WO2013082511A9 - Procédés pour surmonter la résistance tumorale aux antagonistes de vegf - Google Patents

Procédés pour surmonter la résistance tumorale aux antagonistes de vegf Download PDF

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WO2013082511A9
WO2013082511A9 PCT/US2012/067419 US2012067419W WO2013082511A9 WO 2013082511 A9 WO2013082511 A9 WO 2013082511A9 US 2012067419 W US2012067419 W US 2012067419W WO 2013082511 A9 WO2013082511 A9 WO 2013082511A9
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tumor
vegf
cells
csf
cancer
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WO2013082511A1 (fr
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Napoleone Ferrara
Vernon PHAN
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Genentech, Inc.
F. Hoffmann-La Roche Ag
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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
    • 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
    • A61K31/44—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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/243—Colony Stimulating Factors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A61K2039/507—Comprising a combination of two or more separate antibodies

Definitions

  • the present invention relates generally to the field of molecular biology and clinical oncology.
  • the invention concerns compositions and methods effective for overcoming tumor resistance to treatment with VEGF antagonists.
  • angiogenesis which involves the formation of new blood vessels from preexisting endothelium, plays a significant role in the pathogenesis of a variety of disorders. These include solid tumors and metastasis, atherosclerosis, retrolental fibroplasia, hemangiomas, chronic inflammation, intraocular neovascular syndromes such as proliferative retinopathies, e.g., diabetic retinopathy, age-related macular degeneration (AMD), neovascular glaucoma, immune rejection of transplanted corneal tissue and other tissues, rheumatoid arthritis, and psoriasis.
  • proliferative retinopathies e.g., diabetic retinopathy, age-related macular degeneration (AMD), neovascular glaucoma, immune rejection of transplanted corneal tissue and other tissues, rheumatoid arthritis, and psoriasis.
  • AMD age-related macular degeneration
  • tumor angiogenesis appears to be crucial for the transition from hyperplasia to neoplasia, and for providing nourishment for the growth and metastasis of the tumor.
  • Neovascularization allows the tumor cells to acquire a growth advantage and proliferative autonomy compared to normal cells.
  • a tumor usually begins as a single aberrant cell which can proliferate only to a size of a few cubic millimeters due to the distance from available capillary beds, and it can stay 'dormant' without further growth and dissemination for a long period of time.
  • VEGF vascular endothelial growth factor
  • Ferrara & Kerbel Nature, 438:967-74 (2005).
  • VEGF has been shown to be a key mediator of neovascularization associated with tumors and intraocular disorders. Ferrara et al., Endocr. Rev., 18: 4-25 (1997). The VEGF mRNA is overexpressed by the majority of human tumors examined. Berkman et al., J. Clin.
  • VEGF vascular endothelial growth factor
  • VEGF as a pleiotropic growth factor, exhibits multiple biological effects in other physiological processes, such as endothelial cell survival, vessel permeability and vasodilation, monocyte chemotaxis and calcium influx.
  • VEGF receptor tyrosine kinase (RTK) inhibitors have all been used in various preclinical models to inhibit tumor angiogenesis and tumor growth. See, e.g., Sieffle et al, Cancer Metastasis Rev., 17:241-248 (1998); Cao, Nat. Rev. Clin.
  • VEGF inhibitors have demonstrated clinical efficacy and a survival advantage in patients with advanced cancer. However, similar to the majority of anti-cancer therapies tested to date, many patients eventually relapse despite the robust initial response. Berger and Hanahan, Nat. Rev. Cancer, 8:592-603 (2008); Ellis and Hicklin, Nat. Rev. Cancer, 8:579-91 (2008). Moreover, in preclinical models, a number of tumors do not respond at all, or respond initially but become resistant at a later stage of VEGF inhibition. Crawford et al., Cancer Cell, 15:21-34 (2009); Shaojaei et al, PNAS USA, 106:6742-47 (2009).
  • tumor stroma various bone marrow-derived cell types have been shown to play important roles in regulating tumor angiogenesis and growth. Recently, research has focused on a population of myeloid cells, identified in the mouse by the expression of the cell surface markers CD1 lb and Grl, that include neutrophils, immature dendritic cells, monocytes, and early myeloid progenitors. In cancer, interest in these cells stems from their ability to promote tumor angiogenesis and facilitate metastasis. Yang et al., Cancer Cell, 13:23-35 (2008).
  • CD1 lb+ Grl+ cells termed myeloid- derived suppressor cells (MDSCs)
  • MDSCs myeloid- derived suppressor cells
  • CD1 lb+Grl+ cells to the tumor stroma mediates tumor refractoriness to anti-VEGF therapy in several murine models.
  • G-CSF or GCSF hematopoietic growth factor granulocyte colony- stimulating factor
  • G-CSF also up-regulates the proangiogenic factor Bv8.
  • G-CSF-mobilized CD1 lb+ Grl+ produce a variety of factors that facilitate primary tumor growth and metastasis, including MMP9, S100A8 and S100A9.
  • the RAS/RAF/MAPK/EPvK pathway plays a maj or role in mediating cell growth and differentiation in response to numerous extracellular signals. Ras-GTP activates Raf kinase, which in turn activates the MEK/ER pathway and drives cellular proliferation. Downward, Nat. Rev. Cancer, 3: 11-22 (2003). To regulate cellular proliferation, activated ER s translocate to the nucleus and regulate gene expression through the activation of several key transcription factors. Abnormal regulation of the RAS/RAF/MEK/ERK pathway contributes to uncontrolled proliferation, invasion, metastasis, angiogenesis, and diminished apoptosis.
  • ERK/MAPK pathway is upregulated in 30% of all tumors and oncogenic activating mutations in K-Ras and B-Raf have been identified in 22% and 18% of all cancers respectively. It has been shown that inhibition of the ERK pathway, and in particular inhibition of MEK kinase activity, results in anti-metastatic effects largely due to a reduction of cell-cell contact and motility.
  • MEK inhibitors have been generated and tested for their turmor inhibition activities in both preclinical models and clinical trials. See, for example, WO 02/06213, WO 03/077855, WO 03/077914, WO09/085983 and US Pat. 7803839.
  • some MEK inhibitors e.g., PD0325901
  • PD0325901 have been shown to downregulate VEGF expression in malignant melanoma cells and thereby inhibit VEGF-induced tumor angiogenesis. Ciuffreda et al, Neoplasia, 11 :720-31 (2009).
  • the present invention is based in part on the discovery that inhibition of the
  • RAS/RAF/MEK signaling pathway can effectively overcome tumor's resistance to anti- VEGF therapy.
  • inhibition e.g., by using a MEK inhibitor, blocks the tumor stroma-induced angiogenesis that is independent of VEGF, via downregulating the transcription and expression of granulocyte colony- stimulating factor (G-CSF), a hematopoietic growth factor known to promote tumor angiogenesis.
  • G-CSF granulocyte colony- stimulating factor
  • the invention provides methods of inhibiting tumor growth by administering an effective amount of a MEK inhibitor to a human subject having a tumor that overexpresses G-CSF and is resistant or refractory to treatment with a vascular endothelial growth factor (VEGF) antagonist.
  • VEGF vascular endothelial growth factor
  • a method of sensitizing tumor to treatment with a VEGF antagonist comprising administering to a human subject having a tumor a MEK inhibitor in an amount effective to overcome tumor's resistance to treatment with the VEGF antagonist.
  • a method of enhancing tumor's response to treatment with a VEGF antagonist comprising administering to a human subject having a tumor an effective amount of a MEK inhibitor, whereby the tumor becomes more responsive to the VEGF antagonist.
  • One aspect of the invention relates to a method of inhibiting tumor
  • angiogenesis comprising administering an effective amount of a MEK inhibitor to a human subject having a tumor that is resistant or refractory to treatment with a VEGF antagonist.
  • the tumor angiogenesis subject to the method can be induced by cells in tumor stroma and independent of VEGF expression level.
  • the cells capable of inducing tumor angiogenesis are derived from bone marrow of the human subject.
  • the cells are of hematopoietic lineage, such as those of myeloblast sub lineage.
  • the method of the invention is directed to inhibiting tumor angiogenesis induced by human cell population that is the counterpart of the murine CDl lb+/Grl+ myeloid cells.
  • CDl lb+/Grl+ myeloid cells and their roles in tumor angiogenesis have been well characterized, e.g., in Shaojaei et al, ibid (2007); Shaojaei et al, ibid (2009).
  • Human cells derived from bone marrow having substantially similar functions and morphologies as the murine CDl lb+/Grl+ myeloid cells are capable of promoting tumor angiogenesis.
  • these cells are responsive to stimulation by certain growth factors such as G-CSF and express and release pro-angiogenic factors such as Bv8.
  • the method of the invention is directed to inhibiting tumor angiogenesis induced by human granulocytes or subset thereof such as CDl lb+/Ly6G+ neutrophils.
  • the invention proides that the presence and level of the CDl lb+/Ly6G+ neutrophil population in tumor surrounding environment can serve as a biomarker for tumor's resisntacne to anti-VEGF therapy as well as for treatment efficacy.
  • a method of inhibiting proliferation and migration of cells in tumor stroma comprising administering an effective amount of a MEK inhibitor to a human subject having a tumor that is resistant or refractory to treatment with a VEGF antagonist.
  • the cells in tumor stroma are derived from bone marrow, are of hematopoietic lineage or subsets thereof as decribed above.
  • the cells in tumor stroma are tumor-associated fibroblast cells.
  • the VEGF antagonist in the methods of the invention is an anti-VEGF antibody or functional fragment thereof, such as bevacizumab.
  • the MEK inhibitor useful in the methods of the invention is a small molecule compound or a pharmaceutically acceptable salt thereof.
  • the small molecule compound is selected from the group consisting of PD325901, PD-181461, AR Y142886 / AZD6244, ARRY-509, GDC0973 (XL518), GDC0987, JTP- 74057, AS-701255, AS-701173, AZD8330, ARRY162, ARRY300, RDEA436, E6201 , R04987655/R-7167, GSK1120212 and AS-703026.
  • the small molecule compound is an azetidine compound.
  • azetidine compounds capable of inhibiting MEK are described in, e.g., US Pat No. 7803839; WO2011/054620.
  • the small molecule compound is an imidazopyridine compound.
  • azetidine compounds capable of inhibiting MEK are described in, e.g., WO2009/085983.
  • the MEK inhibitor of the invention is the compound GDC-0973 or GDC-0987.
  • the human subject in the methods of the invention has been previously treated with chemotherapy, a VEGF antagonist or both.
  • the human subject has been previously treated with bevacizumab but the tumor therein has relapsed.
  • the methods contemplated herein further comprise administering to the human subject a VEGF antagonist such as an anti-VEGF antibody or functional fragment thereof.
  • a VEGF antagonist such as an anti-VEGF antibody or functional fragment thereof.
  • the anti-VEGF antibody useful for the invention can be bevacizumab, G6 or B20 series antibodies that are further described in the Definitions herein.
  • Combination therapies using a MEK inhibitor or a G-CSF antagnonist (e.g., anti-G-CSF antibody) and an anti-VEGF antibody or functional fragment thereof are contemplated.
  • the two agents can be administered concurrently, separately or sequentially.
  • the MEK inhibitor is administered first, in an amount and for a duration effective to sensitize the tumor for a subsequenct treatment with an anti-VEGF antibody or functional fragment thereof.
  • a combination therapy using bevacizumab and GDC-0973 is contemplated.
  • a combination therapy using anti-G-CSF antibody and bevacizumab is contemplated.
  • the methods of the invention are further combined with chemotherapy or radiation therapy.
  • Tumors or cancers suitable to be targeted by the methods of the invention can be of any type or form, including those listed under Definitions herein.
  • the tumor is in the colon, rectum, liver, lung, prostate, breast, bladder, skin, brain, thyroid, pancreas or ovary of the human subject.
  • the tumors are resistant to anti- VEGF therapy, especially to bevacizumab. Certain means of identifying and selecting such resistant tumors are further described in the Detailed Description herein.
  • the cancer to be treated by the methods of the invention is pancreatic ductal adenocarcinoma (PDAC), which is known to be resistant to anti-VEGF therapeutics such as bevacizumab.
  • PDAC pancreatic ductal adenocarcinoma
  • the methods contemplated herein further comprise monitoring the efficacy of the MEK inhibitor by determining the amount of bone marrow- derived cells, such as neutrophil cells, in a tumor sample or a peripheral blood sample obtained from the human subject, relative to the amount of the cells in a tumor sample or a peripheral blood sample obtained from the human subject prior to administration of the MEK inhibitor, wherein a reduced amount of the tested cells indicates efficacy of the MEK inhibitor.
  • bone marrow- derived cells such as neutrophil cells
  • the efficacy of the MEK inhibitor is monitored by measuring the proportion of bone marrow-derived cells, such as neutrophil cells, in a tumor sample or a peripheral blood sample obtained from the human subject, relative to the proportion of the cells in a tumor sample or a peripheral blood sample obtained from the human subject prior to administration of the MEK inhibitor, wherein a reduced proportion of the tested cells indicates efficacy of the MEK inhibitor.
  • bone marrow-derived cells such as neutrophil cells
  • the efficacy of the MEK inhibitor is monitored by measuring the expression level of the angiogenic factor Bv8 in a tumor sample or a peripheral blood sample obtained from said human subject, relative to the Bv8 expression level in a tumor sample or a peripheral blood sample obtained from the human subject prior to administration of the MEK inhibitor, wherein a reduced Bv8 expression indicates efficacy of the MEK inhibitor.
  • the invention provides a novel combination of a) a MEK inhibitor and b) a VEGF antagonist for concurrent, separate or sequential use in treating tumor. Also provided is a pharmaceutical preparation comprising an effective amount of the above combination and at least one pharmaceutically acceptable carrier.
  • Figure 1 depicts the transcriptional regulation of G-CSF expression in cancer cells constitutively expressing G-CSF.
  • G-CSF promoter is active in a subset of mouse mammary cancer cell lines.
  • G-CSF promoter driving Luciferase cDNA was expressed in 4T1 -related cell lines. Luciferase activity is detected in the metastatic 4T1 and 4T07 cell lines but not in the non-metastatic 67NR or 168FARN cell lines. *p ⁇ 0.001.
  • Ets2 directly regulates G-CSF expression.
  • FIG. 2 depicts the signaling mechanisms controlling G-CSF expression in cancer cells.
  • (2B) Enforced expression of mutant BRAF (V600E) induces G-CSF expression in 67NR cells. *p 0.0004.
  • (2C) MEK inhibitor (GDC-0973) inhibits ERK phosphorylation and G-CSF expression in 4T1 cells. *p ⁇ 0.002.
  • ELISA shows the effects of MEKi and Pi3K inhibitor LY294002 (Pi3Ki) on G-CSF release. *p ⁇ 0.001.
  • Figure 3 depicts growth factors that positively regulate G-CSF secretion in
  • FIG. 4 depicts effects of MEKi, anti-G-CSF, anti-VEGF antibodies and combinations thereof on 4T1 tumor growth.
  • (4A-B) MEKi reduces G-CSF and Bv8 levels in 4T1 tumor-bearing mice, *p ⁇ 0.001.
  • (4F) Reduced angiogenesis in 4T1 tumors treated with MEKi plus anti-VEGF, or anti-G-CSF plus anti-VEGF combination treatments. Treatment groups are indicated in the figure. Tumor sections were stained with anti-CD31 (red). Scale bar is lOOum.
  • (4G) Quantitative analysis of tumor vascular surface area. Whole tumor cross-sections were stained with CD31 and analyzed as described in Methods (n 4, *p ⁇ 0.05).
  • FIG. 5 depicts effects of MEKi, anti-G-CSF, anti-VEGF antibodies and combinations thereof on LLC tumor growth.
  • Figure 6 depicts effects of RAF and MEK inhibitors on G-CSF expression in cancer cells.
  • MEKi GDC-0973 inhibits G-CSF
  • RAFi GDC-0879 induces G-CSF in mouse lung cell lines with KRAS mutation.
  • D DMSO
  • P PI3Ki LY-294002 5uM
  • R RAFi GDC-0879 luM
  • GDC-0973 MEKi: O.OluM, O. luM and l .OuM
  • G-CSF release was analyzed by ELISA.
  • Ten different human cancer cell lines were analyzed for G-CSF release by ELISA.
  • AKT and ERK phosphorylations were assessed in total lysates.
  • Cells were treated with DMSO (D), PBKi LY-294002 5uM (P), RAFi GDC-0879 luM (R), or GDC-0973 (MEKi : O.OluM, O. luM and l .OuM) for 24hrs.
  • FIG. 7 depicts effects of MEKi, anti-VEGF antibody and combination thereof on LLC tumor growth.
  • MEKi plus anti-VEGF combination treatment reduces growth of LLC tumors by approximately 64% at day 26 when compared to either MEKi or anti- VEGF as a single agent.
  • Figure 8 shows a marked reduction in total white blood cells counts in the peripheral blood of the animals that received either the MEKi alone, or MEKi plus anti- VEGF combination treatments compared to controls (8A). Reduction in white blood cells was correlated with a decrease in Cdl lb + Ly6G + cells in peripheral blood and in G-CSF and Bv8 plasma levels in animals that received MEKi as a single agent or MEKi plus anti- VEGF (8B-D).
  • FIG. 9 illustrates results from an Anti-VEGF resistant PDAC allograft mouse model.
  • G-CSFR wild-type (G-CSFR +/+ ) or G-CSFR knockout (G-CSFR 7 ) mice were crossed with RAG2 knockout (RAG2 ⁇ ⁇ ) mice to generate G-CSFR /+ RAG2 ⁇ ⁇ and (G-CSFR ⁇ / ⁇ RAG2 ⁇ / ⁇ .
  • aRAG anti-Ragweed
  • aVEGF anti-VEGF
  • Figure 10 illustrates results from a Kras-driven PDAC GEMM. (10A)
  • Figure 11 shows G-CSF expression and its correlation with phospho-MEK activation and neutrophil recruitment in human PDAC biopsies.
  • Figure 12 illustrates effects of MEKi GDC-0973 on G-CSF release and neutrophil mobilization in a Kras-driven PDAC GEMM.
  • (12A) ELISA analysis of G-CSF release in PDAC mice after received MEKi GDC-0973 at different time points, as indicated. Each number corresponds to an animal (n 5/group).
  • (12B) Cytokines and growth factors changes in plasma were monitored by Luminex. Time points were analyzed at day 7 after MEKi GDC-0973 administration. Wildtype (Naive), PDAC treated with control (Vehicle) or MEKi GDC-0973, (n 5/group) *p ⁇ 0.05. Error bars indicate SD.
  • tumor refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.
  • cancer refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.
  • cancer refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.
  • tumor are not mutually exclusive as referred to herein.
  • cancer and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth.
  • examples of cancer include but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies.
  • squamous cell cancer e.g., epithelial squamous cell cancer
  • 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, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer and gastrointestinal stromal cancer
  • pancreatic cancer including pancreatic ductal adenocarcinoma (PDAC), intraductal papillary mucinous neoplasm (IPMN), signet ring cell carcinoma, hepatoid carcinoma, colloid carcinoma, pancreatic cystic neoplasm, pancreatic neuroendocrine carcinoma (PNEC), glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, cancer of the urinary tract, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma
  • PDAC pancreatic ductal
  • intermediate grade/follicular NHL intermediate grade diffuse NHL
  • high grade follicular NHL intermediate grade diffuse NHL
  • immunoblastic NHL high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and
  • ALL lymphoblastic leukemia
  • PTLD post-transplant lymphoproliferative disorder
  • cancers that are amenable to treatment by the anti-G-CSF antibody, anti-Bv8-antibody, anti-PK l antibody or any combination thereof include breast cancer, colorectal cancer, rectal cancer, non-small cell lung cancer, glioblastoma, non-Hodgkins lymphoma (NHL), renal cell cancer, prostate cancer, melanoma, liver cancer, pancreatic cancer, soft-tissue sarcoma, kaposi's sarcoma, carcinoid carcinoma, head and neck cancer, ovarian cancer and multiple myeloma.
  • breast cancer colorectal cancer, rectal cancer, non-small cell lung cancer, glioblastoma, non-Hodgkins lymphoma (NHL), renal cell cancer, prostate cancer, melanoma, liver cancer, pancreatic cancer, soft-tissue sarcoma, kaposi's sarcoma, carcinoid carcinoma, head and neck cancer, ovarian cancer and multiple myelom
  • the cancer is selected from the group consisting of small cell lung cancer, gliblastoma, neuroblastomas, melanoma, breast carcinoma, gastric cancer, colorectal cancer (CRC), and hepatocellular carcinoma. Yet, in some embodiments, the cancer is selected from the group consisting of non-small cell lung cancer, colorectal cancer, renal cell cancer, ovarian cancer, prostate cancer, glioblastoma and breast carcinoma, including metastatic forms of those cancers.
  • cancer embraces a collection of proliferative disorders, including but not limited to pre-cancerous growths, benign tumors, malignant tumors and dormant tumors.
  • Benign tumors remain localized at the site of origin and do not have the capacity to infiltrate, invade, or metastasize to distant sites.
  • Malignant tumors will invade and damage other tissues around them. They can also gain the ability to break off from where they started and spread to other parts of the body (metastasize), usually through the bloodstream or through the lymphatic system where the lymph nodes are located.
  • Dormant tumors are quiescent tumors in which tumor cells are present but tumor progression is not clinically apparent.
  • Cancer cells Primary tumors are classified by the type of tissue from which they arise; metastatic tumors are classified by the tissue type from which the cancer cells are derived. Over time, the cells of a malignant tumor become more abnormal and appear less like normal cells. This change in the appearance of cancer cells is called the tumor grade and cancer cells are described as being well-differentiated, moderately-differentiated, poorly-differentiated, or undifferentiated. Well-differentiated cells are quite normal appearing and resemble the normal cells from which they originated. Undifferentiated cells are cells that have become so abnormal that it is no longer possible to determine the origin of the cells.
  • Epithelial cancers generally evolve from a benign tumor to a preinvasive stage
  • carcinoma in situ e.g., carcinoma in situ
  • dysplasia is meant any abnormal growth or development of tissue, organ, or cells. In certain embodiments, the dysplasia is high grade or precancerous.
  • the term "tumor that is resistant to” or “tumor's resistance to” treatment with a VEGF antagonist refers to cancer, cancerous cells, or a tumor that shows little or no response to a cancer therapy comprising a VEGF antagonist.
  • a resistant tumor also refers to a tumor diagnosed as resistant herein (also referred to herein as "anti-VEGF resistant tumor”).
  • anti-VEGF resistant tumor there is an elevated level of G-CSF expression and/or an increase in bone marrow-derived stromal cells in a resistant tumor compared to a tumor that is sensitive to therapy comprising a VEGF antagonist.
  • resistant tumor is a tumor that is resistant to anti-VEGF antibody therapy.
  • the anti-VEGF antibody is bevacizumab.
  • resistant tumor is a tumor that is unlikely to respond to a cancer therapy comprising a VEGF antagonist.
  • resistant tumor is a tumor that is intrinsically non-responsive or resistant to a cancer therapy comprising a VEGF antagonist.
  • adenocarcinoma (PDAC) investigated in the present invention is a type of cancer resistant to anti-VEGF therapy.
  • a cancer refers to cancer, cancerous cells, or a tumor that initially responded to a cancer therapy comprising at least a VEGF antagonist, but eventually reinitiates growth despite ongoing cancer therapy.
  • a cancer is relapse tumor growth or relapse cancer cell growth where the number of cancer cells has not been significantly reduced, or has increased, or tumor size has not been significantly reduced, or has increased, or fails any further reduction in size or in number of cancer cells.
  • the determination of whether the cancer cells are relapse tumor growth or relapse cancer cell growth can be made either in vivo or in vitro by any method known in the art for assaying the effectiveness of treatment on cancer cells, using the art-accepted meanings of "relapse” or “refractory” or “non-responsive” in such a context.
  • a cancer or tumor is said to "overexpress G-CSF" when an abnormally high level of G-CSF is detected from the tumor or its surrounding, when compared to a reference tissue or cell population wherein the G-CSF expression is not up-regulated.
  • G-CSF G-CSF expression level at least 20%. 30%. 40%, 50%, 60% 70%), 80%), 90%), 100%) higher than the G-CSF expression level from a reference tissue or cell population.
  • the "MEK inhibitor” refers to compositions capable of interfering, blocking or significantly deminising the RAS/RAF/MEK signaling pathway in a target cell, by acting directly or indirectly on the MEK kinase.
  • the MEK inhibitor is a small molecule compound with defined chemical structure. Non-limiting examples of such compound are further described herein below.
  • the MEK inhibitor is [3,4-Difluoro-2-(2-fluoro-4-iodo-phenylamino)-phenyl]-((S)-3-hydroxy-3- piperidin-2-yl-azetidin-l-yl)-methanone also known as GDC-0973/XL-518.
  • sensitizing tumor to treatment with a VEGF antagonist herein is meant a step by which the targeted tumor becomes more sensitive, vulnerable or susceptable to treatment with a VEGF antagonist than a non-sensitized tumor control.
  • Cells in tumor stroma refers to the cell population in stroma, the microenvironment surrounding and supporting a tumor.
  • the cell population in tumor stroma often comprises tumor-associated fibroblasts, pericytes, mesenchymal stem cells and inflammatory-imuune cells.
  • Some cells in tumor stroma are derived from bone marrow and are of hematopoietic lineage, including but not limited to, myeloid cells, granulocytes and neutropils.
  • Bone marrow-derived cells that are "human counterpart of the murine
  • CD1 lb+/Grl+ myeloid cells are those cells of myeloblast lineage in human that behave morphologically and/or functionally similar to the murine CD1 lb+/Grl+ myeloid cells.
  • they are cells recruited to the tumor stroma and capable of promoting tumor angiogenesis.
  • these cells are responsive to stimulation by certain growth factors such as G-CSF and express and release pro-angiogenic factors such as Bv8.
  • a polypeptide "variant" i.e. a variant of any polypeptide disclosed herein
  • a variant means a biologically active polypeptide having at least about 80% amino acid sequence identity with the corresponding native sequence polypeptide.
  • Such variants include, for instance, polypeptides wherein one or more amino acid (naturally occurring amino acid and/or a non-naturally occurring amino acid) residues are added, or deleted, at the N- and/or C-terminus of the polypeptide.
  • a variant will have at least about 80% amino acid sequence identity, or at least about 90%> amino acid sequence identity, or at least about 95% or more amino acid sequence identity with the native sequence polypeptide.
  • Variants also include polypeptide fragments (e.g., subsequences, truncations, etc.), typically biologically active, of the native sequence.
  • Antagonist when used herein refers to a molecule capable of neutralizing, blocking, inhibiting, abrogating, reducing or interfering with the activities of a protein of the invention including its binding to one or more receptors in the case of a ligand or binding to one or more ligands in case of a receptor.
  • Antagonists include antibodies and antigen-binding fragments thereof, proteins, peptides, glycoproteins, glycopeptides, glycolipids, polysaccharides, oligosaccharides, nucleic acids, bioorganic molecules, peptidomimetics, pharmacological agents and their metabolites, transcriptional and
  • Antagonists also include small molecule inhibitors of a protein of the invention, and fusions proteins, receptor molecules and derivatives which bind specifically to protein thereby sequestering its binding to its target, antagonist variants of the protein, antisense molecules directed to a protein of the invention, R A aptamers, and ribozymes against a protein of the invention.
  • a "blocking" antibody or an “antagonist” antibody is one which inhibits or reduces biological activity of the antigen it binds. Certain blocking antibodies or antagonist antibodies substantially or completely inhibit the biological activity of the antigen.
  • inhibiting tumor angiogenesis refers to the inhibition of the ability of tumors to induce new blood-vessel formation, or to inhibit a tumor's ability to recruit existing vasculature.
  • tumor growth refers to a tumor that does not grow further after treatment and/or does not metastasize. Tumor growth can be inhibited when tumor angiogenesis is inhibited as described herein.
  • inhibition of tumor growth decreases tumor volume in said human subject by at least 5%, or at least 10%, or at least 15%, or at least 20%, or at least 25%, or at least 30%, or at least 35%, or at least 40%, or at least 45%, or at least 50%, or at least 55%, or at least 60%, or at least 65% or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95% or at least 99%, as compared to a tumor in a human subject that was not administered an effective amount of an MEK inhibitor.
  • said decrease in tumor volume is measured by computerized axial tomography (CAT Scan), magnetic resonance imaging (MRI), positron emission tomography (PET), or
  • SPECT tomography
  • VEGF vascular endothelial cell growth factor
  • VEGF- A the native sequence 165 -amino acid vascular endothelial cell growth factor and related 121-, 145-, 183-, 189-, and 206- amino acid vascular endothelial cell growth factors, as described by Leung et al. Science, 246: 1306 (1989), Houck et al. Mol. Endocrin., 5: 1806 (1991), and, Robinson & Stringer, Journal of Cell Science, 144(5):853-865 (2001), together with the naturally occurring allelic and processed forms thereof, as well as variants thereof.
  • VEGF -A is part of a gene family including VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F, and P1GF.
  • VEGF -A primarily binds to two high affinity receptor tyrosine kinases, VEGFR-1 (Flt-1) and VEGFR-2 (Flk-l/KDR), the latter being the major transmitter of vascular endothelial cell mitogenic signals of VEGF -A.
  • VEGFR-1 Flt-1
  • VEGFR-2 Flk-l/KDR
  • VEGF vascular endothelial growth factor
  • VEGF 165 The amino acid positions for a "truncated" native VEGF are numbered as indicated in the native VEGF sequence. For example, amino acid position 17 (methionine) in truncated native VEGF is also position 17 (methionine) in native VEGF.
  • the truncated native VEGF has binding affinity for the KDR and Flt-1 receptors comparable to native sequence VEGF.
  • a "VEGF antagonist” refers to a molecule (peptidyl or non-peptidyl) capable of neutralizing, blocking, inhibiting, abrogating, reducing or interfering with activities of a native sequence VEGF including its binding to one or more VEGF receptors.
  • VEGF antagonists include anti-VEGF antibodies and antigen-binding fragments thereof, receptor molecules and derivatives which bind specifically to VEGF thereby sequestering its binding to one or more receptors (e.g., soluble VEGF receptor proteins, or VEGF binding fragments thereof, or chimeric VEGF receptor proteins), anti-VEGF receptor antibodies and VEGF receptor antagonists such as small molecule inhibitors of the VEGFR tyrosine kinases, and fusions proteins, e.g., VEGF-Trap (Regeneron), VEGFm-gelonin (Peregine).
  • receptors e.g., soluble VEGF receptor proteins, or VEGF binding fragments thereof, or chimeric VEGF receptor proteins
  • anti-VEGF receptor antibodies and VEGF receptor antagonists such as small molecule inhibitors of the VEGFR tyrosine kinases, and fusions proteins, e.g., VEGF-Trap (Regeneron), VEGF
  • VEGF antagonists also include antagonists of VEGF, antisense molecules directed to VEGF, RNA aptamers, and ribozymes against VEGF or VEGF receptors.
  • VEGF antagonists useful in the methods of the invention further include peptidyl or non-peptidyl compounds that specifically bind VEGF, such as anti-VEGF antibodies and antigen-binding fragments thereof, polypeptides, antibody variants or fragments thereof that specifically bind to VEGF;
  • the VEGF antagonist reduces or inhibits, by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more, the expression level or biological activity of VEGF.
  • the VEGF inhibited by the VEGF antagonist is VEGF (8-109), VEGF (1-109), or VEGFi 65 .
  • anti-VEGF antibody or "an antibody that binds to VEGF” refers to an antibody that is capable of binding to VEGF with sufficient affinity and specificity that the antibody is useful as a diagnostic and/or therapeutic agent in targeting VEGF.
  • the anti-VEGF antibody of the invention can be used as a therapeutic agent in targeting and interfering with diseases or conditions wherein the VEGF activity is involved. See, e.g., U.S. Patents 6,582,959, 6,703,020; W098/45332; WO 96/30046; WO94/10202,
  • the antibody selected will normally have a sufficiently strong binding affinity for VEGF, for example, the antibody may bind hVEGF with a Ka value of between 100 nM-1 pM.
  • Antibody affinities may be determined by a surface plasmon resonance based assay (such as the BIAcoreTM assay as described in PCT Application Publication No. WO2005/012359); enzyme-linked
  • ELISA immunoabsorbent assay
  • competition assays e.g. RIA's
  • the antibody may be subjected to other biological activity assays, e.g., in order to evaluate its effectiveness as a therapeutic.
  • assays are known in the art and depend on the target antigen and intended use for the antibody. Examples include the HUVEC inhibition assay; tumor cell growth inhibition assays (as described in WO 89/06692, for example); antibody- dependent cellular cytotoxicity (ADCC) and complement-mediated cytotoxicity (CDC) assays (US Patent 5,500,362); and agonistic activity or hematopoiesis assays (see WO 95/27062).
  • anti-VEGF antibodies will usually not bind to other VEGF homologues such as VEGF-B, VEGF-C, VEGF-D or VEGF-E, nor other growth factors such as P1GF, PDGF or bFGF.
  • anti-VEGF antibodies include a monoclonal antibody that binds to the same epitope as the monoclonal anti-VEGF antibody A4.6.1 produced by hybridoma ATCC HB 10709; a recombinant humanized anti-VEGF monoclonal antibody generated according to Presta et al. (1997) Cancer Res. 57:4593-4599, including but not limited to the antibody known as "bevacizumab (BV),” also known as "rhuMAb VEGF” or
  • Bevacizumab comprises mutated human IgGl framework regions and antigen-binding complementarity-determining regions from the murine anti-hVEGF monoclonal antibody A.4.6.1 that blocks binding of human VEGF to its receptors.
  • Bevacizumab Approximately 93% of the amino acid sequence of bevacizumab, including most of the framework regions, is derived from human IgGl , and about 7% of the sequence is derived from the murine antibody A4.6.1. Bevacizumab has a molecular mass of about 149,000 daltons and is glycosylated. Bevacizumab and other humanized anti-VEGF antibodies are further described in U.S. Pat. No. 6,884,879 issued February 26, 2005.
  • VEGF antibody may be substituted with a VEGF specific antagonist, e.g., a VEGF receptor molecule or chimeric VEGF receptor molecule as described herein.
  • the anti-VEGF antibody is bevacizumab.
  • the anti-VEGF antibody, or antigen-binding fragment thereof can be a monoclonal antibody, a chimeric antibody, a fully human antibody, or a humanized antibody.
  • Exemplary antibodies useful in the methods of the invention include bevacizumab (AVASTIN®), a G6 antibody, a B20 antibody, and fragments thereof, as described in WO2005/012359. For additional preferred antibodies see U.S. Pat. Nos.
  • a "G6 series antibody” is an anti-VEGF antibody that is derived from a sequence of a G6 antibody or G6-derived antibody according to any one of Figures 7, 24-26, and 34-35 of PCT Publication No. WO2005/012359, the entire disclosure of which is expressly incorporated herein by reference. See also PCT Publication No. WO2005/044853, the entire disclosure of which is expressly incorporated herein by reference.
  • the G6 series antibody binds to a functional epitope on human VEGF comprising residues F17, Y21, Q22, Y25, D63, 183 and Q89.
  • a "B20 series antibody” according to this invention is an anti-VEGF antibody that is derived from a sequence of the B20 antibody or a B20-derived antibody according to any one of Figures 27-29 of PCT Publication No. WO2005/012359, the entire disclosure of which is expressly incorporated herein by reference. See also PCT Publication No.
  • the B20 series antibody binds to a functional epitope on human VEGF comprising residues F17, M18, D19, Y21, Y25, Q89, 191, K101, E103, and C104.
  • a “hematopoietic stem/progenitor cell” or “primitive hematopoietic cell” is one which is able to differentiate to form a more committed or mature blood cell type.
  • Lymphoid blood cell lineages are those hematopoietic precursor cells which are able to differentiate to form lymphocytes (B-cells or T-cells). Likewise, “lymphopoeisis” is the formation of lymphocytes. “Erythroid blood cell lineages” are those hematopoietic precursor cells which are able to differentiate to form erythrocytes (red blood cells) and
  • erythropoeisis is the formation of erythrocytes.
  • myeloblast lineages encompasses all hematopoietic progenitor cells, other than lymphoid and erythroid blood cell lineages as defined above, and “myelopoiesis” involves the formation of blood cells (other than lymphocytes and erythrocytes).
  • a myeloid cell population can be enriched in myeloid immune cells that are
  • Grl+/CD1 lb+ (or CDl lb+Grl+) or Grl+/Mac-1+. These cells express a marker for myeloid cells of the macrophage lineage, CDl lb, and a marker for granulocytes, Grl .
  • Grl+/CD1 lb+ can be selected by immunoadherent panning, for example, with an antibody to Grl+.
  • biological sample refers to a body sample from any animal, but preferably is from a mammal, more preferably from a human.
  • samples include biological fluids such as blood, serum, plasma, bone marrow, vitreous fluid, lymph fluid, synovial fluid, follicular fluid, seminal fluid, amniotic fluid, milk, whole blood, urine, cerebro-spinal fluid, saliva, sputum, tears, perspiration, mucus, and tissue culture medium, as well as tissue extracts such as homogenized tissue, and cellular extracts.
  • biological fluids such as blood, serum, plasma, bone marrow, vitreous fluid, lymph fluid, synovial fluid, follicular fluid, seminal fluid, amniotic fluid, milk, whole blood, urine, cerebro-spinal fluid, saliva, sputum, tears, perspiration, mucus, and tissue culture medium, as well as tissue extracts such as homogenized tissue, and cellular extracts.
  • tissue extracts such as homogenized tissue,
  • antibody is used in the broadest sense and and specifically covers monoclonal antibodies (including full length or intact monoclonal antibodies), polyclonal antibodies, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, and antibody fragments (see below) so long as they exhibit the desired biological activity.
  • Antibody fragments comprise only a portion of an intact antibody, generally including an antigen binding site of the intact antibody and thus retaining the ability to bind antigen.
  • Examples of antibody fragments encompassed by the present definition include: (i) the Fab fragment, having VL, CL, VH and CHI domains; (ii) the Fab' fragment, which is a Fab fragment having one or more cysteine residues at the C-terminus of the CHI domain; (iii) the Fd fragment having VH and CHI domains; (iv) the Fd' fragment having VH and CHI domains and one or more cysteine residues at the C-terminus of the CHI domain; (v) the Fv fragment having the VL and VH domains of a single arm of an antibody; (vi) the dAb fragment (Ward et al, Nature 341, 544-546 (1989)) which consists of a VH domain; (vii) isolated CDR regions; (viii) F(ab')2 fragments
  • treatment and grammatical variations thereof such as
  • treat refers to clinical intervention in an attempt to alter the natural course of the individual being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis.
  • antibodies of the invention are used to delay development of a disease or to slow the progression of a disease.
  • the treatment may directly prevent, slow down or otherwise decrease the pathology of cellular degeneration or damage, such as the pathology of a disease or conditions associated with the mobilization of myeloid cells and/or with tumor angiogenesis.
  • an "effective amount" of an agent refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic, sensitizing or prophylactic result.
  • anti-neoplastic composition refers to a composition useful in treating cancer comprising at least one active therapeutic agent, e.g., "anti-cancer agent.”
  • therapeutic agents include, but are limited to, e.g., chemotherapeutic agents, growth inhibitory agents, cytotoxic agents, agents used in radiation therapy, anti-angiogenesis agents, apoptotic agents, anti-tubulin agents, toxins, and other- agents to treat cancer, e.g., anti-VEGF neutralizing antibody, VEGF antagonist, anti-G-CSF antagonist, interferons, cytokines, including VEGF receptor antagonists (e.g., neutralizing antibodies), and other bioactive and organic chemical agents, etc.
  • chemotherapeutic agents include, but are limited to, e.g., chemotherapeutic agents, growth inhibitory agents, cytotoxic agents, agents used in radiation therapy, anti-angiogenesis agents, apoptotic agents, anti-tubulin agents, toxins, and other- agents to treat cancer, e
  • cytostatic agent refers to a compound or composition which arrests growth of a cell either in vitro or in vivo.
  • a cytostatic agent may be one which significantly reduces the percentage of cells in S phase.
  • Further examples of cytostatic agents include agents that block cell cycle progression by inducing G0/G1 arrest or M-phase arrest.
  • the humanized anti-Her2 antibody trastuzumab (HERCEPTIN®) is an example of a cytostatic agent that induces G0/G1 arrest.
  • Classical M-phase blockers include the vincas (vincristine and vinblastine), taxanes, and topoisomerase II inhibitors such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin.
  • Certain agents that arrest Gl also spill over into S-phase arrest, for example, DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C.
  • Paclitaxel and docetaxel promote the assembly of microtubules from tubulin dimers and stabilize microtubules by preventing depolymerization, which results in the inhibition of mitosis in cells.
  • the term is intended to include radioactive isotopes (e.g., 211 At, 131 1, 125 1, 90 Y, 186 Re, 188 Re, 153 Sm, 212 Bi, 32 P and radioactive isotopes of Lu), chemotherapeutic agents, and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof.
  • a “growth inhibitory agent” when used herein refers to a compound or composition which inhibits growth of a cell in vitro and/or in vivo.
  • the growth inhibitory agent may be one which significantly reduces the percentage of cells in S phase.
  • growth inhibitory agents include agents that block cell cycle progression (at a place other than S phase), such as agents that induce Gl arrest and M-phase arrest.
  • Classical M-phase blockers include the vincas (vincristine and vinblastine), TAXOL®, and topo II inhibitors such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin.
  • DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5- fluorouracil, and ara-C. Further information can be found in The Molecular Basis of Cancer, Mendelsohn and Israel, eds., Chapter 1, entitled “Cell cycle regulation, oncogenes, and antineoplastic drugs” by Murakami et al. (WB Saunders: Philadelphia, 1995), especially p. 13.
  • a "chemotherapeutic agent” refers to a chemical compound useful in the treatment of cancer.
  • chemotherapeutic agents include alkylating agents such as thiotepa and cyclosphosphamide (CYTOXAN®); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylomelamine;
  • alkylating agents such as thiotepa and cyclosphosphamide (CYTOXAN®)
  • alkyl sulfonates such as busulfan, improsulfan and piposulfan
  • aziridines such as benzodopa, carboquone, meturedopa, and ure
  • acetogenins especially bullatacin and bullatacinone
  • dronabinol, MARINOL® beta-lapachone
  • lapachol colchicines
  • betulinic acid a camptothecin (including the synthetic analogue topotecan (HYCAMTIN®), CPT-11
  • spongistatin nitrogen mustards such as chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard;
  • nitrogen mustards such as chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard;
  • nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e. g., calicheamicin, especially calicheamicin gammall and calicheamicin omegall (see, e.g., Nicolaou et al., Angew. Chem Intl. Ed.
  • CDP323 an oral alpha-4 integrin inhibitor
  • dynemicin including dynemicin A
  • esperamicin as well as neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomysins, actinomycin,
  • doxorubicin including ADRIAMYCIN®, morpholino-doxorubicin, cyanomorpholino- doxorubicin, 2-pyrrolino-doxorubicin, doxorubicin HC1 liposome injection (DOXIL®), liposomal doxorubicin TLC D-99 (MYOCET®), peglylated liposomal doxorubicin
  • CAELYX® deoxy doxorubicin
  • epirubicin esorubicin
  • idarubicin idarubicin
  • marcellomycin mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins
  • peplomycin porfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate,
  • gemcitabine GEMZAR®
  • tegafur UTORAL®
  • capecitabine XELODA®
  • an epothilone 5-fluorouracil
  • 5-fluorouracil 5-fluorouracil
  • folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate
  • purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine
  • pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine
  • androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone
  • anti-adrenals such as aminoglutethimide, mitotane, trilostane
  • aceglatone aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine;
  • elliptinium acetate an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan;
  • lonidainine lonidainine
  • maytansinoids such as maytansine and ansamitocins
  • mitoguazone lonidainine
  • mitoxantrone mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; 2- ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2'- trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine (ELDISINE®, FILDESIN®); dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); thiotepa; taxoid, e.g., paclit
  • mercaptopurine mercaptopurine
  • methotrexate platinum agents such as cisplatin, oxaliplatin (e.g.,
  • ELOXATIN® etoposide
  • carboplatin etoposide
  • vincas which prevent tubulin polymerization from forming microtubules, including vinblastine (VELBAN®), vincristine (ONCOVIN®), vindesine (ELDISINE®, FILDESIN®), and vinorelbine (NAVELBINE®); etoposide (VP- 16);
  • ifosfamide mitoxantrone; leucovorin; novantrone; edatrexate; daunomycin; aminopterin; ibandronate; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid, including bexarotene (TARGRETIN®); bisphosphonates such as clodronate (for example, BONEFOS® or OSTAC®), etidronate (DIDROCAL®), NE-58095, zoledronic acid/zoledronate (ZOMETA®), alendronate (FOSAMAX®), pamidronate
  • AREDIA® tiludronate
  • SKELID® tiludronate
  • ACTONEL® risedronate
  • troxacitabine a 1,3- dioxolane nucleoside cytosine analog
  • antisense oligonucleotides particularly those that inhibit expression of genes in signaling pathways implicated in aberrant cell proliferation, such as, for example, PKC-alpha, Raf, H-Ras, and epidermal growth factor receptor (EGF- R)
  • vaccines such as THERATOPE® vaccine and gene therapy vaccines, for example, ALLOVECTIN® vaccine, LEUVECTIN® vaccine, and VAXID® vaccine
  • topoisomerase 1 inhibitor e.g., LURTOTECAN®
  • rmRH e.g., ABARELIX®
  • BAY439006 asorafenib; Bayer
  • SU-11248 subunitinib, SUTENT®, Pfizer
  • celecoxib or etoricoxib proteosome inhibitor
  • proteosome inhibitor e.g. PS341
  • bortezomib VELCADE®
  • CCI-779 tipifarnib (Rl 1577); orafenib, ABT510
  • Bcl-2 inhibitor such as oblimersen sodium
  • GENESENSE® pixantrone; EGFR inhibitors (see definition below); tyrosine kinase inhibitors (see definition below); serine -threonine kinase inhibitors such as rapamycin (sirolimus, RAPAMUNE®); farnesyltransferase inhibitors such as lonafarnib (SCH 6636, SARASARTM); and pharmaceutically acceptable salts, acids or derivatives of any of the above; as well as combinations of two or more of the above such as CHOP, an abbreviation for a combined therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone; and FOLFOX, an abbreviation for a treatment regimen with oxaliplatin (ELOXATINTM) combined with 5-FU and leucovorin.
  • ELOXATINTM oxaliplatin
  • Chemotherapeutic agents as defined herein include “anti-VEGF antagonists” or “endocrine therapeutics” which act to regulate, reduce, block, or inhibit the effects of hormones that can promote the growth of cancer. They may be hormones themselves, including, but not limited to: anti-estrogens with mixed agonist/antagonist profile, including, tamoxifen (NOLVADEX®), 4-hydroxytamoxifen, toremifene (FARESTON®), idoxifene, droloxifene, raloxifene (EVISTA®), trioxifene, keoxifene, and selective estrogen receptor modulators (SERMs) such as SERM3; pure anti-estrogens without agonist properties, such as fulvestrant (FASLODEX®), and EM800 (such agents may block estrogen receptor (ER) dimerization, inhibit DNA binding, increase ER turnover, and/or suppress ER levels);
  • anti-estrogens with mixed agonist/antagonist profile including, tam
  • aromatase inhibitors including steroidal aromatase inhibitors such as formestane and exemestane (AROMAS IN®), and nonsteroidal aromatase inhibitors such as anastrazole (ARIMIDEX®), letrozole (FEMARA®) and aminoglutethimide, and other aromatase inhibitors include vorozole (RIVISOR®), megestrol acetate (MEGASE®), fadrozole, and 4(5)-imidazoles; lutenizing hormone-releaseing hormone agonists, including leuprolide (LUPRON® and ELIGARD®), goserelin, buserelin, and tripterelin; sex steroids, including progestines such as megestrol acetate and medroxyprogesterone acetate, estrogens such as diethylstilbestrol and premarin, and androgens/retinoids such as fluoxymesterone, all transretionic acid and fenretinide; on
  • cytokine is a generic term for proteins released by one cell population which act on another cell as intercellular mediators.
  • cytokines are lymphokines, monokines, and traditional polypeptide hormones. Included among the cytokines are growth hormone such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor-alpha and -beta; mullerian-inhibiting substance; mouse gonadotropin-associated peptide; inhibin; activin;
  • growth hormone such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone
  • parathyroid hormone such as thyroxine
  • insulin pro
  • vascular endothelial growth factors e.g., VEGF, VEGF-B, VEGF-C, VEGF-D, VEGF-E
  • placental derived growth factor PIGF
  • platelet derived growth factors PDGF, e.g., PDGFA, PDGFB, PDGFC, PDGFD
  • integrin thrombopoietin
  • nerve growth factors such as NGF-alpha; platelet-growth factor; transforming growth factors (TGFs) such as TGF-alpha and TGF-beta; insulin-like growth factor-I and -II; erythropoietin (EPO); osteoinductive factors; interferons such as interferon-alpha, -beta and -gamma, colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-C
  • an "angiogenic factor or agent” is a growth factor which stimulates the development of blood vessels, e.g., promotes angiogenesis, endothelial cell growth, stability of blood vessels, and/or vasculogenesis, etc.
  • angiogenic factors include, but are not limited to, e.g., VEGF and members of the VEGF family, PIGF, PDGF family, fibroblast growth factor family (FGFs), TIE ligands (Angiopoietins), ephrins, ANGPTL3, ANGPTL4, etc.
  • IGF-I insulin-like growth factor-I
  • VIGF insulin-like growth factor
  • EGF epidermal growth factor
  • CTGF CTGF and members of its family
  • TGF-a and TGF- ⁇ factors that accelerate wound healing, such as growth hormone, insulin-like growth factor-I (IGF-I), VIGF, epidermal growth factor (EGF), CTGF and members of its family
  • IGF-I insulin-like growth factor-I
  • VIGF insulin-like growth factor-I
  • EGF epidermal growth factor
  • CTGF tumor necrosis factor-a and TGF- ⁇ .
  • an "anti-angiogenesis agent” or “angiogenesis inhibitor” refers to a small molecular weight substance, a polynucleotide, a polypeptide, an isolated protein, a recombinant protein, an antibody, or conjugates or fusion proteins thereof, that inhibits angiogenesis, vasculogenesis, or undesirable vascular permeability, either directly or indirectly.
  • an anti-angiogenesis agent is an antibody or other antagonist to an angiogenic agent as defined above, e.g., antibodies to VEGF, antibodies to VEGF receptors, small molecules that block VEGF receptor signaling (e.g., PTK787/ZK2284, SU6668, SUTENT/SU11248 (sunitinib malate), AMG706).
  • Anti-angiogensis agents also include native angiogenesis inhibitors, e.g., angiostatin, endostatin, etc. See, e.g., Klagsbrun and D'Amore, Annu. Rev.
  • immunosuppressive agent refers to substances that act to suppress or mask the immune system of the mammal being treated herein. This would include substances that suppress cytokine production, down-regulate or suppress self-antigen expression, or mask the MHC antigens. Examples of such agents include 2-amino-6-aryl-5- substituted pyrimidines (see U.S. Pat. No.
  • nonsteroidal antiinflammatory drugs NSAIDs
  • ganciclovir tacrolimus, glucocorticoids such as Cortisol or aldosterone
  • antiinflammatory agents such as a cyclooxygenase inhibitor, a 5 -lipoxygenase inhibitor, or a leukotriene receptor antagonist
  • purine antagonists such as azathioprine or mycophenolate mofetil (MMF)
  • alkylating agents such as cyclophosphamide; bromocryptine; danazol;
  • dapsone dapsone; glutaraldehyde (which masks the MHC antigens, as described in U.S. Pat. No. 4,120,649); anti-idiotypic antibodies for MHC antigens and MHC fragments; cyclosporin A; steroids such as corticosteroids or glucocorticosteroids or glucocorticoid analogs, e.g., prednisone, methylprednisolone, and dexamethasone; dihydrofolate reductase inhibitors such as methotrexate (oral or subcutaneous); hydroxycloroquine; sulfasalazine; leflunomide; cytokine or cytokine receptor antibodies including anti-interferon-alpha, -beta, or -gamma antibodies, anti-tumor necrosis factor-alpha antibodies (infliximab or adalimumab), anti- TNF-alpha immunoahesin (
  • T-cell receptor Cohen et al, U.S. Pat. No. 5,114,721
  • T-cell- receptor fragments Offner et al, Science, 251 : 430-432 (1991); WO 1990/11294; Ianeway, Nature, 341 : 482 (1989); and WO 1991/01133
  • T-cell-receptor antibodies EP 340,109
  • Administration "in combination with” one or more further therapeutic agents includes simultaneous (concurrent) and consecutive administration in any order.
  • Carriers as used herein include pharmaceutically acceptable carriers, excipients, or stabilizers and refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject.
  • a pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.
  • an "effective amount" of an agent refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic, sensitizing or prophylactic result.
  • the expressions "cell,” “cell line,” and “cell culture” are used interchangeably and all such designations include progeny.
  • the words “transformants” and “transformed cells” include the primary subject cell and cultures derived therefrom without regard for the number of transfers. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations.
  • progeny refers to any and all offspring of every generation subsequent to an originally transformed cell or cell line. Mutant progeny that have the same function or biological activity as screened for in the originally transformed cell are included. Where distinct designations are intended, it will be clear from the context.
  • MEK inhibitors are available in the field and can be used for the purposes of the invention. Of particular relevance are small molecule compounds currently in preclinical studies or clinical trials. Several small molecule MEK inhibitors and methods of making/screening the same have been discussed in, e.g., WO02/06213, WO 03/077855, WO03/077914, WO09/085983, WOl 1/054620, WO2010/006225 and US Pat No 7803839.
  • MEK inhibitor of the present invention can be selected from the group consisting of PD325901, PD-181461, ARRY142886 / AZD6244, ARRY-509, GDC0973 (XL518), GDC0987, JTP-74057, AS-701255, AS-701173, AZD8330, ARRY162, ARRY300, RDEA436, E6201 , R04987655/R-7167, GSKl 120212 and AS-703026.
  • the MEK inhibitor is one of the azetidine compounds such as those described in US Pat No 7803839, including but not limited to those listed in Table 1 of the patent.
  • the MEK inhibitor is one of the imidazopyridine compounds such as those described in WO09/085983.
  • the MEK inhibitor is [3,4-Difluoro-2-(2-fluoro-4-iodo-phenylamino)-phenyl]-((S)-3-hydroxy-3-piperidin-2- yl-azetidin-l -yl)-methanone also known as GDC-0973/XL-518.
  • the MEK inhibitor or G-CSF antagonist of the present invention can be used, alone or in combination with a VEGF antagonist or other therapeutic agent(s) for the inhibition of tumor growth.
  • Primary targets for the treatment methods of the present invention are tumors that have shown or are known to be resistant to treatment with VEGF antagonists, in particular anti-VEGF antibodies.
  • tumors or neoplastic conditions are described herein under the terms “tumor”, “cancer” and “cancerous.”
  • the neoplastic condition is characterized by pathological disorder associated with aberrant or undesired angiogenesis that is resistant to VEGF antagonist treatment.
  • Anti-angiogenic therapy in relationship to cancer is a cancer treatment strategy aimed at inhibiting the development of tumor blood vessels required for providing nutrients to support tumor growth. Because angiogenesis is involved in both primary tumor growth and metastasis, the antiangiogenic treatment provided by the invention is capable of inhibiting the neoplastic growth of tumor at the primary site as well as preventing metastasis of tumors at the secondary sites, therefore allowing attack of the tumors by other therapeutics.
  • anti-cancer agent or therapeutic is an anti-angiogenic agent.
  • anti-cancer agent is a chemotherapeutic agent.
  • an MEK inhibitor of the invention is used in combination with an anti-VEGF neutralizing antibody (or fragment) and/or another VEGF antagonist or a VEGF receptor antagonist including, but not limited to, for example, soluble VEGF receptor (e.g., VEGFR-1, VEGFR-2, VEGFR-3, neuropillins (e.g., NRP1, NRP2)) fragments, aptamers capable of blocking VEGF or VEGFR, neutralizing anti-VEGFR antibodies, low molecule weight inhibitors of VEGFR tyrosine kinases (RTK), antisense strategies for VEGF, ribozymes against VEGF or VEGF receptors, antagonist variants of VEGF; and any combinations thereof.
  • soluble VEGF receptor e.g., VEGFR-1, VEGFR-2, VEGFR-3, neuropillins (e.g., NRP1, NRP2)
  • aptamers capable of blocking VEGF or VEGFR e.g., VEGFR
  • angiogenesis inhibitors may optionally be co-administered to the patient in addition to VEGF antagonist and other agent of the invention.
  • one or more additional therapeutic agents e.g., anti-cancer agents, can be administered in combination with agent of the invention, the VEGF antagonist, and/or an anti-angiogenesis agent.
  • the invention provides a method of blocking or reducing resistant tumor growth or growth of a cancer cell, by administering effective amounts of a MEK inhibitor and one or more chemotherapeutic agents to a patient susceptible to, or diagnosed with, cancer.
  • chemotherapeutic agents may be used in the combined treatment methods of the invention.
  • An exemplary and non-limiting list of chemotherapeutic agents contemplated is provided herein under "Definition.”
  • the appropriate doses of chemotherapeutic agents will be generally around those already employed in clinical therapies wherein the chemotherapeutics are administered alone or in combination with other chemotherapeutics. Variation in dosage will likely occur depending on the condition being treated. The physician administering treatment will be able to determine the appropriate dose for the individual subject.
  • the invention also provides methods and compositions for inhibiting or preventing relapse tumor growth or relapse cancer cell growth. Relapse tumor growth or relapse cancer cell growth is used to describe a condition in which patients undergoing or treated with one or more currently available therapies (e.g., cancer therapies, such as chemotherapy, radiation therapy, surgery, hormonal therapy and/or biological
  • a cancer is relapse tumor growth or relapse cancer cell growth where the number of cancer cells has not been significantly reduced, or has increased, or tumor size has not been significantly reduced, or has increased, or fails any further reduction in size or in number of cancer cells.
  • the determination of whether the cancer cells are relapse tumor growth or relapse cancer cell growth can be made either in vivo or in vitro by any method known in the art for assaying the effectiveness of treatment on cancer cells, using the art-accepted meanings of "relapse” or “refractory” or “non-responsive” in such a context.
  • a tumor resistant to anti-VEGF treatment is an example of a relapse tumor growth.
  • the invention provides methods of blocking or reducing relapse tumor growth or relapse cancer cell growth in a subject by administering one or more compositions of the invention to block or reduce the relapse tumor growth or relapse cancer cell growth in subject.
  • the MEK inhibitor can be administered subsequent to the cancer therapeutic.
  • the MEK inhibitors of the invention are administered simultaneously with cancer therapy, e.g., chemotherapy.
  • the MEK inhibitor therapy alternates with another cancer therapy, which can be performed in any order.
  • the invention also encompasses methods for administering one or more inhibitory antibodies to prevent the onset or recurrence of cancer in patients predisposed to having cancer. Generally, the subject was or is concurrently undergoing cancer therapy.
  • the cancer therapy is a combination treatment with an anti-angiogenesis agent, e.g. , a VEGF antagonist.
  • an anti-angiogenesis agent includes those known in the art and those found under the Definitions herein.
  • the anti-angiogenesis agent is an anti-VEGF neutralizing antibody or fragment thereof (e.g., humanized A4.6.1, AVASTIN ® (Genentech, South San Francisco, CA), Y0317, M4, G6, B20, 2C3, etc.). See, e.g., U.S.
  • Additional agents can be administered in combination with an MEK inhibitor for blocking or reducing relapse tumor growth or relapse cancer cell growth, e.g., see section entitled Combination Therapies herein.
  • compositions or formulations of the present invention include MEK inhibitors, G-CSF antagonits, VEGF antagonists and combinations thereof, and one or more pharmaceutically acceptable carrier, glidant, diluent, or excipient.
  • MEK inhibitors, G-CSF antagonits and VEGF antagonists of the present invention may exist in unsolvated as well as solvated forms with pharmaceutically acceptable solvents such as water, ethanol, and the like, and it is intended that the invention embrace both solvated and unsolvated forms.
  • compositions of the present invention may also exist in different tautomeric forms, and all such forms are embraced within the scope of the invention.
  • tautomer or "tautomeric form” refers to structural isomers of different energies which are interconvertible via a low energy barrier.
  • proton tautomers also known as prototropic tautomers
  • Valence tautomers include interconversions by reorganization of some of the bonding electrons.
  • compositions encompass both the bulk composition and individual dosage units comprised of more than one (e.g., two) pharmaceutically active agents including a MEK inhibitor and a VEGF antagonist selected from the lists of the additional agents described herein, along with any pharmaceutically inactive excipients, diluents, carriers, or glidants.
  • the bulk composition and each individual dosage unit can contain fixed amounts of the aforesaid pharmaceutically active agents.
  • the bulk composition is material that has not yet been formed into individual dosage units.
  • An illustrative dosage unit is an oral dosage unit such as tablets, pills, capsules, and the like.
  • the herein-described method of treating a patient by administering a pharmaceutical composition of the present invention is also intended to encompass the administration of the bulk composition and individual dosage units.
  • compositions also embrace isotopically-labeled compounds of the present invention which are identical to those recited herein, but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. All isotopes of any particular atom or element as specified are contemplated within the scope of the compounds of the invention, and their uses. Exemplary isotopes that can be incorporated into compounds include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, chlorine and iodine, such as 2 H, 3 H, U C, 13 C, 14 C, 13 N, 15 N, 15 0, 17 0, 18 0, 32 P, 33 P,
  • Certain isotopically-labeled compounds of the present invention are useful in compound and/or substrate tissue distribution assays. Tritiated ( 3 H) and carbon-14 ( 14 C) isotopes are useful for their ease of preparation and detectability. Further, substitution with heavier isotopes such as deuterium ( H) may afford certain therapeutic advantages resulting from greater metabolic stability (e.g., increased in vivo half-life or reduced dosage requirements) and hence may be
  • Positron emitting isotopes such as O, N, C and F are useful for positron emission tomography (PET) studies to examine substrate receptor occupancy.
  • Isotopically labeled compounds of the present invention can generally be prepared by following procedures analogous to those disclosed in the Schemes and/or in the Examples herein below, by substituting an isotopically labeled reagent for a non- isotopically labeled reagent.
  • MEK inhibitors and VEGF antagonists are formulated in accordance with standard pharmaceutical practice for use in a therapeutic combination for therapeutic treatment (including prophylactic treatment) of hyperproliferative disorders in mammals including humans.
  • the invention provides a pharmaceutical composition comprising a MEK inhibitor in association with one or more pharmaceutically acceptable carrier, glidant, diluent, or excipient.
  • Suitable carriers, diluents and excipients are well known to those skilled in the art and include materials such as carbohydrates, waxes, water soluble and/or swellable polymers, hydrophilic or hydrophobic materials, gelatin, oils, solvents, water and the like.
  • the particular carrier, diluent or excipient used will depend upon the means and purpose for which the compound of the present invention is being applied.
  • Solvents are generally selected based on solvents recognized by persons skilled in the art as safe (GRAS) to be administered to a mammal.
  • safe solvents are non-toxic aqueous solvents such as water and other non-toxic solvents that are soluble or miscible in water.
  • Suitable aqueous solvents include water, ethanol, propylene glycol, polyethylene glycols (e.g., PEG 400, PEG 300), etc. and mixtures thereof.
  • the formulations may also include one or more buffers, stabilizing agents, surfactants, wetting agents, lubricating agents, emulsifiers, suspending agents, preservatives, antioxidants, opaquing agents, glidants, processing aids, colorants, sweeteners, perfuming agents, flavoring agents and other known additives to provide an elegant presentation of the drug (i.e., a compound of the present invention or pharmaceutical composition thereof) or aid in the manufacturing of the pharmaceutical product (i.e., medicament).
  • the formulations may be prepared using conventional dissolution and mixing procedures.
  • the bulk drug substance i.e., compound of the present invention or stabilized form of the compound (e.g., complex with a cyclodextrin derivative or other known complexation agent) is dissolved in a suitable solvent in the presence of one or more of the excipients described above.
  • the compound of the present invention is typically formulated into pharmaceutical dosage forms to provide an easily controllable dosage of the drug and to enable patient compliance with the prescribed regimen.
  • the pharmaceutical composition (or formulation) for application may be packaged in a variety of ways depending upon the method used for administering the drug.
  • an article for distribution includes a container having deposited therein the pharmaceutical formulation in an appropriate form.
  • Suitable containers are well known to those skilled in the art and include materials such as bottles (plastic and glass), sachets, ampoules, plastic bags, metal cylinders, and the like.
  • the container may also include a tamper-proof assemblage to prevent indiscreet access to the contents of the package.
  • the container has deposited thereon a label that describes the contents of the container. The label may also include appropriate warnings.
  • compositions of the present invention may be prepared for various routes and types of administration.
  • a MEK inhibitor having the desired degree of purity may optionally be mixed with pharmaceutically acceptable diluents, carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences (1995) 18th edition, Mack Publ. Co., Easton, PA), in the form of a lyophilized formulation, milled powder, or an aqueous solution.
  • Formulation may be conducted by mixing at ambient temperature at the appropriate pH, and at the desired degree of purity, with physiologically acceptable carriers, i.e., carriers that are non-toxic to recipients at the dosages and concentrations employed.
  • physiologically acceptable carriers i.e., carriers that are non-toxic to recipients at the dosages and concentrations employed.
  • the pH of the formulation depends mainly on the particular use and the concentration of compound, but may range from about 3 to about 8.
  • the pharmaceutical formulation is preferably sterile.
  • formulations to be used for in vivo administration must be sterile. Such sterilization is readily accomplished by filtration through sterile filtration membranes.
  • the pharmaceutical formulation ordinarily can be stored as a solid
  • composition a lyophilized formulation or as an aqueous solution.
  • the pharmaceutical formulations will be dosed and administered in a fashion, i.e., amounts, concentrations, schedules, course, vehicles and route of administration, consistent with good medical practice.
  • Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners.
  • the "therapeutically effective amount" of the compound to be administered will be governed by such considerations, and is the minimum amount necessary to prevent, ameliorate, or treat the coagulation factor mediated disorder. Such amount is preferably below the amount that is toxic to the host or renders the host significantly more susceptible to bleeding.
  • MEK inhibitor administered orally or parenterally per dose will be in the range of about 0.01-1000 mg/kg, namely about 0.1 to 20 mg/kg of patient body weight per day, with the typical initial range of compound used being 0.3 to 15 mg/kg/day.
  • the dose of the MEK inhibitor and the dose of the VEGF antagonist to be administered may range for each from about 1 mg to about 1000 mg per unit dosage form, or from about 10 mg to about 100 mg per unit dosage form.
  • the doses of MEK inhibitor and the VEGF antagonist may administered in a ratio of about 1 :50 to about 50: 1 by weight, or in a ratio of about 1 : 10 to about 10: 1 by weight.
  • Acceptable diluents, carriers, excipients and stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride;
  • hexamethonium chloride benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol;
  • cyclohexanol 3-pentanol; and m-cresol
  • low molecular weight polypeptides such as serum albumin, gelatin, or immunoglobulins
  • hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionic surfactants such as TWEENTM, PLURONICSTM or polyethylene glycol (PEG).
  • amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine
  • monosaccharides, disaccharides and other carbohydrates including glucose, mannose, or dextrins such as EDTA
  • sugars such as sucrose, mannitol, trehalose or
  • the active pharmaceutical ingredients may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres,
  • microemulsions nano-particles and nanocapsules
  • macroemulsions Such techniques are disclosed in Remington's Pharmaceutical Sciences 18th edition, (1995) Mack Publ. Co., Easton, PA.
  • Sustained-release preparations of MEK inhibitors may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing a MEK inhibitor, which matrices are in the form of shaped articles, e.g., films, or microcapsules.
  • sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly( vinyl alcohol)), polylactides (US 3773919), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate, non-degradable ethylene -vinyl acetate, degradable lactic acid- glycolic acid copolymers such as the LUPRON DEPOTTM (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate) and poly-D (-) 3- hydroxybutyric acid.
  • the pharmaceutical formulations include those suitable for the administration routes detailed herein. The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Techniques and formulations generally are found in Remington's
  • Such methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients.
  • the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.
  • Formulations of a MEK inhibitor and/or VEGF antagonist suitable for oral administration may be prepared as discrete units such as pills, hard or soft e.g., gelatin capsules, cachets, troches, lozenges, aqueous or oil suspensions, dispersible powders or granules, emulsions, syrups or elixirs each containing a predetermined amount of a MEK inhibitor and/or a VEGF antagonist.
  • the amount of MEK inhibitor and the amount of VEGF antagonist may be formulated in a pill, capsule, solution or suspension as a combined formulation.
  • the MEK inhibitor and the VEGF antagonist may be formulated separately in a pill, capsule, solution or suspension for administration by alternation.
  • Formulations may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions may contain one or more agents including sweetening agents, flavoring agents, coloring agents and preserving agents, in order to provide a palatable preparation.
  • Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, preservative, surface active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered active ingredient moistened with an inert liquid diluent. The tablets may optionally be coated or scored and optionally are formulated so as to provide slow or controlled release of the active ingredient therefrom.
  • Tablet excipients of a pharmaceutical formulation may include: Filler (or diluent) to increase the bulk volume of the powdered drug making up the tablet;
  • Disintegrants to encourage the tablet to break down into small fragments, ideally individual drug particles, when it is ingested and promote the rapid dissolution and absorption of drug; Binder to ensure that granules and tablets can be formed with the required mechanical strength and hold a tablet together after it has been compressed, preventing it from breaking down into its component powders during packaging, shipping and routine handling; Glidant to improve the flowability of the powder making up the tablet during production; Lubricant to ensure that the tableting powder does not adhere to the equipment used to press the tablet during manufacture.
  • Tablets containing the active ingredient in admixture with non-toxic pharmaceutically acceptable excipient which are suitable for manufacture of tablets are acceptable.
  • excipients may be, for example, inert diluents, such as calcium or sodium carbonate, lactose, calcium or sodium phosphate; granulating and disintegrating agents, such as maize starch, or alginic acid; binding agents, such as starch, gelatin or acacia; and lubricating agents, such as magnesium stearate, stearic acid or talc. Tablets may be uncoated or may be coated by known techniques including microencapsulation to delay disintegration and adsorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate alone or with a wax may be employed.
  • the formulations are preferably applied as a topical ointment or cream containing the active ingredient(s) in an amount of, for example, 0.075 to 20% w/w.
  • the active ingredients may be employed with either a paraffinic or a water- miscible ointment base.
  • the active ingredients may be formulated in a cream with an oil-in-water cream base.
  • the aqueous phase of the cream base may include a polyhydric alcohol, i.e., an alcohol having two or more hydroxyl groups such as propylene glycol, butane 1 ,3-diol, mannitol, sorbitol, glycerol and polyethylene glycol (including PEG 400) and mixtures thereof.
  • the topical formulations may desirably include a compound which enhances absorption or penetration of the active ingredient through the skin or other affected areas. Examples of such dermal penetration enhancers include dimethyl sulfoxide and related analogs.
  • the oily phase of the emulsions of this invention may be constituted from known ingredients in a known manner, including a mixture of at least one emulsifier with a fat or an oil, or with both a fat and an oil.
  • a hydrophilic emulsifier is included together with a lipophilic emulsifier which acts as a stabilizer.
  • the emulsifier(s) with or without stabilizer(s) make up an emulsifying wax, and the wax together with the oil and fat comprise an emulsifying ointment base which forms the oily dispersed phase of cream formulations.
  • formulation include Tween® 60, Span® 80, cetostearyl alcohol, benzyl alcohol, myristyl alcohol, glyceryl mono-stearate and sodium lauryl sulfate.
  • Aqueous suspensions of the pharmaceutical formulations contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions.
  • excipients include a suspending agent, such as sodium carboxymethylcellulose, croscarmellose, povidone, methylcellulose, hydroxypropyl methylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia, and dispersing or wetting agents such as a naturally occurring phosphatide (e.g., lecithin), a condensation product of an alkylene oxide with a fatty acid (e.g., polyoxyethylene stearate), a condensation product of ethylene oxide with a long chain aliphatic alcohol (e.g., heptadecaethyleneoxycetanol), a condensation product of ethylene oxide with a partial ester derived from a fatty acid and a hexitol anhydride (e.g., polyoxyethylene sorbitan mono
  • the aqueous suspension may also contain one or more preservatives such as ethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents and one or more sweetening agents, such as sucrose or saccharin.
  • preservatives such as ethyl or n-propyl p-hydroxybenzoate
  • coloring agents such as a coloring agent
  • flavoring agents such as sucrose or saccharin.
  • sweetening agents such as sucrose or saccharin.
  • compositions may be in the form of a sterile injectable preparation, such as a sterile injectable aqueous or oleaginous suspension.
  • a sterile injectable preparation such as a sterile injectable aqueous or oleaginous suspension.
  • This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above.
  • the sterile injectable preparation may be a solution or a suspension in a non-toxic parenterally acceptable diluent or solvent, such as a solution in 1,3-butanediol or prepared from a lyophilized powder.
  • the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution.
  • sterile fixed oils may conventionally be employed as a solvent or suspending medium.
  • any bland fixed oil may be employed including synthetic mono- or diglycerides.
  • fatty acids such as oleic acid may likewise be used in the
  • the amount of active ingredient that may be combined with the carrier material to produce a single dosage form will vary depending upon the host treated and the particular mode of administration.
  • a time -release formulation intended for oral administration to humans may contain approximately 1 to 1000 mg of active material compounded with an appropriate and convenient amount of carrier material which may vary from about 5 to about 95% of the total compositions (weight: weight).
  • an aqueous solution intended for intravenous infusion may contain from about 3 to 500 ⁇ g of the active ingredient per milliliter of solution in order that infusion of a suitable volume at a rate of about 30 mL/hr can occur.
  • Formulations suitable for parenteral administration include aqueous and nonaqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.
  • Formulations suitable for topical administration to the eye also include eye drops wherein the active ingredient is dissolved or suspended in a suitable carrier, especially an aqueous solvent for the active ingredient.
  • the active ingredient is preferably present in such formulations in a concentration of about 0.5 to 20% w/w, for example about 0.5 to 10%) w/w, for example about 1.5% w/w.
  • Formulations suitable for topical administration in the mouth include lozenges comprising the active ingredient in a flavored basis, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia; and mouthwashes comprising the active ingredient in a suitable liquid carrier.
  • Formulations for rectal administration may be presented as a suppository with a suitable base comprising for example cocoa butter or a salicylate.
  • Formulations suitable for intrapulmonary or nasal administration have a particle size for example in the range of 0.1 to 500 microns (including particle sizes in a range between 0.1 and 500 microns in increments microns such as 0.5, 1, 30 microns, 35 microns, etc.), which is administered by rapid inhalation through the nasal passage or by inhalation through the mouth so as to reach the alveolar sacs.
  • Suitable formulations include aqueous or oily solutions of the active ingredient.
  • Formulations suitable for aerosol or dry powder administration may be prepared according to conventional methods and may be delivered with other therapeutic agents such as compounds heretofore used in the treatment or prophylaxis disorders as described below.
  • Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations containing in addition to the active ingredient such carriers as are known in the art to be appropriate.
  • the formulations may be packaged in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water, for injection immediately prior to use.
  • sterile liquid carrier for example water
  • Extemporaneous injection solutions and suspensions are prepared from sterile powders, granules and tablets of the kind previously described.
  • Preferred unit dosage formulations are those containing a daily dose or unit daily sub-dose, as herein above recited, or an appropriate fraction thereof, of the active ingredient.
  • the MEK inhibitor or a pharmaceutically acceptable salt thereof may be employed in combination with VEGF antagonists for the treatment of a hyperproliferative disease or disorder, including tumors, cancers, and neoplastic tissue, along with pre- malignant and non-neoplastic or non-malignant hyperproliferative disorders.
  • a MEK inhibitor or a pharmaceutically acceptable salt thereof is combined in a dosing regimen as combination therapy, with a second compound that has anti- hyperproliferative properties or that is useful for treating the hyperproliferative disorder.
  • the second compound of the dosing regimen preferably has complementary activities to the MEK inhibitor or a pharmaceutically acceptable salt thereof, and such that they do not adversely affect each other.
  • Such compounds may be administered in amounts that are effective for the purpose intended.
  • the therapeutic combination is administered by a dosing regimen wherein the therapeutically effective amount of a MEK inhibitor, or a pharmaceutically acceptable salt thereof is administered in a range from twice daily to once every three weeks (q3wk), and the therapeutically effective amount of the VEGF antagonist is administered in a range from twice daily to once every three weeks.
  • the combination therapy may be administered as a simultaneous or sequential regimen.
  • the combination When administered sequentially, the combination may be administered in two or more administrations.
  • the combined administration includes coadministration, using separate formulation, and consecutive administration in either order, wherein preferably there is a time period while both (or all) active agents simultaneously exert their biological activities.
  • the pharmaceutically acceptable salt thereof can be administered for a time period of about 1 to about 10 days after administration of the one or more agents begins.
  • the MEK inhibitor or the pharmaceutically acceptable salt thereof can be administered for a time period of about 1 to 10 days before administration of the combination begins.
  • administration of the MEK inhibitor or the pharmaceutically acceptable salt thereof and administration of the VEGF antagonist begin on the same day.
  • Suitable dosages for any of the above coadministered agents are those presently used and may be lowered due to the combined action (synergy) of the newly identified agent and other VEGF antagonists or treatments, such as to increase the therapeutic index or mitigate toxicity or other side-effects or consequences.
  • a MEK inhibitor, or pharmaceutically acceptable salt thereof may be combined with an anti-VEGF antibody such as bevacizumab, as well as combined with chemotherapy, surgical therapy and radiotherapy.
  • an anti-VEGF antibody such as bevacizumab
  • the amounts of the MEK inhibitor or a pharmaceutically acceptable salt thereof and the other pharmaceutically active VEGF antagonist(s) and the relative timings of administration will be selected in order to achieve the desired combined therapeutic effect.
  • the efficacy of the treatment of the invention can be measured by various endpoints commonly used in evaluating neoplastic or non-neoplastic disorders.
  • cancer treatments can be evaluated by, e.g., but not limited to, tumor regression, tumor weight or size shrinkage, time to progression, duration of survival, progression free survival, overall response rate, duration of response, quality of life, protein expression and/or activity.
  • the anti-angiogenic agents described herein target the tumor vasculature and not necessarily the neoplastic cells themselves, they represent a unique class of anticancer drugs, and therefore can require unique measures and definitions of clinical responses to drugs.
  • tumor shrinkage of greater than 50% in a 2-dimensional analysis is the standard cutoff for declaring a response.
  • the inhibitors of the invention may cause inhibition of metastatic spread without shrinkage of the primary tumor, or may simply exert a
  • tumouristatic effect approaches to determining efficacy of the therapy can be employed, including for example, measurement of plasma or urinary markers of angiogenesis and measurement of response through radiological imaging.
  • kits containing a MEK inhibitor or pharmaceutically acceptable salt thereof useful for the treatment of the diseases and disorders described above.
  • the kit comprises a container and a MEK inhibitor or pharmaceutically acceptable salt thereof.
  • the kit may further comprise a label or package insert, on or associated with the container.
  • package insert is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, contraindications and/or warnings concerning the use of such therapeutic products.
  • Suitable containers include, for example, bottles, vials, syringes, blister pack, etc.
  • the container may be formed from a variety of materials such as glass or plastic.
  • the container may hold a MEK inhibitor or
  • At least one active agent in the composition is a MEK inhibitor or a
  • the label or package insert indicates that the composition is used for treating the condition of choice, such as cancer.
  • the condition of choice such as cancer.
  • the label or package inserts indicates that the composition comprising a MEK inhibitor or pharmaceutically acceptable salt thereof can be used to treat a disorder resulting from abnormal cell growth.
  • the label or package insert may also indicate that the composition can be used to treat other disorders.
  • the article of manufacture may further comprise a second container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate -buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.
  • the kit may further comprise directions for the administration of the compound of a MEK inhibitor or pharmaceutically acceptable salt thereof , and, if present, the second pharmaceutical formulation.
  • the kit may further comprise directions for the simultaneous, sequential or separate administration of the first and second pharmaceutical compositions to a patient in need thereof.
  • kits are suitable for the delivery of solid oral forms of a MEK inhibitor or pharmaceutically acceptable salt thereof, such as tablets or capsules.
  • a kit preferably includes a number of unit dosages.
  • Such kits can include a card having the dosages oriented in the order of their intended use.
  • An example of such a kit is a "blister pack".
  • Blister packs are well known in the packaging industry and are widely used for packaging pharmaceutical unit dosage forms.
  • a memory aid can be provided, for example in the form of numbers, letters, or other markings or with a calendar insert, designating the days in the treatment schedule in which the dosages can be administered.
  • a kit may comprise (a) a first container with a
  • the kit may further comprise a third container comprising a pharmaceutically-acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate -buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.
  • BWFI bacteriostatic water for injection
  • phosphate -buffered saline such as bacteriostatic water for injection (BWFI), phosphate -buffered saline, Ringer's solution and dextrose solution.
  • BWFI bacteriostatic water for injection
  • phosphate -buffered saline such as bacteriostatic water for injection (BWFI), phosphate -buffered saline, Ringer's solution and dextrose solution.
  • BWFI bacteriostatic water for injection
  • the kit may comprise a container for containing the separate compositions such as a divided bottle or a divided foil packet, however, the separate compositions may also be contained within a single, undivided container.
  • the kit comprises directions for the administration of the separate components.
  • the kit form is particularly advantageous when the separate components are preferably administered in different dosage forms (e.g., oral and parenteral), are administered at different dosage intervals, or when titration of the individual components of the combination is desired by the prescribing physician.
  • Kras LSL ⁇ G12D mice were from Dr. T. Jacks (MIT, Boston, MA).
  • pl6/plf /fl mice were from Dr. A. Berns (NKI, Netherlands) and Pdxl-Cre mice from Dr. A. Lowy (University of Ohio).
  • G-CSF-K ' mice were obtained from Dr. D. Link (Washington
  • RAG2 ⁇ ' ⁇ mice were purchased from Taconic.
  • Female Nude/Nude Balb/c-mice were from Charles River Laboratory (Hollister, CA). Animals were housed and cared for according to guidelines from the Institutional Animal Care and Use Committee (IACUC) at Genentech, Inc.
  • Mouse lung cancer cells lines LKPH2, LKP9 and LKP10 were isolated from individual Kras induced Lewis lung carcinomas (LLCs).
  • Mouse pancreatic cancer cell lines KPP388 and KPP449 were generated from tumors harvested from Kras G12D/+ ;pl6/pl ⁇ /fl ;pdxCre animals. Tumors were minced and dissociated in RPMI media supplemented with 2.5% FBS and collagenase (0.5 mg/mL). The reaction was quenched with 0.02% EDTA after 20 minute incubation at room temperature.
  • the dissociated cells were passed through a 70-100 micron filter, pelleted and rinsed with RPMI supplemented with 2.5% FBS twice. Cells were sorted twice for EpCam positivity utilizing fluorescence activated cell sorter. Tumor cells were cultured in IMDM. Human cancer lines were from the ATCC and were grown in DMEM (Invitrogen, Carlsbad, CA). Media were supplemented with 10% FBS (Sigma, St. Louis, MO). Cells were cultured and maintained at 37°C in a 5% C02, 80% humidity incubator.
  • Isolated tumor cells (1.0 10 7 ) were subcutaneously inoculated in the dorsal flank of nu/nu immunodeficient mice. Antibodies were IP injected at various doses as described in the figure legends. Treatments with the anti-VEGF mAb B20-4.1.1 (Liang et al., J. Biol. Chem., 281 :951-61 (2006)), anti-G-CSF mAb from R&D Systems, or MEKi GDC- 0973 were initiated 3 or 6 days after tumor cell inoculation. All tumor growth experiments were performed at least three times and conducted in accordance with the Guide for the Care and Use of Laboratory Animals. An Institutional Animal Care and Use Committee (IACUC) approved all animal protocols.
  • IACUC Institutional Animal Care and Use Committee
  • qRT-PCR quantitative reverse transcription-PCR
  • TaqMan Gene Expression Assay primers and probe mixes were used: Bv8 (assay ID: Mm00450080_ml), mouse G-CSF (assay ID: Mm00438334_ml), mouse Ets2 (assay ID: mm00468972_ml), mouse GAPDH (assay ID: Mm99999915_gl), human G-CSF (hs00738431_gl), human GAPDH (assay ID: hs99999905_ml). Analyses were carried out on a standard ABI 7500 machine (Invitrogen) according to the manufacturer's recommended protocols. Immunoblotting
  • Tumor cells were seeded at a density of 5x105 per 2ml of 10% FBS plus
  • Protein samples were separated on precast NuPAGE Novex 4%-12% Bis-Tris gradient gels (Invitrogen), transferred onto a PVDF membrane (Millipore) and incubated overnight with one of the following primary antibodies: phospho-ERKl/2, ERK1/2, AKT, pAKT (Cell Signaling); Ets2, BRAF, or Actin (Santa Cruz Biotechnologies); Membranes were incubated with secondary HRP antibodies for 1 hr followed by signal detection detected using a western blotting detection system (GE Healthcare Bio- Science).
  • Pancreatic tumor volumes were estimated using in vivo high-resolution micro- ultrasound imaging. Imaging was performed with a Visualsonics Vevo2100 microimaging system employing an array transducer (MS550D) with a 40 MHz center frequency, 12 x 10 mm fief d-of- view (FOV), axial resolution of 40 ⁇ , and lateral resolution of 100 ⁇ .
  • MS550D array transducer
  • FOV d-of- view
  • axial resolution of 40 ⁇ axial resolution of 40 ⁇
  • lateral resolution 100 ⁇ .
  • Red blood cells were lysed using ACK (Lonza, Basel, Switzerland) lysis buffer, followed by staining with anti-mouse CD1 lb (BD Biosciences, San Jose, CA) conjugated to fluorescein isothiocyanate rat (FITC), anti-Ly6G conjugated to phycoerythrin (Clone 1A8).
  • aSMA+CD105+CD31- cells were sorted twice for CD 105 positivity and CD31 negativity utilizing fluorescence activated cell sorter. Data were acquired in the FACS instrument (BD Biosciences, San Jose, CA) and analyzed BD Biosciences software.
  • Tumor samples were lysed in RIPA buffer and total protein contents were measured by the Bradford method, according to the manufacturer's protocol (Pierce).
  • Levels of G-CSF, GM- CSF, IL-6, IL-17, M-CSF, PDGF-AB, P1GF, TNFa, IL1B, KC in tumors lysates and/or animals plasma were measured using ELISA Kits from R&D Systems or Invitrogen Inc., for G-CSF, according to the manufacturer's instruction.
  • Bv8 concentrations were measured by ELISA.
  • Luminex assays were performed using Luminex multiplex system (BioRad).
  • 4T07 and 4T1 were shown to express high G-CSF levels whereas the non-metastatic 67NR and 168FARN cells expressed undetectable G-CSF levels.
  • a G-CSF promoter-driven luciferase reporter was expressed in the 4Tl-related cell lines. Strong luciferase activation was detected in 4T07 and 4T1 but not in 67NR or 168FARN cells ( Figure 1 A). Potential transcriptional factors binding sites were identified approximately 500 base pair upstream of the ATG initiation codon.
  • Ets2 mRNA levels were significantly higher in 4T1 cells compared to 67NR (>5fold). Furthermore, expression of Ets2 was shown to increase G-CSF protein and RNA copy numbers in 4T1 cells (Figure 1C). To assess the role of Ets2 in G-CSF gene expression, shRNA was expressed to silence Ets2 transcription. As illustrated in Figure ID, down-regulation of Ets2 was directly correlated with reduction in G-CSF expression in 4T1 cells. To assess the role of Ets2 in G-CSF gene expression, shRNA was expressed to silence Ets2 transcription. As illustrated in Figure ID, down-regulation of Ets2 was directly correlated with reduction in G-CSF expression in 4T1 cells. To
  • MEKi MEK inhibitor GDC-0973/XL518
  • Figure 3A shows the structure of this compound.
  • EGF could also increase G-CSF release in LLC, although it resulted in a lower induction (Figure 3B).
  • FGFs stimulation resulted in FGFRs phosphorylation and activation of the RAS signaling pathway. Accordingly, ERK phosphorylation was readily detected when LLC cells were stimulated with the different FGFs.
  • MEKi strongly inhibited ERK phosphorylation in the presence of FGFs ( Figure 3C).
  • different FGFs were incubated with MEKi or DMSO and FGFs-induced G-CSF expression were assessed in these cells.
  • FGF-induced G- CSF expression was inhibited when LLC cells were treated with MEKI compared to DMSO-treated controls (Figure 3C).
  • FGFs were also tested for their ability to induce G- CSF release in 168FARN, a cancer cell line that, as noted above, does not express G-CSF. Stimulations of 168FARN cells with the different FGFs resulted in high G-CSF release ( Figure 3D) or expression ( Figure 3E). Similarly, MEKi treatment resulted in significant G-CSF inhibition ( Figure 3D-E).
  • mice receiving MEKi or combination of MEKi + anti-VEGF or anti-G-CSF + anti-VEGF treatments had a significant reduction in both G-CSF and Bv8 levels in the plasma compared to anti-VEGF or control groups ( Figures 4A and 4B).
  • mice that received either MEKi or anti-G-CSF had significantly fewer circulating Cdl lb + Ly6G + cells ( Figure 4C), and reduced peripheral white blood cells count ( Figure 4D) compared to control or anti-VEGF treated groups.
  • mice that received combination treatment of MEKi + anti-VEGF or anti-G-CSF + anti-VEGF had significantly decreased tumor and spleen weights. Analysis of tumor growth showed that 4T1 tumors are indeed resistant to anti-VEGF, anti-G-CSF or MEKi as monotherapy. However, combination treatment of MEKi + anti-VEGF or anti-G-CSF + anti-VEGF significantly reduced tumor growth compared to anti-ragweed treated group (Figure 4E).
  • G-CSF and Bv8 plasma levels in LLC tumor bearing mice were directly correlated with the recruitment and mobilization of CD1 lb + Grl + cells and tumor resistance to anti-VEGF antibodies.
  • This experiment tests whether MEKi or anti-G-CSF antibody treatment could inhibit G-CSF release and G-CSF-induced Cdl lb + Ly6G + myeloid cells mobilization in LLC tumor-bearing mice. Animals were treated with the different drug regimens as indicated in Figure 5E.
  • MEKi, anti-G-CSF, combination of MEKi plus anti-VEGF or anti-G-CSF plus anti-VEGF treatment significantly reduced G-CSF and Bv8 levels (Figure 5A and 5B) compared to anti-ragweed or anti-VEGF treated groups.
  • Cdl lb + Ly6G + cells were significantly decreased in the peripheral blood (Figure 5C), accompanied by marked reduction in total white blood cell counts ( Figure 5D).
  • MEKi treatment resulted in approximately 50% inhibition in tumor growth, whereas anti-G-CSF resulted in approximately 25% inhibition compared to anti-ragweed control group (Figure 5E).
  • mice and human tumor cell lines with activation of RAS pathway were examined as to whether they have enhanced G-CSF expression.
  • MEKi treatment inhibited G-CSF expression in a dose- dependent manner ( Figure 6A).
  • PI3Ki treatment had no effect, again confirming that the PI3K pathway does not control G-CSF expression.
  • BRAFi GDC-0879 (luM) was found to further increase ERK phosphorylation and induce G-CSF expression in LKPH2, LKP9 or LKP10 cells ( Figure 6A).
  • 31 human cancer cell lines representing 6 different cancer types were screened for G-CSF expression. As shown in Table 1, 13 out of 31 (approximately 42%) express G- CSF in a RAS/RAF/MEK pathway activation-dependent manner. Eight have mutations in KRAS (Calu-1, Calu-3, Calu-6, EBC-1, HCC-15, SW1463, H2122, MDA-MB231). The bladder cell line BFTC-095 has NRAS mutation. Three cell lines possess receptor tyrosine kinase amplifications or mutations that lead to activation of the RAS pathway, as measured by ERK phosphorylation.
  • EGFR mutation in the HI 975 lung cancer cell line include EGFR mutation in the HI 975 lung cancer cell line, EGFR and FGF amplification in the 5637 bladder cancer cell line and EpoR amplification the H838 lung carcinoma cell line.
  • the remaining cell line expressing G-CSF (UM-UC-1) is responsive to MEKi (Fig. 6B).
  • Colon cancer Colon cancer ;Colo201 BRAF (V600E) No 1 0
  • Example 7 Combined inhibition of MEK or G-CSF with Anti-VEGF Therapy
  • Pancreatic ductal adenocarcinoma the predominant form of pancreatic neoplasm, remains one of the most aggressive and lethal malignacies. It is recently ranked as the fourth-leading cause of cancer death in the US with a median survival of less than 6 months and an average 5-year survival rate of less than 5%. Hidalgo, New Eng. J. Med. 362: 1605-17 (2010). Its lethal nature is largely due to its propensity to rapidly disseminate to the lymphatic system and distant organs. PDAC is known to be resistant to many conventional and targeted therapies, making it almost incurable at the time of diagnosis.
  • GEMM Genetically engineered mouse model
  • GEMM Kras LSL ⁇ G12D ; pl6/pl ⁇ ' fl ;Pdx-Cre
  • Human PDACs have a large component of stromal cells (6), including alpha-smooth muscle actin (aSMA)- positive myofibroblast-like stellate cells.
  • aSMA alpha-smooth muscle actin
  • mouse PDAC tumors were stained positively for aSMA. Since the stroma has been proposed to be responsible for PDAC pathogenesis and resistance to chemotherapeutic treatments, it is hypothesized that FGFs could stimulate aSMA+ cells to release G-CSF.
  • aSMA/CD105 double positive
  • myofibroblast-like cell fractions (29) that are negative for CD31 were purified to exclude endothelial cell contamination from tumors. Antibody staining confirmed that these cells express aSMA and CD 105 and are negative for CD31. Incubation of aSMA+CD105+CD31- cells with FGFs resulted in G-CSF release in a MEK-dependent manner.
  • CDl lb+Grl+ myeloid cells are mixed population of cells, consisting of immature dendritic cells, early myeloid progenitors, Ly6C+ granulocytic monocytes and Ly6G+ neutrophils.
  • CDl lb+Grl+ myeloid population drives resistance to anti-VEGF therapy, antibodies that specifically recognize Ly6G+ neutrophils and Ly6C+ monocytes were used, as well as G-CSF-R'RAGT'- mice, which exhibit reduced Ly6G+ neutrophil population.
  • naive G-CSF-K 1' RAG2 ⁇ ' ⁇ mice have a significant reduction in CDl lb+Ly6G+ neutrophils when compared to G-CSF-R +/+ RA GT'- mice, but show no significant differences in the percentages of CDl lb+Ly6C+ monocytes.
  • KPP388 cells generated from primary tumors of the Kras-driven PDAC GEMM, were subcutaneously implanted into immunodeficient G- CSF-R +/+ RA G2 ⁇ ⁇ and G-CSF-R'RA GT' ' animals.
  • mice Four days after implantation, mice were treated with either anti- Ragweed or anti-VEGF (B20-4.1.1) antibodies and tumor volumes were measured.
  • Anti-VEGF treatment had little effect on tumor growth in wild-type G- CSF-R +/+ RAG2 ⁇ F ⁇ icQ (Fig. 9A).
  • CDl lb+Ly6G+ neutrophil reduction alone was not sufficient at reducing tumor growth.
  • anti-VEGF antibody treatment
  • CDl lb+Ly6G+ neutrophil subpopulation can serve as a biomarker for tumors resistant to anti-VEGF therapy (Fig. 9B).
  • VEGF or anti-G-CSF and anti-VEGF were investigated in a PDAC allograft mouse model.
  • Anti-G-CSF alone had no significant effect on tumor growth (Fig. 9C), despite reducing CDl lb+Ly6G+ neutrophils (Fig. 9E).
  • MEKi GDC-0973 plus anti-G-CSF combination treatment significantly reduced CDl lb+Ly6G+ neutrophils but had no added effect on tumor growth when compared to GDC-0973 alone.
  • MEKi GDC-0973 and anti-VEGF or anti-G-CSF and anti-VEGF could prolong overall survival (OS) in the Kras LSL-G12D; P 16/pl9fl/fl;Pdx-Cre PDAC GEMM.
  • the dynamics of the myeloid cell subpopulations in the PDAC GEMM were first examined at day 7 post drug treatments (Fig. IOC and 10D).
  • Inhibition of G-CSF with either MEKi GDC-0973 or anti-G-CSF significantly reduced CDl lb+Ly6G+ neutrophils (Fig. IOC) in the peripheral blood.
  • neutralizing G-CSF did not have a significant effect on the
  • CDl lb+Ly6C+ monocyte are not included in the GCSF-induced myeloid cell mobilization in the PDAC GEMM.
  • the cohorts were first stratified by performing G-CSF ELISA and micro-ultrasound analysis. Consistent with the allograft studies above, PDAC GEMM cohorts that received GDC-0973 or anti-G- CSF as single agent had no significant survival benefit, despite a marked reduction in the CDl lb+Ly6G+ neutrophil population (Fig. 10B). The data confirmed that the PDAC GEMM is resistant to anti-VEGF monotherapy, which is consistent with previous reports. In contrast, combination therapies significantly improved median survival.
  • High-resolution micro-ultrasound imaging was also performed to measure tumor volumes in the cohorts and calculated the daily fold change in the treated animals.
  • Anti-G-CSF plus anti-VEGF or MEKi GDC-0973 plus anti-VEGF combination therapy resulted in significantly slower tumor growth as compared to single treatment arms (Fig. 10B).
  • the Kras-driven PDAC GEMM was also used to determine whether targeting MEK activation could inhibit G-CSF release in anti-VEGF resistant PDAC.
  • PDAC tumor-bearing mice were found to have high G-CSF plasma levels relative to naive wild-type animals (Fig. 12B).
  • cytokines and growth factors released in the plasma of PDAC mice were profiled and compared to MEKi-treated or naive wild-type animals.
  • significant increases in the levels of inflammatory growth factors and cytokines including bFGF, TNFa, GM-CSF, KC, IL17, and IL-lb were measured in the plasma of PDAC tumor-bearing mice (Fig. 12B).

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Abstract

La présente invention concerne d'une manière générale l'inhibition de la croissance tumorale. En particulier, l'invention concerne la prévention ou le traitement d'angiogenèse de tumeur et l'inhibition de la croissance tumorale dans des tumeurs résistant au traitement anti-VEGF, à l'aide d'inhibiteurs de MEK ou d'antagonistes de G-CSF, soit individuellement soit en combinaison avec un antagoniste de VEGF.
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