HK1194974A - Combination treatments comprising c-met antagonists and b-raf antagonists - Google Patents

Description

Combination therapy comprising a C-MET antagonist and a B-RAF antagonist

Cross reference to related applications

Priority claims to U.S. patent application No. 61/536,436 filed on 9/19/2011, U.S. patent application No. 61/551,328 filed on 10/25/2011, U.S. patent application No. 61/598,783 filed on 2/14/2012, and U.S. patent application No. 61/641,139 filed on 5/1/2012 are hereby incorporated herein by reference in their entirety.

Sequence listing

This application contains a sequence listing, has been filed in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. The ASCII copy created on day 28/8/2012 was named p47361wo. txt and was 16,669 bytes in size.

Technical Field

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

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. For example, breast cancer is the second most common form of cancer and is the second cancer killer in american women. 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.

Despite major advances in cancer treatment, improved therapies are still being sought.

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 a disease for the treatment of the disease, optionally in combination with a c-met antagonist and a B-raf antagonist. In particular, the results reported demonstrate that combination treatment with the B-raf antagonist vemurafenib (PLX-4032) and the c-met antagonist results in a statistically significant improvement in tumor regression, including a dramatic improvement in partial response, compared to treatment with vemurafenib alone. c-met expression correlates negatively with sensitivity to treatment with vemurafenib. In addition, patients with B-raf mutant melanoma with higher levels of circulating Hepatocyte Growth Factor (HGF) exhibit substantially shortened progression-free survival and overall survival when treated with a B-raf antagonist relative to patients with lower levels of circulating HGF treated with a B-raf antagonist.

The present invention provides combination therapies for treating pathological conditions such as cancer, wherein a c-met antagonist is combined with a B-raf antagonist, thereby providing significant anti-tumor activity.

In one aspect, methods are provided for treating a cancer patient with an increased likelihood of developing resistance to a B-raf antagonist, comprising administering an effective amount (in combination) of a B-raf antagonist and a c-met antagonist.

In one aspect, methods are provided for increasing and/or restoring sensitivity to a B-raf antagonist comprising administering to a cancer patient an effective amount of a B-raf antagonist and a c-met antagonist.

In one aspect, methods are provided for extending the period of B-raf antagonist sensitivity comprising administering to a cancer patient an effective amount of a B-raf antagonist and a c-met antagonist.

In one aspect, methods are provided for treating a patient having a B-raf resistant (B-raf antagonist resistant) cancer comprising administering an effective amount of a B-raf antagonist and a c-met antagonist.

In one aspect, methods are provided for extending the duration of response of a B-raf antagonist comprising administering an effective amount of a B-raf antagonist and a c-met antagonist.

In one aspect, methods are provided for delaying or preventing the development of an HGF-mediated B-raf resistant cancer comprising administering an effective amount of a B-raf antagonist and a c-met antagonist.

In one aspect, a method for determining c-met biomarker expression is provided, comprising the step of determining whether a patient's cancer expresses c-met biomarker, wherein c-met biomarker expression indicates that the patient is likely to have a B-raf antagonist resistant cancer. In some embodiments, the patient's cancer has been shown to express a B-raf biomarker. In some embodiments, the c-met biomarker is expressed as protein expression and is determined in a sample from the patient using IHC. In some embodiments, a high number of c-met biomarkers (e.g., as determined using c-met IHC or using HGF detection, e.g., ELISA or IHC) indicates that the patient is likely to have a B-raf antagonist resistant cancer. As used herein, "elevated" or "high" c-met refers to the amount of c-met that is associated with the responsiveness of a patient to treatment. In some embodiments, a small amount of a c-met biomarker (e.g., as determined using c-met IHC or using HGF detection, e.g., ELISA or IHC) indicates that the patient is unlikely to have a B-raf antagonist resistant cancer. In some embodiments, high c-met is low, moderate, or high as measured c-met expression, e.g., relative to the c-met staining intensity of control cell pellets a549, H441, H1155, and HEK-293 as described herein. In some embodiments, high c-met is moderate or high assay c-met expression, e.g., relative to the c-met staining intensity of control cell aggregates a549, H441, H1155, and HEK-293 as described herein. As used herein, a "small" amount of c-met refers to an amount of c-met associated with a lack of response to treatment, or in some embodiments, an amount of c-met associated with a poorer response to treatment (e.g., reduced clinical benefit compared to no treatment). In some embodiments, "low" c-met is low or no measured c-met expression, e.g., relative to the c-met staining intensity of control cell aggregates a549, H441, H1155, and HEK-293 as described herein. In some embodiments, a "low" c-met expression is no measured c-met expression, e.g., relative to the c-met staining intensity of control cell aggregates a549, H441, H1155, and HEK-293 as described herein.

In one aspect, a method for determining c-met biomarker expression is provided, comprising the step of determining whether a patient's cancer expresses c-met biomarker, wherein c-met biomarker expression indicates that the patient is likely to form a B-raf resistant cancer. In some embodiments, the patient's cancer has been shown to express a B-raf biomarker. In some embodiments, the c-met biomarker is expressed as protein expression and is determined in a sample from the patient using IHC. In some embodiments, the patient is treated with a B-raf antagonist and a c-met antagonist. In some embodiments, a high number of c-met biomarkers (e.g., as determined using c-met IHC or using HGF detection, e.g., ELISA or IHC) indicates that the patient is likely to have a B-raf antagonist resistant cancer. In some embodiments, high c-met is low, moderate, or high as measured c-met expression, e.g., relative to the c-met staining intensity of control cell pellets a549, H441, H1155, and HEK-293 as described herein. In some embodiments, high c-met is moderate or high assay c-met expression, e.g., relative to the c-met staining intensity of control cell aggregates a549, H441, H1155, and HEK-293 as described herein. In some embodiments, "low" c-met is low or no measured c-met expression, e.g., relative to the c-met staining intensity of control cell aggregates a549, H441, H1155, and HEK-293 as described herein. In some embodiments, a "low" c-met expression is no measured c-met expression, e.g., relative to the c-met staining intensity of control cell aggregates a549, H441, H1155, and HEK-293 as described herein.

In one aspect, a method for determining c-met biomarker expression is provided, comprising the step of determining whether a cancer of a patient expresses c-met biomarker, wherein c-met biomarker expression indicates that the patient is a candidate for treatment with a c-met antagonist and a B-raf antagonist: increasing the sensitivity of the patient's cancer to a B-raf antagonist, restoring the sensitivity of the patient's cancer to a B-raf antagonist, extending the period of time the sensitivity of the patient's cancer to a B-raf antagonist, and/or preventing the patient's cancer from developing HGF-mediated B-raf antagonist resistance. In some embodiments, the patient's cancer has been shown to express a B-raf biomarker. In some embodiments, the c-met biomarker is expressed as protein expression and is determined in a sample from the patient using IHC. In some embodiments, the patient is treated with a B-raf antagonist and a c-met antagonist. In some embodiments, a high number of c-met biomarkers (e.g., as determined using c-met IHC or using HGF detection, e.g., ELISA or IHC) indicates that the patient is likely to have a B-raf antagonist resistant cancer. In some embodiments, high c-met is low, moderate, or high as measured c-met expression, e.g., relative to the c-met staining intensity of control cell pellets a549, H441, H1155, and HEK-293 as described herein. In some embodiments, high c-met is moderate or high assay c-met expression, e.g., relative to the c-met staining intensity of control cell aggregates a549, H441, H1155, and HEK-293 as described herein. In some embodiments, "low" c-met is low or no measured c-met expression, e.g., relative to the c-met staining intensity of control cell aggregates a549, H441, H1155, and HEK-293 as described herein. In some embodiments, a "low" c-met expression is no measured c-met expression, e.g., relative to the c-met staining intensity of control cell aggregates a549, H441, H1155, and HEK-293 as described herein.

In one aspect, a method for selecting a therapy for a patient having a cancer that has been shown to express a B-raf biomarker is provided, comprising determining the expression of a c-met biomarker in a sample from the patient, and selecting a cancer drug based on the level of expression of the biomarker. In some embodiments, the patient is selected for treatment with a c-met antagonist in combination with a B-raf antagonist if the cancer sample expresses a c-met biomarker. In some embodiments, the patient is treated for cancer with a therapeutically effective amount of a c-met antagonist and a B-raf antagonist. In some embodiments, the patient is selected for treatment with a cancer drug other than a c-met antagonist if the cancer sample expresses substantially undetectable levels of the c-met biomarker. In some embodiments, a high number of c-met biomarkers (e.g., as determined using c-met IHC or using HGF detection, e.g., ELISA or IHC) indicates that the patient is likely to have a B-raf antagonist resistant cancer. In some embodiments, high c-met is low, moderate, or high as measured c-met expression, e.g., relative to the c-met staining intensity of control cell pellets a549, H441, H1155, and HEK-293 as described herein. In some embodiments, high c-met is moderate or high assay c-met expression, e.g., relative to the c-met staining intensity of control cell aggregates a549, H441, H1155, and HEK-293 as described herein. In some embodiments, "low" c-met is low or no measured c-met expression, e.g., relative to the c-met staining intensity of control cell aggregates a549, H441, H1155, and HEK-293 as described herein. In some embodiments, a "low" c-met expression is no measured c-met expression, e.g., relative to the c-met staining intensity of control cell aggregates a549, H441, H1155, and HEK-293 as described herein.

In one aspect, methods are provided for identifying a patient as a candidate for treatment with a B-raf antagonist and a c-met antagonist, comprising determining that the patient's cancer expresses a c-met biomarker. In some embodiments, a high number of c-met biomarkers (e.g., as determined using c-met IHC or using HGF detection, e.g., ELISA or IHC) indicates that the patient is likely to have a B-raf antagonist resistant cancer. In some embodiments, high c-met is low, moderate, or high as measured c-met expression, e.g., relative to the c-met staining intensity of control cell pellets a549, H441, H1155, and HEK-293 as described herein. In some embodiments, high c-met is moderate or high assay c-met expression, e.g., relative to the c-met staining intensity of control cell aggregates a549, H441, H1155, and HEK-293 as described herein. In some embodiments, "low" c-met is low or no measured c-met expression, e.g., relative to the c-met staining intensity of control cell aggregates a549, H441, H1155, and HEK-293 as described herein. In some embodiments, a "low" c-met expression is no measured c-met expression, e.g., relative to the c-met staining intensity of control cell aggregates a549, H441, H1155, and HEK-293 as described herein.

In one aspect, methods are provided for identifying a patient as at risk for developing resistance to a B-raf antagonist comprising determining that the patient's cancer expresses a c-met biomarker.

In one aspect, a method of determining the efficacy of a treatment with a B-raf antagonist for treating cancer in a patient is provided, comprising determining the presence of a c-met biomarker and/or a B-raf biomarker in a sample obtained from the patient by immunoassay, ELISA, hybridization assay, PCR, 5' nuclease assay, IHC, and/or RT-PCR, wherein the presence of a c-met biomarker indicates that the B-raf antagonist is therapeutically effective to treat cancer in the subject. In some embodiments, the patient's cancer has been shown to express a B-raf biomarker. In some embodiments, the B-raf biomarker is mutant B-raf. In some embodiments, the mutant B-raf is constitutively activated B-raf. In some embodiments, the mutant B-raf is B-raf V600. In some embodiments, B-raf V600 is B-raf V600E. In some embodiments, the mutant B-raf is one or more of B-raf V600K (GTG > AAG), V600R (GTG > AGG), V600E (GTG > GAA) and/or V600D (GTG > GAT). In some embodiments, mutant B-raf biomarker expression is determined using a method comprising: (a) performing one or more of gene expression profiling, PCR (such as rtPCR or allele-specific PCR), RNA-seq, microarray analysis, SAGE, MassARRAY technique, or FISH on a sample (such as a patient cancer sample); and (B) determining the expression of the mutated B-raf biomarker in the sample. In some embodiments, mutant B-raf biomarker expression is determined using a method comprising: (a) performing PCR on nucleic acids extracted from a patient cancer sample (such as an FFPE-fixed patient cancer sample); and (B) determining the expression of the mutated B-raf biomarker in the sample. In some embodiments, the patient's cancer has been shown to express a c-met biomarker. In some embodiments, Immunohistochemistry (IHC) is used to determine c-met biomarker expression. In some embodiments, c-met expression is measured relative to the c-met staining intensity of control cell pellets, and high c-met expression is low, moderate and strong measured c-met expression relative to cell lines HEK-293, a549 and H441. In some embodiments, c-met expression is measured relative to the c-met staining intensity of control cell pellets, and high c-met expression is intermediate and strong measured c-met expression relative to cell line a549 and cell line H441. In some embodiments, c-met expression is low c-met expression. In some embodiments, c-met expression is determined relative to the c-met staining intensity of control cell pellets, and low c-met expression is no or low determined c-met expression relative to cell line H1155 and cell line HEK-293. In some embodiments, c-met expression is determined relative to the c-met staining intensity of control cell pellets, and low c-met expression is no c-met expression determined relative to cell line H1155. In some embodiments, c-met 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. In some embodiments, c-met biomarker expression is determined using a phosphate-ELISA. In some embodiments, the c-met biomarker is expressed as phospho-met expression. In some embodiments, c-met biomarker expression is determined by determining expression of Hepatocyte Growth Factor (HGF), e.g., using ELISA. In some embodiments, HGF is expressed as autocrine. In some embodiments, HGF is expressed in a tumor or tumor stroma (e.g., using an IHC assay). In some embodiments, expression is determined in the serum of the patient (e.g., using an ELISA assay). In some embodiments, the cancer is melanoma, colorectal cancer, breast cancer, ovarian cancer, or thyroid cancer. In some embodiments, the cancer is melanoma. In some embodiments, the cancer is papillary thyroid carcinoma.

In one aspect, the invention provides a method for determining the prognosis of a melanoma patient comprising determining expression of a c-met biomarker in a sample from the patient, wherein c-met biomarker is HGF and HGF expression is predictive of cancer in the subject. In some embodiments, increased HGF expression is predictive of decreased progression-free survival and/or decreased overall survival, e.g., when a patient is treated with a B-raf inhibitor (e.g., vemurafenib). In some embodiments, HGF expression is determined in the serum of a patient, e.g., using ELISA. In some embodiments, HGF expression in patient serum exceeds a median HGF expression level (such as the median HGF expression level in a population). In some embodiments, HGF expression in the serum of a patient exceeds, for example, about 330 ng/ml. In some embodiments, HGF expression in serum of a patient exceeds about 300ng/ml, 310ng/ml, 320ng/ml, 330ng/ml, 340ng/ml, 350ng/ml, 360ng/ml, 370ng/ml, 380ng/ml, 390ng/ml, 400ng/ml, 420ng/ml, 440ng/ml, 460ng/ml, 480ng/ml, 500ng/ml, or more. In some embodiments, the patient is selected for treatment with an effective amount of a c-met antagonist and a B-raf antagonist. In some embodiments, the patient is treated with an effective amount of a c-met antagonist and a B-raf antagonist. In some embodiments, the melanoma expresses (has shown to express) B-raf V600.

In some embodiments, the patient's cancer has been shown to express a B-raf biomarker. The B-raf biomarker may be a mutant B-raf. The mutant B-raf is constitutively activated B-raf. In some embodiments, the mutant B-raf is B-raf V600. The B-raf V600 can be B-raf V600E. A non-limiting exemplary list of mutants B-raf is: b-raf V600K (GTG > AAG), V600R (GTG > AGG), V600E (GTG > GAA) and/or V600D (GTG > GAT). In some embodiments, a mutant B-raf polypeptide is detected. In some embodiments, a mutant B-raf nucleic acid is detected. "V600E" refers to a mutation at nucleotide 1799 of BRAF resulting in the substitution of glutamine for valine at amino acid 600 of B-raf (T > A). "V600E" was also referred to as "V599E" (1796T > A) under the previous numbering system (Kumar et al, Clin. cancer Res.9: 3362-.

In some embodiments, mutant B-raf biomarker expression is determined using a method comprising: (a) performing one or more of gene expression profiling, PCR (such as rtPCR or allele-specific PCR), RNA-seq, 5' nuclease assay (e.g., TaqMan), microarray analysis, SAGE, MassARRAY technology, or FISH on a sample (such as a patient cancer sample); and (B) determining the expression of the mutant B-raf biomarker in the sample. In some embodiments, mutant B-raf biomarker expression is determined using a method comprising: (a) performing RT-PCR on nucleic acids extracted from a patient cancer sample (such as an FFPE-fixed patient cancer sample); and (B) determining the expression of the mutant B-raf biomarker in the sample. In some embodiments, mutant B-raf biomarker expression is determined using a method comprising: (a) performing PCR on nucleic acids extracted from a patient cancer sample (such as an FFPE-fixed patient cancer sample); and (B) determining the expression of the mutant B-raf biomarker in the sample. In some embodiments, mutant B-raf biomarker expression is determined using a method comprising: (a) hybridizing the first and second oligonucleotides to at least one variant of the B-raf target sequence; wherein the first oligonucleotide is complementary to at least a portion of one or more variants of the target sequence and the second oligonucleotide is complementary to at least a portion of one or more variants of the target sequence and has at least one internal selective nucleotide that is complementary to only one variant of the target sequence; (b) extending the second oligonucleotide with a nucleic acid polymerase; wherein the polymerase is capable of preferentially extending the second oligonucleotide when the selective nucleotide forms a base pair with the target and is substantially less when the selective nucleotide does not form a base pair with the target; and (c) detecting the product of extension of said oligonucleotide, wherein extension indicates the presence of a variant of the target sequence to which the oligonucleotide has a complementary selective nucleotide. In some embodiments, the one or more variants of a B-raf target sequence are wild-type B-raf and V600E B-raf.

In some embodiments, the patient's cancer has been shown to express a c-met biomarker. The c-met biomarker may be a c-met polypeptide. In some embodiments, Immunohistochemistry (IHC) is used to determine c-met biomarker expression. In some embodiments, a high number of c-met biomarkers (e.g., as determined using c-met IHC or using HGF detection, e.g., ELISA or IHC) indicates that the patient is likely to have a B-raf antagonist resistant cancer. In some embodiments, high c-met is low, moderate, or high as measured c-met expression, e.g., relative to the c-met staining intensity of control cell pellets a549, H441, H1155, and HEK-293 as described herein. In some embodiments, high c-met is moderate or high assay c-met expression, e.g., relative to the c-met staining intensity of control cell aggregates a549, H441, H1155, and HEK-293 as described herein. In some embodiments, "low" c-met is low or no measured c-met expression, e.g., relative to the c-met staining intensity of control cell aggregates a549, H441, H1155, and HEK-293 as described herein. In some embodiments, a "low" c-met expression is no measured c-met expression, e.g., relative to the c-met staining intensity of control cell aggregates a549, H441, H1155, and HEK-293 as described herein. In some embodiments, the IHC score is 2. In some embodiments, the IHC score is 3. In some embodiments, the IHC score is 1. In some embodiments, the IHC score is 0. In some embodiments, a high c-met biomarker is expressed as 50% or more of tumor cells having moderate c-met staining intensity, combined moderate/high c-met staining intensity or high c-met staining intensity. In some embodiments, c-met biomarker expression is determined using a phosphate-ELISA. In some embodiments, the c-met biomarker is expressed as phospho-met expression, and in some embodiments, is detected using an anti-phospho-c-met antibody.

The c-met biomarker expression may be nucleic acid expression. In some embodiments, the c-met biomarker is determined in a sample from the patient using PCR (such as rtPCR or allele-specific PCR), RNA-seq, microarray analysis, SAGE, MassARRAY technique, or FISH.

C-met biomarkers can be determined by determining the expression of Hepatocyte Growth Factor (HGF). Thus, in some embodiments, the c-met biomarker is HGF expression, and HGF expression is detected, e.g., in serum (e.g., using ELISA) or by IHC (e.g., tumor or tumor stroma). HGF expression may be autocrine. HGF may be expressed in the tumor stroma. In some embodiments, HGF expression is determined in the serum of the patient. In some embodiments, the HGF expression level exceeds the median HGF expression level. In some embodiments, the median HGF expression level is about 330 pg/mL. In some embodiments, HGF expression in serum is greater than the median HGF expression level. In some embodiments, HGF expression in serum is greater than about 330 pg/ml. In some embodiments, HGF expression in serum of a patient exceeds about 300ng/ml, 310ng/ml, 320ng/ml, 330ng/ml, 340ng/ml, 350ng/ml, 360ng/ml, 370ng/ml, 380ng/ml, 390ng/ml, 400ng/ml, 420ng/ml, 440ng/ml, 460ng/ml, 480ng/ml, 500ng/ml, or more.

The c-met antagonist may be an antagonistic anti-c-met antibody. In some embodiments, the anti-c-met antibody comprises: (a) HVR1-HC comprising the sequence set forth in SEQ ID NO: 1; (b) HVR2-HC comprising the sequence set forth in SEQ ID NO. 2; (c) HVR3-HC comprising the sequence set forth in SEQ ID NO. 3; (d) HVR1-LC comprising the sequence shown in SEQ ID NO. 4; (e) HVR2-LC comprising the sequence shown in SEQ ID NO. 5; and (f) HVR3-LC comprising the sequence shown in SEQ ID NO: 6. In some embodiments, the anti-c-met antibody is monovalent and comprises: (a) a first polypeptide comprising a heavy chain, said polypeptide comprising the sequence set forth in SEQ ID NO 11; (b) a second polypeptide comprising a light chain, the polypeptide comprising the sequence set forth in SEQ ID NO 12; and a third polypeptide comprising an Fc sequence, the polypeptide comprising the sequence set forth in SEQ ID No. 13, wherein the heavy chain variable domain and the light chain variable domain are present as a complex and form a single antigen binding arm, wherein the first and second Fc polypeptides are present in the complex and form an Fc region that increases the stability of the antibody fragment as compared to a Fab molecule comprising the antigen binding arm.

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, GDC-0712, and/or LA 480. In some embodiments, the c-met antagonist is crizotinib. In some embodiments, the c-met antagonist is tivatinib. In some embodiments, the c-met antagonist is GDC-0712.

In some embodiments, the B-RAF antagonist is one or more of sorafenib, PLX4720, PLX-3603, GSK2118436, GDC-0879, N- (3- (5- (4-chlorophenyl) -1H-pyrrolo [2,3-B ] pyridine-3-carbonyl) -2, 4-difluorophenyl) propane-1-sulfonamide, vemurafenib, GSK2118436, RAF265(Novartis), XL281, ARQ736, BAY 73-4506. In still other embodiments, the B-raf antagonist is vemurafenib. In yet other embodiments, the B-raf antagonist is GSK 2118436. The B-raf antagonist may be selective for B-raf V600E.

The B-raf antagonist and the c-met antagonist can be administered simultaneously. The B-raf antagonist and the c-met antagonist may be administered sequentially. In some embodiments, the B-raf antagonist is administered prior to the c-met antagonist. In some embodiments, the c-met antagonist is administered prior to the B-raf antagonist.

In one aspect, methods are provided that include administering at least one additional therapy (such as a cancer drug) to the subject.

The cancer may be melanoma, colorectal cancer, ovarian cancer, breast cancer or papillary thyroid cancer. Other cancers are described herein. In some embodiments, the cancer is melanoma. In some embodiments, the cancer is resistant to a B-raf antagonist. In some embodiments, the patient has been previously treated with a B-raf antagonist. In some embodiments, the patient has not been previously treated with a B-raf antagonist. In some embodiments, the patient is refractory to a B-raf antagonist.

Furthermore, the present invention relates to methods of advertising a cancer drug (e.g., a c-met antagonist), comprising promoting the cancer drug to a target audience for use in treating a cancer patient based on expression of a c-met biomarker (and, in some embodiments, further based on expression of a B-raf biomarker (e.g., a mutant B-raf biomarker)). Promotion may be by any available means. In some embodiments, the promotion is by a package insert accompanying a commercial formulation of a c-met antagonist (such as an anti-c-met antibody). The promotion may also be by a package insert accompanying a commercial formulation of the second drug (when the treatment is a combination therapy using a c-met antagonist and a second drug (e.g., a B-raf antagonist, such as vemurafenib)). The promotion may be by written or oral notification to a physician or health care provider. In some embodiments, the promotion is by a package insert, wherein the package insert provides instructions to receive the c-met antagonist, and in some embodiments, in combination therapy with a second drug, such as a B-raf antagonist (such as vemurafenib). In some embodiments, the promotion is followed by treatment of the patient with a c-met antagonist with or without a second drug (e.g., vemurafenib). In some embodiments, the promotion is followed by treatment of the patient with a second drug with or without treatment with a c-met antagonist. In some embodiments, the package insert indicates that the patient is treated with a c-met antagonist in a case where the cancer sample of the patient expresses a high c-met biomarker. In some embodiments, the package insert indicates that the patient is not being treated with a c-met antagonist in instances where the cancer sample of the patient expresses a low c-met biomarker.

In some aspects, the invention features methods of guiding a cancer (such as melanoma) patient expressing a c-met biomarker by providing instructions to receive treatment with an anti-c-met antibody (e.g., an anti-c-met antibody), and in some embodiments, a second drug (such as a B-raf antagonist, e.g., vemurafenib) to, for example, prolong survival of the patient, reduce the risk of cancer recurrence in the patient, and/or increase the likelihood of survival of the patient. In some embodiments, the treatment comprises administering to a melanoma patient an anti-c-met antibody (e.g., MetMAb) in combination with a B-raf antagonist (such as vemurafenib). In some embodiments, the method further provides instructions to receive at least one chemotherapeutic agent treatment. In certain embodiments, the patient is treated in accordance with the guidance of the guidance method.

The invention also provides a commercial method comprising marketing a c-met antagonist (e.g., an anti-c-met antibody) for use in treating cancer (e.g., melanoma) in a human patient, e.g., for prolonging survival, reducing the likelihood of cancer recurrence in a patient, and/or increasing the likelihood of survival of a patient, wherein the cancer expression in the patient is high (elevated) c-met biomarker expression. In some embodiments, the treatment comprises administering an anti-c-met antibody (e.g., onartuzumab (metmab)) to a cancer patient, and in some embodiments, administering a second drug (e.g., a B-raf antagonist, such as vemurafenib).

In one aspect, the invention provides a diagnostic kit comprising one or more reagents for determining the expression of a c-met biomarker in a sample from a cancer (e.g., melanoma) patient. The diagnostic kit is suitable for use with any of the methods described herein. In some embodiments, the kit further comprises instructions for using the kit to select a c-met drug to treat a melanoma patient. In some embodiments, the treatment comprises administering an anti-c-met antibody (e.g., onartuumab (metmab)), and in some embodiments, a second drug (e.g., a B-raf antagonist, such as vemurafenib) to the cancer patient.

The invention also relates to an article of manufacture comprising a c-met antagonist in a pharmaceutically acceptable carrier and a package insert indicating that the c-met antagonist is for use in treating a cancer patient based on expression of a c-met biomarker. Methods of treatment include any of the methods of treatment disclosed herein.

Brief Description of Drawings

FIG. 1: RTK ligands attenuate kinase inhibition in oncogene addicted cancer cell lines. a, graph depicting results from RTK ligand matrix screening (matrix screen). Cancer cell lines with kinase addiction were treated with increasing concentration ranges of appropriate kinase inhibitors in the presence or absence of RTK ligands (50 ng/mL). b, summary of matrix screening results from 41 kinase-addicted cancer cell lines treated with potent kinase inhibitors and each of six unique RTK ligands. NR represents no rescue, P represents partial rescue, and R represents complete rescue. c, cell viability assay, which demonstrates the diversity of the effects of drug-treated cancer cell lines (72h) of RTK ligands. As indicated, cells were co-treated with 50ng/mL RTK ligand, with three different outcomes observed-no rescue, partial rescue or complete rescue. Error bars represent mean +/-s.e.m.

FIG. 2: survival-promoting pathway reactivation is associated with RTK ligand rescue. a, immunoblot showing the effect of acute RTK ligand treatment (50ng/mL) on sAKT and ERK phosphorylation after kinase inhibition (1 μ M, 2 h). RTK ligand rescue is indicated, grey squares indicate complete rescue, and black squares indicate partial rescue, as determined by initial screening (fig. 1 b). b, cell viability assay, which demonstrated suppression of cell proliferation in three kinase-addicted cancer cell lines after drug treatment (72 h). As indicated, cells were co-treated with 50ng/mL RTK ligand in the presence of the appropriate second kinase inhibitor (0.5 μ M). PD: PD173074, Lap: lapatinib, Criz: crizotinib. Error bars represent mean +/-s.e.m. c, immunoblot showing the effect of acute kinase inhibition (1 μ M) on AKT and ERK phosphate phosphorylation in the presence and absence of RTK ligand (50ng/mL, 2 h). The cell cells were co-treated with a second kinase inhibitor (0.5. mu.M) as appropriate. And Sun: sunitinib, PD: PD173074, PLX: PLX4032, Lap: lapatinib, Erl: erlotinib, Criz: crizotinib.

FIG. 3: HGF promotes lapatinib resistance in HER2 expanded cell lines. a, immunoblot showing suppression of apoptosis (cleaved PAPR) in breast cancer cells amplified with AU565HER2 after treatment with lapatinib (Lap, 1 μ M), HGF (50ng/mL) and crizotinib (Criz, 0.5 μ M) as indicated. b, immunoblot showing pMET and MET expression in a panel of HER2 amplified breast cancer cell lines. HGF rescue was indicated, black squares indicated partial rescue as determined by initial screening (fig. 1 b). c, as indicated, Syto60 staining of breast cancer cells expanded with AU565HER2 treated with either lapatinib (Lap, 1 μ M), HGF (50ng/mL) or crizotinib (Criz, 0.5 μ M). Cells were processed every three days for the indicated data. Mutations represent 3 independent experiments, and values indicate mean +/-s.d. d, immunoblot showing reactivation of pAKT and pERK in two MET positive (AU565, HCC1954) and one MET negative (BT474) HER2 expanded cell lines. Cells were treated with either lapatinib (Lap, 1. mu.M), HGF (50ng/mL) or crizotinib (Criz, 0.5. mu.M) as indicated (2 h). e, representative slides showing MET expression in HER2 positive (3+) breast cancer tissues. f, selection of AU565 cells with higher MET expression after 3 treatments with lapatinib (1 μ M) and HGF (50 ng/mL). g, as indicated, Syto60 staining of HCC1954HER 2-expanded breast cancer cells treated with either lapatinib (5. mu.M) or crizotinib (1. mu.M). Cells were treated twice weekly for the indicated time. Images represent 3 independent experiments and values indicate mean +/-s.d.

FIG. 4: HGF promotes PLX4032 resistance in BRAF mutant melanoma cell lines. a, left, immunoblot showing pMET and MET expression in a panel of BRAF mutant melanoma cell lines. HGF rescue is shown, with gray squares representing complete rescue, black squares representing partial rescue, and white squares representing no rescue. To the right, the association between MET expression and percent rescue as determined by densitometry in PLX4032(1 μ M) treated BRAF mutant melanoma lines in the presence of HGF (72 h). b, immunoblot showing reactivation of pERK in three MET positive (NAE, 624MEL, a375) and two MET negative (M14, Hs693T) BRAF mutant cell lines. Cells were treated with PLX4032(PLX, 1 μ M), HGF (50ng/mL) or crizotinib (Criz, 0.5 μ M) as indicated (2 h). c, as indicated, Syto60 staining of 624MEL BRAF mutant melanoma cells treated with either PLX4032(5 μ M) and/or crizotinib (1 μ M). Cells were treated twice weekly for the indicated time. Images represent 3 independent experiments and values indicate mean +/-s.d. D, tumor growth assay showing the effect of activating MET receptors using 3D6MET agonistic antibodies on the effect of PLX4032 treatment in 928MEL xenografts. As indicated, mice were treated with either control antibody (anti-gp 120), 3D6 (anti-MET agonistic antibody), RG7204(PLX4032) or GDC-0712(MET small molecule inhibitor), 10 per group, for 4 weeks. Error bars represent mean +/-s.e.m.

FIG. 5: a, immunoblot showing activation of PDGFR following stimulation with PDGF (50ng/mL, 30 min). b, summary of screening results from six kinase-addicted cancer cell lines co-treated with cisplatin and six unique RTK ligands. NR represents no rescue. c, cell viability assay, which demonstrates suppression of cell proliferation in three kinase-addicted cancer cell lines after drug treatment (72 h). As indicated, cells were co-treated with 50ng/mL RTK ligand in the presence of the appropriate second kinase inhibitor (0.5 μ M). PD: PD173074, Lap: lapatinib, Criz: crizotinib. Error bars represent mean +/-s.e.m. d, immunoblot, showing the effect of acute kinase inhibition (1 μ M) on AKT and ERK phosphorylation in the presence or absence of RTK ligand (50ng/mL, 2 h). Cells were co-treated with a second kinase inhibitor (0.5 μ M) as appropriate. Criz: crizotinib, PD: PD173074, Lap: lapatinib.

FIG. 6: a, immunoblot showing expression of MET, PDGFR α, IGF1R β, EGFR, HER2, HER3, FGFR1, FGFR2, and FGFR3 in a panel of 41 kinase-addicted cancer cell lines from matrix screening. Indicating RTK ligand rescue; grey squares represent complete rescue, black squares represent partial rescue, white squares represent no rescue, and hatched squares represent ligand-associated kinases. X represents the removed sample, amp represents amplified, and mut represents mutated. Equal loading was determined using β -tubulin. b, correlating RTK expression with the ability of the RTK ligand to rescue kinase-addicted cells from kinase inhibition. Statistical significance was determined using a 2x2 tabulation. The p-value is given.

FIG. 7: a, immunoblot demonstrating activation of the receptor without coupling to downstream survival signals in receptor-expressing, non-RTK ligand-rescued cells. PLX: PLX4032, Lap: lapatinib. b, immunoblotting demonstrating activation of the receptor in cells rescued by the receptor-expressing, non-RTK ligand, coupled with at least one downstream survival signal. PLX: PLX4032, TAE: TAE684, Erl: erlotinib. c, immunoblotting, which demonstrates that RTK ligands fail to activate the appropriate receptor and corresponding downstream survival signals in receptor-expressing, non-RTK ligand-rescued cells. PLX: PLX4032, TAE: TAE684, Erl: erlotinib.

FIG. 8: a, cell viability assay, which demonstrates the suppression of cell proliferation in NSCLC cancer cell lines translocated by H3122EML4-ALK following treatment with TAE684 or crizotinib (72H). Cells were co-treated with 50ng/mL HGF. Error bars represent mean +/-s.e.m. b, immunoblot showing the effect of acute TAE684 or criptotritinib (1 μ M) treatment on AKT and ERK phosphorylation in the presence or absence of HGF (50ng/mL, 2 h). c, as indicated, Syto60 staining of H2228EML4-ALK translocated NSCLC cells treated with TAE684(2 μ M) in the presence or absence of HGF (50 ng/mL). Cells were treated every 3 days for 9 days. d, as indicated, Syto60 staining of H358 EGF-like ligand-driven NSCLC cells treated with Erlotinib (5 μ M) in the presence and absence of HGF (50 ng/mL). Cells were treated every 3 days for 9 days. Images represent 3 independent experiments and values indicate mean +/-s.d.

Fig. 9 shows a, a cell viability assay, demonstrating suppression of cell proliferation in two BRAF mutant cell lines after treatment with PLX4032(72 h). Cells were co-treated with 50ng/mL RTK ligand and cribtinib (Criz, 0.5. mu.M) as indicated. Error bars represent mean +/-s.e.m. b, time course, which shows the sustained survival signal (pAKT and pERK) following HGF (50ng/mL) stimulation in breast cancer cells expanded with AU565HER2 treated with lapatinib (1 μ M).

FIG. 10: rescue results for various RTK ligands in cells with BRAF V600F.

FIG. 11: as indicated, Syto60 cell staining of HCC1954HER 2-expanded breast cancer cells treated with either lapatinib (5 μ M) or crizotinib (1 μ M). Cells were treated twice weekly for the indicated time. Images represent 3 independent experiments and values indicate mean +/-s.d.

FIG. 12: a, immunoblot showing reactivation of ERK in MET positive (NAE, 624MEL, 928MEL, a375) and MET negative (M14, Hs693T) BRAF mutant cell lines. Cells were treated with PLX4032(PLX, 1 μ M), HGF (50ng/mL) or crizotinib (Criz, 0.5 μ M) as indicated (2 h). b, tumor growth assay showing the effect of activating MET using 3D6MET agonistic antibodies on the growth inhibitory activity of PLX4032 in 928MEL and 624MEL xenografts. As indicated, mice (10 per group) were treated with either control antibody (anti-gp 120), 3D6 (anti-MET agonistic antibody), RG7204(PLX4032) or GDC-0712(MET small molecule inhibitor) for 4 weeks and tumor volume was measured at regular times. Error bars represent mean +/-s.e.m. Differences between the 2 groups (═ 0.0008) were determined using two-way ANOVA.

FIG. 13: progression-free survival and overall survival in metastatic melanoma in patients treated with PLX 4032. Patients were stratified into two groups based on their plasma HGF levels (green < median HGF; red > median HGF).

FIG. 14: a, cell viability assay, which demonstrates additive rescue from kinase inhibition by activating both PI3K and MAPK pathways (72 h). AU565 cells were co-treated with lapatinib (1. mu.M) in combination with 10ng/mL NRG1 or FGF. b, cell viability assay, which demonstrates that inhibition of the PI3K pathway is more potent than the MAPK pathway in reversing ligand-induced rescue. Cells were treated with the appropriate kinase inhibitor in the presence of HGF (50 ng/mL). Cells were then treated with either 100nM PI3K inhibitor (BEZ235) or MAPK inhibitor (AZD 6244). Error bars represent mean +/-s.e.m.

FIG. 15: immunoblots showing expression of MET, PDGFR α, IGF1R β, EGFR, HER2, HER3, FGFR1, FGFR2, and FGFR3 in a panel of 41 kinase-addicted cancer cell lines from matrix screening. Indicating an RTK ligand; grey squares represent complete rescue, dark grey squares represent partial rescue, white squares represent no rescue, and black squares represent ligand-associated kinase. X represents the removed sample, amp represents amplified, and mut represents mutated. Equal loading was determined using β -tubulin.

FIG. 16: as indicated, Syto60 staining of H2228EML4-ALK translocated NSCLC cells treated with TAE684(2 μ M) in the presence or absence of HGF (50 ng/mL). Cells were treated every 3 days for 9 days. Images represent 3 independent experiments and values indicate mean +/-s.d.

FIG. 17: a, a graph depicting the analysis of PLX4032 sensitivity to 446 test secreted factors in SK-MEL-28 cells. b, summary of results from analysis of 446 test secreted factors on SK-MEL-28 cells in the presence of 5. mu.M PLX4032(72h) in the presence of 50ng/mL ligand. The figure presents the ligands that formed the initial assay and newly identified soluble factors that rescue SK-MEL-28 cells from PLX4032 sensitivity. Error bars represent mean +/-s.e.m.

FIG. 18: as indicated, Syto60 cells of the a375 and 928MEL BRAF mutant melanoma cell lines treated with either PLX4032(5 μ M) and/or crizotinib (1 μ M) were stained. Cells were treated twice weekly for the indicated time. Images represent 3 independent experiments and values indicate mean +/-s.d.

Fig. 19A and 19B: table summarizing results from 928MEL and 624MEL xenograft studies.

Fig. 20 shows a summary of ELISA results for HGF protein levels in plasma from 126 metastatic melanoma patients (before dosing cycle 1).

FIG. 21: IHC staining of MET in cultured BRAF mutant melanoma cancer cells.

FIG. 22: cell viability assay, which demonstrated suppression of cell proliferation in HCC1954 and AU565 following treatment with crizotinib (72h syto60 assay). Cells were co-treated with 50ng/mL HGF in the presence of crizotinib (0.5. mu.M) (MET TKI) and lapatinib (EGFR/HER2 TKI).

FIG. 23: immunoblots showing the effect of lapatinib (1 μ M) on AKT and ERK phosphorylation in the presence of NRG1(50ng/mL, 2 h). Cells were co-treated with erlotinib (0.5 μ M) as indicated. And Lap: lapatinib, Erl: erlotinib.

FIG. 24: a, bar graph (hatched) showing the frequency distribution of log (hgf) levels superimposed with empirical density (black) from 126 metastatic melanoma patients enrolled in the BRIM2 trial prior to dosing cycle 1 (Kolmogorov-Smirnoff p value of 0.18 off normalcy). b, Progression Free Survival (PFS) and Overall Survival (OS) in metastatic melanoma patients treated with PLX 4032. Patients were stratified into three groups based on their plasma HGF levels. The number of events per group, patients and the time to median event are shown. The hazard ratio and corresponding p-value are calculated using a cox-proportional model of the results over successive results.

FIG. 25: a, structure of GDC-0712. b, cMet and enzyme IC50 of the selected kinase. The cMet potency was determined using poly (Glu, Tyr) phosphorylation by activated cMet kinase domain, detected by ELISA. Data are the geometric mean of multiple assays (n-5). Other kinase assays were performed using the Invitrogen selected screening service according to the Invitrogen standard protocol. All IC50, [ ATP ] were determined at the appropriate value for Km. c, potency and selectivity of GDC-0712 against a selected RTK in a cell-based assay. All assays measure RTK autophosphorylation in the cell lines defined in the table after 2 hours incubation with compound in the presence of 10% FBS. d, kinase selectivity profiling data. GDC-0712 was assayed at 0.1 μ M against a panel of 210 kinases using Invitrogen selected screening services. All kinases with > 50% inhibition are listed. e, graphic representation of GDC-0712 kinase selectivity. The percent inhibition of a particular kinase at 0.1 μ M compound is indicated by the size and color of the circles overlaid on the human kineme.

FIG. 26: according to what is outlined in international patent application WO2007103308A2Protocol preparation GDC-0712. Reagents and conditions: (a) (EtO)₃CH, Meldrum's acid, 80 ℃, 76%; (b) dowtherm, 220 ℃, 45%; (c)3, 4-difluoronitrobenzene, Cs ₂CO₃，DMF，100℃，88％；(d)TFA，70℃，99％；(e)I₂，KOH，DMF，50℃，88％；(f)PMBCl，K₂CO₃，DMF，rt，61％；(g)SnCl₂-2H₂O, EtOH, 65 ℃; (h) CuI, indole-2-carboxylic acid, DMSO, K₂CO₃，115℃，56％；(i)EDCI，HOBt，ipr₂EtNH，DMF，92％；(j)TFA，CH₂Cl₂，rt；(k)CH₃CHO，NaHB(OAc)₃77% (by two steps); (l) TFA, 70 ℃, 73%.

Detailed Description

I. Definition of

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 "melanoma cancer patient", i.e. a patient suffering from or at risk of suffering from one or more symptoms of melanoma. In addition, the patient may be a previously treated melanoma patient.

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.

As used herein, the term "c-met variant" refers to a c-met polypeptide that includes one or more amino acid mutations in the native c-met sequence. Optionally, the one or more amino acid mutations comprise amino acid substitutions.

An "anti-c-met antibody" refers to an antibody that binds c-met with sufficient affinity and specificity. The selected antibody will normally have a sufficiently strong binding affinity for c-met, e.g., the antibody can bind human c-met with a Kd value of between 100nM and 1 pM. Antibody affinity can be determined by, for example, a surface plasmon resonance-based assay (such as a BIAcore assay, as described in PCT application publication No. wo 2005/012359); enzyme-linked immunosorbent assay (ELISA); and competition assays (e.g., RIA). In certain embodiments, anti-c-met antibodies are useful as therapeutic agents in targeting and interfering with diseases or conditions involving c-met activity. Also, the antibody may be subjected to other biological activity assays, for example, to assess its efficacy as a therapeutic agent. Such assays are known in the art and depend on the target antigen of the antibody and the intended use.

"c-met antagonist" (interchangeably referred to as "c-met inhibitor") refers to an agent that interferes with c-met activation or function. In a specific embodiment, the inhibitor of c-met has a binding affinity (dissociation constant) for c-met of about 1,000nM or less. In another embodiment, the inhibitor of c-met has a binding affinity for c-met of about 100nM or less. In another embodiment, the inhibitor of c-met has a binding affinity for c-met of about 50nM or less. In another embodiment, the inhibitor of c-met has a binding affinity for c-met of about 10nM or less. In another embodiment, the inhibitor of c-met has a binding affinity for c-met of about 1nM or less. In a specific embodiment, the inhibitor of c-met is covalently bound to c-met. In a specific embodiment, the c-met inhibitor inhibits c-met signaling with an IC50 of 1,000nM or less. In another embodiment, the c-met inhibitor inhibits c-met signaling with an IC50 of 500nM or less. In another embodiment, the c-met inhibitor inhibits c-met signaling with an IC50 of 50nM or less. In another embodiment, the c-met inhibitor inhibits c-met signaling with an IC50 of 10nM or less. In another embodiment, the c-met inhibitor inhibits c-met signaling with an IC50 of 1nM or less.

"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.

"B-raf activation" refers to the activation or phosphorylation of B-raf kinase. In general, B-raf activation leads to signal transduction.

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

As used herein, the term "B-raf variant" refers to a B-raf polypeptide that includes one or more amino acid mutations in the native B-raf sequence. Optionally, the one or more amino acid mutations comprise amino acid substitutions.

"B-raf antagonist" (interchangeably referred to as "B-raf inhibitor") refers to an agent that interferes with B-raf activation or function. In a specific embodiment, the B-raf inhibitor has a binding affinity (dissociation constant) for B-raf of about 1,000nM or less. In another embodiment, the B-raf inhibitor has a binding affinity for B-raf of about 100nM or less. In another embodiment, the B-raf inhibitor has a binding affinity for B-raf of about 50nM or less. In another embodiment, the B-raf inhibitor has a binding affinity for B-raf of about 10nM or less. In another embodiment, the B-raf inhibitor has a binding affinity for B-raf of about 1nM or less. In a specific embodiment, the B-raf inhibitor inhibits B-raf signaling with an IC50 of 1,000nM or less. In another embodiment, the B-raf inhibitor inhibits B-raf signaling with an IC50 of 500nM or less. In another embodiment, the B-raf inhibitor inhibits B-raf signaling with an IC50 of 50nM or less. In another embodiment, the B-raf inhibitor inhibits B-raf signaling with an IC50 of 10nM or less. In another embodiment, the B-raf inhibitor inhibits B-raf signaling with an IC50 of 1nM or less.

"V600E" refers to a mutation in the BRAF gene that results in the substitution of glutamine for valine at amino acid position 600 of B-Raf. "V600E" was also referred to under the previous numbering system as "V599E" (Kumar et al, Clin. cancer Res.9: 3362-.

"affinity" refers to the strength of the sum of all non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). As used herein, unless otherwise indicated, "binding affinity" refers to an intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be expressed in terms of the dissociation constant (Kd). Affinity can be measured by common methods known in the art, including the methods described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described below.

"Selective" or "greater affinity" is intended to mean that the antagonist binds more tightly (with a lower dissociation constant) to the mutant protein than to the wild-type protein. In some embodiments, the greater affinity or selectivity is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 200, 300, 400, 500, or more fold greater binding. As used herein, the term "B-raf targeting agent" refers to a therapeutic agent that binds to B-raf and inhibits B-raf activation.

As used herein, the term "c-met targeting drug" refers to a therapeutic agent that binds c-met and inhibits c-met activation.

As used herein, the term "constitutive" as applied, for example, to receptor kinase activity refers to sustained signaling activity of the receptor independent of the presence of a ligand or other activating molecule. Depending on the nature of the receptor, all activities may be constitutive, or the activity of the receptor may be further activated by the binding of other molecules (e.g., ligands). Cellular events leading to receptor activation are well known to those of ordinary skill in the art. For example, activation can include oligomerization (e.g., dimerization, trimerization, etc.) to a higher receptor complex. The complex may comprise a single species of protein, i.e., a homomeric complex. Alternatively, the complex may comprise at least two different protein species, i.e. a heteromeric complex. Complex formation can be caused, for example, by overexpression of normal or mutant forms of the receptor on the cell surface. Complex formation can also be caused by specific mutations in the receptor.

The phrase "gene amplification" refers to the process of forming multiple copies of a gene or gene fragment in a particular cell or cell line. The replication region (the segment of amplified DNA) is often referred to as an "amplicon. Generally, the amount of messenger rna (mrna) produced, i.e., the level of gene expression, also increases in proportion to the number of copies made by the particular gene expressed.

A "tyrosine kinase inhibitor" is a molecule that inhibits to some extent the tyrosine kinase activity of tyrosine kinases such as the c-met receptor or B-raf.

A cancer or biological sample that "displays c-met and/or B-raf expression, amplification, or activation" refers to a cancer or biological sample that expresses (including overexpresses) c-met and/or B-raf in a diagnostic test, amplifies the c-met and/or B-raf genes, and/or otherwise indicates c-met and/or B-raf activation or phosphorylation.

A cancer or biological sample that "does not exhibit c-met and/or B-raf expression, amplification, or activation" refers to a cancer or biological sample that does not express (including overexpress) c-met and/or B-raf in a diagnostic test, does not amplify c-met and/or B-raf genes, and/or does not otherwise indicate c-met and/or B-raf activation or phosphorylation.

A cancer or biological sample "exhibiting C-met and/or B-raf activation" refers to a cancer or biological sample that exhibits C-met and/or B-raf activation or phosphorylation in a diagnostic test. Such activation can be determined directly (e.g., by measuring C-met and/or B-raf phosphorylation by ELISA or IHC) or indirectly.

A cancer or biological sample that "does not exhibit c-met and/or B-raf activation" refers to a cancer or biological sample that does not exhibit c-met and/or B-raf activation or phosphorylation in a diagnostic test. Such activation can be determined directly (e.g., by measuring C-met and/or B-raf phosphorylation by ELISA or IHC) or indirectly.

A cancer or biological sample that "exhibits constitutive c-met and/or B-raf activation" refers to a cancer or biological sample that exhibits constitutive c-met and/or B-raf activation or phosphorylation in a diagnostic test. Such activation can be determined directly (e.g., by measuring c-met and/or B-raf phosphorylation by ELISA) or indirectly.

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.

A cancer or biological sample "displaying c-met" refers to a cancer or biological sample having an amplified c-met gene in a diagnostic test.

A cancer or biological sample that "does not exhibit constitutive c-met and/or B-raf activation" refers to a cancer or biological sample that does not exhibit constitutive c-met and/or B-raf activation or phosphorylation in a diagnostic test. Such activation can be determined directly (e.g., by measuring c-met and/or B-raf phosphorylation by ELISA) or indirectly.

"phosphorylation" refers to the addition of one or more phosphate groups to a protein, such as B-raf and/or c-met, or a substrate thereof.

"phospho-ELISA assay" (phosho-ELISA assay) refers herein to an assay that evaluates phosphorylation of one or more c-met and/or B-raf in an enzyme-linked immunosorbent assay (ELISA), using reagents (typically antibodies) that detect phosphorylated c-met and/or B-raf, a substrate, or a downstream signal molecule. Preferably, antibodies are used which detect phosphorylated c-met and/or B-raf. The assay can be performed on a cell lysate, preferably from a fresh or frozen biological sample.

A cancer cell that "overexpresses or amplifies c-met" refers to a cancer cell that has significantly higher levels of c-met protein or gene as compared to a noncancerous cell of the same tissue type. Such overexpression may be caused by gene amplification or increased transcription or translation. C-met overexpression or amplification can be determined in a diagnostic or prognostic assay by assessing elevated levels of c-met protein present on the surface of a cell (e.g., by immunohistochemistry assay; IHC). Alternatively or additionally, the level of c-met encoding nucleic acid in the cell may be measured, for example, by fluorescence in situ hybridization (FISH; see WO98/45479 published 10.1998), Southern blotting or Polymerase Chain Reaction (PCR) techniques such as quantitative real-time PCR (qRT-PCR). In addition to the above assays, a variety of in vivo assays may be utilized by the skilled practitioner. For example, cells within the body of a patient may be exposed to an antibody that is optionally labeled with a detectable label, such as a radioisotope, and binding of the antibody to cells within the body of the patient may be assessed, such as by external scanning for radioactivity or by analyzing a biopsy taken from a patient that has been previously exposed to the antibody.

A cancer cell that "does not overexpress or amplify c-met" refers to a cancer cell that does not have a higher than normal level of c-met protein or gene as compared to a noncancerous cell of the same tissue type.

The term "mutation" as used herein refers to the difference in amino acid or nucleic acid sequence of a particular protein or nucleic acid (gene, RNA) relative to the wild-type protein or nucleic acid, respectively. The mutated protein or nucleic acid may be expressed by or found in one allele (heterozygous) or both alleles (homozygous) of a gene, and it may be somatic or germline. 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).

"inhibit" refers to a decrease or decrease in activity, function, and/or amount as compared to a reference.

Protein "expression" refers to the conversion of information encoded in a gene into messenger rna (mrna) and then into a protein.

As used herein, a sample or cell that "expresses" a protein of interest (such as a c-met receptor) refers to a sample or cell in which mRNA or protein (including fragments thereof) encoding the protein is determined to be present.

A "blocking" antibody or antibody "antagonist" refers to an antibody that inhibits or reduces the biological activity of the antigen to which it binds. Preferred blocking or antagonistic antibodies completely inhibit the biological activity of the antigen.

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

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 refers herein to the promotion of a therapeutic agent, such as an anti-c-met antibody and/or a B-raf antagonist, for an indication, such as melanoma treatment, where such promotion is approved by the Food and Drug Administration (FDA) and is considered to have been 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 medication). Sales specifically include packaging, advertising, and any commercial activity that commercializes a product.

For purposes herein, a "previously treated" cancer patient has received a prior cancer therapy.

Progress has occurred even when antineoplastic agents, such as chemotherapeutic agents, are administered to cancer patients, "refractory" or "refractory" (refractory) cancers.

"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 MetMAb; b-raf antagonists.

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.

An "amount" or "level" of a biomarker associated with a decreased clinical benefit in a patient with cancer (e.g., melanoma) refers to the absence of a detectable biomarker or a low detectable level in a biological sample, wherein the level of the biomarker is associated with a decreased 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 IHC (e.g., of a patient tumor sample) and/or ELISA and/or 5' nuclease assay and/or PCR (e.g., allele-specific PCR).

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 amount of protein in a biological sample, as determined using IHC.

"patient sample" refers to a collection of similar cells 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, circulating tumor cells, serum or plasma, circulating plasma proteins, ascites fluid, 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 melanoma tumor sample.

"effective response" or "responsiveness" of a patient to drug treatment and the like refer to the clinical or therapeutic benefit given to a patient at risk for or having cancer (e.g., melanoma) following administration of a cancer drug. Such benefits include any one or more of the following: extended survival (including overall survival and progression-free survival); results in objective responses (including complete responses or partial responses); or ameliorating signs or symptoms of cancer, etc. In one embodiment, biomarkers are used to identify patients who are expected to have greater Progression Free Survival (PFS) relative to patients not expressing the biomarker at the same level when treated with a drug (e.g., an anti-c-met antibody).

"survival" (survival) means that the patient remains alive and includes overall survival (overall survival) and progression free survival (progress free survival).

"overall survival" refers to patients who remain alive for a period of time, such as 1 year, 5 years, etc., calculated from the time of diagnosis or treatment.

"progression-free survival" refers to a patient that remains alive without progression or worsening of cancer.

By "extended survival" is meant an increase in 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 (such as a chemotherapeutic regimen for erlotinib).

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

"complete response" (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 decreases in response to treatment.

"treatment" and "treatment" (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 are to be prevented from developing or relapsing.

The term "therapeutically effective amount" refers to an amount of a therapeutic agent that treats or prevents a disease or condition in a mammal. In the case of cancer, a therapeutically effective amount of a therapeutic agent can 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.

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 cancer include, but are not limited to, carcinoma, lymphoma, blastoma (including medulloblastoma and retinoblastoma), sarcoma (including liposarcoma and synovial cell sarcoma), neuroendocrine tumor (including carcinoid tumor, gastrinoma and islet cell carcinoma), mesothelioma, schwannoma (including acoustic neuroma), meningioma, adenocarcinoma, melanoma, and leukemia or lymphoid malignancies. More specific examples of such cancers include melanoma, colorectal cancer, thyroid cancer (e.g., papillary thyroid cancer), non-small cell lung cancer (NSCLC), peritoneal cancer, hepatocellular cancer, gastric cancer (gastic or stomach cancer) including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer (liver cancer or hepatoma), bladder cancer, hepatoma (hepatoma), breast cancer (including metastatic breast cancer), colon cancer, rectal cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer (kidney or renal cancer), prostate cancer, vulval cancer, thyroid cancer, anal cancer, penile cancer, testicular cancer, esophageal cancer, biliary tract cancer, and head and neck cancer. In some embodiments, the cancer is melanoma; colorectal cancer; thyroid cancer, such as papillary thyroid cancer; or ovarian cancer.

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.

"chemotherapeutic agent" refers to a chemical compound useful for the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents (alkylating agents), such as thiotepa and cyclophosphamide (cyclophosphamide) (TM)) () (ii) a Alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines (aziridines), such as benzotepa (benzodepa), carboquone (carboquone), metoclopramide (meteredepa), and uretepa (uredepa); ethyleneimines and methylmelamines, including altretamine, triethylenemelamine, and triethylphosphorylAmines (triethylenephosphoramide), triethylenethiophosphoramide (triethylenethiophosphamide), and trimethlamelamine (trimethlomelamine); annonaceous acetogenins (especially bullatacin and bullatacin); delta-9-tetrahydrocannabinol (dronabinol),) (ii) a Beta-lapachone (lapachone); lapachol (lapachol); colchicines (colchicines); betulinic acid (betulinic acid); camptothecin (camptothecin) (including the synthetic analogue topotecan (topotecan) (a)) ) CPT-11 (irinotecan),) Acetyl camptothecin, scopoletin (scopoletin), and 9-aminocamptothecin); bryostatin; callystatin; CC-1065 (including its adozelesin (adozelesin), carvelesin (carzelesin), and bizelesin (bizelesin) synthetic analogs); podophyllotoxin (podophylotoxin); podophyllinic acid (podophyllic acid); teniposide (teniposide); 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 (nitrosgen 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), triamcinolone (trofosfamide), uracil mustard (uracil mustard); nitrosoureas such as carmustine (carmustine), chlorouretocin (chlorozotocin), fotemustine (fotemustine) ne), lomustine (lomustine), nimustine (nimustine) and ranimustine (ranimustine); antibiotics such as enediynes antibiotics (enediynes) (e.g., calicheamicins, especially calicheamicin γ 1I and calicheamicin ω I1 (see, e.g., Nicolaou et al, Angew. chem. int. Ed. Engl. 33:183-186 (1994)); CDP323, an oral α -4 integrin inhibitor; anthracyclines (dynemicin), including dynemicin A; esperamicin), and neocarzinostain (neocarzinostatin) and related chromophorins of chromenes enediynes), aclacinomycin (aclacinomycin), actinomycin (actinomycin), amphomycin (amphomycin), anthranomycin (anthramycin), azaserine (azaserine), bleomycin (eomycin), actinomycin C (acanthomycin), carmycin (carbamycin), carmycin (monocrotamycin), daunomycin (6-erythromycin (monocrotamycin), daunomycin (monocrotamycin), daunomycin (daunomycin), daunomycin (5-6-D), daunomycin (daunomycin), daunomycin (daunom, Doxorubicin (doxorubicin) (includingMorpholino doxorubicin, cyanomorpholino doxorubicin, 2-pyrrol doxorubicin, doxorubicin hydrochloride liposome injection (doxorubicin hydrochloride) ) Liposomal doxorubicin TLC D-99() PEGylated liposomal doxorubicin: () And doxorubicin), epirubicin (epirubicin), esorubicin (esorubicin), idarubicin (idarubicin), marijuomycin (marcellomycin), mitomycins (mitomycins) such as mitomycin C, mycophenolic acid (mycophenolic acid), norramycin (nogalamycin), olivomycin (olivomycin), pelomycin (peplomycin), pofiomycin (potfiromycin), puromycin (puromycin), and ferrirubicin (que)lamycin), rodobicin (rodorubicin), streptonigrin (streptonigrin), streptozocin (streptozocin), tubercidin (tubicidin), ubenimex (ubenimex), abstaine (zinostatin), zorubicin (zorubicin); antimetabolites, such as methotrexate, gemcitabine (gemcitabine) (iii)) Tegafur (tegafur) ((tegafur))) Capecitabine (capecitabine) (iii)) Epothilone (epothilone) and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteroyltriglutamic acid (pteropterin), trimetrexate (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); 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 (defosfamide); dimecorsine (demecolcine); diazaquinone (diaziqutone); elfornitine; ammonium etitanium acetate; etoglut (etoglucid); gallium nitrate; hydroxyurea (hydroxyurea); lentinan (lentinan); lonidamine (lonidamine); maytansinoids (maytansinoids), such as maytansine (maytansine) and ansamitocins (ansamitocins); rice support Guanylhydrazone (mitoguzone); mitoxantrone (mitoxantrone); mopidamol (mopidamol); diamine nitracridine (nitrarine); pentostatin (pentostatin); methionine mustard (phenamett); pirarubicin (pirarubicin); losoxantrone (losoxantrone); 2-ethyl hydrazide (ethylhydrazide); procarbazine (procarbazine);polysaccharide complex (JHS natural products, Eugene, OR); razoxane (rizoxane); rhizomycin (rhizoxin); sisofilan (sizofiran); helical germanium (spirogermanium); tenuazonic acid (tenuazonic acid); triimine quinone (triaziquone); 2,2',2' ' -trichlorotriethylamine; trichothecenes (trichothecenes), especially the T-2 toxin, verrucin A, rorodin A and snake-fish (anguidin); urethane (urethan); dacarbazine (dacarbazine); mannitol mustard (mannomustine); dibromomannitol (mitobronitol); dibromodulcitol (mitolactol); pipobromane (pipobroman); a polycytidysine; cytarabine (arabine) ("Ara-C"); thiotepa (thiotepa); taxols (taxoids), e.g. paclitaxel (paclitaxel) ((R))) Albumin engineered nanoparticle dosage form paclitaxel (ABRAXANE)^TM) And docetaxel (doxetaxel) ((doxetaxel)) ) (ii) a Chlorambucil (chlorambucil); 6-thioguanine (thioguanine); mercaptopurine (mercaptoprine); methotrexate (methotrexate); platinum analogs such as cisplatin (cissplatin), oxaliplatin (oxaliplatin), and carboplatin (carboplatin); vinblastines (vincas), which prevent tubulin polymerization to form microtubules, include vinblastine (vinblastine) (vinblastine)) Vincristine (vincristine) ((vincristine))) Vindesine (vindesine) ((B)),) And vinorelbine (vinorelbine) ((vinorelbine))) (ii) a Etoposide (VP-16); ifosfamide (ifosfamide); mitoxantrone (mitoxantrone); leucovorin (leucovovin); oncostatin (novantrone); edatrexate (edatrexate); daunomycin (daunomycin); aminopterin (aminopterin); ibandronate (ibandronate); topoisomerase inhibitor RFS 2000; difluoromethyl ornithine (DMFO); retinoids, such as tretinoin acid (Retinoic acid), including bexarotene (bexarotene) ((R))) (ii) a Diphosphonates (bisphosphates), such as clodronate (e.g. clodronate)Or) Etidronate (etidronate) ((ii))) NE-58095, zoledronic acid/zoledronate (zoledronic acid/zoledronate) ((R))) Alendronate (alendronate) (II) ) Pamidronate (pamidronate) ((a))) Tilurophonate (tirudronate) ((A) and (B))) Or risedronate (risedronate) ((R))) (ii) a And troxacitabine (a 1, 3-dioxolane nucleoside cytosine analogue); antisense oligonucleotides, particularly antisense oligonucleotides that inhibit gene expression in signaling pathways involved in abnormal cell proliferation, such as, for example, PKC- α, Raf, H-Ras and epidermal growth factor receptor (EGF-R); vaccines, e.g.Vaccines and gene therapy vaccines, e.g.A vaccine,A vaccine anda vaccine; topoisomerase 1 inhibitors (e.g. topoisomerase 1 inhibitors)) (ii) a rmRH (e.g. rmRH)) (ii) a BAY439006 (sorafenib; Bayer); SU-11248 (Pfizer); perifosine (perifosine), COX-2 inhibitors (such as celecoxib (celecoxib) or etoricoxib (etoricoxib)), proteosome inhibitors (such as PS 341); bortezomib (a), (b), (c), (d), () (ii) a CCI-779; tipifarnib (R11577); orafenaib, ABT 510; bcl-2 inhibitors, such as oblimersen sodium (C.)) (ii) a pixantrone; EGFR inhibitors (see definition below); tyrosine kinase inhibitors (see definition below); and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing; and combinations of two or more of the above, such as CHOP (abbreviation for cyclophosphamide, doxorubicin, vincristine and prednisolone combination therapy) and FOLFOX (oxaliplatin) ^TM) Abbreviation for treatment regimen combining 5-FU and folinic acid).

As used herein, a chemotherapeutic agent includes the class of "anti-hormonal agents" or "endocrine therapeutic agents" that act to modulate, reduce, block or inhibit the effects of hormones that promote cancer growth. They may themselves be hormones, including but not limited to: antiestrogens with mixed agonist/antagonist properties including tamoxifen (tamoxifen) (NOLVADEX), 4-hydroxytamoxifen, toremifene (toremifene) ((R))) Idoxifene (idoxifene), droloxifene (droloxifene), raloxifene (raloxifene) ((ii))) Trovaxifene (trioxifene), naloxifene (keoxifene), and Selective Estrogen Receptor Modulators (SERMs), such as SERM 3; pure antiestrogens without agonist properties, such as fulvestrant (fulvestrant) ((r))) And EM800 (such agents may block Estrogen Receptor (ER) dimerization, inhibit DNA binding, increase ER turnover, and/or suppress ER levels); aromatase inhibitors, including steroidal aromatase inhibitors, such as formestane (formestane) and exemestane (exemestane) ((C))) And non-steroidal aromatase inhibitors such as anastrozole (anastrozole) ((R))) Letrozole (letrozole) (iii) ) And aminoglutethimide, and other aromatase inhibitors, including vorozole (vorozole) ((r))) Megestrol acetate (megestrol acetate) (ii)) Fadrozole and 4(5) -imidazole; luteinizing hormone releasing hormone agonists, including leuprolide (leuprolide) ((leuprolide))And) Goserelin (goserelin), buserelin (buserelin) and triptorelin (triptorelin); sex steroids (sex steroids) including pregnanins (progestines) such as megestrol acetate and medroxyprogesterone acetate (medroxyprogesterone), estrogens such as diethylstilbestrol (diethylstilbestrol) and pramlins (premarin), and androgens/retinoids such as fluoxymesterone (fluoroxymesterone), all trans retinoic acid (transretinic acid) and fenretinide (fenretinide); onapristone (onapristone); anti-pregnenones; estrogen receptor down-regulators (ERD); anti-androgens such as flutamide (flutamide), nilutamide (nilutamide), and bicalutamide (bicalutamide); and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing; and combinations of two or more of the foregoing.

Specific examples of chemotherapeutic agents or chemotherapeutic regimens herein include: alkylating agents (e.g., chlorambucil, bendamustine (bendamustine), or cyclophosphamide); nucleoside analogues or antimetabolites (e.g., fludarabine), Fludarabine and Cyclophosphamide (FC); prednisone or prednisolone (prednisolone); combination therapies with alkylating agents include cyclophosphamide, vincristine, prednisolone (CHOP), or cyclophosphamide, vincristine, prednisolone (CVP), and the like.

"target audience" refers to a group or institution of people who receive or intend to receive a particular 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.

An "affinity matured" antibody refers to an antibody that has one or more alterations in one or more hypervariable regions (HVRs) which result in an improved affinity of the antibody for an antigen compared to a parent antibody that does not possess such alterations.

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.

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 IgA₂. The constant domains of heavy chains corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

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.

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 healthcare service 5 th 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.

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 and/or which forms structurally defined loops ("hypervariable loops"). Typically, a native 4 chain antibody comprises 6 HVRs; three in VH (H1, H2, H3) and three in VL (L1, L2, L3). HVRs typically comprise amino acid residues from hypervariable loops and/or from "complementarity determining regions" (CDRs), the latter being of highest sequence variability and/or involved in antigen recognition. Exemplary hypervariable loops are 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)). Exemplary CDRs (CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3) exist at amino acid residues 24-34 of L1, 50-56 of L2, 89-97 of L3, 50-65 of 31-35B, H2 of H1, and 95-102 of H3 (Kabat et al, Sequences of Proteins of Immunological Interest, 5 th edition Public Health service, National Institutes of Health, Bethesda, Md (1991)). In addition to CDR1 in VH, the CDRs generally comprise amino acid residues that form hypervariable loops. CDRs also contain "specificity determining residues", or "SDRs", which are residues that contact the antigen. SDR is contained within a CDR region called a shortened-CDR, or a-CDR. Exemplary a-CDRs (a-CDR-L1, a-CDR-L2, a-CDR-L3, a-CDR-H1, a-CDR-H2, and a-CDR-H3) are present at amino acid residues 31-34 of L1, 50-55 of L2, 89-96 of L3, 50-58 of 31-35B, H2 of H1, and 95-102 of H3 (see Almagro and Fransson, Front. biosci. 13:1619-1633 (2008)). Unless otherwise indicated, HVR residues and other residues (e.g., FR residues) in the variable domains are numbered herein according to Kabat et al, supra. In one embodiment, the c-met antibody herein comprises SEQ ID NO: 1-6 HVRs.

"affinity" refers to the strength of the sum of all non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). As used herein, unless otherwise indicated, "binding affinity" refers to an intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be expressed in terms of the dissociation constant (Kd). Affinity can be measured by common methods known in the art, including the methods described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described below.

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.

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 (e.g., a package or container) comprising at least one reagent (e.g., a drug for treating cancer (e.g., melanoma, colorectal cancer), or a reagent (e.g., an antibody) for specifically detecting a biomarker gene or protein). The article of manufacture is preferably advertised, distributed, or sold in a unit for carrying out the method of the 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.

II cancer medicine

In one aspect, the invention features the use of a c-met antagonist and a B-raf antagonist in a combination therapy for treating a pathological condition (such as cancer) in a subject. In another aspect, the invention relates to 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;

-a B-raf antagonist;

-chemotherapeutic agents and chemotherapeutic regimens;

other drugs in development or approved for the treatment of cancer (e.g. melanoma) or combinations thereof.

Examples of c-met antagonists include, but are not limited to, soluble c-met receptor, soluble HGF variants, aptamers (aptamers) or peptibodies (peptibodies) that bind c-met or HGF, c-met small molecules, anti-c-met antibodies, and anti-HGF antibodies.

In one embodiment, the antagonist of c-met is an antibody, e.g., an antibody directed against or binding to c-met. 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 another embodiment, the anti-c-met antibody is metmab (onartuzumab) or a biologically similar form thereof. MetMAb is disclosed, for example, in WO 2006/015371; jin et al, Cancer Res (2008)68: 4360. In another embodiment, the anti-c-met antibody comprises a heavy chain variable domain comprising one or more of: (a) HVR1-HC comprising sequence GYTFTSYWLH (SEQ ID NO: 1); (b) HVR2-HC 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-HC comprising sequence GYTFTSYWLH (SEQ ID NO: 1); (b) HVR2-HC 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 of 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, an anti-c-met antibody is provided, wherein the antibody comprises a VH in any of the embodiments provided above and a VL in any of the embodiments provided above.

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 or are competitively inhibited by an anti-c-met antibody comprising the VH sequence SEQ ID NO:7 and the VL sequence SEQ ID NO: 8.

In yet another aspect of the present invention, the anti-c-met antibody according to any of the above embodiments 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 (or consists of or consists essentially of): (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, wherein the heavy chain variable domain and the light chain variable domain are present as a complex and form a single antigen binding arm, wherein the first and second Fc polypeptides are present in the complex and form an Fc region that increases the stability of the antibody fragment as compared to a Fab molecule comprising the antigen binding arm. 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, the polypeptide comprising sequence DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:13), wherein the heavy chain variable domain and the light chain variable domain are present as a complex and form a single antigen-binding arm.

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 one aspect, the anti-c-met antibody comprises at least one feature that promotes heterodimerization of Fc sequences within the antibody fragment while minimizing homodimerization. Such features improve the yield and/or purity and/or homogeneity of the immunoglobulin population. In one embodiment, the antibody comprises Fc mutations that constitute "knots" 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 hole mutation may be T366W.

In some embodiments, the c-met antagonist is an anti-Hepatocyte Growth Factor (HGF) antibody, e.g., 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. The small molecule inhibitor is preferably an organic molecule that binds (preferably specifically binds c-met) other than a binding polypeptide or antibody as defined herein.

C-met receptor molecules or fragments thereof that specifically bind HGF can be used in the methods of the invention, e.g., for binding and sequestering HGF protein, thereby preventing signaling thereof. Preferably, the c-met receptor molecule or HGF binding fragment thereof is in soluble form. In some embodiments, the soluble form of the receptor exerts an inhibitory effect on the biological activity of c-met protein by binding HGF, thereby preventing it from binding to its native receptor present on the surface of a target cell. Also included are c-met receptor fusion proteins, examples of which are described below.

The soluble or chimeric c-met receptor protein of the present invention includes a c-met receptor protein that is not immobilized to the cell surface via a transmembrane domain. Thus, soluble forms of c-met receptor (including chimeric receptor proteins) do not contain a transmembrane domain and as such do not generally become bound to the cell membrane of cells expressing the molecule while being capable of binding and inactivating HGF. See, e.g., Kong-Beltran, M et al Cancer Cell (2004)6(1): 75-84.

HGF molecules or fragments thereof that specifically bind c-met and block or reduce c-met activation, thereby preventing it from signaling, can be used in the methods of the invention.

Aptamers are nucleic acid molecules that form a tertiary structure that specifically binds to a target molecule, such as HGF or c-met polypeptide. The generation and therapeutic use of aptamers is well established in the art. See, for example, U.S. Pat. No.5,475,096. HGF aptamers are pegylated modified oligonucleotides that adopt a three-dimensional conformation that enables them to bind extracellular HGF. Additional information regarding aptamers can be found in U.S. patent application publication No. 20060148748.

Peptibodies are peptide sequences linked to amino acid sequences encoding fragments or portions of immunoglobulin molecules. The polypeptide may be derived from a randomized sequence, and specific binding selected by any method, including but not limited to phage display techniques. In a preferred embodiment, the selected polypeptide may be linked to an amino acid sequence encoding an immunoglobulin Fc region. Peptide bodies that specifically bind and antagonize HGF or c-met are also useful in the methods of the invention.

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 antagonist of c-met competes for c-met binding with Hepatocyte Growth Factor (HGF). In some embodiments, the c-met antagonist binds HGF.

In certain embodiments, the c-met antagonist is any one of: GDC-0712, SGX-523, Crizotinib (PF-02341066; 3- [ (1R) -1- (2, 6-dichloro-3-fluorophenyl) ethoxy ] -5- (1-piperidin-4-ylpyrazol-4-yl) pyridin-2-amine; CAS No. 877399-52-5); 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 (GSK1363089), XL (CAS No. 849217-64-7; XL880 is an inhibitor of MET and VEGFR2 and KDR), MGCD-265 (Methgene; MGCD 265 targeting c-MET, VEGFR1, VEGFR2, VEGFR3, Ron and Tie-2 receptors; CAS No.875337-44-3), tivantiniib (ARQ 197; (-) - (3R,4R) -3- (5, 6-dihydro-4H-pyrrolo [3,2,1-ij ] quinolin-1-yl) -4- (1H-indol-3-yl) pyrrolidine-2, 5-dione; see Munchi et al, Mol cancer ther June 20109; 1544; CAS No.905854-02-6), LY-2801653(Lilly), LY2875358(Lilly), MP-470, Ritulomama (AMG102, 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 (XL-184, CAS No. 849217-68-1; carbozantinib is a dual inhibitor of MET and VEGFR 2), MP-470 (SuperGen; is a novel 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-cyclohepta [4,5] cyclohepta [1, 5] cyclohepta, 2-b ] pyridin-7-yl ] sulfonamide; CAS No. 917879-39-1); MK8066(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. In some embodiments, the c-met antagonist is GDC-0712.

B-raf antagonists are known in the art and include, for example, sorafenib, PLX4720, PLX-3603, GSK2118436, GDC-0879, N- (3- (5- (4-chlorophenyl) -1H-pyrrolo [2,3-B ] B]Pyridine-3-carbonyl) -2, 4-difluorophenyl) propane-1-sulfonamide, and those described in WO2007/002325, WO2007/002433, WO2009111278, WO2009111279, WO2009111277, WO2009111280, and U.S. patent No.7,491,829. Other B-raf antagonists include vemurafenib (also known as vemurafenib)And PLX-4032), GSK2118436, RAF265(Novartis), XL281, ARQ736, BAY 73-4506. In some embodiments, the B-raf antagonist is a selective B-raf antagonist. At one endIn some embodiments, the B-raf antagonist is a selective antagonist of B-raf V600. In some embodiments, the B-raf antagonist is a selective antagonist of B-raf V600E. In some embodiments, B-rafV600 is B-raf V600E, B-raf V600K, and/or V600D. In some embodiments, B-rafV600 is B-rafV 600R.

The B-raf antagonist can be a small molecule inhibitor. The small molecule inhibitor is preferably an organic molecule other than a polypeptide or antibody that binds (preferably specifically) B-raf as defined herein. In some embodiments, the B-raf antagonist is a kinase inhibitor. In some embodiments, the B-raf antagonist is an antibody, peptide, peptidomimetic, aptamer (aptamer), or polynucleotide.

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')₂Fv, and scFv fragments, single-armed antibodies, and other fragments described below. For a review of certain antibody fragments, see Hudson et al nat. Med.9: 129-. For reviews on scFv fragments, see, for example, Pluckth ü n, eds (Springer-Verlag, N.Y.) on the Pharmacology of Monoclonal Antibodies, Vol.113, Rosenburg and Moore, (Springer-Verlag, N.Y.), p.269-315 (1994); also visible are WO 93/16185; and U.S. Pat. Nos. 5,571,894 and 5,587,458. With respect to Fab and F (ab') which contain salvage receptor binding epitope residues and have 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., EP404,097; WO 1993/01161; hudson et al, nat. Med.9: 129-; and Hollinger et al, Proc. Natl. Acad. Sci. USA90:6444-6448 (1993). 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). MetMAb 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, Nature332:323-329 (1988); queen et al, Proc.nat' l Acad.Sci.USA86:10029-10033 (1989); U.S. Pat. Nos. 5,821,337,7,527,791,6,982,321, and 7,087,409; kashmiri et al, Methods36:25-34(2005) (SDR (a-CDR) grafting is described); padlan, mol.Immunol.28:489-498(1991) (describes "resurfacing"); dall' Acqua et al, Methods36:43-60(2005) (describing "FR shuffling"); and Osbourn et al, Methods36:61-68(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 fully antibodies with human variable regions in response to an antigenic challenge. 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. US2007/0061900, which describes A 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 Brandlens, Histology and Histopathology,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, Methods in Molecular Biology178:1-37 (ed by O' Brien et al, Human Press, Totowa, NJ,2001), and further described, for example, in McCafferty et al, Nature348: 552-; clackson et al, Nature352: 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.USA101(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 (e.g., B-raf). 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-light chain pairs with different specificities (see Milstein and Cuello, Nature305:537 (1983)), WO93/08829, and Traunecker et al, EMBO J.10:3655(1991)), and "bump-in-hole" 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., US2006/0025576a 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 TIBTECH15:26-32 (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, heterozygous 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. us2003/0157108(Presta, L.); US2004/0093621(Kyowahakko 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.249:533-545 (1986); U.S. patent application No. US2003/0157108A1, Presta, L; and WO2004/056312A1, Adams et al, inter alia, in example 11), and knock-out cell lines such as alpha-1, 6-fucosyltransferase gene FUT8 knock-out CHO cells (see, e.g., Yamane-Ohnuki et al Biotech. Bioeng.87:614 (2004); Kanda, Y. et al, Biotechnol. Bioeng. 94(4):680-688 (2006); and WO 2003/085107).

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 WO2003/011878(Jean-Mairet et al); U.S. Pat. No.6,602,684(Umana et al); and US2005/0123546(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 WO1997/30087(Patel et al); WO1998/58964(Raju, S.); and WO1999/22764(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; WO2004/056312, 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. No.6,194,551, WO99/51642, 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, Nature322:738-40 (1988); U.S. Pat. Nos. 5,648,260; U.S. Pat. Nos. 5,624,821; and WO94/29351, 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, polyvinyl pyrrolidone, poly-1, 3-dioxolane, poly-1, 3, 6-tris Alkanes, ethylene/maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers), and dextran or poly (n-vinyl pyrrolidone) 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. USA102: 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 EP0425235B 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 (modeccin) a chain, α -sarcin (sarcin), aleurites (aleurites fordii) toxic protein, dianthus caryophyllus (dianthin) toxic protein, phytolacca americana (phytolaccai americana) protein (papapi, PAPII and PAP-S), Momordica charantia (mordica charrantia) localized inhibitor, curcin (curcin), crotin (crotin), saponaria officinalis (sapacicularia), leptinolide (phycin) inhibitor, gelonin (gelonin) inhibitor, gelonin (gelonin), gelonin (e) localized protein (S), gelonin (trichomycin (sancin), or a protein (sanmycin), or fragment thereof, or a toxin (sanmycin) or a) inhibitor, or a toxin (sanmycin) or a) or, 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 At ²¹¹、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 I123, or a spin label 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, Science238:1098 (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 Res52: 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.).

Chemotherapeutic agents

The combination therapy of the present invention may additionally comprise treatment with one or more chemotherapeutic agents. Combined administration includes co-administration or simultaneous administration and sequential administration in either order using separate formulations or a single pharmaceutical formulation, wherein preferably all active agents exert their biological activity simultaneously for a period of time. Chemotherapeutic agents, if administered, are generally administered at doses known or optionally reduced with respect to them due to the combined effects of the drugs or negative side effects attributable to their administration. The preparation and dosing schedule for such chemotherapeutic agents can be used according to the manufacturer's instructions or as determined empirically by the practitioner.

Various chemotherapeutic agents that can be combined are disclosed above. In some embodiments, the chemotherapeutic agents to be combined are selected from the group consisting of: paclitaxel (taxoid) (including docetaxel (docetaxel) and paclitaxel (paclitaxel)), vinca (vinca) (such as vinorelbine (vinorelbine) or vinblastine (vinblastine)), platinum compounds (such as carboplatin or cisplatin (cispin)), aromatase inhibitors (such as letrozole (letrozole), anastrozole (anastrozole), or exemestane (exemestane)), antiestrogens (such as fulvestrant (fulvestrant) or tamoxifen (tamoxifen)), etoposide (etoposide), thiotepa (thiotepa), cyclophosphamide (cyclophosphamide), methotrexate (methotrexate), liposomal doxorubicin (lipodoxorubicin), pegylated liposomal doxorubicin (pegylated lipodoxorubicin), pegylated liposomal doxorubicin (COX), gemcitabine (paclitaxel (gemcitabine), e.g., paclitaxel (gemcitabine), or gemcitabine (paclitaxel), e.g., carboplatin inhibitors (capicin), or (cistatin). In some embodiments, the chemotherapeutic agent is temozolomide (temozolomide) and/or dacarbazine (dacarbazine).

Combination therapy

In one aspect, methods are provided for treating a patient having cancer comprising administering an effective (e.g., therapeutically effective) amount of a B-raf antagonist and a c-met antagonist. In some embodiments, the c-met antagonist is an anti-c-met antibody (e.g., MetMAb). In some embodiments, the treatment comprises administering an anti-c-met antibody (e.g., MetMAb) in combination with a B-raf antagonist (such as vemurafenib). In some embodiments, the anti-c-met antibody is metmab (onartuzumab).

In another aspect, methods are provided for treating a cancer patient with an increased likelihood of developing resistance to a B-raf antagonist, comprising administering an effective amount of a B-raf antagonist and a c-met antagonist.

In another aspect, methods for increasing sensitivity to a B-raf antagonist are provided, comprising administering to a cancer patient an effective amount of a B-raf antagonist and a c-met antagonist.

In another aspect, methods for restoring sensitivity to a B-raf antagonist are provided, comprising administering to a cancer patient an effective amount of a B-raf antagonist and a c-met antagonist.

In another aspect, methods for extending the period of B-raf antagonist sensitivity are provided, comprising administering to a cancer patient an effective amount of a B-raf antagonist and a c-met antagonist.

In another aspect, methods for treating a patient with a B-raf resistant cancer comprising administering an effective amount of a B-raf antagonist and a c-met antagonist are provided.

In another aspect, methods for prolonging response to a B-raf antagonist are provided, comprising administering an effective amount of a B-raf antagonist and a c-met antagonist.

In another aspect, methods of delaying or preventing the development of an HGF-mediated B-raf resistant cancer are provided, comprising administering an effective amount of a B-raf antagonist and a c-met antagonist.

In another aspect, a method is provided for treating a patient whose cancer has been shown to express a B-raf biomarker (e.g., a mutant B-raf biomarker), comprising determining whether the patient's cancer expresses a c-met biomarker, and administering a B-raf antagonist and a c-met antagonist if the patient's cancer expresses a c-met biomarker.

In another aspect, there is provided a method for treating a patient whose cancer has been shown to express a B-raf biomarker (e.g., a mutant B-raf biomarker), comprising: (i) monitoring a patient being treated with a B-raf antagonist to determine whether the patient's cancer develops c-met biomarker expression, and (ii) modifying the patient's treatment regimen in the event that the patient's cancer shows expression of a c-met biomarker, including a c-met antagonist in addition to the B-raf antagonist.

In another aspect, there is provided a method for treating a patient whose cancer has been shown to express a B-raf biomarker (e.g., a mutant B-raf biomarker), comprising: (i) monitoring a patient being treated with a B-raf antagonist to determine whether the patient's cancer develops resistance to the antagonist, (ii) testing the patient to determine whether the patient's cancer expresses a c-met biomarker, and (iii) modifying the patient's treatment regimen in the event that the patient's cancer shows expression of a c-met biomarker, including a c-met antagonist in addition to the B-raf antagonist.

The term cancer encompasses a collection of proliferative disorders including, but not limited to, pre-cancerous growths, benign tumors, and malignant tumors. Benign tumors remain localized to the site of origin and have no ability to penetrate, invade, or metastasize to distant sites. Malignant tumors can invade and damage other tissues around them. They also gain the ability to break free from the site of origin and spread to other parts of the body (metastasis), usually via the bloodstream or via the lymphatic system where lymph nodes are located. Primary tumors are classified by the type of tissue in which they occur; metastatic tumors are classified by the tissue type from which the cancer cells are derived. Over time, malignant cells become more and more abnormal and appear less like normal cells. This change in the appearance of cancer cells is called tumor grade (tumor grade), and cancer cells are described as fully-differentiated (low grade), moderately-differentiated (modular-differentiated), poorly-differentiated (pore-differentiated), or undifferentiated (high grade). Fully differentiated cells are fairly normal, appearing as normal cells of similar origin. Undifferentiated cells refer to cells that have become so abnormal that it is no longer possible to determine their origin.

Cancer staging system describes how far the cancer has spread anatomically and attempts are made to place patients with similar prognosis and treatment in the same staging group. Several tests may be performed to aid in staging cancer, including biopsies and certain image detections such as chest x-rays, mammograms, bone scans, CT scans, and MRI scans. Blood tests and clinical assessments are also used to assess the overall health of a patient and to detect whether cancer has spread to certain organs.

To stage cancer, the American Joint Committee on cancer first places the cancer (particularly solid tumors) into a letter classification using the TNM classification system. Cancers are assigned the letters T (tumor size), N (palpable nodules), and/or M (metastasis). T1, T2, T3, and T4 describe increasing primary lesion sizes; n0, N1, N2, N3 indicate node involvement in progressive progression; and M0 and M1 reflect the presence or absence of distant metastasis.

In a second Staging approach, also known as Overall Stage Grouping or Roman numerical Staging, the cancer is divided into stages 0 to IV, incorporating the size of the primary disorder and the presence of node spread and distant metastases. In this system, cancers are grouped into four stages, represented by roman numerals I to IV, or classified as "relapsed". For some cancers, stage 0 is referred to as "in situ" or "Tis," an in situ ductal carcinoma such as breast cancer or in situ lobular carcinoma. High-grade adenomas may also be classified as stage 0. Generally, stage I cancer is a small localized cancer that is usually curable, while stage IV usually represents an inoperable or metastatic cancer. Stage II and III cancers are often locally advanced and/or exhibit involvement of regional lymph nodes. In general, a higher number of stages indicates more severe (extensive) disease, including larger tumor size and/or spread of the cancer to nearby lymph nodes and/or organs adjacent to the primary tumor. These stages are precisely defined, but the definition is different for each cancer and is known to the skilled artisan.

Many cancer registers such as the NCI "watch Epidemiology and End Results Program" (SEER) use generalized staging. Such systems are used for all types of cancer. It divides cancer medical records into five major categories:

"in situ" refers to an early stage cancer that is present only in the cell layer from which it originated.

By "localized" is meant that the cancer is confined to the organ in which it begins, with no evidence of spread.

By "regional" is meant that the cancer has spread beyond the site of origin (primary) to nearby lymph nodes or organs and tissues.

"distal" refers to the spread of the cancer from the primary site to a distant organ or a distant lymph node.

"unknown" is used to describe the situation where there is insufficient information to indicate a stage.

In addition, it is common for cancer to recur months or years after the primary tumor has cleared. Cancers that recur after all visible tumors have been eradicated are referred to as recurrent disease. Recurrent disease in the area of the primary tumor is locally recurrent, while recurrent disease as a metastasis is referred to as distant recurrence.

The tumor may be a solid tumor or a non-solid tumor or a soft tissue tumor. Examples of soft tissue tumors include leukemias (e.g., chronic myelogenous leukemia (chronic myelogenous leukemia), acute myelogenous leukemia (acute myelogenous leukemia), adult acute lymphoblastic leukemia (adult acute lymphoblastic leukemia), acute myelogenous leukemia (acute myelogenous leukemia), mature B-cell acute lymphoblastic leukemia (mass B-cell acute lymphoblastic leukemia), chronic lymphocytic leukemia (chronic lymphocytic leukemia), polymorphonuclear leukemia (polymorphonuclear leukemia), or hairy cell leukemia (hairy cell leukemia)) or lymphomas (e.g., non-Hodgkin's lymphoma, cutaneous T-cell lymphoma, or Hodgkin's disease). Solid tumors include any cancer of body tissues other than the blood, bone marrow, or lymphatic system. Solid tumors can be further divided into those of epithelial cell origin and those of non-epithelial cell origin. Examples of solid epithelial tumors include tumors of the gastrointestinal tract, colon, breast, prostate, lung, kidney, liver, pancreas, ovary, head and neck, oral cavity, stomach, duodenum, small intestine, large intestine, anus, gall bladder, lip (labium), nasopharynx, skin, uterus, male reproductive organs, urinary organs, bladder, and skin. Solid tumors of non-epithelial origin include sarcomas, brain tumors, and bone tumors. In some embodiments, the cancer is melanoma (e.g., B-raf mutant melanoma). In some embodiments, the cancer is colorectal cancer. In some embodiments, the cancer is breast cancer (e.g., Her2 positive breast cancer). In some embodiments, the cancer is papillary thyroid carcinoma. Other examples of cancer are provided in the definition.

In some embodiments, the patient's cancer has been shown to express a B-raf biomarker. In some embodiments, the B-raf biomarker is mutant B-raf. In some embodiments, the mutant B-raf is B-raf V600. In some embodiments, B-raf V600 is B-raf V600E. In some embodiments, the mutant B-raf is constitutively active.

In some embodiments, the patient's cancer has been shown to express a c-met biomarker. Assays for c-met activity and expression are described herein.

In some embodiments, a B-raf resistant cancer means that the cancer patient has progressed on B-raf antagonist therapy (i.e., the patient is "B-raf refractory"), or the patient has progressed within 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or more after completing a B-raf antagonist based therapy regimen.

In some embodiments, a vemurafenib-resistant cancer means that the cancer patient has progressed while receiving vemurafenib-based therapy (i.e., the patient is "vemurafenib refractory"), and the patient has progressed within 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or more after completing a B-raf antagonist-based therapy regimen.

In some embodiments, resistance to, e.g., a B-raf inhibitor develops (acquires) upon treatment with a B-raf antagonist or, e.g., upon exposure to HGF (e.g., HGF-mediated resistance). In other embodiments, the patient (e.g., a patient with a B-raf resistant cancer) has not been previously treated with a B-raf antagonist.

In some embodiments, the patient is currently being treated with a B-raf antagonist. In some embodiments, the patient has been previously treated with a B-raf antagonist. In some embodiments, the patient has not been previously treated with a B-raf antagonist.

In one aspect, a cancer patient is treated with another cancer drug. In some embodiments, the additional cancer drug is a chemotherapeutic agent. In some embodiments, the additional cancer drug is yrervoy. In some embodiments, the additional cancer drug is a cancer immunotherapy agent. In some embodiments, the additional cancer drug is a different (additional) B-raf antagonist. In some embodiments, the additional cancer drug is a different (additional) c-met antagonist.

In one aspect, methods for reducing B-raf phosphorylation in cancer cells are provided, comprising contacting the cells with a B-raf antagonist and a c-met antagonist. In some embodiments, the cell is resistant to a B-raf antagonist (in some embodiments, resistance to a B-raf antagonist has already developed). In some embodiments, the cell expresses a c-met biomarker.

In one aspect, methods are provided for reducing PI 3K-mediated signaling in a cancer cell, comprising contacting the cell with a B-raf antagonist and a c-met antagonist. In some embodiments, the cell is resistant to a B-raf antagonist (in some embodiments, resistance to a B-raf antagonist has already developed). In some embodiments, the cell expresses a c-met biomarker.

In one aspect, methods are provided for reducing PI 3K-mediated signaling in a cancer cell, comprising contacting the cell with a B-raf antagonist and a c-met antagonist. In some embodiments, the cell is resistant to a B-raf antagonist (in some embodiments, resistance to a B-raf antagonist has already developed). In some embodiments, the cell expresses a c-met biomarker.

In one aspect, methods for reducing MAPk-mediated signaling in a cancer cell are provided, comprising contacting the cell with a B-raf antagonist and a c-met antagonist. In some embodiments, the cell is resistant to a B-raf antagonist (in some embodiments, resistance to a B-raf antagonist has already developed). In some embodiments, the cell expresses a c-met biomarker.

In one aspect, methods for reducing AKT-mediated signaling in a cancer cell are provided, comprising contacting the cell with a B-raf antagonist and a c-met antagonist. In some embodiments, the cell is resistant to a B-raf antagonist (in some embodiments, resistance to a B-raf antagonist has already developed). In some embodiments, the cell expresses a c-met biomarker.

In one aspect, methods for reducing ERK-mediated signaling in a cancer cell are provided, comprising contacting the cell with a B-raf antagonist and a c-met antagonist. In some embodiments, the cell is resistant to a B-raf antagonist (in some embodiments, resistance to a B-raf antagonist has already developed). In some embodiments, the cell expresses a c-met biomarker.

In one aspect, methods for reducing B-raf mediated signaling in a cancer cell are provided, comprising contacting the cell with a B-raf antagonist and a c-met antagonist. In some embodiments, the cell is resistant to a B-raf antagonist (in some embodiments, resistance to a B-raf antagonist has already developed). In some embodiments, the cell expresses a c-met biomarker.

In one aspect, methods for reducing growth and/or proliferation, or increasing apoptosis of a cancer cell are provided, comprising contacting the cell with a B-raf antagonist and a c-met antagonist. In some embodiments, the cell is resistant to a B-raf antagonist (in some embodiments, resistance to a B-raf antagonist has already developed). In some embodiments, the cell expresses a c-met biomarker.

In one aspect, methods for increasing apoptosis in cancer cells are provided, comprising contacting the cells with a B-raf antagonist and a c-met antagonist. In some embodiments, the cell is resistant to a B-raf antagonist (in some embodiments, resistance to a B-raf antagonist has already developed). In some embodiments, the cell expresses a c-met biomarker.

The therapeutic agents used in the present invention will be formulated, dosed, and administered in a manner consistent with good medical practice. Factors considered in this context include the particular condition being treated, the particular subject being treated, the clinical condition of the individual patient, the cause of the condition, the site at which the agent is delivered, the method of administration, the schedule of administration, the drug-drug interactions of the agents to be combined, and other factors known to medical practitioners.

Therapeutic formulations are prepared by mixing the active ingredient of the desired purity with optional physiologically acceptable carriers, excipients or stabilizers using standard methods known in the art (Remington's pharmaceutical Sciences (20 th edition), a.gennaro eds., 2000, Lippincott, Williams&Wilkins, philiadelphia, PA). Acceptable carriers include saline, or buffers such as phosphate, citrate, and other organic acids; antioxidants, including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents, such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions, such as sodium; and/or nonionic surfactants, such as TWEEN ^TM、PLURONICS^TMOr PEG.

Optionally but preferably, the formulation contains a pharmaceutically acceptable salt, preferably sodium chloride, and preferably at about physiological concentrations. Optionally, the formulations of the present invention may contain a pharmaceutically acceptable preservative. In some embodiments, the concentration of the preservative ranges from 0.1 to 2.0%, typically v/v. Suitable preservatives include those known in the pharmaceutical arts. Benzyl alcohol, phenol, m-cresol, methyl paraben, and propyl paraben are preferred preservatives. Optionally, the formulation of the present invention may include a pharmaceutically acceptable surfactant at a concentration of 0.005-0.02%.

The formulations herein may also contain more than one active compound necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. Suitably, such molecules are combined in amounts effective for the intended purpose.

The active ingredients may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (for example, hydroxymethylcellulose or gelatin-microcapsules and poly (methylmethacylate) microcapsules, respectively), in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules), or in macroemulsions. Such techniques are disclosed in Remington's pharmaceutical sciences, supra.

The therapeutic agents of the invention are administered to a human patient according to known methods, such as intravenous administration (like bolus injection or by continuous infusion over a period of time), by intramuscular, intraperitoneal, intracerobrospinal, subcutaneous, intraarticular, intrasynovial, intrathecal, oral, topical, or inhalation routes. Ex vivo strategy may also be used for therapeutic applications. Ex vivo strategies involve transfecting or transducing cells obtained from a subject with a polynucleotide encoding a c-met or B-raf antagonist. The transfected or transduced cells are then returned to the subject. The cells can be any of a wide variety of types, including but not limited to hematopoietic cells (e.g., bone marrow cells, macrophages, monocytes, dendritic cells, T cells, or B cells), fibroblasts, epithelial cells, endothelial cells, keratinocytes, or muscle cells.

For example, if the c-met or B-raf antagonist is an antibody, the antibody is administered by any suitable means, including parenteral, subcutaneous, intraperitoneal, intrapulmonary, and intranasal, and, if local immunosuppressive therapy is desired, intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. In addition, it is suitable to administer the antibody by pulse infusion, in particular with decreasing doses of the antibody. Preferably, the dosage is administered by injection, most preferably, the dosage is administered by intravenous or subcutaneous injection, depending in part on whether the administration is short-term or long-term.

In another example, the c-met or B-raf antagonist compound is administered topically, e.g., by direct injection, as permitted at the site of the disorder or tumor, and the injection can be repeated periodically. C-met or B-raf antagonists can also be delivered systemically to a subject or directly to tumor cells, such as a tumor or tumor bed, following surgical removal of the tumor, to prevent or reduce local recurrence or metastasis.

Administration of the therapeutic agents in the combination is typically carried out over a defined period of time (typically minutes, hours, days or weeks, depending on the combination selected). Combination therapy is intended to encompass administration of these therapeutic agents in a sequential manner (that is, where each therapeutic agent is administered at a different time), as well as administration of at least two of these therapeutic agents or therapeutic agents in a substantially simultaneous manner.

The therapeutic agents may be administered by the same route or by different routes. For example, the B-raf and/or c-met antagonist in the combination may be administered by intravenous injection, while the protein kinase inhibitor in the combination may be administered orally. Alternatively, for example, the two therapeutic agents may be administered orally, or the two therapeutic agents may be administered by intravenous injection, depending on the particular therapeutic agent. The order of administration of the therapeutic agents also varies with the particular agent.

Depending on the type and severity of the disease, the initial candidate dose for administration to the patient is about 1 μ g/kg to 100mg/kg (e.g., 0.1-30mg/kg) of each therapeutic agent, e.g., either by one or more separate administrations or by continuous infusion. Typical daily dosages may range from about 1 μ g/kg to about 100mg/kg or more, depending on the factors discussed above. For repeated administrations over several days or longer, depending on the condition, the treatment is continued until the cancer is treated as measured according to the methods described above. However, other dosage regimens may also be useful. In one example, if the c-met or B-raf antagonist is an antibody, the antibody of the invention is administered every two to three weeks at a dose ranging from about 5mg/kg to about 150 mg/kg. If the c-met or B-raf antagonist is an oral small molecule compound, the drug may be administered daily at a dosage ranging from about 25mg/kg to about 50 mg/kg. Furthermore, the oral compounds of the present invention may be administered under traditional high dose intermittent regimens, or using lower and more frequent doses without scheduled interruptions, known as "metronomic therapy". Where an intermittent regimen is used, for example, the drug may be administered daily for 2-3 weeks, followed by 1 week of interruption; alternatively, the drug may be administered daily for 4 weeks followed by 2 weeks of discontinuation, depending on the daily dose and the particular indication. The progress of the therapy of the invention is readily monitored by conventional techniques and assays.

The application encompasses administration of c-met and/or B-raf antagonists by gene therapy. See, e.g., WO96/07321 published 3/14/1996, which focuses on the use of gene therapy to generate intrabodies.

Diagnostic method

In some embodiments, the patient herein is subjected to a diagnostic test, e.g., before and/or during and/or after treatment.

In one aspect, a method for determining c-met biomarker expression is provided, comprising the step of determining whether a patient's cancer expresses c-met biomarker, wherein c-met biomarker expression indicates that the patient is likely to have a B-raf antagonist resistant cancer. In some embodiments, the patient's cancer has been shown to express a B-raf biomarker (such as a mutant B-raf). In some embodiments, the c-met biomarker is expressed as protein expression and is determined in a sample from the patient using IHC. In some embodiments, the patient is treated with a B-raf antagonist and a c-met antagonist.

In one aspect, a method for determining c-met biomarker expression is provided, comprising the step of determining whether a patient's cancer expresses c-met biomarker, wherein c-met biomarker expression indicates that the patient is likely to form a B-raf resistant cancer. In some embodiments, the patient's cancer has been shown to express a B-raf biomarker (such as a mutant B-raf). In some embodiments, the c-met biomarker is expressed as protein expression and is determined in a sample from the patient using IHC. In some embodiments, the patient is treated with a B-raf antagonist and a c-met antagonist.

In one aspect, a method for determining c-met biomarker expression is provided, comprising the step of determining whether a cancer of a patient expresses c-met biomarker, wherein c-met biomarker expression indicates that the patient is a candidate for treatment with a c-met antagonist and a B-raf antagonist: for increasing the sensitivity of a patient's cancer to a B-raf antagonist, restoring the sensitivity of a patient's cancer to a B-raf antagonist, prolonging the period of sensitivity of a patient's cancer to a B-raf antagonist, and/or preventing the development of HGF-mediated B-raf drug resistance in a patient's cancer. In some embodiments, the patient's cancer has been shown to express a B-raf biomarker (such as a mutant B-raf). In some embodiments, the c-met biomarker is expressed as protein expression and is determined in a sample from the patient using IHC. In some embodiments, the patient is treated with a B-raf antagonist and a c-met antagonist.

The invention also relates to a method for selecting a therapy for a patient having a cancer that has been shown to express a B-raf biomarker (e.g., a mutant B-raf biomarker), comprising determining the expression of a c-met biomarker in a sample from the patient, and selecting a cancer drug based on the level of expression of the biomarker. In one embodiment, if the cancer sample expresses a c-met biomarker, the patient is selected for treatment with a c-met antagonist (e.g., an anti-c-met antibody) in combination with a B-raf antagonist. In some embodiments, the patient is treated for cancer with a therapeutically effective amount of a c-met antagonist and a B-raf antagonist. Thus, in some embodiments, if a cancer sample from a patient expresses a c-met biomarker, the patient is selected for treatment with a c-met antagonist (e.g., an anti-c-met antibody) and (after selection) the patient is treated for cancer with a therapeutically effective amount of the c-met antagonist and a B-raf antagonist. In another embodiment, the patient is selected for treatment with a cancer drug other than a c-met antagonist if the cancer sample expresses the c-met biomarker at a substantially undetectable level. In some embodiments, the patient is treated for cancer with a therapeutically effective amount of a cancer drug other than a c-met antagonist (e.g., treatment with a B-raf antagonist). Thus, in some embodiments, if the cancer sample expresses the c-met biomarker at a substantially undetectable level, the patient is selected for treatment with a cancer drug other than a c-met antagonist (e.g., a B-raf antagonist, e.g., vemurafenib), and the patient is treated for cancer (after selection) with a therapeutically effective amount of the c-met antagonist.

In another aspect, the invention provides a method for identifying a patient as a candidate for treatment with a B-raf antagonist and a c-met antagonist, comprising determining that the patient's cancer expresses a c-met biomarker. In some embodiments, the patient has been (previously) treated with a B-raf antagonist. In some embodiments, the patient's cancer is resistant (e.g., acquired resistant) to the B-raf antagonist.

In another aspect, the invention provides a method for identifying a patient as at risk for developing resistance to a B-raf antagonist comprising determining that the patient's cancer expresses a c-met biomarker. In some embodiments, the patient has been (previously) treated with a B-raf antagonist. In some embodiments, the patient is being treated with a B-raf antagonist.

In one aspect, the invention provides a method for determining prognosis of a melanoma patient comprising determining expression of a c-met biomarker in a sample from the patient, wherein the c-met biomarker is HGF and expression of HGF is predictive of cancer in the subject. In some embodiments, elevated HGF expression is predictive of, e.g., shortened progression-free survival and/or shortened overall survival when a patient is treated with a B-raf inhibitor (e.g., vemurafenib). In some embodiments, HGF expression is determined in patient serum, e.g., using ELISA. In some embodiments, HGF expression in patient serum exceeds a median HGF expression level (such as a median HGF expression level in a population). In some embodiments, HGF expression in the serum of a patient exceeds, for example, about 330 ng/ml. In some embodiments, HGF expression in serum of a patient exceeds about 300ng/ml, 310ng/ml, 320ng/ml, 330ng/ml, 340ng/ml, 350ng/ml, 360ng/ml, 370ng/ml, 380ng/ml, 390ng/ml, 400ng/ml, 420ng/ml, 440ng/ml, 460ng/ml, 480ng/ml, 500ng/ml, or more. In some embodiments, the patient is selected for treatment with an effective amount of a c-met antagonist and a B-raf antagonist. In some embodiments, the patient is treated with an effective amount of a c-met antagonist and a B-raf antagonist. HGF expression is detected, for example, by IHC (e.g., to a tumor or tumor stroma).

Methods for detecting c-met expression, activation and amplification are known in the art. In one aspect, c-met biomarker expression is determined using a method comprising the steps of: (a) performing an IHC analysis on a sample (such as a patient cancer sample) with an anti-c-met antibody; and (b) determining c-met biomarker expression in the sample. In some embodiments, the c-met IHC staining intensity is determined relative to a reference value. In some embodiments, a high number of c-met biomarkers (e.g., as determined using c-met IHC or using HGF detection, e.g., ELISA or IHC) indicates that the patient is likely to have a B-raf antagonist resistant cancer. In some embodiments, high c-met is low, moderate, or high as measured c-met expression, e.g., relative to the c-met staining intensity of control cell pellets a549, H441, H1155, and HEK-293 as described herein. In some embodiments, high c-met is moderate or high assay c-met expression, e.g., relative to the c-met staining intensity of control cell aggregates a549, H441, H1155, and HEK-293 as described herein. In some embodiments, "low" c-met is low or no measured c-met expression, e.g., relative to the c-met staining intensity of control cell aggregates a549, H441, H1155, and HEK-293 as described herein. In some embodiments, a "low" c-met expression is no measured c-met expression, e.g., relative to the c-met staining intensity of control cell aggregates a549, H441, H1155, and HEK-293 as described herein. In some embodiments, c-met biomarker expression is determined using a c-met staining intensity scoring protocol disclosed herein (e.g., in table a). In some embodiments, the method further comprises stratifying the patient based on IHC score. In some embodiments, the IHC score is 1. In some embodiments, the IHC score is 0 and c-met expression is observed in the patient's cancer.

In some embodiments, c-met is expressed as a polynucleotide expression. In some embodiments, the polynucleotide is RNA. In some embodiments, the polynucleotide is DNA. In some embodiments, the patient's cancer has shown to express a copy number of c-met (e.g., by FISH analysis) that is greater than 2, greater than 3, greater than 4, greater than 5, greater than 6, greater than 7, greater than 8, or higher. In some embodiments, the c-met copy number is less than 8, less than 7, less than 6, less than 5, less than 4, less than 3.

The invention contemplates that HGF can be detected in accordance with the methods of the invention. As such, in some embodiments, the c-met biomarker is HGF, and in yet other embodiments, HGF is expressed as autocrine expression. In some embodiments, HGF expression is detected in a cancer of a patient. In some embodiments, HGF expression is detected in the tumor stroma of the patient. In some embodiments, HGF expression is detected in patient serum, e.g., using ELISA.

In one aspect, c-met biomarker expression is determined using a method comprising the step of determining c-met biomarker expression in a sample (such as a cancer sample of a patient) wherein the sample of the patient has been subjected to an IHC assay using an anti-c-met antibody. In some embodiments, the c-met IHC staining intensity is determined relative to a reference value. In some embodiments, a high number of c-met biomarkers (e.g., as determined using c-met IHC or using HGF detection, e.g., ELISA or IHC) indicates that the patient is likely to have a B-raf antagonist resistant cancer. In some embodiments, high c-met is low, moderate, or high as measured c-met expression, e.g., relative to the c-met staining intensity of control cell pellets a549, H441, H1155, and HEK-293 as described herein. In some embodiments, high c-met is moderate or high assay c-met expression, e.g., relative to the c-met staining intensity of control cell aggregates a549, H441, H1155, and HEK-293 as described herein. In some embodiments, "low" c-met is low or no measured c-met expression, e.g., relative to the c-met staining intensity of control cell aggregates a549, H441, H1155, and HEK-293 as described herein. In some embodiments, a "low" c-met expression is no measured c-met expression, e.g., relative to the c-met staining intensity of control cell aggregates a549, H441, H1155, and HEK-293 as described herein. In some embodiments, c-met biomarker expression is determined using a c-met staining intensity scoring protocol disclosed herein (e.g., in table a).

In some embodiments, the 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. The use of IHC for the detection of c-met biomarkers is disclosed herein, and a c-met staining intensity scoring protocol, such as in table a, is disclosed herein. Other biomarkers may be detected as noted herein. Exemplary other biomarkers are disclosed herein. In some embodiments of any of the inventions disclosed herein, a high c-met biomarker is expressed as a met diagnostic positive clinical status, as defined in accordance with table a herein. In some embodiments of any of the inventions disclosed herein, a low c-met biomarker is expressed as a met diagnostic negative clinical status, as defined in accordance with table a herein.

In one aspect, c-met biomarker expression is determined using a method comprising the steps of: (a) performing one or more of Western blot, ELISA, phospho-ELISA, IHC using phospho-met antibody, IHC using anti-HGF antibody; and (b) determining expression of c-met biomarkers, including for example HGF, in the sample.

In one aspect, c-met activation is determined using a method comprising the steps of: (a) performing one or more of IHC or phospho-ELISA using phospho-c-met antibodies; and (b) determining the presence of a phospho-c-met biomarker (e.g., phospho-c-met) in the sample.

In one aspect, c-met biomarker expression is determined using a method comprising the step of determining the expression or activity of a c-met downstream signaling pathway molecule, e.g., the expression or activity of AKT (e.g., phospho-AKT), the expression or activity of ERK (e.g., phospho-ERK).

In one aspect, c-met biomarker expression is determined using a method comprising the steps of: (a) performing gene expression profiling, PCR (such as rtPCR or allele specific PCR), 5' nuclease assay (e.g., Taq-man), RNA-seq, microarray analysis, SAGE, MassARRAY techniques, in situ hybridization (e.g., to c-met and/or HGFmRNA), IHC (e.g., to c-met and/or HGF polypeptide), or FISH on a sample (such as a patient cancer sample); and (b) determining c-met biomarker expression in the sample.

Other biomarkers may be detected as noted herein. Exemplary other biomarkers are disclosed herein. In some embodiments, ALK biomarkers are detected. In some embodiments, one or more of the FGF, FGFR, PDGF, and/or PGFR biomarkers are detected.

Methods for detecting B-raf and mutant B-raf are known in the art and are commercially available. See, e.g., Hailat et al, Diagn Mol Pathol.2012Mar; 21(1):1-8. In some embodiments, the V600E mutation (also referred to as V599E (1796T > a)) is detected using a method comprising determining the presence of a single base mutation at position 1799 of the nucleotide in codon 600 of exon 15 (T > a). This mutation can also be derived from the double base mutation TG > AA at nucleotide positions 1799-1800. The double base mutation can also be examined by evaluating position 1799. In some embodiments, the nucleic acid may also be evaluated for the presence of a substitution at position 1800. Other mutations may also be present at codon 600. These include V600K, V600D, and V600R. In some embodiments, the feature of detecting the V600E mutation also enables detection of other codon 600 mutations, such as V600D, V600K and/or V600R. In some embodiments, the probe is also capable of detecting a mutation at codon 601.

The presence of the V600E mutation can be determined by assessing the nucleic acid (e.g., genomic DNA or mRNA) for the presence of a base substitution at position 1799. In some embodiments, the nucleic acid analysis method is one or more of: hybridization using allele-specific oligonucleotides, primer extension, allele-specific ligation, sequencing, or electrophoretic separation techniques, such as single-stranded conformation polymorphism (SSCP) and heteroduplex analysis. Exemplary assays include 5' nuclease assays, allele-specific PCR, template-directed dye-terminator incorporation, molecular beacon allele-specific oligonucleotide assays, single base extension assays, and mutation analysis using real-time pyrosequencing. Analysis of the amplified sequences can be performed using various techniques, such as microchips, fluorescence polarization assays, and matrix-assisted laser desorption ionization (MALDI) mass spectrometry. Two additional methods that can be used are assays based on invasive cleavage of Flap nuclease and methods employing padlock probes.

In some embodiments, the mutant B-raf is B-raf V600E (B-raf polypeptide comprising a V600E mutation (GTG > GAG)). In some embodiments, the mutant B-raf is one or more of B-raf V600K (GTG > AAG), V600R (GTG > AGG), V600E (GTG > GAA) and/or V600D (GTG > GAT). In some embodiments, the mutant B-raf is a mutant at residue V600. In some embodiments, the mutant B-raf polynucleotide comprises a T1799A mutation. In some embodiments, the mutant B-raf polynucleotide comprises a mutation in exon 11 and/or exon 15. In some embodiments, the mutant B-raf expression is detected using a method comprising the steps of: (a) performing one or more of gene expression profiling, PCR (such as rtPCR or allele specific PCR), 5' nuclease assay, IHC, hybridization assay, RNA-seq, microarray analysis, SAGE, MassARRAY technology, or FISH on a sample (such as a patient cancer sample); and (B) determining the expression of the mutated B-raf biomarker in the sample. In some embodiments, the mutant B-raf biomarker expression is detected using a method comprising the steps of: (a) performing PCR on nucleic acids extracted from a patient cancer sample (such as an FFPE-fixed patient cancer sample); and (B) determining the expression of the mutated B-raf biomarker in the sample.

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; 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, tumor cells, serum or plasma, circulating plasma proteins, ascites fluid, 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 patient sample is a formalin-fixed, paraffin-embedded (FFPE) tumor sample (e.g., a melanoma tumor sample or a colorectal cancer tumor sample or a sample of tumor stroma). The sample may be obtained prior to treatment of the patient with a cancer drug, such as an anti-c-met antagonist. 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. In some embodiments, the tumor sample is of lung, skin, lymph node, bone, liver, colon, thyroid, and/or ovary.

Various methods for determining gene amplification, protein, or mRNA expression include, but are not limited to, gene expression profiling, Polymerase Chain Reaction (PCR), including quantitative real-time PCR (qRT-PCR), allele specific PCR, RNA-Seq, FISH, microarray analysis, gene expression Sequential Analysis (SAGE), MassARRAY, proteomics, Immunohistochemistry (IHC), and the like. In some embodiments, protein expression is quantified. Such protein analysis may be performed using IHC, for example on a patient tumor sample.

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

1. Gene expression profiling

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 Biology106:247-283 (1999)); RNase protection assay (Hod, Biotechniques13:852-854 (1992)); and Polymerase Chain Reaction (PCR) (Weis et al, Trends in genetics8:263-264 (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).

2. Polymerase Chain Reaction (PCR) and 5' nuclease assay

One sensitive and flexible quantitative method is PCR, which can be used, for example, to compare mRNA levels in different sample populations, normal and tumor tissues, with or without drug treatment, characterize gene expression patterns, distinguish closely related mrnas, and analyze RNA structure. It should be noted, however, that other nucleic acid amplification protocols (i.e., other than PCR) can also be used in the nucleic acid analysis methods described herein. For example, suitable amplification methods include ligase chain reactions (see, e.g., Wu & Wallace, Genomics4: 560-; strand displacement assays (see, e.g., Walker et al, Proc. Natl. Acad. Sci. USA89: 392-; and several transcription-based amplification systems, including the methods described in U.S. Pat. Nos. 5,437,990, 5,409,818, and 5,399,491; the Transcription Amplification System (TAS) (Kwoh et al, Proc. Natl. Acad. Sci. USA86: 1173-; and self-sustained sequence replication (3SR) (Guatelli et al, Proc. Natl. Acad. Sci. USA87: 1874-. Alternatively, methods for amplifying the probe to detectable levels can be used, such as Q.beta. -replicase amplification (Kramer & Lizardi, Nature339:401-402, 1989; Lomeli et al, Clin. chem.35:1826-1831, 1989). For example, Abramson and Myers in Current Opinion in Biotechnology4:41-47,1993 provide reviews of known amplification methods.

mRNA can be isolated from a starting tissue 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 tumors or tumor cell lines of the breast, lung, colon, prostate, brain, liver, kidney, pancreas, spleen, thymus, testis, ovary, uterus, and the like, as well as pooled DNA from healthy donors. 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, BioTechniques18:42044 (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 example, total RNA from cultured cells can be isolated using qiagen rneasy mini columns. Other commercial RNA isolation The kit comprisesComplete 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, in some embodiments, 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. In other embodiments, a combination reverse transcription-polymerase chain reaction (RT-PCR) reaction may be used, for example, as described in U.S. Pat. nos. 5,310,652; 5,322,770; 5,561,058; 5,641,864, respectively; and 5,693,517. 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.

Can useOr 5' -nuclease assays, such as those described in U.S. Pat. Nos. 5,210,015; 5,487,972; and 5,804,375; and Holland et al, 1988, Proc.Natl.Acad.Sci.USA88: 7276-.PCR typically utilizes the 5' -nuclease activity of Taq or Tth polymerase to hydrolyze hybridization probes bound to their target amplicons, but allowsAny enzyme having equivalent 5' nuclease activity is 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 Taq DNA polymerase non-extendable and labeled with a reporter fluorescent dye and a quencher fluorescent dye. 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. The hybridization probes employed in the assay may be allele-specific probes that distinguish between mutant and wild-type BRAF alleles, for example at the V600E mutation site. Alternatively, the method can be performed using allele-specific primers and labeled probes that bind to the amplification products.

Any method suitable for detecting degradation products can be used in the 5' nuclease assay. Typically, the detection probe is labeled with two fluorescent dyes, one dye being capable of quenching the fluorescence of the other dye. The dyes are attached to the probe, preferably one to the 5' end and the other to an internal site, such that the probe is quenched when in an unhybridized state and such that cleavage of the probe by the 5' to 3 ' exonuclease activity of the DNA polymerase occurs between the two dyes. Amplification results in cleavage of the probe between the dyes, with elimination of quenching and an increase in fluorescence observable from the initially quenched dye. Accumulation of degradation products was monitored by measuring the increase in fluorescence of the reaction. U.S. Pat. Nos. 5,491,063 and 5,571,673 (both incorporated herein by reference) describe alternative methods for detecting probe degradation that occurs with amplification. The 5' -nuclease assay data can be initially expressed as Ct, or cycle threshold (threshold cycle). As discussed above, 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. The ideal internal standard is expressed at a constant level between different tissues and is not affected by experimental treatments. The most frequently used RNAs for gene expression pattern normalization are the mRNA for the housekeeping genes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and P-actin.

In some embodiments, probes that detect V600E (e.g., TTS155-BRAF _ MU) also detect V600D (1799-1800 TG > AT) and V600K (1798-1799 GT > AA). In some embodiments, probes that detect the mutation of V600E also detect K601E (1801A > G) and V600R (1798-1799 GT > AG).

In some embodiments, sequences that are substantially identical to the probe sequence may be used. Sequences that are substantially identical to the probe sequence include those that hybridize to the same complementary sequence as the probe. Thus, in some embodiments, probe sequences for use in the present invention comprise at least 15 contiguous nucleotides, sometimes at least 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 contiguous nucleotides. In some embodiments, the primer has at least 27, 28, 29, or 30 consecutive nucleotides of TTS155-BRAF _ MU or TTS148-BRAF _ WT. In other embodiments, the primers used in the present invention are at least 80% identical to TTS155-BRAF MU or TTS148-BRAF WT, in some embodiments at least 85% identical, and in other embodiments at least 90% or more identical.

Multiple 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., Hailat et al, Diagn Mol pathol.2012mar; 21(1): 1-8; god freeet al, j.molec. diagnostics2:84-91 (2000); Specht et al, am.j.pathol.158:419-29 (2001)). Briefly, in some embodiments, one representative method begins by cutting out an approximately 10 micron thick section of a paraffin-embedded tumor tissue sample. 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.

PCR primers and probes are designed based on the 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 DNA BLAT software or BLAST software, including variations thereof, developed by Kent, W., Genome Res.12(4):656-64 (2002). The subsequent steps follow a fully established PCR primer and probe design approach.

To avoid non-specific signals, it may be important to mask the repeated sequences within the (mask) intron when designing the primers and probes. This can be easily accomplished by using the Repeat Masker program, which is available on-line by the Baylor College of Medicine (Baylor College of Medicine), which screens DNA sequences against libraries of Repeat elements and returns query sequences with Repeat elements masked therein. The masked intron sequences can then be used to design Primer and probe sequences using any commercially available Primer/probe design package or by other means, such as Primer Express (Applied Biosystems); MGBassay-by-design (applied biosystems); primer3(Rozen and Squaletsky (2000) Primer3 is available on the world Wide Web for general users and biologist programmers in "Bioinformatics Methods and Protocols: Methods in Molecular Biology", by Krawetz S, Miseners, Humana Press, Totowa, N.J., pp. 365-.

Factors considered in PCR primer design include primer length, melting temperature (Tm), G/C content, specificity, complementary primer sequence, and 3' -end sequence. In general, optimal PCR primers are typically 17-30 bases long, comprising about 20-80% such as, for example, about 50-60% G + C bases. Typical preferred Tm is between 50 and 80 ℃, e.g., about 50-70 ℃.

For further guidance on PCR Primer and probe Design see, e.g., Dieffenbach et al, "General Concepts for PCR Primer Design" (General concept for PCR Primer Design), in PCR Primer, A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, 1995, p. 133-; innis and Gelfand, "Optimization of PCRs" (Optimization of PCR), in PCR Protocols, A Guide to Methods and Applications, CRC Press, London, 1994, pages 5-11; and Plaster, T.N., Primeselect: Primerand probe design. methods mol. biol.70:520-527(1997), the entire disclosure of which is expressly incorporated herein by reference.

Alternatively, allele-specific amplification of a target nucleic acid can be used to detect the presence or absence of a nucleic acid mutation. Amplification involves the use of allele-specific primers.

In one embodiment, the invention is a method of allele-specific amplification of variants of a target sequence (which exist in the form of several variant sequences), the method comprising: providing a sample, which may contain at least one variant of the target sequence; providing a first oligonucleotide that is at least partially complementary to one or more variants of the target sequence; providing a second oligonucleotide that is at least partially complementary to one or more variants of the target sequence but has at least one internal selective nucleotide that is complementary to only one variant of the target sequence; providing conditions suitable for hybridization of the first and second oligonucleotides to at least one variant of a target sequence; providing conditions suitable for extension of the oligonucleotide by a nucleic acid polymerase; wherein the polymerase is capable of extending the second oligonucleotide when the second oligonucleotide has the complementary internally selective nucleotide for the target sequence variant to which it hybridizes, and substantially less when the second oligonucleotide has a non-complementary internally selective nucleotide for the target sequence variant to which it hybridizes; and the procedure of the hybridization and extension steps is repeated several times.

In some embodiments of the invention, amplification involves a polymerase chain reaction, i.e., repeated cycles of template denaturation, annealing (hybridization) of oligonucleotide primer to template, and nucleic acid polymerase extension of primer. In some embodiments, annealing and extension occur at the same temperature step.

In some embodiments, the amplification reaction involves a hot start protocol. The background for allele-specific amplification is good and the selectivity of allele-specific primers with respect to mismatched targets can be enhanced by using a hot start protocol. Various hot start protocols are known in the art, such as the use of waxes, separation of key reagents from the rest of the reaction mixture (U.S. Pat. No.5,411,876), the use of nucleic acid polymerases that are reversibly inactivated by antibodies (U.S. Pat. No.5,338,671), the use of nucleic acid polymerases that are reversibly inactivated by oligonucleotides designed to specifically bind to their active temperature (U.S. Pat. No.5,840,867), or the use of nucleic acid polymerases with reversible chemical modifications (as described, for example, in U.S. Pat. Nos. 5,677,152 and 5,773,528).

In some embodiments of the invention, the allele-specific amplification assay is a real-time PCR assay. In real-time PCR assays, the measurement of amplification is the "threshold cycle" or Ct value. In the context of allele-specific real-time PCR assays, the difference in Ct values between matched and mismatched templates is a measure of discrimination between alleles or assay selectivity. A larger difference indicates a larger delay in amplification of the mismatched template and thus a larger distinction between alleles. Mismatched templates are often present in much larger amounts than matched templates. For example, in tissue samples, only a small fraction of the cells may be malignant and carry mutations targeted by the allele-specific amplification assay ("matched templates"). Mismatched templates present in normal cells may amplify less efficiently, but the overwhelming number of normal cells will overcome any delay in amplification and eliminate any advantage of mutant templates. To detect rare mutations in the presence of wild-type template, the specificity of the allele-specific amplification assay is crucial. The 4800BRAF V600 mutation test is commercially available and utilizes real-time PCR technology. Each target-specific oligonucleotide probe in the reaction is labeled with a fluorescent dye (which acts as a reporter) and a quencher molecule (which absorbs (quenches) the fluorescent emission from the reporter dye within the intact probe). During each amplification cycle, probes complementary to single-stranded DNA sequences in the amplicon bind and are subsequently cleaved by the 5 'to 3' nuclease activity of Z05DNA polymerase. Once the reporter dye is separated from the quencher by this nuclease activity, fluorescence at a characteristic wavelength can be measured when the reporter dye is excited by a suitable spectrum. Target-specific BRAF wild-type (WT) probe and BRAF V600E mutant probe were labeled using two different reporter dyes. Amplification of two BRAF sequences can be detected independently in one reaction well by measuring fluorescence at two characteristic wavelengths in a dedicated optical channel.

In some embodiments, primers that distinguish between B-raf and V600E B-raf are utilized in accordance with U.S. patent publication No. 2011/0311968.

In some embodiments, a mutant B-raf polynucleotide (e.g., DNA) is detected using a method comprising: (a) performing PCR on nucleic acids (e.g., genomic DNA) extracted from a patient cancer sample (such as an FFPE-fixed patient cancer sample); and (B) determining the expression of the mutant B-raf polynucleotide in the sample. In some embodiments, the mutant B-raf polynucleotide expression is detected using a method comprising: (a) hybridizing the first and second oligonucleotides to at least one variant of the B-raf target sequence; wherein the first oligonucleotide is complementary to at least a portion of one or more variants of the target sequence and the second oligonucleotide is complementary to at least a portion of one or more variants of the target sequence and has at least one internal selective nucleotide that is complementary to only one variant of the target sequence; (b) extending the second oligonucleotide with a nucleic acid polymerase; wherein the polymerase is capable of preferentially extending the second oligonucleotide when the selective nucleotide forms a base pair with the target and is substantially less when the selective nucleotide does not form a base pair with the target; and (c) detecting the product of extension of said oligonucleotide, wherein extension indicates the presence of a variant of the target sequence to which the oligonucleotide has a complementary selective nucleotide. In some embodiments, a mutant B-raf polynucleotide (e.g., DNA) is detected using a method comprising: (a) performing PCR on nucleic acids (e.g., genomic DNA) extracted from a patient cancer sample (e.g., an FFPE-fixed patient cancer sample); and (B) determining the expression of the mutant B-raf polynucleotide in the sample. In some embodiments, a mutant B-raf polynucleotide (e.g., DNA) is detected using a method comprising: (a) isolating DNA (e.g., genomic DNA) from a patient cancer sample (such as an FFPE-fixed patient cancer sample); (b) performing PCR on DNA extracted from a patient cancer sample; and (c) determining the expression of the mutant B-raf polynucleotide in the sample.

In some embodiments, the mutant B-raf polynucleotide expression is detected using a method comprising: (a) isolating DNA (e.g., genomic DNA) from a patient cancer sample (such as an FFPE-fixed patient cancer sample); (b) hybridizing the first and second oligonucleotides to at least one variant of a B-raf target sequence in DNA; wherein the first oligonucleotide is complementary to at least a portion of one or more variants of the target sequence and the second oligonucleotide is complementary to at least a portion of one or more variants of the target sequence and has at least one internal selective nucleotide that is complementary to only one variant of the target sequence; (c) extending the second oligonucleotide with a nucleic acid polymerase; wherein the polymerase is capable of preferentially extending the second oligonucleotide when the selective nucleotide forms a base pair with the target and is substantially less when the selective nucleotide does not form a base pair with the target; and (d) detecting the product of extension of said oligonucleotide, wherein extension indicates the presence of a variant of the target sequence to which the oligonucleotide has a complementary selective nucleotide. In some embodiments, the mutant B-raf polynucleotide expression is detected using a method comprising: (a) hybridizing the first and second oligonucleotides to at least one variant of the B-raf target sequence; wherein the first oligonucleotide is complementary to at least a portion of one or more variants of the target sequence and the second oligonucleotide is complementary to at least a portion of one or more variants of the target sequence and has at least one internal selective nucleotide that is complementary to only one variant of the target sequence; (b) extending the second oligonucleotide with a nucleic acid polymerase; wherein the polymerase is capable of preferentially extending the second oligonucleotide when the selective nucleotide forms a base pair with the target and is substantially less when the selective nucleotide does not form a base pair with the target; and (c) detecting the product of extension of said oligonucleotide, wherein extension indicates the presence of a variant of the target sequence to which the oligonucleotide has a complementary selective nucleotide.

In some embodiments, a mutant B-raf polynucleotide (e.g., DNA) is detected using a method comprising: (a) performing PCR on nucleic acids (e.g., genomic DNA) extracted from a patient cancer sample (such as an FFPE-fixed patient cancer sample); (b) the expression of the mutant B-raf polynucleotides was determined by sequencing the PCR amplified nucleic acids. In some embodiments, sequencing (e.g., Sanger sequencing or pyrosequencing) is used to detect mutant B-raf polynucleotides (e.g., DNA).

3. Other nucleic acid mutation detection methods

Nucleic acid mutations can also be detected by direct sequencing (e.g., at nucleotide 1799 (GTG)>GAA) that results in the presence (or absence) of glutamine in place of valine at amino acid 600 of B-raf). Methods include dideoxy sequencing-based methods and methods such as Pyrosequencing^TMOligonucleotide long products, and the like. Such methods often employ amplification techniques, such as PCR. Another similar method for sequencing does not require the use of complete PCR, but typically only uses primers to extend a fluorescently labeled dideoxyribonucleic acid molecule (ddNTP) that is complementary to the nucleotide to be investigated. The nucleotide at the polymorphic site can be identified by detecting a primer that is extended by one base and fluorescently labeled (e.g., Kobayashi et al, mol. cell. probes,9: 175-.

Amplification products generated using an amplification reaction (e.g., PCR) can also be analyzed by using denaturing gradient gel electrophoresis. Different alleles can be identified based on different sequence-dependent melting properties of DNA in solution and electrophoretic migration (see, e.g., Erlich, ed., PCR Technology, Principles and applications for DNA Amplification, W.H.Freeman and Co, New York,1992, Chapter 7).

In other embodiments, single-strand conformational polymorphism analysis (which identifies base differences by electrophoretic migration changes of single-strand PCR products) can be used to distinguish between alleles of a target sequence, as described, for example, in Orita et al, proc.nat.acad.sci.86,2766-2770 (1989). Amplified PCR products can be generated as described above and heated or otherwise denatured to form single-stranded amplification products. Single-stranded nucleic acids can refold or form secondary structures, which are dependent in part on the base sequence. Different electrophoretic mobilities of single-stranded amplification products can be correlated with sequence differences between the target region's alignment.

Allele-specific amplification or primer extension methods can be used to detect the presence or absence of a mutation (e.g., a nucleic acid mutation). These reactions typically involve the use of primers designed to specifically target the mutant (or wild-type) site via mismatches at the 3 ' end of the primer (e.g., the 3 ' nucleotide or the next to last 3 ' nucleotide). When the polymerase lacks error correcting activity, the presence of a mismatch affects the ability of the polymerase to extend the primer. For example, to detect the V600E mutant sequence using an allele-specific amplification or extension based method, a primer complementary to the mutant a allele at nucleotide 1799 in codon 600 of BRAF is designed such that the 3' terminal nucleotide hybridizes at the mutant position. The presence of a mutant allele can be determined by the ability of the primer to initiate extension. If the 3' end is mismatched, extension is hindered. Thus, for example, if a primer matches a mutant allele nucleotide at the 3' end, the primer will be extended efficiently. Amplification can also be performed using allele-specific primers specific for the sequence from the wild-type sequence of BRAF at position 1799.

Typically, the primer is used in conjunction with a second primer in an amplification reaction. The second primer hybridizes at a site unrelated to the mutation position. Amplification from both primers produces a detectable product indicative of the presence of a particular allelic form. Methods based on allele-specific amplification or extension are described, for example, in WO 93/22456; U.S. Pat. Nos. 5,137,806; U.S. Pat. Nos. 5,595,890; U.S. patent nos. 5,639,611; and U.S. patent No.4,851,331.

Using genotype determination based on allele-specific amplification, identification of the allele requires only detection of the presence or absence of the amplified target sequence. Methods for detecting amplified target sequences are well known in the art. For example, gel electrophoresis and probe hybridization assays are described which are often used to detect the presence of nucleic acids.

In an alternative probe-less method, amplified nucleic acids are detected by monitoring an increase in the total amount of double-stranded DNA in the reaction mixture, as described, for example, in U.S. Pat. nos. 5,994,056; and European patent publication Nos. 487,218 and 512,334. Detection of double-stranded target DNA relies on an increase in fluorescence exhibited by various DNA binding dyes (e.g., SYBR green) when bound to double-stranded DNA.

As will be appreciated by those skilled in the art, allele-specific amplification methods can be performed in reactions employing a variety of allele-specific primers to target a particular allele. Primers for such multiplex applications are typically labeled with distinguishable markers or are selected such that amplification products generated from the alleles are distinguishable by size. Thus, for example, one amplification reaction can be used to identify both the wild type and mutant V600E alleles in a sample by gel analysis of the amplification products.

The allele-specific oligonucleotide primer may be exactly complementary to one of the alleles in the hybridizing region, or may have some mismatch at a position other than the 3' end of the oligonucleotide. For example, the penultimate 3' nucleotide in an allele-specific oligonucleotide may be mismatched. In other embodiments, mismatches may occur at (non-mutant) sites of both allelic sequences.

In some embodiments, allele-specific hybridization is performed in an assay format that utilizes an immobilized target or immobilized probe. Such formats are well known in the art and include, for example, dot blot formats and reverse dot blot assay formats, described in U.S. patent nos. 5,310,893; no.5,451,512; no.5,468,613; and No.5,604,099, each incorporated herein by reference.

4.RNN-Seq

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: wang et al, "RNA-Seq: a Revolitional tool for Transcriptotomics" Nature reviews Genetics10(1):57-63(January 2009); ryan et al BioTechniques45(1):81-94 (2008); and Maher et al, "transaction sequencing to detection gene fusions inventor". Nature458(7234):97-101(January 2009).

5. Microarray

Differential gene expression can also be identified or verified using microarray technology. Thus, the expression profile of breast cancer-associated genes 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 10,000The seed nucleotide sequence is applied to a 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. USA93(2):106-149 (1996)). 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.

6. Continuous analysis of Gene expression (SAGE)

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, Science270: 484-; velculescu et al, Cell88:243-51 (1997).

MassARRAY technology

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.

8. Immunohistochemistry

Immunohistochemistry ("IHC") methods are also suitable for detecting the expression level of the biomarkers of the invention. Immunohistochemical staining of tissue sections has proven to be a reliable method of assessing or detecting 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. Thus, expression is detected using antibodies or antisera, preferably polyclonal antisera, most preferably 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.

There are two commonly used IHC methods: direct and indirect assays. According to the first assay, 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 (primary antibody), which is visualized without further antibody interaction. In a typical indirect assay, unconjugated primary antibody binds to the antigen, and then binds to the primary antibody via a labeled secondary antibody (secondary 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 a radioisotope using 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, fluorescent labels can be conjugated to antibodies using Current Protocols in Immunology, see the techniques disclosed above. 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 coupling 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, 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 coupled 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 mentioned above is conjugated to an anti-hapten antibody. Thus, indirect coupling 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 enzymatic 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.

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.

anti-c-MET Antibodies suitable for IHC are well known in the art and include SP-44(Ventana), DL-21(Upstate), MET4, ab27492(Abcam), PA1-37483(Pierce Antibodies). Those skilled in the art understand that other suitable anti-c-met antibodies can be identified and characterized by comparison to c-met antibodies, for example, using the IHC protocol disclosed herein. Anti-phosphoc-met antibodies are known in the art and include anti-phosphoc-met antibody Y1234/5 from Cell Signalling Technologies. anti-HGF antibodies suitable for use in IHC are well known in the art and include: ab24865(Abcam), H00003082-A01(Abnova), MA1-24767(Thermo Fisher), LS-C123743(Life span). As used herein, it is to be understood that detecting HGF in a sample of a patient tumor encompasses, for example, detecting HGF in the tumor stroma present in the sample of the patient tumor as well as detecting HGF in the tumor cells. Assays for detecting HGF in serum, such as ELISA assays, are commercially available and known in the art. See, e.g., Catenacci et al, Cancer Discovery (2011)1: 573.

In some embodiments, control cell pellets with various staining intensities can be utilized as controls for IHC analysis as well as scoring controls. For example, H441 (strong c-met staining intensity); a549 (moderate c-met staining intensity); h1703 (weak c-met staining intensity), HEK-293(293) (weak c-met staining intensity); and TOV-112D (negative c-met staining intensity) or H1155 (negative c-met staining intensity). In some embodiments, exemplary c-met IHC score strengths may be referenced in fig. 1 and/or 2 herein. In some embodiments, fig. 1 depicts exemplary 0, 1+, 2+, and 3+ c-met IHC scoring strengths, such as the scoring scheme in accordance with table a below.

In some embodiments, the C-met staining intensity criteria may be evaluated according to table a:

TABLE A

In some embodiments, the "clinical Met diagnostic positive" and "clinical Met diagnostic negative" classifications are defined as follows:

positive clinical diagnosis of c-met: IHC score 2 or 3 (as defined in Table A), and

clinical c-met negative diagnosis: IHC score 0 or 1 (as defined in table a).

In some embodiments, the high c-met biomarker of interest is IHC score 2, IHC score 3, or IHC score 2 or 3.

In some embodiments, the low c-met biomarker is IHC score 0, IHC score 1, or IHC score 0 or 1.

9. Proteomics

The term "proteome" is defined as the totality of proteins present in a sample (e.g., a tissue, organism or cell culture) at a point in time. Proteomics includes, among other things, the global variation of protein expression in a study sample (also referred to as "expression proteomics"). Proteomics typically includes the following steps: (1) separating the various proteins in the sample by two-dimensional gel electrophoresis (2-D PAGE); (2) identifying the various proteins recovered from the gel, for example by mass spectrometry or N-terminal sequencing; and (3) analyzing the data using bioinformatics. Proteomic methods are useful supplements to other methods of gene expression profiling, either alone or in combination with other methods, for detecting the products of the diagnostic markers of the invention.

10. Gene amplification

Detection of c-met gene amplification is accomplished using certain techniques known to those skilled in the art. For example, comparative genomic hybridization can be used to generate a map of DNA sequence copy number as a function of chromosomal location. See, for example, Kallioniemi et al (1992) Science258: 818-. Amplification of the c-met gene can also be detected, for example, by Southern hybridization (using a probe specific for the c-met gene) or by real-time quantitative PCR.

In certain embodiments, detection of amplification of c-met gene is achieved by directly evaluating copy number of c-met gene, for example, by using a probe that hybridizes to c-met gene. For example, a FISH assay can be performed. In certain embodiments, detecting amplification of the c-met gene is achieved by indirectly assessing the copy number of the c-met gene, for example by assessing the copy number of a chromosomal region located outside of but co-amplified with the c-met gene. Biomarker expression may also be assessed using in vivo diagnostic assays, for example by administering a molecule (such as an antibody) that binds to the molecule to be detected and is labeled with a detectable label (e.g., a radioisotope), and then externally scanning the patient to locate the label.

11. Other exemplary methods

Biomarkers can be detected by a variety of immunoassay methods (including IHC described herein, e.g., see above). For a review of the immunological and immunoassay protocols, see Basic and clinical Immunology (Stits & Terr eds.,7th ed.1991). Furthermore, the immunoassays of the present invention can be carried out in any of several configurations, for an extensive review of which see EnzymeImmunoassay (Maggio, ed.,1980) and Harlow & Lane, supra. For an overview of general immunoassays see also Methods in Cell Biology: Antibodies in Cell Biology, volume37(Asai, ed.1993); basic and Clinical Immunology (Stits & Ten, eds.,7th ed.1991).

Commonly used assays include non-competitive assays (e.g., sandwich/sandwich assays) and competitive assays. Typically, assays such as ELISA assays may be used. ELISA assays are known in the art, for example, for the determination of various tissues and samples, including plasma or serum. An ELISA assay for determining HGF in serum is exemplified herein. anti-HGF antibodies suitable for use in ELISA are known in the art.

A wide range of immunoassay techniques using such assay formats are available, see, for example, U.S. Pat. nos. 4,016,043, 4,424,279, and 4,018,653. These include both non-competitive types of single-point and two-point or "sandwich/sandwich" assays as well as traditional competitive binding assays. These assays also include direct binding of labeled antibodies to the target biomarkers. Sandwich/sandwich assays are commonly used assays. There are many variations of the sandwich/sandwich assay technique. For example, in a typical forward assay, an unlabeled antibody is immobilized on a solid substrate and the sample to be tested is contacted with the bound molecule. After a suitable period of incubation, i.e., a period of time sufficient to allow formation of an antibody-antigen complex, a second antibody specific for the antigen, labeled with a reporter molecule capable of producing a detectable signal, is then added and incubated for a period of time sufficient to allow formation of another complex of antibody-antigen-labeled antibody. Any unreacted material is washed away and the presence of the antigen is determined by observing the signal generated by the reporter molecule. The results may be qualitative (by simple observation of a visual signal) or may be quantitative (by comparison to a control sample containing a known amount of biomarker).

Variations on the forward assay include a simultaneous assay (simultaneous assay), in which both the sample and the labeled antibody are added to the bound antibody at the same time. These techniques are well known to those skilled in the art and include any minor variations that would be readily apparent. In a typical forward sandwich assay, a first antibody specific for the biomarker is either covalently or passively bound to a solid surface. The solid surface may be glass or a polymer, the most commonly used polymers being cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene. The solid support may take the form of a tube, bead, microplate tray, or any other surface suitable for conducting an immunoassay. Binding protocols are well known in the art and generally involve cross-linking, covalent bonding, or physical adsorption, washing the polymer-antibody complex, and preparing it for testing. An aliquot of the test sample is then added to the solid phase complex and incubated for a sufficient period of time (e.g., 2-40 minutes or overnight, if more convenient) under suitable conditions (e.g., from room temperature to 40 ℃, such as between 25 ℃ and 32 ℃ inclusive) that are suitable or sufficient to allow binding of any subunits present in the antibody. After the incubation period, the antibody subunit solid phase is washed, dried, and incubated with a second antibody specific for a portion of the biomarker. The second antibody is linked to a reporter molecule that indicates binding of the second antibody to the molecular marker.

An alternative method comprises: target biomarkers in a sample are immobilized and the immobilized target is then exposed to specific antibodies, which may or may not be labeled with a reporter molecule. Depending on the amount of target and the strength of the reporter molecule signal, the bound target can be made detectable by direct labeling with the antibody. Alternatively, a labeled second antibody specific for the first antibody is exposed to the target-first antibody complex to form a target-first antibody-second antibody ternary complex. The complex is detected by a signal emitted by a labeled reporter.

In the case of enzyme immunoassays, the enzyme is conjugated to a second antibody, typically by glutaraldehyde or periodate. However, it will be readily appreciated that there are a wide variety of different coupling techniques, which are readily available to the skilled person. Commonly used enzymes include horseradish peroxidase, glucose oxidase, beta-galactosidase, and alkaline phosphatase, and other enzymes are disclosed herein. The substrate used with a particular enzyme is generally selected to produce a detectable color change upon hydrolysis by the corresponding enzyme. Examples of suitable enzymes include alkaline phosphatase and peroxidase. It is also possible to use a fluorogenic substrate that produces a fluorescent product instead of the chromogenic substrate noted above. In all cases, an enzyme-labeled antibody is added to the first antibody-molecular marker complex, allowed to bind, and then the excess reagent is washed away. A solution containing a suitable substrate is then added to the antibody-antigen-antibody complex. The substrate will react with the enzyme linked to the second antibody, giving a qualitative visual signal, which can be further quantified (usually by spectrophotometry) to give an indication of the amount of biomarker present in the sample. Alternatively, fluorescent compounds (such as fluorescein and rhodamine) can be chemically coupled to antibodies without altering the binding capacity of the antibodies. Upon excitation by illumination with light of a particular wavelength, the fluorochrome-labeled antibody absorbs light energy, inducing an excited state in the molecule, followed by emission of light, the characteristic color of which is visually detectable with an optical microscope. As in the EIA, a fluorescently labeled antibody is allowed to bind to the first antibody-molecular marker complex. After washing away unbound reagents, the remaining ternary complex is then exposed to light of the appropriate wavelength, and the observed fluorescence indicates the presence of the molecular marker of interest. Immunofluorescence and EIA techniques are both well established in the art and are discussed herein.

Other detection techniques (e.g., MALDI) can be used to directly detect the presence of a biomarker (e.g., mutant Braf) in a sample.

V. product

In another embodiment of the invention, an article of manufacture for treating cancer (such as melanoma or papillary thyroid carcinoma) is provided. 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 articles of manufacture of the invention also include information, e.g., in the form of a package insert, indicating that the composition is for use in treating cancer based on the expression levels of the biomarkers 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. Methods include any of the therapeutic and diagnostic methods herein.

In accordance with one embodiment of the present invention, an article of manufacture is provided comprising a c-met antagonist (e.g., an anti-c-met antibody) in a pharmaceutically acceptable carrier and a package insert indicating that the c-met antagonist is for use in treating a cancer (such as melanoma) patient based on expression of a c-met biomarker, packaged together. In some embodiments, the treatment is in combination with a B-raf antagonist. In some embodiments, the package insert indicates that the c-met antagonist is used in combination with a B-raf antagonist to treat a patient having cancer (such as melanoma) based on the expression of the c-met biomarker and the B-raf biomarker. In some embodiments, the B-raf biomarker is B-raf V600E.

The 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) and a package insert indicating that the pharmaceutical composition is for treating a cancer (such as NSCLC) patient based on expression of a c-met biomarker. In some embodiments, the treatment is in combination with a B-raf antagonist. In some embodiments, the package insert indicates that the c-met antagonist is used in combination with a B-raf antagonist to treat a patient having cancer (such as melanoma) based on the expression of the c-met biomarker and the B-raf biomarker. In some embodiments, the B-raf biomarker is B-raf V600. In some embodiments, the B-raf biomarker is B-raf V600E.

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 a method for detecting anyA diagnostic kit for 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 of c-met and B-raf (such as B-raf V600) biomarkers in a sample from a cancer 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, in combination with a B-raf antagonist) to treat a cancer patient if the patient expresses a c-met biomarker and/or the patient expresses a B-raf biomarker. In some embodiments, the B-raf biomarker is B-raf V600. In some embodiments, the B-raf biomarkers are detected using a method comprising: (a) performing PCR or sequencing on nucleic acids (e.g., DNA) extracted from a patient melanoma sample; and (b) determining BRAF in the sample^V600Expression of (2). In some embodiments, the melanoma sample is formalin fixed, paraffin coated. In some embodiments, the c-met biomarker is HGF and expression is detected in a patient melanoma (or melanoma stroma) sample using IHC. In some embodiments, the c-met biomarker is HGF and expression is detected in a serum sample of the patient using ELISA. The diagnostic method includes any of the diagnostic methods herein.

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) to a target audience for treating a cancer patient based on the expression of a c-met biomarker and/or a B-raf biomarker.

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 (sales promotion). 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 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, 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.

Examples

Example 1: growth factor-driven resistance to anticancer kinase inhibitors

Method of producing a composite material

And (4) screening an RTK ligand matrix. Cell viability was assessed using the nucleic acid dye Syto60 (Invitrogen). Cells (3000-. The next day, cells were treated with (or without) 50ng/mL RTK ligand and concomitantly exposed to increasing concentration ranges of the relevant kinase inhibitor. After 72 hours drug exposure, cells were fixed in 4% formaldehyde, stained with Syto60, and cell number was assessed using an Odyssey scanner (Li-Cor). Cell viability was calculated by dividing the fluorescence obtained from drug-treated cells by the fluorescence obtained from control (no drug) treated cells.

A cell line. Human cancer cell lines were obtained and tested for sensitivity using an automated platform as previously described (Johannessen, c.m. et al. cotdrive resistance to RAF inhibition route reactivity. nature468,968-972, doi:10.1038/nature09627 (2010)). At 5% CO₂Maintained at 37 ℃ and cultured in RPMI1640 or DMEM/F12 growth medium (GIBCO) supplemented with 10% fetal bovine serum (GIBCO), 50U/mL penicillin and 50. mu.g/mL streptomycin (GIBCO).

And (3) a reagent. Lapatinib, sunitinib and erlotinib were purchased from LC Laboratories. Crizotinib, TAE684, AZD6244 and BEZ235 were purchased from Selleck Chemicals. PD173074 was purchased from tocris bioscience. PLX4032 was purchased from Active Biochem. Recombinant human (rh) HGF, EGF, FGF-basic, IGF-1 and PDGF-AB were purchased from Peprotech. rhNRG 1-. beta.1 was purchased from R and D Systems. For in vivo studies, 3D6 anti-MET agonistic antibodies, RG7204(PLX4032) and GDC-0712, were generated in Genentech. GDC-0712 was used in xenograft experiments because it has a similar kinase profile to crizotinib (Liederer, b.m.et al. xenobiotica41,327-339, doi:10.3109/00498254.2010.542500(2011)) (fig. 25 and 26).

Immunoblotting. Cell lysates were harvested using Nonidet-P40 lysis buffer supplemented with a mixture of Halt protease and phosphatase inhibitors (ThermoScientific) and protein immunodetection was performed using standard protocols. phospho-HER 2 (Y1248; #2247), HER2(#2242), phospho-HER 3 (Y1289; #4791), phospho-MET (Y1234/5; #3126), PDGFR α (#5241), phospho-FRS 2 α (Y196; #3864), IGF-1R β (#3027), phospho-ALK (Y1604; #3341), AKT (#9272), phospho-ERK (T202/Y204; #9101), ERK (#9102), GAPDH (#2118), and β -tubulin 2146) antibodies were purchased from Cell Signaling Technologies. Antibodies against HER3(SC-285), MET (SC-10), phospho-PDGFR α (SC-12911), FRS2 α (SC-8318), FGFR1(SC-7945), FGFR2(SC-122), FGFR3(SC-13121) and ALK (SC-25447) were purchased from Santa Cruz Biotechnologies. phospho-AKT (S473; #44-621G) antibody was purchased from Invitrogen. phospho-EGFR (Y1068; ab5644) antibodies were purchased from Abcam. EGFR (#610017) antibody was purchased from BD Biosciences. PARP (#14-6666-92) antibody was purchased from eBioscience. Densitometry was performed using ImageJ software.

A tissue sample. Primary (Primary) breast tumor samples were obtained from the following sources, with appropriate IRB approval and patient informed consent: cureline (South San Francisco, Calif.), ILSbi (Chestertown, MD) and the national cancer society human organizational cooperative network (USA). Metastatic melanoma tumor samples were obtained from BRIM2 assay. Human tissue samples used in the study were de-identified (double-coded) prior to use, and thus the study using these samples was not considered a human subject study according to the U.S. health and human services guidelines and related guidelines. MET immunohistochemistry was performed on formalin fixed, paraffin embedded sections cut at 4 μm thickness onto positively charged glass slides. Staining was performed on a Discovery XT autostainer (VMSI, Tucson, AZ) equipped with Ultraview detection using the MET rabbit monoclonal antibody SP44(Spring BioScience, Pleasanton, CA; # M3441) and CC1 standard antigen retrieval. Sections were counterstained with hematoxylin and scored for specific staining (e.g., membrane staining) of c-MET on a scale from 0 (no staining) to 3+ (strong staining).

Scoring protocols are described in commonly owned U.S. patent publication No. us20120089541a1, the contents of which are hereby incorporated by reference in their entirety. Briefly, tumor cells were scored for c-Met staining. Staining was assigned as strong (3+), moderate (2+), weak (1+), suspicious (+/-) or negative (-) staining intensity relative to control cell pellets with various staining intensities that could be used as IHC assay controls as well as scoring controls. H441 (strong c-met staining intensity) was used; a549 (moderate c-met staining intensity); h1703 (weak c-met staining intensity), HEK-293(293) (weak c-met staining intensity); and TOV-112D (negative c-met staining intensity) or H1155 (negative c-met staining intensity). In addition to assessing staining intensity, the percentage of various staining intensities/patterns in samples with heterogeneous signals was also assessed visually.

Hepatocyte Growth Factor (HGF) ELISA. Plasma was obtained from metastatic melanoma patients prior to the first dosing cycle and HGF concentrations in patient-derived plasma were quantitatively measured using a sandwich enzyme-linked immunosorbent assay (ELISA). The wells of NUNC MaxiSorp microtiter plates were coated (ON, 4 ℃) with 0.5. mu.g/mL affinity purified goat anti-human hepatocyte growth factor polyclonal antibody in 100. mu.L coating buffer (0.05M sodium carbonate buffer, pH9.6) and then blocked with 0.5% Bovine Serum Albumin (BSA) in assay buffer (PBS, 0.5% BSA, 0.05% P20, 0.25% CHAPS, 0.35M NaCl, 5mM EDTA, 10ppm Proclin300, pH7.4) for 1 hour at room temperature. Diluted human hepatocyte growth factor control and plasma samples (100 μ L) in assay buffer were loaded in duplicate and incubated for 2 hours at room temperature, after which 100 μ L of affinity purified goat anti-human hepatocyte growth factor biotin (150ng/mL) was added and incubated for an additional 1 hour at room temperature. PBS, 0.5% BSA, 0.05% P20, 10ppm Proclin300, horseradish peroxidase (40ng/mL) conjugated with avidin at pH7.4 (1 h, room temperature) was added and the reaction was visualized by adding 100. mu.L of chromogenic substrate (TMB) for 15 min. The reaction was stopped with 1M phosphoric acid and absorbance at 450nm was measured using an ELISA plate reader, minus absorbance at 630 nm. Each step was followed by washing the plate 3 times with wash buffer (0.05% Tween 20/PBS). As a quantitative reference, a standard curve was established by serial dilution of human hepatocyte growth factor (CritRS CR 67; 2000-15.625 pg/mL).

Xenograft studies. All procedures were in compliance with guidelines and principles set by the Genentech scientific research animal care and use committee and were conducted in an AAALAC (joint laboratory animal care assessment and certification) certified facility. At CRL C.B-17Bg mice (Charles River Laboratories) in the right side inoculated with 1x10⁷928MEL or 624MEL BRAF mutant melanoma cells (suspended in HBSS/Matrigel (e.g., 1:1 mixture)). When the tumor reached an average volume of 200mm³In time, mice (10 groups) were treated with either control antibody (anti-gp 120; 10mg/kg once weekly; intraperitoneally), 3D6 (anti-MET agonistic antibody; 10mg/kg once weekly; intraperitoneally), RG7204(PLX 4032; 50mg/kg twice daily, periocular), GDC-0712(MET small molecule inhibitor, 100mg/kg daily, periocular) for 4 weeks as indicated. Tumors were measured twice weekly using a digital caliper (Fred v. Tumor volume was calculated using the formula (Lx (WxW))/2. The Partial Response (PR) in this example is defined as a reduction in tumor volume of greater than 50% but less than 100%. The Complete Response (CR) in this example is defined as a 100% reduction in tumor volume. The difference between the control antibody groups treated with PLX4032 and GDC-0712 was determined using a two-way ANOVA (═ 0.0008).

And (4) screening secretion factors. Where appropriate, recombinant purified secretion factors were purchased from Peprotech and R and DSystems and reconstituted in PBS/0.1% BSA. Secreted factors were transferred into 96-well plates at a concentration of 1 μ g/mL and subsequently diluted to 100ng/mL in medium containing either no drug or 5 μ M PLX 4032. Equal volumes of diluted factor (final concentration 50ng/mL) were arrayed into 384-well plates pre-seeded with SK-MEL-28 cells (500 cells per well seeded the previous day) using an Oasis liquid manipulator. After 72 hours incubation, Cell viability was determined using Cell Titer Glo (Promega).

And (5) statistics. Error bars in cell viability assays represent standard errors of mean plus or minus means (s.e.m.). To associate the receptor with the ligand, rescue was performed using a 2x2 listing with the following group: receptor positive, rescuing RTK ligands; receptor positive, not rescuing RTK ligands; receptor negative, rescuing RTK ligands; receptor negative, not rescuing RTK ligands. Significance was determined using the two-tailed Fisher exact probability test.

Statistical analysis of BRIM2 clinical samples. HGF levels were logarithmically scaled and the resulting distributions were tested for deviation from Gaussian distributions using the Kolmogorov-Smirnoff test. Logarithmically scaled HGF levels were examined for association with Progression Free Survival (PFS) and Overall Survival (OS) using a cox-scale model. The association between response and HGF levels was tested using Wilcoxon rank sum test. The Kaplan-meier (km) curve was used to show the correlation between HGF levels and time to event results (PFS and OS). The number of events/patients per group and median time to event are displayed. The hazard ratio and corresponding p-value are calculated using a cox-proportional model of the results (as a function of continuous HGF levels).

Results

Use of 41 different human tumor-derived cell lines with a previously defined kinase dependence^7-9We adopted "matrix analysis" to examine 6 different RTK ligands (HGF, EGF, FGF, PDGF, NRG1, IGF) -known to be widely expressed in cancer cells and tumor stroma¹⁰-effect on drug response. Specifically, we quantified the effect of exposing these cancer cell lines (e.g., AU565(HER2amp)) to IC50 of each ligand on kinase inhibitors (e.g., lapatinib) that potently repressed their growth within 72 hours in other cases (fig. 1 a). Almost all cells tested for kinase-dependent cancer cell lines, including cells derived from multiple tissue types and with different kinase-dependencies (EGFR, HER2, BRAF, MET, ALK, PDGFR, and FGFR), could be rescued from drug-induced growth inhibition by one or more RTK ligands, highlighting the potential broad contribution of these ligands to the response to selective kinase inhibitors in kinase-addicted tumor cells (fig. 1 b).

The consequences of ligand exposure to drug response can be divided into three categories (fig. 1 c); "do not save": addition of ligand had no detectable effect on drug response; "partial rescue": ligand partial elimination treatment response, or "complete rescue": the ligand "right-shifted" the IC50 curve by > 10-fold, or completely suppressed the drug response. HGF, FGF and NRG1 are the most widely active ligands in conferring drug resistance, followed by EGF; whereas IGF and PDGF have relatively minor effects, despite their ability to activate their respective receptors (fig. 5a and 7 a). Notably, most of the cell lines tested could be rescued from treatment sensitivity by exposure to two or even three different ligands, highlighting the apparent ability of such cells to be involved in redundant survival pathways when exposed to multiple RTK ligands. Importantly, none of the RTK ligands tested in the several cell lines tested were able to rescue cells from the growth inhibitory effect of the chemotherapeutic drug cisplatin, suggesting that the observed ligand rescue effect does not reflect extensive protection from the general toxic agents, but is limited to pathway-specific signal disruption (fig. 5 b).

To further explore the signaling kinetics associated with ligand-mediated rescue from kinase-dependence, we evaluated the status of two key downstream survival signaling pathways, PI3K/AKT and MAPK/ERK pathway-, that are normally involved in RTKs¹¹. In the case of achieving ligand-mediated rescue, RTK ligands are effective to "reactivate" at least one of these pathways despite the presence of kinase inhibitors (fig. 2 a). Pathway reactivation is not due to reactivation of oncogenic kinases, as autophosphorylation of addictive kinases remains repressed after RTK ligand co-treatment. In the various models tested, HGF reactivates both the PI3K and MAPK pathways, IGF and NRG1 only reactivate PI3K, while FGF and EGF only reactivate the MAPK pathway.

Activation of the "redundant RTKs" and the resulting downstream survival signaling lasted at least 48 hours as exhibited by AU565 cells co-treated with lapatinib and HGF (fig. 9 b). A "additive" effect of reactivation of both PI3K and MAPK pathways was observed in AU565 cells treated with lapatinib in the presence of NRG1, FGF or a combination (fig. 14 a). However, specific inhibition of the PI3K pathway (but not MAPK) attenuated HGF-driven drug resistance, which was associated with the involvement of both survival pathways (fig. 14 b).

As expected, the observed cell survival rescue and pathway signaling induced by RTK ligands could be reversed by co-targeting a second activated kinase, confirming that potent ligands act via their associated RTKs (fig. 2b, c, fig. 5c, d, fig. 22). Importantly, inhibitors of the mediator-driven salvaged "second" RTK in the various models tested had little or no effect when treated as a single agent in these cell lines, indicating that kinase-addicted cells were not initially dependent on multiple different RTKs in the absence of available ligands. Similarly, RTK ligand stimulation had little or no effect on cell proliferation in the absence of kinase inhibitors (fig. 1c and 2 b).

Analysis of baseline RTK expression among cell line panels confirmed that all of these kinase-dependent cancer cells expressed multiple RTKs, suggesting that many cancer cells are "primed" to receive survival signals from extracellular ligands. Notably, in some cases (e.g., MET/HGF, EGFR/EGF and HER3/NRG1), ligand-induced rescue correlated well with the expression of certain RTKs (p < 0.01; fig. 6), suggesting that the RTK profile of tumors prior to treatment could inform the optimal therapeutic strategy, which is expected to require co-targeting of two or more kinases that may contribute to cancer cell survival, depending on the availability of the respective ligands in the tumor microenvironment.

In some cases, the ligand cannot rescue the cell from drug sensitivity despite the expression of the ligand-associated RTK. We identified two different biochemical events associated with failure of ligand-induced rescue in this background (figure 7). In a few cases, RTK ligands are capable of activating its receptor, as evidenced by RTK phosphorylation; however, no resulting downstream signaling via PI3K or MAPK was observed. This was seen, for example, in COLO-201 and BT474 cell lines when treated with IGF (FIG. 7 a). In other cases, the RTK ligand activates its receptor and at least one downstream survival effector; however, that is not sufficient to rescue cells from kinase inhibition. This was observed, for example, with H2228 and H358 cells (upon exposure to HGF) or with COLO-201 cells (upon exposure to NRG 1) (fig. 7 b). However, H2228 and H358 cells were "rescued" by HGF after longer term treatment, possibly suggesting the presence of subpopulations of cells capable of responding to HGF and possibly being selected over time in the presence of inhibitory kinases, as detailed below (fig. 8c, d).

Cell line analysis yielded several itemsIt was found to have potentially important clinical implications. For example, one of two NSCLC cell lines tested that contained an ALK-associated chromosomal translocation (NCI-H3122) and exhibited ALK kinase addiction could be effectively rescued from ALK inhibition by brief exposure to HGF (fig. 8). In these cells, HGF promotes ERK and AKT activation in the presence of the HGF receptor MET expression, even in the presence of the ALK selective inhibitor TAE 684. Importantly, however, survival of these cells was achieved by treatment with crizotinib (a dual ALK/MET kinase inhibitor, recently demonstrating impressive clinical activity in ALK translocation NSCLC) ¹²Treatment was effectively suppressed even in the presence of HGF. In view of the observed ability of these cells to respond to HGF, the relatively long-lasting clinical response observed in many ALK-translocating NSCLC patients may be due in part to the dual inhibitory nature of crizotinib, which is effective in suppressing both ALK-and MET-mediated survival signals. Interestingly, the second ALK translocation NSCLC line (NCI-H2228) also expressed detectable MET, both without HGF rescue from ALK inhibition at the 72 hour time point tested. However, HGF treatment was able to reactivate AKT and ERK activity in the presence of TAE684 (fig. 7b), and longer term TAE684 treatment prevented acquired resistance to TAE684 in these cells in the presence of HGF (fig. 8 c). This finding reminds that the previously documented subpopulation of existing MET-expressing tumor cells has been shown to be present in some patients with EGFR mutant NSCLC¹³。

The ability of HGF to rescue 3 of the 9 HER2 amplified breast cancer cell lines tested against growth inhibition by the HER2 kinase inhibitor lapatinib was also unexpected (fig. 3 a). These 3 cell lines all expressed MET and expression correlated well with HGF's ability to attenuate lapatinib responses (fig. 3 b). Longer co-treatment (12 days) of partially HGF-rescued AU565MET expressing cells revealed that HGF rapidly promoted resistance to lapatinib, as in the NCI-H228 cell line, presumably by driving selection of MET expressing cell subsets (fig. 3c, 9 b). Indeed, 9-day lapatinib and HGF co-treatment of AU565 cells yielded a population of cells with increased MET expression, suggesting HGF exposure to select a subset of MET expressing cells (fig. 3 f). Biochemical analysis indicated that HGF reactivated PI3K and MAPK signaling pathways exclusively in MET-positive cells, but not MET-negative cells (fig. 3 d).

We next determined whether HER2 positive primary breast tumors detectably expressed MET protein (fig. 3 e). Of the 10 samples analyzed, 1 sample exhibited moderate and high MET expression in-30% of the tumor cells, and 5 samples exhibited MET expression in approximately 10% of the tumor cells. One HER 2-expanded breast cancer cell line (HCC1954) displayed elevated phospho-MET in the absence of exogenous HGF, suggesting an autocrine mechanism (fig. 3b), and MET kinase inhibition in these cells delayed the emergence of lapatinib resistance (fig. 3 g). Taken together, these results suggest that MET expressing HER2 positive breast tumors could potentially circumvent HER2 kinase inhibition by involving MET in a subpopulation of "primed" tumor cells, resulting in resistance to targeted therapies, and that this shift towards MET dependence could be driven by the availability of HGF. Consistent with this possibility, SKBR3 and AU565 cells were derived from the same patient, highlighting the possible heterogeneity of MET expression within the patient's tumor. We also found that 9 to 8 HER2 expanded breast cell lines tested were able to be rescued from lapatinib sensitivity by exposure to HER3 ligand NRG1, suggesting a potentially important role for NRG1 expression in the tumor microenvironment in the variable response to HER2 targeted therapy (figure 23).

Another observation that immediately had potential clinical implications was the unexpected finding that HGF exposure significantly attenuated the response to the BRAF kinase inhibitor PLX4032 in several tested BRAF mutant PLX4032 sensitive melanoma and colorectal cell lines. PLX4032 recently exhibited significant clinical efficacy in BRAF mutant melanoma, resulting in its recent approval for clinical use¹⁴。

To determine the potential role of growth factors and other cytokines other than HGF similarly affecting PLX4032 sensitivity, we compared SK-MEL-28 cells for sensitivity to PLX4032 in the presence of each of 446 different recombinantly purified secreted factors. This analysis revealed that very few factors, including HGF, reduced PLX4032 sensitivity (fig. 17).

We examined another 12 BRAF mutant melanoma cell lines to explore the potentially broader role of HGF-MET signaling in response to PLX4032 (fig. 4 a). HGF significantly attenuated PLX4032 sensitivity in 5 of 12 germline. 8 of the 10 HGF rescued cell lines displayed detectable MET expression, whereas MET was undetectable or barely detectable in non-rescued cells. Notably, MET expression was negatively associated with PLX4032 sensitivity in HGF-rescuable cell lines, and HGF was able to reactivate MAPK signaling in HGF-rescued cell lines, but not in MET-negative HGF-undeployed cells (fig. 4 b). As expected, when MET was inhibited by criptiotinib, survival rescue by HGF was reversed (fig. 4b and fig. 9 a). A BRAF mutant cell line (624MEL) exhibited elevated phospho-MET in the absence of exogenous HGF, consistent with an autocrine mechanism (fig. 4a), and MET kinase inhibition in these cells delayed the emergence of PLX4032 resistance (fig. 4 c).

Strizotinib co-treatment also prevented resistance to PLX4032 in two undetectable phospho-MET cell lines (a375 and 928MEL), further supporting the potential role of HGF-activated MET in mediating resistance to PLX4032 (figure 18).

To validate the potential role of HGF-MET signaling in resistance to BRAF inhibition in vivo, we performed xenograft studies with BRAF mutant 928MEL melanoma cells. Importantly, activation of MET using agonistic antibody 3D6 in these tumors abrogated the growth inhibitory effect of PLX4032 (fig. 4D). The relevance of 3D 6-induced MET activation in attenuating the response to PLX4032 was demonstrated by co-treatment with MET small molecule kinase inhibitors. Taken together, these results suggest that MET kinase (activated via HGF) can contribute to the clinical response to PLX4032 in a subset of BRAF mutant melanoma.

The general findings highlight the broad nature of signaling communication between RTKs that are co-expressed in most tumor cells, and the use of RTK ligands in contributing to innate and acquired resistance to selective kinase inhibitors, such as cancer therapeuticsHas potential broad effects. Such ligands may be generated by the tumor cells themselves to drive autocrine survival mechanisms, or may be generated by the tumor stroma to influence drug responses in tumor cells via paracrine effects on survival signaling ^15,16。

The heterogeneity of human tumors is increasingly recognized, which significantly complicates the elucidation of drug resistance mechanisms^17-19. Our findings highlight the potentially broad role of RTK ligands, and in this context we envisage different mechanisms by which such heterogeneous properties contribute to acquired resistance. Thus, it may be the case that a subpopulation of tumor cells capable of responding to a survival-promoting RTK ligand exists prior to treatment, and that this subpopulation expands via the selective pressure of drug treatment if such ligands become available within the tumor microenvironment. Indeed, IHC analysis of MET expression in BRAF mutant melanoma cells revealed a heterogeneous cell population (fig. 21). In the case of EGFR mutant NSCLC, a subpopulation of tumor cells that exhibit MET drive when exposed to HGF during treatment with an EGFR kinase inhibitor¹³. Notably, activation of multiple RTKs was reported in glioblastoma, and suppression of pro-survival signals and cell death was observed only after co-targeting multiple activated receptors (Stommel, j.m. et al. science318,287-290, doi:10.1126/science.1142946 (2007)). It is also possible to select a subpopulation of tumor cells by virtue of the ability to obtain the production of RTK ligands. In a variety of preclinical models of acquired resistance to kinase inhibitors, the observed resistance mechanism involves a "shift" towards new RTK dependence " ^20-25This may be attributed in some cases to increased production of RTK ligands. Such increased ligand production could potentially be achieved by either mutation or epigenetic mechanisms.

Although genomic biomarkers (such as BRAF and EGFR mutations) are crucial in identifying patients most likely to benefit from therapy, there is a wide range of initial clinical responses to kinase inhibitory drugs in such patients that have not yet been explained to date-both in magnitude and duration of response^12,14. Meridian swelling of stomachThe potential role of RTK ligands secreted by neoplastic cells, expressed in the tumor microenvironment, or even systemically provided has been heretofore largely unexplored. Since tumor-derived cell lines have been shown to be a powerful model for capturing genotype-related sensitivity to selective kinase inhibitors in a subset of mutation definitions^7,8The findings from this matrix analysis therefore support the potential broad role of RTK ligands in the overall clinical benefit from such therapies, and provide the basis for the use of biomarkers to inform treatment strategies that anticipate both innate and acquired resistance mechanisms associated with redundant survival signaling (which is via key effectors common to many widely expressed RTKs) based on the expression of RTKs and their associated ligands.

Example 2: rescue results for various PTK ligands in cells with BRAF V600E

The method used here is similar to that described in example 1. We examined the effect of 6 different RTK ligands (HGF, EGF, FGF, PDGF, NRG1, IGF) on drug response (PLX4032) in cells with BRAF V600E. Figure 10 shows the rescue results for various PTK ligands in cells treated with PLX 4032.

Example 3: role of MET kinase inhibition in delaying lapatinib resistance

The method used here is similar to that described in example 1. The effect of MET kinase inhibition on delayed lapatinib resistance was examined in HCC1954 cells. HCC1954HER2 expanded breast cancer cells were treated with lapatinib (5. mu.M) and/or crizotinib (1. mu.M) and stained with Syto 60. Figure 11 shows MET kinase inhibition delays the emergence of lapatinib resistance in HCC1954 cells.

Example 4: role of HGF-MET signaling in cellular response to PLX4032

The method used here is similar to that described in example 1. We examined the role of HGF-MET signaling in the cellular response to PLX 4032. We observed that HGF was able to reactivate MAPK signaling in cell lines rescued by HGF, but not in MET negative, non-HGF rescued cells (fig. 4a, 12 a). To validate the potential role of HGF-MET signaling in resistance to BRAF inhibition in vivo, we performed xenograft studies with BRAF mutant 928MEL and 624MEL melanoma cells. Importantly, activation of MET in these tumors using MET agonistic antibody 3D6 strongly abrogated the growth inhibitory effect of PLX4032 (fig. 12 b). The relevance of 3D 6-induced MET activation in attenuating responses to PLX4032 was demonstrated by co-treatment with MET small molecule kinase inhibitors. Similar to in vitro findings, we observed a greater effect of inhibiting MET kinase activity on tumor regression in xenografts treated with PLX4032, with more partial responses observed (928 MEL: 1 to 8; fig. 12b and fig. 19). Taken together, these results suggest that MET kinase (activated via HGF) can contribute to the clinical response to PLX4032 in a subset of BRAF mutant melanomas.

Example 5: role of HGF-MET signaling in clinical settings

The method used here is similar to that described in example 1. To examine the potential role of HGF-MET signaling in the clinical setting, we examined the hypothesis that circulating HGF in BRAF mutant melanoma patients may contribute to clinical outcome. Thus, pretreatment plasma HGF levels were measured from 126 of 132 metastatic melanoma patients enrolled in the BRIM2 clinical trial (BRAF mutant metastatic melanoma patients treated with PLX 4032). HGF levels ranged from 33pg/mL to 7200pg/mL, with a median level of 334pg/mL (fig. 20). Patients treated with PLX4032 with HGF levels above median exhibited substantially shorter progression-free survival (p ═ 0.005) and overall survival (p <0.001) than patients with HGF levels below median (fig. 13). Elevated HGF was associated with poorer results as measured by progression free survival (PFS, hazard ratio of 1.42 and p <0.005) and overall survival (OS, hazard ratio of 1.8 and p < 0.001). Segregating patients into three groups (tertiary) revealed a continuous correlation between HGF levels and outcomes, rather than a threshold effect (fig. 24B). These studies link HGF-MET signaling with disease progression and overall survival, and possibly clinical response to BRAF inhibition (in BRAF mutant melanoma).

Example 6: ligand-induced rescue in cells

The method used here is similar to that described in example 1. We analyzed RTK expression and ligand-induced rescue in cells. The results are shown in FIG. 15. In some cases (e.g., MET/HGF, EGFR/EGF and HER3/NRG1), ligand-induced rescue correlated well with the expression of certain RTKs (p < 0.01; fig. 15), suggesting that the RTK profile of tumors prior to treatment could inform the optimal therapeutic strategy, which is expected to require co-targeting of two or more kinases that may contribute to cancer cell survival, depending on the availability of the respective ligands in the tumor microenvironment.

Example 7: role of HGF in preventing acquired resistance to TAE684

The method used here is similar to that described in example 1. We examined the role of HGF in H2228 cells treated with TAE 684. Fig. 16 shows that longer term treatment of TAE684 in the presence of HGF prevented acquired resistance to TAE684 in these cells.

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Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, the description and examples should not be construed as limiting the scope of the invention. The disclosures of all patent and scientific literature cited herein are expressly incorporated by reference in their entirety.

Claims (43)

1. A method for treating a patient having cancer comprising administering an effective amount of a B-raf antagonist and a c-met antagonist.

2. A method for treating a cancer patient with an increased likelihood of developing resistance to a B-raf antagonist, comprising administering an effective amount of a B-raf antagonist and a c-met antagonist.

3. A method for increasing and/or restoring sensitivity to a B-raf antagonist comprising administering to a cancer patient an effective amount of a B-raf antagonist and a c-met antagonist.

4. A method for extending a period of B-raf antagonist sensitivity comprising administering to a cancer patient an effective amount of a B-raf antagonist and a c-met antagonist.

5. A method for treating a patient having a B-raf antagonist resistant cancer comprising administering an effective amount of a B-raf antagonist and a c-met antagonist.

6. A method for extending the duration of B-raf antagonist response comprising administering an effective amount of a B-raf antagonist and a c-met antagonist.

7. A method for delaying or preventing the development of an HGF-mediated B-raf antagonist resistant cancer in a patient comprising administering an effective amount of a B-raf antagonist and a c-met antagonist.

8. The method of any one of claims 1-7, wherein the patient's cancer exhibits expression of a B-raf biomarker.

9. The method of claim 8, wherein the B-raf biomarker is B-raf V600.

10. The method of claim 8, wherein the B-raf biomarker is B-raf V600E.

11. The method of any one of claims 8-10, wherein mutant B-raf biomarker expression in the patient's cancer is determined using a method comprising: (a) performing one or more of gene expression profiling, PCR hybridization assay, in situ hybridization, 5' nuclease assay mutation detection assay, RNA-seq, microarray analysis, SAGE, MassARRAY technique, or FISH on the sample, and (B) determining expression of mutant B-raf biomarkers in the sample.

12. The method of claim 11, wherein mutant B-raf biomarker expression in the patient's cancer is determined using a method comprising: (a) performing PCR on genomic DNA extracted from a patient cancer sample, and (B) determining the expression of mutant B-raf biomarkers in the sample.

13. The method of any one of claims 1-12, wherein the patient's cancer shows expression of a c-met biomarker.

14. The method of claim 13, wherein the c-met biomarker is a polypeptide.

15. The method of claim 14, wherein c-met biomarker expression is determined using Immunohistochemistry (IHC).

16. The method of claim 15, wherein c-met biomarker expression is determined by determining expression of Hepatocyte Growth Factor (HGF).

17. The method of claim 16, wherein HGF is expressed in a tumor or tumor stroma.

18. The method of claim 16, wherein HGF expression is determined in the serum of the patient.

19. The method of any one of claims 1-18, wherein the c-met antagonist is an antagonistic anti-c-met antibody.

20. The method of any one of claims 1-19, wherein the c-met antagonist is one or more of onartuzumab, 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.

21. The method of any one of claims 1-20, wherein the B-RAF antagonist is one or more of sorafenib, PLX4720, PLX-3603, GSK2118436, GDC-0879, N- (3- (5- (4-chlorophenyl) -1H-pyrrolo [2,3-B ] pyridine-3-carbonyl) -2, 4-difluorophenyl) propane-1-sulfonamide, vemurafenib, GSK2118436, RAF265(Novartis), XL281, ARQ736, BAY 73-4506.

22. The method of claim 21, wherein the B-raf antagonist is vemurafenib.

23. The method of claim 21, wherein the B-raf antagonist is GSK 2118436.

24. The method of any one of claims 1-23, wherein the B-raf antagonist and the c-met antagonist are administered simultaneously.

25. The method of any one of claims 1-23, wherein the B-raf antagonist and the c-met antagonist are administered sequentially.

26. The method of claim 25, wherein the B-raf antagonist is administered prior to the c-met antagonist.

27. The method of claim 26, wherein the c-met antagonist is administered prior to the B-raf antagonist.

28. The method of any one of claims 1-27, further comprising administering at least one additional treatment to the subject.

29. The method of any one of claims 1-28, wherein the cancer is melanoma, colorectal cancer, ovarian cancer, breast cancer, or papillary thyroid cancer.

30. The method of claim 29, wherein the cancer is melanoma that exhibits expression of B-raf V600.

31. The method of any one of claims 1-30, wherein the cancer is resistant to a B-raf antagonist.

32. The method of any one of claims 1-30, wherein the patient has not been previously treated with a B-raf antagonist.

33. A method for determining c-met biomarker expression, comprising the step of determining whether a patient's cancer expresses c-met biomarker, wherein c-met biomarker expression indicates that the patient is a candidate for treatment with a c-met antagonist and a B-raf antagonist: increasing the sensitivity of the patient's cancer to a B-raf antagonist, restoring the sensitivity of the patient's cancer to a B-raf antagonist, extending the period of the sensitivity of the patient's cancer to a B-raf antagonist, and/or preventing the development of HGF-mediated B-raf antagonist resistance in the patient's cancer.

34. A method for identifying a patient as a candidate for treatment with a B-raf antagonist and a c-met antagonist, comprising: (a) determining the patient's cancer expresses a c-met biomarker; and (B) identifying the patient as a candidate for treatment with a B-raf antagonist and a c-met antagonist.

35. A method for identifying a patient at risk for developing resistance to a B-raf antagonist, comprising: (a) determining the patient's cancer expresses a c-met biomarker; and (B) identifying the patient as at risk for developing resistance to a B-raf antagonist.

36. The method of claim 34 or 35, wherein after steps (a) and (B), the patient is treated with an effective amount of a c-met antagonist and a B-raf antagonist.

37. A method of determining the efficacy of a treatment with a B-raf antagonist for treating cancer in a patient, comprising determining the presence of a c-met biomarker and/or a B-raf biomarker in a sample obtained from said patient by immunoassay, ELISA, hybridization assay, PCR, 5' nuclease assay, IHC, and/or RT-PCR, and selecting said patient for treatment with a B-raf antagonist.

38. The method of claim 37, further comprising selecting the patient for treatment with a c-met antagonist.

39. The method of claim 38, further comprising treating the patient with an effective amount of a B-raf antagonist and a c-met antagonist.

40. A method of determining the prognosis of a melanoma patient comprising determining expression of a c-met biomarker in a sample from the patient, wherein c-met biomarker is HGF and HGF expression is predictive of cancer in the subject.

41. A kit comprising a c-met antagonist and a B-raf antagonist.

42. The kit of claim 41, further comprising instructions for a method for treating a melanoma patient comprising administering to the patient an effective amount of a c-met antagonist and a B-raf antagonist.

43. An article of manufacture comprising a c-met antagonist and a package insert in a pharmaceutically acceptable carrier packaged together, the package insert indicating that the c-met antagonist is for use in treating a patient having melanoma based on expression of a B-raf biomarker, wherein the treatment is in combination with the B-raf antagonist.