HU0303340A2 - Methods for preventing or treating inflammatory or autoimmune disorders integrin alpha v beta 3 antagonists in combination with other prophylactic or therapeutic agents by administering an effective - Google Patents

Methods for preventing or treating inflammatory or autoimmune disorders integrin alpha v beta 3 antagonists in combination with other prophylactic or therapeutic agents by administering an effective Download PDF

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HU0303340A2
HU0303340A2 HU0303340A HU0303340A HU0303340A2 HU 0303340 A2 HU0303340 A2 HU 0303340A2 HU 0303340 A HU0303340 A HU 0303340A HU 0303340 A HU0303340 A HU 0303340A HU 0303340 A2 HU0303340 A2 HU 0303340A2
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cd
method
mg
antibody
subject
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HU0303340A
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Christine Dingivan
Ronald Wilder
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Medimunne, Inc.
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Priority to US31632101P priority
Priority to US34691801P priority
Priority to US35842402P priority
Application filed by Medimunne, Inc. filed Critical Medimunne, Inc.
Priority to PCT/US2002/006679 priority patent/WO2002070007A1/en
Publication of HU0303340A2 publication Critical patent/HU0303340A2/en

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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2839Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the integrin superfamily
    • C07K16/2848Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the integrin superfamily against integrin beta3-subunit-containing molecules, e.g. CD41, CD51, CD61
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K39/395Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/39541Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against normal tissues, cells
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/241Tumor Necrosis Factors
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2806Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD2
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K2039/505Medicinal preparations containing antigens or antibodies comprising antibodies
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K2039/545Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2317/00Immunoglobulins specific features
    • C07K2317/20Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered

Abstract

The present invention relates to methods for preventing or treating one or more symptoms associated with an autoimmune or inflammatory disorder. More specifically, the present invention provides a method of preventing, treating or alleviating one or more of the symptoms associated with an autoimmune or inflammatory disorder, comprising administering to a subject in need of such treatment one or more integrin avb3 antagonists and at least one other prophylactic or therapeutic agent. The invention further provides compositions and industrial products for the prevention, treatment or alleviation of one or more symptoms associated with an autoimmune or inflammatory disorder. HE

Description

EXAMPLE pQ3 03340

77 449 / HF

S. Ö. G. & K.

Patent Attorney's Office H-1062 Budapest, Ardrássy ut U3.

Methods for Preventing or Treating Inflammatory or Autoimmune Disorders by Administration of Integrin ct v P3 Antagonists with Other Prophylactic or Therapeutically Effective Agents

The present invention relates to methods for the prevention, treatment or alleviation of one or more symptoms associated with an autoimmune or inflammatory disorder. More specifically, the present invention relates to methods for the prevention, treatment or alleviation of one or more symptoms associated with an autoimmune or inflammatory disorder, characterized in that the patient in need of such treatment has one or more integrin α ν β3 antagonists and at least one other proactive or therapeutic agent. agent is administered. The present invention also provides compositions and articles for the prevention, treatment or alleviation of one or more symptoms associated with an autoimmune or inflammatory disorder.

Inflammation is a process in which the body's white blood cells and chemicals protect the body from infections caused by foreign substances such as bacteria or viruses. This is usually characterized by pain, swelling, warming and reddening of the affected area. Compounds known as cytokines and prostaglandins regulate this process, they are released into the blood or the affected tissues in a controlled, self-limiting cascade. This release of chemicals increases blood flow to the area of injury or infection, resulting in redness and warming. Some chemicals provide fluid to these tissues, resulting in swelling. This defense process can stimulate the nerves and cause pain. These changes, if kept for a limited time in the affected area, will benefit the organization.

In autoimmune and / or inflammatory disorders, the immune system triggers an inflammatory response even when there is no foreign substance to fight, and the body's normally protective immune system causes damage to its own tissues, inadvertently attacking itself. There are many different autoimmune disorders that affect the body in different ways. For example, the brain is the affected organ in patients with multiple sclerosis, the intestine is the affected organ in Crohn's disease, and joint joints, bone and cartilage, and various joints are involved in patients with rheumatoid arthritis. As the autoimmune disorder progresses, it may be the result of the destruction of one or more tissues in the body, abnormal growth of an organ, or altered organ function. An autoimmune disorder may affect a single organ or tissue type, but may also affect multiple organs and tissue types. Organs and tissues commonly affected by autoimmune disorders include red blood cells, blood vessels, connective tissues, endocrine glands (e.g., thyroid or pancreas), muscles, joints, and skin. Autoimmune disorders include, but are not limited to, Hashimoto thyroiditis, anemia, Adison's disease, type 1 diabetes, rheumatoid arthritis, systemic lupus erythematosus, dermatomyositis, Sjogren's symptom, dermatomyositis, a. lupus erythematosus, multiple sclerosis, autoimmune disease of the inner ear, severe muscle weakness, Reiter symptom, Graves' disease, autoimmune hepatitis, family adenomatous polyposis, and ulcerative colitis.

Rheumatoid arthritis (RA) and juvenile rheumatoid arthritis are types of inflammatory arthritis. Arthritis is a generic term for arthritis. Some types of arthritis, but not all, are the result of inflammation to the wrong place. In addition to rheumatoid arthritis, other inflammatory arthritis may include psoriatic arthritis, Reiter's syndrome, stiffening arthritis, and gout arthritis. Rheumatoid arthritis is a form of chronic arthritis that occurs in the joints on both sides of the body (ie in both hands, wrists, or knees). This symmetry helps to distinguish rheumatoid arthritis from other types of arthritis. In addition to touching the joints, rheumatoid arthritis occasionally affects the skin, eyes, lungs, heart, blood and nerves.

Rheumatoid arthritis affects about 1% of the world's population and potentially makes it immobilized. The American

In the United States, about 2.9 million rheumatic arthritic diseases occur. Among them, 2-3 times more women than men. Rheumatoid arthritis typically occurs between 25-50 years of age. The juvenile rheumatic arthritis affects 71,000 young Americans (up to 18 years of age), and six times as many as the boy.

Rheumatoid arthritis is an autoimmune disorder where the body's immune system incorrectly identifies the arthritis that secretes lubricants into the joints. This results in inflammation and tissue damage around the cartilage, joints, and joints. In severe cases, this inflammation also extends to other articular tissues and surrounding cartilage, where it erodes or destroys bone and cartilage, causing joint deformities. The body replaces the damaged tissue with scar tissue, resulting in a narrowing of the normal spaces between the joints and the fusion of the bones. Rheumatoid arthritis causes tightness, swelling, fatigue, anemia, weight loss, fever and often paralyzing pain. Common symptoms of rheumatoid arthritis include tightness of the joints upon awakening, which may take several hours; swelling of a specific finger or wrist joint; swelling of soft tissues surrounding the joints and swelling on both sides of the joint. Swelling may occur without pain and pain, and may continue to deteriorate or remain on the same level for years before it begins to deteriorate. The diagnosis of rheumatoid arthritis is based on a combination of the following factors: the specific location and symmetry of the painful joints, the presence of joint stiffness in the morning, the presence of bumps and nodules under the skin (rheumatic nodules), rheumatoid arthritis, and / or rheumatism factor positive blood test. Many but not all patients with rheumatoid arthritis have rheumatoid factor antibodies in their blood. Rheumatic factor can also occur in the blood of people who do not suffer from rheumatoid arthritis. Other diseases also have the potential to deliver rheumatic factor into the blood. That is why the diagnosis of rheumatoid arthritis is based on a combination of several factors and not just the presence of rheumatic factor in the blood.

The typical course of the disease is the presence of permanent but fluctuating joint symptoms, and about 10 years later, 90% of sufferers suffer from structural damage in the bone and cartilage. A small percentage of patients have a short-term illness that is completely gone, and another small percentage has a severe illness, with a number of joint deformations and occasionally other manifestations of the disease. The inflammatory process causes erosion or destruction of the bone and cartilage in the joints. In rheumatoid arthritis, there is an autoimmune cycle of constant antigen presentation, T cell stimulation, cytokine secretion, activation of the cells in the joints, and destruction of the joint. The disease has a very serious impact on both the individual and society, causes severe pain, reduced function and disability, and costs millions of dollars in health spending.

·. ···. • · · · and lost in wages (see, for example, the NIH website and the NIAID website).

Currently available therapy for arthritis focuses on reducing inflammation of the joints by anti-inflammatory or immunosuppressive medication. In the treatment of any arthritis, anti-inflammatory agents, such as aspirin, ibuprofen, and Cox-2 inhibitors, such as celeoxib and rofecoxib, are generally present in the first line of treatment. "Second line" drugs include gold, methotrexate, and steroids. Although these are well-developed treatments for arthritis, it causes relief for very few patients if only these lines of treatment are used. Recent advances in the pathogenesis of rheumatoid arthritis have led to the use of methotrexate in combination with cytokines or recombinant soluble receptors. For example, tumor necrosis factor (TNFα) is used as a recombinant soluble receptor in combination with methotrexate in the treatment of arthritis. However, only about 50% of patients exhibit clinically significant improvement with methotrexate and an anti-TNFa agent such as TNFα recombinant soluble receptor. Despite the treatment, there are no positive changes in many patients. Patients with rheumatoid arthritis still have severe treatment problems. Many of the treatments currently in use have very common side effects, or they are not able to completely prevent the development of the disease. Until now, there is no ideal treatment, no cure «

••• Curing. New therapeutic agents are needed to treat rheumatoid arthritis and other autoimmune disorders more effectively.

Citation of any reference in this or any other part is not to be construed as acknowledging that such reference is available in the art as a novelty of the present invention.

The present invention is based, in part, on the recognition that integrin α ν β3 antagonists have potential for certain anti-inflammatory treatments and synergies with these, including anti-TNFα agents and methotrexate. Thus, the present invention relates to treatment protocols that provide superior prophylactic and therapeutic profiles in autoimmune and / or inflammatory disorders as currently used single-agent therapies. The present invention further provides combination therapies for the prevention, treatment or alleviation of one or more symptoms associated with an autoimmune or inflammatory disorder in a subject and said combination therapies comprising one or more integrin α ν β3 antagonists and one or more of said subjects. several prophylactic or therapeutic agents other than integrin α ν β3 antagonists are administered. More specifically, the present invention relates to combination therapies for the prevention, treatment or alleviation of one or more symptoms associated with an autoimmune or inflammatory disorder in a subject and said combination therapies comprising one or more integrin α ν β3 antagonists, preferably VITAXIN ™ and at least one other prophylactic or therapeutic agent · ♦ * · ··· · »are administered, the mechanism of action of which is different from that of integrin α ν β3 antagonists.

A combination of one or more integrin α ν β3 antagonists and a prophylactic or therapeutic agent other than one or more integrin α ν β3 antagonists in a subject provides a better prophylactic or therapeutic result than any of the treatments alone. In some embodiments, a combination of a prophylactic or therapeutic agent other than an integrin α ν β3 antagonist and an integrin α ν β3 antagonist has a prophylactic or therapeutic effect twice, preferably three, four, five, six, seven, seven, eight, nine, ten, fifteen, or twenty times. in a subject suffering from an autoimmune or inflammatory disorder as any treatment alone. In other embodiments, a combination of a prophylactic or therapeutic agent other than an integrin α ν β3 antagonist and an integrin α ν β3 antagonist is 10%, preferably 15%, 20%, 25%, 30%, 35%. %, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% with 90%, 95%, 100%, 125%, 150% or 200% better prophylactic or therapeutic effects in a subject suffering from an autoimmune or inflammatory disorder than with any treatment itself. In a particular embodiment, the combination of integrin α ν β3 antagonists and a prophylactic or therapeutic agent other than an integrin α ν β3 antagonist is 25%, 30%, 35%, 40%, 45%, 50%. %, 55%, 60%, 65% ... ·· ♦ · / ·. • · «.

. ·. ···. '... ... »· Kai, 70%, 75%, 80%, 85%, 90%, 95%, or 98% greater reduction of a given in an organ, tissue, or joint in a subject suffering from an autoimmune disorder associated with inflammation, an inflammatory disorder or an inflammatory disorder, such as any of the treatments alone. In other embodiments, a combination of one or more integrin α ν β3 antagonists and one or more of the integrin α ν β3 antagonists has a more than additive effect or has a synergistic effect in a subject suffering from autoimmune or inflammatory disorders.

The combination therapies of the present invention allow the use of lower doses of integrin α ν β3 antagonists and / or less frequent administration of integrin α ν β3 antagonists (preferably VITAXIN ™) to a patient suffering from an autoimmune or inflammatory disorder with a view to being prophylactic. therapeutic effect. The combination therapies of the present invention allow the use of a lower dose of prophylactic or therapeutic agents in combination with integrin α ν β3 antagonists for the prevention and / or treatment of an autoimmune or inflammatory disorder and / or administration of such prophylactic or therapeutic agents at a lower frequency. to a subject suffering from an autoimmune or inflammatory disorder with a view to achieving a prophylactic or therapeutic effect. The combination therapies of the present invention reduce or avoid unwanted or detrimental effects associated with single-agent therapies and / or existing combination therapies currently being used in the treatment of autoimmune or inflammatory disorders, and are further improved. patient cooperation with the treatment protocol.

The prophylactic or therapeutic agents of the combination therapies of the present invention may be administered to a subject simultaneously or sequentially. The prophylactic or therapeutic agents of the present invention may be administered cyclically. Cyclical therapy includes administering a first prophylactic or therapeutic agent for a period of time, then administering a second prophylactic or therapeutic agent for a period of time and repeating this sequential administration, i.e. the cycle. to reduce the development of resistance to one agent and to avoid or reduce the side effects of one agent and / or to improve the efficacy of the treatment.

The prophylactic or therapeutic agents of the combination therapies of the present invention may be administered simultaneously to a subject. The term "simultaneously" is not limited to administering prophylactic or therapeutic agents at exactly the same time, but rather to administering an antagonist of integrin α ν β3 and the other agent in the order and within a time interval. that the integrin α ν β3 antagonist can interact with the other agent at the same time, thereby increasing the beneficial effect of the agents being administered differently. For example, each prophylactic or therapeutic agent (such as VITAXIN ™, an anti-TNFα antibody, or methotrexate) can be administered at the same time or sequentially, in any order, at different times; however, if not administered at the same time, the administrations should be close enough to each other in time to obtain the desired therapeutic or prophylactic effect. Each prophylactic or therapeutic agent may be administered separately, in any suitable form, and in any appropriate manner. In various embodiments, prophylactic or therapeutic agents are within 15 minutes, within 30 minutes, within 1 hour, within about 1-2 hours, within about 2-3 hours, about 3-4 hours, within about 4-5 hours, within about 5-6 hours, about 6-7 hours, about 7-8 hours, about 8-9 hours, about 9-10 hours, about 10-11 hours, about 11-12 hours, maximum It can be given within 24 hours or within 48 hours. In preferred embodiments, the two or more prophylactic or therapeutic agents are co-administered when the patient is present.

The prophylactic or therapeutic agents of the combination therapies may be administered to a subject in the same formulation. Alternatively, the prophylactic or therapeutic agents of the combination therapies may be administered to a subject in succession in different therapeutic compositions. Prophylactic or therapeutic agents may be administered to the subject by the same route of administration or by a different route of administration.

The present invention relates to methods for the prevention, management or alleviation of an autoimmune or inflammatory disorder, or one or more of its symptoms, characterized in that said method comprises administering one or more integrin α ν β3 antagonists to a subject in need of such treatment. one or more prophylactic or therapeutic agents other than the integrin α ν β3 antagonist, which are currently used, used or known to be useful in the prophylaxis or treatment of one or more symptoms associated with an autoimmune or inflammatory disorder; or mitigating it. Examples of integrin α ν β3 antagonists include, but are not limited to, proteins, polypeptides, peptides, fusion proteins, antibodies, antibody fragments, large molecules, or small molecules (less than 10 kDa) that inhibit, reduce, or neutralize the function, activity and / or expression of α ν β of integrin. In a specific embodiment, the present invention relates to a method for the prevention, treatment, management or alleviation of an autoimmune or inflammatory disorder, or one or more of its symptoms, characterized in that said method comprises administering one or more integrin α to a subject in need of such treatment. ν β3 antagonist and one or more non-integrin α ν β3 antagonist prophylactic or therapeutic agents wherein at least one integrin α ν β3 antagonist is an antibody or fragment thereof that binds immune-specific to integrin α 3. In a preferred embodiment, the present invention relates to a method for the prevention, treatment, management or alleviation of an autoimmune or inflammatory disorder, or one or more of its symptoms, characterized in that said method comprises administering one or more of the following to a subject in need of such treatment. an integrin α ν β3 antagonist and one or more non-integrin α ν β3 antagonist prophylactic or therapeutic agents wherein at least one integrin α ν β3 antagonist is MEDI-522 human monoclonal antibody (commercial name VITAXIN ™) or an antigen thereof. -binding fragment.

Autoimmune disorders include, but are not limited to, spotted baldness, paralysis of the vertebrae, antifungal phospholipid syndrome, autoimmune Addison's disease, autoimmune diseases of the adrenal gland, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune oophoritis and orchitis, autoimmune thrombocytopenia, Behcet's disease, bullous pemphigoid, cardiomyopathy, abdominal sprue dermatitis, chronic fatigue immune dysfunction symptom (CFIDS), chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, scarred pemphigism, CREST syndrome, cold agglutinin disease, Crohn's disease, pancreatic lupus, essential \ t mixed cryoglobulinemia, fibromyalgia fibromyositis, glomerulonephritis, Graves' disease, Guillan-Barre, Hashimoto thyroiditis, idiopathic lung fibrosis, idiopathic thrombocytopenia purpurea (ITP), IgA neuropathy, juvenile arthritis, lichen planus, lupus erythematosus, Meniere's disease, mixed binder tissue disease, sclerosis14 • ··· ·· · · · · · ·· «! ··· · · · * · ·· · · · zis multiplex, Type 1, or Immunological Diabetes, Severe Muscle Weakness, Pemphigus Vulgar, Injury Anemia, Lumpy Polyaritis, Polycondritis, Polyglandular Syndrome, Polymalgia Rheumatic, Polymeritis and Dermatomyositis, Primary agammaglobulinemia, primary epecirosis, psoriasis, psoriatic arthritis, Raynauld phenomenon, Reiter syndrome, rheumatoid arthritis, sarcoidosis, scleroderma, Sjögren's syndrome, stiffman syndrome, systemic lupus erythematosus, lupus erythematosus, takayasu arteritis, glandular arteritis / giant cell arteritis, ulcerative colitis, uveitis, vasculitis such as dermatitis herpetiformis vasculitis, vitiligo, and Wegener granulomatosis. Inflammatory diseases include, but are not limited to, asthma, encephalitis, inflammatory bowel disease, chronic obstructive pulmonary disease (COPD), allergic disorders, septic shock, pulmonary fibrosis, undifferentiated spondyloarthropathy, undifferentiated arthropathy, arthritis, inflammatory osteolysis, and chronic inflammation. resulting from chronic viral or bacterial infection. As described below, some autoimmune disorders are associated with an inflammatory condition. So there is an overlap between what we consider to be an autoimmune disorder and an inflammatory disorder. Therefore, some autoimmune disorders can be characterized as inflammatory disorders.

The present invention relates to a method for treating an autoimmune or inflammatory disorder or one or more symptoms thereof, wherein said method comprises:

comprising administering to a patient in need of such treatment one or more integrin α ν β3 antagonists and one or more immunomodulatory agents. Immunomodulatory agents are preferably not administered to subjects with autoimmune or inflammatory disorders whose mean absolute lymphocyte count is less than 500 cells / mm 3 , less than 550 cells / mm 3 , less than 600 cells / mm 3 , less than 650 cells / mm 3 , less than 700 cells / mm 3 , less than 750 cells / mm 3 , less than 800 cells / mm 3 , less than 850 cells / mm 3 , or less than 900 cells / mm 3 . Thus, in a preferred embodiment, before or after administering one or more immunomodulatory agents to an autoimmune or inflammatory patient, the subject's absolute lymphocyte count is determined by techniques known to those skilled in the art, including, for example, flow cytometry or trypan blue number.

Immunomodulatory agents include, but are not limited to, methotrexate, leflunomide, cyclophosphamide, cyclosporin A, and macrolide antibiotics (such as FK506, tacrolimus), methylprednisolone (MP), corticosteroids, steroids, and the like. mycophenolate mofetil, rapamycin (sirolimus), mizoribine, deoxypergualin, brequinar, malononitrile amides (such as leflunamide), T cell receptor modulators, and cytokine receptor modulators. For clarification of the role of T cell receptor modulators and cytokine receptor modulators see below. T cell receptors include, but are not limited to, soluble cytokine receptors (e.g., the extracellular domain of a TNFα receptor or a fragment thereof, an extracellular domain of an interleukin-1 β receptor, or a fragment thereof, and an interleukin). Extracellular domain of 6 receptors or fragments thereof), cytokines and fragments thereof (e.g., interleukin2, interleukin-4, interleukin-5, interleukin-6, interleukin-7, interleukin-8, interleukin-9, interleukin-10, interleukin-11) , interleukin-12, interleukin-15, TNFα, TNFβ, interferon-a, interferon-β, IFNγ and granulocyte macrophage colony stimulating factor), anti-cytokine receptor antibodies (e.g., anti-interleukin-2 receptor antibodies, anti-interleukin-4) receptor antibodies), anti-interleukin-6 receptor antibodies, anti-interleukin-10 receptor antibodies, and anti-interleukin-12 receptor antibodies), anti-cytokine antibodies (e.g., anti-in \ t terferon receptor antibodies, anti-TNFα antibodies, anti-interleukin1β antibodies, anti-interleukin-6 antibodies, and anti-interleukin 12 antibodies).

In a specific embodiment, the present invention relates to a method for the prevention, treatment, management or alleviation of an autoimmune or inflammatory disorder, or one or more of its symptoms, characterized in that said method comprises treating a subject in need of such treatment prophylactically or therapeutically. administering one or more integrin α ν β3 antagonists in an amount and administering one or more immunomodulatory agents in a prophylactically or therapeutically effective amount. In another embodiment, the present invention relates to a method for the prevention, treatment, management or alleviation of an autoimmune or inflammatory disorder, or one or more of its symptoms, characterized in that said method comprises treating a subject in need of such treatment prophylactically or therapeutically. administering one or more integrin α ν β3 antagonists in an amount and administering one or more immunomodulatory agents in a prophylactically or therapeutically effective amount, wherein at least one of the integrin α ν β3 antagonists is an antibody or fragment thereof that specifically binds to integrin α ν β3. -όοζ. In a preferred embodiment, the present invention relates to a method for the prevention, treatment, management or alleviation of an autoimmune or inflammatory disorder, or one or more of its symptoms, characterized in that said method comprises treating a subject in need of such treatment prophylactically or therapeutically. administering an effective amount of one or more integrin α ν β3 antagonists and administering one or more immunomodulatory agents in a prophylactically or therapeutically effective amount, wherein at least one of the integrin α ν β3 antagonists is VITAXIN ™ or an antigen-binding fragment thereof. In another preferred embodiment, the present invention relates to a method for the prevention, treatment, management or alleviation of an autoimmune or inflammatory disorder, or one or more of its symptoms, characterized in that said method comprises the prevention or prophylaxis of a subject in need of such treatment. administering a therapeutically effective amount of VITAXIN ™, or

an antigen-binding fragment thereof and a prophylactically or therapeutically effective amount of one or more immunomodulatory agents.

In a specific embodiment, the present invention relates to a method for the prevention, treatment, management or alleviation of an autoimmune or inflammatory disorder, or one or more of its symptoms, characterized in that said method comprises treating a subject in need of such treatment prophylactically or therapeutically. administering one or more integrin α ν β3 antagonists in an amount and administering a prophylactically or therapeutically effective amount of methotrexate or cyclosporin. In another embodiment, the present invention relates to a method for the prevention, treatment, management or alleviation of an autoimmune or inflammatory disorder, or one or more of its symptoms, characterized in that said method comprises treating a subject in need of such treatment prophylactically or therapeutically. VITAXIN ™ is administered in an amount which is administered prophylactically or in a therapeutically effective amount to methotrexate or cyclosporin. In another embodiment, the present invention relates to a method for the prevention, treatment, management or alleviation of an autoimmune or inflammatory disorder, or one or more of its symptoms, characterized in that said method comprises treating a subject in need of such treatment prophylactically or therapeutically. administering one or more integrin α ν β3 antagonists and administering methotrexate in a prophylactically or therapeutically effective amount and administering a cyclosporin in a prophylactically or therapeutically effective amount.

The present invention further relates to a method for the prevention, treatment, management or alleviation of an autoimmune or inflammatory disorder, or one or more of its symptoms, characterized in that said method comprises administering to a subject in need thereof a therapeutically or therapeutically effective amount of a subject. or more integrin α ν β3 antagonists and one or more CD2 antagonists. More specifically, the present invention relates to a method for the prevention, treatment, management or alleviation of an autoimmune or inflammatory disorder, or one or more of its symptoms, characterized in that said method comprises administering to a subject in need thereof a therapeutically or therapeutically effective amount. VITAXIN ™ or an antigen-binding fragment thereof and one or more CD2 antagonists.

The present invention further relates to a method for the prevention, treatment, management or alleviation of an autoimmune or inflammatory disorder, or one or more of its symptoms, characterized in that said method comprises administering to a subject in need thereof a therapeutically or therapeutically effective amount of a subject. or more integrin α ν β3 antagonists, and one or more CD2 binding molecules (e.g., peptides, polypeptides, proteins, antibodies (MEDI-507), and fusion proteins that bind immunoprecipitatively to a CD2 polypeptide 20 · ··· * · · · · · · · · · · · · · · · · 9 »Λ * · affects tide and directly or indirectly affects the disappearance of peripheral blood lymphocytes. Preferably, CD2 binding molecules are not administered to a subject suffering from an autoimmune or inflammatory disorder with an average absolute value. lymphocyte count less than 500 cells / mm 3 , less than 5 50 cells / mm 3 , less than 600 cells / mm 3 , less than 650 cells / mm 3 , less than 700 cells / mm 3 , less than 750 cells / mm 3 , less than 800 cells / mm 3 , less than 850 cells / mm 3 or less than 900 cells / mm 3 . Thus, in a preferred embodiment, before or after administering one or more immunomodulatory agents to an autoimmune or inflammatory patient, the subject's absolute lymphocyte count is determined by techniques known to those skilled in the art, including, for example, flow cytometry or trypan blue number.

In a specific embodiment, the percentage of CD2 polypeptides bound by the CD2 binding molecules is estimated after administration of the first dose of one or more CD2 binding molecules to a subject suffering from an autoimmune or inflammatory disorder and prior to administering one or more subsequent doses of one or more CD2 binding molecules. In another embodiment, the percentage of CD2 polypeptides bound by CD2 binding molecules is regularly evaluated (e.g., weekly, bi-weekly, three-weekly, four-weekly, weekly, eight-week, or 12-week), one or more doses of CD2 binding molecules in said autoimmune or inflammatory disorder. after administration to a subject. Preferably, a patient with an autoimmune or inflammatory disorder is dosed with the next one or more CD2 molecules when the percentage of CD2 polypeptides bound by CD2 binding molecules is less than 80%, preferably less than 75%, less than 70%, less than 65%. less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25% or less than 20%. The percentage of CD2 polypeptides bound by CD2 binding molecules can be determined in the manner known to those of ordinary skill in the art, or as described herein.

In a specific embodiment, the present invention relates to a method for the prevention, treatment, management or alleviation of an autoimmune or inflammatory disorder, or one or more of its symptoms, characterized in that said method comprises administering to one subject in need of such treatment one or more of the following: integrin α ν β3 antagonist and is administered prophylactically or therapeutically effective amounts of one or more integrin α ν β3 antagonists and is administered prophylactically or therapeutically effective amounts of one or more CD2 binding molecules. In one embodiment, the present invention relates to a method for the prevention, treatment, management or alleviation of an autoimmune or inflammatory disorder, or one or more of its symptoms, characterized in that said method comprises treating a subject in need of such treatment in a prophylactically or therapeutically effective amount. administering one or more integrin α ν β3 antagonists and administering one or more CD2 binding molecules in a prophylactically or therapeutically effective amount, wherein at least one of the integrin α ν β3 antagonists is an antibody or fragment thereof that binds immune-specific to the integrin α ν β3 antagonist. . In a preferred embodiment, the present invention relates to a method for the prevention, treatment, management or alleviation of an autoimmune or inflammatory disorder, or one or more of its symptoms, characterized in that said method comprises treating a subject in need of such treatment prophylactically or therapeutically. an effective amount of one or more integrin α ν β3 antagonists is administered and a prophylactically or therapeutically effective amount of one or more CD2 binding molecules is administered, wherein at least one integrin α ν β3 antagonist is VITAXIN ™ or an antigen-binding fragment thereof. In another embodiment, the present invention relates to a method for the prevention, treatment, management or alleviation of an autoimmune or inflammatory disorder, or one or more of its symptoms, characterized in that said method comprises treating a subject in need of such treatment prophylactically or therapeutically. VITAXIN ™ or an antigen-binding fragment thereof is administered in an amount, and one or more CD2 binding molecules are administered prophylactically or therapeutically.

In another embodiment, the present invention relates to a method for the prevention, treatment, management or alleviation of an autoimmune or inflammatory disorder, or one or more of its symptoms, characterized in that said method comprises treating a subject in need of such treatment prophylactically or therapeutically. administering one or more integrin α ν β3 antagonists in an amount and administering one or more CD2 binding molecules in a prophylactically or therapeutically effective amount, wherein at least one of the CD2 molecules is a soluble LFA-3 polypeptide or LFA3TIP. In another embodiment, the present invention relates to a method for the prevention, treatment, management or alleviation of an autoimmune or inflammatory disorder, or one or more of its symptoms, characterized in that said method comprises treating a subject in need of such treatment prophylactically or therapeutically. administering one or more integrin α ν β3 antagonists in an amount and administering one or more immunomodulatory agents in a prophylactically or therapeutically effective amount, wherein at least one of the CD2 binding molecules is an antibody or fragment thereof that immunospecifically binds to a CD2 polypeptide. In a preferred embodiment, the present invention relates to a method for the prevention, treatment, management or alleviation of an autoimmune or inflammatory disorder, or one or more of its symptoms, characterized in that said method comprises treating a subject in need of such treatment prophylactically or therapeutically. an effective amount of one or more integrin α ν β3 antagonists is administered and administered in a prophylactically or therapeutically effective amount to one or more im24.

an modulator agent comprising at least one CD2 binding molecule MEDI-507 or an antigen-binding fragment thereof.

In another embodiment, the present invention relates to a method for the prevention, treatment, management or alleviation of an autoimmune or inflammatory disorder, or one or more of its symptoms, characterized in that said method comprises treating a subject in need of such treatment prophylactically or therapeutically. administering one or more integrin α ν β3 antagonists in an amount and administering one or more CD2 binding molecules in a prophylactically or therapeutically effective amount, wherein at least one of the integrin α ν ββ antagonists is an antibody or fragment thereof that immunospecifically binds to integrin α ν β3. And wherein at least one CD2 binding molecule is a soluble LFA-3 polypeptide or LFA3TIP.

In a preferred embodiment, the present invention relates to a method for the prevention, treatment, management or alleviation of an autoimmune or inflammatory disorder, or one or more of its symptoms, characterized in that said method comprises treating a subject in need of such treatment prophylactically or therapeutically. an effective amount of one or more integrin α ν β3 antagonists is administered and a prophylactically or therapeutically effective amount of one or more CD2 binding molecules is administered, wherein at least one integrin α ν β3 antagonist is VITAXIN ™, or an antigen-binding fragment thereof, and CD2. at least one of the binding molecules or an antigen-binding fragment thereof. According to another preferred embodiment, the present invention relates to a method of treating an autoimmune or inflammatory disease. characterized in that said method comprises administering to a subject in need of such treatment a prophylactically or therapeutically effective amount of VITAXIN ™, or an antigen-binding fragment thereof; and administering one or more CD2 binding molecules in a prophylactically or therapeutically effective amount, and at least one of the CD2 binding molecules or an antigen-binding fragment thereof. In a further preferred embodiment, the present invention relates to a method for the prevention, treatment, management or alleviation of an autoimmune or inflammatory disorder, or one or more of its symptoms, characterized in that said method comprises the prophylactic use of a subject in need of such treatment. administering a therapeutically effective amount of VITAXIN ™, or an antigen-binding fragment thereof, and administering a prophylactically or therapeutically effective amount of MEDI-507 or an antigen-binding fragment thereof.

The present invention relates to a method for the prevention, treatment, management or alleviation of an autoimmune or inflammatory disorder or one or more of its symptoms, characterized in that said method comprises administering to a subject in need of such treatment a prophylactically or therapeutically effective amount of one or more of the following: multiple integrin α ν β3 antagonists and one or more TNFa antagonists. Among TNFα antagonists • J ·· «« · · * · · · · · ·

Belong to the «« »» · · · · · · · · · · · · 4 · ♦ · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · (REMICADE ™; Centacor), D2E7 (Abbott Laboratories, Knoll Pharmaceuticals Co., Mt. Olive, NJ), CDP571, also known as HUMICADE ™ and CDP-870 (all Celltech / Pharmacia, Slough, UK), and TN3-19.12 (Williams et al., 1994, Proceedings of the National Academy of Sciences, USA 91: 2762-2766; Thorbecke et al., Proceedings of the National Academy of Sciences, USA 89, 7375-7379 (1994)). soluble TNFα receptors (e.g., sTNF-RI (Amgen), etanercept (ENBREL ™; Immunex) and its rat homologue, RENBREL ™, TNFα and TNFrI inhibitors of TNFα [Kohno et al., Proceedings of the National Academy of Sciences , USA 87, 8331-8335 (1990)] and TNFα Inh (Seckinger et al., Proceedings of the National Academy of Sciences, USA 87, 5188-5192 (199)). 0)], interleukin10, TNFR-IgG [Ashkenazi et al., Proceedings of the National Academy of Sciences, USA 88, 10535-10539 (1991)], rodent TBP (Serono / Yeda), Cyto / TAB vaccine (Protherics), 104838 antisense molecule (ISIS), RDP-58 peptide (SangStat), thalidomide (Celgene), CDC-801 (Celgene), DPC-333 (Dupont), VX-745 (Vertex) , AGIX-4207 (AtheroGenetics), ITF-2357 (Italfarmaco), NPI-13021-31 (Nereus), SCIO-469 (Scios), Tace targeting (Immunix / AHP), CLX-120500 (Calyx ), Thiazolopyrim (Dynavax), auranofin (Ridaura) (SmithKline Beecham Pharmaceuticals), quinacrine (mepacrine dihydro27 • · „· · • · • • · · η η * * * * or át át át át át), tenidap (Enablex) , Melanin (Large Scale Biological) and anti-p38 MAPK Agents (Uriach).

In a specific embodiment, the present invention relates to a method for the prevention, treatment, management or alleviation of an autoimmune or inflammatory disorder, or one or more of its symptoms, characterized in that said method comprises treating a subject in need of such treatment prophylactically or therapeutically. administering one or more integrin v 03 antagonists in an amount and administering one or more TNFα antagonists in a prophylactically or therapeutically effective amount. In another embodiment, the present invention relates to a method for the prevention, treatment, management or alleviation of an autoimmune or inflammatory disorder, or one or more of its symptoms, wherein said method comprises prophylactically or therapeutically effective for a subject in need of such treatment. administering one or more integrin α ν β3 antagonists in an amount and administering one or more TNFα antagonists in a prophylactically or therapeutically effective amount, wherein at least one of the integrin α ν β3 antagonists is an antibody or fragment thereof that binds immune-specific to integrin α ν. β3 ~ 0οζ.

In a preferred embodiment, the present invention relates to a method for the prevention, treatment, management or alleviation of an autoimmune or inflammatory disorder, or one or more of its symptoms, characterized in that said method comprises the use of such a treatment. * * * ·> V * · **

T t ·. · W * «·« «- ·· · * * * * ·« '* í »· an subject in need of prophylactic or therapeutically administered one or more integrin α ν β3 antagonist in an effective amount, and is prophylactically or therapeutically effective amount. comprising one or more TNFα antagonists, wherein at least one of the integrin α ν β3 antagonists is VITAXIN ™ or an antigen-binding fragment thereof. In another preferred embodiment, the present invention relates to a method for the prevention, treatment, management or alleviation of an autoimmune or inflammatory disorder, or one or more of its symptoms, characterized in that said method comprises the prevention or prophylaxis of a subject in need of such treatment. administering a therapeutically effective amount of VITAXIN ™ and administering one or more TNFα antagonists in a prophylactically or therapeutically effective amount.

In another embodiment, the present invention relates to a method for the prevention, treatment, management or alleviation of an autoimmune or inflammatory disorder, or one or more of its symptoms, wherein said method comprises prophylactically or therapeutically effective for a subject in need of such treatment. administering one or more integrin α ν β3 antagonists in an amount and administering one or more TNFα antagonists in a prophylactically or therapeutically effective amount, wherein at least one of the TNFα antagonists is a soluble TNFα receptor, such as etanercept (ENBREL ™; Immunex), or a fragment, derivative, or analog thereof, or an antibody that specifically binds to TNFα29, such as infliximab (REMICADE ™; Centacor), or a derivative, analogue, or antigen-binding fragment thereof.

In a preferred embodiment, the present invention relates to a method for the prevention, treatment, management or alleviation of an autoimmune or inflammatory disorder, or one or more of its symptoms, characterized in that said method comprises treating a subject in need of such treatment prophylactically or therapeutically. an effective amount of one or more integrin α ν β3 antagonists is administered and a prophylactically or therapeutically effective amount of one or more TNFα antagonists is administered, wherein at least one of the integrin α ν β3 antagonists is an antibody or fragment thereof that binds immune-specific to the integrin. α ν β3-1ιοζ, and at least one of the TNFα antagonists is a soluble TNFα receptor, such as etanercept (ENBREL ™; Immunex), or a fragment, derivative or analog thereof, or an antibody that binds immune-specific to TNFα, e.g. the infliximab (REMICADE ™; Centacor) or a derivative, analogue or antigen-binding fragment thereof.

In a preferred embodiment, the present invention relates to a method for the prevention, treatment, management or alleviation of an autoimmune or inflammatory disorder, or one or more of its symptoms, characterized in that said method comprises treating a subject in need of such treatment prophylactically or therapeutically. an effective amount of one or more integrin α ν β3 antagonists is administered and a prophylactically or therapeutically effective amount of one or more TNFα antagonists is administered wherein at least one of the integrin α ν β3 antagonists is VITAXIN? at least one of which is a soluble TNFα receptor, such as etanercept (ENBREL ™; Immunex), or a fragment, derivative or analog thereof, or an antibody that binds immune-specific to TNFα, such as infliximab (REMICADE ™; Centacor), or or an antigen-binding fragment thereof.

The present invention relates to a method for the prevention, treatment, management or alleviation of an autoimmune or inflammatory disorder, or one or more of its symptoms, characterized in that said method comprises administering to a subject in need of such treatment a prophylactically or therapeutically effective amount of one or more of the following: multiple integrin α ν β3 antagonists and one or more anti-inflammatory agents. Examples of anti-inflammatory agents include, but are not limited to, non-steroidal anti-inflammatory drugs (such as aspirin, ibuprofen, celecoxib (CELEBREX ™), diclofenac (VOLTAREN ™), etodolac (LODINE ™), feoprofen (NALFON ™)), indomethacin (INDOCIN ™), ketoralac (TORADOL ™), oxaprozine (DAYPRO ™), rofecoxib (VIOXX ™), naproxen (ALEVE ™, NAPROSYN ™), ketoprofen (ACTRON ™) and nabumetone (RELAFEN ™)), and steroidal anti-inflammatory drugs (e.g., glucocorticoids, dexamethasone (DECADRON ™), cortisone, hydrocortisone, prednisone (DELTASONE ™), prednisolone, triamc31noneone, azulfidine and eicosanoids such as prostaglandins, thromboxanes and leukotrienes).

In a specific embodiment, the present invention relates to a method for the prevention, treatment, management or alleviation of an autoimmune or inflammatory disorder, or one or more of its symptoms, characterized in that said method comprises treating a subject in need of such treatment in a prophylactically or therapeutically effective amount. one or more integrin α ν β3 antagonists and one or more anti-inflammatory agents are administered. In another embodiment, the present invention relates to a method for the prevention, treatment, management or alleviation of an autoimmune or inflammatory disorder, or one or more of its symptoms, wherein said method comprises prophylactically or therapeutically effective for a subject in need of such treatment. an amount of one or more integrin α ν β3 antagonists and one or more anti-inflammatory agents are administered, wherein at least one of the integrin α ν β3 antagonists is an antibody or fragment thereof that is immunospecifically bound to integrin a v p3.

In another embodiment, the present invention relates to a method for the prevention, treatment, management or alleviation of an autoimmune or inflammatory disorder, or one or more of its symptoms, wherein said method comprises prophylactically or therapeutically effective for a subject in need of such treatment. administering one or more integrin α ν β3 antagonists and one or more anti-inflammatory agents, wherein at least one of the integrin α ν β3 antagonists is VITAXIN ™ or an antigen-binding fragment thereof. In another preferred embodiment, the present invention relates to a method for the prevention, treatment, management or alleviation of an autoimmune or inflammatory disorder, or one or more of its symptoms, characterized in that said method comprises the prevention or prophylaxis of a subject in need of such treatment. administering a therapeutically effective amount of VITAXIN ™ or an antigen-binding fragment thereof and administering in a prophylactically or therapeutically effective amount one or more anti-inflammatory agents.

The present invention relates to a method for the prevention, treatment, management or alleviation of an autoimmune or inflammatory disorder, or one or more of its symptoms, characterized in that said method comprises administering to a subject in need of such treatment a prophylactically or therapeutically effective amount of one or more of the following: multiple integrin α ν β3 antagonists, one or more TNFα antagonists and one or more immunomodulatory agents. In a specific embodiment, the present invention relates to a method for the prevention, treatment, management or alleviation of an autoimmune or inflammatory disorder, or one or more of its symptoms, characterized in that said method comprises treating a subject in need of such treatment prophylactically or therapeutically. VITAXIN ™ or an antigen-binding fragment thereof is administered in an amount, a soluble TNFα receptor (e.g., etanercept) is administered prophylactically or in a therapeutically effective amount, and methotrexate is administered in a prophylactically or therapeutically effective amount. In another embodiment, the present invention relates to a method for the prevention, treatment, management or alleviation of an autoimmune or inflammatory disorder, or one or more of its symptoms, wherein said method comprises prophylactically or therapeutically effective for a subject in need of such treatment. in an amount of VITAXIN ™ administered in an prophylactically or therapeutically effective amount of an antibody that specifically binds to TNFα (i.e., infliximab or an antigen-binding fragment thereof) and is administered prophylactically or therapeutically effective amount of methotrexate.

The present invention relates to a method for the prevention, treatment, management or alleviation of one or more symptoms of an autoimmune or inflammatory disorder, characterized in that said method comprises administering to a subject in need of such treatment a prophylactically or therapeutically effective amount of one or more integrin α. ν β3 antagonist, one or more TNFα antagonists and one or more CD2 binding molecules. In a specific embodiment, the present invention relates to a method for the prevention, treatment, management or alleviation of one or more symptoms of an autoimmune or inflammatory disorder, characterized in that said method comprises the prevention or prophylaxis of a subject in need of such treatment. In a therapeutically effective amount of VITAXIN ™ or an antigen-binding fragment thereof, a soluble TNFa receptor (e.g., etanercept) is administered in a prophylactically or therapeutically effective amount. -et) and a prophylactically or therapeutically effective amount of MEDI-507 or an antigen-binding fragment thereof. In another embodiment, the present invention relates to a method for the prevention, treatment, management or alleviation of one or more symptoms of an autoimmune or inflammatory disorder, characterized in that said method comprises administering to a subject in need of such treatment a prophylactically or therapeutically effective amount. VITAXIN ™ or an antigen-binding fragment thereof is administered prophylactically or in a therapeutically effective amount to an antibody that binds immune-specific to TNFα (e.g., infliximab or an antigen-binding fragment thereof) and is administered in a prophylactically or therapeutically effective amount. -507 or an antigen-binding fragment thereof.

The present invention relates to a method for the prevention, treatment, management or alleviation of one or more symptoms of an autoimmune or inflammatory disorder, characterized in that said method comprises administering to a subject in need of such treatment a prophylactically or therapeutically effective amount of one or more integrin α. ν β3 antagonist, one or more TNFα antagonists and one or more anti-inflammatory agents. In a specific embodiment, the present invention relates to a method for the prevention, treatment, management or alleviation of an autoimmune or inflammatory disorder, or one or more of its symptoms, characterized in that said method comprises treating a subject in need of such treatment prophylactically or therapeutically. to administer VITAXIN ™ or an antigen-binding fragment thereof in a prophylactically or therapeutically effective amount (e.g., etanercept) and to administer a steroid or non-steroidal anti-inflammatory drug in a prophylactically or therapeutically effective amount. In another embodiment, the present invention relates to a method for the prevention, treatment, management or alleviation of one or more symptoms of an autoimmune or inflammatory disorder, characterized in that said method comprises administering to a subject in need of such treatment a prophylactically or therapeutically effective amount. VITAXIN ™ or an antigen-binding fragment thereof is administered prophylactically or in a therapeutically effective amount of an antibody that binds immune-specific to TNFα (e.g., infliximab or an antigen-binding fragment thereof) and is administered in a prophylactically or therapeutically effective amount. a steroid or non-steroidal anti-inflammatory drug.

The present invention relates to a method for the prevention, treatment, management or alleviation of one or more symptoms of an autoimmune or inflammatory disorder, characterized in that said method comprises administering one or more integrin α ν β3 antagonists to one or more subjects in need of such treatment. TNFα antagonist, one or more immunomodulatory agents and one or more anti-inflammatory agents are administered. In a specific embodiment, the present invention relates to a method for the prevention, treatment, management or alleviation of one or more symptoms of an autoimmune or inflammatory disorder, said method comprising administering to a subject in need of such treatment a prophylactically or therapeutically effective amount. VITAXIN ™ or an antigen-binding fragment thereof is administered prophylactically or therapeutically effective amounts of a soluble TNFα receptor (e.g., etanercept) or an antibody that specifically binds to TNFα (e.g., infliximab or an antigen-binding fragment thereof) ), a prophylactically or therapeutically effective amount of methotrexate is administered and a therapeutically effective amount of a steroid or non-steroidal anti-inflammatory drug is administered. In another embodiment, the present invention relates to a method for the prevention, treatment, management or alleviation of one or more symptoms of an autoimmune or inflammatory disorder, characterized in that said method comprises administering to a subject in need of such treatment a prophylactically or therapeutically effective amount. VITAXIN ™ or an antigen-binding fragment thereof, in a prophylactically or therapeutically effective amount, is administered as a soluble TNFα receptor (e.g., etanercept) or an antibody that specifically binds to · TNFα (e.g., infliximab). or an antigen-binding fragment thereof), a prophylactically or therapeutically effective amount of a CD2 binding molecule (e. g., MEDI-507 or an antigen-binding fragment thereof) is administered in a prophylactically or therapeutically effective amount of a steroid or non-steroid. gyulladáscs medication.

The present invention relates to a method for the prevention, treatment, management or alleviation of one or more symptoms of an autoimmune or inflammatory disorder, characterized in that said method comprises administering one or more integrin α ν β3 antagonists to a subject in need of such treatment, and one or more of them. more nucleic acid molecules are encoded to prophylactic or therapeutic agents other than one or more integrin α ν β3 antagonists. The present invention further relates to a method for the prevention, treatment, management or alleviation of one or more symptoms of an autoimmune or inflammatory disorder, characterized in that said method comprises administering to a subject in need of such treatment one or more nucleic acid molecules which have been administered to a subject. or more than one integrin α ν β3 is encoded and one or more nucleic acid molecules encoding a prophylactic or therapeutic agent other than one or more integrin α ν β3 antagonists are administered.

The present invention relates to pharmaceutical compositions comprising a pharmaceutically acceptable carrier, one or more integrin α ν β3 antagonists, and one or more other than integrin α ν β3 antagonists.

................

Agent · · · · · · · · · · · · · · · · · · · · · · · · · · · · · The pharmaceutical composition of the present invention may be used in the methods of the present invention for the prevention, treatment or alleviation of one or more symptoms associated with an autoimmune or inflammatory disorder. The pharmaceutical compositions of the present invention are preferably sterile and have the form to be administered to a patient suffering from an autoimmune or inflammatory disorder in a particular manner.

In one embodiment, the pharmaceutical composition comprises a pharmaceutically acceptable carrier, one or more integrin α ν β3 antagonists, and one or more immunomodulatory agents. In another embodiment, a pharmaceutical composition comprises a pharmaceutically acceptable carrier, VITAXIN ™ and one or more immunomodulatory agents. In another embodiment, a pharmaceutical composition comprises a pharmaceutically acceptable carrier, VITAXIN ™ and methotrexate.

In a specific embodiment, the pharmaceutical composition comprises a pharmaceutically acceptable carrier, one or more integrin α ν β3 antagonists, and one or more CD2 binding molecules. In another embodiment, a pharmaceutical composition comprises a pharmaceutically acceptable carrier, VITAXIN ™ or an antigen-binding fragment thereof, and one or more CD2 binding molecules. In a preferred embodiment, the pharmaceutical composition comprises a pharmaceutically acceptable carrier, VITAXIN ™ or an antigen-binding fragment thereof, and a soluble TNFα receptor (e.g., etanercept), or an antibody that binds immune-specific to TNFα. .

In a specific embodiment, the pharmaceutical composition comprises a pharmaceutically acceptable carrier, one or more integrin α ν β3 antagonists, and one or more anti-inflammatory agents. In another embodiment, a pharmaceutical composition comprises a pharmaceutically acceptable carrier, VITAXIN ™, or an antigen-binding fragment thereof, and one or more anti-inflammatory agents. In a preferred embodiment, the pharmaceutical composition comprises a pharmaceutically acceptable carrier, VITAXIN ™ or an antigen-binding fragment thereof, and a steroid or non-steroidal anti-inflammatory drug.

In one embodiment, the pharmaceutical composition comprises a pharmaceutically acceptable carrier, one or more integrin α ν β3 antagonists, one or more immunomodulatory agents, and one or more TNFα antagonists. In another embodiment, the pharmaceutical composition comprises a pharmaceutically acceptable carrier, one or more integrin α ν β3 antagonists, one or more CD2 binding molecules, and one or more TNFα antagonists. In another embodiment, the pharmaceutical composition comprises a pharmaceutically acceptable carrier, one or more integrin α ν βδ antagonists, one or more CD2 binding molecules, and one or more TNFα antagonists. In another embodiment, the pharmaceutical composition comprises a pharmaceutically acceptable carrier, one or more integrin α ν β3 antagonists, one or more anti-inflammatory agents, and one or more anti-inflammatory agents. or more TNFα antagonists. According to these embodiments, at least one of the integrin α ν β3 antagonists is preferably VITAXIN ™ or its antigen-binding fragment.

The compositions and methods described herein are particularly useful for the prevention of rheumatoid arthritis, spondyloarthropathies (e.g., psoriatic arthritis, paralytic vertebrae, Reiter's syndrome (also known as reactive arthritis), arthritis associated with inflammatory bowel disease, and undifferentiated spondyloarthropathy), psoriasis, undifferentiated arthropathy and arthritis. or treat it. Examples of the types of psoriasis that can be treated with the compositions and methods of the present invention include, but are not limited to, plaque psoriasis, bladder psoriasis, erythrodermic psoriasis, patchy psoriasis, and inverse psoriasis. The compositions and methods of the present invention may also be used for the prevention, treatment, management or alleviation of one or more symptoms associated with inflammatory osteolysis, other disorders characterized by abnormal bone reabsorption, or bone loss (e.g., osteoporosis). In a preferred embodiment, the compositions and methods described herein are used in prophylactic or therapeutic protocols for the prevention, treatment, management or alleviation of one or more symptoms associated with rheumatoid arthritis. In another preferred embodiment, the compositions and methods described herein are for the prophylaxis, prevention, treatment, or amelioration of one or more symptoms associated with psoriasis or psoriatic arthritis. or in therapeutic protocols. In another preferred embodiment, the compositions and methods described herein are used in prophylactic or therapeutic protocols for the prevention, treatment, management or alleviation of one or more symptoms of osteoporosis associated with rheumatoid arthritis, psoriasis or psoriatic arthritis.

The present invention relates to industrial products comprising a packaging material and a pharmaceutical composition of the present invention in a form suitable for administration to a subject packaged in said packaging material. More specifically, the present invention relates to industrial products comprising a packaging material and a pharmaceutical composition of the present invention in a form suitable for administration to a subject packaged in said packaging material, said pharmaceutical composition comprising one or more integrins α ν βββ. an antagonist, one or more prophylactic or therapeutic agents other than an integrin α ν β3 antagonist, and a pharmaceutically acceptable carrier. The industrial products of the present invention may include instructions for the administration of a pharmaceutical composition or other information material informing the physician, technician, or patient how to properly treat the disease or disorder in question.

In a specific embodiment, the industrial product comprises a packaging material and a pharmaceutical composition in a form suitable for administration to a subject packaged in said packaging material, said pharmaceutical composition comprising an integrin α ν β3 antagonist, an anti-inflammatory agent, and a pharmaceutically acceptable carrier. . In another embodiment, the industrial product comprises a packaging material and a pharmaceutical composition, in a form suitable for administration to a subject, preferably a human, and most preferably a human suffering from an autoimmune or inflammatory disorder, packaged in said packaging material, and said pharmaceutical composition comprising an integrin α ν β3 antagonist, an anti-inflammatory agent, and a pharmaceutically acceptable carrier.

In one embodiment, the industrial product comprises a packaging material and a pharmaceutical composition, in a form suitable for administration to a subject, preferably a human, and most preferably to a human suffering from an autoimmune or inflammatory disorder, packaged in said packaging material, and said pharmaceutical composition comprises an integrin α ν. a β3 antagonist, a CD2 binding molecule, and a pharmaceutically acceptable carrier. In a preferred embodiment, the industrial product comprises a packaging material and a pharmaceutical composition for a subject, preferably a human, and air43, preferably in a form suitable for administration to a human suffering from an autoimmune or inflammatory disorder, packaged in said packaging material, and said pharmaceutical composition comprising VITAXIN. ™ antagonist, MEDI-507 and a pharmaceutically acceptable carrier.

In another embodiment, the industrial product comprises a packaging material and a pharmaceutical composition, in a form suitable for administration to a subject, preferably a human, and most preferably to a human suffering from an autoimmune or inflammatory disorder, packaged in said packaging material, and said pharmaceutical composition comprising an integrin α. ν β3 antagonist, a TNFα antagonist and a pharmaceutically acceptable carrier. In a preferred embodiment, the industrial product comprises a packaging material and a pharmaceutical composition, in a form suitable for administration to a subject, preferably a human, and most preferably to a human suffering from an autoimmune or inflammatory disorder, packaged in said packaging material, and said pharmaceutical composition comprising an integrin. α ν β3 antagonist, ENBREL ™ or REMICADE ™ and a pharmaceutically acceptable carrier.

Hereinafter, the terms "incidental, incidental," and "relationship" are used interchangeably with the terms "in combination" or "combinatorial".

Hereinafter, the term "analog", when used in connection with the polypeptides, is for a polypeptide • · «· · ·« «· ··· ·· ·« · ····· «·· ··· *« · · Refers to a similar or identical function to another polypeptide, but not necessarily the same or similar amino acid sequence to the second polypeptide, or to the same or similar structure to the second polypeptide. A polypeptide having a similar amino acid sequence refers to a second polypeptide that satisfies at least one of the following requirements: a) a polypeptide having an amino acid sequence of at least 30%, at least 35%, at least 40% at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85% %, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence of a second polypeptide; b) a polypeptide encoded by a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence encoding a second polypeptide comprising at least 5 contiguous amino acid residues, of at least 10 contiguous amino acid residues, of at least 15 contiguous amino acid residues at least 20 from a contiguous amino acid group of at least 25 contiguous amino acid groups of at least 40 contiguous amino acid groups of at least 50 contiguous amino acid groups of at least 60 contiguous amino acid residues of at least 70 contiguous amino acid groups of at least 80 contiguous amino acid residues of at least 90 contiguous amino acid residues an amino acid residue of at least 100 contiguous amino acid residues comprising at least 125 contiguous amino acid residues, or * at least 150 contiguous amino acid residues; and c) a polypeptide encoded by a nucleotide sequence which is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60% at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% is identical to a nucleotide sequence encoding a second polypeptide. A polypeptide having a structure similar to a second polypeptide is a polypeptide having a secondary, tertiary or quaternary structure similar to the second polypeptide. The structure of a polypeptide may be determined in a manner known to those skilled in the art, including, but not limited to, sequencing of the peptide, X-ray crystallography, nuclear magnetic resonance, circular dichroism, and crystallographic electron microscopy.

In order to determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned side by side for optimal comparison (i.e., insertion into the first amino acid sequence or nucleic acid sequence to optimally align with a second amino acid sequence or nucleic acid sequence). The amino acid residues or nucleotides at the appropriate amino acid positions or nucleotide positions are then compared. If a position in the first sequence is occupied by the same amino acid group or nucleotide as in the corresponding position of the second chair46 ··· • ft · encia, the molecules are identical at that position. The percent identity between the two sequences depends on the number of common positions in the sequences. (ie% identity = the number of identical overlapping positions / positions closed * 100%). In one embodiment, the length of the two sequences is the same.

The percent identity between the two sequences can also be determined using a mathematical algorithm. A preferred non-limiting example of a mathematical algorithm used to compare the two sequences is the Kariin and Altschul algorithms [Karin and Altscul, 1990, Proc. Altschul: Proceedings of the National Academy of Sciences, USA 90, 5873-5877 (1993). This algorithm is used in NBLAST and XBLAST programs (Altschul et al., Journal of Molecular Biology 215, 403 (1990)). For example, BLAST nucleotide searches may be performed with the parameters of the following NBLAST nucleotide program: value = 100, word length = 12 to obtain nucleotide sequences homologous to the nucleic acid molecule of the present invention. For example, BLAST protein searches can be performed using the following XBLAST program parameters: value = 50, word length = 3 to obtain amino acid sequences homologous to the protein molecule of the present invention. Gappped Blast can be used for alignment purposes, as described by Altschul et al., Altschul et al.

Nucleic Acids Research 25: 3389-3402 (1997)]. Alternatively, PSI-BLAST can be used to perform an iterative search that detects distant connections between molecules (Altschul et al., 1997, Nucleic Acids Research 25: 33893402). When using BLAST, Gapped-BLAST and PSI-BLAST, you can use the basic parameters of the appropriate programs (ie XBLAST and NBLAST) (see, for example, the NCBI site). Another preferred non-limiting example of a mathematical algorithm used to compare sequences is the Myers and Miller algorithm [Myers and Miller, CABIOS 4, 11-17 (1988)]. Such an algorithm exists in ALIGN (version 2.0), which is part of the GCG sequence alignment suite. If the ALIGN program is used to compare amino acid sequences, a PAM120 weight group table, slot 12 penalty and slot 4 can be used.

The percent identity between the two sequences can be determined by techniques similar to those described above, by enabling or disabling gaps. Typically, only exact matches are counted in the percent identity calculation.

In the following, the term "analog" for non-protein-like analogs refers to a second organic or inorganic molecule having a similar or identical function to a first organic or inorganic molecule and structurally similar to the first organic or inorganic molecule.

«·· £ | · 4 · ·· *!

«4 · * · * ·« Λ <· * · <Μ * · · «

As used herein, the term "agonist" and "antagonist" refers to any protein, polypeptide, peptide, antibody, antibody fragment, large molecule or small molecule (size less than 10 kDa) which blocks, inhibits, reduces or neutralizes the activity of another molecule, and / or expression. In various embodiments, an antagonist has the function, activity, and / or expression of another molecule at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35% , at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% , at least 80%, at least 85%, at least 90%, 9 5%, or at least 99% relative to a control such as phosphate buffered saline (PBS).

Hereinafter, the term "antibody" and "antibodies" are defined as monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, single chain FVs (scFV), single chain antibodies, Fab fragments, F (ab + ) fragments, disulfide-linked Fv (sdFV) and anti-idiotypic (anti-Id) antibodies (including, for example, anti-Id antibodies against antibodies of the present invention) and epitope-binding fragments thereof. More specifically, the antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules containing an antigen binding site. Immunoglobin · molecules may be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY) and of any class (i.e., IgGi, IgG 2 , IgG 3 , IgG 4 , IgAi and IgA 2 ) or subclass.

The term "anti-TNFα agent", "TNFa antagonist" and the like herein may mean any protein, polypeptide, peptide, fusion protein, antibody, antibody fragment, large molecule or small molecule that blocks, inhibits, reduces or neutralizes the tumor. necrosis factor alpha (TNFα) activity and / or expression. TNFα antagonists include, but are not limited to, REMICADE ™ and ENBREL ™. In various embodiments, TNFα antagonizes TNFα function, activity and / or expression by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35% - at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, \ t at least 80%, at least 85%, at least 90%, 95% or at least 99% relative to a control such as phosphate buffered saline (PBS).

As used herein, the term "CD2 polypeptide" refers to a CD2 glycoprotein (also known as Tll or LFA-2) or a fragment thereof. In a preferred embodiment, a CD2 polypeptide is a 50-55 kDa cell surface glycoprotein expressed by immune cells such as T cells and natural killer cells ("NK"). The CD2 polypeptide may be derived though50

......, ··. What kind of "I · * · · · * · · · ···· · · · * * · · · · ··. The nucleotide and / or amino acid sequence of the CD2 polypeptides can be found in the literature or in public databases, or the nucleotide and / or amino acid sequences can be determined by cloning and sequencing techniques known to those skilled in the art. For example, the nucleotide sequence of human CD2 can be found in the GenBank database (see, for example, Accession No. X06413, AH002740, and M16455).

As used herein, the term "cytokine receptor modulator" refers to an agent that affects the phosphorylation of a cytokine receptor, the activation of a signal transduction biosynthesis pathway by a cytokine receptor, and / or the expression of a particular protein, such as a cytokine. Such an agent can directly and indirectly affect the phosphorylation of a cytokine receptor, the activation of a cytokine receptor-binding signal transduction biosynthesis pathway, and / or the expression of a particular protein, such as a cytokine. Thus, for example, cytokine receptor modulators include, but are not limited to, cytokines, cytokine fragments, fusion proteins, and antibodies that immunoprecipitate specifically to a cytokine receptor or a fragment thereof. Examples of cytokine receptor modulators include, but are not limited to, peptides, polypeptides (e.g., soluble cytokine receptors), fusion proteins, and antibodies that specifically bind to a cytokine or fragment thereof.

As used herein, the term "dermatological agent" and analogous terms refer to an agent that helps to treat skin diseases and complaints. A dermatological agent is preferably a topical agent used to prevent, treat, or alleviate a skin condition associated with a skin condition, particularly increased T cell infiltration, increased T cell activation and / or abnormal antigen presentation. In a particularly preferred embodiment, a dermatological agent term is a topical agent used to prevent, treat or alleviate psoriasis or one or more of its symptoms.

As used herein, the term "derivative" refers to a polypeptide comprising a sequence that has been altered by inserting, cutting, or substituting an amino acid residue for polypeptides. The term "derivative" is hereinafter defined as a polypeptide that has been modified, for example, by covalently attaching any type of molecule to the polypeptide. For example, but without being limited thereto, an antibody may be modified, for example, by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protein / blocking groups, proteolytic cleavage, coupling to a cellular ligand or other protein, and the like. A polypeptide derivative may be prepared by chemical modifications using techniques known to those skilled in the art, which may include, but are not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, and the like. In addition, a polypeptide derivative may contain one or more non-classic amino acids. A polypeptide derivative has similar or identical functions to the polypeptide from which it is derived.

As used herein, the term "derivative" refers to a second organic or inorganic molecule for a non-protein derivative, which is derived from the structure of a first organic or inorganic molecule. An organic molecule can be a derivative, without being limited thereto, by a molecule that can be produced, for example, by the addition or removal of a hydroxy, methyl, ethyl, carboxy or amine group. An organic molecule may be further esterified, alkylated and / or phosphorylated.

Hereinafter, the terms "disorder" and "disease" can be used to replace one another if we want to talk about the condition of a subject. More specifically, the term "autoimmune disease" may be used interchangeably with the term "autoimmune disorder" when a subject is characterized by cellular, tissue, and / or organ damage caused by the subject's own cells, tissues and / or organs by an immunological reaction. .

As used herein, the term "epitope" refers to a fragment of a polypeptide or protein having antigenic or immunogenic activity, preferably in a mammal, most preferably in humans. An epitope having an immunogenic activity is a fragment of a polypeptide or protein that elicits an antibody response in an animal. An epitope with an antigenic activity is a fragment of a polypeptide or protein for which an antibody is immunospecifically bound, which can be determined by a method well known to those skilled in the art, such as by immunoassay. The antigenic epitopes need not necessarily be immunogenic.

Hereinafter, the term "fragment" refers to a peptide or polypeptide comprising at least 5 contiguous amino acid residues of another amino acid sequence of another polypeptide, a group of at least 10 contiguous amino acids, a group of at least 15 contiguous amino acids, at least 20 contiguous amino acid residues, at least 25 contiguous amino acid residues, at least 40 contiguous amino acid residues, at least 50 contiguous amino acid residues, at least 60 contiguous amino acid residues, at least 70 contiguous amino acid residues, at least 80 contiguous amino acid residues, at least 90 contiguous amino acid residues, at least 100 contiguous amino acid residues, at least 125 contiguous amino acid residues, at least 150 of at least 175 contiguous amino acid residues, at least 200 contiguous amino acid residues, or at least 250 contiguous amino acid residues; is amino group. In a specific embodiment, a polypeptide fragment retains at least one function of the polypeptide.

• · • · ** · • · ···

Hereinafter, the term "functional fragment" refers to a peptide or polypeptide that is a group of at least 5 contiguous amino acid sequences of the amino acid sequence of a second, different polypeptide, a group of at least 10 contiguous amino acids, a group of at least 15 contiguous amino acids, at least 20 contiguous amino acid residues, at least 25 contiguous. from at least 40 contiguous amino acid residues, at least 50 contiguous amino acid residues, at least 60 contiguous amino acid residues, at least 70 contiguous amino acid residues, at least 80 contiguous amino acid residues, at least 90 contiguous amino acid residues, at least 100 contiguous amino acid residues, at least 125 contiguous amino acid residues, , from at least 150 contiguous amino acid residues, from at least 175 contiguous amino acid residues, from at least 200 contiguous amino acid residues, or it comprises at least 250 contiguous amino acid residues and said peptide or polypeptide retains at least one of the functions of the second, different polypeptide.

As used herein, the term "fusion protein" means a polypeptide comprising an amino acid sequence of a first protein, or a functional fragment, analogue or derivative thereof, and a heterologous protein (e.g., a second protein or a functional fragment, analogue or derivative thereof) amino acid sequence. In one embodiment, a fusion protein comprises a prophylactic or therapeutic agent linked to a heterologous protein, polypeptide or peptide. According to this embodiment, the heterologous protein, polypeptide or peptide may be the same but may be a different type of prophylactic or therapeutic agent. For example, two different proteins, polypeptides or peptides having immunomodulatory activity can be fused with the formation of a fusion protein. In some embodiments, a fusion protein comprises a protein, polypeptide, or peptide having immunomodulatory activity and comprises a heterologous protein, polypeptide or peptide. In other embodiments, a fusion protein comprises a CD2 binding molecule and a heterologous protein, polypeptide or peptide. In other embodiments, a fusion protein comprises a protein, polypeptide or peptide having a TNFα antagonist activity, and a heterologous protein, polypeptide or peptide. In a preferred embodiment, the fusion proteins retain or obtain integrin α ν β3 antagonist activity, immunomodulatory activity, or TNFα antagonist activity relative to the activity of the original protein, polypeptide or peptide prior to fusion with a heterologous protein, or to confirm it.

As used herein, the term &quot; host cell &quot; refers to a particular cell that has been transfected with a nucleic acid molecule as well as progeny or potential progeny of such a cell. The progeny of such a cell may not be identical to the original cell transfected with the nucleic acid molecule due to mutations or environmental influences that occur in subsequent generations or during the integration of the nucleic acid molecule into the host cell genome.

Hereinafter, the term "hybridizes under stringent conditions describes conditions for hybridization and washing under which nucleic acid sequences of at least 60% (65, 70, preferably 75%) are typically hybridized to one another. Such stringent conditions are well known to those skilled in the art [Current Protocols in Molecular Biology, 6.3.1-6.3.6, ed. Ausubel et al., Greene Publishing and Wiley-Interscience: New York (1987)]. In one non-limiting example, the stringent hybridization condition for hybridization in 6X sodium chloride / sodium citrate (SSC) solution at about 45 ° C is followed by one or more washings of 0.1XSSC in about 0.2% SDS. ° C, then one or more washings in 0.2XSSC, 0.1% SDS at 50-65 ° C (i.e. one or more washings at 50 ° C, 55 ° C, 60 ° C on or at 65 ° C). Obviously, the nucleic acids of the present invention do not contain nucleic acids that hybridize under such conditions to a sequence containing only A or T nucleotides.

As used herein, the term "immunomodulatory agent" and variants thereof include, but are not limited to, immunomodulatory agents, an agent that affects the immune system of the host. In some embodiments, an immunomodulatory agent is an immunostimulatory agent. According to the present invention, the immunomodulatory agent used in the combination therapies of the present invention cannot be an integrin α ν β3 antagonist. Immunomodulatory agents include, but are not limited to, small molecules, peptides, polypeptides, fusion proteins, antibodies, inorganic molecules, mimetic agents, and organic molecules. In some embodiments, the immunomodulatory agent used in the combination therapy of the present invention is a CD2 binding molecule. In another embodiment, the immunomodulatory agent used in the combination therapy of the present invention is not a CD2 binding molecule. In another embodiment, the immunomodulatory agent used in the combination therapies of the present invention is a TNFα antagonist. In other embodiments, the immunomodulatory agent used in the combination therapies of the present invention is not a TNFα antagonist. In other embodiments, the immunomodulatory agent used in the combination therapy of the present invention is methotrexate. In other embodiments, the immunomodulatory agent used in the combination therapies of the present invention is not methotrexate.

The term &quot; immune-specific binding to an antigen &quot; is now defined, and the analogous terms refer to peptides, polypeptides, fusion proteins, and antibodies, or fragments thereof, that specifically bind to an antigen or fragments thereof but do not specifically bind to other antigens. A peptide or polypeptide58. The tid that binds immune-specific to an antigen can bind to other peptides or polypeptides with less affinity, which can be determined, for example, by immunoassay, BIAcore, or other methods known to those skilled in the art. Antibodies or fragments that bind immunoprecipitatively to an antigen may cross-react with related antigens. Preferably, the antibodies or fragments that immunoprecipitate specifically bind to an antigen do not cross-react with other antigens. In some embodiments, the antigen to which a peptide, polypeptide or antibody is immunospecifically bound is a cytokine receptor or a T cell receptor.

The term &quot; immune-specific binding to a CD2 polypeptide &quot;, and the analogous terms refer to peptides, polypeptides, fusion proteins, and antibodies, or fragments thereof, that specifically bind to a CD2 polypeptide or fragment thereof and are not specifically bound to other polypeptides. A peptide or polypeptide that is immunospecifically bound to a CD2 polypeptide may have a lower affinity to bind to other peptides or polypeptides, which may be determined, for example, by immunoassay, BIAcore, or other assays known in the art. Antibodies or fragments that specifically bind to a CD2 polypeptide may cross-react with related antigens. Antibodies or fragments thereof that immunoprecipitate specifically to a CD2 polypeptide or fragment thereof, preferably do not cross-react with other antigens. Antibodies or fragments specifically binding to a CD2 polypeptide may be identified, for example, by immunoassay, BIAcore, or other techniques known to those skilled in the art. An antibody or fragment thereof specifically binds to a CD2 polypeptide when it binds to a VD2 polypeptide with greater affinity than any cross-reacting antigen that can be determined by experimental techniques such as radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA). Immunology Second Edition, 332-336. , Paul, Raven Press, New York (1989), where antibody specificity is discussed].

In the following, the term "immune-specific binds to integrin a v p3" and the analogous terms refer to peptides, polypeptides, fusion proteins and antibodies, or fragments thereof, that specifically bind to an integrin α ν β3 polypeptide or an integrin α ν β3 polypeptide and do not specifically bind to other polypeptides. Antibodies or fragments that immunoprecipitatively bind to an integrin α ν β3 polypeptide or fragment thereof preferably do not cross-react with other antigens. Antibodies or fragments thereof that bind immunoprecipitatively to an integrin α ν β3 polypeptide may be identified, for example, by immunoassay or other techniques known to those skilled in the art. Elena60 fragments or fragments thereof that bind to an integrin α ν β3 polypeptide, or fragment thereof, preferably only antagonize the α ν β3 activity of integrin and do not significantly antagonize the activity of other integrins.

As used herein, the term "in combination" means that more than one prophylactic and / or therapeutic agent is used. The use of the term "in combination" does not limit the order in which prophylactic and / or therapeutic agents are administered to a subject suffering from an autoimmune or inflammatory disorder. A first prophylactic or therapeutic agent may be administered to a patient suffering from an autoimmune or inflammatory disorder prior to administration of a second prophylactic or therapeutic agent (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours). hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), or at the same time (eg. 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks , 5 weeks, 6 weeks, 8 weeks, or 12 weeks later).

Hereinafter, "integrin α ν β3 antagonist" and analogue terms refer to any protein, polypeptide, fusion protein, antibody, antibody fragment, large molecule or small molecule (size less than 10 kDa) that blocks, inhibits, reduces or neutralizes function, activity and / or expression of integrin α ν β3. A preferred non-limiting example of an integrin α ν β3 • μ antagonist is VITAXIN ™. In various embodiments, an integrin α ν β3 antagonist has the function, activity and / or expression of integrin α ν β at least 10%, at least 15%, at least 20%, at least 25%, at least 30% - with at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, 95%, or at least 99%, relative to a control such as phosphate buffered saline (PBS).

As used herein, the term "isolated" refers to a peptide, polypeptide, fusion protein, or antibody that is substantially free of cellular materials or contaminating proteins of the cell source or source of tissue from which it is derived or substantially free of chemicals. precursors or other chemical substances if chemically prepared. The term "substantially free of cellular materials" means a peptide, polypeptide, fusion protein, or antibody that is isolated from the cellular components of the cell from which it is derived, or is produced by a recombinant method. Thus, a peptide, polypeptide, fusion protein, or antibody substantially free of cellular material is a peptide, polypeptide, fusion protein, or antibody composition with less than 30%, 20%, 10%, or 5%, a heterologous protein based on dry weight • 9 • τ »« · * · • t ...

If the peptide, polypeptide, fusion protein or antibody is produced by a recombinant method, it is preferably free from the culture medium, for example, the culture medium is less than 20% of the volume of the protein composition, 10% or 5% of the total. If the peptide, polypeptide, fusion protein or antibody is prepared by chemical synthesis, it is preferably substantially free of chemical precursors or other chemicals, i.e., separated from chemical precursors or other chemicals that are included in the synthesis of the peptide, polypeptide, fusion protein, or antibody . Accordingly, such compositions of a peptide, polypeptide, fusion protein, or antibody contain less than 30%, 20%, 10%, 5% (based on dry weight) of other chemical precursors or compounds than for us an interesting peptide, polypeptide, fusion protein, or antibody. In a preferred embodiment, an integrin α ν β3 antagonist is isolated. In another preferred embodiment, an immunomodulatory agent is isolated. In yet another preferred embodiment, a TNFα antagonist is isolated.

In the following, the term "isolated" refers to a nucleic acid molecule separated from other nucleic acid molecules in the natural environment of the nucleic acid molecule, relative to the nucleic acid molecules. In addition, an "isolated" nucleic acid molecule, such as a cDNA molecule, may be substantially free of other cellular material or culture medium by recombinant techniques or substantially free of chemical precursors or other chemicals when synthesized chemically. In a preferred embodiment, a nucleic acid molecule encoding an integrin α ν β3 antagonist is isolated. In another preferred embodiment, a nucleic acid molecule encoding an immunomodulatory agent is isolated. In yet another preferred embodiment, a nucleic acid molecule encoding a TNFα antagonist is isolated.

In the following, the term "low tolerance" refers to a condition in which the patient suffers from side effects of the treatment and therefore does not see the benefit of the patient and / or does not continue therapy because of adverse side effects.

Hereinafter, the term "manager", "management" refers to the beneficial effects that a subject enjoys from a prophylactic or therapeutic treatment, but does not result in the cure of the disease. In some embodiments, a subject is administered one or more prophylactic or therapeutic agents to "manage" an abnormality in order to prevent further development or worsening of the disorder.

In the following, the term "mild disease" refers to an arthritic patient having at least 2 swollen joints but a maximum of 10 delicate, sore joints.

Hereinafter, the term "non-reactive" and "difficult to treat" refers to patients treated with the currently available prophylactic or therapeutic agent due to an autoimmune or inflammatory disorder (e.g., methotrexate alone or an anti-TNFa agent) that is not clinically relevant. suitable for alleviating one or more of the symptoms associated with autoimmune or inflammatory disorders. Typically, such patients suffer from severe, persistent disease and require further therapy to alleviate symptoms associated with autoimmune or inflammatory disorders.

As used herein, the term "nucleic acids" and "nucleotide sequence" refers to DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), DNA and RNA molecules, or hybridized DNA / RNA molecules, and DNA or RNA analogues of molecules. These analogs can be prepared, for example, by nucleotide analogues, which include, but are not limited to, inosine or tritilated bases. These analogues may be DNA or RNA molecules that have a backbone modified to provide preferred properties to the molecules, such as nuclease resistance or increased ability to penetrate cellular membranes. The nucleic acids or nucleotide sequences may be single-stranded, double-stranded, may contain single-chain and double-stranded moieties, and may also comprise three-stranded portions but preferably double-stranded DNAs.

In the following, the term "potential" refers to an improvement in the effectiveness of a prophylactic or therapeutic agent at a conventional or accepted dose.

In the following, the term "prophylactic agent" and "prophylactic agents" refers to any agent useful in the prevention of an autoimmune or inflammatory disorder. In some embodiments, the term "prophylactic agent" means an integrin α ν β3 antagonist (such as VITAXIN ™). In some other embodiments, the term "prophylactic agent" does not refer to an integrin α ν β3 antagonist (such as VITAXIN ™). A prophylactic agent is preferably an agent that is known to be useful or is currently used to prevent or hinder the development, action or progression of an autoimmune or inflammatory disorder.

As used herein, the terms "preventive", "prevention" means preventing the occurrence or occurrence of one or more symptoms of an autoimmune or inflammatory disorder in a subject as a result of the administration of a prophylactic or therapeutic agent.

As used herein, the term "prophylactically effective amount" refers to an amount of a prophylactic agent sufficient to prevent the occurrence or occurrence of one or more symptoms of a disorder.

In the following, the term "prophylactic protocol" refers to the dose range and timing of administration of one or more prophylactic agents.

In the following, the term "protocol" refers to dosage plans and dosage ranges. The protocols used here are methods of application and include prophylactic and therapeutic protocols.

As used herein, the term "side effects" refers to the undesirable and harmful effects of a prophylactic or therapeutic agent. Adverse effects are always undesirable effects, but undesirable effects are not necessarily harmful. The detrimental effect of a prophylactic or therapeutic agent can be abnormal or uncomfortable or risky. The side effects of the administration of REMICADE ™ include, but are not limited to, the risk of serious infection and hypersensitivity reactions. Other side effects may include non-specific symptoms such as fever or chills, itching or hives and cardiopulmonary reactions such as chest pain, low blood pressure, high blood pressure, or breathing problems such as muscle pain, arthralgia, rash, face , hand or lip edema, difficulty swallowing, sore throat and headache. Other side effects include, but are not limited to, abdominal hernia, spleen infarction, splenic enlargement, dizziness, upper motor lesions, lupus erythematosus symptom, rheumatic nodules, ceruminosis, abdominal pain, diarrhea, gastric ulcer, intestinal obstruction, intestinal perforation, intestinal obstruction 67 nausea, pancreatitis, vomiting, back pain, fracture, tendon disorder or injury, heart damage, myocardial ischemia, lymphoma, thrombocytopenia, cellulitis, anxiety, confusion, delirium, depression, abnormal sleepiness, suicide attempt, anemia, abscess, bacterial infection and sepsis. Adverse reactions from the administration of ENBREL ™ include, but are not limited to, the risk of severe infection and sepsis, including fatal outcomes. The harmful side effects of a serious infection spread from severe infections such as pyelonephritis, bronchitis, septic arthritis, abdominal abscess, cellulitis, osteomyelitis, wound infection, pneumonia, foot abscess, leg ulcer, diarrhea, sinusitis, sepsis, headache, nausea, nasal mucosa inflammation, dizziness, throat inflammation, cough, weakness, abdominal pain, rash, peripheral edema, respiratory disorder, indigestion, nasal mucosa, vomiting, oral cavity, baldness and pneumonia to other less common adverse effects such as heart attack, heart infection, myocardium ischemia, cerebral ischemia, hypertension, low blood pressure, gall bladder inflammation, pancreatitis, gastrointestinal hematoma, mucositis, depression respiratory disorders, deep vein thrombosis, pulmonary embolism, membrane glomerulonephropathy, polymiositis and thrombophlebitis. Adverse reactions from methotrexate administration include, but are not limited to, severe toxic reactions that are fatal • · · ·

They may be outcome I, such as unexpectedly severe bone marrow suppression, gastrointestinal toxicity, fibrosis and cirrhosis after prolonged use, lung diseases, diarrhea and ulcerative inflammation of the mouth, malignant lymphomas and occasionally fatal severe skin reactions.

As used herein, the term "small molecules", as used herein, and the like, without being limited to, peptides, peptidomimetics, amino acids, amino acid analogues, polynucleotides, polynucleotide analogs, nucleotides, nucleotide analogues, organic or inorganic compounds (including hetero-organic and organometallic compounds) having a molecular weight of less than about 10,000 grams per mole of organic or inorganic compounds having a molecular weight of less than about 5,000 grams per mole of organic or inorganic compounds having a molecular weight of less than about 1000 grams per mole of organic or inorganic compounds having a molecular weight of less than about 500 grams per mole, and salts, esters, and other pharmaceutically acceptable forms thereof.

Hereinafter, the terms "subject" and "patient" can be used interchangeably. In the following, the term "subject" and "subject" refers to an animal, preferably a mammal, including a non-primate (e.g., cattle, pig, horse, cat, dog, rat, and mouse), a primate (e.g., a monkey, such as cynomolgous monkey and ······ · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ''. In one embodiment, the subject is a non-immunocompromised or immunosuppressed mammal, preferably a human (e. G., A TEA patient). In another embodiment, the subject is not a mammal, preferably a human having a lymphocyte count of about 500 cells / mm 3 . In one embodiment, the subject is a mammal, preferably a human, previously treated with one or more TNFα antagonists and methotrexate. In another embodiment, the subject is a mammal, preferably a human, who is not currently treated with a TNFα antagonist or methotrexate. In another embodiment, the subject is a mammal, preferably a human, with an autoimmune or inflammatory disorder that is resistant to a TNFα antagonist with a non-steroidal anti-inflammatory agent. or methotrexate treatment. In a preferred embodiment, the subject is a human. In another embodiment, the subject is suffering from non-differentiated arthropathy or psoriasis in rheumatoid arthritis, spondyloarthropathy (e.g., psoriatic arthritis, paralyzing spondylitis, Reiter syndrome (also referred to as reactive arthritis), arthritis associated with inflammatory bowel disease, or undifferentiated spondyloarthropathy. the subject is rheumatoid arthritis, psoriatic arthritis or psoriasis.

In the following, the term "synergistic" refers to a combination of prophylactic or therapeutic agents that are more effective than either additive alone agent.

• · • · effect. The synergistic effect of a combination of prophylactic or therapeutic agents allows the use of lower doses of one or more agents and / or less frequent administration of said agents to an autoimmune or inflammatory subject. The possibility of using lower dose prophylactic or therapeutic agents and / or administering said agents less frequently reduces the toxicity associated with the administration of said agents to a subject without reducing the effectiveness of said agents in the prevention or treatment of an autoimmune or inflammatory disorder. In addition, the synergistic effect may result in increased efficacy of agents in the prevention or treatment of autoimmune or inflammatory disorders. Finally, a synergistic effect of a combination of prophylactic or therapeutic agents can eliminate or reduce adverse or unwanted side effects associated with a single agent-based therapy.

Hereinafter, the term "T cell receptor modulator" refers to an agent that affects the phosphorylation of a T cell receptor, a pathway of signal transduction associated with a T cell receptor, and / or the expression of a particular protein, such as a cytokine. . Such an agent directly or indirectly modulates phosphorylation of a T cell receptor, activation of a T-cell receptor-associated signal transduction biosynthesis pathway, and / or expression of a particular protein, such as a cytokine. Thus, for example, T cell receptor modulators include, but are not limited to, peptides, polypeptides, fusion proteins, and antibodies that immunoprecipitate specifically to a T cell receptor or a fragment thereof. Furthermore, T cell receptor modulators, for example, without limiting them, are peptides, polypeptides (e.g., soluble T cell receptors), fusion proteins, and antibodies that bind immunoprecipitatively to the ligand of a T cell receptor or fragment thereof.

As used herein, the term "therapeutic agent" and "therapeutic agent" refers to any agent that can be used to prevent, treat, manage or alleviate one or more symptoms associated with an autoimmune or inflammatory disorder. In some embodiments, the term "therapeutic agent" means an integrin α ν β3 antagonist (such as VITAXIN ™). In some other embodiments, the term "therapeutic agent" does not include an integrin α ν β3 antagonist (such as VITAXIN ™). Preferably, the therapeutic agent is an agent known to be useful for treating or alleviating one or more symptoms associated with an autoimmune or inflammatory disorder, or is already being used for this purpose.

In the following, the term "therapeutically effective amount" refers to an amount of therapeutic agent sufficient to alleviate one or more symptoms of a disorder.

result. As far as the treatment of psoriasis is concerned, the therapeutically effective amount preferably means the amount of therapeutic agent that has a Psoriasis Area and Severity Index (PASI) value of at least 20%, at least 25%, at least 30%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% by at least 80% or at least 85%. Alternatively, in the treatment of psoriasis, the therapeutically effective amount is preferably the amount of the therapeutic agent that has a human overall estimate of at least 20%, at least 25%, at least 30%, at least 35%. , at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95%.

Hereinafter, the term "therapeutic protocol" refers to the dosage range and schedule of administration of one or more therapeutic agents.

As used herein, the terms "treat", "treatment" and "treat" refer to the alleviation of one or more symptoms associated with an autoimmune or inflammatory disorder, which results from the administration of one or more prophylactic or therapeutic agents. In some embodiments, the term means a reduction in the swelling of one or more joints, or pain tubes associated with an autoimmune or inflammatory disorder · · · · · · · ··· ·· · · · · · · · · Lubrication resulting from the administration of one or more prophylactic or therapeutic agents to a patient suffering from such a disorder. In another embodiment, the term denotes a decrease in human PASi. In other embodiments, the term results in an improvement in the overall estimation value of the human being.

The attached figures are briefly described below.

1A-1B. Figure 4A: The nucleotide sequence and deduced amino acid sequence of the variable region of the VITAXIN ™ antibody. The IA. Figure 4 shows the nucleotide sequence and deduced amino acid sequence of the VITAXIN ™ heavy chain variable region (SEQ ID NO: 7 and SEQ ID NO: 8). The IB. Figure 4 shows the nucleotide sequence and deduced amino acid sequence of the VITAXIN ™ light chain variable region (SEQ ID NO: 9 and SEQ ID NO: 10).

The present invention relates to treatment protocols which result in better prophylactic and therapeutic profiles than the one-agent therapies currently used to treat autoimmune or inflammatory disorders. SUMMARY OF THE INVENTION The present invention provides a combination therapy for the prevention, treatment or alleviation of one or more symptoms associated with an autoimmune or inflammatory disorder in a subject, and said combination therapies comprising one or more integrin α ν β3 antagonists and one or more of said integrins. a prophylactic or therapeutic agent other than integrin α ν β3 antagonists is administered. More specifically, the present invention relates to combinatorial therapies, one of which is a · · · «" · · · · · · · · · · · · · ·

". ·· ·" for the prevention, treatment or alleviation of one or more symptoms associated with an autoimmune or inflammatory disorder, and said combination therapies comprising said integrin α ν β3 antagonist, preferably VITAXIN? another prophylactic or therapeutic agent that has a mechanism of action different from the mechanism of action of the integrin α ν β3 antagonist.

A combination of one or more integrin α ν β3 antagonists and one or more of the prophylactic or therapeutic agents other than the integrin α ν β3 antagonist results in a better prophylactic or therapeutic effect in a subject than any treatment alone. In some embodiments, a combination of a prophylactic or therapeutic agent other than an integrin α ν β3 antagonist and an integrin α ν β3 antagonist is 20%, preferably 25%, 30%, 35%, 40%, 45% , 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% results in a prophylactic or therapeutic effect in a subject suffering from an autoimmune or inflammatory disorder as any treatment alone. In some embodiments, a combination of an integrin α ν β3 antagonist and a prophylactic or therapeutic agent other than an α ν β3 antagonist is 20%, preferably 25%, 30%, 35%, 40%, 45% %, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% reduces inflammation in a particular organ, tissue or joint in an autoimmune or inflammatory disorder

than any treatment alone. In other embodiments, a combination of one or more integrin α ν β3 antagonists and one or more of the integrin α ν β3 antagonists has a more than additive or synergistic effect in an autoimmune or inflammatory disorder subject.

The combination therapies of the present invention allow smaller doses of integrin α ν β3 antagonists to be used, and / or less frequent administration of integrin α ν β3 antagonists, preferably VITAXI ™, to a subject suffering from an autoimmune or inflammatory disorder, and yet to be achieved. a prophylactic or therapeutic effect. The combination therapies of the present invention allow lower doses of prophylactic or therapeutic agents used in conjunction with integrin α ν β3 antagonists to be used to prevent or treat an autoimmune or inflammatory disorder, and / or to rarely administer such prophylactic or therapeutic agents to an autoimmune or inflammatory agent. and having a prophylactic or therapeutic effect. The combination therapies of the present invention reduce or avoid unwanted or harmful side effects associated with the presently present single-agent therapies for autoimmune or inflammatory disorders and / or existing combination therapies, which in turn increases patient cooperation.

The prophylactic or therapeutic agents of the combination therapies of the present invention may be administered sequentially, in parallel or sequentially. The prophylactic or therapeutic agents of the combination therapies of the present invention may also be administered cyclically. Cyclic therapy consists of administering a first prophylactic or therapeutic agent for a period of time, then administering a second prophylactic or therapeutic agent for a period of time, and then repeating this sequential administration, i.e., a cycle, to reduce any of the agents. the development of resistance to avoid side effects of one of the agents and / or increase the effectiveness of the treatment.

The present invention relates to a method for the prevention, treatment, management or alleviation of an autoimmune or inflammatory disorder, or one or more of its symptoms, characterized in that said method comprises administering to a subject in need thereof one or more integrin α ν β3 antagonists and one or more integrins. or more prophylactic or therapeutic agents other than integrin α ν β3 antagonists, which are currently used, used or known to be prophylactic or therapeutic agents, are useful for the prevention, treatment or treatment of one or more symptoms associated with an autoimmune or inflammatory disorder. (see, for example, non-limiting examples of prophylactic or therapeutic agents that can be administered to a subject in combination with one or more integrin α ν β3 antagonists, such that one or more autoimmune drugs are administered to a subject;

ΊΊ

to prevent, treat, manage or alleviate the symptom associated with an inflammatory disorder).

The present invention relates to a method for the prevention, treatment, management or alleviation of an autoimmune or inflammatory disorder, or one or more of its symptoms, characterized in that said method comprises administering to a subject in need thereof one or more integrin α ν β3 antagonists and one or more integrins. or more immunomodulatory agents are administered. Preferably, the immunomodulatory agent is not administered to a patient suffering from an autoimmune or inflammatory disorder whose absolute lymphocyte count is less than 500 cells / mm 3 , less than 550 cells / mm 3 , less than 600 cells / mm 3 , less than 650 cells / mm 3 , less than 700 cells / mm 3 , less than 750 cells / mm 3 , less than 800 cells / mm 3 , less than 850 cells / mm 3 , or less than 900 cells / mm 3 .

The present invention relates to a method for the prevention, treatment, management or alleviation of an autoimmune or inflammatory disorder, or one or more of its symptoms, characterized in that said method comprises administering to a subject in need thereof one or more integrin α ν β3 antagonists and one or more integrins. or more CD2 antagonists are administered. More specifically, the present invention relates to a method for the prevention, treatment, management or alleviation of an autoimmune or inflammatory disorder or one or more symptoms thereof, characterized in that said method comprises administering one or more VITAXIN ™ to a subject in need of such treatment. its anti78

a gene-binding fragment thereof and a prophylactically or therapeutically effective amount of one or more CD2 antagonists.

The present invention relates to a method for the prevention, treatment, management or alleviation of an autoimmune or inflammatory disorder, or one or more of its symptoms, characterized in that said method comprises administering to a subject in need thereof one or more integrin α ν β3 antagonists and one or more integrins. or multiple CD2 binding molecules (e.g., peptides, polypeptides, proteins, antibodies (MEDI-507), and fusion proteins that specifically bind to a CD2 polypeptide that directly or indirectly affect the disappearance of peripheral blood lymphocytes). Preferably, the CD2 binding molecule is not administered to patients with autoimmune or inflammatory disorders whose absolute lymphocyte count is less than 500 cells / mm 3 , less than 550 cells / mm 3 , less than 600 cells / mm 3 , less than 650 cells / mm 3 , less than 700 cells / mm 3 , less than 750 cells / mm 3 , less than 800 cells / mm 3 , less than 850 cells / mm 3 , or less than 900 cells / mm 3 . More specifically, the present invention relates to a method for the prevention, treatment, management or alleviation of an autoimmune or inflammatory disorder, or one or more of its symptoms, characterized in that said method comprises administering to a subject in need thereof a VITAXIN ™ or antigen-binding fragment thereof. a prophylactically or therapeutically effective amount of a prophylactically or therapeutically effective amount of MEDI-507 or an antigen-binding fragment thereof.

• · ···· · ·· '

The present invention relates to a method for the prevention, treatment, management or alleviation of an autoimmune or inflammatory disorder, or one or more of its symptoms, characterized in that said method comprises administering to a subject in need thereof one or more integrin α ν β3 antagonists and one or more integrins. or more anti-angiogenic agents are administered. More specifically, the present invention relates to a method for the prevention, treatment, management or alleviation of an autoimmune or inflammatory disorder, or one or more of its symptoms, characterized in that said method comprises administering to a subject in need thereof a VITAXIN ™ or antigen-binding fragment thereof. a prophylactically or therapeutically effective amount and a prophylactically or therapeutically effective amount of one or more anti-angiogenic agents.

The present invention relates to a method for the prevention, treatment, management or alleviation of an autoimmune or inflammatory disorder, or one or more of its symptoms, characterized in that said method comprises administering to a subject in need thereof one or more integrin α ν β3 antagonists and one or more integrins. or more TNFα antagonists are administered. More specifically, the present invention relates to a method for the prevention, treatment, management or alleviation of an autoimmune or inflammatory disorder, or one or more of its symptoms, characterized in that said method comprises administering to a subject in need thereof a VITAXIN ™ or antigen-binding fragment thereof. a prophylactically or therapeutically effective amount of a prophylactically or therapeutically effective amount of one or more TNFα antagonists.

The present invention relates to a method for the prevention, treatment, management or alleviation of an autoimmune or inflammatory disorder, or one or more of its symptoms, characterized in that said method comprises administering to a subject in need thereof one or more integrin α ν βδ antagonists and one or more integrins. or more anti-inflammatory agents are administered. More specifically, the present invention relates to a method for the prevention, treatment, management or alleviation of an autoimmune or inflammatory disorder, or one or more of its symptoms, characterized in that said method comprises administering to a subject in need thereof a VITAXIN ™ or antigen-binding fragment thereof. or a therapeutically effective amount of one or more anti-inflammatory agents is administered.

The present invention relates to a method for the prevention, treatment, management or alleviation of an autoimmune or inflammatory disorder or one or more symptoms thereof, characterized in that said method comprises administering one or more integrin α ν β3 antagonists to a subject in need of such treatment. or more TNFα antagonists and one or more anti-inflammatory agents. More specifically, the present invention relates to a method for the prevention, treatment, management or alleviation of an autoimmune or inflammatory disorder, or one or more of its symptoms, characterized in that said method comprises administering to a subject in need thereof a VITAXIN ™ or antigen-binding fragment thereof. a prophylactically or therapeutically effective amount of a prophylactically or therapeutically effective amount of one or more TNFα antagonists and a prophylactically or therapeutically effective amount of methotrexate or cyclosporin.

The present invention relates to a method for the prevention, treatment, management or alleviation of an autoimmune or inflammatory disorder or one or more symptoms thereof, characterized in that said method comprises administering one or more integrin α ν β3 antagonists to a subject in need of such treatment. or more TNFα antagonists and one or more CD2 binding molecules. More specifically, the present invention relates to a method for the prevention, treatment, management or alleviation of an autoimmune or inflammatory disorder, or one or more of its symptoms, characterized in that said method comprises administering to a subject in need thereof a VITAXIN ™ or antigen-binding fragment thereof. a prophylactically or therapeutically effective amount of a prophylactically or therapeutically effective amount of one or more TNFα antagonists and a prophylactically or therapeutically effective amount of MEDI-507 or an antigen-binding fragment thereof.

The present invention relates to a method for the prevention, treatment, management or alleviation of an autoimmune or inflammatory disorder or one or more symptoms thereof, characterized in that said method comprises administering one or more integrin α ν β3 antagonists to a subject in need of such treatment. or more TNFα antagonists and one or more anti-inflammatory agents. More specifically, the present invention relates to a method for the prevention, treatment, management or alleviation of an autoimmune or inflammatory disorder, or one or more of its symptoms, characterized in that said method comprises administering to a subject in need thereof a VITAXIN ™ or antigen-binding fragment thereof. a prophylactically or therapeutically effective amount of a prophylactically or therapeutically effective amount of one or more TNFα antagonists and a prophylactically or therapeutically effective amount of a steroid or non-steroidal anti-inflammatory drug.

The present invention relates to a method for the prevention, treatment, management or alleviation of an autoimmune or inflammatory disorder or one or more symptoms thereof, characterized in that said method comprises administering one or more integrin α ν β3 antagonists to a subject in need of such treatment. or more TNFα antagonists, one or more immunomodulatory agents and one or more anti-inflammatory agents. More specifically, the present invention relates to a method for the prevention, treatment, management or alleviation of an autoimmune or inflammatory disorder, or one or more of its symptoms, characterized in that said method comprises administering to a subject in need thereof a VITAXIN ™ or antigen-binding fragment thereof. a prophylactically or therapeutically effective amount of a prophylactically or therapeutically effective amount of one or more TNFα antagonists, a prophylactically or therapeutically effective amount of methotrexate, and a prophylactically or therapeutically effective amount of a steroid or non-steroidal anti-inflammatory drug.

The present invention relates to a method for the prevention, treatment, management or alleviation of an autoimmune or inflammatory disorder, or one or more of its symptoms, characterized in that said method comprises the prophylactic or preventive treatment of one or more integrin α ν β3 antagonists in a subject in need of such treatment. a therapeutically effective amount of a prophylactically or therapeutically effective amount of one or more TNFα antagonists, a prophylactically or therapeutically effective amount of one or more CD2 binding molecules, and a prophylactically or therapeutically effective amount of one or more anti-inflammatory agents. More specifically, the present invention relates to a method for the prevention, treatment, management or alleviation of an autoimmune or inflammatory disorder, or one or more of its symptoms, characterized in that said method comprises administering to a subject in need thereof a VITAXIN ™ or antigen-binding fragment thereof. a prophylactically or therapeutically effective amount of a prophylactically or therapeutically effective amount of one or more TNFα antagonists, a prophylactically or therapeutically effective amount of MEDI-507 or an antigen-binding fragment thereof, and a prophylactically or therapeutically effective amount of a steroid or non-steroidal anti-inflammatory drug.

• ·· ·

The present invention relates to pharmaceutical compositions comprising a pharmaceutically acceptable carrier, one or more integrin α ν β3 antagonists, and a prophylactic or therapeutic agent other than one or more integrin α ν β3 antagonists. Any prophylactic or therapeutic agent currently used or used or known to be useful for the prophylaxis, treatment of one or more symptoms associated with an autoimmune or inflammatory disorder, may be combined with one or more integrin α ν β3 antagonists to produce a pharmaceutical composition suitable for administration to a subject. The following non-limiting examples illustrate prophylactic and / or therapeutic agents that can be combined with one or more integrin α ν β3 antagonists to produce a pharmaceutical composition suitable for administration to a subject. The pharmaceutical compositions of the present invention may be used in the methods of the present invention for the prevention, treatment or alleviation of one or more symptoms associated with an autoimmune or inflammatory disorder. The pharmaceutical compositions of the present invention are preferably sterile and are formulated to be administered to a patient suffering from an autoimmune or inflammatory disorder by a suitable method.

The compositions and methods of the present invention described herein are useful for the prevention and / or treatment of autoimmune and / or inflammatory disorders. The Autoimmune Order 85 * ······· · · ··· · «· · · · · · · · · · · · · · · is one of the nations, without limiting ourselves, the spotted baldness, paralyzing vertebrae, antifospholipid syndrome, autoimmune Addison's disease, adrenal autoimmune diseases, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune oophoritis and orchitis, autoimmune thrombocytopenia, Behcet's disease, bullous pemphigoid, cardiomyopathy, abdominal sprue dermatitis, chronic fatigue immune dysfunction symptom (CFIDS), chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, sciatic pemphigism, CREST syndrome, cold agglutinin disease, Crohn's disease, pancreatic lupus, essential mixed cryoglobulinemia, fibromyalgia bromyositis, glomerulonephritis, Graves' disease, Guillan-Barre, Hashimoto thyroiditis, idiopathic lung fibrosis, idiopathic thrombocytopenia purpurea (ITP), IgA neuropathy, juvenile arthritis, lichen plans lupus erythematosus, Meni erectile disease, mixed connective tissue disease, multiple sclerosis, type 1 or immunological type diabetes, severe muscle weakness, pemphigic vulgar, severe anemia, lumpy polar arthritis, polycondritisism, polyglandular syndromes, polymialgia rheumatic, polymyositis and dermatomyositis, primary agammaglobulinemia, primary epecirrosis, primary epecirrosis, psoriasis, psoriatic arthritis, Raynauld phenomenon, Reiter's syndrome, rheumatoid arthritis, sarcoidosis, scleroderma, Sjögren's syndrome, stiffman syndrome, systemic lupus erythematosus, lupus erythematosus, takayasu arteritis, temporal arteritis / giant cell arteritis, ulcerative colitis, uveitis, vasculitis such as dermatitis herpetiformis • ··· ···

vasculitis, vitiligo, and Wegener granulomatosis. Inflammatory diseases include, but are not limited to, asthma, encephalitis, inflammatory bowel disease, chronic obstructive pulmonary disease (COPD), allergic disorders, septic shock, pulmonary fibrosis, undifferentiated spondyloarthropathy, undifferentiated arthropathy, arthritis, inflammatory osteolysis, and chronic inflammation. resulting from chronic viral or bacterial infection. The compositions and methods of the present invention can be used in combination with conventional therapy for the management or treatment of one or more of the above diseases.

The compositions and methods described herein are particularly useful for the prevention of rheumatoid arthritis, spondyloarthropathies (e.g., psoriatic arthritis, paralytic vertebrae, Reiter's syndrome (also known as reactive arthritis), arthritis associated with inflammatory bowel disease, and undifferentiated spondyloarthropathy), psoriasis, undifferentiated arthropathy and arthritis. or treat it. The compositions and methods described herein may also be used for the prevention, treatment, management or alleviation of one or more symptoms associated with inflammatory osteolysis, other disorders characterized by abnormal bone reabsorption, or bone loss (e.g., osteoporosis).

The present invention relates to industrial products comprising a packaging material and a pharmaceutical composition of the present invention in a form suitable for administration to a subject packaged in said packaging material. More specifically, the present invention relates to industrial products comprising a packaging material and a pharmaceutical composition of the present invention in a form suitable for administration to a subject packaged in said packaging material, said pharmaceutical composition comprising one or more integrin α ν β3. an antagonist, one or more prophylactic or therapeutic agents other than an integrin α ν β3 antagonist, and a pharmaceutically acceptable carrier. The industrial products of the present invention may include instructions for the administration of a pharmaceutical composition or other information material informing the physician, technician or patient how to properly treat the disease or disorder in question.

Any integrin α ν β3 antagonist well known to those skilled in the art can be used in the methods and compositions of the present invention. The present invention relates to the use of one or more integrin α ν β3 antagonists in the compositions and methods of the present invention. Integrin α ν ν β3 antagonists include, but are not limited to, protein-like agents such as non-catalytic metalloproteinase fragments, at RGD peptides, peptidomimetics, fusion proteins, desintegrins, or derivatives or fragments thereof, and those antibodies. which bind immunoprecipitatively to an integrin α ν β3 antagonist nucleic acid molecule, organic molecule, and inorganic molecule. Integrin is one of the RGD peptides recognized by α ν β3 without </ * «· · • · · · · · · · to limit ourselves to this, Triflavin. Immune-specific binding antibodies to integrin α ν β3 include, but are not limited to, 11D2 (Searle), LM609 (Scripps), and VITAXIN ™ (Medimmune, Inc.). Examples of small molecule peptidomimetics include, but are not limited to, S836 (Searle) and S448 (Searle). Desintegrins include, but are not limited to, Accutin. The present invention further relates to any of the integrin α ν β3 antagonists described in the following U.S. patent, in the compositions and methods of the present invention.

agents: 5,149,780; 5,196,511; 5,204,445; 5,262,520;
5,306,620; 5,478,725; 5,498,694; 5,523,209; 5,578,704;
5,598,570; 5,652,109; 5,652,110; 5,693,612; 5,705,481;
5,767,071; 5,770,565; 5,780,426; 5,817,457; 5,830,678;
5,849,692; 5,955,572; 5,985,278; 6,048,861; 6,090,944;
6,096,707; 6,130,231; 6,153,628; 6,160,099; and 6,171,588,

which publications will now be incorporated by reference in their entirety.

In some embodiments, an integrin α ν β3 antagonist is a small organic molecule. In other embodiments, the integrin α ν β3 antagonist is not a small organic molecule. In a preferred embodiment, an integrin α ν β3 antagonist is an antibody that binds immune-specific to integrin α ν β3-ύοζ. In another preferred embodiment, an integrin α ν β3 antagonist is VITAXIN ™, or a derivative, analogue, or antigen-binding fragment thereof.

In a preferred embodiment, the integrin α ν β3 antagonists inhibit or reduce angiogenesis.

In a preferred embodiment, the proteins, polypeptides or peptides (including antibodies and fusion proteins) that are used as integrin α ν β3 antagonists are derived from the same species as the proteins, polypeptides or peptides, thereby reducing the likelihood of the proteins. that an immune response is generated against said proteins, polypeptides or peptides. In another preferred embodiment, if the subject is a human, the integrin α ν β3 antagonist is a protein, polypeptide or peptide that is human or humanized.

According to the present invention, one or more integrin α ν β3 antagonists are administered to a subject suffering from an autoimmune or inflammatory disorder before, after, or simultaneously with the administration of one or more other prophylactic or therapeutic agents that are known to be well known or well known. can be used to prevent or treat said autoimmune or inflammatory disorder.

Integrin α ν β3 antagonist functional proteins, polypeptides or peptides, nucleic acid molecules encoding or integrin α ν β3 antagonist functional proteins, polypeptides or peptides may be administered to a subject an autoimmune or inflammatory disorder methods of the present invention. In addition, nucleic acid molecules encoding derivatives, analogs, fragments or variants of proteins, polypeptides or peptides that act as integrin α ν β3 antagonists, or derivatives, analogs, fragments or variants of proteins, polypeptides or peptides acting as integrin α ν β3 antagonists in an autoimmune or inflammatory disorder subject to the methods of the present invention. Such derivatives, analogs, variants and fragments preferably retain the full-length, wild-type protein, polypeptide or peptide integrin α ν β3 antagonist activity.

It is evident that integrin α ν β3-1ιοζ is an immuno-specific binding and antagonist antibody is known in the art. Examples of integrin α ν β3-1ιοζ immuno-specific binding antibodies include, but are not limited to, 11D2 (Searle), LM609 (Scripps), LM609 murine antibody (International Publication Number WO 89/015155, which is hereby incorporated by reference in its entirety). and the MEDI-522 humanized antibody [also known as VITAXIN ™; Medlmmune, Inc., Gaithersburg, MD; Wu et al., Proceedings of the National Academy of Sciences, USA 95 (11), 6037-6042 (1998); International Patent Publication No. WO 90/33919 and WO 00/78815; and U.S. Patent No. 5,753,230, which are hereby incorporated by reference in their entirety.

«· Integrin is an antibody specifically binding to v p3 without being restricted to it, monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, single chain Fvs (scFV-). k), single-chain antibodies, Fab fragments, (F (ab ') fragments, disulfide-linked Fvs (sdFvs), and anti-idiotypic (anti-Id) antibodies (including, for example, the present invention); Specifically, the antibodies of the present invention include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that bind immunoprecipitatively to integrin α ν β3. The immunoglobulins of the present invention may be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY) and of any type. (e.g. IgGi, IgG2, IgG3, IgG 4 , IgAi and IgA2) or subclasses. In a preferred embodiment, the integrin α ν β3-ύοζ immune-specific binding antibodies are antagonists of integrin α ν β3. In another preferred embodiment, the integrin α ν β3-ύοζ immune-specific binding antibodies inhibit or reduce angiogenesis.

Antibodies that bind immunoprecipitatively to integrin ανβ3-1ιοhatnak can be derived from any animal, including birds and mammals (such as humans, rodents, donkeys, sheep, rabbits, goats, golden hamsters, • · · · · · · · · · · · · · · · · · · · · · · · · ~ ~ ~ ~ ~). from a horse or chicken). Antibodies specifically binding to integrin ct v P3 are preferably human or humanized antibodies. As used herein, &quot; human &quot; antibodies are those whose amino acid sequence is an amino acid sequence of a human immunoglobulin, including antibodies isolated from human immunoglobulin libraries, or antibodies isolated from mice expressing antibodies against human genes.

The integrin α ν β3-ύοζ immune-specific binding antibodies may be monospecific, bispecific, trispecific, or multispecific antibodies. The multispecific antibody may be specific for different epitopes of an α ν β3 integrin, or may be specific for both an integrin α ν ββ epitope and a heterologous epitope, such as a heterologous polypeptide or solid support (see, e.g., WO 93/17715, WO 92 / PCT patent applications 08802, WO 90/00360 and WO 92/05793; Tutt et al., J. of Immunol. 147, 60-69 (1991); U.S. Patent Nos. 4,474,893, 4,714,681, 4,925,648, 5,573,920, and 5,601,819; Kostelny et al., J. of Immunol. 148: 1547-1553 (1993).

The present invention relates to antibodies having high affinity for integrin α ν β3-ύοζ. In a specific embodiment, an integrin α ν β3-ύοζ specific binding antibody constant or k on rate (antibody (Ab) + antigen (Ag) ^ -> Ab-Ag) is at least

10 5 / Molxsec; at least 5 * 10 5 / Molxsec, at least 10 6 / Molxsec; at least 5 x 10 6 / Mol x sec, at least 10 7 / Molxsec, at least 5 x 10 7 / Molxsec, or at least 10 8 / Molxsec. In a preferred embodiment, a 33 v integrin antibody binds immunospecifically to k on is at least 5xl0 5 / Molxsec, at least 10 6 / Molxsec; at least 5 x 10 6 / Molxsec, at least 10 7 / Mol x sec, at least 5 x 10 7 / Molxsec, or at least 10 8 / Molxsec.

In another embodiment, a v? 3 integrin antibody to immunospecifically bind k O ff rate (antibody (Ab) + antigen (Ag) h ° ff <-AB-Ag) of less than LCH / sec; less than 5x10 ^ sec; less than 10- 2 / sec; less than about 5xlO- 2 / sec; less than 10 3 / sec; less than 5x10- 3 / sec; less than 10 4 / sec; less than 5x10 ' 4 / sec; less than 10- 5 / sec; less than 5x10- 5 / sec; less than 10 6 / sec; less than 5 x 10 -6 / sec; less than 10 7 / sec; less than 5x10 7 / sec; less than 10 ' 8 / sec; less than 5 x 10 -8 / sec; less than 10 ' 9 / sec; less than 5x10 9 / sec; or less than 10 10 / sec.

In one embodiment, an integrin a v P3 immunospecifically binding antibody affinity constant or K a = (k on / k 0 ff) of at least 10 2 / mol; at least 5 x 10 2 / mol; at least 10 3 / Mole; at least 5x10 3 / mol; at least 10 4 / mol; at least

5x10 4 / mol; at least 10 5 / mol; at least 5x10 5 / mol; at least 10 6 / mol; at least 5x10 6 / mol; at least 10 7 / Mole; at least

5x10 7 / Mole; at least 10 8 / mol; at least 5x10 8 / mol; at least 10 9 / mol; at least 5x10 9 / mol; at least 10 10 Moles; at least

5x10 10 / mol; at least 10 n / mole; at least 5x10 n / mole; at least 10 12 Moles; at least 5x10 12 / Mole; at least 10 13 / mol; at least 5 x 10 13 / mol; at least 10 14 / Mole; at least 5x10 14 / Mole; at least 10 15 Moles; or at least 5x10 15 / Mole. In another embodiment, an antibody that binds immune-specific to a CD2 polypeptide has a dissociation constant or Kd (k 0 ff / kon) of less than 10 2 moles, less than 5x10 2 moles, less than 10 3 moles, less than 5 χ 10- 3 mol and less than 4 · HI mol, less than 4 · 5xlCl mol, less than l (l · 5 mol, less than 5XL (l · 5 mol, less than LCL · 6M, less than 5XL (l · 6 moles, less than l (l · 7 moles, less than 5xl (l · 7 moles, less than l (l · 8 moles, less than 5xl (l · 8 moles, less than Hl · 9 moles, less than 5xl) (l · 9 moles, less than Hl · 10 moles, less than 5xl (l · 10 moles, less than Hl · 11 moles, less than 5x10 n moles, less than l (l · 12 moles, less than 5x10 12 moles, less than l (l · 13 moles, less than 5χ10 13 moles, less than l (l · 14 moles, less than 5x10 14 moles, less than Hl · 15 moles or less than 5x10 15 moles).

In a specific embodiment, the antibody that is immunospecifically bound to integrin ? V ? Is the LM609 or antigen-binding fragment thereof (e.g., one or more complementarity determining regions (CDR) of LM619). The amino acid sequence of LM609 has already been disclosed (International Publication No. WO 89/05155, which is hereby incorporated by reference in its entirety, for all purposes, as reference), and communicated to the American Type Culture Collection (ATCC®, 1081 University Boulevard, Manassas, Virginia 20110). -2209) HB 9537

the amino acid sequence of the monoclonal antibody produced by the deposited cell line. In another embodiment, the integrin α ν β3-1ιοζ immune-specific binding antibody is not an antigen-binding fragment of LM609 or LM609.

In a preferred embodiment, the antibody that binds immune-specific to integrin α. ν β3-1ιοζ, VITAXIN ™, or antigen-binding fragment thereof (such as one or more complementarity determining regions (CDR) of VITAXIN ™). VITAXIN ™ has been described in WO 98/33919 and WO 00/78815, U.S. Patent Application Serial No. 09 / 339,822 and U.S. Patent No. 5,753,230, which are hereby incorporated by reference in their entirety. treated as a reference. In an alternative embodiment, the antibody that immunoprecipitates binding to the integrin α ν β3-1ιοζ, non-VITAXIN ™ or VITAXIN ™ antigen-binding fragment.

The present invention further relates to antibodies that immunoprecipitate specifically to integrin α ν β3-1ιοζ and said antibodies comprise a variable heavy ("VH") domain having the amino acid sequence of the LM609 or VITAXIN ™ VH domain. . The present invention further relates to antibodies that immunoprecipitate specifically to integrin α ν β3-1ιοζ and said antibodies have a VH complementarity determining region having the same amino acid sequence as t.

With the complementarity region of one of the VHs listed in Table 1.

Table 1
CDR sequences for LM609
CDR Sequence Chair. ID. Woman
VH1 SYDMS 1
VH2 KVSSGGG 2
VH3 HNYGSFAY 3
VL1 QASQSISNHLH 4
VL2 YRSQSIS 5
VL3 QQSGSWPHT 6

In one embodiment, the integrin a v 33 immune-specific binding antibodies comprise VH CDR1, the amino acid sequence of which is shown in SEQ ID NO: 1. In another embodiment, the integrin α v β 3 immune-specific binding antibodies comprise VH CDR2, the amino acid sequence of which is shown in SEQ ID NO: 2. In another embodiment, the integrin α v β 3 immune-specific binding antibodies comprise VH CDR3, the amino acid sequence of which is shown in SEQ ID NO: 3. In a preferred embodiment, the integrin is an antibody specifically binding to v 33 containing a VH CDR1, the amino acid sequence of which is shown in SEQ ID NO: 1, a VH CDR2 having the amino acid sequence shown in SEQ ID NO: 2 and a VH CDR3 having the amino acid sequence shown in SEQ ID NO: 3.

The present invention further relates to antibodies that bind immunoprecipitatively to integrin α ν β3-1ιοζ and said antibodies comprise a variable light ("VL") domain having the amino acid sequence identical to the amino acid sequence of the LML9 or VITAXIN ™ VL domain. The present invention further provides antibodies that immunoprecipitate specifically to integrin c3 and said antibodies comprise a VL complementarity determining region having the amino acid sequence of any of the VL CDRs listed in Table 1.

In one embodiment, the antibodies specifically binding to integrin ^ 3 contain VL CDR1, the amino acid sequence of which is shown in SEQ ID NO: 4. In another embodiment, the antibodies specifically binding to integrin ou 3 contain a VL CDR2, the amino acid sequence of which is shown in SEQ ID NO: 5. In another embodiment, the antibodies that bind immunoprecipitatively to integrin? 3 contain a VL CDR3 having the amino acid sequence shown in SEQ ID NO: 6. In a preferred embodiment, the antibodies specifically binding to integrin? 3 contain a VL CDR1 having an amino acid sequence as shown in SEQ ID NO: 4;

VL CDR2, whose amino acid sequence is shown in SEQ ID NO: 5, and a VL CDR3 having the amino acid sequence shown in SEQ ID NO: 6.

The present invention further relates to antibodies that immunoprecipitate specifically to integrin α ν β3-1ιοζ and said antibodies comprise a VH domain as described herein, in combination with a VL domain described herein, or other VL domain. The present invention further relates to antibodies that immunoprecipitate specifically to integrin α ν β3-1ιοζ and said antibodies comprise a VL domain as described herein, in combination with a VH domain described herein, or other VH domain.

The present invention further relates to antibodies that immunoprecipitate specifically to integrin (Xvfo, and said antibodies include one or more of the VH CDRs and VL CDRs listed in Table 1). which immune-specific binds to an integrin α ν β3-ύοζ, and said antibody comprises a VH CDR1 and a VL CDR1, a VH CDR1 and a VL CDR1, a VH CDR1, and a VL CDR1- , a VH CDR2 and a VL CDR1, a VH CDR2 and a VL CDR-2, a VH CDR2 and a VL CDR3, a VH CDR3 and a VH CDR1. et al., a VH CDR3 and a VL CDR2, a VH CDR3 and a VL CDR3, or any combination of the VH CDRs and VL CDRs listed in Table 1.

In one embodiment, the antibody that is immunospecifically bound to integrin α ν β3-ύοζ comprises a VH CDR1 having the amino acid sequence of SEQ ID NO: 1 and a VL CDR1 having the amino acid sequence as shown in SEQ ID NO: 4. SEQ ID NO. In another embodiment, the antibody that binds immune-specific to integrin α ν β3-ύοζ comprises a VH CDR1 having the amino acid sequence of SEQ ID NO: 1 and a VL CDR2 having the amino acid sequence of SEQ ID NO: 5. SEQ ID NO. In another embodiment, the antibody that is immunospecifically bound to integrin a v p3 comprises a VH CDR1 having the amino acid sequence of SEQ ID NO: 1 and a VL CDR3 having the amino acid sequence as shown in Figure 6. SEQ ID NO.

In one embodiment, the antibody that is immunospecifically bound to integrin c3 comprises a VH CDR2 having the same amino acid sequence as SEQ ID NO: 2 and a VL CDR1 having the amino acid sequence as shown in Figure 4. SEQ ID NO. In another embodiment, the antibody that is immunospecifically bound to integrin α ν β3-ύο tartalmaz comprises a VH CDR2 having the amino acid sequence of SEQ ID NO: 2 and a VL CDR2 having the amino acid sequence of SEQ ID NO: 5. No.

With 100 sequences. In another embodiment, the antibody that specifically binds to the integrin α ν β3-1ιοζ comprises a VH CDR2 having the amino acid sequence of SEQ ID NO: 2 and a VL CDR3 having the amino acid sequence of 6 SEQ ID NO.

In one embodiment, the antibody that binds immune-specific to integrin α ν β3-1ιοζ contains a VH CDR3 having the amino acid sequence of SEQ ID NO: 3, and a VL CDR1 having the amino acid sequence of SEQ ID NO: 4. SEQ ID NO. In another embodiment, the antibody that binds immune-specific to integrin α ν β3-1τοζ comprises a VH CDR3 having an amino acid sequence identical to SEQ ID NO: 3 and a VL CDR2 having the amino acid sequence of SEQ ID NO: 5. SEQ ID NO. In another embodiment, the antibody that binds immune-specific to integrin α ν β3-1ιοζ comprises a VH CDR3 having an amino acid sequence identical to SEQ ID NO: 3 and a VL CDR3 having the amino acid sequence as defined in SEQ ID NO: 6. SEQ ID NO.

The present invention further provides a nucleic acid molecule, generally an isolated nucleic acid molecule, which encodes an antibody that binds immune-specific to integrin α ν β3. In a specific embodiment, an isolated nucleic acid molecule encodes an antibody that binds immune-specific to integrin c 3, and said antibody is an amino acid.

· · · · · · · · · E «• This is the sequence of the amino acid sequence LM609 or VITAXIN ™.

In one embodiment, an isolated nucleic acid molecule encodes an antibody that binds immune-specific to integrin ? V ? And said antibody comprises a VH domain having the amino acid sequence identical to the amino acid sequence of LM609 or VITAXIN ?. In another embodiment, an isolated nucleic acid molecule encodes an antibody that binds immune-specific to integrin α ν β3-1ιοζ, and said antibody comprises a VH domain having an amino acid sequence at the American Type Culture Collection deposited by HB 9537. the amino acid sequence of the VH domain of the monoclonal antibody produced. In another embodiment, an isolated nucleic acid molecule encodes an antibody that specifically binds to integrin α ν β3-1ιοζ, and said antibody comprises a VH CDR1 having the amino acid sequence of the VH CDR1 amino acid sequence listed in Table 1. In another embodiment, an isolated nucleic acid molecule encodes an antibody that specifically binds to integrin? 3, and said antibody comprises a VH CDR2 having the amino acid sequence of the VH CDR2 amino acid sequence listed in Table 1. In another embodiment, an isolated nucleic acid molecule encodes an antibody that is immunospecificly bound to integrin? 3, and said antibody is tar102? *? having the amino acid sequence corresponding to the amino acid sequence of the VH CDR3 listed in Table 1.

In one embodiment, an isolated nucleic acid molecule encodes an antibody that binds immune-specific to integrin ? V ? And said antibody comprises a VL domain having the amino acid sequence identical to the amino acid sequence of the LML9 or VITAXIN? VL domain. In another embodiment, an isolated nucleic acid molecule encodes an antibody that binds immune-specific to integrin α ν β3-1ιοζ, and said antibody comprises a VL domain having the same amino acid sequence as the cell line deposited at HB 9537 at the American Type Culture Collection. by the amino acid sequence of the VL domain of the monoclonal antibody produced by it. In another embodiment, an isolated nucleic acid molecule encodes an antibody that binds immune-specific to integrin α ν β3 ~ 1ιοζ, and said antibody comprises a VL CDR1 having the amino acid sequence of the VL CDR1 amino acid sequence listed in Table 1. In another embodiment, an isolated nucleic acid molecule encodes an antibody that binds immune-specific to integrin α ν β3-1ιοζ, and said antibody comprises a VL CDR2 having the amino acid sequence of the VL CDR2 amino acid sequence listed in Table 1. In another embodiment, an isolated nucleic acid molecule encodes an antibody that is immunospecific?

binds integrin a to v p3, and said antibody contains a VL CDR3 having the amino acid sequence of the VL CDR3 amino acid sequence listed in Table 1.

In another embodiment, an isolated nucleic acid molecule encodes an antibody that binds immune-specific to the integrin, and said antibody comprises a VH domain having the amino acid sequence of LM609 or VITAXIN ™ and comprising a VL. domain having the amino acid sequence identical to the amino acid sequence of the VL domain of LM609 or VITAXIN ™. In another embodiment, an isolated nucleic acid molecule encodes an antibody that binds immune-specific to integrin a v p3, and said antibody comprises a VH CDR1, a VL CDR1, a VH CDR2, a VL CDR2t. , a VH CDR3, a VL CDR3, or any combination thereof, whose amino acid sequence is listed in Table 1.

The present invention further provides antibodies that immunoprecipitate specifically to integrin a v p3, and said antibodies include derivatives of VH domains, VH CDRs, VL domains, or VL CDRs, as described herein, which bind immunoprecipitatively to integrin for v p3. Standard techniques known to those skilled in the art may be used to introduce mutations into the nucleotide sequence encoding the antibodies of the present invention.

Including, for example, site-specific mutagenesis and polymerase chain reaction mutagenesis, resulting in amino acid substitutions. Preferably, the derivatives have less than 25 amino acid substitutions, less than 20 amino acid substitutions, less than 15 amino acid substitutions, less than 10 amino acid substitutions less than 5 amino acid substitutions, less than 4 amino acid substitutions, less than 3 amino acid substitutions, or less than 2 amino acid substitutions relative to the original molecule. In a preferred embodiment, the derivatives are subjected to conservative amino acid substitutions at one or more presumably non-essential amino acid residues (e.g., amino acids that are not critical to the antibody-specific binding of integrin α ν β3). "Conservative amino acid substitution" is an amino acid substitution when an amino acid residue is replaced by an amino acid residue having a similarly charged side chain. Families of amino acid residues having a similarly charged side chain have already been defined in the art. Such families include amino acids having a basic side chain (e.g., lysine, arginine, histidine), amino acid residues with an acid side chain (e.g., aspartic acid, glutamic acid), amino acids with an uncharged polar side chain (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with an apolar side chain (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tripto105) · · · · · · · · · · ·· · · · ·

tree), beta-branched-chain amino acids (e.g., threonine, valine, isoleucine), and amino acid residues with an aromatic side chain (e.g., tyrosine, phenylalanine, tryptophan, histidine). Alternatively, mutations may be introduced at random, in whole or in part of the coding sequence, such as saturation mutagenesis, and the resulting mutants may be assayed for their biological activity to identify mutants that have retained their activity. Following mutagenesis, the encoded antibody can be expressed and the activity of the antibody can be determined.

The present invention relates to antibodies that bind immunoprecipitatively to integrin α v β3, and said antibodies comprise the amino acid sequence of LM609 or VITAXIN ™, with one or more amino acid substitutions in the variable light (VL) domain and / or variable. heavy (VH) domain. The present invention further provides antibodies that immunoprecipitatively bind to integrin α ν β3, and said antibody comprises the amino acid sequence of LM609 or VITAXIN ™ by one or more amino acid substitutions in one or more VL CDRs and / or one or more amino acid residues. VH in CDR. The antibodies produced by replacing substitutions in the VH domain, VH CDRs, VL domain and / or VL CDRs of the LM609 or VITAXIN ™ can be assayed in vitro and in vivo, e.g. integrin α ν β3-1ιοζ (for example, by immunoassay including, without limitation, by ELISA).

106 · ··· ·· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · * - · IA IA IA IA IA IA IA ük ük ük ük ük ük ük ük ük ük ük ük ük ük ük ük ük ük ük ük ük ük ük ük ük ük ük ük ük ük ük ük ük ük ük ük ük ük ük ük *. associated with one or more symptoms.

In a specific embodiment, the antibody that binds immune-specific to the integrin α ν β3-ύο tartalmaz contains a nucleotide sequence that hybridizes to the American Type Culture Collection (ATCC®, 1081 University Boulevard, Manassas, Virginia 20110-2209) HB 9537 the nucleotide sequence encoding the amino acid sequence of the monoclonal antibody produced by the deposited cell line, i.e. hybridizes to the filter-bound DNA in a 6 * sodium chloride / sodium citrate (SSC) solution at about 45 ° C followed by one or more washes; 0.2 x SSC / 0.1% SDS at about 50-65 ° C under stringent conditions, i.e. hybridization to the filter sheet bound nucleic acid in 6 x SSC at about 45 ° C followed by one or more washings 0, 1 x SSC / 0.2% SDS in a solution at about 68 ° C or other types of stringent conditions known to those skilled in the art well known to mber [Current Protocols in Molecular Biology, 6.3. .6.3.6 and 2.10.3, edited by Ausubel et al., Greene Publishing and Wiley-Interscience: New York (1987)].

In a specific embodiment, the antibody that binds immune-specific to integrin c3 contains a nucleotide sequence that hybridizes under stringent conditions to the LM609 or VITAXIN ™ coding sequence, i.e., hybridization to the filter-bound nucleic acid 6 x SSC in

107 at about 45 ° C, followed by one or more washings in a 0.1xSSC / 0.2% SDS composition at about 68 ° C, or under other stringent conditions well known to those skilled in the art [Current Protocols] in Molecular Biology, 6.3.1-6.3.6 and 2.10.3, edited by Ausubel et al., Greene Publishing and Wiley-Interscience: New York (1987)].

In a specific embodiment, the antibody that binds immune-specific to the integrin ? V? 3 has an amino acid sequence of a VH domain or an amino acid sequence of a VL domain that hybridizes under stringent conditions to the DNA bound to the filter sheet by 6? Sodium chloride / sodium citrate (SSC) solution at about 45 ° C followed by one or more washes in 0.2xSSC / 0.1% SDS at about 50-65 ° C under highly stringent conditions, i.e., hybridization to the filter sheet bound nucleic acid at 6xSSC at about 45 ° C, followed by one or more washings in a 0.1xSSC / 0.2% SDS composition at about 68 ° C, or under other stringent conditions well known to those skilled in the art. are known [Current Protocols in Molecular Biology, 6.3.1-6.3.6 and 2.10.3, ed. Ausubel et al., Greene Publishing and Wiley-Interscience: New York (1987)] .

In another embodiment, the antibody that is immunospecifically bound to integrin v 33, which has an amino acid sequence of a VH CDR, or an amino acid sequence of a VL CDR encoded by a nucleotide sequence that hybridizes under stringent conditions nucleic acid sequence encoding any of the VH CDRs or VL CDRs listed in Table 1, i.e., hybridizing to the filter-bound DNA in a 6 * sodium chloride / sodium citrate (SSC) solution at about 45 ° C, this is followed by one or more washes in a solution of 0.2 x SSC / 0.1% SDS at about 50-65 ° C under highly stringent conditions, i.e. hybridization to the filter sheet bound nucleic acid in 6 * SSC at about 45 ° C, is followed by one or more washings in a solution of 0.1 x SSC / 0.2% SDS at about 68 ° C or under other stringent conditions known to those skilled in the art. [Current Protocols in Molecular Biology, 6.3. .6.3.6 and 2.10.3, edited by Ausubel et al., Greene Publishing and Wiley-Interscience: New York (1987)].

In another embodiment, the antibody that is immunospecifically bound to integrin a v p3, which has an amino acid sequence of a VH CDR, or an amino acid sequence of a VL CDR encoded by a nucleotide sequence that hybridizes under stringent conditions to the nucleic acid sequence is 9537 number monoclonal antibody produced by the deposited cell lines encodes HB any of the VH CDRs or VL CDRs Collection at the American Type Culture, that hybridizes to DNA were added with the filter plate 6 χ sodium chloride / sodium citrate (SSC) in solution at about 45 ° C followed by one or more washes of 0.2 x SSC / 0, 1% SDS

109 in a solution at about 50-65 ° C under highly stringent conditions, i.e., hybridization to the filter sheet-bound nucleic acid at 6 ° SSC at about 45 ° C followed by one or more washings of 0.1xSSC / 0.2% SDS at about 68 ° C or under other stringent conditions well known to those skilled in the art [Current Protocols in Molecular Biology, 6.3.1-6.3.6 and 2.10.3, edited by Ausubel et al. Greene Publishing and Wiley-Interscience: New York (1987)].

In another embodiment, the antibody that is immunospecifically bound to integrin a v p3, which has an amino acid sequence of a VH CDR, or an amino acid sequence of a VL CDR encoded by a nucleotide sequence that hybridizes under stringent conditions to the nucleic acid that encodes any of the VH CDRs or VL CDRs listed in Table 1, i.e., hybridizes to the filter-bound DNA in a 6 * sodium chloride / sodium citrate (SSC) solution at about 45 ° C. followed by one or more washes in a solution of 0.2 * SSC / 0.1% SDS at about 50-65 ° C under highly stringent conditions, i.e. hybridization to the filter sheet bound nucleic acid in 6 * SSC at about 45 ° C, followed by one or more washings in a solution of 0.1 to <SSC / 0.2% SDS at about 68 ° C, or under other stringent conditions as will be apparent to one skilled in the art to well known [Current Protocols in Molecular Biology, 06.03.1110

6.3.6 and 2.10.3, edited by Ausubel et al., Greene Publishing and Wiley-Interscience: New York (1987)].

In another embodiment, the antibody that is immunospecifically bound to integrin α ν β3-1ιοζ, which has an amino acid sequence of a VH CDR, or an amino acid sequence of a VL CDR encoded by a nucleotide sequence that hybridizes under stringent conditions to the nucleic acid to encode a CDR or VL CDR of any of the VH CDRs of a monoclonal antibody produced by the cell line deposited by the American Type Culture Collection at HB 9537, i.e., hybridizes to the filter-bound DNA with 6 * sodium chloride / sodium citrate (SSC) at about 45 ° C, followed by one or more washings in 0.2 x SSC / 0.1% SDS at about 50-65 ° C under highly stringent conditions, i.e. hybridization to the nucleic acid bound to the filter plate 6 x At about 45 ° C in SSC followed by one or more washes in a solution of 0.1 x SSC / 0.2% SDS sits at 68 ° C or under other stringent conditions well known to those skilled in the art [Current Protocols in Molecular Biology, 6.3.1-6.3.6 and 2.10.3, edited by Ausubel et al., Greene Publishing and Wiley-Interscience: New York (1987)].

In a specific embodiment, the antibody that is immunospecifically bound to integrin a v p3 contains an amino acid sequence that is at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, air111 • ··· below 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% , at least 95% or at least 99% identical to the sequence of the monoclonal antibody produced by the cell line deposited at HB 9537 at the American Type Culture Collection. In another embodiment, the antibody that is immunospecifically bound to integrin ? V p3 comprises an amino acid sequence that is at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical to the VITAXIN ™ amino acid sequence.

In another embodiment, the antibody that is immunospecifically bound to integrin a v p3 comprises an amino acid sequence of a VH domain that is at least 35%, at least 40%, at least 45%, at least 50% at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95 % or at least 99% identical to the amino acid sequence of the VITAXIN ™ VH domain. In another embodiment, the antibody that binds immune-specific to integrin v 33 comprises an amino acid sequence of a VH domain that is at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, air121 • · · · * · · ··· · · · · · ····· w · · · at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the sequence of the VH domain of the monoclonal antibody used by the American Type Culture Collection produces a cell line deposited under HB 9537.

In another embodiment, the antibody that immunoprecipitatively binds to one or more VR CDR amino acid sequences of integrin α ν β3-1ιοζ, which is at least 35%, at least 40%, at least 45%, at least 50% at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical to any of the VH CDR amino acid sequences listed in Table 1. In another embodiment, the antibody that immunoprecipitatively binds to one or more VR CDR amino acid sequences of integrin α ν β3-1ιοζ, which is at least 35%, at least 40%, at least 45%, at least 50% at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical to the VH CDR sequence of the monoclonal antibody produced by the cell line deposited at HB 9537 at the American Type Culture Collection.

In another embodiment, the antibody that is immunospecifically bound to the integrin cu contains an amino acid sequence of a VL domain that is at least 35%, at least 41%, below 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% at least 95% or at least 99% identical to the amino acid sequence of the VITAXIN ™ VL domain. In another embodiment, the antibody that is immunospecifically bound to integrin a v p3 comprises an amino acid sequence of a VH domain that is at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95 % or at least 99% identical to the VL domain sequence of the monoclonal antibody produced by the cell line deposited at HB 9537 at the American Type Culture Collection.

In another embodiment, the antibody that binds immunoprecipitatively comprises the amino acid sequence of one or more VL CDRs of integrin α ν β3-3, which is at least 35%, at least 40%, at least 45%, at least 50% at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical to the amino acid sequences of any of the VL CDRs listed in Table 1. In another embodiment, the antibody that binds immune-specific comprises the amino acid sequences of one or more VL CDRs of integrin α ν β3β, which is \ t

1) 4 at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70% , at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the VL CDR sequence of the monoclonal antibody as described in the American Type Culture Collection The cell line deposited under HB 9537 is produced.

The present invention relates to antibodies that compete with an antibody described herein for binding to integrin v p3. In a specific embodiment, the present invention relates to antibodies that compete with LM609 or an antigen-binding fragment thereof for binding to integrin v p3. In a preferred embodiment, the present invention relates to antibodies that compete with VITAXIN ™ or an antigen-binding fragment thereof for binding to integrin v p3.

The present invention relates to VH CDRs that are

The Vin CDR of the monoclonal antibody produced by the VH CDR described in Table 1 or the cell line produced by the American Type Culture Collection HB 9537 is competed for integrin v (binding to b. The present invention relates to a VL CDR). which compete with the VL CDR of the monoclonal antibody produced by the VL CDR described in Table 1 or by the cell line deposited by the American Type Culture Collection HB 9537, for binding to integrin v p3.

115

Antibodies specifically binding to integrin ct v p3 include derivatives that are modified, for example, by covalently attaching any type of molecule to the antibody. For example, without limiting it, antibody derivatives include antibodies that are modified, such as glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, a cellular ligand, or other protein. switching, etc. Any of a number of different chemical modifications may be performed by known techniques, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, and the like. In addition, the derivatives may also contain one or more non-classic amino acids.

The present invention also relates to antibodies that immunoprecipitate specifically to integrin α v β3, and said antibodies comprise a framework known to those skilled in the art. The framework of the antibody of the present invention is preferably human. In a specific embodiment, integrin is an antibody specifically binding to v p3 comprising the VITAXIN ™ framework region.

The present invention also relates to antibodies that bind immunoprecipitatively to integrin α ν β3-1ιοζ and said antibodies comprise the amino acid sequence of VITAXIN ™ with one or more mutations (i.e., one or more amino acids).

116 substitution in the framework regions). In some embodiments, the integrin is an antibody specifically binding to v p3 comprising the amino acid sequence of VITAXIN ™ with one or more mutations in the framework regions of the VH and / or VL domains.

The present invention also provides antibodies that bind immunoprecipitatively to integrin α ν β3-1ιοζ, and said antibodies comprise the amino acid sequence of VITAXIN ™, with one or more mutations (i.e., one or more amino acid substitutions) in the variable and framework regions.

The present invention further provides fusion proteins comprising an antibody that is immunospecificly bound to integrin? 3 and a heterologous polypeptide. The heterologous polypeptide to which the antibody is fused is preferably useful for directing the antibody to platelets, monocytes, endothelial cells and / or B cells.

The present invention relates to antibodies that bind immunoprecipitatively to integrin α ν β3-1ιοζ and have a longer in vivo half-life. More specifically, the present invention relates to antibodies that bind immunoprecipitatively to integrin α ν β3-1ιοζ and have a half-life in an animal, preferably a mammal, and most preferably a human being greater than 3 days, greater than 7 days, greater than 10 days, preferably greater than 15 days, greater than 25 days, greater than 30 days, greater than 35 days, greater than 40 days, greater than 45 days

117% $ day, greater than 2 months, greater than 3 months, greater than 4 months, or greater than 5 months.

In order to extend antibodies (e.g., monoclonal antibodies, single chain antibodies, and Fab fragments) circulating in the serum, for example, inert polymer molecules such as high molecular weight polyethylene glycol (PEG), can be coupled to the antibodies, with or without a multifunctional linker, or by site-specific conjugation of PEG via the N-terminus or C-terminus of the antibody, or through the epilone amino groups on the lysine moieties. Linear or branched polymer derivatization is used which results in minimal loss of biological activity. The extent of conjugation is closely monitored by SDS-PAGE and mass spectroscopy to ensure proper conjugation of PEG molecules to antibodies. The unreacted PEG can be separated from the antibody PEG conjugates by size exclusion or ion exchange chromatography. The PEG derivatives of the antibodies can be assayed for binding activity and in vivo efficacy by methods known to those skilled in the art, such as the immunoassay described below.

The present invention relates to antibodies or antigen-binding fragments thereof that bind immunoprecipitatively to integrin α v β3 and are fused or recombinantly chemically conjugated (including both covalent and non-covalent conjugation) by a hetero118. hanging polypeptide (or a fragment thereof, preferably at least 5, at least 10, at least 15, at least 20, at least 25, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100 contiguous amino acid residues) for fusion protein production. The fusion need not necessarily be direct, but may occur through linker sequences. For example, antibodies can be used to target heterologous polypeptides to selected cell types (e.g., platelets, endothelial cells, B cells or monocytes), in vitro or in vivo, fusing or conjugating antibodies to antibodies to the particular cell surface receptors such as CD11c. specific, CD14, CD17, CD19, CD25, CD36, CD41, CD42, CD51, CD61 and CD78.

The present invention further provides antibodies or antigen-binding fragments thereof that immunoprecipitate specifically for marker sequences, such as integrin α ν β3-1ιοζ fused to a peptide for ease of purification. In a preferred embodiment, the marker amino acid sequence is a hexa-histidine peptide, such as the one provided by the pQE vector (Quiagen, Inc., 9259 Eton Avenue, Chatsworth, CA, 91311), and many of these are commercially available from Gentz et al. for convenient purification of the fusion protein, hexa-histidine can be used (Gentz et al., Proceedings of the National Academy of Sciences, USA 86, 821119).

824 (1989)]. Other peptide labels used for purification include, without limitation to one thousand, hemagglutinin &quot; HA &quot; which corresponds to one epitope derived from influenza hemagglutinin protein (Wilson et al., Cell, 37, 767 (1984)).

The present invention further provides antibodies or antigen-binding fragments thereof that bind immunoprecipitatively to integrin α ν β3-1ιοζ conjugated to an agent having a potential therapeutic advantage. An antibody or antigen-binding fragment thereof that specifically binds to integrin α ν β3 may be conjugated to a therapeutic unit, such as a cytotoxin, such as a cytostatic or cytocidal agent, an agent having potential advantages, or a radioactive metal ion, e.g. alpha issuer. The cytotoxin or cytotoxic agent may be any agent that is harmful to cells. Cytotoxin or cytotoxic agents may include, but are not limited to, paclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetin, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxythracinone, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol and puromycin, and analogues and homologs thereof. Agents with potential therapeutic benefits include, but are not limited to, antimetabolites (such as methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5120 fluorouracil decarbazine), alkylating agents (e.g. mechloretamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulphan, dibromannite, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DPP, cisplatin), anthracyclines (e.g. daunorubicin) (formerly daunomycin) and doxorubicin), antibodies (such as dactinomycin (formerly actinomycin), bleomycin, mithramycin and anthramycin (AMC)), and anti-mitotic agents (such as vincristine and vinblastine).

In addition, an antibody or antigen-binding fragment thereof that immunospecifically binds to integrin α v p3 may be conjugated to a therapeutic agent or drug substance that modifies a particular biological response. Agents with a potential therapeutic advantage, or pharmaceutical agents, should not be limited to classical chemical therapy agents. The drug agent may be, for example, a protein or polypeptide having the desired biological activity. Examples of such proteins include a toxin such as abrin, ricin A, Pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor, interferon-α ("IFN-α"), interferon-β ("IFN-β"), nerve growth factor ("NGF"), platelet-derived growth factor ("PDGF") , tissue plasminogen activator ("TPA"), an apoptotic agent such as TNFα, Fβ, AIM I (WO 97/33899), AIM II (International Patent Publication No. 97/34911),

121 • ··· ·· · · · · · ··· · · · ···

Woody Ligand [Takahashi et al., J. of Immunol. 6, 1567-1574 (1994)] and VEGF (see, for example, WO 99/23105), a thrombotic agent or an anti-angiogenesis agent such as angiostatin or endostatin; or a biological response modifier, such as a lymphokine (e.g., interleukin-1 ("IL-1"), interleukin-2, interleukin-6, interleukin-10, granulocyte macrophage colony stimulating factor ("GMCSF"), and granulocyte colony stimulating growth factor ("G-CSF") or a growth factor (such as growth hormone ("GH")).

Techniques used to conjugate such therapeutic units to antibodies are well known in the art (Armon et al., "Monoclonal Antibodies Forum for Immunodeficiency Drugs In Cancer Therapy", In: Monoclonal Antibodies and Cancer Therapy, ed. Reisfeld et al., 243-256. , Alan R. Liss, Inc., (1985); Hellstrom et al., "Antibodies Forum Drug Delivery", In: Controlled Drug Delivery, 2nd Edition, by Robinson et al., 623-653. page, Marcel Dekker, Inc. (1987); Thorpe: "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review", In: Monoclonal Antibodies '84: Biological and Clinical Applications, Pinchera et al., 1985, 475-506; "Analysis, Results, and Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Canver Therapy", In: Monoclonal Antibodies Forum Cancer Detection And Therapy, eds. Baldwin et al., 303-316. page, Academic Press

122 • · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · (1983) Immunological Reviews 62: 119-158.

An antibody or antigen-binding fragment thereof that binds immune-specific to integrin α ν β3-1ιοι can be conjugated to a second antibody to form an antibody heteroconjugate (U.S. Patent No. 4,676,980, which is hereby incorporated by reference in its entirety. , we treat all purposes as reference).

The antibodies, or antigen-binding fragments thereof, that bind immunoprecipitatively to the integrin α ν β3-1ιοζ can be bound to a solid support that is particularly useful for purifying cells such as platelets and endothelial cells. Such solid carriers include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene.

The present invention relates to compositions comprising one or more integrin α ν β3 antagonists and one or more prophylactic or therapeutic agents other than integrin α ν β3 antagonists, and to one or more autoimmune subjects of the present invention. or methods for preventing, treating or ameliorating a symptom associated with an inflammatory disorder, comprising administering one or more of said compositions to said subject. Prophylactic or therapeutic agents include, but are not limited to, peptides, polypeptides, fusion proteins, nucleic acid molecules, small molecules123 ·· ·· «·· · ··

Drugs, mimetic agents, synthetic drugs, inorganic molecules and organic molecules. Any agent known to be useful or currently used for the prevention, treatment or alleviation of one or more symptoms associated with an autoimmune or inflammatory disorder may be used in combination with an integrin α ν β3 antagonist as described herein. Examples of such agents include, but are not limited to, dermatological agents used to treat rashes and swelling (e.g., phototherapy (e.g., ultraviolet B irradiation), photochemotherapy (e.g., PUVA) and topical agents such as softening agents. agents, salicylic acid, coal tar, topical steroids, topical corticosteroids, topical vitamin D3 analogues (such as calcipotriene), tazarotene and topical retinoids), anti-inflammatory agents (e.g., corticosteroids (e.g., prednisone and hydrocortisone), glucocorticoids, steroids, non-steroidal anti-inflammatory drugs drugs (such as aspirin, ibuprofen, diclofenac, and COX2 inhibitors), beta-antagonists, anticholinergic agents, and methyl xanthines), immunomodulatory agents (e.g., small organic molecules, T-cell receptor modulators, cytokine receptor modulators, T-cell depleting agents) genes, cytokine antagonists, monokine antagonists, lymphocyte inhibitors, or anticancer agents), gold injections, sulphasalazine, penicillamine, anti-angiogenic agents (e.g., angiostatin, TNFα antagonists (such as anti-TNFα antibodies) and endostatin), dapsone,

124 psoralenes (such as methoxalen and trioxsalen), anti-malarial agents (e.g., hydroxycloroquine), antiviral agents and antibiotics (such as erythromycin and penicillin).

Any immunomodulatory agent well known to those skilled in the art may be used in the methods and compositions of the present invention. Immunomodulatory agents can affect one or more aspects of the immune response in a subject or all of them. Aspects of the immune response include, but are not limited to, inflammatory response, complement cascade, leukocyte and lymphocyte differentiation, proliferation and / or effector function, monocyte and / or basophil numbers, and cellular communication to immune cells between. In certain embodiments of the present invention, an immunomodulatory agent affects one aspect of the immune response. In other embodiments, an immunomodulatory agent affects one or more aspects of the immune response. In a preferred embodiment of the present invention, the administration of an immunomodulatory agent to a subject inhibits or reduces one or more aspects of the subject's immune response. In a specific embodiment of the present invention, the immunomodulatory agent inhibits or suppresses the immune response in a subject. According to the present invention, the immunomodulatory agent is not an integrin α ν β3 antagonist. In some embodiments, an immunomodulatory agent is not an anti-inflammatory agent. In other embodiments, the immunomodulatory agent is not a CD2 antagonist. Other

In embodiments, the immunomodulatory agent is not a CD2 binding molecule. In other embodiments, the immunomodulatory agent is not MEDI-507.

An immunomodulatory agent may be selected to interfere with the interaction between T helper subgroups (TH1 and TH2) and B cells to inhibit the formation of neutralizing antibody. An immunomodulatory agent can be selected to inhibit the interaction between TH1 cells and CTL cells, thereby causing the occurrence of CTL-induced death. An immunomodulatory agent may be selected to alter (e.g., inhibit or suppress) the proliferation, differentiation, activity and / or function of CD4 + and / or CD8 + T cells. For example, T-cell specific antibodies may be used as immunomodulatory agents to attenuate or alter the proliferation, differentiation, activity and / or function of CD4 + and / or CD8 + T cells.

Immunomodulatory agents include, but are not limited to, proteinaceous agents such as cytokines, peptidomimetics, and antibodies (e.g., human, humanized, chimeric, monoclonal, polyclonal, Fvs, ScFvs, Fab or F (ab ) 2 fragments or epitope-binding fragments), nucleic acid molecules (such as antisense nucleic acid molecules and triple helix), small molecules, organic compounds, and inorganic compounds. More specifically, immunomodulatory agents include, but are not limited to, methotrexate, leflunomide, cyclophosphamide;

Cytoxan, Immura, cyclosporin A, minocycline, azathiopyrin, antibiotics (such as FK506 (tacrolimus)), methylprednisolone (MP), corticosteroids, steroids, mycophenolate mofetil , rapamycin (sirolimus), mizoribine, deoxypergualin, brequinar, malononitrile amides (such as leflunamide), T cell receptor modulators, and cytokine receptor modulators. T cell receptor modulators and cytokine receptor modulators. For clarification of the role of T cell receptor modulators and cytokine receptors, see above. T-cell receptor modulators include, but are not limited to, anti-T cell receptor antibodies (e.g., antiCD4 antibodies (e.g., cM-T412 (Boehringer), IDEC-CE9.1 (IDEC and SKB), mAB 4162W94, Orthoclone and OKTcdr4a (Janssen-Cilag)), anti-CD3 antibodies, anti-CD5 antibodies (e.g., anti-CD5 ricin-linked immunoconjugate), anto-CD7 antibodies (e.g. 380 (Novartis)), anti-CD8 antibodies and anti-CD40 ligand monoclonal antibodies, anti-CD52 antibodies (such as CAMPATH 1H (Ilex)), and anti-CD2 monoclonal antibodies) and CTLA4 immunoglobulin. In a specific embodiment, a T cell receptor modulator is a CD2 antagonist. In other embodiments, a T cell receptor modulator is not a CD2 antagonist. In another specific embodiment, a T cell receptor modulator is not a CD2 binding molecule.

127

Examples of cytokine receptor modulators include, but are not limited to, soluble cytokine receptors (such as the extracellular domain of a TNFα receptor or fragment thereof, an extracellular domain of an interleukin-β receptor, or a fragment thereof, and an interleukin-6 receptor or a fragment thereof). cytokines or fragments thereof (such as interleukin-2, interleukin-3, interleukin-4, interleukin-5, interleukin-6, interleukin-7, interleukin-8, interleukin-9, interleukin-10, interleukin-9, interleukin-10) 11, interleukin-12, interleukin-15, TNFα, TNFβ, interferon-a, interferon-β, interferon-γ and granulocyte macrophage colony stimulating factor), anti-cytokine receptor antibodies (e.g., anti-interleukin2 receptor antibodies, anti-interleukin) -4 receptor antibodies, anti-interleukin-6 receptor antibodies, anti-interleukin-10 receptor antibodies, and anti-interleukin-12 receptor antibodies), anti-cit okin antibodies (e.g., anti-interferon receptor antibodies, anti-TNFα antibodies, anti-interleukin ββ antibodies, anti-interleukin-6 antibodies and antiazine interleukin-12 antibodies). In a specific embodiment, the cytokine receptor modulator is interleukin-4, interleukin-10, or a fragment thereof. In another embodiment, a cytokine receptor modulator is an anti-interleukin ΐβ antibody, an anti-interleukin-6 antibody, an anti-interleukin-12 receptor antibody, an anti-TNFα antibody. In another embodiment, a cytokine receptor modulator is an extracellular domain of a TNFα receptor or fragment thereof. Certain

According to embodiments, the cytokine receptor modulator is not a TNFα antagonist.

In a preferred embodiment, the proteins, polypeptides or peptides (including antibodies) used as the immunomodulatory agent are derived from the same species as the recipient of the proteins, polypeptides or peptides to reduce the likelihood of an immune response to said proteins, polypeptides or peptides. . In another preferred embodiment, when the subject is a human, the proteins, polypeptides or peptides used as the immunomodulatory agent are of human origin or humanized.

According to the present invention, one or more immunomodulatory agents are administered to a subject suffering from an autoimmune or inflammatory disorder prior to, during or after the prophylactic or therapeutic agent of the present invention. Preferably, one or more immunomodulatory agents are administered to a subject suffering from an autoimmune or inflammatory disorder to reduce or inhibit, as necessary, one or more aspects of the immune response. Any technique well known to those skilled in the art may be used to measure one or more aspects of an immune response in a particular subject, and thereby determine when an immunomodulatory agent should be administered to said subject. In a preferred embodiment, a subject has 500 cells / mm 3 , preferably 600 cells / mm 3 , more preferably 700 cells / mm 3 .

129 cells / mm 3 , and most preferably 800 cells / mm 3, maintain an absolute lymphocyte count. In another preferred embodiment, an immunomodulatory agent is not administered to an autoimmune or inflammatory subject if the absolute lymphocyte count is less than 500 cells / mm 3 , less than 550 cells / mm 3 , less than 600 cells / mm 3 , less than 650 cell / mm 3 , less than 700 cells / mm 3 , less than 750 cells / mm 3 , less than 800 cells / mm 3 .

In a preferred embodiment, one or more immunomodulatory agents are administered to a patient suffering from an autoimmune or inflammatory disorder to transiently reduce or inhibit one or more aspects of the immune response. Such transient inhibition or reduction of one or more aspects of the immune response may last for hours, days, weeks or months. Preferably, the transient inhibition or reduction of one or more aspects of the immune response lasts for a few hours (2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 14 hours, 16 hours, 18 hours, 24 hours) , For 36 hours or 48 hours), lasts for a few days (e.g., 3 days, 4 days, 5 days, 6 days, 7 days, or 14 days), or a few weeks (e.g., 3 weeks, 4 weeks, 5 weeks or 6 weeks). Transient reduction or inhibition of one or more aspects of the immune response enhances the prophylactic or therapeutic capabilities of an integrin α ν β3 antagonist.

130 · · · · · · · · ··· ·· · * · · · · · · · ·· · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·

In one embodiment of the present invention, an immunomodulatory agent that reduces or attenuates T cells, preferably memory T cells, is administered to a subject suffering from an autoimmune or inflammatory disorder by the methods of the present invention (see, e.g., U.S. Pat. No. 4,658,019). United States Patent Specification). In another embodiment of the present invention, an immunomodulatory agent that inactivates CD8 + eket cells is administered to a subject suffering from an autoimmune or inflammatory disorder according to the method of the present invention. In a specific embodiment, anti-CD8 antibodies are used to attenuate CD8 + cells.

Antibodies that interfere with or block the interactions required for activation with B cells (T helper cells), thereby blocking neutralizing antibodies, are useful as immunomodulatory agents in the methods of the present invention. To activate B cells with T cells, certain interactions have to be made (Durie et al., Immunology Today. 15 (9), 406-410), such as CD40 ligand on T cells on CD40 antigen on B cells. should bind, and CD28 and / or CTLA4 ligands on T cells should bind to B7 antigen on B cells. Without both interactions, B cells cannot be activated to induce the production of neutralizing antibodies.

131 • · • ···

The CD40 ligand (CD40L) -CD40 interaction is a desirable point in blocking the immune response because of its wide activity in both T cell activation and function and, in the absence of redundancy, in the signaling biosynthesis pathway. Thus, in a specific embodiment of the present invention, the interaction of CD40L with CD40 is transiently blocked at the time of administration of one or more immunomodulatory agents. This is achieved by treating with a substance that blocks the CD40 ligand on the TH cell and interferes with the normal binding of CD40 ligand on T cells to CD40 antigen on B cells. An antibody to CD40 ligand (anti-CD40L) (available from Bristol-Myers Squibb, Co.; see, for example, European Patent Publication No. 555,880, published August 18, 1993), or a soluble CD40 molecule may be selected and used as an immunomodulatory agent. by the methods of the present invention.

In another embodiment, an immunomodulatory agent that reduces or inhibits one or more biological activities (e.g., differentiation, proliferation, and / or effector function) of the TH4, TH1 and / or TH2 subgroups of CD4 + T helper cells in an autoimmune or inflammatory disorder is administered to a patient suffering from the method of the present invention. An example of such an immunomodulatory agent is interleukin-4. Interleukin-4 enhances the antigen-specific activity of TH2 cells at the expense of TH1 cell function (see, for example, Yokota et al., Proceedings of the National Academy of

132 · «« »« ··· ·· * · · α · * · · · · · · · · · · · · · · · · ··· <· · ···

Sciences, USA 83, 5894-5898 (1986); and U.S. Patent No. 5,017,691]. Other examples of immunomodulatory agents that affect the biological activity (e.g., proliferation, differentiation, and / or effector functions) of auxiliary cells (more specifically, TH1 and / or TH2 cells), without being limited thereto, are interleukin-6, interleukin-10, \ t interleukin-12 and IFNγ.

In one embodiment, an immunomodulatory agent administered to a patient suffering from an autoimmune or inflammatory disorder by the methods of the present invention is a cytokine that prevents antigen presentation. In a preferred embodiment, an immunomodulatory agent used in the method of the present invention is interleukin-10. In addition, interleukin-10 reduces or inhibits macrophage action, which includes bacterial elimination.

Other examples of immunomodulatory agents useful in the present invention include, but are not limited to, corticosteroids, azathiopyrine, mycophenolate mofetil, cyclosporin A, hydrocortisone, FK506, methotrexate, leflunomide, and cyclophosphamide. The short-term treatment of cyclophosphamide has been shown to successfully interrupt both CD4 + and CD8 + T cell activation for adenoviral capsid protein (Jooss et al., Human Gene Therapy 7, 15551566 (1996)), and at higher doses for neutralizing antibody formation was also hindered. Hydrocortisone or Ciclosporin \ tTreatment has also been successfully used to reduce

133 • •• fc «·· · · · ♦ <·« · * · · · · · · · · · · · · · · · · · · · · · · · · · · · Induction of cytokines, some of which may play a role in the elimination of bacterial infection.

Nucleic acid molecules encoding proteins, polypeptides or peptides having immunomodulatory activity, or proteins, polypeptides or peptides having immunomodulatory activity may be administered to a patient suffering from an autoimmune or inflammatory disorder using the methods of the present invention. In addition, nucleic acid molecules encoding the analogs, fragments or variants of proteins, polypeptides, or variants of proteins having immunomodulatory activity, or derivatives, analogs, fragments or variants of proteins, polypeptides or peptides having immunomodulatory activity may be administered to a patient suffering from an autoimmune or inflammatory disorder. using the methods of the present invention. Such derivatives, analogs, variants and fragments preferably retain the activity of a full-length wild-type protein, polypeptide or peptide immunomodulator.

Proteins, polypeptides or peptides useful as immunomodulatory agents can be prepared by any technique well known in the art or described herein [Short Protocols in Molecular Biology, Chapter 16, edited by Ausubel et al., John Wiley and Sons New York (1999)]. discloses methods for the preparation of proteins, polypeptides or peptides, the disclosure of which is incorporated herein by reference in its entirety, for all purposes. Fabrics for use as immunomodulatory agents may be prepared as described in U.S. Pat. No. 6,245,527; Harlow et al., "Antibodies: A Laboratory Manual,

2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1988), which is hereby incorporated by reference in its entirety. Commercial agents known to act as immunomodulatory agents are used in the compositions and methods of the present invention. The activity of an immunomodulator of an agent can be determined in vitro and / or by any of the techniques well known to those skilled in the art, including, for example, CTL assays, reproductive assays, and immunoassays (e.g., ELISAs) that can be used to determine a particular drug. protein, such as co-stimulator molecules and cytokines.

In some embodiments, the CD2 antagonists directly or indirectly induce the depletion of peripheral blood lymphocytes, preferably T lymphocytes and / or NK cells. In other embodiments, a CD2 antagonist has a T cell proliferation of at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%. %, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, 95% or at least 98% reduced in an in vivo or in vitro assay known to those skilled in the art or known to those skilled in the art. In other embodiments, one is

135 · * · <·· «» · · * «* · - '· ·» ««

CD2 antagonist has a T cell proliferation of at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, 95%, or at least 98% and induces cytolysis of peripheral blood T cells in an in vitro or in vitro assay known to those skilled in the art or known to those skilled in the art. In other embodiments, a CD2 antagonist has a T cell activation of at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%. %, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, 95% or at least 98% reduced in an in vivo or in vitro assay known to those skilled in the art or known to those skilled in the art.

In some embodiments, a CD2 antagonist has an interaction between a CD2 polypeptide and LFA-3 by at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% , 95%, or at least 98%, is reduced in an in vivo or in vitro assay as described herein (e. G., ELISA) or in the art. In other embodiments, the CD2 antagonists do not inhibit CD2 polypeptide and LFA-3.

Interaction between 136 j * *·· * <· * · · * ν. In other embodiments, the CD2 antagonists inhibit the interaction between CD2 polypeptide and LFA-3 by less than 20%, less than 15%, less than 10%, or less than 5%.

In some embodiments, a CD2 antagonist does not induce or reduce cytokine expression and / or release in an in vitro or in vitro assay as described herein or well known to those skilled in the art. In a specific embodiment, a CD2 antagonist does not induce cytokines such as IFNγ, interleukin-2 ("IL2"), interleukin-4 ("IL-4"), interleukin-6 ("IL-6"), an increase in the concentration of interleukin-9 ("IL-9"), interleukin-12 ("IL-12"), and interleukin-15 ("IL-15") in a subject receiving a CD2 antagonist. In alternative embodiments, a CD2 antagonist induces cytokine expression and / or release in an in vitro or in vitro assay as described herein or known to those skilled in the art. In a specific embodiment, a CD2 antagonist is a cytokine such as IFN ?, interleukin-2, interleukin-7 (&quot; IL-7 &quot;), interleukin-9 (&quot; IL-9 &quot;), interleukin-10 (&quot;"IL-10") and induces an increase in tumor necrosis factor alpha ("TNFa") concentration in the serum of a subject treated with a CD2 binding molecule. Serum concentrations of cytokines can be measured by any technique known to those skilled in the art, such as, for example, immunoassays, including, for example, ELISA.

137

In some embodiments, a CD2 antagonist induces T cell anergy in an in vivo or in vitro assay known to those skilled in the art or known to those skilled in the art. In alternative embodiments, a CD2 antagonist does not induce T-cell anergy in an in vivo or in vitro assay known to the person skilled in the art or known to the person skilled in the art. In other embodiments, the CD2 antagonist induces an antigen-specific non-response or hypo-responsive state for at least 2 hours, at least 6 hours, for at least 12 hours, at least 24 hours, for at least 2 days, at least 5 days, at least 7 for at least 10 days or more, in an in vitro assay as described herein or known to those skilled in the art.

In other embodiments, the CD2 antagonist activates the Tse cell by at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55% - with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, 95%, or \ t at least 98% inhibition, and T cell proliferation at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, 95% - or inhibits at least 98% in an in vitro or in vitro assay as described herein or well known to those skilled in the art.

138

In some embodiments, a CD2 antagonist is not a small molecule. In other embodiments, a CD2 antagonist is not an antisense nucleic acid molecule or triple helix. In a preferred embodiment, a CD2 antagonist is a CD2 binding molecule.

In a preferred embodiment, the proteins, polypeptides or peptides (including antibodies and fusion proteins) used as the CD2 antagonist are derived from the same species as the recipient of the proteins, polypeptides, peptides to reduce one of said proteins, polypeptides, or peptides. probability of developing an immune response against peptides. In another preferred embodiment, if the subject is a human, the proteins, polypeptides or peptides used as the CD2 antagonist are human or humanized.

Nucleic acid molecules encoding CD2 antagonist proteins, polypeptides or peptides, or proteins, polypeptides or peptides acting as CD2 antagonists may be administered to a patient suffering from an autoimmune or inflammatory disorder by the methods of the present invention. In addition, nucleic acids encoding derivatives, analogs, fragments or variants of proteins, polypeptides or peptides that act as CD2 antagonists, or derivatives, analogs, fragments or variants of proteins, polypeptides or peptides acting as CD2 antagonists in a patient suffering from an autoimmune or inflammatory disorder.

139 ·· · · · can be administered using the methods of the present invention. Such derivatives, analogs, variants and fragments preferably retain CD2 antagonist activity of the full-length wild-type protein, polypeptide or peptide.

The term &quot; CD2 binding molecule &quot; and analogous terms refer to a biologically active molecule that binds immune-specific to a CD2 polypeptide and directly or indirectly affects an activity or function of lymphocytes, more particularly peripheral blood T cells. In a specific embodiment, CD2 binding molecules directly or indirectly affect the depletion of lymphocytes, or more specifically, peripheral blood T cells. Preferably, the CD2 binding molecule binds to a CD2 polypeptide and preferentially affects the depletion of memory T cells (i.e., CD45RO + T cells), but does not affect naive T cells. In a specific embodiment, a CD2 binding molecule is immunospecifically bound to a CD2 polypeptide expressed by an immune cell, such as a T cell or an NK cell. In a preferred embodiment, a CD2 binding molecule binds immune-specific to a CD2 polypeptide expressed by a T cell and / or an NK cell. CD2 binding molecules may be identified, for example, by immunosuppression or by other techniques well known to those skilled in the art. CD2 binding molecules include, but are not limited to, peptides, polypeptides, fusion proteins, small molecules, mimetic agents, synthetic drug active ingredients, organic molecules, inorganic molecules, and antibodies.

In one embodiment, a CD2 binding molecule depletes the T-cells of the peripheral blood such that the T cell proliferation is at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85% by at least 90%, 95%, or at least 98% in an in vitro or in vitro assay known to those skilled in the art or known to those skilled in the art. In another embodiment, a CD2 binding molecule affects the depletion of peripheral blood T cells, inducing cytolysis of T cells. In another embodiment, a CD2 binding molecule affects the depletion of peripheral blood T cells, T cell proliferation by at least 25%, at least 30%, at least 35%, at least 40%, at least 45% , at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85% , reduces by at least 90%, 95%, or at least 98%, and induces cytolysis of peripheral blood T cells in an in vivo or in vitro assay as described herein or known to those skilled in the art.

In a specific embodiment, a CD2 binding molecule binds immune-specific to a CD2 polypeptide and does not specifically bind to other polypeptides. In another embodiment, a CD2 binding molecule binds immune-specific to a CD2 polypeptide and shows cross-reactivity with other antigens. In a preferred embodiment, a CD2 binding molecule binds immune-specific to a CD2 polypeptide and does not cross-react with other antigens.

In one embodiment, a CD2 binding molecule is present in an in vivo or in vitro assay as described herein or known to those skilled in the art by about 25%, 30%, 35%, 40%, 45% , 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% inhibits or decreases the CD2 polypeptide and a naturally occurring CD2 binding partner. In an alternative embodiment, a CD2 binding molecule does not inhibit the interaction between a CD2 polypeptide and a naturally-occurring carrier of carrier CD2 (e.g., LFA-3 molecule), a carrier described herein or known to one of ordinary skill in the art. or in an in vitro assay. In another embodiment, a CD2 binding molecule inhibits an interaction of a CD2 polypeptide with LFA-3 by less than 20%, less than 15%, less than 10%, or less than 5%. It can be a naturally occurring in vivo CD2 binding partner, without being limited to a peptide, a polypeptide, and an organic molecule that binds to the CD2 polypeptide. A naturally occurring in-carrier CD2 binder

142 partner binds to an extracellular domain of a CD2 polypeptide or a fragment thereof.

In a specific embodiment, a CD2 binding molecule increases T cell activity by at least 25%, by at least 30%, by at least 35%, by at least 40%, by at least 45%, by at least 50% , at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, 95 or by at least 98% in an in vitro or in vitro assay known to those skilled in the art or known to those skilled in the art.

In another embodiment, a CD2 binding molecule does not induce or reduce cytokine expression and / or release in an in vitro or in vitro assay as described herein or known to one skilled in the art. In a specific embodiment, a CD2 binding molecule does not induce cytokines, such as IFNγ, interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-6 (IL-6). ”), Interleukin-9 (" IL-9 "), interleukin-12 (" IL-12 "), and interleukin-15 (" IL-15 ") in the subject receiving a CD2 binding molecule. In alternative embodiments, a CD2 binding molecule induces cytokine expression and / or release in an in vitro or in vitro assay as described herein or known to those skilled in the art. In a specific embodiment, a CD2 binding molecule is a cytokine such as IFN ?, interleukin-2, interleukin-7 (&quot; IL-7 &quot;), interleukin-9 (&quot; IL-9 &quot;), interleukin-10.

143 ("IL-10") and induces an increase in tumor necrosis factor alpha ("TNFα") concentration in the serum of a subject treated with a CD2 binding molecule. Serum concentrations of cytokines can be measured by any technique known to those skilled in the art, such as, for example, immunoassays, including, for example, ELISA.

In a specific embodiment, a CD2 binding molecule induces T cell anergy in an in vivo or in vitro assay known to those skilled in the art or known to those skilled in the art. In alternative embodiments, a CD2 binding molecule does not induce T-cell anergy in an in vivo or in vitro assay known to those skilled in the art or known to those skilled in the art. In other embodiments, the CD2 binding molecule induces an antigen-specific non-response or hypo-responsive state for at least 2 hours, at least 6 hours, for at least 12 hours, for at least 24 hours, for at least 2 days, for at least 5 days, for at least 7 days, for at least 10 days or more, in an in vitro assay disclosed herein or known to those skilled in the art.

In other embodiments, the CD2 binding molecule activates the Tsex by at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55% , at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, 95%, or at least 98% and inhibits T cell proliferation by at least 25%, at least 30%, at least

144

35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% inhibits at least 80%, at least 85%, at least 90%, 95%, or at least 98% in an in vitro or in vitro assay as described herein or well known to one skilled in the art .

In one embodiment, a CD2 binding molecule or antigen-binding fragment thereof that binds immune-specific to a CD2 polypeptide. In a preferred embodiment, a CD2 binding molecule is an antibody or antigen-binding fragment thereof that binds immune-specific to a CD2 polypeptide expressed by an immune cell, such as a T cell or NK cell. In another embodiment, a CD2 binding molecule is an LFA-3 peptide, polypeptide, or derivative or analogue thereof that binds immune-specific to a CD2 polypeptide. In another embodiment, a CD2 binding molecule is a fusion protein that specifically binds to a CD2 polypeptide. In a preferred embodiment, a CD2 binding molecule is a fusion protein that is immunospecificly bound to a CD2 polypeptide expressed by an immune cell, such as a T cell or NK cell. In some embodiments, a CD2 binding molecule is a small organic molecule. In other embodiments, a CD2 binding molecule is not an organic molecule.

Obviously, antibodies specifically binding to the CD2 polypeptide are known in the art. Known antibodies specifically binding to CD2 po145 lipeptide include, but are not limited to, the murine monoclonal antibody produced by the UMCD2 cell line [Ancell Immunology Research Products, Bayport, MN; Kozarsky et al., Cell Immunol. 150, 235-246 (1993)], a murine monoclonal antibody produced by the RPA2.10 cell line [Zymed Laboratories, Inc., San Francisco, CA; Rabinowitz et al., Cancer Immunol. &> Immunpathol. 76 (2), 148-154], rat LO-CD2b monoclonal antibody (WO 00/78814 A2), rat LO-CD2a / BTI-322 monoclonal antibody [Latinne et al., Int. Immunol. 8 (7), 1113-1119 (1996)] and the humanized MEDI-507 monoclonal antibody [Medlmmune, Inc., Gaithersburg, MD; Branco et al., Transplantation 68 (10), 1588-1596 (1999).

The present invention relates to antibodies that bind immunoprecipitatively to a CD2 polypeptide expressed by an immune cell, such as a Tse or NK cell, and that said antibodies affect the activity or function of lymphocytes, preferably peripheral blood T cells. In a specific embodiment, antibodies specifically immunoprecipitating to a CD2 polypeptide directly or indirectly affect the depletion of lymphocytes, preferably peripheral blood T cells. More specifically, the present invention relates to antibodies that bind immunoprecipitatively to a CD2 polypeptide expressed by a T cell and / or NK cell, and that said antibodies affect the depletion of peripheral blood T cells.

146

In a specific embodiment, the antibodies that immunoprecipitate specifically to a CD2 polypeptide, in an in vitro or in vitro assay known to those skilled in the art or known to those skilled in the art, are about 25%, 30%, 35%, 40% - , 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% - inhibit or reduce the interaction between a CD2 polypeptide and LFA-3 by 95% or 98%. In an alternative embodiment, the antibodies that immunoprecipitate specifically to a CD2 polypeptide do not reduce the interaction between the CD2 polypeptide and LFA-3 in in vitro or in vitro assays known to those skilled in the art. In another embodiment, the antibodies that immunoprecipitate specifically bind to a CD2 polypeptide, less than 20%, less than 15%, less than 10%, or less than 5%, inhibit a CD2 polypeptide and LFA. 3 interactions.

In a specific embodiment, the antibodies that bind immunoprecipitatively to a CD2 polypeptide are in at least 25%, at least 30%, at least 35%, in at least 25%, in in vitro or in vitro assays known to those skilled in the art; at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80 %, at least 85%, at least 90%, 95%, or at least 98% reduction in T cell activation.

147

In another embodiment, the antibodies that immunoprecipitate specifically to a CD2 polypeptide do not induce or reduce cytokine expression in the in vitro or in vitro assays described herein or known to those skilled in the art. In a specific embodiment, antibodies that immunoprecipitate specifically to a CD2 polypeptide do not induce cytokines such as IFNγ, interleukin-2, interleukin-4, interleukin-6, interleukin-9, and interleukin-12. and the concentration of interleukin-15 in the serum of a subject having given a CD2 binding molecule. In an alternative embodiment, the antibodies that immunoprecipitate specifically to a CD2 polypeptide are induced or reduced by cytokine expression and / or release in in vitro or in vitro assays known to those skilled in the art. In a specific embodiment, antibodies that immunoprecipitate specifically to a CD2 polypeptide induce cytokines such as IFNγ, interleukin-2, interleukin-4, interleukin-6, interleukin-9, interleukin-10, and \ t concentrations of interleukin-12 and TNFa in a subject having a CD2 binding molecule. The concentration of a cytokine in serum can be measured by any technique well known to those skilled in the art, including, for example, ELISA.

In another embodiment, the antibodies that immunoprecipitate specifically to a CD2 polypeptide are those described herein or known to those skilled in the art.

• ••• fc ·· «• · In vitro or in vitro assays induce T-cell anergy. In an alternative embodiment, the antibodies that bind immunoprecipitatively to a CD2 polypeptide do not induce T-cell anergy in an in vitro or in vitro assay as described herein or known to those skilled in the art. In another embodiment, the antibodies that immunoprecipitate specifically bind to a CD2 polypeptide induce an antigen-specific non-response or hypo-responsiveness state for at least 30 minutes in at least one of the in vitro or in vivo assays known to those skilled in the art. 1 hour, at least 2 hours, for at least 6 hours, at least 12 hours, for at least 24 hours, for at least 2 days, for at least 5 days, for at least 7 days, for at least 10 days.

In one embodiment, the antibodies that immunoprecipitate specifically for a CD2 polypeptide, the proliferation of T cells in at least 25%, at least 30%, in at least 25% of the in vitro or in vitro assays described herein or known to those skilled in the art. 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% with at least 80%, at least 85%, at least 90%, 95%, or at least 98%, on the depletion of peripheral blood T cells. In another embodiment, the antibodies that bind immunoprecipitatively to a CD2 polypeptide, the proliferation of T cells is described herein.

149 •• «••« a ·

or at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, in at least 25%, at least 45%, at least 50%, of in vitro or in vitro assays known to those skilled in the art; 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, 95% - or at least 98% reduces the depletion of peripheral blood T cells and induces cytolysis of peripheral blood T cells.

In another embodiment, the antibodies that bind immunoprecipitatively to a CD2 polypeptide, activate T cells in at least 25%, at least 30%, of in vitro or in vitro assays described herein or known to those skilled in the art. at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% , at least 80%, at least 85%, at least 90%, 95%, or at least 98% inhibit, and proliferation of T cells by one skilled in the art or as skilled in the art. at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, in at least 50%, at least 55%, in in vitro or in-vitro assays; 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, 95%, or at least 98% inhibition.

In another embodiment, an Fc domain of an antibody specifically binding to the CD2 polypeptide binds to a

Immune cells such as an NK cell, a monocyte and a FcyRIII expressed by a macrophage. In another embodiment, a fragment (e.g. CH2 and / or CH3 of the Fc domain) of an Fc domain of an antibody specifically binding to the CD2 polypeptide binds to an FcR which is an immune cell, such as an NK cell, a monocyte and a \ t macrophage expresses.

Antibodies specifically binding to a CD2 polypeptide include, but are not limited to, monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, single chain FVS (scFvs), single chain antibodies, Fab fragments, F (ab ') fragments, disulfide-bonded Fvs (sdFv), and anti-idiotypic (anti-Id) antibodies (including, for example, anti-Id antibodies against antibodies of the present invention), and above also mentioned epitope-binding fragments. More specifically, antibodies specifically binding to the CD2 polypeptide include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen-binding site that binds to a CD2 polypeptide. Immunoglobulin molecules of the present invention may be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY) or any class (i.e. IgG, IgG 2, IgG3, IgG 4, IgAi and IgA 2) or subclass. In a specific embodiment, it is immunospecific for a CD2 molecule

Binds, and antibodies that affect the depletion of T cells contain an Fc domain, or a fragment thereof (e.g., an Fc domain CH2, CH3, and / or an Fc domain). or wrist region). In a preferred embodiment, the antibodies specifically binding to a CD2 polypeptide and affecting the depletion of T cells comprise an Fc domain or a fragment thereof that binds to an FcR, preferably an FcγRIII that is expressed by an immune cell.

Antibodies specifically binding to a CD2 polypeptide may be derived from any animal, including birds and mammals (e.g., human, rodent, donkey, sheep, rabbit, goat, golden hamster, camel, horse or chicken). Human antibodies that specifically bind to a CD2 polypeptide include antibodies that have the amino acid sequence of a human immunoglobulin, or antibodies isolated from human immunoglobulin libraries, or mice expressing antibodies to human genes.

Antibodies specifically binding to a CD2 polypeptide may be monospecific, bispecific, trispecific, or multispecific. The multispecific antibodies may be specific for different epitopes of a CD2 polypeptide, or they may be specific for both a CD2 polypeptide and a heterologous epitope, such as a heterologous polypeptide or solid support (see, for example, WO 93/17715, WO 92/08802, WO 91 / PCT Publication No. 00360 and WO 92/05793; Tutt et al., J. of Immunol. 147, 60-69 (1991);

152 · · · · · · ······ · · · · · · · · · · · · · · · ·

4,474,893; 4,714,681; 4,925,648; U.S. Patent Nos. 5,573,920 and 5,601,819; Kostelny et al., J. of Immunol. 148: 1547-1553 (1992).

The present invention relates to antibodies which exhibit high binding affinity for a CD2 polypeptide. In a specific embodiment, an antibody-specific binding rate constant or rate of an antibody specific for a CD2 polypeptide (antibody (Ab) + antigen (Ag) k ° n-> Ab-Ag) is at least 10 5 / Mol * sec; at least 5 * 10 5 / Mol * sec, at least 10 6 / Mol * sec; at least 5 * 10 6 / Mol * sec, at least 10 7 / Mol * sec, at least 5 * 10 7 / Mol * sec, or at least 10 8 / Mol * sec. In a preferred embodiment, a polypeptide immunospecifically binds CD2 antibody k on is at least 2 * 10 5 / sec * Mol, 5 x 10 5 / sec * Mol at least 10 6 / mole * sec; at least 5 * 10 6 / Mol * sec, at least 10 7 / Mol * sec, at least 5 * 10 7 / Mol * sec, or at least 10 8 / mol * sec.

In another embodiment, immunospecifically bind to a CD2 polypeptide antibody k O ff rate (antibody (Ab) + antigen (Ag) Ko £ f <-AB-Ag) of less than lO Vsec; less than 5 * 10Vsec; less than 10 2 / sec; less than 5 * 10- 2 / sec; less than IO 3 / sec; less than 5 * 10- 3 / sec; less than 10 4 / sec; less than 5 * 10 4 / sec; less than 10 5 / sec; less than 5 * 10- 5 / sec; less than 10- 6 / sec; less than 5 * 10 6 / sec; less than 10 7 / sec; less than 5 x 10- 7 / sec; less than 10 8 / sec; less than 5 * 10 ' 8 / sec; less than 10 9 / sec; less than 5 x 10 9 / sec; or less than 10 10 / sec. A preferred embodiment

According to a method 153 immunospecifically binds CD2 polypeptide antibody kon value than 5 * 10 4 / sec; less than 10- 5 / sec; less than 5 x 10 5 / sec; less than 10 6 / sec; less than 5 χ 10 6 / sec; less than 10 7 / sec; less than 5 x 10 -7 / sec; less than 10 8 / sec; less than 5 x 10 8 / sec; less than 10 9 / sec; less than 5 x 10 -9 / sec; or less than 10 10 / sec.

In another embodiment, an antibody specific binding to a CD2 polypeptide is an affinity constant or a K a (ko n / koff) of at least 10 2 / mole; at least 5 x 10 2 / Mole; at least 10 3 / Mole; at least 5 χ 10 3 / Μό1; at least 10 4 / mol; at least 5 χ 10 4 / Μό1; at least 10 5 / mol; at least 5 χ 10 5 / Μό1; at least 10 6 / mol; at least 5 x 10 6 / Mole; at least 10 7 / Mole; at least 5 χ 10 7 / Μό1; at least 10 8 / mol; at least 5 χ 10 8 / Μό1; at least 10 9 / mol; at least 5 χ 10 9 / Μό1; at least 10 10 Moles; at least 5 χ 10 / Μό1; at least 10 11 / mol; at least 5 x 10 xl / mol; at least 10 12 Moles; at least 5 χ 10 12 / Μό1; at least 10 13 / mol; at least 5 χ 10 13 / Μό1; at least 10 14 / Mole; at least 5 χ 10 14 / Μό1; at least 10 15 Moles; or at least 5 χ 10 15 / Μό1. In another embodiment, an antibody that immunospecifically binds to integrin α ν β3-1ιοζ dissociation constant or Kd (koff / O k n) of less than 10 2 M, less than 5 χ 10 2 M, of less than 10 3 M, less than 5 χ 10- 3 M, less than IO 4 M, less than 5 χ 10 -4 mol, less than about 10 5 M, less than 5 χ 10 5 M, less than IO 6 M, less than 5 χ 10 6 Mole, less than 7 7 Mole, less than 5 χ 10 7 Mole, less than 8 8 Mole, less than 5 χ 10- 8 Mole, less than 10 9 Mole, less than 5x1 Cl · 9 Mole, less than

154

10 Mole, less than 5 χ 10 1, Mole, less than 10 11 Mole, less than 5 χ 10 η mole, less than 10 12 Mole, less than 5 χ 10 12 Mole, less than 1O 13 Mole, less than 5 χ 1 Ο 13 Mol, less than 10 14 Mole, less than 5 χ 10 14 Mole, less than 10 15 Mole, more dense than 5 χ15 Mole.

In a specific embodiment, an antibody specifically binding to a CD2 polypeptide is LO-CD2a / BTI322, or an antigen-binding fragment thereof (e.g., one or more complementarity determining regions (CDR) of LOCD2a / BTI-322). The amino acid sequence of LO-CD2a / BTI-322 has been published in the literature, e.g., U.S. Patent Nos. 5,730,979, 5,817,311 and 5,951,983, and U.S. Patent Application Serial Nos. 09 / 056,072 and 09 / 462,140 (which is incorporated herein by reference). published as a reference in its entirety), or a monoclonal antibody produced by a cell line deposited at the American Type Culture Collection (ATCC?), 10801 University Boulevard, Manassas, Virginia 20110-2209, July 28, 1993, deposit number HB 11423. amino acid sequence. In an alternative embodiment, an antibody specifically binding to a CD2 polypeptide is not an LO-CD2a / BTI-322 or an antigen-binding fragment of LO-CD2a / BTI-322.

In another specific embodiment, an antibody specifically binding to a CD2 polypeptide is LOCD2b or an antigen-binding fragment thereof (e.g., LO155).

CD2b one or more complementarity determining regions). The amino acid sequence of the LO-CD2b is the same as the amino acid sequence of the antibody produced by the cell line deposited under the PTA-802 deposit number at the American Type Culture Collection (ATCC?), 10801 University Boulevard, Manassas, Virginia 20110-2209, June 22, 1999; or with the sequence disclosed, for example, by Dehoux et al. (Dehoux et al., Transplantation 69 (12), 2622-2633 (2000); and WO 00/78814, which are hereby incorporated by reference in their entirety). In an alternative embodiment, an antibody specifically binding to a CD2 polypeptide is not an antigen-binding fragment of LO-CD2b or LO-CD2b.

In a preferred embodiment, an antibody specifically binding to a CD2 polypeptide is MEDI-507 or an antigen-binding fragment thereof (e.g., one or more complementarity determining regions of MEDI-507). For example, MEDI-507 has been described in WO 99/03502 and in U.S. Patent Application Serial No. 09 / 462,140, which are hereby incorporated by reference in their entirety. In an alternative embodiment, an antibody of the present invention is not an antigen-binding fragment of MEDI-607 or MEDI-507.

The present invention further provides an antibody that binds immune-specific to a CD2 polypeptide.

156 and said antibodies comprise a variable heavy (&quot; VH &quot;) domain having the amino acid sequence of the amino acid sequence of the VH domain of LOCD2a / BTI-322 or MEDI-507. The present invention further relates to antibodies specifically binding to a CD2 polypeptide, and said antibodies comprise a VH CDR having the amino acid sequence of any of the VH CDR amino acid sequences listed in Table 2.

Table 2

CDR sequences for LO-CD2a / BTI-322

CDR Sequence Chair, ID. WOMAN.
VH1 EYYMY 11
VH2 RIDPEDGSIDYVEKFKK 12
VH3 GKNYRFAY 13
VL1 RSQSLLHSSGNTLNW 14
VL2 LVSKLES 15
VL3 MQFTHYPYT 16

In one embodiment, an antibody specifically binding to a CD2 polypeptide comprises a VH CDR1, the amino acid sequence of which is shown in SEQ ID NO: 11. In another embodiment, the antibodies specifically binding to a CD2 polypeptide comprise a VH CDR2 having an amino acid sequence of

SEQ ID NO: 12. Another embodiment

157

In another embodiment, antibodies specifically binding to a CD2 polypeptide comprise a VH CDR3 having the amino acid sequence shown in SEQ ID NO: 13. In a preferred embodiment, the antibodies specifically binding to a CD2 polypeptide comprise a VH CDR1, the amino acid sequence of which is shown in SEQ ID NO: 11, contains a VH CDR2, the amino acid sequence of which is shown in SEQ ID NO: 12, and contain a VH CDR3, the amino acid sequence of which is shown in SEQ ID NO: 13.

The present invention further provides an antibody that specifically binds to a CD2 polypeptide, and said antibodies comprise a variable light ("VL") domain having the amino acid sequence corresponding to the amino acid sequence of the VL domain of LOCD2a / BTI-322 or MEDI-507. . The present invention further relates to antibodies specifically binding to a CD2 polypeptide, and said antibodies comprise a VL CDR having the amino acid sequence of any of the VL CDR amino acid sequences listed in Table 2.

In one embodiment, an antibody specifically binding to a CD2 polypeptide comprises a VL CDR1, the amino acid sequence of which is shown in SEQ ID NO: 14. In another embodiment, the antibodies specifically binding to a CD2 polypeptide comprise a VL CDR2 having an amino acid sequence of

158

SEQ ID NO: 15. In another embodiment, the antibodies specifically binding to a CD2 polypeptide comprise a VL CDR3 whose amino acid sequence is shown in SEQ ID NO: 16. In a preferred embodiment, the antibodies specifically binding to a CD2 polypeptide comprise a VL CDR1, the amino acid sequence of which is shown in SEQ ID NO: 14, contains a VL CDR2, the amino acid sequence of which is shown in SEQ ID NO: 15, and contain a VL CDR3, the amino acid sequence of which is shown in SEQ ID NO: 163.

The present invention further relates to antibodies that immunoprecipitatively bind to a CD2 polypeptide and said antibody comprises a VH domain as described herein, in combination with a VL domain described herein, or other VL domain. The present invention further relates to antibodies that immunoprecipitatively bind to a CD2 polypeptide, and said antibody comprises a VL domain as described herein, in combination with a VH domain described herein or with another VH domain.

The present invention relates to antibodies that bind immunoprecipitatively to a CD2 polypeptide and said antibody comprises one or more VH CDRs and one or more VL CDRs listed in Table 2. More specifically, the present invention relates to an antibody that binds immune-specific to a CD2 polypeptide, and that

This antibody contains a VH CDR1 and a VL CDR1, a VH CDR1, and a VL CDR1, a VH CDR1, and a VL CDR1. CDR3, a VH CDR2 and a VL CDR1, a VH CDR2 and a VL CDR-2, a VH CDR2 and a VL CDR3, a VH CDR3 and a VH CDR1, a VH CDR3 and a VL CDR2, a VH CDR3, and a VL CDR3, or any combination of the VH CDRs and VL CDRs listed in Table 2.

In one embodiment, the antibody specifically binding to a CD2 polypeptide comprises a VH CDR1 having an amino acid sequence. and VL CDR1, the amino acid sequence of which is shown in SEQ ID NO: 14. In another embodiment, an antibody that binds immune-specific to a CD2 polypeptide comprises a VH CDR1 having the amino acid sequence shown in SEQ ID NO: 11 and VL CDR2, the amino acid sequence of which is shown in SEQ ID NO: 15. In another embodiment, an antibody that binds immune-specific to a CD2 polypeptide comprises a VH CDR1 under its amino acid sequence. SEQ ID NO: 4 and VL CDR3, which has an amino acid sequence low. SEQ ID NO.

In one embodiment, the antibody specifically binding to a CD2 polypeptide comprises a VH CDR2 having the amino acid sequence shown in SEQ ID NO: 12 and VL CDR1, the amino acid sequence of which is shown in SEQ ID NO: 14. In another embodiment, an antibody that binds immune-specific to a CD2 polypeptide comprises a VH CDR2 having the amino acid sequence shown in SEQ ID NO: 12 and VL CDR2, the amino acid sequence of which is shown in SEQ ID NO: 15. In another embodiment, an antibody that binds immune-specific to a CD2 polypeptide comprises a VH CDR2 having the amino acid sequence shown in SEQ ID NO: 12 and VL CDR3, the amino acid sequence of which is shown in SEQ ID NO: 16.

In one embodiment, the antibody specifically binding to a CD2 polypeptide comprises a VH CDR3 having the amino acid sequence shown in SEQ ID NO: 13 and VL CDR1, the amino acid sequence of which is shown in SEQ ID NO: 14. In another embodiment, an antibody that binds immune-specific to a CD2 polypeptide comprises a VH CDR3 having the amino acid sequence shown in SEQ ID NO: 13 and VL CDR2, the amino acid sequence of which is shown in SEQ ID NO: 15. In another embodiment, an antibody that binds immune-specific to a CD2 polypeptide comprises a VH CDR3 having the amino acid sequence shown in SEQ ID NO: 13 and VL CDR3, the amino acid sequence of which is shown in SEQ ID NO: 16.

161

The present invention further relates to a nucleic acid molecule, usually an isolated molecule, which encodes an antibody specifically binding to a CD2 polypeptide. In a specific embodiment, an isolated nucleic acid molecule encodes an antibody that binds to a CD2 polypeptide, and the amino acid sequence of said antibody is LO-CD2a / BTI-322, LO-CD2b, or the MEDI-507 amino acid sequence.

In one embodiment, an isolated nucleic acid molecule encodes an antibody that specifically binds to a CD2 polypeptide, and said antibody comprises a VH domain having the amino acid sequence identical to the amino acid sequence of LO-CD2a / BTI-322 or MEDI-507. In another embodiment, an isolated nucleic acid molecule encodes an antibody that specifically binds to a CD2 polypeptide, and said antibody comprises a VH domain having the amino acid sequence of the VH domain of the monoclonal antibody produced by the cell line deposited at ATCC® HB141423. amino acid sequence. In a further embodiment, an isolated nucleic acid molecule encodes an antibody specifically binding to a CD2 polypeptide, said antibody comprising a VH CDR1 having the same amino acid sequence as

VR CDR1 amino acid sequence listed in Table 2. In another embodiment, an isolated nucleic acid molecule encodes an antibody specifically binding to a CD2 polypeptide, said antibody comprising a VH CDR2,

162 having the amino acid sequence identical to the VH CDR2 amino acid sequence listed in Table 2. In another embodiment, an isolated nucleic acid molecule encodes an antibody specifically binding to a CD2 polypeptide, said antibody comprising a VH CDR3 having the amino acid sequence of the VH CDR3 amino acid sequence listed in Table 2.

In one embodiment, an isolated nucleic acid molecule encodes an antibody that specifically binds to a CD2 polypeptide, and said antibody comprises a VL domain having the amino acid sequence identical to the amino acid sequence of LO-CD2a / BTI-322 or MEDI-507. In another embodiment, an isolated nucleic acid molecule encodes an antibody that specifically binds to a CD2 polypeptide, and said antibody comprises a VL domain having the amino acid sequence of the VL domain of the monoclonal antibody produced by the cell line deposited at ATCC® HB141423. amino acid sequence. In a further embodiment, an isolated nucleic acid molecule encodes an antibody specifically binding to a CD2 polypeptide, said antibody comprising a VL CDR1 having the amino acid sequence identical to that of an antibody.

VL CDR1 amino acid sequence listed in Table 2. In another embodiment, an isolated nucleic acid molecule encodes an antibody specifically binding to a CD2 polypeptide, said antibody comprising a VL CDR2,

The amino acid sequence of the VL CDR2 amino acid sequence listed in Table 2 is identical to that of the amino acid sequence. In another embodiment, an isolated nucleic acid molecule encodes an antibody specifically binding to a CD2 polypeptide, said antibody comprising a VL CDR3 having the amino acid sequence of the VL CDR3 amino acid sequence listed in Table 2.

In one embodiment, an isolated nucleic acid molecule encodes an antibody that specifically binds to a CD2 polypeptide, and said antibody comprises a VH domain having the amino acid sequence corresponding to the amino acid sequence LO-CD2a / BTI-322 or MEDI-507, and a VL domain having the amino acid sequence identical to the amino acid sequence of LO-CD2a / BTI-322 or MEDI-507. In another embodiment, an isolated nucleic acid molecule encodes an antibody specifically binding to a CD2 polypeptide, and said antibody comprises a VH CDR1, a VL CDR1, a VH CDR2, a VL CDR2, a VH CDR3- and a VL CDR3 or any combination thereof, the amino acid sequence of which is listed in Table 2.

The present invention further provides antibodies that immunoprecipitate specifically to a CD2 polypeptide, and said antibodies include the derivatives of VH domains, VH CDRs, VL domains, or VL CDRs described herein that bind immunoprecipitatively to integrin α ν β3164. »« · Ο »« ·· • ·

9 · »· * * · * * Standard techniques known to those skilled in the art may be used to introduce mutations into the nucleotide sequence encoding the antibodies of the present invention, including, for example, site-specific mutagenesis and mutagenesis by polymerase chain reaction resulting in amino acid substitutions. Preferably, the derivatives have less than 25 amino acid substitutions, less than 20 amino acid substitutions, less than 15 amino acid substitutions, less than 10 amino acid substitutions, less than 5 amino acid substitutions, less than 4 amino acid substitutions, less than 3 amino acid substitutions, or less than 2 amino acid substitutions relative to the original molecule. In a preferred embodiment, the derivatives are subjected to conservative amino acid substitutions at one or more presumably non-essential amino acid residues (e.g., amino acids that are not critical for binding antibody to CD2 polypeptide). "Conservative amino acid substitution" is an amino acid substitution when an amino acid residue is replaced by an amino acid residue having a similarly charged side chain. Families of amino acid residues having a similarly charged side chain have already been defined in the art. Such families include amino acids with a basic side chain (e.g., lysine, arginine, histidine), amino acid residues with an acid side chain (such as aspartic acid, glutamic acid), an amino · · · · · with an uncharged polar side chain:

165 acids (such as glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids having an apolar side chain (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), having a branched beta branch. amino acids (e.g., threonine, valine, isoleucine), and amino acids having an aromatic side chain (e.g., tyrosine, phenylalanine, tryptophan, histidine). Alternatively, mutations may be introduced at random, in whole or in part of the coding sequence, such as saturation mutagenesis, and the resulting mutants may be assayed for their biological activity to identify mutants that have retained their activity. Following mutagenesis, the encoded antibody can be expressed and the activity of the antibody can be determined.

The present invention relates to antibodies that bind immunoprecipitatively to a CD2 polypeptide, and said antibodies comprise the amino acid sequence of LO-CD2a / BTI-322 or MEDI-507 by one or more amino acid substitutions in the variable light (VL) domain and / or \ t in the variable heavy (VH) domain. The present invention further provides antibodies that immunoprecipitate specifically for the CD2 polypeptide, and said antibodies comprise the sequence of LO-CD2a / BTI-322 or MEDI-507 amino acid by one or more amino acid substitutions in one or more VL CDRs and / or \ t or one or more VH CDRs. The antibodies produced by LO-CD2a / BTI-322 or MEDI-507

Ν k k · «*« k k k k k «« «« k k k «« «

Substitutions have been introduced into VH domain, VH CDRs, VL domain and / or VL CDRs, in vitro and in vivo, for example, the ability to bind to a CD2 polypeptide (e.g., by immunoassay including, but not limited to, immunostaining). ELISA and BIAcore) or their ability to prevent, treat or alleviate one or more symptoms associated with an autoimmune or inflammatory disorder.

In a specific embodiment, the antibody that immunoprecipitates specifically to the CD2 polypeptide comprises a nucleotide sequence that hybridizes to the American Type Culture Collection (ATCC®, 1081 University Boulevard, Manassas, Virginia 20110-2209) deposited under the deposit number HB 9537. the nucleotide sequence encoding the amino acid sequence of the monoclonal antibody produced by the cell line, i.e., hybridizes to the filter-bound DNA in a 6 * sodium chloride / sodium citrate (SSC) solution at about 45 ° C followed by one or more washes of 0.2xSSC / ml. In a solution of 0.1% SDS at about 50-65 ° C under stringent conditions, i.e. hybridization to the filter sheet-bound nucleic acid at 6xSSC at about 45 ° C, followed by one or more washings with a 0.1xSSC / 0.2% SDS composition in a solution at about 68 ° C, or under other stringent conditions that are well known to those skilled in the art are well known (Current Protocols in Molecular Biology, 6.3.Ιό.3.6 and 2.10.3, ed. Ausubel et al., Greene Publishing and Wiley-Interscience: New York (1987)).

167 ·· «

In a specific embodiment, the antibody that immunoprecipitates specifically to the CD2 polypeptide comprises a nucleotide sequence that hybridizes under stringent conditions to the sequence encoding MEDI-507, i.e., hybridization to the filter sheet-bound nucleic acid in 6? SSC at about 45 ° C, followed by one or more washes in a solution of 0.1 * SSC / 0.2% SDS at about 68 [deg.] C. or under other stringent conditions well known to those skilled in the art [Current Protocols in Molecular Biology, 6.3. .6.3.6 and 2.10.3, edited by Ausubel et al., Greene Publishing and Wiley-Interscience: New York (1987)].

In a specific embodiment, the antibody that is immunospecifically bound to the CD2 polypeptide has an amino acid sequence of a VH domain or an amino acid sequence of a VL domain encoded by a nucleic acid sequence that hybridizes under stringent conditions to LO-CD2a / BTI-322. or the nucleotide sequence encoding the VH or VL domain of mEDI-507, i.e., hybridization to the supernatant-bound DNA in a 6 * sodium chloride / sodium citrate (SSC) solution at about 45 ° C followed by one or more washings 0, 2 * SSC / 0.1% SDS in a solution at about 50-65 ° C under highly stringent conditions, i.e. hybridization to the filter membrane bound nucleic acid in 6xSSC at about 45 ° C followed by one or more washings of 0.1>< SSC / 0.2% solution in SDS at about 68 ° C, or under other stringent conditions, which will be apparent to one skilled in the art168 • ··· · Well known [Current Protocols in Molecular Biology, 6.3.Ιό.3.6 and 2.10.3, edited by Ausubel et al., Greene Publishing and Wiley-Interscience: New York (1987)].

In another embodiment, the antibody that is immunospecifically bound to the CD2 polypeptide having an amino acid sequence of a VH CDR or an amino acid sequence of a VL CDR encoded by a nucleotide sequence that hybridizes under stringent conditions to the nucleic acid sequence that is Any of the VH CDRs or VL CDRs listed in Table 1, i.e., hybridizes to the filter-bound DNA in a 6 * sodium chloride / sodium citrate (SSC) solution at about 45 ° C, is followed by one or more of these. washing in a solution of 0.2 * SSC / 0.1% SDS at about 50-65 ° C under highly stringent conditions, i.e., hybridization to the filter plate bound nucleic acid in 6? SSC at about 45 ° C followed by one or more washing in a solution of 0.1 * SSC / 0.2% SDS at about 68 [deg.] C or under other stringent conditions as will be appreciated by one of ordinary skill in the art. are well known (Current Protocols in Molecular Biology, 6.3.Ιό.3.6 and 2.10.3, ed. Ausubel et al., Greene Publishing and Wiley-Interscience: New York (1987)).

In another embodiment, the antibody that is immunospecifically bound to the CD2 polypeptide having an amino acid sequence of a VH CDR or an amino acid sequence of a VL CDR encoded by a nucleotide sequence that hybridizes under stringent conditions

169 nucleic acid sequences encoding any of the VH CDRs or VL CDRs of any of the VH CDRs produced by the monoclonal antibody produced by the American Type Culture Collection deposited under HB 11423, i.e., hybridizing to the filter-bound DNA by 6 * sodium chloride / sodium citrate ( SSC) in solution at about 45 ° C followed by one or more washings in a solution of 0.2> SSC / 0.1% SDS at about 50-65 ° C under highly stringent conditions, i.e. hybridization to the filter sheet nucleic acid at 6 ° C in SSC at about 45 ° C, followed by one or more washings in 0.1% SSC / O, 2% SDS at about 68 ° C, or under other stringent conditions in the art. is well known to those skilled in the art [Current Protocols in Molecular Biology, 6.3.1-6.3.6 and 2.10.3, ed. Ausubel et al., Greene Publishing and Wiley-Interscience: New York (1987)].

In another embodiment, the antibody that is immunospecifically bound to the CD2 polypeptide having an amino acid sequence of a VH CDR and an amino acid sequence of a VL CDR encoded by a nucleotide sequence that hybridizes under stringent conditions to the nucleic acid sequence that is Any of the VH CDRs or VL CDRs listed in Table 1, i.e., hybridizes to the filter-bound DNA in a 6 * sodium chloride / sodium citrate (SSC) solution at about 45 ° C, is followed by one or more of these. washing in a solution of 0.2 * SSC / 0.1% SDS at about 50-65 ° C under highly stringent conditions, i.

170 for filter-bound nucleic acid in 6 x SSC at about 45 ° C, followed by one or more washings in a 0.1xSSC / 0.2% SDS composition at about 68 ° C or under other stringent conditions as will be appreciated by one of ordinary skill in the art. [Current Protocols in Molecular Biology, 6.3. .6.3.6 and 2.10.3, edited by Ausubel et al., Greene Publishing and Wiley-Interscience: New York (1987)].

In another embodiment, the antibody that is immunospecifically bound to integrin a V P3 having an amino acid sequence of a VH CDR and an amino acid sequence of a VL CDR encoded by a nucleotide sequence that hybridizes under stringent conditions to the nucleic acid to encode any CDR or VL CDR of a monoclonal antibody produced by a cell line deposited by the American Type Culture Collection at HB 11423, i.e., hybridizes to the filter-bound DNA with 6 X sodium chloride / sodium citrate (SSC) at about 45 ° C, followed by one or more washings in 0.2xSSC / 0.1% SDS at about 50-65 ° C under highly stringent conditions, i.e., hybridization to the filter-bound nucleic acid at about 6xSSC. At 45 ° C, followed by one or more washes in a 0.1xSSC / 0.2% SDS solution of about 68 ° C n or other stringent conditions well known to those skilled in the art [Current Protocols in Molecular Biology, 6.3.1-6.3.6 and 2.10.3, eds.

171

Ausubel et al., Greene Publishing and Wiley-Interscience: New York (1987)].

In a specific embodiment, the antibody that binds immune-specific to the CD2 polypeptide comprises an amino acid sequence that is at least 35%, at least 40%, at least 45%, at least 50%, at least 55% , at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% is identical to the sequence of the monoclonal antibody produced by the cell line deposited at HB 11423 at the American Type Culture Collection. In another embodiment, the antibody that is immunospecifically bound to a CD2 polypeptide comprises an amino acid sequence that is at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99 % identical to the MEDI-507 amino acid sequence.

In another embodiment, the antibody that is immunospecifically bound to a CD2 polypeptide comprises an amino acid sequence of a VH domain that is at least 35%, at least 40%, at least 45%, at least 50%, at least 55% at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%

• identical to the amino acid sequence of the MEDI-507 VH domain. In another embodiment, the antibody that is immunospecifically bound to a CD2 polypeptide comprises an amino acid sequence of a VH domain that is at least 35%, at least 40%, at least 45%, at least 50%, at least 55% at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the sequence of the VH domain of the monoclonal antibody produced by the cell line deposited at the American Type Culture Collection HB 11423.

In another embodiment, the antibody that is immunospecifically bound to a CD2 polypeptide comprises one or more VR CDR amino acid sequences that are at least 35%, at least 40%, at least 45%, at least 50%, at least 55%. at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical to the amino acid sequence of any of the VH CDRs listed in Table 2. In another embodiment, the antibody that is immunospecifically bound to a CD2 polypeptide comprises one or more VR CDR amino acid sequences that are at least 35%, at least 40%, at least 45%, at least 50%, at least 55%. at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% Or • at least 99% identical to the VH CDR sequence of the monoclonal antibody produced by the cell line deposited at HB 11423 at the American Type Culture Collection.

In another embodiment, the antibody that is immunospecifically bound to the CD2 polypeptide comprises an amino acid sequence of a VL domain that is at least 35%, at least 40%, at least 45%, at least 50%, at least 55% at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence of the VL domain of MEDI-507. In another embodiment, the antibody that binds immune-specific to the CD2 polypeptide comprises an amino acid sequence of a VH domain that is at least 35%, at least 40%, at least 45%, at least 50%, at least 55% - at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the sequence of the VL domain of the monoclonal antibody produced by the cell line deposited at HB 11423 at the American Type Culture Collection.

In another embodiment, the antibody that binds immune-specific to the CD2 polypeptide comprises an amino acid sequence of one or more VL CDRs that are at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least

174

70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to any of the VL CDRs listed in Table 2 . In another embodiment, the antibody that is immunospecifically bound to integrin a v p3 contains one or more VL CDR amino acid sequences that are at least 35%, at least 40%, at least 45%, at least 50% at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical to the VL CDR sequence of the monoclonal antibody produced by the cell line deposited at HB 11423 at the American Type Culture Collection.

The present invention relates to antibodies that compete with an antibody described herein for binding to a CD2 polypeptide. In a specific embodiment, the present invention relates to antibodies that compete with LOCD2a / BTI-322 or an antigen-binding fragment thereof for binding to a CD2 polypeptide. In a specific embodiment, the present invention relates to antibodies that compete with MEDI-507 or an antigen-binding fragment thereof for binding to a CD2 polypeptide.

The present invention also relates to VH domains that compete with the VH domain of LO-CD2a / BTI-322 or MEDI-507 for binding to a CD2 polypeptide. The present invention also relates to VL domains which are LO-CD2a / BTI-322

175 or with the VL domain of MEDI-507 for binding to a CD2 polypeptide.

The present invention relates to VH CDRs that compete with the VH CDR of the monoclonal antibody produced by the VH CDR described in Table 2 or by the cell line deposited by the American Type Culture Collection HB 11423 for binding to the CD2 polypeptide. The present invention relates to VL CDRs that compete with the VL CDR of the monoclonal antibody produced by the VL CDR described in Table 2 or by the cell line deposited by the American Type Culture Collection HB 11423 for binding to the CD2 polypeptide.

Antibodies specifically binding to the CD2 polypeptide include derivatives that are modified, for example, by covalently attaching any type of molecule to the antibody. For example, without limiting it, antibody derivatives include antibodies that are modified, such as glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, a cellular ligand, or other protein. switching, etc. Any of a number of different chemical modifications may be carried out by known techniques including, but not limited to, specific chemical cleavage, acetylation, formylation, tunicamycin mimetic 176 bolic synthesis, and the like. In addition, the derivatives may also contain one or more non-classic amino acids.

The present invention also provides antibodies that bind immunoprecipitatively to a CD2 polypeptide, and said antibodies comprise a framework known to those skilled in the art. The framework of the antibody of the present invention is preferably human. In a specific embodiment, the antibody specifically binding to the CD2 polypeptide comprises the MEDI-507 framework region.

The present invention also provides antibodies that bind immunoprecipitatively to the CD2 polypeptide, and said antibodies comprise the amino acid sequence of MEDI-507 with one or more mutations (i.e., one or more amino acid substitutions in the framework regions). In some embodiments, the antibodies specifically binding to the CD2 polypeptide comprise the amino acid sequence of MEDI-507 with one or more mutations in the framework regions of the VH and / or VL domains.

The present invention also provides antibodies that bind immunoprecipitatively to a CD2 polypeptide, and said antibodies comprise the amino acid sequence of MEDI-507, with one or more mutations (i.e., one or more amino acid substitutions) in the variable and framework regions.

The present invention further provides fusion proteins comprising an antibody that binds immune-specific to the CD2 polypeptide and comprises a heterologous polypeptide. The heterologous polypeptide to which the ellena177 is fused is preferably useful for directing the antibody to platelets, monocytes, endothelial cells and / or B cells.

The present invention relates to antibodies that bind immunoprecipitatively to the CD2 polypeptide and have a longer carrier half-life. More specifically, the present invention relates to antibodies that bind immunoprecipitatively to a CD2 polypeptide and have a half-life in an animal, preferably a mammal, and most preferably a human being greater than 3 days, greater than 7 days, greater than 10 days, preferably greater than 15 days , greater than 25 days, greater than 30 days, greater than 35 days, greater than 40 days, greater than 45 days, greater than 2 months, greater than 3 months, greater than 4 months or greater than 5 months.

In order to extend antibodies (e.g., monoclonal antibodies, single chain antibodies, and Fab fragments) circulating in the serum, for example, inert polymer molecules such as high molecular weight polyethylene glycol (PEG), can be coupled to the antibodies, with or without a multifunctional linker, or by site-specific conjugation of PEG via the N-terminus or C-terminus of the antibody, or through the epilone amino groups on the lysine moieties. Linear or branched polymer derivatization is used which results in minimal loss of biological activity. The extent of conjugation is closely monitored by SDS-PAGE and mass spectroscopy to ensure correct conjugation of the PEG molecules to the antibodies. The unreacted PEG can be separated from the antibody PEG conjugates by size exclusion or ion exchange chromatography. The PEG derivatives of the antibodies can be assayed for binding activity and in vivo efficacy by methods known to those skilled in the art, such as the immunoassay described below.

Antibodies having an increased half-life can also be produced by introducing one or more amino acid modifications (e.g., substitution, insertion or deletion) into an IgG constant domain or its FcRn binding fragment (preferably into an Fc or hinge Fc domain fragment) (see for example, WO 98/23289; WO 97/34631; and U.S. Pat. No. 6,277,375, which are hereby incorporated by reference in their entirety.

The present invention relates to antibodies or antigen-binding fragments thereof that bind immunoprecipitatively to a CD2 polypeptide and are fused or recombinantly chemically conjugated (including both covalent and non-covalent conjugation) to a heterologous polypeptide (or a covalent conjugate). for a fragment, the polypeptide preferably has at least 5, at least 10, at least 15, at least 20, at least 25, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100 contiguous amino acid tubes • * · * «· «· ··

179) to produce a fusion protein. The fusion need not necessarily be direct, but may occur through linker sequences. For example, antibodies can be used to target heterologous polypeptides to selected cell types (e.g., platelets, endothelial cells, B cells or monocytes), in vitro or in vivo, to antibody-linked or conjugated antibodies to the particular cell surface receptors such as CD11c. CD14, CD17, CD19, CD25, CD36, CD41, CD42, CD51, CD61 and CD78.

The present invention further relates to antibodies or antigen-binding fragments thereof that immunoprecipitate specifically to a CD2 polypeptide fused to a marker sequence, such as a peptide for ease of purification. In a preferred embodiment, the marker amino acid sequence is a hexa-histidine peptide, such as the one provided by the pQE vector (Quiagen, Inc., 9259 Eton Avenue, Chatsworth, CA, 91311), and many of these are commercially available. According to Gentz et al., The hexa-histidine can be used for convenient purification of the fusion protein (Gentz et al., Proceedings of the National Academy of Sciences, USA 86, 821824 (1989)). Other peptide labels used for purification include, but are not limited to, the haemagglutinin &quot; HA &quot;

180 epitopes derived from 180 haemagglutinin proteins (Wilson et al., Cell 37: 767 (1984)).

The present invention further provides antibodies or antigen-binding fragments thereof that bind immunoprecipitatively to a CD2 polypeptide conjugated to an agent having a potential therapeutic advantage. An antibody or antigen-binding fragment thereof that specifically binds to a CD2 polypeptide may be conjugated to a therapeutic moiety, such as a cytotoxin, such as a cytostatic or cytocidal agent, an agent having potential advantages, or a radioactive metal ion, e.g. alpha issuer. The cytotoxin or cytotoxic agent may be any agent that is harmful to cells. Cytotoxin or cytotoxic agents may include, but are not limited to, paclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetin, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxythracinone, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol and puromycin, and analogues and homologs thereof. Agents with potential therapeutic benefits include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5fluorouracil decarbazine), alkylating agents (e.g. , thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide,

Busulphan, dibromannite, streptozotocin, mitomycin C and cysticlordiamine platinum (II) (DPP, cisplatin), anthracyclines (such as daunorubicin (formerly daunomycin) and doxorubicin), antibodies (i. E. for example, dactinomycin (formerly actinomycin), bleomycin, mithramycin and anthramycin (AMC)), and anti-mitotic agents (such as vincristine and vinblastine).

In addition, an antibody or antigen-binding fragment thereof that specifically binds to the CD2 polypeptide may be conjugated to a therapeutic agent or drug that modifies a particular biological response. Agents with a potential therapeutic advantage, or pharmaceutical agents, should not be limited to classical chemical therapy agents. The drug agent may be, for example, a protein or polypeptide having the desired biological activity. Examples of such proteins include a toxin such as abrin, ricin A, Pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor, interferon-α ("IFN-α"), interferon-β ("IFN-β"), nerve growth factor ("NGF"), platelet-derived growth factor ("PDGF") , tissue plasminogen activator ("TPA"), an apoptotic agent such as TNFα, Fβ, AIM I (WO 97/33899), AIM II (International Patent Publication No. 97/34911), Woody Ligand [Takahashi et al., J. of Immunol. 6, 1567-1574 (1994)] and VEGF (see, for example, WO 99/23105), a thrombotic agent or an anti-an182 »4 · · ··· ·· · · * ♦ · · · 44? ·· * · a genesis agent such as angiostatin or endostatin; or a biological response modifier, such as a lymphokine (e.g., interleukin-1 ("IL-1"), interleukin-2, interleukin-6, interleukin-10, granulocyte macrophage colony stimulating factor ("GMCSF"), and granulocyte colony stimulating growth factor ("G-CSF") or a growth factor (such as growth hormone UGH)).

Techniques used to conjugate such therapeutic units to antibodies are well known in the art (Armon et al., "Monoclonal Antibodies Forum for Immunodeficiency Drugs In Cancer Therapy", In: Monoclonal Antibodies and Cancer Therapy, ed. Reisfeld et al., 243-256. , Alan R. Liss, Inc., (1985); Hellstrom et al., "Antibodies Forum Drug Delivery", In: Controlled Drug Delivery, 2nd Edition, by Robinson et al., 623-653. page, Marcel Dekker, Inc. (1987); Thorpe: "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review", In: Monoclonal Antibodies '84: Biological and Clinical Applications, Pinchera et al., 1985, 475-506; "Analysis, Results, and Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Canver Therapy", In: Monoclonal Antibodies Forum Cancer Detection And Therapy, eds. Baldwin et al., 303-316. page, Academic Press (1985); (1983) Immunological Reviews 62: 119-158.

An antibody, or antigen-binding fragment thereof, that binds to a CD2 polypeptide immunoprecipitatively is conjugated.

183 · - ·% <··· «·« · • * * ·· * · · · · · · · · · · · · · · · · · · · a second antibody to form an antibody heteroconjugate (Segal: 4,676,980)

United States Patent Publication, which is incorporated herein by reference in its entirety, for all purposes.

Antibodies or antigen-binding fragments thereof that bind immunoprecipitatively to a CD2 polypeptide can be bound to a solid carrier that is particularly useful for purifying CD2 + cells and T cells. Such solid carriers include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene.

The present invention relates to LFA-3 peptides, polypeptides, derivatives and analogues thereof that immunoprecipitate specifically to a CD2 polypeptide for use in the prevention, treatment or alleviation of one or more symptoms associated with an autoimmune or inflammatory disorder. Soluble LFA-3 polypeptides that are immunospecifically bound to a CD2 binding molecule preferably have at least 5, at least 10, at least 15, at least 20, at least 25, at least 40, at least 50, at least 60, at least 70, at least 80, at least 80, at least 80, at least 80, LFA-3. It contains 90 groups of at least 100 contiguous amino acids. Soluble LFA-3 peptides, polypeptides, derivatives, and analogues thereof that are immunospecifically binding to a CD2 binding molecule are derived from any species.

184 * »r« · ·· · ·> ♦

The nucleotide and / or amino acid sequences of LFA-3 can be found in the literature or in public databases, or the nucleic acid and / or amino acid sequences can be determined by cloning and sequencing techniques well known to those skilled in the art. For example, the nucleotide and amino acid sequences of human LFA-3 can be found in GenBank databases (see, for example, deposit numbers E12817 and CAA29622).

In a specific embodiment, a soluble LFA-3 polypeptide that binds to a CD2 polypeptide comprises an extracellular domain of naturally occurring LFA-3, or amino acid residues 1-187 of SEQ ID NO: 17. In another embodiment, a soluble LFA-3 polypeptide that binds immune-specific to a CD2 polypeptide comprises the extracellular domain of LFA-3 (e.g., residues 1-92 of SEQ ID NO: 17, amino acid residues 1-85, amino acids 1-75). groups of amino acids 1-70, amino acid residues 1-65, or amino acid residues 1-60).

In a specific embodiment, a soluble LFA-3 polypeptide that is immunospecifically bound to a CD2 polypeptide is about 25%, 30%, 35%, 40%, in an in vitro or in vitro assay known to the person skilled in the art, or in vitro. , 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% - inhibits or decreases the interaction between CD2 polypeptide and LFA3 by 95% or 98%. In another embodiment, one is:

A soluble LFA-3 polypeptide that immunoprecipitatively binds to a CD2 polypeptide does not inhibit the interaction between a CD2 polypeptide and LFA-3 in an in vivo or in vitro assay known to those skilled in the art or known to those skilled in the art. In another embodiment, a soluble LFA-3 polypeptide that binds immune-specific to a CD2 polypeptide is less than 20%, less than 15%, less than 10%, or less than 10%, less than 10% of the interaction between the CD2 polypeptide and LFA-3. less than 5%.

In a specific embodiment, the soluble LFA-3 polypeptide that binds to the CD2 polypeptide is at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% , at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, 95 % or at least 98% inhibits T cell activation in an in vitro or in vitro assay known to those skilled in the art or known to those skilled in the art. In another embodiment, the soluble LFA-3 polypeptide that binds to the CD2 polypeptide is at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% , at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, 95 % or at least 98% inhibits T cell proliferation in an in vivo or in vitro assay known to those skilled in the art or known to those skilled in the art. One

In another embodiment, the soluble LFA-3 polypeptide that binds to the CD2 polypeptide is at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% , at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, 95 % or at least 98% inhibits T cell activation and at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% , 95% or at least 98% inhibits T cell proliferation in an in vitro or in vitro assay known to those skilled in the art or known to those skilled in the art.

In another embodiment, a soluble LFA-3 polypeptide that binds to a CD2 polypeptide does not induce or reduce cytokine expression and / or release in an in vitro or in vitro assay as described herein or known to those skilled in the art. In a specific embodiment, a LFA-3 polypeptide binding to a CD2 binding molecule does not induce cytokines such as IFNγ, interleukin-2 ("IL-2"), interleukin-4 ("IL-4"), interleukin-6. ("IL-6"), interleukin-9 ("IL-9"), interleukin-12 ("IL-12"), and interleukin-15 ("IL-15") in a subject who has a \ t Get a CD2 binding molecule. In alternative embodiments, a CD2 binding molecule induces the expression and / or release of the cytokine kinase in an in vitro or in vitro assay as described herein or known to one skilled in the art. In a specific embodiment, a CD2 binding molecule is a cytokine such as IFNγ, interleukin2, interleukin-7 ("IL-7"), interleukin-9 ("IL-9"), interleukin-10 (" IL-10 ”) and induces an increase in tumor necrosis factor alpha (" TNFα ") concentration in the serum of a subject treated with a CD2 binding molecule. Serum concentrations of cytokines can be measured by any technique known to those skilled in the art, such as, for example, immunoassays, including, for example, ELISA.

In a specific embodiment, a soluble LFA-3 T cell anergic binding to a CD2 polypeptide induces an in vivo or in vitro assay described herein or known to those skilled in the art. In alternative embodiments, a soluble LFA-3 that binds immune-specific to a CD2 polypeptide does not induce T cell anergy in an in vivo or in vitro assay known to the person skilled in the art. In other embodiments, a soluble LFA-3 that binds immune-specific to a CD2 polypeptide induces an antigen-specific non-response or hypovarity state for at least 2 hours, at least 6 hours, for at least 12 hours, at least 24 hours, for at least 2 days, at least For 5 days, at least 7 days, for at least 10 days or more, in an in vitro assay as described herein or known to those skilled in the art.

188

In a specific embodiment, soluble LFA-3 polypeptides that bind immunoprecipitatively to the CD2 polypeptide affect the clearance of peripheral blood T cells by inducing T-cell cytolysis. In another preferred embodiment, soluble LFA-3 polypeptides that bind immunoprecipitatively to the CD2 polypeptide affect the clearance of peripheral blood T cells by at least 25%, at least 30%, or at least 35%, of at least 25%, of T cell proliferation. 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80% with at least 85%, at least 90%, 95%, or at least 98% inhibition, and inducing cytolysis of peripheral blood T cells in an in vitro as described herein or known to one skilled in the art. essay.

The present invention relates to soluble LFA-3 polypeptides that bind immunoprecipitatively to a CD2 polypeptide which has a longer in vivo half-life of the carrier. More specifically, the present invention relates to soluble LFA-3 polypeptides that immunoprecipitate specifically for a CD2 polypeptide and have a half-life in an animal, preferably a mammal, and most preferably a human being greater than 3 days, greater than 7 days, greater than 10 days, preferably greater than 15 days, greater than 25 days, greater than 30 days, greater than 35 days, greater than 40 days, greater than 45 days, greater than 2 months, greater than 3 months, greater than 4 months or greater than 5 months .

189

In order to prolong the in vivo circulation of LFA-3 polypeptides that are immunospecifically binding to the CD2 polypeptide, for example, inert polymer molecules such as high molecular weight polyethylene glycol (PEG) can be coupled to the antibodies via a multifunctional linker, or with the PEG. by site-specific conjugation of the antibody at its N-terminus or C-terminus or through the epilone amino groups on the lysine moieties. Linear or branched polymer derivatization is used which results in minimal loss of biological activity. The extent of conjugation is closely monitored by SDS-PAGE and mass spectroscopy to ensure correct conjugation of PEG molecules to soluble LFA-3 polypeptides. The unreacted PEG can be separated by size exclusion or ion exchange chromatography from the LFA-3 polypeptide PEG conjugates. The LFA-3 polypeptides can be tested for PEG derivatives for binding activity and in vivo efficacy by methods known to those skilled in the art, such as the immunoassay described below.

The present invention relates to soluble LFA-3 peptides and polypeptides that bind immunoprecipitatively to a marker sequence, such as a CD2 polypeptide fused to a peptide, to facilitate purification. In a preferred embodiment, the marker amino acid sequence is a hexa-histidine peptide, such as the one provided by the pQE vector (Quiagen, Inc., 9259).

190 ···· ·· * ··· ·· · «· · · · ··· ·· · · · · · · · · ·

Eton Avenue, Chatsworth, CA, 91311), and many of them are commercially available. According to Gentz et al., Hexa-histidine can be used for convenient purification of the fusion protein (Gentz et al., Proceedings of the National Academy of Sciences, USA 86, 821-824 (1989)). Other peptide labels used for purification include, but are not limited to, haemagglutinin "HA", which corresponds to one of the epitopes derived from influenza hemagglutinin protein (Wilson et al., Cell 37, 767 (1984)).

The present invention further relates to LFA-3 peptides and polypeptides that bind immunoprecipitatively to a CD2 polypeptide conjugated to an agent having a potential therapeutic advantage. A soluble LFA-3 polypeptide that binds immune-specific to the CD2 polypeptide may be conjugated to a therapeutic moiety, such as a cytotoxin, such as a cytostatic or cytocidal agent, an agent with potential advantages, or a radioactive metal ion such as an alpha-emitter. . The cytotoxin or cytotoxic agent may be any agent that is harmful to cells. Cytotoxin or cytotoxic agents may include, but are not limited to, paclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetin, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxythracinone, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol and puromycin;

191 · «·» · 99 · * <

analogues and homologues. Agents with potential therapeutic benefits include, but are not limited to, antimetabolites (such as methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechloretamine). , thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulphan, dibromannite, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DPP, cisplatin), anthracyclines (e.g. daunorubicin ( previously daunomycin) and doxorubicin), antibodies (such as dactinomycin (formerly actinomycin), bleomycin, mithramycin and anthramycin (AMC)), and anti-mitotic agents (such as vincristine and vinblastine).

In addition, a soluble LFA-3 polypeptide that binds immune-specific to the CD2 polypeptide may be conjugated to a therapeutic agent or drug that modifies a particular biological response. Agents with a potential therapeutic advantage, or pharmaceutical agents, should not be limited to classical chemical therapy agents. The drug agent may be, for example, a protein or polypeptide having the desired biological activity. Examples of such proteins include a toxin such as abrin, ricin A, Pseudomonas exotoxin, or diphtheria toxin; a protein, such as tumor necrosis factor, interferon-α ("IFN-α"), interferon-β ("IFN-β"), nerve growth factor ("NGF"), platelet-derived growth factor

192 ("PDGF"), tissue plasminogen activator ("TPA"), an apoptotic agent such as TNF ?, TNF ?, AIM I (WO 97/33899), AIM II (97/34911) , U.S. Pat. No. 4,196,196), Woody Ligand [Takahashi et al., J. of Immunol. 6, 1567-1574 (1994)] and VEGF (see, for example, WO 99/23105), a thrombotic agent or an anti-angiogenesis agent such as angiostatin or endostatin; or a biological response modifier, such as a lymphokine (e.g., interleukin-1 ("IL-1"), interleukin-2, interleukin-6, interleukin-10, granulocyte macrophage colony stimulating factor ("GM-CSF"), and granulocyte colony stimulating growth factor ("G-CSF") or a growth factor (such as growth hormone ("GH")).

The present invention relates to fusion proteins that bind immunoprecipitatively to CD2 polypeptide and affect the activity or function of lymphocytes, preferably peripheral blood T cells, for the prevention, treatment or alleviation of one or more symptoms associated with an autoimmune or inflammatory disorder. course. Preferably, such fusion proteins directly or indirectly affect the depletion of lymphocytes, more specifically, peripheral blood T cells. More specifically, the present invention relates to fusion proteins that bind immunoprecipitatively to a CD2 polypeptide expressed by an immune cell, such as a T cell or an MK cell, and which affects the depletion of lymphocytes, more particularly, peripheral blood T cells.

193

In a specific embodiment, a fusion protein that binds immune-specific to a CD2 polypeptide is in an in vitro or in vitro assay described herein or well known to those skilled in the art, by about 25%, 30%, 35%, 40%, 40%, 40%, 40%, 40%, 40% %, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90 %, 95%, or 98% inhibits or reduces the interaction between a CD2 polypeptide and LFA-3. In an alternative embodiment, a fusion protein that binds immune-specific to a CD2 polypeptide is not inhibited by the interaction between the CD2 polypeptide and LFA-3 in an in vitro or in vitro assay as described herein or well known to those skilled in the art. In another embodiment, a fusion protein that binds immune-specific to a CD2 polypeptide is less than 20%, less than 15%, less than 10% or less than 5% interaction between CD2 polypeptide and LFA-3. % reduction.

In another embodiment, a fusion protein specifically binding to a CD2 polypeptide does not induce or reduce cytokine expression in an in vitro or in vitro assay as described herein or well known to those skilled in the art. In a specific embodiment, a fusion protein specifically binding to a CD2 polypeptide does not induce or increase cytokines such as IFNγ, interleukin-2, interleukin-7 ("IL-7"), interleukin-9 ("IL-9"). ”), Interleukin-10 (“ IL-10 ”), and tumor necrosis factor alpha (“ TNFa ”) kon194 ·· *:

• * * ► Increase in the center of a subject treated with a CD2 binding molecule. In an alternative embodiment, a fusion protein that binds to a CD2 polypeptide induces or increases cytokines such as IFNγ, interleukin-2, interleukin-7 ("IL-7"), interleukin-9 ("IL-9"). ), an increase in the concentration of interleukin-10 ("IL-10") and tumor necrosis factor alpha ("TNFa") in the serum of a subject treated with a CD2 binding molecule. Serum concentrations of cytokines can be measured by any technique known to those skilled in the art, such as, for example, immunoassays, including, for example, ELISA.

In another embodiment, a fusion protein that binds to a CD2 polypeptide induces anergy in an in vivo or in vitro assay known to those skilled in the art or known to those skilled in the art. In an alternative embodiment, a fusion protein specifically binding to a CD2 polypeptide does not induce T cell anergy in an in vivo or in vitro assay known to the person skilled in the art. In another embodiment, a fusion protein specifically binding to a CD2 polypeptide induces an antigen-specific non-response or hypo-responsive state for at least 2 hours, at least 6 hours, for at least 12 hours, at least 24 hours, for at least 2 days, at least 5 for at least 7 days, for at least 10 days or more, in an in vitro assay as described herein or known to those skilled in the art.

195 • • 9.

In a specific embodiment, the CD2 polypeptide immunoprecipitates fusion proteins to affect peripheral blood T cell depletion by at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% %, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% - 95% or at least 98% inhibiting T cell proliferation in an in vitro or in vitro assay known to those skilled in the art or known to those skilled in the art. In a preferred embodiment, the CD2 polypeptide has immunospecific fusion proteins that affect the depletion of peripheral blood T cells by inducing cytolysis of T cells. In another preferred embodiment, the fusion proteins specifically binding to the CD2 polypeptide affect the depletion of peripheral blood T cells by at least 25%, at least 30%, at least 35%, at least 40%, at least 45% - with at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, \ t at least 90%, 95%, or at least 98% inhibiting T cell proliferation and cytolysis of peripheral blood T cells in an in vitro or in vitro assay known to those skilled in the art or known to those skilled in the art.

In another embodiment, the fusion protein specifically binding to the CD2 polypeptide is at least 25%, at least 30%, at least 35%, at least 40%, at least

196

45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85% with at least 90%, 95%, or at least 98% inhibition of T-cell activation and at least 25%, at least 30%, at least 35%, at least 40% , at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, 95%, or at least 98% inhibition of T cell activation in an in vitro or in vitro assay known to the person skilled in the art.

In another embodiment, a fusion protein specifically binding to a CD2 polypeptide binds to an FcR expressed by an immune cell, such as an NK cell, a monocyte, and a macrophage. In a preferred embodiment, a fusion protein that binds to the CD2 polypeptide is bound to an FcγRIII expressed by an immune cell, such as an NK cell, a monocyte, and a macrophage.

In one embodiment, a fusion protein that specifically binds to the CD2 polypeptide comprises a biologically active molecule, an immunoglobulin molecule, or a Fc domain of a fragment thereof, fused. In another embodiment, a fusion protein specifically binding to a CD2 polypeptide comprises a biologically active molecule, a CH2 and / or CH3 region of an Fc domain of an immunoglobulin molecule. In a further embodiment, one, a

197 »· ·· ···· • · • · · ·

A fusion protein specifically binding to a CD2 polypeptide comprises a biologically active molecule, fused to the CH2, CH3 and hinge regions of the Fc domain of an immunoglobulin molecule. Thus, in embodiments, the biologically active molecule binds immune-specific to a CD2 polypeptide. Biologically active molecules that bind immunoprecipitatively to a CD2 polypeptide include, but are not limited to, peptides, polypeptides, small molecules, mimetic agents, synthetic drug agents, inorganic molecules, and organic molecules. Preferably, a biologically active molecule that is immunospecifically bound to a CD2 polypeptide is a polypeptide comprising at least 5, preferably at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 contiguous amino acid residues and is heterologous to the Fc domain of an immunoglobulin molecule or fragment thereof.

In a specific embodiment, a fusion protein specifically binding to a CD2 polypeptide comprises a LFA-3, or a fragment thereof, that binds immune-specific to an Fc domain of an immunoglobulin molecule or a fragment thereof fragmented into a CD2 polypeptide. , the fusion protein specifically binding to the CD2 polypeptide comprises a LFA-3, or a fragment thereof, that binds immunoprecipitatively to a CD2 polypeptide fused to the CH2 and / or CH3 region of an Fc domain of an immunoglobulin molecule or fragment thereof.

198

In another embodiment, a fusion protein specifically binding to the CD2 polypeptide comprises an extracellular domain of LFA-3 (i.e., SEQ ID NO: 17).

Amino acids 1-187), fused to an Fc domain of an immunoglobulin molecule or fragment thereof. In another embodiment, a fusion protein specifically binding to the CD2 polypeptide comprises an extracellular domain of LFA-3 (i.e., amino acids 1-187 of SEQ ID NO: 17) fused to the CH2 and / or CH3 regions of the Fc domain of an immunoglobulin molecule. In another embodiment, a fusion protein specifically binding to a CD2 polypeptide comprises an extracellular domain of LFA-3 (i.e., amino acids 1-187 of SEQ ID NO: 17) fused to the CH2, CH3 and hinge regions of the Fc domain of an immunoglobulin molecule.

In another embodiment, a soluble fusion protein that binds to a CD2 polypeptide comprises the extracellular domain of LFA-3 (e.g., amino acid residues 1-92 of SEQ ID NO: 17, amino acid residues 1-85;

Amino acid residues 1-75, amino acid residues 1-70, amino acid residues 165, or amino acid residues 1-60), fused to an Fc domain of an immunoglobulin molecule or fragment thereof. In another embodiment, a soluble fusion protein that binds to a CD2 polypeptide comprises the extracellular domain of LFA-3 (e.g., 1-92 amino acid residues of SEQ ID NO: 17, amino acid residues 1-85, amino acid residues 1-75; Amino acid residues 1-70, groups 1-65 of amino acids, or amino acid residues 1-60), fusion of an immunoglobulin molecule or fragment thereof with the CH2 and / or CH3 region of the CH2 and / or CH3 regions. In another embodiment, a soluble LFA-3 polypeptide that binds immune-specific to a CD2 polypeptide comprises the extracellular domain of LFA-3 (e.g., residues 1-92 of SEQ ID NO: 17, amino acid residues 1-85;

Amino acid residues 1-75, amino acid residues 1-70, amino acid residues 165, or amino acid residues 1-60), fused to the CH2, CH3 and hinge regions of the Fc domain of an immunoglobulin molecule.

In a specific embodiment, a CD2 binding molecule is LFA-3TIP (Biogen, Inc., Cambridge, MA). In an alternative embodiment, the CD2 binding molecule is not LFA-3TIP.

In another embodiment, a fusion protein that specifically binds to the CD2 polypeptide comprises a polypeptide having an amino acid sequence of at least 35%, at least 40%, at least 45%, at least 50%, at least 55%. , at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% is identical to the amino acid sequence of LFA-3 or a fragment thereof fused to the Fc domain of an immunoglobulin molecule or fragment thereof. In another embodiment, a fusion protein specifically binding to a CD2 polypeptide comprises a polypeptide having an amino acid sequence number of at least 35%, at least 40%, at least 45%, at least 50%, at least 55% - at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence of LFA-3 or a fragment thereof fused to the CH2 and / or CH3 region of the Fc domain of an immunoglobulin molecule or fragment thereof. In another embodiment, a fusion protein specifically binding to a CD2 polypeptide comprises a polypeptide having an amino acid sequence of at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least It is 99% identical to the amino acid sequence of a LFA-3 or a fragment thereof fused to the CH2, CH3 and hinge regions of the Fc domain of an immunoglobulin molecule or fragment thereof.

In another embodiment, a fusion protein that specifically binds to the CD2 polypeptide comprises a polypeptide having an amino acid sequence of at least 35%, at least 40%, at least 45%, at least 50%, at least 55%. , at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least In 99% the amino acid of an extracellular domain of LFA-3 is identical

201 * ··· ···· ·· • · (i.e., amino acid residues 1-187 of SEQ ID NO: 17), fused to the Fc domain of an immunoglobulin molecule or fragment thereof. In another embodiment, a fusion protein that specifically binds to the CD2 polypeptide comprises a polypeptide having an amino acid sequence of at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical to the amino acid sequence of an extracellular domain of LFA-3 (i.e., residues 1-187 of SEQ ID NO: 17), fused to the CH2 and / or CH3 regions of the Fc domain of an immunoglobulin molecule or fragment thereof. In another embodiment, a fusion protein that specifically binds to the CD2 polypeptide comprises a polypeptide having an amino acid sequence of at least 35%, at least 40%, at least 45%, at least 50%, at least 55%. , at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical to the amino acid sequence of an extracellular domain of LFA-3 (i.e., residues 1-187 of SEQ ID NO: 17), fused to the CH2, CH3 and hinge regions of the Fc domain of an immunoglobulin molecule or fragment thereof.

202

In another embodiment, a fusion protein that specifically binds to the CD2 polypeptide comprises a polypeptide having an amino acid sequence of at least 35%, at least 40%, at least 45%, at least 50%, at least 55%. , at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence of an extracellular domain of LFA-3 (e.g., 1-92 amino acid residues of SEQ ID NO: 17, amino acid residues 1-85, amino acid residues 1-75, amino acid residues 1-70) , Amino acid residues 1-65, or amino acid residues 1-60), fused to an Fc domain of an immunoglobulin molecule or a fragment thereof.

In another embodiment, a fusion protein that specifically binds to the CD2 polypeptide comprises a polypeptide having an amino acid sequence of at least 35%, at least 40%, at least 45%, at least 50%, at least 55%. , at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence of an extracellular domain of LFA-3 (e.g., 1-92 amino acid residues of SEQ ID NO: 17, amino acid residues 1-85, amino acid residues 1-75, amino acid residues 1-70) , Amino acid residues 1-65, or amino acid residues 1-60);

Fused to the CH2 and / or CH3 regions of the Fc domain of the immunoglobulin molecule. \ T

In another embodiment, a fusion protein that specifically binds to the CD2 polypeptide comprises a polypeptide having an amino acid sequence of at least 35%, at least 40%, at least 45%, at least 50%, at least 55%. , at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence of an extracellular domain of LFA-3 (e.g., 1-92 amino acid residues of SEQ ID NO: 17, amino acid residues 1-85, amino acid residues 1-75, amino acid residues 1-70) With amino acid residues 1-65 or amino acid residues 1-60) fused to the CH2, CH3 and hinge regions of the Fc domain of an immunoglobulin molecule.

The present invention relates to fusion proteins which bind immunoprecipitatively to a CD2 polypeptide comprising an Fc domain of an immunoglobulin molecule or a fragment thereof, fused to a polypeptide encoded by a nucleic acid molecule that hybridizes to LFA-3 or a thereof. for the nucleic acid sequence encoding the fragment thereof.

The present invention relates to fusion proteins which bind immunoprecipitatively to a CD2 polypeptide comprising an Fc domain of an immunoglobulin molecule or a fragment thereof, fused to a polypeptide that is \ t

204 ···· ···· encodes a nucleic acid molecule that hybridizes under stringent conditions to the nucleic acid sequence encoding LFA-3 or a fragment thereof, i.e., hybridizes to the filter-bound DNA with 6 * sodium chloride / sodium citrate ( SSC) in solution at about 45 ° C followed by one or more washings in a 0.2xSSC / 0.1% SDS composition at about 50-65 ° C under highly stringent conditions, i.e., hybridization to the filter-bound nucleic acid 6xSSC- at about 45 ° C, followed by one or more washings in a solution of 0.1 * SSC / 0.2% SDS at about 68 ° C or under other stringent conditions well known to those skilled in the art [ Current Protocols in Molecular Biology, 6.3.1-6.3.6 and 2.10.3, edited by Ausubel et al., Greene Publishing and Wiley-Interscience: New York (1987)].

The present invention relates to fusion proteins which bind immunoprecipitatively to a CD2 polypeptide comprising an Fc domain of an immunoglobulin molecule or a fragment thereof, fused to a polypeptide encoded by a nucleic acid molecule that hybridizes under stringent conditions to LFA-3. a nucleic acid sequence encoding its extracellular domain (i.e., amino acid residues 1-187 of SEQ ID NO: 17), i.e. hybridizing to the filter-bound DNA in a 6 * sodium chloride / sodium citrate (SSC) solution at about 45 ° C At C, followed by one or more washings in 0.2> SSC / 0.1% SDS at about 50-65 ° C under highly stringent conditions, i.e., hybridization to '' ·· * · ·· J ~ * · · - *. · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·

205 * 1. under conditions well known to those skilled in the art [Current Protocols in Molecular Biology, 6.3.Ιό.3.6 and 2.10.3, ed. Ausubel et al., Greene Publishing and Wiley-Interscience: New York (1987)].

The present invention relates to fusion proteins which bind immunoprecipitatively to a CD2 polypeptide comprising an Fc domain of an immunoglobulin molecule or a fragment thereof, fused to a polypeptide encoded by a nucleic acid molecule that hybridizes under stringent conditions to a LFA-3. extracellular domain (i.e., 1-92 amino acid residues of SEQ ID NO: 17, amino acid residues 1-85, amino acid residues 1-75, amino acid residues 1 to 70, amino acid residues 1-65, or 1 to 65 amino acid residues; 60), i.e., hybridizes to the filter-bound DNA in a 6x sodium chloride / sodium citrate (SSC) solution at about 45 ° C followed by one or more washings of 0.2 * SSC / In a solution of 0.1% SDS at about 50-65 ° C under highly stringent conditions, i.e. hybridization to the filter plate bound nucleic acid in 6xSSC at about 45 ° Con, followed by one or more washings in a solution of 0, 1xSSC / 0.2% SDS at about 68 ° C, or under other stringent conditions well known to those skilled in the art [Current Protocols in Molecular Biology, 6.3. 1206 • ·, '* · · * «· ·

6.3.6 and 2.10.3, edited by Ausubel et al., Greene Publishing and Wiley-Interscience: New York (1987)].

The present invention relates to fusion proteins that bind immunoprecipitatively to a marker sequence, such as a CD2 polypeptide fused to a peptide, to facilitate purification. In preferred embodiments, the marker amino acid sequence is a hexa-histidine peptide, such as the one provided by the pQE vector (Quiagen, Inc., 9259 Eton Avenue, Chatsworth, CA, 91311), and many of these are is in commercial circulation. According to Gentz et al., The hexa-histidine can be used for convenient purification of the fusion protein (Gentz et al., Proceedings of the National Academy of Sciences, USA 86, 821824 (1989)). Other peptide labels used for purification include, but are not limited to, haemagglutinin &quot; HA &quot; corresponding to one epitope derived from influenza hemagglutinin protein (Wilson et al., Cell 37, 767 (1984)), and &quot; flag &quot; ”Marking.

The present invention further relates to fusion proteins which bind immunoprecipitatively to a CD2 polypeptide conjugated to a therapeutic agent. A fusion protein that binds immune-specific to the CD2 polypeptide may be conjugated to a therapeutic unit, such as a cytotoxin, such as a cytostatic or cytocidal agent, an agent that has potential advantages, or a radioactive metal ion, such as an alpha-emitter. Cytotoxin may be a cytotoxic agent

207 · «···« «· ··· 'J. ♦ · any agent that is harmful to cells. Cytotoxin or cytotoxic agents may include, but are not limited to, paclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetin, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxythracinone, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol and puromycin, and analogues and homologs thereof. Agents with potential therapeutic benefits include, but are not limited to, antimetabolites (such as methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechloretamine). , thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulphan, dibromannite, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DPP, cisplatin), anthracyclines (e.g. daunorubicin ( previously daunomycin) and doxorubicin), antibodies (such as dactinomycin (formerly actinomycin), bleomycin, mithramycin and anthramycin (AMC)), and anti-mitotic agents (such as vincristine and vinblastine).

Furthermore, a fusion protein that binds immune-specific to the CD2 polypeptide may be conjugated to a therapeutic agent or drug that modifies a particular biological response. A potential therapeutic benefit

208

agents or pharmaceutical agents may not be limited to classical chemical therapy agents. The drug agent may be, for example, a protein or polypeptide having the desired biological activity. Examples of such proteins include a toxin such as abrin, ricin A, Pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor, interferon-α ("IFN-α"), interferon-β ("IFN-β"), nerve growth factor ("NGF"), platelet-derived growth factor ("PDGF )), Tissue plasminogen activator ("TPA"), an apoptotic agent such as TNFα, ββ, AIM I (WO 97/33899), AIM II (International Patent Publication 97/34911) , Woody Ligand [Takahashi et al., J. of Immunol. 6, 1567-1574 (1994)] and VEGF (see, for example, WO 99/23105), a thrombotic agent or an anti-angiogenesis agent such as angiostatin or endostatin; or a biological response modifier, such as a lymphokine (e.g., interleukin-1 ("IL1"), interleukin-2, interleukin-6, interleukin-10, granulocyte macrophage colony stimulating factor ("GM-CSF"), and granulocyte colony stimulating growth factor ("G-CSF") or a growth factor (such as growth hormone ("GH")).

Any anti-angiogenic agent well known to those skilled in the art may be used in the compositions and methods of the present invention. Non-limiting examples include proteins, polypeptides, peptides, fusion proteins, antibodies (e.g., human, humanized, chimeric, fusion proteins).

209 's monoclonal, polyclonal, Fvs, ScFvs, Fab fragments, F (ab) 2 fragments and antigen-binding fragments thereof), i.e., antibodies specifically binding to TNFα, nucleic acid molecules (e.g., antisense molecules or triple helix), organic molecules, inorganic molecules, and small molecules that reduce, inhibit, or neutralize angiogenesis. More specifically, anti-angiogenesis agents may be, for example, without limitation, endostatin, angiostatin, apomigren, anti-angiogenic antithrombin III, 29 kDa N-terminal fibronectin and 40 kDa C-terminal proteolytic fragments. , an uPa receptor antagonist, a 16 kDa proteolytic fragment of prolactin, a 7.8 kDa proteolytic fragment of platelet factor 4, an anti-angiogenic fragment of platelet factor 24 having 24 amino acids, anti-angiogenesis 13.40 , thrombospondin I anti-angiogenic, 22 amino acid peptide fragment, SPARC 20 amino acid anti-angiogenic peptide fragment, RGD and NGR containing peptides, laminin, fibronectin, procollagan and EGF small anti-angiogenic peptide, integrin α ν β3 antagonists (such as anti-integrin α ν β ?, antibodies), acid fibroblast growth factor (aFGF) antagonists, basic fibroblast growth factor (bFGF) ant agonists, vascular endothelial growth factor (VEGF) antagonists, and vascular endothelial growth factor receptor (VEGFR) antagonists such as anti-VEGFR antibodies).

In a specific embodiment of the present invention, the anti-angiogenic agent is endostatin. The natural range210 »« · »* · <

endostatin contains the C-terminus of collagen XVIII of about 180 amino acids (cDNAs encoding two forms of collagen XVIII fusion, GenBank deposit number AF18081 and AF18082). The angiostatin proteins naturally contain the four kringle domains of plasminogen, from kringle 1 to kringle 4. It has been shown that the recombinant kringle domains 1, 2 and 3 have antiangiogenic properties of the natural peptide, while kringle 4 has no such property [Cao et al., Journal of Biological Chemistry 271, 29461-29467 ( 1996)]. Accordingly, the angiostatin peptides comprise at least one, and preferably more than one kringle domain, from kringle 1, 2, and 3. In a specific embodiment, the anti-angiogenic peptide is the 40 kDa isoform of the human angiostatin molecule, the 42 kDa isoform of the human angiostatin molecule, the 45 kDa isoform of the human angiostatin molecule, or a combination thereof. In another embodiment, an anti-angiogenic agent is kringle domain 5 of plasminogen, which is a more potent inhibitor of angiogenesis than angiostatin (angiostatin contains kringle domains 1-4). In one embodiment of the present invention, antithrombin III is an anti-angiogenic agent. Antithrombin III, hereinafter referred to as antithrombin, contains a heparin binding domain that binds the protein to the wall of the blood vessel and an active site loop that interacts with thrombin. When antithrombin is bound to heparin, the protein undergoes a conformational change that is

211 allows the active loop to interact with thrombin, resulting in proteolytic cleavage of said loop with thrombin. The proteolytic cleavage event also results in another conformational change of antithrombin, which i) alters the interaction interface between thrombin and antithrombin, and ii) liberates the complex from heparin [Carrell: Science 285, 1861-1862 (1999); references cited therein]. O'Reilly et al. Discovered that cleaved antithrombin has potent anti-angiogenic activity. Accordingly, in one embodiment, an anti-angiogenic agent is an anti-angiogenic form of antithrombin. In one embodiment of the present invention, an anti-angiogenic agent is a 40 kDa and / or 29 kDa proteolytic fragment of fibronectin.

In one embodiment of the present invention, the antiangiogenic agent is a urokinase plasminogen activator (uPa) receptor antagonist. In one embodiment of the present invention, the antagonist is the dominant negative mutant of uPa (see, for example, Crowley et al., Proceedings of the National Academy of Sciences, USA 90, 5021-5025 (1993)). In one embodiment of the present invention, the antagonist is a peptide antagonist or a fusion protein thereof (Goodson, et al., Proceedings of the National Academy of Sciences, USA 91: 7129-7133 (1994)). In another embodiment, the antagonist is a dominant negative soluble uPa receptor [Min et al., Cancer Research 56: 2428-2433 (1996)]. In another embodiment of the present invention, the therapeutic molecule of the present invention is a

A 16 kDa N-terminal fragment of prolactin 212 · * · · containing about 120 amino acids or a biologically active fragment thereof (the coding sequence of prolactin can be found in GenBank, accession no. NM_000948). In another embodiment of the present invention, an anti-angiogenic factor is the 7.8 kDa platelet factor fragment 4. In another embodiment of the present invention, the therapeutic molecule of the present invention is a small peptide which is a 13-amino acid anti-angiogenic fragment of platelet factor 4, an anti-angiogenic factor 13.40, a 22-amino acid anti-angiogenic peptide of thrombospondin I , the 20 amino acid anti-angiogenic peptide fragment of SPARC, small anti-angiogenic peptides of laminin, fibronectin, procollagen or EGF, or small peptide antagonists of integrin α ν β3, or the VEGF receptor. In another embodiment, the small rat comprises an RGD or NGR motif. In some embodiments, the anti-angiogenic agent is a TNFα antagonist. In other embodiments, the anti-angiogenic agent is not a TNFα antagonist.

Any TNFα antagonist well known in the art can be used in the compositions and methods of the present invention. TNFα antagonists may be proteins, polypeptides, peptide fusion proteins, antibodies (e.g., human, humanized, chimeric, monoclonal, polyclonal, Fvs, ScFvs, Fab fragments, F (ab) 2 fragments, and antigen-binding fragments thereof), i. Antibodies specifically binding to TNFα, nucleic acid molecules (e. G

213 antisense molecules or triple helix), organic molecules, inorganic molecules, and small molecules that reduce, inhibit, or neutralize the function, activity, and / or expression of TNFα. In various embodiments, a TNFα antagonist has a TNFα function, activity, and / or expression of at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35% , at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% , at least 80%, at least 85%, at least 90%, 95%, or at least 99%, relative to a control such as phosphate buffered saline (PBS).

Antibodies specifically binding to TNFα include, but are not limited to, infliximab (REMICADE ™; Centacor), D2E7 (Abbott Laboratories, Knoll Pharmaceuticals Co., Mt. Olive, NJ), CDP571, which is HUMICADE ™ and Also known as CDP-870 (both Celltech / Pharmacia, Slough, UK), and TN3-19.12 [Williams et al., Proceedings of the National Academy of Sciences, USA 91: 2762-2766 (1994); Thorbecke et al., Proceedings of the National Academy of Sciences, USA 89: 73757379 (1994). The present invention also relates to the use of antibodies specific for immunofluorescence of TNFahos, which antibodies are described in U.S. Patent Nos. 5,136,021; 5,147,638; 5,223,395; 5,231,024; 5,334,380;

5,360,716; 5,426,181; 5,436,154; 5,610,279; 5,644,034;

214

5,656,272; 5,658,746; 5,698,195; 5,736,138; 5,741,488; 5,808,029; 5,919,452; 5,958,412; 5,959,087; 5,968,741; 5,994,510; 6,036,978; 6,114,517; and U.S. Patent Nos. 6,171,787, which are hereby incorporated by reference in their entirety. Soluble TNFα receptors include, but are not limited to, sTNF-R1 (Amgen), etanercept (ENBREL ™; Immunex) and its rat homologue, RENBREL ™, TNFα and TNFrII inhibitors from TNF? Kohno et al., Proceedings of the National Academy of Sciences, USA 87: 83318335 (1990)] and TNFα Inh (Seckinger et al., 1990, Proceedings of the National Academy of Sciences, USA 87, 5188-5192).

In one embodiment, the TNFα antagonist used in the compositions and methods of the present invention is a soluble TNFα receptor. In a specific embodiment, the TNFα antagonist used in the compositions and methods of the present invention is etanercept (ENBREL ™; Immunex), or fragments, derivatives, or analogs thereof. In another embodiment, the TNFα antagonist used in the compositions and methods of the present invention is an antibody that specifically binds to TNFα. The TNFα antagonist used in compositions and methods of a specific embodiment is infliximab (REMICADE ™; Centacor), or fragments, derivatives, or analogs thereof.

215

Other TNFα antagonists of the present invention, without being limited thereto, are interleukin-10, which is known to cross-block TNFα production on interferon-γ-activated macrophages [Oswald et al., Proceedings of the National Academy of Sciences. USA 89: 86768680 (1992); TNFR-IgG (Ashkenazi et al., 1991, Proceedings of the National Academy of Sciences, USA 88, 10535-10539), rodent product TBP-1 (Serono / Yeda), CytoTAB vaccine (Protherics), 104838 antisense molecule (ISIS), RDP-58 peptide (SangStat), thalidomide (Celgene), CDC-801 (Celgene), DPC-333 (Dupont), VX-745 (Vertex), AGIX-4207 ( AtheroGenics), ITF-2357 (Italfarmaco), NPI-13021-31 (Nereus), SCIO-469 (Scios), Tace targeting (Immunix / AHP), CLX-120500 (Calyx), Thiazolopyrim (Dynavax ), auranofin (Ridaura) (SmithKline Beecham Pharmaceuticals), quinacrine (mepacrine dichlorohydrate), tenidap (Enablex), Melanin (Large Scale Biological), and Uriach anti-p38 MAPK.

Nucleic acid molecules encoding proteins, polypeptides or peptides having TNFα antagonist activity, or proteins, polypeptides or peptides having TNFα antagonist activity may be administered to a patient suffering from an autoimmune or inflammatory disorder by the methods of the present invention. In addition, nucleic acid molecules encoding derivatives, analogs, fragments, or variants of proteins, polypeptides or peptides having TNFα antagonist activity, or derivatives, analogs, fragments or variants of white aliquots, polypeptides or peptides having TNFα antagonist activity may be administered by the methods of the present invention by an autoimmune or patient with an inflammatory disorder. Such derivatives, analogs, variants and fragments preferably retain the full-length wild-type protein, polypeptide or peptide TNFα antagonist activity.

Proteins, polypeptides or peptides having TNFα antagonist activity can be prepared by any of the methods well known in the art or by the method described herein. Proteins, polypeptides or peptides with TNFα antagonist activity can be altered by biotechnological methods to increase the in vivo half-life of such proteins, polypeptides or peptides by any of the methods well known in the art or by the method described herein. The compositions and methods of the present invention preferably employ commercially available agents having known TNFα antagonist activity. TNFα antagonist activity of an agent may be determined in vitro and / or by any of the methods well known in the art.

Anti-inflammatory agents have been shown to be successful in the treatment of autoimmune or inflammatory disorders, and are now a common and accepted treatment of such disorders. Any anti-inflammatory agent well known to those skilled in the art may be used in the compositions and methods of the present invention. They may be anti-inflammatory agents without limitation, non-steroidal anti-inflammatory drugs (NSAIDs), anti-steroidal anti-inflammatory drugs, beta-agonists, anticholinergic agents, and anti-inflammatory drugs. methyl xanthines. NSAIDs include, but are not limited to, aspirin, ibuprofen, celecoxib (CELEBREX ™), diclofenac (VOLTAREN ™), etodolac (LODINE ™), feoprofen (NALFON ™), indomethacin (INDOCIN ™), ketoralac (TORADOL ™), oxaprozine (DAYPRO ™), nabumetone (RELAFEN ™), sulindac (CLINORIL ™), tolmentin (TOLECTIN ™) rofecoxib (VIOXX ™), naproxen (ALEVE ™, NAPROSYN ™), ketoprofen (ACTRON ™) and a nabumetone (RELAFEN ™)). Such NSAIDs act by inhibiting the cyclooxygenase enzyme (such as COX-1 and / or COX-2). Examples of steroid anti-inflammatory drugs include, but are not limited to, glucocorticoids, dexamethasone (DECADRON ™), cortisone, hydrocortisone, prednisone (DELTASONE ™), prednisolone, triamcinolone, azulfidine, and eicosanoids such as prostaglandins, thromboxanes, and the like. leukotrienes).

The present invention relates to one or more of a patient methods for the prevention, treatment, management or amelioration of symptoms associated with autoimmune or inflammatory disorder comprising the said methods comprising administering to said subject one or more integrin α ν β3 antagonist and one or more than one prophylactic or therapeutic agent other than the integrin α ν β3 antagonist, which is prophylactic

Or therapeutic agents are currently being used or used or known to be useful for the prevention, treatment or alleviation of one or more symptoms associated with an autoimmune or inflammatory disorder. Non-limiting examples of prophylactic or therapeutic agents that can be used in conjunction with integrin α ν β3 antagonists for the prevention, treatment or alleviation of one or more symptoms associated with an autoimmune or inflammatory disorder are set forth above.

In a specific embodiment, the present invention relates to a method for the prevention, severity, management or alleviation of an autoimmune or inflammatory disorder or one or more of its symptoms, wherein said method comprises administering one or more integrin α to a subject in need of such treatment. ν β3 antagonist and one or more non-integrin α ν β3 antagonist prophylactic or therapeutic agents wherein at least one integrin α ν β3 antagonist is an antibody or fragment thereof that binds immune-specific to the integrin cujU. In a preferred embodiment, the present invention relates to a method for the prevention, severity, management or alleviation of an autoimmune or inflammatory disorder or one or more of its symptoms, characterized in that said method comprises administering one or more integrins to a subject in need of such treatment. α ν β3 antagonist and one or more non-integrin α ν β3 antagonist prophylactic or therapeutic agents, wherein at least one of the in219 tegrin οίνββ antagonist is MEDI-522 humanized monoclonal antibody (commercial name VITAXIN ™) or an antigen-binding agent thereof. fragment.

The methods of the present invention include, but are not limited to, autoimmune disorders that can be prevented, treated, or managed without: blemish bleeding, paralysis of the vertebrae, anphospholipid syndrome, autoimmune Addison's disease, autoimmune diseases of the adrenal gland, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune oophoritis and orchitis, autoimmune thrombocytopenia, Behcet's disease, bullous pemphigoid, cardiomyopathy, abdominal sprue dermatitis, chronic fatigue immune dysfunction symptom (CFIDS), chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, scarred pemphigism, CREST syndrome, cold agglutinin disease, Crohn's disease, lupus lupus, essential mixed cryoglobulinemia, fibromyalgia fibromyositis, glomerulonephritis, Graves's disease, Guillan-Barre, Hashimoto thyroiditis, idiopathic lung fibrosis, idiopathic thrombocytopenia purpurea (ITP), IgA neuropathy , juvenile arthritis, lichen planus, lupus erythematosus, Meniere's disease, mixed connective tissue disease, multiple sclerosis, type 1 or immunological type diabetes, severe muscle weakness, pemphigic vulgar, severe anemia, lumpy polar arthritis, polycondritisism, polyglandular syndromes, polyalgia rheumatic, \ t polymyositis and dermatomyositis, primary agammaglobulinemia, primary epecirrosis, psoriasis, psoriatic arthritis,

220

Raynauld phenomenon, Reiter syndrome, rheumatoid arthritis, sarcoidosis, scleroderma, Sjögren's syndrome, stiffman's syndrome, systemic lupus erythematosus, lupus erythematosus, takayasu arteritis, temporal arterial / giant cell arteritis, ulcerative colitis, uveitis, vasculitis such as dermatitis herpetiformis vasculitis, vitiligo, and Wegener granulomatosis. Inflammatory diseases include, but are not limited to, asthma, encephalitis, inflammatory bowel disease, chronic obstructive pulmonary disease (COPD), allergic disorders, septic shock, pulmonary fibrosis, undifferentiated spondyloarthropathy, undifferentiated arthropathy, arthritis, inflammatory osteolysis, and chronic inflammation. resulting from chronic viral or bacterial infection.

The present invention relates to a method for treating an autoimmune or inflammatory disorder or one or more of its symptoms, wherein said method comprises administering one or more integrin α ν β3 antagonists and one or more immunomodulatory agents to a patient in need of such treatment. are given. Immunomodulatory agents are preferably not administered to subjects with autoimmune or inflammatory disorders whose mean absolute lymphocyte count is less than 500 cells / mm 3 , less than 550 cells / mm 3 , less than 600 cells / mm 3 , less than 650 cells / mm 3 , less than 700 cells / mm 3 , less than 750 cells / mm 3 , less than 800 cells / mm 3 , less than 850 cells / mm 3 , or less than 900 cells / mm 3 . Thus, in a preferred embodiment, before,

221 · * ·· «...,.

...... ...: ·:>

or after administering one or more doses of an immunomodulatory agent to a patient suffering from an autoimmune or inflammatory disorder, the absolute lymphocyte number of said subject is determined by techniques known to those skilled in the art, including, for example, flow cytometry or trypan blue. The immunomodulatory agents mentioned above have been defined without being limited to the methods of the present invention.

In a specific embodiment, the present invention relates to a method for the prevention, severity, management or alleviation of an autoimmune or inflammatory disorder or one or more of its symptoms, characterized in that said method comprises treating a subject in need of such treatment with a prophylactically or therapeutically effective amount. one or more integrin α ν β3 antagonists and a prophylactically or therapeutically effective amount of one or more immunomodulatory agents. In another embodiment, the present invention relates to a method for the prevention, severity, management or alleviation of an autoimmune or inflammatory disorder or one or more of its symptoms, characterized in that said method comprises treating a subject in need of such treatment with a prophylactically or therapeutically effective amount. one or more integrin α ν β3 antagonists and a prophylactically or therapeutically effective amount of one or more immunomodulatory agents, wherein at least one of the integrin α ν β3 antagonists is a counter-agent, or

222 fragments that bind immunoprecipitatively to integrin α ν β3-1ιοζ. In a preferred embodiment, the present invention relates to a method for the prevention, severity, management or alleviation of an autoimmune or inflammatory disorder or one or more symptoms thereof, wherein said method comprises prophylactically or therapeutically effective for a subject in need of such treatment. of one or more integrin α ν β 3 antagonist and administering one or more immunomodulatory agents prophylactically or therapeutically effective amount of wherein the α ν β3 integrin antagonists of at least one of the Vitaxin ™, or antigen-binding fragment. In another preferred embodiment, the present invention relates to a method for the prevention, severity, management or alleviation of an autoimmune or inflammatory disorder or one or more of its symptoms, characterized in that said method comprises the prevention or prophylaxis of a subject in need of such treatment. a therapeutically effective amount of VITAXIN ™ or an antigen-binding fragment thereof is administered, or more immunomodulatory agents are administered prophylactically or in a therapeutically effective amount.

In a specific embodiment, the present invention relates to a method for the prevention, severity, management or alleviation of an autoimmune or inflammatory disorder or one or more of its symptoms, characterized in that said method comprises:

223; : · ♦ · ·.

···. Or a therapeutically effective amount of one or more integrin α ν ββ antagonists and a prophylactically or therapeutically effective amount of methotrexate or cyclosporin is administered. In another embodiment, the present invention relates to a method for the prevention, severity, management or alleviation of an autoimmune or inflammatory disorder or one or more of its symptoms, characterized in that said method comprises treating a subject in need of such treatment with a prophylactically or therapeutically effective amount. VITAXIN ™ and a prophylactically or therapeutically effective amount of methotrexate or cyclosporin is administered. In a specific embodiment, the present invention relates to a method for the prevention, severity, management or alleviation of an autoimmune or inflammatory disorder or one or more of its symptoms, characterized in that said method comprises treating a subject in need of such treatment with a prophylactically or therapeutically effective amount. one or more integrin α ν β3 antagonists, prophylactically or therapeutically effective amounts of methotrexate or prophylactically or therapeutically effective amounts of cyclosporin.

The present invention relates to methods for the prevention, treatment, management or alleviation of one or more symptoms associated with an autoimmune or inflammatory disorder, characterized in that said method comprises administering to said subject one or more integrin α ν β3 antagonists. and one or more CD2 binding molecules (e.g., CD2)

224 polypeptide-like immunospecific binding peptides, polypeptides, proteins, antibodies (MEDI-507) and fusion proteins are administered which directly or indirectly affect the depletion of peripheral blood lymphocytes). Preferably, the CD2 binding molecules are not administered to a subject suffering from an autoimmune or inflammatory disorder whose absolute lymphocyte count is less than 500 cells / mm 3 , less than 550 cells / mm 3 , less than 600 cells / mm 3 , less than 650 cells / mm 3 , less than 700 cells / mm 3 , less than 750 cells / mm 3 , less than 800 cells / mm 3 , less than 850 cells / mm 3 , or less than 900 cells / mm 3 . Thus, in a preferred embodiment, before or after administering to a patient suffering from an autoimmune or inflammatory disorder one or more doses of one or more CD2 binding molecules, the absolute lymphocyte number of said subject is determined by techniques known to those skilled in the art, including, e.g. flow cytometry or trypan blue number.

In a specific embodiment, the percentage of CD2 polypeptides bound by the CD2 binding molecules is estimated after administration of the first dose of one or more CD2 binding molecules to a subject suffering from an autoimmune or inflammatory disorder and prior to administering one or more subsequent doses of one or more CD2 binding molecules. In another embodiment, the percentage of CD2 polypeptides bound by CD2 binding molecules is regularly evaluated (e.g., weekly, every two weeks, every three weeks, every week, every five weeks,

225

every eight weeks or every 12 weeks) after administration of one or more doses of CD2 binding molecules to said subject having an autoimmune or inflammatory disorder. Preferably, a patient with an autoimmune or inflammatory disorder is dosed with the next one or more CD2 molecules when the percentage of CD2 polypeptides bound by CD2 binding molecules is less than 80%, preferably less than 75%, less than 70%, less than 65%. less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25% or less than 20%. The percentage of CD2 polypeptides bound by CD2 binding molecules can be determined in the manner known to those of ordinary skill in the art, or as described herein.

In a specific embodiment, the present invention relates to a method for the prevention, treatment, management or alleviation of an autoimmune or inflammatory disorder, or one or more of its symptoms, characterized in that said method comprises treating a subject in need of such treatment prophylactically or therapeutically. administering one or more integrin α ν β3 antagonists in an amount and administering one or more CD2 binding molecules in a prophylactically or therapeutically effective amount. In another embodiment, the present invention provides a method of preventing, treating, managing or alleviating an autoimmune or inflammatory disorder, or one or more of its symptoms, comprising:

That said method comprises administering to a subject in need of such treatment a prophylactically or therapeutically effective amount of one or more integrin α ν β3 antagonists and administering a prophylactically or therapeutically effective amount of one or more integrin α ν β3 antagonists. a plurality of CD2 binding molecules wherein at least one integrin α ν β3 antagonist is an antibody or fragment thereof that immunoprecipitates the integrin α ν β3 antagonist. In a preferred embodiment, the present invention relates to a method for the prevention, treatment, management or alleviation of an autoimmune or inflammatory disorder, or one or more of its symptoms, characterized in that said method comprises treating a subject in need of such treatment prophylactically or therapeutically. an effective amount of one or more integrin α ν β3 antagonists is administered and a prophylactically or therapeutically effective amount of one or more CD2 binding molecules is administered, wherein at least one integrin α ν β3 antagonist is VITAXIN ™ or an antigen-binding fragment thereof. In another preferred embodiment, the present invention relates to a method for the prevention, treatment, management or alleviation of an autoimmune or inflammatory disorder, or one or more of its symptoms, characterized in that said method comprises the prevention or prophylaxis of a subject in need of such treatment. administering a therapeutically effective amount of VITAXIN ™ or an antigen-binding fragment thereof and administering one or more CD2 binding molecules in a prophylactically or therapeutically effective amount.

227

In another embodiment, the present invention relates to a method for the prevention, treatment, management or alleviation of an autoimmune or inflammatory disorder, or one or more of its symptoms, characterized in that said method comprises treating a subject in need of such treatment prophylactically or therapeutically. administering one or more integrin α ν β3 antagonists in an amount and administering one or more CD2 binding molecules in a prophylactically or therapeutically effective amount, wherein at least one of the CD2 molecules is a soluble LFA-3 polypeptide or LFA3TIP. In another embodiment, the present invention relates to a method for the prevention, treatment, management or alleviation of an autoimmune or inflammatory disorder, or one or more of its symptoms, characterized in that said method comprises treating a subject in need of such treatment prophylactically or therapeutically. administering one or more integrin α ν β3 antagonists in an amount and administering one or more immunomodulatory agents in a prophylactically or therapeutically effective amount, wherein at least one of the CD2 binding molecules is an antibody or fragment thereof that immunospecifically binds to a CD2 polypeptide. In a preferred embodiment, the present invention relates to a method for the prevention, treatment, management or alleviation of an autoimmune or inflammatory disorder, or one or more of its symptoms, characterized in that said method comprises treating a subject in need of such treatment prophylactically or therapeutically. effective amounts are administered

228 one or more integrin α ν β3 antagonists and a prophylactically or therapeutically effective amount of one or more immunomodulatory agents comprising at least one CD2 binding molecule MEDI-507 or an antigen-binding fragment thereof.

In another embodiment, the present invention relates to a method for the prevention, treatment, management or alleviation of an autoimmune or inflammatory disorder, or one or more of its symptoms, characterized in that said method comprises treating a subject in need of such treatment prophylactically or therapeutically. administering one or more integrin α ν β3 antagonists in an amount and administering one or more CD2 binding molecules in a prophylactically or therapeutically effective amount, wherein at least one of the integrin α ν β3 antagonists is an antibody or fragment thereof that immunospecifically binds to integrin <x v p3, and wherein at least one CD2 binding molecule is a soluble LFA-3 polypeptide or LFA3TIP.

In a preferred embodiment, the present invention relates to a method for the prevention, treatment, management or alleviation of an autoimmune or inflammatory disorder, or one or more of its symptoms, characterized in that said method comprises treating a subject in need of such treatment prophylactically or therapeutically. administering one or more integrin α ν β3 antagonists in an effective amount and administering one or more CD2 binding molecules in a prophylactically or therapeutically effective amount, wherein at least one of the integrin α ν β3 antagonists is VITAXIN ™, or an antigen-binding fragment thereof, and

229

At least one of the CD2 binding molecules or an antigen-binding fragment thereof. In another preferred embodiment, the present invention relates to a method for the prevention, treatment, management or alleviation of an autoimmune or inflammatory disorder, or one or more of its symptoms, characterized in that said method comprises the prevention or prophylaxis of a subject in need of such treatment. administering a therapeutically effective amount of VITAXIN ™, or an antigen-binding fragment thereof, and administering one or more CD2 binding molecules in a prophylactically or therapeutically effective amount, and at least one of the CD2 binding molecules or an antigen-binding fragment thereof. In a further preferred embodiment, the present invention relates to a method for the prevention, treatment, management or alleviation of an autoimmune or inflammatory disorder, or one or more of its symptoms, characterized in that said method comprises the prevention or prophylaxis of a subject in need of such treatment. administering a therapeutically effective amount of VITAXIN ™, or an antigen-binding fragment thereof, and administering a prophylactically or therapeutically effective amount of MEDI-507 or an antigen-binding fragment thereof.

The present invention relates to a method for the prevention, treatment, management or alleviation of an autoimmune or inflammatory disorder, or one or more of its symptoms, characterized in that said method comprises prophylactically or therapeutically effective treatment of a subject in need of such treatment.

230 or more of one or more integrin α ν β3 antagonists and one or more TNFα antagonists are administered. Non-limiting examples of TNFα antagonists that can be used in the methods of the present invention are described above.

In a specific embodiment, the present invention relates to a method for the prevention, treatment, management or alleviation of an autoimmune or inflammatory disorder, or one or more of its symptoms, characterized in that said method comprises treating a subject in need of such treatment prophylactically or therapeutically. administering one or more integrin α ν β3 antagonists in an amount and administering one or more TNFα antagonists in a prophylactically or therapeutically effective amount. In another embodiment, the present invention relates to a method for the prevention, treatment, management or alleviation of an autoimmune or inflammatory disorder, or one or more of its symptoms, characterized in that said method comprises treating a subject in need of such treatment prophylactically or therapeutically. administering one or more integrin α ν β3 antagonists in an amount and administering one or more TNFα antagonists in a prophylactically or therapeutically effective amount, wherein at least one of the integrin α ν ββ antagonists is an antibody or fragment thereof that binds immune-specific to integrin α ν β3-1ιοζ.

In a preferred embodiment, the present invention relates to a method for preventing an autoimmune or inflammatory disorder or one or more symptoms thereof,

Characterized in that said method comprises administering to a subject in need of such treatment a prophylactically or therapeutically effective amount of one or more integrin α ν β3 antagonists and administering it in a prophylactically or therapeutically effective amount to one or more of said compounds. TNFα antagonist, wherein at least one of the integrin α ν β 3 antagonists is VITAXIN ™ or an antigen-binding fragment thereof. In another preferred embodiment, the present invention relates to a method for the prevention, treatment, management or alleviation of an autoimmune or inflammatory disorder, or one or more of its symptoms, characterized in that said method comprises the prevention or prophylaxis of a subject in need of such treatment. administering a therapeutically effective amount of VITAXIN ™ or an antigen-binding fragment thereof and administering one or more TNFα antagonists in a prophylactically or therapeutically effective amount.

In another embodiment, the present invention relates to a method for the prevention, treatment, management or alleviation of one or more symptoms associated with an autoimmune or inflammatory disorder in a subject, wherein said method comprises prophylactically or therapeutically treating said subject. administering one or more integrin α ν β 3 antagonists in an effective amount and administering one or more TNFα antagonists in a prophylactically or therapeutically effective amount, wherein at least one of the TNFα antagonists is a soluble TNFα receptor, e.g.

232 etanercept (ENBREL ™; Immunex), or fragments, derivatives or analogs thereof, or an antibody that specifically binds to TNFα, such as infliximab (REMICADE ™; Centacor), or a derivative, analogue, or antigen-binding fragment thereof. .

In another embodiment, the present invention relates to a method for the prevention, treatment, management, or amelioration of one or more symptoms associated with an autoimmune or inflammatory disorder in a subject, said method comprising administering said subject to a prophylactic or therapeutic route. administering one or more integrin α ν β3 antagonists in an effective amount and administering one or more TNFα antagonists in a prophylactically or therapeutically effective amount, wherein at least one of the integrin α ν β3 antagonists is an antibody, or a fragment thereof, and at least one of the TNFα antagonists; one is a soluble TNFa receptor, such as etanercept (ENBREL ™; Immunex), or a fragment, derivative or analog thereof, or an antibody that specifically binds to TNFα, such as infliximab (REMICADE ™; Centacor), or its derivative, anal an antigen or antigen-binding fragment thereof.

In another embodiment, the present invention provides a method of preventing, treating, managing, or ameliorating one or more symptoms associated with an autoimmune or inflammatory disorder in a subject, said method comprising the steps of:

233 subjects are administered prophylactically or therapeutically effective amounts of one or more integrin α ν β3 antagonists and are administered prophylactically or therapeutically effective amounts of one or more TNFα antagonists, wherein at least one of the integrin α ν β3 antagonists is VITAXIN ™ or a fragment thereof, and at least one of the TNFα antagonists is a soluble TNFα receptor, such as etanercept (ENBREL ™; Immunex), or a fragment, derivative or analog thereof, or an antibody that binds immune-specific to TNFα, such as infliximab (REMICADE ™). ; Centacor), or a derivative, analogue, or antigen-binding fragment thereof.

The present invention relates to a method for the prevention, treatment, management or amelioration of one or more symptoms associated with an autoimmune or inflammatory disorder in a subject, said method comprising administering to said subject a prophylactically or therapeutically effective amount of a subject. or more integrin α ν β3 antagonists and one or more anti-inflammatory agents. In the foregoing, non-limiting examples of anti-inflammatory agents are provided which are useful in the methods of the present invention.

In another embodiment, the present invention provides a method of preventing, treating, managing, or ameliorating one or more symptoms associated with an autoimmune or inflammatory disorder in a subject, said method comprising the steps of said * · »· ·

234 • · • ·· '·· · One or more integrin α ν β3 antagonists are administered prophylactically or therapeutically to a subject in a prophylactically or therapeutically effective amount and one or more anti-inflammatory agents are administered prophylactically or therapeutically, wherein at least one of the integrin α ν β3 antagonists is administered. one is VITAXIN ™ or a fragment thereof.

In another embodiment, the present invention relates to a method for the prevention, treatment, management or alleviation of one or more symptoms associated with an autoimmune or inflammatory disorder in a subject, wherein said method comprises prophylactically or therapeutically treating said subject. administering an effective amount of VITAXIN ™ or an antigen-binding fragment thereof and administering a prophylactically or therapeutically effective amount of one or more anti-inflammatory agents.

The present invention relates to a method for the prevention, treatment, management or amelioration of one or more symptoms associated with an autoimmune or inflammatory disorder in a subject, said method comprising administering to said subject a prophylactically or therapeutically effective amount of a subject. or multiple integrin α ν ββ antagonists, one or more TNFα antagonists and one or more immunomodulatory agents. In a specific embodiment, the present invention relates to a method for the prevention, treatment, management or alleviation of one or more symptoms associated with an autoimmune or inflammatory disorder in a subject, said method comprising:

235 ** ·· ···· <«·« • · · ♦ ·· ♦ · *. · · · <

that said subject is administered a prophylactically or therapeutically effective amount of VITAXIN ™, a prophylactically or therapeutically effective amount of a soluble TNFα receptor (such as etanercept) is administered and methotrexate is administered in a prophylactically or therapeutically effective amount. In another embodiment, the present invention relates to a method for the prevention, treatment, management or alleviation of one or more symptoms associated with an autoimmune or inflammatory disorder in a subject, wherein said method comprises prophylactically or therapeutically treating said subject. an effective amount of VITAXIN ™ is administered in a prophylactically or therapeutically effective amount of an antibody specifically binding to TNFα (such as infliximab or an antigen-binding fragment thereof) and methotrexate is administered in a prophylactically or therapeutically effective amount.

In another embodiment, the present invention relates to a method for the prevention, treatment, management or alleviation of one or more symptoms associated with an autoimmune or inflammatory disorder in a subject, wherein said method comprises prophylactically or therapeutically treating said subject. administering an effective amount of one or more integrin α ν β3 antagonists, one or more TNFα antagonists, and one or more CD2 binding molecules. In a specific embodiment, the present invention relates to a method for the prevention, treatment, management or alleviation of one or more symptoms associated with an autoimmune or inflammatory disorder in a subject, said method comprising: by administering to said subject a prophylactically or therapeutically effective amount of VITAXIN ™, a prophylactically or therapeutically effective amount of a TNFα receptor (e. g., etanercept) is administered prophylactically or in a therapeutically effective amount to either MEDI-507 or antigenic binding fragment. In another embodiment, the present invention relates to a method for the prevention, treatment, management or alleviation of one or more symptoms associated with an autoimmune or inflammatory disorder in a subject, wherein said method comprises prophylactically or therapeutically treating said subject. administering an effective amount of VITAXIN ™, prophylactically or therapeutically effective amounts of an antibody that binds to TNFα immune-specific (e.g., infliximab or antigen-binding fragment thereof) and is administered in a prophylactically or therapeutically effective amount. or an antigen-binding fragment thereof.

The present invention relates to a method for the prevention, treatment, management or alleviation of one or more symptoms associated with an autoimmune or inflammatory disorder in a subject, said method comprising administering to said subject one or more integrin α ν β3 antagonists. , one or more TNFα antagonists and one or more anti-inflammatory agents are administered. A specific embodiment «·· V

237 • · · · ··· »« · · · · · ··· W * · «* 4« • »

According to the present invention, there is provided a method of preventing, treating, managing or ameliorating one or more symptoms associated with an autoimmune or inflammatory disorder in a subject, said method comprising administering to said subject a prophylactically or therapeutically effective amount. VITAXIN ™ is administered as a prophylactically or therapeutically effective amount of a soluble TNFα receptor (such as etanercept) and is administered prophylactically or therapeutically in an effective amount of a steroid or non-steroidal anti-inflammatory drug. In another embodiment, the present invention relates to a method for the prevention, treatment, management or alleviation of one or more symptoms associated with an autoimmune or inflammatory disorder in a subject, wherein said method comprises prophylactically or therapeutically treating said subject. adding an effective amount of VITAXIN ™, administering prophylactically or therapeutically effective amounts of an antibody that specifically binds to TNF? drug active ingredient.

The present invention relates to a method for the prevention, treatment, management or alleviation of one or more symptoms associated with an autoimmune or inflammatory disorder in a subject, wherein said method comprises administering to said subject one or more integrin α ν β3 antago238 nistate, one or more TNFα antagonists, one or more immunomodulatory agents and one or more anti-inflammatory agents. In a specific embodiment, the present invention relates to a method for the prevention, treatment, management or alleviation of one or more symptoms associated with an autoimmune or inflammatory disorder in a subject, said method comprising: prophylactically or therapeutically treating said subject. by administering an effective amount of VITAXIN ™, a prophylactically or therapeutically effective amount of a soluble TNFα receptor (such as etanercept) or an antibody specifically binding to TNFα (e.g., infliximab or an antigen-binding fragment thereof) and prophylactically or therapeutically. administering an effective amount of methotrexate and administering a prophylactically or therapeutically effective amount of a steroid or non-steroidal anti-inflammatory drug. In another embodiment, the present invention relates to a method of preventing, treating, managing or ameliorating one or more symptoms associated with an autoimmune or inflammatory disorder in a subject, said method comprising: prophylactically or therapeutically treating said subject; by administering an effective amount of VITAXIN ™, a prophylactically or therapeutically effective amount of a soluble TNFα receptor (such as etanercept) or an antibody specifically binding to TNFα (e.g., infliximab or an antigen-binding fragment thereof), prophylactically or therapeutically.

an effective amount of a CD2 binding molecule (such as MEDI-507 or an antigen-binding fragment thereof) is administered in a dummy amount and a steroid or non-steroidal anti-inflammatory drug is administered prophylactically or therapeutically.

The present invention relates to a method for the prevention, treatment, management or alleviation of one or more symptoms associated with an autoimmune or inflammatory disorder in a subject, said method comprising administering to said subject one or more integrin α ν β3 antagonists. and administering one or more nucleic acid molecules encoding a prophylactic or therapeutic agent other than one or more integrin α ν β3 antagonists. The present invention also relates to a method for the prevention, treatment, management or alleviation of one or more symptoms associated with an autoimmune or inflammatory disorder in a subject, said method comprising the step of administering to said subject one or more of one or more of said symptoms. a nucleic acid molecule encoding integrin α ν β3 and one or more nucleic acid molecules encoding a prophylactic or therapeutic agent other than one or more integrin α ν β3 antagonists. . The present invention also relates to a method for the prevention, treatment, management or alleviation of one or more symptoms associated with an autoimmune or inflammatory disorder in a subject, said method comprising the step of administering to said subject one or more of one or more of said symptoms. integrin α ν β3 antagonist

240 • · »| A nucleic acid molecule encoding a nucleic acid molecule and one or more nucleic acid molecules that are prophylactic or therapeutic agents other than one or more of the integrin α ν β3 antagonists; encoded.

The methods of the present invention are particularly useful for the prevention or prevention of rheumatoid arthritis, spondyloarthropathies (e.g., psoriatic arthritis, paralytic vertebrae, Reiter's syndrome (also known as reactive arthritis), arthritis associated with inflammatory bowel disease, and undifferentiated spondyloarthropathy). treatment. The compositions and methods of the present invention may also be used for the prevention, treatment, management or alleviation of one or more symptoms associated with inflammatory osteolysis, other disorders characterized by abnormal bone reabsorption, or bone loss (e.g., osteoporosis).

In another preferred embodiment, the compositions and methods described herein are used to prevent, treat, manage or alleviate one or more symptoms associated with rheumatoid arthritis, psoriasis, or psoriatic arthritis. In another preferred embodiment, the compositions and methods described herein are used for the prevention, treatment, management or alleviation of one or more symptoms associated with psoriasis or psoriatic arthritis. In another preferred embodiment, the compositions and methods described herein are described in one or more of the claims. reu241 is used to prevent, treat, manage or alleviate symptoms of osteoporosis associated with other arthritis, psoriasis, psoriatic arthritis and juvenile chronic arthritis.

The present invention relates to a method for the prevention, treatment, management or alleviation of one or more symptoms associated with an autoimmune or inflammatory disorder in a subject, said method comprising administering to said subject one or more integrin α ν β3 antagonists. or a pharmaceutical composition comprising one or more integrin α ν β3 antagonists. The present invention further relates to a method for the prevention, treatment, management or alleviation of one or more symptoms associated with an autoimmune or inflammatory disorder in a subject who is resistant to a single agent treatment of such therapies, characterized in that said method comprises: that one or more integrin α ν β3 antagonists and one or more prophylactic or therapeutic agents other than integrin α ν β3 antagonists are administered to said subject. The present invention also relates to a method for the prevention, treatment, management or alleviation of one or more symptoms associated with an autoimmune or inflammatory disorder in a subject, which is resistant to a single agent treatment of such therapies, characterized in that said method comprises: said subject having one or more integrin α ν β3 antagonists containing 242 and one or more integrin a prophylactic or therapeutic agents other than v? 3 antagonists.

The invention also relates to a method of treating an autoimmune or inflammatory disorder in a subject treated with methotrexate and a TNFα antagonist. These include those with active persistent disease (ie, resistant patients) and those with mild disease despite being treated with methotrexate and a TNFα antagonist. The present invention further provides a method of preventing the re-proliferation of one or more symptoms of an autoimmune or inflammatory disorder, comprising administering to a patient treated with methotrexate or a TNFα antagonist (such as REMICADE ™ or ENBREL ™) an integrin α ν β3 antagonist. and do not show abnormal activity.

The invention also relates to a method of treating an autoimmune or inflammatory disorder comprising administering an integrin α ν β3 antagonist to subjects taking methotrexate and not receiving a TNFα antagonist. Among these subjects are those who do not show abnormal activity who suffer from a constant illness and suffer from mild pathological activity. Among these subjects are subjects who are simultaneously treated with other prophylactic or therapeutic agents, but not with a TNFα antagonist. These subjects include those treated with methotrexate alone.

243 • · «· ·« β · · · · · · ··· ·· · · ··· ·· · * ·

The invention also relates to a method of treating an autoimmune or inflammatory disorder, comprising administering an integrin α ν β 3 antagonist to subjects treated with a prophylactic or therapeutic agent other than methotrexate. These subjects include those treated with a TNFα antagonist (such as REMICADE ™ or ENBREL ™) and those who are not treated with a TNFα antagonist but with some other prophylactic or therapeutic agent.

The present invention relates to a method of preventing the occurrence of an autoimmune or inflammatory disorder or one or more symptoms associated with an autoimmune or inflammatory disorder in a subject susceptible to said disorder, characterized in that said method comprises one or more of the following: integrin α ν β 3 antagonist, and one or more different integrin α ν β3 antagonist prophylactic or therapeutic agent is administered. In a specific embodiment, the present invention relates to a method for preventing the occurrence of rheumatoid arthritis, psoriatic arthritis or psoriasis, or to preventing one or more symptoms thereof in a subject susceptible to such disorder, characterized in that said method comprises one or more integrins. An α ν β3 antagonist and one or more prophylactic or therapeutic agents other than the integrin α ν β3 antagonist are administered.

The present invention relates to a method for the occurrence of an autoimmune or inflammatory disorder, or one or more of one

To prevent a symptom associated with an autoimmune or inflammatory disorder in a subject susceptible to said disorder, characterized in that said method comprises administering one or more integrin α ν β3 to said subject. a composition comprising an antagonist and one or more prophylactic or therapeutic agents other than the integrin α ν β3 antagonist. In a specific embodiment, the present invention relates to a method for preventing rheumatoid arthritis, psoriatic arthritis or psoriasis, or preventing one or more symptoms thereof, in a subject susceptible to such disorder, characterized in that said method comprises: a composition comprising one or more integrin α ν β3 antagonists and one or more prophylactic or therapeutic agents other than the integrin α ν β3 antagonist.

The present invention relates to methods for treating, preventing and alleviating one or more symptoms associated with an autoimmune or inflammatory disorder. In a specific embodiment, a composition comprises one or more integrin α ν β3 antagonists. In another embodiment, the composition comprises one or more nucleic acid molecules comprising one or more integrin α ν β3 antagonists. In another embodiment, a composition comprises one or more integrin α ν β3 antagonists and one or more of the integrin α ν β3 antagonists has a prophylactic or therapeutic agent which is a prophylactic or therapeutic agent.

245 is known to be useful, or used, or is currently used to prevent, treat or alleviate one or more symptoms associated with an autoimmune or inflammatory disorder. In another embodiment, a composition comprises one or more nucleic acid molecules encoding one or more integrin α ν β3 antagonists, and comprises one or more prophylactic or therapeutic agents other than the integrin α ν β3 antagonist known for prophylactic or therapeutic agents. are useful, or have been used, or are currently being used to prevent, treat or alleviate one or more symptoms associated with an autoimmune or inflammatory disorder. In another embodiment, a composition comprises one or more integrin α ν β3 antagonists and one or more nucleic acid molecules encoding a prophylactic or therapeutic agent other than an integrin α ν β3 antagonist, known for prophylactic or therapeutic agents, that are useful or useful; has been or is being used to prevent, treat or alleviate one or more symptoms associated with an autoimmune or inflammatory disorder. In another embodiment, a composition comprises one or more nucleic acid molecules encoding one or more integrin α ν β3 antagonists, and a nucleic acid molecule encoding one or more other than one or more integrin α ν β3 antagonists, which is prophylactic or therapeutic. agents are known to be useful, used, or are currently being used in one or more autoimmune or inflammatory conditions.

246 to prevent, treat or alleviate a symptom associated with an abnormality.

In a specific embodiment, a composition comprises one or more integrin α ν β3 antagonists and one or more immunomodulatory agents. In another embodiment, the composition comprises VITAXIN ™ and one or more immunomodulatory agents. In another embodiment, a composition comprises one or more integrin α ν β3 antagonists and one or more CD2 antagonists. In another embodiment, a composition comprises one or more integrin α ν β3 antagonists and one or more CD2 binding molecules. In yet another embodiment, the composition comprises VITAXIN ™ or an antigen-binding fragment thereof and comprises one or more CD2 binding molecules. In a preferred embodiment, the composition comprises VITAXIN ™, or an antigen-binding fragment thereof, and MEDI-507, or an antigen-binding fragment thereof.

In a specific embodiment, a composition is one or more integrin α ν β3 antagonists and one or more antiangiogenic agents. In another embodiment, a composition comprises VITAX1N ™, or an antigen-binding fragment thereof, and one or more anti-angiogenic agents.

In a specific embodiment, a composition comprises one or more integrin α ν β3 antagonists and one or more TNFα antagonists. In another embodiment, a composition comprises VITAXIN ™ or an antigen-binding fragment thereof, and one or more TNFα antagonists. In a preferred embodiment, the composition comprises VITAXIN ™ or an antigen-binding fragment thereof, and a soluble TNFα receptor (e.g., etanercept), or an antibody that binds immune-specific to TNFα.

In a specific embodiment, a composition comprises one or more integrin α ν β3 antagonists and one or more anti-inflammatory agents. In another embodiment, the composition comprises VITAXIN ™ or an antigen-binding fragment thereof and one or more anti-inflammatory agents. In a preferred embodiment, the composition comprises VITAXIN ™ or an antigen-binding fragment thereof, and a steroid or non-steroidal anti-inflammatory drug.

In one embodiment, the composition comprises one or more integrin α ν β3 antagonists, one or more immunomodulatory agents, and one or more TNFα antagonists. In another embodiment, the composition comprises one or more integrin α ν β3 antagonists, one or more CD2 binding molecules, and one or more TNFα antagonists. In another embodiment, a composition comprises one or more integrin α ν β3 antagonists, one or more anti-inflammatory agents, and one or more TNFα antagonists. Thus, according to embodiments, integrin α ν β3 antagonists have at least one VITAXIN ™ or antigen-binding fragment thereof.

In a preferred embodiment, the composition of the present invention is a pharmaceutical composition. Such

Compositions include a prophylactically or therapeutically effective amount of one or more prophylactic or therapeutic agents (e.g., an integrin α ν β3 antagonist or other prophylactic or therapeutic agent) as well as a pharmaceutically acceptable carrier. In a specific embodiment, the term "pharmaceutically acceptable" means that it has been adopted by a federal or state government agency, or by a state agency in the US Pharmacopoeia, or by another agency listed in a generally recognized pharmacopoeia for use in animals, or more specifically, in humans. The term "carrier" refers to a diluent, an adjuvant (e.g., Freund's adjuvant (complete or incomplete)), a filler, or a carrier to administer the active ingredient. Such pharmaceutical carriers may include sterile liquids such as water and oils, including mineral oil, animal oil, vegetable oil, or synthetic oil derivatives such as peanut oil, soybean oil, mineral oil, sesame oil and the like. The preferred carrier is water when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose as well as glycerol solutions can also be used as liquid carriers, especially for injectable solutions. Suitable pharmaceutical fillers include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride , dried skimmed milk, glycerol, propylene, glycol, water, ethanol and the like. The composition may, if necessary, contain mi249 native wetting or emulsifying agents, or pH buffering agents. The composition may be in the form of a solution, suspension, emulsion, tablet, pills, capsules, powders, controlled release formulations, and the like. The oral composition may contain standard carriers such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, saccharin sodium salt, cellulose, magnesium carbonate, and the like. Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" (EW Martin). Such compositions comprise a prophylactically or therapeutically effective amount of a prophylactic or therapeutic agent, preferably in a purified form, with an appropriate amount of carrier to provide the form for proper administration to the patient. The formulation should be adapted to the route of administration. In a preferred embodiment, the pharmaceutical compositions are sterile and must be in a suitable form for administration to a subject, preferably an animal, more preferably a mammal, and most preferably a human.

In a specific embodiment, it may be desirable for the pharmaceutical composition of the present invention to be administered topically to the area in need of treatment. This can be achieved, for example, without limiting it, by local infusion, by injection, by means of an implanted device, which implant is a porous, non-porous or gelatinous material, including membranes, such as sialistic membranes or yarns. If one or more prophylactic or therapeutic

250 agents are administered, it is preferable to use substances that are not adsorbed by prophylactic or therapeutic agents.

In another embodiment, the composition may be administered in a vesicle, more specifically, in a liposome [Langer, Science 249, 1527-1533 (1990); Treat et al.

Liposomes in Therapy of Infectious Disease and Cancer, 353365, ed., Lopez-Berestein and Fidler, Liss, New York (1989); Lopez-Berestein, ibid, 317-327].

In another embodiment, the composition may be administered in a controlled release or sustained release system. In one embodiment, a pump may be used to obtain controlled or sustained release [Langer, Science 249, 1527-1533 (1990); Sefton, CRC Crit. Ref. Biomed. Eng. 14, 20 (1987); Buchwald et al., Surgery 88, 507 (1980); Saudek et al., The New England Journal of Medicine 321, 574 (1989)]. In another embodiment, the polymeric material may be used to provide controlled or continuous release of the antibodies or fragments thereof of the present invention (Medical Applications of Controlled Release, Langer and Wise, CRC Press, Boca Raton, Florida (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Vol. Smolen and Ball, Wiley, New York (1984); Ranger and Peppas: J. Macromol. Sci. Macromol. Chem. 1983, 23, 61; Levy et al., Science 228: 190 (1985); During, et al., Ann. Neurol. 25, 351 (1989); Howard and

251, J. Neurosurg. 71: 105 (1989); 5,679,377; 5,916,597; 5, 912,015; 5,989,463; U.S. Patent No. 5,128,326

United States Patent Specification; PCT Publication No. WO 99/15154 and WO 99/20253]. The sustained release formulations include, but are not limited to, poly (2-hydroxyethyl methacrylate), poly (methyl methacrylate), polyacrylic acid, poly (ethylene-co-vinyl acetate), poly (methacrylic acid), pliglycolides (PLG), polyanhydrides, poly (N-vinyl pyrrolidone), polyvinyl alcohol, polyacrylamide, polyethylene glycol, polylactides, poly (lactide-co-glycolides) (PLGA), and polyorthoesters. In a preferred embodiment, the polymer used in a sustained release formulation is inclined, free of soluble impurities, stable, sterile and biodegradable during storage. In another embodiment, a controlled or sustained release system can be placed in the vicinity of a therapeutic target, such as the lungs, so that only a fraction of the systemic dose is required (see, e.g., Goodson, Medical Applications of Controlled Release, 2, 115-138, eds. Langer and Wise, CRC Press, Boca Raton, Florida (1984)].

Controlled release formulations were summarized by Langer in a review [Langer, Science 249, 1527-1533 (1990)]. Any technique known to those skilled in the art may be used to prepare controlled release formulations comprising one or more antibodies of the present invention or fragments thereof (see, e.g., U.S. Patent No. 4,526,938).

252 ·· «description; PCT Publication WO 91/05548 and WO 96/20698; Ning et al., Intratumoral Radioimmunotherapy of the Human Colon Cancer Xenograft Using the Sustained-Release Gel, Radiotherapy & Oncology 39, 179-189 (1996); Song et al.

, Antibody Mediated Lung Targeting of Long-Circulating Emulsions, PDA Journal of Pharmaceutical Science & Technology 50, 372-397 (1995); Cleek et al., "Biodegradable Polymeric Carriers Forum for bFGF Antibody Forum Cardiovascular Application", Pro. Int'l Symp. Control Rel. Bioact. Mater. 24: 853-854 (1997); Lám et al., Microencapsulation of Recombinant Humanized

Monoclonal Antibody Forum Local Deliveiy, Pro. Int'l Symp. Control Rel. Bioact. Mater 759-760 (1997); which publications will be treated as reference in their entirety).

In a specific embodiment, where one or more nucleic acid molecules encoding one or more prophylactic or therapeutic agents of the present invention are administered, the nucleic acids may be administered in vivo to promote the expression of the prophylactic or therapeutic agents encoded by them, as part of an appropriate nucleic acid expression vector. and then administering it to be intracellular, for example using a retroviral vector (see, e.g., U.S. Patent No. 4,980,286), or by direct injection or microparticle bombardment (e.g., a gene gun; Biolistic, Dupont), or coated with lipids or cell surface receptors, or with transfecting agents, or coupled to a homeobox-like peptide known in the art;

253 ··· · · «·· ·· ···· ♦ ·. To enter the nucleus [see, for example, Joliét et al., Proceedings of the National Academy of Sciences, USA, 88: 1864-1868 (1991)]; etc Alternatively, a nucleic acid may be introduced intracellularly and incorporated into the host cell DNA by homologous recombination.

A pharmaceutical composition of the present invention should be formulated to be compatible with the intended mode of administration. Methods of administration include, but are not limited to, parenteral, i.e., intravenous, intradermal, subcutaneous, oral (e.g., by inhalation), intranasal, transdermal (topical), transmucosal, and rectal. In a specific embodiment, the composition is formulated according to routine methods, for example, for intravenous, subcutaneous, intramuscular, oral, intranasal or topical administration to humans. In a preferred embodiment, a pharmaceutical composition is formulated by routine methods for subcutaneous administration to humans. Formulations for intravenous administration are typically solutions in sterile isotonic buffer. If necessary, the composition may further comprise a solubilizing agent and a local anesthetic such as lignocam to relieve pain at the injection site.

If the compositions of the present invention are to be administered topically, the compositions may be formulated as ointment, cream, transdermal patch, lotion, gel, shampoo, spray, aerosol, solution, emulsion, or a person skilled in the art.

Well known in other forms (see, for example, Remington's Pharmaceutical Sciences and Introductionto Pharmaceutical Dosage Forms, 4th Ed., Lea & Febiger, Philadelphia, PA (1985)]. For non-sprayable topical topical dosage forms, the viscous viscosity is typically used in the form of solid forms containing a carrier or one or more fillers compatible with the topical route of administration and having a dynamic viscosity greater than that of water. The method includes formulations without being limited to solutions, suspensions, emulsions, creams, ointments, powders, lubricants, balms, and the like, if necessary sterilized or supplemented agents (such as preservatives, stabilizers, wetting agents, buffers, or salts) are mixed to influence the different properties, such as osmotic pressure. Other suitable topical dosage forms may be a sprayable aerosol formulation in which the active ingredient, preferably in combination with a solid or liquid, excipient carrier, is packaged in combination with a pressurizing volatile material (e.g., gaseous propellant such as freon) or into a pressure bottle. Wetting agents or soaking agents may also be added to pharmaceutical compositions and dosage forms if necessary. Such additional additives are well known in the art.

If the compositions of the present invention are to be administered intranasally, the compositions may be formulated as an aerosol,

255

Spray, smoke, or droplets. More specifically, prophylactic or therapeutic agents may be conveniently delivered for use in the present invention in the form of an aerosol spray, a pressurized package, or a nebulizer. using a suitable propellant such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. For a pressurized aerosol, the unit dose can be determined by using a valve that emits a measured amount. Capsules and cartridges, such as gelatin, may be formulated for use in an inhaler or insufflator containing a mixture of the compound and a suitable powder base such as lactose or starch.

When the compositions of the present invention are to be administered orally, the compositions should be formulated accordingly, e.g. in the form of tablets, capsules, cachets, gel capsules, solutions, suspensions, and the like. Tablets or capsules may be prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pre-gelatinized corn starch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or CaHPO4); lubricants (e.g., magnesium stearate, talc or silicate); disintegrating agents (e.g., potato starch or starch glycolate sodium salt); or wetting agents (e.g., sodium do256 decylsulfate). Tablets may be coated by methods well known in the art. Liquid preparations for oral administration may take the form of, for example, a solution, syrup or suspension, or may be prepared as a dry product, reconstituted with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional methods such as pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); with non-aqueous carriers (e.g., methyl or propyl p-hydroxybenzoates or spray acid). The compositions may contain buffering salts, perfuming, coloring and sweetening agents as needed. Formulations for compositions for oral administration may be suitable for the slow release, controlled, or sustained release of one or more prophylactic or therapeutic agents.

The compositions of the present invention may be formulated for parenteral administration, e.g., by bolus injection or continuous infusion. Formulations for injection may be provided in unit dosage form, such as in ampoules or in multi-dose containers, with an added preservative. The formulations may take the form of a suspension, solution or emulsion in an oily or aqueous vehicle, and may contain formulating agents such as suspending, stabilizing and / or dispersing agents. Another

According to variant 257, the active ingredient may be in powder form for reconstitution with a suitable carrier such as sterile pyrogen-free water before use.

The compositions of the present invention may be presented in the form of rectal formulations, such as suppositories or retention enemas, for example containing conventional suppository bases such as cocoa butter or other glycerides.

In addition to the formulations described above, the compositions of the present invention may be formulated as depot formulations. Such sustained release formulations may be administered by implantation (e.g., subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the formulations may be formulated with suitable polymeric or hydrophobic materials (e. G., As an emulsion in an acceptable oil) or with ion exchange resins or poorly soluble derivatives, such as a low soluble salt.

The compositions of the present invention may also be presented in the form of a neutral salt. Pharmaceutically acceptable salts include salts formed with anions, such as hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, and the like. and salts formed with cations, such as sodium, potassium, ammonium, calcium, iron hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, and the like. trained salts.

258

The additives of the compositions of the present invention are provided either separately or mixed with one another in unit dosage form, for example, in the form of lyophilized powder or anhydrous concentrate, in a hermetically sealed container such as an ampoule or sachet indicating the amount of active ingredient. If the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. If the composition is to be administered by injection, sterile water or saline solution for injection can be provided by mixing the additives prior to administration.

More specifically, the present invention relates to the incorporation of one or more prophylactic or therapeutic agents or pharmaceutical compositions of the present invention into a hermetically sealed container, such as an ampoule or bag, wherein the amount of active ingredient is indicated. In one embodiment, one or more prophylactic or therapeutic agents or pharmaceutical compositions of the present invention are provided in the form of a sterilized lyophilized powder or anhydrous concentrate in a hermetically sealed container and can be reconstituted, for example, with water or saline, for administration to a subject. Preferably, one or more prophylactic or therapeutic agents or pharmaceutical compositions of the present invention are provided in the form of a dry sterile lyophilized powder in a hermetically sealed container of at least 5 mg, more preferably at least 10 mg, at least 15 mg.

259 unit doses of at least 25 mg, at least 35 mg, at least 45 mg, at least 50 mg, at least 75 mg or at least 100 mg. The prophylactic or therapeutic agent or pharmaceutical composition of the present invention should be stored between 2 and 8 ° C in its original container and a prophylactic or therapeutic agent or pharmaceutical composition of the present invention within at least 1 week after recovery, preferably within 5 days, Within 72 hours, within 48 hours, within 24 hours, 12 hours, 6 hours, 5 hours, 3 hours, or within 1 hour. In an alternative embodiment, the prophylactic or therapeutic agent or pharmaceutical composition of the present invention is provided in a liquid form in a hermetically sealed container having an amount and concentration of agent. The liquid form of the administered composition is provided in a hermetically sealed container which is at least 25 mg / ml, more preferably at least 0.5 mg / ml, at least 1 mg / ml, at least

2.5 mg / ml, at least 5 mg / ml, at least 8 mg / ml, at least 10 mg / ml, at least 15 mg / ml, at least 25 mg / ml, at least 50 mg / ml, at least 75 mg / ml, or at least 100 mg / ml. The liquid form should be stored between 2 ° C and 8 ° C in the original container.

In a preferred embodiment, REMICADE ™ is provided as a sterile and lyophilized powder for intravenous infusion, which is reconstituted with 10 ml of sterile water for injection. Each single-use REMICADE ™ vial contains 100 mg infliximab, 500 mg sucrose, 0.5 mg \ t

260 polysorbate 80, 2.2 mg monobasic sodium phosphate and 6.1 mg dibasic sodium phosphate. According to The Physician Desk Reference (55th edition, 2001), the total dose of reconstituted product should be further diluted with 250 ml of 0.9% sodium chloride injection (USP) and the infusion concentration is 0.4 mg / ml and 4 ml. mg / ml.

In another preferred embodiment of the present invention, ENBREL ™ is provided as a sterile and lyophilized powder for parenteral administration after reconstitution with 1 ml of sterile bacteriostatic injection water (USP) (containing 0.9% benzyl alcohol). According to The Physician Desk Reference (55th edition, 2001), each disposable ENBREL ™ vial contains 25 mg etanercept, 40 mg mannitol, 10 mg sucrose and 1.2 mg tromethamin.

In a further preferred embodiment of the present invention, VITAXIN ™ is prepared at a concentration of 1 mg / ml, 5 mg / ml, 10 mg / ml and 25 mg / ml for intravenous injection and 5 mg / ml, 10 mg / ml, 80 mg / ml, or 100 mg / ml are prepared for repeated subcutaneous administration.

In other preferred embodiments of the present invention, methotrexate is formulated at a concentration of 25 mg / ml and delivered in vials, for example, in a 1 ml, 2 ml and 10 ml vial. Methotrexate for injection contains the sodium salt of methotrexate equivalent to 50 mg or methotrexate, with 90% w / v benzyl alcohol as a preservative, 0.260% w / v sodium chloride and for injection.

261

made with water. Methotrexate can be administered by intramuscular, intravenous, intra-arterial injection using a preservative formulation containing benzyl alcohol. Methotrexate can also be administered intrathecally using a non-preservative formulation. In another embodiment of the present invention, methotrexate is provided as a tablet in the form of a unit dose of 2.5 mg of methotrexate sodium salt.

In further preferred embodiments, according to the present invention, MEDI-507 is packaged in a hermetically sealed container, such as an ampoule or sachet, indicated by the amount of MEDI-507. In one embodiment, MEDI-507 is provided as a sterilized lyophilized powder or an anhydrous concentrate in a hermetically sealed container and can be reconstituted, for example, with water or saline at a suitable concentration to be administered to a subject. Preferably, MEDI-507 is provided as a dry sterile lyophilized powder in a hermetically sealed container of at least 5 mg, more preferably at least 10 mg, at least 15 mg, at least 25 mg, at least 35 mg. containing at least 45 mg, at least 50 mg, at least 75 mg or at least 100 mg. In an alternative embodiment, MEDI-507 is provided in a liquid form in a hermetically sealed container with the amount and concentration of MEDI-507 indicated. The liquid form of MEDI-507 is provided in a hermetically sealed container, at least 25 mg / ml, more preferably at least 0.5 mg / ml, at least 1 mg / ml, at least 2.5

262 mg / ml, at least 5 mg / ml, at least 8 mg / ml, at least 10 mg / ml, at least 15 mg / ml, at least 25 mg / ml, at least 50 mg / ml, at least 75 mg / ml, or at least 100 mg / ml.

The compositions may, if necessary, be presented in a package or dispensing apparatus, which may comprise one or more unit dosage forms containing the active ingredient. The package may comprise, for example, a metal or plastic film, such as a blister pack. The packing or dispensing apparatus is accompanied by instructions for administration. In certain preferred embodiments, the package or distributor comprises one or more unit dosage forms containing up to 25 mg of ENBREL ™, 2.5 mg METOTREXAT, 100 mg REMICADE ™ and 5 mg / ml VITAXIN ™.

The additives of the compositions of the present invention are generally derived from a subject belonging to the same species as a subject receiving such compositions. Thus, in a preferred embodiment, human or humanized antibodies are administered to a human for therapy or prophylaxis.

An effective amount of a composition of the present invention for treating, preventing or ameliorating one or more symptoms associated with an autoimmune or inflammatory disorder may be determined by standard clinical techniques. The exact dosage to be used in the formulation will depend on the route of administration and the severity of the condition, and this should

263 and the circumstances of each patient. Effective dosages can be determined based on dose-response curves from in vitro or animal model test systems.

For antibodies, proteins, polypeptides, peptides and fusion proteins of the present invention, the dosage administered to a patient will typically range from 0.0001 mg / kg to 100 mg / kg of patient weight. The dose administered to a patient is preferably 0.0001 mg / kg and 20 mg / kg, 0.0001 mg / kg and 10 mg / kg, 0.0001 mg / kg and 5 mg / kg, 0.0001 mg / kg and 2 mg / kg, 0.0001 mg / kg and 1 mg / kg, 0.0001 mg / kg and 0.75 mg / kg, 0.0001 mg / kg and 0.5 mg / kg, 0.0001 mg / kg and 0.25 mg / kg, 0.0001 mg / kg and 0.25 mg / kg, 0.0001 mg / kg and 0.10 mg / kg, 0.001 mg / kg and 0.5 mg / kg, 0, Between 01 mg / kg and 0.25 mg / kg or 0.01 mg / kg to 0.10 mg / kg of patient weight. Human antibodies generally have a higher half-life in the human body than antibodies from other species because of the immune response to foreign polypeptides. Thus, it is often possible to administer lower doses of anti-human antibodies or less frequently. Thus, the dose and frequency of administration of the antibodies or fragments thereof of the present invention may be reduced by altering the uptake and tissue penetration of the antibody by modifications such as lipidation.

In a specific embodiment, the composition of the present invention or a prophylactic or therapeutic agent administered to prevent a patient is treated with:

Or to alleviate one or more symptoms associated with an autoimmune or inflammatory disorder, doses of 150 pg / kg or less, preferably 125 pg / kg or less, 100 pg / kg or less, 95 pg / kg or less, 90 pg / kg, or less than 85 pg / kg or less, 80 pg / kg or less, 75 pg / kg or less, 70 pg / kg or less, 65 pg / kg or less, 60 pg / kg or less, 55 pg / kg or less , 50 pg / kg or less, 45 pg / kg or less, 40 pg / kg or less, 35 pg / kg or less, 30 pg / kg or less, 25 pg / kg or less, 20 pg / kg or less, 15 pg / kg or less, 10 pg / kg or less, 5 pg / kg or less, 2.5 pg / kg or less, 2 pg / kg or less, 1.5 pg / kg or less, 1 pg / kg or less, or 0.5 pg / kg or less per patient's weight. In another embodiment, the composition of the present invention or a prophylactic or therapeutic agent administered to prevent, treat, or ameliorate one or more symptoms associated with an autoimmune or inflammatory disorder in a patient is 0.1 mg and 20 mg. , 0.1 mg and 15 mg, 0.1 mg and 12 mg, 0.1 mg and 10 mg, 0.1 mg and 8 mg, 0.1 mg and 7 mg, 0.1 mg and 5 mg, 0 , 1 mg and 2.5 mg, 0.25 mg and 20 mg, 0.25 mg and 15 mg, 0.25 mg and 12 mg, 0.25 and 10 mg, 0.25 and 8 mg, 0.25 mg and 7 mg, 0.25 mg and 5 mg, 0.5 mg and 2.5 mg, 1 mg and 20 mg, 1 mg and 15 mg, 1 mg and 12 mg, 1 mg and 10 mg, 1 mg and \ t Between 8 mg, 1 mg and 7 mg, 1 mg and 5 mg, or 1 mg to 2.5 mg.

265 ·· «· ♦ ··· • ·

In one embodiment, the recommended dose of ENBREL ™ is 0.01 mg / kg to 10 mg / kg, preferably 0.1 mg / kg to 10 mg / kg, more preferably 0.1 mg / kg and 5 mg / kg, moreover more preferably from 0.5 mg / kg to 2 mg / kg. In one embodiment of the present invention, the recommended dose of ENBREL ™ is 0.01 mg / kg / week and 10 mg / kg / week, more preferably 0.1 mg / kg to 5 mg / kg / week, even more preferably 0.5 mg / kg / week and more preferably between 0.5 mg / kg / week and 2 mg / kg / week. In a most preferred embodiment, the weekly dose does not exceed 50 mg / week. In preferred embodiments, ENBREL ™ is administered twice a week by subcutaneous injection.

In a preferred embodiment of the present invention, ENBREL ™ is administered at a dosage of from about 1 mg to about 50 mg, more preferably from about 10 mg to about 40 mg, most preferably from about 20 mg to about 30 mg. In some embodiments, an antagonist of integrin α ν β3 is ENBREL ™ weekly 0.1 mg and 1 mg, 1 mg and 5 mg, 5 mg and 10 mg, 10 mg and 15 mg, 15 mg and 20 mg, 20 mg, and 25 mg, 25 mg and 30 mg, 30 mg and 35 mg, 35 mg and 40 mg, 40 mg and 45 mg, 45 mg and 50 mg, 50 mg and 60 mg, 60 mg and 65 mg, 65 mg and 70 mg , 70 mg, 75 mg, 75 mg and 80 mg, 80 mg and 85 mg, 85 mg and 90 mg, 90 mg and 95 mg, 95 mg and 100 mg, 100 mg and 105 mg, 105 mg and 110 mg, 110 mg or 115 mg or 115 mg to 120 mg. ENBREL ™ is preferably administered twice a week, subcutaneously in266 ·· ·· ···· jekection. The injections should preferably be 72-96 hours. In one embodiment, the injections are administered at a difference of 36 to 132 hours, preferably 48 to 114 hours, more preferably at a difference of 72 to 96 hours, even more preferably at about 48 hours apart. In a preferred embodiment, the dose of ENBREL ™ is less than typical when administered alone. See The Physician Desk Reference (55th Edition, 2001). Accordingly, in a preferred embodiment, administration of an antagonist of integrin α ν β 3 is combined with administration of up to 25 mg / kg of ENBREL ™. In preferred embodiments, less than 25 mg, less than 20 mg, less than 15 mg, or less than 5 mg of ENBREL are administered per dose. In accordance with the methods of the present invention, ENBREL ™ is 1 mg, 1 mg, and 5 mg. mg, 5 mg and 10 mg, 10 mg and 15 mg, 15 mg and 20 mg, 20 mg and 25 mg, or 25 mg twice a week. The integrin α ν β 3 antagonist is preferably VITAXIN ™.

In another embodiment of the present invention, an integrin α ν β3 antagonist is administered in combination with anti-TNFα antibodies. The anti-TNFα antibody is preferably infliximab (REMICADE ™). In one embodiment of the present invention, the recommended dose of REMICADE ™ is 0.1 mg / kg to 10 mg / kg, more preferably 1 mg / kg to 7 mg / kg, more preferably 2 mg / kg and 6 mg / kg, and most preferably between 3 mg / kg and 5 mg / kg. In a most preferred embodiment, the dose is

267 does not exceed 3 mg / kg. In some preferred embodiments, REMICADE ™ is administered by intravenous infusion, followed by a second dose given 2-6 weeks after the first infusion, and then every 8 weeks thereafter.

In a preferred embodiment, REMICADE ™ is administered at a dose of from about 1 mg to about 600 mg, more preferably from about 100 mg to 500 mg, most preferably from about 200 mg to about 400 mg. In a particular embodiment of the present invention, an integrin α ν β3 antagonist is 1 mg and 10 mg, 10 mg and 50 mg, 50 mg and 100 mg, 100 mg and 150 mg, 150 mg and 200 mg, 200 mg and 250 mg,

250 mg and 300 mg, 300 mg and 350 mg, 350 mg and 400 mg, 400 mg and 450 mg, 450 mg and 500 mg, 550 mg and 600 mg, 600 mg and 650 mg, 650 mg and 700 mg, 700 mg and 750 mg, 750 mg and 800 mg, 800 mg and 850 mg, 850 mg and 900 mg, 900 mg and 950 mg,

It is given in combination with REMICADE ™ between 950 mg and 1000 mg, first and then 2-6 weeks after the first dose, followed by eight weeks thereafter. In a preferred embodiment, the doses of REMICADE ™ are less than the typical doses when administered alone. See The Physician Desk Reference (55th Edition, 2001). Accordingly, in a preferred embodiment, up to 600 mg of REMICADE ™ is administered as an intravenous fusion followed by a second dose at 2 and 6 weeks after the first infusion, followed by a further eight weeks. In other embodiments, further doses are administered every 1-12 weeks, preferably 4-12 weeks, more preferably 6-12

It is administered every 268 weeks, more preferably every 8-12 weeks. The integrin α ν β3 antagonist is preferably VITAXIN ™.

According to certain methods of validation of the invention, four integrin α ν β3 antagonists are administered in combination with methotrexate alone or in combination with other prophylactic or therapeutic agents. In certain embodiments, the recommended dose of methotrexate is from 0.01 to 3 mg / kg, more preferably from 0.1 mg / kg to 2 mg / kg, most preferably from 0.5 mg / kg to 1 mg / kg. In some preferred embodiments, the recommended dose of methotrexate is 0.01 mg / kg / week and 3 mg / kg / week, more preferably 0.1 mg / kg / week and 2 mg / kg / week, and most preferably 0.5 mg / kg / week to 0.1 mg / kg / week. In a most preferred embodiment, the weekly dose does not exceed 20 g / week.

In a preferred embodiment, methotrexate is administered from about 0.01 mg to about 70 mg, preferably about 1 mg to 60 mg, most preferably about 10 mg to 60 mg. Methotrexate is 0.5 mg and 1 mg, 1 mg and 1.5 mg, 1.5 mg and 2 mg, 2 mg and 2.5 mg, 2.5 mg and 3 mg, 3 mg and \ t

3.5 mg, 3.5 mg and 4 mg, 4 mg and 4.5 mg, 4.5 mg and 5 mg, 5 mg and 5.5 mg, 5.5 mg and 6 mg, 6 mg and 6.5 mg , 6.5 mg and 7 mg, 7 mg and 7.5 mg, 7.5 mg and 8 mg, 0.5 mg and 1 mg, 1 mg and 1.5 mg,

1.5 mg and 2 mg, 2 mg and 2.5 mg, 2.5 mg and 3 mg, 3 mg and 3.5 mg, 3.5 mg and 4 mg, 4 mg and 4.5 mg, 4.5 mg and 5 mg, 5 mg and

5.5 mg, 5.5 mg and 6 mg, 6 mg and 6.5 mg, 6.5 mg and 7 mg, 7 mg and 7.5 mg, 7.5 mg and 8 mg, 8 mg and 8.5 mg , 8.5 mg and 9 mg, 9

269 rf »« <rf Χ rf rf rf rf £ £

*>»»> ♦ r · · * · mg and 9.5 mg, 9.5 mg and 10 mg, 10 mg and 10.5 mg, 10.5 mg and 11 mg, 11 mg and 12 mg, 12 mg and 13 mg, 13 mg and 14 mg, 14 mg and 15 mg, 15 mg and 20 mg, 20 mg and 25 mg, 25 mg and 30 mg, 30 mg and 35 mg, 35 mg and 40 mg, 40 mg and 45 mg. mg, 45 mg and 50 mg, 50 mg and 60 mg, 60 mg and 70 mg, 70 mg and 80 mg. In a preferred embodiment, the dose of methotrexate administered is less than the typical dose if administered alone. See The Physician Desk Reference (55th Edition, 2001). Accordingly, in a preferred embodiment of the present invention, an integrin α ν β3 antagonist is administered in combination with oral or intramuscular administration of up to 57 mg methotrexate, up to a maximum of 2.5 mg, every 12 hours, in three doses per week. In a more preferred embodiment of the present invention, an integrin α ν β3 antagonist is administered in combination with oral or intramuscular administration of up to 20 mg methotrexate at weekly intervals. In certain embodiments of the present invention, methotrexate is administered at a difference of 6 to 12 hours, with a difference of 12 to 18 hours, with a difference of 18 to 24 hours, with a difference of 24 to 36 hours, with a difference of 36 to 48 hours, with a difference of 48 to 52 hours, with a difference of 52 to 60 hours. With 60-72 hours difference, 72-84 hours difference, 84-96 hours difference, or 96-120 hours difference. In a most preferred embodiment of the present invention, an integrin α ν β3 antagonist is administered by co-administration of up to 15-20 mg methotrexate at a single weekly dose. Other embodiments

270 • «Μ» · «· · · · '····· * *. · ·· "» "According to which methotrexate is given once a week, once every two weeks, once every three weeks, or once a month.

In some embodiments, the dose of VITAXIN ™ administered to a subject is from 0.1 to 10 mg / kg, preferably from 1 to 9 mg / kg, more preferably from 2 to 8 mg / kg, more preferably from 3 to 7 mg / kg, and most preferably from 4 to 7 mg / kg. 6 mg / kg. In other preferred embodiments, VITAXIN ™ is from 0.1 to 10 mg / kg / week, preferably from 1 to 9 mg / kg / week, more preferably from 2 to 8 mg / kg / week, more preferably from 3 to 7 mg / kg / week, and most preferably 4-6 mg / kg / week.

In other embodiments, a subject has 200 pg / kg or less, preferably 125 pg / kg or less, 100 pg / kg or less, 95 pg / kg or less, 90 pg / kg or less, 85 pg / kg or less, 80 pg / kg or less, 75 pg / kg or less, 70 pg / kg or less, 65 pg / kg or less, 60 pg / kg or less, 55 pg / kg or less, 50 pg / kg or less, 45 pg / kg or less, 40 pg / kg or less, 35 pg / kg or less, 30 pg / kg or less, 25 pg / kg or less, 20 pg / kg or less, 15 pg / kg or less, 10 pg / kg kg or less, 5 pg / kg or less

2.5 pg / kg or less, 2 pg / kg or less, 1.5 pg / kg or less, 1 pg / kg or less, 0.5 pg / kg or less, or 0.4 pg / kg or less in amounts of MEDI-507. for the prevention, treatment or alleviation of one or more symptoms associated with an autoimmune or inflammatory disorder. Such doses are preferably administered intravenously

271

X is administered to a patient suffering from an autoimmune or inflammatory disorder.

In a specific embodiment, one or more, 0.1 mg and 20 mg, 0.1 mg and 15 mg, 0.1 mg and 12 mg, 0.1 mg and 10 mg, 0.1 mg and 8 mg, 0 , 1 mg and 7 mg, 0.1 mg and 5 mg, 0.1 mg and

2.5 mg, 0.25 mg and 20 mg, 0.25 mg and 15 mg, 0.25 mg and 12 mg, 0.25 and 10 mg, 0.25 and 8 mg, 0.25 mg and 7 mg , 0.25 mg and 5 mg, 0.5 mg and 2.5 mg, 1 mg and 20 mg, 1 mg and 15 mg, 1 mg and 12 mg, 1 mg and 10 mg, 1 mg and 8 mg, 1 mg and 7 mg, 1 mg and 5 mg, or 1 mg and 2.5 mg MEDI-507 are administered in a unit dose to prevent, treat or alleviate one or more symptoms associated with an autoimmune or inflammatory disorder. In another embodiment, a subject has one or more 0.1 mg, 0.25 mg, 0.5 mg, 1 mg, 1.5 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg , 8mg, 9mg, 10mg, 11mg, 12mg, 13mg, 14mg, 15mg, or 16mg doses of MEDI-507 are administered to prevent one or more symptoms associated with an autoimmune or inflammatory disorder; treat or relieve. The unit doses of MEDI-507 are preferably administered subcutaneously to a patient suffering from an autoimmune or inflammatory disorder.

In another embodiment, a subject is administered one or more doses of MEDI-507 in a prophylactically or therapeutically effective amount, wherein the prophylactically or therapeutically effective amount is not the same in all doses. In another embodiment, a subject, preferably a human, is

272

One or more doses of MEDI-507 that are prophylactically or therapeutically effective are administered, wherein the prophylactically or therapeutically effective amount of the MEDI-507 dose administered to said subject is increased, e.g., 0.01 pg / kg, 0.02 pg. / kg, 0.04 pg / kg, 0.05 pg / kg, 0.06 pg / kg, 0.08 pg / kg, 0.1 pg / kg, 0.2 pg / kg, 0.25 µg / kg , 0.5 pg / kg, 0.75 pg / kg, 1 pg / kg, 1.5 pg / kg, 2 pg / kg, 4 pg / kg, 5 pg / kg, 10 pg / kg, 15 pg / kg kg, 20 pg / kg, 25 pg / kg, 30 pg / kg, 35 pg / kg, 40 pg / kg, 45 pg / kg, 50 pg / kg, 55 pg / kg, 60 pg / kg, 65 pg / kg kg, 70 pg / kg, 75 pg / kg, 80 pg / kg, 85 pg / kg, 90 pg / kg, 95 pg / kg, 100 pg / kg, or 125 pg / kg as treatment progresses.

In another embodiment, a subject, preferably a human, is administered one or more doses of MEDI-507 which are prophylactically or therapeutically effective, wherein the prophylactically or therapeutically effective amount of MEDI-507 administered to said subject is reduced, e.g., 0.01 pg / kg , 0.02 pg / kg, 0.04 pg / kg, 0.05 pg / kg, 0.06 pg / kg, 0.08 pg / kg, 0.1 pg / kg, 0.2 pg / kg, 0.25 µg / kg, 0.5 µg / kg, 0.75 µg / kg, 1 µg / kg, 1.5 µg / kg, 2 µg / kg, 4 µg / kg, 5 µg / kg, 10 µg / kg kg, 15 pg / kg, 20 pg / kg, 25 pg / kg, 30 pg / kg, 35 pg / kg, 40 pg / kg, 45 pg / kg, 50 pg / kg, 55 pg / kg, 60 pg / kg kg, 65 pg / kg, 70 pg / kg, 75 pg / kg, 80 pg / kg, 85 Pg / kg, 90 pg / kg, 95 pg / kg, 100 pg / kg, or 125 pg / kg as described in treatment progresses.

In a further embodiment, a subject is administered one or more doses of a prophylactically or therapeutically effective amount of one or more immunomodulatory agents, wherein:

273

The dose of said prophylactically or therapeutically effective amount administered to said subject in said subject is about 500 cells / mm below 3 to 1500 cells / mm 3 , preferably below 1500 cells / mm 3, below 1300 cells / mm 3 , 1250 cells / mm 3 less than 1200 cells / mm under 3, 1100 cells / mm 3 below or reaches 1,000 cells / mm, a mean absolute lymphocyte count of less than the third In another embodiment, a subject is administered a prophylactically or therapeutically effective amount of one or more CD2 binding molecules wherein said prophylactically or therapeutically effective amount of said subject is administered to said subject in an amount of about 500 cells / mm 3 to 1500 cells / mm 3 below, preferably 1500 cells / mm 3 below 1300 cells / mm under 3, 1250 cells / mm 3 below 1,200 cells / mm under 3, 1100 cells / mm 3 below, or 1000 cells / mm, a mean absolute lymphocyte count of less than 3 reaches. In a preferred embodiment, a subject is administered a prophylactically or therapeutically effective amount of MEDI-507, wherein said prophylactically or therapeutically effective amount of said subject is administered to said subject in an amount of about 500 cells / mm 3 to 1500 cells / mm. 3 below, preferably 1500 cells / mm 3 below 1300 cells / mm under 3, 1250 cells / mm 3 below 1,200 cells / mm under 3, 1100 cells / mm 3 below, or 1000 cells / mm, a mean absolute lymphocyte count of less than 3 reaches.

In other embodiments, a subject is administered one or more doses of one or more CD2 binding molecules that are prophylactically or therapeutically effective, wherein the dose of the CD2 binding molecule is at least 2025%, 25-30, at the dose of the prophylactically or therapeutically effective amount of the CD2 binding molecule. %, 30-35%, 35-40%, 40-45%, 50-55%, 55-60%, 6065%, 65-70%, 70-75%, 75-80%, up to 80% it binds to CD2 binding molecules. In yet another embodiment, the subject is administered one or more doses of MEDI-507 which are prophylactically or therapeutically effective, wherein the dose of MEDI507 administered, prophylactically or therapeutically effective amounts to at least 20-25%, 2530%, 30-50% of the CD2 polypeptide. 35%, 35-40%, 40-45%, 50-55%, 55-60%, 60-65%, 6570%, 70-75%, 75-80%, up to 80% CD2 binder for m moles.

In a specific embodiment, nucleic acids comprising one or more sequences encoding prophylactic or therapeutic agents are administered with a view to treating, preventing or alleviating one or more symptoms associated with an autoimmune or inflammatory disorder. Gene therapy means that the therapy is performed by administering an expression or expression nucleic acid to the subject. In this embodiment of the present invention, the nucleic acids produce encoded prophylactic or therapeutic agents that exert a prophylactic or therapeutic effect.

Any gene therapy method known in the art may be used in the present invention. As examples, the following methods are mentioned.

275

General summaries of gene therapy are available in Goldspiel et al., Clin. Pharmacy 12: 488-505 (1993); Wu and Wu, Biotherapy 3, 87-95 (1991); Tolstoshev: Ann. Port. Pharmacol. Toxicol. 32: 573-596 (1993); Mulligan, Science 260: 926-932 (1993); Morgan and Anderson: Annu. Port. Biochem. 62, 191-217 (May 1993); TIBTECH 11 (5), 155-215 (1993)]. Methods commonly used in the field of recombinant DNA technology and useful in the present invention are summarized in the literature (Current Protocols in Molecular Biology, ed. Ausubel et al., Greene Publishing and WileyInterscience: New York (1987); Kriegler: Gene Transfer and Expression, A Laboratory Manual, Stockton Press, New York (1990)].

In a preferred embodiment, the composition of the present invention comprises nucleic acids encoding a prophylactic or therapeutic agent, and said nucleic acids form part of an expression vector that expresses the prophylactic or therapeutic agent in a suitable host. Specifically, such nucleic acids have a promoter, preferably a heterologous promoter, operably linked to the antibody coding region, and said promoter may be inducible or constitutive and optionally tissue specific. In another embodiment, nucleic acid molecules are used in which the sequences encoding the prophylactic or therapeutic agent are delimited by any other desired sequence in regions that promote the ho &apos;

276 • Mologic recombination at a desired point in the genome to provide intracromosomal expression of the antibody encoding the nucleic acids [Roller and Smithies, Proceedings of the National Academy of Sciences, USA 86, 8932-8935 (1989); Zijlstra et al., Nat. 342: 435-438 (1989)]. In certain embodiments, the prophylactic or therapeutic agents are expressed. In other embodiments, the expressed prophylactic or therapeutic agent is an agent known to be useful, or used, or is currently used to prevent, treat, or ameliorate one or more symptoms associated with an autoimmune or inflammatory disorder. In a preferred embodiment, the expressed prophylactic or therapeutic agent is VITAXIN ™.

The introduction of nucleic acids into a subject may be direct, in which case the subject is brought into direct contact with the nucleic acid, or with the nucleic acid carrying vectors, or indirectly when the cells are first transformed with nucleic acids in vitro and then transplanted into the subject. The two approaches are well known as in-carrier or ex-carrier gene therapy.

In a specific embodiment, the nucleic acid sequences are administered directly by the in vivo carrier when expressed and producing the encoded product. This can be accomplished by any of a number of methods known in the art, e.g., by creating them as part of an appropriate nucleic acid expression vector, and administering them to become intracellular, such as infection, defective or attenuated retroviral, or

277., .... »··· using other viral vectors (see, for example, U.S. Patent No. 4,980,286) or direct injection of pure DNA, or microparticle bombardment (e.g., gene pouch; Biolistic, DuPont) or with a matrix in vitro fixation containing the nucleic acid sequence (see, for example, EP 0 741 785 B1 and U.S. Patent No. 5,962,427) or coated with lipids or cell surface receptors or transfecting agents; encapsulated in liposomes, microparticles, or microcapsules, or administered by coupling to a peptide known to enter the nucleus or linked to a ligand dumbbell known to be subject to endocytosis by the receptors [see, for example, Wu and Wu: Journal of Biological Chemistry 262: 4429-4432 (1987) (which is used herein) can be used to target cell types expressing the receptors, etc.). In another embodiment, nucleic acid ligand complexes may be formulated in which the ligand comprises a fusogenic viral peptide to disrupt the endosomes by allowing the nucleic acids to avoid lysosomal degradation. In yet another embodiment, the nucleic acids may be in vivo predicted for cell-specific uptake and expression by targeting a specific receptor (see, for example, WO 92/06080; WO 92/22635; WO 92/203; WO 093/14188 and WO 93/20221). PCT publications). Alternatively, the nucleic acid enters

278 can be incorporated into the host cell DNA by expression by homologous recombination (Roller and Smithies, Proceedings of the National Academy of Sciences, USA 86, 89328935 (1989)); Zijlstra et al., Nat. 342: 435-438 (1989)].

In a specific embodiment, viral vectors comprising nucleic acid sequences encoding a prophylactic or therapeutic agent are used. For example, a retroviral vector may be used (see, e.g., Miller et al., Methods in Enzymology 217, 581-599 (1993)). These retroviral vectors contain the components that are required for proper packing of the viral genome and integration into the host cell DNA. Nucleic acid sequences encoding the antibody to be used in gene therapy are cloned into one or more vectors, which facilitates the entry of the gene into the subject. Details of retroviral vectors can be found in the literature (Boesen et al., 1994, Biotherapy 6: 291-302), which describes the use of the retroviral vector for the introduction of the mdrl gene into hematopoietic stem cells with the aim of making stem cells resistant to chemotherapy. . There are other publications in the literature that illustrate the use of retroviral vectors in gene therapy [Clowes et al., J. Clinic. Invest. 93, 644-651 (1994); Klein et al., Blood 83, 1467-1473 (1994); Salmons and Gunzberg, Human Gene Therapy 4: 129-141 (1993); Grossman and Wilson: Current Opinion Gene. Develop. 3, 110-114 (1993)].

• ··· ·· · ·· · · ·

279

Adenoviruses are other viral vectors that can be used in gene therapy. Adenoviruses are particularly attractive carriers for introducing genes into respiratory epithelium. Adenoviruses naturally infect the respiratory epithelium, where they cause mild illness. Other targets for adenovirus delivery systems are the liver, the central nervous system, endothelial cells and muscle. Adenoviruses have the advantage of being able to infect non-dividing cells. Kozarsky and Wilson have published a summary of adenovirus-based gene therapy [Kozarsky and Wilson: Current Opinion Gene. Develop. 3, 499-503 (1993). Bout et al. Have demonstrated: the use of adenoviral vectors for introducing genes into rhesus monkeys in respiratory epithelium (Bout et al., Human Gene Therapy 5, 3-10 (1994)). Other examples of the use of adenoviruses in gene therapy can be found in Rosenfeld et al., Science 252: 431-434 (1991); Rosenfeld et al., Cell 68: 143155 (1992); Mastrangeli et al., J. Clinic. Invest. 91: 225-234 (1993); PCT Publication No. WO 94/12649; Wang et al., Gene Ther. 2, 775-738 (1995). In a preferred embodiment, adenoviral vectors are used.

Adeno-associated viruses (AAV) have also been proposed for use in gene therapy (Walsh et al., Proc. Soc. Exp. Bioi. Med. 204, 289-294-300 (1993); and U.S. Patent No. 5,436,146.

··

280

Another approach to gene therapy involves the delivery of a gene in cells in tissue cultures, such as electroporation, lipofection, calcium phosphate transfection, or viral transfection. The transfer method generally involves the transfer of a selection marker. The cells are then selected for isolation of the cells that have received and express the transferred gene. These cells are then administered to a subject.

In this embodiment, the nucleic acid is introduced into a cell prior to administration of the resulting recombinant cell in vivo. This input can be carried out by any method known to those skilled in the art, including, but not limited to, infection by transfection, electroporation, microinjection, viral or bacteriophage vector containing nucleic acid sequences, cell fusion, gene transfer with chromosomes, microcells gene transfer, spheroplast fusion, and the like. Several techniques are known in the art for introducing foreign genes into cells (see, e.g., Loeffler and Behr, Methods in Enzymology 217, 599618 (1993); Cohen et al., Methods in Enzymology 217: 618644 (1993)]; Clin. Pharmacol. Ther. 29, 69-92 (1985)] and may be used in accordance with the present invention, provided that the necessary developmental and physiological functions of the recipient cells are not impaired. The technique must ensure stable delivery of the nucleic acid into the cell such that the nucleic acid is a

It can be expressed by cell 281 and is preferably heritable and expressed by the progeny of the cell.

The resulting recombinant cells can be introduced into a subject by various methods known in the art. Recombinant blood cells (e.g., hematopoietic stem cells or stem cells) are preferably administered intravenously. The number of cells to be used for use depends on the desired effect, the condition of the patient, etc., and can be determined by one skilled in the art.

The cells into which a nucleic acid may be introduced for gene therapy may be any available cell types, including, but not limited to, epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytes; blood cells such as T lymphocytes, Blimfocytes, natural killer cells (NK cells), monocytes, macrophages, neutrophils, eosinophils, megakaryocytes, granulocytes; different stem cells, particularly hematopoietic stem cells, such as those from bone marrow, umbilical vein, peripheral blood, and fetal liver.

In a preferred embodiment, the cell used in gene therapy is autologous to the subject.

In one embodiment, in which recombinant cells are used in gene therapy, the nucleic acid sequences encoding the prophylactic or therapeutic agent are introduced into the cells so that they can be expressed by the cells or their progeny, and the recombinant cells are then introduced into the cells.

• ·

282 is administered for prophylactic or therapeutic effects. In a specific embodiment, stem cells are used. Any stem cell that can be isolated and capable of being sustainably utilized in vitro may be used according to this embodiment of the present invention (see, for example, PCT Publication WO 94/08598; Stemple and Anderson, Cell, 7: 973-985 (1992); Rheinwald: Meth. Cell Bio. 21A, 229 (1980); Pittelkow and Scott, Mayo Clinic Proc. 61, 771 (1986)].

In a specific embodiment, the nucleic acid introduced for gene therapy comprises a constitutive, tissue-specific or inducible promoter linked operatively to the coding region. In a preferred embodiment, the nucleic acid introduced for gene therapy comprises a constitutive, tissue-specific or inducible promoter linked operatively to the coding region, such that the nucleic acid can be induced for transcription by the presence or absence of the appropriate inducer.

Many aspects of the pharmaceutical compositions of the present invention or prophylactic or therapeutic agents may be tested in vitro, in a cell culture system, and in an animal model organism, such as a rodent animal model system, prior to application in humans to obtain the desired therapeutic effect. For example, the assays that can be used to determine whether a specific pharmaceutical composition can be administered may be cell culture assays in which the patient's tissue sample is propagated in culture, then brought into contact with, or otherwise contacted by, the culture.

Contact with a pharmaceutical composition and observe the effect of such a composition on the tissue sample. The tissue sample can be obtained from the patient by biopsy. This test allows us to identify the therapeutically most effective prophylactic or therapeutic molecule (s) for each patient. In various specific embodiments, in vitro assays may be performed on representative cells or cell types that play a role in an autoimmune or inflammatory disorder (e.g., T cells) to determine whether a pharmaceutical composition of the present invention has the desired effect on such a cell type. .

Combinations of prophylactic and / or therapeutic agents can be tested in appropriate animal model systems before being administered to humans. Such animal model systems include, but are not limited to, rats, mice, chickens, cattle, pigs, dogs, rabbits, and the like. Any animal model system well known in the art can be used. In a specific embodiment of the present invention, combinations of prophylactic and / or therapeutic agents are tested in a mouse model system. Such model systems have been widely used and are well known to those skilled in the art. Prophylactic and / or therapeutic agents may be administered repeatedly. Many aspects of the process may change. Such an aspect is, for example, the timing of administration of prophylactic and / or therapeutic agents, and whether such agents are administered separately or mixed.

· ··· · * · ···

The anti-inflammatory activity of the combination therapies of the present invention can be determined using various animal models known in the art for inflammatory arthritis [Crofford LJ and Wilder RL: "Arthritis and Autoimmunity in Animals", In: Arthitis and Allied Conditions: The Textbook of Rheomatology, Chapter 30, (McCarty et al., Lee and Febiger, 1993)]. Experimental and spontaneous animal models of inflammatory arthritis and autoimmune rheumatic diseases may also be used to estimate the anti-inflammatory activity of the combination therapies of the present invention. Here are a few essays to illustrate, without limiting ourselves to these.

The main animal models of arthritis or inflammatory disease known and widely used in the art are: adjuvant-induced arthritis rat models, collagen-induced arthritis rat and mouse models, and antigen-induced arthritis rat, rabbit and hamster models [Crofford LJ and Wilder RL: "Arthritis and Autoimmunity in Animals, In: Arthitis and Allied Conditions: The Textbook of Rheomatology, Chapter 30, McCarty et al. (Lee and Febiger, 1993), the disclosure of which is incorporated herein by reference in its entirety, for all purposes.

The anti-inflammatory activity of the combination therapies of the present invention can be estimated in a carrageenan-induced arthritis rat model. Carrageenan-induced arthritis is used in rabbits, dogs and pigs, with

285 • · «·« 2 · * ·· ·· · ♦ · ··· »« '· · «while studying chronic arthritis or inflammation. Quantitative histomorphometric estimation is used to determine therapeutic efficacy Methods using such a carrageenan-induced arthritis model have been described by Hansra P. et al., Hansra P. et al., Carrageenan-Induced Arthritis in the Rat, Inflammation 24 (2), 141 -155 (2000)].

In addition, zymosan-induced inflammatory animal models have also been commonly used, as is known and published in the art.

The anti-inflammatory activity of the combination therapies of the present invention can be determined by measuring the inhibition of carrageenan-induced paw edema in a rat using a modification of the method described by Winter CA et al. [Winter CA et al., Carrageenan-Induced Edema in Hind Paw of the Rat as an Assay Forum Anti-Inflammatory Drugs, Proc. Soc. Exp. Bioi. Med. 111: 544-547 (1962). This assay is used as a primary in υίνο screening test for the anti-inflammatory activity of most NSAIDs and is considered as a predictor of human efficacy. The anti-inflammatory activity of the test prophylactic or therapeutic agents is expressed as a percentage of the increase in the weight of the hind paw of the test group relative to the vehicle treated group.

In a specific embodiment of the present invention, when the experimental animal model used is an adjuvant-induced arthritis rat model, body weight can be measured relative to a control group in order to determine the presence of the present · · ♦ * · * * «· · · · · · · · · · • · ·

The anti-inflammatory activity of the combination therapies of the present invention. Combined therapies include, but are not limited to, the following combinations: any integrin α ν ββ antagonist that is functionally homologous to VITAXIN ™, a TNFα inhibitor, and a chemotherapeutic agent. RENBREL ™, which is a rat homolog of ENBREL ™ and acts as a TNFa inhibitor, can also be tested in combination therapies in rat models.

Alternatively, the efficacy of the combination therapies of the present invention may be determined using assays that determine bone loss. Animal models, such as overiectomy-induced bone resorption in mouse, rat, and rabbit models are known in the art, and dynamic bone formation parameters can be obtained. Using methods such as those published by Yositake et al. Or Yamamoto et al. of Sciences, USA 96, 8156-8160 (1999); Yamamoto et al., "The Integrin Ligand Echistatin Prevents Bonus Loss in Ovariectomized Mice and Rats" Endocrinology 139 (3), 1411-1419 (1998), the disclosures of which are hereby incorporated by reference in their entirety.

287; ··· · · 4 · ♦ · 99 • *

In addition, animal models of inflammatory bowel disease may also be used to estimate the efficacy of the combination therapies of the present invention [Kim et al., Scand. J. Gastroenterol. 27, 529-537 (1992); Strober et al., Dig. Dis. Sci. 30 (12 burns.), 3S10S (1985)]. Ulcerative colitis and Crohn's disease are human inflammatory bowel diseases that can be induced in animals. Sulphated polysaccharides include, but are not limited to, amylopectin, carrageenan, amylopectin sulfate and dextran sulfate, or chemical irritants, including, but not limited to, trinitrobenzenesulfonic acid (TNBS) and acetic acid. which can be administered orally to animals to induce inflammatory bowel diseases.

Animal models of asthma may also be used to evaluate the efficacy of the combination therapies of the present invention. An example of such a model is the rodent adoptive transfer model in which the aeroallergen provocation of the TH1 or TH2 recipient mice results in TH effector cells migrating into the airways and associating with an intense neutrophil (TH1) and eosinophil (TH2) lung mucosal inflammatory response [Cohn] J. Exp. Med. 186: 1737-1747 (1997).

Animal models of autoimmune disorders may also be used to evaluate the efficacy of the combination therapies of the present invention. Autoimmune disorders, such as type 1 diabetes, thyroid autoimmunity, systemic lupus erythematosis, and glomerulonephritis are animals288 * »** · · · · · ·« ϊ · f · «·» «·· · · · Delhi has been developed [Flanders et al., Autoimmunity 29, 235246 (1999); Krogh et al., Biochimie 81: 511-515 (1999); Foster: Smin. Nephrol. 19: 12-24 (1999).

In addition, any of the assays known to those skilled in the art may be used to estimate the prophylactic and / or therapeutic utility of the combination therapy disclosed herein for the treatment of autoimmune and / or inflammatory diseases.

The effect of the combination therapies of the present invention on peripheral blood lymphocyte numbers can be monitored / estimated using standard techniques known to those skilled in the art. The number of peripheral blood lymphocytes in a subject can be determined, for example, by taking a peripheral blood sample from said subject, lymphocytes are separated from other components of peripheral blood, such as plasma, using Ficoll-Hypaque (Pharmacia) gradient centrifugation and lymphocytes. counted with trypan blue. For example, T-cell numbers for peripheral blood can be determined by lymphocytes being separated from other peripheral blood components, such as plasma, using Ficoll-Hypaque (Pharmacia) gradient centrifugation, and T cells from a T cell antigen. such as, for example, CD3, CD4, and CD8 antibodies conjugated to FITC or phycoerythrin, and the number of T cells measured by FACS.

The percentage of CD2 polypeptides expressed by the peripheral blood T cells bound by the CD2 binding molecules is the administration of one or more doses of the CD2 binding molecule and one or more doses of the other pro289 • * · · *> ♦ · · · ♦ · filactic or therapeutic agents. before or after, or both before and after, can be estimated using standard techniques known to those skilled in the art. The percentage of CD2 polypeptides expressed by CD2 binding molecules expressed by peripheral blood T cells can be determined, for example, by taking a peripheral blood sample from a subject, lymphocytes are separated from other components of peripheral blood, such as plasma, for this purpose. Using Hypaque (Pharmacia) gradient centrifugation, T cells are labeled with an FITC conjugated anti-CD2 binding molecule antibody and an antibody directed against CD4 conjugated to a T cell antigen, such as CD3, CD4, or phycoerythrin. and determining FACS the number of T cells labeled with the anti-CD2 binding molecule relative to the number of T cells labeled with an antibody to a T cell antigen.

The toxicity and / or efficacy of the prophylactic and / or therapeutic protocols of the present invention can be determined by standard pharmaceutical methods in cell cultures or in experimental animals, such as determining the LD50 (lethal dose to 50% of the population) and ED50 (50% of the population). therapeutically effective dose). The ratio between doses of toxic and therapeutic effects is the therapeutic index and is expressed as the LD50 / ED50 ratio. Prophylactic and / or therapeutic agents having a high therapeutic index are preferred. Although prophylactic and / or therapeutic agents with toxic side effects may be used, care should be taken

290 «« sky * ·· * <* · »* · *

- · »··

· "To design a delivery agent that directs such agents into the affected tissue to minimize potential damage to uninfected cells and thereby reduce side effects.

Data from cell culture studies and animal studies can be used to design a dose range for prophylactic and / or therapeutic agents for use in humans. The dosage of such agents is preferably within the range of circulating concentration that contains ED 50 with little or no toxicity. The dose may vary within this range depending on the dosage form used and the route of administration. For any agent used in the methods of the present invention, a therapeutically effective dose should first be estimated in cell culture. Dosage can be formulated in animal models to achieve a circulating plasma concentration range that contains the IC50 value (i.e., the concentration of test compound that results in half-maximal inhibition of symptoms as determined in cell cultures. Such information may be used to more accurately determine the usefulness in humans). Plasma levels can be measured, for example, by high pressure liquid chromatography.

Antibodies specifically binding to an antigen may be prepared in the art by methods known to synthesize antibodies, for example, by chemical synthesis, or more preferably by recombinant expression techniques.

291 £ ♦ »« r ·. r • * · · - »

- V «» «'· ·

Polyclonal antibodies specific for an antigen may be prepared by various methods well known in the art. For example, a human antigen may be administered to various host animals, including, but not limited to, rabbits, mice, rats, and the like. to induce serum production containing polyclonal antibodies specific for the human antigen. Various adjuvants may be used to enhance the immune response, depending on the host species, and include, but are not limited to, Freund's adjuvant (complete and incomplete), mineral gels such as aluminum hydroxide, surfactants, e.g. for example, lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, borehole hemocyanin, dinitrophenol, and potentially useful human adjuvants such as BCG (Bacillus Calmette-Guerin) and Corynebacterium parvum. Such adjuvants are well known in the art.

Monoclonal antibodies can be prepared by a wide variety of techniques known in the art, including hybridoma, recombinant and phage display technologies, or a combination thereof. For example, monoclonal antibodies may be produced by hybridoma techniques, including those known in the art, as described by Harlow et al., "Antibodies: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1988) ; Hammerling et al., In: Monoclonal Antibodies and T-cell Hybridomas 563-681, Elsevier, New York (1981)] (which are hereby incorporated by reference in their entirety). The term "monoclonal antibody" is no longer limited to antibodies produced by hybridoma technology. The term "monoclonal antibody" refers to an antibody derived from a single clone, including any eukaryotic, prokaryotic or phage clones, and not including the method that has been prepared.

Methods for producing and screening specific antibody using hybridoma technology are well known in the art. Briefly, mice may be immunized with a non-rodent antigen and when the immune response is detected, i.e., antigen specific antibodies are detected in the mouse serum, the spleen of the mice is harvested and the splenocytes are isolated. The splenocytes are then fused with well-known techniques with cells of any of the myeloma cells, such as the SP21 cell line available from the American Type Culture Collection. Hybridomas are selected and cloned with limited dilution. The hybridoma clones are then analyzed by methods known in the art, looking for cells that secrete antibodies capable of binding to the polypeptide of the present invention. Ascites fluid, which generally contains high levels of antibodies, can be produced by immunizing mice with positive hybridoma clones.

Accordingly, the present invention provides methods for producing monoclonal antibodies as well as monoclonal antibodies produced by the method, characterized in that a hybridoma cell secreting an antibody of the present invention is cultured, wherein the hybridoma is preferably prepared by immunizing a non-rodent antigen. mouse splenocytes are fused with myeloma cells, and the fusion hybridomas are screened for hybridoma clones secreting the antibody capable of binding to the antigen.

Antibody fragments recognizing each specific epitope can be prepared by any technique known to those skilled in the art. For example, the Fab and F (ab ') 2 fragments of the present invention may be produced by proteolytic cleavage of immunoglobulin molecules, using enzymes such as papain (to produce Fab fragments) or pepsin (F (ab') 2 fragments). At F (ab ') 2 fragments include the heavy chain variable region, the light chain constant region, and the CH1 domain. Further, the antibodies of the present invention may also be prepared by various phage display methods known in the art.

In phage display methods, there are functional antibody domains on the surface of phage particles that carry the polynucleotide sequences encoding them. Specifically, DNA sequences encoding VH and VL domains are amplified from animal cDNA libraries (e.g., from human or rodent cDNA libraries of affected tissues). The DNA encoding the VH and VL domains were recombined by using a polymerase chain reaction with a scFv linker and cloned into a phagemid vector. The vector

294

It is electroporated into Escherichia coli and infected with a phage that aids Escherichia coli. The phages used in these methods are generally filamentous phages, including fd and M13 phage, and the VH and VL domains are generally fused to recombinant methods to the phage III gene or VlII gene. A phage expressing an antigen-binding domain that binds to a particular antigen may be selected or identified with an antigen, such as a labeled antigen, or a antigen bound to a solid surface or bead or captured. Examples of phage display methods for preparing antibodies of the present invention are described in the literature (Brinkman et al., J. Immunological Methods 182: 41-50 (1995); Ames et al., J. Immunological Methods 184, 177-186 (1995);

Kettkeborough et al., Eur. J. Immunol. 24, 952-958 (1994); Persic et al., Gene 187, 9-18 (1997); Burton et al., Advances in Immunology 57, 191-280 (1994); PCT / GB91 / O1 134 PCT Submission; PCT publications WO 90/02809, WO 91/10737, WO 92/01047, WO 92/18619, WO 93/11236, WO 95/15982, WO 95/20401 and WO 97/13844, and 5,698,426, 5,403,484, 5,580,717, 5,427,908, 5,750,753,

5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225,

U.S. Patent Nos. 5,658,727, 5,733,743, and 5,969,108; which publications will be treated as reference in their entirety].

As described in the previous references, after the phage selection, the regions encoding the antibody can be isolated from the phage, and f

These may be used to produce complete antibodies, including human antibodies, and may be used to produce any other desired antigen-binding fragment and may be expressed in any desired host including mammalian cells, insect cells, plant cells, yeasts and bacteria as described below. Techniques for recombinant production of Fab, Fab 'and F (ab') 2 fragments can also be used using methods well known in the art [PCT Publication No. WO 92/22324; Mullinax et al., Bio / Techniques 12 (6), 864-869 (1992); Sawai et al., AJRI 34, 26-34 (1995); Better et al., Science 240: 1041-1043 (1988), the disclosures of which are hereby incorporated by reference in their entirety.

For the production of complete antibodies, polymerase chain reaction primers containing VH and VL nucleotide sequences, a restriction cleavage site and a delimiting sequence can be used to amplify the VH and VL sequences in the scFv clone. Using cloning techniques known to those skilled in the art, VH domains amplified by polymerase chain reaction may be cloned into vectors expressing the VH constant region, such as the human gamma 4 constant region, and VL domains amplified by polymerase chain reaction may be cloned into the VL constant region, such as human kappa. or vectors expressing lambda constant regions. Vectors expressing VH or VL domains preferably contain an EFla promoter, a secretion signal, and a cloning site for vari?

296 for bile domain, constant domains, and a selection marker such as neomycin. VH and VL domains can also be cloned into a vector that expresses the required constant regions. Heavy chain conversion vectors and heavy chain conversion vectors are then co-transfected into cell lines to generate stable or transient cell lines expressing full-length antibodies, such as IgG, using techniques known to those skilled in the art.

In some uses, including the use of antibodies in humans and in vitro detection assays, it may be advantageous to use human or chimeric antibodies. Fully human antibodies are particularly desirable in the therapeutic treatment of human subjects. Human antibodies can be prepared by a variety of methods known to those skilled in the art using antibody libraries from human immunoglobulin sequences (U.S. Patent Nos. 4,444,887 and 4,716,111; WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735 and WO 91/10741, the disclosures of which are hereby incorporated by reference.

Human antibodies can be produced by transgenic mice that are unable to express functional endogenous immunoglobulins, but are capable of expressing human immunoglobulin genes. For example, human heavy chain and light chain immunoglobulin gene complexes are random or homologous

297 can be introduced into mouse embryonic stem cells by recombination. Alternatively, the human variable region, constant region, and diversity region may be introduced into mouse embryonic stem cells along with human light chain and heavy chain genes. Mouse heavy chain and light chain genes can be made either separately or simultaneously by the introduction of human immunoglobulin loci by homologous recombination. Specifically, homozygous deletion of the Jh region prevents the production of endogenous antibody. Modified embryonic stem cells are propagated and microinjected into blastocytes to produce chimeric mice. The chimeric mice are then cultured to obtain homozygous progeny expressing human antibodies. The transgenic mice are then normally immunized with a selected antigen, such as a polypeptide of the present invention, or a portion thereof. Monoclonal antibodies against antigen can be obtained from transgenic skies by conventional hybridoma technology. Human immunoglobulin transgenes carried by transgenic mice are rearranged during B-cell differentiation, followed by class change and somatic mutation. Thus, by using such a technique, therapeutically useful IgG, IgA, IgM and IgE antibodies can be produced. A summary of this technology for the production of human antibodies can be found in the literature [Lonberg and Huszar: Int. Immunol. 13, 65-93 (1995). A detailed discussion of the pro298 tokens of human antibodies and human monoclonal antibodies and the preparation of such antibodies is disclosed in the literature (WO 98/24893, WO 96/34096 and WO 96/33735; 5,413,923, 5,625,126, 5,633,425, 5,569,825,

U.S. Patent Nos. 5,661,016, 5,545,806, 5,814, 318, and 5,939,598, which are hereby incorporated by reference in their entirety. In addition, various companies, such as Abgenix, Inc. (Freemont, CA) and Genpharm (San Jose, CA), may be involved in providing human antibodies against a selected antigen using the technologies described above.

A chimeric antibody is a molecule in which different portions of the antibody are derived from different immunoglobulin molecules, such as a variable region of a human antibody and a constant region of a non-human immunoglobulin. Methods for producing chimeric antibodies are known in the art [Morrison, Science 229, 1202 (1985); Oi et al. BioTechniques 4, 214 (1986); Gillies et al., J. Immunological Methodsl25, 191-202 (1989); and U.S. Patent Nos. 5,807,715, 4,816,567, and 4,816,397, which are hereby incorporated by reference in their entirety. The chimeric antibodies containing one or more CDRs from one human and framework regions from a non-human immunoglobulin molecule can be prepared by a variety of techniques known in the art, including, for example, CDR grafting (EP 239,400; PCT Pub. U.S. Patent Nos. 5,225,539, 5,530,101 and 5,585,089), obscuring, or re-forming the surface [EP 592,106; EP 519,596; Padlan: Molecular Immunology 28 (4/5), 489-498 (1991); Studnicka et al., Protein Engineering 7 (6), 805-814 (1994); Roguska et al., Proceedings of the National Academy of Sciences, USA 91, 969-973 (1994), and chain blending (U.S. Patent No. 5,565,332). In a preferred embodiment, the chimeric antibodies comprise a human CDR3 having an amino acid sequence identical to the amino acid sequence of any of the CDR3s listed in Tables 1 or 2 and comprising non-human framework regions. In the framework regions, the framework region groups are often replaced by the appropriate group derived from the CDR donor antibody to alter, preferably improve antigen binding. Such framework region substitutions can be identified by means of the wobble method, such as modeling interactions between the CDR and the framework region to identify framework regions that are important for antigen binding and also to compare sequences to identify unusual framework regions at a given position [see for example, Qeen et al., U.S. Patent No. 5,585,089; Riechmann et al., Nat. 332, 323 (1988), the disclosures of which are hereby incorporated by reference in their entirety.

* ·· ····

300

J.

··

However, antibodies specifically binding to an antigen (e.g., a CD2 polypeptide) may be used to produce anti-idiotypic antibodies that "mimic" an antigen using techniques well known to those skilled in the art (see, for example, Greenspan & Bona, FASEB J. 7 (5)) 437-444 (1989); Nissinoff, J. of Immunol. 147 (8): 2429-2438 (1991).

The present invention relates to polynucleotides comprising a nucleotide sequence encoding an antibody or fragment thereof that specifically binds to an antigen. The present invention further provides polynucleotides that hybridize under stringent, moderate, or low stringency conditions (e.g., as noted above) to polynucleotides encoding the antibody of the present invention.

The polynucleotides may be prepared by any of the methods known in the art and the nucleotide sequence of the polynucleotides may be determined. The nucleotide sequence of antibodies specific for an antigen may be obtained, for example, from the literature or from a database such as GenBank. Since the amino acid sequence of VITAXIN ™ is known, the nucleotide sequences encoding this antibody may be determined by methods well known in the art, such as nucleotide codons encoding the selected amino acids being generated to generate the nucleic acid encoding the antibody. Such a polynucleotide encoding the antibody is chemically synthesized. ···: "· gonucleotides can be constructed [e.g. Kutmeier et al., BioTechniques 17: 242 (1994)], which briefly include the synthesis of overlapping oligonucleotides containing details of the sequence of the antibody sequence, and fitting and ligating these oligonucleotides. the oligonucleotides are amplified by polymerase chain reaction.

Alternatively, a polynucleotide encoding an antibody may be generated from a nucleic acid from a suitable source. If a clone containing a nucleic acid encoding a particular antibody is not available, but the antibody molecule sequence is known, the immunoglobulin encoding nucleic acid may be chemically synthesized or produced from a suitable source (e.g., from a cDNA library generated from an antibody cDNA library or a polyA + RNA, or such a nucleic acid, which can be isolated from any antibody-expressing tissue or cells, such as hybridoma cells, which are selected on the basis of expression of the antibody of the present invention by using polymerase chain reaction amplification using synthetic primers hybridizing to the 3 ' and 5 ' ends of the sequence or by cloning using an oligonucleotide probe specific for a gene sequence to be identified, such as cDNA clone from a cDNA library encoding the antibody. The amplified nucleic acids generated by the polymerase chain reaction can then be cloned into replicating cloning vectors by any of the methods known in the art.

302 • · «· ··· · <« ··:

Once the nucleotide sequence of the antibody is determined, the nucleotide sequence of the antibody can be manipulated using methods well known in the art for manipulating nucleotide sequences, such as recombinant DNA techniques, site-specific mutagenesis, polymerase chain reactions, and the like. see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual; 2nd Edition, Cold Spring Harbor Press, Cold Spring Harbor, NY (1989); Current Protocols in Molecular Biology, eds. Ausubel et al., Greene Publishing and WileyInterscience: New York (1987)], which are hereby incorporated by reference in their entirety in order to generate antibodies having different amino acid sequences, such as amino acid substitutions, deletions and / or insertions are made.

In a specific embodiment, one or more CDRs are inserted into the framework regions using routine recombinant DNA techniques. The framework regions may be natural or consensus framework regions, preferably human framework regions (see, for example, Chothia et al., Journal of Molecular Biology 278, 457-479 (1998), where human framework regions are listed). Preferably, the polynucleotide generated by a combination of framework and CDRs encodes an antibody that specifically binds to a particular antigen. As discussed above, one or more amino acid substitutions may be made in the framework regions, and preferably, the amino acid substitutions improve the binding of the antibody to the antigen. In addition, such methods can be used to

303 **; j · * · ·· ••• j »

. »··· ·. · To 4 amino acid substitutions, deletions or execute one or more variable region cysteine groups in place, which are involved in a disulfide bridge between the chains, thereby producing an antibody molecules that make up one or more disulfide bond is missing. Other variations of the polynucleotide are also contemplated by the present invention and are within the skill of those skilled in the art.

Recombinant expression of an antibody specifically binding to an antigen requires the generation of an expression vector comprising the polynucleotide encoding the antibody. Once the polynucleotide encoding the antibody molecule of the present invention is obtained, the vector used to produce the antibody molecule can be produced by recombinant DNA technology using methods well known in the art (see, for example, U.S. Patent No. 6,331,415, which is hereby incorporated by reference in its entirety. , we treat all purposes as reference). Thus, methods for expressing a polynucleotide comprising a nucleotide sequence encoding an antibody are described. Methods well known to those skilled in the art can be used to construct expression vectors containing antibody coding sequences and appropriate transcriptional and translational control signals. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Thus, the present invention relates to replicating vectors, ·· ♦ ·

- · ··· • * · ·· »···

304 containing an antibody molecule of the present invention, a heavy chain and light chain of an antibody, a heavy chain or light chain variable domain of an antibody or portion thereof, or a nucleotide sequence encoding a heavy chain or light chain CDR, operably linked to a promoter . Such vectors contain the nucleotide sequence encoding the constant region of the antibody molecule (see, for example, WO 86/05807, PCT Publication WO 89/01036, and U.S. Patent No. 5,122,464, and the variable domain of antibody). such a vector may be cloned to express the entire heavy chain or light chain, or both the complete heavy chain and the complete light chain.

The expression vector can be introduced into a host cell by conventional techniques, and the transfected cells are propagated by conventional techniques to produce the antibody of the present invention. Thus, the present invention relates to host cells comprising an antibody of the present invention, or a fragment thereof, or a heavy chain or light chain thereof, or a portion thereof, or a single chain antibody coding polynucleotide, operably linked to a heterologous promoter. In preferred embodiments of the expression of two-chain antibodies, both the heavy chain and the light chain may be co-expressed in the host cell to express the entire immunoglobulin molecule as described below.

305 • í: * ♦ · »··· ··· ♦» · • 9 «

Many different host vector systems can be used to express antibody molecules of the present invention (see, for example, U.S. Patent No. 5,807,715). Such host expression systems are carriers that can be produced by coding sequences of interest to us, but also represent cells that, when transformed or transfected with the appropriate nucleotide coding sequences, express the antibody molecule of the present invention in situ. These include, but are not limited to, transforming microorganisms such as bacteria (e.g., Eschenchia coli and Bacillus subtilis), recombinant bacteriophage DNA containing antibody coding sequences, plasmid DNA, or cosmid DNA expression vectors; yeasts (e.g., Saccharomyces, Pichia) transformed with recombinant yeast vectors containing the antibody coding sequence; insect cell systems infected with recombinant viral expression vectors (e.g., baculovirus) that contain antibody coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV, tobacco mosaic virus, TMV), or recombinant plasmid expression vectors (e.g., Ti plasmid) containing antibody coding sequences or mammalian cell systems ( such as COS, CHO, BHK, 293, NSO, and 3T3 cells, which are recombinant exp • fc · ♦ · · · · · ·> · · · · ·

306 · «:,.

They carry carrying structures (such as a metallothionein promoter) or a promoter derived from mammalian viruses (such as the late promoter of the adenovirus, the 7.5 K promoter of the vaccine virus). Preferably, bacterial cells such as Escherichia coli, or more preferably eukaryotic cells, are preferably used to produce a recombinant antibody molecule. For example, mammalian cells, such as golden hamster ovary cells (CHO), are an effective expression system for antibodies to a antibody, such as a major direct gene promoter element derived from a vector, such as human cytomegalovirus (Foecking et al., Gene 45, 101 (1986); Cockett et al., Bio / Technology 8: 2 (1990). In a specific embodiment, expression of nucleotide sequences encoding antibodies specifically binding to one or more antigens is regulated by a constitutive promoter, an inducible promoter, or a tissue-specific promoter.

In bacterial systems, a number of expression vectors may be advantageously selected, depending on the purpose for which the expressed antibody is to be used. For example, if a large amount of such protein is to be produced to generate a pharmaceutical composition of an antibody molecule, those vectors that control the high-level expression of the easily purified fusion protein products are desirable. Such vectors include, but are not limited to, the Escherichia coli pUR278 vector [Ruther et al., EMBO Journal.

307

12, 1791 (1983)], in which the antibody coding sequence may be ligated separately to the vector in a reading frame with the lacZ coding region such that a fusion protein is produced; pIN vectors [Inouye & Inouye: Nucleic Acids Research 13: 31013109 (1985); Van Heeke & Schuster, Journal of Biological Chemistry 24, 5503-5509 (1989), and the like. The pGEX vectors can be used to express foreign polypeptides as a fusion protein formed by glutathione-5 transferase (GST). In general, such fusion proteins are soluble and can be easily purified from lysed cells by adsorption and binding to a glutathione agarose bead matrix followed by elution in the presence of free glutathione. The pGEX vectors are designed to contain factor Xa protease cleavage sites, thereby releasing the cloned gene product from the GST unit.

In an insect system, Autographa californica nuclear polyhedrosis (AcNPV) is used as a vector to express foreign genes. The virus multiplies in Spodoptera frugiperda cells. The antibody coding sequence may be cloned separately into non-essential regions of the virus (e.g., the polyhedrin gene) and placed under the control of an AcNPV promoter (e.g., a polyhedrin promoter).

Numerous viral-based expression systems can be used in mammalian host cells. In cases where an adenovirus is used as an expression vector, the antibody coding sequence of interest is ligated into an adenovirus transcription / translation control complex, e.g.

308 promoters and three-part leader sequences. This chimeric gene can then be inserted into the genome of the adenovirus by in vitro or in vivo recombination. Insertion into the non-essential region of the viral genome (i.e., the region of E1 or E3) results in a recombinant virus that is viable and capable of expressing the antibody molecule in infected hosts (see, for example, Logan & Shenk, Proceedings of the National Academy of Sciences, USA 81: 355-359 (1984)]. Specific initiation signals may also be required for efficient translation of the sequences encoding the built-in antibody. These signals include the ATG initiation codon and adjacent sequences. In addition, the initiation codon must be in a reading phase with the reading frame of the desired coding sequence to ensure translation of the complete insert. These exogenous translational control signals and initiation codons may be of any origin, including natural and synthetic origin. The efficiency of expression can be enhanced by suitable transcription enhancing elements, transcription terminators, and the like. (see, for example, Bittner et al., Methods in Enzymology 153: 51-544 (1987)).

In addition, a host cell can be selected that modulates the expression of the inserted sequences or modifies and processes the gene product in the desired specific manner. Such modifications of protein products (e.g., glycosylation) and processing (e.g. cleavage) may be important for the function of the protein. Different host cells are specific and specific

There are 309 mechanisms for post-translational modification of proteins and gene products. Suitable cell lines or host cells may be selected to provide correct modification and processing of the expressed foreign gene. For this purpose, eukaryotic host cells having the cellular mechanism necessary for the proper processing of the primary transcript, glycosylation and phosphorylation of the gene product can be used. Such mammalian host cells include, but are not limited to, CHO, VERY, BHK, HeLa, COS, MDCK, 293, 3T3, W138, BT483, Hs578T, HTB2, BT2O and T47D, NSO (a murine myeloma cell line that does not produce endogenously any immunoglobulin chain), CRL7O3O and HsS78Bst cells.

Long-term stable expression of recombinant proteins is preferred. For example, biotechnological methods can produce cell lines that stably express antibody molecules. Instead of using expression vectors containing a viral origin of replication, host cells can be transformed with appropriate expression control elements (e.g., promoter, enhancer sequences, transcription terminators, polyadenylation sites, etc.) and a selection marker. Following the introduction of foreign DNA, it may be possible for the altered cells to grow for 1-2 days in a complete medium and then transferred to selective media. Selection marker in the recombinant plasmid provides resistance to selection and allows cells to stably integrate plas310 mid into their chromosome and grow focal points, which are then cloned and cultured into cell lines. This method can advantageously be used to produce cell lines that express the antibody molecule. Such altered cell lines are particularly useful for the preparation of compositions that interact directly or indirectly with the antibody molecule.

Many different selection systems can be used, including, but not limited to, the herpes simplex virus thymidine kinase (Wigler et al., Cell 11: 223 (1977)), hypoxantinguanine phosphoribosyl transferase [Szybalska & Szybalski: Proceedings of the National Academy of Sciences, USA 48, 202 (1992)] and adenine phosphoribosyltransferase (Lowry et al., Cell 22, 8-17 (1980)) can be used in tk, hgprt or cells. In addition, antimetabolite resistance can be used as the basis for selection of the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., Proceedings of the National Academy of Sciences, USA 77, 357 (1980); O'Hare et al., Proceedings of the National Academy of Sciences, USA 78: 1527 (1981)]; gpt, which confers resistance to mycophenolic acid [Mulligan & Berg, Proceedings of the National Academy of Sciences, USA 78, 2072 (1981)]; neo, which confers resistance to G-418 aminoglycoside [Wu and Wu: Biotherapy 3, 8795 (1991); Tolstoshev: Ann. Port. Pharmacol. Toxicol. 32: 573-596 (1993); Mulligan, Science 260: 926-932 (1993); Morgan and Anderson: Annu. Port. Biochem. 62: 191-217 (1993); TIBTECH

311 ♦♦ 999 · «

11 (5), 155-215 (May 1993)]; and hygro, which provides hygromycin resistance (Santerre et al., Gene 30: 147 (1984)). Methods commonly known in recombinant DNA technology can be routinely used to select the desired recombinant clone and are described in Current Protocols in Molecular Biology, ed. Ausubel et al., Greene Publishing and Wiley-Interscience: New York (1987); Kriegler: Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990); Current Protocols in Human Genetics, Vol

Chapter 13, Dracopoli et al., John Wiley and Sons New York (1994); Colberre-Garapin et al., Journal of Molecular Biology 150, 1 (1981)], the disclosures of which are hereby incorporated by reference in their entirety.

Expression levels of an antibody molecule are increased by vector amplification [Bebbington and Hentschel: The expression of cloned genes in mammalian cells in DNA cloning, 3, Academic Press, New York (1987)]. If the antibody can be amplified by a marker in an expression vector system, an increase in the level of inhibitor in the host cell culture increases the copy number of the marker gene. Since the amplified region is associated with the antibody gene, antibody production is also increased (Crouse et al., Molecular & Cellular Biology 3: 257 (1983)).

The host cell may be co-transfected with two expression vectors of the present invention, the first vector encoding the heavy chain polypeptide, the second vector encoding the light chain po312 lipeptide. The two vectors may contain the same selection markers, which allow the same expression of the light chain and heavy chain polypeptide. Alternatively, a single vector can be used to encode and express both heavy chain and light chain polypeptides. In such situations, the light chain must be placed in front of the heavy chain to avoid the excess of the toxic free heavy chain [Proudfoot: Nature 322: 52 (1986); Kohler: Proceedings of the National Academy of Sciences, USA 77: 2197 (1980)]. The heavy chain and light chain coding sequences may contain cDNA or genomic DNA.

If the antibody of the present invention is produced by recombinant expression, it can be purified in the art by any of the methods known for purifying an immunoglobulin molecule, such as chromatography (e.g., ion exchange, affinity, in particular affinity for specific antigen after Protein A, and size exclusion column chromatography), centrifugation, solubility. or by any other standard technique used to purify proteins. In addition, the antibodies of the present invention, or fragments thereof, may be fused to heterologous polypeptide sequences described herein or known in the art to facilitate purification.

The polypeptides and fusion proteins may be prepared by standard recombinant DNA techniques, or protein synthesis techniques, or by a peptide synthesizer. For example, a polypeptide

Nucleic acid molecules encoding 313 or fusion proteins can also be synthesized by conventional techniques with automated DNA synthesizers. Alternatively, the gene fragments can be made by primers that fix polymerase chain reaction amplification, providing complementary overhanging ends between two consecutive gene fragments that can then be matched and amplified to produce a chimeric gene sequence [Current Protocols in Molecular Biology, ed. et al., Greene Publishing and Wiley-Interscience: New York (1992)]. In addition, a nucleic acid encoding a biologically active molecule may be cloned into an expression vector containing the Fc domain or a fragment thereof, such that the biologically active molecule is bound to the Fc domain or Fc domain fragment in a reading frame.

Methods for fusion or conjugation of polypeptides to constant regions of antibodies are known in the art [5,336,603, 5,622,929, 5,359,046, 5,349,053, 5,447,851, 5,723,125, 5,783,181, 5,909,626,

U.S. Patent Nos. 5,844,095 and 5,112,946; EP 307,434; EP 367,166; EP 394,827; PCT Publication WO 91/06570, WO 96/04388, WO 96/22024, WO 97/34631 and WO 99/04813; Ashkenazi et al., Proceedings of the National Academy of Sciences, USA 88, 10535-10539 (1991); Traunecker et al., Natura 331, 84-86 (1988); Zheng et al., J. of Immunol. 154, 5590-5600 (1995); Vil et al., Proceedings of the National Academy of Sciences,

314

USA 89: 11337-11341 (1992); which publications will be treated as reference in their entirety].

Nucleotide sequences encoding a biologically active molecule and an Fc domain or fragment thereof may be prepared based on information available to one of ordinary skill in the art (e.g., Genbank, literature, or routine cloning). The nucleotide sequence encoding a polynucleotide of a fusion protein may be incorporated into a suitable expression vector, such as a vector containing the elements required for transcription and translation of the inserted protein coding sequence. Various host-vector systems can be used in the present invention to express the protein coding sequence. These include, but are not limited to, mammalian cell systems infected with the virus (e.g., vaccine virus, adenovirus scar, etc.); virus infected insect cells (e.g., baculovirus); microorganisms, such as yeast, containing a yeast vector; or bacteria transformed with bacteriophage, DNA, plasmid DNA, or cosmid DNA. The expression elements of the vectors vary in their strength and specificity. Depending on the host vector systems used, any of the appropriate transcriptional and translational elements may be used.

Expression of a polypeptide or fusion protein can be controlled by any of the promoters or enhancer elements known in the art. Promoters for regulating the expression of a gene encoding a fusion protein include, but are not limited to

315, the SV40 early promoter region [Bernoist and Chambon: Natúré 290, 304-310 (1981)], a promoter of the 3 'long terminal repeat of the Rous sarcoma virus (Yamamoto et al., Cell 22, 787-797 (1980)). )], the herpes thymidine kinase promoter (Wagner et al., Proc. of the National Academy of Sciences, USA 78: 1441-1445 (1981)), regulatory sequences of the metallothionein gene (Brinster et al., Natura 296, 39-42). 1982)], the tetracycline (Tet) promoter [Gossen et al., 1995, Proceedings of the National Academy of Sciences, USA 89: 5547-5551], prokaryotic expression vectors, such as the beta-lactamase promoter [Villa-Kamaroff, et al. Proceedings of the National Academy of Sciences, USA 75, 37273731 (1978)] or the tac promoter [DeBoer et al., Proceedings of the National Academy of Sciences, USA 80, 21-25 (1983), see also: Useful proteins from recombinant bacteria, Scientific American 242, 74-94 (1980); plant expression vectors containing the nopaline synthase promoter region (HerreraEstrella et al., Natura 303, 209-213) or cauliflower mosaic virus 35S promoter (Gardner et al., Nucleic Acids Research 9, 2781 (1981)), and photosynthetic ribulose promoter of 1,5-bisphosphate carboxylase enzyme (Herrera-Estrella et al., Nat. 310, 115120 (1984)); promoter elements from yeast or other fungi, such as the Gal4 promoter, the ADC (alcohol dehydrogenase) promoter, the PGK (phosphoglycerokinase) promoter, the alkaline phosphatase promoter, and the following animal transcriptional control regions having tissue specificity, and in transgenic animals

316 were used: elastase I gene control region active in pancreatic acinar cells (Swift et al., Cell 38, 639-646 (1984); Ornitz et al., Cold Spring Harbor Symp. Quant. Biol. 50, 399-409 (1986); MacDonald: Hepatology 7: 425-515 (1987); the control region of the insulin gene active in pancreatic beta cells (Hanahan: Natré 315, 115-122 (1985)), the control region of the immunoglobulin gene active in lymphoid cells (Grosschedl et al., Cell 38: 647-658 (1984)) ); Adames et al., 1985, Natura 318, 533-538; Alexander et al., Molecular & Cellular Biology 7: 1436-1444 (1987)], a mouse breast tumor virus control region active in testicular, breast, lymphoid and mast cells [Leder et al., Cell 45, 485-495 (1986)] , albumin gene control region active in the liver [Pinkert et al. Genes and Development J_, 268-276 (1987)], the control region of the alpha-fetoprotein gene active in the liver [Krumlauf et al., Molecular & Cellular Biology 5: 1639 -1648 (1985); Hammer et al., Science 235: 53-58 (1987); the control region of the alpha-1-antitrypsin gene that is active in the liver [Kelsey et al., Genes and Development, 1987, 161-171], the control region of the beta-globin gene active in myeloid cells [Mogram et al. Natura 315, 338-340 (1985); Kollias et al., Cell 46, 89-94 (1986)], the control region of the basic protein of myelin, which is active in oligodendrocyte cells in the brain (Readhead et al., Cell 48: 703-712 (1987)), a light chain of myosin. 2 gene control region active in skeletal muscles [Sani: Natúré 314, 283-286 (1985)], neuron-specific enolase (NSE) active in neuronal cells [Morelli et al., Gen. Virol.

317 • ·· ·

80, 571-583 (1999)], the control region of the brain-derived neurotrophic factor (BDNF) gene that is active in neuronal cells (Tabuchi et al., 1998, Biochemical and Biophysical Research Communications 253, 818-823), glia yarn acid Protein (GFAP) promoter active in astrocytes [Gomes et al., Braz. J. Med. Bio. Gap. 32 (5), 619-631 (1999); Morelli et al., Gen. Virol. 80, 571583 (1999)], and the control region of the gonadotrophic releasing gene active in the hypothalamus (Mason et al., Science 234, 13721378). 1986)].

In a specific embodiment, expression of a polypeptide or a fusion protein is regulated by a constitutive promoter. In another embodiment, expression of a polypeptide or fusion protein is controlled by an inducible promoter. In another embodiment, expression of a polypeptide or fusion protein is controlled by a tissue-specific promoter.

In a specific embodiment, a vector comprising a promoter, operably linked to a nucleic acid encoding a polypeptide or fusion protein, contains one or more origin of replication and optionally contains one or more selection markers (e.g., an antibiotic resistance gene).

Several viral-based expression systems can be used in mammalian host cells. In cases where an adenovirus is used as an expression vector, the sequence encoding the polypeptide or fusion protein can be ligated to an adenovirus trans318 • · · · · · · · · · · · · · · · · · · · · · · · · · · · · control complex, such as the late promoter and the three-part leader sequence. This chimeric gene is then inserted into the adenoviral genome by in vitro or in vivo recombination. Insertion into a non-essential region of the viral genome (e. G., E1 or E3) results in a recombinant virus that is viable and can express the antibody molecule in an infected host [see, for example, Logan & Shenk, Proceedings of the National Academy of Sciences, USA 81: 355-359 (1984)]. Specific initiation signals may also be required for efficient translation of sequences encoding the embedded fusion proteins. These signals include the ATG initiation codon and adjacent sequences. In addition, the initiation codon must be in a reading phase with the reading frame of the desired coding sequence to ensure translation of the complete insert. These exogenous translational control signals and initiation codons may be of any origin, including natural and synthetic origin. The efficiency of expression can be enhanced by suitable transcription enhancing elements, transcription terminators, and the like. (see, for example, Bittner et al., Methods in Enzymology 153: 51-544 (1987)).

Expression vectors containing the inserts of a gene encoding a polypeptide or fusion protein can be identified by three general approaches: a) nucleic acid hybridization,

b) presence or absence of "marker" gene functions; and c) expression of inserted sequences. The first approach is one

The presence of a gene encoding a polypeptide or fusion protein in an expression vector can be detected by nucleic acid hybridization using probes with a sequence homologous to the sequence of the inserted gene encoding the polypeptide or fusion protein. According to the second approach, the recombinant host-vector system can be identified and certain "marker" gene functions (e.g., thymidine kinase activity, antibiotic resistance, transformation phenotype, inclusion body formation in baculovirus, etc.) can be selected which is encoded by the nucleotide encoding the polypeptide or fusion protein. insertion of the sequence into the vector. For example, if the nucleotide sequence encoding the fusion protein is inserted into the sequence of the vector marker gene, recombinants containing the gene encoding the fusion protein insert may be identified based on the lack of a marker gene function. In the third approach, recombinant expression vectors can be identified by examining the gene product (e.g., the fusion protein) expressed by the recombinant. Such an assay may be based, for example, on the physical or functional properties of the fusion protein in an in vitro assay system; i.e., an anti-biologically active molecule bound to an antibody.

In addition, host cell strains can be selected that affect expression of the inserted sequences or modify and process the gene products in the desired specific manner. Expression from certain promoters may increase in the presence of certain inducers; Thus, expression of genetically engineered fusion proteins can be controlled.

320 • · · · · · · · · · · · · · · · · · ·

In addition, different host cells have specific and specific mechanisms for post-translational modification of proteins and gene products (e.g., for glycosylation, phosphorylation of the protein). For example, expression in a bacterial system results in a glycosylated product, expression in yeast results in a glycosylated product. For this purpose, eukaryotic host cells having the cellular mechanism necessary for the proper processing of the primary transcript, glycosylation and phosphorylation of the gene product can be used. Such mammalian host cells include, but are not limited to, CHO, VERY, BHK, HeLa, COS, MDCK, 293, 3T3, WI38, NSO, and especially neuronal cell lines. such as SK-N-AS, SK-N-FI, and SK-N-DZ human neuroblastoma [Sugimoto et al., Journal of the National Cancer Institute 73, 51-57 (1984)], SK-N -SH Human Neuroblastoma [Biochimica et Biophysica Acta 704, 450-460], Daoy Human Cerebellar Medullal Blastoma (He et al., Cancer Research 52, 1144-1148 (1992)), DBTRG-05MG glioblastoma cells [Kruse et al. . Dev. Biol. 28A, 609-614 (1992)], human neuroblastoma of IMR-32 (Cancer Research 30, 2110-2118 (1970)), human astrocytoma 1321N1 (Proceedings of the National Academy of Sciences, USA 74, 4816 (1977)) , MOG-G-GCM Human Astrocytoma [Br. J. Cancer 49, 269 (1984)], U87MG human glioblastoma astrocytoma [Acta Pathol. Microbiol. Scand. 74, 465-486 (1968) J, human glioblastoma A172 [Olopade et al., Cancer Research 52, 2523321 · ♦ · ···

2529 (1992), rat rat glioma cells C6 (Benda et al., Science 161: 370-371 (1968)), Neuro-2a mouse neuroblastoma (Proceedings of the National Academy of Sciences, USA 65, 129136 (1970)) , NB41A3 mouse neuroblastoma [Proceedings of the National Academy of Sciences, USA 48, 1184-1190 (1962)], SCP sheep choroid plexus [Bolin et al., J. Virol. Meth. 48, 211221 (1994)], G355-5, PG-4 cat normal astrocyte [Haapala et al., J. Virol. 53, 827-833 (1985)], the Mpf ferret brain (Towbridge et al., In Vitro 18: 952-960 (1982)), and normal cell lines, such as CTX TNA2, normal cortex brain [Radany et al. Proceedings of the National Academy of Sciences, USA 89: 6467-6471 (1992), such as CRL7030 and Hs678Bst. In addition, different host vector expression systems can vary the degree of processing response.

A long term, high yield production of recombinant proteins requires a stable expression system. For example, cell lines can be produced that stably express a polypeptide or a fusion protein. Instead of using expression vectors containing a viral origin of replication, host cells can be regulated by suitable expression control elements (e.g., promoter, enhancer sequences, transcription terminators, polyadenylation sites, etc.) transformed with DNA and a selection marker. After the introduction of foreign DNA, the genetically engineered cells are left for 1-2 days in complete media

322 and then transferred to selective media. The selection marker in the recombinant plasmid provides resistance to selection and allows the cells to integrate stably into the chromosome and form colonies that can then be cloned and propagated into cell lines. This method is preferably used to produce cell lines expressing a polypeptide or fusion protein that binds immune-specific to a CD2 polypeptide. Such genetically engineered cell lines may be particularly useful in screening and evaluating compounds that affect the activity of a polypeptide or fusion protein that binds to a CD2 polypeptide.

Many different selection systems can be used, including, but not limited to, the herpes simplex virus thymidine kinase (Wigler et al., Cell 11: 223 (1977)), hypoxantinguanine phosphoribosyl transferase [Szybalska & Szybalski: Proceedings of the National Academy of Sciences, USA 48, 202 (1992)] and adenine phosphoribosyltransferase (Lowry et al., Cell 22, 8-17 (1980)) can be used in tk, hgprt or cells. In addition, antimetabolite resistance can be used as the basis for selection of the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., Proceedings of the National Academy of Sciences, USA 77, 357 (1980); O'Hare et al., Proceedings of the National Academy of Sciences, USA 78: 1527 (1981)]; gpt, which provides resistance to mycophenolic acid

323 [Mulligan & Berg: Proceedings of the National Academy of Sciences, USA 78: 2072 (1981)]; neo, which confers resistance to G-418 aminoglycoside [Wu and Wu: Biotherapy 3, 8795 (1991); Tolstoshev: Ann. Port. Pharmacol. Toxicol. 32: 573-596 (1993); Mulligan, Science 260: 926-932 (1993); Morgan and Anderson: Annu. Port. Biochem. 62: 191-217 (1993); TIBTECH 11 (5), 155-215 (May 1993)]; and hygro, which provides hygromycin resistance (Santerre et al., Gene 30: 147 (1984)).

If the polypeptide or fusion protein of the present invention has been produced by recombinant expression, it can be purified by any of the methods known in the art for purifying a protein, such as chromatography (e.g., ion exchange, affinity, particularly affinity for specific antigen after Protein A, and size exclusion column chromatography) by centrifugation. , solubility difference, or any other standard technique used to purify proteins.

The present invention also relates to an end product and a labeled pharmaceutical product. The present invention further relates to articles containing a packaging material and a pharmaceutical composition of the present invention in a form suitable for administration to the subject in said packaging material. More particularly, the present invention also relates to articles containing a packing material and a pharmaceutical composition of the present invention in a form suitable for administration to a subject.

In said packaging material, wherein said pharmaceutical composition is one or more integrin α ν β3 antagonists, one or more of the integrin α ν β3 antagonists is a prophylactic agent. or a therapeutic agent and a pharmaceutically acceptable carrier.

In a specific embodiment, an article comprising a packing material and a pharmaceutical composition of the present invention is in a form suitable for administration to the subject in said packaging material, wherein said pharmaceutical composition is one or more integrin α ν β3 antagonists, one or more. an anti-inflammatory agent and a pharmaceutically acceptable carrier. In another embodiment, an article comprising a packing material and a pharmaceutical composition of the present invention is in a form suitable for administration to a subject, preferably a human, most preferably an autoimmune or inflammatory disorder, in said packaging material, wherein said pharmaceutical composition is one or more integrin α ν β3 antagonists, an immunomodulatory agent, and a pharmaceutically acceptable carrier.

In another embodiment, an article comprising a packing material and a pharmaceutical composition of the present invention is in a form suitable for administration to a subject, preferably a human, most preferably an autoimmune or inflammatory disorder, in said packaging material, wherein said medicament is a pharmaceutical composition. Contains one or more integrin α ν β3 antagonists, a CD2 binding molecule, and a pharmaceutically acceptable carrier. In a preferred embodiment, an article comprising a packing material and a pharmaceutical composition of the present invention is in a form suitable for administration to a subject, preferably a human, most preferably an autoimmune or inflammatory disorder, in said packaging material, wherein said pharmaceutical composition is VITAXIN ™, MEDI-507, and a pharmaceutically acceptable carrier.

In another embodiment, an article comprising a packing material and a pharmaceutical composition of the present invention is in a form suitable for administration to a subject, preferably a human, most preferably an autoimmune or inflammatory disorder, in said packaging material, wherein said pharmaceutical composition is one an integrin α ν β ββ antagonist, a TNFα antagonist, and a pharmaceutically acceptable carrier. In a preferred embodiment, an article comprising a packing material and a pharmaceutical composition of the present invention is in a form suitable for administration to a subject, preferably a human, most preferably an autoimmune or inflammatory disorder, in said packaging material, wherein said pharmaceutical composition is an integrin α ν β3 antagonist, ENBREL ™ or REMICADE ™, and a pharmaceutically acceptable carrier.

326

As with any other pharmaceutical composition, the packaging material and container of the articles of the present invention are designed to protect the stability of the product during storage and transport. More specifically, the present invention relates to an industrial product comprising a packing material, such as a box, bottle, tube, ampoule, container, sprayer, inhaler, intravenous bag, wrapper, and the like; and a unit dosage form of a pharmaceutical agent packaged in said packaging material. More specifically, the present invention relates to an industrial product comprising a packing material, such as a box, bottle, tube, ampoule, container, sprayer, inhaler, intravenous bag, wrapper, and the like; and at least one unit dosage form of each pharmaceutical agent packaged in said packaging material. The present invention relates to an industrial product comprising a packing material, such as a box, bottle, tube, ampoule, container, sprayer, inhaler, intravenous bag, cover and the like; and at least one unit dosage form of each pharmaceutical agent packaged in said packaging material. This industrial product contains the unit dosage form in a suitable container or container such as a glass ampoule or other container that can be hermetically sealed. For suitable dosage forms for parenteral administration, the active ingredient is sterile and is a particulate solution suitable for administration. In other words, the present invention provides both parenteral solutions and lyophilized solutions

327 powders, each sterile, and the latter is capable of preparing a solution prior to injection. Alternatively, the unit dosage form may be a solid suitable for oral, transdermal, topical or mucosal administration. In a preferred embodiment, the unit dosage form is suitable for intravenous, intramuscular or subcutaneous administration.

The industrial products of the present invention may include instructions for the administration of a pharmaceutical composition or other information material advising the physician, nurse or patient on how to properly prevent or treat the disease or disorder in question. In other words, the industrial product contains instructions for use that indicate dose ranges or suggestions, including, without limitation, actual dosages, monitoring procedures, total lymphocyte and T cell numbers, and other monitoring information.

The present invention provides that adverse effects that can be reduced or avoided by the methods of the present invention are indicated in an information material provided with an industrial product to prevent, treat, or alleviate one or more symptoms associated with an autoimmune or inflammatory disorder. The methods of the present invention include methods that can be avoided or reduced without limiting themselves to the normality of life functions ab328 • ··· ·· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · / by: blood pressure, low blood pressure), hematological events (anemia, lymphopenia, thrombocytopenia), headache, colds, dizziness, nausea, back pain, chest pain (chest pressure), diarrhea, muscle pain, itching, psoriasis, rhinitis, sweating, injection site reaction and vasodilation. Since some of the prophylactic or therapeutic agents used in the present invention may be immunosuppressive, prolonged immunosuppression may increase the risk of infection, including opportunistic infection. Prolonged and sustained immunosuppression may result in an increased risk of developing certain types of cancer.

Further, the information material that is placed in an industrial product used to prevent, treat, or ameliorate one or more symptoms associated with an autoimmune or inflammatory disorder may indicate that foreign proteins may result in allergic reactions, including anaphylaxis or cytokine. release syndrome. The information material should indicate that allergic reactions occur only in the form of mild itchy rashes or may be severe, such as erythroderma, Steve-Jonson syndrome, vasculitis or anaphylaxis. The information material should indicate that anaphylactic reactions (anaphylaxis) are severe and occasionally fatal hypersensitivity reactions. Allergic reactions, including anaphylaxis, may occur when any foreign protein is injected into the body.

329 ι

They can range from mild appearance, such as hives or rashes to lethal systemic reactions. Anaphylactic reactions usually occur within 10 minutes after contact. Patients experience paraesthesia, low blood pressure, laryngeal edema, altered mental status, facial or pharyngeal edema, airway obstruction, bronchospasm, urticaria and itching, serum sickness, arthritis, allergic nephritis, glomerulonephritis, temporary arthritis or eosinophilia.

The information material may also indicate that the cytokine release syndrome is an acute clinical syndrome, temporarily associated with the administration of certain activating anti-T cell antibodies. Cytokine release syndrome is attributed to cytokines emitted by activated lymphocytes or monocytes. Clinical manifestations of cytokine release syndrome range from a wide range of mild, limited, influenza-like illnesses, to a more severe, life-threatening, shock-like reaction that can be severe cardiovascular, pulmonary, and central nervous system manifestations. Typically, the symptom begins about 30-60 minutes after administration (but may occur later) and may last for several hours. The frequency and severity of this symptom complex is generally the highest at the first dose. At each new dose, the incidence and severity of the syndrome tend to decrease. If we increase the amount of a dose or start the treatment again after an interruption, this symptom is re-introduced.

330 may occur. As mentioned above, the present invention relates to methods of treatment and prevention which avoid or reduce one or more of the adverse effects described herein.

The following example is given by way of illustration and is not to be construed as limiting the scope of the present invention.

Example

Treatment of patients with rheumatoid arthritis

A phase I, open-label, dose escalation study was designed to evaluate the pharmacokinetics and safety of VITAXIN ™ in patients with active rheumatoid arthritis. Active rheumatic arthritis is defined as the presence of 2 swollen joints, including hands, wrists, knees, or ankles. Patients with rheumatoid arthritis are currently receiving methotrexate therapy, with or without additional anti-rheumatic agents such as etanercept, infliximab, sulfasalazine or hydroxycloroquine. Patients who are currently receiving stable doses of non-steroidal anti-inflammatory drugs or prednisone (<10 mg / day) should continue with these treatments. In patients receiving ciclosporin A, leflunomide or gold, these drugs should be discontinued at least 4 weeks before starting VITAXIN ™.

Patients are given a single intravenous dose and then analyzed 4 weeks later for repeated intravenous administration.

331 doses at the same dose for 12 weeks. Changes in the safety and potential of VITAXIN ™ should be estimated in the activity of the disease during 26 weeks of intravenous administration. Different groups of patients are treated similarly but receive doses of 1 mg / kg, 2 mg / kg, 4 mg / kg or 8 mg / kg.

VITAXIN ™ is given by intravenous injection of 5 mg / ml and 10 mg / ml. Repeated subcutaneous administration requires a formulation of 80 mg / ml.

The change in disease activity is estimated based on the total number of soft and swollen joints, total score of the patient and physician for pain and disease activity, and ESR / CRP. The progression of structural joint damage is estimated based on the quantitative X-ray evaluation of hands, joints, and feet (Sharp methods). The functional status is evaluated based on the Health Assessment Questionnaire (HAQ) and the quality of life is estimated on the basis of SF-36.

VITAXIN ™ is prepared and prepared according to U.S. Patent No. 09 / 339,922 (filed June 24, 1999), the disclosure of which is hereby incorporated by reference in its entirety, for all purposes.

It is not intended to limit the scope of the present invention to exemplary embodiments illustrating only one aspect of the invention. Indeed, the various modifications of this ta332 ························································· by of those described above will be apparent to those skilled in the art from the description described above. These changes are intended to be within the scope of the appended claims.

All patents, patent applications, and non-patent publications cited herein are incorporated herein by reference in their entirety, as is the case for each patent, patent application, and non-patent publication.

The sequences described herein are described in detail below and in computer readable form.

SEQUENCE LIST <110> Medimmune, Inc <120> Procedures for the Prevention or Treatment of Inflammatory or Autoimmune Disorders by Combination with Integrin α ν β 3 Antagonists and Other Prophylactic or Therapeutic Agents <130> 10271-053-228 <150> US 60 / 273,098 <151> 2001-03-02

333 • · · · ····. ··.

• ··· «· · ·· <150> US 60 / 316,321 <151> 2001-08-31 <160> 17 <170> Patent Version 3.1 <210> 1 <211> 5 <212> WHITE sp.

<400> 1

Ser Tyr Asp Met Ser 1 5 <210> 2 <211> WHITE <213> Mus sp.

<400> 2

Lys Val Ser Ser Gly Gly Gly 1 5 <210> 3 <211> 8

334

<212> WHITE <213> Mus sp.

<400> 3

His Asn Tyr Gly Ser Phe Ala Tyr 1 5 <210> 4 <211> 11 <212> WHITE <213> Mus sp.

<400> 4

Gin Alá Ser Gin Ser Ile Ser Asn His Leu His 15 10 <210> 5 <211> WHITE <213> Mus sp.

<400> 5

Tyr Arg Ser Gin Ser Ile Ser Asn His Leu His 15 10 <210> 6 <211> WHITE

335 • · · <

• · · · ··· ··· <213> Mus sp.

<400> 6

Gin Gin Ser Gly Ser Trp Pro His Thr 1 5 <210> 7 <211> 351 <212> The DNA <213> Mus sp.

<220>

<221> CDS <222> (1) .. (351) <223>

<400> 7

cag GTG cag cOT GTG gag TCT ggg GGA GGC gtt GTG cag cetacean GGA agg 48
Gin With Gin Leu With Glu Ser Gly Gly Gly With With Gin Pro Gly Arg
1 5 10 15
tcc cat aga CTC tcc tgt GCA gcc TCT GGA ttc acc ttc agt AGC poop 96
Ser His Arg Leu Ser Cys below below Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
gac atg TCT TGG gtt ege cag get CCG GGC aag GGT cOT gag TGG gtc 199
asp Met Ser Trp With Arg Gin below Pro Gly Lys Gly Leu Glu Trp With
35 40 45
GCA aaa gtt agt agt GGT GGT GGT AGC acc ATC over the tta gac act GTG 192
below Lys With Ser Ser Gly Gly Gly Ser Thr Ile Ile Leu asp Thr With
50 55 60
cag GGC cga ttc acc ATC tcc aga gac aat agt aag aac acc cta Tac 290
Gin Gly Arg Phe Thr Ile Ser Arg asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80

336

cOT caa atg aac TCT cOT aga gcc gag gac aca gcc GTG poop
Leu Gin Met Asn Ser Leu Arg below Glu asp Thr below With Tyr
85 90 95
GCA aga cat aac Tac GGC agt ttt get Tac TGG GGC caa ggg
below Arg His Asn Tyr Gly Ser Phe below Tyr Trp Gly Gin Gly
100 105 110
GTG act gtt TCT agt
With Thr With Ser Ser

115 tac tgt 288

Tyr Cys act aca 336 Thr Thr

351 <210> 8 <211> 117 <212> WHITE <213> Mus sp.

<400> 8
Gin With Gin Leu With Glu Ser Gly Gly Gly With With Gin Pro Gly Arg
1 5 10 15
Ser His Arg Leu Ser Cys below below Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
asp Met Ser Trp With Arg Gin below Pro Gly Lys Gly Leu Glu Trp With
35 40 4 5
below Lys With Ser Ser Gly Gly Gly Ser Thr ile Ile Leu asp Thr With
50 55 60
Gin Gly Arg Phe Thr Ile Ser Arg asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gin Met Asn Ser Leu Arg below Glu asp Thr below With Tyr Tyr Cys
85 90 95
below Arg His Asn Tyr Gly Ser Phe below Tyr Trp Gly Gin Gly Thr Thr
100 105 110
With Thr With Ser Ser

<210> 9 <211> 321

115 t

337 • ♦ ·· · · · · · · · · ♦>

·· ··· ht · <212> DNS <213> Mus sp.

<220>

<221> CDS <222> (1) .. (321) <223>

<400> 9

gag att GTG cta act cag bet circa gcc acc cOT bet CTC AGC circa GGA 98
Glu Ile With Leu Thr Gin Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly
1 5 10 15
gaa agg GCG act ett tcc TGC cag gcc AGC gaa agt att AGC aac cac 96
Glu Arg Ala Thr Leu Ser Cys Gin Ala Ser Glu Ser Ile Ser Asn His
20 25 30
cta cac TGG poop caa caa agg cetacean GGT caa gcc circa agg ett CTC ATC 199
Leu His Trp Tyr Gin Gin Arg Pro Gly Gin Ala Pro Arg Leu Leu Ile
35 40 45
aag poop CGT tcc cag tcc ATC bet ggg ATC ccc gcc agg ttc agt GGC 192
Lys Tyr Arg Ser Gin Ser Ile Ser Gly Ile Pro Ala Arg Phe Ser Gly
50 55 60
agt GGA tea ggg aca dam ttc acc CTC act ATC tcc agt cOT gag cetacean 290
Ser Gly Ser Gly Thr asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro
65 70 75 80
gaa dam ttt GCA gtc poop Tac tgt caa cag agt GGC AGC TGG cetacean cac 288
Glu asp Phe Ala With Tyr Tyr Cys Gin Gin Ser Gly Ser Trp Pro His
85 90 95
ACG ttc GGA ggg ggg acc aag GTG gaa att aag 321
Thr Phe Gly Gly Gly Thr Lys With Glu Ile Lys
100 105
<210> 10
<211> 107

<212> WHITE · «· * ···· · · · ·« * ··· ·· ·

338 ··· · · · · · · <213> Mus sp.

<400> 10

Glu 1 Ile With Leu Thr 5 Gin Ser Pro Ala Thr 10 Leu Ser Leu Ser Pro 15 Gly
Glu Arg Ala Thr Leu Ser Cys Gin Ala Ser Glu Ser Ile Ser Asn His
20 25 30
Leu Hi s Trp Tyr Gin Gin Arg Pro Gly Gin Ala Pro Arg Leu Leu Ile
35 40 45
Lys Tyr Arg Ser Gin Ser Ile Ser Gly Ile Pro Ala Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr asp Phe Thr Leu Thr He Ser Ser Leu Glu Pro
65 70 75 80
Glu asp Phe Ala With Tyr Tyr Cys Gin Gin Ser Gly Ser Trp Pro His
85 90 95
Thr Phe Gly Gly Gly Thr Lys With Glu Ile Lys
100 105

<210> 11 <211> 5 <212> WHITE <213> Mus sp.

<400> 11

Glu Tyr Tyr Met Tyr 1 5 <210> 12 <211> 17 <212> WHITE <213> Mus sp.

339

- · 4 · 4 · »* ·· »4 · · · · · · · · · · · · · 4 ··· ·· · 4» <400> 12

Arg Ile Asp Pro Glu Asp Gly Ser Ile Asp Tyr Val Glu Lys Phe Lys 15 10 15

Lys <210> 13 <211> 9 <212> WHITE <213> Mus sp.

<400> 13

Gly Lys Phe Asn Tyr Arg Phe Ala Tyr 1 5 <210> 14 <211> 16 <212> WHITE <213> Mus sp.

<400> 14

Arg Ser Ser Gin Ser Leu Leu His Ser Ser Gly Asn Thr Leu Asn Trp 15 10 15 <210> 15 <211> 7 <212> WHITE <213> Mus sp.

<400> 15

340 • * · * ·

• «. • Aj Aj Aj Aj Aj Aj Aj Aj Aj Aj Aj

Leu Val Ser Lys Leu Glu Ser 1 5 <210> 16 <211> WHITE <213> Mus sp.

<400> 16

Met Gin Phe Thr His Tyr Pro Tyr Thr 1 5 <210> 17 <211> 347 <212> WHITE <213> Mus sp.

<400> 17

Met 1 With below Gly Ser 5 asp below Gly Arg Ala 10 Leu Gly With Leu Ser 15 With
With Cys Leu Leu His Cys Phe Gly Phe Ile Ser Cys Phe Ser Gin Gin
20 25 30
Ile Tyr Gly With With Tyr Gly Asn With Thr Phe His With Pro Ser Asn
35 40 45
With Pro Leu Lys Glu With Leu Trp Lys Lys Gin Lys asp Lys With below
50 55 60
Glu Leu Glu Asn Ser Glu Phe Arg below Phe Ser Ser Phe Lys Asn Arg
65 70 75 80
With Tyr Leu asp Thr With Ser Gly Ser Leu Thr He Tyr Asn Leu Thr
85 90 95
Ser Ser asp Glu asp Glu Tyr Glu Met Glu Ser Pro Asn Ile Thr asp
100 105 110
Thr Met Lys Phe Phe Leu Tyr With asp Lys Thr His Thr Cys Pro Pro

341

115 120 125

Cys Pro 130 below Pro Glu Leu Leu 135 Gly Gly Pro Ser Val 140 Phe Leu Phe Pro
Pro Lys Pro Lys asp Thr Leu Met Ile Ser Arg Thr Pro Glu With Thr
145 150 155 160
Cys With With With asp With Ser His Glu asp Pro Glu With Lys Phe Asn
165 170 175
Trp Tyr With asp Gly With Glu With His Asn below Lys Thr Lys Pro Arg
180 185 190
Glu Glu Gin Tyr Asn Ser Thr Tyr Arg With With Ser With Leu Thr With
195 200 205
Leu His Gin asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys With Ser
210 215 220
Asn Lys below Leu Pro below Pro Ile Glu Lys Thr Ile Ser Lys below Lys
225 230 235 240
Gly Gin Pro Arg Glu Pro Gin With Tyr Thr Leu Pro Pro Ser Arg asp
245 250 255

I

». · «·» * · * ···. - »

342

Claims (1)

  1. PATIENT INDIVIDUAL POINTS
    A method of treating an inflammatory disorder or an autoimmune disorder or one or more of these symptoms, wherein said method comprises administering to said subject in need thereof a therapeutically or therapeutically effective amount of one or more integrin α ν β3 antagonists and proactively. or a therapeutically effective amount of one or more immunomodulatory agents.
    2. A method of treating an inflammatory disorder or an autoimmune disorder or one or more of these symptoms, wherein said method comprises administering to a subject in need thereof a therapeutically or therapeutically effective amount of VITAXIN ™ and in a proactive or therapeutically effective amount. one or more immunomodulatory agents are administered.
    3. A method of treating an inflammatory disorder or an autoimmune disorder or one or more of these symptoms, wherein said method comprises administering to said subject in need thereof a therapeutically or therapeutically effective amount of one or more integrin α ν β3 antagonists and proactively. or administering a therapeutically effective amount of one or more anti-inflammatory agents
    4. A method of treating an inflammatory disorder or an autoimmune disorder or one or more of these symptoms, wherein said method comprises administering to said subject in need of prophylactic or therapeutically effective amount of VITAXIN ™ and having a prophylactic or therapeutic effect. one or more anti-inflammatory agents are administered in an amount.
    5. A method of treating an inflammatory disorder or an autoimmune disorder or one or more of these symptoms, wherein said method comprises administering to a subject in need thereof a therapeutically effective amount of one or more integrin α ν β3 antagonists and prophylactically. or a therapeutically effective amount of one or more TNFα antagonists.
    6. A method of treating an inflammatory disorder or an autoimmune disorder or one or more of these symptoms, wherein said method comprises administering to a subject in need of such treatment a prophylactically or therapeutically effective amount of VITAXIN ™ and in a prophylactically or therapeutically effective amount. one or more TNFα antagonists are administered.
    7. A method of treating an inflammatory disorder or autoimmune disorder, or a one or more symptoms, characterized in that said method comprises administering one or more integrin α ν β3 antagonist and prophylactically prophylactically to a subject in need of such treatment or therapeutically effective amount of or administering one or more CD2 binding molecules in a therapeutically effective amount.
    344
    8. A method of treating an inflammatory disorder or an autoimmune disorder or one or more of these symptoms, wherein said method comprises administering to said subject in need thereof a prophylactically or therapeutically effective amount of VITAXIN ™ in a prophylactically or therapeutically effective amount. one or more CD2 binding molecules are administered.
    A method according to claim 1 or 2, wherein the at least one immunomodulatory agent is a small organic molecule.
    The method of claim 1 or 2, wherein the at least one immunomodulatory agent is a T cell receptor modulator or a cytokine receptor modulator.
    11. The method of claim 9 wherein the small organic molecule is methotrexate, leflunomide, cyclophosphamide, cyclosporin A, FK506, mycophenolate mofetil, rapamycin, mizoribine, deoxyspergualin, brequinar, malonitrileamide, steroid or corticosteroid.
    12. The method of claim 10, wherein the T cell receptor is an antibody, peptide or fusion protein that binds immune-specific to a T cell receptor.
    13. The method of claim 12, wherein the antibody is a monoclonal antibody or an antigen-binding fragment thereof that is immunospecificly binding to a T cell receptor.
    345
    14. The method of claim 13, wherein the monoclonal antibody is a human or humanized monoclonal antibody.
    15. The method of claim 13, wherein the monoclonal antibody is an anti-CD2 monoclonal antibody, an anti-CD4 monoclonal antibody, an anti-CD8 monoclonal antibody, or an anti-CD40 monoclonal antibody.
    16. The method of claim 12, wherein the fusion protein is CTLA4-Ig.
    17. The method of claim 10, wherein the cytokine receptor modulator is a cytokine, a fragment of the cytokine, a fusion protein, or an antibody that specifically binds to a cytokine receptor.
    18. The method of claim 10, wherein the cytokine receptor modulator is a peptide, polypeptide, fusion protein, or antibody that binds immune-specific to a cytokine.
    19. The method of claim 17, wherein the antibody that is immunospecifically bound to a cytokine receptor is a monoclonal antibody or antigen-binding fragment thereof.
    20. The method of claim 19, wherein the monoclonal antibody is a human or humanized monoclonal antibody.
    346
    21. The method of claim 17, wherein the antibody is an anti-IL2 receptor antibody and anti-IL-12 receptor antibodies.
    22. The method of claim 18, wherein the antibody that is immunospecifically bound to a cytokine is a monoclonal antibody or an antibody-binding fragment thereof.
    23. The method of claim 22, wherein the monoclonal antibody is a human or humanized monoclonal antibody.
    24. The method of claim 18, wherein the antibody is an anti-TNFα antibody, an anti-ILp antibody, or an anti-IL6 antibody.
    25. The method of claim 17, wherein the cytokine is IL-4 or IL-10.
    26. The method of claim 18, wherein the polypeptide is a fragment of a cytokine receptor that binds to a cytokine immunospecificly.
    27. The method of claim 26, wherein the fragment is a portion of an extracellular domain of a TNFα receptor.
    28. The method of claim 3 or 4, wherein the at least one anti-inflammatory agent is a non-steroidal anti-inflammatory agent.
    347
    29. The method of claim 27, wherein the non-steroidal anti-inflammatory agent is aspirin, ibuprofen, diclofenac, nabunetone, naproxen or ketoprofen.
    30. The method of claim 5 or 6, wherein the TNFα antagonist is ENBREL ™ or REMICADE ™.
    31. The method of claim 5 or 6, further comprising the step of administering to said subject a prophylactically or therapeutically effective amount of methotrexate.
    32. The method of claim 7 or 8, wherein the CD2 binding molecule is a peptide, polypeptide, fusion protein, or antibody that binds immune-specific to a CD2 binding molecule.
    33. The method of claim 32, wherein the fusion protein is LFA-3TIP.
    34. The method of claim 7 or 8, further comprising the step of administering to said subject in a prophylactically or therapeutically effective amount a non-steroidal anti-inflammatory agent.
    35. The method of claim 34, wherein said non-steroidal anti-inflammatory drug is aspirin, ibuprofen, diclofenac, nabumetone, naproxen or ketoprofen.
    36. The method of claim 7 or 8, further comprising the step of providing said subject with a prophylactic or therapeutic effective amount.
    One or more immunomodulatory agents other than one CD2 binding molecule are administered.
    37. A method of treating an inflammatory disorder or an autoimmune disorder, or one or more of these symptoms, comprising administering to a patient in need thereof a prophylactically or therapeutically effective amount of one or more integrin α ν β3 antagonists and administered in a prophylactically or therapeutically effective amount. include MEDI-507 or an antigen-binding fragment thereof.
    38. A method of treating an inflammatory disorder or an autoimmune disorder, or one or more of these symptoms, comprising administering to a patient in need of such treatment a prophylactically or therapeutically effective amount of VITAXIN ™ and administering it in a prophylactically or therapeutically effective amount. 507 or an antigen-binding fragment thereof.
    39. The method of claim 37 or 38, further comprising the step of administering to said subject a prophylactically or therapeutically effective amount of one or more TNFα antagonists.
    40. The method of claim 37 or 38, further comprising the step of administering to said subject a prophylactically or therapeutically effective amount of methotrexate.
    41. The method of claim 37 or 38, further comprising the step of: said &quot; β &quot;
    349 subjects are administered prophylactically or therapeutically effective amounts of one or more TNFα antagonists and administered methotrexate in a prophylactically or therapeutically effective amount.
    42. The method of claim 37 or 38, further comprising the step of administering to said subject in a prophylactically or therapeutically effective amount a non-steroidal anti-inflammatory drug.
    43. The method of claim 39, wherein at least one TNFα antagonist is ENBREL ™ or REMICADE ™.
    A method according to claim 1, 3, 5 or 7, wherein at least one integrin α ν β 3 antagonist is an antiintegrin α ν β 3 antibody.
    45. The method of claim 44, wherein the anti-αvβ 3 antibody is a monoclonal antibody, or antigen-binding fragment.
    46. The method of claim 45, wherein the monoclonal antibody is a human or humanized antibody.
    47. The method according to claim 1, 2, 3, 4, 5, 6, 7, 8, 37 or 38, wherein the inflammatory disorder is asthma, encephalitis, inflammatory bowel disease, chronic obstructive pulmonary disease (COPD), arthritis or an allergic disorder.
    48. The method of claim 1, 2, 3, 4, 7, 8, 37 or 38, wherein the autoimmune disorder is rheumatoid arthritis, psoriatic arthritis, paralytic vertebrae,
    350
    Reiter syndrome, arthritis associated with inflammatory bowel disease, undifferentiated spondyloarthropathy, psoriasis, or undifferentiated arthropathy.
    A method according to claim 1, 2, 3, 4, 5, 6, 7, 8, 37 or 38, wherein the subject is human.
    50. The method of claim 5, 6, 7, 8, 37 or 38, wherein the subject is a human being treated or treated with one or more TNFα antagonists.
    51. The method of claim 5, 6, 7, 8, 37 or 38, wherein the subject is a human being treated or treated with one or more TNFα antagonists and methotrexate.
    52. The method of claim 5, 6, 7, 8, 37 or 38, wherein the subject is a human who has not been treated with a TNFα antagonist or methotrexate.
    53. The method of claim 5, 6, 7, 8, 37 or 38, wherein the subject is a human being resistant to treatment with a TNFα antagonist, a non-steroidal anti-inflammatory agent or methotrexate.
    54. The method of claim 2, 4, 6, 8 or 38, wherein the VITAXIN ™ or antigen-binding fragment thereof is administered orally, topically, intravenously, intramuscularly or subcutaneously. to the girl.
    55. The method of claim 37 or 38, wherein the MEDI-507 or antigen-binding fragment thereof is:
    The subject is administered orally, topically, intravenously, intramuscularly or subcutaneously to the subject.
    56. The method of claim 1, 3, 5, 7 or 37, wherein said integrin α ν β3 antagonists are not small organic molecules.
    57. The method of claim 1, 3, 5, 7 or 37, wherein the at least one integrin α ν β3 is a small organic molecule.
    58. The method of claim 7 or 8, wherein said CD2 binding molecules are not small organic molecules.
    59. The method of claim 7 or 8, wherein at least one CD2 binding molecule is a small organic molecule.
    60. A method of treating or alleviating an inflammatory disorder or an autoimmune disorder or one or more of its symptoms, wherein said method comprises administering to said subject in need thereof a prophylactically or therapeutically effective amount of VITAXIN? an antigen-binding fragment is administered in a prophylactically or therapeutically effective amount of REMICADE ™ or ENBREL ™ and is administered in a prophylactically or therapeutically effective amount of methotrexate.
    61. The method of claim 60, wherein the amount of VITAXIN ™ administered to said subject or an antigen-binding fragment thereof is from about 0.1 mg / kg to about 10 mg / kg.
    «7 * f
    352 ·:
    62. The method of claim 60, wherein the amount of REMICADE ™ administered to said subject is from about 0.1 mg / kg to about 10 mg / kg.
    63. The method of claim 60, wherein the amount of ENBREL ™ administered to said subject is from about 0.01 mg / kg to about 10 mg / kg.
    64. The method of claim 60, wherein the amount of methotrexate administered to said subject is from about 0.01 mg / kg to about 3 mg / kg.
    65. A pharmaceutical composition comprising an integrin α ν β3 antagonist, a TNFα antagonist, and a pharmaceutically acceptable carrier.
    66. A pharmaceutical composition comprising an integrin α ν β3 antagonist, a CD2 binding molecule, and a pharmaceutically acceptable carrier.
    67. The composition of claim 65, further comprising methotrexate.
    68. The composition of claim 65 or 67, wherein the integrin α ν is a β3 antagonist VITAXIN ™, or an antigen-binding fragment thereof.
    69. The composition of claim 65, wherein the TNFα antagonist is REMICADE ™ or ENBREL ™.
    70. The composition of claim 66, wherein the CD2 binding molecule is an LFA3TIP, MEDI-507 or an antigen-binding fragment of MEDI-507.
    353 »> Ί
    71. The composition of claim 65 or 66, wherein the integrin α ν β3 antagonist is not a small organic molecule.
    72. The composition of claim 65 or 66, wherein the integrin α ν β3 is a small organic molecule.
    73. The composition of claim 66 wherein the CD2 binding molecule is not a small organic molecule.
    74. The composition of claim 66, wherein the CD2 binding molecule is a small organic molecule.
    75. A pharmaceutical composition comprising VITAXIN ™ or an antigen-binding fragment thereof, MEDI-507, or an antigen-binding fragment thereof, and a pharmaceutically acceptable carrier.
    76. An industrial product comprising a packing material and a pharmaceutical composition in said ligation in a form suitable for administration to a human, wherein said pharmaceutical composition comprises VITAXIN ™ or an antigen-binding fragment thereof, MEDI-507 or an antigen-binding fragment thereof. and a pharmaceutically acceptable carrier.
    77. The industrial product of claim 73, further comprising instructions for use in said packaging material, which propose a dosage range for use in the prevention or treatment of an inflammatory disorder or autoimmune disorder.
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HU0303340A 2001-03-02 2002-03-04 Methods for preventing or treating inflammatory or autoimmune disorders integrin alpha v beta 3 antagonists in combination with other prophylactic or therapeutic agents by administering an effective HU0303340A2 (en)

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EP1372720A4 (en) 2006-07-26
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NO20033862D0 (en) 2003-09-01
AU2002306651B2 (en) 2007-12-13
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US20020168360A1 (en) 2002-11-14
CA2439852A1 (en) 2002-09-12

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