WO2012100045A2 - Procédés de prédiction de la réaction à une thérapie par un inhibiteur de jnk - Google Patents

Procédés de prédiction de la réaction à une thérapie par un inhibiteur de jnk Download PDF

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WO2012100045A2
WO2012100045A2 PCT/US2012/021857 US2012021857W WO2012100045A2 WO 2012100045 A2 WO2012100045 A2 WO 2012100045A2 US 2012021857 W US2012021857 W US 2012021857W WO 2012100045 A2 WO2012100045 A2 WO 2012100045A2
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cancer
jnk
peptide
jnk inhibitor
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WO2012100045A9 (fr
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Lynda Chin
Ronald A. Depinho
Giulio Draetta
Timothy P. HEFFERNAN
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Dana Farber Cancer Institute Inc
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Dana Farber Cancer Institute Inc
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/415—1,2-Diazoles
    • A61K31/416—1,2-Diazoles condensed with carbocyclic ring systems, e.g. indazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/425—Thiazoles
    • A61K31/427—Thiazoles not condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/425—Thiazoles
    • A61K31/428—Thiazoles condensed with carbocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/005—Enzyme inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/575—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/5752—Immunoassay; Biospecific binding assay; Materials therefor for cancer of the lungs
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/575—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57535—Immunoassay; Biospecific binding assay; Materials therefor for cancer of the large intestine, e.g. colon, rectum or anus
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • C—CHEMISTRY; METALLURGY
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the invention relates to generally to the predicting a subjects response to JNK inhibitor therapy.
  • Mammalian cells respond to extracellular stimuli by activating signaling cascades that are mediated by members of the mitogen-activated protein (MAP) kinase family, which include the extracellular signal regulated kinases (ERKs), the p38 MAP kinases and the c-Jun N-terminal kinases (JNKs).
  • MAP kinases are activated by a variety of signals including growth factors, cytokines, UV radiation, and stress-inducing agents.
  • MAPKs are serine/threonine kinases and their activation occurs by dual phosphorylation of threonine and tyrosine at the Thr-X-Tyr segment in the activation loop.
  • MAPKs phosphorylate various substrates including transcription factors, which in turn regulate the expression of specific sets of genes and thus mediate a specific response to the stimulus.
  • JNKs c-Jun NH 2 -terminal protein kinases
  • JNKl Three distinct genes, JNKl, JNK2, and JNK3 have been identified and at least ten different splicing isoforms of JNKs exist in mammalian cells.
  • JNKs along with other MAPKs, have been implicated in having a role in mediating cellular response to cancer, thrombin-induced platelet aggregation, immunodeficiency disorders, autoimmune diseases, cell death, allergies, osteoporosis and heart disease.
  • the invention features method of accessing the effectiveness of a JNK inhibitor treatment regimen of a subject having cancer by obtaining a sample from the subject and detecting c-JUN expression or the presence or absence of a mutation in the EGFR or KRAS polypeptide.
  • the presence of c-JUN expression or a KRAS mutation indicates that the subject is responsive to JNK inhibitor treatment.
  • the presence of an EGFR mutation indicates the subject is non- responsive to JNK inhibitor treatment.
  • JNK inhibitors suitable for therapy include for example JIP-1, CC-410, 1,9-pyrazoloanthrone, 2- benzothiazoleacetonitrile, XG-102, BI78D3 and BI87G9.
  • the cancer is any cancer capable of being treated with a JNK inhibitor.
  • the cancer is melanoma, non-small cell lung cancer or colorectal cancer.
  • the subject has not received treatment for the cancer.
  • the subject has received treatment for the cancer.
  • FC flow-cytometry
  • IHC immuno-histochemistry
  • IF immuno-fluorescence
  • PCR polymerase chain reaction
  • JNK2 is required to maintain the growth of established melanoma xenografts.
  • A. M619 melanoma cells, expressing DOX-inducible JNK2 shRNAs, were injected subcutaneously into NUDE mice. When xenografts reached 150mm 3 in size the animals were fed +/- DOX to induce JNK2 shRNA expression. Tumor growth was then measured until control tumors reached 2cm 3 .
  • B. Weight of tumors at study endpoint.
  • D. QPCR of the same tumor lysates measuring RNA expression of JNK2.
  • FIG. 1 cJUN expression predicts sensitivity of melanoma cell lines to JNK inhibition.
  • A. M619 melanoma cells were transduced with lentivirus expressing shRNAs against JNK2 and anchorage independent growth measured in soft agar.
  • B. Western immunoblot p-cJUN expression in melanoma cell lines.
  • KRAS mutation predicts sensitivity to JNK inhibition.
  • A. A549 cells were transduced with lentivirus expressing shRNAs against KRAS or JNK1/JNK2 and cell growth measured in 2D (A.) and 3D (B.). NTC refers to the scrambled non-targeting control shRNA.
  • C. Compilation data showing the response of 7 NSCLC cell lines to JNK inhibition. Red: Responder (sensitive to JNK inhibition); Blue: Non-responder.
  • adenocarcinoma specimens from genetically engineered mouse models were processed and stained for p-cJUN expression by IHC.
  • the present invention relates to the identification of biomarkers associated with the responsiveness to c-Jun N-terminal kinase (JNK) inhibitor treatment.
  • JNK c-Jun N-terminal kinase
  • NSCLC non-small cell lung cancer
  • CRC colorectal cancer
  • JNK inhibitors suitable for human administration include for example, JIP-1, CC-410, 1,9-pyrazoloanthrone, 2-benzothiazoleacetonitrile, XG-102, BI78D3 and BI87G9.
  • JNK inhibitor therapy alone or in combination has shown a clinical benefit in patients having cancer, currently there are no known biomarkers capable of stratifying responders and non-responders. Accordingly a need exist is identify biomarkers that are predictive of responsiveness JNK inhibitor therapy.
  • JNK pathway functions in a context specific manner to promote either cell growth or cell death.
  • a correlation between KRAS expression and JNK pathway activation was also observed in lung adenocarcinomas from genetically engineered mouse models.
  • RAS mutant cancers may express, and rely upon the JNK pathway for transformation, and thus JNK inhibitors are effective in treating RAS mutant NSCLC and CRC.
  • the invention provides methods of determining the responsiveness, e.g., sensitivity or resistance, of a cancer cell to treatment with JNK inhibitors. These methods are also useful for monitoring subjects undergoing treatments and therapies for cancer such as melanoma, NSCLC and CRC, and for selecting therapies and treatments that would be efficacious in subjects having cancer, wherein selection and use of such treatments and therapies slow the progression of cancer. More specifically, the invention provides methods of determining the whether a patient with melanoma, NSCLC or CRC will be responsive to JNK inhibitor therapy.
  • “Accuracy” refers to the degree of conformity of a measured or calculated quantity (a test reported value) to its actual (or true) value. Clinical accuracy relates to the proportion of true outcomes (true positives (TP) or true negatives (TN) versus misclassified outcomes (false positives (FP) or false negatives (FN)), and may be stated as a sensitivity, specificity, positive predictive values (PPV) or negative predictive values (NPV), or as a likelihood, odds ratio, among other measures.
  • Biomarker in the context of the present invention encompasses, without limitation, proteins, nucleic acids, and metabolites, together with their polymorphisms, mutations, variants, modifications, subunits, fragments, protein-ligand complexes, and degradation products, protein-ligand complexes, elements, related metabolites, and other analytes or sample-derived measures. Biomarkers can also include mutated proteins or mutated nucleic acids. Biomarkers also encompass non-blood borne factors or non-analyte physiological markers of health status, such as "clinical parameters” defined herein, as well as “traditional laboratory risk factors”, also defined herein.
  • Biomarkers also include any calculated indices created mathematically or combinations of any one or more of the foregoing measurements, including temporal trends and differences. Where available, and unless otherwise described herein, biomarkers which are gene products are identified based on the official letter abbreviation or gene symbol assigned by the international Human Genome Organization Naming Committee (HGNC) and listed at the date of this filing at the US National Center for Biotechnology Information (NCBI) web site.
  • HGNC Human Genome Organization Naming Committee
  • NCBI National Center for Biotechnology Information
  • a “Clinical indicator” is any physiological datum used alone or in conjunction with other data in evaluating the physiological condition of a collection of cells or of an organism. This term includes pre-clinical indicators.
  • “Clinical parameters” encompasses all non-sample or non-analyte biomarkers of subject health status or other characteristics, such as, without limitation, age (Age), ethnicity (RACE), gender (Sex), or family history (FamHX).
  • FN is false negative, which for a disease state test means classifying a disease subject incorrectly as non-disease or normal.
  • FP is false positive, which for a disease state test means classifying a normal subject incorrectly as having disease.
  • a “formula,” “algorithm,” or “model” is any mathematical equation, algorithmic, analytical or programmed process, or statistical technique that takes one or more continuous or categorical inputs (herein called “parameters”) and calculates an output value, sometimes referred to as an "index” or “index value.”
  • “formulas” include sums, ratios, and regression operators, such as coefficients or exponents, biomarker value transformations and normalizations (including, without limitation, those normalization schemes based on clinical parameters, such as gender, age, or ethnicity), rules and guidelines, statistical classification models, and neural networks trained on historical populations.
  • biomarkers Of particular use in combining biomarkers are linear and non-linear equations and statistical classification analyses to determine the relationship between biomarkers detected in a subject sample and the subject's responsiveness to chemotherapy.
  • panel and combination construction of particular interest are structural and synactic statistical classification algorithms, and methods of risk index construction, utilizing pattern recognition features, including established techniques such as cross-correlation, Principal Components Analysis (PCA), factor rotation, Logistic Regression (LogReg), Linear Discriminant Analysis (LDA), Eigengene Linear Discriminant Analysis (ELDA), Support Vector Machines (SVM), Random Forest (RF), Recursive Partitioning Tree (RPART), as well as other related decision tree classification techniques, Shrunken Centroids (SC), StepAIC, Kth-Nearest Neighbor, Boosting, Decision Trees, Neural Networks, Bayesian Networks, Support Vector Machines, and Hidden Markov Models, among others.
  • PCA Principal Components Analysis
  • LogReg Logistic Regression
  • LDA Linear Discriminant Analysis
  • AIC Akaike's Information Criterion
  • BIC Bayes Information Criterion
  • the resulting predictive models may be validated in other studies, or cross-validated in the study they were originally trained in, using such techniques as Bootstrap, Leave-One-Out (LOO) and 10-Fold cross-validation (10-Fold CV).
  • LEO Leave-One-Out
  • 10-Fold cross-validation 10-Fold CV.
  • false discovery rates may be estimated by value permutation according to techniques known in the art.
  • a "health economic utility function" is a formula that is derived from a combination of the expected probability of a range of clinical outcomes in an idealized applicable patient population, both before and after the introduction of a diagnostic or therapeutic intervention into the standard of care.
  • a cost and/or value measurement associated with each outcome, which may be derived from actual health system costs of care (services, supplies, devices and drugs, etc.) and/or as an estimated acceptable value per quality adjusted life year (QALY) resulting in each outcome.
  • the sum, across all predicted outcomes, of the product of the predicted population size for an outcome multiplied by the respective outcomes expected utility is the total health economic utility of a given standard of care.
  • the difference between (i) the total health economic utility calculated for the standard of care with the intervention versus (ii) the total health economic utility for the standard of care without the intervention results in an overall measure of the health economic cost or value of the intervention.
  • This may itself be divided amongst the entire patient group being analyzed (or solely amongst the intervention group) to arrive at a cost per unit intervention, and to guide such decisions as market positioning, pricing, and assumptions of health system acceptance.
  • Such health economic utility functions are commonly used to compare the cost-effectiveness of the intervention, but may also be transformed to estimate the acceptable value per QALY the health care system is willing to pay, or the acceptable cost-effective clinical performance characteristics required of a new intervention.
  • a health economic utility function may preferentially favor sensitivity over specificity, or PPV over NPV based on the clinical situation and individual outcome costs and value, and thus provides another measure of health economic performance and value which may be different from more direct clinical or analytical performance measures.
  • Measurement or “measurement,” or alternatively “detecting” or “detection,” means assessing the presence, absence, quantity or amount (which can be an effective amount) of either a given substance within a clinical or subject-derived sample, including the derivation of qualitative or quantitative concentration levels of such substances, or otherwise evaluating the values or categorization of a subject's non-analyte clinical parameters.
  • NDV Neuronal predictive value
  • hazard ratios and absolute and relative risk ratios within subject cohorts defined by a test are a further measurement of clinical accuracy and utility. Multiple methods are frequently used to defining abnormal or disease values, including reference limits, discrimination limits, and risk thresholds.
  • “Analytical accuracy” refers to the reproducibility and predictability of the measurement process itself, and may be summarized in such measurements as coefficients of variation, and tests of concordance and calibration of the same samples or controls with different times, users, equipment and/or reagents. These and other considerations in evaluating new biomarkers are also summarized in Vasan, 2006.
  • “Performance” is a term that relates to the overall usefulness and quality of a diagnostic or prognostic test, including, among others, clinical and analytical accuracy, other analytical and process characteristics, such as use characteristics (e.g., stability, ease of use), health economic value, and relative costs of components of the test. Any of these factors may be the source of superior performance and thus usefulness of the test, and may be measured by appropriate "performance metrics," such as AUC, time to result, shelf life, etc. as relevant. "Positive predictive value” or “PPV” is calculated by TP/(TP+FP) or the true positive fraction of all positive test results. It is inherently impacted by the prevalence of the disease and pre-test probability of the population intended to be tested.
  • “Risk” in the context of the present invention relates to the probability that an event will occur over a specific time period, as in the responsiveness to treatment, cancer recurrence or survival and can mean a subject's "absolute” risk or “relative” risk.
  • Absolute risk can be measured with reference to either actual observation post-measurement for the relevant time cohort, or with reference to index values developed from statistically valid historical cohorts that have been followed for the relevant time period.
  • Relative risk refers to the ratio of absolute risks of a subject compared either to the absolute risks of low risk cohorts or an average population risk, which can vary by how clinical risk factors are assessed.
  • Odds ratios the proportion of positive events to negative events for a given test result, are also commonly used (odds are according to the formula p/(l-p) where p is the probability of event and (1- p) is the probability of no event) to no-conversion.
  • Risk evaluation or “evaluation of risk” in the context of the present invention encompasses making a prediction of the probability, odds, or likelihood that an event or disease state may occur, the rate of occurrence of the event or conversion from one disease state.
  • Risk evaluation can also comprise prediction of future clinical parameters, traditional laboratory risk factor values, or other indices of cancer, either in absolute or relative terms in reference to a previously measured population.
  • the methods of the present invention may be used to make continuous or categorical measurements of the responsiveness to treatment thus diagnosing and defining the risk spectrum of a category of subjects defined as being responders or non-responders. In the categorical scenario, the invention can be used to discriminate between normal and other subject cohorts at higher risk for responding.
  • sample in the context of the present invention is a biological sample isolated from a subject and can include, by way of example and not limitation, tissue biopsies, whole blood, serum, plasma, blood cells, endothelial cells, lymphatic fluid, ascites fluid, interstitital fluid (also known as "extracellular fluid” and encompasses the fluid found in spaces between cells, including, inter alia, gingival crevicular fluid), bone marrow, cerebrospinal fluid (CSF), saliva, mucous, sputum, sweat, urine, or any other secretion, excretion, or other bodily fluids.
  • a “sample” may include a single cell or multiple cells or fragments of cells.
  • the sample is also a tissue sample.
  • the sample is or contains a circulating endothelial cell or a circulating tumor cell.
  • the sample includes a primary tumor cell, primary tumor, a recurrent tumor cell, or a metastatic tumor cell.
  • Specificity of an assay is calculated by TN/(TN+FP) or the true negative fraction of non-disease or normal subjects.
  • Statistical significance can be determined by any method known in the art. Commonly used measures of significance include the p-value, which presents the probability of obtaining a result at least as extreme as a given data point, assuming the data point was the result of chance alone. A result is considered highly significant at a p-value of 0.05 or less. Preferably, the p-value is 0.04, 0.03, 0.02, 0.01, 0.005, 0.001 or less.
  • a "subject" in the context of the present invention is preferably a mammal.
  • the mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but are not limited to these examples. Mammals other than humans can be advantageously used as subjects that represent animal models of cancer.
  • a subject can be male or female.
  • TN is true negative, which for a disease state test means classifying a non-disease or normal subject correctly.
  • TP j s lrue positive, which for a disease state test means correctly classifying a disease subject.
  • Traditional laboratory risk factors correspond to biomarkers isolated or derived from subject samples and which are currently evaluated in the clinical laboratory and used in traditional global risk assessment algorithms.
  • Traditional laboratory risk factors for tumor recurrence include for example Proliferative index, tumor infiltrating lymphocytes. Other traditional laboratory risk factors for tumor recurrence known to those skilled in the art.
  • Effectiveness is the capability of producing a desired result in a subject when administered a JNK inhibitor, e.g., the tumor cells or tumor tissues of the subject undergo apoptosis and/or necrosis, or any other desired results for cancer treatment known in the art.
  • the methods disclosed herein are used with subjects undergoing treatment and/or therapies for cancer, subjects who are at risk for developing a reoccurrence of cancer (e.g. melanoma, non-small cell lung cancer (NSCLC) or colorectal cancer (CRC), and subjects who have been diagnosed with cancer.
  • the methods of the present invention are to be used to monitor or select a treatment regimen for a subject who has cancer, and to evaluate the predicted survivability and/or survival time of a cancer-diagnosed subject.
  • Treatment regimens include JNK inhibitor therapies such as JIP-1, CC-410, 1,9-pyrazoloanthrone, 2- benzothiazoleacetonitrile, XG-102, BI78D3 and BI87G9.
  • Responsiveness e.g., resistance or sensitivity
  • Responsiveness of a melanoma patient to JNK inhibitor therapy is determined by detecting cJUN expression in a test sample (e.g., a subject derived sample such as for example a cancer cell).
  • the expression of cJUN indicates the patient will be responsive (i.e., sensitive) to JNK inhibitor therapy.
  • the absence of cJUN expression indicates the patient will be non- responsive (i.e., resistant) to JNK inhibitor therapy.
  • Responsiveness e.g., resistance or sensitivity
  • Responsiveness of a NSCLC or CRC patient to JNK inhibitor therapy is determined by detecting a KRAS mutation in a test sample (e.g., a subject derived sample such as for example a cancer cell).
  • a test sample e.g., a subject derived sample such as for example a cancer cell.
  • the presence of a KRAS mutation indicates the patient will be responsive (i.e., sensitive) to JNK inhibitor therapy.
  • the absence of a KRAS mutation indicates the patient will be non- responsive (i.e., resistant) to JNK inhibitor therapy.
  • Responsiveness (e.g., resistance or sensitivity) of a NSCLC to JNK inhibitor therapy is determined by detecting an EGFR mutation in a test sample (e.g., a subject derived sample such as for example a cancer cell).
  • a test sample e.g., a subject derived sample such as for example a cancer cell.
  • the presence of an EGFR mutation indicates the patient will be non-responsive (i.e., resistant) to JNK inhibitor therapy.
  • the absence of an EGFR mutation indicates the patient will be responsive (i.e., sensitive) to JNK inhibitor therapy.
  • resistance to a therapy it is meant that a patient fails to respond to an agent.
  • resistance to JNK inhibitor therapy means the cancer cell is not damaged or killed by the dmg.
  • sensitivity to a therapy it is meant that that the cell responds to an agent.
  • sensitivity to JNK inhibitor therapy means the cell is damaged or killed by the drug.
  • the methods of the present invention are used to identify and/or diagnose subjects who are asymptomatic for a cancer recurrence.
  • “Asymptomatic” means not exhibiting the traditional symptoms.
  • Identification of cJUN expression, RAS mutation or EGFR mutation allows for the determination of whether a subject will derive a benefit from a particular course of treatment.
  • a biological sample is provided from a subject before undergoing treatment, e.g., JNK inhibitor therapy or combinations thereof.
  • recipient a benefit it is meant that the subject will respond to the course of treatment.
  • responding it is meant that the treatment decreases in size, prevalence, or metastatic potential of a cancer (e.g., melanoma, NSCLC or CRC) in a subject.
  • “responding” means that the treatment retards or prevents a cancer (e.g., melanoma, NSCLC or CR) recurrence from forming or retards, prevents, or alleviates a symptom.
  • a cancer e.g., melanoma, NSCLC or CR
  • Assessments of cancers are made using standard clinical protocols.
  • the present invention can also be used to screen patient or subject populations in any number of settings.
  • a health maintenance organization, public health entity or school health program can screen a group of subjects to identify those requiring interventions, as described above, or for the collection of epidemiological data.
  • Insurance companies e.g., health, life or disability
  • Data collected in such population screens, particularly when tied to any clinical progression to conditions like cancer, will be of value in the operations of, for example, health maintenance organizations, public health programs and insurance companies.
  • Such data arrays or collections can be stored in machine- readable media and used in any number of health-related data management systems to provide improved healthcare services, cost effective healthcare, improved insurance operation, etc. See, for example, U.S.
  • Such systems can access the data directly from internal data storage or remotely from one or more data storage sites as further detailed herein.
  • Each program can be implemented in a high level procedural or object oriented programming language to communicate with a computer system. However, the programs can be implemented in assembly or machine language, if desired. The language can be a compiled or interpreted language. Each such computer program can be stored on a storage media or device (e.g., ROM or magnetic diskette or others as defined elsewhere in this disclosure) readable by a general or special purpose programmable computer, for configuring and operating the computer when the storage media or device is read by the computer to perform the procedures described herein.
  • the health-related data management system of the invention may also be considered to be implemented as a computer-readable storage medium, configured with a computer program, where the storage medium so configured causes a computer to operate in a specific and predefined manner to perform various functions described herein.
  • Differences in the genetic makeup of subjects can result in differences in their relative abilities to metabolize various drugs, which may modulate the symptoms or risk factors of cancer or metastatic events.
  • Subjects that have cancer, or at risk for developing cancer or a metastatic event can vary in age, ethnicity, and other parameters. Accordingly, detection of the bio markers disclosed herein, both alone and together in combination with known genetic factors for drug metabolism, allow for a pre-determined level of predictability that a putative therapeutic or prophylactic to be tested in a selected subject will be suitable for treating cancer in the subject. Performance And Accuracy Measures Of The Invention
  • the performance and thus absolute and relative clinical usefulness of the invention may be assessed in multiple ways as noted above.
  • the invention is intended to provide accuracy in clinical diagnosis and prognosis.
  • the accuracy of a diagnostic, predictive, or prognostic test, assay, or method concerns the ability of the test, assay, or method to distinguish between subjects responsive to therapeutic treatment and those that are not, is based on whether the subjects express c-JUN or has a KRAS or EGFR mutation.
  • an "acceptable degree of diagnostic accuracy” is herein defined as a test or assay in which the AUC (area under the ROC curve for the test or assay) is at least 0.60, desirably at least 0.65, more desirably at least 0.70, preferably at least 0.75, more preferably at least 0.80, and most preferably at least 0.85.
  • a “very high degree of diagnostic accuracy” it is meant a test or assay in which the AUC (area under the ROC curve for the test or assay) is at least 0.80, desirably at least 0.85, more desirably at least 0.875, preferably at least 0.90, more preferably at least 0.925, and most preferably at least 0.95.
  • the predictive value of any test depends on the sensitivity and specificity of the test, and on the prevalence of the condition in the population being tested. This notion, based on Bayes' theorem, provides that the greater the likelihood that the condition being screened for is present in an individual or in the population (pre-test probability), the greater the validity of a positive test and the greater the likelihood that the result is a true positive.
  • pre-test probability the greater the likelihood that the condition being screened for is present in an individual or in the population
  • a positive result has limited value (i.e., more likely to be a false positive).
  • a negative test result is more likely to be a false negative.
  • ROC and AUC can be misleading as to the clinical utility of a test in low disease prevalence tested populations (defined as those with less than 1 % rate of occurrences (incidence) per annum, or less than 10% cumulative prevalence over a specified time horizon).
  • absolute risk and relative risk ratios as defined elsewhere in this disclosure can be employed to determine the degree of clinical utility.
  • Populations of subjects to be tested can also be categorized into quartiles by the test's measurement values, where the top quartile (25% of the population) comprises the group of subjects with the highest relative risk for therapeutic unresponsiveness, and the bottom quartile comprising the group of subjects having the lowest relative risk for therapeutic unresponsiveness.
  • values derived from tests or assays having over 2.5 times the relative risk from top to bottom quartile in a low prevalence population are considered to have a "high degree of diagnostic accuracy," and those with five to seven times the relative risk for each quartile are considered to have a "very high degree of diagnostic accuracy.” Nonetheless, values derived from tests or assays having only 1.2 to 2.5 times the relative risk for each quartile remain clinically useful are widely used as risk factors for a disease; such is the case with total cholesterol and for many inflammatory biomarkers with respect to their prediction of future events. Often such lower diagnostic accuracy tests must be combined with additional parameters in order to derive meaningful clinical thresholds for therapeutic intervention, as is done with the aforementioned global risk assessment indices.
  • a health economic utility function is yet another means of measuring the performance and clinical value of a given test, consisting of weighting the potential categorical test outcomes based on actual measures of clinical and economic value for each.
  • Health economic performance is closely related to accuracy, as a health economic utility function specifically assigns an economic value for the benefits of correct classification and the costs of misclassification of tested subjects.
  • As a performance measure it is not unusual to require a test to achieve a level of performance which results in an increase in health economic value per test (prior to testing costs) in excess of the target price of the test.
  • diagnostic accuracy In general, alternative methods of determining diagnostic accuracy are commonly used for continuous measures, when a disease category or risk category has not yet been clearly defined by the relevant medical societies and practice of medicine, where thresholds for therapeutic use are not yet established, or where there is no existing gold standard for diagnosis of the pre-disease.
  • measures of diagnostic accuracy for a calculated index are typically based on curve fit and calibration between the predicted continuous value and the actual observed values (or a historical index calculated value) and utilize measures such as R squared, Hosmer- Lemeshow P-value statistics and confidence intervals.
  • c-JUN expression or the KRAS or EGFR mutation can be determined at the protein or nucleic acid level using any method known in the art.
  • c-Jun or mutation -specific reagents useful in the practice of the disclosed methods include, among others, mutant polypeptide specific antibodies and AQUA peptides (heavy- isotope labeled peptides) corresponding to, and suitable for detection and quantification of, polypeptide expression in a biological sample.
  • a polypeptide- specific reagent is any reagent, biological or chemical, capable of specifically binding to, detecting and/or quantifying the presence/level of expressed polypeptide in a biological sample.
  • a mutant polypeptide-specific reagent is any reagent, biological or chemical, capable of specifically binding to, detecting and/or quantifying the presence/level of expressed mutant polypeptide (i.e., KRAS or EGFR mutation) in a biological sample, while not binding to or detecting wild type.
  • the term includes, but is not limited to, the preferred antibody and AQUA peptide reagents discussed below, and equivalent reagents are within the scope of the present invention.
  • Reagents suitable for use in practice of the methods of the invention include a polypeptide-specific antibody.
  • Polypeptide-specific antibodies generated against human mutant may also bind to highly homologous and equivalent epitopic peptide sequences in other mammalian species, for example murine, rat, feline, pig, or rabbit, and vice versa.
  • Antibodies useful in practicing the methods of the invention include (a) monoclonal antibodies, (b) purified polyclonal antibodies that specifically bind to the target polypeptide (e.g. an epitope comprising the KRAS or EGFR mutation point , (c) antibodies as described in (a)-(b) above that bind equivalent and highly homologous epitopes or phosphorylation sites in other non-human species (e.g. mouse, rat), and (d) fragments of (a)-(c) above that bind to the antigen (or more preferably the epitope) bound by the exemplary antibodies disclosed herein.
  • antibody or “antibodies” as used herein refers to all types of
  • the antibodies may be monoclonal or polyclonal and may be of any species of origin, including (for example) mouse, rat, rabbit, horse, or human, or may be chimeric antibodies. See, e.g., M. Walker et al., Molec. Immunol. 26: 403-11 (1989); Morrision et al, Proc. Nat'l. Acad. Sci. 81:6851 (1984); Neuberger et al., Nature 312:604 (1984)).
  • the antibodies may be recombinant monoclonal antibodies produced according to the methods disclosed in U.S. Pat. No.
  • the antibodies may also be chemically constructed specific antibodies made according to the method disclosed in U.S. Pat. No. 4,676,980 (Segel et al.)
  • the invention is not limited to use of antibodies, but includes equivalent molecules, such as protein binding domains or nucleic acid aptamers, which bind, in a mutant-protein or truncated-protein specific manner, to essentially the same epitope to which a mutant polypeptide-specific antibody useful in the methods of the invention binds. See, e.g., Neuberger et al., Nature 312: 604 (1984). Such equivalent non-antibody reagents may be suitably employed in the methods of the invention further described below.
  • Polyclonal antibodies useful in practicing the methods of the invention may be produced according to standard techniques by immunizing a suitable animal (e.g., rabbit, goat, etc.) with an antigen encompassing a desired mutant-protein specific epitope (e.g. the sequence comprising the G724S mutation site) collecting immune serum from the animal, and separating the polyclonal antibodies from the immune serum, and purifying polyclonal antibodies having the desired specificity, in accordance with known procedures.
  • the antigen may be a synthetic peptide antigen comprising the desired epitopic sequence, selected and constructed in accordance with well-known techniques. See, e.g., ANTIBODIES: A
  • Monoclonal antibodies may also be beneficially employed in the methods of the invention, and may be produced in hybridoma cell lines according to the well-known technique of Kohler and Milstein. Nature 265:495-97 (1975); Kohler and Milstein, Eur. J.
  • Monoclonal antibodies so produced are highly specific, and improve the selectivity and specificity of assay methods provided by the invention.
  • a solution containing the appropriate antigen e.g. a synthetic peptide comprising the mutant junction of polypeptide
  • the mouse sacrificed and spleen cells obtained.
  • the spleen cells are then immortalized by fusing them with myeloma cells, typically in the presence of polyethylene glycol, to produce hybridoma cells.
  • Rabbit mutant hybridomas for example, may be produced as described in U.S. Pat. No. 5,675,063, K. Knight, Issued Oct. 7, 1997.
  • the hybridoma cells are then grown in a suitable selection media, such as
  • hypoxanthine-aminopterin-thymidine HAT
  • HAT hypoxanthine-aminopterin-thymidine
  • the secreted antibody may be recovered from tissue culture supernatant by conventional methods such as precipitation, ion exchange or affinity chromatography, or the like.
  • Monoclonal Fab fragments may also be produced in Escherichia coli by recombinant techniques known to those skilled in the art. See, e.g., W. Huse, Science 246:1275-81 (1989); Mullinax et al., Proc. Nat'l Acad. Sci. 87: 8095 (1990). If monoclonal antibodies of one isotype are preferred for a particular application, particular isotypes can be prepared directly, by selecting from the initial mutant, or prepared secondarily, from a parental hybridoma secreting a monoclonal antibody of different isotype by using the sib selection technique to isolate class-switch variants (Steplewski, et al., Proc. Nat'l. Acad.
  • the antigen combining site of the monoclonal antibody can be cloned by PCR and single-chain antibodies produced as phage-displayed recombinant antibodies or soluble antibodies in E. coli (see, e.g., ANTIBODY
  • U.S. Pat. No. 5,194,392 Geysen (1990) describes a general method of detecting or determining the sequence of monomers (amino acids or other compounds) which is a topological equivalent of the epitope (i.e., a "mimotope") which is complementary to a particular paratope (antigen binding site) of an antibody of interest. More generally, this method involves detecting or determining a sequence of monomers which is a topographical equivalent of a ligand which is complementary to the ligand binding site of a particular receptor of interest.
  • U.S. Pat. No. 5,480,971, Houghten et al., (1996) discloses linear C.sub.
  • l-C-alkyl peralkylated oligopeptides and sets and libraries of such peptides, as well as methods for using such oligopeptide sets and libraries for determining the sequence of a peralkylated oligopeptide that preferentially binds to an acceptor molecule of interest.
  • non-peptide analogs of the epitope -bearing peptides of the invention also can be made routinely by these methods.
  • Antibodies useful in the methods of the invention may be screened for epitope and mutant protein specificity according to standard techniques. See, e.g. Czernik et al., Methods in Enzymology, 201: 264-283 (1991).
  • the antibodies may be screened against a peptide library by ELISA to ensure specificity for both the desired antigen and, if desired, for reactivity only with a mutant polypeptide of the invention and not with wild type.
  • the antibodies may also be tested by Western blotting against cell preparations containing target protein to confirm reactivity with the only the desired target and to ensure no appreciable binding to other mutants not containing the G724S point mutation.
  • the production, screening, and use of mutant protein-specific antibodies are known to those of skill in the art, and have been described.
  • Antibodies employed in the methods of the invention may be further characterized by, and validated for, use in a particular assay fonnat, for example FC, IHC, and/or ICC.
  • a particular assay fonnat for example FC, IHC, and/or ICC.
  • the use of polypeptide-specific antibodies in such methods is further described below.
  • Antibodies may also be advantageously conjugated to fluorescent dyes (e.g. Alexa488, PE), or labels such as quantum dots, for use in multi-parametric analyses along with other signal transduction (phospho-AKT, phospho-Erk 1/2) and/or cell marker (cytokeratin) antibodies, as further described below.
  • the AQUA methodology employs the introduction of a known quantity of at least one heavy-isotope labeled peptide standard (which has a unique signature detectable by LC-SRM chromatography) into a digested biological sample in order to determine, by comparison to the peptide standard, the absolute quantity of a peptide with the same sequence and protein modification in the biological sample.
  • the AQUA methodology has two stages: peptide internal standard selection and validation and method development; and
  • the method is a powerful technique for detecting and quantifying a given peptide/protein within a complex biological mixture, such as a cell lysate, and may be employed, e.g., to quantify change in protein phosphorylation as a result of drug treatment, or to quantify differences in the level of a protein in different biological states.
  • a particular peptide (or modified peptide) within a target protein sequence is chosen based on its amino acid sequence and the particular protease to be used to digest.
  • the peptide is then generated by solid-phase peptide synthesis such that one residue is replaced with that same residue containing stable isotopes
  • the result is a peptide that is chemically identical to its native counterpart formed by proteolysis, but is easily distinguishable by MS via a 7-Da mass shift.
  • the newly synthesized AQUA internal standard peptide is then evaluated by LC-MS/MS. This process provides qualitative information about peptide retention by reverse-phase chromatography, ionization efficiency, and fragmentation via collision-induced dissociation. Informative and abundant fragment ions for sets of native and internal standard peptides are chosen and then specifically monitored in rapid succession as a function of chromatographic retention to form a selected reaction monitoring (LC-SRM) method based on the unique profile of the peptide standard.
  • LC-SRM reaction monitoring
  • the second stage of the AQUA strategy is its implementation to measure the amount of a protein or modified protein from complex mixtures.
  • Whole cell lysates are typically fractionated by SDS-PAGE gel electrophoresis, and regions of the gel consistent with protein migration are excised. This process is followed by in-gel proteolysis in the presence of the AQUA peptides and LC-SRM analysis.
  • AQUA peptides are spiked in to the complex peptide mixture obtained by digestion of the whole cell lysate with a proteolytic enzyme and subjected to immunoaffinity purification as described above.
  • the retention time and fragmentation pattern of the native peptide formed by digestion e.g.
  • trypsinization is identical to that of the AQUA internal standard peptide determined previously; thus, LC-MS/MS analysis using an SRM experiment results in the highly specific and sensitive measurement of both internal standard and analyte directly from extremely complex peptide mixtures.
  • the ratio of the areas under the curve can be used to determine the precise expression levels of a protein or phosphorylated form of a protein in the original cell lysate.
  • the internal standard is present during in-gel digestion as native peptides are formed, such that peptide extraction efficiency from gel pieces, absolute losses during sample handling (including vacuum centrifugation), and variability during introduction into the LC-MS system do not affect the determined ratio of native and AQUA peptide abundances.
  • An AQUA peptide standard is developed for a known sequence previously identified by the IAP-LC-MS/MS method within in a target protein. If the site is modified, one AQUA peptide incorporating the modified form of the particular residue within the site may be developed, and a second AQUA peptide incorporating the unmodified form of the residue developed. In this way, the two standards may be used to detect and quantify both the modified an unmodified forms of the site in a biological sample. Peptide internal standards may also be generated by examining the primary amino acid sequence of a protein and determining the boundaries of peptides produced by protease cleavage.
  • proteases include, but are not limited to, serine proteases (e.g. trypsin, hepsin), metallo proteases (e.g. PUMP1), chymotrypsin, cathepsin, pepsin, thermolysin, carboxypeptidases, etc.
  • a peptide sequence within a target protein is selected according to one or more criteria to optimize the use of the peptide as an internal standard.
  • the size of the peptide is selected to minimize the chances that the peptide sequence will be repeated elsewhere in other non-target proteins.
  • a peptide is preferably at least about 6 amino acids.
  • the size of the peptide is also optimized to maximize ionization frequency.
  • peptides longer than about 20 amino acids are not preferred.
  • the preferred ranged is about 7 to 15 amino acids.
  • a peptide sequence is also selected that is not likely to be chemically reactive during mass spectrometry, thus sequences comprising cysteine, tryptophan, or methionine are avoided.
  • a peptide sequence that does not include a modified region of the target region may be selected so that the peptide internal standard can be used to determine the quantity of all forms of the protein.
  • a peptide internal standard encompassing a modified amino acid may be desirable to detect and quantify only the modified form of the target protein.
  • Peptide standards for both modified and unmodified regions can be used together, to determine the extent of a modification in a particular sample (i.e. to determine what fraction of the total amount of protein is represented by the modified form).
  • peptide standards for both the phosphorylated and unphosphorylated form of a protein known to be phosphorylated at a particular site can be used to quantify the amount of phosphorylated form in a sample.
  • the peptide is labeled using one or more labeled amino acids (i.e. the label is an actual part of the peptide) or less preferably, labels may be attached after synthesis according to standard methods.
  • the label is a mass-altering label selected based on the following considerations: The mass should be unique to shift fragments masses produced by MS analysis to regions of the spectrum with low background; the ion mass signature component is the portion of the labeling moiety that preferably exhibits a unique ion mass signature in MS analysis; the sum of the masses of the constituent atoms of the label is preferably uniquely different than the fragments of all the possible amino acids.
  • the labeled amino acids and peptides are readily distinguished from unlabeled ones by the ion/mass pattern in the resulting mass spectrum.
  • the ion mass signature component imparts a mass to a protein fragment that does not match the residue mass for any of the 20 natural amino acids.
  • the label should be robust under the fragmentation conditions of MS and not undergo unfavorable fragmentation. Labeling chemistry should be efficient under a range of conditions, particularly denaturing conditions, and the labeled tag preferably remains soluble in the MS buffer system of choice.
  • the label preferably does not suppress the ionization efficiency of the protein and is not chemically reactive.
  • the label may contain a mixture of two or more isotopically distinct species to generate a unique mass spectrometric pattern at each labeled fragment position. Stable isotopes, such as 2 H, 1 C, l ⁇ N, 17 O, 18 O, or 34 S, are among preferred labels. Pairs of peptide internal standards that incorporate a different isotope label may also be prepared. Preferred amino acid residues into which a heavy isotope label may be incorporated include leucine, proline, valine, and phenylalanine.
  • Peptide internal standards are characterized according to their mass-to-charge (m/z) ratio, and preferably, also according to their retention time on a chromatographic column (e.g. an HPLC column). Internal standards that co-elute with unlabeled peptides of identical sequence are selected as optimal internal standards.
  • the internal standard is then analyzed by fragmenting the peptide by any suitable means, for example by collision-induced dissociation (CID) using, e.g., argon or helium as a collision gas.
  • CID collision-induced dissociation
  • the fragments are then analyzed, for example by multi-stage mass spectrometry (MS) to obtain a fragment ion spectrum, to obtain a peptide fragmentation signature.
  • MS mass spectrometry
  • peptide fragments have significant differences in m/z ratios to enable peaks corresponding to each fragment to be well separated, and a signature is that is unique for the target peptide is obtained. If a suitable fragment signature is not obtained at the first stage, additional stages of MS are performed until a unique signature is obtained.
  • Fragment ions in the MS/MS and MS 3 spectra are typically highly specific for the peptide of interest, and, in conjunction with LC methods, allow a highly selective means of detecting and quantifying a target peptide/protein in a complex protein mixture, such as a cell lysate, containing many thousands or tens of thousands of proteins.
  • a complex protein mixture such as a cell lysate, containing many thousands or tens of thousands of proteins.
  • Any biological sample potentially containing a target protein peptide of interest may be assayed. Crude or partially purified cell extracts are preferably employed.
  • the sample has at least 0.01 mg of protein, typically a concentration of 0.1-10 mg/mL, and may be adjusted to a desired buffer concentration and pH.
  • a known amount of a labeled peptide internal standard, preferably about 10 femtomoles, corresponding to a target protein to be detected/quantified is then added to a biological sample, such as a cell lysate.
  • the spiked sample is then digested with one or more protease(s) for a suitable time period to allow digestion.
  • a separation is then performed (e.g. by HPLC, reverse-phase HPLC, capillary electrophoresis, ion exchange chromatography, etc.) to isolate the labeled internal standard and its corresponding target peptide from other peptides in the sample.
  • Microcapillary LC is a preferred method.
  • Each isolated peptide is then examined by monitoring of a selected reaction in the MS. This involves using the prior knowledge gained by the characterization of the peptide internal standard and then requiring the MS to continuously monitor a specific ion in the MS/MS or MS" spectrum for both the peptide of interest and the internal standard. After elution, the area under the curve (AUC) for both peptide standard and target peptide peaks is calculated. The ratio of the two areas provides the absolute quantification that can be normalized for the number of cells used in the analysis and the protein's molecular weight, to provide the precise number of copies of the protein per cell. Further details of the AQUA methodology are described in Gygi et al., and Gerber et al. supra.
  • AQUA internal peptide standards may desirably be produced, as described above, to detect and quantify any unique site (e.g. the KRAS or EGFR mutation point) within a polypeptide of the invention.
  • an AQUA phosphopeptide may be prepared that corresponds to the KRAS or EGFR peptide sequence immediately encompassing the mutation point.
  • Peptide standards may be produced and such standards employed in the AQUA methodology to detect and quantify the presence of mutant KRAS or EGFR (i.e. the presence of the peptide sequence encompassing the point mutation) in a biological sample.
  • an exemplary AQUA peptide of the invention comprises the amino acid sequence EVA, which corresponds to the three amino acids immediately flanking each side of the mutation point in the polypeptide.
  • EVA amino acid sequence
  • larger AQUA peptides comprising the mutant junction sequence (and additional residues downstream or upstream of it) may also be constructed.
  • a smaller AQUA peptide comprising less than all of the residues of such sequence (but still comprising the point of mutant junction itself) may alternatively be constructed.
  • Such larger or shorter AQUA peptides are within the scope of the present invention, and the selection and production of preferred AQUA peptides may be carried out as described above (see Gygi et al., Gerber et al., supra.).
  • Mutant-specific reagents provided by the invention also include nucleic acid probes and primers suitable for detection of a mutant EGFR and KRAS polynucleotide.
  • the specific use of such probes in assays includes such as fluorescence in-situ hybridization (FISH) or polymerase chain reaction (PGR) amplification.
  • Immunoassays useful in the practice of the methods of the invention may be homogenous immunoassays or heterogeneous immunoassays.
  • the immunological reaction usually involves a polypeptide-specific reagent, a labeled analyte, and the biological sample of interest.
  • the signal arising from the label is modified, directly or indirectly, upon the binding of the antibody to the labeled analyte. Both the immunological reaction and detection of the extent thereof are carried out in a homogeneous solution.
  • Immunochemical labels that may be employed include free radicals, radio-isotopes, fluorescent dyes, enzymes, bacteriophages, coenzymes, and so forth.
  • Semi-conductor nanocrystal labels, or “quantum dots”, may also be advantageously employed, and their preparation and use has been well described. See generally, K. Barovsky, Nanotech. Law & Bus. 1(2): Article 14 (2004) and patents cited therein.
  • the reagents are usually the biological sample, a polypeptide-specific reagent, and suitable means for producing a detectable signal.
  • Biological samples as further described below may be used.
  • the antibody is generally immobilized on a support, such as a bead, plate or slide, and contacted with the sample suspected of containing the antigen in a liquid phase.
  • the support is then separated from the liquid phase and either the support phase or the liquid phase is examined for a detectable signal employing means for producing such signal.
  • the signal is related to the presence of the analyte in the biological sample.
  • Means for producing a detectable signal include the use of radioactive labels, fluorescent labels, enzyme labels, quantum dots, and so forth.
  • an antibody which binds to that site can be conjugated to a detectable group and added to the liquid phase reaction solution before the separation step.
  • the presence of the detectable group on the solid support indicates the presence of the antigen in the test sample.
  • suitable immunoassays are the radioimmunoassay, immunofluorescence methods, enzyme-linked immunoassays, and the like.
  • reagent-antibody complexes Conditions suitable for the formation of reagent-antibody complexes are well known to those of skill in the art. See id. Polypeptide-specific monoclonal antibodies may be used in a "two- site” or “sandwich” assay, with a single hybridoma cell line serving as a source for both the labeled monoclonal antibody and the bound monoclonal antibody. Such assays are described in U.S. Pat. No. 4,376,110. The concentration of detectable reagent should be sufficient such that the binding of polypeptide is detectable compared to background.
  • Antibodies useful in the practice of the methods disclosed herein may be conjugated to a solid support suitable for a diagnostic assay (e.g., beads, plates, slides or wells formed from materials such as latex or polystyrene) in accordance with known techniques, such as precipitation.
  • a diagnostic assay e.g., beads, plates, slides or wells formed from materials such as latex or polystyrene
  • Antibodies or other polypeptide-binding reagents may likewise be conjugated
  • radiolabels e.g., S, I, I
  • enzyme labels e.g., horseradish peroxidase, alkaline phosphatase
  • fluorescent labels e.g., fluorescein
  • Cell-based assays such flow cytometry (FC), immuno-histochemistry (IHC), or immunofluorescence (IF) are particularly desirable in practicing the methods of the invention, since such assay formats are clinically-suitable, allow the detection of polypeptide expression in vivo, and avoid the risk of artifact changes in activity resulting from manipulating cells obtained from, e.g. a tumor sample in order to obtain extracts. Accordingly, in some preferred embodiment, the methods of the invention are implemented in a flow-cytometry (FC), immuno-histochemistry (IHC), or immunofluorescence (IF) assay format.
  • FC flow cytometry
  • IHC immuno-histochemistry
  • IF immunofluorescence
  • FC Flow cytometry
  • tumor cells from a bone marrow sample may be analyzed by flow cytometry for polypeptide expression and/or activation, as well as for markers identifying cancer cell types, etc., if so desired.
  • 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). Briefly and by way of example, the following protocol for cytometric analysis may be employed: fixation of the cells with 2% paraformaldehyde for 10 minutes at 37. degree. C. followed by permeabilization in 90% methanol for 30 minutes on ice.
  • Cells may then be stained with the primary polypeptide-specific antibody, washed and labeled with a fluorescent-labeled secondary antibody.
  • the cells would then be analyzed on a flow cytometer (e.g. a Beckman Coulter FC500) according to the specific protocols of the instrument used. Such an analysis would identify the level of expressed polypeptide in the tumor.
  • Immunohistochemical (IHC) staining may be also employed to determine the expression and/or activation status of polypeptide in a mammalian cancer (e.g. AML) before, during, and after treatment with a drug targeted at inhibiting JNK activity.
  • IHC may be carried out according to well-known techniques. See, e.g., ANTIBODIES; A
  • paraffin-embedded tissue e.g. tumor tissue from a biopsy
  • paraffin-embedded tissue is prepared for immunohistochemical staining by deparaffinizing tissue sections with xylene followed by ethanol; hydrating in water then PBS; unmasking antigen by heating slide in sodium citrate buffer; incubating sections in hydrogen peroxide; blocking in blocking solution; incubating slide in primary anti-polypeptide antibody and secondary antibody; and finally detecting using ABC avidin/biotin method according to manufacturer's instructions.
  • Immunofluorescence assays may be also employed to determine the expression and/or activation status of polypeptide in a mammalian cancer before, during, and after treatment with a drug targeted at inhibiting JNK activity.
  • IF may be carried out according to well-known techniques. See, e.g., J. M. Polak and S. Van Noorden (1997) INTRODUCTION TO IMMUNOCYTOCHEMISTRY, 2nd Ed.; ROYAL MICROSCOPY SOCIETY
  • patient samples may be fixed in paraformaldehyde followed by methanol, blocked with a blocking solution such as horse serum, incubated with the primary antibody against polypeptide followed by a secondary antibody labeled with a fluorescent dye such as Alexa 488 and analyzed with an epifluorescent microscope.
  • Antibodies employed in the above-described assays may be advantageously conjugated to fluorescent dyes (e.g. Alexa488, PE), or other labels, such as quantum dots, for use in multi-parametric analyses along with other signal transduction (, phospho-AKT, phospho-Erk 1/2) and/or cell marker (cytokeratin) antibodies.
  • ELISA enzyme-linked immunosorbent assay
  • RIA radio-Immunoassay
  • FACS fluorescent- activated cell sorting
  • polypeptide-specific reagent comprises a heavy isotope labeled phosphopeptide (AQUA peptide) corresponding to a peptide sequence comprising the mutant junction of polypeptide, as described above.
  • Polypeptide-specific reagents useful in practicing the methods of the invention may also be mRNA, oligonucleotide or DNA probes that can directly hybridize to, and detect, mutant or tmncated polypeptide expression transcripts in a biological sample.
  • formalin-fixed, paraffin-embedded patient samples may be probed with a fluorescein-labeled RNA probe followed by washes with formamide, SSC and PBS and analysis with a fluorescent microscope.
  • Polynucleotides encoding polypeptide may also be used for diagnostic purposes.
  • the polynucleotides that may be used include oligonucleotide sequences, antisense RNA and DNA molecules.
  • the polynucleotides may be used to detect and quantitate gene expression in biopsied tissues in which expression of polypeptide or mutated KRAS or EGFR polypeptide may be correlated with disease.
  • the diagnostic assay may be used to distinguish between absence, presence, and excess expression of polypeptide, and to monitor regulation of polypeptide levels during therapeutic intervention.
  • hybridization with PCR probes which are capable of detecting polynucleotide sequences, including genomic sequences, encoding polypeptide or mutated KRAS or EGFR polypeptide, or closely related molecules, may be used to identify nucleic acid sequences which encode polypeptide.
  • genomic sequences including genomic sequences, encoding polypeptide or mutated KRAS or EGFR polypeptide, or closely related molecules.
  • PCR probes which are capable of detecting polynucleotide sequences, including genomic sequences, encoding polypeptide or mutated KRAS or EGFR polypeptide, or closely related molecules, may be used to identify nucleic acid sequences which encode polypeptide. The construction and use of such probes is described above.
  • the specificity of the probe whether it is made from a highly specific region, e.g., 10 unique nucleotides in the mutant junction, or a less specific region, e.g., the 3' coding region, and the stringency of the hybridization or amplification (maximal, high, intermediate, or low) will determine whether the probe identifies only naturally occurring sequences encoding polypeptide, alleles, or related sequences.
  • Probes may also be used for the detection of related sequences, and should preferably contain at least 50% of the nucleotides from any of the polypeptide encoding sequences.
  • the hybridization probes of the subject invention may be DNA or RNA and derived from the nucleotide sequence and encompassing the mutation point, or from genomic sequence including promoter, enhancer elements, and introns of the naturally occurring polypeptides but comprising the mutation point sequence.
  • a mutant EGFR of KRAS polynucleotide may be used in Southern or northern analysis, dot blot, or other membrane-based technologies; in PCR technologies; or in dip stick, pin, ELISA or chip assays utilizing fluids or tissues from patient biopsies to detect altered EGFR or KRAS polypeptide expression. Such qualitative or quantitative methods are well known in the art.
  • Mutant EGFR or KRAS polynucleotides may be labeled by standard methods, and added to a fluid or tissue sample from a patient under conditions suitable for the formation of hybridization complexes. After a suitable incubation period, the sample is washed and the signal is quantitated and compared with a standard value.
  • nucleotide sequences have hybridized with nucleotide sequences in the sample, and the presence of altered levels of nucleotide sequences encoding polypeptide in the sample indicates the presence of the associated disease.
  • assays may also be used to evaluate the efficacy of a particular therapeutic treatment regimen in animal studies, in clinical trials, or in monitoring the treatment of an individual patient. In order to provide a basis for the diagnosis of disease characterized by expression of polypeptide, a normal or standard profile for expression is established.
  • Standard hybridization may be quantified by comparing the values obtained from normal subjects with those from an experiment where a known amount of a substantially purified polynucleotide is used.
  • Standard values obtained from normal samples may be compared with values obtained from samples from patients who are symptomatic for disease. Deviation between standard and subject values is used to establish the presence of disease.
  • hybridization assays may be repeated on a regular basis to evaluate whether the level of expression in the patient begins to approximate that which is observed in the normal patient.
  • the results obtained from successive assays may be used to show the efficacy of treatment over a period ranging from several days to months.
  • PCR polymerase chain reaction
  • PCR oligomers may be chemically synthesized, generated enzymatically, or produced from a recombinant source. Oligomers will preferably consist of two nucleotide sequences, one with sense orientation (5' to 3') and another with antisense (3' to 5'), employed under optimized conditions for identification of a specific gene or condition. The same two oligomers, nested sets of oligomers, or even a degenerate pool of oligomers may be employed under less stringent conditions for detection and/or quantitation of closely related DNA or RNA sequences.
  • Methods which may also be used to quantitate the expression of polypeptide include radiolabeling or biotinylating nucleotides, coamplification of a control nucleic acid, and standard curves onto which the experimental results are interpolated (Melby et al., J.
  • nucleic acid detection such as minor groove-binding conjugated oligonucleotide probes (see, e.g. U.S. Pat. No. 6,951,930, "Hybridization- Triggered Fluorescent Detection of Nucleic Acids") are known to those of skill in the art.
  • HMEL hTERT/CDK4(R24C)/p53DD/BRAF(V600E) melanocytes
  • HMEL melanocytes
  • Mouse melanocytes were isolated from Ink/Arf -/- mice according to standard protocols and grown in RPMI 1640 medium supplemented with 10 % heat-inactivated FBS, 2 nM TPA (Sigma), and 2 nM cholera toxin (Sigma). Mouse melanocytes were propagated at 37 °C and 10% C0 2 in humidified atmosphere.
  • Inducible JNK2 shRNAs were purchased from Open Biosy stems. Lentivius was prepared and cells transduced according to established protocols.
  • Soft-agar assays were performed on 6-well plates in triplicate. For each well, lxlO 4 cells were mixed thoroughly in cell growth medium containing 0.4% SeaKem LE agarose (Fisher) in RPMI plus 10% FBS, followed by plating onto bottom agarose prepared with
  • agarose 0.65% agarose in RPMI and 10% FBS. Each well was allowed to solidify and subsequently covered in 1 ml RPMI and 10% FBS, which was refreshed every 4 days. When appropriate, doxycycline was added to agarose and growth medium at a final concentration of 2 ug/ml.
  • Colonies were stained with 0.05% (w/v) iodonitrotetrazolium chloride (Sigma) and scanned at 1,200 dots per inch (d.p.i.) using a flatbed scanner, and counted.
  • melanoma xenogaft cells stably expressing inducible JNK2 shRNA were subcutaneously implanted into female nude animals (Taconic) at 1x106 cells per site on both flanks.
  • Tumor growth mice were fed normal H20 or H20 containing 2mg/ml doxycycline and 2% sucrose.
  • JNK expression was required for tumor maintenance, cells were implanted and tumors allowed to reach approximately 200 mm 3 , after which time animals were randomized into separate cohorts for treatment with H20 or H20 containing 2mg/ml doxycycline and 2% sucrose. Tumor volumes were measured after dox administration.
  • JNK pathway deregulation was found to cooperate with BRAF V600E to transform human melanocytes in vivo.
  • 3 NRAS mutant melanoma cell lines were found to be sensitive to JNK knockdown, suggesting that RAS mutation may predict response to JNK inhibition.
  • Example 4 EGFR Mutation Predicts Resistance to JNK Inhibition.
  • EGFR mutant NSCLC lines failed to respond to JNK inhibition (Fig. 3). The lack of response suggests EGFR mutation may serve as a mechanism to stratify NSCLC for treatment with JNK inhibitors.

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Abstract

Cette présente invention concerne des méthodes de traitement, d'estimation/de surveillance de la réactivité d'une cellule cancéreuse à une thérapie par un inhibiteur de JNK.
PCT/US2012/021857 2011-01-21 2012-01-19 Procédés de prédiction de la réaction à une thérapie par un inhibiteur de jnk Ceased WO2012100045A2 (fr)

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