TW201019961A - Combination therapy - Google Patents

Abstract

The present invention relates generally to the fields of molecular biology and growth factor regulation. More specifically, the invention relates to combination therapies for the treatment of pathological conditions, such as cancer.

Description

201019961 VI. Description of the Invention: TECHNICAL FIELD OF THE INVENTION The present invention relates generally to the field of molecular biology and growth factor regulation. More specifically, the present invention relates to a combination therapy for treating a pathological condition such as cancer. * This application claims priority to U.S. Provisional Patent Application Serial No. 61/106,513, filed on Jan. 27, 2008, the entire disclosure of which is hereby incorporated by reference. ❹ [Prior Art] HGF is a pleiotropic factor obtained from mesenchyme, which has mitogenic, motogenic, and morphogenic activities against several different cell types. The HGF effector is mediated via the specific valine kinase c-met, and abnormal HGF and c-met expression is often observed in a variety of tumors. See, for example, Maulik et al., Cytokine & Growth Factor

Reviews (2002), 13:41-59; Danilkovitch-Miagkova and Zbar, J. Clin. Invest. (2002), 109(7): 863-867. Tumor progression and metastasis ❹. Involves the regulation of the HGF/c-Met signaling pathway. See, for example, Trusolino and Comoglio, Nature Rev. (2002), 2: 289-300 ° HGF binds to the extracellular domain of Met receptor tyrosine kinase (RTK) and regulates various biological processes such as cell dispersion, proliferation and survival. HGF-Met signaling is essential for normal embryonic development (especially muscle progenitor migration and liver and nervous system development) (Bladt et al, Nature (1995), 376, 768-771; Hamanoue et al., Faseb J ( 2000), 14, 399-406; Maina et al., Cell (1996), 87, 531-542; Schmidt et al., 143939.doc 201019961

Nature (1995), 373, 699-702; Uehara et al, Nature (1995), 373, 702-705). The developmental phenotype of Met and HGF knockout mice is very similar, indicating that HGF is a cognate ligand for the Met receptor (Schmidt et al, 1995, supra; Uehara et al, 1995, supra). HGF-Met also plays a role in liver regeneration, angiogenesis and wound healing (Bussolino et al, J Cell Biol (1992), 119, 629-641; Matsumoto and Nakamura, Exs (1993), 65, 225-249; Nusrat Et al, J Clin Invest (1994) 93, 2056-2065). The precursor Met receptor undergoes proteolytic cleavage and forms an extracellular alpha subunit and a transmembrane beta subunit linked by a disulfide bond (Tempest et al, Br J Cancer (1988), 58, 3-7). The beta subunit contains a cytoplasmic kinase domain and has a multi-substrate docking site at the C-terminus, where the adaptor protein binds and initiates signaling (Bardelli et al., Oncogene (1997) ), 15, 3103-3111; Nguyen et al, J Biol Chem (1997), 272, 20811-20819; Pelicci et al, Oncogene (1995), 10, 1631-1638; Ponzetto et al, Cell (1994), 77 , 261-271; Weidner et al, Nature (1996), 384, 173-176). After HGF binding, activation of Met causes tyrosine phosphorylation and downstream signaling via Gabl and Grb2/Sos-mediated activation of PI3 kinase and Ras/MAPK, respectively, thereby driving cell movement and proliferation (Furge et al., Oncogene (2000) , 19, 5582-5589; Hartmann et al, J Biol Chem (1994), 269, 21936-21939; Ponzetto et al, J Biol Chem (1996), 271, 14119-14123; Royal and Park, J Biol Chem (1995) ), 270, 27780-27787) 〇 has been shown to transform Met in carcinogen-treated osteosarcoma cell lines (Cooper 143939.doc 201019961 et al, Nature (1984), 311, 29-33; Park et al, Cell (1986) ), 45, 895-904). Met overexpression or gene amplification was observed in a variety of human cancers. For example, Met proteins are at least 5-fold overexpressed in colorectal cancer and have been reported to be genetically amplified in liver metastases (Di Renzo et al, Clin Cancer Res (1995), 1, 147-154; Liu et al. , Oncogene (1992), 7, 181-185). Met protein has also been reported to be overexpressed in oral squamous cell carcinoma, hepatocellular carcinoma, renal cell carcinoma, breast cancer, and lung cancer (Jin et al, Cancer (1997), 79, 749-760; Morello et al, J Cell Physiol ( 2001), 189, 285-290; Natali et al, Int J Cancer (1996), 69, 212-217; Olivero et al, Br J Cancer (1996), 74, 1862-1868; Suzuki et al, Br J Cancer (1996), 74, 1862-1868). In addition, excessive mRNA expression has been observed in hepatocellular carcinoma, gastric cancer, and colorectal cancer (Boix et al, Hepatology (1994), 19, 88-91; Kuniyasu et al, Int J Cancer (1993), 55, 72- 75; Liu et al., Oncogene (1992), 7, 181-185). Several mutations that cause constitutive receptor activation have been found in the kinase domain of Met in renal papillary carcinoma (Olivero et al, Int J Cancer (1999), 82, 640-643; Schmidt et al, Nat Genet (1997), 16, 68-73; Schmidt et al., Oncogene (1999), 18, 2343-2350). Such viable mutations phosphorylate constitutive Met tyrosine and cause MAPK activation, focus formation, and tumor formation (Jeffers et al, Proc Natl Acad Sci U S A (1997), 94, 11445-11450). In addition, such mutations enhance cell movement and invasion (Giordano et al, Faseb J (2000), 14, 399-406; Lorenzato et al, Cancer Res (2002), 143939. doc 201019961 62, 7025-703 0). HGF-dependent Met activation in transformed cells mediates increased movement, dispersion, and migration, ultimately leading to invasive tumor growth and metastasis (Jeffers et al, Mol Cell Biol (1996), 16, 1115-1125; Meiners et al, Oncogene ( 1998), 16, 9-20). Met has been shown to interact with other proteins that drive receptor activation, transformation, and invasion. In neoplastic cells, it has been reported that Met interacts with the receptor α6β4 integrin of the extracellular matrix (ECM) component (such as laminin) to promote HGF-dependent invasive growth (Trusolino et al., Cell (2001), 107, 643-654). In addition, the extracellular domain of Met has been shown to interact with plexin B1, a member of the semaphorin family, and enhance invasive growth (Giordano et al, Nat Cell Biol (2002), 4, 720-724). In addition, it has also been reported that CD44v6 involved in tumor formation and metastasis forms a complex with Met and HGF and causes Met receptor activation (Orian-Rousseau et al., Genes Dev (2002), 16, 3074-3086).

Met is a member of the subfamily of receptor tyrosine kinase (RTK), including Ron and Sea (Maulik et al., Cytokine Growth Factor Rev (2002), 13, 41-59). Prediction of the extracellular domain structure of Met indicates that it shares homology with signaling proteins and plexifins. The N-terminus of Met contains a Sema domain of about 500 amino acids, which is conserved across all signaling proteins and plexifins. Signal proteins and plexins are a large family of secreted and membrane-bound proteins that were first described for their role in neurodevelopment (Van Vactor and Lorenz, Curr Bio (1999), 19, R201-204). However, recent overexpression of signaling proteins has been associated with tumor invasion and metastasis. The PSI domain of cysteine enriched in plexin, signal protein and integrin (also known as 143939.doc 201019961)

The Met Related Sequence domain is adjacent to the Sema domain, which is followed by four IPT repeat units, which are immunoglobulin-like regions visible in the plexus and transcription factors. Recent studies have shown that the Met Sema domain is sufficient for HGF and heparin binding (Gherardi et al, Proc Natl Acad Sci U S A (2003), 100(21): 12039-44). As explained above, Met-induced tyrosine kinase is activated by its cognate ligand HGF, and receptor citrate activates the downstream pathways of MAPK, PI-3 kinase and PLC-γ (L. Trusolino and Ρ·M. Comoglio, Nat Rev Cancer 2, 289 (2002); C. Birchmeier et al, Nat Rev Mol Cell Biol 4, 915 (2003)). Phosphorylation of Y1234/Y1235 in the kinase domain is critical for Met kinase activation, whereas Y1349 and Y1356 in the multi-substrate docking site for src homology-2 (SH2) binding, phosphotyrosine binding (PTB) and Met binding domain (MBD) protein (C. Ponzetto et al, Cell 77, 261 (1994); K. Μ Weidner et al, Nature 384, 173 (1996); G. Pelicci et al, Oncogene 10, 16; (1995)) It is important to mediate the activation of downstream signaling pathways. The binding of another juxtamembrane phosphorylation site, Y1003, to the lysine kinase binding (TKB) domain of Cbl E3 ligase has been well characterized (P. Peschard et al, Mol Cell 8, 995 (2001); P. Peschard, N. Ishiyama, T. Lin, S. Lipkowitz, M. Park, J Biol Chem 279, 29565 (2004)). It has been reported that Cbl binds to endophilin-mediated receptor endocytosis, ubiquitination, and subsequent receptor degradation (A. Petrem et al, Nature 416, 187 (2002)). This receptor down-regulation mechanism has previously been described in the EGFR family, which also has a Cbl binding site (K. Shtiegman, Y. Yarden, Semin Cancer Biol 143939. doc 201019961 13, 29 (2003); MD Marmor, Y. Yarden , Oncogene 23, 2057 (2004); P. Peschard, M. Park, Cancer Cell 3, 519 (2003)). Regulatory abnormalities in Met and HGF have been reported in various tumors. Ligand-driven Met activation was observed in several cancers. Elevated serum and intratumoral HGF are observed in lung cancer, breast cancer, and multiple myeloma (JM Siegfried et al, Ann Thorac Surg 66, 1915 (1998); Ρ·C. Ma et al, Anticancer Res 23, 49 (2003); BE Elliott et al, Can J Physiol Pharmacol 80, 91 (2002); C. Seidel et al, Med Oncol 15, 145 (1998)). Overexpression of Met and/or HGF, Met amplification or mutation has been reported in various cancers such as colorectal cancer, lung cancer, gastric cancer and renal cancer, and is believed to drive non-ligand-dependent receptor activation (C. Birchmeier Et al., Nat Rev Mol Cell Biol 4, 915 (2003); G. Maulik et al., Cytokine Growth Factor Rev 13, 41 (2002)). In addition, inducible overexpression of Met in a mouse model of liver triggers hepatocellular carcinoma, suggesting that receptor overexpression drives non-ligand-dependent tumor formation (R· Wang et al, J Cell Biol 153, 1023 (2001)) . For patients with familial and sporadic renal papillary carcinoma (RPC), the report suggests the strongest evidence for Met in cancer. Mutations that cause constitutive activation of the receptor in the Met kinase domain have been identified in the RPC as germline and somatic mutations (L. Schmidt et al, Nat Genet 16, 68 (1997)). Introduction of such mutations in a mouse model of transgenic mice causes tumor formation and metastasis. (M. Jeffers et al., Proc Natl Acad Sci U S A 94, 11445 (1997)). The epidermal growth factor receptor (EGFR) family contains four closely related receptors involved in cellular responses such as differentiation and proliferation (HER1/EGFR, HER2, 143939.doc 201019961 HER3 and HER4). Overexpression of EGFR kinase or its ligand TGF-α is often associated with many cancers, including breast cancer, lung cancer, colorectal cancer, ovarian cancer, renal cell carcinoma, bladder cancer, head and neck cancer, and glioblastoma. Tumors and astrocytoma, and the excessive expression of this tumor promotes the malignant growth of these tumors. It has also been found that specific deletion mutations in the EGFR gene (EGFRvIII) increase cell tumor formation. Activation of the EGFR-stimulated signaling pathway promotes multiple processes that potentially promote cancer (e.g., proliferation, angiogenesis, cell motility, and invasion), reduces apoptosis, and induces resistance. Increased HER1/EGFR performance is often associated with advanced disease, metastasis, and poor prognosis. For example, in NSCLC and gastric cancer, increased HER1/EGFR performance is associated with high metastatic rate, poor tumor differentiation, and increased tumor proliferation. Mutations in the intrinsic protein tyrosine kinase activity of activated receptors and/or increased downstream signaling are observed in NSCLC and glioblastoma. However, mutations serve as the primary mechanism in conferring EGF receptor inhibitors (eg, erlotidine). The role of erlotinib (TARCEVA®) or gefitinib (gefitinib) is controversial. Mutant forms of the full-length EGF receptor have been reported to predict reactivity to the EGF receptor tyrosine kinase inhibitor gefitinib (Paez, JG et al, (2004) Science 304: 1497-1500; Lynch, T. J. Et al., (2004) Ν Engl. J. Med. 3 50:2129-2139). Cell culture studies have shown that cell lines exhibiting these mutated forms of EGF receptor (ie, H3255) are more sensitive to growth inhibition by the EGF receptor tyrosine kinase inhibitor gefitinib and require higher concentrations. Gefitinib inhibits tumor cell lines expressing wild-type EGF receptors. These observations suggest that although specific mutations in the EGF receptor may reflect higher sensitivity to EGF receptor inhibitors 143939.doc 201019961 , but does not identify a completely unresponsive phenotype. The use of compounds that directly inhibit the kinase activity of EGFR and antibodies that reduce EGFR kinase activity by blocking EGFR activation has been a hot research area as an anti-tumor agent (de Bono JS and Rowinsky, EK (2002) Trends in Mol. Medicine 8 :S19-S26 ; Dancey, J. and

Sausville, E.A. (2003) Nature Rev. Drug Discovery 2: 92-313). A number of studies have shown, revealed or suggested that some EGFR kinase inhibitors can improve tumor cell killing or tumor formation killing when used in combination with certain other anti-cancer or chemotherapeutic agents or treatments (eg Herbst, RS et al., 2001). ) Expert Opin. Biol. Ther· 1:719-732 ; Solomon, B. et al., (2003) Int. J. Radiat. Oncol. Biol. Phys· 55:713-723 ; Krishnan, S. et al. 2003) Frontiers in Bioscience 8, el-13; Grunwald, V. and Hidalgo, M. (2003) J. Nat· Cancer Inst. 95:851-867; Seymour L. (2003) Current Opin. Investig. Drugs 4( 6): 658-666; Khalil, Μ·Υ. et al., (2003) Expert Rev. Anticancer Ther. 3:367-380; Bulgaru, AM· et al. (2003) Expert Rev. Anticancer Ther. 3:269 -279; Dancey, J. & Sausville, EA (2003) NatureRev. DrugDiscovery 2: 92-313; Ciardiello, F. et al., (2000) Clin. Cancer Res. 6: 2053-2063; and Patent Publication No. US 2003/0157104). Erlotinib (such as erlotinib hydrochloride, also known as TARCEVA® or OSI-774) is an orally administrable inhibitor of EGFR kinase. In vitro, erlotinib has been shown to have substantial inhibitory activity against EGFR kinase in several human tumor cell lines, including colorectal cancer and breast cancer (Moyer 143939.doc -10- 201019961 JD· et al., (1997) Cancer Res. 57:4838), and preclinical evaluation showed activity against several human tumor xenografts expressing EGFR (Pollack, VA et al, (1999) J. Pharmacol. Exp. Ther. 291:739). In clinical trials, erlotinib has been shown to be active in several indications, including head and neck cancer (Soulieres, D. et al., (2004) J. Clin. Oncol. 22:77), NSCLC (Perez). - Soler R et al. (2001) Proc· Am. Soc. Clin Oncol. 20:310a, Abstract 1235), CRC (Oza, M. et al., (2003) Proc. Am. Soc. Clin. Oncol. 22 : 196a, Abstract 785) and MBC (Winer, E. et al., (2002) Breast Cancer Res. Treat. 76: 5115a, Abstract 445; Jones, RJ et al., (2003) Proc. Am. Soc. Clin. Oncol 22:45a, abstract 180). In a phase III trial, erlotinib monotherapy significantly prolonged survival in patients with advanced refractory NSCLC, delaying disease progression and delaying the progression of lung cancer-related symptoms (Shepherd, F. et al. (2004) J. Clin. Oncology , 22:14S (Supplementary Supplement, July 15), Abstract 7022). In November 2004, the US Food and Drug Administration (FDA) approved TARCEVA® for the treatment of patients with locally advanced or metastatic non-small cell lung cancer (NSCLC) after failure of at least one prior chemotherapy regimen. The development of the vascular system is a fundamental requirement for many physiological and pathological processes. Organizations such as embryos and tumors that are actively growing need adequate blood supply. These tissues meet this need by producing pro-angiogenic factors that promote neovascularization via a process called angiogenesis. Angiogenesis is a complex but ordered biological event that includes all or more of the following steps: a) endothelial cell (EC) proliferation from existing EC or differentiation from progenitor cells; 143. EC migrates and coalesces to form a cord-like structure; c) subsequent vascular cord tubulogenesis to form a gold tube with a central lumen; d) existing vascular cord or blood vessel sprouts to form a secondary blood vessel; e) The original vascular plexus is further remodeled and reshaped; and f) the peri-endothelial cells are recruited to encapsulate the endothelial tube, thereby providing maintenance and regulation to the blood vessels; the cells include cells outside the small capillaries ( Pericyte), smooth muscle cells of larger blood vessels and cardiomyocytes in the heart. Hanahan, D. Seience 277:48-50 (1997); Hogan, BL· and Kolodziej, PA Nature Reviews Genetics·3:513-23 (2002); Lubarsky, B. and Krasnow, MA Cell·112:19-28 (2003). It has been well established that the pathogenesis of a variety of conditions involves angiogenesis. These conditions include solid tumors and metastases; atherosclerosis; post-lens fibrous tissue hyperplasia; hemangioma; chronic inflammation; intraocular neovascular diseases such as proliferative retinopathy (eg, 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); and Garner A., "Vascular diseases", Pathobiology A Dynamic Approach, Garner A., Klintworth GK, 2nd ed. (Marcel Dekker, NY, 1994), pp. 1625-1710. In the case of tumor growth, angiogenesis appears to be essential for the conversion of hyperplasia into tumor formation and nutrition for tumor growth and metastasis. Folkman 143939.doc -12- 201019961 et al, Nature 339:58 (1989). Neovascularization allows tumor cells to acquire growth advantage and pr〇liferative autonomy compared to normal cells. Tumors usually begin with a single abnormal cell that may only proliferate to a size of a few cubic millimeters due to the distance from the available capillary bed' and the tumor may remain "sleeping" for a long time without further growth and dissemination. Some tumor cells then transform into an angiogenic phenotype that activates endothelial cells, which proliferate and mature into new capillary blood vessels. These newly formed blood vessels not only enable the primary tumor to grow continuously, but also allow the metastatic tumor cells to spread and re-contribute (rec〇1〇nizati〇n). Accordingly, there has been a correlation between microvessel density in tumor sections and patient survival observed in breast cancer as well as in several other tumors. Take the example of N. Engl. J. Med 324:1-6 (1991); Horak et al., Lancet 340:1120-1124 (1992); Macchiarini et al., 1^11.61 340:145- 146 (1992). Although the exact mechanism controlling angiogenic transformation is not fully understood, the new gold tube production of the Xianxin tumor block is caused by a large number of chemistries generating a net balance of stimulating agents and inhibitors (Folkman, 1995, Nat. Med. 1(1) :27-31) The process of vascular development is strictly regulated. To date, a large number of molecules (mainly secreted factors produced by surrounding cells) have been shown to regulate EC differentiation, proliferation, migration and coalescence into a cord-like structure. For example, vascular endothelial growth factor (VEGF) has been identified as a key factor involved in stimulating angiogenesis and inducing vascular permeability. Ferrara et al., Endocr. Rev. 18:4_25 (1997). It was found that even a single VEGF allele loss can lead to embryonic death, indicating that this factor plays an irreplaceable role in vascular system development and differentiation. 143939.doc -13- 201019961 Role. In addition, VEGF has been shown to be a key mediator of neovascularization associated with tumors and intraocular disorders. Ferrara et al., Endocr. Rev. Ibid. Most human tumors examined overexpress VEGF mRNA. 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). Anti-VEGF neutralizing antibodies inhibit the growth of various human tumor cell lines in nude mice (Kim et al, Nature 362: 841-844 (1993); Warren et al, J. Clin. Invest. 95: 1789-1797 (1995) BorgstrSm et al, Cancer Res. 56:4032-4039 (1996); Melnyk et al, Cancer Res. 56:921-924 (1996)) and also inhibits intraocular angiogenesis in a model of ischemic retinopathy. Adamis et al., Arch. Ophthalmol. 114: 66-71 (1996). Thus, anti-VEGF monoclonal antibodies or other inhibitors of VEGF action are promising candidates for the treatment of tumors and various intraocular neovascular disorders. Such antibodies are described in, for example, EP 817,648, published Jan. 14, 998, and WO 98/45331 and WO 98/45332, issued Oct. 15, 1998. Cancer is one of the most deadly threats to human health. In the United States alone, nearly 1.3 million new patients suffer from cancer each year, and cancer is the second leading cause of death after cardiovascular disease, which accounts for about a quarter of the cause of death. Most of these deaths are caused by solid tumors. Although the medical treatment of certain cancers has made significant progress, the total five-year survival rate of all cancers has only increased by about 143939.doc •14·201019961 1 〇% over the past 20 years. Even if the treatment of cancer has made significant progress, it is still necessary to seek improved treatment. All references (including patent applications and publications) cited herein are hereby incorporated by reference in their entirety. SUMMARY OF THE INVENTION The present invention provides a combination therapy for treating a pathological condition such as cancer, wherein a c-met antagonist is combined with a VEGF antagonist, or wherein a c-met antagonist is combined with a VEGF antagonist and an EGFR antagonist, thereby Provides significant anti-tumor activity. In one aspect, the invention provides a method of treating cancer in a subject, the method comprising administering to the individual a therapeutically effective amount of a c-met antagonist and a VEGF antagonist. In another aspect, the invention provides a method of treating cancer in a subject, the method comprising administering to the individual a therapeutically effective amount of a c-met antagonist, a VEGF antagonist, and an EGFR antagonist. In certain embodiments, the VEGF antagonist is a compound that interferes with the binding of VEGF to a cellular receptor. Examples of such antagonists that block VEGF include, but are not limited to, soluble VEGF receptors, aptamers or peptides specific for VEGF, and anti-VEGF antibodies. In one embodiment, the anti-VEGF antibody is bevacizumab. In certain embodiments, when used in combination, administration of bevacizated in the range of from about 0.05 mg/kg to about 15 mg/kg Monoclonal antibody. In one embodiment, one or more of the following doses can be administered to an individual: about 0.5 mg/kg, 1 · 0 mg/kg, 2.0 mg/kg, 3.0 mg/kg '4.0 mg/kg, 5.0 mg/kg , 6.0 mg/kg, 143939.doc -15- 201019961 7.0 mg/kg, 7.5 mg/kg, 8.0 mg/kg, 9.0 mg/kg, 10 mg/kg or 15 mg/kg (or any combination thereof). The doses can be administered intermittently, for example once a day, every three days, every week or every two to three weeks. Examples of c-met antagonists include, but are not limited to, soluble c-met receptors, soluble HGF variants, aptamers or peptibodies that are specific for c-met or HGF, c-met small molecules, anti-c- Met antibody and anti-HGF antibody. In some embodiments, the c-met antagonist is an anti-c-met antibody. In some embodiments, the anti-c-met antibody is a one-armed antibody comprising an Fc region (ie, a heavy-bond variable domain and a light-bond variable domain form a single antigen-binding arm), wherein the Fc region comprises The first and second Fc polypeptides, wherein the first and second Fc polypeptides are present as a complex and form an Fc region that increases the stability of the antibody fragment compared to a Fab molecule comprising the antigen binding arm.

In some embodiments, the anti-c-met antibody comprises (a) a first polypeptide comprising a heavy chain variable domain comprising: EVQLVESGGGLVQPGGS LRLSCAASGYTFTSYWLHWVRQAPGKGLEWVGMIDPSNS DTRFNPNFKDRFTISADTSKNTAYLQMNSLRAEDTAVYYC ATYRSYVTPLDYWGQGTLVTVSS (SEQ ID NO: 10) > CHI sequence and first Fc polypeptide; (b) a second polypeptide comprising a light chain variable domain having the sequence: DIQMTQSPSSLSASVGDRVTITCKSSQSLLYT SSQKNYLAWYQQKPGKAPKLLIYWASTRESGVPSRFSGSG SGTDFTLTISSLQPEDFATYYCQQYYAYPWTFGQGTKVEIK R (SEQ ID NO: 11) and CL1 sequence; and (c) third comprising the second Fc polypeptide a polypeptide, wherein the heavy chain variable domain and the light chain variable domain are present in a complex form 143939.doc •16·201019961 and form a single antigen binding arm, wherein the first and second Fc polypeptides are present in a complex form and form An Fab molecule comprising the antigen binding arm is compared to an Fc region that increases the stability of the antibody fragment. In some embodiments, the first polypeptide comprises the Fc sequence depicted in Figure 1 (SEQ ID NO: 12) and the second polypeptide comprises the Fc sequence depicted in Figure 2 (SEQ ID NO: 13). In some embodiments, the first polypeptide comprises the Fc sequence depicted in Figure 2 (SEQ ID NO: 13) and the second polypeptide comprises the Fc sequence depicted in Figure 1 (SEQ ID NO: 12).

In some embodiments, the anti-c-met antibody comprises (a) a first polypeptide comprising a heavy chain variable domain, the polypeptide comprising the sequence EVQLVESGGGLVQPGGSLR LSCAASGYTFTSYWLHWVRQAPGKGLEWVGMIDPSNSDT RFNPNFKDRFTISADTSKNTAYLQMNSLRAEDTAVYYCAT YRSYVTPLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTS GGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPE VTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 14) ; (b) a second polypeptide comprising a light chain variable domain of the polypeptide comprising the sequence DIQMTQSPSSLSASVGDRVTITCKSSQSLLYTSSQKNYLAW YQQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFTLTIS SLQPEDFATYYCQQYYAYPWTFGQGTKVEIKRTVAAPSVFI 143939.doc -17 · 201019961 FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC (SEQ ID NO: 15); and the third polypeptide comprising a FC sequence , the polypeptide comprises the sequence CPPCPAPELLGGPSVFLFP P KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK VSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQV SLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK (SEQ ID NO: 13), wherein the heavy chain variable domain and a light chain variable domain form and form a single antigen binding arm is a complex form, wherein the first and second Fc polypeptides form complexes in the form of There is and forms an Fc region that increases the stability of the antibody fragment compared to a Fab molecule comprising the antigen binding arm. In one embodiment, the anti-c-met antibody comprises a heavy chain variable domain comprising the CDR1-HC, CDR2-HC and CDR3-HC sequences depicted in Figure 1 (SEQ ID NO: 4, 5 And/or 9) one or more. In some embodiments, the antibody comprises a light chain variable domain comprising the CDR1-LC, CDR2-LC and CDR3-LC sequences depicted in FIG. 1 (SEQ ID NO: 1, 2, and/or One or more of 3). In some embodiments, the heavy chain variable domain comprises the FR1-HC, FR2-HC, FR3-HC, and FR4-HC sequences depicted in Figure 1 (SEQ ID NO: 21-24). In some embodiments, the light chain variable domain comprises the FR1-LC, FR2-LC, FR3-LC, and FR4-LC sequences depicted in Figure 1 (SEQ ID NOS: 16-19). Other anti-c-met antibodies suitable for use in the methods of the invention are described herein in 143939.doc • 18 - 201019961 and are known in the art. In one aspect, the anti-c-met antibody comprises at least one feature that promotes heterodimerization of the Fc sequence within the antibody fragment while minimizing homodimerization. This (etc.) feature improves the yield and/or purity and/or homogeneity of the immunoglobulin population. In one embodiment, the antibody comprises an Fc mutation that constitutes "knobs" and "holes" as described in WO 2005/063816. For example, the mutation can be one or more of T366A, L368A and/or Y407V in the Fc polypeptide, and the cavity mutation can be T366W. In certain embodiments, the administered dose is about 15 mg/kg of 杬c_ met antibody every three weeks. In certain embodiments, the EGFR antagonist is erlotinib. In certain embodiments, a dose of 15 mg of erlotinib is administered daily for a three week period. In certain embodiments, a dose of 1 mg of erlotinib is administered daily for a three week period. In certain embodiments, a dose of 5 mg of erlotinib is administered daily for a three week period. The methods of the invention can be used to affect any suitable pathological condition. By way of example, the methods of the invention can be used to treat different cancers, solid tumors and fluid tumors, as well as soft tissue tumors. Non-limiting examples of cancers that can be treated with the present invention include breast cancer, colorectal cancer, rectal cancer, non-small cell lung cancer, non-Hodgkin's lymphoma (n〇n_H〇dgkins, renal cell carcinoma, prostate cancer, liver cancer ( Such as hepatocellular carcinoma), pancreatic cancer, soft, atherosclerosis, Kaposi's sarcoma (kap〇si, s... (3) off), carcinoid carcinoma, head and neck cancer, melanoma, ovarian cancer, stomach cancer , mesothelioma, and multiple myeloma. In some aspects, the cancer is metastatic 143939.doc • 19-201019961. In other aspects, the cancer is non-metastatic. In some embodiments, the resistance A c_met antibody (such as MetMAb) and an anti-VEGF antibody (such as bevacizumab) are used in combination therapy such as cancer of non-small cell lung cancer. In another embodiment, an anti-det antibody (such as MetMAb), an anti-VEGF antibody ( Such as bevacizumab) and egFr inhibitors (such as erlotinib) are used in combination therapies such as cancer of non-small cell lung cancer. Depending on the particular cancer indication to be treated, the combination therapy of the present invention may Other therapeutic agents A combination such as a chemotherapeutic agent or other therapy such as radiation therapy or surgery. Many known chemotherapeutic agents are useful in the group therapy of the present invention. In certain embodiments, the combination therapy of the present invention can be combined with a Combination of the above chemotherapeutic agents. In certain embodiments, a standard chemotherapeutic agent for treating a particular indication is used. In another embodiment, the dosage or frequency of each therapeutic agent used in the combination is equal to or less than the corresponding agent at Dosage or frequency when used under other agents. [Embodiment] L Definitions The term "hepatocyte growth factor" or "HGF" as used herein, unless otherwise indicated, refers to the ability to activate HGF/c-met signals. Any native or variant (native or synthetic) HGF polymorphism of the process under the conditions of the conduction pathway. The term "wild-type HGF" refers broadly to a polypeptide comprising the amino acid sequence of a naturally occurring HGF protein. The term "wild type" Sequence" - the amino acid sequence visible in naturally occurring HGF. (>111 is 11 (the known receptor for 317, biologically via this receptor) Now HGF intracellular signaling. 143939.doc • 20· 201019961 The term “HGF variant” as used herein refers to an HGF polypeptide comprising one or more amino acid mutations in the native HGF sequence. Amino acid mutagenesis includes, as appropriate, amino acid substitution. A "native sequence" polypeptide comprises a polypeptide having the same amino acid sequence as a polypeptide derived from nature. Thus, a native sequence polypeptide can have an amine of any mammalian naturally occurring polypeptide. The native sequence polypeptide may be isolated from nature or may be produced by recombinant or synthetic methods. The term "native sequence" polypeptide specifically encompasses the naturally occurring truncated or secreted form of the polypeptide (eg, extracellular domain sequences), the natural presence of the polypeptide Variant forms (or, for example, spliced forms) and naturally occurring allelic variants. A polypeptide "variant" means a biologically active polypeptide having at least about 80% amino acid sequence identity to a native sequence polypeptide. Such variants include, for example, polypeptides having one or more amino acid residues added or deleted at the N-terminus or C-terminus of the polypeptide. The variant will typically have at least about 80% amino acid sequence identity to the native sequence polypeptide, more preferably at least about 90% amino acid sequence identity, and even more preferably at least about 95% amino acid sequence identity. . "EGFR" means the receptor tyrosine kinase polypeptide epidermal growth factor receptor' is described in Ullrich et al, Nature (1984) 309:418425 'or as the Her-1 and c-erbB gene products; and Variants, such as EGFRvIII. Variants of EGFR also include deletions, substitutions, and insertional variants such as Lynch et al. (New England Journal of Medicine 2004, 350: 2129), Paez et al. (Science 2004, 304: 1497), Pao et al. (PNAS 2004). , 101:13306) variants described. “Biological samples” (interchangeable as r samples) or “tissue or cell-like 143939.doc •21 - 201019961 products” cover a wide range of sample types obtained from individual vessels and can be used for diagnostic or monitoring tests. It is intended to encompass blood and other liquid samples of biological origin, solid tissue samples (such as biopsy samples) or tissue cultures or cells obtained therefrom and their progeny. This definition also includes samples that have been manipulated in any manner after they have been obtained, such as manipulations, dissolution, or enrichment of certain components (such as proteins or polynucleotides) or entrapment for slicing purposes. In a semi-solid or solid matrix. The term "biological sample" encompasses clinical samples' and also includes cells in culture, cell supernatants, cell lysate beta plasma, biological fluids, and tissue samples. The source of the biological sample may be a solid tissue from fresh, cold; eastern and / or preserved organ or tissue samples or biological specimens or extracts; blood or any blood components; body fluids, such as cerebrospinal fluid, amniotic fluid, peritoneal fluid Or interstitial fluid; cells of an individual during pregnancy or at any time during development. In some embodiments, the biological sample is obtained from a primary or metastatic tumor. The biological sample may contain compounds which are not naturally intermixed with the tissue in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics or the like. An "anti-c-met antibody" is an antibody that binds with sufficient affinity and specificity. The selected antibody should generally be sufficient for c_met to have a strong binding affinity'. For example, §, antibody binding to human "met Kd value can be between 1 〇〇 nM-1 pM. Antibody affinity can be determined by the following method, for example based on surface A plasma resonance assay (such as the BlAcore assay described in PCT Application Publication No. WO 2005/012359), an enzyme-linked immunosorbent assay (ELISA), and a competition assay (eg, RIA). In some embodiments, An anti-c-met antibody is used as a therapeutic agent for dryness and interference with diseases or conditions involving c-met activity 143939. doc -22-201019961. Additionally, antibodies can be subjected to other biological activity assays, for example to assess their use as therapeutic agents The utility of these assays is known in the art and depends on the intended use of the target antigen and antibody. "c-met antagonists" (interchangeably referred to as "c-met inhibitors") are interference c -met activation or functional agent. Examples of c-met inhibitors include c-met antibodies, HGF antibodies, small molecule c-met antagonists, c-met tyrosine kinase inhibitors, antisense and inhibitory RNA (eg, shRNA) molecules (see, eg, WO 2004). /87207). The c-met inhibitor is preferably an antibody or a small molecule that binds to c-met. In a particular embodiment, the binding affinity (dissociation constant) of the c-met inhibitor to c-met is about 1, 〇〇〇 nM or lower. In another embodiment, the binding affinity of the c-met inhibitor to c-met is about 100 nM or less. In another embodiment, the binding affinity of the c-met inhibitor to c-met is about 50 nM or less. In a specific embodiment, the c-met inhibitor is covalently bound to c-met. In a specific embodiment, the c-met inhibitor inhibits c-met signaling with an IC50 of 1,000 nM or less. In another embodiment, the c-met inhibitor inhibits c-met signaling with an IC50 of 500 nM or less. In another embodiment, the c-met inhibitor inhibits c-met signaling with a 1 C50 of 50 nM or less. In certain embodiments, the c-met antagonist reduces or inhibits the expression level or biological activity of c-met by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or higher. "C-met activation" refers to the activation or filling of the c-met receptor. C-met activation generally results in signal transduction (e. g., caused by c-met or the intracellular kinase domain of the c-met receptor phosphorylating tyrosine residues in the polypeptide). C-met activation can be mediated by a c-met ligand (HGF) that binds to the relevant c-met receptor. Binding of HGF to c-143939.doc •23-201019961 met activates the kinase domain of c-met, resulting in phosphorylation of valine residues in c-met and/or tyrosine residues in other receptor polypeptides Phosphorylation. The term "VEGF" or "VEGF-A" is used to mean 165-amino acid human as described by Leung et al, Science, 246: 1306 (1989) and Houck et al, Mol. Endocrin., 5: 1806 (1991). Vascular endothelial growth factor and related 12 amino acid, 189-amino acid and 206-amino acid human vascular endothelial growth factor, as well as naturally occurring allelic forms and processed forms thereof. VEGF-A is part of a gene family including VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F and P1GF. VEGF-A mainly binds to two high-affinity receptor tyrosine kinases, namely VEGFR-1 (Flt-1) and VEGFR-2 (Flk-1/KDR), which is the cell division signal of vascular endothelial cells of VEGF-A. The main pass quality. In addition, neuropilin-1 has been identified as a receptor for heparin-binding VEGF-A isoforms and may play a role in vascular development. The term "VEGF" or "VEGF-A" also refers to VEGF from non-human species such as mice, rats or primates. VEGF from a particular species is sometimes indicated by terms such as hVEGF (referred to as human VEGF) or mVEGF (referred to as murine VEGF). The term "VEGF" is also used to refer to a truncated form or fragment of a polypeptide comprising amino acid 8 to 109 or 1 to 109 of the 165-amino acid human endothelium cell growth factor. In the present application, the reference to any such form of VEGF can be identified, for example, by "VEGF (8-109)", "VEGF (1-109)" or "VEGF165". The amino acid position of the native VEGF that is "truncated" is numbered as indicated in the native VEGF sequence. For example, the amino acid position 17 (methionine) in the native VEGF of 143939.doc -24-201019961 is also position 17 (methionine) in native VEGF. The binding affinity of the truncated native VEGF to KDR and Flt-1 receptors is comparable to native VEGF. The term "VEGF variant" as used herein refers to a VEGF polypeptide comprising one or more amino acid mutations in the native VEGF sequence. The one or more amino acid mutations optionally include amino acid substitutions. It should be noted that for the purpose of abbreviating the designation of the VEGF variant described herein, the numbering refers to the position of the amino acid residue along the putative amino acid sequence of the native VEGF (provided in Leung et al., supra and Houck et al. Ibid.) "VEGF biological activity" includes binding to any VEGF receptor' or any VEGF signaling activity, such as regulation of normal and abnormal angiogenesis and vasculogenesis (Ferrar a and Davis-Smyth (1997) Endocrine Rev. 18 : 4-25; Ferrara (1999) J. Mo/. Mei 77: 527-543); promotes embryonic angiogenesis and angiogenesis (Carmeliet et al., (1996) iVaiwre 380: 435-439; Ferrara et al., 1996 380: 439-442); and regulation of periodic vascular proliferation, bone growth and cartilage formation in the female reproductive tract (Ferrara et al., (1998) MeA 4:336-340; Gerber et al., (1999) iVaiwre

Mei 5: 623-628). As a pleiotropic growth factor, VEGF, in addition to its angiogenic factors in angiogenesis and angiogenesis, also exhibits a variety of biological effects in other physiological processes such as endothelial cell survival, vascular infiltration, and ductless expansion. Nuclear cell chemotaxis and maternal inflow (Ferrara and Davis-Smyth (1997), supra; and Cebe-Suarez et al., Ce//. Mo/, h/e & ζ·. 63:601-615 (2006) ). In addition, recent studies have reported that 143939.doc -25-201019961 VEGF has a cell division effect on a few non-endothelial cell types such as retinal pigment epithelial cells, pancreatic duct cells, and Schwann cells. Guerrin et al., (1995) 乂Ce// Ρ;2>;5ζ·ο/. 164:385-394; Oberg-Welsh^ AJ (1997) Mol. Cell. Endocrinol. 126:125-132 ; Sondell^- A * (1999) Neurosci. 19:573 1-5740 o "Angiogenesis inhibitor" or "anti-angiogenic agent" refers to a small molecular weight substance that directly or indirectly inhibits angiogenesis, angiogenesis or undesired vascular penetration. Nucleic acid, polymorphism, isolated protein, recombinant protein, antibody or combination thereof or fusion protein. It will be appreciated that an anti-angiogenic agent includes an agent that binds to and blocks the angiogenic activity of an angiogenic factor or its receptor. For example, an anti-angiogenic agent is an antibody or other antagonist of an angiogenic agent as defined above, such as an antibody to a VEGF-A or VEGF-A receptor (eg, a KDR receptor or a Flt-Ι receptor), Anti-PDGFR inhibitor (such as GLEEVEC® (Imatinib Mesylate)). Anti-angiogenic agents also include pro-angiogenic inhibitors such as angiostatin, endostatin and the like. See, for example, Klagsbrun and D'Amore, P/jyno/., 53:217-39 (1991); Streit and Detmar, 22:3172-3179 (2003) (eg, Table 3 lists anti-angiogenic therapies for malignant melanoma) ); Ferrara and Alitalo, iVaiwre Me Hawthorn 5: 1359-1364 (1999); Tonini et al, 22: 6549-6556 (2003) (for example, Table 2 lists known anti-angiogenic factors); and Sato. «ί· «/. C/ζ'π. 8:200-206 (2003) (for example, Table 1 lists anti-jk tube generators used in clinical trials). "VEGF antagonist" means a molecule that is capable of neutralizing, blocking, inhibiting, eliminating, 143939.doc -26-201019961 reducing or interfering with VEGF activity, including its binding to one or more VEGF receptors (peptidyl or non-peptide) Peptidyl). In certain embodiments, the VEGF antagonist reduces or inhibits the expression level or biological activity of VEGF by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more high. In one embodiment, the VEGF that is inhibited by the VEGF antagonist is VEGF (8-109), VEGF (1-109), or VEGF165. VEGF antagonists suitable for use in the methods of the invention include peptidyl or non-peptidyl compounds that specifically bind to VEGF, such as anti-VEGF antibodies and antigen-binding fragments thereof, polypeptides that specifically bind VEGF or fragments thereof, and specifically bind to VEGF Isolates a receptor molecule and derivative thereof that binds to one or more receptors (eg, a soluble VEGF receptor protein or a VEGF-binding fragment thereof, or a chimeric VEGF receptor protein); is complementary to at least a fragment of a nucleic acid molecule encoding a VEGF polypeptide An antisense nucleobase oligomer; a small RNA complementary to at least a fragment of a nucleic acid molecule encoding a VEGF polypeptide; a ribozyme that targets VEGF; a peptibody of VEGF; and a VEGF aptamer. An "anti-VEGF antibody" is an antibody that binds VEGF with sufficient affinity and specificity. The selected anti-sputum generally has a sufficiently strong binding affinity for VEGF, e.g., the antibody binding to hVEGF may have a Kd value between 100 nM and 1 pM. Antibody affinity can be determined by methods such as surface plasma resonance based assays (such as the BIAcore assay described in PCT Application Publication No. WO 2005/012359), enzyme-linked immunosorbent assay (ELISA), and competition assays (eg, RIA). ). In certain embodiments, an anti-VEGF antibody of the invention can be used as a therapeutic agent that targets and interferes with a disease or condition involving VEGF activity. Alternatively, the antibody can be subjected to other biological activity assays, e.g., as 143939.doc -27-201019961 for its utility as a therapeutic. Such assays are known in the art and depend on the intended use of the antigen and antibody. Examples include HUVEC inhibition assays (as described in the Examples below); tumor cell growth inhibition assays (eg, as described in WO 89/06692); antibody-dependent cellular cytotoxicity (ADCC) and complement-mediated cytotoxicity (CDC) Assay (US Patent 5,5,0,362); and agonistic activity or hematopoietic assay (see w〇95/27062) anti-VEGF antibodies should generally not bind to other VEGF homologs (such as VEGF-B or VEGF-C), nor Binding to other growth factors (such as P1GF, PDGF or bFGF). In certain embodiments, the anti-VEGF antibody comprises a monoclonal antibody that binds to the same epitope as a monoclonal anti-VEGF antibody A4.6.1 produced using the fusionoma ATCC HB 10709. According to Presta et al., and recombinant humanized anti-VEGF monoclonal antibody produced at 57:4593-4599 (1997). In one embodiment, the anti-VEGF antibody is "bevacizumab (BV)", also known as "rhuMAb VEGF" or "AVASTIN®", which comprises a mutant human IgG1 framework region and an antigen binding complementarity determining region from a murine anti-hVEGF monoclonal antibody A.4.6.1 that blocks binding of human VEGF to its receptor. Approximately 93% of the amino acid sequence resistant to the package The majority of the framework regions are derived from human IgG1, and about 7% of the sequence is derived from murine antibody A4.6.1. The molecular mass of bevacizumab is about 149,000 Daltons, and it is glycosylated. Valamycin has been approved by the FDA in combination with a chemotherapy regimen for the treatment of metastatic colorectal cancer (CRC) and non-small cell lung cancer (NSCLC). Hurwitz et al., N. Engl. J. Med. 350:2335-42 ( 2004); Sandler# A » N. Engl. J. Med. 3 55:2542-50 (2006). Currently, bevacizumab is in the process of many 143939.doc -28 · 201019961 ongoing treatment for various cancer adaptations In the study of clinical trials, Kerbel, */. C/z«. Owco/· 19:45S-5 IS (2001); De Vore et al. A 5 Proc. Am. Soc. Clin. Oncol. 19:485a. (2000); Hurwitz et al., C//«· Cawcer 6:66-69 (2006); Johnson et al., Proc. Jm. C7i«. 20:315a (2001); Kabbinavar et al., /. Once? /· 21:60-65 (2003); Miller et al.

Ca«. and ej. 7>eai. 94: Zengzi ij 1:S6 (2005) o Bevacizumab and other humanized anti-VEGF antibodies are further described in US Patent No. 6,884,879, issued February 26, 2005 No. Other antibodies include G6 or Β20 series antibodies (e.g., G6-31, Β20-4.1) as described in PCT Publication No. WO 2005/012359, PCT Publication No. WO 2005/044853, and U.S. Patent Application Serial No. 60/991,302. The contents of these patent applications are expressly incorporated herein by reference. For other antibodies, see U.S. Patent Nos. 7,060,269, 6,582,959, 6, 703, 020, 6, 054, 297, WO 98/45332, WO 96/30046, WO 94/10202, ΕΡ 0666868Β1, and U.S. Patent Application Publication No. 2006009360, No. 9 20050186208, No. 20030206899, No. 20030190317, No. 20030203409, and No. 20050112126; and Popkov et al, Journal of Immunological Methods 288: 149-164 (2004) ° Other antibodies including binding to human VEGF containing residues F17 M18, D19, Y21, Y25, Q89, 191, K101, E103 and C104 or an antibody comprising functional epitopes of residues F17, Y21, Q22, Y25, D63, 183 and Q89. The "G6 series antibody" according to the present invention is a G6 antibody or G6 derived from any one of Figures 7, 24-26 and 34-35 according to PCT Publication No. 143939.doc -29-201019961 WO 2005/012359. Anti-VEGF antibodies that are derived from the sequence of the antibody, the entire disclosure of which is expressly incorporated herein by reference. See also PCT Publication No. WO 2005/044853, the entire disclosure of which is expressly incorporated herein by reference. In one embodiment, the G6 series antibody binds to a functional epitope of residues F17, Y21, Q22, Y25, D63, 183 and Q89 on human VEGF. The "B20 series anti-caries" according to the present invention is an anti-VEGF antibody derived from the sequence of the B20 antibody or the B20-derived antibody according to any one of Figures 27-29 of PCT Publication No. WO 2005/012359, the disclosure of which is The entire disclosure is expressly incorporated herein by reference. See also PCT Publication No. WO 2005/044853 and U.S. Patent Application Serial No. 60/991, the disclosure of each of which is expressly incorporated by reference. In one embodiment, the B20 series of antibodies bind to functional determinants of residues F17, M18, D19, Y21, Y25, Q89, 191, K101, E103 and C104 on human VEGF. The "functional epitope" according to the present invention means an amino acid residue in an antigen which contributes to antibody binding in energy. Mutations in residues that are energy-dependent in any of the antigens (eg, mutations in wild-type VEGF due to mutations in alanine or homologs) will disrupt antibody binding, resulting in a relative affinity ratio of antibodies (IC50/ of mutant VEGF) The wild type VEGF will have an IC50) greater than 5 (see Example 2 of WO 2005/012359). In one embodiment, the relative affinity ratio is determined by the solution-bound phage displaying an ELISA. Briefly, the antibody to be tested at a concentration of 2 pg/ml in PBS at 4 ° C 143939.doc -30· 201019961

The Fab format was coated with 96-well Maxisorp immunoplate (NUNC) overnight and blocked with PBS, 0.5 〇/〇 BSA and 0·05ο/〇 Tween 20 (PBT) for 2 hours at room temperature. First, phage were incubated on Fab-coated plates at room temperature to present serial dilutions of hVEGF alanine point mutant (residue 8-109 format) or wild-type hVEGF (8-109) in PBT for 15 minutes, and The plates were washed with PBS, 0.05% Tween 20 (PBST). The anti-M13 monoclonal antibody horseradish peroxidase (Amer sham Pharmacia) conjugate diluted 1:5000 in PBT was used to detect bound phage using 3,3',5,5'-tetramethylbenzidine (TMB, Kirkegaard & Perry Labs, Gaithersburg, MD) was subjected to color development for about 5 minutes, stopped with 1.0 M H3P〇4 and read spectrophotometrically at 45 0 nm. The ratio of IC50 values (IC50, ala/IC50, wt) represents a fold reduction in binding affinity (relative binding affinity). "EGFR antagonists" (interchangeably referred to as "EGFR inhibitors") are agents that interfere with c-met activation or function. Examples of EGFR inhibitors include EGFR antibodies, EGFR ligand antibodies, small molecule EGFR antagonists, EGFR tyrosine kinase inhibitors, antisense and inhibitory RNA (e.g., shRNA) molecules (see, e.g., WO 2004/87207). The EGFR inhibitor is preferably an antibody or small molecule that binds to EGFR. In some embodiments, the EGFR inhibitor is an EGFR-targeted drug. In a specific embodiment, the binding affinity (dissociation constant) of the EGFR inhibitor to EGFR is about 1,000 nM or less. In another embodiment, the binding affinity of the EGFR inhibitor to EGFR is about 100 nM or less. In another embodiment, the binding affinity of the EGFR inhibitor to EGFR is about 50 nM or less. In a specific embodiment, the EGFR inhibitor is covalently bound to EGFR. In a specific embodiment, 143939.doc -31-201019961 EGFR inhibitors inhibit EGFR signaling with an IC50 of 1,000 nM or less. In another embodiment, the EGFR inhibitor inhibits EGFR signaling with an IC50 of 500 nM or less. In another embodiment, the EGFR inhibitor inhibits EGFR signaling with an IC50 of 50 nM or less. In certain embodiments, the EGFR antagonist reduces or inhibits the expression level or biological activity of EGFR by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more high. "EGFR activation" refers to activation or phosphorylation of EGFR. EGFR activation generally results in signal transduction (e. g., caused by the intracellular kinase domain of EGFR receptor phosphorylated tyrosine residues in EGFR or a polypeptide of interest). EGFR activation can be mediated by binding of an EGFR ligand to an EGFR dimer comprising EGFR. Binding of an EGFR ligand to an EGFR dimer activates one or more kinase domains of a EGFR in a dimer, thereby causing phosphorylation of tyrosine residues in one or more EGFR and/or other receptor polypeptides Phosphorylation of tyrosine residues. The term "drug targeted to EGFR" as used herein refers to a therapeutic agent that binds to EGFR and inhibits EGFR activation. Examples of such agents include antibodies and small molecules that bind EGFR. Examples of antibodies that bind to EGFR include MAb 579 (ATCC CRL HB 8506), MAb 455 (ATCC CRL HB8507), MAb 225 (ATCC CRL 8508), MAb 528 (ATCC CRL 8509) (see US Patent No. 4,943,533 to Mendelsohn et al.) And its variants, such as chimeric 225 (C225 or Cetuximab; ERBUTIX®) and reshaped human 225 (H225) (see WO 96/40210, Imclone Systems Inc.); IMC-11F8, A fully 143939.doc-32-201019961 human antibody (Imcl〇ne) to EGFR; an antibody that binds to a π-type mutant EGFR (U.S. Patent No. 5,212,290); as described in U.S. Patent No. 5,891,996, incorporated herein by reference. Humanized and chimeric antibodies; and human antibodies that bind to EGFR, such as ABX-EGF (see WO 98/50433, Abgenix); EMD 55900 (Stragliotto et al, */. Cawcer 32A: 636-640 (1996)); EMD7200 (Matuzumab), a humanized EGFR antibody against EGFR that competes with EGF and TGF-α for binding to EGFR; and mAb 806 or humanized mAb 806 (Johns et al., */··δζ· ο/· C/iewi· 279(29): 30375-30384 (2004)). The anti-EGFR antibody can be combined with a cytotoxic agent to produce an immunoconjugate (see, e.g., EP 659, 439 A2, Merck Patent GmbH). Examples of small molecules that bind to EGFR include ZD1839 or gefitinib (IRESSA; Astra Zeneca); CP-358774 or erlotinib (TARCEVATM; Genentech/OSI); and AG1478, AG1571 (SU 5271; Sugen); EMD- 7200. The phrase "gene amplification" refers to a method of forming multiple copies of a gene or gene fragment in a particular cell or cell line. The amount of messenger RNA (mRNA) produced by the replication region (a piece of amplified DNA), often referred to as the "amplicon" (ie, the amount of gene expression), is usually also the number of copies produced by the particular gene being expressed. Proportionate increase. A "tyrosine kinase inhibitor" is a molecule that inhibits the tyrosine kinase activity of a tyrosine kinase (such as a c-met receptor) to some extent. "A cancer or biological sample showing c-met and/or EGFR expression, amplification or activation is a manifestation (including overexpression) of c_met and/or EGFR in a diagnostic test, with an amplified c-met and/or EGFR gene And/or show c-met and / 143939.doc • 33· 201019961 or a sample of activation or phosphorylation of EGFR. “A cancer or biological sample that does not show c-met and/or EGFR expression, amplification or activation” is Samples that do not exhibit (including overexpression) C-met and/or EGFR, have no amplified c_inet; and/or EGFR genes and/or do not display activation or phosphorylation of c-met and/or EGFR. A cancer or biological sample that shows "c-met and/or EGFR activation" is a sample that exhibits activation or acidification of c-met and/or EGFR in a diagnostic test. This activation can be determined directly (e.g., by measuring c-met and/or EGFR acidification using ELISA) or indirectly. A cancer or biological sample that does not display c-met and/or EGFR activation is a sample that does not exhibit activation or acidification of c-met & / / EGFR in diagnostic tests. This activation can be determined directly (e.g., by measuring c-met and/or EGFR squaring using ELISA) or indirectly. A cancer or biological sample that does not display c-met and/or EGFR amplification is a sample that does not have an amplified c-met and/or EGFR gene in a diagnostic test. A cancer or biological sample showing "c-met and/or EGFR amplification" is a sample having an amplified c-met and/or EGFR gene in a diagnostic test. As used herein, "phosphoryl-ELIS A assay" is the use of a reagent, typically an antibody, in an enzyme-linked immunosorbent assay (ELISA) to detect c-met & / or EGFR, a receptor or a downstream signaling molecule, thereby A assay to assess phosphorylation of one or more c-mets and/or EGFR. Preferably, antibodies that detect phosphorylated c-met and/or EGFR are used. This assay can be performed on cell lysates preferably from fresh or frozen biological samples. The cancer cells of "c-met and/or EGFR overexpressed or expanded" have significant levels of c-met and/or EGFR protein or gene compared to non-cancerous cells of the same 143939.doc -34-201019961 tissue type. Higher cells. This overexpression can be caused by gene amplification or increased transcription or translation. Overexpression or amplification of c-met and/or EGFR can be determined in a diagnostic or prognostic assay by assessing the increase in the amount of c-met and/or EGFR protein present on the cell surface (eg, via immunohistochemical assay; IHC) . Alternatively or additionally, for example, via fluorescence in situ hybridization (FISH; see WO 98/45479, published October 1998), southern blotting or polymerase chain reaction (PCR) techniques. (such as quantitative real-time PCR (qRT-PCR)) to measure the amount of c-met and/or EGFR-encoding nucleic acid in a cell. In addition to the above tests, skilled individuals can also use a variety of in vivo tests. For example, cells in a patient can be exposed to antibodies that optionally detect detectable labels (eg, radioisotopes) and can be obtained by, for example, external scanning of radioactivity or by analysis of patients previously exposed to antibodies. The assay assesses the binding of the antibody to the patient's cells. A cancer cell that does not overexpress or amplify c-met and/or EGFR is a cell with a c-met and/or EGFR protein or gene content that is not higher than normal cells compared to a non-cancerous cell of the same tissue type. . The term "mutation" as used herein means that the amino acid or nucleic acid sequence of a particular protein or nucleic acid (gene, RNA) differs from the wild type protein or nucleic acid, respectively. A mutein or nucleic acid can be expressed from or can be found on one or both alleles of a gene (homozygous) or both alleles, and can be a somatic cell line or a germ cell line . In the present invention, the mutation is generally a somatic mutation. Mutations include sequence rearrangements, such as insertions, deletions, and point mutations (including single nucleotides/amino acid polymorphisms). "Inhibition" is the reduction or decrease in activity, function and/or amount compared to a reference value. Protein "performance" refers to the conversion of information encoded in a gene into messenger RNA (mRNA), which is then converted to protein. As used herein, a sample or cell that "expresses" a related protein (such as a HER receptor or a HER ligand) is a sample or cell that determines the presence of mRNA or protein (including fragments thereof) encoding the protein. "Immunoconjugate" (interchangeably referred to as "antibody-drug conjugate" or "ADC") means an antibody that binds to one or more cytotoxic agents, such as chemotherapeutics, drugs, growth. Inhibitors, toxins (eg, protein toxins, bacterial, fungal, enzymatically active toxins or fragments thereof of plant or animal origin) or radioisotopes (ie, radioconjugates). The phrase "antigen-binding arm" as used herein refers to a component of an antibody fragment of the present invention which is capable of specifically binding to a related target molecule. The antigen binding arm is generally and preferably a complex of immunoglobulin polypeptide sequences (e.g., CDRs and/or variable domain sequences of immunoglobulin light and heavy chains). As used herein, the phrase "N-terminal truncated heavy chain" refers to a polypeptide comprising a portion, but not all, of the full-length immunoglobulin heavy chain, wherein the deleted portion is the portion of the heavy chain end region. The missing portion may include, but is not limited to, a variable domain, a CH1, and a portion or all of the hinge sequence. In general, if no wild-type hinge sequence is present, the remaining constant domain of the N-terminal truncated heavy chain should comprise a component capable of binding another Fc sequence (ie, a "first" Fc polypeptide as described herein). The component may be a modified residue capable of forming a double 143939.doc -36·201019961 sulfur bond or an added cysteine residue. The term "Fc region" as used herein generally refers to a heavy immunoglobulin-containing molecule. A dimeric complex of a C-terminal polypeptide sequence of a chain, wherein the c-terminal polypeptide sequence is a sequence obtainable by papain digestion of an intact antibody. The Fc region may comprise a native or variant Fc sequence. Although the immunoglobulin heavy chain is pc The boundaries of the sequences may vary, but the human IgG heavy chain Fc sequence is generally defined as an amino acid residue at position Cys226 or from the approximate position pr〇230 to the carboxy terminus of the Fc sequence. Generally, two constant domains, namely a CH2 domain and a CH3 domain, and optionally a CH4 domain are included. Herein, "Fc polypeptide" means a polypeptide constituting an Fc region. The Fc polymorphism can be derived from, for example, IgG 1, IgG2, IgG3 or IgG4 subtype, IgA, IgE Any suitable immunoglobulin of IgD or IgM is obtained. In some embodiments, the Fc polypeptide comprises part or all of a wild-type hinge sequence (generally at the N-terminus). In some embodiments, the Fc polypeptide does not comprise a functional or wild type Hinge sequences. The terms "Fc receptor" and "FcR" are used to describe a receptor that binds to the Fc region of an antibody. For example, 'FcR can be a native sequence human FcR. In general, FcR is a binding IgG antibody (gamma receptor) And includes Fc (RI, Fc (RII and Fc) receptors of the RIII subclass (including allelic variants of these receptors and additional spliced forms) FcR. Fc (RII receptors include Fc (RIIA ( "Activated receptors" and Fc (RIIB ("inhibiting receptors"), Fc (RIIA and Fc (RIIB has a similar amino acid sequence' mainly differs in its cytoplasmic domain. Other isotypes of immunoglobulins may also be caused by certain FcRs Binding (see, for example, Jane Way et al, Immuno Biology. The immune system in health and disease, (Elsevier Science Ltd., NY) (4th ed., 1999)). Activated receptor Fc 143939.doc 37 · 201019961 (RIIA Immunoreceptor based on its cytoplasmic domain The immunoreceptor tyrosine-based activation motif (ITAM) inhibits receptor Fc (RIIB contains immunoreceptor tyrosine-based inhibition motifs (ITIM) in its cytoplasmic domain). (.^ idL Daeron, Annu. Rev. Immunol. 15:203-234 (1997)). FcR is reviewed in Ravetch and Kinet, .*

Immunol 9:457-92 (1991); Capel^A. » Immunomethods 4:25-34 (1994); and de Haas et al, J. Lab. Clin. Med. 126:330-41 (1995). The term "FcR" as used herein encompasses other FcRs, including FcR to be identified in the future. The term also includes neonatal receptor FcRn, which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., /. 117:587 (19*76); and Kim et al., X 24:249). (1994)) 0 "Hinge region", "hinge sequence" and variations thereof as used herein includes meanings known in the art, which are illustrated, for example, by Jane Way et al., Immuno Biology · the immune system in health And disease, (Elsevier Science Ltd., NY) (4th ed., 1999); Bloom et al, Protein Science (1997), 6: 407-415; Humphreys et al, J. Immunol. Methods (1997), 209: 193-202. An "agonist antibody" as used herein is an antibody that mimics at least one functional activity of a related polypeptide (e.g., HGF). Throughout this specification and the scope of the patent application, the numbering of residues in the immunoglobulin heavy chain is Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, 143939.doc -38-201019961

National Institutes of Health, Bethesda, Md. (1991), index number, which is expressly incorporated herein by reference. "EU index in Kabat" refers to the number of residues in human [gGi EU antibodies. The term "antibody" is used in its broadest sense and specifically covers monoclonal antibodies ' (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (eg, bispecific antibodies), monovalent antibodies, multivalent antibodies, and antibody fragments. As long as it exhibits the desired biological activity. The "antibody fragment" contains only a portion of the intact antibody, wherein the portion preferably retains at least one, preferably most or all of the functions normally associated with the portion when present in the intact antibody. In one embodiment, the antibody fragment comprises the antigen binding site of the intact antibody, thereby retaining the ability to bind antigen. In another embodiment, an antibody fragment (eg, an antibody fragment comprising an Fc region) retains at least one biological function normally associated with a region when present in an intact antibody, such as FcRn binding, antibody half-life regulation, ADC (: function and In one embodiment, the antibody fragment is a monovalent antibody having a half-life in vivo substantially similar to an intact antibody. For example, the antibody fragment may comprise a sequence that is capable of conferring in vivo stability to the fragment. The antigen binding arm. In one embodiment, the antibody of the invention is a one-armed antibody as described in W〇2〇〇5/〇63816. In the embodiment, the one-armed antibody comprises a composition such as

The Fc mutations of "杵" and "白" described in W〇 2005/063816. For example, the canine can be one or more of T366A, L368A&/ or Y4〇7V in the Fc polypeptide, and the hole mutation can be T366W. A "blocking" antibody or antibody "antagonist" is an antibody that inhibits or reduces the biological activity of the original antibody against 143939.doc-39-201019961. Preferably, the blocking antibody or antagonist antibody completely inhibits the biological activity of the antigen. The "functional antigen binding site" of an antibody is a site capable of binding to an antigen-free site. Although the antigen binding affinity of the antigen binding site does not have to be as strong as the parent antibody from which the antigen binding site is derived, the ability to bind the antigen must be able to use any of the methods known for assessing the binding of the antibody to the antigen. Measure. Furthermore, the antigen binding affinities of the various antigen binding sites of the multivalent antibodies herein need not be identical in number. For the multimeric antibodies herein, the number of functional antigen binding sites can be assessed using ultracentrifugation analysis as described in Example 2 of U.S. Patent Application Publication No. 20050186208. According to this analytical method, different ratios of dry antigen to multimeric antibody are combined, and assuming a different number of functional binding sites to calculate the average molecular weight of the complex. These theoretical values are compared to the actual experimental values obtained to assess the number of functional binding sites. A "species-dependent antibody" is an antibody that binds to an antigen from a first mammalian species more strongly than to an antigenic homologue from a second mammalian species. Although species-dependent antibodies typically "specifically bind" to a human antigen (ie, binding affinity (a value of κ〇 does not exceed about 〇X 1〇·7 Μ, preferably does not exceed about lxl0_8 M, and optimally does not exceed about ^1) 〇 9 M), but the binding affinity for an antigen homolog from a second non-human mammalian species is at least about 5 fold or at least about 500 fold, or at least about 1000 fold weaker than the binding affinity for the human antigen. A sex antibody can be any of the various types of antibodies as defined above. In one embodiment, the 'species-dependent antibody is a humanized or human antibody. 143939.doc • 40- 201019961 "Antibody" as used herein "Mutant" or "antibody variant" refers to an amino acid sequence variant of a species-dependent antibody in which one or more amino acid residues of a species-dependent antibody have been modified. These mutants are necessarily species dependent The antibody has a sequence identity or similarity of less than 100%. In one embodiment, the antibody mutant will have at least 75% of the amino acid sequence of the heavy or light chain variable domain of the species dependent antibody. More preferably an amino acid sequence of at least 80%, more preferably at least 85%, more preferably at least 90%, and most preferably at least 95% amino acid sequence identity or similarity. For the sequence or similarity In this context, it is defined that after sequence alignment and, if necessary, introducing a gap to achieve a maximum sequence identity percentage, the candidate sequence is identical (ie the same residue) or similar (ie from a The percentage of amino acid residues of the same group of amino acid residues of the common side chain character, see below). N-terminal, C-terminal or internal extension, deletion or insertion in the antibody sequence outside the variable domain It should be considered to affect sequence identity or similarity. A "chimeric VEGF receptor protein" is a VEGF receptor molecule having an amino acid sequence derived from at least two different proteins, at least one of which is a VEGF receptor protein. In certain embodiments, a chimeric VEGF receptor protein is capable of binding to VEGF and inhibiting its biological activity. Unless otherwise indicated, the expression "multivalent antibody" is used throughout the specification to indicate that it comprises three or more An antibody to the original binding site. The multivalent antibody is preferably engineered to have three or more antigen binding sites' and is generally not a native sequence IgM or IgA antibody. The "Fv" fragment is a complete antigen recognition and binding. Site antibody fragment 143939.doc -41 · 201019961. This region consists of a tightly associated heavy chain variable domain and a light chain variable domain dimer, which can be a total of Valuable, for example in scFv. In this configuration, the three CDRs of each variable domain interact to define an antigen binding site on the surface of the Vh-V1 dimer. Overall, six CDRs or their sub- The set confers antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three antigen-specific CDRs) has the ability to recognize and bind antigen, but its affinity is generally lower than the entire binding site. As used herein, "antibody variable domain" refers to a portion of the light and heavy chains of an antibody molecule, including the complementarity determining regions (CDRs; ie, CDR1, CDR2 and CDR3) and the amino acid sequence of the framework region (FR). . VH refers to the variable domain of the heavy chain. VL refers to the variable domain of the light chain. According to the method used in the present invention, the position of the amino acid assigned to the CDR and FR can be defined according to Kabat (Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md., 1987 and 1991)). The amino acid number of the antibody or antigen-binding fragment is also numbered according to Kabat. The term "complementarity determining region" (CDR; i.e., CDR1, CDR2, and CDR3) as used herein refers to an amino acid residue that is necessary for antigen binding in an antibody variable domain. Each variable domain typically has three CDR regions identified as CDR1, CDR2 and CDR3. Each of the complementarity determining regions may comprise an amino acid residue from a "complementarity determining region" as defined by Kabat (i.e., about residues 24-34 (L1), 50-56 (L2) and 89 in the light chain variable domain. -97 (L3), and heavy chain variable domains 31-35 (HI), 50-65 (H2), and 95-102 (H3); Kabat et al., Sequences of Proteins of Immunological 143939.doc -42· 201019961 TVziereW, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD. (1991)) and/or residues from the "hypervariable loop" (ie, about 26-32 residues in the light chain variable domain) (L1), 50-52 (L2) and 91-96 (L3), and heavy chain variable domains 26-32 (HI), 53-55 (H2) and 96-101 (H3); Chothia and Lesk, ·/. Mo/· 5/〇/. 196:901-917 (1987)). In some cases, the complementarity determining region can include an amino acid from a CDR region and a hypervariable loop as defined by Kabat. For example, the CDRH1 of the heavy chain of antibody 4D5 includes amino acids 26 to 35. A "framework region" (hereinafter referred to as FR) is a variable domain residue other than a CDR residue. Each variable domain typically has four FRs identified as FR1, FR2, FR3, and FR4. If the CDRs are defined according to Kabat, the light chain FR residues are located at about residues 1-23 (LCFR1), 35-49 (LCFR2), 57-88 (LCFR3), and 98-107 (LCFR4), and the heavy chain FR remains The base is located at about residues 1-30 (HCFR1), 36-49 (HCFR2), 66-94 (HCFR3), and 103-113 (HCFR4) in the heavy chain residue. If the CDR comprises an amino acid residue from a hypervariable loop, the light chain FR residue is located in the light chain at residues 1-25 (LCFR1), 33-49 (LCFR2), 53-90 (LCFR3) and 97- 107 (LCFR4), and the heavy chain FR residue is located in the heavy chain residue about residues 1-25 (HCFR.1), 33-52 (HCFR2), 56-95 (HCFR3), and 102-113 (HCFR4) At the office. In some cases, when the CDRs comprise an amino acid from a CDR and a hypervariable ring as defined by Kabat, the FR residue should be adjusted accordingly. For example, when CDRH1 comprises the amino acid H26-H35, the heavy chain FR1 residue is at positions 1-25 and the FR2 residue is at position 36-49. The "Fab" fragment contains a light chain variable domain and a constant domain, and a heavy chain variable domain 143939.doc -43 - 201019961 and a first constant domain (CH1). The F(ab')2 antibody fragment comprises a pair of Fab fragments which are typically covalently linked to their carboxy terminus by a hinged cysteine in between. Other chemical couplings of antibody fragments are also known in the art. A "single-chain Fv" or "scFv" antibody fragment comprises the VH and VL domains of an antibody, wherein such domains are present in a single polypeptide chain. Fv polypeptides typically additionally comprise a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding. For a review of scFv, see Pluckthun, The Pharmacology of Monoclonal Antibodies, vol. 3, ed. Rosenburg and Moore, Springer-Verlag, New York, pp. 269-315 (1994). The term "bifunctional antibody" refers to a small antibody fragment having two antigen binding sites comprising a heavy chain variable domain linked to a light chain variable domain (vL) in the same polypeptide chain (VH and VL). (VH). By using a linker that is too short to be paired between two domains on the same strand, the domains are forced to pair with complementary domains on the other strand and create two antigen-binding sites. Bifunctional antibodies are described in more detail in, for example, EP 404,097; WO 93/11161; and Hollinger et al, Proc. iVai/. Sci. USA, 90 6444-6448 (1993). The expression "linear antibody" refers to an antibody as described in Zapata et al., /Voieiw 8(10): 1057-1062 (1995). Briefly, the antibodies comprise a pair of tandem Fd segments (VH-CH1-VH-CH1) which together with the complementary light chain polypeptide form a pair of antigen binding regions. Linear antibodies can have bispecific or monospecificity. 143939.doc -44- 201019961 The modifier "single plant" refers to the characteristics of an antibody obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies to be used in accordance with the present invention can be prepared using a variety of techniques including, for example, fusion knob methods (e.g., Kohler and Milstein, Nature, 256:495-97 (1975); Hongo et al.

Hybridoma, 14 (3): 253-260 (1995); Harlow et al.

Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd edition, 1988); Hammerling et al, Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N_Y., 1981)), recombinant DNA methods (see for example U.S. Patent No. 4,816,567, phage display technology (see, e.g., Clackson et al, 352: 624-628 (1991); Marks et al., / / Μ ο ο ο ο ο ο ο ο ο ο ο ο ο ο ο ο Person, "/· Mo/·扪〇/· 338(2): 299-310 (2004); Lee et al., "/. Mo/. Price 〇/. 340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al., /////21^〇/.^/6 from 〇心284(1- 2): 119-132 (2004)), and techniques for producing human or human-like antibodies having partial or all human immunoglobulin loci or genes encoding human immunoglobulin sequences in animals (see, for example, WO 1998/24893 WO 1996/34096; WO 1996/33735; WO 1991/10741 ; Jakobovits et al., household roc· ΛΓαί/. 90:2551 (1993);

Jakobovits^ Λ. ? Nature 362:255-258 (1993); Bruggemann et al., Fear ζ·« 7:33 (1993); US Patent No. 5,545,807; 5,545,806; 5,569,825; 5,625,126; .doc -45-201019961 5,633,425; and 5,661,016; Marks et al., price -o/rec/mo/o illusion; 10:779-783 (1992); Lonberg et al., 368:856-859 ( 1994); Morrison, iVaiMre 368:812-813 (1994); Fishwild et al, Nature Biotechnol. 14:845-851 (1996); Neuberger, Nature Biotechnol. 14:826 (1996); and Lonberg and Huszar, 13: 65-93 (1995)). The monoclonal antibodies herein specifically include "chimeric" antibodies in which one of the heavy and/or light chains is identical or homologous to the corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, and the chain The remainder is identical or homologous to the corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass; and fragments of such antibodies as long as they exhibit the desired biological activity (see, for example, US patents) No. 4,816,567; and Morrison et al., Ρτ-oc. Natl. Acad. Sci. USA 81:6851-6855 (1984)). Chimeric antibodies include PRIMATIZED® antibodies wherein the antigen binding region of the antibody is derived from an antibody produced by, for example, immunizing a macaque monkey with a related antigen. The "humanized" form of a non-human (e.g., murine) antibody is a chimeric antibody containing minimal sequence derived from a non-human immunoglobulin. Humanized antibodies are primarily human immunoglobulins (recipient antibodies) where residues from the recipient's hypervariable region are derived from, for example, mice, rats, rabbits or non-human primates. Residue replacement of the hypervariable region of an animal's non-human species (donor antibody) with the desired specificity, affinity, and ability. In some instances, the Fv framework region (FR) residues of human immunoglobulin are replaced by corresponding non-human residues. In addition to 143939.doc -46-201019961, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further improve antibody potency. In general, a humanized antibody should comprise substantially all of at least one, usually two variable domains 'where the hypervariable loops wholly or substantially all correspond to the hypervariable loop of the non-human immunoglobulin' and the FR regions are all or substantially All of them are FR regions of human immunoglobulin sequences. The humanized antibody will also optionally comprise at least a portion of an immunoglobulin binding region (Fc), typically a human immunoglobulin constant region. For more details, see Jones et al., 321:522-525 (1986); Riechmann et al., Waiwre 332:323-329 (1988); &Presta, Cwr. (9/7. «Sirwci. 2:593 -596 (1992) ° "Human antibody" is an amino acid sequence having an amino acid sequence corresponding to an antibody produced by a human and/or an antibody which has been prepared using any of the techniques for preparing a human antibody as disclosed herein. The definition of such human antibodies specifically excludes humanized antibodies comprising non-human antigen-binding residues. Human antibodies can be produced using various techniques known in the art. In one embodiment, the human anti-system is selected from the group of bacteria. The bacillus library exhibits human antibodies (Vaughan^ A » Nature Biotechnology 14:309-314 (1996);

Sheets et al., iVa". Heart/. 95:6157-6162 (1998);

Hoogenboom and Winter, «/. Mol. Biol., 227:381 (1991); Marks et al., "/· M〇/· 5ζ·ο/·, 222:581 (1991)). Human antibodies can also be prepared by introducing a human immunoglobulin locus into a transgenic animal, such as a mouse in which the endogenous immunoglobulin gene has been partially or completely inactivated. Human antibody production was observed after stimulation, which is very similar to that seen in humans in all aspects including gene rearrangement, assembly, and antibody lineage. The method is described in, for example, U.S. Patent Nos. 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425, 5,661,016 and below: Marks et al. 5io/TecA«o/6>Magic; 10:779-783 (1992); Lonberg et al, Nature 368:856-859 (1994); Morrison, 368:812-13 (1994); Fishwild et al. Biotechnology 14: 845-51 (1996) » Neuberger, JVaiwre 14:826 (1996); Lonberg and Huszar, 13:65-93 (1995). Alternatively, human antibodies can be prepared by immortalization of human B lymphocytes that produce antibodies against the target antigen (the B lymphocytes can be recovered from the individual or may have been immunized in vitro). See, for example, Cole et al. 'Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, page 77 (1985); Boerner et al, J. Immunol, 147 (1): 86-95 (1991); and U.S. Patent 5,750,373 No. "Naked antibody" is an antibody that does not bind to a heterologous molecule, such as a cytotoxic moiety or a radioactive label. "Affinity maturation" antibodies are antibodies that have one or more changes in one or more CDRs that alter the affinity of the antibody for the antigen as compared to a parent antibody that does not have this (equal) change. Preferred affinity matured antibodies will have nanomolar or even picomolar affinity for the target antigen. Affinity mature resistance systems are generated using procedures known in the art. Marks et al., Bio/Technology 10:779-783 (1992) describe affinity maturity achieved by VH and VL domain shuffling. Random mutation induction of CDR and/or framework residues is described in Barbas et al. 143939.doc • 48-201019961 A » Proc Nat. Acad. Sci, USA 91:3809-3813 (1994); Schier et al., Gene 169:147- 155 (1995); Yelton et al., ///wwwwo/. 155:1994-2004 (1995); Jackson et al., /. 154(7):3310-9 (1995); and Hawkins et al. Mi>/· 5zo/· 226:889-896 (1992) The antibody having the "biological characteristics" of the specified antibody is an antibody having one or more of the antibodies which are different from the biological characteristics of other antibodies that bind to the same antigen. To screen for antibodies that bind to an epitope on an antigen that binds to the relevant antibody, routine cross-blocking assays can be performed, such as those described in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Harlow, and David Lane (1988). .

To increase the half-life of an antibody or polypeptide comprising an amino acid sequence of the invention, a salvage receptor binding epitope can be linked to an antibody (especially an antibody fragment) as described in, for example, U.S. Patent 5,739,277. For example, a nucleic acid molecule encoding a rescue receptor binding epitope can be ligated in-frame with a nucleic acid encoding a polypeptide sequence of the invention such that the fusion protein represented by the engineered nucleic acid molecule comprises a rescue receptor binding epitope and Polypeptide Sequence of the Invention As used herein, the term "responsible receptor binding epitope" refers to an antigenic epitope in the Fc region of an IgG molecule (eg, IgG1, IgG2, IgG3, or IgG4) that is responsible for increasing the in vivo serum half-life of an IgG molecule. Base (e.g., Ghetie et al, Rev. Immunol. 18:739-766 (2000), Table 1). Antibodies having substitutions and increased serum half-life in the Fe region are also described in the following documents: WO 00/42072; WO 143939.doc • 49-201019961 02/060919; Shields et al., J. Biol. Chem. 276:6591-6604 ( 2001) Hinton, J. Biol. Chem. 279: 6213-6216 (2004). In another embodiment, the serum half-life can also be increased, e.g., by ligation of other polypeptide sequences. For example, an antibody or other polypeptide suitable for use in the methods of the invention may be linked to a portion of a serum albumin or serum albumin that binds to an FcRn receptor or a serum albumin binding peptide such that serum albumin binds to the antibody or polypeptide, such polypeptides Sequences are for example disclosed in WO 01/45746. In a preferred embodiment, the serum albumin peptide to be linked comprises the amino acid sequence DICLPRWGCLW (SEQ ID NO: 32). In another embodiment, the half-life of the Fab is increased using such methods. For serum albumin binding peptide sequence 歹1J, see also Dennis et al, J. Biol. Chem. 277:35035-35043 (2002). An "isolated" polypeptide or an "isolated" antibody is one which has been identified and isolated and/or recovered from components of the natural environment. Contaminants in their natural environment are substances that interfere with the diagnostic or therapeutic use of the polypeptide or antibody, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In a preferred embodiment, the polypeptide or antibody will be purified to: (1) the polypeptide or antibody comprises 95% by weight or more and preferably 99% by weight or more as determined by the Lowry method; (2) by using a spin The cup sequencer is sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence; or (3) by Coomassie blue or preferably silver staining under reducing or non-reducing conditions The SDS-PAGE of (silver stain) is determined to be homogeneity. Since at least one component of the polypeptide natural environment will not be present, the isolated polypeptide or antibody comprises a polypeptide or antibody that is in situ in a recombinant cell. However, 143939.doc -50-201019961 will often be prepared by at least one purification step to prepare an isolated polypeptide or antibody. "Fragment" means a portion of a polypeptide or nucleic acid molecule that preferably contains at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90 of the total length of the reference nucleic acid molecule or polypeptide. 〇/〇, 95°/〇 or longer. Fragments may contain 10, 20, 30, 40, 50, 60, 70, 80, 90 or 1 〇〇, 200, 300, 400, 500, 600 or more nucleotides, or 1 〇, 2 〇, 3 〇, 40, 50, 60, 70, 80, 90, 1 〇〇, 120, 140, 160, 180, 190, 200 or more amino acids. Treatment refers to therapeutic treatment and preventive measures. Those in need of treatment include those who have developed benign, precancerous or non-metastatic tumors, as well as those who need to prevent cancer from developing or recurring. The term "therapeutically effective amount" refers to an amount of a therapeutic agent that treats or prevents a disease or condition in a mammal. In the case of cancer, a therapeutically effective amount of the therapeutic agent can reduce the number of cancer cells; reduce the size of the primary tumor; inhibit (i.e., slow down and better terminate) cancer cells infiltrate into peripheral organs; (i.e., to some extent slow and preferably terminate) tumor metastasis; to some extent inhibit tumor growth; and/or to some extent alleviate one or more symptoms associated with the condition. It inhibits cell growth and/or is cytotoxic insofar as the drug prevents growth and/or kills existing cancer cells. For cancer therapy, in vivo efficacy can be measured, for example, by assessing duration of survival, time to disease progression (TTP), response rate (RR), duration of response, and/or quality of life. The terms "cancer" and "cancerous" or describe the physiological condition in which the growth of cells is usually characterized by unregulated cell growth. This definition includes good I43939.doc •51- 201019961 ^•born cancer and malignant cancer^ Γearly cancer” or early stage tumor means “non-invasive or metastatic cancer, or classified as stage 0, stage I or η Cancer of cancer cancer Examples include, but are not limited to, carcinoma, lymphoma, blastoma (including chorioblastoma and retinoblastoma), sarcoma (including liposarcoma and synovial sarcoma), nerve Endocrine tumors (including carcinoid tumors, gastrinoma and islet cell carcinoma), mesothelioma, schwannomas (including hearing neoplasms), meningioma, adenocarcinoma, melanoma, and leukemia or lymphoid malignancies. More specific examples of such cancers include squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer (including small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), lung adenocarcinoma, and lung squamous cell carcinoma), Peritoneal cancer, hepatocellular carcinoma, lunar cancer (including gastrointestinal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer (hepatoma) Including metastatic breast cancer), colon cancer, rectal cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, genital cancer, squamous cell carcinoma, liver cancer (4) heart (10) in_), anal cancer 35 Cancer, testicular cancer, esophageal cancer, biliary tract cancer, and head and neck cancer. The term "pre-cancer" refers to the condition or growth that usually precedes cancer or develops into cancer. "Precancerous" growth will have cells characterized by abnormal cell cycle regulation, proliferation or differentiation, which can be determined by indicators of cell cycle regulation, cell proliferation or differentiation. Plant development plant dysplasia means any abnormal growth of tissue, organ, or cell. Dysplasia is preferably high grade or precancerous. “Transfer” means that the cancer spreads from its original site to the rest of the body. 143939.doc -52· 201019961. Cancer cells can detach from the primary tumor, penetrate into the lymph and blood vessels, and grow (metastasize) through the bloodstream and in distant lesions in normal tissues in other parts of the body. The transfer can be local or distant. The metastasis is a continuous process, in which the tumor cells are detached from the primary tumor, and the blood is prevalent, and stays at a distant site. At this new site, the cells establish blood supply and grow to form life-threatening substances. The stimulatory and inhibitory molecular pathways within tumor cells regulate this behavior and the interaction between tumor cells and host cells in distant sites is also evident. "Non-metastatic" means that the cancer is benign, or stays at the primary site and does not penetrate into the lymphatic or vascular system or in tissues other than the primary site. Non-metastatic cancers are generally any cancer that is stage 0, J or π cancer and sometimes stage III cancer. "Primary tumor" or "primary cancer" means an initial cancer and does not mean a metastatic disease in another tissue, organ or location within the individual. A "benign tumor" or "benign cancer" means a tumor that is still at the site of origin and does not have the ability to infiltrate, invade, or metastasize to a distant site. "Tumor burden" means the number of cancer cells in the body, the size of the tumor, or the amount of cancer. The tumor burden is also called the tumor load (load). "Number of tumors" means the number of tumors. "Individual" means a mammal, including but not limited to a human or non-human mammal, such as a cow, horse, dog, sheep, or an individual, preferably a human. 143939.doc -53- 201019961 The term "anticancer therapy" refers to a therapy that is suitable for the treatment of cancer. Examples of anti-cancer therapeutics include, but are not limited to, for example, chemotherapeutic agents, growth inhibitors, cytotoxic agents, agents used in radiation therapy, anti-angiogenic agents, apoptotic agents, anti-tubulin agents, and cancer treatments. Other agents, anti-CD20 antibodies, platelet-derived growth factor inhibitors (eg GleevecTM (imatinib mesylate)), COX-2 inhibitors (eg celecoxib), interferons, cytokines An antagonist (eg, a neutralizing antibody) that binds to one or more of the following standard ErbB2, ErbB3, ErbB4, PDGFR-β, BlyS, APRIL, BCMA or VEGF receptors, TRAIL/Apo2, and other bioactive agents and organic chemistry Agents, etc. Combinations of the invention are also included in the invention. The term "cytotoxic agent" as used herein refers to a substance that inhibits or blocks cellular function and/or causes cell destruction. The term is intended to include radioisotopes (e.g., I131, I125, Y9G, and Re186), chemotherapeutic agents, and toxins (such as enzymatically active toxins of bacterial, fungal, plant or animal origin or fragments thereof). A "chemotherapeutic agent" is a compound suitable for treating cancer. Examples of chemotherapeutic agents include compounds suitable for the treatment of cancer. Examples of chemotherapeutic agents include: alkylating agents such as thiotepa and CYTOXAN® cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan and Piposulfan; gas C. Aziridine, such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimine and methyl Methylamine (methylamelamine), including six 143939.doc -54- 201019961

Alkretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide, and trimethylolomelamine ; acetogenin (especially bullatacin and bullatacinone); camptothecin (including synthetic analogue topotecan); moss Bryostatin; callistatin; CC-1065 (including its adozelesin, carzelesin, and bizelesin synthetic analogues); rosary Algae cryptophycin (especially Candida cyclic peptide 1 and Nostoccal cyclic peptide 8); dolastatin; doocarmycin (including synthetic analogues KW-2189 and CB1-TM1); Eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustard, such as chlorambucil, naphthyl mustard (chlornaphazine), chlorolamine (cholophosphamide), Estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride 'methodamine, new novembichin, cholesterol phenylacetic acid Phenosterine, prednimustine, trofosfamide, uracil mustard, and nitrosurea, such as carmustine Carmustine), chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics, such as diacetylene antibiotics (eg calicheamicin, especially card 143939.doc -55- 201019961 gibberellin gamma 卡 and kawachi ω ΐΐ (see eg Angew, C/^ew/wi/·五五五《茗/·, 33: 183-186 (1994)); dynemicin, including damicin A; bisphosphonate, such as clodronate; esperamicin ; and neocarzinostatin chromophores and related colored eggs Olefin diacetyl antibiotic chromophore), aclacinomysin, actinomycin, authramycin, azaserine, bleomycins ), actinomycin C (cactinomycin), carabincin, carminomycin, carzinophilin, chromomycini, dactinomycin, daunorubicin (daunorubicin), detorubicin, 6-diazo-5-oxo-L-positral acid, ADRIAMYCIN® doxorubicin (including morpholinyl-doxorubicin, cyanide) Benzolinyl-doxorubicin, 2-pyrroline-doxorubicin and deoxydoxombicin), epirubicin, esorubicin, idarubicin , marcellomycin (marcellomycin), mitomycin (such as mitomycin C (mitomycin (1:)), mycophenolic acid (1] 1) ^ 〇卩 1 ^ 11 〇 1 ^ ^ (^ ( 1), nogamycin (11〇§ &1&1]1>^11) 'olivomycin, peplomycin, potfiromycin, 嗓吟Puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex ), ketostatin, zorubicin; antimetabolites such as amidoxime and 5-fluorouracil (5-FU); folic acid analogues such as denoptinin, Methotrexate, pteropterin, 143939.doc -56- 201019961 Trimetrexate; 嗓呤 analogs, such as fludarabine, 6-weig 嗓吟, 嗟π Thiamiprine, thioguanine; ° bite analogs, such as amphetamine

(ancitabine), azacitidine, hexazone, carmofur, cytarabine, dideoxyuridine, doxifluridine, Enocitabine, floxuridine; androgens, such as caluterone, dromostanolone propionate, epitiostolol, mepitiostane , testolactone; anti-adrenal, such as aminoglutethimide, mitotane, trilostane; folic acid supplements, such as furolin Folinic acid; aceglatone; awake aldophosphamide glycoside; alanine levulinic acid; eniluracil; (amsacrine); bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; Elfornithine; elliptinium acetate ; epothilone; etoglucid; gallium wall; transbasin; lentinan; lonidainine; maytansinoids, such as Maytansine and ansamitocin; mitoguazone; mitoxantrone; mopidanmol; bowl of nitraerine; pentastatin (pentostatin); phennamet; 143939.doc -57- 201019961 0 pirarubicin; losoxantrone; podophyllinic acid; 2-ethyl hydrazine ( 2-ethylhydrazide); procarbazine; PSK® complex (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; sizofiran; Syrrogermanium; tenuazonic acid; triaziquone; 2,2',2M-trichlorotriethylamine; trichothecene (trichothecene) In particular, T-2 toxin, verracurin A, roridin A, and anguidine; (urethan); vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; 0 pospobroman ); gacytosine; arabinose ("Ara-C"); cyclophosphamide; thiotepa; taxoid, such as TAXOL® paclitaxel (Bristol-Myers Squibb Oncology, Princeton, NJ), ABRAXANETM Crepes-free, pacliphor-free, paclitaxel-based albumin engineered nanoparticle formulation (American Pharmaceutical Partners, Schaumberg, Illinois) and TAXOTERE® docetaxel (docetaxel 5 Rhone- Poulenc Rorer, Antony, France); chloranbucil; GEMZAR® gemcitabine; 6-thioguanine; guanidinium; amidoxime; platinum analogues such as cisplatin (cisplatin) and carboplatin; vintin; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; NAVELBINE® vinorelbine Bin (vinorelbi Ne); Novartron 143939.doc -58- 201019961

(novantrone); teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate ); irinotecan (Camptosar, CPT-11), including irinotecan and 5-FU and leucovorin; topoisomerase inhibitor RFS 2000 Difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; combretastatin; formazan tetrahydrofolate (LV) Oxaliplatin, including Osili Platinum treatment plan (卩01^0 again); (: -α, Raf, H-Ras, EGFR (eg, erlotinib (TarcevaTM)) and an inhibitor of VEGF-A, which reduces cell proliferation; and a pharmaceutically acceptable salt of any of the above, Acids or derivatives. Also included in the definition are anti-hormonal agents for regulating or inhibiting the effects of hormones on tumors, such as anti-estrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (tamoxifen). (including NOLVADEX® tamoxifen), raloxifene, droloxifene, 4-pyroxyzin, trioxifene, keoxifene, LY117018, onapristone and FARES TON· toremifene (FARESTON 'toremifene); an aromatase inhibitor that inhibits aromatase, which regulates estrogen production in the adrenal gland, such as 4(5)-imidazole, amine Lumet, MEGASE® megestrol acetate, AROMASIN® exemestane, formestanie, fadrozole, RIVISOR® vorozole, FEMARA® Letrozole and ARIMIDEX® (A) Nastrozole); 143939.doc -59- 201019961 and antiandrogens, such as flutamide, nilutamide, bicalutamide, leuprolide and goserelin (goserelin); and troxacitabine (a 1,3-dioxolan nucleoside cytosine analog); antisense oligonucleotides, particularly in the signal transduction pathway involved in the inhibition of abnormal cell proliferation Antisense nucleotides expressed by genes such as PKC-ct, Raf and H-Ras; ribonucleases such as VEGF expression inhibitors (eg ANGIOZYME® ribonuclease) and HER2 expression inhibitors; vaccines, such as gene therapy Vaccines such as ALLOVECTIN® vaccine, LEUVECTIN® vaccine and VAXID® vaccine; PROLEUKIN® rIL-2; LURTOTECAN® topoisomerase 1 inhibitor; ABARELIX® rmRH; vinorelbine and espartame (see US Patent 4,675,187) And a pharmaceutically acceptable salt, acid or derivative of any of the above. The term "prodrug" as used in this application refers to a precursor or derivative form of a pharmaceutically active substance, and Maternal medicine It is small compared to the cytotoxicity of tumor cells, and is capable of being enzymatically activated or converted into the more active parent form of the present. See, for example, Wilman, "Prodrugs in Cancer Chemotherapy" Biochemical Society Transactions, , pp. 375-382, 615th Meeting Belfast (1986); and Stella et al., "Prodrugs: A Chemical Approach to Targeted Drug Delivery", Direcied Drwg Borchardt et al. (eds.), pp. 247-267, Humana Press (1985). Prodrugs of the present invention include, but are not limited to, pre-phosphate containing drugs, prodrugs containing thiophosphates, prodrugs containing sulfates, prodrugs containing peptides, modified with D-amino acids 143939.doc -60-201019961 Drug, glycosylation prodrug, prodrug containing β-namidoxime, prodrug containing optionally substituted phenoxyacetamide or prodrug containing phenethylamine optionally substituted, 5-fluoro Cytosine and other 5-fluorouridine prodrugs that can be converted to more active, non-cytotoxic drugs. Examples of cytotoxic drugs which may be derivatized for use in the prodrug form of the present invention include, but are not limited to, the above chemotherapeutic agents.

"Radiotherapy" means the use of directional gamma rays or beta rays to induce sufficient cellular damage to limit its ability to function normally or to completely destroy cells. It should be understood that a number of methods are known in the art for determining the therapeutic dose and duration. Typical treatments are administered in a single administration with a typical dose ranging from 10 to 200 units per day (Gray). "Reducing or inhibiting" means causing an overall decrease of 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or higher. Reduced or inhibited may refer to the symptoms of the condition being treated, the presence or size of metastases, the size of the primary tumor, or the size or number of blood vessels in the angiogenic condition. Therapeutic Agents The present invention relates to the use of a combination therapy with a c-met antagonist and a VEGF antagonist for the treatment of a pathological condition such as a tumor. In another aspect, the invention relates to the use of a combination therapy to treat a pathological condition, such as a tumor, in a subject in combination with a c-met antagonist, a VEGF antagonist, and an EGFR antagonist. C-met antagonists C-met antagonists suitable for use in the methods of the invention include polypeptides that specifically bind c-met, anti-c-met antibodies, c-met small molecules, receptor molecules that specifically bind c-met, and derivatives And fusion proteins. C-met antagonists also include c-143939.doc • 61 - 201019961 met polypeptide, antagonistic variants of RNA aptamers and peptibodies against c-met and HGF. As c-met antagonists suitable for use in the methods of the invention, anti-HGF antibodies, anti-HGF polypeptides, c-met receptor molecules and derivatives which specifically bind to HGF are also included. Examples of each of these antagonists are described below. Anti-c-met antibodies suitable for use in the methods of the invention include any antibody which binds c-met with sufficient affinity and specificity and which reduces or inhibits c-met activity. The selected antibody should generally have sufficient binding affinity for c-met, for example, the antibody binding to human c-met may have a Kd value between 100 nM and 1 pM. Antibody affinities can be determined by methods such as surface plasmon resonance based assays (such as the BIAcore assay described in PCT Application Publication No. WO 2 〇 05/012359), enzyme-linked immunosorbent assay (ELISA), and competition assays ( For example, RIA). Preferably, the anti-c-met antibody of the present invention can be used as a therapeutic agent for targeting and interfering with diseases or conditions involving c-met/HGF activity. Alternatively, the antibody can be subjected to other biological activity assays, e.g., to assess its utility as a therapeutic. Such assays are known in the art and depend on the intended use of the target antigen and antibody.

Anti-c-met antibodies, which may be provided in the form of a one-armed antibody, are known in the art. See, for example, Martens, T et al, (2006) Clin Cancer Res 12 (20 Pt 1): 6144; US 6,468,529; WO 2006/015371; WO 2007/063816; US 7,408,043; WO 2009/007427; WO 2005/016382; WO 2007/126799. In some embodiments, the anti-c-met antibody is MetMAb. The sequence of MetMAb is shown in Figures 9 and 10. MetMAb (also known as OA5D5v2) is also described in WO 2006/015371 and Jin et al, Cancer Res (2008) 68:4360. 143939.doc -62-201019961 In some embodiments, the anti-c-met antibody comprises (a) a first polypeptide comprising a heavy chain variable domain comprising: EVQLVESGGGLVQPGGSL RLSCAASGYTFTSYWLHWVRQAPGKGLEWVGMIDPSNSD TRFNPNFKDRFTISADTSKNTAYLQMNSLRAEDTAVYYCA TYRSYVTPLDYWGQGTLVTVSS (SEQ ID NO: 10) a CH1 sequence and a first Fc polypeptide; (b) a second polypeptide comprising a light chain variable domain having the sequence: DIQMTQSPSSLSASVGDRVTITCKSSQSLLYTS SQKNYLAWYQQKPGKAPKLLIYWASTRESGVPSRFSGSGS GTDFTLTISSLQPEDFATYYCQQYYAYPWTFGQGTKVEIKR (SEQ ID NO: 11) and CL1 sequence; and (c) comprising a second a third polypeptide of an Fc polypeptide, wherein the heavy chain variable domain and the light chain variable domain are present in a complex form and form a single antigen binding arm, wherein the first and second Fc polypeptides are present in a complex form and are formed and included The Fab molecule of the antigen binding arm is an Fc region that increases the stability of the antibody fragment. In some embodiments, the first polypeptide comprises the Fc sequence depicted in Figure 1 (SEQ ID NO: 12) and the second polypeptide comprises the Fc sequence depicted in Figure 2 (SEQ ID NO: 13). In some embodiments, the first polypeptide comprises the Fc sequence depicted in Figure 2 (SEQ ID NO: 13) and the second polypeptide comprises the Fc sequence depicted in Figure 1 (SEQ ID NO: 12).

In some embodiments, the anti-c-met antibody comprises (a) a first polypeptide comprising a heavy chain variable domain, the polypeptide comprising the sequence EVQLVESGGGLVQPGGSLR LSCAASGYTFTSYWLHWVRQAPGKGLEWVGMIDPSNSDT RFNPNFKDRFTISADTSKNTAYLQMNSLRAEDTAVYYCAT YRSYVTPLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTS GGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS 143939.doc • 63- 201019961 SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPE VTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSD: IAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSR : WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 14); (b) a second polypeptide comprising a light chain variable domain, the polypeptide comprising the sequence DIQMTQSPSSLSASVGDRVTITCKSSQSLLYTSSQKNYLAW ❿ YQQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFTLTIS SLQPEDFATYYCQQYYAYPWTFGQGTKVEIKRTVAAPSVFI FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC (SEQ ID NO: 15); and FC comprises a first sequence of a tripeptide comprising the sequence CPPCPAPELL GGPSVFLFP PKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK VSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQV SLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK (SEQ ID NO: 13), wherein the heavy chain variable domain and a light chain variable domain as a form of a complex and form a single antigen binding arm, wherein the first and second Fc polypeptides form complexes The form exists and forms an Fc region that increases the stability of the antibody fragment compared to a Fab molecule comprising the antigen binding arm. 143939.doc -64 - 201019961 Anti-c-met antibodies (available in the form of a one-armed antibody) are known in the art (see, for example, Martens, T et al. (2006) Clin Cancer Res 12 (20 Pt 1 ): 6144; US 6,468,529; WO 2006/015371; WO 2007/063 816). In one embodiment, the anti-c-met antibody comprises a heavy chain variable domain comprising the CDR1-HC, CDR2-HC and CDR3-HC sequences depicted in Figure 1 (SEQ ID NO: 4, 5) And/or 9) one or more. In some embodiments, the antibody comprises a light chain variable domain comprising the CDR1-LC, CDR2-LC and CDR3-LC sequences depicted in FIG. 1 (SEQ ID NO: 1, 2, and/or One or more of 3). In some embodiments, the heavy chain variable domain comprises the FR1-HC, FR2-HC, FR3-HC, and FR4-HC sequences depicted in Figure 1 (SEQ ID NO: 21-24). In some embodiments, the light chain variable domain comprises the FR1-LC, FR2-LC, FR3-LC, and FR4-LC sequences depicted in Figure 1 (SEQ ID NOS: 16-19). In other embodiments, the antibody comprises the accession number ATCC HB-11894 (fusion tumor 1A3.3.13) or HB-11895 (fusion tumor 5D5.11.6) deposited in the American Type Culture Collection. One or more CDR sequences of a monoclonal antibody produced by the fusion of the tumor cell line. In one aspect, the anti-c-met antibody comprises: (a) at least one, two, three, four or five hypervariable region (CDR) sequences selected from the group consisting of: (1) comprising sequence A1- CDR-L1 of A17, wherein A1-A17 is KSSQSLLYTSSQKNYLA (SEQ ID NO: 1), (ii) comprises CDR-L2 of sequence B1-B7, wherein B1-B7 is WASTRES (SEQ ID NO: 2), 143939.doc -65- 201019961 (iii) CDR-L3 comprising the sequence C1-C9, wherein C1-C9 is QQYYAYPWT (SEQ ID NO: 3) > (iv) CDR-H1 comprising the sequence D1-D10, wherein D1-D10 are GYTFTSYWLH (SEQ ID NO: 4), (v) comprises CDR-H2 of sequence E1-E18, wherein E1-E18 is GMIDPSNSDTRFNPNFKD (SEQ ID NO: 5), and (vi) CDR-H3 comprising sequence F1-F11, Wherein F1-F11 is XYGSYVSPLDY (SEQ ID NO: 6) and X is not R; and (b) at least one variant CDR, wherein the variant CDR sequence comprises SEQ ID NO: 1, 2, 3, 4, 5 or 6. Modification of at least one residue of the sequence of the strand. In one embodiment, the CDR-L1 of an antibody of the invention comprises the sequence SEQ ID NO: 1. In one embodiment, the CDR-L2 of an antibody of the invention comprises the sequence SEQ ID NO: 2. In one embodiment, the CDR-L3 of the antibody of the invention comprises the sequence of SEQ ID NO: 3. In one embodiment, the CDR-H1 of an antibody of the invention comprises the sequence of SEQ ID NO: 4. In one embodiment, the CDR-H2 of the antibody of the invention comprises the sequence of SEQ ID NO: 5. In one embodiment, the CDR-H3 of the antibody of the invention comprises the sequence of SEQ ID NO: 6. In one embodiment, CDR-H3 comprises TYGSYVSPLDY (SEQ ID NO: 7). In one embodiment, CDR-H3 comprises SYGSYVSPLDY (SEQ ID NO: 8). In one embodiment, an antibody of the invention comprising such sequences (as described herein;) is a humanized or human antibody. In one aspect, the invention provides an antibody comprising 1, 2, 3, 4, 5 or 6 CDRs, wherein each CDR comprises a SEQ ID NO: 1, 2, 3, 143939. doc • 66-201019961 4 a sequence consisting of, consisting of, or consisting essentially of the sequence of 5, 6, 7, and 8 and wherein SEQ ID NO: 1 corresponds to CDR-L1 'SEQ ID NO: 2 corresponds to CDR-L2, SEQ ID NO: 3 corresponds to CDR-L3 'SEQ ID NO: 4 corresponds to CDR-H1, SEQ ID NO: 5 corresponds to CDR-H2, and SEQ ID NO: 6, 7 or 8 corresponds to CDR-H3. In one embodiment, an antibody of the invention comprises CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2 and CDR-H3, wherein each sequence comprises SEQ ID NO: 1, 2, 3, 4 , 5 and 7. In one embodiment, the antibody of the invention comprises CDR-❹ LI, CDR-L2, CDR-L3, CDR-H1, CDR-H2 and CDR-H3, wherein each sequence comprises SEQ ID NO: 1, 2, 3, 4, 5 and 8. A variant CDR in an antibody of the invention may have a modification of one or more residues in the CDR. In one embodiment, the CDR-L2 variant comprises 1-5 (1, 2, 3, 4 or 5) substitutions at any of the following positions: B1 (M or L), B2 (P, T, G or S), B3 (N, G, R or T), B4 (I, N or F), B5 (P, I, L or G), B6 (A, D, T or V) and B7 (R) , I, Μ or G). In one embodiment, the CDR-H1 variant comprises 1-5 (1, 2, 3, 4 or 5) substitutions at any position below any combination: D3 (N, Ρ, L, S, A, I), D5 (I, S or Y), D6 (G, D, T, K, R), D7 (F, Η, R, . S, T or V) and D9 (M or V). In one embodiment, the 'CDR-H2 variant comprises 1-4 (1, 2, 3 or 4) substitutions at any of the following positions: E7 (Y), E9 (I), E10 (I), E14 (T or Q), E15 (D, K, S, T or V), E16 (L), E17 (E, Η, N or D) and E18 (Y, E or H). In one embodiment, the CDR-H3 variant comprises 1-5 (1, 2, 3, 4 or 5) substitutions at any of the following positions: F1 (T, S), F3 (R, S, Η, 143939.doc •67- 201019961 Τ, A, Κ), F4(G), F6 (R, F, M, T, E, K, A, L, W), F7 (L, I, T, R, K, V), F8 (S, A), F10 (Y, N), and F11 (Q, S, H, F). The letters in parentheses after each position indicate an illustrative substitution (i.e., replacement) of the amino acid; it will be apparent to those skilled in the art that it can be routinely evaluated using techniques known in the art and/or described herein. The suitability of other amino acids as substituted amino acids in the case described. In one embodiment, CDR-L1 comprises the sequence SEQ ID NO: 1. In one embodiment, F1 in the variant CDR-H3 is T. In one embodiment, F1 in the variant CDR-H3 is S. In one embodiment, F3 in the variant CDR-H3 is R. In one embodiment, F3 in the variant CDR-H3 is S. In one embodiment, F7 in the variant CDR-H3 is T. In one embodiment, an antibody of the invention comprises a variant CDR-H3, wherein F1 is T or S, F3 is R or S, and F7 is T. In one embodiment, an antibody of the invention comprises a variant CDR-H3, wherein F1 is T, F3 is R, and F7 is T. In one embodiment, an antibody of the invention comprises a variant CDR-H3, wherein F1 is S. In one embodiment, an antibody of the invention comprises a variant CDR-H3' wherein F1 is T and F3 is R. In one embodiment, an antibody of the invention comprises a variant CDR-H3, wherein F1 is S and F3 is RXF7 is T. In one embodiment, an antibody of the invention comprises a variant cdr_H3, wherein F1 is T, F3 is S, F7 is T, and F8 is S. In one embodiment, an antibody of the invention comprises a variant CDR-H3, wherein F1 is T, F3 is S, F7 is T, and F8 is A. In some embodiments, the variant CDR-H3 antibody further comprises CDR-L1, CDR-L2, CDR-L3, CDR-H1 and CDR-H2, wherein each sequence comprises SEQ ID NO: i, 2, 3, 4 And the sequence depicted in 5. In a 143939.doc • 68-201019961 embodiment, the antibodies further comprise a human subgroup III heavy chain framework consensus sequence. In one embodiment of such antibodies, the framework consensus sequence comprises a substitution at positions 71, 73 and/or 78. In some embodiments of such antibodies, position 71 is A, 73 is T and/or 78 is A. In one embodiment of such antibodies, the antibodies further comprise a human kappa light chain framework consensus sequence. In one embodiment, the antibody of the invention comprises a variant CDR-L2, wherein Β6 is V. In some embodiments, the variant CDR-L2 antibody further comprises CDR-L1, CDR-L3, CDR-H1, CDR-H2 and CDR-H3, wherein each sequence comprises SEQ ID NO: 1, 3, 4, 5 And the sequence depicted in 6. In some embodiments, the variant CDR-L2 antibody further comprises CDR-L1, CDR-L3, CDR-H1, CDR-H2 and CDR-H3, wherein each sequence comprises SEQ ID NO: 1, 3, 4, 5 And the sequence depicted in 7. In some embodiments, the variant CDR-L2 antibody further comprises CDR-L1, CDR-L3, CDR-H1, CDR-H2 and CDR-H3, wherein each sequence comprises SEQ ID NO: 1, 3, 4, 5 And the sequence depicted in 8. In some embodiments, the antibodies further comprise a human subgroup III heavy chain framework consensus sequence. In one embodiment of such antibodies, the framework consensus sequence comprises a substitution at positions 71, 73 and/or 78. In some embodiments of such antibodies, position 71 is A, 73 is T and/or 78 is A. In one embodiment of such antibodies, the antibodies further comprise a human kappa light linkage framework consensus sequence. In one embodiment, an antibody of the invention comprises a variant CDR-H2, wherein Ε14 is Τ' Ε15 is Κ' and Ε17 is Ε. In one embodiment, an antibody of the invention comprises a variant CDR-H2, wherein Ε17 is Ε» In some embodiments, the variant CDR-H3 antibody further comprises CDR-L1, CDR-L2, CDR-L3, 143939.doc - 69-201019961 CDR-H1 and CDR-H3, each of which comprises the sequence depicted in SEQ ID NO: 1, 2, 3, 4 and 6. In some embodiments, the variant CDR-H2 antibody further comprises CDR-L1, CDR-L2, CDR-L3, CDR-H1 and CDR-H3, wherein each sequence comprises SEQ ID NO: 1, 2, 3, 4 And the sequence depicted in 7. In some embodiments, the variant CDR-H2 antibody further comprises CDR-L1, CDR-L2, CDR-L3, CDR-H1 and CDR-H3, wherein each sequence comprises SEQ ID NO: 1, 2, 3, 4 And the sequence depicted in 8. In some embodiments, the antibodies further comprise a human subgroup III heavy chain framework consensus sequence. In one embodiment of such antibodies, the framework common sequence comprises a substitution at positions 71, 73 and/or 78. In some embodiments of such antibodies, position 71 is A, 73 is T and/or 78 is A. In one embodiment of such antibodies, the antibodies further comprise a human kappa light chain framework consensus sequence. In other embodiments, a c-met antibody of the invention specifically binds at least a portion of a c-met Sema domain or a variant thereof. In one example, an antagonist antibody of the invention specifically binds to at least one sequence selected from the group consisting of LDAQT (SEQ ID NO: 25) (eg, residue 269-273 of c_met), LTEKRKKRS (SEQ ID NO: 26) (eg residue 3GO-308 of c_met), KPDSAEPM (SEQ ID NO: 27) (eg residues 350-357 of c-met) and NVRCLQHF (SEQ ID NO: 28) (eg residues of c-met) 381-388). In one embodiment, an antagonist antibody of the invention specifically binds to a conformational epitope formed by at least one or a portion of a sequence selected from the group consisting of LDAQT (SEQ ID NO: 25) (eg, c -met residue 269-273), LTEKRKKRS (SEQ ID NO: 26) (eg, 143939.doc • 70. 201019961 c_met residue 300-308), KPDSAEPM (SEQ ID NO: 27) (eg c-met Residues 350-357) and NVRCLQHF (SEQ ID NO: 28) (e.g., residues 381-388 of c-met). In one embodiment, an antagonist antibody of the invention specifically binds to an amino acid having at least 50%, 60%, 70%, 80%, 90%, 95%, 98% sequence identity or similarity to the following sequences Sequence: LDAQT (SEQ ID NO: 25), LTEKRKKRS (SEQ ID NO: 26), KPDSAEPM (SEQ ID NO: 27) and/or NVRCLQHF (SEQ ID NO: 28). In one aspect, the anti-c-met antibody comprises at least one feature that promotes heterodimerization of the Fc sequence within the antibody fragment while minimizing homodimerization. This (etc.) feature improves the yield and/or purity and/or homogeneity of the immunoglobulin population. In one embodiment, the antibody comprises an Fc mutation that constitutes "杵" and "white" as described in the following literature: WO 2005/063816; Ridgeway, J et al, Prot Eng (1996) 9: 617-21; Zhu Z et al., Prot Sci (1997) 6:781-8. For example, the 臼 mutation can be one or more of T366A, L368A, and/or Y407V in the Fc polypeptide, and the hole mutation can be T366W. Anti-HGF antibodies are well known in the art. See, for example, Kim KJ et al, Clin Cancer Res. (2006) 12(4): 1292-8; WO 2007/115049; WO 2009/002521; WO 2007/143098; WO 2007/017107; WO 2005/017107; L2G7; AMG-102. A c-met receptor molecule or a fragment thereof that specifically binds to HGF can be used in the method of the present invention, for example, to bind and isolate the HGF protein' to prevent its conduction signal. The c-met receptor molecule or its HGF binding fragment is preferably in a soluble form. In some embodiments, the soluble form of the receptor has an inhibitory effect on the biological activity of the c-143939.doc-71-201019961 met protein by binding to HGF, thereby preventing its binding to its natural presence on the surface of the stem cell. Receptor. Also included are c-met receptor fusion proteins, examples of which are described below. The soluble c-met receptor protein or the entrapped c-met receptor protein of the present invention includes a c-met receptor protein that is not immobilized on the cell surface via a transmembrane domain. Thus, the soluble form of the c-met receptor (including chimeric receptor proteins), although capable of binding to HGF and inactivating it, does not comprise a transmembrane domain and is therefore generally not associated with cell membranes of cells expressing the molecule. See, for example, Kong-Beltran, et al., Cancer Cell (2004) 6(1): 75-84. HGF molecules or fragments thereof can be used in the methods of the invention, and such HGF molecules or fragments thereof specifically bind to c-met and block or reduce the activation of c-met, thereby preventing its conduction signal. An aptamer is a nucleic acid molecule that forms a tertiary structure that specifically binds to a target molecule, such as an HGF polypeptide. The production of aptamers and their therapeutic use are well established in this technology. See, for example, U.S. Patent No. 5,475,096. The HGF aptamer is a poly(ethylene glycol) modified nucleotide that adopts a three-dimensional configuration that enables it to bind to extracellular HGF. Further information on aptamers can be found in U.S. Patent Application Publication No. 20060148748. A peptidic body is a peptide sequence linked to an amino acid sequence encoding a fragment or a portion of an immunoglobulin molecule. The polypeptide may be derived from any random sequence selected for any method of specific binding, including but not limited to phage display technology. In a preferred embodiment, the selected polypeptide can be linked to an amino acid sequence encoding the Fc portion of an immunoglobulin. Peptides that specifically bind to and antagonize HGF or c-met are also suitable for use in the methods of the invention. 143939.doc -72- 201019961

C-met antagonists include small molecules such as US 5,792,783, US 5,834,504, US 5,880,141, US 6,297,238, US 6,599,902, US 6,790,852 > US 2003/0125370 ' US 2004/0242603, US 2004/0198750, US 2004/0110758, US 2005/0009845, US 2005/0009840, US 2005/0245547, US 2005/0148574, US 2005/0101650, US 2005/0075340, US 2006/0009453, US 2006/0009493, WO 98/007695 'WO 2003/000660 ' WO 2003/087026, WO 2003/097641, WO 2004/076412, WO 2005/004808, WO 2005/121125, WO 2005/030140, WO 2005/070891, WO 2005/080393, WO 2006/014325, WO 2006/021886 ' WO Compounds described in 2006/021 881, WO 2007/103308. PHA-665752 is a small molecule ATP competitive active site inhibitor of c-met catalytic activity and cell growth, cell movement, invasion and morphology of various tumor cells (Ma et al., (2005) Clin. Cancer Res 11:2312 -2319; Christensen et al, (2003) Cancer Res· 63:7345-7355) ° EGFR antagonist EGFR antagonists include antibodies, such as humanized monoclonal antibodies called nimotuzumab (YM Biosciences) Complete human ABX-EGF (panitumumab, Abgenix Inc.); and known as Ε1·1, E2.4, Ε2·5, E6.2, E6.4, E2.ll, Ε6·3 And fully human antibody of E7.6.3 and described in US 6,235,883; MDX-447 (Medarex Inc.). Pertuzumab (2C4) is a humanized antibody that binds directly to HER2 but interferes with the dimerization of HER2-EGFR, thereby inhibiting EGFR signaling 143939.doc-73-201019961. Other examples of antibodies that bind to EGFR include MAb 579 (ATCC CRL HB 8506), MAb 455 (ATCC CRL HB8507), MAb 225 (ATCC CRL 8508), MAb 528 (ATCC CRL 8509) (see U.S. Patent No. 4,943,533 to Mendelsohn et al. And its variants, such as chimeric 225 (C225 or cetuximab; ERBUTIX®) and reshaped human 225 (H225) (see WO 96/40210, Imclone Systems Inc.); IMC-11F8, a target A fully human antibody to EGFR (Imclone); an antibody that binds to a type 11 mutation of 〇11 (U.S. Patent No. 5,212,290); a humanized and chimeric antibody that binds to EGFR as described in U.S. Patent No. 5,891,996; Human antibodies that bind to EGFR, such as ABX-EGF (see WO 98/50433, Abgenix); EMD 55900 (Stragliotto et al., £*/·32A: 636-640 (1996)); EMD7200 (martuzumab) , a humanized EGFR antibody against EGFR that competes with EGF and TGF-α for binding to EGFR; and mAb 806 or humanized mAb 806 (Johns et al., X-valent ·/· 279(29): 30375-30384 (2004) ). The anti-EGFR antibody can bind to a cytotoxic agent to produce an immunoconjugate (see, for example, EP 659, 439 A2, Merck Patent GmbH). Anti-EGFR antibodies suitable for use in the methods of the invention include binding to EGFR with sufficient affinity and specificity and can reduce or inhibit Any antibody that is active in EGFR. The selected antibody will generally have a sufficiently strong binding affinity for EGFR', e.g., the antibody binding to human EGFR may have a Kd value between 1 〇〇 nM-1 pM. Antibody affinity can be determined by methods such as surface plasma resonance based assays (such as the BIAcore assay described in PCT Application Publication No. WO 2005/012359), enzyme-linked immunosorbent assay (ELISA), and competition assays (example 143939). .doc • 74- 201019961 as RIA). Preferably, the anti-c-met antibody of the present invention can be used as a therapeutic agent for targeting and interfering with diseases or conditions involving EGFR/EGFR ligand activity. In addition, the antibody can be subjected to other biological activity assays, e.g., to assess its utility as a therapeutic agent. Such assays are known in the art and depend on the intended use of the target antigen and antibody. Bispecific antibodies are antibodies that have binding specificities for at least two different epitopes. Exemplary bispecific antibodies can bind to EGFR and c-met. In another example, an exemplary bispecific antibody can bind to two different epitopes of the same protein (e. g., c-met protein). Alternatively, the c-met or EGFR arm can be combined with an arm that binds to a trigger molecule on a white blood cell to aggregate a cellular defense mechanism in a cell that exhibits c-met or EGFR, such as a T cell receptor molecule (eg, CD2 or CD3). Or Fc receptors of IgG (FcyR) (such as FcyRI (CD64), FcyRII (CD32) and FcyRIII (CD1 6)). Bispecific antibodies can also be used to localize cytotoxic agents in cells that exhibit EGFR or c-met. These antibodies have EGFR or c-met binding arms and binding cytotoxic agents (eg saporin, anti-interferon-α, vinca alkaloid, ricin A chain, methotrexate) The arm of a sputum or radioisotope hapten. Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g., F(ab')2 bispecific antibodies). EGFR antagonists also include small molecules such as: US5616582 'US5457105, US5475001, US5654307, US5679683, US6084095, US6265410, US6455534, US6521620, US6596726, US6713484, US5770599, US6140332, US5866572, US6399602 'US6344459 ' US6602863, 143939.doc-75- The compounds described in 201019961 US Pat. No. 6,391, 874, WO 9814451, WO 9850038, WO 9909016, WO 9924037, WO 9935146, WO 0132651, US6344455, US5760041, US6002008, US5747498. Specific small molecule EGFR antagonists include OSI-774 (CP-358774, erlotinib, OSI Pharmaceuticals); PD 183805 (CI 1033, N-[4-[(3-fluoro-4-fluorophenyl)amino) -7-[3-(4-Mercapto)propoxy]-6-啥σ 琳 ]]-2-propenylamine dihydrochloride, Pfizer Inc.; Iressa® (ZD 1839, Ji Fei Azin, AstraZeneca); ZM 105180 ((6-Amino-4-(3-mercaptophenyl-amino)-quinazoline, Zeneca); BIBX-1382 (N8-(3-chloro-4-fluoro) -phenyl)-N2-(l-methyl-piperidin-4-yl)-pyrimido[5,4-d]pyrimidine-2,8-diamine, Boehringer Ingelheim); PKI-166 ((R) -4-[4-[(l-phenylethyl)amino]-1Η-πpiro[2,3-d] mouth bite-6-yl]-benzene discretion·);(R)-6 -(4-Phenylphenyl)-4-[(1-phenylethyl)amino]-7H-° ratio slightly [2,3-d]°^ bite); CL-387785 (N-[ 4-[(3- >odor phenyl)amino]-6-啥 琳 基 ]]-2-butyl fast-acting amine); EKB-569 (N-[4-[(3- gas_4_fluorine) Phenyl)amino]-3-cyano-7-ethoxy-6-wowenyl]-4-(dimethylamino)-2-butanthene); lapatinib (Tykerb, GlaxoSmithKline) ; ZD6474 (Zactima, AstraZeneca) ; CUDC-lOl (Curis); Carnamate (canertinib) (CI-1033) ; AEE788 (6·[4-[(4-ethyl-1-piperazinyl)indolyl]phenyl]-1^-[(11^)-1-phenyl) Base]-711-11 than 0 each [2,3-(1] mouth bite-4-amine, 1^〇2003013541, Novartis) and ΡΚΙ166 (4-[4-[[(lR)-l-phenyl) Ethyl]amino]-7H-pyrrolo[2,3-d]pyrimidin-6-yl]-indolol, WO 9702266, Novartis) °

In a specific embodiment, the EGFR antagonist has according to US 143939.doc -76· 201019961

5,75 7,498 (incorporated herein by reference)

Wherein: m is 1, 2 or 3; halo, hydroxy, hydroxytrifluoromethyl and -(Cr S or NH; each R1 is independently selected from the group consisting of: aminino, carboxyl, nitro, fluorenyl , ureido, cyano C4 alkyl) -W-(phenyl), wherein w is a single bond, hydrazine or each R1 is independently selected from R9 and cyano substituted 2Ci_C4 alkyl, wherein R9 is selected from the group consisting of Group: R5, _〇R6, _NR6R6, _c(〇)r7, -NHOR5, -0C(0)R6, cyano, Aj_YR5; r, Ci C4^, R is independently hydrogen or R5; , _〇r6 or -Nr6r6; a is selected from the group consisting of N-piperidinyl, N-morpholinyl, N-pyrrolidinyl, 4_R6_piperazine-丨-yl, imidazole-1-f, 4-»pyridinium _ small group, _(CiC4 alkylene) (c〇2H), phenoxyphenyl, phenylthio, CVC4 alkenyl and -(CVC4 alkyl) C(0)NR R, and Y is s , 8〇 or 8〇2; wherein the calcined moiety of 6 in r5, _〇6 and Wei R6 is substituted by (1) a dentate substituent, and the thiol moiety of R 〇R and NR R is optionally 1 or 2 R9 groups are taken as appropriate. The alkyl group of the substituent is selected as the case where the two heteroatoms are not connected to the same carbon source. 43939.doc -77· 201019961 or each R1 is independently selected from -NHS02R5, phthalimido-(Cl_c4)-alkylsulfonylamino, benzhydrylamine, phenylsulfonylamino, a 3-phenylphenyl group, a 2-sided oxypyrrolidinyl group, a 2,5-di-oxypyrrolidinyl group, and an R1G-(c2-c4)-alkylmercaptoamine group, wherein Ri is selected from the group consisting of Halogen, _or6, c2_e4 alkoxy, -C(〇)R7 and _NR6R6; and wherein the groups _nhso2r5, phthalic acid iodide-(Cl_c4)-alkylsulfonylamino group, Benzoylamino, phenylsulfonylamino, 3-phenylureido, 2-sided oxypyrrole, 2,5-di-oxypyrrolidine, and Ri〇_( The C2_C4)-alkylamino group is optionally substituted by 1 or 2 substituents independently selected from halo, Ci-C4 alkyl, cyano, methanesulfonyl and Ci-C* alkoxy; or The R1 group together with the carbon to which it is attached forms a 5-8 membered ring comprising 1 or 2 heteroatoms selected from the group consisting of ruthenium, s and N; R2 is hydrogen or a CrC6 base, the CrC6 alkyl group being subjected to 1 Substituting to three substituents independently selected from the group consisting of i group, CVC4 alkoxy group, _NR6R6 and -so2r5; η is 1 or 2' and each R3 is independently selected from hydrogen and halogen a radical, a radical, a c-alkyl group, an alkoxy group, wherein the alkyl moiety of the R3 group is optionally one to three substituents independently selected from the group consisting of halo, alkoxy, _NR6R6 and -SO2R Substituting; and R4 is azido or -(ethynyl)-Rn ' wherein R11 is hydrogen or Cl-C6 alkyl, and the C"C6 alkyl group is optionally substituted with hydroxy, _R6 or _NR6R6. In a specific embodiment, the EGFR antagonist is a compound of formula I selected from the group consisting of: (6,7-dimethoxyquinazolin-4-yl)-(3-ethynylphenyl)-amine (6,7-two 143939.doc -78 · 201019961 methoxyquinazolin-4-yl)-[3-(3'-hydroxypropyn-1-yl)phenyl]-amine; [3_ ( 2-(Aminoguanidino)-ethynyl)phenyl]-(6,7-dimethoxy-isocyanin- 4-yl)-amine; (3-ethynylphenyl)-(6-wall (6,7-dimethoxyquinazolin-4-yl)-(4-ethynylphenyl)-amine; (6,7-dimethoxy Quinazolin-4-yl)-(3-ethylidyl-2-mercaptophenyl)-amine; (6-amino 啥β坐琳-4-yl)-(3-ethynylphenyl)- Amine; (3-ethynylphenyl)-(6-methanesulfonylaminoquinazolin-4-yl)-amine; (3-ethynylphenyl)-(6,7-methylenedioxy (喧, -4-yl)-amine; (6,7-dimethoxy quinazolin-4-yl)-(3-ethynyl-6-methylphenyl)-amine; (3-acetylene Phenyl)-(7-nitroindole _4_yl)-amine; (3-ethynylphenyl H6-(f-indolesulfonylamino)quinazoline-4-yl]- Amine; (3-ethynylphenyl)-{6-[2'-ortho Di-n-imino-ethyl-7'-yl-y-glycosylamino] 啥β sitin _4-yl}-amine, (3-ethyl-bromophenyl)-(6-fluorenyl 喧β sitting mouth _4_yl)-amine; (7-aminoquinazolin-4-yl)-(3.ethynylphenyl)-amine; (3-ethynylphenyl)-(7-methoxyquinazoline) 4-yl)-amine; (6-methoxycarbonylquinazolin-4-yl)-(3-ethynylphenyl)-amine; (7-fluorenyloxyquinazoline-4-yl)-( 3_B-phenylphenyl)-amine; [6,7-bis(2-decyloxyethoxy) 啥 琳 · 4 4 4 4 ; ; ; ; 3- 3- Azidophenyl)-(6,7-dimethoxyquinazolin-4-yl)-amine, (3-isole-based gas-based)-(6,7-dimethyllacyl oxime- 4-yl)-amine; (4-azidophenylphosphonium 6,7-dimethoxyoxyquinazolin-4-yl)-amine; (3-ethynylphenyl)-(6-methanesulfonyl) -quinazolin-4-yl)amine; (6-ethanethio-indolyl-4-yl)-(3-ethynylphenyl)-amine; (6,7-dimethoxy -喧β坐琳_ 4-yl)-(3-ethynyl-4-fluoro-phenyl.)-amine; (6,7-dimethoxy-quinazoline-4-yl)-[3-( Propyne-1,-yl)-phenyl]-amine; [6,7-bis-(2-methoxy-ethoxy)-啥°琳琳-4-yl]-(5-ethylhexyl-2-methyl-phenyl)amine; [6,7-bis-(2-oxo 143939.doc • 79- 201019961 base-ethoxyl )-quinazolin-4-yl]-(3-ethynyl-4-fluoro-phenyl)-amine; [6,7-bis-(2-a-ethoxy)quinazoline_4_ [7-(3-ethane-ethoxy)-7-(2-methoxy-ethoxy)-quinazoline-4-yl]- (3-ethynyl-phenyl)-amine; [6,7-bis-(2-acetoxy-ethoxy)-quinazoline-4-yl]-(3-ethynyl-phenyl) -amine; 2-[4-(3-ethynyl-phenylamino)-7-(2-hydroxy-ethoxy)-quinoxalin-6-yloxy]-ethanol; [6-(2 -Ethyloxy-ethoxy)-7-(2-decyloxy-ethoxy)quinazoline-4-yl]-(3-ethynyl-phenyl)-amine '[7-( 2-gas-ethoxy)_6-(2-methoxy-ethoxy)-oxime! 1 sits _4-yl]-(3-ethynyl-phenyl)-amine; [7_(2_ Ethyloxy-ethoxy)-6-(2-methoxy-ethoxy)-quinazolin-4-yl]-(3-ethynylphenyl)-amine; 2-[4-(3 Ethyl-phenylamino)-6-(2-hydroxy-ethoxy)-quinazolin-7-yloxy]-ethanol; 2-[4-(3-ethynyl-phenylamino) Methoxy-ethoxy) quinazoline -6-yloxy]-ethanol; 2-[4-(3-ethynyl-phenylamino)-6-(2-decyloxy-ethoxy)-quinazolin-7-yloxy ]-ethanol; [6-(2-acetoxy-ethoxy)-7-(2-decyloxy-ethoxy)-quinazoline·4-yl]-(3-ethynyl-benzene ()) amine; (3-ethynyl-phenyl fluorenyloxy-ethoxy)_7-[2-(4-methyl-Octosin-1-yl)-ethoxy]-quinazoline _ 4_Ketamine; (3-ethynyl-phenyl[7-(2-methylsulfanyl-ethoxy)-6-(2-morphin-4-yl)-ethoxy)-isophilic _ 4_ kibamine: ^^-diethoxyquinazoline-丨-kib^-ethynylphenyl-amine; (6,7-dibutoxyquinazoline·丨-yl^, B block Phenyl)-amine; (6,7•diisopropoxyquinazolin-1·yl)-(3-ethynylphenyl)-amine; (6,7-diethoxyoxazoline-1 -yl)-(3-ethynyl-2-indenyl-phenyl)-amine; [6,7-bis-(2-decyloxy-ethoxy)-quinazolin-1-yl]-( 3-ethynyl-2-indenyl-phenyl)-amine; (3_ethynylphenylphosphonium 6-(2-hydroxy-ethoxy)-7-(2-methoxy-ethoxy)·quino 143939 .doc •80- 201019961 ° sitin-1-yl]-amine; [6,7-bis-(2-trans-ethoxy-)-啥" sit Lin-i_基]_( 3-_ ethynylphenyl)-amine; 2-[4-(3-ethynyl-phenylamino)-6-(2-methoxy-ethoxy)-喧°坐琳-7-yloxy ]_Ethanol; (6,7-dipropoxy-hydrazinyl-4-yl)-(3-ethyl-p-phenyl)-amine; (6,7-diethoxy-啥β sitting _4_基)_(3-ethynyl-5-fluoro-phenyl)-amine; (6,7-diethoxyquinazolin-4-yl)-(3_ethynyl-4-fluoro-benzene (6,7-diethoxy-oxime-cylin-4-yl)-(5-ethylidyl-2-methyl-phenyl)-amine; (6,7-diethyl) Oxy-喧°坐琳-4-yl)-(3-ethynyl-4-methyl-phenyl)-amine; (6-aminomethyl-7-methoxy-嗟η sitting _4_ (6-Aminoalkynyl-7-methoxy-quinazolyl-4-yl)-(3-ethoxyphenyl)-amine; (6-Aminomethyl-7-methoxy-Nytyl-4-yl)-(3-ethoxyphenyl)-amine; (6-Aminoethylethyl-7- Methoxy-quinazolin-4-yl)-(3-ethynylphenyl)-amine; (6-aminocarbonylmethyl-7-ethoxy-quinazolin-4-yl)-(3 -ethynylphenyl)-amine; (6-aminocarbonylethyl-7-ethoxy-quinazolin-4-yl)-(3-ethynylphenyl) -amine; (6-aminomethylmethyl-7-isopropoxy-indolyl-4-yl)-(3-ethynylphenyl)amine; (6-aminocarbonylmethyl-7) -propoxy-quinazolin-4-yl)-(3-ethynylphenyl)-amine; (6-aminocarbonylmethyl-7-decyloxy-quinazoline-4-yl)-( 3-(ethynylphenyl)-amine, (6-aminoethylethyl-7-isopropoxy-oxiolin-4-yl)-(3-ethynylphenyl)-amine; -Amino-t- ethylethyl-7-propoxy-??-?? _4·yl)_(3_ethynylphenyl)-amine; (6,7-diethoxy 啥 琳 _1 _1 _ base )-(3·B-ylphenyl)-amine; (3-ethynylphenyl)-[6-(2-hydroxy-ethoxy)-7-(2-decyloxy-ethoxy)- Quinazolin-1-yl]-amine; [6,7-bis-(2-hydroxy-ethoxy)-quinazoline-1-yl]-(3-ethynylphenyl)-amine; [6 ,7-bis-(2-methoxy-ethoxy)-quinazolin-1-yl]-(3-ethynylphenyl)-amine; (6,7-dimethoxyquinazoline _ Ip_ 143939.doc • 81 · 201019961 base M3-ethyl-bromophenyl)-amine·'(3-ethyl-based phenyl)-(6-methanesulfonylamino-hydrazin-1-yl)- Amine; and (6-amino-saltyl-l-yl)-(3B-b-phenylene)-amineIn a particular embodiment, the EGFR antagonist of Formula I is ethoxylated phenyl)-6,7-bis(2-decyloxyethoxy) 4-indolylamine. In a specific embodiment, the EGFR antagonist Ν-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy quinazolinamine is in the form of the hydrochloride salt. In the examples, the EGFR antagonist Ν-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)_4_ 坐 ° sitalamine is a substantially homogeneous crystalline polymorph Form (described as polymorph 〇 in W〇01/34,574), which exhibits a characteristic peak of the X-ray powder diffraction pattern of 2 Θ, expressed at about 6.26, 12.48, 13.39, 16.96, 20.20, 21.10, 22.98, 24.46, 25.14 and 26.91. The polymorphic form of Ν-(3-ethyl-bromophenyl)-6,7-bis(2-decyloxyethoxy)-4-indenylamine is called TarcevaTM and OSI-774, CP-358774 and erlotinib. The compounds of formula I, their pharmaceutically acceptable salts and prodrugs (hereinafter referred to as active compounds) may be any of those known to be suitable for the preparation of chemically related compounds. Process Preparation. The active compounds can generally be prepared from the appropriately substituted quinazolines using the appropriate substituted amines as shown in General Scheme I, as disclosed in US 5,747,498: 143939.doc -82· 201019961

As shown in Scheme I, a suitable 4-substituted quinazoline 2 (wherein X is a suitable dissociable leaving group such as halo, aryloxy, alkylsulfinyl, alkylsulfonyl) Such as trifluoromethanesulfonyloxy), arylsulfinyl, aryl fluorenyl, oxime, cyano, π-salt, triterpene or tetra-β, and 143939.doc-83 - 201019961 Preferably 4-oxoquinazoline and the appropriate amine or amine hydrochloride 4 or 5 (wherein R4 is as described above and Y is Βι, I or trifluoromethane) in a solvent (such as (ci·:) alcohol, dimethylformamide (DMF), N_methyl bite_2, chloroform, ^ guess, tetrahydroanthracene (THF), M-dioxime " Reaction in an aprotic solvent). The reaction can be carried out in the presence of a test, preferably a metal or soil test metal carbonate or hydroxide, or a tertiary amine test such as ruthenium, 2,6-lutidine, trimethylpyridine, N _Methyl morpholine, triethylamine, decylamino-pyridine or N,N-dimethylaniline. These bases are hereinafter referred to as suitable bases. Maintaining the reaction mixture at a temperature from about ambient temperature to about the reflux temperature of the solvent, preferably from about 35 ° C to about reflux temperature, until substantially no residual 4-dentate quinazoline is detected, typically from about 2 to about 24 hour. The reaction is preferably carried out under an inert atmosphere such as dry nitrogen. The reactants are typically mixed stoichiometrically. When an amine base is used as the compound using the salt of the amine 4 or 5 (usually the hydrochloride salt), it is preferred to use an excess of the amine base, usually 1 equivalent of an excess of the amine base. (Alternatively, if an amine base is not used, an excess of amine 4 or 5 can be used). For compounds using a hindered amine 4 (such as 2·alkyl-3_ethynylaniline) or a highly reactive 4-aminoquinazoline, it is preferred to use a third butanol or a polar aprotic solvent (such as DMF or N-methylpyrrolidone) as a solvent. Alternatively, a 4-substituted quinazoline 2 (wherein X is a hydroxy or pendant oxy group (and 2 • nitrogen is hydrogenated)) and a tetra-carbonated carbon and, optionally, a substituted triaryl group supported on an inert polymer Phosphine (eg, polymer supported triphenylphosphine, Alddch Cat. No. 36, 645-5, which contains 2 millimoles of phosphorus per 2 grams of phosphorus crosslinked with divinylbenzene) in a solvent such as four gases Carbon, 143939.doc • 84 · 201019961 Gas, di-ethane, tetrahydrofuran, acetonitrile or other aprotic solvent or its reaction in the sputum. The reaction mixture is maintained at about ambient temperature to reflux temperature, preferably about 35. (: 2 to 24 hours at the temperature of the reflux temperature, the mixture is removed directly or by evaporation, for example, by vacuum and a suitable replacement solvent (such as (CVC6) alcohol, DMF, N-methylpyrrolidine) is added. The ketone, pyridine or 1,4-dioxane is then reacted with the appropriate amine or amine hydrochloride 4 or 5. Subsequently, the reaction mixture is maintained at about ambient temperature to the reflux temperature of the solvent, preferably from about 35 ° C to about reflux. The temperature is formed until the product formation is substantially complete, usually for about 2 to about 24 hours. The reaction is preferably carried out under an inert atmosphere such as dry nitrogen. When compound 4 is used (wherein γ is Br,] [or trifluoro When decanesulfonyloxy) is reacted as a starting material with quinazoline 2, a compound of formula 3 (wherein Ri, R2, R3 and Y are as described above) is formed by compound 3 with a suitable palladium reagent (such as Ruthenium (triphenylphosphine) palladium or bis(triphenylphosphine) dipalladium palladium) in a suitable Lewis acid (such as vaporized cuprous) and a suitable alkyne (such as tri-φ methyl decyl) In the presence of acetylene, propargyl alcohol or 3-(N,N-dimethylamino)-propyne Reacting in a reagent such as diethylamine or triethylamine to convert compound 3 to a compound of formula 1 wherein R4 is Rn ethynyl and Rn is as defined above. It may be by using a diazotizing agent such as an acid and Nitrite (eg, acetic acid and NaN〇2)) treats compound 3 (where γ is NH2), followed by treatment of the resulting product with an azide (such as NaNs) to convert compound 3 to a compound where r4 is an azide) . To prepare a compound of formula I wherein R1 is an amine or hydroxylamine group, the corresponding compound of formula I is reduced. 143939.doc -85 - 201019961 The restoration should be made using any of a number of procedures known for such conversion. The reduction can be carried out, for example, by reacting an inert solvent bath with a hydrogen (tetra) based compound in the presence of a suitable metal catalyst such as p, 10 or nickel. Another suitable reducing agent is, for example, an activated metal such as activated iron (produced by washing iron powder with a dilute acid solution such as hydrochloric acid). Thus, for example, a mixture of a nitro compound and an activated metal with concentrated hydrochloric acid in a solvent such as a mixture of water and an alcohol: the alcohol such as methanol or ethanol is heated to, for example, 5 (rc to 15 (temperature in the range of TC) (It should be heated to ... or reduced by thief. Another suitable class of reducing agent is alkali metal dithionite (such as sodium dithionite), which can be in (Cl_C4) alkanoic acid, (Ci_C6) alkanol, water or For use in the preparation of a mixture of R2 or R3 having a - or a secondary amine moiety (other than an amine group intended to react with an oxazoline), preferably protecting the free amine group prior to the above reaction, The protecting group is then removed after the above reaction with 4_(substituted)quinazoline 2. Several well-known nitrogen protecting groups may be used. These groups include (CiC6) alkoxycarbonyl, optionally substituted benzoyl Oxycarbonyl, aryloxycarbonyl, triphenylsulfonyl, ethyleneoxycarbonyl, fluorenyl-nitrophenylsulfonyl, diphenylphosphinyl, p-toluenesulfonyl and benzyl. Nitrogen protecting group The addition can be at a temperature of from about 01 to about 50 t: Between ambient temperature, in the presence or absence of a tertiary amine such as triethylamine, diisopropylethylamine or pyridine, preferably triethylamine, in a gasified hydrocarbon solvent (such as di-methane or hydrazine) , 2_di-ethane) or ether solvent (such as ethylenediamine dimethyl ether, diethylene glycol dioxime ether or THF). Or, and use Schoonton_Baumann 143939.doc -86-201019961 Conditional attachment of protecting groups. After the above coupling reactions of compounds 2 and 5, the protecting groups can be removed by methods known to those skilled in the art, such as for products protected by a third butoxy group. Treatment with trifluoroacetic acid in di-methane.

For a description of the protecting groups and their uses, see T. w. Greene & p G M. Wuts, "Protective Groups in Organic Synthesis", 2nd edition 'John Wiley & Sons, New York, 1991. For the preparation of a compound of the formula wherein R or R2 is hydroxy, it is preferred to cleave a compound of the formula wherein R1*R2 is a (C1-C4) alkoxy group. The cleavage reaction is preferably carried out using any of a number of procedures known for use in this transformation. The oxime-dealkylation can be carried out by treating the protected formula with 15% rc to 17.5 with molten pyridine hydrochloride (20-30 equivalents). Alternatively, the cleavage reaction can be vulcanized, for example, with an alkali metal (Cl_C4) alkyl group. Treatment of the protected quinazoline derivative or treatment with an alkali metal diaryl phosphide such as lithium diphenylphosphinate is carried out by treatment with a substance such as sodium ethoxide. The cleavage reaction is also preferably trihalogenated or trihalide. Treatment of the protected quinazoline derivative with φ aluminum (such as boron tribromide) is preferably carried out at a suitable temperature in the presence of a reaction inert solvent. R1 or R2 is (Cl-C: 4) alkane The compound of the formula I of the sulfinyl group or the (a.)alkylsulfonyl group is preferably prepared by oxidizing a compound of the formula (I) or a caliper of the formula (Ci_C4)alkylthio. Suitable oxidizing agents for the group and/or sulfonyl group are known in the art, such as hydrogen peroxide, peracids (such as 3-hydroxy peroxybenzoic acid or peracetic acid), alkali metal peroxysulfates (such as peroxygen). Potassium monosulfate), chromium dioxide or gaseous oxygen in the presence of platinum, generally under mildest conditions Gasification with a stoichiometric amount of oxidant to reduce the risk of excess oxygen and damage to other functional groups. The reaction is generally in a suitable solvent (such as di-methane, gas, acetone, tetrahydrofuran or In the third butyl fluorenyl bond) and at a temperature of about -25 C to 50 C (preferably at or near ambient temperature, i.e., in the range of 15 ° C to 35 ° C). When it is desired to have a sulfinyl group In the case of a compound, a milder oxidizing agent (such as sodium metaperiodate or potassium molybdate) should be used. It is preferably in a polar solvent such as acetic acid or ethanol. The compound of formula I containing (Ci_c4)alkylsulfonyl can be borrowed. Obtained by oxidizing the corresponding (Ci_c4) alkylsulfinyl compound and the corresponding (CrCU) alkylthio compound. R is optionally substituted (C2-C4) decylamino group, gland base, 3-benzene The compound of formula j wherein the ureido group, the phenyl oxime group or the sulfonamide group can be prepared by deuteration or sulfonation of the corresponding compound wherein R1 is an amine group. Suitable oximation agents are known in the art. Any reagent which forms a guanamine group on a guanidinium group, in the presence of a suitable base, for example Base teeth, such as (C2_C4) alkane or (C2_C4) alkane bromine, or benzamidine chloride or benzyl bromine; alkanoic anhydride or mixed anhydride, such as acetic anhydride or from alkanoic acid and (C-C4) alkane A mixed acid anhydride formed by the reaction of an oxycarbonyl halide (for example, a (C1_C4) alkoxycarbonyl gas). When preparing a compound of the formula I which is a ureido group or a 3-phenylureido group, a suitable oximation agent is, for example, a cyanate salt. (for example an alkali metal cyanate such as sodium cyanate) or an isocyanate (such as phenyl isocyanate p N_sulfonation can be carried out in the presence of a tertiary amine base using a suitable sulfonyl or sulfonyl anhydride. Alternatively, the sulfonation is carried out in a reaction inert solvent and at a temperature in the range of from about -30 <>> to 120 C (preferably at ambient temperature or about ambient temperature). R is a (C1-C4) alkoxy group or a substituted ((^_C4) alkoxy group, or R is a (CrC4) alkylamino group or a substituted mono-N_(C)_CJ alkylamino group or _ 143939.doc • 88 - 201019961 NA-CCi-C4) Alkylamino compounds of formula I are prepared by alkylating R1 to a corresponding compound of a hydroxy or amine group, preferably in the presence of a suitable base. In the presence of a suitable base 'in a reaction inert solvent and at a temperature in the range of from about 1 Torr to about 4 〇〇c (preferably at ambient temperature or about ambient temperature), suitable alkylating agents include alkyl groups _ Or substituted alkyl, such as optionally substituted (CVC4) alkyl gas, ((: 丨-(:4) alkyl bromide or (Cl_c4) alkyl iodide. For the preparation of R1 is substituted by an amine group, The compound of formula I which is substituted by an oxy group or substituted with a cyano group (Ci-COalkyl substituent) preferably has R1 as a group which may be substituted by an amine group, an alkoxy group or a cyano group in the presence of a suitable test. The corresponding compound of the (Ci_c4)alkyl substituent is reacted with an appropriate amine 'alcohol or cyanide. The reaction is preferably carried out in a reaction inert solvent or diluent at a temperature in the range of from about 1 ° C to about 1 Torr ( Preferably, the compound of formula j wherein R is a carboxy substituent or a substituent comprising a carboxy group is hydrolyzed by R1 to a (Ci-C4) alkoxycarbonyl substituent or includes (Cl_c4). The corresponding compound of the substituent of the alkoxy group is prepared. The hydrolysis is preferably carried out, for example, under basic conditions (for example, in the presence of a metal hydroxide). R1 is an amine group, (CVC4) alkylamino group, two [ (CVCJ alkyl)amino, "pyrrolidin-1-yl, N-piperidinyl, N-morpholinyl, piperazin-1-yl, '(CVCO pyrrolizin-1-yl or The Ci-Cd alkylthio group of the formula I can be prepared by reacting a corresponding compound wherein R1 is an amine or a thiol replaceable group with a suitable amine or thiol in the presence of a suitable test. The reaction is preferably carried out in a reaction inert solvent. Or in a diluent and in the range of about 10 ° C to 180 T: (preferably in the range of 100 ° C to 150 ° (in range). R1 is 2 · side oxy-pyrrolidine _ 丨 _ group or 2 _ Side Oxypiperidine 丨 基 基 143 143939.doc -89 - 201019961 The compound is prepared by cyclizing Rl to the corresponding compound of the functional group _(cvc4) in the presence of a suitable test. Preferably, the reaction is carried out in a reaction inert solvent or diluent at a temperature in the range of from about KTC to (10) C (preferably at ambient temperature or about ambient temperature). In the preparation of R1 is an amine methyl group, substituted In the case of a formula of an amine methyl sulfhydryl group, a fluorenyloxy group or a hydrazine substituted alkoxy group, it is preferred to catalyze or oxime the corresponding compound of the hydroxy group. Suitable oximation agents for the hydroxyaryl moiety to form an alkoxy aryl group typically include, for example, (c2_c4)alkyl fluorenyl halide, (C^-C:4) alkanoic acid anhydride, and mixtures as described above, in the presence of a suitable base An acid anhydride, and a suitable substituted derivative thereof can be used. Alternatively, the (C2-C4) succinic acid or a suitably substituted derivative thereof can be coupled with a compound of formula I wherein R is hydroxy by means of a condensing agent such as a carbamide. In the preparation of a compound of formula I wherein Ri is an aminomethyl sulfhydryl group or a substituted amine mercapto group, a suitable amine oximation agent is, for example, a cyanate or an alkyl isocyanate or an aryl isocyanate, usually in the presence of a suitable base. . Alternatively, a suitable intermediate such as a chloroformate or a carbonylimidazole derivative of the compound of the formula [wherein R1 is hydroxy" may be treated, for example, by treatment with phosgene (or phosgene 4) or a few bases of miso. produce. The resulting intermediate can then be reacted with the appropriate amine or substituted amine to yield the desired amine mercapto derivative. A compound wherein R1 is an aminocarbonyl group or a substituted aminocarbonyl group can be prepared by subjecting R1 to a suitable intermediate of a carboxyl group. Activation and coupling of a compound of formula I wherein R1 is a carboxy group can be carried out by a variety of methods known to those skilled in the art. Suitable methods include activating the carboxyl group as an acid halide, azide, symmetrical or mixed anhydride or active ester having the appropriate reactivity with the desired amine coupling of 143939.doc-90.201019961. Examples of such intermediates and their preparation and their use for coupling with amines are widely found in the literature; for example, M. Bodansky and A. Bodansky, "The Practice of Peptide Synthesis", Springer-Verlag, New York, 1984 The compound of formula I can be isolated and purified by standard methods such as solvent removal and recrystallization or chromatography. The starting materials of the reaction scheme I (e.g., amines, quinazolines, and amine protecting groups) are readily available or can be readily synthesized by those skilled in the art using conventional organic methods of synthesis. For example, the preparation of 2,3-dihydro-1,4-benzoxazine derivatives is described in RC ElderHeld, W. H. Todd, S_ Gerber, Chapter 12, "Heterocyclic Compounds", Volume 6 , ed. Elderfield, John Wiley and Sons, Inc., Ν·Υ·, 1957, 0 RC Elderfield and EE Harris, in Elderfield, “Heterocyclic Compounds”, Volume 6, Chapter 13, Chapter 2, 3 a -dihydrobenzothiazinyl compound. In another specific embodiment, the EGFR antagonist has the formula II as described in US 5,457,105 (incorporated herein by reference)

II 143939.doc -91 - 201019961 wherein: m is 1, 2 or 3; and each R1 is independently 6-hydroxy, 7-hydroxy, amine, carboxyl, aminemethanyl, ureido, (1-4C) Alkoxycarbonyl, N-(1-4C)alkylamine fluorenyl, hydrazine, hydrazine bis[(1-4C)alkyl]amine fluorenyl, hydroxylamine, (4C) alkoxyamino , 4C) alkanomethoxyamino group, trifluoromethoxy group, 〇4C) alkyl group, 6-(1-4C) alkoxy group, 7-(l-4C) alkoxy group, (1-3C) Alkyldioxy, (1-4C)alkylamino, bis[(1-4C)alkyl]amino, pyrrolidin-1-yl, N-piperidinyl, N-morpholinyl , piperazin-1-yl, 4-(l-4C)alkylpiperazin-1-yl, (1-4C)alkylthio, (1-4C)alkylphosphonium, (1-4C)alkyl Sulfonyl, bromomethyl, dibromoindolyl, hydroxy-(1-4C)alkyl, (2-4C) decyloxy-(1-4C)alkyl, (1-4C)alkoxy- (1-4C)alkyl, carboxy-U-4C)alkyl, (1-4C)alkoxycarbonyl-(1-4C)alkyl, aminyl-(b40)alkyl, N-( 1-4C)alkylamine fluorenyl-(1-4C)alkyl, N,N-bis[(1-4C)alkyl]aminocarboxamido-(1-4C)alkyl, amine-U -4C)alkyl, (1-4C)alkylamino-(1_4C)alkyl, bis[(1-4C)alkyl] Base (Bu 4C)alkyl, N_decridinyl-(1,4-tetraalkyl, N-morpholinyl-(1-4C)alkyl, piperazin-1-yl-(b 4C) Base, 4-(1-40) yard base, bottom 嗓-1-yl-U-4C), ketone-(2-4C)alkoxy-(1-4C)alkyl, (1-4C Alkoxy-(2-4C)alkoxy-(Bu 4C)alkyl, hydroxy-(2-4C)alkylamino-(Bu 4C)Hyun, (1-4C) aerated base - (2-4C)alkylamino-(1-4C)alkyl, (1-4C)-indolyl-(1-4C) leukoyl, hydroxy-(2-4C)alkylthio-(1_4C)alkane , (1-4C) alkoxy-(2-4C)alkylthio-(1-4C)alkyl, phenoxy-(1-4C)alkyl, anilino-(i-4C)alkyl , phenylthio-0-4C)alkyl, cyano-(1-4C)alkyl, _yl_(2_ 143939.doc • 92· 201019961 4C) alkoxy, hydroxy-(2-4C) alkoxy , (2-4C) alkanomethoxy-(2-4C) alkoxy, (1-4C) alkoxy-(2-4C)alkoxy, carboxy-(1-4C)alkoxy, (1-4C) alkoxycarbonyl-(1-4C)alkoxy, amidino-(1-4C)alkoxy, N-(1-4C)alkylaminemethanyl-(1- 4C) alkoxy, anthracene, fluorenyl-bis[(Ια)alkyl]aminoindolyl-(1-4C)alkoxy, amino-(2-4C)alkoxy, (1-4C)alkane Amino-(2-4C)alkoxy, bis[(1-4C)alkane Amino-(2-4C)alkoxy, (2-4C)alkoxy, hydroxy-(2-4C)alkyloxy, (1-4C)alkoxy-(2-4C) Alkyloxy, phenyl-(1-4C)alkoxy, phenoxy-(2-4C)alkoxy, anilino-(2-4C)alkoxy, phenylthio-(2-4C Alkoxy, hydrazine-piperidinyl-(2-4C)alkoxy, indole-morpholinyl-(2-4C)alkoxy, piperazin-1-yl-(2-4C)alkoxy 4-(l-4C)alkylpiperazin-1-yl-(2-4C)alkoxy, halo-(2-4C)alkylamino, hydroxy-(2-4C)alkylamino (2-4C) alkanomethoxy-(2-4C)alkylamino, (1-4C) alkoxy-(2-4C)alkylamino, carboxy-(1-4C)alkylamine , (1-4C) alkoxycarbonyl-(1-4C)alkylamino, amidino-(1-4C)alkylamino, N-(1-4C)alkylaminecarbamyl -(1-4C)alkylamino, N,N-bis[(1-4C)alkyl]aminoindolyl-(1-4C)alkylamino, amino-(2-4C)alkyl Amino, (1-4C)alkylamino-(2-4C)alkylamino, bis[(1-4C)alkyl]amino-(2-4C)alkylamino, phenyl-( 1-4C) alkylamino group, phenoxy-(2-4C)alkylamino group, anilino-(2-4C)alkylamino group, phenylthio-(2-4C)alkylamino group, (2-4C) alkanoylamino, (1-4C) alkoxy Carbonylamino, (1-4C)alkylsulfonylamino, benzoylamino, benzenesulphonamido, 3-phenylureido, 2-oxooxy η -yl, 2,5-di-tertiary oxyl-l-l-l-yl, aryl-(2-4C)alkylhydrazino, hydroxy-(2-4C)alkylamino, (1- 4C) alkane 143939.doc -93- 201019961 oxy-(2-4C)alkylmercaptoamine, carboxy-(2-4C)alkylalkylamino, (1-4C) alkoxycarbonyl-(2- 4C) alkyl mercaptoamine, amine mercapto-(2-4C)alkylhydrazino, N-(1-4C)alkylamine, mercapto-(2-4C)alkyldecylamine, N , N-bis[(1-4C)alkyl]aminecarbamyl-(2-4C)alkylhydrazino, amino-(2-4C)alkylhydrazino, (1-4C)alkyl Amino-(2-4C)alkylhydrazine-amine or bis[(bu-4C)alkyl]amino-(2-4C)alkylmercaptoamine, and wherein the benzhydrylamine or benzenesulfonamide The substituent or any of the anilino, phenoxy or phenyl groups in the R1 substituent may optionally have one or two halo, (1-4C)alkyl or (1-4C) alkoxy substituents; Is 1 or 2; and each R2 is independently hydrogen, hydroxy, halo, trifluoromethyl, amine, nitro, cyano, (1-4C) , (1-4C) alkoxy, (1-4C)alkylamino, bis[(1-4C)alkyl]amino, (1-4C)alkylthio, (1-4C)alkyl Sulfosyl or (1-4C)alkylsulfonyl; or a pharmaceutically acceptable salt thereof; the exception is the exclusion of 4-(4'-hydroxyanilino)-6-methoxyquinazoline, 4 -(4,-hydroxyanilino)-6,7-methylenedioxyquinazoline, 6-amino-4-(4'-aminoanilino)quinazoline, 4-anilino-6 -methylquinazoline or its hydrochloride and 4-anilino-6,7-dimethoxyoxyquinazoline or its hydrochloride. In a specific embodiment, the EGFR antagonist is a compound of formula II selected from the group consisting of 4-(3·-chloro-4'-fluoroanilino)-6,7-dimethoxy quinazoline; -(3',4·-dianiline)-6,7-dimethoxyquinazoline; 6,7-dimethoxy-4-(3'-nitroanilino)-quinazoline ;6,7-diethoxy-4-(3,-methylanilino)-quinazoline; 6-decyloxy-4-(3'-methylanilino)-quinazoline; 4- (3,-chloroanilino)-6-methoxy quinazoline; 6,7-extended ethyldioxy-4-(3,- 143939.doc 94 201019961 methylanilino)-喧 琳 琳; & Insurance i 7 Listen, 6, Amino-7-methoxy-4·(3,-Methylanilino)-quinazoline; 4-(3'-Methylaminoamino)-6-ureido Quinazoline; 6-(2-methoxyethoxymethyl)_4·(3,·methylanilino)_啥sai; 6,7-di-(2-methoxyethoxy)- 4-(3,-decylamino)-(iv); 6-dimethylamino _4·(31-methylanilino)quinazoline; 6-benzamide _4_(3,_A Benzylamino) quinazoline, 6,7-dimethoxy_4_(3,-trifluoromethylanilino)-quinazoline; 6-hydroxy-7-methoxy-4-(3,-曱Alkylamino) _ quinazoline; 7-hydroxy-6-methoxy-4-(3'-methyl amidoquinazoline; 7-amino-4-(3,-methylanilino)-quinazoline; 6_Amino_4_(3,-methylanilino)quinazoline; amidino_4_(3,-chloroanilino)-quinazoline; 6-acetamido_4-(3'- Methylanilino)-quinazoline; 6-(2-methoxyethylamino)_4_(3,-mercaptoanilino)-quinazoline; 7-(2-methoxyethenylamino) _4·(3Ι_mercaptoanilide)_quinazoline; 7-(2-hydroxyethoxy)-6-methoxy-4-(3methylanilino)·啥啥琳; 7_(2_甲Oxyethoxyethoxy)-6-methoxy-4-(3·-methylanilino)-啥.Shen Lin; 6_Amino_4·(3'-methylanilino)-喧π The quinone derivative of the formula II or a pharmaceutically acceptable salt thereof can be prepared by any method known to be suitable for the preparation of a chemically related compound. A suitable method is, for example, the method used in US 4,322,420. Starting materials are commercially available or can be obtained using standard organic chemistry procedures. (a) The quinazoline (1) which is a displaceable group is preferably reacted with aniline (ii) in the presence of a suitable base. c -95· 201019961

Suitable displaceable groups z are, for example, a southern group, an alkoxy group, an aryloxy group or a sulfonyloxy group such as a gas group, a bromo group, a methyl group, a phenoxy group, a phenoxy group, a methanesulfonyloxy group. Or f benzene-p-sulfonyloxy. Suitable bases are, for example, organic amine bases, such as pyridine, 2,6-lutidine, trimethylpyridine, 4-dimethylamino 咻a & 签 丞 丞 、, diethylamine , morpholine, Ν-methylmorpholine or diazabicyclo[54〇]undecene butene, or for example an alkali metal or soil test metal carbonate or hydroxide, ^ such as sodium carbonate, potassium carbonate, calcium carbonate , sodium hydroxide or potassium hydroxide. The reaction is preferably carried out in the presence of a suitable inert solvent or diluent such as an alcohol or vinegar such as methanol, ethanol, isopropanol or ethyl acetate; a solvent such as a gas. Aromatic, gas-like or tetra-vaporized carbon; ethers, such as tetrahydrofurfury or 1,4-dioxane; aromatic solvents such as toluene; or dipolar non-premature, F-negative In the cold agent, such as hydrazine, hydrazine dimethyl dimethyl carbamide, hydrazine, hydrazine, methyl ketone, Ν-methyl Λ η 4 4 4 4 甲基 甲基 甲基 甲基 _2 _2 _2 _2 _2 _2 _2 _2 _2 _2 . The reaction is preferably, for example, from 1 〇 to 1 $ 〇. It is carried out at the temperature of the main range (preferably 20〇c to the cost of the ribs). The salicin derivative of formula II can be obtained as a free base by using the method of 5 hai, or it can be formed as a salt with an acid of the formula HZ (wherein 7 i, ^, , as defined above) Form obtained. When it is desired to obtain the free base from the salt, the salt can be treated with a suitable base as defined above using conventional procedures. 143939.doc •96· 201019961 (b) is a quinazoline derivative of the formula π which is a ruthenium compound in which R1 or R2 is a hydroxyl group, which is cleaved or otherwise is a U-^c) alkoxy group. The cleavage reaction is preferably carried out using any of a number of procedures known for such transformation. The quinazoline derivative can be treated, for example, by treatment with an alkali metal (1_4C) alkyl sulfide such as sodium ethanethiolate or, for example, with an alkali metal diaryl phosphating agent such as lithium diphenylphosphide. To react. Alternatively, the cleavage reaction is preferably carried out, for example, by treating the quinazoline derivative with boron trioxide or aluminum trichloride (such as boron tribromide). Preferably, the reactions are carried out in the presence of a suitable inert solvent or diluent as defined above and at a suitable temperature. (c) is a compound of formula II wherein R or R is (1·4 C)alkyl or a (1 _ 4 c)alkylsulfonyl group, and the Ri*R2 is oxidized to (1_4C)alkylthio a quinazoline derivative. Suitable oxidizing agents are, for example, any of the agents known in the art for oxidizing sulfur groups to form sulfinyl groups and/or sulfonyl groups, such as hydrogen peroxide, peracids such as 3·gas peroxybenzoic acid or peroxygen Acetic acid), alkali metal peroxosulfate φ (such as potassium peroxymonosulfate), chromium trioxide or gaseous oxygen in the presence of platinum. Oxidation is generally carried out using the stoichiometric amount of oxidizing agent as mildly as possible to reduce the risk of excessive oxidation and damage to other functional groups. The reaction is generally carried out in a suitable solvent or diluent such as dichloromethane, gas, propionium, tetra-n- or tri-decyl ether and at a temperature of, for example, _25 to 50 C (preferably around) The temperature is about or about ambient temperature, that is, in the range of 15C to 35C. When a compound having a sulfinyl group is desired, it is also preferred to use a milder oxidizing agent (e.g., sodium metaperiodate or potassium metaperiodate) in a polar solvent such as acetic acid or ethanol. It should be understood that when a compound of the formula π containing 143939.doc -97· 201019961 (1-4C)alkylsulfonyl group is required, the corresponding (1_4C) alkyl group can be oxidized and the corresponding (1-4C) An alkylthio compound to obtain the compound of the formula II. (d) A quinazoline derivative of the formula wherein R is a nitro group for the preparation of a compound of formula II wherein R1 is an amine group. The reduction should be carried out using any of a number of procedures known for such transformations. The reduction can be carried out, for example, by hydrogenating a solution of the nitro compound in the presence of a suitable metal catalyst such as palladium or platinum in an inert solvent or diluent as defined above. Another suitable reducing agent is, for example, an activated metal such as activated iron (produced by washing iron powder with a dilute acid solution such as hydrochloric acid). Thus, for example, a mixture of the nitro compound and the activating metal in a suitable solvent or diluent such as a mixture of water and an alcohol, such as decyl alcohol or ethanol, can be heated to, for example, 50. (: to a temperature in the range of 150 ° C (preferably heated to 7 (rc or about 70 ° C) for reduction. (e) For the preparation of R1 is (2-4C) decylamino group or substituted (2_4 〇 a compound of the formula hydrazino, ureido, 3-phenylureido or benzylideneamine, or a compound wherein R2 is an acetamino group or a benzylamino group, which is an amine group or a ruler 2 A quinazoline derivative of the formula II. Suitable oximation agents are, for example, any of the agents known in the art for the oximation of an amine group to form a guanamine group, for example, in the presence of a suitable base as defined above. Alkyl halides such as (2_4C) alkane sulfhydryl or (2_4C) alkanoyl bromide, or phenylhydrazine or benzamidine bromide; alkanoic anhydride or mixed anhydride in the presence of a suitable base as defined above, for example ( 2_4C) an alkanoic anhydride (such as acetic anhydride) or a mixed acid anhydride formed by an alkanoic acid and a (1-4C) alkoxycarbonyl halide (for example, (1-4C) alkoxycarbonyl gas) 143939.doc • 98-201019961. When preparing a compound of formula II wherein R1 is a ureido group or a 3-phenylureido group, suitable oximation agents are, for example, cyanates (for example metal citrates such as sodium cyanate) or for example isocyanic acid An ester (such as phenyl isocyanate). The oximation is generally in a suitable inert solvent or diluent as defined above and at a temperature in the range of, for example, -3 ° C to 120 ° C (preferably at ambient temperature or about (at ambient temperature) (0 is the preparation of R1 is (1-4C) alkoxy or substituted (1-4C) alkoxy' or R1 is (1-4C) alkylamino group or substituted ( 1-4C) Alkylamino group of a compound of formula II, preferably alkylated R1, in the presence of a suitable base as defined above, as defined above as a hydroxy or a quinazoline derivative of formula II. In the presence of a suitable base, in a suitable inert solvent or diluent as defined above and at, for example, 1 (TC to 140 ° C temperature (preferably at ambient temperature or about ambient temperature), a suitable alkyl group The agent is, for example, any of those known in the art for alkylating a hydroxyl group to form an alkoxy group or a substituted alkoxy group or for alkylating an amine group to form an alkylamino group or a substituted alkylamine group. a reagent such as an alkyl i or a substituted alkyl group, for example, (1-4C), a base gas, a (1 to 4C) alkyl bromide or a (14C) alkyl iodide or a substituted (1- 4C) House-based gas, substituted (丨_4(:) alkyl bromide or substituted (14(:)) alkyl moth. (g) Formula for preparing R1 as a carboxyl substituent or a substituent including a carboxyl group The π compound 'hydrolyzes W is a 〇-4C) alkoxycarbonyl substituent or a quinazoline derivative of the formula 包括 including a substituent of the (1-4C) alkoxycarbonyl group. The hydrolysis is preferably carried out, for example, under the conditions of the test. (h) for the preparation of a compound of formula II wherein R1 is substituted by an amine group, substituted by an oxy group, substituted by a thio group, 143939.doc • 99· 201019961 or substituted by a cyano group (1-4C) alkyl group, preferably A quinazoline derivative of formula II wherein R1 is a (1-4C)alkyl substituent having a replaceable group as defined above in the presence of a suitable base as defined above, together with an appropriate amine, alcohol, thiol Or cyanide reaction. The reaction is preferably carried out in a suitable inert solvent or diluent as defined above and at a temperature in the range of, for example, 10 ° C to the loot (preferably at ambient temperature or about ambient temperature). When a pharmaceutically acceptable salt of a quinazoline derivative of the formula II is desired, the salt can be obtained, for example, by reacting the compound with, for example, a suitable acid using a conventional procedure. In a particular embodiment, the EGFR antagonist is a compound of formula II, as disclosed in US 5,770,599, which is incorporated herein by reference.

Wherein: η is 1, 2 or 3; each R2 is independently halo or trifluoromethyl; R3 is (1-4C) alkoxy; and R1 is bis[(1-4C)alkyl]amino -(2-4C) alkoxy, pyrrolidin-buki-(2-4C) alkoxy, oxime-branches-(2-4C) alkoxy, morphine-(2-4C) *oxy, 派-l-yl-(2-4C) alkoxy, 4-(l-4C) 炫" base 11 桊-1_ 143939.doc -100- 201019961 base-(2-4C) Alkoxy, imidazolium-hydrazino-yl-hydroxyl, bis[(1_4C)alkoxy-(2-4C)alkyl]amino-(2-4C)alkoxy, thiophene Phytyl-(2_4C) alkoxy, 1-o-oxy-N-indolyl-(2-4C)alkoxy or di-oxy-N-singly-based-(2-4C) An oxy group, and any of the above-mentioned Ri substituents comprising a CH2 (methylene group) not bonded to a N or a ruthenium atom, optionally having a hydroxy substituent on the CH2 group; or pharmaceutically acceptable Accept the salt. In a specific embodiment, the EGFR antagonist is a hydrazine compound selected from the group consisting of 4-(3,-gas-4,-fluoroanilino)-7-decyloxy-6-(2-pyrrolidine) -1-ylethoxy)-quinazoline; 4-(3,-gas-4,-fluoroanilino)-7-methoxy-6-(2-N-morpholinylethoxyquinazoline ; 4_(3,_Chloro-4, fluoroanilino 6-(3-diethylaminopropoxy)_7-methoxyquinazoline; 4_(3, gas·4, fluoroanilino)- 7-Methoxy-6-(3-pyridin-1-ylpropoxy)-quinazoline; 4_(3'-gas-4'-fluoroanilino)_6-(3-dimethylamine Propyloxy)-7-methoxyquinoxaline; 4-(3',4'-difluoroanilino)-7-methoxy-6-(3-indole-morpholinopropoxy)-indole °Situline; 4-(3'-gas-4,-fluoroanilino)-7-decyloxy-6-(3-indole-piperidinylpropoxy)-quinazoline; 4-(3, - gas-4,-fluoroanilino)-7-decyloxy-6-(3-indole-morpholinopropoxy)-quinazoline; 4_(31_gas_41_fluoroanilino)_6_( 2-didecylaminoethoxy)-7-methoxyquinazoline; 4-(2,,4'-difluoroanilino)-6-(3.dimethylaminopropylpropoxy) _7_曱oxyquinazoline; 4_(2,,41-difluoroanilinyl -7-methoxy-6-(3-indolyl-morpholinylpropoxy)-quinazoline; 4-(3,-aero-4-fluoroanilino)-6-(2-imidazole-1 -ylethoxy)-7-methoxy quinazoline; 4-(3·-gas-41-fluoroanilino)-6-(3-imidazol-1-ylpropoxy)-7-methoxy Quinazoline; 4-(3'-gas-41-fluoroanilino)-6-(2·didecylaminoethoxy)-7-methoxy 143939.doc -101- 201019961 quinazoline, 4-(2',4'-difluoroanilino)-6-(3-didecylaminopropoxy)-7-methoxyquinazoline; 4_(2,4,-difluoroaniline _7_methoxy_6_(3_n_morpholinylpropoxy)-quinazoline; 4_(31_gas_4,-fluoroanilino)6(2-imidazolium-1-ylethoxy)- 7-methoxyquinazoline; and 4_(3,_qi_4'-fluoroanilino)_6_(3-imidazolium-1-ylpropoxy)_7-methoxyquinazoline. In a specific implementation In the case, the EGFR antagonist is a compound of the formula, that is, 4_(3H-anilino)·7_methoxy_6_(3_Ν_morphine-propoxy-oxazoline, or ZD 1839, Gefitse And Iressa®. The quinazoline derivative of formula II or a pharmaceutically acceptable salt thereof is known to be suitable for use in any method for preparing chemically related compounds. Suitable methods include, for example, the methods described in us 5616582, us 558〇87〇, us 5475〇〇1 and still 5569658. Unless otherwise stated, t _n R2 ^ R1 has any meaning as defined by llUn above. The necessary starting materials are commercially available or obtained using standard organic chemical procedures. (4) It is preferred to react (4) with a cyclizable group (Hi) with an aniline (iv) in the presence of a suitable test.

Suitable substituted bases Z are, for example, a southern group, an alkoxy group, an aryloxy group or a hydrazine group such as a gas group, a bromo group, a methoxy group, a phenoxy group, a methyl group or a toluene-4-sulfonate. Alkoxy. 143939.doc •102· 201019961

Suitable tests are, for example, organic amine bases such as ... 2 each of dimethyl hydrazine, trimethyl hydrazine, and I dimethylamino. Than mouth, triethylamine, morphine, N-methylmorpholine or diazabicyclo [5.4.0] tens - ''' 7_浠, or for example metal or alkaline earth metal carbonate or hydroxide, such as J Such as sodium carbonate, potassium carbonate, carbonic acid, sodium hydroxide or hydroxide. Or human *, a suitable base is, for example, an alkali metal or alkaline earth metal amine, such as sodium amination or a bis(dimethylalkylalkyl) aminated nano

The reaction is preferably carried out in the presence of a suitable inert solvent or diluent such as a sterol or an ester such as methanol, ethanol, isopropanol or ethyl acetate; a solvent such as dichloro A hospital, gas-like or carbon tetrachloride; an ether such as tetrahydrofuran or 154-dioxane; an aromatic solvent such as toluene; or a dipolar aprotic solvent such as N,N-dimethylformamide, N , N-dimethylacetamide, N_decylpyrrolidine-2-one or dimethyl sulfoxide. The reaction is preferably, for example, 1 Torr. 〇 to 150. 〇; in the range (preferably in the range of 2 (^ to 8〇1). The quinazoline derivative of the formula can be obtained as a free base by this method, or it can be represented by the formula HZ (wherein Z) The salt form of the acid formed having the meaning defined above is obtained. When it is desired to obtain the free base from the salt, a suitable procedure can be used to prepare the salt as defined above using a conventional procedure. (b) Preparation of R1 as an amine a quinazoline derivative of the formula 2-4, wherein the quinazoline derivative of the formula 1 is a hydroxy group, in the presence of a suitable base as defined above. Where appropriate, in the presence of a suitable inert solvent or diluent as defined above and at a temperature in the range of, for example, lot: to 140 ° C, 143939.doc -103.201019961 degrees (preferably at 80. (: below or about At 80 ° C, 'suitable singly-based agents are, for example, any of the reagents known in the art for alkylating a hydroxyl group to form an amino-substituted alkoxy group, such as an amine-substituted alkyl halide. For example, an amine group substituted (2-4C) alkyl gas, an amine substituted (2-4C) alkyl bromide or an amine Substituted (2-4C) alkyl iodide. (c) For the preparation of a compound of formula II' wherein R1 is an amino substituted (2-4C) alkoxy group, it is preferred to have a suitable base as defined above. R1 is a hydroxy-(2-4C) alkoxy compound of the formula or a reactive derivative thereof, which is reacted with a suitable amine. R is a suitable reactive derivative of a hydrazine compound of the group -(2-4C) alkoxy group. For example, halo-(2-4C)alkoxy or sulfo-oxy-(2-4C)alkoxy, such as bromo-(2-4C)alkoxy or decanesulfonyloxy_(2_4C) Alkoxy. The reaction is preferably carried out in the presence of a suitable inert solvent or diluent as defined above and at a temperature in the range of, for example, 10 ° C to 150 (suitably at 5 (rc or about 50 C) (d) For the preparation of a compound of the formula: wherein R1 is hydroxy-amino-(2_4C)alkoxy, R1 is 2,3-epoxypropoxy or 3,4-epoxybutenyl, π, The compound is reacted with a suitable amine. The reaction is preferably carried out in the presence of a suitable inert solvent or diluent as defined above and at a temperature in the range of, for example, H)m5 (preferably at or about 70 ° C). When the pharmaceutically acceptable salt of the desired organism is (e.g., a mono- or diacid addition salt of a hydrazine derivative), which can be obtained, for example, by reacting the compound with, for example, a suitable acid using conventional procedures to obtain the 143939.doc 201019961 in a particular implementation In an embodiment, the EGFR antagonist is a hydrazine compound, or a salt or solvate thereof, as disclosed in WO 993 5146, which is incorporated herein by reference.

/U HN

III X is N or CH; Y is CR1 and v is N; or Y is N and V is CR1; or Y is CR1 and V is CR2; or Y is CR2 and V is CR1;

R1 represents a group CH3S02CH2CH2NHCH2-Ar-, wherein the Ar is selected from the group consisting of phenyl, decyl, thiophene, pyrrole and thiazole, each of which may be optionally substituted by one or two dentate groups, Cw alkyl or Cw alkoxy; R2 Selected from the group consisting of hydrogen, halo, hydroxy, Cl.4 alkyl, alkoxy, Cl.4 alkylamine tomb and bis[Ci 4 alkyl]amine; U represents phenyl, oxaridinyl , 3H-imidazolyl, fluorenyl, isodecyl, dolantyl, isoindolinyl, 1H-carbazolyl, 2,3-dihydro-1H_, oxazolyl, 1H-benzimidazolyl, 2,3-dioxin-1H-benzimidazolyl or 1H-benzodisalyl' is substituted by an R3 group and optionally substituted with at least one independently selected R4 group; 143939.doc •105- 201019961 R3 is selected from the group consisting of benzyl, halobenzyl, dihalobenzoyl and tridentylmethyl, benzoinyl, 0-pyridylthio, and pyridyl a methoxy group, a phenoxy group, a phenoxy group, a benzoyl methoxy group, a dihalo benzyloxy group, a trihalophenyl fluorenyloxy group, and a benzene sulfonyl group; or a calilem 3 represents a trihalo fluorenyl group Benzyl or trihalomethylbenzyloxy·, or R3 A group of formula

Wherein each R5 is independently selected from the group consisting of halogen, C1-4 alkyl and C1-4 alkoxy; and η is 0 to 3; and each R4 is independently hydroxy, halogen, Cl-4 alkyl, c2-4 alkenyl , C2.4 alkynyl, c 4·alkoxy, amine, Cl 4 alkylamino, bis[Cl 4 alkyl]amine, Cw thiol, Cl 4 alkylsulfinyl, Cl 4 alkyl Sulfonic acid group, Cl 4 alkyl group, ruthenium, amine carbaryl group, Cl-4 alkoxycarbonyl group, Cl 4 alkylalkylamino group, N-%-4 alkyl group, amine fluorenyl group, n, N - bis(Cw alkyl)amine fluorenyl, cyano, nitro and trifluoromethyl. In a specific embodiment, the antagonist of the formula ni2 EGFR is excluded: (1-benzoinyl 1H-indolyl-5-yl)-(6-(5-((2-methylpyrene)ethylamino) _Methyl)-furan-2-yl)-pyrido[3,4-d]pyrimidin-4-yl-amine; (4-benzoyloxyphenyl)-(6-(5-((2-decane)) Sulfhydryl-ethylamino)-methyl)-furan-2-yl)-. than biting and [3,4-d] nits 4-yl-amine; (1-phenylmethyl-1H-吲.Sit_5_yl)-(6-(5·((2-decanesulfonyl)ethyl)ethyl)-furan-2-ylquinazoline core - 143939.doc • 106· 201019961 Amine (1 benzyl hydrazino-indazol-5-yl)-(7-(5-((2-methanesulfonyl)ethylamino)-methyl)-furan-2-yl) _ quinazoline _4_ yl-amine; and (1 benzyl-_ιη_carbazolyl)-(6-(5-((2-methanesulfonyl-ethylamino)-methyl)) _ Pyrrolyl)-quinazolin-4-yl-amine. In a particular embodiment, the EGFR antagonist of Formula III is selected from the group consisting of: 4·(4-fluorobenzyloxy)-phenyl Η6-(5-((2·甲烧烧基基基基基基基)_〇夫喃-2-yl)-η ratio bite [3,4-d] bite _4_ base)_ Amine; ((fluoro*benzyloxy)-benzyl)-(6 -(5-((2-methylpropioninyl-ethylamino)methyl)furan-2-yl)-D-pyrido[3,4-d]pyrimidin-4-yl)-amine; (4- Phenylsulfonyl-phenyl)-(6-(5-((2-decanesulfonyl)ethylamino)-methyl)-furan-2_pyridinium[3,4-d]pyrimidine -4-yl)-amine; (4-phenyloxy-phenyl)-(6-(3-((2-methanesulfonyl-ethylamino))methyl)phenyl)pyridinium[ 34d] pyrimidin-4-yl)-amine; (4-benzoyloxy-phenyl)-(6_(5·((2 methanesulfonyl)ethylamino)-indenyl)-» (2) quinazoline _4_yl)-amine; (4-(3-fluorobenzyloxy-phenyl)-(6-(4-((2-methanesulfonyl-ethylamino)) ) _methyl)-furan-2-yl)-n-pyrido[34_d]pyrimidin-4-yl)amine; (4-benzoyloxyphenyl)_(6-(2-((2-methanesulfonyl)) Ethylethylamino)-methylthiazole-4-yl)quinazoline-4-yl)-amine; N-{4-[(3-fluoromethyl)oxy]phenyl}_6_[5· ({[2_(decane fluorescein) ethyl]amino}methyl)_2_furanyl]_4_quinazolinamine; Ν-{4-[(3-fluorobenzyl)oxy] -3-methoxyphenyl}_6_[5·({[2_(methanesulfonyl)ethyl]amino}methyl)-2-furanyl]_4_ quin啉-[4-(phenylhydroxy)phenyl]-7-methoxy-6-[5-({[2-(methanesulfonyl)ethyl)amino}methyl)· 2·furanyl]-4-quinazolinamine; Ν_[4_(benzyloxy)phenyl]_6_[4_({[2-(methanesulfonyl)ethyl]amino}indenyl)_2_ Furanyl]_4_quinazoline 143939.doc -107- 201019961 Amine; Ν-{4-[(3·fluorobenzyl)oxy]-3.methoxyphenyl}-6-[2-( {[2-(decanesulfonyl)ethyl]amino}indolyl)-l,3-thiazol-4-yl]-4-quinazolinamine; N-{4-[(3-bromo) Mercapto)oxy]phenyl}_6-[2-({|;2•(decanesulfonyl)ethyl]amino}methyl)-1,3-thiasen-4-yl]-4 - quinazoline; N-{4-[(3-fluorophenylindenyl)oxy]phenyl)_6-[2-({[2-(methanesulfonyl)ethyl)amino}}} 1,3-thiazol-4-yl]-4-quinazolinamine; N-[4-(benzyloxy)-3-fluorophenyl]-6-[2-({[2-(methane) Sulfhydryl)ethyl]amino)indenyl)_1,3-thiazole-4-yl]-4-quinazolinamine; N-(l-benzyl-1H-carbazol-5-yl)- 7-decyloxy-6-[5-({[2-(methanesulfonyl)ethyl)amino)indolyl)_2_furanyl]_4_quinazolinamine·' 6-[5-( {[2-(decanesulfonyl)ethyl]amine;) Base)_2_furanyl]_N-(4-{[3-(trifluoromethyl)benzyl]oxy)phenyl)-4-quinazolinamine; N_ {3-fluoro-4-[( 3-fluorobenzyl)oxy]phenyl}_6_[5_({[2_(decanesulfonyl)ethyl]amino)methyl)-2-furanyl]-4-quinalin; N-{4-[(3-Bromobenzyl)oxy]phenyl)-6-[5-({[2-(decanesulfonyl)ethyl;]amino)indolyl)_2_ bite喃 ]]-4-quinazolinamine; N-[4-(benzyloxy)phenyl]-6_[3-({[2-(A))]]]}}}} _2_furanyl]_4_quinazoline; n-[1-(3-fluorobenzyl)·ιη-carbazol-5-yl]-6-[2-({[2-(decane) Sulfonic acid)ethyl]amino}indenyl)-1,3-thiazol-4-yl]-4-quinazolinamine; 6-[5-({[2-( sulfonyl)) Amino]methyl)methyl)_2_furanylΝ[[(phenylsulfonyl)phenyl]-4-quinazolinamine; 6-[2-({[2-(methanesulfonyl))ethyl) Amino) sulfhydryl)ethyl]amino]amino]amino]amino]amino]amino]amino]amino]amino]amino]amino]amino]amino]amino]amino] }methylhydrazine, thiazole·4_yl]_Ν_(Μ[3·(difluoroindolyl)benzyl)oxy)phenyl)_4_quinazolinamine;Ν_{ 3 -fluoro-4-[ (3- Benzyl)oxy]phenyl)·6_[2_({[2(methanesulfonyl)ethyl)amine 143939.doc -108- 201019961 yl}methyl)-l,3-thiazol-4-yl ]-4-quinazolinamine; N-(l-benzyl-1H-indazol-5-yl)-6-[2-({[2-(methanesulfonyl)))]] )methyl)_13_thiazol-4-yl]-4-quinazolinamine; ^_(3_Fluoro-4_benzyloxyphenyl (methyl sulphonate) ethyl]amino)methyl)_, 3_嗟Sept. 4_yl]_4_ quinazoline; N-(3-chloro-4-phenoxyphenyl; (methanesulfonyl)ethyl]amino)methyl)-1,3-thiazol-4-yl]-4-quinazolinamine; N-{3-Ga-4-[(3-fluorobenzyl)oxy]phenyl}-6-[5-({[2-(methanesulfonyl)ethyl)amino)methyl) -2-furyl]-4-quinazolinamine; 6-[5-({[2-(decanesulfonyl)ethyl]amino)methyl)-2-)) Methoxy-N-(4-phenylsulfonyl)phenyl-4-quinoxalin; N-[4-(benzyloxy)phenyl]_7_fluoro_6_[5_({[2_( Methanesulfonyl)ethyl]amino)methyl)-2-furyl]-4-quinazolinamine; N-(l-benzyl-1H-indole-5-yl)-7-fluoro -6-[5-({[2-(methanesulfonyl)ethyl)amino}methyl)-2-furanyl]-4- quinacamine; N-[4-(phenyl hydrazino) Phenyl]-7-fluoro-6-[5-({[2-(methanesulfonyl)ethyl)amino}methyl)_2-furanyl]_4_quinazolinamine; N-(3) -trifluoromethyl-4-benzyloxyphenyl)-6-[5-({[2_(decanesulfonyl)ethyl]amino)methyl)-4-oxime]-4 - 啥 啥 胺 amine; and its salts and solvents Thereof. In a specific embodiment, the EGFR antagonist is N-[3- gas-4-[(3-fluorophenyl)decyloxy]phenyl]-6-[5-[[[2-(indenyl) Indenyl)ethyl]amino]methyl]-2-furanyl]-4-quinazolinamine xylene sulfonate (lapatinib). In a particular embodiment, the EGFR antagonist is a compound of formula IV as disclosed in WO 0132651 (incorporated herein by reference), 143939.doc -109.

N

IV where: m is an integer from 1 to 3;

R1 represents a halo group or a C.3 alkyl group; X1 represents -0-; R2 is selected from the following three groups: 1) Cw alkyl R3 (wherein R3 is a pipe or two W-hydroxyalkyl groups) And a core group of C alkoxy group, which may have a group selected from the group consisting of a hydroxyl group, a halogen group, a C alkyl group, a c substituent; 2) a C 2 ·5 rare group R 3 (wherein R 3 is as herein described) 3) CM alkynyl R3 (wherein R3 is as defined herein and any of the alkyl, alkenyl or block groups may have one or more substituents selected from the group consisting of fluorenyl, dentate and amine groups Or a salt thereof. In a particular embodiment, the EGFR antagonist is selected from the group consisting of 4-(4-Gas-2-fluoroanilino)-6-methoxy-7-(i• fluorenyl) 0辰咬_4_yloxy)quinazoline; 4-(2-fluoro-4-methylanilino)_6_methoxy_7_(i-mercaptopiperidin-4-ylmethoxy a quinazoline; 4-(4-bromo-2-fluoroanilino)-6-mercapto-7-(1-indolylpiperidin-4-ylindoleoxy)quinazoline; 4-(4) - gas-2,6-difluoroanilino)-6-decyloxy-7-(1-methyl-singe-4-ylmethylindenyl) hydrazine. sitting-in; 4-(4- desert- 143939. Doc -110- 201019961 2,6-Difluoroanilino)-6-methoxy-7-(1-mercaptopiperidone 4--4-mercaptooxy)quinoquinone, 4-(4-milo-2-oxoanilino)-6-indolyloxy-7-(Big -4-ylmethoxy) 喧ί sitting Lin, 4-(2- gas-4-methylanilino)-6-methoxy-7-((唆-4-基曱ethoxy)啥β坐淋,4-(4 -》臭-2 - aza-anilino)-6-decyloxy-7-(pyrylene-4-ylmethoxy)quinazoline; 4-(4-gas-2,6-difluoroanilino)-6-methoxy -7-(piperidin-4-ylmethoxy)quinazoline; 4-(4-bromo-2,6-difluoroanilino)-6-decyloxy-7-(piperidin-4- Methoxy)quinazoline; and pharmaceutically acceptable salts and solvates thereof. In a particular embodiment, the EGFR antagonist is 4-(4-bromo-2-fluoroanilino)-6- Methoxy-7-(1-mercaptopiperidin-4-ylindoleoxy)quinazoline (Zactima) and its salts. VEGF antagonist VEGF antagonist refers to the ability to bind VEGF, reduce VEGF expression content or neutralize a molecule that blocks, inhibits, eliminates, reduces or interferes with VEGF activity, including binding of VEGF to one or more VEGF receptors, and VEGF-mediated angiogenesis and endothelial cell survival or proliferation. VEGF antagonism suitable for use in the methods of the invention Agent includes specific binding to VEGF Peptides, anti-VEGF antibodies and antigen-binding fragments thereof, receptor molecules and derivatives which specifically bind to VEGF to isolate their binding to one or more receptors, fusion proteins (such as VEGF-Trap (Regeneron)) and VEGFm-white ( Peregrine). VEGF antagonists also include VEGF polypeptides, antagonists of RNA aptamers against VEGF, and antagonists of peptibodies. Examples of each of these materials are described below. Anti-VEGF antibodies suitable for use in the methods of the invention include any of the 143939.doc-111-201019961 antibodies or antigen-binding fragments thereof that bind to VEGF with sufficient affinity and specificity and which reduce or inhibit the biological activity of VEGF. Anti-VEGF antibodies should generally not bind to other VEGF homologs (such as VEGF-B or VEGF-C) or other growth factors (such as P1GF, PDGF or bFGF). Examples of such anti-VEGF antibodies include, but are not limited to, the anti-VEGF antibodies provided in the "Definitions" herein. Two well characterized VEGF receptors are VEGFR1 (also known as Flt-Ι) and VEGFR2 (for murine homologs, also known as KDR and FLK-1). Although the specificity of each receptor of each VEGF family member is different, VEGF-A binds to Flt-1 and KDR. The full-length Flt-1 receptor includes an extracellular domain having seven Ig domains, a transmembrane domain, and an intracellular domain having tyrosine kinase activity. The extracellular domain is involved in the binding of VEGF, while the intracellular domain is involved in signal transduction. A VEGF receptor molecule or a fragment thereof that specifically binds to VEGF can be used in the method of the invention to bind and sequester the VEGF protein, thereby preventing its conduction signal. In certain embodiments, the VEGF receptor molecule or VEGF binding fragment thereof is in a soluble form, such as sFlt-1. The soluble form of the receptor has an inhibitory effect on the biological activity of the VEGF protein by binding to VEGF, thereby preventing its binding to its natural receptor present on the cell surface. VEGF receptor fusion proteins are also included, examples of which are described below. A chimeric VEGF receptor protein is a receptor molecule having an amino acid sequence derived from at least two different proteins, wherein at least one of the proteins is a VEGF receptor protein capable of binding to VEGF and inhibiting its biological activity (eg, fit-1 or KDR) Receptor). In certain embodiments, a chimeric VEGF receptor protein of the invention consists of an amino acid sequence derived only from two different VEGF receptor molecules; however, a cell comprising a fit-1 and/or KDR receptor The amino acid sequence of 1, 2, 3, 4, 5, 6, or all 7 Ig-like domains of the outer coordination 艎 binding region may be associated with its amino acid sequence (eg, immunization) with its 143939.doc •112-201019961 The globin sequence) is linked. Other amino acid sequences in combination with an Ig-like domain will be apparent to those of ordinary skill in the art. Examples of chimeric VEGF receptor proteins include soluble Flt-1/Fc, KDR/Fc or Flt-1/KDR/Fc (also known as VEGF Trap). (See, for example, PCT Application Publication No. WO 97/44453). The soluble VEGF receptor protein or chimeric VEGF receptor protein of the invention comprises a VEGF receptor protein that is not immobilized on the cell surface via a transmembrane domain. Thus, soluble forms of the VEGF receptor (including chimeric receptor proteins), while capable of binding to and inactivating VEGF, do not comprise a transmembrane domain and are therefore generally not associated with cell membranes of cells expressing the molecule. An aptamer is a nucleic acid molecule that forms a tertiary structure that specifically binds to a target molecule, such as a VEGF polypeptide. The production and therapeutic use of aptamers is well established in the art. See, for example, U.S. Patent No. 5,475,096. The VEGF aptamer is a PEGylated oligonucleotide that is configured to bind to the three-dimensional configuration of extracellular VEGF. An example of a therapeutically effective aptamer that targets VEGF to treat age-related macular degeneration is pegaptanib (MacugenTM, OSI). Further information on aptamers can be found in U.S. Patent Application Publication No. 20060148748. A peptidic body is a peptide sequence linked to an amino acid sequence encoding a fragment or a portion of an immunoglobulin molecule. The polypeptide may be derived from any random sequence selected for any method of specific binding, including but not limited to phage display technology. In one embodiment, the selected polypeptide can be linked to an amino acid sequence encoding the Fc portion of an immunoglobulin. Peptides that specifically bind to and antagonize VEGF are also suitable for use in the methods of the invention. 143939.doc • 113-201019961 Combination Therapy The present invention relates to the use of a combination of a paw antagonist and a VEGF antagonist, and in some aspects 'for the combined use of a c-met antagonist, a VEGF antagonist and an EGFR antagonist as intended The beneficial effects of a combination of specific therapeutic regimens that provide a beneficial effect of such therapeutic agents include, but are not limited to, pharmacokinetic or pharmacodynamic effects resulting from a combination of therapeutic agents. The invention is particularly useful for treating various types of cancer at various stages of the disease. The term cancer includes a collection of proliferative disorders including, but not limited to, precancerous growth, benign tumors, and malignant tumors. Benign tumors remain localized at the site of origin and do not have the ability to infiltrate, invade, or metastasize to distant sites. A malignant tumor will invade and damage other tissues around it. It can also be detached from the original site and typically spread to other parts of the body (metastasis) via the bloodstream or via the lymphatic system in which the lymph nodes are located. Primary tumors are classified according to the type of tissue from which the tumors are produced; metastatic tumors are classified according to the type of tissue from which the cancer cells are produced. Cells of malignant tumors become more abnormal over time and appear to be less like normal cells. This change in appearance of cancer cells is called tumor grade, and the cancer cell line is described as sufficiently differentiated (lower), moderately differentiated, poorly differentiated, or undifferentiated (advanced). Fully differentiated cells appear to be quite normal and similar to the normal cells from which they are produced. "Undifferentiated cells are cells that have become abnormal so that it is no longer possible to determine the origin of the cell. The cancer staging system describes how far the cancer has been anatomically spread and attempts to classify patients with similar prognosis and treatment into the same disease group. Several tests can be performed to help with cancer staging' including biopsy and some imaging tests 143939.doc •114- 201019961 (imaging test) (such as chest x-ray, mammogram, Bone scan, CT scan and MRI scan). Blood tests and clinical assessments are also used to assess the overall health of the patient and to detect if the cancer has spread to certain organs. To stage cancer, the American Joint Committee on Cancer first uses a TNM classification system to sort cancers (especially solid tumors) in alphabetical order. Specify the letter T (tumor size), sputum (tactile nodules), and/or sputum (metastasis) for cancer. ΤΙ, Τ2, Τ3, and Τ4 describe incremental lesions of primary lesions; NO, Nl, Ν2, Ν3 indicate progressive involvement of node involvement; and M0 and Ml reflect the absence of distant metastasis or the presence of distant sites Transfer. In the second staging method, also known as Overall Stage Grouping or Roman Numeral Staging, the cancer is divided into the size of the primary lesion and the presence of nodular spread and distant metastasis. 0 to IV. In this system, cases are grouped into four phases, represented by Roman numerals I through IV, or classified as "recurrent." For some cancers, stage 0 is called "in situ" or "Tis", such as breast ductal carcinoma in situ or lobular carcinoma in situ. Advanced adenomas can also be classified as stage 0. Stage I cancers are generally small, localized cancers that are usually curable, while stage IV usually indicates cancer or metastatic cancer that cannot be operated on. Stage II and III cancers are usually locally advanced and/or exhibit local lymph node involvement. Higher grades generally indicate a broader range of diseases, including larger tumor sizes and/or cancer spreading to nearby lymph nodes and/or organs adjacent to the primary tumor. Although these stages are precisely defined, the definitions of each type of cancer are not the same and are known to those skilled in the art. H3939.doc -115- 201019961 Many cancer registries (such as the National Cancer Institute's Surveillance, Epidemiology, and End Results Program (SEER)) use summary staging. This system is used for all types of cancer. It divides cancer cases into five main categories: and is an early cancer that exists only in the initial cell layer. Xin Xin is a cancer that is limited to the beginning of the organ without signs of spread.厘域# is a cancer that has spread beyond the original (primary) site to nearby lymph nodes or organs and tissues. The study is a cancer that has spread from the primary site to the distal or distant lymph nodes. Not as read to describe cases where there is not enough information to indicate the stage of the disease. In addition, cancer usually recurs months or years after the primary tumor is removed. A cancer that recurs after all visible tumors have been removed is called a recurrent disease. The disease that recurs in the primary tumor area is locally recurrent, and the disease that metastasizes and relapses is called distant recurrence. The tumor can be a solid tumor or a non-solid or soft tissue tumor. Examples of soft tissue tumors include leukemia (eg, chronic osteogenic leukemia, acute osteogenic leukemia, adult acute lymphoblastic leukemia, acute myeloid leukemia, mature B cell acute lymphoblastic leukemia, chronic lymphocytic leukemia, anterior lymphocytes) Leukemia or hairy cell leukemia) or lymphoma (eg, non-Hodgkin's lymphoma, cutaneous T-cell lymphoma, or Hodgkin's disease). Shi Zhao Zhao Tu includes any cancer of body tissue other than blood, bone forging or lymphatic system. Real 143939.doc -116· 201019961

Body tumors can be further divided into solid tumors of epithelial origin and solid limb tumors of non-epithelial origin. Examples of epithelial solid lumbar tumors include gastrointestinal tumors, colon tumors, breast tumors, prostate tumors, lung tumors, kidney tumors, hepatic tumors, pancreatic tumors, ovarian tumors, head and neck tumors, oral tumors, stomach tumors, and eleven fingers. Intestinal limb tumor, small intestine tumor, large intestine tumor, anal tumor, gallbladder tumor, lip tumor, nasopharyngeal tumor, skin tumor, uterine tumor, male genital tumor, urinary tumor, bladder tumor and skin tumor. Solid tumors of non-epithelial origin include sarcomas, brain tumors, and bone tumors. In addition to the treatment regimens described herein, patients can also undergo surgical removal of cancer cells and/or radiation therapy. A chemotherapeutic agent, which may be combined with other therapeutic regimens, third, fourth, etc., wherein the second chemotherapeutic agent is a different anti-metabolite chemotherapeutic agent, or a chemical that is not an antimetabolite The therapeutic agent is exemplified by s 'the second chemotherapeutic agent may be a taxane (such as yew (plus (4) or paclitaxel or docetaxel), an antimetabolite drug (such as gemcitabine or 5. fluorourethane (four)), card Peitabin thiol chemotherapy = agent (such as card turn, cistern or Osereco cobalt), anthracycline (such as amylin liposomal doxorubicin), topotecan, pemei (four) 'Changchun flower test (such as vinorelbine) and 286. can be administered a mixture of different chemotherapeutic agents. </ RTI>;: anti-body and coffee antagonist combination of other therapeutic agents including examples: antibodies against tumor-associated antigens '·Anti-hormone compounds such as ft estrogen compounds (such as tamoxifen) or aromatase inhibits the heart (to prevent or reduce any myocardial function associated with therapy: 143939.doc -117- 201019961); cytokines; Anti-angiogenic agents (especially by Genen Tech is sold as bevacizumab under the trademark AVASTIN J; glutamate kinase inhibitors such as sunutinib (SUTENT) and sorafenib; COX inhibitors (eg COX-1 or COX) -2 inhibitor); non-steroidal anti-inflammatory drug, celecoxib (CELEBREX7); farnesyltransferase inhibitor (eg, tipifani/ZARNESTRA7 R115777 available from Johnson and Johnson or available from Schering-Plough Lonfarnib SCH66336); mTOR inhibitors such as RAD001 and temsirolimus; antibodies that bind to carcinoembryonic protein CA 125, such as Oregovomab (MoAb B43.13) HER2 vaccine (such as the HER2 AutoVac vaccine from Pharmexia, or the APC8024 protein vaccine from Dendreon, or the HER2 peptide vaccine from GSK/Corixa); another HER targeted therapy (eg trastuzumab, cetuximab) , ABX-EGF, EMD7200, gefitinib, erlotinib, panitumumab, CP724714, CI1033, GW572016, IMC-11F8, TAK165, etc.); Raf and/or ras inhibitors (see eg WO 2003/86467 ); doxorubicin hydrochloride Liposomal injection (DOXIL®); topoisomerase I inhibitors such as topotecan; taxane; HER2 and EGFR dual tyrosine kinase inhibitors such as lapatinib/GW572016; TLK286 (TELCYTA® EMD-7200; a drug for treating nausea, such as a serotonin antagonist, a steroid or a benzodiazepine; a drug for preventing or treating dermatitis or a standard acne treatment, including topical or oral antibiotics; a drug for treating or preventing diarrhea; A drug that lowers body temperature, such as acetaminophen phenol, diphenhydramine or meperidine; hematopoietic growth factor. 143939.doc -118· 201019961 The appropriate dose of any of the above co-administered agents is the currently used dose, and due to the combination of the agent with anti-e_met antibody and egfr antagonist (synergy), the dose can be The dosage may be decreased, or as determined by the treating physician, for example. When the inhibitor is an antibody, the antibody administered is preferably a naked antibody. However, the inhibitor administered can be combined with a cytotoxic agent. The combined inhibitor and/or the antigen to which it binds is preferably intracellularly internalized such that the therapeutic effect of the conjugate to kill the cancer cells to which it binds is increased. In a preferred embodiment, the cytotoxic agent is targeted. Or interfere with nucleic acids in cancer cells. Examples of such cytotoxic agents include maytansinoids, calicheamicin, ribonucleases, and DNA endonucleases. In some embodiments, the patient herein is subjected to a diagnostic test, such as prior to and/or during therapy and/or after therapy. In general, if a # break test is performed, the sample can be obtained from a patient in need of therapy. When the individual has cancer, the sample can be a tumor sample or other biological sample, such as a biological fluid, including but not limited to blood, urine, saliva, ascites or derivatives (such as serum and plasma) and the like. In some embodiments, the cancer of an individual exhibits c-met and/or EGFR. Methods for measuring 疋c-met or EOFR performance are known in the art and some methods are described herein. In some embodiments, the serum of the individual exhibits a high level of IL8. In some embodiments, the serum of an individual exhibits more than about 15 〇 pg/mliIL8, or in some embodiments, more than about 5 〇 pg/mli IL8. In some embodiments, the individual's jk clear performance exceeds about 1 〇 pg/m b 2 〇 pg/m b 3 〇 pg/ml 143939.doc - 119 · 201019961 or higher IL8. Methods for determining the serum concentration of IL8 are known in the art, and a method is described in the examples of the present invention. In some embodiments, the individual's serum exhibits a high level of HGF. In some embodiments, the individual's serum exhibits an HGF of more than about 5,000 pg/ml, 10,000 pg/ml, or 50,000 pg/ml. In some embodiments, for example, from a c-metase treatment (and in some implementations) In the case of a tumor or serum sample of a patient further treated with a VEGF antagonist, the mRNA or protein exhibits a reduced prognosis such as a response to treatment or a c-met antagonist activity, and in some embodiments, a prognosis c- Activity of met antagonists and VEGF antagonists. In some embodiments, several angiogenic factors (such as interleukin 8 (IL8), vascular endothelial growth factor A (VEGFA), EPH receptor A2 (EphA2), angiopoietin-like protein 4 (Angptl4), and Ephrin The performance of B2 (EFNB2)) reduces the prognosis, for example, the response to treatment or the activity of a c-met antagonist (and in some embodiments, the activity of a prognostic VEGF antagonist). The decrease in performance can be determined relative to the untreated sample or reference standard value or relative to the level of performance of the patient prior to treatment with c-met antagonist (or treatment with c-met antagonist and VEGF antagonist). In some embodiments, for example, HGF or IL8 in a sample from a patient's tumor or serum exhibits a reduced prognosis such as a response to treatment or a c-met antagonist (and in some embodiments, a VEGF antagonist). In one embodiment, a decrease of more than 50% of the performance of IL8 in serum or a decrease of more than 70% (e.g., relative to the level of IL8 performance of the patient prior to treatment) is indicative of a response to treatment. Performance may be reduced relative to untreated sample or reference standard values or relative to patients treated with c-met antagonist (or treated with c-met antagonist and VEGF antagonist 143939.doc-120-201019961) The content is determined. In some embodiments, for example, an increase in mRNA or protein expression in a sample of a tumor or serum from a patient treated with a c-met antagonist (and in some embodiments, further treated with a VEGF antagonist), eg, for treatment The activity of the reaction or c-met antagonist (and in some embodiments, a VEGF antagonist). The decrease in performance can be determined relative to the untreated sample or reference standard value or relative to the performance level of the patient prior to treatment with c-met antagonist (or treatment with c-met antagonist and VEGF antagonist). In some embodiments, the FDG-PET imaging prognosis is, for example, a response to treatment or an activity of a c-met antagonist (and in some embodiments, the activity of a VEGF antagonist). Accordingly, the present invention also provides a prognostic method. Thus, the disclosed method can provide a simple, efficient, and potentially cost-effective method of obtaining information and information suitable for assessing the future course of a disease, including the selection of an appropriate therapy for treating the patient. In another aspect, the invention provides a method of assessing a patient having or suspected of having cancer, the method comprising: based on comparing a patient's biological sample with vascular endothelial growth factor A (VEGFA), EPH receptor A2 (EphA2) , angiopoietin-like protein 4 (Angptl4) and / or Ephrin B2 (EFNB2) and vascular endothelial growth factor A (VEGFA), EPH receptor A2 (EphA2), angiopoietin-like protein 4 in control samples Angptl4) and/or Ephrin B2 (EFNB2) is used to predict the prognosis of a patient's cancer; wherein the patient's biological sample is vascular endothelial growth factor A (VEGFA), EPH receptor A2 (EphA2), angiopoietin-like relative to the control sample. Protein 4 (Angptl4) and / or 143939.doc •121 · 201019961

Ephrin B2 (EFNB2) represents cancer in prognosis patients. In some embodiments, the method further comprises (a) obtaining a biological sample from the patient (eg, prior to treatment and/or during treatment); and (b) detecting vascular endothelial growth factor A (VEGFA), EPH in the biological sample Expression of receptor A2 (EphA2), angiopoietin-like protein 4 (Angptl4) and/or Ephrin B2 (EFNB2). In some embodiments, the patient biological sample exhibits vascular endothelial growth factor A (VEGFA), EPH receptor A2 (EphA2), angiopoietin-like protein 4 (Angptl4), and/or Ephrin B2 (EFNB2) relative to the control sample. Increase the cancer of patients with prognosis. In some embodiments, the patient biological sample exhibits vascular endothelial growth factor A (VEGFA), EPH receptor A2 (EphA2), angiopoietin-like protein 4 (Angptl4), and/or Ephrin B2 (EFNB2) relative to the control sample. Reduce the cancer of patients with prognosis. In another aspect, the invention provides a method of assessing a patient undergoing cancer therapy, the method comprising: based on comparing a patient's biological sample (eg, Qiqing) with vascular endothelial growth factor A (VEGFA), EPH receptor A2 ( EphA2), angiopoietin-like protein 4 (Angptl4) and/or Ephrin B2 (EFNB2), and vascular endothelial growth factor A (VEGFA), EPH receptor A2 (EphA2), in patient biological samples obtained prior to treatment, The expression of angiopoietin-like protein 4 (Angptl4) and/or Ephrin B2 (EFNB2) is used to predict the prognosis of cancer in patients. The serum of patients undergoing treatment includes vascular endothelial growth factor A (VEGFA) and EPH receptor A2 (EphA2). , the performance of angiopoietin-like protein 4 (Angptl4) and / or Ephrin B2 (EFNB2) relative to the performance of the pre-treatment sample to reduce cancer in prognosis patients. In some embodiments, the prognostic cancer comprises 143939.doc-122-201019961 predicting or predicting (prognosis) any one or more of the following: for treatment (eg, with a c-met antagonist (such as an anti-c-met antibody) or Reaction of c_met antagonist and VEGF antagonist therapy, activity of c-met antagonist (such as anti-e-met antibody) or c-met antagonist and VEGF antagonist, for treatment (eg with c-met antagonist Or the response of a c-met antagonist and a VEGF antagonist treatment, treatment (eg, treatment with a c-met antagonist or c_·-met antagonist and a VEGF antagonist), survival of a patient susceptible to or diagnosed with cancer Duration, duration of recurrence-free survival, duration of progression-free survival in patients who are predisposed to or diagnosed with cancer, response rate in patients with or susceptible to cancer, predisposition, or diagnosis The duration of response to a patient or group of patients with cancer, and/or the likelihood of metastasis in a patient susceptible to or diagnosed with cancer. In some embodiments, the predicted or predicted survival duration is increased. In some embodiments, the predicted or predicted survival duration is reduced. In some embodiments, an indication of an increase in the duration of recurrence-free survival is predicted or predicted. In some embodiments, the predictive or predictive reduction in the duration of recurrence-free survival, in some embodiments, the predictive or pre-announced increase in response rate, in some embodiments, predicts or predicts a decrease in response rate. In some embodiments, the predicted or predicted duration of the reaction is increased. In some embodiments, the predicted or predicted duration of the reaction is reduced. In some embodiments, the prediction or predictive increase in transfer probability is increased. In some embodiments, the prediction or predictive transfer likelihood is reduced. In another example, the present invention provides a method for selecting a treatment for a patient suffering from or suspected of having cancer, such as squatting, earthy, and sputum. (a) based on comparing biological samples of patients. Expression of vascular endothelial growth factor A receptor (EphA2) angiopoietin-like protein 4 (Angptl4) and/or Ephrin 143939.doc-123-201019961 B2 (EFNB2) and vascular endothelial growth factor A in control samples VEGFA), EPH receptor A2 (EphA2), angiopoietin-like protein 4 (Angptl4) and/or Ephrin B2 (EFNB2) are used to predict the prognosis of a patient's cancer, wherein the patient's biological sample grows relative to the control sample. Factor A (VEGFA), EPH receptor A2 (EphA2), angiopoietin-like protein 4 (Angptl4) and/or Ephrin B2 (EFNB2) exhibit cancer in prognosis patients, and (b) after step (a), for patients Cancer therapy is selected wherein the choice of treatment is based on the prognosis of the patient as determined in step (a). In some embodiments, the methods further comprise (c) obtaining a patient biological sample; (d) detecting vascular endothelial growth factor A (VEGFA), EPH receptor A2 (EphA2), angiopoietin-like protein in the biological sample 4 (Angptl4) and/or Ephrin B2 (EFNB2), in which vascular endothelial growth factor A (VEGFA), EPH receptor A2 (EphA2), angiopoietin-like protein 4 (Angptl4) and/or Ephrin B2 (EFNB2) is a prognostic cancer. In some embodiments, the patient biological sample exhibits vascular endothelial growth factor A (VEGFA), EPH receptor A2 (EphA2), angiopoietin-like protein 4 (Angptl4), and/or Ephrin B2 (EFNB2) relative to the control sample. Increase the cancer of patients with prognosis. In some embodiments, the patient biological sample exhibits vascular endothelial growth factor A (VEGFA), EPH receptor A2 (EphA2), angiopoietin-like protein 4 (Angptl4), and/or Ephrin B2 (EFNB2) relative to the control sample. Reduce the cancer of patients with prognosis. In another aspect, the invention provides methods of selecting a treatment for a patient undergoing cancer treatment, the methods comprising: (a) comparing vascular endothelial growth factor A (VEGFA), EPH in a patient biological sample (eg, serum) Receptor 143939.doc -124- 201019961 A2 (EphA2), Angiopoietin-like Protein 4 (Angptl4) and/or Ephrin B2 (EFNB2) and vascular endothelial growth factor A (VEGFA) in patient biological samples obtained prior to treatment , the expression of EPH receptor A2 (EphA2), angiopoietin-like protein 4 (Angptl4) and / or Ephrin B2 (EFNB2) to predict the prognosis of patients with cancer, wherein the vascular endothelial growth factor A in the serum of patients undergoing treatment (VEGFA), EPH receptor A2 (EphA2), angiopoietin-like protein 4 (Angptl4) and/or Ephrin B2 (EFNB2) are expressed relative to cancer in patients with prognosis in pre-treatment samples; and (b) in steps (a) Thereafter, the patient is selected for cancer treatment, wherein the choice of treatment is based on the patient's prognosis as determined in step (a). In some embodiments, the methods further comprise (c) obtaining a patient biological sample; (d) detecting vascular endothelial growth factor A (VEGFA), EPH receptor A2 (EphA2), angiopoietin-like protein in the biological sample 4 (Angptl4) and/or Ephrin B2 (EFNB2), in which the patient's biological sample is vascular endothelial growth factor A (VEGFA), EPH receptor A2 (EphA2), angiopoietin-like protein 4 (Angptl4) and/or Ephrin B2 (EFNB2) is a prognostic cancer. In some embodiments, the patient biological sample exhibits vascular endothelial growth factor A (VEGFA), EPH receptor A2 (EphA2), angiopoietin-like protein 4 (Angptl4), and/or Ephrin B2 (EFNB2) relative to the control sample. Increase the cancer of patients with prognosis. In some embodiments, the patient biological sample exhibits vascular endothelial growth factor A (VEGFA), EPH receptor A2 (EphA2), angiopoietin-like protein 4 (Angptl4), and/or Ephrin B2 (EFNB2) relative to the control sample. Reduce the cancer of patients with prognosis. The biological sample herein may be a fixed sample (e.g., a formalin fixed stone 143939.doc-125-201019961 wax-embedded (FFPE) sample) or a frozen sample. Various methods for determining mRNA or protein expression include, but are not limited to, determining gene expression profiles, polymerase chain reaction (PCR) (including quantitative real-time PCR (qRT-PCR)), microarray analysis, and serial analysis of Gene expression, SAGE), MassARRAY, gene expression analysis, proteomic research, immunohistochemistry (IHC), etc. by Massively Parallel Signature Sequencing (MPSS). Preferably, the mRNA is quantified. Such mRNA analysis is preferably carried out using polymerase chain reaction (PCR) techniques or by microarray analysis. When PCR is employed, the preferred form of PCR is quantitative real-time PCR (qRT-PCR). In one embodiment, a performance of one or more of the indicated genes is considered positive if, for example, is above or below the median of other samples of the same tumor type. The determination of the median performance level can be performed substantially simultaneously with the measurement of the gene performance, or may have been previously determined. A number of published journal articles (eg, Godfrey et al., J. Molec. Diagnostics 2: 84-91 (2000); Specht et al., Am. J. Pathol. 158: 419-29 (2001)) The paraffin-embedded tissue serves as a source of RNA to determine the steps of a representative protocol for gene expression profiling, including mRNA isolation, purification, primer extension, and amplification. Briefly, a representative method begins with the cutting of about 10 micrograms of a slice of a paraffin-embedded tumor tissue sample. The RNA is then extracted and the protein and DNA are removed. After analyzing the RNA concentration, an RNA repair and/or amplification step may be included as necessary, and the RNA is reverse transcribed using a gene-specific promoter, followed by PCR. Finally, the data was analyzed to determine the optimal treatment options available to the patient based on the characteristic gene expression pattern identified in the tumor sample examined 143939.doc-126-201019961. Detection of gene or protein expression can be determined directly or indirectly. The expression or amplification (direct or indirect) of c-met and/or EGFR in cancer can be determined. This can take advantage of various diagnostic/prognostic assays. In one embodiment, IHC can be used to analyze excessive performance of c-met and/or EGFR. Paraffin-embedded tissue sections from tumor biopsies can be subjected to IHC assays and matched to the following c-met and/or EGFR protein staining intensity criteria: Fraction 〇: No staining or less than 10% of tumor cells were observed Membrane staining was observed. Score 1+: Blurring/reluctantly detectable membrane staining was detected in more than 10% of tumor cells. Only part of the cell membrane was stained on the cells. Fraction 2+: Weak to moderate complete membrane staining was observed in more than 10% of tumor cells. Score 3+: Moderate to strong complete membrane staining was observed in more than 10% of tumor cells. In some embodiments, a tumor with a score of 0 or 1 + with an over-expression of c-met and/or EGFR may be characterized as not overexpressing c-met and/or EGFR, and a tumor having a score of 2 + or 3 + may Characterized by overexpression of c-met and/or EGFR. In some embodiments, tumors that overexpress c-met and/or EGFR can be ranked according to an immunohistochemical fraction corresponding to the number of copies of c-met and/or EGFR molecules exhibited by each cell, and can be biochemically Ways to determine: 0 = 0 - 10,000 replicates / cell, 1 + = at least about 200,000 copies / cell, 143939.doc -127 - 201019961 2+ = at least about 500,000 copies / cell, 3 + = at least about 2,000,000 Replica / cell. Alternatively or additionally, a fumarin-fixed paraffin-embedded tumor group can be subjected to FISH assay to determine the extent of c-met and/or EGFR expansion in the tumor, if any. ·- c-met or EGFR activation can be directly (for example by sulphate-ELISA test or other means of detecting phosphorylated receptors) or indirectly (eg downstream signal transduction pathway components activated by debt testing, Detective Determination of receptor dimers (eg, homodimers, heterodimers, detection of gene expression profiles, and the like). Similarly, the presence of c-met or EGFR constitutively activated or non-ligand-dependent EGFR or c-met can be directly or indirectly (eg, by detecting receptor mutations associated with constitutive activity, by detecting Receptor amplification associated with constitutive activity and similar methods) detection. Methods for detecting nucleic acid mutations are well known in the art. Although not required, the target nucleic acid in the sample is often amplified to provide the desired amount of material to determine if a mutation is present. Amplification techniques are well known in the art. For example, S, the amplification product may or may not encompass all nucleic acid sequences encoding the relevant protein, as long as the amplification product contains a specific amino acid/nucleic acid sequence position suspected of having a mutation. In an example, the presence of a mutated can be determined by contacting a nucleic acid from S-like S with a nucleic acid that is capable of specifically hybridizing to a nucleic acid encoding a mutated nucleic acid and detecting the hybridization. In one embodiment, the probe is labeled, for example, with a radioisotope (H, p, p, etc.), a luminescent agent (rhodamine, fluorescent, etc.) or a color developing agent. In some embodiments, the probe is an antisense polymer, 143939.doc -128-201019961 such as PNA, N-morpholinyl-amino phosphate, LNA or 2,-alkoxy alkoxy The needle can have from about 8 nucleotides to about 100 nucleotides, or from about 1 to about 75 nucleotides, or from about 15 to about 5 nucleotides, or from about 2 to about 3 inches. Nuclear acid. In another aspect, the nucleic acid probe of the present invention is provided in a kit for identifying a c-met mutation in a sample. 'The kit comprises or is specific to a mutation site in a nucleic acid encoding c_met Sexual hybridization raises bitter acid. The kit may further comprise instructions for treating a patient suffering from a limb tumor containing a c-met mutation with a c-met antagonist based on the results of the hybridization test using the kit. Mutations can also be detected by comparing the electrophoretic mobility of the amplified nucleic acid to the electrophoretic mobility of the corresponding nucleic acid encoding the wild type cardiac met. The difference in mobility indicates the presence of a mutation in the amplified nucleic acid sequence. Electrophoretic mobility can be measured using any suitable molecular separation technique (e.g., on a silicone gel gel). Enzyme-induced mutation detection (EMD) can also be used to detect nucleic acids to detect mutations (Del Tito, Clinical Chemistry 44: 731-739, 1998). EMD uses the phage resolving enzyme D4 endonuclease VII, which scans along double-stranded DNA until it detects structural deformation caused by base pair mismatches caused by nucleic acid variations (such as point mutations, insertions and deletions) and It cleaves. The use of, for example, gel electrophoresis to detect two short fragments formed by cleavage of the dissociation enzyme indicates the presence of a mutation. The advantage of the EMD method is that a single protocol is used to identify point mutations, deletions, and insertions directly from the amplification reaction assay, thereby eliminating the need for sample purification, shortening the hybridization time, and increasing the signal-to-noise ratio. A mixed sample containing up to a 20-fold excess of normal nucleic acid and a fragment of up to 4 kb in size can be assayed. However, EMD scans are unable to identify specific base 143939.doc -129· 201019961 changes that occur in mutation-positive samples, so other sequencing procedures are often needed to identify specific mutations when necessary. The CEL I enzyme can be used similarly to the dissociation enzyme T4 endonuclease VII as indicated in U.S. Patent No. 5,869,245. Another simple cluster for detecting mutations is a reverse hybridization test strip similar to Haemochromatosis StripAssayTM (Viennalabs » http://www.bamburghmarrsh.com/pdf74220.pdf), which is used for debt testing. Multiple mutations in the HF E, TFR2 and FPN1 genes of the disease. Such assays are based on sequence-specific hybridization following PCR amplification. For single mutation assays, a microplate-based detection system can be applied, and for multiple mutation assays, a test strip can be used as a "macro-array." Kits can include ready-to-use reagents for sample preparation, amplification, and mutation detection. The complex amplification protocol provides simplicity and allows for the testing of very limited volumes of samples. Using the direct StripAssay format, 20 or more mutations can be tested in less than 5 hours without the need for expensive equipment. DNA is isolated from the sample, and the target nucleic acid is typically amplified in vitro (e.g., using PCR) and labeled with biotin in a single ("complex") amplification reaction. The amplified product is then selectively hybridized to a nucleotide probe (wild type and mutant specific) immobilized on a solid support such as a test strip, wherein the probe is immobilized in solid parallel or strip form on the solid support On the object. Biotin-labeled amplicons were detected using streptavidin-alkaline phosphatase and colored receptors. Such assays can detect all mutations or any subset thereof of the invention. Regarding a particular mutant probe band, it may be one of the following three signaling patterns: (1) a band of wild-type probes only, indicating a normal nucleic acid sequence, (ii) a band of wild-type and mutant probes, indicating miscellaneous A genomic form, and (iii) a band of only mutant probes indicating a homozygous mutant genotype. Thus, in a 143939.doc-130-201019961 aspect, the invention provides a method of detecting a mutation of the invention, the method comprising: isolating and/or amplifying a target from a sample (: 〇1 in a nucleic acid sequence such that amplification The product comprises a ligand; the amplification product is contacted with a probe comprising a detectable binding partner of the ligand and capable of specifically hybridizing to the mutation of the invention; and subsequently detecting the probe and the amplification Hybridization of the product. In one embodiment, the ligand is biotin' and the binding partner comprises avidin or streptavidin. In one embodiment, the binding partner comprises a detectable color acceptor Streptavidin_basic. In one embodiment, the probe is immobilized, for example, on a test strip, wherein probes complementary to different mutations are separated from each other. Alternatively, the nucleic acid is amplified by radioisotope labeling, in which case The probe need not contain a detectable marker. The variation of the wild-type gene covers all mutant forms, such as insertions, inversions, deletions, and/or point mutations. In one embodiment, the mutation is a somatic mutation. Somatic mutation Mutations that occur only in certain tissues (eg, tumor tissue) and are not inherited in the germline. Germline mutations can be found in any body tissue. Samples containing target nucleic acids are well known in the art and are suitable for the particular type and location of the tumor. Methods obtained. Tissue biopsy is often used to obtain representative fragments of tumor tissue. Alternatively, tumor cells can be obtained indirectly from tissue/fluid forms known or believed to contain relevant tumor cells. For example, resection, bronchi can be used Mirror, fine needle aspiration (flne needle aspimi〇n), bronchial brushing or a sample of lung cancer lesions obtained from saliva, pleural fluid or blood. Can be from a tumor or other body sample (such as urine, saliva or Serum) detection of a mutated gene or gene product. The same technique for detecting a mutant target gene or gene product in a tumor sample as discussed above in 143939.doc-131 - 201019961 can be applied to other body samples. In such body samples, diseases such as cancer can be achieved by screening such body samples Simple early diagnosis. In addition, the progress of the therapy can be more easily monitored by testing mutant target genes or gene products of such body samples. Methods for enriching tissue preparations of tumor cells are known in the art. Tissues are isolated from paraffin or cryosections. Flow cytometry or laser capture microdissection can also be used to isolate cancer cells from normal cells. These and other isolated tumor cells and normal cells are well known in the art. Techniques. If tumor tissue is highly contaminated by normal cells, although techniques for minimizing contamination and/or false positive/negative results are known (some of which are described below), mutation detection may still be more difficult. For example, the presence of biological indicators (including mutations) known to be associated with associated tumor cells (but not corresponding normal cells) in the sample may also be assessed, or vice versa. Detection of point mutations in the target nucleic acid can be accomplished by molecularly cloning the target nucleic acid and determining the nucleic acid sequence using techniques well known in the art. Alternatively, the target nucleic acid sequence can be directly amplified from a genomic DNA preparation from tumor tissue using an amplification technique such as polymerase chain reaction (PCR). The nucleic acid sequence of the amplified sequence can then be determined and the mutation identified therefrom. Amplification techniques are well known in the art, such as the polymerase linkage reactions described in Saiki et al., Science 239: 487, 1988; U.S. Patent Nos. 4,683,203 and 4,683,195. It should be noted that the design and selection of appropriate primers is a well-established technique in this technology. 143939.doc -132- 201019961 A ligase chain reaction known in the art can also be used to amplify a target nucleic acid sequence. See, for example, Wu et al., Genomics, Vol. 4, pp. 560-569 (1989). Alternatively, a technique called allele-specific PCR can be used. See, for example, Ruano and Kidd, Nucleic Acids Research, Vol. 17, p. 8392, 1989. According to this technique, a primer that hybridizes to a specific target nucleic acid mutation at the 3' end is used. If no specific mutation is present, the amplification product cannot be observed. Amplification Refractory Mutation System (ARMS) can also be used as disclosed in European Patent Application Publication No. 0332435 and Newton et al., Nucleic Acids Research, Vol. 17, p. 7, 1989. Gene insertions and deletions can also be detected by colonization, sequencing and amplification. Alternatively, a restriction fragment length polymorphism (RFLP) probe of the gene or surrounding marker gene can be used to score allelic variation, or insertion, in a polymorphic fragment. Single stranded conformation polymorphism (SSCP) analysis can also be used to detect base variation variants of alleles. See, for example, Orita et al, Proc. Natl. Acad. Sci. USA, Vol. 86, pp. 2766-2770, 1989; and Genomics, Vol. 5, pp. 874-879, 1989. Other techniques for detecting insertions and deletions known in the art can also be used. Mutations in the wild-type gene can also be detected based on variations in the wild-type performance product of the gene. Such performance products include mRNA and protein products. Point mutations can be detected by amplification and sequencing of mRNA or by cDNA-derived cDNA produced from mRNA. The sequence of the cloned cDNA can be determined using DNA sequencing techniques well known in the art. The sequence of the 143939.doc • 133-201019961 cDNA can also be determined by polymerase chain reaction (PCR). Mismatches are non-100% complementary hybrid nucleic acid duplexes. Lack of total complementarity can be attributed to deletions, insertions, inversions, substitutions or frameshift mutations. Mismatch detection can be used to detect point mutations in target nucleic acids. While these techniques may be less sensitive than sequencing techniques, they are easier to perform on large numbers of tissue samples. An example of a mismatch cleavage technique is the RNase protection method, which is described in detail in Winter et al, Proc. Natl. Acad. Sci. USA, Vol. 82, p. 7575, 1985; and Meyers et al. Science, Vol. 230, p. 1242, 1985. For example, the methods of the invention can include the use of a labeled riboprobe that is complementary to a human wild-type target nucleic acid. The ribonucleic acid probe derived from the tissue sample and the dry nucleic acid are ligated (hybridized) together, followed by digestion with ribonuclease A which is capable of detecting some mismatch in the duplex RNA structure. If RNase A detects a mismatch, it cleaves at the mismatch site. Therefore, when the immobilized RNA preparation is separated on the electrophoresis gel matrix, if a mismatch has been detected and cleavage by ribonuclease A, the visible RNA product is smaller than the full-length duplex RNA of the riboprobe, and mRNA or DNA. The riboprobe is not required to be the full length of the target nucleic acid mRN A or the gene, but may be a part of the target nucleic acid as long as it covers the position of the suspected mutation. If the riboprobe contains only the target nucleic acid mRNA or a segment of the gene, it may be desirable to use several such probes to screen for mismatches throughout the target nucleic acid sequence, if desired. DNA probes can be used in a similar manner to detect mismatches, such as via enzymatic or chemical cleavage. See, for example, Cotton et al., Proc. Natl. Acad. Sci. USA, Vol. 85, 4397, 1988; and Shenk et al., Proc. Natl. 143939.doc • 134-201019961

Acad. Sci. USA, Vol. 72, vol. 989, 1975. Alternatively, mismatches can be detected by detecting the difference in electrophoretic mobility of the mismatched duplex relative to the matched duplex. See, for example, Cariello, Human Genetics, Vol. 42, p. 726, 1988. When a riboprobe or DNA probe is used, the target nucleic acid mRNA or DNA which can be mutated can be amplified prior to hybridization. Southern hybrids can also be used to detect changes in target nucleic acid DNA, especially when the change is gr〇ss rearrangement (such as deletions and insertions).等 Allele-specific probes can also be used to screen amplified target nucleic acid DNA sequences. These probes are nucleic acid oligomers each containing a target nucleic acid gene region having a known mutation. For example, an oligomer can be about 3 nucleotides long, corresponding to a portion of a target gene sequence. By using a set of such allele-specific probes, the target nucleic acid amplification product can be screened to identify the presence of a previously identified mutation in the target gene. For example, hybridization of an allele-specific probe to an amplified target nucleic acid sequence on a nylon filter will hybridize to a specific probe under stringent hybridization conditions to indicate the presence and allele in the tumor φ tissue. The same mutation in the specific probe. Variations in wild-type target genes can also be detected by screening for variations in the corresponding wild-type proteins. For example, monoclonal antibodies that are immunoreactive with a target gene product can be used to screen for tissue, such as antibodies known to bind to specific mutational positions of a gene product (protein). For example, the antibody used can be an antibody that binds to a deletion exon (e.g., exon 14) or binds to a conformational epitope comprising a deletion portion of the target protein. A lack of a homologous antigen will indicate a mutation. An antibody specific for the product of the mutant allele can also be used to (4) mutate the gene product. It can be used to identify the anti-bacteria Zhao Zhao 143939.doc • 135- 201019961 body. Such immunological assays can be performed in any convenient format known in the art, including jtb format, Western blotting, immunohistochemistry, and ELISA assays. The variation of the wild type dry gene can be extracted by the method of (4) variant protein. A primer pair is suitable for use in nucleic acid amplification techniques, such as polymerase chain reaction, to determine the nucleotide sequence of a target nucleic acid. A single-stranded DNA primer pair can be affixed to a sequence within the target nucleic acid sequence or around the target nucleic acid sequence to initiate amplification of the target sequence. Allele-specific primers can also be used. These primers only bind the sequence to a particular mutation, and thus will only amplify the product under the mutated target sequence as a template. To facilitate subsequent selection of the amplified sequence, the primer may have a restriction enzyme site sequence appended to its terminus. Such enzymes and sites are well known in the art. The primers themselves can be synthesized using techniques well known in the art. Primers are typically produced using commercially available nucleotide synthesizers. The design of a particular primer is well within the skill of this technology. Nucleic acid probes are suitable for several purposes. It can be used to hybridize to the southern part of the genomic DNA and to be used in the ribonuclease protection method to detect the point mutations discussed above. A probe can be used to detect a target nucleic acid amplification product. It can also be used to detect mismatches in wild-type genes or mRNA using other techniques. The mismatch can be detected using an enzyme (eg, S1 nuclease), a chemical (eg, hydroxylamine or tetrazolium and piperidine) or a mismatched hybrid electrophoretic shift change compared to a perfectly matched hybrid. These techniques are known. See Novack et al., Proc.

Natl. Acad. Sci. USA, Vol. 83, p. 586, 1986. The probe is generally complementary to a sequence external to the kinase domain. The entire set of nucleic acid probes can be used to construct a set that detects mutations in the target nucleic acid. This set allows for hybridization to a large region of the relevant target sequence I43939.doc • 136·201019961. The probes can overlap or abut each other. If a ribonucleic acid probe is used to detect a mismatch in an mRNA, it is generally complementary to the mRNA of the target gene. Because an RNA probe is complementary to a sense strand, it does not encode the corresponding gene product and is therefore an antisense probe. The riboprobe probe will typically be labeled with a radioactive, colorimetric or fluorescent material, which can be accomplished by any method known in the art. If a riboprobe is used to detect a mismatch in DNA, it can be polar or antisense. Similarly, DNA probes can also be used to detect mismatches. In some cases, cancer overexpresses or does not overexpress c-met receptors and/or EGFR. Overexpression of the receptor can be determined in a diagnostic or prognostic assay by assessing the increase in the amount of receptor protein present on the cell surface (e.g., via immunohistochemical assay; IHC). Alternatively or additionally, for example, via fluorescence in situ hybridization (FISH; see WO 98/45479, published October 1998), southern blotting or polymerase chain reaction (PCR) techniques (such as real-time quantitative PCR) (RT-PCR)) to measure the amount of nucleic acid encoding a receptor in a cell. In addition to the above tests, skilled personnel can also use a variety of in vivo tests. For example, cells in a patient can be exposed to antibodies that detect a detectable label (eg, a radioisotope) as appropriate, and can be obtained by, for example, external scanning radioactivity or analysis of a biopsy obtained from a patient previously exposed to the antibody. The binding of the antibody to the patient's cells is assessed. Chemotherapeutic Agents The combination therapies of the invention may further comprise one or more chemotherapeutic agents. Combination administration includes co-administration or concurrent administration using separate formulations or individual pharmaceutical formulations, and continuous administration in either order, with preferably 143939.doc -137-201019961 present-stage period 'at this time two (or All) active agents simultaneously exert their biological activity. If a chemotherapeutic agent is administered, the administered dose will usually be its known dose, or may be due to a combination of drugs or may be negative due to administration of an antimetabolite chemotherapeutic agent. The preparation of the chemical treatment (4) and the time course of administration can be used according to the manufacturer's instructions or as determined by a skilled practitioner. Various chemotherapeutic agents that can be combined have been disclosed above. In some embodiments, the chemotherapeutic agent to be combined is selected from the group consisting of: purple (including multi-riding (four) and Pacific (tetra) alcohol), periwinkle (such as long rubin or vinblastine), Platinum compounds (such as carboplatin or cisplatin), aromatase inhibitors (such as Letrozin, Ammet or Exemestane), antiestrogens (such as fulvestrant or tamoxifen), Etoposide, thiotecycline, methotrexate, liposomal doxorubicin, pegylated liposomal doxorubicin, capecitabine, gemcitabine, COX 2 inhibitor (eg necenab) Or a proteosome inhibitor (eg PS342). Formulations, Dosage, and Administration The therapeutic agents used in the present invention should be formulated, dosed, and administered in a manner consistent with good medical practice. Factors considered herein include the particular condition being treated, the particular individual being treated, the clinical condition of the individual patient, the cause of the condition, the site of the agent, the method of administration, the time course of administration, the drug-drug interaction of the combination agent, and Other factors known to the physician. Therapeutic formulations are prepared using standard methods known in the art, and the active ingredients of the desired purity and the physiologically selected 143939.doc-138-201019961 carrier, excipient or stabilizer are used (Remington's) Pharmaceutical Sciences (20th Edition), edited by A. Gennaro, 2000, Lippincott, Williams &amp; Wilkins, Philadelphia, PA). Acceptable carriers include physiological saline or buffers such as phosphates, citrates and other organic acids; antioxidants, including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin Or immunoglobulin; a hydrophilic polymer such as polyvinylpyrrolidone; an amino acid such as glycine, glutamic acid, aspartame, arginine or lysine; monosaccharide, disaccharide and Other carbohydrates, including glucose, mannose or dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming ions, such as sodium; and/or non-ionic interfacial ligands Preferably, however, it preferably contains a pharmaceutically acceptable salt, preferably sodium chloride, and preferably about physiological concentration. The formulations of the present invention may optionally contain a pharmaceutically acceptable preservative. In some embodiments, the concentration of the preservative ranges from 0.1% to 2.0%, typically in v/v. Suitable preservatives include those known in the pharmaceutical arts. Phenylmethyl alcohol, phenol, m-nonylphenol, methyl p-hydroxybenzoate and propyl p-hydroxybenzoate are preferred preservatives. The formulations of the present invention may optionally comprise from 5% to 0.02% by weight of a pharmaceutically acceptable surfactant. The formulations herein may also contain more than one active compound, preferably a compound having complementary activities that do not adversely affect each other, depending on the particular indication being treated. The molecules are suitably present in combination in an amount effective for the intended purpose. 143939.doc -139- 201019961 It is also possible to entrap active ingredients in colloidal drug delivery systems (eg liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or macroemulsions, for example using agglomeration techniques or interfaces. The microcapsules prepared by polymerization (for example, hydroxymethylcellulose or gelatin microcapsules and poly(methyl sulfonium methacrylate microcapsules, respectively) are polymerized. These techniques are disclosed in Remingt〇n, s pharmaceutical Sciences, supra. Sustained release formulations. Suitable examples of sustained release formulations include solid hydrophobic polymeric semipermeable matrices containing antibodies, which are in the form of shaped articles, such as films or microcapsules. Examples of sustained release matrices include polyesters; hydrogels (eg poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol)); polylactide (U.S. Patent No. 3,773,919); L-glutamic acid and γ-ethyl-L-glutamine Copolymer of acid ester; non-degradable ethylene vinyl acetate S曰' degradable lactic acid-glycolic acid copolymer, such as lupron DEPOT (including lactic acid-glycolic acid copolymer and Liu Pei Lin (〗 〖eUpr Injectable microspheres; and polyhydroxybutyrate. While polymers such as ethylene vinyl acetate and lactic acid-glycolic acid are capable of releasing molecules for more than 100 days, certain hydrogels release proteins for a shorter period of time. When the encapsulated antibody remains in the sputum for a long time, it may be degraded or aggregated due to its exposure to water, resulting in loss of biological activity and possibly altering immunogenicity. Depending on the mechanism involved, it can be designed reasonably. Strategy to achieve stabilization. For example, if the aggregation mechanism is found to form intermolecular SS bonds via thio-disulfide interchange, the sulphur residue can be modified, lyophilized from an acidic solution, and moisture content controlled. Stabilization is achieved using appropriate additives and development of specific polymer matrix compositions. 143939.doc 201019961 Therapeutic agents of the invention are administered to human patients according to known methods, such as intravenously in the form of bolus Inject, or by continuous infusion for a while, by intramuscular, intraperitoneal, intracranial (intracer〇br〇Spinai), subcutaneous, intra-articular, synovial Intrathecal, oral, topical or inhalation routes. In the case of VEGF antagonists, if extensive side effects or toxicity are associated with VEGF antagonism, then topical administration is particularly desirable. Therapeutic applications may also use the off-campus strategy. The cells obtained from the individual are transfected or transduced with a polynucleotide encoding a paw or an EGFR or VEGF antagonist. The transfected or transduced cells are then returned to the individual. The cells can be any of a variety of types. 'Includes, but is not limited to, hematopoietic cells (eg, bone marrow cells, macrophages, monocytes, dendritic cells, T cells, or B cells), fibroblasts, epithelial cells, endothelial cells, keratinocytes, or muscle cells. For example, if the c-met or EGFR or VEGf antagonist is an antibody, the antibody is administered by any suitable means, including parenteral, subcutaneous, intraperitoneal, intrapulmonary, and intranasal administration, if desired. Immunosuppressive therapy 'includes intralesional administration'. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. In addition, antibodies are administered by pulse infusion, particularly by decreasing doses of the antibody. Preferably, it is administered by injection, preferably by intravenous or subcutaneous injection, depending in part on whether the administration is short or long term. In another example, a c_met or EGFR4 VEGF antagonist compound is administered topically by, for example, direct injection, when the condition or tumor location permits, and the injection can be repeated periodically. C_met or EGFR or VEGF antagonists can also be delivered systemically to the individual or directly to tumor cells, for example to tumor 143939.doc-141 - 201019961 or to the tumor bed after surgical removal of the tumor to prevent or reduce local recurrence or Transfer. When the inhibitor is an antibody, the antibody administered is preferably a naked antibody. However, the inhibitor to which the inhibitor is administered can bind to the cytotoxic agent and/or the antigen to which it binds is preferably intracellularly internalized, thereby allowing the binding to kill the cancer cells to which it binds. Increased efficacy. In a preferred embodiment, the cytotoxic agent is dry or interferes with nucleic acids in cancer cells. Examples of such cytotoxic agents include maytansinoids, calicheamicin, ribonucleases, and DNA endonucleases.合适 Any suitable dosage of the therapeutic agents listed herein is the amount currently employed and may be reduced or increased as the treating physician believes. In some embodiments, an anti-c-met antibody is administered in an amount sufficient to obtain a serum trough concentration of 15 μg/ml or 15 μg/ml or more. In some embodiments, the administered dose is about 5 mg/kg or more anti-omet antibody every three weeks. In some embodiments, the anti-c-met antibody is administered at a dose of about 15-20 mg/kg for three weeks. In some embodiments, an anti-c-met antibody is administered at a total dose of about ©5 © mg/kg or higher over a three week period. In one embodiment, the EGFR antagonist is erlotinib and a dose of 150 mg of erlotinib can be administered on each of the three week weeks. In some embodiments, an erlotinib dose of 1 mg is administered. In some embodiments, a dose of 50 mg of erlotinib is administered. It is expected that the dose of erlotinib will be reduced as indicated by the label of erlotinib. In certain embodiments, when used in combination, the shellfish is administered at a dose of about 5 mg/kg 143939.doc -142.201019961 to about 15 mg/kg. In one embodiment, one or more of the following doses can be administered to an individual: 巧〇.5 mg/kg, 1.0 mg/kg, 2 〇mg/kg, 3.0 mg/kg, 4·〇., Λ &quot; g/kg, 5_G mg/kg, 6.0 mg/kg, 7.0 mg/kg, 7.5 mg/kg, 8 ft n 8 g 8·〇mg/kg' 9.0 mg/kg, w or 15 mg/kg (or Any combination) The special dose $ can be administered intermittently, for example once a day, every three days, every week or every two to two weeks. In another embodiment, when used in combination, the parent is administered intravenously to the individual one week apart.

MB/kg of bevacizumab, 戋善=, and parental administration of mg/kg of bevacizumab to the individual intravenously. The combination of therapeutic agents is usually administered within a specified period of time (generally within minutes, hours, days or weeks depending on the combination selected). Combination therapies are intended to include administering such therapeutic agents in a continuous manner (i.e., each therapeutic agent is administered at different times) and administering the therapeutic agents or at least two therapeutic agents in a substantially simultaneous manner. The therapeutic agent can be administered via the same route or by different routes. For example, a combined form of a VEGF, EGFR or c-met antagonist can be administered by intravenous injection&apos; and a combined form of a protein kinase inhibitor can be administered orally. Alternatively, for example, depending on the particular therapeutic agent, both therapeutic agents can be administered orally or both therapeutic agents can be administered by intravenous injection. The order in which the therapeutic agent is administered will also vary depending on the particular agent. This application contemplates the use of gene therapy to administer VEGF, c-met and/or EGFR antagonists. For the use of gene therapy to produce intracellular antibodies, see, for example, WO 96/07321, published March 14, 1996. There are two main methods for introducing nucleic acids (as appropriate in a vector) into a patient's cells. 143939.doc • 143-201019961; in vivo and ex vivo. For in vivo delivery, the nucleic acid is typically injected directly into the patient at the site where the antibody is desired. For ex vivo treatment, the patient cells are removed, nucleic acids are introduced into the isolated cells, and the modified cells are administered directly to the patient, or, for example, encapsulated in a porous membrane to be implanted in a patient (see, e.g., U.S. Patent No. 4,892,538 And No. 5 283 187). There are a variety of techniques available for introducing nucleic acids into living cells. Such techniques may vary depending on whether the nucleic acid is transferred in vitro to the cultured cells, or the nucleic acid is transferred in vivo to the cells of the intended host. Techniques suitable for the in vitro transfer of nucleic acids into mammalian cells include the use of liposomes, electroporation, microinjection, cell fusion, DEAE-dextran, calcium phosphate precipitation, and the like. A commonly used vector for in vitro delivery of genes is a retrovirus. Currently preferred in vivo nucleic acid transfer techniques include transfection with viral vectors (such as adenovirus, simple blister type I virus or adeno-associated virus) and lipid-based systems (lipid-mediated gene transfer for lipids such as DOTMA, DOPE and DC-Chol). In some cases, it may be desirable to provide a nucleic acid source with an agent that targets a target cell, such as an antibody specific for a cell surface membrane protein or stem cell, a ligand for a receptor on a target cell, and the like. When a liposome is used, a protein that binds to a cell surface membrane protein associated with endocytosis can be used to target and/or facilitate uptake, for example, a capsid protein or fragment thereof that is tropic for a particular cell type, in circulation. An antibody that undergoes internalization of the protein, and a protein that localizes within the cell and enhances intracellular half-life. Techniques for receptor-mediated endocytosis are described, for example, in Wu et al., j. 〇/. 262:4429-4432 (1987); and Wagner et al., Pr.

Jcac/· «Sci. 87:3410-3414 (1990). For a review of currently known 143939.doc 201019961 known gene signatures and gene therapy protocols, see Anderson et al, Sdewce 256:808-813 (1992). See also WO 93/25673 and references cited therein. The following are examples of the methods and compositions of the present invention. It will be appreciated that in view of the general description provided above, various other embodiments can be implemented. EXAMPLES Materials and Methods Cell lines: KP4 cell lines were obtained from the RIKEN BioResource Center of the Institute of Physical and Chemical Research (Ibaraki, Japan). The NCI-H596 cell line was obtained from the American Type Culture Collection (Manassas, VA). NCI-H441-shMet-3.ll and EBC-l-shMet-3.15 and 4.5 are previously described in commonly-owned co-pending U.S. Patent Publication No. 2009/0226443. Briefly, retroviral constructs were prepared as follows: shRNA sequences encoding c-met (5,-GATCCCCGAACAG AATCACTGACATATTCAAGAGATATGTCAGTGATTCTGTT CTTTTTTGGAAA-3' (SEQ ID NO: 29) (shMet 3) and 5'GATCCCCGAAACTGTATGCTGGATGATTCAAGAGATC ATCCAGCATACAGTTTCTTTTTTGGAAA (SEQ ID NO: 30) (shMet 4)) The oligonucleotide was cloned in the Bglll/Hindlll site of the pShuttle-Hl vector downstream of the HI promoter (David Davis, GNE). Bold letters indicate dry hybridization sequences. Recombination of these constructs with the retroviral pHUSH-GW vector (Gray D et al, BMC Biotechnology. 143939. doc-145-201019961 2007; 7:61) using the Clonase II enzyme (Invitrogen) to generate shRNA expression inducibility The construct of the promoter control. Treatment with the tetracycline analog doxycycline caused shRNA expression. A shGFP2 control retroviral construct containing shRNA directed against GFP (Hoeflich et al, Cancer Res. (2006) 66(2): 999-1006) was supplied by David Davis, Genentech, Inc. shGFP2 contains the following oligonucleotides:

(EGFP) shRNA (sense) 5'-GATCCCCAGATCCGCCACAACATCGATTCAA GAGATCGATGTTGTGGCGGATCTTGTTTTTGGGAAA-3 (SEQ ID NO: 31). The pure line stably transduced by the retroviral construct was grown in appropriate medium +/- 1 pg/ml doxycycline (Clontech) to induce shRNA expression, and anti-c-met C-12 antibody (Santa Cruz Biotech) was used. Screening for c-met knockouts by Western blotting. Establishment of luciferyl-c-met (also known as pmet or p-pc-met) ink using anti-acid-based-c-met Y1003 (Biosource) and anti-phospho-c-met Y1234/1234 (Cell Signaling) antibodies point. As a control group, an actin ink dot was established using an anti-actin 1-19 antibody (Santa Cruz Biotech). EBC pure line 3.15 and EBC pure line 4.12 showed a sharp decrease in c-met performance and selenate-c-met content. H441 pure line 3.11 and H441 pure line 3.1 showed a moderate decrease in c-met performance and phosphate-c-met performance. EBC pure line 4.5 showed less reduction in c-met and androstyl-c-met performance. The cell line EBC pure line 4.5 and the EBC pure line 4.12 contained the construct shMet4, and the cell line H441 pure line 3.1, H441 pure line 3.11 and EBC pure line 3.15 contained the construct 811]^613. Western blotting method: To separate proteins, all cell lysates were run over a 4-143939.doc-146-201019961 12% Tris-Glyceric acid gel (Invitrogen). The gel was transferred to a 0.45 m cellulose acetate membrane (Invitrogen). The membrane was blocked in Odyssey blocking buffer (LiCor Biosciences) for 30 minutes at room temperature, followed by incubation in primary antibody diluted in blocking buffer plus 0.1% Tween 20 at 4 °C Overnight. The membrane was washed with TBST, followed by goat anti-mouse IR800 (Rockland), goat anti-rabbit Alexa Fluor 680 (Molecular Probes) according to the appropriate primary antibody in blocking buffer plus 0.1% Tween 20 and 0.01% SDS at room temperature. And cockroach anti-goat Alexa Fluor 680 (Molecular Probes) - incubated for 2 hours. The antibody was lysed by using the Odyssey System scan: (in vitro) the cells were washed with cold TBS. On ice, in lysis buffer (TBS + 1% NP40 + Complete Mini-EDTA-Free protease inhibitor (Roche) + lx phosphatase inhibitor cocktail 2 (Sigma)) Dissolve the cells for 10 minutes (on the plate). The lysate was centrifuged at 10 ° C for 10 minutes at 4 ° C and the lysate was transferred to a new tube. The lysate was quantified using a BCA assay (Pierce). Tumor lysate··Use a hand-held homogenizer in lysis buffer (PBS+1% Triton X-100+ EDTA-free complete minimum protease inhibitor (R〇che) + 3 X phosphatase inhibitor mixture 2 ( The tumor is homogenized in sigina)). The lysate was incubated on ice for 1 hour under occasional vortexing. The lysate was centrifuged at 10000 xg for 10 minutes at 4 ° C and the lysate was transferred to a new tube. The lysate was quantified using a BCA assay (Pierce). Modified medium: After the cells were treated, the medium was removed and centrifuged for 1 minute at l〇〇〇〇xg 143939.doc -147·201019961. The modified medium is then transferred to a new tube. Microarray treatment: EBC-l shMet4-12 and shGFP-2 cells were seeded in 10 cm plates (3 parts each). 10% without Tet FBS RPMI 1640 and incubated overnight. The medium was replaced with fresh medium +/- 100 ng/ml doxycylin (dox). The plates were incubated for 24 hours or 48 hours. After 24 hours of dox treatment, HGF-treated cells were treated with 100 ng/ml HGF for 2 hours. The cells were then trypsinized and centrifuged. The pellet was frozen and stored at -80 °C. RNA was extracted from the cells using the Qiagen RNeasy kit as described in the manufacturer's instructions. IL8 assay • Dilute anti-human IL8 (R&amp;D Systems, Minneapolis, MN, USA) to 5 pg/mL in PBS (pH 7.4) and plate on Maxisorp 384 well flat bottom plate (Nunc, Neptune, NJ) 25 μΐ per well), incubated overnight (16-18 hours) at 4 °C. Plates were washed 3 times in wash buffer (PBS, pH 7.4, 0.05% Tween 20) and blocking buffer (PBS, pH 7.4, 0.5% BSA) was added to each well at 50 μM per well. The plates were blocked for 1-3 hours; the incubation and all subsequent incubations were performed at room temperature on a rotary shaker. Samples and standards were prepared in sample diluent (PBS/0.5% BSA/0.05% T-20/5 mM EDTA/0.25% CHAPS/0.2% BGG/10 ppM Proclin) during the blocking step. The blocked plates were washed three times (as described above) and samples and standards (25 μM per well) were added to the ELISA plate. After 1-2 hours of incubation, the ELISA plate was washed three times (as described above) and diluted by adding the assay buffer (PBS/0.5% BSA/0.05% Tween 20, pH 7.4) to 50 ng/mL. Labeled anti-human IL8 antibody (R&amp;D Systems,. Minneapolis, MN, USA) (25 μM per well) for 143939.doc -148- 201019961

Human IL8 combined with a plate. The plates were incubated for 1-2 hours and washed as described above. HRP-bound streptavidin (Amersham BioSciences, Piscataway, NJ, US A) (25 μM per well) diluted 1:40,000 was then added to the ELISA plate and the plates were incubated for 0.5 hours. After the final wash, ΤΜΒ substrate (Kirkegaard &amp; Perry Laboratories, Gaithersburg, MD) was added to the ELISA plate at 25 μM per well. After about 5 minutes, color development was stopped by adding 25 μΐ 1.0 Μ phosphoric acid per well. Absorbance at 450 nm and 650 nm was measured using a SpectraMax 250 microtiter plate reader (Molecular Devices, Sunnyvale, CA). Human VEGF165-206 ELISA (detection of VEGF165): MaxiSorp 96-well microwells were coated with 0.4 μg/ml of 3.5?8 antibody in 50 mM carbonate buffer (pH 9.6) at 100 μΐ per well at 4 °C. Plates (Nunc, Roskilde, Denmark) were overnight and used for 0.05% polysorbate S 20 (pH 7.4) wash in PBS. The plates were incubated with 0.5% bovine serum albumin, 10 ppm Proclin 300 (Supelco, Bellefonte, PA) in PBS for 1 hour at room temperature and washed. VEGF standard in 0.5% bovine serum albumin, 0.05% polysorbate 20, 10 ppm Proclin 300, 5 mM EDTA and 0.35 NNaCl (pH 6.3) (assay buffer) added to PBS in 100 μM per well ( 1.56-200 pg/ml VEGF165 or 0.041-5.2 pM VEGF, two-fold serial dilution; Genentech) and sample (minimum 1:10 dilution) "The plates were incubated for 2 hours at room temperature and washed. Plate-bound VEGF was detected by biotinylated A4.6.1 added to assay buffer. After incubation for 2 hours at room temperature, the plates were washed and added to the assay buffer for streptavidin-HRP (GE Healthcare, Piscataway, 143939.doc • 149. 201019961 NJ) and incubated for 30 minutes. The plate was washed, followed by the addition of the 3,3',5,5'-tetradecylbenzidine (Kirkegaard &amp; Perry Laboratories, Gaithersburg, MD). The reaction was stopped by the addition of 1 M phosphoric acid and the absorbance was read at 450 nm. The titration curve was analyzed using a four parameter regression curve fitting program (Genentech). The VEGF concentration in the sample is calculated using data points that fall within the range of the standard curve. VEGF110-206 ELISA (Total VEGF Detection): MaxiSorp 96-well microplates were coated with 0.5 μg/ml of antibody 2E3 in 50 mM carbonate buffer (pH 9.6) at 100 μΐ per well at 4 °C ( Nunc, Roskilde, Denmark) was washed overnight with 0.05% polysorbate 20 (pH 7.4) in PBS. 0.5% bovine serum albumin, 10 ppm Proclin 300 (Supelco, Bellefonte, PA) blocking plates (150 μl per well) in PBS for 1 hour at room temperature and washed. 0.5% bovine serum albumin, 0.05% polysorbate 20, 10 ppm Proclin 300, 5 mM EDTA, 0.25% CHAPS, 0.2% bovine gamma globulin (Sigma) added to PBS in 100 μM per well. , St. Louis, MO) and 0.35 N NaCl (pH 7.4) (assay buffer) VEGF standard (1.56-200 pg/ml VEGF1654 0.0409_ 5.24 pM VEGF, two-fold serial dilution; Genentech) and sample (minimum 1:10 dilution). The plates were incubated for 2 hours at room temperature and washed. The plate-bound VEGF was detected by biotinylated Α4·6·1 (100 μM per well) added to the sample buffer. After incubating for 1 hour at room temperature, the plates were washed, and anti-protein-HRP (GE Healthcare, Piscataway, NJ) was added and incubated for 30 minutes (100 μM per well). The plate was washed and then biotinylated amide salt was added to the plate (ELAST ELISA Amplification System, Perkin Elmer Life 143939.doc -150· 201019961)

Sciences Inc., ΜΑ) (100 μM per well). After 15 minutes of incubation, the plate was washed and the streptavidin-HRP (100 μM per well) was added to the sample buffer. After incubation for 30 minutes, the plates were washed and the substrate was added with 3,3,5,5-tetramethylbenzidine (Kirkegaard &amp; Perry Laboratories) (100 μM per well). The reaction was stopped by the addition of 1 guanidine phosphate (100 μM per well). Read the absorbance at 450 nm. The titration curve was analyzed using a four parameter regression curve fitting program (Genentech). The VEGF concentration in the sample is calculated using data points that fall within the range of the standard curve. Mouse VEGF ELISA (Total VEGF Detection): goat anti-mouse VEGF (R&amp;D, Minneapolis, MN) at 0.5 pg/ml in 50 mM carbonate buffer (pH 9.6) at 4 °C MaxiSorp 96-well microplates (Nunc, Roskilde, Denmark) were coated overnight at 100 μM per well and washed with 0.05% polysorbate 20 (pH 7.4) in PBS. 0.5% bovine serum albumin, 10 ppm Proclin 300 (Supelco, Bellefonte, PA) blocking plates (150 μl per well) in PBS for 1 hour at room temperature and washed. 0.5% bovine serum albumin, 0.05% polysorbate 20, 10 ppm Proclin 300, 5 mM EDTA, 0.25% CHAPS, 0.2% bovine gamma globulin (Sigma, St.) added to PBS at 100 μM per well. Louis, MO) and mouse VEGF standard in 0_35 N NaCl (pH 7.4) (sample buffer) (1.0-128 pg/ml VEGF164 or 0.026-3.4 pMVEGF, two-fold serial dilution; R&amp;D Systems, McKinley Place, NE) and sample (minimum dilution 1:10). After 2 hours of incubation, the plate-bound VEGF was detected by biotinylated goat anti-mouse VEGF antibody added to the sample buffer. After incubation for 1 hour at room temperature, the plates were washed and added to PBS 143939.doc • 151 · 201019961 0.5% bovine serum albumin, 0.05% polysorbate 20, 10 ppm Pro cl in 300 (pH 7.4 Streptavidin-HRP (GE Healthcare, Piscataway, NJ). After 45 minutes of incubation, the plates were washed and the substrate was added with 3,3',5,5'-tetramethylbenzidine (Kirkegaard &amp; Perry Laboratories, Gaithersburg, MD) (100 μM per well). The reaction was stopped by the addition of 1 guanidine phosphate (100 μM per well) and the absorbance was read at 450 nm. The titration curve was analyzed using a four parameter regression curve fitting program (Genentech). The VEGF concentration in the sample is calculated using data points that fall within the range of the standard curve. In vivo xenograft tumor research you test #于·· Provided anti-VEGF antibody binding to mouse and human VEGF in a clear liquid form by the Antibody Engineering Department of Genentech, Inc. (B20-4.1 and B20-4.1 .1), and diluted in 1 x PBS. The anti-c-met monovalent monoclonal antibody MetMAb (rhuOA5D5v2; WO 2007/063816) was also provided in a clear liquid form by the antibody engineering department of Genentech, Inc., and in MetMAb buffer (10 mM histidine sulphate ( Diluted in histidine succinate), 4% trehalose dihydrate, 0.02% polysorbate 20, pH 5.7). Control antibodies include the mouse IgG2a isotype 10D9-1E11-1F12 (anti-porcine) antibody and the human IgG1 homotype hu5B6 (anti-gD) antibody, both obtained in clear liquid form from the Antibody Engineering Division of Genentech, Inc., and Dilute in lxPBS. Ertotinib (TarcevaTM) was supplied by OSI Pharmaceuticals to the Genentech formulation group and was found to be a sufficient amount of vehicle (methylcellulose tween (MCT)). 0.5 or 1 mg/mL of doxycycline (Dox) was freshly prepared in 5% sucrose water and changed regularly every 3 days 143939.doc -152· 201019961. In the Dox study, control animals were given 5% sucrose water, which was changed every 3 days. The material is stored in a refrigerator set to maintain a temperature in the range of 4 ° C to 8 ° C. #礼自 Charles River Laboratories (CRL) obtained nude mice (nu/nu) 6 to 8 weeks old and domesticated at the Genentech's feeding center (vivarium) for at least one week prior to the study. Four to six week old hu-HGF-Tg-C3H-SCID mice were generated at the Van Andel Institute, and the mice were maintained in the study and all studies were performed using this line. Animals are housed in a ventilated cage system in a room with a filter that supplies high efficiency particulate air (HEPA). The study used only animals that appeared healthy and had no significant abnormalities. Study Design Study 1: Treatment with a combination of MetMAb舆 anti-VEGF antibody in a KP4 pancreatic xenograft tumor model: consisting of RPMI 1640 medium (In vitro gen), 2 mM L-face valeric acid and 10% fetal bovine serum KP4 cells were cultured in growth medium. To prepare cells for inoculation into mice, the cells were trypsinized and washed with 10 ml of sterile lx phosphate buffered saline (PBS). A subset of cells was counted using tryptan blue exclusion and the remaining cells were resuspended in 100 μL sterile 1 X PBS to a concentration of 5χ107 cells per ml. Mice were inoculated subcutaneously in the lower right scapular region with 5χ106 ΚΡ4 cells. Tumors were monitored until their average volume reached 230 mm3. Mice were randomized into 7 groups of 10 mice each and treatment was started (summarized in Table 1). The mice in Group 1 were treated with 100 μM MetMAb buffer (IP, once) and anti-porcine control antibody (5 mg/kg, IP, twice a week for 3 weeks) at 143939.doc -153· 201019961. Mice in Group 2 were treated with B20-4.1 (5 mg/kg, IP, twice a week for 3 weeks). Mice in group 3 were treated with a sub-therapeutic dose of MetMAb (2.5 mg/kg, IP, once). Mice in group 4 were treated with MetMAb (ED5: 7.5 mg/kg, IP, once) at a dose of about 50% tumor inhibition for 21 days. Mice in group 5 were treated with high doses of MetMAb' (30 mg/kg, ip, once). What is the sub-therapeutic dose of the mice in group 6? MetMAb plus anti-VEGF antibody (2.5 mg/kg MetMAb, IP, once, plus 5 mg/kg B20-4.1, IP, twice a week for 3 weeks). MetMAb of mouse ED50 in group 7 was administered anti-VEGF antibody (7.5 mg/kg Φ MetMAb, IP, once, plus 5 mg/kg B20-4.1, IP' twice a week for 3 weeks). Tumor volume was measured twice a week and animals were monitored for 25 days. Table 1 Number of combinations of MetMAb and anti-VEGF antibodies in KP4 pancreatic xenograft tumor model / Sex test material route Dose frequency dose (mg/kg) Dose concentration (mg/ml) Dose volume (μ〇1 10/F MetMAb buffer Control antibody IP once; twice weekly x3 weeks 0 0 100 2 10/F anti-VEGF antibody (B20-4.1) IP twice a week (MetMAb buffer once) X3 weeks 5 1.25 100 3 10/F MetMAb IP once (Control antibody twice a week) 2.5 0.625 100 4 10/F MetMAb IP once (control antibody twice weekly x3 weeks) 7.5 1.875 100 5 10/F MetMAb IP once (control antibody twice a week x 3 weeks) 30 7.5 100 6 10/F MetMAb+anti-VEGF antibody (B20-4.1) IP once (MetMAb); twice weekly CB20-4.1) 2.5 (MetMAb); 5 (B20-4.1) 0.625 (MetMAb); 1·25 (Β2( Μ.1) 100 7 10/F MetMAb+anti-VEGF antibody fB20-4.1) IP once (MetMAb); twice a week (B20-4.1) 2.5 (MetMAb); 5 (B20-4.1) 0.625 (MetMAb); 1.25 ( B20-4.1) 100 143939.doc -154- 201019961 Study 2: Combination treatment with c-met knockout anti-VEGF anti-sputum in NCI-H441 NSCLC xenograft tumor model: in RPMI 1640 culture Yl growth medium (Invitrogen), 2 mM L- roughness acid amide, and 10% fetal bovine serum in the culture composition of the NCI-H44l-shMet-3.11 cells. To prepare cells for inoculation in mice, the cells were trypsinized and washed with 10 ml of sterile 1X phosphate buffered saline (PBS). A subset of the cells was counted using trypan blue exclusion and the remaining cells were resuspended in 100 μL sterile 1X PBS to a concentration of 5χ107 cells per ml. Mice were inoculated subcutaneously in the lower right scapular region with 5 x 106 NCI-H441-shMet-4.11 cells. Tumors were monitored until their average volume reached 200 mm3. Mice were randomized into 4 groups of 10 mice each and treatment was started (summarized in Table 2). Mice in group 1 were treated with anti-porcine control antibody (5 mg/kg, IP, twice a week for 4 weeks) and given 5% sucrose drinking water. The small anti- ragweed control antibody (5 mg/kg, IP, twice a week for 4 weeks) in group 2 was treated with 'doxcycline (丨mg/mL) in 5% sucrose drinking water. . Mice in Group 3 were treated with anti-VEGF antibody (B20-4.1.1, 5 mg/kg, IP, twice a week for 4 weeks) and given 5% sucrose drinking water. The mice in Group 4 were treated with anti-VEGF antibody (B20-4.1.1 '5 mg/kg, IP, twice a week for 4 weeks) and given doxycycline in 5% sucrose drinking water (1 The antibody was continued to be administered for four weeks at mg/mLh, at which time the administration of docecycline was maintained, but antibody administration was stopped. Tumor volume was monitored until day 57. 0 143939.doc -155-201019961 Table 2 NCI-441 NSCLC xenogene Transplanted tumor model te_meti|j^|^ VEGF antibody combination group 1 number / sex 10/F test material sucrose; anti-porcine control antibody pathway drinking water; IP dose frequency arbitrary feeding (whole process); twice weekly x4 Weekly dose (mg/kg) 5 (anti-porcion) Dose concentration (mg/ml) 1.25 (anti-porcelain) Dose volume (μΐ) 100 2 10/F doxycycline; Control antibody drinking water; IP Any feeding (whole process); twice weekly x4 weeks 5 (anti-porcelain) 1.25 (anti-porcelain); 1 (Dox) 100 3 10/F sucrose; anti-VEGF (B20-4.1.1) drinking water; IP arbitrary Food (whole process); twice weekly x4 weeks 5 (anti-VEGF) 1.25 (anti-VEGF) 100 4 10/F doxycycline; anti-VEGF (B20-4.1.1) drinking water; IP Arbitrary feeding (whole process); twice weekly X4 weeks 5 (anti-VEGF) 1.25 (anti-VEGF); 1 (Dox) 100 studies 3 and 4: c-met in the EBC-l-shMet NSCLC xenograft model Knockout combination of anti-VEGF anti-sputum treatment: cultured in growth medium consisting of RPMI 1640 medium (Invitrogen), 2 mM L-branched tyrosine and 1% fetal bovine serum: £801-311^461;-3.15 And £801-3111^161;-4.5 cell strain. To prepare cells for inoculation in mice, the cells were trypsinized and washed with 10 ml of sterile lx phosphate buffered saline (PBS). Dye a subset of counted cells' and resuspend the remaining cells in 100 μl sterile lxPBS to a concentration of 5 x 107 cells per ml. Use 5xl06 EBC-l-shMet-3.1 5 and EBC-l-shMet-4.5 cells in the lower right Mice were subcutaneously inoculated in the scapular region. Tumors were monitored until their average volume reached 300 mm3 °. For EBC-l-shMet-3.15 study and EBC-l-shMet-4.5 study, mice were randomly divided into 2 groups, each group consisting of 10 small The rats 'total 4 groups' and began processing 143939.doc -156-201019961 (summarized in Table 3). The mice in Group 1 had an EBC-l-shMet-3.15 tumor and were given 5% sucrose drinking water. The mice in Group 2 had EBC-1-shMet-3.15 tumors and were administered to doxycycline (1 mg/mL) in 5% sucrose drinking water. The mice in Group 3 had EBC-l-shMet-4.5 tumors and were given 5% sucrose drinking water. The mice in Group 4 had EBC-l-shMet-4.5 tumors and were administered to doxycycline (丨mg/mL) in 53⁄4 sucrose drinking water. The tumor volume was monitored for 21 days.

Table 3 NCI-441 NSCLC xenograft tumor model (4) Combination of knockout and anti-sense antibody group 1 cell line

Number / test material sex 10/F route dose frequency drinking water free feeding (whole process) 10/F Dorset drinking water free feeding (whole process) 3 EBC-l-shMet-4.5 10/F Sucrose drinking water can be eaten freely (the whole process) 4 EBC-l-shMet·4.5 10/F Dorsetian drinking water can be fed ad libitum (complete __ process) 2 EBC-1-shMet- 3.15 EBC-1-shMet- 3.15 sucrose dose dose concentration dose volume (mg/kg) (mg/ml) (μ〇

EBC-l-shMet-4.5 NSCLC xenograft tumors were further tested with anti-VEGF antibodies selective for mouse and human VEGF (B20-4.1) or human VEGF (bevacizumab) alone to test human or mouse VEGF Relative contribution to driving tumor growth. Although mice were inoculated as described above, mice were instead randomly divided into 3 groups of 10 mice each and treatment was started (summarized in Table 4). 143939.doc •157- 201019961 Table 4 Characterization of the contribution of mouse or human VEGF to EBC-l-shMet-4.5 NSCLC xenograft tumor growth Group number / sex test material route agent Dong frequency dose (mg/kg) dose concentration ( Mg/ml) Dosage volume (μΐ) 1 10/F Anti-gD control antibody (hu5B6) IP twice weekly x3 weeks 5 (anti-gD) 】·25 (anti-gD) 100 2 10/F anti-VEGF (B20-4.1 IP twice weekly x3 weeks 5 (anti-mouse and human VEGF) 1.25 (anti-mouse and human 100 3 10/F anti-VEGF (bevacizumab) IP twice weekly for 3 weeks 5 (anti-human VEGF) 1.25 (anti-human VEGF) 100 Study 5: Combination of c-met knockout, EGFR antagonist erlotinib and anti-VEGF anti-spasm in EBC-l-shMet-4.5 NSCLC xenograft tumor model •· The EBC-l-shMet-4.5 cells were inoculated, and the mice were randomly divided into 8 groups of 10 mice each and started treatment (summarized in Table 5). Preliminary tolerance study indicated that doxycycline and angstrom The combination of rotinib can significantly reduce body weight. Therefore, the concentration of doxycycline is reduced to 0.5 mg/mL with drinking water. This concentration of doxycycline is sufficient for induction in vivo. -met shRNA. The mice in group 1 (control group) were treated with anti-porcine control antibody (5 mg/kg, IP, twice a week for 4 weeks) plus MCT (l〇〇μι, PO, QD, χ28) Day) treated with 5% sucrose drinking water. Group 2 mice were treated with control antibody (5 mg/kg, IP, twice a week for 4 weeks) plus MCT (100 μί, PO, QD, x28 days) Treated and given doxycycline (0.5 mg/mL) in 5% sucrose drinking water. The mice in group 3 were treated with anti-VEGF antibody (5 mg/kg, IP twice a week for 4 weeks) MCT (100 pL, P〇, QD, x28 days) was treated with 5% sucrose drinking water. Group 4 mice were treated with anti-porcine control antibody (5 mg/kg, IP, twice a week for 4 weeks) Treated with erlotinib (100 143939.doc -158-201019961 mg/kg, PO, QD, x28 days) and given 5% sucrose drinking water. Group 5 mice were treated with anti-VEGF antibody (5 mg/ Kg, IP 'twice a week for 4 weeks) plus MCT (100 μί, P〇, QD, x28 days) and given doxycycline (〇·5 mg/mL) in 5% sucrose drinking water The mice in group 6 were treated with anti-porcine control antibody (5 mg/kg 'IP, twice a week) 4 weeks) Jiaai Luo erlotinib (100 mg / kg, PO, QD, χ28 day) treated, and administered in a 5% sugar strict drinking water as much as doxycycline (square · 5 mg / mL). Group 7 mice were treated with anti-VEGF antibody (5 mg/kg, IP, twice a week for 4 weeks) plus erlotinib (100 mg/kg, PO, QD, χ28 days) and given 5% Strict sugar drinking water. Furthermore, mice in group 8 were treated with anti-VEGF antibody (5 mg/kg, IP twice a week for 4 weeks) plus erlotinib (100 mg/kg, PO, QD, χ28 days) and at 5% Treatment with doxycycline (0.5 mg/mL) in sucrose drinking water. Table 5 Combination of c-met knockout erlotinib and anti-VEGF antibody in EBC-l-shMet-4.5 NSCLC xenograft tumor model Test material route Sword frequency dose (mg/kg) Dose concentration (mg/ml) Dosage volume (μΐ) against ragweed control antibody; MCT; sucrose IP; P0; drinking water twice a week for x4 weeks; QD; arbitrary feeding 5 (anti-porcine) 1.25 (anti-gD) 100 anti-porcino antibody; ; doxycycline IP; PO; drinking water every two X4 weeks; QD; arbitrary feeding 5 (anti-porcine) 1.25 (anti-gD); 0.5 (Dox) 100 anti-VEGF antibody (B20-4.1.1) MCT; sucrose IP; PO; drinking water twice a week X4 weeks; QD; arbitrary feeding 5 (Β20-4.Π) 1·25 (anti-gD) 100 anti-porcino antibody; erlotinib; ; PO; drinking water every two X4 encounters; QD; arbitrary feeding 5 (anti-grass); 10 〇 (erlotinib) 1.25 (anti-gD); 25 (erlotinib) 100 number / ~ group Gender 143939.doc • 159- 201019961 5 6 7 8 10/F anti-VEGF antibody (B20-4.1_1); MCT; doxycycline IP; PO; drinking water twice a week x4 weeks; QD; (B20-4.1.1) 1.25 (anti-gD); 0.5 (Dox) 10 0 10/F anti-porcino antibody; erlotinib; doxycycline IP; PO; drinking water twice a week for 4 weeks; QD; any feeding 5 (B20-4.1.1); Rotinib) 1.25 (anti-gD); 25 (erlotinib); 0.5 (Dox) 100 10/F anti-VEGF antibody (B20-4.1.1); erlotinib; sucrose IP; PO; Weeks twice χ 4 weeks; QD; arbitrarily feeding 5 (B20-4.1.1); 100 (erlotinib) 1.25 (anti-gD); 25 (erlotinib) 100 10/F anti-VEGF antibody (B20- 4.1.1); erlotinib; doxycycline IP; PO; drinking water twice a week X4 weeks; QD; arbitrary feeding 5 (B20-4.1.1); 100 (erlotinib) 1.25 ( Anti-g〇); 25 (Errotinib); 0.5 (Dox) 100 Study 6: Combination of c-met knockout, erlotinib and anti-VEGF antispasmodic in NCI-H596 NSCLC xenograft tumor model : NCI-H596 cells were cultured in growth medium consisting of RPMI 1640 medium (Invitrogen), 2 mM L-face valine and 10% fetal bovine serum. To prepare cells for inoculation into mice, the cells were trypsinized and washed with 1 ml of sterile lx phosphate buffered saline (PBS). A subset of cells were counted using trypan blue exclusion and the remaining cells were resuspended in 100 μl sterile 1 X PBS to a concentration of 5 x 106 cells per ml. Human HGF transgenic C3H-SCID mice (hu-HGF-Tg-SCID) were used to mimic paracrine human HGF stimulation (Zhang YW et al., £111^11〇6§1*〇\^1;11〇£ &gt;1111111&amp;11&lt;:-11161;-expressing xenografts in a new strain of immunocompromised mice transgenic for human hepatocyte growth factor/scatter factor. Oncogene 24:101-6, 2005). Since mouse HGF is a poor ligand for human HGF, these mice are produced, and thus the growth of tumor cell lines that respond to HGF can be enhanced in vivo. Because NCI-H596 cells cannot form tumors in normal nude or SCID mice, but can be in hu-HGF- 143939.doc -160- 201019961

Tumors are formed in Tg-SCID mice, so this model represents a tumor model driven by HGF paracrine. After shaving the right side of the animals, NCI-H596 cells were inoculated subcutaneously in the lower right scapular region with 0.5 x 106 NCI-H596 cells. The tumors were monitored until their average volume reached 100 mm3.

Mice were randomized into 9 groups of 10 mice each and treatment was started (summarized in Table 6). Mice in group 1 (untreated group) contained tumor-bearing mice, and although the mice were not treated, tumors were monitored in parallel with the treated group. Mice in group 2 (control group) were treated with anti-porcine control antibody (5 mg/kg, IP twice a week for 12 weeks) plus MCT (100 μιη, PO, QD, x84 days) and MetMAb buffer ( 100 μΐ^, IP, once every three weeks χ 4) treatment. Group 3 mice were treated with control antibody (5 mg/kg, IP, twice a week for 12 weeks) plus MCT (100 μιη, PO, QD, x84 days) and MetMAb (30 mg/kg, IP, every three) Once a week X4) processing. Group 4 mice were treated with anti-VEGF antibody (B20-4.1, 5 mg/kg, IP, twice a week for 12 weeks) plus MCT (100 pL, PO, QD, x84 days) and MetMAb buffer (100 pL) , IP, once every three weeks χ 4) processing. Group 5 mice were treated with anti-porcine control antibody (5 mg/kg, IP twice a week for 12 weeks) plus erlotinib (100 mg/kg, PO, QD, x84 days) and MetMAb buffer (100 pL, IP, x4 every three weeks). Group 6 mice were treated with anti-VEGF antibody (B20-4.1, 5 mg/kg, IP, twice a week for 12 weeks) plus MCT (100 μί, PO, QD, x84 days) and MetMAb (30 mg/kg) , IP, once every three weeks χ 4) processing. Group 7 mice were treated with ragweed control antibody (5 mg/kg, IP twice a week for 12 weeks) plus erlotinib (100 mg/kg, PO, QD, x84 days) and MetMAb (30 Mg/kg, IP, once every three weeks χ 4) treatment. Group 8 mice were treated with anti-VEGF 143939.doc •161- 201019961 antibody (B20-4.1, 5 mg/kg, IP, twice a week for 12 weeks) plus erlotinib (100 mg/kg, PO, QD, χ84 days) and MetMAb buffer (100 μΐ^ 'IP, x4 every three weeks). Group 9 mice were treated with anti-VEGF antibody (B20-4.1, 5 mg/kg, IP, twice a week for 12 weeks) plus erlotinib (100 mg/kg, PO, QD, χ84 days) and MetMAb (30 mg/kg, IP, x4 every three weeks). Table 6 Number of combinations of MetMAb, erlotinib and anti-VEGF antibodies in NCI-H596 NSCLC xenograft tumor models grown in hu-HGF-Tg-C3H-SCID mice / test material dose concentration dose volume group sex route dose Frequency dose (mg/kg) (mg/ml) (μΐ) 1 10/F untreated - - _ - - 2 10/F anti-porcino antibody; IP; twice a week χ 12 5 (anti-porcine) 1· 25 (anti-porcelain) 100 MCT; MetMAb PO; week; QDx84 buffer IP days; Q3Wx4 3 10/F anti-porcino antibody; IP; twice weekly xl2 5 (anti-porcelain); 1.25 (anti-porcelain) 100 MCT; MetMAb PO; IP week; QDx84 days; Q3Wx4 30 (MetMAb) 7.5 (MetMAb) 4 10/F anti-VEGF antibody IP; twice weekly xl2 5 (anti-VEGF) 1.25 (anti-VEGF) 100 (B20- 4.1); PO; week; QDx84 MCT; MetMAb IP day; Q3Wx4 buffer 5 10/F anti-porcino antibody; Ai IP; twice weekly xl2 5 (anti-porcelain); 1.25 (anti-porcelain); TIN; PO; week; QDx84 100 (erlotinib) 25 (erlotinib) MetMAb buffer IP days; Q3Wx4 6 10/F anti-VEGF antibody IP; Twice X12 5 (anti-VEGF); 1.25 (anti-100 (B20-4.1); PO; week; QDx84 30 (MetMAb) VEGF); 7.5 MCT; MetMAb IP day; Q3Wx4 (MetMAb) 7 10/F anti-porcine antibody埃IP; twice a week xl2 5 (anti-porcelain); 1.25 (anti-porcelain); 100 rotinib; PO; week; QDx84 1〇〇 (erlotidine 25 (erlotinib); MetMAb IP Day; Q3Wx4 Ni;); 30 (MetMAb) 7.5 (MetMAb) 8 10/F anti-VEGF antibody IP; every two times xl2 5 (anti-VEGF); 1.25 (anti-100 (B20-4.1); Ero PO; ; QDx84 1 〇〇 (erlotinib) VEGF); 25 (Etinib; MetMAb IP days; Q3Wx4 rotinib) Buffer 9 10/F anti-VEGF antibody IP; twice weekly X12 5 (anti-VEGF) 1.25 (anti-100 (B2 (Ml); Ero PO; week; QDx84 1 〇〇 (Errot VEGF); 25 (Etinib; AetMAb IP days; Q3Wx4 Ni); 30 rotinib); 7.5 ( MetMAb) (MetMAb) -162- 143939.doc 201019961 For all studies reported here, if the tumor reached 2000 mm3 or more or the tumor showed signs of necrotic lesions, the study of the animals was cancelled. If more than 50% of the animals in any given group have been canceled, the treatment of the group is stopped and the study of all animals is cancelled. All studies and treatments in mice follow the guidelines of the Institutional Animal Care and Use Committee (IACUC).廑痨和浚# f琢··Measure the two dimensions (length and width) of the tumor volume using the UltraCal-IV caliper (model 54-10-111, Fred V· Fowler Company, Inc.; Newton, ΜΑ) ). Tumor volume was calculated by Excel vl 1.2 (Microsoft Corporation; Redmond, WA) using the following formula: Tumor volume (mm3) = (length x width 2) x 0.5 must be f-segmentation · use KaleidaGraph 3.6 (Synergy Software; Reading , PA) for tumor inhibition curve. The percentage of growth inhibition (%Inh) was calculated as follows: %Ihn=100x (l-[tumor size (treated)/tumor size (vehicle))) The incidence of tumors was the number of tumors that could be measured in each group at the end of the study. to make sure. Partial degeneration (PR) is defined as tumor regression of more than 50% but less than 100% of the initial tumor volume on any day during the study. Complete degeneration (CR) is defined as the tumor regression of 1% of the initial starting tumor volume on any day during the study period. JMP software version 5.1.2 (SAS Institute; Cary, NC) was used to calculate the mean tumor volume and the standard error of the mean (SEM). Data analysis was also performed using JMP software version 5.1.2 and p values were generated using Student's t-test or Dunnett's test. 143939.doc • 163· 201019961 Results using the cell line depicted in Figure 1, by treatment with hgf (hu-HGF-Tg-SCID) in vitro or in vivo and by inhibition of c-met (using anti-c-met antibody MetMAb or shRNA for c-met) to study c-met function. These cell lines are representative models of ligand-induced (NCI-H596 and KP4) or non-ligand-dependent (NCI-H441 and EBC-1) c-met activities, selected based on the c-met activation pattern and included Paracrine (NSCLC cell line NCI-H596), autocrine (pancreatic cell line KP4), c-met overexpression (NSCLC cell line NCI-H441) and locally amplified and overexpressed c-met type (NSCLC cells) Strain EBC-1). The relative performance of the c-met protein is shown in Figure 1B. These results indicate that the ligand-reactive cell lines KP4 and NCI H596 exhibit a much lower level of c-met than non-ligand-dependently activated cell lines. The EBC-1 cell line exhibited the highest level of c-met. KP4 and NCI-H596 are ligand-reactive (Fig. 1C), and this reaction is inhibited by treatment with MetMAb. KP4 is an autocrine cell line and shows a decrease in p-c-met after treatment with MetMAb in the absence of other HGF. A stable cell line expressing dasicyclin-inducible shRNA to c-met mRN A was generated for NCI-H441 (Fig. 1D) and EBC-1 (Fig. 1E). Although 4, 5 or 7 days after treatment with doxycycline, &gt;1 (^1-11441-311^461 cell lines 3.1 and 3.11 showed a significant decrease in 〇-11161; and ?-〇11^1, but in No change in phospho-c-met expression was observed in the control NCI-H441-shGFP1 cell line (Fig. 1D). EBC-l-shMet-3.5 and 4.12 cell lines were treated 1 or 2 days after doxycycline treatment. A complete blockade of c-met performance was shown, and within this same time frame, EBC-l-shMet-4.5 only showed partial blockade of c-met performance (Fig. 1E). Treatment of control EBC-1 with doxycycline - 143939.doc 201019961 shGFP2 has no effect on c-met performance. To study the effect of activating c-met or disrupting active c-met signaling in NSCLC cell lines. First, treatment of ligand-dependent cell lines (such as NCI-) with HGF. H596 and HOP92) were expressed for 6 hours and analyzed by microarray analysis. Treatment of ligand-dependent cell lines with HGF caused several angiogenic factors: (including interleukin 8 (IL8), vascular endothelial growth factor A ( The gene expression of VEGFA), EPH receptor A2 (EphA2), angiopoietin-like protein 4 (Angptl4) and Ephrin B2 (EFNB2) was significantly upregulated (Fig. 2A). Secondly, ® was more Treatment of non-ligand-dependent NSCLC cell lines EBC-l-shMet-3.15 and EBC-l-shMet-4.12 in the presence or absence of cyclin to reduce c-met content, thereby assessing visually active c-met signaling Gene expression. Several angiogenic factors (including interleukin 8 (IL8), vascular endothelial growth factor A (VEGFA), EPH receptor A2 (EphA2), angiopoietin-like protein 4 (Angptl4) and Ephrin B2 were observed. The decrease in performance (EFNB2)) indicates that in non-ligand-dependent EBC-1 cells, the expression of these factors is dependent on c-metxin transmission (Fig. 2B). These results indicate activation of c-met It activates a group of angiogenic genes. c-met activation can indirectly promote angiogenesis by positively regulating angiogenic molecules. Because of c-met activation in ligand-dependent and non-ligand-dependent NSCLC cell lines. Both of them caused up-regulation of VEGF and IL8, so the expression levels of VEGF and IL8 protein were studied in cell culture and xenograft tumors. EBC-l-shMet-3.15 and EBC-l-shMet-4_12 cell lines were treated with doxycycline. Inducing c-met knockdown, resulting in secreted VEG at 1 and 2 days after treatment The F protein content was significantly reduced, while the control 143939.doc-165-201019961 EBC-l-shGFP2 cell line treated with doxycycline had no effect on VEGF content (Fig. 3A). A similar effect was observed in IL8 protein content in xenograft tumors formed from EBC-l-shMet-3.15, EBC-l-shMet-4.12 and EBC-l-shGFP2 cell lines (Fig. 3D). Treatment of the ligand-dependent cell line NCI-H596 with HGF also caused a significant decrease in protein content of VEGF (Fig. 3B) and IL8 (Fig. 3E). In addition, in culture, c-met knockout caused NCI-H441-shMet-3.ll and NCI-H441-shMet-3.1 cell lines treated with doxycycline (but no NCI-H441-shGFP1 cell line) The VEGF protein content was significantly reduced, however this effect was only apparent after 4 days of treatment with doxycycline (Fig. 3C). Since HGF treatment does not increase VEGF or IL8 levels in adeno-gland (KP4) cells, this effect may be unique to NSCLC cell lines. These results indicate that VEGF and IL8 are common results of Met activation in NSCLC cell lines and that c-met inhibition in NSCLC cell lines reduces the expression of VEGF and IL8. To test whether the combination of c-met and VEGF inhibitor produced better anti-tumor activity, the HGF/Met autocrine KP4 pancreatic tumor model was used to evaluate the treatment of the c_ met antagonist antibody MetMAb and the anti-VEGF antibody. The dose of MetMAb was selected based on the dose range study, which was based on administration once every three weeks, defining a therapeutic dose of 2.5 mg/kg and a minimum effective dose (MED) of 75 mg/kg (corresponding to a 50% effective dose (ΕΕ&gt ; 5 〇)), and 3 〇 mg / kg is the best effective dose. Anti-VEGF antibody (B20-4.1) binding to mouse and human VEGF was used at the recommended dose and time course (5 mg/kg twice a week), and administration of anti-VEGF antibody alone partially inhibited κρ4 xenograft tumor growth (about 22%) (Figure 4). When administered alone, when administered at 3〇mg/kg, it showed good tumor suppression (about 65%); when it was 7.5939.doc -166-201019961 with 7·5 111§/1^, partial inhibition Tumor growth (about 24%); and when administered at 2.5 mg/kg, did not inhibit tumor growth.

The efficacy of the combination of MetMAb and anti-VEGF antibody exceeded the additive efficacy (about 81% and about 83%, respectively, for the sub-therapeutic dose of MetMAb or MED MetMAb) (Figure 4). An increase in the number of partial (PR) and complete (CR) reactions was observed in the combination group compared to the group treated with MetMAb or anti-VEGF alone. Specifically, the anti-VEGF antibody plus a therapeutic dose of MetMAb (2.5 mg/kg) produced 3 PRs and 1 CR, whereas each agent alone did not have PR or CR. Anti-VEGF antibody plus 7.5 mg/kg of MetMAb produced 5 PR and 2 CR, while 7.5 mg/kg of MetMAb alone produced 5 PR and 1 CR. These results indicate that the sub-therapeutic dose or the lowest effective dose of MetMAb enhances the anti-tumor effect of the anti-VEGF antibody, resulting in tumor suppression over treatment alone. In human pancreatic tumor models, VEGF and c-met inhibitors significantly improve each other's activity. The effect of the combination of c-met and VEGF inhibitors in non-ligand (HGF)-dependent cell lines was further investigated using the NCI-H441-shMet-3.ll NSCLC xenograft model. The mice were given doxycycline via drinking water, thereby blocking c-met performance in vivo. Dox treatment induces shRNA expression against c-met, thereby reducing the performance of c-met. Treatment with Dox alone partially inhibited tumor growth (Figure 5). Treatment of animals with anti-VEGF antibodies alone also partially inhibited NCI-H441-shMet-3.ll tumor growth (Fig. 5), indicating that the growth and survival of these tumors depend on VEGF. The combination of c-met knockdown and VEGF antibody treatment significantly improved anti-tumor activity, resulting in tumor stagnation (Figure 5). An increase in the number of PR and CR was observed in the combination treatment group compared to the group treated with Dox or anti-VEGF alone at 143939.doc-167. 201019961. With Zhao Yanzhi, it was observed that the Dox plus anti-VEGF antibody treatment group had 1 PR and 2 CR, while the control treatment group, D〇x treatment group or anti-VEGF antibody treatment group had no PR and had 1 CR. These results indicate that VEGF and c-met inhibitors significantly improve each other's activity in a human NSCLC tumor model. To further investigate the effect of c-met knockdown on tumor growth, EBC-1-shMet NSCLC cell lines were generated (Fig. 1). Some pure lines are completely knocked out of c-met like EBC-1-shMet-3.15, while other pure lines are only partially knocked out of c-met like EBC-l-shMet-4.5 (Fig. 1; see also co-owned U.S. Patent Publication No. 2009/0226443, filed. When these cell lines were grown as xenograft tumors in nude mice, treatment of mice with doxycycline (via drinking water) caused tumor suppression reflecting c-met knockout levels, in EBC-l-shMet- 3.15 Complete knockout of c-met in the pure line caused tumor regression, whereas partial knockdown of c-met in the EBC-lshMet-4.5 pure line caused a delay in tumor growth (Fig. 6A). These results indicate that tumor growth and survival of EBC-1 cell lines are highly dependent on c-met performance. EBC-l-shMet-4.5 pure is the ideal model for studying the activity of other therapeutic agents in combination with shRNA-mediated c-met knockout. Tumor angiogenesis can be affected by VEGF provided by the tumor or by the host in the case of tumor xenografts. The EBC-1-shMet cell line expresses VEGF in a c-met-dependent manner (Figures 2 and 3). To determine whether c-met-dependent VEGF production is required for tumor progression, anti-VEGF antibodies (bevacizumab, AVASTIN®) that bind to human VEGF alone or bind human and mouse VEGF to 143939.doc -168- 201019961 VEGF antibody (B20-4.1) was treated with EBC-l-shMet-3.15 tumor mice. Inhibition of VEGF by human anti-VEGF against AVASTIN® (Genentech, Inc., South San Francisco, CA) and human and mouse cross-reactive antibody B20-4.1 in EBC-l-shMet-3.15 NSCLC xenografts Equivalent, indicating that functional VEGF is tumor cell-derived in the EBC-1 model (Fig. 6B). Furthermore, the mechanism of 'c-met inhibitory activity is not limited to the induction of VEGF expression by c-met, as knocking out c-met in the EBC-l-shMet-3.15 xenograft tumor model causes tumor regression (Fig. 6A). Anti-VEGF antibody treatment caused tumor stagnation (Fig. 6B). The effect of c-met knockdown combined with treatment with erlotinib and anti-VEGF antibody was tested and compared to treatment with a double combination and treatment with each agent alone. Previous studies have shown that. &quot;&quot;&quot;&quot;&quot;&quot;&quot;&quot;&quot;&quot;&quot;&quot;&lt;RTIID=0.0&gt;&gt;&gt; The combination of -met knockout, erlotinib and anti-VEGF antibodies produced an immediate and sustained tumor response (-92%); Figure 7) 'Maximum efficacy in any of the protocols tested in this experiment. The combination of either c-met knockout with anti-VEGF antibody treatment or erlotinib treatment also resulted in a significant increase in anti-tumor activity (_77% and -82%, respectively; Figure 7). Appropriate controls (sucrose, MCT and anti-porcine antibody; open circles) treated mice with EBC-l-shMet-4.5 tumors did not produce significant inhibition. Similarly, erlotinib alone showed minimal effect on tumor growth (positive Triangle) (_9〇/〇; Figure 7). Consistent with the studies shown above, c_met knockout (closed squares) and 143939.doc -169-201019961 treatment with anti-VEGF antibodies (inverted triangles) can cause delayed tumor growth or Stagnant (-59% and -70%, respectively; Figure 7). In contrast, 'erlotinib The combination with the anti-VEGF antibody showed no improvement compared to the efficacy observed with the anti-VEGF antibody alone (-61%; Figure 7). The combination of c-met knockdown and anti-VEGF antibody treatment also caused partial reaction (PR) and Increase in the number of complete responses (CR): combination treatment yielded 4 PRs and 1 CR, whereas c-met knockout alone produced 2 PRs and no CR, and treatment with anti-VEGF antibodies alone yielded 1 PR with no CR 0 The erlotinib plus c-met knockdown and anti-VEGF antibody protocol increased the number of reactions to 7 PR. The effect of treatment with anti-c-met antibody MetMAb, erlotinib and anti-VEGF antibody was tested and used separately Drug treatment or comparison with two-two combination treatment. NSCLC NCI-H596 xenografts were produced in hu-HGF-Tg-SCID mice, and MetMAb, anti-VEGF antibody (B20-4·1) and/or Ero were used. Treatment with nyani.

The combination of MetMAb, erlotinib and anti-VEGF antibodies produced the most immediate and significant tumor growth inhibition observed (about 95% at day 25; Figure 9). Immediate tumor volume regression was observed in this group, corresponding to 9 PRs and 1 CR in this group. In comparison, the MetMAb plus anti-VEGF antibody treated group had 2 PRs and no CR. No tumor growth inhibition was observed in control-treated mice (MetMAb buffer, MCT, and anti-porcine antibody) compared to untreated mice (Fig. 8). Consistent with previous studies, erlotinib (1 〇〇 mg/kg, administered daily throughout the course) had no effect on NCI-H596 tumor growth, whereas MetMAb (30 mg/kg once every 3 weeks) caused partial tumors Inhibition (approximately 59% at day 25) 143939.doc • 170-201019961 and the combination of erlotinib and MetMAb produced an effect that was stronger than the additive effect (about 89% at day 25; Figure 8). Anti-VEGF antibody (B20-4.1 '5 mg/kg twice weekly; inverted triangle) causes significant tumor growth inhibition when used alone (about 67% at day 25), however when MetMAb is added 'There is a significant additive effect (about 90% in Daysday; Figure 8) » This effect is unique for c-met inhibition, because when the anti-VEGF antibody is combined with erlotinib, no superposition effect is observed (in the first About 71% at 25 days; Figure 8). At the subsequent time point, the difference between the response curve of the MetMAb plus erlotinib combination and the response curve of the MetMAb plus anti-VEGF antibody increased, indicating that C-met and VEGF signaling were used when used in a two-two combination. Inhibition provides the most significant and long lasting anti-tumor 7 tongue. These data indicate that the combination of a c-met antagonist, a VEGF antagonist and an EGFR antagonist, and a combination of a c-met antagonist and a VEGF antagonist each significantly inhibited tumor growth in non-small cell lung cancer. The above description of the invention has been described in detail by way of illustration and example, and the description and examples should not be construed as limiting the scope of the invention. [Simplified illustration] Figure 1: Summary of cell lines selected for xenograft studies ° Cell lines are selected based on c-met activation pattern and include paracrine (NSCLC^® cell line NCH-H596), autocrine (pancreatic cell line KP4), c_met overexpression (NSCLC cell line NCI-H441), and locally amplified and overexpressed C-met type (NSCLC cell line EBC-1). (BE) Western ink dot method 'Shows as follows 143939.doc -171 - 201019961 shows acid-based-c-met and c-met protein content: (B) 4 cell lines under basic conditions, (C) KP4 and H596 cells were treated with HGF (100 ng/ml) and/or monovalent anti-c-met monoclonal antibody MetMAb (200 nM), and (D) showed Dox-inducible shRNA against c-met (shMet 3-1 and shMet) 3-11) or Dox-inducible shRNA (shGFP 1) for NCI-H441 cells, stable pure after Dox treatment, and (E) dox-induced shRNA for c-met (shMet 3-15) The stable pure EBC-1 cells of shMet 4-12, shMet 4-5) or dox-inducible shRNA (shGFP2) against GFP were treated with Dox. Dox-treated EBC-1 shMet pure lines 3-15 and 4-12 completely blocked c-met performance, while EBC-1 shMet pure line 4-5 partially blocked c-met expression. Nd = untreated; Dox = doxycycline; NSCLC = non-small cell lung cancer; Figure 2: mRNA content of c-met regulated angiogenic factors. (A) c-met activity associated with angiogenic molecules after treatment of a ligand-dependent cell line with HGF. (B) Treatment with HGF at 24 and 48 hours after Dox treatment restored gene expression in cells containing EBC-I Met shRNA. HGF = hepatocyte growth factor, Dox = doxycycline, RMA = robust multi-chip average, IL8 = interleukin 8, VEGF A = vascular endothelial growth factor A, EPHA2 = EPH receptor A2, ANGPTL4 = angiopoietin-like protein 4, EFNB2 = ephrin B2; Figure 3: c-met regulated VEGF and IL8 protein content. (A, C) (A) EBC-1, (C) Regulation of VEGF protein content in NCI-H441 cells. (D) IL-8 protein content in EBC-1 tumors after knockdown of c-met by c-met shRNA against Dox inducible. (B, E) Treatment of NCI-H596 cells with HGF 143939.doc -172- 201019961, (B) Regulation of VEGF and (E) IL8 protein content. Dox = doxycycline, VEGF = vascular endothelial growth factor; Figure 4: Treatment with anti-c-met antibody MetMAb enhances the efficacy of anti-VEGF antibodies in pancreatic tumors. Nude mice bearing subcutaneous KP4 pancreatic xenograft tumors were treated as indicated. Control treated animals showed no response to treatment. MetMAb was administered at the optimal dose (30 mg/kg, IP, once), the lowest effective dose (7.5 mg/kg, 10, once) and the sub-therapeutic dose (2.5 mg/kg, once). Human and mouse VEGF anti-VEGF antibody (B20-4.1) was administered at the optimal dose (5 mg/kg, IP twice a week), or with MetMAb (2.5 mg/kg once) or the lowest effective dose MetMAb (7.5 kg/kg, once) was administered in combination. A graph of the mean tumor volume for each group (n=10). Tumor suppression was calculated at day 25 (TI = 100x (l-(TV*341/TC* ***))). For the purposes of this experiment, PR and CR were each determined to reduce tumor volume by more than 50% or tumor volume by 100%. *PS〇.05; PR=partial response, CR=complete response, VEGF=vascular endothelial growth factor, IP=intraperitoneal; Figure 5: c-met inhibition in NCI-H441-shMet-3.ll NSCLC transmembrane It will enhance the anti-tumor effect of anti-VEGF antibodies. Nude mice with subcutaneous NCI-H441 shMet 3-11 NSCLC xenograft tumors were treated as follows: treatment with c-met shRNA in vivo using treatment with Dox alone (1 mg/ml in 5% sucrose in drinking water) Treatment with a control (5% sucrose in drinking water); or Dox treatment in combination with anti-VEGF (B20-4.1) antibody treatment. A graph of the mean tumor volume of each group (n=10). Dox = doxycycline, NSCLC = non-small cell lung cancer, VEGF = vascular endothelial growth factor, Ab = antibody; 143939.doc -173· 201019961 Figure 6: Characterization of c-met and VEGF inhibition in the EBC-1-shMet model . Nude mice bearing subcutaneous EBC-l-shMet-3.15 or EBC-l-shMet-4.5 NSCLC tumors were treated as indicated. (A) Mice with EBC-l-shMet-3.15 and EBC-1-shMet-4.5 tumors were treated with Dox (1 mg/ml in 5% sucrose in drinking water) or sucrose (in drinking water) 5% sucrose) treatment. (B) Animals with EBC-1-shmet-3.15 tumors were treated with control antibody (hu5B6), anti-human VEGF antibody (bevacizumab) or anti-human/mouse VEGF antibody (B20-4.1) (both are 5 mg/kg, IP, twice a week). A graph of the mean tumor volume for each group (n=10). IP = intraperitoneal, Dox = doxycycline, NSCLC = non-small cell lung cancer, VEGF = vascular endothelial growth factor; Figure 7: c-met, EGFR and VEGF in the EBC-l-shMet-4.5 NSCLC xenograft model The combination of inhibition significantly inhibits tumor growth. Nude mice bearing subcutaneous EBC-l-shMet-4.5 NSCLC tumors were treated with sucrose (5% sucrose in drinking water) or Dox (0.5 mg/ml in 5% sucrose in drinking water) to induce The performance of c-met shRNA. Erlotinib (100 mg/kg, PO, daily) and/or anti-VEGF antibody (B20-4.1.1, 5 mg/kg, IP, twice a week) are either alone or in combination (Dox+B20 -4.1.1; Dox+ erlotinib; B20-4.1.1+ erlotinib) or three combinations (Dox+B20-4.1 + erlotinib) form the administration. A graph of the mean tumor volume of each group (n = 10) was made. Tumor inhibition was calculated on day 27 (ΤΙ=100χ(1-(ΤνΑ817Τ(:*^))). For the purposes of this experiment, PR and CR were determined to reduce tumor volume by more than 50% or tumor volume by 100, respectively. %.PR=Partial response 'CR=complete response, KD=knockout, Dox=doxycycline, EGFR=EGFR, NSCLC=non-small cell lung cancer, VEGF=vascular endothelial growth factor' 143939.doc • 174·201019961 IP=intraperitoneal, PO=oral; FIG. 8: In the NCI-H596 NSCLC animal model, treatment with a combination of anti-c-met antibody MetMAb, erlotinib and anti-VEGF antibody significantly inhibited tumor growth. (A) Human HGF-transgenic gene SCID (hu-' HGF-Tg-SCID) mice with subcutaneous tumors were untreated, treated with control, anti-c- : met antibody MetMAb (30 mg/kg) , IP, every three weeks), EGFR inhibitor erlotinib (100 mg/kg, PO, daily) or anti-VEGF antibody (B20-4.1, 5 mg/kg, IP, twice a week) Or treated by its two-in-one combination (MetMAb+B20-4.1; MetMAb+ erlotinib; B20-4.1 + erlotinib) or in combination of three (B) Me The anti-tumor effect of tMAb + erlotinib, MetMAb + anti-VEGF antibody, and MetMAb + erlotinib + anti-VEGF antibody group highlights the magnitude of the difference. The graph of the mean tumor volume of each group (n = 10) Tumor suppression was calculated on day 25 (TI = 100x (l-(TV*3te/TC#s**))). PR and CR were determined to reduce tumor volume by more than 50% or tumor volume by 100%, respectively. PR = partial response, CR = complete response, EGFR = epidermal growth factor receptor, NSCLC = 9 non-small cell lung cancer, VEGF = vascular endothelial growth factor, IP = intraperitoneal, PO = oral 〇 * P &lt; 0.02, ** P = 0.005; • Figure 9: depicts the amino acid sequence of the framework (FR), CDRs, first constant domain (CL or CH1) and Fc region (Fc) of MetMAb (OA5D5v2). The depicted Fc sequence comprises as WO "臼" (hole) mutations T366S, L368A and Y407V as described in 2005/063816; and Figure 10: depicts the sequence of an Fc polypeptide comprising a "杵" (prominent) mutation as described in WO 2005/063816 T366W. In an embodiment 'comprising 143939.doc • 175. 201019961 Fc polypeptide of this sequence forms complex with Fc polypeptide comprising the Fc sequence of Figure 9 Thereof, thereby generating an Fc region. 143939.doc 176- 201019961 Sequence Listing &lt;110&gt;US-based Nandek Corporation &lt;120&gt; Combination Therapy&lt;130&gt;P4277R1 &lt;140&gt;098134878 &lt;141&gt;2009-10-14 &lt;150&gt;US 61 /106,513 &lt;151&gt; 2008-10-17 &lt;160&gt;33 &lt;210&gt;1 &lt;211&gt; 17 &lt;212&gt;PRT &lt;213&gt;Artificial sequence&lt;220&gt;&lt;223&gt; Synthesis sequence &lt;400&gt;;1

Lys Ser Ser Gin Ser Leu Leu Tyr Thr Ser Ser Gin Lys Asn Tyr 1 5 10 15

Leu Ala &lt;210&gt;2 &lt;211&gt;7 &lt;212&gt; PRT &lt;213&gt; artificial sequence &lt;220&gt;&lt;223&gt; synthetic sequence &lt;400&gt;2

Trp Ala Ser Thr Arg Glu Ser &lt;210&gt;3 &lt;211&gt;9 &lt;212&gt;PRT &lt;213&gt;Artificial sequence &lt;220&gt;&lt;223&gt; Synthesis sequence &lt;400&gt;3

Gin Gin Tyr Tyr Ala Tyr Pro Trp Thr 5 &lt;210&gt;4 &lt;211&gt;10 &lt;212&gt; PRT &lt;213&gt;Artificial sequence &lt;220&gt;&lt;223&gt; Synthesis sequence &lt;40Q&gt;4

Gly Tyr Thr Phe Thr Ser Tyr Trp Leu His 5 10 &lt;210&gt;5 &lt;211&gt; 18 &lt;212&gt;PRT &lt;213&gt; Artificial sequence &lt;220&gt;&lt;223&gt; Synthesis sequence &lt;400&gt;

Gly Met lie Asp Pro Ser Asn Ser Asp Thr Arg Phe Asn Pro Asn 143939.doc 201019961 1 5 10 15

Phe Lys Asp &lt;210&gt;6 &lt;211&gt;11 &lt;212&gt; PRT &lt;213&gt; Synthetic sequence &lt;220&gt;&lt;223&gt; X is any amino acid other than R &lt;400&gt;

Xaa Tyr Gly Ser Tyr Val Ser Pro Leu Asp Tyr 5 10 &lt;210&gt;7 &lt;211&gt;11 &lt;212&gt; PRT &lt;213&gt;Artificial Sequence &lt;220&gt;&lt;223&gt; Synthesis Sequence &lt;400&gt;

Thr Tyr Gly Ser Tyr Val Ser Pro Leu Asp Tyr 5 10 &lt;210&gt;8 &lt;211&gt;11 &lt;212&gt; PRT &lt;213&gt;Artificial Sequence &lt;220&gt;&lt;223&gt; Synthesis Sequence &lt;400&gt;8

Ser Tyr Gly Ser Tyr Val Ser Pro Leu Asp Tyr 5 10 &lt;210&gt;9 &lt;211&gt; 12 &lt;212&gt;PRT &lt;213&gt;Artificial Sequence &lt;220&gt;&lt;223&gt; Synthesis Sequence &lt;400&gt;9

Ala Thr Tyr Arg Ser Tyr Val Thr Pro Leu Asp Tyr 5 10 &lt;210&gt; 10 &lt;211&gt;119 &lt;212&gt; PRT &lt;213&gt;Artificial Sequence&lt;220&gt;&lt;223&gt; Synthesis Sequence &lt;400&gt; GIu Val Gin Leu Val Glu Ser Gly Gly Gly Leu Val Gin Ρω Gly 1 5 10 15

Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr 20 25 30

Ser Tyr Trp Leu His Trp Val Axg Gin Ala Pro Gly Lys Gly Leu 35 40 45

Glu Trp Val Gly Met lie Asp Pro Ser Asn Ser Asp Thr Arg Phe 50 55 60

Asn Pro Asn Phe Lys Asp Arg Phe Thr lie Ser Ala Asp Thr Ser •2· 143939.doc 201019961 65 70 75

Lys Asn Thr Ala Tyr Leu Gin Met Asn Ser Leu Arg Ala Glu Asp 80 85 90

Thr AJa Val Tyr Tyr Cys Ala Thr Tyr Arg Ser Tyr Val Thr Pro 95 100 105

Leu Asp Tyr Trp Gly Gin Gly Thr Leu Val Thr Val Ser Ser 110 115 &lt;210&gt;11 &lt;211&gt;114

&lt;212&gt;PRT &lt;213&gt; Artificial sequence &lt;220&gt;&lt;223&gt; Synthesis sequence &lt;400&gt;

Asp He Gin Met Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val 1 5 10 15

Gly Asp Arg Val Thr lie Thr Cys Lys Ser Ser Gin Ser Leu Leu 20 25 30

Tyr Thr Ser Ser Gin Lys Asn Tyr Leu Ala Trp Tyr Gin Gin Lys 35 40 45

Pro Gly Lys Ala Pro Lys Leu Leu lie Tyr Trp Ala Ser Thr Arg 50 55 60

Glu Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr 65 70 75

Asp Phe Thr Leu Thr lie Ser Ser Leu Gin Pro Glu Asp Phe Ala 80 85 90

Thr Tyr Tyr Cys Gin Gin Tyr Tyr Ala Tyr Pro Trp Thr Phe Gly 95 100 105

Gin Gly Thr Lys Val Glu lie Lys Arg 110 &lt;210&gt; 12 &lt;211&gt;222 &lt;212&gt; PRT &lt;213&gt; Artificial Sequence &lt;220&gt;&lt;223&gt; Synthesis Sequence

&lt;400&gt;12 Cys Pro I

Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val - 10 15

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met lie Ser Arg 20 25 30

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 35 40 45

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 50 55 60

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gin Tyr Asn Ser Thr Tyr 65 70 75

Arg Val Val Ser Val Leu Thr Val Leu His Gin Asp Trp Leu Asn 80 85 90

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 95 100 105

Pro lie Glu Lys Thr lie Scr Lys Ala Lys Gly Gin Pro Arg Glu 143939.doc 115 201019961 110

Pro Gin Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys 125 130 135

Asn Gin Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser 140 145 150

Asp lie Ala Val Glu Trp Glu Ser Asn Gly Gin Pro Glu Asn Asn 155 160 165

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 170 175 180

Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gin Gin Gly 185 190 195

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 2(X) 205 210

Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro Gly Lys 215 220 &lt;210&gt; 13 &lt;211&gt;222 Artificial Sequence &lt;212&gt; PRT &lt;213&gt;&lt;220&gt;&lt;223&gt; Synthesis Sequence &lt;400&gt; Cys Pro 】

Pro Cys Pro Ak Pro Glu Leu Leu Gly Gly Pro Ser Val a 10 15

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met lie Ser Arg 20 25 30

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 35 40 45

Pro Glu Val Lys Phe Asn Tip Tyr Val Asp Gly Val Glu Val His 50 55 60

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gin Tyr Asn Ser Thr Tyr 65 70 75

Arg Val Val Ser Val Leu Thr Val Leu His Gin Asp Trp Leu Asn 80 85 90

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 95 100 105

Pro lie Glu Lys Thr lie Ser Lys Ala Lys Gly Gin Pro Arg Glu 110 115 120

Pro Gin Va3 Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys 125 130 135

Asn Gin Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser 140 145 150

Asp lie Ala Val Glu Trp Glu Ser Asn Gly Gin Pro Glu Asn Asn 155 160 165

Tyr Lys Thr Hir Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 170 175 180

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Tip Gin Gin Gly 185 190 195

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 200 205 210

Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro Gly Lys -4- 143939.doc 201019961 215 220 &lt;210&gt; 14 &lt;211&gt;449

&lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;&lt;223&gt; Synthesis sequence &lt;400&gt;14

Glu Val Gin Leu Val Glu Set Gly Gly Gly Leu Val Gin Pro Gly 1 5 10 15

Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr 20 25 30

Ser Tyr Trp Leu His Trp Val Arg Gin Ala Pro Gly Lys Gly Leu 35 40 45

Glu Trp Val Gly Met lie Asp Pro Ser Asn Ser Asp Thr Arg Phe 50 55 60

Asn Pro Asn Phe Lys Asp Arg Phe Thr lie Ser Ala Asp Thr Ser 65 70 75

Lys Asn Thr Ala Tyr Leu Gin Met Asn Ser Leu Arg Ala Glu Asp 80 85 90

Thr Ala Val Tyr Tyr Cys Ala Thr Tyr Arg Ser Tyt Val Thr Pro 95 100 105

Leu Asp Tyr Trp Gly Gin Gly Thr Leu Val Thr Val Ser Ser Ala 110 115 120

Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Set Lys 125 130 135

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp 140 145 150

Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu 155 160 165

Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gin Ser Ser Gly 170 175 180

Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu 185 190 195

Gly Thr Gin Thr Tyr lie Cys Asn Val Asn His Lys Pro Ser Asn 200 205 210

Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 215 220 225

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 230 235 240

Ser Val Phe Leu Pbe Pro Pro Lys Pro Lys Asp Thr Leu Met lie 245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gin Tyr Asn Ser 290 295 300

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gin Asp Trp 305 310 315

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 143939.doc 201019961 320 325 330

Pro Ala Pro lie Glu Lys Tb: lie Ser Lys Ala Lys Gly Gin Pro 335 340 345

Arg Glu Pro Gin Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met 350 355 360

Thr Lys Asn Gin Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr 365 370 375

Pro Ser Asp lie Ala Val Glu Trp Glu Ser Asn Gly Gin Pro Glu 380 385 390

Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 395 400 405

Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gin 410 415 420

Gin Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 425 430 435

Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro Gly Lys 440 445 &lt;210&gt;15 &lt;211&gt;220

&lt;212&gt;PRT &lt;213&gt; artificial sequence &lt;220&gt;&lt;223&gt; synthetic sequence &lt;400>15

Asp lie Gin Met Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val 1 5 10 15

Gly Asp Arg Val Thr lie Thr Cys Lys Ser Ser Gin Ser Leu Leu 20 25 30

Tyr Thr Ser Ser Gin Lys Asn Tyr Leu Ala Trp Tyr Gin Gin Lys 35 40 45

Pro Gly Lys Ala Pro Lys Leu Leu lie Tyr Trp Ala Ser Thr Arg 50 55 60

Glu Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr 65 70 75

Asp Phe Thr Leu Thr lie Ser Ser Leu Gin Pro Glu Asp Phe Ala 80 85 90

Thr Tyr Tyr Cys Gin Gin Tyr Tyr Ala Tyr Pro Trp Thr Phe Gly 95 100 105

Gin Gly Thr Lys Val Glu He Lys Arg Thr Val Ala Ala Pro Ser 110 115 120

Val Phe lie Phe Pro Pro Ser Asp Glu Gin Leu Lys Ser Gly Thr 125 130 135

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 140 145 150

Lys Val Gin Trp Lys Vai Asp Asn Ala Leu Gin Ser Gly Asn Ser 155 160 165

Gin Glu Ser Val Thr Glu Gin Asp Ser Lys Asp Scr Thr Tyr Ser 170 175 180

Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His 185 190 195

Lys Val Tyr Ala Cys Glu Val Thr His Gin Gly Leu Ser Ser Pro -6- 143939.doc 201019961 200 205 210

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 215 220 &lt;210&gt; 16 &lt;211&gt; 23 &lt;212&gt; PRT &lt;213&gt; artificial sequence &lt;220&gt;&lt;223&gt; synthetic sequence. &lt;400&gt;

Asp lie Gin Met Thr Gin Ser Pro Ser Ser Leu Ser Ala Ser Val 1 5 10 15

Gly Asp Arg Val Thr He Thr Cys 20 &lt;210&gt; 17 &lt;211&gt;15 &lt;212&gt; PRT &lt;213&gt; Artificial sequence

&lt;220&gt;&lt;223&gt;Synthesis sequence &lt;400&gt;17 Trp Tyr Gin Gin Lys

Pro Gly Lys Ala Pro Lys Leu Leu lie Tyr 10 15 &lt;210&gt; 18 &lt;211&gt;32 &lt;212&gt; PRT &lt;213&gt;Artificial sequence &lt;220&gt;&lt;223&gt;Synthesis sequence &lt;4O0&gt;

Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe 1 5 10 15

Thr Leu Thr He Ser Ser Leu Gin Pro Glu Asp Phe Ala Thr Tyr 20 25 30

Tyr Cys &lt;210&gt; 19 &lt;211&gt;11 &lt;212&gt; PRT &lt;213&gt; artificial sequence &lt;220&gt;&lt;223&gt; synthetic sequence &lt;400&gt;

Phe Gly Gin Gly Thr Lys Val Glu lie Lys Arg 5 10 &lt;210&gt; 20 &lt;211&gt; 106 &lt;212&gt;PRT &lt;213&gt;Artificial sequence &lt;220&gt;&lt;223&gt; Synthesis sequence &lt;400&gt;

Thr Val Ala Ala Pro Ser Va! Phe lie Pbe Pro Pro Ser Asp Glu 1 5 10 15

Gin Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn 143939.doc 201019961 20 25 30

Phe Tyr Pro Arg Glu Ala Lys Val Gin Trp Lys Val Asp Asn Ala 35 40 45

Leu Gin Ser Gly Asn Set Gin Glu Ser Val Thr Glu Gin Asp Ser 50 55 60

Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Tlir Leu Ser Lys 65 70 75

Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His 80 85 90

Gin Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu 95 100 105 &lt;210&gt;21 &lt;211&gt;25 &lt;212&gt;PRT &lt;213&gt;Artificial Sequence&lt;220&gt;&lt;223&gt;SynthesisSequence&lt;;400&gt;21

Glu Val Gin Leu Val Glu Ser Gly Gly Gly Leu Val Gin Pro Gly 1 5 10 15

Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser 20 25 &lt;210&gt; 22 &lt;211&gt; 13 &lt;212&gt;PRT &lt;213&gt;Artificial sequence &lt;220&gt;&lt;223&gt; Synthesis sequence &lt;400&gt;22

Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 5 10 &lt;210&gt; 23 &lt;211&gt;30 &lt;212&gt;PRT &lt;213&gt;Artificial Sequence&lt;220&gt;&lt;223&gt;SynthesisSequence&lt;400&gt; twenty three

Arg Phe Thr lie Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu 1 5 10 15

Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 20 25 30 &lt;210&gt;24 &lt;211&gt;11 &lt;212&gt;PRT &lt;213&gt;Artificial Sequence&lt;220&gt;&lt;223&gt;SynthesisSequence&lt;;400&gt;24

Trp Gly Gin Gly Thr Leu Val Thr Val Ser Ser 5 10 &lt;210&gt;25 143939.doc 201019961 &lt;211&gt;5 &lt;212&gt; PRT &lt;213&gt; Homo sapiens &lt;400&gt;25

Leu Asp Ala Gin Thr &lt;210&gt;26 &lt;211&gt;9 &lt;212&gt;PRT &lt;213&gt; Homo sapiens &lt;400&gt;26

Leu Thr Glu Lys Arg Lys Lys Arg Ser et MIGlu Λα Al ser T human asp5 278PR 智27ΓΟ 0&gt;1&gt;2&gt;3&gt;&lt;21&lt;21&lt;21&lt;21&lt;210&gt;28&lt;211&gt;8&lt;212&gt;PRT&lt;213&gt; Homo sapiens &lt;400&gt; 28

Asn Val Arg Cys Leu Gin His Phe &lt;210&gt;29 &lt;211&gt;65 &lt;212&gt; DNA &lt;213&gt;Artificial sequence&lt;220&gt;&lt;223&gt;Synthesissequence&lt;400&gt;29 gatccccgaa cagaatcact gacatattca agagatatgt cagtgattct 50 Gttctttttt ggaaa65 &lt;210&gt;30 &lt;211&gt;65 &lt;212&gt;DNA &lt;213&gt;Artificial sequence&lt;220&gt;&lt;223&gt;Synthesissequence&lt;400&gt; 30 gatccccgaa actgtatgct ggatgattca agagatcatc cagcatacag 50 tttctttttt ggaaa65 &lt;210&gt; 31 &lt;211&gt;67 &lt;212&gt; DNA &lt;213&gt; artificial sequence &lt;220&gt;&lt;223&gt; synthetic sequence &lt;400&gt;31 gatccccaga tccgccacaa catcgattca agagatcgat gttgtggcgg 50 atcttgtttt ttggaaa67 &lt;210&gt;32 -9- 143939. Doc 201019961 &lt;212&gt;PRT &lt;213&gt;Artificial sequence&lt;220&gt;&lt;223&gt;Synthesis sequence&lt;400&gt;32 Asp lie Cys Leu Pro Arg Trp Gly Cys Leu Trp 5 10 b&gt; Λ &gt; lx «1 lx ^3⁄4^ &lt; A1,

Glyuu n As 5 &gt; 10 &gt; iRl &gt; 33serserTyrThrLeuGlyTl: Hi serLysAlaserserserPLV sersuvalGlysersersSPTOsAS proLeserGlnserLVrcy AlacyslLeupronHissse uuGlyTrpvalvalASDOLVs ^ 5¾¾¾¾¾ ^ pheAlavalprovall I valAlaThrphevalcyssval sers ΐ sThrMserw sSLyloo proGlyproHisserTyrPLyM;:. Lar 143939.doc 10-

Claims (1)

201019961 VII. Scope of Application: 1. A use of a combination of a c-met antagonist and a VEGF antagonist for the manufacture of a medicament for treating cancer in an individual. 2. The use of claim 1, wherein the c-met antagonist is an antibody. 3. The use of claim 2, wherein the antibody is a monovalent antibody. 4. The use of claim 3, wherein the antibody is monovalent and comprises an Fc region, wherein the Fc region comprises a first polypeptide and a second polypeptide, wherein the first polypeptide comprises the SEQ ID NO: 12 The Fc sequence and the second polypeptide comprise the sequence set forth in SEQ ID NO: 13. 5. The use of claim 4, wherein the antibody comprises (a) a first polypeptide comprising a heavy chain variable domain having the sequence: EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWLHW VRQAPGKGLEWVGMIDPSNSDTRFNPNFKDRFTISADTS KNTAYLQMNSLRAEDTAVYYCATYRSYVTPLDYWGQGT LVTVSS (SEQ ID NO: 10), CH1 sequence and first Fc polypeptide; (b) a second polypeptide comprising a light chain variable domain having the sequence: • DIQMTQSPSSLSASVGDRVTITCKSSQSLLYTSSQKN YLAWYQQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTD FTLTISSLQPEDFATYYCQQYYAYPWTFGQGTKVEIKR (SEQ ID NO: 11) and CL1 sequence; and (c) including the second Fc polypeptide a tripeptide, wherein the heavy chain variable domain and the light chain variable domain are present in a complex form and form a single antigen binding arm, wherein the first Fc polypeptide and the second Fc polypeptide are present in a complex form and form an Fc The region, which increases the stability of the antibody fragment compared to the Fab molecule comprising the antigen binding arm, 143939.doc 201019961. 6. The use of claim 5, wherein the first polypeptide comprises the Fc sequence set forth in SEQ ID NO: 12, and the second polypeptide comprises the Fc sequence set forth in SEQ ID NO: 13. 7. The use of claim 5, wherein the first polypeptide comprises the Fc sequence set forth in SEQ ID NO: 13, and the second polypeptide comprises the Fc sequence set forth in SEQ ID NO: 12. 8. The use of claim 4, wherein the antibody is MetMAb. The use according to any one of claims 1 to 8, wherein the VEGF antagonist is an aptamer capable of specifically binding to VEGF. The use according to any one of claims 1 to 8, wherein the VEGF antagonist is a soluble VEGF receptor protein, or a VEGF-binding fragment thereof, or a chimeric VEGF receptor protein. 11. The use of claim 10, wherein the chimeric VEGF receptor protein comprises at least the extracellular domain 2 of Flt-Ι or KDR. 12. The use of any one of claims 1 to 8, wherein the VEGF antagonist is an anti-VEGF antibody. 13. The use of claim 12, wherein the anti-VEGF antibody is a monoclonal antibody. 14. The use of claim 13, wherein the monoclonal antibody is a chimeric antibody, a fully human antibody or a humanized antibody. 15. The use of claim 14, wherein the anti-VEGF antibody is bevacizumab, a G6 series antibody, a B20 series antibody or a VEGF binding fragment thereof. 16. The use of claim 15, wherein the anti-VEGF antibody is bevacizumab. The use of any one of claims 1 to 8, wherein the medicament further comprises or is used in combination with an EGFR antagonist. 18. The use of claim 17, wherein the eGFR antagonist has a general formula: m is 1, 2 or 3; each R1 is independently selected from the group consisting of hydrogen, halo, hydroxy, hydroxyamino, thiol, nitro, decyl, ureido, cyano, trifluoromethyl and (CVC4 alkylene)_w-(phenyl), wherein W is a single bond, hydrazine, s or NH; or each R1 is independently selected from R9 and cyano substituted 匚丨-匚4 alkyl, wherein R9 is The following components are selected: R5, -OR6, -NR6R6, -C(〇)R7, -NHOR5, -〇C(0)R6, cyano, A and ·YR5; R5 is CVC4 alkyl; R6 independently Is hydrogen or R5; R7 is R5, -〇R6 or _NR6R6; A is selected from N-β piperidino, N-?1# morpholino, N-pyrrolidino, 4 -R6-piperazin-1-yl, imidazol-1-yl, 4_bistidin-1-yl, -(CVC4alkylene) (C02H), phenoxy, phenyl, phenylthio, C2- C4 alkenyl and -(CVC4alkylene)c(o)nr6r6; and Y is S, SO or S〇2; wherein the alkyl moiety of R5, _〇6 and _NR6R6 is optionally 1 to 3 Substituted by a substituent, and the alkyl moiety in R5, -OR6 and -NR6R6 is optionally substituted with 1 or 2 R9 groups, and wherein the 143939.doc 201019961 The alkyl moiety is optionally substituted with a halo group or R9, with the proviso that the two heteroatoms are not attached to the same carbon atom; or each R is independently selected from _NHS〇2R5, phthalimid0- (ci-C4)-alkylsulfonylamino, benzammonium, benzenesulfonylamino, triphenylphenylurea, 2-tertoxy (fluorene) pipyridyl, 2,5-di-side oxypyrrolidinyl and Rn)-(C2_C4)-alkylalkylamino, wherein R10 is selected as a free radical, _OR6, C2_C4 alkyloxy, _c(〇)r7 and -nr6r6; And wherein the R1 groups _nhs〇2rS, phthalimido-(Ci-C4)alkylsulfonylamino, benzammonium, benzenesulfonylamino, 3-phenyl Glandyl, 2, oxypyrazine, 2,5-dioxyl, butyl-1-yl and R1G_(C2-C4)-calcium-based amines, 1 or 2 Independently selected from halo, C丨-C4 alkyl, cyano, methanesulfonyl and oxime. Substituent substitution of alkoxy; or two R1 groups together with the carbon to which they are attached form a 5-8 ring , the ring comprises 1 or 2 heteroatoms selected from the group consisting of hydrazine, s and N; R is hydrogen or C--C6 alkyl group - the C--C6 alkyl group is optionally 1 3 substituents independently selected from _ group, C!-C4 alkoxy, -NR6R6 and -S02R5; η is 1 or 2' and each R3 is independently selected from hydrogen, halo, thio, c丨_c6 alkyl, -NR6R6 and CVC4 alkoxy' wherein the alkyl portion of the R3 ring is optionally independently selected from halo, (^-(24 alkoxy, -NR6R6 and -) Substituent for S02R; and R4 is azido or -(ethynyl)-Rn, wherein R11 is hydrogen or Cl-C6 alkyl'. The C"C6 alkyl group is optionally substituted with hydroxy, -OR6 or -NR6R6. The use of claim 17, wherein the EGFR antagonist is a compound of formula I selected from the group consisting of: (6,7-dimethoxyquinazolin-4-yl)-(3) -ethynylphenyl)-amine; (6,7-dimethoxyquinazolin-4-ylhydroxypropyn-1-yl)phenyl]-amine; [3-(2'-(amino hydrazine) ()ethynyl)phenyl μ(6,7-dioxaoxyquinazoline-4_:yl)-amine; (3-ethynylphenyl)-(6-nitroquinazolin-4-yl) )-amine; (6,7-dimethoxy quinazolin-4-yl)-(4-ethynylphenyl)-amine; (6,7-dimethoxy quinazolin-4-yl) -(3-ethynyl-2-methylphenyl)-amine; (6-aminoquinazolyl-4-phenyl)-(3-ethynylphenyl)-amine; (3-ethynylphenyl) )-(6-nonanesulfonylaminoquinazolin-4-yl)-amine; (3-ethynylphenyl)-(6,7-methylenedioxyquinazolin-4-yl )-amine; (6,7-dimethoxyquinazolin-4-yl)-(3-ethynyl-6-methylphenyl)-amine; (3-ethynylphenyl)_(7_ Nitroquinazolin-4-yl)-amine; (3-ethynylphenyl)-[6-(4'-toluenesulfonylamino)quina. (3_ethynylphenyl)_{6_[2,_o-phenylenedimino]ethylidene-yl-ylideneamino]quinazoline_4_yl} Amine; (3-ethynylphenyl)_ φ (6. decyl quinazolin-4-yl)-amine; (7-aminoquinazolin-4-yl)-(3-ethynylphenyl) -amine; (3-ethynylphenyl)-(7-methoxyquinazolin-4-yl)-amine; (6-indoleoxycarbonyl quinazoline-4-yl)-(3-ethynylbenzene (7-曱. oxycarbonylquinoxalin-4-yl)-(3-ethynylphenyl)-amine; [6,7-bis(2-decyloxyethoxy) quinazoline 4-(4-ethynylphenyl)-amine; (3-azidophenyl)-(6,7-dimethoxyquinazolin-4-yl)-amine; -azido-5-gas phenyl hydrazine 6,7-dimethoxyquinazolin-4-yl)-amine; (4-azidophenyl)-(6,7-dimethoxyquinazoline (4-ethynylphenyl)-(6.methanesulfonic acid-oxime "sopolinyl"-amine; (6. ethanethio-quinazolin-4-yl )-(3- 143939.doc 201019961 ethynylphenyl)-amine; (6,7-dimethoxy-anthracene) sit--4-yl)-(3-ethyl-furyl-4-fluoro-phenyl )·amine; (6,7-dimethoxy-quinazoline _4_yl)-[ 3 ((propyne-fluorenyl)-phenyl]-amine; [6,7-bis-(2-methoxy-ethoxy)-quinazoline-4-yl]-(5-ethynyl) -2-methyl-phenyl)-amine·,[6,7-bis-(2-decyloxy-ethoxy)-quinazolin-4-yl]-(3-ethynyl-4-fluoro -phenyl)-amine; [6,7-bis-(2-chloro-ethoxy)quinazoline-4(yl)-(3-ethynyl-phenyl)-amine; [6-(2 - gas-ethoxy)-7-(2-decyloxy-ethoxy)-oxime-4-yl]-(3-ethylidyl-phenyl)-amine; [6,7-double -(2-ethoxycarbonyl.ethoxy)-quinazolin-4-yl M3-ethynyl-phenyl)-amine; 2-[4-(3-ethynyl-phenylamino)_7_ ( 2-hydroxy-ethoxy)-quinazoline-6-yloxy]·ethanol; [6_(2_acetoxy-ethoxy)-7-(2-decyloxy-ethoxy) - (7-(2-carboyl-phenyl)-amine; [7-(2-methoxy-ethoxy)-6-(2-methoxy-ethoxy)-quina Oxazolin-4-yl]-(3-ethynyl-phenyl)amine; [7-(2-acetoxy-ethoxy)-6-(2-methoxy-ethoxy)- Quinoline _4_yl]_(3_ethynyl-styl)_amine; 2-[4-(3-ethynyl-phenylamino)_6_(2_carbyl-ethoxy)-啥.琳琳-7-yloxy]-ethanol; 2-[4-(3-ethynyl-phenylamino)-7-(2-methoxy-ethoxy)-quinazolin-6-yloxy ]-ethanol; 2_[4-(3-ethynyl-phenylamino)-6-(2-methoxy-ethoxy)-quinazoline-7-yloxy]-ethanol; [6· (2-acetoxy-ethoxy)-7-(2-decyloxyethoxy)-quinazolin-4-yl]-(3-ethynyl-phenyl)-amine; (3 -ethynyl-phenyl)_6_(2-methoxy-ethoxy)-7-[2-(4-indolyl-n-indolyl-indoleyl)-ethoxy]quinoline_ 4-yl}-amine; (3-ethylfastyl-phenyl)-[7.(2.methoxy-ethoxy)_6_(2·hryn-4-yl)-ethoxy)-oxime (6,7-diethoxyanthracene-1-yl)-(3-ethynylphenyl)-amine; (6,7-dibutoxyanthracene坐琳_ι· 143939.doc -6- 201019961 base)-(3-ethynylphenyl)-amine; (6,7-diisopropoxy quinazoline-anthraceyl)_ (3-B Block phenyl)-amine, (6,7--ethoxyphenyl-1-yl)-(3-ethylidyl-2-methyl-phenyl)-amine; [6,7- Bis-(2-decyloxy-ethoxy)-quinazoline-1-yl]-(3-ethynyl-2-methyl-phenyl)-amine; (3-ethynylbenzene) (6)-[6-(2-hydroxy-ethoxy)-7-(2-methoxyethoxy)-quinazolin-1-yl]-amine; [6,7-bis-(2- Hydroxy-ethoxy)-quinazolin-1-yl]-(3-ethynylphenyl)-amine; 2-[4-(3-ethynyl-phenylamino)-6-(2-A Oxy-ethoxy)-quinazoline-7-yloxy]-ethanol; (6,7-dipropoxy-quinazoline-4-yl;)_(3-ethynyl-phenyl) -amine; (6,7-diethoxy-quinazolin-4-yl)-(3-ethynyl-5-fluoro-phenyl)-amine; (6,7-diethoxy-quinazoline Benzyl-4-yl)-(3-ethylidyl-4-fluoro-phenyl)-amine; (6,7-diethyllacyl-indenyl-4-yl)-(5-ethyl-based) 2-methyl-phenyl)-amine; (6,7-diethoxy-indolyl-4-yl)-(3-ethynyl-4-methyl-phenyl)-amine; (6- Aminomethyl-7-methoxy-quinazoline-4-yl)-(3-ethylidyl-phenyl)-amine; (6-aminomethyl-7-methoxy-carbazole (6-aminocarbonylindenyl-7-nonyloxy-quinazolin-4-yl)-(3-ethynylphenyl) -amine; (6-aminocarbonylethyl-7-methoxy-quinazolin-4-yl)-(3-ethynylphenyl)-amine; (6. Aminomethyl-7-- Ethoxy- Quinazolin-4-yl)-(3-ethynylphenyl)-amine; (6-aminoethylethyl-7-ethoxy-quino-azidin-4-yl)-(3-acetylene (6-aminocarbonylcarbonylindol-7-isopropoxy-quinazolin-4-yl)-(3•ethylphenylphenyl)-amine; (6-amino group Methyl-7-propoxy-oxime 1&gt;sitin_4-yl)-(3-ethynylphenyl)-amine; (6-aminocarbonylmethyl-7-methoxy-quinazoline _ 4-yl)-(3-ethynylphenyl)-amine; (6-aminocarbonylethyl-7-isopropoxy-quinoxalin-4-yl)-(3-ethynylphenyl) -amine; and (6-aminocarbonylethyl_7_143939.doc 201019961 propoxyquinazolin-4-yl)-(3-ethynylphenyl)-amine; (6,7·diethoxy Quinazolin-1-yl)-(3-ethynylphenyl)-amine; (3-ethynylphenyl)-[6-(2-hydroxy-ethoxy)-7-(2-methoxy -ethoxy)-quinazolin-1-yl]-amine; [6,7-bis-(2.hydroxy-ethoxy)-quinazolin-1-yl]-(3-ethynylphenyl -Amine; [6,7-bis-(2-decyloxy-ethoxy)-quinazolin-1-yl]-(3-ethynylphenyl)-amine; (6,7-dioxin) Oxyquinazolin-1-yl)-(3-ethynylphenyl)-amine; (3-acetylene Phenyl)-(6-nonanesulfonylamino-quinazolin-1-yl)-amine; and (6-amino-quinazoline-yl)-(3-ethynylphenyl) amine. 20. 21. 22. 23. 24. 25. The use of claim 17, wherein the EGFR antagonist of formula I is Ν-(3-ethynylphenyl)-6,7-bis(2-methoxy Ethoxy)-4-quinazolinamine. The use of claim 17 wherein the EGFR antagonist Ν·(3·ethynylphenyl)-6,7-bis(2-methoxyethoxy)_ quinazoline is in the form of the hydrochloride salt. The use of claim 17, wherein the EGFR antagonist ^ (i-ethynylphenyl)-6,7-bis(2-methoxyethoxy)_4_啥 坐 坐 呈 is substantially uniform A crystalline polymorph form that exhibits a characteristic peak of the diffraction pattern of the calendering powder at 2 Θ, expressed at about 6.26, 12.48, 13.39, 16.96, 20.20, 21'1〇, 22.98, 24.46, 25.14, and 26.91. The use of Item 17, wherein the £ (31 Å antagonist is 4 (3 | fluoroanilino) ~ 7-decyloxy-6-(3-N-morpholinylpropoxy) quinazoline. The use of claim 17, wherein the EgfR antagonist is N_[3_gas_4_fluorophenyl)nonyloxy]phenyl]_6-[5-[[[2-(methylsulfanyl)ethyl]] Amino]methyl]-2-furanyl]quinazolinamine. ^ Item 17 (IV), wherein - (iv) antagonist forest (4 bromo-2-fluorobenzamide)-6-methoxy _7♦ A The use of the anti-cmet antibody is MetMAb and the EGFR antagonist is N-(3-ethynylphenyl)-6. 143939.doc 201019961 26. The use of claim 17, wherein the anti-cmet antibody is MetMAb , 7-bis(2-methoxyethoxy)-4-quinazolinamine, the VEGF antagonist is bevacizumab. 27. As claimed in claim 1. The use according to any one of the preceding claims, wherein the cancer is selected from the group consisting of breast cancer, colorectal cancer, rectal cancer, non-small cell lung cancer, non-Hodgkins lymphoma, kidney cells Cancer, prostate cancer, liver cancer, pancreatic cancer, soft tissue sarcoma, kaposi's sarcoma, carcinoid carcinoma, head and neck cancer, stomach cancer, melanoma, ovarian cancer, mesothelioma and multiple bone marrow 28. The use of claim 27, wherein the cancer is non-small cell lung cancer. The use of any one of claims 1 to 8, wherein the medicament further comprises or is used in combination with a chemotherapeutic agent A pharmaceutical composition for treating cancer in a subject, comprising a therapeutically effective amount of a c-met antagonist and a VEGF antagonist. The pharmaceutical composition according to claim 30, wherein the c-met antagonist is an antibody 32. The pharmaceutical composition of claim 31, wherein the antibody is a monovalent antibody. The pharmaceutical composition of claim 32, wherein the antibody is monovalent and comprises an Fc region, wherein the Fc region comprises a first polypeptide A second polypeptide, wherein the first polypeptide comprises the Fc sequence set forth in Figure 9 (SEQ ID NO: 12) and the second polypeptide comprises the sequence set forth in Figure 10 (SEQ ID NO: 13). The pharmaceutical composition of claim 33, wherein the antibody comprises (a) a first polypeptide comprising a heavy chain variable domain having the sequence: EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWLHW 143939.doc 201019961 VRQAPGKGLEWVGMIDPSNSDTRFNPNFKDRFTISADTS KNTAYLQMNSLRAEDTAVYYCATYRSYVTPLDYWGQGT LVTVSS (SEQ ID NO: 10), a CHI sequence and a first Fc polypeptide; (b) a second polypeptide comprising a light chain variable domain having the sequence: DIQMTQSPSSLSASVGDRVTITCKSSQSLLYTSSQKN YLAWYQQKPGKAPKLLIYWASTRESGVPSRFSGSGSGT DFTLTISSLQPEDFATYYCQQYYAYPWTFGQGTKVEIKR (SEQ ID NO: 11) and CL1 sequence; and (c) comprising a second Fc a third polypeptide of the polypeptide, wherein the heavy chain variable domain and the light chain variable domain are present in a complex form and form a single antigen binding arm, wherein the first Fc polypeptide and the second Fc polypeptide are present in a complex form And forming an Fe region that increases the resistance compared to the Fab molecule comprising the antigen binding arm Stability fragments. 35. The pharmaceutical composition of claim 34, wherein the first polypeptide comprises the Fc sequence set forth in SEQ ID NO: 12, and the second polypeptide comprises the Fc sequence set forth in SEQ ID NO: 13. The pharmaceutical composition of claim 34, wherein the first polypeptide comprises the Fc sequence set forth in SEQ ID NO: 13, and the second polypeptide comprises the F c sequence set forth in SEQ ID NO: . 37. The pharmaceutical composition of claim 33, wherein the antibody is MetMAb. The pharmaceutical composition according to any one of claims 30 to 37, wherein the VEGF antagonist is an aptamer capable of specifically binding to VEGF. The pharmaceutical composition according to any one of claims 30 to 37, wherein the VEGF antagonist is a soluble VEGF receptor protein, or a VEGF-binding fragment thereof, 143939.doc-10-201019961 or a chimeric VEGF receptor protein. 40. The pharmaceutical composition of claim 39, wherein the chimeric VEGF receptor protein comprises at least the extracellular domain 2 of Fit-1 or KDR. The pharmaceutical composition according to any one of claims 30 to 37, wherein the VEGF antagonist is an anti-VEGF antibody. 42. The pharmaceutical composition of claim 41, wherein the anti-VEGF antibody is a monoclonal antibody. 43. The pharmaceutical composition of claim 42, wherein the monoclonal antibody is a chimeric antibody, a fully human antibody, or a humanized antibody. 44. The pharmaceutical composition of claim 43, wherein the anti-VEGF antibody is bevacizumab, a G6-series antibody, a B20-series antibody or a VEGF-binding fragment thereof. 45. The pharmaceutical composition of claim 44, wherein the anti-VEGF antibody is bevacizumab. The pharmaceutical composition according to any one of claims 30 to 37, which further comprises an EGFR antagonist. 47. The pharmaceutical composition of claim 46, wherein the EGFR antagonist has the general formula I: Wherein: m is 1, 2 or 3; 143939.doc 201019961 Each is independently selected from the group consisting of hydrogen, halo, mu, amine, county, wall, sulfhydryl, gland, cyano , trifluoromethyl and -(cvcaalkyl)-W_(phenyl), wherein w is a single bond, hydrazine, s or ie; or each R1 is independently selected from r9 and substituted by a cyano group 2Ci_C4 alkyl, wherein R9 It is selected from the group consisting of r5, _〇r6, _nr6r6, _c(〇)r7, -NHOR, -〇c(〇)r6, cyano, A and _yr5; R, c _C4 alkyl; R6 independent The ground is hydrogen or R5; R7sR5, _〇r4_nr6r6; A is selected from N-piperidinyl, N-morpholinyl, N-pyrrolidinyl, 4_r6-piperazinyl, imidazol-1-yl, 4-pyridone ·〗 _ base, _ (Ci_C4 alkylene) (C〇2H), phenoxy, phenyl, phenylthio, C2-C4 alkenyl and • (CVC: 4 extended alkyl) c (〇) NR6R6 And γ are S, 8〇 or s〇2: wherein the alkyl moiety of R, -OR6 and -NR6R6 is optionally substituted with 1 to 3 mercapto substituents, and R5, _〇R6 and -Nr6r6 The alkyl moiety is optionally substituted with 1 or 2 R9 groups, and wherein the alkyl moiety of the substituent is optionally halogenated Or R9 is substituted by the 'restriction condition that two heteroatoms are not attached to the same carbon atom; or each R1 is independently selected from -NHS02R5, phthalic acid imine-(Cl_C4)-alkylsulfonylamino group, benzene Formamyl, phenylsulfonylamino, 3-phenylurea, 2-oxopyrrolidin-1-yl, 2,5-di-oxypyrrolidin-1-yl and R10-(C2- a C4)-alkylalkylamino group, wherein R10 is selected from the group consisting of dentate, _〇r6, C2-C4 alkoxy, _(:(0)117 and _NR6R6; and wherein the R1 group -NHSC^R5 , phthalimide imino-(Cl_C4)alkylsulfonylamino, benzylamino, phenylsulfonylamino, 3-phenylureido, 2-oxopyrrolidine-1- Base, 2,5-di-oxypyrrolidin-1-yl and R1Q-(C2-C4)- 143939.doc • 12-201019961 The mercaptoamine group is optionally selected from halo by 1 or 2 , a Ci_C alkyl group, a cyano group, a decanesulfonyl group, and a substituent of (^-(: 4 methoxy); or two R1 groups together with the carbon to which they are attached form a 5-8 ring, which The ring includes 1 or 2 heteroatoms selected from the group consisting of hydrazine, s and N; R 2 is hydrogen or CrC6 alkyl, and the CrCe alkyl group is optionally selected from the group consisting of dentate groups, C "C4" Substituted by a substituent of oxy, -NR6R6 and -S02R5; π is 1 or 2, and each R3 is independently selected from the group consisting of hydrogen, halo, hydroxy, Cl-c6 alkyl, alkoxy, wherein the r3 group The alkyl moiety is optionally substituted with from 1 to 3 substituents independently selected from halo, CrCU alkoxy, -NR6R6 and -S02R; and R4 is azido or -(ethynyl)-rh, wherein R&quot; Is a hydrogen or Cl-c6 alkyl group. The CVC6 alkyl group is optionally substituted with a hydroxy group, 〇R6 or _NR6R6. The pharmaceutical composition of claim 46, wherein the EGFR antagonist is a compound of formula I selected from the group consisting of: (6,7-dimethoxyquinazolin-4-yl)-(3-acetylene (phenyl))-amine; (6,7-dimethoxyquinazolin-4-yl)-[3-(3,-hydroxypropyn-1-yl)phenyl]-amine; [3-( 2'-(Aminomethyl)-ethynyl)phenyl (6 7-dimethoxyquinazolin-4-yl)-amine; (3-ethynylphenyl)-(6-nitroquinazoline) (6,7-dimethoxy quinazolin-4-yl H4-ethynylphenyl)-amine; (6,7-dimethoxyquinazolin-4- (6-aminoacetylquin-2-yl)-(3-ethynylphenyl)-amine; (3-ethynylbenzene) (6)-(6-methanesulfonylaminoquinazolin-4-yl)-amine; (3-ethyl-bromophenyl)-(6,7-methylenedioxyquinazolin-4- (6,7-dimethoxy quinazoline _4_yl 143939.doc •13· 201019961 ethynyl-6-methylphenylamine; (3_ethynylphenyl)_(7 _Nitroquinazolin-4-yl)-amine; (3-ethynylphenyl)-[6-(4'-toluenesulfonylamino)quinazolin-4-yl]-amine; (3 _acetylene Phenyl)_{6-[2,-o-phenylenediminoimido-ethylidene-yl-sulfonylamino]quinazolin-4-yl}•amine; (3-ethynylphenyl) -(6-nonylquinazolin-4-yl)-amine; (7-aminoquinazoline-4-yl)-(3-ethynylphenyl)-amine; (3-ethynylphenyl) -(7-oxime quinazolin-4-yl)-amine; (6-fluorenyloxyquinazolin-4-yl)-(3-ethynylphenyl)-amine; (7-oxiranylcarbonyl) Quinazolin-4-yl)-(3-ethynylphenyl)-amine; [6,7-bis(2-decyloxyethoxy)quinazolin-4-yl]-(3-ethynyl Phenyl)-amine; (3-azidosulfonylphenyl)-(6,7-dimethoxyquinazolin-4-ylamine; (3_azido-5-phenylphenylphosphonium 6,7) -dimethoxyquinazolin-4-yl)-amine; (4. azidophenyl)-(6,7-dimethoxyquinazolin-4-yl)-amine; (3_acetylene (6-decane fluorenyl-quinoxaline _4_yl)-amine; (6-ethanethio-quinazoline-4-yl)-(3_ethynylphenyl)- Amine; (6,7-dimethoxy-quinazoline-4-yl)-(3-ethynyl-4-fluoro-phenyl)-amine; (6,7-dimethoxy-quinazoline _4_基)_[3_(C-fast-1'-yl)-phenyl]-amine; [6,7_double_(2_methoxy -Ethoxy)-quinazoline-4-yl]-(5-ethynyl-2-indoleyl-phenyl)-amine; [6,7-bis-(2-methoxy-g-oxygen) ))-喧 喧 啉 -4- -4-yl]-(3-ethynyl-4-fluoro-phenyl)-amine; [6,7·bis-(2-gas-ethoxy)·quinazolium _4 Alkyl]-(3-ethynyl-phenyl)amine; [6_(2_g-ethoxy)-7-(2-methoxy-ethoxy)-quinazolin-4-yl]-(3 -ethynyl-styl)-amine; [6,7-bis-(2-acetoxy-ethoxy)-quinazolin-4-yl]ethynyl-phenyl)-amine; 2-[ 4-(3-ethynyl-phenylamino)-7-(2-hydroxy-ethoxy)-quinazolin-6-yloxy]-ethanol; [6-(2-ethyloxy)- Ethoxy)-7-(2-methoxyethoxymethyl)-oxazole-4-yl]-(3-acetylene 143939.doc •14-201019961 phenyl-phenyl)-amine;[7- (2-Gas-ethoxy)-6-(2-methoxy-ethoxy)-quinoxalin-4-yl]-(3-ethynyl-phenyl)-amine; [7-(2 -ethoxycarbonyl-ethoxy)-6-(2-methoxy-ethoxy)-oxazolidine-4-yl]-(3-ethynyl-phenyl)-amine, 2-[4 -(3-ethyl-fast-n-ylamino)-6-(2-carbo-ethoxy)-ββ-lin-7-yloxy]-ethanol; 2-[4-(3-acetylene) Phenyl-phenylamino)-7-(2-decyloxy -ethoxy)-quinazolin-6-yloxy]-ethanol; 2-[4-(3-ethynyl-phenylamino)-6-(2-decyloxy-ethoxy)-啥嗤琳_7_基基]-ethanol][6-(2-ethoxy-ethoxy-ethoxy)-7-(2-methoxy-ethoxy)-啥β-s- s- -4- (3-(3-ethynyl-phenyl)-amine; (3-ethynyl-phenylindole 6_(2-methoxy-ethoxy)-7-[2-(4-mercapto-L. -1-yl)-ethoxy]-啥啥琳_ 4-ylbumin; (3_ethynyl-phenyl)·[7-(2-methoxy-ethoxy)_6_(2_morpholine 4-yl)-ethoxy)-quinazolin-4-yl]-amine; (6,7-diethoxyoxazolin-1-yl)-(3-ethynylphenyl)-amine (6,7-dibutoxyquinazolinyl)-(3-ethynylphenyl)-amine; (6,7-diisopropoxyquinazoline-yl)·(3-ethyl (phenyl))-amine; (6,7-diethoxyhydrazinyl-indoleyl)-(3-ethynyl-2-methyl-phenyl)-amine; [6,7-bis-( 2-methoxy-ethoxy)-quinazoline-1-yl]-(3-ethynyl-2-methyl-phenyl)-amine; (3-ethylphenylphenyl) (2-an -ethoxy)-7-(2-methoxy-ethoxy)-噎 琳 _ ι ι ι ι ι ι ι 胺 胺 胺 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 Spit ·_1-yl]_(3_ethynylphenyl)-amine; 2-[4-(3-ethylidyl-phenylamino)-6-(2-methoxy-ethoxyxo) 啥β (7,7-dipropoxy-hydrazinyl) (3-ethyl-p-phenyl)-amine; (6,7-diethoxy- Squatting _4_yl)_(3_ethynyl-5-fluoro-phenyl)-amine; (6,7-diethoxy-hydrazinyl)_(3_ethynyl-4- Gas-phenyl)-amine; (6,7-diethoxy-hydrazino- 4_yl)-(5_B 143939.doc -15- 201019961 alkynyl-2-methyl-phenyl)- Amine; (6,7-diethoxy-quinazolin-4-yl)-(3-ethynyl-4-methyl-phenyl)-amine; (6-aminomethyl-7-methoxy (quinoquinazolin-4-yl)-(3-ethynyl-phenyl)-amine; (6-aminomethyl-7-decyloxy-quino-[beta]-yl-4-yl)-(3- Ethyl phenyl)-amine; (6-amino benzyl-7-methoxy-indolyl-4-yl)-(3-ethyl-bromophenyl)-amine; (6-amine (6-Amino-indenyl-7-ethoxyquine) (oxazolin-4-yl)-(3-ethynylphenyl)-amine; (6-aminocarbonylethyl-7-ethoxy-quinazoline 4-(4-yl)-(3-ethynylphenyl)-amine; (6-aminocarbonylindol-7-isopropoxy-quinazolin-4-yl)-(3-ethynylphenyl) -amine; (6-aminocarbonylmethyl-7-propoxy-quinazolin-4-yl)-(3-ethynylphenyl)-amine; (6-aminocarbonylmethyl-7-- Oxy-quinazolin-4-yl)-(3.ethynylphenyl)-amine; (6-aminocarbonylethyl-7-isopropoxy-quinazolin-4-yl)-(3 -ethynylphenyl)-amine; and (6-aminocarbonylethyl-7-propoxy-quinazolin-4-yl)-(3-ethynylphenyl)-amine; (6,7- Diethoxyquinazolin-1-yl)-(3-ethynylphenyl)-amine; (3-ethynylphenyl)-[6-(2-hydroxy-ethoxy)-7-(2 -methoxy-ethoxy)-quinazolin-1-yl]-amine; [6,7-bis-(2-hydroxyethyloxy)-quinazolin-1-yl]-(3- Ethynylphenyl)-amine; [6,7-bis-(2-decyloxy-ethoxy)-quinazolin-1-yl]-(3-ethynylphenyl)-amine; (6, 7-Dimethoxyquinazolin-1-yl)-(3-ethynylphenyl)-amine; (3-ethynylphenyl)·(6·decanesulfonylamino-quinazoline- 1-yl)-amine; and (6-amino-quinazolin-1-yl)-(3-ethynylphenyl)-amine49. The pharmaceutical composition of claim 46, wherein the EGFR antagonist of formula I is Ν-(3. ethynylphenyl)-6,7-bis(2-methoxyethoxy)-4-quinoline Oxazoline amine. The pharmaceutical composition of claim 46, wherein the EGFR antagonist N-(3-ethynylphenyl)-6,7-bis(2-decyloxyethoxy)- 4-quinazolinamine is in the form of the hydrochloride salt. 51. The pharmaceutical composition of claim 46, wherein the EGFR antagonist N-(3-ethyl 'alkynylphenyl)-6,7-bis(2-decyloxyethoxy)-4-quinazoline The amine is in the form of a substantially uniform crystalline polymorph which exhibits a characteristic peak of the X-ray powder diffraction pattern of 2 Å. Expressed at approximately 6.26, 12.48, 13.39, 16.96, 20.20, 21.10, 22.98, 24.46, 25.14 and 26.91. 52. The pharmaceutical composition of claim 46, wherein the EGFR antagonist is 4-(3,-gas-4'-fluoroanilino)-7-methoxy-6-(3-N-morpholinylpropionate Oxy) oxazole. 53. The pharmaceutical composition of claim 46, wherein the EGFR antagonist is N-[3- gas-4-[(3.fluorophenyl)decyloxy]phenyl]_6_[5_[[[2_(A Alkylsulfonyl)ethyl]amino]methyl]-2-indolyl]-4-quino 54. The pharmaceutical composition of claim 46, wherein the EGFR antagonist is 4-(4-^bromo-2-fluoroanilino)-6-methoxy-7-(1-indenyl-4-yl-4-yl Methoxy) 啥吐琳. 55. The pharmaceutical composition of claim 46, wherein the anti-cmet antibody is; MetMAb, the EGFR antagonist is N-(3-ethynylphenyl)-6,7-bis(2-decyloxyethoxy) -4- quinazolinamine, the antagonist is a bevacizumab. The pharmaceutical composition according to any one of claims 30 to 37, wherein the cancer is selected from the group consisting of breast cancer , colorectal cancer, rectal cancer, non-small cell lung cancer, non-Hodgkin's lymphoma, renal cell carcinoma, prostate 143939.doc 17 201019961 cancer, liver cancer, pancreatic cancer, soft tissue sarcoma, Kaposi's sarcoma, carcinoid tumor , head and neck cancer, gastric cancer, melanoma, ovarian cancer, mesothelioma, and multiple myeloma. The pharmaceutical composition of claim 56, wherein the cancer is non-small cell lung cancer. The pharmaceutical composition according to any one of 37, which further comprises a chemotherapeutic agent. 143939.doc 18-