HK1241936A1

HK1241936A1 - Cancer treatment with c-met antagonists and correlation of the latter with hgf expression

Info

Publication number: HK1241936A1
Application number: HK18101344.1A
Authority: HK
Inventors: Hartmut Koeppen; See PHAN; Sandra ROST; Wei Yu; David SHAMES
Original assignee: 豪夫迈．罗氏有限公司
Priority date: 2014-03-24
Filing date: 2015-03-24
Publication date: 2018-06-15

Description

Cancer treatment using C-MET antagonists and correlation of the former with HGF expression

Cross reference to related applications

This application claims priority to provisional patent No.61/985,316 filed on day 4/28 2014 and provisional application No.61/969,706 filed on day 3/24 2014, the contents of each of which are incorporated herein by reference.

Sequence listing

This application contains a sequence listing, which has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy created 3/20/2015 was named P5805R1-WO sl. txt and was 31,028 bytes in size.

Technical Field

The present invention concerns methods of therapeutic treatment. In particular, the invention concerns the treatment of human cancer patients with c-met antagonists. In addition, the present invention concerns biomarkers, such as hepatocyte growth factor.

Background

Cancer remains one of the most fatal threats to human health. Cancer affects nearly 130 million new patients each year in the united states and is the second cause of death after heart disease, accounting for approximately 1 of 4 deaths. It is also predicted that cancer may be the first cause of death in 5 years beyond cardiovascular disease. Solid tumors are responsible for most of these deaths. Although significant advances have been made in the medical treatment of certain cancers, the overall 5-year survival rate for all cancers has improved by only about 10% over the last 20 years. Cancer (or malignant tumor) rapidly grows and metastasizes in an uncontrolled manner, making timely detection and treatment extremely difficult.

Gliomas account for 81% of all malignant brain and CNS tumors. Glioblastoma, a World Health Organization (WHO) grade IV astrocytoma, accounts for 60% to 70% of malignant gliomas and remains the most aggressive subtype of glioma. It occurs mostly in adults (median age at diagnosis: 64 years) and its incidence is estimated to be 3.05/100,000 in the U.S. and less than 2/100,000 in Europe. Given that the overall survival was 29% and 3% for 1 and 5 years, respectively, the prognosis of glioblastoma remained particularly poor (central brain tumor registry in the united states (2005) (CBTRUS; http:// www.cbtrus.org).

Although there have been some advances in the treatment of glioblastoma, this disease faces highly unmet medical needs with limited treatment options.

Mesothelioma is a form of cancer that occurs from cells of the mesothelium (the protective lining that covers many internal organs). The incidence of malignant mesothelioma indicates significant variation from country to country. In countries with the highest incidence, australia, belgium, and the uk, the incidence is estimated to be around 3/100,000. There is evidence indicating a correlation between exposure to asbestos and the development of mesothelioma. The latency between the first exposure to asbestos and the diagnosis of mesothelioma varies greatly, possibly as a result of a change in the intensity of the asbestos exposure. Malignant mesothelioma remains a serious Health problem due to the poor outcome of current therapies (Bianchi, C.andDianchi, T., Industrial Health,45:379-387 (2007)).

Hepatocellular carcinoma (HCC, also known as malignant hepatoma) is the most common type of liver cancer. Most cases of HCC are secondary to either viral hepatitis infection (hepatitis b or c) or cirrhosis. HCC is one of the most common tumors worldwide. It occurs more frequently in men than women, and is often seen in people 50 years of age or older. HCC usually results in death within 3-6 months if the cancer cannot be completely removed by surgery (Medlineplus (2013); http:// www.nlm.nih.gov/MedlinePlus/ency/article/000280. htm).

Gastric cancer is most commonly caused by infection with the bacterium helicobacter pylori. About 90-95% of gastric cancers are adenocarcinomas. Gastric cancer is most often developed in adults (mean age at diagnosis: 69 years). The incidence of gastric cancer is about 1 in 111 people. The overall 5-year relative survival rate in all people with gastric Cancer in the United states is about 29% (American Cancer Society (2014); http:// www.cancer.org/Cancer/stomachcancer/index).

Renal cell carcinoma is the most common type of kidney cancer, accounting for about 90% of kidney cancers. Renal cell carcinoma occurs mostly in adults (mean age at diagnosis: 64). The lifetime risk of developing renal cancer is about 1 in 63. The 5-year survival rate of people diagnosed with renal Cancer varies with the stage of Cancer, from 81% of the 5-year survival rate of people with stage I renal Cancer to 8% of the 5-year survival rate of people with stage IV renal Cancer (American Cancer Society (2015); http:// www.cancer.org/Cancer/kidneycancer/index).

Sarcomas are cancers derived from transformed cells of mesenchymal origin. Sarcomas can be derived from a variety of tissues, including bone, cartilage, fat, muscle, blood vessels, and hematopoietic tissues. There are approximately 15,000 new cases of sarcoma in the united states each year. Osteosarcoma has a 5-year survival rate of about 70% (Longi, a., et al, Cancer treat. rev.,32 (6); 423-36 (2006)).

All references (including patent applications and publications) cited herein are incorporated by reference in their entirety.

Summary of The Invention

Use of c-met antagonists for the effective treatment of cancer patients is provided. The present application also provides better methods for diagnosing diseases and for optionally treating diseases with c-met antagonists. c-met antagonists are optionally used in combination with VEGF antagonists to effectively treat cancer.

In particular, hepatocyte growth factor (interchangeably referred to as "HGF") biomarkers are utilized to identify patient populations in which treatment with an anti-c-met antagonist, optionally plus a VEGF antagonist, provides clinically meaningful benefits. In particular, the present invention provides data from one randomized phase II clinical trial of anti-c-met antibody MetMAb (onartuzumab) in combination with an anti-VEGF antibody (bevacizumab) in subjects with relapsed glioblastoma. HGF biomarkers were used to identify patient populations in which MetMAb plus bevacizumab treatment provided clinically meaningful benefit, assessed by progression free survival and overall survival. In this clinical trial, treatment with MetMAb and bevacizumab provided clinically meaningful benefit to patients with recurrent glioblastoma that expressed high levels of HGF biomarkers. The results show that efficacy assessed by Progression Free Survival (PFS) and Overall Survival (OS) is positive, especially when compared to PFS and OS data for bevacizumab alone treatment. The differences were statistically significant, and addition of MetMab to bevacizumab extended both progression free and overall survival in patients with recurrent glioblastoma that expressed high levels of HGF biomarkers. Clinical trial data also show that treatment with MetMAb in combination with bevacizumab increases the risk of progression and death in patients with recurrent glioblastoma expressing low levels of HGF biomarkers relative to the risk of progression and death in such patients treated with bevacizumab alone. The results show that efficacy assessed by PFS and OS is worse in patients treated with MetMAb and bevacizumab when compared to PFS and OS data in patients treated with bevacizumab alone with glioblastoma expressing low levels of HGF biomarkers. The differences were statistically significant.

In one aspect, provided are methods for treating a patient with cancer comprising administering an effective amount of a c-met antagonist to the patient if the patient's cancer has been found to have a high amount of HGF biomarker.

In some embodiments, the patient's cancer overexpresses c-met. In some embodiments, the patient's cancer exhibits c-met amplification. In some embodiments, the patient's cancer does not exhibit c-met amplification.

In some embodiments, the patient's cancer expresses both c-met and HGF. In some embodiments, HGF secreted from a cell binds c-met on the surface of the cell that secretes it in an autocrine manner. In some embodiments, the patient's cancer expresses both c-met and HGF and signals in an autocrine manner. In some embodiments, HGF expression is determined in a cancer of a patient using IHC or ISH or other methods known in the art.

In some embodiments, the c-met antagonist is an antagonistic anti-c-met antibody. In some embodiments, the anti-c-met antibody comprises (a) HVR1 comprising sequence GYTFTSYWLH (SEQ ID NO: 1); (b) HVR2 comprising sequence GMIDPSNSDTRFNPNFKD (SEQ ID NO: 2); (c) HVR3-HC comprising sequence ATYRSYVTPLDY (SEQ ID NO: 3); (d) HVR1-LC comprising sequence KSSQSLLYTSSQKNYLA (SEQ ID NO: 4); (e) HVR2-LC comprising sequence WASTRES (SEQ ID NO: 5); and (f) HVR3-LC comprising sequence QQYYAYPWT (SEQ ID NO: 6). In some embodiments, the anti-c-met antibody binds an onartuzumab (onartuzumab) epitope. In some embodiments, the anti-c-met antibody is obinutuzumab. In some embodiments, the effective amount of the anti-c-met antibody is 15mg/kg every three weeks. In some embodiments, the effective amount of the anti-c-met antibody is 10mg/kg every two weeks. In some embodiments, the c-met antagonist is one or more of crizotinib, tivtinib, carbozantinib, MGCD-265, ficlatuzumab, humanized TAK-701, rilotumumab, foretinib, h224G11, DN-30, MK-2461, E7050, MK-8033, PF-4217903, AMG208, JNJ-38877605, EMD1204831, INC-280, LY-2801653, SGX-126, RP1040, LY2801653, BAY-853474, and/or LA 480.

In some embodiments, the treatment is with an effective amount of a combination of a c-met antagonist and a VEGF antagonist. In some embodiments, the VEGF antagonist is an anti-VEGF antibody. In some embodiments, the anti-VEGF antibody binds the a4.6.1 epitope. In some embodiments, the anti-VEGF antibody is bevacizumab (bevacizumab). In some embodiments, the anti-VEGF antibody comprises a variable heavy chain (VH) and a variable light chain (VL), wherein the VH has amino acid sequence EVQLVESGGGLVQPGGSLRL SCAASGYTFT NYGMNWVRQA PGKGLEWVGW INTYTGEPTY AADFKRRFTF SLDTSKSTAYLQMNSLRAED TAVYYCAKYP HYYGSSHWYF DVWGQGTLVT VSS (SEQ ID NO:14) and the VL has amino acid sequence DIQMTQSPSS LSASVGDRVT ITCSASQDIS NYLNWYQQKP GKAPKVLIYF TSSLHSGVPSRFSGSGSGTD FTLTISSLQP EDFATYYCQQ YSTVPWTFGQ GTKVEIKR (SEQ ID NO: 15). In some embodiments, the effective amount of the anti-VEGF antibody is 10mg/kg intravenously every two weeks. In some embodiments, the effective amount of the anti-VEGF antibody is 15mg/kg intravenously every three weeks. In some embodiments, the effective amount of the anti-VEGF antibody is administered initially intravenously over 90 minutes, with subsequent infusions over 60 minutes, then over 30 minutes. In some embodiments, the anti-VEGF antibody is administered to the patient second during the first cycle. In some embodiments, the subsequent administration of the anti-VEGF antibody is either before or after the c-met antagonist. In some embodiments, the VEGF antagonist is administered concurrently with the c-met antagonist.

In some embodiments, the patient is less than 50 years of age. In some embodiments, the patient is equal to or greater than 50 years of age. In some embodiments, the patient has a Karnofsky performance status of 70% to 80%. In some embodiments, the patient has a Karnofsky performance status of 90% to 100%.

In some embodiments, the patient has greater PFS and/or OS relative to a patient not having a high HGF biomarker. In some embodiments, the patient has greater PFS and/or OS relative to a patient treated with a VEGF antagonist alone.

In some embodiments, the HGF biomarker is HGF mRNA, and HGF biomarker mRNA expression is determined in a sample from the patient using In Situ Hybridization (ISH). In some embodiments, high HGF biomarker is an ISH score of 2+ and/or 3 +. In some embodiments, high HGF biomarker is an ISH score of 2+ and 3 +. In some embodiments, high HGF mRNA biomarker is presence of about 12 or more HGF ISH signal positive cells in the sample. In some embodiments, high HGF mRNA biomarker is presence of about 15 or more HGF ISH signal positive cells in the sample. In some embodiments, high HGF mRNA biomarker is presence of about 20 or more HGF ISH signal positive cells in the sample. In some embodiments, high HGF mRNA biomarker is the presence of about 25 or more HGFISH signal positive cells in the sample. In some embodiments, high HGF mRNA biomarker is presence of about 30 or more HGF ISH signal positive cells in the sample. In some embodiments, high HGF mRNA biomarker is presence of about 35 or more HGF ISH signal positive cells in the sample. In some embodiments, high HGF mRNA biomarker is 1% or more of HGF ISH signal positive cells in the sample. In some embodiments, high HGF mRNA biomarker is 2% or more HGF ISH signal positive cells in the sample. In some embodiments, high HGF mRNA biomarker is 3% or more of HGF ISH signal positive cells in the sample. In some embodiments, high HGF mRNA biomarker is 4% or more HGF ISH signal positive cells in the sample. In some embodiments, high HGF mRNA biomarker is 5% or more of HGF ISH signal positive cells in the sample. In some embodiments, high HGF mRNA biomarker is 10% or more HGF ISH signal positive cells in the sample.

In some embodiments, the HGF biomarker expression is nucleic acid expression and is determined in a sample from the patient using an amplification-based assay, RNA-seq, microarray analysis, SAGE, MassARRAY technique, or FISH. In some embodiments, the amplification-based assay is a Polymerase Chain Reaction (PCR) -based assay (e.g., quantitative PCR, real-time PCR, quantitative real-time PCR (qRT-PCR), reverse transcriptase PCR (rt-PCR), and reverse transcription quantitative PCR (rt-qPCR)).

In some embodiments, the HGF biomarker is HGF mRNA, and HGF biomarker mRNA expression is determined in a sample from the patient using an amplification-based assay, RNA-seq, microarray analysis, SAGE, MassARRAY technique, or FISH. In some embodiments, the amplification-based assay is a PCR-based assay (e.g., quantitative PCR, real-time PCR, quantitative real-time PCR (qRT-PCR), reverse transcriptase PCR (rt-PCR), and reverse transcription quantitative PCR (rt-qPCR)). In some embodiments, the PCR-based assay is rt-qPCR. In some embodiments, high HGF biomarker is an HGF expression level in the upper 50% of a reference patient population. In some embodiments, high HGF biomarker is an HGF expression level in the upper 40% of a reference patient population. In some embodiments, high HGF biomarker is an HGF expression level in the upper 35% of a reference patient population. In some embodiments, high HGF biomarker is an HGF expression level in the upper 30% of a reference patient population. In some embodiments, high HGF biomarker is an HGF expression level in the upper 25% of a reference patient population. In some embodiments, high HGF biomarker is an HGF expression level in the upper 20% of a reference patient population.

In some embodiments, the sample is of a cancer of the patient. The sample of the patient's cancer may include cancer cells, lymphocytes, leukocytes, stroma, blood vessels, connective tissue, basal lamina, and any other cell type associated with the cancer. In some embodiments, the sample comprises cancer cells and benign stromal cells. In some embodiments, the cancer is glioblastoma, mesothelioma, hepatocellular carcinoma, renal cell carcinoma, gastric cancer, sarcoma (e.g., osteosarcoma), non-small cell lung cancer, breast cancer, gallbladder cancer, or pancreatic cancer. In some embodiments, the cancer is glioblastoma, mesothelioma, renal cell carcinoma, gastric cancer, hepatocellular carcinoma, or sarcoma. In some embodiments, the cancer is glioblastoma. In some embodiments, the cancer is a previously treated glioblastoma. In some embodiments, the sample comprises glioblastoma cells and benign stromal cells. In some embodiments, the benign stromal cells are one or more of reactive astrocytes, glial cells, pericytes and endothelial cells. In some embodiments, the cancer is mesothelioma. In some embodiments, the cancer is previously treated mesothelioma. In some embodiments, the sample comprises mesothelioma cells and benign stromal cells. In some embodiments, the cancer is gastric cancer. In some embodiments, the cancer is previously treated gastric cancer. In some embodiments, the cancer comprises gastric cancer cells and benign stromal cells. In some embodiments, the benign stromal cells are one or more of fibroblasts, macrophages, and endothelial cells. In some embodiments, the cancer is renal cell carcinoma. In some embodiments, the cancer is previously treated renal cell carcinoma. In some embodiments, the sample comprises renal cell carcinoma cells and benign stromal cells. In some embodiments, the cancer is hepatocellular carcinoma. In some embodiments, the cancer is previously treated hepatocellular carcinoma. In some embodiments, the sample comprises hepatocellular carcinoma cells and benign stromal cells. In some embodiments, the cancer is a sarcoma (e.g., osteosarcoma). In some embodiments, the cancer is a previously treated sarcoma (e.g., a previously treated osteosarcoma). In some embodiments, the sample comprises sarcoma cells and benign stromal cells. In some embodiments, the sample is of a tumor of the patient. Tumor samples may include cancer cells, lymphocytes, leukocytes, stroma, blood vessels, connective tissue, basal lamina, and any other cell type associated with the tumor.

In some embodiments, the sample is obtained prior to treatment with the c-met antagonist. In some embodiments, the sample is obtained prior to treatment with a VEGF antagonist. In some embodiments, the sample is obtained prior to treatment with a cancer drug.

In some embodiments, the sample is formalin fixed and paraffin embedded. In some embodiments, the ISH is detected using hybridization-based signal amplification.

In some embodiments, RNA is isolated from the sample. In some embodiments, RNA is isolated from the formalin-fixed paraffin-embedded sample. In some embodiments, the isolated RNA is purified. In some embodiments, the purified RNA is used as a source of RNA for an amplification-based assay. In some embodiments, the amplification-based assay is a PCR-based assay. In some embodiments, the PCR-based assay is rt-qPCR.

In some embodiments, the cancer is glioblastoma, mesothelioma, hepatocellular carcinoma, renal cell carcinoma, gastric cancer, sarcoma (e.g., osteosarcoma), non-small cell lung cancer, breast cancer, gallbladder cancer, or pancreatic cancer. In some embodiments, the cancer is glioblastoma, mesothelioma, renal cell carcinoma, gastric cancer, hepatocellular carcinoma, or sarcoma. In some embodiments, the cancer is a previously treated glioblastoma. In some embodiments, the cancer is previously treated mesothelioma. In some embodiments, the cancer is previously treated renal cell carcinoma. In some embodiments, the cancer is previously treated gastric cancer. In some embodiments, the cancer is previously treated hepatocellular carcinoma. In some embodiments, the cancer is a previously treated sarcoma.

In one aspect, provided is a method for treating a patient with cancer comprising administering to the patient a therapeutically effective amount of a drug other than a c-met antagonist if the patient's cancer has been found to have a small amount of HGF biomarker.

In one aspect, the invention provides a method for identifying a cancer patient who is likely to be responsive to treatment with a c-met antagonist, comprising the step of determining whether the patient's cancer has a high amount of HGF biomarker, wherein the HGF biomarker expression indicates that the patient is likely to be responsive to treatment with the c-met antagonist.

In some embodiments, the HGF biomarker is HGF mRNA, and HGF biomarker mRNA expression is determined in a sample from the patient using In Situ Hybridization (ISH). In some embodiments, high HGF biomarker is an ISH score of 2+ and/or 3 +. In some embodiments, high HGF biomarker is an ISH score of 2+ and 3 +. In some embodiments, high HGF mRNA biomarker is presence of about 12 or more HGF ISH signal positive cells in the sample. In some embodiments, high HGF mRNA biomarker is presence of about 15 or more HGF ISH signal positive cells in the sample. In some embodiments, high HGF mRNA biomarker is presence of about 20 or more HGF ISH signal positive cells in the sample. In some embodiments, high HGF mRNA biomarker is the presence of about 25 or more HGFISH signal positive cells in the sample. In some embodiments, high HGF mRNA biomarker is presence of about 30 or more HGF ISH signal positive cells in the sample. In some embodiments, high HGF mRNA biomarker is presence of about 35 or more HGF ISH signal positive cells in the sample. In some embodiments, there is 1% or more HGF ISH signal positive cells in the sample. In some embodiments, high HGF mRNA biomarker is 2% or more HGF ISH signal positive cells in the sample. In some embodiments, high HGF mRNA biomarker is 3% or more of HGF ISH signal positive cells in the sample. In some embodiments, high HGF mRNA biomarker is 4% or more HGF ISH signal positive cells in the sample. In some embodiments, high HGF mRNA biomarker is 5% or more of HGF ISH signal positive cells in the sample. In some embodiments, high HGF mRNA biomarker is 10% or more HGF ISH signal positive cells in the sample.

In some embodiments, the HGF biomarker is HGF mRNA, and HGF biomarker mRNA expression is determined in a sample from the patient using an amplification-based assay, RNA-seq, microarray analysis, SAGE, MassARRAY technique, or FISH. In some embodiments, the amplification-based assay is a Polymerase Chain Reaction (PCR) -based assay (e.g., quantitative PCR, real-time PCR, quantitative real-time PCR (qRT-PCR), reverse transcriptase PCR (rt-PCR), and reverse transcription quantitative PCR (rt-qPCR)). In some embodiments, high HGF biomarker is an HGF expression level in the upper 50% of a reference patient population. In some embodiments, high HGF biomarker is an HGF expression level in the upper 40% of a reference patient population. In some embodiments, high HGF biomarker is an HGF expression level in the upper 35% of a reference patient population. In some embodiments, high HGF biomarker is an HGF expression level in the upper 30% of a reference patient population. In some embodiments, high HGF biomarker is an HGF expression level in the upper 25% of a reference patient population. In some embodiments, high HGF biomarker is an HGF expression level in the upper 20% of a reference patient population.

In one aspect, provided is a method for identifying a cancer patient who is not likely to be responsive to treatment with a c-met antagonist, comprising the step of determining whether the patient's cancer has a low amount of HGF biomarker, wherein the HGF biomarker expression indicates that the patient is not likely to be responsive to treatment with the c-met antagonist. In some embodiments, HGF biomarker nucleic acid expression is determined in a sample from the patient using In Situ Hybridization (ISH). In some embodiments, a low HGFmRNA biomarker is an ISH score of less than 2 +. In some embodiments, low HGF mRNA biomarker is an ISH score of less than 1 +. In some embodiments, low HGF mRNA biomarker is an ISH score of 0 or 1 +. In some embodiments, low HGF mRNA biomarker is an ISH score of 0. In some embodiments, low HGF biomarker is presence of HGF ISH positive signal in 10 or fewer cells. In some embodiments, low HGF biomarker is presence of HGF ISH positive signal in 5 or fewer cells. In some embodiments, low HGF biomarker is presence of HGF ISH positive signal in no cells.

In one aspect, provided is a method for identifying a cancer patient who is not likely to be responsive to treatment with a c-met antagonist, comprising the step of determining whether the patient's cancer has a low amount of HGF biomarker, wherein the HGF biomarker expression indicates that the patient is not likely to be responsive to treatment with the c-met antagonist. In some embodiments, HGF biomarker nucleic acid expression is determined in a sample from the patient using an amplification-based assay, RNA-seq, microarray analysis, SAGE, MassARRAY technique, or FISH. In some embodiments, the amplification-based assay is a Polymerase Chain Reaction (PCR) -based assay (e.g., quantitative PCR, real-time PCR, quantitative real-time PCR (qRT-PCR), reverse transcriptase PCR (rt-PCR), and reverse transcription quantitative PCR (rt-qPCR)). In some embodiments, low HGF mRNA biomarker is an HGF expression level in the lower 50% of a reference patient population. In some embodiments, low HGF mRNA biomarker is an HGF expression level in the lower 60% of a reference patient population. In some embodiments, low HGF mRNA biomarker is an HGF expression level in the lower 65% of a reference patient population. In some embodiments, low HGF mRNA biomarker is an HGF expression level in the lower 70% of a reference patient population. In some embodiments, low HGF mRNA biomarker is an HGF expression level in the lower 75% of a reference patient population. In some embodiments, low HGF mRNA biomarker is an HGF expression level in the lower 80% of a reference patient population.

In some embodiments, the patient is a human patient. The patient may be a cancer patient, i.e. suffering from or at risk of suffering from one or more symptoms of cancer. Moreover, the patient may be a previously treated cancer patient. The patient may be a glioblastoma patient, i.e. suffering from or at risk of suffering from one or more symptoms of glioblastoma. Moreover, the patient may be a previously treated glioblastoma patient. In some embodiments, the patient has been treated with no more than one prior chemotherapy. In some embodiments, the patient has been previously treated with temozolomide. In some embodiments, the patient has previously been treated with temozolomide in combination with radiation. In some embodiments, the patient has previously been treated with temozolomide in combination with another agent. In some embodiments, the glioblastoma is a second-line glioblastoma. The patient may be a mesothelioma patient, i.e. suffering from or at risk of suffering from one or more symptoms of mesothelioma. Moreover, the patient may be a previously treated mesothelioma patient. In some embodiments, the patient has been treated with no more than one prior chemotherapy. In some embodiments, the patient has previously been treated with a combination of chemotherapy and radiation. In some embodiments, the patient has previously been treated with chemotherapy in combination with another agent. In some embodiments, the mesothelioma is a second-line mesothelioma. The patient may be a gastric cancer patient, i.e. suffering from or at risk of suffering from one or more symptoms of gastric cancer. Moreover, the patient may be a previously treated gastric cancer patient. In some embodiments, the patient has been treated with no more than one prior chemotherapy. In some embodiments, the patient has previously been treated with a combination of chemotherapy and radiation. In some embodiments, the patient has previously been treated with chemotherapy in combination with another agent. In some embodiments, the gastric cancer is second-line gastric cancer. The patient may be a renal cell carcinoma patient, i.e., suffering from or at risk of suffering from one or more symptoms of renal cell carcinoma. Moreover, the patient may be a previously treated renal cell carcinoma patient. In some embodiments, the patient has been treated with no more than one prior chemotherapy. In some embodiments, the patient has been previously treated with chemotherapy. In some embodiments, the patient has previously been treated with a combination of chemotherapy and radiation. In some embodiments, the patient has previously been treated with chemotherapy in combination with another agent. In some embodiments, the renal cell carcinoma is a second-line renal cell carcinoma. The patient may be a hepatocellular carcinoma patient, i.e. suffering from or at risk of suffering from one or more symptoms of hepatocellular carcinoma. Moreover, the patient may be a previously treated hepatocellular carcinoma patient. In some embodiments, the patient has been treated with no more than one prior chemotherapy. In some embodiments, the patient has been previously treated with chemotherapy. In some embodiments, the patient has previously been treated with a combination of chemotherapy and radiation. In some embodiments, the patient has previously been treated with chemotherapy in combination with another agent. In some embodiments, the hepatocellular carcinoma is a second-line hepatocellular carcinoma.

In some embodiments, the sample is a collection of cells or fluids obtained from a cancer patient. The source of the tissue or cell sample may be a solid tissue, such as from a fresh, frozen and/or preserved organ or tissue sample or biopsy or aspirate; blood or any blood component; body fluids such as cerebrospinal fluid, amniotic fluid (amniotic fluid), peritoneal fluid (ascites), or interstitial fluid; cells from a subject at any time during pregnancy or development. The tissue sample may contain compounds that are not naturally intermixed with the tissue in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, and the like. Examples of tumor samples herein include, but are not limited to, tumor biopsies, fine needle aspirates, bronchial lavage, pleural fluid (pleural fluid), sputum, urine, surgical specimens, circulating tumor cells, serum, plasma, circulating plasma proteins, ascites, primary cell cultures or cell lines derived from tumors or exhibiting tumor-like properties, and preserved tumor samples, such as formalin-fixed, paraffin-embedded tumor samples or frozen tumor samples. Tumor samples may include cancer cells, lymphocytes, leukocytes, stroma, blood vessels, connective tissue, basal lamina, and any other cell type associated with a tumor. In one embodiment, the sample comprises a glioblastoma tumor sample (e.g., a glioblastoma tumor sample comprising benign stroma, e.g., reactive astrocytes, glial cells, pericytes, and/or endothelial cells). In some embodiments, the sample comprises a macro-dissected glioblastoma tumor sample (e.g., where morphologically normal brain tissue has been removed from the tumor sample). In some embodiments, the macro-dissected glioblastoma tumor sample comprises benign stroma (e.g., reactive astrocytes, glial cells, pericytes, and/or endothelial cells). In some embodiments, the sample is a glioblastoma biopsy. In some embodiments, the sample is glioblastoma cancer resected. In some embodiments, the sample is obtained after recurrence of glioblastoma in the patient. In some embodiments, the sample is obtained prior to recurrence of glioblastoma in the patient. In one embodiment, the sample comprises a mesothelioma tumor sample (e.g., a mesothelioma tumor sample comprises benign stroma). In some embodiments, the sample comprises a macroscopically-dissected mesothelioma tumor sample (e.g., where morphologically normal mesothelial tissue has been removed from the tumor sample). In some embodiments, the macro-dissected mesothelioma tumor sample comprises benign stroma. In some embodiments, the sample is a mesothelioma biopsy. In some embodiments, the sample is resected for mesothelioma cancer. In some embodiments, the sample is obtained after recurrence of mesothelioma in the patient. In some embodiments, the sample is obtained prior to recurrence of mesothelioma in the patient. In one embodiment, the sample comprises a gastric cancer tumor sample (e.g., a gastric cancer tumor sample comprising benign stroma, e.g., fibroblasts, macrophages and/or endothelial cells). In some embodiments, the sample comprises a macroscopically-dissected gastric cancer tumor sample (e.g., where morphologically normal gastric tissue has been removed from the tumor sample). In some embodiments, the macro-dissected gastric cancer tumor sample comprises benign stroma (e.g., fibroblasts, macrophages, and/or endothelial cells). In some embodiments, the sample is a gastric cancer biopsy. In some embodiments, the sample is resected with gastric cancer. In some embodiments, the sample is obtained after recurrence of gastric cancer in the patient. In some embodiments, the sample is obtained prior to recurrence of gastric cancer in the patient. In one embodiment, the sample comprises a renal cell carcinoma tumor sample (e.g., a renal cell carcinoma tumor sample comprises benign stroma). In some embodiments, the sample comprises a macroscopically-dissected renal cell carcinoma tumor sample (e.g., where morphologically normal renal tissue has been removed from the tumor sample). In some embodiments, the macro-dissected renal cell carcinoma tumor sample comprises benign stroma. In some embodiments, the sample is a renal cell carcinoma biopsy. In some embodiments, the sample is resected for renal cell carcinoma cancer. In some embodiments, the sample is obtained after recurrence of the renal cell carcinoma in the patient. In some embodiments, the sample is obtained prior to recurrence of the renal cell carcinoma in the patient. In one embodiment, the sample comprises a hepatocellular carcinoma tumor sample (e.g., the hepatocellular carcinoma tumor sample comprises benign stroma). In some embodiments, the sample comprises a macroscopically-dissected hepatocellular carcinoma tumor sample (e.g., where morphologically normal liver tissue has been removed from the tumor sample). In some embodiments, the macro-dissected hepatocellular carcinoma tumor sample comprises benign stroma. In some embodiments, the sample is a hepatocellular carcinoma biopsy. In some embodiments, the sample is resected for hepatocellular carcinoma cancer. In some embodiments, the sample is obtained after recurrence of hepatocellular carcinoma in the patient. In some embodiments, the sample is obtained prior to recurrence of hepatocellular carcinoma in the patient.

In some embodiments, the sample is of a cancer of the patient. In some embodiments, the sample is of a glioblastoma of the patient. In some embodiments, the glioblastoma is previously treated. In some embodiments, the sample comprises glioblastoma cells and benign stromal cells. In some embodiments, the benign stromal cells are one or more of reactive astrocytes, glial cells, pericytes and endothelial cells. In some embodiments, the sample is of mesothelioma of the patient. In some embodiments, the mesothelioma is previously treated. In some embodiments, the sample comprises mesothelioma cells and benign stromal cells. In some embodiments, the sample is of gastric cancer in the patient. In some embodiments, the gastric cancer is previously treated. In some embodiments, the sample comprises gastric cancer cells and benign stromal cells. In some embodiments, the benign stromal cells are one or more of fibroblasts, macrophages, and endothelial cells. In some embodiments, the cancer is renal cell carcinoma. In some embodiments, the cancer is previously treated renal cell carcinoma. In some embodiments, the sample comprises renal cell carcinoma cells and benign stromal cells. In some embodiments, the cancer is hepatocellular carcinoma. In some embodiments, the cancer is previously treated hepatocellular carcinoma. In some embodiments, the sample comprises hepatocellular carcinoma cells and benign stromal cells. In some embodiments, the cancer is a sarcoma (e.g., osteosarcoma). In some embodiments, the cancer is a previously treated sarcoma (e.g., a previously treated osteosarcoma). In some embodiments, the sample comprises sarcoma cells and benign stromal cells.

In some embodiments, a patient previously treated for a glioblastoma has received a prior cancer therapy for a glioblastoma. In some embodiments, the patient has been treated with no more than one prior chemotherapy. In some embodiments, the patient has been previously treated with temozolomide. In some embodiments, the patient has previously been treated with temozolomide in combination with radiation. In some embodiments, the patient has previously been treated with temozolomide in combination with another agent. In some embodiments, the glioblastoma is a second-line glioblastoma.

In some embodiments, a patient previously treated for mesothelioma has received a prior cancer therapy for mesothelioma. In some embodiments, the patient has been treated with no more than one prior chemotherapy. In some embodiments, the patient has previously been treated with a combination of chemotherapy and radiation. In some embodiments, the patient has previously been treated with chemotherapy in combination with another agent. In some embodiments, the mesothelioma is a second-line mesothelioma.

In some embodiments, a previously treated gastric cancer patient has received a previous cancer therapy for gastric cancer. In some embodiments, the patient has been treated with no more than one prior chemotherapy. In some embodiments, the patient has previously been treated with a combination of chemotherapy and radiation. In some embodiments, the patient has previously been treated with chemotherapy in combination with another agent. In some embodiments, the gastric cancer is second-line gastric cancer.

In some embodiments, a patient previously treated for renal cell carcinoma has received a prior cancer therapy for renal cell carcinoma. In some embodiments, the patient has been treated with no more than one prior chemotherapy. In some embodiments, the patient has previously been treated with a combination of chemotherapy and radiation. In some embodiments, the patient has previously been treated with chemotherapy in combination with another agent. In some embodiments, the renal cell carcinoma is a second-line renal cell carcinoma.

In some embodiments, a previously treated patient for hepatocellular carcinoma has received a prior cancer therapy for hepatocellular carcinoma. In some embodiments, the patient has been treated with no more than one prior chemotherapy. In some embodiments, the patient has previously been treated with a combination of chemotherapy and radiation. In some embodiments, the patient has previously been treated with chemotherapy in combination with another agent. In some embodiments, the hepatocellular carcinoma is a second-line hepatocellular carcinoma.

In some embodiments, the sample is obtained prior to treatment with the c-met antagonist. In some embodiments, the sample is obtained prior to treatment with a VEGF antagonist. In some embodiments, the sample is obtained prior to treatment with the c-met antagonist and the VEGF antagonist. In some embodiments, the sample is obtained prior to treatment with a cancer drug. In some embodiments, the sample is formalin fixed and paraffin embedded. In some embodiments, the ISH is examined using hybridization-based signal amplification. In some embodiments, RNA is isolated from the sample. In some embodiments, RNA is isolated from the formalin-fixed paraffin-embedded sample. In some embodiments, the isolated RNA is purified. In some embodiments, the purified RNA is used as a source of RNA for an amplification-based assay. In some embodiments, the amplification-based assay is a PCR-based assay. In some embodiments, the PCR-based assay is rt-qPCR.

In some embodiments, the c-met antagonist is an antagonistic anti-c-met antibody. In some embodiments, the anti-c-met antibody comprises (a) HVR1 comprising sequence GYTFTSYWLH (SEQ ID NO: 1); (b) HVR2 comprising sequence GMIDPSNSDTRFNPNFKD (SEQ ID NO: 2); (c) HVR3-HC comprising sequence ATYRSYVTPLDY (SEQ ID NO: 3); (d) HVR1-LC comprising sequence KSSQSLLYTSSQKNYLA (SEQ ID NO: 4); (e) HVR2-LC comprising sequence WASTRES (SEQ ID NO: 5); and (f) HVR3-LC comprising sequence QQYYAYPWT (SEQ ID NO: 6). In some embodiments, the anti-c-met antibody binds an onartuzumab epitope. In some embodiments, the anti-c-met antibody is obinutuzumab. In some embodiments, the effective amount of the anti-c-met antibody is 15mg/kg every three weeks. In some embodiments, the effective amount of the anti-c-met antibody is 10mg/kg every two weeks. In some embodiments, the c-met antagonist is one or more of crizotinib, tivtinib, carbozantinib, MGCD-265, ficlatuzumab, humanized TAK-701, rilotumumab, foretinib, h224G11, DN-30, MK-2461, E7050, MK-8033, PF-4217903, AMG208, JNJ-38877605, EMD1204831, INC-280, LY-2801653, SGX-126, RP1040, LY2801653, BAY-853474, and/or LA 480.

In some embodiments, the VEGF antagonist is an anti-VEGF antibody. In some embodiments, the anti-VEGF antibody binds the a4.6.1 epitope. In some embodiments, the anti-VEGF antibody is bevacizumab. In some embodiments, the anti-VEGF antibody comprises a variable heavy chain (VH) and a variable light chain (VL), wherein the VH has amino acid sequence EVQLVESGGGLVQPGGSLRL SCAASGYTFT NYGMNWVRQA PGKGLEWVGW INTYTGEPTY AADFKRRFTF SLDTSKSTAYLQMNSLRAED TAVYYCAKYP HYYGSSHWYF DVWGQGTLVT VSS (SEQ ID NO:14) and the VL has amino acid sequence DIQMTQSPSS LSASVGDRVT ITCSASQDIS NYLNWYQQKP GKAPKVLIYF TSSLHSGVPSRFSGSGSGTD FTLTISSLQP EDFATYYCQQ YSTVPWTFGQ GTKVEIKR (SEQ ID NO: 15). In some embodiments, the effective amount of the anti-VEGF antibody is 10mg/kg intravenously every two weeks. In some embodiments, the effective amount of the anti-VEGF antibody is 15mg/kg intravenously every three weeks. In some embodiments, the effective amount of the anti-VEGF antibody is administered initially intravenously over 90 minutes, with subsequent infusions over 60 minutes, then over 30 minutes. In some embodiments, the anti-VEGF antibody is administered to the patient second during the first cycle. In some embodiments, the administration of the anti-VEGF antibody is before or after said c-met antagonist. In some embodiments, the VEGF antagonist is administered concurrently with the c-met antagonist.

In one aspect, provided is a method of identifying a patient having a glioblastoma (e.g., a previously treated glioblastoma) as likely to respond to a therapy comprising (a) an anti-c-met antibody (e.g., onartuzumab) and (b) an anti-VEGF antibody (e.g., bevacizumab), the method comprising: (i) measuring HGF biomarker in a sample from the patient, wherein the HGF biomarker is HGF nucleic acid (e.g., mRNA) and measuring is by ISH; and (ii) identifying the patient as more likely to respond to therapy comprising (a) a c-met antagonist antibody (e.g., onartuzumab) and (b) an anti-VEGF antibody (e.g., bevacizumab) when the sample has high HGF biomarker. In some embodiments, the method further comprises (iii) selecting for the patient a therapy comprising (a) a c-met antagonist antibody (e.g., onartuzumab) and (b) an anti-VEGF antibody (e.g., bevacizumab) or recommending a therapy comprising (a) a c-met antagonist antibody (e.g., onartuzumab) and (b) an anti-VEGF antibody (e.g., bevacizumab). In some embodiments, the method further comprises (iv) treating the patient with a therapy comprising (a) a c-met antagonist antibody (e.g., onartuzumab) and (b) an anti-VEGF antibody (e.g., bevacizumab).

In one aspect, provided is a method of identifying a patient having a glioblastoma (e.g., a previously treated glioblastoma) as likely to respond to a therapy comprising an anti-c-met antibody (e.g., onartuzumab), the method comprising: (i) measuring HGF biomarker in a sample from the patient, wherein the HGF biomarker is HGF nucleic acid (e.g., mRNA) and measuring is by ISH; and (ii) identifying the patient as more likely to respond to therapy comprising a c-met antagonist antibody (e.g., onartuzumab) when the sample has high HGF biomarker. In some embodiments, the method further comprises (iii) selecting a therapy comprising a c-met antagonist antibody (e.g., onartuzumab) or recommending a therapy comprising a c-met antagonist antibody (e.g., onartuzumab) for the patient. In some embodiments, the therapy further comprises a second cancer drug. In some embodiments, the method further comprises (iv) treating the patient with a therapy comprising a c-met antagonist antibody (e.g., onartuzumab).

In one aspect, provided is a method of identifying a patient having a mesothelioma (e.g., a previously treated mesothelioma) as likely to respond to therapy comprising (a) an anti-c-met antibody (e.g., onartuzumab) and (b) an anti-VEGF antibody (e.g., bevacizumab), the method comprising: (i) measuring HGF biomarker in a sample from the patient, wherein the HGF biomarker is HGF nucleic acid (e.g., mRNA) and measuring is by ISH; and (ii) identifying the patient as more likely to respond to therapy comprising (a) a c-met antagonist antibody (e.g., onartuzumab) and (b) an anti-VEGF antibody (e.g., bevacizumab) when the sample has high HGF biomarker. In some embodiments, the method further comprises (iii) selecting for the patient a therapy comprising (a) a c-met antagonist antibody (e.g., onartuzumab) and (b) an anti-VEGF antibody (e.g., bevacizumab) or recommending a therapy comprising (a) a c-met antagonist antibody (e.g., onartuzumab) and (b) an anti-VEGF antibody (e.g., bevacizumab). In some embodiments, the method further comprises (iv) treating the patient with a therapy comprising (a) a c-met antagonist antibody (e.g., onartuzumab) and (b) an anti-VEGF antibody (e.g., bevacizumab).

In one aspect, provided is a method of identifying a patient having mesothelioma (e.g., previously treated mesothelioma) as likely to respond to therapy comprising an anti-c-met antibody (e.g., onartuzumab), the method comprising: (i) measuring HGF biomarker in a sample from the patient, wherein the HGF biomarker is HGF nucleic acid (e.g., mRNA) and measuring is by ISH; and (ii) identifying the patient as more likely to respond to therapy comprising a c-met antagonist antibody (e.g., onartuzumab) when the sample has high HGF biomarker. In some embodiments, the method further comprises (iii) selecting a therapy comprising a c-met antagonist antibody (e.g., onartuzumab) or recommending a therapy comprising a c-met antagonist antibody (e.g., onartuzumab) for the patient. In some embodiments, the therapy further comprises a second cancer drug. In some embodiments, the method further comprises (iv) treating the patient with a therapy comprising a c-met antagonist antibody (e.g., onartuzumab).

In one aspect, provided is a method of identifying a patient with gastric cancer (e.g., previously treated gastric cancer) as likely to respond to therapy comprising (a) an anti-c-met antibody (e.g., onartuzumab) and (b) an anti-VEGF antibody (e.g., bevacizumab), the method comprising: (i) measuring HGF biomarker in a sample from the patient, wherein the HGF biomarker is HGF nucleic acid (e.g., mRNA) and measuring is by ISH; and (ii) identifying the patient as more likely to respond to therapy comprising (a) a c-met antagonist antibody (e.g., onartuzumab) and (b) an anti-VEGF antibody (e.g., bevacizumab) when the sample has high HGF biomarker. In some embodiments, the method further comprises (iii) selecting for the patient a therapy comprising (a) a c-met antagonist antibody (e.g., onartuzumab) and (b) an anti-VEGF antibody (e.g., bevacizumab) or recommending a therapy comprising (a) a c-met antagonist antibody (e.g., onartuzumab) and (b) an anti-VEGF antibody (e.g., bevacizumab). In some embodiments, the method further comprises (iv) treating the patient with a therapy comprising (a) a c-met antagonist antibody (e.g., onartuzumab) and (b) an anti-VEGF antibody (e.g., bevacizumab).

In one aspect, provided is a method of identifying a patient having gastric cancer (e.g., previously treated gastric cancer) as likely to respond to therapy comprising an anti-c-met antibody (e.g., onartuzumab), the method comprising: (i) measuring HGF biomarker in a sample from the patient, wherein the HGF biomarker is HGF nucleic acid (e.g., mRNA) and measuring is by ISH; and (ii) identifying the patient as more likely to respond to therapy comprising a c-met antagonist antibody (e.g., onartuzumab) when the sample has high HGF biomarker. In some embodiments, the method further comprises (iii) selecting a therapy comprising a c-met antagonist antibody (e.g., onartuzumab) or recommending a therapy comprising a c-met antagonist antibody (e.g., onartuzumab) for the patient. In some embodiments, the therapy further comprises a second cancer drug. In some embodiments, the method further comprises (iv) treating the patient with a therapy comprising a c-met antagonist antibody (e.g., onartuzumab).

In one aspect, provided is a method of identifying a patient having renal cell carcinoma (e.g., a previously treated renal cell carcinoma) as likely to respond to therapy comprising (a) an anti-c-met antibody (e.g., onartuzumab) and (b) an anti-VEGF antibody (e.g., bevacizumab), the method comprising: (i) measuring HGF biomarker in a sample from the patient, wherein the HGF biomarker is HGF nucleic acid (e.g., mRNA) and measuring is by ISH; and (ii) identifying the patient as more likely to respond to therapy comprising (a) a c-met antagonist antibody (e.g., onartuzumab) and (b) an anti-VEGF antibody (e.g., bevacizumab) when the sample has high HGF biomarker. In some embodiments, the method further comprises (iii) selecting for the patient a therapy comprising (a) a c-met antagonist antibody (e.g., onartuzumab) and (b) an anti-VEGF antibody (e.g., bevacizumab) or recommending a therapy comprising (a) a c-met antagonist antibody (e.g., onartuzumab) and (b) an anti-VEGF antibody (e.g., bevacizumab). In some embodiments, the method further comprises (iv) treating the patient with a therapy comprising (a) a c-met antagonist antibody (e.g., onartuzumab) and (b) an anti-VEGF antibody (e.g., bevacizumab).

In one aspect, provided is a method of identifying a patient having renal cell carcinoma (e.g., previously treated renal cell carcinoma) as likely to respond to a therapy comprising an anti-c-met antibody (e.g., onartuzumab), the method comprising: (i) measuring HGF biomarker in a sample from the patient, wherein the HGF biomarker is HGF nucleic acid (e.g., mRNA) and measuring is by ISH; and (ii) identifying the patient as more likely to respond to therapy comprising a c-met antagonist antibody (e.g., onartuzumab) when the sample has high HGF biomarker. In some embodiments, the method further comprises (iii) selecting a therapy comprising a c-met antagonist antibody (e.g., onartuzumab) or recommending a therapy comprising a c-met antagonist antibody (e.g., onartuzumab) for the patient. In some embodiments, the therapy further comprises a second cancer drug. In some embodiments, the method further comprises (iv) treating the patient with a therapy comprising a c-met antagonist antibody (e.g., onartuzumab).

In one aspect, provided is a method of identifying a patient with hepatocellular carcinoma (previously treated hepatocellular carcinoma) as likely to respond to therapy comprising (a) an anti-c-met antibody (e.g., onartuzumab) and (b) an anti-VEGF antibody (e.g., bevacizumab), the method comprising: (i) measuring HGF biomarker in a sample from the patient, wherein the HGF biomarker is HGF nucleic acid (e.g., mRNA) and measuring is by ISH; and (ii) identifying the patient as more likely to respond to therapy comprising (a) a c-met antagonist antibody (e.g., onartuzumab) and (b) an anti-VEGF antibody (e.g., bevacizumab) when the sample has high HGF biomarker. In some embodiments, the method further comprises (iii) selecting for the patient a therapy comprising (a) a c-met antagonist antibody (e.g., onartuzumab) and (b) an anti-VEGF antibody (e.g., bevacizumab) or recommending a therapy comprising (a) a c-met antagonist antibody (e.g., onartuzumab) and (b) an anti-VEGF antibody (e.g., bevacizumab). In some embodiments, the method further comprises (iv) treating the patient with a therapy comprising (a) a c-met antagonist antibody (e.g., onartuzumab) and (b) an anti-VEGF antibody (e.g., bevacizumab).

In one aspect, provided is a method of identifying a patient having hepatocellular carcinoma (e.g., previously treated hepatocellular carcinoma) as likely to respond to therapy comprising an anti-c-met antibody (e.g., onartuzumab), the method comprising: (i) measuring HGF biomarker in a sample from the patient, wherein the HGF biomarker is HGF nucleic acid (e.g., mRNA) and measuring is by ISH; and (ii) identifying the patient as more likely to respond to therapy comprising a c-met antagonist antibody (e.g., onartuzumab) when the sample has high HGF biomarker. In some embodiments, the method further comprises (iii) selecting a therapy comprising a c-met antagonist antibody (e.g., onartuzumab) or recommending a therapy comprising a c-met antagonist antibody (e.g., onartuzumab) for the patient. In some embodiments, the therapy further comprises a second cancer drug. In some embodiments, the method further comprises (iv) treating the patient with a therapy comprising a c-met antagonist antibody (e.g., onartuzumab).

In one aspect, provided is a method of identifying a patient having a sarcoma (e.g., a previously treated sarcoma) as likely to respond to therapy comprising (a) an anti-c-met antibody (e.g., onartuzumab) and (b) an anti-VEGF antibody (e.g., bevacizumab), the method comprising: (i) measuring HGF biomarker in a sample from the patient, wherein the HGF biomarker is HGF nucleic acid (e.g., mRNA) and measuring is by ISH; and (ii) identifying the patient as more likely to respond to therapy comprising (a) a c-met antagonist antibody (e.g., onartuzumab) and (b) an anti-VEGF antibody (e.g., bevacizumab) when the sample has high HGF biomarker. In some embodiments, the method further comprises (iii) selecting for the patient a therapy comprising (a) a c-met antagonist antibody (e.g., onartuzumab) and (b) an anti-VEGF antibody (e.g., bevacizumab) or recommending a therapy comprising (a) a c-met antagonist antibody (e.g., onartuzumab) and (b) an anti-VEGF antibody (e.g., bevacizumab). In some embodiments, the method further comprises (iv) treating the patient with a therapy comprising (a) a c-met antagonist antibody (e.g., onartuzumab) and (b) an anti-VEGF antibody (e.g., bevacizumab).

In one aspect, provided is a method of identifying a patient having a sarcoma (e.g., a previously treated sarcoma) as likely to respond to therapy comprising an anti-c-met antibody (e.g., onartuzumab), the method comprising: (i) measuring HGF biomarker in a sample from the patient, wherein the HGF biomarker is HGF nucleic acid (e.g., mRNA) and measuring is by ISH; and (ii) identifying the patient as more likely to respond to therapy comprising a c-met antagonist antibody (e.g., onartuzumab) when the sample has high HGF biomarker. In some embodiments, the method further comprises (iii) selecting a therapy comprising a c-met antagonist antibody (e.g., onartuzumab) or recommending a therapy comprising a c-met antagonist antibody (e.g., onartuzumab) for the patient. In some embodiments, the therapy further comprises a second cancer drug. In some embodiments, the method further comprises (iv) treating the patient with a therapy comprising a c-met antagonist antibody (e.g., onartuzumab).

In one aspect, provided is a method of identifying a patient having a glioblastoma (e.g., a previously treated glioblastoma) as likely to respond to a therapy comprising (a) an anti-c-met antibody (e.g., onartuzumab) and (b) an anti-VEGF antibody (e.g., bevacizumab), the method comprising: (i) measuring HGF biomarker in a sample from the patient, wherein the HGF biomarker is HGF nucleic acid (e.g., mRNA) and measuring is by a PCR-based (e.g., rt-qPCR) assay; and (ii) identifying the patient as more likely to respond to therapy comprising (a) a c-met antagonist antibody (e.g., onartuzumab) and (b) an anti-VEGF antibody (e.g., bevacizumab) when the sample has high HGF biomarker. In some embodiments, the method further comprises (iii) selecting for the patient a therapy comprising (a) a c-met antagonist antibody (e.g., onartuzumab) and (b) an anti-VEGF antibody (e.g., bevacizumab) or recommending a therapy comprising (a) a c-met antagonist antibody (e.g., onartuzumab) and (b) an anti-VEGF antibody (e.g., bevacizumab). In some embodiments, the method further comprises (iv) treating the patient with a therapy comprising (a) a c-met antagonist antibody (e.g., onartuzumab) and (b) an anti-VEGF antibody (e.g., bevacizumab).

In one aspect, provided is a method of identifying a patient having a glioblastoma (e.g., a previously treated glioblastoma) as likely to respond to a therapy comprising an anti-c-met antibody (e.g., onartuzumab), the method comprising: (i) measuring HGF biomarker in a sample from the patient, wherein the HGF biomarker is HGF nucleic acid (e.g., mRNA) and measuring is by a PCR-based (e.g., rt-qPCR) assay; and (ii) identifying the patient as more likely to respond to therapy comprising a c-met antagonist antibody (e.g., onartuzumab) when the sample has high HGF biomarker. In some embodiments, the method further comprises (iii) selecting a therapy comprising a c-met antagonist antibody (e.g., onartuzumab) or recommending a therapy comprising a c-met antagonist antibody (e.g., onartuzumab) for the patient. In some embodiments, the therapy further comprises a second cancer drug. In some embodiments, the method further comprises (iv) treating the patient with a therapy comprising a c-met antagonist antibody (e.g., onartuzumab).

In one aspect, provided is a method of identifying a patient having a mesothelioma (e.g., a previously treated mesothelioma) as likely to respond to therapy comprising (a) an anti-c-met antibody (e.g., onartuzumab) and (b) an anti-VEGF antibody (e.g., bevacizumab), the method comprising: (i) measuring HGF biomarker in a sample from the patient, wherein the HGF biomarker is HGF nucleic acid (e.g., mRNA) and measuring is by a PCR-based (e.g., rt-qPCR) assay; and (ii) identifying the patient as more likely to respond to therapy comprising (a) a c-met antagonist antibody (e.g., onartuzumab) and (b) an anti-VEGF antibody (e.g., bevacizumab) when the sample has high HGF biomarker. In some embodiments, the method further comprises (iii) selecting for the patient a therapy comprising (a) a c-met antagonist antibody (e.g., onartuzumab) and (b) an anti-VEGF antibody (e.g., bevacizumab) or recommending a therapy comprising (a) a c-met antagonist antibody (e.g., onartuzumab) and (b) an anti-VEGF antibody (e.g., bevacizumab). In some embodiments, the method further comprises (iv) treating the patient with a therapy comprising (a) a c-met antagonist antibody (e.g., onartuzumab) and (b) an anti-VEGF antibody (e.g., bevacizumab).

In one aspect, provided is a method of identifying a patient having mesothelioma (e.g., previously treated mesothelioma) as likely to respond to therapy comprising an anti-c-met antibody (e.g., onartuzumab), the method comprising: (i) measuring HGF biomarker in a sample from the patient, wherein the HGF biomarker is HGF nucleic acid (e.g., mRNA) and measuring is by a PCR-based (e.g., rt-qPCR) assay; and (ii) identifying the patient as more likely to respond to therapy comprising a c-met antagonist antibody (e.g., onartuzumab) when the sample has high HGF biomarker. In some embodiments, the method further comprises (iii) selecting a therapy comprising a c-met antagonist antibody (e.g., onartuzumab) or recommending a therapy comprising a c-met antagonist antibody (e.g., onartuzumab) for the patient. In some embodiments, the therapy further comprises a second cancer drug. In some embodiments, the method further comprises (iv) treating the patient with a therapy comprising a c-met antagonist antibody (e.g., onartuzumab).

In one aspect, provided is a method of identifying a patient with gastric cancer (e.g., previously treated gastric cancer) as likely to respond to therapy comprising (a) an anti-c-met antibody (e.g., onartuzumab) and (b) an anti-VEGF antibody (e.g., bevacizumab), the method comprising: (i) measuring HGF biomarker in a sample from the patient, wherein the HGF biomarker is HGF nucleic acid (e.g., mRNA) and measuring is by a PCR-based (e.g., rt-qPCR) assay; and (ii) identifying the patient as more likely to respond to therapy comprising (a) a c-met antagonist antibody (e.g., onartuzumab) and (b) an anti-VEGF antibody (e.g., bevacizumab) when the sample has high HGF biomarker. In some embodiments, the method further comprises (iii) selecting for the patient a therapy comprising (a) a c-met antagonist antibody (e.g., onartuzumab) and (b) an anti-VEGF antibody (e.g., bevacizumab) or recommending a therapy comprising (a) a c-met antagonist antibody (e.g., onartuzumab) and (b) an anti-VEGF antibody (e.g., bevacizumab). In some embodiments, the method further comprises (iv) treating the patient with a therapy comprising (a) a c-met antagonist antibody (e.g., onartuzumab) and (b) an anti-VEGF antibody (e.g., bevacizumab).

In one aspect, provided is a method of identifying a patient having gastric cancer (e.g., previously treated gastric cancer) as likely to respond to therapy comprising an anti-c-met antibody (e.g., onartuzumab), the method comprising: (i) measuring HGF biomarker in a sample from the patient, wherein the HGF biomarker is HGF nucleic acid (e.g., mRNA) and measuring is by a PCR-based (e.g., rt-qPCR) assay; and (ii) identifying the patient as more likely to respond to therapy comprising a c-met antagonist antibody (e.g., onartuzumab) when the sample has high HGF biomarker. In some embodiments, the method further comprises (iii) selecting a therapy comprising a c-met antagonist antibody (e.g., onartuzumab) or recommending a therapy comprising a c-met antagonist antibody (e.g., onartuzumab) for the patient. In some embodiments, the therapy further comprises a second cancer drug. In some embodiments, the method further comprises (iv) treating the patient with a therapy comprising a c-met antagonist antibody (e.g., onartuzumab).

In one aspect, provided is a method of identifying a patient having renal cell carcinoma (e.g., a previously treated renal cell carcinoma) as likely to respond to therapy comprising (a) an anti-c-met antibody (e.g., onartuzumab) and (b) an anti-VEGF antibody (e.g., bevacizumab), the method comprising: (i) measuring HGF biomarker in a sample from the patient, wherein the HGF biomarker is HGF nucleic acid (e.g., mRNA) and measuring is by a PCR-based (e.g., rt-qPCR) assay; and (ii) identifying the patient as more likely to respond to therapy comprising (a) a c-met antagonist antibody (e.g., onartuzumab) and (b) an anti-VEGF antibody (e.g., bevacizumab) when the sample has high HGF biomarker. In some embodiments, the method further comprises (iii) selecting for the patient a therapy comprising (a) a c-met antagonist antibody (e.g., onartuzumab) and (b) an anti-VEGF antibody (e.g., bevacizumab) or recommending a therapy comprising (a) a c-met antagonist antibody (e.g., onartuzumab) and (b) an anti-VEGF antibody (e.g., bevacizumab). In some embodiments, the method further comprises (iv) treating the patient with a therapy comprising (a) a c-met antagonist antibody (e.g., onartuzumab) and (b) an anti-VEGF antibody (e.g., bevacizumab).

In one aspect, provided is a method of identifying a patient having renal cell carcinoma (e.g., previously treated renal cell carcinoma) as likely to respond to a therapy comprising an anti-c-met antibody (e.g., onartuzumab), the method comprising: (i) measuring HGF biomarker in a sample from the patient, wherein the HGF biomarker is HGF nucleic acid (e.g., mRNA) and measuring is by a PCR-based (e.g., rt-qPCR) assay; and (ii) identifying the patient as more likely to respond to therapy comprising a c-met antagonist antibody (e.g., onartuzumab) when the sample has high HGF biomarker. In some embodiments, the method further comprises (iii) selecting a therapy comprising a c-met antagonist antibody (e.g., onartuzumab) or recommending a therapy comprising a c-met antagonist antibody (e.g., onartuzumab) for the patient. In some embodiments, the therapy further comprises a second cancer drug. In some embodiments, the method further comprises (iv) treating the patient with a therapy comprising a c-met antagonist antibody (e.g., onartuzumab).

In one aspect, provided is a method of identifying a patient with hepatocellular carcinoma (e.g., previously treated hepatocellular carcinoma) as likely to respond to therapy comprising (a) an anti-c-met antibody (e.g., onartuzumab) and (b) an anti-VEGF antibody (e.g., bevacizumab), the method comprising: (i) measuring HGF biomarker in a sample from the patient, wherein the HGF biomarker is HGF nucleic acid (e.g., mRNA) and measuring is by a PCR-based (e.g., rt-qPCR) assay; and (ii) identifying the patient as more likely to respond to therapy comprising (a) a c-met antagonist antibody (e.g., onartuzumab) and (b) an anti-VEGF antibody (e.g., bevacizumab) when the sample has high HGF biomarker. In some embodiments, the method further comprises (iii) selecting for the patient a therapy comprising (a) a c-met antagonist antibody (e.g., onartuzumab) and (b) an anti-VEGF antibody (e.g., bevacizumab) or recommending a therapy comprising (a) a c-met antagonist antibody (e.g., onartuzumab) and (b) an anti-VEGF antibody (e.g., bevacizumab). In some embodiments, the method further comprises (iv) treating the patient with a therapy comprising (a) a c-met antagonist antibody (e.g., onartuzumab) and (b) an anti-VEGF antibody (e.g., bevacizumab).

In one aspect, provided is a method of identifying a patient having hepatocellular carcinoma (e.g., previously treated hepatocellular carcinoma) as likely to respond to therapy comprising an anti-c-met antibody (e.g., onartuzumab), the method comprising: (i) measuring HGF biomarker in a sample from the patient, wherein the HGF biomarker is HGF nucleic acid (e.g., mRNA) and measuring is by a PCR-based (e.g., rt-qPCR) assay; and (ii) identifying the patient as more likely to respond to therapy comprising a c-met antagonist antibody (e.g., onartuzumab) when the sample has high HGF biomarker. In some embodiments, the method further comprises (iii) selecting a therapy comprising a c-met antagonist antibody (e.g., onartuzumab) or recommending a therapy comprising a c-met antagonist antibody (e.g., onartuzumab) for the patient. In some embodiments, the therapy further comprises a second cancer drug. In some embodiments, the method further comprises (iv) treating the patient with a therapy comprising a c-met antagonist antibody (e.g., onartuzumab).

In one aspect, provided is a method of identifying a patient having a sarcoma (e.g., a previously treated sarcoma) as likely to respond to therapy comprising (a) an anti-c-met antibody (e.g., onartuzumab) and (b) an anti-VEGF antibody (e.g., bevacizumab), the method comprising: (i) measuring HGF biomarker in a sample from the patient, wherein the HGF biomarker is HGF nucleic acid (e.g., mRNA) and measuring is by a PCR-based (e.g., rt-qPCR) assay; and (ii) identifying the patient as more likely to respond to therapy comprising (a) a c-met antagonist antibody (e.g., onartuzumab) and (b) an anti-VEGF antibody (e.g., bevacizumab) when the sample has high HGF biomarker. In some embodiments, the method further comprises (iii) selecting for the patient a therapy comprising (a) a c-met antagonist antibody (e.g., onartuzumab) and (b) an anti-VEGF antibody (e.g., bevacizumab) or recommending a therapy comprising (a) a c-met antagonist antibody (e.g., onartuzumab) and (b) an anti-VEGF antibody (e.g., bevacizumab). In some embodiments, the method further comprises (iv) treating the patient with a therapy comprising (a) a c-met antagonist antibody (e.g., onartuzumab) and (b) an anti-VEGF antibody (e.g., bevacizumab).

In one aspect, provided is a method of identifying a patient having a sarcoma (e.g., a previously treated sarcoma) as likely to respond to therapy comprising an anti-c-met antibody (e.g., onartuzumab), the method comprising: (i) measuring HGF biomarker in a sample from the patient, wherein the HGF biomarker is HGF nucleic acid (e.g., mRNA) and measuring is by a PCR-based (e.g., rt-qPCR) assay; and (ii) identifying the patient as more likely to respond to therapy comprising a c-met antagonist antibody (e.g., onartuzumab) when the sample has high HGF biomarker. In some embodiments, the method further comprises (iii) selecting a therapy comprising a c-met antagonist antibody (e.g., onartuzumab) or recommending a therapy comprising a c-met antagonist antibody (e.g., onartuzumab) for the patient. In some embodiments, the therapy further comprises a second cancer drug. In some embodiments, the method further comprises (iv) treating the patient with a therapy comprising a c-met antagonist antibody (e.g., onartuzumab).

In one aspect, provided is a method for determining HGF biomarker expression, comprising the step of determining whether a cancer of a patient has a high level of HGF biomarker, wherein the HGF biomarker expression is mRNA expression and is determined in a sample from the patient using ISH, wherein high HGF biomarker expression is an ISH score greater than 2+, wherein the high HGF biomarker expression indicates that the patient is likely to have prolonged OS and/or PFS when the patient is treated with an anti-c-met antibody (e.g., onartuzumab) in combination with an anti-VEGF antibody (e.g., bevacizumab).

In one aspect, provided is a method for determining HGF biomarker expression, comprising the step of determining whether a cancer of a patient has a high level of HGF biomarker, wherein the HGF biomarker expression is mRNA expression and is determined in a sample from the patient using ISH, wherein high HGF biomarker expression is an ISH score greater than 2+, wherein the high HGF biomarker expression indicates that the patient is likely to have prolonged OS and/or PFS when the patient is treated with an anti-c-met antibody (e.g., onartuzumab). In some embodiments, the patient is treated with an anti-c-met antibody (e.g., onartuzumab) optionally in combination with a second cancer drug.

In one aspect, provided is a method for determining HGF biomarker expression, comprising the step of determining whether a cancer of a patient has a high level of HGF biomarker, wherein the HGF biomarker expression is mRNA expression and is determined in a sample from the patient using a PCR-based (e.g., rt-qPCR) assay, wherein high HGF biomarker expression is an HGF expression level in the upper 25% of a reference patient population, wherein the high HGF biomarker expression indicates that the patient is likely to have prolonged OS and/or PFS when the patient is treated with an anti-c-met antibody (e.g., onartuzumab) in combination with an anti-VEGF antibody (e.g., bevacizumab).

In one aspect, provided is a method for determining HGF biomarker expression, comprising the step of determining whether a cancer of a patient has a high level of HGF biomarker, wherein the HGF biomarker expression is mRNA expression and is determined in a sample from the patient using a PCR-based (e.g., rt-qPCR) assay, wherein high HGF biomarker expression is an HGF expression level in the upper 25% of a reference patient population, wherein the high HGF biomarker expression indicates that the patient is likely to have prolonged OS and/or PFS when the patient is treated with an anti-c-met antibody (e.g., onartuzumab). In some embodiments, the patient is treated with an anti-c-met antibody (e.g., onartuzumab) optionally in combination with a second cancer drug.

In some embodiments, recommending treatment refers to using information or data generated relating to the level or presence of c-met in a sample of a patient to identify the patient as suitable or unsuitable for treatment with a certain therapy. In some embodiments, the therapy may comprise a c-met antibody (e.g., onartuzumab). In some embodiments, the therapy may comprise a VEGF antagonist (e.g., bevacizumab). In some embodiments, the therapy may comprise an anti-c-met antibody (e.g., onartuzumab) in combination with a VEGF antagonist (e.g., bevacizumab). The information or data may be in any form, written, spoken, or electronic. In some embodiments, using the generated information or data includes communicating, presenting, reporting, storing, sending, transferring, provisioning, transmitting, delivering, distributing, or a combination thereof. In some embodiments, the communicating, presenting, reporting, storing, sending, transferring, provisioning, transmitting, delivering, distributing, or a combination thereof, is performed by a computing device, an analysis unit, or a combination thereof. In some further embodiments, the communicating, presenting, reporting, storing, sending, transferring, supplying, transmitting, distributing, or a combination thereof, is performed by an individual (e.g., a laboratory or medical professional). In some embodiments, the information or data comprises a comparison of the level of HGF to a reference level. In some embodiments, the information or data comprises an indication of the presence or absence of HGF in the sample. In some embodiments, the information or data includes an indication that HGF ISH signal strength is present at a particular level (e.g., 0, 1+, 2+, 3 +). In some embodiments, the information or data includes an indication that HGF ISH signal intensity is present in a particular percentage of cells (e.g., glioblastoma tumor cells and benign stromal cells, mesothelioma tumor cells and benign stromal cells, gastric cancer tumor cells and benign stromal cells, hepatocellular carcinoma tumor cells and benign stromal cells, renal cell carcinoma tumor cells and benign stromal cells, or sarcoma tumor cells and benign stromal cells). In some embodiments, the information or data includes an indication that the HGFmRNA expression level is in a particular percentile compared to the HGF mRNA expression level in tumors obtained from a reference patient population comprising a representative number of patients, including patients with a particular cancer (e.g., the first 50%, the first 40%, the first 35%, the first 30%, the first 25%, the first 20%, the second 50%, the second 60%, the second 65%, the second 70%, the second 75%, the second 80%). In some embodiments, the information or data includes an indication that the patient is suitable or unsuitable for treatment with a therapy comprising a c-met antagonist antibody (e.g., onartuzumab). In some embodiments, the information or data includes an indication that the patient is suitable or unsuitable for treatment with a therapy comprising a c-met antagonist antibody (e.g., onartuzumab) in combination with a second cancer drug. In some embodiments, the information or data includes an indication that the patient is suitable or unsuitable for treatment with a therapy comprising a c-met antagonist antibody (e.g., onartuzumab) in combination with a VEGF antagonist (e.g., bevacizumab).

In one aspect, provided are methods for advertising c-met antibodies, comprising promoting treatment of a patient with cancer with a c-met antibody based on expression of HGF biomarker to a target audience. In some embodiments, the promotion is by a package insert accompanying a commercial formulation of the anti-c-met antibody. In some embodiments, the promotion is by a package insert accompanying a commercial formulation of the second medicament. In some embodiments, the second drug is a chemotherapeutic agent. In some embodiments, the second drug is a VEGF antagonist. In some embodiments, the anti-c-met antibody is onartuzumab and the VEGF antagonist is bevacizumab. In some embodiments, the patient is selected for treatment with a c-met antagonist if the cancer sample expresses the biomarker at a high level. In some embodiments, the promotion is by a package insert, wherein the package insert provides instructions to receive anti-c-met antibody therapy in combination with a VEGF antagonist. In some embodiments, the promotion is followed by treating the patient with the anti-c-met antibody with or without a second drug.

In some embodiments, the promotion comprises promotion of a therapeutic agent, such as an anti-c-met antagonist (e.g., onartuzumab) and/or a VEGF antagonist (e.g., bevacizumab), for a therapeutic indication, such as glioblastoma (e.g., recurrent glioblastoma), mesothelioma (e.g., recurrent mesothelioma), gastric cancer (e.g., recurrent gastric cancer), renal cell cancer (e.g., recurrent renal cell carcinoma), hepatocellular carcinoma (e.g., recurrent hepatocellular carcinoma), or sarcoma (e.g., recurrent sarcoma), wherein such promotion is approved by the Food and Drug Administration (FDA) as having been demonstrated to be associated with statistically significant therapeutic efficacy and acceptable safety in a population of subjects.

In one aspect, provided are diagnostic kits comprising one or more reagents for determining expression of an HGF biomarker in a sample from a cancer patient, wherein detection of a high amount of the HGF biomarker means extended survival (e.g., PFS and/or OS) when the patient is treated with an effective amount of a c-met antagonist. In some embodiments, the cancer is glioblastoma, mesothelioma, hepatocellular carcinoma, renal cell carcinoma, gastric cancer, sarcoma (e.g., osteosarcoma), non-small cell lung cancer, breast cancer, gallbladder cancer, or pancreatic cancer. In some embodiments, the cancer is a previously treated glioblastoma, mesothelioma, renal cell carcinoma, gastric cancer, hepatocellular carcinoma, or sarcoma (e.g., osteosarcoma). In some embodiments, detection of a high amount of the HGF biomarker means extended survival (e.g., PFS and/or OS) when the patient is treated with an effective amount of a combination of a c-met antagonist and a second cancer drug. In some embodiments, detection of a high amount of the HGF biomarker means extended survival (e.g., PFS and/or OS) when the patient is treated with a combination of an effective amount of a c-met antagonist and a standard of care antineoplastic agent. In some embodiments, detection of a high amount of the HGF biomarker means extended survival (e.g., PFS and/or OS) when the patient is treated with an effective amount of a combination of a c-met antagonist and a VEGF antagonist. In some embodiments, the kit further comprises instructions for using the kit to select a c-met antagonist to treat the previously treated cancer patient if a high amount of HGF biomarker is determined.

In one aspect, provided is a method of making any of the diagnostic kits provided herein, comprising combining in one package a pharmaceutical composition comprising a cancer drug and a package insert indicating that the pharmaceutical composition is for treating a patient having cancer based on expression of HGF biomarker.

In some embodiments of any of the methods of the invention, the method further comprises testing a sample of the patient for a biomarker. In some embodiments, the biomarker is a c-met biomarker. In some embodiments, the high c-met biomarker is determined using any of the methods provided herein. In some embodiments, the biomarker is HGF biomarker. In some embodiments, high HGF biomarker is determined using any of the methods provided herein.

Brief Description of Drawings

FIG. 1: an overview of the study design is shown.

FIG. 2: overall survival was shown according to a subgroup analysis of HGF ISH status. 41 patients (approximately 32% of all patients) had HGF 2+ or 3+ samples. HR was not stratified.

FIG. 3: Kaplan-Meier analysis showing overall survival in HGF ISH low (0/1+) patients and HGF ISH high (2+/3+) patients. Bevacizumab + placebo arm ═ solid line. Bevacizumab + onartuzumab branch ═ dashed line.

FIG. 4: showing progression free survival was analyzed according to a subgroup of HGF ISH status. HR was not stratified.

FIG. 5: Kaplan-Meier analysis showing progression free survival in HGF ISH low (0/1+) patients and HGF ISH high (2+/3+) patients. Bevacizumab + placebo arm ═ solid line. Bevacizumab + onartuzumab ═ dashed line.

FIG. 6: analysis of overall survival in patients randomized to bevacizumab + placebo (solid line) compared to patients randomized to bevacizumab + onartuzumab (dashed line) is shown. HR was from stratified analysis.

FIG. 7: analysis of progression free survival in patients randomized to bevacizumab + placebo (solid line) compared to patients randomized to bevacizumab + onartuzumab (dashed line). HR was from stratified analysis.

FIG. 8: exemplary photomicrographs of glioblastoma sections showing 3+ HGF ISH signal. The sections were viewed using the 10-fold objective lens and positive cells were easily identified. Arrows indicate exemplary HGF ISH signal positive cells.

FIG. 9: an exemplary micrograph of a glioblastoma section shown in fig. 10 when viewed at high magnification (roughly equivalent to a 40-fold objective lens). HGF ISH signal was observed in multiple cells spread throughout the field of view.

FIG. 10: exemplary photomicrographs of glioblastoma sections showing 1+ HGF ISH signal. Sections were viewed using low magnification (roughly equivalent to a 10-fold objective lens) and it was difficult to identify HGF ISH signal positive cells.

FIG. 11: an exemplary micrograph of a glioblastoma section shown in fig. 10 when viewed at high magnification (roughly equivalent to a 40-fold objective lens). Weak HGF ISH signals were observed in cells scattered throughout the field of view. Arrows indicate exemplary HGFISH signal positive cells.

FIG. 12: an exemplary micrograph of a glioblastoma section showing 3+ HGF ISH signal, viewed at medium magnification (roughly equivalent to a 20-fold objective). HGF ISH positive signals were observed in multiple cells at the invasive margin of the tumor.

FIG. 13: representative in situ hybridization of HGF RNA in gastric cancer showing focal (arrow) high expression (3+) in stromal cells. Probe hybridization was visualized by brown chromogen spots against blue hematoxylin counterstain. Bar 100 um.

FIG. 14: representative in situ hybridization of HGF RNA in mesothelioma cancer is shown. Probe hybridization was visualized by red chromogen against blue hematoxylin counterstain.

FIG. 15: representative in situ hybridization of HGF RNA in mesothelioma cancers showing HGF expression with intratumoral heterogeneity. Probe hybridization was visualized by red chromogen against blue hematoxylin counterstain.

FIG. 16: representative in situ hybridization of HGF in mesothelioma cancers displaying autocrine HGF expression is shown. Probe hybridization was visualized by red chromogen against blue hematoxylin counterstain.

FIG. 17: overall survival was shown according to a subgroup analysis of HGF-PCR status. HR was not stratified.

FIG. 18: Kaplan-Meier analysis showing overall survival in patients with low HGF-PCR (last 75%) and in patients with high HGF-PCR (first 25%). Bevacizumab + placebo arm ═ solid line. Bevacizumab + onartuzumab branch ═ dashed line.

FIG. 19: progression free survival was shown to follow a subgroup analysis of HGF-PCR status. HR was not stratified.

FIG. 20: Kaplan-Meier analysis showing progression free survival in patients with low HGF-PCR (last 75%) and in patients with high HGF-PCR (first 25%). Bevacizumab + placebo arm ═ solid line. Bevacizumab + onartuzumab branch ═ dashed line.

FIG. 21: the Overall Response Rate (ORR) was shown to be high in HGF-PCR in the bevacizumab + onartuzumab arm (top 25%) patients compared to patients in the bevacizumab + placebo arm.

FIG. 22: prognostic effect of progression free survival (top) and overall survival (bottom) in HGF-PCR low (last 75%) and HGF-PCR high (first 25%) patients in bevacizumab + placebo arms is shown.

Detailed Description

I. Definition of

The term "anti-angiogenic agent" or "angiogenesis inhibitor" refers to a small molecular weight substance, polynucleotide, polypeptide, isolated protein, recombinant protein, antibody, or conjugate or fusion protein thereof that inhibits, either directly or indirectly, angiogenesis (vasculogenesis) or unwanted vascular permeability. It is understood that anti-angiogenic agents include those agents that bind to and block the angiogenic activity of angiogenic factors or their receptors. For example, an anti-angiogenic agent is an antibody or other antagonist of an angiogenic agent as defined throughout the specification or known in the art, such as, but not limited to, an antibody to VEGF-a, an antibody to a VEGF-a receptor (e.g., KDR receptor or Flt-1 receptor), a VEGF trap, an anti-PDGFR inhibitor such as Gleevec^TM(Imatinib Mesylate). Anti-angiogenic agents also include natural angiogenesis inhibitors such as angiostatin (angiostatin), endostatin (endostatin), and the like. See, e.g., Klagsbrun and D' Amore, Annu. Rev. physiol.,53:217-39 (1991); streit and Detmar, Oncogene,22: 3172-; ferrara&Alitalo, Nature medicine 5: 1359-; tonnii et al, Oncogene,22: 6549-; and sato. int.j.clin.oncol.,8: 200-.

The term "Bevacizumab" (Bevacizumab) refers to a recombinant humanized anti-VEGF monoclonal antibody produced according to Presta et al (1997) Cancer Res.57:4593-4599, also known as "rhuMAb VEGF" orIt contains the mutated human IgG1 framework regions and antigen binding complementarity determining regions from the murine anti-hVEGF monoclonal antibody a.4.6.1 (which blocks the binding of human VEGF to its receptor). Bevacizumab derives approximately 93% of the amino acid sequence (including most of the framework regions) from human IgG1, and approximately 7% of the sequence from the murine antibody a4.6.1. Bevacizumab and hybridoma ATCC HB 10709, and a4.6.1 binds to the same epitope.

"epitope A4.6.1" refers to bevacizumab which is an anti-VEGF antibody(see Muller Y et., Structure,15September 1998,6: 1153-1167). In certain embodiments of the invention, anti-VEGF antibodies include, but are not limited to, monoclonal antibodies that bind the same epitope as the monoclonal anti-VEGF antibody a4.6.1 produced by hybridoma ATCC HB 10709; recombinant humanized anti-VEGF monoclonal antibodies generated according to Presta et al (1997) Cancer Res.57: 4593-4599.

The term "intravenous infusion" refers to the introduction of a drug into the vein of an animal or human subject over a period of time in excess of about 5 minutes, preferably about 30-90 minutes, although intravenous infusion may alternatively be administered for 10 hours or less in accordance with the present invention.

A "maintenance" (maintenance) dose refers herein to one or more doses of a therapeutic agent administered to a subject during or after treatment. Typically, the maintenance dose is administered at therapeutic intervals, such as at intervals of about every week, about every two weeks, about every three weeks, or about every four weeks. By "maintenance therapy" is meant a treatment regimen that is administered in order to reduce the likelihood of disease recurrence or progression. Maintenance therapy may last for any extended period of time, including for extended periods of time up to the lifetime of the subject. Maintenance therapy can be provided after the initial therapy or in combination with the initial or additional therapy. The dosage for the maintenance therapy may vary and may include a reduced dosage compared to the dosage used for other types of therapy. Also see "maintenance" herein.

Herein, a "patient" is a human patient. The patient may be a "cancer patient", i.e. a patient suffering from or at risk of suffering from one or more symptoms of cancer. Furthermore, the patient may be a previously treated cancer patient. The patient may be a "glioblastoma patient," i.e., a patient suffering from or at risk of suffering from one or more symptoms of glioblastoma. In addition, the patient may be a previously treated glioblastoma patient. In some embodiments, the patient has been treated with no more than one prior chemotherapy. In some embodiments, the patient has been previously treated with temozolomide. In some embodiments, the patient has been previously treated with temozolomide in combination with radiation. In some embodiments, the patient has been previously treated with temozolomide in combination with another agent. In some embodiments, the glioblastoma is a second-line glioblastoma.

As used herein, unless otherwise indicated, the term "c-Met" or "Met" refers to any natural or variant (whether natural or synthetic) c-Met polypeptide. The term "wild-type c-met" generally refers to a polypeptide comprising the amino acid sequence of a naturally occurring c-met protein. The term "wild-type c-met sequence" generally refers to the amino acid sequence found in naturally occurring c-met.

As used herein, unless otherwise indicated, the term "hepatocyte growth factor" or "HGF" refers to any natural or variant (whether natural or synthetic) HGF polypeptide. The term "wild-type HGF" generally refers to a polypeptide comprising the amino acid sequence of a naturally occurring HGF protein. The term "wild-type HGF sequence" generally refers to the amino acid sequence found in naturally occurring HGF.

The terms "anti-c-met antibody" and "antibody that binds c-met" refer to an antibody that is capable of binding c-met with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent in targeting c-met. In one embodiment, the extent of binding of the anti-c-met antibody to unrelated, non-c-met proteins is less than about 10% of the binding of the antibody to c-met, as measured, for example, by Radioimmunoassay (RIA). In certain embodiments, an antibody that binds c-met has ≦ 1 μ M ≦ 100nM, ≦ 10nM, ≦ 1nM, ≦ 0.1nM, ≦ 0.01nM, or ≦ 0.001nM (e.g., 10 nM)^-8M or less, e.g. 10^-8M to 10^-13M, e.g. 10^-9M to 10^-13M) dissociation constant (Kd). In certain embodiments, the anti-c-met antibody binds to a c-met epitope that is conserved among c-met from different species.

The terms "anti-HGF antibody" and "antibody that binds HGF" refer to an antibody that is capable of binding HGF with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent in targeting HGF. In one embodiment, the anti-HGF antibody binds to an unrelated, non-HGF protein to less than about 10% of the binding of the antibody to HGF as measured, for example, by a Radioimmunoassay (RIA). In certain embodiments, an antibody that binds HGF has a concentration of ≦ 1 μ M ≦ 100nM, ≦ 10nM, ≦ 1nM, ≦ 0.1nM, ≦ 0.01nM, or ≦ 0.001nM (e.g., 10 nM)^-8M or less, e.g. 10^-8M to 10^-13M, e.g. 10^-9M to 10^-13M) dissociation constant (Kd). In certain embodiments, the anti-HGF antibody binds an epitope of HGF that is conserved among HGFs from different species.

"c-met activation" refers to the activation or phosphorylation of c-met receptor. In general, c-met activation results in signal transduction (e.g., by phosphorylation of tyrosine residues in c-met or substrate polypeptides caused by the intracellular kinase domain of the c-met receptor). c-met activation can be mediated by c-met ligand (HGF) binding to the c-met receptor of interest. Binding of HGF to c-met activates the kinase domain of c-met and thereby leads to phosphorylation of tyrosine residues in c-met and/or in other substrate polypeptides.

A subject "population" refers to a group of cancer subjects, such as in a clinical trial, or as seen by an oncologist after approval for a particular indication, such as glioblastoma therapy FDA.

For the methods of the present invention, the term "instructing" a patient means providing instructions regarding applicable therapy, medication, treatment regimens, and the like, by any means, but preferably in writing, such as in the form of a package insert or other written promotional material.

For the methods of the present invention, the term "promoting" means providing, advertising, selling, or describing a particular drug, combination of drugs, or treatment modality by any means, including in writing, such as in the form of a package insert. Promotion herein refers to the promotion of a therapeutic agent, such as an anti-c-met antagonist (e.g., onartuzumab) and/or a VEGF antagonist (e.g., bevacizumab), for an indication, such as treatment of glioblastoma (e.g., relapsed glioblastoma), where such promotion is approved by the Food and Drug Administration (FDA) as having been demonstrated to be associated with statistically significant therapeutic efficacy and acceptable safety in a population of subjects.

The term "sale" is used herein to describe the promotion, sale, or distribution of a product (e.g., a drug). Sales specifically include packaging, advertising, and any commercial activity that commercializes a product.

For purposes herein, a "previously treated" glioblastoma patient has received a prior cancer therapy for a glioblastoma. In some embodiments, the patient has been treated with no more than one prior chemotherapy. In some embodiments, the patient has been previously treated with temozolomide. In some embodiments, the patient has been previously treated with temozolomide in combination with radiation. In some embodiments, the patient has been previously treated with temozolomide in combination with another agent. In some embodiments, the glioblastoma is a second-line glioblastoma.

"cancer drug" refers to a drug effective in treating cancer. Examples of cancer drugs include chemotherapeutic agents and chemotherapeutic regimens described below; c-met antagonists, including anti-c-met antibodies, such as onartuzumab; and VEGF antagonists, including anti-VEGF antibodies, such as bevacizumab.

As used herein, the term "biomarker" or "marker" generally refers to a molecule, including a gene, mRNA, protein, carbohydrate structure, or glycolipid, whose expression or secretion in or on mammalian tissue or cells can be detected by known methods (or methods disclosed herein) and is predictive of or useful in predicting (or aiding in predicting) the responsiveness of a cell, tissue, or patient to a therapeutic regimen. A biomarker of particular interest herein is HGF.

As used herein, "negative c-met staining intensity" or "negative staining intensity" refers to the c-met staining intensity of TOV-112D, H522, H1155, LXFL529 and/or H23. In some embodiments, a negative c-met staining intensity is indicative of the c-met staining intensity of the control cell line TOV-112D. In some embodiments, a negative c-met staining intensity is indicative of c-met staining intensity of control cell line H522. In some embodiments, a negative c-met staining intensity is indicative of the c-met staining intensity of the control cell line H1155. In some embodiments, a negative c-met staining intensity is indicative of the c-met staining intensity of control cell line LXFL 529. In some embodiments, a negative c-met staining intensity is indicative of the c-met staining intensity of the control cell line H23. Methods for c-met IHC are known in the art. In some embodiments, the c-met staining intensity is determined using a c-met antibody (e.g., SP44) to stain formalin-fixed paraffin-embedded cells (e.g., prepared in a tissue microarray) of a control cell pellet.

As used herein, "weak c-met staining intensity" or "weak staining intensity" refers to the c-met IHC staining intensity of control cell lines H1703, HEK-293, and/or H460. In some embodiments, weak c-met staining intensity is indicative of c-met staining intensity of control cell line H1703. In some embodiments, weak c-met staining intensity is indicative of c-met staining intensity of the control cell line HEK-293. In some embodiments, weak c-met staining intensity is indicative of c-met staining intensity of control cell line H460. Methods for c-met IHC are known in the art. In some embodiments, the c-met staining intensity is determined using a c-met antibody (e.g., SP44) to stain formalin-fixed paraffin-embedded cells (e.g., prepared in a tissue microarray) of a control cell pellet.

As used herein, "moderate c-met staining intensity" or "moderate staining intensity" refers to the c-met IHC staining intensity of control cell lines A549 and/or SKMES 1. In some embodiments, the moderate c-met staining intensity represents the c-met staining intensity of control cell line a 549. In some embodiments, the moderate c-met staining intensity is indicative of the c-met staining intensity of control cell line SKMES 1. Methods for c-met IHC are known in the art. In some embodiments, the c-met staining intensity is determined using a c-met antibody (e.g., SP44) to stain formalin-fixed paraffin-embedded cells (e.g., prepared in a tissue microarray) of a control cell pellet.

As used herein, "strong c-met staining intensity" or "strong staining intensity" refers to the c-met IHC staining intensity of the control cell lines EBC-1 and/or H441. In some embodiments, the strong c-met staining intensity is indicative of the c-met staining intensity of the control cell line EBC-1. In some embodiments, the strong c-met staining intensity is indicative of the c-met staining intensity of control cell line H441. Methods for c-met IHC are known in the art. In some embodiments, the c-met staining intensity is determined using a c-met antibody (e.g., SP44) to stain formalin-fixed paraffin-embedded cells (e.g., prepared in a tissue microarray) of a control cell pellet.

"patient sample" refers to a collection of cells or fluids obtained from a cancer patient. The source of the tissue or cell sample may be a solid tissue, like from a fresh, frozen and/or preserved organ or tissue sample or biopsy sample or punch sample; blood or any blood component; body fluids such as cerebrospinal fluid, amniotic fluid (amniotic fluid), peritoneal fluid (ascites), or interstitial fluid; cells from a subject at any time of pregnancy or development. Tissue samples may contain compounds that are not naturally intermixed with tissue in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, and the like. Examples of tumor samples herein include, but are not limited to, tumor biopsies, fine needle aspirates, bronchial lavage, pleural fluid (pleural fluid), sputum, urine, surgical specimens, circulating tumor cells, serum, plasma, circulating plasma proteins, ascites, primary cell cultures or cell lines derived from tumors or exhibiting tumor-like properties, and preserved tumor samples, such as formalin-fixed, paraffin-embedded tumor samples or frozen tumor samples. In one embodiment, the sample comprises a glioblastoma tumor sample (e.g., a glioblastoma tumor sample comprising benign stroma, e.g., reactive astrocytes, glial cells, pericytes, and/or endothelial cells). In some embodiments, the sample comprises a macro-dissected glioblastoma tumor sample (e.g., where morphologically normal brain tissue has been removed from the tumor sample). In some embodiments, the macro-dissected glioblastoma tumor sample comprises benign stroma (e.g., reactive astrocytes, glial cells, pericytes, and/or endothelial cells). In some embodiments, the sample is a glioblastoma biopsy. In some embodiments, the sample is glioblastoma cancer resected. In some embodiments, the sample is obtained after recurrence of glioblastoma in the patient. In some embodiments, the sample is obtained prior to recurrence of glioblastoma in the patient.

"effective response" or "responsiveness" of a patient to drug treatment, and the like, refers to the clinical or therapeutic benefit given to a patient at risk for or having cancer (e.g., glioblastoma) following administration of a cancer drug. Such benefits include any one or more of the following: extended survival (e.g., extended overall and/or progression-free survival); results in objective responses (including complete responses or partial responses); or ameliorating cancer signs or symptoms, etc., including prolonging the time to worsening of clinically significant disease-related symptoms experienced by patients having glioblastoma (e.g., previously treated glioblastoma). In some embodiments, the symptom is any one or more (any combination) of epilepsy, neurocognitive function (including but not limited to the orientation of a person, time, and/or place), reading, writing, and understanding. In one embodiment, biomarkers (e.g., HGF mRNA expression, e.g., determined using ISH and/or qPCR) are used to identify patients expected to have extended survival (e.g., extended overall and/or progression-free survival) when treated with c-met antagonist and VEGF antagonist relative to patients treated with VEGF antagonist alone. The incidence of biomarkers herein (e.g., as determined by HGF mRNA ISH and/or rtPCR analysis) is effective to predict or predict with high sensitivity such effective responses.

By "extended survival" is meant that the overall or progression-free survival of a patient treated according to the invention is extended relative to a patient not receiving treatment and/or relative to a patient treated with one or more approved antineoplastic agents but not receiving treatment according to the invention. In a specific example, "extending survival" means extending Progression Free Survival (PFS) and/or Overall Survival (OS) of a cancer patient receiving a combination therapy of the invention (e.g., treatment with a combination of a c-met antagonist (e.g., onartuzumab) and a VEGF antagonist (e.g., bevacizumab)) relative to a patient treated with bevacizumab alone. In another specific example, "extending survival" means extending Progression Free Survival (PFS) and/or Overall Survival (OS) of a cancer patient (e.g., a cancer patient population) receiving a combination therapy of the invention (e.g., treatment with a combination of obinutuzumab and bevacizumab) relative to a patient (e.g., a cancer patient population) treated with bevacizumab alone.

"survival" (survival) means that the patient remains alive and includes overall survival (overall survival) and progression free survival (progress free survival). In the studies underlying the present invention, the event used for survival analysis was death of any cause.

"overall survival" refers to patients who remain alive for a period of time, such as 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 1 year, 2 years, 3 years, etc., as calculated from the time of diagnosis or treatment. Survival can be assessed by the Kaplan-Meier method.

"progression-free survival" refers to a patient that remains alive without progression or worsening of cancer. In some embodiments, the patient remains alive for 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, or more, without cancer progression or worsening. In one aspect of the invention, the PFS of glioblastoma can be assessed by Neuro-Oncology Response Assessment (RANO) criteria. Wen et al, JClin Oncol 2010; 28:1963-72. In some embodiments, the PFS is evaluated using the rest criteria.

By "extended survival" is meant an extension of the overall survival or progression-free survival of a patient receiving treatment relative to a patient not receiving treatment (i.e., relative to a patient not treated with a drug), or relative to a patient not expressing a biomarker at a specified level, and/or relative to a patient treated with an approved anti-neoplastic agent. In some embodiments, overall or progression-free survival is extended by 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, or more.

"Objective response" refers to a measurable response, including a Complete Response (CR) or a Partial Response (PR).

"complete response" or "CR" means that all signs of cancer disappear in response to treatment. This does not always mean that the cancer has cured.

"partial response" or "PR" means that the size of one or more tumors or lesions or the extent of cancer in vivo is reduced in response to treatment.

"overall response rate" or "objective response rate" refers to the percentage of people who experience a reduction in the size of a cancer (or the amount of a hematological cancer) for a minimum amount of time.

Hazard Ratio (HR) is a statistical definition of the event rate. For the purposes of the present invention, hazard ratio is defined to represent the probability of an event in an experimental branch divided by the probability of an event in a control branch at any particular point in time. The "hazard ratio" in the progression free survival assay is a summary of the difference between the two progression free survival curves, representing a reduced risk of mortality for the treatment compared to the control over the follow-up period.

The term "VEGF" or "VEGF-a" is used to refer to 165 amino acid human vascular endothelial cell growth factor and related 121, 145, 189 and 206 amino acid human vascular endothelial cell growth factors, as described, for example, in Leung et al science,246:1306 (1989); and Houck et al mol Endocrin, 5:1806(1991), and naturally occurring allelic and processed forms thereof. VEGF-A is part of a gene family that includes VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F and PlGF. VEGF-A binds primarily to two high affinity receptor tyrosine kinases, VEGFR-1(Flt-1) and VEGFR-2(Flk-1/KDR), the latter being the major transmitters of the mitotic signals of VEGF-A vascular endothelial cells. In addition, neuropilin-1 has been identified as a receptor for heparin-binding VEGF-a isoforms (isofom) and may play a role in vascular development. The term "VEGF" or "VEGF-a" also refers to VEGF from non-human species such as mouse, rat, or primate. Sometimes, VEGF from a particular species is represented as follows, hVEGF for human VEGF and mVEGF for murine VEGF. Typically, VEGF refers to human VEGF. The term "VEGF" is also used to refer to truncated forms or polypeptide fragments comprising 165 amino acids from amino acid positions 8-109 or positions 1-109 of human vascular endothelial growth factor. It is possible in the present application to identify any such form of VEGF by, for example, "VEGF (8-109)", "VEGF (1-109)" or "VEGF 165". The amino acid positions of a "truncated" native VEGF are numbered as indicated in the native VEGF sequence. For example, amino acid 17 (methionine) in truncated native VEGF is also amino acid 17 (methionine) in native VEGF. The truncated native VEGF has comparable binding affinity to the KDR and Flt-1 receptors as native VEGF.

An "anti-VEGF antibody" refers to an antibody that binds VEGF with sufficient affinity and specificity. The selected antibody will typically have binding affinity for VEGF, for example, the antibody may have a K between 100nM and 1pM_dValues bound to hVEGF. Antibody affinity can be determined by, for example, a surface plasmon resonance-based assay (such as the BIAcore assay described in PCT application publication No. wo 2005/012359); enzyme-linked immunosorbent assay (ELISA); and competition assays (e.g., RIA). In certain embodiments, the anti-VEGF antibodies of the invention are useful as therapeutic agents for targeting and interfering with diseases or conditions in which VEGF activity is implicated. Also, the antibody may be subjected to other biological activity assays, such as those conducted to assess its efficacy as a therapeutic agent. Such assays are known in the artAnd depends on the target antigen of the antibody and the intended use. Examples include HUVEC inhibition assays; tumor cell growth inhibition assays (as described, for example, in WO 89/06692); antibody-dependent cellular cytotoxicity (ADCC) and complement-mediated cytotoxicity (CDC) assays (U.S. patent 5,500,362); and agonist activity or hematopoietic assays (see WO 95/27062). anti-VEGF antibodies typically do not bind to other VEGF homologs, such as VEGF-B or VEGF-C, nor to other growth factors, such as PlGF, PDGF or bFGF.

"VEGF antagonist" refers to a molecule that is capable of neutralizing, blocking, inhibiting, abrogating, reducing, or interfering with VEGF activity, including its binding to one or more VEGF receptors. VEGF antagonists include anti-VEGF antibodies and antigen-binding fragments thereof, receptor molecules and derivatives that specifically bind to VEGF thereby sequestering it from binding to one or more receptors, anti-VEGF receptor antibodies, and VEGF receptor antagonists such as small molecule inhibitors of VEGFR tyrosine kinase.

"chimeric VEGF receptor protein" refers to a VEGF receptor molecule having amino acid sequences derived from at least two different proteins, at least one of which is a VEGF receptor protein. In certain embodiments, the chimeric VEGF receptor protein is capable of binding VEGF and inhibiting the biological activity of VEGF.

The term "gene amplification" refers to the process of forming multiple copies of a gene or gene fragment in a particular cell or cell line.

The terms "level of expression" or "expression level" are generally used interchangeably and generally refer to the amount of a polynucleotide, mRNA, or amino acid product or protein in a biological sample. "expression" generally refers to the process by which information encoded by a gene is converted into structures present and operating in a cell. Thus, according to the present invention, "expression" of a gene may refer to transcription into a polynucleotide, translation into a protein, or even post-translational modification of a protein. Fragments of the transcribed polynucleotide, of the translated protein, or of the post-translationally modified protein should also be considered expressed, whether they are derived from transcripts generated or degraded by alternative splicing, or from post-translational processing of the protein (e.g., by proteolysis). In some embodiments, "level of expression" refers to the presence or absence or amount or prevalence of HGF mRNA (e.g., the percentage of cells expressing HGF mRNA), as assessed using ISH and/or rtPCR.

As used herein, the phrase "… … -based expression" means that information regarding the level of expression or the presence or absence of expression of one or more biomarkers herein (e.g., the presence or absence or prevalence of HGFISH signals (e.g., the percentage of cells displaying HGF ISH signal)) in glioblastoma tumor cells and related benign matrices is used to inform treatment decisions, information provided on package inserts, or marketing/promotion guidance, among others.

As used herein, the phrase "without substantial biomarker expression" or "substantially no biomarker expression" with respect to a biomarker means that the biomarker does not exhibit an expression level above background levels (in some embodiments, above background levels of statistical significance). As used herein, the phrase "at least no biomarker expression" with respect to a biomarker means that the biomarker does not exhibit expression in a biologically meaningful amount. As will be understood in the art, the amount of expression can be determined quantitatively or qualitatively, so long as a comparison between the biomarker sample and the reference counterpart can be made. Expression can be measured or detected according to any assay or technique known in the art, including, for example, those described herein (such as ISH).

An "amount" or "level" of a biomarker associated with increased clinical benefit in a patient with cancer (e.g., glioblastoma) refers to a detectable level in a biological sample, wherein the level of the biomarker is associated with increased clinical benefit in the patient. These can be measured by methods known to those skilled in the art and also disclosed in the present invention. The level or amount of expression of the biomarker assessed can be used to determine response to treatment. In some embodiments, the amount or level of a biomarker is determined using ISH (e.g., of a patient cancer sample, such as a glioblastoma sample comprising tumor cells and benign stromal cells). In some embodiments, high HGF mRNA is associated with increased clinical benefit. In some embodiments, high HGF mRNA is determined using ISH. In some embodiments, high HGF mRNA is HGF ISH signal intensity of at least + 2. In some embodiments, high HGF mRNA is HGF ISH signal intensity of at least + 3. In some embodiments, high HGF mRNA is HGF ISH signal intensity of +2 or + 3. In some embodiments, high HGF mRNA is determined using PCR (e.g., rtPCR).

An "amount" or "level" of a biomarker associated with reduced clinical benefit in a patient with cancer (e.g., glioblastoma) refers to a low detectable level or no detectable biomarker in a biological sample, wherein the level of the biomarker is associated with reduced clinical benefit in the patient. These can be measured by methods known to those skilled in the art and also disclosed in the present invention. The level or amount of expression of the biomarker assessed can be used to determine response to treatment. In some embodiments, the amount or level of a biomarker is determined using ISH (e.g., of a patient cancer sample, e.g., comprising tumor cells and benign stromal cells). In some embodiments, low HGF mRNA is associated with decreased clinical benefit. In some embodiments, the low HGFmRNA is determined using ISH. In some embodiments, low HGF mRNA is an HFG ISH signal intensity of 0. In some embodiments, low HGF mRNA is HGF ISH signal intensity of + 1. In some embodiments, low HGF mRNA is HGF ISH signal intensity of 0 or + 1. In some embodiments, low HGF mRNA is determined using PCR (e.g., rtPCR).

A cancer or biological sample "displaying HGF mRNA expression" refers to a cancer or biological sample that expresses (including overexpresses) HGFmRNA in a diagnostic test. A glioblastoma sample "exhibiting HGF mRNA expression" refers to a glioblastoma sample expressing (including over-expressing) HGF mRNA in a diagnostic test. In some embodiments, the glioblastoma sample includes tumor cells and benign stromal cells.

A cancer or biological sample "exhibiting c-met amplification" refers to a cancer or biological sample in which the c-met gene is amplified in a diagnostic test. In some embodiments, the amplified c-met gene is on average (in the cell population) greater than or equal to 5 or more copies of the c-met gene, or on average 8 or more copies of the c-met gene, or more, such as 10 or more, 15 or more, or 20 or more copies of the c-met gene.

A cancer or biological sample that "does not exhibit c-met amplification" refers to a cancer or biological sample that does not have the c-met gene amplified in the diagnostic test.

As used herein, the term "mutation" refers to a difference in the amino acid or nucleic acid sequence of a particular protein or nucleic acid (e.g., DNA, RNA) relative to the wild-type protein or nucleic acid, respectively. Mutant proteins or nucleic acids can be expressed from or found in one allele (heterozygous) or both alleles (homozygous) of a gene. In the present invention, the mutation is generally somatic. Mutations include sequence rearrangements such as insertions, deletions, and point mutations (including single nucleotide/amino acid polymorphisms).

The term "primer" refers to a single-stranded polynucleotide capable of hybridizing to a nucleic acid and allowing polymerization of the complementary nucleic acid, typically by providing a free 3' -OH group.

The term "array" or "microarray" refers to an ordered arrangement of hybridizable array elements, preferably polynucleotide probes (e.g., oligonucleotides), on a substrate. The substrate may be a solid substrate, such as a glass slide, or a semi-solid substrate, such as a nitrocellulose membrane.

The term "amplification" refers to the process of generating one or more copies of a reference nucleic acid sequence or its complement. Amplification may be linear or exponential (e.g., PCR). "copy" does not necessarily mean complete sequence complementarity or identity with respect to the template sequence. For example, the copies may comprise nucleotide analogs (such as deoxyinosine), intentional sequence changes (such as sequence changes introduced via primers comprising sequences that are hybridizable, but not fully complementary, to the template), and/or sequence errors that occur during amplification.

The term "housekeeping biomarker" refers to a biomarker or a group of biomarkers (e.g., polynucleotides and/or polypeptides) that are typically similarly present in all cell types. In some embodiments, the housekeeping biomarker is a "housekeeping gene. "housekeeping gene" refers herein to a gene or set of genes that encode a protein whose activity is essential for the maintenance of cell function, and is typically similarly present in all cell types.

As used herein, "amplification" generally refers to the process of generating multiple copies of a desired sequence. "multicopy" means at least 2 copies. "copy" does not necessarily mean complete sequence complementarity or identity to the template sequence. For example, the copies may comprise nucleotide analogs such as deoxyinosine, intentional sequence changes (such as sequence changes introduced via primers comprising sequences that are hybridizable but not complementary to the template), and/or sequence errors that occur during amplification.

The term "multiplex PCR" refers to a single PCR reaction that is performed on nucleic acids (e.g., DNA or RNA) obtained from a single source (e.g., an individual) using more than one set of primers for the purpose of amplifying two or more DNA sequences in a single reaction.

"ISH" or "in situ hybridization" refers to a type of hybridization that uses a complementary DNA or RNA strand (e.g., a primer or probe) to locate a particular DNA or RNA sequence in a portion or section of a tissue or cell (in situ). In some embodiments, complementary DNA strands are used to locate a particular RNA sequence in situ in a portion or section of a tissue or cell. In some embodiments, the ISH further comprises hybridization-based amplification.

The "stringency" of the hybridization reaction can be readily determined by one of ordinary skill in the art, and is generally calculated empirically based on probe length, washing temperature, and salt concentration. In general, longer probes require higher temperatures for proper annealing, while shorter probes require lower temperatures. Hybridization generally depends on the ability of denatured DNA or RNA to reanneal when the complementary strand is present in an environment below its melting temperature. The higher the degree of desired homology between the probe and the hybridizable sequence, the higher the relative temperature that can be used. As a result, it can be concluded that higher relative temperatures will tend to make the reaction conditions more stringent, while lower temperatures are less stringent. For additional details and explanations of the stringency of hybridization reactions, see Ausubel et al, Current Protocols in Molecular Biology, Wiley Interscience Publishers, (1995).

By "slice" of a tissue sample is meant a piece or sheet of the tissue sample, e.g., a thin slice of tissue or cells cut from the tissue sample. It should be understood that multiple slices of tissue sample may be made and analyzed. In some embodiments, the same section of tissue sample may be used for both morphological and molecular level analysis. In some embodiments, the same section of the tissue sample may be analyzed for both polypeptides and polynucleotides.

"correlating" or "correlating" means comparing the performance and/or results of a first analysis or protocol with the performance and/or results of a second analysis or protocol in any manner. For example, the results of a first analysis or protocol may be used to implement a second protocol, and/or the results of a first analysis or protocol may be used to decide whether a second analysis or protocol should be implemented. For embodiments of polynucleotide analysis or protocols, the results of the polynucleotide expression analysis or protocol can be used to determine whether a particular treatment protocol should be implemented.

As used herein, the term "substantially the same" means a sufficiently high degree of similarity between two numerical values such that one of skill in the art would consider the difference between the two numerical values to be of little or no biological and/or statistical significance within the context of the biological characteristic measured by the numerical values (e.g., Kd values or expressions). The difference between the two values is, for example, less than about 50%, less than about 40%, less than about 30%, less than about 20%, and/or less than about 10% as a function of the reference/comparison value.

As used herein, the phrase "substantially different" means a sufficiently high degree of difference between two numerical values such that one of skill in the art would consider the difference between the two numerical values to be statistically significant within the context of the biological characteristic measured with the numerical values (e.g., Kd values). The difference between the two values is, for example, greater than about 10%, greater than about 20%, greater than about 30%, greater than about 40%, and/or greater than about 50% as a function of the reference/comparative molecular value.

The word "label" as used herein refers to a detectable compound or composition. The label is typically conjugated or fused, directly or indirectly, to an agent, such as a polynucleotide probe or antibody, and facilitates detection of the agent to which it is conjugated or fused. The label may be detectable by itself (e.g., a radioisotope label or a fluorescent label), or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition that produces a detectable product.

"polymerase chain reaction" or "PCR" techniques, as used herein, generally refer to procedures in which minute amounts of specific fragments of nucleic acid, RNA and/or DNA are amplified. Generally, it is necessary to know sequence information at or beyond the end of the region of interest so that oligonucleotide primers can be designed; these primers will be identical or similar in sequence to the opposite strand of the template to be amplified. The 5' terminal nucleotides of both primers may coincide with the ends of the amplified material. PCR can be used to amplify specific RNA sequences, specific DNA sequences, etc. from total genomic DNA and cDNA, phage or plasmid sequences transcribed from total cellular RNA. See generally Mullis et al, Cold spring harbor Symp. Quant. biol.51:263 (1987); erlich ed., PCR Technology, Stockton Press, NY, 1989. As used herein, PCR is considered to be one, but not the only, example of a nucleic acid polymerase reaction method for amplifying a nucleic acid test sample, including the use of a known nucleic acid (DNA or RNA) as a primer and the use of a nucleic acid polymerase to amplify or generate a specific nucleic acid fragment, or to amplify or generate a specific nucleic acid fragment complementary to a specific nucleic acid.

"treatment" and "treatment" refer to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those that already have benign, precancerous, or non-metastatic tumors and those that are to be prevented from developing or relapsing.

The term "therapeutically effective amount" refers to the amount of a therapeutic agent (drug) that treats or prevents a disease or condition in a mammal. In the case of a cancer (e.g., a glioblastoma, such as a previously treated glioblastoma), a therapeutically effective amount of a therapeutic agent may reduce the number of cancer cells; reducing the size of the primary tumor; inhibit (i.e., slow to some extent, preferably prevent) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent, preferably prevent) tumor metastasis; inhibit tumor growth to some extent; and/or to alleviate one or more symptoms associated with the condition to some extent. Depending on the extent to which the drug can prevent growth and/or kill existing cancer cells, it can be cytostatic and/or cytotoxic. For cancer therapy, in vivo efficacy can be measured by, for example, assessing survival duration, time to disease progression (TTP), Response Rate (RR), response duration, and/or quality of life and/or TDD.

The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Included in this definition are benign and malignant cancers. "early cancer" or "early tumor" refers to a cancer that is non-invasive or metastatic, or is classified as a stage 0, stage I, or stage II cancer. Examples of cancers include, but are not limited to, carcinoma, lymphoma, blastoma (including medulloblastoma and retinoblastoma), sarcoma (including liposarcoma and synovial cell sarcoma), neuroendocrine tumors (including carcinoid tumors, gastrinoma and islet cell carcinoma), mesothelioma, schwannoma (including acoustic neuroma), meningioma, adenocarcinoma, melanoma, and leukemia or lymphoid malignancies. Additional examples of such cancers include, but are not limited to, glioblastoma (e.g., recurrent glioblastoma, second-line glioblastoma). In some embodiments, the cancer is lung cancer (e.g., NSCLC), renal cell carcinoma, gastric cancer, melanoma, breast cancer (e.g., triple negative breast cancer), colorectal cancer, sarcoma (e.g., osteosarcoma), cancer, bladder cancer, hepatocellular carcinoma, prostate cancer.

The term "concurrently" is used herein to refer to the administration of two or more therapeutic agents, wherein at least some of the administrations overlap in time. Thus, concurrent administration includes a dosing regimen in which administration of one or more agents is discontinued and administration of one or more other agents continues.

The term "polynucleotide" when used in the singular or plural generally refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. Thus, for example, a polynucleotide as defined herein includes, but is not limited to, single-and double-stranded DNA, DNA comprising single-and double-stranded regions, single-and double-stranded RNA, and RNA comprising single-and double-stranded regions, hybrid molecules comprising DNA and RNA, which may be single-stranded or, more typically, double-stranded, or comprise single-and double-stranded regions. In addition, the term "polynucleotide" as used herein refers to a triple-stranded region comprising RNA or DNA or both RNA and DNA. The chains in such regions may be from the same molecule or from different molecules. The region may comprise the entire population of one or more molecules, but more typically is a region comprising only some molecules. One of the molecules of the triple-helical region is often an oligonucleotide. The term "polynucleotide" specifically includes cDNA. The term includes DNA (including cDNA) and RNA that contain one or more modified bases. Thus, a DNA or RNA whose backbone is modified for stability or other reasons is also a "polynucleotide" for which the term is intended herein. In addition, DNA or RNA comprising rare bases such as inosine or modified bases such as tritiated bases are also included within the term "polynucleotide" as defined herein. In general, the term "polynucleotide" encompasses all chemically, enzymatically and/or metabolically modified forms of unmodified polynucleotides, as well as chemical forms of DNA and RNA characteristic of viruses and cells, including simple and complex cells.

The term "oligonucleotide" refers to relatively short polynucleotides, including but not limited to single-stranded deoxyribonucleotides, single-or double-stranded ribonucleotides, RNA, DNA hybrids, and double-stranded DNA. Oligonucleotides, such as single-stranded DNA probe oligonucleotides, are often synthesized by chemical methods, for example using a commercially available automated oligonucleotide synthesizer. However, oligonucleotides can be prepared by a variety of other methods, including in vitro recombinant DNA-mediated techniques and by expression of DNA in cells and organisms.

An antibody having the "biological characteristics" of a given antibody refers to an antibody that possesses one or more biological characteristics that distinguish the given antibody from other antibodies that bind the same antigen.

An "antibody that binds to the same epitope" as a reference antibody refers to an antibody that blocks binding of the reference antibody to its antigen by 50% or more in a competition assay, and conversely, the reference antibody blocks binding of the antibody to its antigen by 50% or more in a competition assay. An exemplary competition assay is provided herein.

As used herein, the phrase "providing a diagnosis" refers to using information or data generated relating to the level or presence of HGF (e.g., the level or presence or prevalence of HGF mRNA (e.g., the percentage of cells that express HGF mRNA)) in a sample of a patient to diagnose a glioblastoma in the patient. The information or data may be in any form, written, spoken, or electronic. In some embodiments, using the generated information or data includes communicating, presenting, reporting, storing, sending, transferring, provisioning, transmitting, delivering, distributing, or a combination thereof. In some embodiments, the communicating, presenting, reporting, storing, sending, transferring, provisioning, transmitting, delivering, distributing, or a combination thereof, is performed by a computing device, an analysis unit, or a combination thereof. In some further embodiments, the communicating, presenting, reporting, storing, sending, transferring, supplying, transmitting, distributing, or a combination thereof, is performed by an individual (e.g., a laboratory or medical professional). In some embodiments, the information or data comprises a comparison of the level of HGF (e.g., the level of HGF mRNA, e.g., measured using ISH or PCR) to a reference level. In some embodiments, the information or data includes the prevalence of HGF ISH signal (e.g., the prevalence of positive HGFISH signal in cells in a glioblastoma tumor sample). In some embodiments, the information or data includes an indication of the presence or absence of HGF (e.g., HGF mRNA) in the sample. In some embodiments, the information or data comprises an indication that the patient is diagnosed with glioblastoma (in some embodiments, HGF positive glioblastoma).

As used herein, the phrase "recommending treatment" refers to using information or data generated relating to the level or presence of c-met in a sample of a patient to identify the patient as suitable or unsuitable for treatment with a certain therapy. In some embodiments, the therapy may comprise a c-met antibody (e.g., onartuzumab). In some embodiments, the therapy may comprise a VEGF antagonist (e.g., bevacizumab). In some embodiments, the therapy may comprise an anti-c-met antibody (e.g., onartuzumab) in combination with a VEGF antagonist (e.g., bevacizumab). The information or data may be in any form, written, spoken, or electronic. In some embodiments, using the generated information or data includes communicating, presenting, reporting, storing, sending, transferring, provisioning, transmitting, delivering, distributing, or a combination thereof. In some embodiments, the communicating, presenting, reporting, storing, sending, transferring, provisioning, transmitting, delivering, distributing, or a combination thereof, is performed by a computing device, an analysis unit, or a combination thereof. In some further embodiments, the communicating, presenting, reporting, storing, sending, transferring, supplying, transmitting, distributing, or a combination thereof, is performed by an individual (e.g., a laboratory or medical professional). In some embodiments, the information or data comprises a comparison of the level of HGF to a reference level. In some embodiments, the information or data comprises an indication of the presence or absence of HGF in the sample. In some embodiments, the information or data includes an indication that HGF ISH signal strength is present at a particular level (e.g., 0, +1, +2, + 3). In some embodiments, the information or data includes an indication that HGF ISH signal intensity is present in a particular percentage of cells (e.g., glioblastoma tumor cells and benign stromal cells). In some embodiments, the information or data includes an indication that the patient is suitable or unsuitable for treatment with a therapy comprising a c-met antagonist antibody (e.g., onartuzumab). In some embodiments, the information or data includes an indication that the patient is suitable or unsuitable for treatment with a therapy comprising a c-met antagonist antibody (e.g., onartuzumab) in combination with a VEGF antagonist (e.g., bevacizumab).

"target audience" refers to a group or institution of people who receive a particular drug promotion or intended drug promotion, such as by promotion or advertising, particularly for a particular use, treatment, or indication, such as individual patients, groups of patients, newspapers, medical literature, and magazine readers, television or internet viewers, radio or internet listeners, physicians, drug companies, and the like.

The term "package insert" is used to refer to instructions for use typically contained in commercial packaging for therapeutic products, which contains information regarding indications, usage, dosage, administration, contraindications, other therapeutic products to be combined with the packaged product, and/or warnings, etc., relating to the use of such therapeutic products.

The term "antibody" herein is used in the broadest sense and encompasses a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.

An "antibody fragment" refers to a molecule distinct from an intact antibody that comprises a portion of the intact antibody that binds to an antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab '-SH, F (ab')₂(ii) a A diabody; a linear antibody; single chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

The term "chimeric" antibody refers to an antibody in which a portion of the heavy and/or light chain is derived from a particular source or species, while the remainder of the heavy and/or light chain is derived from a different source or species.

The "class" of an antibody refers to the type of constant domain or constant region that its heavy chain possesses. There are 5 major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG₁、IgG₂、IgG₃、IgG₄、IgA₁And IgA₂The constant domains of heavy chains corresponding to different classes of immunoglobulins are designated α, γ, and μ, respectively.

The term "Fc region" is used herein to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of a constant region. The term includes native sequence Fc regions and variant Fc regions. In one embodiment, the human IgG heavy chain Fc region extends from Cys226, or from Pro230, to the carboxy-terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also known as the EU index, as described in Kabat et al, Sequences of Proteins of Immunological Interest, published Health Service 5th edition, National Institutes of Health, Bethesda, MD, 1991.

"framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. In general, the FRs of a variable domain consist of 4 FR domains: FR1, FR2, FR3, and FR 4. Thus, HVR and FR sequences typically occur in the following order in VH (or VL): FR1-H1(L1) -FR2-H2(L2) -FR3-H3(L3) -FR 4.

The terms "full length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to a native antibody structure or having a heavy chain comprising an Fc region as defined herein.

"human antibody" refers to an antibody having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human or human cell or derived from a non-human source using a repertoire of human antibodies or other human antibody coding sequences. This definition of human antibodies specifically excludes humanized antibodies comprising non-human antigen binding residues.

"human consensus framework" refers to a framework representing the amino acid residues most commonly found in the selection of human immunoglobulin VL or VH framework sequences. Typically, the selection of human immunoglobulin VL or VH sequences is from a subset of variable domain sequences. Typically, the sequence subgroups are subgroups as in Kabat et al, Sequences of Proteins of Immunological Interest, fifth edition, NIHPublication 91-3242, Bethesda MD (1991), volumes 1-3. In one embodiment, for VL, the subgroup is as in Kabat et al, supra for subgroup kappa I. In one embodiment, for the VH, the subgroup is as in Kabat et al, supra, subgroup III.

A "humanized" antibody is a chimeric antibody comprising amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody will comprise at least one, and typically two, substantially the entire variable domains, in which all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody. Optionally, the humanized antibody may comprise at least a portion of an antibody constant region derived from a human antibody. An antibody, e.g., a "humanized form" of a non-human antibody, refers to an antibody that has undergone humanization.

As used herein, the term "hypervariable region" or "HVR" refers to each region of an antibody variable domain which is hypervariable in sequence ("complementarity determining regions" or "CDRs") and/or which forms structurally defined loops ("hypervariable loops") and/or which contains antigen-contacting residues ("antigen contacts"). Generally, an antibody comprises 6 HVRs: three in VH (H1, H2, H3) and three in VL (L1, L2, L3). Exemplary HVRs herein include:

(a) hypervariable loops present at amino acid residues 26-32(L1), 50-52(L2), 91-96(L3), 26-32(H1), 53-55(H2), and 96-101(H3) (Chothia and Lesk, J.mol.biol.196:901-917 (1987));

(b) CDRs present at amino acid residues 24-34(L1), 50-56(L2), 89-97(L3), 31-35b (H1), 50-65(H2), and 95-102(H3) (Kabat et al, Sequences of Proteins of immunological interest,5th Ed. public Health Service, National Institutes of Health, Bethesda, MD (1991));

(c) antigen contacts, present at amino acid residues 27c-36(L1), 46-55(L2), 89-96(L3), 30-35b (H1), 47-58(H2), and 93-101(H3) (MacCallum et al.J.mol.biol.262:732-745 (1996)); and

(d) a combination of (a), (b), and/or (c) comprising HVR amino acid residues 46-56(L2), 47-56(L2), 48-56(L2), 49-56(L2), 26-35(H1), 26-35b (H1), 49-65(H2), 93-102(H3), and 94-102 (H3). In one embodiment, HVR residues comprise those identified elsewhere in the specification. Unless otherwise indicated, HVR residues and other residues in the variable domain (e.g., FR residues) are numbered herein according to Kabat et al, supra.

As used herein, the term "cytotoxic agent" refers to a substance that inhibits or prevents cellular function and/or causes cell death or destruction. Cytotoxic agents include, but are not limited to: radioisotope (e.g. At)²¹¹、I¹³¹、I¹²⁵、Y⁹⁰、Re¹⁸⁶、Re¹⁸⁸、Sm¹⁵³、Bi²¹²、P³²、Pb²¹²And radioactive isotopes of Lu); chemotherapeutic agents or drugs (e.g., methotrexate), doxorubicin (adriamycin), vinca alkaloids (vinca alkaloids) (vincristine), vinblastine (vinblastine), etoposide (etoposide)), doxorubicin (doxorubicin), melphalan (melphalan), mitomycin (mitomycin) C, chlorambucil (chlorembucil), daunorubicin (daunorubicin), or other intercalating agents); a growth inhibitor; enzymes and fragments thereof, such as nucleolytic enzymes; (ii) an antibiotic; toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof; and various antitumor or anticancer agents disclosed hereinafter.

"Effector function" refers to those biological activities attributable to the Fc region of an antibody and which vary with the antibody isotype. Examples of antibody effector functions include: c1q binding and Complement Dependent Cytotoxicity (CDC); fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down-regulation of cell surface receptors (e.g., B cell receptors); and B cell activation.

An "immunoconjugate" refers to an antibody conjugated to one or more heterologous molecules, including but not limited to cytotoxic agents.

As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and/or bind the same epitope, except, for example, for possible variant antibodies containing naturally occurring mutations or occurring during the production of a monoclonal antibody preparation, such variants are typically present in very small amounts. Unlike polyclonal antibody preparations, which typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on the antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a population of substantially homogeneous 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 generated by a variety of techniques, including but not limited to hybridoma methods, recombinant DNA methods, phage display methods, and methods that utilize transgenic animals containing all or part of a human immunoglobulin locus, such methods and other exemplary methods for generating monoclonal antibodies are described herein.

"naked antibody" refers to an antibody that is not conjugated to a heterologous moiety (e.g., a cytotoxic moiety) or a radioactive label. The naked antibody may be present in a pharmaceutical formulation.

"Natural antibody" refers to a naturally occurring immunoglobulin molecule having a different structure. For example, a native IgG antibody is an heterotetrameric glycan protein of about 150,000 daltons, consisting of two identical light chains and two identical heavy chains that are disulfide-bonded. From N to C-terminus, each heavy chain has one variable region (VH), also called variable or heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH 3). Similarly, from N-to C-terminus, each light chain has a variable region (VL), also known as the variable light domain or light chain variable domain, followed by a Constant Light (CL) domain. Antibody light chains can be classified into one of two types, called kappa (κ) and lambda (λ), based on their constant domain amino acid sequences.

For purposes herein, "Onartuzumab" (Onartuzumab) and "MetMAb" are used interchangeably to refer to nucleic acid molecules comprising the amino acid sequences set forth in SEQ ID NOs: 8 and 7, and the Fc sequence of SEQ ID NO: 13. In some embodiments, it comprises SEQ ID NO:12 and the light chain amino acid sequence in SEQ ID NO:11 and the Fc sequence of SEQ ID NO: 13. The antibody is optionally produced by E.coli cells. The terms "onartuzumab" and "MetMAb" are encompassed herein with names Adopted in the United States (United States addressed Name, USAN) or International non-proprietary Name (INN): biologically similar forms of onartuzumab drug.

"onartuzumab epitope" refers to an epitope recognized by onartuzumab, an anti-c-met antibody (see Merchant, m.et al, PNAS (2013)110(32): E2987-E2996).

The term "pharmaceutical formulation" refers to a sterile preparation in a form that allows the biological activity of a drug to be effective, and that is free of other ingredients that would cause unacceptable toxicity to a subject to whom the formulation is administered.

"sterile" formulations are sterile or free of all living microorganisms and their spores.

"kit" refers to any article of manufacture (e.g., a package or container) comprising at least one reagent (e.g., a drug for treating cancer (e.g., glioblastoma), or a reagent (e.g., an antibody) for specifically detecting a biomarker gene or protein of the invention). The articles are preferably distributed, or sold in a unit for practicing the methods of the present invention.

"pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation that is different from the active ingredient and is not toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

"percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with amino acid residues in the reference polypeptide sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and without considering any conservative substitutions as part of the sequence identity. Comparison for the purpose of determining percent amino acid sequence identity can be performed in a variety of ways within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or megalign (dnastar) software. One skilled in the art can determine suitable parameters for aligning sequences, including any algorithms necessary to achieve maximum alignment over the full length of the sequences being compared. However, for purposes of the present invention,% amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was written by Genentech, inc and the source code has been submitted to the US Copyright Office (US Copyright Office, Washington d.c.,20559) along with the user document, where it is registered with US Copyright registration number TXU 510087. ALIGN-2 programs are publicly available from Genentech, Inc., South San Francisco, Calif., or may be compiled from source code. The ALIGN2 program should be compiled for use on UNIX operating systems, including digital UNIX V4.0D. All sequence comparison parameters were set by the ALIGN-2 program and were not changed.

In the case of employing ALIGN-2 to compare amino acid sequences, the% amino acid sequence identity of a given amino acid sequence a relative to (to), with (with), or against (against) a given amino acid sequence B (or may be stated as having or comprising a given amino acid sequence a with respect to, with, or against a given amino acid sequence B) is calculated as follows:

fractional X/Y times 100

Wherein X is the number of amino acid residues scored as identical matches in the A and B alignments of the sequence alignment program by the program ALIGN-2, and wherein Y is the total number of amino acid residues in B. It will be appreciated that if the length of amino acid sequence a is not equal to the length of amino acid sequence B, then the% amino acid sequence identity of a relative to B will not equal the% amino acid sequence identity of B relative to a. Unless otherwise specifically indicated, all% amino acid sequence identity values used herein are obtained using the ALIGN-2 computer program as described in the preceding paragraph.

"chemotherapeutic agent" refers to a chemical compound useful for the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents such as, for example, temozolomide, an imidazole tetrazine derivative of the alkylating agent dacarbazine. Further examples of chemotherapeutic agents include, for example, paclitaxel or topotecan or Pegylated Liposomal Doxorubicin (PLD). Other examples of chemotherapeutic agents include alkylating agents (alkylating agents), such as thiotepa andcyclophosphamide (cyclophosphamide); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines (aziridines), such as benzotepa (benzodopa), carboquone (carboquone), metoclopramide (meteredopa) and uretepa (uredpa); ethyleneimines and methylmelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethlamelamine; annonaceous acetogenins (especially bullatacin and bullatacin); camptothecin (camptothecin); bryostatin; callystatin; CC-1065 (including its adozelesin (adozelesin), carvelesin (carzelesin) and bizelesin (bizelesin) synthetic analogs); cryptophycins (especially cryptophycins 1 and 8); dolastatin (dolastatin); duocarmycins (including synthetic analogs, KW-2189 and CB1-TM 1); eiscosahol (eleutherobin); pancratistatin; sarcodictyin; spongistatin (spongistatin); nitrogen mustards (nitrogen mustards), such as chlorambucil (chlorambucil), chlorambucil (chlorenaphazine), cholorophosphamide (cholorophosphamide), estramustine (estramustine), ifosfamide (ifosfamide), mechlorethamine (mechlorethamine), mechlorethamine hydrochloride (mechlorethamine oxide hydrochloride), melphalan (melphalan), neomustard (novembichin), benzene mustard cholesterol (phenylesterine), prednimustine (prednimustine), trofosfamide (trofosfamide), uracil mustard (uracil mustard); nitrosoureas such as carmustine (carmustine), chlorouretocin (chlorozotocin), fotemustine (fotemustine), lomustine (lomustine), nimustine (nimustine) and ramustine (ranirnustine); antibiotics such as enediynes antibiotics (enediynes) (e.g., calicheamicin, especially calicheamicin γ 1I and calicheamicin ω I1 (see, e.g., Agnew, chem. int. Ed. Engl.,33:183-186(1994)), anthracyclines (dynemicin), including dynamicin A, bisphosphonates (bisphosphonates), such as clodronate), esperamicin (esperamicin), and neocarzinostain (neocarzinostatin) and related chromophoric protein enediynes, aclacinomycin (acrinomycin), actinomycin (actinomycin), anidamycin (anthramycin), azaserine (serzanine), bleomycin (bleomycin), actinomycin C (actinomycin C), actinomycin, carmycin (carbamycin), daunomycin (doxorubicin-5), rhodomycin (rhodomycin), rhodomycin (monocrotamycin),doxorubicin (doxorubicin) (including morpholinodoxorubicin, cyanomorpholinodoxorubicin, 2-pyrrolodoxorubicin and deoxydoxorubicin), epirubicin (epirubicin), esorubicin (esorubicin), idarubicin (idarubicin), marijumycin (marcellomomycin), mitomycins (mitomycins) such as mitomycin C, mycophenolic acid (mycophenolic acid), norramycin (nogalamycin), olivomycin (olivomycin), peyromycinMycin (peplomycin), pofiomycin (potfiromycin), puromycin (puromycin), triiron doxorubicin (quelamycin), rodobicin (rodorubicin), streptonigrin (streptonigrin), streptozocin (streptozocin), tubercidin (tubicidin), ubenimex (ubenimex), restatin (zinostatin), zorubicin (zorubicin); antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteroyltriglutamic acid (pteropterin), trimetrexate; purine analogs such as fludarabine (fludarabine), 6-mercaptopurine (mercaptoprine), thiamiprine (thiamiprine), thioguanine (thioguanine); pyrimidine analogs such as ancitabine (ancitabine), azacitidine (azacitidine), 6-azauridine, carmofur (carmofur), cytarabine (cytarabine), dideoxyuridine (dideoxyuridine), deoxyfluorouridine (doxifluridine), enocitabine (enocitabine), floxuridine (floxuridine); androgens such as carotinone (calusterone), dromostanolone propionate, epitioandrostanol (epitiostanol), mepiquitane (mepiquitane), testolactone (testolactone); anti-adrenal agents such as aminoglutethimide (aminoglutethimide), mitotane (mitotane), trilostane (trilostane); folic acid supplements such as folinic acid (folinic acid); acetoglucurolactone (acegultone); (ii) an aldophosphamide glycoside; aminolevulinic acid (aminolevulinic acid); eniluracil (eniluracil); amsacrine (amsacrine); bestrabuucil; bisantrene; edatrexate (edatraxate); desphosphamide (defofamine); dimecorsine (demecolcine); diazaquinone (diaziqutone); elformithine; ammonium etitanium acetate; epothilone (epothilone); etoglut (etoglucid); gallium nitrate; hydroxyurea (hydroxyurea); lentinan (lentinan); lonidamine (lonidainine); maytansinoids (maytansinoids), such as maytansine (maytansine) and ansamitocins (ansamitocins); mitoguazone (mitoguzone); mitoxantrone (mitoxantrone); mopidanol (mopidanmol); diamine nitracridine (nitrarine); pentostatin (pentostatin); methionine mustard (phenamett); pirarubicin (pirarubicin); losoxant (losoxant)rone); podophyllinic acid (podophyllic acid); 2-ethyl hydrazide (ethylhydrazide); procarbazine (procarbazine);polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane (rizoxane); rhizomycin (rhizoxin); sisofilan (sizofuran); helical germanium (spirogermanium); tenuazonic acid (tenuazonic acid); triimine quinone (triaziquone); 2, 2' -trichlorotriethylamine; trichothecenes (trichothecenes), especially the T-2 toxin, verrucin (verrucin) A, bacillocin (roridin) A and snakes (anguidine); urethane (urethan); vindesine (vindesine); dacarbazine (dacarbazine); mannitol mustard (mannomustine); dibromomannitol (mitobronitol); dibromodulcitol (mitolactol); pipobromane (pipobroman); a polycytidysine; cytarabine (arabine) ("Ara-C"); cyclophosphamide (cyclophosphamide); thiotepa (thiotepa); taxols (taxoids), e.g.Paclitaxel (paclitaxel) (Bristol-Myers Squibb Oncology, Princeton, N.J.),paclitaxel (American pharmaceutical Partners, Schaumberg, Ill.) and paclitaxel in nanoparticulate dosage form without Cremophor, albumin modification, anddocetaxel (docetaxel) (Rhone-Poulenc Rorer, Antony, France); chlorambucil (chlorenbucil);gemcitabine (gemcitabine); 6-thioguanine (thioguanine); mercaptopurine (mercaptoprine); methotrexate (methotrexate); platinum analogs such as cisplatin (cissplatin), oxaliplatin (oxaliplatin) and carboplatin (carboplatin); vinblastine (vinblastine); platinum; etoposide (VP-16)) (ii) a Ifosfamide (ifosfamide); mitoxantrone (mitoxantrone); vincristine (vincristine);vinorelbine (vinorelbine); oncostatin (novantrone); teniposide (teniposide); edatrexate (edatrexate); daunomycin (daunomycin); aminopterin (aminopterin); (xiloda); ibandronate (ibandronate); irinotecan (irinotecan) (Camptosar, CPT-11) (treatment regimens including irinotecan with 5-FU and leucovorin); topoisomerase inhibitor RFS 2000; difluoromethyl ornithine (DMFO); retinoids (retinoids), such as retinoic acid (retinoic acid); capecitabine (capecitabine); combretastatin (combretastatin); leucovorin (LV); lapatinib (lapatinib)Which reduces cell proliferation; and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing. In some embodiments, the chemotherapeutic agent is temozolomide; procarbazine; lomustine; vincristine (PCV), carmustine implanted wafer, cisplatin; and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing.

II cancer medicine

In one aspect, provided are methods of treating cancer comprising administering a c-met antagonist. In some embodiments, the method of treating cancer comprises administering a c-met antagonist, optionally in combination with a second cancer drug. In some embodiments, the method of treating cancer comprises administering a combination of a c-met antagonist and a VEGF antagonist. In one aspect, provided are methods of selecting a patient treatable with a cancer drug based on the expression of one or more biomarkers disclosed herein. Examples of cancer drugs include, but are not limited to:

-c-met antagonists, including anti-c-met antibodies;

-VEGF antagonists, including anti-VEGF antibodies;

-chemotherapeutic agents and chemotherapeutic regimens;

other drugs or combinations thereof in the development or approved for the treatment of cancer (e.g. glioblastoma, mesothelioma, gastric cancer, hepatocellular carcinoma, renal cell carcinoma, and sarcoma).

c-Met antagonists

In one embodiment, the agent is an antibody, including but not limited to an antibody that binds human c-met. In some embodiments, the antibody interferes with (e.g., blocks) the binding of c-met to Hepatocyte Growth Factor (HGF). In some embodiments, the antibody binds c-met. In some embodiments, the antibody binds HGF. In one embodiment, the extent of binding of the anti-c-met antibody to unrelated, non-c-met proteins is less than about 10% of the binding of the antibody to c-met, as measured, for example, by Radioimmunoassay (RIA). In one embodiment, the extent of binding of the anti-HGF antibody to unrelated, non-c-met protein is less than about 10% of the binding of the antibody to HGF as measured, for example, by a Radioimmunoassay (RIA). The antibodies herein include: monoclonal antibodies, including chimeric, humanized or human antibodies. In one embodiment, the antibody is an antibody fragment, such as an Fv, Fab ', a one-armed antibody, an scFv, a diabody, or F (ab')₂And (3) fragment. In another embodiment, the antibody is a full length antibody, e.g., a complete IgG1 antibody or other antibody class or isotype, as defined herein. In one embodiment, the antibody is monovalent. In another embodiment, the antibody is a one-armed antibody comprising an Fc region (i.e., the heavy chain variable domain and the light chain variable domain form a single antigen binding arm), wherein the Fc region comprises a first and a second Fc polypeptide, wherein the first and second Fc polypeptides are present in a complex and form an Fc region that increases the stability of said antibody fragment compared to a Fab molecule comprising said antigen binding arm. The one-armed antibody may be monovalent.

In one embodiment, the anti-c-met antibody is obinutuzumab. In another embodiment, the anti-c-met antibody comprises a heavy chain variable domain comprising one or more of: (a) HVR1, comprising sequence GYTFTSYWLH (SEQ ID NO: 1); (b) HVR2, comprising sequence GMIDPSNSDTRFNPNFKD (SEQ ID NO: 2); and/or (c) HVR3-HC comprising sequence ATYRSYVTPLDY (SEQ ID NO: 3). In some embodiments, the antibody comprises a light chain variable domain comprising one or more of: (a) HVR1-LC comprising sequence KSSQSLLYTSSQKNYLA (SEQ ID NO: 4); HVR2-LC comprising the sequence WASTRES (SEQ ID NO: 5); and/or (c) HVR3-LC comprising sequence QQYYAYPWT (SEQ ID NO: 6). In some embodiments, the anti-c-met antibody comprises a heavy chain variable domain comprising: (a) HVR1, comprising sequence GYTFTSYWLH (SEQ ID NO: 1); (b) HVR2, comprising sequence GMIDPSNSDTRFNPNFKD (SEQ ID NO: 2); and (c) an HVR3-HC comprising sequence ATYRSYVTPLDY (SEQ ID NO:3), the light chain variable domain comprising: (a) HVR1-LC comprising sequence KSSQSLLYTSSQKNYLA (SEQ ID NO: 4); HVR2-LC comprising the sequence WASTRES (SEQ ID NO: 5); and (c) HVR3-LC comprising sequence QQYYAYPWT (SEQ ID NO: 6).

In any of the above embodiments, for example, the anti-c-met antibody can be humanized. In one embodiment, the anti-c-met antibody comprises the HVR of any of the above embodiments, and further comprises an acceptor human framework, e.g., a human immunoglobulin framework or a human consensus framework.

In another aspect, the anti-c-met antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO. 7. In certain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-c-met antibody comprising that sequence retains the ability to bind human c-met. In certain embodiments, a total of 1 to 10 amino acids are substituted, altered, inserted and/or deleted in SEQ ID NO. 7. In certain embodiments, substitutions, insertions, or deletions are present in regions outside of the HVR (i.e., in the FR). Optionally, the anti-c-met antibody comprises the VH sequence of SEQ ID NO 7, including post-translational modifications of this sequence.

In another aspect, anti-c-met antibodies are provided, wherein the antibodies comprise a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO. 8. In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-c-met antibody comprising that sequence retains the ability to bind c-met. In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted and/or deleted in SEQ ID NO 8. In certain embodiments, substitutions, insertions, or deletions are present in regions outside of the HVR (i.e., in the FR). Optionally, the anti-c-met antibody comprises the VL sequence of SEQ ID NO 8, including post-translational modifications of this sequence.

In yet another embodiment, the anti-c-met antibody comprises a VL region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO.8 and a VH region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO. 7. In yet another embodiment, the anti-c-met antibody comprises: HVR-L1 comprising the amino acid sequence SEQ ID NO 1; HVR-L2 comprising the amino acid sequence SEQ ID NO 2; HVR-L3 comprising the amino acid sequence SEQ ID NO 3; HVR-H1 comprising the amino acid sequence SEQ ID NO 4; HVR-H2 comprising the amino acid sequence SEQ ID NO 5; and HVR-H3, comprising the amino acid sequence SEQ ID NO 6.

In another aspect, the anti-c-met antibody comprises a VH in any of the embodiments provided above and a VL in any of the embodiments provided above. In some embodiments, the anti-c-met antibody is monovalent and further comprises an Fc polypeptide.

In another aspect, the c-met antagonist binds to an onartuzumab epitope. In some embodiments, the c-met antagonist (e.g., an anti-c-met antibody) comprises at least one peptide from 1) a319-a 347; 2) S360-V427; 3) L439-T457; or 4) binding to human c-met at the binding site of amino acid residues of R461-L480, wherein the position of the amino acid residue is based on (or according to) the position in SEQ ID NO: 16. In some embodiments, the binding site comprises at least one amino acid residue selected from the group consisting of: c-met A327, Q328, R331, Q332, I333, G334, A335, S336, L337, N338, D339, K368, Y369, R426, I446, G448, D449, or R469, wherein the position of the amino acid residue is based on the position in SEQ ID NO 16. In some embodiments, the binding site comprises at least one amino acid residue from a319-a 347. In some embodiments, the binding site comprises at least one of amino acid residues a327, Q328, R331, Q332, I333, G334, a335, S336, L337, N338, or D339. In some embodiments, the binding site comprises at least one of amino acid residues Q328, R331, L337, and N338. In some embodiments according to any of the embodiments in this paragraph, the binding site further comprises at least one peptide from 1) S360-V427; 2) L439-T457; or 3) the amino acid residues of R461-L480. In some embodiments according to any of the embodiments in this paragraph, the binding site comprises at least one amino acid residue selected from the group consisting of: k368, Y369, R426, I446, G448, D449, and R469. In some embodiments according to any of the above-described embodiments, the binding site comprises amino acid residues Q328, R331, L337, and N338. In some embodiments, the binding site further comprises amino acid residues R331, Q332, I333, G334, a335, S336, D339, K368, Y369, R426, I446, G448, D449, and R469.

In yet another aspect, the invention provides antibodies that bind to the same epitope as the anti-c-met antibodies provided herein. For example, in certain embodiments, antibodies are provided that bind to the same epitope as an anti-c-met antibody comprising the VH sequence SEQ ID NO 7 and the VL sequence SEQ ID NO 8.

In another aspect, the present invention provides anti-c-met antibodies having the same biological characteristics as onartuzumab.

In yet another aspect of the invention, the anti-c-met antibody according to any embodiment herein may be a monoclonal antibody, including a monovalent antibody, a chimeric antibody, a humanized antibody, or a human antibody. In one embodiment, the anti-c-met antibody is an antibody fragment, such as a single arm, Fv, Fab ', scFv, diabody, or F (ab')₂And (3) fragment. In another embodiment, the antibody is a full length antibody, e.g., a complete IgG1 or IgG4 antibody or other antibody class or isotype, as defined herein. According to another embodiment, the antibody is a bispecific antibody. In one embodiment, the bispecific antibody comprises an HVR as described above or comprises a VH and VL region as described above.

In some embodiments, the anti-c-met antibody is monovalent and comprises the following: (a) a first polypeptide comprising a heavy chain variable domain having the sequence EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWLHWVRQAPGKGLEWVGMIDPSNSDTRFNPNFKDRFTISADTSKNTAYLQMNSLRAEDTAVYYCATYRSYVTPLDYWGQGTLVTVSS (SEQ ID NO:7), a CH1 sequence, and a first Fc polypeptide; (b) a second polypeptide comprising a light chain variable domain having the sequence DIQMTQSPSSLSASVGDRVTITCKSSQSLLYTSSQKNYLAWYQQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYAYPWTFGQGTKVEIKR (SEQ ID NO:8), and a CL1 sequence; and (c) a third polypeptide comprising a second Fc polypeptide. In some embodiments, the first polypeptide comprises Fc sequence CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:9) and the second polypeptide comprises Fc sequence CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 10).

In other embodiments, the anti-c-met antibody is monovalent and comprises: (a) a first polypeptide comprising a heavy chain, said polypeptide comprising the sequence: EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWLHWVRQAPGKGLEWVGMIDPSNSDTRFNPNFKDRFTISADTSKNTAYLQMNSLRAEDTAVYYCATYRSYVTPLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 11); (b) a second polypeptide comprising a light chain, said polypeptide comprising sequence DIQMTQSPSSLSASVGDRVTITCKSSQSLLYTSSQKNYLAWYQQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYAYPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 12); and a third polypeptide comprising an Fc sequence, said polypeptide comprising sequence DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 13).

The use of node-in-pocket is well known in the art as a method for generating multispecific antibodies and/or single-armed antibodies and/or immunoadhesins. See U.S. Pat. No.5,731,168, PCT publication No. WO2009089004, and U.S. patent publication No. 20090182127. See also Marvin and Zhu, Acta Pharmacologa Sincia (2005)26(6): 649-. In one embodiment, the antibody comprises Fc mutations that constitute "nodes" and "holes", as described in WO 2005/063816. For example, the hole mutation may be one or more of T366A, L368A, and/or Y407V in the Fc polypeptide, and the cavity mutation may be T366W in the Fc polypeptide.

Other anti-c-met antibodies are described herein and known in the art to be suitable for use in the methods of the invention. For example, anti-c-met antibodies disclosed in WO05/016382 (including but not limited to antibody 13.3.2, 9.1.2, 8.70.2, 8.90.3); an anti-c-met antibody that generates or recognizes an epitope on the extracellular domain of HGF receptor β chain (said epitope being the same as the epitope recognized by the monoclonal antibody) from the hybridoma cell line deposited at cba (genoa) with ICLC number PD 03001; anti-c-met antibodies disclosed in WO2007/126799 (including but not limited to 04536, 05087, 05088, 05091, 05092, 04687, 05097, 05098, 05100, 05101, 04541, 05093, 05094, 04537, 05102, 05105, 04696, 04682); anti-c-met antibodies disclosed in WO2009/007427 (including but not limited to antibodies deposited at CNCM (Institut Pasteur, Paris, France) at 14.3.2007 with number I-3731, at 14.3.2007 with number I-3732, at 6.7.2007 with number I-3786, at 14.3.2007 with number I-3724); 20110129481; anti-c-met antibodies disclosed in US 20110104176; anti-c-met antibodies disclosed in WO 2009/134776; anti-c-met antibodies disclosed in WO 2010/059654; anti-c-met antibodies disclosed in WO2011020925 (including but not limited to antibodies secreted by hybridomas deposited at CNCM (Institut Pasteur, Paris, France) from 2008 at 12.3 and 14.2010 at number I-4273).

In some embodiments, the c-met antagonist is an anti-Hepatocyte Growth Factor (HGF) antibody, including but not limited to the humanized anti-HGF antibody TAK701, rilotumumab, Ficlatuzumab, and/or humanized antibody 2B8 described in WO 2007/143090. In some embodiments, the anti-HGF antibody is an anti-HGF antibody described in US7718174B 2.

In some embodiments, the c-met antagonist is a small molecule inhibitor of c-met. In some embodiments, the c-met small molecule inhibitor is a selective c-met small molecule inhibitor.

In one embodiment, the antagonist of c-met binds to the extracellular domain of c-met. In some embodiments, the c-met antagonist binds to a c-met kinase domain. In some embodiments, the c-met antagonist competes for c-met binding with HGF. In some embodiments, the c-met antagonist competes for HGF binding with c-met. In some embodiments, the c-met antagonist binds HGF.

In certain embodiments, the c-met antagonist is any one of: SGX-523, Crizotinib; JNJ-38877605(CAS No.943540-75-8), BMS-698769, PHA-665752(Pfizer), SU5416, INC-280 (Incyte; SU11274 (Sugen; [ (3Z) -N- (3-chlorophenyl) -3- ({3, 5-dimethyl-4- [ (4-methylpiperazin-1-yl) carbonyl ] -1H-pyrrol-2-yl } methylene) -N-methyl-2-oxoindoline-5-sulfonamide; CAS No.658084-23-2]), Foretinib, MGCD-265 (MethylGene; MGCD-265 targets c-MET, VEGFR1, VEGFR2, VEGFR3, Ron and Tie-2 receptors; CAS No.875337-44-3), Tivantinib (ARQ 197), LY-2801653(Lilly), LY2875358(Lilly), MP-470, Rilotumumab (AMG 102, anti-HGF monoclonal antibody), antibody 223C4 or humanized antibody 223C4(WO2009/007427), humanized L2G7 (humanized TAK 701; humanized anti-HGF monoclonal antibody); EMD1214063(Merck Sorono), EMD1204831 (Merck Serono), NK4, Cabozantinib (carbozantinib is a dual inhibitor of MET and VEGFR 2), MP-470 (SuperGen; is an inhibitor of c-KIT, MET, PDGFR, Flt3, and AXL), Comp-1, Ficlatuzumab (AV-299; anti-HGF monoclonal antibody), E7050(Cas No. 1196681-49-8; E7050 is a dual c-MET and VEGFR2 inhibitor (Esai), MK-2461 (Merck; N- ((2R) -1, 4-dioxan-2-ylmethyl) -N-methyl-N' - [3- (1-methyl-1H-pyrazol-4-yl) -5-oxo-5H-benzo [4,5] cyclohepta [1,2-b ] pyridine-7-yl ] pyridine-3- (1-methyl-1H-pyrazol-4-yl) -5-oxo-5H-benzo [4,5] cyclohepta [1,2-b ] pyridine-yl ] CAS 7-yl ] pyridine-6739; MK-917879; Merck), PF4217903(Pfizer), AMG208(Amgen), SGX-126, RP1040, LY2801653, AMG458, EMD637830, BAY-853474, DP-3590. In certain embodiments, the c-met antagonist is any one or more of crizotinib, tivatinib, carbozantinib, MGCD-265, ficlatuzumab, humanized TAK-701, rilotumumab, foretinib, h224G11, DN-30, MK-2461, E7050, MK-8033, PF-4217903, AMG208, JNJ-38877605, EMD1204831, INC-280, LY-2801653, SGX-126, RP1040, LY2801653, BAY-853474, and/or LA 480. In certain embodiments, the c-met antagonist is any one or more of crizotinib, tivatinib, carbozantinib, MGCD-265, ficlatuzumab, humanized TAK-701, rilotumumab, and/or foretinib.

anti-VEGF antibodies and antagonists

The VEGF antigen to be used for the production of VEGF antibodies may be, for example, VEGF₁₆₅Molecules as well as other isoforms of VEGF or fragments thereof containing the desired epitope. In one embodiment, the desired epitope is an epitope recognized by bevacizumab, which binds to the same epitope (referred to as the "a.4.6.1 epitope" as defined herein) as the monoclonal anti-VEGF antibody a4.6.1 produced by hybridoma ATCC HB 10709. Other forms of VEGF that may be used to generate the anti-VEGF antibodies of the invention will be apparent to those skilled in the art.

Human VEGF was first obtained by screening a cDNA library prepared from human cells using bovine VEGF cDNA as a hybridization probe. Leung et al (1989) Science, 246: 1306. one such identified cDNA encodes a 165 amino acid protein having more than 95% homology to bovine VEGF; this 165 amino acid protein is commonly referred to as human VEGF (hVEGF) or VEGF₁₆₅. The mitogenic activity of human VEGF was verified by expression of human VEGF cDNA in mammalian host cells. Media conditioned by cells transfected with human VEGF cDNA promoted capillary endothelial cell proliferation, while control cells did not. Leung et al (1989) Science, supra. Further efforts were made to clone and express VEGF via recombinant DNA techniques. (see, e.g., Ferrara, Laboratory Investigation 72: 615-618(1995) and references cited therein).

VEGF is expressed in various tissues as multiple homodimeric forms (121, 145, 165, 189, and 206 amino acids per monomer) resulting from alternative RNA splicing. VEGF₁₂₁Is a soluble mitogen, does not bind heparin; longer forms of VEGF bind heparin with increasingly higher affinity. The heparin-bound form of VEGF can be cleaved by plasmin at the carboxy terminus to release the diffusible form of VEGF. Amino acid sequencing of the carboxy-terminal peptide identified after plasmin cleavage was Arg₁₁₀-Ala₁₁₁. Amino-terminal "core" proteins isolated as homodimers, VEGF (1-110) to be used with intact VEGF₁₆₅Homodimers bind neutralizing monoclonal antibodies (such as the antibodies designated 4.6.1 and 3.2 e3.1.1) and soluble forms of the VEGF receptor with similar affinity.

Several molecules that are structurally related to VEGF have also been recently identified, including placental growth factor (PIGF), VEGF-B, VEGF-C, VEGF-D and VEGF-E. Ferrara and Davis-Smyth (1987) endocr.rev., supra; ogawa et al, j.biological chem.273: 31273-31281 (1998); meyer et al, EMBO j., 18: 363-374(1999). The receptor tyrosine kinase Flt-4(VEGFR-3) was identified as a receptor for VEGF-C and VEGF-D. Joukov et al, EMBO.J.15: 1751 (1996); lee et al, PNAS USA 93: 1988 1992 (1996); achen et al (1998) PNAS USA 95: 548-553. VEGF-C has been shown to be involved in the regulation of lymphangiogenesis. Jeltsch et al, Science 276: 1423-1425(1997).

Two VEGF receptors have been identified, Flt-1 (also known as VEGFR-1) and KDR (also known as VEGFR-2). Shibuya et al (1990) Oncogene 8: 519-; de Vries et al (1992) Science 255: 989-; terman et al (1992) biochem. Biophys. Res. Commun.187: 1579-1586. Neuropilin-1 is shown to be a selective VEGF receptor, capable of binding heparin-binding VEGF isoforms (Soker et al (1998) Cell 92: 735-45).

anti-VEGF antibodies useful in the methods of the invention include any antibody or antigen-binding fragment thereof that binds VEGF with sufficient affinity and specificity and is capable of reducing or inhibiting the biological activity of VEGF. anti-VEGF antibodies typically do not bind to other VEGF homologs, such as VEGF-B or VEGF-C, nor to other growth factors, such as PlGF, PDGF, or bFGF.

In certain embodiments of the invention, anti-VEGF antibodies include, but are not limited to, antibodies directed against the monoclonal anti-VEGF antibody a4.6.1 produced by hybridoma ATCC HB 10709; according to Presta et al (1997) Cancer Res.57: the recombinant humanized anti-VEGF monoclonal antibody generated by 4593-4599 can be combined with monoclonal antibody with same epitope. In one embodiment, the anti-VEGF antibody is "Bevacizumab (BV)", also known as "rhuMAb VEGF" orIt contains mutated human IgG1 frame region and antigen binding complementarity determining region from mouse anti-hVEGF monoclonal antibody A.4.6.1, and blocks human VEGF bindingIts receptor. Approximately 93% of the amino acid sequence of bevacizumab (including most of the framework regions) was derived from human IgG1, and approximately 7% of the sequence was derived from the mouse antibody a4.6.1.

BevacizumabIs the first anti-angiogenic therapy to be FDA approved and is approved for the treatment of metastatic colorectal cancer (first and second line therapy, in combination with intravenous 5-FU based chemotherapy), advanced non-squamous, non-small cell lung cancer (NSCLC) (unresectable, locally advanced, recurrent or metastatic first line therapy of NSCLC, in combination with carboplatin and paclitaxel) and metastatic HER2 negative breast cancer (previously untreated, metastatic HER2 negative breast cancer, in combination with paclitaxel).

Bevacizumab and other humanized anti-VEGF antibodies are further described in U.S. patent No.6,884,879, published on 26/2/2005. Additional antibodies include antibodies of the G6 or B20 series (e.g., G6-31, B20-4.1), as described in PCT publication No. WO2005/012359, PCT publication No. WO2005/044853, and U.S. patent application 60/991,302, the contents of which are expressly incorporated herein by reference. For additional antibodies, see U.S. Pat. nos. 7,060,269, 6,582,959, 6,703,020; 6,054,297; WO 98/45332; WO 96/30046; WO 94/10202; EP0666868B 1; U.S. patent application publication nos. 2006009360, 20050186208, 20030206899, 20030190317, 20030203409, and 20050112126; and Popkov et al, Journal of Immunological Methods 288: 149-164(2004). Other antibodies include those that bind to a functional epitope on human VEGF that comprises residues F17, M18, D19, Y21, Y25, Q89, I91, K101, E103, and C104 or that comprises residues F17, Y21, Q22, Y25, D63, I83, and Q89.

In one embodiment of the invention, the anti-VEGF antibody has a light chain variable region comprising the following amino acid sequence: DIQMTQSPSS LSASVGDRVT ITCSASQDIS NYLNWYQQKP GKAPKVLIYF TSSLHSGVPSRFSGSGSGTD FTLTISSLQP EDFATYYCQQ YSTVPWTFGQ GTKVEIKR (SEQ ID NO: 15); and a heavy chain variable region comprising the amino acid sequence: EVQLVESGGG LVQPGGSLRL SCAASGYTFT NYGMNWVRQA PGKGLEWVGWINTYTGEPTY AADFKRRFTF SLDTSKSTAY LQMNSLRAED TAVYYCAKYP HYYGSSHWYF DVWGQGTLVTVSS (SEQ ID NO: 14).

The "G6 series antibody" according to the present invention refers to an anti-VEGF antibody derived from the sequence of the G6 antibody or a G6-derived antibody according to any one of fig. 7, 24-26, and 34-35 of PCT publication No. wo2005/012359, the complete disclosure of which is expressly incorporated herein by reference. See also PCT publication No. WO2005/044853, the entire disclosure of which is expressly incorporated herein by reference. In one embodiment, the G6 series antibody binds to a functional epitope on human VEGF, which comprises residues F17, Y21, Q22, Y25, D63, I83, and Q89.

The "B20 series antibody" according to the present invention refers to an anti-VEGF antibody derived from the sequence of the B20 antibody or a B20 derived antibody according to any one of fig. 27-29 of PCT publication No. wo2005/012359, the entire disclosure of which is expressly incorporated herein by reference. See also PCT publication No. WO2005/044853 and U.S. patent application 60/991,302, the contents of which are expressly incorporated herein by reference. In one embodiment, the B20 series antibody binds to a functional epitope on human VEGF that comprises residues F17, M18, D19, Y21, Y25, Q89, I91, K101, E103, and C104.

"functional epitope" according to the present invention refers to an amino acid residue in an antigen that strongly facilitates antibody binding. Mutations in any of the contributing residues in the antigen (e.g., alanine or homolog mutations to wild-type VEGF) disrupt antibody binding such that the relative affinity ratio of the antibody (IC50 mutant VEGF/IC50 wild-type VEGF) is greater than 5 (see example 2 of WO 2005/012359). In one embodiment, the relative affinity ratio is determined by a solution-binding phage display ELISA. Briefly, 96-well Maxisorp immunoplates (NUNC) were coated with the test antibody in Fab form at a concentration of 2ug/ml in PBS overnight at 4 ℃ and blocked with PBS, 0.5% BSA, and 0.05% Tween20(PBT) for 2 hours at room temperature. Phage displaying hVEGF alanine spot mutants (residues 8-109 forms) or wild type hVEGF (8-109) were first incubated in PBT serial dilutions in Fab coated plates for 15 min at room temperature and the plates were washed with PBS, 0.05% Tween20 (PBST). Bound phage were detected with a 1:5000 dilution of anti-M13 monoclonal antibody horseradish peroxidase (Amersham Pharmacia) conjugate in PBT, developed with 3,3 ', 5, 5' -tetramethylbenzidine (TMB, Kirkegaard & Perry Labs, Gaithersburg, Md.) substrate for approximately 5 minutes, quenched with 1.0M H3PO4, and read spectrophotometrically at 450 nm. The ratio of the IC50 values (IC50, ala/IC50, wt) represents the fold reduction in binding affinity (relative binding affinity).

VEGF receptor molecules

The two most well characterized VEGF receptors are VEGFR1 (also known as Flt-1) and VEGFR2 (murine homologs also known as KDR and FLK-1). The specificity of each receptor for each VEGF family member varies, but VEGF-A binds both Flt-1 and KDR. Both Flt-1 and KDR belong to the Receptor Tyrosine Kinase (RTK) family. RTKs comprise a large family of transmembrane receptors with diverse biological activities. At least nineteen (19) unique RTK subfamilies were identified. The family of Receptor Tyrosine Kinases (RTK) includes receptors that are essential for the growth and differentiation of a variety of Cell types (Yarden and Ullrich (1988) Ann. Rev. biochem. 57: 433-478; Ullrich and Schlessinger (1990) Cell 61: 243-254). The intrinsic function of RTKs is activated upon ligand binding, leading to phosphorylation of receptors and various cellular substrates, and subsequently to a variety of cellular responses (Ullrich and Schlessinger (1990) Cell 61: 203-212). Thus, receptor tyrosine kinase mediated signal transduction is initiated by extracellular interaction with specific growth factors (ligands), usually followed by receptor dimerization, stimulating intrinsic protein tyrosine kinase activity and receptor transphosphorylation. Thereby creating binding sites for intracellular signaling molecules and resulting in complex formation with a range of cytoplasmic signaling molecules, driving the appropriate cellular response. (e.g., cell division, differentiation, metabolic effects, changes in the extracellular microenvironment) see Schlesssinger and Ullrich (1992) Neuron 9: 1-20. Structurally, both Flt-1 and KDR have seven immunoglobulin-like domains in the extracellular domain, a single transmembrane region, and a consensus tyrosine kinase sequence interrupted by a kinase insertion domain. Matthews et al (1991) proc.natl.acad.sci.usa 88: 9026-; terman et al (1991) Oncogene 6: 1677-1683. The extracellular domain is involved in VEGF binding, while the intracellular domain is involved in signal transduction.

VEGF receptor molecules or fragments thereof that specifically bind VEGF can be used in the methods of the invention to bind to and sequester VEGF protein, thereby preventing it from signaling. In certain embodiments, the VEGF receptor molecule or VEGF-binding fragment thereof is a soluble form, such as sFlt-1. The soluble form of the receptor exerts an inhibitory effect on the biological activity of the VEGF protein by binding to VEGF, thereby preventing it from binding to its native receptor present on the surface of the target cell. Also included are VEGF receptor fusion proteins, examples of which are described below.

Chimeric VEGF receptor proteins refer to receptor molecules having amino acid sequences derived from at least two different proteins, at least one of which is a VEGF receptor protein, such as the flt-1 or KDR receptor, and which are capable of binding to and inhibiting the biological activity of VEGF. In certain embodiments, the chimeric VEGF receptor proteins of the invention consist of amino acid sequences derived from only two different VEGF receptor molecules; however, amino acid sequences comprising one, two, three, four, five, six, or all seven Ig-like domains from the extracellular ligand binding region of the flt-1 and/or KDR receptor may be linked to amino acid sequences from other unrelated proteins, such as immunoglobulin sequences. Other amino acid sequences in combination with an Ig-like domain will be apparent to one of ordinary skill in the art. Examples of chimeric VEGF receptor proteins include, for example, soluble Flt-1/Fc, KDR/Fc, or FLt 1/KDR/Fc (also known as VEGF Trap) (see, e.g., PCT application publication No. WO97/44453).

Soluble or chimeric VEGF receptor proteins of the invention include VEGF receptor proteins that are not immobilized to the surface of a cell via a transmembrane domain. Thus, soluble forms of VEGF receptors (including chimeric receptor proteins), while capable of binding to and inactivating VEGF, do not contain a transmembrane domain and as such do not generally become bound to the cell membrane of the cell in which the molecule is expressed.

In one embodiment, an antibody (e.g., an antibody used in the methods herein) can incorporate any single or combination of features, as described in sections 1-6 below.

1. Antibody fragments

In certain embodiments, the antibodies provided herein are antibody fragments. Antibody fragments include, but are not limited to, Fab '-SH, F (ab')₂For reviews of certain antibody fragments, see Hudson et al, nat. Med.9:129-134(2003) for reviews of scFv fragments, see for example Pluckth ü n, in The Pharmacology of Monoclonal Antibodies, Vol.113, Rosenburg and Moore, (Springer-Verlag, New York), p.269-315 (1994), see also WO 93/16185, and U.S. Pat. Nos. 5,571,894 and 5,587,458 for Fab and F (ab') containing salvage receptor binding epitope residues and having an extended in vivo half-life₂See U.S. Pat. No.5,869,046 for a discussion of fragments.

Diabodies are antibody fragments with two antigen binding sites, which may be bivalent or bispecific. See, e.g., EP 404,097; WO 1993/01161; hudson et al, nat. Med.9: 129-; and Hollinger et al, Proc. Natl. Acad. Sci. USA 90: 6444-. Tri-and tetrabodies are also described in Hudson et al, nat. Med.9: 129-.

Single domain antibodies are antibody fragments that comprise all or part of the heavy chain variable domain or all or part of the light chain variable domain of the antibody. In certain embodiments, the single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Pat. No.6,248,516B1).

Single-arm antibodies (i.e., the heavy chain variable domain and the light chain variable domain form a single antigen-binding arm) are disclosed in, for example, WO 2005/063816; martens et al, Clin Cancer Res (2006),12: 6144. For the treatment of pathological conditions where antagonistic function is required and where antibody bivalent properties lead to unwanted agonistic effects, the monovalent character of a one-armed antibody (i.e. an antibody comprising a single antigen binding arm) leads to and/or ensures antagonistic function when the antibody binds to a target molecule. In addition, the Fc region-containing one-armed antibodies are characterized by superior pharmacokinetic properties (such as reduced clearance rate and/or extended half-life in vivo) compared to Fab forms with similar/substantially identical antigen binding characteristics, thus overcoming the major disadvantages of using conventional monovalent Fab antibodies. Techniques for making single-armed antibodies include, but are not limited to, "pocket-entry" engineering (see, e.g., U.S. Pat. No.5,731,168). Onartuzumab is an example of a one-armed antibody.

Antibody fragments can be generated by a variety of techniques, including but not limited to proteolytic digestion of intact antibodies and production of recombinant host cells (e.g., e.coli or phage), as described herein.

2. Chimeric and humanized antibodies

In certain embodiments, the antibodies provided herein are chimeric antibodies. Certain chimeric antibodies are described, for example, in U.S. Pat. nos. 4,816,567; and Morrison et al, Proc. Natl. Acad. Sci. USA,81: 6851-. In one example, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate, such as a monkey) and a human constant region. In yet another example, a chimeric antibody is a "class-switched" antibody in which the class or subclass has been altered from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

In certain embodiments, the chimeric antibody is a humanized antibody. Typically, non-human antibodies are humanized to reduce immunogenicity to humans, while retaining the specificity and affinity of the parent non-human antibody. Generally, a humanized antibody comprises one or more variable domains in which HVRs, e.g., CDRs (or portions thereof), are derived from a non-human antibody and FRs (or portions thereof) are derived from a human antibody sequence. Optionally, the humanized antibody will also comprise at least a portion of a human constant region. In some embodiments, some FR residues in the humanized antibody are replaced with corresponding residues from a non-human antibody (e.g., an antibody from which HVR residues are derived), e.g., to restore or improve antibody specificity or affinity.

Humanized antibodies and methods for their production are reviewed, for example, in Almagro and Fransson, front.biosci.13:1619-1633(2008), and further described, for example, in Riechmann et al, Nature 332:323-329 (1988); queen et al, Proc.nat' l Acad.Sci.USA 86:10029-10033 (1989); U.S. Pat. Nos. 5,821,337,7,527,791,6,982,321, and 7,087,409; kashmiri et al, Methods 36:25-34(2005) (SDR (a-CDR) grafting is described); padlan, mol.Immunol.28:489-498(1991) (describes "resurfacing"); dall' Acqua et al, Methods 36:43-60(2005) (describing "FR shuffling"); and Osbourn et al, Methods 36:61-68(2005) and Klimka et al, Br.J. cancer,83:252-260(2000) (describing the "guided selection" method of FR shuffling).

Human framework regions that may be used for humanization include, but are not limited to: framework regions selected using the "best-fit" method (see, e.g., Sims et al, J.Immunol.151:2296 (1993)); framework regions derived from consensus sequences of a specific subset of human antibodies from the light or heavy chain variable regions (see, e.g., Carter et al, Proc. Natl. Acad. Sci. USA,89:4285 (1992); and Presta et al, J.Immunol.,151:2623 (1993)); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, front.biosci.13:1619-1633 (2008)); and framework regions derived by screening FR libraries (see, e.g., Baca et al, J.biol.chem.272:10678-10684(1997) and Rosok et al, J.biol.chem.271:22611-22618 (1996)).

3. Human antibodies

In certain embodiments, the antibodies provided herein are human antibodies. Human antibodies can be generated using a variety of techniques known in the art. In general, human antibodies are described in van Dijk and van de Winkel, Curr, Opin, Pharmacol.5:368-74(2001), and Lonberg, Curr, Opin, Immunol.20: 450-.

Human antibodies can be made by administering an immunogen to a transgenic animal that has been modified to produce fully human antibodies or human antibodies in response to an antigenic challengeIntact antibodies of the variable region. Such animals typically contain all or part of a human immunoglobulin locus, which replaces an endogenous immunoglobulin locus, or which exists extrachromosomally or is randomly integrated into the chromosome of the animal. In such transgenic mice, the endogenous immunoglobulin locus has typically been inactivated. For an overview of the method of obtaining human antibodies from transgenic animals, see Lonberg, nat. Biotech.23:1117-1125 (2005). See also, for example, U.S. Pat. Nos. 6,075,181 and 6,150,584, which describe XENOMOUSE^TMA technique; U.S. Pat. No.5,770,429, which describesA technique; U.S. Pat. No.7,041,870, which describes K-MTechnology, and U.S. patent application publication No. us 2007/0061900, which describesA technique). The human variable regions from the whole antibodies generated by such animals may be further modified, for example by combination with different human constant regions.

Human antibodies can also be generated by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for the Production of human Monoclonal antibodies have been described (see, e.g., Kozbor J.Immunol.,133:3001 (1984); Brodeur et al, Monoclonal Antibody Production Techniques and Applications, pp 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al, J.Immunol.,147:86 (1991)). Human antibodies generated via human B-cell hybridoma technology are also described in Li et al, Proc.Natl.Acad.Sci.USA,103:3557-3562 (2006). Other methods include those described, for example, in U.S. Pat. No.7,189,826, which describes the production of monoclonal human IgM antibodies from hybridoma cell lines, and Ni, Xiaondai Mianyixue,26(4):265-268(2006), which describes human-human hybridomas. The human hybridoma technique (Trioma technique) is also described in Vollmers and Brandlein, Histologyand Histopathlogy, 20(3): 927-.

Human antibodies can also be generated by isolating Fv clone variable domain sequences selected from a human-derived phage display library. Such variable domain sequences can then be combined with the desired human constant domains. Techniques for selecting human antibodies from antibody libraries are described below.

4. Library-derived antibodies

Antibodies of the invention can be isolated by screening combinatorial libraries for antibodies having a desired activity or activities. For example, various methods for generating phage display libraries and screening such libraries for antibodies possessing desired binding characteristics are known in the art. Such Methods are reviewed, for example, in Hoogenboom et al, in Methods in molecular biology 178:1-37 (O' Brien et al, eds., Human Press, Totowa, NJ,2001), and further described, for example, in McCafferty et al, Nature 348: 552-; clackson et al, Nature 352: 624-; marks et al, J.mol.biol.222:581-597 (1992); marks and Bradbury, in Methods in Molecular Biology248:161-175(Lo eds., Human Press, Totowa, NJ, 2003); sidhu et al, J.mol.biol.338(2):299-310 (2004); lee et al, J.mol.biol.340(5):1073-1093 (2004); fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-; and Lee et al, J.Immunol.Methods284(1-2):119-132 (2004).

In some phage display methods, the repertoire of VH and VL genes, respectively, is cloned by Polymerase Chain Reaction (PCR) and randomly recombined in a phage library, which can then be screened for antigen-binding phages, as described in Winter et al, Ann. Rev. Immunol.,12:433-455 (1994). Phage typically display antibody fragments either as single chain fv (scfv) fragments or as Fab fragments. Libraries from immunized sources provide high affinity antibodies to the immunogen without the need to construct hybridomas. Alternatively, the natural repertoire can be cloned (e.g., from humans) to provide a single source of antibodies to a large panel of non-self and also self-antigens in the absence of any immunization, as described by Griffiths et al, EMBO J,12: 725-. Finally, non-rearranged V gene segments can also be synthesized by cloning non-rearranged V gene segments from stem cells and using PCR primers containing random sequences to encode the highly variable CDR3 regions and effecting rearrangement in vitro, as described by Hoogenboom and Winter, J.mol.biol.,227:381-388 (1992). Patent publications describing human antibody phage libraries include, for example: U.S. Pat. No.5,750,373, and U.S. patent publication Nos. 2005/0079574,2005/0119455,2005/0266000,2007/0117126,2007/0160598,2007/0237764,2007/0292936 and 2009/0002360.

Antibodies or antibody fragments isolated from a human antibody library are considered to be human antibodies or human antibody fragments herein.

5. Multispecific antibodies

In certain embodiments, the antibodies provided herein are multispecific antibodies, e.g., bispecific antibodies. Multispecific antibodies are monoclonal antibodies that have binding specificities for at least two different sites. In certain embodiments, one of the binding specificities is for c-met and the other is for any other antigen. In certain embodiments, a bispecific antibody can bind two different epitopes of c-met. Bispecific antibodies can also be used to localize cytotoxic agents to cells expressing c-met. Bispecific antibodies can be prepared as full length antibodies or antibody fragments.

Techniques for generating multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs with different specificities (see Milstein and Cuello, Nature 305:537 (1983)), WO 93/08829, and Traunecker et al, EMBO J.10:3655(1991)), and "protuberance-into-cavity" engineering (see, e.g., U.S. Pat. No.5,731,168). Effects can also be manipulated electrostatically by engineering for the generation of antibody Fc-heterodimer molecules (WO2009/089004a 1); crosslinking two or more antibodies or fragments (see, e.g., U.S. Pat. No.4,676,980, and Brennan et al, Science,229:81 (1985)); the use of leucine zippers to generate bispecific antibodies (see, e.g., Kostelny et al, J.Immunol.,148(5):1547-1553 (1992)); the "diabody" technique used to generate bispecific antibody fragments is used (see, e.g., Hollinger et al, Proc. Natl. Acad. Sci. USA,90: 6444-; and the use of single chain fv (sFv) dimers (see, e.g., Gruber et al, J.Immunol.,152:5368 (1994)); and making a trispecific antibody to generate a multispecific antibody as described, for example, in Tutt et al, j.

Also included herein are engineered antibodies having three or more functional antigen binding sites, including "octopus antibodies" (see, e.g., US 2006/0025576a 1).

Antibodies or fragments herein also include "dual action fabs" or "DAFs" comprising an antigen binding site that binds c-met and another different antigen (such as EGFR) (see, e.g., US 2008/0069820).

6. Antibody variants

In certain embodiments, amino acid sequence variants of the antibodies provided herein are encompassed. For example, it may be desirable to improve the binding affinity and/or other biological properties of an antibody. Amino acid sequence variants of an antibody can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into, and/or substitutions of, residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can be made to arrive at the final construct, so long as the final construct possesses the desired characteristics, e.g., antigen binding.

In certain embodiments, antibody variants having one or more amino acid substitutions are provided. Sites of interest for substitutional mutagenesis include HVRs and FRs. Amino acid substitutions can be introduced into the antibody of interest and the product screened for a desired activity, such as retained/improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

One class of surrogate variants involves replacing one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variants selected for further study will have an alteration (e.g., an improvement) in certain biological properties (e.g., increased affinity, decreased immunogenicity) relative to the parent antibody and/or will substantially retain certain biological properties of the parent antibody. Exemplary surrogate variants are affinity matured antibodies, which can be conveniently generated, for example, using phage display-based affinity maturation techniques such as those described herein. Briefly, one or more HVR residues are mutated and the variant antibodies are displayed on phage and screened for a particular biological activity (e.g., binding affinity).

Amino acid sequence insertions include amino and/or carboxy-terminal fusions ranging in length from 1 residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include fusions of the N-or C-terminus of the antibody with an enzyme (e.g., for ADEPT) or a polypeptide that extends the serum half-life of the antibody.

In certain embodiments, the antibodies provided herein are altered to increase or decrease the degree of glycosylation of the antibody. Addition or deletion of glycosylation sites of an antibody can be conveniently achieved by altering the amino acid sequence such that one or more glycosylation sites are created or eliminated.

In the case of antibodies comprising an Fc region, the carbohydrate to which they are attached may be altered. Natural antibodies produced by mammalian cells typically comprise branched, bi-antennary oligosaccharides, which are typically N-linked to Asn297 of the CH2 domain attached to the Fc region. See, e.g., Wright et al, TIBTECH 15:26-32 (1997). Oligosaccharides may include various carbohydrates, for example, mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to GlcNAc in the "backbone" of the bi-antennary oligosaccharide structure. In some embodiments, the oligosaccharides in the antibodies of the invention may be modified to create antibody variants with certain improved properties.

In one embodiment, antibody variants are provided that have a carbohydrate structure that lacks fucose attached (directly or indirectly) to an Fc region. For example, the amount of fucose in such antibodies may be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297, relative to the sum of all sugar structures (e.g. complexed, hybrid and high mannose structures) attached to Asn297, as measured by MALDI-TOF mass spectrometry, e.g. as described in WO 2008/077546. Asn297 refers to the asparagine residue located at about position 297 in the Fc region (Eu numbering of Fc region residues); however, Asn297 may also be located about ± 3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in the antibody. Such fucosylated variants may have improved ADCC function. See, e.g., U.S. patent publication No. us 2003/0157108(Presta, L.); US 2004/0093621(Kyowa Hakko Kogyo co., Ltd). Examples of publications relating to "defucosylated" or "fucose-deficient" antibody variants include: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO 2005/035778; WO 2005/053742; WO 2002/031140; okazaki et al, J.mol.biol.336:1239-1249 (2004); Yamane-Ohnuki et al, Biotech.Bioeng.87:614 (2004). Examples of cell lines capable of producing defucosylated antibodies include protein fucosylation deficient Lec13CHO cells (Ripka et al, Arch. biochem. Biophys.249: 533-.

Further provided are antibody variants having bisected oligosaccharides, for example, wherein biantennary oligosaccharides attached to the Fc region of the antibody are bisected by GlcNAc. Such antibody variants may have reduced fucosylation and/or improved ADCC function. Examples of such antibody variants are described, for example, in WO 2003/011878(Jean-Mairet et al); U.S. Pat. No.6,602,684(Umana et al); and US 2005/0123546(Umana et al). Antibody variants having at least one galactose residue in an oligosaccharide attached to an Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO 1997/30087(Patel et al); WO 1998/58964(Raju, S.); and WO 1999/22764(Raju, S.).

In certain embodiments, one or more amino acid modifications can be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4Fc region) comprising an amino acid modification (e.g., substitution) at one or more amino acid positions.

In certain embodiments, the invention encompasses antibody variants possessing some, but not all, effector functions that make them desirable candidates for applications where the in vivo half-life of the antibody is important, while certain effector functions (such as complement and ADCC) are unnecessary or detrimental.

Antibodies with reduced effector function include those having substitutions in one or more of residues 238,265,269,270,297,327 and 329 of the Fc region (U.S. Pat. No.6,737,056). Such Fc mutants include Fc mutants having substitutions at two or more of amino acid positions 265,269,270,297 and 327, including so-called "DANA" Fc mutants having substitutions of residues 265 and 297 to alanine (U.S. Pat. No.7,332,581).

Certain antibody variants with improved or reduced binding to FcR are described (see, e.g., U.S. Pat. No.6,737,056; WO 2004/056312, and Shields et al, J.biol.chem.9(2):6591-6604 (2001)).

In certain embodiments, an antibody variant comprises an Fc region with one or more amino acid substitutions that improve ADCC, e.g., substitutions at positions 298, 333, and/or 334 (EU numbering of residues) of the Fc region.

In some embodiments, alterations are made to the Fc region that result in altered (i.e., improved or reduced) C1q binding and/or Complement Dependent Cytotoxicity (CDC), e.g., as described in U.S. Pat. Nos. 6,194,551, WO 99/51642, and Idusogene et al, J.Immunol.164: 4178-.

Antibodies with extended half-life and improved binding to neonatal Fc receptor (FcRn) responsible for the transfer of maternal IgG to the fetus are described in US2005/0014934A1(Hinton et al), the neonatal Fc receptor (FcRn) and are responsible for the transfer of maternal IgG to the fetus (Guyer et al, J.Immunol.117:587(1976) and Kim et al, J.Immunol.24:249 (1994)). Those antibodies comprise an Fc region having one or more substitutions therein that improve the binding of the Fc region to FcRn. Such Fc variants include those having substitutions at one or more of residues 238,256,265,272,286,303,305,307,311,312,317,340,356,360,362,376,378,380,382,413,424 or 434 of the Fc region, for example, at residue 434 of the Fc region (U.S. patent No.7,371,826).

Also found in Duncan and Winter, Nature 322:738-40 (1988); U.S. Pat. Nos. 5,648,260; U.S. Pat. Nos. 5,624,821; and WO 94/29351, which concerns other examples of Fc region variants.

In certain embodiments, it may be desirable to create cysteine engineered antibodies, e.g., "thiomabs," in which one or more residues of the antibody are replaced with cysteine residues. In particular embodiments, the substituted residues are present at accessible sites of the antibody. By replacing those residues with cysteine, the reactive thiol groups are thus localized at accessible sites of the antibody and can be used to conjugate the antibody with other moieties, such as drug moieties or linker-drug moieties, to create immunoconjugates, as further described herein. In certain embodiments, cysteine may be substituted for any one or more of the following residues: v205 of the light chain (Kabat numbering); a118 of the heavy chain (EU numbering); and S400 of the heavy chain Fc region (EU numbering). Cysteine engineered antibodies can be produced as described, for example, in U.S. patent No.7,521,541.

In certain embodiments, the antibodies provided herein can be further modified to contain additional non-proteinaceous moieties known in the art and readily available. Suitable moieties for derivatization of the antibody include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1, 3-dioxolane, poly-1, 3, 6-trioxane, ethylene/maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers), and dextran or poly (n-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymers, propylene oxide/ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in production due to its stability in water. The polymer may be of any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody can vary, and if more than one polymer is attached, they can be the same or different molecules. In general, the number and/or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the specific properties or functions of the antibody to be improved, whether the antibody derivative will be used for therapy under specified conditions, and the like.

In another embodiment, conjugates of an antibody and a non-proteinaceous moiety that can be selectively heated by exposure to radiation are provided. In one embodiment, the non-proteinaceous moiety is a carbon nanotube (Kam et al, Proc. Natl. Acad. Sci. USA 102: 11600-. The radiation can be of any wavelength and includes, but is not limited to, wavelengths that are not damaging to normal cells, but heat the non-proteinaceous moiety to a temperature at which cells in the vicinity of the antibody-non-proteinaceous moiety are killed.

In one embodiment, the drug is an immunoconjugate comprising an antibody (such as a c-met antibody) conjugated to one or more cytotoxic agents, such as a chemotherapeutic agent or drug, a growth inhibitory agent, a toxin (e.g., a protein toxin, an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or a fragment thereof), or a radioisotope.

In one embodiment, the immunoconjugate is an antibody-drug conjugate (ADC) in which the antibody is conjugated to one or more drugs, including but not limited to maytansinoids (see U.S. Pat. nos. 5,208,020, 5,416,064, and european patent EP 0425235B 1); auristatins such as monomethyl auristatin drug modules DE and DF (MMAE and MMAF) (see U.S. Pat. nos. 5,635,483 and 5,780,588 and 7,498,298); dolastatin (dolastatin); calicheamicin (calicheamicin) or a derivative thereof (see U.S. Pat. Nos. 5,712,374,5,714,586,5,739,116,5,767,285,5,770,701,5,770,710,5,773,001 and 5,877,296; Hinman et al, Cancer Res.53:3336-3342 (1993); and Lode et al, Cancer Res.58:2925-2928 (1998)); anthracyclines such as daunomycin (daunomycin) or doxorubicin (doxorubicin) (see Kratz et al, Current Med. chem.13: 477-; methotrexate; vindesine (vindesine); taxanes (taxanes) such as docetaxel (docetaxel), paclitaxel, larotaxel, tesetaxel, and ortataxel; trichothecenes (trichothecenes); and CC 1065.

In another embodiment, the immunoconjugate comprises an antibody as described herein conjugated to an enzymatically active toxin, or fragment thereof, including, but not limited to, diphtheria a chain, a non-binding active fragment of diphtheria toxin, exotoxin a chain (from Pseudomonas aeruginosa), ricin (ricin) a chain, abrin (abrin) a chain, modeccin a chain, α -sarcin (sarcin), aleurites (aleutis fordii) toxic protein, dianthus caryophyllus (dianthin) toxic protein, phytolacca americana (phytolaccai americana) protein (papapi, PAPII and PAP-S), Momordica charantia (mordicacharantia) localized inhibitor, curcin (curcin), crotin (crotin), saponaria officinalis (sapacicularia), leptinorella salicina (sapacia), fumonis officinalis (sapicilin), trichothecin (resmycin), trichothecin (trichomycin), or fragment thereof, or a salicin (gelonin), or fragment thereof, or a fumonisin (trichothecin) inhibitor, or fragment thereof, or a fragment thereof, enomycin (enomycin) and trichothecenes (trichothecenes).

In another embodiment, the immunoconjugate comprises an antibody as described herein conjugated to a radioactive atom to form a radioconjugate. A variety of radioisotopes are available for use in generating radioconjugates. Examples include At²¹¹、I¹³¹、I¹²⁵、Y⁹⁰、Re¹⁸⁶、Re¹⁸⁸、Sm¹⁵³、Bi²¹²、P³²、Pb²¹²And radioactive isotopes of Lu. Where a radioconjugate is used for detection, it may contain a radioactive atom for scintigraphic studies, for example tc⁹⁹m or I¹²³Or spin labels for Nuclear Magnetic Resonance (NMR) imaging (also known as magnetic resonance imaging, mri), such as again iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron.

A variety of bifunctional protein coupling agents may be used to generate conjugates of the antibody and cytotoxic agent, such as N-succinimidyl 3- (2-pyridyldithio) propionate (SPDP), succinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), Iminothiolane (IT), imidoesters (such as dimethyl adipimidate hcl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis (p-diazoniumbenzoyl) -ethylenediamine), diisothiocyanates (such as toluene 2, 6-diisocyanate), and bis-active fluorine compounds (such as 1, 5-difluoro-2, 4-dinitrobenzene) is used. For example, a ricin immunotoxin may be prepared as described in Vitetta et al, Science 238:1098 (1987). Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelator for conjugating radionucleotides to antibodies. See WO 94/11026. The linker may be a "cleavable linker" that facilitates release of the cytotoxic drug in the cell. For example, acid-labile linkers, peptidase-sensitive linkers, photolabile linkers, dimethyl linkers, or disulfide-containing linkers can be used (Chari et al, Cancer Res 52: 127-.

Immunoconjugates or ADCs herein expressly encompass, but are not limited to, such conjugates prepared with crosslinking agents including, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB (succinimidyl- (4-vinylsulfone) benzoate), which are commercially available (e.g., from Pierce Biotechnology, inc., Rockford, il., u.s.a.).

Binding assays and other assays

In one aspect, antibodies are tested for antigen binding activity, for example, by known methods such as ELISA, Western blot, and the like.

In another aspect, a competition assay can be used to identify a polypeptide comprising an amino acid sequence of SEQ ID NO: HVR-L1 of 1; comprises the amino acid sequence of SEQ ID NO: HVR-L2 of 2; comprises the amino acid sequence of SEQ ID NO: 3 HVR-L3; comprises the amino acid sequence of SEQ ID NO: 4 HVR-H1; comprises the amino acid sequence of SEQ ID NO: HVR-H2 of 5; and a polypeptide comprising the amino acid sequence of SEQ ID NO: 6 and/or an anti-c-met antibody comprising the VH sequence SEQ ID NO 7 and the VL sequence SEQ ID NO 8 competes for binding to human c-met. In some embodiments, such competition assays may be used to identify antibodies that compete with obinutuzumab for binding to human c-met.

Exemplary methods for locating epitopes to which antagonists (e.g., antibodies) bind are well known in the art. See, e.g., Merchant, M.et al, PNAS (2013)110(32): E2987-E2996, and Morris (1996) "epitomeping Protocols," in Methods in Molecular Biology vol.66(Humana Press, Totowa, N.J.). For example, agents can be screened for the ability to bind both human c-met or fragments thereof and altered forms of c-met or fragments thereof (where the amino acid residues at the binding site are altered). A c-met antagonist is determined to bind human c-met or a fragment thereof if its binding to the altered form of c-met is reduced (e.g., significantly reduced) as compared to human c-met or a fragment thereof. Binding assays for altered forms of c-met or fragments thereof can be performed simultaneously with binding assays for human c-met or fragments thereof, e.g., as a counter-screen in a high throughput screening background. Alternatively, a binding assay for an altered form of c-met can be performed after the agent has been identified/confirmed to bind human c-met or a fragment thereof. In some embodiments, the method comprises: comparing a) binding of a c-met antagonist (e.g., a c-met antibody) to human c-met or a fragment thereof to b) binding of a c-met antagonist to an altered form of c-met or a fragment thereof comprising an alteration of at least one amino acid residue (including, e.g., at least 2,3, or 4 amino acid residues) of Q328, R331, L337, and N338, wherein a c-met antagonist that exhibits greater binding affinity for human c-met or a fragment thereof than the altered form is selected as a c-met antagonist that selectively binds to a binding site on human c-met comprising such amino acid residue altered in the altered form.

Diagnostic method

In one aspect, the invention provides diagnostic methods, e.g., for identifying a cancer patient who is likely to respond to treatment with a c-met antagonist. In some embodiments, the c-met antagonist is an anti-c-met antibody. In some embodiments, the anti-c-met antibody is obinutuzumab.

Provided are methods of identifying a patient having a glioblastoma (e.g., a previously treated glioblastoma) as likely to respond to therapy comprising a c-met antagonistic antibody (e.g., onartuzumab), the method comprising: (i) measuring the level or presence or absence or prevalence of HGF (e.g., the percentage of cells expressing HGF mRNA) in a sample from the patient; (ii) identifying the patient as more likely to respond to therapy comprising a c-met antagonist antibody (e.g., onartuzumab) when the sample has high HGF biomarker expression. In some embodiments, the method further comprises (iii) selecting a therapy comprising a c-met antagonist antibody (e.g., onartuzumab) or recommending a therapy comprising a c-met antagonist antibody (e.g., onartuzumab) for the patient. In some embodiments, the therapy comprises a c-met antagonist antibody (e.g., onartuzumab), optionally in combination with a second cancer drug. In some embodiments, the method is an in vitro method. In some embodiments, the method further comprises (iv) treating the patient with a therapy comprising a c-met antagonist antibody (e.g., onartuzumab).

Provided are methods of identifying a patient having mesothelioma (e.g., previously treated mesothelioma) as likely to respond to therapy comprising a c-met antagonist antibody (e.g., onartuzumab), the method comprising: (i) measuring the level or presence or absence or prevalence of HGF (e.g., the percentage of cells expressing HGF mRNA) in a sample from the patient; (ii) identifying the patient as more likely to respond to therapy comprising a c-met antagonist antibody (e.g., onartuzumab) when the sample has high HGF biomarker expression. In some embodiments, the method further comprises (iii) selecting a therapy comprising a c-met antagonist antibody (e.g., onartuzumab) or recommending a therapy comprising a c-met antagonist antibody (e.g., onartuzumab) for the patient. In some embodiments, the therapy comprises a c-met antagonist antibody (e.g., onartuzumab), optionally in combination with a second cancer drug. In some embodiments, the method is an in vitro method. In some embodiments, the method further comprises (iv) treating the patient with a therapy comprising a c-met antagonist antibody (e.g., onartuzumab).

Provided are methods of identifying a patient having gastric cancer (e.g., previously treated gastric cancer) as likely to respond to therapy comprising a c-met antagonist antibody (e.g., onartuzumab), the method comprising: (i) measuring the level or presence or absence or prevalence of HGF (e.g., the percentage of cells expressing HGF mRNA) in a sample from the patient; (ii) identifying the patient as more likely to respond to therapy comprising a c-met antagonist antibody (e.g., onartuzumab) when the sample has high HGF biomarker expression. In some embodiments, the method further comprises (iii) selecting a therapy comprising a c-met antagonist antibody (e.g., onartuzumab) or recommending a therapy comprising a c-met antagonist antibody (e.g., onartuzumab) for the patient. In some embodiments, the therapy comprises a c-met antagonist antibody (e.g., onartuzumab), optionally in combination with a second cancer drug. In some embodiments, the method is an in vitro method. In some embodiments, the method further comprises (iv) treating the patient with a therapy comprising a c-met antagonist antibody (e.g., onartuzumab).

Provided are methods of identifying a patient having renal cell carcinoma (e.g., a previously treated renal cell carcinoma) as likely to respond to therapy comprising a c-met antagonist antibody (e.g., onartuzumab), the method comprising: (i) measuring the level or presence or absence or prevalence of HGF (e.g., the percentage of cells expressing HGF mRNA) in a sample from the patient; (ii) identifying the patient as more likely to respond to therapy comprising a c-met antagonist antibody (e.g., onartuzumab) when the sample has high HGF biomarker expression. In some embodiments, the method further comprises (iii) selecting a therapy comprising a c-met antagonist antibody (e.g., onartuzumab) or recommending a therapy comprising a c-met antagonist antibody (e.g., onartuzumab) for the patient. In some embodiments, the therapy comprises a c-met antagonist antibody (e.g., onartuzumab), optionally in combination with a second cancer drug. In some embodiments, the method is an in vitro method. In some embodiments, the method further comprises (iv) treating the patient with a therapy comprising a c-met antagonist antibody (e.g., onartuzumab).

Provided are methods of identifying a patient having hepatocellular carcinoma (e.g., a previously treated hepatocellular carcinoma) as likely to respond to therapy comprising a c-met antagonist antibody (e.g., onartuzumab), the method comprising: (i) measuring the level or presence or absence or prevalence of HGF (e.g., the percentage of cells expressing HGF mRNA) in a sample from the patient; (ii) identifying the patient as more likely to respond to therapy comprising a c-met antagonist antibody (e.g., onartuzumab) when the sample has high HGF biomarker expression. In some embodiments, the method further comprises (iii) selecting a therapy comprising a c-met antagonist antibody (e.g., onartuzumab) or recommending a therapy comprising a c-met antagonist antibody (e.g., onartuzumab) for the patient. In some embodiments, the therapy comprises a c-met antagonist antibody (e.g., onartuzumab), optionally in combination with a second cancer drug. In some embodiments, the method is an in vitro method. In some embodiments, the method further comprises (iv) treating the patient with a therapy comprising a c-met antagonist antibody (e.g., onartuzumab).

In one aspect, the invention provides diagnostic methods, e.g., for identifying cancer patients who are likely to respond to treatment with a c-met antagonist and a VEGF antagonist (e.g., bevacizumab).

Provided are methods of identifying a patient having a glioblastoma (e.g., a previously treated glioblastoma) as likely to respond to therapy comprising (a) a c-met antagonist antibody (e.g., onartuzumab) and (b) a VEGF antagonist (e.g., bevacizumab), the method comprising: (i) measuring the level or presence or absence or prevalence of HGF (e.g., the percentage of cells expressing HGF mRNA) in a sample from the patient; (ii) identifying the patient as more likely to respond to therapy comprising (a) a c-met antagonist antibody (e.g., onartuzumab) and (b) a VEGF antagonist (e.g., bevacizumab) when the sample has high HGF biomarker expression. In some embodiments, the method further comprises (iii) selecting the therapy comprising (a) the c-met antagonist antibody (e.g., onartuzumab) and (b) the VEGF antagonist (e.g., bevacizumab) for the patient or recommending a therapy comprising (a) the c-met antagonist antibody (e.g., onartuzumab) and (b) the VEGF antagonist (e.g., bevacizumab). In some embodiments, the method is an in vitro method. In some embodiments, the method further comprises (iv) treating the patient with a therapy comprising (a) a c-met antagonist antibody (e.g., onartuzumab) and (b) an anti-VEGF antibody (e.g., bevacizumab).

Provided are methods of identifying a patient having a mesothelioma (e.g., a previously treated mesothelioma) as likely to respond to therapy comprising (a) a c-met antagonist antibody (e.g., onartuzumab) and (b) a VEGF antagonist (e.g., bevacizumab), the method comprising: (i) measuring the level or presence or absence or prevalence of HGF (e.g., the percentage of cells expressing HGF mRNA) in a sample from the patient; (ii) identifying the patient as more likely to respond to therapy comprising (a) a c-met antagonist antibody (e.g., onartuzumab) and (b) a VEGF antagonist (e.g., bevacizumab) when the sample has high HGF biomarker expression. In some embodiments, the method further comprises (iii) selecting the therapy comprising (a) the c-met antagonist antibody (e.g., onartuzumab) and (b) the VEGF antagonist (e.g., bevacizumab) for the patient or recommending a therapy comprising (a) the c-met antagonist antibody (e.g., onartuzumab) and (b) the VEGF antagonist (e.g., bevacizumab). In some embodiments, the method is an in vitro method. In some embodiments, the method further comprises (iv) treating the patient with a therapy comprising (a) a c-met antagonist antibody (e.g., onartuzumab) and (b) an anti-VEGF antibody (e.g., bevacizumab).

Provided are methods of identifying a patient having a gastric cancer (e.g., a previously treated gastric cancer) as likely to respond to therapy comprising (a) a c-met antagonist antibody (e.g., onartuzumab) and (b) a VEGF antagonist (e.g., bevacizumab), the method comprising: (i) measuring the level or presence or absence or prevalence of HGF (e.g., percentage of HGFmRNA-expressing cells) in a sample from the patient; (ii) identifying the patient as more likely to respond to therapy comprising (a) a c-met antagonist antibody (e.g., onartuzumab) and (b) a VEGF antagonist (e.g., bevacizumab) when the sample has high HGF biomarker expression. In some embodiments, the method further comprises (iii) selecting the therapy comprising (a) the c-met antagonist antibody (e.g., onartuzumab) and (b) the VEGF antagonist (e.g., bevacizumab) for the patient or recommending a therapy comprising (a) the c-met antagonist antibody (e.g., onartuzumab) and (b) the VEGF antagonist (e.g., bevacizumab). In some embodiments, the method is an in vitro method. In some embodiments, the method further comprises (iv) treating the patient with a therapy comprising (a) a c-met antagonist antibody (e.g., onartuzumab) and (b) an anti-VEGF antibody (e.g., bevacizumab).

Provided are methods of identifying a patient having renal cell carcinoma (e.g., a previously treated renal cell carcinoma) as likely to respond to therapy comprising (a) a c-met antagonist antibody (e.g., onartuzumab) and (b) a VEGF antagonist (e.g., bevacizumab), the method comprising: (i) measuring the level or presence or absence or prevalence of HGF (e.g., the percentage of cells expressing HGF mRNA) in a sample from the patient; (ii) identifying the patient as more likely to respond to therapy comprising (a) a c-met antagonist antibody (e.g., onartuzumab) and (b) a VEGF antagonist (e.g., bevacizumab) when the sample has high HGF biomarker expression. In some embodiments, the method further comprises (iii) selecting the therapy comprising (a) the c-met antagonist antibody (e.g., onartuzumab) and (b) the VEGF antagonist (e.g., bevacizumab) for the patient or recommending a therapy comprising (a) the c-met antagonist antibody (e.g., onartuzumab) and (b) the VEGF antagonist (e.g., bevacizumab). In some embodiments, the method is an in vitro method. In some embodiments, the method further comprises (iv) treating the patient with a therapy comprising (a) a c-met antagonist antibody (e.g., onartuzumab) and (b) an anti-VEGF antibody (e.g., bevacizumab).

Provided are methods of identifying a patient having hepatocellular carcinoma (e.g., a previously treated hepatocellular carcinoma) as likely to respond to therapy comprising (a) a c-met antagonist antibody (e.g., onartuzumab) and (b) a VEGF antagonist (e.g., bevacizumab), the method comprising: (i) measuring the level or presence or absence or prevalence of HGF (e.g., the percentage of cells expressing HGF mRNA) in a sample from the patient; (ii) identifying the patient as more likely to respond to therapy comprising (a) a c-met antagonist antibody (e.g., onartuzumab) and (b) a VEGF antagonist (e.g., bevacizumab) when the sample has high HGF biomarker expression. In some embodiments, the method further comprises (iii) selecting the therapy comprising (a) the c-met antagonist antibody (e.g., onartuzumab) and (b) the VEGF antagonist (e.g., bevacizumab) for the patient or recommending a therapy comprising (a) the c-met antagonist antibody (e.g., onartuzumab) and (b) the VEGF antagonist (e.g., bevacizumab). In some embodiments, the method is an in vitro method. In some embodiments, the method further comprises (iv) treating the patient with a therapy comprising (a) a c-met antagonist antibody (e.g., onartuzumab) and (b) an anti-VEGF antibody (e.g., bevacizumab).

Provided is a method of providing a cancer diagnosis, comprising: (i) measuring HGF biomarker (e.g., level or presence or absence or prevalence of HGF (e.g., percentage of cells expressing HGF mRNA)) in a sample from the patient; (ii) diagnosing the patient as having a cancer comprising a high HGF biomarker when the sample has a high HGF biomarker expression. In some embodiments, the method further comprises (iii) selecting a therapy comprising a c-met antagonist antibody (e.g., onartuzumab) or recommending a therapy comprising a c-met antagonist antibody (e.g., onartuzumab) for the patient. In some embodiments, the therapy comprises a c-met antagonist antibody (e.g., onartuzumab), optionally in combination with a second cancer drug. In some embodiments, the method is an in vitro method. In some embodiments, the method further comprises (iv) treating the patient with a therapy comprising a c-met antagonist antibody (e.g., onartuzumab).

Provided is a method of providing a cancer diagnosis, comprising: (i) measuring HGF biomarker (e.g., level or presence or absence or prevalence of HGF (e.g., percentage of cells expressing HGF mRNA)) in a sample from the patient; (ii) diagnosing the patient as having a cancer comprising a high HGF biomarker when the sample has a high HGF biomarker expression. In some embodiments, the method further comprises (iii) selecting a therapy comprising (a) a c-met antagonist antibody (e.g., onartuzumab) and (b) a VEGF antagonist (e.g., bevacizumab) for the patient or recommending a therapy comprising (a) a c-met antagonist antibody (e.g., onartuzumab) and (b) a VEGF antagonist (e.g., bevacizumab). In some embodiments, the method is an in vitro method. In some embodiments, the method further comprises (iv) treating the patient with a therapy comprising (a) a c-met antagonist antibody (e.g., onartuzumab) and (b) an anti-VEGF antibody (e.g., bevacizumab).

Provided are methods of providing a glioblastoma diagnosis, comprising: (i) measuring HGF biomarker (e.g., level or presence or absence or prevalence of HGF (e.g., percentage of cells expressing HGF mRNA)) in a sample from the patient; (ii) diagnosing the patient as having a glioblastoma comprising high HGF biomarker when the sample has high HGF biomarker expression. In some embodiments, the method further comprises (iii) selecting a therapy comprising a c-met antagonist antibody (e.g., onartuzumab) or recommending a therapy comprising a c-met antagonist antibody (e.g., onartuzumab) for the patient. In some embodiments, the therapy comprises a c-met antagonist antibody (e.g., onartuzumab), optionally in combination with a second cancer drug. In some embodiments, the method is an in vitro method. In some embodiments, the method further comprises (iv) treating the patient with a therapy comprising a c-met antagonist antibody (e.g., onartuzumab).

Provided are methods of providing a glioblastoma diagnosis, comprising: (i) measuring HGF biomarker (e.g., level or presence or absence or prevalence of HGF (e.g., percentage of cells expressing HGF mRNA)) in a sample from the patient; (ii) diagnosing the patient as having a glioblastoma comprising high HGF biomarker when the sample has high HGF biomarker expression. In some embodiments, the method further comprises (iii) selecting a therapy comprising (a) a c-met antagonist antibody (e.g., onartuzumab) and (b) a VEGF antagonist (e.g., bevacizumab) for the patient or recommending a therapy comprising (a) a c-met antagonist antibody (e.g., onartuzumab) and (b) a VEGF antagonist (e.g., bevacizumab). In some embodiments, the method is an in vitro method. In some embodiments, the method further comprises (iv) treating the patient with a therapy comprising (a) a c-met antagonist antibody (e.g., onartuzumab) and (b) an anti-VEGF antibody (e.g., bevacizumab).

Provided is a method of providing a diagnosis of mesothelioma, comprising: (i) measuring HGF biomarker (e.g., level or presence or absence or prevalence of HGF (e.g., percentage of cells expressing HGF mRNA)) in a sample from the patient; (ii) diagnosing the patient as having mesothelioma comprising high HGF biomarker when the sample has high HGF biomarker expression. In some embodiments, the method further comprises (iii) selecting a therapy comprising a c-met antagonist antibody (e.g., onartuzumab) or recommending a therapy comprising a c-met antagonist antibody (e.g., onartuzumab) for the patient. In some embodiments, the therapy comprises a c-met antagonist antibody (e.g., onartuzumab), optionally in combination with a second cancer drug. In some embodiments, the method is an in vitro method. In some embodiments, the method further comprises (iv) treating the patient with a therapy comprising a c-met antagonist antibody (e.g., onartuzumab).

Provided is a method of providing a diagnosis of mesothelioma, comprising: (i) measuring HGF biomarker (e.g., level or presence or absence or prevalence of HGF (e.g., percentage of cells expressing HGF mRNA)) in a sample from the patient; (ii) diagnosing the patient as having mesothelioma comprising high HGF biomarker when the sample has high HGF biomarker expression. In some embodiments, the method further comprises (iii) selecting a therapy comprising (a) a c-met antagonist antibody (e.g., onartuzumab) and (b) a VEGF antagonist (e.g., bevacizumab) for the patient or recommending a therapy comprising (a) a c-met antagonist antibody (e.g., onartuzumab) and (b) a VEGF antagonist (e.g., bevacizumab). In some embodiments, the method is an in vitro method. In some embodiments, the method further comprises (iv) treating the patient with a therapy comprising (a) a c-met antagonist antibody (e.g., onartuzumab) and (b) an anti-VEGF antibody (e.g., bevacizumab).

Provided is a method for providing a diagnosis of gastric cancer, comprising: (i) measuring HGF biomarker (e.g., level or presence or absence or prevalence of HGF (e.g., percentage of cells expressing HGF mRNA)) in a sample from the patient; (ii) diagnosing the patient as having gastric cancer comprising a high HGF biomarker when the sample has a high HGF biomarker expression. In some embodiments, the method further comprises (iii) selecting a therapy comprising a c-met antagonist antibody (e.g., onartuzumab) or recommending a therapy comprising a c-met antagonist antibody (e.g., onartuzumab) for the patient. In some embodiments, the therapy comprises a c-met antagonist antibody (e.g., onartuzumab), optionally in combination with a second cancer drug. In some embodiments, the method is an in vitro method. In some embodiments, the method further comprises (iv) treating the patient with a therapy comprising a c-met antagonist antibody (e.g., onartuzumab).

Provided is a method for providing a diagnosis of gastric cancer, comprising: (i) measuring HGF biomarker (e.g., level or presence or absence or prevalence of HGF (e.g., percentage of cells expressing HGF mRNA)) in a sample from the patient; (ii) diagnosing the patient as having gastric cancer comprising a high HGF biomarker when the sample has a high HGF biomarker expression. In some embodiments, the method further comprises (iii) selecting a therapy comprising (a) a c-met antagonist antibody (e.g., onartuzumab) and (b) a VEGF antagonist (e.g., bevacizumab) for the patient or recommending a therapy comprising (a) a c-met antagonist antibody (e.g., onartuzumab) and (b) a VEGF antagonist (e.g., bevacizumab). In some embodiments, the method is an in vitro method. In some embodiments, the method further comprises (iv) treating the patient with a therapy comprising (a) a c-met antagonist antibody (e.g., onartuzumab) and (b) an anti-VEGF antibody (e.g., bevacizumab).

Provided are methods of providing a diagnosis of renal cell carcinoma, comprising: (i) measuring HGF biomarker (e.g., level or presence or absence or prevalence of HGF (e.g., percentage of cells expressing HGF mRNA)) in a sample from the patient; (ii) diagnosing the patient as having a renal cell carcinoma comprising a high HGF biomarker when the sample has a high HGF biomarker expression. In some embodiments, the method further comprises (iii) selecting a therapy comprising a c-met antagonist antibody (e.g., onartuzumab) or recommending a therapy comprising a c-met antagonist antibody (e.g., onartuzumab) for the patient. In some embodiments, the therapy comprises a c-met antagonist antibody (e.g., onartuzumab), optionally in combination with a second cancer drug. In some embodiments, the method is an in vitro method. In some embodiments, the method further comprises (iv) treating the patient with a therapy comprising a c-met antagonist antibody (e.g., onartuzumab).

Provided are methods of providing a diagnosis of renal cell carcinoma, comprising: (i) measuring HGF biomarker (e.g., level or presence or absence or prevalence of HGF (e.g., percentage of cells expressing HGF mRNA)) in a sample from the patient; (ii) diagnosing the patient as having a renal cell carcinoma comprising a high HGF biomarker when the sample has a high HGF biomarker expression. In some embodiments, the method further comprises (iii) selecting a therapy comprising (a) a c-met antagonist antibody (e.g., onartuzumab) and (b) a VEGF antagonist (e.g., bevacizumab) for the patient or recommending a therapy comprising (a) a c-met antagonist antibody (e.g., onartuzumab) and (b) a VEGF antagonist (e.g., bevacizumab). In some embodiments, the method is an in vitro method. In some embodiments, the method further comprises (iv) treating the patient with a therapy comprising (a) a c-met antagonist antibody (e.g., onartuzumab) and (b) an anti-VEGF antibody (e.g., bevacizumab).

Provided are methods of providing a diagnosis of hepatocellular carcinoma, comprising: (i) measuring HGF biomarker (e.g., level or presence or absence or prevalence of HGF (e.g., percentage of cells expressing HGF mRNA)) in a sample from the patient; (ii) diagnosing the patient as having hepatocellular carcinoma comprising a high HGF biomarker when the sample has a high HGF biomarker expression. In some embodiments, the method further comprises (iii) selecting a therapy comprising a c-met antagonist antibody (e.g., onartuzumab) or recommending a therapy comprising a c-met antagonist antibody (e.g., onartuzumab) for the patient. In some embodiments, the therapy comprises a c-met antagonist antibody (e.g., onartuzumab), optionally in combination with a second cancer drug. In some embodiments, the method is an in vitro method. In some embodiments, the method further comprises (iv) treating the patient with a therapy comprising a c-met antagonist antibody (e.g., onartuzumab).

Provided are methods of providing a diagnosis of hepatocellular carcinoma, comprising: (i) measuring HGF biomarker (e.g., level or presence or absence or prevalence of HGF (e.g., percentage of cells expressing HGF mRNA)) in a sample from the patient; (ii) diagnosing the patient as having hepatocellular carcinoma comprising a high HGF biomarker when the sample has a high HGF biomarker expression. In some embodiments, the method further comprises (iii) selecting a therapy comprising (a) a c-met antagonist antibody (e.g., onartuzumab) and (b) a VEGF antagonist (e.g., bevacizumab) for the patient or recommending a therapy comprising (a) a c-met antagonist antibody (e.g., onartuzumab) and (b) a VEGF antagonist (e.g., bevacizumab). In some embodiments, the method is an in vitro method. In some embodiments, the method further comprises (iv) treating the patient with a therapy comprising (a) a c-met antagonist antibody (e.g., onartuzumab) and (b) an anti-VEGF antibody (e.g., bevacizumab).

Provided is a method of recommending treatment for a patient, comprising: (i) measuring HGF biomarker (e.g., level or presence or absence or prevalence of HGF (e.g., percentage of cells expressing HGF mRNA)) in a sample from the patient; (ii) treatment with a c-met antagonist (optionally with a combination of a c-met antagonist and a VEGF antagonist) is recommended when the sample has high HGF biomarker expression. In some embodiments, the method further comprises (iii) selecting a therapy comprising a c-met antagonist antibody (e.g., onartuzumab). In some embodiments, the method further comprises (iv) treating the patient with a therapy comprising a c-met antagonist antibody (e.g., onartuzumab). In some embodiments, the therapy comprises a c-met antagonist antibody (e.g., onartuzumab), optionally in combination with a second cancer drug. In some embodiments, the therapy comprises a c-met antagonist antibody (e.g., onartuzumab) in combination with a VEGF antagonist. In some embodiments, the method is an in vitro method.

Provided are methods of recommending treatment for a glioblastoma patient, comprising: (i) measuring HGF biomarker (e.g., level or presence or absence or prevalence of HGF (e.g., percentage of cells expressing HGF mRNA)) in a sample from the patient; (ii) treatment with a c-met antagonist (optionally with a combination of a c-met antagonist and a VEGF antagonist) is recommended when the sample has high HGF biomarker expression. In some embodiments, the method further comprises (iii) selecting a therapy comprising a c-met antagonist antibody (e.g., onartuzumab). In some embodiments, the method further comprises (iv) treating the patient with a therapy comprising a c-met antagonist antibody (e.g., onartuzumab). In some embodiments, the therapy comprises a c-met antagonist antibody (e.g., onartuzumab), optionally in combination with a second cancer drug. In some embodiments, the therapy comprises a c-met antagonist antibody (e.g., onartuzumab) in combination with a VEGF antagonist. In some embodiments, the method is an in vitro method.

Provided is a method of recommending treatment for a mesothelioma patient, comprising: (i) measuring HGF biomarker (e.g., level or presence or absence or prevalence of HGF (e.g., percentage of cells expressing HGF mRNA)) in a sample from the patient; (ii) treatment with a c-met antagonist (optionally with a combination of a c-met antagonist and a VEGF antagonist) is recommended when the sample has high HGF biomarker expression. In some embodiments, the method further comprises (iii) selecting a therapy comprising a c-met antagonist antibody (e.g., onartuzumab). In some embodiments, the method further comprises (iv) treating the patient with a therapy comprising a c-met antagonist antibody (e.g., onartuzumab). In some embodiments, the therapy comprises a c-met antagonist antibody (e.g., onartuzumab), optionally in combination with a second cancer drug. In some embodiments, the therapy comprises a c-met antagonist antibody (e.g., onartuzumab) in combination with a VEGF antagonist. In some embodiments, the method is an in vitro method.

Provided is a method of recommending treatment for a gastric cancer patient, comprising: (i) measuring HGF biomarker (e.g., level or presence or absence or prevalence of HGF (e.g., percentage of cells expressing HGF mRNA)) in a sample from the patient; (ii) treatment with a c-met antagonist (optionally with a combination of a c-met antagonist and a VEGF antagonist) is recommended when the sample has high HGF biomarker expression. In some embodiments, the method further comprises (iii) selecting a therapy comprising a c-met antagonist antibody (e.g., onartuzumab). In some embodiments, the method is an in vitro method. In some embodiments, the method further comprises (iv) treating the patient with a therapy comprising a c-met antagonist antibody (e.g., onartuzumab). In some embodiments, the therapy comprises a c-met antagonist antibody (e.g., onartuzumab), optionally in combination with a second cancer drug. In some embodiments, the therapy comprises a c-met antagonist antibody (e.g., onartuzumab) in combination with a VEGF antagonist.

Provided is a method of recommending treatment for a patient with renal cell carcinoma, comprising: (i) measuring HGF biomarker (e.g., level or presence or absence or prevalence of HGF (e.g., percentage of cells expressing HGF mRNA)) in a sample from the patient; (ii) treatment with a c-met antagonist (optionally with a combination of a c-met antagonist and a VEGF antagonist) is recommended when the sample has high HGF biomarker expression. In some embodiments, the method further comprises (iii) selecting a therapy comprising a c-met antagonist antibody (e.g., onartuzumab). In some embodiments, the method is an in vitro method. In some embodiments, the method further comprises (iv) treating the patient with a therapy comprising a c-met antagonist antibody (e.g., onartuzumab). In some embodiments, the therapy comprises a c-met antagonist antibody (e.g., onartuzumab), optionally in combination with a second cancer drug. In some embodiments, the therapy comprises a c-met antagonist antibody (e.g., onartuzumab) in combination with a VEGF antagonist.

Provided is a method of recommending treatment for a hepatocellular carcinoma patient, comprising: (i) measuring HGF biomarker (e.g., level or presence or absence or prevalence of HGF (e.g., percentage of cells expressing HGF mRNA)) in a sample from the patient; (ii) treatment with a c-met antagonist (optionally with a combination of a c-met antagonist and a VEGF antagonist) is recommended when the sample has high HGF biomarker expression. In some embodiments, the method further comprises (iii) selecting a therapy comprising a c-met antagonist antibody (e.g., onartuzumab). In some embodiments, the method further comprises (iv) treating the patient with a therapy comprising a c-met antagonist antibody (e.g., onartuzumab). In some embodiments, the therapy comprises a c-met antagonist antibody (e.g., onartuzumab), optionally in combination with a second cancer drug. In some embodiments, the therapy comprises a c-met antagonist antibody (e.g., onartuzumab) in combination with a VEGF antagonist. In some embodiments, the method is an in vitro method.

In some embodiments of any of the inventions provided herein, the sample is obtained prior to treatment with the c-met antagonist. In some embodiments of any of the inventions provided herein, the sample is obtained prior to treatment with a VEGF antagonist. In some embodiments of any of the inventions provided herein, the sample is obtained prior to treatment with the c-met antagonist and the VEGF antagonist. In some embodiments, the sample is obtained prior to treatment with the cancer drug. In some embodiments, the sample is obtained after the cancer has metastasized. In some embodiments, the sample is Formalin Fixed and Paraffin Embedded (FFPE). In some embodiments, the first sample is tested for HGF expression (e.g., using ISH or PCR). In some embodiments, the sample is a biopsy (e.g., core biopsy), a surgical specimen (e.g., a specimen from a surgical resection), or a fine needle aspirate.

A sample from a patient is tested for expression of one or more biomarkers herein. The source of the tissue or cell sample may be solid tissue, such as from a fresh, frozen and/or preserved organ or tissue sample or biopsy or aspirate (including but not limited to a fine needle aspirate); blood or any blood component; body fluids such as cerebrospinal fluid, amniotic fluid (amniotic fluid), peritoneal fluid (ascites), bronchial lavage fluid, pleural fluid (pleural fluid), sputum, or interstitial fluid; cells from a subject at any time of pregnancy or development. Tissue samples may contain compounds that are not naturally intermixed with tissue in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, and the like. Examples of tumor samples herein include, but are not limited to, tumor biopsies, tumor cells, serum, plasma, circulating plasma proteins, ascites, primary cell cultures or cell lines derived from tumors or exhibiting tumor-like properties, bronchial lavage, pleural fluid (pleural fluid), sputum, cerebrospinal fluid, urine, and preserved tumor samples, such as formalin-fixed, paraffin-embedded tumor samples (including, but not limited to, formalin-fixed paraffin-embedded fine needle aspirate samples) or frozen tumor samples. In one embodiment, the patient sample is a formalin-fixed, paraffin-embedded (FFPE) tumor sample (e.g., a glioblastoma tumor sample, a mesothelioma tumor sample, or a gastric cancer tumor sample). In one embodiment, the patient sample is a biopsy (e.g., needle biopsy). In one embodiment, the patient sample is a formalin-fixed paraffin-embedded sample from a fine needle aspirate. In one embodiment, the sample is an FFPE tumor sample from a core biopsy (e.g., glioblastoma core biopsy, mesothelioma core biopsy, gastric cancer core biopsy, or renal cell carcinoma core biopsy). In one embodiment, the patient sample is a surgical resection sample. The sample may be obtained before or during treatment of the patient with a cancer drug, such as an anti-c-met antagonist. The sample may be obtained before or during treatment of the patient with the cancer drug. Samples can be obtained from primary tumors or from metastatic tumors. The sample may be obtained at the first diagnosis of cancer or, for example, after a tumor has metastasized. Tumor samples may include cancer cells, lymphocytes, leukocytes, stroma, blood vessels, connective tissue, basal lamina, and any other cell type associated with a tumor. In some embodiments, the tumor sample is of the lung, lymph node, stomach, liver, brain, or kidney. In some embodiments, the tumor is macroscopically dissected, for example, to remove morphologically normal brain tissue from a glioblastoma tumor sample. In some embodiments, a macro-dissected glioblastoma tumor sample comprises benign stromal cells (e.g., reactive astrocytes, glial cells, pericytes, and/or endothelial cells). In some embodiments, the tumor is macroscopically dissected, for example, to remove morphologically normal mesothelial tissue from a mesothelioma tumor sample. In some embodiments, the macro-dissected mesothelioma tumor sample comprises benign stromal cells. In some embodiments, the tumor is macroscopically dissected, for example, to remove morphologically normal stomach tissue from a gastric cancer tumor sample. In some embodiments, the macroscopically-dissected gastric cancer tumor sample comprises benign stromal cells (e.g., fibroblasts, macrophages, and/or endothelial cells). In some embodiments, the tumor is macroscopically dissected, for example, to remove morphologically normal renal tissue from the renal cell carcinoma tumor sample. In some embodiments, the macroscopically dissected renal cell carcinoma tumor sample comprises benign stromal cells. In some embodiments, the tumor is macroscopically dissected, for example, to remove morphologically normal liver tissue from a hepatocellular carcinoma tumor sample. In some embodiments, the macro-dissected hepatocellular carcinoma tumor sample comprises benign stromal cells.

A cancer or biological sample exhibiting HGF mRNA expression refers to a cancer or biological sample that expresses (including overexpresses) HGFmRNA in a diagnostic test. A glioblastoma sample exhibiting HGF mRNA expression refers to a glioblastoma sample expressing (including overexpressing) HGF mRNA in a diagnostic test. In some embodiments, the glioblastoma sample includes tumor cells and benign stromal cells. A mesothelioma sample exhibiting HGF mRNA expression refers to a mesothelioma sample that expresses (including overexpresses) HGF mRNA in a diagnostic test. In some embodiments, the mesothelioma sample comprises tumor cells and benign stromal cells. A gastric cancer sample exhibiting HGF mRNA expression refers to a gastric cancer sample that expresses (including overexpresses) HGF mRNA in a diagnostic test. In some embodiments, the gastric cancer sample comprises tumor cells and benign stromal cells. A renal cell carcinoma sample exhibiting HGF mRNA expression refers to a renal cell carcinoma sample that expresses (including overexpresses) HGF mRNA in a diagnostic test. In some embodiments, the renal cell carcinoma sample comprises tumor cells and benign stromal cells. A hepatocellular carcinoma sample exhibiting HGF mRNA expression refers to a hepatocellular carcinoma sample expressing (including overexpressing) HGF mRNA in a diagnostic test. In some embodiments, the hepatocellular carcinoma sample includes tumor cells and benign stromal cells. A sarcoma sample displaying HGF mRNA expression refers to a sarcoma sample expressing (including overexpressing) HGF mRNA in a diagnostic test. In some embodiments, the sarcoma sample comprises tumor cells and benign stromal cells.

A cancer or biological sample exhibiting c-met amplification refers to a cancer or biological sample having an amplified c-met gene in a diagnostic test. In some embodiments, the amplified c-met gene is an average (in a population of cells) of greater than or equal to 4 or more copies of the c-met gene, 5 or more copies of the c-met gene, or an average of 8 or more copies of the c-met gene, or more, such as 10 or more, 12 or more, 15 or more, or 20 or more copies of the c-met gene.

Various methods for determining gene amplification, protein, or mRNA expression include, but are not limited to, gene expression profiling analysis, Polymerase Chain Reaction (PCR) (including quantitative real-time PCR (qRT-PCR)), reverse transcriptase quantitative PCR (rt-qPCR), RNA-Seq, FISH, CISH, microarray analysis, gene expression Sequencing (SAGE), MassARRAY, proteomics, Immunohistochemistry (IHC), Northern and Southern blot analysis, in situ hybridization (e.g., single or multiple nucleic acid in situ hybridization techniques such as the RNAscope technique of Advanced Cell diagnostics), RNAse protection assays, and microarrays (e.g., Illumina BeadArray)^TMA technique; bead array for detection of gene expression (BADG E)). Biomarkers can also be measured by Polymerase Chain Reaction (PCR) based assays, such as quantitative PCR, real-time PCR, quantitative real-time PCR (qRT-PCR), reverse transcriptase PCR (RT-PCR), and reverse transcriptase quantitative PCR (RT-qPCR). Other amplification-based methods include, for example, transcript-mediated amplification (TMA), Strand Displacement Amplification (SDA), nucleic acid sequence-based amplification (NASBA), and signal amplification methods such as bDNA. Nucleic acid biomarkers can also be measured by, for example, NanoString nCounter and high coverage expression profiling (HiCEP). Analysis of the amplified nucleic acid sequences can be performed using various techniques such as microchips, fluorescence polarization assays, sequencing, and matrix-assisted laser desorption ionization (MALDI) mass spectrometry. In some embodimentsThe amplified nucleic acids are analyzed by sequencing. In some embodiments, nucleic acid expression is measured and/or quantified. In some embodiments, protein expression is measured and/or quantified.

Various exemplary methods for determining biomarker expression will now be described in more detail.

PCR assays are well known in the art and include, but are not limited to, real-time PCR (RT-PCR) assays such as quantitative PCR assays, including reverse transcriptase quantitative PCR (RT-qPCR). Platforms for performing quantitative PCR assays include: fluidigm (e.g. BioMark)^TMHD System), Roche Molecular System (e.g., cobas 4800 System).

In one embodiment, HGF nucleic acid (e.g., HGF mRNA) is detected using a method comprising (a) generating cDNA from a sample by reverse transcription using at least one primer; (b) amplifying the cDNA; and (c) detecting the presence of the amplified cDNA. In addition, such methods can include one or more steps that allow for the determination of the level of mRNA in a sample (e.g., by simultaneously or separately examining the level of a comparative control mRNA sequence for a certain gene (e.g., a housekeeping gene, such as an actin family member)). Optionally, the sequence of the amplified cDNA may be determined.

In some embodiments, HGF nucleic acid (e.g., HGF mRNA) is detected using a method comprising (a) performing PCR on nucleic acid (e.g., mRNA) extracted from a patient cancer sample, such as an FFPE-fixed patient cancer sample; and (b) determining the expression of the nucleic acid in the sample.

At the nucleic acid level, the detection can be carried out by electrophoresis, Northern and Southern blot analysis, in situ hybridization (e.g., single or multiple nucleic acid in situ hybridization), RNase protection assay, and microarray (e.g., Illumina bead array)^TMA technique; bead arrays for detecting gene expression (BADG E)) to measure biomarkers. It can also be performed by Polymerase Chain Reaction (PCR) based assays, such as quantitative PCR, real-time PCR, quantitative real-time PCR (qRT-PCR), reverse transcriptase PCR (rt-PCR), and reverse transcriptase quantitative PCR (rt-qPCR)Measuring the biomarker. Other amplification-based methods include, for example, transcript-mediated amplification (TMA), Strand Displacement Amplification (SDA), nucleic acid sequence-based amplification (NASBA), and signal amplification methods such as bDNA. It may also be performed by sequencing-based techniques such as, for example, Gene expression Sequencing (SAGE), RNA-Seq, and high throughput sequencing techniques (e.g., massively parallel sequencing), and SequenomTechniques to measure nucleic acid biomarkers. Nucleic acid biomarkers can also be measured by, for example, NanoString nCounter and high coverage expression profiling (HiCEP).

Among the techniques listed above, one sensitive and flexible quantification method is rt-qPCR, which can be used to compare mRNA levels in different sample populations, in normal and tumor tissues, with or without drug treatment, characterize gene expression patterns, distinguish closely related mrnas, and analyze RNA structure.

The first step is to isolate mRNA from the target sample. The starting material is typically total RNA isolated from human tumors or tumor cell lines and corresponding normal tissues or cell lines, respectively. Thus, RNA can be isolated from a variety of primary tumors, including breast, lung, colon, prostate, brain, liver, kidney, pancreas, stomach, gall bladder, spleen, thymus, testis, ovary, uterus, etc., corresponding normal tissues, or tumor cell lines. If the source of the mRNA is a primary tumor, the mRNA can be extracted from, for example, a frozen or archived paraffin-embedded and fixed (e.g., formalin-fixed) tissue sample. General methods for extracting mRNA are well known in the art and are disclosed in standard textbooks of molecular Biology, including Ausubel et al, Current Protocols of molecular Biology, John Wiley and Sons, 1997. Methods for extracting RNA from paraffin-embedded tissue are disclosed, for example, in Rupp and Locker, Lab invest.56: A67 (1987); de Andre et al, BioTechniques 18:42044 (1995). Specifically, RNA isolation can be performed using purification kits, buffer sets, and proteases from commercial manufacturers such as Qiagen, according to the manufacturer's instructions. For exampleTotal RNA from cultured cells can be isolated using Qiagen RNeasy mini-columns. Other commercial RNA isolation kits includeComplete DNA and RNA purification kit (Madison, Wis.) and paraffin block RNA isolation kit (Ambion, Inc.). Total RNA from tissue samples can be isolated using RNA Stat-60 (Tel-Test). RNA prepared from tumors can be isolated by, for example, cesium chloride density gradient centrifugation.

Since RNA cannot serve as a template for PCR, the first step in gene expression profiling by PCR is reverse transcription of the RNA template into cDNA, followed by its exponential amplification in a PCR reaction. The two most commonly used reverse transcriptases are avian myeloblastosis Virus reverse transcriptase (AMV-RT) and Moloney murine leukemia Virus reverse transcriptase (MMLV-RT). The reverse transcription step is typically primed using specific primers, random hexamers, or oligo-dT primers, depending on the circumstances and goals of the expression profiling analysis. For example, the extracted RNA can be GENEAMP^TMThe RNA PCR kit (Perkin Elmer, calif., USA) was reverse transcribed following the manufacturer's instructions. The derived cDNA can then be used as a template for subsequent PCR reactions. Although the PCR step may employ a variety of thermostable DNA-dependent DNA polymerases, typically Taq DNA polymerase is used, which has 5 '-3' nuclease activity but lacks 3 '-5' proofreading endonuclease activity. In this manner, the user can easily and accurately select the desired target,PCR typically utilizes the 5 '-nuclease activity of Taq or Tth polymerase to hydrolyze hybridization probes bound to their target amplicons, but any enzyme with equivalent 5' nuclease activity can be used. Two oligonucleotide primers are used to generate the amplicon, typically of a PCR reaction. A third oligonucleotide or probe is designed to detect the nucleotide sequence located between the first two PCR primers. The probe is non-extensible with Taq DNA polymerase and is quenched with a reporter fluorescent dyeAnd (5) extinguishing the fluorescent dye mark. When the two dyes are in close proximity as they are on the probe, any laser-induced emission from the reporter dye is quenched by the quenching dye. During the amplification reaction, Taq DNA polymerase cleaves the probe in a template-dependent manner. The resulting probe fragments dissociate in solution and the signal from the released reporter dye is no longer subject to the quenching effect of the second fluorophore. The detection of the unquenched reporter dye provides the basis for quantitative elucidation of the data.

PCR can be performed using commercially available equipment, such as, for example, ABI PRISM(Perkin-Elmer-Applied Biosystems, Foster City, Calif., USA) or Lightcycler (Roche Molecular Biochemicals, Mannheim, Germany). In one embodiment, the 5' nuclease protocol is run on a real-time quantitative PCR device, such as the ABI PRISMA sequence detection system. The system consists of a thermal cycler, a laser, a Charge Coupled Device (CCD), a camera and a computer. The system amplifies samples in 96-well format on a thermal cycler. During amplification, laser-induced fluorescence signals were collected in all 96 wells in real time via fiber optic cables and detected at the CCD. The system includes software for running the device and analyzing the data.

The 5' -nuclease assay data can be initially expressed as Ct, or cycle threshold (threshold cycle). Fluorescence values were recorded during each cycle and represent the amount of product amplified up to that point in the amplification reaction. The first point at which a statistically significant fluorescence signal is recorded is the cycle threshold (Ct).

To minimize the effects of errors and sample-to-sample variation, PCR is typically performed using internal standards that are expressed at constant levels across different tissues and are not affected by experimental treatments. The most frequently used RNAs for gene expression pattern normalization are the mrnas for the housekeeping genes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β -actin.

One recent variation of the PCR technique is quantitative real-time PCR (qRT-PCT), which generates probes by dual-labeled fluorescence (i.e., double-labeled-PCR)Probe) to measure PCR product accumulation. Quantitative real-time polymerase chain reaction technology refers to a form of PCR in which the amount of PCR product is measured at each step of the PCR reaction. This technique has been described in a number of publications including Cronin et al, am.j.pathol.164(1):35-42 (2004); ma et al, Cancer cell.5: 607-. Real-time PCR is compatible with both quantitative competitive PCR (where internal competitors for each target sequence are used for normalization) and quantitative comparative PCR (where normalization genes or housekeeping genes contained within a sample are used for PCR). For more details see, for example, Held et al, Genome Research 6: 986-. The reverse transcription quantitative polymerase chain reaction (rt-qPCR) technique is a form of PCR in which the nucleic acid to be amplified is RNA, the RNA is first reverse transcribed into cDNA, and the amount of PCR product is measured at each step of the PCR reaction.

Several published journal papers show steps of representative protocols for gene expression profiling using fixed, paraffin-embedded tissues as a source of RNA, including mRNA isolation, purification, primer extension and amplification (e.g., godfreyeet al, j.molec. diagnostics 2:84-91 (2000); Specht et al, am.j. pathol.158:419-29 (2001)). Briefly, one representative method begins by cutting out approximately 10 micron thick sections of paraffin-embedded tumor tissue samples. Then, mRNA is extracted, and protein and DNA are removed. After analysis of the RNA concentration, RNA repair and/or amplification steps may be included, if necessary, and the RNA is reverse transcribed using gene specific promoters, followed by PCR.

According to one aspect of the invention, PCR primers and probes are designed based on intron sequences present in the gene to be amplified. In this embodiment, the first step in primer/probe design is to delineate intron sequences within the gene. This may be done by publicly available software, such as DNABLAT software or BLAST software, including variants thereof, developed by Kent, W.S., Genome Res.12(4):656-64 (2002). The subsequent steps follow a fully established PCR primer and probe design approach.

Thus, in one embodiment, HGF biomarker may be determined using a method comprising: (a) providing a sample comprising or suspected of comprising a target nucleic acid; (b) isolating mRNA from the sample; (c) purifying mRNA from the sample; (d) performing reverse transcription of RNA into cDNA; (e) providing at least one set of two PCR probes capable of hybridizing to the cDNA of the target nucleic acid; (f) providing a third probe designed to hybridize between the two PCR probes to the target nucleic acid, wherein the third probe is non-extendable by Taq DNA polymerase and labeled with a reporter fluorescent dye and a quencher fluorescent dye; (g) amplifying cDNA of the target nucleic acid using PCR; (h) quantifying the amount of the target nucleic acid in the sample by detecting the amount of unquenched reporter dye; (i) comparing the amount of the target nucleic acid in the sample to the expression level of an internal standard.

In one embodiment, HGF biomarkers can be determined using a method comprising: (a) providing a sample comprising or suspected of comprising HGF nucleic acid, wherein the sample comprises a paraffin-embedded, formalin-fixed tissue sample (e.g., a paraffin-embedded, formalin-fixed glioblastoma, mesothelioma, gastric cancer, renal cell carcinoma, hepatocellular carcinoma, or sarcoma tissue sample); (b) isolating HGF mRNA from the sample; (c) purifying HGF mRNA from the sample; (d) performing reverse transcription of RNA into cDNA; (e) providing at least one set of two PCR probes capable of hybridizing to cDNA of HGF (f) providing a third probe designed to hybridize between the two PCR probes to the target nucleic acid, wherein the third probe is non-extendable by Taq DNA polymerase and labeled with a reporter fluorescent dye and a quencher fluorescent dye; (g) amplifying the cDNA of HGF using PCR; (h) quantifying the amount of HGF nucleic acid in the sample by detecting the amount of unquenched reporter dye; (i) comparing the amount of HGF nucleic acid in the sample to the expression level of one or more internal standards (e.g., the expression level of GAPDH, β -actin, AL-1377271, and/or VPS-33B) based on the difference in the Ct value of HGF and the mean Ct value of the internal standards.

In some embodiments, a high amount of HGF biomarker (high HGF biomarker) refers to a high HGF mRNA (e.g., in a sample, e.g., in a tumor section of a cancer (e.g., glioblastoma, mesothelioma, gastric cancer, renal cell carcinoma, hepatocellular carcinoma, or sarcoma) of a patient). In some embodiments, HGF mRNA expression is determined using an amplification-based assay. In some embodiments, the amplification-based assay is a PCR-based assay. In some embodiments, the PCR-based assay is quantitative PCR, real-time PCR, quantitative real-time PCR (qRT-PCR), reverse transcriptase PCR (rt-PCR), or reverse transcription quantitative PCR (rt-qPCR). In some embodiments, HGF mRNA expression is determined using rt-qPCR. In some embodiments, HGF mRNA expression is determined using Fluidigm gene expression analysis. In some embodiments, high HGF mRNA is determined based on relative expression levels compared to a standard established by measuring HGF mRNA levels in tumor samples obtained from a reference population of patients comprising a representative number of patients, including patients with a particular cancer (e.g., glioblastoma, mesothelioma, gastric cancer, renal cell carcinoma, hepatocellular carcinoma, or sarcoma). In some embodiments, the representative number of patients is 10 or more patients. In some embodiments, the representative number of patients is 25 or more patients. In some embodiments, the representative number of patients is 50 or more patients. In some embodiments, the representative number of patients is 100 or more patients. In some embodiments, a reference population of patients described herein comprises a representative number of glioblastoma patients (e.g., relapsed glioblastoma). In some embodiments, a reference population of patients described herein comprises a representative number of mesothelioma patients (e.g., relapsed mesothelioma). In some embodiments, a reference population of patients described herein comprises a representative number of gastric cancer patients (e.g., recurrent gastric cancer). In some embodiments, a reference population of patients described herein comprises a representative number of renal cell carcinoma patients (e.g., recurrent renal cell carcinoma). In some embodiments, a reference population of patients described herein comprises a representative number of hepatocellular carcinoma patients (e.g., recurrent hepatocellular carcinoma). In some embodiments, a reference population of patients described herein comprises a representative number of sarcoma patients (e.g., recurrent sarcomas). In some embodiments, the high HGF mRNA expression level of the patient tumor sample is an HGF mRNA expression level that is greater than 50% of the HGF mRNA expression level in a tumor sample obtained from a reference patient population comprising patients having a particular cancer (e.g., glioblastoma, mesothelioma, gastric cancer, renal cell carcinoma, hepatocellular carcinoma, or sarcoma). In some embodiments, the high HGF mRNA expression level of the patient tumor sample is an HGFmRNA expression level that is greater than 60% of the HGF mRNA expression level in a tumor sample obtained from a reference patient population comprising patients having a particular cancer (e.g., glioblastoma, mesothelioma, gastric cancer, renal cell carcinoma, hepatocellular carcinoma, or sarcoma). In some embodiments, the high HGF mRNA expression level of the patient tumor sample is an HGF mRNA expression level that is greater than 65% of the HGF mRNA expression level in a tumor sample obtained from a reference patient population comprising patients having a particular cancer (e.g., glioblastoma, mesothelioma, gastric cancer, renal cell carcinoma, hepatocellular carcinoma, or sarcoma). In some embodiments, the high HGF mRNA expression level of the patient tumor sample is an HGF mRNA expression level that is greater than 70% of the HGF mRNA expression level in a tumor sample obtained from a reference patient population comprising patients having a particular cancer (e.g., glioblastoma, mesothelioma, gastric cancer, renal cell carcinoma, hepatocellular carcinoma, or sarcoma). In some embodiments, the high HGFmRNA expression level of the patient tumor sample is an HGF mRNA expression level that is greater than 75% of an HGF mRNA expression level in a tumor sample obtained from a reference patient population comprising patients having a particular cancer (e.g., glioblastoma, mesothelioma, gastric cancer, renal cell carcinoma, hepatocellular carcinoma, or sarcoma). In some embodiments, the high HGF mRNA expression level of the patient tumor sample is an HGF mRNA expression level that is greater than 80% of an HGF mRNA expression level in a tumor sample obtained from a reference patient population comprising patients having a particular cancer (e.g., glioblastoma, mesothelioma, gastric cancer, renal cell carcinoma, hepatocellular carcinoma, or sarcoma). In some embodiments, the tumor sample comprises glioblastoma tumor cells and benign stromal cells. In some embodiments, the tumor sample comprises mesothelioma tumor cells and benign stromal cells. In some embodiments, the tumor sample comprises gastric cancer tumor cells and benign stromal cells. In some embodiments, the tumor sample comprises renal cell carcinoma tumor cells and benign stromal cells. In some embodiments, the tumor sample comprises hepatocellular carcinoma tumor cells and benign stromal cells. In some embodiments, the tumor sample comprises sarcoma tumor cells and benign stromal cells.

In some embodiments, high HGF mRNA biomarker is determined using an amplification-based assay. In some embodiments, the amplification-based assay is a PCR-based assay. In some embodiments, the PCR-based assay is quantitative PCR, real-time PCR, quantitative real-time PCR (qRT-PCR), reverse transcriptase PCR (rt-PCR), or reverse transcription quantitative PCR (rt-qPCR). In some embodiments, the PCR-based assay is rt-qPCR. In some embodiments, the high HGFmRNA biomarker is determined using Fluidigm gene expression analysis. In some embodiments, high HGF mRNA biomarker is determined by determining the Ct of HGF mRNA compared to the Ct of mRNA from a reference gene. In some embodiments, the reference gene is a gene that is stably expressed at equivalent levels across multiple cell lines, in freshly frozen tissue samples, and in formalin-fixed paraffin-embedded tissue samples. In some embodiments, the Ct of several reference genes is determined and the mean Ct is compared to the Ct of HGF mRNA. In some embodiments, high HGF mRNA biomarker is determined by determining a Δ Ct for HGF expression, wherein the Δ Ct is equal to the mean Ct for HGF minus the mean Ct for the target gene.

In some embodiments, a small amount of HGF biomarker (low HGF biomarker) refers to a low HGF mRNA biomarker (e.g., in a sample, e.g., in a tumor slice of a cancer (e.g., glioblastoma, mesothelioma, gastric cancer, renal cell carcinoma, hepatocellular carcinoma, or sarcoma) of a patient). In some embodiments, low mRNA expression is determined using an amplification-based assay. In some embodiments, the amplification-based assay is a PCR-based assay. In some embodiments, the PCR-based assay is quantitative PCR, real-time PCR, quantitative real-time PCR (qRT-PCR), reverse transcriptase PCR (rt-PCR), or reverse transcription quantitative PCR (rt-qPCR). In some embodiments, HGF mRNA expression is determined using rt-qPCR. In some embodiments, HGFmRNA expression is determined using Fluidigm gene expression analysis. In some embodiments, the low HGFmRNA is determined based on the relative expression level compared to a standard established by measuring HGF mRNA levels in tumor samples obtained from a reference population of patients comprising a representative number of patients, including patients with a particular cancer (e.g., glioblastoma, mesothelioma, gastric cancer, renal cell carcinoma, hepatocellular carcinoma, or sarcoma). In some embodiments, the representative number of patients is 10 or more patients. In some embodiments, the representative number of patients is 25 or more patients. In some embodiments, the representative number of patients is 50 or more patients. In some embodiments, the representative number of patients is 100 or more patients. In some embodiments, a reference population of patients described herein comprises a representative number of glioblastoma patients (e.g., relapsed glioblastoma). In some embodiments, a reference population of patients described herein comprises a representative number of mesothelioma patients (e.g., relapsed mesothelioma). In some embodiments, a reference population of patients described herein comprises a representative number of gastric cancer patients (e.g., recurrent gastric cancer). In some embodiments, a reference population of patients described herein comprises a representative number of renal cell carcinoma patients (e.g., recurrent renal cell carcinoma). In some embodiments, a reference population of patients described herein comprises a representative number of hepatocellular carcinoma patients (e.g., recurrent hepatocellular carcinoma). In some embodiments, a reference population of patients described herein comprises a representative number of sarcoma patients (e.g., recurrent sarcomas). In some embodiments, the low HGFmRNA expression level of the patient tumor sample is an HGF mRNA expression level that is less than 50% of an HGF mRNA expression level in a tumor sample obtained from a reference patient population comprising patients having a particular cancer (e.g., glioblastoma, mesothelioma, gastric cancer, renal cell carcinoma, hepatocellular carcinoma, or sarcoma). In some embodiments, a low HGF mRNA expression level of a patient tumor sample is an HGF mRNA expression level that is less than 60% of the HGF mRNA expression level in a tumor sample obtained from a reference patient population comprising patients with a particular cancer (e.g., glioblastoma, mesothelioma, gastric cancer, renal cell carcinoma, hepatocellular carcinoma, or sarcoma). In some embodiments, a low HGF mRNA expression level of a patient tumor sample is an HGF mRNA expression level that is less than 65% of the HGF mRNA expression level in a tumor sample obtained from a reference patient population comprising patients having a particular cancer (e.g., glioblastoma, mesothelioma, gastric cancer, renal cell carcinoma, hepatocellular carcinoma, or sarcoma). In some embodiments, a low HGF mRNA expression level of a patient tumor sample is an HGF mRNA expression level that is less than 70% of the HGF mRNA expression level in a tumor sample obtained from a reference patient population comprising patients with a particular cancer (e.g., glioblastoma, mesothelioma, gastric cancer, renal cell carcinoma, hepatocellular carcinoma, or sarcoma). In some embodiments, a low HGF mRNA expression level of a patient tumor sample is an HGF mRNA expression level that is less than 75% of an HGF mRNA expression level in a tumor sample obtained from a reference patient population comprising patients having a particular cancer (e.g., glioblastoma, mesothelioma, gastric cancer, renal cell carcinoma, hepatocellular carcinoma, or sarcoma). In some embodiments, a low HGF mRNA expression level of a patient tumor sample is an HGF mRNA expression level that is less than 80% of an HGF mRNA expression level in a tumor sample obtained from a reference patient population comprising patients having a particular cancer (e.g., glioblastoma, mesothelioma, gastric cancer, renal cell carcinoma, hepatocellular carcinoma, or sarcoma). In some embodiments, the tumor sample comprises glioblastoma tumor cells and benign stromal cells. In some embodiments, the tumor sample comprises mesothelioma tumor cells and benign stromal cells. In some embodiments, the tumor sample comprises gastric cancer tumor cells and benign stromal cells. In some embodiments, the tumor sample comprises renal cell carcinoma tumor cells and benign stromal cells. In some embodiments, the tumor sample comprises hepatocellular carcinoma tumor cells and benign stromal cells. In some embodiments, the tumor sample comprises sarcoma tumor cells and benign stromal cells.

In some embodiments, low HGF mRNA biomarker is determined using an amplification-based assay. In some embodiments, the amplification-based assay is a PCR-based assay. In some embodiments, the PCR-based assay is quantitative PCR, real-time PCR, quantitative real-time PCR (qRT-PCR), reverse transcriptase PCR (rt-PCR), or reverse transcription quantitative PCR (rt-qPCR). In some embodiments, the PCR-based assay is rt-qPCR. In some embodiments, the low HGFmRNA biomarker is determined using Fluidigm gene expression analysis. In some embodiments, low HGF mRNA biomarker is determined by determining the Ct of HGF mRNA compared to the Ct of mRNA from a reference gene. In some embodiments, the reference gene is a gene that is stably expressed at equivalent levels across multiple cell lines, in a fresh frozen tissue sample, and/or in a formalin-fixed paraffin-embedded tissue sample. In some embodiments, the Ct of several reference genes is determined and the mean Ct is compared to the Ct of HGF mRNA. In some embodiments, low HGF mRNA biomarker is determined by determining a Δ Ct for HGF expression, wherein the Δ Ct is equal to the mean Ct for HGF minus the mean Ct for the target gene.

ISH refers to a type of hybridization that uses a complementary DNA or RNA strand (i.e., probe) to locate a specific DNA or RNA sequence in a portion or section of tissue (in situ). Primer and probe types includeBut are not limited to, double-stranded dna (dsdna), single-stranded dna (ssdna), single-stranded complementary rna (sscrna), messenger rna (mrna), microrna (mirna), and synthetic oligonucleotides. In some embodiments, the probe is labeled, for example with a fluorescent label (e.g., FISH, or fluorescent in situ hybridization). In some embodiments, the probe is labeled, e.g., with a chromogenic label (e.g., CISH, or chromogenic in situ hybridization). In some embodiments, ISH is performed (e.g., using DNA primers) and then the ISH signal is amplified using hybridization-based signal amplification (e.g., using an amplification probe and a label probe). Examples of hybridization-based signal amplification include the use of branched DNA molecules to amplify ISH signals. Exemplary platforms utilizing hybridization-based signal amplification include:(Affymetrix)；(Advanced Cell Technology). ISH can be performed in single pass (single target) or in multiple passes (multiple targets). For example, for the QuantiGene assay, a probe set is used to hybridize to a target mRNA. A typical probe set utilizes up to 20 or more pairs of oligonucleotide probes. After hybridization of the probe sets, preamplifiers, amplifiers and label probes are added to generate signals for visualization. The preamplifier probe binds to the target-specific probe, then the amplifier probe binds to the preamplifier probe, followed by the label probe binding to the amplifier probe. For example, forAssay Technology (Advanced Cell Technology), two or more capture probes are hybridized sequentially to a target mRNA. The capture probe may contain, for example, a region of 18-25 bases complementary to the target RNA, a spacer sequence, and a 15 base tail. A pair of target probes, each possessing a different type of tail sequence, are used and hybridize sequentially to the target region (about 50 bp). Two "tail" sequences on the probe form a 28 base preamplifier probe hybridization site, the preamplifier probe contains multiple(e.g., 20) amplifier probe binding sites, the amplifier probe in turn contains a plurality (e.g., 20) of label probe binding sites. Preamplifiers, amplifiers and label probes hybridize sequentially to each pair of capture probes, resulting in the accumulation of up to 8,000 label molecules per 1kb target RNA. The label probe may be conjugated to a fluorophore or a chromogenic enzyme (e.g., horseradish peroxidase or alkaline phosphatase) to enable viewing of the hybridization signal under a standard bright field or epifluorescence microscope, respectively. Thus, in one embodiment, HGF biomarker may be determined using a method comprising: (a) providing a sample comprising or suspected of comprising the target nucleic acid; (b) providing at least one set of two or more capture probes capable of hybridizing to the target nucleic acid; (c) providing: (i) an amplifier capable of hybridizing to the label probe; (ii) a preamplifier capable of hybridizing to an amplifier and capable of hybridizing to a set of said two or more capture probes; (iii) a label probe; (d) hybridizing the set of two or more capture probes to the target nucleic acid; (e) capturing a preamplifier, an amplifier and a label probe to the set of two or more capture probes, thereby capturing the label probe to the target nucleic acid; and (f) detecting the presence, absence, or amount of label associated with the captured label probe.

In some embodiments, a high amount of HGF biomarker (high HGF biomarker) refers to a high HGF mRNA biomarker (e.g., in a sample, e.g., in a tumor slice of a cancer (e.g., glioblastoma, mesothelioma, gastric cancer, renal cell carcinoma, hepatocellular carcinoma, or sarcoma) of a patient). In some embodiments, HGF mRNA expression is determined using ISH. In some embodiments, high HGF mRNA biomarker is 1% or more of HGF ISH signal positive cells in the sample. In some embodiments, high HGF mRNA biomarker is 2% or more of HGF ISH signal positive cells in the sample. In some embodiments, high HGF mRNA biomarker is 3% or more of HGF ISH signal positive cells in the sample. In some embodiments, high HGF mRNA biomarker is 4% or more of HGF ISH signal positive cells in the sample. In some embodiments, high HGF mRNA biomarker is 5% or more of HGF ISH signal positive cells in the sample. In some embodiments, high HGF mRNA biomarker is 6% or more of HGF ISH signal positive cells in the sample. In some embodiments, high HGF mRNA biomarker is 7% or more of HGF ISH signal positive cells in the sample. In some embodiments, high HGF mRNA biomarker is 8% or more of HGF ISH signal positive cells in the sample. In some embodiments, high HGF mRNA biomarker is 9% or more of HGF ISH signal positive cells in the sample. In some embodiments, high HGF mRNA biomarker is 10% or more of HGF ISH signal positive cells in the sample. In some embodiments, high HGF mRNA biomarker is 12% or more of HGF ISH signal positive cells in the sample. In some embodiments, high HGF mRNA biomarker is 15% or more of HGF ISH signal positive cells in the sample. In some embodiments, high HGF mRNA biomarker is 20% or more of HGF ISH signal positive cells in the sample. In some embodiments, high HGF mRNA biomarker is 25% or more of HGF ISH signal positive cells in the sample. In some embodiments, high HGF mRNA biomarker is 30% or more of HGF ISH signal positive cells in the sample. In some embodiments, high HGF mRNA biomarker is 35% or more of HGF ISH signal positive cells in the sample. In some embodiments, high HGF mRNA biomarker is 40% or more of HGF ISH signal positive cells in the sample. In some embodiments, high HGF mRNA biomarker is 50% or more of HGF ISH signal positive cells in the sample. In some embodiments, high HGF mRNA biomarker is 55% or more of HGF ISH signal positive cells in the sample. In some embodiments, high HGF mRNA biomarker is 60% or more of HGF ISH signal positive cells in the sample. In some embodiments, high HGF mRNA biomarker is 65% or more of HGF ISH signal positive cells in the sample. In some embodiments, high HGF mRNA biomarker is 70% or more of HGF ISH signal positive cells in the sample. In some embodiments, high HGF mRNA biomarker is 75% or more of HGF ISH signal positive cells in the sample. In some embodiments, the cells are glioblastoma tumor cells and benign stromal cells. In some embodiments, the cells are mesothelioma tumor cells and benign stromal cells. In some embodiments, the cells are gastric cancer tumor cells and benign stromal cells. In some embodiments, the cells are renal cell carcinoma tumor cells and benign stromal cells. In some embodiments, the cells are hepatocellular carcinoma tumor cells and benign stromal cells. In some embodiments, the cells are sarcoma tumor cells and benign stromal cells.

In some embodiments, high HGF mRNA biomarker is the presence of (e.g., in a sample, e.g., in a tumor section of a cancer (e.g., glioblastoma, mesothelioma, gastric cancer, renal cell carcinoma, hepatocellular carcinoma, or sarcoma) in a patient) about 10 or more HGF ISH signal positive cells. In some embodiments, high HGF mRNA biomarker is the presence of about 11 or more HGF ISH signal positive cells. In some embodiments, high HGF mRNA biomarker is the presence of about 12 or more HGF ISH signal positive cells. In some embodiments, high HGF mRNA biomarker is the presence of about 13 or more HGF ISH signal positive cells. In some embodiments, high HGF mRNA biomarker is the presence of about 14 or more HGF ISH signal positive cells. In some embodiments, high HGF mRNA biomarker is the presence of about 15 or more HGF ISH signal positive cells. In some embodiments, high HGF mRNA biomarker is the presence of about 16 or more HGF ISH signal positive cells. In some embodiments, high HGF mRNA biomarker is the presence of about 20 or more HGF ISH signal positive cells. In some embodiments, high HGF mRNA biomarker is the presence of about 25 or more HGF ISH signal positive cells. In some embodiments, high HGF mRNA biomarker is the presence of about 30 or more HGF ISH signal positive cells. In some embodiments, high HGF mRNA biomarker is the presence of about 35 or more HGF ISH signal positive cells. In some embodiments, high HGF mRNA biomarker is the presence of about 40 or more HGF ISH signal positive cells. In some embodiments, high HGF mRNA biomarker is the presence of about 45 or more HGF ISH signal positive cells. In some embodiments, high HGF mRNA biomarker is the presence of about 50 or more HGF ISH signal positive cells. In some embodiments, high HGF mRNA biomarker is the presence of about 55 or more HGF ISH signal positive cells. In some embodiments, high HGF mRNA biomarker is the presence of about 60 or more HGF ISH signal positive cells. In some embodiments, high HGF mRNA biomarker is the presence of about 70 or more HGF ISH signal positive cells. In some embodiments, high HGF mRNA biomarker is the presence of about 75 or more HGF ISH signal positive cells. In some embodiments, high HGF mRNA biomarker is the presence of about 80 or more HGF ISH signal positive cells. In some embodiments, the cells are glioblastoma tumor cells and benign stromal cells. In some embodiments, the cells are mesothelioma tumor cells and benign stromal cells. In some embodiments, the cells are gastric cancer tumor cells and benign stromal cells. In some embodiments, the cells are renal cell carcinoma tumor cells and benign stromal cells. In some embodiments, the cells are hepatocellular carcinoma tumor cells and benign stromal cells. In some embodiments, the cells are sarcoma tumor cells and benign stromal cells.

In some embodiments, high HGF mRNA biomarker is an ISH score of greater than 2 +. In some embodiments, high HGF mRNA biomarker is an ISH score of greater than 3 +. In some embodiments, high HGF mRNA biomarker is an ISH score of 2+ or 3 +. In some embodiments, high HGF mRNA biomarker is an ISH score of greater than 1 +.

In some embodiments, high HGF mRNA biomarker is presence of HGF ISH positive signal in a plurality of cells (e.g., as observed using an optical microscope equipped with a low power objective lens). In some embodiments, high HGF mRNA biomarker is the presence of HGF ISH positive signal in frequent cells (e.g., as observed using light microscopy using a medium or high power objective lens).

In some embodiments, high HGF mRNA biomarker is an HGF ISH positive signal that is readily observed in the presence of a sample viewed with an optical microscope equipped with a low power objective lens (e.g., a 10 power objective lens). In some embodiments, high HGF mRNA biomarker is an HGF ISH positive signal that is readily observed in the presence of a sample viewed with an optical microscope equipped with a medium power objective lens (e.g., a 20 power objective lens) or a high power objective lens (e.g., a 40 power objective lens).

In some embodiments, high HGF mRNA biomarker is the presence of more than 2 foci comprising HGF ISH signal positive cells (e.g., in a sample, e.g., in a tumor section of a patient's cancer (e.g., glioblastoma, mesothelioma, gastric cancer, renal cell carcinoma, hepatocellular carcinoma, or sarcoma)). As used herein, a "foci" refers to one or more HGF ISH signal positive cells surrounded by HGF mRNA ISH signal negative cells. In some embodiments, high HGF mRNA biomarker is the presence of more than 3 foci comprising HGF ISH signal positive cells. In some embodiments, high HGF mRNA biomarker is the presence of more than 4 foci comprising HGF ISH signal positive cells. In some embodiments, high HGF mRNA biomarker is the presence of more than 5 foci comprising HGF ISH signal positive cells. In some embodiments, high HGF mRNA biomarker is the presence of more than 6 foci comprising HGF ISH signal positive cells. In some embodiments, high HGF mRNA biomarker is the presence of more than 7 foci comprising HGF ISH signal positive cells. In some embodiments, high HGF mRNA biomarker is the presence of more than 8 foci comprising HGF ISH signal positive cells. In some embodiments, high HGF mRNA biomarker is the presence of more than 9 foci comprising HGF ISH signal positive cells. In some embodiments, high HGF mRNA biomarker is the presence of more than 10 foci comprising HGF ISH signal positive cells. In some embodiments, high HGF mRNA biomarker is the presence of more than 11 foci comprising HGF ISH signal positive cells. In some embodiments, high HGF mRNA biomarker is the presence of more than 12 foci comprising HGF ISH signal positive cells. In some embodiments, high HGF mRNA biomarker is the presence of more than 13 foci comprising HGF ISH signal positive cells. In some embodiments, a high HGFmRNA biomarker is the presence of more than 14 foci comprising HGF ISH signal positive cells. In some embodiments, high HGF mRNA biomarker is the presence of more than 15 foci comprising HGF ISH signal positive cells. In some embodiments, high HGF mRNA biomarker is the presence of more than 16 foci comprising HGF ISH signal positive cells. In some embodiments, high HGF mRNA biomarker is the presence of more than 17 foci comprising HGF ISH signal positive cells. In some embodiments, high HGF mRNA biomarker is the presence of more than 18 foci comprising HGF ISH signal positive cells. In some embodiments, high HGF mRNA biomarker is the presence of more than 19 foci (or more, such as more than 20, 25, 30, 35, 40, 45, 50 or more foci) comprising HGF ISH signal positive cells. In some embodiments, the cells are glioblastoma tumor cells and benign stromal cells. In some embodiments, the cells are mesothelioma tumor cells and benign stromal cells. In some embodiments, the cells are gastric cancer tumor cells and benign stromal cells. In some embodiments, the cells are renal cell carcinoma tumor cells and benign stromal cells. In some embodiments, the cells are hepatocellular carcinoma tumor cells and benign stromal cells. In some embodiments, the cells are sarcoma tumor cells and benign stromal cells.

In some embodiments, the foci are visible when the slide is viewed using an optical microscope at low magnification (e.g., approximately equivalent to a 10-fold objective lens). In some embodiments, the foci are visible when the slide is viewed using an optical microscope at moderate magnification (e.g., approximately equivalent to a 20-fold objective lens). In some embodiments, the foci are visible when the slide is viewed using an optical microscope at high magnification (e.g., approximately equivalent to a 40-fold objective lens).

In some embodiments, low HGF mRNA biomarker is an ISH score of less than 2 +. In some embodiments, low HGF mRNA biomarker is an ISH score of less than 1 +. In some embodiments, low HGF mRNA biomarker is an ISH score of 0 or 1 +. In some embodiments, low HGF mRNA biomarker is an ISH score of 0.

In some embodiments, low HGF mRNA biomarker is the presence of HGF ISH positive signal in a minority of cells, e.g., 10 or less, such as 9,8, 7, 6, or less (e.g., as observed using an optical microscope equipped with a medium or high power objective lens), for example in a section of a patient's cancer (e.g., glioblastoma, mesothelioma, gastric cancer, renal cell carcinoma, hepatocellular carcinoma, or sarcoma). In some embodiments, low HGF mRNA biomarker is the absence of HGF ISH positive signal in the cell (e.g., as observed using an optical microscope equipped with a medium or high power objective lens).

In some embodiments, HGF mRNA ISH positive signal is compared to a positive control, e.g., HGFISH performed on KP4 pancreatic tumor cells known to express and secrete HGF (Riken BioResource Center, order number RCB 1005). In some embodiments, HGF mRNA ISH positive signal is compared to a negative control, e.g., KP4 cells probed with a DapB ISH probe.

In some embodiments, IHC (discussed further below) and ISH assay formats include performing a series of processing steps on tissue sections mounted for microscopic examination on a suitable solid support (e.g., a slide or other flat support), highlighting certain morphological indicators of disease status or detecting biological markers by selective staining.

In some embodiments, a pre-detection protocol is performed prior to performing detection of a target in an ISH or IHC (discussed further below) assay format. It may involve, for example, the following steps: cutting and trimming tissue, fixing, dehydrating, paraffin infiltrating, cutting into thin sections, sealing on a glass slide, baking, removing paraffin, rehydrating, antigen repairing, sealing, applying primary antibody, cleaning, applying secondary antibody-enzyme conjugate and cleaning.

Many methods of fixing and embedding tissue specimens are known, such as alcohol fixation and formalin fixation and subsequent paraffin embedding (FFPE). The method of fixing and embedding tissue specimens is further discussed below with respect to IHC.

In some embodiments, the target antigen is repaired or uncovered via pretreatment of the target to increase the reactivity of most targets. For an extensive review of antigen retrieval (antigen discovery), see Shi et al, 1997, J Histochem Cytochem,45(3): 327. Antigen retrieval includes various methods to maximize the accessibility of the target for reactivity with specific detection reagents. The most common techniques are enzymatic digestion with proteolytic enzymes (e.g., protease, pronase, pepsin, papain, trypsin or neuraminidase) in a suitable buffer or heat-induced epitope retrieval (HIER) using microwave irradiation, water bath, steam box, oven, autoclave or autoclave heating in a suitable pH stable buffer (usually containing EDTA, EGTA, Tris-HCl, citrate, urea, glycine-HCl or boric acid). The antigen retrieval buffer may be aqueous, but may also contain other solvents, including solvents with boiling points above that of water. In addition, in some embodiments, the signal-to-noise ratio can be improved by different physical methods, including freezing and thawing the slices, or applying vacuum and ultrasound, before or during reagent incubation. As a step in the detection protocol, the endogenous biotin binding site or endogenous enzyme activity (e.g., phosphatase, catalase, or peroxidase) can be removed, for example, by treatment with peroxide. Endogenous phosphatase activity can be removed by treatment with levamisole. Endogenous phosphatases and esterases can be destroyed by heating. Blocking of non-specific binding sites with inert proteins like Horse Serum Albumin (HSA), casein, Bovine Serum Albumin (BSA), and ovalbumin, fetal bovine serum or other serum, or detergents like Tween20, Triton X-100, Saponin, Brij or Pluronics can be used. Non-specific binding sites in tissues or cells can also be blocked using specific reagents with unlabeled and non-specific versions of the target.

In some embodiments, the melting temperature T at the probe_mThe hybridization is carried out at a temperature of about 15 to about 25 ℃. By passingHybridization is carried out using a hybridization buffer containing the probe and other components (e.g., organic solvent, ionic solution). Post-hybridization washes may be performed to remove unbound probes and only partially bound probes. For example, can be at T_mThe cleaning is performed at a temperature of about 10 to about 15 c below and/or by using a solution with decreasing salt concentration.

In some embodiments, the procedure of the method (a) can be followed to seal tissue sections on slides after critical incubation with immunospecific reagents. The mounted tissue sections on the slides are then subjected to the remainder of the testing process. In some embodiments, free-floating techniques can also be used to prepare samples and detect target molecules. In this method, tissue sections are contacted with various reagents and wash buffers in a suitable container (e.g., a microcentrifuge tube) in a suspended or free-floating state.

RNA-Seq (also known as Whole Transcriptome Shotgun Sequencing (WTSS)) refers to sequencing cDNA using high-throughput sequencing techniques to obtain information about the RNA content of a sample. Publications describing RNA-Seq include: "RNA-Seq: an innovative tool for transcriptotomics" Nature Reviews Genetics 10(1):57-63(January 2009); ryan et al BioTechniques 45(1):81-94 (2008); and "transfer sequence to detection gene fusions in cancer" by Maher et al, Nature 458(7234), 97-101(January 2009).

Differential gene expression can also be identified or verified using microarray technology. As such, the expression profile of a glioblastoma-related gene, mesothelioma-related gene, gastric cancer-related gene, renal cell carcinoma-related gene, hepatocellular carcinoma-related gene, or sarcoma-related gene can be measured in fresh or paraffin-embedded tumor tissue using microarray technology. In this method, polynucleotide sequences of interest (including cDNAs and oligonucleotides) are coated (plate) or arrayed (array) on a microchip substrate. The aligned sequences are then hybridized to specific DNA probes from the cell or tissue of interest. As in the PCR method, the source of mRNA is typically total RNA isolated from human tumors or tumor cell lines and corresponding normal tissues or cell lines. Thus, RNA can be isolated from a variety of primary tumors or tumor cell lines. If the source of the mRNA is a primary tumor, the mRNA can be extracted from, for example, frozen or archived paraffin-embedded and fixed (e.g., formalin-fixed) tissue samples that are routinely prepared and stored in routine clinical practice.

In one specific embodiment of microarray technology, inserts of PCR amplified cDNA clones are applied to a substrate in a dense array. Preferably, at least 10,000 nucleotide sequences are applied to the substrate. Microarray genes immobilized on a microchip in 10,000 components each are suitable for hybridization under stringent conditions. Fluorescently labeled cDNA probes can be generated by incorporating fluorescent nucleotides into reverse transcription of RNA extracted from a tissue of interest. Labeled cDNA probes applied to the chip hybridize specifically to each DNA spot on the array. After stringent washing to remove non-specifically bound probes, the chip is scanned by confocal laser microscopy or by other detection methods such as a CCD camera. Quantification of hybridization of each array component allows assessment of the abundance of the corresponding mRNA. Separately labeled cDNA probes generated from both RNA sources were hybridized in pairs to the array by virtue of dual colored fluorescence. The relative abundance of transcripts from both sources corresponding to each designated gene was thus determined simultaneously. Miniaturized scale hybridization provides convenient and rapid evaluation of large numbers of gene expression patterns. Such methods have shown at least about 2-fold differences in the sensitivity and reproducible detection expression levels required to detect rare transcripts expressed in a small number of copies per cell (Schena et al, Proc. Natl. Acad. Sci. USA 93(2): 106-. Microarray analysis can be performed using commercial equipment following the manufacturer's protocol, such as using Affymetrix GENCHIP^TMThe technique, or Incyte's microarray technique.

Microarray methods developed for large-scale analysis of gene expression make it possible to systematically search for molecular markers for cancer classification and outcome prediction in a variety of tumor types.

Sequential Analysis of Gene Expression (SAGE) is a method that allows simultaneous and quantitative analysis of a large number of gene transcripts without the need to provide individual hybridization probes for each transcript. First, a short sequence tag (about 10-14bp) is generated that contains sufficient information to uniquely identify a transcript, provided that the tag is derived from a unique position within each transcript. Many transcripts are then joined to form long, contiguous molecules that can be sequenced to reveal the identity (identity) of multiple tags simultaneously. The expression pattern of any transcript population can be quantitatively assessed by determining the abundance of individual tags and identifying the genes corresponding to each tag. For more details see, e.g., Velculescu et al, Science 270: 484-; velculescu et al, Cell 88:243-51 (1997).

The MassARRAY (Sequenom, San Diego, Calif.) technique is an automated, high-throughput gene expression analysis method using Mass Spectrometry (MS) for detection. According to this method, after RNA isolation, reverse transcription and PCR amplification, the cDNA is subjected to primer extension. The cDNA-derived primer extension products were purified and distributed onto a chip array preloaded with the components required for MALDI-TOF MS sample preparation. The various cDNAs present in the reaction were quantified by analyzing the peak areas in the obtained mass spectra.

In general, methods of gene expression profiling can be divided into two major groups: methods based on polynucleotide hybridization analysis, and methods based on polynucleotide sequencing. The most commonly used Methods known in the art for the quantification of mRNA expression in a sample include Northern blotting and in situ hybridization (Parker & Barnes, Methods in Molecular Biology 106:247-283 (1999)); RNase protection assay (Hod, Biotechniques 13:852-854 (1992)); and Polymerase Chain Reaction (PCR) (Weis et al, Trends in Genetics 8:263-264 (1992)). Alternatively, antibodies that recognize specific duplexes (including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes) may be used. Representative methods of sequencing-based gene expression analysis include gene expression Sequential Analysis (SAGE) and gene expression analysis by Massively Parallel Signature Sequencing (MPSS).

HGF proteins can be determined from patient samples (e.g., plasma, serum, urine, cerebrospinal fluid, sputum, feces, respiratory condensate, tumors, other tissues.) methods for determining HGF proteins are known in the art and include ELISA, mass spectrometry, surface plasmon resonance, western blot, IHC, and other well-known methods see, e.g., Mai et al, molecular cancer Ther (2013)13(2): 540-52. kits for detecting HGF are commercially available.

Immunohistochemical staining (IHC) of tissue sections has proven to be a reliable method to assess or detect the presence of proteins in a sample. Immunohistochemical techniques utilize antibodies to probe and visualize cellular antigens in situ, usually by chromogenic or fluorescent methods. As such, expression is detected using antibodies or antisera, in some embodiments, polyclonal antisera, and in some embodiments, monoclonal antibodies, specific for each marker. As discussed in greater detail below, the antibody may be detected by directly labeling the antibody itself, for example with a radioactive label, a fluorescent label, a hapten label such as biotin, or an enzyme such as horseradish peroxidase or alkaline phosphatase. Alternatively, unlabeled primary antibodies are used in combination with labeled secondary antibodies, including antisera, polyclonal antisera, or monoclonal antibodies specific for the primary antibody. Immunohistochemistry protocols and kits are well known in the art and are commercially available.

In some embodiments, the IHC assay is a direct assay, wherein the binding of an antibody to a target antigen is determined directly. This direct assay uses a labeled reagent, such as a fluorescent label or an enzyme-labeled primary antibody, which is visualized without further antibody interaction. In some embodiments, the IHC assay is an indirect assay. In a typical indirect assay, an unconjugated primary antibody binds to the antigen, and then binds to the primary antibody via a labeled secondary antibody (second antibody). If the second antibody is conjugated to an enzyme label, a chromogenic or fluorogenic substrate is added to provide visualization of the antigen. Signal amplification occurs because several secondary antibodies can react with different epitopes on the primary antibody.

The primary and/or secondary antibodies used for immunohistochemistry are typically labeled with a detectable moiety. Many markers are available and can be generally classified into the following categories:

(a) radioisotopes, e.g.³⁵S、¹⁴C、¹²⁵I、³H and¹³¹I. for example, antibodies can be labeled with radioisotopes using the techniques described in Current Protocols in immunology, volumes 1 and 2, Coligen et al, Wiley-Interscience, New York, N.Y., Pubs.1991, and radioactivity can be measured using scintillation counting.

(b) Colloidal gold particles.

(c) Fluorescent labels, including but not limited to, rare chelates (europium chelates), Texas Red, rhodamine, fluorescein, dansyl, lissamine, umbelliferone, phycoerythrin, phycocyanin, or commercial fluorophores such as SPECTRUMAnd SPECTRUMAnd/or derivatives of one or more of the foregoing. For example, the fluorescent label may be conjugated to the antibody using the techniques disclosed above in Current protocols in Immunology. Fluorescence can be quantified using a fluorometer.

(d) Various enzyme-substrate labels are available and a review of some of them is provided in U.S. Pat. No.4,275,149. Enzymes generally catalyze chemical changes in a chromogenic substrate that can be measured using a variety of techniques. For example, the enzyme may catalyze a color change in the substrate that can be measured spectrophotometrically. Alternatively, the enzyme may alter the fluorescence or chemiluminescence of the substrate. Techniques for quantifying changes in fluorescence are described above. The chemiluminescent substrate becomes electronically excited by a chemical reaction and can then emit light that can be measured (e.g., using a chemiluminometer) or used to energize a fluorescent acceptor. Examples of enzymatic labels include luciferases (e.g., firefly luciferase and bacterial luciferases; U.S. Pat. No.4,737,456), luciferin, 2, 3-dihydrophthalazinediones, malate dehydrogenase, urease, peroxidases such as horseradish peroxidase (HRPO), alkaline phosphatase, beta-galactosidase, glucoamylase, lysozyme, carbohydrate oxidases (e.g., glucose oxidase, galactose oxidase and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (such as uricase and xanthine oxidase), lactoperoxidase, microperoxidase, and the like. Techniques for conjugating enzymes to antibodies are described in O' Sullivan et al, Methods for the preparation of Enzyme-Antibody Conjugates for use in Enzyme Immunoassay, Methods in Enzyme, ed.J.Langon and H.Van Vunakis, Academic Press, New York,73:147-166 (1981).

Examples of enzyme-substrate combinations include, for example:

(i) horseradish peroxidase (HRPO), which uses hydrogen peroxide as a substrate, wherein the hydrogen peroxide oxidizes a dye precursor (e.g., o-phenylenediamine (OPD) or 3,3 ', 5, 5' -tetramethylbenzidine hydrochloride (TMB)). 3, 3-Diaminobenzazidine (DAB) can also be used to visualize HRP-labeled antibodies;

(ii) alkaline Phosphatase (AP) with p-nitrophenyl phosphate as chromogenic substrate; and

(iii) beta-D-galactosidase (. beta. -D-Gal) with either a chromogenic substrate (e.g., p-nitrophenyl-. beta. -D-galactoside) or a fluorogenic substrate (e.g., 4-methylumbelliferyl-. beta. -D-galactoside).

Many other enzyme-substrate combinations are available to those skilled in the art. For a general review of these, see U.S. Pat. Nos. 4,275,149 and 4,318,980.

Sometimes, the label is indirectly conjugated to the antibody. The skilled artisan is aware of a variety of techniques for achieving this. For example, an antibody may be conjugated to biotin and any of the four broad classes of labels described above may be conjugated to avidin, or vice versa. Biotin binds selectively to avidin, whereby the label can be conjugated to the antibody in this indirect manner. Alternatively, to achieve indirect conjugation of the label to the antibody, the antibody is conjugated to a small hapten and one of the different types of labels described above is conjugated to an anti-hapten antibody. Thereby, indirect conjugation of the label to the antibody can be achieved.

In addition to the sample preparation protocol discussed above, further processing of the tissue slices before, during, or after IHC may be required. For example, epitope retrieval methods can be performed, such as heating tissue samples in citrate buffer (see, e.g., Leong et al, appl. Immunohistochem.4(3):201 (1996)).

Following the optional blocking step, the tissue section is exposed to the first antibody under suitable conditions for a time sufficient for the first antibody to bind to the target protein antigen in the tissue sample. Suitable conditions for achieving this can be determined by routine experimentation.

The extent of binding of the antibody to the sample is determined by using any of the detectable labels discussed above. Preferably, the label is an enzymatic label (e.g., HRPO) that catalyzes a chemical change in a chromogenic substrate such as 3, 3' -diaminobenzidine chromogen. Preferably, the enzyme label is conjugated to an antibody that specifically binds to the first antibody (e.g., the first antibody is a rabbit polyclonal antibody and the second antibody is a goat anti-rabbit antibody).

The specimen thus prepared can be placed and covered with a cover slip. Slide evaluation is then performed, for example, using a microscope.

IHC may be combined with morphological staining, either before or after. After deparaffinization, the sections mounted on the slides can be stained with a morphological dye for evaluation. The morphological dye used provides an accurate morphological assessment of the tissue section. The sections may be stained with one or more dyes, each dye uniquely staining a different cellular component. In one embodiment, hematoxylin is used to stain the nuclei on the slide. Hematoxylin is widely available. An example of a suitable hematoxylin is hematoxylin ii (ventana). When a bluish nucleus is desired, a bluing reagent may be used after hematoxylin staining. One skilled in the art will appreciate that staining of a given tissue can be optimized by extending or shortening the length of time the slide remains in the dye.

Automated systems for slide preparation and IHC processing are commercially available.The BenchMark XT system is an example of such an automated system.Autostainer Plus and Leica Bond III are also examples of such automated systems.

After staining, the tissue sections can be analyzed by standard techniques of microscopy. Typically, a pathologist or similar person assesses the tissue for the presence of abnormal or normal cells or specific cell types and provides a location for the cell type of interest. Thus, for example, a pathologist or the like would examine the slide and identify normal cells (such as normal lung cells) and abnormal cells (such as abnormal or neoplastic lung cells). Any means of defining the location of the cell of interest (e.g., coordinates on the X-Y axis) may be used.

In some embodiments, IHC is performed using an anti-HGF antibody.

In some embodiments, c-met biomarkers are detected, e.g., using IHC. anti-C-Met Antibodies suitable for use in IHC are well known in the art and include SP-44(Ventana), DL-21(Upstate), D1C2(Cell Signaling technologies), ab27492(Abcam), PA1-37483(Pierce Antibodies), Met4 (a monoclonal antibody produced by the hybridoma Cell line deposited with the American type culture Collection under accession number PTA-7680; see, e.g., U.S. Pat. No.6,548,640). In some embodiments, the anti-c-met antibody is SP 44. In some embodiments, the anti-c-met antibody is DL-21. In some embodiments, the anti-C-met antibody is D1C 2. In some embodiments, the anti-c-Met antibody is Met 4. The ordinarily skilled artisan understands that, for example, using the IHC protocols and examples disclosed herein, additional suitable anti-c-met antibodies can be identified and characterized by comparison to c-met antibodies.

Control cell lines (e.g., centrifuged into cell pellets and formalin-fixed and paraffin-embedded, e.g., prepared into tissue microarrays, and stained with SP44, for example) with various staining intensities (e.g., when stained with the c-met antibody SP 44) can be used as controls for IHC analysis. For example, H441 (strong c-met staining intensity); EBC1 (Strong c-met staining intensity); a549 (moderate c-met staining intensity); SKMES1 (moderate c-met staining intensity); h1703 (weak c-met staining intensity), HEK 293 (weak c-met staining intensity); h460 (weak c-met staining intensity); and TOV-112D (negative c-met staining intensity), LXFL529 (negative c-met staining intensity), H522 (negative c-met staining intensity), H23 (negative c-met staining intensity) or H1155 (negative c-met staining intensity). The ordinarily skilled artisan understands that other control cell aggregates having negative, weak, medium, and high c-met staining intensity can be readily identified using the teachings of the present application and methods well known in the art and disclosed herein. Thus, in some embodiments, the strong c-met staining intensity is that of a control cell having a c-met staining intensity of H441 and/or EBC 1. In some embodiments, the moderate c-met staining intensity is the c-met staining intensity of control cells having the c-met staining intensity of a549 and/or SKMES 1. In some embodiments, the weak c-met staining intensity is that of a control cell having a c-met staining intensity of HEK-293 and/or H460. In some embodiments, the negative c-met staining intensity is the c-met staining intensity of a control cell with a c-met staining intensity of LXFL529, H522, H23, and/or H1155. The use of control cell pellets with different staining intensities for IHC analysis (e.g., the scoring and analysis of c-met IHC for cancer samples) is well known in the art. The c-met immunohistochemistry and scoring protocol is exemplified herein. In some embodiments, c-met IHC is analyzed using the following protocol:

TABLE A

In some embodiments, c-Met IHC is analyzed using the following protocol:

TABLE B

In some embodiments, c-met IHC is analyzed according to table X or table B, and the cancer is glioblastoma, mesothelioma, hepatocellular carcinoma, renal cell carcinoma, gastric cancer, sarcoma (e.g., osteosarcoma), non-small cell lung cancer, breast cancer, gallbladder cancer, or pancreatic cancer. In some embodiments, c-met is analyzed according to table B, and the cancer is glioblastoma. In some embodiments, c-met is analyzed according to table B and the cancer is mesothelioma. In some embodiments, c-met is analyzed according to table B and the cancer is gastric cancer. In some embodiments, c-met is analyzed according to table B, and the cancer is renal cell carcinoma. In some embodiments, c-met is analyzed according to table B and the cancer is hepatocellular carcinoma. In some embodiments, c-met is analyzed according to table B and the cancer is sarcoma.

In some embodiments, the patient's tumor is c-met positive when 1% or more of the tumor cells in the sample express c-met protein (e.g., express c-met protein at any intensity). In some embodiments, the patient's tumor is c-met positive when more than 1% of the tumor cells in the sample express c-met protein (e.g., express c-met protein at any intensity). In some embodiments, the patient's tumor is c-met positive when 5% or more of the tumor cells in the sample express c-met protein (e.g., express c-met protein at any intensity). In some embodiments, the patient's tumor is c-met positive when 10% or more of the tumor cells in the sample express c-met protein (e.g., express c-met protein at any intensity). In some embodiments, the patient's tumor is c-met positive when 15% or more of the tumor cells in the sample express c-met protein. In some embodiments, the patient's tumor is c-met positive when 20% or more of the tumor cells in the sample express c-met protein (e.g., express c-met protein at any intensity). In some embodiments, the patient's tumor is c-met positive when 25% or more of the tumor cells in the sample express c-met protein (e.g., express c-met protein at any intensity). In some embodiments, the patient's tumor is c-met positive when 30% or more of the tumor cells in the sample express c-met protein (e.g., express c-met protein at any intensity). In some embodiments, the patient's tumor is c-met positive when 35% or more of the tumor cells in the sample express c-met protein (e.g., express c-met protein at any intensity). In some embodiments, the patient's tumor is c-met positive when 40% or more of the tumor cells in the sample express c-met protein (e.g., express c-met protein at any intensity). In some embodiments, the patient's tumor is c-met positive when 45% or more of the tumor cells in the sample express c-met protein (e.g., express c-met protein at any intensity). In some embodiments, the patient's tumor is c-met positive when 50% or more of the tumor cells in the sample express c-met protein (e.g., express c-met protein at any intensity). In some embodiments, the patient's tumor is c-met positive when 55% or more of the tumor cells in the sample express c-met protein (e.g., express c-met protein at any intensity). In some embodiments, the patient's tumor is c-met positive when 60% or more of the tumor cells in the sample express c-met protein (e.g., express c-met protein at any intensity). In some embodiments, the patient's tumor is c-met positive when 65% or more of the tumor cells in the sample express c-met protein (e.g., express c-met protein at any intensity). In some embodiments, the patient's tumor is c-met positive when 70% or more of the tumor cells in the sample express c-met protein (e.g., express c-met protein at any intensity). In some embodiments, the patient's tumor is c-met positive when 75% or more of the tumor cells in the sample express c-met protein (e.g., express c-met protein at any intensity). In some embodiments, the patient's tumor is c-met positive when 80% or more of the tumor cells in the sample express c-met protein (e.g., express c-met protein at any intensity). In some embodiments, the patient's tumor is c-met positive when 85% or more of the tumor cells in the sample express c-met protein (e.g., express c-met protein at any intensity). In some embodiments, the patient's tumor is c-met positive when 90% or more of the tumor cells in the sample express c-met protein (e.g., express c-met protein at any intensity). In some embodiments, the patient's tumor is c-met positive when 95% or more of the tumor cells in the sample express c-met protein (e.g., express c-met protein at any intensity). In some embodiments, the c-met expression is membranous. In some embodiments, c-met expression is cytoplasmic. In some embodiments, c-met-expression is membrane and cytosolic. In some embodiments, the cancer is glioblastoma, mesothelioma, hepatocellular carcinoma, renal cell carcinoma, gastric cancer, sarcoma (e.g., osteosarcoma), non-small cell lung cancer, breast cancer, gallbladder cancer, or pancreatic cancer. In some embodiments, the cancer is a glioblastoma (e.g., relapsed glioblastoma). In some embodiments, the cancer is mesothelioma. In some embodiments, the cancer is gastric cancer. In some embodiments, the cancer is renal cell carcinoma. In some embodiments, the cancer is hepatocellular carcinoma. In some embodiments, the cancer is a sarcoma.

In some embodiments, the patient's tumor is c-met positive when 1% or more of the tumor cells in the sample express c-met protein with moderate and/or strong staining intensity. In some embodiments, the patient's tumor is c-met positive when more than 1% of the tumor cells in the sample express c-met protein with moderate and/or strong staining intensity. In some embodiments, the patient's tumor is c-met positive when 5% or more of the tumor cells in the sample express c-met protein with moderate and/or strong staining intensity. In some embodiments, the patient's tumor is c-met positive when 10% or more of the tumor cells in the sample express c-met protein with moderate and/or strong staining intensity. In some embodiments, the patient's tumor is c-met positive when 15% or more of the tumor cells in the sample express c-met protein with moderate and/or strong staining intensity. In some embodiments, the patient's tumor is c-met positive when 20% or more of the tumor cells in the sample express c-met protein with moderate and/or strong staining intensity. In some embodiments, the patient's tumor is c-met positive when 25% or more of the tumor cells in the sample express c-met protein with moderate and/or strong staining intensity. In some embodiments, the patient's tumor is c-met positive when 30% or more of the tumor cells in the sample express c-met protein with moderate and/or strong staining intensity. In some embodiments, the patient's tumor is c-met positive when 35% or more of the tumor cells in the sample express c-met protein with moderate and/or strong staining intensity. In some embodiments, the patient's tumor is c-met positive when 40% or more of the tumor cells in the sample express c-met protein with moderate and/or strong staining intensity. In some embodiments, the patient's tumor is c-met positive when 45% or more of the tumor cells in the sample express c-met protein with moderate and/or strong staining intensity. In some embodiments, the patient's tumor is c-met positive when 50% or more of the tumor cells in the sample express c-met protein with moderate and/or strong staining intensity. In some embodiments, the patient's tumor is c-met positive when 55% or more of the tumor cells in the sample express c-met protein with moderate and/or strong staining intensity. In some embodiments, the patient's tumor is c-met positive when 60% or more of the tumor cells in the sample express c-met protein with moderate and/or strong staining intensity. In some embodiments, the patient's tumor is c-met positive when 65% or more of the tumor cells in the sample express c-met protein with moderate and/or strong staining intensity. In some embodiments, the patient's tumor is c-met positive when 70% or more of the tumor cells in the sample express c-met protein with moderate and/or strong staining intensity. In some embodiments, the patient's tumor is c-met positive when 75% or more of the tumor cells in the sample express c-met protein with moderate and/or strong staining intensity. In some embodiments, the patient's tumor is c-met positive when 80% or more of the tumor cells in the sample express c-met protein with moderate and/or strong staining intensity. In some embodiments, the patient's tumor is c-met positive when 85% or more of the tumor cells in the sample express c-met protein with moderate and/or strong staining intensity. In some embodiments, the patient's tumor is c-met positive when 90% or more of the tumor cells in the sample express c-met protein with moderate and/or strong staining intensity. In some embodiments, the patient's tumor is c-met positive when 95% or more of the tumor cells in the sample express c-met protein with moderate and/or strong staining intensity. In some embodiments, the c-met expression is membranous. In some embodiments, c-met expression is cytoplasmic. In some embodiments, c-met expression is membrane and cytosolic. In some embodiments, the cancer is glioblastoma, mesothelioma, hepatocellular carcinoma, renal cell carcinoma, gastric cancer, sarcoma (e.g., osteosarcoma), non-small cell lung cancer, breast cancer, gallbladder cancer, or pancreatic cancer. In some embodiments, the cancer is a glioblastoma (e.g., relapsed glioblastoma). In some embodiments, the cancer is mesothelioma. In some embodiments, the cancer is gastric cancer. In some embodiments, the cancer is renal cell carcinoma. In some embodiments, the cancer is hepatocellular carcinoma. In some embodiments, the cancer is a sarcoma.

In some embodiments, the tumor of the patient is c-met positive when the maximum staining intensity of the tumor is 1. In some embodiments, the tumor of the patient is c-met positive when the maximum staining intensity of the tumor is 2. In some embodiments, the tumor of the patient is c-met positive when the maximum staining intensity of the tumor is 3. In some embodiments, the c-met expression is membranous. In some embodiments, c-met expression is cytoplasmic. In some embodiments, c-met expression is membrane and cytosolic. In some embodiments, the cancer is glioblastoma, mesothelioma, hepatocellular carcinoma, renal cell carcinoma, gastric cancer, sarcoma (e.g., osteosarcoma), non-small cell lung cancer, breast cancer, gallbladder cancer, or pancreatic cancer. In some embodiments, the cancer is a glioblastoma (e.g., relapsed glioblastoma). In some embodiments, the cancer is mesothelioma. In some embodiments, the cancer is gastric cancer. In some embodiments, the cancer is renal cell carcinoma. In some embodiments, the cancer is hepatocellular carcinoma. In some embodiments, the cancer is a sarcoma. In some embodiments, c-met polypeptide or HGF is measured using IHC. In some embodiments, the c-met polypeptide is measured using IHC and the patient sample is formalin fixed and paraffin embedded. In some embodiments, the c-met polypeptide is measured by contacting the sample with an agent that binds (in some embodiments, specifically binds) to the c-met polypeptide, thereby forming a complex between the agent and the c-met biomarker, wherein the tumor is c-met positive when 50% or more of the tumor cells in the sample have medium or high c-met staining intensity. In some embodiments, a tumor is c-met positive when 50% or more of the tumor cells in the sample have high c-met staining intensity. In some embodiments, a tumor is c-met positive when 50% or more of the tumor cells in the sample have moderate c-met staining intensity. In some embodiments, a tumor is c-met positive when 50% or more of the tumor cells in the sample have low, medium, or high c-met staining intensity. In some embodiments, the agent that binds c-met is an anti-c-met antibody SP 44. In some embodiments, the agent that binds C-met is anti-C-met antibody D1C 1. In some embodiments, the agent that binds c-Met is an anti-c-Met antibody Met 4. In some embodiments, the agent that binds c-met is anti-c-met antibody DL 21. In some embodiments, the c-met intensity is determined by comparing c-met staining in the sample to a reference level. In some embodiments, the reference level is c-met staining of a control cell pellet (e.g., a control cell line a549, SKMES1, EBC-1, H441, or a cell or cell line having an intensity comparable to any of a549, SKMES1, EBC-1, H441). In some embodiments, the moderate c-met staining intensity represents the c-met staining intensity of control cell line a 549. In some embodiments, the moderate c-met staining intensity is indicative of the c-met staining intensity of control cell line SKMES 1. In some embodiments, the strong c-met staining intensity is indicative of the c-met staining intensity of the control cell line EBC-1. In some embodiments, the strong c-met staining intensity is indicative of the c-met staining intensity of control cell line H441. In some embodiments, the patient sample is obtained prior to treatment with the c-met antagonist and/or VEGF antagonist. In some embodiments, the sample is obtained after the cancer has metastasized. In some embodiments, the sample is obtained prior to treatment with the cancer drug. In some embodiments, the sample is a biopsy, surgical specimen, or fine needle aspirate. In some embodiments, the sample is formalin fixed and paraffin embedded. In some embodiments, wherein the control cell pellet is formalin fixed and paraffin embedded. In some embodiments, the control cell pellet is prepared as a tissue microarray. In some embodiments, the c-met expression is membranous. In some embodiments, c-met expression is cytoplasmic. In some embodiments, c-met expression is membrane and cytosolic. In some embodiments, the cancer is glioblastoma, mesothelioma, hepatocellular carcinoma, renal cell carcinoma, gastric cancer, sarcoma (e.g., osteosarcoma), non-small cell lung cancer, breast cancer, gallbladder cancer, or pancreatic cancer. In some embodiments, the cancer is a glioblastoma (e.g., relapsed glioblastoma). In some embodiments, the cancer is mesothelioma. In some embodiments, the cancer is gastric cancer. In some embodiments, the cancer is renal cell carcinoma. In some embodiments, the cancer is hepatocellular carcinoma. In some embodiments, the cancer is a sarcoma. In some embodiments, the c-met IHC is scored using an H-score (e.g., is c-met positive). In some embodiments, HGF IHC is scored using an H-score (e.g., is HGF positive). Methods for calculating H-scores are disclosed in the art. Briefly, the proportion of tumor cells that show staining at weak, medium, and strong intensities (e.g., using the cell line controls discussed herein) can be counted or estimated as a percentage of the total number of tumor cells in a given glioblastoma sample. Calculating a composite H-score based on the formula: (% tumor cells x1 stained with weak intensity) + (% tumor cells x2 stained with medium intensity) + (% tumor cells x3 stained with strong intensity). Following this formula, a given tumor can be associated with a value between "0" (no tumor cells show any staining) and "300" (100% of tumor cells show strong staining). In some embodiments of any of the methods herein, high c-Met or HGF expression corresponds to an H score of about 160 or more (about 161, 162, 163, 164, 165, 166, 167, 168, 169, or more), 160 or more, about 160 to about 230, about 160 to 230, about 160 (about any of 161, 162, 163, 164, 165, 166, 167, 168, 169, or more to about any of 220, 221, 223, 224, 225, 226, 227, 228, 229, 230 or more), 230 or more, about 220, 221, 223, 224, 225, 226, 227, 228, 229, 230 or more), about 170 or more, or 170 or more (e.g., about any of 171, 172, 173, 175, 175, 176, 177, 178, 179, 180 or more). In one embodiment, the H score is about 180 or higher. In some embodiments, the H score is greater than about 10. In some embodiments, the H score is greater than about 25. In some embodiments, the H score is greater than about 50. In some embodiments, the H score is greater than about 75. In some embodiments, the H score is greater than about 100. In some embodiments, the H score is greater than about 125. In some embodiments, the H score is greater than about 150. In some embodiments, the H score is greater than about 175. In some embodiments, the H score is greater than about 200. In some embodiments, the c-met expression is membranous. In some embodiments, c-met expression is cytoplasmic. In some embodiments, c-met expression is membrane and cytosolic. In some embodiments, the cancer is glioblastoma, mesothelioma, hepatocellular carcinoma, renal cell carcinoma, gastric cancer, sarcoma (e.g., osteosarcoma), non-small cell lung cancer, breast cancer, gallbladder cancer, or pancreatic cancer. In some embodiments, the cancer is a glioblastoma (e.g., relapsed glioblastoma). In some embodiments, the cancer is mesothelioma. In some embodiments, the cancer is gastric cancer. In some embodiments, the cancer is renal cell carcinoma. In some embodiments, the cancer is hepatocellular carcinoma. In some embodiments, the cancer is a sarcoma.

In some embodiments, the analysis (e.g., IHC analysis) further comprises morphological staining, either before or after. In one embodiment, the slide is stained for nuclei using hematoxylin. Hematoxylin is widely available. An example of a suitable hematoxylin is hematoxylin ii (ventana). When a bluish nucleus is desired, a bluing reagent may be used after hematoxylin staining.

Therapeutic treatment of

Use of c-met antagonists for the effective treatment of cancer patients is provided. Use of c-met antagonists and VEGF antagonists for the effective treatment of cancer patients is provided. In particular, HGF biomarkers are used to identify a population of patients in which onartuzumab, onartuzumab plus a second cancer drug, onartuzumab plus a chemotherapeutic agent, or onartuzumab plus a VEGF antagonist treatment provides clinically meaningful benefits.

The cancer drug may be used in combination with other cancer drugs. For example, the c-met antibody may be co-administered with another c-met antagonist. Such combination therapies described above encompass both combined administration (where two or more therapeutic agents are included in the same formulation or in separate formulations) and separate administration, in which case the administration of the first drug may be prior to, concurrent with, and/or subsequent to the administration of the second drug. Examples of cancer drugs include, but are not limited to, surgery, radiation therapy (radiotherapy), biological therapy, immunotherapy, chemotherapy (e.g., temozolomide), or a combination of these therapies. In addition, cytotoxic agents, anti-angiogenic agents and antiproliferative agents may be used in combination with anti-VEGF antagonists and/or c-met antagonists.

An exemplary and non-limiting list of contemplated chemotherapeutic agents is provided herein under the "definitions" or described herein. In one embodiment, the chemotherapeutic agent is temozolomide. In another embodiment, the chemotherapeutic agent is administered concomitantly with the radiation therapy.

The medicaments herein may be administered by any suitable means, including parenteral, intrapulmonary, and intranasal, and, if desired for topical treatment, intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing may be by any suitable route, for example by injection, such as intravenous or subcutaneous injection, depending in part on whether administration is transient or chronic. Various dosing schedules are contemplated herein, including but not limited to a single administration or multiple administrations over multiple time points, bolus administration, and pulse infusion.

For the prevention or treatment of disease, the appropriate dosage of the antibody of the invention (when used alone or in combination with one or more other therapeutic agents) will depend on the type of disease to be treated, the type of antibody, the severity and course of the disease, whether the antibody is administered for prophylactic or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody, and the discretion of the attending physician. The antibody is suitable for administration to a patient in one or a series of treatments. For repeated administrations over several days or longer, depending on the condition, treatment will generally continue until the desired suppression of disease symptoms occurs. However, other dosage regimens may be used. Progress of treatment is readily monitored by conventional techniques and assays.

Depending on the type and severity of the disease, about 1. mu.g/kg to 100mg/kg (e.g., 0.1-20mg/kg) of anti-c-met antibody is administered to the subject as an initial candidate dose, whether, for example, by one or more divided administrations or by continuous infusion. In one embodiment, desirable doses include, for example, 6mg/kg, 8mg/kg, 10mg/kg, and 15 mg/kg. For repeated administrations or cycles lasting days or longer, depending on the condition, the treatment is continued until the cancer is treated, as measured by the methods described above or known in the art. However, other dosage regimens may also be useful. In one example, the anti-c-met antibody is administered once weekly, biweekly, or every three weeks at a dose ranging from about 6mg/kg to about 15mg/kg, including but not limited to 6mg/kg, 8mg/kg, 10mg/kg, or 15 mg/kg. The progress of the therapy of the invention is readily monitored by conventional techniques and assays. In other embodiments, such dosing regimens are used in combination with a chemotherapeutic regimen in a glioblastoma. Further information on suitable dosages is provided in the examples below. In other embodiments, such dosing regimens are used in combination with a chemotherapeutic regimen in mesothelioma. In other embodiments, such dosing regimens are used in combination with a chemotherapeutic regimen in gastric cancer. In other embodiments, such dosing regimens are used in combination with a chemotherapeutic regimen in renal cell carcinoma. In other embodiments, such dosing regimens are used in combination with a chemotherapeutic regimen in hepatocellular carcinoma. In other embodiments, such dosing regimens are used in combination with a chemotherapeutic regimen in a sarcoma. In some embodiments, an effective amount of an anti-c-met antibody is 15mg/kg every three weeks, e.g., administered intravenously. In some embodiments, an effective amount of an anti-c-met antibody is 10mg/kg biweekly, e.g., administered intravenously.

Depending on the type and severity of the disease, about 1 μ g/kg to 100mg/kg (e.g., 0.1-20mg/kg) of the anti-VEGF antibody is administered to the subject as an initial candidate dose, whether, for example, by one or more separate administrations or by continuous infusion. In one embodiment, desirable doses include, for example, 6mg/kg, 8mg/kg, 10mg/kg, and 15 mg/kg. For repeated administrations or cycles lasting days or longer, depending on the condition, the treatment is continued until the cancer is treated, as measured by the methods described above or known in the art. However, other dosage regimens may also be useful. In one example, the anti-VEGF antibody is administered once weekly, biweekly, or every three weeks at a dose ranging from about 6mg/kg to about 15mg/kg, including but not limited to 6mg/kg, 8mg/kg, 10mg/kg, or 15 mg/kg. The progress of the therapy of the invention is readily monitored by conventional techniques and assays. In other embodiments, such dosing regimens are used in combination with a chemotherapeutic regimen in a glioblastoma. Further information on suitable dosages is provided in the examples below. In other embodiments, such dosing regimens are used in combination with a chemotherapeutic regimen in mesothelioma. In other embodiments, such dosing regimens are used in combination with a chemotherapeutic regimen in gastric cancer. In other embodiments, such dosing regimens are used in combination with a chemotherapeutic regimen in renal cell carcinoma. In other embodiments, such dosing regimens are used in combination with a chemotherapeutic regimen in hepatocellular carcinoma. In other embodiments, such dosing regimens are used in combination with a chemotherapeutic regimen in a sarcoma. In some embodiments, the effective amount of the anti-VEGF antibody is 10mg/kg intravenously every two weeks, e.g., initially administered intravenously over 90 minutes, with subsequent infusions over 60 minutes, then over 30 minutes. In some embodiments, the effective amount of the anti-VEGF antibody is 15mg/kg intravenously every three weeks, e.g., initially administered intravenously over 90 minutes, with subsequent infusions over 60 minutes, then over 30 minutes. In the methods described above, the anti-VEGF antibody is administered to the patient second in the first cycle, and then the subsequent administration of the anti-VEGF antibody is either before or after the chemotherapy. In another embodiment, the anti-VEGF antibody is administered concurrently with said chemotherapy and radiotherapy. In some embodiments, administration of the steroid to the patient is discontinued.

In some embodiments, the effective amount of obinutuzumab is 15mg/kg intravenously every three weeks, and the effective amount of bevacizumab is 15mg/kg intravenously every three weeks.

In some other aspects of any of the methods and uses, the treating further comprises administering an additional cancer drug. Exemplary cancer drugs include antagonists of other factors involved in tumor growth, such as EGFR, ErbB3, ErbB4, or TNF. Sometimes, it may be beneficial to also administer one or more cytokines to the subject. In one embodiment, the anti-VEGF antibody is co-administered with a growth inhibitory agent. For example, the growth inhibitory agent may be administered first, followed by the VEGF antibody. However, simultaneous or prior administration of the VEGF antibody is also contemplated. Suitable dosages of growth inhibitory agents are those currently used and may be reduced by the combined action (synergy) of the growth inhibitory agent and the anti-VEGF antibody.

The formulations herein may also contain more than one active compound necessary for the particular indication being treated, preferably with complementary activities that are not detrimental to each otherThe influence of the magnetic field. For example, it may be desirable to further provide in one formulation an antibody that binds EGFR, VEGF (e.g., binds a different epitope or the same epitope on VEGF), VEGFR, or ErbB2 (e.g.) The antibody of (1). Alternatively, or in addition, the composition may comprise a chemotherapeutic agent or cytotoxic agent. Suitably, such molecules are present in combination in amounts effective for the intended purpose.

In certain aspects of any of the methods and uses, other therapeutic agents useful for the combination cancer therapy of the antibodies of the invention include other anti-angiogenic agents. A number of anti-angiogenic agents have been identified and are known in the art, including those listed by Carmeliet and Jain (2000). In one embodiment, the anti-VEGF antibody is used in combination with another VEGF antagonist or VEGF receptor antagonist, such as a VEGF variant, a soluble VEGF receptor fragment, an aptamer capable of blocking VEGF or VEGFR, a neutralizing anti-VEGFR antibody, a low molecular weight inhibitor of VEGFR tyrosine kinase, and any combination thereof. Alternatively, or in addition, two or more anti-VEGF antibodies may be co-administered to the subject.

As will be appreciated by those of ordinary skill in the art, suitable dosages of chemotherapeutic agents or other anti-cancer agents are generally up to date dosages that have been earlier adopted in clinical therapy (e.g., administration of chemotherapeutic agents alone or in combination with other chemotherapeutic agents). Dose variation may occur depending on the condition being treated. The physician administering the treatment will be able to determine the appropriate dosage for the individual subject.

In some embodiments, the treatment results in a clinical or therapeutic benefit being conferred upon a patient at risk of or suffering from cancer (e.g., glioblastoma, mesothelioma, gastric cancer, renal cell carcinoma, hepatocellular carcinoma, or sarcoma) following administration of the cancer drug. Such benefits include any one or more of the following: extending survival (e.g., improving overall and/or progression-free survival); results in objective responses (including complete responses or partial responses); or ameliorating the signs or symptoms of a cancer, etc., including extending the time to exacerbation of a clinically significant disease-related symptom experienced by a patient having a glioblastoma (e.g., a previously treated glioblastoma), extending the time to exacerbation of a clinically significant disease-related symptom experienced by a patient having a mesothelioma (e.g., a previously treated mesothelioma), extending the time to exacerbation of a clinically significant disease-related symptom experienced by a patient having a gastric cancer (e.g., a previously treated gastric cancer), extending the time to exacerbation of a clinically significant disease-related symptom experienced by a patient having a renal cell carcinoma (e.g., a previously treated renal cell carcinoma), extending the time to exacerbation of a clinically significant disease-related symptom experienced by a patient having a hepatocellular carcinoma (e.g., a previously treated hepatocellular carcinoma), or extending the time to exacerbation of a clinically significant disease-related symptom experienced by a patient having a sarcoma (e.g., a previously treated sarcoma) The previous time. In some embodiments, the symptom is any one or more (any combination) of epilepsy, neurocognitive function (including, but not limited to, the orientation of a person, time, and/or place), reading, writing, and understanding. In some embodiments, the symptom is any one or more (any combination) of chest wall pain, pleural effusion, shortness of breath, fatigue, anemia, wheezing, hoarseness, cough, bloody sputum, abdominal pain, ascites, abdominal mass, bowel function problems, weight loss, blood clots, disseminated intravascular coagulation, jaundice, low blood glucose levels, and pulmonary emboli. In some embodiments, the symptom is any one or more (any combination) of dyspepsia, heartburn, weakness, fatigue, abdominal distension, abdominal pain, nausea, vomiting, diarrhea, constipation, weight loss, bleeding, anemia, and dysphagia. In some embodiments, the symptom is any one or more (any combination) of hematuria (or bloody urine), flank pain, abdominal or flank mass, weight loss, loss of appetite, fever, hypertension, malaise, night sweats, anemia, polycythemia, varicocele, hypertension, and hypercalcemia. In some embodiments, the symptom is any one or more (any combination) of yellowing of skin, abdominal distension due to abdominal fluids, easy congestion due to blood clotting abnormalities, loss of appetite, unintended weight loss, abdominal pain, nausea, vomiting, and malaise. In one embodiment, the biomarker (e.g., HGF mRNA expression, e.g., as determined using ISH and/or rt-qPCR) is used to identify patients expected to have extended survival (e.g., extended overall and/or progression-free survival) when treated with a c-met antagonist. In some embodiments, the biomarker (e.g., HGF mRNA expression, e.g., determined using ISH and/or rt-qPCR) is used to identify patients expected to have extended survival (e.g., extended overall and/or progression-free survival) when treated with c-met antagonist and VEGF antagonist relative to patients treated with VEGF antagonist alone. The incidence of biomarkers herein (e.g., as determined by HGF mRNA ISH and/or rt-qPCR analysis) are effective in predicting or predicting with high sensitivity such effective responses.

In some embodiments, extending survival means extending the overall or progression-free survival of a patient treated according to the invention relative to a patient not receiving treatment and/or relative to a patient treated with one or more approved antineoplastic agents but not receiving treatment according to the invention. In a specific example, extended survival means extending Progression Free Survival (PFS) and/or Overall Survival (OS) of a cancer patient receiving therapy of the invention (e.g., treatment with a c-met antagonist (e.g., onartuzumab)) relative to an untreated patient and/or relative to a patient treated with one or more approved antineoplastic agents but not treated with a c-met antagonist. In another specific example, extended survival means extending the Progression Free Survival (PFS) and/or Overall Survival (OS) of a cancer patient (e.g., a cancer patient population) receiving a therapy of the invention (e.g., treatment with a c-met antagonist (e.g., onartuzumab)) relative to untreated patients (e.g., a cancer patient population) and/or relative to patients treated with one or more approved antineoplastic agents but not treated with a c-met antagonist (e.g., a cancer patient population). In another specific example, extended survival means that Progression Free Survival (PFS) and/or Overall Survival (OS) of a cancer patient receiving a combination therapy of the invention (e.g., treatment with a combination of a c-met antagonist (e.g., onartuzumab) and a VEGF antagonist (e.g., bevacizumab)) is extended relative to a patient treated with bevacizumab alone. In another specific example, extended survival means that Progression Free Survival (PFS) and/or Overall Survival (OS) of a cancer patient (e.g., a cancer patient population) receiving a combination therapy of the invention (e.g., treatment with a combination of obinutuzumab and bevacizumab) is extended relative to a patient (e.g., a cancer patient population) treated with bevacizumab alone.

In some embodiments, treatment results in an improvement in signs or symptoms of cancer, and the like, including an increase in the time to worsening of clinically significant disease-related symptoms experienced by patients having glioblastoma (e.g., previously treated glioblastoma). In some embodiments, the symptom is any one or more (any combination) of epilepsy, neurocognitive function (including, but not limited to, the orientation of a person, time, and/or place), reading, writing, and understanding. In some embodiments, methods are provided for preventing such signs or symptoms of cancer from becoming exaggerated/heavy.

In some embodiments, treatment results in an improvement in signs or symptoms of cancer, and the like, including an extension of the pre-exacerbation time of clinically significant disease-related symptoms experienced by patients having mesothelioma (e.g., previously treated mesothelioma). In some embodiments, the symptom is any one or more (any combination) of chest wall pain, pleural effusion, shortness of breath, fatigue, anemia, wheezing, hoarseness, cough, bloody sputum, abdominal pain, ascites, abdominal mass, bowel function problems, weight loss, blood clots, disseminated intravascular coagulation, jaundice, low blood glucose levels, and pulmonary emboli. In some embodiments, methods are provided for preventing such signs or symptoms of cancer from becoming exaggerated/heavy.

In some embodiments, treatment results in an improvement in signs or symptoms of cancer, and the like, including extending the time to worsening of clinically significant disease-related symptoms experienced by patients with gastric cancer (e.g., previously treated gastric cancer). In some embodiments, the symptom is any one or more (any combination) of dyspepsia, heartburn, weakness, fatigue, abdominal distension, abdominal pain, nausea, vomiting, diarrhea, constipation, weight loss, bleeding, anemia, and dysphagia. In some embodiments, methods are provided for preventing such signs or symptoms of cancer from becoming exaggerated/heavy.

In some embodiments, treatment results in an improvement in signs or symptoms of cancer, and the like, including extending the time to worsening of clinically significant disease-related symptoms experienced by patients with hepatocellular carcinoma (e.g., previously treated hepatocellular carcinoma). In some embodiments, the symptom is any one or more (any combination) of yellowing of skin, abdominal distension due to abdominal fluids, easy congestion due to blood clotting abnormalities, loss of appetite, unintended weight loss, abdominal pain, nausea, vomiting, and malaise. In some embodiments, methods are provided for preventing such signs or symptoms of cancer from becoming exaggerated/heavy.

In some embodiments, treatment results in an improvement in signs or symptoms of cancer, and the like, including extending the time to worsening of clinically significant disease-related symptoms experienced by patients with renal cell carcinoma (e.g., previously treated renal cell carcinoma). In some embodiments, the symptom is any one or more (any combination) of hematuria (or bloody urine), flank pain, abdominal or flank mass, weight loss, loss of appetite, fever, hypertension, malaise, night sweats, anemia, polycythemia, varicocele, hypertension, and hypercalcemia. In some embodiments, methods are provided for preventing such signs or symptoms of cancer from becoming exaggerated/heavy.

In some embodiments, the patient is a glioblastoma patient. In some embodiments, the patient has not received prior treatment with a c-met antagonist. In some embodiments, the patient has not received prior treatment with the agent in the brain. In some embodiments, the patient does not have proteinuria of greater than 1.0g protein in 24 hours as determined using a urine dipstick test for proteinuria. In some embodiments, the patient does not have uncontrolled hypertension (e.g., systolic blood pressure greater than 150mmHg and/or diastolic blood pressure greater than 100mmHg on antihypertensive medication). In some embodiments, the patient has no prior history of hypertensive crisis or hypertensive encephalopathy. In some embodiments, the patient has no prior history of myocardial infarction (e.g., within 12 months) or unstable angina (e.g., within 6 months). In some embodiments, the patient has no history of stroke or transient ischemic attack (e.g., within 6 months). In some embodiments, the patient is free of significant vascular disease (e.g., an aortic aneurysm requiring surgical repair or recent peripheral arterial thrombosis, e.g., within 6 months). In some embodiments, the patient has no history of abdominal fistulas or gastrointestinal perforations (e.g., within 6 months). In some embodiments, the patient has no evidence of hemorrhagic diathesis or coagulopathy (under therapeutic anticoagulant depletion). In some embodiments, the patient has no history of intracranial abscesses (e.g., within 6 months).

In some embodiments, the patient has received prior treatment with temozolomide. In some embodiments, the patient has received no more than one prior chemotherapy (e.g., a prior temozolomide, e.g., concurrent or adjunctive temozolomide). In some embodiments, the patient has a Karnofsky performance status of greater than or equal to 70%.

In another aspect, provided is a method for assessing adverse events associated with treatment of a previously treated glioblastoma, mesothelioma, gastric cancer, renal cell carcinoma, hepatocellular carcinoma, or sarcoma using any of the methods described herein in a patient, wherein the treatment is with a c-met antagonist (e.g., onartuzumab), comprising the step of monitoring the patient for one or more adverse events. In some embodiments, the patient is monitored for the number and/or severity of one or more adverse events. Exemplary adverse events are disclosed herein, and include, but are not limited to: peripheral edema.

In another aspect, provided is a method for assessing adverse events associated with treatment of a previously treated glioblastoma, mesothelioma, gastric cancer, renal cell carcinoma, hepatocellular carcinoma, or sarcoma using any of the methods described herein in a patient, wherein the treatment is with a c-met antagonist (e.g., onartuzumab) and a chemotherapeutic agent, the method comprising the step of monitoring the patient for one or more adverse events. In some embodiments, the patient is monitored for the number and/or severity of one or more adverse events. Exemplary adverse events are disclosed herein, and include, but are not limited to: peripheral edema.

In another aspect, provided is a method for assessing adverse events associated with treatment of a previously treated glioblastoma or renal cell carcinoma with any of the methods described herein in a patient, wherein the treatment is with a c-met antagonist (e.g., onartuzumab) and a VEGF antagonist (e.g., bevacizumab), the method comprising the step of monitoring the patient for one or more adverse events. In some embodiments, the patient is monitored for the number and/or severity of one or more adverse events. Exemplary adverse events are disclosed herein, and include, but are not limited to: peripheral edema.

It is understood that any of the formulations or methods of treatment described herein can be practiced using an immunoconjugate of the antibody as a drug, in place of or in addition to the antibody.

V. product

In another embodiment of the invention, an article of manufacture is provided for treating cancer (such as glioblastoma, mesothelioma, gastric cancer, renal cell carcinoma, hepatocellular carcinoma, or sarcoma). In some embodiments, the cancer is a previously treated cancer (such as a previously treated (e.g., second line) glioblastoma, mesothelioma, gastric cancer, renal cell carcinoma, hepatocellular carcinoma, or sarcoma). The article includes a container and a label or package insert on or accompanying the container. Suitable containers include, for example, bottles (bottles), vials (vitamins), syringes (syringees), and the like. The container may be made of a variety of materials such as glass or plastic. The container contains or contains a composition comprising a cancer drug as an active agent and may have a sterile access port (e.g., the container may be an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle).

The article of manufacture may further comprise a second container containing a pharmaceutically acceptable dilution buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution, and dextrose solution. The article of manufacture may also include other materials as desired from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

The article of manufacture of the invention further comprises information, e.g., in the form of a package insert, indicating that the composition is for use in treating cancer based on the expression of the biomarker as disclosed herein. The insert or label may take any form, such as paper or on an electronic medium, such as a magnetic recording medium (e.g., floppy disk) or a CD-ROM. The label or insert may also include other information regarding the pharmaceutical composition and dosage form in the kit or article of manufacture.

The present invention also relates to a method for making an article of manufacture comprising combining in a package a pharmaceutical composition comprising a c-met antagonist (e.g., an anti-c-met antibody, e.g., onartuzumab) and a package insert indicating that the pharmaceutical composition is for treating a patient having a cancer (e.g., glioblastoma, mesothelioma, gastric cancer, renal cell carcinoma, hepatocellular carcinoma, or sarcoma) based on expression of HGF biomarkers as disclosed herein. In some embodiments, the cancer is a previously treated cancer (e.g., second-line glioblastoma, mesothelioma, gastric cancer, renal cell carcinoma, hepatocellular carcinoma, or sarcoma).

The invention also relates to a method for making an article of manufacture comprising combining in a package a pharmaceutical composition comprising a VEGF antagonist (e.g., bevacizumab) and a package insert indicating that the pharmaceutical composition is for treating a patient having cancer (e.g., glioblastoma, mesothelioma, gastric cancer, renal cell carcinoma, hepatocellular carcinoma, or sarcoma) based on expression of HGF biomarkers as disclosed herein. In some embodiments, the cancer is a previously treated cancer (e.g., second-line glioblastoma, mesothelioma, gastric cancer, renal cell carcinoma, hepatocellular carcinoma, or sarcoma).

The article of manufacture may further comprise another container containing a pharmaceutically acceptable dilution buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution, and/or dextrose solution. The article of manufacture may also include other materials as desired from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

VI. diagnostic kit

The invention also relates to diagnostic kits useful for detecting any one or more of the biomarkers identified herein. Thus, a diagnostic kit is provided comprising one or more reagents for determining the expression of one or more HGF biomarkers in a sample from a cancer patient (e.g., a glioblastoma, mesothelioma, gastric cancer, renal cell carcinoma, hepatocellular carcinoma, or sarcoma patient). Optionally, the kit further comprises instructions for using the kit to select a cancer drug (e.g., a c-met antagonist, such as an anti-c-met antibody, e.g., onartuzumab) to treat a glioblastoma, mesothelioma, gastric cancer, renal cell carcinoma, hepatocellular carcinoma, or sarcoma patient if the patient's cancer has been determined to have a high amount of HGF biomarker (e.g., by ISH, or PCR). In some embodiments, the cancer patient is a previously treated cancer patient (e.g., a previously treated glioblastoma, mesothelioma, gastric cancer, renal cell carcinoma, hepatocellular carcinoma, or sarcoma patient). Optionally, the kit further comprises instructions for using the kit to select a cancer drug (e.g., a c-met antagonist, such as an anti-c-met antibody, e.g., onartuzumab) to treat the previously treated glioblastoma, mesothelioma, gastric cancer, renal cell carcinoma, hepatocellular carcinoma, or sarcoma patient if the patient's cancer has been determined to have a high amount of HGF biomarker (e.g., by ISH, or PCR). In another embodiment, the kit further comprises instructions for using the kit to select a treatment with a c-met antagonist antibody (e.g., onartuzumab) and a VEGF antagonist (e.g., bevacizumab) if the patient's cancer (e.g., glioblastoma, mesothelioma, gastric cancer, renal cell carcinoma, hepatocellular carcinoma, or sarcoma) has been determined to have a high amount of HGF biomarker. In some embodiments, the kit comprises primers and/or probes (e.g., 1, 2,3, 4, or more) complementary to HGF mRNA.

VII. advertising method

The invention herein also concerns a method for advertising a cancer drug comprising promoting the use of the cancer drug (e.g., an anti-c-met antibody, optionally in combination with an anti-VEGF antibody) to a target audience for treating a cancer patient based on expression of HGF biomarkers, as disclosed herein.

Advertising is a communication, typically paid for, via non-personal media, where the originator is authenticated and the information is controlled. Advertising for purposes herein includes promotions (publicity), public relations (public relations), product placement (product placement), sponsorships (sponsorship), insurance (underpurwritting), and promotions (salespromotation). The term also includes commercial information announcements appearing in any printed distribution medium designed to appeal to the general public to persuade, inform, promote, motivate, or otherwise alter behavior toward buying, supporting, or approving the advantageous modes of the invention herein.

The advertising and promotion of the diagnostic methods herein may be accomplished by any means. Examples of advertising media used to deliver such information include television, radio, movies, magazines, newspapers, the internet, and billboards, including commercial, i.e., information that appears in broadcast media. Advertisements also include those on food truck seats, on airport walkway walls, and on the sides of buses, or on telephone waiting messages or heard in the in-store PA system, or anywhere where visual or auditory communications may be placed.

More specific examples of promotional or advertising means include television, radio, movies, the internet such as the web posters and web seminars, interactive computer networks intended for synchronous users, fixed or electronic billboards and other public signs, posters, traditional or electronic documents such as magazines and newspapers, other media channels, lectures, or personal contact, for example by email, telephone, instant messaging, mail, courier, mass, or carrier mail (carrier mail), in person access, and the like.

The type of advertising used may depend on many factors, such as the nature of the targeted audience to be communicated, e.g., hospitals, insurance companies, clinics, doctors, nurses, and patients, and the relevant jurisdictional laws and regulations governing cost considerations and advertising of drugs and diagnostic agents. The advertising may be personalized or customized based on user characterizations defined by service interactions and/or other data, such as user demographics and geographic positioning.

In some embodiments, promotion refers to promotion of a therapeutic agent, such as an anti-c-met antagonist (e.g., onartuzumab) and/or a VEGF antagonist (e.g., bevacizumab), for a therapeutic indication, such as glioblastoma (e.g., recurrent glioblastoma), mesothelioma (e.g., recurrent mesothelioma), gastric cancer (e.g., recurrent gastric cancer), renal cell carcinoma (e.g., recurrent renal cell carcinoma), hepatocellular carcinoma (e.g., recurrent hepatocellular carcinoma), or sarcoma (e.g., recurrent sarcoma), wherein such promotion is approved by the Food and Drug Administration (FDA) as having been demonstrated to be associated with statistically significant therapeutic efficacy and acceptable safety in a population of subjects.

Sequence of

SEQ ID NO:16

MKAPAVLAPGILVLLFTLVQRSNGECKEALAKSEMNVNMKYQLPNFTAETPIQNVILHEHHIFLGATNYIYVLNEEDLQKVAEYKTGPVLEHPDCFPCQDCSSKANLSGGVWKDNINMALVVDTYYDDQLISCGSVNRGTCQRHVFPHNHTADIQSEVHCIFSPQIEEPSQCPDCVVSALGAKVLSSVKDRFINFFVGNTINSSYFPDHPLHSISVRRLKETKDGFMFLTDQSYIDVLPEFRDSYPIKYVHAFESNNFIYFLTVQRETLDAQTFHTRIIRFCSINSGLHSYMEMPLECILTEKRKKRSTKKEVFNILQAAYVSKPGAQLARQIGASLNDDILFGVFAQSKPDSAEPMDRSAMCAFPIKYVNDFFNKIVNKNNVRCLQHFYGPNHEHCFNRTLLRNSSGCEARRDEYRTEFTTALQRVDLFMGQFSEVLLTSISTFIKGDLTIANLGTSEGRFMQVVVSRSGPSTPHVNFLLDSHPVSPEVIVEHTLNQNGYTLVITGKKITKIPLNGLGCRHFQSCSQCLSAPPFVQCGWCHDKCVRSEECLSGTWTQQICLPAIYKVFPNSAPLEGGTRLTICGWDFGFRRNNKFDLKKTRVLLGNESCTLTLSESTMNTLKCTVGPAMNKHFNMSIIISNGHGTTQYSTFSYVDPVITSISPKYGPMAGGTLLTLTGNYLNSGNSRHISIGGKTCTLKSVSNSILECYTPAQTISTEFAVKLKIDLANRETSIFSYREDPIVYEIHPTKSFISGGSTITGVGKNLNSVSVPRMVINVHEAGRNFTVACQHRSNSEIICCTTPSLQQLNLQLPLKTKAFFMLDGILSKYFDLIYVHNPVFKPFEKPVMISMGNENVLEIKGNDIDPEAVKGEVLKVGNKSCENIHLHSEAVLCTVPNDLLKLNSELNIEWKQAISSTVLGKVIVQPDQNFTGLIAGVVSISTALLLLLGFFLWLKKRKQIKDLGSELVRYDARVHTPHLDRLVSARSVSPTTEMVSNESVDYRATFPEDQFPNSSQNGSCRQVQYPLTDMSPILTSGDSDISSPLLQNTVHIDLSALNPELVQAVQHVVIGPSSLIVHFNEVIGRGHFGCVYHGTLLDNDGKKIHCAVKSLNRITDIGEVSQFLTEGIIMKDFSHPNVLSLLGICLRSEGSPLVVLPYMKHGDLRNFIRNETHNPTVKDLIGFGLQVAKGMKYLASKKFVHRDLAARNCMLDEKFTVKVADFGLARDMYDKEYYSVHNKTGAKLPVKWMALESLQTQKFTTKSDVWSFGVLLWELMTRGAPPYPDVNTFDITVYLLQGRRLLQPEYCPDPLYEVMLKCWHPKAEMRPSFSELVSRISAIFSTFIGEHYVHVNATYVNVKCVAPYPSLLSSEDNADDEVDTRPASFWETS

Examples

Example 1: in situ hybridization analysis of HGF mRNA expression in glioblastoma samples

Sample preparation: a pre-treatment patient glioblastoma sample from a blinded, phase II, randomized, multicenter trial designed to assess the primary activity and safety of onartuzumab plus bevacizumab treatments over bevacizumab plus placebo in patients with relapsed glioblastoma (described further below) was analyzed. All patients enrolled in the study were asked to submit formalin-fixed paraffin-embedded tumor specimens representing glioblastoma.

In Situ Hybridization (ISH): the ISH assay is performed using a set of target DNA probes to hybridize to a target RNA of interest, followed by hybridization-based signal amplification. The target probes are oligonucleotides designed to hybridize in pairs, each pair creating a binding site for the preamplifier. The preamplifier hybridizes to the target probes at a temperature that facilitates hybridization to the target probe pair, but not to the target probe individual. This ensures that no signal amplification occurs if the unpaired target probe non-specifically hybridizes to non-specific RNA. The amplifier then hybridizes to the preamplifier. Finally, the label probes conjugated with the chromophoric molecules are hybridized to amplifiers.

The Assay was performed according to the manufacturer's instructions (RNAscope 2.0 Manual Assay) except that the pretreatment steps were changed relative to the manufacturer's instructions. The change of the pre-processing step is important for e.g. optimizing the signal.

The scheme is as follows:

1. the slides were baked at 60 ℃ for 60 minutes.

2. Deparaffinization/rehydration: slides were treated with 3 Xxylene (EMD Millipore Chemicals; Cat. XX0060-4) for 5 minutes followed by 2X 100% reagent alcohol (Thermo Scientific; Cat. 9111) for 2 minutes.

All reagents stored at 4 ℃ were allowed to warm to room temperature. The probe was preheated to 40 ℃ approximately 10 minutes before use.

3. Slides were air dried and a hydrophobic barrier was placed around the tissue.

4. Add pre-treatment (PT)1 solution (endogenous peroxidase, Advanced Cell Diagnostics, catalog No. 310020) to each slide by pipette to cover the specimen, then incubate the sample at room temperature for 10 minutes, then transfer the slide to H₂O2 for 2 minutes each

5. Preparation of 1.5 liters of Pre-treatment (PT)2 solution (Advanced Cell Diagnostics; Cat. No. 320043; 10 Xstock from solution, before use in dH₂O to 1x) and transferred to PT module, then the slide was placed in PT module filled with PT2 solution and boiled therein at 92 ℃ for 20 minutes, then the slide was placed in PT module (LabVision)^TMPT Module，Thermo Scientific, part No. A80400112) and left at 92 ℃ for 20 minutes, and then the slides were transferred to H₂O to cool the slide 2 times for 2 minutes each.

6. Pretreatment (PT)3(Advanced Cell Diagnostics; catalog number 310020) was added to each slide by pipette to cover the specimen, the slides were placed at 40 ℃ for 20 minutes and then transferred to phosphate buffered saline 2 times for 2 minutes each.

7. Slides were fixed in 4% paraformaldehyde in PBS pH7.4(Genentech Media Prep) for 5 minutes at room temperature, and then washed 2 times for 1-5 minutes each in PBS to rinse out the paraformaldehyde.

8. Probe hybridization was performed according to the manufacturer's instructions (see RNAscope 2.0 Manual Assay protocol). The probe (HGF probe or control probe) was hybridized for 2 hours at 40 ℃ and then washed in wash buffer (RNAscope 50 XFFPE wash buffer; Advanced Cell Diagnostics, Cat. No. 310091; in dH₂Diluted to 1x in O) and washed 2 times for 2 minutes each.

8. An amplification step 1: slides were incubated at 40 ℃ for 30 minutes and then washed 2 times in wash buffer for 2 minutes each.

9. And (2) amplification step: slides were incubated at 40 ℃ for 15 minutes and then washed 2 times in wash buffer for 2 minutes each.

10. And 3, an amplification step: slides were incubated at 40 ℃ for 30 minutes and then washed 2 times in wash buffer for 2 minutes each.

11. And 4, an amplification step: slides were incubated at 40 ℃ for 15 minutes and then washed 2 times in wash buffer for 2 minutes each.

12. And 5, an amplification step: slides were incubated at room temperature for 30 minutes and then washed 2 times in wash buffer for 2 minutes each.

13. And 6, an amplification step: slides were incubated for 15 minutes at room temperature and then washed 2 times for 2 minutes each in wash buffer.

14. And (3) detection: DAB A and DAB (both from2.0 detection kit-brown; advanced cell Diagnostics; order number 310033)1:1 mix, added to slides, then incubate slides at room temperature for 10 minutes, then at dH₂And (4) rinsing in O.

15. Counterdyeing: gill's hematoxylin (Gill's hematoxylin # 2; Polysciences, Inc.; order No. 24243-₂Wash slide in O. Slides were washed in ammonium water (ammonium hydroxide; Sigma Aldrich; 221228-25 ML-A-at dH₂Diluted to 0.01%) in O, immersed 5 times, and slides in dH₂O was immersed 5 times.

16. And (3) dehydrating: slides were immersed in 70% reagent alcohol (70% reagent alcohol, # ALREA70GAL, American Master Tech Scientific, Inc.; # ALREA70GAL) 1 time 2 minutes, then 100% reagent alcohol 2 times 2 minutes each, then xylene 1 time 5 minutes.

17. In Permount mounting Medium (Tissue)Glas^TMSealing the medium; sakura; order No. 6419) cover slides with coverslips.

Step 6-11 in humidity control Panel (HybEZ)^TMA humidity control panel; advanced Cell Diagnostics; subscription number 310012, and dH₂O-soaked moisturizing paper, HybEZ^TMMoistening the paper; ACD; subscription number 310015) and is implemented in HybEZ^TMIncubate in oven (Advanced Cell Diagnostics; order number 241000 ACD).

The probes used in HGF ISH were: HGF probe: Hs-HGF probes (Advanced Cell Diagnostics; order number 310761); positive control probe Hs-UBC probe (ubiquitin C) (Advanced Cell Diagnostics; order No. 310041); and a negative control probe, DapB probe (dihydrodipicolinate reductase) (Advanced CellDiagnostics; order No. 310043).

Glioblastoma samples were scored with ISH of HGF mRNA: samples displaying positive HGF ISH signal show punctate brown spots in the nucleus and/or cytoplasm of the cells. Positive HGF ISH signals are observed in tumor cells and benign stromal cells (e.g., reactive astrocytes, glial cells, pericytes, and endothelial cells), and never observed in morphologically normal brain tissue (e.g., in cases where a broad portion of normal brain exists on sections away from the tumor). HGF ISH signal is focal in most, if not all, samples, such that positive HGF ISH signal in tumors and/or benign stroma can be observed in some parts of the section and not in other parts of the section. In fact, sections with positive HGF ISH signal in some fields and lacking HGF ISH signal in other fields (sometimes several fields lack HGF ISH signal) are not abnormal. Thus, the entire section is scored for the presence or absence and prevalence of positive ISH signals in tumors and benign stromal cells, except that morphologically normal brain tissue present on the section distal to the tumor is not scored. In some cases, normal brain tissue may already be present in the region included in the tissue field in which the scoring analysis is performed (e.g., when normal brain tissue is contained within the tumor region).

Sections from the same tumor were also hybridized with positive (UBC) and negative (DapB) control probes as controls. Any cases without UBC positive control ISH positive signal were excluded from the analysis.

The ISH assay has very low levels of non-specific (or background) signal, which helps detect positive HGF ISH signal at low levels and prevalence. The level of background signal in this assay was assessed by including positive and negative controls for HGF expression and background staining in each experiment: KP4 cell lines are known to express and secrete HGF. Slides were prepared with sections of FFPE-fixed KP4 cell pellets and analyzed using HGF probe (positive control) and DapB probe (negative control). DapB is a bacterial gene that is not expressed in mammalian cells, and thus a positive DapB ISH signal is not expected to be observed in KP4 cells.

Scoring was performed by scanning the entire section on an optical microscope using a 10-fold objective lens, and then scanning the entire section using a 20-or 40-fold objective lens. Samples were scored on a scale from 0 to 3+ according to the prevalence of cells with positive HGF ISH signal as described below. To determine the presence or absence of signals in rare, occasional or numerous cells, the following approach was taken: the entire tumor section was scanned using a 10-fold objective lens (as described above, focusing on the tumor and adjacent benign stroma in the sample and excluding a broad portion of morphologically normal brain tissue from the tumor). If a positive HGF ISH signal is readily observed using a 10-fold objective lens, then this sample is characterized as showing HGF ISH signal in numerous cells and scored as HGF ISH 3 +. Fig. 8 shows a micrograph of a glioblastoma section showing 3+ HGF ISH signal, viewed at low magnification. Fig. 9 shows a micrograph of the same section, viewed at high magnification.

If a positive HGF ISH signal is not readily observed using a 10-fold objective, then the entire tumor section is scanned using a 20X and/or 40-fold objective. If a positive HGF ISH signal is observed in multiple cells, viewed at 20 or 40X (typically in several fields of the slide; sometimes several fields of the slide have to be scanned before fields with positive signals are located), then the sample is characterized as showing HGF ISH signal in occasional cells and scored as HGF ISH 2 +. If few cells (typically about 10 or less in the whole slice) are observed with a positive HGF ISH signal, e.g., multiple fields of the slide generally need to be searched before a positive HGFISH signal is observed, then the sample is characterized as showing HGF ISH signal in rare cells and scored as HGF ISH 1 +. Fig. 10 shows an exemplary micrograph of a glioblastoma section showing 1+ HGF ISH signal. Sections were viewed using low magnification (roughly equivalent to a 10-fold objective lens) and it was difficult to identify HGF ISH signal positive cells. Fig. 11 shows an exemplary micrograph of the same glioblastoma section, viewed at high magnification (roughly equivalent to a 40-fold objective). Weak HGF ISH signals were observed in cells scattered throughout the field of view. Arrows indicate exemplary HGF ISH signal positive cells. Fig. 12 shows an exemplary micrograph of a glioblastoma section showing 3+ HGF ISH signal, viewed at medium magnification (roughly equivalent to a 20-fold objective). HGFISH positive signals were observed in multiple cells at the invasive margin of the tumor.

If no HGF ISH signal was observed in the sections, the samples were scored as HGF ISH 0.

128 samples were evaluated. The tissues of 4 samples were not sufficient for evaluation. 8 samples were excluded due to insufficient RNA quality (negative staining of the positive control UBC gene ISH). 27 samples were negative for HGF (23%), 49 samples were 1+ (42%), 34 samples were 2+ (29%) and 6 samples were 3+ (5%) for HGF. 34% scored as HGF diagnostic positive (HGF ISH 2+ and 3 +). 22 samples showed positive HGF ISH signal in tumor cells that were apparently malignant according to cytological criteria (cell/nuclear atypia).

Example 2: in situ hybridization assay for HGF mRNA expression in gastric cancer and mesothelioma samples

ISH analysis for HGF RNA was performed on formalin-fixed paraffin-embedded gastric cancer samples as described above for glioblastoma samples, and the samples were scored essentially as described above for glioblastoma samples, except that the stromal cells found in gastric cancer samples included fibroblasts, macrophages, endothelial cells. In some cases, a tumor sample can include cancer cells, lymphocytes, leukocytes, stroma, blood vessels, connective tissue, basal lamina, and any other cell type associated with a tumor. FIG. 13: representative in situ hybridization of HGF in gastric cancer showing high focal (arrowhead) expression (3+) in stromal cells. Probe hybridization was visualized by brown chromogen against blue hematoxylin counterstain. Bar 100 um.

The formalin fixed paraffin embedded mesothelioma sample was subjected to ISH analysis for HGF RNA as described above for the glioblastoma sample, and the sample was scored essentially as described above for the glioblastoma sample. FIG. 14: representative in situ hybridization of HGF RNA in mesothelioma cancers showing focal high expression (3+) in stromal cells. Probe hybridization was visualized by red chromogen against blue hematoxylin counterstain. FIG. 15: representative in situ hybridization of HGF RNA in mesothelioma cancers showing HGF expression with intratumoral heterogeneity. Probe hybridization was visualized by red chromogen against blue hematoxylin counterstain. FIG. 16: representative in situ hybridization of HGF RNA in mesothelioma cancers displaying autocrine HGF expression is shown. Probe hybridization was visualized by red chromogen against blue hematoxylin counterstain.

Example 3: a randomized, double-blind, placebo-controlled, multicenter phase II study to evaluate the efficacy and safety of onartuzumab in combination with bevacizumab in patients with relapsed glioblastoma

This is a randomized, double-blind, placebo-controlled, multicenter phase II trial to assess the efficacy and safety of onartuzumab + bevacizumab in first relapse in patients with glioblastoma versus placebo + bevacizumab.

Background: the standard treatment for newly diagnosed glioblastoma is surgical debulking (debulking), followed by radiotherapy and Temozolomide (TMZ) and additional maintenance of TMZ. Although such treatments are associated with survival benefits, almost all patients relapse after initial therapy. Patients with relapsed glioblastoma had a median Progression Free Survival (PFS) of about 4 months and an OS of less than 10 months. Optimal management of patients with recurrent glioblastoma remains unclear, as there is no randomized trial directly comparing active intervention with supportive care. The most important prognostic factors from the benefits of re-intervention are pre-treatment performance status and patient age. Active intervention includes repeated surgery, re-irradiation, or systemic therapy with the aim of improving or preserving neurological function and prolonging Progression Free Survival (PFS) and Overall Survival (OS). TMZ chemotherapy exhibited prolonged survival as a second line therapy in initial trials. However, TMZ is now commonly used as a component of first-line therapy, and thus no established chemotherapeutic regimen is available for recurrent glioblastoma.

Research and design: patients were randomly assigned (1:1) to one of two treatment arms: placebo + bevacizumab (branch a) or obinutuzumab + bevacizumab (branch B). Patients were stratified based on Karnofsky performance status (70% -80% versus 90% -100%) and age (<50 versus > 50 years) because these features have been identified using the Cox proportional hazards model as prognostic factors in patients with relapsed glioblastoma receiving active treatment. The availability of paraffin-embedded tumor samples representing glioblastoma diagnosis is mandatory for randomized inclusion studies. Tissue from recurrent surgery is preferred, but tissue from initial surgery is sufficient for entry into the study. Study treatment was continued until disease progression, unacceptable toxicity, patient or physician decision to abort, or death. A switch from placebo bevacizumab (branch a) to obinutuzumab treatment was not tolerated. After discontinuation of treatment, patients were followed every 6 weeks for survival. Fig. 1 shows an overview of the study design.

To characterize the safety and tolerability profiles of placebo + bevacizumab and obinutuzumab + bevacizumab, patients were monitored throughout the study for adverse events (all grades), severe adverse events, any adverse event requiring discontinuation or discontinuation of the drug, changes in laboratory values, and physical findings.

And (3) measuring the effect outcome: the efficacy outcome of this study was measured as follows.

The main outcome measures of this study were:

progression Free Survival (PFS), defined as the time from the day of randomization to the day of first recording of disease progression or death (whichever occurs first). Disease progression will be determined based on investigator assessment using the RANO criteria. Because glioblastoma typically does not exhibit prolonged disease inactivity (as opposed to low-grade gliomas), the duration of no tumor progression is often clinically significant. Secondary outcome measures of the study were:

total survival (OS), defined as the time from randomization until death of any cause

Total survival-9 (OS-9), defined as the percentage of patients alive at 9 months after randomization

Progression free survival-6 (PFS-6), defined as the percentage of patients alive and progression free at 6 months after randomization

Overall Response Rate (ORR), defined as the percentage of patients in each treatment arm that the investigator judges to have an objective response (as determined using the RANO criteria)

Duration of response (DOR), defined as the time from the first recorded objective response to disease progression (as determined by investigators using the RANO criteria) or death of any cause during the study, the safety outcome measure of this study is as follows:

adverse events according to NCI CTCAE version 4.0, including incidence, nature, and severity of Severe Adverse Events (SAE)

Changes in clinical laboratory results during and after study drug administration

Incidence and serum levels of ATA against onartuzumab

PK outcome measures for this study were as follows:

minimum concentrations of onartuzumab and bevacizumab in serum before first infusion on days 1, 2, 3 and 4 of cycles 1 and at Study Drug Discontinuation Visit (SDDV) (C)_min)

Maximum concentration of onartuzumab and bevacizumab in serum 30 minutes after the last infusion on days 1, 2, 3, and 4 of cycles 1, 2, 3, and 4 (C)_max)

The exploratory outcome of this study was measured as follows:

use of corticosteroids

Changes in biomarkers, and correlation of biomarkers to PFS, ORR, and OS

Neurocognitive function as determined using MMSE

Outcome of patient reports of glioblastoma and treatment-related symptom severity and intervention, as determined using MDASI-BT

Materials and methods

The patients: patients were potentially eligible for this study if they had glioblastomas at the first recurrence following concurrent or adjuvant radiotherapy. Patients in the study entered the study meeting the following criteria:

disease characteristics include the following:

histologically confirmed glioblastoma at the first recurrence after concurrent or adjuvant radiotherapy

Imaging confirmation of first tumor progression or regrowth as defined by the RANO standard

Temozolomide (TMZ) prior treatment.

No more than one of the previous chemotherapies. Concurrent and adjuvant TMZ-based chemotherapy (including TMZ in combination with investigational agents) is considered a line of chemotherapy.

Bevacizumab-free or other agent targeting VEGF or VEGF receptor prior treatment

Experimental treatment prior exposure without targeting HGF or Met pathway

Allowance for prior therapy with gamma knife or other focal high-dose radiotherapy, but the patient must have subsequent histological evidence of recurrence, unless the recurrence is a new lesion outside the field of irradiation

Prolifeprospan 20 pretreatment without carmustine-accompanied wafers

Non-anticipatory intracerebral agent

Recovery of toxic effects from prior therapy

No evidence of recent bleeding on baseline brain MRI

No need for urgent palliative intervention against major diseases (e.g. impending hernia)

Formalin-fixed paraffin-embedded tumor tissue representing glioblastoma

Patient characteristics include the following:

willingness and ability to provide informed consent and compliance with the study protocol at the discretion of the investigator

Age ≥ 18 years

Karnofsky Performance State > 70%

Stable or decreasing doses of corticosteroid over 5 days before randomization

Patients who met any of certain criteria were excluded from study entry, including the following:

patient could not undergo a brain MRI scan of IV gadolinium

Absolute Neutrophil Count (ANC) within 7 days before enrollment<1.5x10⁹L; platelet count<100x10⁹L; or hemoglobin (Hb)<9.0 g/dL. Note that: it is acceptable to use blood transfusion or other intervention to achieve Hb ≧ 9 g/dL.

Total bilirubin ≥ 1.5x ULN (except for patients diagnosed with Gilbert's disease)

AST (SGOT), ALT (SGPT), or alkaline phosphatase (ALP) ≥ 2.5 x ULN

Serum creatinine > 1.5 × ULN or calculated creatinine clearance (CrCl) <60mL/min (Cockcroft and Gault)

Urine dipstick test for proteinuria no less than 2 +; it was found that patients with ≥ 2+ proteinuria should undergo 24-hour urine collection and must exhibit ≤ 1.0g protein in 24 hours)

International Normalized Ratio (INR), Prothrombin Time (PT), or activated partial thromboplastin time

(APTT) is as follows:

in the absence of a therapeutic agent intended to anticoagulate the patient: INR > 1.5 or PT > 1.5XULN or aPTT > 1.5XULN

Or

In the presence of a therapeutic agent intended to anticoagulate the patient: INR or PT and aPTT were not within therapeutic limits (according to medical standards in the regimen) or patients had not taken a stable dose of anticoagulant for at least 2 weeks prior to randomization. (Note: following the ASCO guidelines, low molecular weight heparin [ LMWH ] should be the preferred approach.)

Improperly controlled hypertension (defined as systolic blood pressure > 150mmHg and/or diastolic blood pressure > 100mmHg on antihypertensive medication)

Uncontrolled diabetes, manifested by fasting serum glucose levels > 200mg/dL

Prior history of hypertensive crisis or hypertensive encephalopathy

New York Heart Association (NYHA) class II or higher congestive heart failure

History of myocardial infarction (within 12 months) or unstable angina (within 6 months) prior to randomization

History of stroke or transient ischemic attack within 6 months prior to randomization

Major angiopathy (e.g. aortic aneurysm requiring surgical repair or recent peripheral arterial thrombosis) within 6 months prior to randomization

Evidence of hemorrhagic diathesis or coagulopathy (under therapeutic anticoagulation deficiency)

History of abdominal fistulas or gastrointestinal perforations within 6 months prior to randomization

History of intracranial abscesses 6 months before randomization

Major surgical procedure, open biopsy, or significant traumatic injury in 28 before randomization

Anticipated need for major surgical procedures during the trial procedure

Severe non-healing wounds, active ulcers, or untreated fractures

History of another malignancy in the previous 3 years, no disease interval <3 years. Patients with a prior history of carcinoma in situ or basal or squamous cell skin cancer are eligible.

Evidence of any active infection requiring hospitalization or IV antibiotics within 2 weeks prior to randomization

Known hypersensitivity to any excipient of obinutuzumab or bevacizumab

Hypersensitivity to Chinese hamster ovary cell products or other recombinant human or humanized antibodies

Study treatment

Onartuzumab/onartuzumab placebo. Obinutuzumab was provided as a sterile liquid in a single use 15-cc vial containing 600mg obinutuzumab. The obinutuzumab drug product is formulated as 60mg/mL obinutuzumab in 10mM histidine acetate, 120mM sucrose, 0.4mg/mL polysorbate 20, pH 5.4. Onartuzumab placebo consists of a 250cc 0.9% saline solution (NSS) IV bag and will be provided by the survey site. Once obinutuzumab is diluted, the solution must be administered within 8 hours.

Bevacizumab. Bevacizumab is supplied as a preservative-free, clear to slightly opaque, colorless to light brown, sterile liquid in a single-use vial for IV infusion. It was supplied in a 20mL (400mg, 25mg/mL) glass vial with a 16mL fill. The formulation contained sodium phosphate, trehalose, polysorbate 20, and sterile water for injection (SWFI), USP.

Dosage, administration

The administration of onartuzumab/bevacizumab/placebo depends on the assigned treatment arm. In this study, onartuzumab/onartuzumab placebo was administered first followed by bevacizumab. Bevacizumab was administered at the end of onartuzumab/onartuzumab placebo infusion followed by a 60 minute recommended observation period.

Patients in arm a received onartuzumab placebo throughout the study. Patients in arm B received 15mg/kg of onartuzumab every three weeks throughout the study. The dose of onartuzumab/onartuzumab placebo is based on the weight of the patient at the time of screening. This dose was administered throughout the study and did not vary by weight. Liquid obinutuzumab was diluted with 0.9% NSS to a total volume of 250 mL. Once onartuzumab is diluted into NSS, the solution is recommended to be used within 8 hours. Onartuzumab was not diluted with dextrose. It is recommended to discard any remaining solution. Onartuzumab/onartuzumab placebo was administered as an IV infusion. The first dose was infused over 60 minutes (+ -10 minutes). For patients experiencing infusion-related symptoms, onartuzumab/onartuzumab placebo infusion may be slowed or interrupted. Patients were observed for fever, chills, or other infusion-related symptoms for at least 60 minutes following onartuzumab/onartuzumab placebo administration. Subsequently, onartuzumab/onartuzumab placebo doses were administered over 30(± 10) minutes, provided the patient tolerated the previous infusion.

Patients in arm a and arm B received 15mg/kg bevacizumab every three weeks throughout the study (after onartuzumab infusion). The dose of bevacizumab is based on the weight of the patient at the time of screening and will remain the same throughout the study unless the weight of the patient varies by more than 10%. Bevacizumab was diluted to a total volume of 100mL in 0.9% sodium chloride injection, USP. The initial dose was delivered over 90 ± 15 minutes. If the first infusion is tolerated without any infusion-related adverse events (fever and/or chills), then the second infusion is delivered within 60 ± 10 minutes. If a 60 minute infusion is well tolerated, all subsequent infusions are delivered within 30 ± 10 minutes.

And (5) carrying out statistical analysis. Therapeutic comparisons of PFS were based on a rank-sum test of stratification at the 0.05 level of significance (bilateral). The stratification factors are Karnofsky performance status (70% -80% versus 90% -100%) and age (<50 versus ≧ 50 years). The median PFS of each treatment arm was assessed using the Kaplan-Meier methodology and a Kaplan-Meier curve was constructed to provide a visual depiction of the difference between onartuzumab + bevacizumab and placebo + bevacizumab. Estimates of treatment efficacy were expressed as Hazard Ratio (HR) via the use of a layered Cox model, including 95% Confidence Intervals (CI). OS is defined as the time from randomization until death of any cause. Data from patients who have died at the time of analysis were not reported for review on the day of last day of living being known; if data is not available after baseline, OS is reviewed on the day of randomization. The analytical method was the same as for PFS. OS-9 is defined as the percentage of patients who were alive at 9 months. OS-9 was evaluated using the Kaplan-Meier method, along with standard error and corresponding 95% CI, using the Greenwood equation. The 95% CI and p values for the difference between OS-9 from branches A and B were determined by the z-test using the standard error estimated from the Greenwood equation. PFS-6 is defined as the percentage of patients who are alive and non-progressing at 6 months (24 weeks). The analytical method was the same as that of OS-9. The objective response is defined as CR or PR. Patients without post-baseline disease assessment were considered non-responders. The analysis population for ORR was all randomized patients with measurable disease at baseline. The ORR estimates for each treatment arm and their 95% CI were calculated using the Blyth-Still-Casella method. The CI of the difference in ORR between the two branches is determined using a normal approximation of the binomial distribution. DOR is defined as the time from initial response to disease progression or death in patients experiencing CR or PR during the study. Patients who did not progress or die at the time of analysis were reviewed on the last day of disease assessment. DOR was estimated using the Kaplan-Meier methodology. Comparisons between treatment arms via the use of non-stratified rank-sum tests are made for descriptive purposes only.

Exploratory analyses included the following:

mini mental state check (MMSE). Changes in neurocognitive function using MMSE were summarized relative to baseline at treatment arms and time points.

A corticosteroid. Corticosteroid use at baseline and change in dexamethasone equivalent dose from baseline were summarized in treatment arm and time points.

A biomarker. Exploratory biomarker analyses were performed in an effort to understand the association of these markers with study drug responses, including efficacy and/or adverse events.

Patient Reported Outcome (PRO). The MD Anderson symptom inventory-brain tumor questionnaire (MDASI-BT) was used to assess disease and PRO for treatment-related symptom severity and symptom intervention. MDASI-BT symptom severity and symptom interdose scale were summarized as means (and 95% CI) and plotted over time for all patients. Changes in mean (and 95% CI) from baseline (day 1 pre-dose for cycle 1) and absolute scores for each time point were reported. The scoring is based on MDASI and MDASI-BT validation files (Cleeland et al, Cancer (2000)89: 1634-46; Armstrong et al, neuroool (2006)80: 27-35). Mean (and 95% CI) changes from baseline (day 1 of cycle 1) were compared between the two main treatment arms (onartuzumab + bevacizumab and bevacizumab + placebo).

And (6) obtaining the result. 129 patients were randomized into both arms. Median survival follow-up in months was 9.9 (placebo + bevacizumab), 9.8 (onartuzumab + bevacizumab). The clinical data expiration date for this analysis was 2013, 11 months, and 07 days.

In patients with relapsed glioblastoma, treatment with a combination of the c-met antagonist ornuzumab and the VEGF antagonist bevacizumab exhibited:

(i) PFS and OS in HGF ISH 2+/3+ that are significantly longer relative to the control branches; and

(ii) significantly shorter PFS and OS in HGF ISH 0/1+ relative to control arm.

FIG. 2: overall survival was shown according to a subgroup analysis of HGF ISH status. Patients with high HGF ISH (2+/3+) had a median overall survival of 6.6 months when treated with placebo + bevacizumab compared to 10.9 months when treated with onartuzumab + bevacizumab (HR ═ 0.39 (95% CI 0.16, 0.96)). Patients with low HGF ISH (0/1+) had a median overall survival of 12.6 months when treated with placebo + bevacizumab compared to 8.6 months when treated with onartuzumab + bevacizumab (HR ═ 2.37 (95% CI 1.21, 4.66)).

FIG. 3: Kaplan-Meier analysis showing overall survival in HGF ISH low (0/1+) patients and HGF ISH high (2+/3+) patients.

FIG. 4: showing progression free survival was analyzed according to a subgroup of HGF ISH status. Patients with high HGF ISH (2+/3+) had a median progression-free survival of 2.8 months when treated with placebo + bevacizumab compared to a median overall survival of 8.3 months when treated with onartuzumab + bevacizumab (HR ═ 0.32 (95% CI 0.15, 0.6)). Patients with low HGFISH (0/1+) had a median progression-free survival of 4.1 months when treated with placebo + bevacizumab compared to a median overall survival of 2.9 months when treated with onartuzumab + bevacizumab (HR ═ 1.63 (95% CI 0.99, 2.68)).

FIG. 5: Kaplan-Meier analysis showing progression free survival in HGF ISH low (0/1+) patients and HGF ISH high (2+/3+) patients.

FIG. 6: analysis of overall survival in all patients randomized to bevacizumab + placebo (solid line) compared to patients randomized to bevacizumab + onartuzumab (dashed line) is shown.

FIG. 7: analysis of progression free survival in all patients randomized to bevacizumab + placebo (solid line) compared to patients randomized to bevacizumab + onartuzumab (dashed line) is shown.

Although the foregoing invention has been described in some detail by way of illustration for purposes of clarity of understanding, the description and examples should not be construed as limiting the scope of the invention.

Example 3 a: randomized, double-blind, placebo-controlled, multicenter phase II study analysis using PCR to assess efficacy and safety of onartuzumab in combination with bevacizumab in patients with relapsed glioblastoma

The randomized, double-blind, placebo-controlled, multicenter phase II trial described above, for assessing the efficacy and safety of onartuzumab + bevacizumab in first relapse in patients with glioblastoma was evaluated using PCR. For this analysis, the HGFmRNA expression level was assessed using Fluidigm gene expression analysis.

The scheme is as follows:

paraffin-embedded, formalin-fixed glioblastoma tumor tissue samples were cut out in 10 micron thick sections. RNA is then extracted and proteins and DNA are removed. Mu.l of total RNA was reverse transcribed to cDNA and pre-amplified in one reaction using Superscript III/Platinum Taq (Invitrogen) and a pre-amplification reaction mix (Invitrogen). Primer/probe sets selected for detection of HGF expression were included in the pre-amplification reaction (which included an additional 95 probe primer pairs) at a final concentration of 0.05-fold the initial Taqman assay concentration (Applied Biosystems). The thermal cycling conditions were as follows: 50 ℃ for 15min for 1 cycle, 70 ℃ for 2min for 1 cycle, followed by 95 ℃ for 15sec and 60 ℃ for 4min for 14 cycles.

The preamplified cDNA was diluted 1.94 fold and then amplified using Taqman Universal PCR Master mix (Applied Biosystems) on BioMark BMK-M-96.96 platform (Fluidigm) according to the manufacturer's instructions. All samples were assayed in triplicate. The expression panel included two custom designed reference genes, AL-1377271 and VPS-33B, that had previously evaluated their expression stability among various cell lines, fresh frozen tissue samples, and FFPE tissue samples. The mean of Ct values for these two reference genes was calculated for each sample and HGF expression levels were determined using the Δ Ct (dct) method as follows: mean Ct (target gene) -mean Ct (reference gene).

(i) PFS and OS significantly longer in patients with the upper 25% HGF-PCR expression relative to control arms; and

(ii) significantly shorter PFS and OS in patients with the latter 75% HGF-PCR expression relative to the control arm.

FIG. 17: overall survival was shown according to a subgroup analysis of HGF-PCR expression. Patients with high HGF-PCR (top 25%) had a median overall survival of 7.3 months in the placebo + bevacizumab arm, compared to that not reached in the onartuzumab + bevacizumab arm (HR ═ 0.29 (95% CI 0.08, 1.06)). Patients with low HGF-PCR (last 75%) had an unattained median overall survival in the placebo + bevacizumab arm compared to 8.6 months median overall survival in the onartuzumab + bevacizumab arm (HR ═ 1.86 (95% CI 1.03, 3.36)).

FIG. 18: Kaplan-Meier analysis showing overall survival in low HGF-PCR (last 75%) and high HGF-PCR (first 25%) patients.

FIG. 19: progression free survival was shown to follow a subgroup analysis of HGF-PCR expression. Patients with high HGF-PCR (top 25%) had a median progression-free survival of 2.8 months in the placebo + bevacizumab arm, compared to 6.1 months in the onartuzumab + bevacizumab arm (HR ═ 0.37 (95% CI 0.16, 0.86)). Patients with low HGF-PCR (last 75%) had a median progression-free survival of 4.1 months in the placebo + bevacizumab arm, compared to 2.9 months in the onartuzumab + bevacizumab arm (HR ═ 1.39 (95% CI 0.87, 2.20)).

FIG. 20: Kaplan-Meier analysis showing progression free survival in low HGF-PCR (last 75%) patients and high HGF-PCR (first 25%).

FIG. 22: prognostic effect of progression-free survival and overall survival in low (posterior 75%) and high (anterior 25%) HGF-PCR patients in bevacizumab + placebo arms is shown.

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Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

145 150 155 160

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

165 170 175

Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

180 185 190

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

195 200 205

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

210 215 220

<210>11

<211>449

<212>PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Note = "description of Artificial sequence: synthetic polypeptide"

<400>11

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Trp Leu His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

3540 45

Gly Met Ile Asp Pro Ser Asn Ser Asp Thr Arg Phe Asn Pro Asn Phe

50 55 60

Lys Asp Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Thr Tyr Arg Ser Tyr Val Thr Pro Leu Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Ser

355 360 365

Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

Lys

<210>12

<211>220

<212>PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Note = "description of Artificial sequence: synthetic polypeptide"

<400>12

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Leu Tyr Thr

20 25 30

Ser Ser Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys

35 40 45

Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln

85 90 95

Tyr Tyr Ala Tyr Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile

100 105110

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

115 120 125

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

130 135 140

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

145 150 155 160

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

165 170 175

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

180 185 190

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

195 200 205

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215 220

<210>13

<211>227

<212>PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Note = "description of Artificial sequence: synthetic polypeptide"

<400>13

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

1 5 10 15

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115120 125

Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser

130 135 140

Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Lys

225

<210>14

<211>123

<212>PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Note = "description of Artificial sequence: synthetic polypeptide"

<400>14

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Asn Tyr

20 25 30

Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Ala Asp Phe

50 55 60

Lys Arg Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Tyr Pro His Tyr Tyr Gly Ser Ser His Trp Tyr Phe Asp Val

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210>15

<211>108

<212>PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Note = "description of Artificial sequence: synthetic polypeptide"

<400>15

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Gln Asp Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Val Leu Ile

35 40 45

Tyr Phe Thr Ser Ser Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Thr Val Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg

100 105

<210>16

<211>1390

<212>PRT

<213> Artificial sequence

<220>

<221> sources

<223 >/Note = "description of Artificial sequence: synthetic polypeptide"

<400>16

Met Lys Ala Pro Ala Val Leu Ala Pro Gly Ile Leu Val Leu Leu Phe

1 5 10 15

Thr Leu Val Gln Arg Ser Asn Gly Glu Cys Lys Glu Ala Leu Ala Lys

20 25 30

Ser Glu Met Asn Val Asn Met Lys Tyr Gln Leu Pro Asn Phe Thr Ala

35 40 45

Glu Thr Pro Ile Gln Asn Val Ile Leu His Glu His His Ile Phe Leu

50 55 60

Gly Ala Thr Asn Tyr Ile Tyr Val Leu Asn Glu Glu Asp Leu Gln Lys

65 70 75 80

Val Ala Glu Tyr Lys Thr Gly Pro Val Leu Glu His Pro Asp Cys Phe

85 90 95

Pro Cys Gln Asp Cys Ser Ser Lys Ala Asn Leu Ser Gly Gly Val Trp

100 105 110

Lys Asp Asn Ile Asn Met Ala Leu Val Val Asp Thr Tyr Tyr Asp Asp

115 120 125

Gln Leu Ile Ser Cys Gly Ser Val Asn Arg Gly Thr Cys Gln Arg His

130 135 140

Val Phe Pro His Asn His Thr Ala Asp Ile Gln Ser Glu Val His Cys

145 150 155 160

Ile Phe Ser Pro Gln Ile Glu Glu Pro Ser Gln Cys Pro Asp Cys Val

165 170 175

Val Ser Ala Leu Gly Ala Lys Val Leu Ser Ser Val Lys Asp Arg Phe

180 185 190

Ile Asn Phe Phe Val Gly Asn Thr Ile Asn Ser Ser Tyr Phe Pro Asp

195 200 205

His Pro Leu His Ser Ile Ser Val Arg Arg Leu Lys Glu Thr Lys Asp

210 215 220

Gly Phe Met PheLeu Thr Asp Gln Ser Tyr Ile Asp Val Leu Pro Glu

225 230 235 240

Phe Arg Asp Ser Tyr Pro Ile Lys Tyr Val His Ala Phe Glu Ser Asn

245 250 255

Asn Phe Ile Tyr Phe Leu Thr Val Gln Arg Glu Thr Leu Asp Ala Gln

260 265 270

Thr Phe His Thr Arg Ile Ile Arg Phe Cys Ser Ile Asn Ser Gly Leu

275 280 285

His Ser Tyr Met Glu Met Pro Leu Glu Cys Ile Leu Thr Glu Lys Arg

290 295 300

Lys Lys Arg Ser Thr Lys Lys Glu Val Phe Asn Ile Leu Gln Ala Ala

305 310 315 320

Tyr Val Ser Lys Pro Gly Ala Gln Leu Ala Arg Gln Ile Gly Ala Ser

325 330 335

Leu Asn Asp Asp Ile Leu Phe Gly Val Phe Ala Gln Ser Lys Pro Asp

340 345 350

Ser Ala Glu Pro Met Asp Arg Ser Ala Met Cys Ala Phe Pro Ile Lys

355 360 365

Tyr Val Asn Asp Phe Phe Asn Lys Ile Val Asn Lys Asn Asn Val Arg

370 375 380

Cys Leu Gln His Phe Tyr Gly Pro Asn His Glu His Cys Phe Asn Arg

385 390 395 400

Thr Leu Leu Arg Asn Ser Ser Gly Cys Glu Ala Arg Arg Asp Glu Tyr

405 410 415

Arg Thr Glu Phe Thr Thr Ala Leu Gln Arg Val Asp Leu Phe Met Gly

420 425 430

Gln Phe Ser Glu Val Leu Leu Thr Ser Ile Ser Thr Phe Ile Lys Gly

435 440 445

Asp Leu Thr Ile Ala Asn Leu Gly Thr Ser Glu Gly Arg Phe Met Gln

450 455 460

Val Val Val Ser Arg Ser Gly Pro Ser Thr Pro His Val Asn Phe Leu

465 470 475 480

Leu Asp Ser His Pro Val Ser Pro Glu Val Ile Val Glu His Thr Leu

485 490 495

Asn Gln Asn Gly Tyr Thr Leu Val Ile Thr Gly Lys Lys Ile Thr Lys

500 505 510

Ile Pro Leu Asn Gly Leu Gly Cys Arg His Phe Gln Ser Cys Ser Gln

515 520 525

Cys Leu Ser Ala Pro Pro Phe Val Gln Cys Gly Trp Cys His Asp Lys

530 535 540

Cys Val Arg Ser Glu Glu Cys Leu Ser Gly Thr Trp Thr Gln Gln Ile

545 550 555 560

Cys Leu Pro Ala Ile Tyr Lys Val Phe Pro Asn Ser Ala Pro Leu Glu

565 570 575

Gly Gly Thr Arg Leu Thr Ile Cys Gly Trp Asp Phe Gly Phe Arg Arg

580 585 590

Asn Asn Lys Phe Asp Leu Lys Lys Thr Arg Val Leu Leu Gly Asn Glu

595 600 605

Ser Cys Thr Leu Thr Leu Ser Glu Ser Thr Met Asn Thr Leu Lys Cys

610 615 620

Thr Val Gly Pro Ala Met Asn Lys His Phe Asn Met Ser Ile Ile Ile

625 630 635 640

Ser Asn Gly His Gly Thr Thr Gln Tyr Ser Thr Phe Ser Tyr Val Asp

645 650 655

Pro Val Ile Thr Ser Ile Ser Pro Lys Tyr Gly Pro Met Ala Gly Gly

660 665 670

Thr Leu Leu Thr Leu Thr Gly Asn Tyr Leu Asn Ser Gly Asn Ser Arg

675 680 685

His Ile Ser Ile Gly Gly Lys Thr Cys Thr Leu Lys Ser Val Ser Asn

690 695 700

Ser Ile Leu Glu Cys Tyr Thr Pro Ala Gln Thr Ile Ser Thr Glu Phe

705 710 715 720

Ala Val Lys Leu Lys Ile Asp Leu Ala Asn Arg Glu Thr Ser Ile Phe

725 730 735

Ser Tyr Arg Glu Asp Pro Ile Val Tyr Glu Ile His Pro Thr Lys Ser

740 745 750

Phe Ile Ser Gly Gly Ser Thr Ile Thr Gly Val Gly Lys Asn Leu Asn

755 760 765

Ser Val Ser Val Pro Arg Met Val Ile Asn Val His Glu Ala Gly Arg

770 775 780

Asn Phe ThrVal Ala Cys Gln His Arg Ser Asn Ser Glu Ile Ile Cys

785 790 795 800

Cys Thr Thr Pro Ser Leu Gln Gln Leu Asn Leu Gln Leu Pro Leu Lys

805 810 815

Thr Lys Ala Phe Phe Met Leu Asp Gly Ile Leu Ser Lys Tyr Phe Asp

820 825 830

Leu Ile Tyr Val His Asn Pro Val Phe Lys Pro Phe Glu Lys Pro Val

835 840 845

Met Ile Ser Met Gly Asn Glu Asn Val Leu Glu Ile Lys Gly Asn Asp

850 855 860

Ile Asp Pro Glu Ala Val Lys Gly Glu Val Leu Lys Val Gly Asn Lys

865 870 875 880

Ser Cys Glu Asn Ile His Leu His Ser Glu Ala Val Leu Cys Thr Val

885 890 895

Pro Asn Asp Leu Leu Lys Leu Asn Ser Glu Leu Asn Ile Glu Trp Lys

900 905 910

Gln Ala Ile Ser Ser Thr Val Leu Gly Lys Val Ile Val Gln Pro Asp

915 920 925

Gln Asn Phe Thr Gly Leu Ile Ala Gly Val Val Ser Ile Ser Thr Ala

930 935 940

Leu Leu Leu Leu Leu Gly Phe Phe Leu Trp Leu Lys Lys Arg Lys Gln

945 950 955 960

Ile Lys Asp Leu Gly Ser Glu Leu Val Arg Tyr Asp Ala Arg Val His

965 970 975

Thr Pro His Leu Asp Arg Leu Val Ser Ala Arg Ser Val Ser Pro Thr

980 985 990

Thr Glu Met Val Ser Asn Glu Ser Val Asp Tyr Arg Ala Thr Phe Pro

995 1000 1005

Glu Asp Gln Phe Pro Asn Ser Ser Gln Asn Gly Ser Cys Arg Gln

1010 1015 1020

Val Gln Tyr Pro Leu Thr Asp Met Ser Pro Ile Leu Thr Ser Gly

1025 1030 1035

Asp Ser Asp Ile Ser Ser Pro Leu Leu Gln Asn Thr Val His Ile

1040 1045 1050

Asp Leu Ser Ala Leu Asn Pro Glu Leu Val Gln Ala Val Gln His

1055 10601065

Val Val Ile Gly Pro Ser Ser Leu Ile Val His Phe Asn Glu Val

1070 1075 1080

Ile Gly Arg Gly His Phe Gly Cys Val Tyr His Gly Thr Leu Leu

1085 1090 1095

Asp Asn Asp Gly Lys Lys Ile His Cys Ala Val Lys Ser Leu Asn

1100 1105 1110

Arg Ile Thr Asp Ile Gly Glu Val Ser Gln Phe Leu Thr Glu Gly

1115 1120 1125

Ile Ile Met Lys Asp Phe Ser His Pro Asn Val Leu Ser Leu Leu

1130 1135 1140

Gly Ile Cys Leu Arg Ser Glu Gly Ser Pro Leu Val Val Leu Pro

1145 1150 1155

Tyr Met Lys His Gly Asp Leu Arg Asn Phe Ile Arg Asn Glu Thr

1160 1165 1170

His Asn Pro Thr Val Lys Asp Leu Ile Gly Phe Gly Leu Gln Val

1175 1180 1185

Ala Lys Gly Met Lys Tyr Leu Ala Ser Lys Lys Phe Val His Arg

1190 1195 1200

Asp Leu Ala Ala Arg Asn Cys Met Leu Asp Glu Lys Phe Thr Val

1205 1210 1215

Lys Val Ala Asp Phe Gly Leu Ala Arg Asp Met Tyr Asp Lys Glu

1220 1225 1230

Tyr Tyr Ser Val His Asn Lys Thr Gly Ala Lys Leu Pro Val Lys

1235 1240 1245

Trp Met Ala Leu Glu Ser Leu Gln Thr Gln Lys Phe Thr Thr Lys

1250 1255 1260

Ser Asp Val Trp Ser Phe Gly Val Leu Leu Trp Glu Leu Met Thr

1265 1270 1275

Arg Gly Ala Pro Pro Tyr Pro Asp Val Asn Thr Phe Asp Ile Thr

1280 1285 1290

Val Tyr Leu Leu Gln Gly Arg Arg Leu Leu Gln Pro Glu Tyr Cys

1295 1300 1305

Pro Asp Pro Leu Tyr Glu Val Met Leu Lys Cys Trp His Pro Lys

1310 1315 1320

Ala Glu Met Arg Pro Ser Phe Ser Glu Leu Val Ser Arg Ile Ser

1325 1330 1335

Ala Ile Phe Ser Thr Phe Ile Gly Glu His Tyr Val His Val Asn

1340 1345 1350

Ala Thr Tyr Val Asn Val Lys Cys Val Ala Pro Tyr Pro Ser Leu

1355 1360 1365

Leu Ser Ser Glu Asp Asn Ala Asp Asp Glu Val Asp Thr Arg Pro

1370 1375 1380

Ala Ser Phe Trp Glu Thr Ser

1385 1390

Claims

1. A method for identifying a cancer patient who is likely to respond to treatment with a c-met antagonist, comprising the step of determining whether the patient's cancer has a high amount of HGF biomarker, wherein the HGF biomarker expression indicates that the patient is likely to respond to treatment with the c-met antagonist.

2. A method for determining the prognosis of a cancer patient comprising the step of determining whether the patient's cancer has a high amount of HGF biomarker, wherein the HGF biomarker expression indicates that the patient is likely to have prolonged Overall Survival (OS) and/or Progression Free Survival (PFS) when the patient is treated with a c-met antagonist.

3. The method of claim 1 or 2, wherein the patient's cancer is previously treated glioblastoma and treatment is with a therapeutically effective combination of a c-met antagonist and a VEGF antagonist.

4. The method of claim 1 or 2, wherein the patient's cancer is previously treated renal cell carcinoma.

5. The method of claim 4, wherein the treatment is with a therapeutically effective combination of a c-met antagonist and a VEGF antagonist.

6. The method of claim 1 or 2, wherein the patient's cancer is previously treated mesothelioma.

7. The method of claim 6, wherein the treatment is with a therapeutically effective combination of a c-met antagonist and a second cancer drug.

8. The method of claim 1 or 2, wherein the patient's cancer is previously treated gastric cancer.

9. The method of claim 8, wherein the treatment is with a therapeutically effective combination of a c-met antagonist and a second cancer drug.

10. The method of claim 1 or 2, wherein the patient's cancer is previously treated hepatocellular carcinoma.

11. The method of claim 10, wherein the treatment is with a therapeutically effective combination of a c-met antagonist and a second cancer drug.

12. The method of any one of the preceding claims, wherein HGF biomarker is HGF mRNA, and HGF biomarker mRNA expression is determined in a sample from the patient using In Situ Hybridization (ISH).

13. The method of claim 12, wherein high HGF biomarker is an ISH score of 2+ and/or 3 +.

14. The method of claim 12, wherein high HGF biomarker is an ISH score of 2+ and 3 +.

15. The method of claim 12, wherein high HGF mRNA biomarker is the presence of about 12 or more HGFISH signal positive cells in the sample.

16. The method of claim 12, wherein high HGF mRNA biomarker is the presence of about 15 or more HGFISH signal positive cells in the sample.

17. The method of claim 12, wherein high HGF mRNA biomarker is the presence of about 20 or more HGFISH signal positive cells in the sample.

18. The method of claim 12, wherein high HGF mRNA biomarker is the presence of about 25 or more HGFISH signal positive cells in the sample.

19. The method of claim 12, wherein high HGF mRNA biomarker is the presence of about 30 or more HGFISH signal positive cells in the sample.

20. The method of claim 12, wherein high HGF mRNA biomarker is the presence of about 35 or more HGFISH signal positive cells in the sample.

21. The method of claim 12, wherein high HGF mRNA biomarker is 1% or more of HGF ISH signal positive cells in the sample.

22. The method of claim 12, wherein high HGF mRNA biomarker is 2% or more of HGF ISH signal positive cells in the sample.

23. The method of claim 12, wherein high HGF mRNA biomarker is 3% or more of HGF ISH signal positive cells in the sample.

24. The method of claim 12, wherein high HGF mRNA biomarker is 4% or more of HGF ISH signal positive cells in the sample.

25. The method of claim 12, wherein high HGF mRNA biomarker is 5% or more of HGF ISH signal positive cells in the sample.

26. The method of claim 12, wherein high HGF mRNA biomarker is 10% or more of HGF ISH signal positive cells in the sample.

27. The method of any one of claims 1-11, wherein HGF biomarker expression is nucleic acid expression and is determined in a sample from the patient using PCR (e.g., rt-qPCR), RNA-seq, microarray analysis, SAGE, MassARRAY technique, or FISH.

28. The method of claim 27, wherein HGF biomarker expression is determined in a sample from the patient using rt-qPCR.

29. The method of claim 27 or 28, wherein high HGF mRNA biomarker is in the upper 25% of a reference patient population.

30. A method for identifying a cancer patient who is not likely to respond to treatment with a c-met antagonist, comprising the step of determining whether the patient's cancer has a low amount of HGF biomarker, wherein the HGF biomarker expression indicates that the patient is not likely to respond to treatment with the c-met antagonist.

31. The method of claim 30, wherein HGF biomarker nucleic acid expression is determined in a sample from the patient using In Situ Hybridization (ISH).

32. The method of claim 31, wherein low HGF mRNA biomarker is an ISH score of less than 2 +.

33. The method of claim 31, wherein low HGF mRNA biomarker is an ISH score of 0 or 1 +.

34. The method of claim 31, wherein low HGF mRNA biomarker is an ISH score of 0.

35. The method of claim 31, wherein low HGF biomarker is presence of HGF ISH positive signal in 10 or fewer cells.

36. The method of claim 31, wherein low HGF biomarker is presence of HGF ISH positive signal in 5 or fewer cells.

37. The method of claim 30, wherein HGF biomarker nucleic acid expression is determined in a sample from the patient using a technique selected from the group consisting of: PCR, RNA-seq, microarray analysis, SAGE, and MassARRAY techniques.

38. The method of claim 37, wherein the PCR is rt-qPCR.

39. The method of claim 37 or 38, wherein low HGF mRNA biomarker is in the lower 75% of a reference patient population.

40. The method of any one of claims 12-29, wherein the sample is of a cancer of the patient.

41. The method of claim 40, wherein the sample comprises glioblastoma cells and benign stromal cells.

42. The method of claim 41, wherein the benign stromal cells are one or more of reactive astrocytes, glial cells, pericytes and endothelial cells.

43. The method of claim 40, wherein the sample comprises mesothelioma cells and benign stromal cells.

44. The method of claim 40, wherein the sample comprises gastric cancer cells and benign stromal cells.

45. The method of claim 44, wherein the benign stromal cells are one or more of fibroblasts, macrophages and endothelial cells.

46. The method of claim 40, wherein the sample comprises hepatocellular carcinoma cells and benign stromal cells.

47. The method of claim 40, wherein the sample comprises renal cell carcinoma cells and benign stromal cells.

48. The method of claim 40, wherein the sample comprises sarcoma cells and benign stromal cells.

49. The method of any one of claims 40-48, wherein the sample is obtained prior to treatment with a c-met antagonist.

50. The method of any one of claims 40-48, wherein the sample is obtained prior to treatment with a VEGF antagonist.

51. The method of any one of claims 40-48, wherein the sample is obtained prior to treatment with a cancer drug.

52. The method of any one of claims 40-51, wherein the sample is formalin fixed and paraffin embedded.

53. The method of any one of claims 12-26, wherein the ISH is detected using hybridization-based signal amplification.

54. The method of claim 40, wherein the cancer is glioblastoma, mesothelioma, hepatocellular carcinoma, renal cell carcinoma, gastric cancer, sarcoma, osteosarcoma, non-small cell lung cancer, breast cancer, gallbladder cancer, or pancreatic cancer.

55. The method of claim 54, wherein the cancer is glioblastoma, mesothelioma, renal cell carcinoma, gastric cancer, hepatocellular carcinoma or sarcoma.

56. The method of claim 55, wherein the cancer is previously treated glioblastoma.

57. The method of claim 55, wherein the cancer is previously treated gastric cancer.

58. The method of claim 55, wherein the cancer is previously treated mesothelioma.

59. The method of claim 55, wherein the cancer is previously treated hepatocellular carcinoma.

60. The method of claim 55, wherein the cancer is previously treated renal cell carcinoma.

61. The method of claim 55, wherein the cancer is a previously treated sarcoma.

62. The method of any one of the preceding claims, wherein the antagonist of c-met is an antagonistic anti-c-met antibody.

63. The method of claim 62, wherein the anti-c-met antibody comprises (a) HVR1 comprising sequence GYTFTSYWLH (SEQ ID NO: 1); (b) HVR2 comprising sequence GMIDPSNSDTRFNPNFKD (SEQ ID NO: 2); (c) HVR3-HC comprising sequence ATYRSYVTPLDY (SEQ ID NO: 3); (d) HVR1-LC comprising sequence KSSQSLLYTSSQKNYLA (SEQ ID NO: 4); (e) HVR2-LC comprising sequence WASTRES (SEQ ID NO: 5); and (f) HVR3-LC comprising sequence QQYYAYPWT (SEQ ID NO: 6).

64. The method of claim 62, wherein the anti-c-met antibody binds an onartuzumab (onartuzumab) epitope.

65. The method of claim 62, wherein the anti-c-met antibody is onartuzumab.

66. The method of any one of claims 62-65, wherein the effective amount of the anti-c-met antibody is 15mg/kg every three weeks.

67. The method of any one of claims 62-65, wherein the effective amount of the anti-c-met antibody is 10mg/kg biweekly.

68. The method of any one of claims 1-61, wherein the c-met antagonist is one or more of crizotinib, tivatinib, carbozantinib, MGCD-265, ficlatuzumab, humanized TAK-701, rilotumumab, foretinib, h224G11, DN-30, MK-2461, E7050, MK-8033, PF-4217903, AMG208, JNJ-38877605, EMD1204831, INC-280, LY-2801653, SGX-126, RP1040, LY2801653, BAY-853474, and/or LA 480.

69. The method of claim 3 or 5, wherein the VEGF antagonist is an anti-VEGF antibody.

70. The method of claim 69, wherein the anti-VEGF antibody binds the A4.6.1 epitope.

71. The method of claim 69, wherein the anti-VEGF antibody is bevacizumab (bevacizumab).

72. The method of claim 69, wherein the anti-VEGF antibody comprises a variable heavy chain (VH) and a variable light chain (VL), wherein the VH has amino acid sequence EVQLVESGGG LVQPGGSLRL SCAASGYTFT NYGMNWVRQA PGKGLEWVGWINTYTGEPTY AADFKRRFTF SLDTSKSTAY LQMNSLRAED TAVYYCAKYP HYYGSSHWYF DVWGQGTLVTVSS (SEQ ID NO:14) and the VL has amino acid sequence DIQMTQSPSS LSASVGDRVT ITCSASQDISNYLNWYQQKP GKAPKVLIYF TSSLHSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ YSTVPWTFGQGTKVEIKR (SEQ ID NO: 15).

73. The method of any one of claims 69-72, wherein said effective amount of said anti-VEGF antibody is 10mg/kg intravenously every two weeks.

74. The method of any one of claims 69-72, wherein said effective amount of said anti-VEGF antibody is 15mg/kg intravenously every three weeks.

75. The method of any one of claims 69-74, wherein said effective amount of said anti-VEGF antibody is administered initially intravenously over 90 minutes with subsequent infusions over 60 minutes and then over 30 minutes.

76. The method of any one of claims 69-75, wherein said anti-VEGF antibody is administered to said patient second in a first cycle.

77. The method of claim 76, wherein subsequent administrations of the anti-VEGF antibody precede or follow the c-met antagonist.

78. The method of any one of claims 3,5 or 69-77, wherein the VEGF antagonist is administered concurrently with the c-met antagonist.

79. The method of any one of the preceding claims, wherein the patient is less than 50 years of age.

80. The method of any one of claims 1-78, wherein the patient is equal to or greater than 50 years of age.

81. The method of any one of the preceding claims, wherein the patient has a Karnofsky performance status of 70% to 80%.

82. The method of any one of claims 1-80, wherein the patient has a Karnofsky performance status of 90% to 100%.

83. The method of any one of claims 1-29 or 40-82, wherein the patient has greater PFS and/or OS relative to a patient not having high HGF biomarker.

84. The method of claim 3, wherein the patient has greater PFS and/or OS relative to a patient treated with the VEGF antagonist alone.

85. The method of claim 5, wherein the patient has greater PFS and/or OS relative to a patient treated with the VEGF antagonist alone.

86. A method of identifying a patient having a previously treated cancer selected from glioblastoma, mesothelioma, gastric cancer, renal cell carcinoma, hepatocellular carcinoma, and sarcoma as likely to respond to a therapy comprising an anti-c-met antibody, wherein the method comprises:

(i) measuring HGF biomarker in a sample from the patient, wherein HGF biomarker is HGF nucleic acid and measuring is by ISH; and are

(ii) Identifying the patient as likely to respond to therapy comprising a c-met antagonist antibody when the sample has high HGF biomarker.

87. The method of claim 86, wherein the method further comprises (iii) selecting a therapy comprising a c-met antagonist antibody for the patient or recommending a therapy comprising a c-met antagonist antibody.

88. A method of identifying a patient having a previously treated cancer selected from glioblastoma and renal cell carcinoma as likely to respond to a therapy comprising (a) an anti-c-met antibody and (b) an anti-VEGF antibody, wherein the method comprises:

(ii) Identifying the patient as likely to respond to the therapy comprising (a) a c-met antagonist antibody and (b) an anti-VEGF antibody when the sample has high HGF biomarker.

89. The method of claim 88, wherein the method further comprises (iii) selecting the therapy comprising (a) the c-met antagonist antibody and (b) the anti-VEGF antibody for the patient or recommending a therapy comprising (a) the c-met antagonist antibody and (b) the anti-VEGF antibody.

90. A method of identifying a patient having a previously treated cancer selected from glioblastoma, mesothelioma, gastric cancer, renal cell carcinoma, hepatocellular carcinoma, and sarcoma as likely to respond to a therapy comprising an anti-c-met antibody, wherein the method comprises:

(i) measuring HGF biomarker in a sample from the patient, wherein HGF biomarker is HGF nucleic acid and measuring is by rt-qPCR; and are

(ii) Identifying the patient as likely to respond to the therapy comprising the c-met antagonist antibody when the sample has high HGF biomarker.

91. The method of claim 90, wherein the method further comprises (iii) selecting the therapy comprising a c-met antagonist antibody or recommending a therapy comprising a c-met antagonist antibody for the patient.

92. A method of identifying a patient having a previously treated cancer selected from glioblastoma and renal cell carcinoma as likely to respond to a therapy comprising (a) an anti-c-met antibody and (b) an anti-VEGF antibody, wherein the method comprises:

93. The method of claim 92, wherein the method further comprises (iii) selecting the therapy comprising (a) the c-met antagonist antibody and (b) the anti-VEGF antibody for the patient or recommending a therapy comprising (a) the c-met antagonist antibody and (b) the anti-VEGF antibody.

94. A method for determining HGF biomarker expression comprising the step of determining whether a cancer of a patient has a high level of HGF biomarker, wherein HGF biomarker expression is mRNA expression and is determined in a sample from the patient using ISH, wherein high HGF biomarker expression is an ISH score greater than 2+, wherein the high HGF biomarker expression indicates that the patient is likely to have prolonged OS and/or PFS when the patient is treated with an anti-c-met antibody.

95. A method for determining HGF biomarker expression comprising the step of determining whether a cancer of a patient has a high level of HGF biomarker, wherein HGF biomarker expression is mRNA expression and is determined in a sample from the patient using ISH, wherein high HGF biomarker expression is an ISH score greater than 2+, wherein the high HGF biomarker expression indicates that the patient is likely to have prolonged OS and/or PFS when the patient is treated with an anti-c-met antibody in combination with an anti-VEGF antibody.

96. A method for determining HGF biomarker expression comprising the step of determining whether a cancer of a patient has a high level of HGF biomarker, wherein HGF biomarker expression is mRNA expression and is determined in a sample from the patient using rt-qPCR, wherein high HGF biomarker expression is HGF biomarker expression in the upper 25% of a reference patient population, wherein the high HGF biomarker expression indicates that the patient is likely to have prolonged OS and/or PFS when the patient is treated with an anti-c-met antibody.

97. A method for determining HGF biomarker expression, comprising the step of determining whether a cancer of a patient has a high level of HGF biomarker, wherein HGF biomarker expression is mRNA expression and is determined in a sample from the patient using rt-qPCR, wherein high HGF biomarker expression is HGF biomarker expression in the upper 25% of a reference patient population, wherein the high HGF biomarker expression indicates that the patient is likely to have prolonged OS and/or PFS when the patient is treated with an anti-c-met antibody in combination with an anti-VEGF antibody.

98. A method for treating a patient having cancer comprising administering an effective amount of a c-met antagonist to the patient if the patient's cancer has been found to have a high amount of HGF biomarker.

99. The method of claim 98, wherein the c-met antagonist is an antagonistic anti-c-met antibody.

100. The method of claim 99, wherein the anti-c-met antibody comprises (a) HVR1 comprising sequence GYTFTSYWLH (SEQ id no: 1); (b) HVR2 comprising sequence GMIDPSNSDTRFNPNFKD (SEQ ID NO: 2); (c) HVR3-HC comprising sequence ATYRSYVTPLDY (SEQ ID NO: 3); (d) HVR1-LC comprising sequence KSSQSLLYTSSQKNYLA (SEQ ID NO: 4); (e) HVR2-LC comprising sequence WASTRES (SEQ ID NO: 5); and (f) HVR3-LC comprising sequence QQYYAYPWT (SEQ ID NO: 6).

101. The method of claim 99, wherein the anti-c-met antibody binds an onartuzumab epitope.

102. The method of claim 99, wherein the anti-c-met antibody is onartuzumab.

103. The method of claim 99, wherein the effective amount of the anti-c-met antibody is 15mg/kg every three weeks.

104. The method of claim 99, wherein the effective amount of the anti-c-met antibody is 10mg/kg biweekly.

105. The method of claim 98, wherein the c-met antagonist is one or more of crizotinib, tivtinib, carbozantinib, MGCD-265, ficlatuzumab, humanized TAK-701, rilotumumab, foretinib, h224G11, DN-30, MK-2461, E7050, MK-8033, PF-4217903, AMG208, JNJ-38877605, EMD1204831, INC-280, LY-2801653, SGX-126, RP1040, LY2801653, BAY-853474, and/or LA 480.

106. The method of any one of claims 98-105, wherein the treatment is with an effective amount of a combination of a c-met antagonist and a VEGF antagonist.

107. The method of claim 106, wherein the VEGF antagonist is an anti-VEGF antibody.

108. The method of claim 107, wherein the anti-VEGF antibody binds the a4.6.1 epitope.

109. The method of claim 107, wherein the anti-VEGF antibody is bevacizumab.

110. The method of claim 107, wherein the anti-VEGF antibody comprises a variable heavy chain (VH) and a variable light chain (VL), wherein the VH has amino acid sequence EVQLVESGGG LVQPGGSLRL SCAASGYTFT NYGMNWVRQA PGKGLEWVGWINTYTGEPTY AADFKRRFTF SLDTSKSTAY LQMNSLRAED TAVYYCAKYP HYYGSSHWYF DVWGQGTLVTVSS (SEQ ID NO:14) and the VL has amino acid sequence DIQMTQSPSS LSASVGDRVT ITCSASQDISNYLNWYQQKP GKAPKVLIYF TSSLHSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ YSTVPWTFGQGTKVEIKR (SEQ ID NO: 15).

111. The method of any one of claims 107-110, wherein said effective amount of said anti-VEGF antibody is 10mg/kg intravenously every two weeks.

112. The method of any one of claims 107-110, wherein said effective amount of said anti-VEGF antibody is 15mg/kg intravenously every three weeks.

113. The method of any one of claims 107-112, wherein the effective amount of the anti-VEGF antibody is administered initially intravenously over 90 minutes, with subsequent infusions over 60 minutes, then over 30 minutes.

114. The method of any one of claims 107-113, wherein said anti-VEGF antibody is administered to said patient second during the first cycle.

115. The method of any one of claims 107-114, wherein subsequent administration of the anti-VEGF antibody is either before or after the c-met antagonist.

116. The method of any one of claims 107-113, wherein the VEGF antagonist and the c-met antagonist are administered concurrently.

117. The method of any one of claims 98-116, wherein the patient is less than 50 years of age.

118. The method of any one of claims 98-116, wherein the patient is equal to or greater than 50 years of age.

119. The method of any one of claims 98-118, wherein the patient has a Karnofsky performance status of 70% to 80%.

120. The method of any one of claims 98-118, wherein the patient has a Karnofsky performance status of 90% to 100%.

121. The method of any one of claims 98-120, wherein the patient has greater PFS and/or OS relative to a patient not having high HGF biomarker.

122. The method of any one of claims 98-120, wherein the patient has greater PFS and/or OS relative to a patient treated with the VEGF antagonist alone.

123. The method of any one of claims 98-122, wherein HGF biomarker is HGF mRNA, and HGF biomarker mRNA expression is determined in a sample from the patient using In Situ Hybridization (ISH).

124. The method of claim 123, wherein high HGF biomarker is an ISH score of 2+ and/or 3 +.

125. The method of claim 123, wherein high HGF biomarker is an ISH score of 2+ and 3 +.

126. The method of claim 123, wherein high HGF mRNA biomarker is presence of about 12 or more HGF ISH signal positive cells in the sample.

127. The method of claim 123, wherein high HGF mRNA biomarker is presence of about 15 or more HGF ISH signal positive cells in the sample.

128. The method of claim 123, wherein high HGF mRNA biomarker is presence of about 20 or more HGF ISH signal positive cells in the sample.

129. The method of claim 123, wherein high HGF mRNA biomarker is presence of about 25 or more HGF ISH signal positive cells in the sample.

130. The method of claim 123, wherein high HGF mRNA biomarker is presence of about 30 or more HGF ISH signal positive cells in the sample.

131. The method of claim 123, wherein high HGF mRNA biomarker is presence of about 35 or more HGF ISH signal positive cells in the sample.

132. The method of claim 123, wherein high HGF mRNA biomarker is 1% or more HGFISH signal positive cells in the sample.

133. The method of claim 123, wherein high HGF mRNA biomarker is 2% or more HGFISH signal positive cells in the sample.

134. The method of claim 123, wherein high HGF mRNA biomarker is 3% or more HGFISH signal positive cells in the sample.

135. The method of claim 123, wherein high HGF mRNA biomarker is 4% or more HGFISH signal positive cells in the sample.

136. The method of claim 123, wherein high HGF mRNA biomarker is 5% or more HGFISH signal positive cells in the sample.

137. The method of claim 123, wherein high HGF mRNA biomarker is 10% or more HGFISH signal positive cells in the sample.

138. The method of any one of claims 98-122, wherein HGF biomarker expression is nucleic acid expression and is determined in a sample from the patient using PCR, RNA-seq, microarray analysis, SAGE, MassARRAY technique, or FISH.

139. The method of claim 138, wherein the PCR is rt-qPCR.

140. The method of claim 138 or 139, wherein high HGF mRNA biomarker is in the upper 25% of a reference patient population.

141. The method of any one of claims 123-140, wherein the sample is of a cancer of the patient.

142. The method of claim 141, wherein the cancer is a previously treated glioblastoma.

143. The method of claim 142, wherein the sample comprises glioblastoma cells and benign stromal cells.

144. The method of claim 143, wherein the benign stromal cells are one or more of reactive astrocytes, glial cells, pericytes and endothelial cells.

145. The method of claim 141, wherein the cancer is previously treated mesothelioma.

146. The method of claim 145, wherein the sample comprises mesothelioma cells and benign stromal cells.

147. The method of claim 141, wherein the cancer is previously treated gastric cancer.

148. The method of claim 147, wherein the sample comprises gastric cancer cells and benign stromal cells.

149. The method of claim 148, wherein the benign stromal cells are one or more of fibroblasts, macrophages and endothelial cells.

150. The method of claim 141, wherein the cancer is previously treated renal cell carcinoma.

151. The method of claim 150, wherein the sample comprises renal cell carcinoma cells and benign stromal cells.

152. The method of claim 141, wherein the cancer is previously treated hepatocellular carcinoma.

153. The method of claim 152, wherein the sample comprises hepatocellular carcinoma cells and benign stromal cells.

154. The method of claim 141, wherein the cancer is a previously treated sarcoma.

155. The method of claim 154, wherein the sample comprises sarcoma cells and benign stromal cells.

156. The method of any one of claims 123-155, wherein the sample is obtained prior to treatment with the c-met antagonist.

157. The method of any one of claims 123-155, wherein the sample is obtained prior to treatment with the VEGF antagonist.

158. The method of any one of claims 123-155, wherein the sample is obtained prior to treatment with the cancer drug.

159. The method of any one of claims 123-158, wherein the sample is formalin fixed and paraffin embedded.

160. The method of any one of claims 123-137 wherein the ISH is detected using hybridization-based signal amplification.

161. A method for treating a patient having cancer comprising administering to the patient a therapeutically effective amount of a drug other than a c-met antagonist if the patient's cancer has been found to have a small amount of HGF biomarker.

162. A method for advertising a c-met antibody, comprising promoting treatment of a patient with cancer with the c-met antibody based on expression of HGF biomarker to a target audience.

163. The method of claim 162, wherein the promotion is by a package insert accompanying a commercial formulation of the anti-c-met antibody.

164. The method of claim 162, wherein the promotion is by a package insert accompanying a commercial formulation of the second medicament.

165. The method of claim 164, wherein the second drug is a VEGF antagonist.

166. The method of claim 165, wherein the anti-c-met antibody is onartuzumab and the VEGF antagonist is bevacizumab.

167. The method of claim 162, wherein the patient is selected for treatment with a c-met antagonist if the cancer sample expresses the biomarker at a high level.

168. The method of claim 162, wherein the promotion is by a package insert, wherein the package insert provides instructions for receiving therapy with an anti-c-met antibody in combination with a VEGF antagonist.

169. The method of claim 162, wherein the promotion is followed by treatment of the patient with the anti-c-met antibody with or without a second agent.

170. A diagnostic kit comprising one or more reagents for determining expression of an HGF biomarker in a sample from a previously treated glioblastoma patient, wherein detection of a high amount of HGF biomarker means extended survival (e.g., PFS and/or OS) when the patient is treated with an effective amount of a c-met antagonist.

171. The diagnostic kit of claim 170, wherein detection of a high amount of HGF biomarker means extended survival when the patient is treated with an effective amount of a combination of a c-met antagonist and a VEGF antagonist.

172. The diagnostic kit of claim 170, further comprising instructions for using the kit to select a c-met antagonist to treat the previously treated glioblastoma patient if a high amount of HGF biomarker is determined.

173. A method of making the diagnostic kit of any one of claims 170-172 comprising combining in a package a pharmaceutical composition comprising a cancer drug and a package insert indicating that the pharmaceutical composition is for treating a patient having cancer based on expression of HGF biomarker.

174. A diagnostic kit comprising one or more reagents for determining expression of an HGF biomarker in a sample from a previously treated mesothelioma patient, wherein detection of a high amount of HGF biomarker means extended survival (e.g., PFS and/or OS) when the patient is treated with an effective amount of a c-met antagonist.

175. The diagnostic kit of claim 174, further comprising instructions for using the kit to select a c-met antagonist to treat the previously treated mesothelioma patient if a high amount of HGF biomarker is determined.

176. A method of making the diagnostic kit of claim 174 or 175, comprising combining in a package a pharmaceutical composition comprising a cancer medicament and a package insert indicating that the pharmaceutical composition is for treating a patient having cancer based on expression of HGF biomarker.

177. A diagnostic kit comprising one or more reagents for determining expression of an HGF biomarker in a sample from a previously treated gastric cancer patient, wherein detection of a high amount of HGF biomarker means extended survival (e.g., PFS and/or OS) when the patient is treated with an effective amount of a c-met antagonist.

178. The diagnostic kit of claim 177, further comprising instructions to use the kit to select a c-met antagonist to treat the previously treated gastric cancer patient if a high amount of HGF biomarker is determined.

179. A method of making the diagnostic kit of claim 177 or 178, comprising combining, in a package, a pharmaceutical composition comprising a cancer drug and a package insert indicating that the pharmaceutical composition is for treating a patient having cancer based on expression of HGF biomarker.

180. A diagnostic kit comprising one or more reagents for determining expression of an HGF biomarker in a sample from a previously treated renal cell carcinoma patient, wherein detection of a high amount of HGF biomarker means extended survival (e.g., PFS and/or OS) when the patient is treated with an effective amount of a c-met antagonist.

181. The diagnostic kit of claim 180, wherein detection of a high amount of HGF biomarker means extended survival when the patient is treated with an effective amount of a combination of a c-met antagonist and a VEGF antagonist.

182. The diagnostic kit of claim 180, further comprising instructions for using the kit to select a c-met antagonist to treat the previously treated renal cell carcinoma patient if a high amount of HGF biomarker is determined.

183. A method of making the diagnostic kit of any one of claims 180-182 comprising combining in a package a pharmaceutical composition comprising a cancer drug and a package insert indicating that the pharmaceutical composition is for treating a patient having cancer based on expression of HGF biomarker.

184. A diagnostic kit comprising one or more reagents for determining expression of an HGF biomarker in a sample from a previously treated hepatocellular carcinoma patient, wherein detection of a high amount of HGF biomarker means extended survival (e.g., PFS and/or OS) when the patient is treated with an effective amount of a c-met antagonist.

185. The diagnostic kit of claim 184 further comprising instructions for using the kit to select a c-met antagonist to treat the previously treated hepatocellular carcinoma patient if a high amount of HGF biomarker is determined.

186. A method of making the diagnostic kit of claim 184 or 185, comprising combining in a package a pharmaceutical composition comprising a cancer medicament and a package insert indicating that the pharmaceutical composition is for treating a patient with cancer based on expression of HGF biomarker.

187. A diagnostic kit comprising one or more reagents for determining expression of an HGF biomarker in a sample from a previously treated sarcoma patient, wherein detection of a high amount of HGF biomarker means extended survival (e.g., PFS and/or OS) when the patient is treated with an effective amount of a c-met antagonist.

188. The diagnostic kit of claim 187 further comprising instructions for using the kit to select a c-met antagonist to treat the previously treated sarcoma patient if a high amount of HGF biomarker is determined.

189. A method of making the diagnostic kit of claim 187 or 188, comprising combining in a package a pharmaceutical composition comprising a cancer medicament and a package insert indicating that the pharmaceutical composition is for treating a patient having cancer based on expression of HGF biomarker.