JP2017501137A - Identification of predictive biomarkers associated with WNT pathway inhibitors - Google Patents

Abstract

Biomarkers are provided to identify tumors that may respond to treatment with Wnt pathway inhibitors. Also provided are methods for identifying tumors and / or patients that may be responsive or non-responsive to treatment with Wnt pathway inhibitors. Methods are provided for treating patients with cancers that are predicted to respond to Wnt pathway inhibitors.

Description

CROSS REFERENCE FOR RELATED APPLICATIONS All the provisional applications are hereby incorporated by reference in their entirety.

The present invention relates to the field of cancer treatment. More specifically, the present invention provides methods for identifying tumors that may be responsive or non-responsive to treatment with Wnt pathway inhibitors. In addition, the present invention also provides a method for identifying, selecting and / or treating patients with cancer that may respond to treatment with either a Wnt pathway inhibitor alone or in combination with other therapeutic agents. provide.

BACKGROUND OF THE INVENTION Cancer is one of the leading causes of death in developed countries, with approximately 1.6 million people diagnosed with cancer each year in the United States alone, and more than 550,000 people die. Overall, it is estimated that more than 1 in 3 people will develop some form of cancer during their lifetime. There are over 200 different types of cancer, four of which, breast cancer, lung cancer, colorectal cancer, and prostate cancer 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 (Non-Patent Document 1)).

  Signal transduction pathways typically lead to the expression of genes that directly or indirectly control cell growth, differentiation, survival, and death by connecting extracellular signals to the nucleus. However, in a wide variety of cancers, signal transduction pathways are dysregulated and can be associated with tumor initiation and / or progression. Signaling pathways that have been implicated in human tumorigenesis include the Wnt pathway, Ras-Raf-MEK-ERK pathway or MAPK pathway, PI3K-AKT pathway, CDKN2A / CDK4 pathway, Bcl-2 / TP53 pathway, and NOTCH paths are included, but not limited to them.

  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 cell fate specification, including cell polarity generation and self-renewal by stem cell populations. Unregulated activation of the Wnt pathway is associated with many human cancers, and it is thought that such activation can alter the developmental fate of cells. It is believed that activation of the Wnt pathway may maintain tumor cells in an undifferentiated state and / or lead to uncontrolled growth. Thus, carcinogenesis can progress by surpassing the homeostasis mechanism that controls normal development and tissue repair (Reya & Clevers, 2005, Nature, 434: 843-50); Beachy et al., 2004, Nature, 432: 324-31 (non-patent document 3)).

  The Wnt signaling pathway was first elucidated in the Drosophila development mutant wingless (wg) and from the mouse proto-oncogene int-1 (now Wnt1) (Nusse & Varmus, 1982, Cell, 31: 99-109 (Non-Patent Document 4); Van Ooyen & Nusse, 1984, Cell, 39: 233-40 (Non-Patent Document 5); Cabrera et al., 1987, Cell, 50: 659-63 (Non-Patent Document 6) Rijsewijk et al., 1987, Cell, 50: 649-57 (Non-Patent Document 7)). The Wnt gene encodes a secreted lipid-modified glycoprotein, and 19 different Wnt proteins have been identified in mammals. These secretory 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). The FZD receptor is a member of the G protein-coupled receptor (GPCR) superfamily and contains a seven-transmembrane domain and a large extracellular N-terminal ligand binding domain. The N-terminal ligand binding domain contains 10 conserved cysteines and is known as the cysteine rich domain (CRD) or “Fri domain”. There are 10 types of human FZD receptors: FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, and FZD10. Different FZD CRDs have different binding affinities for specific Wnt proteins (Wu & Nusse, 2002, J. Biol. Chem., 277: 41762-9). In addition, FZD receptors can be divided into those that activate the classical β-catenin pathway and those that activate the nonclassical pathway (Miller et al., 1999, Oncogene, 18: 7860-72 ( Non-patent document 9)).

  The role of Wnt signaling in cancer was first revealed by the identification of Wnt1 (originally int1) as an oncogene in breast tumors transformed by insertion of mouse viruses in the vicinity (Nusse & Varmus , 1982, Cell, 31: 99-109 (Non-Patent Document 4)). Since these early observations, further evidence has accumulated about the role of Wnt signaling in breast cancer. For example, overexpression of β-catenin in the mammary gland of transgenic mice results in hyperplasia and adenocarcinoma (Imbert et al., 2001, J. Cell Biol., 153: 555-68); Michaelson & Leder, 2001, Oncogene, 20: 5093-9 (Non-Patent Document 11)), while loss of Wnt signaling interferes with normal mammary development (Tepera et al., 2003, J. Cell Sci., 116: 1137-49 (non-patent document 12); Hatsell et al., 2003, J. Mammary Gland Biol. Neoplasia, 8: 145-58 (non-patent document 13)). In human breast cancer, β-catenin accumulation has been shown to activate Wnt signaling in more than 50% of carcinomas, and up-regulation of Frizzled receptor expression has been observed, although no specific mutation has been identified (Brennan & Brown, 2004, J. Mammary Gland Biol. Neoplasia, 9: 119-31 (Non-Patent Document 14); Malovanovic et al., 2004, Int. J. Oncol, 25: 1337-42 (Non-Patent Document 15)) .

  Activation of the Wnt pathway is also associated with colorectal cancer, lung cancer, pancreatic cancer, and melanoma. Approximately 5-10% of all colorectal cancers are hereditary, and one of the major cancer types is familial colorectal adenomatosis (FAP). FAP is an autosomal dominant disease, and approximately 80% of affected individuals contain germline mutations in the colorectal adenomatosis (APC) gene. Mutations have also been identified in other Wnt pathway components including axin and β-catenin. Individual adenomas are clonal expansions of epithelial cells containing a second inactivating allele, and many FAP adenoma cancers are inevitably adenocarcinoma due to additional mutations in oncogenes and / or tumor suppressor genes Cause the occurrence of. Furthermore, activation of the Wnt signaling pathway, including loss-of-function mutations in APC and stabilizing mutations in β-catenin, can induce hyperplasia and tumor growth in a mouse model (Oshima et al, 1997, Cancer Res., 57: 1644-9 (Non-Patent Document 16); Harada et al., 1999, EMBO J., 18: 5931-42 (Non-Patent Document 17)).

  Thus, the Wnt pathway has been recognized as a target for cancer therapy and treatment. As drug discovery and drug development advances, particularly in the cancer field, the “one drug fits all” approach is moving to a “personalized medicine” strategy. Personalized medical strategies can include treatment regimens based on cancer biomarkers, including prognostic markers, pharmacodynamic markers, and predictive markers. In general, predictive biomarkers assess the likelihood that a tumor or cancer is responsive or sensitive to a particular therapeutic agent and identify patients most likely to benefit from the use of that agent. / Or may allow selection.

  The present invention provides for the identification of predictive biomarkers associated with the use of Wnt pathway inhibitors in the treatment of cancer. There are also methods that use predictive biomarkers to identify, select, and / or classify cancer patients and / or tumors as potentially responsive or non-responsive to treatment with Wnt pathway inhibitors. provide. Also provided are methods for treating patients predicted to be responsive to treatment with Wnt inhibitors.

Siegel et al., 2012, CA: A Cancer J. for Clin., 62: 10-29 Reya & Clevers, 2005, Nature, 434: 843-50 Beachy et al., 2004, Nature, 432: 324-31 Nusse & Varmus, 1982, Cell, 31: 99-109 Van Ooyen & Nusse, 1984, Cell, 39: 233-40 Cabrera et al., 1987, Cell, 50: 659-63 Rijsewijk et al., 1987, Cell, 50: 649-57 Wu & Nusse, 2002, J. Biol. Chem., 277: 41762-9 Miller et al., 1999, Oncogene, 18: 7860-72 Imbert et al., 2001, J. Cell Biol., 153: 555-68 Michaelson & Leder, 2001, Oncogene, 20: 5093-9 Tepera et al., 2003, J. Cell Sci., 116: 1137-49 Hatsell et al., 2003, J. Mammary Gland Biol. Neoplasia, 8: 145-58 Brennan & Brown, 2004, J. Mammary Gland Biol. Neoplasia, 9: 119-31 Malovanovic et al., 2004, Int. J. Oncol, 25: 1337-42 Oshima et al, 1997, Cancer Res., 57: 1644-9 Harada et al., 1999, EMBO J., 18: 5931-42

  Biomarkers are provided for identifying patients who are likely to respond to treatment with Wnt pathway inhibitors. In addition, methods for identifying tumors and / or patients that may be responsive or non-responsive to treatment with Wnt pathway inhibitors are also provided. Further provided are methods of treating cancer in a patient who is predicted to be responsive to a Wnt pathway inhibitor or identified as such as a Wnt pathway inhibitor.

  In one aspect, the present invention provides: (a) obtaining a sample of a human tumor; (b) measuring the expression level of each biomarker of the biomarker signature in the sample, wherein the biomarker signature is biomarker FBXW2 Including one or more of: CCND2, RHOU, CRBP2, WIF1, and DKK1, and (c) the tumor may be responsive or non-responsive to treatment based on the expression level of the biomarker A method of identifying a human tumor that may be responsive or non-responsive to treatment with a Wnt pathway inhibitor, comprising identifying as being. In some embodiments, a method of identifying a human tumor that may be responsive or non-responsive to treatment with a Wnt pathway inhibitor comprises: (a) obtaining a sample of a human tumor; (b) a bio in the sample Measuring the expression level of each biomarker of the marker signature, wherein the biomarker signature comprises one or more of the biomarkers FBXW2, CCND2, RHOU, CRBP2, WIF1, and DKK1; and (c) bio Calculating a decision value based on the normalized expression of the biomarker in the marker signature, wherein a positive decision value indicates that the tumor is predicted to be responsive to a Wnt pathway inhibitor, and A negative decision value indicates that the tumor is predicted to be non-responsive to Wnt pathway inhibitors. As used herein, “standardized” and “normalized” can be used interchangeably. In some embodiments, the method includes identifying a human tumor that may be responsive or non-responsive to treatment with a Wnt pathway inhibitor in combination with paclitaxel.

  In another aspect, the present invention includes (a) obtaining a sample of a human tumor; (b) measuring the expression level of each biomarker of the biomarker signature in the sample, wherein the biomarker signature is a biomarker Including one or more of FBXW2, CCND2, RHOU, CTBP2, WIF1, and DKK1, and (c) the tumor may be responsive or non-responsive to treatment based on the expression level of the biomarker A method is provided for classifying a human tumor as potentially responsive or non-responsive to treatment with a Wnt pathway inhibitor, comprising the step of classifying as being. In some embodiments, a method of classifying a human tumor as potentially responsive or non-responsive to treatment with a Wnt pathway inhibitor comprises: (a) obtaining a sample of a human tumor; (b) in the sample Measuring the expression level of each biomarker of the biomarker signature, wherein the biomarker signature comprises one or more of the biomarkers FBXW2, CCND2, RHOU, CTBP2, WIF1, and DKK1; and (c) Calculating a decision value based on the normalized expression of the biomarker in the biomarker signature, wherein a positive decision value indicates that the tumor is predicted to be responsive to a Wnt pathway inhibitor; And a negative decision value indicates that the tumor is predicted to be non-responsive to Wnt pathway inhibitors. In some embodiments, the method includes categorizing the human tumor as potentially responsive or non-responsive to treatment with a Wnt pathway inhibitor in combination with paclitaxel.

  In another aspect, the present invention relates to (a) obtaining a sample of a human tumor; (b) measuring the expression level of each biomarker of the biomarker signature in the sample, wherein the biomarker signature is the gene FBXW2 Treatment with a Wnt pathway inhibitor comprising: one or more of: CCND2, RHOU, CTBP2, WIF1, and DKK1; and (c) determining tumor responsiveness to treatment based on the expression level of the biomarker A method of determining the responsiveness (or sensitivity) of a human tumor to. In some embodiments, a method of determining human tumor responsiveness or sensitivity to treatment with a Wnt pathway inhibitor comprises: (a) obtaining a sample of a human tumor; (b) for each biomarker of the biomarker signature in the sample. Measuring expression levels, wherein the biomarker signature comprises one or more of the genes FBXW2, CCND2, RHOU, CTBP2, WIF1, and DKK1; and (c) normalization of the biomarker in the biomarker signature Calculating a decision value based on the expressed expression, wherein a positive decision value indicates that the tumor is predicted to be responsive or sensitive to a Wnt pathway inhibitor. In some embodiments, the method includes determining the responsiveness or sensitivity of the human tumor to treatment with a Wnt pathway inhibitor in combination with paclitaxel.

  In another aspect, the present invention provides (a) obtaining a sample of a patient's tumor; (b) measuring the expression level of each biomarker of the biomarker signature in the sample, wherein the biomarker signature is bio Comprising one or more of the markers FBXW2, CCND2, RHOU, CTBP2, WIF1, and DKK1, and (c) identifying a patient that may respond to treatment based on the expression level of the biomarker, Methods are provided for identifying patients with cancer that may respond to treatment with a Wnt pathway inhibitor. In some embodiments, a method of identifying a patient with cancer that may respond to treatment with a Wnt pathway inhibitor comprises: (a) obtaining a sample of the patient's tumor; (b) a biomarker in the sample Measuring the expression level of each biomarker of the signature, wherein the biomarker signature comprises one or more of the biomarkers FBXW2, CCND2, RHOU, CTBP2, WIF1, and DKK1; and (c) a biomarker Calculating a decision value based on the normalized expression of the biomarker in the signature, where a positive decision value indicates that the patient is expected to respond to treatment with a Wnt pathway inhibitor. In some embodiments, the method includes identifying a patient with cancer that may respond to treatment with a Wnt pathway inhibitor in combination with paclitaxel.

  In another aspect, the present invention provides (a) obtaining a sample of a patient's tumor; (b) measuring the expression level of each biomarker of the biomarker signature in the sample, wherein the biomarker signature is bio Treatment with a Wnt pathway inhibitor comprising the step of comprising one or more of the markers FBXW2, CCND2, RHOU, CTBP2, WIF1, and DKK1; (c) selecting a patient for treatment based on the expression level of the biomarker A method of selecting a patient with cancer for. In some embodiments, a method of selecting a patient with cancer for treatment with a Wnt pathway inhibitor comprises: (a) obtaining a sample of the patient's tumor; (b) each biomarker of a biomarker signature in the sample Measuring the expression level of wherein the biomarker signature comprises one or more of the biomarkers FBXW2, CCND2, RHOU, CTBP2, WIF1, and DKK1; (c) the biomarker in the biomarker signature Calculating a decision value based on the normalized expression; and (d) selecting a patient for treatment when the patient's tumor sample has a positive decision value. In some embodiments, the method comprises selecting a patient with cancer for treatment with a Wnt pathway inhibitor in combination with paclitaxel.

  In another aspect, the present invention provides a method of treating cancer in a patient comprising the following steps: (a) identifying whether the patient is likely to respond to treatment with a Wnt pathway inhibitor. The identification is a step of (i) obtaining a cancer sample of a patient; (ii) measuring the expression level of each biomarker of the biomarker signature in the sample, wherein the biomarker signature is a biomarker Including one or more of the markers FBXW2, CCND2, RHOU, CTBP2, WIF1, and DKK1; and (iii) identifying a patient who may respond to treatment based on the expression level of the biomarker. And (b) administering an effective amount of a Wnt pathway inhibitor to a patient who may respond to the treatment. In some embodiments, the method of treating cancer in a patient comprises the steps of (a) identifying whether the patient is likely to respond to treatment with a Wnt pathway inhibitor, wherein the identification comprises (i) Obtaining a patient cancer sample; (ii) measuring the expression level of each biomarker biomarker signature in the sample, wherein the biomarker signature is biomarker FBXW2, CCND2, RHOU, CTBP2, WIF1, And (iii) calculating a decision value based on the normalized expression of the biomarker in the signature, wherein the positive decision value is when the patient responds to treatment Including the steps of indicating what is expected; and (b) administering an effective amount of a Wnt pathway inhibitor to a patient predicted to respond to treatment. In some embodiments, the method includes identifying whether the patient is likely to respond to treatment with a Wnt pathway inhibitor in combination with paclitaxel. In some embodiments, the method comprises administering to the patient a Wnt pathway inhibitor in combination with paclitaxel.

  In another aspect, the invention provides a method of treating cancer in a patient comprising administering to the patient an effective amount of a Wnt pathway inhibitor, wherein the patient expresses a biomarker signature in the patient's tumor sample. A method is provided that is predicted to respond to treatment with a Wnt inhibitor based on level, wherein the signature comprises one or more of the biomarkers FBXW2, CCND2, RHOU, CTBP2, WIF1, and DKK1. In some embodiments, the method of treating cancer in a patient comprises administering to the patient an effective amount of a Wnt pathway inhibitor, wherein the patient is normalized to a biomarker in a biomarker signature in a patient tumor sample. A set of biomarkers that includes one or more of the following biomarkers: FBXW2, CCND2, RHOU, CTBP2, WIF1, and DKK1, Process. In some embodiments, the patient is expected to respond to treatment with a Wnt pathway inhibitor in combination with paclitaxel. In some embodiments, the method comprises administering to the patient a Wnt pathway inhibitor in combination with paclitaxel.

  In another aspect, the invention provides (a) identifying whether a patient has a tumor that may respond to treatment with a Wnt pathway inhibitor, the identification comprising: (i) the patient's cancer (Ii) measuring the expression level of each biomarker biomarker signature in the sample, wherein the biomarker signature is one of the biomarkers FBXW2, CCND2, RHOU, CTBP2, WIF1, and DKK1. And (iii) identifying a patient who may respond to treatment based on the expression level of the biomarker; and (b) providing the patient with an effective amount of a Wnt pathway inhibitor Methods are provided for increasing the likelihood of effective treatment with a Wnt pathway inhibitor comprising the step of administering. In some embodiments, a method for increasing the likelihood of effective treatment with a Wnt pathway inhibitor comprises: (a) identifying whether the patient has a tumor that may respond to treatment with a Wnt pathway inhibitor The identification is a step of (i) obtaining a cancer sample of a patient; (ii) measuring the expression level of each biomarker of the biomarker signature in the sample, wherein the biomarker signature is a biomarker Comprising one or more of the markers FBXW2, CCND2, RHOU, CTBP2, WIF1, and DKK1, and (iii) calculating a decision value based on the normalized expression of the biomarker in the biomarker signature Showing that a positive decision value is predicted that the patient will respond to treatment; and (b) administering an effective amount of a Wnt pathway inhibitor to a patient whose tumor has a positive decision value Including a process. In some embodiments, the method includes identifying whether the patient has a tumor that may respond to treatment with a Wnt pathway inhibitor in combination with paclitaxel. In some embodiments, the method comprises administering to the patient a Wnt pathway inhibitor in combination with paclitaxel.

  In another aspect, the invention provides a method for increasing the likelihood of effective treatment with a Wnt pathway inhibitor comprising the step of administering to the patient an effective amount of a Wnt pathway inhibitor, wherein the patient has a patient tumor sample. Identified as likely to respond to treatment with a Wnt inhibitor based on the level of expression of the biomarker signature in, wherein the signature comprises one or more of the biomarkers FBXW2, CCND2, RHOU, CTBP2, WIF1, and DKK1 I will provide a. In some embodiments, a method for increasing the likelihood of effective treatment with a Wnt pathway inhibitor comprises administering to a patient an effective amount of a Wnt pathway inhibitor, wherein the patient is in a patient tumor sample. A set of biomarkers identified as potential biomarkers FBXW2, CCND2, RHOU was identified as likely to respond to treatment based on a positive decision value calculated from the weighted sum of normalized expression of biomarkers in the biomarker signature , CTBP2, WIF1, and DKK1. In some embodiments, the patient is identified as likely to respond to treatment with a Wnt pathway inhibitor in combination with paclitaxel. In some embodiments, the method comprises administering to the patient a Wnt pathway inhibitor in combination with paclitaxel.

  In certain embodiments of each aspect described above, and in other aspects and / or embodiments described elsewhere in this specification, the biomarker signature is biomarker FBXW2, CCND2, RHOU, CTBP2, WIF1, DKK1, EP300, And one or more of CTBP1. In some embodiments, the biomarker signature is biomarker FBXW2, CCND2, RHOU, CTBP2, WIF1, DKK1, EP300, CTBP1, WNT6, WNT3, FZD2, APC, TLE2, DVL2, PITX2, WISP1, GSK3B, WNT9A, FZD7 , And one or more of LEF1. In some embodiments, the biomarker signature comprises one or more of the biomarkers FBXW2, CCND2, RHOU, CTBP2, WIF1, and DKK1, and at least one additional biomarker from Table 2.

を含む重鎖CDR1、

を含む重鎖CDR2、および

を含む重鎖CDR3、ならびに(b)

を含む軽鎖CDR1、

を含む軽鎖CDR2、および

を含む軽鎖CDR3を含む抗体である。 In certain embodiments of each of the aforementioned aspects, as well as other aspects and / or embodiments described elsewhere in this specification, the Wnt pathway inhibitor is an antibody. In some embodiments, the Wnt pathway inhibitor is an antibody that specifically binds to at least one Frizzled (FZD) protein or fragment thereof. In some embodiments, the Wnt pathway inhibitor is an antibody that specifically binds to at least one FZD protein selected from the group consisting of FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, and FZD10. It is. In some embodiments, the Wnt pathway inhibitor is an antibody that specifically binds to at least one FZD protein selected from the group consisting of FZD1, FZD2, FZD5, FZD7, and FZD8. In certain embodiments, the Wnt pathway inhibitor comprises (a)

Heavy chain CDR1, including

A heavy chain CDR2 comprising, and

A heavy chain CDR3 comprising, and (b)

A light chain CDR1,

A light chain CDR2 comprising, and

An antibody comprising a light chain CDR3 comprising

  In certain embodiments, the Wnt pathway inhibitor is an antibody comprising a heavy chain variable region comprising SEQ ID NO: 7 and a light chain variable region comprising SEQ ID NO: 8. In certain embodiments, the Wnt pathway inhibitor is an antibody comprising a heavy chain variable region and a light chain variable region encoded by a plasmid deposited with the ATCC as PTA-9541. In certain embodiments, the Wnt pathway inhibitor is an antibody comprising a heavy and light chain encoded by a plasmid deposited with the ATCC as PTA-9541. In some embodiments, the Wnt pathway inhibitor is antibody OMP-18R5.

  In certain embodiments of each of the aforementioned aspects, as well as other aspects and / or embodiments described elsewhere in this specification, the Wnt pathway inhibitor is a soluble receptor. In some embodiments, the Wnt pathway inhibitor comprises the extracellular domain of a FZD receptor protein. In some embodiments, the Wnt pathway inhibitor comprises the Fri domain of the FZD protein. In some embodiments, the Wnt pathway inhibitor comprises the Fri domain of FZD8. In certain embodiments, the Wnt pathway inhibitor comprises a Fri domain of FZD8 and a human Fc domain. In some embodiments, the Wnt pathway inhibitor is the soluble receptor OMP-54F28.

  In some embodiments, the tumor is a breast tumor, lung tumor, colon tumor, glioma, gastrointestinal tumor, renal tumor (renal tumor), ovarian tumor, liver tumor, colorectal tumor, endometrial tumor, kidney tumor ( kidney tumor), prostate tumor, thyroid tumor, neuroblastoma, pancreatic tumor, glioblastoma multiforme, cervical tumor, stomach tumor, bladder tumor, hepatoma, melanoma, and head and neck tumor Is done. In some embodiments, the tumor is a breast tumor.

  In some embodiments, the cancer is breast cancer, lung cancer, colon cancer, glioma, gastrointestinal cancer, renal cancer, ovarian cancer, liver cancer, colorectal cancer, endometrium. Cancer, kidney cancer, prostate cancer, thyroid cancer, neuroblastoma, pancreatic cancer, glioblastoma multiforme, cervical cancer, stomach cancer, bladder cancer, hepatoma, melanoma And selected from the group consisting of head and neck cancer. In some embodiments, the cancer is breast cancer.

  In some embodiments, the method further comprises administering a second therapeutic agent to the patient. In some embodiments, the second therapeutic agent is a chemotherapeutic agent. In some embodiments, the second therapeutic agent is paclitaxel.

  In certain embodiments of each aspect described above, and in other aspects and / or embodiments described in other sections of the specification, the sample includes a tumor biopsy material, a core biopsy tissue sample, a fine needle aspirate, hair Includes, but is not limited to, any clinically relevant tissue sample such as a sac or a sample of body fluid such as blood, plasma, serum, lymph, ascites, cyst fluid, or urine. In some embodiments, the sample is taken from a patient having a tumor or cancer. In some embodiments, the sample is a primary tumor. In some embodiments, the sample is a metastatic cancer. In some embodiments, the sample is a tissue sample. In some embodiments, the sample is a tumor 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 a cell. In some embodiments, the sample is circulating tumor cells (CTC).

  In certain embodiments of each aspect described above, and in other aspects and / or embodiments described in other sections of the specification, the expression level of the biomarker is not limited to these, but includes reverse transcription PCR ( (RT-PCR), quantitative RT-PCR (qPCR), TaqMan ™, or TaqMan ™ low density arrays (TLDA). In some embodiments, the level of biomarker expression is determined using a microarray.

  In certain embodiments of each aspect described above, and in other aspects and / or embodiments described in other sections of the specification, the normalized expression of each biomarker is to measure the expression level for each biomarker; And multiplying it by the corresponding weight value, where the weight value for each biomarker is determined by the biomarker expression. In certain embodiments, the decision value is calculated according to the formula: 0.4560427 * FBXW2 + 0.3378467 * CCND2−0.4809354 * RHOU + 0.409029 * CTBP2 + 0.3291529 * WIF1 + 0.2926374 * DKK1 + 0.04662682.

  In some embodiments, the expression level of the biomarker is measured or determined by a PCR-based assay. In some embodiments, the expression level of FBXW2, CCND2, RHOU, CTBP2, WIF1, and DKK1 is measured using a polynucleotide selected from the group consisting of SEQ ID NOs: 62-79. In some embodiments, the expression levels of FBXW2, CCND2, RHOU, CTBP2, WIF1, and DKK1 are (a) a forward primer of SEQ ID NO: 62, a reverse primer of SEQ ID NO: 63, and SEQ ID NO: 64 (B) a forward primer of SEQ ID NO: 65, a reverse primer of SEQ ID NO: 66, and a probe comprising SEQ ID NO: 67; (c) a forward primer of SEQ ID NO: 68, SEQ ID NO : A reverse primer of 69 and a probe comprising SEQ ID NO: 70; (d) a forward primer of SEQ ID NO: 71, a reverse primer of SEQ ID NO: 72 and a probe comprising SEQ ID NO: 73; (e) A forward primer of SEQ ID NO: 74, a reverse primer of SEQ ID NO: 75, and a probe comprising SEQ ID NO: 76; and (f) a forward primer of SEQ ID NO: 77, a reverse primer of SEQ ID NO: 78, And a probe comprising SEQ ID NO: 79.

  In some embodiments, the level of biomarker expression is determined by multiple analyte profile testing, radioimmunoassay (RIA), Western blot assay, immunofluorescence assay, enzyme immunoassay, enzyme linked immunosorbent assay (ELISA), immunoprecipitation. Measured or determined by assay, chemiluminescence assay, immunohistochemistry assay, dot blot assay, or slot blot assay. In some embodiments where the assay uses an antibody, the antibody is detectably labeled. In some embodiments, the label is selected from the group consisting of immunofluorescent labels, chemiluminescent labels, phosphorescent labels, enzyme labels, radiolabels, avidin / biotin labels, colloidal gold particles, colored particles, and magnetic particles.

  The present invention also provides a kit comprising a container containing at least one reagent for specifically detecting the expression of at least one biomarker of the present invention. In certain embodiments, the reagent is an antibody or nucleic acid probe that binds to a biomarker of the invention.

  In some embodiments, the kit comprises a polynucleotide selected from the group consisting of SEQ ID NOs: 62-79. In some embodiments, the kit comprises (a) a forward primer of SEQ ID NO: 62, a reverse primer of SEQ ID NO: 63, and a probe comprising SEQ ID NO: 64; (b) a forward of SEQ ID NO: 65 A primer, a reverse primer of SEQ ID NO: 66, and a probe comprising SEQ ID NO: 67; (c) a forward primer of SEQ ID NO: 68, a reverse primer of SEQ ID NO: 69, and SEQ ID NO: 70 A probe comprising (d) a forward primer of SEQ ID NO: 71, a reverse primer of SEQ ID NO: 72, and SEQ ID NO: 73; (e) a forward primer of SEQ ID NO: 74, SEQ ID NO: 75 And a probe comprising SEQ ID NO: 76; and (f) a forward primer of SEQ ID NO: 77, a reverse primer of SEQ ID NO: 78, and a probe comprising SEQ ID NO: 79.

  Where an aspect or embodiment of the invention is described in the form of a Markush group or other optional group, the invention not only encompasses the entire listed group together, but each member of the group also individually And includes all possible subgroups of the main group, including a main group that lacks one or more of the group members. The present invention also contemplates the explicit exclusion of one or more of any of the group members in the claimed invention.

Classification of reactive or non-reactive breast tumors. Breast tumor OMP-B34 cells were injected subcutaneously into NOD / SCID mice. Mice were treated with OMP-18R5 antibody (-■-), taxol (-▲-), a combination of OMP-18R5 and taxol (-▼-), or a control antibody (-●-). Data are shown as tumor volume (mm ³ ) versus days after treatment. Classification of reactive or non-reactive breast tumors. Breast tumor OMP-B39 cells were injected subcutaneously into NOD / SCID mice. Mice were treated with OMP-18R5 antibody (-■-), taxol (-▲-), a combination of OMP-18R5 and taxol (-▼-), or a control antibody (-●-). Data are shown as tumor volume (mm ³ ) versus days after treatment. Classification of reactive or non-reactive breast tumors. Breast tumor OMP-B44 cells were injected subcutaneously into NOD / SCID mice. Mice were treated with OMP-18R5 antibody (-■-), taxol (-▲-), a combination of OMP-18R5 and taxol (-▼-), or a control antibody (-●-). Data are shown as tumor volume (mm ³ ) versus days after treatment. Classification of reactive or non-reactive breast tumors. Breast tumor OMP-B59 cells were injected subcutaneously into NOD / SCID mice. Mice were treated with OMP-18R5 antibody (-■-), taxol (-▲-), a combination of OMP-18R5 and taxol (-▼-), or a control antibody (-●-). Data are shown as tumor volume (mm ³ ) versus days after treatment. Classification of reactive or non-reactive breast tumors. Breast tumor OMP-B60 cells were injected subcutaneously into NOD / SCID mice. Mice were treated with OMP-18R5 antibody (-■-), taxol (-▲-), a combination of OMP-18R5 and taxol (-▼-), or a control antibody (-●-). Data are shown as tumor volume (mm ³ ) versus days after treatment. Classification of reactive or non-reactive breast tumors. Breast tumor UM-T01 cells were injected subcutaneously into NOD / SCID mice. Mice were treated with OMP-18R5 antibody (-■-), taxol (-▲-), a combination of OMP-18R5 and taxol (-▼-), or a control antibody (-●-). Data are shown as tumor volume (mm ³ ) versus days after treatment. Classification of reactive or non-reactive breast tumors. Breast tumor UM-T03 cells were injected subcutaneously into NOD / SCID mice. Mice were treated with OMP-18R5 antibody (-■-), taxol (-▲-), a combination of OMP-18R5 and taxol (-▼-), or a control antibody (-●-). Data are shown as tumor volume (mm ³ ) versus days after treatment. Classification of reactive or non-reactive breast tumors. Breast tumor UM-PE13 cells were injected subcutaneously into NOD / SCID mice. Mice were treated with OMP-18R5 antibody (-■-), taxol (-▲-), a combination of OMP-18R5 and taxol (-▼-), or a control antibody (-●-). Data are shown as tumor volume (mm ³ ) versus days after treatment. The performance curve of the top 20 genes. PCA plot of 6 selected genes. Correlation between 6-gene biomarker signature and tumor volume ratio. Predicting tumor reactivity based on classification probability analysis. Tumor used in training set for establishment of T = 6 gene signature. In vivo validation of predictive biomarkers. Breast tumor OMP-B29 cells were injected subcutaneously into NOD / SCID mice. Mice were treated with OMP-18R5 antibody (-■-), taxol (-▲-), a combination of OMP-18R5 and taxol (-▼-), or a control antibody (-●-). Data are shown as tumor volume (mm ³ ) versus days after treatment. In vivo validation of predictive biomarkers. Breast tumor OMP-B71 cells were injected subcutaneously into NOD / SCID mice. Mice were treated with OMP-18R5 antibody (-■-), taxol (-▲-), a combination of OMP-18R5 and taxol (-▼-), or a control antibody (-●-). Data are shown as tumor volume (mm ³ ) versus days after treatment. In vivo validation of predictive biomarkers. Breast tumor OMP-B84 cells were injected subcutaneously into NOD / SCID mice. Mice were treated with OMP-18R5 antibody (-■-), taxol (-▲-), a combination of OMP-18R5 and taxol (-▼-), or a control antibody (-●-). Data are shown as tumor volume (mm ³ ) versus days after treatment. In vivo validation of predictive biomarkers. Breast tumor OMP-B90 cells were injected subcutaneously into NOD / SCID mice. Mice were treated with OMP-18R5 antibody (-■-), taxol (-▲-), a combination of OMP-18R5 and taxol (-▼-), or a control antibody (-●-). Data are shown as tumor volume (mm ³ ) versus days after treatment. In vivo validation of predictive biomarkers. Breast tumor UM-T02 cells were injected subcutaneously into NOD / SCID mice. Mice were treated with OMP-18R5 antibody (-■-), taxol (-▲-), a combination of OMP-18R5 and taxol (-▼-), or a control antibody (-●-). Data are shown as tumor volume (mm ³ ) versus days after treatment. In vivo validation of predictive biomarkers. Breast tumor UM-T06 cells were injected subcutaneously into NOD / SCID mice. Mice were treated with OMP-18R5 antibody (-■-), taxol (-▲-), a combination of OMP-18R5 and taxol (-▼-), or a control antibody (-●-). Data are shown as tumor volume (mm ³ ) versus days after treatment. Estimating population ownership of 6-gene biomarker signatures using 3 public databases.

Detailed Description of the Invention
I. Definitions In order to facilitate the understanding of the present invention, several terms and expressions are defined below.

  The term “biomarker” as used herein can include, but is not limited to, nucleic acids and proteins, and variants and fragments thereof. Biomarkers can include DNA comprising all or part of a nucleic acid sequence encoding a biomarker, or the complement of such a sequence. Biomarker nucleic acids useful in the present invention are considered to include both DNA and RNA containing the entire or partial sequence of any nucleic acid sequence of interest. A biomarker protein is considered to include the entire or partial amino acid sequence of any biomarker protein or polypeptide.

  As used herein, the term “antibody” refers to a target such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or a combination of the foregoing, at least one antigen binding within the variable region of an immunoglobulin molecule. Refers to an immunoglobulin molecule that is recognized by and binds specifically to a site. As used herein, the term includes intact polyclonal antibodies, intact monoclonal antibodies, single chain antibodies, antibody fragments (eg, 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 is included so long as the antibody exhibits the desired biological activity. Antibodies are five major immunoglobulin classes based on the uniqueness of heavy chain constant domains, called alpha, delta, epsilon, gamma, and mu, respectively: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes) (For example, IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) may be used. Different classes of immunoglobulins have different well-known subunit structures and three-dimensional configurations. The antibody may be intact or conjugated to other molecules including, but not limited to, toxins and radioisotopes.

  The term “antibody fragment” refers to a portion of an intact antibody and refers to the antigenic variable region of the intact antibody. Examples of antibody fragments include Fab, Fab ′, F (ab ′) 2, and Fv fragments, linear antibodies, single chain antibodies, and multispecific antibodies formed from antibody fragments. However, it is not limited to them. As used herein, an “antibody fragment” includes at least one antigen binding site or epitope binding site.

  The term “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. A heavy or light chain variable region generally consists of four framework regions (FR) connected by three complementarity determining regions (CDRs), also known as “hypervariable regions”. The CDRs in each chain are grouped closer together by the framework region, and contribute to the formation of the antigen binding site of the antibody. There are at least two techniques for determining CDRs: (1) an approach based on sequence variability between different species (ie 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, in the art, CDRs may be determined using a combination of these two approaches.

  As used herein, the term “monoclonal antibody” refers to a homogeneous antibody population involved in markedly specific recognition and binding of a single antigenic determinant or epitope. This is in contrast to polyclonal antibodies, which typically contain a mixture of different antibodies against a variety of different antigenic determinants. The term “monoclonal antibody” refers to both intact and full-length monoclonal antibodies, as well as antibody fragments (eg, Fab, Fab ′, F (ab ′) 2, Fv), single chain (scFv) antibodies, antibody Includes fusion proteins comprising a portion, and any other modified immunoglobulin molecule comprising an antigen binding site. Furthermore, “monoclonal antibody” refers to an antibody made by a number of techniques including, but not limited to, hybridoma production, phage selection, recombinant expression, and transgenic animals.

  As used herein, the term “humanized antibody” refers to an antibody that is a specific immunoglobulin chain, chimeric immunoglobulin, or fragment thereof containing minimal non-human sequences. The methods used to make humanized antibodies are well known in the art.

  The term “human antibody” as used herein refers to an antibody produced by a human or an antibody having an amino acid sequence corresponding to an antibody produced by a human. Human antibodies may be made using any technique known in the art.

  The term “chimeric antibody” as used herein refers to an antibody in which the amino acid sequence of an immunoglobulin molecule is derived from more than one species. Typically, the variable regions of the light and heavy chains are variable for antibodies from one mammalian species (eg, mouse, rat, rabbit, etc.) that have the desired specificity, affinity, and / or binding ability. The constant region corresponds to a region from an antibody derived from another species (usually human).

  As used herein, the term “affinity matured antibody” is one that results in an improved affinity of an antibody for an antigen in one or more CDRs of that antibody compared to the parent antibody without modification. Or it refers to an antibody with multiple modifications. This definition also includes modifications in non-CDR residues made together with modifications of CDR residues. Preferred affinity matured antibodies have nanomolar affinity for the target antigen, and even picomolar affinity. Affinity matured antibodies are produced by techniques known in the art. For example, techniques may include affinity maturation by VH domain and VL domain shuffling, random mutagenesis of CDR and / or framework residues, and site-directed mutagenesis.

  The terms “epitope” and “antigenic determinant” are used interchangeably herein and refer to that portion of an antigen that has the ability to be recognized and specifically bound by a particular antibody. Where the antigen is a polypeptide, the epitope can be formed from contiguous amino acids or from discrete amino acids juxtaposed by tertiary folding of the protein. Epitopes formed from consecutive amino acids (also referred to as linear epitopes) are typically maintained during protein denaturation, whereas epitopes formed by tertiary folding (also referred to as conformational epitopes) are typically Specifically, it is lost during protein denaturation. An epitope typically includes at least 3, and more usually, at least 5 or 8-10 amino acids in a unique spatial conformation.

The term “selectively binds” or “specifically binds” refers to a binding agent or antibody that is more than a surrogate that includes an irrelevant or related protein to an epitope, protein, or target molecule. It means reacting or associating at a higher frequency, more quickly, over a longer duration, with a stronger affinity, or some combination of the foregoing. In certain embodiments, "specifically binds", for example, the antibody protein, about 0.1 mM or less, which means that more usually to bind with a K _D of less than about 1 [mu] M. In certain embodiments, "specifically binds", antibody, when there is at least about 0.1 [mu] M or less with a K _D, or at least about 0.01 [mu] M following _the K _D when there, and also when there is at least It means to bind to the target of about 1 nM or less of the K _D. Because of sequence identity between homologous proteins in different species, specific binding can include antibodies that recognize two or more species of proteins (eg, human FZD and mouse FZD). Similarly, due to the homology within certain regions of polypeptide sequences of different proteins, specific binding can be achieved by antibodies (or other polypeptides or binding agents) that recognize two or more proteins (eg, human FZD1 and human FZD7). ). It will be appreciated that in certain embodiments, an antibody or binding agent that specifically binds to a first target may or may not specifically bind to a second target. Thus, “specific binding” does not necessarily require (although it can include) binding exclusively to a single target. Thus, a binding agent may specifically bind to more than one target in certain embodiments. In certain embodiments, multiple targets may be bound by the same binding site on the agent or antibody. For example, an antibody can include, in one example, two identical antigen binding sites, each of which specifically binds to the same epitope on two or more proteins. In certain alternative embodiments, the antibody may be bispecific or multispecific and comprise at least two antigen binding sites that differ in specificity. As a non-limiting example, a bispecific agent includes one binding site that recognizes a target on one protein (e.g., human FZD) and a different target on a second protein (e.g., human WNT protein). A second different binding site that recognizes can be included. In general, reference to binding means specific binding, but not necessarily.

  The terms “polypeptide” and “peptide” and “protein” are used interchangeably herein to refer to polymers of amino acids of any length. The polymer may be linear or branched, may contain modified amino acids, and may be interrupted with non-amino acids. These terms are naturally modified or by intervention, such as disulfide bond formation, glycosylation, lipid addition, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included are modified amino acid polymers. This definition also includes polypeptides containing, for example, one or more amino acid analogs (such as unnatural amino acids) as well as other modifications known in the art. Since the polypeptides of the present invention can be based on antibodies, it is understood that in certain embodiments, the polypeptides may exist as single chains or as associated chains (eg, dimers).

  The terms “polynucleotide” and “nucleic acid” are used interchangeably herein and refer to a polymer of nucleotides of any length, including DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or modified bases, and / or their analogs, or any substrate that can be incorporated by DNA or RNA polymerase.

  “High stringency conditions” (1) utilize low ionic strength and high temperature for washing, eg, 15 mM sodium chloride / 1.5 mM sodium citrate / 0.1% sodium dodecyl sulfate at 50 ° C .; (2) During hybridization, a denaturing agent such as formamide, for example 50% (v / v) formamide, together with 0.1% bovine serum albumin / 0.1% Ficoll / 0.1% polyvinylpyrrolidone / 50 mM sodium phosphate buffer at pH 6.5 Utilize in 5 × SSC (0.75 M NaCl, 75 mM sodium citrate) at 42 ° C .; or (3) During hybridization, 50% formamide is added to 5 × SSC, 50 mM sodium phosphate (pH 6.8), Use at 42 ° C in 0.1% sodium pyrophosphate, 5x Denhardt's solution, sonicated salmon sperm DNA (50 μg / ml), 0.1% SDS, and 10% dextran sulfate in 0.2x SSC and 50% formamide Wash at 42 ° C with Then, it can be specified by the condition of performing washing consisting of 0.1 × SSC containing EDTA at 55 ° C.

  The terms “identical” or percent “identity” with respect to two or more nucleic acids or polypeptides are compared such that any conservative amino acid substitutions are not considered part of the sequence identity and are maximally matched. Refers to two or more sequences or subsequences that are the same or have the specified percentage of the same nucleotide or amino acid residues when aligned (introducing gaps if necessary). Percent identity can be measured by software or algorithms for sequence comparison or by visual inspection. Various algorithms and software that can be used to obtain alignments of amino acid or nucleotide sequences are well known in the art. These include, but are not limited to, BLAST, ALIGN, Megalign, BestFit, GCG Wisconsin Package, and variations thereof. In some embodiments, the two nucleic acids or polypeptides of the invention are substantially identical. That is, they are at least 70%, at least 75%, at least 80% when compared and aligned for maximum match, as measured using a sequence comparison algorithm, or as measured by visual inspection. It has at least 85%, at least 90%, and in some embodiments at least 95%, 96%, 97%, 98%, 99% nucleotide or amino acid residue identity. In some embodiments, the identity is at least about 10 residues long, at least about 20 residues long, at least about 40-60 residues long, at least about 60-80 residues long of the sequences, or their Exists over a region that is any integer value in between. In some embodiments, identity exists over a region longer than 60-80 residues, such as a region of at least about 80-100 residues, and in some embodiments, the sequence being compared, e.g., coding for a nucleotide sequence The sequence is substantially identical over the entire length of the region.

  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. The art has defined a family of amino acid residues with similar side chains, including basic side chains (eg lysine, arginine, histidine), acidic side chains (eg aspartic acid, glutamic acid), Uncharged polar side chains (eg glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (eg alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (Eg threonine, valine, isoleucine) and aromatic side chains (eg tyrosine, phenylalanine, tryptophan, histidine). For example, substitution of tyrosine with phenylalanine is a conservative substitution. Preferably, conservative substitutions in the polypeptide and antibody sequences of the invention do not deter binding of a polypeptide or antibody containing the amino acid sequence to an antigen to which the polypeptide or antibody binds. Methods for identifying conservative substitutions of nucleotides and amino acids that do not eliminate antigen binding are well known in the art.

  As used herein, the term “vector” refers to a construct that has the ability to deliver and usually express one or more genes or sequences of interest to a host cell. Examples of vectors include viral vectors, naked DNA or RNA expression vectors, plasmids, cosmids, or phage vectors, DNA or RNA expression vectors associated with cationic flocculants, and DNA or RNA expression vectors encapsulated in liposomes, etc. There are, but are not limited to them.

  As used herein, the term “soluble receptor” refers to the extracellular domain (or fragment thereof) of a receptor protein that precedes the first transmembrane domain of the receptor, which can be secreted in a soluble form from the cell. Point to. In general, this is the N-terminal part of the receptor protein.

  As used herein, the term "FZD soluble receptor" or "soluble FZD receptor" refers to an FZD receptor protein that precedes the first transmembrane domain of the receptor that can be secreted from the cell in a soluble form. Refers to the N-terminal extracellular fragment. The term encompasses FZD soluble receptors that contain the entire N-terminal extracellular domain (ECD) as well as smaller fragments. Thus, FZD soluble receptors that include the FZD Fri domain are also included in this term.

  An “isolated” polypeptide, antibody, polynucleotide, vector, cell, or composition is a polypeptide, antibody, polynucleotide, vector, cell, or composition that is in a form not found in nature. . Isolated polypeptides, antibodies, polynucleotides, vectors, cells, or compositions include those purified to the extent that they are no longer found in nature. In some aspects, the isolated polypeptide, antibody, polynucleotide, vector, cell, or composition is substantially pure.

  The term “substantially pure” as used herein is at least 50% pure (ie, free of impurities), at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure. Refers to material.

  As used herein, the terms “cancer” and “cancerous” refer to or refer to a physiological condition in a mammal where the population of cells is characterized by unregulated cell growth. Is described. Examples of cancer include, but are not limited to, carcinomas, blastomas, sarcomas, and blood cancers such as lymphomas and leukemias.

  As used herein, the terms “tumor” and “neoplasm”, whether benign (non-cancerous) or malignant (cancerous) including precancerous lesions, excessive cell growth or proliferation. Refers to any tissue mass produced by.

  As used herein, the term “metastasis” refers to the process by which a cancer spreads or migrates from its original site to another area of the body, producing a similar cancerous lesion in a new location. A “metastatic” or “metastatic” cell is one that enters a secondary site by losing adhesive contact with neighboring cells and migrating from the primary site of disease (eg, by blood flow or lymph).

  The terms "cancer stem cell" and "CSC" and "tumor stem cell" and "tumor progenitor cell" are used interchangeably herein and (1) have a high proliferative capacity and (2) asymmetric cell division Have the ability to produce one or more types of differentiated cell progeny that have reduced and / or restricted proliferative or developmental potential, and (3) symmetrical for self-renewal or self-maintenance Refers to cells from a cancer or tumor that have the ability to undergo cell division. These properties confer to cancer stem cells the ability to form or establish tumors or cancers when compared to the majority of tumor cells that cannot form tumors during successive transplants into immunodeficient hosts (eg mice). To do. Cancer stem cells form a tumor with an abnormal cell type that can change over time as mutations occur by randomly causing self-renewal and differentiation.

  The terms “cancer cell” and “tumor cell” refer to the entire population of cells derived from cancer or a tumor or precancerous lesion, and non-tumorigenic cells that make up the majority of the cancer cell population; Includes both tumorigenic stem cells (cancer stem cells). As used herein, the term “cancer cell” or “tumor cell” refers exclusively to cells that lack the ability to regenerate and differentiate to distinguish them from cancer stem cells. Modified with the term “non-tumorigenic”.

  As used herein, the term “tumorogenic” refers to the self-renewal nature of giving rise to new tumorigenic cancer stem cells and the generation of all other tumor cells by proliferation (differentiated and thus non- Refers to the functional characteristics of cancer stem cells, including the property of producing tumorigenic tumor cells.

  As used herein, the term “tumorigenic” refers to the ability of a random cell sample from a tumor to form a palpable tumor upon successive transplantation into an immunodeficient host (eg, a mouse). This definition also encompasses a population of enriched and / or isolated cancer stem cells that form palpable tumors upon successive transplantation into an immunodeficient host (eg, a mouse).

  The term “patient” refers to any animal (eg, mammal) that is the recipient of a particular treatment, including but not limited to humans, non-human primates, dogs, cats, rodents, and the like. I don't mean. Typically, the terms “patient” and “subject” are used interchangeably herein with respect to a human patient.

  The term “pharmaceutically acceptable” refers to a product or compound that has been (or can be) approved by a federal or state government regulatory authority, or the United States Pharmacopeia or other commonly used for use in animals, including humans. A product or compound listed in a recognized pharmacopoeia.

  The term “pharmaceutically acceptable excipient, carrier or adjuvant” or “acceptable pharmaceutical carrier” can be administered to a subject together with at least one agent of the present disclosure (eg, an antibody), Refers to an excipient, carrier or adjuvant that does not destroy the activity of the agent. The excipient, carrier, or adjuvant should be non-toxic when the agent is administered at a dosage sufficient to deliver a therapeutic effect.

  The term “effective amount” or “therapeutically effective amount” or “therapeutic effect” refers to a binding agent, antibody, polypeptide, polynucleotide, organic small molecule that is effective to “treat” a disease or disorder in a subject or mammal. Refers to the amount of a molecule or other drug. In the case of cancer, a therapeutically effective amount of a drug (eg, an antibody) has a therapeutic effect, thus reducing the number of cancer cells, reducing tumorigenicity, tumorigenesis frequency, or tumorigenic potential, Reduce the number or frequency of cancer stem cells, reduce tumor size, reduce cancer cell population, and inhibit and / or stop cancer cell invasion to surrounding organs, such as cancer spread to soft tissue or bone Inhibit and / or stop metastasis of tumor cells or cancer cells, inhibit and / or stop the growth of tumor cells or cancer cells, and to some extent eliminate one or more of the symptoms associated with cancer, It can reduce morbidity and mortality, improve quality of life, or provide a combination of these effects. To the extent an agent, such as an antibody, prevents growth and / or kills existing cancer cells, it can be referred to as cytostatic and / or cytotoxic.

  The terms “treat” or “treatment” or “treating” or “relieve” or “relieve” are: 1) cure, reduce, reduce the symptoms of the diagnosed morbidity or disorder, And / or refers to both therapeutic means to stop the progression and 2) prophylactic or preventative means to prevent or delay the occurrence of the targeted morbidity or disorder. Thus, those in need of treatment include those who have already been diagnosed with a disorder, those who are likely to suffer from a disorder, and those who should be prevented. In some embodiments, a subject has been successfully “treated” by the methods of the invention if the patient exhibits one or more of the following effects: a reduction in the number of cancer cells and / or Complete lack of cancer cells; Reduction of tumor size; Inhibition of tumor growth; Inhibition and / or lack of cancer cell invasion to surrounding organs including propagation of cancer cells to soft tissue and bone; Tumor cells or cancer cells Inhibition and / or lack of metastasis; inhibition and / or lack of cancer growth; elimination of one or more symptoms associated with the specific cancer; reduction of morbidity and mortality; improvement of quality of life Reduced tumorigenicity; reduced number or frequency of cancer stem cells; or some combination of such effects.

  As used in this disclosure and the claims, the singular forms “a”, “an”, and “the” include the plural unless the context clearly dictates otherwise.

  Whenever an embodiment is described herein using the word “comprising”, it is said to be “consisting of” and / or “consisting essentially of”. It is understood that other aspects similar to those described in terms are also provided. Also, whenever an embodiment is described herein using the word “consisting essentially of”, an otherwise similar embodiment described in the term “consisting of” is also provided. It is understood.

  The term “and / or” as used herein in expressions such as “A and / or B” encompasses both A and B, A or B, A (only), and B (only). Shall. Similarly, the term “and / or” used in expressions such as “A, B, and / or C” is intended to encompass each of the following embodiments: A, B, and C; B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (only); B (only); and C (only).

II. Methods of Using Predictive Biomarkers As used herein, patients with cancer that may be responsive (“sensitive”) or non-responsive (“resistant”) to treatment with Wnt pathway inhibitors and / or Alternatively, a method for identifying, classifying, and / or selecting a tumor is provided. In addition, methods for treating patients with cancer that are likely to respond to treatment with a Wnt pathway inhibitor, are predicted to respond to treatment, and / or have been identified to respond to treatment. Provided.

  In the present specification, (a) obtaining a sample of human tumor; (b) measuring the expression level of each biomarker biomarker signature in the sample, wherein the biomarker signature is biomarker FBXW2, CCND2, Including one or more of RHOU, CRBP2, WIF1, and DKK1; and (c) identifying the tumor as potentially responsive or non-responsive to treatment based on the expression level of the biomarker A method of identifying a human tumor that may be responsive or non-responsive to treatment with a Wnt pathway inhibitor is provided. In some embodiments, a method of identifying a human tumor that may be responsive or non-responsive to treatment with a Wnt pathway inhibitor comprises: (a) obtaining a sample of a human tumor; (b) a bio in the sample Measuring the expression level of each biomarker of the marker signature, wherein the biomarker signature comprises one or more of the biomarkers FBXW2, CCND2, RHOU, CRBP2, WIF1, and DKK1; and (c) bio Calculating a decision value based on the normalized expression of the biomarker in the marker signature, wherein a positive decision value indicates that the tumor is predicted to be responsive to a Wnt pathway inhibitor, and A negative decision value indicates that the tumor is predicted to be non-responsive to Wnt pathway inhibitors.

  In the present specification, (a) obtaining a sample of human tumor; (b) measuring the expression level of each biomarker biomarker signature in the sample, wherein the biomarker signature is biomarker FBXW2, CCND2, Including one or more of RHOU, CTBP2, WIF1, and DKK1; and (c) classifying the tumor as potentially responsive or non-responsive to treatment based on the expression level of the biomarker A method of classifying a human tumor as being potentially reactive or non-responsive to treatment with a Wnt pathway inhibitor is provided. In some embodiments, a method of classifying a human tumor as potentially responsive or non-responsive to treatment with a Wnt pathway inhibitor comprises: (a) obtaining a sample of a human tumor; (b) in the sample Measuring the expression level of each biomarker of said biomarker signature, wherein said biomarker signature comprises one or more of biomarkers FBXW2, CCND2, RHOU, CTBP2, WIF1, and DKK1; and (c ) Calculating a decision value based on the normalized expression of the biomarker in the biomarker signature, where a positive decision value indicates that the tumor is predicted to be responsive to a Wnt pathway inhibitor And a negative decision value indicates that the tumor is predicted to be non-responsive to Wnt pathway inhibitors.

  In the present specification, (a) obtaining a human tumor sample; (b) measuring the expression level of each biomarker biomarker signature in the sample, wherein the biomarker signature is the gene FBXW2, CCND2, RHOU , Comprising one or more of: CTBP2, WIF1, and DKK1; and (c) determining the responsiveness of the tumor to the treatment based on the expression level of the biomarker, A method for determining reactivity (or sensitivity) is provided. In some embodiments, a method of determining human tumor responsiveness or sensitivity to treatment with a Wnt pathway inhibitor comprises: (a) obtaining a sample of a human tumor; (b) for each biomarker of the biomarker signature in the sample. Measuring expression levels, wherein the biomarker signature comprises one or more of the genes FBXW2, CCND2, RHOU, CTBP2, WIF1, and DKK1; and (c) normalization of the biomarker in the biomarker signature Calculating a decision value based on the expressed expression, wherein a positive decision value indicates that the tumor is predicted to be responsive to a Wnt pathway inhibitor.

  In the present specification, (a) obtaining a sample of a patient's tumor; (b) measuring the expression level of each biomarker of the biomarker signature in the sample, wherein the biomarker signature is biomarker FBXW2, CCND2 By one or more of: RHOU, CTBP2, WIF1, and DKK1, and (c) identifying a patient who may respond to treatment based on the expression level of the biomarker Methods are provided for identifying patients with cancer that may respond to treatment. In some embodiments, a method of identifying a patient with cancer that may respond to treatment with a Wnt pathway inhibitor comprises: (a) obtaining a sample of the patient's tumor; (b) a biomarker in the sample Measuring the expression level of each biomarker of the signature, wherein the biomarker signature comprises one or more of the biomarkers FBXW2, CCND2, RHOU, CTBP2, WIF1, and DKK1; and (c) a biomarker Calculating a decision value based on the normalized expression of the biomarker in the signature, where a positive decision value indicates that the patient is expected to respond to treatment with a Wnt pathway inhibitor. In some aspects, the method further comprises selecting a patient for treatment when the patient's tumor sample has a positive determination. In some embodiments, the method further comprises administering to the patient a therapeutically effective amount of a Wnt pathway inhibitor.

  In the present specification, (a) obtaining a sample of a patient's tumor; (b) measuring the expression level of each biomarker of the biomarker signature in the sample, wherein the biomarker signature is biomarker FBXW2, CCND2 Comprising: one or more of: RHOU, CTBP2, WIF1, and DKK1; (c) selecting a patient for treatment based on the expression level of the biomarker; A method for selecting patients with cancer is provided. In some embodiments, a method of selecting a patient with cancer for treatment with a Wnt pathway inhibitor comprises: (a) obtaining a sample of the patient's tumor; (b) each biomarker of a biomarker signature in the sample Measuring the expression level of wherein the biomarker signature comprises one or more of the biomarkers FBXW2, CCND2, RHOU, CTBP2, WIF1, and DKK1; (c) the biomarker in the biomarker signature Calculating a decision value based on the normalized expression; and (d) selecting a patient for treatment when the patient's tumor sample has a positive decision value. In some embodiments, the method further comprises administering to the patient a therapeutically effective amount of a Wnt pathway inhibitor.

  As used herein, (a) identifying whether a patient is likely to respond to treatment with a Wnt pathway inhibitor, wherein the identification comprises (i) obtaining a patient's cancer sample; ii) measuring the expression level of each biomarker in the biomarker signature in the sample, wherein the biomarker signature comprises one or more of the biomarkers FBXW2, CCND2, RHOU, CTBP2, WIF1, and DKK1 And (iii) identifying a patient who is likely to respond to treatment based on the expression level of the biomarker; and (b) an effective amount of a Wnt pathway inhibitor for patients who are likely to respond to treatment; A method of treating cancer in a patient is provided comprising the step of administering In some embodiments, the method of treating cancer in a patient comprises the steps of (a) identifying whether the patient is likely to respond to treatment with a Wnt pathway inhibitor, wherein the identification comprises (i) Obtaining a patient cancer sample; (ii) measuring the expression level of each biomarker biomarker signature in the sample, wherein the biomarker signature is biomarker FBXW2, CCND2, RHOU, CTBP2, WIF1, And (iii) calculating a decision value based on the normalized expression of the biomarker in the signature, wherein the positive decision value is when the patient responds to treatment Including the steps of indicating what is expected; and (b) administering an effective amount of a Wnt pathway inhibitor to a patient predicted to respond to treatment.

  In another aspect, the invention provides a method of treating cancer in a patient comprising administering to the patient an effective amount of a Wnt pathway inhibitor, wherein the patient expresses a biomarker signature in the patient's tumor sample. Based on levels, methods are provided that are predicted to be responsive to treatment with a Wnt inhibitor and the signature comprises one or more of the biomarkers FBXW2, CCND2, RHOU, CTBP2, WIF1, and DKK1. In some embodiments, a method of treating cancer in a patient comprises administering to the patient an effective amount of a Wnt pathway inhibitor, wherein the patient is a biomarker in a biomarker signature in the patient's tumor sample. Based on a positive decision value calculated from the weighted sum of the normalized expression of, predicted to respond to treatment, the set of biomarkers is one of the biomarkers FBXW2, CCND2, RHOU, CTBP2, WIF1, and DKK1 Includes multiple.

  As used herein, (a) identifying whether a patient has a tumor that may respond to treatment with a Wnt pathway inhibitor, wherein the identification comprises (i) obtaining a sample of the patient's cancer Step (ii) measuring the expression level of each biomarker biomarker signature in the sample, wherein the biomarker signature comprises one or more of the biomarkers FBXW2, CCND2, RHOU, CTBP2, WIF1, and DKK1 And (iii) identifying a patient that may respond to treatment based on the expression level of the biomarker; and (b) administering an effective amount of a Wnt pathway inhibitor to the patient. Methods are provided for increasing the likelihood of effective treatment with a Wnt pathway inhibitor. In some embodiments, a method for increasing the likelihood of effective treatment with a Wnt pathway inhibitor comprises: (a) identifying whether the patient has a tumor that may respond to treatment with a Wnt pathway inhibitor The identification is a step of (i) obtaining a cancer sample of a patient; (ii) measuring the expression level of each biomarker of the biomarker signature in the sample, wherein the biomarker signature is a biomarker Comprising one or more of the markers FBXW2, CCND2, RHOU, CTBP2, WIF1, and DKK1, and (iii) calculating a decision value based on the normalized expression of the biomarker in the biomarker signature Showing that a positive decision value is predicted that the patient will respond to treatment; and (b) administering an effective amount of a Wnt pathway inhibitor to a patient whose tumor has a positive decision value Including a process.

  In another aspect, the invention provides a method for increasing the likelihood of effective treatment with a Wnt pathway inhibitor comprising the step of administering to the patient an effective amount of a Wnt pathway inhibitor, wherein the patient has a patient tumor sample. Based on the level of expression of the biomarker signature in, identified as likely to respond to treatment with a Wnt inhibitor, the signature comprising one or more of the biomarkers FBXW2, CCND2, RHOU, CTBP2, WIF1, and DKK1, Provide a method. In some embodiments, a method for increasing the likelihood of effective treatment with a Wnt pathway inhibitor comprises administering to a patient an effective amount of a Wnt pathway inhibitor, wherein the patient is in a patient tumor sample. Based on the positive decision value calculated from the weighted sum of the normalized expression of the biomarkers in the biomarker signature, the set of biomarkers identified as biomarkers FBXW2, CCND2, Contains one or more of RHOU, CTBP2, WIF1, and DKK1. In some embodiments, the patient is identified as likely to respond to treatment with a Wnt pathway inhibitor in combination with paclitaxel. In some embodiments, the method comprises administering to the patient a Wnt pathway inhibitor in combination with paclitaxel.

  In the present specification, (a) obtaining a sample of human tumor; (b) measuring the expression level of each biomarker biomarker signature in the sample, wherein the biomarker signature is biomarker FBXW2, CCND2, Including one or more of RHOU, CRBP2, WIF1, and DKK1, and (c) calculating a decision value based on the normalized expression of the biomarker in the biomarker signature, wherein the positive decision value is Treatment with a Wnt pathway inhibitor, indicating that the tumor is predicted to be responsive to a Wnt pathway inhibitor, and a negative decision value indicates that the tumor is predicted to be non-responsive to a Wnt pathway inhibitor Use is provided for identifying human tumors that may be reactive or non-reactive.

  In the present specification, (a) obtaining a sample of human tumor; (b) measuring the expression level of each biomarker biomarker signature in the sample, wherein the biomarker signature is biomarker FBXW2, CCND2, Including one or more of RHOU, CTBP2, WIF1, and DKK1; and (c) calculating a decision value based on the normalized expression of the biomarker in the biomarker signature, including a positive decision value A human tumor wherein the tumor is predicted to be responsive to a Wnt pathway inhibitor and a negative decision value indicates that the tumor is predicted to be non-responsive to a Wnt pathway inhibitor, Use is provided to classify as potentially reactive or non-responsive to treatment with Wnt pathway inhibitors.

  In the present specification, (a) obtaining a human tumor sample; (b) measuring the expression level of each biomarker biomarker signature in the sample, wherein the biomarker signature is the gene FBXW2, CCND2, RHOU Including one or more of: CTBP2, WIF1, and DKK1, and (c) calculating a decision value based on the normalized expression of the biomarker in the biomarker signature, wherein the positive decision value is Provided is a use for determining the susceptibility of a human tumor to treatment with a Wnt pathway inhibitor, indicating that the tumor is predicted to be responsive to the Wnt pathway inhibitor.

  In the present specification, (a) obtaining a sample of a patient's tumor; (b) measuring the expression level of each biomarker of the biomarker signature in the sample, wherein the biomarker signature is biomarker FBXW2, CCND2 Including one or more of: RHOU, CTBP2, WIF1, and DKK1; and (c) calculating a decision value based on the normalized expression of the biomarker in the biomarker signature, comprising a positive decision value Is provided for use in identifying patients with cancer that may respond to treatment with a Wnt pathway inhibitor, indicating that the patient is expected to respond to treatment with a Wnt pathway inhibitor. In some aspects, the use further comprises selecting a patient for treatment when the patient's tumor sample has a positive determination. In some embodiments, the use further comprises administering to the patient a therapeutically effective amount of a Wnt pathway inhibitor.

  In the present specification, (a) obtaining a sample of a patient's tumor; (b) measuring the expression level of each biomarker of the biomarker signature in the sample, wherein the biomarker signature is the biomarker FBXW2, Comprising one or more of CCND2, RHOU, CTBP2, WIF1, and DKK1; (c) calculating a decision value based on the normalized expression of the biomarker in the biomarker signature; and (d) the patient's Use is provided for selecting a patient with cancer for treatment with a Wnt pathway inhibitor comprising the step of selecting the patient for treatment when the tumor sample has a positive determination. In some embodiments, the use further comprises administering to the patient a therapeutically effective amount of a Wnt pathway inhibitor.

  As used herein, (a) identifying whether a patient is likely to respond to treatment with a Wnt pathway inhibitor, the identification comprising: (i) obtaining a patient's cancer sample; ii) measuring the expression level of each biomarker of the biomarker signature in the sample, wherein the biomarker signature comprises one or more of the biomarkers FBXW2, CCND2, RHOU, CTBP2, WIF1, and DKK1, And (iii) calculating a decision value based on the normalized expression of the biomarker in the signature, wherein the positive decision value indicates that the patient is expected to respond to treatment. A Wnt pathway inhibitor for use in the treatment of cancer in a patient, comprising: (b) administering an effective amount of a Wnt pathway inhibitor to the patient predicted to be responsive to the treatment.

  As used herein, (a) identifying whether a patient has a tumor that may respond to treatment with a Wnt pathway inhibitor, wherein the identification comprises (i) obtaining a sample of the patient's cancer Step (ii) measuring the expression level of each biomarker of the biomarker signature in the sample, wherein the biomarker signature is one or more of the biomarkers FBXW2, CCND2, RHOU, CTBP2, WIF1, and DKK1 And (iii) calculating a decision value based on the normalized expression of the biomarker in the biomarker signature, wherein a positive decision value is predicted that the patient is expected to respond to treatment. Increasing the likelihood of effective treatment with a Wnt pathway inhibitor comprising: (b) administering an effective amount of a Wnt pathway inhibitor to a patient whose tumor has a positive determination value Use is provided for.

  As used herein, a Wnt pathway inhibitor for use in the treatment of cancer in a patient identified as potentially responsive to treatment with a Wnt pathway inhibitor, wherein the identification of the patient is obtained from a patient (i) Measuring the expression level of each biomarker in a biomarker signature in a given cancer sample, wherein the biomarker signature includes one or more of the biomarkers FBXW2, CCND2, RHOU, CTBP2, WIF1, and DKK1 And (ii) calculating a decision value based on the normalized expression of the biomarker in the signature, wherein a positive decision value indicates that the patient is expected to respond to treatment Wnt pathway inhibitors are provided.

  As used herein, a Wnt pathway inhibitor for use in the treatment of cancer in a patient, wherein the patient is positive based on the normalized expression of each biomarker of the biomarker signature in the patient's cancer sample. A Wnt pathway inhibitor is provided wherein the determination value is calculated and the biomarker signature comprises one or more of the biomarkers FBXW2, CCND2, RHOU, CTBP2, WIF1, and DKK1.

  In some embodiments of the methods described herein, the biomarker signature comprises two or more of the biomarkers FBXW2, CCND2, RHOU, CTBP2, WIF1, and DKK1. In some embodiments, the biomarker signature comprises three or more of the biomarkers FBXW2, CCND2, RHOU, CTBP2, WIF1, and DKK1. In some embodiments, the biomarker signature comprises four or more of the biomarkers FBXW2, CCND2, RHOU, CTBP2, WIF1, and DKK1. In some embodiments, the biomarker signature comprises five or more of the biomarkers FBXW2, CCND2, RHOU, CTBP2, WIF1, and DKK1. In some embodiments, the biomarker signature comprises FBXW2, CCND2, RHOU, CTBP2, WIF1, and DKK1. In some embodiments, the biomarker signature consists of FBXW2, CCND2, RHOU, CTBP2, WIF1, and DKK1.

  In some embodiments, the biomarker signature includes one or more additional biomarkers in addition to at least one of the biomarkers FBXW2, CCND2, RHOU, CTBP2, WIF1, and DKK1. In some embodiments, the biomarker signature includes one or more additional biomarkers selected from the genes listed in Table 2, in addition to at least one of the biomarkers FBXW2, CCND2, RHOU, CTBP2, WIF1, and DKK1. Contains a marker. In some embodiments, the biomarker signature is biomarkers EP300, CTBP1, WNT6, WNT9A, SNT3, FZD2, FZD7, APC in addition to at least one of the biomarkers FBXW2, CCND2, RHOU, CTBP2, WIF1, and DKK1. , TLE2, DVL2, PITX2, WISP1, GSK3B, and LEF1. In some embodiments, the biomarker signature comprises one or more of the biomarkers FBXW2, CCND2, RHOU, CTBP2, WIF1, DKK1, EP300, and CTBP1. In some embodiments, the biomarker signature is biomarker FBXW2, CCND2, RHOU, CTBP2, WIF1, DKK1, EP300, CTBP1, WNT6, WNT3, FZD2, APC, TLE2, DVL2, PITX2, WISP1, GSK3B, WNT9A, FZD7 , And one or more of LEF1.

  In some embodiments of the methods described herein, the biomarker signature comprises FBXW2. In some embodiments, the biomarker signature comprises CCND2. In some embodiments, the biomarker signature comprises RHOU. In some embodiments, the biomarker signature comprises CTBP2. In some embodiments, the biomarker signature comprises WIF1. In some embodiments, the biomarker signature comprises DKK1.

  In some embodiments of the methods described herein, the biomarker signature comprises FBXW2 and CCND2. In some embodiments, the biomarker signature comprises FBXW2 and RHOU. In some embodiments, the biomarker signature comprises FBXW2 and CTBP2. In some embodiments, the biomarker signature comprises FBXW2 and WIF1. In some embodiments, the biomarker signature comprises FBXW2 and DKK1. In some embodiments, the biomarker signature comprises CCND2 and RHOU. In some embodiments, the biomarker signature comprises CCND2 and CTBP2. In some embodiments, the biomarker signature comprises CCND2 and WIF1. In some embodiments, the biomarker signature comprises CCND2 and DKK1. In some embodiments, the biomarker signature comprises RHOU and CTBP2. In some embodiments, the biomarker signature comprises RHOU and WIF1. In some embodiments, the biomarker signature comprises RHOU and DKK1. In some embodiments, the biomarker signature comprises CTBP2 and WIF1. In some embodiments, the biomarker signature comprises CTBP2 and DKK1. In some embodiments, the biomarker signature comprises WIF1 and DKK1.

  In some embodiments of the methods described herein, the biomarker signature comprises FBXW2, CCND2, and RHOU. In some embodiments, the biomarker signature comprises FBXW2, CCND2, and CTBP2. In some embodiments, the biomarker signature comprises FBXW2, CCND2, and WIF1. In some embodiments, the biomarker signature comprises FBXW2, CCND2, and DKK1. In some embodiments, the biomarker signature comprises FBXW2, RHOU, and CTBP2. In some embodiments, the biomarker signature comprises FBXW2, RHOU, and WIF1. In some embodiments, the biomarker signature comprises FBXW2, RHOU, and DKK1. In some embodiments, the biomarker signature comprises FBXW2, CTBP2, and WIF1. In some embodiments, the biomarker signature comprises FBXW2, CTBP2, and DKK1. In some embodiments, the biomarker signature comprises FBXW2, WIF1, and DKK1. In some embodiments, the biomarker signature comprises CCND2, RHOU, and CTBP2. In some embodiments, the biomarker signature comprises CCND2, RHOU, and WIF1. In some embodiments, the biomarker signature comprises CCND2, RHOU, and DKK1. In some embodiments, the biomarker signature comprises CCND2, CTBP2, and WIF1. In some embodiments, the biomarker signature comprises CCND2, CTBP2, and DKK1. In some embodiments, the biomarker signature comprises CCND2, WIF1, and DKK1. In some embodiments, the biomarker signature comprises RHOU, CTBP2, and WIF1. In some embodiments, the biomarker signature comprises RHOU, CTBP2, and DKK1. In some embodiments, the biomarker signature comprises RHOU, WIF1, and DKK1. In some embodiments, the biomarker signature comprises CTBP2, WIF1, and DKK1.

  In some embodiments of the methods described herein, the biomarker signature comprises FBXW2, CCND2, RHOU, and CTBP2. In some embodiments, the biomarker signature comprises FBXW2, CCND2, RHOU, and WIF1. In some embodiments, the biomarker signature comprises FBXW2, CCND2, RHOU, and DKK1. In some embodiments, the biomarker signature comprises FBXW2, RHOU, CTBP2, and WIF1. In some embodiments, the biomarker signature comprises FBXW2, RHOU, CTBP2, and DKK1. In some embodiments, the biomarker signature comprises FBXW2, CTBP2, WIF1, and DKK1. In some embodiments, the biomarker signature comprises CCND2, RHOU, CTBP2, and WIF1. In some embodiments, the biomarker signature comprises CCND2, RHOU, CTBP2, and DKK1. In some embodiments, the biomarker signature comprises CCND2, CTBP2, WIF1, and DKK1. In some embodiments, the biomarker signature comprises RHOU, CTBP2, WIF1, and DKK1. In some embodiments, any of these signatures may include one or more additional biomarkers.

  In some embodiments of the methods described herein, the biomarker signature comprises FBXW2, CCND2, RHOU, CTBP2, and WIF1. In some embodiments, the biomarker signature comprises FBXW2, CCND2, RHOU, CTBP2, and DKK1. In some embodiments, the biomarker signature comprises FBXW2, CCND2, CTBP2, WIF1, and DKK1. In some embodiments, the biomarker signature comprises FBXW2, CCND2, RHOU, WIF1, and DKK1. In some embodiments, the biomarker signature comprises FBXW2, RHOU, CTBP2, WIF1, and DKK1. In some embodiments, the biomarker signature comprises CCND2, RHOU, CTBP2, WIF1, and DKK1.

  In some embodiments, the sample includes a tumor biopsy, core biopsy tissue sample, fine needle aspirate, hair follicle, or body fluid such as blood, plasma, serum, lymph, ascites, cyst fluid, or urine. This includes, but is not limited to, any clinically relevant tissue sample, such as a sample. In some embodiments, the sample is taken from a patient having a tumor or cancer. In some embodiments, the sample is a primary tumor. In some embodiments, the sample is a metastatic cancer. The sample may be taken from a human or a non-human mammal such as a mouse, rat, non-human primate, dog, cat, ruminant, pig, or sheep. In some embodiments, the sample is taken from the subject at multiple time points, eg, before treatment, at the time of treatment, and / or after treatment. In some embodiments, the samples are taken from different sites in the subject, such as a sample from the primary tumor and a sample from a metastatic cancer at a distal site.

In some embodiments, the sample is a paraffin-embedded fixed tissue sample. In some embodiments, the sample is a formalin fixed paraffin embedded (FFPE) tissue sample. In some embodiments, the sample is a fresh tissue (eg, tumor) sample. In some embodiments, the sample is a frozen tissue sample. In some embodiments, the sample is a fresh frozen (FF) tissue (eg, tumor) sample. In some embodiments, the sample is a cell isolated from a body fluid. In some embodiments, the sample comprises circulating tumor cells (CTC). In some embodiments, the sample is a stored tissue sample. In some embodiments, the sample is a stored tissue sample with a known diagnostic history, treatment history, and / or prognostic history. In some embodiments, the sample is a tissue mass. In some embodiments, the sample is a dispersed cell. In some embodiments, the sample size is from about 1 cell to about 1 × 10 ⁶ cells or more. In some embodiments, the sample size is from about 10 cells to about 1 × 10 ⁵ cells. In some embodiments, the sample size is from about 10 cells to about 10,000 cells. In some embodiments, the sample size is from about 10 cells to about 1,000 cells. In some embodiments, the sample size is from about 10 cells to about 100 cells. In some embodiments, the sample size is from about 1 cell to about 10 cells. In some embodiments, the sample size is 1 cell.

  In some embodiments, the sample is processed into DNA or RNA. In some embodiments, the RNA is isolated from the sample. In some embodiments, the mRNA is isolated from the sample. In some embodiments, RNA is isolated from cells by techniques that include cell lysis and denaturation of the proteins contained therein. In some embodiments, DNase is added and DNA is removed. In some embodiments, an RNase inhibitor is added to the lysis buffer. In some embodiments, protein denaturation / digestion steps are added to the protocol. Methods for preparing total RNA and mRNA are well known in the art and RNA isolation kits are commercially available (eg, RNeasy mini kit, Qiagen, USA). In some embodiments, RNA is amplified by PCR-based techniques.

  The determination of biomarker expression levels can be performed by any suitable method, including but not limited to, methods based on analysis of polynucleotide expression, sequencing of polynucleotides, and / or analysis of protein expression. For example, determination of the biomarker expression level can be performed by detecting the expression of mRNA expressed from the gene of interest and / or detecting the expression of the polypeptide encoded by the gene.

  Commonly used methods for polynucleotide analysis include Southern blot analysis, Northern blot analysis, and in situ hybridization, RNAse protection assays, and, for example, known as reverse transcription polymerase chain reaction (RT-PCR), real-time PCR Polymerase chain reaction (PCR) based methods such as quantitative PCR (qPCR), TaqMan (TM), TaqMan (TM) low density array (TLDA), anchor PCR, competitive PCR, rapid amplification of cDNA ends (RECE method) , And microarray analysis. RT-PCR is a quantitative method that can be used to compare mRNA levels in different samples and examine gene expression profiles. A variation of RT-PCR is real-time quantitative PCR, where PCR product accumulation is measured with a double-labeled fluorogenic probe (eg, TaqMan ™ probe). Other PCR-based techniques known to those skilled in the art including, but not limited to, differential display, amplified fragment length polymorphism, BeadArray ™ technology, high coverage expression profiling (HiCEP) and digital PCR There are many. Typical methods for sequencing-based gene expression analysis include NexGen, including gene expression chain analysis (SAGE), massively parallel signature sequencing (MPSS), and mRNA sequencing. Sequencing analysis is included.

  In certain embodiments, biomarker expression is determined using a qPCR assay. For example, total RNA is extracted from fresh frozen (FF) samples, or total RNA is extracted from macro-dissected formalin-fixed paraffin-embedded (FFPE) tissue samples. The amount and quality of total RNA is assessed by standard spectrophotometry and / or any other suitable method (eg, Agilent Bioanalyzer). Following RNA extraction, RNA samples are reverse transcribed using standard methods and / or commercially available cDNA synthesis kits (eg, Roche Transcriptor First Strand cDNA synthesis kit). The resulting cDNA is preamplified using, for example, an ABI preamplification kit. Expression of biomarkers (eg, FBXW2, CCND2, RHOU, CTBP2, WIF1, and / or DKK1) is assessed using, for example, the ABI TaqMan Gene Expression Mastermix by the Roche Lightcycler 480 system (Roche Diagnostics). qPCR reactions are performed in triplicate. For each assay, a subset of samples is run without reverse transcription (RT negative control), and similarly, control samples are run without template. A universal human reference RNA sample is included on each plate and serves as a positive control. Appropriate reference genes are identified from a standard panel of reference genes. Candidate reference genes are selected by different cellular functions to eliminate the risk of simultaneous control. The optimal reference gene is evaluated and selected using specific software and algorithms (eg, Genex software; GeNorm and Normfinder algorithms). The expression level of each biomarker is normalized using the selected optimal reference gene. In some embodiments, these normalized (normalized) expression values for each biomarker are used to calculate a determination value for the sample. In some embodiments, these normalized (normalized) values for each biomarker are used to calculate expression levels.

  In some embodiments, biomarker expression uses a PCR-based assay that includes primers and / or probes specific for each biomarker (e.g., FBXW2, CCND2, RHOU, CTBP2, WIF1, and / or DKK1). Determined. As used herein, the term “probe” refers to any molecule capable of selectively binding to a specifically intended target biomolecule. Probes can be synthesized by those skilled in the art using known techniques or can be derived from biological preparations. Probes can include, but are not limited to, RNA, DNA, proteins, peptides, aptamers, antibodies, and organic molecules. The term “primer” or “probe” encompasses an oligonucleotide having a sequence of a specific SEQ ID NO or an oligonucleotide having a sequence complementary to a specific SEQ ID NO. In some embodiments, the probe is modified. In some embodiments, the probe is modified with a quencher. In some embodiments, the probe is labeled. Labels can include, but are not limited to, colorimetric labels, fluorescent labels, chemiluminescent labels, or bioluminescent labels.

の配列を含むポリヌクレオチド、

の配列を含むポリヌクレオチド、および

の配列を含むポリヌクレオチド、またはそれら相補体を用いて決定される。いくつかの態様では、CCND2発現は、配列

のフォワードプライマーおよび配列

のリバースプライマーを用いて決定される。いくつかの態様では、CCND2発現は、配列

のプローブを用いて決定される。 In some embodiments, biomarker expression for each biomarker is determined using specific primer sets and probes. In some embodiments, the specific primer set consists of a forward primer and a reverse primer. In some embodiments, CCND2 expression is

A polynucleotide comprising the sequence of

A polynucleotide comprising the sequence of

Are determined using a polynucleotide comprising the sequence of or a complement thereof. In some embodiments, the CCND2 expression is a sequence

Forward primer and sequence

Of the reverse primer. In some embodiments, the CCND2 expression is a sequence

It is determined using the probe.

の配列を含むポリヌクレオチド、

の配列を含むポリヌクレオチド、および

の配列を含むポリヌクレオチド、またはそれらの相補体を用いて決定される。いくつかの態様では、CTBP2発現は、配列

のフォワードプライマーおよび

の配列のリバースプライマーを用いて決定される。いくつかの態様では、CTBP2発現は、配列

のプローブを用いて決定される。 In some embodiments, CTBP2 expression is isolated,

A polynucleotide comprising the sequence of

A polynucleotide comprising the sequence of

Are determined using a polynucleotide comprising the sequence of or a complement thereof. In some embodiments, CTBP2 expression is a sequence

Forward primer and

The reverse primer of the sequence of In some embodiments, CTBP2 expression is a sequence

It is determined using the probe.

の配列を含むポリヌクレオチド、

の配列を含むポリヌクレオチド、および

の配列を含むポリヌクレオチド、またはそれらの相補体を用いて決定される。いくつかの態様では、DKK1発現は、配列

のフォワードプライマーおよび

の配列のリバースプライマーを用いて決定される。いくつかの態様では、DKK1発現は、配列

のプローブを用いて決定される。 In some embodiments, DKK1 expression is isolated,

A polynucleotide comprising the sequence of

A polynucleotide comprising the sequence of

Are determined using a polynucleotide comprising the sequence of or a complement thereof. In some embodiments, the DKK1 expression is a sequence

Forward primer and

The reverse primer of the sequence of In some embodiments, the DKK1 expression is a sequence

It is determined using the probe.

の配列を含むポリヌクレオチド、

の配列を含むポリヌクレオチド、および

の配列を含むポリヌクレオチド、またはそれらの相補体を用いて決定される。いくつかの態様では、FBXW2発現は、配列

のフォワードプライマーおよび配列

のリバースプライマーを用いて決定される。いくつかの態様では、FBXW2発現は、配列

のプローブを用いて決定される。 In some embodiments, FBXW2 expression is

A polynucleotide comprising the sequence of

A polynucleotide comprising the sequence of

Are determined using a polynucleotide comprising the sequence of or a complement thereof. In some embodiments, the FBXW2 expression is a sequence

Forward primer and sequence

Of the reverse primer. In some embodiments, the FBXW2 expression is a sequence

It is determined using the probe.

の配列を含むポリヌクレオチド、

の配列を含むポリヌクレオチド、および

の配列を含むポリヌクレオチド、またはそれらの相補体を用いて決定される。いくつかの態様では、RHOU1発現は、配列

のフォワードプライマーおよび配列

のリバースプライマーを用いて決定される。いくつかの態様では、RHOU1発現は、配列

のプローブを用いて決定される。 In some embodiments, RHOU1 expression is

A polynucleotide comprising the sequence of

A polynucleotide comprising the sequence of

Are determined using a polynucleotide comprising the sequence of or a complement thereof. In some embodiments, the RHOU1 expression is a sequence

Forward primer and sequence

Of the reverse primer. In some embodiments, the RHOU1 expression is a sequence

It is determined using the probe.

の配列を含むポリヌクレオチド、

の配列を含むポリヌクレオチド、および

の配列を含むポリヌクレオチド、またはそれらの相補体を用いて決定される。いくつかの態様では、WIF1発現は、配列

のフォワードプライマーおよび配列

のリバースプライマーを用いて決定される。いくつかの態様では、WIF1発現は、配列

のプローブを用いて決定される。 In some embodiments, WIF1 expression is

A polynucleotide comprising the sequence of

A polynucleotide comprising the sequence of

Are determined using a polynucleotide comprising the sequence of or a complement thereof. In some embodiments, the WIF1 expression is a sequence

Forward primer and sequence

Of the reverse primer. In some embodiments, the WIF1 expression is a sequence

It is determined using the probe.

  In some embodiments of any of the methods described herein, the expression level of FBXW2, CCND2, RHOU, CTBP2, WIF1, and DKK1 is a polynucleotide selected from the group consisting of SEQ ID NOs: 62-79 Is measured. In some embodiments of any of the methods described herein, the expression level of FBXW2, CCND2, RHOU, CTBP2, WIF1, and DKK1 is (a) a forward primer of SEQ ID NO: 62, SEQ ID NO: 63 reverse primers, and a probe comprising SEQ ID NO: 64; (b) a forward primer of SEQ ID NO: 65, a reverse primer of SEQ ID NO: 66, and a probe comprising SEQ ID NO: 67; (c) SEQ A forward primer of ID NO: 68, a reverse primer of SEQ ID NO: 69, and a probe comprising SEQ ID NO: 70; (d) a forward primer of SEQ ID NO: 71, a reverse primer of SEQ ID NO: 72, and SEQ A probe comprising ID NO: 73; (e) a forward primer of SEQ ID NO: 74, a reverse primer of SEQ ID NO: 75, and a probe comprising SEQ ID NO: 76; and (f) a forward of SEQ ID NO: 77 Includes primer, reverse primer of SEQ ID NO: 78, and SEQ ID NO: 79 It is measured using a probe.

  In some embodiments, the expression level of each biomarker (eg, FBXW2, CCND2, RHOU, CTBP2, WIF1, and / or DKK1) is determined in a separate assay (eg, 6 assays). In some embodiments, the reference gene and normalization method for each assay is the same for all six assays. In some embodiments, the expression levels of multiple biomarkers (eg, FBXW2, CCND2, RHOU, CTBP2, WIF1, and / or DKK1) are detected in a single multiplex assay.

  Alternatively, biomarker expression levels can be determined by amplifying complementary DNA (cDNA) or complementary RNA (cRNA) made from mRNA and analyzing it using a microarray. Microarray technology allows simultaneous analysis of the expression of thousands of genes. Several different array arrangements and methods for making them are known to those skilled in the art. In addition, microarrays are commercially available (eg, Affymetrix GeneChips) or can be customized. Currently widely used microarrays include cDNA arrays and oligonucleotide arrays. Generally, a polynucleotide of interest (eg, a probe or probe set) is placed or aligned on a microchip substrate. In some embodiments, probes for at least 10, 25, 50, 100, 500, 1000, 5000, 10,000, 20,000, or 25,000 genes or more are immobilized on the array substrate. The substrate can be a porous or non-porous support, such as a glass, plastic or gel surface. Probes can include DNA, RNA, DNA and RNA copolymer sequences, DNA and / or RNA analogs, or combinations thereof. In some embodiments, the microarray includes a support having a regular arrangement of binding sites for each individual gene. The microarray may be an addressable array or a position addressable array, e.g., each probe of the array is on a solid support so that the identity of each probe can be determined from the position of the array. Are arranged in known predetermined positions.

  Each probe on the microarray can be 10-50,000 nucleotides in length. In some embodiments, the microarray probes are less than about 1,000 nucleotides in length, less than about 750 nucleotides in length, less than about 500 nucleotides in length, less than about 250 nucleotides in length, less than about 100 nucleotides in length, or less than about 50 nucleotides in length. It can consist of a nucleotide sequence having In general, the array includes a positive control probe and a negative control probe.

  In certain embodiments, biomarker expression is determined using a microarray. For example, total RNA is extracted from a fresh frozen (FF) tissue sample, or total RNA is extracted from a macrodissected formalin-fixed paraffin-embedded (FFPE) tissue sample. The amount and quality of total RNA is assessed by standard spectrophotometry and / or any other suitable technique (eg, Agilent Bioanalyzer). Following RNA extraction, the RNA sample is amplified using standard methods and / or commercially available amplification systems (eg, NuGEN Ovation RNA Amplification System V2). Amplified cDNA is fragmented, labeled and hybridized to a microarray according to standard techniques (eg, using NuGEN Encore Biotin Module and Affymetrix GeneChip arrays). The array is washed, stained and scanned according to the microarray instructions. Microarray data is preprocessed and probe-level intensity measurements are background corrected, normalized, and aggregated as expression measurements using the Robust Multichip algorithm (RMA). Probe level data is aggregated to obtain the expression level of each biomarker (eg, FBXW2, CCND2, RHOU, CTBP2, WIF1, and / or DKK1). A combination of quality parameter thresholds and data reduction techniques (eg, principal component analysis) is applied to the data set to establish profile quality and identify samples that may be outside of a specified range. These normalized (or standardized) expression values for each biomarker are used to calculate a sample decision value.

  In some embodiments, biomarker expression is analyzed by examining protein expression of one or more genes of interest. Methods commonly used for analysis of protein expression include, but are not limited to, immunohistochemistry (IHC) based methods, antibody based methods, and mass spectrometry based methods. Antibodies, generally monoclonal antibodies, can be used to detect the expression of gene products (eg, proteins). In some embodiments, the antibody can be detected by direct labeling of the antibody itself. In other embodiments, an unlabeled primary antibody is used in combination with a labeled secondary antibody. Tissue immunochemistry methods and / or kits are well known in the art and are commercially available.

  In some embodiments, biomarker expression is determined by multiple analyte profile testing, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay, western blot assay, immunofluorescence assay, enzyme immunoassay, immunoprecipitation assay, chemiluminescence assay Determined by assays known to those skilled in the art including, but not limited to, immunohistochemical assays, dot blot assays, or slot blot assays. In some embodiments, an antibody is used in the assay and the antibody is detectably labeled. Antibody labels may include, but are not limited to, immunofluorescent labels, chemiluminescent labels, phosphorescent labels, enzyme labels, radiolabels, avidin / biotin, colloidal gold particles, colored particles, and magnetic particles. .

  Other methods suitable for analyzing biomarker expression include proteomics-based methods. Proteomics specifically includes testing for global changes in protein expression in a sample. In some embodiments, the proteomic method comprises the following steps: (1) separation of individual proteins in the sample by 2-D electrophoresis (2-D PAGE), (2) individual proteins recovered from the gel Identification (eg, by mass spectrometry or N-terminal sequencing) and (3) analysis of data using bioinformatics. In some embodiments, the proteomic method comprises using a tissue microarray (TMA). The tissue array can be constructed by various techniques known to those skilled in the art. In certain embodiments, a manual tissue array is used to remove a “core” from a paraffin block prepared from a tissue sample. The core is then inserted into another paraffin block at a specified position on the grid. A core from about 400 samples can be inserted into one recipient block. The resulting tissue array can be processed into thin sections for analysis. In some embodiments, the proteomic method comprises an antibody microarray. In some embodiments, the proteomic method includes using mass spectrometry, including but not limited to SELDI, MALDI, electrospray, and surface plasmon resonance. In some embodiments, proteomic methods include bead-based techniques, including but not limited to antibodies on beads in an array format. In some embodiments, the proteomic method comprises a reverse phase protein microarray (RPPM). In some embodiments, the proteomic method comprises multiplex protein profiling, including but not limited to the Global Proteome Survey (GPS) method.

  In some embodiments, the biomarker signature is identified by differential gene expression between the two samples. In some embodiments, the biomarker signature includes genes that are identified by differential gene expression between the two samples and that are differentially expressed in cancer cells compared to normal cells. In some embodiments, the biomarker signature comprises a gene that is differentially expressed in tumorigenic cancer stem cells compared to non-tumorigenic cancer cells. In some embodiments, the biomarker signature is a gene that is differentially expressed in tumor-derived cells that are responsive to the same treatment as compared to tumor-derived cells that are non-responsive to a particular treatment. including.

  In some embodiments, the expression profile is determined using microarray analysis. The microarray data identifies a gene profile that includes similarly expressed and differentially expressed genes between the two samples. In some embodiments, the expression profile is refined, sorted, and / or subdivided into biomarker signatures based on fold expression change. In some embodiments, all genes that exceed a certain fold expression change are included in the biomarker signature. The expression change fold can be increased, decreased, or both increased and decreased. In some embodiments, all genes with more than a 2-fold change in expression are included in the biomarker signature. In some embodiments, all genes with an expression change of 2.5 fold or more are included in the biomarker signature. In some embodiments, all genes with an expression change of 3 fold or more are included in the biomarker signature. In some embodiments, all genes with an expression change of 3.5 fold or more are included in the biomarker signature. In some embodiments, all genes with an expression change of 4 fold or more are included in the biomarker signature.

  In some embodiments, the gene expression profile is refined, sorted, and / or subdivided into biomarker signatures based on statistical analysis. Statistical methods include cluster analysis, support vector machine (SVM) analysis, support vector machine-recursive feature elimination (SVM-RFE) analysis, Platt scaling, neural networks, and other algorithms. However, it is not limited to these. In some embodiments, the gene expression profile is analyzed using t-test analysis. In some embodiments, gene expression profiles are analyzed using empirical Bayes analysis of corresponding samples. In some embodiments, a combination of statistical analysis is used. In some aspects, an SVM model is used to obtain a decision value based on the training data. In some embodiments, the decision value is calculated by a weighted sum of the normalized expression of the set of biomarkers. In some embodiments, a positive decision value indicates a tumor that is predicted to be a responder, and a negative decision value indicates a tumor that is predicted to be a non-responder. In some embodiments, classification probabilities for responders and non-responders are obtained using Platt scaling (Platt, 1999, Advances in Large Margin Classifiers, pp. 61-74, MIT Press). Platt scaling may include fitting a logistic distribution using the maximum likelihood to the decision value obtained, for example by an SVM model. In some embodiments, tumors associated with a probability higher than 0.5 are expected to be responders and tumors with a probability lower than 0.5 are expected to be non-responders.

  In some embodiments of any of the methods or uses described herein, the tumor classification probability (for responder status or non-responder status) is obtained based on the decision value. In some aspects, the probability is obtained by fitting a logistic regression to the decision value. In some embodiments, tumors associated with a probability higher than 0.5 are predicted to be responders, and tumors with a probability lower than 0.5 are predicted to be non-responders.

  In some embodiments, the biomarker signature is obtained by a series of analytical steps. For example, expression data from a training sample set is obtained from microarray analysis. The data is reprocessed to obtain an expression matrix with specific genes. Genes whose variation is close to zero are removed because they are genes with expression values below a predetermined level. The remaining genes are ranked using SVM-RFE analysis. The single predictive cross-validation (LOOCV) method is used to identify and select the best predictive gene, and also measure positive predictive value (PPV), negative predictive value (NPV), sensitivity and specificity.

  In some embodiments, all genes with an inter-sample P value less than or equal to 0.01 and having increased expression, all genes having decreased expression, or both are included in the biomarker signature. In some embodiments, all genes with an inter-sample P-value of 0.005 or less and having increased expression, all genes having decreased expression, or both are included in the biomarker signature. In some embodiments, all genes having an inter-sample P value of 0.001 or less and having an increased expression, all genes having a decreased expression, or both are included in the biomarker signature. In some embodiments, all genes with an increased expression, FDR (false discovery rate) of 0.25 or less, all genes with decreased expression, or both are included in the biomarker signature. In some embodiments, all genes with increased expression, all genes with decreased expression, or both with an FDR of 0.1 or less, 0.01 or less, or 0.001 or less are included in the biomarker signature.

In some embodiments, gene expression profiles and / or biomarker signatures are refined, sorted and / or subdivided based on statistical models. In some embodiments, gene expression profiles and / or biomarker signatures are refined, sorted and / or subdivided based on survival analysis models. These models include Kaplan-Meier survival model, Cox proportional model, Cox proportional hazard model, χ ² analysis, univariate logistic regression model, multivariate competitive risk model, linear discriminant analysis model, parametric regression model and correlation analysis model Can be included, but is not limited to these.

  In some embodiments, gene expression profiles and / or biomarker signatures are filtered, sorted, fragmented and / or tested using gene expression array data sets with relevant clinical results. There are a plurality of databases including publicly available data sets such as Gene Expression Omnibus (GEO) and ArrayExpress.

  In some embodiments, gene expression profiles and / or biomarker signatures are refined using biological function parameters and / or gene sets. For example, in some embodiments, gene expression profiles and / or biomarker signatures are refined using Gene Set Enrichment Analysis (GSEA) (Subramanian et al., 2005, PNAS, 102: 15545-15550). In some embodiments, gene expression profiles are refined based on their ability to predict clinical outcome.

  In some embodiments of the methods described herein, the Wnt pathway inhibitor is an anti-FZD antibody described herein. In some embodiments of the methods described herein, the Wnt pathway inhibitor is an antibody that specifically binds to at least one Frizzled (FZD) protein or portion thereof. In some embodiments, the anti-FZD antibody specifically binds to at least one FZD protein selected from the group consisting of FZD1, FZD2, FZD5, FZD7, and FZD8. In other embodiments, 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 NFIKYVFAN (SEQ ID NO: 3) And (b) a light chain CDR1 containing SGDNIGSFYVH (SEQ ID NO: 4), a light chain CDR2 containing DKSNRPSG (SEQ ID NO: 5), and a light chain containing QSYANTLSL (SEQ ID NO: 6). Contains chain CDR3. In some embodiments, the anti-FZD antibody comprises a heavy chain variable region comprising amino acids of SEQ ID NO: 7. In some embodiments, the anti-FZD antibody comprises a light chain variable region comprising amino acids of SEQ ID NO: 8. In some embodiments, the anti-FZD antibody comprises a heavy chain variable region comprising amino acids of SEQ ID NO: 7 and a light chain variable region comprising 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 a plasmid having ATCC deposit number 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 a plasmid deposited with the ATCC having deposit number PTA-9541.

  In some aspects of the methods described herein, the tumor is a breast tumor, lung tumor, colon tumor, glioma, gastrointestinal tumor, kidney tumor, ovarian tumor, liver tumor, colorectal tumor, endometrial tumor , Kidney tumor, prostate tumor, thyroid tumor, neuroblastoma, pancreatic tumor, glioblastoma multiforme, cervical tumor, stomach tumor, bladder tumor, hepatoma, melanoma, and head and neck tumor Is done. In some embodiments, the tumor is a breast tumor. In some embodiments, the tumor is a HER-2 negative breast tumor. In some embodiments, the tumor is a triple negative breast cancer (TNBC) tumor.

  In some aspects of the methods described herein, the cancer is breast cancer, lung cancer, colon cancer, glioma, gastrointestinal cancer, kidney cancer, ovarian cancer, liver cancer, colorectal cancer. , Endometrial cancer, kidney cancer, prostate cancer, thyroid cancer, neuroblastoma, pancreatic cancer, glioblastoma multiforme, cervical cancer, stomach cancer, bladder cancer, hepatoma, melanoma And selected from the group consisting of head and neck cancer. In some embodiments, the cancer is breast cancer. In some embodiments, the cancer is HER2-negative breast cancer. In some embodiments, the cancer is triple negative breast cancer (TNBC).

  In some embodiments of the methods described herein, the methods include a Wnt pathway inhibitor (e.g., an anti-tumor agent) described herein, particularly after the patient has been identified as responsive to treatment with a Wnt pathway inhibitor. Treating the patient with an FZD antibody). In some embodiments, treatment comprises administering at least one additional therapeutic agent in combination with a Wnt pathway inhibitor. The additional therapeutic agent can be administered prior to, concurrently with, and / or subsequent to administration of the Wnt pathway inhibitor. In some embodiments, the at least one additional therapeutic agent comprises one, two, three, 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., cisplatin, mono (platinum) complexes, bis (platinum) complexes, 3 (Platinum complexes such as nuclear platinum complexes and carboplatin), anthracyclines, antibiotics, antifolates, antimetabolites, chemotherapy sensitizers, duocarmycin, etoposide, fluoropyrimidine, ionophore, lexitropsin, nitroso Examples include urea, platinol, purine antimetabolite, puromycin, radiosensitizer, steroids, taxanes, topoisomerase inhibitors, or vinca alkaloids. In certain embodiments, the second therapeutic agent is an alkylating agent, antimetabolite, mitotic inhibitor, topoisomerase inhibitor, or angiogenesis inhibitor.

  Therapeutic agents that can be administered in combination with Wnt pathway inhibitors include chemotherapeutic agents. Thus, in some embodiments, the method or treatment involves administration of a Wnt pathway inhibitor of the invention in combination with a chemotherapeutic agent or a cocktail of multiple different chemotherapeutic agents. Treatment with a Wnt pathway inhibitor (eg, an anti-FZD antibody) can occur prior to chemotherapy, at the same time as chemotherapy, or after chemotherapy. Co-administration includes co-administration in a single pharmaceutical formulation, or co-administration in separate formulations, or in either order, but generally all active agents have their respective biological activities. Sequential administration can be included within a period in which can be exerted simultaneously. Preparation and dosing schedules for such chemotherapeutic agents can be used according to the manufacturer's instructions or based on the experience of a skilled physician. Preparation and administration schedules for such chemotherapy are also described in The Chemotherapy Source Book, 4th Edition, 2008, edited by M.C. Perry, Lippincott, Williams & Wilkins (Philadelphia, PA).

  Chemotherapeutic agents useful in the present invention include alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN); alkyl sulfonates such as busulfan, improsulfan and piperosulphane; aziridines such as benzodopa, carbocon, methredopa (Meturedopa), and uredopa; ethyleneimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine; nitrogen mustards, such as Chlorambucil, chlornafazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, meclomethamine oxide hydrochloride, melphalan, nobuen Bikin, phenesterine, prednisotin, trophosphamide, uracil mustard; nitrosoureas, such as carmustine, chlorozotocin, hotemustine, lomustine, nimustine, ranimustine; antibiotics, such as aclacinomycins, actinomycin, anthromycin, azaserine, cactomycin, cactomycin, cactomycin , Calicheamicin, carabicin, carminomycin, cardinophylline, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcelomycin , Mitomycin, mycophenolic acid, nogaramycin, olivomycin, peplomycin, potfiromycin, Uromycin, quelamycin, rhodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, dinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, Methotrexate, pteropterin, trimethrexate; purine analogues such as fludarabine, 6-mercaptopurine, thiamidpurine, thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytosine arabinoside, dideoxyuridine, doxifluridine, enocitabine Floxuridine, 5-FU; androgens such as carsterone, drostanolone propionate, epithiostanol, mepitiosta , Test lactones; antiadrenals, such as aminoglutethimide, mitotane, trilostane; folic acid supplements, such as folinic acid; acegraton; aldophosphamide glycoside; aminolevulinic acid; amsacrine; vesturacil; bisantrene; Defofamine; defofamine; demecoltin; diazicon; elformithine; ellipticine acetate; etoglucid; gallium nitrate; hydroxyurea; Acid; 2-ethylhydrazide; procarbazine; PSK; razoxan; sizofuran; spirogermanium; tenuazonic acid; triadicon; 2,2 ', 2' '-trichlorotriethylamine; urethane; vindesine; dacal Gin; Mannomustine; Mitobronitol; Mitractol; Piperobroman; Gacytosine; Arabinoside (Ara-C); Taxoids such as paclitaxel (TAXORE) and docetaxel (TAXOTERE); Chlorambucil; Gemcitabine; 6-thiopurine; Cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantron; teniposide; daunomycin; aminopterin; Difluoromethylornithine (DMFO); retinoic acid; esperamicins; capecitabine (XELODA); and any of the above pharmaceutically acceptable salts, acids or derivatives , But it is not limited to these. Chemotherapeutic agents also include antihormonal agents such as antiestrogens that act to regulate or inhibit hormonal effects on tumors, such as tamoxifen, raloxifene, aromatase inhibitory 4 (5) -imidazoles, 4-hydroxy tamoxifen , Trioxyphene, keoxifen, LY117018, onapristone, and toremifene (FARESTON); and antiandrogens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and any pharmaceutically acceptable salt, acid or derivative of the above Is included. In certain embodiments, the additional therapeutic agent is paclitaxel (taxol).

  In certain embodiments, the chemotherapeutic agent is a topoisomerase inhibitor. Topoisomerase inhibitors are chemotherapeutic agents that interfere with the action of topoisomerase enzymes (eg, topoisomerase I or II). Topoisomerase inhibitors include doxorubicin HCl, daunorubicin citrate, mitoxantrone HCl, actinomycin D, etoposide, topotecan HCl, teniposide (VM-26), and irinotecan, and pharmaceutically acceptable salts of any of these, Examples include, but are not limited to acids, or derivatives.

  In certain embodiments, the chemotherapeutic agent is an antimetabolite. Antimetabolites are chemicals that resemble metabolites required for normal biochemical reactions, but have structures that are different enough to interfere with one or more normal cell functions such as cell division. Antimetabolites include gemcitabine, fluorouracil, capecitabine, methotrexate sodium, raltitrexed, pemetrexed, tegafur, cytosine arabinoside, thioguanine, 5-azacytidine, 6-mercaptopurine, azathioprine, 6-thioguanine, pentostatin, fludarabine phosphate, And cladribine, as well as any pharmaceutically acceptable salt, acid or derivative thereof.

  In certain embodiments, the chemotherapeutic agent is a mitotic inhibitor that includes, but is not limited to, an agent that binds to tubulin. In some embodiments, the agent is a taxane. In certain embodiments, the agent is paclitaxel or docetaxel, or a pharmaceutically acceptable salt, acid, or derivative of paclitaxel or docetaxel. In certain embodiments, the agent is paclitaxel (TAXOL), docetaxel (TAXOTERE), albumin-bound paclitaxel (nab-paclitaxel; ABRAXANE), DHA-paclitaxel, or PG-paclitaxel. In certain alternative embodiments, the mitotic inhibitor comprises a vinca alkaloid, such as vincristine, vinblastine, vinorelbine, or vindesine, or a pharmaceutically acceptable salt, acid, or derivative thereof. In some embodiments, the mitotic inhibitor is an inhibitor of kinesin Eg5 or an inhibitor of mitotic kinases such as Aurora A or Plk1. In certain embodiments where the chemotherapeutic agent administered in combination with the Wnt pathway inhibitor is a mitotic inhibitor, the cancer or tumor being treated is a breast cancer or breast tumor. In certain embodiments, the additional therapeutic agent is paclitaxel (taxol) or albumin-bound paclitaxel.

  In some embodiments, the additional therapeutic agent comprises an agent such as a small molecule. For example, treatment includes a Wnt pathway inhibitor of the present invention and a small molecule that acts as an inhibitor against additional tumor-associated antigens, including but not limited to EGFR, ErbB2, HER2, and / or VEGF. In combination. In certain embodiments, the additional therapeutic agent is a small molecule that inhibits the cancer stem cell pathway. In some embodiments, the additional therapeutic agent is an inhibitor of the Notch pathway. In some embodiments, the additional therapeutic agent is an inhibitor of the Wnt pathway. In some embodiments, the additional therapeutic agent is an inhibitor of the BMP pathway.

  Certain embodiments of the invention are responsive or non-reactive to treatment with an antibody that specifically binds to at least one human frizzled (FZD) selected from the group consisting of FZD1, FZD2, FZD5, FZD7, and FZD8. A method for identifying a human breast tumor that may be: (a) obtaining a sample of a human breast tumor; (b) measuring the expression level of each biomarker of the biomarker signature in the sample The biomarker signature includes one or more of the biomarkers FBXW2, CCND2, RHOU, CTBP2, WIF1, and DKK1, and (c) determined based on the normalized expression of the biomarker in the biomarker signature Calculating a value, wherein a positive decision value indicates that a breast tumor is predicted to be responsive to antibody treatment, and a negative decision value indicates that the tumor is non-responsive to antibody treatment. Expected to be And a method comprising the steps of: Some embodiments may be responsive to treatment with an antibody that specifically binds to at least one human frizzled (FZD) selected from the group consisting of FZD1, FZD2, FZD5, FZD7, and FZD8. A method for identifying a patient having breast cancer, comprising: (a) obtaining a sample of breast cancer; (b) measuring the expression level of each biomarker in the biomarker signature in the sample, Including one or more of the biomarkers FBXW2, CCND2, RHOU, CTBP2, WIF1, and DKK1, and (c) calculating a criterion based on the normalized expression of the biomarker in the biomarker signature And wherein the positive decision value includes a method comprising indicating that the breast cancer is predicted to be responsive to antibody treatment. Some embodiments select patients with breast cancer for treatment with an antibody that specifically binds to at least one human frizzled (FZD) selected from the group consisting of FZD1, FZD2, FZD5, FZD7, and FZD8. (A) obtaining a breast cancer sample; (b) measuring the expression level of each biomarker biomarker signature in the sample, wherein the biomarker signature is biomarker FBXW2, CCND2, Including one or more of RHOU, CTBP2, WIF1, and DKK1; (c) calculating a decision value based on the normalized expression of the biomarker in the biomarker signature, wherein the positive decision value is Including a step of indicating that breast cancer is expected to be responsive to treatment with the antibody; and selecting a patient for treatment when the patient's tumor sample has a positive determination. .

  Some embodiments of the invention include (a) treatment with an antibody that specifically binds to at least one human frizzled (FZD) selected from the group consisting of FZD1, FZD2, FZD5, FZD7, and FZD8. Identifying whether there is a possibility of reaction, wherein the identification comprises (i) obtaining a sample of a patient's breast cancer, (ii) measuring the expression level of each biomarker of the biomarker signature in the sample Determining whether the biomarker signature includes one or more of the biomarkers FBXW2, CCND2, RHOU, CTBP2, WIF1, and DKK1, and (iii) standardized expression of the biomarker in the signature Calculating a value comprising indicating that a positive decision value is predicted that the patient will respond to the treatment; and (b) an effective amount of antibody in the patient predicted to respond to the treatment Including the step of administering It includes a method of treating a definitive breast cancer.

  Certain aspects of the invention include: A method of identifying a human breast tumor that may be responsive or non-responsive to treatment with an anti-FZD antibody OMP-18R5 in combination with paclitaxel, comprising: (a) obtaining a sample of a human breast tumor; (b) measuring the expression level of each biomarker of the biomarker signature in the sample, Biomarker sign is biomarker FBXW2, CCND2, RHOU, CTBP2, WIF1, And including DKK1 Process; And (c) calculating a decision value based on the normalized expression of the biomarker in the biomarker signature, Positive judgment value is Showing that breast tumors are expected to be responsive to treatment, Negative judgment value is Indicating that the tumor is expected to be non-responsive to treatment; Including a method comprising the steps. Some aspects are A method of identifying patients with breast cancer that may be responsive to treatment with anti-FZD antibody OMP-18R5 in combination with paclitaxel, comprising: (a) obtaining a breast cancer sample; (b) measuring the expression level of each biomarker of the biomarker signature in the sample, Biomarker sign is biomarker FBXW2, CCND2, RHOU, CTBP2, WIF1, And including DKK1 Process; And (c) calculating a decision value based on the normalized expression of the biomarker in the biomarker signature, Positive judgment value is Indicates that breast cancer is expected to be responsive to treatment, Including a method comprising the steps. Some aspects are A method of selecting patients with breast cancer for treatment with an anti-FZD antibody OMP-18R5 in combination with paclitaxel comprising: (a) obtaining a breast cancer sample; (b) measuring the expression level of each biomarker of the biomarker signature in the sample, Biomarker sign is biomarker FBXW2, CCND2, RHOU, CTBP2, WIF1, And including DKK1 Process; (c) calculating a determination value based on the normalized expression of the biomarker in the biomarker signature, Positive judgment value is Indicates that breast cancer is expected to be responsive to treatment, Process; And selecting a patient for treatment when the patient's tumor sample has a positive decision value, Including a method comprising the steps.

  Some embodiments of the invention include (a) identifying whether a patient is likely to respond to treatment with the anti-FZD antibody OMP-18R5 in combination with paclitaxel, wherein the identification comprises (i ) Obtaining a patient's breast cancer sample, (ii) measuring the expression level of each biomarker biomarker signature in the sample, the biomarker signature is biomarker FBXW2, CCND2, RHOU, CTBP2, WIF1, And (iii) calculating a decision value based on the normalized expression of the biomarker in the signature, wherein a positive decision value is predicted that the patient is expected to respond to treatment. And (b) a method of treating breast cancer in a patient, comprising: (b) administering an effective amount of an antibody and paclitaxel to the patient predicted to be responsive to the treatment.

III. Wnt Pathway Inhibitors The present invention provides methods for identifying patients with cancer and / or tumors that may be responsive or sensitive to treatment with Wnt pathway inhibitors. As used herein, “Wnt pathway inhibitors” include, but are not limited to, Frizzled (FZD) binding agents and Wnt binding agents. An FZD binding agent can include an antibody that specifically binds to an FZD protein. Wnt binding agents can include antibodies that specifically bind to Wnt proteins, as well as soluble FZD receptors that bind to Wnt proteins.

  In certain embodiments, the Wnt pathway inhibitor is an agent that binds to one or more human FZD proteins. In some embodiments, the FZD binding agent specifically binds 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 FZD proteins. In some embodiments, the FZD binding agent binds to one or more FZD proteins selected from the group consisting of FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, and FZD10. . In some embodiments, the FZD binding agent binds to one or more FZD proteins including FZD1, FZD2, FZD5, FZD7, and / or FZD8. In certain embodiments, the FZD binding agent binds to FZD7. In certain embodiments, the FZD binding agent binds to FZD5 and / or FZD8. In certain embodiments, the FZD binding agent specifically binds to FZD1, FZD2, FZD5, FZD7, and FZD8. Non-limiting examples of FZD binding agents can be found in US Pat. No. 7,982,013.

  In certain embodiments, the FZD binding agent is an 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 classical Wnt signaling.

  In some embodiments, 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, the antigen binding site of an FZD binding antibody or FZD binding polypeptide described herein has the ability to bind 1, 2, 3, 4, 5 or more human FZD proteins ( Or combine). In certain embodiments, the antigen binding site of the FZD binding antibody or FZD binding polypeptide is selected from the group consisting of FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9 and FZD10. Have the ability to specifically bind to 4 or 5 human FZD proteins. In some embodiments, if the FZD binding agent is an antibody that binds to two or more FZD proteins, it can be referred to as a “pan-FZD antibody”.

  In certain embodiments, the FZD binding agent (eg, antibody) specifically binds to the extracellular domain (ECD) of one or more human FZD proteins to which it binds. In certain embodiments, the FZD binding agent specifically binds within the Fri domain (also known as the cysteine rich domain (CRD)) of the human FZD protein to which it binds. The sequence of the Fri domain of each human FZD protein is known in the art and includes SEQ ID NO: 13 (FZD1), SEQ ID NO: 14 (FZD2), SEQ ID NO: 15 (FZD3), SEQ ID NO : 16 (FZD4), SEQ ID NO: 17 (FZD5), SEQ ID NO: 18 (FZD6), SEQ ID NO: 19 (FZD7), SEQ ID NO: 20 (FZD), SEQ ID NO: 21 (FZD9) And as SEQ ID NO: 22 (FZD10).

  In certain embodiments, the FZD binding agent binds to 1, 2, 3, 4, 5 or more FZD proteins. In some embodiments, the FZD binding agent specifically binds to 1, 2, 3, 4, or 5 FZD proteins selected from the group consisting of FZD1, FZD2, FZD5, FZD7, and FZD8. . In some embodiments, the FZD binding agent specifically binds at least FZD5 and FZD8.

In some embodiments, the FZD binding agent comprises at least one human FZD protein of about 1 μM or less, about 100 nM or less, about 40 nM or less, about 20 nM or less, about 10 nM or less, about 1 nM or less, or about 0.1 nM or less. The dissociation constant (K _D ) In some embodiments, FZD binding agent is at least one FZD protein, binds about 10nM or less of K _D. In some embodiments, FZD binding agent is at least one FZD protein, binds about 1nM or less of K _D. In some embodiments, FZD binding agent is at least one FZD protein, binds about 0.1nM following K _D. In certain embodiments, the FZD binding agent is about 40 nM or less for each of one or more of FZD1, FZD2, FZD5, FZD7, and FZD8 (eg, 1, 2, 3, 4, or 5) _Bind with KD. In certain embodiments, FZD binding agent is, FZD1, FZD2, FZD5, FZD7 , and each of one or more of the FZD8, binds about 10nM or less of K _D. In certain embodiments, FZD binding agent is, FZD1, FZD2, FZD5, FZD7 , and each FZD8, binds with approximately 10nM of K _D. In some embodiments, K _D of the binding agent (e.g. an antibody) against FZD protein, using the FZD-Fc fusion protein comprising at least a portion of the immobilized FZD extracellular domain or FZD-Fri domain on Biacore chips a K _D determined Te.

In certain embodiments, the FZD binding agent is present in one or more (eg, 2 or more, 3 or more, or 4 or more) human FZD proteins of about 1 μM or less, about 100 nM or less, about 40 nM or less, It binds with an EC ₅₀ of about 20 nM or less, about 10 nM or less, or about 1 nM or less. In certain embodiments, an FZD binding agent binds to two or more FZD proteins with an EC ₅₀ of about 40 nM or less, about 20 nM or less, or about 10 nM or less. In certain embodiments, the FZD binding agent has an EC _{50 of} about 20 nM or less for one or more of the following FZD proteins (eg, 1, 2, 3, 4, or 5): FZD1, FZD2, FZD5, FZD7, and FZD8. In certain embodiments, the FZD binding agent has an EC _{50 of} about 10 nM or less for one or more of the following FZD proteins (eg, 1, 2, 3, 4, or 5): FZD1, FZD2, FZD5, FZD7, and FZD8. In certain embodiments, the FZD binding agent has an EC ₅₀ of about 40 nM or less or 20 nM or less for binding of FZD5 and / or FZD8.

  In certain embodiments, the Wnt pathway inhibitor is an FZD binding agent that is an antibody. In some embodiments, the antibody is a recombinant antibody. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a chimeric antibody. In some embodiments, the antibody is a humanized antibody. In some embodiments, the antibody is a human antibody. In certain embodiments, the antibody is an IgG1 antibody. In certain embodiments, the antibody is an IgG2 antibody. In certain embodiments, the antibody is an antibody fragment that includes an antigen binding site. In some embodiments, the antibody is monovalent, monospecific, or bivalent. In some embodiments, the antibody is a bispecific antibody or a multispecific antibody. In some embodiments, the antibody is conjugated to a cytotoxic moiety. In some embodiments, the antibody is isolated. In some embodiments, the antibody is substantially pure.

  The FZD binding agents (eg, antibodies) of the present invention can be assayed for specific binding by any method known in the art. Immunoassays that can be used include Biacore analysis, FACS analysis, immunofluorescence, immunocytochemistry, Western blot analysis, radioimmunoassay, ELISA, “sandwich” immunoassay, immunoprecipitation assay, precipitation reaction, gel diffusion sedimentation reaction, immunoassay Examples include, but are not limited to, competitive and non-competitive assay systems using techniques such as diffusion assays, agglutination assays, complement binding assays, immunoradiation assays, fluorescent immunoassays, and protein A immunoassays. Such assays are routine and well known in the art (see, eg, “Current Protocols in Molecular Biology” edited by Ausubel et al. (1994-present), John Wiley & Sons, Inc., New York, NY). ).

を含む重鎖CDR1、

を含む重鎖CDR2、

を含む重鎖CDR3を含むFZD結合作用物質（例えば抗体）である、Wnt経路インヒビターを提供する。いくつかの態様では、FZD結合作用物質がさらに、

を含む軽鎖CDR1、

を含む軽鎖CDR2、および

を含む軽鎖CDR3を含む。いくつかの態様では、FZD結合作用物質が、

を含む軽鎖CDR1、

を含む軽鎖CDR2、および

を含む軽鎖CDR3を含む。一定の態様では、FZD結合作用物質が、（a）

を含む重鎖CDR1、

を含む重鎖CDR2、および

を含む重鎖CDR3、ならびに（b）

を含む軽鎖CDR1、

を含む軽鎖CDR2、および

を含む軽鎖CDR3を含む。 In certain embodiments, the present invention provides:

Heavy chain CDR1, including

Heavy chain CDR2, including

A Wnt pathway inhibitor is provided that is an FZD binding agent (eg, antibody) comprising a heavy chain CDR3 comprising In some embodiments, the FZD binding agent further comprises

A light chain CDR1,

A light chain CDR2 comprising, and

Including light chain CDR3. In some embodiments, the FZD binding agent is

A light chain CDR1,

A light chain CDR2 comprising, and

Including light chain CDR3. In certain embodiments, the FZD binding agent comprises (a)

Heavy chain CDR1, including

A heavy chain CDR2 comprising, and

Heavy chain CDR3 containing, and (b)

A light chain CDR1,

A light chain CDR2 comprising, and

Including light chain CDR3.

を含む重鎖CDR1、または1、2、3、もしくは4個のアミノ酸置換を含むその変異体;（b）

を含む重鎖CDR2、または1、2、3、もしくは4個のアミノ酸置換を含むその変異体;（c）

を含む重鎖CDR3、または1、2、3、もしくは4個のアミノ酸置換を含むその変異体;（d）

を含む軽鎖CDR1、または1、2、3、もしくは4個のアミノ酸置換を含むその変異体;（e）

を含む軽鎖CDR2、または1、2、3、もしくは4個のアミノ酸置換を含むその変異体;および（f）

を含む軽鎖CDR3、または1、2、3、もしくは4個のアミノ酸置換を含むその変異体。一定の態様では、アミノ酸置換が保存的置換である。 In certain embodiments, the present invention provides FZD binding agents (eg, antibodies) comprising the following elements: (a)

A heavy chain CDR1 comprising, or a variant thereof comprising 1, 2, 3, or 4 amino acid substitutions; (b)

A heavy chain CDR2 comprising, or a variant thereof comprising 1, 2, 3, or 4 amino acid substitutions; (c)

A heavy chain CDR3 comprising or a variant thereof comprising 1, 2, 3, or 4 amino acid substitutions; (d)

A light chain CDR1 comprising or a variant thereof comprising 1, 2, 3, or 4 amino acid substitutions; (e)

A light chain CDR2 comprising, or a variant thereof comprising 1, 2, 3, or 4 amino acid substitutions; and (f)

A light chain CDR3 comprising or a variant thereof comprising 1, 2, 3 or 4 amino acid substitutions. In certain embodiments, the amino acid substitution is a conservative substitution.

  In certain embodiments, the invention provides a heavy chain variable region having at least about 80% sequence identity to SEQ ID NO: 7, and / or at least 80% sequence identity to SEQ ID NO: 8 FZD binding agents (eg, antibodies) are provided that comprise a light chain variable region having In certain embodiments, the FZD binding agent has 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 Contains the heavy chain variable region. In certain embodiments, the FZD binding agent has 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 Contains the light chain variable region. In certain embodiments, the FZD binding agent comprises a heavy chain variable region having at least about 95% sequence identity to SEQ ID NO: 7, and / or at least about 95% to SEQ ID NO: 8. It includes a light chain variable region with sequence identity. 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. In certain embodiments, 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 present invention provides FZD binding agents (eg, antibodies) comprising the following elements: (a) SEQ ID NO: 9 (with or without signal sequence) or SEQ ID NO: 11 Heavy chains with at least 90% sequence identity to; and / or (b) at least 90% sequence identity to SEQ ID NO: 10 (with or without signal sequence) or SEQ ID NO: 12 A light chain. In some embodiments, the FZD binding agent comprises the following elements: (a) SEQ ID NO: 9 (with or without signal sequence) or at least 95% sequence identity to SEQ ID NO: 11 And / or (b) a light chain having at least 95% sequence identity to SEQ ID NO: 10 (with or without signal sequence) or SEQ ID NO: 12. In some embodiments, the FZD binding agent is SEQ ID NO: 9 (with or without signal sequence) or heavy chain comprising SEQ ID NO: 11, and / or SEQ ID NO: 10 (with or without signal sequence) No light sequence) 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. In some embodiments, the FZD binding agent comprises a heavy chain consisting essentially of amino acids 20-463 of SEQ ID NO: 9 and a light chain consisting essentially of amino acids 20-232 of SEQ ID NO: 10. In some embodiments, the FZD binding agent comprises a heavy chain consisting essentially of SEQ ID NO: 11 and a light chain consisting essentially of SEQ ID NO: 12.

  In certain embodiments, the invention provides a Wnt pathway inhibitor that is a FZD binding agent (eg, an antibody) that specifically binds to at least one of FZD1, FZD2, FZD5, FZD7, and / or FZD8, wherein Provided are FZD binding agents (eg, antibodies) comprising one, two, three, four, five, and / or six of the CDRs of antibody OMP-18R5. The antibody OMP-18R5 (also known as 18R5 and bunchikumab) has been previously described in US Pat. No. 7,982,013, along with other FZD binding agents. DNA encoding the heavy and light chains of the OMP-18R5 IgG2 antibody was deposited with the ATCC on September 29, 2008 under the provisions of the Budapest Treaty and assigned ATCC accession number PTA-9541. In some embodiments, the FZD binding agent is 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, OMP Includes four or more of the CDRs of -18R5, five or more of the CDRs of OMP-18R5, or all six of the CDRs of OMP-18R5.

  The present invention provides polypeptides that are Wnt pathway inhibitors. The polypeptide includes, but is not limited to, an antibody that specifically binds to human FZD protein. In some embodiments, the polypeptide binds to one or more FZD proteins selected from the group consisting of FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, and FZD10. In some embodiments, the polypeptide binds to FZD1, FZD2, FZD5, FZD7, and / or FZD8. In some embodiments, the polypeptide binds to FZD1, FZD2, FZD5, FZD7, and FZD8.

  In certain embodiments, the polypeptide comprises one, two, three, four, five, and / or six of the CDRs of antibody OMP-18R5. In some embodiments, the polypeptide comprises a CDR with up to 4 amino acid substitutions per CDR (ie, 0, 1, 2, 3, or 4). In certain embodiments, heavy chain CDRs are included within the heavy chain variable region. In certain embodiments, the light chain CDRs are contained within the light chain variable region.

  In some embodiments, the invention provides a polypeptide that specifically binds to one or more human FZD proteins, wherein the polypeptide is at least about 80% sequence identical to SEQ ID NO: 7 And / or amino acid sequences having at least about 80% sequence identity to SEQ ID NO: 8. In certain embodiments, the polypeptide has an amino acid sequence that has 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 including. In certain embodiments, the polypeptide has an amino acid sequence that has 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 including. In certain embodiments, the polypeptide has an amino acid sequence having at least about 95% sequence identity to SEQ ID NO: 7, and / or at least about 95% sequence identity to SEQ ID NO: 8. Amino acid sequence having 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.

  In some embodiments, the FZD binding agent consists 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 comprising a sequence selected from the group.

  In certain embodiments, the FZD binding agent comprises a heavy chain variable region and a light chain variable region of an OMP-18R5 antibody. In certain embodiments, the FZD binding agent comprises the heavy and light chains (with or without a leader sequence) of an OMP-18R5 antibody.

  In certain embodiments, the FZD binding agent comprises, consists essentially of, or consists of the antibody OMP-18R5.

  In certain embodiments, the FZD binding agent (eg, antibody) comprises a heavy chain variable region comprising SEQ ID NO: 7 and SEQ ID NO: 8 for specific binding to one or more human FZD proteins. Competes with an antibody comprising a light chain variable region. In certain embodiments, the FZD binding agent (eg, antibody) comprises a heavy chain comprising SEQ ID NO: 9 (with or without signal sequence) for specific binding to one or more human FZD proteins; Competes with an antibody comprising a light chain comprising SEQ ID NO: 10 (with or without signal sequence). In certain embodiments, the FZD binding agent (eg, antibody) comprises a heavy chain comprising SEQ ID NO: 11 and a light chain comprising SEQ ID NO: 12 for specific binding to one or more human FZD proteins. Compete with antibodies containing In certain embodiments, the FZD binding agent competes with antibody OMP-18R5 for specific binding to one or more human FZD proteins. In some embodiments, the FZD binding agent or FZD binding antibody competes for specific binding to one or more human FZD proteins in an in vitro competitive binding assay.

  In certain embodiments, an FZD binding agent (eg, antibody) binds to the same or essentially the same epitope on one or more human FZD proteins as the antibodies of the invention. In another embodiment, the FZD binding agent is an antibody that binds to an epitope on one or more human FZD proteins that overlaps with an epitope on the FZD protein to which an antibody of the invention binds. In certain embodiments, the FZD binding agent (eg, antibody) binds to the same or essentially the same epitope on one or more FZD proteins as antibody OMP-18R5. In another embodiment, the FZD binding agent is an antibody that binds to an epitope on one or more human FZD proteins that overlaps with an epitope on the FZD protein to which antibody OMP-18R5 binds.

  In certain embodiments, the Wnt pathway inhibitor is an agent that binds to one or more human Wnt proteins. In certain embodiments, the agent specifically binds to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more Wnt proteins. In some embodiments, the Wnt binding agent is Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, Wnt10a, Wnt10b, Wnt11, It binds to one or more human Wnt proteins selected from the group consisting of In certain embodiments, the Wnt binding agent is one or more (or two) selected from the group consisting of Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt10a, and Wnt10b. It binds to Wnt proteins (3 or more, 4 or more, 5 or more). In certain embodiments, one or more (or two or more, three or more, four or more, five or more, etc.) Wnt proteins are Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt8a, Wnt8b, Wnt10a , And Wnt10b.

  In certain embodiments, the Wnt binding agent is a Wnt antagonist. In certain embodiments, the Wnt binding agent is a Wnt pathway antagonist. In certain embodiments, the Wnt binding agent inhibits Wnt signaling. In some embodiments, the Wnt binding agent inhibits classical Wnt signaling.

  In some embodiments, the Wnt binding agent is an antibody. In some embodiments, the Wnt binding agent is a polypeptide. In certain embodiments, the Wnt binding agent is an antibody or a polypeptide comprising an antigen binding site. In certain embodiments, an antigen binding site of a Wnt binding antibody or polypeptide described herein has the ability to bind 1, 2, 3, 4, 5 or more human Wnt proteins ( Or combine). In certain embodiments, the antigen binding site of the Wnt binding antibody or Wnt binding polypeptide is selected from the group consisting of Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt10a, and Wnt10b, Has the ability to specifically bind 2, 3, 4, or 5 human Wnt proteins. Non-limiting examples of Wnt binding agents can be found in WO 2011/088127.

  In certain embodiments, the Wnt binding agent binds to the C-terminal cysteine rich domain of one or more human Wnt proteins. In certain embodiments, the Wnt binding agent binds to a domain in one or more Wnt proteins selected from the group consisting of: SEQ ID NO: 46 (Wnt1), SEQ ID NO: 47 ( Wnt2), SEQ ID NO: 48 (Wnt2b), SEQ ID NO: 49 (Wnt3), SEQ ID NO: 50 (Wnt3a), SEQ ID NO: 51 (Wnt7a), SEQ ID NO: 52 (Wnt7b), SEQ ID NO: 53 (Wnt8a), SEQ ID NO: 54 (Wnt8b), SEQ ID NO: 55 (Wnt10a), and SEQ ID NO: 56 (Wnt10b).

In certain embodiments, the Wnt binding agent comprises about 1 μM or less, about 100 nM or less, about 40 nM or less, about 1 or more (eg, 2 or more, 3 or more, or 4 or more) Wnt proteins. 20nM or less, or binds about 10nM or less of K _D. For example, in certain embodiments, Wnt binding agents described herein that binds to two or more Wnt proteins, their Wnt protein, about 100nM or less, binds about 20nM or less, or about 10nM or less a K _D To do. In certain embodiments, Wnt binding agent is, in each of the Wnt protein of one or more (e.g. 1, 2, 3, 4 or five), binds at about 40nM or less of K _D, where The Wnt protein is selected from the group consisting of Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt10a, and Wnt10b. In some embodiments, K _D of the binding agent on Wnt proteins, such as antibodies, is determined using the Wnt fusion proteins comprising at least a portion of the immobilized Wnt C-terminal cysteine-rich domain on Biacore chips K _D.

In certain embodiments, the Wnt binding agent is present in one or more (eg, 2 or more, 3 or more, or 4 or more) human Wnt proteins of about 1 μM or less, about 100 nM or less, about 40 nM or less, It binds with an EC ₅₀ of about 20 nM or less, about 10 nM or less, or about 1 nM or less. In certain embodiments, a Wnt binding agent binds to two or more Wnts with an EC ₅₀ of about 40 nM or less, about 20 nM or less, or about 10 nM or less. In certain embodiments, the Wnt binding agent is a Wnt protein Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, Wnt10a, Wnt10b, Wnt10b, Wnt10b, For one or more of Wnt16 (eg, 1, 2, 3, 4, or 5), it has an EC _{50 of} about 20 nM or less. In certain embodiments, the Wnt binding agent has an EC _{50 of} about 10 nM or less for one or more of the following Wnt proteins (eg, 1, 2, 3, 4, or 5): Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt8a, Wnt8b, Wnt10a, and / or Wnt10b.

  In certain embodiments, the Wnt pathway inhibitor is a Wnt binding agent that is an antibody. In some embodiments, the antibody is a recombinant antibody. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a chimeric antibody. In some embodiments, the antibody is a humanized antibody. In some embodiments, the antibody is a human antibody. In certain embodiments, the antibody is an IgG1 antibody. In certain embodiments, the antibody is an IgG2 antibody. In certain embodiments, the antibody is an antibody fragment that includes an antigen binding site. In some embodiments, the antibody is monovalent, monospecific, or bivalent. In some embodiments, the antibody is a bispecific antibody or a multispecific antibody. In some embodiments, the antibody is conjugated to a cytotoxic moiety. In some embodiments, the antibody is isolated. In some embodiments, the antibody is substantially pure.

  The Wnt binding agents (eg, antibodies) of the present invention can be assayed for specific binding by any method known in the art, as described herein for FZD binding agents.

  In certain embodiments, the Wnt binding agent is a soluble receptor. In certain embodiments, the Wnt binding agent comprises the extracellular domain of an FZD receptor protein. In some embodiments, the Wnt binding agent comprises the Fri domain of the FZD protein. In some embodiments, a soluble receptor comprising an FZD Fri domain can exhibit altered biological activity (eg, increased protein half-life) compared to a soluble receptor comprising an entire FZD ECD. Protein half-life can be further modified (ie, increased) by covalent modification with polyethylene glycol (PEG) or polyethylene oxide (PEO). In certain embodiments, the FZD protein is a human FZD protein. In certain embodiments, the human FZD protein is FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, or FZD10. Non-limiting examples of soluble FZD receptors can be found in US Pat. Nos. 7,723,477 and 7,947,277 and US Patent Application Publication No. 2013/0034551.

  The predicted Fri domains of each of the human FZD1-10 proteins are listed as SEQ ID NOs: 13-22. The predicted minimal Fri domains of each of the human FZD1-10 proteins are listed as SEQ ID NOs: 23-32. There may be differences in understanding among those skilled in the art regarding the exact amino acids corresponding to the various Fri domains. Thus, the N-terminus and / or C-terminus of the domains outlined above and herein may be extended by 1, 2, 3, 4, 5, 6, 7, 8, 9, or even 10 amino acids. It is done.

  In certain embodiments, the Wnt binding agent comprises a Fri domain of a human FZD protein, or a fragment or variant of a Fri domain that binds to one or more human Wnt proteins. In certain embodiments, the human FZD protein is FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, or FZD10. In certain embodiments, the human FZD protein is FZD4. In certain embodiments, the human FZD protein is FZD5. In certain embodiments, the human FZD protein is FZD8. In certain embodiments, the human FZD protein is FZD10. In certain embodiments, the FZD protein is FZD4 and the Wnt binding agent comprises SEQ ID NO: 16. In certain embodiments, the FZD protein is FZD5 and the Wnt binding agent comprises SEQ ID NO: 17. In certain embodiments, the FZD protein is FZD7 and the Wnt binding agent comprises SEQ ID NO: 19. In certain embodiments, the FZD protein is FZD8 and the Wnt binding agent comprises SEQ ID NO: 20. In certain embodiments, the FZD protein is FZD10 and the Wnt binding agent comprises SEQ ID NO: 22. In certain embodiments, the FZD protein is FZD8 and the Wnt binding agent comprises SEQ ID NO: 33.

  In some embodiments, Wnt binding agent Minimum Fri domain of FZD1 (SEQ ID NO: twenty three), FZD2 minimal Fri domain (SEQ ID NO: twenty four), FZD3 minimal Fri domain (SEQ ID NO: twenty five), FZD4 minimal Fri domain (SEQ ID NO: 26), FZD5 minimal Fri domain (SEQ ID NO: 27), FZD6 minimal Fri domain (SEQ ID NO: 28), FZD7 minimal Fri domain (SEQ ID NO: 29), FZD8 minimal Fri domain (SEQ ID NO: 30), FZD9 minimal Fri domain (SEQ ID NO: 31), Or the minimal Fri domain of FZD10 (SEQ ID NO: 32) including Fri domain. In some embodiments, Wnt binding agent FZD8 (SEQ ID NO: 30) including the Fri domain including the minimal Fri domain.

  In some embodiments, the Wnt binding agent is FZD1 Fri domain, FZD2 Fri domain, FZD3 Fri domain, FZD4 Fri domain, FZD5 Fri domain, FZD6 Fri domain, FZD7 Fri domain, FZD8 Fri domain A Fri domain consisting essentially of the domain, the Fri domain of FZD9, or the Fri domain of FZD10. In some embodiments, the Wnt binding agent comprises a Fri domain consisting essentially of the Fri domain of FZD8.

  In some embodiments, the Wnt binding agent comprises a sequence selected from the group consisting of the following sequences: SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, and SEQ ID NO : 33. In some embodiments, the Wnt binding agent comprises a Fri domain consisting essentially of SEQ ID NO: 20. In some embodiments, the Wnt binding agent comprises a Fri domain consisting essentially of SEQ ID NO: 33.

  In certain embodiments, storage of one or more Wnt binding agents (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) It includes a variant of any one of the aforementioned FZD Fri domain sequences that includes a general substitution and has the ability to bind to the Wnt protein.

  In certain embodiments, a Wnt binding agent, eg, an agent comprising a Fri domain of a human FZD receptor, further comprises a non-FZD polypeptide. In some embodiments, the FZD soluble receptor is a non-FZD functional and structural polypeptide such as, but not limited to, a human Fc region, a protein tag (eg, myc, FLAG, GST), other endogenous proteins Alternatively, it may comprise a FZD EGD or Fri domain linked to a protein fragment, or any other useful protein sequence including any linker region between the FZD ECD or Fri domain and the second polypeptide. In certain embodiments, the non-FZD polypeptide comprises a human Fc region. The Fc region can be obtained from any of the immunoglobulin classes, IgG, IgA, IgM, IgD and IgE. In some embodiments, the Fc region is a human IgG1 Fc region. In some embodiments, the Fc region is a human IgG2 Fc region. In some embodiments, the Fc region is a wild type Fc region. In some embodiments, the Fc region is a mutant Fc region. In some embodiments, the N-terminal portion of the Fc region is truncated by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids (eg, in the hinge domain). In some embodiments, amino acids in the hinge domain are altered to prevent unwanted disulfide bond formation. In some embodiments, cysteine is replaced with serine to prevent or prevent undesired disulfide bond formation. In some embodiments, the C-terminal portion of the Fc region is truncated by 1, 2, 3 amino acids or more. In some embodiments, the C-terminal portion of the Fc region is truncated by one amino acid. In certain embodiments, the non-FZD polypeptide comprises SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, or SEQ ID NO: 38. In certain embodiments, the non-FZD polypeptide consists essentially of SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, or SEQ ID NO: 38. In certain embodiments, the non-FZD polypeptide consists essentially of SEQ ID NO: 36 or SEQ ID NO: 37.

  In certain embodiments, the Wnt binding agent is a fusion protein comprising at least the minimal Fri domain and Fc region of the FZD receptor. As used herein, a “fusion protein” is a hybrid protein expressed by a nucleic acid molecule comprising nucleotide sequences of at least two genes. In some embodiments, the C-terminus of the first polypeptide is linked to the N-terminus of the immunoglobulin Fc region. In some embodiments, the first polypeptide (eg, FZD Fri domain) is linked directly (ie, without an intervening linker) to the Fc region. In some embodiments, the first polypeptide is linked to the Fc region via a linker.

を挙げることができるが、それらに限定されるわけではない。本明細書にいうリンカーは、第1ポリペプチド（例えばFZD Friドメイン）のC末端からのアミノ酸残基も第2ポリペプチド（例えばFc領域）のN末端からのアミノ酸残基も含まない介在ペプチド配列である。 As used herein, the term “linker” refers to a linker that is inserted between a first polypeptide (eg, an FZD component) and a second polypeptide (eg, an Fc region). In some embodiments, the linker is a peptide linker. The linker must not adversely affect the expression, secretion or biological activity of the polypeptide. The linker must not be antigenic and must not elicit an immune response. Suitable linkers are known to those skilled in the art and often include a combination of glycine and serine residues and often include amino acids with low steric hindrance. Other amino acids that can be incorporated into useful linkers include threonine and alanine residues. The linker length can range, for example, from 1 to 50 amino acids, from 1 to 22 amino acids, from 1 to 10 amino acids, from 1 to 5 amino acids, or from 1 to 3 amino acids. As the linker, SerGly, GGSG, GSGS, GGGS, S (GGS) _n (where n is 1 to 7), GRA, poly (Gly), poly (Ala),

But are not limited to these. As used herein, a linker is an intervening peptide sequence that does not contain amino acid residues from the C-terminus of the first polypeptide (eg, FZD Fri domain) nor from the N-terminus of the second polypeptide (eg, Fc region). It is.

を含む。 In some embodiments, the Wnt binding agent comprises an FZD Fri domain, an Fc region, and a linker that connects the FZD Fri domain to the Fc region. In some embodiments, the FZD Fri domain comprises SEQ ID NO: 20, SEQ ID NO: 30, or SEQ ID NO: 33. In some embodiments, the linker is

including.

  In some embodiments, the Wnt binding agent is SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, or SEQ ID NO: 33 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, or a second polypeptide comprising SEQ ID NO: 38, wherein the first polypeptide is directly linked to the second polypeptide Yes. In some embodiments, the Wnt binding agent comprises a first polypeptide comprising SEQ ID NO: 20 and SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, or And a second polypeptide comprising SEQ ID NO: 38. In some embodiments, the Wnt binding agent comprises a first polypeptide comprising SEQ ID NO: 20 and a second polypeptide comprising SEQ ID NO: 36 or SEQ ID NO: 37. In some embodiments, the Wnt binding agent comprises a first polypeptide consisting essentially of SEQ ID NO: 20 and a second polypeptide consisting essentially of SEQ ID NO: 36 or SEQ ID NO: 37. Including. In some embodiments, the Wnt binding agent comprises a first polypeptide comprising SEQ ID NO: 30 and SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, or And a second polypeptide comprising SEQ ID NO: 38. In some embodiments, the Wnt binding agent comprises a first polypeptide comprising SEQ ID NO: 30 and a second polypeptide comprising SEQ ID NO: 36 or SEQ ID NO: 37. In some embodiments, the Wnt binding agent comprises a first polypeptide comprising SEQ ID NO: 33 and SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, or And a second polypeptide comprising SEQ ID NO: 38. In some embodiments, the Wnt binding agent comprises a first polypeptide comprising SEQ ID NO: 33 and a second polypeptide comprising SEQ ID NO: 36, SEQ ID NO: 37, or SEQ ID NO: 35. including. In some embodiments, the Wnt binding agent comprises a first polypeptide consisting essentially of SEQ ID NO: 33 and essentially consisting of SEQ ID NO: 36, SEQ ID NO: 37, or SEQ ID NO: 35. And a second polypeptide.

  In some embodiments, the Wnt binding agent is SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, or SEQ ID NO: 33 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, or a second polypeptide comprising SEQ ID NO: 38, wherein the first polypeptide is connected to the second polypeptide by a linker. ing. In some embodiments, the Wnt binding agent comprises a first polypeptide comprising SEQ ID NO: 20 and SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, or And a second polypeptide comprising SEQ ID NO: 38. In some embodiments, the Wnt binding agent comprises a first polypeptide comprising SEQ ID NO: 20 and a second polypeptide comprising SEQ ID NO: 36 or SEQ ID NO: 37. In some embodiments, the Wnt binding agent comprises a first polypeptide consisting essentially of SEQ ID NO: 20 and a second polypeptide consisting essentially of SEQ ID NO: 36 or SEQ ID NO: 37. Including. In some embodiments, the Wnt binding agent comprises a first polypeptide comprising SEQ ID NO: 30 and SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, or And a second polypeptide comprising SEQ ID NO: 38. In some embodiments, the Wnt binding agent comprises a first polypeptide comprising SEQ ID NO: 33 and SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, or And a second polypeptide comprising SEQ ID NO: 38. In some embodiments, the Wnt binding agent comprises a first polypeptide comprising SEQ ID NO: 33 and a second polypeptide comprising SEQ ID NO: 36, SEQ ID NO: 37, or SEQ ID NO: 35. including. In some embodiments, the Wnt binding agent comprises a first polypeptide consisting essentially of SEQ ID NO: 33 and essentially consisting of SEQ ID NO: 36, SEQ ID NO: 37, or SEQ ID NO: 35. And a second polypeptide.

  In some embodiments, the Wnt binding agent is SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, or first poly that is at least 95% identical to SEQ ID NO: 33 And a second polypeptide comprising SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, or SEQ ID NO: 38, wherein the first polypeptide is the second It is directly linked to the polypeptide. In some embodiments, the Wnt binding agent is a first polypeptide that is at least 95% identical to SEQ ID NO: 20, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, or a second polypeptide comprising SEQ ID NO: 38. In some embodiments, the Wnt binding agent is a first polypeptide that is at least 95% identical to SEQ ID NO: 30, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, or a second polypeptide comprising SEQ ID NO: 38. In some embodiments, the Wnt binding agent is a first polypeptide that is at least 95% identical to SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, or a second polypeptide comprising SEQ ID NO: 38.

  In some embodiments, the Wnt binding agent is SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, or first poly that is at least 95% identical to SEQ ID NO: 33 A peptide and a second polypeptide comprising SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, or SEQ ID NO: 38, wherein the first polypeptide is a linker To the second polypeptide. In some embodiments, the Wnt binding agent is a first polypeptide that is at least 95% identical to SEQ ID NO: 20, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, or a second polypeptide comprising SEQ ID NO: 38. In some embodiments, the Wnt binding agent is a first polypeptide that is at least 95% identical to SEQ ID NO: 30, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, or a second polypeptide comprising SEQ ID NO: 38. In some embodiments, the Wnt binding agent is a first polypeptide that is at least 95% identical to SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, or a second polypeptide comprising SEQ ID NO: 38.

  The FZD protein contains a signal sequence that directs the transport of the protein. A signal sequence (also called a signal peptide or leader sequence) is located at the N-terminus of the nascent polypeptide. They direct the polypeptide to the endoplasmic reticulum, and the proteins are separated to their destination, for example, the internal space of the organelle, the inner membrane, the outer membrane, or the outside of the cell by secretion. Most signal sequences are excised from the protein by signal peptidase after the protein is transported to the endoplasmic reticulum. The excision of a signal sequence from a polypeptide usually occurs at a specific site in the amino acid sequence and depends on amino acid residues in the signal sequence. Although the specific cleavage site is usually one, more than one cleavage site may be recognized and / or used by the signal peptidase, resulting in a heterogeneous N-terminus of the polypeptide. For example, the use of different cleavage sites within the signal sequence can result in different N-terminal amino acids of the expressed polypeptide. Thus, in some embodiments, a polypeptide described herein can comprise a mixture of polypeptides having different N-termini. In some embodiments, the N-terminus is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acids or more different in length. In some embodiments, the N-terminus is 1, 2, 3, 4, or 5 amino acids different in length. In some embodiments, the polypeptides are substantially uniform, i.e., the polypeptides have the same N-terminus. In some embodiments, the signal sequence of the polypeptide is one or more (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) amino acid substitutions and / or amino acid deletions including. In some embodiments, the amino acid substitutions and / or amino acids allow the signal sequence of the polypeptide to result in a substantially uniform polypeptide with one type of N-terminus by predominating one cleavage site. Includes deletions.

  In some embodiments, the Wnt binding agent is SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, and SEQ ID NO: 44. An amino acid sequence selected from the group consisting of ID NO: 45 is included.

  In certain embodiments, the Wnt binding agent comprises the sequence of SEQ ID NO: 39. In certain embodiments, the sequence is SEQ ID NO: 39, wherein the agent comprises one or more (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 etc.) conservative substitutions including. In certain embodiments, the agent comprises a sequence having at least about 90%, about 95%, or about 98% sequence identity with SEQ ID NO: 39. In certain embodiments, the variant of SEQ ID NO: 39 maintains the ability to bind to one or more human Wnt proteins.

  In certain embodiments, the Wnt binding agent comprises the sequence of SEQ ID NO: 40. In some embodiments, the Wnt binding agent is SEQ ID NO: 40. In certain alternative embodiments, the agent is of SEQ ID NO: 40 comprising one or more (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) conservative substitutions Contains an array. In certain embodiments, the agent comprises a sequence having at least about 90%, about 95% or about 98% sequence identity with SEQ ID NO: 40. In certain embodiments, the variant of SEQ ID NO: 40 maintains the ability to bind to one or more human Wnt proteins.

  In certain embodiments, the Wnt binding agent comprises the sequence of SEQ ID NO: 41. In some embodiments, the Wnt binding agent is SEQ ID NO: 41. In certain alternative embodiments, the agent is of SEQ ID NO: 41 comprising one or more (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) conservative substitutions Contains an array. In certain embodiments, the agent comprises a sequence having at least about 90%, about 95%, or about 98% sequence identity with SEQ ID NO: 41. In certain embodiments, the variant of SEQ ID NO: 41 retains the ability to bind to one or more human Wnt proteins.

  In some embodiments, the Wnt binding agent is OMP-54F28.

  In certain embodiments, the Wnt binding agent is SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, and SEQ ID A polypeptide comprising an amino acid sequence selected from the group consisting of NO: 45. In certain embodiments, the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 39, SEQ ID NO: 40, and SEQ ID NO: 41. In some embodiments, the polypeptide consists essentially of an amino acid sequence selected from the group consisting of SEQ ID NO: 39, SEQ ID NO: 40, and SEQ ID NO: 41. In certain embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO: 39. In some embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO: 40. In certain embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO: 41. In certain embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO: 42. In certain embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO: 43. In certain embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO: 44. In certain embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO: 45.

  In some embodiments, the polypeptide is a substantially purified polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 39, SEQ ID NO: 40, and SEQ ID NO: 41. In some embodiments, the polypeptide is a substantially purified polypeptide comprising SEQ ID NO: 41. In certain embodiments, the substantially purified polypeptide consists of at least 90% of the polypeptide having the N-terminal sequence ASA. In some embodiments, the nascent polypeptide includes a signal sequence that results in a substantially uniform polypeptide product with one N-terminal sequence.

  In certain embodiments, the Wnt binding agent comprises an immunoglobulin Fc region. Some of the binding agents of the present invention have desirable biochemical characteristics, such as increased cancer, when compared to approximately the same immunogenic fusion protein comprising a native, i.e. unmodified, constant region. At least a portion of the Fc region is deleted or otherwise modified to provide cellular localization, increased tumor penetration, reduced serum half-life, or increased serum half-life It will be understood by those skilled in the art to include such fusion proteins. Modification of the Fc region can include addition, deletion, or substitution of one or more amino acids in one or more domains. The modified fusion proteins disclosed herein can include one or more alterations or changes in the two heavy chain constant domains (CH2 or CH3), or alterations or alterations in the hinge region. In another embodiment, the entire CH2 domain can be removed (ΔCH2 construct). In some embodiments, omitted constant region domains are replaced with short amino acid spacers (eg, 10 aa residues) that provide some of the mobility of the molecule that is typically conferred by the missing constant region domain.

  In some embodiments, the modified fusion protein is engineered to link the CH3 domain directly to the hinge region. In another embodiment, a peptide spacer is inserted between the hinge region and the modified CH2 and / or CH3 domain. For example, a construct can be expressed in which the CH2 domain is deleted and the remaining CH3 domain (modified or unmodified) is joined to the hinge region with a 5-20 amino acid spacer. Such spacers can be added to ensure that the regulatory elements of the constant domain remain free and accessible or that the hinge region remains mobile. However, it should be noted that amino acid spacers may in some cases be immunogenic and elicit unwanted immune responses against the construct. Thus, in certain embodiments, any spacer added to the construct will be relatively non-immunogenic so that the desired biological quality of the fusion protein is maintained.

  In some embodiments, the modified fusion protein may have only a partial deletion of the constant domain, or may have a few or only one amino acid substitution. For example, single amino acid mutations in selected areas of the CH2 domain may be sufficient to substantially reduce Fc binding and thus increase cancer cell localization and / or tumor penetration. Similarly, it may be desirable to simply delete portions of one or more constant region domains that control specific effector functions (eg, complement C1q binding). Such partial deletions of the constant region may improve selected characteristics (eg, serum half-life) of the binding agent while leaving other desirable functions associated with the subject constant region domain intact. . Moreover, as noted above, the constant regions of the fusion proteins of the present disclosure may be modified by one or more amino acid mutations or substitutions that enhance the profile of the resulting construct. In this regard, it may be possible to interfere with the activity (eg, Fc binding) provided by a conserved binding site while substantially maintaining the configuration and immunogenic profile of the modified fusion protein. In certain embodiments, the modified fusion protein has one or more of the constant regions to enhance desirable characteristics such as reduced or increased effector function or to provide more cytotoxin or carbohydrate attachment sites. Addition of the amino acid.

  It is known in the art that the constant region mediates several types of effector functions. For example, binding of the C1 component of complement to the Fc region of an IgG antibody or IgM antibody (conjugated to an antigen) activates the complement system. Complement activation is important in the cytopathogen opsonization and lysis. Complement activation also stimulates inflammatory responses and may be involved in autoimmune hypersensitivity. In addition, the Fc region of an immunoglobulin can bind to cells that express the Fc receptor (FcR). There are several Fc receptors specific for different antibody classes, such as IgG (gamma receptor), IgE (epsilon receptor), IgA (alpha receptor) and IgM (mu receptor). Antibody binding to Fc receptors on the cell surface involves phagocytosis and destruction of antibody-coated particles, clearance of immune complexes, lysis of antibody-coated target cells by killer cells, release of inflammatory mediators, placental translocation, and immunoglobulins It triggers several important and diverse biological responses, such as production control.

  In some embodiments, the modified fusion protein provides altered effector function, which in turn affects the biological profile of the administered agent. For example, in some embodiments, deletion of constant region domains or inactivation (by point mutation or other means) reduces Fc receptor binding of circulating modulators, thereby causing cancer cell localization and / or Or it can increase tumor penetration. In another embodiment, the constant region modification increases or decreases the serum half-life of the agent. In some embodiments, the constant region is modified such that a disulfide linkage or oligosaccharide moiety is eliminated.

  In certain embodiments, the modified fusion protein does not have one or more effector functions normally associated with the Fc region. In some embodiments, the agent does not have antibody dependent cellular cytotoxicity (ADCC) activity and / or complement dependent cytotoxicity (CDC) activity. In certain embodiments, the agent does not bind to Fc receptors and / or complement factors. In certain embodiments, the agent does not have an effector function.

  In some embodiments, a Wnt binding agent (eg, a soluble receptor) described herein has been modified to reduce immunogenicity. In general, immune responses against fully normal human proteins are rare when using these proteins as therapeutic agents. However, although many fusion proteins contain the same polypeptide sequence as found in nature, some therapeutic fusion proteins have been shown to be immunogenic in mammals. In some studies, fusion proteins containing a linker have been found to be more immunogenic than fusion proteins that do not contain a linker. Thus, in some embodiments, the polypeptides of the invention are analyzed by computational techniques to predict immunogenicity. In some embodiments, the polypeptide is analyzed for the presence of T cell epitopes and / or B cell epitopes. Once a T cell epitope or B cell epitope has been identified and / or predicted, modifications (eg, amino acid substitutions) can be made to those regions to interfere with or destroy that epitope. Various algorithms and software that can be used to predict T cell epitopes and / or B cell epitopes are known in the art. For example, the software programs SYFPEITHI, HLA Bind, PEPVAC, RANKPEP, DiscoTope, ElliPro, and Antibody Epitope Prediction can all be used publicly.

  In some embodiments, cells that produce any of the Wnt binding agents (eg, soluble receptors) or Wnt binding polypeptides described herein are provided. In some embodiments, compositions comprising any of the Wnt binding agents (eg, soluble receptors) or Wnt binding polypeptides described herein are provided. In some embodiments, the composition comprises at least 80%, 90%, 95%, 97%, 98%, or 99% polypeptide having the N-terminal sequence ASA. In some embodiments, the composition comprises a polypeptide in which 100% of the polypeptide has an N-terminal sequence of ASA. In some embodiments, the composition comprises a polypeptide in which at least 80% of the polypeptide has an N-terminal sequence of ASA. In some embodiments, the composition comprises a polypeptide in which at least 90% of the polypeptide has an N-terminal sequence of ASA. In some embodiments, the composition comprises a polypeptide in which at least 95% of the polypeptide has an N-terminal sequence of ASA.

  The polypeptides described herein can be recombinant polypeptides, natural polypeptides, or synthetic polypeptides. It will be recognized in the art that some amino acid sequences of the present invention can be altered without significantly affecting the structure or function of the protein. It should be noted that if such sequence differences are possible, there will be important areas on the protein that determine activity. Thus, the present invention further encompasses variations of the polypeptide that exhibit substantial activity of the FZD protein or that comprise a region of the FZD protein, eg, the protein portion discussed herein. Such mutants include deletions, insertions, inversions, repeats, and type substitutions.

  Of course, the number of amino acid substitutions that one skilled in the art will make depends on many factors, including those described above. In certain embodiments, for any given soluble receptor polypeptide, the number of substitutions will be no more than 50, 40, 30, 25, 20, 15, 10, 5 or 3.

  A fragment or portion of a polypeptide of the invention can be used to produce the corresponding full-length polypeptide by peptide synthesis. Therefore, the fragment can be used as an intermediate to produce a full-length polypeptide. These fragments or portions of the polypeptide may also be referred to as “protein fragments” or “polypeptide fragments”.

A “protein fragment” of the present invention is a portion or all of a protein that has the ability to bind to one or more human Wnt proteins or one or more human FZD proteins. In some embodiments, the fragment has a high affinity for one or more human Wnt proteins. In some embodiments, the fragment has a high affinity for one or more human FZD proteins. Some fragments of Wnt binding agents described herein are protein fragments that include at least a portion of the extracellular portion of the FZD protein linked to at least a portion of an immunoglobulin constant region (eg, an Fc region). is there. The binding affinity of the protein fragment can be in the range of about 10 ⁻¹¹ to 10 ⁻¹² M. However, as the fragment size changes, the affinity can vary considerably and can range from 10 ⁻⁷ to 10 ⁻¹³ M. In some embodiments, the fragment is from about 100 to about 200 amino acids in length and comprises a binding domain linked to at least a portion of an immunoglobulin constant region.

  In some embodiments, the Wnt pathway inhibitor is a polyclonal antibody. Polyclonal antibodies can be prepared by any known method. In some embodiments, an animal (eg, rabbit, rat, mouse, goat) is injected multiple times subcutaneously or intraperitoneally with an antigen of interest (eg, a purified peptide fragment, full-length recombinant protein, or fusion protein). , Immunize the donkey) to generate polyclonal antibodies. The antigen can optionally be conjugated to a carrier such as keyhole limpet hemocyanin (KLH) or serum albumin. Antigens (with or without carrier protein) are diluted in sterile saline and usually mixed with an adjuvant (eg, complete or incomplete Freund's adjuvant) to form a stable emulsion. After a sufficient period of time, polyclonal antibodies are recovered from the blood and / or ascites of the immunized animal. Polyclonal antibodies can be purified from serum or ascites by standard methods in the art, such as, but not limited to, affinity chromatography, ion exchange chromatography, gel electrophoresis, and dialysis.

  In some embodiments, the Wnt pathway inhibitor is a monoclonal antibody. Monoclonal antibodies can be prepared using hybridoma methods known to those skilled in the art (see, for example, Kohler and Milstein, 1975, Nature, 256: 495-497). In some embodiments using the hybridoma method, immunization of mice, hamsters, or other suitable host animals as described above causes lymphocytes to produce antibodies that will specifically bind to the immunizing antigen. Cause it to occur. In some embodiments, lymphocytes can be immunized in vitro. In some embodiments, the immunizing antigen can be a human protein or a portion thereof. In some embodiments, the immunizing antigen can be a mouse protein or a portion thereof.

  After immunization, lymphocytes are isolated and fused with an appropriate myeloma cell line using, for example, polyethylene glycol to form hybridoma cells, which are then selected from unfused lymphocytes and myeloma cells. Can do. Hybridomas that produce monoclonal antibodies specifically directed to a selected antigen include, but are not limited to, immunoprecipitation, immunoblotting, and in vitro binding assays (eg, flow cytometry, FACS, ELISA, and radioimmunoassay). It can be identified by a variety of methods including: Hybridomas can be grown using standard methods in in vitro culture (JW Goding, 1996 “Monoclonal Antibodies: Principles and Practice” 3rd Edition, Academic Press, San Diego, Calif.) Or in vivo as ascites tumors in animals. it can. Monoclonal antibodies can be purified from culture medium or ascites according to standard methods in the art including, but not limited to, affinity chromatography, ion exchange chromatography, gel electrophoresis, and dialysis.

  In certain embodiments, monoclonal antibodies can be made using recombinant DNA techniques known to those of skill in the art. Polynucleotides encoding monoclonal antibodies are isolated from mature B cells or hybridoma cells, such as by RT-PCR using oligonucleotide primers that specifically amplify the genes encoding the antibody heavy and light chains, and their The sequence is determined using conventional techniques. The isolated polynucleotides encoding the heavy and light chains are then purified from E. coli, monkey COS cells, Chinese hamster ovary (CHO) cells, or bone marrow, which would otherwise not produce immunoglobulin proteins. Cloning into an appropriate expression vector that produces monoclonal antibodies when transfected into a host cell such as a tumor cell. In another aspect, the recombinant monoclonal antibody, or fragment thereof, can be isolated from a phage display library.

  Polynucleotides encoding monoclonal antibodies can be further modified in a number of different ways using recombinant DNA techniques to generate surrogate antibodies. In some embodiments, generating a chimeric antibody or replacing a non-immunoglobulin polypeptide, eg, by replacing the same region of a human antibody with, for example, the light and heavy chain constant domains of a murine monoclonal antibody. Thus, a fusion antibody can be generated. In some embodiments, the constant region is truncated or removed to produce the desired antibody fragment of the monoclonal antibody. In order to optimize the specificity, affinity, etc. of the monoclonal antibody, site-directed mutagenesis or high-density mutagenesis of the variable region can be used.

  In some embodiments, the Wnt pathway inhibitor is a humanized antibody. Typically, a humanized antibody is a non-human species (eg, mouse, rat) in which the amino acid residues of the CDRs have the desired specificity, affinity, and / or binding ability using methods known to those skilled in the art. , Rabbits, hamsters, etc.) are human immunoglobulins that have been replaced with the amino acid residues of the CDRs of immunoglobulins. In some embodiments, Fv framework region amino acid residues of a human immunoglobulin are replaced with corresponding amino acid residues from an antibody of a non-human species that has the desired specificity, affinity, and / or binding ability. In some embodiments, additional amino acid residues in the Fv framework region and / or within the replaced non-human amino acid residues to refine and optimize the specificity, affinity, and / or ability of the antibody. The humanized antibody can be further modified by substitution. In general, a humanized antibody will comprise at least one, typically substantially all of the two variable domain regions, containing all or substantially all of the CDRs corresponding to the non-human immunoglobulin, On the other hand, all or substantially all of the framework regions are of human immunoglobulin sequences. In some embodiments, a humanized antibody can also comprise at least a portion of an immunoglobulin constant region or constant domain (Fc), typically that of a human immunoglobulin. In certain embodiments, such humanized antibodies are used for therapy. This is because they can reduce antigenicity and HAMA (human anti-mouse antibody) response when administered to human subjects. The methods used to generate humanized antibodies are well known in the art.

  In certain embodiments, the Wnt pathway inhibitor is a human antibody. Human antibodies can be prepared directly using various techniques known in the art. In some embodiments, immortalized human B lymphocytes producing antibodies against the target antigen, immunized in vitro, or isolated from an immunized individual can be generated. In some embodiments, human antibodies can be selected from phage libraries that express human antibodies. Alternatively, human antibodies and antibody fragments can be produced in vitro using phage display technology from an immunoglobulin variable domain gene repertoire of non-immunized donors. Techniques for generating and using antibody phage libraries are well known in the art. Affinity maturation strategies such as, but not limited to, chain shuffling (Marks et al., 1992, Bio / Technology, 10: 779-783) and site-directed mutagenesis are known in the art and have high affinity. It can be used for the production of sex human antibodies.

  In some embodiments, human antibodies can be made in transgenic mice with human immunoglobulin loci. When immunized, these mice have the ability to produce a full repertoire of human antibodies in the absence of endogenous immunoglobulin production. This approach is described in US Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016.

  The invention also encompasses bispecific antibodies that specifically recognize at least one human FZD protein or at least one Wnt protein. Bispecific antibodies have the ability to specifically recognize and bind to at least two different epitopes. Different epitopes may be in the same molecule (eg two different epitopes on human FZD5) or on different molecules (eg one epitope on FZD5 and different epitopes on a second protein) . In some embodiments, the bispecific antibody is a monoclonal human antibody or a monoclonal humanized antibody. In some embodiments, the antibody has a first antigen target (eg, FZD protein) and a second antigen target, eg, an effector molecule on leukocytes, to focus a cellular defense mechanism on cells that express the first antigen target. It can specifically recognize and bind to (eg, CD2, CD3, CD28, CD80, or CD86) or Fc receptors (eg, CD64, CD32, or CD16). In some embodiments, antibodies can be used to direct cytotoxic agents to cells that express a particular target antigen. These antibodies have an antigen-binding arm and an arm that binds to a cytotoxic agent or radionuclide chelator such as EOTUBE, DPTA, DOTA, or TETA.

  Bispecific antibodies can be intact antibodies or antibody fragments. Antibodies with three or more valencies are also contemplated. For example, trispecific antibodies can be prepared (Tutt et al., 1991, J. Immunol., 147: 60). Thus, in certain embodiments, the antibody is multispecific. Techniques for making bispecific and multispecific antibodies are known to those skilled in the art.

  In certain embodiments, the antibodies (or other polypeptides) described herein can be monospecific. For example, in certain embodiments, each of the one or more antigen binding sites that an antibody contains has the ability to bind (or bind) a homologous epitope on a different protein. In certain embodiments, the antigen binding sites of the monospecific antibodies described herein have the ability to bind (or bind) to, for example, FZD5 and FZD7 (ie, the same epitope is present in FZD5 and FZD7 proteins). Is also found).

  In certain embodiments, the Wnt pathway inhibitor is an antibody fragment that includes an antigen binding site. Antibody fragments can have different functions or abilities than intact antibodies. For example, antibody fragments can have increased tumor penetration. Various techniques for producing antibody fragments are known, including but not limited to proteolytic digestion of intact antibodies. In some embodiments, antibody fragments include F (ab ′) 2 fragments produced by pepsin digestion of antibody molecules. In some embodiments, antibody fragments include Fab fragments produced by reducing disulfide bridges of F (ab ′) 2 fragments. In another embodiment, antibody fragments include Fab fragments generated by treating antibody molecules with papain and a reducing agent. In certain embodiments, antibody fragments are produced recombinantly. In some embodiments, antibody fragments include Fv fragments 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, thereby allowing these fragments to be produced in large quantities. In some embodiments, antibody fragments are isolated from antibody phage libraries as discussed herein. For example, Fab expression libraries can be used to allow rapid and effective identification of monoclonal Fab fragments with the desired specificity for FZD protein or Wnt protein or derivatives, fragments, analogs or homologues thereof. A method for building can be used. In some embodiments, the antibody fragment is a linear antibody fragment. In certain embodiments, the antibody fragment is monospecific or bispecific. In certain embodiments, the Wnt pathway inhibitor is scFv. Various techniques can be used for the production of single chain antibodies specific for one or more human FZD proteins or one or more human Wnt proteins.

  Furthermore, it may be preferred to modify an antibody to increase its serum half-life, particularly in the case of antibody fragments. This can be accomplished, for example, by incorporating a salvage receptor binding epitope into the antibody fragment by mutagenesis into the appropriate region in the antibody fragment, or by incorporating the epitope into a peptide tag and placing it in one or the middle of the antibody fragment. It can be achieved by fusing (eg by DNA synthesis or peptide synthesis). In some embodiments, the antibody is modified so that its serum half-life is reduced.

  Heteroconjugate antibodies are also within the scope of the present invention. Heteroconjugate antibodies are composed of two antibodies joined by a covalent bond. Such antibodies have been proposed, for example, to target immune cells to unwanted cells. It is contemplated that heteroconjugate antibodies can also be prepared in vitro using known methods in synthetic protein chemistry, such as those requiring a cross-linking agent. 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.

  For the purposes of the present invention, it should be understood that the modified antibody can comprise any type of variable region that results in the association of the antibody with the target (ie, human FZD protein or human Wnt protein). In this regard, the variable region can include or be derived from any type of mammal capable of triggering the initiation of a humoral response and the production of immunoglobulins against the desired tumor associated antigen. sell. Thus, the variable region of the modified antibody can be derived from, for example, a human, mouse, non-human primate (eg, cynomolgus monkey, macaque, etc.) or rabbit. In some embodiments, both the variable and constant regions of the modified immunoglobulin are human regions. In another aspect, the engineered or specific engineered variable region of a compatible antibody (typically derived from a non-human source) to improve the binding properties of the molecule or reduce immunogenicity. Can be processed. In this regard, variable regions useful in the present invention can be humanized or otherwise modified by including an imported amino acid sequence.

  In certain embodiments, both the heavy and light chain variable domains are both at least partially replaced by one or more CDRs and, if necessary, replaced by a partial framework region and sequence modifications and / or Or it is modified by sequence modification. Although CDRs can be from the same class as the antibody from which the framework region is derived, and even from the same subclass, it is likely that the CDRs will preferably be derived from antibodies from different species. In order to transfer the antigen binding ability of one variable domain to another, it may not be necessary to replace all of the CDRs with all of the CDRs from the donor variable region. Rather, it will only be necessary to transfer the residues necessary to maintain the activity of the antigen binding site.

  Apart from variable region modifications, the modified antibodies of the present invention may have a desired biochemical characteristic, such as increased, when compared to approximately the same immunogenic antibody comprising a native, i.e. unmodified, constant region. Antibodies in which at least one or more fragments of the constant region domain have been deleted or otherwise modified so as to obtain improved tumor localization and / or increased serum half-life (eg, full length It will be understood by those skilled in the art that the antibody or immunoreactive fragment thereof). In some embodiments, the constant region of the modified antibody will comprise a human constant region. Constant region modifications compatible with the present invention include one or more amino acid additions, deletions or substitutions in one or more domains. The modified antibodies disclosed herein can include alterations or modifications to one or more of the three heavy chain constant domains (CH1, CH2 or CH3) and / or to the light chain constant domain (CL). In some embodiments, one or more domains are partially or completely deleted from the constant region of the modified antibody. In some embodiments, the modified antibody will comprise a domain deletion construct or domain deletion mutant (ΔCH2 construct) in which the entire CH2 domain has been removed. In some embodiments, omitted constant region domains are replaced with short amino acid spacers (eg, 10 amino acid residues) that provide some of the mobility of the molecule typically conferred by the missing constant region domain. .

  In some embodiments, the modified antibody is engineered to fuse the CH3 domain directly to the hinge region of the antibody. In another embodiment, a peptide spacer is inserted between the hinge region and the modified CH2 domain and / or CH3 domain. For example, a construct can be expressed in which the CH2 domain is deleted and the remaining CH3 domain (modified or unmodified) is joined to the hinge region with a 5-20 amino acid spacer. Such spacers can be added to ensure that the regulatory elements of the constant domain remain free and accessible or that the hinge region remains mobile. However, it should be noted that amino acid spacers may in some cases be immunogenic and elicit unwanted immune responses against the construct. Thus, in certain embodiments, any spacer added to the construct will be relatively non-immunogenic so that the desired biological quality of the modified antibody is maintained.

  In some embodiments, the modified antibody may have only a partial deletion of the constant domain, or may have a few or only one amino acid substitution. For example, single amino acid mutations in selected areas of the CH2 domain may be sufficient to substantially reduce Fc binding and thus increase cancer cell localization and / or tumor penetration. Similarly, it may be desirable to simply delete portions of one or more constant region domains that control specific effector functions (eg, complement C1q binding). Such partial deletion of the constant region may improve selected characteristics (serum half-life) of the antibody while leaving other desirable functions associated with the subject constant region domain intact. Moreover, as noted above, the constant regions of the antibodies of the present disclosure may be modified by one or more amino acid mutations or substitutions that enhance the profile of the resulting construct. In this regard, it may be possible to interfere with the activity (eg, Fc binding) provided by a conserved binding site while substantially maintaining the configuration and immunogenic profile of the modified antibody. In certain embodiments, the modified antibody may include one or more of the constant regions to enhance desirable characteristics such as reduced or increased effector function or to provide more cytotoxin or carbohydrate attachment sites. Includes addition of amino acids.

  It is known in the art that the constant region mediates several types of effector functions. For example, binding of the C1 component of complement to the Fc region of an IgG antibody or IgM antibody (conjugated to an antigen) activates the complement system. Complement activation is important in the cytopathogen opsonization and lysis. Complement activation also stimulates inflammatory responses and may be involved in autoimmune hypersensitivity. In addition, the Fc region of an antibody can bind to cells that express the Fc receptor (FcR). There are several Fc receptors specific for different antibody classes, such as IgG (gamma receptor), IgE (epsilon receptor), IgA (alpha receptor) and IgM (mu receptor). Antibody binding to Fc receptors on the cell surface involves phagocytosis and destruction of antibody-coated particles, clearance of immune complexes, lysis of antibody-coated target cells by killer cells, release of inflammatory mediators, placental translocation, and immunoglobulins It triggers several important and diverse biological responses, such as production control.

  In certain embodiments, the Wnt pathway inhibitor is an antibody that confers altered effector function. These altered effector functions can affect the biological profile of the administered antibody. For example, in some embodiments, deletion of constant region domains or inactivation (by point mutation or other means) reduces Fc receptor binding of circulating modified antibodies (eg, anti-FZD antibodies), thereby causing cancer Cell localization and / or tumor penetration may be increased. In another embodiment, the constant region modification increases or decreases the serum half life of the antibody. In some embodiments, the constant region is modified such that a disulfide linkage or oligosaccharide moiety is eliminated. Modification of the constant region according to the present invention can be easily performed using well-known biochemical techniques or molecular engineering techniques well understood by those skilled in the art.

  In certain embodiments, the Wnt pathway inhibitor is an antibody that does not have one or more effector functions. For example, in some embodiments, the antibody does not have ADCC activity and / or CDC activity. In certain embodiments, the antibody does not bind to Fc receptor and / or complement factor. In certain embodiments, the antibody does not have an effector function.

  The invention further encompasses variants and equivalents that are substantially homologous to the chimeric, humanized, and human antibodies, or antibody fragments thereof, described herein. These can contain, for example, conservative substitution mutations, ie substitution of one or more amino acids with similar amino acids. For example, a conservative substitution is the substitution of one amino acid with another amino acid within the same general class, for example, replacing one acidic amino acid with another acidic amino acid, or replacing one basic amino acid with another basic amino acid. Substitution or replacement of one neutral amino acid with another neutral amino acid, and the like. What conservative amino acid substitutions are intended are well known in the art and are also described herein.

  In certain embodiments, the antibodies described herein are isolated. In certain embodiments, the antibodies described herein are substantially pure.

  In some embodiments of the invention, the Wnt pathway inhibitor is a polypeptide. The polypeptide can be a recombinant polypeptide, a natural polypeptide, or a synthetic polypeptide, including an antibody or fragment thereof that binds to 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 present invention can be altered without significantly affecting the structure or function of the protein. Accordingly, the present invention further encompasses variations of the polypeptide that exhibit substantial activity of the antibody or fragment thereof against human FZD protein or Wnt protein or that include regions of the antibody or fragment thereof. In some embodiments, the amino acid sequence variation of the FZD-binding polypeptide or Wnt-binding polypeptide comprises a deletion, insertion, inversion, repeat, and / or other type of substitution.

Polypeptides, analogs and variants thereof can be further modified to contain additional chemical moieties not normally part of the polypeptide. The derivatized moiety can improve the solubility, biological half-life and / or absorption of the polypeptide. These moieties can also reduce or eliminate any undesirable side effects of the polypeptides and variants. A review on the chemical part can be found in “Remington: The Science and Practice of Pharmacy” 22 ^st Edition, 2012, Pharmaceutical Press (London).

  Isolated polypeptides that can be used in the methods described herein can be produced by any suitable method known in the art. Such methods vary from direct protein synthesis methods to methods of constructing a DNA sequence encoding a polypeptide sequence and expressing the sequence in a suitable host. In some embodiments, the DNA sequence is constructed using recombinant techniques by isolating or synthesizing DNA encoding the wild type protein of interest. Optionally, functional analogues can be obtained by introducing mutations into the sequence by site-directed mutagenesis.

  In some embodiments, a DNA sequence encoding a polypeptide of interest can be constructed by chemical synthesis using an oligonucleotide synthesizer. Oligonucleotides are designed by selecting preferred codons in the host cell that will produce the recombinant polypeptide of interest based on the amino acid sequence of the desired polypeptide. By applying standard methods, a polynucleotide sequence encoding the isolated polypeptide of interest can be synthesized. For example, the reverse amino acid sequence can be constructed using the complete amino acid sequence. In addition, DNA oligomers containing a nucleotide sequence encoding a particular isolated polypeptide can be synthesized. For example, several small oligonucleotides that encode portions of the desired polypeptide can be synthesized and then ligated. Individual oligonucleotides typically contain a 5 ′ or 3 ′ overhang for complementary assembly.

  Once assembled (by synthesis, site-directed mutagenesis, or other methods), a polynucleotide sequence encoding a particular polypeptide of interest is inserted into an expression vector suitable for expression of the protein in the desired host. Can be operably linked to different expression control sequences. Proper assembly can be ascertained by nucleotide sequencing, restriction enzyme mapping, and / or expression of the biologically active polypeptide in a suitable host. As is well known in the art, to obtain high expression levels of a gene transfected in the host, the gene is operably linked to transcriptional and translational expression control sequences that are functional in the chosen expression host. There must be.

  In certain embodiments, a recombinant expression vector is used to amplify and express DNA encoding a binding agent (eg, antibody or soluble receptor) or fragment thereof for human FZD protein or Wnt protein. For example, a recombinant expression vector can be an FZD binding agent, Wnt binding agent, anti-antigen, operably linked to appropriate transcriptional and / or translational regulatory elements derived from mammalian genes, microbial genes, viral genes or insect genes. It can be a replicable DNA construct having a synthetic DNA fragment or a DNA fragment derived from cDNA encoding an FZD antibody or fragment thereof, an anti-Wnt antibody or fragment thereof, or a polypeptide chain of FZD-Fc soluble receptor . A transcription unit generally comprises (1) one or more genetic elements that have a regulatory role in gene expression, such as transcription promoters or enhancers, (2) structural or coding sequences that are transcribed into mRNA and translated into protein. And (3) assembly of appropriate transcriptional and translational initiation and termination sequences. Regulatory elements can include operator sequences for controlling transcription. A selection gene that facilitates recognition of transformants and the ability to replicate in the host, usually conferred by the origin of replication, can also be incorporated. DNA regions are “operably linked” when they are in a functional relationship with each other. For example, the signal peptide (secretory leader) DNA is operably linked to the polypeptide DNA if it is expressed as a precursor involved in the secretion of the polypeptide, and the promoter is If it controls the transcription of a coding sequence, it is operably linked to that coding sequence, or the ribosome binding site is in the coding sequence if it is in a position that allows translation. Operatively linked. In some embodiments, a structural element intended for use in a yeast expression system includes a leader sequence that allows extracellular secretion of the translated protein by the host cell. In another embodiment, where the recombinant protein is 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 give the final product.

  The choice of expression control sequence and expression vector will depend on the choice of host. A wide variety of expression host / vector combinations can be used. Useful expression vectors for eukaryotic hosts include, for example, vectors containing 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 containing pCR1, pBR322, pMB9 and their derivatives, and plasmids with a broader host range, such as M13 phage and other filamentous forms. A single-stranded DNA phage is mentioned.

  Suitable host cells for expressing FZD binding agents or Wnt binding agents (or proteins for use as antigens) include prokaryotes, yeast cells, insect cells, or higher under the control of appropriate promoters. Examples include eukaryotic cells. Prokaryotes include gram negative or gram positive organisms such as E. coli or Bacillus. Examples of higher eukaryotic cells include established mammalian cell lines described later. Cell-free translation systems can also be used. Suitable cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cell hosts are well known in the art. Additional information regarding protein production, including antibody production, can be found, for example, in US Patent Application Publication No. 2008/0187954, US Patent Nos. 6,413,746 and 6,660,501, and International Publication No. 2004/009823.

  Various mammalian culture systems are used to express recombinant polypeptide. Expression of recombinant proteins in mammalian cells will be preferred because such proteins are generally correctly folded, appropriately modified and biologically functional. Examples of suitable mammalian host cell lines include COS-7 (monkey kidney derived) cell line, L-929 (mouse fibroblast derived) cell line, C127 (mouse breast tumor derived) cell line, 3T3 (mouse fibroblast) Cell line), cell line, CHO (derived from Chinese hamster ovary) cell line, HeLa (derived from human cervical cancer) cell line, BHK (derived from hamster kidney fibroblast) cell line, HEK-293 (derived from human fetal kidney) cell line Strains and variants thereof. Mammalian expression vectors contain non-transcribed elements, such as origins of replication, appropriate promoters and enhancers linked to the gene to be expressed, and other 5 'or 3' flanking non-transcribed sequences, and 5 'or 3' untranslated It can contain sequences such as necessary ribosome binding sites, polyadenylation sites, splice donor and splice acceptor sites, and transcription termination sequences.

  Recombinant protein expression in insect cell culture systems (eg, baculovirus) is also a robust way to produce correctly folded biologically functional proteins. Baculovirus systems for producing heterologous proteins in insect cells are well known to those skilled in the art (see, eg, Luckow and Summers, 1988, Bio / Technology, 6:47).

  Accordingly, the present invention provides a cell comprising a FZD binding agent or Wnt binding agent as described herein. In some embodiments, the cell produces a binding agent described herein (eg, an antibody or a soluble receptor). In certain embodiments, the cells produce antibodies. In certain embodiments, the cell produces antibody OMP-18R5. In some embodiments, the cell produces a soluble receptor. In some embodiments, the cell produces a FZD-Fc soluble receptor. In some embodiments, the cell produces a FZD8-Fc soluble receptor. In some embodiments, the cell produces FZD8-Fc soluble receptor 54F28.

  The protein produced by the transformed host can be purified according to any suitable method. Standard methods include methods by chromatography (eg, ion exchange, affinity, and size fractionation column chromatography), centrifugation, differential solubility, or other standard protein purification techniques. 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 through a suitable affinity column. Isolated proteins can also be physically characterized using techniques such as proteolysis, mass spectrometry (MS), nuclear magnetic resonance (NMR), high performance liquid chromatography (HPLC), and x-ray crystallography. it can.

  In some embodiments, the supernatant from an expression system that secretes the recombinant protein into the culture medium can be first concentrated using a commercially available protein concentration filter, such as an Amicon or Millipore Pellicon ultrafiltration unit. . Following the concentration step, the concentrate can be applied to a suitable purification matrix. In some embodiments, an anion exchange resin such as a matrix or substrate having pendant diethylaminoethyl (DEAE) groups can be used. The matrix can be acrylamide, agarose, dextran, cellulose, or other types commonly used for protein purification. In some embodiments, a cation exchange step can be used. Suitable cation exchangers include various insoluble matrices that contain sulfopropyl or carboxymethyl groups. In some embodiments, a hydroxyapatite medium such as, but not limited to, ceramic hydroxyapatite (CHT) can be used. In certain embodiments, the binding agent can be further purified using one or more reverse phase HPLC steps using hydrophobic RP-HPLC media such as silica gel with pendant methyl or other aliphatic groups. . In order to obtain a homogeneous recombinant protein, some or all of the purification steps described above can be used in various combinations.

  In some embodiments, the recombinant protein produced in the bacterial culture is subjected to one or more concentration steps, salting out steps, aqueous ion exchange chromatography steps, or size exclusion chromatography, for example after initial extraction from the cell pellet. It can be isolated by performing a process or the like. HPLC can be used for the final purification step. The microbial cells used for recombinant protein expression can be disrupted by any conventional method such as freeze-thaw cycling, sonication, mechanical disruption, or the use of cytolytic agents.

  Methods known in the art for purifying antibodies and other proteins include those described, for example, in US Patent Publication Nos. 2008/0312425, 2008/0177048, and 2009/0187005. Can be mentioned.

  In certain embodiments, the Wnt binding agent or FZD binding agent is a polypeptide that is not an antibody. Various methods are known in the art for identifying and producing non-antibody polypeptides that bind with high affinity to protein targets. For example, Skerra, 2007, Curr. Opin. Biotechnol., 18: 295-304; Hosse et al., 2006, Protein Science, 15: 14-27; Gill et al., 2006, Curr. Opin. Biotechnol, 17: 653 -658; Nygren, 2008, FEBS J., 275: 2668-76; and Skerra, 2008, FEBS J., 275: 2677-83. In certain embodiments, phage display technology can be used to produce and / or identify FZD-binding polypeptides or Wnt-binding polypeptides. In certain embodiments, the polypeptide comprises a protein scaffold of a type selected from the group consisting of protein A, protein G, lipocalin, fibronectin domain, ankyrin consensus repeat domain, and thioredoxin.

  In certain embodiments, the binding agent can be used in any one of several conjugated (ie, immunoconjugate or radioconjugate) forms or unconjugated forms. In certain embodiments, antibodies are used in unconjugated form to take advantage of the subject's natural defense mechanisms, including complement-dependent cytotoxicity and antibody-dependent cytotoxicity, to eliminate malignant cells or cancer cells. Can do.

In some embodiments, the binding agent is conjugated to a cytotoxic agent. In some embodiments, the cytotoxic agent is a chemotherapeutic agent, such as, but not limited to, methotrexate, adriamicin, doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents. In some embodiments, the cytotoxic agent is an enzymatically active toxin from bacteria, fungi, plants, or animals, or a fragment thereof, such as but not limited to diphtheria A chain, non-diphtheria toxin. Binding active fragment, exotoxin A chain, ricin A chain, abrin A chain, modesin A chain, alpha-sarcin, Aleurites fordii protein, diantin protein, Phytolaca americana protein (PAPI, PAPII) , And PAP-S), momordica charantia inhibitors, crucine, clotin, Sapaonaria officinalis inhibitors, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and trichothecenes It is. In some embodiments, the cytotoxic agent is a radioisotope for producing a radioconjugate or radioconjugated antibody. Various radionuclides are produced for the production of radioconjugated antibodies, including but not limited to ⁹⁰ Y, ¹²⁵ I, ¹³¹ I, ¹²³ I, ¹¹¹ In, ¹³¹ In, ¹⁰⁵ Rh, ¹⁵³ Sm, ⁶⁷ Cu, ⁶⁷ Ga ¹⁶⁶ Ho, ¹⁷⁷ Lu, ¹⁸⁶ Re, ¹⁸⁸ Re and ²¹² Bi can be used. In some embodiments, a conjugate of an antibody and one or more small molecule toxins, such as calicheamicin, maytansinoids, trichothene, and CC1065, and derivatives of these toxins that have toxin activity Can be produced. In certain embodiments, the conjugate of the antibody and cytotoxic agent may be a variety of bifunctional protein coupling agents such as N-succinimidyl-3- (2-pyridyldithiol) propionate (SPDP), iminothiolane (IT). , Bifunctional derivatives of imide esters (eg dimethyl adipoimidate HCL), active esters (eg disuccinimidyl suberate), aldehydes (eg glutaraldehyde), bis-azide compounds (eg bis (p-azidobenzoyl)) Hexanediamine), bis-diazonium derivatives (eg bis- (p-diazoniumbenzoyl) -ethylenediamine), diisocyanates (eg toluene 2,6-diisocyanate), and bis-active fluorine compounds (eg 1,5-difluoro-2,4- It is made using dinitrobenzene.

  In certain embodiments, the Wnt pathway inhibitor (eg, antibody or soluble receptor) is at least one Wnt protein (ie 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 Wnt proteins). ) Antagonists. In certain embodiments, the Wnt pathway inhibitor inhibits the activity of the Wnt protein to which it binds. In certain embodiments, the Wnt pathway inhibitor inhibits the activity of the human Wnt protein to which it binds 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% inhibition.

  In certain embodiments, a Wnt pathway inhibitor (eg, an antibody or soluble receptor) inhibits binding of at least one human Wnt to a suitable receptor. In certain embodiments, the Wnt pathway inhibitor inhibits binding of at least one human Wnt protein to one or more human FZD proteins. In some embodiments, at least one Wnt protein is Wnt1, Wnt2, Wnt2b / 13, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, Wnt10a, Wnt10b, It is selected from the group consisting of Wnt11 and Wnt16. In some embodiments, 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. In certain embodiments, the Wnt pathway inhibitor inhibits binding of one or more Wnt proteins to FZD1, FZD2, FZD4, FZD5, FZD7, and / or FZD8. In certain embodiments, the Wnt pathway inhibitor inhibits binding of one or more Wnt proteins to FZD8. In certain embodiments, inhibition of binding of a particular Wnt to 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 %. In certain embodiments, an agent that inhibits Wnt binding to the FZD protein also inhibits Wnt pathway signaling. In certain embodiments, the Wnt pathway inhibitor that inhibits human Wnt pathway signaling is an antibody. In certain embodiments, the Wnt pathway inhibitor that inhibits human Wnt pathway signaling is a FZD-Fc soluble receptor. In certain embodiments, the Wnt pathway inhibitor that inhibits human Wnt pathway signaling is a FZD8-Fc soluble receptor. In certain embodiments, the Wnt pathway inhibitor that inhibits human Wnt pathway signaling is soluble receptor 54F28.

  In certain embodiments, a Wnt pathway inhibitor (eg, an antibody or soluble receptor) described herein is an antagonist of at least one human Wnt protein and inhibits Wnt activity. In certain embodiments, the Wnt pathway inhibitor inhibits Wnt 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 the activity of 1, 2, 3, 4, 5, or more Wnt proteins. In some embodiments, Wnt pathway inhibitors are Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, Wnt10a, Wnt10b, Wnt11 and Wnt11, and Wnt11 Inhibits the activity of at least one human Wnt protein selected from the group consisting of In some embodiments, the Wnt binding agent binds to at least one Wnt protein selected from the group consisting of Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt10a, and Wnt10b. . In certain embodiments, the at least one Wnt protein is selected from the group consisting of Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt8a, Wnt8b, Wnt10a, and Wnt10b. In certain embodiments, the Wnt pathway inhibitor that inhibits human Wnt activity is an antibody. In certain embodiments, the Wnt pathway inhibitor that inhibits human Wnt activity is a FZD-Fc soluble receptor. In certain embodiments, the Wnt pathway inhibitor that inhibits human Wnt activity is a FZD8-Fc soluble receptor. In certain embodiments, the Wnt pathway inhibitor that inhibits human Wnt activity is soluble receptor 54F28.

  In certain embodiments, a 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 the activity of 1, 2, 3, 4, 5, or more FZD proteins. In some embodiments, the Wnt pathway inhibitor inhibits the activity of at least one human FZD protein selected from the group consisting of FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, and FZD10. . In certain embodiments, the Wnt pathway inhibitor inhibits the activity of FZD1, FZD2, FZD4, FZD5, FZD7, and / or FZD8. In certain embodiments, the Wnt pathway inhibitor inhibits the activity of FZD8. In some embodiments, the Wnt pathway inhibitor is an anti-FZD antibody. In certain embodiments, the Wnt pathway inhibitor is the anti-FZD antibody OMP-18R5.

  In certain embodiments, a Wnt pathway inhibitor described herein is an antagonist of at least one human Wnt protein and inhibits Wnt signaling. In certain embodiments, the Wnt pathway inhibitor inhibits Wnt 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%. In some embodiments, the Wnt pathway inhibitor inhibits signal transduction by 1, 2, 3, 4, 5 or more Wnt proteins. In some embodiments, the Wnt pathway inhibitor is capable of signaling at least one Wnt protein selected from the group consisting of Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt10a, and Wnt10b. Inhibit. In certain embodiments, the Wnt pathway inhibitor that inhibits Wnt signaling is an antibody. In certain embodiments, the Wnt pathway inhibitor that inhibits Wnt signaling is a soluble receptor. In certain embodiments, the Wnt pathway inhibitor that inhibits Wnt signaling is a FZD-Fc soluble receptor. In certain embodiments, the Wnt pathway inhibitor that inhibits Wnt signaling is a FZD8-Fc soluble receptor. In certain embodiments, the Wnt pathway inhibitor that inhibits Wnt signaling is soluble receptor 54F28.

  In certain embodiments, the Wnt pathway inhibitor described herein is an antagonist of β-catenin signaling. In certain embodiments, the Wnt pathway inhibitor inhibits β-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% To do. In certain embodiments, the Wnt pathway inhibitor that inhibits β-catenin signaling is an antibody. In certain embodiments, the Wnt pathway inhibitor that inhibits β-catenin signaling is an anti-FZD antibody. In certain embodiments, the Wnt pathway inhibitor that inhibits β-catenin signaling is antibody OMP-18R5. In certain embodiments, the Wnt pathway inhibitor that inhibits β-catenin signaling is a soluble receptor. In certain embodiments, the Wnt pathway inhibitor that inhibits β-catenin signaling is a FZD-Fc soluble receptor. In certain embodiments, the Wnt pathway inhibitor that inhibits β-catenin signaling is a FZD8-Fc soluble receptor.

  In certain embodiments, a 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, FZD2, FZD3, FZD4, FDZ5, FDZ6, FDZ7, FDZ8, FDZ9, and / or FZD10. In certain embodiments, 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%. In certain embodiments, 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 β-catenin signaling. In certain embodiments, the Wnt pathway inhibitor that inhibits binding of at least one human Wnt to at least one FZD protein is an antibody. In certain embodiments, the 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, the Wnt pathway inhibitor that inhibits binding of at least one human Wnt to at least one FZD protein is antibody OMP-18R5. In certain embodiments, a Wnt pathway inhibitor that inhibits binding of at least one human Wnt to at least one FZD protein is a soluble receptor. In certain embodiments, the Wnt pathway inhibitor that inhibits binding of at least one human Wnt to at least one FZD protein is a FZD-Fc soluble receptor. In certain embodiments, the Wnt pathway inhibitor that inhibits binding of at least one human Wnt to at least one FZD protein is a FZD8-Fc soluble receptor. In certain embodiments, the Wnt pathway inhibitor that inhibits binding of at least one human Wnt to at least one FZD protein is FZD8-Fc soluble receptor 54F28.

  In certain embodiments, a Wnt pathway inhibitor described herein blocks binding of at least one Wnt to a receptor. In certain embodiments, the Wnt pathway inhibitor blocks binding of at least one human Wnt protein to one or more of its receptors. In some embodiments, the Wnt pathway inhibitor blocks binding of at least one Wnt to at least one FZD protein. In some embodiments, the Wnt pathway inhibitor blocks binding of at least one Wnt protein to FZD1, FZD2, FZD3, FZD4, FDZ5, FDZ6, FDZ7, FDZ8, FDZ9, and / or FZD10. In certain embodiments, 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%. In certain embodiments, 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 β-catenin signaling. In certain embodiments, the Wnt pathway inhibitor that blocks binding of at least one human Wnt to at least one FZD protein is an antibody. In certain embodiments, the Wnt pathway inhibitor that blocks binding of at least one human Wnt to at least one FZD protein is an anti-FZD antibody. In certain embodiments, the Wnt pathway inhibitor that blocks binding of at least one human Wnt to at least one FZD protein is antibody OMP-18R5. In certain embodiments, the Wnt pathway inhibitor that blocks binding of at least one human Wnt to at least one FZD protein is a soluble receptor. In certain embodiments, the Wnt pathway inhibitor that blocks binding of at least one human Wnt to at least one FZD protein is a FZD-Fc soluble receptor. In certain embodiments, the Wnt pathway inhibitor that blocks binding of at least one human Wnt to at least one FZD protein is a FZD8-Fc soluble receptor. In certain embodiments, the Wnt pathway inhibitor that blocks binding of at least one human Wnt to at least one FZD protein is soluble receptor 54F28.

  In certain embodiments, a Wnt pathway inhibitor described herein inhibits Wnt pathway signaling. 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 does not necessarily inhibit signaling by all receptors. It is understood that this is not possible. In certain alternative embodiments, Wnt pathway signaling by all human receptors can be inhibited. In certain embodiments, 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. . In certain embodiments, inhibition of Wnt pathway signaling 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 the level of Wnt pathway signaling, or A reduction of at least about 95%. In some embodiments, the Wnt pathway inhibitor that inhibits Wnt pathway signaling is an antibody. In some embodiments, the Wnt pathway inhibitor that inhibits Wnt pathway signaling is an anti-FZD antibody. In some embodiments, the Wnt pathway inhibitor that inhibits Wnt pathway signaling is antibody OMP-18R5. In some embodiments, the Wnt pathway inhibitor that inhibits Wnt pathway signaling is a soluble receptor. In some embodiments, the Wnt pathway inhibitor that inhibits Wnt pathway signaling is a FZD-Fc soluble receptor. In some embodiments, the Wnt pathway inhibitor that inhibits Wnt pathway signaling is a FZD8-Fc soluble receptor. In some embodiments, the Wnt pathway inhibitor that inhibits Wnt pathway signaling is soluble receptor 54F28.

  In certain embodiments, Wnt pathway inhibitors described herein inhibit β-catenin activation. A Wnt pathway inhibitor that inhibits the activation of β-catenin may, in certain embodiments, inhibit β-catenin activation by one or more receptors, but not necessarily all receptors by β-catenin. It is understood that it cannot inhibit activation. In certain alternative embodiments, the activation of β-catenin by all human receptors can be inhibited. In certain embodiments, activation of β-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 In certain embodiments, inhibition of β-catenin activation by a Wnt binding agent is at least about 10%, at least about 25%, at least about 50%, at least about 75%, at least about the level of β-catenin activation. About 90%, or at least about 95% reduction. In some embodiments, the Wnt pathway inhibitor that inhibits activation of β-catenin is an antibody. In some embodiments, the Wnt pathway inhibitor that inhibits activation of β-catenin is an anti-FZD antibody. In some embodiments, the Wnt pathway inhibitor that inhibits activation of β-catenin is antibody OMP-18R5. In some embodiments, the Wnt pathway inhibitor that inhibits activation of β-catenin is a soluble receptor. In some embodiments, the Wnt pathway inhibitor that inhibits activation of β-catenin is a FZD-Fc soluble receptor. In some embodiments, the Wnt pathway inhibitor that inhibits activation of β-catenin is a FZD8-Fc soluble receptor. In some embodiments, the Wnt pathway inhibitor that inhibits activation of β-catenin is soluble receptor 54F28.

  In vivo and in vitro assays to determine whether Wnt pathway inhibitors inhibit β-catenin signaling are known in the art. For example, to measure β-catenin signaling levels in vitro, use a cell-based luciferase reporter assay that utilizes a TCF / Luc reporter vector containing multiple copies of the TCF binding domain upstream of the firefly luciferase reporter gene. (Gazit et al., 1999, Oncogene, 18; 5959-66; TOPflash, Millipore, Billerica, Mass.). Binds the level of β-catenin signaling in the presence of one or more Wnt proteins (eg, Wnt expressed by transfected cells or Wnt provided by Wnt conditioned medium) and in the presence of a binding agent Compare to the level of signaling in the absence of the agent. In addition to the TCF / Luc reporter assay, the effect of binding agents (or candidate agents) on β-catenin signaling is influenced by genes that are regulated by β-catenin, such as c-myc (He et al., 1998, Science , 281: 1509-12), cyclin D1 (Tetsu et al., 1999, Nature, 398: 422-6), and / or fibronectin (Gradl et al., 1999, Mol. Cell Biol., 19: 5576-87). ) Can be measured in vitro or in vivo. In certain embodiments, the effect of the binding agent on β-catenin signaling is determined by the effect of the agent on the phosphorylation state of dishebeled-1, dish-2, dish-3, LRP5, LRP6, and / or β-catenin. It can also be evaluated by measuring.

  In certain embodiments, the Wnt pathway inhibitor has one or more of the following effects: inhibit tumor cell proliferation, inhibit tumor growth, reduce the frequency of cancer stem cells in the tumor, Reduce tumorigenicity, reduce tumor tumorigenicity by reducing the frequency of cancer stem cells in the tumor, cause tumor cell death, induce tumor differentiation, tumorigenic cells Is differentiated into a non-tumorigenic state, induces the expression of differentiation markers in tumor cells, prevents tumor cell metastasis, or decreases tumor cell survival.

  In certain embodiments, the Wnt pathway inhibitor has the ability to inhibit tumor growth. In certain embodiments, the Wnt pathway inhibitor has the ability to inhibit tumor growth in vivo (eg, in a xenograft mouse model and / or in a human with cancer). In some embodiments, the tumor is a colorectal tumor, colon tumor, pancreatic tumor, lung tumor, ovarian tumor, liver tumor, breast tumor, kidney tumor, prostate tumor, gastrointestinal tumor, melanoma, cervical tumor, bladder tumor, A tumor selected from the group consisting of glioblastoma and head and neck tumors. In certain embodiments, the tumor is melanoma. In certain embodiments, the tumor is a colorectal tumor. In certain embodiments, the tumor is a pancreatic tumor. In certain embodiments, the tumor is a breast tumor. In certain embodiments, the tumor is a Wnt-dependent tumor.

  In certain embodiments, the Wnt pathway inhibitor has the ability to reduce the tumorigenicity of the tumor. In certain embodiments, the Wnt pathway inhibitor has the ability to reduce the tumorigenicity of a tumor comprising cancer stem cells in an animal model, such as a mouse xenograft model. In certain embodiments, the number or frequency of cancer stem cells in the tumor is at least about 1/2, about 1/3, about 1/5, about 1/10, about 1/50, about 100 minutes Of 1 or about 1/1000. In certain embodiments, a reduction in the number or frequency of cancer stem cells is determined by a limiting dilution assay using an animal model. Additional examples and guidance on determining a reduction in the number or frequency of cancer stem cells in a tumor using a limiting dilution assay can be found in, for example, WO 2008/042236 and US Patent Publication Nos. 2008/0064049 and 2008. You can find it in / 0178305.

  In certain embodiments, a Wnt pathway inhibitor described herein is 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, Active in vivo for at least about 1 week, or at least about 2 weeks. In certain embodiments, the Wnt pathway inhibitor is 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 is an IgG (eg, IgG1 or IgG2) antibody that is active in vivo. In certain embodiments, the Wnt pathway inhibitor is 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 The fusion protein is active in vivo for at least about 2 weeks.

  In certain embodiments, the Wnt pathway inhibitor described herein is 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 in mice, cynomolgus monkeys, or humans. Has a circulation half-life of about 1 week, or at least about 2 weeks. In certain embodiments, the Wnt pathway inhibitor is 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 in mice, cynomolgus monkeys, or humans. An IgG (eg, IgG1 or IgG2) antibody with a circulating half-life of about 2 weeks. In certain embodiments, the Wnt pathway inhibitor is 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 in mice, cynomolgus monkeys, or humans. A fusion protein with a circulating half-life of about 2 weeks. Methods for increasing (or decreasing) the half-life of agents such as polypeptides and antibodies are known in the art. For example, known methods for increasing the circulating half-life of IgG antibodies include the introduction of mutations into the Fc region that increase the pH-dependent binding of antibodies to neonatal Fc receptors (FcRn) at pH 6.0 ( For example, see US Patent Publication Nos. 2005/0276799, 2007/0148164, and 2007/0122403). Known methods for increasing the circulating half-life of antibody fragments lacking the Fc region include techniques such as PEGylation.

IV. Kits Further provided are kits for performing the methods of the present invention. “Kit” contemplates any product (eg, package or container) that includes at least one reagent for specifically detecting expression of at least one biomarker of the invention, such as an antibody, nucleic acid probe, and the like. The kit can be promoted, distributed, and / or sold as a unit for performing the methods of the present invention. In addition, the kit may include a package insert that describes the kit and includes instructions for its use.

  In some embodiments, the kit includes reagents for performing the methods of the invention using microarray technology. In some embodiments, the kit includes reagents for performing the methods of the invention using a qPCR assay. Positive controls and / or negative controls can be included in the kit to confirm the activity and correct use of the reagents utilized by the present invention. Controls can include samples that are known to be either positive or negative for the presence of the biomarker of interest, or other samples that contain the biomarker of interest. The control design and use is standard and well within the normal capabilities of one skilled in the art.

  In some embodiments, the kit comprises a polynucleotide selected from the group consisting of SEQ ID NOs: 62-79. In some embodiments, the kit comprises (a) a forward primer of SEQ ID NO: 62, a reverse primer of SEQ ID NO: 63, and a probe comprising SEQ ID NO: 64; (b) a forward of SEQ ID NO: 65 A primer, a reverse primer of SEQ ID NO: 66, and a probe comprising SEQ ID NO: 67; (c) a forward primer of SEQ ID NO: 68, a reverse primer of SEQ ID NO: 69, and SEQ ID NO: 70 A probe comprising (d) a forward primer of SEQ ID NO: 71, a reverse primer of SEQ ID NO: 72, and SEQ ID NO: 73; (e) a forward primer of SEQ ID NO: 74, SEQ ID NO: 75 And a probe comprising SEQ ID NO: 76; and (f) a forward primer of SEQ ID NO: 77, a reverse primer of SEQ ID NO: 78, and a probe comprising SEQ ID NO: 79.

  It is further understood that any or all of the steps of the method of the present invention may be performed by a person or alternatively in an automated fashion. Thus, steps such as sample preparation and detection of biomarker expression can be automated.

  Aspects of the present disclosure can be further demonstrated by reference to the following non-limiting examples that detail the preparation of certain antibodies of the present disclosure and methods for using the antibodies of the present disclosure. . It will be apparent to those skilled in the art that many changes, both in materials and methods, can be made without departing from the scope of the disclosure.

Example 1
Identification of tumors that are responsive to treatment with a combination of OMP-18R5 and Taxol Breast tumor xenograft models OMP-B34, OMP-B39, OMP-B44, OMP-B59, OMP-B60, UM-T01, UM-T03, And UM-PE13 were established from tumor specimens from patients that had been minimally passaged with OncoMed Pharmaceuticals or the University of Michigan. 2-4 x 10 ⁴ cells of OMP-B34, OMP-B39, OMP-B44, OMP-B59, OMP-B60, UM-T01, UM-T03, or UM-PE13 tumors, 6-8 weeks old NOD / SCID mice were injected subcutaneously. Tumors were allowed to grow until they reached an average volume of 100-150 mm ³ . Randomly divided tumor-bearing mice into 4 groups (each group n = 10), control antibody 1B711 (15 mg / kg), anti-FZD antibody OMP-18R5 (15 mg / kg), taxol (10 mg / kg), Or treated with OMP-18R5 (15 mg / kg) in combination with taxol (10 mg / kg). Treatment with antibody and / or taxol was given weekly. Tumor growth was monitored and tumor volume was measured by electronic calipers at designated time points. Data are expressed as mean ± SEM.

  To determine whether tumors were responsive to the anti-FZD antibody OMP-18R5, single agent tumor volume data was compared to controls and combined treatment with OMP-18R5 and taxol was compared to taxol as a single agent. For this study, “responder” tumors were defined as tumors that showed significantly better tumor growth inhibition with OMP-18R5 and taxol compared to tumor growth inhibition with taxol as a single agent.

  The results for each xenograft model are shown in FIGS. A t-test was performed at each time point. Multiple comparisons used two-factor repeated measures ANOVA followed by Bonferroni correction. t-test and two-factor repeated measures ANOVA were performed using GraphPad Prism5 (GraphPad Software Inc.). Tumors OMP-B59, OMP-B60, UM-T03, and UM-PE13 are shown to be responders, and tumors OMP-B34, OMP-B39, OMP-B44, and UM-T01 are non-responders It has been shown. The results are summarized in Table 1.

Example 2
Predictive biomarker identification
Breast tumors that did not respond to treatment with a combination of OMP-18R5 and taxol OMP-B34, OMP-B39, OMP-B44 untreated breast tumors (“non-responders”), and UM-T01, and OMP-18R5 Microarray analysis was performed on untreated tumors of tumors OMP-B59, OMP-B60, UM-T03, and UM-PE13 (“responders”) in response to treatment with a combination of and taxol. RNA was isolated from each tumor by DNAse treatment using RNeasy Fibrous Tissue Mini Kit (Qiagen, Valencia CA) according to the manufacturer's instructions. Samples were stored at -80 ° C. RNA was visualized with an Agilent 2100 Bioanalyzer and integrity was confirmed by the presence of intact 28S and 18S ribosomal peaks. All RNA samples had a 260/280 ratio> 1.8. Total RNA isolated from each tumor was amplified using the Ovation RNA Amplification System V2 (NuGEN, San Carlos, CA). The amplified antisense single-stranded cDNA was fragmented and biotinylated using FL-Ovation cDNA Biotin Module V2 (NuGEN). Prior to hybridization to the array, the quality of the cDNA and fragmented cDNA was assessed with a spectrophotometer and Bioanalyzer. Treated RNA was hybridized to an Affymetrix HG-U133 plus 2.0 microarray (Affymetrix, Santa Clara, Calif.) As outlined in the manufacturer's technical manual. After hybridization, the microarray was washed, scanned and analyzed. Microarray data was processed into probe set level data using GeneChip-RMA (Wu et al., 2004, J. Amer. Stat. Assn., 99: 909-917). Probe sets that could cross-hybridize with mouse markers were excluded. To summarize the data for the gene level and confirm that the probe with the strongest signal was selected, maximum expression was used among all probe sets mapping one gene. Genes with low expression (<5 on log2 scale) or near zero variation (<0.01) were removed. Genes were normalized to N (0,1) by subtracting log2 scale expression from the mean and dividing by the standard deviation of each gene.

  Analysis includes multiple pathways including classical pathways, in-plane cell polarity, Wnt / Ca + 2, negative regulation of Wnt signaling, cell fate, tissue polarity, cell growth and proliferation, cell migration, cell cycle, and cell homeostasis This was done using genes from the signal transduction pathway (see Table 2).

  Support vector machine-recursive feature elimination (SVM-RFE) method (Guyon et al, 2002, Machine Learning, 46: 389-422) is used to identify genes that can distinguish between responder and non-responder tumors, The support vector machine (SVM) method (Cortes and Vapnik, 1995, Machine Learning, 20: 273-297) was used for classification. A single cross validation (LOOCV) method was used to select the number of genes and to measure the positive predictive value (PPV), negative predictive value (NPV), sensitivity and specificity of the model. Biomarker signatures including FBXW2, CCND2, RHOU, CTBP2, WIF1, and DKK1 achieved the best performance of PPV = NPV = sensitivity = specificity = 100% using 8 breast tumors (see Figure 2) . As shown in FIG. 3, principal component analysis (PCA) showed that the 6-gene biomarker signature resulted in almost complete differentiation of 8 breast tumors. In addition, a strong correlation was observed between the 6-gene biomarker signature and the tumor volume ratio (RTV) from the in vivo experiments described in Example 1 (correlation = 0.95, p-value = 0.0003; cross-validation) Correlation = 0.89, p-value = 0.00027; Fig. 4).

Based on the training data, decision values were determined from the SVM model. For the 6 gene biomarker signature, the decision value can be calculated by the weighted sum of the normalized expression of 6 genes: 0.4560427 ^* FBXW2 + 0.3378467 ^* CCND2-0.4809354 ^* RHOU + 0.409029 ^* CTBP2 + 0.3291529 ^* WIF1 + 02926374 ^* DKK1 + 0.04662682. A positive decision value indicated that the tumor was predicted to be a responder, and a negative decision value indicated that the tumor was predicted to be a non-responder. In addition, classification probabilities can be obtained by fitting logistic regression to the decision values. Tumors associated with a probability higher than 0.5 were predicted to be responders, and tumors with a probability lower than 0.5 were predicted to be non-responders.

Example 3
In vivo validation of predictive biomarkers
Six additional breast cancer tumors were selected from OncoMed Tumor Bank and microarray analysis was performed as described in Example 1. The six breast cancer tumors were OMP-B29, OMP-B71, OMP-B84, OMP-B90, UM-T02, and UM-T06. As described herein, classification probability analysis with 6-gene biomarker signatures was used to predict the response of each of these tumors to treatment with anti-FZD antibody OMP-18R5 in combination with taxol (see FIG. 5). In parallel, six tumors were evaluated in an in vivo xenograft model as described in Example 1 (see FIGS. 6A-F). As described in Example 1, a “responder” in an in vivo model is a tumor that exhibits significantly superior tumor growth inhibition with the combination of OMP-18R5 and taxol compared to tumor growth inhibition with taxol as a single agent. is there. The prediction based on classification probability was compared with the result of in vivo xenograft model. The results are summarized in Table 3.

  As shown in Table 3, the response of each of the 6 types of breast cancer tumors was accurately predicted by a 6-gene biomarker signature using decision values and classification probabilities.

Example 4
Estimating the prevalence of 6-gene biomarker signatures The prevalence of biomarker signatures can be defined as the proportion of the population predicted to be a responder based on biomarker signatures. By applying the 6-gene biomarker signature to three publicly available breast cancer microarray datasets, the prevalence of the 6-gene biomarker signature in the HER2-negative (HER2-) and triple-negative breast cancer (TNBC) populations Estimated. The Cremoux 2001 data set was aggregated based on the Affymetrix U133plus2 microarray by 226 patients, including 145 Her2- and 81 HER2 +, where 51 TNBCs were included in the HER2-group. The Wang2011 data set was aggregated based on the Affymetrix U133plus2 microarray by 115 patients, including 79 HER2- and 36 HER2 +, where 28 TNBCs were included in the HER2-group. The Prat2010 data set was compiled based on the Agilent Human 1A microarray by 333 patients, including 215 HER2- and 118 HER2 +, where 57 TNBCs were included in the HER2-group . Public data pre-processing included downloading data, extracting probe set mappings for 6 genes, and combining probe sets into 6 genes. Gene level expression data was further normalized by subtracting the average and dividing by the standard deviation of each gene in the public data. The public data was classified using the SVM model built by the training data. Classification probabilities were obtained and the proportion of predicted responders (probability> 0.5) was calculated based on the 6-gene biomarker signature.

  As shown in FIG. 7, the predicted prevalence of 6-gene biomarker signatures in the three databases was very similar (approximately 60%). This prediction suggests that there is a large population of breast cancer patients who are responsive to treatment with the anti-FZD antibody OMP-18R5 in combination with taxol.

Example 5
QPCR assay for 6-gene biomarker signature
A qPCR assay was developed to determine the expression levels of FBXW2, CCND2, RHOU, CTBP2, WIF1, and DKK1 in the sample. Primers and probes were designed using publicly available mRNA sequences. Primers and probes were made and used in optimization and validation studies using human fresh frozen (FF) tissue samples and formalin-fixed paraffin embedded (FFPE) human tissue samples. Specific primers and probes are listed in Table 4 (all sequences in the 5 'to 3' direction). Four reference genes were used for normalization, including TOP1 (topoisomerase 1), GUSB (β-glucuronidase), SDHA (succinate dehydrogenase), and PUM1 (pumilio homolog 1).

  QPCR was performed on total RNA from 18 xenograft breast tumors. Tumor specimens were collected, rapidly snap frozen and stored at -80 ° C prior to RNA isolation. Total RNA was extracted by TissueLyzer homogenization and DNase I treatment using the RNeasy Fibrous Mini Kit (Qiagen, Valencia CA, PN # 74704) according to the manufacturer's protocol. RNA was visualized with Bioanalyzer 2100 (Agilent, Santa Clara, Calif.) And verified to be intact RNA with RIN value> 6.0. All RNAs had an A260 / A280 ratio of> 1.8.

  qPCR was performed in a two-step method. First, cDNA was synthesized from total RNA using random hexamers as described in Applied Biosystems User Bulletin 2. In the next qPCR reaction, TaqMan Universal PCR Master Mix (Applied Biosystems, Foster City, CA. Cat # 4304437 and 4326708) was used according to the manufacturer's protocol. The amount of gene expression was determined from triplicate reactions using the Ct (cycle threshold) method. The cycle threshold is generally considered to be the number of cycles required for the signal to cross the detection threshold. Ct levels are inversely proportional to the amount of target nucleic acid in the sample. The Ct of 6 genes is normalized using the Ct levels of 4 reference genes. The 6-gene signature Ct normalized for 18 xenograft samples is shown in Table 5.

  The decision value can be calculated by the weighted sum of the expression of the six genes normalized from the data generated from the qPCR assay. These decision values differed from the decision values resulting from the analysis based on microarray data, but the predictive capabilities of the two models are very similar.

  The examples and embodiments described herein are for illustrative purposes only, and various changes or modifications will be suggested to one skilled in the art in light of them, which are intended to be included in the spirit and scope of the present application. It will be understood that.

  All publications, patents, and patent applications mentioned herein are hereby incorporated by reference to the same extent as if each individual publication, patent, or patent application was individually indicated to be incorporated by reference in their entirety. Incorporated into the book.

OMP-18R5重鎖CDR2（SEQ ID NO:2）

OMP-18R5重鎖CDR3（SEQ ID NO:3）

OMP-18R5軽鎖CDR1（SEQ ID NO:4）

OMP-18R5軽鎖CDR2（SEQ ID NO:5）

OMP-18R5軽鎖CDR3（SEQ ID NO:6）

OMP-18R5重鎖可変領域アミノ酸配列（SEQ ID NO:7）

OMP-18R5軽鎖可変領域アミノ酸配列（SEQ ID NO:8）

OMP-18R5重鎖アミノ酸配列（下線部は予測シグナル配列）（SEQ ID NO:9）

OMP-18R5軽鎖アミノ酸配列（下線部は予測シグナル配列）（SEQ ID NO:10）

OMP-18R5重鎖アミノ酸配列（予測シグナル配列なし）（SEQ ID NO:11）

OMP-18R5軽鎖アミノ酸配列（予測シグナル配列なし）（SEQ ID NO:12）

ヒトFZD1 Friドメインアミノ酸配列（予測シグナル配列なし）（SEQ ID NO:13）

ヒトFZD2 Friドメインアミノ酸配列（予測シグナル配列なし）（SEQ ID NO:14）

ヒトFZD3 Friドメインアミノ酸配列（予測シグナル配列なし）（SEQ ID NO:15）

ヒトFZD4 Friドメインアミノ酸配列（予測シグナル配列なし）（SEQ ID NO:16）

ヒトFZD5 Friドメインアミノ酸配列（予測シグナル配列なし）（SEQ ID NO:17）

ヒトFZD6 Friドメインアミノ酸配列（予測シグナル配列なし）（SEQ ID NO:18）

ヒトFZD7 Friドメインアミノ酸配列（予測シグナル配列なし）（SEQ ID NO:19）

ヒトFZD8 Friドメインアミノ酸配列（予測シグナル配列なし）（SEQ ID NO:20）

ヒトFZD9 Friドメインアミノ酸配列（予測シグナル配列なし）（SEQ ID NO:21）

ヒトFZD10 Friドメインアミノ酸配列（予測シグナル配列なし）（SEQ ID NO:22）

ヒトFZD1アミノ酸116〜227（SEQ ID NO:23）

ヒトFZD2アミノ酸39〜150（SEQ ID NO:24）

ヒトFZD3アミノ酸28〜133（SEQ ID NO:25）

ヒトFZD4アミノ酸48〜161（SEQ ID NO:26）

ヒトFZD5アミノ酸33〜147（SEQ ID NO:27）

ヒトFZD6アミノ酸24〜129（SEQ ID NO:28）

ヒトFZD7アミノ酸49〜160（SEQ ID NO:28）

ヒトFZD8アミノ酸35〜148（SEQ ID NO:30）

ヒトFZD9アミノ酸39〜152（SEQ ID NO:31）

ヒトFZD10アミノ酸34〜147（SEQ ID NO:32）

ヒトFZD8 Friドメインアミノ酸配列（予測シグナル配列なし）（変異体）（SEQ ID NO:33）

ヒトIgG₁ Fc領域（SEQ ID NO:34）

ヒトIgG₁ Fc領域（変異体）（SEQ ID NO:35）

ヒトIgG₁ Fc領域（SEQ ID NO:36）

ヒトIgG₁ Fc領域（SEQ ID NO:37）

ヒトIgG₂ Fc領域（SEQ ID NO:38）

FZD8-Fc変異体54F03アミノ酸配列（予測シグナル配列なし）（SEQ ID NO:39）

FZD8-Fc変異体54F16、54F17、54F18、54F23、54F25、54F27、54F29、54F31、および54F34アミノ酸配列（予測シグナル配列なし）（SEQ ID NO:40）

FZD8-Fc変異体54F19、54F20、54F24、54F26、54F28、54F30、54F32、54F34および54F35アミノ酸配列（予測シグナル配列なし）（SEQ ID NO:41）

FZD8-Fc変異体54F03アミノ酸配列（シグナル配列あり）（SEQ ID NO:42）

FZD8-Fc変異体54F16アミノ酸配列（シグナル配列あり）（SEQ ID NO:43）

FZD8-Fc変異体54F26（シグナル配列あり）（SEQ ID NO:44）

FZD8-Fc変異体54F28（シグナル配列あり）（SEQ ID NO:45）

ヒトWnt1 C末端システインリッチドメイン（aa288〜370）（SEQ ID NO:46）

ヒトWnt2 C末端システインリッチドメイン（aa267〜360）（SEQ ID NO:47）

ヒトWnt2b C末端システインリッチドメイン（aa298〜391）（SEQ ID NO:48）

ヒトWnt3 C末端システインリッチドメイン（aa273〜355)（SEQ ID NO:49）

ヒトWnt3a C末端システインリッチドメイン（aa270〜352）（SEQ ID NO:50）

ヒトWnt7a C末端システインリッチドメイン（aa267〜359）（SEQ ID NO:51）

ヒトWnt7b C末端システインリッチドメイン（aa267〜349）（SEQ ID NO:52）

ヒトWnt8a C末端システインリッチドメイン（aa248〜355）（SEQ ID NO:53）

ヒトWnt8b C末端システインリッチドメイン（aa245〜351）（SEQ ID NO:54）

ヒトWnt10a C末端システインリッチドメイン（aa335〜417）（SEQ ID NO:55）

ヒトWnt10b C末端システインリッチドメイン（aa307〜389）（SEQ ID NO:56）

リンカー（SEQ ID NO:57）

リンカー（SEQ ID NO:58）

リンカー（SEQ ID NO:59）

リンカー（SEQ ID NO:60）

リンカー（SEQ ID NO:61）

CCND2フォワードプライマー (SEQ ID NO:62)

CCND2リバースプライマー (SEQ ID NO:63)

CCND2 プローブ (SEQ ID NO:64)

CTBP2フォワードプライマー (SEQ ID NO:65)

CTBP2リバースプライマー (SEQ ID NO:66)

CTBP2 プローブ (SEQ ID NO:67)

DKK1フォワードプライマー (SEQ ID NO:68)

DKK1リバースプライマー (SEQ ID NO:69)

DKK1 プローブ (SEQ ID NO:70)

FBXW2フォワードプライマー (SEQ ID NO:71)

FBXW2リバースプライマー (SEQ ID NO:72)

FBXW2 プローブ (SEQ ID NO:73)

RHOU1フォワードプライマー (SEQ ID NO:74)

RHOU1リバースプライマー (SEQ ID NO:75)

RHOU1 プローブ (SEQ ID NO:76)

WIF1フォワードプライマー (SEQ ID NO:77)

WIF1リバースプライマー (SEQ ID NO:78)

WIF1 プローブ (SEQ ID NO:79)

The following are the sequences disclosed in this application.
OMP-18R5 heavy chain CDR1 (SEQ ID NO: 1)

OMP-18R5 heavy chain CDR2 (SEQ ID NO: 2)

OMP-18R5 heavy chain CDR3 (SEQ ID NO: 3)

OMP-18R5 light chain CDR1 (SEQ ID NO: 4)

OMP-18R5 light chain CDR2 (SEQ ID NO: 5)

OMP-18R5 light chain CDR3 (SEQ ID NO: 6)

OMP-18R5 heavy chain variable region amino acid sequence (SEQ ID NO: 7)

OMP-18R5 light chain variable region amino acid sequence (SEQ ID NO: 8)

OMP-18R5 heavy chain amino acid sequence (underlined is the predicted signal sequence) (SEQ ID NO: 9)

OMP-18R5 light chain amino acid sequence (underlined is the predicted signal sequence) (SEQ ID NO: 10)

OMP-18R5 heavy chain amino acid sequence (no predicted signal sequence) (SEQ ID NO: 11)

OMP-18R5 light chain amino acid sequence (no predicted signal sequence) (SEQ ID NO: 12)

Human FZD1 Fri domain amino acid sequence (no predicted signal sequence) (SEQ ID NO: 13)

Human FZD2 Fri domain amino acid sequence (no predicted signal sequence) (SEQ ID NO: 14)

Human FZD3 Fri domain amino acid sequence (no predicted signal sequence) (SEQ ID NO: 15)

Human FZD4 Fri domain amino acid sequence (no predicted signal sequence) (SEQ ID NO: 16)

Human FZD5 Fri domain amino acid sequence (no predicted signal sequence) (SEQ ID NO: 17)

Human FZD6 Fri domain amino acid sequence (no predicted signal sequence) (SEQ ID NO: 18)

Human FZD7 Fri domain amino acid sequence (no predicted signal sequence) (SEQ ID NO: 19)

Human FZD8 Fri domain amino acid sequence (no predicted signal sequence) (SEQ ID NO: 20)

Human FZD9 Fri domain amino acid sequence (no predicted signal sequence) (SEQ ID NO: 21)

Human FZD10 Fri domain amino acid sequence (no predicted signal sequence) (SEQ ID NO: 22)

Human FZD1 amino acids 116-227 (SEQ ID NO: 23)

Human FZD2 amino acids 39-150 (SEQ ID NO: 24)

Human FZD3 amino acids 28-133 (SEQ ID NO: 25)

Human FZD4 amino acids 48-161 (SEQ ID NO: 26)

Human FZD5 amino acids 33-147 (SEQ ID NO: 27)

Human FZD6 amino acids 24-129 (SEQ ID NO: 28)

Human FZD7 amino acids 49-160 (SEQ ID NO: 28)

Human FZD8 amino acids 35-148 (SEQ ID NO: 30)

Human FZD9 amino acids 39-152 (SEQ ID NO: 31)

Human FZD10 amino acids 34 to 147 (SEQ ID NO: 32)

Human FZD8 Fri domain amino acid sequence (no predicted signal sequence) (mutant) (SEQ ID NO: 33)

Human IgG ₁ Fc region (SEQ ID NO: 34)

Human IgG ₁ Fc region (mutant) (SEQ ID NO: 35)

Human IgG ₁ Fc region (SEQ ID NO: 36)

Human IgG ₁ Fc region (SEQ ID NO: 37)

Human IgG ₂ Fc region (SEQ ID NO: 38)

FZD8-Fc variant 54F03 amino acid sequence (no predicted signal sequence) (SEQ ID NO: 39)

FZD8-Fc variants 54F16, 54F17, 54F18, 54F23, 54F25, 54F27, 54F29, 54F31, and 54F34 amino acid sequence (no predicted signal sequence) (SEQ ID NO: 40)

FZD8-Fc variant 54F19, 54F20, 54F24, 54F26, 54F28, 54F30, 54F32, 54F34 and 54F35 amino acid sequence (no predicted signal sequence) (SEQ ID NO: 41)

FZD8-Fc variant 54F03 amino acid sequence (with signal sequence) (SEQ ID NO: 42)

FZD8-Fc variant 54F16 amino acid sequence (with signal sequence) (SEQ ID NO: 43)

FZD8-Fc variant 54F26 (with signal sequence) (SEQ ID NO: 44)

FZD8-Fc variant 54F28 (with signal sequence) (SEQ ID NO: 45)

Human Wnt1 C-terminal cysteine-rich domain (aa288-370) (SEQ ID NO: 46)

Human Wnt2 C-terminal cysteine-rich domain (aa267-360) (SEQ ID NO: 47)

Human Wnt2b C-terminal cysteine-rich domain (aa298-391) (SEQ ID NO: 48)

Human Wnt3 C-terminal cysteine-rich domain (aa273-355) (SEQ ID NO: 49)

Human Wnt3a C-terminal cysteine-rich domain (aa270-352) (SEQ ID NO: 50)

Human Wnt7a C-terminal cysteine-rich domain (aa267-359) (SEQ ID NO: 51)

Human Wnt7b C-terminal cysteine-rich domain (aa267-349) (SEQ ID NO: 52)

Human Wnt8a C-terminal cysteine-rich domain (aa248-355) (SEQ ID NO: 53)

Human Wnt8b C-terminal cysteine-rich domain (aa245-351) (SEQ ID NO: 54)

Human Wnt10a C-terminal cysteine-rich domain (aa335-417) (SEQ ID NO: 55)

Human Wnt10b C-terminal cysteine-rich domain (aa307-389) (SEQ ID NO: 56)

Linker (SEQ ID NO: 57)

Linker (SEQ ID NO: 58)

Linker (SEQ ID NO: 59)

Linker (SEQ ID NO: 60)

Linker (SEQ ID NO: 61)

CCND2 forward primer (SEQ ID NO: 62)

CCND2 reverse primer (SEQ ID NO: 63)

CCND2 probe (SEQ ID NO: 64)

CTBP2 forward primer (SEQ ID NO: 65)

CTBP2 reverse primer (SEQ ID NO: 66)

CTBP2 probe (SEQ ID NO: 67)

DKK1 forward primer (SEQ ID NO: 68)

DKK1 reverse primer (SEQ ID NO: 69)

DKK1 probe (SEQ ID NO: 70)

FBXW2 forward primer (SEQ ID NO: 71)

FBXW2 reverse primer (SEQ ID NO: 72)

FBXW2 probe (SEQ ID NO: 73)

RHOU1 forward primer (SEQ ID NO: 74)

RHOU1 reverse primer (SEQ ID NO: 75)

RHOU1 probe (SEQ ID NO: 76)

WIF1 forward primer (SEQ ID NO: 77)

WIF1 reverse primer (SEQ ID NO: 78)

WIF1 probe (SEQ ID NO: 79)

Claims (23)

A method of treating breast cancer in a patient comprising the following steps:
(a) identifying whether the patient is likely to respond to treatment with a Wnt pathway inhibitor, wherein the Wnt pathway inhibitor comprises:

Heavy chain CDR1, including

Heavy chain CDR2, including

Heavy chain CDR3, including

A light chain CDR1,

A light chain CDR2 comprising, and

An antibody that specifically binds to human FZD1, FZD2, FZD5, FZD7, and FZD8, comprising a light chain CDR3 comprising:
The identification
(i) obtaining a patient's breast cancer sample;
(ii) measuring the expression level of each biomarker of the biomarker signature in the sample, the signature comprising the biomarkers FBXW2, CCND2, RHOU, CTBP2, WIF1, and DKK1; and
(iii) identifying a patient likely to respond to treatment based on the expression level of the biomarker; and
(b) administering an effective amount of a Wnt pathway inhibitor to a patient who may respond to the treatment. A method of identifying a human breast tumor that may be responsive or non-responsive to treatment with a Wnt pathway inhibitor, wherein the Wnt pathway inhibitor comprises:

Heavy chain CDR1, including

Heavy chain CDR2, including

Heavy chain CDR3, including

A light chain CDR1,

A light chain CDR2 comprising, and

An antibody that specifically binds to human FZD1, FZD2, FZD5, FZD7, and FZD8, comprising a light chain CDR3 comprising:
The method is
(a) obtaining a sample of a breast tumor;
(b) measuring the expression level of each biomarker of the biomarker signature in the sample, the signature comprising the biomarkers FBXW2, CCND2, RHOU, CTBP2, WIF1, and DKK1; and
(c) identifying the tumor as potentially responsive or non-responsive to treatment based on the expression level of the biomarker. A method of classifying human breast tumors as potentially responsive or non-responsive to treatment with a Wnt pathway inhibitor, wherein the Wnt pathway inhibitor comprises:

Heavy chain CDR1, including

Heavy chain CDR2, including

Heavy chain CDR3, including

A light chain CDR1,

A light chain CDR2 comprising, and

An antibody that specifically binds to human FZD1, FZD2, FZD5, FZD7, and FZD8, comprising a light chain CDR3 comprising:
The method is
(a) obtaining a sample of a breast tumor;
(b) measuring the expression level of each biomarker of the biomarker signature in the sample, the signature comprising the biomarkers FBXW2, CCND2, RHOU, CTBP2, WIF1, and DKK1; and
(c) categorizing the tumor as potentially responsive or non-responsive to treatment based on expression of the biomarker. A method of determining the responsiveness of a human breast tumor to treatment with a Wnt pathway inhibitor, wherein the Wnt pathway inhibitor comprises:

Heavy chain CDR1, including

Heavy chain CDR2, including

Heavy chain CDR3, including

A light chain CDR1,

A light chain CDR2 comprising, and

An antibody that specifically binds to human FZD1, FZD2, FZD5, FZD7, and FZD8, comprising a light chain CDR3 comprising:
The method is
(a) obtaining a sample of a breast tumor;
(b) measuring the expression level of each biomarker of the biomarker signature in the sample, the signature comprising the biomarkers FBXW2, CCND2, RHOU, CTBP2, WIF1, and DKK1; and
(c) determining the responsiveness of the tumor to treatment based on expression of the biomarker. A method of identifying a patient with breast cancer that may respond to treatment with a Wnt pathway inhibitor, wherein the Wnt pathway inhibitor comprises:

Heavy chain CDR1, including

Heavy chain CDR2, including

Heavy chain CDR3, including

A light chain CDR1,

A light chain CDR2 comprising, and

An antibody that specifically binds to human FZD1, FZD2, FZD5, FZD7, and FZD8, comprising a light chain CDR3 comprising:
The method is
(a) obtaining a sample of a patient's breast tumor;
(b) measuring the expression level of each biomarker of the biomarker signature in the sample, the signature comprising the biomarkers FBXW2, CCND2, RHOU, CTBP2, WIF1, and DKK1; and
(c) identifying the patient who is likely to respond to treatment based on the expression level of the biomarker. A method of selecting patients with breast cancer for treatment with a Wnt pathway inhibitor, wherein the Wnt pathway inhibitor comprises:

Heavy chain CDR1, including

Heavy chain CDR2, including

Heavy chain CDR3, including

A light chain CDR1,

A light chain CDR2 comprising, and

An antibody that specifically binds to human FZD1, FZD2, FZD5, FZD7, and FZD8, comprising a light chain CDR3 comprising:
The method is
(a) obtaining a sample of a patient's breast tumor;
(b) measuring the expression level of each biomarker of the biomarker signature in the sample, wherein the biomarker signature comprises biomarkers FBXW2, CCND2, RHOU, CTBP2, WIF1, and DKK1; and
(c) selecting the patient for treatment based on the expression level of the biomarker.   7. The method of any one of claims 1-6, wherein the expression of each biomarker is measured by a PCR-based assay, qPCR assay, microarray, or RNA sequencing.   The expression level of FBXW2, CCND2, RHOU, CTBP2, WIF1, and DKK1 is measured using a polynucleotide selected from the group consisting of SEQ ID NOs: 62-79. The method described in 1. The expression levels of FBXW2, CCND2, RHOU, CTBP2, WIF1, and DKK1 are
(a) a probe comprising a forward primer of SEQ ID NO: 62, a reverse primer of SEQ ID NO: 63, and SEQ ID NO: 64;
(b) a forward primer of SEQ ID NO: 65, a reverse primer of SEQ ID NO: 66, and a probe comprising SEQ ID NO: 67;
(c) a probe comprising a forward primer of SEQ ID NO: 68, a reverse primer of SEQ ID NO: 69, and SEQ ID NO: 70;
(d) a forward primer of SEQ ID NO: 71, a reverse primer of SEQ ID NO: 72, and a probe comprising SEQ ID NO: 73;
(e) a probe comprising SEQ ID NO: 74 forward primer, SEQ ID NO: 75 reverse primer, and SEQ ID NO: 76;
(f) The method of claim 8, measured using a forward primer of SEQ ID NO: 77, a reverse primer of SEQ ID NO: 78, and a probe comprising SEQ ID NO: 79.   10. The method of any one of claims 1-9, wherein the Wnt pathway inhibitor is an antibody comprising a heavy chain variable region comprising SEQ ID NO: 7 and a light chain variable region comprising SEQ ID NO: 8.   10. The method of any one of claims 1-9, wherein the Wnt pathway inhibitor is an antibody comprising a heavy chain variable region and a light chain variable region encoded by a plasmid deposited with the ATCC as PTA-9541.   The antibody according to any one of claims 1 to 11, wherein the antibody is a monoclonal antibody, a recombinant antibody, a chimeric antibody, a bispecific antibody, a humanized antibody, a human antibody, or an antibody fragment comprising an antigen binding site. Method.   10. The method according to any one of claims 1 to 9, wherein the Wnt pathway inhibitor is antibody OMP-18R5.   The method according to any one of claims 1 to 13, wherein the breast tumor or breast cancer is a HER2-negative breast tumor.   14. The method according to any one of claims 1 to 13, wherein the breast tumor or breast cancer is a triple negative breast cancer (TNBC) tumor.   16. The method of any one of claims 1-15, wherein treatment with a Wnt pathway inhibitor is in combination with one or more additional therapeutic agents.   17. The method of claim 16, wherein the additional therapeutic agent is a chemotherapeutic agent.   17. The method of claim 16, wherein the additional therapeutic agent is paclitaxel.   17. The method of claim 16, wherein the additional therapeutic agent is nab-conjugated paclitaxel (ABRAXANE).   20. The method according to any one of claims 1 to 19, wherein the sample is a tissue sample or a tumor biopsy material.   21. A method according to any one of claims 1 to 20, wherein the sample is a formalin fixed paraffin embedded (FFPE) sample.   A kit for detecting FBXW2, CCND2, RHOU, CTBP2, WIF1, and DKK1 in a breast tumor sample or a breast cancer sample, comprising a polynucleotide selected from the group consisting of SEQ ID NOs: 62-79, kit. (a) a probe comprising a forward primer of SEQ ID NO: 62, a reverse primer of SEQ ID NO: 63, and SEQ ID NO: 64;
(b) a forward primer of SEQ ID NO: 65, a reverse primer of SEQ ID NO: 66, and a probe comprising SEQ ID NO: 67;
(c) a probe comprising a forward primer of SEQ ID NO: 68, a reverse primer of SEQ ID NO: 69, and SEQ ID NO: 70;
(d) a forward primer of SEQ ID NO: 71, a reverse primer of SEQ ID NO: 72, and a probe comprising SEQ ID NO: 73;
(e) a probe comprising SEQ ID NO: 74 forward primer, SEQ ID NO: 75 reverse primer, and SEQ ID NO: 76;
23. The kit of claim 22, comprising a probe comprising (f) a forward primer of SEQ ID NO: 77, a reverse primer of SEQ ID NO: 78, and SEQ ID NO: 79.

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