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WO2006122053A2 - Biomarqueurs permettant d'evaluer la probabilite selon laquelle une tumeur est sensible a un inhibiteur de mtor - Google Patents

Biomarqueurs permettant d'evaluer la probabilite selon laquelle une tumeur est sensible a un inhibiteur de mtor Download PDF

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Publication number
WO2006122053A2
WO2006122053A2 PCT/US2006/017770 US2006017770W WO2006122053A2 WO 2006122053 A2 WO2006122053 A2 WO 2006122053A2 US 2006017770 W US2006017770 W US 2006017770W WO 2006122053 A2 WO2006122053 A2 WO 2006122053A2
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Prior art keywords
protein
tumor
level
mtor inhibitor
fkbp
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PCT/US2006/017770
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WO2006122053A3 (fr
Inventor
Camille L. Bedrosian
Timothy P. Clackson
Victor Rivera
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Ariad Gene Therapeutics Inc
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Ariad Gene Therapeutics Inc
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Priority to US11/913,964 priority Critical patent/US20090215812A1/en
Priority to EP06759344A priority patent/EP1899463A4/fr
Publication of WO2006122053A2 publication Critical patent/WO2006122053A2/fr
Anticipated expiration legal-status Critical
Publication of WO2006122053A3 publication Critical patent/WO2006122053A3/fr
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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
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • 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
    • 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
    • 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

  • cancer remains a highly lethal disease. Although notable successes have been achieved in treating some cancers such as childhood leukemia and Hodgkin's lymphoma, options for treating many types of cancer remain far from satisfactory. In many cases, the mainstays of therapy remain surgery, radiation, and conventional chemotherapeutic agents that exhibit nonspecific cytostatic or cytotoxic activity, particularly against rapidly dividing cells. As a result, side effects are a significant issue and limit the maximum tolerated dose and thus the efficacy of these agents.
  • mTOR mimmalian target of rapamycin, also known as FKBP-rapamycin associated protein, or FRAP
  • FKBP-rapamycin associated protein FKBP-rapamycin associated protein
  • FRAP FKBP-rapamycin associated protein
  • the present invention provides a variety of methods and reagents for evaluating the likelihood that a tumor is sensitive to an mTOR inhibitor.
  • the invention is related in part to the discovery that the expression level of FKBP 12 protein varies significantly among tumor cell lines derived from a variety of different tumor types and that the level of FKBP 12 expression correlates with the sensitivity of the tumor cell lines to an mTOR inhibitor.
  • tumor cell lines having lower FKBP 12 expression levels exhibited reduced sensitivity to treatment with an mTOR inhibitor relative to tumor cell lines with higher FKBP 12 expression.
  • the invention therefore establishes FKBP 12 as a biomarker for assessing the likelihood that a tumor is sensitive to an mTOR inhibitor and for assessing the likelihood that a subject suffering from a tumor will respond to treatment with an mTOR inhibitor.
  • the invention provides a method for evaluating the likelihood that a tumor is sensitive to an mTOR inhibitor comprising a step of assessing the expression level or activity of an FKBP protein in a sample derived from a subject in need of such evaluation, wherein the subject has a tumor, or assessing an indicator of the expression or activity of the FKBP protein in the tumor in vivo, wherein the value of the indicator, relative to a reference value for the indicator, is predictive of the likelihood that the tumor is sensitive to the mTOR inhibitor.
  • the FKBP protein is FKBP 12.
  • the invention is also related to the identification of a variety of additional biomarkers that are of use individually, in conjunction with one another, and/or in conjunction with FKBP expression or activity level for assessing the likelihood that a tumor is sensitive to an mTOR inhibitor and for assessing the likelihood that a subject suffering from a tumor will respond to treatment with an mTOR inhibitor.
  • the method for evaluating the likelihood that a tumor is sensitive to an mTOR inhibitor comprises the further step of assessing at least one additional indicator of the likelihood that the tumor is sensitive to an mTOR inhibitor, wherein the value of the indicator, relative to a reference value for the indicator, is predictive of the likelihood that the tumor is sensitive to the mTOR inhibitor.
  • the at least one additional indicator is preferably selected from the group consisting of: (a) the proportion or level of TSC2 protein that is phosphorylated; (b) the proportion or level of AKT protein that is phosphorylated; (c) the proportion or level of S6 protein that is phosphorylated; (d) the proportion or level of S6 kinase protein that is phosphorylated; (e) the proportion or level of 4E-BP1 protein that is phosphorylated; (f) the proportion or level of mTOR protein that is phosphorylated; (g) the level of cyclin Dl mRNA or protein, cyclin D 3 mRNA or protein, myc mRNA or protein, or any combination of these (h) the level of HIF-I ⁇ mRNA or protein, HIF -2q mRNA or protein; HIF-I ⁇ mRNA or protein, or any combination of the foregoing; (i) the level of VHL mRNA or protein or a mutation affecting VHL expression or activity; (j)
  • the additional indicator is obtained by performing one or more functional imaging studies.
  • one or more indicators is assessed prior to and following administration of an mTOR inhibitor, and an alteration in the value of the indicator is indicative of the likelihood that the tumor is sensitive to an mTOR inhibitor.
  • the invention provides a method of evaluating the likelihood that a subject with a tumor will exhibit a favorable response to treatment with an mTOR inhibitor.
  • the method comprises the step of evaluating the likelihood that the tumor is sensitive to an mTOR inhibitor by assessing the expression level or activity of an FKBP protein in a sample derived from the tumor, or assessing an indicator of the expression or activity of the FKBP protein in the tumor in vivo, wherein the value of the indicator, relative to a reference value for the indicator, is predictive of the likelihood that the tumor is sensitive to the mTOR inhibitor.
  • the FKBP protein is FKBP 12.
  • the method comprises the further step assessing at least one additional indicator of the likelihood that the tumor is sensitive to an mTOR inhibitor, wherein the value of the indicator, relative to a reference value for the indicator, is predictive of the likelihood that the tumor is sensitive to the mTOR inhibitor.
  • the invention provides a method of selecting a subject for treatment with an mTOR inhibitor, wherein the subject has a tumor. The method comprises the step of evaluating the likelihood that the tumor is sensitive to an mTOR inhibitor by assessing any of a variety of indicators, and/or evaluating the likelihood that the subject will exhibit a favorable response to the mTOR inhibitor by assessing any of a variety of indicators.
  • the invention provides a method of selecting an appropriate therapeutic agent for a subject with a tumor.
  • the invention further provides a method for treating a subject suffering from a tumor, the method comprising the steps of: (a) evaluating the likelihood that the subject will exhibit a favorable response to an mTOR inhibitor by a method that comprises evaluating the likelihood that the tumor is sensitive to an mTOR inhibitor by assessing one or more of a variety of indicators and (b) administering an mTOR inhibitor to the subject.
  • the invention provides a variety of kits.
  • the invention provides a kit for evaluating the likelihood that a tumor is sensitive to an mTOR inhibitor, the kit comprising a reagent for assessing an indicator of the expression or activity of an FKBP protein in a sample obtained from a subject with a tumor, wherein the reagent has been demonstrated to be of use in evaluating the likelihood that a tumor is sensitive to an mTOR inhibitor.
  • the invention further provides a kit comprising a reagent that specifically binds to an FKBP protein, e.g., FKBP 12 and (b) an additional item selected from the group consisting of: (a) an antibody that specifically binds to a protein selected from the group consisting of: mTOR, PTEN, Akt, Raptor, G ⁇ L, or any other component of a pathway involving mTOR.
  • a reagent that specifically binds to an FKBP protein e.g., FKBP 12
  • an additional item selected from the group consisting of: (a) an antibody that specifically binds to a protein selected from the group consisting of: mTOR, PTEN, Akt, Raptor, G ⁇ L, or any other component of a pathway involving mTOR.
  • at least one of the antibodies is specific for a phosphorylated form of the protein.
  • the invention further provides automated staining instruments of use in evaluating the likelihood that a tumor is sensitive to an mTOR inhibitor.
  • the invention further provides computer-readable medium on which is stored information related to samples, tumors, and/or subjects, the value of indicators in said samples, tumors, and/or subjects, and, optionally, the sensitivity or responsiveness of such tumors and/or subjects to administration of an mTOR inhibitor.
  • the invention makes use of standard methods of molecular biology, cell culture, flow cytometry, histology, immunology, immunohistochemistry, etc. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
  • Figure 1 shows a Western blot of FKBP 12 protein expression in a panel of tumor cell lines.
  • Antibody refers to an immunoglobulin that binds to an antigen.
  • An antibody may be natural or wholly or partially synthetically produced.
  • An antibody may be derived from natural sources, e.g., purified from an animal such as a rodent, rabbit, or chicken, that has been immunized with an antigen or a construct that encodes the antigen.
  • An antibody may be a member of any immunoglobulin class, including any of the human classes: IgG 5 IgM, IgA, IgD, and IgE.
  • the antibody may be an antibody fragment such as an Fab', F(ab') 2 , scFv (single-chain variable) or other fragment that retains an antigen binding site, or a recombinantly produced scFv fragment, including recombinantly produced fragments that comprise an immunoglobulin antigen binding domain.
  • an antibody fragment such as an Fab', F(ab') 2 , scFv (single-chain variable) or other fragment that retains an antigen binding site, or a recombinantly produced scFv fragment, including recombinantly produced fragments that comprise an immunoglobulin antigen binding domain.
  • Antibodies, antibody fragments, and/or protein domains comprising an antigen binding site may be generated and/or selected in vitro, e.g., using techniques such as phage display (Winter, G. et all 994. Annu, Rev. Immunol.
  • An antibody may be a "humanized” antibody in which for example, a variable domain of rodent origin is fused to a constant domain of human origin, thus retaining the specificity of the rodent antibody.
  • the domain of human origin need not originate directly from a human in the sense that it is first synthesized in a human being. Instead, "human" domains may be generated in rodents whose genome incorporates human immunoglobulin genes.
  • An antibody may be polyclonal (e.g., an affinity-purified polyclonal antibody) or monoclonal.
  • the terms “approximately” or “about” in reference to a number are generally include numbers that fall within a range of 5% in either direction (greater than or less than) of the number unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).
  • the terms “assess”, “assessing”, and the like are understood broadly and include obtaining information, e.g., determining a value, whether through direct examination or by receiving information from another party that performs the examination.
  • biomarker is used herein as understood in the art.
  • the term can refer to an indicator that provides information about a process, phenotype, or outcome of interest, e.g., the sensitivity of a tumor to a chemotherapeutic agent, or the response of a subject to a chemotherapeutic agent. In general, the value of such an indicator is correlated with the process, phenotype, or outcome of interest.
  • biomarker can also refer to a molecule that is the subject of an assay or measurement the result of which provides information about a process, phenotype, or outcome of interest.
  • an elevated expression level of a particular protein can be an indicator that a subject has a disease.
  • the expression level of the protein, an elevated expression level of the protein, and the protein itself can all be referred to as "biomarkers”.
  • an "effective amount" of an active agent refers to the amount of the active agent sufficient to elicit a desired biological response, e.g., to treat or prevent a disease or condition in a subject.
  • a desired biological response e.g., to treat or prevent a disease or condition in a subject.
  • the absolute amount of a particular agent that is effective may vary depending on such factors as the desired biological endpoint, the agent to be delivered, the target tissue, etc.
  • an "effective amount” may be administered in a single dose, or may be achieved by administration of multiple doses.
  • an effective amount may reduce the growth rate of the tumor (e.g., reduce the proliferation rate of tumor cells), reduce or prevent local spread of the tumor, reduce or prevent invasion of other organs, reduce or prevent metastasis, alleviate one or more symptoms associated with the tumor, etc.
  • An effective amount of a therapeutic agent for a tumor may kill tumor cells (i.e., may be cytotoxic) or may prevent their further proliferation (i.e., may be cytostatic).
  • An effective amount of a therapeutic agent may be an amount sufficient to elicit a partial or complete response, cause stabilization of disease, increase time to progression, etc.
  • “Expression” refers to the process by which a polynucleotide is transcribed to produce mRNA (mRNA expression) and by which the mRNA is translated to produce a polypeptide (protein expression). Expression may include processing, such as splicing of an mRNA.
  • gene has its meaning as understood in the art.
  • a gene is understood to include gene regulatory sequences (e.g., promoters, enhancers, etc.) and/or intron sequences and typically also includes a sequence that encodes a polypeptide.
  • gene regulatory sequences e.g., promoters, enhancers, etc.
  • intron sequences typically also includes a sequence that encodes a polypeptide.
  • a “gene” can refer to a nucleic acid that does not encode a protein but rather encodes a functional RNA molecule such as an rRNA, short hairpin RNA (shRNA), tRNA, etc.
  • a "gene product” or “expression product” is an RNA transcribed from the gene (e.g., either pre- or post-processing) or a polypeptide encoded by an RNA transcribed from the gene (e.g., either pre- or post-modification).
  • the term “hybridize” refers to the interaction between two or more nucleic acid segments comprising or consisting of complementary portions such that a complex stabilized by hydrogen bonds is formed.
  • a first and second nucleic acid segment will hybridize to each other if the Tm of a duplex formed by the first and second nucleic acid segments is less than 15°C below, preferably less than 1O 0 C below the Tm of a duplex that would be formed by the second nucleic acid and a third nucleic acid that is the same length as, and 100% complementary to, the second nucleic acid and contains nucleosides and internucleosidic linkages of the same type.
  • Hybridization conditions suitable for various applications are known in the art and are found in standard reference works, e.g., Ausubel, supra, and Sambrook, supra. Hybridization reactions can be performed under conditions of different "stringency".
  • stringent hybridization conditions comprise 6 X sodium chloride/sodium citrate (SSC) and 0.1% SDS at a temperature 10-15 0 C below the Tm of a perfectly complementary duplex, followed by washing 1-2 times for 30 minutes in 2 X SSC and 0.1% SDS at a temperature 25°C below the Tm of a perfectly complementary duplex.
  • SSC sodium chloride/sodium citrate
  • the Tm is defined as the temperature at which 50% of a nucleic acid and its perfect complement are in duplex in solution.
  • Imaging Methods for calculating or experimentally determining Tm are known in the art.
  • Imaging imaging study, imaging procedure and like terms are used consistently with their meaning in the art to refer to a process that includes producing one or more "pictures” or representations of one or more body structures or tissues (in which blood is considered a tissue), typically one or more internal body structures or tissues, including an abnormal growth such as a tumor, a foreign body, etc., that may exist within such body structure or tissue.
  • a wide variety of imaging procedures are known in the art and are commonly used for diagnostic and/or therapeutic purposes.
  • the image may reflect any of a variety of properties and/or features of the tissue or body structure including physical, anatomical, biochemical, and/or metabolic features.
  • imaging modalities include, but are not limited to, radiographic (e.g., X- rays, computed tomography (CT), magnetic resonance imaging (MRI), positron emission tomography (PET), ultrasound, scintigraphy).
  • CT computed tomography
  • MRI magnetic resonance imaging
  • PET positron emission tomography
  • Some imaging methods involve injection of small amounts of radioactively labeled (or otherwise detectably labeled) substances (e.g., radio-isotopes) into a subject.
  • the radioactive substances may concentrate directly in an organ/tissue or may be attached to a targeting agent such as an antibody.
  • An image produced by an imaging procedure may be analysed and/or interpreted visually and/or using automated image analysis.
  • indicator is used broadly herein to refer to any parameter, attribute, or characteristic of a molecule, sample, or subject that can be detected, determined, and/or quantitated to obtain a value that can be compared with another value, e.g., a reference value.
  • a value can be qualitative or quantitative and can be a range of values.
  • An indicator can be, for example, the level of expression or activity of a protein, the proportion of a protein that is phosphorylated, the presence or number of cells of a particular type, presence or absence of a mutation, a parameter obtained from an imaging study, etc.
  • inhibitor is used herein to refer to any molecule or other agent capable of inhibiting (e.g., partially or completely blocking, retarding, interfering with) one or more biological activities (e.g., a physiologically significant enzymatic activity) of a target molecule such as mTOR.
  • a target molecule such as mTOR.
  • examples include small molecules such as rapamycin and rapamycin analogs, antibodies, short interfering RNA (siRNA), short hairpin RNA (shRNA), antisense molecules, ribozymes, etc.
  • An inhibitor may inhibit synthesis of a target polypeptide (e.g., by inhibiting synthesis of, or causing destabilization of, an mRNA that encodes the polypeptide, or by inhibiting translation of the polypeptide), may accelerate degradation of the polypeptide, may inhibit activation of the polypeptide (e.g., by inhibiting an activating modification such as phosphorylation or cleavage), may block an active site of the polypeptide, may cause a conformational change in the polypeptide that reduces its activity, may cause dissociation of an active complex containing the polypeptide, etc.
  • An inhibitor may act directly by physical interaction with a target molecule, or indirectly, for example by interacting with a second molecule whose activity contributes to activation of the target molecule (e.g., a molecule that activates the target molecule, e.g., by phosophorylating it), by competing with the target molecule for binding to a substrate, activator, or binding partner needed for activity of the target molecule, etc.
  • a second molecule whose activity contributes to activation of the target molecule
  • a second molecule e.g., molecule that activates the target molecule, e.g., by phosophorylating it
  • the term "isolated” means 1) separated from at least some of the components with which it is usually associated in nature; 2) prepared or purified by a process that involves the hand of man; and/or 3) not occurring in nature.
  • operably linked refers to a relationship between two nucleic acid sequences wherein the expression of one of the nucleic acid sequences is controlled by, regulated by, modulated by, etc., the other nucleic acid sequences, or a relationship between two polypeptides wherein the expression of one of the polypeptides is controlled by, regulated by, modulated by, etc., the other polypeptide.
  • the transcription of a nucleic acid sequence is directed by an operably linked promoter sequence; post-transcriptional processing of a nucleic acid is directed by an operably linked processing sequence; the translation of a nucleic acid sequence is directed by an operably linked translational regulatory sequence; the transport, stability, or localization of a nucleic acid or polypeptide is directed by an operably linked transport or localization sequence; and the post-translational processing of a polypeptide is directed by an operably linked processing sequence.
  • nucleic acid sequence that is operably linked to a second nucleic acid sequence, or a polypeptide that is operatively linked to a second polypeptide is covalently linked, either directly or indirectly, to such a sequence, although any effective three-dimensional association is acceptable.
  • Polynucleotide or “oligonucleotide” refers to a polymer of nucleotides, typically comprising at least three nucleotides. As used herein, an oligonucleotide is typically less than 100 nucleotides in length. A polynucleotide or oligonucleotide may also be referred to as a nucleic acid. Naturally occurring nucleic acids include DNA and RNA.
  • a nucleotide comprises a nitrogenous base, a sugar molecule, and a phosphate group.
  • a nucleoside comprises a nitrogenous base linked to a sugar molecule.
  • nucleosides in their 2'-deoxy and 2'-hydroxyl forms as described in Kornberg and Baker, DNA Replication, 2nd Ed. (Freeman, San Francisco, 1992) and nucleoside analogs.
  • natural nucleosides include adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine.
  • Nucleoside “analogs” refers to synthetic nucleosides having modified base moieties and/or modified sugar moieties, e.g.
  • nucleosides designed to enhance binding properties, reduce degeneracy, increase specificity, and the like.
  • DNA or RNA naturally occurring nucleic acids
  • polynucleotides and oligonucletides containing modified backbones or non-naturally occurring internucleoside linkages can also be used in the present invention. Examples include, but are not limited to, phosphorothioate and 5'-N-phosphoramidite linkages. See U.S. Pub. No.
  • a polynucleotide may be of any size or sequence and may be single- or double-stranded.
  • DNA may be double-stranded or single-stranded, and if single-stranded may be a coding (sense) strand or non-coding (anti-sense) strand.
  • a polynucleotide may be, for example, a modified or unmodified circular plasmid, a linearized plasmid, a cosmid, a modified or unmodified viral genome, a gene or gene fragment, messenger RNA, a short interfering RNA (siRNA) or short hairpin RNA (shRNA), an antisense oligonucleotide, a ribozyme, etc.
  • the polynucleotide has been engineered using recombinant techniques (for a more detailed description of these techniques, please see Ausubel et al., Current Protocols in Molecular Biology, supra, and Molecular Cloning: A Laboratory Manual, supra.
  • a polynucleotide may be obtained from natural sources and purified from contaminating components found normally in nature.
  • a polynucleotide may be synthesized using enzymatic techniques, either within cells or in vitro.
  • the polynucleotide may also be chemically synthesized in a laboratory.
  • the polynucleotide is synthesized using standard solid phase chemistry.
  • the polynucleotide may be modified by chemical or biological means. In certain preferred embodiments, these modifications lead to increased stability of the polynucleotide. Modifications include methylation, phosphorylation, end-capping, etc.
  • a polynucleotide may be labeled, e.g., by incorporation or attachment of one or more detectable labels.
  • Polypeptide refers to a polymer of amino acids.
  • a protein is composed of one or more polypeptides.
  • a peptide is a relatively short polypeptide, typically between about 2 and 60 amino acids in length.
  • Peptide, polypeptide, or protein may refer to an individual peptide, polypeptide, or protein, or a collection thereof.
  • Polypeptides used herein preferably contain only natural amino acids, although non-natural amino acids (i.e., compounds that do not occur in nature but that can be incorporated into a polypeptide chain) and/or amino acid analogs as are known in the art may alternatively be employed. Also, one or more of the amino acids in an inventive peptide may be modified, for example, by the addition of a chemical entity such as a carbohydrate group, a phosphate group, a farnesyl group, an isofarnesyl group, a fatty acid group, a linker for conjugation, functionalization, or other modification, etc.
  • a chemical entity such as a carbohydrate group, a phosphate group, a farnesyl group, an isofarnesyl group, a fatty acid group, a linker for conjugation, functionalization, or other modification, etc.
  • the modifications of the peptide lead to a more stable peptide (e.g., greater half-life in vivo). These modifications may include cyclization of the peptide, the incorporation of D-amino acids, etc. None of the modifications should substantially interfere with the desired biological activity of the peptide.
  • Preventing refers to causing a disease, disorder, condition, or symptom or manifestation of such, or worsening of the severity of such, not to occur.
  • Purified means separated from many other compounds or entities. A compound or entity may be partially purified, substantially purified, or pure. A compound or entity is considered pure when it is removed from substantially all other compounds or entities, i.e., is preferably at least about 90%, more preferably at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99% pure.
  • a partially or substantially purified compound or entity may be removed from at least 50%, at least 60%, at least 70%, or at least 80% of the material with which it is naturally found, e.g., cellular material such as cellular proteins and/or nucleic acids.
  • the term "primer” refers to an oligonucleotide, whether natural or synthetic, that hybridizes to a nucleic acid "target” or “template” present in a sample of interest and is capable of acting as a point of initiation of nucleic acid synthesis under conditions in which primer extension, e.g., polymerase-catalyzed primer extension can occur.
  • primer extension e.g., polymerase-catalyzed primer extension can occur.
  • the appropriate length of a primer depends on the intended use of the primer, but typically ranges from about 15 to about 35 nt.
  • a primer may be longer, e.g., up to about 60 nt in length. Short primer molecules generally require cooler temperatures to form sufficiently stable hybrid complexes with a template. A primer need not be 100% complementary to the template but must be sufficiently complementary to hybridize with the template for primer elongation to occur.
  • the primer can be extended by one or more nucleotides, depending on the particular application. Primers are typically used for replication of a nucleic acid (amplification) and/or for detecting a mutation by any of a variety of methods. A primer can also be used for a variety of purposes that do not require polymerase- catalyzed polymerization. For example, a primer can be used in a reaction such as ligation.
  • a primer can also be used as a probe for detection of a complementary nucleic acid.
  • Methods for selecting primers, e.g., using the Tm value, are well known in the art and computer programs for doing so are widely available.
  • the term "probe”, when referring to a nucleic acid refers to a nucleic acid that can hybridize with and thereby detect the presence of a complementary nucleic acid The probe should be sufficiently complementary to the nucleic acid being detected so that specific hybridization can occur under the hybridization stringency conditions used.
  • the probe may comprise a label or means for attachment of a label. Suitable labels include, but are not limited to radioisotopes, fluorescent moieties, chemiluminescent compounds, dyes, and proteins, including enzymes.
  • reference value is used broadly herein to refer to a value for any parameter, attribute, or characteristic of a molecule, sample, or subject that can be detected, determined, and/or quantitated and can be compared with another value, e.g., a value obtained for that parameter, attribute, or characteristic from a molecule, sample, or subject of interest.
  • a reference value can be qualitative or quantitative and can be a range of values.
  • a reference value can be, for example, the level of expression or activity of a protein, the proportion of a protein that is phosphorylated, the presence or number of cells of a particular type, presence or absence of a mutation, a parameter obtained from an imaging study, etc.
  • a reference value is obtained from a population of molecules, samples, or subjects (reference population) known to exhibit one or more characteristics or features, and a comparison of the reference value for a particular parameter, characteristic or attribute with and a value for that parameter, characteristic, or attribute obtained from a molecule, sample, or subject of interest provides information regarding the similarity between the reference population and the molecule, sample, or subject of interest with respect to those characteristics or features.
  • regulatory element or "regulatory sequence” in reference to a nucleic acid is generally used herein to describe a portion of nucleic acid that directs or increases one or more steps in the expression (particularly transcription, but in some cases other events such as splicing or other processing) of nucleic acid sequence(s) with which it is operatively linked.
  • the term includes promoters and can also refer to enhancers and other transcriptional control elements. Promoters are regions of nucleic acid that include a site to which RNA polymerase binds before initiating transcription and that are typically necessary for even basal levels of transcription to occur. Generally such elements comprise a TATA box.
  • Enhancers are regions of nucleic acid that encompass binding sites for protein(s) that elevate transcriptional activity of a nearby or distantly located promoter, typically above some basal level of expression that would exist in the absence of the enhancer. Regulatory sequences may direct constitutive expression of a nucleotide sequence (e.g., expression in most or all cell types under typical physiological conditions in culture or in an organism) or may direct cell or tissue-specific and/or inducible expression. Regulatory elements may also inhibit or decrease expression of an operatively linked nucleic acid.
  • a “response” is a change in a tumor or subject relative to a previous state.
  • a response to a therapeutic agent occurs following, and related to, administration of one or more doses of the agent.
  • a "favorable response” as used herein refers to any biological, chemical, or physical response that is recognized by those skilled in the art as being beneficial to a subject, e.g., as indicating a decreased rate of tumor growth or progression.
  • a favorable response can be a partial or complete response, stabilization of disease, increased time to progression, etc., or any therapeutically beneficial change in the tumor or subject, relative to the condition of the tumor or subject that would exist in the absence of treatment.
  • a variety of criteria are available to determine whether a tumor or subject is exhibiting or has exhibited a favorable response.
  • criteria include clinical criteria, imaging criteria, criteria based on tissue biopsy, etc. Standardized criteria can be used and may be preferred for clinical trials.
  • RECIST Response Evaluation Criteria in Solid Tumors
  • Additional criteria include, but are not limited to, inhibition of cancer cell growth, proliferation and/or survival; tumor shrinkage; etc.
  • sample refers to any specimen that contains cellular material, e.g., contains a cell or collection of cells or a cell lysate. A sample is typically obtained from a subject.
  • the sample can be a body fluid such as blood, lymph, ascites fluid, cerebrospinal fluid, urine; a washing or lavage such as a ductal lavage or broncheoalveolar lavage; an aspirate such as a fine needle aspirate; a scraping; a bone marrow specimen; a tissue biopsy specimen; a surgical specimen, etc.
  • the sample when obtained, contains intact cells.
  • the cells typically originate from a tumor and may be isolated directly from the tumor or may be circulating in the blood. In certain embodiments the cells originate from blood vessels that supply a tumor.
  • the sample contains circulating endothelial cells, circulating endothelial progenitor cells, or both.
  • the sample, or a portion thereof may be subjected to any of a variety of processing steps and is still considered to be a sample derived from the subject.
  • a tumor or tumor cell line is "sensitive" to a therapeutic agent if the agent inhibits (i.e., reduces) the growth rate of the tumor or tumor cell line.
  • the growth rate of the tumor or tumor cell line is detectably lower following exposure to the agent and/or in the presence of the agent (e.g., after administration of the agent to a subject, addition of the agent to tissue culture medium, etc.) than it was prior to the exposure and/or in the absence of the agent.
  • the growth rate e.g., cell proliferation rate, is decreased by at least a predetermined amount.
  • a cell line or tumor is considered sensitive to an agent if the proliferation rate following exposure to the agent is reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150% (1.5 fold), at least 200% (2-fold), at least 3- fold, at least 5-fold, at least 10-fold, at least 20-fold, or more, relative to the growth rate prior to exposure to the agent.
  • the proliferation rate is reduced to 0, or the number of cells decreases.
  • the number of cells may decline at a rate that is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150% (1.5 fold), at least 200% (2-fold), at least 3-fold, at least 5-fold, at least 10-fold, or at least 20-fold, as great as the proliferation rate prior to exposure to the agent.
  • a predetermined amount may be any other value that falls within any subrange, as has any specific value (specified to the tenths place), within the limits of the values set forth above.
  • the exposure can be a single exposure or can be ongoing exposure, e.g., as when a patient is administered a course of a chemotherapeutic agent that includes administration of multiple doses over a period of time.
  • Growth typically refers to cell proliferation.
  • cell proliferation typically results in an increase in volume of the tumor.
  • a tumor or tumor cell line that is sensitive to a therapeutic agent is said to "respond” to the agent.
  • a tumor or tumor cell line that is not sensitive to a therapeutic agent is said to be "resistant” or "non-responsive" to the agent.
  • a variety of methods can be used to measure the ability of an agent to inhibit cell proliferation.
  • Typical in vitro assays measure the metabolic rate of the cells or the synthesis of macromolecules such as DNA. Measuring incorporation of radiolabeled thymidine into DNA (e.g., using autoradiography or flow cytometry) has long been considered the gold standard for measuring DNA synthesis.
  • Commercially available assays for measuring cell proliferation include the MTT, XTT, or WST-I assays (Roche Applied Science; Promega), which are based on the conversion of colored tetrazolium salts into compounds of different colors by metabolically active cells.
  • IC50 or IC25
  • concentration that reduces cell number by 50% or 25%, respectively.
  • Small molecule refers to organic compounds, whether naturally- occurring or artificially created (e.g., via chemical synthesis) that have relatively low molecular weight and that are not proteins, polypeptides, or nucleic acids. Typically, small molecules have a molecular weight of less than about 1500 g/mol. Also, small molecules typically have multiple carbon-carbon bonds.
  • Specific binding generally refers to a physical association between a target polypeptide (or, more generally, a target molecule) and a binding molecule such as an antibody or ligand.
  • the association is typically dependent upon the presence of a particular structural feature of the target such as an antigenic determinant or epitope recognized by the binding molecule. For example, if an antibody is specific for epitope A, the presence of a polypeptide containing epitope A or the presence of free unlabeled A in a reaction containing both free labeled A and the binding molecule that binds thereto, will reduce the amount of labeled A that binds to the binding molecule.
  • specificity need not be absolute but generally refers to the context in which the binding occurs.
  • numerous antibodies cross-react with other epitopes in addition to those present in the target molecule.
  • Such cross-reactivity may be acceptable depending upon the application for which the antibody is to be used.
  • One of ordinary skill in the art will be able to select antibodies or Iigands having a sufficient degree of specificity to perform appropriately in any given application (e.g., for detection of a target molecule, for therapeutic purposes, etc).
  • specificity may be evaluated in the context of additional factors such as the affinity of the binding molecule for the target versus the affinity of the binding molecule for other targets, e.g., competitors.
  • binding molecule exhibits a high affinity for a target molecule that it is desired to detect and low affinity for nontarget molecules, the antibody will likely be an acceptable reagent.
  • specificity of a binding molecule may be employed in other, preferably similar, contexts without necessarily re-evaluating its specificity. Binding of two or more molecules with one another may be considered specific if the affinity (equilibrium dissociation constant, Kd) is at least 10 "3 M, preferably 10 '4 M, more preferably 10 '5 M, e.g., 10 '6 M, 10 "7 M, 10 '8 M, or 10 "9 M under the conditions tested.
  • Subject refers to an individual who is a candidate for administration of an mTOR inhibitor or to whom an mTOR inhibitor is or has been administered.
  • the subject may have or be at risk of developing a tumor.
  • a "normal subject” is an individual not known to have or to be at elevated risk for developing a tumor.
  • Preferred subjects are mammals, particularly humans. Other subjects include domesticated mammals (e.g., dogs, cats, etc.) and non-human primates.
  • Treating as used herein, can generally include reversing, alleviating, inhibiting the progression of, or reducing the likelihood of the disease, disorder, or condition to which such term applies, or one or more symptoms or manifestations of such disease, disorder or condition.
  • Tumor refers to an abnormal mass of tissue that results from excessive cell division.
  • a tumor can be benign (not cancerous) or malignant (cancerous).
  • Tumor includes disorders characterized by excessive division of hematopoietic cells. Such disorders include malignant and premalignant hematologic disorders such as leukemia, lymphoma, myeloma, and myeloproliferative disorders. Tumors can be diagnosed using any of a variety of art-accepted methods including physical diagnosis, imaging studies, histopathology (e.g., performed on a cell or tissue sample), biochemical tests, etc.
  • tumors include sarcomas, prostate cancer, breast cancer, endometrial cancer, hematologic tumors (e.g., leukemia, Hodgkin's and non-Hodgkin's lymphoma, multiple myeloma and other plasma cell disorders, myeloproliferative disorders), brain tumors (e.g., low grade astrocytoma, anaplastic astrocytoma, glioblastoma multiforme, oligodendroglioma, and ependymoma), and gastrointestinal stromal tumors (GIST).
  • hematologic tumors e.g., leukemia, Hodgkin's and non-Hodgkin's lymphoma, multiple myeloma and other plasma cell disorders, myeloproliferative disorders
  • brain tumors e.g., low grade astrocytoma, anaplastic astrocytoma, glioblastoma multiforme, oli
  • Sarcomas include osteosarcoma, Ewing's sarcoma, soft tissue sarcoma, and leiomyosarcoma. Additional examples of malignant tumors include small cell and non-small cell lung cancer, kidney cancer (e.g., renal cell carcinoma), hepatocellular carcinoma, pancreatic cancer, esophageal cancer, colon cancer, rectal cancer, stomach cancer, breast cancer, ovarian cancer, bladder cancer, testicular cancer, thyroid cancer, head and neck cancer, thyroid cancer, etc.
  • "Vector" as used herein, refers to a nucleic acid molecule capable of mediating entry of, e.g., transferring, transporting, etc., a second nucleic acid molecule into a cell.
  • the transferred nucleic acid is generally linked to, e.g., inserted into, the vector nucleic acid molecule.
  • a vector may include sequences that direct autonomous replication, or may include sequences sufficient to allow integration into host cell DNA.
  • Useful vectors include, for example, plasmids (typically DNA molecules although RNA plasmids are also known), cosmids, and viral vectors.
  • viral vector may refer either to a nucleic acid molecule (e.g., a plasmid) that includes virus-derived nucleic acid elements that typically facilitate transfer or integration of the nucleic acid molecule (examples include retroviral or lentiviral vectors) or to a virus or viral particle that mediates nucleic acid transfer (examples include retroviruses or lentiviruses).
  • viral vectors may include various viral components in addition to nucleic acid(s).
  • Expression vectors are vectors that include regulatory sequence(s), e.g., expression control sequences (e.g., a promoter and/or other expression signals and, optionally, 3' sequences, such as 3' regulatory sequences or termination signals), sufficient to direct transcription of an operably linked nucleic acid segment.
  • the nucleic acid segment may, but need not be, a protein coding sequence.
  • the nucleic acid segment may be chimeric, meaning that it includes more than one sequence of distinct origin that are fused together by recombinant DNA techniques, resulting in a nucleotide sequence that does not occur naturally.
  • expression vector can refer to a vector either before or after insertion of the operably linked nucleic acid segment.
  • An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or by an in vitro expression system.
  • Such vectors typically include one or more appropriately positioned sites for restriction enzymes, e.g., to facilitate introduction of the nucleic acid segment to be expressed into the vector.
  • the present invention relates to the identification of new biomarkers and biomarker combinations that are useful for predicting the likelihood that a tumor is sensitive to an mTOR inhibitor.
  • mTOR is a serine/threonine kinase that has emerged as a central regulator of a number of important cellular processes.
  • mTOR signaling is regulated by several upstream pathways that include both positive and negative regulators, and signaling through mTOR regulates a number of downstream pathways that affect cell size and proliferation.
  • mTOR phosphorylates a variety of proteins involved in translation initiation, resulting in increased cap-dependent translation and increased translation of ribosomal proteins.
  • mTOR Activation of mTOR may result in increased expression of various proteins involved in cell cycle progression. These features, among others, have made mTOR an attractive target for cancer therapy.
  • mTOR inhibitors have demonstrated promising results in a variety of tumor types (Sawyers, C. Cancer Cell, 4:343-348, 2003). However, the response has been variable even among tumors that are classified into similar types on clinical and/or histopathologic grounds, and it has heretofore not been possible to predict which tumors are likely to be sensitive.
  • the present invention provides methods and reagents for evaluating the likelihood that a tumor is sensitive to an mTOR inhibitor, e.g., a rapamycin analog.
  • the invention is based in part on the discovery of a correlation between the expression level of an FK506 binding protein (FKBP protein) in a panel of human tumor cell lines and the likelihood that the tumor cell lines are sensitive to an mTOR inhibitor.
  • FKBPs are the cytosolic receptors for macrolides such as FK506, FK520, rapamycin, and rapamycin analogs, and are highly conserved across species lines. When bound to these ligands, FKBPs bind to and inhibit a variety of cellular proteins.
  • rapamycin binds to FKBP 12, and the FKBP12-rapamycin complex binds to mTOR, thereby inhibiting mTOR activity.
  • Rapamycin analogs such as temsirolimus (CCI-779), everolimus (RADOOl ; Certican), and AP23573 behave in a similar manner.
  • the inventors treated a variety of different tumor cell lines with the mTOR inhibitor AP23573, a rapamycin analog that has shown promise as a cancer chemotherapeutic agent in a number of clinical trials, determined the sensitivity of these cell lines to the agent, and measured their FKBP 12 expression level.
  • FKBP 12 is generally considered to be ubiquitously expressed at high levels, with particularly high expression in the brain (Snyder, S., et al., Neuron, 21 :283-294).
  • the tumors displayed an unexpected degree of heterogeneity with respect to the expression of FKBP 12. Certain tumor cell lines displayed high expression while others displayed low or close to undetectable expression.
  • This discovery provides the basis for use of FKBP expression or activity levels as biomarkers for evaluating the likelihood that a tumor is sensitive to an mTOR inhibitor and for evaluating the likelihood that a tumor or subject will exhibit a favorable response to treatment with an mTOR inhibitor.
  • the fact that expression of FKBP 12 was significantly reduced in cell lines from tumors of a variety of types suggests that these biomarkers will have broad utility for evaluating the likelihood of tumor sensitivity and/or patient response to an mTOR inhibitor.
  • the term "evaluating" is intended to mean that the method provides information useful in forming a judgement as to the likelihood that a tumor is sensitive to an mTOR inhibitor and/or that a subject suffering from a tumor is responding or will respond to treatment with an mTOR inhibitor.
  • the methods may be used to identify subjects who are likely to respond to an mTOR inhibitor, relative to the likelihood of response in the overall population of subjects. It will be appreciated that the methods do not necessarily predict with complete accuracy whether any particular tumor or subject will exhibit a favorable response but rather indicate that tumors or subjects having certain features are more likely or less likely to exhibit a favorable response than tumors or subjects not having such features. While FKBP 12 is exemplified herein, the invention encompasses similar use of the expression level or activity of related FKBP proteins, e.g., FKBP proteins that bind to rapamycin or a rapamycin analog and, when so bound, inhibit the mTOR kinase.
  • expression or activity of an FKBP protein is measured in a sample derived from a tumor (referred to as a "test sample").
  • expression or activity of an FKBP protein in a tumor is measured in vivo, e.g., using a functional imaging method.
  • An expression or activity level can be qualitative or quantitative. Thus a determination of whether a polynucleotide or polypeptide is present or absent (e.g., detectable or undetectable) constitutes determining its expression level in various embodiments of the invention while in other embodiments a quantitative level is determined.
  • Determining whether or not a polypeptide exhibits a particular activity constitutes determining the activity of the polypeptide in certain embodiments of the invention. In other embodiments a quantitative determination of activity is performed.
  • the phrase "expression or activity" is not intended to indicate that measurements of these parameters are mutually exclusive. A single measurement can provide information about the level of expression, activity, or both.
  • evaluating the level of expression or activity of a protein includes evaluating one or more parameters or features that provide information about the level of expression of the protein, the activity of the protein, or both.
  • the invention therefore provides methods for evaluating the likelihood that a tumor is sensitive to an mTOR inhibitor comprising assessing the expression or activity of an FKBP protein in a tumor, e.g., in a sample derived from a subject with a tumor or using an in vivo imaging method.
  • the level of expression or activity of the FKBP protein is compared with a reference value for the expression or activity of the FKBP protein, and the result of the comparison is predictive of the likelihood that the tumor is sensitive to an mTOR inhibitor.
  • a reduced level of expression or activity of an FKBP protein (or its absence) in a tumor indicates a decreased likelihood that the tumor is sensitive to an mTOR inhibitor.
  • an increased level of expression or activity of the FKBP protein (or its presence) in a tumor indicates an increased likelihood that the tumor is sensitive to an mTOR inhibitor.
  • the methods may be applied in situations in which a tumor has not been previously exposed to an mTOR inhibitor and in situations in which a tumor has been previously exposed to an mTOR inhibitor and may still be undergoing exposure to the mTOR inhibitor.
  • a tumor is considered to be sensitive if it is currently displaying sensitivity to an mTOR inhibitor or if it possesses characteristics such that it will display sensitivity to an mTOR inhibitor when exposed to the mTOR inhibitor.
  • a tumor is considered to be resistant if it is currently displaying resistance (lack of sensitivity) to an mTOR inhibitor or if it possesses characteristics such that it will display resistance to the mTOR inhibitor when exposed to the mTOR inhibitor.
  • a method for evaluating the likelihood that a subject will exhibit a favorable response to an mTOR inhibitor also evaluates the likelihood that the subject will not exhibit a favorable response to an mTOR inhibitor.
  • the present application refers primarily to methods for evaluating the likelihood that a tumor is sensitive to an mTOR inhibitor and/or that a subject will exhibit a favorable response to an mTOR inhibitor. Such methods are considered equivalent to methods for evaluating the likelihood that a tumor is resistant to an mTOR inhibitor and/or that a subject will not exhibit a favorable response to an mTOR inhibitor since the information obtained by practicing the methods can be expressed in any of these various terminologies.
  • a sample is obtained from a subject suffering from a tumor.
  • sample comprises tumor cells obtained directly from the tumor, e.g., by fine needle aspiration biopsy, tissue biopsy, etc.
  • the sample may be a fresh frozen section, a paraffin embedded section (e.g., a formalin fixed, paraffin embedded section), etc.
  • a tissuesample can be physically disrupted, enzymatically or chemically digested, etc., to obtain individual cells free from surrounding tissue components.
  • the sample may comprise tumor cells isolated from the blood.
  • Methods for identifying and isolating circulating tumor cells are known in the art (see, e.g., Guadagni, F., et al, Cancer Res. 61:2523-32, 2001; Taback, B., et al., Cancer Res., 61 :8845-50. 2001).
  • Such methods may, for example, involve detection of mRNAs (e.g., using RT-PCR) or proteins that are expressed selectively by tumor cells.
  • tumor specific antigens include, for example, prostate specific antigen (PSA), CA- 125, etc.
  • PSA prostate specific antigen
  • cytokeratins can also be used to identify tumor cells from epithelial tumors. The level of expression or activity of the FKBP protein in the sample is assessed, and the likelihood that the tumor is sensitive to an mTOR inhibitor is evaluated as described herein.
  • Isolated cells obtained either from a tissue sample, blood sample, aspirate, washing, etc. can be fixed, if desired, and dispersed on a slide or other surface for examination, subjected to flow cytometry, etc.
  • a reference value for an indicator such as the level of expression or activity of an FKBP protein may be obtained in a variety of ways. In certain embodiments of the invention the reference value is obtained by assessing the indicator in a reference sample or samples. Reference values obtained from any of a number of different reference samples can be used as a basis for comparison with values obtained from a test sample. The nature of the comparison and the interpretation of the result will differ depending on the nature of the reference sample.
  • cells of the same cell type as the cell type of origin of the tumor are used as a reference sample.
  • the tumor is a breast cancer
  • cells obtained from normal breast tissue can be used as the reference sample.
  • the tumor is a metastatic (secondary) tumor
  • cells from the tissue of origin of the primary tumor can be used as a reference sample.
  • the tumor is a metastasis derived from a primary breast cancer
  • cells obtained from normal breast tissue may be used as the reference sample.
  • a reference sample comprising normal cells may be obtained from an individual. Typically the individual does not suffer from a tumor.
  • the sample may be obtained from an individual who is undergoing surgery for a condition other than a tumor.
  • normal (non-tumor) cells from a subject with a tumor can be used.
  • the tumor is less likely to be sensitive to an mTOR inhibitor (and thus more likely to be resistant to the mTOR inhibitor) than if the expression or activity of the FKBP protein in the test sample is equivalent to or greater than the reference value.
  • the tumor is more likely to be sensitive to the mTOR inhibitor than if the expression or activity of the FKBP protein in the test sample is less than the reference value.
  • cells obtained from a cell line that is sensitive to an mTOR inhibitor in vitro are used as a reference sample.
  • the cell line is a tumor cell line, although any immortalized cell line can be used.
  • the tumor is less likely to be sensitive to an mTOR inhibitor (and thus more likely to be resistant to the mTOR inhibitor) than if the expression or activity of the FKBP protein in the test sample is equivalent to or greater than the reference value.
  • the tumor is more likely to be sensitive to the mTOR inhibitor than if the expression or activity of the FKBP protein in the test sample is less than the reference value.
  • cells obtained from a cell line that is resistant to an mTOR inhibitor in vitro are used as a reference sample.
  • the cell line is a tumor cell line.
  • the tumor is less likely to be sensitive to an mTOR inhibitor (and thus more likely to be resistant to the mTOR inhibitor) than if the expression or activity of the FKBP protein in the test sample is greater than the reference value.
  • tumor cells obtained from a subject who exhibited a favorable response to an mTOR inhibitor following administration of the mTOR inhibitor are used as a reference sample.
  • the tumor is less likely to be sensitive to an mTOR inhibitor (and thus more likely to be resistant to the mTOR inhibitor) than if the expression or activity of the FKBP protein in the test sample is equivalent to or greater than the reference value. If, on the other hand, the expression or activity of the FKBP protein in the reference sample is equivalent to or greater than the reference value, the tumor is more likely to be sensitive to the mTOR inhibitor than if the expression or activity of the FKBP protein in the test sample is less than the reference value.
  • tumor cells obtained from a subject who did not exhibit a favorable response to an mTOR inhibitor following administration of the mTOR inhibitor are used as a reference sample.
  • the tumor is less likely to be sensitive to an mTOR inhibitor (and thus more likely to be resistant to the mTOR inhibitor) than if the expression or activity of the FKBP protein in the test sample is greater than the reference value.
  • the tumor is more likely to be sensitive to the mTOR inhibitor than if the expression or activity of the FKBP protein in the test sample is less than or equivalent to the reference value.
  • the reference value is obtained by an in vivo imaging method that is applied, e.g., to normal tissue, to tumors in subjects that exhibited a favorable response to an mTOR inhibitor, or to tumors in subjects that did not exhibit a favorable response to an mTOR inhibitor.
  • an in vivo imaging method that is applied, e.g., to normal tissue, to tumors in subjects that exhibited a favorable response to an mTOR inhibitor, or to tumors in subjects that did not exhibit a favorable response to an mTOR inhibitor.
  • a variety of different imaging modalities may be used to assess expression or activity of an FKBP protein in vivo.
  • a labeled FKBP ligand e.g., labeled FK506, labeled rapamycin, or a labeled rapamycin analog
  • a subject with a tumor or to a normal individual and its presence in a tumor or in normal tissue may be detected using, e.g., computed tomography (CT) scans, positron emission tomography (PET) scans, etc.
  • CT computed tomography
  • PET positron emission tomography
  • Reference values obtained in vivo from normal tissue, from tumors in subjects that exhibited a favorable response to an mTOR inhibitor, or from samples obtained from tumors in subjects that did not exhibit a favorable response to an mTOR inhibitor are used similarly to reference values obtained from samples obtained from normal tissue, from samples obtained from tumors in subjects that exhibited a favorable response to an mTOR inhibitor, or from tumors in subjects that did not exhibit a favorable response to an mTOR inhibitor, respectively.
  • the determination of a reference value may be performed initially and the value used thereafter for practicing the methods. Thus it is not necessary to determine a reference value or assess a reference sample each time a particular method is practiced. However, in certain embodiments of the invention the test sample is compared with one or more reference or control samples known to display particular expression levels, staining patterns, etc., which are characteristic of sensitive or resistant tumors. Methods for determining a reference value are discussed further below (see Section VII).
  • the reference value is a range of values.
  • a tumor may be deemed sensitive to an mTOR inhibitor if a value obtained for an indicator, e.g., the level of expression or activity of the FKBP protein, is within a range of values or is outside a range of values.
  • a tumor may be deemed resistant to an mTOR inhibitor if a value obtained for an indicator, e.g., the level of expression or activity of the FKBP protein, is within a range of values or is outside a range of values.
  • Terms such as “compare”, “comparison” and the like are used broadly herein and include determining whether a value is greater than, equal to, or less than a reference value, determining whether a value falls within a range, etc.
  • An assessment can include determining the extent to which a value differs from a reference value or falls outside a range.
  • An assessment can include determining whether a difference between a value and a reference value is statistically significant.
  • the difference is considered informative in term of evaluating the likelihood that a tumor is sensitive to an mTOR inhibitor and/or that a subject will exhibit a favorable response to an mTOR inhibitor.
  • a test value is equal to approximately 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 250%, 300%, 400%, 500%, 1000%, etc.
  • the difference is considered informative, i.e., it indicates an increased or decreased likelihood that the tumor is sensitive and/or that the subject will respond.
  • any difference between a test value and a reference value is considered informative.
  • a test value that is approximately 5, 10, 15, 20, 25, 50, or more times as large as a reference value is considered informative.
  • Test values that fall within any subrange or have any specific value (specified to the tenths place) within the limits of the values set forth above are considered informative according to various embodiments of the invention.
  • the predetermined amount is independent of the reference and/or test value(s).
  • Some assessment methods such as immunohistochemistry (discussed further below) utilize a scoring system, e.g., samples are assigned a score ranging from 0 - 3, from 0 - 12, etc. When such a scoring system is used, a difference of 1 scoring unit is informative. In other embodiments a difference of 2, 3, 4, 5, or 6 scoring units is considered informative, up to the maximum difference possible according to the scoring system.
  • a relationship is determined between values of an indicator for a tumor or subject and the likelihood that a tumor is sensitive or the subject will exhibit a favorable response.
  • Results of an evaluation can be expressed in terms of the probability (ranging from 0% to 100%) that a tumor having a particular value for an indicator or indicator(s) (e.g., a value for an indicator or indicator(s) that fall(s) within a particular range) is sensitive to an mTOR inhibitor or in terms of the probability that a subject will exhibit a favorable response to an mTOR inhibitor.
  • a particular value for an indicator or indicator(s) e.g., a value for an indicator or indicator(s) that fall(s) within a particular range
  • reference values are established for each mTOR inhibitor of interest.
  • reference values established for one or more mTOR inhibitors may be employed for other mTOR inhibitors, e.g., other mTOR inhibitors that have a similar structure, mechanism of action, etc.
  • a reference value or values established for one rapamycin analog may be used to evaluate the likelihood that a tumor is sensitive to a second rapamycin analog.
  • an assessment of the level of an indicator e.g., the level of expression or activity of an FKBP protein
  • results in a value indicative of an increased likelihood that a tumor is sensitive to an mTOR inhibitor results in a value indicative of an increased likelihood that a tumor is sensitive to an mTOR inhibitor
  • the value is referred to as a "favorable value”.
  • an assessment of an indicator e.g., the level of expression or activity of an FKBP protein
  • results in a value indicative of a decreased likelihood that a tumor is sensitive to an mTOR inhibitor results in a value indicative of a decreased likelihood that a tumor is sensitive to an mTOR inhibitor
  • the value is referred to as an "unfavorable value”.
  • the fact that a value is not favorable does not necessarily mean that it is unfavorable, and the fact that a value is not unfavorable does not necessarily mean that it is favorable. In some embodiments of the invention some values are considered uninformative.
  • the invention further provides methods to evaluate the likelihood that a subject with a tumor will exhibit a favorable response to administration of an mTOR inhibitor.
  • the methods comprise applying any of the methods for evaluating the likelihood that a tumor is sensitive to an mTOR inhibitor to the subject or to a sample derived from the tumor.
  • the likelihood that the tumor is sensitive to an mTOR inhibitor is indicative of the likelihood that the subject will exhibit a favorable response to the mTOR inhibitor or will continue to exhibit a favorable response if he or she is already exhibiting a favorable response.
  • the methods for evaluating the likelihood that a tumor is sensitive to an mTOR inhibitor and/or for evaluating the likelihood that a subject suffering from a tumor will exhibit a favorable response to an mTOR inhibitor or will continue to respond to an mTOR inhibitor may be performed prior to administering an mTOR inhibitor to a subject or after administration of one or more doses or courses of an mTOR inhibitor to a subject.
  • the invention therefore provides methods for selecting a subject who is a suitable candidate for initiating or continuing treatment with an mTOR inhibitor either as sole therapy or in combination with one or more other agents or treatment modalities such as radiotherapy (gamma-radiation, neutron beam radiotherapy, electron beam radiotherapy, proton therapy, brachytherapy, systemic radioactive isotopes, etc.), surgery, etc., or for selecting an appropriate therapeutic agent or combination of agents and/or other therapeutic modalities for a subject with a tumor.
  • radiotherapy gamma-radiation, neutron beam radiotherapy, electron beam radiotherapy, proton therapy, brachytherapy, systemic radioactive isotopes, etc.
  • mTOR inhibitors may be administered concurrently with one or more additional chemotherapeutic agents either together in single formulation or as individual agents.
  • a "'chemotherapeutic agent” is an agent recognized by those of skill in the art as being useful for treating a tumor and/or under investigation or contemplated for such use.
  • the methods of the invention may be used to assess the likelihood that a tumor is sensitive to an mTOR inhibitor and/or the likelihood that a subject will exhibit a favorable response to an mTOR inhibitor when the mTOR inhibitor is administered by itself or concurrently with another chemotherapeutic agent and/or an agent such as an anti-emetic that is administered to counteract side effects of chemotherapy.
  • Such agents include, but are not limited to, alkylating drugs (mechlorethamine, chlorambucil, Cyclophosphamide, Melphalan, Ifosfamide), antimetabolites (Methotrexate), purine antagonists and pyrimidine antagonists (6-Mercaptopurine, 5-Fluorouracil, Cytarabile, Gemcitabine), tubulin inhibitors (Vinblastine, Vincristine, Vinorelbine, Paclitaxel (taxol), Docetaxel, epithilones, discodermolides), podophyllotoxins (Etoposide, Irinotecan, Topotecan), antibiotics (Doxorubicin, Bleomycin, Mitomycin), nitrosoureas (Carmustine, Lomustine), inorganic ions (Cisplatin, Carboplatin), enzymes (Asparaginase), and hormones (Tamoxifen, Leuprolide, Flutamide, and Megestrol), other endocrine
  • the methods are used to predict tumor sensitivity and/or patient response to an mTOR inhibitor administered concurrently with an angiogenesis inhibitor or a kinase inhibitor.
  • the angiogenesis inhibitor or kinase inhibitor is an anti-VEGF or anti- VEGF-R antibody, small molecule VEGF-R antagonist, anti-angiogenic peptide such as endostatin, an EGF-R antagonist (e.g., SU5416 or SU6668 or an antibody that binds to an EGF-R family member).
  • a sample is obtained from a subject who is suffering from a tumor.
  • the likelihood that the tumor is sensitive to the mTOR inhibitor is evaluated and is used as a basis on which to determine whether the subject is a suitable candidate for initiating or continuing treatment with the mTOR inhibitor or for enrolling in or remaining in a clinical trial of the mTOR inhibitor. For example, if the likelihood is greater than a predetermined value, then the subject may be considered a suitable candidate for initiating or continuing treatment with the mTOR inhibitor. If the likelihood is less than a predetermined value, then the subject may be considered not suitable as a candidate.
  • a variety of factors may be considered in determining whether a subject is a suitable candidate for therapy with an mTOR inhibitor.
  • the subject's response to other therapies, or the results of tests to evaluate the likelihood that the subject will respond to other therapies may be considered as may the side effect profile of the mTOR inhibitor or of any available alternative therapy.
  • Any of a variety of other indicators may be assessed and used in conjunction with the assessment of the expression or activity of an FKBP protein in order to evaluate the the likelihood that a subject having a tumor that is deemed either sensitive or resistant to the mTOR inhibitor will in fact exhibit a favorable response.
  • any of the additional biomarkers described in further detail below may be used.
  • a variety of other parameters and clinical factors may be considered including, but not limited to, the metabolic activity of the tumor (which may be measured, for example, using functional imaging methods known in the art and discussed below), the size of the tumor, the vascularization of the tumor, whether the tumor has metastasized, the overall health of the subject, prior therapies that the subject has received, etc.
  • the methods may be used to monitor expression or activity of the FKBP protein over time to determine whether it is appropriate to continue therapy with an mTOR inhibitor and/or to determine whether another agent should be added to the therapeutic regimen. For example, if the level of expression or activity of the FKBP protein decreases over time, or if tumor cell clones emerge that exhibit reduced FKBP expression or activity, it may be advantageous to add another agent to the patient's therapeutic regimen.
  • FKBP 12 is FKBP 12, also known as FKBPlA.
  • FKBP 12 is described, for example, in the following references (Brown,EJ., et al., Nature 369, 756-758,1994; Hidalgo et al., Oncogene 19: 6680-6686, 2000).
  • FKBP protein is FKBP 12.6 (Deivanayagam et al., Acta. Crystallogr. D. Biol. Crystallog 56: 266-271, 2000).
  • FKBP proteins whose expression or activity may be assessed in accordance with the invention include FKBP51, also referred to as FKBP54 (Nair, et al., MoI. Cell. Biol, 17(2): 594- 595, 1997; U.S. Pat. No. 6,821,731) and FKBP52 (Yamamoto-Yamaguchi et al., Exp Hematol 29:582-588, 2001 ; Genbank Accession No. M88279).
  • FKBP proteins of use in the present invention preferably bind to rapamycin or a rapamycin analog and inhibit the mTOR protein kinase when so bound.
  • Such methods may be used, e.g., to express an FKBP in vitro, e.g., to obtain an antigen preparation for immunization of an animal to obtain an antibody to the FKBP protein.
  • An expression vector that encodes an FKBP protein can be used to generate a cell line, e.g., a tumor cell line, that expresses FKBP protein at higher levels than the original cell line.
  • a cell line e.g., a tumor cell line
  • an FKBP protein can be expressed in a tumor cell line that exhibits low sensitivity to an mTOR inhibitor, e.g., is resistant to the mTOR inhibitor, and the sensitivity of the tumor cell line thereby increased.
  • An FKBP gene sequence can be used to design nucleic acids that inhibit expression of the FKBP protein when introduced into or expressed in cells.
  • examples include nucleic acids that inhibit gene expression by RNA interference such as short interfering RNA (siRNA) and short hairpin RNA (shRNA).
  • siRNA short interfering RNA
  • shRNA short hairpin RNA
  • Antisense nucleic acids which frequently act by inhibiting translation of a complementary mRNA or inducing its degradation by RNase H can also be designed. These inhibitory agents are well known in the art. See, e.g., Dyxhoorn, D., et al., Nat Rev MoI Cell Biol.
  • siRNAs are typically a short RNA duplex, usually with an ⁇ 19 nucleotide duplex region and symmetric 2-3 nucleotide 3' overhangs.
  • One strand of the duplex (the antisense or "guide" strand) is complementary to a target mRNA transcript.
  • siRNAs are incorporated into the endogenous RNA-induced silencing complex (RISC).
  • siRNA duplex is unwound, and the antisense strand guides RISC to the target mRNA, which is then degraded.
  • siRNA are typically chemically synthesized using known methods, and numerous variations on the basic siRNA structure are effective including agents that incorporate a variety of nucleotide analogs and modifications. See, e.g., Crooke, S. (ed.) "Antisense Drug Technology: Principles, Strategies, and Applications” (1 st ed), Marcel Dekker; ISBN: 0824705661; 2001) and references therein for discussion of antisense nucleic acids and their mechanism of action.
  • An inhibitory nucleic acid e.g., an siRNA or shRNA that inhibits expression of an FKBP protein can be introduced into or expressed in cells, e.g., cells of a tumor cell line that is sensitive to an mTOR inhibitor, and the sensitivity of the tumor cell line is thereby decreased.
  • Tumor cells and tumor cell lines that contain a range of different FKBP expression levels can be generated. Such cells and cell lines can be used, e.g., in screens to identify new chemotherapeutic agents or to explore the effect of varying FKBP expression level on the activity of known chemotherapeutic agents including, but not limited to, mTOR inhibitors such as rapamycin analogs. Such screens may identify therapeutic agents that act synergistically with mTOR inhibitors, that help overcome or prevent resistance to mTOR inhibitors, or that are effective in situations in which mTOR inhibitors are ineffective.
  • the present invention encompasses tumor cells and tumor cell lines with an artificially altered (increased or decreased) expression level of an FKBP protein and methods of using such cells to evaluate candidate therapeutic agents, e.g., by contacting the cells with such agent(s) in the presence or absence of an mTOR inhibitor and evaluating the effect of the agent or combination thereof on cell proliferation.
  • the tumor cells are implanted into an animal, e.g., a nude mouse, and the effect of one or more candidate anti-tumor agents (e.g., an mTOR inhibitor) is evaluated. Similar methods may be applied for various other indicators described herein.
  • methods of evaluating the likelihood that a tumor is sensitive to an mTOR inhibitor can be applied to tumor cell lines, tumor explants, etc., cultured in vitro, e.g., to aid in the development of new chemotherapeutic agents, in the reanalysis of known agents that did not exhibit sufficient efficacy in clinical trials to merit further development, in the analysis of chemotherapeutic agents currently in use to identify tumor types and phenotypes that are likely to be sensitive, etc.
  • the GeneID search is performed by selecting "Gene” from the pull-down menu at the top left (below “nucleotide”, “protein”, etc.).
  • the entry for each gene includes considerable information about the gene including links to mRNA and protein sequence entries, links to scientific literature, etc.
  • the following list presents the names and Gene ID numbers for the human forms of various FKBP proteins.
  • the list also presents names and Gene ID numbers for the human forms of a variety of upstream and downstream components of pathways involving mTOR and also for other polypeptides that are useful as biomarkers for evaluating the likelihood that a tumor is sensitive to an mTOR inhibitor and/or that a subject will exhibit a favorable response to an mTOR inhibitor.
  • probes and primers e.g., amplification primers
  • siRNA sequences e.g., antisense sequences, etc.
  • VHL 7428
  • IGF-IR 3480
  • VEGF-A 7422
  • VEGF-B 7423
  • VEGF-C 7424
  • VEGFR-I 2321
  • VEGFR-2 3791
  • any such name e.g., AKT
  • AKT is intended to encompass any individual family member, isoform, or splice variant, and any combination of such family members or isoforms.
  • each embodiment of the instant invention includes variations in which a specific family member, isoform, or splice variant is detected and optionally quantitated to provide information useful for evaluating the likelihood that a tumor is sensitive to an mTOR inhibitor and/or that a subject will exhibit a favorable response to an mTOR inhibitor.
  • Expression of the protein can be assessed by a direct method, by which is meant any method that is based on detecting and optionally quantitating the protein. In other embodiments expression is assessed by detecting and optionally quantitating mRNA that encodes the protein, In other embodiments expression is assessed by determining whether the genomic DNA of a cell or subject contains a mutation that affects expression of the protein. Typically such a mutation will be in a gene that encodes the protein, e.g., in an expression control region. The mutation may be an insertion, substitution, or deletion of one or more nucleotides. In certain embodiments the mutation is a deletion of part or all of a gene that encodes the FKBP protein.
  • Methods for detecting and optionally quantitating an FKBP protein typically involve use of a reagent that specifically binds to the FKBP protein.
  • the reagent may be, e.g., an antibody, antibody fragment, aptamer, affibody, polypeptide, small molecule ligand, or the like. Any reagent that specifically binds to the FKBP protein can be used. Such reagents are referred to collectively as "binding agents".
  • the binding agent may be labeled, e.g., with a radioactive moiety, fluorophore, colorimetric agent, enzyme, magnetically responsive atom or group, etc.
  • the FKBP protein is detected and optionally quantitated using an immunological method.
  • immunological methods include, but are not limited to, Western blots; immunoassays, e.g., enzyme-linked immunosorbent assays (ELISA); flow cytometry; immunohistochemistry, reverse phase assays, etc. These methods are known to one of ordinary skill in the art and are described, e.g., in U.S. Pat. Nos. 6,821,731; US Pub. Nos. 20030190689, 20040106141, and 20040248151.
  • Immunohistochemistry is a preferred method for detecting and optionally quantitating the level of expression of an FKBP protein and can be performed either manually or using automatic staining instruments.
  • Immunohistochemistry is a method that utilizes monoclonal or polyclonal antibodies to detect cells or specific epitopes. Typically the method detects a protein antigen.
  • Methods for preparing and processing samples for IHC are known in the art. For example, a paraffin-embedded tissue (e.g.
  • tumor tissue can be prepared for IHC staining by deparaffinizing tissue sections with xylene followed by ethanol; hydrating in water and then PBS; unmasking the antigen by heating the slide in sodium citrate buffer; incubating sections in hydrogen peroxide; blocking in blocking solution; incubating the slide in primary antibody (e.g., antibodies that bind to an FKBP protein, phospho-specific antibodies) and secondary antibody; and finally detecting using an ABC avidin/biotin detection system according to manufacturer's instructions.
  • primary antibody e.g., antibodies that bind to an FKBP protein, phospho-specific antibodies
  • secondary antibody e.g., antibodies that bind to an FKBP protein, phospho-specific antibodies
  • Immunohistochemistry protocols employ detection systems that make the epitope detectable to the naked eye, e.g., visible, or detectable to an automated detection system.
  • an antibody or mixture of antibodies that binds to a specific protein or other antigen is labeled with a fluorescent or luminescent compound, prosthetic group, radioactive moiety, or an enzyme, e.g., an enzyme that can convert a substrate to a visible dye.
  • the labeled antibody is incubated with the tissue and after washing unbound antibody away the bound antibody distribution is revealed by fluorescence microscopy or incubation with a chromogenic substrate.
  • the first antibody primary antibody
  • a second antibody with an attached label
  • Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials.
  • suitable enzymes include horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, or acetylcholinesterase;
  • suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin;
  • suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material is luminol;
  • examples of bioluminescent materials include luciferase, luciferin, and aequorin.
  • Suitable radioactive material are 125 1, 131 1, 35 S and 3 H.
  • Suitable fluorogenic or chromogenic substrates include nitro blue tetrazolium (NBT) in combination with the phosphatase substrate 5-bromo-4-chloro-3-indolyl phosphate (BCIP), diaminobenzidine (DAB), etc.
  • IHC results can be qualitative, semi-quantitative, or quantitative.
  • results are presented in terms of a semiquantitative scoring system, e.g., ranging from 0 (no staining), to 3+.
  • the score can reflect the percentage of cells that stain, the intensity of staining, the pattern of staining, or any combination of the foregoing.
  • a sample can be considered "positive” or "negative".
  • a score of 1+, 2+, or 3+ can be considered positive, or 2+ and 3+ can be considered positive with 0 or 1+ being considered negative, etc.
  • Staining in tumor cells can be scored by comparing their intensity of staining with that of endothelial cells present in the sample.
  • an automated, quantitative IHC method in which a sample is assigned a score that is a numerical representation of the intensity of the immunohistochemical staining of the sample and represents the amount of the antigen to be detected that is present in the portion of the sample analyzed.
  • the score can be an optical density (OD).
  • Suitable automated IHC sample processing, scanning, and analysis systems are known in the art and are available, e.g., from Ventana Medical Systems.
  • the BenchmarkTM system (Ventana Medical Systems, Tuscon AZ) performs automated sample preparation and processing. Samples can be assessed visually or optical imaging and computer analysis, e.g., using a system such as the Accumed Accell 2000 Image Analyzer, equipped with appropriate image analysis software, can be used to automatically acquire an image and provide a quantitative measurement of OD.
  • Flow cytometry is another method that is well known in the art and is useful for assessing expression levels, phosphorylation state, etc., using appropriate antibodies. Flow cytometry may be carried out according to standard methods. See, e.g. Chow et al., Cytometry (Communications in Clinical Cytometry) 46: 72-78 (2001). In certain embodiments of the invention flow cytometry is used to assess the level or proportion of CECs and/or CEPs in a blood sample. Antibodies to cell surface markers that are characteristic of these cell types are known in the art. For example, CD31 and VEGFR-I identify cells as endothelialcells.
  • CEPs are distinguished by the presence of one or more stem cell antigens on the cell surface such as CDl 17, Sca-1 or, in humans, CD 133.
  • Antibodies to a number of these antigens are commercially available, e.g., from Becton Dickinson.
  • IHC can also be used to assess the level or proportion of CECs and CEPs in a sample.
  • Laser-scanning cytometry-mediated analysis can be used in conjunction with immunofluorescence on tissue biopsy samples to quantify polypeptide expression levels and phosphorylation state on a single cell level (Davis, DW, et al,. Clinical Cancer Res., 11 :678-689, 2005).
  • Tumor microvessel density (MVD) and vessel sizes can also be measured by LSC (Davis, DW, et al., Cancer- Res., 64:4601-10, 2004).
  • Endothelial and/or tumor cell apoptosis can be measured using terminal deoxynucleotidyl transferase-mediated nick end labeling (TlHSfEL) assays. These assays may provide additional information about the sensitivity of a tumor to an mTOR inhibitor and/or subject response thereto, particularly when administered in conjunction with an angiogenesis inhibitor.
  • Immunoassays such as ELISA assays or modifications thereof are well known in the art.
  • Such detection techniques can be divided into (1) antibody capture assays; (2) antigen capture assays; (3) two-antibody capture assays, any of which can be configured by one of ordinary skill in the art to accomplish detection in a qualitative, semi-quantitative, or quantitative mode.
  • One of ordinary skill in the art will be able to select an appropriate assay taking into consideration factors such as the abundance of the molecule to be detected and the relative sensitivity of different assay formats.
  • the level of expression or activity of a protein is assessed by measuring the expression level of an mRNA that encodes the protein, by determining the presence or absence of a mutation in, or amplification of, a gene that encodes the protein; by determining the presence or absence of a mutation that affects the expression level of the protein and is located outside the gene that encodes the protein.
  • other methods for assessing the expression or activity of a protein can include determining the existence, number, location, and/or nature of a post-translational modification such as phosphorylation, glycosylation, acetylation, etc.; determining the localization of the protein; or detecting or measuring any biological or chemical activity of the protein (e.g., binding activity, enzymatic activity towards a substrate) etc.
  • a mutation that affects the expression or activity of a protein can be a substitution, deletion, or addition of one or more nucleotides, a chromosomal abnormality such as an inversion, translocation, deletion, rearrangement, amplification, etc.
  • Suitable methods that may be used to detect and optionally quantitate mRNA include Northern blots, RT-PCR, cDNA or oligonucleotide microarray analysis, in situ hybridization (e.g., fluorescent in situ hybridization), etc.
  • a number of the methods for nucleic acid detection and/or analysis make use of nucleic acid hybridization to detect a nucleic acid of interest, e.g., mRNA, cDNA, or genomic DNA.
  • Suitable probes can readily be designed based on known sequences.
  • the nucleic acid of interest can be amplified using methods known in the art.
  • amplification method including exponential amplification, linked linear amplification, ligation-based amplification, and transcription-based amplification.
  • An example of an exponential nucleic acid amplification method is the polymerase chain reaction (PCR) which is described, for example, in Mullis et al. Cold Spring Harbor Symp. Quant. Biol. 51 :263-273 (1986); PCR Cloning Protocols: From Molecular Cloning to Genetic Engineering, Methods in Molecular Biology, White, B. A., ed., vol. 67 (1998); Mullis EP 201,184; Mullis et al., U.S. Pat. Nos.
  • PCR polymerase chain reaction
  • Isothermal target amplification methods include transcription mediated amplification (TMA), self-sustained sequence replication (3SR), Nucleic Acid Sequence Based Amplification (NASBA), and variations thereof.
  • TMA transcription mediated amplification
  • NASBA nucleic Acid Sequence Based Amplification
  • TMA transcription mediated amplification
  • NASBA Nucleic Acid Sequence Based Amplification
  • others e.g., as described in Malek et al., U.S. Pat. No. 5,130,238; Kacian and Fultz, U.S. Pat. No. 5,399,491; Burg et al., U.S. Pat. No. 5,437,990).
  • a wide variety of methods are available to detect mutations, e.g., in an FKBP protein or mTOR pathway component. Southern blots and restriction fragment analysis, etc., represent traditional methods. Mutation detection and/or sequence comparison can be performed using any of a variety of methods known in the art, e.g., amplification-based assays, hybridization assays, primer extension assays (e.g., allele- specific primer extension assays), oligonucleotide ligation assays (U.S. Pat. Nos. 5,185,243, 5,679,524 and 5,573,907), cleavage assays , heteroduplex tracking analysis (HTA) assays, etc.
  • amplification-based assays e.g., hybridization assays
  • primer extension assays e.g., allele- specific primer extension assays
  • oligonucleotide ligation assays U.S. Pat. Nos. 5,185,243, 5,
  • Examples include the Taqman ® assay, Applied Biosystems (U.S. Pat. No. 5,723,591).
  • the probe oligonucleotide contains two fluorescent moieties.
  • the polymerase cleaves the probe oligonucleotide. The cleavage causes the two fluorescent moieties to become physically separated, which causes a change in the wavelength of the fluorescent emission. As more PCR product is created, the intensity of the novel wavelength increases.
  • Cycling probe technology CPT which is a nucleic acid detection system based on signal or probe amplification rather than target amplification (U.S. Pat. Nos.
  • Invasive cleavage assays e.g., Invader ® assays (Third Wave Technologies), described in Eis, P. S. et al., Nat. Biotechnol. 19:673, 2001, can also be used to detect mutations and allelic variants.
  • Assays based on molecular beacons U.S. Pat. Nos. 6,277,607; 6,150,097; 6,037,130
  • FRET fluorescence energy transfer
  • the conformational change of the oligonucleotide increases the physical distance between a fluorophore moiety and a quencher moiety present on the oligonucleotide. This increase in physical distance causes the effect of the quencher to be diminished, thus increasing the signal derived from the fluorophore.
  • U.S. Pub. No. 20050069908 and references therein describe a variety of other methods that can be used for the detection and analysis of nucleic acids.
  • U.S. Pat. Nos. 5,854,033, 6,143,495, and 6,239,150 describe compositions and a method for amplification of and multiplex detection of molecules of interest involving rolling circle replication.
  • the method is useful for simultaneously detecting multiple specific nucleic acids in a sample,
  • direct sequencing using any available method known in the art can be used. Examples include the chain termination or dideoxynucleotide method (Sanger, et al., Proc. Natl. Acad. Sci. 74:5463-5467, 1977) and the chemical degradation method (Maxam & Gilbert, Proc. Natl. Acad. Sci. 74:560-564, 1977), of which the former has been most extensively employed, improved upon, and automated.
  • Other sequencing approaches include pyrosequencing (see, e.g., U.S. Pat. Nos. 6,210,891 and 6,258,568; sequencing by hybridization (U.S. Pat. No.
  • the activity is binding activity of the FKBP protein towards mTOR in the presence of an FKBP ligand, e.g., rapamycin or a rapamycin analog.
  • FKBP ligand e.g., rapamycin or a rapamycin analog.
  • a labeled small molecule FKBP ligand is employed, e.g. fluorescently labeled form of FK506 such as AP1491.
  • Suitable methods for assaying the binding activity of an FKBP protein, e.g., in a cell lysate, are described in U.S. Pat. Nos. 6,150,527 and 6,649,595.
  • Similar methods may be employed to assay binding activity of an FKBP protein in a sample such as a tissue biopsy specimen, or in vivo using a suitable imaging technique, e.g., an imaging technique that detects labeled molecules.
  • a sample e.g., a tissue biopsy sample
  • Suitable incubation conditions include, e.g., those described in U.S. Pat. Nos. 6,150,527 and 6,649,595. After a period of time the sample is washed to remove unbound label and is visualized, e.g., using a fluorescence microscope, automated image acquisition system, etc.
  • mTOR is a protein kinase that is a component of a number of signal transduction pathways that regulate a range of cellular processes.
  • the invention provides a variety of additional indicators that may be assessed either independently or in conjunction with an assessment of the expression or activity of an FKBP protein to evaluate the likelihood that a tumor is sensitive to an mTOR inhibitor. The indicators can be used to evaluate the likelihood that a subject with a tumor will exhibit a favorable response to an mTOR inhibitor.
  • the phosphorylation state and/or level of one or more mTOR pathway components is measured, e.g., in a sample derived from a tumor, or using an in vivo imaging method.
  • a value is obtained for an indicator. The value may be compared with a reference value. General considerations regarding reference values are described in Sections I and VII.
  • the reference value is obtained from a reference sample containing cellular material from any of the sources discussed above, e.g., normal cells (which may be of the same cell type as the cell type of origin of the tumor or of the same cell type as cells presenting the tissue in which the tumor is found), tumor cell lines that are sensitive to an mTOR inhibitor, tumor cell lines that are resistant to an mTOR inhibitor, tumors from subjects who exhibited a favorable response to an mTOR inhibitor, tumors from subjects who did not exhibit a favorable response to an mTOR inhibitor, etc.
  • normal cells which may be of the same cell type as the cell type of origin of the tumor or of the same cell type as cells presenting the tissue in which the tumor is found
  • tumor cell lines that are sensitive to an mTOR inhibitor tumor cell lines that are resistant to an mTOR inhibitor
  • tumors from subjects who exhibited a favorable response to an mTOR inhibitor tumors from subjects who did not exhibit a favorable response to an mTOR inhibitor, etc.
  • the reference value is obtained from blood samples from individuals not suffering from a tumor, subjects that exhibited a favorable response to an mTOR inhibitor, or subjects who did not exhibit a favorable response to an mTOR inhibitor.
  • the reference value can be obtained from normal tissue, from tumors in subjects who exhibited a favorable response to an mTOR inhibitor, or from tumors in subjects who did not exhibit a favorable response to an mTOR inhibitor. If an assessment of an indicator results in a value indicative of an increased likelihood that a tumor is sensitive to an mTOR inhibitor, the value is referred to as a "favorable value".
  • any of a variety of indicators may be assessed and used in conjunction with an assessment of the level of expression or activity of an FKBP protein in order to evaluate the likelihood that a tumor is sensitive to an mTOR inhibitor.
  • the assessment of the level of expression or activity of the FKBP protein and the assessment(s) of one or more additional indicators are considered in conjunction to arrive at an overall assessment of the likelihood that a tumor is sensitive to an mTOR inhibitor.
  • "In conjunction" encompasses a variety of different situations. In various embodiments of the invention the results of each assessment are considered individually, but each of the assessments is used to arrive at an overall assessment.
  • the likelihood that the tumor is sensitive is greater than if either or both of the assessments results in an unfavorable value.
  • results of each assessment are combined (e.g., numerically) to produce a combined value, which is then used to arrive at an overall assessment, e.g., by comparison with a reference value.
  • none of two or more assessments considered individually is predictive of the likelihood that a tumor is sensitive, but taken collectively the assessments are predictive.
  • upstream components of a pathway involving mTOR include IGF-IR, PTEN, AKT, TSC2, and RHEB.
  • Other indicators relate to polypeptides that are downstream components of a pathway involving mTOR.
  • downstream components of a pathway involving mTOR include S6, S6 kinase, 4E-BP1, eIF4E, cyclin Dl, cyclin D3, myc, p27Kipl, and HIF-I ⁇ .
  • Other biomarkers relate to polypeptides that form a complex with mTOR. These polypeptides include Raptor and G ⁇ L.
  • the proportion or level of certain upstream or downstream components, or of mTOR itself, that is phosphorylated is used to evaluate the likelihood that a tumor is sensitive to an mTOR inhibitor.
  • the proportion of that protein that is phosphorylated in a sample can be determined by dividing the amount of that protein that is phosphorylated in the sample by the total amount of that protein present in the sample and multiplying by 100, i.e., by computing 100[Prot p /(Prot p + Prot UP )], where Prot p indicates a phosphorylated form of the protein and Prot up indicates an unphosphorylated form of the protein.
  • the level of that protein that is phosphorylated in a sample refers to the total amount or concentration of the phosphorylated form of the protein present in a sample.
  • phosphorylation can occur at certain serine, threonine, or tyrosine residues in a protein.
  • Phosphorylation at specific positions is of particular interest in certain embodiments of the invention.
  • phosphorylation at serine 473 of AKT, serine 235 of S6, or serine 2481 of mTOR may be of particular importance.
  • Forms of these proteins that are specifically phosphorylated on these positions are detected in various embodiments of the invention.
  • the proteins may or may not be phosphorylated at one or more other positions in different embodiments.
  • At least one indicator is assessed in a tumor in addition to assessing the level of expression or activity of an FKBP protein.
  • the at least one additional indicator is selected from the group consisting of: (a) the proportion or level of TSC2 protein that is phosphorylated in a tumor; (b) the proportion or level of AKT protein that is phosphorylated in a tumor; (c) the proportion or level of S 6 protein that is phosphorylated in a tumor; (d) the proportion or level of S6 kinase protein that is phosphorylated in a tumor; (e) the proportion or level of 4E-BP1 protein that is phosphorylated; (f) the proportion or level of mTOR protein that is phosphorylated; (g) the level of cyclin Dl mRNA or protein, cyclin D3 mRNA or protein, myc mRNA or protein, or any combination of these; (h) the level of HIF-I ⁇ mRNA or protein, HIF- l ⁇
  • Indicators (a) - (q) would typically be assessed in a sample containing tumor cells or cellular material.
  • Indicators (r) and (s) refer to cells that are circulating in the bloodstream and would typically be assessed in a blood sample from a subject with a tumor.
  • Certain of the above indicators relate to assessing the proportion or level of a phosphorylated form of a polypeptide.
  • the total level of these polypeptides including both phosphorylated and unphosphorylated forms is assessed instead of, or in addition to, assessing the proportion or level of a phosphorylated form.
  • this difference in conjunction with a favorable value for the level of expression or activity of an FKBP protein, is indicative of an increased likelihood that the tumor is sensitive to an mTOR inhibitor, relative to the likelihood if the value for that indicator in the tumor is equal to or lower than that in normal cells or in tumors that are resistant to an mTOR inhibitor.
  • the indicators include (a) the proportion or level of TSC2 protein that is phosphorylated; (b) the proportion or level of AKT protein that is phosphorylated; (c) the proportion or level of S6 protein that is phosphorylated; (d) the proportion or level of S 6 kinase protein that is phosphorylated; (e) the proportion or level of 4E-BP1 protein that is phosphorylated; (f) the proportion or level of mTOR protein that is phosphorylated; (g) the level of cyclin Dl mRNA or protein, cyclin D3 mRNA or protein, myc mRNA or protein, or any combination of these; (h) the level of HIF-l ⁇ mRNA or protein, HIF-l ⁇ mRNA or protein, HIF -2 ⁇ mRNA or protein or any combination of the foregoing; (i) the level of VHL mRNA or protein or or a mutation affecting VHL expression; (j) the level of RHEB mRNA or protein or the activity of RHE
  • this difference is indicative of an increased likelihood that the tumor is sensitive to the mTOR inhibitor relative to a situation in which the level of PTEN expression or activity in the tumor is equivalent to that in normal cells or in tumors that are resistant to an mTOR inhibitor.
  • any of the above-mentioned indicators, or any group of two or more of the indicators is assessed and used in conjunction with an assessment of the level of expression or activity of an FKBP protein to evaluate the likelihood that a tumor is sensitive to an mTOR inhibitor.
  • One or more indicators may be assessed in a single sample, or different samples may be used to assess different indicators.
  • one or more of the above-mentioned indicators is assessed and used to evaluate the likelihood that a tumor is sensitive to an mTOR inhibitor and/or that a subject will exhibit a favorable response to an mTOR inhibitor.
  • the invention provides a method for evaluating the likelihood that a tumor is sensitive to an mTOR inhibitor comprising assessing the level of myc mRNA or protein, or any combination of these is assessed.
  • the level of HIF-I ⁇ mRNA or protein, HIF- l ⁇ mRNA or protein, HIF-2 ⁇ mRNA or protein or any combination of the foregoing is assessed.
  • the level of VHL mRNA or protein or presence of a mutation affecting VHL expression is assessed.
  • the level of RHEB mRNA or protein or the activity of RHEB protein is assessed.
  • the level of Raptor mRNA, protein, or activity, or presence of a mutation affecting Raptor expression or activity is assessed.
  • the level of G ⁇ L mRNA, protein, or activity, or presence of a mutation affecting G ⁇ L expression or activity is assessed.
  • an indicator involving an upstream component of an mTOR pathway is assessed, and the assessment is used in conjunction with an assessment of the level of expression or activity of an FKBP protein to predict the likelihood that a tumor is sensitive to an mTOR inhibitor.
  • an indicator involving a downstream component of an mTOR pathway is assessed, and the assessment is used in conjunction with an assessment of the level of expression or activity of an FKBP protein to predict the likelihood that a tumor is sensitive to an mTOR inhibitor.
  • at least two indicators are assessed in addition to assessing the level of expression or activity of an FKPB protein. For example, a first indicator involving an upstream component and a second indicator involving a downstream component may be used in conjunction with the level of expression or activity of an FKBP protein to predict the likelihood that a tumor is sensitive to an mTOR inhibitor.
  • VEGF vascular endothelial growth factor
  • HIF-l ⁇ HIF-I ⁇
  • HIF-2 ⁇ HIF-2 ⁇
  • VEGF vascular endothelial growth factor
  • the term "VEGF” is used herein to refer to any VEGF polypeptide, or any combination of VEGF polypeptides (e.g., VEGF-A, VEGF-B, VEGF-C, VEGF-D, or any combination of the foregoing). In other words, in various embodiments of the invention any VEGF polypeptide or combination thereof may be assessed.
  • Angiogenesis-related biomarkers include indicators related to the level of expression of HIF-l ⁇ , HIF- l ⁇ , HIF -2 ⁇ , or VEGF in a tumor, the level of a circulating VEGF polypeptide in a subject, the proportion or level of circulating endothelial cells (CECs) in a subject, and the proportion or level of circulating endothelial progenitor cells (CEPs) in a subject.
  • CECs circulating endothelial cells
  • CEPs circulating endothelial progenitor cells
  • an angiogenesis-related indicator and an assessment of the level of expression or activity of an FKBP protein are used in conjunction to evaluate the likelihood that a tumor is sensitive to an mTOR inhibitor and/or the likelihood that a subject will respond to an mTOR inhibitor.
  • any group of two or more of the indicators is assessed and the assessments are used in conjunction with one another to evaluate the likelihood that a tumor is sensitive to an mTOR inhibitor.
  • an assessment of the expression or activity of an FKBP protein is used in conjunction with assessments of two or more indicators.
  • Particular groups that can be utilized include (a) any indicator related to an upstream component of an mTOR pathway and any indicator related to a downstream component of an mTOR pathway; (b) any indicator related to an upstream component of an mTOR pathway and any angiogenesis-related indicator; (c) any indicator related to a downstream component of an mTOR pathway and any angiogenesis-related indicator; (d) the level of expression or activity of mTOR and any other indicator.
  • any of the above methods may be used to evaluate the likelihood that a subject having a tumor will exhibit a favorable response to an mTOR inhibitor.
  • FKBP protein e.g., FKBP 12
  • FKBP 12 FKBP 12
  • FKBP protein e.g., FKBP 12
  • FKBP 12 FKBP protein
  • FKBP protein e.g., FKBP 12
  • FKBP 12 FKBP 12
  • mTOR protein e.g., FKBP 12
  • p the proportion or level of mTOR protein that is phosphorylated
  • p the level of Raptor mRNA, protein, or activity
  • q the level of G ⁇ L mRNA, protein, or activity.
  • FKBP protein e.g., FKBP 12
  • FKBP 12 FKBP protein
  • CECs circulating endothelial cells
  • CEPs circulating endothelial progenitor cells
  • one or more of the indicators listed below is/are assessed prior to initiation of treatment with an mTOR inhibitor (e.g., typically within a relatively short time window such as days to 1-2 weeks prior to initiation of therapy) and then again one or times at a relatively early time point in the treatment, e.g., within several hours, days, or weeks after administration of the initial dose of the agent.
  • the indicator is measured after a period of time sufficient to allow the mTOR inhibitor to reach a steady-state plasma concentration, or shortly thereafter.
  • the values of an indicator obtained prior to and following administration of an mTOR inhibitor to a subject are compared.
  • the comparison is used to evaluate the likelihood that the tumor is sensitive to an mTOR inhibitor and/or the likelihood that the subject will experience a favorable response to the mTOR inhibitor.
  • the result of the comparison e.g., the extent to which the value of the indicator changes following administration of the mTOR inhibitor, is compared with a reference value.
  • the reference value in this case is the change in the value of the indicator that occurs following administration of an mTOR inhibitor to subjects who did not exhibit a favorable response to an mTOR inhibitor, or the change in the indicator that occurs following administration of an mTOR inhibitor to subjects who did exhibit a favorable response to the mTOR inhibitor.
  • Certain changes in the value of an indicator following administration of an mTOR inhibitor are indicative of an increased likelihood that the tumor is sensitive to the mTOR inhibitor and/or that the subject is exhibiting or will exhibit a favorable response to the mTOR inhibitor. Such changes are referred to as "favorable alterations". For example, if a tumor is not sensitive to an mTOR inhibitor, then an indicator of the rate of cell proliferation in the tumor would not be expected to decrease following administration of the mTOR inhibitor, i.e., there would be no alteration, or there would be an increase in the value of the indicator. If the tumor is sensitive to the mTOR inhibitor, then an indicator of the rate of cell proliferation in the tumor would be expected to decrease following administration of the mTOR inhibitor.
  • a favorable alteration in one or more indicators is indicative of an increased likelihood that the tumor is sensitive to the mTOR inhibitor and, in general, is indicative of an increased likelihood that the subject is exhibiting or will exhibit a favorable response to the mTOR inhibitor, relative to the likelihood of such response if the favorable alteration is not observed.
  • one or more of the following indicators is/are evaluated in a tumor or in a subject with a tumor: (a) the proportion or level of TSC2 protein that is phosphorylated; (b) the proportion or level of AKT protein that is phosphorylated; (c) the proportion or level of S6 protein that is phosphorylated; (d) the proportion or level of S6 kinase protein that is phosphorylated; (e) the proportion or level of 4E-BP1 protein that is phosphorylated; (f) the proportion or level of mTOR protein that is phosphorylated; (g) the level of cyclin D 1 mRNA or protein, cyclin D3 mRNA or protein, myc mRNA or protein, or any combination of these; (h) the level of HIF-I ⁇ mRNA or protein, HIF-2 ⁇ mRNA or protein; HIF- l ⁇ mRNA or protein, or any combination of the foregoing (i) the level of VHL mRNA or protein or
  • the following alterations following administration of an mTOR inhibitor are indicative of an increased likelihood that a tumor is sensitive to the mTOR inhibitor and are considered favorable alterations: (a) a decrease in the proportion or level of TSC2 protein that is phosphorylated; (b) a decrease in the proportion or level of AKT protein that is phosphorylated; (c) a decrease in the proportion or level of S6 protein that is phosphorylated; (d) a decrease in the proportion or level of S6 kinase protein that is phosphorylated; (e) a decrease in the proportion or level of 4E-BP1 protein that is phosphorylated; (f) a decrease in the proportion or level of mTOR protein that is phosphorylated; (g) a decrease in the level of cyclin Dl mRNA or protein, cyclin D3 mRNA or protein, myc mRNA or protein, or any combination of these; (h) a decrease in the level of HIF-I ⁇ mRNA or protein, HIF -2
  • an indicator based on detecting an upstream component of an mTOR pathway e.g., TSC2, AKT, mTOR
  • an indicator based on detecting a downstream component of an mTOR pathway e.g., S6, S6 kinase, cyclin Dl, cyclin D3, myc, 4E-BP1
  • Any combination of indicators can be assessed.
  • the level of expression or activity of an FKBP protein is assessed and its value is used in conjunction with a favorable or unfavorable alteration in one or more of the above indicators to evaluate the likelihood that a tumor is sensitive to an mTOR inhibitor.
  • the size of a favorable alteration can vary.
  • a favorable alteration can be an increase or decrease of at least approximately 5%, 10%, 20%, 30%, 40%, 50%, 60%, 60%, 80%, 90%, 100%, 150%, 200% (2-fold), 300% (3-fold), 4-fold, 5-fold, 10-fold, etc., of the pre-administration value for the indicator. Any subrange or specific value (specified to the tenths place) within these ranges is a favorable alteration according to various embodiments of the invention.
  • a favorable alteration can be a change from a value that indicates cell proliferation to a value that indicates a lower degree of cell proliferation, i.e., a lower cell proliferation rate. For example, a decline in standard uptake value in a PET scan is a favorable alteration in certain embodiments of the invention.
  • an FKBP protein e.g., FKBP 12
  • FKBP 12 an FKBP protein, e.g., FKBP 12
  • an mTOR inhibitor (a) the proportion or level of TSC2 protein that is phosphorylated; (b) the proportion or level of AKT protein that is phosphorylated; (c) the proportion or level of S 6 protein that is phosphorylated; (d) the proportion or level of S6 kinase protein that is phosphorylated; (e) the proportion or level of 4E-BP1 protein that is phosphorylated; (f) the proportion or level of mTOR protein that is phosphorylated.
  • an FKBP protein e.g., FKBP 12
  • FKBP 12 in conjuction with assessing a change that occurs in one or more of the following after administration of an mTOR inhibitor: (g) the level of cyclin Dl mRNA or protein, cyclin D3 mRNA or protein, myc mRNA or protein, or any combination of these; (h) the level of HIF-l ⁇ mRNA or protein, HIF- l ⁇ mRNA or protein, HIF-2 ⁇ mRNA or protein or any combination of the foregoing.
  • an FKBP protein e.g., FKBP 12
  • an FKBP inhibitor e.g., FKBP 12
  • a change that occurs in one or more of the following after administration of an mTOR inhibitor (f) the proportion or level of mTOR protein that is phosphorylated; (j) the level of RHEB mRNA or protein or the activity of RHEB protein; (o) the level of PTEN mRNA, protein, or activity, or a mutation affecting PTEN expression; (p) the level of Raptor mRNA, protein, or activity, or a mutation affecting Raptor expression; (q) the level of G ⁇ L mRNA, protein, or activity, or a mutation affecting G ⁇ L expression.
  • Methods for assessing the proportion or level of a phosphorylated protein include immunological methods that make use of phospho-specific antibodies, i.e., antibodies that detect a phosphorylated form of a protein but generally do not substantially detect unphosphorylated forms. Suitable immunological methods include Western blots, immunoassays, and IHC, which are described above. A phospho-specific antibody may specifically detect a form of the protein that is phosphorylated at a particular position. Suitable phospho-specific antibodies are known in the art and are described, e.g., in U.S. Pub.
  • Various of the indicators involve detecting or measuring an activity of one or more components of an mTOR pathway. Methods of assessing an activity are selected in accordance with the particular activity. For example, AKT, IGF-IR, S6 kinase, and mTOR are kinases, and their kinase activity can be measured by performing a kinase assay using an appropriate substrate or by examining the amount of a phosphorylated substrate in a sample using a phosphor-specific antibody. Relevant natural and synthetic substrates and methods for performing kinase assays are known in the art.
  • RHEB is a GTPase, and an appropriate method for assessing its activity is a GTPase assay.
  • PTEN is a phosphatase
  • an appropriate method for assessing its activity is a phosphatase assay. See, e.g.,Rahman, A., et al., Bioassay Techniques for Drug Development, Harwood Academic Publishers, Amsterdam, 2001, for description of a large number of cell-based assays.
  • Raptor and G ⁇ L form complexes with mTOR, and an appropriate assay for their activity is a binding assay, e.g., a co-immunoprecipitation assay, immunoaffinity assay, competition assay, etc.
  • Activity can also be assessed by determining whether a mutation exists in an active site or in a region of the protein needed for activity. For example, phosphorylation sites are known to be important for activity of certain of the kinases mentioned above. Mutations at these sites will generally diminish activity of the kinase. Mutations that affect the activity of other molecules mentioned above are known in the art or can be determined by sequencing DNA from tumors in which such activity is altered.
  • An exemplary mutation is a mutation in a region of mTOR that interacts with the FKBP12/rapamycin complex, e.g., the FRB region (Chen, et al., Proc. Natl. Acad. Sci. USA, 92: 4947-4951, 1995; Choi, et al., Science, 273:239-42, 1996), which may alter the binding of an FKBP/inhibitor complex to mTOR. [00175] B. AnRJogenesis-Related Biomarkers
  • the invention provides a variety of biomarkers that reflect the degree of vascularization of a tumor or the degree of angiogenic activity of the tumor. Certain of these biomarkers are of use for evaluating the likelihood that a tumor is sensitive to an mTOR inhibitor. Certain additional angiogenesis-related biomarkers are of use for monitoring the response of a subject to therapy with an mTOR inhibitor.
  • the invention provides a method of evaluating the likelihood that tumor is sensitive to an mTOR inhibitor comprising measuring the level of CEPs in a subject prior to administration of an mTOR inhibitor, administering the mTOR inhibitor to the subject, and measuring the level of CEPs in the subject within a short time window (e.g., days to weeks) following administration of the mTOR inhibitor, wherein a decrease in the number of CEPs following administration of the mTOR inhibitor is indicative of an increased likelihood that the subject has a tumor that is sensitive to the mTOR inhibitor.
  • a short time window e.g., days to weeks
  • the invention also provides a method of evaluating the likelihood that tumor is sensitive to an mTOR inhibitor comprising measuring the level of circulating VEGF polypeptide in a subject prior to administration of an mTOR inhibitor, administering the mTOR inhibitor to the subject, and measuring the level of circulating VEGF polypeptide in the subject within a short time window (e.g., days to weeks) following administration of the mTOR inhibitor, wherein a decrease in the amount of VEGF polypeptide following administration of the mTOR inhibitor is indicative of an increased likelihood that the subject has a tumor that is sensitive to the mTOR inhibitor.
  • a short time window e.g., days to weeks
  • the level of VEGF in the tumor is measured, wherein a decrease in VEGF in the tumor is indicative of an increased likelihood that the subject has a tumor that is sensitive to the mTOR inhibitor.
  • Methods for evaluating the angiogenesis-related indicators include methods for detecting mRNA or protein discussed above, and functional imaging methods such as those discussed below.
  • an angiogenesis-related indicator and a second indicator based on detecting an alteration in the proportion or level of phosphorylation of an mTOR pathway component are measured.
  • a favorable alteration in both indicators indicates an increased likelihood that the tumor is sensitive to the mTOR inhibitor relative to the likelihood in which there is a favorable alteration in only one of the indicators.
  • FKBP protein e.g., FKBP 12
  • FKBP 12 FKBP 12
  • the invention encompasses the use of various imaging techniques to assess the ability of an mTOR inhibitor to inhibit tumor growth in vivo.
  • the methods may be used to identify a subject who is a suitable candidate for therapy with an mTOR inhibitor and/or to evaluate or predict the likelihood that a subject will exhibit a favorable response to an mTOR inhibitor.
  • Conventional imaging modalities including conventional X-rays, conventional CT scans, or conventional magnetic resonance imaging (MRI) scans, rely on detecting changes in tumor mass and volume that can take weeks or months to become evident.
  • MRI magnetic resonance imaging
  • a functional imaging technique is used.
  • a functional imaging technique detects a feature of the tumor that is indicative of its growth rate or state such as cell metabolism, cell proliferation, blood flow, etc.
  • the invention provides the recognition that functional imaging is of particular use in selecting patients for therapy with an mTOR inhibitor, for predicting the likelihood of response, and/of for monitoring therapy.
  • a functional imaging technique may also detect changes in tumor mass and volume in addition to providing functional information, e.g., information related to the physiological state and/or physiological processes of the tumor cells.
  • Positron emission tomography using a labeled metabolic substrate or precursor is a preferred functional imaging technique.
  • 18 F 2-fluoro-2- deoxyglucose ([ 18 F]-FDG) positron emission tomography (FDG-PET) can be used to measure cellular metabolic activity since the rate of [ 18 F]-FDG accumulation in tumors is proportional to the rate of glucose utilization (Gupta, N., et al., Eur. J. Cancer, 38:2094-2107, 2002).
  • EORTC European Organization for Research and Treatment of Cancer
  • these guidelines or updated versions thereof are used to assess tumor response to administration of an mTOR inhibitor.
  • PET methodology is also available for measuring radiolabeled thymidine (e.g., 2-[ 1 1 C] thymidine or 3'-deoxy-3'-[ ! 8 F] fluorothymidine (FLT) incorporation into DNA to provide an assessment of cell proliferation and thus tumor growth rate in vivo.
  • radiolabeled thymidine e.g., 2-[ 1 1 C] thymidine or 3'-deoxy-3'-[ ! 8 F] fluorothymidine (FLT) incorporation into DNA to provide an assessment of cell proliferation and thus tumor growth rate in vivo.
  • the invention also encompasses use of PET to measure angiogenesis, hypoxia, and/or apoptosis in a tumor prior to and/or following administration of an mTOR inhibitor.
  • VEGF is upregulated in a number of tumor types.
  • Angiogenesis can be measured, e.g., by PET, after administering a labeled antibody to VEGF.
  • Blood flow can be measured by PET after administration of labeled (e.g., 15 O-labeled) H 2 O and/or CO.
  • labeled e.g., 15 O-labeled
  • functional CT and functional MRI are capable of detecting blood flow and metabolic activity in tumors using appropriate contrast agents, etc.
  • an image that provides functional information about a tumor is obtained from a subject prior to administration of an mTOR inhibitor.
  • the image is obtained within a relatively short window of time prior to administration of the mTOR inhibitor (e.g., immediately before (within hours), up to several (e.g., 1-4) days before, up to 1 week before, or up to several weeks before administration of an mTOR inhibitor.
  • the mTOR inhibitor is then administered to the subject, and a second functional image is obtained.
  • the second functional image is obtained a relatively short time after administration of the mTOR inhibitor, e.g., after the mTOR inhibitor has reached a steady-state plasma concentration, after several days, after approximately 1 week, 1- 2 weeks, up to 4 weeks, etc., after administration of the mTOR inhibitor.
  • a significant decrease in a parameter indicative of cell metabolism and/or cell proliferation is indicative of an increased likelihood that the tumor is sensitive to the mTOR inhibitor and is indicative of an increased likelihood that the subject is exhibiting or will exhibit a favorable response to the mTOR inhibitor, relative to the likelihood of sensitivity and/or response if there is no decrease in the parameter.
  • the invention encompasses any method in which a functional image of a tumor is obtained prior to and following administration of an mTOR inhibitor, e.g., a rapamycin analog, to a subject with the tumor.
  • FKBP protein e.g., FKBP 12
  • FKBP12 FKBP12
  • a change in tumor metabolism and/or cell proliferation e.g., using PET.
  • a favorable value for FKBP protein expression or activity in conjunction with a favorable alteration in SUV may indicate an increased likelihood that a subject will exhibit a favorable response to an mTOR inhibitor, relative to the likelihood in a situation in which the subject does not have a favorable value for the FKBP protein and/or does not exhibit a favorable alteration in SUV (or another measurement of tumor cell metabolism and/or proliferation)
  • an FKBP protein e.g., FKBP 12
  • FKBP 12 FKBP 12
  • a second indicator of the likelihood that a tumor is sensitive to an mTOR inhibitor and/or that a subject will exhibit a favorable response to an mTOR inhibitor wherein the second indicator is any of the non image-based indicators discussed herein.
  • the present invention may be used to evaluate the likelihood that a tumor is sensitive to any of a variety of mTOR inhibitors and/or to evaluate the likelihood that a subject will respond to any of a variety of mTOR inhibitors.
  • the mTOR inhibitor is a rapamycin analog, which term includes rapamcyin derivatives such as those described below.
  • Rapamycin is a macrolide antibiotic produced by Streptomyces hygroscopicus and discovered in the 1970's. Rapamycin is a potent immunosuppressive agent and is used clinically to prevent rejection of transplanted organs.
  • rapamycin and everolimus are used as immunosuppressants in organ transplant recipients, and rapamycin and a number of the C-43-modified rapamycin analogs are being used, evaluated or developed for use on stents as anti- restenotic agents following interventional cardiology.
  • rapamycin derivatives modified at position 43 and/or at one or more other positions are known.
  • certain other O-substituted rapamycins are disclosed in WO 94/02136, U.S. Pat. No. 5,258,389 and WO 94/09010 (O-aryl and O-alkyl rapamycins); see also WO 92/05179 (carboxylic acid esters), U.S. Pat. No. 5,118,677 (amide esters), U.S. Pat. No. 5, 118,678 (carbamates), U.S. Pat. No. 5,100,883 (fluorinated esters), U.S. Pat. No.
  • rapamycin derivatives are described in PCT application number EP96/02441, for example 32-deoxorapamycin as described in Example 1 therein, and 16pent-2-ynyloxy-32(S)-dihydrorapamycin as described in Examples 2 and 3 therein (using that document's numbering system).
  • EP96/02441 32-deoxorapamycin as described in Example 1 therein
  • 16pent-2-ynyloxy-32(S)-dihydrorapamycin as described in Examples 2 and 3 therein (using that document's numbering system).
  • analogs and derivatives of rapamycin see, e.g., WO 01/144387 and the references in Table 2.
  • Table 2 Table 2
  • mTOR inhibitors include, but are not limited to, small molecule or antibody inhibitors of any of the kinases in the mTOR pathway (e.g., AKT, PI3 kinase), e.g., wortmannin, LY compounds, etc. Other inhibitors disrupt the interaction between G ⁇ L and mTOR, or between Raptor and mTOR. Agents such as siRNA or antisense molecules that reduce expression of mTOR, AKT, eIF4E, Raptor, or G ⁇ L, are additional examples of mTOR inhibitors. [00196] VI. Kits, Databases, and Diafinostic Instruments
  • the invention provides reagents and kits for evaluating the likelihood that a tumor is sensitive to an mTOR inhibitor and/or for evaluating the likelihood that a subject with a tumor will exhibit a favorable response to an mTOR inhibitor.
  • the kits comprise a validated reagent for assessing the expression or activity of an FKBP protein in a sample or in a subject.
  • validated is meant that the reagent has been used to assess the level of expression or activity of an FKBP protein in a plurality of samples derived from tumors that have been exposed to an mTOR inhibitor, and/or in a plurality of subjects with tumors to whom an mTOR inhibitor has been administered.
  • the results obtained using the reagent are reproducible and consistent.
  • the results obtained using the reagent are of use for evaluating the likelihood that a tumor is sensitive to the mTOR inhibitor and/or that a subject will experience a favorable response to an mTOR inhibitor.
  • the reagent is an antibody, antibody fragment, antibody derivative, or ligand that specifically binds to the FKBP protein.
  • the reagent is a nucleic acid that hybridizes to a nucleic acid that encodes the FKBP protein.
  • the FKBP protein is FKBP 12.
  • the antibody, antibody fragment, antibody derivative, or ligand may be labeled.
  • labeled FK506, labeled rapamycin, or a labeled rapamycin analog such as AP 1491 (fluoresceinated FK506) can be used.
  • the kit further comprises at least one item selected from the group consisting of: a positive control sample, a negative control sample, one or more reference samples, a substrate, an enzyme, a wash solution, a reagent for assessing a second indicator of the likelihood that a tumor is sensitive to an mTOR inhibitor; and instructions for use of the kit.
  • the reagent for assessing a second indicator of the likelihood that a tumor is sensitive to an mTOR inhibitor may be a validated reagent.
  • the kit comprises a reagent for assessing the expression or activity of an FKBP protein in a sample or in a subject and further comprises at least one item selected from the group consisting of: a reagent for assessing a second indicator of the likelihood that the tumor is sensitive to an mTOR inhibitor, a positive control sample, a negative control sample, one or more reference samples, a substrate, an enzyme, a wash solution, and instructions for use of the kit.
  • a reagent for assessing a second indicator of the likelihood that the tumor is sensitive to an mTOR inhibitor a positive control sample, a negative control sample, one or more reference samples, a substrate, an enzyme, a wash solution, and instructions for use of the kit.
  • Either or both of the reagents may be a validated reagent.
  • a kit contains one or more antibodies or other binding agents, each of which specifically binds to a polypeptide selected from the group consisting of: IGF-Rl, PTEN, AKT, TSC2, S6, S6 kinase, mTOR, G ⁇ L, Raptor, 4E-BP1 , HIF-I ⁇ , or any of the other components of an mTOR pathway discussed herein.
  • one or more of the antibodies is a phospho-specif ⁇ c antibody.
  • an antibody that binds to a phosphorylated form of AKT, S6, S6 kinase, 4E-BP1, TSC2, mTOR, etc. can be included.
  • an antibody that recognizes phosphorylated or unphosphorylated forms of one or more of the afore-mentioned polypeptides are available, e.g., from Cell Signaling Technologies (Beverly, MA).
  • the positive control sample may be, e.g., cells that express the FKBP protein.
  • the negative control may be, e.g., cells in which the FKBP protein is not expressed at a level that is detectable using the kit or is expressed at a low level.
  • the reference sample may be, e.g., cells from a tumor that is sensitive to an mTOR inhibitor, cells from a tumor that is resistant to an mTOR inhibitor, etc.
  • the positive, negative, and/or reference sample may comprise cells provided on a slide.
  • Kits will generally include one or more vessels, tubes, or containers so that certain of the individual kit components may be separately housed.
  • the kits may also include a means for enclosing the individual containers in relatively close confinement for commercial sale, e.g., a plastic or Styrofoam box, in which instructions, packaging materials, etc., may be enclosed.
  • Containers or substrates, e.g., slides, microliter dishes, etc., for performing an assay may be included.
  • the kits and/or reagent containers may have a bar code or radio frequency (RF) ID tag.
  • RF radio frequency
  • the invention therefore provides a computer- readable medium (e.g., hard or floppy disk, zip disk, CD, flash memory, or the like) on which is stored: (a) a value for each of one or more indicators selected from the group consisting of: the level of expression or activity of an FKBP protein; the proportion or level of TSC2 protein that is phosphorylated; the proportion or level of AKT protein that is phosphorylated; the proportion or level of S 6 protein that is phosphorylated; the proportion or level of S6 kinase protein that is phosphorylated; the proportion or level of 4E-BP1 protein that is phosphorylated; the proportion or level of mTOR protein that is phosphorylated; the level of cyclin Dl mRNA or protein, cyclin D3 mRNA or protein, myc mRNA or protein, or any combination of these; the level of HIF-I ⁇ mRNA or protein, HIF-2 ⁇ mRNA or protein; HIF-I ⁇ mRNA or protein, or any combination
  • the values and information may be stored in any convenient format.
  • the computer-readable medium will store values for one or more indicators, wherein the values are obtained from a plurality of different samples derived from tumors or from subjects having a tumor.
  • the values and information are associated with each other in the sense that the computer-readable medium stores sufficient information so that it is possible to determine for each tumor, sample, or subject for which an indicator value is stored, the tumor-related information relevant to that tumor, sample, or subject.
  • the values and tumor-related information are stored in a database, e.g., a relational database.
  • a database e.g., a relational database.
  • the samples, tumors, and/or subjects will be identified by an identifier, e.g., an alphanumeric code, bar code reference number or bar code character string, name, social security number, etc.
  • the invention encompasses a computer or other instrument that comprises a computer-readable medium as described above.
  • the invention further encompasses an automated staining machine, e.g., for performing IHC to assess one or more of the indicators described herein, e.g., to assess the level or activity of an FKBP protein, e.g., FKBP12.
  • the indicators described herein are generally based on the identification of differences between groups, such that a difference in the indicator between different groups correlates with a physiologically relevant characteristic or feature, e.g., a difference in sensitivity to an mTOR inhibitor.
  • groups of tumors that express different levels of an FKBP protein may display differential sensitivity to an mTOR inhibitor
  • groups of subjects with tumors that expess different levels of an FKBP protein may display differential responsiveness to an mTOR inhibitor such that there is a difference in the response rate between groups of subjects whose tumors express different levels of an FKBP protein.
  • Statistical methods known to one of ordinary skill in the art can be used to demonstrate a statistically significant difference for an indicator between groups and to demonstrate a correlation with a characteristic or feature of interest, e.g., a therapeutically relevant characteristic such as sensitivity to an mTOR inhibitor.
  • a statistically significant difference is one that has a less than 5% likelihood of occurring by chance.
  • Suitable methods for demonstrating correlations include t-tests (e.g., Fisher t test), chi square tests, determination of the Pearson correlation coefficient, linear regression, cluster analysis, etc. See, e.g., Motulsky, H., Intuitive Biostatistics (ISBN 0-19-508607-4), Oxford University Press Inc., 1995; and US Pub. No. 20030190689.
  • t-tests e.g., Fisher t test
  • chi square tests determination of the Pearson correlation coefficient, linear regression, cluster analysis, etc. See, e.g., Motulsky, H., Intuitive Biostatistics (ISBN 0-19-508607-4), Oxford University Press Inc., 1995; and US Pub. No. 20030190689.
  • selection of a suitable statistical test will depend upon the question being asked, type of data, etc., and that suitable tests are selected for any particular experiment or trial. Exemplary tests and appropriate situations for their use are listed in Table 3. Software for performing statistical tests is widely available.
  • Cluster analysis can be performed.
  • Programs for performing hierarchical clustering are publicly available at the Stanford University web site or at the web site having URL rana.lbl.gov/EisenSoftware.htm and widely known in the art (initially described in Eisen M.B., et al. Proc. Natl. Acad. Sci. USA , 95:14863-14868, 1998 and subsequently modified).
  • a relationship between the value(s) of one or more indicator(s) and the likelihood that a tumor is sensitive to an mTOR inhibitor and/or the likelihood that a subject with a tumor will exhibit a favorable response to an mTOR inhibitor may be determined using regression in the case of a single indicator or multiple regression in the case of multiple indicators.
  • the relationship can be linear or nonlinear.
  • a multivariate logistic regression model is used (Choe, G., et al., Cancer Res., 63:2742- 2746, 2003).
  • a variety of statistical methods known to one of ordinary skill in the art may likewise be used to determine reference values for practicing the methods herein.
  • determination of a reference value involves assessing a plurality of samples having certain characteristics of interest in common and combining the values obtained therefrom in any of a number of different ways to arrive at a value that is characteristic of the plurality and that correlates with a characteristic or feature of interest, e.g., a therapeutically relevant characteristic such as sensitivity to an mTOR inhibitor.
  • a mean, median, mode, or other statistic can be computed. Not all of the values need to be considered in arriving at the reference value. For example, outliers may be excluded.
  • One of ordinary skill in the art is aware of appropriate statistical methods for obtaining a reference value from a plurality of values.
  • a reference value for an indicator is obtained from one or more groups of samples or subjects.
  • a reference value may be obtained from a plurality of (a) samples containing normal cells; (b) samples from tumors that are sensitive to an mTOR inhibitor; (c) samples from tumors that are resistant to an mTOR inhibitor; (d) samples from tumors in subjects who did exhibited a favorable response to an mTOR inhibitor; (e) samples from tumors in subjects who did not exhibit a favorable response to an mTOR inhibitor, (f) blood samples from subjects not having a tumor; (g) blood samples from subjects who exhibited a favorable response to an mTOR inhibitor; (h) blood samples from subjects who did not exhibit a favorable response to an mTOR inhibitor, etc.
  • reference values can be obtained from (i) normal tissue; (j) tumors in subjects who exhibited a favorable response to an mTOR inhibitor; (k) tumors in subjects who did not exhibit a favorable response to an mTOR inhibitor.
  • the number of samples or subjects assessed in each group can vary, e.g., from as few as 2 to dozens, hundreds, thousands, etc. While it is generally preferred to obtain reference values from multiple samples or subjects, in some embodiments of the invention a reference value is obtained from a single sample or subject.
  • a reference value for an indicator is obtained from a tumor cell line, e.g., any of the tumor cell lines listed in Example 1.
  • a reference value obtained from a tumor cell line that is relatively sensitive to an mTOR inhibitor can be used, or a reference value obtained from a tumor cell line that is relatively resistant to an mTOR inhibitor can be used.
  • an indicator e.g., an FKPB expression or activity level, is measured in archived tumor samples, particularly from subjects who have been treated with an mTOR inhibitor. The results are correlated with the likelihood that a subject having the tumor exhibited a favorable response to treatment and are used to obtain appropriate reference values for practicing one or more of the methods herein.
  • tissue microarrays typically comprise dozens to hundreds of cylindrical tissue biopsies (diameter 0.6 mm) from morphologically representative regions of individual tumors, which are arrayed in a single paraffin block. Consecutive sections from such arrays provide targets for parallel in situ visualization and/or quantitation of DNA, RNA or protein targets. See, e.g., U.S. Pub. Nos. 20020192702 and 20030215936.
  • the samples may or may not be from subjects who were treated with an mTOR inhibitor.
  • Analysing large numbers of tumor samples e.g., by assessing the level of FKBP protein or assessing one or more of the other indicators described herein, allows the identification of correlations between values for these indicators and particular histopathologic types, grades, clinical stages, etc., and/or allows the identification of correlations between indicators. [00217] VIIL Methods of Treatment
  • This invention thus also provides a method for treating a subject suffering from a tumor, the method comprising the steps of (1) evaluating the likelihood that the tumor is sensitive to an mTOR inhibitor; and (2) administering an mTOR inhibitor to the subject.
  • the invention further provides a method for treating a subject suffering from a tumor, the method comprising the steps of (1) evaluating the likelihood that the subject will exhibit a favorable response to an mTOR inhibitor; and (2) administering an mTOR inhibitor to the subject.
  • the step of evaluating is performed according to one of the methods described herein.
  • the step of evaluating may comprise evaluating one or more of any of the indicators described herein.
  • the step of evaluating may comprise assessing the expression level or activity of an FKBP protein, e.g., FKBP12, in a sample derived from the subject in need of such evaluation, or assessing an indicator of the expression or activity of the FKBP protein in the tumor in vivo, wherein the value of the indicator, relative to a reference value for the indicator, is predictive of the likelihood that the tumor is sensitive to the mTOR inhibitor.
  • FKBP protein e.g., FKBP12
  • the step of evaluating comprises (a) assessing the expression level or activity of an FKBP protein, e.g., FKBP 12, in a sample derived from the subject in need of such evaluation, or assessing an indicator of the expression or activity of the FKBP protein in the tumor in vivo, wherein the value of the indicator, relative to a reference value for the indicator, is predictive of the likelihood that the tumor is sensitive to the mTOR inhibitor; and (b) assessing at least one additional indicator of the likelihood that the tumor is sensitive to an mTOR inhibitor, wherein the value of the at least one additional indicator, relative to a reference value for the indicator, is predictive of the likelihood that the tumor is sensitive to the mTOR inhibitor.
  • an FKBP protein e.g., FKBP 12
  • the at least one additional indicator may be any of the indicators mentioned herein.
  • the practice of the above methods of treatment will constitute treating a subject selected for a higher likelihood that his or her tumor is sensitive to an mTOR inhibitor and/or that the subject will respond to an mTOR inhibitor relative, for example, to the likelihood of such sensitivity and/or response in the overall population of tumor and subjects.
  • the result of such treatment is beneficial to the overall health of the subject.
  • Preferred mTOR inhibitors include but are not limited to Serolimus, Temsirolimus, Everolimus, and AP23573.
  • the mTOR inhibitor of steps (1) and (2) of the above methods is the same, e.g., the likelihood of sensitivity to a specific mTOR inhibitor is evaluated, and that particular mTOR inhibitor is administered to the subject.
  • the application of the foregoing methods to patients with one of the various sarcomas; breast cancer; endometrial cancer; prostate cancer; a leukemia, lymphoma or other hematologic cancer; or a brain cancer, although the invention is applicable to a far wider range of cancers as discussed herein.
  • mTOR inhibitors may be administered as part of a therapeutic regimen that includes one or more additional chemotherapeutic agents and/or as part of a therapeutic regimen that includes a treatment modality such as radiation therapy, surgery, etc.
  • the methods of treatment encompass these situations.
  • the methods may involve administration of one or more additional chemotherapeutic agents.
  • Example 1 Correlation Between FKBP12 Expression Level and Sensitivity to a Rapamycin Analog in Tumor Cell Lines
  • the number of cells in each well was determined using a CellTiter 96® Aqueous One Solution Cell Proliferation metabolic activity assay kit (Promega). This assay is based on the use of a reagent that monitors the total reducing environment of a population of cells. The quantity of reduced products, which is determined spectrophotometrically, is directly proportional to the number of viable cells in the culture. The percentage of cells remaining after treatment with each concentration of AP23573 was then determined relative to the number of cells remaining in the untreated wells. [00227] For each cell line the percentage cells remaining after treatment with AP23573 reached a plateau between 1 and 10 nM.
  • FKBP 12 Proteins were transferred to a membrane for immunoblotting, and the presence of FKBP 12 was determined by probing with an anti-FKBP12 antibody (Affinity Bioreagents # PAl -026A) followed by incubation with a labelled secondary antibody and autoradiography, using a standard immunoblotting procedure. The membranes were stripped and reprobed with an antibody against the GAPDH protein (Abeam # ab9485) as a control. Quantitation was performed by scanning the X-ray film and analyzing the data using ID Image Analysis Software (Kodak). [00230] Results
  • Figure 1 shows the FKBP 12 expression levels in the cell lines.
  • Table 5 shows the correlation between expression levels of FKBP 12 and sensitivity to AP23573.
  • cell lines having higher expression of FKBP 12 relative to other cell lines were more sensitive to AP23573 than cell lines with lower levels of FKBP 12 expression as evidenced by the fact that a greater percentage of cells remained after treatment in the case of cell lines that had lower levels of FKBP 12 expression and a lower percentage of cells remained after treatment in the case of cell lines that had higher levels of FKBP12.
  • HT1080, PC3, ES2, SKBR3 display relatively high levels of FKBP 12 expression and relatively high levels of sensitivity
  • HEClA, BT474, MDA468, DU145, and HCTl 16 display lower levels of FKBP12 expression and lower levels of sensitivity to AP23573.
  • Cell lines having lower levels of expression of FKBP 12 relative to other cell lines were generally less sensitive to AP23573.
  • higher expression of FKBP 12 is positively correlated with sensitivity to an AP23573.
  • Example 2 Effect of Expressing FKBP12 in a Tumor Cell Line that is Resistant to a Rapamycin Analog on mTOR Sensitivity
  • Cell line HCTl 16 is relatively resistant to AP23573 and displays a low FKBP 12 expression level.
  • HCTl 16 cells are transfected with an FKBP 12 expression vector.
  • the human FKBP 12 ORF is obtained by PCR using the cDNA IMAGE clone, CSODGOOl YF135, as a template (Invitrogen).
  • the hFKBP ORF is ligated into the Sail and Notl sites of p4694 to generate pFKBP12.
  • the plasmid is purified using the Qiagen (Valencia, CA) Megaprep endotoxin-free kit according to the manufacturer's directions and verified by sequencing analysis. Transfection is performed using standard procedures. Populations of pFKBP12-transfected and control cells are divided into aliquots for parallel analysis of FKBP 12 expression and AP23573 sensitivity.
  • RNA from aliquots of pFKBP12-transfected cells is isolated a using Nucleospin RNAII kit (Clontech). Five ⁇ g of total RNA is treated with 20 units of DNase and reverse transcribed with oligo dT:random primer (1 :1 at 1.5 ⁇ g each) by using Superscript II (Invitrogen). Fifty nanograms of first-strand cDNA is used in subsequent real-time PCR carried out with GeneAmp 5700 (Applied Biosystems) by using SyBr green dye (Applied Biosystems) as the fluorescent probe.
  • the following gene-specific primer sets generated by using PRIMER EXPRESS (Applied Biosystems) are used: FKBP12 sense primer 5'- AGATGAGTGTGGGTCAGAGA-3', FKBP 12 antisense primer 5'-TAGAAG CTCCACATCGAAGAC-3'.
  • the following parameters are used in the PCR: 10 min denaturation at 95 0 C, 35 cycles of 15 sec at 95°C, and 1 min at 55°C.
  • Cy values are used to quantitate expression in pFKBP12-transfected cells. Cy is the point at which the fluorescence signal rises above the baseline fluorescence and begins to increase exponentially.
  • the Cy value is in logarithmic inverse relationship with the abundance of the transcripts, based on the assumption that CT values increase by « 1 for each twofold dilution. Detection of an greater level of FKBP 12 niRNA in pFKBP12-transfected cells relative to controls (cells transfected with p4694 lacking an insert) confirms that the transfectants express high levels of FKBP 12 mRNA and, presumptively, high levels of FKBP 12 protein.
  • an in vitro binding assay is performed on lysates from aliquots of pFKBP12-transfected cells and control cells using the fluorescein-labeled FKBP 12 ligand AP 1491.
  • the assay is performed essentially as described in U.S. Pat. Nos. 6,150,527 and 6,649,595.
  • a series of in vitro proliferation assays are performed on populations of control cells and populations of pFBKP12- transfected cells exposed to AP23573 at concentrations ranging from 0.01 nM to 100 nM. The assays are performed as described in Example 1.
  • the percentage of cells remaining is determined. A lower percentage of pFKBP12-transfected cells remaining than control cells remaining after treatment at a particular AP23573 concentration is an indication that higher expression of FKBP 12 results in increased sensitivity to AP23573.
  • the correlation between FKBP 12 expression levels and sensitivity to AP23573 is evaluated using linear regression.
  • pFKBP 12-transfected and control cells are harvested for DNA content analysis as described (Lamm, G.M., et al., Nucleic Acids Res. 25, 4855-4857, 1997). Cells are analyzed to determine the proportion of cells arrested in the Gl phase of the cell cycle. Cell-cycle analysis was performed using FACScan (Becton Dickinson) and CELLQUEST and MODFITLT analysis programs (Becton Dickinson). A higher proportion of FKBPl 2-transfected cells in Gl phase than control cells in Gl phase is an indication that higher expression of FKBP12 results in increased sensitivity to AP23573.
  • FKBP 12 expression levels and sensitivity to AP23573 is evaluated using a linear regression model. Differences between sensitivity in pFKBP12-tranfected and control cells are analysed using a t test to determine whether an increase in FKBP 12 expression results in an increase in sensitivity. A P value ⁇ 0.05 is considered statistically significant. Statistical analysis is performed using InStat Statistical Software version 3.05 (San Diego, CA).
  • Example 3 Effect of Reducing Expression ofFKBPU Using siRNA Targeted to FKBP12 in a Tumor Cell Line that is Sensitive to a Rapamycin Analog on mTOR Sensitivity
  • FKBP 12 expression in HTl 080 cells is inhibited using RNA interference by transfecting the cells with a FKBP12-specific siRNA.
  • siRNA construction and transfection is performed as follows: FKBP 12 siRNA is synthesized in vitro using the Silencer short interfering RNA (siRNA) construction kit (Ambion, Austin, TX), according to the manufacturer's instructions. The sense DNA oligo used in in vitro transcription is: 5'- AATAGGCATAGTCTGAGGAGACCTGTCTC-3'.
  • a negative control siRNA sequence is designed by randomizing the above oligonucleotide sequence to obtain 5'- CCTGGGTAGTCTAAATAGAAGGTAGCCTC-3' and the negative control siRNA is synthesized according to the same protocol as the FKBP 12 siRNA.
  • a total of 25 nM siRNA is transfected using the siPORT lipid transfection reagent (Ambion) according to the manufacturer's instructions.
  • FKBP 12 siRN A-transfected cells and control cells are divided into aliquots for parallel analysis of FKBP 12 expression and AP23573 sensitivity.
  • FKBP 12 expression is assessed as described in Example 2.
  • Aliquots of cells are treated with AP23573 and their sensitivity is assessed using a cell proliferation assay and cell cycle analysis as described in Example 2.
  • a higher percentage of FKBP 12 siRNA-transfected cells remaining than control cells remaining after treatment at a particular AP23573 concentration is an indication that decreasing the expression of FKBP12 results in decreased sensitivity to AP23573.
  • the correlation between FKBP 12 expression levels and sensitivity to AP23573 is evaluated using linear regression.
  • a lower proportion of FKBP 12 siRNA-transfected cells in Gl phase than control cells in Gl phase is an indication that lower expression of FKBP12 results in decreased sensitivity to AP23573.
  • the correlation between FKBP 12 expression levels and sensitivity to AP23573 is evaluated using a linear regression model. Differences between sensitivity in pFKBP12-tranfected and control cells are analysed using a t test to determine whether an increase in FKBP 12 expression results in an increase in sensitivity. A P value ⁇ 0.05 is considered statistically significant. Statistical analysis is performed using InStat Statistical Software version 3.05 (San Diego, CA). [00244] Example 4: Correlation Between FKBPl 2 Expression Level and Patient Response to a Rapamycin Analog
  • FKBP 12 expression is evaluated in 50 tumor samples (tissue biopsy samples) from subjects who responded favorably to an mTOR inhibitor and 50 tumor samples from subjects who did not exhibit a favorable response after similar treatment.
  • the experiment is repeated several times using either mixed sets of samples that included samples from tumors of various types or using sets of samples from a single tumor type (prostate cancer, leiomyosarcoma, osteosarcoma).
  • N those patients with at least one response assessment
  • NE not evaluable or treatment ended
  • Samples are prepared for IHC using standard procedures as described (Higgins, JP, et al., Am J. Clin Pathol, 112:241-7, 1999) using microwave heat- induced epitope retrieval in citrate buffer, and FKBP 12 staining is detected using an anti-FKBP12 antibody (Affinity Bioreagents # PAl -026A). The signal is detected using SuperSignal West Pico Chemiluminescent Substrate (Pierce, Rockford, IL). Samples are assigned a score ranging from 0 to 3+, based on the level of staining as observed visually. Scores are assigned by three independent observers and the results averaged.
  • the analyses are performed multiple times. In some analyses the scores are reduced to "positive” or “negative", with 0 being considered negative and 1+, 2+, and 3+ being considered positive. In other analyses 0 and 1+ are considered negative with 2+ and 3+ being considered positive. In other analyses only 3+ is considered positive. Ten visual fields from different areas of each tumor are used for evaluation of the score. Negative control slides without primary antibody are included for each staining. Normal epithelium of the relevant tissue type or vascular endothelium known to express FKBP 12 is used as a positive control.
  • Example 5 Use of Multiple Biomarkers to Evaluate Likelihood of Sensitivity to a Rapamycin Analog in Tumor Cell Lines
  • PTEN expression is also assessed.
  • PTEN is detected by Western blot using PTEN-specific antibodies (A2B1) from Santa Cruz Biotechnology (Santa Cruz, CA) as described (Nagata, Y., et al, Cancer Cell, 6:117-127, 2004). Expression levels are quantitated as for FKBP 12.
  • Multivariate analysis is used to analyse the association between FKBP12 levels, PTEN levels, and sensitivity to AP23573.
  • PTEN expression is also assessed using IHC.
  • slides of formalin- fixed, paraffin-embedded tissue sections are incubated with PTEN antibody (Ab-2, 1 :500) as described (Podsypanina et al, Proc. Natl. Acad Sci. USA, 98:10320-10325, 2001).
  • PTEN antibody Ab-2, 1 :500
  • IHC is performed with the LSAB2 kit (DAKO, Carpentaria, CA), color development with 3-3'-diaminobenzidine, and counterstaining using hematoxylin.
  • IRS immunoreactive score
  • Multivariate analysis is used to analyse the association between FKBP 12 levels, PTEN levels, and sensitivity to AP23573.
  • the data are analysed to determine whether use of FKBP 12 and PTEN levels in conjunction is better able to evaluate the likelihood that a tumor cell line is sensitive to AP23573, e.g., to determine whether an increased FKBP 12 level relative to a reference value, in conjunction with a decreased PTEN level relative to a reference value, is more strongly correlated with sensitivity to AP23573 than either biomarker alone.

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Abstract

L'invention concerne des compositions et des procédés permettant d'évaluer la probabilité selon laquelle une tumeur est sensible à un inhibiteur de mTOR, p. ex. la rapamycine ou un analogue de la rapamycine. L'invention concerne des protéines FKBP utilisées comme biomarqueurs pour prédire la probabilité selon laquelle une tumeur est sensible à un inhibiteur de mTOR. Les procédés comprennent l'évaluation de l'expression ou de l'activité d'une protéine FKBP, p. ex. FKBP 12, chez un sujet présentant une tumeur ou dans un échantillon provenant d'une tumeur. L'invention concerne aussi d'autres biomarqueurs et combinaisons de biomarqueurs. L'invention concerne également des trousses contenant par exemple un anticorps ou un ligand validé permettant d'évaluer l'expression ou l'activité d'une protéine FKBP.
PCT/US2006/017770 2005-05-09 2006-05-08 Biomarqueurs permettant d'evaluer la probabilite selon laquelle une tumeur est sensible a un inhibiteur de mtor Ceased WO2006122053A2 (fr)

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WO2010091354A3 (fr) * 2009-02-06 2011-04-07 H. Lee Moffitt Cancer Center And Research Institute, Inc. Biomarqueur de phosphorylation de la tyrosine 176 d'akt pour le cancer
US20120028972A1 (en) * 2010-07-30 2012-02-02 Lilly Wong Biomarker assays for detecting or measuring inhibition of tor kinase activity
WO2012130720A2 (fr) 2011-03-28 2012-10-04 Cemm - Forschungszentrum Für Molekulare Medizin Gmbh Prédiction de la sensibilité vis-à-vis d'inhibiteurs de pi3k/mtor
EP2510121A1 (fr) * 2009-12-11 2012-10-17 Wyeth LLC Biomarqueurs de la voie de la phosphatidylinotisol-3-kinase
WO2012138789A3 (fr) * 2011-04-04 2012-12-27 Netherlands Cancer Institute Procédés et compositions pour prédire une résistance à un traitement anti-cancéreux
JP2013074866A (ja) * 2011-09-30 2013-04-25 Osaka Univ ｍＴＯＲ阻害剤の投与が有効ながん患者の選択方法
WO2014144451A2 (fr) 2013-03-15 2014-09-18 Memorial Sloan-Kettering Cancer Center Biomarqueurs pour réponse à des analogues de rapamycine
WO2014184734A1 (fr) * 2013-05-14 2014-11-20 Novartis Ag Marqueurs associés à l'inhibition de mtor
WO2015123377A1 (fr) * 2014-02-12 2015-08-20 Dana-Farber Cancer Institute, Inc. P13k-mtorc1-s6k1 voie de signalisation biomarqueurs prédictifs de réponses anti-cancer
FR3028955A1 (fr) * 2014-11-26 2016-05-27 Centre Leon Berard Procede de prediction de la sensibilite d'un patient a un traitement inhibiteur de la voie mtor
US11096940B2 (en) 2017-06-22 2021-08-24 Celgene Corporation Treatment of hepatocellular carcinoma characterized by hepatitis B virus infection

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Cited By (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010091354A3 (fr) * 2009-02-06 2011-04-07 H. Lee Moffitt Cancer Center And Research Institute, Inc. Biomarqueur de phosphorylation de la tyrosine 176 d'akt pour le cancer
US8557516B2 (en) 2009-02-06 2013-10-15 H. Lee Moffitt Cancer Center And Research Institute, Inc. AKT tyrosine 176 phosphorylation cancer biomarker
EP2510121A1 (fr) * 2009-12-11 2012-10-17 Wyeth LLC Biomarqueurs de la voie de la phosphatidylinotisol-3-kinase
US20120028972A1 (en) * 2010-07-30 2012-02-02 Lilly Wong Biomarker assays for detecting or measuring inhibition of tor kinase activity
WO2012130720A2 (fr) 2011-03-28 2012-10-04 Cemm - Forschungszentrum Für Molekulare Medizin Gmbh Prédiction de la sensibilité vis-à-vis d'inhibiteurs de pi3k/mtor
WO2012130720A3 (fr) * 2011-03-28 2012-12-20 Cemm - Forschungszentrum Für Molekulare Medizin Gmbh Prédiction de la sensibilité vis-à-vis d'inhibiteurs de pi3k/mtor
WO2012138789A3 (fr) * 2011-04-04 2012-12-27 Netherlands Cancer Institute Procédés et compositions pour prédire une résistance à un traitement anti-cancéreux
JP2013074866A (ja) * 2011-09-30 2013-04-25 Osaka Univ ｍＴＯＲ阻害剤の投与が有効ながん患者の選択方法
WO2014144451A2 (fr) 2013-03-15 2014-09-18 Memorial Sloan-Kettering Cancer Center Biomarqueurs pour réponse à des analogues de rapamycine
EP2971122A4 (fr) * 2013-03-15 2017-02-22 Memorial Sloan Kettering Cancer Center Biomarqueurs pour réponse à des analogues de rapamycine
US10610521B2 (en) 2013-03-15 2020-04-07 Memorial Sloan Kettering Cancer Center Biomarkers for response to rapamycin analogs
AU2014229108B2 (en) * 2013-03-15 2020-07-02 Memorial Sloan-Kettering Cancer Center Biomarkers for response to rapamycin analogs
WO2014184734A1 (fr) * 2013-05-14 2014-11-20 Novartis Ag Marqueurs associés à l'inhibition de mtor
WO2015123377A1 (fr) * 2014-02-12 2015-08-20 Dana-Farber Cancer Institute, Inc. P13k-mtorc1-s6k1 voie de signalisation biomarqueurs prédictifs de réponses anti-cancer
US11366100B2 (en) 2014-02-12 2022-06-21 Dana-Farber Cancer Institute, Inc. P13K-MTORC1-S6K1 signaling pathway biomarkers predictive of anti-cancer responses
FR3028955A1 (fr) * 2014-11-26 2016-05-27 Centre Leon Berard Procede de prediction de la sensibilite d'un patient a un traitement inhibiteur de la voie mtor
WO2016083741A1 (fr) * 2014-11-26 2016-06-02 Centre Leon Berard Procede de prediction de la sensibilite d'un patient a un traitement inhibiteur de la voie mtor
US11096940B2 (en) 2017-06-22 2021-08-24 Celgene Corporation Treatment of hepatocellular carcinoma characterized by hepatitis B virus infection

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