Compositions and methods for diagnosing and treating cancer

[0001] The invention relates to the field of oncology and provides new compositions and methods for the diagnosis and treatment of cancer. In particular, the invention provides tumor antagonists, and in particular tumor stem cell markers, including receptor fusion proteins useful for the study, diagnosis and treatment of solid tumors.

Background of the Invention [0002] Cancer is one of the leading causes of death in developed countries, causing over 500,000 deaths annually in the United States alone. Every year in the US, more than a million people are diagnosed with cancer, and it is generally estimated that more than 1 in 3 people will develop some form of cancer in their lifetime. Although there are over 200 different types of cancer, four of them - breast, lung, large intestine and prostate constitute more than half of all new cases (Jemal et al., Cancer J. Clin. 53: 5-26 (2003)).

[0003] Breast cancer is the most common cancer in women and an estimated 12% of women are at risk of developing the disease during life. Although mortality rates have decreased due to early detection and better treatment, breast cancer remains one of the main causes of death in middle-aged women. Furthermore, metastatic breast cancer is still an incurable disease. At admission, most patients with metastatic breast cancer have only one or two organ systems affected, but usually as the disease progresses, it begins to spread across multiple sites. The most common metastatic sites are local relapses in the skin and soft tissues of the chest wall, as well as in the armpit and supraclavicular areas. The most common site for distant metastases is bone (30-40% of distant metastases), followed by lungs and liver. Although only about 1-5% of women with newly diagnosed breast cancer have distant metastases at the time of diagnosis, within five years about 50% of patients with local disease eventually have a metastatic recurrence. The median survival from the appearance of distant metastases is currently about three years.

[0004] Current methods for diagnosing and determining the stage of breast cancer include a tumor-node-metastasis (TNM) system that is based on tumor size, the presence of tumors in lymph nodes, and the presence of distant metastases, as described in The American Joint Committee on Cancer, AJCC Cancer Staging Manual, Philadelphia, PA, Lippincott-Raven Publishers, 5th ed. (1997), pp. 171-180, and in Harris, JR: "Staging of breast carcinoma" in Harris, J. R., et al., Eds., Breast Diseases, Philadelphia, Lippincott (1991). These parameters are used to provide prognosis and select appropriate therapy. The morphological appearance of the tumor can also be assessed, but since tumors with a similar histopathological picture may show significant clinical variability, this approach has

-2 severe restrictions. Finally, cell surface marker tests can be used to divide certain types of tumors into subclasses. For example, one of the factors taken into account in the prognosis and treatment of breast cancer is the presence of the estrogen receptor (ER) because ER-positive breast cancers usually respond better to hormone therapies such as tamoxifen or aromatase inhibitors than ER-negative tumors. However, these analyzes, while useful, only partially predict the clinical behavior of breast tumors and there is a large phenotypic variation in breast cancers that cannot be detected by current diagnostic tools or cured by current therapies.

[0005] Prostate cancer is the most common cancer in men in developed countries and accounts for an estimated 33% of all new cancer cases in the US and is the second leading cause of death (Jemal et al., CA Cancer J. Clin. 53: 5-26 (2003) ). Since the introduction of the prostate-specific antigen (PSA) blood test, early detection of prostate cancer has significantly improved survival rates, and the five-year survival rate for patients with local or local prostate cancer at the time of diagnosis is approaching 100%. However, still more than 50% of patients eventually develop locally advanced or metastatic disease (Muthuramalingam et al., Clin. Oncol. 16: 505-516 (2004)).

[0006] Radical prostatectomy and radiation therapy currently provide effective treatment for most localized prostate tumors. However, these therapeutic options are very limited to advanced cases. For metastatic disease, androgen ablation using a luteinizing hormone releasing hormone (LHRH) agonist alone or in combination with antiandrogens is standard treatment. However, despite maximal blocking of androgens, the disease almost always progresses and in most cases the disease develops independent of androgens. There is currently no generally accepted treatment for hormone refractory prostate cancer and chemotherapeutic systems are commonly used (Muthuramalingam et al., Clin. Oncol. 16: 505-516 (2004); Trojan et al., Anticancer Res. 25: 551-561 (2005) ).

[0007] Colorectal cancer is the third most common cancer and the fourth most common cause of cancer-related deaths in the world (Weitz et al., 2005, Lancet 365: 153-65). Approximately 5-10% of all colorectal cancers are hereditary, with familial adenomatous polyposis (FAP) being the most common form, a dominant autosomal disease in which 80% of affected individuals contain germline mutations in the adenomatous colon polyp (APC) gene . Colorectal cancer has a tendency to locally invade through peripheral growth and other sites through lymphatic, haematopoietic, peritoneal and perineural spread. The most common non-lymphatic site is the liver, and the lungs are the most commonly affected organ outside the abdomen. Other sites of blood-borne spread include bones, kidneys, adrenal glands and the brain.

[0008] The current colorectal cancer assessment system is based on the extent of tumor penetration through the intestinal wall and the presence or absence of node involvement. The system of stage assessment is defined by three main Duke's classifications: Duke's disease is limited to layers

-3 submucosal colon or rectum; Duke's disease has tumors that invade through the dermis and can penetrate the wall of the colon or rectum; and Duke's C disease includes any degree of intestinal wall invasion with local lymph node metastases. Surgical resection is very effective in the early stages of colorectal cancer and provides a healing rate of 95% among patients with Duke A disease, while in patients with Duke B disease this ratio is reduced to 75%, and in the presence of positive lymph node in Duke C disease is predicted to be 60% likely to recur over five years. Treatment of patients with Duke's C disease as part of post-operative chemotherapy reduces the relapse rate to 40% -50% and is now the standard of care for these patients.

[0009] Lung cancer is the most common cancer in the world, the third most-diagnosed cancer in the United States, and by far the most common cause of cancer-related deaths (Spiro et al., Am. J. Respir. Crit. Care Med. 166: 1166-1196 (2002 ); Jemal et al., CA Cancer J. Clin. 53: 5-26 (2003)). Cigarette smoking is thought to be responsible for an estimated 87% of all lung cancer cases, making it the most deadly disease that can be avoided. Lung cancer is divided into two main types that account for over 90% of all lung cancers: small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). SCLC accounts for 15-20% of cases and is characterized by its onset in the large central airways and the histological composition of small cell layers with a small amount of cytoplasm. SCLC is more aggressive than NSCLC, it grows quickly and early and often metastasizes. NSCLC accounts for 80-85% of all cases and based on histology is further divided into three main subtypes: adenocarcinoma, squamous cell carcinoma (epidermal carcinoma) and undifferentiated large cell carcinoma.

[0010] Lung cancer usually manifests itself late in the course and therefore has a median survival of only 6-12 months after diagnosis and an overall 5-year survival rate of only 5-10%. Although surgery offers the best chance of recovery, only a small fraction of lung cancer patients are eligible, and most are treated with chemotherapy and radiation. Despite attempts to manipulate time and intensity of dosing in these therapies, survival rates have increased slightly over the past 15 years (Spiro et al., Am. J. Respir. Crit. Care Med. 166: 1166-1196 (2002)).

[0011] Cancer arises from dysregulation of mechanisms that control normal tissue development and persistence, and stem cells are increasingly thought to play a major role (Beachy et al., Nature 432: 324 (2004)). During the normal development of the animal, cells of most or all tissues arise from normal precursors called stem cells (Morrison et al., Cell 88: 287298 (1997); Morrison et al., Curr. Opin. Immunol. 9: 216-221 (1997); Morrison et al., Annu. Rev. Cell. Dev. Biol. 11: 35-71 (1995)). Stem cells are cells that: (1) have extensive proliferative capacity; (2) are capable of asymmetric cell division to produce one or more types of progeny with reduced proliferative and / or developmental potential; and (3) are capable of

-4 symmetrical cell divisions for self-renewal or self-retention. The best known example of renewing adult cells by differentiating stem cells is a hematopoietic system in which immature precursors (hematopoietic stem and progenitor cells) respond to molecular signals and create different types of blood cells and lymphoid cells. Other cells, including the cells of the intestine, breast and skin ducts, are constantly replenished with a small population of stem cells in each tissue, and recent studies suggest that most other adult tissues are also a stem cell habitat, including the brain.

[0012] Solid tumors are composed of heterogeneous cell populations. For example, breast cancers are a mixture of cancer cells and normal cells, including mesenchymal (stromal) cells, inflammatory cells and endothelial cells. According to classical tumor models, all phenotypically different tumor cell populations have the ability to proliferate and cause a new tumor to form. In the classical model, heterogeneity of tumor cells results from environmental factors, as well as from mutations occurring in tumor cells, which results in a diverse population of tumorigenic cells. This model is based on the belief that all tumor cell populations have the same degree of tumorigenic potential (Pandis et al., Genes, Chromosomes & Cancer 12: 122-129 (1998); Kuukasjrvi et al., Cancer Res. 57: 15971604 (1997); Bonsing et al., Cancer 71: 382-391 (1993); Bonsing et al., Genes Chromosomes & Cancer 82: 173-183 (2000); Beerman H. et al., Cytometry. 12: 147-154 (1991); Aubele M & Werner M, Analyst. Cell. Path. 19:53 (1999); Shen Let al., Cancer Res. 60: 3884 (2000)).

[0013] An alternative model of the observed heterogeneity of solid tumor cells is that solid tumors arise from a "solid tumor stem cell" (or "solid tumor cancer stem cell") that then develops chaotically through rounds of both symmetrical and asymmetrical divisions phones. In this stem cell model, solid tumors contain a distinctive and limited (possibly even rare) subset of cells that have the properties of normal "stem cells" in that they proliferate extensively and efficiently produce both additional solid tumor stem cells (self-renewing) and most non-tumorigenic solid tumor cells. Indeed, mutations in long-lived stem cell populations can initiate the formation of cancer stem cells that underpin the growth and persistence of tumors and whose presence contributes to the failure of current therapeutic approaches.

[0014] The properties of cancer stem cells were first disclosed for blood cancer - acute myeloid leukemia (AML) (Lapidot et al., Nature 17: 645-648 (1994)). Recently, human malignant breast tumors have been shown to similarly contain a small distinguishable population of cancer stem cells richer in their ability to form tumors in immunodeficient mice. The populations of ESA +, CD44 +, CD24- / low, Lin- cells were found to be 50 times richer in tumor cells compared to unfractionated tumor cells (Al-Hajj et al., PNAS

-5100: 3983-3988 (2003)). The ability to prospectively isolate tumorigenic cancer cells enables the study of key biological pathways that underpin tumorigenicity in these cells, and thus provides the opportunity to develop better diagnostic tests and therapeutic measures for cancer patients. The invention is geared precisely for this purpose.

[0015] US2002 / 0137129 discloses the use of FZD4 in fusion with Fc for therapy.

BRIEF DESCRIPTION OF THE INVENTION [0016] The invention provides a pharmaceutical composition comprising a soluble receptor and a pharmaceutically acceptable carrier, excipient and / or stabilizer, wherein the amino acid sequence of the soluble receptor consists of residues 28 to 158 of SEQ ID NO: 7 linked to a non-sequence sequence FZD receptor, wherein the non-FZD receptor sequence comprises human Fc.

[0017] The invention also provides the claimed composition for use in a method of therapy of the human body by means of as set out in claim 4, and for use in a method of treatment of cancer as set out in any of claims 5 to 7.

[0018] Examples of solid tumors that can be treated using the therapeutic composition of the invention include but are not limited to sarcomas and cancers such as, but not limited to: fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, osteosarcoma, angiosarcoma, endothelial sarcoma, lymphosarcoma, lymphosarcoma, lymphoma, myosarcoma, leiomyosarcoma, leiomyosarcoma, fibrosarcoma , prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland cancer, sebaceous gland cancer, papillary carcinoma, papillary adenocarcinomas, cystic adenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct cancer, chorionic malignant, seminoma, germ cell carcinoma, Wilms tumor, cervical cancer, testicular tumor, lung cancer, small cell cancer urinary tract cancer, epithelial carcinoma, glioma, astrocytoma, spinal cord, cranial scleroma, ependymoma, pineal gland, embryonic hemangioma, auditory neuroma, oligoma, meningioma, melanoma, neuroblastoma, and retinoblastoma. The invention is applicable to sarcomas and epithelial cancers such as ovarian and breast cancers.

BRIEF DESCRIPTION OF THE DRAWINGS / FIGURES [0019]

FIG. 1: Half-life of soluble FZD.Fc receptors. Each of the purified Fc fusion proteins was administered ip to 2 mice and blood samples were taken at different times after administration. The FZD4 Fri.Fc, FZD5 Fri.Fc and FZD8 Fri.Fc proteins are still present in the blood serum 72 hours after injection, and the FZD5 Fri.Fc and FZD8 Fri.Fc proteins are present in the blood serum up to 96 hours after administration. For comparison, FZD5 BCD.Fc is undetectable in serum after 24 hours (top).

-6FIG. 2: Soluble FZD Fc receptors inhibit Wnt3a signaling. Increasing concentrations (2 nM, 5 nM and 60 nM) of FZD Fc fusion proteins, including FZD4 Fri.Fc, FZD5 ECD.Fc, FZD5 Fri.Fc and FZD8 Fri.Fc, were incubated with L cells in the presence or absence of ligand Wnt3a and β-catenin stabilization was determined by immunoblotting. Β-catenin (LCM) could not be detected in the absence of the Wnt3a ligand. In the presence of Wnt3a, β-catenin was stabilized and this stabilization was blocked by increasing amounts of soluble FZD5, FZD8 and FZD4 Fc receptor protein, but not by the control Fc protein (Kont Fc).

FIG. 3: Soluble FZD Fc receptors inhibit Wnt signaling. Hek 293 cells stably transfected with the 8xTCF-luciferase reporter were incubated with increasing concentrations of soluble FZD: Fc receptors in the presence of various Wnt ligands, including Wnt1, Wnt2, Wnt3, Wnt3a and Wnt7b. FZD4 Fc, FZD5 Fc and FZD8 Fc fusion proteins inhibited Wnt signaling dependent on all five Wnt ligands, as indicated by a loss of luciferase activity.

FIG. 4: Reduction of tumor growth by soluble FZDFc receptor proteins. NOD / SCID mice injected subcutaneously with dissociated colon tumor cells (10,000 cells per animal; n = 10) two days later were administered soluble FZD7ECD.Fc receptor, soluble FZD10ECD.Fc receptor or control injections. Total tumor volume was shown for days 21, 24, 28 and 30. The reduction in tumor volume by FZD7ECD.Fc was statistically significant on day 28 and day 30 (*).

FIG. 5: Prevention of Wnt-dependent tumor growth by soluble FZD8 receptor protein Fri.Fc. NOD / SCID mice injected with 50,000 cells derived from MMTV WNT1 tumor (n = 10) were administered the next day with soluble FZD8 Fri.Fc or PBS receptor as a control. Tumor growth was monitored weekly until growth was detected, then tumor growth was measured twice a week. Tumor growth in FZD Fri.Fc-treated animals (left bar) was properly suppressed compared to that observed in control animals (right bar).

FIG. 6: Reduction of PE13 tumor xenograft growth by soluble FZD8 receptor protein Fri.Fc. NOD / SCID mice injected with 50,000 PE13 breast tumor cells (n = 10) were administered the next day with soluble FZD8 Fri.Fc or PBS receptor as a control. Tumor growth was monitored weekly until growth was detected, then tumor growth was measured twice a week. Tumor growth in FZD Fri.Fc-treated animals (left bar) was significantly smaller compared to that observed in control animals (right bar).

FIG. 7: Treatment of Wnt-dependent tumor growth by soluble FZD receptor protein Fri.Fc. Female rag-2 / γ chain double knockout mice were implanted with 50,000 cells derived from MMTV Wnt1 breast tumor. Treatment with 5 mg / kg FZD8 Fri.Fc reduced tumor growth, as measured by total tumor volume over time, relative to PBS-treated mice (white bars). Treatment with 10 mg / kg and 30 mg / kg FZD8 Fri.Fc yet

Was more effective in reducing the size of previously developed tumors. In contrast, FZD5 Fri.Fc showed no anti-tumor activity against developed breast tumors that require Wnt1 for growth.

DETAILED DESCRIPTION OF THE INVENTION

Definitions [0020] The term "antagonist" herein includes any molecule that partially or completely blocks, inhibits or neutralizes the expression or biological activity of a cancer stem cell marker disclosed herein, and such biological activity includes, but is not limited to, inhibition of tumor growth. The term "antagonist" includes any molecule that partially or completely blocks, inhibits or neutralizes the biological activity of the FZD. Suitable antagonist molecules include, but are not limited to, fragments or variants of the amino acid sequence of native FZD receptor proteins, including soluble FZD receptors.

[0021] The terms "isolated" or "purified" refer to material that is substantially or substantially free of components that normally accompany it in its native state. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. The protein (e.g. soluble receptor) or nucleic acid that dominates the preparation is essentially purified. In particular, the isolated nucleic acid is separated from open reading frames that naturally flank a given gene and encode proteins other than the protein encoded by that gene. The isolated antibody is separated from other non-immunoglobulin proteins and from other immunoglobulin proteins with different antigen binding specificity. It may also mean that the nucleic acid or protein is at least 85% pure, at least 95% pure, and in some embodiments at least 99% pure.

[0022] The terms "soluble receptor" and "soluble FZD receptor" refer to the N-terminal extracellular fragment of the human FZD receptor protein preceding the first transmembrane domain of the receptor that can be secreted from the cell in a soluble form. Soluble FZD receptors containing the entire N-terminal extracellular domain (ECD) (referred to herein as "FCD ECD") and smaller fragments are predicted. Further disclosed are soluble FZD receptors containing the Fri domain (referred to herein as "FZD Fri"). Soluble FZD Fri receptors may exhibit altered biological activity (e.g., increased half-life of protein) compared to soluble receptors containing all FZD ECD. The half-life of the protein can be further extended by covalent modifications with poly (ethylene glycol) or poly (ethylene oxide (both referred to as PEG). Soluble FZD receptors include FZD ECD or Fri domains fused in the same reading frame with other functional and structural proteins including but not limited to human Fc (e.g., human Fc derived from IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE, IgE ); protein tags (e.g. myc, FLAG, GST); other endogenous proteins or protein fragments; or

Any other useful protein sequences, including any linker region between the FZD ECD or Fri domain and the attached protein. In some embodiments, the FZD receptor Fri domain is linked to human IgG1 Fc (referred to herein as "FZD Fri.Fc").

Soluble FZD receptors also include proteins with insertions, deletions, substitutions and conservative amino acid changes, etc.

[0023] The terms "cancer" and "cancerous" mean or describe the physiological condition in mammals in which the cell population is characterized by uncontrolled cell growth. Examples of cancers include but are not limited to cancer, lymphoma, germ cell tumor, sarcoma and leukemia. More specific examples of such cancers include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma and squamous cell lung cancer, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioma, cervical cancer, ovarian cancer, liver cancer, cancer bladder, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer and various types of head and neck cancer.

[0024] The terms "proliferative disorder" or "proliferative disease" means disorders associated with abnormal cell proliferation, such as cancer.

[0025] "Tumor" herein refers to any tissue mass that results from excessive cell growth or proliferation, benign or malignant, including pre-cancerous lesions.

[0026] "Metastasis" refers here to a process that results in the cancer spreading or moving from its origin to other regions of the body with the development of similar cancerous lesions at a new location. A "metastatic" or "metastatic" cell is one that loses adhesive contact with neighboring cells and migrates through the bloodstream or lymph from the original disease location and invades adjacent body structures.

[0027] The term "subject" herein means any animal (e.g., mammal), including but not limited to humans, non-human primates, rodents and the like that is to receive specific treatment. Typically, the terms "subject" and patient "are used interchangeably herein to refer to a human subject.

[0028] The terms "cancer stem cell", "tumor stem cell" or "solid tumor stem cell" are used interchangeably herein and refer to a population of solid tumor cells that: (1) have extensive proliferative capacity; (2) are capable of asymmetric cell division to produce one or more types of differentiated progeny with reduced proliferative or developmental potential; and (3) are capable of symmetrical cell division for self-renewal or self-retention. The properties of "cancer stem cells", "tumor stem cells" or "solid tumor stem cells" give these cancer stem cells the ability to form palpable tumors when serially transplanting mice with reduced immunity in

-9 compared to most tumor cells that fail to form tumors. Tumor cells, i.e. non-tumorigenic cells, may form a tumor a limited number of times (e.g., one or two times) after obtaining tumor cells from solid tumor, but will not maintain the ability to form palpable tumors in serial transplantation of immunocompromised mice. Cancer stem cells undergo self-renewal or differentiation in a chaotic manner to form tumors with abnormal cell types that can change over time due to mutations. Stem cells of the solid tumors of the invention differ from the "stem tumor line" provided in Pat. USA No. 6,004,528. In this patent, the "parent tumor line" is defined as a type of slow-growing progenitor cell that itself contains several mutations, but which undergoes symmetrical rather than asymmetrical cell divisions as a result of tumorigenic changes that occur in the cell environment. The "parent tumor line" hypothesis therefore suggests that highly mutated, rapidly proliferating tumor cells arise mainly due to an abnormal environment that causes relatively normal stem cells to accumulate and then undergo mutations that cause them to become tumor cells. In Pat. No. 6,004,528 has been proposed to use such a model to improve cancer diagnosis. The solid tumor stem cell model differs fundamentally from the "parent tumor line" model and, as a result, shows utility not provided by the "parent tumor line" model. First, solid tumor stem cells are not "mutatively spared". "Mutationally spared parental tumor line" described in Pat. US No. 6,004,528 can be considered a precancerous lesion, while the solid tumor stem cells described in this invention are cancer cells that themselves contain mutations that are responsible for tumorigenesis. That is, the solid tumor stem cells ("cancer stem cells") of the invention would belong to the highly mutated cells that distinguish themselves from the "cancer stem line" of Pat. USA No. 6,004,528. Secondly, genetic mutations that lead to cancer can be mostly native to solid tumor stem cells, as well as environmental. The solid tumor stem cell model predicts that isolated solid tumor stem cells can cause additional tumors after transplantation (which explains metastasis), while the "parent tumor line" model would predict that transplanted "cancer stem line" cells would not be able to cause a new tumor to form. tumor because their abnormal environment was tumorigenic. Indeed, the possibility of transplanting dissociated and phenotypically isolated human solid tumor stem cells into mice (into an environment that is very different from the normal tumor environment), where they still form new tumors, sets the invention apart from the "parent tumor line" model. Third, solid tumor stem cells probably divide both symmetrically and asymmetrically, with symmetrical cell division not an obligatory property. Fourthly, solid tumor stem cells can divide quickly or slowly, depending on many variables, with a slow proliferation rate not a distinguishing feature.

[0029] The terms "cancer cell", "tumor cell" and their grammatical equivalents refer to the entire population of tumor-derived cells, including both non-tumorigenic cells that constitute a large proportion of the tumor cell population and tumorigenic stem cells, called here also cancer stem cells.

[0030] "Tumorogenic" refers here to the functional properties of a solid tumor stem cell, including self-renewing (causing the formation of additional tumorigenic cancer stem cells) and proliferation to produce all other tumor cells (causing the formation of differentiated and therefore non-tumorigenic cells), which allow solid tumor stem cells to form a tumor. These self-renewing and proliferative properties to produce all other tumor cells ensure that these cancer stem cells of the invention are able to form palpable tumors in serial transplantation in immunocompromised mice compared to most tumor cells that are unable to form tumors in serial transplantation. Tumor cells, i.e. non-tumorigenic tumor cells, when transplanted, immunocompromised mice may form a tumor a limited number of times (e.g., once or twice) after obtaining tumor cells from a solid tumor.

[0031] The terms "cancer stem cell marker (s)", "cancer stem cell marker (s)", "tumor stem cell marker (s)" or "solid tumor stem cell marker (s)" refer to the gene or genes or a protein, polypeptide or peptide expressed by a gene or genes whose expression level, alone or in combination with other genes, is correlated with the presence of tumorigenic cells compared to non-tumorigenic cells. Correlation may refer to increased or decreased gene expression (e.g., increased or decreased levels of mRNA or gene encoded peptide).

[0032] The terms "biopsy" and "tissue biopsy" refer to a tissue or fluid sample that is taken from an individual to determine if the sample contains tumor tissue. In some embodiments, a tissue biopsy or fluid is taken because the subject is suspected of having cancer. A tissue or fluid biopsy is then examined for the presence or absence of cancer.

[0033] "Acceptable pharmaceutical carrier" herein means any material which, when combined with an active ingredient of a pharmaceutical composition, such as an antibody, allows the antibody to, for example, maintain its biological activity. In addition, the "acceptable pharmaceutical carrier" does not elicit an immune response in the individual to whom it is administered. Examples include, but are not limited to, any of the standard pharmaceutical carriers such as phosphate buffered saline, water and various oil-in-water emulsions. Examples of diluents for aerosol or parenteral administration are phosphate buffered saline or saline (0.9%).

[0034] The term "therapeutically effective amount" refers to the amount of a soluble receptor or other drug effective in "treating" a disease or disorder in an individual or mammal. In the case of cancer, a therapeutically effective amount of the drug may reduce the number of cancer cells; reduce tumor size; inhibit or stop the penetration of cancer cells into peripheral organs; inhibit or stop tumor metastasis; inhibit and stop tumor growth; and / or ameliorate one or more of the symptoms associated with the cancer to some extent. To some extent, the drug prevents growth and / or kills existing cancer cells; it may be called a cytostatic and / or cytotoxic drug.

[0035] The term "inhibiting tumor growth" refers herein to any mechanism by which tumor cell growth may be inhibited. In some embodiments, tumor cell growth is inhibited by slowing tumor cell proliferation. In some embodiments, tumor cell growth is inhibited by stopping tumor cell proliferation. In some embodiments, tumor cell growth is inhibited by killing tumor cells. In some embodiments, tumor cell growth is inhibited by inducing tumor cell apoptosis. In some embodiments, tumor cell growth is inhibited by depriving the tumor cells of nutrients. In some embodiments, tumor cell growth is inhibited by preventing tumor cell migration. In some embodiments, tumor cell growth is inhibited by preventing tumor cell invasion.

[0036] "Providing a diagnosis" or "diagnostic information" refers to any information that is useful in determining whether a patient has a disease or condition, and / or when classifying a disease or condition into a phenotypic category or any category relevant to forecasting or likely response to treatment (general treatment or any specific treatment) of the disease or condition. Similarly, diagnosis refers to the provision of any type of diagnostic information including, but not limited to, whether an individual may have a condition (such as a tumor), information related to the properties or classification of a tumor, such as, for example, a high-risk or low-risk tumor, information related to forecast and / or information useful when selecting the appropriate treatment. Choosing treatment may include choosing a particular chemotherapeutic agent or other therapeutic method, such as surgery or radiation, or deciding whether to withhold or provide therapy.

[0037] The terms "providing prognosis", "prognostic information" or "predictive information" as used herein refer to providing information regarding the effect of the presence of cancer (e.g., as determined by the diagnostic methods of the invention) on the future health of the subject (e.g., predicted morbidity or mortality, cancer probability and metastasis risk).

[0038] The terms "treating", "treating", "treating", "soothing", "alleviating" and "ameliorate" refer to both 1) therapeutic agents that treat, slow down, reduce symptoms and / or stop the progression diagnosed pathological condition or disorder, and 2) preventive or preventive measures that prevent or

-12 slow down the development of a particular pathological condition or disorder. Those in need of treatment therefore include those already suffering from the disorder; those prone to disturbance; and those in whom the disorder should be prevented. The subject is effectively "treated" according to the methods of the invention if the patient exhibits one or more of the following: reduction in number or total absence of cancer cells; reduction in tumor size; inhibiting or not penetrating cancer cells into peripheral organs, including the spread of the cancer to soft tissue and bone; inhibition or absence of tumor metastasis; inhibition or absence of tumor growth; resolution of one or more symptoms associated with the specific cancer; reduced morbidity and mortality; and improving the quality of life.

[0039] The terms "polynucleotide" and "nucleic acid" mean a polymer composed of many nucleotide units (ribonucleotides or deoxyribonucleotides or related structural variants) linked by phosphodiester linkages, including, but not limited to, DNA or RNA. The term includes sequences that include any of the known DNA and RNA base analogues including but not limited to 4-acetylcytosine, 8-hydroxy-N6-methyloadenosine, aziridinylcytosine, pseudoisocytosine, 5- (carboxyhydroxylmethyl) uracil, 5-fluorouracil, 5-bromouracyl, 5-carboxylacetyl 5-carboxymethylaminomethyluracil, dihydrouracil, inosine, N6-isopentenyloadenine, 1-methyloadenine, 1-methylpseudouracil, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyloadenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyloadenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylcososine, 5'-methoxycarbonyl-2-methyluracyl methylthio-N6-isopentenyl adenine, uracyl-5-oxoacetic acid methyl ester, uracyl-5-oxoacetic acid, oxybutoxosine, pseudouracil, quesosine, 2-thiocytosine, 5-methyl-2-thiouracyl, 2-thiouracyl, 4-thiouracyl methyluracil, N-uracyl-5-oxyacetic acid methyl ester, uracyl-5-oxyacetic acid, pseudouracil, quweosine, 2-thiocytosine and 2,6-diaminopurine.

[0040] The term "gene" means a nucleic acid molecule (e.g., DNA) that contains coding sequences necessary for the production of a polypeptide, precursor or RNA (e.g., rRNA, tRNA). The polypeptide may be encoded by the full-length coding sequence or by any part of the coding sequence as long as the desired activity or functional properties (e.g. enzymatic activity, ligand binding, signal transduction, immunogenicity etc.) of the full length or fragment are maintained. The term further encompasses coding regions of the structural gene and sequences adjacent to the coding region, both at the 5 'and 3' end, at about 1 kb or longer than any end, such that the gene corresponds to the length of full length mRNA. Sequences lying 5 'to the coding region and present in the mRNA are called 5' untranslated sequences. Sequences lying 3 'to or below the coding region and present in the mRNA are called 3' untranslated sequences. The term "gene" includes both cDNA and genomic forms of the gene. The genomic form or clone of the gene contains a coding region interrupted by non-coding sequences called "introns" or "interruption regions," or "interruption sequences." Introns are episodes

- genes that are transcribed into nuclear RNA (hnRNA); introns may contain regulatory elements such as enhancers. Introns are removed or "excised" from the nuclear or primary transcript; introns are therefore absent in the messenger RNA transcript (mRNA). During translation, mRNA is used to determine the sequence or order of amino acids in the nascent polypeptide. In addition to introns, the genomic form of the gene may also contain sequences at both the 5 'and 3' ends of the sequence that are present in RNA transcription. These sequences are called "flanking" sequences or regions (these flanking sequences are 5 'or 3' away from the untranslated sequences present in the mRNA transcript). The 5 'flanking region may contain regulatory sequences, such as promoters and enhancers, that control or affect gene transcription. The 3 'flanking region may contain sequences that control transcription termination, post-translational cleavage and polyadenylation.

[0041] The term "recombinant", when used in reference to a cell, nucleic acid, protein or vector, indicates that the cell, nucleic acid, protein or vector has been modified by introducing a heterologous nucleic acid or protein, altering the native nucleic acid or protein or that the cell is derived from such a modified cell. So e.g. recombinant cells express genes that are not found in the native (non-recombinant) form of the cell, or express native genes that are overexpressed or expressed in other non-usual ways, such as, for example, expressed as non-naturally occurring fragments or splice variants. The term "recombinant nucleic acid" herein means a nucleic acid originally created in vitro, generally by manipulating nucleic acid e.g. using polymerases and endonucleases, in a form not normally found in nature. In this way, an operational combination of different sequences is achieved. For the purposes of the invention, an isolated nucleic acid molecule in linear form or an expression vector formed in vitro by ligation of DNA molecules that are not normally linked are recombinant. It is understood that when a recombinant nucleic acid molecule is formed and introduced into a host cell or organism, it will replicate non-recombinantly, i.e. in vivo using cellular machinery machinery of the host cell and not in vitro treatments; however, such recombinantly produced nucleic acids, although subsequently replicating non-recombinantly, are still considered recombinant for the purposes of the invention. Similarly, "recombinant protein" is a protein made using recombinant techniques, i.e., by expressing a recombinant nucleic acid molecule as set out above.

[0042] "Heterologous gene" here means a gene that is not in its natural environment. For example, a heterologous gene includes a gene from one species introduced into another species. The heterologous gene also includes a native gene that has been somehow altered (e.g., mutated, added in multiple copies, linked to non-native regulatory sequences, etc.). Heterologous genes differ from endogenous genes in that the sequences of the heterologous genes are usually attached to DNA sequences that are not normally associated with the sequences of these genes on

Chromosome, or are associated with parts of the chromosome not found in nature (e.g., genes expressed in loci where the gene is not normally expressed).

[0043] The term "vector" is used herein to refer to nucleic acid molecules that transfer DNA segment (s) from one cell to another. The term "carrier" is sometimes used interchangeably with "vector". Vectors are often derived from plasmids, bacteriophages or plant or animal viruses.

[0044] "Ligation" means the process of forming phosphodiester linkages between two double-stranded nucleic acid fragments. Unless otherwise specified, ligation can be performed using known buffers and conditions using 10 units of T4 DNA ligase ("ligase") per 0.5 μg of ligation intended for DNA fragments present in approximately equimolar amounts. Nucleic acid ligation can be used to combine two proteins in one reading frame to produce one protein or fusion protein.

[0045] The terms "gene expression" or "gene expression" refers to the process of converting gene encoded gene information into RNA (e.g., mRNA, rRNA, tRNA or snRNA) by "transcribing" the gene (e.g., by the enzymatic action of RNA polymerase) , and in the case of genes encoding proteins - into protein, by "translation" of mRNA. Gene expression can be regulated in many stages in the process. "Upregulation" or "activation" refers to regulation that increases the production of gene expression products (e.g., RNA or protein), while "downregulation" or "repression" refers to regulation that reduces production. Molecules (e.g., transcription factors) that bind to up-regulation or down-regulation are often referred to as "activators" or "repressors," respectively.

[0046] The terms "polypeptide", "peptide", "protein"

and "protein fragment" is used interchangeably herein to refer to a polymer of amino acid residues. The term refers to amino acid polymers in which one or more amino acid residues is an artificial chemical mimetic of the corresponding naturally occurring amino acid, as well as to polymers of naturally occurring amino acids and non-naturally occurring amino acids.

[0047] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, gamma-carboxyglutamate and O-phosphoserine. Amino acid analogues refer to compounds that have the same basic chemical structure as the naturally occurring amino acid, e.g. alpha carbon, which is bonded to hydrogen, hydroxyl group, amino group and R group, e.g. homoserine, norleucine, methionine sulfoxide, methylsulfonium [derivative] methionine. Such analogues may have modified R groups (e.g. norleucine) or modified peptide backbones, but retain the same basic chemical structure as the naturally occurring amino acid. mimetics

-15 amino acids refer to chemical compounds that have a structure that differs from the overall chemical structure of amino acids, but which act similarly to a naturally occurring amino acid.

[0048] "Conservatively modified variants" refers to both amino acid and nucleic acid sequences. "Amino acid variants" refer to amino acid sequences. With respect to specific nucleic acid sequences, conservatively modified variants refer to those nucleic acids that encode identical or substantially identical amino acid sequences, or - where the nucleic acid does not encode the amino acid sequence - to substantially identical or associated (e.g., naturally adjacent) sequences . Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode most proteins. For example, the codons GCA, GCC, GCG and GCU encode the amino acid alanine. Thus, at any position where alanine is designated by a codon, the codon can be replaced with another of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid changes are "silent changes", which are one of the varieties of conservatively modified changes. Any nucleic acid sequence that encodes a polypeptide also describes silent nucleic acid changes herein. One skilled in the art will recognize that in certain contexts, any codon in a nucleic acid (except AUG, which is normally the only methionine codon, and TGG, which is normally the only codon of tryptophan) can be modified to obtain a functionally identical molecule. Accordingly, the silent changes of the nucleic acid that encodes the polypeptide are obvious in the described sequence with respect to the expression product, but not with respect to actual probe sequences. Regarding amino acid sequences, one of ordinary skill in the art will know that individual substitutions, deletions or additions in a nucleic acid, peptide, polypeptide or protein sequence that change, add or remove a single amino acid or a small percentage of amino acids in the coded sequence form a "conservatively modified variant" involving a situation in which a change results in an amino acid substitution with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are additional to and do not exclude polymorphic variants, interspecies homologs and alleles of the invention. Conservative substitutions typically include: 1) alanine (A), glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) cysteine ​​(C), methionine (M) (see e.g. Creighton, Proteins (1984)).

[0049] The term "epitope tag / tag" herein refers to a chimeric polypeptide comprising a cancer stem cell marker protein, or a domain sequence or portion thereof, fused to an "epitope tag / tag". The epitope tag / tag polypeptide contains a sufficient number of amino acid residues to provide the epitope for recognition by the antibody, and at the same time is short enough not to interfere with the activity of the cancer stem cell marker protein.

[0050] "About" refers to plus or minus 10% of the indicated number / quantity. For example, "about 10%" indicates a range from 9% to 11%.

Detailed Description [0051] Compositions and methods for researching, diagnosing, characterizing, and treating cancer are described herein. In particular, solid tumor stem cell marker antagonists and methods for using these antagonists to inhibit tumor growth and treat cancer in human patients are described. Antagonists include soluble receptor proteins including cancer stem cell markers.

[0052] In some embodiments, the invention provides a pharmaceutical composition comprising a soluble receptor as defined in the appended claim 1.

[0053] The composition may be provided for use in a method of treatment of cancer as described herein.

[0054] The method of treating cancer may comprise administering a soluble receptor in an amount effective to inhibit tumor cell growth.

[0055] The method of treating cancer may comprise administering a soluble receptor in an amount effective to inhibit tumor cell growth in combination with radiation therapy. The method of treating cancer may comprise administering a soluble receptor in an amount effective to inhibit tumor cell growth in combination with chemotherapy. The method of treating cancer may comprise administering a soluble receptor in an amount effective to inhibit the growth of tumor cells from a breast tumor, colorectal tumor, lung tumor, pancreatic tumor, prostate tumor, or head and neck tumor.

Stem cells and stem cells of solid tumor [0056] The most common cancers arise in tissues that contain a subpopulation of proliferating cells that are responsible for replenishing short-lived mature cells. In such organs, cell maturation is organized hierarchically, where a rare population of stem cells gives rise to more diverse cells and is maintained through a process called self-renewal (Akashi & Weissman, Developmental Biology of Hematopoiesis, Oxford Univ. Press, NY (2001); Spangrude et al. , Science 241: 5861 (1988); Baum et al., PNAS 89: 2804-2808 (1992); Morrison et al., PNAS 92: 10302-20306 (1995); Morrison et al., Immunity 5: 207-216 (1996); Morrison et al., Annu. Rev. Cell dev. Biol. 11: 35-71 (1995); Morrison et al., Dev. 124: 1929-1939 (1997); Morrison & Weissman, Immunity 1: 661. (1994); Morrison et al., Cell 88: 287-298 (1997); Uchida et al., PNAS 97: 14720-14725 (2000); Morrison et al., Cell 101: 499-510 (2000)). Although it is likely that most tissues contain stem cells, because of their rarity, these cells

Have been rigorously identified and purified for testing their biological, molecular and biochemical properties in only a few tissues. The best characterized stem cells are those that give rise to the hematopoietic system, called hematopoietic stem cells (HSCs). HSC has been shown to be useful in cancer therapy by extensive use in bone marrow transplantation to regenerate the blood-lymphatic system after myeloablative protocols (Baum et al., Bone Marrow Transplantation, Blackwell Scientific Publications, Boston (1994)). Understanding the cellular biology of tissues in which cancers arise, and in particular stem cells found in these tissues, provides a new perspective on cancer biology.

[0057] Like the tissues from which they originate, solid tumors consist of a heterogeneous population of cells. The fact that most of these cells are not tumorigenic suggests that the development and maintenance of solid tumors is also based on a small population of stem cells (i.e. tumorigenic cells) with the ability to proliferate and successfully give rise to additional tumor stem cells (self-renewing) and most of the more diverse tumor cells that have no tumorigenic potential (i.e. non-tumorigenic cells). The idea of ​​cancer stem cells was first introduced shortly after the discovery of HSC and was established experimentally in acute myeloid leukemia (AML) (Park et al., J. Natl. Cancer Inst. 46: 411-422 (1971); Lapidot et al., Nature 367: 645-648 (1994); Bonnet & Dick, Nat. Med. 3: 730-737 (1997); Hope et al., Nat. Immunol. 5: 738-743 (2004)). Stem cells from solid tumors have recently been isolated based on their unique expression pattern of surface cell receptors and on the evaluation of their self-renewal and proliferative properties in culture in animal xenograft models. The population of ESA +, CD44 +, CD24- / low Lineage- with 50-fold higher tumor formation compared to unfractionated tumor cells was detected (Al-Hajj et al., PNAS 100: 3983-3988 (2003)). The ability to isolate tumor stem cell tumors from non-tumor cell mass has led to the identification of tumor stem cell markers, genes with different expression in cancer stem cells compared to tumor-free tumor cells or normal breast epithelium using microarray analysis. The invention uses knowledge of these identified cancer stem cell markers to study, characterize, diagnose and treat cancer.

Cancer stem cell marker protein [0058] Normal stem cells and cancer stem cells have the ability to proliferate and self-renew, so it is not surprising that a number of genes that regulate the development of normal stem cells contribute to tumorigenicity (discussed in Reya et al., Nature 414: 105-111 (2001) and Taipale & Beachy, Nature 411: 349-354 (2001). The invention identifies Fzd receptors, including, for example, Fzd4, Fzd5 and Fzd8 as markers of cancer stem cells and implies that it is the Wnt signaling pathway

Is involved in the maintenance of cancer stem cells, and is indicated as a target in the treatment of cancer by eliminating these tumorigenic cells.

[0059] The Wnt signaling pathway is one of several key regulators of embryonic pattern formation, embryonic tissue persistence, and stem cell biology. More particularly, Wnt signaling plays an important role in producing cell polarity and determining cell destination, including self-renewal using a stem cell population. Uncontrolled activation of the Wnt pathway is associated with many human cancers in which it can alter the development of tumor cells to maintain them in an undifferentiated and proliferative state. Carcinogenesis can therefore occur by taking over homeostatic mechanisms that control the normal development and repair of tissues by stem cells (discussed in Reya & Clevers, Nature 434: 843 (2005); Beachy et al., Nature 432: 324 (2004)).

[0060] The Wnt signaling pathway was first explained in the wingless developmental mutant (by) Drosophila and based on the mouse prot-oncogene int-1, now Wnt1 (Nusse & Varmus, Cell 31: 99-109 (1982); Van Ooyen & Nusse, Cell 39 : 233-240 (1984); Cabrera et al., Cell 50: 659-663 (1987); Rijsewijk et al., Cell 50: 649-657 (1987)). Wnt genes encode secreted lipid-modified glycoproteins, 19 of which have been identified in mammals. These secreted ligands activate a receptor complex consisting of a member of the Frizzled (Fzd) family of receptors and a 5 or 6 (LPR5 / 6) protein associated with the low density lipoprotein (LDL) receptor. Fzd receptors are seven proteins with transmembrane domains from the G protein-coupled receptor superfamily (GPCR) and they contain a large extracellular N-terminal ligand-binding domain with conserved cysteines, known as the cysteine-rich domain (CRD) or the Fri domain. There are ten human FZD receptors: FZD1-10. Different CRDs from Fzd have different binding affinities for specific Wnt (Wu & Nusse, J. Biol. Chem. 277: 41762-41769 (2002)) and Fzd receptors are divided into those that activate the canonical β-catenin pathway, and those that activate the non-canonical pathways described below (Miller et al., Oncogene 18: 7860-7872 (1999)). LRP5 / 6 are single transmembrane proteins with four extracellular EGF-like domains separated by six YWTD amino acid repeats that contribute to Fzd and ligand binding (Johnson et al., J. Bone Mineral Res 19: 1749 (2004)).

[0061] The canonical Wnt signaling pathway activated after receptor binding is dependent on the cytoplasmic Disheveled (Dsh) protein interacting directly with the Fzd receptor and causes stabilization and accumulation of β-catenin in the cytoplasm. In the absence of the Wnt signal, β-catenin is transferred to the cytoplasmic degradation complex, which includes tumor suppression proteins - adenomatous colon polyp (APC) and auxin. These proteins serve as critical scaffolds enabling glycogen synthase (GSK) -3e to bind to and phosphorylate β-catenin, which directs it to degradation in the ubiquitin / proteasome pathway. Activation of Dsh causes GSK3e phosphorylation and dissociation of the degradation complex. Accumulated cytoplasmic β-catenin is

Then transported to the nucleus, where it interacts with DNA binding proteins of the Tcf / Lef family to activate transcription.

[0062] In addition to the canonical signaling pathway, Wnt ligands also activate pathways independent of β-catenin (Veeman et al., Dev. Cell 5: 367-377 (2003)). Non-canonical Wnt signaling was implicated in many processes, but most convincingly in gastrulatory movements through a mechanism similar to the planar cell polarization (PCP) pathway from Drosophila. Other potential mechanisms for non-canonical Wnt signaling include calcium flow, JNK and both small and heterotrimeric G proteins. Antagonism between canonical and non-canonical pathways is often observed and some evidence suggests that non-canonical signaling may suppress tumor formation (Olson & Gibo, Exp. Cell Res. 241: 134 (1998); Topol et al., J. Cell Biol. 162: 899-908 (2003)).

[0063] Haematopoietic stem cells (HSCs) are the body's best known stem cells, and Wnt signaling implies both in their normal maintenance and in leukemic transformation (Reya & Clevers, 2005, Nature 434: 843). HSCs are a rare population of cells that occur in a porous niche within mature bone marrow. These cells are characterized by both their unique gene expression profile and the ability to continuously give rise to more differentiated progenitor cells to restore the entire hematopoietic system. Both HSC and cells from their porous microenvironment express Wnt ligands and reporter Wnt activation is present in HSC in vivo. Furthermore, both β-catenin and purified Wnt3A promote self-renewal of mouse HSG in vitro and enhance their ability to restore the hematopoietic system in vivo, while Wnt5A promotes the expansion of human hematopoietic progenitors in vitro and re-colonization in the NOD-SCID xenograft model (Reya et al ., Nature 423: 409-414 (2003); Willert et al., Nature 423: 448-452 (2003); Van Den Berg et al., Blood 92: 3189-3202 (1998); Murdoch et al., PNAS 100: 34223427 (2003)).

[0064] Recently, Wnt signaling has been found to play a role in oncogenic growth of both myeloid and lymphoid lines. For example, macrophage macrophage (GMP-y) progenitors from chronic myelogenous leukemia are characterized by activated Wnt signaling, on which their growth and recovery depends (Jamieson et al., N. Engl. J. Med. 351: 657-667 (2004)). Although leukemias do not appear to contain mutations in the Wnt pathway, autocrine and / or paracrine Wnt signaling may support tumor self-renewal (Reya & Clevers, Nature 434: 843 (2005)).

[0065] The Wnt canonical signaling pathway also plays a central role in maintaining stem cell populations in the small intestine and colon, and inappropriate activation of this pathway plays an important role in colorectal cancer (Reya & Clevers, Nature 434: 843 (2005)). Absorbing intestinal epithelium is organized into villi and crypts. Stem cells are in the crypts and slowly divide and produce rapidly proliferating cells that give rise to all populations of differentiated cells that move from the crypts and occupy intestinal villi. The Wnt signaling cascade plays a dominant role in regulating cell destiny along the crypt-villus axis and is

-20 necessary to maintain stem cell population. The Wnt signaling disorder resulting from the genetic loss of Tcf7 / 2 resulted from homologous recombination (Korinek et al.,

Nat. Genet. 19: 379 (1998)) or overexpression of Dickkopf-1 (Dkkl), a strong secreted Wnt antagonist (Pinto et al., Genes Dev. 17: 1709-1713 (2003); Kuhnert et al., PNAS

101: 266-271 (2004)) causes depletion of intestinal stem cell populations.

[0066] Colorectal cancer is most often initiated by the activation of mutations in the Wnt signaling cascade. Approximately 5-10% of all colorectal cancers are hereditary, with familial adenomatous polyposis (FAP) being the most common form, a dominant autosomal disease in which 80% of affected individuals contain germline mutations in the adenomatous colon polyp (APC) gene . Mutations have also been identified in other components of the Wnt pathway, including auxin and β-catenin. Individual adenomas are clonal hyperplasia of the epithelial cell containing the second inactivated allele, and a large number of FAP adenomas inevitably cause the development of adenocarcinomas through additional mutations in oncogenes and / or tumor suppression genes. Furthermore, activation of the Wnt signaling pathway, including mutations in the acquisition of function in APC and β-catenin, may induce hyperplastic tumor development and growth in mouse models (Oshima et al., Cancer Res. 57: 1644-1649 (1997); Harada et al. , EMBO J. 18: 5931-5942 (1999)).

[0067] The role of Wnt signaling in cancer was first recognized by identifying Wnt1 (originally int1) as an oncogen in breast tumors transformed as a result of the recent insertion of mouse virus (Nusse & Varmus, Cell 31: 99-109 (1982)). Since then, additional evidence has been added indicating the role of Wnt signaling in breast cancer. For example, transgenic overexpression of β-catenin in mammary glands causes hyperplasia and the formation of adenocarcinomas (Imbert et al., J. Cell Biol. 153: 555-568 (2001); Michaelson & Leder, Oncogene 20: 5093-5099 (2001)), while the loss of Wnt signaling interferes with the normal development of the mammary gland (Tepera et al., J. Cell Sc. 116: 1137-1149 (2003); Hatsell et al., J Mammary Gland Biol. Neoplasia 8: 145-158 (2003)). Recently, breast stem cells have been shown to be activated by Wnt signaling (Liu et al., PNAS 101: 4158 (2004)). In human breast cancer, the accumulation of β-catenin implies activated Wnt signaling in over 50% of cancers, and although no specific mutations have been identified, upregulation of Frizzled receptor expression has been observed (Brennan & Brown, J. Mammary Gland Neoplasia 9: 119-131 (2004) ; Malovanovic et al., Int. J. Oncol. 25: 1337-1342 (2004)).

[0068] FZD10, FZD8, FZD7, FZD4 and FZD5 are five of the ten identified human Wnt receptors. In the mouse embryo, Fzd10 is expressed with Wnt7a in the neural tube, in limb buds and Mullerian ducts (Nunnally & Parr, Dev. Genes Evol. 214: 144-148 (2004)) and can serve as a receptor for Wnt7a during the development of bud buds (Kawakami et al., Dev. Growth Duffer. 42: 561-569 (2000)). Fzd10 is co-expressed with Wnt7b in the lung and cell transfection studies have shown that the Fzd10 / LRP5 co-receptor activates the Wnt canonical signaling pathway in response to Wnt7b (Wang et al., Mol. Cell Biol. 25: 5022-5030 (2005)). FZD10 mRNA is up regulated in

-21 many cancer cell lines, including cervical, stomach and glioma cell lines, and in primary cancers, including about 40% of primary gastric cancers, colon cancers, and synovial sarcomas (Saitoh et al., Int. J. Oncol. 20 : 117-120 (2002); Terasaki et al., Int. J. Mol. Med. 9: 107-112 (2002); Nagayama et al., Oncogene 1-12 (2005)). FZD8 is upregulated in several human cancer cell lines, primary gastric and kidney cancers (Saitoh et al., Int. J. Oncol. 18: 991996 (2001); Kirikoshi et al., Int. J. Oncol. 19: 111-115 (2001); Janssens et al., Tumor Biol. 25: 161-171 (2004)). FZD7 is expressed in the gastrointestinal tract and is upregulated in one case for six primary gastric cancer in humans (Kirikoshi et al., Int. J. Oncol. 19: 111-115 (2001)). Expression of the FZD7 ectodomain by a colon cancer cell line induced morphological changes and reduced tumor growth in a xenograft model (Vincan et al., Differentiation 73: 142-153 (2005)). FZD5 plays an important role in yolk sac and placental angiogenesis (Ishikawa et al., Dev. 128: 25-33 (2001)) and is upregulated in renal cancer in connection with activation of Wnt / e-catenin signaling (Janssens et al ., Tumor Biology 25: 161-171 (2004)). FZD4 is expressed at high levels in intestinal crypt epithelial cells and is one of several factors that show different expression in normal tissue compared to cancer (Gregorieff et al., Gastroenterology 129: 626-638 (2005)). The identification of FZD4, 5, 7, 8 and 10 as markers of cancer stem cells thus makes these proteins ideal targets for cancer therapy agents.

Tumor stem cell marker antagonists [0069] In the context of the disclosure, a suitable antagonist is an agent that may have one or more of the following effects, for example: interfering with the expression of a tumor stem cell marker; interferes with activation of the cancer stem cell signal transduction pathway by, for example, sterically inhibiting the interaction between the cancer stem cell marker and its ligand, receptor or co-receptors; or binds to a cancer stem cell marker and causes cell death or inhibits tumor cell proliferation.

[0070] The tumor stem cell marker antagonists act extracellularly and affect or inhibit the function of the cancer stem cell marker.

[0071] The antagonist is a soluble cancer stem cell protein receptor or a soluble receptor protein. Extracellular binding of a cancer stem cell marker antagonist may inhibit the signaling of a cancer stem cell marker protein by inhibiting its proper activation (e.g. kinase activity) of a cancer stem cell marker and / or sterically inhibiting the interaction of, for example, a cancer stem cell marker with its ligand, a cancer stem cell marker with its receptor, a cancer stem cell marker with a co-receptor or a cancer stem cell marker with an extracellular matrix. Furthermore, extracellular binding of a cancer stem cell marker antagonist can down regulate the expression of a cancer stem cell marker on the cell surface, such as, for example, by

Internalizing the cancer stem cell marker protein and / or reducing the transport of the cancer stem cell marker to the cell surface.

[0072] Antagonists of cancer stem cell markers can indirectly cause cell death by inhibiting angiogenesis. Angiogenesis is a process in which new blood vessels form from existing vessels, and it is a fundamental process required for normal growth, for example during embryonic development, wound healing and in response to ovulation. For the growth of solid tumors above 1-2 mm² angiogenesis is also required for the supply of nutrients and oxygen, without which the tumor cells die. The tumor stem cell marker antagonist may thus be directed against vascular cells that express this cancer stem cell marker, including, for example, endothelial cells, smooth muscle cells, or extracellular matrix components required for vessel formation. A tumor stem cell marker antagonist may inhibit growth factor signaling required for the recruitment, grouping, maintenance or survival of vascular cells.

Polynucleotides [0073] Also described herein are isolated polynucleotides encoding polypeptides comprising SEQ ID NO: 1-9. Polynucleotides can exist in the form of RNA or in the form of DNA, wherein the DNA comprises cDNA, genomic DNA and artificial DNA. DNA can be double-stranded or single-stranded, and if single-stranded, it may be the coding strand or the non-coding (anti-sense) strand. Thus, the term "polynucleotide encoding a polypeptide" includes a polynucleotide that includes only sequences encoding the polypeptide, as well as a polynucleotide that includes additional coding and / or non-coding sequences.

[0074] Variants of the above-described polynucleotides, encoding, for example, fragments, analogs and derivatives are also described. The polynucleotide variant may be a naturally occurring allelic variant of the polynucleotide or a non-naturally occurring variant of the polynucleotide. As indicated above, the polynucleotide may have a coding sequence that is a naturally occurring allelic variant of the sequence encoding the disclosed polypeptide. As known in the art, an allelic variant is an alternative form of a polynucleotide sequence that has the substitution, deletion or addition of one or more nucleotides that do not substantially alter the function of the encoded polypeptide.

[0075] Polynucleotides are described in which the coding sequence of the mature polypeptide can be fused in the same reading frame with a polynucleotide that helps, for example, in the expression, secretion, protein stability of the polypeptide from a host cell, including, for example, a leader sequence that acts as secretory sequence for controlled transport of the polypeptide from the cell. A polypeptide having a leader sequence is a preprotein and may have a leader sequence cleaved by the host cell to produce the mature form of the polypeptide. Polynucleotides can also encode proprotein, which is a mature protein with additional 5 'amino acid residues. Mature Protein

23 having pro-sequence is a proprotein and is an inactive form of protein. After cutting off the sequence, active mature protein is formed. Thus, for example, a polynucleotide may encode a mature protein or protein having a pro-sequence or a protein having both a pro-sequence and a pre-sequence (leader sequence).

[0076] Polynucleotides may also have a coding sequence fused in one reading frame with a marker sequence that allows purification of the polypeptide of the invention. The marker sequence may be the six histidine tag provided by the pQE-9 vector to allow purification of the mature polypeptide fused to the marker in the case of a bacterial host, or for example the marker sequence may be a hemagglutinin (HA) tag when a mammalian host is used, e.g., COS cells -7. The HA tag corresponds to an epitope derived from the flu protein hemagglutinin (Wilson, I., et al., Cell 37: 767 (1984)).

[0077] The isolated nucleic acid molecules may contain a polynucleotide with a nucleotide sequence at least 90% identical, 95% identical, and in some embodiments at least 96%, 97%, 98%, or 99% identical to the nucleotide that encodes the disclosed sequences .

[0078] Polynucleotide variants may include changes in coding regions, non-coding regions, or both. Polynucleotide variants may contain changes that create silent substitutions, additions or deletions, but do not change the properties or activities of the encoded polypeptide. Nucleotide variants can be produced by silent substitutions due to the degeneracy of the genetic code. Polynucleotide variants can be produced for a variety of reasons, e.g. to optimize codon expression for a particular host (changing codons in human mRNA to those beneficial for a bacterial host such as E. coli).

Soluble receptor polypeptides [0079] Described herein are recombinant polypeptides, natural polypeptides or synthetic polypeptides with the sequence SEQ ID NO: 1-9. It will be understood from the prior art that some amino acid sequences can be altered without significantly affecting the structure or function of the protein. If such sequence differences are contemplated, it should be remembered that there are key areas in the protein that determine activity. Described herein are variants of the polypeptides that exhibit significant activity or that include regions of the FZD protein, such as parts of the protein discussed herein. Such mutants include deletions, insertions, inversions, repeats and types of substitution. As indicated below, guidelines on which amino acid changes are likely to be phenotypically silent can be found in Bowie, et al., Science 247: 1306-1310 (1990).

[0080] Fragments, derivatives or analogues of a polypeptide may thus be: (i) in which one or more amino acid residues are replaced by a conservative or non-conservative amino acid residue and such replaced amino acid residue may or may not be encoded by the genetic code; or (ii) those in which one or more amino acid residues comprise a substituted group; or (iii) those in which

The mature polypeptide is fused to another compound, such as a compound extending the half life of the polypeptide (e.g., polyethylene glycol); or (iv) those wherein, when fused to the mature polypeptide are additional amino acids, such as a leader or secretory sequence, or a sequence that is used to purify the mature polypeptide or proprotein sequence. Such fragments, derivatives and analogues are considered to be within the knowledge of experts in the field due to the guidelines contained herein.

[0081] Of particular interest are the substitutions of charged amino acids with other charged amino acids and neutral or negatively charged amino acids. The latter result in a protein with a reduced positive charge to improve the properties of the soluble receptor protein. Aggregation prevention is highly desirable because protein aggregation not only causes loss of activity, but can also be problematic when preparing pharmaceutical formulations because [aggregates] can be immunogenic (Pinckard et al., Clin. Exp. Immunol. 2: 331340 (1967) ; Robbins et al., Diabetes 36: 838-845 (1987); Cleland et al. Crit. Rev. Therapeutic Drug Carrier Systems 10: 307-377 (1993)).

[0082] As indicated, the changes are usually minor, such as conservative amino acid substitutions that do not significantly affect folding or white activity (see Tables 1 and 2).

TABLE 1. Conservative amino acid substitutions

<td>aromatic</td><td>phenylalanine tryptophan tyrosine</td>

<td>hydrophobic</td><td>leucine isoleucine valine</td>

<td>polar</td><td>glutamine asparagine</td>

<td>basic</td><td>arginine lysine histidine</td>

<td>acidic</td><td>Aspartic acid</td>

<td>small</td><td>Glutamic acid alanine serine threonine methionine glycine</td>

Table. 2. Ammic acid substitutions

<td>The rest is original</td><td>substitutions</td><td>Examples of substitutions</td>

<td>Ala (A)</td><td>val</td><td>Val; Leu; How much</td>

<td>Arg (R)</td><td>lys</td><td>Lys; Gln; own</td>

<td>Asn (N)</td><td>Gln</td><td>Gln; His; Lys; Arg</td>

<td>Asp (D)</td><td>Glu</td><td>Glu</td>

<td>Cys (C)</td><td>Cheese</td><td>Cheese</td>

<td>Gln (Q)</td><td>own</td><td>own</td>

<td>Glu (E)</td><td>Asp</td><td>Asp</td>

<td>Gly (G)</td><td>Pro</td><td>Pro</td>

<td>His (H)</td><td>Arg</td><td>Asn; Gln; Lys; Arg</td>

<td>How many (i)</td><td>Leu</td><td>Leu; Val; Underworld; Ala; Phe; norleucine</td>

<td>Leu (L)</td><td>How much</td><td>norleucine; How much; Val; Underworld; Ala; phe</td>

<td>^Ls^{s (K)}</td><td>Arg</td><td>Arg; Gln; own</td>

<td>Met (M)</td><td>Leu</td><td>Leu; Phe; How much</td>

<td>Phe (F)</td><td>Leu</td><td>Leu; Val; How much; ala</td>

<td>Pro (P)</td><td>Gly</td><td>Gly</td>

<td>Ser (S)</td><td>Thr</td><td>Thr</td>

<td>Thr (T)</td><td>Cheese</td><td>Cheese</td>

<td>Trp (W)</td><td>Tyr</td><td>Tyr</td>

<td>Tyr (Y)</td><td>phe</td><td>Trp; Phe; Thr; Cheese</td>

<td>The rest is original</td><td>substitutions</td><td>Examples of substitutions</td>

<td>Val (V)</td><td>Leu</td><td>How much; Leu; Underworld; Phe; Ala; norleucine</td>

[0083] The number of amino acid substitutions to be made by one of skill in the art depends, of course, on many factors, including those described above. Generally speaking, the number of substitutions for any soluble receptor polypeptide will not be greater than 50, 40, 30, 25, 20, 15, 10, 5 or 3.

[0084] Disclosed herein are polypeptides of SEQ ID NO: 1-9, as well as polypeptides that at some points have at least 90% similarity to polypeptides of SEQ ID NO: 1-9, and at some times at least 95% similarity to polypeptides SEQ ID NO: 1-9, and at some points at least 96%, 97%, 98% or 99% similarity to polypeptides of SEQ ID NO: 1-9. As is known in the art, the "similarity" between two polypeptides is determined by comparing the amino acid sequence and conserved amino acid substitutions of one polypeptide with that of the other polypeptide.

[0085] Fragments or portions of the polypeptides can be used to produce the corresponding full-length polypeptide using peptide synthesis; therefore, the fragments can be used as intermediates for the production of full-length polypeptides. Fragments or portions of polynucleotides can be used to synthesize full-length polynucleotides of the invention.

[0086] A protein fragment is part or all of a protein that is capable of binding to a cancer stem cell marker protein or a cancer stem cell binding protein partner (e.g., receptor, co-receptor, ligand or colligand). This fragment has a high affinity for a cancer stem cell marker protein or cancer stem cell binding protein partner (e.g., receptor, co-receptor, ligand or colligand). Some fragments of fusion proteins are protein fragments comprising at least a portion of the extracellular portion of a cancer stem cell marker protein or a cancer stem cell binding protein partner associated with at least a portion of an immunoglobulin constant region. Affinities can range from about 10^-11 up to 10^-12 M, although the affinity may be very different for fragments of different sizes and may range from 10^-7 up to 10^-13 M. The fragment may be about 10-255 amino acids in length and contains a ligand binding site of a cancer stem cell marker protein associated with at least a portion of an immunoglobulin constant region.

[0087] Polypeptides and analogues may further be modified to contain additional chemical moieties that are not normally part of the protein. These derivatized moieties can improve the solubility, biological half life or absorption of the protein. The groups may also reduce or eliminate

Any desired side effects of proteins and the like. A discussion of these moieties can be found in Remington's Pharmaceutical Sciences, 20th ed., Mack Publishing Co., Easton, PA (2000).

[0088] The chemical moieties most suitable for derivatization include water-soluble polymers. A water-soluble polymer is desirable because the protein to which it is attached does not precipitate in an aqueous environment, such as a physiological environment. In some embodiments, the polymer will be pharmaceutically acceptable for the preparation of a therapeutic product or composition. The skilled person will be able to select the desired polymer based on such requirements as whether the polymer / protein conjugate will be used therapeutically and if so based on the desired dosage, circulation time, resistance to proteolysis and other requirements. The derivatization efficiency can be determined by providing the derivative in the desired form (i.e. using an osmotic pump or injection or infusion or additionally formulated for oral delivery, pulmonary delivery or other delivery methods) and determining its effectiveness. Suitable water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene anhydride , polyamino acids (homopolymers or random copolymers) and dextran or poly (n-vinylpyrrolidone) - polyethylene glycol, homopolymers of propylene glycol, polypropylene oxide / ethylene oxide copolymers, polyoxyethylene polyols (e.g. glycerol), polyvinyl alcohol and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in production due to its stability in water.

[0089] The number of polymer molecules attached in this way may vary and one of ordinary skill in the art will be able to determine its effect on function. Monodivatization or di-, tri-, tetra- or other derivatization combinations using the same or different chemical moieties (e.g. polymers such as polyethylene glycols of different weight) can be provided. The ratio of polymer molecules to protein (or peptide) molecules will vary, as will their concentrations in the reaction mixture. The optimum ratio (in terms of efficiency of reaction in terms of the fact that there is no excess unreacted protein or polymer) will generally be determined by factors such as the desired degree of derivatization (eg. Mono-, di-, tri- etc.), the molecular weight and the polymer selected, whether the polymer is branched or unbranched, and the reaction conditions.

[0090] Polyethylene glycol (or other chemical moiety) molecules should be attached to the protein taking into account the effect on the functional or antigenic domain of the protein. There are many ways to attach available to those skilled in the art. See, for example, EP 0 401 384 (PEG conjugation to G-CSF), see also Malik et at., Exp. Hematol 20: 10281035 (1992) (pegylation of GM-CSF using tresyl chloride is shown). For example, polyethylene glycol may be covalently bonded through amino acid residues via a reactive group, such as a free amino or carboxyl group. Reactive groups are those with which the activated molecule can be bound

Polyethylene glycol. Amino acid residues having a free amino group may include lysine residues and an N-terminal amino acid residue. Those having a free carboxyl group may include aspartic acid residues, glutamic acid residues, and a C-terminal amino acid residue. Sulfhydryl groups can also be used as a reactive group for attachment of a polyethylene glycol molecule (s). For therapeutic purposes, attachment on an amino group, such as attachment on an N-terminal or lysine group, can be performed. Attachment in residues important for receptor binding should be avoided if receptor binding is desired.

[0091] In particular, one may want to obtain a protein with a chemically modified amino terminus. If we use polyethylene glycol to illustrate the composition of the invention, we can choose from various polyethylene glycol molecules (in terms of molecular weight, branching, etc.), [we can choose] the ratio of polyethylene glycol molecules to protein (or peptide) molecules in the reaction mixture, type of pegylation reaction, to be carried out, and a method for obtaining the selected pegylated protein at the N-terminus. Method of preparation of the preparation with pegylation at the N-terminal (i.e. separating this moiety from other monopegylated moieties (if necessary) may involve purifying the material with N-terminus pegylation from a population of pegylated protein molecules. Selective chemical N-terminal modification can be achieved by reductive alkylation, which uses the different reactivity of the different types of primary amine groups (lysine and N-terminal) available for derivatization in a particular protein. Under appropriate reaction conditions, substantially selective derivatization of the protein at the N-terminus using a carbonyl-containing polymer is achieved. For example, you can selectively pegylate the protein at the N-terminal by performing a pH reaction that allows you to use the pKa differences of the epsilonamine group of lysine residues and that of the alpha-amino group of the N-terminal residue of a protein. Using this selective derivatization, the attachment of the water-soluble polymer to the protein is controlled: polymer conjugation usually occurs at the N-terminus of the protein and no significant modification of other reactive groups, such as lysine side chain amino groups, occurs. By using reductive alkylation, the water-soluble polymer may be of the type described above and should have one reactive aldehyde for protein conjugation. Polyethylene glycol propionaldehyde containing one reactive aldehyde may be used.

[0092] Pegylation can be carried out using any of the pegylation reactions known in the art. See for example: Focus on Growth Factors, 3 (2): 4-10 (1992); EP 0 154 316, EP 0 401 384; and other publications cited herein that relate to pegylation. Pegylation can be carried out via an acylation reaction or an alkylation reaction using a reactive polyethylene glycol molecule (or equivalent water-soluble reactive polymer).

[0094] It is therefore contemplated that the soluble receptor polypeptides intended for use in the present invention may include pegylated proteins or

Variant soluble receptors in which the PEG group (s) is / are attached via acyl or alkyl groups. Such products may be monopegylated or polypegylated (e.g., they contain 2 6, and typically 2 5 PEG groups). PEG groups are generally attached to a protein in a- or e-amino groups of amino acids, but it is also contemplated that PEG groups may be attached to any amino group attached to a protein that is sufficiently reactive to be attached to the group PEG under appropriate reaction conditions.

[0095] The polymer molecules used in both acylation and alkylation may be selected from water-soluble polymers as described above. The selected polymer should be modified to have a single reactive group, such as an active ester for acylation or an aldehyde for alkylation, so that the degree of polymerization can be controlled as provided in the present methods. An example of a PEG reactive aldehyde is polyethylene glycol propionaldehyde, which is stable in water, or its mono-C10-alkoxy or aryloxy derivatives (see US Pat. No. 5,252,714). The polymer may be branched or unbranched. In the case of an acylation reaction, the polymer (s) selected should have one reactive ester group. In the case of reductive alkylation, the chosen polymer (s) should have one reactive aldehyde group. In general, the water-soluble polymer will not be selected from naturally occurring glycosyl residues because they are usually more conveniently created by mammalian recombinant expression systems. The polymer may have any molecular weight and may be branched or unbranched. One of the water-soluble polymers to be used here is polyethylene glycol. Polyethylene glycol herein is intended to include any form of PEG used to derivatize other proteins, such as mono (C1-C10) alkoxy or polyethylene aryloxyglycol.

[0096] Other reaction parameters, such as solvent, reaction times, temperatures, etc., and product purification agents, can be determined in each case based on published information related to protein derivatization using water-soluble polymers (see publications cited herein).

[0097] The isolated polypeptides described herein can be produced by any suitable method known in the art. Such methods range from direct protein synthesis methods for constructing DNA sequences encoding isolated polypeptide sequences and expressing these sequences in a suitable transformed host. For example, cDNA can be obtained by screening a human cDNA library using a labeled DNA fragment encoding the polypeptide of SEQ ID NO: 1 and identifying positive clones by autoradiography. Further rounds of plaque purification and hybridization are carried out using conventional methods.

[0098] In some embodiments of the recombinant method, the DNA sequence is constructed by isolating or synthesizing a DNA sequence encoding the wild-type protein of interest. Optionally, the sequence may be mutagenized by site-specific mutagenesis to ensure its functional

-30analogów. See, e.g., Zoeller et al., Proc. Natl. Acad. Sci. USA 81: 5662-5066 (1984) and Pat. USA No. 4,588,585. Another way to construct a DNA sequence encoding a polypeptide of interest would be chemical synthesis using an oligonucleotide synthesizer. Such oligonucleotides can be designed based on the amino acid sequence of the desired polypeptide, selecting those codons that are beneficial in the host cell in which the recombinant polypeptide of interest will be produced.

[0099] Standard methods can be used to synthesize sequences of isolated polynucleotides encoding an isolated polypeptide of interest. For example, the complete amino acid sequence can be used to construct a gene as part of reverse translation. Furthermore, a DNA oligomer containing a nucleotide sequence encoding a specific isolated polypeptide can be synthesized. For example, several small oligonucleotides encoding parts of the desired polypeptide can be synthesized and then ligated. Individual oligonucleotides typically contain 5 'or 3' overhangs for complementary assembly.

[0100] After assembly (by synthesis, site-directed mutagenesis or other method) the mutated DNA sequences encoding the specific isolated polypeptide of interest will be inserted into the expression vector and operably linked to an expression control sequence suitable for expression of the protein in the desired host. Proper structure can be confirmed by nucleotide sequencing, restriction mapping, and expression of a biologically active polypeptide in a suitable host. As is well known in the art, in order to obtain high expression levels of the transfected gene in the host, the gene must be operably linked to transcriptional and translational expression control sequences functional in the selected expression host.

[0101] Recombinant expression vectors can be used to amplify and express DNA encoding a stem cell tumor marker polypeptide fusion. Recombinant expression vectors are replication-capable DNA constructs that have artificial or cDNA-derived DNA fragments encoding a tumor stem cell marker polypeptide fusion or a biologically equivalent analog operably linked to appropriate transcriptional or translational regulatory elements derived from mammalian, microbial, viral or insect genes. The transcription unit generally comprises a set of (1) genetic element (s) with a regulatory role in gene expression, e.g., promoters or transcriptional enhancers, (2) a structural or coding sequence that is transcribed into mRNA and translated into protein, and ( 3) appropriate transcription and translation initiation and termination sequences, as described in detail below. Such regulatory elements may include an operator sequence for transcription control. In addition, a host's replication capacity, which typically conferring an origin of replication, and a selection gene to recognize transformants can be provided. DNA regions are operably linked if they are functionally linked to each other. For example, signal peptide DNA

-31 (secretory leader) is operably linked to polypeptide DNA if it is expressed as a precursor that participates in the secretion of the polypeptide; a promoter is operably linked to a coding sequence if it controls the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned in such a way that it allows translation. Operatively related generally means contiguous, and in the case of secretory leaders means contiguous and in one reading frame. Structural elements intended for use in yeast expression systems may include a leader sequence enabling extracellular secretion of the translated protein by the host cell. Alternatively, if the recombinant protein is expressed without a leader or transport sequence, it may include an N-terminal methionine residue. Optionally, this residue may then be cleaved from the expressed recombinant protein to provide the final product.

[0102] The choice of expression control sequence and expression vector will depend on the choice of host. A wide variety of host / expression vector combinations can be used. Expression vectors useful for eukaryotic hosts include, for example, vectors containing expression control sequences from SV40, bovine papilloma virus, adenovirus and cytomegalovirus. Expression vectors useful for bacterial hosts include known bacterial plasmids, such as plasmids from Escherichia coli, including pCR1, pBR322, pMB9 and derivatives thereof, broader host plasmids such as M13, and filamentous phage with single-stranded DNA.

[0103] Host cells suitable for expressing a cancer stem cell marker protein include prokaryote, yeast, insect or higher eukaryotic cells under the control of appropriate promoters. Prokaryotes include gram-negative or gram-positive organisms, for example E. coli or bacilli. Higher eukaryotic cells include established cell lines of mammalian origin as described below. Extracellular translation systems can also be used. Suitable cloning and expression vectors for use with bacterial, fungal, yeast and mammalian cell hosts are described in Pouwels et al., Cloning Vectors: A Laboratory Manual, Elsevier, NY (1985).

[0104] Various mammalian or insect cell culture systems are also preferably used to express the recombinant protein. Recombinant proteins can be expressed in mammalian cells because such proteins are generally correctly folded, properly modified, and completely functional. Examples of suitable mammalian host cell lines include the COS-7 monkey kidney cell lines described by Gluzman, Cell 23: 175 (1981) and other cell lines capable of expressing from a suitable vector, including, for example, L cells, C127, 3T3, ovary cell lines Chinese hamster (CHO), HeLa and BHK. Mammalian expression vectors may contain non-transcribed elements such as the origin of replication, the appropriate promoter and enhancer associated with the gene to be expressed and other non-transcribed 5 'or 3' side flanking sequences and 5 'or 3' untranslated sequences such

- necessary ribosome binding sites, polyadenylation sites, donor and acceptor splice sites, and transcriptional termination sequences. Baculovirus systems for producing heterologous proteins in insect cells are discussed in Luckow and Summers, Bio / Technology 6:47 (1988).

[0105] Proteins produced by the transformed host can be purified according to any suitable method. Such standard methods include chromatography (e.g., ion exchange, affinity and column size chromatography), centrifugation, differential solubility, or any other standard protein purification technique. Affinity tags, such as hexahistidine, maltose binding domain, influenza envelope sequence and glutathione S-transferase, can be attached to the protein to allow easy purification as it passes through the appropriate affinity column. Isolated proteins can also be physically characterized using techniques such as proteolysis, nuclear magnetic resonance and X-ray crystallography.

[0106] For example, supernatants from systems that secrete recombinant protein into culture media can first be concentrated using a commercially available protein concentration filter, for example the Amicon or Millipore Pellicon ultrafiltration assembly. After the concentration step, the concentrate can be applied to a suitable purification matrix. Alternatively, anion exchange resin, e.g. matrix or diethylaminoethyl (DEAE) side support can be used. The matrices can be acrylamide, agarose, dextran, cellulose or other types commonly used in purifying protein. Alternatively, a cation exchange step may be used. Suitable cation exchangers include various insoluble matrices containing sulfopropyl or carboxymethyl groups. Finally, one or more reverse phase high performance liquid chromatography (RP-HPLC) steps using a hydrophobic RP-HPLC medium, e.g. Silica gel with methyl or other aliphatic side groups can be used to further purify the cancer stem cell Fc protein composition. Some or all of the above purification steps, in various combinations, can also be used to provide a homogeneous recombinant protein.

[0107] Recombinant protein produced in bacterial culture is usually isolated using the initial extraction from cell pellets followed by one or more concentration, salting out, aqueous ion exchange or size exclusion chromatography steps. High performance liquid chromatography (HPLC) can be used for the final purification steps. The microbial cells used to express the recombinant protein can be disrupted by any convenient method, including freezing and thawing cycles, sonication, mechanical disruption or the use of cell lysing agents.

Inhibiting tumor cell growth

[0108] Also described herein are methods for inhibiting the growth of tumorigenic cells expressing a cancer stem cell marker using antagonists of the cancer stem cell marker described herein. A method of inhibiting the growth of tumorigenic cells expressing a cancer stem cell marker involves contacting the cell with a cancer stem cell marker antagonist in vitro. For example, an immortalized cell line or cancer cell line that expresses a cancer stem cell marker is cultured in medium to which an antagonist of the expressed cancer stem cell marker has been added to inhibit cell growth. Alternatively, tumor cells and / or tumor stem cells are isolated from a patient sample, such as, for example, tissue biopsy, pleural effusion or blood sample, and cultured in medium to which a stem cell tumor marker antagonist has been added to inhibit cell growth. The antagonist may be a fusion of a cancer stem cell marker protein that specifically binds to a cancer stem cell marker protein or to a cancer stem cell marker protein (e.g., receptor, co-receptor, ligand or colligand). For example, a purified tumor stem cell marker protein fusion is added to an isolated tumor stem cell culture medium to inhibit cell growth.

[0109] A method of inhibiting the growth of tumorigenic cells expressing a cancer stem cell marker includes contacting the cell with a cancer stem cell marker antagonist in vivo. Contact of the tumorigenic cell with the tumor stem cell marker antagonist in an animal model may be undertaken. For example, xenografts expressing a cancer stem cell marker are cultured in immunocompromised mice (e.g. NOD / SCID mice) that are administered a tumor stem cell marker antagonist to inhibit tumor growth. Alternatively, cancer stem cells that express a cancer stem cell marker are isolated from a patient sample, such as, for example, tissue biopsy, pleural effusion or blood sample, and are injected with immunocompromised mice, which are then administered a tumor cell marker antagonist. parent to inhibit tumor cell growth. The tumor stem cell marker antagonist may be administered at the same time or shortly after introducing tumorigenic cells into the animal to prevent tumor growth. The tumor stem cell marker antagonist can be administered as a therapeutic agent after the tumorigenic cells have grown to a certain size. The antagonist may be a fusion of a cancer stem cell marker protein that specifically binds to a cancer stem cell marker protein or to a cancer stem cell marker protein (e.g., receptor, co-receptor, ligand or colligand). Contact of a tumorigenic cell with a cancer stem cell antagonist can be undertaken in a human patient diagnosed with cancer.

Pharmaceutical compositions

[0110] Pharmaceutical compositions containing antagonists that are directed against a cancer stem cell marker find use in inhibiting tumor cell growth and treating cancer in human patients.

[0111] Formulations are prepared for storage and use by combining the purified antagonist (e.g., antibody) of the invention with a pharmaceutically acceptable carrier, excipient and / or stabilizer, in the form of a sterile lyophilized powder, aqueous solution, etc. (Remington, The Science and Practice of Pharmacy, 20th Edition, Mack Publishing (2000)). Suitable carriers, excipients or stabilizers include non-toxic buffers such as phosphate, citric and other organic acids; salts such as sodium chloride; antioxidants, including ascorbic acid and methionine; preservatives (e.g. octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzetonium chloride; phenol; butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol and m-cresol); low molecular weight polypeptides (such as having less than about 10 amino acid residues); proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; carbohydrates such as monosaccharides, disaccharides, glucose, mannose or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol, salt-forming counterions such as sodium [ion]; metal complexes (e.g., Zn-protein complexes) and / or nonionic surfactants, such as TWEEN or polyethylene glycol (PEG).

[0112] The pharmaceutical composition of the invention may be administered in a variety of ways for local or systemic treatment. Administration can be topical administration (such as mucosal delivery, including intravaginal and rectal delivery), such as transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders; pulmonary (e.g. by inhalation or insufflation of powders or aerosols, including with a nebulizer; intratracheal, nasal, epidermal or transdermal); oral; or parenteral, including intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial (e.g. intrathecal or intraventricular).

[0113] Therapeutic formulations may be in unit dosage form. Such formulations include tablets, pills, capsules, powders, granules, solutions or suspensions in water or non-aqueous media, or suppositories for oral, parenteral or rectal administration, or for administration by inhalation. In solid compositions, such as tablets, the primary active ingredient is mixed with the pharmaceutical carrier. Conventional tableting ingredients include corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate or gums and other diluents (e.g. water) to form a solid preformulation composition containing a homogeneous mixture of a compound of the invention or a non-toxic pharmaceutically acceptable compound salt. The composition of solid preformulation is

Then divided into unit dosage forms of the type described above. Tablets, pills etc. of the new composition may be coated or otherwise combined to provide a dosage form that benefits the prolonged action. For example, a tablet or pill may contain an inner composition coated with an outer component. Moreover, the two components can be separated by an enteric layer, which serves to suppress disintegration and allows the internal component to pass intact through the stomach or delayed release. A variety of materials may be used for such enteric layers or coatings, such materials include a plurality of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol and cellulose acetate.

[0114] Pharmaceutical formulations include the antagonists of the invention complexed with liposomes (Epstein, et al., Proc. Natl. Acad. Sci. USA 82: 3688 (1985); Hwang, et al., Proc. Natl. Acad. Sci. USA 77: 4030 (1980); and US Patent 4,485,045 and 4,544,545). Liposomes with extended circulation time are disclosed in US Patent 5,013,556. Liposomes can be produced using reverse phase evaporation with a lipid composition containing phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded with filters of defined pore size to obtain liposomes with the desired diameter.

[0115] The antagonist may also be encapsulated in microcapsules. Such microcapsules are produced, for example, by coacervation techniques or by means of interfacial polymerization, for example, hydroxymethylcellulose or gelatin, respectively, microcapsules and poly (methylmethacrylate) microcapsules, in colloidal drug delivery systems (e.g. liposomes, albumin microspheres, nanoparticles, nanoparticles) or in macroemulsions as described in Remington, The Science and Practice of Pharmacy, 20th Ed. Mack Publishing (2000).

[0116] In addition, sustained release formulations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, these matrices being in the form of shaped articles (e.g., films or microcapsules). Examples of sustained release matrices include polyesters, hydrogels such as poly (2-hydroxyethyl methacrylate) or poly (vinyl alcohol), polylactides (US Patent 3,773,919), copolymers of L-glutamic acid and ethyl L-glutamate, non-degradable ethylene vinyl acetate, degraded lactic acid copolymers - glycolic acid, such as LUPRON DEPOT TM (injectable microspheres made of lactic acid glycolic acid and leuprolide acetate copolymer), sucrose isobutyrate acetate and poly-D - (-) - 3-hydroxybutyric acid.

Antagonist Treatment [0117] Antagonists of the invention are anticipated to be used to treat various conditions characterized by expression and / or increased cell reactivity to a cancer stem cell marker. In particular, antagonists are anticipated

-36 (e.g., antibodies) against a cancer stem cell marker will be used to treat proliferative disorders, including but not limited to benign and malignant tumors of the kidney, liver, bladder, breast, stomach, ovary, colon, rectum, prostate, lung, vulva, thyroid, head and neck, brain (glioma, astrocytoma, medulla etc.), blood and lymph (leukemia and lymphoma).

[0118] Antagonists are administered to a human patient in the form of a suitable pharmaceutical composition according to known methods. A suitable mode of administration includes intravenous bolus or continuous infusion over a period of time and includes, but is not limited to, intramuscular, intraperitoneal, intravenous, intrathecal, subcutaneous, intraarterial, intrasynovial, intrathecal, oral, topical or inhalation.

[0119] In some embodiments, the treatment comprises the combined administration of the antagonist of the invention and a chemotherapeutic agent or cocktail of a wide variety of chemotherapeutic agents. Antagonist treatment may occur before, simultaneously with or after chemotherapy. The chemotherapy contemplated by the invention includes chemicals or drugs that are known in the art and are commercially available, such as doxorubicin, 5-fluorouracil, cytosine arabinoside ("Ara-C"), cyclophosphamide, thiotepa, busulfan, cytoxin, taxol, methotrexate, cisplatin, melphalan, vinblastine and carboplatin. Combined administration may include simultaneous administration, in one pharmaceutical formulation or using separate formulations, or successive administration in any order, but preferably in such a time interval that all active agents can simultaneously display their biological activities. For such chemotherapeutic agents, production and dosage plans may be used as recommended by the manufacturers, or empirically determined by one of ordinary skill in the art. Production and dosage plans for such chemotherapy are also described in the Chemotherapy Service, MC Perry, ed., Williams &amp; Wilkins, Baltimore, MD (1992).

[0120] In some embodiments, the treatment comprises the combined administration of the antagonist of the invention and radiation therapy. Antagonist therapy may occur before, simultaneously with or after radiotherapy. Any dosage plan for such radiation therapy may be used empirically determined by one of ordinary skill in the art.

[0121] In some embodiments, the treatment may comprise combined administration of the antagonists of the invention with antibodies against additional tumor associated antigens, including but not limited to antibodies that bind to EGFR, HER2 and VEGF. Furthermore, the treatment may involve the administration of one or more cytokines, may be accompanied by surgical removal of the tumor cells, or any other therapy deemed necessary by the attending physician.

[0122] For treating a disease, the appropriate dosage of the antagonist of the invention depends on the type of disease being treated, the severity and course of the disease, the response of the disease, whether the antagonist is administered for therapeutic or preventive purposes, prior therapy, the patient's clinical history and so on. all at the discretion of the attending physician. The antagonist may be administered once or in a series

-Treatment lasting from several days to several months or until healing or reaching reduction of the disease status (e.g. reduction of tumor size). Optimal dosing plans can be calculated from measurements of drug accumulation in the patient's body and will vary depending on the relative potency of the individual antagonists. The attending physician can easily determine the optimal doses, dosage methodologies, and repetition rate. In general, the dose ranges from 0.01 μg to 100 mg per kg body weight and can be given once or more times per day, week, month or year. Your doctor may estimate repetition rates for dosing based on measured residence times and drug concentration in body fluids or tissues.

Kits [0123] Kits may be used to implement the methods described herein. Kits may contain purified soluble stem cell tumor marker receptor in one or more containers. The kits may contain all components necessary and / or sufficient to perform the detection test, including all controls, test guidelines and any necessary software for analyzing and presenting the results.

[0124] A multi-chamber kit is described in which reagents are contained in separate containers. Such containers allow efficient transfer of reagents from one compartment to another compartment, so that samples and reagents are not cross-contaminated, and the agents or solutions from each container can be added in a quantitative manner from one compartment to another. Such containers will include a container that will accept the test sample, a container that contains the soluble receptor used in the methods, containers that contain washing reagents (such as phosphate buffered saline, Tris buffers, etc.), and containers that contain reagents used for detection bound antibody or probe. One skilled in the art will readily recognize that the disclosed polynucleotides, polypeptides and antibodies can be easily incorporated into one of the established forms of the kit that are well known in the art.

EXAMPLES

Example 1

Production of soluble FZD Fc receptor proteins and in vivo half-life determination [0125] Soluble versions of the N-terminal extracellular domain (ECD) of human FZD receptors bind Wnt ligands and act as antagonists of the Wnt signaling pathway (He et al., (1997) Science 275: 1652-54; Tanaka et al., (1998) Proc. Natl. Acad. Sci. 95: 10164-69; Holmen et al., (2002) JBC 277: 34727-35; Vincan et al., (2005) Differentiation 73: 142-53). Soluble FZD receptors were generated by ligation of 1) ECD domain or 2) Fri with FZD10, FZD7, FZD5, FZD4 or FZD8 in one reading frame with human Fc IgG1 isolated from a human B cell library (SEQ ID NO: 4) in a vector for expression in insect cells and HEK 293 cells. Standard recombinant DNA technology was used to isolate polynucleotides encoding ECD domains of FZD receptors

- comprising: amino acids from about 21 to 227 of FZD10 (FZD10 ECD.Fc); amino acids from about 32 to 255 from FZD7 (FZD7 ECD.Fc); amino acids from about 27 to 233 of FZD5 (FZD5 ECD.Fc); and amino acids from about 37 to 224 of FZD4 (FZD4 ECD.Fc), as well as the Fri domains of FZD receptors including: amino acids from about 21 to 154 of FZD10 (FZD10 Fri.Fc); amino acids from about 32 to 171 of FZD7 (FZD7 Fri.Fc); amino acids from about 27 to 157 from FZD5 (FZD5 Fri.Fc); amino acids from about 37 to 170 of FZD4 (FZD4 Fri.Fc); and amino acids from about 28 to 158 from FZD8 (FZD8 Fri.Fc). Soluble receptor proteins were purified on a protein A column.

[0126] In vivo experiments were performed to determine the half-life of soluble FZD receptors. In particular, 200 μg of purified FZD4 Fri.Fc, FZD8 Fri.Fc, FZD5 Fri.Fc and FZD5 ECD.Fc were given to mice (n = 3) ip and blood samples were taken at the indicated time points (Fig. 1). Serum proteins retained on Protein A agarose beads were separated on an SDS-PAGE gel, transferred to nitrocellulose membranes and tested using HRP conjugated goat [antibody] against human IgG Fc fragment to detect hFc fusion proteins. The FZD4 Fri.Fc, FZD5 Fri.Fc and FZD8 Fri.Fc proteins are all present in the blood serum 72 hours after injection, and FZD5 Fri.Fc and FZD8 Fri.Fc are present in the blood serum 96 hours after injection (Fig. 1) . For comparison, FZD5 ECD.Fc is undetectable after 24 hours (Fig. 1).

Example 2

In vitro tests for evaluation of soluble FZD Fc receptor protein [0127] This example describes methods for testing in vitro tests for FZD Fc receptor activity on cell proliferation and pathway activation.

Proliferation assay [0128] Expression of the FZD receptor by various tumor cell lines is quantified using Taqman analysis. Cell lines identified as expressing the FZD receptor are plated at a density of 10⁴ cells per well in tissue well culture 96 well microplates and allowed to grow for 24 hours. Cells are then cultured for an additional 12 hours in fresh DMEM with 2% FCS and soluble FZD Fc receptor protein or control protein in the presence of 10 μmol / L BrdU is added to the culture medium. After labeling with BrdU, the culture medium is removed and the cells fixed at room temperature for 30 min in ethanol and reacted for 90 min with peroxidase-conjugated anti-BrdU monoclonal antibody (BMG clone 6H8, Fab fragments). The substrate is developed in a solution containing tetramethylbenzidine and retained after 15 min using 25 μl 1 mol / LH₂SO₄. The color reaction is measured using an automatic ELISA plate reader using a 450 nm filter (UV Microplate Reader; Bio-Rad Laboratories, Richmond, CA). All experiments were carried out in triplicate. The ability of the soluble FZD Fc receptor protein to inhibit cell proliferation is determined.

Trail activation test

[0129] The ability of soluble FZD Fc receptor protein to block activation of the Wnt signaling pathway is determined in vitro. In one embodiment of the cell

HEK 293 cultured in DMEM supplemented with antibiotics and 10% FCS is co-transfected with

1) Wnt7B and FZD10 expression vectors for activation of the Wnt signaling pathway; 2) wild type or mutant TCF / Luc reporter vector containing three copies of the TCF binding domain upstream of the firefly luciferase reporter gene for measuring levels of canonical Wnt signaling (Gazit et al., 1999, Oncogene 18: 5959-66); and 3) [gene] Renilla luciferase reporter (Promega; Madison, WI) as an internal control of transfection efficiency. FZD Fc protein is then added to the cell culture medium. Forty-eight hours after transfection, luciferase levels are measured using a double luciferase assay kit (Promega; Madison, WI) with firefly luciferase activity normalized to Renilla luciferase activity. Three independent experiments were carried out in triplicate. The ability of the soluble FZD10 Fc protein to inhibit the activation of the Wnt pathway is thus determined.

[0130] In some embodiments, increasing amounts of FZD Fc fusion proteins were incubated with L cells in the presence or absence of Wnt3a ligand and Wnt3a-induced β-catenin stabilization was determined by immunoblotting. Only in the presence of Wnt3a β-catenin was detectable and this stabilization was blocked by increasing amounts of soluble FZD5 ECD.Fc, FZD8 Fri.Fc and FZD4 Fri.Fc receptor protein (Fig.

2), which showed that soluble FZD Fc receptor proteins antagonize the Wnt signaling pathway activated by the Wnt3a ligand.

[0131] The ability of FZD: Fc fusion proteins to antagonize signal transduction by different Wnt ligands was then determined. HEK 293 cells stably transfected with the 8xTCF-luciferase reporter were incubated with increasing amounts of soluble FZD Fri: Fc receptors in the presence of various Wnt ligands, including Wnt1, Wnt2, Wnt3, Wnt3a and Wnt7b. FZD4 Fri.Fc, FZD5 Fri.Fc and FZD8 Fri.Fc fusion proteins inhibited Wnt signaling dependent on all five Wnt ligands (Fig. 3).

Example 3

Prevention of tumor growth in vivo using soluble FZD Fc receptor protein [0132] This example describes the use of soluble FZD Fc receptor to prevent tumor growth in a xenograft model.

[0133] Tumor cells from a patient sample (solid tumor biopsy or pleural effusion) that were passaged as xenograft in mice were prepared for repassaging to experimental animals as detailed above. Dissociated tumor cells (&lt;10,000 cells per animal; n = 10) were then injected subcutaneously into the fat pads of the NOD / SCID mouse nipples to induce tumor growth.

[0134] In some embodiments, dissociated tumor cells are first sorted into tumorigenic and non-tumorigenic cells based on cell surface markers prior to injection into experimental animals. IN

In particular, dissociated tumor cells as described above are washed twice with Hepes buffered saline (HBSS) containing 2% heat inactivated calf serum (HICS) and resuspended at 10⁶ cells per 100 pl. Antibodies are added and the cells are incubated for 20 min on ice after two washes with HBSS / 2% HICS. Antibodies include anti-ESA (Biomeda, Foster City, CA), anti-CD44, anti-CD24 and Lineage markers - anti-CD2, -CD3, -CD10, -CD16, -CD18, CD31, -CD64 and -CD140b (collectively referred to as Lin; PharMingen, San Jose, CA). Antibodies are directly conjugated to fluorochromes for positive or negative selection of cells expressing these markers. Mouse cells are eliminated as a result of selection against H2Kd + cells, and dead cells are eliminated using live cell 7AAD dye. Flow cytometry is performed on FACSVantage (Becton Dickinson, Franklin Lakes, NJ). Lateral scatter and forward scatter profiles are used to eliminate cell aggregates. Isolated tumorigenic ESA +, CD44 +, CD24- / low, Lin- cells are then injected subcutaneously into the fat pads of the nipples to obtain breast tumors or sideways to obtain tumors other than the breast in NOD / SCID mice to induce tumor growth.

[0135] In some embodiments, two days after injection of tumor cells, animals were treated with soluble FZD7 ECD.Fc receptor, soluble FZD10 ECD.Fc receptor or soluble FZD5 ECD.Fc receptor. Each injected test animal received intraperitoneally (ip) 10 mg / kg of FZD4 ECD.Fc, FZD5 ECD.Fc or FZD10 ECD.Fc 2-3x per week for a total of 4 weeks. Animals given control injections were given injections 2x per week for a total of 4 weeks. Tumor size was assessed on days 21, 24, 28 and 30. Treatment with both soluble FZD10 ECD.Fc and FZD7 ECD.Fc reduced the total tumor volume compared to control treated animals (Fig. 4). The tumor volume reduction by FZD7 ECD.Fc was statistically significant on day 28 and day 30 (Fig. 4).

[0136] The effect of soluble FZD Fc receptor treatment on the presence of cancer stem cells in the tumor was then evaluated. Tumor samples from FZD Fc-treated and control mice were cut into small pieces, completely milled using sterile blades, and single cell suspensions were obtained by enzymatic digestion and mechanical disruption. Dissociated tumor cells were then analyzed by FACS analysis for the presence of tumor stem cancer cells based on the expression of cell surface markers ESA +, CD44 +, CD24- / low, Lin-, as described in detail above.

[0137] The tumorigenicity of cells isolated based on ESA +, CD44 +, CD24- / low, Lin- expression after FZD Fc treatment was then evaluated. 5,000, 1,000, 500 and 100 isolated ESA +, CD44 +, CD24- / low, Lin tumor tumor stem cells from FZD Fc-treated or control mice were re-injected subcutaneously into the breast pads of NOD / SCID mice. Tumorigenicity of cancer cells

The mothers were therefore assessed based on the number of cells that had to be injected for consistent tumor formation.

[0138] In some embodiments, female rag2 / γ chain double knockout mice were injected at 5-7 weeks of age with 50,000 tumor derived cells with murine mammary tumor virus (MMTV) -WNT1 tumor in the upper right mammary fat pad. Transgenic mice (MMTV) -Wnt-1 exhibit separate neoplastic tumor stages, including hyperplasia, invasive ductal carcinoma and distant metastasis, and therefore this mouse model of breast cancer is a useful tool for analyzing the role of Wnt in tumor formation and growth (Nusse and Varmus (1982) Cell 31: 99-109). Tumors from these mice were dissociated and these dissociated tumor cells were used to propagate the tumor. Mice with tumor cells implanted into nipple fat pads were treated 5x a week with 200 μΐ PBS (n = 10) or soluble FZD8 Fri.Fc receptor (10 mg / kg) diluted in PBS. When tumors were palpable, tumor sizes were measured twice a week. Soluble FZD8 Fri.Fc receptor treatment strongly reduced tumor growth compared to control PBS treatment (Fig. 5).

[0139] To re-examine the ability of soluble FZD receptors to inhibit tumor growth, NOD / SCID mice were injected with 50,000 PE13 breast tumor cells. One day after injecting the cells with 200 μl soluble FZD8 Fri.Fc receptor diluted in PBS, ip was injected at 10 mg / kg or 200 μl PBS was injected and treatment was continued 5x a week (n = 10 per experimental group). Tumor growth was monitored weekly until growth was detected, then tumor growth was measured twice a week. Treatment of animals using FZD8 Fri.Fc significantly reduced the growth of breast tumor cells compared to controls injected with PBS (Fig. 6). Example 4

In vivo treatment of tumor growth using soluble FZD Fc receptor protein.

[0140] This example describes the use of soluble FZD Fc receptor for the treatment of tumors in a xenograft model.

[0141] In certain embodiments, 50,000 cells derived from MMTV Wnt1 breast tumor in Matrigel were implanted subcutaneously in 5-7 week old female mice with double knockout rag-2 / y chain. On the nineteenth day, tumor-bearing mice were randomly assigned to groups with an average tumor volume of 65 mm³and on day twenty-six, treatment with FZD8 Fri.Fc or FZD5 Fri.Fc. fusion proteins was started. In particular, FZD8 Fri.Fc fusion protein was administered five times a week at increasingly higher concentrations (5 mg / kg, 10 mg / kg and 30 mg / kg) and FZD5 Fri.Fc was administered at 10 mg / kg. Control animals were treated with PBS.

[0142] Dose-dependent anti-tumor activity of the FZD8 fusion protein was observed

Fri.Fc (Fig. 7). At the lowest dose - 5 mg / kg - FZD8 Fri.Fc reduced tumor growth relative to PBS-treated mice, but treatment plans using 10 mg / kg and 30 mg / kg FZD8 Fri.Fc were significantly more effective in reducing size earlier

-42 developed tumors. In contrast, FZD5 Fri.Fc did not show any anti-tumor activity against developed breast tumors that require Wnt1 for growth.

Example 5

In vivo treatment of tumors using soluble FZD Fc receptor protein [0143] This example describes the use of soluble FZD Fc receptor for cancer treatment in a xenograft model.

[0144] Tumor cells from a patient sample (solid tumor biopsy or pleural effusion) that were passaged as xenograft in mice were prepared for repassaging to experimental animals. Tumor tissue was removed, cut into small pieces, completely ground using sterile blades and single cell suspensions were obtained by enzymatic digestion and mechanical disruption. Dissociated tumor cells were then injected subcutaneously into the fat pads of the nipples to obtain breast tumors or sideways to obtain tumors other than the breast in NOD / SCID mice to induce tumor growth. Alternatively, ESA +, CD44 +, CD24 / low, Lin- tumorigenic tumor cells were isolated as described in detail above and injected.

[0145] After tumor cell injection, the animals were monitored for tumor growth. When the tumors reached an average size of approximately 150 to 200 mm, treatment with FZD Fc protein was started. Each animal received 10 mg / kg FZD Fc or ip control protein two to five times a week for a total of 6 weeks. During these 6 weeks, tumor size was assessed twice a week. The FZD Fc's ability to prevent further tumor growth or reduce tumor size compared to control antibodies was thus determined.

Example 6

Treatment of human cancer using soluble FZD Fc receptor protein [0146] This example describes methods of treating cancer using soluble FZD Fc receptor directed against tumors containing cancer stem cells and / or tumor cells in which FZD receptor expression has been detected.

[0147] The presence of tumor stem cell marker expression can first be determined by tumor biopsy. Tumor cells from a biopsy from a patient diagnosed with cancer are removed under sterile conditions. In one embodiment, the tissue biopsy is freshly frozen in liquid nitrogen, embedded in OCT and sliced ​​on a cryostat into 10 μm sections for slides. Alternatively, the tissue biopsy is fixed in formalin, embedded in paraffin and microtome cut into 10 μm sections for slides. Sections are incubated with anti-FZD receptor antibodies to detect protein expression. In addition, the presence of cancer stem cells can be determined. Tissue biopsy samples are cut into small ones

Pieces, completely ground using sterile blades and the cells are subjected to enzymatic digestion and mechanical disruption to obtain a single cell suspension. Dissociated tumor cells are then incubated with anti-ESA, CD44, -CD24, -Lin and -FZD antibodies to detect cancer stem cells and the presence of ESA +, CD44 +, CD24- / low, Lin-, FZD + tumor stem cells are determined by flow cytometry as described in detail above.

[0148] Cancer patients who have been diagnosed with tumors with cancer stem cells are treated with soluble FZD: Fc receptor. The FZD Fc human fusion protein produced as described above is purified and formulated with a suitable pharmaceutical carrier in PBS for injection. Patients are treated with FZD Fc preferably once a week for at least 10 weeks, but more preferably once a week for at least about 14 weeks. A pharmaceutically effective dose of FZD Fc of about 2 to about 100 mg / ml or about 5 to about 40 mg / ml should be administered each time. FZD Fc can be administered before, simultaneously with or after standard radiotherapy regimens or chemotherapy regimens using one or more chemotherapeutic agents such as oxaliplatin, fluorouracil, leucovorin or streptozocin. Patients are monitored to determine whether such treatment has elicited an anti-tumor response, for example based on tumor regression, reduction of new tumors, lower tumor antigen expression, reduced cancer stem cell numbers, or other means of assessing disease prognosis.

SEQ ID NO: 1

N-terminal extracellular domain FZD10

MQRPGPRLWLVLQVMGSCAAJSSMDMERPGDGKCQPIEIPMCKI&gt; IGYNMTRMPNLMGHENQRBA

AIQLHEFAPLVEYGCHGHLRFFLCSLYAPMCTEQVSTPrPACRVMCEQARLKCSPIMEQFNFKWPD

SLDCRKLPNKNDPNYLCMEAPNNGSDEPTRGSGLFPPLFRPQRPHSAQEHPLKDGGPGRGGCDNP

GKFHHVEKSASCAPLCTPGVDVYWSREDKRFA

SEQ ID NO: 2

N-terminal extracellular domain FZD7

MRDPGAAAPLSSLGLCALVLALLGALSAGAGAQPYHGEKGISVPDHGFCQPISIPLCTDIAYNQTIL

PNLLGHTNQEDAGLEVHQFYPLVKVQCSPELRFFLCSMYAPVCTVLDQAIPPCRSLCERARQGCEA

LMNKFGFQWPERLRCENFPVHGAGEICVGQNTSDGSGGPGGGPTAYPTAPYLPDLPFTALPPGASD

GRGRPAFPFSCPRQLKVPPYLGYRFLGERDCGAPCEPGRANGLMYFKBEERRFARL

SEQ ID NO: 3

N-terminal extracellular domain of FZD5

MARPDFSAPPSLLLLLLAQLVGRAAAASKAPVCQEITVPMCRGIGYNLTHMPNQFNHDTQDEAGL

EVHQFWPLVEIQCSPDLRFFLCSMYTP1CLPDYHKPLPPCRSVCERAKAGCSPLMRQYGFAWPERM

SCDRLPVLORDAEVLCMDYNRSEATTAPPRPFPAKPTLPGPPGAPASOGECPAGGPFVCK.CREPFV

PILKESHPLYNKVRTGQVPNCAVPCYQPSFSADERT

SEQ ID NO: 4

Human IgG1 Fc

-44DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLM] SRTPEVTCVWDVSHEDPEVKFNWYVDGVBVHN

AKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPrEKTISKAKGQPREPQVYTL

PPSRDELTKNQVSLTCLVKGFYPSDIAVBWESNGQPENNYKTTPPVLDSDGSFFLYSICLTVDKSRW

QQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO: 5

N-terminal extracellular domain FZD6

MEMFTFLLTCIFLPLLRGHSLFTCEPITVPRCMKMAYNMTFFPNLMGHYDQSIAAVEMEHFLPLAN

LECSPNIETFLCKAFVPTCIEQIHVVPPCRKI, CEK.VYSDCKK.LIDTFGIRWPEELECDRLQYCDETVP

VTFDPHTEFLGPQKKTEQVQKDIGFWCPRHLKTSGGQGYKFLGIDQCAPPCPNMYFKSDELEFAKS

FIGTVSI

SEQ ID NO: 6

N-terminal extracellular domain of FZD

MLAMAWRGAGPSVPGAPGGVGLSLGLLLQLLLLLGPARGFGDEEERRCDPIRJSMCQNLGYNVTK

MPNLVGHELQTDAELQLTTFTPLIQYGCSSQLQFFLCSVYVPMCTEKINIPIGPCGGMCLSVKRRCE

PVLKEFGFAWPESLNCSKFPPQNDHNHMCMEGPGDEEVPLPHKTPIQPGEECHSVGTNSDQYrWV

KRSLNCVLKCGYDAGLYSRSAKEFTDI

SEQ ID NO: 7

Fri FZD8 domain

MEWGYLLEVTSLLAALALLQRSSGAAAASAKELACQEITVPLCKGIGYNYTYMPNQFNHDTQDE

AGLEVHQFWPLVEIQCSPDLKFFLCSMYTPICLEDYKKPLPPCRSVCERAKAGCAPLMRQYGFAWP

DRMRCDRLPEQGNPDTLCMDYNRTDLTT

SEQ ID NO: 8

Fri FZD4 domain

MLAMAWRGAGPSVPGAPGGVGLSLGLLLQLLLLLGPARGFGDEEERRCDPIRISMCQNLGYNVTK

MPNLVGHELQTDAELQLTTFTPLIQYGCSSQLQFFLCSVYVPMCTEKINIPIGPCGGMCLSVKRRCE

PVLKEFGFAWPBSLNCSKFPPQNDHNHMCMEGPGDEEV

SEQ ID NO: 9

Fri FZD5 domain

MARPDPSAPPSLLLLLLAQLVGRAAAASKAPVCQEITVPMCRG [GYNLTHMPNQFNHDTQDBAOL

EVHQFWPLVEIQCSPDLRFFLCSMYTP1CLPDYHKPLPPCRSVCERAKAGCSPLMRQYGFAWPERM

SCDRLPVLGRDAEVLCMDYNRSEATT

SEQUENCE LIST [0149] &lt;110&gt; OncoMed Pharmaceuticals, Inc.

&lt;120&gt; Compositions and methods for diagnosis and treatment of cancer &lt;130&gt; HMK / FP6810626 &lt;140&gt; EP &lt;141&gt; 2006-10-31 &lt;150&gt; EP 07752161.5 &lt;151&gt; 2006-10-31

-45 &lt;150&gt; PCT / US2007 / 005443 &lt;151&gt; 2006-10-31 &lt;150&gt; US 60 / 812,966 &lt;151&gt; 2006-06-13 &lt;150&gt; US 60 / 731,468 &lt;151&gt; 2005-10-31 &lt;160 &gt; 9 &lt;170&gt; PatentIn version 3.3 &lt;210&gt; 1 &lt;211&gt; 227 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; N-terminal extracellular domain of human FZD10 &lt;400&gt; 1

<td>Underworld 1</td><td>Gln</td><td>Arg</td><td>Pro</td><td>Gly 5</td><td>Pro</td><td>Arg</td><td>Leu</td><td>Trp</td><td>Leu 10</td><td>val</td><td>Leu</td><td>Gln</td><td>val</td><td>Underworld 15</td><td>Gly</td>

<td>Cheese</td><td>Cys</td><td>ala</td><td>ala twenty</td><td>How much</td><td>Cheese</td><td>Cheese</td><td>Underworld</td><td>Asp 25</td><td>Underworld</td><td>Glu</td><td>Arg</td><td>Pro</td><td>Gly thirty</td><td>Asp</td><td>Gly</td>

<td>lys</td><td>Cys</td><td>Gln 35</td><td>Pro</td><td>How much</td><td>Glu</td><td>How much</td><td>Pro 40</td><td>Underworld</td><td>Cys</td><td>lys</td><td>Asp</td><td>How much 45</td><td>Gly</td><td>Tyr</td><td>own</td>

<td>Underworld</td><td>Thr 50</td><td>Arg</td><td>Underworld</td><td>Pro</td><td>own</td><td>Leu 55</td><td>Underworld</td><td>Gly</td><td>His</td><td>Glu</td><td>own 60</td><td>Gln</td><td>Arg</td><td>Glu</td><td>ala</td>

<td>ala 65</td><td>How much</td><td>Gln</td><td>Leu</td><td>His</td><td>Glu 70</td><td>phe</td><td>ala</td><td>Pro</td><td>Leu</td><td>val 75</td><td>Glu</td><td>Tyr</td><td>Gly</td><td>Cys</td><td>His 80</td>

<td>Gly</td><td>His</td><td>Leu</td><td>Arg</td><td>phe 85</td><td>phe</td><td>Leu</td><td>Cys</td><td>Cheese</td><td>Leu 90</td><td>Tyr</td><td>ala</td><td>Pro</td><td>Underworld</td><td>Cys 95</td><td>Thr</td>

<td>Glu</td><td>Gln</td><td>val</td><td>Cheese</td><td>Thr</td><td>Pro</td><td>How much</td><td>Pro</td><td>ala</td><td>Cys</td><td>Arg</td><td>val</td><td>Underworld</td><td>Cys</td><td>Glu</td><td>Gln</td>

100 105 110

<img file="PL2500360T3_D0001.tif" />

Arg Phe Ala 225 &lt;210&gt; 2 &lt;211&gt; 255 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; N-terminal extracellular domain of human FZD7 &lt;400&gt; 2

-47Met Arg Asp Pro Gly Ala Ala Ala 1 5

Pro Leu Cheese Cheese Leu Gly Leu Cys 10 15

Ala Leu Val Leu Ala Leu Leu Gly 20

Ala Leu Cheese Ala Gly Ala Gly Ala 25 30

Gln Pro Tyr His Gly Glu Lys Gly 35 .40

Ile Ser Val Pro Asp His Gly Phe I 45

Cys Gln Pro Ile Cheese Ile Pro Leu 50 55

Cys Thr Asp Ile Ala Tyr Asn Gln 60

Thr Ile Leu Pro Asn Leu Leu Gly 65 70

His Thr Asn Gln Glu Asp Ala Gly 75 80

Leu Glu Val His Gln Phe Tyr Pro 85

Leu Val Lys Val Gln Cys Ser Pro 90 95

-48Glu Leu Arg Phe Phe Leu Cys Ser Met Tyr Ala Pro Val Cys Thr Val 100 105 110

Leu Asp Gin Ala Ile Pro Pro Cys Arg Cheese Leu Cys Glu Arg Ala Arg 115 120 125

Gin Gly Cys Glu Ala Leu Met Asn Lys Phe Gly Phe Gin Trp Pro Glu 130 135 140

Arg Leu Arg Cys Glu Asn Phe Pro Val His Gly Ala Gly Glu Ile Cys 145 150 155 160

Val Gly Gin Asn Thr Ser Asp Gly Ser Gly Gly Pro Gly Gly Gly Pro 165 170 175

Thr Ala Tyr Pro Thr Ala Pro Tyr Leu Pro Asp Leu Pro Phe Thr Ala 180 185 190

Leu Pro Pro Gly Ala Cheese Asp Gly Arg Gly Arg Pro Ala Phe Pro Phe 195 200 205

Cheese Cys Pro Arg Gin Leu Lys Val Pro Pro Tyr Leu Gly Tyr Arg Phe 210 215 220

Leu Gly Glu Arg Asp Cys Gly Ala Pro CyS Glu Pro Gly Arg Ala Asn 225 230 235 240

Gly Leu Met Tyr Phe Lys Glu Glu Glu Arg Arg Phe Ala Arg Leu 245 250 255 &lt;210&gt; 3 &lt;211&gt; 233 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; N-terminal extracellular domain of human FZD5 &lt;400&gt; 3

<img file="PL2500360T3_D0002.tif" />

-50Leu Glu Val His Gin Phe Trp Pro Leu Val Glu Ile Gin Cys Ser Pro 65 70 75 80

Asp Leu Arg Phe Phe Leu Cys Ser Met Tyr Thr Pro Ile Cys Leu Pro 85 90 95

Asp Tyr His Lys Pro Leu Pro Pro Cys Arg Ser Val Cys Glu Arg Ala 100 105 110

Lys Ala Gly Cys Ser Pro Leu Met Arg Gin Tyr Gly Phe Ala Trp Pro 115 120 125

Glu Arg Met Cheese Cys Asp Arg Leu Pro Val Leu Gly Arg Asp Ala Glu

130

135

140

Val Leu Cys Met Asp Tyr Asn Arg Ser Glu Ala Thr Thr Ala Pro Pro 145 150 155 160

Arg Pro Phe Pro Ala Lys Pro Thr Leu Pro Gly Pro Pro Gly Ala Pro 165 170 175

Ala Ser Gly Gly Glu Cys Pro Ala Gly Gly Pro Phe Val Cys Lys Cys 180 185 190

Arg Glu Pro Phe Val Pro Ile Leu Lys Glu Ser His Pro Leu Tyr Asn 195 200 205

Lys Val Arg Thr Gly Gin Val Pro Asn Cys Ala Val Pro Cys Tyr Gin "210 215 220

Pro Ser Phe Ser Ala Asp Glu Arg Thr 225 230 &lt;210&gt; 4 &lt;211&gt; 227 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Human Fc IgG1 &lt;400&gt; 4

<img file="PL2500360T3_D0003.tif" />

-5235

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gin Tyr Asn Ser Thr Tyr 65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gin Asp Trp Leu Asn Gly 85 90 95

Lys Glu Tyr Lys Cys Lys Val Cheese Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110

Glu Lys Thr Ile Ser Lys Alii Lys Gly Gin Pro Arg Glu Pro Gin Val

115

120

125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gin Val Ser 130 135 140

Leu Thr Cyś Leu Val Lys Gly Phe Tyr Pro Cheese Asp Ile Ala Val Glu 145 150 155 160

Trp Glu Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190

Asp Lys Ser Arg Trp Gin Gin Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser 210 215 220

Pro Gly Lys 225 &lt;210&gt; 5 &lt;211&gt; 207 &lt;212&gt; PRT

-53 &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; N-terminal extracellular domain of human FZD6 &lt;400&gt; 5

Met Glu Met Phe Thr Phe Leu Leu Thr Cys Ile Phe Leu Pro Leu Leu 15 10 15

<img file="PL2500360T3_D0004.tif" />

&lt;210&gt; 6 &lt;211&gt; 224

-54 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; N-terminal extracellular domain of human FZD &lt;400&gt; 6

Met Leu Ala Met Ala Trp Arg Gly Ala Gly Pro Ser Val Pro Gly Ala

5 10 15

Pro Gly Gly Val Gly Leu Cheese Leu Gly Leu Leu Lein Gin Leu Leu Leu

25 30

-55Leu Leu Gly Pro Ala Arg Gly Phe 35 40

Gly Asp Glu Glu Glu Arg Arg Cys 45

Asp Pro Ile Arg Ile Ser Met Met Cys 50 55

Gln Asn Leu Gly Tyr Asn Val Thr 60

Lys Met Pro Asn Leu Val Gly His 65 70

Glu Leu Gln Thr Asp Ala Glu Leu 75 80

Gln Leu Thr Thr Phe Thr Pro Leu 85

Ile Gln Tyr Gly Cys Cheese Cheese Gln 90 95

Leu Gln Phe Phe Leu Cys Cheese Val 100

Tyr Val Pro Met Cys Thr Glu Lys 105 110

Ile Asn Ile Pro Ile Gly Pro Cys 115 120

Gly Gly Met Cys Leu Cheese Val Lys 125

Arg Arg Cys Glu Pro Val Leu Lys 130 135

Glu Phe Gly Phe Ala Trp Pro Glu 140

Leu Asn Cys cheese Lys Phe Pro 145 150 cheese

Pro Gln Asn Asp His Asn His Met 155 160

Cys Met Glu Gly Pro Gly Asp Glu 165

Glu Val Pro Leu Pro His Lys Thr 170 175

Pro Ile Gln Pro Gly Glu Glu Cys 180

His Ser Val Gly Thr Asn Ser Asp 185 190

Gln Tyr Ile Trp Val Lys Arg Ser 195 200

Leu Asn Cys Val Leu Lys Cys Gly 205

Tyr Asp Ala Gly Leu Tyr Ser Arg 210 215 &lt;210&gt; 7 &lt;211&gt; 158 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Fri domain of human FZD8

Ala Lys Glu Phe Thr Asp Ile 220 cheese

-56&lt;400&gt; 7

Met Glu Trp Gly Tyr Leu Leu Glu Val Thr Ser Leu Leu Ala Ala Leu 15 10 15

Ala Leu Leu Gin Arg Cheese Cheese Gly Ala Ala Ala Ala Cheese Ala Lys Glu 20 25 30

Leu Ala Cys Gin Glu Ile Thr Val Pro Leu Cys Lys Gly Ile Gly Tyr 35 40 45

Asn Tyr Thr Tyr Met Pro Asn Gin Phe Asn His Asp Thr Gin Asp Glu 50 55 60 '

Ala Gly Leu Glu Val His Gin Phe Trp Pro Leu Val Glu Ile Gin Cys 65 70 75 80

Ser Pro Asp Leu Lys Phe Phe Leu Cys Ser Met Tyr Thr Pro Ile Cys 85 90 95

Leu Glu Asp Tyr Lys Lys Pro Leu Pro Pro Cys Arg Ser Val Cys Glu 100 I 105 110

Arg Ala Lys Ala Gly Cys Ala Pro Leu Met Arg Gin Tyr Gly Phe Ala 115 120 125

Trp Pro Asp Arg Met Arg Cys Asp Arg Leu Pro Glu Gin Gly Asn Pro 130 135 140

Asp Thr Leu Cys Met Asp Tyr Asn Arg Thr Asp Leu Thr Thr 145 150 155 &lt;210&gt; 8 &lt;211&gt; 170 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Fri human FZD4 domain &lt;400&gt; 8

-57Met Leu Ala Met Ala Trp Arg Gly Ala Gly Pro Cheese Val Pro Gly Ala

5 10 ' 15

Pro Gly Gly Val Gly Leu Cheese Leu Gly Leu Leu Leu Gin Leu Leu Leu 20 25 30

Leu Leu Gly Pro Ala Arg Gly Phe Gly Asp Glu Glu Glu Arg Arg Cys 35 40 45

Asp Pro Ile Arg Ile Ser Met Cys Gin Asn Leu Gly Tyr Asn Val Thr 50 55 60 "

Lys Met Pro Asn Leu Val Gly His Glu Leu Gin Thr Asp Ala Glu Leu 65 70 75 80

Gin Leu Thr Thr Phe Thr Pro Leu Ile Gin Tyr Gly Cys Ser Ser Gin

90 95

Leu Gin Phe Phe Leu Cys Ser Val Tyr Val Pro Met Cys Thr Glu Lys 100 105 110

Ile Asn Ile Pro Ile Gly Pro Cys Gly Gly Met Cys Leu Ser Val Lys 115 120 125

Arg Arg Cys Glu Pro Val Leu Lys Glu Phe Gly Phe Ala Trp Pro Glu 130 135 140

Leu Asn Cys Cheese Lys Phe Pro Pro Gin Asn Asp His Asn His Met 145 150 155 160

Cys Met Glu Gly Pro Gly Asp Glu Glu Val 165 170 &lt;210&gt; 9 &lt;211&gt; 157 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Fri human FZD5 &lt;400&gt; domain 9

-58Met Ala Arg Pro Asp Pro Cheese Ala Pro Pro Cheese Leu Leu Leu Leu Leu 1 5 10 15

Leu Ala Gin Leu Val Gly Arg Ala Ala Ala Ala Cheese Lys Ala Pro Val 20 25 30

Cys Gin Glu Ile Thr Val Pro Met Cys Arg Gly Ile Gly Tyr Asn Leu 35 40 45

Thr His Met Pro Asn Gin Phe Asn His Asp Thr Gin Asp Glu Ala Gly 50 55 60

Leu Glu Val His Gin Phe Trp Pro Leu Val Glu Ile Gin Cys Ser Pro 65 70 75 80

Asp Leu Arg Phe Phe Leu Cys Ser Met Tyr Thr Pro Ile Cys Leu Pro 85 90 95

Asp Tyr His Lys Pro Leu Pro Pro Cys Arg Ser Val Cys Glu Arg Ala 100 105 110

Lys Ala Gly Cys Ser Pro Leu Met Arg Gin Tyr Gly Phe Ala Trp Pro 115 120 125

Glu Arg Met Cheese Cys Asp Arg Leu Pro Val Leu Gly Arg Asp Ala Glu 130 135 140

Val Leu Cys Met Asp Tyr Asn Arg Ser Glu Ala Thr Thr 145 150 155

Piotr Godlewski

Patent Attorney