WO2009151908A1 - Marqueurs biologiques de l'efficacité de médicaments basés sur egfr/her/erbb - Google Patents

Marqueurs biologiques de l'efficacité de médicaments basés sur egfr/her/erbb Download PDF

Info

Publication number
WO2009151908A1
WO2009151908A1 PCT/US2009/044771 US2009044771W WO2009151908A1 WO 2009151908 A1 WO2009151908 A1 WO 2009151908A1 US 2009044771 W US2009044771 W US 2009044771W WO 2009151908 A1 WO2009151908 A1 WO 2009151908A1
Authority
WO
WIPO (PCT)
Prior art keywords
erbb3
cell
phosphorylation
erbb2
compound
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2009/044771
Other languages
English (en)
Inventor
Thomas W. Grunt
Waheed Shabbir
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Wyeth LLC
Original Assignee
Wyeth LLC
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Wyeth LLC filed Critical Wyeth LLC
Priority to AU2009257802A priority Critical patent/AU2009257802A1/en
Priority to CA2722890A priority patent/CA2722890A1/fr
Priority to BRPI0911451A priority patent/BRPI0911451A2/pt
Publication of WO2009151908A1 publication Critical patent/WO2009151908A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/475—Assays involving growth factors
    • G01N2333/485—Epidermal growth factor [EGF] (urogastrone)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
    • G01N2333/912—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • G01N2333/91205—Phosphotransferases in general
    • G01N2333/9121—Phosphotransferases in general with an alcohol group as acceptor (2.7.1), e.g. general tyrosine, serine or threonine kinases

Definitions

  • the invention relates generally to biomarkers and their use in assessing or predicting cancer therapy efficacy in a patient.
  • ErbB transmembrane proteins belong to the family of growth factor receptor protein tyrosine kinases (RTK).
  • RTK growth factor receptor protein tyrosine kinases
  • ErbB1 EGFR
  • ErbB2 HER-2
  • ErbB3 HER-3
  • ErbB4 HER-4
  • ErbB3 In the case of ErbB3, it has no endogenous tyrosine kinase activity of its own. When ErbB3 binds to its cognate ligand, heregulin, a ErbB2/ErbB3 kinase-active heterodimer is formed, resulting in the activation of the PI3K/Akt cascade, which enables cell proliferation.
  • Deregulation and hyperactivation of signaling through the EGF family of receptors is associated with many types of cancers.
  • Deregulation of ErbB1 is associated with head and neck, breast, lung, bladder, prostate, brain, pancreatic, ovary, colon and kidney cancers.
  • Deregulation of Erb2 is associated with breast, ovary, lung, prostate, gastric and oral cancers.
  • ErbB1-directed therapeutic antibodies include e.g. cetuximab (Erbitux®, Imclone, Branchburg, NJ), ABX-EGF (Abgenix), MDX-447 (Mederex) and EMD 72000 (Merck KgA).
  • ErbB1 TKIs which show some level of inhibiting ErbB2, include e.g. gefitinib, erlotinib and pilitinib (a.k.a.
  • ErbB2-directed therapeutic antibodies include e.g. trastuzumab (Herceptin®, Genentech) and pertuzumab (OmnitargTM, Genentech).
  • ErbB2 TKIs include e.g. CP-654,577 (European Pat. No.
  • Tyrosine kinase inhibitors of multiple ErbBs include e.g. canertinib (CI-1033; a 4-anilinoquinazoline acrylamide derivative), HKI-272 (a 6,7-disubstituted-4-anilinoquinoline-3-carbonitrile), lapatinib (GW572016, PCT Pub. No.
  • ErbB1 and 2 are overexpressed or hyperactivated in many tumors, including ovarian and breast cancer. They stimulate carcinogenesis and malignant progression, and confer unfavorable prognoses. Clinical success has recently been obtained by targeting ErbB2 in ErbB2+ breast cancers. However, only approximately 30% of ErbB2+ breast cancers respond to targeted ErbB2 blockade and most of the responders eventually develop secondary resistance.
  • the invention provides molecular markers (biomarkers) that enable the determination or prediction of whether a particular cancer having an ErbB phenotype can respond favorably to a particular ErbB modulating drug.
  • biomarkers in the ErbB3 pathway are useful in determining the sensitivity of cells to ErbB1/ErbB2 blockers and kinase inhibitors.
  • the invention provides a method of determining the sensitivity of a cell to a compound that modulates ErbB2 activity, comprising the steps of (a) contacting the cell with the compound; (b) determining the phosphorylation status of one or more components of an ErbB3 signaling pathway; and (c) comparing the phosphorylation status of the one or more components of the ErbB3 signaling pathway obtained in step (b) to a reference phosphorylation status of the one or more components of the ErbB3 signaling pathway, wherein a difference between the phosphorylation status of the one or more component of an ErbB3 signaling pathway obtained in step (b) and the reference phosphorylation status of the one or more components of the ErbB3 signaling pathway indicates that the cell is sensitive to the compound.
  • the result of the comparison of step (c) is provided to a user in a readable format, such as a written report or digital format.
  • the compound inhibits ErbB2 kinase activity.
  • the compound is (2E)-N- ⁇ 4-[(3-chloro-4-fluorophenyl)amino]-3-cyano-7- ethoxyquinolin-6-yl ⁇ -4-(dimethylamino)but-2-enamide (EKB-569, a.k.a. pilitinib.)
  • the cell is a cancer cell, such as a breast cancer cell or an ovary cancer cell.
  • the cancer cell is from a cell line, such as e.g. BT-20, BT-474, MCF-7, MDA-MB-231 , MDA-MB-361 , MDA-MB-453, MDA-MB- 468, SKBR-3, SW-527, T-47D, ZR-75-30, A-2774, A-2780, A-2780-ADR, CAOV3, H- 134, HEY, HOC-7, OVCAR3, PA1 , SKOV3, and TR-170.
  • the cell is obtained from a tumor in a patient.
  • the components of the ErbB3 pathway include ErbB3, protein kinase B (Akt), phosphatidylinositol-3-OH kinase (PI3K), glycogen synthase 3 (GSK3), and phosphoinositide phosphatase (PTEN).
  • Akt protein kinase B
  • PI3K phosphatidylinositol-3-OH kinase
  • GSK3 glycogen synthase 3
  • PTEN phosphoinositide phosphatase
  • the phosphorylation status of ErbB3 is determined by detecting the relative level of pTyr1289-ErbB3 in the cell. A reduction in relative ErbB3 phosphorylation indicates that the cell is predicted to be sensitive to the antiproliferative effects of the ErbB2 kinase inhibitor.
  • the phosphorylation status of Akt is determined by detecting the relative level of pSer473pThr308-Akt in the cell. A reduction in relative Akt phosphorylation indicates that the cell is predicted to be sensitive to the antiproliferative effects of the ErbB2 kinase inhibitor.
  • the phosphorylation status of GSK3 ⁇ is determined by detecting the relative level of pSer9-GSK3 ⁇ in the cell. A reduction in relative GSK3 ⁇ phosphorylation indicates that the cell is predicted to be sensitive to the antiproliferative effects of the ErbB2 kinase inhibitor.
  • the phosphorylation status of PTEN is determined by detecting the relative level of pSer380-PTEN in the cell. An increase in relative PTEN phosphorylation indicates that the cell is predicted to be sensitive to the antiproliferative effects of the ErbB2 kinase inhibitor.
  • the reference phosphorylation status of one or more components of the ErbB3 pathway is established in cells treated with a ErbB2 modulating compound, wherein the cells are resistant to the ErbB2 modulating compound.
  • a the level of pSer9- GSK3 ⁇ is determined in the test cell after contact with the compound
  • the level of pSer9-GSK3 ⁇ is determined in a compound-resistant cell after contact with the compound
  • the levels of pSer9-GSK3 ⁇ determined in each cell are compared.
  • the levels of the phosphorylated ErbB3 pathway components are standardized to a standard.
  • the standard can be a constitutive protein such as e.g. actin or GAPDH.
  • the standard can be the level of unphosphorylated ErbB3 pathway component, or the total level of phosphorylated and unphosphorylated ErbB3 pathway component.
  • the invention provides for the use of an ErbB3 pathway phosphorylation assay to determine whether a tumor can respond to the antiproliferative effects of an ErbB2 blocking compound.
  • the phosphorylation assay determines the phosphorylation status of members of the
  • ErbB2 blocking compound which in turn correlates to the tumor's sensitivity to the ErbB2 blocking compound.
  • kits that is useful in assessing the sensitivity of a cell to an ErbB2 blocking agent.
  • the kits includes (a) at least one antibody that specifically binds to the phosphorylated form ErbB3.
  • Figure 1 depicts a line graph showing the proliferation response of breast cancer cell lines to increasing doses of pelitinib.
  • Figure 2 depicts a line graph showing the proliferation response of ovarian cancer cell lines to increasing doses of pelitinib.
  • Figure 3 depicts Western blots indicating the levels of EGFR and actin in breast cancer cell lines (upper panel) and ovarian cancer cell lines (lower two panels.)
  • Figure 4 depicts Western blots indicating the levels of ErbB2 and actin in breast cancer cell lines (upper panel) and ovarian cancer cell lines (lower two panels.)
  • Figure 5 depicts the correlation between relative EGFR and ErbB2 protein expression level and antiproliverative efficacy of pelitinib in breast (upper panels) and ovarian (lower panels) cancer cell lines.
  • Figure 6 depicts the dose-response relationship of in vitro growth inhibition
  • SKBR-3 pelletitinib-sensitive - upper left panel
  • T47D pelletitinib-resistant - upper right panel
  • the lower panels depict Western blots of the time-dependent effects of pelitinib (0.1 ⁇ M) in pelitinib-sensitive SKBR3 (left panels) and pelitinib-resistant T47D (right panels) breast cancer cell lines.
  • Figure 7 depicts a line graph showing the proliferation response of transfected SKBR3 cells, which contain either myr-AKT, an empty vector, or a GFP construct, to increasing doses of pelitinib.
  • the lower panel depicts a Western blot showing phosphor-AKT expression, AKT, and actin expression in the transfected
  • An object of the invention is the assessment of a tumor cell's ability to respond to targeted ErbB2 blockade.
  • the benefit to being able to predict whether a particular cancer will respond to a given type of therapy is useful for determining the safety and effectiveness of cancer treatment.
  • the invention provides a method for determining or predicting the ability of a cell to respond to therapy that blocks ErbB2 signaling, by assessing the phosphorylation status of components of the ErbB3 pathway.
  • the cell that is tested can be any cell.
  • the cell is a tumor cell, such as e.g. an ovarian cancer cell or breast cancer cell.
  • the cell can be from a cell line, a primary cell line or from a biopsy of a tumor from a patient.
  • the therapy can be any compound or set of conditions that blocks the ErbB2 pathway.
  • the invention provides for the use of a phosphorylation assay for ErbB3 pathway components in the diagnosis or prognosis of a tumor's response to ErbB2 blocking compounds.
  • a cell is obtained from a tumor in a patient and contacted with an ErbB2 blocking compound.
  • the phosphorylation status of one or more ErbB3 pathway components in the cell is determined.
  • an activating component of the ErbB3 pathway shows reduced phosphorylation
  • the diagnosis or prognosis of the tumor's response to ErbB2 blocking compounds is positive, i.e., the tumor is sensitive to the compound.
  • a reduced level of pSer9- GSK3 ⁇ indicates that the tumor is sensitive to the compound.
  • the diagnosis or prognosis of the tumor's response to ErbB2 blocking compounds is positive, i.e., the tumor is sensitive to the compound.
  • an increased level of pSer380-PTEN indicates that the tumor is sensitive to the compound.
  • the invention provides a kit useful in the determination of a cell's sensitivity to an ErbB2 blocking agent.
  • the kit includes an agent that ascertains the phosphorylation status of an ErbB3 pathway component and instructions for determining whether the test cell is sensitive to an ErbB2 blocking compound.
  • the kit includes an antibody specific to a phosphorylated form of an ErbB3 pathway component.
  • component of an ErbB3 pathway means any one or more of an upstream ligand of ErbB3, binding partner of ErbB3, and/or downstream effector molecule that is modulated through ErbB3.
  • Non-limiting examples of ErbB3 pathway components include heregulin, ErbB3/HER3, phosphatidylinositol-3-OH kinase
  • PI3K protein kinase B
  • Akt or PKB protein kinase B
  • HER3 phosphatase human epidermal growth factor receptor tyrosine kinase inhibitor
  • HER TKI human epidermal growth factor receptor tyrosine kinase inhibitor
  • HER2 phosphatase and tensin homolog
  • PTEN phosphatidylinositol 4,5-bisphosphate
  • PEP 2 phosphatidylinositol
  • PIP3 3,4,5-trisphosphate
  • p27 phosphoinositide-dependent kinase 1 and 2
  • Activating components of the ErbB3 pathway are those components, which when phosphorylated, stimulate cell proliferation and/or, protein sysnthesis. Examples include PI3K, GSK3 and Akt.
  • Inactivating components of the ErbB3 pathway are those components, which when phosphorylated, inhibit cell proliferation and/or, protein synthesis. An example of an inactivating component is PTEN. See also Planchon et al., Journal of Cell science, 121 (3):249-253, 2008; Menedez and Lupu, Breast Cancer Research, 9:1 11-115, 2007; and Osaki et al., Apoptosis, 9:667-676, 2004, which are incorporated in their entirety herein by reference.
  • phosphorylation status refers to whether a molecular entity is phosphorylated, and/or to what extent the molecular entity is phosphorylated.
  • phosphorylation and dephosphorylation of proteins, lipids and carbohydrates can determine the activity of the protein, lipid or carbohydrate.
  • Kinases catalyze the addition of a phosphate group to a protein, such as on a tyrosine, serine and/or threonine residue, or on a lipid, such as a phosphoinositol, whereas phosphatases catalyze the removal of phosphate groups.
  • ErbB proteins are tyrosine kinases that activate kinase/phosphatase signaling cascades.
  • ErbB3 (a) activated ErbB3 is phosphorylated at least at tyrosine 1289, which (b) phosphorylates and activates PI3K, which (c) mediates the phosphorylation of PIP2 to PIP3, which (d) recruits PKB/Akt and PDK1 to the plasma membrane.
  • PKB/Akt is activated by being phosphorylated at serine 473 by PDK2 and at threonine 308 by PDK1.
  • PKB/Akt phosphorylates the constitutively active GSK3 at least at serine 9, thereby inactivating it. Active non-phosphorylated GSK3 degrades mitogenic ⁇ -catenin and inhibits protein synthesis, hence, phosphorylated (inactivated) GSK3 allows for transcription and protein sysnthesis to proceed.
  • PTEN which is a phosphoinositide phosphatase, See also Farrar et al., Aging Cell, 4:1-12, 2005, which is incorporated herein by reference.
  • Phosphorylation status can be determined in myriad ways. For example, it is well known in the art that phosphorylated proteins can be detected via immunoassays using antibodies that specifically recognize the phosphorylated form of the protein
  • Immunoassays generally include immunoblotting (e.g., Western blotting), RIAs and ELISAs. More specific types of immunoassays include antigen capture/antigen competition, antibody capture/antigen competition, two-antibody sandwiches, antibody capture/antibody excess, and antibody capture/antigen excess. Immunoassays and methods of making antibodies are described in Harlow and Lane, Antibodies: A Laboratory Manual, 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, which is incorporated herein by reference.
  • Phospho- specifc antibodies can be made de novo or obtained from commercial or noncommercial sources.
  • anti-phospho-GSK-3 ⁇ (Ser 9) can be obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Examples of the effective use of that antibody are depicted in Endo et al., Stroke, 37: 2140-2146, 2006; Capozza et al., Am. J. Physiol. Cell Physiol. 288: C1317-C1331 , 2005; Rauch et al., Am. J. Physiol. Cell Physiol.
  • Phosphorylation status can also be determined by metabolically labeling cells with radioactive phosphate in the form of [ ⁇ - 32 P]ATP or [ ⁇ - 33 P]ATP. Phosphorylated proteins or lipids become radioactive and hence traceable and quantifiable through scintillation counting, radiography, and the like (see Wang and Koshland, J. Biol.
  • metabolically labeled proteins can be extracted from cells, separated by gel electrophoresis, transferred to a membrane, probed with an antibody specific for a particular ErbB3 pathway component and subjected to autoradiography to detect 32 P or 33 P.
  • the gel can be subjected to autoradiography prior to membrane transference and antibody probing.
  • compound that modulates ErbB2 activity means a compound that either activates the ErbB2 pathway or inhibits the ErbB2 pathway.
  • ⁇ rbB2 blocking compound or “compound that blocks the ErbB2 pathway” means a compound that blocks signaling through ErbB2.
  • Examples include antibodies such as trastuzumab and pertuzumab, and small molecule RTK-inhibitors such as gefitinib, erlotinib, pilitinib, CP-654,577, CP-724,714, canertinib, HKI-272, lapatinib, PKI-166, and AEE788.
  • EXAMPLE 1 CELL PROLIFERATION
  • IC 50 -values vary over a wide range among the individual breast and ovarian cancer cell lines tested.
  • the data presented in Table 1 were obtained from MTT assays after 72 h of drug exposure. The means of 3 separate experiments are provided, the standard deviations of which were always less than 25% of the mean.
  • SKBR3 and T47D were identified as most sensitive and most resistant breast cancer cell lines, respectively. In contrast, the sensitivity of the ovarian cancer cell lines was more evenly distributed (Tables 2 & 3; Figs. 1 & 2).
  • the small ErbB tyrosine kinase inhibitory drug pelitinib (EKB-569) inhibited the in vitro growth of breast cancer cell lines in a dose- dependent manner, but the individual cell lines revealed distinct sensitivities against the inhibitor.
  • the cell numbers were determined by MTT assay after 72 hours of drug exposure. Results are means of three independent experiments. SD was always less than 30% of the mean.
  • pelitinib also inhibited the in vitro growth of ovarian cancer cell lines in a dose-dependent manner, but the individual cell lines revealed distinct sensitivities against the inhibitor.
  • the cell numbers were determined by MTT assay after 72 hours of drug exposure. Results are means of three independent experiments. SD was always less than 30% of the mean.
  • Figure 3 depicts the baseline levels of EGFR protein expression in breast (upper panel) and ovarian cancer cell lines (lower panels) as demonstrated by Western blot analysis using enhanced chemiluminescence.
  • Figure 4 depicts the baseline levels of ErbB2 protein expression in breast (upper panel) and ovarian cancer cell lines (lower panels) as demonstrated by Western blot analysis using enhanced chemiluminescence.
  • BT-474 breast cancer cells were used as reference EGFR-positive and ErbB2-positive cells, respectively, in all Western analyses. Membranes were semi- quantitatively evaluated by densitometry.
  • EGFR and ErbB2 bands were related to actin bands.
  • the resulting EGFR/actin and ErbB2/actin ratios were arbitrarily set at 1.0 and the ratios of all other cell lines were related to it and are shown below each autoradiograph.
  • the upper panel of Figure 6 provides a dose-response relationship of in vitro growth inhibition of SKBR-3 (pelitinib-sensitive - left panel) and T47D (pelitinib- resistant - right panel) breast cancer cell lines.
  • SKBR-3 pelletitinib-sensitive - left panel
  • T47D pelletitinib- resistant - right panel
  • the lower panels provide a time-dependent effect of the ErbB tyrosine kinase inhibitor pelitinib (0.1 ⁇ M) on the expression and activity (phosphorylation) of the ErbB receptors and of the ErbB downstream signaling mediators PTEN, AKT, GSK-3 ⁇ , and ERK in pelitinib-sensitive SKBR3 (left panels) and pelitinib-resistant T47D (right panels) breast cancer cell lines. In both cell lines, pelitinib stably repressed the phosphorylation of EGFR (pEGFR), whereas it downregulated pErbB2 only moderately.
  • pelitinib stably repressed the phosphorylation of EGFR (pEGFR), whereas it downregulated pErbB2 only moderately.
  • AKT confered resistance against the ErbB tyrosine kinase inhibitor pelitinib on SKBR3 breast cancer cells.
  • pelitinib sensitive SKBR3 cells were transfected with pCMV6 containing no insert (empty vector), myristoylated AKT (myr- AKT), or green fluorescent protein (GFP). MTT assays revealed that myr-AKT transfected SKBR3 cells continued to grow in the presence of increasing concentrations of pelitinib, whereas growth of vector and GFP transfected cells remained inhibited by pelitinib (upper panel).
  • the following human breast carcinoma cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA): BT20, BT474, MCF7, MDA-MB-231 , MDA-MB-361 , MDA-MB-453, MDA-MB-468, SKBR3, SW527, T47D, ZR-75-30.
  • the human ovarian carcinoma cell lines CAOV3 and H 134, as well as the ovarian teratocarcinoma cell line PA1 were also obtained from the ATCC. Additional human ovarian carcinoma cell lines used were: OVCAR3 and SKOV3, from the National Institutes of Health; A2774 from C. Marth, Med. Univ. Innsbruck, Austria; A2780 and A2780ADR (Adriamycin resistant) from M. Krainer, Med. Univ. Vienna, Austria; HEY and HOC7 from R. Buick, Univ. Toronto, Canada; and TR170 from B. Hill, Imperial Cancer Research Fund, London, UK.
  • Cell lines were stored in liquid nitrogen. Cells were trypsinized when confluence reached 70%, centrifuged at IOOOrpm for 5 min, resuspended in ice cold freezing solution (growth medium containing 15% FCS and 10% (v/v) DMSO) and aliquoted in cryotubes, which were put into a styrofoam box and transferred into a -8O 0 C freezer for gradual cooling. The following day, the frozen tubes were transferred into liquid nitrogen for long-term storage.
  • Frozen cell lines were thawed at room temperature in hand-warm water.
  • the cell solution was transferred into a 50 ml centrifuge tube and resuspended in medium with 10% FCS and centrifuged at IOOOrpm for
  • the proliferation rate of all cell lines was assayed by the EZ4U Nonradioactive Cell Proliferation Assay (a.k.a. MTT assay; Biomedica, Vienna, Austria).
  • This assay depends on the reduction of non-toxic tetrazolium salt into intensely coloured formazan derivatives. This reduction requires functional mitochondria, which are inactivated within a few minutes after cell death.
  • cultured cells were incubated for the last 3 - 4 h with EZ4U, and formazan production, which is linearly related to cell number, was assayed by measuring absorbance at 490/620 nm wavelength in a microplate photometer. Calibration curves showed that a highly significant correlation occurred between optical density and the number of cells.
  • the p-value summary gives the answer to the question whether the correlation is significant. The analysis was done using GRAPHPADTM PRISM software (GraphPad Software, Inc., La JoIIa, CA.)
  • 3x10 5 cells were plated in 60 mm dishes in DMEM containing 10% FCS, 100 IU ( ⁇ g)/ml penicillin-streptomycin, and 2 mM glutamine. After overnight incubation, drugs were added in serum-free medium in order to obtain a final concentration of 5% FCS. After treatment, cells were washed twice with ice-cold PBS.
  • Cold modified RIPA buffer (15OmM NaCI, 5OmM Tris pH 7.4, 0.5% Na-deoxycholate, 2mM EGTA, 5mM EDTA, pH 7.4, 3OmM NaF, 4OmM ⁇ -Glycerophosphate, pH7.2, 1OmM tetrasodium pyrophosphate, 3mM Benzamidine, 1% Nonidet P-40, 2 mM Na- Orthovanadate) was applied. After 5 minutes incubation on ice, the cells were scraped using a cell scraper and transferred into 1.5 - 2.0 ml Eppendorf tubes, vortexed several times and left on ice for another 5 minutes.
  • the cell lysate was centrifuged at 12,500 rpm in a microfuge at 4°C for 30 minutes. The supernatant was transferred into new Eppendorf tubes and stored at -80 0 C. After having determined the protein concentrations of each sample using the Bradford method (Bradford, Anal. Biochem. 72:248-254, 1976), 4 x sample buffer (50% Glycerol, 125mM Tris- HCI, pH 6.8, 4% SDS, 0.125% Bromophenol blue, 5% Beta-mercaptoethanol) was added to the lysate and suitable amounts of protein were loaded onto an SDS- polyacrylamide gel for electrophoresis.
  • 4 x sample buffer 50% Glycerol, 125mM Tris- HCI, pH 6.8, 4% SDS, 0.125% Bromophenol blue, 5% Beta-mercaptoethanol
  • BSA solutions with concentrations between 0.156 and 10 ⁇ g/ ⁇ l were used. Each standard and sample was measured 3 times and the average values were calculated. The final concentration of the protein samples was adjusted to 1 ⁇ g/ ⁇ l with 3 volumes modified RIPA and 1 volume of 4x sample buffer. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was used to separate proteins by molecular weight. The MINI PROTEAN IITM equipment from Bio-Rad, Inc. was used for polyacryamide gel electrophoresis. 20 ⁇ g of samples (1 ⁇ g/ ⁇ l) were loaded in each slot. 1 ⁇ l of marker (MAGIC MARK XP, Invitrogen) was used in the first slot.
  • marker MAGIC MARK XP, Invitrogen
  • PVDF polyvinylidenedifluoride
  • the membrane was transferred to a small container with blocking solution (4% BSA, 5OmM Tris pH7.5, 15OmM NaCI, 0.1 % Tween20) and incubated for 1 hour at room temperature on a shaker to decrease unspecific binding of the primary antibody.
  • the next step included three washings in TBS-T (5OmM Tris pH7.5, 15OmM NaCI, 0.1 % Tween20) for 15 minutes.
  • TBS-T 5OmM Tris pH7.5, 15OmM NaCI, 0.1 % Tween20
  • the diluted primary antibody was added and incubated in a closed plastic container at room temperature for 2 hours. After that, 2 x 5 minutes and 2 x 10 minutes washing with TBS-T was done followed by incubation in the secondary antibody for 1 hour at room temperature.
  • the membrane was then washed again 2 x 5 minutes and 2 x 10 minutes in TBS-T, and then 2 x 5 min and 2 x 10 minutes in TBS.
  • phosphospecific antibodies were always used in the first round of immunoblotting.
  • the WESTERN-BLOT ECL DETECTION KIT was used according to the manufacturer (Amersham Biosciences). After 5 minutes, the blot was drained on paper towel, warped in saran wrap, and exposed in an X-ray cartridge to a sheet of autoradiography film until optimum signals were obtained. The autoradiographs were scanned and processed using ADOBE PHOTOSHOP 6.0 software (Adobe).
  • Antibodies used to detect the phosphorylated proteins were provided in Phospho- Erk1/2 Pathway Sampler Kit and the Phospho-Akt Pathway Sampler Kit (Cell
  • the antibodies were diluted in TBS-T plus 1 % bovine serum albumin (BSA). Primary antibody solutions also contained 0.05% Na-azide. Table 4 provides a list of the antibodies used in this study: TABLE 4

Landscapes

  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biomedical Technology (AREA)
  • Immunology (AREA)
  • Hematology (AREA)
  • Chemical & Material Sciences (AREA)
  • Urology & Nephrology (AREA)
  • Molecular Biology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Analytical Chemistry (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Toxicology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Cell Biology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

La présente invention concerne des marqueurs biologiques utiles dans la détermination de la sensibilité d'une cellule à un composé bloquant ErbB2, des procédés d'utilisation des marqueurs biologiques et des nécessaires comprenant des outils pour détecter les marqueurs biologiques. Les marqueurs biologiques comprennent des composants de ErbB3/PI3K/Akt et leurs formes phosphorylées.
PCT/US2009/044771 2008-05-25 2009-05-21 Marqueurs biologiques de l'efficacité de médicaments basés sur egfr/her/erbb Ceased WO2009151908A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
AU2009257802A AU2009257802A1 (en) 2008-05-25 2009-05-21 Biomarkers for EGFR/HER/ErbB drug efficacy
CA2722890A CA2722890A1 (fr) 2008-05-25 2009-05-21 Marqueurs biologiques de l'efficacite de medicaments bases sur egfr/her/erbb
BRPI0911451A BRPI0911451A2 (pt) 2008-05-25 2009-05-21 biomarcadores para eficácia medicamentosa de egfr/her/erb

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US5601608P 2008-05-25 2008-05-25
US61/056,016 2008-05-25

Publications (1)

Publication Number Publication Date
WO2009151908A1 true WO2009151908A1 (fr) 2009-12-17

Family

ID=41078344

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2009/044771 Ceased WO2009151908A1 (fr) 2008-05-25 2009-05-21 Marqueurs biologiques de l'efficacité de médicaments basés sur egfr/her/erbb

Country Status (4)

Country Link
AU (1) AU2009257802A1 (fr)
BR (1) BRPI0911451A2 (fr)
CA (1) CA2722890A1 (fr)
WO (1) WO2009151908A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2013517476A (ja) * 2010-01-13 2013-05-16 ワイス・エルエルシー 腫瘍を正確に識別し、かつＰＡＮ‐ＥｒｂＢ阻害剤に対する薬物応答を予測するＰＴＥＮタンパク質発現におけるカットポイント

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007077028A2 (fr) * 2005-12-30 2007-07-12 U3 Pharma Ag Anticorps dirigés contre le her-3 et leurs utilisations
WO2007106503A2 (fr) * 2006-03-13 2007-09-20 Osi Pharmaceuticals, Inc. Traitement combiné avec un inhibiteur de kinase egfr et un agent sensibilisant les cellules tumorales aux effets des inhibiteurs de kinase egfr
US20080108091A1 (en) * 2006-08-07 2008-05-08 Hennessy Bryan T Proteomic Patterns of Cancer Prognostic and Predictive Signatures

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007077028A2 (fr) * 2005-12-30 2007-07-12 U3 Pharma Ag Anticorps dirigés contre le her-3 et leurs utilisations
WO2007106503A2 (fr) * 2006-03-13 2007-09-20 Osi Pharmaceuticals, Inc. Traitement combiné avec un inhibiteur de kinase egfr et un agent sensibilisant les cellules tumorales aux effets des inhibiteurs de kinase egfr
US20080108091A1 (en) * 2006-08-07 2008-05-08 Hennessy Bryan T Proteomic Patterns of Cancer Prognostic and Predictive Signatures

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
HSIEH A C ET AL: "Targeting HER proteins in cancer therapy and the role of the non-target HER3", BRITISH JOURNAL OF CANCER, vol. 97, no. 4, August 2007 (2007-08-01), pages 453 - 457, XP009123247, ISSN: 0007-0920(print) 1532-1827(ele *
SERGINA NATALIA V ET AL: "Escape from HER-family tyrosine kinase inhibitor therapy by the kinase-inactive HER3.", NATURE 25 JAN 2007, vol. 445, no. 7126, 25 January 2007 (2007-01-25), pages 437 - 441, XP002548476, ISSN: 1476-4687 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2013517476A (ja) * 2010-01-13 2013-05-16 ワイス・エルエルシー 腫瘍を正確に識別し、かつＰＡＮ‐ＥｒｂＢ阻害剤に対する薬物応答を予測するＰＴＥＮタンパク質発現におけるカットポイント

Also Published As

Publication number Publication date
BRPI0911451A2 (pt) 2019-09-24
CA2722890A1 (fr) 2009-12-17
AU2009257802A1 (en) 2009-12-17

Similar Documents

Publication Publication Date Title
Wang et al. Acylglycerol kinase promotes cell proliferation and tumorigenicity in breast cancer via suppression of the FOXO1 transcription factor
Chen et al. VEGFA upregulates FLJ10540 and modulates migration and invasion of lung cancer via PI3K/AKT pathway
Nadler et al. Growth factor receptor-bound protein-7 (Grb7) as a prognostic marker and therapeutic target in breast cancer
Tripathi et al. Receptor tyrosine kinase activation of RhoA is mediated by AKT phosphorylation of DLC1
Atienza et al. Label-free and real-time cell-based kinase assay for screening selective and potent receptor tyrosine kinase inhibitors using microelectronic sensor array
Peng et al. PTK6/BRK is expressed in the normal mammary gland and activated at the plasma membrane in breast tumors
He et al. Prognostic impact of H3K27me3 expression on locoregional progression after chemoradiotherapy in esophageal squamous cell carcinoma
MX2013007429A (es) Seleccion de farmacos para terapia de cancer maligno usando disposiciones a base de anticuerpos.
Stauffer et al. CDK1-mediated mitotic phosphorylation of PBK is involved in cytokinesis and inhibits its oncogenic activity
Sahoo et al. Coordinate expression of the PI3-kinase downstream effectors serum and glucocorticoid-induced kinase (SGK-1) and Akt-1 in human breast cancer
JP6126382B2 (ja) 腫瘍を正確に識別し、かつＰＡＮ‐ＥｒｂＢ阻害剤に対する薬物応答を予測するＰＴＥＮタンパク質発現におけるカットポイント
He et al. Overexpression of Hexokinase 1 as a poor prognosticator in human colorectal cancer
AU2003302821A1 (en) Method for predicting the response to her2-directed therapy
He et al. Expression of anoctamin 1 is associated with advanced tumor stage in patients with non-small cell lung cancer and predicts recurrence after surgery
Wu et al. Overexpression of p62 induces autophagy and promotes proliferation, migration and invasion of nasopharyngeal carcinoma cells through promoting ERK signaling pathway
Radhakrishnan et al. Inhibition of rapamycin-induced AKT activation elicits differential antitumor response in head and neck cancers
Brunetto et al. CDC25A protein stability represents a previously unrecognized target of HER2 signaling in human breast cancer: implication for a potential clinical relevance in trastuzumab treatment
Bu et al. Inhibition of STAT3 reduces proliferation and invasion in salivary gland adenoid cystic carcinoma
Ding et al. Expression of focal adhesion kinase and phosphorylated focal adhesion kinase in human gliomas is associated with unfavorable overall survival
Saldivar-Cerón et al. p21-Activated kinase 1 promotes breast tumorigenesis via phosphorylation and activation of the Calcium/Calmodulin-Dependent protein kinase II
Sawyers Making progress through molecular attacks on cancer
Choi et al. Prognostic significance of AMP-dependent kinase alpha expression in cervical cancer
Mizuguchi et al. Breast tumor kinase/protein tyrosine kinase 6 (Brk/PTK6) activity in normal and neoplastic biliary epithelia
Zhang et al. The prognostic significance of tyrosine-protein phosphatase nonreceptor type 12 expression in nasopharyngeal carcinoma
WO2009151908A1 (fr) Marqueurs biologiques de l'efficacité de médicaments basés sur egfr/her/erbb

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 09763199

Country of ref document: EP

Kind code of ref document: A1

WWE Wipo information: entry into national phase

Ref document number: 2009257802

Country of ref document: AU

WWE Wipo information: entry into national phase

Ref document number: 2722890

Country of ref document: CA

ENP Entry into the national phase

Ref document number: 2009257802

Country of ref document: AU

Date of ref document: 20090521

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

WWE Wipo information: entry into national phase

Ref document number: 2010144996

Country of ref document: RU

122 Ep: pct application non-entry in european phase

Ref document number: 09763199

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: PI0911451

Country of ref document: BR

Kind code of ref document: A2

Effective date: 20101025