EP1572103A2 - Utilisations anti-angiogeniques d'antagonistes de il-6 - Google Patents

Utilisations anti-angiogeniques d'antagonistes de il-6

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Publication number
EP1572103A2
EP1572103A2 EP03783264A EP03783264A EP1572103A2 EP 1572103 A2 EP1572103 A2 EP 1572103A2 EP 03783264 A EP03783264 A EP 03783264A EP 03783264 A EP03783264 A EP 03783264A EP 1572103 A2 EP1572103 A2 EP 1572103A2
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EP
European Patent Office
Prior art keywords
angiogenesis
mammal
monoclonal antibody
antibody
fragment
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP03783264A
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German (de)
English (en)
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EP1572103A4 (fr
Inventor
Mohit Trikha
Zhao Zhou
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Janssen Biotech Inc
Original Assignee
Centocor Inc
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Publication of EP1572103A2 publication Critical patent/EP1572103A2/fr
Publication of EP1572103A4 publication Critical patent/EP1572103A4/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/244—Interleukins [IL]
    • C07K16/248—IL-6
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/04—Antipruritics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/10—Anti-acne agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A61P27/06—Antiglaucoma agents or miotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

  • the present invention relates to a method of using IL-6 antagonists to treat pathological processes associated with proliferative diseases, such as cancer, by specifically preventing or inhibiting the ability of new tissue to develop a blood supply.
  • the invention more specifically relates to methods of treating such diseases by the use of IL-6 antagonists such as antibodies directed toward IL-6, including specified portions or variants, specific for at least one lnterleukin-6 (IL-6 also known as interferon ⁇ 2)) protein or fragment thereof, in an amount effective to inhibit angiogenesis.
  • IL-6 antagonists such as antibodies directed toward IL-6, including specified portions or variants, specific for at least one lnterleukin-6 (IL-6 also known as interferon ⁇ 2)) protein or fragment thereof, in an amount effective to inhibit angiogenesis.
  • IL-6 antagonists such as antibodies directed toward IL-6, including specified portions or variants, specific for at least one lnterleukin-6 (IL-6 also known as interferon ⁇ 2)) protein or fragment thereof, in an amount effective
  • IL-6 (interleukin 6) is a 22-27 kDa secreted glycoprotein formerly known as monocyte- derived human B-cell growth factor, B-cell stimulatory factor 2, BSF-2, interferon beta-2, and hybridoma growth factor, which has growth stimulatory and proinflammatory activities (Hirano et al. Nature 324: 73-76, 1986).
  • IL-6 belongs to the granulocyte colony-stimulating factor (G-CSF) and myelomonocytic growth factor (MGF) family which includes leukemia inhibitory factor (LIF), oncostatin M (OSM), ciliary neurotropic factor (CNTF), cardiotropin-1 (CT-1), IL-1 , and IL-11.
  • G-CSF granulocyte colony-stimulating factor
  • MMF myelomonocytic growth factor family which includes leukemia inhibitory factor (LIF), oncostatin M (OSM), ciliary neurotropic factor (CNTF), cardiotropin-1
  • Squamous cell carcinoma is the most common malignancy of the larynx and of the head and neck. Often correlated to smoking the, common primary site is the vocal cords (particularly the anterior portion), epiglottis, pyriform sinus, and postcricoid area. Angiogenesis is correlated with regional recurrence (Ch. 88 The Merck Manual 17 1h Ed. 1999). Kaposi's Sarcoma, common to HIV infected patients, is likely a vascular or dysplastic endothelial cell derived from a mesenchymal precursor cell, which may be transformed by exposure to an infectious agent.
  • Murine monocolonal antibodies to IL-6 are known as in, for example, U.S. Patent
  • U.S. Patent 5,856,135 discloses reshaped human antibodies to human IL-6 drived from a mouse monoclonal antibody SK2 in which the complementary determining regions (CDR's) from the variable region of the mouse antibody SK2 are transplanted into the variable region of a human antibody and joined to the constant region of a human antibody.
  • CDR's complementary determining regions
  • CLB-6/8 Another murine IL-6 monoclonal antibody referred to as CLB-6/8 capable of inhibiting receptor signaling was reported (Brakenhoff et al, J. Immunol. (1990) (145:561). A chimerized form of this antibody called cCLB8 was constructed (Centocor, Leiden, The Netherlands) and has been given to multiple myeloma patients (Van Zaanen, et al. 1996 supra). The method of making the resulting antibody from the murine antigen binding domains has been fully described in the applicants' copending application USSN 60/332,743.
  • anti-lL6 Mabs have the potential to impact tumor cell survival and disease progression including: inhibiting growth of human brain tumor cells (Goswami et al. (1998) J Neurochem 71 : 1837-1845) or tumors (Mauray et al. 2000), human renal carcinoma tumors and serum calcium concentrations (Weisglass et al. (1995) Endocrinology 138(5):1879-8), and human hormone refractory prostate tumor xenografts (Smith et al. (2001) Prostate; 48(1):47-53).
  • Chronic inflammatory diseases such as arthritis; dermatological diseases such as psoriasis, telangiectasis, pyogenic granuloma, seborrheic dermatitis, venous ulcers, acne, rosacea (acne rosacea or erythematosa), warts (verrucas), eczema, hemangiomas, lymphangiogenesis are also angiogenesis-dependent.
  • Diabetic retinopathy can take one of two forms, non- proliferative or proliferative.
  • Proliferative retinopathy is characterized by abnormal new vessel formation (neovascularization), which grows on the vitreous surface or extends into the vitreous cavity.
  • neovascularization In advanced disease, neovascular membranes can occur, resulting in a traction retinal detachment.
  • Vitreous hemorrhages may result from neovascularization. Visual symptoms vary. A sudden severe loss of vision can occur when there is intravitreal hemorrhage.
  • Macular degeneration likewise takes two forms, dry and wet. In exudative macular degeneration (wet form), which is much less common, there is formation of a subretinal network of choroidal neovascularization often associated with intraretinal hemorrhage, subretinal fluid, pigment epithelial detachment, and hyperpigmentation.
  • Rheumatoid arthritis an inflammatory disease, also results in inappropriate angiogenesis.
  • the growth of vascular endothelial cells in the synovial cavity is activated by the inflammatory cytokines, and results in cartilage destruction and replacement with pannus in the articulation (Koch AK, Polverini PJ and Leibovich SJ, Arth; 15 Rhenium, 29, 471-479(1986); Stupack DG, Storgard CM and Cheresh DA, Braz. J. Med. Biol. Res., 32, 578-581(1999); Koch AK, Arthritis Rheum, 41 , 951 962(1998)).
  • Psoriasis is caused by uncontrolled proliferation of skin cells. Fast growing cell requires sufficient blood supply, and abnormal angiogenesis is induced in psoriasis (Folkrnan J., J. Invest. Derrnatol., 59, 40- 48(1972)).
  • integrins vascular endothelial growth factor
  • TNFalpha TNFalpha
  • bFGF vascular endothelial growth factor
  • cytokines including IL-6 and IL-12.
  • integrins vascular endothelial growth factor
  • TNFalpha TNFalpha
  • bFGF vascular endothelial growth factor
  • cytokines including IL-6 and IL-12.
  • An antibody generated against aVb3 blocked basic fibroblast growth factor (bFGF) induced angiogenesis
  • VEGF vascular endothelial growth factor
  • VEGF vascular endothelial growth factor
  • IL-6 is elevated in tissues undergoing angiogenesis and can induce VEGF in
  • A431 cells a human epidermoid carcinoma cell line (Cohen, et al. J. Biol. Chem. 271: 736-741 , 1996).
  • IL-6 has been implicated in angiogenesis as a direct or indirect actor also because of its action on a number of cell types and observed expression in gonadotropin-primed hyperstimulated ovaries during a period of formation of a capillary network and vasculature extending fromm the ovarian medulla to growing follicles (Motro, B. et al. PNAS 87:3092-3096, 1990).
  • Angiogenesis is know to be a contributing factor in number of pathological conditions including the ability of tumors to grow and metastasize, disorders of the eye including retinopathies, and disorders of the skin including Kaposi's Sarcoma. While numerous factors have been shown to be associated with these processes, including IL-6, it has not heretofore been demonstrated that an IL-6 antagonist with the ability to prevent IL-6 activation of receptor signaling has a direct effect on angiogenesis.
  • Figure 5 A graph showing the data points representing the mean length of microvessels in plugs injected into Nude mice with added murine IL6. Each point represents average length/view from one Matrigel plug, with the line representing the mean from 10 plugs (2 plugs/animal). A two-tailed unpaired t-test analysis calculated p ⁇ 0.001 for murine IL6 groups compared to the control group (0 ng/mL IL6).
  • Figure 6 A graph showing the data points representing the mean number of microvessels in plugs injected into nude mice with added murine IL6. Each point represents vessel number/view from one Matrigel plug, with the line representing the mean from 10 plugs (2 plugs/animal). A two-tailed unpaired t-test analysis calculated p ⁇ 0.001 for murine IL6 groups compared to the control group (0 ng/mL IL6).
  • FIG. 11 A graph showing the relationship between IL6 concentration and migration of HUVECs and U373 towards vitronectin in the presence of IL6. Each data point is the mean ⁇ SD of 3 measurements and all are relative to no added IL6.
  • the anti-angiogenic IL-6 antagonists of the invention are useful in inhibiting and preventing angiogenesis in so far as they blocking the stimulatory effects of IL6 on endothelial cells, reduce endothelial cell division, decrease endothelial cell migration, and impair the activity of the proteolytic enzymes secreted by the endothelium.
  • a number of pathologies including various forms of solid primary tumors and the metastases, lesions of the eye and disorders of the skin are improved by treatment with 11-6 antagonists in the method of the present invention.
  • Sarcoma, basal cell carinoma and squamous cell carcinoma, small cell lung cancer, choriocarcinoma, rhabdomyosarcoma, angiosarcoma, hemangioendothelioma, Wilms Tumor, neuroblastoma, mouth/pharynx, esophageal, larynx, kidney and lymphoma, among others may be treated using anti- IL6 antibodies of the present invention.
  • conditions such as neurofibromatosis, tuberous sclerosis (each of which conditions produces benign tumors of the skin), hemangiomas and lymphangiogenesis, among others, may be treated effectively with IL-6 antagonists according to the present invention
  • a secondary tumor is a tumor which originated in a primary site elsewhere in the body, but has now spread to a distant organ.
  • the common routes for metastasis are direct growth into adjacent structures, spread through the vascular or lymphatic systems, and tracking along tissue planes and body cavaties with, for example, peritoneal fluid or cerebrospinal fluid.
  • Secondary hepatic tumors are one of the most common causes of death in cancer patients and are by far and away the most common form of liver tumor.
  • Corneal neovascularization can result from corneal ulcers.
  • a wide variety of etiologies may produce corneal ulcers including for example corneal infections (trachoma, herpes simplex keratitis, leishmaniasis and onchocerciasis), immunological processes (graft rejection and Stevens-Johnson's syndrome), alkali bums, trauma, inflammation (of any cause), toxic and Vitamin A or protein deficiency states, and as a complication of wearing contact lenses.
  • Topical therapy with IL-6 antibodies may also be useful prophylactically in corneal lesions which are known to have a high probability of inducing an angiogenic response (such as chemical burns). In these instances the treatment, likely in combination with steroids, may be instituted immediately to help prevent subsequent complications.
  • Angiogenesis is characterized by the invasion, migration and proliferation of smooth muscle and endothelial cells.
  • the ⁇ v ⁇ 3 integrin also known as the vitronectin receptor
  • the ⁇ v ⁇ 3 integrin is known to play a role in various conditions or disease states including tumor metastasis, solid tumor growth (neoplasia), osteoporosis, Paget's disease, humoral hypercalcemia of malignancy, angiogenesis, including tumor angiogenesis, retinopathy, including macular degeneration, arthritis, including rheumatoid arthritis, periodontal disease, psoriasis and smooth muscle cell migration (e.g. restenosis).
  • a method of treating a disease or condition associated with angiogenesis which comprises administering a combination of an integrin antagonist and an anti-IL-6 antibody to inhibit angiogenesis in a patient in need of such treatment.
  • Other antibodies which selectively bind integrins or integrin subunits, especially those that bind the alphaV subunit, are disclosed in U.S. Patents 5,985,278 and 6,160,099. Mabs that inhibit binding of alphaVbeta3 to its natural ligands containing the tripeptide argininyl-glycyl- aspartate (RGD) are disclosed in US 5,766,591 and WO0078815.
  • Anti-IL-6 Antibodies Any of the anti-IL-6 antibodies known it the art may be employed in the method of the present invention.
  • Murine monocolonal antibodies to IL-6 are known as in, for example, U.S. Patent 5,618,700.
  • U.S. Patent 5,856,135 discloses reshaped human antibodies to human IL-6 derived from a mouse monoclonal antibody SK2 in which the complementary determining regions (CDR's) from the variable region of the mouse antibody SK2 are transplanted into the variable region of a human antibody and joined to the constant region of a human antibody.
  • CDR's complementary determining regions
  • Adminstration may be affect by the implantation of a device whose primary function may not be as a drug delivery vehicle as, for example, a vascular stent.
  • methods are provided for treating corneal neovascularization (including corneal graft neovascularization), comprising the step of administering a therapeutically effective amount of an anti-angiogenic anti-IL6 antibodies of the invention directly to the cornea or systemically to the patient, such that the formation of blood vessels is inhibited.
  • VEGF vascular endothelial growth factor While having described the invention in general terms, the embodiments of the invention will be further disclosed in the following examples.
  • the primary angiogenic stimulus was IL6 added to the Matrigel plug.
  • the mice received a subcutaneous injection of Matrigel with and without human or murine IL6 (Table 1).
  • Matrigel forms a gelatinous plug once it reaches body temperature and this plug is stable within the body of the animal.
  • Liquid Matrigel was maintained at 4°C.
  • IL6 was added to Matrigel to the final concentration indicated, mixed thoroughly and stored overnight at 4°C.
  • the injection sites were located on the dorsal side approximately 0.25 inches caudal to the last rib and 0.25 inches from the backbone on each side.
  • the mice were injected in two sites with 0.5 mL of Matrigel. The area swelled if the injection was done properly.
  • mice Nude female mice (4-6 weeks old) obtained from Charles River (Raleigh, N.C.) were used in the study. Matrigel prepared from the Engelbreth-Holm-Swarm tumor was obtained from Becton Dickinson (Bedford, MA). C57 antibody (human IgG specific for CMV) from Centocor
  • Human IL6 (hlL6) and murine IL6 (mlL6) was purchased (R&D Systems, Minneapolis,
  • Ketamine 80 mg/kg, IP. Animals were injected in two sites with 0.5 mL of
  • mice were euthanized by C0 2 asphyxiation. Plugs were surgically removed and weighed, photographed and graded for angiogenesis. Two plugs per animal were assayed for hemoglobin content using a Drabkin kit (Sigma, ST Louis, MO).
  • Phase 3 To measure the total area of neovessels, a computerized digitizer called the Phase 3
  • Matrigel plugs with 2X magnification objective of the inverted phase contrast microscope were calculated using the trace program of Phase 3 Image System. The mean value from all photos of entire Matrigel plugs was calculated with standard deviation from the mean.
  • the Matrigel plug was lysed with lysis buffer (1% SDS, 0.5%Triton). The hemoglobin content of the gels was quantitated using Drabkin reagent kit (Sigma, St. Louis, MO). The concentration of hemoglobin in the gels was determined from a standard curve of hemoglobin. Hemoglobin content was expressed as milligrams hemoglobin per gram Matrigel. Means ⁇ SEM were calculated using the Student's unpaired t test; p ⁇ 0.05 was considered 5 statistically significant.
  • mice received an IV injection of cCLB ⁇ Mab, anti-human IL6, also o called chimeric CLB8 (Centocor, Malvern, PA) or control antibody (C57) immediately following injection of Matrigel spiked with human IL6 at 200 ng/mL, as indicated in Table 2.
  • chimeric CLB8 also o called chimeric CLB8 (Centocor, Malvern, PA) or control antibody (C57)
  • Matrigel spiked with human IL6 200 ng/mL, as indicated in Table 2.
  • Antibodies or vehicle were injected IV immediately after Matrigel injections.
  • mice were euthanized by C0 2 5 asphyxiation. Plugs were surgically removed and weighed.
  • cCLB ⁇ Inhibited Angiogenesis IL6 induced blood vessel formation in Matrigel plugs and cCLB8 decreased the vessel formation induced by IL6.
  • Matrigel plugs with IL6 incorporated have more red color than Matrigel plugs with no IL6, and C57 Matrigel plugs have more color than cCLB ⁇ Matrigel plugs.
  • IL6 significantly increased vessel formation in Matrigel plugs and cCLB ⁇ significantly inhibited vessel formation induced by IL6 ( Figure 7 - 9), whether cCLB8 was included in the Matrigel or injected IV after Matrigel plug injection.
  • DNA fragmentation was analyzed by the Cell Death Detection ELISA Kit (Roche Diagnostics GmBH, Mannheim, Germany) as recommended by the manufacturer.
  • the assay is a quantitative sandwich-enzyme-immunoassays which uses mouse Mabs capable of detecting DNA and histones allowing for detection of mono- and oligonucleosomes in cell lysates.
  • Cell apoptosis is directly proportional to the final absorbance at 405 nm. All determinations were performed in triplicate.
  • DNA fragment was measured in HUVEC cells after incubation in the presence of IL6 with and without CNTO 328 (10 ⁇ g/mL) for 3 days.
  • the datas shown in Fig. 10 DNA are mean ⁇ SD of triplicate determinations, expressed as percent of control, with cells in serum free medium without IL6 or CNTO 328 defined as 100%.
  • the experiment shows cCLB ⁇ is capable of inducing apoptosis as measured by DNA fragmentation in HUVEC in the absence of IL6 and can reverse the protective effect of IL6 seen at high concentrations.
  • EXAMPLE 4 IL6 induced Migration of HUVEC
  • HUVEC as described and cultured in the previous example.
  • Sub-confluent 24-hr cell cultures of HUVECS were starved with serum free medium overnight, harvested with trypsin-EDTA, washed twice, and resuspended in serum free media containing 0.1% BSA.
  • Cells (100,000/500 microL) were added to the upper chamber.
  • 750 microL of medium containing 0.1% BSA and different concentrations of IL6 or cCLB8 was added to the bottom chambers and the plate was placed in a tissue culture incubator. Migration was terminated after the specified elapsed time by removing the cells in the upper chamber with a cotton swab.
  • the undersides of migration chamber filters were coated with 0.5 microg/mL vitronectin, and the assay was performed as described in methods. Cells were allowed to migrate for 6 h. Each data point is the mean ⁇ SD of 3 transwell filters (FIG 11-12). The data in Fig. 11 show a dose dependent response of the HUVEC cells to IL-6 with maximal activity at about 100 ng/ml. In the presence of a neutralizing anti-IL6 Mab, cCLB8, amount of migration is suppressed.
  • the experiments described herein demonstrate that IL6 induced angiogenesis and related functions of endothelial cells which are stimulated by IL6 can be reduced by a specific Mab that prevents IL6 signaling through a receptor complex which includes gp130.
  • the process of angiogenesis as it occurs in new tissue forming in vivo was simulated in Matrigel plugs in nude mice, and as measured by increased number and length of microvessels, and increased Hb content.
  • the major component is laminin, but Matrigel also contains trace amounts of fibroblast growth factor, TGF-beta, tissue plasminogen activator, and other growth factors that occur naturally in the EHS tumor.
  • Matrigel is the basis for several types of tumor cell invasion assays and provides the necessary substrate for the study of angiogenesis.
  • Matrigel forms a soft gel plug when injected subcutaneously into mice or rats and supports an intense vascular response when supplemented with angiogenic factors.
  • cCLB ⁇ inhibited angiogenesis in Matrigel when it was incorporated in the Matrigel.
  • Experimental results demonstrated that a single injection of cCLB8 almost completely inhibited IL-6 mediated angiogenesis in the Matrigel plug model in nude mice.
  • cCLB ⁇ inhibited angiogenesis in Matrigel when it was injected IV following injection of Matrigel.

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Abstract

L'invention concerne un procédé d'utilisation d'antagonistes de IL-6 pour traiter les processus pathologiques associés à des maladies prolifératives, notamment le cancer. Le procédé consiste à inhiber ou à empêcher spécifiquement la capacité d'un nouveau tissu à développer une réserve sanguine. L'invention concerne en particulier des méthodes de traitement de telles maladies faisant appel à l'utilisation d'antagonistes de IL-6, notamment des anticorps dirigés contre IL-6, y compris des parties ou des variants spécifiés, spécifiques d'au moins une protéine d'interleukine-6 (IL-6 également connu sous le nom d'interféron beta 2) ou un fragment de celle-ci, dans une quantité efficace pour inhiber l'angiogenèse.
EP03783264A 2002-11-15 2003-11-10 Utilisations anti-angiogeniques d'antagonistes de il-6 Withdrawn EP1572103A4 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US42690102P 2002-11-15 2002-11-15
US426901P 2002-11-15
PCT/US2003/035651 WO2004045507A2 (fr) 2002-11-15 2003-11-10 Utilisations anti-angiogeniques d'antagonistes de il-6

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EP1572103A2 true EP1572103A2 (fr) 2005-09-14
EP1572103A4 EP1572103A4 (fr) 2008-02-13

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AU (1) AU2003290682A1 (fr)
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WO (1) WO2004045507A2 (fr)

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Publication number Priority date Publication date Assignee Title
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WO2004045507A2 (fr) 2004-06-03
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WO2004045507A3 (fr) 2006-01-26
JP2006516957A (ja) 2006-07-13

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