WO2014150819A2 - Biomarkers and methods of treatment - Google Patents

Biomarkers and methods of treatment Download PDF

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Publication number
WO2014150819A2
WO2014150819A2 PCT/US2014/024316 US2014024316W WO2014150819A2 WO 2014150819 A2 WO2014150819 A2 WO 2014150819A2 US 2014024316 W US2014024316 W US 2014024316W WO 2014150819 A2 WO2014150819 A2 WO 2014150819A2
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WO
WIPO (PCT)
Prior art keywords
met
staining intensity
cancer
score
patient
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PCT/US2014/024316
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French (fr)
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WO2014150819A3 (en
Inventor
Robert Yauch
Hartmut Koeppen
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Genentech, Inc.
F. Hoffmann-La Roche Ag
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Publication of WO2014150819A2 publication Critical patent/WO2014150819A2/en
Publication of WO2014150819A3 publication Critical patent/WO2014150819A3/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/5748—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving oncogenic proteins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/71—Assays involving receptors, cell surface antigens or cell surface determinants for growth factors; for growth regulators
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the present invention concerns cancer biomarkers.
  • the invention concerns c-met as a biomarker for patient selection and prognosis in cancer, as well as methods of therapeutic treatment, articles of manufacture and methods for making them, diagnostic kits, methods of detection and methods of advertising related thereto.
  • Cancer remains to be one of the most deadly threats to human health.
  • cancer affects nearly 1.3 million new patients each year, and is the second leading cause of death after heart disease, accounting for approximately 1 in 4 deaths.
  • breast cancer is the second most common form of cancer and the second leading cancer killer among American women. It is also predicted that cancer may surpass cardiovascular diseases as the number one cause of death within 5 years. Solid tumors are responsible for most of those deaths.
  • the overall 5 -year survival rate for all cancers has improved only by about 10% in the past 20 years. Cancers, or malignant tumors, metastasize and grow rapidly in an uncontrolled manner, making timely detection and treatment extremely difficult.
  • a c-met antagonist for effectively treating cancer patients are provided.
  • This application also provides better methods for diagnosing disease for use in treating the disease optionally with c-met antagonist.
  • c-met (interchangeably termed "Met) biomarker was used to identify a patient population in which Onartuzumab (interchangeably termed "MetMAb”) plus erlotinib treatment provided clinically meaningful benefit, evaluated by progression-free survival and overall survival, and a patient population in which
  • c-met antagonist e.g., in combination with EGFR antagonist
  • a patient with cancer comprising administering a therapeutically effective amount of a c-met antagonist to the patient if the patient's cancer has been found to have an H-score about 160 or higher (about 161, 162, 163, 164, 165, 166, 167, 168, 169, or more), about 160 to about 230, about 230 or higher, or about 170 or higher, wherein the H-score is determined using the formula (% cancer cells staining with weak c-met staining intensity) + (% cancer cells staining with moderate c-met staining intensity) + (% cancer cells staining with high c-met staining intensity).
  • the patient has greater PFS and/or OS (e.g., relative to a patient who has an H score less than 160). In some embodiments, the patient has greater PFS (e.g., relative to a patient who has an H score less than 160). In some embodiments, the patient has greater OS (e.g., relative to a patient who has an H score less than 160). In some embodiments, the H-score is about 160 or higher. In some embodiments, the H-score is 160 or higher. In some embodiments, H-score is about 160 to about 230. In some embodiments, the H-score is 160-230. In some embodiments, the H score is greater than about 10. In some embodiments, the H score is greater than about 25.
  • the H score is greater than about 50. In some embodiments, the H score is greater than about 75. In some embodiments, the H score is greater than about 100. In some embodiments, the H score is greater than about 125. In some embodiments, the H score is greater than about 150. In some embodiments, the H score is greater than about 175. In some embodiments, the H score is greater than about 200.
  • c-met expression is membranous. In some embodiments, c-met expression is cytoplasmic. In some embodiments, c-met- expression is membranous and cytoplasmic.
  • a patient with cancer comprising administering a therapeutically effective amount of a medicament other than a c-met antagonist to the patient if the patient's cancer has been found to have an H-score of less than 160, wherein the H-score is determined using the formula (% cancer cells staining with weak c-met staining intensity) + (% cancer cells staining with moderate c-met staining intensity) + (% cancer cells staining with high c-met staining intensity).
  • the H-score is less than 160.
  • the H-score is less than 150.
  • the H score is greater than about 10.
  • the H score is greater than about 25. In some embodiments, the H score is greater than about 50. In some embodiments, the H score is greater than about 75. In some embodiments, the H score is greater than about 100. In some embodiments, the H score is greater than about 125. In some embodiments, the H score is greater than about 150. In some embodiments, the H score is greater than about 175. In some embodiments, the H score is greater than about 200.
  • c-met expression is membranous. In some embodiments, c-met expression is cytoplasmic. In some embodiments, c-met- expression is membranous and cytoplasmic.
  • kits for selecting a therapy for a patient with cancer comprising determining an H-score from a sample of the patient's cancer using the formula (% cancer cells staining with weak c-met staining intensity) + (% cancer cells staining with moderate c-met staining intensity) + (% cancer cells staining with high c-met staining intensity), and selecting a cancer medicament based on the H-score.
  • the patient is selected for treatment with a c-met antagonist if the H-score is about 160 or higher, about 160 to about 230, about 230 or higher, or about 170 or higher.
  • the H score is greater than about 10. In some embodiments, the H score is greater than about 25.
  • the H score is greater than about 50. In some embodiments, the H score is greater than about 75. In some embodiments, the H score is greater than about 100. In some embodiments, the H score is greater than about 125. In some embodiments, the H score is greater than about 150. In some embodiments, the H score is greater than about 175. In some embodiments, the H score is greater than about 200.
  • c-met expression is membranous. In some embodiments, c-met expression is cytoplasmic. In some embodiments, c-met- expression is membranous and cytoplasmic. In some embodiments, the patient is selected for treatment with a cancer medicament other than a c-met antagonist if the H-score is lower than 160.
  • the patient is selected for treatment with a cancer medicament other than a c-met antagonist if the H-score is lower than 159, 158, 157, 156, 155, 154, 153, 152, 151, or 150.
  • provided method for identifying a cancer patient who is likely to respond to treatment with a c-met antagonist comprising the step of determining an H-score from a sample of the patient's cancer using the formula (% cancer cells staining with weak c- met staining intensity) + (% cancer cells staining with moderate c-met staining intensity) + (% cancer cells staining with high c-met staining intensity), wherein an H-score of about 160 or higher, about 160 to about 230, about 230 or higher, or about 170 or higher indicates that the patient is likely to response to treatment with a c-met antagonist.
  • the H score is greater than about 10. In some embodiments, the H score is greater than about 25.
  • the H score is greater than about 50. In some embodiments, the H score is greater than about 75. In some embodiments, the H score is greater than about 100. In some embodiments, the H score is greater than about 125. In some embodiments, the H score is greater than about 150. In some embodiments, the H score is greater than about 175. In some embodiments, the H score is greater than about 200.
  • c-met expression is membranous. In some embodiments, c-met expression is cytoplasmic. In some embodiments, c-met- expression is membranous and cytoplasmic.
  • a method for determining cancer patient prognosis comprising the step of determining an H-score from a sample of the patient's cancer using the formula (% cancer cells staining with weak c-met staining intensity) + (% cancer cells staining with moderate c-met staining intensity) + (% cancer cells staining with high c-met staining intensity), wherein an H-score of about 160 or higher, about 160 to about 230, about 230 or higher, or about 170 or higher indicates that the patient is likely to have increased overall survival (OS) and/or progression-free survival (PFS) when the patient is treated with a c-met antagonist.
  • OS overall survival
  • PFS progression-free survival
  • the H score is greater than about 10.
  • the H score is greater than about 25. In some embodiments, the H score is greater than about 50. In some embodiments, the H score is greater than about 75. In some embodiments, the H score is greater than about 100. In some embodiments, the H score is greater than about 125. In some embodiments, the H score is greater than about 150. In some embodiments, the H score is greater than about 175. In some embodiments, the H score is greater than about 200.
  • c-met expression is membranous. In some embodiments, c-met expression is cytoplasmic. In some embodiments, c-met- expression is membranous and cytoplasmic.
  • a cancer patient who is less likely to be respond to treatment with a c-met antagonist comprising the step of determining an H-score from a sample of the patient's cancer using the formula (% cancer cells staining with weak c-met staining intensity) + (% cancer cells staining with moderate c-met staining intensity) + (% cancer cells staining with high c-met staining intensity), wherein an H-score of less than about 160 indicates that the patient is less likely to respond to treatment with the c- met antagonist.
  • the H score is greater than about 10. In some embodiments, the H score is greater than about 25. In some embodiments, the H score is greater than about 50.
  • the H score is greater than about 75. In some embodiments, the H score is greater than about 100. In some embodiments, the H score is greater than about 125. In some embodiments, the H score is greater than about 150. In some embodiments, the H score is greater than about 175. In some embodiments, the H score is greater than about 200.
  • c-met expression is membranous. In some embodiments, c-met expression is cytoplasmic. In some embodiments, c-met- expression is membranous and cytoplasmic.
  • determining cancer patient prognosis comprising the step of determining an H-score from a sample of the patient's cancer using the formula (% cancer cells staining with weak c-met staining intensity) + (% cancer cells staining with moderate c-met staining intensity) + (% cancer cells staining with high c-met staining intensity), wherein an H-score of about 160 or higher, about 160 to about 230, about 230 or higher, or about 170 or higher indicates that the patient is likely to have increased overall survival (OS) and/or progression-free survival (PFS) when the patient is treated with a c-met antagonist.
  • OS overall survival
  • PFS progression-free survival
  • the H score is greater than about 10.
  • the H score is greater than about 25. In some embodiments, the H score is greater than about 50. In some embodiments, the H score is greater than about 75. In some embodiments, the H score is greater than about 100. In some embodiments, the H score is greater than about 125. In some embodiments, the H score is greater than about 150. In some embodiments, the H score is greater than about 175. In some embodiments, the H score is greater than about 200.
  • c-met expression is membranous. In some embodiments, c-met expression is cytoplasmic. In some embodiments, c-met- expression is membranous and cytoplasmic.
  • an H-score from a sample of the patient's cancer using the formula (% cancer cells staining with weak c-met staining intensity) + (% cancer cells staining with moderate c-met staining intensity) + (% cancer cells staining with high c-met staining intensity).
  • an H-score of about 160 or higher, about 160 to about 230, about 230 or higher, or about 170 or higher indicates that the patient is likely to have increased overall survival (OS) and/or progression-free survival (PFS) when the patient is treated with a c-met antagonist.
  • OS overall survival
  • PFS progression-free survival
  • the patient has greater PFS and/or OS (e.g., relative to a patient who has an H score less than 160). In some embodiments, the patient has greater PFS (e.g., relative to a patient who has an H score less than 160). In some embodiments, the patient has greater OS (e.g., relative to a patient who has an H score less than 160). In some embodiments, the H-score is about 160 or higher. In some embodiments, the H-score is 160 or higher. In some embodiments, H-score is about 160 to about 230. In some embodiments, H- score is 160-230. . In some embodiments, the H score is greater than about 10. In some embodiments, the H score is greater than about 25.
  • the H score is greater than about 50. In some embodiments, the H score is greater than about 75. In some embodiments, the H score is greater than about 100. In some embodiments, the H score is greater than about 125. In some embodiments, the H score is greater than about 150. In some embodiments, the H score is greater than about 175. In some embodiments, the H score is greater than about 200.
  • c-met expression is membranous. In some embodiments, c-met expression is cytoplasmic. In some embodiments, c-met- expression is membranous and cytoplasmic.
  • the invention provides a method of determining patient prognosis, comprising determining an H-score from a sample of the patient's cancer using the formula (% cancer cells staining with weak c-met staining intensity) + (% cancer cells staining with moderate c-met staining intensity) + (% cancer cells staining with high c-met staining intensity), wherein an H-score of about 160 or higher, about 160 to about 230, about 230 or higher, or about 170 or higher indicates that the patient is likely to have poor prognosis. .
  • the H score is greater than about 10. In some embodiments, the H score is greater than about 25. In some embodiments, the H score is greater than about 50.
  • the H score is greater than about 75. In some embodiments, the H score is greater than about 100. In some embodiments, the H score is greater than about 125. In some embodiments, the H score is greater than about 150. In some embodiments, the H score is greater than about 175. In some embodiments, the H score is greater than about 200.
  • c-met expression is membranous. In some embodiments, c-met expression is cytoplasmic. In some embodiments, c-met- expression is membranous and cytoplasmic.
  • the invention provides a method of optimizing therapeutic efficacy comprising determining an H-score from a sample of the patient's cancer using the formula (% cancer cells staining with weak c-met staining intensity) + (% cancer cells staining with moderate c-met staining intensity) + (% cancer cells staining with high c-met staining intensity).
  • an H-score of about 160 or higher, about 160 to about 230, about 230 or higher, or about 170 or higher indicates that the patient is likely to have increased overall survival (OS) and/or progression- free survival (PFS) when the patient is treated with a c-met antagonist.
  • OS overall survival
  • PFS progression- free survival
  • the patient has greater PFS and/or OS (e.g., relative to a patient who has an H score less than 160). In some embodiments, the patient has greater PFS (e.g., relative to a patient who has an H score less than 160). In some embodiments, the patient has greater OS (e.g., relative to a patient who has an H score less than 160). In some embodiments, the H-score is about 160 or higher. In some embodiments, the H-score is 160 or higher. In some embodiments, H-score is about 160 to about 230. In some embodiments, H- score is 160-230. . In some embodiments, the H score is greater than about 10. In some embodiments, the H score is greater than about 25.
  • the H score is greater than about 50. In some embodiments, the H score is greater than about 75. In some embodiments, the H score is greater than about 100. In some embodiments, the H score is greater than about 125. In some embodiments, the H score is greater than about 150. In some embodiments, the H score is greater than about 175. In some embodiments, the H score is greater than about 200.
  • c-met expression is membranous. In some embodiments, c-met expression is cytoplasmic. In some embodiments, c-met- expression is membranous and cytoplasmic.
  • the c-met antagonist is an antagonist anti-c-met antibody.
  • the anti-c-met antibody comprises a (a) HVR1 comprising sequence GYTFTSYWLH (SEQ ID NO: 1); (b) HVR2 comprising sequence GMIDPSNSDTRFNPNFKD (SEQ ID NO: 2); (c) HVR3-HC comprising sequence ATYRS YVTPLDY (SEQ ID NO : 3); (d) HVR1 -LC comprising sequence
  • the anti-c-met antibody is monovalent and comprises (a) a first polypeptide comprising a heavy chain, said polypeptide comprising the sequence:
  • the c-met antagonist is one or more of crizotinib, tivantinib, carbozantinib, MGCD-265, ficlatuzumab, humanized TAK-701, rilotumumab, foretinib, h224Gl 1, 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 LA480.
  • the c-met antagonist is rilotumumab.
  • the c-met antagonist is crizotinib.
  • the c-met antagonist is tivantinib.
  • treatment with c-met antagonist is in combination with treatment with an EGFR antagonist.
  • the EGFR antagonist is erlotinib.
  • the c-met antagonist is onartuzumab and treatment further comprises treatment with erlotinib.
  • c-met antagonist is crizotinib, tivantinib, carbozantinib, MGCD-265, ficlatuzumab, humanized TAK-701, or foretinib, and treatment further comprises treatment with erlotinib.
  • the patient has NSCLC and is treated with a combination of anti-c-met antibody and an EGFR antagonist.
  • the EGFR antagonist is erlotinib.
  • the patient has NSCLC and is treated with (a) onartuzumab at a dose of about 15 mg/kg every three weeks; and (b) erlotinib (N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)-4-quinazolinamine HC1) at a dose of 150 mg, each day of a three week cycle.
  • methods for advertising a c-met antibody comprising promoting, to a target audience, the use of the c-met antibody for treating a patient with cancer based on an H-score from a sample of the patient's cancer, wherein the H-score is calculated using the formula (% cancer cells staining with weak c-met staining intensity) + (% cancer cells staining with moderate c-met staining intensity) + (% cancer cells staining with high c- met staining intensity).
  • the promotion is by a package insert accompanying a commercial formulation of an anti-c-met antibody.
  • promotion is by a package insert accompanying a commercial formulation of a second medicament.
  • the promotion is by a package insert accompanying a c- met antibody suitable for use in c-met IHC assay.
  • the second medicament is an EGFR antagonist.
  • the anti-c-met antibody is
  • the patient is selected for treatment with a c-met antagonist if the H-score is about 160 or higher, about 160 to about 230, about 230 or higher, or about 170 or higher.
  • the patient is treated with the c-met antibody if the H-score is about 160 or higher, about 160 to about 230, about 230 or higher, or about 170 or higher.
  • the H score is greater than about 10. In some embodiments, the H score is greater than about 25. In some embodiments, the H score is greater than about 50. In some embodiments, the H score is greater than about 75. In some embodiments, the H score is greater than about 100.
  • the H score is greater than about 125. In some embodiments, the H score is greater than about 150. In some embodiments, the H score is greater than about 175. In some embodiments, the H score is greater than about 200.
  • c-met expression is membranous. In some embodiments, c-met expression is cytoplasmic. In some embodiments, c-met- expression is membranous and cytoplasmic. In some embodiments, the promotion is by a package insert where the package insert provides instructions to receive therapy with anti-c-met antibody in combination with an EGFR antagonist. In some embodiments, the promotion is followed by the treatment of the patient with the anti-c-met antibody with or without the second
  • diagnostic kits comprising one or more reagent for determining an H-score from a sample of the patient's cancer using the formula (% cancer cells staining with weak c-met staining intensity) + (% cancer cells staining with moderate c-met staining intensity) + (% cancer cells staining with high c-met staining intensity), wherein an H- score of about 160 or higher, about 160 to about 230, about 230 or higher, or about 170 or higher indicates that the patient is likely to response to treatment with a c-met antagonist.
  • the patient has greater PFS and/or OS (e.g., relative to a patient who has an H score less than 160).
  • the patient has greater PFS (e.g., relative to a patient who has an H score less than 160). In some embodiments, the patient has greater OS (e.g., relative to a patient who has an H score less than 160). In some embodiments, the H- score is about 160 or higher. In some embodiments, the H-score is 160 or higher. In some embodiments, H-score is about 160 to about 230. In some embodiments, H-score is 160-230. . In some embodiments, the H score is greater than about 10. In some embodiments, the H score is greater than about 25. In some embodiments, the H score is greater than about 50. In some embodiments, the H score is greater than about 75.
  • the H score is greater than about 100. In some embodiments, the H score is greater than about 125. In some embodiments, the H score is greater than about 150. In some embodiments, the H score is greater than about 175. In some embodiments, the H score is greater than about 200.
  • c-met expression is membranous. In some embodiments, c-met expression is cytoplasmic. In some embodiments, c-met- expression is membranous and cytoplasmic.
  • diagnostic kits comprising one or more reagent for determining an H-score from a sample of the patient's cancer using the formula (% cancer cells staining with weak c-met staining intensity) + (% cancer cells staining with moderate c-met staining intensity) + (% cancer cells staining with high c-met staining intensity), wherein an H- score of about 160 or higher, about 160 to about 230, about 230 or higher, or about 170 or higher means the patient has greater PFS and/or OS (e.g., relative to a patient who has an H score less than 160). In some embodiments, the patient has greater PFS (e.g., relative to a patient who has an H score less than 160).
  • the patient has greater OS (e.g., relative to a patient who has an H score less than 160).
  • the diagnostic kits herein further comprise instructions to use the kit to select a c-met medicament to treat the NSCLC patient if the H-score is about 160 or higher, about 160 to about 230, about 230 or higher, or about 170 or higher. .
  • the H score is greater than about 10.
  • the H score is greater than about 25.
  • the H score is greater than about 50.
  • the H score is greater than about 75.
  • the H score is greater than about 100.
  • the H score is greater than about 125.
  • the H score is greater than about 150. In some embodiments, the H score is greater than about 175. In some embodiments, the H score is greater than about 200. In some embodiments, c-met expression is membranous. In some embodiments, c-met expression is cytoplasmic. In some embodiments, c-met- expression is membranous and cytoplasmic.
  • kits comprising a c-met antagonist, and instructions to use the c-met medicament to treat a patient if an H-score determined from a sample of the patient's cancer is about 160 or higher, about 160 to about 230, about 230 or higher, or about 170 or higher, wherein the H-score is calculated using the formula (% cancer cells staining with weak c-met staining intensity) + (% cancer cells staining with moderate c-met staining intensity) + (% cancer cells staining with high c-met staining intensity).
  • the patient has greater PFS and/or OS (e.g., relative to a patient who has an H score less than 160).
  • the patient has greater PFS (e.g., relative to a patient who has an H score less than 160). In some embodiments, the patient has greater OS (e.g., relative to a patient who has an H score less than 160). In some embodiments, the H-score is about 160 or higher. In some embodiments, the H-score is 160 or higher. In some embodiments, H-score is about 160 to about 230. In some embodiments, H-score is 160-230. In some embodiments, the H score is greater than about 10. In some embodiments, the H score is greater than about 25. In some embodiments, the H score is greater than about 50. In some embodiments, the H score is greater than about 75. In some embodiments, the H score is greater than about 100.
  • the H score is greater than about 125. In some embodiments, the H score is greater than about 150. In some embodiments, the H score is greater than about 175. In some embodiments, the H score is greater than about 200.
  • c-met expression is membranous. In some embodiments, c-met expression is cytoplasmic. In some embodiments, c-met- expression is membranous and cytoplasmic.
  • the invention also concerns articles of manufacture comprising, packaged together, a c- met antagonist in a pharmaceutically acceptable carrier and a package insert indicating that the c-met antagonist is for treating a patient with cancer based on expression of c-met biomarker.
  • Treatment methods include any of the treatment methods disclosed herein.
  • the package insert indicates that the c-met antagonist is to be used to treat the patient if the patient's cancer sample c-met antagonist, and instructions to use the c-met medicament to treat a patient if an H-score determined from a sample of the patient's cancer is about 160 or higher, about 160 to about 230, about 230 or higher, or about 170 or higher, wherein the H-score is calculated using the formula (% cancer cells staining with weak c-met staining intensity) + (% cancer cells staining with moderate c-met staining intensity) + (% cancer cells staining with high c-met staining intensity).
  • the patient has greater PFS and/or OS (e.g., relative to a patient who has an H score less than 160). In some embodiments, the patient has greater PFS (e.g., relative to a patient who has an H score less than 160). In some embodiments, the patient has greater OS (e.g., relative to a patient who has an H score less than 160). In some embodiments, the H-score is about 160 or higher. In some embodiments, the H-score is 160 or higher. In some embodiments, H-score is about 160 to about 230. In some embodiments, H-score is 160-230. In some embodiments, the H score is greater than about 10. In some embodiments, the H score is greater than about 25.
  • the H score is greater than about 50. In some embodiments, the H score is greater than about 75. In some embodiments, the H score is greater than about 100. In some embodiments, the H score is greater than about 125. In some embodiments, the H score is greater than about 150. In some embodiments, the H score is greater than about 175. In some embodiments, the H score is greater than about 200.
  • c-met expression is membranous. In some embodiments, c-met expression is cytoplasmic. In some embodiments, c-met- expression is membranous and cytoplasmic.
  • an H-score less than 160, or less than 159, 158, 157, 156, 155, 154, 153, 152, 151 or 150 means that the patient has lower PFS and/or OS (e.g., relative to a patient who has an H score greater than 160).
  • the patient has lower PFS (e.g., relative to a patient who has an H score less than 160).
  • the patient has lower OS (e.g., relative to a patient who has an H score greater than 160).
  • the H score is greater than about 10.
  • the H score is greater than about 25. In some embodiments, the H score is greater than about 50.
  • the H score is greater than about 75. In some embodiments, the H score is greater than about 100. In some embodiments, the H score is greater than about 125. In some embodiments, the H score is greater than about 150. In some embodiments, the H score is greater than about 175. In some embodiments, the H score is greater than about 200.
  • c-met expression is membranous. In some embodiments, c-met expression is cytoplasmic. In some embodiments, c-met- expression is membranous and cytoplasmic.
  • the invention concerns methods for manufacturing an article of manufacture comprising combining in a package a pharmaceutical composition comprising a cancer medicament and a package insert indicating that the pharmaceutical composition is for treating a patient with cancer based on expression of c-met biomarker.
  • Treatment methods include any of the treatment methods disclosed herein.
  • the package insert indicates that the c-met antagonist is to be used to treat the patient if the patient's cancer sample c-met antagonist, and instructions to use the c-met medicament to treat a patient if an H-score determined from a sample of the patient's cancer is about 160 or higher, about 160 to about 230, about 230 or higher, or about 170 or higher, wherein the H-score is calculated using the formula (% cancer cells staining with weak c-met staining intensity) + (% cancer cells staining with moderate c-met staining intensity) + (% cancer cells staining with high c-met staining intensity).
  • the patient has greater PFS and/or OS (e.g., relative to a patient who has an H score less than 160). In some embodiments, the patient has greater PFS (e.g., relative to a patient who has an H score less than 160). In some embodiments, the patient has greater OS (e.g., relative to a patient who has an H score less than 160). In some embodiments, the H-score is about 160 or higher. In some embodiments, the H-score is 160 or higher. In some embodiments, H-score is about 160 to about 230. In some embodiments, H- score is 160-230. In some embodiments, the H score is greater than about 10. In some embodiments, the H score is greater than about 25.
  • the H score is greater than about 50. In some embodiments, the H score is greater than about 75. In some embodiments, the H score is greater than about 100. In some embodiments, the H score is greater than about 125. In some embodiments, the H score is greater than about 150. In some embodiments, the H score is greater than about 175. In some embodiments, the H score is greater than about 200.
  • c-met expression is membranous. In some embodiments, c-met expression is cytoplasmic. In some embodiments, c-met- expression is membranous and cytoplasmic.
  • an H-score less than 160, or less than 159, 158, 157, 156, 155, 154, 153, 152, 151 or 150 means that the patient has lower PFS and/or OS (e.g., relative to a patient who has an H score greater than 160).
  • the patient has lower PFS (e.g., relative to a patient who has an H score less than 160).
  • the patient has lower OS (e.g., relative to a patient who has an H score greater than 160).
  • negative c-met staining intensity means c-met staining intensity of control cell line TOV-112D.
  • negative c-met staining intensity means c-met staining intensity of control cell line H522. In some embodiments, negative c-met staining intensity means c-met staining intensity of control cell line HI 155. In some embodiments, negative c-met staining intensity refers to c-met staining intensity of control cell line LXFL529. In some embodiments, negative c-met staining intensity means c-met staining intensity of control cell line H23. In some embodiments, weak c-met staining intensity means c-met staining intensity of control cell line HI 703. In some embodiments, weak c-met staining intensity means c-met staining intensity of control cell line HEK-293.
  • moderate c-met staining intensity means c-met staining intensity of control cell line A549. In some embodiments, moderate c-met staining intensity means c-met staining intensity of control cell line SKMES1. In some embodiments, strong c- met staining intensity means c-met staining intensity of control cell line EBC-1. In some embodiments, strong c-met staining intensity means c-met staining intensity of control cell line H441.
  • the sample is obtained prior to treatment with c-met antagonist. In some embodiments, the sample is obtained prior to treatment with a cancer medicament. In some embodiments, the sample is obtained after the cancer has metastasized. In some embodiments, the sample is formalin fixed and paraffin embedded. In some embodiments, the sample is of a biopsy, a surgical specimen, or a fine needle aspirate.
  • control cell pellets are formalin fixed and paraffin embedded. In some embodiments, the control cell pellets are prepared as a tissue microarray.
  • the c-met IHC used to determine c-met staining intensity is performed using anti-c-met antibody SP44. In some embodiments, c-met IHC used to determine c-met staining intensity is performed using anti-c-met antibody D1C1. In some embodiments, c-met IHC used to determine c-met staining intensity is performed using anti-c-met antibody Met4. In some embodiments, c-met IHC used to determine c-met staining intensity is performed using anti-c-met antibody DL-21.
  • the cancer is non-small cell lung cancer, renal cell cancer, pancreatic cancer, gastric carcinoma, bladder cancer, esophageal cancer, mesothelioma, melanoma, breast cancer, thyroid cancer, colorectal cancer, head and neck cancer, osteosarcoma, prostate cancer, or glioblastoma.
  • cancer is non-small cell lung cancer (NSCLC).
  • NSCLC is second-line or third-line locally advanced or metastatic non-small cell lung cancer.
  • the NSCLC is adenocarcinoma.
  • the NSCLC is squamous cell carcinoma.
  • the patient did not receive more than two prior treatments for Stage IIIB/IV. In some embodiments, the patient did not receive more than 30 days of exposure to an investigational or marketed agent that can act by EGFR inhibition, or a known EGFR-related toxicity resulting in dose modifications.
  • EGFR inhibitors include (but are not limited to) gefitinib, erlotinib, and cetuximab.
  • the patient did not receive chemotherapy, biologic therapy, radiotherapy or investigational drug within 28 days prior to randomization (except that optionally, kinase inhibitors may be used within two weeks prior to randomization provided any drug related toxicity was adequately resolved).
  • the patient is not a patient with untreated and/or active (progressing or requiring anticonvulsants or corticosteroids for symptomatic control) CNS metastasis.
  • a sample of the patient's cancer has been shown to have wildtype EGFR.
  • a sample of the patient's cancer has not been shown to have mutated EGFR.
  • Other patient exclusion criteria are described in the Examples, and the present inventions contemplate use of one or more of the exclusions described therein.
  • a patient's tumor is c-met positive when 1% or more of the tumor cells in the sample express c-met protein (e.g., express c-met protein at any intensity, e.g., low, moderate and high). In some embodiments, a patient's tumor is c-met positive when more than 1% of the tumor cells in the sample express c-met protein (e.g., express c-met protein at any intensity). In some embodiments, a patient's tumor is c-met positive when 5% or more of the tumor cells in the sample express c-met protein (e.g., express c-met protein at any intensity).
  • a patient's tumor is c-met positive when 10% or more of the tumor cells in the sample express c-met protein (e.g., express c-met protein at any intensity). In some embodiments, a patient's tumor is c-met positive when 15% or more of the tumor cells in the sample express c-met protein. In some embodiments, a patient's tumor is c-met positive when 20% or more of the tumor cells in the sample express c-met protein (e.g., express c-met protein at any intensity). In some embodiments, a patient's tumor is c-met positive when 25% or more of the tumor cells in the sample express c-met protein (e.g., express c-met protein at any intensity).
  • a patient's tumor is c-met positive when 30% or more of the tumor cells in the sample express c-met protein (e.g., express c-met protein at any intensity). In some embodiments, a patient's tumor is c-met positive when 35% or more of the tumor cells in the sample express c-met protein (e.g., express c-met protein at any intensity). In some embodiments, a patient's tumor is c-met positive when 40% or more of the tumor cells in the sample express c-met protein (e.g., express c-met protein at any intensity). In some
  • a patient's tumor is c-met positive when 45% or more of the tumor cells in the sample express c-met protein (e.g., express c-met protein at any intensity).
  • c-met protein e.g., express c-met protein at any intensity
  • a patient's tumor is c-met positive when 50% or more of the tumor cells in the sample express c-met protein (e.g., express c-met protein at any intensity).
  • c-met protein e.g., express c-met protein at any intensity.
  • a patient's tumor is c-met positive when 55% or more of the tumor cells in the sample express c-met protein (e.g., express c-met protein at any intensity).
  • c-met protein e.g., express c-met protein at any intensity.
  • a patient's tumor is c-met positive when 60% or more of the tumor cells in the sample express c-met protein (e.g., express c-met protein at any intensity).
  • c-met protein e.g., express c-met protein at any intensity.
  • a patient's tumor is c-met positive when 65% or more of the tumor cells in the sample express c-met protein (e.g., express c-met protein at any intensity).
  • c-met protein e.g., express c-met protein at any intensity.
  • a patient's tumor is c-met positive when 70% or more of the tumor cells in the sample express c-met protein (e.g., express c-met protein at any intensity).
  • c-met protein e.g., express c-met protein at any intensity.
  • a patient's tumor is c-met positive when 75% or more of the tumor cells in the sample express c-met protein (e.g., express c-met protein at any intensity).
  • c-met protein e.g., express c-met protein at any intensity.
  • a patient's tumor is c-met positive when 80% or more of the tumor cells in the sample express c-met protein (e.g., express c-met protein at any intensity).
  • c-met protein e.g., express c-met protein at any intensity.
  • a patient's tumor is c-met positive when 85% or more of the tumor cells in the sample express c-met protein (e.g., express c-met protein at any intensity).
  • c-met protein e.g., express c-met protein at any intensity.
  • a patient's tumor is c-met positive when 90% or more of the tumor cells in the sample express c-met protein (e.g., express c-met protein at any intensity).
  • c-met protein e.g., express c-met protein at any intensity.
  • a patient's tumor is c-met positive when 95% or more of the tumor cells in the sample express c-met protein (e.g., express c-met protein at any intensity).
  • c-met protein e.g., express c-met protein at any intensity.
  • c-met expression is membranous. In some embodiments, c-met expression is cytoplasmic. In some embodiments, c-met- expression is membranous and cytoplasmic.
  • a patient's tumor is c-met positive when a maximum staining intensity of the tumor is 1. In some embodiments, a patient's tumor is c-met positive when a maximum staining intensity of the tumor is 2. In some embodiments, a patient's tumor is c-met positive when a maximum staining intensity of the tumor is 3. In some embodiments, c-met expression is membranous. In some embodiments, c-met expression is cytoplasmic. In some embodiments, c-met- expression is membranous and cytoplasmic.
  • the c-met antagonist is an antagonist anti-c-met antibody.
  • the anti-c-met antibody comprises a (a) HVR1 comprising sequence GYTFTSYWLH (SEQ ID NO: 1); (b) HVR2 comprising sequence GMIDPSNSDTRFNPNFKD (SEQ ID NO: 2); (c) HVR3-HC comprising sequence ATYRSYVTPLDY (SEQ ID NO: 3); (d) HVRl-LC comprising sequence KSSQSLLYTSSQKNYLA (SEQ ID NO: 4); (e) HVR2-LC comprising sequence WASTRES (SEQ ID NO: 5); and (f) HVR3-LC comprising sequence QQYYAYPWT (SEQ ID NO: 6).
  • the anti-c-met antibody is monovalent and comprises (a) a first polypeptide comprising a heavy chain, said polypeptide comprising the sequence of SEQ ID NO: 11; (b) a second polypeptide comprising a light chain, the polypeptide comprising the sequence of SEQ ID NO: 12; and a third polypeptide comprising a Fc sequence, the polypeptide comprising the sequence of 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 a complex and form a Fc region that increases stability of said antibody fragment compared to a Fab molecule comprising said antigen binding arm.
  • the c-met antibody is monovalent and comprises (a) a first polypeptide comprising a heavy chain, said polypeptide comprising the sequence of SEQ ID NO: 11; (b) a second polypeptide comprising a light chain, the polypeptide comprising the sequence of
  • the c-met antagonist is any one or more of crizotinib, tivantinib, carbozantinib, MGCD-265, ficlatuzumab, humanized TAK-701, rilotumumab, foretinib, h224Gl l, 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 LA480.
  • Other c-met antagonists suitable for use in the present inventions are described herein.
  • Cancer medicaments can be used either alone or in combination with other cancer medicaments.
  • a c-met antagonist e.g., anti-c-met antibody
  • an EGFR antagonist e.g., erlotinib
  • erlotinib is administered at a dose of 150 mg, each day of a three week cycle.
  • erlotinib is administered at a dose of 100 mg, each day of a three week cycle.
  • erlotinib is administered at a dose of 50 mg, each day of a three week cycle.
  • An exemplary protocol is administering to a NSCLC patient (a) an anti-c-met antibody (such as Onartuzumab) at a dose of about 15 mg/kg every three weeks; and (b) erlotinib (N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)-4-quinazolinamine) at a dose of 150 mg, each day of a three week cycle.
  • a c-met antagonist e.g., anti-c-met antibody
  • an anti-VEGF antibody and chemotherapy e.g., a taxane
  • An exemplary protocol is administering to a triple-negative metastatic breast cancer patient an anti-c-met antibody (e.g., Onartuzumab) administered at a dose of 10 mg/kg on Day 1 and Day 15 of a 28-day cycle, anti-VEGF antibody (e.g., bevacizumab) administered at a dose of 10 mg/kg on Day 1 and Day 15 of the 28-day cycle and paclitaxel administered at a dose of 90 mg/m by IV infusion on Day 1, Day 8, and Day 15 of the 28-day cycle.
  • an anti-c-met antibody e.g., Onartuzumab
  • anti-VEGF antibody e.g., bevacizumab
  • paclitaxel administered at a dose of 90 mg/m by IV infusion on Day 1, Day 8, and Day 15 of the 28-day cycle.
  • Another exemplary protocol is administering to a triple-negative metastatic breast cancer patient an anti-c-met antibody (e.g., Onartuzumab) administered at a dose of 10 mg/kg on Day 1 and Day 15 of a 28-day cycle, and paclitaxel administered at a dose of 90 mg/m by IV infusion on Day 1, Day 8, and Day 15 of the 28-day cycle.
  • an anti-c-met antibody e.g., Onartuzumab
  • paclitaxel administered at a dose of 90 mg/m by IV infusion on Day 1, Day 8, and Day 15 of the 28-day cycle.
  • Onartuzumab is an anti-c-met antibody administered at a dose of 10 mg/kg on Day 1 and Day 15 of a 28-day cycle
  • paclitaxel administered at a dose of 90 mg/m by IV infusion on Day 1, Day 8, and Day 15 of the 28-day cycle.
  • Onartuzumab is an anti-c-met antibody administered at a dose of 10 mg/
  • crizotinib is used in combination with an EGFR antagonist (in some embodiments, erlotinib).
  • carbozantinib is used in combination with an EGFR antagonist (in some embodiments, erlotinib).
  • foretinib is used in combination with an EGFR antagonist (in some
  • erlotinib In some embodiments, erlotinib). In some embodiments, tivantinib is used in combination with an EGFR antagonist (in some embodiments, erlotinib). In some embodiments, MGCD-265 is used in combination with an EGFR antagonist (in some embodiments, erlotinib). In some embodiments, rilotumumab is used in combination with an EGFR antagonist (in some embodiments, erlotinib). In some embodiments, ficlatuzumab is used in combination with an EGFR antagonist (in some embodiments, erlotinib). In some embodiments, humanized anti- HGF antibody TAK-701 is used in combination with an EGFR antagonist (in some embodiments,
  • additional biomarkers may be detected in a patient's sample.
  • the patient's cancer has been found to express wildtype EGFR (in some embodiments, further expresses c-met gene amplification, and in still further embodiments, does not express c-met gene amplification).
  • the patient's cancer has been found to express mutated EGFR.
  • the patient's cancer has been found to express a biomarker selected from kras and EGFR.
  • the patient's cancer has been found to express mutated kras.
  • the patient's cancer has been found to express wildtype kras.
  • the patient's cancer e.g., the patient's NSCLC
  • the patient's cancer has been found to express an anaplastic lymphoma kinase (ALK) translocation.
  • ALK anaplastic lymphoma kinase
  • the ALK translocation is an EML4-ALK translocation.
  • the patient's cancer has been found to express mutated c-met.
  • the patient's cancer has been found to express wildtype c-met.
  • the invention provides a method for evaluating adverse events in a patient associated with treatment of a cancer that expresses a high amount of c-met biomarker using any of the methods disclosed herein, wherein treatment is with a c-met antagonist (e.g., onartuzumab) and the method comprises the steps of monitoring the number and/or severity of one or more adverse events.
  • a c-met antagonist e.g., onartuzumab
  • Figures 1A-C show examples of anti-c-met antibody SP44 IHC staining patterns in human lung tissues. Representative images depicting the range of SP44 staining intensities (A, negative, weak, moderate and strong) and the frequently observed heterogeneity in this staining pattern (B) in malignant NSCLC tissues. C, Representative images of SP44 staining of benign lung tissues depicting reactivity in respiratory mucosa and pneumocytes.
  • Figure 2 shows relationship of H-score with clinical scoring metric.
  • Figures 3A-C show detailed breakdown of anti-c-met antibody SP44 staining intensity and proportion in NSCLC specimens, with H-score.
  • a "patient” is a human patient.
  • the patient may be a "cancer patient,” i.e. one who is suffering or at risk for suffering from one or more symptoms of cancer.
  • the patient may be a previously treated cancer patient.
  • the patient may be a "NSCLC cancer patient,”/. e. one who is suffering or at risk for suffering from one or more symptoms of NSCLC.
  • the patient may be a previously treated NSCLC patient.
  • c-met or “Met”, as used herein, refers, unless indicated otherwise, to any native or variant (whether native or synthetic) c-met polypeptide.
  • wild type c-met generally refers to a polypeptide comprising the amino acid sequence of a naturally occurring c-met protein.
  • wild type c-met sequence generally refers to an amino acid sequence found in a naturally occurring c-met.
  • an "anti-c-met antibody” is an antibody that binds to c-met with sufficient affinity and specificity.
  • c-met antibodies bind human c-met.
  • the antibody selected will normally have a sufficiently strong binding affinity for c-met, for example, the antibody may bind human c-met with a 3 ⁇ 4 value of between 100 nM-1 pM.
  • Antibody affinities may be determined by a surface plasmon resonance based assay (such as the BIAcore assay as described in PCT Application Publication No. WO2005/012359); enzyme-linked
  • the anti-c-met antibody can be used as a therapeutic agent in targeting and interfering with diseases or conditions wherein c-met activity is involved.
  • the antibody may be subjected to other biological activity assays, e.g., in order to evaluate its effectiveness as a therapeutic.
  • Such assays are known in the art and depend on the target antigen and intended use for the antibody.
  • c-met antagonist is an agent that interferes with c-met activation or function.
  • c-met inhibitors include c-met antibodies; HGF antibodies; small molecule c-met antagonists; c-met tyrosine kinase inhibitors; antisense and inhibitory RNA (e.g., shRNA) molecules (see, for example,
  • the c-met inhibitor is an antibody or small molecule which binds to c-met.
  • a c-met inhibitor has a binding affinity (dissociation constant) to c-met of about 1 ,000 nM or less.
  • a c-met inhibitor has a binding affinity to c-met of about 100 nM or less.
  • a c-met inhibitor has a binding affinity to c-met of about 50 nM or less.
  • a c-met inhibitor is covalently bound to c-met.
  • a c-met inhibitor inhibits c- met signaling with an IC50 of 1 ,000 nM or less. In another embodiment, a c-met inhibitor inhibits c-met signaling with an IC50 of 500 nM or less. In another embodiment, a c-met inhibitor inhibits c-met signaling with an IC50 of 50 nM or less.
  • C-met activation refers to activation, or phosphorylation, of the c-met receptor.
  • c-met activation results in signal transduction (e.g. that caused by an intracellular kinase domain of a c-met receptor phosphorylating tyrosine residues in c-met or a substrate polypeptide).
  • C-met activation may be mediated by c-met ligand (HGF) binding to a c-met receptor of interest.
  • HGF binding to c-met may activate a kinase domain of c-met and thereby result in phosphorylation of tyrosine residues in the c-met and/or phosphorylation of tyrosine residues in additional substrate polyp eptides(s).
  • a "population" of subjects refers to a group of subjects with cancer, such as in a clinical trial, or as seen by oncologists following FDA approval for a particular indication, such as breast cancer therapy.
  • biomarker does not exhibit an expression level that is above background level that is of statistical significance.
  • little to no biomarker expression with respect to a biomarker, as used herein, means the biomarker does not display a biologically meaningful amount of expression.
  • amount of expression may be determined quantitatively or qualitatively, so long as a comparison between a biomarker sample and a reference counterpart can be done.
  • the expression can be measured or detected according to any assay or technique known in the art, including, e.g., those described herein (such as IHC).
  • gene amplification refers to a process by which multiple copies of a gene or gene fragment are formed in a particular cell or cell line.
  • the term "instructing" a patient means providing directions for applicable therapy, medication, treatment, treatment regimens, and the like, by any means, but preferably in writing, such as in the form of package inserts or other written promotional material.
  • the term "promoting" means offering, advertising, selling, or describing a particular drug, combination of drugs, or treatment modality, by any means, including writing, such as in the form of package inserts. Promoting herein refers to promotion of therapeutic agent(s), such as an anti-c-met antibody (e.g., onartuzumab) and/or erlotinib, for an indication, such as NSCLC treatment, where such promoting is authorized by the Food and Drug Administration (FDA) as having been demonstrated to be associated with statistically significant therapeutic efficacy and acceptable safety in a population of subjects
  • FDA Food and Drug Administration
  • marketing is used herein to describe the promotion, selling or distribution of a product (e.g., drug). Marketing specifically includes packaging, advertising, and any business activity with the purpose of commercializing a product.
  • cancer medicament is a drug effective for treating cancer.
  • cancer medicaments include the chemotherapeutic agents and chemotherapy regimens noted below; c- met antagonists, including anti-c-met antibodies, such as onartuzumab.
  • biomarker refers generally to a molecule, including a gene, mRNA, protein, carbohydrate structure, or glycolipid, the expression of which in or on a tissue or cell or secreted can be detected by known methods (or methods disclosed herein) and is predictive or can be used to predict (or aid prediction) for a cell, tissue, or patient's responsiveness to treatment regimes.
  • the biomarker of particular interest herein is c-met.
  • negative c-met staining intensity or “negative staining intensity” means c-met staining intensity of TOV-112D, H522, HI 155, LXFL529 and/or H23.
  • negative c-met staining intensity means c-met staining intensity of control cell line TOV-112D.
  • negative c-met staining intensity means c-met staining intensity of control cell line H522.
  • negative c-met staining intensity means c-met staining intensity of control cell line HI 155.
  • negative c- met staining intensity refers to c-met staining intensity of control cell line LXFL529.
  • negative c-met staining intensity means c-met staining intensity of control cell line H23.
  • Methods for c-met IHC are known in the art and exemplified herein.
  • c-met staining intensity is determined using c-met antibody (e.g., SP44) staining of formalin-fixed paraffin embedded cell control cell pellets (e.g., prepared in a tissue microarray).
  • weak c-met staining intensity or “weak staining intensity” means c- met IHC staining intensity of control cell line H1703, HEK-293, and/or H460.
  • weak c-met staining intensity means c-met staining intensity of control cell line HI 703.
  • weak c-met staining intensity means c-met staining intensity of control cell line HEK-293.
  • weak c-met staining intensity means c-met staining intensity of control cell line H460.
  • Methods for c-met IHC are known in the art and exemplified herein.
  • c-met staining intensity is determined using c-met antibody (e.g., SP44) staining of formalin-fixed paraffin embedded cell control cell pellets (e.g., prepared in a tissue microarray).
  • moderate c-met staining intensity or “moderate staining intensity” means c-met IHC staining intensity of control cell line A549 and/or SKMES1.
  • moderate c-met staining intensity means c-met staining intensity of control cell line A549.
  • moderate c-met staining intensity means c-met staining intensity of control cell line SKMES1.
  • Methods for c-met IHC are known in the art and exemplified herein.
  • c-met staining intensity is determined using c-met antibody (e.g., SP44) staining of formalin-fixed paraffin embedded cell control cell pellets (e.g., prepared in a tissue microarray).
  • strong c-met staining intensity or “strong staining intensity” means c- met IHC staining intensity of control cell line EBC-1 and/or H441.
  • strong c-met staining intensity means c-met staining intensity of control cell line EBC-1.
  • strong c-met staining intensity means c-met staining intensity of control cell line H441.
  • Methods for c-met IHC are known in the art and exemplified herein.
  • c-met staining intensity is determined using c-met antibody (e.g., SP44) staining of formalin-fixed paraffin embedded cell control cell pellets (e.g., prepared in a tissue microarray).
  • tissue sample a collection of cells obtained from a cancer patient.
  • the source of the tissue or cell sample may be solid tissue as from a fresh, frozen and/or preserved organ or tissue sample or biopsy or aspirate; blood or any blood constituents; bodily fluids such as cerebral spinal fluid, amniotic fluid, peritoneal fluid, or interstitial fluid; cells from any time in gestation or development of the subject.
  • the tissue sample may contain compounds which are not naturally intermixed with the tissue in nature such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, or the like.
  • tumor samples herein include, but are not limited to, tumor biopsies, fine needle aspirate, bronchiolar lavage, pleural fluid, sputum, a surgical specimen, circulating tumor cells, serum or plasma, circulating plasma proteins, ascitic fluid, primary cell cultures or cell lines derived from tumors or exhibiting tumor-like properties, as well as preserved tumor samples, such as formalin-fixed, paraffin- embedded tumor samples or frozen tumor samples.
  • the sample comprises NSCLC (e.g., squamous subtype or nonsquamous subtype) tumor sample.
  • an "effective response" of a patient or a patient's “responsiveness” to treatment with a medicament and similar wording refers to the clinical or therapeutic benefit imparted to a patient at risk for, or suffering from, cancer (e.g., NSCLC) upon administration of the cancer medicament.
  • Such benefit includes any one or more of: extending survival (including overall survival and progression free survival); resulting in an objective response (including a complete response or a partial response); or improving signs or symptoms of cancer, etc.
  • the biomarker (e.g., c-met expression, for example, as determined using IHC) is used to identify the patient who is expected to have greater progression free survival (PFS) when treated with a medicament (e.g., anti-c-met antibody), relative to a patient who does not express the biomarker at the same level.
  • PFS progression free survival
  • the biomarker is used to identify the patient who is expected to have reduced PFS when treated with a medicament, relative to a patient treated with the medicament who does not express the biomarker at the same level, or relative to a patient who is not treated with the medicament who does not express the biomarker at the same level.
  • the biomarker is used to identify the patient who is expected to have greater overall survival (OS) when treated with a medicament, relative to a patient who does not express the biomarker at the same level. In one embodiment, the biomarker is used to identify the patient who is expected to have reduced overall survival (OS), relative to a patient who is treated with the medicament who does not express the biomarker at the same level, or relative to a patient who is not treated with the medicament who does not express the biomarker at the same level.
  • OS overall survival
  • “Survival” refers to the patient remaining alive, and includes overall survival as well as progression free survival.
  • “Overall survival” refers to the patient remaining alive for a defined period of time, such as 1 year, etc from the time of diagnosis or treatment.
  • progression free survival refers to the patient remaining alive, without the cancer progressing or getting worse.
  • extending survival is meant increasing overall or progression free survival in a treated patient relative to an untreated patient (i.e. relative to a patient not treated with the medicament), or relative to a patient who does not express a biomarker at the designated level, and/or relative to a patient treated with an approved anti-tumor agent (such as chemotherapy regimen of erlotinib.
  • an approved anti-tumor agent such as chemotherapy regimen of erlotinib.
  • An “objective response” refers to a measurable response, including complete response (CR) or partial response (PR).
  • Partial response refers to a decrease in the size of one or more tumors or lesions, or in the extent of cancer in the body, in response to treatment.
  • level of expression or “expression level” in general are used interchangeably and generally refer to the amount of a polynucleotide, mR A, or an amino acid product or protein in a biological sample.
  • Expression generally refers to the process by which gene-encoded information is converted into the structures present and operating in the cell. Therefore, according to the invention "expression” of a gene may refer to transcription into a polynucleotide, translation into a protein, or even posttranslational modification of the protein.
  • Fragments of the transcribed polynucleotide, the translated protein, or the post- translationally modified protein shall also be regarded as expressed whether they originate from a transcript generated by alternative splicing or a degraded transcript, or from a post- translational processing of the protein, e.g., by proteolysis.
  • level of expression refers to amount of a protein in a biological sample as determined using IHC.
  • based on expression of when used herein means that information about expression level of the one or more biomarkers herein is used to inform a treatment decision, information provided on a package insert, or marketing/promotional guidance etc.
  • a cancer e.g. NSCLC
  • a c-met antagonist e.g., anti-c-met antibody, e.g., Onartuzumab
  • patients may be treated with a cancer medicament other than c-met antagonist (e.g., other than anti-c-met antibody(e.g.,
  • Treatment refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already having a benign, pre-cancerous, or non-metastatic tumor as well as those in which the occurrence or recurrence of cancer is to be prevented.
  • therapeutically effective amount refers to an amount of a therapeutic agent (medicament) to treat or prevent a disease or disorder in a mammal.
  • the therapeutically effective amount of the therapeutic agent may reduce the number of cancer cells; reduce the primary tumor size; inhibit (i.e., slow to some extent and preferably stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and preferably stop) tumor metastasis; inhibit, to some extent, tumor growth; and/or relieve to some extent one or more of the symptoms associated with the disorder.
  • the drug may prevent growth and/or kill existing cancer cells, it may be cytostatic and/or cytotoxic.
  • efficacy in vivo can, for example, be measured by assessing the duration of survival, time to disease progression (TTP), the response rates (RR), duration of response, and/or quality of life.
  • 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.
  • head stage cancer or “early stage tumor” is meant a cancer that is not invasive or metastatic or is classified as a Stage 0, 1, or II cancer. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma (including
  • medulloblastoma and retinoblastoma include sarcoma (including liposarcoma and synovial cell sarcoma), neuroendocrine tumors (including carcinoid tumors, gastrinoma, and islet cell cancer), mesothelioma, schwannoma (including acoustic neuroma), meningioma,
  • adenocarcinoma melanoma
  • leukemia or lymphoid malignancies More particular examples of such cancers include squamous cell cancer (e.g. epithelial squamous cell cancer), lung cancer including small-cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer (including metastatic breast cancer), colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, testicular cancer, esophageal cancer, tumors of the biliary tract, as well
  • the cancer is triple-negative metastatic breast cancer, including any histologically confirmed triple-negative (ER-, PR-, HER2-) adenocarcinoma of the breast with locally recurrent or metastatic disease (where the locally recurrent disease is not amenable to resection with curative intent).
  • a cancer or biological sample which "displays c-met expression" is one which, in a diagnostic test, expresses (including overexpresses) a c-met receptor.
  • a cancer or biological sample which "displays c-met amplification" is one which, in a diagnostic test, has amplified c-met gene.
  • amplified c-met gene is an average (in a population of cell) of greater than or equal to 5 or more copies of the c-met gene, or an average of eight or more copies of a c-met gene, or more.
  • a cancer or biological sample which "does not display c-met amplification" is one which, in a diagnostic test, does not have amplified c-met gene.
  • a sample which does not display c-met amplification is a sample which has an average of fewer than 4 copies of c-met gene.
  • EGFR is meant the receptor tyrosine kinase polypeptide Epidermal Growth Factor Receptor which is described in Ullrich et al, Nature (1984) 309:418425, alternatively referred to as Her-1 and the c-erbB gene product, as well as variants thereof such as EGFRvIII.
  • Variants of EGFR also include deletional, substitutional and insertional variants, for example those described in Lynch et al (New England Journal of Medicine 2004, 350:2129), Paez et al (Science 2004, 304: 1497), Pao et al (PNAS 2004, 101 : 13306).
  • EGFR antagonist is an agent that interferes with EGFR activation or function.
  • EGFR inhibitors include EGFR antibodies; EGFR ligand antibodies; small molecule EGFR antagonists; EGFR tyrosine kinase inhibitors; antisense and inhibitory RNA (e.g., shRNA) molecules (see, for example,
  • the EGFR inhibitor is an antibody or small molecule which binds to EGFR.
  • the EGFR inhibitor is an EGFR-targeted drug.
  • an EGFR inhibitor has a binding affinity (dissociation constant) to EGFR of about 1 ,000 nM or less.
  • an EGFR inhibitor has a binding affinity to EGFR of about 100 nM or less.
  • an EGFR inhibitor has a binding affinity to EGFR of about 50 nM or less.
  • an EGFR inhibitor is covalently bound to EGFR.
  • an EGFR inhibitor inhibits EGFR signaling with an IC50 of 1 ,000 nM or less. In another embodiment, an EGFR inhibitor inhibits EGFR signaling with an IC50 of 500 nM or less. In another embodiment, an EGFR inhibitor inhibits EGFR signaling with an IC50 of 50 nM or less. In certain embodiments, the EGFR antagonist reduces or inhibits, by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more, the expression level or biological activity of EGFR.
  • EGFR activation refers to activation, or phosphorylation, of EGFR. Generally, EGFR activation results in signal transduction (e.g. that caused by an intracellular kinase domain of EGFR receptor phosphorylating tyrosine residues in EGFR or a substrate polypeptide). EGFR activation may be mediated by EGFR ligand binding to a EGFR dimer comprising EGFR.
  • EGFR ligand binding to a EGFR dimer may activate a kinase domain of one or more of the EGFR in the dimer and thereby results in phosphorylation of tyrosine residues in one or more of the EGFR and/or phosphorylation of tyrosine residues in additional substrate
  • EGFR-targeted drug refers to a therapeutic agent that binds to EGFR and inhibits EGFR activation.
  • agents include antibodies and small molecules that bind to EGFR.
  • antibodies which bind to EGFR include MAb 579 (ATCC CRL HB 8506), MAb 455 (ATCC CRL HB8507), MAb 225 (ATCC CRL 8508), MAb 528 (ATCC CRL 8509) (see, US Patent No.
  • EGFR human antibodies that bind EGFR
  • human antibodies that bind EGFR such as ABX-EGF (see WO98/50433, Abgenix); EMD 55900 (Stragliotto et al. Eur. J. Cancer 32A:636-640 (1996)); EMD7200 (matuzumab) a humanized EGFR antibody directed against EGFR that competes with both EGF and TGF- alpha for EGFR binding; and mAb 806 or humanized mAb 806 (Johns et al., J. Biol. Chem. 279(29):30375-30384 (2004)).
  • the anti-EGFR antibody may be conjugated with a cytotoxic agent, thus generating an immunoconjugate (see, e.g., EP659,439A2, Merck Patent GmbH).
  • a cytotoxic agent see, e.g., EP659,439A2, Merck Patent GmbH.
  • small molecules that bind to EGFR include ZD 1839 or Gefitinib (IRESSA; Astra Zeneca); CP-358774 or Erlotinib (TARCEVATM; Genentech/OSI); and AG1478, AG1571 (SU 5271; Sugen); EMD-7200.
  • polynucleotide when used in singular or plural, generally refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA.
  • polynucleotides as defined herein include, without limitation, single- and double-stranded DNA, DNA including single- and double-stranded regions, single- and double-stranded RNA, and RNA including single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or include single- and double-stranded regions.
  • polynucleotide refers to triple- stranded regions comprising RNA or DNA or both RNA and DNA.
  • the strands in such regions may be from the same molecule or from different molecules.
  • the regions may include all of one or more of the molecules, but more typically involve only a region of some of the molecules.
  • One of the molecules of a triple- helical region often is an oligonucleotide.
  • polynucleotide specifically includes cDNAs.
  • the term includes DNAs (including cDNAs) and RNAs that contain one or more modified bases.
  • DNAs or RNAs with backbones modified for stability or for other reasons are “polynucleotides” as that term is intended herein.
  • DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritiated bases are included within the term “polynucleotides” as defined herein.
  • polynucleotide embraces all chemically, enzymatically and/or metabolically modified forms of unmodified polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including simple and complex cells.
  • oligonucleotide refers to a relatively short polynucleotide, including, without limitation, single-stranded deoxyribonucleotides, single- or double-stranded
  • Oligonucleotides such as single- stranded DNA probe oligonucleotides, are often synthesized by chemical methods, for example using automated oligonucleotide synthesizers that are commercially available.
  • oligonucleotides can be made by a variety of other methods, including in vitro recombinant DNA-mediated techniques and by expression of DNAs in cells and organisms.
  • chemotherapeutic agent is a chemical compound useful in the treatment of cancer.
  • examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclosphosphamide (CYTOXAN®); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa;
  • ethylenimines and methylamelamines including altretamine, triethylenemelamine,
  • acetogenins especially bullatacin and bullatacinone
  • camptothecin including the synthetic analogue topotecan (HYCAMTIN®), CPT-11
  • calicheamicin especially calicheamicin gamma II and calicheamicin omegall (see, e.g., Nicolaou et al., Angew. Chem Intl. Ed. Engl., 33: 183-186 (1994));
  • ADRIAMYCIN® morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino- doxorubicin, doxorubicin HC1 liposome injection (DOXIL®), liposomal doxorubicin TLC D- 99 (MYOCET®), peglylated liposomal doxorubicin (CAELYX®), and deoxydoxorubicin
  • epirubicin esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate, gemcitabine (GEMZAR®), tegafur
  • folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate
  • purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine
  • pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine
  • anti-adrenals such as aminoglutethimide, mitotane, trilostane
  • folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defof
  • paclitaxel TAXOL®
  • albumin- engineered nanoparticle formulation of paclitaxel ABRAXANETM
  • docetaxel TAXOL®
  • TXOTERE® chloranbucil; 6-thioguanine; mercaptopurine; methotrexate; platinum agents such as cisplatin, oxaliplatin, and carboplatin; vincas, which prevent tubulin polymerization from forming microtubules, including vinblastine (VELBAN®), vincristine (ONCOVIN®), vindesine (ELDISINE®, FILDESIN®), and vinorelbine (NAVELBINE®); etoposide (VP- 16); ifosfamide; mitoxantrone; leucovovin; novantrone; edatrexate; daunomycin; aminopterin; ibandronate; topoisomerase inhibitor RFS 2000; difluorometlhylornithine (DMFO); retinoids such as retinoic acid, including bexarotene (TARGRETIN®); bisphosphonates such as clodronate (for example, BONEFOS
  • AREDIA® tiludronate
  • SKELID® tiludronate
  • ACTONEL® risedronate
  • troxacitabine a 1 ,3- dioxolane nucleoside cytosine analog
  • antisense oligonucleotides particularly those that inhibit expression of genes in signaling pathways implicated in aberrant cell proliferation, such as, for example, PKC-alpha, Raf, H-Ras, and epidermal growth factor receptor (EGF-R)
  • vaccines such as THERATOPE® vaccine and gene therapy vaccines, for example,
  • ALLOVECTIN® vaccine, LEUVECTIN® vaccine, and VAXID® vaccine ; topoisomerase 1 inhibitor (e.g., LURTOTECAN®); rmRH (e.g., ABARELLX®); BAY439006 (sorafenib; Bayer); SU- 1 1248 (Pfizer); perifosine, COX-2 inhibitor (e.g. celecoxib or etoricoxib), proteosome inhibitor (e.g.
  • ELOXATINTM oxaliplatin
  • chemotherapeutic agents include “anti-hormonal agents” or “endocrine therapeutics” which act to regulate, reduce, block, or inhibit the effects of hormones that can promote the growth of cancer. They may be hormones themselves, including, but not limited to: anti-estrogens with mixed agonist/antagonist profile, including, tamoxifen
  • SERMs such as SERM3
  • pure anti-estrogens without agonist properties such as fulvestrant (FASLODEX®), and EM800 (such agents may block estrogen receptor (ER) dimerization, inhibit DNA binding, increase ER turnover, and/or suppress ER levels); aromatase inhibitors, including steroidal aromatase inhibitors such as formestane and exemestane (AROMASIN®), and nonsteroidal aromatase inhibitors such as anastrazole (ARIMIDEX®), letrozole
  • FEMARA® aminoglutethimide
  • other aromatase inhibitors include vorozole (RIVISOR®), megestrol acetate (MEGASE®), fadrozole, and 4(5)-imidazoles; lutenizing hormone-releaseing hormone agonists, including leuprolide (LUPRON® and ELIGARD®), goserelin, buserelin, and tripterelin; sex steroids, including progestines such as megestrol acetate and medroxyprogesterone acetate, estrogens such as diethylstilbestrol and premarin, and androgens/retinoids such as fluoxymesterone, all transretionic acid and fenretinide;
  • progestines such as megestrol acetate and medroxyprogesterone acetate
  • estrogens such as diethylstilbestrol and premarin
  • androgens/retinoids such as fluoxymesterone, all transretionic acid and
  • chemotherapeutic agents or chemotherapy regimens herein include: alkylating agents (e.g. chlorambucil, bendamustine, or cyclophosphamide); nucleoside analogues or antimetabolites (e.g. fludarabine), fludarabine and cyclophosphamide (FC);
  • alkylating agents e.g. chlorambucil, bendamustine, or cyclophosphamide
  • nucleoside analogues or antimetabolites e.g. fludarabine), fludarabine and cyclophosphamide (FC)
  • prednisone or prednisolone akylator-containing combination therapy, including
  • cyclophosphamide vincristine, prednisolone (CHOP), or cyclophosphamide, vincristine, prednisolone (CVP), etc.
  • a "target audience” is a group of people or an institution to whom or to which a particular medicament is being promoted or intended to be promoted, as by marketing or advertising, especially for particular uses, treatments, or indications, such as individual patients, patient populations, readers of newspapers, medical literature, and magazines, television or internet viewers, radio or internet listeners, physicians, drug companies, etc.
  • a "package insert” is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, contraindications, other therapeutic products to be combined with the packaged product, and/or warnings concerning the use of such therapeutic products, etc.
  • antibody herein is used in the broadest sense and encompasses various 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.
  • antibody fragment refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds.
  • antibody fragments include but are not limited to Fv, Fab, Fab', Fab'-SH, F(ab') 2 ; diabodies; linear antibodies; single-chain antibody molecules (e.g. scFv); and multispecific antibodies formed from antibody fragments.
  • an “affinity matured” antibody refers to an antibody with one or more alterations in one or more hypervariable regions (HVRs), compared to a parent antibody which does not possess such alterations, such alterations resulting in an improvement in the affinity of the antibody for antigen.
  • HVRs hypervariable regions
  • 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 in a competition assay by 50% or more, and conversely, the reference antibody blocks binding of the antibody to its antigen in a competition assay by 50% or more.
  • 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 possessed by its heavy chain.
  • the heavy chain constant domains that correspond to the different classes of immunoglobulins are called ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ , respectively.
  • cytotoxic agent refers to a substance that inhibits or prevents a cellular function and/or causes cell death or destruction.
  • Cytotoxic agents include, but are not limited to, radioactive isotopes (e.g., At 211 , 1 131 , 1 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212 and radioactive isotopes of Lu); chemotherapeutic agents or drugs (e.g., methotrexate, adriamicin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents); growth inhibitory agents; enzymes and fragments thereof such as nucleolytic enzymes; antibiotics; toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal
  • Antibody effector functions refer to those biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype. Examples of antibody effector functions include: Clq 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 receptor); and B cell activation.
  • Fc region herein is used to define a C-terminal region of an
  • immunoglobulin heavy chain that contains at least a portion of the constant region.
  • the term includes native sequence Fc regions and variant Fc regions.
  • a human IgG heavy chain Fc region extends from Cys226, or from Pro230, to the carboxyl-terminus of the heavy chain.
  • the C-terminal lysine (Lys447) of the Fc region may or may not be present.
  • numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.
  • FR Framework or "FR” refers to variable domain residues other than hypervariable region (HVR) residues.
  • the FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the HVR and FR sequences generally appear in the following sequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.
  • full length antibody “intact antibody,” and “whole antibody” are used herein interchangeably to refer to an antibody having a structure substantially similar to a native antibody structure or having heavy chains that contain an Fc region as defined herein.
  • a "human antibody” is one which possesses an amino acid sequence which corresponds to that of an antibody produced by a human or a human cell or derived from a non-human source that utilizes human antibody repertoires or other human antibody-encoding sequences. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues.
  • a “humanized” antibody refers to a chimeric antibody comprising amino acid residues from non-human HVRs and amino acid residues from human FRs.
  • a humanized antibody will comprise substantially all of at least one, and typically two, 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.
  • a humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody.
  • a "humanized form" of an antibody, e.g., a non-human antibody refers to an antibody that has undergone humanization.
  • hypervariable region refers to each of the regions of an antibody variable domain which are hypervariable in sequence and/or form structurally defined loops ("hypervariable loops").
  • native four-chain antibodies comprise six HVRs; three in the VH (HI, H2, H3), and three in the VL (LI, L2, L3).
  • HVRs generally comprise amino acid residues from the hypervariable loops and/or from the "complementarity determining regions" (CDRs), the latter being of highest sequence variability and/or involved in antigen recognition.
  • CDRs complementarity determining regions
  • Exemplary hypervariable loops occur at amino acid residues 26-32 (LI), 50-52 (L2), 91-96 (L3), 26-32 (HI), 53-55 (H2), and 96-101 (H3).
  • Exemplary CDRs CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 occur at amino acid residues 24-34 of LI, 50-56 of L2, 89-97 of L3, 31-35B of HI, 50-65 of H2, and 95-102 of H3.
  • CDRs generally comprise the amino acid residues that form the hypervariable loops.
  • CDRs also comprise "specificity determining residues,” or "SDRs,” which are residues that contact antigen. SDRs are contained within regions of the CDRs called abbreviated-CDRs, or a-CDRs.
  • Exemplary a-CDRs (a-CDR-Ll, a- CDR-L2, a-CDR-L3, a-CDR-Hl, a-CDR-H2, and a-CDR-H3) occur at amino acid residues 31- 34 of LI, 50-55 of L2, 89-96 of L3, 31-35B of HI, 50-58 of H2, and 95-102 of H3. (See Almagro and Fransson, Front. Biosci. 13: 1619-1633 (2008)). Unless otherwise indicated, HVR residues and other residues in the variable domain (e.g., FR residues) are numbered herein according to Kabat et al., supra. In one embodiment, the c-met antibody herein comprises the HVRs of SEQ ID NOs: 1-6.
  • an “immunoconjugate” is an antibody conjugated to one or more heterologous molecule(s), including but not limited to a cytotoxic agent.
  • 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 possible variant antibodies, e.g., containing naturally occurring mutations or arising during production of a monoclonal antibody preparation, such variants generally being present in minor amounts.
  • polyclonal antibody preparations typically include different antibodies directed against different determinants (epitopes)
  • each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen.
  • the modifier "monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
  • the monoclonal antibodies to be used in accordance with the present invention may be made by a variety of techniques, including but not limited to the hybridoma method, recombinant DNA methods, phage-display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, such methods and other exemplary methods for making monoclonal antibodies being described herein.
  • a “naked antibody” refers to an antibody that is not conjugated to a heterologous moiety (e.g., a cytotoxic moiety) or radiolabel. The naked antibody may be present in a pharmaceutical formulation.
  • Native antibodies refer to naturally occurring immunoglobulin molecules with varying structures.
  • native IgG antibodies are heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light chains and two identical heavy chains that are disulfide-bonded. From N- to C-terminus, each heavy chain has a variable region (VH), also called a variable heavy domain or a heavy chain variable domain, followed by three constant domains (CHI, CH2, and CH3).
  • VH variable region
  • VL variable region
  • the light chain of an antibody may be assigned to one of two types, called kappa ( ⁇ ) and lambda ( ⁇ ), based on the amino acid sequence of its constant domain.
  • pharmaceutical formulation refers to a sterile preparation that is in such form as to permit the biological activity of the medicament to be effective, and which contains no additional components that are unacceptably toxic to a subject to which the formulation would be administered.
  • a "sterile" formulation is aseptic or free from all living microorganisms and their spores.
  • a “kit” is any manufacture (e.g a package or container) comprising at least one reagent, e.g., a medicament for treatment of cancer (e.g., NCSLC or triple-negative breast cancer), or a reagent (e.g., antibody) for specifically detecting a biomarker gene or protein of the invention.
  • the manufacture is preferably promoted, distributed, or sold as a unit for performing the methods of the present invention.
  • a “pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject.
  • pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.
  • 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 the 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 not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
  • % amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2.
  • the ALIGN-2 sequence comparison computer program was authored by Genentech, Inc., and the source code has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087.
  • the ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or may be compiled from the source code.
  • the ALIGN-2 program should be compiled for use on a UNLX operating system, including digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.
  • % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B is calculated as follows:
  • the invention concerns selecting patients who can be treated with cancer medicaments based on expression of one or more of the biomarkers disclosed herein.
  • cancer medicaments include, but are not limited to: - c-met antagonists, including anti-c-met antibodies.
  • the medicament is an antibody, including but not limited to directed against or which binds to human c-met.
  • the antibody interfered with c-met binding to hepatocyte growth factor (HGF).
  • HGF hepatocyte growth factor
  • the antibody is an antibody fragment, e.g., a Fv, Fab, Fab', one-armed antibody, scFv, diabody, or F(ab') 2 fragment.
  • the antibody is a full length antibody, e.g., an intact IgGl antibody or other antibody class or isotype as defined herein.
  • the antibody is monovalent.
  • the antibody is a one- armed antibody (i.e., the heavy chain variable domain and the light chain variable domain form a single antigen binding arm) comprising an Fc region, 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 a Fc region that increases stability of said antibody fragment compared to a Fab molecule comprising said antigen binding arm.
  • the one-armed antibody may be monovalent.
  • the c-met antagonist is an anti-c-met antibody.
  • the anti-c-met antibody is onartuzumab or a biosimilar version thereof.
  • the anti-c-met antibody comprises a heavy chain variable domain comprising one or more of (a) HVR1 comprising sequence GYTFTSYWLH (SEQ ID NO: l ); (b) HVR2 comprising sequence GMIDPSNSDTRFNPNFKD (SEQ ID NO: 2); and/or (c) HVR3-HC comprising sequence ATYRSYVTPLDY (SEQ ID NO: 3).
  • HVR1 comprising sequence GYTFTSYWLH (SEQ ID NO: l )
  • HVR2 comprising sequence GMIDPSNSDTRFNPNFKD
  • HVR3-HC comprising sequence ATYRSYVTPLDY (SEQ ID NO: 3).
  • 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 sequence WASTRES (SEQ ID NO: 5); and/or (c) HVR3-LC comprising sequence QQYYAYPWT (SEQ ID NO: 6).
  • the anti-c-met antibody comprises a heavy chain variable domain comprising (a) HVR1 comprising sequence GYTFTSYWLH (SEQ ID NO: 1); (b) HVR2 comprising sequence GMIDPSNSDTRFNPNFKD (SEQ ID NO: 2); and (c) HVR3-HC comprising sequence ATYRSYVTPLDY (SEQ ID NO: 3) and a light chain variable domain comprising (a) HVR1-LC comprising sequence KSSQSLLYTSSQK YLA (SEQ ID NO: 4); HVR2-LC comprising sequence WASTRES (SEQ ID NO: 5); and (c) HVR3-LC comprising sequence QQYYAYPWT (SEQ ID NO: 6).
  • an anti-c-met antibody can be humanized.
  • an anti- c-met antibody comprises HVRs as in any of the above embodiments, and further comprises an acceptor human framework, e.g. a human immunoglobulin framework or a human consensus framework.
  • an 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.
  • VH heavy chain variable domain
  • 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 to human c-met.
  • the anti-c-met antibody comprises the VH sequence in SEQ ID NO:7, including post-translational modifications of that sequence.
  • an anti- c-met antibody comprising 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.
  • VL light chain variable domain
  • 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 to c-met.
  • the anti- c-met antibody comprises the VL sequence in SEQ ID NO: 8, including post-translational modifications of that sequence.
  • 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.
  • the anti- c-met antibody comprises a HVR-L1 comprising amino acid sequence SEQ ID NO: 1; an HVR-L2 comprising amino acid sequence SEQ ID NO: 2; an HVR-L3 comprising amino acid sequence SEQ ID NO: 3; an HVR-H1 comprising amino acid sequence SEQ ID NO: 4; an HVR-H2 comprising amino acid sequence SEQ ID NO: 5; and an HVR-H3 comprising amino acid sequence SEQ ID NO: 6.
  • an anti- c-met antibody comprising a
  • VH as in any of the embodiments provided above
  • VL as in any of the embodiments provided above
  • the invention provides an antibody that binds to the same epitope as an anti- c-met antibody provided herein.
  • an antibody is provided that binds to the same epitope as or can by competitively inhibited by an anti- c-met antibody comprising a VH sequence of SEQ ID NO: 7 and a VL sequence of SEQ ID NO:8.
  • an anti- c-met antibody can be a monoclonal antibody, including a monovalent, chimeric, humanized or human antibody.
  • an anti- c-met antibody is an antibody fragment, e.g., a one-armed, Fv, Fab, Fab', scFv, diabody, or F(ab') 2 fragment.
  • the antibody is a full length antibody, e.g., an intact IgGl or IgG4 antibody or other antibody class or isotype as defined herein.
  • the antibody is a bispecific antibody.
  • the bispecific antibody comprises the HVRs or comprises the VH and VL regions described above.
  • the anti-c-met antibody is monovalent, and comprises (a) a first polypeptide comprising a heavy chain variable domain having the sequence:
  • the first polypeptide comprises Fc sequence CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 9) and the second polypeptide comprises the Fc sequence
  • the anti-c-met antibody is monovalent and comprises (a) a first polypeptide comprising a heavy chain, said polypeptide comprising the sequence:
  • anti-c-met antibodies suitable for use in the methods of the invention are described herein and known in the art.
  • WO05/016382 (including but not limited to antibodies 13.3.2, 9.1.2, 8.70.2, 8.90.3); an anti-c- met antibodies produced by the hybridoma cell line deposited with ICLC number PD 03001 at the CBA in Genoa, or that recognizes an epitope on the extracellular domain of the ⁇ chain of the HGF receptor, and said epitope is the same as that recognized by the monoclonal antibody); 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 an antibody deposited at CNCM, Institut Pasteur, Paris, France, on March 14, 2007 under the number 1-3731, on March 14, 2007 under the number
  • WO2010/059654 an anti-c-met antibody disclosed in WO2011020925 (including but not limited to an antibody secreted from a hybridoma deposited at the CNCM, Institut Pasteur, Paris, France, on march 12, 2008 under the number 1-3949 and the hybridoma deposited on January 14, 2010 under the number 1-4273).
  • the anti-c-met antibody comprises at least one characteristic that promotes heterodimerization, while minimizing homodimerization, of the Fc sequences within the antibody fragment. Such characteristic(s) improves yield and/or purity and/or homogeneity of the immunoglobulin populations.
  • the antibody comprises Fc mutations constituting "knobs" and "holes” as described in WO2005/063816.
  • a hole mutation can be one or more of T366A, L368A and/or Y407V in an Fc polypeptide
  • a cavity mutation can be T366W.
  • the c-met antagonist is an anti-hep atocyte growth factor (HGF) antibody, including but not limited to, humanized anti-HGF antibody TAK701, rilotumumab, Ficlatuzumab, and/or humanized antibody 2B8 described in WO2007/143090.
  • HGF humanized anti-HGF antibody
  • the anti-HGF antibody is an anti-HGF antibody described in US7718174B2.
  • the c-met antagonist is a c-met small molecule inhibitor.
  • the c-met small molecule inhibitor is a selective c-met small molecule inhibitor.
  • the c-met antagonist binds c-met extracellular domain. In some embodiments, the c-met antagonist binds c-met kinase domain. In some embodiments, the c- met antagonist competes for c-met binding with HGF. In some embodiments, the c-met antagonist competes for HGF binding to c-met. In some embodiments, the c-met antagonist binds HGF. In certain embodiments, the c-met antagonist inhibits cell proliferation, e.g., HGF- induced cell proliferation of cell line EBC-1, H441 and/or KP4.
  • cell proliferation is inhibited with a Ki of 600 nM or less (more potent), 500 nM or less, 400 nM or less, 300 nM or less or more potent.
  • the c-met antagonist inhibits c- met signaling (e.g., phospho-c-met, phospho-AKT, phospho-MAPK) when EBC-1 cells are treated with c-met antagonist in the presence of 10% fetal bovine serum.
  • c- met signaling e.g., phospho-c-met, phospho-AKT, phospho-MAPK
  • c-met signaling is inhibited with a Ki of 600 nM or less (more potent), 500 nM or less, 400 nM or less, 300 nM or less (more potent).
  • the c-met antagonist treats (is capable of treating) squamous cell carcinoma.
  • the c-met antagonist treats (is capable of treating) NSCLC that expresses wild-type k-ras.
  • the c-met inhibitor is not a non-ATP-competitive small molecule.
  • the c-met antagonist inhibits cell proliferation, e.g., HGF- induced cell proliferation of cell line EBC-1, H441 and/or KP4.
  • the c- met antagonist inhibits c-met signaling (e.g., phospho-c-met, and downstream c-met signaling pathways, e.g., phospho-AKT, phospho-MAPK) when EBC-1 cells are treated with c-met antagonist in the presence of 10% fetal bovine serum.
  • the c-met antagonist does not inhibit cell proliferation of cell lines MDA-MB-231 and HT29 (in the presence or absence of exogenous HGF). In certain embodiments, the c-met antagonist does not inhibit cell proliferation of cell lines MDA-MB-231 and HT29 in the presence of 10% fetal bovine serum. Methods for assaying cell proliferation are well-known in the art, and some methods are described in WO2009/111691; WO2006/015371; and Jin et al, Cancer Res (2008) 68:4360. In certain embodiments, the c-met antagonist treats (is capable of treating) squamous cell carcinoma.
  • the c-met antagonist treats (is capable of treating) NSCLC that expresses wild-type k-ras.
  • the c-met antagonist is not Tivantinib (ARQ-197).
  • a c-met antagonist inhibits c-met signaling with an IC50 of 1,000 nM or less (i.e., more potent).
  • a c-met antagonist inhibits c-met signaling with an IC50 of 400 nm or less, 500 iiM or less, 600 nm or less, 700 nm or less.
  • a c-met antagonist inhibits c-met signaling with an IC50 of 50 iiM or less.
  • the c-met antagonist is not crizotinib. In certain embodiments, the c-met antagonist is not foretinib. In certain embodiments, the c-met antagonist is not ficlatuzumab. In certain embodiments, the c-met antagonist is not
  • the c-met antagonist is any one of: SGX-523, Crizotinib (PF- 02341066; 3-[(lR)- 1 -(2,6-dichloro-3-fluorophenyl)ethoxy]-5-(l -piperidin-4-ylpyrazol-4- yl)pyridin-2-amine; CAS no. 877399-52-5); JNJ-38877605 (CAS no.
  • XL880 is a inhibitor of met and VEGFR2 and KDPv); MGCD-265 (MethylGene; MGCD-265 targets the c-MET, VEGFR1, VEGFR2, VEGFR3, Ron and Tie-2 receptors; CAS no. 875337-44-3), Tivantinib (ARQ 197; (-)- (3R,4R)-3-(5,6-dihydro-4H-pyrrolo[3,2,l-ij]quinolin-l-yl)-4-(lH-indol-3-yl)pyrrolidm ⁇ dione; see Munchi et al, Mol Cancer Ther June 2010 9; 1544; CAS no.
  • LY- 2801653 (Lilly), LY2875358 (Lilly), MP-470, Rilotumumab (AMG 102, anti-HGF monoclonal antibody), antibody 223 C4 or humanized antibody 223 C4 (WO2009/007427), humanized L2G7 (humanized TAK701 ; humanized anti-HGF monoclonal antibody); EMD 1214063 (Merck Sorono), EMD 1204831 (Merck Serono), NK4, Cabozantinib (XL- 184, CAS no.
  • carbozantinib is a dual inhibitor of met and VEGFR2), 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.
  • E7050 is a dual c-met and VEGFR2 inhibitor (Esai); MK-2461 (Merck; N-((2R)- 1 ,4-Dioxan-2-ylmethyl)-N-methyl-N * -[3- (l-methyl-lH-pyrazol-4-yl)-5-oxo-5H-benzo[4,5]cyclohepta[l,2-b]pyridin-7-yl]sulfamide; CAS no.
  • the c-met antagonist is any one or more of crizotinib, tivantinib, carbozantinib, MGCD-265, ficlatuzumab, humanized TAK-701, rilotumumab, foretinib, h224Gl 1, 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 LA480.
  • the c-met antagonist is any one or more of crizotinib, tivantinib, carbozantinib, MGCD-265, ficlatuzumab, humanized TAK-701, rilotumumab, and/or foretinib.
  • EGFR antagonists include antibodies such as humanized monoclonal antibody known as nimotuzumab (YM Biosciences), fully human ABX-EGF (panitumumab, Abgenix Inc.) as well as fully human antibodies known as El . l, E2.4, E2.5, E6.2, E6.4, E2.11, E6. 3 and E7.6. 3 and described in US 6,235,883; MDX-447 (Medarex Inc).
  • Pertuzumab (2C4) is a humanized antibody that binds directly to HER2 but interferes with HER2-EGFR dimerization thereby inhibiting EGFR signaling.
  • antibodies which bind to EGFR include MAb 579 (ATCC CRL HB 8506), MAb 455 (ATCC CRL HB8507), MAb 225 (ATCC CRL 8508), MAb 528 (ATCC CRL 8509) (see, US Patent No. 4,943, 533, Mendelsohn et al.) and variants thereof, such as chimerized 225 (C225 or Cetuximab; ERBUTLX®) and reshaped human 225 (H225) (see, WO 96/40210, Imclone Systems Inc.); IMC-11F8, a fully human, EGFR-targeted antibody (Imclone); antibodies that bind type II mutant EGFR (US Patent No.
  • the anti-EGFR antibody may be conjugated with a cytotoxic agent, thus generating an immunoconjugate (see, e.g., EP659,439A2, Merck Patent GmbH).
  • Anti-EGFR antibodies that are useful in the methods of the invention include any antibody that binds with sufficient affinity and specificity to EGFR and can reduce or inhibit EGFR activity.
  • the antibody selected will normally have a sufficiently strong binding affinity for EGFR, for example, the antibody may bind human c-met with a Kd value of between 100 nM-1 pM.
  • Antibody affinities may be determined by a surface plasmon resonance based assay (such as the BIAcore assay as described in PCT Application Publication No.
  • the anti-EGFR antibody of the invention can be used as a therapeutic agent in targeting and interfering with diseases or conditions wherein
  • Bispecific antibodies are antibodies that have binding specificities for at least two different epitopes. Exemplary bispecific antibodies may bind to EGFR and to c-met. In another example, an exemplary bispecific antibody may bind to two different epitopes of the same protein, e.g., c-met protein. Bispecific antibodies may also be used to localize cytotoxic agents to cells which express EGFR or c-met.
  • bispecific antibodies possess a EGFR or c-met- binding arm and an arm which binds the cytotoxic agent (e.g. saporin, anti-interferon-a, vinca alkaloid, ricin A chain, methotrexate or radioactive isotope hapten).
  • cytotoxic agent e.g. saporin, anti-interferon-a, vinca alkaloid, ricin A chain, methotrexate or radioactive isotope hapten.
  • Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g. F(ab')2 bispecific antibodies).
  • the bispecific antibody is any of the bispecific MET-EGFR antibodies disclosed in US20100254989A.
  • EGFR antagonists also include small molecules such as compounds described in US5616582, US5457105, US5475001, US5654307, US5679683, US6084095, US6265410, US6455534, US6521620, US6596726, US6713484, US5770599, US6140332, US5866572, US6399602, US6344459, US6602863, US6391874, W09814451, WO9850038, WO9909016, WO9924037, W09935146, WO0132651, US6344455, US5760041, US6002008, US5747498.
  • EGFR antagonists include OSI-774 (CP-358774, erlotinib, OSI Pharmaceuticals); PD 183805 (CI 1033, 2-propenamide, N-[4-[(3-chloro-4- fluorophenyl)amino] -7-[3 -(4-morpholinyl)propoxy] -6-quinazolinyl] -, dihydrochloride, Pfizer Inc.); Iressa ® (ZD 1839, gefitinib, AstraZeneca); ZM 105180 ((6-amino-4-(3-methylphenyl- amino)-quinazoline, Zeneca); BIBX-1382 (N8-(3-chloro-4-fluoro-phenyl)-N2-(l-methyl- piperidin-4-yl)-pyrimido[5,4-d]pyrimidine-2,8-diamine, Boehringer Ingelheim); PKI-166 ((R)- 4-
  • the antibody e.g. the antibody used in the methods herein may incorporate any of the features, singly or in combination, as described in Sections 1-6 below: 1. Antibody Fragments
  • an antibody provided herein is an antibody fragment.
  • Antibody fragments include, but are not limited to, Fab, Fab', Fab'-SH, F(ab') 2 , Fv, and scFv fragments, a one-armed antibody, and other fragments described below.
  • Fab fragment antigen
  • Fab' fragment antigen binding protein
  • Fab'-SH fragment antigen binding protein
  • Fv fragment antigen binding protein
  • scFv fragments fragment antigen binding protein fragments
  • a one-armed antibody and other fragments described below.
  • scFv fragments see, e.g., Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer- Verlag, New York), pp. 269-315 (1994); see also WO 93/16185; and U.S. Patent Nos.
  • Diabodies are antibody fragments with two antigen-binding sites that may be bivalent or bispecific. See, for example, EP 404,097; WO 1993/01161; Hudson et al, Nat. Med. 9: 129- 134 (2003); and Hollinger et al, Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al, Nat. Med. 9: 129-134 (2003).
  • Single-domain antibodies are antibody fragments comprising all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody.
  • a single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Patent No. 6,248,516 Bl).
  • One-armed antibodies i.e., the heavy chain variable domain and the light chain variable domain form a single antigen binding arm
  • the monovalent trait of a one-armed antibody results in and/or ensures an antagonistic function upon binding of the antibody to a target molecule.
  • the one-armed antibody comprising a Fc region is characterized by superior pharmacokinetic attributes (such as an enhanced half life and/or reduced clearance rate in vivo) compared to Fab forms having similar/substantially identical antigen binding characteristics, thus overcoming a major drawback in the use of conventional monovalent Fab antibodies.
  • Techniques for making one-armed antibodies include, but are not limited to, "knob-in-hole” engineering (see, e.g., U.S. Patent No.
  • Onartuzumab is an example of a one-armed antibody.
  • Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody as well as production by recombinant host cells (e.g. E. coli or phage), as described herein.
  • an antibody provided herein is a chimeric antibody.
  • Certain chimeric antibodies are described, e.g., in U.S. Patent No. 4,816,567; and Morrison et al, Proc. Natl. Acad. Sci. USA, 81 :6851-6855 (1984)).
  • 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.
  • a chimeric antibody is a "class switched" antibody in which the class or subclass has been changed from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.
  • a chimeric antibody is a humanized antibody.
  • a non-human antibody is humanized to reduce immunogenicity to humans, while retaining the specificity and affinity of the parental non-human antibody.
  • 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 human antibody sequences.
  • HVRs e.g., CDRs, (or portions thereof) are derived from a non-human antibody
  • FRs or portions thereof
  • a humanized antibody optionally will also comprise at least a portion of a human constant region.
  • some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the HVR residues are derived), e.g., to restore or improve antibody specificity or affinity.
  • a non-human antibody e.g., the antibody from which the HVR residues are derived
  • 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 the consensus sequence of human antibodies of a particular subgroup of 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.
  • an antibody provided herein is a human antibody.
  • Human antibodies can be produced using various techniques known in the art. Human antibodies are described generally in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5: 368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20:450-459 (2008).
  • Human antibodies may be prepared by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigenic challenge. Such animals typically contain all or a portion of the human immunoglobulin loci, which replace the endogenous
  • immunoglobulin loci or which are present extrachromosomally or integrated randomly into the animal's chromosomes. In such transgenic mice, the endogenous immunoglobulin loci have generally been inactivated.
  • endogenous immunoglobulin loci have generally been inactivated.
  • Human antibodies can also be made 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-ceil hybridoma technology are also described in Li et aL, Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006).
  • Additional methods include those described, for example, in U.S. Patent No. 7,189,826 (describing production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (describing human-human hybridomas).
  • Human hybridoma technology Trioma technology
  • Vollmers and Brandlein, Histology and Histopathology, 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3): 185-91 (2005).
  • Human antibodies may also be generated by isolating Fv clone variable domain sequences selected from human-derived phage display libraries. Such variable domain sequences may then be combined with a desired human constant domain. Techniques for selecting human antibodies from antibody libraries are described below. 4. Library-Derived Antibodies
  • Antibodies of the invention may be isolated by screening combinatorial libraries for antibodies with the desired activity or activities. For example, a variety of methods are known in the art for generating phage display libraries and screening such libraries for antibodies possessing the desired binding characteristics. Such methods are reviewed, e.g., in
  • naive repertoire can be cloned (e.g., from human) to provide a single source of antibodies to a wide range of non-self and also self antigens without any immunization as described by Griffiths et al, EMBO J 12: 725-734 (1993).
  • naive libraries can also be made synthetically by cloning unrearranged V-gene segments from stem cells, and using PCR primers containing random sequence to encode the highly variable CDR3 regions and to accomplish 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: US Patent No. 5,750,373, and US 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 human antibody libraries are considered human antibodies or human antibody fragments herein.
  • an antibody provided herein is a multispecific antibody, e.g. a bispecific antibody.
  • 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.
  • bispecific antibodies may bind to two different epitopes of c-met. Bispecific antibodies may also be used to localize cytotoxic agents to cells which express c-met. Bispecific antibodies can be prepared as full length antibodies or antibody fragments.
  • Multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs having different specificities (see Milstein and Cuello, Nature 305: 537 (1983), WO 93/08829, and Traunecker et al, EMBO J. 10: 3655 (1991)), and "knob-in-hole” engineering (see, e.g., U.S. Patent No. 5,731,168).
  • Multi-specific antibodies may also be made by engineering electrostatic steering effects for making antibody Fc-heterodimeric molecules (WO 2009/089004A1); cross- linking two or more antibodies or fragments (see, e.g., US Patent No.
  • the antibody or fragment herein also includes a "Dual Acting FAb” or “DAF” comprising an antigen binding site that binds to c-met as well as another, different antigen, such as EGFR (see, US 2008/0069820, for example).
  • DAF Double Acting FAb
  • EGFR EGFR
  • amino acid sequence variants of the antibodies provided herein are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of the antibody.
  • Amino acid sequence variants of an antibody may 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 sequences of the antibody. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., antigen-binding .
  • antibody variants having one or more amino acid substitutions are provided.
  • Sites of interest for substitutional mutagenesis include the HVRs and FRs.
  • Amino acid substitutions may be introduced into an antibody of interest and the products screened for a desired activity, e.g., retained/improved antigen binding, decreased
  • substitutional variant involves substituting one or more hypervariable region residues of a parent antibody ⁇ e.g. a humanized or human antibody).
  • a parent antibody ⁇ e.g. a humanized or human antibody.
  • the resulting variant(s) selected for further study will have modifications (e.g., improvements) in certain biological properties (e.g., increased affinity, reduced immunogenicity) relative to the parent antibody and/or will have substantially retained certain biological properties of the parent antibody.
  • An exemplary substitutional variant is an affinity matured antibody, which may be conveniently generated, e.g., using phage display-based affinity maturation techniques such as those described herein. Briefly, one or more HVR residues are mutated and the variant antibodies displayed on phage and screened for a particular biological activity (e.g. binding affinity).
  • Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues.
  • terminal insertions include an antibody with an N-terminal methionyl residue.
  • Other insertional variants of the antibody molecule include the fusion to the N- or C -terminus of the antibody to an enzyme (e.g. for ADEPT) or a polypeptide which increases the serum half-life of the antibody.
  • an antibody provided herein is altered to increase or decrease the extent to which the antibody is glycosylated. Addition or deletion of glycosylation sites to an antibody may be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites is created or removed.
  • the carbohydrate attached thereto may be altered.
  • Native antibodies produced by mammalian cells typically comprise a branched, biantennary oligosaccharide that is generally attached by an N-linkage to Asn297 of the CH2 domain of the Fc region. See, e.g., Wright et al. TIBTECH 15:26-32 (1997).
  • oligosaccharide may include various carbohydrates, e.g., mannose, N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as a fucose attached to a GlcNAc in the "stem" of the biantennary oligosaccharide structure.
  • modifications of the oligosaccharide in an antibody of the invention may be made in order to create antibody variants with certain improved properties.
  • antibody variants having a carbohydrate structure that lacks fucose attached (directly or indirectly) to an Fc region.
  • the amount of fucose in such antibody may be from 1% to 80%, from 1% to 65%, from 5% to 65%> or from 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 glycostructures attached to Asn 297 (e. g. complex, hybrid and high mannose structures) as measured by MALDI-TOF mass spectrometry, as described in WO 2008/077546, for example.
  • 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 antibodies.
  • Such fucosylation variants may have improved ADCC function. See, e.g., US Patent Publication Nos. US 2003/0157108 (Presta, L.); US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications related to "defucosylated” or "fucose-deficient" antibody variants include: US 2003/0157108; WO 2000/61739; WO 2001/29246; US
  • Examples of cell lines capable of producing defucosylated antibodies include Lecl3 CHO cells deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); US Pat Appl No US 2003/0157108 Al, Presta, L; and WO 2004/056312 Al, Adams et al, especially at Example 11), and knockout cell lines, such as alpha- 1,6-fucosyltransferase gene, FUT8, knockout 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 WO2003/085107).
  • Antibodies variants are further provided with bisected oligosaccharides, e.g., in which a biantennary oligosaccharide attached to the Fc region of the antibody is bisected by GlcNAc. Such antibody variants may have reduced fucosylation and/or improved ADCC function.
  • antibody variants examples include WO 2003/011878 (Jean-Mairet et al); US Patent No. 6,602,684 (Umana et al); and US 2005/0123546 (Umana et al).
  • Antibody variants with at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such antibody variants may have improved CDC function.
  • Such antibody variants are described, e.g., in WO 1997/30087 (Patel et al); WO 1998/58964 (Raju, S.); and WO 1999/22764 (Raju, S.).
  • one or more amino acid modifications may 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 IgGl, IgG2, IgG3 or IgG4 Fc region) comprising an amino acid modification ⁇ e.g. a substitution) at one or more amino acid positions.
  • the invention contemplates an antibody variant that possesses some but not all effector functions, which make it a desirable candidate for applications in which the half life of the antibody in vivo is important yet certain effector functions (such as complement and ADCC) are unnecessary or deleterious.
  • Antibodies with reduced effector function include those with substitution of one or more of Fc region residues 238, 265, 269, 270, 297, 327 and 329 (U.S. Patent No. 6,737,056).
  • Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including the so-called "DANA" Fc mutant with substitution of residues 265 and 297 to alanine (US Patent No. 7,332,581).
  • an antibody variant comprises an Fc region with one or more amino acid substitutions which improve ADCC, e.g., substitutions at positions 298, 333, and/or 334 of the Fc region (EU numbering of residues).
  • alterations are made in the Fc region that result in altered (i.e., either improved or diminished) Clq binding and/or Complement Dependent Cytotoxicity (CDC), e.g., as described in US Patent No. 6,194,551, WO 99/51642, and Idusogie et al. J. Immunol. 164: 4178-4184 (2000).
  • Those antibodies comprise an Fc region with one or more substitutions therein which improve binding of the Fc region to FcRn.
  • Fc variants include those with substitutions at one or more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434, e.g., substitution of Fc region residue 434 (US Patent No. 7,371,826).
  • cysteine engineered antibodies e.g., "thioMAbs”
  • one or more residues of an antibody are substituted with cysteine residues.
  • the substituted residues occur at accessible sites of the antibody.
  • reactive thiol groups are thereby positioned at accessible sites of the antibody and may be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties, to create an immunoconjugate, as described further herein.
  • any one or more of the following residues may be substituted with cysteine: V205 (Kabat numbering) of the light chain; Al 18 (EU numbering) of the heavy chain; and S400 (EU numbering) of the heavy chain Fc region.
  • Cysteine engineered antibodies may be generated as described, e.g., in U.S. Patent No.
  • an antibody provided herein may be further modified to contain additional nonproteinaceous moieties that are known in the art and readily available.
  • the moieties suitable for derivatization of the antibody include but are not limited to water soluble polymers.
  • water soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol/propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1, 3- dioxolane, poly-1, 3, 6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymers or random copolymers), and dextran or poly(n-vinyl pyrrolidone)polyethylene glycol, propropylene glycol homopolymers, prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylated polyols (e.g., glycerol
  • Polyethylene glycol propionaldehyde may have advantages in manufacturing 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 may vary, and if more than one polymer are 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 particular properties or functions of the antibody to be improved, whether the antibody derivative will be used in a therapy under defined conditions, etc.
  • conjugates of an antibody and nonproteinaceous moiety that may be selectively heated by exposure to radiation are provided.
  • the nonproteinaceous moiety is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102: 11600-11605 (2005)).
  • the radiation may be of any wavelength, and includes, but is not limited to, wavelengths that do not harm ordinary cells, but which heat the nonproteinaceous moiety to a temperature at which cells proximal to the antibody-nonproteinaceous moiety are killed.
  • the medicament is an mmunoconjugate comprising an antibody (such as a c-met antibody) conjugated to one or more cytotoxic agents, such as
  • chemotherapeutic agents or drugs growth inhibitory agents, toxins (e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof), or radioactive isotopes.
  • toxins e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof
  • radioactive isotopes e.g., radioactive isotopes.
  • an immunoconjugate is an antibody-drug conjugate (ADC) in which an antibody is conjugated to one or more drugs, including but not limited to a maytansinoid (see U.S. Patent Nos. 5,208,020, 5,416,064 and European Patent EP 0 425 235 Bl); an auristatin such as monomethylauristatin drug moieties DE and DF (MMAE and MMAF) (see U.S. Patent Nos. 5,635,483 and 5,780,588, and 7,498,298); a dolastatin; a calicheamicin or derivative thereof (see U.S. Patent 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.
  • ADC antibody-drug conjugate
  • an immunoconjugate comprises an antibody as described herein conjugated to an enzymatically active toxin or fragment thereof, including but not limited to diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha- sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.
  • an enzymatically active toxin or fragment thereof including but not limited to diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain
  • an immunoconjugate comprises an antibody as described herein conjugated to a radioactive atom to form a radioconjugate.
  • a radioactive atom to form a radioconjugate.
  • isotopes are available for the production of radioconjugates. Examples include At , 1 , 1 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212 and radioactive isotopes of Lu.
  • radioconjugate is used for detection, it may comprise a radioactive atom for scintigraphic studies, for example tc99m or 1123, or a spin label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, mri), such as iodine- 123 again, iodine- 131, indium-I l l, fluorine- 19, carbon- 13, nitrogen- 15, oxygen- 17, gadolinium, manganese or iron.
  • NMR nuclear magnetic resonance
  • Conjugates of an antibody and cytotoxic agent may be made using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), succinimidyl-4-(N-maleimidomethyl) cyclohexane-l-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HC1), 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), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such
  • a ricin immunotoxin can be prepared as described in Vitetta et al, Science 238: 1098 (1987).
  • Carbon- 14-labeled l-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX- DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See W094/11026.
  • the linker may be a "cleavable linker" facilitating release of a cytotoxic drug in the cell.
  • an acid-labile linker, peptidase-sensitive linker, photolabile linker, dimethyl linker or disulfide-containing linker (Chari et al., Cancer Res. 52: 127-131 (1992); U.S. Patent No. 5,208,020) may be used.
  • the immunuoconjugates or ADCs herein expressly contemplate, but are not limited to such conjugates prepared with cross-linker reagents 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).
  • cross-linker reagents including, but not limited to, BMPS, EMCS, GMBS, HBVS,
  • the invention provides diagnostic methods, e.g. for identifying a cancer patient who is likely to respond to treatment with a c-met antagonist.
  • IHC analysis further comprises morphological staining, either prior to or thereafter.
  • hematoxylin is use for staining cellular nucleic of the slides. Hematoxylin is widely available. An example of a suitable hematoxylin is
  • Hematoxylin II (Ventana). When lighter blue nuclei are desired, a bluing reagent may be used following hematoxylin staining.
  • Detection of c-met biomarker using IHC is disclosed herein, and a c-met staining intensity scoring scheme is disclosed herein.
  • Disclosed herein is a method for determining an H-score, wherein the H-score is determined using the formula (% cancer cells staining with weak c-met staining intensity) + (% cancer cells staining with moderate c-met staining intensity) + (% cancer cells staining with high c-met staining intensity).
  • Methods for calculating the H-score are disclosed and exemplified herein.
  • the H-score is about 160 or higher (about 161, 162, 163, 164, 165, 166, 167, 168, 169, or higher), 160 or higher, about 160 to about 230, about 160 ro 230, about 160 (any of about 161, 162, 163, 164, 165, 166, 167, 168, 169, or higher to any of abour 220, 221,
  • the H- score is about 180 orhigher.
  • the H-score is less than about 160, less than 160 (e.g., 159, 158, 157, 156, 155, 154, 143, 142, 141 or less), less than about 150, less than 150 (e.g., 149, 148, 147, 146, 145, 144, 143, 142, 141, or less).
  • the H score is greater than about 10. In some embodiments, the H score is greater than about 25. In some embodiments, the H score is greater than about 50. In some embodiments, the H score is greater than about 75. In some embodiments, the H score is greater than about 100. In some embodiments, the H score is greater than about 125. In some embodiments, the H score is greater than about 150. In some embodiments, the H score is greater than about 175. In some embodiments, the H score is greater than about 200. In some embodiments, c-met expression is membranous. In some embodiments, c-met expression is cytoplasmic. In some embodiments, c-met- expression is membranous and cytoplasmic.
  • biomarkers may be detected. Exemplary other biomarkers are disclosed herein.
  • the source of the tissue or cell sample may be solid tissue as from a fresh, frozen and/or preserved organ or tissue sample or biopsy or aspirate (including but not limited to a fine needle aspirate); blood or any blood constituents; bodily fluids such as cerebral spinal fluid, amniotic fluid, peritoneal fluid, bronchiolar lavage, pleural fluid, sputum, or interstitial fluid; cells from any time in gestation or development of the subject.
  • the tissue sample may contain compounds which are not naturally intermixed with the tissue in nature such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, or the like.
  • tumor samples herein include, but are not limited to, tumor biopsies, tumor cells, serum or plasma, circulating plasma proteins, ascitic fluid, primary cell cultures or cell lines derived from tumors or exhibiting tumor-like properties, bronchiolar lavage, pleural fluid, sputum, as well as preserved tumor samples, such as formalin- fixed, paraffin-embedded tumor samples (including, but not limited to formalin- fixed paraffin-embedded fine needle aspirate samples) or frozen tumor samples.
  • the patient sample is a formalin-fixed paraffin- embedded (FFPE) tumor sample (e.g., a NSCLC tumor sample or a breast cancer tumor sample).
  • FFPE formalin-fixed paraffin- embedded
  • the patient sample is a formalin-fixed paraffin-embedded sample from a fine needle aspirate.
  • the sample may be obtained prior to or during the patient's treatment with a cancer medicament (such as an anti-c-met antagonist).
  • the sample may be obtained prior to or during the patient's prior treatment with a cancer medicament.
  • the sample may be obtained from the primary tumor or from a metastatic tumor.
  • the sample may be obtained when the cancer is first diagnosed or, for example, after the tumor has metastasized.
  • the tumor sample is of lung, lymph node, liver or brain.
  • Immunohistochemical staining of tissue sections has been shown to be a reliable method of assessing or detecting presence of proteins in a sample.
  • Immunohistochemistry techniques utilize an antibody to probe and visualize cellular antigens in situ, generally by chromogenic or fluorescent methods.
  • antibodies or antisera in some embodiments, polyclonal antisera, and in some embodiments, monoclonal antibodies specific for each marker are used to detect expression.
  • the antibodies can be detected by direct labeling of the antibodies themselves, for example, with radioactive labels, fluorescent labels, hapten labels such as, biotin, or an enzyme such as horse radish peroxidase or alkaline phosphatase.
  • unlabeled primary antibody is used in conjunction with a labeled secondary antibody, comprising antisera, polyclonal antisera or a monoclonal antibody specific for the primary antibody.
  • a labeled secondary antibody comprising antisera, polyclonal antisera or a monoclonal antibody specific for the primary antibody. Immunohistochemistry protocols and kits are well known in the art and are commercially available.
  • IHC Two general methods of IHC are available; direct and indirect assays.
  • binding of antibody to the target antigen is determined directly.
  • This direct assay uses a labeled reagent, such as a fluorescent tag or an enzyme-labeled primary antibody, which can be visualized without further antibody interaction.
  • a labeled reagent such as a fluorescent tag or an enzyme-labeled primary antibody, which can be visualized without further antibody interaction.
  • unconjugated primary antibody binds to the antigen and then a labeled secondary antibody binds to the primary antibody.
  • a chromagenic or fluorogenic substrate is added to provide visualization of the antigen. Signal amplification occurs because several secondary antibodies may react with different epitopes on the primary antibody.
  • the primary and/or secondary antibody used for immunohistochemistry typically will be labeled with a detectable moiety.
  • Numerous labels are available which can be generally grouped into the following categories:
  • Radioisotopes such as S, C, I, H, and I.
  • the antibody can be labeled with the radioisotope using the techniques described in Current Protocols in Immunology, Volumes 1 and 2, Coligen et al, Ed. Wiley-Interscience, New York, N.Y., Pubs. (1991) for example and radioactivity can be measured using scintillation counting.
  • Fluorescent labels including, but are not limited to, rare earth chelates (europium chelates), Texas Red, rhodamine, fluorescein, dansyl, Lissamine, umbelliferone,
  • the fluorescent labels can be conjugated to the antibody using the techniques disclosed in Current Protocols in Immunology, supra, for example. Fluorescence can be quantified using a fluorimeter.
  • Various enzyme-substrate labels are available and U.S. Pat. No. 4,275,149 provides a review of some of these.
  • the enzyme generally catalyzes a chemical alteration of the chromogenic substrate that can be measured using various techniques. For example, the enzyme may catalyze a color change in a substrate, which can be measured
  • the enzyme may alter the fluorescence or
  • chemiluminescence of the substrate involves techniques for quantifying a change in fluorescence.
  • the chemiluminescent substrate becomes electronically excited by a chemical reaction and may then emit light which can be measured (using a chemiluminometer, for example) or donates energy to a fluorescent acceptor.
  • enzymatic labels include luciferases (e.g., firefly luciferase and bacterial luciferase; U.S. Pat. No.
  • luciferin 2,3-dihydrophthalazinediones, malate dehydrogenase, urease, peroxidase such as horseradish peroxidase (HRPO), alkaline phosphatase, ⁇ -galactosidase, glucoamylase, lysozyme, saccharide 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.
  • HRPO horseradish peroxidase
  • alkaline phosphatase alkaline phosphatase
  • ⁇ -galactosidase glucoamylase
  • lysozyme saccharide oxidases
  • glucose oxidase galactose oxidas
  • enzyme-substrate combinations include, for example:
  • HRPO Horseradish peroxidase
  • OPD orthophenylene diamine
  • TMB 3,3',5,5'-tetramethyl benzidine hydrochloride
  • DAB 3,3-Diaminobenzidine
  • ⁇ -D-galactosidase ( ⁇ -D-Gal) with a chromogenic substrate (e.g., p-nitrophenyl- ⁇ - D-galactosidase) or fluorogenic substrate (e.g., 4-methylumbelliferyl ⁇ -D-galactosidase).
  • a chromogenic substrate e.g., p-nitrophenyl- ⁇ - D-galactosidase
  • fluorogenic substrate e.g., 4-methylumbelliferyl ⁇ -D-galactosidase
  • the label is indirectly conjugated with the antibody.
  • the antibody can be conjugated with biotin and any of the four broad categories of labels mentioned above can be conjugated with avidin, or vice verse. Biotin binds selectively to avidin and thus, the label can be conjugated with the antibody in this indirect manner.
  • the antibody is conjugated with a small hapten and one of the different types of labels mentioned above is conjugated with an anti-hapten antibody.
  • indirect conjugation of the label with the antibody can be achieved.
  • tissue section prior to, during or following IHC may be desired.
  • epitope retrieval methods such as heating the tissue sample in citrate buffer may be carried out [see, e.g., Leong et al. Appl. Immunohistochem. 4(3):201 (1996)].
  • the tissue section is exposed to primary antibody for a sufficient period of time and under suitable conditions such that the primary antibody binds to the target protein antigen in the tissue sample. Appropriate conditions for achieving this can be determined by routine experimentation.
  • the label is an enzymatic label (e.g. HRPO) which catalyzes a chemical alteration of the chromogenic substrate such as 3,3'- diaminobenzidine chromogen.
  • the enzymatic label is conjugated to antibody which binds specifically to the primary antibody (e.g. the primary antibody is rabbit polyclonal antibody and secondary antibody is goat anti-rabbit antibody).
  • Specimens thus prepared may be mounted and coverslipped. Slide evaluation is then determined, e.g. using a microscope.
  • IHC may be combined with morphological staining, either prior to or thereafter.
  • the sections mounted on slides may be stained with a morphological stain for evaluation.
  • the morphological stain to be used provides for accurate morphological evaluation of a tissue section.
  • the section may be stained with one or more dyes each of which distinctly stains different cellular components.
  • hematoxylin is use for staining cellular nucleic of the slides. Hematoxylin is widely available.
  • An example of a suitable hematoxylin is Hematoxylin II (Ventana).
  • a bluing reagent may be used following hematoxylin staining.
  • staining may be optimized for a given tissue by increasing or decreasing the length of time the slides remain in the dye.
  • the Ventana® BenchMark XT system is an example of such an automated system. After staining, the tissue section may be analyzed by standard techniques of
  • a pathologist or the like assesses the tissue for the presence of abnormal or normal cells or a specific cell type and provides the loci of the cell types of interest.
  • a pathologist or the like would review the slides and identify normal cells (such as normal lung cells) and abnormal cells (such as abnormal or neoplastic lung cells).
  • Any means of defining the loci of the cells of interest may be used (e.g., coordinates on an X-Y axis).
  • Anti-c-met antibodies suitable for use in IHC are well known in the art, and include SP- 44 (Ventana), DL-21 (Upstate), D1C2 (Cell Signaling Technologies), ab27492 (Abeam), PA1- 37483 (Pierce Antibodies), Met4 (see, e.g, US Patent No. 6,548, 640).
  • the anti-c-met antibody is SP44.
  • the anti-c-met antibody is DL-21.
  • the anti-c-met antibody is D1C2.
  • the anti-c-met antibody is Met4.
  • additional suitable anti-c-met antibodies may be identified and characterized by comparing with c-met antibodies using the IHC protocol and examples disclosed herein, for example.
  • Control cell lines e.g., centrifuged into a pellet and formalin fixed and paraffin embedded, e.g., and prepared as a tissue microarray, and e.g., stained with SP44
  • various staining intensities e.g., when stained with c-met antibody SP44
  • H441 strong c-met staining intensity
  • EBCl strong c-met staining intensity
  • A549 moderate c-met staining intensity
  • SKMES1 moderate c-met staining intensity
  • HI 703 weak c-met staining intensity
  • HEK-293 weak c-met staining intensity
  • H460 weak c-met staining intensity
  • TOV-112D negative c-met staining intensity
  • LXFL529 negative c-met staining intensity
  • H522 negative c-met staining intensity
  • H23 negative c-met staining intensity
  • HI 155 negative c-met staining intensity
  • strong c-met staining intensity is c-met staining intensity of a control cell having c-met staining intensity of H441 and/or EBCl .
  • moderate c-met staining intensity is c-met staining intensity of a control cell having c-met staining intensity of A549 and/or SKMES 1.
  • weak c-met staining intensity is c-met staining intensity of a control cell having c-met staining intensity of HEK-293 and/or H460.
  • negative c-met staining intensity is c-met staining intensity of a control cell having c-met staining intensity of LXFL529, H522, H23, and/or HI 155.
  • c-met IHC is analyzed using the scheme shown in Table X:
  • c-Met IHC is analyzed using the scheme shown in Table Y:
  • a patient's tumor is c-met positive when 1% or more of the tumor cells in the sample express c-met protein (e.g., express c-met protein at any intensity, e.g., low, moderate and high). In some embodiments, a patient's tumor is c-met positive when more than 1% of the tumor cells in the sample express c-met protein (e.g., express c-met protein at any intensity). In some embodiments, a patient's tumor is c-met positive when 5% or more of the tumor cells in the sample express c-met protein (e.g., express c-met protein at any intensity).
  • a patient's tumor is c-met positive when 10% or more of the tumor cells in the sample express c-met protein (e.g., express c-met protein at any intensity). In some embodiments, a patient's tumor is c-met positive when 15% or more of the tumor cells in the sample express c-met protein. In some embodiments, a patient's tumor is c-met positive when 20% or more of the tumor cells in the sample express c-met protein (e.g., express c-met protein at any intensity). In some embodiments, a patient's tumor is c-met positive when 25% or more of the tumor cells in the sample express c-met protein (e.g., express c-met protein at any intensity).
  • a patient's tumor is c-met positive when 30% or more of the tumor cells in the sample express c-met protein (e.g., express c-met protein at any intensity). In some embodiments, a patient's tumor is c-met positive when 35% or more of the tumor cells in the sample express c-met protein (e.g., express c-met protein at any intensity). In some embodiments, a patient's tumor is c-met positive when 40% or more of the tumor cells in the sample express c-met protein (e.g., express c-met protein at any intensity).
  • a patient's tumor is c-met positive when 45% or more of the tumor cells in the sample express c-met protein (e.g., express c-met protein at any intensity). In some embodiments, a patient's tumor is c-met positive when 50% or more of the tumor cells in the sample express c-met protein (e.g., express c-met protein at any intensity). In some embodiments, a patient's tumor is c-met positive when 55% or more of the tumor cells in the sample express c-met protein (e.g., express c-met protein at any intensity).
  • a patient's tumor is c-met positive when 60% or more of the tumor cells in the sample express c-met protein (e.g., express c-met protein at any intensity). In some embodiments, a patient's tumor is c-met positive when 65% or more of the tumor cells in the sample express c-met protein (e.g., express c-met protein at any intensity). In some embodiments, a patient's tumor is c-met positive when 70% or more of the tumor cells in the sample express c-met protein (e.g., express c-met protein at any intensity).
  • a patient's tumor is c-met positive when 75% or more of the tumor cells in the sample express c-met protein (e.g., express c-met protein at any intensity). In some embodiments, a patient's tumor is c-met positive when 80% or more of the tumor cells in the sample express c-met protein (e.g., express c-met protein at any intensity). In some embodiments, a patient's tumor is c-met positive when 85% or more of the tumor cells in the sample express c-met protein (e.g., express c-met protein at any intensity).
  • a patient's tumor is c-met positive when 90% or more of the tumor cells in the sample express c-met protein (e.g., express c-met protein at any intensity). In some embodiments, a patient's tumor is c-met positive when 95% or more of the tumor cells in the sample express c-met protein (e.g., express c-met protein at any intensity). In some embodiments, c-met expression is membranous. In some embodiments, c-met expression is cytoplasmic. In some embodiments, c-met- expression is membranous and cytoplasmic.
  • a patient's tumor is c-met positive when a maximum staining intensity of the tumor is 1. In some embodiments, a patient's tumor is c-met positive when a maximum staining intensity of the tumor is 2. In some embodiments, a patient's tumor is c-met positive when a maximum staining intensity of the tumor is 3. In some embodiments, c-met expression is membranous. In some embodiments, c-met expression is cytoplasmic. In some
  • c-met- expression is membranous and cytoplasmic.
  • the tumor is c-met positive when 50% or more of the tumor cells in the sample have high c-met staining intensity.
  • the tumor is c-met positive when 50% or more of the tumor cells in the sample have moderate c-met staining intensity.
  • the tumor is c-met positive when 50% or more of the tumor cells in the sample have low, moderate or high c-met staining intensity.
  • biomarker detection includes but not limited to nucleic acid detection methods (including but not limited to PCR, sequencing, rtPCT, RNA- seq, microarray analysis, SAGE, Mass ARRAY technique and FISH) and protein detection methods (including but not limited to mass spec, western blotting).
  • Detecting amplification of the c-met gene is achieved using certain techniques known to those skilled in the art. For example, comparative genome hybridization may be used to produce a map of DNA sequence copy number as a function of chromosomal location. See, e.g., Kallioniemi et al. (1992) Science 258:818-821.
  • Amplification of the c-met gene may also be detected, e.g., by Southern hybridization using a probe specific for the c-met gene or by real-time quantitative PCR.
  • detecting amplification of the c-met gene is achieved by directly assessing the copy number of the c-met gene, for example, by using a probe that hybridizes to the c-met gene.
  • a FISH assay may be performed.
  • 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 that lies outside the c-met gene but is co-amplified with the c-met gene.
  • Biomarker expression may also be evaluated using an in vivo diagnostic assay, e.g. by administering a molecule (such as an antibody) which binds the molecule to be detected and is tagged with a detectable label ⁇ e.g. a radioactive isotope) and externally scanning the patient for localization of the label.
  • a detectable label e.g. a radioactive isotope
  • the invention provides treatment (e.g., therapeutic treatment) methods, for treating a cancer patient who is likely to respond to treatment with a c-met antagonist.
  • treatment e.g., therapeutic treatment
  • Cancer medicaments can be used either alone or in combination with other cancer medicaments.
  • an anti-c-met antibody for example, Onartuzumab
  • a c-met antibody may be co-administered with at least one additional therapeutic agent, e.g. with a chemotherapeutic agent, with other c-met antagonists (such as other c-met antibodies), with an EGFR antagonist (such as erlotinib), or with an anti-VEGF antibody (such a bevacizumab).
  • a c-met antibody may be co-administered with an additional c-met antagonist.
  • Such combination therapies noted above encompass combined administration (where two or more therapeutic agents are included in the same or separate formulations), and separate administration, in which case, administration of a first medicament can occur prior to, simultaneously, and/or following, administration of a second medicament.
  • an anti-c-met antibody such as Onartuzumab
  • erlotinib N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)-4- quinazolinamine
  • a pharmaceutically acceptable salt at a dose of 150 mg, each day of a three week cycle.
  • a c-met antagonist e.g., anti-c-met antibody
  • an anti-VEGF antibody and chemotherapy e.g., a taxane
  • An exemplary protocol is administering to a triple-negative metastatic breast cancer patient an anti-c-met antibody (e.g., Onartuzumab) administered at a dose of 10 mg/kg on Day 1 and Day 15 of a 28- day cycle, anti-VEGF antibody (e.g., bevacizumab) administered at a dose of 10 mg/kg on Day 1 and Day 15 of the 28-day cycle and paclitaxel administered at a dose of 90 mg/m by IV infusion on Day 1, Day 8, and Day 15 of the 28-day cycle.
  • an anti-c-met antibody e.g., Onartuzumab
  • anti-VEGF antibody e.g., bevacizumab
  • paclitaxel administered at a dose of 90 mg/m by IV infusion on Day 1, Day 8, and Day 15 of the 28-day cycle.
  • An exemplary protocol is administering to a triple-negative metastatic breast cancer patient an anti-c-met antibody (e.g., Onartuzumab) administered at a dose of 10 mg/kg on Day 1 and Day 15 of a 28-day cycle, and paclitaxel administered at a dose of 90 mg/m by IV infusion on Day 1, Day 8, and Day 15 of the 28 -day cycle.
  • an anti-c-met antibody e.g., Onartuzumab
  • paclitaxel administered at a dose of 90 mg/m by IV infusion on Day 1, Day 8, and Day 15 of the 28 -day cycle.
  • Cancer medicaments can also be used in combination with radiation therapy.
  • the medicament(s) herein can be administered by any suitable means, including parenteral, intrapulmonary, and intranasal, and, if desired for local treatment, intralesional administration.
  • Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration.
  • Dosing can be by any suitable route, e.g. by injections, such as intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic.
  • Various dosing schedules including but not limited to single or multiple administrations over various time-points, bolus administration, and pulse infusion are contemplated herein.
  • an antibody of the invention when used alone or in combination with one or more other additional therapeutic agents, will depend on the type of disease to be treated, the type of antibody, the severity and course of the disease, whether the antibody is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody, and the discretion of the attending physician.
  • the antibody is suitably administered to the patient at one time or over a series of treatments.
  • One typical daily dosage might range from about 1 ⁇ g/kg to 100 mg/kg or more, depending on the factors mentioned above.
  • the treatment would generally be sustained until a desired suppression of disease symptoms occurs.
  • other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.
  • the patient did not receive more than two prior treatments for Stage IIIB/IV. In some embodiments, the patient did not receive more than 30 days of exposure to an investigational or marketed agent that can act by EGFR inhibition, or a known EGFR- related toxicity resulting in dose modifications.
  • EGFR inhibitors include (but are not limited to) gefitinib, erlotinib, and cetuximab.
  • the patient did not receive chemotherapy, biologic therapy, radiotherapy or investigational drug within 28 days prior to randomization (except that optionally, kinase inhibitors may be used within two weeks prior to randomization provided any drug related toxicity was adequately resolved).
  • the patient is not a patient with untreated and/or active (progressing or requiring anticonvulsants or corticosteroids for symptomatic control) CNS metastasis.
  • Other patient exclusion criteria are described in the Examples, and the present inventions contemplate use of one or more of the exclusions described therein.
  • a sample of the patient's cancer has been shown to have wildtype EGFR.
  • a sample of the patient's cancer has been shown to have mutant EGFR.
  • a sample of the patient's cancer has not been shown to have mutated EGFR.
  • an article of manufacture for use in treating cancer (such as NSCLC or breast cancer) is provided.
  • the article of manufacture comprises a container and a label or package insert on or associated with the container.
  • Suitable containers include, for example, bottles, vials, syringes, etc.
  • the containers may be formed from a variety of materials such as glass or plastic.
  • the container holds or contains a composition comprising the cancer medicament as the active agent and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle).
  • the article of manufacture may further comprise a second container comprising a pharmaceutically-acceptable diluent buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution.
  • a pharmaceutically-acceptable diluent buffer such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution.
  • BWFI bacteriostatic water for injection
  • phosphate-buffered saline such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution.
  • BWFI bacteriostatic water for injection
  • phosphate-buffered saline such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution.
  • the article of manufacture may further include other
  • the article of manufacture of the present invention also includes information, for example in the form of a package insert, indicating that the composition is used for treating cancer based on expression of biomarker(s) as disclosed herein.
  • the insert or label may take any form, such as paper or on electronic media such as a magnetically recorded medium (e.g., floppy disk) or a CD-ROM.
  • the label or insert may also include other information concerning the pharmaceutical compositions and dosage forms in the kit or article of manufacture.
  • the invention also concerns a method for manufacturing 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 patient with cancer (such as NSCLC) based on expression of a c- met biomarker as disclosed herein.
  • a c-met antagonist e.g., an anti-c-met antibody
  • a package insert indicating that the pharmaceutical composition is for treating a patient with cancer (such as NSCLC) based on expression of a c- met biomarker as disclosed herein.
  • the article of manufacture may further comprise an additional container comprising a pharmaceutically acceptable diluent buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and/or dextrose solution.
  • a pharmaceutically acceptable diluent buffer such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and/or dextrose solution.
  • BWFI bacteriostatic water for injection
  • phosphate-buffered saline such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and/or dextrose solution.
  • BWFI bacteriostatic water for injection
  • phosphate-buffered saline such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and/or dextrose solution.
  • kits useful for detecting any one or more of the biomarker(s) identified herein comprising one or more reagents for determining expression of one or more of c-met, k-ras, ALK and EGFR biomarker in a sample from a cancer patient.
  • the kit further comprises instructions to use the kit to select a cancer medicament (e.g. a c-met antagonist, such as an anti-c-met antibody) for treating the cancer patient if the patient expresses the c-met biomarker at a high level.
  • a cancer medicament e.g. a c-met antagonist, such as an anti-c-met antibody
  • the instructions are to use the kit to select a cancer medicament other than c-met antagonist (or other than an anti-c-met antibody) if the patient expresses the biomarker at a reduced level.
  • the invention herein also concerns a method for advertising a cancer medicament comprising promoting, to a target audience, the use of the cancer medicament (e.g. anti-c-met antibody) for treating a patient with cancer based on expression of c-met biomarker as disclosed herein.
  • the cancer medicament e.g. anti-c-met antibody
  • Advertising is generally paid communication through a non-personal medium in which the sponsor is identified and the message is controlled. Advertising for purposes herein includes publicity, public relations, product placement, sponsorship, underwriting, and sales promotion. This term also includes sponsored informational public notices appearing in any of the print communications media designed to appeal to a mass audience to persuade, inform, promote, motivate, or otherwise modify behavior toward a favorable pattern of purchasing, supporting, or approving the invention herein.
  • the advertising and promotion of the diagnostic method herein may be accomplished by any means.
  • Examples of advertising media used to deliver these messages include television, radio, movies, magazines, newspapers, the internet, and billboards, including commercials, which are messages appearing in the broadcast media. Advertisements also include those on the seats of grocery carts, on the walls of an airport walkway, and on the sides of buses, or heard in telephone hold messages or in-store PA systems, or anywhere a visual or audible communication can be placed.
  • promotion or advertising means include television, radio, movies, the internet such as webcasts and webinars, interactive computer networks intended to reach simultaneous users, fixed or electronic billboards and other public signs, posters, traditional or electronic literature such as magazines and newspapers, other media outlets, presentations or individual contacts by, e.g., e-mail, phone, instant message, postal, courier, mass, or carrier mail, in-person visits, etc.
  • the type of advertising used will depend on many factors, for example, on the nature of the target audience to be reached, e.g., hospitals, insurance companies, clinics, doctors, nurses, and patients, as well as cost considerations and the relevant jurisdictional laws and regulations governing advertising of medicaments and diagnostics.
  • the advertising may be individualized or customized based on user characterizations defined by service interaction and/or other data such as user demographics and geographical location.
  • Onartuzumab is a monoclonal monovalent (one-armed) antibody specifically designed to inhibit HGF-stimulated activation of the Met signaling pathway.
  • a recent phase II study (OAM4558g) was conducted to evaluate the activity of onartuzumab in combination with the EGFR pathway inhibitor, erlotinib, compared to erlotinib + placebo in patients with recurrent NSCLC.
  • a tissue specimen was required for all patients enrolled on this study to evaluate Met expression by immunohistochemistry (IHC).
  • Co-primary endpoints were progression-free survival (PFS) in the intent-to-treat (ITT) population and in the population of patients defined as Met-positive based upon the IHC test result, respectively.
  • Tumors with >50% of tumor cells exhibiting moderate to strong staining intensity were pre-defined as Met positive, prior to unblinding the treatment assignment.
  • the primary efficacy endpoint was progression free survival (PFS) with PFS in the Met-positive population as co-primary endpoint.
  • PFS progression free survival
  • the other key endpoints included safety and overall survival (OS).
  • TFS progression free survival
  • OS safety and overall survival
  • FFPE cell pellets were produced by growing eight T175 flasks for each cell line, dislodging cells with 10 mmol/L EDTA (pH 8.0) followed by centrifugation, washing in PBS, and then fixation overnight in 10% neutral buffered formalin. Fixed pellets were processed in a Tissue-Tek processor (Sakura Finetek, CA, USA) and then embedded in a paraffin block prior to constructing a tissue microarray.
  • Met expression levels were evaluated by IHC analysis of archival tissue specimens using the CONFIRM anti-total cMet (SP44) rabbit monoclonal primary antibody (cat no. 790- 4430, Ventana Medical Systems, Arlington, AZ), according to the manufacturer's instructions.
  • the staining was performed on the Ventana Benchmark XT instrument using CC1 standard antigen retrieval (Ventana Medical Systems, Arlington, AZ).
  • Incubation with primary antibody was performed for 16 minutes at 37°C using a concentration of primary antibody of 9.75 ⁇ g/ml.
  • Specifically-bound primary antibody was detected using ultra View methodology with diaminobenzidine (Ventana Medical Systems, Arlington, AZ); sections were counterstained with hematoxylin.
  • MET IHC staining intensity in tumor samples in H-score format, the proportion of tumor cells showing staining at weak, moderate, and strong intensity was estimated as percentage of the total number of tumor cells in a given NSCLC case.
  • a single intensity score was generated for each sample (i.e., both membranous and cytoplasmic staining were combined into a single intensity score). If staining of multiple intensities was present in a sample, the highest intensity staining of the sample was recorded as the sample intensity.
  • the composite score was calculated based on the formula:
  • Tumor cells showing no staining for Met were not considered for this calculation. Following this formula, a given tumor can be associated with a value between "0" (none of the tumor cells show any staining) and "300" (100% of the tumor cells show strong staining).

Abstract

The present invention concerns cancer biomarkers. In particular, the invention concerns c-met as biomarkers for patient selection and patient prognosis in cancer, as well as methods of therapeutic treatment, articles of manufacture and methods for making them, diagnostic kits, methods of detection and methods of advertising related thereto.

Description

BIOMARKERS AND METHODS OF TREATMENT
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims priority to U.S. patent application number 61/801,276, filed 15 March 2013, which is hereby incorporated by reference in its entirety.
SEQUENCE LISTING
The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on March 11, 2014, is named P5591RlWO_PCTSequenceListing.txt and is 16,509 bytes in size.
FIELD OF THE INVENTION
The present invention concerns cancer biomarkers. In particular, the invention concerns c-met as a biomarker for patient selection and prognosis in cancer, as well as methods of therapeutic treatment, articles of manufacture and methods for making them, diagnostic kits, methods of detection and methods of advertising related thereto.
BACKGROUND
Cancer remains to be one of the most deadly threats to human health. In the U.S., cancer affects nearly 1.3 million new patients each year, and is the second leading cause of death after heart disease, accounting for approximately 1 in 4 deaths. For example, breast cancer is the second most common form of cancer and the second leading cancer killer among American women. It is also predicted that cancer may surpass cardiovascular diseases as the number one cause of death within 5 years. Solid tumors are responsible for most of those deaths. Although there have been significant advances in the medical treatment of certain cancers, the overall 5 -year survival rate for all cancers has improved only by about 10% in the past 20 years. Cancers, or malignant tumors, metastasize and grow rapidly in an uncontrolled manner, making timely detection and treatment extremely difficult.
Despite the significant advancement in the treatment of cancer, improved therapies are still being sought. All references cited herein, including patent applications and publications, are incorporated by reference in their entirety.
SUMMARY OF THE INVENTION
Uses of a c-met antagonist for effectively treating cancer patients are provided. This application also provides better methods for diagnosing disease for use in treating the disease optionally with c-met antagonist. In particular, c-met (interchangeably termed "Met") biomarker was used to identify a patient population in which Onartuzumab (interchangeably termed "MetMAb") plus erlotinib treatment provided clinically meaningful benefit, evaluated by progression-free survival and overall survival, and a patient population in which
Onartuzumab plus erlotinib treatment significantly increased the risk of cancer progression and death (compared to treatment with erlotinib alone). This worse outcome underscores the need to select patients who will benefit from treatment with c-met antagonist (e.g., in combination with EGFR antagonist).
In one aspect, provided are methods for treating (e.g., therapeutically treating) a patient with cancer comprising administering a therapeutically effective amount of a c-met antagonist to the patient if the patient's cancer has been found to have an H-score about 160 or higher (about 161, 162, 163, 164, 165, 166, 167, 168, 169, or more), about 160 to about 230, about 230 or higher, or about 170 or higher, wherein the H-score is determined using the formula (% cancer cells staining with weak c-met staining intensity) + (% cancer cells staining with moderate c-met staining intensity) + (% cancer cells staining with high c-met staining intensity). In some embodiments, the patient has greater PFS and/or OS (e.g., relative to a patient who has an H score less than 160). In some embodiments, the patient has greater PFS (e.g., relative to a patient who has an H score less than 160). In some embodiments, the patient has greater OS (e.g., relative to a patient who has an H score less than 160). In some embodiments, the H-score is about 160 or higher. In some embodiments, the H-score is 160 or higher. In some embodiments, H-score is about 160 to about 230. In some embodiments, the H-score is 160-230. In some embodiments, the H score is greater than about 10. In some embodiments, the H score is greater than about 25. In some embodiments, the H score is greater than about 50. In some embodiments, the H score is greater than about 75. In some embodiments, the H score is greater than about 100. In some embodiments, the H score is greater than about 125. In some embodiments, the H score is greater than about 150. In some embodiments, the H score is greater than about 175. In some embodiments, the H score is greater than about 200. In some embodiments, c-met expression is membranous. In some embodiments, c-met expression is cytoplasmic. In some embodiments, c-met- expression is membranous and cytoplasmic.
In one aspect, provided are methods for treating (e.g., therapeutically treating) a patient with cancer comprising administering a therapeutically effective amount of a medicament other than a c-met antagonist to the patient if the patient's cancer has been found to have an H-score of less than 160, wherein the H-score is determined using the formula (% cancer cells staining with weak c-met staining intensity) + (% cancer cells staining with moderate c-met staining intensity) + (% cancer cells staining with high c-met staining intensity). In some embodiments, the H-score is less than 160. In some embodiments, the H-score is less than 150. In some embodiments, the H score is greater than about 10. In some embodiments, the H score is greater than about 25. In some embodiments, the H score is greater than about 50. In some embodiments, the H score is greater than about 75. In some embodiments, the H score is greater than about 100. In some embodiments, the H score is greater than about 125. In some embodiments, the H score is greater than about 150. In some embodiments, the H score is greater than about 175. In some embodiments, the H score is greater than about 200. In some embodiments, c-met expression is membranous. In some embodiments, c-met expression is cytoplasmic. In some embodiments, c-met- expression is membranous and cytoplasmic.
In one aspect, provided are methods for selecting a therapy for a patient with cancer comprising determining an H-score from a sample of the patient's cancer using the formula (% cancer cells staining with weak c-met staining intensity) + (% cancer cells staining with moderate c-met staining intensity) + (% cancer cells staining with high c-met staining intensity), and selecting a cancer medicament based on the H-score. In some embodiments, the patient is selected for treatment with a c-met antagonist if the H-score is about 160 or higher, about 160 to about 230, about 230 or higher, or about 170 or higher. In some embodiments, the H score is greater than about 10. In some embodiments, the H score is greater than about 25. In some embodiments, the H score is greater than about 50. In some embodiments, the H score is greater than about 75. In some embodiments, the H score is greater than about 100. In some embodiments, the H score is greater than about 125. In some embodiments, the H score is greater than about 150. In some embodiments, the H score is greater than about 175. In some embodiments, the H score is greater than about 200. In some embodiments, c-met expression is membranous. In some embodiments, c-met expression is cytoplasmic. In some embodiments, c-met- expression is membranous and cytoplasmic. In some embodiments, the patient is selected for treatment with a cancer medicament other than a c-met antagonist if the H-score is lower than 160. In some embodiments, the patient is selected for treatment with a cancer medicament other than a c-met antagonist if the H-score is lower than 159, 158, 157, 156, 155, 154, 153, 152, 151, or 150.
In another aspect, provided method for identifying a cancer patient who is likely to respond to treatment with a c-met antagonist comprising the step of determining an H-score from a sample of the patient's cancer using the formula (% cancer cells staining with weak c- met staining intensity) + (% cancer cells staining with moderate c-met staining intensity) + (% cancer cells staining with high c-met staining intensity), wherein an H-score of about 160 or higher, about 160 to about 230, about 230 or higher, or about 170 or higher indicates that the patient is likely to response to treatment with a c-met antagonist. . In some embodiments, the H score is greater than about 10. In some embodiments, the H score is greater than about 25. In some embodiments, the H score is greater than about 50. In some embodiments, the H score is greater than about 75. In some embodiments, the H score is greater than about 100. In some embodiments, the H score is greater than about 125. In some embodiments, the H score is greater than about 150. In some embodiments, the H score is greater than about 175. In some embodiments, the H score is greater than about 200. In some embodiments, c-met expression is membranous. In some embodiments, c-met expression is cytoplasmic. In some embodiments, c-met- expression is membranous and cytoplasmic.
In another aspect, provided are method for determining cancer patient prognosis, comprising the step of determining an H-score from a sample of the patient's cancer using the formula (% cancer cells staining with weak c-met staining intensity) + (% cancer cells staining with moderate c-met staining intensity) + (% cancer cells staining with high c-met staining intensity), wherein an H-score of about 160 or higher, about 160 to about 230, about 230 or higher, or about 170 or higher indicates that the patient is likely to have increased overall survival (OS) and/or progression-free survival (PFS) when the patient is treated with a c-met antagonist. . In some embodiments, the H score is greater than about 10. In some embodiments, the H score is greater than about 25. In some embodiments, the H score is greater than about 50. In some embodiments, the H score is greater than about 75. In some embodiments, the H score is greater than about 100. In some embodiments, the H score is greater than about 125. In some embodiments, the H score is greater than about 150. In some embodiments, the H score is greater than about 175. In some embodiments, the H score is greater than about 200. In some embodiments, c-met expression is membranous. In some embodiments, c-met expression is cytoplasmic. In some embodiments, c-met- expression is membranous and cytoplasmic.
In another aspect, provided are methods for identifying a cancer patient who is less likely to be respond to treatment with a c-met antagonist comprising the step of determining an H-score from a sample of the patient's cancer using the formula (% cancer cells staining with weak c-met staining intensity) + (% cancer cells staining with moderate c-met staining intensity) + (% cancer cells staining with high c-met staining intensity), wherein an H-score of less than about 160 indicates that the patient is less likely to respond to treatment with the c- met antagonist. . In some embodiments, the H score is greater than about 10. In some embodiments, the H score is greater than about 25. In some embodiments, the H score is greater than about 50. In some embodiments, the H score is greater than about 75. In some embodiments, the H score is greater than about 100. In some embodiments, the H score is greater than about 125. In some embodiments, the H score is greater than about 150. In some embodiments, the H score is greater than about 175. In some embodiments, the H score is greater than about 200. In some embodiments, c-met expression is membranous. In some embodiments, c-met expression is cytoplasmic. In some embodiments, c-met- expression is membranous and cytoplasmic.
In another aspect, provided are methods for determining cancer patient prognosis, comprising the step of determining an H-score from a sample of the patient's cancer using the formula (% cancer cells staining with weak c-met staining intensity) + (% cancer cells staining with moderate c-met staining intensity) + (% cancer cells staining with high c-met staining intensity), wherein an H-score of about 160 or higher, about 160 to about 230, about 230 or higher, or about 170 or higher indicates that the patient is likely to have increased overall survival (OS) and/or progression-free survival (PFS) when the patient is treated with a c-met antagonist. . In some embodiments, the H score is greater than about 10. In some embodiments, the H score is greater than about 25. In some embodiments, the H score is greater than about 50. In some embodiments, the H score is greater than about 75. In some embodiments, the H score is greater than about 100. In some embodiments, the H score is greater than about 125. In some embodiments, the H score is greater than about 150. In some embodiments, the H score is greater than about 175. In some embodiments, the H score is greater than about 200. In some embodiments, c-met expression is membranous. In some embodiments, c-met expression is cytoplasmic. In some embodiments, c-met- expression is membranous and cytoplasmic. In another aspect, provided are methods for determining c-met biomarker expression, comprising determining an H-score from a sample of the patient's cancer using the formula (% cancer cells staining with weak c-met staining intensity) + (% cancer cells staining with moderate c-met staining intensity) + (% cancer cells staining with high c-met staining intensity). In some embodiments, an H-score of about 160 or higher, about 160 to about 230, about 230 or higher, or about 170 or higher indicates that the patient is likely to have increased overall survival (OS) and/or progression-free survival (PFS) when the patient is treated with a c-met antagonist. In some embodiments, the patient has greater PFS and/or OS (e.g., relative to a patient who has an H score less than 160). In some embodiments, the patient has greater PFS (e.g., relative to a patient who has an H score less than 160). In some embodiments, the patient has greater OS (e.g., relative to a patient who has an H score less than 160). In some embodiments, the H-score is about 160 or higher. In some embodiments, the H-score is 160 or higher. In some embodiments, H-score is about 160 to about 230. In some embodiments, H- score is 160-230. . In some embodiments, the H score is greater than about 10. In some embodiments, the H score is greater than about 25. In some embodiments, the H score is greater than about 50. In some embodiments, the H score is greater than about 75. In some embodiments, the H score is greater than about 100. In some embodiments, the H score is greater than about 125. In some embodiments, the H score is greater than about 150. In some embodiments, the H score is greater than about 175. In some embodiments, the H score is greater than about 200. In some embodiments, c-met expression is membranous. In some embodiments, c-met expression is cytoplasmic. In some embodiments, c-met- expression is membranous and cytoplasmic.
In one aspect, the invention provides a method of determining patient prognosis, comprising determining an H-score from a sample of the patient's cancer using the formula (% cancer cells staining with weak c-met staining intensity) + (% cancer cells staining with moderate c-met staining intensity) + (% cancer cells staining with high c-met staining intensity), wherein an H-score of about 160 or higher, about 160 to about 230, about 230 or higher, or about 170 or higher indicates that the patient is likely to have poor prognosis. . In some embodiments, the H score is greater than about 10. In some embodiments, the H score is greater than about 25. In some embodiments, the H score is greater than about 50. In some embodiments, the H score is greater than about 75. In some embodiments, the H score is greater than about 100. In some embodiments, the H score is greater than about 125. In some embodiments, the H score is greater than about 150. In some embodiments, the H score is greater than about 175. In some embodiments, the H score is greater than about 200. In some embodiments, c-met expression is membranous. In some embodiments, c-met expression is cytoplasmic. In some embodiments, c-met- expression is membranous and cytoplasmic.
In one aspect, the invention provides a method of optimizing therapeutic efficacy comprising determining an H-score from a sample of the patient's cancer using the formula (% cancer cells staining with weak c-met staining intensity) + (% cancer cells staining with moderate c-met staining intensity) + (% cancer cells staining with high c-met staining intensity). In some embodiments, an H-score of about 160 or higher, about 160 to about 230, about 230 or higher, or about 170 or higher indicates that the patient is likely to have increased overall survival (OS) and/or progression- free survival (PFS) when the patient is treated with a c-met antagonist. In some embodiments, the patient has greater PFS and/or OS (e.g., relative to a patient who has an H score less than 160). In some embodiments, the patient has greater PFS (e.g., relative to a patient who has an H score less than 160). In some embodiments, the patient has greater OS (e.g., relative to a patient who has an H score less than 160). In some embodiments, the H-score is about 160 or higher. In some embodiments, the H-score is 160 or higher. In some embodiments, H-score is about 160 to about 230. In some embodiments, H- score is 160-230. . In some embodiments, the H score is greater than about 10. In some embodiments, the H score is greater than about 25. In some embodiments, the H score is greater than about 50. In some embodiments, the H score is greater than about 75. In some embodiments, the H score is greater than about 100. In some embodiments, the H score is greater than about 125. In some embodiments, the H score is greater than about 150. In some embodiments, the H score is greater than about 175. In some embodiments, the H score is greater than about 200. In some embodiments, c-met expression is membranous. In some embodiments, c-met expression is cytoplasmic. In some embodiments, c-met- expression is membranous and cytoplasmic.
In some embodiments of any of the inventions herein, the c-met antagonist is an antagonist anti-c-met antibody. In some embodiments, the anti-c-met antibody comprises a (a) HVR1 comprising sequence GYTFTSYWLH (SEQ ID NO: 1); (b) HVR2 comprising sequence GMIDPSNSDTRFNPNFKD (SEQ ID NO: 2); (c) HVR3-HC comprising sequence ATYRS YVTPLDY (SEQ ID NO : 3); (d) HVR1 -LC comprising sequence
KSSQSLLYTSSQKNYLA (SEQ ID NO: 4); (e) HVR2-LC comprising sequence WASTRES (SEQ ID NO: 5); and (f) HVR3-LC comprising sequence QQYYAYPWT (SEQ ID NO: 6). In some embodiments, the anti-c-met antibody is monovalent and comprises (a) a first polypeptide comprising a heavy chain, said polypeptide comprising the sequence:
EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWLHWVRQAPGKGLEWVGMIDPSNS DTRFNPNFKDRFTISADTSKNTAYLQMNSLRAEDTAVYYCATYRSYVTPLDYWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDK VEPKSCDKTHTCPPC PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNA KTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRE PQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 11); (b) a second polypeptide comprising a light chain, the polypeptide comprising the sequence
DIQMTQSPSSLSASVGDRVTITCKSSQSLLYTSSQKNYLAWYQQKPGKAPKLLIYWAST RESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYAYPWTFGQGTKVEIKRTVAA PSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKD STYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 12); and a third polypeptide comprising a Fc sequence, the polypeptide comprising the sequence
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (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 a complex and form a Fc region that increases stability of said antibody fragment compared to a Fab molecule comprising said antigen binding arm. In some embodiments, the c-met antagonist is one or more of crizotinib, tivantinib, carbozantinib, MGCD-265, ficlatuzumab, humanized TAK-701, rilotumumab, foretinib, h224Gl 1, 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 LA480. In some embodiments, the c-met antagonist is rilotumumab. In some embodiments, the c-met antagonist is crizotinib. In some embodiments, the c-met antagonist is tivantinib.
In some embodiments of any of the inventions herein, treatment with c-met antagonist is in combination with treatment with an EGFR antagonist. In some embodiments, the EGFR antagonist is erlotinib. In some embodiments, the c-met antagonist is onartuzumab and treatment further comprises treatment with erlotinib. In some embodiments, c-met antagonist is crizotinib, tivantinib, carbozantinib, MGCD-265, ficlatuzumab, humanized TAK-701, or foretinib, and treatment further comprises treatment with erlotinib.
In some embodiments of any of the inventions herein, the patient has NSCLC and is treated with a combination of anti-c-met antibody and an EGFR antagonist. In some embodiments, the EGFR antagonist is erlotinib. In some embodiments, the patient has NSCLC and is treated with (a) onartuzumab at a dose of about 15 mg/kg every three weeks; and (b) erlotinib (N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)-4-quinazolinamine HC1) at a dose of 150 mg, each day of a three week cycle.
In one aspect, methods are provided for advertising a c-met antibody comprising promoting, to a target audience, the use of the c-met antibody for treating a patient with cancer based on an H-score from a sample of the patient's cancer, wherein the H-score is calculated using the formula (% cancer cells staining with weak c-met staining intensity) + (% cancer cells staining with moderate c-met staining intensity) + (% cancer cells staining with high c- met staining intensity). In some embodiments, the promotion is by a package insert accompanying a commercial formulation of an anti-c-met antibody. In some embodiments, promotion is by a package insert accompanying a commercial formulation of a second medicament. In some embodiments, the promotion is by a package insert accompanying a c- met antibody suitable for use in c-met IHC assay. In some embodiments, the second medicament is an EGFR antagonist. In some embodiments, the anti-c-met antibody is
Onartuzumab and the EGFR antagonist is erlotinib. In some embodiments, the patient is selected for treatment with a c-met antagonist if the H-score is about 160 or higher, about 160 to about 230, about 230 or higher, or about 170 or higher. In some embodiments, the patient is treated with the c-met antibody if the H-score is about 160 or higher, about 160 to about 230, about 230 or higher, or about 170 or higher. . In some embodiments, the H score is greater than about 10. In some embodiments, the H score is greater than about 25. In some embodiments, the H score is greater than about 50. In some embodiments, the H score is greater than about 75. In some embodiments, the H score is greater than about 100. In some embodiments, the H score is greater than about 125. In some embodiments, the H score is greater than about 150. In some embodiments, the H score is greater than about 175. In some embodiments, the H score is greater than about 200. In some embodiments, c-met expression is membranous. In some embodiments, c-met expression is cytoplasmic. In some embodiments, c-met- expression is membranous and cytoplasmic. In some embodiments, the promotion is by a package insert where the package insert provides instructions to receive therapy with anti-c-met antibody in combination with an EGFR antagonist. In some embodiments, the promotion is followed by the treatment of the patient with the anti-c-met antibody with or without the second
medicament.
In one aspect, provided are diagnostic kits comprising one or more reagent for determining an H-score from a sample of the patient's cancer using the formula (% cancer cells staining with weak c-met staining intensity) + (% cancer cells staining with moderate c-met staining intensity) + (% cancer cells staining with high c-met staining intensity), wherein an H- score of about 160 or higher, about 160 to about 230, about 230 or higher, or about 170 or higher indicates that the patient is likely to response to treatment with a c-met antagonist. In some embodiments, the patient has greater PFS and/or OS (e.g., relative to a patient who has an H score less than 160). In some embodiments, the patient has greater PFS (e.g., relative to a patient who has an H score less than 160). In some embodiments, the patient has greater OS (e.g., relative to a patient who has an H score less than 160). In some embodiments, the H- score is about 160 or higher. In some embodiments, the H-score is 160 or higher. In some embodiments, H-score is about 160 to about 230. In some embodiments, H-score is 160-230. . In some embodiments, the H score is greater than about 10. In some embodiments, the H score is greater than about 25. In some embodiments, the H score is greater than about 50. In some embodiments, the H score is greater than about 75. In some embodiments, the H score is greater than about 100. In some embodiments, the H score is greater than about 125. In some embodiments, the H score is greater than about 150. In some embodiments, the H score is greater than about 175. In some embodiments, the H score is greater than about 200. In some embodiments, c-met expression is membranous. In some embodiments, c-met expression is cytoplasmic. In some embodiments, c-met- expression is membranous and cytoplasmic.
In one aspect, provided are diagnostic kits comprising one or more reagent for determining an H-score from a sample of the patient's cancer using the formula (% cancer cells staining with weak c-met staining intensity) + (% cancer cells staining with moderate c-met staining intensity) + (% cancer cells staining with high c-met staining intensity), wherein an H- score of about 160 or higher, about 160 to about 230, about 230 or higher, or about 170 or higher means the patient has greater PFS and/or OS (e.g., relative to a patient who has an H score less than 160). In some embodiments, the patient has greater PFS (e.g., relative to a patient who has an H score less than 160). In some embodiments, the patient has greater OS (e.g., relative to a patient who has an H score less than 160). In some embodiments, the diagnostic kits herein further comprise instructions to use the kit to select a c-met medicament to treat the NSCLC patient if the H-score is about 160 or higher, about 160 to about 230, about 230 or higher, or about 170 or higher. . In some embodiments, the H score is greater than about 10. In some embodiments, the H score is greater than about 25. In some embodiments, the H score is greater than about 50. In some embodiments, the H score is greater than about 75. In some embodiments, the H score is greater than about 100. In some embodiments, the H score is greater than about 125. In some embodiments, the H score is greater than about 150. In some embodiments, the H score is greater than about 175. In some embodiments, the H score is greater than about 200. In some embodiments, c-met expression is membranous. In some embodiments, c-met expression is cytoplasmic. In some embodiments, c-met- expression is membranous and cytoplasmic.
In one aspect, provided are kits comprising a c-met antagonist, and instructions to use the c-met medicament to treat a patient if an H-score determined from a sample of the patient's cancer is about 160 or higher, about 160 to about 230, about 230 or higher, or about 170 or higher, wherein the H-score is calculated using the formula (% cancer cells staining with weak c-met staining intensity) + (% cancer cells staining with moderate c-met staining intensity) + (% cancer cells staining with high c-met staining intensity). In some embodiments, the patient has greater PFS and/or OS (e.g., relative to a patient who has an H score less than 160). In some embodiments, the patient has greater PFS (e.g., relative to a patient who has an H score less than 160). In some embodiments, the patient has greater OS (e.g., relative to a patient who has an H score less than 160). In some embodiments, the H-score is about 160 or higher. In some embodiments, the H-score is 160 or higher. In some embodiments, H-score is about 160 to about 230. In some embodiments, H-score is 160-230. In some embodiments, the H score is greater than about 10. In some embodiments, the H score is greater than about 25. In some embodiments, the H score is greater than about 50. In some embodiments, the H score is greater than about 75. In some embodiments, the H score is greater than about 100. In some embodiments, the H score is greater than about 125. In some embodiments, the H score is greater than about 150. In some embodiments, the H score is greater than about 175. In some embodiments, the H score is greater than about 200. In some embodiments, c-met expression is membranous. In some embodiments, c-met expression is cytoplasmic. In some embodiments, c-met- expression is membranous and cytoplasmic.
The invention also concerns articles of manufacture comprising, packaged together, a c- met antagonist in a pharmaceutically acceptable carrier and a package insert indicating that the c-met antagonist is for treating a patient with cancer based on expression of c-met biomarker. Treatment methods include any of the treatment methods disclosed herein. In some embodiments, the package insert indicates that the c-met antagonist is to be used to treat the patient if the patient's cancer sample c-met antagonist, and instructions to use the c-met medicament to treat a patient if an H-score determined from a sample of the patient's cancer is about 160 or higher, about 160 to about 230, about 230 or higher, or about 170 or higher, wherein the H-score is calculated using the formula (% cancer cells staining with weak c-met staining intensity) + (% cancer cells staining with moderate c-met staining intensity) + (% cancer cells staining with high c-met staining intensity). In some embodiments, the patient has greater PFS and/or OS (e.g., relative to a patient who has an H score less than 160). In some embodiments, the patient has greater PFS (e.g., relative to a patient who has an H score less than 160). In some embodiments, the patient has greater OS (e.g., relative to a patient who has an H score less than 160). In some embodiments, the H-score is about 160 or higher. In some embodiments, the H-score is 160 or higher. In some embodiments, H-score is about 160 to about 230. In some embodiments, H-score is 160-230. In some embodiments, the H score is greater than about 10. In some embodiments, the H score is greater than about 25. In some embodiments, the H score is greater than about 50. In some embodiments, the H score is greater than about 75. In some embodiments, the H score is greater than about 100. In some embodiments, the H score is greater than about 125. In some embodiments, the H score is greater than about 150. In some embodiments, the H score is greater than about 175. In some embodiments, the H score is greater than about 200. In some embodiments, c-met expression is membranous. In some embodiments, c-met expression is cytoplasmic. In some embodiments, c-met- expression is membranous and cytoplasmic.
In some embodiments, an H-score less than 160, or less than 159, 158, 157, 156, 155, 154, 153, 152, 151 or 150 means that the patient has lower PFS and/or OS (e.g., relative to a patient who has an H score greater than 160). In some embodiments, the patient has lower PFS (e.g., relative to a patient who has an H score less than 160). In some embodiments, the patient has lower OS (e.g., relative to a patient who has an H score greater than 160). . In some embodiments, the H score is greater than about 10. In some embodiments, the H score is greater than about 25. In some embodiments, the H score is greater than about 50. In some embodiments, the H score is greater than about 75. In some embodiments, the H score is greater than about 100. In some embodiments, the H score is greater than about 125. In some embodiments, the H score is greater than about 150. In some embodiments, the H score is greater than about 175. In some embodiments, the H score is greater than about 200. In some embodiments, c-met expression is membranous. In some embodiments, c-met expression is cytoplasmic. In some embodiments, c-met- expression is membranous and cytoplasmic.
In a related aspect, the invention concerns methods for manufacturing an article of manufacture comprising combining in a package a pharmaceutical composition comprising a cancer medicament and a package insert indicating that the pharmaceutical composition is for treating a patient with cancer based on expression of c-met biomarker. Treatment methods include any of the treatment methods disclosed herein. In some embodiments, the package insert indicates that the c-met antagonist is to be used to treat the patient if the patient's cancer sample c-met antagonist, and instructions to use the c-met medicament to treat a patient if an H-score determined from a sample of the patient's cancer is about 160 or higher, about 160 to about 230, about 230 or higher, or about 170 or higher, wherein the H-score is calculated using the formula (% cancer cells staining with weak c-met staining intensity) + (% cancer cells staining with moderate c-met staining intensity) + (% cancer cells staining with high c-met staining intensity). In some embodiments, the patient has greater PFS and/or OS (e.g., relative to a patient who has an H score less than 160). In some embodiments, the patient has greater PFS (e.g., relative to a patient who has an H score less than 160). In some embodiments, the patient has greater OS (e.g., relative to a patient who has an H score less than 160). In some embodiments, the H-score is about 160 or higher. In some embodiments, the H-score is 160 or higher. In some embodiments, H-score is about 160 to about 230. In some embodiments, H- score is 160-230. In some embodiments, the H score is greater than about 10. In some embodiments, the H score is greater than about 25. In some embodiments, the H score is greater than about 50. In some embodiments, the H score is greater than about 75. In some embodiments, the H score is greater than about 100. In some embodiments, the H score is greater than about 125. In some embodiments, the H score is greater than about 150. In some embodiments, the H score is greater than about 175. In some embodiments, the H score is greater than about 200. In some embodiments, c-met expression is membranous. In some embodiments, c-met expression is cytoplasmic. In some embodiments, c-met- expression is membranous and cytoplasmic. In some embodiments, an H-score less than 160, or less than 159, 158, 157, 156, 155, 154, 153, 152, 151 or 150 means that the patient has lower PFS and/or OS (e.g., relative to a patient who has an H score greater than 160). In some embodiments, the patient has lower PFS (e.g., relative to a patient who has an H score less than 160). In some embodiments, the patient has lower OS (e.g., relative to a patient who has an H score greater than 160). In some embodiments of any of the inventions provided herein, negative c-met staining intensity means c-met staining intensity of control cell line TOV-112D. In some embodiments, negative c-met staining intensity means c-met staining intensity of control cell line H522. In some embodiments, negative c-met staining intensity means c-met staining intensity of control cell line HI 155. In some embodiments, negative c-met staining intensity refers to c-met staining intensity of control cell line LXFL529. In some embodiments, negative c-met staining intensity means c-met staining intensity of control cell line H23. In some embodiments, weak c-met staining intensity means c-met staining intensity of control cell line HI 703. In some embodiments, weak c-met staining intensity means c-met staining intensity of control cell line HEK-293. In some embodiments, moderate c-met staining intensity means c-met staining intensity of control cell line A549. In some embodiments, moderate c-met staining intensity means c-met staining intensity of control cell line SKMES1. In some embodiments, strong c- met staining intensity means c-met staining intensity of control cell line EBC-1. In some embodiments, strong c-met staining intensity means c-met staining intensity of control cell line H441.
In some embodiments of any of the inventions provided herein, the sample is obtained prior to treatment with c-met antagonist. In some embodiments, the sample is obtained prior to treatment with a cancer medicament. In some embodiments, the sample is obtained after the cancer has metastasized. In some embodiments, the sample is formalin fixed and paraffin embedded. In some embodiments, the sample is of a biopsy, a surgical specimen, or a fine needle aspirate.
In some embodiments of any of the inventions provided herein, control cell pellets are formalin fixed and paraffin embedded. In some embodiments, the control cell pellets are prepared as a tissue microarray. In some embodiments, the c-met IHC used to determine c-met staining intensity is performed using anti-c-met antibody SP44. In some embodiments, c-met IHC used to determine c-met staining intensity is performed using anti-c-met antibody D1C1. In some embodiments, c-met IHC used to determine c-met staining intensity is performed using anti-c-met antibody Met4. In some embodiments, c-met IHC used to determine c-met staining intensity is performed using anti-c-met antibody DL-21.
In some embodiments of any of the inventions provided herein, the cancer is non-small cell lung cancer, renal cell cancer, pancreatic cancer, gastric carcinoma, bladder cancer, esophageal cancer, mesothelioma, melanoma, breast cancer, thyroid cancer, colorectal cancer, head and neck cancer, osteosarcoma, prostate cancer, or glioblastoma. In some embodiments, cancer is non-small cell lung cancer (NSCLC). In some embodiments, NSCLC is second-line or third-line locally advanced or metastatic non-small cell lung cancer. In some embodiments, the NSCLC is adenocarcinoma. In some embodiments, the NSCLC is squamous cell carcinoma.
In certain embodiments of any of the inventions provided herein, the patient did not receive more than two prior treatments for Stage IIIB/IV. In some embodiments, the patient did not receive more than 30 days of exposure to an investigational or marketed agent that can act by EGFR inhibition, or a known EGFR-related toxicity resulting in dose modifications. EGFR inhibitors include (but are not limited to) gefitinib, erlotinib, and cetuximab. In some embodiments, the patient did not receive chemotherapy, biologic therapy, radiotherapy or investigational drug within 28 days prior to randomization (except that optionally, kinase inhibitors may be used within two weeks prior to randomization provided any drug related toxicity was adequately resolved). In some embodiments, the patient is not a patient with untreated and/or active (progressing or requiring anticonvulsants or corticosteroids for symptomatic control) CNS metastasis. In some embodiments, a sample of the patient's cancer has been shown to have wildtype EGFR. In some embodiments, a sample of the patient's cancer has not been shown to have mutated EGFR. Other patient exclusion criteria are described in the Examples, and the present inventions contemplate use of one or more of the exclusions described therein.
In some embodiments, a patient's tumor is c-met positive when 1% or more of the tumor cells in the sample express c-met protein (e.g., express c-met protein at any intensity, e.g., low, moderate and high). In some embodiments, a patient's tumor is c-met positive when more than 1% of the tumor cells in the sample express c-met protein (e.g., express c-met protein at any intensity). In some embodiments, a patient's tumor is c-met positive when 5% or more of the tumor cells in the sample express c-met protein (e.g., express c-met protein at any intensity). In some embodiments, a patient's tumor is c-met positive when 10% or more of the tumor cells in the sample express c-met protein (e.g., express c-met protein at any intensity). In some embodiments, a patient's tumor is c-met positive when 15% or more of the tumor cells in the sample express c-met protein. In some embodiments, a patient's tumor is c-met positive when 20% or more of the tumor cells in the sample express c-met protein (e.g., express c-met protein at any intensity). In some embodiments, a patient's tumor is c-met positive when 25% or more of the tumor cells in the sample express c-met protein (e.g., express c-met protein at any intensity). In some embodiments, a patient's tumor is c-met positive when 30% or more of the tumor cells in the sample express c-met protein (e.g., express c-met protein at any intensity). In some embodiments, a patient's tumor is c-met positive when 35% or more of the tumor cells in the sample express c-met protein (e.g., express c-met protein at any intensity). In some embodiments, a patient's tumor is c-met positive when 40% or more of the tumor cells in the sample express c-met protein (e.g., express c-met protein at any intensity). In some
embodiments, a patient's tumor is c-met positive when 45% or more of the tumor cells in the sample express c-met protein (e.g., express c-met protein at any intensity). In some
embodiments, a patient's tumor is c-met positive when 50% or more of the tumor cells in the sample express c-met protein (e.g., express c-met protein at any intensity). In some
embodiments, a patient's tumor is c-met positive when 55% or more of the tumor cells in the sample express c-met protein (e.g., express c-met protein at any intensity). In some
embodiments, a patient's tumor is c-met positive when 60% or more of the tumor cells in the sample express c-met protein (e.g., express c-met protein at any intensity). In some
embodiments, a patient's tumor is c-met positive when 65% or more of the tumor cells in the sample express c-met protein (e.g., express c-met protein at any intensity). In some
embodiments, a patient's tumor is c-met positive when 70% or more of the tumor cells in the sample express c-met protein (e.g., express c-met protein at any intensity). In some
embodiments, a patient's tumor is c-met positive when 75% or more of the tumor cells in the sample express c-met protein (e.g., express c-met protein at any intensity). In some
embodiments, a patient's tumor is c-met positive when 80% or more of the tumor cells in the sample express c-met protein (e.g., express c-met protein at any intensity). In some
embodiments, a patient's tumor is c-met positive when 85% or more of the tumor cells in the sample express c-met protein (e.g., express c-met protein at any intensity). In some
embodiments, a patient's tumor is c-met positive when 90% or more of the tumor cells in the sample express c-met protein (e.g., express c-met protein at any intensity). In some
embodiments, a patient's tumor is c-met positive when 95% or more of the tumor cells in the sample express c-met protein (e.g., express c-met protein at any intensity). In some
embodiments, c-met expression is membranous. In some embodiments, c-met expression is cytoplasmic. In some embodiments, c-met- expression is membranous and cytoplasmic.
In some embodiments, a patient's tumor is c-met positive when a maximum staining intensity of the tumor is 1. In some embodiments, a patient's tumor is c-met positive when a maximum staining intensity of the tumor is 2. In some embodiments, a patient's tumor is c-met positive when a maximum staining intensity of the tumor is 3. In some embodiments, c-met expression is membranous. In some embodiments, c-met expression is cytoplasmic. In some embodiments, c-met- expression is membranous and cytoplasmic.
C-met antagonists, e.g., suitable for use in any of the inventions described herein, are known in the art and some are further described herein. In certain embodiments, the c-met antagonist is an antagonist anti-c-met antibody. In certain embodiments, the anti-c-met antibody comprises a (a) HVR1 comprising sequence GYTFTSYWLH (SEQ ID NO: 1); (b) HVR2 comprising sequence GMIDPSNSDTRFNPNFKD (SEQ ID NO: 2); (c) HVR3-HC comprising sequence ATYRSYVTPLDY (SEQ ID NO: 3); (d) HVRl-LC comprising sequence KSSQSLLYTSSQKNYLA (SEQ ID NO: 4); (e) HVR2-LC comprising sequence WASTRES (SEQ ID NO: 5); and (f) HVR3-LC comprising sequence QQYYAYPWT (SEQ ID NO: 6). In certain embodiments, the anti-c-met antibody is monovalent and comprises (a) a first polypeptide comprising a heavy chain, said polypeptide comprising the sequence of SEQ ID NO: 11; (b) a second polypeptide comprising a light chain, the polypeptide comprising the sequence of SEQ ID NO: 12; and a third polypeptide comprising a Fc sequence, the polypeptide comprising the sequence of 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 a complex and form a Fc region that increases stability of said antibody fragment compared to a Fab molecule comprising said antigen binding arm. In certain embodiments, the c-met antibody is
Onartuzumab. In certain embodiments, the c-met antagonist is any one or more of crizotinib, tivantinib, carbozantinib, MGCD-265, ficlatuzumab, humanized TAK-701, rilotumumab, foretinib, h224Gl l, 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 LA480. Other c-met antagonists suitable for use in the present inventions are described herein.
Cancer medicaments can be used either alone or in combination with other cancer medicaments. For example, in some embodiments, a c-met antagonist (e.g., anti-c-met antibody) is used in combination with an EGFR antagonist (e.g., erlotinib). In certain embodiments, erlotinib is administered at a dose of 150 mg, each day of a three week cycle. In certain embodiments, erlotinib is administered at a dose of 100 mg, each day of a three week cycle. In certain embodiments, erlotinib is administered at a dose of 50 mg, each day of a three week cycle. An exemplary protocol is administering to a NSCLC patient (a) an anti-c-met antibody (such as Onartuzumab) at a dose of about 15 mg/kg every three weeks; and (b) erlotinib (N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)-4-quinazolinamine) at a dose of 150 mg, each day of a three week cycle. In other embodiments, a c-met antagonist (e.g., anti-c-met antibody) is used in combination with an anti-VEGF antibody and chemotherapy (e.g., a taxane). An exemplary protocol is administering to a triple-negative metastatic breast cancer patient an anti-c-met antibody (e.g., Onartuzumab) administered at a dose of 10 mg/kg on Day 1 and Day 15 of a 28-day cycle, anti-VEGF antibody (e.g., bevacizumab) administered at a dose of 10 mg/kg on Day 1 and Day 15 of the 28-day cycle and paclitaxel administered at a dose of 90 mg/m by IV infusion on Day 1, Day 8, and Day 15 of the 28-day cycle. Another exemplary protocol is administering to a triple-negative metastatic breast cancer patient an anti-c-met antibody (e.g., Onartuzumab) administered at a dose of 10 mg/kg on Day 1 and Day 15 of a 28-day cycle, and paclitaxel administered at a dose of 90 mg/m by IV infusion on Day 1, Day 8, and Day 15 of the 28-day cycle. In certain embodiments, Onartuzumab is
administered at a dose of about 15 mg/kg every three weeks, or at a dose of about 10 mg/kg every two weeks. In some embodiments, crizotinib is used in combination with an EGFR antagonist (in some embodiments, erlotinib). In some embodiments, carbozantinib is used in combination with an EGFR antagonist (in some embodiments, erlotinib). In some
embodiments, foretinib is used in combination with an EGFR antagonist (in some
embodiments, erlotinib). In some embodiments, tivantinib is used in combination with an EGFR antagonist (in some embodiments, erlotinib). In some embodiments, MGCD-265 is used in combination with an EGFR antagonist (in some embodiments, erlotinib). In some embodiments, rilotumumab is used in combination with an EGFR antagonist (in some embodiments, erlotinib). In some embodiments, ficlatuzumab is used in combination with an EGFR antagonist (in some embodiments, erlotinib). In some embodiments, humanized anti- HGF antibody TAK-701 is used in combination with an EGFR antagonist (in some
embodiments, erlotinib). Other cancer medicaments are described herein.
Optionally, additional biomarkers may be detected in a patient's sample. In some embodiments, the patient's cancer has been found to express wildtype EGFR (in some embodiments, further expresses c-met gene amplification, and in still further embodiments, does not express c-met gene amplification). In some embodiments, the patient's cancer has been found to express mutated EGFR. In certain embodiments, the patient's cancer has been found to express a biomarker selected from kras and EGFR. In some embodiments, the patient's cancer has been found to express mutated kras. In some embodiments, the patient's cancer has been found to express wildtype kras. In some embodiments, the patient's cancer (e.g., the patient's NSCLC) has been found to express an anaplastic lymphoma kinase (ALK) translocation. In some embodiments, the ALK translocation is an EML4-ALK translocation. In some embodiments, the patient's cancer has been found to express mutated c-met. In some embodiments, the patient's cancer has been found to express wildtype c-met.
In another aspect, the invention provides a method for evaluating adverse events in a patient associated with treatment of a cancer that expresses a high amount of c-met biomarker using any of the methods disclosed herein, wherein treatment is with a c-met antagonist (e.g., onartuzumab) and the method comprises the steps of monitoring the number and/or severity of one or more adverse events. Exemplary adverse events are disclosed herein. BRIEF DESCRIPTION OF THE FIGURES
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
Figures 1A-C: show examples of anti-c-met antibody SP44 IHC staining patterns in human lung tissues. Representative images depicting the range of SP44 staining intensities (A, negative, weak, moderate and strong) and the frequently observed heterogeneity in this staining pattern (B) in malignant NSCLC tissues. C, Representative images of SP44 staining of benign lung tissues depicting reactivity in respiratory mucosa and pneumocytes.
Figure 2: shows relationship of H-score with clinical scoring metric.
Figures 3A-C: show detailed breakdown of anti-c-met antibody SP44 staining intensity and proportion in NSCLC specimens, with H-score.
DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION
I. DEFINITIONS
Herein, a "patient" is a human patient. The patient may be a "cancer patient," i.e. one who is suffering or at risk for suffering from one or more symptoms of cancer. Moreover, the patient may be a previously treated cancer patient. The patient may be a "NSCLC cancer patient,"/. e. one who is suffering or at risk for suffering from one or more symptoms of NSCLC. Moreover, the patient may be a previously treated NSCLC patient.
The term "c-met" or "Met", as used herein, refers, unless indicated otherwise, to any native or variant (whether native or synthetic) c-met polypeptide. The term "wild type c-met" generally refers to a polypeptide comprising the amino acid sequence of a naturally occurring c-met protein. The term "wild type c-met sequence" generally refers to an amino acid sequence found in a naturally occurring c-met.
An "anti-c-met antibody" is an antibody that binds to c-met with sufficient affinity and specificity. Generally, c-met antibodies bind human c-met. The antibody selected will normally have a sufficiently strong binding affinity for c-met, for example, the antibody may bind human c-met with a ¾ value of between 100 nM-1 pM. Antibody affinities may be determined by a surface plasmon resonance based assay (such as the BIAcore assay as described in PCT Application Publication No. WO2005/012359); enzyme-linked
immunoabsorbent assay (ELISA); and competition assays (e.g. RIA's), for example. In certain embodiments, the anti-c-met antibody can be used as a therapeutic agent in targeting and interfering with diseases or conditions wherein c-met activity is involved. Also, the antibody may be subjected to other biological activity assays, e.g., in order to evaluate its effectiveness as a therapeutic. Such assays are known in the art and depend on the target antigen and intended use for the antibody.
A "c-met antagonist" (interchangeably termed "c-met inhibitor") is an agent that interferes with c-met activation or function. Examples of c-met inhibitors include c-met antibodies; HGF antibodies; small molecule c-met antagonists; c-met tyrosine kinase inhibitors; antisense and inhibitory RNA (e.g., shRNA) molecules (see, for example,
WO2004/87207). Preferably, the c-met inhibitor is an antibody or small molecule which binds to c-met. In a particular embodiment, a c-met inhibitor has a binding affinity (dissociation constant) to c-met of about 1 ,000 nM or less. In another embodiment, a c-met inhibitor has a binding affinity to c-met of about 100 nM or less. In another embodiment, a c-met inhibitor has a binding affinity to c-met of about 50 nM or less. In a particular embodiment, a c-met inhibitor is covalently bound to c-met. In a particular embodiment, a c-met inhibitor inhibits c- met signaling with an IC50 of 1 ,000 nM or less. In another embodiment, a c-met inhibitor inhibits c-met signaling with an IC50 of 500 nM or less. In another embodiment, a c-met inhibitor inhibits c-met signaling with an IC50 of 50 nM or less.
"C-met activation" refers to activation, or phosphorylation, of the c-met receptor.
Generally, c-met activation results in signal transduction (e.g. that caused by an intracellular kinase domain of a c-met receptor phosphorylating tyrosine residues in c-met or a substrate polypeptide). C-met activation may be mediated by c-met ligand (HGF) binding to a c-met receptor of interest. HGF binding to c-met may activate a kinase domain of c-met and thereby result in phosphorylation of tyrosine residues in the c-met and/or phosphorylation of tyrosine residues in additional substrate polyp eptides(s).
A "population" of subjects refers to a group of subjects with cancer, such as in a clinical trial, or as seen by oncologists following FDA approval for a particular indication, such as breast cancer therapy.
The phrase "does not possess substantial biomarker expression" or "substantially no biomarker expression" with respect to a biomarker, as used herein, means the biomarker does not exhibit an expression level that is above background level that is of statistical significance. The phrase "little to no biomarker expression" with respect to a biomarker, as used herein, means the biomarker does not display a biologically meaningful amount of expression. As would be understood in the art, amount of expression may be determined quantitatively or qualitatively, so long as a comparison between a biomarker sample and a reference counterpart can be done. The expression can be measured or detected according to any assay or technique known in the art, including, e.g., those described herein (such as IHC).
The term "gene amplification" refers to a process by which multiple copies of a gene or gene fragment are formed in a particular cell or cell line.
For the methods of the invention, the term "instructing" a patient means providing directions for applicable therapy, medication, treatment, treatment regimens, and the like, by any means, but preferably in writing, such as in the form of package inserts or other written promotional material.
For the methods of the invention, the term "promoting" means offering, advertising, selling, or describing a particular drug, combination of drugs, or treatment modality, by any means, including writing, such as in the form of package inserts. Promoting herein refers to promotion of therapeutic agent(s), such as an anti-c-met antibody (e.g., onartuzumab) and/or erlotinib, for an indication, such as NSCLC treatment, where such promoting is authorized by the Food and Drug Administration (FDA) as having been demonstrated to be associated with statistically significant therapeutic efficacy and acceptable safety in a population of subjects
The term "marketing" is used herein to describe the promotion, selling or distribution of a product (e.g., drug). Marketing specifically includes packaging, advertising, and any business activity with the purpose of commercializing a product.
For the purposes herein, a "previously treated" cancer patient has received prior cancer therapy. A "cancer medicament" is a drug effective for treating cancer. Examples of cancer medicaments include the chemotherapeutic agents and chemotherapy regimens noted below; c- met antagonists, including anti-c-met antibodies, such as onartuzumab.
The term "biomarker" or "marker" as used herein refers generally to a molecule, including a gene, mRNA, protein, carbohydrate structure, or glycolipid, the expression of which in or on a tissue or cell or secreted can be detected by known methods (or methods disclosed herein) and is predictive or can be used to predict (or aid prediction) for a cell, tissue, or patient's responsiveness to treatment regimes. The biomarker of particular interest herein is c-met.
As used herein, "negative c-met staining intensity" or "negative staining intensity" means c-met staining intensity of TOV-112D, H522, HI 155, LXFL529 and/or H23. In some embodiments, negative c-met staining intensity means c-met staining intensity of control cell line TOV-112D. In some embodiments, negative c-met staining intensity means c-met staining intensity of control cell line H522. In some embodiments, negative c-met staining intensity means c-met staining intensity of control cell line HI 155. In some embodiments, negative c- met staining intensity refers to c-met staining intensity of control cell line LXFL529. In some embodiments, negative c-met staining intensity means c-met staining intensity of control cell line H23. Methods for c-met IHC are known in the art and exemplified herein. In some embodiments, c-met staining intensity is determined using c-met antibody (e.g., SP44) staining of formalin-fixed paraffin embedded cell control cell pellets (e.g., prepared in a tissue microarray).
As used herein, "weak c-met staining intensity" or "weak staining intensity" means c- met IHC staining intensity of control cell line H1703, HEK-293, and/or H460. In some embodiments, weak c-met staining intensity means c-met staining intensity of control cell line HI 703. In some embodiments, weak c-met staining intensity means c-met staining intensity of control cell line HEK-293. In some embodiments, weak c-met staining intensity means c-met staining intensity of control cell line H460. Methods for c-met IHC are known in the art and exemplified herein. In some embodiments, c-met staining intensity is determined using c-met antibody (e.g., SP44) staining of formalin-fixed paraffin embedded cell control cell pellets (e.g., prepared in a tissue microarray).
As used herein, "moderate c-met staining intensity" or "moderate staining intensity" means c-met IHC staining intensity of control cell line A549 and/or SKMES1. In some embodiments, moderate c-met staining intensity means c-met staining intensity of control cell line A549. In some embodiments, moderate c-met staining intensity means c-met staining intensity of control cell line SKMES1. Methods for c-met IHC are known in the art and exemplified herein. In some embodiments, c-met staining intensity is determined using c-met antibody (e.g., SP44) staining of formalin-fixed paraffin embedded cell control cell pellets (e.g., prepared in a tissue microarray).
As used herein, "strong c-met staining intensity" or "strong staining intensity" means c- met IHC staining intensity of control cell line EBC-1 and/or H441. In some embodiments, strong c-met staining intensity means c-met staining intensity of control cell line EBC-1. In some embodiments, strong c-met staining intensity means c-met staining intensity of control cell line H441. Methods for c-met IHC are known in the art and exemplified herein. In some embodiments, c-met staining intensity is determined using c-met antibody (e.g., SP44) staining of formalin-fixed paraffin embedded cell control cell pellets (e.g., prepared in a tissue microarray).
By "patient sample" is meant a collection of cells obtained from a cancer patient. The source of the tissue or cell sample may be solid tissue as from a fresh, frozen and/or preserved organ or tissue sample or biopsy or aspirate; blood or any blood constituents; bodily fluids such as cerebral spinal fluid, amniotic fluid, peritoneal fluid, or interstitial fluid; cells from any time in gestation or development of the subject. The tissue sample may contain compounds which are not naturally intermixed with the tissue in nature such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, or the like. Examples of tumor samples herein include, but are not limited to, tumor biopsies, fine needle aspirate, bronchiolar lavage, pleural fluid, sputum, a surgical specimen, circulating tumor cells, serum or plasma, circulating plasma proteins, ascitic fluid, primary cell cultures or cell lines derived from tumors or exhibiting tumor-like properties, as well as preserved tumor samples, such as formalin-fixed, paraffin- embedded tumor samples or frozen tumor samples. In one embodiment the sample comprises NSCLC (e.g., squamous subtype or nonsquamous subtype) tumor sample.
An "effective response" of a patient or a patient's "responsiveness" to treatment with a medicament and similar wording refers to the clinical or therapeutic benefit imparted to a patient at risk for, or suffering from, cancer (e.g., NSCLC) upon administration of the cancer medicament. Such benefit includes any one or more of: extending survival (including overall survival and progression free survival); resulting in an objective response (including a complete response or a partial response); or improving signs or symptoms of cancer, etc. In one embodiment, the biomarker (e.g., c-met expression, for example, as determined using IHC) is used to identify the patient who is expected to have greater progression free survival (PFS) when treated with a medicament (e.g., anti-c-met antibody), relative to a patient who does not express the biomarker at the same level. In one embodiment, the biomarker is used to identify the patient who is expected to have reduced PFS when treated with a medicament, relative to a patient treated with the medicament who does not express the biomarker at the same level, or relative to a patient who is not treated with the medicament who does not express the biomarker at the same level. In one embodiment, the biomarker is used to identify the patient who is expected to have greater overall survival (OS) when treated with a medicament, relative to a patient who does not express the biomarker at the same level. In one embodiment, the biomarker is used to identify the patient who is expected to have reduced overall survival (OS), relative to a patient who is treated with the medicament who does not express the biomarker at the same level, or relative to a patient who is not treated with the medicament who does not express the biomarker at the same level. The incidence of biomarker(s) herein (i.e. as determined by H-score analysis as disclosed herein) effectively predicts, or predicts with high sensitivity, such effective response.
"Survival" refers to the patient remaining alive, and includes overall survival as well as progression free survival.
"Overall survival" refers to the patient remaining alive for a defined period of time, such as 1 year, etc from the time of diagnosis or treatment.
"Progression free survival" refers to the patient remaining alive, without the cancer progressing or getting worse.
By "extending survival" is meant increasing overall or progression free survival in a treated patient relative to an untreated patient (i.e. relative to a patient not treated with the medicament), or relative to a patient who does not express a biomarker at the designated level, and/or relative to a patient treated with an approved anti-tumor agent (such as chemotherapy regimen of erlotinib.
An "objective response" refers to a measurable response, including complete response (CR) or partial response (PR).
By "complete response" or "CR" is intended the disappearance of all signs of cancer in response to treatment. This does not always mean the cancer has been cured.
"Partial response" or "PR" refers to a decrease in the size of one or more tumors or lesions, or in the extent of cancer in the body, in response to treatment. The terms "level of expression" or "expression level" in general are used interchangeably and generally refer to the amount of a polynucleotide, mR A, or an amino acid product or protein in a biological sample. "Expression" generally refers to the process by which gene-encoded information is converted into the structures present and operating in the cell. Therefore, according to the invention "expression" of a gene may refer to transcription into a polynucleotide, translation into a protein, or even posttranslational modification of the protein. Fragments of the transcribed polynucleotide, the translated protein, or the post- translationally modified protein shall also be regarded as expressed whether they originate from a transcript generated by alternative splicing or a degraded transcript, or from a post- translational processing of the protein, e.g., by proteolysis. In some embodiments, "level of expression" refers to amount of a protein in a biological sample as determined using IHC.
The phrase "based on expression of when used herein means that information about expression level of the one or more biomarkers herein is used to inform a treatment decision, information provided on a package insert, or marketing/promotional guidance etc. In the case of high expression of the biomarker, patients may be treated with a cancer (e.g. NSCLC) medicament such as a c-met antagonist (e.g., anti-c-met antibody, e.g., Onartuzumab). In the case of reduced level of expression of the biomarker, patients may be treated with a cancer medicament other than c-met antagonist (e.g., other than anti-c-met antibody(e.g.,
Onartuzumab)).
"Treatment" refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already having a benign, pre-cancerous, or non-metastatic tumor as well as those in which the occurrence or recurrence of cancer is to be prevented.
The term "therapeutically effective amount" refers to an amount of a therapeutic agent (medicament) to treat or prevent a disease or disorder in a mammal. In the case of cancers, the therapeutically effective amount of the therapeutic agent may reduce the number of cancer cells; reduce the primary tumor size; inhibit (i.e., slow to some extent and preferably stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and preferably stop) tumor metastasis; inhibit, to some extent, tumor growth; and/or relieve to some extent one or more of the symptoms associated with the disorder. To the extent the drug may prevent growth and/or kill existing cancer cells, it may be cytostatic and/or cytotoxic. For cancer therapy, efficacy in vivo can, for example, be measured by assessing the duration of survival, time to disease progression (TTP), the response rates (RR), duration of response, 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. By "early stage cancer" or "early stage tumor" is meant a cancer that is not invasive or metastatic or is classified as a Stage 0, 1, or 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 tumors (including carcinoid tumors, gastrinoma, and islet cell cancer), mesothelioma, schwannoma (including acoustic neuroma), meningioma,
adenocarcinoma, melanoma, and leukemia or lymphoid malignancies. More particular examples of such cancers include squamous cell cancer (e.g. epithelial squamous cell cancer), lung cancer including small-cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer (including metastatic breast cancer), colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, testicular cancer, esophageal cancer, tumors of the biliary tract, as well as head and neck cancer. In some embodiments, the cancer is triple-negative metastatic breast cancer, including any histologically confirmed triple-negative (ER-, PR-, HER2-) adenocarcinoma of the breast with locally recurrent or metastatic disease (where the locally recurrent disease is not amenable to resection with curative intent).
A cancer or biological sample which "displays c-met expression" is one which, in a diagnostic test, expresses (including overexpresses) a c-met receptor.
A cancer or biological sample which "displays c-met amplification" is one which, in a diagnostic test, has amplified c-met gene. In some embodiments, amplified c-met gene is an average (in a population of cell) of greater than or equal to 5 or more copies of the c-met gene, or an average of eight or more copies of a c-met gene, or more.
A cancer or biological sample which "does not display c-met amplification" is one which, in a diagnostic test, does not have amplified c-met gene. In some embodiments, a sample which does not display c-met amplification is a sample which has an average of fewer than 4 copies of c-met gene.
By "EGFR" is meant the receptor tyrosine kinase polypeptide Epidermal Growth Factor Receptor which is described in Ullrich et al, Nature (1984) 309:418425, alternatively referred to as Her-1 and the c-erbB gene product, as well as variants thereof such as EGFRvIII.
Variants of EGFR also include deletional, substitutional and insertional variants, for example those described in Lynch et al (New England Journal of Medicine 2004, 350:2129), Paez et al (Science 2004, 304: 1497), Pao et al (PNAS 2004, 101 : 13306).
An "EGFR antagonist" (interchangeably termed "EGFR inhibitor") is an agent that interferes with EGFR activation or function. Examples of EGFR inhibitors include EGFR antibodies; EGFR ligand antibodies; small molecule EGFR antagonists; EGFR tyrosine kinase inhibitors; antisense and inhibitory RNA (e.g., shRNA) molecules (see, for example,
WO2004/87207). Preferably, the EGFR inhibitor is an antibody or small molecule which binds to EGFR. In some embodiments, the EGFR inhibitor is an EGFR-targeted drug. In a particular embodiment, an EGFR inhibitor has a binding affinity (dissociation constant) to EGFR of about 1 ,000 nM or less. In another embodiment, an EGFR inhibitor has a binding affinity to EGFR of about 100 nM or less. In another embodiment, an EGFR inhibitor has a binding affinity to EGFR of about 50 nM or less. In a particular embodiment, an EGFR inhibitor is covalently bound to EGFR. In a particular embodiment, an EGFR inhibitor inhibits EGFR signaling with an IC50 of 1 ,000 nM or less. In another embodiment, an EGFR inhibitor inhibits EGFR signaling with an IC50 of 500 nM or less. In another embodiment, an EGFR inhibitor inhibits EGFR signaling with an IC50 of 50 nM or less. In certain embodiments, the EGFR antagonist reduces or inhibits, by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more, the expression level or biological activity of EGFR.
"EGFR activation" refers to activation, or phosphorylation, of EGFR. Generally, EGFR activation results in signal transduction (e.g. that caused by an intracellular kinase domain of EGFR receptor phosphorylating tyrosine residues in EGFR or a substrate polypeptide). EGFR activation may be mediated by EGFR ligand binding to a EGFR dimer comprising EGFR. EGFR ligand binding to a EGFR dimer may activate a kinase domain of one or more of the EGFR in the dimer and thereby results in phosphorylation of tyrosine residues in one or more of the EGFR and/or phosphorylation of tyrosine residues in additional substrate
polypeptides(s). As used herein, the term "EGFR-targeted drug" refers to a therapeutic agent that binds to EGFR and inhibits EGFR activation. Examples of such agents include antibodies and small molecules that bind to EGFR. Examples of antibodies which bind to EGFR include MAb 579 (ATCC CRL HB 8506), MAb 455 (ATCC CRL HB8507), MAb 225 (ATCC CRL 8508), MAb 528 (ATCC CRL 8509) (see, US Patent No. 4,943, 533, Mendelsohn et al.) and variants thereof, such as chimerized 225 (C225 or Cetuximab; ERBUTIX®) and reshaped human 225 (H225) (see, WO 96/40210, Imclone Systems Inc.); IMC-11F8, a fully human, EGFR-targeted antibody (Imclone); antibodies that bind type II mutant EGFR (US Patent No. 5,212,290); humanized and chimeric antibodies that bind EGFR as described in US Patent No. 5,891,996; and human antibodies that bind EGFR, such as ABX-EGF (see WO98/50433, Abgenix); EMD 55900 (Stragliotto et al. Eur. J. Cancer 32A:636-640 (1996)); EMD7200 (matuzumab) a humanized EGFR antibody directed against EGFR that competes with both EGF and TGF- alpha for EGFR binding; and mAb 806 or humanized mAb 806 (Johns et al., J. Biol. Chem. 279(29):30375-30384 (2004)). The anti-EGFR antibody may be conjugated with a cytotoxic agent, thus generating an immunoconjugate (see, e.g., EP659,439A2, Merck Patent GmbH). Examples of small molecules that bind to EGFR include ZD 1839 or Gefitinib (IRESSA; Astra Zeneca); CP-358774 or Erlotinib (TARCEVA™; Genentech/OSI); and AG1478, AG1571 (SU 5271; Sugen); EMD-7200.
The term "polynucleotide," when used in singular or plural, generally refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. Thus, for instance, polynucleotides as defined herein include, without limitation, single- and double-stranded DNA, DNA including single- and double-stranded regions, single- and double-stranded RNA, and RNA including single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or include single- and double-stranded regions. In addition, the term "polynucleotide" as used herein refers to triple- stranded regions comprising RNA or DNA or both RNA and DNA. The strands in such regions may be from the same molecule or from different molecules. The regions may include all of one or more of the molecules, but more typically involve only a region of some of the molecules. One of the molecules of a triple- helical region often is an oligonucleotide. The term "polynucleotide" specifically includes cDNAs. The term includes DNAs (including cDNAs) and RNAs that contain one or more modified bases. Thus, DNAs or RNAs with backbones modified for stability or for other reasons are "polynucleotides" as that term is intended herein. Moreover, DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritiated bases, are included within the term "polynucleotides" as defined herein. In general, the term
"polynucleotide" embraces all chemically, enzymatically and/or metabolically modified forms of unmodified polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including simple and complex cells.
The term "oligonucleotide" refers to a relatively short polynucleotide, including, without limitation, single-stranded deoxyribonucleotides, single- or double-stranded
ribonucleotides, R A:DNA hybrids and double- stranded DNAs. Oligonucleotides, such as single- stranded DNA probe oligonucleotides, are often synthesized by chemical methods, for example using automated oligonucleotide synthesizers that are commercially available.
However, oligonucleotides can be made by a variety of other methods, including in vitro recombinant DNA-mediated techniques and by expression of DNAs in cells and organisms.
A "chemotherapeutic agent" is a chemical compound useful in the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclosphosphamide (CYTOXAN®); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa;
ethylenimines and methylamelamines including altretamine, triethylenemelamine,
trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine;
acetogenins (especially bullatacin and bullatacinone); delta-9-tetrahydrocannabinol
(dronabinol, MARINOL®); beta-lapachone; lapachol; colchicines; betulinic acid; a
camptothecin (including the synthetic analogue topotecan (HYCAMTIN®), CPT-11
(irinotecan, CAMPTOSAR®), acetylcamptothecin, scopolectin, and 9-aminocamptothecin); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); podophyllotoxin; podophyllinic acid; teniposide; cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamme, mechlorethamme oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e. g., calicheamicin, especially calicheamicin gamma II and calicheamicin omegall (see, e.g., Nicolaou et al., Angew. Chem Intl. Ed. Engl., 33: 183-186 (1994));
CDP323, an oral alpha-4 integrin inhibitor; dynemicin, including dynemicin A; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including
ADRIAMYCIN®, morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino- doxorubicin, doxorubicin HC1 liposome injection (DOXIL®), liposomal doxorubicin TLC D- 99 (MYOCET®), peglylated liposomal doxorubicin (CAELYX®), and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate, gemcitabine (GEMZAR®), tegafur
(UFTORAL®), capecitabine (XELODA®), an epothilone, and 5-fiuorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"- trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman;
gacytosine; arabinoside ("Ara-C"); thiotepa; taxoid, e.g., paclitaxel (TAXOL®), albumin- engineered nanoparticle formulation of paclitaxel (ABRAXANE™), and docetaxel
(TAXOTERE®); chloranbucil; 6-thioguanine; mercaptopurine; methotrexate; platinum agents such as cisplatin, oxaliplatin, and carboplatin; vincas, which prevent tubulin polymerization from forming microtubules, including vinblastine (VELBAN®), vincristine (ONCOVIN®), vindesine (ELDISINE®, FILDESIN®), and vinorelbine (NAVELBINE®); etoposide (VP- 16); ifosfamide; mitoxantrone; leucovovin; novantrone; edatrexate; daunomycin; aminopterin; ibandronate; topoisomerase inhibitor RFS 2000; difluorometlhylornithine (DMFO); retinoids such as retinoic acid, including bexarotene (TARGRETIN®); bisphosphonates such as clodronate (for example, BONEFOS® or OSTAC®), etidronate (DIDROCAL®), NE-58095, zoledronic acid/zoledronate (ZOMETA®), alendronate (FOSAMAX®), pamidronate
(AREDIA®), tiludronate (SKELID®), or risedronate (ACTONEL®); troxacitabine (a 1 ,3- dioxolane nucleoside cytosine analog); antisense oligonucleotides, particularly those that inhibit expression of genes in signaling pathways implicated in aberrant cell proliferation, such as, for example, PKC-alpha, Raf, H-Ras, and epidermal growth factor receptor (EGF-R); vaccines such as THERATOPE® vaccine and gene therapy vaccines, for example,
ALLOVECTIN® vaccine, LEUVECTIN® vaccine, and VAXID® vaccine; topoisomerase 1 inhibitor (e.g., LURTOTECAN®); rmRH (e.g., ABARELLX®); BAY439006 (sorafenib; Bayer); SU- 1 1248 (Pfizer); perifosine, COX-2 inhibitor (e.g. celecoxib or etoricoxib), proteosome inhibitor (e.g. PS341); bortezomib (VELCADE®); CCI-779; tipifarnib (R1 1577); orafenib, ABT510; Bcl-2 inhibitor such as oblimersen sodium (GENASENSE®); pixantrone; EGFR inhibitors (see definition below); tyrosine kinase inhibitors (see definition below); and pharmaceutically acceptable salts, acids or derivatives of any of the above; as well as combinations of two or more of the above such as CHOP, an abbreviation for a combined therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone, and FOLFOX, an abbreviation for a treatment regimen with oxaliplatin (ELOXATIN™) combined with 5-FU and leucovovin.
Herein, chemotherapeutic agents include "anti-hormonal agents" or "endocrine therapeutics" which act to regulate, reduce, block, or inhibit the effects of hormones that can promote the growth of cancer. They may be hormones themselves, including, but not limited to: anti-estrogens with mixed agonist/antagonist profile, including, tamoxifen
(NOLVADEX®), 4-hydroxytamoxifen, toremifene (FARESTON®), idoxifene, droloxifene, raloxifene (EVISTA®), trioxifene, keoxifene, and selective estrogen receptor modulators
(SERMs) such as SERM3; pure anti-estrogens without agonist properties, such as fulvestrant (FASLODEX®), and EM800 (such agents may block estrogen receptor (ER) dimerization, inhibit DNA binding, increase ER turnover, and/or suppress ER levels); aromatase inhibitors, including steroidal aromatase inhibitors such as formestane and exemestane (AROMASIN®), and nonsteroidal aromatase inhibitors such as anastrazole (ARIMIDEX®), letrozole
(FEMARA®) and aminoglutethimide, and other aromatase inhibitors include vorozole (RIVISOR®), megestrol acetate (MEGASE®), fadrozole, and 4(5)-imidazoles; lutenizing hormone-releaseing hormone agonists, including leuprolide (LUPRON® and ELIGARD®), goserelin, buserelin, and tripterelin; sex steroids, including progestines such as megestrol acetate and medroxyprogesterone acetate, estrogens such as diethylstilbestrol and premarin, and androgens/retinoids such as fluoxymesterone, all transretionic acid and fenretinide;
onapristone; anti-progesterones; estrogen receptor down-regulators (ERDs); anti-androgens such as flutamide, nilutamide and bicalutamide; and pharmaceutically acceptable salts, acids or derivatives of any of the above; as well as combinations of two or more of the above.
Specific examples of chemotherapeutic agents or chemotherapy regimens herein include: alkylating agents (e.g. chlorambucil, bendamustine, or cyclophosphamide); nucleoside analogues or antimetabolites (e.g. fludarabine), fludarabine and cyclophosphamide (FC);
prednisone or prednisolone; akylator-containing combination therapy, including
cyclophosphamide, vincristine, prednisolone (CHOP), or cyclophosphamide, vincristine, prednisolone (CVP), etc.
A "target audience" is a group of people or an institution to whom or to which a particular medicament is being promoted or intended to be promoted, as by marketing or advertising, especially for particular uses, treatments, or indications, such as individual patients, patient populations, readers of newspapers, medical literature, and magazines, television or internet viewers, radio or internet listeners, physicians, drug companies, etc.
A "package insert" is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, contraindications, other therapeutic products to be combined with the packaged product, and/or warnings concerning the use of such therapeutic products, etc.
The term "antibody" herein is used in the broadest sense and encompasses various 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 other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g. scFv); and multispecific antibodies formed from antibody fragments.
An "affinity matured" antibody refers to an antibody with one or more alterations in one or more hypervariable regions (HVRs), compared to a parent antibody which does not possess such alterations, such alterations resulting in an improvement in the affinity of the antibody for antigen.
An "antibody that binds to the same epitope" as a reference antibody refers to an antibody that blocks binding of the reference antibody to its antigen in a competition assay by 50% or more, and conversely, the reference antibody blocks binding of the antibody to its antigen in a competition assay by 50% or more.
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 possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgGi, IgG2, IgG3, IgG4, IgAi, and IgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.
The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents a cellular function and/or causes cell death or destruction. Cytotoxic agents include, but are not limited to, radioactive isotopes (e.g., At211, 1131, 1125, Y90, Re186, Re188, Sm153, Bi212, P32, Pb212 and radioactive isotopes of Lu); chemotherapeutic agents or drugs (e.g., methotrexate, adriamicin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents); growth inhibitory agents; enzymes and fragments thereof such as nucleolytic enzymes; antibiotics; toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof; and the various antitumor or anticancer agents disclosed below.
"Effector functions" refer to those biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype. Examples of antibody effector functions include: Clq 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 receptor); and B cell activation.
The term "Fc region" herein is used to define a C-terminal region of an
immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. In one embodiment, a human IgG heavy chain Fc region extends from Cys226, or from Pro230, to the carboxyl-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, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.
"Framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the HVR and FR sequences generally appear in the following sequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.
The terms "full length antibody," "intact antibody," and "whole antibody" are used herein interchangeably to refer to an antibody having a structure substantially similar to a native antibody structure or having heavy chains that contain an Fc region as defined herein.
A "human antibody" is one which possesses an amino acid sequence which corresponds to that of an antibody produced by a human or a human cell or derived from a non-human source that utilizes human antibody repertoires or other human antibody-encoding sequences. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues.
A "humanized" antibody refers to 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 substantially all of at least one, and typically two, 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. A humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization.
The term "hypervariable region" or "HVR," as used herein, refers to each of the regions of an antibody variable domain which are hypervariable in sequence and/or form structurally defined loops ("hypervariable loops"). Generally, native four-chain antibodies comprise six HVRs; three in the VH (HI, H2, H3), and three in the VL (LI, L2, L3). HVRs generally comprise amino acid residues from the hypervariable loops and/or from the "complementarity determining regions" (CDRs), the latter being of highest sequence variability and/or involved in antigen recognition. Exemplary hypervariable loops occur at amino acid residues 26-32 (LI), 50-52 (L2), 91-96 (L3), 26-32 (HI), 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) occur at amino acid residues 24-34 of LI, 50-56 of L2, 89-97 of L3, 31-35B of HI, 50-65 of H2, and 95-102 of H3. (Kabat et al., Sequences of Proteins of
Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)) With the exception of CDR1 in VH, CDRs generally comprise the amino acid residues that form the hypervariable loops. CDRs also comprise "specificity determining residues," or "SDRs," which are residues that contact antigen. SDRs are contained within regions of the CDRs called abbreviated-CDRs, or a-CDRs. Exemplary a-CDRs (a-CDR-Ll, a- CDR-L2, a-CDR-L3, a-CDR-Hl, a-CDR-H2, and a-CDR-H3) occur at amino acid residues 31- 34 of LI, 50-55 of L2, 89-96 of L3, 31-35B of HI, 50-58 of H2, and 95-102 of H3. (See Almagro and Fransson, Front. Biosci. 13: 1619-1633 (2008)). Unless otherwise indicated, HVR residues and other residues in the variable domain (e.g., FR residues) are numbered herein according to Kabat et al., supra. In one embodiment, the c-met antibody herein comprises the HVRs of SEQ ID NOs: 1-6.
An "immunoconjugate" is an antibody conjugated to one or more heterologous molecule(s), including but not limited to a cytotoxic agent.
The term "monoclonal antibody" as used herein 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 possible variant antibodies, e.g., containing naturally occurring mutations or arising during production of a monoclonal antibody preparation, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by a variety of techniques, including but not limited to the hybridoma method, recombinant DNA methods, phage-display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, such methods and other exemplary methods for making monoclonal antibodies being described herein. A "naked antibody" refers to an antibody that is not conjugated to a heterologous moiety (e.g., a cytotoxic moiety) or radiolabel. The naked antibody may be present in a pharmaceutical formulation.
"Native antibodies" refer to naturally occurring immunoglobulin molecules with varying structures. For example, native IgG antibodies are heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light chains and two identical heavy chains that are disulfide-bonded. From N- to C-terminus, each heavy chain has a variable region (VH), also called a variable heavy domain or a heavy chain variable domain, followed by three constant domains (CHI, CH2, and CH3). Similarly, from N- to C-terminus, each light chain has a variable region (VL), also called a variable light domain or a light chain variable domain, followed by a constant light (CL) domain. The light chain of an antibody may be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of its constant domain.
The term "pharmaceutical formulation" refers to a sterile preparation that is in such form as to permit the biological activity of the medicament to be effective, and which contains no additional components that are unacceptably toxic to a subject to which the formulation would be administered.
A "sterile" formulation is aseptic or free from all living microorganisms and their spores.
A "kit" is any manufacture (e.g a package or container) comprising at least one reagent, e.g., a medicament for treatment of cancer (e.g., NCSLC or triple-negative breast cancer), or a reagent (e.g., antibody) for specifically detecting a biomarker gene or protein of the invention. The manufacture is preferably promoted, distributed, or sold as a unit for performing the methods of the present invention.
A "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject. A
pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.
"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 the 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 not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For purposes herein, however, % amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc., and the source code has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or may be compiled from the source code. The ALIGN-2 program should be compiled for use on a UNLX operating system, including digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.
In situations where ALIGN-2 is employed for amino acid sequence comparisons, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows:
100 times the fraction X/Y where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in that program's alignment of A and B, and where Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. Unless specifically stated otherwise, all % amino acid sequence identity values used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program.
II. CANCER MEDICAMENTS
In one aspect, the invention concerns selecting patients who can be treated with cancer medicaments based on expression of one or more of the biomarkers disclosed herein. Examples of cancer medicaments include, but are not limited to: - c-met antagonists, including anti-c-met antibodies.
-Chemotherapeutic agents and chemotherapy regimens.
- Other medicaments or combinations thereof in development, or approved, for treating cancer, e.g., NSCLC.
In one embodiment, the medicament is an antibody, including but not limited to directed against or which binds to human c-met. In some embodiments, the antibody interfered with c-met binding to hepatocyte growth factor (HGF). The antibody herein includes:
monoclonal antibodies, including a chimeric, humanized or human antibodies. In one embodiment, the antibody is an antibody fragment, e.g., a Fv, Fab, Fab', one-armed antibody, scFv, diabody, or F(ab')2 fragment. In another embodiment, the antibody is a full length antibody, e.g., an intact IgGl 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 (i.e., the heavy chain variable domain and the light chain variable domain form a single antigen binding arm) comprising an Fc region, 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 a Fc region that increases stability of said antibody fragment compared to a Fab molecule comprising said antigen binding arm. The one-armed antibody may be monovalent.
In one embodiment, the c-met antagonist is an anti-c-met antibody. In another embodiment, the anti-c-met antibody is onartuzumab or a biosimilar version thereof.
Onartuzumab is disclosed in, for example, WO2006/015371; 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 comprising sequence GYTFTSYWLH (SEQ ID NO: l ); (b) HVR2 comprising sequence GMIDPSNSDTRFNPNFKD (SEQ ID NO: 2); and/or (c) HVR3-HC comprising sequence ATYRSYVTPLDY (SEQ ID NO: 3). In some
embodiments, the antibody comprises a light chain variable domain comprising one or more of (a) HVR1-LC comprising sequence KSSQSLLYTSSQKNYLA (SEQ ID NO: 4); HVR2-LC comprising sequence WASTRES (SEQ ID NO: 5); and/or (c) HVR3-LC comprising sequence QQYYAYPWT (SEQ ID NO: 6). In some embodiments the anti-c-met antibody comprises a heavy chain variable domain comprising (a) HVR1 comprising sequence GYTFTSYWLH (SEQ ID NO: 1); (b) HVR2 comprising sequence GMIDPSNSDTRFNPNFKD (SEQ ID NO: 2); and (c) HVR3-HC comprising sequence ATYRSYVTPLDY (SEQ ID NO: 3) and a light chain variable domain comprising (a) HVR1-LC comprising sequence KSSQSLLYTSSQK YLA (SEQ ID NO: 4); HVR2-LC comprising 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, an anti-c-met antibody can be humanized. In one embodiment, an anti- c-met antibody comprises HVRs as in 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, an 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 to human c-met. In certain embodiments, a total of 1 to 10 amino acids have been substituted, altered inserted and/or deleted in SEQ ID NO:7. In certain embodiments, substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., in the FRs). Optionally, the anti-c-met antibody comprises the VH sequence in SEQ ID NO:7, including post-translational modifications of that sequence.
In another aspect, an anti- c-met antibody is provided, wherein the antibody comprises 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 to c-met. In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO:8. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., in the FRs). Optionally, the anti- c-met antibody comprises the VL sequence in SEQ ID NO: 8, including post-translational modifications of that 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 a further embodiment, the anti- c-met antibody comprises a HVR-L1 comprising amino acid sequence SEQ ID NO: 1; an HVR-L2 comprising amino acid sequence SEQ ID NO: 2; an HVR-L3 comprising amino acid sequence SEQ ID NO: 3; an HVR-H1 comprising amino acid sequence SEQ ID NO: 4; an HVR-H2 comprising amino acid sequence SEQ ID NO: 5; and an HVR-H3 comprising amino acid sequence SEQ ID NO: 6.
In another aspect, an anti- c-met antibody is provided, wherein the antibody comprises a
VH as in any of the embodiments provided above, and a VL as in any of the embodiments provided above.
In a further aspect, the invention provides an antibody that binds to the same epitope as an anti- c-met antibody provided herein. For example, in certain embodiments, an antibody is provided that binds to the same epitope as or can by competitively inhibited by an anti- c-met antibody comprising a VH sequence of SEQ ID NO: 7 and a VL sequence of SEQ ID NO:8.
In a further aspect of the invention, an anti- c-met antibody according to any of the embodiments herein can be a monoclonal antibody, including a monovalent, chimeric, humanized or human antibody. In one embodiment, an anti- c-met antibody is an antibody fragment, e.g., a one-armed, Fv, Fab, Fab', scFv, diabody, or F(ab')2 fragment. In another embodiment, the antibody is a full length antibody, e.g., an intact IgGl 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 the HVRs or comprises the VH and VL regions described above.
In some embodiments, the anti-c-met antibody is monovalent, and comprises (a) a first polypeptide comprising a heavy chain variable domain having the sequence:
EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWLHWVRQAPGKGLEWVGMIDPSNS DTRFNPNFKDRFTISADTSKNTAYLQMNSLRAEDTAVYYCATYRSYVTPLDYWGQGT LVTVSS (SEQ ID NO:7), CHI sequence, and a first Fc polypeptide; (b) a second polypeptide comprising a light chain variable domain having the sequence:
DIQMTQSPSSLSASVGDRVTITCKSSQSLLYTSSQKNYLAWYQQKPGKAPKLLIYWAST R ESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYAYPWTFGQGTKVEIKR (SEQ ID NO: 8), and 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 a complex and form a Fc region that increases stability of said antibody fragment compared to a Fab molecule comprising said antigen binding arm. In some embodiments, the first polypeptide comprises Fc sequence CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 9) and the second polypeptide comprises the Fc sequence
CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 10).
In another embodiments, the anti-c-met antibody is monovalent and comprises (a) a first polypeptide comprising a heavy chain, said polypeptide comprising the sequence:
EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWLHWVRQAPGKGLEWVGMIDPSNS DTRFNPNFKDRFTISADTSKNTAYLQMNSLRAEDTAVYYCATYRSYVTPLDYWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDK VEPKSCDKTHTCPPC PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNA KTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRE PQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 11); (b) a second polypeptide comprising a light chain, the polypeptide comprising the sequence DIQMTQSPSSLSASVGDRVTITCKSSQSLLYTSSQKNYLAWYQQKPGKAPKLLIYWAST RESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYAYPWTFGQGTKVEIKRTVAA PSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKD STYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 12); and a third polypeptide comprising a Fc sequence, the polypeptide comprising the sequence DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (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 a complex and form a Fc region that increases stability of said antibody fragment compared to a Fab molecule comprising said antigen binding arm).
Other anti-c-met antibodies suitable for use in the methods of the invention are described herein and known in the art. For example, anti-c-met antibodies disclosed in
WO05/016382 (including but not limited to antibodies 13.3.2, 9.1.2, 8.70.2, 8.90.3); an anti-c- met antibodies produced by the hybridoma cell line deposited with ICLC number PD 03001 at the CBA in Genoa, or that recognizes an epitope on the extracellular domain of the β chain of the HGF receptor, and said epitope is the same as that recognized by the monoclonal antibody); 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 an antibody deposited at CNCM, Institut Pasteur, Paris, France, on March 14, 2007 under the number 1-3731, on March 14, 2007 under the number 1-3732, on July 6, 2007 under the number 1-3786, on March 14, 2007 under the number 1-3724; an anti-c-met antibody disclosed in 20110129481 ; an anti-c-met antibody disclosed in US20110104176; an anti-c-met antibody disclosed in WO2009/134776; an anti-c-met antibody disclosed in
WO2010/059654; an anti-c-met antibody disclosed in WO2011020925 (including but not limited to an antibody secreted from a hybridoma deposited at the CNCM, Institut Pasteur, Paris, France, on march 12, 2008 under the number 1-3949 and the hybridoma deposited on January 14, 2010 under the number 1-4273).
In one aspect, the anti-c-met antibody comprises at least one characteristic that promotes heterodimerization, while minimizing homodimerization, of the Fc sequences within the antibody fragment. Such characteristic(s) improves yield and/or purity and/or homogeneity of the immunoglobulin populations. In one embodiment, the antibody comprises Fc mutations constituting "knobs" and "holes" as described in WO2005/063816. For example, a hole mutation can be one or more of T366A, L368A and/or Y407V in an Fc polypeptide, and a cavity mutation can be T366W.
In some embodiments, the c-met antagonist is an anti-hep atocyte growth factor (HGF) antibody, including but not limited to, humanized anti-HGF antibody TAK701, rilotumumab, Ficlatuzumab, and/or humanized antibody 2B8 described in WO2007/143090. In some embodiments, the anti-HGF antibody is an anti-HGF antibody described in US7718174B2. In some embodiments, the c-met antagonist is a c-met small molecule inhibitor. In some embodiments, the c-met small molecule inhibitor is a selective c-met small molecule inhibitor.
In one embodiment, the c-met antagonist binds c-met extracellular domain. In some embodiments, the c-met antagonist binds c-met kinase domain. In some embodiments, the c- met antagonist competes for c-met binding with HGF. In some embodiments, the c-met antagonist competes for HGF binding to c-met. In some embodiments, the c-met antagonist binds HGF. In certain embodiments, the c-met antagonist inhibits cell proliferation, e.g., HGF- induced cell proliferation of cell line EBC-1, H441 and/or KP4. In some embodiments, cell proliferation is inhibited with a Ki of 600 nM or less (more potent), 500 nM or less, 400 nM or less, 300 nM or less or more potent. In certain embodiments, the c-met antagonist inhibits c- met signaling (e.g., phospho-c-met, phospho-AKT, phospho-MAPK) when EBC-1 cells are treated with c-met antagonist in the presence of 10% fetal bovine serum. In some
embodiments, c-met signaling is inhibited with a Ki of 600 nM or less (more potent), 500 nM or less, 400 nM or less, 300 nM or less (more potent). In certain embodiments, the c-met antagonist treats (is capable of treating) squamous cell carcinoma. In certain embodiments, the c-met antagonist treats (is capable of treating) NSCLC that expresses wild-type k-ras.
In certain embodiments, the c-met inhibitor is not a non-ATP-competitive small molecule. In certain embodiments, the c-met antagonist inhibits cell proliferation, e.g., HGF- induced cell proliferation of cell line EBC-1, H441 and/or KP4. In certain embodiments, the c- met antagonist inhibits c-met signaling (e.g., phospho-c-met, and downstream c-met signaling pathways, e.g., phospho-AKT, phospho-MAPK) when EBC-1 cells are treated with c-met antagonist in the presence of 10% fetal bovine serum. In certain embodiments, the c-met antagonist does not inhibit cell proliferation of cell lines MDA-MB-231 and HT29 (in the presence or absence of exogenous HGF). In certain embodiments, the c-met antagonist does not inhibit cell proliferation of cell lines MDA-MB-231 and HT29 in the presence of 10% fetal bovine serum. Methods for assaying cell proliferation are well-known in the art, and some methods are described in WO2009/111691; WO2006/015371; and Jin et al, Cancer Res (2008) 68:4360. In certain embodiments, the c-met antagonist treats (is capable of treating) squamous cell carcinoma. In certain embodiments, the c-met antagonist treats (is capable of treating) NSCLC that expresses wild-type k-ras. In certain embodiments, the c-met antagonist is not Tivantinib (ARQ-197). In a particular embodiment, a c-met antagonist inhibits c-met signaling with an IC50 of 1,000 nM or less (i.e., more potent). In another embodiment, a c-met antagonist inhibits c-met signaling with an IC50 of 400 nm or less, 500 iiM or less, 600 nm or less, 700 nm or less. In another embodiment, a c-met antagonist inhibits c-met signaling with an IC50 of 50 iiM or less. In certain embodiments, the c-met antagonist is not crizotinib. In certain embodiments, the c-met antagonist is not foretinib. In certain embodiments, the c-met antagonist is not ficlatuzumab. In certain embodiments, the c-met antagonist is not
rilotumumab.
In certain embodiments, the c-met antagonist is any one of: SGX-523, Crizotinib (PF- 02341066; 3-[(lR)- 1 -(2,6-dichloro-3-fluorophenyl)ethoxy]-5-(l -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] - 1 H-pyrrol-2- yl}methylene)-N-methyl-2-oxoindoline-5 -sulfonamide; CAS no. 658084-23-2]), Foretinib (GSK1363089), XL880 (CAS no. 849217-64-7; XL880 is a inhibitor of met and VEGFR2 and KDPv); MGCD-265 (MethylGene; MGCD-265 targets the c-MET, VEGFR1, VEGFR2, VEGFR3, Ron and Tie-2 receptors; CAS no. 875337-44-3), Tivantinib (ARQ 197; (-)- (3R,4R)-3-(5,6-dihydro-4H-pyrrolo[3,2,l-ij]quinolin-l-yl)-4-(lH-indol-3-yl)pyrrolidm^ dione; see Munchi et al, Mol Cancer Ther June 2010 9; 1544; CAS no. 905854-02-6), LY- 2801653 (Lilly), LY2875358 (Lilly), MP-470, Rilotumumab (AMG 102, anti-HGF monoclonal antibody), antibody 223 C4 or humanized antibody 223 C4 (WO2009/007427), humanized L2G7 (humanized TAK701 ; humanized anti-HGF monoclonal antibody); EMD 1214063 (Merck Sorono), EMD 1204831 (Merck Serono), NK4, Cabozantinib (XL- 184, CAS no.
849217-68-1; carbozantinib is a dual inhibitor of met and VEGFR2), 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- (l-methyl-lH-pyrazol-4-yl)-5-oxo-5H-benzo[4,5]cyclohepta[l,2-b]pyridin-7-yl]sulfamide; 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, tivantinib, carbozantinib, MGCD-265, ficlatuzumab, humanized TAK-701, rilotumumab, foretinib, h224Gl 1, 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 LA480. In certain embodiments, the c-met antagonist is any one or more of crizotinib, tivantinib, carbozantinib, MGCD-265, ficlatuzumab, humanized TAK-701, rilotumumab, and/or foretinib.
EGFR antagonists include antibodies such as humanized monoclonal antibody known as nimotuzumab (YM Biosciences), fully human ABX-EGF (panitumumab, Abgenix Inc.) as well as fully human antibodies known as El . l, E2.4, E2.5, E6.2, E6.4, E2.11, E6. 3 and E7.6. 3 and described in US 6,235,883; MDX-447 (Medarex Inc). Pertuzumab (2C4) is a humanized antibody that binds directly to HER2 but interferes with HER2-EGFR dimerization thereby inhibiting EGFR signaling. Other examples of antibodies which bind to EGFR include MAb 579 (ATCC CRL HB 8506), MAb 455 (ATCC CRL HB8507), MAb 225 (ATCC CRL 8508), MAb 528 (ATCC CRL 8509) (see, US Patent No. 4,943, 533, Mendelsohn et al.) and variants thereof, such as chimerized 225 (C225 or Cetuximab; ERBUTLX®) and reshaped human 225 (H225) (see, WO 96/40210, Imclone Systems Inc.); IMC-11F8, a fully human, EGFR-targeted antibody (Imclone); antibodies that bind type II mutant EGFR (US Patent No. 5,212,290); humanized and chimeric antibodies that bind EGFR as described in US Patent No. 5,891,996; and human antibodies that bind EGFR, such as ABX-EGF (see WO98/50433, Abgenix); EMD 55900 (Stragliotto et al. Eur. J. Cancer 32A:636-640 (1996)); EMD7200 (matuzumab) a humanized EGFR antibody directed against EGFR that competes with both EGF and TGF- alpha for EGFR binding; and mAb 806 or humanized mAb 806 (Johns et al., J. Biol. Chem. 279(29):30375-30384 (2004)). The anti-EGFR antibody may be conjugated with a cytotoxic agent, thus generating an immunoconjugate (see, e.g., EP659,439A2, Merck Patent GmbH).
Anti-EGFR antibodies that are useful in the methods of the invention include any antibody that binds with sufficient affinity and specificity to EGFR and can reduce or inhibit EGFR activity. The antibody selected will normally have a sufficiently strong binding affinity for EGFR, for example, the antibody may bind human c-met with a Kd value of between 100 nM-1 pM. Antibody affinities may be determined by a surface plasmon resonance based assay (such as the BIAcore assay as described in PCT Application Publication No.
WO2005/012359); enzyme-linked immunoabsorbent assay (ELISA); and competition assays (e.g. RIA's), for example. Preferably, the anti-EGFR antibody of the invention can be used as a therapeutic agent in targeting and interfering with diseases or conditions wherein
EGFR/EGFR ligand activity is involved. Also, the antibody may be subjected to other biological activity assays, e.g., in order to evaluate its effectiveness as a therapeutic. Such assays are known in the art and depend on the target antigen and intended use for the antibody. Bispecific antibodies are antibodies that have binding specificities for at least two different epitopes. Exemplary bispecific antibodies may bind to EGFR and to c-met. In another example, an exemplary bispecific antibody may bind to two different epitopes of the same protein, e.g., c-met protein. Bispecific antibodies may also be used to localize cytotoxic agents to cells which express EGFR or c-met. These antibodies possess a EGFR or c-met- binding arm and an arm which binds the cytotoxic agent (e.g. saporin, anti-interferon-a, vinca alkaloid, ricin A chain, methotrexate or radioactive isotope hapten). Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g. F(ab')2 bispecific antibodies). In one embodiment, the bispecific antibody is any of the bispecific MET-EGFR antibodies disclosed in US20100254989A.
EGFR antagonists also include small molecules such as compounds described in US5616582, US5457105, US5475001, US5654307, US5679683, US6084095, US6265410, US6455534, US6521620, US6596726, US6713484, US5770599, US6140332, US5866572, US6399602, US6344459, US6602863, US6391874, W09814451, WO9850038, WO9909016, WO9924037, W09935146, WO0132651, US6344455, US5760041, US6002008, US5747498. Particular small molecule EGFR antagonists include OSI-774 (CP-358774, erlotinib, OSI Pharmaceuticals); PD 183805 (CI 1033, 2-propenamide, N-[4-[(3-chloro-4- fluorophenyl)amino] -7-[3 -(4-morpholinyl)propoxy] -6-quinazolinyl] -, dihydrochloride, Pfizer Inc.); Iressa® (ZD 1839, gefitinib, AstraZeneca); ZM 105180 ((6-amino-4-(3-methylphenyl- amino)-quinazoline, Zeneca); BIBX-1382 (N8-(3-chloro-4-fluoro-phenyl)-N2-(l-methyl- piperidin-4-yl)-pyrimido[5,4-d]pyrimidine-2,8-diamine, Boehringer Ingelheim); PKI-166 ((R)- 4-[4-[(l-phenylethyl)amino]-lH-pyrrolo[2,3-d]pyrimidin-6-yl]-phenol); (R)-6-(4- hydroxyphenyl)-4-[(l-phenylethyl)amino]-7H-pyrrolo[2,3-d]pyrimidine); CL-387785 (N-[4- [(3 -bromophenyl)amino] -6-quinazolinyl] -2 -butynamide); EKB-569 (N-[4-[(3-chloro-4- fluorophenyl)amino]-3-cyano-7-ethoxy-6-quinolinyl]-4-(dimethylamino)-2-butenamide); lapatinib (Tykerb, GlaxoSmithKline); ZD6474 (Zactima, AstraZeneca); CUDC-101 (Curis); canertinib (CI-1033); AEE788 (6-[4-[(4-ethyl-l-piperazinyl)methyl]phenyl]-N-[(lR)-l- phenylethyl]-7H-pyrrolo[2,3-d]pyrimidin-4-amine, WO2003013541, Novartis) and PKI166 4- [4-[[(lR)-l-phenylethyl]amino]-7H-pyrrolo[2,3-d]pyrimidin-6-yl]-phenol, WO9702266 Novartis).
In one embodiment, the antibody, e.g. the antibody used in the methods herein may incorporate any of the features, singly or in combination, as described in Sections 1-6 below: 1. Antibody Fragments
In certain embodiments, an antibody provided herein is an antibody fragment.
Antibody fragments include, but are not limited to, Fab, Fab', Fab'-SH, F(ab')2, Fv, and scFv fragments, a one-armed antibody, and other fragments described below. For a review of certain antibody fragments, see Hudson et al. Nat. Med. 9: 129-134 (2003). For a review of scFv fragments, see, e.g., Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer- Verlag, New York), pp. 269-315 (1994); see also WO 93/16185; and U.S. Patent Nos. 5,571,894 and 5,587,458. For discussion of Fab and F(ab*)2 fragments comprising salvage receptor binding epitope residues and having increased in vivo half-life, see U.S. Patent No. 5,869,046.
Diabodies are antibody fragments with two antigen-binding sites that may be bivalent or bispecific. See, for example, EP 404,097; WO 1993/01161; Hudson et al, Nat. Med. 9: 129- 134 (2003); and Hollinger et al, Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al, Nat. Med. 9: 129-134 (2003).
Single-domain antibodies are antibody fragments comprising all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain embodiments, a single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Patent No. 6,248,516 Bl).
One-armed 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, WO2005/063816; Martens et al, Clin Cancer Res (2006), 12: 6144. For treatment of pathological conditions requiring an antagonistic function, and where bivalency of an antibody results in an undesirable agonistic effect, the monovalent trait of a one-armed antibody (i.e., an antibody comprising a single antigen binding arm) results in and/or ensures an antagonistic function upon binding of the antibody to a target molecule. Furthermore, the one-armed antibody comprising a Fc region is characterized by superior pharmacokinetic attributes (such as an enhanced half life and/or reduced clearance rate in vivo) compared to Fab forms having similar/substantially identical antigen binding characteristics, thus overcoming a major drawback in the use of conventional monovalent Fab antibodies. Techniques for making one-armed antibodies include, but are not limited to, "knob-in-hole" engineering (see, e.g., U.S. Patent No.
5,731,168). Onartuzumab is an example of a one-armed antibody. Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody as well as production by recombinant host cells (e.g. E. coli or phage), as described herein.
2. Chimeric and Humanized Antibodies
In certain embodiments, an antibody provided herein is a chimeric antibody. Certain chimeric antibodies are described, e.g., in U.S. Patent No. 4,816,567; and Morrison et al, Proc. Natl. Acad. Sci. USA, 81 :6851-6855 (1984)). 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 a further example, a chimeric antibody is a "class switched" antibody in which the class or subclass has been changed from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.
In certain embodiments, a chimeric antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce immunogenicity to humans, while retaining the specificity and affinity of the parental 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 human antibody sequences. A humanized antibody optionally will also comprise at least a portion of a human constant region. In some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the HVR residues are derived), e.g., to restore or improve antibody specificity or affinity.
Humanized antibodies and methods of making them are reviewed, e.g., in Almagro and Fransson, Front. Biosci. 13: 1619-1633 (2008), and are further described, e.g., in Riechmann et al, Nature 332:323-329 (1988); Queen et al, Proc. Nat 'l Acad. Sci. USA 86: 10029-10033 (1989); US Patent Nos. 5, 821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al, Methods 36:25-34 (2005) (describing SDR (a-CDR) grafting); Padlan, Mol. Immunol. 28:489- 498 (1991) (describing "resurfacing"); DaU'Acqua et al, Methods 36:43-60 (2005) (describing "FR shuffling"); and Osbourn et al, Methods 36:61-68 (2005) and Klimka et al, Br. J. Cancer, 83 :252-260 (2000) (describing the "guided selection" approach to 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 the consensus sequence of human antibodies of a particular subgroup of 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 from 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, an antibody provided herein is a human antibody. Human antibodies can be produced using various techniques known in the art. Human antibodies are described generally in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5: 368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20:450-459 (2008).
Human antibodies may be prepared by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigenic challenge. Such animals typically contain all or a portion of the human immunoglobulin loci, which replace the endogenous
immunoglobulin loci, or which are present extrachromosomally or integrated randomly into the animal's chromosomes. In such transgenic mice, the endogenous immunoglobulin loci have generally been inactivated. For review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23: 1117-1125 (2005). See also, e.g., U.S.
Patent Nos. 6,075,181 and 6,150,584 describing XENOMOUSE™ technology; U.S. Patent No. 5,770,429 describing HUMAB® technology; U.S. Patent No. 7,041,870 describing K-M
MOUSE® technology, and U.S. Patent Application Publication No. US 2007/0061900, describing VELOCIMOUSE® technology). Human variable regions from intact antibodies generated by such animals may be further modified, e.g., by combining with a different human constant region.
Human antibodies can also be made 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-ceil hybridoma technology are also described in Li et aL, Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006). Additional methods include those described, for example, in U.S. Patent No. 7,189,826 (describing production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (describing human-human hybridomas). Human hybridoma technology (Trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology, 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3): 185-91 (2005).
Human antibodies may also be generated by isolating Fv clone variable domain sequences selected from human-derived phage display libraries. Such variable domain sequences may then be combined with a desired human constant domain. Techniques for selecting human antibodies from antibody libraries are described below. 4. Library-Derived Antibodies
Antibodies of the invention may be isolated by screening combinatorial libraries for antibodies with the desired activity or activities. For example, a variety of methods are known in the art for generating phage display libraries and screening such libraries for antibodies possessing the desired binding characteristics. Such methods are reviewed, e.g., in
Hoogenboom et al. in Methods in Molecular Biology 178: 1-37 (O'Brien et al, ed., Human
Press, Totowa, NJ, 2001) and further described, e.g., in the McCafferty et al, Nature 348:552- 554; Clackson et al, Nature 352: 624-628 (1991); Marks et al, J. Mol. Biol. 222: 581-597 (1992); Marks and Bradbury, in Methods in Molecular Biology 248: 161-175 (Lo, ed., Human Press, Totowa, NJ, 2003); Sidhu et al, J. Mol. Biol. 338(2): 299-310 (2004); Lee et al, J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al, J. Immunol. Methods 284(1-2): 119-132(2004).
In certain phage display methods, repertoires of VH and VL genes are separately cloned by polymerase chain reaction (PCR) and recombined randomly in phage libraries, which can then be screened for antigen-binding phage 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 requirement of constructing hybridomas.
Alternatively, the naive repertoire can be cloned (e.g., from human) to provide a single source of antibodies to a wide range of non-self and also self antigens without any immunization as described by Griffiths et al, EMBO J 12: 725-734 (1993). Finally, naive libraries can also be made synthetically by cloning unrearranged V-gene segments from stem cells, and using PCR primers containing random sequence to encode the highly variable CDR3 regions and to accomplish 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: US Patent No. 5,750,373, and US 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 human antibody libraries are considered human antibodies or human antibody fragments herein.
5. Multispecific Antibodies
In certain embodiments, an antibody provided herein is a multispecific antibody, e.g. a bispecific antibody. Multispecific antibodies are monoclonal antibodies that have binding specificities for at least two different sites. In certain embodiments, one of the binding specificities is for c-met and the other is for any other antigen. In certain embodiments, bispecific antibodies may bind to two different epitopes of c-met. Bispecific antibodies may also be used to localize cytotoxic agents to cells which express c-met. Bispecific antibodies can be prepared as full length antibodies or antibody fragments.
Techniques for making multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs having different specificities (see Milstein and Cuello, Nature 305: 537 (1983), WO 93/08829, and Traunecker et al, EMBO J. 10: 3655 (1991)), and "knob-in-hole" engineering (see, e.g., U.S. Patent No. 5,731,168). Multi-specific antibodies may also be made by engineering electrostatic steering effects for making antibody Fc-heterodimeric molecules (WO 2009/089004A1); cross- linking two or more antibodies or fragments (see, e.g., US Patent No. 4,676,980, and Brennan et al, Science, 229: 81 (1985)); using leucine zippers to produce bi-specific antibodies (see, e.g., Kostelny et al, J. Immunol., 148(5): 1547-1553 (1992)); using "diabody" technology for making bispecific antibody fragments (see, e.g., Hollinger et al, Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain Fv (sFv) dimers (see,e.g. Gruber et al, J.
Immunol, 152:5368 (1994)); and preparing trispecific antibodies as described, e.g., in Tutt et al. J. Immunol. 147: 60 (1991).
Engineered antibodies with three or more functional antigen binding sites, including "Octopus antibodies," are also included herein (see, e.g. US 2006/0025576A1).
The antibody or fragment herein also includes a "Dual Acting FAb" or "DAF" comprising an antigen binding site that binds to c-met as well as another, different antigen, such as EGFR (see, US 2008/0069820, for example). 6. Antibody Variants
In certain embodiments, amino acid sequence variants of the antibodies provided herein are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of the antibody. Amino acid sequence variants of an antibody may 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 sequences of the antibody. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that 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 the HVRs and FRs.
Amino acid substitutions may be introduced into an antibody of interest and the products screened for a desired activity, e.g., retained/improved antigen binding, decreased
immunogenicity, or improved ADCC or CDC.
One type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody {e.g. a humanized or human antibody). Generally, the resulting variant(s) selected for further study will have modifications (e.g., improvements) in certain biological properties (e.g., increased affinity, reduced immunogenicity) relative to the parent antibody and/or will have substantially retained certain biological properties of the parent antibody. An exemplary substitutional variant is an affinity matured antibody, which may be conveniently generated, e.g., using phage display-based affinity maturation techniques such as those described herein. Briefly, one or more HVR residues are mutated and the variant antibodies displayed on phage and screened for a particular biological activity (e.g. binding affinity).
Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an antibody with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include the fusion to the N- or C -terminus of the antibody to an enzyme (e.g. for ADEPT) or a polypeptide which increases the serum half-life of the antibody. In certain embodiments, an antibody provided herein is altered to increase or decrease the extent to which the antibody is glycosylated. Addition or deletion of glycosylation sites to an antibody may be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites is created or removed.
Where the antibody comprises an Fc region, the carbohydrate attached thereto may be altered. Native antibodies produced by mammalian cells typically comprise a branched, biantennary oligosaccharide that is generally attached by an N-linkage to Asn297 of the CH2 domain of the Fc region. See, e.g., Wright et al. TIBTECH 15:26-32 (1997). The
oligosaccharide may include various carbohydrates, e.g., mannose, N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as a fucose attached to a GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some embodiments, modifications of the oligosaccharide in an antibody of the invention may be made in order to create antibody variants with certain improved properties.
In one embodiment, antibody variants are provided having a carbohydrate structure that lacks fucose attached (directly or indirectly) to an Fc region. For example, the amount of fucose in such antibody may be from 1% to 80%, from 1% to 65%, from 5% to 65%> or from 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 glycostructures attached to Asn 297 (e. g. complex, hybrid and high mannose structures) as measured by MALDI-TOF mass spectrometry, as described in WO 2008/077546, for example. 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 antibodies. Such fucosylation variants may have improved ADCC function. See, e.g., US Patent Publication Nos. US 2003/0157108 (Presta, L.); US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications related 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; WO2005/053742; WO2002/031140; 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 Lecl3 CHO cells deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); US Pat Appl No US 2003/0157108 Al, Presta, L; and WO 2004/056312 Al, Adams et al, especially at Example 11), and knockout cell lines, such as alpha- 1,6-fucosyltransferase gene, FUT8, knockout 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 WO2003/085107).
Antibodies variants are further provided with bisected oligosaccharides, e.g., in which a biantennary oligosaccharide attached to the Fc region of the antibody is bisected by GlcNAc. Such antibody variants may have reduced fucosylation and/or improved ADCC function.
Examples of such antibody variants are described, e.g., in WO 2003/011878 (Jean-Mairet et al); US Patent No. 6,602,684 (Umana et al); and US 2005/0123546 (Umana et al). Antibody variants with at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, e.g., in WO 1997/30087 (Patel et al); WO 1998/58964 (Raju, S.); and WO 1999/22764 (Raju, S.).
In certain embodiments, one or more amino acid modifications may 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 IgGl, IgG2, IgG3 or IgG4 Fc region) comprising an amino acid modification {e.g. a substitution) at one or more amino acid positions.
In certain embodiments, the invention contemplates an antibody variant that possesses some but not all effector functions, which make it a desirable candidate for applications in which the half life of the antibody in vivo is important yet certain effector functions (such as complement and ADCC) are unnecessary or deleterious.
Antibodies with reduced effector function include those with substitution of one or more of Fc region residues 238, 265, 269, 270, 297, 327 and 329 (U.S. Patent No. 6,737,056). Such Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including the so-called "DANA" Fc mutant with substitution of residues 265 and 297 to alanine (US Patent No. 7,332,581).
Certain antibody variants with improved or diminished binding to FcRs are described. (See, e.g., U.S. Patent No. 6,737,056; WO 2004/056312, and Shields et al, J. Biol. Chem. 9(2): 6591-6604 (2001).)
In certain embodiments, an antibody variant comprises an Fc region with one or more amino acid substitutions which improve ADCC, e.g., substitutions at positions 298, 333, and/or 334 of the Fc region (EU numbering of residues). In some embodiments, alterations are made in the Fc region that result in altered (i.e., either improved or diminished) Clq binding and/or Complement Dependent Cytotoxicity (CDC), e.g., as described in US Patent No. 6,194,551, WO 99/51642, and Idusogie et al. J. Immunol. 164: 4178-4184 (2000).
Antibodies with increased half lives and improved binding to the neonatal Fc receptor
(FcRn), which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al., J.
Immunol. 117:587 (1976) and Kim et al, J. Immunol. 24:249 (1994)), are described in
US2005/0014934A1 (Hinton et al.). Those antibodies comprise an Fc region with one or more substitutions therein which improve binding of the Fc region to FcRn. Such Fc variants include those with substitutions at one or more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434, e.g., substitution of Fc region residue 434 (US Patent No. 7,371,826).
See also Duncan & Winter, Nature 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO 94/29351 concerning 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 an antibody are substituted with cysteine residues. In particular embodiments, the substituted residues occur at accessible sites of the antibody. By substituting those residues with cysteine, reactive thiol groups are thereby positioned at accessible sites of the antibody and may be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties, to create an immunoconjugate, as described further herein. In certain embodiments, any one or more of the following residues may be substituted with cysteine: V205 (Kabat numbering) of the light chain; Al 18 (EU numbering) of the heavy chain; and S400 (EU numbering) of the heavy chain Fc region.
Cysteine engineered antibodies may be generated as described, e.g., in U.S. Patent No.
7,521,541.
In certain embodiments, an antibody provided herein may be further modified to contain additional nonproteinaceous moieties that are known in the art and readily available. The moieties suitable 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), copolymers of ethylene glycol/propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1, 3- dioxolane, poly-1, 3, 6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymers or random copolymers), and dextran or poly(n-vinyl pyrrolidone)polyethylene glycol, propropylene glycol homopolymers, prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.
Polyethylene glycol propionaldehyde may have advantages in manufacturing 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 may vary, and if more than one polymer are 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 particular properties or functions of the antibody to be improved, whether the antibody derivative will be used in a therapy under defined conditions, etc.
In another embodiment, conjugates of an antibody and nonproteinaceous moiety that may be selectively heated by exposure to radiation are provided. In one embodiment, the nonproteinaceous moiety is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102: 11600-11605 (2005)). The radiation may be of any wavelength, and includes, but is not limited to, wavelengths that do not harm ordinary cells, but which heat the nonproteinaceous moiety to a temperature at which cells proximal to the antibody-nonproteinaceous moiety are killed.
In one embodiment, the medicament is an mmunoconjugate comprising an antibody (such as a c-met antibody) conjugated to one or more cytotoxic agents, such as
chemotherapeutic agents or drugs, growth inhibitory agents, toxins (e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof), or radioactive isotopes.
In one embodiment, an immunoconjugate is an antibody-drug conjugate (ADC) in which an antibody is conjugated to one or more drugs, including but not limited to a maytansinoid (see U.S. Patent Nos. 5,208,020, 5,416,064 and European Patent EP 0 425 235 Bl); an auristatin such as monomethylauristatin drug moieties DE and DF (MMAE and MMAF) (see U.S. Patent Nos. 5,635,483 and 5,780,588, and 7,498,298); a dolastatin; a calicheamicin or derivative thereof (see U.S. Patent 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)); an anthracycline such as daunomycin or doxorubicin (see Kratz et al, Current Med. Chem. 13:477-523 (2006); Jeffrey et al, Bioorganic & Med. Chem. Letters 16:358-362 (2006); Torgov et al, Bioconj. Chem. 16:717-721 (2005); Nagy et al, Proc. Natl. Acad. Sci. USA 97:829-834 (2000);
Dubowchik et al, Bioorg. & Med. Chem. Letters 12: 1529-1532 (2002); King et al, J. Med. Chem. 45:4336-4343 (2002); and U.S. Patent No. 6,630,579); methotrexate; vindesine; a taxane such as docetaxel, paclitaxel, larotaxel, tesetaxel, and ortataxel; a trichothecene; and CC1065.
In another embodiment, an immunoconjugate comprises an antibody as described herein conjugated to an enzymatically active toxin or fragment thereof, including but not limited to diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha- sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.
In another embodiment, an immunoconjugate comprises an antibody as described herein conjugated to a radioactive atom to form a radioconjugate. A variety of radioactive
211 131 125 isotopes are available for the production of radioconjugates. Examples include At , 1 , 1 , Y90, Re186, Re188, Sm153, Bi212, P32, Pb212 and radioactive isotopes of Lu. When the
radioconjugate is used for detection, it may comprise a radioactive atom for scintigraphic studies, for example tc99m or 1123, or a spin label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, mri), such as iodine- 123 again, iodine- 131, indium-I l l, fluorine- 19, carbon- 13, nitrogen- 15, oxygen- 17, gadolinium, manganese or iron.
Conjugates of an antibody and cytotoxic agent may be made using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), succinimidyl-4-(N-maleimidomethyl) cyclohexane-l-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HC1), 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), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as l,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al, Science 238: 1098 (1987). Carbon- 14-labeled l-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX- DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See W094/11026. The linker may be a "cleavable linker" facilitating release of a cytotoxic drug in the cell. For example, an acid-labile linker, peptidase-sensitive linker, photolabile linker, dimethyl linker or disulfide-containing linker (Chari et al., Cancer Res. 52: 127-131 (1992); U.S. Patent No. 5,208,020) may be used.
The immunuoconjugates or ADCs herein expressly contemplate, but are not limited to such conjugates prepared with cross-linker reagents 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).
III. DIAGNOSTIC METHODS
In one aspect, the invention provides diagnostic methods, e.g. for identifying a cancer patient who is likely to respond to treatment with a c-met antagonist.
In some embodiments, IHC analysis further comprises morphological staining, either prior to or thereafter. In one embodiment, hematoxylin is use for staining cellular nucleic of the slides. Hematoxylin is widely available. An example of a suitable hematoxylin is
Hematoxylin II (Ventana). When lighter blue nuclei are desired, a bluing reagent may be used following hematoxylin staining.
Detection of c-met biomarker using IHC is disclosed herein, and a c-met staining intensity scoring scheme is disclosed herein. Disclosed herein is a method for determining an H-score, wherein the H-score is determined using the formula (% cancer cells staining with weak c-met staining intensity) + (% cancer cells staining with moderate c-met staining intensity) + (% cancer cells staining with high c-met staining intensity). Methods for calculating the H-score are disclosed and exemplified herein. In some embodiments of any of the methods herein, the H-score is about 160 or higher (about 161, 162, 163, 164, 165, 166, 167, 168, 169, or higher), 160 or higher, about 160 to about 230, about 160 ro 230, about 160 (any of about 161, 162, 163, 164, 165, 166, 167, 168, 169, or higher to any of abour 220, 221,
223, 224, 225, 226, 227, 228, 229, 230 orhigher), 230 or higher, any of about 220, 221, 223,
224, 225, 226, 227, 228, 229, 230 or higher), about 170 or higher, or 170 or higher (e.g, any of about 171, 172, 173, 175, 175, 176, 177, 178, 179 180 or higher). In one embodiment, the H- score is about 180 orhigher. In some embodiments of any of the methods of the invention, the H-score is less than about 160, less than 160 (e.g., 159, 158, 157, 156, 155, 154, 143, 142, 141 or less), less than about 150, less than 150 (e.g., 149, 148, 147, 146, 145, 144, 143, 142, 141, or less). . In some embodiments, the H score is greater than about 10. In some embodiments, the H score is greater than about 25. In some embodiments, the H score is greater than about 50. In some embodiments, the H score is greater than about 75. In some embodiments, the H score is greater than about 100. In some embodiments, the H score is greater than about 125. In some embodiments, the H score is greater than about 150. In some embodiments, the H score is greater than about 175. In some embodiments, the H score is greater than about 200. In some embodiments, c-met expression is membranous. In some embodiments, c-met expression is cytoplasmic. In some embodiments, c-met- expression is membranous and cytoplasmic.
As is noted herein, other biomarkers may be detected. Exemplary other biomarkers are disclosed herein.
A sample from the patient is tested for expression of one or more of the biomarkers herein. The source of the tissue or cell sample may be solid tissue as from a fresh, frozen and/or preserved organ or tissue sample or biopsy or aspirate (including but not limited to a fine needle aspirate); blood or any blood constituents; bodily fluids such as cerebral spinal fluid, amniotic fluid, peritoneal fluid, bronchiolar lavage, pleural fluid, sputum, or interstitial fluid; cells from any time in gestation or development of the subject. The tissue sample may contain compounds which are not naturally intermixed with the tissue in nature such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, or the like. Examples of tumor samples herein include, but are not limited to, tumor biopsies, tumor cells, serum or plasma, circulating plasma proteins, ascitic fluid, primary cell cultures or cell lines derived from tumors or exhibiting tumor-like properties, bronchiolar lavage, pleural fluid, sputum, as well as preserved tumor samples, such as formalin- fixed, paraffin-embedded tumor samples (including, but not limited to formalin- fixed paraffin-embedded fine needle aspirate samples) or frozen tumor samples. In one embodiment, the patient sample is a formalin-fixed paraffin- embedded (FFPE) tumor sample (e.g., a NSCLC tumor sample or a breast cancer tumor sample). In one embodiment, the patient sample is a formalin-fixed paraffin-embedded sample from a fine needle aspirate. The sample may be obtained prior to or during the patient's treatment with a cancer medicament (such as an anti-c-met antagonist). The sample may be obtained prior to or during the patient's prior treatment with a cancer medicament. The sample may be obtained from the primary tumor or from a metastatic tumor. The sample may be obtained when the cancer is first diagnosed or, for example, after the tumor has metastasized. In some embodiments, the tumor sample is of lung, lymph node, liver or brain.
Immunohistochemical (IHC) staining of tissue sections has been shown to be a reliable method of assessing or detecting presence of proteins in a sample. Immunohistochemistry techniques utilize an antibody to probe and visualize cellular antigens in situ, generally by chromogenic or fluorescent methods. Thus, antibodies or antisera, in some embodiments, polyclonal antisera, and in some embodiments, monoclonal antibodies specific for each marker are used to detect expression. As discussed in greater detail below, the antibodies can be detected by direct labeling of the antibodies themselves, for example, with radioactive labels, fluorescent labels, hapten labels such as, biotin, or an enzyme such as horse radish peroxidase or alkaline phosphatase. Alternatively, unlabeled primary antibody is used in conjunction with a labeled secondary antibody, comprising antisera, polyclonal antisera or a monoclonal antibody specific for the primary antibody. Immunohistochemistry protocols and kits are well known in the art and are commercially available.
Two general methods of IHC are available; direct and indirect assays. According to the first assay, binding of antibody to the target antigen is determined directly. This direct assay uses a labeled reagent, such as a fluorescent tag or an enzyme-labeled primary antibody, which can be visualized without further antibody interaction. In a typical indirect assay, unconjugated primary antibody binds to the antigen and then a labeled secondary antibody binds to the primary antibody. Where the secondary antibody is conjugated to an enzymatic label, a chromagenic or fluorogenic substrate is added to provide visualization of the antigen. Signal amplification occurs because several secondary antibodies may react with different epitopes on the primary antibody.
The primary and/or secondary antibody used for immunohistochemistry typically will be labeled with a detectable moiety. Numerous labels are available which can be generally grouped into the following categories:
35 14 125 3 131
(a) Radioisotopes, such as S, C, I, H, and I. The antibody can be labeled with the radioisotope using the techniques described in Current Protocols in Immunology, Volumes 1 and 2, Coligen et al, Ed. Wiley-Interscience, New York, N.Y., Pubs. (1991) for example and radioactivity can be measured using scintillation counting.
(b) Colloidal gold particles.
(c) Fluorescent labels including, but are not limited to, rare earth chelates (europium chelates), Texas Red, rhodamine, fluorescein, dansyl, Lissamine, umbelliferone,
phycocrytherin, phycocyanin, or commercially available fluorophores such SPECTRUM ORANGE® and SPECTRUM GREEN® and/or derivatives of any one or more of the above. The fluorescent labels can be conjugated to the antibody using the techniques disclosed in Current Protocols in Immunology, supra, for example. Fluorescence can be quantified using a fluorimeter. (d) Various enzyme-substrate labels are available and U.S. Pat. No. 4,275,149 provides a review of some of these. The enzyme generally catalyzes a chemical alteration of the chromogenic substrate that can be measured using various techniques. For example, the enzyme may catalyze a color change in a substrate, which can be measured
spectrophotometrically. Alternatively, the enzyme may alter the fluorescence or
chemiluminescence of the substrate. Techniques for quantifying a change in fluorescence are described above. The chemiluminescent substrate becomes electronically excited by a chemical reaction and may then emit light which can be measured (using a chemiluminometer, for example) or donates energy to a fluorescent acceptor. Examples of enzymatic labels include luciferases (e.g., firefly luciferase and bacterial luciferase; U.S. Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones, malate dehydrogenase, urease, peroxidase such as horseradish peroxidase (HRPO), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, saccharide oxidases (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (such as uricase and xanthine oxidase), lactoperoxidase, microperoxidase, and the like. Techniques for conjugating enzymes to antibodies are described in O'Sullivan et al. Methods for the Preparation of Enzyme-Antibody Conjugates for use in Enzyme Immunoassay, in Methods in Enzym. (ed J. Langone & H. Van Vunakis), Academic press, New York, 73: 147-166 (1981).
Examples of enzyme-substrate combinations include, for example:
(i) Horseradish peroxidase (HRPO) with hydrogen peroxidase as a substrate, wherein the hydrogen peroxidase oxidizes a dye precursor [e.g., orthophenylene diamine (OPD) or 3,3',5,5'-tetramethyl benzidine hydrochloride (TMB)]. 3,3-Diaminobenzidine (DAB) may also be used to visualize the HRP-labeled antibody;
(ii) alkaline phosphatase (AP) with para-Nitrophenyl phosphate as chromogenic substrate; and
(iii) β-D-galactosidase (β-D-Gal) with a chromogenic substrate (e.g., p-nitrophenyl-β- D-galactosidase) or fluorogenic substrate (e.g., 4-methylumbelliferyl^-D-galactosidase).
Numerous 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 with the antibody. The skilled artisan will be aware of various techniques for achieving this. For example, the antibody can be conjugated with biotin and any of the four broad categories of labels mentioned above can be conjugated with avidin, or vice verse. Biotin binds selectively to avidin and thus, the label can be conjugated with the antibody in this indirect manner. Alternatively, to achieve indirect conjugation of the label with the antibody, the antibody is conjugated with a small hapten and one of the different types of labels mentioned above is conjugated with an anti-hapten antibody. Thus, indirect conjugation of the label with the antibody can be achieved.
Aside from the sample preparation procedures discussed above, further treatment of the tissue section prior to, during or following IHC may be desired. For example, epitope retrieval methods, such as heating the tissue sample in citrate buffer may be carried out [see, e.g., Leong et al. Appl. Immunohistochem. 4(3):201 (1996)].
Following an optional blocking step, the tissue section is exposed to primary antibody for a sufficient period of time and under suitable conditions such that the primary antibody binds to the target protein antigen in the tissue sample. Appropriate conditions for achieving this can be determined by routine experimentation.
The extent of binding of antibody to the sample is determined by using any one of the detectable labels discussed above. Preferably, the label is an enzymatic label (e.g. HRPO) which catalyzes a chemical alteration of the chromogenic substrate such as 3,3'- diaminobenzidine chromogen. Preferably the enzymatic label is conjugated to antibody which binds specifically to the primary antibody (e.g. the primary antibody is rabbit polyclonal antibody and secondary antibody is goat anti-rabbit antibody).
Specimens thus prepared may be mounted and coverslipped. Slide evaluation is then determined, e.g. using a microscope.
IHC may be combined with morphological staining, either prior to or thereafter. After deparaffinization, the sections mounted on slides may be stained with a morphological stain for evaluation. The morphological stain to be used provides for accurate morphological evaluation of a tissue section. The section may be stained with one or more dyes each of which distinctly stains different cellular components. In one embodiment, hematoxylin is use for staining cellular nucleic of the slides. Hematoxylin is widely available. An example of a suitable hematoxylin is Hematoxylin II (Ventana). When lighter blue nuclei are desired, a bluing reagent may be used following hematoxylin staining. One of skill in the art will appreciate that staining may be optimized for a given tissue by increasing or decreasing the length of time the slides remain in the dye.
Automated systems for slide preparation and IHC processing are available
commercially. The Ventana® BenchMark XT system is an example of such an automated system. After staining, the tissue section may be analyzed by standard techniques of
microscopy. Generally, a pathologist or the like assesses the tissue for the presence of abnormal or normal cells or a specific cell type and provides the loci of the cell types of interest. Thus, for example, a pathologist or the like would review the slides and identify normal cells (such as normal lung cells) and abnormal cells (such as abnormal or neoplastic lung cells). Any means of defining the loci of the cells of interest may be used (e.g., coordinates on an X-Y axis).
Anti-c-met antibodies suitable for use in IHC are well known in the art, and include SP- 44 (Ventana), DL-21 (Upstate), D1C2 (Cell Signaling Technologies), ab27492 (Abeam), PA1- 37483 (Pierce Antibodies), Met4 (see, e.g, US Patent No. 6,548, 640). In some embodiments, the anti-c-met antibody is SP44. In some embodiments, the anti-c-met antibody is DL-21. In some embodiments, the anti-c-met antibody is D1C2. In some embodiments, the anti-c-met antibody is Met4. One of ordinary skill understands that additional suitable anti-c-met antibodies may be identified and characterized by comparing with c-met antibodies using the IHC protocol and examples disclosed herein, for example.
Control cell lines (e.g., centrifuged into a pellet and formalin fixed and paraffin embedded, e.g., and prepared as a tissue microarray, and e.g., stained with SP44) with various staining intensities (e.g., when stained with c-met antibody SP44) may be utilized as controls for IHC analysis. For example, H441 (strong c-met staining intensity); EBCl (strong c-met staining intensity), A549 (moderate c-met staining intensity); SKMES1 (moderate c-met staining intensity) HI 703 (weak c-met staining intensity), HEK-293 (weak c-met staining intensity); H460 (weak c-met staining intensity), and TOV-112D (negative c-met staining intensity), LXFL529 (negative c-met staining intensity), H522 (negative c-met staining intensity), H23 (negative c-met staining intensity) or HI 155 (negative c-met staining intensity). One of ordinary skill understands that other control cell pellets with negative, weak, moderate and high c-met staining intensity may readily be identified using the teachings of the present application and methods well known in the art and disclosed herein. Accordingly, in some embodiments, strong c-met staining intensity is c-met staining intensity of a control cell having c-met staining intensity of H441 and/or EBCl . In some embodiments, moderate c-met staining intensity is c-met staining intensity of a control cell having c-met staining intensity of A549 and/or SKMES 1. In some embodiments, weak c-met staining intensity is c-met staining intensity of a control cell having c-met staining intensity of HEK-293 and/or H460. In some embodiments, negative c-met staining intensity is c-met staining intensity of a control cell having c-met staining intensity of LXFL529, H522, H23, and/or HI 155. Use of control cell pellets with different staining intensity for IHC analysis, e.g., while scoring and analyzing c- met IHC of cancer samples, is well known in the art. A c-met immunohistochemistry protocol and scoring scheme is exemplified herein.
In some embodiments, c-met IHC is analyzed using the scheme shown in Table X:
Table X
Figure imgf000066_0001
In some embodiments, c-Met IHC is analyzed using the scheme shown in Table Y:
Table Y
Figure imgf000067_0001
In some embodiments, a patient's tumor is c-met positive when 1% or more of the tumor cells in the sample express c-met protein (e.g., express c-met protein at any intensity, e.g., low, moderate and high). In some embodiments, a patient's tumor is c-met positive when more than 1% of the tumor cells in the sample express c-met protein (e.g., express c-met protein at any intensity). In some embodiments, a patient's tumor is c-met positive when 5% or more of the tumor cells in the sample express c-met protein (e.g., express c-met protein at any intensity). In some embodiments, a patient's tumor is c-met positive when 10% or more of the tumor cells in the sample express c-met protein (e.g., express c-met protein at any intensity). In some embodiments, a patient's tumor is c-met positive when 15% or more of the tumor cells in the sample express c-met protein. In some embodiments, a patient's tumor is c-met positive when 20% or more of the tumor cells in the sample express c-met protein (e.g., express c-met protein at any intensity). In some embodiments, a patient's tumor is c-met positive when 25% or more of the tumor cells in the sample express c-met protein (e.g., express c-met protein at any intensity). In some embodiments, a patient's tumor is c-met positive when 30% or more of the tumor cells in the sample express c-met protein (e.g., express c-met protein at any intensity). In some embodiments, a patient's tumor is c-met positive when 35% or more of the tumor cells in the sample express c-met protein (e.g., express c-met protein at any intensity). In some embodiments, a patient's tumor is c-met positive when 40% or more of the tumor cells in the sample express c-met protein (e.g., express c-met protein at any intensity). In some embodiments, a patient's tumor is c-met positive when 45% or more of the tumor cells in the sample express c-met protein (e.g., express c-met protein at any intensity). In some embodiments, a patient's tumor is c-met positive when 50% or more of the tumor cells in the sample express c-met protein (e.g., express c-met protein at any intensity). In some embodiments, a patient's tumor is c-met positive when 55% or more of the tumor cells in the sample express c-met protein (e.g., express c-met protein at any intensity). In some embodiments, a patient's tumor is c-met positive when 60% or more of the tumor cells in the sample express c-met protein (e.g., express c-met protein at any intensity). In some embodiments, a patient's tumor is c-met positive when 65% or more of the tumor cells in the sample express c-met protein (e.g., express c-met protein at any intensity). In some embodiments, a patient's tumor is c-met positive when 70% or more of the tumor cells in the sample express c-met protein (e.g., express c-met protein at any intensity). In some embodiments, a patient's tumor is c-met positive when 75% or more of the tumor cells in the sample express c-met protein (e.g., express c-met protein at any intensity). In some embodiments, a patient's tumor is c-met positive when 80% or more of the tumor cells in the sample express c-met protein (e.g., express c-met protein at any intensity). In some embodiments, a patient's tumor is c-met positive when 85% or more of the tumor cells in the sample express c-met protein (e.g., express c-met protein at any intensity). In some embodiments, a patient's tumor is c-met positive when 90% or more of the tumor cells in the sample express c-met protein (e.g., express c-met protein at any intensity). In some embodiments, a patient's tumor is c-met positive when 95% or more of the tumor cells in the sample express c-met protein (e.g., express c-met protein at any intensity). In some embodiments, c-met expression is membranous. In some embodiments, c-met expression is cytoplasmic. In some embodiments, c-met- expression is membranous and cytoplasmic.
In some embodiments, a patient's tumor is c-met positive when a maximum staining intensity of the tumor is 1. In some embodiments, a patient's tumor is c-met positive when a maximum staining intensity of the tumor is 2. In some embodiments, a patient's tumor is c-met positive when a maximum staining intensity of the tumor is 3. In some embodiments, c-met expression is membranous. In some embodiments, c-met expression is cytoplasmic. In some
embodiments, c-met- expression is membranous and cytoplasmic. In some embodiments, the tumor is c-met positive when 50% or more of the tumor cells in the sample have high c-met staining intensity. In some embodiments, the tumor is c-met positive when 50% or more of the tumor cells in the sample have moderate c-met staining intensity. In some embodiments, the tumor is c-met positive when 50% or more of the tumor cells in the sample have low, moderate or high c-met staining intensity.
Other techniques for biomarker detection are known in the art, including but not limited to nucleic acid detection methods (including but not limited to PCR, sequencing, rtPCT, RNA- seq, microarray analysis, SAGE, Mass ARRAY technique and FISH) and protein detection methods (including but not limited to mass spec, western blotting). Detecting amplification of the c-met gene is achieved using certain techniques known to those skilled in the art. For example, comparative genome hybridization may be used to produce a map of DNA sequence copy number as a function of chromosomal location. See, e.g., Kallioniemi et al. (1992) Science 258:818-821. Amplification of the c-met gene may also be detected, e.g., by Southern hybridization using a probe specific for the c-met gene or by real-time quantitative PCR. In certain embodiments, detecting amplification of the c-met gene is achieved by directly assessing the copy number of the c-met gene, for example, by using a probe that hybridizes to the c-met gene. For example, a FISH assay may 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 that lies outside the c-met gene but is co-amplified with the c-met gene. Biomarker expression may also be evaluated using an in vivo diagnostic assay, e.g. by administering a molecule (such as an antibody) which binds the molecule to be detected and is tagged with a detectable label {e.g. a radioactive isotope) and externally scanning the patient for localization of the label.
IV. THERAPEUTIC METHODS
In one aspect, the invention provides treatment (e.g., therapeutic treatment) methods, for treating a cancer patient who is likely to respond to treatment with a c-met antagonist.
Cancer medicaments can be used either alone or in combination with other cancer medicaments. For instance, an anti-c-met antibody (for example, Onartuzumab) may be administered at a dose of about 15 mg/kg every three weeks, or at a dose of about 10 mg/kg every two weeks.
For instance, a c-met antibody may be co-administered with at least one additional therapeutic agent, e.g. with a chemotherapeutic agent, with other c-met antagonists (such as other c-met antibodies), with an EGFR antagonist (such as erlotinib), or with an anti-VEGF antibody (such a bevacizumab). A c-met antibody may be co-administered with an additional c-met antagonist. Such combination therapies noted above encompass combined administration (where two or more therapeutic agents are included in the same or separate formulations), and separate administration, in which case, administration of a first medicament can occur prior to, simultaneously, and/or following, administration of a second medicament. In one embodiment, an anti-c-met antibody (such as Onartuzumab) at a dose of about 15 mg/kg every three weeks; is used in combination with erlotinib (N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)-4- quinazolinamine), or a pharmaceutically acceptable salt, at a dose of 150 mg, each day of a three week cycle. In other embodiments, a c-met antagonist (e.g., anti-c-met antibody) is used in combination with an anti-VEGF antibody and chemotherapy (e.g., a taxane). An exemplary protocol is administering to a triple-negative metastatic breast cancer patient an anti-c-met antibody (e.g., Onartuzumab) administered at a dose of 10 mg/kg on Day 1 and Day 15 of a 28- day cycle, anti-VEGF antibody (e.g., bevacizumab) administered at a dose of 10 mg/kg on Day 1 and Day 15 of the 28-day cycle and paclitaxel administered at a dose of 90 mg/m by IV infusion on Day 1, Day 8, and Day 15 of the 28-day cycle. An exemplary protocol is administering to a triple-negative metastatic breast cancer patient an anti-c-met antibody (e.g., Onartuzumab) administered at a dose of 10 mg/kg on Day 1 and Day 15 of a 28-day cycle, and paclitaxel administered at a dose of 90 mg/m by IV infusion on Day 1, Day 8, and Day 15 of the 28 -day cycle.
Cancer medicaments can also be used in combination with radiation therapy.
The medicament(s) herein can be administered by any suitable means, including parenteral, intrapulmonary, and intranasal, and, if desired for local treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing can be by any suitable route, e.g. by injections, such as intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic. Various dosing schedules including but not limited to single or multiple administrations over various time-points, bolus administration, and pulse infusion are contemplated herein.
For the prevention or treatment of disease, the appropriate dosage of an antibody of the invention (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the type of antibody, the severity and course of the disease, whether the antibody is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody, and the discretion of the attending physician. The antibody is suitably administered to the patient at one time or over a series of treatments. One typical daily dosage might range from about 1 μg/kg to 100 mg/kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, the treatment would generally be sustained until a desired suppression of disease symptoms occurs. However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.
It is understood that any of the above formulations or therapeutic methods may be carried out using an immunoconjugate of an antibody in place of or in addition to the antibody as the medicament.
In some embodiments, the patient did not receive more than two prior treatments for Stage IIIB/IV. In some embodiments, the patient did not receive more than 30 days of exposure to an investigational or marketed agent that can act by EGFR inhibition, or a known EGFR- related toxicity resulting in dose modifications. EGFR inhibitors include (but are not limited to) gefitinib, erlotinib, and cetuximab. In some embodiments, the patient did not receive chemotherapy, biologic therapy, radiotherapy or investigational drug within 28 days prior to randomization (except that optionally, kinase inhibitors may be used within two weeks prior to randomization provided any drug related toxicity was adequately resolved). In some embodiments, the patient is not a patient with untreated and/or active (progressing or requiring anticonvulsants or corticosteroids for symptomatic control) CNS metastasis. Other patient exclusion criteria are described in the Examples, and the present inventions contemplate use of one or more of the exclusions described therein. In some embodiments, a sample of the patient's cancer has been shown to have wildtype EGFR. In some embodiments, a sample of the patient's cancer has been shown to have mutant EGFR. In some embodiments, a sample of the patient's cancer has not been shown to have mutated EGFR.
V. ARTICLES OF MANUFACTURE
In another embodiment of the invention, an article of manufacture for use in treating cancer (such as NSCLC or breast cancer) is provided. The article of manufacture comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. The containers may be formed from a variety of materials such as glass or plastic. The container holds or contains a composition comprising the cancer medicament as the active agent and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle).
The article of manufacture may further comprise a second container comprising a pharmaceutically-acceptable diluent buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution. The article of manufacture may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.
The article of manufacture of the present invention also includes information, for example in the form of a package insert, indicating that the composition is used for treating cancer based on expression of biomarker(s) as disclosed herein. The insert or label may take any form, such as paper or on electronic media such as a magnetically recorded medium (e.g., floppy disk) or a CD-ROM. The label or insert may also include other information concerning the pharmaceutical compositions and dosage forms in the kit or article of manufacture.
The invention also concerns a method for manufacturing 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 patient with cancer (such as NSCLC) based on expression of a c- met biomarker as disclosed herein.
The article of manufacture may further comprise an additional container comprising a pharmaceutically acceptable diluent buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and/or dextrose solution. The article of manufacture may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.
VI. DIAGNOSTIC KITS
The invention also concerns diagnostic kits useful for detecting any one or more of the biomarker(s) identified herein. Accordingly, a diagnostic kit is provided which comprises one or more reagents for determining expression of one or more of c-met, k-ras, ALK and EGFR biomarker in a sample from a cancer patient. Optionally, the kit further comprises instructions to use the kit to select a cancer medicament (e.g. a c-met antagonist, such as an anti-c-met antibody) for treating the cancer patient if the patient expresses the c-met biomarker at a high level. In another embodiment, the instructions are to use the kit to select a cancer medicament other than c-met antagonist (or other than an anti-c-met antibody) if the patient expresses the biomarker at a reduced level. VII. METHODS OF ADVERTISING
The invention herein also concerns a method for advertising a cancer medicament comprising promoting, to a target audience, the use of the cancer medicament (e.g. anti-c-met antibody) for treating a patient with cancer based on expression of c-met biomarker as disclosed herein.
Advertising is generally paid communication through a non-personal medium in which the sponsor is identified and the message is controlled. Advertising for purposes herein includes publicity, public relations, product placement, sponsorship, underwriting, and sales promotion. This term also includes sponsored informational public notices appearing in any of the print communications media designed to appeal to a mass audience to persuade, inform, promote, motivate, or otherwise modify behavior toward a favorable pattern of purchasing, supporting, or approving the invention herein.
The advertising and promotion of the diagnostic method herein may be accomplished by any means. Examples of advertising media used to deliver these messages include television, radio, movies, magazines, newspapers, the internet, and billboards, including commercials, which are messages appearing in the broadcast media. Advertisements also include those on the seats of grocery carts, on the walls of an airport walkway, and on the sides of buses, or heard in telephone hold messages or in-store PA systems, or anywhere a visual or audible communication can be placed.
More specific examples of promotion or advertising means include television, radio, movies, the internet such as webcasts and webinars, interactive computer networks intended to reach simultaneous users, fixed or electronic billboards and other public signs, posters, traditional or electronic literature such as magazines and newspapers, other media outlets, presentations or individual contacts by, e.g., e-mail, phone, instant message, postal, courier, mass, or carrier mail, in-person visits, etc.
The type of advertising used will depend on many factors, for example, on the nature of the target audience to be reached, e.g., hospitals, insurance companies, clinics, doctors, nurses, and patients, as well as cost considerations and the relevant jurisdictional laws and regulations governing advertising of medicaments and diagnostics. The advertising may be individualized or customized based on user characterizations defined by service interaction and/or other data such as user demographics and geographical location. EXAMPLES
Examples
Onartuzumab is a monoclonal monovalent (one-armed) antibody specifically designed to inhibit HGF-stimulated activation of the Met signaling pathway. A recent phase II study (OAM4558g) was conducted to evaluate the activity of onartuzumab in combination with the EGFR pathway inhibitor, erlotinib, compared to erlotinib + placebo in patients with recurrent NSCLC. A tissue specimen was required for all patients enrolled on this study to evaluate Met expression by immunohistochemistry (IHC). Co-primary endpoints were progression-free survival (PFS) in the intent-to-treat (ITT) population and in the population of patients defined as Met-positive based upon the IHC test result, respectively. Tumors with >50% of tumor cells exhibiting moderate to strong staining intensity were pre-defined as Met positive, prior to unblinding the treatment assignment.
Whereas no improvement in either PFS or overall survival (OS) was observed in the overall (ITT) population treated with onartuzumab+erlotinib (n=137, PFS hazard ratio (HR): 1.09; P=0.69; OS HR: 0.80; P=0.34), a statistically significant improvement in both PFS and OS was observed in Met-positive patients (n=66, PFS HR: 0.53; P=0.04; OS HR: 0.37;
P=0.002). Conversely, clinical benefit was not observed in Met-negative patients treated with onartuzumab plus erlotinib (n=62, PFS HR: 1.82; P=0.05; OS HR: 1.78; P=0.16). In patients treated with erlotinib alone, Met-positive patients had a worse outcome compared to Met- negative patients (PFS HR: 1.71; P=0.06; OS HR: 2.61; P=0.004), consistent with the previously reported poor prognostic effect of Met expression. Patients randomized to erlotinib + placebo performed similarly to historic controls and sensitivity analyses of this study demonstrated that the clinical benefit was not exclusively driven by any biomarker subgroup examined, including EGFR mutant or MET gene amplified patients.
These results showed that the addition of onartuzumab to erlotinib nearly tripled the survival compared with placebo in Met-positive patients (median OS 12.6 months versus 3.8 months). The addition of onartuzumab to erlotinib treatment also resulted in a 47% reduction in the risk of disease progression that was statistically significant in the c-met overexpression group (median PFS 2.9 months vs. 1.5 months). In Met-negative patients, those treated with onartuzumab had worse survival outcomes than those on placebo (OS 8.1 months vs. 15.3 months; PFS 1.4 months vs. 2.7 months). Overall, these data provided early proof-of-concept that combined inhibition of Met and EGFR can result in a survival benefit, and more importantly, highlight the importance of integrating diagnostic tests in early clinical studies. See, e.g., co-owned PCT Patent Publication No. WO2012031027.
In the present work, we evaluated the H-score method of semi-quantitatively assessing Met expression as an alternative Met biomarker analysis method.
Materials and methods
Tissue specimens
OAM4558g, a phase II, double-blind, randomized (1 : 1), global study enrolled 137 patients with locally-advanced or metastatic NSCLC to compare the survival of patients who receive onartuzumab plus erlotinib (n=69) compared with placebo plus erlotinib (n=68). The primary efficacy endpoint was progression free survival (PFS) with PFS in the Met-positive population as co-primary endpoint. The other key endpoints included safety and overall survival (OS). Tumor tissue specimens in the form of a tissue block or unstained slides were an eligibility requirement. Evaluable specimens were obtained from 128 (93.4%) patients.
Cell Lines
Cell lines were obtained from American Type Culture Collection (Manassas, VA),
National Cancer Institute Division of Cancer Treatment and Diagnosis Tumor Repository, or Japan Health Sciences Foundation. Cell lines were grown in RPMI 1640 supplemented with 10% fetal bovine. FFPE cell pellets were produced by growing eight T175 flasks for each cell line, dislodging cells with 10 mmol/L EDTA (pH 8.0) followed by centrifugation, washing in PBS, and then fixation overnight in 10% neutral buffered formalin. Fixed pellets were processed in a Tissue-Tek processor (Sakura Finetek, CA, USA) and then embedded in a paraffin block prior to constructing a tissue microarray.
IHC
Met expression levels were evaluated by IHC analysis of archival tissue specimens using the CONFIRM anti-total cMet (SP44) rabbit monoclonal primary antibody (cat no. 790- 4430, Ventana Medical Systems, Tucson, AZ), according to the manufacturer's instructions. The staining was performed on the Ventana Benchmark XT instrument using CC1 standard antigen retrieval (Ventana Medical Systems, Tucson, AZ). Incubation with primary antibody was performed for 16 minutes at 37°C using a concentration of primary antibody of 9.75 μg/ml. Specifically-bound primary antibody was detected using ultra View methodology with diaminobenzidine (Ventana Medical Systems, Tucson, AZ); sections were counterstained with hematoxylin. Met IHC Scoring
The performance of anti-c-met antibody staining was examined on human lung tissue specimens. Similar to NSCLC cell lines, a range of staining intensities was detected in NSCLC tumor specimens that could be characterized as negative, weak, moderate or strong intensity (Figure 1 A). Heterogeneity in the staining intensity was frequently observed in individual NSCLC tumors (Figure IB and Figure 3). In benign lung tissue, staining was observed in the respiratory mucosa and reactive pneumocytes, often reaching a moderate staining intensity (Figure 1C). To account for the heterogeneity of Met expression seen in NSCLC tissue, a comprehensive clinical scoring system was developed. The scoring system evaluates both membranous and cytoplasmic staining intensity and percent cells staining at a given intensity level, as shown in Table A
Table A
Cell line Anti-c-met antibody (SP44)
IHC staining intensity
H1155 negative
LXFL529 negative
H522 negative
H23 negative
H661 negative
H460 weak
A549 moderate
SKMES1 moderate
H441 strong
EBC1 strong
A proportional cut-off of >50% was selected to ensure that a majority of the cells within a given specimen express Met at either a weak (clinical score=l+), moderate (clinical score=2+), or strong (clinical score=3+) intensity level. Specimens with no or equivocal staining in tumor cells or <50% of tumor cells staining at any given intensity (could be membrane and/or cytoplasmic staining) were considered negative (clinical score=0). NSCLC cases were classified as Met-positive (Met-diagnostic positive) or Met-negative (Met-diagnostic negative) according to the scoring scheme shown in Table B. See also
Koeppen et al, "Characterization and Clinical Validation of an Immunohistochemical Assay for Met in Non-Small Cell Lung Cancer", presented at United States and Canadian Academy of Pathology Annual Meeting, March 17-23, 2012, Vancouver Canada.
Table B
Diagnostic Soere Seeing Gsieria
a 50% <&imOf cells wit m smbrans arid/or c¾j1ap¾& ii-ik. sialm sg will's
3 +
I <ΐΐΐ H.'
'31
& 50% tumor with
2 +
moderate or ■i§ Mam !¾y bat « 50% tumor eel a wit %t;0 *¾g interssst a 50% tumor cells with m smbram ami&tf eytapla isYsie s!sini f¾g with
1 H mk or hsqhi sr intensity fc >ui 50% tumor cells wt Ji modern e or higher
M&rssi y
si
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z Bampto. m si&m ^ ar «50%· turner cells it membr we a^d or
0
,ta¾rs§ {COL Id be com fRSfso ©f m nterisi ies) H-score
In order to express MET IHC staining intensity in tumor samples in H-score format, the proportion of tumor cells showing staining at weak, moderate, and strong intensity was estimated as percentage of the total number of tumor cells in a given NSCLC case. A single intensity score was generated for each sample (i.e., both membranous and cytoplasmic staining were combined into a single intensity score). If staining of multiple intensities was present in a sample, the highest intensity staining of the sample was recorded as the sample intensity. The composite score was calculated based on the formula:
(% tumor cells staining at weak intensity xl) + (% tumor cells staining at moderate intensity x2) + (% tumor cells staining at strong intensity x3).
Tumor cells showing no staining for Met were not considered for this calculation. Following this formula, a given tumor can be associated with a value between "0" (none of the tumor cells show any staining) and "300" (100% of the tumor cells show strong staining).
Results
We observed a range of H-scores from 0 to 300, with a median H-score = 160 in the set of tissue samples collected from patients in OAM4558g. Most specimens within each clinical score (0, 1+, 2+, and 3+) were distributed around the H-scores of 0, 100, 200 and 300, respectively (Figures 2 and 3) as would be expected if staining was completely homogenous. When heterogeneity in staining intensity was present (n=64), positivity was primarily distributed amongst 2 different staining intensities (n=48). There were four specimens below the median H-score that were considered Met-positive when applying the clinical scoring algorithm; conversely, there was one specimen at the median H-score that was Met-negative by the clinical scoring system. Exploratory analysis did not show that H-score could alter the interpretation of the outcomes from previous analyses that used the scoring scheme shown in Table B.

Claims

WHAT IS CLAIMED IS:
1. A method for identifying a cancer patient who is likely to respond to treatment with a c-met antagonist comprising the step of determining an H-score from a sample of the patient's cancer using the formula (% cancer cells staining with weak c-met staining intensity) + (% cancer cells staining with moderate c-met staining intensity) + (% cancer cells staining with high c-met staining intensity), wherein an H-score of about 160 or higher, about 160 to about 230, about 230 or higher, or about 170 or higher indicates that the patient is likely to response to treatment with a c-met antagonist.
2. A method for determining cancer patient prognosis, comprising the step of determining an H-score from a sample of the patient's cancer using the formula (% cancer cells staining with weak c-met staining intensity) + (% cancer cells staining with moderate c-met staining intensity) + (% cancer cells staining with high c-met staining intensity), wherein an H-score of about 160 or higher, about 160 to about 230, about 230 or higher, or about 170 or higher indicates that the patient is likely to response to treatment with a c-met antagonist.
3. A method for identifying a cancer patient who is less likely to be respond to treatment with a c-met antagonist comprising the step of determining an H-score from a sample of the patient's cancer using the formula (% cancer cells staining with weak c-met staining intensity) + (% cancer cells staining with moderate c-met staining intensity) + (% cancer cells staining with high c-met staining intensity), wherein an H-score of less than about 160 indicates that the patient is less likely to respond to treatment with the c-met antagonist.
4. The method of any of claims 1-2, wherein the H-score is about 160 or higher.
5. The method of claim 4, wherein the H-score is 160 or higher.
6. The method of any of claims 1-2, wherein the H-score is about 160 to about 230.
7. The method of claim 6, wherein the H-score is 160-230.
8. The method of claim 3, wherein the H-score is less than 160.
9. The method of claim 3, wherein the H-score is less than 150.
10. The method of any of the preceding claims, wherein negative c-met staining intensity means c- met staining intensity of control cell line TOV-112D.
11. The method of any of claims 1-9, wherein negative c-met staining intensity means c-met staining intensity of control cell line H522.
12. The method of any of claims 1-9, wherein negative c-met staining intensity means c-met staining intensity of control cell line HI 155.
13. The method of any of claims 1-9, wherein negative c-met staining intensity refers to c-met staining intensity of control cell line LXFL529.
14. The method of any of claims 1-9, wherein negative c-met staining intensity means c-met staining intensity of control cell line H23.
15. The method of any of the preceding claims, wherein weak c-met staining intensity means c-met staining intensity of control cell line HI 703.
16. The method of any of claims 1-14, wherein weak c-met staining intensity means c-met staining intensity of control cell line HEK-293.
17. The method of any of the preceding claims, wherein moderate c-met staining intensity means c-met staining intensity of control cell line A549.
18. The method of any of claims 1-16, wherein moderate c-met staining intensity means c-met staining intensity of control cell line SKMES 1.
19. The method of any of the preceding claims, wherein strong c-met staining intensity means c- met staining intensity of control cell line EBC-1.
20. The method of any of claims 1-19, wherein strong c-met staining intensity means c-met staining intensity of control cell line H441.
21. The method of any of the preceding claims, wherein the sample is obtained prior to treatment with c-met antagonist.
22. The method of any of the preceding claims, wherein the sample is obtained prior to treatment with a cancer medicament.
23. The method of any of the preceding claims, wherein the sample is obtained after the cancer has metastasized.
24. The method of any of the preceding claims, wherein the sample is formalin fixed and paraffin embedded.
25. The method of any of the preceding claims, wherein the sample is of a biopsy, a surgical specimen, or a fine needle aspirate.
26. The method of any of the preceding claims, wherein control cell pellets are formalin fixed and paraffin embedded.
27. The method of claim 26, wherein the control cell pellets are prepared as a tissue microarray.
28. The method of any of the preceding claims, wherein the c-met IHC used to determine c-met staining intensity is performed using anti-c-met antibody SP44.
29. The method of any of claims 1-27, wherein the c-met IHC used to determine c-met staining intensity is performed using anti-c-met antibody D1C1.
30. The method of any of claims 1-27, wherein the c-met IHC used to determine c-met staining intensity is performed using anti-c-met antibody Met4.
31. The method of any of claims 1-27, wherein the c-met IHC used to determine c-met staining intensity is performed using anti-c-met antibody DL-21.
32. The method of any of the preceding claims, wherein the cancer is non-small cell lung cancer, renal cell cancer, pancreatic cancer, gastric carcinoma, bladder cancer, esophageal cancer, mesothelioma, melanoma, breast cancer, thyroid cancer, colorectal cancer, head and neck cancer, osteosarcoma, prostate cancer, or glioblastoma.
33. The method of claim 32, wherein the cancer is non-small cell lung cancer (NSCLC).
34. The method of claim 33, wherein the NSCLC is second-line or third-line locally advanced or metastatic non-small cell lung cancer.
35. The method of claim 33 or 34, wherein the NSCLC is adenocarcinoma.
36. The method of claim 33 or 34, wherein the NSCLC is squamous cell carcinoma.
37. The method of any of the preceding claims, wherein the c-met antagonist is an antagonist antic-met antibody.
38. The method of claim 37, wherein the anti-c-met antibody comprises a (a) HVR1 comprising sequence GYTFTSYWLH (SEQ ID NO: 1); (b) HVR2 comprising sequence
GMIDPSNSDTRFNPNFKD (SEQ ID NO: 2); (c) HVR3-HC comprising sequence
ATYRSYVTPLDY (SEQ ID NO: 3); (d) HVR1-LC comprising sequence
KSSQSLLYTSSQKNYLA (SEQ ID NO: 4); (e) HVR2-LC comprising sequence WASTRES (SEQ ID NO: 5); and (f) HVR3-LC comprising sequence QQYYAYPWT (SEQ ID NO: 6).
39. The method of claim 37, wherein the anti-c-met antibody is monovalent and comprises (a) a first polypeptide comprising a heavy chain, said polypeptide comprising the sequence:
EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWLHWVRQAPGKGLEWVGMIDPSNSDTR FNPNFKDRFTISADTSKNTAYLQMNSLRAEDTAVYYCATYRSYVTPLDYWGQGTLVTVS SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGP SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEM TKNQVSLSCAVKGFYPSDIAVEWESNGQPE NYKTTPPVLDSDGSFFLVSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 11); (b) a second polypeptide comprising a light chain, the polypeptide comprising the sequence
DIQMTQSPSSLSASVGDRVTITCKSSQSLLYTSSQKNYLAWYQQKPGKAPKLLIYWASTRE SGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYAYPWTFGQGTKVEIKRTVAAPSVFI FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSS TLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 12); and a third polypeptide comprising a Fc sequence, the polypeptide comprising the sequence
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA KGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (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 a complex and form a Fc region that increases stability of said antibody fragment compared to a Fab molecule comprising said antigen binding arm.
40. The method of any of claims 1-36, wherein the c-met antagonist is one or more of crizotinib, tivantinib, carbozantinib, MGCD-265, ficlatuzumab, humanized TAK-701, rilotumumab, foretinib, h224Gl l, 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 LA480.
41. The method of claim 40, wherein the c-met antagonist is rilotumumab.
42. The method of claim 40, wherein the c-met antagonist is crizotinib.
43. The method of claim 40, wherein the c-met antagonist is tivantinib.
44. The method of any of the preceding claims, wherein treatment is in combination with treatment with an EGFR antagonist.
45. The method of claim 44, wherein the EGFR antagonist is erlotinib or gefitinib.
46. The method of any of claims 1-44, wherein the c-met antagonist is onartuzumab and treatment further comprises treatment with erlotinib.
47. The method of any of claims 1-44, wherein the c-met antagonist is rilotumumab, crizotinib, tivantinib, carbozantinib, MGCD-265, ficlatuzumab, humanized TAK-701, or foretinib, and treatment further comprises treatment with erlotinib or gefitinib.
48. A method for treating a patient with cancer comprising administering a therapeutically effective amount of a c-met antagonist to the patient if the patient's cancer has been found to have an H-score of about 160 or higher, about 160 to about 230, about 230 or higher, or about 170 or higher, wherein the H-score is determined using the formula (% cancer cells staining with weak c- met staining intensity) + (% cancer cells staining with moderate c-met staining intensity) + (% cancer cells staining with high c-met staining intensity).
49. The method of claim 48, wherein the patient has greater PFS and/or OS (e.g., relative to a patient who has an H score less than 160).
50. The method of claim 49, wherein the patient has greater PFS (e.g., relative to a patient who has an H score less than 160).
51. The method of claim 49, wherein the patient has greater OS (e.g., relative to a patient who has an H score less than 160).
52. A method for treating a patient with cancer comprising administering a therapeutically effective amount of a medicament other than a c-met antagonist to the patient if the patient's cancer has been found to have an H-score of less than 160, wherein the H-score is determined using the formula (% cancer cells staining with weak c-met staining intensity) + (% cancer cells staining with moderate c-met staining intensity) + (% cancer cells staining with high c-met staining intensity).
53. The method of any of claims 48-51, wherein the H-score is about 160 or higher.
54. The method of any of claims 47-50, wherein the H-score is about 160 to about 230.
55. The method of claim 54, wherein the H-score is 160-230.
56. The method of claim 51, wherein the H-score is less than 160.
57. The method of claim 51, wherein the H-score is less than 150.
58. The method of any of claims 47-57, wherein negative c-met staining intensity means c-met staining intensity of control cell line TOV-112D.
59. The method of any of claims 47-57, wherein negative c-met staining intensity means c-met staining intensity of control cell line H522.
60. The method of any of claims 47-57, wherein negative c-met staining intensity means c-met staining intensity of control cell line HI 155.
61. The method of any of claims 47-57, wherein negative c-met staining intensity refers to c-met staining intensity of control cell line LXFL529.
62. The method of any of claims 47-57, wherein negative c-met staining intensity means c-met staining intensity of control cell line H23.
63. The method of any of claims 47-62, wherein weak c-met staining intensity means c-met staining intensity of control cell line HI 703.
64. The method of any of claims 47-62, wherein weak c-met staining intensity means c-met staining intensity of control cell line HEK-293.
65. The method of any of claims 47-64, wherein moderate c-met staining intensity means c-met staining intensity of control cell line A549.
66. The method of any of claims 47-64, wherein moderate c-met staining intensity means c-met staining intensity of control cell line SKMES 1.
67. The method of any of claims 47-66, wherein strong c-met staining intensity means c-met staining intensity of control cell line EBC-1.
68. The method of any of claims 47-66, wherein strong c-met staining intensity means c-met staining intensity of control cell line H441.
69. The method of any of claims 47-68, wherein the sample is obtained prior to treatment with c- met antagonist.
70. The method of any of claims 47-68, wherein the sample is obtained prior to treatment with a cancer medicament.
71. The method of any of claims 47-68, wherein the sample is obtained after the cancer has metastasized.
72. The method of any of claims 47-68, wherein the sample is formalin fixed and paraffin embedded.
73. The method of any of claims 47-68, wherein the sample is of a biopsy, a surgical specimen, or a fine needle aspirate.
74. The method of any of claims 47-68, wherein control cell pellets are formalin fixed and paraffin embedded.
75. The method of claim 74, wherein the control cell pellets are prepared as a tissue microarray.
76. The method of any of claims 47-75, wherein the c-met IHC used to determine c-met staining intensity is performed using anti-c-met antibody SP44.
77. The method of any of claims 47-75, wherein the c-met IHC used to determine c-met staining intensity is performed using anti-c-met antibody D1C1.
78. The method of any of claims 47-75, wherein the c-met IHC used to determine c-met staining intensity is performed using anti-c-met antibody Met4.
79. The method of any of claims 47-75, wherein the c-met IHC used to determine c-met staining intensity is performed using anti-c-met antibody DL-21.
80. The method of any of claims 47-79, wherein the cancer is non-small cell lung cancer, renal cell cancer, pancreatic cancer, gastric carcinoma, bladder cancer, esophageal cancer,
mesothelioma, melanoma, breast cancer, thyroid cancer, colorectal cancer, head and neck cancer, osteosarcoma, prostate cancer, or glioblastoma.
81. The method of claim 80, wherein the cancer is non-small cell lung cancer (NSCLC).
82. The method of claim 81, wherein the NSCLC is second-line or third-line locally advanced or metastatic non-small cell lung cancer.
83. The method of claim 81 or 82, wherein the NSCLC is adenocarcinoma.
84. The method of claim 81 or 82, wherein the NSCLC is squamous cell carcinoma.
85. The method of any of claims 47-84, wherein the c-met antagonist is an antagonist anti-c-met antibody.
86. The method of claim 85, wherein the anti-c-met antibody comprises a (a) HVR1 comprising sequence GYTFTSYWLH (SEQ ID NO: 1); (b) HVR2 comprising sequence
GMIDPSNSDTRFNPNFKD (SEQ ID NO: 2); (c) HVR3-HC comprising sequence
ATYRSYVTPLDY (SEQ ID NO: 3); (d) HVR1-LC comprising sequence
KSSQSLLYTSSQKNYLA (SEQ ID NO: 4); (e) HVR2-LC comprising sequence WASTRES (SEQ ID NO: 5); and (f) HVR3-LC comprising sequence QQYYAYPWT (SEQ ID NO: 6).
87. The method of claim 85, wherein the anti-c-met antibody is monovalent and comprises (a) a first polypeptide comprising a heavy chain, said polypeptide comprising the sequence:
EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWLHWVRQAPGKGLEWVGMIDPSNSDTR FNPNFKDRFTISADTSKNTAYLQMNSLRAEDTAVYYCATYRSYVTPLDYWGQGTLVTVS SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGP SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEM TKNQVSLSCAVKGFYPSDIAVEWESNGQPE NYKTTPPVLDSDGSFFLVSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 11); (b) a second polypeptide comprising a light chain, the polypeptide comprising the sequence
DIQMTQSPSSLSASVGDRVTITCKSSQSLLYTSSQKNYLAWYQQKPGKAPKLLIYWASTRE SGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYAYPWTFGQGTKVEIKRTVAAPSVFI FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSS TLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 12); and a third polypeptide comprising a Fc sequence, the polypeptide comprising the sequence
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA KGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (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 a complex and form a Fc region that increases stability of said antibody fragment compared to a Fab molecule comprising said antigen binding arm.
88. The method of any of claims 47-84, wherein the c-met antagonist is one or more of crizotinib, tivantinib, carbozantinib, MGCD-265, ficlatuzumab, humanized TAK-701, rilotumumab, foretinib, h224Gl l, 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 LA480.
89. The method of claim 88, wherein the c-met antagonist is rilotumumab.
90. The method of claim 88, wherein the c-met antagonist is crizotinib.
91. The method of claim 88, wherein the c-met antagonist is tivantinib.
92. The method of any of the preceding claims, wherein treatment is in combination with treatment with an EGFR antagonist.
93. The method of claim 92, wherein the EGFR antagonist is erlotinib.
94. The method of any of claims 47-85, wherein the c-met antagonist is onartuzumab and treatment further comprises treatment with erlotinib.
95. The method of any of claims 47-84, wherein the c-met antagonist is crizotinib, tivantinib, carbozantinib, MGCD-265, ficlatuzumab, humanized TAK-701, or foretinib, and treatment further comprises treatment with erlotinib.
96. The method of any of claims 47-80, wherein the patient has NSCLC and is treated with a combination of anti-c-met antibody and an EGFR antagonist.
97. The method of claim 96, wherein the EGFR antagonist is erlotinib.
98. The method of claim 97, wherein the patient has NSCLC and is treated with (a) onartuzumab at a dose of about 15 mg/kg every three weeks; and (b) erlotinib (N-(3-ethynylphenyl)-6,7-bis(2- methoxyethoxy)-4-quinazolinamine HC1) at a dose of 150 mg, each day of a three week cycle.
99. A method for selecting a therapy for a patient with cancer comprising determining an H-score from a sample of the patient's cancer using the formula (% cancer cells staining with weak c-met staining intensity) + (% cancer cells staining with moderate c-met staining intensity) + (% cancer cells staining with high c-met staining intensity), and selecting a cancer medicament based on the H-score.
100. The method of claim 99 wherein the patient is selected for treatment with a c-met antagonist if the H-score is about 160 or higher, about 160 to about 230, about 230 or higher, or about 170 or higher.
101. The method of claim 99, wherein the patient is selected for treatment with a cancer medicament other than a c-met antagonist if the H-score is lower than 160.
102. The method of claim 99, wherein the patient is selected for treatment with a cancer medicament other than a c-met antagonist if the H-score is lower than 159, 158, 157, 156, 155, 154, 153, 152, 151, or 150.
103. A method for advertising a c-met antibody comprising promoting, to a target audience, the use of the c-met antibody for treating a patient with cancer based on an H-score from a sample of the patient's cancer, wherein the H-score is calculated using the formula (% cancer cells staining with weak c-met staining intensity) + (% cancer cells staining with moderate c-met staining intensity) + (% cancer cells staining with high c-met staining intensity).
104. The method of claim 103, wherein the promotion is by a package insert accompanying a commercial formulation of an anti-c-met antibody.
105. The method of claim 103, wherein the promotion is by a package insert accompanying a commercial formulation of a second medicament.
106. The method of claims 103, wherein the promotion is by a package insert accompanying a c- met antibody suitable for use in c-met IHC assay.
107. The method of claim 105, wherein the second medicament is an EGFR antagonist.
108. The method of claim 103, wherein the anti-c-met antibody is Onartuzumab and the EGFR antagonist is erlotinib.
109. The method of claim 108, wherein the patient is selected for treatment with a c-met antagonist if the H-score is about 160 or higher, about 160 to about 230, about 230 or higher, or about 170 or higher..
110. The method of claim 108,wherein the patient is treated with the c-met antibody if the H-score is about 160 or higher, about 160 to about 230, about 230 or higher, or about 170 or higher.
111. The method of claim 110, wherein the promotion is by a package insert where the package insert provides instructions to receive therapy with anti-c-met antibody in combination with an EGFR antagonist.
112. The method of claim 108, wherein the promotion is followed by the treatment of the patient with the anti-c-met antibody with or without the second medicament.
113. A diagnostic kit comprising one or more reagent for determining an H-score from a sample of the patient's cancer using the formula (% cancer cells staining with weak c-met staining intensity) + (% cancer cells staining with moderate c-met staining intensity) + (% cancer cells staining with high c-met staining intensity), wherein an H-score of about 160 or higher, about 160 to about 230, about 230 or higher, or about 170 or higher indicates that the patient is likely to response to treatment with a c-met antagonist.
114. A diagnostic kit comprising one or more reagent for determining an H-score from a sample of the patient's cancer using the formula (% cancer cells staining with weak c-met staining intensity) + (% cancer cells staining with moderate c-met staining intensity) + (% cancer cells staining with high c-met staining intensity), wherein an H-score of about 160 or higher, about 160 to about 230, about 230 or higher, or about 170 or higher means the patient has greater PFS and/or OS (e.g., relative to a patient who has an H score less than 160).
115. The method of claim 114, wherein the patient has greater PFS (e.g., relative to a patient who has an H score less than 160).
116. The method of claim 114, wherein the patient has greater OS (e.g., relative to a patient who has an H score less than 160).
117. The diagnostic kit of any of claims 113-116, further comprising instructions to use the kit to select a c-met medicament to treat the NSCLC patient if the H-score is about 160 or higher, about 160 to about 230, about 230 or higher, or about 170 or higher.
118. A method for determining c-met biomarker expression, comprising determining an H-score from a sample of the patient's cancer using the formula (% cancer cells staining with weak c-met staining intensity) + (% cancer cells staining with moderate c-met staining intensity) + (% cancer cells staining with high c-met staining intensity).
119. The method of claim 118, wherein an H-score of about 160 or higher, about 160 to about 230, about 230 or higher, or about 170 or higher indicates that the patient is likely to response to treatment with a c-met antagonist.
120. The method of claim 118, wherein an H-score of less than about 160 indicates that the patient is less likely to respond to treatment with the c-met antagonist.
121. The method of any of claims 118-119, wherein the H-score is about 160 or higher.
122. The method of claim 121, wherein the H-score is 160 or higher.
123. The method of any of claims 118-119, wherein the H-score is about 160 to about 230.
124. The method of claim 123, wherein the H-score is 160-230.
125. The method of claim 121, wherein the H-score is less than 160.
125. The method of claim 121, wherein the H-score is less than 150.
126. The method of any of claims 118-125, wherein negative c-met staining intensity means c-met staining intensity of control cell line TOV-112D.
127. The method of any of claims 118-125, wherein negative c-met staining intensity means c-met staining intensity of control cell line H522.
128. The method of any of claims 118-125, wherein negative c-met staining intensity means c-met staining intensity of control cell line HI 155.
129. The method of any of claims 118-125, wherein negative c-met staining intensity refers to c- met staining intensity of control cell line LXFL529.
130. The method of any of claims 118-125, wherein negative c-met staining intensity means c-met staining intensity of control cell line H23.
131. The method of any of claims 118-130, wherein weak c-met staining intensity means c-met staining intensity of control cell line HI 703.
132. The method of any of claims 118-130, wherein weak c-met staining intensity means c-met staining intensity of control cell line HEK-293.
133. The method of any of claims 118-132, wherein moderate c-met staining intensity means c- met staining intensity of control cell line A549.
134. The method of any of claims 118-132, wherein moderate c-met staining intensity means c- met staining intensity of control cell line SKMES1.
135. The method of any of claims 118-134, wherein strong c-met staining intensity means c-met staining intensity of control cell line EBC-1.
136. The method of any of claims 118-134, wherein strong c-met staining intensity means c-met staining intensity of control cell line H441.
137. The method of any of claims 118-136, wherein the sample is obtained prior to treatment with c-met antagonist.
138. The method of any of claims 118-136, wherein the sample is obtained prior to treatment with a cancer medicament.
139. The method of any of claims 118-136, wherein the sample is obtained after the cancer has metastasized.
140. The method of any of claims 118-136, wherein the sample is formalin fixed and paraffin embedded.
141. The method of any of claims 118-136, wherein the sample is of a biopsy, a surgical specimen, or a fine needle aspirate.
142. The method of any of claims 118-141, wherein control cell pellets are formalin fixed and paraffin embedded.
143. The method of claim 142, wherein the control cell pellets are prepared as a tissue microarray.
144. The method of any of claims 118-143, wherein the c-met IHC used to determine c-met staining intensity is performed using anti-c-met antibody SP44.
145. The method of any of claims 118-143, wherein the c-met IHC used to determine c-met staining intensity is performed using anti-c-met antibody D1C1.
146. The method of any of claims 118-143, wherein the c-met IHC used to determine c-met staining intensity is performed using anti-c-met antibody Met4.
147. The method of any of claims 118-143, wherein the c-met IHC used to determine c-met staining intensity is performed using anti-c-met antibody DL-21.
148. The method of any of claims 118-147, wherein the cancer is non-small cell lung cancer, renal cell cancer, pancreatic cancer, gastric carcinoma, bladder cancer, esophageal cancer,
mesothelioma, melanoma, breast cancer, thyroid cancer, colorectal cancer, head and neck cancer, osteosarcoma, prostate cancer, or glioblastoma.
149. The method of claim 148, wherein the cancer is non-small cell lung cancer (NSCLC).
150. The method of claim 149, wherein the NSCLC is second-line or third-line locally advanced or metastatic non-small cell lung cancer.
151. The method of claim 149 or 150, wherein the NSCLC is adenocarcinoma.
152. The method of claim 149 or 150, wherein the NSCLC is squamous cell carcinoma.
153. The method of any of claims 118-152, wherein the c-met antagonist is an antagonist anti-c- met antibody.
154. The method of claim 153, wherein the anti-c-met antibody comprises a (a) HVR1 comprising sequence GYTFTSYWLH (SEQ ID NO: 1); (b) HVR2 comprising sequence
GMIDPSNSDTRFNPNFKD (SEQ ID NO: 2); (c) HVR3-HC comprising sequence
ATYRSYVTPLDY (SEQ ID NO: 3); (d) HVR1-LC comprising sequence
KSSQSLLYTSSQKNYLA (SEQ ID NO: 4); (e) HVR2-LC comprising sequence WASTRES (SEQ ID NO: 5); and (f) HVR3-LC comprising sequence QQYYAYPWT (SEQ ID NO: 6).
155. The method of claim 153, wherein the anti-c-met antibody is monovalent and comprises (a) a first polypeptide comprising a heavy chain, said polypeptide comprising the sequence:
EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWLHWVRQAPGKGLEWVGMIDPSNSDTR FNPNFKDRFTISADTSKNTAYLQMNSLRAEDTAVYYCATYRSYVTPLDYWGQGTLVTVS SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGP SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEM TKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 11); (b) a second polypeptide comprising a light chain, the polypeptide comprising the sequence
DIQMTQSPSSLSASVGDRVTITCKSSQSLLYTSSQKNYLAWYQQKPGKAPKLLIYWASTRE SGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYAYPWTFGQGTKVEIKRTVAAPSVFI FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSS TLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 12); and a third polypeptide comprising a Fc sequence, the polypeptide comprising the sequence
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA KGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (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 a complex and form a Fc region that increases stability of said antibody fragment compared to a Fab molecule comprising said antigen binding arm.
156. The method of any of claims 118-151, wherein the c-met antagonist is one or more of crizotinib, tivantinib, carbozantinib, MGCD-265, ficlatuzumab, humanized TAK-701,
rilotumumab, foretinib, h224Gl l, 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 LA480.
157. The method of claim 156, wherein the c-met antagonist is rilotumumab.
158. The method of claim 156, wherein the c-met antagonist is crizotinib.
159. The method of claim 156, wherein the c-met antagonist is tivantinib.
160. The method of any of claims 118-159, wherein treatment is in combination with treatment with an EGFR antagonist.
161. The method of claim 160, wherein the EGFR antagonist is erlotinib.
162. The method of any of claims 118-160, wherein the c-met antagonist is onartuzumab and treatment further comprises treatment with erlotinib.
163. The method of any of claims 118-160, wherein the c-met antagonist is rilotumumab, crizotinib, tivantinib, carbozantinib, MGCD-265, ficlatuzumab, humanized TAK-701, or foretinib, and treatment further comprises treatment with erlotinib.
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