WO2013082511A1 - Methods for overcoming tumor resistance to vegf antagonists - Google Patents

Abstract

The present invention relates generally to the inhibition of tumor growth. In particular, the invention concerns the prevention or treatment of tumor angiogenesis and the inhibition of tumor growth in tumors resistant to anti-VEGF treatment, using MEK inhibitors or G-CSF antagonists, either alone or in combination with a VEGF antagonist.

Description

METHODS FOR OVERCOMING TUMOR RESISTANCE TO VEGF ANTAGONISTS

RELATED APPLICATION

[0001] This application claims the benefit of the U.S. Provisional Patent Application

Serial No. 61/566,428, filed on December 2, 2011, which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

[0002] The present invention relates generally to the field of molecular biology and clinical oncology. In particular, the invention concerns compositions and methods effective for overcoming tumor resistance to treatment with VEGF antagonists.

BACKGROUND OF THE INVENTION

[0003] It has been well established that 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. Folkman et al., J. Biol. Chem., 267: 10931-10934 (1992); Klagsbrun et al, Annu. Rev. Physiol, 53: 217-239 (1991).

[0004] In the case of tumor growth, 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. Folkman et al., Nature, 339: 58 (1989). 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. Some tumor cells then switch to the angiogenic phenotype to activate endothelial cells, which proliferate and mature into new capillary blood vessels. These newly formed blood vessels not only allow for continued growth of the primary tumor, but also for the dissemination and recolonization of metastatic tumor cells. Accordingly, a correlation has been observed between density of microvessels in tumor sections and patient survival in breast cancer as well as in several other tumors. Weidner et al., N. Engl. J. Med, 324: 1-6 (1991); Horak et al., Lancet, 340: 1120-1124 (1992); Macchiarini et al, Lancet, 340: 145-146 (1992).

[0005] Many molecules, mostly secreted factors produced by tumor or surrounding stromal cells, have been identified as proangiogenic factors involved in various stages of tumor angiogenesis. The most potent and best characterized angiogenic factor is vascular endothelial growth factor (VEGF), a growth factor secreted by both tumor and stromal cells. 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. Invest., 91 : 153-159 (1993); Brown et al., Human Pathol., 26: 86-91 (1995); Brown et al, Cancer Res., 53: 4727-4735 (1993); Mattern et al, Brit. J. Cancer, 73: 931-934 (1996); Dvorak et al, Am. J. Pathol, 146: 1029-1039 (1995). In addition to being an angiogenic factor in angiogenesis and vasculogenesis, 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. Ferrara and Davis-Smyth, Endocrine Rev., 18:4-25 (1997). Moreover, studies have reported mitogenic effects of VEGF on a few non-endothelial cell types, such as retinal pigment epithelial cells, pancreatic duct cells and Schwann cells. See, e.g., Guerrin et al, J. Cell Physiol, 164:385-394 (1995); Oberg- Welsh et al, Mol. Cell. Endocrinol,

126: 125-132 (1997); and, Sondell et al, J. Neurosci., 19:5731-5740 (1999).

[0006] Recognition of VEGF as a primary regulator of angiogenesis in pathological conditions has led to numerous attempts to block VEGF activities. Inhibitory anti-VEGF receptor antibodies, soluble receptor constructs, antisense strategies, RNA aptamers against VEGF and low molecular weight VEGF receptor tyrosine kinase (RTK) inhibitors have all been used in various preclinical models to inhibit tumor angiogenesis and tumor growth. See, e.g., Siemeister et al, Cancer Metastasis Rev., 17:241-248 (1998); Cao, Nat. Rev. Clin.

Oncol, 7:604-8 (2010). The effect of anti-VEGF strategy has been further validated in randomized clinical trials as well. Disruption of VEGF signaling pathway by using bevacizumab, a humanized anti-VEGF antibody or sorafenib and sunitinib, two small molecule inhibitors of receptor tyrosine kinases (RTK) including VEGF receptors have yielded a significant improvement in progression- free survival for patients with several types of cancers including metastatic colorectal cancer, advanced non-small cell lung cancer, metastatic breast cancer, advanced clear-cell renal cell carcinoma and advanced hepatocellular carcinoma. Hurwitz et al, N. Eng. J. Med., 350:2335-42 (2004); Sandler et al., N. Engl. J. Med., 355:2542-50 (2006); Miller et al, N. Engl. J. Med., 357:2666-76 (2007); Escudlier et al, N. Engl. J. Med., 356: 125-34 (2007); Motzer et al, N. Engl. J. Med., 356: 115-24 (2007); Llover et al, N. Engl. J. Med., 359:378-90 (2008).

[0007] 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). Thus, tumor resistance to VEGF inhibition has become a major obstacle to the effectiveness of antiangiogenic agents in the clinic. Cao, Adv. Cancer Res., 100: 113-31 (2008); Li et al., Cancer Res., 71 :6073-83 (2011).

[0008] Intensive studies are underway to elucidate cellular and molecular

mechanisms underlying reduced response to VEGF inhibitors. Shojaei and N. Ferrara, Cancer Res., 68: 5501-5504 (2008); Shojaei et al, Trends in Cell Biol, 18:372-8 (2008); Li et al., ibid (2011). Traditionally, tumor cells have been thought to be the major source of angiogenic factors. However, compelling evidence now supports the notion that the tumor stroma, composed of numerous cell types including fibroblasts, infiltrating immune cells, endothelial cells and pericytes, can also facilitate tumor development and metastasis through a variety of mechanisms, de Visser et al., Nat. Rev. Cancer, 6:24-37 (2006); Hahn and Weinberg, Nat. Rev. Cancer, 2:331-41 (2002); Shchors and Evan, Cancer Res., 67:7059-61 (2007); Yu et al, Nat. Rev. Cancer, 9:798-809 (2009).

[0009] Withinin 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). Furthermore, subsets of CD1 lb+ Grl+ cells, termed myeloid- derived suppressor cells (MDSCs), have the ability to suppress T-cell responses and thus can promote tumor progression and escape from immune surveillance. Gabrilovich and Nagaraj, Nat. Rev. Immunol, 9: 162-74 (2009); Talmadge, Semin. Cancer Biol, 21 : 131-8 (2011).

[0010] Previous studies have demonstrated that recruitment and mobilization of

CD1 lb+Grl+ cells to the tumor stroma mediates tumor refractoriness to anti-VEGF therapy in several murine models. Shojaei et al., Nat. Biotech., 25:911-20 (2007). While a variety of pro-inflammatory cytokines and growth factors might be responsible for mobilization of CD1 lb+Grl+ myeloid cells, the key hematopoietic growth factor granulocyte colony- stimulating factor (G-CSF or GCSF) has been shown to be the major mediator of expansion and mobilization of these cells. G-CSF also up-regulates the proangiogenic factor Bv8.

Shojaei et al., ibid (2009). G-CSF-mobilized CD1 lb+ Grl+ produce a variety of factors that facilitate primary tumor growth and metastasis, including MMP9, S100A8 and S100A9.

Kowanetz et al, PNAS USA, 107:21248-55 (2010). Also, anti-G-CSF treatment resulted in a dramatic reduction in CD1 lb⁺Grl⁺ cells and Bv8 expression in tumor and plasma of tumor- bearing mice. In human tumors, elevated G-CSF expression have been reported in plasma from patients with a variety of solid tumors and severe leukocytosis, and are often associated with a poor prognosis. Beekman and Touw, Blood, 115:5131-6 (2010); Hasegawa et al, Ann. Thorac. Surg., 83:308-10 (2007); Kawaguchi et al, Int. J. Clin. Oncol, 15: 191-5 (2010);

Yamamoto et al, J. Gastroenterol, 34:640-4 (1999). The precise signaling transduction pathways regulating G-CSF in cancer cells have not been elucidated.

[0011] 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.

[0012] It has been demonstrated that MEK is a critical effector of Ras function. The

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.

[0013] Many small molecule 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. In addition to inhibition of tumor proliferation, some MEK inhibitors, e.g., 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).

[0014] In view of the role of angiogenesis in neoplastic diseases and the clinical value of anti-VEGF therapies, there exists a need for novel therapeutic approaches to overcome tumor resistance to VEGF antagonists. All references cited herein, including patent applications and publications, are incorporated by reference in their entirety.

SUMMARY OF THE INVENTION

[0015] 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. Not wishing to be bound by theory, it is suggested that such 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.

[0016] In certain aspects, 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.

[0017] In one embodiment, a method of sensitizing tumor to treatment with a VEGF antagonist is contemplated, such method 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.

[0018] In another embodiment, a method of enhancing tumor's response to treatment with a VEGF antagonist is contemplated, 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.

[0019] 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. In one aspect, the cells capable of inducing tumor angiogenesis are derived from bone marrow of the human subject. For example, the cells are of hematopoietic lineage, such as those of myeloblast sub lineage. In one aspect, 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. In one aspect, these cells are responsive to stimulation by certain growth factors such as G-CSF and express and release pro-angiogenic factors such as Bv8. In one aspect, the method of the invention is directed to inhibiting tumor angiogenesis induced by human granulocytes or subset thereof such as CDl lb+/Ly6G+ neutrophils. In one aspect, 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.

[0020] In another embodiment, a method of inhibiting proliferation and migration of cells in tumor stroma is contemplated, such method 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. In some aspects, the cells in tumor stroma are derived from bone marrow, are of hematopoietic lineage or subsets thereof as decribed above. In another aspect, the cells in tumor stroma are tumor-associated fibroblast cells.

[0021] In one embodiment, the VEGF antagonist in the methods of the invention is an anti-VEGF antibody or functional fragment thereof, such as bevacizumab.

[0022] In another embodiment, the MEK inhibitor useful in the methods of the invention is a small molecule compound or a pharmaceutically acceptable salt thereof. In one aspect, 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. In another aspect, the small molecule compound is an azetidine compound. Examples of azetidine compounds capable of inhibiting MEK are described in, e.g., US Pat No. 7803839; WO2011/054620. In yet another aspect, the small molecule compound is an imidazopyridine compound. Examples of azetidine compounds capable of inhibiting MEK are described in, e.g., WO2009/085983. In one aspect, the MEK inhibitor of the invention is the compound GDC-0973 or GDC-0987.

[0023] In one aspect, the human subject in the methods of the invention has been previously treated with chemotherapy, a VEGF antagonist or both. In one aspect, the human subject has been previously treated with bevacizumab but the tumor therein has relapsed.

[0024] In some embodiments, the methods contemplated herein further comprise administering to the human subject 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. In some aspects, especially when sensitizing tumor for anti-VEGF therapy is desired, 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.

[0025] In one embodiment, a combination therapy using bevacizumab and GDC-0973 is contemplated. In another embodiment, a combination therapy using anti-G-CSF antibody and bevacizumab is contemplated.

[0026] In some aspects, the methods of the invention are further combined with chemotherapy or radiation therapy.

[0027] 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. In one aspect, the tumor is in the colon, rectum, liver, lung, prostate, breast, bladder, skin, brain, thyroid, pancreas or ovary of the human subject. In some aspects, 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. In one aspect, 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.

[0028] In some embodiments, 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. In other embodiments, 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. In still other embodiments, 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.

[0029] In another aspect, 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.

BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 depicts the transcriptional regulation of G-CSF expression in cancer cells constitutively expressing G-CSF. (1A) 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. (IB) Ets2 directly regulates G-CSF expression. Site-directed mutagenesis of Ets2 transcriptional binding sites - 231 and -101 prior to G-CSF ATG start codon drastically reduces luciferase activity in 4T1 cells. *p <0.001; wildtype (WT), mutated binding sites (M1-M3). (1C) Enforced expression of Ets2 further increases G-CSF expression in 4T1 cells. G-CSF protein levels were assessed by immunoblot and RNA expression was measured by quantitative PCR. *p<0.000003. (ID) shRNA targeting Ets2 expression reduces G-CSF levels in 4T1 cells. shRNAs targeting Ets2 or shRNAs control were transfected into 4T1 cells. Ets2 and G-CSF expressions were detected by quantitative PCR. *p <0.01. (IE) ChIP analysis reveals that Ets2 transcription factor directly binds to G-CSF promoter in 4T1 cells compared to 67NR cells. *p <0.03. (IF) Dominant-negative Ets2 inhibits G-CSF-promoter driven luceferase reporter. Luceferase activity is detected in 4T1 cells co-expressing either GFP plus Ets2 wildtype (Ets2) or GFP plus dominant negative Ets2 (Ets2DN) that has truncation at the N-terminus. *p<1.0xl0^~5.

[0031] Figure 2 depicts the signaling mechanisms controlling G-CSF expression in cancer cells. (2A) RAS signaling pathway activation correlates with increased G-CSF levels. Immunoblot analysis shows BRAF and ERK phosphorylations in 4T1 compared to 67NR cell lines. (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. (2D) The RAF/MEK pathway regulates G- CSF release in 4T1 cells. ELISA shows the effects of MEKi and Pi3K inhibitor LY294002 (Pi3Ki) on G-CSF release. *p<0.001.

[0032] Figure 3 depicts growth factors that positively regulate G-CSF secretion in

LLC cells or NIH 3T3 fibroblasts in vitro. (3 A) Structure of the MEK inhibitor GDC-0973. (3B) Growth factors enhance G-CSF expression. G-CSF levels were measured by ELISA after 12hrs of stimulation (n=3/group, *p<0.001). Data are representative of at least two independent experiments. (3C) Growth factor-induced G-CSF release in LLC cells is dependent on MAPK activation, *p<0.001. (3D-E) MEKi GDC-0973 blocks FGF induced G-CSF in mouse breast cell line (168FARN). G-CSF release and RNA expression are shown, *p<0.01. (3F) A subset of growth factors enhance G-CSF expression in mouse fibroblasts NIH 3T3 in a MAPK activation-dependant manner. MEKi effectively blocks growth factor- induced G-CSF expression in fibroblasts.

[0033] Figure 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. (4C) MEKi reduces Cdl lb+Ly6G+ cells in the peripheral blood of 4T1 tumor-bearing mice, n=5 mice/group. *p<0.001. (4D) MEKi reduces total white blood cells in the peripheral blood of 4T1 tumor-bearing mice, n=5 mice/group. *p<0.001. (4E) 4T1 tumor growth in response to MEKi, anti-VEGF, anti-G-CSF or combination treatments. Beige nude mice were transplanted with 4T1 (n=9-10 mice/group). Three days after tumor cell inoculation, different treatments were initiated as indicated in the figure, p-values are relative to control treated group, *p<0.001, **p<0.006 and *** p<2.6xl0^~9. (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).

[0034] Figure 5 depicts effects of MEKi, anti-G-CSF, anti-VEGF antibodies and combinations thereof on LLC tumor growth. (5A) MEKi and anti-G-CSF reduce G-CSF levels in the plasma of LLC tumor bearing mice; n=10 mice/group, *p<0.001. (5B) MEKi and anti-G-CSF reduce Bv8 levels in the plasma of LLC tumor bearing mice, n=10 mice/group, *p<0.001. (5C) FACs analysis of Cdl lb+Ly6G+ cells in peripheral blood of mice implanted with LLC, n=5 mice/group, *p<0.001. (5D) Total white blood cell counts in peripheral blood of LLC tumor bearing mice, n=10 mice/group, *p<0.001. (5E) MEKi plus anti-VEGF, or anti-G-CSF plus anti-VEGF combination treatments reduce growth of LLC tumors compared to monotherapies. Beige nude mice were transplanted with LLC (n=9-10 mice/group) and at day 3 animals were treated with anti-ragweed (aRagweed), GDC-0973 (MEKi), anti-VEGF (aVEGF), anti-G-CSF (aG-CSF) as a single agent or in combination as indicated (data shown is a representative of at least 3 independent experiments, *p<0.001, ** p<0.00001 and *** p<1.0xl0^"10. (5F) MEKi plus anti-VEGF, or anti-G-CSF plus anti-VEGF combination treatments reduce tumor angiogenesis. Treatment groups are indicated in the figure. Tumor sections were stained with anti-CD31 (red). Scale bar is lOOum. (5G)

Quantitative analysis of tumor vascular surface area. Whole tumor cross-sections were stained with CD31 and analyzed as described in Methods (n=5, *p<0.05).

[0035] Figure 6 depicts effects of RAF and MEK inhibitors on G-CSF expression in cancer cells. (6 A) G-CSF is detected in mouse lung cancer cells with KRAS mutation. MEKi GDC-0973 inhibits G-CSF, whereas RAFi GDC-0879 induces G-CSF in mouse lung cell lines with KRAS mutation. Following treatment with DMSO (D), PI3Ki LY-294002 5uM (P), RAFi GDC-0879 luM (R), or GDC-0973 (MEKi: O.OluM, O. luM and l .OuM) for 24hrs, total lysates were analyzed for pAKT and pER . G-CSF release was analyzed by ELISA. (6B) G-CSF release in human cancer cells is dependent on RAS signaling pathway activation. 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.

[0036] Figure 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.

[0037] 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).

[0038] Figure 9 illustrates results from an Anti-VEGF resistant PDAC allograft mouse model. (9 A) 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^~/~. Mice were transplanted with KPP388 PDAC cells (n=8-9 mice/group) and treated with anti-Ragweed (aRAG) control or anti-VEGF (aVEGF). Three days after cell inoculation, tumor volumes were measured at several time points as indicated,

*p<1.0xl0^~u . Error bars indicate SD. (9B) Flow cytometry analysis of peripheral blood for the presence of CD1 lb⁺Ly6G⁺ neutrophils. Data is representative of each group as indicated. Myeloid cells were gated for CD45⁺, followed by analysis with an antibody that specifically recognizes Ly6G⁺ neutrophils. (9C) KPP388 PDAC tumor growth in response to MEKi GDC-0973, anti-VEGF, anti-G-CSF, or combination treatments. Nu/Nu mice were transplanted with KPP388 cells (n=10 mice/group). Three days after tumor cell inoculation, different treatments were initiated as indicated, *p <0.001. Error bars indicate SD. (9D) G-CSF levels in the plasma of KPP388 PDAC tumor-bearing mice; n=10 mice/group, *p<0.001. Error bars indicate SD. (9E) Flow cytometry analysis of peripheral blood of KPP388 tumor-bearing mice were monitored for CD1 lb⁺Ly6G⁺ neutrophils (n=5 mice/group), *p<0.001. Error bars indicate SD. (9F) KPP388 PDAC tumor sections immunostained with anti-CD31 (red). Mice were treated with anti-Ragweed (aRag), GDC- 0973 (MEKi), anti-VEGF (aVEGF), anti-G-CSF (aG-CSF) or combination treatment as indicated. Scale bar, 100 micron. (9G) Quantitative analysis of tumor vascular surface area (microvessel density). Whole tumor cross-sections were stained with anti-CD31 and analyzed as described in Methods (n=4/group), *p<0.05. Error bars indicate SD.

[0039] Figure 10 illustrates results from a Kras-driven PDAC GEMM. (10A)

Kaplan-Meier plots showing overall survival for the different treatment groups. Animals were treated as indicated: Control (anti-Ragweed and/or vehicle), (GDC-0973 (15 mg/kg), aVEGF (10 mg/kg), aG-CSF (50 ug/mouse). Overall survival was assessed by either mortality or severe morbidity. The number of animals per group are shown, *p=0.01.

(10B) Quantification of daily fold changes in tumor burden by treatment regimen with approximate 95% confidence intervals. Tumor growth analysis is based on serial ultrasounds taken at day 0, 7, 14 and 28; *p<0.01. Error bars indicate SD. (IOC) Flow cytometry analysis of peripheral blood for the presence of CD1 lb⁺Ly6G⁺ neutrophils.

Total myeloid cells were gated for CD45⁺ and then quantified for CD1 lb⁺Ly6G⁺

neutrophils. Naive (n=7), aRagweed (n=7), aVEGF (n=10), aG-CSF (n=10), MEKi (n=9), aVEGF+aG-CSF (n=8), and aVEGF+MEKi (n=5); *p=0.0001. Error bars indicate SD. (10D) Flow cytometry analysis of mouse peripheral blood for the presence of

CD1 lb⁺Ly6C⁺ monocytes. Total myeloid cells were gated for CD45⁺. Quantitative analysis of CD1 lb⁺Ly6C⁺ monocytes is presented. Na^'ive (n=7), aRagweed (n=7), aVEGF (n=10), aG-CSF (n=4), MEKi (n=4), aVEGF+aG-CSF (n=3), and aVEGF+MEKi (n=5); * p=0.05. Error bars indicate SD.

[0040] Figure 11 shows G-CSF expression and its correlation with phospho-MEK activation and neutrophil recruitment in human PDAC biopsies. (HA)

Immunohistochemical staining of (11B) G-CSF, (11C) pMEK, (11D) pFGFR, and (HE) neutrophils in human PDAC biopsies. (11F) Quantification of tumors that are double positive for G-CSF and pMEK or G-CSF and pFGFR. Scale bar, 20 micron.

[0041] 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. (12C) Flow cytometry analysis of peripheral blood for the presence of CD 1 lb⁺Ly6G⁺ neutrophils. Myeloid cells were gated for CD45⁺; cells were analyzed with an antibody that specifically recognized Ly6G⁺ neutrophils. Wildtype (Naive), PDAC treated with control (Vehicle) or MEKi GDC- 0973. Quantitative analysis of the CD1 lb⁺Ly6G⁺ neutrophil population is presented.

Na^'ive, n=7; Control and MEKi GDC-0973 (n=5/group), * p=0.003. Error bars indicate SD.

DETAILED DESCRIPTION OF THE INVENTION

[0042] Before describing the present invention in detail, it is to be understood that this invention is not limited to particular compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. As used in this specification and the appended claims, the singular forms "a", "an" and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a molecule" optionally includes a combination of two or more such molecules, and the like.

Definitions

[0043] The term "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer," "cancerous," "cell proliferative disorder," "proliferative disorder" and

"tumor" are not mutually exclusive as referred to herein.

[0044] The terms "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. More particular examples of such cancers include, but not limited to, 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, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, melanoma, superficial spreading melanoma, lentigo maligna melanoma, acral lentiginous melanomas, nodular melanomas, multiple myeloma and B-cell lymphoma (including low grade/follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL;

intermediate grade/follicular NHL; intermediate grade diffuse NHL; high grade

immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and

Waldenstrom's Macroglobulinemia); chronic lymphocytic leukemia (CLL); acute

lymphoblastic leukemia (ALL); hairy cell leukemia; chronic myeloblastic leukemia; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with phakomatoses, edema (such as that associated with brain tumors), Meigs' syndrome, brain, as well as head and neck cancer, and associated metastases. In certain embodiments, 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. In some embodiments, 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.

[0045] The term 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.

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.

[0046] Epithelial cancers generally evolve from a benign tumor to a preinvasive stage

(e.g., carcinoma in situ), to a malignant cancer, which has penetrated the basement membrane and invaded the subepithelial stroma.

[0047] By "dysplasia" is meant any abnormal growth or development of tissue, organ, or cells. In certain embodiments, the dysplasia is high grade or precancerous.

[0048] 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"). In certain embodiments, 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. In certain embodiments, resistant tumor is a tumor that is resistant to anti-VEGF antibody therapy. In one embodiment, the anti-VEGF antibody is bevacizumab. In certain embodiments, resistant tumor is a tumor that is unlikely to respond to a cancer therapy comprising a VEGF antagonist. In certain embodiments, resistant tumor is a tumor that is intrinsically non-responsive or resistant to a cancer therapy comprising a VEGF antagonist. As a non-limiting example, the pancreatic ductal

adenocarcinoma (PDAC) investigated in the present invention is a type of cancer resistant to anti-VEGF therapy.

[0049] The term "tumor that is refractory to" or "tumor's refracteriness to" treatment with a VEGF antagonist 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. In various embodiments, 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.

[0050] 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. For example, certain tumors resistant to anti-VEGF therapy exhibit elevated level of G-CSF expression compared to other tumors that are responsive to anti-VEGF therapy. In one aspect, a tumor that overexpresses G-CSF has a 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.

[0051] 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. In one aspect, the MEK inhibitor is a small molecule compound with defined chemical structure. Non-limiting examples of such compound are further described herein below. In one embodiment of the invention, 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.

[0052] By "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.

[0053] The term "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.

[0054] 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. In particular, they are cells recruited to the tumor stroma and capable of promoting tumor angiogenesis. In one aspect, these cells are responsive to stimulation by certain growth factors such as G-CSF and express and release pro-angiogenic factors such as Bv8.

Characterization of murine CD1 lb+/Grl+ myeloid cells are described in, for example, PCT Application Publications WO2009/039337, WO2011/014750, Shaojaei et al., ibid (2007).

[0055] In general, a polypeptide "variant" (i.e. a variant of any polypeptide disclosed herein) 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. Ordinarily, 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.

[0056] The term "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

translation control sequences, and the like. 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. [0057] 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.

[0058] The term "inhibiting tumor angiogenesis" as used herein 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.

[0059] The term "inhibiting tumor growth" as used herein 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. As used 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. Further, as used herein said decrease in tumor volume is measured by computerized axial tomography (CAT Scan), magnetic resonance imaging (MRI), positron emission tomography (PET), or single-photon emission computed

tomography (SPECT), which are all well-known techniques in the art.

[0060] The terms "VEGF" and "VEGF- A" are used interchangeably to refer to 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. The term "VEGF" or "VEGF -A" also refers to VEGFs from non-human species such as mouse, rat, or primate. Sometimes the VEGF from a specific species is indicated by terms such as hVEGF for human VEGF or mVEGF for murine VEGF. The term "VEGF" is also used to refer to truncated forms or fragments of the polypeptide comprising amino acids 8 to 109 or 1 to 109 of the 165 -amino acid human vascular endothelial cell growth factor. Reference to any such forms of VEGF may be identified in the present application, e.g., by "VEGF (8-109)," "VEGF (1-109)" or

"VEGF₁₆₅." 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.

[0061] 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). 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;

antisense nucleobase oligomers complementary to at least a fragment of a nucleic acid molecule encoding a VEGF polypeptide; small RNAs complementary to at least a fragment of a nucleic acid molecule encoding a VEGF polypeptide; ribozymes that target VEGF; peptibodies to VEGF; and VEGF aptamers. In one embodiment, 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. In another embodiment, the VEGF inhibited by the VEGF antagonist is VEGF (8-109), VEGF (1-109), or VEGFi₆₅.

[0062] The term "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. For example, 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,

WO2005/044853; ; EP 0666868B1; US Patent Applications 20030206899, 20030190317, 20030203409, 20050112126, 20050186208, and 20050112126; Popkov et al, Journal of Immunological Methods 288: 149-164 (2004); and WO2005012359. 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 BIAcore™ assay as described in PCT Application Publication No. WO2005/012359); enzyme-linked

immunoabsorbent assay (ELISA); and competition assays (e.g. RIA's), for example. The antibody may be subjected to other biological activity assays, e.g., in order to evaluate its effectiveness as a therapeutic. Such 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). An anti-VEGF antibody 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. In one embodiment, 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

"AVASTIN^®." 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.

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.

[0063] In any of the methods, uses and compositions provided herein, the anti-

VEGF antibody may be substituted with a VEGF specific antagonist, e.g., a VEGF receptor molecule or chimeric VEGF receptor molecule as described herein. In certain embodiments of the methods, uses and compositions provided 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.

7,060,269, 6,582,959, 6,703,020; 6,054,297; WO98/045331; WO98/045332; WO 96/30046; WO94/10202; EP 0666868B1; U.S. Patent Application Publication Nos. 2006009360, 20050186208, 20030206899, 20030190317, 20030203409, and 20050112126; and Popkov et al, Journal of Immunological Methods 288: 149-164 (2004).

[0064] A "G6 series antibody" according to this invention, 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. In one embodiment, the G6 series antibody binds to a functional epitope on human VEGF comprising residues F17, Y21, Q22, Y25, D63, 183 and Q89.

[0065] 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.

WO2005/044853, and US Patent Application 60/991,302, the content of these patent applications are expressly incorporated herein by reference. In one embodiment, 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.

[0066] 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. [0067] The phrase "myeloblast lineages", for the purposes herein, 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).

[0068] 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 . A

Grl+/CD1 lb+ can be selected by immunoadherent panning, for example, with an antibody to Grl+.

[0069] The term "biological sample" refers to a body sample from any animal, but preferably is from a mammal, more preferably from a human. Such 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. The preferred biological sample herein is serum, plasma, urine or a bone marrow sample.

[0070] The term "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.

[0071] "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, a bivalent fragment including two Fab' fragments linked by a disulphide bridge at the hinge region; (ix) single chain antibody molecules (e.g. single chain Fv; scFv) (Bird et al, Science 242:423-426 (1988); and Huston et al, PNAS (USA) 85:5879-5883 (1988)); (x) "diabodies" with two antigen binding sites, comprising a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (see, e.g., EP 404,097; WO 93/11161; and Hollinger et al, Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); (xi) "linear antibodies" comprising a pair of tandem Fd segments (VH-CH1-VH-CH1) which, together with complementary light chain polypeptides, form a pair of antigen binding regions (Zapata et al. Protein Eng.

8(10): 1057 1062 (1995); and US Patent No. 5,641,870).

[0072] As used herein, "treatment" (and grammatical variations thereof such as

"treat" or "treating") 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. In some embodiments, antibodies of the invention are used to delay development of a disease or to slow the progression of a disease. Specifically, 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.

[0073] An "effective amount" of an agent, e.g., a pharmaceutical formulation, refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic, sensitizing or prophylactic result.

[0074] The term "anti-neoplastic composition" refers to a composition useful in treating cancer comprising at least one active therapeutic agent, e.g., "anti-cancer agent." Examples of therapeutic agents (anti-cancer 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. Combinations thereof are also included in the invention. [0075] The term "cytostatic agent" refers to a compound or composition which arrests growth of a cell either in vitro or in vivo. Thus, 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. Further information can be found in Mendelsohn and Israel, eds., The Molecular Basis of Cancer, Chapter 1, entitled "Cell cycle regulation, oncogenes, and antineoplastic drugs" by Murakami et al. (W.B. Saunders, Philadelphia, 1995), e.g., p. 13. The taxanes (paclitaxel and docetaxel) are anticancer drugs both derived from the yew tree. Docetaxel (TAXOTERE®, Rhone- Poulenc Rorer), derived from the European yew, is a semisynthetic analogue of paclitaxel (TAXOL®, Bristol-Myers Squibb). 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., ²¹¹At, ¹³¹1, ¹²⁵1, ⁹⁰Y, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁵³Sm, ²¹²Bi, ³²P 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.

[0076] 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. Thus, the growth inhibitory agent may be one which significantly reduces the percentage of cells in S phase. Examples of 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. Those 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. 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.

[0077] A "chemotherapeutic agent" refers to a chemical compound useful in the treatment of cancer. Examples of 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;

acetogenins (especially bullatacin and bullatacinone); delta-9-tetrahydrocannabinol

(dronabinol, MARINOL®); beta-lapachone; lapachol; colchicines; betulinic acid; a camptothecin (including the synthetic analogue topotecan (HYCAMTIN®), CPT-11

(irinotecan, CAMPTOSAR®), acetylcamptothecin, scopolectin, and 9-aminocamptothecin); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); podophyllotoxin; podophyllinic acid; teniposide; cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin;

spongistatin; 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. Engl, 33: 183-186 (1994)); CDP323, an oral alpha-4 integrin inhibitor; dynemicin, including dynemicin A; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomysins, actinomycin,

authramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, 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®), and deoxy doxorubicin), epirubicin, esorubicin, 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 (UFTORAL®), capecitabine (XELODA®), an epothilone, and 5-fluorouracil (5-FU); 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; folic acid replenisher such as frolinic acid;

aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine;

bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfornithine;

elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan;

lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone;

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., paclitaxel (TAXOL®), albumin-engineered nanoparticle formulation of paclitaxel (ABRAXANETM), and docetaxel (TAXOTERE®); chloranbucil; 6-thioguanine;

mercaptopurine; methotrexate; platinum agents such as cisplatin, oxaliplatin (e.g.,

ELOXATIN®), and carboplatin; 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®), or risedronate (ACTONEL®); 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 (sorafenib; Bayer); SU-11248 (sunitinib, SUTENT®, Pfizer); perifosine, COX-2 inhibitor (e.g. celecoxib or etoricoxib), proteosome inhibitor (e.g. PS341); bortezomib (VELCADE®); CCI-779; tipifarnib (Rl 1577); orafenib, ABT510; Bcl-2 inhibitor such as oblimersen sodium

(GENASENSE®); 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.

[0078] 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);

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; onapristone; anti-progesterones; estrogen receptor down- regulators (ERDs); anti-androgens such as flutamide, nilutamide and bicalutamide; and pharmaceutically acceptable salts, acids or derivatives of any of the above; as well as combinations of two or more of the above.

[0079] The term "cytokine" is a generic term for proteins released by one cell population which act on another cell as intercellular mediators. Examples of such 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;

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 (TPO); 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-CSF); interleukins (ILs) such as IL-1, IL-lalpha, IL-lbeta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL- 18, IL-19, IL-20-IL-30; secretoglobin/uteroglobin; oncostatin M (OSM); a tumor necrosis factor such as TNF-alpha or TNF-beta; and other polypeptide factors including LIF and kit ligand (KL). As used herein, the term cytokine includes proteins from natural sources or from recombinant cell culture and biologically active equivalents of the native sequence cytokines.

[0080] 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. For example, 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. It would also include 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, and TGF-a and TGF-β. See, e.g., Klagsbrun and D'Amore, Annu. Rev. Physiol, 53:217-39 (1991); Streit and Detmar, Oncogene, 22:3172-3179 (2003); Ferrara & Alitalo, Nature Medicine 5(12): 1359-1364 (1999); Tonini et al, Oncogene, 22:6549-6556 (2003) (e.g., Table 1 listing angiogenic factors); and, Sato /nt. J. Clin. Oncol, 8:200-206 (2003).

[0081] 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. For example, 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. Physiol, 53 :217-39 (1991); Streit and Detmar, Oncogene, 22:3172- 3179 (2003) (e.g., Table 3 listing anti-angiogenic therapy in malignant melanoma); Ferrara & Alitalo, Nature Medicine 5(12): 1359-1364 (1999); Tonini et al, Oncogene, 22:6549-6556 (2003) (e.g., Table 2 listing antiangiogenic factors); and, Sato Int. J. Clin. Oncol, 8:200-206 (2003) (e.g., Table 1 lists Anti-angiogenic agents used in clinical trials).

[0082] The term "immunosuppressive agent" as used herein 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. 4,665,077); 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; 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 (etanercept), anti-tumor necrosis factor-beta antibodies, anti- interleukin-2 antibodies and anti-IL-2 receptor antibodies; anti-LFA-1 antibodies, including anti-CDl la and anti-CD 18 antibodies; anti-L3T4 antibodies; heterologous anti-lymphocyte globulin; pan-T antibodies, preferably anti-CD3 or anti-CD4/CD4a antibodies; soluble peptide containing a LFA-3 binding domain (WO 1990/08187 published Jul. 26, 1990); streptokinase; TGF-beta; streptodornase; RNA or DNA from the host; FK506; RS-61443; deoxyspergualin; rapamycin; 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); and T-cell-receptor antibodies (EP 340,109) such as T10B9.

[0083] Administration "in combination with" one or more further therapeutic agents includes simultaneous (concurrent) and consecutive administration in any order.

[0084] "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.

[0085] An "effective amount" of an agent, e.g., a pharmaceutical formulation, refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic, sensitizing or prophylactic result.

[0086] As used herein, the expressions "cell," "cell line," and "cell culture" are used interchangeably and all such designations include progeny. Thus, 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. The term "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

[0087] Many 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. For example, 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. In one embodiment of the invention, 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. In another embodiment, the MEK inhibitor is one of the imidazopyridine compounds such as those described in WO09/085983. In yet another embodiment, 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.

Uses of the Compositions and Methods

[0088] 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.

[0089] Examples of tumors or neoplastic conditions are described herein under the terms "tumor", "cancer" and "cancerous." In one embodiment, the neoplastic condition is characterized by pathological disorder associated with aberrant or undesired angiogenesis that is resistant to VEGF antagonist treatment.

[0090] 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. In one embodiment of the invention, anti-cancer agent or therapeutic is an anti-angiogenic agent. In another embodiment, anti-cancer agent is a chemotherapeutic agent.

[0091] Many anti-angiogenic agents have been identified and are known in the arts, including those listed herein, e.g., listed under Definitions, and by, e.g., Carmeliet and Jain, Nature 407:249-257 (2000); Ferrara et al, Nature Reviews :Drug Discovery, 3:391-400 (2004); and Sato to. J. Clin. Oncol, 8:200-206 (2003). See also, US Patent Application US20030055006. In one embodiment, 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. Alternatively, or additionally, two or more

angiogenesis inhibitors may optionally be co-administered to the patient in addition to VEGF antagonist and other agent of the invention. In certain embodiment, 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.

[0092] In certain aspects, 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. A variety of 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."

[0093] As will be understood by those of ordinary skill in the art, 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. [0094] 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

therapy/immunotherapy, anti-VEGF antibody therapy, particularly a standard therapeutic regimen for the particular cancer) is not clinically adequate to treat the patients or the patients are no longer receiving any beneficial effect from the therapy such that these patients need additional effective therapy. As used herein, the phrase can also refer to a condition of the "non-responsive/refractory" patient, e.g., which describe patients who respond to therapy yet suffer from side effects, develop resistance, do not respond to the therapy, do not respond satisfactorily to the therapy, etc. In various embodiments, 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.

[0095] 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. In certain embodiments, the MEK inhibitor can be administered subsequent to the cancer therapeutic. In certain embodiments, the MEK inhibitors of the invention are administered simultaneously with cancer therapy, e.g., chemotherapy. Alternatively, or additionally, 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. In one embodiment, the cancer therapy is a combination treatment with an anti-angiogenesis agent, e.g. , a VEGF antagonist. The anti-angiogenesis agent includes those known in the art and those found under the Definitions herein. In one embodiment, 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. Patents 6,582,959, 6,884,879, 6,703,020; W098/45332; WO 96/30046; WO94/10202; EP 0666868B1; US Patent Applications 20030206899, 20030190317, 20030203409, and 20050112126; Popkov et al, Journal of Immunological Methods 288: 149-164 (2004); and, WO2005012359. 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.

Pharmaceutical Compositions and Formulations

[0096] Pharmaceutical 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.

[0097] The 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.

[0098] The compositions of the present invention may also exist in different tautomeric forms, and all such forms are embraced within the scope of the invention. The term "tautomer" or "tautomeric form" refers to structural isomers of different energies which are interconvertible via a low energy barrier. For example, proton tautomers (also known as prototropic tautomers) include interconversions via migration of a proton, such as keto-enol and imine-enamine isomerizations. Valence tautomers include interconversions by reorganization of some of the bonding electrons.

[0099] Pharmaceutical 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. Similarly, 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.

[0100] Pharmaceutical 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 ²H, ³H, ^UC, ¹³C, ¹⁴C, ¹³N, ¹⁵N, ¹⁵0, ¹⁷0, ¹⁸0, ³²P, ³³P,

35 18 36 123 125

S, F, CI, I and I. Certain isotopically-labeled compounds of the present invention (e.g., those labeled with ³H and ¹⁴C) are useful in compound and/or substrate tissue distribution assays. Tritiated (³H) and carbon-14 (¹⁴C) 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

15 13 11 18 preferred in some circumstances. 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.

[0101] 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.

[0102] 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. In general, 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).

[0103] The formulations may be prepared using conventional dissolution and mixing procedures. For example, 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.

[0104] The pharmaceutical composition (or formulation) for application may be packaged in a variety of ways depending upon the method used for administering the drug. Generally, 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. In addition, the container has deposited thereon a label that describes the contents of the container. The label may also include appropriate warnings.

[0105] Pharmaceutical formulations of the compositions of the present invention may be prepared for various routes and types of administration. For example, 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. 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.

[0106] The pharmaceutical formulation is preferably sterile. In particular, formulations to be used for in vivo administration must be sterile. Such sterilization is readily accomplished by filtration through sterile filtration membranes.

[0107] The pharmaceutical formulation ordinarily can be stored as a solid

composition, a lyophilized formulation or as an aqueous solution.

[0108] 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.

[0109] As a general proposition, the initial pharmaceutically effective amount of the

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.

[0110] 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 (less than about 10 residues) polypeptides; proteins, 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 TWEEN™, PLURONICS™ or polyethylene glycol (PEG). 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) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 18th edition, (1995) Mack Publ. Co., Easton, PA.

[0111] 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. Examples of 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 DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate) and poly-D (-) 3- hydroxybutyric acid. [0112] 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

Pharmaceutical Sciences 18^th Ed. (1995) Mack Publishing Co., Easton, PA. Such methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients. In general 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.

[0113] 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. Alternatively, the MEK inhibitor and the VEGF antagonist may be formulated separately in a pill, capsule, solution or suspension for administration by alternation.

[0114] 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.

[0115] 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. They improve the flow of the powder mixes through the presses and minimize friction and breakage as the finished tablets are ejected from the equipment; Antiadherent with function similar to that of the glidant, reducing adhesion between the powder making up the tablet and the machine that is used to punch out the shape of the tablet during manufacture; Flavor incorporated into tablets to give them a more pleasant taste or to mask an unpleasant one, and Colorant to aid identification and patient compliance.

[0116] Tablets containing the active ingredient in admixture with non-toxic pharmaceutically acceptable excipient which are suitable for manufacture of tablets are acceptable. These 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.

[0117] For treatment of the eye or other external tissues, e.g., mouth and skin, 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. When formulated in an ointment, the active ingredients may be employed with either a paraffinic or a water- miscible ointment base. Alternatively, the active ingredients may be formulated in a cream with an oil-in-water cream base.

[0118] If desired, 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.

[0119] 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. Preferably, a hydrophilic emulsifier is included together with a lipophilic emulsifier which acts as a stabilizer. Together, 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. Emulsifiers and emulsion stabilizers suitable for use in the

formulation include Tween® 60, Span® 80, cetostearyl alcohol, benzyl alcohol, myristyl alcohol, glyceryl mono-stearate and sodium lauryl sulfate.

[0120] Aqueous suspensions of the pharmaceutical formulations contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions. Such 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 monooleate). 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.

[0121] Pharmaceutical compositions may be in the form of 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. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile fixed oils may conventionally be employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid may likewise be used in the preparation of injectables.

[0122] 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. For example, 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). The

pharmaceutical composition can be prepared to provide easily measurable amounts for administration. For example, an aqueous solution intended for intravenous infusion may contain from about 3 to 500 μg 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.

[0123] 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.

[0124] 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.

[0125] 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.

[0126] Formulations for rectal administration may be presented as a suppository with a suitable base comprising for example cocoa butter or a salicylate. [0127] 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.

[0128] 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.

[0129] 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. 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.

Combination Therapy

[0130] 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. In certain embodiments, 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. In one embodiment, 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.

[0131] The combination therapy may be administered as a simultaneous or sequential regimen. 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.

[0132] In one specific aspect of the invention, the MEK inhibitor or 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. In another specific aspect of the invention, 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. In another specific aspect of the invention, administration of the MEK inhibitor or the pharmaceutically acceptable salt thereof and administration of the VEGF antagonist begin on the same day.

[0133] 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.

[0134] In a particular embodiment of anti-cancer therapy, 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. 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.

[0135] The efficacy of the treatment of the invention can be measured by various endpoints commonly used in evaluating neoplastic or non-neoplastic disorders. For example, 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. Because 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. For example, tumor shrinkage of greater than 50% in a 2-dimensional analysis is the standard cutoff for declaring a response. However, the inhibitors of the invention may cause inhibition of metastatic spread without shrinkage of the primary tumor, or may simply exert a

tumouristatic effect. Accordingly, 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.

Article of Manufacture

[0136] In another embodiment of the invention, an article of manufacture, or "kit", containing a MEK inhibitor or pharmaceutically acceptable salt thereof useful for the treatment of the diseases and disorders described above is provided. In one embodiment, the kit comprises a container and a MEK inhibitor or pharmaceutically acceptable salt thereof.

[0137] The kit may further comprise a label or package insert, on or associated with the container. The term "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

pharmaceutically acceptable salt thereof, or a formulation thereof which is effective for treating the condition and may have a sterile access port (for example, the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is a MEK inhibitor or a

pharmaceutically acceptable salt thereof. The label or package insert indicates that the composition is used for treating the condition of choice, such as cancer. In one

embodiment, 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. Alternatively, or additionally, 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.

[0138] 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. For example, if the kit comprises a first composition comprising a MEK inhibitor or pharmaceutically acceptable salt thereof and a 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.

[0139] In another embodiment, the kits are suitable for the delivery of solid oral forms of a MEK inhibitor or pharmaceutically acceptable salt thereof, such as tablets or capsules. Such 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. If desired, 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.

[0140] According to one embodiment, a kit may comprise (a) a first container with a

MEK inhibitor or pharmaceutically acceptable salt thereof contained therein; and optionally (b) a second container with a second pharmaceutical formulation contained therein, wherein the second pharmaceutical formulation comprises a second compound with anti- hyperproliferative activity. Alternatively, or additionally, 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. [0141] Where the kit comprises a composition of a MEK inhibitor or pharmaceutically acceptable salt thereof and a second therapeutic agent, i.e. the VEGF antagonist, 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. Typically, 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.

[0142] Further details of the invention are illustrated by the following non-limiting examples.

EXAMPLES

Experimental Procedures Mouse Strains

[0143] 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

University, St. Louis). 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.

Cell Lines

[0144] Mouse mammary tumor cell lines 67NR, 168FARN, 4T07 and 4T1 were from

F. Miller (Karmanos Cancer Institute, Detroit, MI). 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.

Tumor Models and Dosing Regimens

[0145] Isolated tumor cells (1.0 10⁷) 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.

Human Tumor Arrays

[0146] The following human biopsy arrays were used: Ovarian cancer, T823572-5

(BioChain Inc); Bladder, Z7020106 (BioChain Inc); Head and Neck, Z7020651 (BioChain Inc); Pancrease, PA! 921 (US Bioniax), Z7020090 (BioChain Inc).

RNA Preparation and qRT-PCR Analysis

[0147] Total DNA-free RNA from cancer cells was isolated with the RNeasy kit

(Qiagen, Germany) according to the manufacturer's protocol. One-step quantitative reverse transcription-PCR (qRT-PCR) was done in a total volume of 50μΙ_^ with Superscript III Platinum One-Step qRT-PCR Kit (Invitrogen, Carlsbad, CA) or with TaqMan One-Step RT- PCR Master Mix (Applied Biosystems, Foster City, CA), and ran with a total of lOOng RNA. The following 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

[0148] Tumor cells were seeded at a density of 5x105 per 2ml of 10% FBS plus

IMDM. Twenty four hours later, media were changed to serum- free IMDM, and cells were starved for 6 hours. Cells were stimulated with lOOng/ml of mouse EGF, FGF 2, 3, 5, 6, 8 and 9 (R&D Systems) for 15 minutes and immediately lysed. Lysates were boiled in Tris- Glycine SDS Sample Buffer (Invitrogen, Carlsbad, CA). 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).

Collection of Conditioned Media from Tumor Cells

[0149] LLC, 168FARN, 4T1, KPP388, KPP449 and human cell lines were cultured in

12 well plates at the density of 1x105. After reaching 80-90% confluence, growth media were changed to serum-free IMDM. Cells were then stimulated with PBS, EGF, or fibroblast growth factors 2, 3, 5, 6, 8, and 9. Small molecule inhibitors (GDC-0973, GDC-0879 or LY294002) were added into the cells with or without growth factor stimulation. Cell viability and total cell number were measured using Vi-Cell XR (Beckman Coulter, Fullerton, CA). Conditioned media were analyzed for G-CSF levels by ELISA. Data were normalized to total cell numbers.

Ultrasound imaging

[0150] 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 μτη, and lateral resolution of 100 μτη.

Animals were anesthetized with 2% isoflurane and maintained on a heated imaging platform at 37 °C for the duration of the imaging. Three-dimensional (3D) images of the pancreas were acquired with a motorized drive mechanism that traverses the z-axis while acquiring two-dimensional (x-y) axis B-mode images at regular spatial intervals (50 μτη); these values ranged between 4 to 20 mm based on tumor length. For overall survival studies, imaged animals were randomized based on calculated tumor burden, body weights and gender using a random number generator in JMP statistical discovery software package version 9.0.2 (SAS).

Flow Cytometry (FACS)

[0151] 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.

ELISA and Luminex

[0152] Plasma was collected from animals and stored in EDTA-containing tubes.

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).

Immunohistochemistry

[0153] Tumor samples were frozen in Optimum Cutting Temperature (OCT, Sakura

Finetek), cut (12um) in a cryostat (Leica Microsystem) and were frozen at -200°C. Sections were immuno-stained with antibodies diluted in DAKO Block solution (DakoCytomation). Tumor sections were stained with the following primary antibodies: anti-mouse CD31 antibody (Chemicon), anti-mouse CD1 lb (eBioscience), and anti-aSMA (Sigma). Sections were anti-CD 105 (eBioscience). Human tumor biopsies where stained with anti-G-CSF (clone 3D1 : Santa Cruz), anti-Ets2 (Lifespan Biosciences), anti-pMEK (Cell Signaling), anti- pFGFRl Y653 Y654 (Abeam). The slides were washed and mounted in DAKO fluorescent mounting medium (DakoCytomation). Vascular Surface Area Measurement

[0154] Digital images were captured on TissueGnostics Slide scanner from tumor sections stained with anti-CD31 antibody using a 20x objective. The pixels corresponding to stained vessels were selected using Defmiens Tissue Studio Software (Defmiens, New Jersey). Whole tumor cross-sections from either 4 or 5 tumors per group were analyzed. The aggregate pixel vessel area, relative to the total live tumor tissue area, were analyzed and expressed as percentage of CD31 -positive pixels of viable tissue field.

Statistical analysis

[0155] Student's t-test was used to determine significant differences in all experiments, except overall survival studies. P values of <0.05 were considered significant. In animal studies, Kaplan-Meier survival curve estimates for PDAC murine model were performed using Cox proportional hazards model fit to the data in the R statistical language4 using a 'survival' library. Baseline tumor burden (logarithmically scaled, with "1" added to each scan's value to specify a baseline of "0" on the logarithmic scale) and treatment group were included for each mouse as explanatory factors. The endpoint for mice was survival time (overall survival, OS). All P values reported for hazard ratios are two-sided, and test the null hypothesis that the hazard ratio is 1 between the two groups specified.

Example 1. Ets2 Transcriptional Regulation of G-CSF in Cancer

[0156] The metastatic variants of the 4Tl-related mouse breast cancer cell lines

4T07 and 4T1 were shown to express high G-CSF levels whereas the non-metastatic 67NR and 168FARN cells expressed undetectable G-CSF levels. To identify transcription factors regulating G-CSF expression, 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. Site- directed mutagenesisthe was performed on the two Ets transcriptional consensuses ACCCg (-232) and TAAAc (-101) binding sites to screen for binding sites that potentially regulate G-CSF transcription. The analysis indicated that the two Ets binding sites are important mediators of G-CSF expression (Figure IB). Ets proteins are important for many cellular processes, including cell proliferation, apoptosis, hematopoiesis, angiogenesis and tumorigenesis. In normal and cancer cells, Ets2 activity is controlled by the RAS signaling pathway. Examination of Ets2 expression in 4T07 and 4T1 cells showed that Ets2 protein levels are higher in 4T07 and 4T1 compared to 67NR and 168FARN cells. 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

determine whether Ets2 directly binds to the G-CSF promoter, chromatin

immunoprecipitation (ChIP) analysis in 4T1 and 67NR cells was performed, which showed direct binding of Ets2 at the G-CSF promoter with qPCR primers detecting -500 to -1 nucleotides region of G-CSF promoter (Figure IE). To confirm the role of Ets2 in G-CSF positive regulation, either a dominant negative form of Ets2 (Ets2DN) or wild-type Ets2 was co-expressed with the G-CSF promoter-driven luciferase reporter construct in 4T1 cells. Ets2 dominant-negative protein abolished G-CSF promoter activity (Figure IF). Together, these data indicate that Ets2 transcription factor is a positive regulator of G-CSF and inhibiting Ets2 activity can significantly reduce G-CSF expression in cancer cells.

Example 2. RAS Signaling Drives G-CSF Expression

[0157] Since the RAS/RAF/MEK pathway controls Ets2 trancriptional activity, it was examined as to whether this pathway is activated in 4T1 -related cell lines. BRAF and ERK phosphorylations were assessed using immunoblotting, since these two kinases are downstream effectors of RAS. Indeed, phosphorylated BRAF and ERK were readily detected in 4T1 but not in 67NR cell lysates (Figure 2A). Enforced expression of a mutant form of BRAF (BRAFV600E) resulted in G-CSF expression in 67NR cells (Figure 2B). To test whether MEK inhibition can suppress G-CSF expression in 4T1 cells, a MEK inhibitor GDC-0973/XL518 (hereinafter MEKi) was used. This agent is a potent, selective, orally active inhibitor of MEK1/2 with an IC₅₀ of <1 nM in vitro and is currently

undergoing clinical trials. Figure 3A shows the structure of this compound.

[0158] G-CSF production by 4T1 cells was directly correlated with ERK

phosphorylation levels, following treatment with different concentrations of MEKi (Figure 2C; *p<0.002), but not with cell death (data not shown). The RAS signaling pathway controls growth, proliferation, and survival of cancer cells by activating multiple

downstream effectors including the RAF/MEK/ERK and the PI3K pathways. However, the data herein indicate that the RAF/MEK/ERK pathway, but not the PI3K pathway, is responsible for G-CSF over-expression in 4T1 cells (Figure 2D).

Example 3. Multiple Growth Factors Induce G-CSF Expression

[0159] The results above indicate that oncogenic activation of the RAS signaling pathway can result in G-CSF up-regulation. It is postulated that growth factors or cytokines, produced within the tumor microenvironment, might also induce G-CSF expression. Indeed, fibroblast growth factors (FGFs) FGF2, FGF3, FGF5, FGF6, FGF8 and FGF9 were found capable of inducing significant increases in G-CSF levels (>4-folds; lOOng ml^"1) in LLC cells (Figure 3B). This tumor cell line produced very low levels of G- CSF in vitro, but secreted high levels of G-CSF when implanted in mice, which in turn lead to myeloid cell mobilization and VEGF-independent angiogenesis. In addition to FGFs, 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). To test whether G-CSF production is dependent on MAPK pathway activation, 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).

[0160] In addition to cancer cells, growth factors were also tested for their stimulation of G-CSF expression in stromal fibroblast cells. NIH 3T3 fibroblasts were used for the study and G-CSF levels were measured by ELISA after 12 hrs of stimulation with different growth factors (n=3/group). Furthermore, MEKi was used to test its inhibition of growth factor-induced G-CSF release in fibroblasts. As shown in Figure 3F, certain growth factors, including FGF8, FGF9, PDGF-A, PDGF-B, and PDGF-D markedly induced increase in G-CSF levels, but such induction is dependent on MAPK pathway activation, which is effectively inhibited by MEKi. Example 4. MEK Inhibition Sensitizes Breast Tumors for Anti-VEGF Treatment

[0161] Breast tumor-bearing mice were treated with anti-ragweed, MEKi, anti-

VEGF, anti-G-CSF or combination therapies as indicated in Figure 4E. 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). In agreement with these findings, 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. Similarly, 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).

[0162] Immunostaining of tumor sections with anti-CD31 antibody revealed that anti-G-CSF, anti-VEGF or MEK inhibitor treatment alone had no significant effect on vascular density. However, combination treatment of MEKi plus anti-VEGF or anti-G-CSF plus anti-VEGF significantly reduced vascular density (Figure 4F, G).

Example 5. MEK Inhibitor or Anti-G-CSF Antibody Overcomes Refractoriness to anti-VEGF Treatment in LLC Tumors

[0163] LLC tumors were previously identified as refractory to anti-VEGF therapy.

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. Similar to 4T1 tumors, 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. Also, 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). The greater effectiveness of MEKi compared to anti-G-CSF in decreased tumor growth likely reflects the fact that targeting MEK activity had a broader inhibition targets downstream of receptor tyrosine kinases compared to anti-G-CSF, which only inhibits G- CSF. MEKi + anti-G-CSF combination treatment was comparable to MEKi treatment alone in reducing tumor growth, suggesting that MEKi might target the same underlying mechanism driven by G-CSF stimulation to provide tumor survival advantage, and that adding anti-G-CSF with MEKi does not further reduce growth (Figure 5E). Importantly, combination treatment of MEKi + anti-VEGF or anti-G-CSF + anti-VEGF significantly reduced angiogenesis when compared to anti-ragweed or anti-VEGF monotherapy (Figure 5F, G).

[0164] To further assess the effects of combining MEKi with anti-VEGF on the growth of LLC tumors, tumor-bearing mice were treated with anti -ragweed, MEKi, anti- VEGF or combination MEKi + anti-VEGF. While anti-VEGF or MEKi monotherapy was effective at reducing tumor growth, combination of MEKi and anti-VEGF decreased tumor growth by approximately 64% at day 26 and prolonged the time it would take for tumors to reach the maximum volume allowed by our institution when compared to either MEKi or anti-VEGF as a single agent (Figure 7). Similarly, total white blood cells counts were markedly reduced in the peripheral blood of the animals that received either the MEKi alone, MEKi plus anti-VEGF combination treatments compared to controls (Figure 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 (Figure 8B-D). Next, myeloid cell recruitments and angiogenesis were examined in LLC tumors. Whole tumor sections immuno-stained with the myeloid cell marker CDl lb and the blood vessel marker CD31 showed that anti-VEGF resulted in increased CDl lb⁺ cell recruitment compared to control treated group. MEKi treatment alone resulted in decreased CDl lb⁺ cells recruitment into the tumor

microenvironment. Importantly, combination treatment of MEKi plus anti-VEGF resulted in marked reductions in CD31⁺ vessel density and CDl 1⁺ myeloid cells recruitment compared to control treated animals. Taken together, these data show that combination treatment of MEKi and anti-VEGF is an effective strategy in treating tumors that are resistant to anti-VEGF therapy. Example 6. G-CSF Expression in Mouse and Human Tumor Cells

[0165] Mutations in the RAS signaling pathway have been detected in approximately

30% of all human cancers. Therefore, mouse and human tumor cell lines with activation of RAS pathway were examined as to whether they have enhanced G-CSF expression. First, three different KRAS mutant mouse lung cancer cell lines, LKPH2, LKP9 and LKP10, were tested (Jackson et al, ibid. (2005)). All three cell lines released G-CSF in the medium by ELISA, albeit at different levels. MEKi treatment inhibited G-CSF expression in a dose- dependent manner (Figure 6A). In contrast, PI3Ki treatment had no effect, again confirming that the PI3K pathway does not control G-CSF expression. Furthermore, in agreement with previous studies showing that the RAF inhibitor GDC-0879 could activate the MAPK pathway in KRAS mutant cancer cell lines (Hatzivassiliou et al.), BRAFi GDC-0879 (luM) was found to further increase ERK phosphorylation and induce G-CSF expression in LKPH2, LKP9 or LKP10 cells (Figure 6A).

[0166] 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. These 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).

[0167] Similar to the mouse lung cell lines (Figure 6A), treatment with MEKi reduced G-CSF release in a dose-dependent fashion in all thirteen G-CSF-positive cell lines tested (Figure 6B). PI3Ki treatment has no effect on G-CSF level. Furthermore, BRAFi GDC-0879 treatment resulted in activation of the MAPK pathway in subset of human cancers. Interestingly, the majority of cell lines that up-regulated G-CSF in response to BRAFi GDC- 0879 treatment are derived from lung cancers (Figure 6B and Table 1). Taken together, the results demonstrate that RAS signaling pathway activation induces G-CSF expression in human cancers. ible 1: G-CSF Production in Human Cancer Cell Lines

RAS/MEK G-CSF Protein

G-CSF RNA S.E.» : signaling pathway Detection

(hG-CSF/GAPDH) Valu status (ELISA)

incer Type i Cell Line mg cancer C alii- 1 KRAS Mutant Yes 89 7.5

: Calu-3 KRAS Mutant Yes 46 8 i Calu-6 KRAS Mutant Yes 230 51

EBCl KRAS Mutant Yes 9 1

I IC C - 15 KRAS Mutant Yes 3982 174

1 12 122 KRAS Mutant Yes 1312 170

1 1838 i EpoR amplification Yes 37 6

1 1 1 75 EGFR mutant Yes 26 7

1 13221 Unknown No 1 0

1 12 195 KRAS Mutant No 1 0

)lon cancer : Colo201 BRAF (V600E) No 1 0

: Colo205 BRAF (V600E) No 1 0

1 1 1 55 BRAF (V600E) No 3.5 0

! LoVo KRAS Mutant No 1 0 m c 1 5 KRAS Mutant No 4 0 i n c i 1 KRAS Mutant No 2.5 0

Dl .D- l KRAS Mutant No 2.3 0

: SW48 KRAS Mutant No 1 0

SW837 KRAS Mutant No 1 0

SW 1463 KRAS Mutant Yes 32.5 5

S1.- 1 80 KRAS Mutant No 1 0

SI .- 1 74T KRAS Mutant No 1 0 elanoma Λ375 BRAF (V600E) No 1 0

: 624 Mel BRAF (V600E) No 1 0 east cancer MDA-MB-231 ! KRAS Mutant Yes 1 0 ncreatic cancer i PI .45 Unknown No 10 0 adder cancer 5637 EGFR mutant Yes 1 0

HI IX -905 NRAS mutant Yes 5123 258

;uM-uc-i Unknown Yes 28 5

: UM-UC-3 Unknown No 14 1

; SW780 Unknown No 1 0 Example 7. Combined inhibition of MEK or G-CSF with Anti-VEGF Therapy

Effectively Inhibits Tumor Growth in Anti-VEGF Resistant Pancreatic Tumors

[0168] Pancreatic ductal adenocarcinoma (PDAC), 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. Indeed, two recent PDAC clinical trials using bevacizumab in combination with other therapeutic agents were discontinued due to lack of significant survival benefits by adding the anti-VEGF therapy. Singh et al. Nature Biotech. 28:585-93 (2010). Thus, novel therapeutic strategies and regimen are urgently needed.

[0169] This example elucidates the signaling pathways and molecules that govern or contribute to PDAC's resistance to therapies such as anti-VEGF therapy, and demonstrates novel approaches to effectively overcome such resistance.

7.1 G-CSF Induces CDllb+Ly6G+ Neutrophil Mobilization in Anti-VEGF Resistant PDAC

Allograft Models

[0170] To investigate the microenvironment of PDAC, the mechanism of its resistance to anti-VEGF therapy as well as therapeutics effective for mitigating or overcoming such resistance, a Kras-driven genetically engineered mouse model (GEMM), Kras^LSL~G12D ; pl6/pl≠'^fl ;Pdx-Cre, was generated to simulate PDAC. Human PDACs have a large component of stromal cells (6), including alpha-smooth muscle actin (aSMA)- positive myofibroblast-like stellate cells. Similarly, 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.

[0171] CDl lb+Grl+ myeloid cells are mixed population of cells, consisting of immature dendritic cells, early myeloid progenitors, Ly6C+ granulocytic monocytes and Ly6G+ neutrophils. To investigate which subset of 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. The results indicate that 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. To investigate the contribution of CDl lb+Ly6G+ neutrophils in tumor resistance to anti-VEGF therapy, 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. 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. In contrast, anti-VEGF antibody treatment

significantly reduced tumor growth in the G-CSF-K ^' RA G2^{~ ~} mice (Fig. 9A). Therefore, these data suggest that the ability of anti-VEGF to reduce tumor growth is directly correlated with decreased CDl lb+Ly6G+ neutrophil mobilization, and that the

CDl lb+Ly6G+ neutrophil subpopulation can serve as a biomarker for tumors resistant to anti-VEGF therapy (Fig. 9B).

7.2 Combining MEKi GDC-0973 or Anti-G-CSF Antibody with Anti-VEGF Antibody Effectively Inhibits Tumor Angiogenesis and Growth in a PDAC Allograft Model

[0172] Next, the impact of combination therapies using MEKi GDC-0973 and anti-

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). Similarly, 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. These data suggest that MEK inhibition might target the same underlying signaling mechanism driven by G-CSF, but is not sufficient at reducing tumor growth (Fig. 9C). In contrast, significant reductions in tumor growth were observed when animals received combination therapies with MEKi GDC-0973 plus anti-VEGF or anti-G-CSF plus anti-VEGF (Fig. 9C). Moreover, targeting G-CSF reduced CDl lb+Ly6G+ neutrophil mobilization (Fig. 9D and 9E), and combination with anti-VEGF therapy significantly reduced angiogenesis as measured by CD31+ endothelial cells in tumors (Fig. 9F). Indeed, quantitative analysis revealed marked reduction in microvessel density in the combination therapies (Fig. 9G) compared to anti- Ragweed or single-agent treated groups.

7.3 Combining MEKi GDC-0973 or anti-G-CSF with anti-VEGF Antibody Improves Survival in a Kras-driven PDAC GEMM

[0173] Another experiment investigated whether combination treatments with either

MEKi GDC-0973 and anti-VEGF or anti-G-CSF and anti-VEGF could prolong overall survival (OS) in the Kras LSL-G12D; _P16/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. However, neutralizing G-CSF did not have a significant effect on the

CDl lb+Ly6C+ monocyte population (Fig. 10D, bars 4&6), suggesting that the

CDl lb+Ly6C+ monocyte are not included in the GCSF-induced myeloid cell mobilization in the PDAC GEMM. To investigate survival benefits in PDAC mice, 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. MEKi GDC- 0973 and anti-VEGF combination treatment resulted in increased survival (median survival 3.6 weeks vs. 2.3 weeks for controls; p = 0.002). Similarly, anti-G-CSF and anti-VEGF combination resulted in a median survival of 3.7 weeks, compared to 2.3 weeks in the control group (p = 0.015) (Fig. 10A). 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).

7.4 MEK Pathway Activation and Neutrophil Recruitment in Human PDAC

[0174] The majority of patients diagnosed with PDAC harbor KRAS mutations, immunohistochemistry were performed on human PDAC biopsies to investigate whether there are any correlations between high G-CSF, phospho-MEK (pMEK), and phospho- FGFR (pFGFR). Antibody-binding specificity to MEK and FGFR phosphorylation was first validated by performing control immunohistochemical staining experiments. In 1 16 patient PDAC biopsies, 83% of the samples were positive for G-CSF (97/1 16), 81% were positive for pMEK (94/116), and 25% were positive for pFGFR (27/1 16) (Figs. 1 1A-D).

Immunohistochemical staining revealed co-expressions of pMEK and G-CSF (82%), or pFGFR and G-CSF (26%) in the human PDAC biopsies (Fig. 1 IF). Similar to the results from the Kras-driven PDAC GEMM, significantly increased neutrophil recruitment was found in patient PDAC biopsies that are G-CSF-positive (Fig. 1 IE).

7.5 MEK Inhibition Markedly Reduces G-CSF Release in a Kras-driven PDAC GEMM

[0175] 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). Administration of the MEKi GDC-0973 significantly reduced G-CSF levels in the plasma of tumor-bearing mice at both 7 hours and 7 days after treatment (Fig. 12A). Next, cytokines and growth factors released in the plasma of PDAC mice were profiled and compared to MEKi-treated or naive wild-type animals. In addition to G-CSF, 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). Among these factors, only KC and G-CSF decreased at day 7 post MEKi GDC-0973 treatment (Fig. 12B). Importantly, MEKi GDC- 0973 treatment resulted in decreased CD1 lb+Ly6G+ neutrophil mobilization in the peripheral blood of Kras-driven PDAC GEMM (Fig. 12C).

[0176] All references cited throughout the disclosure are hereby expressly

incorporated by reference in their entirety. While the present invention has been described with reference to what are considered to be the specific embodiments, it is to be understood that the invention is not limited to such embodiments. To the contrary, the invention is intended to cover various modifications and equivalents included within the spirit and scope of the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A method of inhibiting tumor growth, comprising administering an effective amount of a MEK inhibitor to a human subject having a tumor that overexpresses granulocyte colony- stimulating factor (G-CSF) and is resistant to treatment with a vascular endothelial growth factor (VEGF) antagonist.

2. A method of inhibiting tumor growth, comprising administering an effective amount of a MEK inhibitor to a human subject having a tumor that overexpresses granulocyte colony- stimulating factor (G-CSF) and is refractory to treatment with a VEGF antagonist.

3. 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.

4. 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.

5. 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.

6. The method of claim 5, wherein the tumor angiogenesis is induced by cells in tumor stroma and is independent of VEGF expression level.

7. The method of claim 6, wherein the cells in tumor stroma are derived from bone marrow.

8. The method of claim 7, wherein the cells in tumor stroma are of hematopoietic lineage.

9. The method of claim 8, wherein the cells in tumor stroma are of myeloblast lineage.

10. The method of claim 9, wherein the cells in tumor stroma are human counterpart of the murine CD1 lb+/Grl+ myeloid cells.

11. The method of claim 9, wherein the cells in tumor stroma are granulocytes.

12. The method of claim 11 , wherein the cells in tumor stroma are neutrophils.

13. 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.

14. The method of claim 13, wherein the cells in tumor stroma are capable of inducing and promoting tumor angiogenesis.

15. The method of claim 14, wherein the cells in tumor stroma are derived from bone marrow.

16. The method of claim 15, wherein the cells in tumor stroma are of hematopoietic lineage.

17. The method of claim 16, wherein the cells in tumor stroma are of myeloblast lineage.

18. The method of claim 17, wherein the cells in tumor stroma are human counterpart of the murine CD1 lb+/Grl+ myeloid cells.

19. The method of claim 17, wherein the cells in tumor stroma are granulocytes.

20. The method of claim 19, wherein the cells in tumor stroma are neutrophils.

21. The method of claim 14, wherein the cells in tumor stroma are fibroblast cells.

22. A method according to any one of claims 1-21, wherein the VEGF antagonist is an anti- VEGF antibody or functional fragment thereof.

23. The method of claim 22, wherein said anti-VEGF antibody is bevacizumab.

24. A method according to any one of claims 1-21, wherein the MEK inhibitor is a small molecule compound or a pharmaceutically acceptable salt thereof.

25. The method of claim 24, wherein the small molecule compound is 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, GSK1120212 and AS-703026.

26. The method of claim 24, wherein the small molecule compound is an azetidine compound.

27. The method of claim 24, wherein the small molecule compound is an imidazopyridine compound.

28. The method of claim 24, wherein the small molecule compound is GDC-0973.

29. The method of claim 24, wherein the small molecule compound is GDC-0987.

30. A method according to any one of claims 1-21, wherein the human subject having a tumor has been previously treated with chemotherapy, a VEGF antagonist or both.

31. A method according to any one of claims 1-21, further comprising administering to said human subject an anti-VEGF antibody or functional fragment thereof.

32. The method of claim 31 , wherein the anti-VEGF antibody is bevacizumab.

33. The method of claim 31 , wherein the anti-VEGF antibody is bevacizumab and the MEK inhibitor is GDC-0973.

34. The method of claim 33, wherein the human subject is a patient having pancreatic ductal adenocarcinoma (PDAC) and wherein the combining administration of bevacizumab and GDC- 0973 significantly improves the patient's overall survival.

35. The method of claim 31 , wherein the anti-VEGF antibody or functional fragment thereof is administered prior to, concurrent with or subsequent to the administration of the MEK inhibitor.

36. A method according to any one of claims 1-21, further comprising subjecting said human subject to chemotherapy or radiation therapy.

37. A method according to any one of claims 1-21, wherein the tumor is in the colon, rectum, liver, lung, prostate, breast, bladder, skin, brain, thyroid, pancreas or ovary of the human subject.

38. The method of claim 37, wherein the human subject is a patient having PDAC.

39. A method according to any one of claims 1-21, further comprising monitoring the efficacy of said administering step by determining the amount of neutrophil cells in a tumor sample or a peripheral blood sample obtained from said human subject, relative to the amount of the nutrophil cells in a tumor sample or a peripheral blood sample obtained from said human subject prior to administration of said MEK inhibitor, wherein a reduced amount of the neutrophil cells indicates efficacy of the administration.

40. A method according to any one of claims 1-21, further comprising monitoring the efficacy of said administering step by measuring the expression level of Bv8 in a tumor sample or a peripheral blood sample obtained from said human subject, relative to the

expression level of Bv8 in a tumor sample or a peripheral blood sample obtained from said human subject prior to administration of said MEK inhibitor, wherein a reduced Bv8

expression level indicates efficacy of the administration.

41. A combination of a) a MEK inhibitor and b) a VEGF antagonist for concurrent, separate or sequential use in treating tumor.

42. A combination of a) a G-CSF antagonist and b) a VEGF antagonist for concurrent, separate or sequential use in treating tumor.

43. A pharmaceutical preparation comprising an effective amount of the combination of claim 41 or 42 and at least one pharmaceutically acceptable carrier.