whether the test sample will respond to an Eg5 inhibitor.
  • control value can be a value predetermined according to a mathematical model.
  • the model also called a classifier, can be built by using the expression levels of in two or more genes listed in Tables 1-5 from a group of Eg5 responsive and non- responsive samples.
  • the mathematical model can be, for example, any class prediction method or its variations (references:Fisher, R.A. (1998). Annals of Eugenics 7, 179-188; Golub et al., Science 286, 531-537; Duda et al., (2000) Pattern Classification, 2ed. John Wiley & Sons, Inc.,New York, NY; Fukunaga, K. (1990) Introduction to Statistical Pattern Recognition, 2ed.
  • the method includes obtaining mRNA from the mammal's tumor sample.
  • RNA may be extracted from tissue or cell samples by a variety of methods, for example, guanidium thiocyanate lysis followed by CsCl centrifugation (Chirgwin, et al., Biochemistry 18:5294-5299, 1979).
  • RNA from single cells may be obtained as described in methods for preparing cDNA libraries from single cells (see, e.g., Dulac, Curr. Top. Dev. Biol. 36:245, 1998; Jena, et al., J. Immunol. Methods 190:199, 1996).
  • RNA sample can be further enriched for a particular species.
  • poly(A)+ RNA may be isolated from an RNA sample.
  • poly -T oligonucleotides may be immobilized on a solid support to serve as affinity ligands for mRNA. Kits for this purpose are commercially available, for example, the MessageMaker kit (Life Technologies, Grand Island, N. Y.).
  • the RNA population may be enriched for sequences of interest, as detailed on Tables 1-5. Enrichment may be accomplished, for example, by primer-specific cDNA synthesis, or multiple rounds of linear amplification based on cDNA synthesis and template-directed in vitro transcription (see, e.g., Wang, et al., Proc. Natl. Acad. Sci. USA 86:9717, 1989; Dulac, et al., supra; Jena, et al., supra).
  • the target molecules may be labeled to permit detection of hybridization of the target molecules to a microarray. That is, the probe may comprise a member of a signal producing system and thus, is detectable, either directly or through combined action with one or more additional members of a signal producing system.
  • directly detectable labels include isotopic and fluorescent moieties incorporated, usually by a covalent bond, into a moiety of the probe, such as a nucleotide monomeric unit (e.g., dNMP of the primer), or a photoactive or chemically active derivative of a detectable label which can be bound to a functional moiety of the probe molecule.
  • Nucleic acids may be labeled during or after enrichment and/or amplification of RNAs.
  • reverse transcription may be carried out in the presence of a dNTP conjugated to a detectable label, for example, a fluorescently labeled dNTP.
  • the cDNA or RNA probe may be synthesized in the absence of detectable label and may be labeled subsequently, for example, by incorporating biotinylated dNTPs or rNTP, or some similar means (e.g., photo-cross-linking a psoralen derivative of biotin to RNAs), followed by addition of labeled streptavidin (e.g., phycoerythrin-conjugated streptavidin) or the equivalent.
  • biotinylated dNTPs or rNTP or some similar means (e.g., photo-cross-linking a psoralen derivative of biotin to RNAs), followed by addition of labeled streptavidin (e.g., phycoerythrin-conjugated streptavidin) or the equivalent.
  • streptavidin e.g., phycoerythrin-conjugated streptavidin
  • Fluorescent moieties or labels of interest include coumarin and its derivatives (e.g., 7- amino-4-methylcoumarin, aminocoumarin); bodipy dyes such as Bodipy FL and cascade blue; fluorescein and its derivatives (e.g., fluorescein isothiocyanate, Oregon green); rhodamine dyes (e.g., Texas red, tetramethylrhodamine); eosins and erythrosins; cyanine dyes (e.g., Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7); FluorX, macrocyclic chelates of lanthanide ions (e.g., quantum dye.TM.); fluorescent energy transfer dyes such as thiazole orange-ethidium heterodimer, TOTAB, dansyl, etc.
  • bodipy dyes such as Bodipy FL and cascade blue
  • fluorescein and its derivatives e.g., fluorescein isothiocyanate, Oregon
  • Chemiluminescent labels include luciferin and 2,3-dihydrophthalazinediones, for example, luminol.
  • Labels may also be members of a signal producing system that act in concert with one or more additional members of the same system to provide a detectable signal.
  • Illustrative of such labels are members of a specific binding pair, such as ligands, for example, biotin, fluorescein, digoxigenin, antigen, polyvalent cations, chelator groups and the like.
  • Members may specifically bind to additional members of the signal producing system, and the additional members may provide a detectable signal either directly or indirectly, for example, an antibody conjugated to a fluorescent moiety or an enzymatic moiety capable of converting a substrate to a chromogenic product (e.g., alkaline phosphatase conjugate antibody and the like).
  • the target nucleic acid may not be labeled.
  • hybridization may be determined, for example, by plasmon resonance (see, e.g., Thiel, et al., Anal. Chem. 69:4948, 1997).
  • Microarrays for use according to the invention include one or more probes of genes listed in Tables 1-5.
  • the microarray comprises one or more probes corresponding to one or more of genes selected from the group consisting of genes that are up-regulated in a tumor cell that is responsive to treatment with an Eg5 inhibitor and genes that are down-regulated in a tumor cell that is responsive to treatment with an Eg5 inhibitor (See Tables 3 and 4 respectively).
  • the method described above results in the production of hybridization patterns of labeled target nucleic acids on the array surface.
  • the resultant hybridization patterns of labeled nucleic acids may be visualized or detected in a variety of ways, with the particular manner of detection selected based on the particular label of the target nucleic acid.
  • Representative detection means include scintillation counting, autoradiography, fluorescence measurement, calorimetric measurement, light emission measurement, light scattering, and the like.
  • One such method of detection utilizes an array scanner that is commercially available (Affymetrix, Santa Clara, Calif.), for example, the 417.TM. Arrayer, the 418.TM. Array Scanner, or the Agilent GeneArray.TM. Scanner.
  • This scanner is controlled from a system computer with an interface and easy-to-use software tools. The output may be directly imported into or directly read by a variety of software applications. Scanning devices are described in, for example, U.S. Pat. Nos. 5,143,854 and 5,424,186.
  • Proteins Detecting for the presence of a protein product encoded by one or more of the biomarker genes listed in Tables 1 -5 can be done by using any appropriate method known in the art.
  • an agent of interest that can be used to detect a particular protein of interest, for example using an antibody.
  • the method for producing polyclonal and/or monoclonal antibodies that specifically bind to polypeptides useful in the present invention is known to those of skill in the art and may be found in, for example, Dymecki, et al., (J. Biol. Chem. 267:4815, 1992); Boersma & Van Leeuwen, (J. Neurosci.
  • an immunoassay can be used to quantitate the levels of proteins in cell samples.
  • the invention is not limited to a particular assay procedure, and therefore, is intended to include both homogeneous and heterogeneous procedures.
  • Exemplary immunoassays that may be conducted according to the invention include fluorescence polarization immunoassay (FPIA) 5 fluorescence immunoassay (FIA), enzyme immunoassay (EIA), nephelometric inhibition immunoassay (NIA), enzyme-linked immunosorbent assay (ELISA), and radioimmunoassay (RIA).
  • FPIA fluorescence polarization immunoassay
  • FIA fluorescence polarization immunoassay
  • EIA enzyme immunoassay
  • NIA enzyme-linked immunosorbent assay
  • RIA radioimmunoassay
  • An indicator moiety, or label group may be attached to the subject antibodies and is selected so as to meet the needs of various uses of the method that are often dictated by the availability of assay equipment and compatible immunoassay procedures.
  • General techniques to be used in performing the various immunoassays noted above are known to those of ordinary skill in the art.
  • reporter molecule is a molecule that provides an analytically identifiable signal allowing one of skill in the art to identify when an antibody has bound to a protein that it is directed against. Detection may be either qualitative or quantitative. Commonly used reporter molecules include fluorophores, enzymes, biotin, chemiluminescent molecules, bioluminescent molecules, digoxigenin, avidin, streptavidin or radioisotopes.
  • Commonly used enzymes include horseradish peroxidase, alkaline phosphatase, glucose oxidase and beta-galactosidase, among others.
  • the substrates to be used with these enzymes are generally chosen for the production, upon hydrolysis by the corresponding enzyme, of a detectable color change.
  • p-nitrophenyl phosphate is suitable for use with alkaline phosphatase reporter molecules; for horseradish peroxidase, 1 ,2- phenylenediamine, 5-aminosalicylic acid or toluidine are commonly used.
  • Incorporation of a reporter molecule onto an antibody can be by any method known to the skilled artisan.
  • the presence of the marker protein in a tissue sample can be determined using immunohistochemical staining.
  • a multiblock of tissue may be taken from the biopsy or other tissue sample and subjected to proteolytic hydrolysis, employing such agents as protease K or pepsin.
  • proteolytic hydrolysis employing such agents as protease K or pepsin.
  • tissue samples are fixed by treatment with a reagent such as formalin, glutaraldehyde, methanol, or the like.
  • a reagent such as formalin, glutaraldehyde, methanol, or the like.
  • the samples are then incubated with an antibody (e.g., a monoclonal antibody) with binding specificity for the marker polypeptides.
  • This antibody may be conjugated to a label for subsequent detection of binding.
  • Samples are incubated for a time sufficient for formation of the immunocomplexes. Binding of the antibody is then detected by virtue of a label conjugated to this antibody.
  • a second labeled antibody may be employed, for example, that is specific for the isotype of the anti-marker polypeptide antibody. Examples of labels that may be employed include radionuclides, fluorescers, chemiluminescers, enzymes, and the like.
  • the substrate for the enzyme may be added to the samples to provide a colored or fluorescent product.
  • suitable enzymes for use in conjugates include horseradish peroxidase, alkaline phosphatase, malate dehydrogenase, and the like. Where not commercially available, such antibody-enzyme conjugates are readily produced by techniques known to those skilled in the art.
  • the invention contemplates using a panel of antibodies that are generated against the marker polypeptides of this invention.
  • a panel of antibodies may be used as a reliable diagnostic probe for determining if a tumor is responsive to treatment with an Eg5 inhibitor.
  • the data obtained by the reader from the device may be analyzed using a digital computer.
  • the computer will be appropriately programmed for receipt and storage of the data from the device, as well as for analysis and reporting of the data gathered, for example, subtraction of the background, deconvolution of multi-color images, flagging or removing artifacts, verifying that controls have performed properly, normalizing the signals, interpreting fluorescence data to determine the amount of hybridized target, normalization of background and single base mismatch hybridizations, and the like.
  • a system comprises a search function that allows one to search for specific patterns, for example, patterns relating to differential gene expression, for example, between the expression profile of the test tumor cell and the expression profile of a tumor cell that is responsive to treatment with an Eg5 inhibitor.
  • a system may also allow one to search for patterns of gene expression between more than two samples. Comparison of the expression levels of one or more genes characteristic of responsiveness to an Eg5 inhibitor with reference expression levels, for example, expression levels that are characteristic of susceptibility to an Eg5 inhibitor may be conducted using computer systems.
  • kits for determining the expression level of genes characteristic of tumors that are responsive to Eg5 inhibitor treatment may be useful for identifying subjects that are responsive to Eg5 inhibitor treatment.
  • the kit comprises a computer readable medium on that is stored one or more gene expression profiles or values representing levels of expression of one or more genes characteristic of susceptibility to Eg5 inhibitor treatment.
  • the kit can comprise expression profile analysis software capable of being loaded into the memory of a computer system.
  • a kit can comprise a microarray comprising probes of genes characteristic of susceptibility to Eg5 treatment.
  • a kit can comprise one or more probes or primers for detecting the expression level of one or more genes as listed in Tables 1-5 and/or a solid support on that probes attached and that can be used for detecting expression of one or more genes characteristic of responsiveness to an Eg5 inhibitor.
  • a kit may further comprise nucleic acid controls, buffers, and instructions for use.
  • the genes identified in Table 1 may be a determining factor in the pathogenesis of a particular cancer.
  • methods to detect higher or lower levels of gene expression in the diseased tissue compared to normal tissue may suggest that the gene, or its gene product plays a role in disease progression.
  • agonists or antagonists against the gene or its gene product might be useful therapeutically in treating, preventing, and/or ameliorating the cancer. Methods of identifying antagonists or agonist of a gene of interest are well known in the art.
  • the method can include exposing a subject having tumor cells in that marker mRNA or protein is detectable to an agent suspected of inhibiting or enhancing production of marker mRNA or protein; and determining the level of marker mRNA or protein in tumor cells of the exposed mammal.
  • a decrease in the level of marker mRNA or protein after exposure of the subject to the agent is indicative of inhibition of marker nucleic acid expression and an increase in the level of marker mRNA or protein is indicative of enhancement of marker nucleic acid expression.
  • the biological function of the encoded polypeptide may be determined by disrupting a homologue of this polypeptide in mice and/or rats and observing the resulting phenotype. Such knock-out experiments are known in the art.
  • the biological function of the proteins encoded by the genes listed in Tables 1-5 can be determined by the application of antisense and/or sense methodology and the resulting generation of transgenic mice and/or rats.
  • the gene can be either over-expressed, under expressed or knocked out, in a cell of a mouse tumor model and the progression of the tumor monitored.
  • DTP Therapeutic Program of the National Cancer Institute (NCI) to screen compounds for potential anti-cancer activities. Included among the 60 are leukemias, melanomas, and cancers of ovarian, breast, prostate, lung, renal, colon, and central nervous system origin.
  • Affymetrix http://www.affymetrix.com
  • GeneChip human genome U133 set from GeneLogic's GeneExpress (Gene Logic Inc. Gaithersburg, Maryland) database.
  • Affymetrix GeneChip human genome U133 set The GeneChip human genome Ul 33 set from Affymetrix are comprised of, on two probe arrays U133A and U133B, more than 45,000 probesets and 1,000,000 distinct oligonucleotide features, that represent over 39,000 transcripts and variants, including over 33,000 well- substantiated human genes. Oligonucleotide probes were synthesized in situ on the array. Each probeset consists of eleven pairs of such oligonucleotide probes, and is used to measure the level of transcription of each sequence represented on the GeneChip Human Genome U133 Set (HG-U133 Set).
  • Step 1 Target Preparation
  • Double-stranded cDNA was synthesized from total RNA isolated from cells. An in vitro transcription (IVT) reaction was then done to produce biotin-labeled cRNA from cDNA. The cRNA was then fragmented ready for hybridization. Step 2: Target Hybridization
  • a hybridization cocktails, including the fragmented target from step I 5 was prepared. It was then hybridized to probe array during a 16-hour incubation.
  • Step 3 Washing and Staining Immediately following hybridization, the probe array underwent an automated washing and staining protocol.
  • the stored image file was analyzed for probe intensities and results are reported.
  • Expression values (Average Diff) of U133 probesets were extracted from Gene Logic database for all NCI60 cell lines.
  • This group of tumor samples include 3 leukemia samples, 8 breast cancer samples, 4 colon cancer samples, 7 kidney cancer samples, 7 lung cancer samples, 4 ovary cancer samples, 3 prostate cancer samples, 3 skin cancer samples, and 1 small intestine cancer samples.
  • NCI60 cell line responses to Eg5 inhibition A human Eg5 modulator compound was submitted to DTP's anti-cancer screening program. Its anti-cancer activities against NCI60 cell lines, measured as GI50 (50% growth inhibition), were reported back (table 1).
  • NCI screening procedures can be performed as described in Monks et al. JNC/, J. Natl Cancer Inst. 1991, 83, 757-766. Briefly, cell suspensions were diluted to provide a target cell density per well based on cell growth characteristics. The cells were added by pipet (100 ⁇ L) into 96-well microtiter plates. Inoculates were allowed a preincubation period of 24 h at 37° C for stabilization. At the time of drug addition, aliquots of 100 ⁇ l of five different anti-Eg5 compound dilutions plus control were added to the appropriate microtiter wells already containing 100 ⁇ l of medium, resulting in the required final drug concentrations.
  • the compound was incubated with the cells for 48 hours in 5% CO2 atmosphere and 100% humidity.
  • the cells were then assayed by using the sulforhodamine B assay (Rubinstein et al., JNC/, J. Natl. Cancer Inst. 1990, 82, 1107-1112).
  • a plate reader was used to read the optical densities, and a microcomputer processed the optical densities into the special concentration parameters defined as GI50, TGI, and LC50.
  • T is the optical density of the test well after a 48-h period of exposure to the Eg5 inhibitor
  • TO is the optical density at time zero
  • C is the control optical density
  • Log 10 values of the GI50 results from the ⁇ CI60 cell lines using a human Eg5 inhibitor are shown in Table A.
  • 9 of them (COLO 205, HCT-116, K-562, MDA-N, NCI-H23, SK-OV-3, SNB-19, SR, T47D) had no GI50 value and were excluded from the analysis.
  • the histogram of loglO(GI50)s for Eg5 inhibitor shows that there are two separate mode.
  • the mean and standard deviation of loglO(GI50)s across cell lines were calculated.
  • Cell lines were classified as Eg5 inhibitor resistant if their loglO(GI50)s are above 0.8 standard deviation of the mean (-6.7) and as sensitive otherwise. 39 cell lines are thus defined as sensitive, and 12 cell lines as resistant (shown in table A).
  • SVM Supporting Vector Machine
  • sample classification and gene selection are two tightly correlated problems in microarray based data analysis. Given the large number of genes monitored by a microarray study and usually limited samples available, gene selection can reduce the risk of overfitting classification model, i.e. the classification model can work better on samples it has not been trained with, and improve the classification accuracy measured by error rate. More importantly, gene selection will help to find genes directly related to the problem under study. The predictive classifier can then be built based on the features selected.
  • RSVM recursive supporting vector machine
  • Gene expression profile clustering of 41 lung cancer cell lines and 43 tumors produced a dendrogram with cell lines and tumor samples falling into two distinct groups. Only after removing genes that are differentially expressed between cell lines and tumors, clustering analysis integrated cell lines and tumors and produced major branches consistent with pathological type.
  • Our training set contains 12 resistant cell lines and 11 sensitive cell line.
  • the resistant cell lines were defined as described in B3, which resulted in 12 resistant cell lines in training set.
  • the sensitive cell lines were selected in a way to balance the tumor types in the training sets of sensitive and resistant cell lines and to have roughly the same number of cell lines in each group to avoid overfitting problem.
  • the cell line(s) of the same tumor types that either has a GI50 ⁇ mean-0.8*std (-7.7) or the lowest GI50 were selected into the sensitive group, which resulted in 11 sensitive cell lines in the training set.
  • RSVM procedure as described in C2. was used to select 26 genes (Table 2.) that had a prediction error rate of 35% based on leave-one-out cross validation of the training set. Table 1. 57 genes that can predict cell line and/or primary tumor response to Eg5 inhibition.
  • Table 2 26 genes from table 1 that can predict cell line and primary tumor response to Eg5 inhibition
  • Table 3 9 genes from table 1 that have higher expressions in Eg5 inhibition sensitive cell lines and lower expressions in Eg5 resistant cell lines.
  • Table 4 48 genes from table 1 that have higher expressions in Eg5 inhibition resistant cell lines and lower expressions in Eg5 sensitive cell lines.

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Abstract

L'invention concerne un procédé destiné à sélectionner un mammifère atteint ou supposé être atteint d'une tumeur en vue d'un traitement au moyen d'un médicament inhibiteur d'Eg5. Ce procédé consiste à fournir un échantillon biologique d'un sujet atteint du cancer et à tester cet échantillon pour l'expression de n'importe quel gène énuméré dans les tableaux 1, 2 et 5, ou leurs produits géniques, afin de prédire ainsi une probabilité augmentée de réaction au médicament inhibiteur d'Eg5.
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2106451A4 (fr) * 2006-12-04 2010-12-15 Abbott Lab Tests de diagnostic d'accompagnement pour un traitement du cancer
WO2011009523A1 (fr) * 2009-07-24 2011-01-27 Merck Patent Gmbh Utilisation de l’eg5 comme biomarqueur dans les tumeurs thoraciques rares
US11769591B2 (en) * 2018-07-06 2023-09-26 Koninklijke Philips N.V. Computer aided diagnosis and monitoring of heart failure patients

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2006521794A (ja) * 2003-02-14 2006-09-28 スミスクライン・ビーチャム・コーポレイション Ｋｓｐ発現と相関関係にある示差発現核酸

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2106451A4 (fr) * 2006-12-04 2010-12-15 Abbott Lab Tests de diagnostic d'accompagnement pour un traitement du cancer
WO2011009523A1 (fr) * 2009-07-24 2011-01-27 Merck Patent Gmbh Utilisation de l’eg5 comme biomarqueur dans les tumeurs thoraciques rares
US11769591B2 (en) * 2018-07-06 2023-09-26 Koninklijke Philips N.V. Computer aided diagnosis and monitoring of heart failure patients

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