WO2015127234A1 - Utilisation d'ibrutinib pour le traitement du cancer à mutation de l'egfr - Google Patents

Utilisation d'ibrutinib pour le traitement du cancer à mutation de l'egfr Download PDF

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WO2015127234A1
WO2015127234A1 PCT/US2015/016859 US2015016859W WO2015127234A1 WO 2015127234 A1 WO2015127234 A1 WO 2015127234A1 US 2015016859 W US2015016859 W US 2015016859W WO 2015127234 A1 WO2015127234 A1 WO 2015127234A1
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cancer
egfr
ibrutinib
inhibitor
therapy
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PCT/US2015/016859
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Bingliang Fang
Jack A. Roth
Michael Wang
Li Wang
Shuhong Wu
John V. HEYMACH
Stephen G. Swisher
Wayne L. HOFSTETTER
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Board Of Regents, The University Of Texas System
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
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    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4375—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a six-membered ring having nitrogen as a ring heteroatom, e.g. quinolizines, naphthyridines, berberine, vincamine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A61K31/4523—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems
    • A61K31/4545—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems containing a six-membered ring with nitrogen as a ring hetero atom, e.g. pipamperone, anabasine
    • 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
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • 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
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
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    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
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    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7135—Compounds containing heavy metals
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    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57492—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds localized on the membrane of tumor or cancer cells
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Definitions

  • the present invention relates generally to the field of oncology. More particularly, it concerns methods of treating EGFR inhibitor-resistant cancer patients with ibrutinib.
  • EGFR mutations are frequently detected in lung adenocarcinoma patients, especially those who have no smoking history (Pao et al, 2004; Ding et al, 2008).
  • the high susceptibilities of EGFR mutant lung cancer cells to gefitinib and erlotinib have made these two agents the first choice for treatment of EGFR mutant cancers.
  • the cancer comprises (i) an EGFR mutation; (ii) HER2 gene amplification; or (iii) HER2 protein overexpression.
  • the irreversible BTK inhibitor is ibrutinib.
  • the EGFR mutation comprises a T790M substitution, a L858R substitution, a deletion in exon 19, a G719x substitution, and/or a L861Q substitution.
  • the cancer is metastatic, recurrent, or multi-drug resistant.
  • the cancer is colorectal, breast, prostate, lung, or pancreatic cancer.
  • the cancer is non-small cell lung cancer.
  • the method further comprises administering at least a second anticancer therapy to the subject.
  • second anticancer therapy may be a surgical therapy, chemotherapy, radiation therapy, cryotherapy, hormonal therapy, toxin therapy, immunotherapy, or cytokine therapy.
  • the chemotherapy may be an NF-KB/STAT-3 inhibitor (e.g., auranofin), an AXL inhibitor (e.g., SGI-7079, cabozantinib), an ALK/MET inhibitor (e.g., crizotinib), a RAF/VEGFR inhibitor (e.g., sorafenib), an AKT inhibitor (e.g., MK2206), a Src inhibitor (e.g., dasatinib), a JAK/STAT-3 inhibitor (e.g., ruxolitinib), or a WNT/ ⁇ - catenin inhibitor (e.g., LGK974135).
  • an NF-KB/STAT-3 inhibitor e.g., auranofin
  • an AXL inhibitor e.g., SGI-7079, cabozantinib
  • an ALK/MET inhibitor e.g., crizotinib
  • a RAF/VEGFR inhibitor
  • methods of treating a patient having cancer comprising administering a therapeutically effective amount of ibrutinib in combination with an AXL inhibitor (e.g., SGI-7079, cabozantinib) to a patient determined to have a cancer that comprises an EGFR mutation (e.g., T790M, exon 19 deletion, L858R, G719x, and/or L861Q) and AXL protein overexpression and/or E-cadherin protein underexpression.
  • Overexpression can be defined as an expression level in the cancer sample that is elevated relative to a control sample (e.g., a non-tumor sample obtaining from the patient or a sample obtained from a healthy patient).
  • Underexpression can be defined as an expression level in the cancer sample that is decreased relative to a control sample.
  • the patient is a human. In some aspects, the patient is a non- human mammal. In some aspects, the patient is treated at least a second time. In some aspects, the patient is treated over a period of 1 week to 6 months. In some aspects, the patient has previously undergone at least one round of anti-cancer therapy.
  • methods of treating a patient having cancer comprising: (a) selecting a patient determined to comprise a cancer comprising (i) an EGFR mutation; (ii) HER2 gene amplification; or (iii) HER2 protein overexpression; and (b) administering a therapeutically effective amount of an irreversible BTK inhibitor, such as any of those disclosed in U.S. Pat. No. 7,514,444, to the patient.
  • the irreversible BTK inhibitor is ibrutinib.
  • selecting a patient comprises obtaining a sample of the cancer and determining whether the cancer comprises (i) an EGFR mutation; (ii) HER2 gene amplification; or (iii) HER2 protein overexpression.
  • the method comprises providing a report of the determining.
  • the report may be a written or electronic report.
  • the report is provided to the patient, a health care payer, a physician, an insurance agent, or an electronic system.
  • the EGFR mutation comprises a T790M substitution.
  • the amino acid present at position 790 of the EGFR protein is determined by mass spectrometry, western blot, ELISA, or sequencing a nucleic acid comprising at least a portion of the protein coding sequence of the EGFR protein.
  • the EGFR mutation further comprises a L858R EGFR mutation, a deletion in exon 19, a G719x substitution, and/or a L861Q substitution.
  • the amino acid present at position 858, 861, and/or 719 of the EGFR protein is determined by mass spectrometry, western blot, ELISA, or sequencing a nucleic acid comprising at least a portion of the protein coding sequence of the EGFR protein.
  • a deletion in exon 19 of EGFR is determined by mass spectrometry, western blot, ELISA, or sequencing a nucleic acid comprising at least a portion of the protein coding sequence of the EGFR protein.
  • the EGFR mutation comprises a substitution at C797.
  • HER2 gene amplification is determined by FISH or quantitative PCR of genomic DNA.
  • HER2 protein overexpression is determined by western blot, ELISA, mass spectrometry.
  • Overexpression can be defined as an expression level in the cancer sample that is elevated relative to a control sample (e.g., a non- tumor sample obtaining from the patient or a sample obtained from a healthy patient).
  • selecting a patient comprises obtaining results from a test that determines whether the cancer comprises (a) an EGFR mutation; (b) HER2 gene amplification; or (c) HER2 protein overexpression; or taking a patient history that reveals that results.
  • the cancer is metastatic, recurrent, or multi-drug resistant.
  • the cancer is colorectal, breast, prostate, lung, or pancreatic cancer.
  • the cancer is non-small cell lung cancer.
  • the method further comprises administering at least a second anticancer therapy to the subject.
  • second anticancer therapy may be a surgical therapy, chemotherapy, radiation therapy, cryotherapy, hormonal therapy, toxin therapy, immunotherapy, or cytokine therapy.
  • the chemotherapy may be an NF-KB/STAT-3 inhibitor (e.g., auranofin), an AXL inhibitor (e.g., SGI-7079, cabozantinib), an ALK/MET inhibitor (e.g., crizotinib), a RAF/VEGFR inhibitor (e.g., sorafenib), an AKT inhibitor (e.g., MK2206), a Src inhibitor (e.g., dasatinib), a JAK/STAT-3 inhibitor (e.g., ruxolitinib), or a W T/ ⁇ - catenin inhibitor (e.g., LGK974135).
  • an NF-KB/STAT-3 inhibitor e.g., auranofin
  • an AXL inhibitor e.g., SGI-7079, cabozantinib
  • an ALK/MET inhibitor e.g., crizotinib
  • a RAF/VEGFR inhibitor
  • the patient is treated at least a second time. In other aspects, the patient is treated over a period of 1 week to 6 months. In certain aspects, the patient is a human. In other aspect, the patient is a non-human mammal.
  • methods of selecting a drug therapy for a cancer patient comprising: (a) obtaining a sample of the cancer; (b) determining the presence of (i) a mutation in the EGFR protein expressed in the cancer; (ii) amplification of the HER2 gene in the cancer; or (iii) overexpression of the HER2 protein in the cancer; and (c) selecting an irreversible BTK inhibitor, such as any of those disclosed in U.S. Pat. No.
  • the irreversible BTK inhibitor is ibrutinib.
  • the method comprises administering a therapeutically effective amount of ibrutinib to the patient.
  • the mutation in the EGFR protein is a T790M and/or L858R substitution.
  • compositions comprising ibrutinib for use in the treatment of a cancer in a subject, wherein the cancer has been determined to comprise (i) an EGFR mutation; (ii) HER2 gene amplification; or (iii) HER2 protein overexpression.
  • the EGFR mutation comprises a T790M substitution, a L858R substitution, a deletion in exon 19 of EGFR, a G719x substitution, or a L861Q substitution.
  • the cancer is colorectal, breast, prostate, lung (e.g. non-small cell lung cancer), or pancreatic cancer.
  • the composition comprises at least a second anticancer therapy.
  • second anticancer therapy may be a surgical therapy, chemotherapy, radiation therapy, cryotherapy, hormonal therapy, toxin therapy, immunotherapy, or cytokine therapy.
  • the chemotherapy may be an NF-KB/STAT-3 inhibitor (e.g., auranofin), an AXL inhibitor (e.g., SGI-7079, cabozantinib), an ALK/MET inhibitor (e.g., crizotinib), a RAF/VEGFR inhibitor (e.g., sorafenib), an AKT inhibitor (e.g., MK2206), a Src inhibitor (e.g., dasatinib), a JAK/STAT-3 inhibitor (e.g., ruxolitinib), or a W T/p-catenin inhibitor (e.g., LGK974135).
  • AXL inhibitor e.g., SGI-7079, cabozantin
  • ibrutinib in the manufacture of a medicament for the treatment of a cancer, wherein the cancer has been determined to comprise (i) an EGFR mutation; (ii) HER2 gene amplification; or (iii) HER2 protein overexpression.
  • ibrutinib is administered systemically.
  • ibrutinib and a second agent are administered by distinct routes.
  • ibrutinib or the second agent are administered orally, intraarterially or intravenously.
  • ibrutinib is administered after the second agent.
  • ibrutinib is administered before and after the second agent.
  • ibrutinib is administered concurrently with the second agent.
  • ibrutinib is administered at least a second time.
  • the second agent is administered at least a second time.
  • essentially free in terms of a specified component, is used herein to mean that none of the specified component has been purposefully formulated into a composition and/or is present only as a contaminant or in trace amounts.
  • the total amount of the specified component resulting from any unintended contamination of a composition is therefore well below 0.05%, preferably below 0.01%.
  • Most preferred is a composition in which no amount of the specified component can be detected with standard analytical methods.
  • FIGS. 1A-D Antitumor activities of ibrutinib in non-small cell lung cancer ( SCLC) cell lines and EGFR T790M mutant tumors.
  • FIG. 1A Calculated 50% inhibitory concentration [IC5 0 ] on a logarithmic scale for 39 NSCLC cell lines by dose-response cell viability assay after ibrutinib treatment for 72 h.
  • FIG. IB Dose-response curves of erlotinib, afatinib and ibrutinib for the HI 975 cell line, which has a T790M mutation. The data are means with standard deviations for two assays performed in quadruplicate.
  • FIG. 1C In vivo growth of HI 795 tumors. The mice were treated as indicated. The values are means ⁇ SD of data from 5 mice per group. * indicates P ⁇ 0.05 when compared with the control group, using a two-sided Student's t-test.
  • FIG. ID Kaplan Meier Survival Curve of the animals shown in FIG. 1C.
  • FIGS. 2A-D Western blot analysis of epidermal growth factor receptor (EGFR) phosphorylation (p-EGFR) and cleavage of poly(ADP-ribose) polymerase (PARP) and caspase-3 (Casp-3).
  • FIG. 2A H1975 and H3255 cells were treated with erlotinib and ibrutinib with the dose as indicated. Phospho-Y1068 and total EGFR were determined at 24 h after treatment.
  • FIG. 2B HCC827, H292 and A549 cells were treated with erlotinib or ibrutinib at the doses as indicated. Cell lysates were harvested for protein phosphorylation analyses at 24 h.
  • FIG. 2C HCC827 cells were treated with 0.5 ⁇ erlotinib or 0.5 ⁇ ibrutinib and tested for EGFR phosphorylation at different time points as indicated.
  • FIG. 2D HCC827 cells were treated with ibrutinib and tested for EGFR phosphorylation and caspase-3 and PARP 1 cleavage at 48 h.
  • FIG. 3. Bruton tyrosine kinase (BTK) expression in lung cancer cell lines.
  • BTK Bruton tyrosine kinase
  • FIGS. 4A-C Activity in acquired erlotinib-resistant cell lines with EGFR mutations. The responses of parental PC9, HCC827, and HCC4006 cell lines are shown in FIGS. 8A-F.
  • FIG. 4A Allele frequencies of T790M mutations in PC9 and PC9ER determined by allele-specific PCR. The values represent the mean + SD of three assays.
  • FIG. 4B Dose response of erlotinib and ibrutinib in PC9ER cells. The data are means with standard deviations for two assays, performed in quadruplicate.
  • FIG. 4C The data are means with standard deviations for two assays, performed in quadruplicate.
  • FIGS. 5A-C Molecular biomarkers associated with responses to the anti- EGFR agents erlotinib, afatinib, and ibrutinib.
  • FIG. 5A Gene mutations associated with response. Colors represent fold change in IC5 0 values.
  • FIGS. 5B-C Protein (B) and mRNA (C) levels associated with response and identified at FDR of 20% and 5%, respectively. Colors represent correlation coefficients. Green, sensitive; red, resistant.
  • FIGS. 6A-C Combination effects of auranofin and ibrutinib on HI 975 and H1650 cell lines.
  • FIGS. 6A-B Dose response to ibrutinib in the absence and presence of 0.25 ⁇ auranofin. The data are the means with standard deviations for two assays, performed in quadruplicate.
  • FIG. 6C Ibrutinib's IC5 0 values in the presence or absence of auranofin.
  • FIGS. 7A-B FIG. 7A. AXL expression is significantly higher in mesenchymal NSCLC cell lines.
  • FIGS. 8A-F HCC827 and HCC4006 NSCLC cells are sensitive to erlotinib, whereas EGFR TKI resistant variants (ER 1-6) are resistant (FIGS. 8A&D). The majority of resistant variants have acquired a mesenchymal phenotype as indicated by loss of E-cadherin and gain of AXL expression (FIGS. 8B&E). Parental HCC827 and HCC4006 cells are sensitive to ibrutinib, while EGFR TKI resistant variants are not (FIGS. 8C&F).
  • EGFR mutant cancer uses reversible EGFR inhibitors (e.g., erlotinib and gefitinib).
  • EGFR inhibitors e.g., erlotinib and gefitinib.
  • a second mutation of EGER in T790M often causes resistance to erlotinib and gefitinib.
  • Ibrutinib (PCI-32765) is an irreversible inhibitor for Bruton tyrosine kinase (BTK) that has been evaluated in humans for treatment of lymphoma and myeloma. Ibrutinib remains effective for cancer cells with the T790M mutation that are resistant to erlotinib.
  • BTK Bruton tyrosine kinase
  • ibrutinib PCI-32765
  • PCI-32765 non- small cell lung cancer cell
  • ibrutinib was found to selectively inhibit the growth of EGFR mutant NSCLC cells, including cancer cells that harbor a T790M mutation and are resistant to conventional erlotinib.
  • NSCLC non- small cell lung cancer cell
  • ibrutinib is a candidate drug for treatment of EGFR mutant cancers, including erlotinib- or gefitinib-resistant tumors.
  • Ibrutinib (PCI-32765) selectively and irreversibly inhibits Bruton tyrosine kinase (BTK) (Honigberg et al, 2010; Pan et al., 2007), which is specifically required for the B-cell antigen receptor signaling pathway (Davis et al, 2010).
  • BTK Bruton tyrosine kinase
  • ibrutinib led to promising in vivo activity against spontaneous B-cell non-Hodgkin lymphoma in dogs and experimental rheumatoid arthritis in mice (Honigberg et al, 2010; Pan et al, 2007). Ibrutinib also inhibited growth of chronic lymphocytic leukemia and multiple myeloma cells inoculated into immune defective mice (Tai et al, 2012; Ponader et al, 2012).
  • Ibrutinib was approved in November 2013 by the FDA for treatment of mantle cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL) due to its remarkable single-agent efficacy in relapsed or refractory disease (objective response rate of 60%-70% and a complete response of 16%-20%) (Advani et al., 2013; Byrd et al, 2013; Wang et al., 2013). Moreover, clinical trials of ibrutinib in lymphoma or CLL patients revealed that it has an excellent safety profile at wide dose ranges (420-840 mg/day).
  • EGFR-mutant NSCLC cells including the T790M mutant cell line HI 975, are highly susceptible to ibrutinib, with 50% growth inhibition at ⁇ 0.2 ⁇ , which is well within clinically achievable concentrations used for the treatment of lymphoma (Byrd et al, 2013; Advani et al, 2013). Moreover, ibrutinib was recently reported to be well tolerated when used in combination with other targeted therapeutics (Burger et al, 2014; Younes et al, 2014).
  • Inhibitors targeting bypass signaling pathways can dramatically sensitize lung cancer cells to EGFR inhibitors, including ibrutinib.
  • Auranofin has been used to treat rheumatoid arthritis (Bernhard, 1982; Chaffman et al, 1984; Larsen et al, 1984; van Riel et al, 1984) for more than 30 years and the AXL inhibitor cabozantinib (Smith et al, 2013; Bowles et al, 2011) has been approved for treatment of thyroid cancer (Viola et al, 2013).
  • repurposing ibrutinib for NSCLC therapy and enhancing its efficacy through combination with other approved drugs has the potential to rapidly and significantly impact the clinical treatment of EGFR-mutant NSCLC patients.
  • ibrutinib In a previous study of ibrutinib' s activity against a panel of kinases, ibrutinib inhibited wild-type EGFR with an IC5 0 value of 5.6 nM (Honigberg et al, 2010). However, its effects on mutant EGFR and in EGFR-mutant lung cancer cells, which do not express BTK, the known target of ibrutinib, have not been previously reported. The present finding that ibrutinib selectively inhibits EGFR-mutant NSCLC cells, including those with T790M mutant EGFR, by directly inhibiting EGFR phosphorylation (Gao et al, 2014) is highly innovative (Haura and Rix, 2014).
  • Epidermal growth factor receptor is a known oncogenic driver in lung tumorigenesis and has been extensively investigated as a therapeutic target in lung cancer. Activating EGFR mutations are detected in about 10%- 17% of lung adenocarcinoma patients in the United States and Europe (Rosell et al, 2009; Pao et al, 2004; Ding et al, 2008; Marchetti et al, 2005; Gahr et al, 2013) and in about 30%-65% of lung cancer patients in Asia (Choi et al, 2013; Li et al, 2011; Gao et al, 2010; Tanaka et al, 2010).
  • gefitinib Pieris et al, 2004
  • Lynch et al, 2004 Philadelphia et al, 2004
  • erlotinib Pieris et al, 2004
  • Both gefitinib and erlotinib have been reported to significantly improve progression-free survival in patients with EGFR-mutant lung cancer (Cappuzzo et al, 2010; Fukuoka et al, 2011; Thatcher et al., 2005).
  • T790M mutations in exon 20 of the EGFR gene (Pao et al, 2005; Kobayashi et al, 2005; Yun et al, 2008; Engelman et al, 2006), which is found in about 50% of erlotinib- or gefitinib-resistant tumors (Bean et al, 2007; Pao et al, 2005; Kosaka et al, 2006).
  • T790M-independent mechanisms of resistance include "bypass" signaling via activation of alternative pathways.
  • HER2 [Takezawa et al., 2012], MET [Bean et al, 2007; Cappuzzo et al, 2009; Engelman et al, 2007]
  • downstream pathways e.g., IL-6R/STAT-3 [Kim et al, 2012; Sen et al, 2012], NF- ⁇ [Bivona et al, 2011], PI3K/AKT via PTEN loss [Sos et al, 2009]
  • EMT epithelial-to-mesenchymal transition
  • EGFRi resistant NSCLC tumors also have enhanced expression of classical EMT markers and the EMT-associated receptor tyrosine kinase AXL is a potential therapeutic target for overcoming anti-EGFR therapy associated with this mesenchymal phenotype (Byers et al, 2013).
  • the resistance to EGFR inhibitors caused by activation of AXL and EMT has also been reported by others (Zhang et al, 2012; Giles et al, 2013; Meyer et al, 2013; Cufi et al, 2013; Shien et al., 2013), suggesting that it is likely a third major subgroup of resistance in T790M negative tumors.
  • afatinib an irreversible dual EGFR/HER2 inhibitor that exhibits some activity against T790M mutant tumors preclinically (Li et al, 2008), has been approved by the U.S. Food and Drug Administration (FDA) (July 2013) for the treatment of metastatic NSCLC with EGFR mutation.
  • FDA U.S. Food and Drug Administration
  • afatinib is only approved for frontline treatment of EGFR mutant NSCLC, not for treatment of previously treated tumors.
  • a phase lb trial for combination therapy with cisplatin/paclitaxel or cisplatin/5-fluorouracil revealed that the maximum tolerated dose for afatinib was reduced to 20-30 mg/day (Vermorken et al, 2013), suggesting that combining afatinib with other therapeutic agents may not be feasible.
  • T790M- negative cancer cells remain resistant to third-generation EGFR inhibitors. Therefore, developing new therapeutic strategies or agents to overcome resistance to conventional EGFR inhibitors is urgently required.
  • Ibrutinib an irreversible BTK inhibitor recently approved by the FDA for the treatment of mantle cell lymphoma and chronic lymphocytic leukemia, can function as an EGFRi and selectively inhibit growth and induce apoptosis in EGFR-mutant NSCLC cells in vitro and in vivo, including erlotinib-resistant cells that harbor a T790M mutation (Gao et al, 2014).
  • a phase I/II clinical trial will test ibrutinib in EGFR-mutant NSCLC.
  • ibrutinib inhibits HER2 signaling in NSCLC cells, indicating that it may have clinical application in resistant tumors with HER2 overexpression.
  • Targeting other mechanisms of resistance can dramatically sensitize lung cancer cells to EGFRi, including ibrutinib.
  • inhibitors of STAT-3, NF- ⁇ , and AXL can dramatically sensitize NSCLC cells to EGFRi.
  • Auranofin (Bernhard, 1982; Chaffman et al., 1984; Larsen et al., 1984; van Riel et al, 1984), an inhibitor of thioredoxin reductase (TXNRD) (Lima and Rodriguez, 2011 ; Madeira et al, 2012; Schuh et al., 2012), STAT-3 (Kim et al, 2007; Nakaya et al, 201 1), and NF- ⁇ (Nakaya et al, 201 1), has been used to treat rheumatoid arthritis (Bernhard, 1982; Chaffman et al, 1984; Larsen et al, 1984; van Riel et al, 1984) since the 1980s.
  • TXNRD thioredoxin reductase
  • AXL inhibitor cabozantinib is also approved for thyroid cancer. Combination therapy using these agents may provide an opportunity to overcome resistance by drug repurposing and targeting multiple mechanisms of EGFRi resistance.
  • Certain embodiments concern detecting, either in vivo or in a sample, a mutation in the EGFR gene. Other embodiments concern detecting amplification of the HER2 gene. Other embodiments concern detecting a change in expression level of HER2, E- cadherin, and/or AXL gene products (e.g., mR A or protein).
  • assessing the presence of a mutation can involve detecting or quantifying a coding nucleic acid, such as an R A or DNA encoding a gene product (e.g., EGFR).
  • a coding nucleic acid such as an R A or DNA encoding a gene product (e.g., EGFR).
  • all or a portion of cancer cell genome is sequenced to detect the presence of a mutation (e.g., a substitution, deletion, inversion or insertion).
  • PCR polymerase chain reaction
  • a mutation in a gene coding sequence can be detected by determining the sequence of all or part of a coding RNA.
  • reverse transcription PCR can be employed to determine the sequence of an EGFR coding sequence.
  • quantitative PCR or RT PCR can be employed to determine whether cells comprise a reduced amount of an E-cadherin coding sequence or an increased amount of an AXL or a HER2 coding sequence.
  • PCR products described above may be subjected to sequence analysis to identify specific kinds of variations using standard sequence analysis techniques.
  • exhaustive analysis of genes is carried out by sequence analysis using primer sets designed for optimal sequencing.
  • the present embodiments provide methods by which any or all of these types of analyses may be used.
  • oligonucleotide primers may be designed to permit the amplification of sequences throughout the EGFR gene and surrounding sequence.
  • DNA sequencing may be used to detect and/or quantify EGFR, AXL, HER2 and/or E-cadherin coding nucleic acids.
  • Methods for such sequence include, but are not limited to, reversible terminator methods (e.g., used by Illumina® and Helicos® Biosciences), pyrosequencing (e.g., 454 sequencing from Roche) and sequencing by ligation (e.g., Life TechnologiesTM SOLiDTM sequencing).
  • reversible terminator methods e.g., used by Illumina® and Helicos® Biosciences
  • pyrosequencing e.g., 454 sequencing from Roche
  • sequencing by ligation e.g., Life TechnologiesTM SOLiDTM sequencing.
  • a curved line of characteristic shape is formed by connecting the plotted points. Beginning with the first cycle, the slope of the line is positive and constant. This is said to be the linear portion of the curve. After a reagent becomes limiting, the slope of the line begins to decrease and eventually becomes zero. At this point the concentration of the amplified target DNA becomes asymptotic to some fixed value. This is said to be the plateau portion of the curve.
  • the concentration of the target DNA in the linear portion of the PCR amplification is directly proportional to the starting concentration of the target before the reaction began.
  • concentration of the amplified products of the target DNA in PCR reactions that have completed the same number of cycles and are in their linear ranges, it is possible to determine the relative concentrations of the specific target sequence in the original DNA mixture.
  • these methods can be employed to detect EGFR sequences that are deleted in a cell, to detect a reduced overall expression of E-cadherin coding sequence, or to detect an increased overall expression of AXL or HER2 coding sequences.
  • nucleic acids are detected or quantified following gel separation and staining with ethidium bromide and visualization under UV light.
  • the products can then be exposed to x-ray film or visualized under the appropriate stimulating spectra, following separation.
  • visualization is achieved indirectly.
  • a labeled nucleic acid is brought into contact with the target sequence.
  • the probe is conjugated to a chromophore or a radiolabel.
  • the probe is conjugated to a binding partner, such as an antibody or biotin, and the other member of the binding pair carries a detectable moiety.
  • a binding partner such as an antibody or biotin
  • RNA and DNA are, for instance, well known to those of skill in the art.
  • Northern and Southern blotting involves the use of RNA or DNA, respectively, as a target. Briefly, a probe is used to target an RNA or DNA species that has been immobilized on a suitable matrix, often a filter of nitrocellulose. The different species should be spatially separated to facilitate analysis. This often is accomplished by gel electrophoresis of nucleic acid species followed by "blotting" on to the filter. Subsequently, the blotted target is incubated with a probe (such as a labeled probe) under conditions that promote denaturation and rehybridization.
  • a probe such as a labeled probe
  • a labeled probe can be used for in situ hybidization to detect the presence of heterozygous mutations EGFR or the presence of increased copies of the HER2 gene.
  • FISH fluorescence in situ hybridization
  • methods of the embodiments concern detection of the expression or activity of HER2, AXL, or E-cadherin proteins.
  • immunodetection methods for binding, purifying, removing, quantifying and/or otherwise generally detecting HER2, AXL, or E-cadherin proteins can be employed.
  • Antibodies may be employed to detect and/or quantify HER2, AXL, or E-cadherin in a subject or sample, e.g., a tumor biopsy.
  • immunodetection methods include, for example, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunoradiometric assay, fluoroimmunoassay, chemiluminescent assay, bioluminescent assay, mass spectrometry, and Western blot.
  • ELISA enzyme linked immunosorbent assay
  • RIA radioimmunoassay
  • immunoradiometric assay fluoroimmunoassay
  • fluoroimmunoassay chemiluminescent assay
  • bioluminescent assay bioluminescent assay
  • mass spectrometry and Western blot.
  • immunoassays in their most simple and/or direct sense, are binding assays.
  • Certain preferred immunoassays are the various types of enzyme linked immunosorbent assays (ELISAs) and/or radioimmunoassays (RIA) known in the art. Immunohistochemical detection using tissue sections is also particularly useful.
  • ELISAs enzyme linked immunosorbent assays
  • RIA radioimmunoassays
  • antigen-specific antibodies are immobilized onto a selected surface exhibiting protein affinity, such as a well in a polystyrene microtiter plate. Then, a test composition suspected of containing the protein antigen, such as a clinical sample, is added to the wells.
  • the bound protein antigen may be detected. Detection is generally achieved by the addition of another antigen-specific antibody that is linked to a detectable label. This type of ELISA is a simple "sandwich ELISA”. Detection and quantification may also be achieved by the addition of a second antigen-specific antibody, followed by the addition of a third antibody that has binding affinity for the second antibody, with the third antibody being linked to a detectable label.
  • the samples suspected of containing the protein antigen are immobilized onto the well surface and/or then contacted with the antigen-specific antibodies of the embodiments. After binding and/or washing to remove non-specifically bound immune complexes, the bound antibodies are detected and quantified. Where the initial antibodies are linked to a detectable label, the immune complexes may be detected directly. Again, the immune complexes may be detected using a second antibody that has binding affinity for the first antibody, with the second antibody being linked to a detectable label.
  • the antigen proteins, polypeptides and/or peptides are immobilized.
  • ELISA involves the use of antibody competition in the detection. In this ELISA, labeled antibodies against an antigen are added to the wells, allowed to bind, and/or detected by means of their label. The amount of antigen in an unknown sample is then determined by mixing the sample with the labeled antibodies against the antigen before and/or during incubation with coated wells. The presence of an antigen in the sample acts to reduce the amount of antibody against wild type or mutant protein available for binding to the well and thus reduces the ultimate signal.
  • ELISAs have certain features in common, such as coating, incubating and binding, washing to remove non-specifically bound species, and detecting the bound immune complexes. These are steps are well known to a skilled artisan.
  • Antigen-specific antibodies may also be used in conjunction with both fresh- frozen and/or formalin- fixed, paraffin-embedded tissue blocks prepared for study by immunohistochemistry (IHC).
  • IHC immunohistochemistry
  • the method of preparing tissue blocks from these particulate specimens has been successfully used in previous IHC studies of various prognostic factors, and/or is well known to those of skill in the art (Brown et ah, 1990; Abbondanzo et ah, 1990; Allred ei a/., 1990).
  • frozen-sections e.g., vascular tissue sections
  • PBS phosphate buffered saline
  • OCT viscous embedding medium
  • inverting the capsule and/or pelleting again by centrifugation snap-freezing in 70°C isopentane; cutting the plastic capsule and/or removing the frozen cylinder of tissue; securing the tissue cylinder on a cryostat microtome chuck; and/or cutting 25-50 serial sections.
  • Permanent-sections may be prepared by a similar method involving rehydration of the 50 mg sample in a plastic microfuge tube; pelleting; resuspending in 10% formalin for 4 hours fixation; washing/pelleting; resuspending in warm 2.5% agar; pelleting; cooling in ice water to harden the agar; removing the tissue/agar block from the tube; infiltrating and/or embedding the block in paraffin; and/or cutting up to 50 serial permanent sections.
  • MS mass spectrometry
  • MALDI matrix assisted laser desorption/ionization
  • TOF time of flight
  • ESI electrospray ionization
  • MALDI matrix assisted laser desorption/ionization
  • TOF time of flight
  • mass spectrometry may be used to look for the levels of proteins particularly.
  • ESI is a convenient ionization technique that is used to produce gaseous ions from highly polar, mostly nonvolatile biomolecules, including lipids.
  • the sample is injected as a liquid at low flow rates (1-10 ⁇ 7 ⁇ ) through a capillary tube to which a strong electric field is applied.
  • the field generates additional charges to the liquid at the end of the capillary and produces a fine spray of highly charged droplets that are electrostatically attracted to the mass spectrometer inlet.
  • the evaporation of the solvent from the surface of a droplet as it travels through the desolvation chamber increases its charge density substantially. When this increase exceeds the Rayleigh stability limit, ions are ejected and ready for MS analysis.
  • a typical conventional ESI source consists of a metal capillary of typically 0.1-0.3 mm in diameter, with a tip held approximately 0.5 to 5 cm (but more usually 1 to 3 cm) away from an electrically grounded circular interface having at its center the sampling orifice.
  • a potential difference of between 1 to 5 kV (but more typically 2 to 3 kV) is applied to the capillary by power supply to generate a high electrostatic field (10 6 to 10 7 V/m) at the capillary tip.
  • a sample liquid carrying the analyte to be analyzed by the mass spectrometer is delivered to tip through an internal passage from a suitable source (such as from a chromatograph or directly from a sample solution via a liquid flow controller).
  • ESI tandem mass spectroscopy In ESI tandem mass spectroscopy (ESI/MS/MS), one is able to simultaneously analyze both precursor ions and product ions, thereby monitoring a single precursor product reaction and producing (through selective reaction monitoring (SRM)) a signal only when the desired precursor ion is present.
  • SRM selective reaction monitoring
  • the internal standard is a stable isotope-labeled version of the analyte
  • quantification by the stable isotope dilution method This approach has been used to accurately measure pharmaceuticals and bioactive. Newer methods are performed on widely available MALDI-TOF instruments, which can resolve a wider mass range and have been used to quantify metabolites, peptides, and proteins.
  • Protein quantification has been achieved by quantifying tryptic peptides.
  • Complex mixtures such as crude extracts can be analyzed, but in some instances sample clean up is required.
  • Secondary ion mass spectroscopy is an analytical method that uses ionized particles emitted from a surface for mass spectroscopy at a sensitivity of detection of a few parts per billion.
  • the sample surface is bombarded by primary energetic particles, such as electrons, ions (e.g., O, Cs), neutrals or even photons, forcing atomic and molecular particles to be ejected from the surface, a process called sputtering. Since some of these sputtered particles carry a charge, a mass spectrometer can be used to measure their mass and charge. Continued sputtering permits measuring of the exposed elements as material is removed. This in turn permits one to construct elemental depth profiles. Although the majority of secondary ionized particles are electrons, it is the secondary ions which are detected and analysis by the mass spectrometer in this method.
  • LD-MS Laser desorption mass spectroscopy
  • LD-MS When coupled with Time-of-Flight (TOF) measurement, LD-MS is referred to as LDLPMS (Laser Desorption Laser Photoionization Mass Spectroscopy).
  • LDLPMS Laser Desorption Laser Photoionization Mass Spectroscopy
  • the LDLPMS method of analysis gives instantaneous volatilization of the sample, and this form of sample fragmentation permits rapid analysis without any wet extraction chemistry.
  • the LDLPMS instrumentation provides a profile of the species present while the retention time is low and the sample size is small.
  • an impactor strip is loaded into a vacuum chamber. The pulsed laser is fired upon a certain spot of the sample site, and species present are desorbed and ionized by the laser radiation. This ionization also causes the molecules to break up into smaller fragment-ions.
  • the positive or negative ions made are then accelerated into the flight tube, being detected at the end by a microchannel plate detector. Signal intensity, or peak height, is measured as a function of travel time. The applied voltage and charge of the particular ion determines the kinetic energy, and separation of fragments is due to different size causing different velocity. Each ion mass will thus have a different flight- time to the detector.
  • MALDI-TOF-MS Since its inception and commercial availability, the versatility of MALDI- TOF-MS has been demonstrated convincingly by its extensive use for qualitative analysis. For example, MALDI-TOF-MS has been employed for the characterization of synthetic polymers, peptide and protein analysis, DNA and oligonucleotide sequencing, and the characterization of recombinant proteins. Recently, applications of MALDI-TOF-MS have been extended to include the direct analysis of biological tissues and single cell organisms with the aim of characterizing endogenous peptide and protein constituents. [0076] The properties that make MALDI-TOF-MS a popular qualitative tool— its ability to analyze molecules across an extensive mass range, high sensitivity, minimal sample preparation and rapid analysis times— also make it a potentially useful quantitative tool.
  • MALDI-TOF-MS also enables non-volatile and thermally labile molecules to be analyzed with relative ease. It is therefore prudent to explore the potential of MALDI-TOF-MS for quantitative analysis in clinical settings, for toxicological screenings, as well as for environmental analysis. In addition, the application of MALDI-TOF-MS to the quantification of peptides and proteins is particularly relevant.
  • Treatment refers to administration or application of a therapeutic agent to a subject or performance of a procedure or modality on a subject for the purpose of obtaining a therapeutic benefit of a disease or health-related condition.
  • a treatment may include administration of a pharmaceutically effective amount of ibrutinib to a cancer patient in need thereof.
  • Subject and “patient” refer to either a human or non-human, such as primates, mammals, and vertebrates. In particular embodiments, the subject is a human.
  • therapeutic benefit or “therapeutically effective” as used throughout this application refers to anything that promotes or enhances the well-being of the subject with respect to the medical treatment of this condition. This includes, but is not limited to, a reduction in the frequency or severity of the signs or symptoms of a disease.
  • treatment of cancer may involve, for example, a reduction in the size of a tumor, a reduction in the invasiveness of a tumor, reduction in the growth rate of the cancer, or prevention of metastasis. Treatment of cancer may also refer to prolonging survival of a subject with cancer.
  • the administration of ibrutinib may be used for the treatment of diseases, including cancers that are resistant to erlotinib and/or gefitinib.
  • the invention specifically discloses treatment methods using ibrutinib in combination with various secondary agents.
  • Tumors for which the present treatment methods are useful include any malignant cell type, such as those found in a solid tumor or a hematological tumor.
  • Exemplary solid tumors can include, but are not limited to, a tumor of an organ selected from the group consisting of pancreas, colon, cecum, stomach, brain, head, neck, ovary, kidney, larynx, sarcoma, lung, bladder, melanoma, prostate, and breast.
  • Exemplary hematological tumors include tumors of the bone marrow, T or B cell malignancies, leukemias, lymphomas, blastomas, myelomas, and the like.
  • cancers that may be treated using the methods provided herein include, but are not limited to, lung cancer (including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung), cancer of the peritoneum, gastric or stomach cancer (including gastrointestinal cancer and gastrointestinal stromal cancer), pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, various types of head and neck cancer, and melanoma.
  • lung cancer including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung
  • cancer of the peritoneum gastric or stomach cancer (including gastrointestinal cancer and gastrointestinal stromal cancer)
  • pancreatic cancer cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon
  • the cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma;
  • compositions may comprise, for example, at least about 0.1% of an active compound.
  • an active compound may comprise between about 2% to about 75% of the weight of the unit, or between about 25% to about 60%, for example, and any range derivable therein.
  • pharmaceutical compositions may comprise, for example, at least about 0.1% of an active compound.
  • an active compound may comprise between about 2% to about 75% of the weight of the unit, or between about 25% to about 60%, for example, and any range derivable therein.
  • pharmaceutical or pharmacologically acceptable refers to molecular entities and compositions that do not produce an adverse, allergic, or other untoward reaction when administered to an animal, such as a human, as appropriate.
  • compositions comprising an antibody or additional active ingredient will be known to those of skill in the art in light of the present disclosure. Moreover, for animal (e.g., human) administration, it will be understood that preparations should meet sterility, pyrogenicity, general safety, and purity standards as required by FDA Office of Biological Standards.
  • aqueous solvents e.g., water, alcoholic/aqueous solutions, saline solutions, parenteral vehicles, such as sodium chloride, Ringer's dextrose, etc.
  • non-aqueous solvents e.g., propylene glycol, polyethylene glycol, vegetable oil, and injectable organic esters, such as ethyloleate
  • dispersion media coatings, surfactants, antioxidants, preservatives (e.g., antibacterial or antifungal agents, anti-oxidants, chelating agents, and inert gases), isotonic agents, absorption delaying agents, salts, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, fluid and nutrient replenishers, such like materials and combinations thereof, as would be known to one of ordinary skill in the art.
  • the pH and exact concentration e.g., water, alcoholic/aqueous solutions,
  • unit dose refers to physically discrete units suitable for use in a subject, each unit containing a predetermined quantity of the therapeutic composition calculated to produce the desired responses discussed above in association with its administration, i.e., the appropriate route and treatment regimen.
  • the quantity to be administered depends on the effect desired.
  • the actual dosage amount of a composition of the present embodiments administered to a patient or subject can be determined by physical and physiological factors, such as body weight, the age, health, and sex of the subject, the type of disease being treated, the extent of disease penetration, previous or concurrent therapeutic interventions, idiopathy of the patient, the route of administration, and the potency, stability, and toxicity of the particular therapeutic substance.
  • a dose may also comprise from about 1 ⁇ g/kg/body weight to about 1000 mg/kg/body weight (this such range includes intervening doses) or more per administration, and any range derivable therein.
  • a derivable range from the numbers listed herein, a range of about 5 ⁇ g/kg/body weight to about 100 mg/kg/body weight, about 5 ⁇ g/kg/body weight to about 500 mg/kg/body weight, etc. , can be administered.
  • the practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject.
  • the active compounds can be formulated for parenteral administration, e.g., formulated for injection via the intravenous, intramuscular, sub-cutaneous, or even intraperitoneal routes.
  • parenteral administration e.g., formulated for injection via the intravenous, intramuscular, sub-cutaneous, or even intraperitoneal routes.
  • One preferred method of administration is hepatic artery infusion.
  • compositions can be prepared as either liquid solutions or suspensions; solid forms suitable for use to prepare solutions or suspensions upon the addition of a liquid prior to injection can also be prepared; and, the preparations can also be emulsified.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil, or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • the form must be sterile and must be fluid to the extent that it may be easily injected. It also should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
  • compositions may be formulated into a neutral or salt form.
  • Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.
  • a pharmaceutical composition can include a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
  • a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
  • the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants.
  • the prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • compositions and methods of the present embodiments involve ibrutinib in combination with a second or additional therapy.
  • compositions including combination therapies, enhance the therapeutic or protective effect, and/or increase the therapeutic effect of another anti-cancer or anti-hyperproliferative therapy.
  • Therapeutic and prophylactic methods and compositions can be provided in a combined amount effective to achieve the desired effect, such as the killing of a cancer cell and/or the inhibition of cellular hyperproliferation. This process may involve contacting the cells with both ibrutinib and a second therapy.
  • a tissue, tumor, or cell can be contacted with one or more compositions or pharmacological formulation(s) comprising one or more of the agents (i.e., ibrutinib or a second anti-cancer agent), or by contacting the tissue, tumor, and/or cell with two or more distinct compositions or formulations, wherein one composition provides 1) ibrutinib, 2) a second anti-cancer agent, or 3) both ibrutinib and an anti-cancer agent.
  • a combination therapy can be used in conjunction with chemotherapy, radiotherapy, surgical therapy, or immunotherapy.
  • contacted and “exposed,” when applied to a cell are used herein to describe the process by which a therapeutic construct and a chemotherapeutic or radiotherapeutic agent are delivered to a target cell or are placed in direct juxtaposition with the target cell.
  • both agents are delivered to a cell in a combined amount effective to kill the cell or prevent it from dividing.
  • Ibrutinib may be administered before, during, after, or in various combinations relative to an anti-cancer treatment.
  • the administrations may be in intervals ranging from concurrently to minutes to days to weeks.
  • ibrutinib is provided to a patient separately from an anti-cancer agent, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the two compounds would still be able to exert an advantageously combined effect on the patient.
  • a course of treatment will last 1-90 days or more (this such range includes intervening days). It is contemplated that one agent may be given on any day of day 1 to day 90 (this such range includes intervening days) or any combination thereof, and another agent is given on any day of day 1 to day 90 (this such range includes intervening days) or any combination thereof. Within a single day (24-hour period), the patient may be given one or multiple administrations of the agent(s). Moreover, after a course of treatment, it is contemplated that there is a period of time at which no anti-cancer treatment is administered.
  • This time period may last 1-7 days, and/or 1-5 weeks, and/or 1-12 months or more (this such range includes intervening days), depending on the condition of the patient, such as their prognosis, strength, health, etc. It is expected that the treatment cycles would be repeated as necessary. [0096] Various combinations may be employed. For the example below ibrutinib therapy is "A” and a second anti-cancer therapy is "B":
  • chemotherapeutic agents may be used in accordance with the present embodiments.
  • the term “chemotherapy” refers to the use of drugs to treat cancer.
  • a “chemotherapeutic agent” is used to connote a compound or composition that is administered in the treatment of cancer.
  • agents or drugs are categorized by their mode of activity within a cell, for example, whether and at what stage they affect the cell cycle.
  • an agent may be characterized based on its ability to directly cross-link DNA, to intercalate into DNA, or to induce chromosomal and mitotic aberrations by affecting nucleic acid synthesis.
  • chemotherapeutic agents include alkylating agents, such as thiotepa and cyclosphosphamide; alkyl sulfonates, such as busulfan, improsulfan, and piposulfan; aziridines, such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines, including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide, and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); do
  • chemotherapeutics that are contemplated for use herein include NF-KB/STAT-3 inhibitors (e.g., auranofin, compounds disclosed in PCT Publn. No. WO2012/142615), AXL inhibitors (e.g., SGI-7079, cabozantinib, BMS 777607, BGB324, ASP2215, compounds disclosed in PCT Publn. Nos.
  • NF-KB/STAT-3 inhibitors e.g., auranofin, compounds disclosed in PCT Publn. No. WO2012/142615
  • AXL inhibitors e.g., SGI-7079, cabozantinib, BMS 777607, BGB324, ASP2215, compounds disclosed in PCT Publn. Nos.
  • ALK/MET inhibitors e.g., crizotinib, compounds disclosed in PCT Publn. No.
  • DNA damaging factors include what are commonly known as ⁇ -rays, X-rays, and/or the directed delivery of radioisotopes to tumor cells.
  • Other forms of DNA damaging factors are also contemplated, such as microwaves, proton beam irradiation (U.S. Patents 5,760,395 and 4,870,287), and UV-irradiation. It is most likely that all of these factors affect a broad range of damage on DNA, on the precursors of DNA, on the replication and repair of DNA, and on the assembly and maintenance of chromosomes.
  • Dosage ranges for X-rays range from daily doses of 50 to 200 roentgens for prolonged periods of time (3 to 4 wk), to single doses of 2000 to 6000 roentgens.
  • Dosage ranges for radioisotopes vary widely, and depend on the half-life of the isotope, the strength and type of radiation emitted, and the uptake by the neoplastic cells. iii. Immunotherapy
  • immunotherapies may be used in combination or in conjunction with methods of the embodiments.
  • immunotherapeutics generally, rely on the use of immune effector cells and molecules to target and destroy cancer cells.
  • Rituximab (Rituxan®) is such an example.
  • the immune effector may be, for example, an antibody specific for some marker on the surface of a tumor cell.
  • the antibody alone may serve as an effector of therapy or it may recruit other cells to actually affect cell killing.
  • the antibody also may be conjugated to a drug or toxin (chemotherapeutic, radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) and serve merely as a targeting agent.
  • the effector may be a lymphocyte carrying a surface molecule that interacts, either directly or indirectly, with a tumor cell target.
  • Various effector cells include cytotoxic T cells and NK cells.
  • the tumor cell must bear some marker that is amenable to targeting, i.e., is not present on the majority of other cells.
  • Common tumor markers include CD20, carcinoembryonic antigen, tyrosinase (p97), gp68, TAG-72, HMFG, Sialyl Lewis Antigen, MucA, MucB, PLAP, laminin receptor, erb B, and pl55.
  • An alternative aspect of immunotherapy is to combine anticancer effects with immune stimulatory effects.
  • Immune stimulating molecules also exist including: cytokines, such as IL-2, IL-4, IL-12, GM-CSF, gamma-IFN, chemokines, such as MIP-1, MCP-1, IL-8, and growth factors, such as FLT3 ligand.
  • cytokines such as IL-2, IL-4, IL-12, GM-CSF, gamma-IFN
  • chemokines such as MIP-1, MCP-1, IL-8
  • growth factors such as FLT3 ligand.
  • immunotherapies currently under investigation or in use are immune adjuvants, e.g., Mycobacterium bovis, Plasmodium falciparum, dinitrochlorobenzene, and aromatic compounds (U.S.
  • cytokine therapy e.g., interferons ⁇ , ⁇ , and ⁇ , IL-1, GM-CSF, and TNF
  • gene therapy e.g., TNF, IL-1, IL-2, and p53 (Qin et al, 1998; Austin- Ward and Villaseca, 1998; U.S.
  • Curative surgery includes resection in which all or part of cancerous tissue is physically removed, excised, and/or destroyed and may be used in conjunction with other therapies, such as the treatment of the present embodiments, chemotherapy, radiotherapy, hormonal therapy, gene therapy, immunotherapy, and/or alternative therapies.
  • Tumor resection refers to physical removal of at least part of a tumor.
  • treatment by surgery includes laser surgery, cryosurgery, electrosurgery, and microscopically-controlled surgery (Mohs' surgery).
  • a cavity may be formed in the body.
  • Treatment may be accomplished by perfusion, direct injection, or local application of the area with an additional anti-cancer therapy. Such treatment may be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, or 12 months. These treatments may be of varying dosages as well. v. Other Agents
  • agents may be used in combination with certain aspects of the present embodiments to improve the therapeutic efficacy of treatment.
  • additional agents include agents that affect the upregulation of cell surface receptors and GAP junctions, cytostatic and differentiation agents, inhibitors of cell adhesion, agents that increase the sensitivity of the hyperproliferative cells to apoptotic inducers, or other biological agents. Increases in intercellular signaling by elevating the number of GAP junctions would increase the anti-hyperproliferative effects on the neighboring hyperproliferative cell population.
  • cytostatic or differentiation agents can be used in combination with certain aspects of the present embodiments to improve the anti-hyperproliferative efficacy of the treatments.
  • Inhibitors of cell adhesion are contemplated to improve the efficacy of the present embodiments.
  • Examples of cell adhesion inhibitors are focal adhesion kinase (FAKs) inhibitors and lovastatin. It is further contemplated that other agents that increase the sensitivity of a hyperproliferative cell to apoptosis, such as the antibody c225, could be used in combination with certain aspects of the present embodiments to improve the treatment efficacy.
  • NSCLC human non-small cell lung cancer
  • Ibrutinib, erlotinib, and afatinib were obtained from Selleck Chemicals (Houston, TX).
  • Monoclonal antibodies (mouse anti- human) for Western blot analysis were obtained from Cell Signaling Technology (Beverly, MA), except for ⁇ -actin, which was acquired from Sigma (St Louis, MO).
  • Secondary antibodies were purchased from Li-Cor Corp (Lincoln, NE).
  • mice were grouped randomly into four groups (5/group) and treated with oral administration of 1) ibrutinib (25 mg/kg/day); 2) erlotinib (50 mg/kg/day); and 3) solvent (5% DMSO, 2% Tween®-80 and 0.2% 2-hydroxypropyl- -cyclodextrin).
  • Tumor volumes were calculated by using the formula a x b 2 x 0.5, where a and b represented the larger and smaller diameters, respectively.
  • Mice were killed by CO 2 asphyxiation when the tumors had grown to 15 mm in diameter or had ulceration.
  • Example 1 Selective antitumor activity of ibrutinib in EGFR-mutant non-small cell lung cancer cells
  • the antitumor activities of ibrutinib were evaluated in a panel of lung cancer cell lines using a cell viability assay (Liu et al, 2012) following 3 days of treatment with 0.03-31 ⁇ ibrutinib.
  • the 50% inhibitory concentration [IC5 0 ] values of ibrutinib ranged from 0.002 to 31 ⁇ .
  • HCC827, H1975, and H292 had IC5 0 values between 2 and 31 ⁇ .
  • the IC5 0 values were between 0.002 and 0.195 ⁇ (FIG. 1A), which were within the clinically achievable concentrations of ibrutinib in the doses used for treatment of lymphoma (Advani et al, 2013; Byrd et al, 2013).
  • HCC827 and H1975 cells are known to harbor epidermal growth factor receptor (EGFR) mutations, whereas the H292 cell line has wild-type EGFR.
  • EGFR epidermal growth factor receptor
  • Erlotinib and ibrutinib antitumor activities were compared in nine other NSCLC cell lines, seven of which have mutations or deletions in the EGFR gene. As shown in FIG. IB and Table 1, ibrutinib induced an antitumor spectrum similar to that of erlotinib in these cell lines. One exception is the HI 975 cell line, which harbors a T790M mutation in EGFR, and was resistant to erlotinib but sensitive to ibrutinib.
  • the dose- response of ibrutinib was compared to that of erlotinib and afatinib in HI 975 cells and it was found that ibrutinib induced antitumor activity similar to that of afatinib (FIG. IB).
  • H2170 has HER2 amplification.
  • Example 2 Ibrutinib effectively suppresses the in vivo growth of T790M mutant tumors [00116] To test whether ibrutinib could elicit in vivo antitumor activity in
  • EGFR-mutant tumors xenograft tumors were established from HI 975 cells in nude mice. When the tumors reached 4-5 mm in diameters, the animals were treated daily with ibrutinib (25 mg/kg), erlotinib (50 mg/kg), or solvent. The results showed that treatment with ibrutinib, but not erlotinib, significantly suppressed HI 975 tumor growth and prolonged survival of the tumor-bearing animals (FIGS. 1C-D).
  • ibrutinib induced growth suppression or apoptosis in HCC827 cells was also tested.
  • Flow cytometric analysis on apoptotic cells and Western blot analyses of poly(ADP-ribose) polymerase (PARP1) and caspase-3 cleavage showed that ibrutinib induced a dose-dependent increase of apoptotic cells (42% of apoptotic cells at 72 h after treatment with 1 ⁇ ibrutinib versus ⁇ 10% of apoptotic cells in the control group) and cleavage of PARP 1 and caspase-3 in HCC827 cells (FIG. 2D), demonstrating that apoptosis is the major mode of action in EGFR mutant NSCLC, such as HCC827 cells.
  • PARP1 poly(ADP-ribose) polymerase
  • EGFR assay kits (Promega or Invitrogen) will be used to determine the direct effect of ibrutinib on wild-type and mutant EGFR.
  • Afatinib or CO- 1686 (Selleck Chemicals, Houston, TX) will be used as a positive control.
  • Varying doses of ibrutinib and positive controls will be tested for their effects on recombinant wild-type and mutant (L858R, T790M, and T790M/L858R) EGFR proteins (BPS Bioscience, San Diego, CA).
  • IC5 0 values will be calculated on the basis of the dose-response curves. The results will allow for a determination of whether ibrutinib has a differential effect on wild-type and mutant EGFR.
  • T790M mutations are commonly seen in patients resistant to anti-EGFR therapies, few T790M mutant cell lines are available. Mutant EGFR can transform both Ba/F3 cells and immortalized human bronchial epithelial cells, rendering them sensitive to anti-EGFR therapy (Li et al., 2008; Greulich et al., 2005; Jiang et al, 2005; Yuza et al, 2007).
  • retroviral plasmids expressing wild-type, mutant, and double-mutant EGFR including G719S/T790M, L858R/T790M), atypical mutations (e.g., L861Q), and various deletions in exon 19 with and without T790M mutations, have been obtained (Li et al, 2008; Greulich et al., 2005; Yuza et al, 2007).
  • EGFR retroviral vectors will be used to stably transfect Ba/F3 (Jiang et al, 2005; Shimamura et al, 2006) and human bronchial epithelial cells (Greulich et al, 2005; Yuza et al, 2007). Transfected cells will be used to compare the activity of ibrutinib and newer generation EGFRi (CO-1686 and AZD9291). Dose-dependent EGFR inhibition, cell viability, and apoptosis induction will be determined in parallel for all three agents in these cell lines.
  • RPPA Proteomic profiling
  • EGFR activation will also be determined in A549 and H460 cells.
  • A549 and H460 cells will be treated with various doses of ibrutinib, CO-1686, and AZD9291 for 6 h.
  • EGF (10-100 ng/ml) will be added to the cultures for 15 min.
  • the cell lysate will then be analyzed for EGFR phosphorylation.
  • the results will allow comparison of the effects of ibrutinib with the newer EGFRi on EGF-stimulated EGFR activation of wild-type EGFR, as previously reported (Gao et al, 2014; Xu et al, 2010; Naumov et al, 2009).
  • ibrutinib is reported to form a covalent complex with BTK through Cys481 and a conserved cysteine is found in EGFR at Cys797 (Pan et al, 2007) and in HER2 at Cys775, it is likely that ibrutinib binds to EGFR through Cys797.
  • This cysteine residue is also the covalent binding site for all second- and third-generation EGFR inhibitors (Ward et al., 2013), including afatinib (Li et al., 2008), AZD9291 (Cross et al, 2014), and CO-1686 (Tjin et al, 2014).
  • CRISPR Clustered regularly interspaced short palindromic repeats
  • Cas9-mediated target-specific genome editing a novel technology that can efficiently induce mutagenesis in target genes in cultured cells and animals (Shalem et al, 2014; Wang et al, 2013; Zhou et al, 2014), will be used to generate EGFR C797 mutations in HCC827 and HI 975 cells.
  • in vitro mutagenesis will be performed on retroviral plasmids that encode mutant EGFRs to generate a C797S mutation in the original plasmid backbones.
  • the C797S mutant EGFR will then be stably transfected into HCC827 and HI 975 cells.
  • the C797 mutant cell lines will allow a determination as to whether putative mutations in this site will cause resistance to ibrutinib and the novel anti- EGFR therapeutics under development.
  • the cell lines may also be used to develop strategies to overcome putative resistance to second- and third-generation anti-EGFR therapeutics.
  • Cell viability assays will be performed to determine whether C797 mutations in EGFR affect the activity of ibrutinib, afatinib, CO- 1686, and AZD9291 in these cell lines.
  • EGFR C797 mutations are expected to cause resistance to afatinib, AZD9297, and CO-1686 because these agents are known to inhibit EGFR through covalent binding to C797. If ibrutinib also inhibits EGFR through C797, its activity is expected to be affected by C797 mutations.
  • C797 mutations may not have a drastic impact on ibrutinib's anti- EGFR activity, although they may have an impact on activities of other C797-binding anti- EGFR agents.
  • the results will allow a determination of the possible impact of a C797 mutation on novel anti-EGFR therapeutics.
  • Example 5 Ibrutinib in cells with acquired resistance to erlotinib and combinatorial approaches to augment anti-EGFR efficacy
  • Alternative or bypass pathways known to be associated with EGFR inhibitor resistance include amplification of MET (Bean et al., 2007; Cappuzzo et al, 2009; Engelman et al., 2007), loss of PTEN (Sos et al, 2009), and activation of the NF- ⁇ (Bivona et al, 201 1) or IL-6R/STAT-3 pathway (Kim et al, 2012; Sen et al, 2012).
  • a third key mechanism of resistance is acquisition of a mesenchymal phenotype (Byers et al, 2013; Zhang et al., 2012; Giles et al, 2013; Meyer et al, 2013; Cufi et al, 2013; Shien et al, 2013).
  • EMT is associated with a loss of cell adhesion proteins, including E-cadherin, and increased invasiveness. Moreover, EMT is associated with increased AXL expression (Byers et al, 2013), which is a potential therapeutic target in overcoming EMT-associated resistance to EGFR TKIs. Studies in NSCLC cell lines and clinical specimens have demonstrated that EMT and AXL activation are significantly associated with resistance to anti-EGFR therapy (Byers et al, 2013).
  • PTEN Sos et al, 2009
  • HCC4006ER and HCC827ER which are T790M negative, were found to be resistant to ibrutinib, afatinib, and CO- 1686.
  • STAT-3/NF-KB inhibitor, auranofin, and AXL inhibitor, SGI-7079 dramatically augmented the therapeutic efficacy of EGFR inhibitors, including ibrutinib, in erlotinib-resistant NSCLC cells.
  • HCC4006ER and HCC827ER were pan-resistant to ibrutinib, afatinib, and CO- 1686 (FIG. 4C).
  • Overexpression and knockdown approaches will be used to determine whether modulating the expression levels of those molecules restores the sensitivity of HI 650 to ibrutinib, erlotinib, and CO- 1686.
  • Western blot and real-time PCR analyses will be used to compare the expression of these molecules in HCC827, HCC827ER, HCC4006, and HCC4006ER cell lines.
  • gene mutations in the exons of 200 cancer-related genes will be determined by exome sequencing, which includes most of the 138 cancer driver genes affected by mutations (Vogelstein et al, 2013), and protein profiling by RPPA in parental and resistant HCC827ER and HCC4006ER cells. New mutations or significant protein changes detected in HCC827ER or HCC4006ER cells will be further tested for their causal relationship with anti-EGFR resistance, as described above for HI 650 cells.
  • Ibrutinib is highly active against T790M mutant and HER2-amplified cell lines, and inhibitors of both AXL and NF-KB/STAT-3 can dramatically sensitize lung cancer cells to EGFR inhibitors, including ibrutinib.
  • ibrutinib alone will be effective for treatment of acquired resistance caused by T790M mutation and HER2 amplifications, whereas combination therapy will be required to overcome the resistance caused by alterations in cooperative or parallel pathways. It will be determined whether MET inhibitor crizotinib, the AXL inhibitor cabozantinib, and/or auranofin sensitize HCC4006ER and HCC827ER cells to ibrutinib or the novel anti-EGFR agents CO- 1686 and AZD9291.
  • ibrutinib, CO- 1686, and AZD9291 can be used in combination with pathway-targeting drugs to treat T790M-negative tumors.
  • Potential therapeutic agents include ALK/MET inhibitor crizotinib, RAF/VEGFR inhibitor sorafenib, AKT inhibitor MK2206, Src inhibitor dasatinib, JAK/STAT-3 inhibitor ruxolitinib, and W T/p-catenin inhibitor LGK974135. All these agents will be obtained from Selleck Chemicals.
  • Single agent activity of these compounds will be determined in HI 650, HCC4006ER, HCC827ER, and HI 975 cell lines using a dose- dependent cell viability assay, and the IC 50 values will be calculated.
  • the combination activity will also be determined using a dose-dependent cell viability assay with a ratio of the two agents on the basis of their IC 50 values.
  • the combined effects of the two agents will then be analyzed by calculating the combination index with CalcuSyn software (Biosoft, Ferguson, MO), as previously described (Huang et al, 2002; Meng et al, 2010).
  • An unbiased RPPA assay will be used to determine the downstream signaling effects of ibrutinib and the optimal combinatorial therapies identified.
  • three cell lines e.g., H1975, H1650 and HCC827ER
  • ICso-ICso the optimal concentrations
  • Cells will be harvested at 0.5, 1, 2, 4, 8, and 24 h after treatment, and lysates analyzed by RPPA.
  • the time-dependent molecular changes identified will be further analyzed using the Ingenuity Pathway Analysis server to identify possible pathways involved in the pharmacological interaction of the combination therapy.
  • the possible contributions of the candidate targets in enhanced efficacy in combination therapies will be further characterized using specific inhibitors and enforced gene overexpression or knockdown, as previously described (Wei et al, 2009; Lu et al, 2013; Wei et al., 2010).
  • the results will allow a determination as to the possible molecular changes associated with the treatment response of combination therapy; these may be useful as biomarkers for monitoring treatment responses.
  • the excellent safety profile observed in clinical trials of ibrutinib in combination therapy suggests that ibrutinib is a good candidate for combinatorial therapies in anti-EGFR therapy.
  • the possible treatment-related toxicity of the combination therapy will be determined in Balb/c or C3H mice.
  • the animals will be treated with doses in combination therapy. Mice will be observed daily, and their weights will be recorded every 2-3 days for 60 days. Five mice from each group will be euthanized on day 3 after the last treatment and at the end of the experiment (day 60). Complete pathological analyses will be performed for all animals, including moribund animals, at necropsy.
  • tissues will be collected and fixed in cold, buffered, neutral 10% formalin for histopathological examination: adrenal gland, bone (femur), bone marrow (femur), brain, colon, intestines, esophagus, eyes, gall bladder, gonads, heart, kidneys, liver, lungs, lymph nodes, pancreas, skin, spleen, spinal cord, stomach, and urinary bladder.
  • Hematology profiles such as blood cell counts for erythrocytes, platelets, reticulocytes, total and differential leukocytes, and nucleated red blood cells and hemoglobin concentration
  • clinical chemistry profiles such as blood urea nitrogen, creatinine, serum aspartate aminotransferase, serum alanine aminotransferase, alkaline phosphatase, total bilirubin, total protein, and albumin
  • Blood samples and changes in histopathological features will be examined microscopically as described previously (Gu et al, 2000).
  • the paired t-test, one-way ANOVA, or Wilcoxon signed-rank test will be used to compare mouse weights and the testing parameters before treatment and at various time points after treatment. The results will allow a determination as to whether the combination therapy causes toxicity and organs susceptible to the combination therapy. If necessary, pharmacokinetic studies will be performed as recently reported (Wu et al, 2014). The information will be useful for patient monitoring in future clinical trials.
  • mice Possible treatment-related toxicity for effective combination therapy will be further evaluated in Balb/c or C3H mice.
  • the animals will be treated with doses in combination therapy.
  • the mice will be observed daily, and their weights will be recorded every 2-3 days for 60 days.
  • Five mice from each group will be euthanized on day 3 after the last treatment and at the end of the experiment (day 60).
  • Moribund animals will be killed immediately. Complete histopathological and clinical pathological analyses will be performed for all animals, including moribund animals, at necropsy.
  • tissues will be collected and fixed in cold, buffered, neutral 10% formalin for histopathological examination: adrenal gland, bone (femur), bone marrow (femur), brain, colon, intestines, esophagus, eyes, gall bladder, gonads, heart, kidneys, liver, lungs, lymph nodes, pancreas, skin, spleen, spinal cord, stomach, and urinary bladder.
  • Hematology profiles such as blood cell counts for erythrocytes, platelets, reticulocytes, total and differential leukocytes, and nucleated red blood cells and hemoglobin concentration
  • clinical chemistry profiles such as blood urea nitrogen, creatinine, serum aspartate aminotransferase, serum alanine aminotransferase, alkaline phosphatase, total bilirubin, total protein, and albumin
  • Blood samples and changes in histopathological features will be examined microscopically as described previously (Gu et al., 2000).
  • the paired t-test, oneway ANOVA, or Wilcoxon signed-rank test will be used to compare mouse weights and the testing parameters before treatment and at various time points after treatment. The results will allow a determination as to whether the combination therapy causes toxicities and what organs are susceptible to the combination therapy. This information will be useful for patient monitoring.
  • Example 6 Targeting EGFR inhibitor resistance via bypass signaling pathways: HER2 amplification
  • HER2 overexpression is detected in 20% (Berghoff et al, 2013; Nakamura et al, 2005; Heinmoller et al, 2003) of lung cancers, and HER2 amplifications or mutations are detected in ⁇ 3% (Stephens et al, 2004; Mazieres et al, 2013; Arcila et al, 2012; Yoshizawa et al., 2014).
  • Amplification of HER2 is thought to account for -10% of NSCLC with acquired resistance to EGFRi and represent a distinct resistance mechanism, independent of T790M mutation (Takezawa et al, 2012).
  • EGFRi resistant NSCLC also upregulates multiple bypass signaling pathways including PI3K/AKT (via loss of PTEN [Sos et al, 2009; She et al, 2005]), NF- ⁇ (Bivona et al., 2011 ; Tanaka et al, 201 1; Sakuma et al, 2012), and STAT-3 (Kim et al, 2012; Chiu et al, 201 1 ; Harada et al, 2012). In this scenario, it may be possible to combine ibrutinib with other targeted therapies to achieve clinical benefit.
  • Inhibition of the STAT-3 pathway has been shown to overcome resistance to EGFRi therapy in lung cancer (Chiu et al, 2011 ; Harada et al, 2012), head and neck cancer (Sen et al, 2012), pancreatic cancer (Nagaraj et al, 2011), and glioma (Lo et al, 2008). Furthermore, inhibition of IL-6/JAK/STAT-3 signaling or NF- ⁇ activity with siRNA or small-molecule inhibitors dramatically sensitizes cancer cells to treatment with EGFR inhibitors (Kim et al, 2012; Sen et al, 2012; Bivona et al, 2011 ; Sakuma et al., 2012; Chiu et al, 201 1).
  • ibrutinib dose responses were examined in HI 975 and HI 650 cell lines in the presence or absence of auranofin. While low concentrations of auranofin (0.25 ⁇ ) alone demonstrate mild activity, its presence dramatically sensitized HI 975 and HI 650 cells to ibrutinib (IC 50 value reduced 50-100 fold) (FIG. 6), demonstrating the feasibility of enhancing ibrutinib's activity by combination therapy. Recently, clinical trials demonstrated that ibrutinib is well tolerated when used in combination with other targeted therapeutics (Burger et al, 2014; Younes et al, 2014).
  • RPPA will be performed to determine time-dependent changes in molecular targets of multiple cancer related pathways in H2170 cells after treatment with ibrutinib.
  • the activity of ibrutinib and the HER2 inhibitor lapatinib in other HER2 amplified lung cancer cell lines, H650 (7.2 copies/cell), H1092 (6.2 copies/cell), and HCC954 (6.6 copies/cell), will be determined.
  • ibrutinib may be used for treatment of HER2 amplified lung cancers.
  • Wild-type and mutant HER2 plasmids (Li et al, 2004; Wang et al, 2004) will be used to stably transfect EGFR mutant NSCLC cell lines PC9, HCC827, and HCC4006.
  • the transfected cells will be used to compare the activity of ibrutinib, afatinib, CO- 1686, and AZD9291. The results will allow a determination as to whether ibrutinib can be used to overcome the resistance caused by HER2 amplification/mutations.
  • ibrutinib The effects of ibrutinib on in vivo growth of NSCLC cells with HER2 amplification/overexpression will be tested. Briefly, in vivo activity of ibrutinib will be determined by treating tumors derived from cell lines described above in nude mice daily with either ibrutinib or solvent, when tumors reach 50-100 mm 3 in volume.
  • Molecular profiling was performed on NSCLC cell lines and clinical specimens from patients treated in the BATTLE trial (Kim et al, 201 1) and it was demonstrated that mesenchymal cells showed significantly greater resistance to EGFR inhibitors, compared to more epithelial cells. Moreover, mesenchymal NSCLC also expressed increased levels of the receptor tyrosine kinase AXL and showed a trend toward greater sensitivity to the AXL inhibitor SGI-7079, whereas the combination of SGI-7079 with erlotinib reversed erlotinib resistance in mesenchymal lines expressing AXL (FIG.
  • EMT/AXL expression may be a mechanism of resistance to EGFRi, including ibrutinib.
  • ibrutinib may be used in combination with the AXL inhibitor cabozantanib, which is approved for use in thyroid cancer.
  • Example 8 Patient-derived xenografts (PDXs) from lung cancer patients
  • ibrutinib The effects of ibrutinib will be tested in multiple NSCLC mouse models, including 1) xenografts established from cell lines, 2) genetically engineered mouse models (GEMMS), and 3) PDXs.
  • GEMMS genetically engineered mouse models
  • PDXs PDXs.
  • in vivo activity of ibrutinib will be determined by treating tumors derived from cell lines (HI 975 and others as described above) in nude mice daily with either ibrutinib (25-50 mg/kg) or solvent when tumors reach 50-100 mm 3 in volume.
  • PDXs exhibit similar response rates for several therapeutic agents compared to those observed clinically (Sivanand et al, 2012; Rubio-Viqueira et al, 2006; Richtner et al, 2008). PDXs can also be used to effectively select targeted therapy treatment regimens for cancer patients bearing specific mutations (Hidalgo et al, 201 1; Morelli et al, 2012), suggesting that PDXs are clinically relevant tumor models for efficacy studies.
  • PDXs Twenty-three PDXs were established from lung cancer surgical specimens over the past two years and gene mutations were determined in these PDXs and their paired primary tumors by ultra-deep exome sequencing (average coverage of about 600 fold) for 202 cancer-related genes, including 138 cancer driver genes often affected by mutation (Vogelstein et al, 2013).
  • Adenocarcinoma develops at about 4 weeks after receiving doxycycline (Politi et al, 2006; Regales et al, 2009). Treatment will start 4-5 weeks after receiving doxycycline. Mice will be sacrificed at 3-6 weeks after treatment and tumor burden will be determined by analysis of lung histopathology.
  • mice will be randomly divided into groups before treatment starts, when tumors reach 50-100 mm 3 in volume. Ibrutinib will be given via oral gavage daily at doses of 25-50 mg/kg. Animals treated with solvent will be used as controls. A subset of tumors from all experiments (vehicle and ibrutinib treated xenografts, GEMMs, and PDXs) will be snap frozen and protein lysates collected and analyzed by RPPA to compare ibrutinib-induced signaling changes between mutation subsets and determine proteomic markers of response to ibrutinib.
  • mice will be monitored for growth of subcutaneous tumors and body weight changes every 2-3 days for up to 16 weeks. Tumors will be measured with calipers to determine the largest and smallest diameters.
  • the results will be subjected to an analysis of variance (A OVA) using SPSS software (Gu et al, 2000).
  • a P-value of ⁇ 05 will be considered significant.
  • the tumor burden will be determined by analysis of lung histopathology at the end of experiment (3-6 weeks after the treatment).
  • mice will be killed when tumors reach 1500-2000 mm 3 in volume; the survival duration of the tumor-bearing animals will be recorded. At the end of the study (16 weeks after the last treatment), any mice that remain alive will be killed and their tumors and various organs (lungs, heart, stomach, liver, kidneys, brain, gonads, and spleen) will be harvested for histopathologic evaluation (Gu et al, 2000). Survival data will be analyzed using a Kaplan-Meier survival analysis with SPSS software. The results will show whether the treatments improve survival in mice bearing xenografts from cell lines or PDXs of lung cancer.
  • Proteomic changes that occur in the effective treatment group will be determined in tumors derived from cell lines and PDXs. Mice will be euthanized 24 h after the three sequential treatments. Tumors and various organs (lungs, heart, stomach, liver, kidneys, brain, gonads, and spleen) will be harvested, snap frozen, and lysates analyzed by RPPA. Proteomic data quality will be assessed as previously described (Gao et al, 2014; Haura and Rix, 2014). Differences in marker levels between treatment arms and time points will be determined by ANOVA.
  • Results will be confirmed by immunohistochemistry for relevant proteins (e.g., EGFR, p-EGFR, HER2, MET, etc.), AXL/EMT pathway, and other key proteins or phospho-proteins identified by RPPA.
  • relevant proteins e.g., EGFR, p-EGFR, HER2, MET, etc.
  • AXL/EMT pathway e.g., IL-12, etc.
  • other key proteins or phospho-proteins identified by RPPA e.g., RPPA.
  • Analyses of in vivo proteomic changes in tumors will allow a determination of whether those molecular changes can be used as markers for monitoring treatment responses in clinical evaluations.
  • Example 9 - Efficacy of ibrutinib in non-small cell lung cancer patients with EGFR- mutant tumors
  • Biopsies will be obtained prior to treatment with ibrutinib to study resistance mechanisms to frontline EGFR inhibition. Patients will then receive treatment with ibrutinib. There will be a dose escalation phase, with initial patients dosed at 560 mg daily (the approved dose for MCL) and dose escalation to 840 mg daily if treatment is well tolerated. Patients will continue treatment until progressive disease, intolerable side effects, or withdrawal of consent. There will also be biopsies performed at progression, to determine mechanisms of resistance to ibrutinib. [00150] Up to 38 patients will be enrolled on this trial— up to 18 on the dose escalation phase, and a total of 20 at the recommended phase II dose.
  • the primary endpoint for the expansion cohort will be response rate by RECIST 1.1 ; secondary endpoints will include progression free survival and overall survival. Although the small numbers of patients will limit the power of this analysis, response to therapy will be correlated with the mechanism of acquired resistance to frontline TKI. Studies on MCL and CLL (Woyach et al, 2014; Chiron et al, 2014) revealed that analysis on specimens from 5-6 patients was able to identify clinically relevant mechanisms of resistance to ibrutinib.
  • ibrutinib will be most effective in patients with secondary resistance mechanisms, such as T790M mutation and in patients who have upregulated bypass signaling pathways (e.g., HER2 amplification), but will be less effective in patients with STAT-3/NF-KB activation or AXL overexpression.
  • Fresh frozen core needle biopsies (2-3) from patients prior to ibrutinib treatment (day 0) and at disease progression will be obtained and analyzed by RPPA and whole-exome/whole-transciptome sequencing. Changes in protein expression from Day 0 to progression for markers in the EGFR/HER2, MET, STAT-3/NF-KB, and AXL/EMT pathways will be assessed by ?-test. Baseline marker expression and the degree of marker change following treatment will be correlated with clinical response for each RPPA marker.
  • RNAlater® Life Technologies
  • RNAlater® Life Technologies
  • lung cancer with EGFR mutations will be sensitive to the ibrutinib treatment in preclinical and clinical studies, and that mutations in the covalent binding site for newer EGFRi (C797) will cause resistance to those agents, including ibrutinib.
  • C797 newer EGFRi
  • cells or tumors that initially respond to ibrutinib may develop resistance after repeated exposure or treatment cycles, as observed for other EGFRi (Politi et al, 2010).
  • in vivo tumors may regrow after treatment stops. Should this occur, the tumor- bearing animals will be treated again to determine whether the tumors still respond to the treatment. If the tumors do not respond, it will indicate that in vivo resistance may have been induced.
  • Those tumors will be harvested to characterize possible mechanisms of resistance.
  • Adzhubei et al A method and server for predicting damaging missense mutations. Nature
  • Cappuzzo et al MET increased gene copy number and primary resistance to gefitinib therapy in non-small-cell lung cancer patients. Ann Oncol, 20(2):298-304, 2009. Cappuzzo et al, Erlotinib as maintenance treatment in advanced non-small-cell lung cancer: a multicentre, randomised, placebo-controlled phase 3 study. Lancet Oncology, 11:521-
  • Silibinin suppresses EMT-driven erlotinib resistance by reversing the high miR-
  • Gao et al Spectrum of LKB1, EGFR, and KRAS mutations in Chinese lung adenocarcinomas. Journal of Thoracic Oncology, 5: 1130-1135, 2010.
  • Gao et al Selective antitumor activity of ibrutinib in EGFR-mutant non-small cell lung cancer cells. Journal of the National Cancer Institute, 106(9):dju204, 2014.
  • Tumor-specific transgene expression from the human telomerase reverse transcriptase promoter enables targeting of the therapeutic effects of the Bax gene to cancers. Cancer Res., 60:5359-5364, 2000.
  • Heinmoller et al, HER2 status in non-small cell lung cancer results from patient screening for enrollment to a phase II study of herceptin. Clinical Cancer Research, 9:5238- 5243, 2003.
  • Hidalgo et al A pilot clinical study of treatment guided by personalized tumorgrafts in patients with advanced cancer. Molecular Cancer Therapeutics, 10: 1311-1316, 2011. Hirsch et al, Epidermal growth factor receptor inhibition in lung cancer: status 2012. Journal of Thoracic Oncology, 8:373-384, 2013.
  • Nanjundan et al Proteomic profiling identifies pathways dysregulated in non-small cell lung cancer and an inverse association of AMPK and adhesion pathways with recurrence.
  • vascular endothelial growth factor receptor and epidermal growth factor receptor (EGFR) blockade inhibits tumor growth in xenograft models of EGFR inhibitor resistance.
  • EGF receptor gene mutations are common in lung cancers from "never smokers" and are associated with sensitivity of tumors to gefitinib and erlotinib. Proc Natl Acad
  • Pao et al Acquired resistance of lung adenocarcinomas to gefitinib or erlotinib is associated with a second mutation in the EGFR kinase domain.
  • Petrelli et al Relationship between skin rash and outcome in non-small-cell lung cancer patients treated with anti-EGFR tyrosine kinase inhibitors: a literature-based metaanalysis of 24 trials. Lung Cancer, 78:8-15, 2012.
  • NF-B signaling is activated and confers resistance to apoptosis in three- dimensionally cultured EGFR-mutant lung adenocarcinoma cells.
  • the BAD protein integrates survival signaling by EGFR/MAPK and PI3K/Akt kinase pathways in PTEN-deficient tumor cells. Cancer Cell, 8:287-297, 2005.
  • HER2 amplification a potential mechanism of acquired resistance to EGFR inhibition in EGFR-mutant lung cancers that lack the second-site EGFRT790M mutation. Cancer Discovery , 2:922-933, 2012.
  • Vermorken et al A phase lb, open-label study to assess the safety of continuous oral treatment with afatinib in combination with two chemotherapy regimens: cisplatin plus paclitaxel and cisplatin plus 5-fluorouracil, in patients with advanced solid tumors.

Abstract

L'invention concerne des méthodes d'utilisation d'inhibiteurs BTK irréversibles, tel que, par exemple, l'ibrutinib (PCI-32765), pour traiter les cancers à mutation de l'EGFR, les cancers à amplification d'HER2 et/ou les cancers surexprimant l'HER2.
PCT/US2015/016859 2014-02-20 2015-02-20 Utilisation d'ibrutinib pour le traitement du cancer à mutation de l'egfr WO2015127234A1 (fr)

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US10751342B2 (en) 2010-06-03 2020-08-25 Pharmacyclics Llc Use of inhibitors of Bruton's tyrosine kinase (Btk)
US9801883B2 (en) 2010-06-03 2017-10-31 Pharmacyclics Llc Use of inhibitors of bruton's tyrosine kinase (Btk)
US9814721B2 (en) 2010-06-03 2017-11-14 Pharmacyclics Llc Use of inhibitors of bruton'S tyrosine kinase (BTK)
US10478439B2 (en) 2010-06-03 2019-11-19 Pharmacyclics Llc Use of inhibitors of bruton's tyrosine kinase (Btk)
US10653696B2 (en) 2010-06-03 2020-05-19 Pharmacyclics Llc Use of inhibitors of bruton's tyrosine kinase (BTK)
US9862722B2 (en) 2011-07-13 2018-01-09 Pharmacyclics Llc Inhibitors of Bruton's tyrosine kinase
US10954567B2 (en) 2012-07-24 2021-03-23 Pharmacyclics Llc Mutations associated with resistance to inhibitors of Bruton's Tyrosine Kinase (BTK)
US10016434B2 (en) 2013-08-12 2018-07-10 Pharmacyclics Llc Methods for the treatment of HER2 amplified cancer
US9724349B2 (en) 2013-08-12 2017-08-08 Pharmacyclics Llc Methods for the treatment of HER2 amplified cancer
EP3033079A1 (fr) * 2013-08-12 2016-06-22 Pharmacyclics LLC Méthodes de traitement d'un cancer amplifié par her2
EP3033079A4 (fr) * 2013-08-12 2017-04-05 Pharmacyclics LLC Méthodes de traitement d'un cancer amplifié par her2
US11045468B2 (en) 2014-02-04 2021-06-29 Astellas Pharma Inc. Pharmaceutical composition comprising diamino heterocyclic carboxamide compound as active ingredient
EP3103453A4 (fr) * 2014-02-04 2017-09-06 Astellas Pharma Inc. Composition médicinale comprenant un composé de carboxamide hétérocyclique diamino en tant qu'agent actif
US11925638B2 (en) 2014-02-04 2024-03-12 Astellas Pharma Inc. Pharmaceutical composition comprising diamino heterocyclic carboxamide compound as active ingredient
US9885086B2 (en) 2014-03-20 2018-02-06 Pharmacyclics Llc Phospholipase C gamma 2 and resistance associated mutations
US9949971B2 (en) 2014-06-17 2018-04-24 Acerta Pharma B.V. Therapeutic combinations of a BTK inhibitor, a PI3K inhibitor and/or a JAK-2 inhibitor
WO2015193740A3 (fr) * 2014-06-17 2016-08-04 Acerta Pharma B.V. Combinaisons thérapeutiques d'un inhibiteur de la tkb, d'un inhibiteur de la pi3k et/ou d'un inhibiteur de la jak-2
US10513509B2 (en) 2016-05-26 2019-12-24 Recurium Ip Holdings, Llc EGFR inhibitor compounds
US11098030B2 (en) 2016-05-26 2021-08-24 Recurium Ip Holdings, Llc EGFR inhibitor compounds
EP3969121A4 (fr) * 2019-05-15 2024-01-03 Univ Texas Procédés et compositions pour le traitement du cancer du poumon non à petites cellules
CN111973570A (zh) * 2019-05-22 2020-11-24 沈阳药科大学 唾液酸衍生物修饰的依鲁替尼纳米复合物及其制备方法

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