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WO2016201199A1 - Identification de biomarqueurs prédictifs associés à des inhibiteurs de la voie wnt - Google Patents

Identification de biomarqueurs prédictifs associés à des inhibiteurs de la voie wnt Download PDF

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WO2016201199A1
WO2016201199A1 PCT/US2016/036850 US2016036850W WO2016201199A1 WO 2016201199 A1 WO2016201199 A1 WO 2016201199A1 US 2016036850 W US2016036850 W US 2016036850W WO 2016201199 A1 WO2016201199 A1 WO 2016201199A1
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antibody
wnt pathway
pathway inhibitor
patient
sample
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Chun Zhang
Ann M. Kapoun
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Oncomed Pharmaceuticals Inc
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Oncomed Pharmaceuticals Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present invention relates to the field of cancer treatment. More particularly, the invention provides methods for identifying tumors that are likely to be responsive or non-responsive to treatment with a Wnt pathway inhibitor. In addition, the invention provides methods for identifying, selecting, and/or treating patients with cancer who are likely to respond to treatment with a Wnt pathway inhibitor, either alone or in combination with other therapeutic agents. BACKGROUND OF THE INVENTION
  • Cancer is one of the leading causes of death in the developed world, with approximately 1.6 million people diagnosed with cancer and over 550,000 deaths per year in the United States alone. Overall it is estimated that more than 1 in 3 people will develop some form of cancer during their lifetime. There are more than 200 different types of cancer, four of which - breast, lung, colorectal, and prostate— account for almost half of all new cases in the United States (Siegel et al., 2012, CA: A Cancer J. for Clin., 62:10-29).
  • Signaling pathways normally connect extracellular signals to the nucleus leading to the expression of genes that directly or indirectly control cell growth, differentiation, survival, and/or death.
  • signaling pathways are dysregulated and may be linked to tumor initiation and/or progression.
  • Signaling pathways implicated in human oncogenesis include, but are not limited to, the Wnt pathway, the Ras-Raf-MEK-ERK or MAPK pathway, the PI3K-AKT pathway, the CDKN2A/CDK4 pathway, the Bcl-2/TP53 pathway, and the NOTCH pathway.
  • the Wnt signaling pathway is one of several critical regulators of embryonic pattern formation, post-embryonic tissue maintenance, and stem cell biology. More specifically, Wnt signaling plays an important role in the generation of cell polarity and cell fate specification including self-renewal by stem cell populations. Unregulated activation of the Wnt pathway is associated with numerous human cancers where it is believed the activation can alter the developmental fate of cells. It is believed that the activation of the Wnt pathway may maintain tumor cells in an undifferentiated state and/or lead to uncontrolled proliferation.
  • the Wnt signaling pathway was first elucidated in the Drosophila developmental mutant wingless (wg) and from the murine proto-oncogene int-1, now Wntl (Nusse & Varmus, 1982, Cell, 31:99-109; Van Ooyen & Nusse, 1984, Cell, 39:233-40; Cabrera et al., 1987, Cell, 50:659-63;
  • Wnt genes encode lipid-modified glycoproteins which are secreted and 19 different Wnt proteins have been identified in mammals. These secreted ligands activate a receptor complex consisting of a Frizzled (FZD) receptor family member and low-density lipoprotein (LDL) receptor-related protein 5 or 6 (LRP5/6).
  • FZD Frizzled
  • LDL low-density lipoprotein
  • the FZD receptors are members of the G-protein coupled receptor (GPCR) superfamily and contain seven transmembrane domains and a large extracellular N-terminal ligand binding domain.
  • the N-terminal ligand binding domain contains 10 conserved cysteines and is known as a cysteine-rich domain (CRD) or a "Fri domain".
  • CRD cysteine-rich domain
  • Different FZD CRDs have different binding affinities for specific Wnt proteins (Wu & Nusse, 2002, J. Biol. Chem., 277:41762-9).
  • FZD receptors may be grouped into those that activate the canonical ⁇ -catenin pathway and those that activate non-canonical pathways (Miller et al., 1999, Oncogene, 18:7860-72).
  • Neoplasia 8: 145-58.
  • ⁇ -catenin accumulation implicates activated Wnt signaling in over 50% of carcinomas, and though specific mutations have not been identified, up- regulation of Frizzled receptor expression has been observed (Brennan & Brown, 2004, J. Mammary Gland Biol. Neoplasia, 9: 119-31; Malovanovic et al., 2004, Int. J. Oncol., 25: 1337-42).
  • Activation of the Wnt pathway is also associated with colorectal cancer, lung cancer, pancreatic cancer, and melanoma.
  • Personalized medicine strategies may include treatment regimens that are based upon cancer biomarkers, including prognostic biomarkers, pharmacodynamic biomarkers, and predictive biomarkers.
  • predictive biomarkers assess the likelihood that a tumor or cancer will be responsive to or sensitive to a specific therapeutic agent or a combination of therapeutic agents, and may allow for the identification and/or the selection of patients most likely to benefit from the use of that agent or agents.
  • the invention provides the identification of predictive biomarkers associated with the use of Wnt pathway inhibitors in the treatment of cancer. Also provided are methods of using the predictive biomarkers for identifying, selecting, and/or classifying tumors and/or patients with cancer as likely to be responsive or non-responsive to treatment with a Wnt pathway inhibitor. Methods for treating patients that are predicted to be responsive to treatment with a Wnt pathway inhibitor are also provided.
  • ⁇ олованн ⁇ е ⁇ о ⁇ исок ⁇ олованн ⁇ е ⁇ оло ⁇ овки ⁇ ⁇ оловки ⁇ ⁇ о ⁇ оловки ⁇ ⁇ олованн ⁇ е ⁇ о ⁇ оловки ⁇ ⁇ оловки ⁇ ⁇ о ⁇ олованн ⁇ е ⁇ о ⁇ оловки ⁇ ⁇ оло ⁇ ованн ⁇ е ⁇ о ⁇ оловки ⁇ ⁇ елениел ⁇ ⁇ е ⁇ е ⁇ е ⁇ е ⁇ е ⁇ е ⁇ е ⁇ а ⁇ о ⁇ и ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇
  • pancreatic tumor or “pancreatic cancer” includes exocrine tumors/cancers and endocrine tumors/cancers, including but not limited to, pancreatic ductal adenocarcinomas (PDA), pancreatic cystic tumors, cancer of the acinar cells, and pancreatic neuroendocrine tumors (PNET).
  • PDA pancreatic ductal adenocarcinomas
  • PNET pancreatic neuroendocrine tumors
  • the invention provides a method of identifying a human pancreatic tumor that is likely to be responsive or non-responsive to treatment with a Wnt pathway inhibitor, the method comprising: (a) obtaining a sample of the human pancreatic tumor; (b) measuring the expression level of each biomarker of a biomarker signature in the sample, wherein the biomarker signature comprises one, two, or three of the biomarkers smoothened (SMO), insulin-like growth factor 2 (IGF2), and transforming growth factor beta-3 (TGFB3); and (c) identifying the tumor as likely to be responsive or non-responsive to treatment based upon the expression level of the biomarkers.
  • the invention provides a method of identifying a human pancreatic tumor that is likely to be responsive or non-responsive to treatment with a Wnt pathway inhibitor, the method comprising:
  • biomarker signature comprises one, two, or three of the biomarkers SMO, IGF2, and TGFB3;
  • the method comprises identifying a human pancreatic tumor that is likely to be responsive or non-responsive to treatment with a Wnt pathway inhibitor in combination with gemcitabine. In some embodiments, the method comprises identifying a human pancreatic tumor that is likely to be responsive or non-responsive to treatment with a Wnt pathway inhibitor in combination with gemcitabine and nab-paclitaxel (e.g., ABRAXANE).
  • nab-paclitaxel e.g., ABRAXANE
  • the invention provides a method of classifying a human pancreatic tumor as likely to be responsive or non-responsive to treatment with a Wnt pathway inhibitor, the method comprising: (a) obtaining a sample of the human pancreatic tumor; (b) measuring the expression level of each biomarker of a biomarker signature in the sample, wherein the biomarker signature comprises one, two, or three of the biomarkers SMO, IGF2, and TGFB3; and (c) classifying the pancreatic tumor as likely to be responsive or non-responsive to treatment based upon the expression level of the biomarkers.
  • the invention provides a method of classifying a human pancreatic tumor as likely to be responsive or non-responsive to treatment with a Wnt pathway inhibitor, the method comprising: (a) measuring the expression level of each biomarker of a biomarker signature in a sample, wherein the biomarker signature comprises one, two, or three of the biomarkers SMO, IGF2, and TGFB3; and (b) classifying the pancreatic tumor as likely to be responsive or non-responsive to treatment based upon the expression level of the biomarkers in the sample.
  • the method comprises classifying a human pancreatic tumor as likely to be responsive or non-responsive to treatment with a Wnt pathway inhibitor in combination with gemcitabine.
  • the method comprises classifying a human pancreatic tumor that is likely to be responsive or non-responsive to treatment with a Wnt pathway inhibitor in combination with gemcitabine and nab-paclitaxel (e.g., ABRAXANE).
  • a Wnt pathway inhibitor in combination with gemcitabine and nab-paclitaxel (e.g., ABRAXANE).
  • the invention provides a method of determining the responsiveness (or sensitivity) of a human pancreatic tumor to treatment with a Wnt pathway inhibitor, the method comprising: (a) obtaining a sample of the human pancreatic tumor; (b) measuring the expression level of each biomarker of a biomarker signature in the sample, wherein the biomarker signature comprises one, two, or three of the biomarkers SMO, IGF2, and TGFB3; and (c) determining the responsiveness of the pancreatic tumor to treatment based upon the expression level of the biomarkers.
  • the invention provides a method of determining the responsiveness (or sensitivity) of a human pancreatic tumor to treatment with a Wnt pathway inhibitor, the method comprising: (a) measuring the expression level of each biomarker of a biomarker signature in a sample, wherein the biomarker signature comprises one, two, or three of the biomarkers SMO, IGF2, and TGFB3; and (b) determining the responsiveness of the pancreatic tumor to treatment based upon the expression level of the biomarkers in the sample.
  • the method comprises determining the responsiveness or sensitivity of a human pancreatic tumor to treatment with a Wnt pathway inhibitor in combination with gemcitabine.
  • the method comprises determining the responsiveness or sensitivity of a human pancreatic tumor to treatment with a Wnt pathway inhibitor in combination with gemcitabine and nab-paclitaxel (e.g., ABRAXANE).
  • the invention provides a method of identifying a patient with pancreatic cancer who is likely to respond to treatment with a Wnt pathway inhibitor, the method comprising: (a) obtaining a sample of the patient's pancreatic tumor; (b) measuring the expression level of each biomarker of a biomarker signature in the sample, wherein the biomarker signature comprises one, two, or three of the biomarkers SMO, IGF2, and TGFB3; and (c) identifying the patient who is likely to respond to treatment based upon the expression level of the biomarkers.
  • the invention provides a method of identifying a patient with pancreatic cancer who is likely to respond to treatment with a Wnt pathway inhibitor, the method comprising: (a) measuring the expression level of each biomarker of a biomarker signature in a sample, wherein the biomarker signature comprises one, two, or three of the biomarkers SMO, IGF2, and TGFB3; and (b) identifying the patient who is likely to respond to treatment based upon the expression level of the biomarkers in the sample.
  • the method comprises identifying a patient with pancreatic cancer who is likely to respond to treatment with a Wnt pathway inhibitor in combination with gemcitabine.
  • the method comprises identifying a patient with pancreatic cancer who is likely to respond to treatment with a Wnt pathway inhibitor in combination with gemcitabine and nab- paclitaxel (e.g., ABRAXANE).
  • the invention provides a method of selecting a patient with pancreatic cancer for treatment with a Wnt pathway inhibitor, the method comprising: (a) obtaining a sample of the patient's pancreatic tumor; (b) measuring the expression level of each biomarker of a biomarker signature in the sample, wherein the biomarker signature comprises one, two, or three of the biomarkers SMO, IGF2, and TGFB3; and (c) selecting the patient for treatment based upon the expression level of the biomarkers.
  • the invention provides a method of selecting a patient with pancreatic cancer for treatment with a Wnt pathway inhibitor, the method comprising: (a) measuring the expression level of each biomarker of a biomarker signature in a sample, wherein the biomarker signature comprises one, two, or three of the biomarkers SMO, IGF2, and TGFB3; and (b) selecting the patient for treatment based upon the expression level of the biomarkers in the sample.
  • the method comprises selecting a patient with pancreatic cancer for treatment with a Wnt pathway inhibitor in combination with gemcitabine.
  • the method comprises selecting a patient with pancreatic cancer for treatment with a Wnt pathway inhibitor in combination with gemcitabine and nab-paclitaxel (e.g.,
  • the invention provides a method of treating pancreatic cancer in a patient, comprising: (a) identifying if the patient is likely to respond to treatment with a Wnt pathway inhibitor, wherein the identification comprises: (i) obtaining a sample of the patient's pancreatic cancer; (ii) measuring the expression level of each biomarker of a biomarker signature in the sample, wherein the biomarker signature comprises one, two, or three of the biomarkers SMO, IGF2, and TGFB3; and (iii) identifying the patient who is likely to respond to treatment based upon the expression level of the biomarkers; and (b) administering a therapeutically effective amount of a Wnt pathway inhibitor to the patient who is likely to response to treatment.
  • the invention provides a method of treating pancreatic cancer in a patient, comprising: (a) identifying if the patient is likely to respond to treatment with a Wnt pathway inhibitor, wherein the identification comprises: (i) measuring the expression level of each biomarker of a biomarker signature in a sample, wherein the biomarker signature comprises one, two, or three of the biomarkers SMO, IGF2, and TGFB3; and (ii) identifying the patient who is likely to respond to treatment based upon the expression level of the biomarkers in the sample; and (b) administering a therapeutically effective amount of a Wnt pathway inhibitor to the patient who is likely to response to treatment.
  • the method comprises administering to the patient the Wnt pathway inhibitor in combination with gemcitabine. In some embodiments, the method comprises administering to the patient the Wnt pathway inhibitor in combination with gemcitabine and nab-paclitaxel (e.g.,
  • the invention provides a method of treating pancreatic cancer in a patient, comprising: administering a therapeutically effective amount of a Wnt pathway inhibitor to the patient; wherein the patient is predicted to respond to treatment with the Wnt pathway inhibitor based upon expression levels of a biomarker signature in a sample of the patient's pancreatic cancer, wherein the signature comprises one, two, or three of the biomarkers SMO, IGF2, and TGFB3.
  • the patient is predicted to respond to treatment with a Wnt pathway inhibitor in combination with gemcitabine.
  • the method comprises administering to the patient the Wnt pathway inhibitor in combination with gemcitabine.
  • the patient is predicted to respond to treatment with a Wnt pathway inhibitor in combination with gemcitabine and nab-paclitaxel.
  • the method comprises administering to the patient the Wnt pathway inhibitor in combination with gemcitabine and nab-paclitaxel (e.g.,
  • the invention provides a method for increasing the likelihood of effective treatment with a Wnt pathway inhibitor, comprising: (a) identifying if a patient has a pancreatic tumor that is likely to respond to treatment with a Wnt pathway inhibitor, wherein the identification comprises: (i) obtaining a sample of the patient's pancreatic tumor; (ii) measuring the expression level of each biomarker of a biomarker signature in the sample, wherein the biomarker signature comprises one, two, or three of the biomarkers SMO, IGF2, and TGFB3; and (iii) identifying the patient who is likely to respond to treatment based upon the expression level of the biomarkers; and (b) administering a therapeutically effective amount of the Wnt pathway inhibitor to the patient.
  • the invention provides a method for increasing the likelihood of effective treatment with a Wnt pathway inhibitor, comprising: (a) identifying if a patient has a pancreatic tumor that is likely to respond to treatment with a Wnt pathway inhibitor, wherein the identification comprises: (i) measuring the expression level of each biomarker of a biomarker signature in a sample, wherein the biomarker signature comprises one, two, or three of the biomarkers SMO, IGF2, and TGFB3; and (iii) identifying the patient who is likely to respond to treatment based upon the expression level of the biomarkers in the sample; and (b) administering a therapeutically effective amount of the Wnt pathway inhibitor to the patient.
  • the method comprises identifying if a patient has a tumor that is likely to respond to treatment with a Wnt pathway inhibitor in combination with gemcitabine. In some embodiments, the method comprises administering to the patient the Wnt pathway inhibitor in combination with gemcitabine. In some embodiments, the method comprises identifying if a patient has a tumor that is likely to respond to treatment with a Wnt pathway inhibitor in combination with gemcitabine and nab-paclitaxel. In some embodiments, the method comprises administering to the patient the Wnt pathway inhibitor in combination with gemcitabine and nab- paclitaxel (e.g., ABRAXANE).
  • nab- paclitaxel e.g., ABRAXANE
  • the invention provides a method for increasing the likelihood of effective treatment with a Wnt pathway inhibitor, comprising: administering a therapeutically effective amount of a Wnt pathway inhibitor to a patient; wherein the patient is identified as likely to respond to treatment with the Wnt pathway inhibitor based upon expression levels of a biomarker signature in a sample of the patient's pancreatic tumor, wherein the signature comprises one, two, or three of the biomarkers SMO, IGF2, and TGFB3.
  • the patient is identified as likely to respond to treatment with a Wnt pathway inhibitor in combination with gemcitabine.
  • the method comprises administering to the patient the Wnt pathway inhibitor in combination with gemcitabine.
  • the patient is identified as likely to respond to treatment with a Wnt pathway inhibitor in combination with gemcitabine and nab-paclitaxel.
  • the method comprises administering to the patient the Wnt pathway inhibitor in combination with gemcitabine and nab-paclitaxel (e.g., ABRAXANE).
  • the biomarker signature comprises one, two, or three of the biomarkers SMO, IGF2, and TGFB3.
  • the Wnt pathway inhibitor is an antibody.
  • the Wnt pathway inhibitor is an antibody that specifically binds at least one human Frizzled (FZD) protein or fragment thereof.
  • the Wnt pathway inhibitor is an antibody that specifically binds at least one FZD protein selected from the group consisting of: FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD 8, FZD9, and FZD10.
  • the Wnt pathway inhibitor is an antibody that specifically binds at least one FZD protein selected from the group consisting of: FZD1, FZD2, FZD5, FZD7, and FZD8. In some embodiments, the Wnt pathway inhibitor is an antibody that specifically binds FZD1, FZD2, FZD5, FZD7, and FZD8. In certain embodiments, the Wnt pathway inhibitor is an antibody which comprises: (a) a heavy chain CDR1 comprising GFTFSHYTLS (SEQ ID NO:l), a heavy chain CDR2 comprising
  • VISGDGSYTYYADSVKG SEQ ID NO:2
  • a heavy chain CDR3 comprising NFIKYVFAN
  • a light chain CDR1 comprising SGDNIGSFYVH (SEQ ID NO:4)
  • a light chain CDR2 comprising DKSNRPSG (SEQ ID NO:5)
  • a light chain CDR3 comprising
  • the FZD-binding agent comprises a heavy chain variable region having at least about 85%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% sequence identity to SEQ ID NO: 7.
  • the FZD-binding agent comprises a light chain variable region having at least about 85%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% sequence identity to SEQ ID NO: 8.
  • the Wnt pathway inhibitor is an antibody which comprises a heavy chain variable region comprising SEQ ID NO: 7 and a light chain variable region comprising SEQ ID NO: 8.
  • the Wnt pathway inhibitor is an antibody which comprises a heavy chain variable region and a light chain variable region encoded by the plasmid deposited with ATCC as PTA-9541. In certain embodiments, the Wnt pathway inhibitor is an antibody which comprises a heavy chain and a light chain encoded by the plasmid deposited with ATCC as PTA-9541. In some embodiments, the Wnt pathway inhibitor is OMP-18R5 or 18R5 (also known as vantictumab).
  • the method further comprises administering at least one additional therapeutic agent to the patient.
  • the additional therapeutic agent is a chemotherapeutic agent.
  • the additional therapeutic agent is gemcitabine.
  • the additional therapeutic agents are gemcitabine and nab-paclitaxel.
  • the additional therapeutic agents are gemcitabine and ABRAXANE.
  • a sample includes, but is not limited to, any clinically relevant tissue sample, such as a tumor biopsy, a core biopsy tissue sample, a fine needle aspirate, a hair follicle, or a sample of bodily fluid, such as blood, plasma, serum, lymph, ascites fluid, cystic fluid, or urine.
  • the sample is taken from a patient having a tumor or cancer.
  • the sample is a primary tumor.
  • the sample is a metastasis.
  • the sample is a tissue sample.
  • the sample is a tumor sample.
  • the sample is a fresh tissue sample. In some embodiments, the sample is a frozen tissue sample. In some embodiments, the sample is a fresh frozen (FF) tissue sample. In some embodiments, the sample is a formalin-fixed paraffin embedded (FFPE) tissue sample. In some embodiments, the sample is whole blood, plasma, or serum. In some embodiments, the sample is cells. In some embodiments, the sample is circulating tumor cells (CTCs).
  • CTCs circulating tumor cells
  • the expression level of a biomarker is determined using methods that detect the expression level of nucleic acids (e.g., DNA or RNA).
  • the expression level of a biomarker is determined using PCR-based methods, such as but not limited to, reverse transcription PCR (RT-PCR), quantitative RT-PCR (qPCR), TaqManTM, or TaqManTM low density array (TLDA).
  • RT-PCR reverse transcription PCR
  • qPCR quantitative RT-PCR
  • TaqManTM TaqManTM low density array
  • TLDA TaqManTM low density array
  • the expression level of a biomarker is determined using a microarray.
  • the expression level of a biomarker is determined using a sequencing method, such as but not limited to, next-generation sequencing (NGS), RNA sequencing (RNA-Seq), and whole transcriptome shotgun sequencing (WTSS).
  • NGS next-generation sequencing
  • RNA sequencing RNA-Seq
  • WTSS whole transcriptome shotgun sequencing
  • NGS allows genome-wide analysis of transcription at single nucleotide resolution, including identification of alternative splicing events and post- transcriptional RNA editing events.
  • the expression level of a biomarker is measured or determined by a PCR-based assay.
  • an assay uses one or more primer pairs and probes specific for amplification of SMO mRNA.
  • the primer pairs are a forward (sense) primer and a reverse (anti-sense) primer that consist essentially of at least eight contiguous nucleotides of SEQ ID NO: 14.
  • a forward and reverse primer pair hybridizes to a nucleotide sequence of SEQ ID NO: 14.
  • an assay uses one or more primer pairs and probes specific for amplification of IGF2 mRNA.
  • the primer pairs are a forward (sense) primer and a reverse (anti-sense) primer that consist essentially of at least eight contiguous nucleotides of SEQ ID NO: 16.
  • a forward and reverse primer pair hybridizes to a nucleotide sequence of SEQ ID NO: 16.
  • an assay uses one or more primer pairs and probes specific for amplification of TGFB3 mRNA.
  • the primer pairs are a forward (sense) primer and a reverse (anti-sense) primer that consist essentially of at least ten contiguous nucleotides of SEQ ID NO: 18.
  • a forward and reverse primer pair hybridizes to a nucleotide sequence of SEQ ID NO: 18.
  • the expression level of a biomarker is measured or determined by multi-analyte profile testing, radioimmunoassay (RIA), Western blot assay, immunofluore scent assay, enzyme immunoassay, enzyme linked immunosorbent assay (ELISA), immunoprecipitation assay, chemiluminescent assay, immunohistochemical assay, dot blot assay, or slot blot assay.
  • RIA radioimmunoassay
  • Western blot assay immunofluore scent assay
  • enzyme immunoassay enzyme linked immunosorbent assay
  • immunoprecipitation assay chemiluminescent assay
  • immunohistochemical assay immunohistochemical assay
  • dot blot assay dot blot assay
  • slot blot assay slot blot assay.
  • the label is selected from the group consisting of an immunofluore scent label, a chemiluminescent label, a phosphorescent label, an enzyme label, a radiolabel, an avidin/biotin label, colloidal gold particles, colored particles, and magnetic particles.
  • the invention also provides a kit comprising a container, wherein the container contains at least one reagent for specifically detecting the expression of at least one biomarker of the invention.
  • the reagent is an antibody or nucleic acid probe that binds a biomarker of the invention.
  • a kit comprises a forward primer that hybridizes to SEQ ID NO: 14, a reverse primer that hybridizes to SEQ ID NO: 14, and a probe.
  • a kit comprises a forward primer that hybridizes to SEQ ID NO: 16, a reverse primer that hybridizes to SEQ ID NO: 16, and a probe.
  • a kit comprises a forward primer that hybridizes to SEQ ID NO: 18, a reverse primer that hybridizes to SEQ ID NO: 18, and a probe.
  • Pancreatic tumor OMP-PN4 cells were injected subcutaneously into NOD/SCID mice.
  • Pancreatic tumor OMP-PN7 cells were injected subcutaneously into NOD/SCID mice.
  • Pancreatic tumor OMP-PN16 cells were injected subcutaneously into NOD/SCID mice.
  • Pancreatic tumor OMP-PN17 cells were injected subcutaneously into NOD/SCID mice.
  • Pancreatic tumor OMP-PN21 cells were injected subcutaneously into NOD/SCID mice F3 ⁇ 4 *ure 11.
  • Pancreatic tumor OMP-PN23 cells were injected subcutaneously into NOD/SCID mice.
  • Figure 1J. Pancreatic tumor OMP-PN25 cells were injected subcutaneously into NOD/SCID mice.
  • Figure IK. Pancreatic tumor OMP-PN31 cells were injected subcutaneously into NOD/SCID mice.
  • Pancreatic tumor OMP-PN33 cells were injected subcutaneously into NOD/SCID mice.
  • Figure 1M Pancreatic tumor OMP-PN38 cells were injected subcutaneously into NOD/SCID mice.
  • Pancreatic tumor OMP-PN40 cells were injected subcutaneously into NOD/SCID mice. For each experiment, mice were treated with a combination of gemcitabine (Gem) and ABRAXANE (ABX) (- ⁇ -), a combination of OMP-18R5, gemcitabine, and ABRAXANE (- -), or a control antibody (- ⁇ -). Data is shown as tumor volume (mm 3 ) over days post-treatment.
  • FIGS 5A-5C In vivo validation of predictive biomarkers.
  • Figure 5A Pancreatic tumor OMP-PN8 cells were injected subcutaneously into NOD/SCID mice.
  • Figure 5B Pancreatic tumor OMP-PN37 cells were injected subcutaneously into NOD/SCID mice.
  • Figure 5C Pancreatic tumor OMP-PN41 cells were injected subcutaneously into NOD/SCID mice.
  • mice were treated with a combination of gemcitabine (Gem) and ABRAXANE (ABX) (- ⁇ -), a combination of OMP-18R5, gemcitabine, and ABRAXANE (- -), or a control antibody (- ⁇ -).
  • Data is shown as tumor volume (mm 3 ) over days post-treatment.
  • Figure 6 Population prevalence estimation of the 3-gene biomarker signature using public datasets.
  • FIG. 7 Kaplan Meier curves of the 3-gene biomarker signature (TGFB3, IGF2, SMO) with progression-free survival (PFS) and overall survival (OS) in pancreatic cancer patients from a Phase lb trial with vantictumab in combination with nab-paclitaxel and gemcitabine (March 20, 2015 data cut).
  • TGFB3, IGF2, SMO progression-free survival
  • OS overall survival
  • FIG. 8 Kaplan Meier curves of the 3-gene biomarker signature (TGFB3, IGF2, SMO) with overall survival (OS) and time to recurrence in pancreatic cancer patients treated with gemcitabine (data from a public dataset).
  • biomarker may include but is not limited to, nucleic acids and proteins, and variants and fragments thereof.
  • a biomarker may be DNA comprising the entire or partial nucleic acid sequence encoding the biomarker, or the complement of such a sequence.
  • Biomarker nucleic acids useful in the invention are considered to include both DNA and RNA comprising the entire or partial sequence of any of the nucleic acid sequences of interest.
  • Biomarker proteins are considered to comprise the entire or partial amino acid sequence of any of the biomarker proteins or polypeptides.
  • antibody refers to an immunoglobulin molecule that recognizes and specifically binds a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or combinations of the foregoing, through at least one antigen-binding site.
  • the term encompasses intact polyclonal antibodies, intact monoclonal antibodies, single chain antibodies, antibody fragments (such as Fab, Fab', F(ab')2, and Fv fragments), single chain Fv (scFv) antibodies, multispecific antibodies such as bispecific antibodies, monospecific antibodies, monovalent antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins comprising an antigen-binding site of an antibody, and any other modified immunoglobulin molecule comprising an antigen-binding site as long as the antibodies exhibit the desired biological binding activity.
  • antibody fragments such as Fab, Fab', F(ab')2, and Fv fragments
  • scFv single chain Fv antibodies
  • multispecific antibodies such as bispecific antibodies, monospecific antibodies, monovalent antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins comprising an antigen-binding site of an antibody, and any other modified immunoglobulin molecule comprising an antigen-binding site as long as the antibodies exhibit the desired biological binding activity.
  • An antibody can be any of the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes) thereof (e.g., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2), based on the identity of their heavy chain constant domains referred to as alpha, delta, epsilon, gamma, and mu, respectively.
  • the different classes of immunoglobulins have different and well-known subunit structures and three- dimensional configurations.
  • Antibodies can be naked or conjugated to other molecules, including but not limited to, toxins and radioisotopes.
  • antibody fragment refers to a portion of an intact antibody and refers to the antigenic determining variable regions of an intact antibody.
  • antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, linear antibodies, single chain antibodies, and multispecific antibodies formed from antibody fragments.
  • Antibody fragment as used herein comprises at least one antigen-binding site or epitope -binding site.
  • variable region of an antibody refers to the variable region of an antibody light chain, or the variable region of an antibody heavy chain, either alone or in combination.
  • the variable region of a heavy chain or a light chain generally consists of four framework regions (FR) connected by three complementarity determining regions (CDRs), also known as “hypervariable regions”.
  • FR framework regions
  • CDRs complementarity determining regions
  • the CDRs in each chain are held together in close proximity by the framework regions and contribute to the formation of the antigen-binding site(s) of the antibody.
  • CDRs There are at least two techniques for determining CDRs: (1) an approach based on cross-species sequence variability (i.e., Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Edition, National Institutes of Health, Bethesda, MD), and (2) an approach based on crystallographic studies of antigen-antibody complexes (Al-Lazikani et al., 1997, J. Mol. Biol., 273:927-948). In addition, combinations of these two approaches are sometimes used in the art to determine CDRs.
  • the term "monoclonal antibody” as used herein refers to a homogeneous antibody population involved in the highly specific recognition and binding of a single antigenic determinant or epitope. This is in contrast to polyclonal antibodies that typically include a mixture of different antibodies directed against a variety of different antigenic determinants.
  • the term “monoclonal antibody” encompasses both intact and full-length monoclonal antibodies as well as antibody fragments (e.g., Fab, Fab', F(ab')2, Fv), single chain (scFv) antibodies, fusion proteins comprising an antibody portion, and any other modified immunoglobulin molecule comprising an antigen-binding site.
  • “monoclonal antibody” refers to such antibodies made by any number of techniques, including but not limited to, hybridoma production, phage selection, recombinant expression, and transgenic animals.
  • humanized antibody refers to antibodies that are specific immunoglobulin chains, chimeric immunoglobulins, or fragments thereof that contain minimal non- human sequences.
  • human antibody refers to an antibody produced by a human or an antibody having an amino acid sequence corresponding to an antibody produced by a human.
  • chimeric antibody refers to an antibody wherein the amino acid sequence of the immunoglobulin molecule is derived from two or more species.
  • the variable region of the light chain and the variable region of the heavy chain correspond to the variable regions of an antibody derived from one species of mammals (e.g., mouse, rat, rabbit, etc.) with the desired specificity, affinity, and/or binding capability, while the constant regions correspond to sequences from an antibody derived from another species (e.g., human).
  • affinity -matured antibody refers to an antibody with one or more alterations in one or more CDRs thereof that result in an improvement in the affinity of the antibody for antigen, compared to a parent antibody that does not possess those alterations(s).
  • the definition also includes alterations in non-CDR residues made in conjunction with alterations to CDR residues.
  • affinity-matured antibodies will have nanomolar or even picomolar affinities for the target antigen.
  • epitopes can be formed both from contiguous amino acids and noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids (also referred to as linear epitopes) are typically retained upon protein denaturing, whereas epitopes formed by tertiary folding (also referred to as conformational epitopes) are typically lost upon protein denaturing.
  • An epitope typically includes at least 3, and more usually, at least 5 or 8-10 amino acids in a unique spatial conformation.
  • binding agent or an antibody reacts or associates more frequently, more rapidly, with greater duration, with greater affinity, or with some combination of the above to the epitope, protein, or target molecule than with alternative substances, including unrelated or related proteins.
  • specifically binds means, for instance, that a binding agent binds a protein with a K D of about 0. ImM or less, but more usually less than about ⁇ .
  • binding agent binds a target at times with a K D of at least about 0.1 ⁇ or less, at other times at least about 0.01 ⁇ or less, and at other times at least about InM or less.
  • specific binding can include a binding agent that recognizes a protein in more than one species (e.g., a human FZD protein and a mouse FZD protein).
  • specific binding can include a binding agent that recognizes more than one protein (e.g., human FZD1 and human FZD7).
  • a binding agent that specifically binds a first target may or may not specifically bind a second target.
  • “specific binding” does not necessarily require (although it can include) exclusive binding, i.e. binding to a single target.
  • a binding agent may, in certain embodiments, specifically bind more than one target.
  • multiple targets may be bound by the same binding site on the binding agent.
  • a binding agent may, in certain instances, comprise two identical binding sites, each of which specifically binds the same target on two or more proteins.
  • a binding agent may be bispecific or multispecific and comprise at least two binding sites with differing specificities.
  • a bispecific agent may comprise one binding site that recognizes an epitope on one protein (e.g., a human FZD) and further comprise a second, different binding site that recognizes a different epitope on a second protein (e.g., a human Wnt protein).
  • a second protein e.g., a human Wnt protein
  • reference to binding means specific binding.
  • polypeptide and “peptide” and “protein” are used interchangeably herein and refer to polymers of amino acids of any length.
  • the polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids.
  • the terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component.
  • polypeptides containing one or more analogs of an amino acid including, for example, unnatural amino acids
  • the polypeptides of this invention may be based upon antibodies or fusion proteins, in certain embodiments, the polypeptides can occur as single chains or associated chains (e.g., dimers).
  • polynucleotide and “nucleic acid” and “nucleic acid molecule” are used interchangeably herein and refer to polymers of nucleotides of any length, and include DNA and RNA.
  • the polynucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase.
  • Constants of high stringency may be identified by conditions that: (1) employ low ionic strength and high temperature for washing, for example 15mM sodium chloride/1.5mM sodium citrate/0.1% sodium dodecyl sulfate at 50°C; (2) employ during hybridization a denaturing agent, such as formamide, for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/5 OmM sodium phosphate buffer at pH 6.5 in 5x SSC (0.75M NaCl, 75mM sodium citrate) at 42°C; or (3) employ during hybridization 50% formamide in 5x SSC, 50mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5x Denhardt's solution, sonicated salmon sperm DNA (50 ⁇ g/ml), 0.1% SDS, and 10% dextran sulfate at 42°C, with washes at 42°C in 0.2x SSC
  • nucleic acids or two or more polypeptides refer to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned (introducing gaps, if necessary) for maximum correspondence, not considering any conservative amino acid substitutions as part of the sequence identity.
  • the percent identity may be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software that may be used to obtain alignments of amino acid or nucleotide sequences are well-known in the art.
  • two nucleic acids or polypeptides of the invention are substantially identical, meaning they have at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, and in some embodiments at least about 95%, 96%, 97%, 98%, 99% nucleotide or amino acid residue identity, when compared and aligned for maximum correspondence, as measured using a sequence comparison algorithm or by visual inspection.
  • identity exists over a region of the sequences that is at least about 10, at least about 20, at least about 40-60 nucleotides or amino acid residues, at least about 60- 80 nucleotides or amino acid residues in length or any integral value therebetween. In some embodiments, identity exists over a longer region than 60-80 nucleotides or amino acid residues, such as at least about 80-100 nucleotides or amino acid residues, and in some embodiments the sequences are substantially identical over the full length of the sequences being compared, such as the coding region of a nucleotide sequence.
  • a "conservative amino acid substitution” is one in which one amino acid residue is replaced with another amino acid residue having a similar side chain.
  • Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
  • basic side chains
  • substitution of a phenylalanine for a tyrosine is a conservative substitution.
  • conservative substitutions in the sequences of the polypeptides and antibodies of the invention do not abrogate the binding of the polypeptide or antibody containing the amino acid sequence, to the antigen to which the polypeptide or antibody binds.
  • Methods of identifying nucleotide and amino acid conservative substitutions which do not eliminate antigen binding are well-known in the art.
  • vector means a construct, which is capable of delivering, and usually expressing, one or more gene(s) or sequence(s) of interest in a host cell.
  • vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmid, cosmid, or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, and DNA or RNA expression vectors encapsulated in liposomes.
  • a polypeptide, antibody, polynucleotide, vector, cell, or composition which is "isolated” is a polypeptide, antibody, polynucleotide, vector, cell, or composition which is in a form not found in nature.
  • Isolated polypeptides, antibodies, polynucleotides, vectors, cells, or compositions include those which have been purified to a degree that they are no longer in a form in which they are found in nature.
  • a polypeptide, antibody, polynucleotide, vector, cell, or composition which is isolated is substantially pure.
  • substantially pure refers to material which is at least 50% pure (i.e., free from contaminants), at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure.
  • cancer and “cancerous” as used herein refer to or describe the physiological condition in mammals in which a population of cells are characterized by unregulated cell growth.
  • examples of cancer include, but are not limited to, carcinoma, blastoma, sarcoma, and hematologic cancers such as lymphoma and leukemia.
  • tumor and tumor refer to any mass of tissue that results from excessive cell growth or proliferation, either benign (non-cancerous) or malignant (cancerous) including pre-cancerous lesions.
  • metalastasis refers to the process by which a cancer spreads or transfers from the site of origin to other regions of the body with the development of a similar cancerous lesion at a new location.
  • a “metastatic” or “metastasizing” cell is one that loses adhesive contacts with neighboring cells and migrates (e.g., via the bloodstream or lymph) from the primary site of disease to secondary sites.
  • cancer stem cell and “CSC” and “tumor stem cell” and “tumor initiating cell” are used interchangeably herein and refer to cells from a cancer or tumor that: (1) have extensive proliferative capacity; 2) are capable of asymmetric cell division to generate one or more types of differentiated cell progeny wherein the differentiated cells have reduced and/or limited proliferative or developmental potential; and (3) are capable of symmetric cell divisions for self-renewal or self- maintenance.
  • CSC cancer stem cell
  • tumor stem cell undergo self-renewal versus differentiation in a chaotic manner to form tumors with abnormal cell types that can change over time as mutations occur.
  • cancer cell and “tumor cell” refer to the total population of cells derived from a cancer or tumor or pre-cancerous lesion, including both non-tumorigenic cells, which comprise the bulk of the cancer cell population, and tumorigenic stem cells (cancer stem cells).
  • cancer stem cells tumorigenic stem cells
  • tumorigenic refers to the functional features of a cancer stem cell including the properties of self-renewal (giving rise to additional tumorigenic cancer stem cells) and proliferation to generate all other tumor cells (giving rise to differentiated and thus non-tumorigenic tumor cells).
  • tumorigenicity refers to the ability of a random sample of cells from the tumor to form palpable tumors upon serial transplantation into immunocompromised hosts (e.g., mice). This definition also includes enriched and/or isolated populations of cancer stem cells that form palpable tumors upon serial transplantation into immunocompromised hosts (e.g., mice).
  • the term "patient” refers to any animal (e.g., a mammal), including, but not limited to, humans, non-human primates, canines, felines, rodents, and the like, which is to be the recipient of a particular treatment.
  • the terms “patient” and “subject” are used interchangeably herein. Typically, the terms “patient” and “subject” are used in reference to a human patient.
  • pharmaceutically acceptable refers to a product or compound approved (or approvable) by a regulatory agency of the Federal government or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, including humans.
  • pharmaceutically acceptable excipient, carrier or adjuvant refers to an excipient, carrier, or adjuvant that can be administered to a subject, together with at least one agent (e.g., an antibody) of the present disclosure, and which does not destroy the activity of the agent.
  • agent e.g., an antibody
  • the excipient, carrier, or adjuvant should be non-toxic when administered with an agent in doses sufficient to deliver a therapeutic effect.
  • an effective amount or “therapeutically effective amount” or “therapeutic effect” refer to an amount of a binding agent, an antibody, polypeptide, polynucleotide, small organic molecule, or other drug effective to "treat” a disease or disorder in a subject or mammal.
  • the therapeutically effective amount of an agent has a therapeutic effect and as such can reduce the number of cancer cells; decrease tumorigenicity, tumorigenic frequency, or tumorigenic capacity; reduce the number or frequency of cancer stem cells; reduce the tumor size; reduce the cancer cell population; inhibit and/or stop cancer cell infiltration into peripheral organs including, for example, the spread of cancer into soft tissue and bone; inhibit and/or stop tumor or cancer cell metastasis; inhibit and/or stop tumor or cancer cell growth; relieve to some extent one or more of the symptoms associated with the cancer; reduce morbidity and mortality; improve quality of life; or a combination of such effects.
  • the agent e.g., an antibody
  • the agent prevents growth and/or kills existing cancer cells, it can be referred to as cytostatic and/or cytotoxic.
  • treating or “treatment” or “to treat” or “alleviating” or “to alleviate” refer to both 1) therapeutic measures that cure, slow down, lessen symptoms of, and/or halt progression of a diagnosed pathologic condition or disorder and 2) prophylactic or preventative measures that prevent or slow the development of a targeted pathologic condition or disorder.
  • prophylactic or preventative measures that prevent or slow the development of a targeted pathologic condition or disorder.
  • a subject is successfully "treated” according to the methods of the present invention if the patient shows one or more of the following: a reduction in the number of and/or complete absence of cancer cells; a reduction in the tumor size; an inhibition of tumor growth; inhibition of and/or an absence of cancer cell infiltration into peripheral organs including the spread of cancer cells into soft tissue and bone; inhibition of and/or an absence of tumor or cancer cell metastasis; inhibition and/or an absence of cancer growth; relief of one or more symptoms associated with the specific cancer; reduced morbidity and mortality; improvement in quality of life; reduction in tumorigenicity; reduction in the number or frequency of cancer stem cells; or some combination of such effects. [0070] As used in the present disclosure and claims, the singular forms "a”, “an” and “the” include plural forms unless the context clearly dictates otherwise.
  • pancreatic tumors that are likely to be responsive (“sensitive”) or non-responsive (“resistant”) to treatment with a Wnt pathway inhibitor.
  • methods for identifying, classifying, and/or selecting patients that have a pancreatic tumor or have pancreatic cancer that are likely to be responsive (“sensitive”) or non-responsive ("resistant”) to treatment with a Wnt pathway inhibitor are provided herein.
  • methods for treating patients with pancreatic cancer who are likely to respond to treatment are predicted to respond to treatment, and/or have been identified to respond to treatment with a Wnt pathway inhibitor.
  • a method of identifying a human pancreatic tumor that is likely to be responsive or non-responsive to treatment with a Wnt pathway inhibitor comprising: (a) obtaining a sample of the pancreatic tumor; (b) measuring the expression level of each biomarker of a biomarker signature in the sample, wherein the biomarker signature comprises one, two, or three of the biomarkers smoothened (SMO), insulin-like growth factor 2 (IGF2), and transforming growth factor-beta 3 (TGFB3); and (c) identifying the pancreatic tumor as likely to be responsive or non- responsive to treatment based upon the expression level of the biomarkers.
  • SMO biomarkers smoothened
  • IGF2 insulin-like growth factor 2
  • TGFB3 transforming growth factor-beta 3
  • a method of identifying a human tumor that is likely to be responsive or non-responsive to treatment with a Wnt pathway inhibitor comprises: (a) obtaining a sample of the pancreatic tumor; (b) measuring the expression level of biomarkers SMO, IGF2, and TGFB3 in the sample; and (c) identifying the pancreatic tumor as likely to be responsive or non-responsive to treatment based upon the expression level of the biomarkers.
  • a method of identifying a human tumor that is likely to be responsive or non-responsive to treatment with a Wnt pathway inhibitor comprises: (a) measuring the expression level of biomarkers SMO, IGF2, and TGFB3 in a sample; and (b) identifying the pancreatic tumor as likely to be responsive or non-responsive to treatment based upon the expression level of the biomarkers in the sample.
  • the method comprises identifying a human pancreatic tumor that is likely to be responsive or non-responsive to treatment with a Wnt pathway inhibitor in combination with gemcitabine.
  • the method comprises identifying a human pancreatic tumor that is likely to be responsive or non- responsive to treatment with a Wnt pathway inhibitor in combination with gemcitabine and nab- paclitaxel. In some embodiments, the method comprises identifying a human pancreatic tumor that is likely to be responsive or non-responsive to treatment with a Wnt pathway inhibitor in combination with gemcitabine and ABRAXANE.
  • a method of classifying a human pancreatic tumor as likely to be responsive or non-responsive to treatment with a Wnt pathway inhibitor comprising: (a) obtaining a sample of the pancreatic tumor; (b) measuring the expression level of each biomarker of a biomarker signature in the sample, wherein the biomarker signature comprises one, two, or three of the biomarkers SMO, IGF2, and TGFB3; and (c) classifying the pancreatic tumor as likely to be responsive or non-responsive to treatment based upon the expression level of the biomarkers.
  • a method of classifying a human pancreatic tumor as likely to be responsive or non- responsive to treatment with a Wnt pathway inhibitor comprises: (a) obtaining a sample of the pancreatic tumor; (b) measuring the expression level of biomarkers SMO, IGF2, and TGFB3 in the sample; and (c) classifying the pancreatic tumor as likely to be responsive or non-responsive to treatment based upon the expression level of the biomarkers.
  • a method of classifying a human pancreatic tumor as likely to be responsive or non-responsive to treatment with a Wnt pathway inhibitor comprises: (a) measuring the expression level of biomarkers SMO, IGF2, and TGFB3 in a sample; and (b) classifying the pancreatic tumor as likely to be responsive or non- responsive to treatment based upon the expression level of the biomarkers in the sample.
  • the method comprises identifying a human pancreatic tumor that is likely to be responsive or non-responsive to treatment with a Wnt pathway inhibitor in combination with gemcitabine.
  • the method comprises identifying a human pancreatic tumor that is likely to be responsive or non-responsive to treatment with a Wnt pathway inhibitor in combination with gemcitabine and nab-paclitaxel. In some embodiments, the method comprises identifying a human pancreatic tumor that is likely to be responsive or non-responsive to treatment with a Wnt pathway inhibitor in combination with gemcitabine and ABRAXANE.
  • a method of determining the responsiveness (or sensitivity) of a human pancreatic tumor to treatment with a Wnt pathway inhibitor comprising: (a) obtaining a sample of the pancreatic tumor; (b) measuring the expression level of each biomarker of a biomarker signature in the sample, wherein the biomarker signature comprises one, two, or three of the biomarkers SMO, IGF2, and TGFB3; and (c) determining the responsiveness of the pancreatic tumor to treatment based upon the expression level of the biomarkers.
  • a method of determining the responsiveness (or sensitivity) of a human pancreatic tumor to treatment with a Wnt pathway inhibitor comprises: (a) obtaining a sample of the pancreatic tumor; (b) measuring the expression level of biomarkers SMO, IGF2, and TGFB3 in the sample; and (c) determining the responsiveness of the pancreatic tumor to treatment based upon the expression level of the biomarkers.
  • a method of determining the responsiveness (or sensitivity) of a human pancreatic tumor to treatment with a Wnt pathway inhibitor comprises: (a) measuring the expression level of biomarkers SMO, IGF2, and TGFB3 in a sample; and (b) determining the responsiveness of the pancreatic tumor to treatment based upon the expression level of the biomarkers in the sample.
  • the method comprises identifying a human pancreatic tumor that is likely to be responsive or non-responsive to treatment with a Wnt pathway inhibitor in combination with gemcitabine.
  • the method comprises identifying a human pancreatic tumor that is likely to be responsive or non-responsive to treatment with a Wnt pathway inhibitor in combination with gemcitabine and nab-paclitaxel. In some embodiments, the method comprises identifying a human pancreatic tumor that is likely to be responsive or non-responsive to treatment with a Wnt pathway inhibitor in combination with gemcitabine and ABRAXANE.
  • a method of identifying a patient with pancreatic cancer who is likely to respond to treatment with a Wnt pathway inhibitor comprising: (a) obtaining a sample of the patient's pancreatic tumor; (b) measuring the expression level of each biomarker of a biomarker signature in the sample, wherein the biomarker signature comprises one, two, or three of the biomarkers SMO, IGF2, and TGFB3; and (c) identifying the patient who is likely to respond to treatment based upon the expression level of the biomarkers.
  • a method of identifying a patient with pancreatic cancer who is likely to respond to treatment with a Wnt pathway inhibitor comprises: (a) obtaining a sample of the patient's pancreatic tumor; (b) measuring the expression level of biomarkers SMO, IGF2, and TGFB3 in the sample; and (c) identifying the patient who is likely to respond to treatment based upon the expression level of the biomarkers.
  • a method of identifying a patient with pancreatic cancer who is likely to respond to treatment with a Wnt pathway inhibitor comprises: (a) measuring the expression level of biomarkers SMO, IGF2, and TGFB3 in a sample; and (b) identifying the patient who is likely to respond to treatment based upon the expression level of the biomarkers in the sample.
  • the method further comprises selecting the patient for treatment.
  • the method further comprises administering a therapeutically effective amount of the Wnt pathway inhibitor to the patient.
  • the method comprises identifying a human pancreatic tumor that is likely to be responsive or non-responsive to treatment with a Wnt pathway inhibitor in combination with gemcitabine.
  • the method comprises identifying a human pancreatic tumor that is likely to be responsive or non-responsive to treatment with a Wnt pathway inhibitor in combination with gemcitabine and nab-paclitaxel. In some embodiments, the method comprises identifying a human pancreatic tumor that is likely to be responsive or non-responsive to treatment with a Wnt pathway inhibitor in combination with gemcitabine and ABRAXANE.
  • a method of selecting a patient with pancreatic cancer for treatment with a Wnt pathway inhibitor comprising: (a) obtaining a sample of the patient's pancreatic tumor; (b) measuring the expression level of each biomarker of a biomarker signature in the sample, wherein the biomarker signature comprises one, two, or three of the biomarkers SMO, IGF2, and TGFB3; and (c) selecting the patient for treatment based upon the expression level of the biomarkers.
  • a method of selecting a patient with pancreatic cancer for treatment with a Wnt pathway inhibitor comprises: (a) obtaining a sample of the patient's pancreatic tumor; (b) measuring the expression level of biomarkers SMO, IGF2, and TGFB3 in the sample; and (c) selecting the patient for treatment based upon the expression level of the biomarkers.
  • a method of selecting a patient with pancreatic cancer for treatment with a Wnt pathway inhibitor comprises: (a) measuring the expression level of biomarkers SMO, IGF2, and TGFB3 in a sample; and (b) selecting the patient for treatment based upon the expression level of the biomarkers in the sample.
  • the method further comprises administering a therapeutically effective amount of the Wnt pathway inhibitor to the patient.
  • the method comprises identifying a human pancreatic tumor that is likely to be responsive or non-responsive to treatment with a Wnt pathway inhibitor in combination with gemcitabine.
  • the method comprises identifying a human pancreatic tumor that is likely to be responsive or non- responsive to treatment with a Wnt pathway inhibitor in combination with gemcitabine and nab- paclitaxel.
  • the method comprises identifying a human pancreatic tumor that is likely to be responsive or non-responsive to treatment with a Wnt pathway inhibitor in combination with gemcitabine and ABRAXANE.
  • a method of treating pancreatic cancer in a patient comprising: (a) identifying if the patient is likely to respond to treatment with a Wnt pathway inhibitor, wherein the identification comprises: (i) obtaining a sample of the patient's pancreatic cancer; (ii) measuring the expression level of each biomarker of a biomarker signature in the sample, wherein the biomarker signature comprises one, two, or three of the biomarkers SMO, IGF2, and TGFB3; and (iii) identifying the patient who is likely to respond to treatment based upon the expression level of the biomarkers; and (b) administering to the patient who is likely to response to treatment a
  • a method of treating pancreatic cancer in a patient comprises: (a) identifying if the patient is likely to respond to treatment with a Wnt pathway inhibitor, wherein the identification comprises: (i) obtaining a sample of the patient's pancreatic cancer; (ii) measuring the expression level of biomarkers SMO, IGF2, and TGFB3 in the sample; and (iii) identifying the patient who is likely to respond to treatment based upon the expression level of the biomarkers; and (b) administering to the patient who is likely to response to treatment a therapeutically effective amount of the Wnt pathway inhibitor.
  • a method of treating pancreatic cancer in a patient comprises: (a) identifying if the patient is likely to respond to treatment with a Wnt pathway inhibitor, wherein the identification comprises: (i) measuring the expression level of biomarkers SMO, IGF2, and TGFB3 in a sample; and (ii) identifying the patient who is likely to respond to treatment based upon the expression level of the biomarkers in the sample; and (b) administering to the patient who is likely to response to treatment a therapeutically effective amount of the Wnt pathway inhibitor.
  • the method comprises identifying a human pancreatic tumor that is likely to be responsive or non-responsive to treatment with a Wnt pathway inhibitor in combination with gemcitabine.
  • the method comprises identifying a human pancreatic tumor that is likely to be responsive or non- responsive to treatment with a Wnt pathway inhibitor in combination with gemcitabine and nab- paclitaxel. In some embodiments, the method comprises identifying a human pancreatic tumor that is likely to be responsive or non-responsive to treatment with a Wnt pathway inhibitor in combination with gemcitabine and ABRAXANE.
  • pancreatic cancer in a patient, comprising:
  • a Wnt pathway inhibitor administered a therapeutically effective amount of a Wnt pathway inhibitor to the patient; wherein the patient is predicted to respond to treatment with a Wnt pathway inhibitor based upon expression levels of a biomarker signature in a sample of the patient's pancreatic cancer, wherein the signature comprises one, two, or three of the biomarkers SMO, IGF2, and TGFB3.
  • a method of treating pancreatic cancer in a patient comprises: administering a therapeutically effective amount of a Wnt pathway inhibitor to the patient; wherein the patient is predicted to respond to treatment with a Wnt pathway inhibitor based upon expression levels of a biomarker signature in a sample of the patient's pancreatic cancer, wherein the signature comprises biomarkers SMO, IGF2, and TGFB3.
  • the method comprises identifying a human pancreatic tumor that is likely to be responsive or non-responsive to treatment with a Wnt pathway inhibitor in combination with gemcitabine.
  • the method comprises identifying a human pancreatic tumor that is likely to be responsive or non-responsive to treatment with a Wnt pathway inhibitor in combination with gemcitabine and nab-paclitaxel. In some embodiments, the method comprises identifying a human pancreatic tumor that is likely to be responsive or non-responsive to treatment with a Wnt pathway inhibitor in combination with gemcitabine and ABRAXANE.
  • a method for increasing the likelihood of effective treatment with a Wnt pathway inhibitor comprising: (a) identifying if a patient has pancreatic cancer that is likely to respond to treatment with a Wnt pathway inhibitor, wherein the identification comprises: (i) obtaining a sample of the patient's pancreatic cancer; (ii) measuring the expression level of each biomarker of a biomarker signature in the sample, wherein the biomarker signature comprises one, two, or three of the biomarkers SMO, IGF2, and TGFB3; and (iii) identifying the patient who is likely to respond to treatment based upon the expression level of the biomarkers; and (b) administering a therapeutically effective amount of the Wnt pathway inhibitor to the patient.
  • a method for increasing the likelihood of effective treatment with a Wnt pathway inhibitor comprises: (a) identifying if a patient has pancreatic cancer that is likely to respond to treatment with a Wnt pathway inhibitor, wherein the identification comprises: (i) obtaining a sample of the patient's pancreatic cancer; (ii) measuring the expression level of biomarkers SMO, IGF2, and TGFB3 in the sample; and (iii) identifying the patient who is likely to respond to treatment based upon the expression level of the biomarkers; and (b) administering a therapeutically effective amount of the Wnt pathway inhibitor to the patient.
  • a method for increasing the likelihood of effective treatment with a Wnt pathway inhibitor comprises: (a) identifying if a patient has pancreatic cancer that is likely to respond to treatment with a Wnt pathway inhibitor, wherein the identification comprises: (i) measuring the expression level of biomarkers SMO, IGF2, and TGFB3 in a sample; and (ii) identifying the patient who is likely to respond to treatment based upon the expression level of the biomarkers in the sample; and (b) administering a therapeutically effective amount of the Wnt pathway inhibitor to the patient.
  • the method comprises identifying a human pancreatic tumor that is likely to be responsive or non-responsive to treatment with a Wnt pathway inhibitor in combination with gemcitabine.
  • the method comprises identifying a human pancreatic tumor that is likely to be responsive or non-responsive to treatment with a Wnt pathway inhibitor in combination with gemcitabine and nab-paclitaxel. In some embodiments, the method comprises identifying a human pancreatic tumor that is likely to be responsive or non- responsive to treatment with a Wnt pathway inhibitor in combination with gemcitabine and
  • the invention provides a method for increasing the likelihood of effective treatment with a Wnt pathway inhibitor, comprising: administering a therapeutically effective amount of a Wnt pathway inhibitor to a patient with pancreatic cancer; wherein the patient is identified as likely to respond to treatment with a Wnt pathway inhibitor based upon expression levels of a biomarker signature in a sample of the patient's pancreatic cancer, wherein the signature comprises one, two, or three of the biomarkers SMO, IGF2, and TGFB3.
  • a method for increasing the likelihood of effective treatment with a Wnt pathway inhibitor comprises: administering a therapeutically effective amount of a Wnt pathway inhibitor to a patient with pancreatic cancer; wherein the patient is identified as likely to respond to treatment with a Wnt pathway inhibitor based upon expression levels of a biomarker signature in a sample of the patient's pancreatic cancer, wherein the signature comprises biomarkers SMO, IGF2, and TGFB3.
  • the patient is identified as likely to respond to treatment with a Wnt pathway inhibitor in combination with gemcitabine.
  • the patient is identified as likely to respond to treatment with a Wnt pathway inhibitor in combination with gemcitabine and nab-paclitaxel.
  • the biomarker signature comprises the biomarkers SMO and IGF2. In some embodiments of the methods described herein, the biomarker signature comprises the biomarkers SMO and TGFB3. In some embodiments of the methods described herein, the biomarker signature comprises the biomarkers TGFB3 and IGF2. In some embodiments of the methods described herein, the biomarker signature comprises the biomarkers SMO, IGF2, and TGFB3. In some embodiments, the biomarker signature consists of SMO, IGF2, and TGFB3.
  • the biomarker signature comprises one or more additional biomarkers, in addition to at least one of the biomarkers SMO, IGF2, and TGFB3.
  • the biomarker signature comprises one or more additional biomarkers selected from the group consisting of FZD2, TWIST1, RBX1, CBY1, SOX18, SFRP2, and COL1A1, in addition to at least one of the biomarkers SMO, IGF2, and TGFB3.
  • the biomarker signature comprises one or more additional biomarkers selected from the group consisting of FZD2, TWIST1, RBX1, CBY1, SOX 18, SFRP2, and COL1A1, in addition the biomarkers SMO, IGF2, and TGFB3.
  • a sample includes, but is not limited to, any clinically relevant tissue sample, such as a tumor biopsy, a core biopsy tissue sample, a fine needle aspirate, a hair follicle, or a sample of bodily fluid, such as blood, plasma, serum, lymph, ascites fluid, cystic fluid, or urine.
  • the sample is taken from a patient having a tumor or cancer.
  • the sample is from a primary tumor.
  • the sample is from a metastasis.
  • the sample may be taken from a human, or from non-human mammals such as, mice, rats, rabbits, non-human primates, canines, felines, ruminants, swine, or sheep. In some
  • samples are taken from a subject at multiple time points, for example, before treatment, during treatment, and/or after treatment.
  • samples are taken from different locations in the subject, for example, a sample from a primary tumor and a sample from a metastasis in a distant location.
  • the sample is a paraffin-embedded fixed tissue sample.
  • the sample is a formalin-fixed paraffin embedded (FFPE) tissue sample.
  • the sample is a fresh tissue (e.g., tumor) sample.
  • the sample is a frozen tissue sample.
  • the sample is a fresh frozen (FF) tissue (e.g., tumor) sample.
  • the sample is a cell isolated from a fluid.
  • the sample comprises circulating tumor cells (CTCs).
  • the sample is an archival tissue sample.
  • the sample is an archival tissue sample with known diagnosis, treatment, and/or outcome history.
  • the sample is a block of tissue. In some embodiments, the sample is dispersed cells. In some embodiments, the sample size is from about 1 cell to about 1 x 10 6 cells or more. In some embodiments, the sample size is about 10 cells to about 1 x 10 5 cells. In some embodiments, the sample size is about 10 cells to about 10,000 cells. In some embodiments, the sample size is about 10 cells to about 1,000 cells. In some embodiments, the sample size is about 10 cells to about 100 cells. In some embodiments, the sample size is about 1 cell to about 10 cells. In some embodiments, the sample size is a single cell.
  • the sample is processed to DNA or RNA.
  • RNA is isolated from the sample.
  • mRNA is isolated from the sample.
  • RNA is isolated from cells by procedures that involve cell lysis and denaturation of the proteins contained therein.
  • DNase is added to remove DNA.
  • RNase inhibitors are added to the lysis buffer.
  • a protein denaturation/digestion step is added to the protocol. Methods for preparing total and mRNA are well known in the art and RNA isolation kits are commercially available (e.g., RNeasy mini kit, Qiagen).
  • the RNA is amplified by PCR-based techniques.
  • Determination of biomarker expression levels may be performed by any suitable method including, but are not limited to, methods based on analyses of polynucleotide expression, sequencing of polynucleotides, and/or analyses of protein expression. For example, determination of biomarker expression levels may be performed by detecting the expression of mRNA expressed from the genes of interest, and/or by detecting the expression of a polypeptide encoded by the genes.
  • Commonly used methods for the analysis of polynucleotides include Southern blot analysis, Northern blot analysis, in situ hybridization, RNAse protection assays, and polymerase chain reaction (PCR)-based methods, microarray analyses, and sequence-based analyses.
  • Representative methods for sequencing-based gene expression analyses include serial analysis of gene expression (SAGE), massively parallel signature sequencing (MPSS), and NexGen sequencing (NGS), including mRNA sequencing.
  • PCR-based analyses include but are not limited to, reverse transcription polymerase chain reaction (RT-PCR), quantitative PCR (qPCR) as known as real-time PCR, TaqManTM, TaqManTM low density array (TLDA), anchored PCR, competitive PCR, rapid amplification of cDNA ends (RACE), differential display, amplified fragment length polymorphism, BeadArrayTM technology, high coverage expression profiling (HiCEP) and digital PCR.
  • RT-PCR is a quantitative method that can be used to compare mRNA levels in different samples to examine gene expression profiles.
  • a variation of RT-PCR is real time quantitative PCR, which measures PCR product accumulation through a dual-labeled fluorigenic probe (e.g., TaqManTM probe).
  • the biomarker expression is determined using RNA sequencing.
  • total RNA is extracted from a fresh sample, from a fresh frozen (FF) tissue sample, or from a macro-dissected formalin-fixed paraffin embedded (FFPE) tissue sample.
  • the quantity and quality of the total RNA is assessed by standard spectrophotometry and/or any other appropriate method (e.g., an Agilent Bioanalyzer).
  • Total RNA is converted into a library of template molecules that include sequencing adapters.
  • the library is hybridized to a flow cell which contains a lawn of covalently bound oligonucleotides complementary to the sequencing adapters. PCR amplification produces discrete clones that can be optically resolved during sequencing.
  • Sequencing by synthesis proceeds through multiple cycles of nucleotide incorporation and detection. After sequencing is completed, the reads are aligned to a reference genome. The aligned reads may be further normalized. The data is then analyzed using algorithms and software known to those of skill in the art.
  • the biomarker expression is determined using a qPCR assay.
  • total RNA is extracted from a fresh sample, from a fresh frozen (FF) tissue sample, or from a macro-dissected formalin-fixed paraffin embedded (FFPE) tissue sample.
  • the quantity and quality of the total RNA is assessed by standard spectrophotometry and/or any other appropriate method (e.g., an Agilent Bioanalyzer).
  • the RNA sample is reverse transcribed using standard methods and/or a commercially available cDNA synthesis kit (e.g., Roche Transcriptor First Strand cDNA synthesis kit).
  • the resultant cDNA is pre-amplified using, for example, an ABI pre- amplification kit.
  • Expression of the biomarkers e.g., SMO, IGF2, and/or TGFB3 are assessed on, for example, a Roche Lightcycler 480 system (Roche Diagnostics) using an ABI TaqMan Gene Expression Mastermix. qPCR reactions are performed in triplicate. For each assay a subset of the samples is run without reverse transcription (the RT-neg control), as well as, control samples run without template. A universal human reference RNA sample is included on each plate to act as a positive control. Suitable reference genes are identified from a standard panel of reference genes. Candidate reference genes are selected with different cellular functions to eliminate risk of co- regulation.
  • the most suitable reference genes are evaluated and selected using specific software and algorithms (e.g., Genex software; GeNorm and Normfinder algorithms).
  • the expression level of each biomarker is normalized using the selected optimum reference genes. In some embodiments, these normalized (or standardized) expression values for each biomarker are used to calculate the decision value of the sample. In some embodiments, these normalized (or standardized) expression values for each biomarker are used to calculate an expression level.
  • biomarker expression is determined using a PCR-based assay comprising specific primers and/or probes for each biomarker (e.g., SMO, IGF2, and/or TGFB3).
  • probe refers to any molecule that is capable of selectively binding a specifically intended target biomolecule. Probes can be synthesized by one of skill in the art using known techniques, or derived from biological preparations. Probes may include but are not limited to, RNA, DNA, proteins, peptides, aptamers, antibodies, and organic molecules.
  • probe encompasses oligonucleotides that have a specific sequence or oligonucleotides that have a sequence complementary to a specific sequence.
  • the probe is modified.
  • the probe is modified with a quencher.
  • the probe is labeled. Labels can include, but are not limited to, colorimetric, fluorescent, chemiluminescent, or biolumine scent labels.
  • biomarker expression of each biomarker is determined using a specific primer set and probe.
  • a specific primer set consists of a forward primer and a reverse primer.
  • biomarker expression is measured or determined by a PCR-based assay.
  • an assay uses one or more primer pairs and probes specific for amplification of SMO mRNA.
  • the primer pairs are a forward (sense) primer and a reverse (anti-sense) primer that consist essentially of at least eight contiguous nucleotides of SEQ ID NO: 14.
  • a forward and reverse primer pair hybridizes to a nucleotide sequence of SEQ ID NO: 14.
  • an assay uses one or more primer pairs and probes specific for amplification of IGF2 mRNA.
  • the primer pairs are a forward (sense) primer and a reverse (anti-sense) primer that consist essentially of at least eight contiguous nucleotides of SEQ ID NO: 16. In some embodiments, a forward and reverse primer pair hybridizes to a nucleotide sequence of SEQ ID NO: 16. In some embodiments, an assay uses one or more primer pairs and probes specific for amplification of TGFB3 mRNA. In some embodiments, the primer pairs are a forward (sense) primer and a reverse (anti-sense) primer that consist essentially of at least ten contiguous nucleotides of SEQ ID NO: 18. In some embodiments, a forward and reverse primer pair hybridizes to a nucleotide sequence of SEQ ID NO: 18.
  • the expression level of each biomarker is determined in a separate assay (e.g., 3 assays).
  • the reference gene(s) and normalization methods for each assay are the same for all assays.
  • the expression levels of several biomarkers are detected in a single multiplex assay.
  • biomarker expression levels may be determined by amplifying complementary DNA (cDNA) or complementary RNA (cRNA) produced from mRNA and analyzing it using a microarray.
  • cDNA complementary DNA
  • cRNA complementary RNA
  • microarray technology allows for simultaneous analysis of the expression of thousands of genes. A number of different array configurations and methods for their production are known to those skilled in the art.
  • microarrays are commercially available (e.g., Affymetrix GeneChips) or can be custom-produced.
  • Microarrays currently in wide use include cDNA arrays and oligonucleotide arrays.
  • polynucleotides of interest e.g., probes or probe sets
  • probes to at least 10, 25, 50, 100, 500, 1000, 5000, 10,000, 20,000, or 25,000 or more genes are immobilized on an array substrate.
  • the substrate may be a porous or nonporous support, such as a glass, plastic or gel surface.
  • the probes can include DNA, RNA, copolymer sequences of DNA and RNA, DNA and/or RNA analogues, or combinations thereof.
  • a microarray includes a support with an ordered array of binding sites for each individual gene.
  • the microarrays can be addressable arrays or positionally addressable arrays, e.g., each probe of the array is located at a known, predetermined position on the solid support such that the identity of each probe can be determined from its position of the array.
  • Each probe on the microarray can be between 10-50,000 nucleotides in length.
  • the probes of the microarray can consist of nucleotide sequences with lengths of less than about 1,000 nucleotides, less than about 750 nucleotides, less than about 500 nucleotides, less than about 250 nucleotides, less than about 100 nucleotides, or less than about 50 nucleotides in length.
  • an array includes positive control probes and negative control probes.
  • the biomarker expression is determined using a microarray.
  • total RNA is extracted from a fresh frozen (FF) tissue sample or total RNA is extracted from a macro-dissected formalin-fixed paraffin embedded (FFPE) tissue sample.
  • FFPE formalin-fixed paraffin embedded
  • the quantity and quality of the total RNA is assessed by standard spectrophotometry and/or any other appropriate technology (e.g., an Agilent Bioanalyzer).
  • the RNA sample is amplified using standard methods and/or a commercially available amplification system (e.g., NuGEN Ovation RNA Amplification System V2).
  • the amplified cDNA is fragmented, labeled, and hybridized to a microarray (e.g., using NuGEN Encore Biotin Module and Affymetrix GeneChip array) following standard procedures.
  • the array is washed, stained, and scanned in accordance with the instructions for the microarray.
  • the microarray data is pre-processed, the probe-level intensity measurements are background corrected, normalized, and summarized as expression measurements using the Robust Multichip algorithm (RMA).
  • RMA Robust Multichip algorithm
  • the probe level data is summarized to get the expression level of each biomarker (e.g., SMO, IGF2, or TGFB3).
  • a combination of quality parameter threshold and data reduction techniques is applied to the data set to establish profile quality and identify potential outlying samples.
  • these normalized (or standardized) expression values for each biomarker are used to calculate the decision value of the sample.
  • biomarker expression is analyzed by studying the protein expression of the gene or genes of interest.
  • Commonly used methods for the analysis of protein expression include but are not limited to, immunohistochemistry (IHC)-based, antibody-based, and mass spectrometry-based methods.
  • Antibodies generally monoclonal antibodies, may be used to detect expression of a gene product (e.g., protein).
  • the antibodies can be detected by direct labeling of the antibodies themselves.
  • an unlabeled primary antibody is used in conjunction with a labeled secondary antibody. Immunohistochemistry methods and/or kits are well known in the art and are commercially available.
  • biomarker expression is determined by an assay known to those of skill in the art, including but not limited to, multi-analyte profile test, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay, Western blot assay, immunofluorescent assay, enzyme immunoassay, immunoprecipitation assay, chemiluminescent assay, immunohistochemical assay, dot blot assay, or slot blot assay.
  • ELISA enzyme-linked immunosorbent assay
  • immunofluorescent label chemiluminescent label, phosphorescent label, enzyme label, radiolabel, avidin/biotin, colloidal gold particles, colored particles, and magnetic particles.
  • a proteomic method comprises the following steps: (1) separation of individual proteins in a sample by 2-D electrophoresis (2-D PAGE), (2) identification of individual proteins recovered from the gel (e.g., by mass spectrometry or N-terminal sequencing), and (3) analysis of the data using bioinformatics.
  • a proteomic method comprises using a tissue microarray (TMA). Tissue arrays may be constructed according to a variety of techniques known to one of skill in the art.
  • a manual tissue arrayer is used to remove a "core" from a paraffin block prepared from a tissue sample. The core is then inserted into a separate paraffin block in a designated location on a grid. Cores from as many as about 400 samples can be inserted into a single recipient block. The resulting tissue array may be processed into thin sections for analysis.
  • a proteomic method comprises an antibody microarray.
  • a proteomic method comprises using mass spectrometry, including but not limited to, SELDI, MALDI, electro spray, and surface plasmon resonance methods.
  • a proteomic method comprises bead-based technology, including but not limited to, antibodies on beads in an array format.
  • the proteomic method comprises a reverse phase protein microarray (RPPM).
  • the proteomic method comprises multiplexed protein profiling, including but not limited to, the Global Proteome Survey (GPS) method.
  • GPS Global Proteome Survey
  • the biomarker signature is identified by differential gene expression between two samples. In some embodiments, the biomarker signature is identified by differential gene expression between two samples which comprise genes differentially expressed in cancer cells as compared to normal cells. In some embodiments, the biomarker signature comprises genes differentially expressed in tumorigenic cancer stem cells as compared to non-tumorigenic cancer cells. In some embodiments, the biomarker signature comprises genes differentially expressed in cells from a tumor which is responsive to a specific treatment as compared to cells from a tumor which is non- responsive to the same treatment.
  • the gene expression data are refined, filtered, and/or subdivided into biomarker signatures based on statistical analyses.
  • the statistical methods may include, but are not limited to, cluster analysis, supported vector machines (SVM) analysis, supported vector machines - recursive feature elimination (SVM-RFE) analysis, Piatt scaling, neural networks, and other algorithms.
  • the gene expression data are analyzed using a t-test analysis.
  • the gene expression data are analyzed using paired-sample empirical Baysian analysis.
  • a combination of statistical analyses is used.
  • SVM models are used to obtain decision values based on the training data.
  • classification probabilities for responders and non-responders are obtained using Piatt scaling (Piatt, 1999, Advances in Large Margin Classifiers, pp. 61-74, MIT Press). Piatt scaling may comprise fitting a logistic distribution using maximum likelihood to decision values obtained, for example, by SVM models.
  • K nearest neighbor KNN; Altaian, 1992, American
  • LOOCV leave-one-out cross- validation
  • LOOCV leave-one- out cross-validation
  • a biomarker signature is obtained by a series of analytical steps. For example, expression data from a training set of samples are obtained from RNA sequencing. The sequencing data are aligned to an annotated human reference genome. The aligned reads are further normalized us a RPKM algorithm. Two-sample Welch's T-test is used for feature selection and KNN is used for classification. Leave-one-out cross-validation (LOOCV) methods are used to identify and select the best predictive genes and also to measure AUC (area under the ROC curve), ACC
  • the gene expression data and/or biomarker signatures are refined, filtered, and/or subdivided based on additional statistical models.
  • the gene expression data and/or biomarker signatures are refined, filtered, and/or subdivided based on survival analysis models.
  • survival analysis models may include, but are not limited to, Kaplan-Meier survival models, Cox proportional models, Cox proportional hazard models, chi-square analysis, univariate logistic regression models, multivariate competing risk models, linear discriminate analysis models, parametric regression models and correlation analysis models.
  • the gene expression data and/or biomarker signatures are refined, filtered, subdivided and/or tested using gene expression array datasets that have associated clinical outcomes.
  • gene expression array datasets that have associated clinical outcomes.
  • GEO Gene Expression Omnibus
  • ArrayExpress ArrayExpress
  • the gene expression data and/or biomarker signatures are refined using biological function parameters, and/or gene sets.
  • gene expression profiles, and/or biomarker signatures are refined using Gene Set Enrichment Analysis (GSEA) (Subramanian et al., 2005, PNAS, 102: 15545-15550).
  • GSEA Gene Set Enrichment Analysis
  • the gene expression profiles are refined based on their ability to predict clinical outcome.
  • the Wnt pathway inhibitor is an anti-FZD antibody as described herein.
  • the Wnt pathway inhibitor is an antibody that specifically binds at least one human Frizzled (FZD) protein or fragment thereof.
  • the anti-FZD antibody specifically binds at least one FZD protein selected from the group consisting of: FZD1, FZD2, FZD5, FZD7, and FZD8.
  • the anti-FZD antibody specifically binds FZD1, FZD2, FZD5, FZD7, and FZD8.
  • the anti-FZD antibody comprises: (a) a heavy chain CDR1 comprising GFTFSHYTLS (SEQ ID NO: 1), a heavy chain CDR2 comprising VISGDGSYTYYADSVKG (SEQ ID NO:2), and a heavy chain CDR3 comprising NFIKYVFAN (SEQ ID NO:3), and (b) a light chain CDR1 comprising SGDNIGSFYVH (SEQ ID NO:4), a light chain CDR2 comprising DKSNRPSG (SEQ ID NO:5), and a light chain CDR3 comprising QSYANTLSL (SEQ ID NO:6).
  • the anti-FZD antibody comprises a heavy chain variable region comprising the amino acids of SEQ ID NO: 7. In some embodiments, the anti-FZD antibody comprises a light chain variable region comprising the amino acids of SEQ ID NO: 8. In some embodiments, the anti-FZD antibody comprises a heavy chain variable region comprising the amino acids of SEQ ID NO:7 and a light chain variable region comprising the amino acids of SEQ ID NO: 8. In some embodiments, the anti-FZD antibody is antibody OMP-18R5. In some embodiments, the anti-FZD antibody is encoded by the plasmid having ATCC deposit no. PTA-9541. In other embodiments, the anti-FZD antibody competes for specific binding to at least one human FZD protein with an antibody encoded by the plasmid deposited with ATCC having deposit no. PTA-9541.
  • the method comprises treating a patient with a Wnt pathway inhibitor described herein (e.g., an anti-FZD antibody), particularly after the patient has been identified as being responsive to treatment with the Wnt pathway inhibitor.
  • the treatment comprises administering at least one additional therapeutic agent in combination with the Wnt pathway inhibitor.
  • the at least one additional therapeutic agent comprises 1, 2, 3, or more additional therapeutic agents.
  • Useful classes of therapeutic agents include, for example, anti-tubulin agents, auristatins, DNA minor groove binders, DNA replication inhibitors, alkylating agents (e.g., platinum complexes such as cisplatin, mono(platinum), bis(platinum) and tri-nuclear platinum complexes and carboplatin), anthracyclines, antibiotics, anti-folates, antimetabolites, chemotherapy sensitizers, duocarmycins, etoposides, fluorinated pyrimidines, ionophores, lexitropsins, nitrosoureas, platinols, purine antimetabolites, puromycins, radiation sensitizers, steroids, taxanes, topoisomerase inhibitors, vinca alkaloids, or the like.
  • the additional therapeutic agent is an alkylating agent, an antimetabolite, an antimitotic, a topoisomerase inhibitor, or an angiogenesis inhibitor.
  • Therapeutic agents that may be administered in combination with a Wnt pathway inhibitor include chemotherapeutic agents.
  • the method or treatment involves the administration of a Wnt pathway inhibitor of the present invention in combination with a
  • Chemotherapeutic agents useful in the instant invention include, but are not limited to, 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, triethylenethiophosphaoramide and trimethylolomelamime; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, calicheamicin, carabicin, caminomycin, carzinophilin, chromomycins, dactinomycin, da
  • mitobronitol mitolactol
  • pipobroman gacytosine
  • arabinoside Ara-C
  • taxoids e.g. paclitaxel
  • TAXOL docetaxel
  • TXOTERE docetaxel
  • chlorambucil gemcitabine
  • 6-thioguanine 6-thioguanine
  • mercaptopurine platinum analogs such as cisplatin and carboplatin
  • vinblastine platinum
  • platinum etoposide (VP-16);
  • ifosfamide mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; ibandronate; CPT11; topoisomerase inhibitor RFS 2000;
  • Chemotherapeutic agents also include anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens including for example tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4- hydroxytamoxifen, trioxifene, keoxifene, LY 117018, onapristone, and toremifene (FARESTON); and anti -androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above.
  • anti-estrogens including for example tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4- hydroxytamoxifen, trioxifene, keoxifene, LY 117018, onapristone, and toremifene (FARESTON); and anti
  • the chemotherapeutic agent is a topoisomerase inhibitor.
  • Topoisomerase inhibitors are chemotherapy agents that interfere with the action of a topoisomerase enzyme (e.g., topoisomerase I or II).
  • Topoisomerase inhibitors include, but are not limited to, doxorubicin HC1, daunorubicin citrate, mitoxantrone HC1, actinomycin D, etoposide, topotecan HC1, teniposide (VM-26), and irinotecan, as well as pharmaceutically acceptable salts, acids, or derivatives of any of these.
  • the chemotherapeutic agent is an anti -metabolite.
  • An anti-metabolite is a chemical with a structure that is similar to a metabolite required for normal biochemical reactions, yet different enough to interfere with one or more normal functions of cells, such as cell division.
  • Anti-metabolites include, but are not limited to, gemcitabine, fluorouracil, capecitabine, methotrexate sodium, ralitrexed, pemetrexed, tegafur, cytosine arabinoside, thioguanine, 5-azacytidine, 6- mercaptopurine, azathioprine, 6-thioguanine, pentostatin, fludarabine phosphate, and cladribine, as well as pharmaceutically acceptable salts, acids, or derivatives of any of these.
  • the chemotherapeutic agent is an antimitotic agent, including, but not limited to, agents that bind tubulin.
  • the agent is a taxane.
  • the agent is paclitaxel or docetaxel, or a pharmaceutically acceptable salt, acid, or derivative of paclitaxel or docetaxel.
  • the agent is paclitaxel (TAXOL), docetaxel (TAXOTERE), albumin-bound paclitaxel (nab-paclitaxel; ABRAXANE), DHA-paclitaxel, or PG-paclitaxel.
  • the antimitotic agent comprises a vinca alkaloid, such as vincristine, vinblastine, vinorelbine, or vindesine, or pharmaceutically acceptable salts, acids, or derivatives thereof.
  • the antimitotic agent is an inhibitor of kinesin Eg5 or an inhibitor of a mitotic kinase such as Aurora A or Plkl .
  • the additional therapeutic agent is gemcitabine. In some embodiments, the additional therapeutic agent is gemcitabine.
  • the additional therapeutic agent is nab-paclitaxel (e.g., ABRAXANE).
  • the additional therapeutic agents are gemcitabine and nab-paclitaxel (e.g.,
  • Treatment with a Wnt pathway inhibitor can occur prior to, concurrently with, or subsequent to administration of chemotherapeutic agents.
  • Combined administration can include co-administration, either in a single pharmaceutical formulation or using separate formulations, or consecutive administration in either order but generally within a time period such that all active agents can exert their biological activities simultaneously.
  • Preparation and dosing schedules for such chemotherapeutic agents can be used according to manufacturers' instructions or as determined empirically by the skilled practitioner. Preparation and dosing schedules for such chemotherapy are also described in The Chemotherapy Source Book, 4th Edition, 2008, M. C. Perry, Editor, Lippincott, Williams & Wilkins, Philadelphia, PA.
  • Certain embodiments of the present invention comprise a method of identifying a human pancreatic tumor that is likely to be responsive to or non-responsive to treatment with an antibody that specifically binds at least one human frizzled (FZD) selected from the group consisting of FZD 1, FZD2, FZD5, FZD7, and FZD8, the method comprising (a) obtaining a sample of the pancreatic tumor; (b) measuring the expression level of each biomarker of a biomarker signature in the sample, wherein the biomarker signature comprises one, two, or three of the biomarkers SMO, IGF2, and TGFB3; and (c) identifying the pancreatic tumor as likely to be responsive or non-responsive to treatment based upon the expression level of the biomarkers.
  • FZD human frizzled
  • a method of identifying a patient with pancreatic cancer that is likely to be responsive to treatment with an antibody that specifically binds at least one human FZD selected from the group consisting of FZD1, FZD2, FZD5, FZD7, and FZD8, comprises: (a) obtaining a sample of the patient's pancreatic cancer; (b) measuring the expression level of each biomarker of a biomarker signature in the sample, wherein the biomarker signature comprises one, two, or three of the biomarkers SMO, IGF2, and TGFB3; and (c) identifying the patient who is likely to respond to treatment based upon the expression level of the biomarkers.
  • a method of selecting a patient with pancreatic cancer for treatment with an antibody that specifically binds at least one human FZD selected from the group consisting of FZD1, FZD2, FZD5, FZD7, and FZD8, comprises: (a) obtaining a sample of the patient's pancreatic cancer; (b) measuring the expression level of each biomarker of a biomarker signature in the sample, wherein the biomarker signature comprises one, two, or three of the biomarkers SMO, IGF2, and TGFB3; (c) selecting the patient for treatment based upon the expression level of the biomarkers.
  • a method of treating pancreatic cancer in a patient comprises: (a) identifying if the patient is likely to respond to treatment with an antibody that specifically binds at least one human FZD selected from the group consisting of FZD1, FZD2, FZD5, FZD7, and FZD8, wherein the identification comprises: (i) obtaining a sample of the patient's pancreatic cancer; (ii) measuring the expression level of each biomarker of a biomarker signature in the sample, wherein the biomarker signature comprises one, two, or three of the biomarkers SMO, IGF2, and TGFB3; and (iii) identifying the patient who is likely to respond to treatment based upon the expression level of the biomarkers; and (b) administering a therapeutically effective amount of the antibody to the patient who is likley to response to treatment.
  • a method of identifying a human pancreatic tumor that is likely to be responsive to or non-responsive to treatment with anti-FZD antibody OMP-18R5 in combination with gemcitabine and nab-paclitaxel comprises (a) obtaining a sample of the pancreatic tumor; (b) measuring the expression level of each biomarker of a biomarker signature in the sample, wherein the biomarker signature comprises the biomarkers SMO, IGF2, and TGFB3; and (c) identifying the pancreatic tumor as likely to be responsive or non-responsive to treatment based upon the expression level of the biomarkers.
  • a method of identifying a patient with pancreatic cancer that is likely to be responsive to treatment with the anti-FZD antibody OMP-18R5 in combination with gemcitabine and nab-paclitaxel comprises: (a) obtaining a sample of the patient's pancreatic cancer; (b) measuring the expression level of each biomarker of a biomarker signature in the sample, wherein the biomarker signature comprises the biomarkers SMO, IGF2, and TGFB3; and (c)identifying the patient who is likely to response to treatment based upon the expression level of the biomarkers.
  • ABRAXANE comprises: (a) obtaining a sample of the patient's pancreatic cancer; (b) measuring the expression level of each biomarker of a biomarker signature in the sample, wherein the biomarker signature comprises the biomarkers SMO, IGF2, and TGFB3; (c) selecting the patient for treatment based upon the expression level of the biomarkers.
  • a method of treating pancreatic cancer in a patient comprises: (a) identifying if the patient is likely to respond to treatment with the anti-FZD antibody OMP-18R5 in combination with gemcitabine and nab-paclitaxel (ABRAXANE), wherein the identification comprises: (i) obtaining a sample of the patient's pancreatic cancer; (ii) measuring the expression level of each biomarker of a biomarker signature in the sample, wherein the biomarker signature comprises the biomarkers SMO, IGF2, and TGFB3; and (iii)identifying the patient who is likely to respond to treatment based upon the expression level of the biomarkers; and (b) administering a therapeutically effective amount of the antibody, gemcitabine, and nab-paclitaxel to the patient who is likely to respond to treatment.
  • ABRAXANE nab-paclitaxel
  • the present invention provides methods for identifying pancreatic tumors and/or patients with pancreatic cancer that are likely to be responsive to or sensitive to treatment with a Wnt pathway inhibitor.
  • Wnt pathway inhibitor includes, but is not limited to, Frizzled (FZD) binding agents and Wnt-binding agents.
  • FZD-binding agents may include antibodies that specifically bind FZD proteins.
  • Wnt-binding agents may include antibodies that specifically bind Wnt proteins as well as soluble FZD receptors that bind Wnt proteins.
  • a Wnt pathway inhibitor is an agent that binds one or more human FZD proteins.
  • a FZD-binding agent specifically binds one, two, three, four, five, six, seven, eight, nine, or ten FZD proteins.
  • a FZD-binding agent binds one or more FZD proteins selected from the group consisting of FZD 1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, and FZD10.
  • the FZD-binding agent binds one or more FZD proteins comprising FZD1, FZD2, FZD5, FZD7, and/or FZD8. In certain embodiments, the FZD-binding agent specifically binds FZD1, FZD2, FZD5, FZD7, and FZD 8.
  • FZD-binding agents can be found in U.S. Patent No. 7,982,013.
  • the FZD-binding agent is a FZD antagonist. In certain embodiments, the FZD-binding agent is a Wnt pathway antagonist. In certain embodiments, the FZD-binding agent inhibits Wnt signaling. In some embodiments, the FZD-binding agent inhibits canonical Wnt signaling.
  • the FZD-binding agent is an antibody. In some embodiments, the FZD-binding agent is a polypeptide. In certain embodiments, the FZD-binding agent is an antibody or a polypeptide comprising an antigen-binding site. In certain embodiments, an antigen-binding site of a FZD-binding antibody or polypeptide described herein is capable of binding (or binds) one, two, three, four, five, or more human FZD proteins.
  • an antigen-binding site of the FZD-binding antibody or polypeptide is capable of specifically binding one, two, three, four, or five human FZD proteins selected from the group consisting of FZD 1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9 and FZD10.
  • FZD-binding agent when the FZD-binding agent is an antibody that binds more than one FZD protein, it may be referred to as a "pan-FZD antibody".
  • the FZD-binding agent e.g., antibody
  • the FZD-binding agent specifically binds the extracellular domain (ECD) of the one or more human FZD proteins to which it binds.
  • the FZD-binding agent specifically binds within the Fri domain (also known as the cysteine-rich domain (CRD)) of the one or more human FZD proteins to which it binds.
  • the FZD-binding agent binds one, two, three, four, five, or more FZD proteins. In some embodiments, the FZD-binding agent specifically binds one, two, three, four, or five FZD proteins selected from the group consisting of FZD 1, FZD2, FZD5, FZD7, and FZD8. In some embodiments, the FZD-binding agent specifically binds FZD1, FZD2, FZD5, FZD7, and FZD8.
  • the FZD-binding agent binds at least one human FZD protein with a dissociation constant (K D ) of about ⁇ or less, about ⁇ or less, about 40nM or less, about
  • a FZD-binding agent binds at least one FZD protein with a K D of about ⁇ or less. In some embodiments, a FZD-binding agent binds at least one FZD protein with a K D of about InM or less. In some embodiments, a FZD-binding agent binds at least one FZD protein with a K D of about O.lnM or less.
  • a FZD-binding agent binds each of one or more (e.g., 1, 2, 3, 4, or 5) of FZD1, FZD2, FZD5, FZD7, and FZD8 with a K D of about 40nM or less. In certain embodiments, the FZD-binding agent binds each of one or more of FZD1, FZD2, FZD5, FZD7, and FZD8 with a K D of about ⁇ or less. In certain embodiments, the FZD-binding agent binds each of FZD 1, FZD2, FZD5, FZD7, and FZD8 with a K D of about ⁇ .
  • the K D of the binding agent (e.g., an antibody) to a FZD protein is the K D determined using a FZD-Fc fusion protein comprising at least a portion of the FZD extracellular domain or FZD-Fri domain immobilized on a Biacore chip.
  • the FZD-binding agent binds one or more (for example, two or more, three or more, or four or more) human FZD proteins with an EC 50 of about ⁇ or less, about ⁇ or less, about 40nM or less, about 20nM or less, about ⁇ or less, or about InM or less.
  • a FZD-binding agent binds to more than one FZD protein with an EC 50 of about 40nM or less, about 20nM or less, or about ⁇ or less.
  • the FZD-binding agent has an EC 50 of about 20nM or less with respect to one or more (e.g., 1, 2, 3, 4, or 5) of the following FZD proteins: FZD1, FZD2, FZD5, FZD7, and FZD8. In certain embodiments, the FZD-binding agent has an EC 50 of about ⁇ or less with respect to one or more (e.g., 1, 2, 3, 4, or 5) of the following FZD proteins: FZD1, FZD2, FZD5, FZD7, and FZD 8.
  • the Wnt pathway inhibitor is a FZD-binding agent which is an antibody.
  • the antibody is a recombinant antibody.
  • the antibody is a monoclonal antibody.
  • the antibody is a chimeric antibody.
  • the antibody is a humanized antibody.
  • the antibody is a human antibody.
  • the antibody is an IgGl antibody.
  • the antibody is an IgG2 antibody.
  • the antibody is an antibody fragment comprising an antigen-binding site.
  • the antibody is monovalent, monospecific, or bivalent.
  • the antibody is a bispecific antibody or a multispecific antibody.
  • the antibody is conjugated to a cytotoxic moiety.
  • the antibody is isolated.
  • the antibody is substantially pure.
  • the FZD-binding agents (e.g., antibodies) of the present invention can be assayed for specific binding by any method known in the art.
  • the immunoassays which can be used include, but are not limited to, competitive and non-competitive assay systems using techniques such as Biacore analyses, FACS analyses, immunofluorescence, immunocytochemistry, Western blot analyses,
  • radioimmunoassays ELISA, "sandwich” immunoassays, immunoprecipitation assays, precipitation reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, and protein A immunoassays.
  • assays are routine and well-known in the art (see, e.g., Ausubel et al., Editors, 1994-present, Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York, NY).
  • the invention provides a FZD-binding agent which is an antibody that comprises a heavy chain CDRl comprising GFTFSHYTLS (SEQ ID NO: 1), a heavy chain CDR2 comprising VISGDGSYTYYADSVKG (SEQ ID NO:2), and a heavy chain CDR3 comprising NFIKYVFAN (SEQ ID NO:3).
  • the FZD-binding agent further comprises a light chain CDRl comprising SGDNIGSFYVH (SEQ ID NO:4), a light chain CDR2 comprising
  • the FZD-binding agent comprises a light chain CDRl comprising
  • the FZD- binding agent is an antibody that comprises: (a) a heavy chain CDRl comprising GFTFSHYTLS
  • SEQ ID NO: 1 a heavy chain CDR2 comprising VISGDGSYTYYADSVKG (SEQ ID NO:2), and a heavy chain CDR3 comprising NFIKYVFAN (SEQ ID NO:3)
  • a light chain CDRl comprising SGDNIGSFYVH (SEQ ID NO:4), a light chain CDR2 comprising DKSNRPSG (SEQ ID NO:5), and a light chain CDR3 comprising QSYANTLSL (SEQ ID NO:6).
  • the invention provides a FZD-binding agent (e.g., an antibody) that comprises: (a) a heavy chain CDRl comprising GFTFSHYTLS (SEQ ID NO: 1), or a variant thereof comprising 1, 2, 3, or 4 amino acid substitutions; (b) a heavy chain CDR2 comprising
  • VISGDGSYTYYADSVKG (SEQ ID NO:2), or a variant thereof comprising 1, 2, 3, or 4 amino acid substitutions;
  • a heavy chain CDR3 comprising NFIKYVFAN (SEQ ID NO:3), or a variant thereof comprising 1, 2, 3, or 4 amino acid substitutions;
  • a light chain CDRl comprising SGDNIGSFYVH (SEQ ID NO: 4), or a variant thereof comprising 1, 2, 3, or 4 amino acid substitutions;
  • a light chain CDR2 comprising DKSNRPSG (SEQ ID NO:5), or a variant thereof comprising 1, 2, 3, or 4 amino acid substitutions; and
  • a light chain CDR3 comprising
  • QSYANTLSL (SEQ ID NO:6), or a variant thereof comprising 1, 2, 3, or 4 amino acid substitutions.
  • the amino acid substitutions are conservative substitutions.
  • the invention provides a FZD-binding agent which is an antibody that comprises a heavy chain variable region having at least about 80% sequence identity to SEQ ID NO:7 and/or a light chain variable region having at least 80% sequence identity to SEQ ID NO:8.
  • the FZD-binding agent comprises a heavy chain variable region having at least about 85%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% sequence identity to SEQ ID NO:7.
  • the FZD-binding agent comprises a light chain variable region having at least about 85%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% sequence identity to SEQ ID NO: 8.
  • the FZD-binding agent comprises a heavy chain variable region having at least about 95% sequence identity to SEQ ID NO:7 and/or a light chain variable region having at least about 95% sequence identity to SEQ ID NO: 8. In certain embodiments, the FZD-binding agent comprises a heavy chain variable region comprising SEQ ID NO: 7 and/or a light chain variable region comprising SEQ ID NO: 8. In certain embodiments, the FZD-binding agent comprises a heavy chain variable region comprising SEQ ID NO: 7 and a light chain variable region comprising SEQ ID NO: 8.
  • the FZD- binding agent comprises a heavy chain variable region consisting essentially of SEQ ID NO:7 and a light chain variable region consisting essentially of SEQ ID NO: 8. In certain embodiments, the FZD- binding agent comprises a heavy chain variable region of SEQ ID NO:7 and a light chain variable region of SEQ ID NO: 8.
  • the invention provides a FZD-binding agent which is an antibody that comprises: (a) a heavy chain having at least 90% sequence identity to SEQ ID NO: 9 or SEQ ID NO: 11, and/or (b) a light chain having at least 90% sequence identity to SEQ ID NO: 10 or SEQ ID NO: 12.
  • the FZD-binding agent comprises: (a) a heavy chain having at least 95% sequence identity to SEQ ID NO: 9 or SEQ ID NO: 11; and/or (b) a light chain having at least 95% sequence identity to SEQ ID NO: 10 or SEQ ID NO: 12.
  • the FZD-binding agent comprises a heavy chain comprising SEQ ID NO: 11 and/or a light chain comprising SEQ ID NO: 12. In some embodiments, the FZD-binding agent comprises a heavy chain comprising SEQ ID NO: 11 and a light chain comprising SEQ ID NO: 12.
  • the invention provides a Wnt pathway inhibitor which is a FZD- binding agent (e.g., an antibody) that specifically binds at least one of FZD1, FZD2, FZD5, FZD7, and FZD8, wherein the FZD-binding agent (e.g., an antibody) comprises one, two, three, four, five, and/or six of the CDRs of antibody OMP-18R5.
  • Antibody OMP-18R5 also known as 18R5 and vantictumab
  • other FZD-binding agents has been previously described in U.S. Patent No. 7,982,013.
  • the FZD-binding agent comprises one or more of the CDRs of OMP-18R5, two or more of the CDRs of OMP-18R5, three or more of the CDRs of OMP-18R5, four or more of the CDRs of OMP-18R5, five or more of the CDRs of OMP-18R5, or all six of the CDRs of OMP-18R5.
  • the invention provides polypeptides which are Wnt pathway inhibitors.
  • the polypeptides include, but are not limited to, antibodies that specifically bind human FZD proteins.
  • a polypeptide binds one or more FZD proteins selected from the group consisting of FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD 8, FZD9, and FZD10.
  • a polypeptide binds FZD1, FZD2, FZD5, FZD7, and/or FZD8.
  • a polypeptide binds FZD1, FZD2, FZD5, FZD 7, and FZD 8.
  • a polypeptide comprises one, two, three, four, five, and/or six of the CDRs of antibody OMP-18R5. In some embodiments, a polypeptide comprises CDRs with up to four (i.e., 0, 1, 2, 3, or 4) amino acid substitutions per CDR. In certain embodiments, the heavy chain CDR(s) are contained within a heavy chain variable region. In certain embodiments, the light chain CDR(s) are contained within a light chain variable region.
  • the invention provides a polypeptide that specifically binds one or more human FZD proteins, wherein the polypeptide comprises an amino acid sequence having at least about 80% sequence identity to SEQ ID NO:7, and/or an amino acid sequence having at least about 80% sequence identity to SEQ ID NO:8.
  • the polypeptide comprises an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% sequence identity to SEQ ID NO:7.
  • the polypeptide comprises an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% sequence identity to SEQ ID NO:8.
  • the polypeptide comprises an amino acid sequence having at least about 95% sequence identity to SEQ ID NO: 7 and/or an amino acid sequence having at least about 95% sequence identity to SEQ ID NO:8. In certain embodiments, the polypeptide comprises an amino acid sequence comprising SEQ ID NO: 7 and/or an amino acid sequence comprising SEQ ID NO: 8.
  • a polypeptide comprises a sequence selected from the group consisting of: SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12.
  • a FZD-binding agent comprises a polypeptide comprising a sequence selected from the group consisting of: SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12.
  • a polypeptide comprises the heavy chain variable region and light chain variable region of the OMP-18R5 antibody. In certain embodiments, a polypeptide comprises the heavy chain and light chain of the OMP-18R5 antibody (with or without the leader sequence). In certain embodiments, a FZD-binding agent comprises a polypeptide comprising the heavy chain variable region and light chain variable region of the OMP-18R5 antibody. In certain embodiments, a FZD-binding agent comprises a polypeptide comprising the heavy chain and light chain of the OMP- 18R5 antibody (with or without the leader sequence).
  • a FZD-binding agent comprises, consists essentially of, or consists of, the antibody OMP-18R5.
  • a FZD-binding agent (e.g., antibody) competes for specific binding to one or more human FZD proteins with an antibody that comprises a heavy chain variable region comprising SEQ ID NO: 7 and a light chain variable region comprising SEQ ID NO: 8.
  • a FZD-binding agent (e.g., antibody) competes for specific binding to one or more human FZD proteins with an antibody that comprises a heavy chain comprising SEQ ID NO:9 (with or without the signal sequence) and a light chain comprising SEQ ID NO: 10 (with or without the signal sequence).
  • a FZD-binding agent (e.g., antibody) competes for specific binding to one or more human FZD proteins with an antibody that comprises a heavy chain comprising SEQ ID NO: 11 and a light chain comprising SEQ ID NO: 12.
  • a FZD-binding agent competes with antibody OMP-18R5 for specific binding to one or more human FZD proteins.
  • a FZD-binding agent or antibody competes for specific binding to one or more human FZD proteins in an in vitro competitive binding assay.
  • a FZD-binding agent binds the same epitope, or essentially the same epitope, on one or more human FZD proteins as an antibody described herein.
  • a FZD-binding agent is an antibody that binds an epitope on one or more human FZD proteins that overlaps with the epitope on a FZD protein bound by an antibody described herein.
  • a FZD-binding agent binds the same epitope, or essentially the same epitope, on one or more FZD proteins as antibody OMP-18R5.
  • the FZD-binding agent is an antibody that binds an epitope on one or more human FZD proteins that overlaps with the epitope on a FZD protein bound by antibody OMP-18R5.
  • the Wnt pathway inhibitors are polyclonal antibodies.
  • Polyclonal antibodies can be prepared by any known method.
  • polyclonal antibodies are raised by immunizing an animal (e.g., a rabbit, rat, mouse, goat, donkey) by multiple subcutaneous or intraperitoneal injections of an antigen of interest (e.g., a purified peptide fragment, full-length recombinant protein, or fusion protein).
  • the antigen can be optionally conjugated to a carrier such as keyhole limpet hemocyanin (KLH) or serum albumin.
  • KLH keyhole limpet hemocyanin
  • the antigen (with or without a carrier protein) is diluted in sterile saline and usually combined with an adjuvant (e.g., Complete or Incomplete Freund's Adjuvant) to form a stable emulsion.
  • an adjuvant e.g., Complete or Incomplete Freund's Adjuvant
  • polyclonal antibodies are recovered from blood and/or ascites of the immunized animal.
  • the polyclonal antibodies can be purified from serum or ascites according to standard methods in the art including, but not limited to, affinity chromatography, ion-exchange chromatography, gel electrophoresis, and dialysis.
  • the Wnt pathway inhibitors are monoclonal antibodies.
  • Monoclonal antibodies can be prepared using hybridoma methods known to one of skill in the art. In some embodiments, using the hybridoma method, a mouse, hamster, or other appropriate host animal, is immunized as described above to elicit the production of antibodies that will specifically bind the immunizing antigen.
  • isolated lymphocytes can be immunized in vitro.
  • the immunizing antigen can be a human protein or a fragment thereof. In some embodiments, the immunizing antigen can be a mouse protein or a fragment thereof.
  • lymphocytes are isolated and fused with a suitable myeloma cell line using, for example, polyethylene glycol, to form hybridoma cells that can then be selected away from unfused lymphocytes and myeloma cells.
  • Hybridomas that produce monoclonal antibodies directed specifically against a chosen antigen may be identified by a variety of methods including, but not limited to, immunoprecipitation, immunoblotting, and in vitro binding assay (e.g., flow cytometry, FACS, ELISA, and radioimmunoassay).
  • the hybridomas can be propagated either in in vitro culture using standard methods or in vivo as ascites tumors in an animal.
  • the monoclonal antibodies can be purified from the culture medium or ascites fluid according to standard methods in the art including, but not limited to, affinity chromatography, ion-exchange chromatography, gel electrophoresis, and dialysis.
  • monoclonal antibodies can be modified to increase affinity, also known as "affinity-matured” or “affinity maturation”.
  • affinity-matured antibodies may be produced by a variety of procedures known in the art. For example, techniques may include affinity maturation by heavy chain and light chain variable region domain shuffling, random mutagenesis of CDRs, random mutagenesis of framework residues, and/or site-directed mutagenesis.
  • monoclonal antibodies can be made using recombinant DNA techniques known to one skilled in the art.
  • the polynucleotides encoding a monoclonal antibody are isolated from mature B-cells or hybridoma cells, such as by RT-PCR using oligonucleotide primers that specifically amplify the genes encoding the heavy and light chains of the antibody, and their sequence is determined using conventional techniques.
  • the isolated polynucleotides encoding the heavy and light chains are then cloned into suitable expression vectors which produce the monoclonal antibodies when transfected into host cells such as E.
  • recombinant monoclonal antibodies, or fragments thereof can be isolated from phage display libraries.
  • the polynucleotide (s) encoding a monoclonal antibody can further be modified in a number of different manners using recombinant DNA technology to generate alternative antibodies.
  • the constant domains of the light and heavy chains of, for example, a mouse monoclonal antibody can be substituted for those regions of, for example, a human antibody to generate a chimeric antibody, or for a non-immunoglobulin polypeptide to generate a fusion antibody.
  • the constant regions are truncated or removed to generate the desired antibody fragment of a monoclonal antibody. Site-directed or high-density mutagenesis of the variable region can be used to optimize specificity, affinity, etc. of a monoclonal antibody.
  • the Wnt pathway inhibitor is a humanized antibody.
  • humanized antibodies are human immunoglobulins in which amino acid residues of the CDRs are replaced by amino acid residues of CDRs from an immunoglobulin of a non-human species (e.g., mouse, rat, rabbit, hamster, etc.) that have the desired specificity, affinity, and/or binding capability.
  • framework variable region amino acid residues of a human immunoglobulin are replaced with corresponding amino acid residues from an antibody of a non-human species.
  • the humanized antibody can be further modified by the substitution of additional amino acid residues either in the framework variable region and/or within the replaced non-human amino acid residues to further refine and optimize antibody specificity, affinity, and/or binding capability.
  • a humanized antibody will comprise all, or substantially all, of the CDRs that correspond to the non-human immunoglobulin whereas all, or substantially all, of the framework variable regions are those of a human immunoglobulin sequence.
  • the humanized antibody can also comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin.
  • Fc immunoglobulin constant region or domain
  • such humanized antibodies are used therapeutically because they may reduce antigenicity and HAMA (human anti- mouse antibody) responses when administered to a human subject. Methods used to generate humanized antibodies are well known in the art.
  • the Wnt pathway inhibitor is a human antibody.
  • Human antibodies can be directly prepared using various techniques known in the art.
  • immortalized human B lymphocytes immunized in vitro or isolated from an immunized individual that produces an antibody directed against a target antigen can be generated.
  • the human antibody can be selected from a phage library, where that phage library expresses human antibodies.
  • phage display technology can be used to produce human antibodies and antibody fragments in vitro, from immunoglobulin variable domain gene repertoires from unimmunized donors. Techniques for the generation and use of antibody phage libraries are well- known in the art. Affinity maturation strategies including, but not limited to, chain shuffling and site- directed mutagenesis, are known in the art and may be employed to generate high affinity human antibodies.
  • human antibodies can be made in transgenic mice that contain human immunoglobulin loci. These mice are capable, upon immunization, of producing the full repertoire of human antibodies in the absence of endogenous immunoglobulin production.
  • This invention also encompasses bispecific antibodies that specifically recognize at least one human FZD protein.
  • Bispecific antibodies are capable of specifically recognizing and binding at least two different epitopes.
  • the different epitopes can either be within the same molecule (e.g., two different epitopes on human FZD7) or on different molecules (e.g., one epitope on FZD7 and a different epitope on a second protein).
  • the bispecific antibodies are monoclonal antibodies.
  • the bispecific antibodies are humanized antibodies.
  • the bispecific antibodies are human antibodies.
  • the antibodies can specifically recognize and bind a first antigen target, (e.g., a FZD protein) as well as a second antigen target, such as an effector molecule on a leukocyte (e.g., CD2, CD3, CD28, CD80, or CD86) or a Fc receptor (e.g., CD64, CD32, or CD16) so as to focus cellular defense mechanisms to the cell expressing the first antigen target.
  • the antibodies can be used to direct cytotoxic agents to cells which express a particular target antigen.
  • the antibodies possess an antigen-binding arm and an arm which binds a cytotoxic agent or a radionuclide chelator, such as EOTUBE, DPTA, DOTA, or TETA.
  • Bispecific antibodies can be intact antibodies or antibody fragments. Antibodies with more than two valencies are also contemplated (e.g., trispecific antibodies). Thus, in certain embodiments the antibodies are multispecific. Techniques for making bispecific and multispecific antibodies are known by those skilled in the art.
  • the antibodies (or other polypeptides) described herein may be monospecific.
  • each of the one or more antigen-binding sites that an antibody contains is capable of binding (or binds) a homologous epitope on different proteins.
  • an antigen-binding site of a monospecific antibody described herein is capable of binding (or binds), for example, FZD5 and FZD7 (i.e., the same epitope is found on both FZD5 and FZD7 proteins).
  • the Wnt pathway inhibitor is an antibody fragment comprising an antigen-binding site.
  • Antibody fragments may have different functions or capabilities than intact antibodies; for example, antibody fragments can have increased tumor penetration.
  • Various techniques are known for the production of antibody fragments including, but not limited to, proteolytic digestion of intact antibodies.
  • antibody fragments include a F(ab')2 fragment produced by pepsin digestion of an antibody molecule.
  • antibody fragments include a Fab fragment generated by reducing the disulfide bridges of an F(ab')2 fragment.
  • antibody fragments include a Fab fragment generated by the treatment of the antibody molecule with papain and a reducing agent.
  • antibody fragments are produced recombinantly.
  • antibody fragments include Fv or single chain Fv (scFv) fragments.
  • Fab, Fv, and scFv antibody fragments can be expressed in and secreted from E. coli or other host cells, allowing for the production of large amounts of these fragments.
  • antibody fragments are isolated from antibody phage libraries as discussed herein. For example, methods can be used for the construction of Fab expression libraries to allow rapid and effective identification of monoclonal Fab fragments with the desired specificity for a FZD protein or derivatives, fragments, analogs or homologs thereof.
  • antibody fragments are linear antibody fragments.
  • antibody fragments are monospecific or bispecific.
  • the Wnt pathway inhibitor is a scFv.
  • Various techniques can be used for the production of single-chain antibodies specific to one or more human FZD proteins.
  • an antibody can further be desirable, especially in the case of antibody fragments, to modify an antibody in order to increase its serum half-life. This can be achieved, for example, by incorporation of a salvage receptor binding epitope into the antibody fragment by mutation of the appropriate region in the antibody fragment or by incorporating the epitope into a peptide tag that is then fused to the antibody fragment at either end or in the middle (e.g., by DNA or peptide synthesis). In some embodiments, an antibody is modified to decrease its serum half-life.
  • Heteroconjugate antibodies are composed of two covalently joined antibodies. Such antibodies have, for example, been proposed to target immune cells to tumor cells. It is also contemplated that the heteroconjugate antibodies can be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents. For example, immunotoxins can be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl -4-mercaptobutyrimidate.
  • modified antibodies can comprise any type of variable region that provides for the association of the antibody with the target (i.e., a human FZD protein).
  • the variable region may comprise or be derived from any type of mammal that can be induced to mount a humoral response and generate
  • variable region of the modified antibodies can be, for example, of human, murine, non-human primate (e.g. cynomolgus monkeys, macaques, etc.), or rabbit origin.
  • both the variable and constant regions of the modified immunoglobulins are human.
  • the variable regions of compatible antibodies (usually derived from a non-human source) can be engineered or specifically tailored to improve the binding properties or reduce the immunogenicity of the molecule.
  • variable regions useful in the present invention can be humanized or otherwise altered through the inclusion of imported amino acid residues or sequences.
  • variable domains in both the heavy and light chains are altered by at least partial replacement of one or more CDRs and, if necessary, by partial framework region replacement and sequence modification and/or alteration.
  • the CDRs may be derived from an antibody of the same class or even subclass as the antibody from which the framework regions are derived, it is envisaged that the CDRs will be derived preferably from an antibody from a different species. It may not be necessary to replace all of the CDRs with all of the CDRs from the donor variable region to transfer the antigen binding capacity of one variable domain to another. Rather, it may only be necessary to transfer those residues that are necessary to maintain the activity of the antigen-binding site.
  • the modified antibodies of this invention will comprise antibodies (e.g., full-length antibodies or immunoreactive fragments thereof) in which at least a fraction of one or more of the constant region domains has been deleted or otherwise altered so as to provide desired biochemical characteristics such as increased tumor localization and/or increased serum half-life when compared with an antibody of approximately the same immunogenicity comprising a native or unaltered constant region.
  • the constant region of the modified antibodies will comprise a human constant region.
  • Modifications to the constant region compatible with this invention comprise additions, deletions or substitutions of one or more amino acids in one or more domains.
  • the modified antibodies disclosed herein may comprise alterations or modifications to one or more of the three heavy chain constant domains (CHI, CH2 or CH3) and/or to the light chain constant domain (CL).
  • one or more domains are partially or entirely deleted from the constant regions of the modified antibodies.
  • the modified antibodies will comprise domain deleted constructs or variants wherein the entire CH2 domain has been removed (ACH2 constructs).
  • the omitted constant region domain is replaced by a short amino acid spacer (e.g., 10 amino acid residues) that provides some of the molecular flexibility typically imparted by the absent constant region.
  • the modified antibodies are engineered to fuse the CH3 domain directly to the hinge region of the antibody.
  • a peptide spacer is inserted between the hinge region and the modified CH2 and/or CH3 domains.
  • constructs may be expressed wherein the CH2 domain has been deleted and the remaining CH3 domain (modified or unmodified) is joined to the hinge region with a 5-20 amino acid spacer.
  • Such a spacer may be added to ensure that the regulatory elements of the constant domain remain free and accessible or that the hinge region remains flexible.
  • amino acid spacers may, in some cases, prove to be immunogenic and elicit an unwanted immune response against the construct.
  • any spacer added to the construct will be relatively non- immunogenic so as to maintain the desired biological qualities of the modified antibodies.
  • the modified antibodies may have only a partial deletion of a constant domain or substitution of a few or even a single amino acid.
  • the mutation of a single amino acid in selected areas of the CH2 domain may be enough to substantially reduce Fc binding and thereby increase cancer cell localization and/or tumor penetration.
  • Such partial deletions of the constant regions may improve selected characteristics of the antibody (serum half-life) while leaving other desirable functions associated with the subject constant region domain intact.
  • the constant regions of the disclosed antibodies may be modified through the mutation or substitution of one or more amino acids that enhances the profile of the resulting construct.
  • the modified antibodies comprise the addition of one or more amino acids to the constant region to enhance desirable characteristics such as decreasing or increasing effector function or provide for more cytotoxin or carbohydrate attachment sites.
  • the constant region mediates several effector functions. For example, binding of the CI component of complement to the Fc region of IgG or IgM antibodies (bound to antigen) activates the complement system. Activation of complement is important in the opsonization and lysis of cell pathogens. The activation of complement also stimulates the inflammatory response and can also be involved in autoimmune hypersensitivity.
  • the Fc region of an antibody can bind a cell expressing a Fc receptor (FcR).
  • Fc receptors There are a number of Fc receptors which are specific for different classes of antibody, including IgG (gamma receptors), IgE (epsilon receptors), IgA (alpha receptors) and IgM (mu receptors).
  • the Wnt pathway inhibitors are antibodies that provide for altered effector functions. These altered effector functions may affect the biological profile of the administered antibody. For example, in some embodiments, the deletion or inactivation (through point mutations or other means) of a constant region domain may reduce Fc receptor binding of the circulating modified antibody (e.g., anti-FZD antibody) thereby increasing cancer cell localization and/or tumor penetration.
  • the deletion or inactivation (through point mutations or other means) of a constant region domain may reduce Fc receptor binding of the circulating modified antibody (e.g., anti-FZD antibody) thereby increasing cancer cell localization and/or tumor penetration.
  • the constant region modifications increase or reduce the serum half-life of the antibody.
  • the constant region is modified to eliminate disulfide linkages or oligosaccharide moieties. Modifications to the constant region in accordance with this invention may easily be made using well known biochemical or molecular engineering techniques well within the purview of the skilled artisan.
  • a Wnt pathway inhibitor is an antibody does not have one or more effector functions.
  • the antibody has no ADCC activity and/or no CDC activity.
  • the antibody does not bind an Fc receptor and/or complement factors.
  • the antibody has no effector function.
  • the present invention further embraces variants and equivalents which are substantially homologous to the chimeric, humanized, and human antibodies, or antibody fragments thereof, set forth herein. These can contain, for example, conservative substitution mutations.
  • the antibodies described herein are isolated. In certain embodiments, the antibodies described herein are substantially pure.
  • the Wnt pathway inhibitors are polypeptides.
  • the polypeptides can be recombinant polypeptides, natural polypeptides, or synthetic polypeptides comprising an antibody, or fragment thereof, that bind at least one human FZD protein or at least one Wnt protein. It will be recognized in the art that some amino acid sequences of the invention can be varied without significant effect on the structure or function of the protein. Thus, the invention further includes variations of the polypeptides which show substantial activity or which include regions of an antibody, or fragment thereof, against one of more human FZD proteins. In some embodiments, amino acid sequence variations of FZD-binding polypeptides include deletions, insertions, inversions, repeats, and/or other types of substitutions.
  • polypeptides, analogs and variants thereof can be further modified to contain additional chemical moieties not normally part of the polypeptide.
  • the derivatized moieties can improve the solubility, the biological half-life, and/or absorption of the polypeptide.
  • the moieties can also reduce or eliminate any undesirable side effects of the polypeptides and variants.
  • An overview for chemical moieties can be found in Remington: The Science and Practice of Pharmacy, 22st Edition, 2012, Pharmaceutical Press, London.
  • the isolated polypeptides that can be used in the methods described herein can be produced by any suitable method known in the art.
  • Such methods range from direct protein synthesis methods to constructing a DNA sequence encoding polypeptide sequences and expressing those sequences in a suitable host.
  • a DNA sequence is constructed using recombinant technology by isolating or synthesizing a DNA sequence encoding a wild-type protein of interest.
  • the sequence can be mutagenized by site-specific mutagenesis to provide functional analogs thereof.
  • a DNA sequence encoding a polypeptide of interest may be constructed by chemical synthesis using an oligonucleotide synthesizer. Oligonucleotides can be designed based on the amino acid sequence of the desired polypeptide and selecting those codons that are favored in the host cell in which the recombinant polypeptide of interest will be produced.
  • Standard methods can be applied to synthesize a polynucleotide sequence encoding an isolated polypeptide of interest. For example, a complete amino acid sequence can be used to construct a back-translated gene. Further, a DNA oligomer containing a nucleotide sequence coding for the particular isolated polypeptide can be synthesized. For example, several small oligonucleotides coding for portions of the desired polypeptide can be synthesized and then ligated. The individual oligonucleotides typically contain 5' or 3' overhangs for complementary assembly.
  • the polynucleotide sequences encoding a particular polypeptide of interest can be inserted into an expression vector and operatively linked to an expression control sequence appropriate for expression of the protein in a desired host. Proper assembly can be confirmed by nucleotide sequencing, restriction enzyme mapping, and/or expression of a biologically active polypeptide in a suitable host. As is well-known in the art, in order to obtain high expression levels of a transfected gene in a host, the gene must be operatively linked to transcriptional and translational expression control sequences that are functional in the chosen expression host.
  • recombinant expression vectors are used to amplify and express
  • DNA encoding binding agents e.g., antibodies or soluble receptors
  • binding agents e.g., antibodies or soluble receptors
  • recombinant expression vectors can be replicable DNA constructs which have synthetic or cDNA-derived DNA fragments encoding a polypeptide chain of a FZD-binding agent or an anti-FZD antibody or fragment thereof, operatively linked to suitable transcriptional and/or translational regulatory elements derived from mammalian, microbial, viral or insect genes.
  • a transcriptional unit generally comprises an assembly of (1) a genetic element or elements having a regulatory role in gene expression, for example, transcriptional promoters or enhancers, (2) a structural or coding sequence which is transcribed into mRNA and translated into protein, and (3) appropriate transcription and translation initiation and termination sequences.
  • DNA regions are "operatively linked" when they are functionally related to each other.
  • DNA for a signal peptide secretory leader
  • a promoter is operatively linked to a coding sequence if it controls the transcription of the sequence
  • a ribosome binding site is operatively linked to a coding sequence if it is positioned so as to permit translation.
  • structural elements intended for use in yeast expression systems include a leader sequence enabling extracellular secretion of translated protein by a host cell.
  • recombinant protein when expressed without a leader or transport sequence, it can include an N-terminal methionine residue. This residue can optionally be subsequently cleaved from the expressed recombinant protein to provide a final product.
  • Useful expression vectors for eukaryotic hosts include, for example, vectors comprising expression control sequences from SV40, bovine papilloma virus, adenovirus, and cytomegalovirus.
  • Useful expression vectors for bacterial hosts include known bacterial plasmids, such as plasmids from E. coli, including pCRl, pBR322, pMB9 and their derivatives, and wider host range plasmids, such as Ml 3 and other filamentous single-stranded DNA phages.
  • Suitable host cells for expression of a FZD-binding agent include prokaryotes, yeast cells, insect cells, or higher eukaryotic cells under the control of appropriate promoters.
  • Prokaryotes include gram-negative or gram -positive organisms, for example E. coli or Bacillus.
  • Higher eukaryotic cells include established cell lines of mammalian origin as described below. Cell-free translation systems may also be employed. Appropriate cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cellular hosts are well-known in the art.
  • mammalian host cell lines include COS-7 (monkey kidney-derived), L-929 (murine fibroblast- derived), C127 (murine mammary tumor-derived), 3T3 (murine fibroblast-derived), CHO (Chinese hamster ovary-derived), HeLa (human cervical cancer-derived), BHK (hamster kidney fibroblast- derived), HEK-293 (human embryonic kidney-derived) cell lines and variants thereof.
  • Mammalian expression vectors can comprise non-transcribed elements such as an origin of replication, a suitable promoter and enhancer linked to the gene to be expressed, and other 5 Or 3' flanking non-transcribed sequences, and 5' or 3' non-translated sequences, such as necessary ribosome binding sites, a polyadenylation site, splice donor and acceptor sites, and transcriptional termination sequences.
  • non-transcribed elements such as an origin of replication, a suitable promoter and enhancer linked to the gene to be expressed, and other 5 Or 3' flanking non-transcribed sequences, and 5' or 3' non-translated sequences, such as necessary ribosome binding sites, a polyadenylation site, splice donor and acceptor sites, and transcriptional termination sequences.
  • Baculovirus systems for production of heterologous proteins in insect cells are well-known to those of skill in the art.
  • the present invention provides cells comprising the FZD-binding agents described herein.
  • the cells produce the binding agents (e.g., antibodies) described herein.
  • the cells produce an antibody.
  • the cells produce antibody OMP- 18R5 (vantictumab) .
  • the proteins produced by a host cell can be purified according to any suitable method.
  • Standard methods include chromatography (e.g., ion exchange, affinity, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for protein purification.
  • Affinity tags such as hexa-histidine, maltose binding domain, influenza coat sequence, and glutathione-S-transferase can be attached to the protein to allow easy purification by passage over an appropriate affinity column.
  • Isolated proteins can also be physically characterized using such techniques as proteolysis, mass spectrometry (MS), nuclear magnetic resonance (NMR), high performance liquid chromatography (HPLC), and x-ray crystallography.
  • supernatants from expression systems which secrete recombinant protein into culture media can be first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. Following the concentration step, the concentrate can be applied to a suitable purification matrix.
  • a suitable purification matrix for example, an anion exchange resin can be employed, for example, a matrix or substrate having pendant diethylaminoethyl (DEAE) groups.
  • the matrices can be acrylamide, agarose, dextran, cellulose, or other types commonly employed in protein purification.
  • a cation exchange step can be employed.
  • Suitable cation exchangers include various insoluble matrices comprising sulfopropyl or carboxymethyl groups.
  • a hydroxyapatite media can be employed, including but not limited to, ceramic hydroxyapatite (CHT).
  • CHT ceramic hydroxyapatite
  • one or more reverse-phase HPLC steps employing hydrophobic RP-HPLC media, e.g., silica gel having pendant methyl or other aliphatic groups can be employed to further purify a binding agent.
  • hydrophobic RP-HPLC media e.g., silica gel having pendant methyl or other aliphatic groups
  • Some or all of the foregoing purification steps, in various combinations, can also be employed to provide a homogeneous recombinant protein.
  • recombinant protein produced in bacterial culture can be isolated, for example, by initial extraction from cell pellets, followed by one or more concentration, salting-out, aqueous ion exchange, or size exclusion chromatography steps. HPLC can be employed for final purification steps.
  • Microbial cells employed in expression of a recombinant protein can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents.
  • the FZD-binding agent is a polypeptide that is not an antibody.
  • a variety of methods for identifying and producing non-antibody polypeptides that bind with high affinity to a protein target are known in the art.
  • phage display technology may be used to produce and/or identify a FZD-binding polypeptide.
  • the polypeptide comprises a protein scaffold of a type selected from the group consisting of protein A, protein G, a lipocalin, a fibronectin domain, an ankyrin consensus repeat domain, and thioredoxin.
  • the binding agents can be used in any one of a number of conjugated (i.e. an immunoconjugate or radioconjugate) or non-conjugated forms.
  • conjugated i.e. an immunoconjugate or radioconjugate
  • non-conjugated forms i.e. antibodies can be used in a non-conjugated form to harness the subject's natural defense mechanisms including complement-dependent cytotoxicity and antibody dependent cellular toxicity to eliminate the malignant or cancer cells.
  • the binding agent is conjugated to a cytotoxic agent.
  • the cytotoxic agent is a chemotherapeutic agent including, but not limited to, methotrexate, adriamycin, doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents.
  • the cytotoxic agent is an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof, including, but not limited to, diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain, 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.
  • diphtheria A chain nonbinding active fragments of diphtheria toxin
  • exotoxin A chain ricin A chain
  • abrin A chain abrin A chain
  • modeccin A chain alpha-s
  • the cytotoxic agent is a radioisotope to produce a radioconjugate or a radioconjugated antibody.
  • a radionuclides are available for the production of
  • radioconjugated antibodies including, but not limited to, 90 Y, 125 I, 131 1, 123 I, m In, 131 In, 105 Rh, 153 Sm, 67 Cu, 67 Ga, 166 Ho, 177 Lu, 186 Re, 188 Re and 212 Bi.
  • conjugates of an antibody and one or more small molecule toxins such as a calicheamicin, maytansinoids, a trichothene, and CC1065, and the derivatives of these toxins that have toxin activity, can be produced.
  • small molecule toxins such as a calicheamicin, maytansinoids, a trichothene, and CC1065
  • conjugates of an antibody and a cytotoxic agent are made using a variety of bifunctional protein-coupling agents such as N-succinimidyl-3-(2-pyridyidithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), 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).
  • SPDP N-succinimidyl
  • the Wnt pathway inhibitor (e.g., an anti-FZD antibody) is an antagonist of at least one Wnt protein (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 Wnt proteins).
  • the Wnt pathway inhibitor inhibits activity of the Wnt protein(s) to which it binds.
  • the Wnt pathway inhibitor inhibits at least about 10%, at least about 20%, at least about 30%, at least about 50%, at least about 75%, at least about 90%, or about 100% of the activity of the human Wnt protein(s) to which it binds.
  • the Wnt pathway inhibitor (e.g., an anti-FZD antibody) inhibits binding of at least one human Wnt to an appropriate receptor. In certain embodiments, the Wnt pathway inhibitor inhibits binding of at least one human Wnt protein to one or more human FZD proteins.
  • the at least one Wnt protein is selected from the group consisting of: Wntl, Wnt2, Wnt2b/13, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, WntlOa, WntlOb, Wntl 1, and Wntl6.
  • the one or more human FZD proteins are selected from the group consisting of: FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, and FZD10.
  • the Wnt pathway inhibitor inhibits binding of one or more Wnt proteins to FZD1, FZD2, FZD5, FZD7, and/or FZD8.
  • the inhibition of binding of a particular Wnt to a FZD protein by a Wnt pathway inhibitor is at least about 10%, at least about 25%, at least about 50%, at least about 75%, at least about 90%, or at least about 95%.
  • an agent that inhibits binding of a Wnt to a FZD protein also inhibits Wnt pathway signaling.
  • a Wnt pathway inhibitor that inhibits human Wnt pathway signaling is an antibody.
  • the Wnt pathway inhibitor described herein is an antagonist of at least one human FZD protein and inhibits FZD activity. In certain embodiments, the Wnt pathway inhibitor inhibits FZD activity by at least about 10%, at least about 20%, at least about 30%, at least about 50%, at least about 75%, at least about 90%, or about 100%. In some embodiments, the Wnt pathway inhibitor inhibits activity of one, two, three, four, five or more FZD proteins.
  • the Wnt pathway inhibitor inhibits activity of at least one human FZD protein selected from the group consisting of: FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD 8, FZD 9, and FZD10. In certain embodiments, the Wnt pathway inhibitor inhibits activity of FZD1, FZD2, FZD5, FZD7, and/or FZD8. In some embodiments, the Wnt pathway inhibitor is an anti-FZD antibody. In certain embodiments, the Wnt pathway inhibitor is anti-FZD antibody OMP- 18R5.
  • a Wnt pathway inhibitor described herein is an antagonist of ⁇ - catenin signaling.
  • the Wnt pathway inhibitor inhibits ⁇ -catenin signaling by at least about 10%, at least about 20%, at least about 30%, at least about 50%, at least about 75%, at least about 90%, or about 100%.
  • a Wnt pathway inhibitor that inhibits ⁇ - catenin signaling is an antibody.
  • a Wnt pathway inhibitor that inhibits ⁇ - catenin signaling is an anti-FZD antibody.
  • a Wnt pathway inhibitor that inhibits ⁇ -catenin signaling is antibody OMP-18R5.
  • the Wnt pathway inhibitor described herein inhibits binding of at least one Wnt protein to a receptor. In certain embodiments, the Wnt pathway inhibitor inhibits binding of at least one human Wnt protein to one or more of its receptors. In some embodiments, the Wnt pathway inhibitor inhibits binding of at least one Wnt protein to at least one FZD protein. In some embodiments, the Wnt-binding agent inhibits binding of at least one Wnt protein to FZD1,
  • the inhibition of binding of at least one Wnt to at least one FZD protein is at least about 10%, at least about 25%, at least about 50%, at least about 75%, at least about 90%, or at least about 95%.
  • a Wnt pathway inhibitor that inhibits binding of at least one Wnt to at least one FZD protein further inhibits Wnt pathway signaling and/or ⁇ -catenin signaling.
  • a Wnt pathway inhibitor that inhibits binding of at least one human Wnt to at least one FZD protein is an antibody.
  • a Wnt pathway inhibitor that inhibits binding of at least one human Wnt to at least one FZD protein is an anti-FZD antibody. In certain embodiments, a Wnt pathway inhibitor that inhibits binding of at least one human Wnt to at least one FZD protein is antibody OMP-18R5.
  • the Wnt pathway inhibitor described herein blocks binding of at least one Wnt to a receptor.
  • the Wnt pathway inhibitor blocks binding of at least one human Wnt protein to one or more of its receptors.
  • the Wnt pathway inhibitor blocks binding of at least one Wnt to at least one FZD protein.
  • the Wnt pathway inhibitor blocks binding of at least one Wnt protein to FZD1, FZD2, FZD3, FZD4,
  • the blocking of binding of at least one Wnt to at least one FZD protein is at least about 10%, at least about 25%, at least about 50%, at least about 75%, at least about 90%, or at least about 95%.
  • a Wnt pathway inhibitor that blocks binding of at least one Wnt protein to at least one FZD protein further inhibits Wnt pathway signaling and/or ⁇ -catenin signaling.
  • a Wnt pathway inhibitor that blocks binding of at least one human Wnt to at least one FZD protein is an antibody.
  • a Wnt pathway inhibitor that blocks binding of at least one human Wnt to at least one FZD protein is an anti-FZD antibody.
  • a Wnt pathway inhibitor that blocks binding of at least one human Wnt to at least one FZD protein is antibody OMP-18R5.
  • the Wnt pathway inhibitor described herein inhibits Wnt pathway signaling. It is understood that a Wnt pathway inhibitor that inhibits Wnt pathway signaling may, in certain embodiments, inhibit signaling by one or more receptors in the Wnt signaling pathway but not necessarily inhibit signaling by all receptors. In certain alternative embodiments, Wnt pathway signaling by all human receptors may be inhibited.
  • Wnt pathway signaling by one or more receptors selected from the group consisting of FZD1, FZD2, FZD3, FZD4, FDZ5, FDZ6, FDZ7, FDZ8, FDZ9, and FZD10 is inhibited.
  • the inhibition of Wnt pathway signaling by a Wnt pathway inhibitor is a reduction in the level of Wnt pathway signaling of at least about 10%, at least about 25%, at least about 50%, at least about 75%, at least about 90%, or at least about 95%.
  • a Wnt pathway inhibitor that inhibits Wnt pathway signaling is an antibody.
  • a Wnt pathway inhibitor that inhibits Wnt pathway signaling is an anti-FZD antibody.
  • a Wnt pathway inhibitor that inhibits Wnt pathway signaling is antibody OMP-18R5.
  • the Wnt pathway inhibitor described herein inhibits activation of ⁇ - catenin. It is understood that a Wnt pathway inhibitor that inhibits activation of ⁇ -catenin may, in certain embodiments, inhibit activation of ⁇ -catenin by one or more receptors, but not necessarily inhibit activation of ⁇ -catenin by all receptors. In certain alternative embodiments, activation of ⁇ - catenin by all human receptors may be inhibited.
  • activation of ⁇ -catenin by one or more receptors selected from the group consisting of FZD1, FZD2, FZD3, FZD4, FDZ5, FDZ6, FDZ7, FDZ8, FDZ9, and FZD10 is inhibited.
  • the inhibition of activation of ⁇ -catenin by a Wnt-binding agent is a reduction in the level of activation of ⁇ -catenin of at least about 10%, at least about 25%, at least about 50%, at least about 75%, at least about 90%, or at least about 95%.
  • a Wnt pathway inhibitor that inhibits activation of ⁇ - catenin is an antibody.
  • a Wnt pathway inhibitor that inhibits activation of ⁇ - catenin is an anti-FZD antibody. In some embodiments, a Wnt pathway inhibitor that inhibits activation of ⁇ -catenin is antibody OMP- 18R5.
  • TCF/Luc reporter vector containing multiple copies of the TCF-binding domain upstream of a firefly luciferase reporter gene may be used to measure ⁇ -catenin signaling levels in vitro (Gazit et al., 1999, Oncogene, 18; 5959-66; TOPflash, Millipore, Billerica MA).
  • the level of ⁇ -catenin signaling in the presence of one or more Wnt proteins e.g., Wnt(s) expressed by transfected cells or provided by Wnt- conditioned media
  • Wnt proteins e.g., Wnt(s) expressed by transfected cells or provided by Wnt- conditioned media
  • the effect of a binding agent (or candidate agent) on ⁇ -catenin signaling may be measured in vitro or in vivo by measuring the effect of the agent on the level of expression of ⁇ -catenin-regulated genes, such as c-myc, cyclin Dl, and/or fibronectin.
  • the effect of a binding agent on ⁇ -catenin signaling may also be assessed by measuring the effect of the agent on the phosphorylation state of Dishevelled- 1,
  • Dishevelled-2 Dishevelled-3, LRP5, LRP6, and/or ⁇ -catenin.
  • a Wnt pathway inhibitor has one or more of the following effects: inhibit proliferation of tumor cells, inhibit tumor growth, reduce the frequency of cancer stem cells in a tumor, reduce the tumorigenicity of a tumor, reduce the tumorigenicity of a tumor by reducing the frequency of cancer stem cells in the tumor, trigger cell death of tumor cells, induce cells in a tumor to differentiate, differentiate tumorigenic cells to a non-tumorigenic state, induce expression of differentiation markers in the tumor cells, prevent metastasis of tumor cells, or decrease survival of tumor cells.
  • a Wnt pathway inhibitor is capable of inhibiting tumor growth.
  • a Wnt pathway inhibitor is capable of inhibiting tumor growth in vivo (e.g., in a xenograft mouse model, and/or in a human having cancer).
  • the tumor is a pancreatic tumor.
  • the tumor is a Wnt-dependent tumor.
  • a Wnt pathway inhibitor is capable of reducing the tumorigenicity of a tumor.
  • a Wnt pathway inhibitor is capable of reducing the tumorigenicity of a tumor comprising cancer stem cells in an animal model, such as a mouse xenograft model.
  • the number or frequency of cancer stem cells in a tumor is reduced by at least about two-fold, about three-fold, about five-fold, about ten-fold, about 50-fold, about 100-fold, or about 1000-fold.
  • the reduction in the number or frequency of cancer stem cells is determined by limiting dilution assay using an animal model.
  • a Wnt pathway inhibitor described herein is active in vivo for at least 1 hour, at least about 2 hours, at least about 5 hours, at least about 10 hours, at least about 24 hours, at least about 2 days, at least about 3 days, at least about 1 week, or at least about 2 weeks.
  • the Wnt pathway inhibitor is an IgG (e.g., IgGl or IgG2) antibody that is active in vivo for at least 1 hour, at least about 2 hours, at least about 5 hours, at least about 10 hours, at least about 24 hours, at least about 2 days, at least about 3 days, at least about 1 week, or at least about 2 weeks.
  • the Wnt pathway inhibitor is a fusion protein that is active in vivo for at least 1 hour, at least about 2 hours, at least about 5 hours, at least about 10 hours, at least about 24 hours, at least about 2 days, at least about 3 days, at least about 1 week, or at least about 2 weeks.
  • a Wnt pathway inhibitor described herein has a circulating half-life in mice, cynomolgus monkeys, or humans of at least about 5 hours, at least about 10 hours, at least about 24 hours, at least about 2 days, at least about 3 days, at least about 1 week, or at least about 2 weeks.
  • the Wnt pathway inhibitor is an IgG (e.g., IgGl or IgG2) antibody that has a circulating half-life in mice, cynomolgus monkeys, or humans of at least about 5 hours, at least about 10 hours, at least about 24 hours, at least about 2 days, at least about 3 days, at least about 1 week, or at least about 2 weeks.
  • IgG e.g., IgGl or IgG2
  • Kits for practicing the methods of the invention are further provided.
  • kit any manufacture (e.g., a package or a container) comprising at least one reagent, e.g., an antibody, a nucleic acid probe, etc. for specifically detecting the expression of at least one biomarker of the invention.
  • the kit may be promoted, distributed, and/or sold as a unit for performing the methods of the present invention. Additionally, the kits may contain a package insert describing the kit and including instructional material for its use.
  • a kit comprises reagents for practicing the methods of the invention using microarray technology.
  • a kit comprises reagents for practicing the methods of the invention using qPCR assays.
  • Positive and/or negative controls may be included in the kits to validate the activity and correct usage of reagents employed in accordance with the invention. Controls may include samples known to be either positive or negative for the presence of the biomarker of interest, or other samples comprising the biomarkers of interest. The design and use of controls is standard and well within the routine capabilities of those in the art.
  • Embodiments of the present disclosure can be further defined by reference to the following non-limiting examples, which describe in detail preparation of certain antibodies of the present disclosure and methods for using antibodies of the present disclosure. It will be apparent to those skilled in the art that many modifications, both to materials and methods, may be practiced without departing from the scope of the present disclosure. EXAMPLES
  • pancreatic tumor xenograft models OMP-PN4, OMP-PN7, OMP-PN9, OMP-PN13, OMP-PN16, OMP-PN17, OMP-PN18, OMP-PN21, OMP-PN23, OMP-PN25, OMP-PN31, OMP- PN33, OMP-PN38 and OMP-PN40 were established at OncoMed Pharmaceuticals from minimally passaged, patient-derived tumor specimens. Six- to 8-week-old NOD/SCID mice were established at OncoMed Pharmaceuticals from minimally passaged, patient-derived tumor specimens. Six- to 8-week-old NOD/SCID mice were
  • Tumors were allowed to grow until they reached an average volume of 100 to 150mm 3 .
  • tumor volume data from mice treated with anti-FZD antibody 18R5 in combination with gemcitabine and ABRAXANE was compared with tumor volume data from mice treated with the combination of gemcitabine and ABRAXANE.
  • a "responder" tumor was defined as a tumor showing significantly greater tumor growth inhibition with the combination of OMP-18R5, gemcitabine, and ABRAXANE as compared to tumor growth inhibition with combination of gemcitabine and ABRAXANE.
  • the combination of gemcitabine and ABRAXANE is considered the "standard of care" (SOC) for treatment of pancreatic cancer in humans.
  • Analyses were performed using 465 curated genes from several signaling pathways including canonical, planar cell polarity, Wnt/Ca+2, Wnt signaling negative regulation, cell fate, tissue polarity, cell growth and proliferation, cell migration, cell cycle, and cellular homeostasis. Genes with a low percent present call ( ⁇ 10%) according to a combination of four public pancreatic data sets compiled from Affymetrix U133 plus 2 microarrays were removed. Therefore, 420 genes were used for the analyses.
  • pancreatic tumors were selected from the OncoMed Tumor Bank and mRNA sequencing analyses were performed as described in Example 1.
  • the three pancreatic tumors were OMP-PN8, OMP-PN37, and OMP-PN41.
  • the classification model based on the 14 pancreatic tumors was applied to the three tumors to predict the response of each of these tumors to treatment with anti- FZD antibody OMP-18R5 in combination with gemcitabine and nab-paclitaxel (ABRAXANE).
  • ABRAXANE nab-paclitaxel
  • the three tumors were evaluated in in vivo xenograft models as described in Example 1 (see Figures 5A-5C).
  • a "responder" in the in vivo models is a tumor showing significantly greater tumor growth inhibition with the combination of OMP-18R5, gemcitabine, and ABRAXANE as compared to tumor growth inhibition with gemcitabine and ABRAXANE.
  • the predictions based on the classification model were compared to the results of the in vivo xenograft models. The results are shown in Table 2.
  • Prevalence of a biomarker signature can be defined as the proportion of a population predicted to be a responder based upon the biomarker signature.
  • the prevalence of the 3-gene biomarker signature in pancreatic cancer populations was estimated by applying the 3-gene biomarker signature to seven publicly available pancreatic cancer microarray data sets.
  • the Collission 2011 dataset was compiled from Affymetrix U133 plus 2.0 microarray data with 27 pancreatic ductal adenocarcinoma (PDA) FFPE samples.
  • the Stratford 2010 dataset was compiled from Agilent microarrays with 132 PDA FF (fresh frozen) samples.
  • the Winter 2011 dataset was compiled from Affymetrix U133 plus 2.0 microarrays with 30 PDA FF samples.
  • Four other public datasets were combined into one large dataset comprising 127 PDA FF samples (Badea et al., 2008,
  • Pre-processing of the public data included downloading the data, extracting the probe sets mapping to the three genes (SMO, IGF2, and TGFB3), and collapsing the probe sets to the three genes.
  • Gene level expression data was further standardized by subtracting the mean and dividing by the standard deviation of each gene in the public data.
  • the KN model built upon the training data was used to classify the public data. Classification predictions were obtained and the proportion of predicted responders was calculated based on the 3-gene biomarker signature.
  • the study is an on-going open-label Phase lb dose-escalation study of vantictumab in combination with nab-paclitaxel (e.g., ABRAXANE) and gemcitabine in patients with previously untreated stage IV pancreatic cancer.
  • the primary objectives of the study are to determine the safety and the maximum tolerated dose of vantictumab in combination with nab-paclitaxel and gemcitabine and to identify a recommended Phase 2 dose for vantictumab in combination with nab-paclitaxel and gemcitabine.
  • the secondary objectives are to characterize the pharmacokinetics (PK) of vantictumab when administered in combination with nab-paclitaxel and gemcitabine, to characterize the immunogenicity of vantictumab when administered in combination with nab-paclitaxel and gemcitabine, and to make preliminary assessment of vantictumab efficacy when administered in combination with nab-paclitaxel and gemcitabine.
  • PK pharmacokinetics
  • vantictumab was administered IV on Days 1 and 15 of each 28-day cycle, and dose levels of vantictumab were 3.5 and 7mg/kg every 2 weeks. Due to fragility fractures observed in the Phase 1 program, vantictumab was discontinued for all patients in Cohorts 1 and 2. Patients of Cohorts 3 and 4 are administered vantictumab at 3mg/kg and 5mg/kg once every 4 weeks, respectively. Depending on safety in these studies, additional higher dose levels may be evaluated. For potential cohorts > 5, the dose will not be increased by more than two-thirds of the prior dose level.
  • Nab-paclitaxel (125mg/m 2 ) and gemcitabine (1000mg/m 2 ) are administered IV on Days 1, 8, and 15 of each cycle. No dose escalation of vantictumab will be allowed within a dose cohort.
  • the signature score equation [0.728* IGF2 - 0.505 * TFGB3 - 0.902 * SMO + 1.150] was developed based on preclinical data from xenograft mouse models with human pancreatic tumors (14 PDX models: 10 responders and 4 non-responders) using a SVM (support vector machine) algorithm. While the 3-gene biomarker signature was developed by using the KNN (k-nearest neighbor) algorithm, the SVM algorithm and KNN algorithm generated the same prediction for all preclinical models (14 training, 4 testing).
  • the 3-gene biomarker signature score was separated into "biomarker high” and “biomarker low” groups using an optimized 30% cut-off.
  • a Cox proportional hazard model was run for overall survival (OS) and progression-free survival (PFS).
  • OS overall survival
  • PFS progression-free survival
  • the p-value for the association of the 3-gene biomarker signature with PFS is 0.004, while the p-value with OS is 0.191.
  • TCGA Cancer Genome Atlas
  • RNAseq data from 63 pancreatic cancer patients treated with gemcitabine was used for a comparative analysis. Overall survival and time-to-recurrence data were provided for these patients as well. Normalized gene expression data for the three biomarker genes (TGFB3, IGF2, and SMO) were extracted and then standardized to N(0,1) for each gene. The signature score equation was then applied to the standardized expression levels of the three genes. The 3-gene biomarker signature score was then separated into biomarker high and biomarker low groups by using a 30% cut-off. A Cox proportional hazard model was run for overall survival and time-to-recurrence.
  • OMP-18R5 Light chain CDR3 (SEQ ID NO: 6)
  • OMP-18R5 Light chain variable region amino acid sequence (SEQ ID NO: 8)
  • OMP-18R5 Light chain amino acid sequence with predicted signal sequence underlined (SEQ ID NO: 10)

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Abstract

La présente invention concerne des biomarqueurs pour l'identification de tumeurs susceptibles de réagir à un traitement avec des inhibiteurs de la voie Wnt. L'invention concerne également des procédés pour l'identification de tumeurs et/ou de patients qui sont susceptibles ou non susceptibles de réagir au traitement avec un inhibiteur de la voie Wnt. L'invention concerne en outre des méthodes pour le traitement d'un patient atteint d'un cancer, le cancer étant prédit pour réagir à un inhibiteur de la voie Wnt.
PCT/US2016/036850 2015-06-12 2016-06-10 Identification de biomarqueurs prédictifs associés à des inhibiteurs de la voie wnt Ceased WO2016201199A1 (fr)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090012018A1 (en) * 2004-09-13 2009-01-08 Matthias Hebrok Inhibition of pancretic cancer cell growth

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090012018A1 (en) * 2004-09-13 2009-01-08 Matthias Hebrok Inhibition of pancretic cancer cell growth

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
DOSCH, JOSEPH SCOTT.: "EXAMINING THE ROLE OF HEDGEHOG SIGNALING IN THE PANCREATIC TUMOR MICROENVIORNMENT.", DISS., 2011, XP055334435 *

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