WO1996041194A1 - Diagnostic, pronostic et suivi de maladies dues a l'angiogenese - Google Patents

Diagnostic, pronostic et suivi de maladies dues a l'angiogenese Download PDF

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WO1996041194A1
WO1996041194A1 PCT/US1996/009660 US9609660W WO9641194A1 WO 1996041194 A1 WO1996041194 A1 WO 1996041194A1 US 9609660 W US9609660 W US 9609660W WO 9641194 A1 WO9641194 A1 WO 9641194A1
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angiogenesis
growth factor
factor
angiogenesis associated
bfgf
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PCT/US1996/009660
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English (en)
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Moses Judah Folkman
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The Children's Medical Center Corporation
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Priority to AU62645/96A priority Critical patent/AU6264596A/en
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/475—Assays involving growth factors
    • G01N2333/50—Fibroblast growth factors [FGF]
    • G01N2333/503—Fibroblast growth factors [FGF] basic FGF [bFGF]

Definitions

  • This relates to the fields of biochemistry and cellular biology and more particularly relates to angiogenesis associated factors for the detection and monitoring of angiogenesis dependent diseases.
  • angiogenesis means the generation of new blood vessels into a tissue or organ. Under normal physiological conditions, humans or animals only undergo angiogenesis in very specific restricted situations. For example, angiogenesis is normally observed in wound healing, fetal and embryonal development and formation of the corpus luteum, endometrium and placenta.
  • the control of angiogenesis is a highly regulated system of angiogenic stimulators and inhibitors. The control of angiogenesis has been found to be altered in certain disease states and, in many cases, the pathological damage associated with the disease is related to the uncontrolled angiogenesis.
  • Endothelial cells and pericytes surrounded by a basement membrane, form capillary blood vessels.
  • Angiogenesis begins with the erosion of the basement membrane by enzymes released by endothelial cells and leukocytes.
  • the endothelial cells which line the lumen of blood vessels, then protrude through the basement membrane.
  • Angiogenic stimulants induce the endothelial cells to migrate through the eroded basement membrane.
  • the migrating cells form a "sprout" off the parent blood vessel, where the endothelial cells undergo mitosis and proliferate.
  • the endothelial sprouts merge with each other to form capillary loops, creating the new blood vessel.
  • Persistent, unregulated angiogenesis occurs in a multiplicity of disease states, tumor metastasis and abnormal growth by endothelial cells and supports the pathological damage seen in these conditions.
  • the diverse pathological states created due to unregulated angiogenesis have been grouped together as angiogenic dependent or angiogenic associated diseases. Methodologies for monitoring the onset or progression of the angiogenic processes could lead to the early recognition of the presence of the disease, or a determination of the severity of the disease.
  • ocular neovascular disease This disease is characterized by invasion of new blood vessels into the structures of the eye such as the retina or cornea. It is the most common cause of blindness and is involved in approximately twenty eye diseases. In age-related macular degeneration, the associated visual problems are caused by an ingrowth of chorioidal capillaries through defects in Bruch's membrane with proliferation of fibrovascular tissue beneath the retinal pigment epithelium. Angiogenic damage is also associated with diabetic retinopathy, retinopathy of prematurity, corneal graft rejection, neovascular glaucoma and retrolental fibroplasia.
  • corneal neovascularization include, but are not limited to, epidemic keratoconjunctivitis, Vitamin A deficiency, contact lens overwear, atopic keratitis, superior limbic keratitis, pterygium keratitis sicca, sjogrens, acne rosacea, • phylectenulosis, syphilis, Mycobacteria infections, lipid degeneration, chemical burns, bacterial ulcers, fungal ulcers, Herpes simplex infections, Herpes zoster infections, protozoan infections, Kaposi sarcoma, Mooren ulcer, Terrien's marginal degeneration, marginal keratolysis, rheumatoid arthritis, systemic lupus, polyarteritis, trauma, Wegeners sarcoidosis, Scleritis, Steven's Johnson disease, periphigoid radial keratotomy, and corneal graph rejection.
  • Diseases associated with retinal/choroidal neovascularization include, but are not limited to, diabetic retinopathy, macular degeneration, sickle cell anemia, sarcoid, syphilis, pseudoxanthoma elasticum, Pagets disease, vein occlusion, artery occlusion, carotid obstructive disease, chronic uveitis/vitritis, mycobacterial infections, Lyme's disease, systemic lupus erythematosis, retinopathy of prematurity, Eales disease, Bechets disease, infections causing a retinitis or choroiditis, presumed ocular histoplasmosis, Bests disease, myopia, optic pits, Stargarts disease, pars planitis, chronic retinal detachment, hyperviscosity syndromes, toxoplasmosis, trauma and post-laser complications.
  • Other diseases include, but are not limited to, diseases associated with rubeosis (n
  • angiogenesis Another disease in which angiogenesis is believed to be involved is rheumatoid cuThritis.
  • the endothelial cells release factors and reactive oxygen species that lead to pannus growth and cartilage destruction.
  • the factors involved in angiogenesis may actively contribute to, and help maintain, the chronically inflamed state of rheumatoid arthritis.
  • Factors associated with angiogenesis may also have a role in osteoarthritis.
  • the activation of the chondrocytes by angiogenic-related factors contributes to the destruction of the joint. At a later stage, the angiogenic factors would promote new bone formation.
  • Therapeutic intervention that prevents the bone destruction could halt the progress of the disease and provide relief for persons suffering with arthritis.
  • Chronic inflammation may also involve pathological angiogenesis.
  • pathological angiogenesis Such disease states as ulcerative colitis and Crohn's disease show histological changes with the ingrowth of new blood vessels into the inflamed tissues. Bartonellosis, a bacterial infection found in South America, can result in a chronic stage that is characterized by proliferation of vascular endothelial cells.
  • Another pathological role associated with angiogenesis is found in atherosclerosis. The plaques formed within the lumen of blood vessels have been shown to have angiogenic stimulatory activity.
  • Hemangioma is a tumor made up of newly-formed blood vessels. In most cases, the tumors are benign and regress without intervention. In more severe cases, the tumors progress to large cavernous and infiltrative forms and create clinical complications. Systemic forms of hemangiomas, the hemangiomatoses, have a high mortality rate. Therapy-resistant hemangiomas exist that cannot be treated with therapeutics currently in use. Angiogenesis is also responsible for damage found in hereditary diseases such as Osier- Weber-Rendu disease, or hereditary hemorrhagic telangiectasia.
  • angiomas This is an inherited disease characterized by multiple small angiomas, tumors of blood or lymph vessels.
  • the angiomas are found in the skin and mucous membranes, often accompanied by epistaxis (nosebleeds) or gastrointestinal bleeding and sometimes with pulmonary or hepatic arteriovenous fistula.
  • Angiogenesis is also involved in normal physiological processes such as reproduction and wound healing. Angiogenesis is an important step in ovulation and also in implantation of the blastula after fertilization. Prevention of angiogenesis could be used to induce amenorrhea, to block ovulation or to prevent implantation by the blastula.
  • Angiogenesis is prominent in solid tumor formation and metastasis. Angiogenic factors have been found associated with several solid tumors such as rhabdomyosarcomas, retinoblastoma, Ewing sarcoma, neuroblastoma, and osteosarcoma. A tumor cannot expand without a blood supply to provide nutrients and remove cellular wastes. Tumors in which angiogenesis is important include solid tumors, and benign tumors such as acoustic neuroma, neurofibroma, trachoma and pyogenic granulomas. Prevention of angiogenesis could halt the growth of these tumors and the resultant damage to the animal due to the presence of the tumor.
  • angiogenesis has been associated with blood-born tumors such as leukemias, any of various acute or chronic neoplastic diseases of the bone marrow in which unrestrained proliferation of white blood cells occurs, usually accompanied by anemia, impaired blood clotting, and enlargement of the lymph nodes, liver, and spleen. It is believed that angiogenesis plays a role in the abnormalities in the bone marrow that give rise to leukemia-like tumors.
  • Angiogenesis is important in two stages of tumor metastasis.
  • the first stage where angiogenesis stimulation is important is in the vascularization of the tumor which allows tumor cells to enter the blood stream and to circulate throughout the body. After the tumor cells have left the primary site, and have settled into the secondary, metastasis site, angiogenesis must occur before the new tumor can grow and expand. Therefore, prevention of angiogenesis could lead to the prevention of metastasis of tumors and possibly contain the neoplastic growth at the primary site.
  • angiogenesis in the maintenance and metastasis of tumors has led to a prognostic indicator for breast cancer.
  • the amount of neovascularization found in the primary tumor was determined by counting the microvessel density in the area of the most intense neovascularization in invasive breast carcinoma. A high level of microvessel density was found to correlate with tumor recurrence. Control of angiogenesis by therapeutic means could possibly lead to cessation of the recurrence of the tumors.
  • Cancer which is defined herein as a pathological neoplastic or hyperplastic growth in or on the body, may be in the form of a solid tumor, such as a carcinoma or sarcoma, or a non-solid tumor, or a hyperplastic growth by cells, such as leukemia.
  • the cancer may develop in a specific kind of cell found within a specific organ or may be found in cells which are located in various sites throughout the body.
  • the first steps in cancer treatment are generally the diagnosis and identification of the type of cancer, followed by prognosis of the disease.
  • an advanced cancer is more difficult to treat with success than a cancer in the early stages of growth. Therefore, the treatment regime prescribed for a cancer in the early stages may be different that the treatment prescribed for the cancer in later stages.
  • Some patients have only one occurrence of cancer and may be treated using standard oncological methods such as surgery, chemotherapy, or radiation, and then never experience a recurrence. Others experience remission after treatment, but the cancer redevelops, either in the same location as the first or in a different location. Even after extensive treatments, some cancers metastasize to multiple locations within the body.
  • Prognostic agents are used to predict the future health status of a patient. Prognostic agents may be used to differentiate between patients who will remain in remission after cancer treatment and those in whom the cancer will recur or metastasize. The ability to differentiate between these two sets of patients could determine the type and duration of initial therapy and the extent of reexamination required after initial therapy.
  • a non-invasive method of determining the effects of the chemotherapy preferably an analysis of the patient's body fluids, such as cerebrospinal fluid or. more preferably, urine would be greatly preferred for economic, simplicity, and safety reasons.
  • a cancer prognosis method to determine the severity of the cancer or terminal illness and to predict recurrence following the removal or treatment of the cancer by standard oncological treatments such as surgery or chemotherapy.
  • Such a method would preferably be non-invasive and would preferably be able to predict the future reoccurrence of the initial cancer and detect the growth of metastatic foci seeded by the initial cancer.
  • compositions and methods for detecting the presence of angiogenesis associated factors which indicate that angiogenesis is occurring in the body. These compositions and methods could be used to detect the presence of angiogenesis dependent diseases in the body. The compositions and methods could also monitor the progression of the disease and also the efficacy of a therapeutic regime for treatment of the disease.
  • compositions, methods and kits for determining the presence of angiogenesis associated factors for the diagnosis and prognosis of angiogenesis dependent diseases and monitoring the diseases in a human or animal are provided herein.
  • the concentration of one or more angiogenesis associated factors in a biological sample of a patient is monitored alone or in combination with other angiogenesis associated factors.
  • Some angiogenesis associated factors have stimulatory effects and promote angiogenesis, and other angiogenesis associated factors have inhibitory effects and either halt or reverse angiogenesis.
  • angiogenesis associated factors which promote angiogenesis are referred to as angiogenesis associated growth factors and angiogenesis associated factors which inhibit angiogenesis are referred to as angiogenesis associated inhibitory factors.
  • angiogenesis associated factors such as bFGF, an angiogenesis associated growth factor
  • concentrations of angiogenesis associated factors may be monitored alone or in combination with other growth factors or with one or more angiogenesis associated inhibitory factors, such as angiostatin.
  • angiogenesis associated inhibitory factors such as angiostatin.
  • the diagnosis method may be used alone or in conjunction with other standard diagnostic tools.
  • the concentration of one or more angiogenesis associated growth factors and one or more angiogenesis associated inhibitory factors, such as angiostatin, are determined.
  • compositions, methods and kits may be used to detect the presence of angiogenesis associated factors for the diagnosis, prognosis and monitoring of the angiogenesis dependent diseases.
  • detection of the presence of angiogenesis associated growth factors is used for the diagnosis of angiogenesis dependent diseases such as hemangiomas.
  • the preferred growth factors to be monitored in accordance with the methods provided herein include angiogenesis associated growth factors that promote angiogenesis such as basic fibroblast growth factor (bFGF), acidic fibroblast growth factor (aFGF), nerve growth factor (NGF), epidermal growth factor (EGF), insulin-like growth factors 1 and 2, (IGF-1 and IGF-2), platelet derived growth factor (PDGF), tumor angiogenesis factor (TAF), vascular endothelial growth factor (VEGF), corticotropin releasing factor (CRF), transforming growth factors ⁇ and ⁇ (TGF- ⁇ and TGF- ⁇ ), interleukin-8 (EL-8); tumor necrosis factors ⁇ and ⁇ (TNF- ⁇ and TNF- ⁇ ), granulocyte-macrophage colony stimulating factor (GM-CSF); the interleukins, the interferons, and oncogene products such as Bence Jones proteins and carcinoembryonic antigen (CEA) and their receptors.
  • bFGF basic fibroblast growth
  • a preferred angiogenesis associated inhibitory factor to be monitored in accordance with the methods provided herein includes angiostatin.
  • concentrations are determined by diagnostic methods well known to those skilled in the art such as immunoassays.
  • the biological sample analyzed to determine the concentration of growth factor or endogenous inhibitor is preferably a biological fluid of the patient that is obtained by a relatively non-invasive means including saliva, gingival secretions, cerebrospinal fluid, gastrointestinal fluid, mucous, urogenital secretions, blood, serum, plasma, urine, cystic fluid, ascites, pleural effusion, interstitial fluid, intracellular fluid, ocular fluids, mammary secretions, and nasal secretions.
  • the angiogenesis associated growth factor concentrations determined from the biological sample are compared with normal, or baseline, growth factor concentrations.
  • An angiogenesis associated growth factor concentration significantly higher than a normal growth factor concentration is an indication that an angiogenesis dependent disease is present. For example, an increased concentration indicates that a primary cancer exists and is in an advanced stage of growth and may be metastatic or terminal.
  • An elevated angiogenesis associated growth factor concentration is also an indication of tumor regrowth or the metastasis of a cancer that had been in remission.
  • An elevated angiogenesis associated growth factor concentration is also an indication of the presence of one or several hemangiomas in the patient.
  • compositions, methods and kits for the detection of angiogenesis associated factors for the diagnosis, prognosis and monitoring of angiogenesis dependent diseases It is a further object of the present invention to provide a non-invasive method for the diagnosis, prognosis and monitoring of angiogenesis dependent diseases, such as cancer, in a human or animal.
  • FIG. 1 is a graph showing the percent distribution of abnormally elevated levels of basic fibroblast growth factor (bFGF) in the urine of patients with various types of cancer.
  • bFGF basic fibroblast growth factor
  • Figure 2 is a chart showing the level of bFGF in the serum of patients having various forms or types of cancer.
  • Figure 3 is chart showing the mortality of cancer patients having high levels of bFGF in serum compared to low levels of bFGF and controls.
  • Figure 4 is chart showing the mortality of breast cancer patients having high levels of bFGF in serum compared to low levels of bFGF and controls.
  • Figure 5 is a histogram of normal humans as controls showing the fraction of controls having various levels of bFGF in urine, normalized for creatinine in the urine.
  • Figure 6 is a chart showing the bFGF level in the urine of cancer patients having clinically active tumor and metastasis compared to inactive and controls.
  • Figure 7 is a chart showing the bFGF level in urine for patients having various angiogenic states.
  • Figure 8 is a chart showing the bFGF level in cerebrospinal fluid for cancer patients having various brain tumors.
  • compositions, methods and kits for determining the presence of angiogenesis associated factors for the diagnosis and prognosis of angiogenesis dependent diseases and monitoring the diseases in a human or animal are described.
  • the concentration of one or more angiogenesis associated factors in a biological sample of a patient is monitored alone or in combination with other angiogenesis associated factors.
  • Some angiogenesis associated factors have stimulatory effects and promote angiogenesis, and other angiogenesis associated factors have inhibitory effects and either halt or reverse angiogenesis.
  • angiogenesis associated factors which promote angiogenesis are referred to as angiogenesis associated growth factors and angiogenesis associated factors which inhibit angiogenesis are referred to as angiogenesis associated inhibitory factors.
  • angiogenesis dependent disease such as cancer
  • the concentration of one or more angiogenesis associated factors such as an angiogenesis associated growth factor
  • the concentration of one or more angiogenesis associated inhibitory factors such as angiostatin.
  • the concentrations of one or more angiogenesis associated growth factors and one or more angiogenesis associated inhibitory factors, such as angiostatin are determined and compared.
  • the concentrations of one or more angiogenesis associated growth factors and one or more angiogenesis associated inhibitory factors, in a patient's fluid are determined to establish the diagnosis or prognosis of the patient's angiogenesis dependent disease or to monitor the condition of the patient's angiogenesis dependent disease or the efficacy of the treatment of the angiogenesis dependent disease.
  • angiogenesis associated growth factor concentrations and angiogenesis associated inhibitory factor concentrations determined from the biological sample are compared with normal growth factor concentrations and endogenous inhibitor concentrations by diagnostic methods well known to those skilled in the art such as immunoassays, as described in more detail below.
  • diagnostic methods well known to those skilled in the art such as immunoassays, as described in more detail below.
  • an angiogenesis associated growth factor concentration higher than a normal, or baseline, or control growth factor concentration is a prognostic indication that a primary cancer is present or that a primary cancer is in an advanced stage of growth.
  • an angiogenesis associated growth factor concentration higher than a normal, or baseline, or control growth factor concentration is a prognostic indication that a primary cancer may be metastatic.
  • An elevated angiogenesis associated growth factor concentration is also an indication of tumor regrowth or the metastasis of a cancer that had been in remission.
  • An elevated angiogenesis associated inhibitory factor concentration alone or in combination with an elevated growth factor concentration diagnostically indicates the presence of a primary tumor.
  • the biological sample analyzed to determine the concentration of angiogenesis associated growth factor or angiogenesis associated inhibitory factor is preferably a biological fluid of the patient obtained by a relatively non- invasive means and includes, but is not limited to, saliva, gingival secretions, cerebrospinal fluid, gastrointestinal fluid, mucous, urogenital secretions, blood, serum, plasma, urine, cystic fluid, ascites, pleural effusion, interstitial fluid, intracellular fluid, ocular fluids, mammary secretions, and nasal secretions.
  • Angiogenesis dependent diseases are pathologies associated with an angiogenic state different from the normal angiogenesis of the healthy body, such as menstruation or wound healing, as described above. Though not wishing to be bound by the following, it theorized that there are angiogenesis associated factors released by the cells or tissues associated with the pathology or affected by the pathology. Some of these factors are growth factors which act to increase the formation of new blood vessels and contribute to the onset or continuation of the angiogenesis dependent disease. Other factors, angiogenesis associated inhibitory factors, are released and inhibit the formation of new blood vessels and either lead to a steady-state in the angiogenesis dependent disease or a cessation of the disease.
  • angiogenesis dependent disease is seen in cancer, such as tumor formation.
  • angiogenesis associated factors such as angiogenic peptides or growth factors, which act locally and target endothelium in the neighborhood of a primary tumor from an extravascular direction, and do not circulate widely (or circulate with a short half-life).
  • angiogenic peptides must be produced in an amount sufficient to overcome the action of endothelial cell inhibitor (inhibitors of angiogenesis) for a primary tumor to continue to increase its population of cells.
  • angiogenesis associated inhibitory factors such as endothelial cell inhibitors
  • these inhibitors act at a distance from the primary tumor, target capillary endothelium of a tumor metastasis from an intravascular direction, and continue to circulate.
  • the capillary endothelium in its neighborhood could be overcome by incoming angiogenesis associated inhibitory factors, such as angiostatin. The tumor metastasis is thus prevented from growing.
  • angiogenesis associated inhibitory factors such as angiostatin
  • a second tumor implant e.g., a tumor implanted experimentally into the subcutaneous space, or into the cornea, or intravenously to the lung
  • a micrometastasis occurs at the same time, or shortly after a primary tumor is implanted
  • the primary tumor will not be able to suppress the secondary tumor because angiogenesis in the secondary tumor will already be well underway and the angiogenesis associated inhibitory factors from the primary tumor cannot suppress the growth of the second tumor.
  • the angiogenesis associated inhibitory factors may have an equivalent inhibiting effect on each other.
  • the diagnostic methods described herein involve testing the body fluids of a human or animal patient for the presence of angiogenesis associated factors to determine if an angiogenesis dependent disease state is occurring in or on the body.
  • the body fluids are tested for the presence of angiogenesis associated growth factors, either individually or as group of growth factors, along with testing for angiogenesis associated inhibitory factors such as angiostatin.
  • the prognostic methods described herein involve testing the bodily fluids of a patient, human or animal, which currently has or formerly had an angiogenesis dependent disease.
  • the initial cancer may have been treated in some way so that the patient is either in remission or exhibits no signs of cancer.
  • angiogenesis associated factors such as a growth factor found to be associated with that cancer, or a series or panel of growth factors, alone or in addition to monitoring the angiogenesis associated inhibitory factors, the prognosis of the patient can be determined.
  • a urine sample taken from a patient who has been diagnosed with cancer may be analyzed for the presence of abnormal levels of acidic and basic fibroblast growth factors (aFGF and bFGF); vascular endothelial growth factor (VEGF); interleukin-8 (IL- 8): granulocyte-macrophage colony stimulating factor (GM- CSF); and an angiogenesis associated inhibitory factor such as angiostatin.
  • aFGF and bFGF acidic and basic fibroblast growth factors
  • VEGF vascular endothelial growth factor
  • IL-8 interleukin-8
  • GM- CSF granulocyte-macrophage colony stimulating factor
  • an angiogenesis associated inhibitory factor such as angiostatin.
  • the patient is found to have abnormally high levels of bFGF, VEGF and angiostatin, and a biopsy indicates that the patient has breast cancer.
  • the tumor is surgically removed and chemotherapy prescribed, and the patient is diagnosed as being in remission.
  • a second urine sample indicates that the levels of VEGF and angiostatin have returned to normal, but the level of bFGF remains abnormally high. This indicates that tumor regrowth is likely to occur. Therefore, the patient may be maintained on the chemotherapy for a longer period of time or the chemotherapeutic agent changed or an angiogenic inhibitory factor, such as angiostatin, may be given to the patient for at least as long as the growth factor concentration is above normal.
  • cancer means neoplastic growth, hyperplastic or proliferative growth or a pathological state of abnormal cellular development and includes solid tumors, non-solid tumors, and any abnormal cellular proliferation, such as that seen in leukemia.
  • the term “prognostic method” means a method that enables a prediction regarding the progression of a disease of a human or animal diagnosed with the disease, in particular, an angiogenesis dependent disease.
  • diagnosis method means a method that enables a determination of the presence or type of angiogenesis dependent disease in or on a human or animal.
  • growth factor means a molecule that stimulates the growth, reproduction or synthetic activity of cells.
  • angiogenesis associated factor means a factor which either inhibits or promotes angiogenesis.
  • An example of an angiogenesis associated factor is an angiogenic growth factor, such as basic fibroblastic growth factor, which is an angiogenesis promoter.
  • Another example of an angiogenesis associated factor is an angiogenesis inhibiting factor such as angiostatin.
  • Growth factors are biochemical molecules, such as hormones, that act locally or at a distant site to stimulate cell growth. Growth factors trigger a variety of morphological and biochemical changes in a cell, often causing the cell to exhibit the characteristics of a transformed cell. Growth factors have been implicated in the abnormal regulation of proliferation demonstrated by transformed and cancer-derived cell lines. One hypothesis for the mechanism of activity of growth factors is that transformed cells synthesize growth factors, which then cause the transformed cells to proliferate.
  • Angiogenesis is the generation of new capillaries.
  • the intensity of angiogenesis in a given tumor at the time of initial diagnosis may be directly related to the risk of future metastasis or risk of recurrence.
  • the transformed cells are not only producing growth factors to stimulate their own growth and the growth of other transformed cells, but may also be releasing stimulating factors that promote angiogenesis and vascular infiltration of the cell mass. Therefore, by measuring the angiogenesis associated factors, such as growth factors that promote angiogenesis, one may be able to predict metastasis, cancer regrowth or the mortality risk to the patient.
  • the preferred growth factors whose concentrations are to be determined, monitored, or compared in accordance with the methods provided herein for angiogenesis dependent disease prognosis, diagnosis and monitoring include basic fibroblast growth factor (bFGF), acidic fibroblast growth factor (aFGF), nerve growth factor (NGF), epidermal growth factor (EGF), insulin-like growth factors 1 and 2, (IGF-1 and IGF-2), platelet derived growth factor (PDGF), tumor angiogenesis factor (TAF), vascular endothelial growth factor (VEGF), corticotropin releasing factor (CRF), transforming growth factors ⁇ and ⁇ (TGF- ⁇ and TGF- ⁇ ), (IL-8); tumor necrosis factors and ⁇ (TNF- and TNF- ⁇ ), insulin-like growth factor (IGF), fibroblast derived growth factor, granulocyte- macrophage colony stimulating factor (GM-CSF); the interleukins, such as interleukin-8, the interferons, and oncogene products such as Bence Jones proteins and car
  • the growth factors that are analyzed in accordance with the prognostic or diagnostic methods described herein are the angiogenesis associated growth factors that stimulate angiogenesis such as acidic and basic fibroblast growth factors (aFGF and bFGF); vascular endothelial growth factor (VEGF); interleukin-8 (IL-8); and granulocyte-macrophage colony stimulating factor (GM-CSF).
  • aFGF and bFGF acidic and basic fibroblast growth factors
  • VEGF vascular endothelial growth factor
  • IL-8 interleukin-8
  • GM-CSF granulocyte-macrophage colony stimulating factor
  • a growth factor may be identified as one that stimulates or promotes angiogenesis by evaluating the growth factor in an in situ angiogenesis assay such as the chick embryo chorioallantoic membrane (CAM) assay or the rabbit corneal assay.
  • an in situ angiogenesis assay such as the chick embryo chorioallantoic membrane (CAM) assay or the rabbit corneal assay.
  • the CAM assay is described in the scientific paper of Crum et al., Science 230: 1375 (1985) and U.S. Patent No. 5,001,116, which is incorporated by reference herein. Briefly, fertilized chick embryos are removed from their shells on day 3 or 4, and a methylcellulose disc containing the growth factor is implanted on the chorioallantoic membrane.
  • a disc containing the growth factor of interest may be used to determine enhanced new capillary growth or angiogenesis and thereby identify the growth factor as an angiogenic growth factor.
  • Basic fibroblast growth factor is one member of a family of heparin binding growth factors that is found in a variety of normal and neoplastic tissues. It is a polypeptide hormone having a molecular weight of about 17,000 daltons and has a basic isoelectric point. bFGF exhibits growth promoting action on almost all tissues derived from the mesoderm and acts as a differentiating factor to a mesoderm system. For example, bFGF induces the proliferation of neuroectoderm-derived cells, the proliferation of vascular endothelial cells resulting in angiogenesis, and the proliferation of tumor cells. The amino acid sequence of bFGF is described in the scientific paper of F. Esch et al., Proc. Natl. Acad. Sci.
  • Basic FGF may be released by tumor cells, a combination of tumor cells and vascular endothelial cells, or some other source.
  • Vascular endothelial cells synthesize bFGF, and in some tumors, endothelial cells appear to stain more intensely for bFGF than do tumor cells.
  • Newly proliferating capillary endothelial cells that become part of a tumor could thus contribute to the abnormally high urinary levels of bFGF.
  • NGF nerve growth factor
  • EGF Epidermal growth factor
  • PDGF platelet derived growth factor
  • EGF may have a role in cell proliferation and differentiation based on studies that show that EGF will induce early eyelid opening and incisor development in newborn mice.
  • PDGF is released from platelets during blood clot formation at wound sites, and may have a role in a repair processes.
  • Tumor angiogenesis factor (TAF) is a growth factor-like molecule that has been isolated from human and animal tumors. TAF is mitogenic to endothelial cells and stimulates rapid formation of new capillaries in animals. TAF is not found in normal tissues with the exception of placenta.
  • VEGF Vascular endothelial growth factor
  • angiogenesis inducer released by a variety of tumor cells and expressed in human tumors in situ.
  • VEGF may be a mediator of normal and pathological angiogenesis.
  • More potential mediators of tumor angiogenesis include bFGF and aFGF, TNF- , TGF ⁇ , and TGF- ⁇ .
  • VEGF may exist in four different homodimeric molecular specifies, each monomer having 121 , 165, 189, or 206 amino acids respectively.
  • VEGF121 and VEGF165 are soluble proteins, whereas VEGF189 and VEGF206 are bound to heparin- containing proteoglycans in the cell surface or in the basement membrane.
  • a variety of transformed cell lines express the VEGF mRNA and secrete VEGF.
  • in situ hybridization studies demonstrate expression of VEGF mRNA at high levels in various human tumors, including the highly vascularized glioblastoma multi-forme and capillary haemangioglastoma.
  • Chinese hamster ovary cells which express VEGF165 or
  • VEGF121 form tumors in nude mice.
  • An antibody specific for VEGF is described by Kim K. J., et al., Inhibition of vascular endothelial growth factor-induced angiogenesis suppresses tumor growth in vivo. Nature 362:841-844 (1993), which is incorporated by reference herein.
  • Angiogenesis associated inhibitory factors suppress the effects* of angiogenesis associated growth factors or stimulators.
  • An angiogenesis associated inhibitory factor, angiostatin has recently been discovered.
  • Angiostatin is described in U.S. Patent Application Serial Nos. 08/326,785, 08/248,629, and 08/429,743 which are incorporated by reference herein.
  • Angiostatin can overcome the angiogenic activity of endogenous growth factors such as bFGF, in vitro.
  • Angiostatin is a protein having a molecular weight of between approximately 38 kilodaltons and 45 kilodaltons as determined by reducing polyacrylamide gel electrophoresis and having an amino acid sequence substantially similar to that of a murine plasminogen fragment beginning at amino acid number 98 of an intact murine plasminogen molecule.
  • substantially similar when used in reference to angiostatin amino acid sequences, means an amino acid sequence having anti- angiogenic activity and having a molecular weight of approximately 38 kD to 45 kD, which also has a high degree of sequence homology to the peptide fragment of mouse plasminogen beginning approximately at amino acid number 98 in mouse plasminogen and weighing 38 kD to 45 kD.
  • a high degree of homology means at least approximately 60% amino acid homology, desirably at least approximately 70% amino acid homology, and more desirably at least approximately 80% amino acid homology.
  • endothelial inhibiting activity means the capability of a molecule to inhibit angiogenesis in general and, for example, to inhibit the growth of bovine capillary endothelial cells in culture in the presence of fibroblast growth factor.
  • the preferred angiogenesis associated inhibitory factor concentrations to be monitored in accordance with the methods provided herein include angiostatin and other proteins having neovascularization inhibiting activity. It has been discovered that a growing primary tumor is associated with the release into the blood stream specific inhibitor(s) of endothelial cell proliferation, including angiostatin, which can suppress angiogenesis within a distant tumor metastasis and thereby inhibit the growth of the metastasis itself.
  • the compound may be produced by degradation of plasminogen by a specific protease, or angiostatin could be produced by expression of a specific gene coding for angiostatin.
  • the angiogenic phenotype of a primary tumor depends on production of angiogenic peptides in excess of endothelial cell inhibitors which are elaborated by normal cells, but are believed to be down-regulated during transformation to neoplasia. While production of angiostatin may be down-regulated in an individual tumor cell relative to production by its parent, the total inhibitor elaborated by the whole tumor may be sufficient to enter the circulation and suppress endothelial growth at remote sites of micrometastases. Angiostatin remains in the circulation for a significantly longer time than the angiogenic peptide(s) released by a primary tumor. Thus, the angiogenic peptides appear to act locally, whereas angiogenesis associated inhibitory factors act globally, circulating in the blood with relatively long half- lives. The half- life of angiostatin is approximately 12 hours to 5 days.
  • Detection of the concentration of angiogenesis associated factors as used herein means using known techniques for detection of biological factors.
  • Such techniques specifically include immunochemical and histological methods, particularly immunological techniques employing monoclonal or polyclonal antibodies to the growth factors or inhibitors, such as enzyme linked immunosorbant assays, radioimmunoassay, chemiluminescent assays, or other types of assays involving antibodies known to those skilled in the art.
  • Competitive or non-competitive assays are also contemplated by this term. There are many techniques known in the art for detecting a component in a mixture or measuring its amount. Immunoassays, which employ antibodies that bind specifically to the compound of interest, are one of the better known measurement techniques.
  • Classical methods involve reacting a sample containing the analyte with a known excess amount of antibody specific for the analyte, separating bound from free antibody, and determining the amount of one or the other. Often the antibody is labeled with a reporter group to aid in the determination of the amount of bound analyte.
  • the reporter group or "label” is commonly a fluorescent, luminescent or radioactive group or an enzyme.
  • Another form of immunoassay employs two antibodies specific for the analyte, one of which is labeled with an appropriate reporter group.
  • the first antibody which is usually bound to a solid support, binds the analyte from the mixture, forming an antibody-analyte complex.
  • the labeled second antibody is added and binds to this complex.
  • the support is washed to remove unbound labeled antibody, and the amount of bound labeled antibody is determined.
  • the label on the second antibody is an enzyme, the amount of analyte is quantitated by determining the amount of enzyme activity present. For example, some enzymes catalyze a reaction in which a colored product is formed. The color change can be monitored by spectrophotometry. If the label on the second antibody is fluorescent or luminescent, the amount of bound analyte is calculated based on the amount of fluorescence or luminescence measured.
  • Biosensors detect antibody and analyte complexes using a variety of physical methods. Some biosensors measure the change in surface charge that occurs when analyte is bound to antibodies or other binding agents, which in turn are bound to a surface. Other biosensors use binding agents attached to a surface and measure a change in a physical property of the support, other than surface charge, upon binding of analyte. Some biosensor techniques use a specific property of a labeled binding agent or antigen to produce a measurable change.
  • Measuring the concentrations of angiogenesis associated factors can aid in the prognosis of the course of an angiogenesis dependent disease.
  • the prognosis of a patient who was previously diagnosed with cancer can be determined by monitoring the body fluids of the patient for angiogenesis associated growth factors, with or without also measuring angiogenesis associated inhibitory factors.
  • Some factors may function as specific prognostic indicators, such as for a particular angiogenesis dependent disease, whereas other factors may function as generalized prognostic indicators, such as indicators of future neoplastic growth.
  • the angiogenesis associated growth factor monitored may be one that is specific for the patient's specific cancer. Alternatively, a panel of two or more specific or non-specific growth factors may be monitored.
  • the concentrations of angiogenesis associated factors are affected by the angiogenesis dependent disease state.
  • concentrations of angiogenesis associated factors are either increased by the cellular activities associated with an angiogenesis dependent disease or the angiogenesis associated factors are produced de novo by the cellular activities associated with an angiogenesis dependent disease.
  • angiogenesis associated growth factors involved in the angiogenesis dependent disease cancer either the growth factor is newly produced by a neoplastic growth and thus the total concentration of growth factor is measured, or the level of the growth factor is increased by a cancer and amount of increase is calculated.
  • the neoplastic cells may either synthesize the growth factor or the growth factor may affect the neoplastic cells in a manner that causes the cells to increase in number, thereby producing a further increase in growth factor concentration. These activities take place prior to the time when the neoplastic growth is detectable by conventional devices such as NMR or CAT scans.
  • angiogenesis inhibiting factor such as angiostatin
  • the presence of or increase in growth factor concentration allows the physician or veterinarian to predict the course of the angiogenesis dependent disease or the efficacy of treatment regimes. If, for example, a patient who had a certain type of cancer, which was treated, subsequently exhibits an increase in the concentration of a growth factor that is associated with that cancer, the physician or veterinarian can predict that the patient may have additional neoplastic growth in the future or predict a higher risk of death of the patient.
  • the amount of growth factor may be predictive of the outcome of the patient, e.g., how well certain chemotherapeutic agents may act.
  • Angiogenesis Dependent Disease Diagnosis The diagnosis of an angiogenesis dependent disease in a patient can be determined by monitoring the biological fluids of a patient and detecting angiogenesis associated growth factors, individually or in combination with other angiogenesis associated growth factors, or in combination with angiogenesis associated inhibitory factors, or by the measurement of angiogenesis associated inhibitory factors alone or in combination with other angiogenesis associated inhibitory factors.
  • Some angiogenesis associated growth factors may function as specific diagnostic indicators, whereas as other angiogenesis associated growth factors may function as generalized indicators of unregulated angiogenesis or angiogenesis dependent disease.
  • the presence of an individual angiogenesis associated factor may be a diagnostic tool for the presence of a particular type of cancer.
  • the detection in a patient's bodily fluids of the presence of basic fibroblastic growth factor is an indicator of bladder cancer.
  • the presence of an individual angiogenesis associated factor may indicate the presence of an undefined cancer.
  • the presence of the angiogenesis associated inhibitory factor, angiostatin in the body fluids of a patient indicates the presence of a tumor, but does not necessarily indicate the type or location of the cancer.
  • the monitoring may be part of a routine physical examination or could be employed when the presence of an angiogenesis associated factor is indicated by other physiological clues or diagnostic tests.
  • the detection of the presence of specific angiogenesis associated growth factors, individually or in a panel of measurements for growth factors is detected in conjunction with the detection of the presence of specific angiogenesis associated inhibitory factors, such as angiostatin.
  • the panel of factors measured may also provide a non- invasive method of confirming or denying a diagnosis of the presence of an angiogenesis dependent disease in a human or animal.
  • angiogenesis dependent diseases are due to stimulation endothelial cell proliferation, and thus angiogenesis, or the proliferation of new blood vessels, in a vascular bed by secreting angiogenesis associated growth factors.
  • angiogenic stimuli or growth factors include, but are not limited to, acidic and basic fibroblast growth factors (aFGF and bFGF); vascular endothelial growth factor (VEGF); interleukin-8 (IL-8); and granulocyte- macrophage colony stimulating factor (GM-CSF).
  • these endothelial growth factors are labile and have a short half- life (minutes to hours), and thus are active locally in stimulating neovascularization of the primary tumor, but are rapidly inactivated in circulation.
  • These growth factors spill over into circulation or are excreted in urine only when produced in great excess, such as by a large tumor mass.
  • These growth factors are detectable in the bodily fluids of a cancer patient.
  • the primary tumor also generates circulating angiogenesis associated inhibitory factors with a much longer half-life (days), which enter the circulation and inhibit neovascularization in micrometastases at distant tissue sites, thereby inhibiting the growth of the metastases themselves.
  • angiogenesis associated inhibitory factors such as angiostatin
  • angiostatin are detectable in the bodily fluids of a cancer patient and aid in the diagnosis of the presence a cancer within the patient.
  • the presence of these inhibitors can also be used to the monitor the inhibition of the metastatic sites and the prognosis of the growth of these cancers.
  • the level of angiostatin is high, the metastatic sites will not grow. If the level of angiostatin is reduced, the prognosis of the patient would be that additional cancerous growth is likely to occur and tumors will most hkely be found in the patient in the future.
  • Tumor tissues contain focal areas of intense neovascularization, or "hot spots". Therefore, subsets of tumor cells within a primary tumor are more highly angiogenic than other tumor cells.
  • a tumor cell that is already angiogenic when it is shed from the primary tumor is more likely to generate a detectable metastasis when it arrives in a target organ than is a nonangiogenic cell, other conditions being similar. This is because the switch to the angiogenic phenotype in small populations of tumor cells takes time (weeks to months in transgenic animals, and possibly years in humans). Tumor cells that are not angiogenic may become dormant micrometastases. Intensity of tumor angiogenesis is also an indicator of risk of local recurrence of brain tumors.
  • bFGF basic fibroblast growth factor
  • bFGF can be better than cytology for detecting new or recurrent local bladder cancer.
  • the number of patients with high urine level of bFGF was higher than that of patients with high serum level of PSA (prostatic specific antigen).
  • PSA is not specific to the prostatic tumor because it also increases in benign prostatic hypertrophy.
  • bFGF is also not specific for prostatic tumor.
  • Combining the measurement of urine bFGF and PSA can be a better method for diagnosing prostatic cancer.
  • Urine bFGF levels showed tendency to decrease following chemotherapeutic treatment.
  • urine level of bFGF can be a useful marker for detecting recurrent cancer.
  • bFGF levels were not significantly different from the bFGF levels in normal control subjects.
  • the local tumor NED group had a median level of 164 pg/g, with a 90th percentile level of 1150 pg/g, while the metastatic NED group had a median level of 253 pg/g with a 90th percentile level of 1 167 pg/g.
  • active patients with local disease had an elevated median level of 312 pg/g with a 90th percentile level of 2,568 pg/g in comparison with the normal control and NED groups (P ⁇ .05).
  • the active patients with metastatic disease had the highest median level, 479 pg/g, with a 90th percentile level of 14,143 pg/g in comparison with the normal control subjects, the NED groups and the active patients with local disease (P ⁇ .05).
  • Active patients with lymphoma had an elevated bFGF median level of
  • the lymphoma NED group had levels approaching those of normal control subjects, with a median level of 262 pg/g and a 90th percentile level of 623 pg/g.
  • the leukemia NED group had a significantly elevated median level of 574 pg/g and a 90th percentile level of 6,905 pg/g compared with the normal control subjects (P ⁇ .05). All of the above results remained statistically significant even when adjusted for gender difference.
  • Elevated bFGF levels are those greater than the 90th percentile value in the normal control subjects, i.e., 571 pg/g for men and 711 pg/g for women.
  • the percentages of patients in the local active and metastatic active groups with elevated bFGF levels were 31% and 45%, respectively.
  • a substantial number of patients with all types of solid cancers studied except cervical cancer had elevated bFGF levels ranging from 15% to 63%.
  • 5% of NED patients versus 63% of active patients had elevated levels.
  • bFGF levels were equally elevated among NED (50%) and active (54%) patients.
  • bFGF is mainly cell associated because it lacks a signal peptide. It is also localized in the subcellular extracellular matrix. However, bFGF can be exported out of animal tumors, although the mechanism of this export is unknown. The finding of extremely high levels of bFGF in the patients analyzed in these studies suggested that their tumors were exporting abnormally large quantities of this angiogenic peptide. It is possible that some tumors, such as human melanoma, may release more than one angiogenic mediator.
  • Hybridomas HbF52 and HbF98 which produce monoclonal antibodies, MAb52 and MAb98, respectively, have been deposited at the Institute for Fermentation (IFO), Osaka, Japan since August 17, 1987 under the following accession numbers: Mouse HbF52 cell - IFO 50143; Mouse HbF98 cell - LFO 50145.
  • IFO Institute for Fermentation
  • the 3H3 antibody described in this and the following examples can be produced by the method described in PCT WO 91/09875, which is incorporated herein.
  • Mouse 3H3 cells which produce the 3H3 antibody were deposited with the IFO under accession number IFO 50216 on November 10, 1989.
  • the mouse 3H3 cells were also deposited with the Fermentation Research Institute, Agency of Industrial Science and Technology, Ministry of International Trade and Industry,
  • rHbFGF human bFGF
  • the MAb52 plus MAb98 coated plate was prepared as follows. Purified MAb52 and MAb98 were each dissolved in 0.1 M carbonate buffer (pH 9.5) at a concentration of 5 ⁇ g/ml. One hundred microliters of that solution was added to each well of 96-well microtiter plate. After overnight incubation at 4°C, the plate was washed with PBS (0.02 M phosphate buffer, pH 7.2, containing 0.15 M NaCl) and each well was then incubated with 300 microliters of Buffer A (PBS containing 25% Block Ace (Snow Brand Milk Products Co., Japan ) overnight at 4°C.
  • PBS 0.02 M phosphate buffer, pH 7.2, containing 0.15 M NaCl
  • Hybridoma 3H3 cells (-2x10 ⁇ cells) were injected intraperitoneally (i.p.) into Balb/c female mice that had received 0.5 ml. of mineral oil i.p. After 7-10 days, ascitic fluid was collected and MAb3H3 was purified by ammonium sulfate precipitation and Mono Q (Pharmacia, Sweden) column chromatography (Pavlu, et al., J. Chromatog., 359:449-460 (1986)). The Fab' fragment of the purified MAb3H3 (7 mg) was conjugated with horseradish peroxidase (HRP) as described by Isikawa, et al, J Immunoassay, 4:209-327 (1983).
  • HRP horseradish peroxidase
  • the plate was washed with PBS, and 100 microliters of 3H3-HRP solution in Buffer A containing 10 ⁇ g/ml mouse IgG were added to each well. After a 2 hour incubation at 25°C, the plate was washed, and the bound peroxidase activity was measured with o- phenylenediamine (Suzuki, et al., Cancer Res., 41Al%2-4 ⁇ %l (1987)).
  • Serum samples were prepared from the blood samples and the level of bFGF in each sample was measured by the assay described in Example 2 above. bFGF was detectable in 6.8% (58 out of 853) of the cancer patients sera but not in any of the normal samples (FIG. 2).
  • Either clean-voided urine or urine collected in a sterile fashion from an indwelling catheter or cystoscope were used. Samples were placed at 4°C initially, stored at -20°C and then thawed at room temperature. A 1 ml portion of each sample was sent to the chemistry laboratory for a creatinine kinase detection. A 23 ml aliquot was centrifuged at 3000 rpm for 8 minutes at 4°C. The sediment was discarded, and the supernatant urine sample was filtered using a low protein binding filter with a pore size of 1.2 ⁇ m.
  • Each urine sample was dialyzed in SpectraporeTM dialysis tubing (molecular weight cutoff 6,000-8,000 daltons) for 24 hours at 4°C against three changes of distilled water. The dialyzed sample was then lyophilized for two days until all of the sample had been converted into fine powder. The lyophilizate was adjusted to 1 % of the original volume with Buffer D (PBS containing 100 ⁇ g/ml heparin and 10 ⁇ g/ml of mouse IgG). From this mixture, 100 microliters were aliquoted into each of two wells of the MAb52 plus MAb98 coated plate prepared as described in Example 2. The sample was allowed to incubate in each microwell for 24 hours at 4°C.
  • Buffer D PBS containing 100 ⁇ g/ml heparin and 10 ⁇ g/ml of mouse IgG
  • the wells were then washed five times with 300 microliters phosphate buffered saline, following which 100 microliters of 3H3-HRP solution in Buffer A were added (see Example 2). This mixture was allowed to incubate for two hours at room temperature. The plate was then washed five times with 300 microliters of PBS. The bound peroxidase activity was measured with ophenylenediamine.
  • the detection limit of this immunoassay was 100 pg of bFGF/liter of urine.
  • any urine sample with a bFGF content below the detection limit was assigned a value of 99 pg/1.
  • each urine sample bFGF value was normalized against urine creatinine (CR) content according to the followinog formula:
  • Normalized bFGF(pg/g) pg of bFGF/liter g of CR/liter Cancer patients were divided into two major groups according to whether they had solid or hematological tumors. The solid cancer group was further subcategorized according to disease extent and clinical status. Metastatic status was defined as disease involving nodes or distant organs.
  • the active category included patients prior to medical treatment or surgery as well as patients undergoing chemotherapy, radiation therapy or hormonal therapy.
  • the inactive category consisted of patients who had successfully completed treatment, whether medical or surgical, and had been classified by the clinician as "No Evidence of Disease" (NED) based on current staging modalities.
  • the hematological cancer group was divided according to clinical status. The active category included patients both prior to and during treatment. The inactive category consisted of patients who had successfully completed treatment protocols and were classified as in "Complete
  • the data was expressed in terms of medians, 75th and 90th percentiles.
  • the Mann Whitney test was used in order to compare the individual groups to the controls.
  • the median urine bFGF level was 197 pg/g of CR with a 75th percentile of 358 pg/g and 90th percentile of 619 pg/g (FIG. 5).
  • an elevated bFGF level was defined as greater than 619 pg/g, which is the 90th percentile in the normal controls.
  • the local clinically active patients had an elevated median of 312 pg/g with a 75th percentile of 847 pg/g and a 90th percentile of 2,538 pg/g (p ⁇ 0.0001).
  • the clinically metastatic active category had the highest median of 500 pg/g with 75th percentile of 2,600 pg/g and a 90th percentile of 14,286 pg/g (p ⁇ 0.0001).
  • the active lymphoma patients had an elevated bFGF median of 966 pg/g with a 75th percentile of 5,000 pg/g and a 90th percentile of 24,735 pg/g (p ⁇ 0.0001).
  • the clinically active leukemia patients also had an elevated median of 6,46 pg/g with a 75th percentile of 2,232 pg/g and a 90th percentile of 7,917 pg/g (p ⁇ 0.0001).
  • the clinically inactive lymphoma group had levels approaching those of normal patients with a median of 167 pg/g and a 75th percentile of 400 pg/g and a 90th percentile of 428 pg/g.
  • the clinically inactive leukemia group had a significantly elevated median of 512 pg/g, a 75th percentile of 2,833 pg/g and a 90th percentile of 6,667 pg/g (p ⁇ 0.0001).
  • lymphoma and leukemia patients undergoing bone marrow transplant had very elevated bFGF levels with a median of 2,929 pg/g, a 75th percentile of 6,667 pg/g and a 90th percentile of 14,130 pg/g (p ⁇ 0.0001).
  • bFGF is an angiogenic molecule
  • Patients who recently underwent non-cancer related operations (Postop) also had elevated bFGF levels
  • the urine bFGF median value was 906 pg/g with a 75th percentile of 3,182 pg/g and a 90th percentile of 6.435 pg/g.
  • CSF cerebrospinal fluid
  • CSF samples were collected from brain tumor patients under evaluation or treatment.
  • the CSFs from brain tumor patients were obtained prior to surgical resection or were drawn intra-operatively. All sample collections were conducted under approved hospital protocol, and with patient consent.
  • bFGF was detectable in all of the brain tumor patients' CSF (except for patients having medulloblastoma), but not in any of the control samples (FIG. 8).
  • CSF was tested from patients with a variety of brain tumors including astrocytoma, pinealblastoma, craniopharyngioma, glioblastoma and choroid plexis papilloma.
  • Hemangioma is one of the most frequent angiogenic diseases of childhood. In most cases, the tumors are benign and regress without intervention. In more severe cases, the tumors progress to large cavernous and infilrrative forms and create clinical complications which must be treated by therapies.
  • hemangioma is in fact a vascular malformation.
  • urinary bFGF levels were uniformly normal.
  • the urine bFGF level is elevated.
  • bFGF concentration levels were determined in the urine of infants.
  • urine levels of bFGF were elevated abnormally.

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Abstract

L'invention concerne des compositions, des procédés et des matériels permettant de faire un diagnostic, un pronostic et également de suivre l'évolution de maladies dues à l'angiogenèse chez l'homme ou chez un animal. Pour réaliser un pronostic ou suivre l'évolution de maladies dues à l'angiogenèse, on mesure la concentration d'un ou de plusieurs facteurs associés à l'angiogenèse dans un échantillon biologique d'un patient chez qui on a diagnostiqué un tel dérèglement, seul ou en combinaison avec d'autres facteurs associés à l'angiogenèse. Pour réaliser un diagnostic, on détermine la concentration d'un ou de plusieurs facteurs de croissance associés à l'angiogenèse et d'un ou de plusieurs facteurs inhibiteurs liés à l'angiogenèse, comme par exemple l'angiostatine.
PCT/US1996/009660 1995-06-07 1996-06-07 Diagnostic, pronostic et suivi de maladies dues a l'angiogenese WO1996041194A1 (fr)

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US6358735B1 (en) 1995-06-30 2002-03-19 University Of Kansas Medical Center Method for inhibiting angiogenesis and tumors with the isolated NC1 α3 chain monomer of type IV collagen
US6498140B1 (en) 1995-06-30 2002-12-24 University Of Kansas Medical Center Use of isolated domains of type IV collagen to modify cell and tissue interactions
US6440729B1 (en) 1995-06-30 2002-08-27 University Of Kansas Medical Center Treating angiogenesis-mediated diseases with the α2 monomer of type IV collagen
US6432706B1 (en) 1995-06-30 2002-08-13 University Of Kansas Medical Center Method for inhibiting angiogenesis and tumors with the isolated NC1 α6 chain monomer of type IV collagen
US6361994B1 (en) 1995-06-30 2002-03-26 University Of Kansas Medical Center Method for inhibiting angiogenesis and tumors with the isolated NC1 α1 chain monomer of type IV collagen
WO1997035032A1 (fr) * 1996-03-21 1997-09-25 Sugen, Inc. Methode de diagnostic moleculaire de l'angiogenese et de la metastase de tumeurs
US5942385A (en) * 1996-03-21 1999-08-24 Sugen, Inc. Method for molecular diagnosis of tumor angiogenesis and metastasis
US6576609B1 (en) 1996-09-17 2003-06-10 Northwestern University Methods and compositions for generating angiostatin
WO1999003498A1 (fr) 1997-07-18 1999-01-28 Novo Nordisk A/S Utilisation de fvii ou fviiai pour le traitement de pathologies liees au processus de transmission des signaux intracellulaires induites par fviia
WO2001011371A3 (fr) * 1999-08-07 2001-09-07 Ali Said Assad Taha Procede et appareil de diagnostic
WO2001011371A2 (fr) * 1999-08-07 2001-02-15 Ali Said Assad Taha Procede et appareil de diagnostic
US11389453B2 (en) 2005-04-29 2022-07-19 Cyrano Therapeutics, Inc. Compositions and methods for treating chemosensory dysfunction
US8506934B2 (en) 2005-04-29 2013-08-13 Robert I. Henkin Methods for detection of biological substances
US7670849B2 (en) 2005-04-29 2010-03-02 Henkin Robert I Method for diagnosing insulin resistance from nasal secretions
US10206927B2 (en) 2005-04-29 2019-02-19 Cyrano Therapeutics, Inc. Compostions and methods for treating chemosensory dysfunction
US8968706B2 (en) 2005-04-29 2015-03-03 Robert I. Henkin Methods for diagnosing and treating loss or distortion of taste
EP1934373A4 (fr) * 2005-09-14 2009-04-29 Univ California Proteine et arn salivaires permettant la detection du cancer du sein
EP1934373A2 (fr) * 2005-09-14 2008-06-25 The Regents of The University of California Proteine et arn salivaires permettant la detection du cancer du sein
US8663938B2 (en) 2007-01-31 2014-03-04 Robert I. Henkin Methods for detection of biological substances
US8293489B2 (en) 2007-01-31 2012-10-23 Henkin Robert I Methods for detection of biological substances
US9719988B2 (en) 2007-01-31 2017-08-01 Cyrano Therapeutics, Inc. Methods for detection of biological substances
US8580801B2 (en) 2008-07-23 2013-11-12 Robert I. Henkin Phosphodiesterase inhibitor treatment
US10555940B2 (en) 2008-07-23 2020-02-11 Robert I. Henkin Phosphodiesterase inhibitor treatment
WO2010096713A3 (fr) * 2009-02-20 2010-12-02 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Procédé pour le diagnostic de troubles vasculaires liés à l'âge
US10598672B2 (en) 2014-02-18 2020-03-24 Cyrano Therapeutics, Inc. Methods and compositions for diagnosing and treating loss and/or distortion of taste or smell
US11125760B2 (en) 2014-02-18 2021-09-21 Cyrano Therapeutics, Inc. Methods and compositions for diagnosing and treating loss and/or distortion of taste or smell
US11774458B2 (en) 2014-02-18 2023-10-03 Cyrano Therapeutics, Inc. Methods and compositions for diagnosing and treating loss and/or distortion of taste or smell

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