EP0980258A1 - Suppression of tumor necrosis factor alpha and vascular endothelial growth factor in therapy - Google Patents

Suppression of tumor necrosis factor alpha and vascular endothelial growth factor in therapy

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Publication number
EP0980258A1
EP0980258A1 EP98920669A EP98920669A EP0980258A1 EP 0980258 A1 EP0980258 A1 EP 0980258A1 EP 98920669 A EP98920669 A EP 98920669A EP 98920669 A EP98920669 A EP 98920669A EP 0980258 A1 EP0980258 A1 EP 0980258A1
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EP
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Prior art keywords
antagonist
antibody
tumor necrosis
necrosis factor
vascular endothelial
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EP98920669A
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German (de)
English (en)
French (fr)
Inventor
Marc Feldmann
Ravinder Nath Maini
Ewa Maria Paleolog
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Kennedy Trust for Rheumatology Research
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Kennedy Institute of Rheumotology
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Priority to EP01117491A priority Critical patent/EP1170017A1/en
Publication of EP0980258A1 publication Critical patent/EP0980258A1/en
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/241—Tumor Necrosis Factors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered

Definitions

  • TNF ⁇ tumor necrosis factor alpha
  • interleukin-1 interleukin-1
  • TNF ⁇ is a soluble homotrimer of 17 kD protein subunits (Smith et al., J. Biol . Chem. , 262:6951-6954 (1987)). A membrane-bound 26 kD precursor form of TNF also exists (Kriegler et a.1., Cell, 53:45-53 (1988)). For reviews of TNF, see Beutler et al., Nature, 320:584 (1986); Old, Science, 230:630 (1986); and Le et al. , Lab. Invest., 56:234 (1987).
  • TNF ⁇ tumor necrosis factor ⁇
  • human non-monccytic tumor cell lines produce TNF ⁇ (Rubin et al., J. Exp. Med. , 164:1350 (1986); Spriggs et al., Proc . Natl . Acad. Sci . USA, 84:6563 (1987)).
  • CD4 + and CD8+ peripheral blood T lymphocytes and some cultured T and B cell lines (Cuturi et al., J. Exp. Med., 165:1581 (1987); Sung et al . , J. Exp. Med., 168:1539 (1988); Turner et al . , Eur. J. Immunol., 17:1807-1814 (1987)) also produce TNF ⁇ .
  • TNF ⁇ causes pro-inflammatory actions which result in tissue injury, such as degradation of cartilage and bone (Saklatvala, Nature, 322:547-549 (1986); Bertolini, Nature, 315:516-518 (1986)), induction of adhesion molecules, inducing procoagulant activity on vascular endothelial cells (Pober et al., J. Immunol., 136: 1680 (1986) ) , increasing the adherence of neutrophils and lymphocytes (Pober et al., J. Immunol., 138:3319 (1987)), and stimulating the release of platelet activating factor from macrophages, neutrophils and vascular endothelial cells (Camussi et al . , J. Exp. Med . , 166 : 1390 (1987) ) .
  • Cachexia The extensive wasting which is associated with ⁇ cancer, and other diseases, is known as "cachexia" (Kern et al . , J. Parent . Enter. Nutr. , 12:286-298 (1988)). Cachexia includes progressive weight loss, anorexia, and persistent erosion of lean body mass in response to a malignant growth. The cachectic state causes much cancer morbidity and mortality. There is evidence that TNF ⁇ is involved in cachexia in cancer, infectious pathology, and other catabolic states (see, e.g., Beutler and Cerami, Ann. Rev. Immunol . , 7:625-655 (1989) ) .
  • TNF ⁇ is believed to play a central role in gram-negative sepsis and endotoxic shock (Michie et al . , Br. J. ⁇ urg. , 76: 610 -611 (1989); Debets et al . , Second Vienna Shock Forum, p. 463-466 (1989); Simpson et al . , Cri t . Care Clin . , 5:27-47 (1989)), including fever, malaise, anorexia, and cachexia. Endotoxin strongly activates monocyte/macrophage production and secretion of TNF ⁇ and other cytokines (Kornbluth et al., J. Immunol .
  • TNF ⁇ and other monocyte-derived cytokines mediate the metabolic and neurohormonal responses to endotoxin (Michie et al . , New Engl . J. Med . , 318 : 1481-1486 (1988)).
  • Endotoxin administration to human volunteers produces acute illness with flu-like symptoms including fever, tachycardia, increased metabolic. rate and stress hormone release (Revhaug et al . , Arch. Surg., 123:162-170 (1988) ) . Circulating TNF ⁇ increases in patients suffering from Gram-negative sepsis ( aage et al .
  • TNF ⁇ has been implicated in inflammatory diseases, autoimmune diseases, viral, bacterial and parasitic infections, malignancies, and/or neurogenerative diseases and is a useful target for specific biological therapy in diseases, such as rheumatoid arthritis and Crohn's disease.
  • VEGF Vascular endothelial growth factor
  • vascular permeability factor also known as vasculotropin
  • vasculotropin is a diffusible endothelial cell-specific itogen and angiogenic factor that can also increase vascular permeability (Ferrara, Breast Cancer Res. Treat., 36(2). -121-131 (1995)).
  • VEGF is a disulfide-linked homodimeric glycoprotein of about 34-45-kDa consisting of four isoforms (containing either 121, 165, 189 or 206 amino acid residues in the mature monomer (see, e.g., Ferrara, Breast Cancer Res . Treat., 36:121-131 (1995); Dvorak et al . , Am. J.
  • VEGF 121 and to a large extent VEGF 1S5 are secreted in soluble form, whereas VEGF 189 and VEGF 206 remain cell-associated (see, e.g., Ferrara, Breast Cancer Res. Treat., 36:127-137 (1995); Dvorak et al . , Am. J. Path.,
  • VEGF stimulates endothelial cell growth and increases microvascular permeability by interacting with membrane-spanning tyrosine kinase receptors, the fms- like tyrosine kinase receptor (Fit) (deVries et al., Science, 255:989-991 (1992); and Shibuya et al., Oncogene, 5:519-524 (1990)) and kinase insert domain- containing receptor (KDR) (Terman et al., Biochem. Biophys. Res. Commun. , 187:1519-1586 (1992)).
  • Fit membrane-spanning tyrosine kinase receptors
  • KDR kinase insert domain- containing receptor
  • the murine homolog of KDR is fetal liver kinase receptor (Flk) (Quinn et al . , Proc. Natl . Acad. Sci . USA, 90:1533-1531 (1993)). (See also, Ferrara, Breast Cancer Res. Treat., 36:121-131 (1995); and Dvorak et al., Am. J. Path., 146(5) . -1029-1039 (1995)).
  • Flk fetal liver kinase receptor
  • VEGF has been implicated in chronic vascular hyperpermeability and angiogenesis of solid and ascites tumors, healing wounds, rheumatoid arthritis, psoriasis and diabetic retinopathy (see, e.g., Brown et al., J. Immunol., 154(6) -.2801-2801 (1995); Dvorak et al., Int. Arch. Allergy Immunol., 107:233-235 (1995); Ferrara, Breast Cancer Res. Treat., 36(2) :127-137 (1995); Aiello et a . , Proc. Natl.
  • VEGF expression has been reported to be elevated in pathological conditions including cancer, proliferative retinopathy, psoriasis and rheumatoid arthritis (RA) (see, e.g., Claffey et al., Cancer Metastasis Rev., 15(2) . -165-116 (1996); and Koch et al., J. Immunol., 152:4149-4156 (1994)).
  • serum VEGF levels have been reported to be higher in individuals with POEMS (polyneuropathy, organomegaly, endocrinopathy, M- protein and skin changes) syndrome than in normal individuals (Kondo et al., Biochim. Biophys. Acta, 1221:211-214 (1994); Soubrier et al., Arth. Rheum., 39:S131 (1996); and Watanabe et al., Lancet, 347:102 (1996)).
  • POEMS polyneuropathy, organomegaly, endocrinopathy, M
  • VEGF protein and mRNA have been localised to macrophages and lining cells in synovial membranes from RA patients, and VEGF receptors are expressed by RA synovial endothelial cells, the putative target of VEGF (Koch et al., J. Immunol, 152:4149-4156 (1994); and Fava et al., J. Exp. Med., 180:341-346 (1994)).
  • Increased production of VEGF in response to chronic hypoxia has been reported (Tuder et al., J. Clin.
  • hypoxic conditions prevail in RA when the elevated intra-articular pressure exceeds synovial capillary pressure (Blake et al., Lancet, 8633:289-293 (1989) ) .
  • the present invention provides methods for treating and/or preventing a tumor necrosis factor-mediated disease in an individual in need thereof comprising co- administering a tumor necrosis factor alpha (TNF ⁇ ) antagonist and a vascular endothelial growth factor (VEGF) antagonist to the individual in therapeutically effective or synergistic amounts.
  • TNF ⁇ tumor necrosis factor alpha
  • VEGF vascular endothelial growth factor
  • the present invention also provides methods for treating and/or preventing recurrence of a TNF-mediated disease in an individual comprising co-administering a TNF ⁇ antagonist and a VEGF antagonist to the individual in therapeutically effective amounts.
  • TNF-mediated diseases include rheumatoid arthritis, Crohn's disease, and acute and chronic immune diseases associated with an allogenic transplantation (e.g., renal, cardiac, bone marrow, liver, pancreatic, small intestine, skin or lung transplantation) .
  • an allogenic transplantation e.g., renal, cardiac, bone marrow, liver, pancreatic, small intestine, skin or lung transplantation
  • the invention relates to a method of treating and/or preventing (such as preventing relapse of) rheumatoid arthritis in an individual comprising co-administering a TNF ⁇ antagonist and a VEGF antagonist to the individual in therapeutically effective amounts.
  • the invention relates to a method of treating and/or preventing (such as preventing relapse of) Crohn's disease in an individual comprising co-administering a TNF ⁇ antagonist and a VEGF antagonist to the individual in therapeutically effective amounts.
  • the invention in a third embodiment, relates to a method of treating and/or preventing acute or chronic immune disease associated with a transplantation in an individual comprising co-administering a TNF ⁇ antagonist and a VEGF antagonist to the individual in therapeutically effective amounts.
  • compositions comprising a TNF ⁇ antagonist and a VEGF antagonist, such as for use in therapy or in the manufacture of a medicament for treating the above diseases.
  • the composition further comprises methotrexate.
  • TNF ⁇ antagonists useful in the invention include anti-TNF ⁇ antibodies and antigen-binding fragments thereof; receptor molecules which bind specifically to TNF ⁇ ; compounds which prevent and/or inhibit TNF ⁇ synthesis, TNF ⁇ release or its action on target cells, such -as thalidomide, tenidap, phosphodiesterase inhibitors (e.g, pentoxifylline and rolipram) , A2b adenosine receptor agonists and A2b adenosine receptor enhancers; compounds which prevent and/or inhibit TNF ⁇ receptor signalling, such as mitogen activated protein (MAP) kinase inhibitors; compounds which block and/or inhibit membrane TNF ⁇ cleavage, such as metalloproteinase inhibitors; compounds which block and/or inhibit TNF ⁇ activity, such as angiotensin converting enzyme (ACE) inhibitors (e.g., captopril) ; and compounds which block and/or inhibit TNF ⁇ production and/or synthesis, such as
  • VEGF antagonists useful in the invention include anti-VEGF antibodies and antigen-binding fragments thereof; receptor molecules which bind specifically to VEGF; compounds which prevent and/or inhibit VEGF function (e.g., suramin, protein tyrosine kinase (PTK) inhibitors (e.g., lavendustin A); compounds which prevent and/or inhibit binding of VEGF to VEGF receptors or extracellular domains thereof (e.g., platelet factor-4 (PF-4)); compounds which block and/or interfere with VEGF receptor signalling; and compounds which block and/or interfere with VEGF activation (e.g., mithramycin) .
  • VEGF antagonists useful in the invention also include agents which are antagonists of signals that drive VEGF production and/or synthesis, such as agents which decrease and/or block TGF ⁇ or its ligand.
  • Figure 1 is a plot showing concentration of VEGF secreted by monocytic cells, endothelial cells and RA fibroblasts after stimulation in absence or presence of TNF ⁇ or IL-l ⁇ , as determined by ELISA. .Values are means of 3 determinations ⁇ SD, and are representative of 3 similar experiments .
  • Figure 2A is a plot showing serum VEGF concentrations in age- and sex-matched non-arthritic individuals and in patients with RA, as measured by ELISA.
  • Figure 2B is a plot showing the degree of correlation between serum VEGF concentrations and C- reactive protein levels.
  • Figure 3 is graph showing serum VEGF levels in RA patients after infusion of either placebo, 1 mg/kg anti-
  • TNF ⁇ antibody cA2 or 10 mg/kg anti-TNF ⁇ antibody cA2, as determined by ELISA.
  • Figure 4A is a graph showing serum VEGF levels in RA patients after treatment with either anti-TNF ⁇ antibody cA2 at 3 mg/kg (infusions at weeks 0, 2, 6, 10 and 14), methotrexate (7.5 mg/week) or combination of cA2 and methotrexate .
  • Figure 4B is a graph showing serum VEGF levels in RA patients after treatment with methotrexate (7.5 mg/week), either alone or in combination with anti- TNF ⁇ antibody cA2 at either 1 mg/kg, 3 mg/kg or 10 mg/kg (infusions at weeks 0, 2, 6, 10 and 14) .
  • VEGF vascular endothelial growth factor
  • pro-inflammatory cytokines such as TNF ⁇ and IL-1
  • blockade of TNF ⁇ activity in vivo decreases circulating concentrations of VEGF.
  • excess VEGF present in the circulation is produced in the diseased tissue.
  • VEGF is a potent and specific inducer of angiogenesis
  • reduction of circulating VEGF reflects a reduced predisposition to angiogenesis. Therefore, long term blockade of TNF ⁇ can reduce neovascularisation and hence the cellular mass in the diseased tissue.
  • VEGF is a potent inducer of angiogenesis and has been implicated in the formation of blood vessels and activation of microvascular endothelium in RA.
  • VEGF serum concentrations are significantly elevated in patients with active RA, relative to VEGF serum concentrations in normal individuals, and correlate with C-reactive protein (CRP) , a marker of inflammation and disease activity in RA.
  • CRP C-reactive protein
  • the production of CRP is regulated by pro-inflammatory cytokines, and its overall production correlates well with the rate of disease progression.
  • treatment of RA patients with anti-TNF ⁇ monoclonal antibody which leads to amelioration of disease symptoms, results in a significant and persistent reduction in serum VEGF concentrations .
  • RA patients with a combination of anti-TNF ⁇ monoclonal antibody and methotrexate results in a more prolonged reduction in serum VEGF levels relative to patients treated with anti-TNF ⁇ antibody alone or with methotrexate alone. It is likely that serum VEGF reflects synthesis of VEGF, thus suggesting that VEGF production in vivo is cytokine-dependent .
  • VEGF results in the induction of increased vascular permeability and the leakage of vascular fluid into surrounding tissues.
  • the presence of extravascular fluid is well documented and is clinically apparent as joint effusion.
  • treatment of RA patients with anti-TNF ⁇ antibody results in a reduction in joint fluid content as determined by MRI imaging (Kalden- Nemeth et al . , Rheumatol . Int . , 16 : 219 (1997)) .
  • This suggests that VEGF also plays a role in joint swelling in RA, which is rapidly reduced after treatment with anti-TNF ⁇ .
  • the data show that monocytic cells, endothelial cells and synovial membrane fibroblasts, which may be hypothesised to contribute to elevated serum VEGF concentrations in RA, secrete VEGF constitutively, and that spontaneous release of VEGF by RA synovial membrane cells is markedly reduced in the presence of inhibitors of cytokine activity, namely anti-TNF ⁇ antibody and IL-1 receptor antagonist (IL-lra) .
  • IL-lra IL-1 receptor antagonist
  • RA synovial membrane cells Isolation of RA synovial membrane cells by collagenase/DNase digestion yields a heterogeneous population of cells, consisting predominantly of mononuclear cells and to a lesser extent fibroblasts (Brennan et al . , Lancet, 2:244-247 (1989); and Buchan et al . , Clin . Exp . Immunol . , 73:449-455 (1988)).
  • Long-terra (up to 9 days) culture of these cells leads to decreased numbers of CD14+, CD45+ and CD3+ cells, accompanied by the appearance of adherent fibroblast-like cells. The earliest detectable release of VEGF into cell culture supernatants was observed at 24 hours.
  • VEGF levels in culture continued to increase even 9 days after isolation, which contrasts with the rapid increase (within 2 hours of plating) in TNF ⁇ levels in culture supernatants of RA synovial membrane cells, which subsequently returned to undetectable levels by days 5-6 of culture (Buchan et al . , Clin . Exp . Immunol . , 73:449- 455 (1988)) .
  • macrophage-like cells are the predominant source of TNF ⁇ in RA synovial membrane explant cultures, VEGF is released both by monocytic cells and fibroblasts. This is in agreement with the data described herein using THP-1 cells and synovial fibroblasts .
  • RA synovial membrane fibroblasts, endothelial cells and monocytic cells in terms of VEGF secretion, suggests that the reduction in serum VEGF is a result of decreased VEGF production by monocyte/ macrophages and microvascular endothelial cells following in vivo blockade of TNF ⁇ activity by anti-TNF ⁇ antibody and of IL-1 activity subsequent to TNF ⁇ blockade.
  • Synovial explant cultures consist of both macrophage-like cells (which release VEGF in response to TNF ⁇ ) and fibroblasts (secretion from which is induced only by IL-1) . These cells contribute to differing degrees to VEGF release into the cell supernatants.
  • optimal inhibition of VEGF ⁇ roduction in RA patients may require the blockade of both TNF ⁇ and IL-1 activities.
  • the invention includes methods for treating and/or preventing a TNF- mediated disease in an individual, comprising co- administering a TNF ⁇ antagonist and a VEGF antagonist to the individual in therapeutically effective or synergistic amounts.
  • the present invention further relates to a method for treating and/or preventing recurrence of a TNF-mediated disease in an individual comprising co-administering a TNF ⁇ antagonist and a VEGF antagonist to the individual in therapeutically effective amounts.
  • the TNF ⁇ antagonist and VEGF antagonist can be administered simultaneously or sequentially.
  • the TNF ⁇ antagonist and VEGF antagonist can each be administered in single or multiple doses. Multiple VEGF antagonists and multiple TNF antagonists can be co-administered.
  • TNF ⁇ antagonists and VEGF antagonists can be used in combination with the therapeutic co-administration of TNF ⁇ antagonists and VEGF antagonists.
  • methotrexate is co-administered with the TNF ⁇ antagonist and the VEGF antagonist in therapeutically effective or synergistic amounts.
  • TNF-mediated disease refers to a TNF related pathology or disease.
  • TNF related pathologies or diseases include, but are not limited to, the following:
  • A inflammatory diseases, including, but not limited to, acute and chronic immune and autoimmune pathologies, such as, but not limited to, rheumatoid arthritis (RA) , juvenile chronic arthritis (JCA) , spondyloarthropathy, thyroiditis, graft versus host disease (GVHD) , scleroderma, diabetes mellitus, Graves' disease, allergy; acute or chronic immune disease associated with an allogenic transplantation, such as, but not limited to, renal transplantation, cardiac transplantation, bone marrow transplantation, liver transplantation, pancreatic transplantation, small intestine transplantation, lung transplantation and skin transplantation; chronic inflammatory pathologies such as, but not limited to, sarcoidosis, chronic inflammatory bowel disease, ulcerative colitis, and Crohn's pathology or disease; vascular inflammatory pathologies, such as, but not limited to, disseminated intravascular coagulation, atherosclerosis, Kawasaki's pathology and vasculitis syndromes,
  • infections including, but not limited to, sepsis syndrome, cachexia, circulatory collapse and shock resulting from acute or chronic bacterial infection, acute and chronic parasitic and/or infectious diseases, bacterial, viral or fungal, such as a human immunodeficiency virus (HIV) , acquired immunodeficiency syndrome (AIDS) (including symptoms of cachexia, autoimmune disorders, AIDS dementia complex and infections) ;
  • HIV human immunodeficiency virus
  • AIDS acquired immunodeficiency syndrome
  • C neurodegenerative diseases, including, but not limited to, demyelinating diseases, such as multiple sclerosis and acute transverse myelitis; myasthenia gravis; extrapyramidal and cerebellar disorders, such as lesions of the corticospinal system; disorders of the basal ganglia or cerebellar disorders; hyperkinetic movement disorders, such as Huntington's chorea and senile chorea; drug-induced movement disorders, such as those induced by drugs which block central nervous system (CNS) dopamine receptors; hypokinetic movement disorders, such as Parkinson's disease; progressive supranuclear palsy; cerebellar and spinocerebellar disorders, such as astructural lesions of the cerebellum; spinocerebellar degenerations (spinal ataxia, Friedreich's ataxia, cerebellar cortical degenerations, multiple systems degenerations (Mencel, Dejerine-Thomas, Shi-Drager, and MachadoJoseph) ) ;
  • TNF ⁇ - secreting tumors or other malignancies involving TNF ⁇ such as, but not limited to, leukemias (acute, chronic myelocytic, chronic lymphocytic and/or myelodyspastic syndrome); lymphomas (Hodgkin's and non-Hodgkin ' s lymphomas, such as malignant lymphomas (Burkitt's lymphoma or Mycosis fungoides) ) ;
  • G diseases in which angiogenesis or VEGF/VPF production plays a part, such as, but not limited to, ocular neovascularization, psoriasis, duodenal ulcers, angiogenesis of the female reproductive tract, and chronic arthritis, including osteoarthritis vilonodular synovitis, and chronic arthritis associated with hemorraghic diseases, such as hemophilic arthritis.
  • recurrence is defined to encompass the reappearance of one or more symptoms of the disease state.
  • a recurrence can include the experience of one or more of swollen joints, morning stiffness or joint tenderness.
  • the invention relates to a method of treating and/or preventing rheumatoid arthritis in an individual comprising co-administering a TNF ⁇ antagonist and a VEGF antagonist to the individual in therapeutically effective or synergistic amounts.
  • the invention in a second embodiment, relates to a method for treating and/or preventing Crohn's disease in an individual comprising co-administering a TNF ⁇ antagonist and a VEGF antagonist to the individual in therapeutically effective or synergistic amounts.
  • the invention relates to a method for treating and/or preventing an acute or chronic immune disease associated with an allogenic transplantation in an individual comprising co- administering a TNF ⁇ antagonist and a VEGF antagonist to the individual in therapeutically effective or synergistic amounts.
  • a TNF ⁇ antagonist and a VEGF antagonist to the individual in therapeutically effective or synergistic amounts.
  • transplantation includes organ, tissue or cell transplantation, such as renal transplantation, cardiac transplantation, bone marrow transplantation, liver transplantation, pancreatic transplantation, small intestine transplantation, skin transplantation and lung transplantation.
  • the methods of the invention further comprise administering methotrexate to the individual in a therapeutically effective or synergistic amount.
  • a benefit of combination therapy with a TNF ⁇ antagonist and a VEGF antagonist- is significantly improved response in comparison with that obtained with treatment with each antagonist alone.
  • Combination therapy with a TNF ⁇ antagonist, methotrexate and a VEGF antagonist also provides a significantly improved response in comparison with that obtained with treatment with each agent alone.
  • a VEGF antagonist can be administered in combination with a TNF ⁇ antagonist or with a TNF ⁇ antagonist and methotrexate to achieve a synergistic effect.
  • lower dosages can be used to provide the same therapeutic response, thus increasing the therapeutic window between a therapeutic and a toxic effect. Lower doses may also result in lower financial costs to the patient, and potentially fewer side effects.
  • methotrexate reduces immunogenicity of anti-TNF ⁇ antibodies, thus permitting administration of anti-TNF ⁇ antibodies with enhanced safety.
  • compositions comprising a TNF ⁇ antagonist and a VEGF antagonist.
  • the compositions further comprise methotrexate.
  • the compositions of the present invention are useful for treating a subject having a pathology or condition associated with abnormal levels of a substance reactive with a TNF ⁇ antagonist, in particular TNF ⁇ in excess of levels present in a normal healthy subject, where such excess or diminished levels occur in a systemic, localized or particular tissue type or location in the body.
  • tissue types can include, but are not limited to, blood, lymph, central nervous system (CNS) , liver, kidney, spleen, heart muscle or blood vessels, brain or spinal cord white matter or grey matter, cartilage, ligaments, tendons, lung, pancreas, ovary, testes, prostate.
  • Increased TNF ⁇ concentrations relative to normal levels can also be localized to specific regions or cells in the body, such as joints, nerve blood vessel junctions, bones, specific tendons or ligaments, or sites of infection/ such as bacterial or viral infections .
  • the compositions of the present invention can also be used in the manufacture of a medicament for treating the above diseases.
  • Tumor necrosis factor alpha antagonists decreases, blocks, inhibits, abrogates or interferes with TNF ⁇ activity in vivo .
  • a suitable TNF ⁇ antagonist can bind TNF ⁇ and includes anti-TNF ⁇ antibodies, antigen-binding fragments thereof, and receptor molecules and derivatives which bind specifically to TNF ⁇ .
  • a suitable TNF ⁇ antagonist can also prevent or inhibit TNF ⁇ synthesis and/or TNF ⁇ release and includes compounds such as thalidomide, tenidap, and phosphodiesterase inhibitors, such as, but not limited to, pentoxifylline and rolipram.
  • a suitable TNF ⁇ antagonist that can prevent or inhibit TNF ⁇ synthesis and/or TNF ⁇ release also includes A2b adenosine receptor enhancers and A2b adenosine receptor agonists (e.g. , 5 ' - (N-cyclopropyl) -carboxamidoadenosine, 5 ' -N-ethylcarboxamidoadenosine, cyclohexyladenosine and R-N 6 -phenyl-2-propyladenosine) .
  • A2b adenosine receptor enhancers and A2b adenosine receptor agonists (e.g. , 5 ' - (N-cyclopropyl) -carboxamidoadenosine, 5 ' -N-ethylcarboxamidoadenosine, cyclohexyladenosine and R-N 6 -phenyl-2-propyladenosine)
  • a suitable TNF ⁇ antagonist can also prevent or inhibit TNF ⁇ receptor signalling and includes mitogen activated protein (MAP) kinase inhibitors (e.g., SB 203580; Lee and Young, J. Leukocyte Biol . , 59 : 152 - 151 (1996), the teachings of which are entirely incorporated herein by reference) .
  • MAP mitogen activated protein
  • TNF ⁇ antagonists include agents which decrease, block, inhibit, abrogate or interfere with membrane TNF ⁇ cleavage, such as, but not limited to, metalloproteinase inhibitors; agents which decrease, block, inhibit, abrogate or interfere with TNF ⁇ activity, such as, but not limited to, angiotensin converting enzyme (ACE) inhibitors, such as captopril, enalapril and lisinopril; and agents which decrease, block, inhibit, abrogate or interfere with TNF ⁇ production and/or synthesis, such as, but not limited to, MAP kinase inhibitors.
  • TNF ⁇ antagonists are also described in U.S. Application No. 08/690,775 (filed August 1, 1996), U.S.
  • an anti-tumor necrosis factor alpha antibody decreases, blocks, inhibits, abrogates or interferes with TNF ⁇ activity in vivo .
  • the antibody specifically binds the antigen.
  • the antibody can be polyclonal or monoclonal, and the term antibody is intended to encompass both polyclonal and monoclonal antibodies.
  • the terms polyclonal and monoclonal refer to the degree of homogeneity of an antibody preparation, and are not intended to be limited to particular methods of production.
  • Suitable antibodies are available, or can be raised against an appropriate immunogen, such as isolated and/or recombinant antigen or portion thereof (including synthetic molecules, such as synthetic peptides) or against a host cell which expresses recombinant antigen.
  • an appropriate immunogen such as isolated and/or recombinant antigen or portion thereof (including synthetic molecules, such as synthetic peptides) or against a host cell which expresses recombinant antigen.
  • cells expressing recombinant antigen such as transfected cells, can be used as immunogens or in a screen for antibody which binds receptor (see e.g., Chuntharapai et al . , J. Immunol . , 152 : 1783-1789 (1994); and Chuntharapai et al., U.S. Patent No. 5,440,021).
  • Preparation of immunizing antigen, and polyclonal and monoclonal antibody production can be performed using any suitable technique.
  • A- variety of methods have been described (see e.g., Kohler et al__, Nature, 256 : 495-497 (1975) and Bur. J. Immunol . , 6: 511-519 (1976); Milstein et al . , Nature, 266 : 550 -552 (1977) ; Koprowski et al., U.S. Patent No. 4,172,124; Harlow, E. and D. Lane, 1988, Antibodies : A Laboratory Manual , (Cold
  • a hybridoma can be produced by fusing a suitable immortal cell line (e.g., a myeloma cell line -such as SP2/0) with antibody producing cells.
  • a suitable immortal cell line e.g., a myeloma cell line -such as SP2/0
  • the antibody producing cell preferably those of the spleen or lymph nodes, can be obtained from animals immunized with the antigen of interest.
  • Hybridomas can be isolated using selective culture conditions, and cloned by limiting dilution.
  • Cells which produce antibodies with the desired specificity can be selected by a suitable assay (e.g., ELISA) .
  • suitable assay e.g., ELISA
  • Other suitable methods of producing or isolating antibodies of the requisite specificity, including human antibodies, can be used, including, for example, methods by which a recombinant antibody or portion thereof are selected from a library, such as, for example, by phage display technology (see, e.g., Winters et al . , Annu . Rev. Immunol . , 12:433-455 (1994); Hoogenboom et al .
  • Single chain antibodies and chimeric, humanized or primatized (CDR-grafted antibodies, with or without framework changes) , or veneered antibodies, as well as chimeric, CDR-grafted or veneered single chain antibodies, comprising portions derived from different species, and the like are also encompassed by the present invention and the term "antibody".
  • the various portions of these antibodies can be joined together chemically by conventional techniques, or can be prepared as a contiguous protein using genetic engineering techniques. For example, nucleic acids encoding a chimeric or humanized chain can be expressed to produce a contiguous protein. See, e.g., Cabilly et al . , U.S. Patent No. 4,816,567; Cabilly et al .
  • antigen binding fragments of antibodies can also be produced.
  • antigen binding fragments include, but are not limited to, fragments such as Fv, Fab, Fab' and F(ab') 2 fragments.
  • Antigen binding fragments can be produced by enzymatic cleavage or by recombinant techniques, for example. For instance, papain or pepsin cleavage can generate Fab or F(ab') 2 fragments, respectively.
  • Antibodies can also be produced in a variety of truncated forms using antibody genes in which one or more stop codons has been introduced upstream of the natural stop site.
  • a chimeric gene encoding a F(ab') 2 heavy chain portion can be designed to include DNA sequences encoding the CH- . domain and hinge region of the heavy chain.
  • Anti-TNF ⁇ antibodies useful in the present invention are characterized by high affinity binding to TNF ⁇ and low toxicity (including human anti-murine antibody (HAMA) and/or human anti-chimeric antibody (HACA) response) .
  • HAMA human anti-murine antibody
  • HACA human anti-chimeric antibody
  • an antibody where the individual components, such as the variable region, constant region and framework, individually and/or collectively possess low immunogenicity is useful in the present invention.
  • the antibodies which can be used in the invention are characterized by their ability to treat patients for extended periods with good to excellent alleviation of symptoms and low toxicity. Low immunogenicity and/or high affinity, as well as other undefined properties, may contribute to the therapeutic results achieved.
  • Low immunogenicity is defined herein as raising significant HACA or HAMA responses in less than about 75%, or preferably less than about 50% of the patients treated and/or raising low titres in the patient treated (less than about 300, preferably less than about 100 measured . with a double antigen enzyme immunoassay) (Elliott et al . , Lancet 344 : 1125- 1121 (1994) , incorporated herein by reference) .
  • the anti-TNF ⁇ antibody is chimeric monoclonal antibody cA2 (or an antigen binding fragment thereof) or murine monoclonal antibody A2 (or an antigen binding fragment thereof) , or has an epitopic specificity similar to that of chimeric antibody cA2, murine monoclonal antibody A2, or antigen binding fragments thereof, including antibodies or antigen binding fragments reactive with the same or a functionally equivalent epitope on human TNF ⁇ as that bound by chimeric antibody cA2 or murine monoclonal antibody A2 , or antigen binding fragments thereof.
  • Antibodies with an epitopic specificity similar to that of chimeric antibody cA2 or murine monoclonal antibody A2 include antibodies which can compete with chimeric antibody cA2 or murine monoclonal antibody A2 (or antigen binding fragments thereof) for binding to human TNF ⁇ . Such antibodies or fragments can be obtained as described above.
  • Chimeric antibody cA2, murine monoclonal antibody A2 and methods of obtaining these antibodies are also described in U.S. Application No. 08/192,093 (filed February 4, 1994), U.S. Application No. 08/192,102 (filed February 4, 1994), U.S. Application No. 08/192,861 (filed February 4,
  • Chimeric antibody cA2 consists of the antigen binding variable region of the high-affinity neutralizing mouse anti-human TNF ⁇ IgGl antibody, designated A2 , and the constant regions of a human IgGl, kappa immunoglobulin.
  • the human IgGl Fc region improves allogeneic antibody effector function, increases the circulating serum half-life and decreases the immunogenicity of the antibody.
  • the avidity and epitope specificity of the chimeric antibody cA2 is derived from the variable region of the murine antibody A2.
  • a preferred source for nucleic acids encoding the variable region of the murine antibody A2 is the A2 hybridoma cell line.
  • Chimeric A2 (cA2) neutralizes the cytotoxic effect of both natural and recombinant human TNF ⁇ in a dose dependent manner. From binding assays of chimeric antibody cA2 and recombinant human TNF ⁇ , the affinity constant of chimeric antibody cA2 was calculated to be 1.04xl0 10 M "1 . Preferred methods for determining monoclonal antibody specificity and affinity by competitive inhibition can be found in Harlow, et al., Antibodies : A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1988; Colligan et al . , eds . , Current Protocols in Immunology, Greene Publishing Assoc.
  • murine monoclonal antibody A2 is produced by a cell line designated cl34A.
  • Chimeric antibody cA2 is produced by a cell line designated cl68A.
  • anti-TNF ⁇ antibodies or antigen-binding fragments thereof are described in the art (see, e.g., U.S. Patent No. 5,231,024; M ⁇ ller, A. et al . , Cytokine, 2 (3 ) -. 162- 169 (1990); U.S. Application No. 07/943,852 (filed September 11, 1992); Rathjen et al . , International Publication No. WO 91/02078 (published February 21, 1991); Rubin et al . , EPO Patent Publication No. 0 218 868 (published April 22, 1987); Yone et al . , EPO Patent Publication No.
  • antigen binding region refers to that portion of an antibody molecule which contains the amino acid residues that interact with an antigen and confer on the antibody its specificity and affinity for the antigen.
  • the antigen binding region includes the "framework" amino acid residues necessary to maintain the proper conformation of the antigen- binding residues.
  • antigen refers to a molecule or a portion of a molecule capable of being bound by an antibody which is additionally capable of inducing an animal to produce antibody capable of selectively binding to an epitope of that antigen.
  • An antigen can have one or more than one epitope.
  • epitope is meant to refer to that portion of the antigen capable of being recognized by and bound by an antibody at one or more of the antibody's antigen binding region.
  • Epitopes usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and have specific three dimensional structural characteristics as well as specific charge characteristics.
  • inhibiting and/or neutralizing epitope is intended an epitope, which, when bound by an antibody, results in loss of biological activity of the molecule containing the epitope, in vivo or in vitro, more preferably in vivo, including binding of TNF ⁇ to a TNF ⁇ receptor.
  • TNF ⁇ Receptor Molecules TNF ⁇ receptor molecules useful in the methods and compositions of the present invention are those that bind TNF ⁇ with high affinity (see, e.g., Feldmann et al., International Publication No. WO 92/07076 (published April 30, 1992); Schall et al_. , Cell , 61 : 361-310 (1990); and Loetscher et a . , Cell, 61:351-359 (1990) , which references are entirely incorporated herein by reference) and possess low immunogenicity.
  • the 55 kDa (p55 TNF-R) and the 75 kDa (p75 TNF-R) TNF ⁇ cell surface receptors are useful in the present invention. Truncated forms of these receptors, comprising the extracellular domains (ECD) of the receptors or functional portions thereof (see, e.g., Corcoran et al . , Eur. J. Biochem. ,
  • TNF ⁇ receptors which can be used in the invention are characterized by their ability to treat patients for extended periods with good to excellent alleviation of symptoms and low toxicity. Low immunogenicity and/or high affinity, as well as other undefined properties, may contribute to the therapeutic results achieved.
  • TNF ⁇ receptor multimeric molecules useful in the present invention comprise all or a functional portion of the ECD of two or more TNF ⁇ receptors linked via one or more polypeptide linkers or other nonpeptide linkers, such as polyethylene glycol (PEG) .
  • the multimeric molecules can further comprise a signal peptide of a secreted protein to direct expression of the multimeric molecule.
  • TNF ⁇ immunoreceptor fusion molecules useful in the methods and compositions of the present invention comprise at least one portion of one or more immunoglobulin molecules and all or a functional portion of one or more TNF ⁇ receptors .
  • These immunoreceptor fusion molecules can be assembled as monomers, or hetero- or homo-multimers .
  • the immunoreceptor fusion molecules can also be monovalent or multivalent.
  • An example of such a TNF ⁇ immunoreceptor fusion molecule is TNF ⁇ receptor/IgG fusion protein.
  • TNF ⁇ immunoreceptor fusion molecules and methods for their production have been described in the art (Lesslauer et al . , Eur. J. Immunol . , 21:2883-2886
  • a functional equivalent, derivative, fragment or region of TNF ⁇ receptor molecule refers to the portion of the TNF ⁇ receptor molecule, or the portion of the TNF ⁇ receptor molecule sequence which encodes TNF ⁇ receptor molecule, that is of sufficient size and sequences to functionally resemble TNF ⁇ receptor molecules that can be used in the present invention (e.g., bind TNF ⁇ with high affinity and possess low immunogenicity) .
  • a functional equivalent of TNF ⁇ receptor molecule also includes modified TNF ⁇ receptor molecules that functionally resemble TNF ⁇ receptor molecules that can be used in the present invention (e.g., bind TNF ⁇ with high affinity and possess low immunogenicity) .
  • a functional equivalent of TNF ⁇ receptor molecule can contain a "SILENT" codon or one or more amino acid substitutions, deletions or additions (e.g., substitution of one acidic amino acid for another acidic amino acid; or substitution of one codon encoding the same or different hydrophobic amino acid for another codon encoding a hydrophobic amino acid).
  • SILENT substitution of one acidic amino acid for another acidic amino acid
  • substitution of one codon encoding the same or different hydrophobic amino acid for another codon encoding a hydrophobic amino acid See Ausubel et al . , Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley- Interscience, New York (1989) .
  • VEGF Antagonists decreases, blocks, inhibits, abrogates or interferes with VEGF activity synthesis or receptor signalling in vivo .
  • VEGF antagonists include anti-VEGF antibodies and antigen-binding fragments thereof; receptor molecules and derivatives which bind specifically to VEGF; and VEGF receptor antagonists.
  • VEGF antagonists include agents which decrease, inhibit, block, abrogate or interfere with VEGF function, such as, but not limited to, suramin and protein tyrosine kinase (PTK) inhibitors (e.g., lavendustin A) . See, e.g., Waltenberger et al . , J. Mol .
  • PTK protein tyrosine kinase
  • VEGF antagonists also include agents which decrease, inhibit, block, abrogate or interfere with binding of VEGF to VEGF receptors or extracellular domains thereof, such as, but not limited to, platelet factor-4 (PF-4) . See, e.g., Gengrinovitch et al., J. Biol . Chem. , 270:15059-15065 (1995).
  • PF-4 platelet factor-4
  • VEGF antagonists include agents which decrease, inhibit, block, abrogate or interfere with VEGF relaxreceptor signalling; and agents which decrease, inhibit, block, abrogate or interfere with VEGF activation, such as, but not limited to, mithramycin. See, e.g., Ryuto et al . , J. Biol . Chem . , 271 (45) : 28220-28228 (1996).
  • VEGF antagonists also include agents which decrease, inhibit, block, abrogate or interfere with VEGF production, such as compounds (e.g. drugs and other agents, including antibodies) which inhibit, block, abrogate or interfere with TGF ⁇ or its ligands .
  • VEGF antagonists further include agents which are antagonists of signals that drive VEGF production and/or synthesis.
  • an anti-VEGF antibody decreases, blocks, inhibits, abrogates or interferes with VEGF activity in vivo.
  • Antibodies and antigen binding fragments are as described above.
  • the antibody can be polyclonal or monoclonal.
  • the antibody can be a single chain chimeric, humanized, primatized or veneered antibody. Such antibodies or fragments can be obtained as described above.
  • anti-VEGF antibodies are characterized by high affinity binding to VEGF (such as high affinity binding to VEGF 121 , VEGF 165 , VEGF 189 or VEGF 206 ) and low toxicity (including HAMA and/or HACA response) .
  • An antibody where the individual components, such as the variable region, constant region and framework, individually _ and/or collectively possess low immunogenicity is particularly useful .
  • anti-VEGF antibodies (and antigen binding fragments thereof) are characterized by their ability to treat patients for extended periods with good to excellent alleviation of symptoms and low toxicity. Low immunogenicity and/or high affinity, as well as other undefined properties, may contribute to the therapeutic results achieved.
  • anti-VEGF antibodies or antigen binding fragments thereof are described in the art (see, e.g., Asano et al . , Hybridoma, 14 (5 ) -.415 -480 (1995); and Kim et al . , Growth Factors, 7:53-64 (1992), which references are entirely incorporated herein by reference) .
  • VEGF receptor molecules useful in the present invention bind specifically to VEGF and possess low immunogenicity.
  • the VEGF receptor molecule is characterized by high affinity binding to VEGF.
  • VEGF receptor molecules include VEGF receptors, such as tyrosine kinase receptors, KDR, Flk (e.g., Flk-1) and Fit (e.g., Flt-1 and Flt-4) (see, e.g., Lee et al . , Proc. Natl . Acad. Sci . USA, 93:1988-1992 (1996); deVries et al . , Science, 255 : 989-991 (1992); Quinn et al . , Proc . Natl .
  • VEGF receptor molecules also include VEGF receptor multimeric molecules and VEGF immunoreceptor fusion molecules, and derivatives and fragments or portions thereof .
  • VEGF receptor multimeric molecules can comprise all or a functional portion of two or more VEGF receptors linked via one or more linkers. VEGF receptor multimeric molecules can further comprise a signal peptide of a secreted protein to direct expression of the multimeric molecule.
  • VEGF immunoreceptor fusion molecules can comprise at least one portion of one or more immunoglobulin molecules and all or a functional portion of one or more VEGF receptor (s).
  • VEGF immunoreceptor -fusion molecules can be assembled as monomers, or hetero- or homo- multimers.
  • VEGF immunoreceptor fusion molecules can also be monovalent or multivalent . Examples of VEGF immunoreceptor fusion molecules are described by Aiello et al . , Proc . Natl . Acad. Sci . USA, 92 (23) : 10457-10461 (1995) , the teaching of which is entirely incorporated herein by reference. See also, Aiello et al . , N. Engl . J. Med. , 331:1480-1487 (1994); and Park et al . , J. Biol . Chem. , 269 : 25646-25654 (1994), the teachings of which are entirely incorporated herein by reference.
  • a functional equivalent, derivative, fragment or region of VEGF receptor molecule refers to the portion of the VEGF receptor molecule, or the portion of the VEGF receptor molecule sequence which encodes VEGF receptor molecule, that is of sufficient size and sequences to functionally resemble VEGF receptor molecules that can be used in the present invention (e.g., bind specifically to VEGF and possess low immunogenicity) .
  • a functional equivalent of VEGF receptor molecule also includes modified VEGF receptor molecules that functionally resemble VEGF receptor molecules that can be used in the present invention (e.g., bind specifically to VEGF and possess low immunogenicity) .
  • a functional equivalent of VEGF receptor molecule can contain a "SILENT" codon or one or more amino acid substitutions, deletions or additions.
  • a functional equivalent of VEGF receptor molecule can contain a substitution of one acidic amino acid for another acidic amino acid, or a substitution of one codon encoding the same or different hydrophobic amino acid for another codon encoding a hydrophobic amino acid. See Ausubel et al . , Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley-Interscience, New York (1989) .
  • TNF ⁇ receptor molecules can be employed in producing VEGF receptor molecules that can be used in -the present invention.
  • Methotrexate Presently available oral and intravenous formulations of methotrexate include Rheumatrex ® methotrexate dose pack (Lederle Laboratories, Wayne, NJ) ; methotrexate tablets (Mylan Pharmaceuticals Inc., Morgantown, WV; Roxane Laboratories, Inc., Columbus, OH) ; and methotrexate sodium tablets, for injection and injection (Immunex Corporation, Seattle, WA) and methotrexate LPF ® sodium (methotrexate sodium injection) (Immunex Corporation, Seattle, WA) .
  • Methotrexate is also available from Pharmacochemie (Netherlands) .
  • Methotrexate prodrugs, homologs and/or analogs e.g., folate antagonists
  • other immunosuppressive agents or drugs that suppress the immune system
  • TNF ⁇ antagonists, VEGF antagonists, methotrexate and compositions of the present invention can be administered to an individual in a variety of ways .
  • the routes of administration include intradermal, transdermal (e.g., in slow release polymers), intramuscular, intraperitoneal, intravenous (including infusion and/or bolus injection) , subcutaneous, oral, topical, epidural, buccal, rectal, vaginal and intranasal routes .
  • Other suitable routes of administration can also be used, for example, to achieve absorption through epithelial or mucocutaneous linings.
  • TNF ⁇ antagonists, VEGF antagonists, and compositions of the present invention can also be administered by gene therapy wherein a DNA molecule encoding a particular therapeutic protein or peptide is administered to the patient, e.g., via a vector, which causes the particular protein or peptide to be expressed and .secreted at therapeutic levels in vivo .
  • TNF ⁇ antagonists, VEGF antagonists, methotrexate and compositions of the present invention can be administered together with other components of biologically active agents, such as pharmaceutically acceptable surfactants (e.g., glycerides) , excipients (e.g., lactose), carriers, diluents and vehicles. If desired, certain sweetening, flavoring and/or coloring agents can also be added.
  • TNF ⁇ antagonists, VEGF antagonists, methotrexate and compositions of the present invention can be administered prophylactically or therapeutically to an individual .
  • TNF ⁇ antagonists can be administered prior to, simultaneously with (in the same or different compositions) or sequentially with the administration of a VEGF antagonist.
  • TNF antagonists and VEGF antagonists can also be administered prior to, simultaneously with (in the same or different compositions) or sequentially with the administration of methotrexate.
  • methotrexate for example,
  • TNF antagonists and VEGF antagonists can be administered as adjunctive and/or concomitant therapy to methotrexate therapy.
  • TNF ⁇ antagonists for parenteral (e.g., intravenous, subcutaneous, intramuscular) administration
  • VEGF antagonists VEGF antagonists
  • methotrexate and compositions of the present invention can be formulated as a solution, suspension, emulsion or lyophilized powder in association with a pharmaceutically acceptable parenteral vehicle.
  • parenteral vehicles are water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Liposomes and nonaqueous vehicles such as fixed oils can also be used.
  • the vehicle or lyophilized powder can contain additives that maintain isotonicity (e.g., sodium chloride, mannitol) and chemical stability (e.g., buffers and preservatives) .
  • the formulation is sterilized by commonly used techniques .
  • Suitable pharmaceutical carriers are described in Gennaro, A.R., Ed., Remington's Pharmaceutical Sciences, 18th edition, Mack Publishing Co., Easton, PA (1990).
  • a parenteral composition suitable for administration by injection is prepared by dissolving 1.5% by weight of active ingredient in 0.9% sodium chloride solution.
  • TNF ⁇ antagonists, VEGF antagonists and methotrexate are administered in therapeutically effective or synergistic amounts; compositions of the present invention are administered in a therapeutically effective or synergistic amount.
  • a therapeutically effective amount is such that co- administration of TNF ⁇ antagonist and VEGF antagonist, or administration of a composition of the present invention, results in inhibition of the biological activity of TNF ⁇ and VEGF relative to the biological activity of TNF ⁇ and VEGF when therapeutically effective amounts of TNF ⁇ antagonist and VEGF antagonist are not co-administered, or relative to the biological activity of TNF ⁇ and VEGF when a therapeutically effective amount of the composition is not administered.
  • a therapeutically effective amount is also an amount such that co-administration of TNF ⁇ antagonist, VEGF antagonist and methotrexate results in inhibition of the biological activity of TNF ⁇ and VEGF relative to the biological activity of TNF ⁇ and VEGF when therapeutically effective amounts of TNF ⁇ antagonist, VEGF antagonist and methotrexate are not co- administered.
  • a therapeutically effective amount is that amount of TNF ⁇ antagonist and VEGF antagonist necessary to synergistically or significantly reduce or eliminate symptoms associated with a particular TNF- mediated disease.
  • a therapeutically effective amount is also that amount of TNF ⁇ antagonist, VEGF antagonist and methotrexate necessary to synergistically or significantly reduce or eliminate symptoms associated with a particular TNF-mediated disease.
  • a therapeutically effective amount is not an amount such that administration of TNF ⁇ antagonist alone, administration of VEGF antagonist alone, or administration of methotrexate alone, must necessarily result in inhibition of the biological activity of TNF ⁇ or VEGF.
  • a maintenance amount of TNF ⁇ antagonist alone, of VEGF antagonist alone, of methotrexate alone, or of a combination thereof can be administered to the individual .
  • a maintenance amount is the amount of TNF ⁇ antagonist, VEGF antagonist, methotrexate, or combination thereof, necessary to maintain the reduction or elimination of symptoms achieved by the therapeutically effective dose.
  • the maintenance amount can be administered in the form of a single dose, or a series or doses separated by intervals of days or weeks .
  • the dosage administered to an individual will vary depending upon a variety of factors, including the pharmacodynamic characteristics of the particular therapeutic agent, and its mode and route of administration; size, age, health, sex, body weight and diet of the recipient; nature and extent of symptoms of the disease being treated, kind of concurrent treatment, frequency of treatment, and the effect desired.
  • Such in vi tro assays can include a TNF cytotoxicity assay (e.g., the WEHI assay or a radioimmunoassay, ELISA) .
  • In vivo methods can include rodent lethality assays, primate pathology model systems (see, e.g.,
  • TNF ⁇ blockade can be monitored by monitoring IL-6 and C-reactive protein levels (Elliott et al . , Arth . Rheum . , 36:1681-1690 (1993)).
  • Methods of determining inhibition of VEGF are also well known to those of skill in the art (e.g., ELISA) .
  • TNF ⁇ antagonists, VEGF antagonists and methotrexate can be administered in single or multiple doses depending upon factors such as nature and extent of symptoms, kind of concurrent treatment and the effect desired.
  • other therapeutic regimens or agents e.g., multiple drug regimens
  • TNF ⁇ antagonists, VEGF antagonists and methotrexate can be used in combination with the therapeutic administration of TNF ⁇ antagonists, VEGF antagonists and methotrexate.
  • a daily dosage of active ingredient can be about 0.01 to 100 milligrams per kilogram of body weight. Ordinarily 1 to 40 milligrams per kilogram per day given in divided doses 1 to 6 times a day or in sustained release form is effective to obtain desired results . Second or subsequent administrations can be administered at a dosage which is the same, less than or greater than the initial or previous dose administered to the individual .
  • a second or subsequent administration is preferably during or immediately prior to relapse or a flare-up of the disease or symptoms of the disease.
  • the second and subsequent administrations can be given between about one day to 30 weeks from the previous administration.
  • Two, three, four or more total administrations can be delivered to the—individual , as needed.
  • Dosage forms (composition) suitable for internal administration generally contain from about 0.1 milligram to about 500 milligrams of active ingredient per unit.
  • the active ingredient will ordinarily be present in an amount of about 0.5-95% by weight based on the total weight of the composition.
  • RESULTS Patients Seventy-three (73) patients with active rheumatoid arthritis (RA) were enrolled in a multi-centre, randomised, placebo-controlled double-blind clinical trial of anti-TNF ⁇ antibody. All patients met the criteria of the American College of Rheumatology (active RA for ⁇ 6 months, failed treatment with at least one disease-modifying drug and evidence of erosive disease on radiography of hands and feet) (Arnett et al . , Arth . Rheum. , 31:315-324 (1988)).
  • Chimeric monoclonal anti-TNF ⁇ antibody cA2 was supplied as a sterile solution containing 5 mg cA2 per ml of 0.01 M phosphate-buffered saline in 0.15 M sodium chloride with 0.01% polysorbate 80, pH 7.2 (Centocor, Inc., Malvern, PA) .
  • the placebo vials contained 0.1% human serum albumin in the same buffer.
  • the appropriate amount of cA2 or placebo was diluted to 300 ml in sterile saline by the pharmacist, and administered intravenously via a 0.2 ⁇ m in-line filter over 2 hours .
  • the characteristics of the placebo and cA2 infusion bags were identical, and the investigators and patients did not know which infusion was being administered.
  • Synovial membrane samples obtained from RA patients undergoing total joint replacement were digested with 5 mg/ml type IV colagenase (Sigma, UK) and 150 ⁇ g/ml type I DNAse (Sigma, UK) (Brennan F.M. et al . , Lancet 2:244-247 (1989)).
  • Tissue was pipetted through a 200 ⁇ l nylon mesh, and cells were cultured in 60mm 2 wells in RPMI plus 5% foetal calf serum (FCS; BioWhittaker, Belgium) in the absence or presence of 10 ⁇ g/ml anti- TNF ⁇ monoclonal antibody cA2, alone or together with 10 ⁇ g/ml IL-1 receptor antagonist (IL-lra, from Dr. A. Berger, Upjohn Laboratories, MI) .
  • Synovial fibroblasts were selected by continuously culturing synovial membrane cells until a confluent onolayer of fibroblasts was obtained.
  • Adherent cells were further passaged using 0.05% trypsin/O .02% EDTA and cultured in RPMI and 10% FCS .
  • the human dermal microvascular endothelial cell line HMEC-1 was a gift from the Center for Disease Control and Prevention (Atlanta, GA)
  • the human monocytic cell line THP-1 was from the
  • Adherent cells endothelial cells and fibroblasts
  • THP-1 cells were cultured at confluent density in 200 mm 2 wells.
  • THP-1 cells were suspended at a density of 0.5 x 10 6 /60 mm 2 well.
  • Cells were stimulated for 72 hours in RPMI plus 5% FCS, in the absence or presence of either TNF ⁇ (gift from Prof. W. Stec, Centre of Molecular and Macromolecular Studies, Lodz, Tru) or IL-l ⁇ (gift from Hoffmann La Roche, USA) .
  • VEGF in culture supernatants and serum samples was assayed by enzyme-linked immunosorbent assay (ELISA) (R&D Systems, UK) .
  • ELISA enzyme-linked immunosorbent assay
  • Patient data were analyzed by Wilcoxon signed rank test for comparisons within groups, using individual data as input variables, and between treatment groups by Mann-Whitney U-test, using % change from week 0 as response variables .
  • Differences in VEGF release by RA synovial membrane cells between treatment groups were assessed by Mann-Whitney U-test. Comparisons between multiple groups were adjusted using the Bonferroni correction.
  • ESR and CRP Assessments were measured with a standard method (Westergen) .
  • CRP levels were measured by rate nephelometry (Abbott fluorescen . polarizing immunoassay) .
  • rate nephelometry Abbott fluorescen . polarizing immunoassay
  • Serum VEGF concentrations were measured by ELISA in a total of 53 age- and sex-matched non-arthritic individuals and 69 patients with active RA. To assess the degree of correlation between CRP and VEGF, the Kendall rank correlation coefficient for non-parametric data was calculated. Median serum VEGF levels in the 53 non-arthritic individuals were equivalent to 160 pg/ml (interquartile range 122-266 pg/ml) .
  • VEGF Secretion by Synovial Membrane Cells Is Dependent On Pro-inflammatory Cytokines.
  • dissociated RA synovial membrane cells (1 x 10 s cells/ml; from RA joints) were cultured for two days in the absence or presence of inhibitors of cytokine bio-activity (10 ⁇ g/ml cA2 , either alone or in combination with 10 ⁇ g/ml IL-lra) .
  • VEGF concentrations in culture supernatants were determined by ELISA.
  • Statistical analyses versus VEGF release in the absence of cytokine inhibitors were performed by Mann-Whitney U-test. Results are shown in Table 1. Values in the Table are ng/ml VEGF, and are representative of 5-6 experiments, with 1-6 determinations per experiment.
  • Synovial membrane cells were found to release VEGF spontaneously in a total of 29/32 experiments, with immunoreactive protein detected in culture supernatants approximately 12 hours after isolation.
  • median release of VEGF on day 2 in culture was decreased by 22%, from a median value of 3.29 ng/ml to 2.56 ng/ml (mean of 6 experiments) , although this reduction was not statistically significant (see Table) .
  • TNF ⁇ Induces VEGF Release From Monocytic And Endothelial Cell, But Not FromSynovial Fibroblasts.
  • RA synovial membrane fibroblasts and monocytic THP-1 released VEGF spontaneously (Figure 1) .
  • microvascular endothelial cells were also found to constitutively release significant amounts of VEGF protein ( Figure 1) .
  • VEGF secretion from THP-1 monocytic cells was markedly increased by TNF ⁇ (fold increase equivalent to 2.61) but not by IL-l ⁇
  • the response of RA synovial membrane fibroblasts to TNF ⁇ was lower (fold increase 1.29) than that induced by IL-l ⁇ (fold increase 2.23; Figure 1).
  • endothelial cells were almost equally responsive to TNF ⁇ and IL-l ⁇ (fold increase 3.37 and 3.22, respectively; Figure 1).
  • TNF ⁇ antibody in adjunct to methotrexate treatment. All patients met the criteria of the American College of Rheumatology (active RA for ⁇ 6 months, failed treatment with at least one disease-modifying drug and evidence of erosive disease on radiography of hands and feet) (Arnett et al . , Arth . Rheum . , 31:315-324 (1988)). Active disease was defined by the presence of six or more swollen joints plus at least three of four secondary criteria (duration of morning stiffness ⁇ 45 minutes; ⁇ 6 tender or painful joints; erythrocyte sedimentation rate (ESR) ⁇ 28 mm/h; C-reactive protein (CRP) ⁇ 20 mg/1 (Elliott M.J. et al . , Lancet, 344 : 1105 - 1110 (1994) ) .
  • ESR erythrocyte sedimentation rate
  • CRP C-reactive protein
  • Chimeric monoclonal anti-TNF ⁇ antibody cA2 was supplied as a sterile solution containing 5 mg cA2 per ml of 0.01 M phosphate-buffered saline in 0.15 M sodium chloride with 0.01% polysorbate 80, pH 7.2 (Centocor, Inc., Malvern, PA).
  • the placebo vials contained 0.1% human serum albumin in the same buffer.
  • the appropriate amount of cA2 or placebo was diluted to 300 ml in sterile saline by the pharmacist, and administered intravenously via a 0.2 ⁇ m in-line filter over 2 hours.
  • the characteristics of the placebo and cA2 infusion bags were identical, and the investigators and patients did not know which infusion was being administered.
  • Treatment Protocol and Serum Samples Patients were randomized to one of seven treatment groups. The number of patients in each dose (or treatment) group is indicated in Table 2. Each of the 43 patients received multiple infusions of either 0, 1, 3 or 10 mg/kg cA2. Infusions were at weeks 0, 2, 6, 10 and 14. Starting at week 0, the patients were receiving 7.5 mg/week methotrexate (Pharmacochemie, Netherlands) or 3 placebo tablets/week (Pharmacochemie, Netherlands) . Patients were monitored for adverse events during infusions and regularly thereafter, by interviews, physical examination and laboratory testing. Serum samples were obtained before and up to 28 weeks after the initial infusion.
  • Examples 1 and 2 demonstrate that pro-inflammatory cytokines, including TNF ⁇ and IL-1, regulate the major mediator of angiogenesis, VEGF, during the pathogenesis of RA, and that blockade of cytokine activity may modulate new blood vessel formation.
  • TNF ⁇ modulates production of VEGF in vivo, suggesting that part of the benefit of anti-TNF ⁇ antibody treatment may be due to reduction in angiogenesis, and that long term TNF ⁇ blockade can reduce neovascularisation and hence the cellular mass of the pannus and its destructive potential .
  • VEGF is an appropriate therapeutic target in RA for achieving synergism with anti-TNF ⁇ therapy, leading to long term benefit.

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EP98920669A 1997-05-12 1998-05-12 Suppression of tumor necrosis factor alpha and vascular endothelial growth factor in therapy Ceased EP0980258A1 (en)

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US7056695B2 (en) 2000-03-02 2006-06-06 Xencor TNF-α variants
AU4541101A (en) * 2000-03-02 2001-09-12 Xencor Inc Design and discovery of protein based tnf-alpha variants for the treatment of tnf-alpha related disorders
US9028822B2 (en) 2002-06-28 2015-05-12 Domantis Limited Antagonists against TNFR1 and methods of use therefor
CN101745112A (zh) * 2002-07-19 2010-06-23 艾博特生物技术有限公司 TNFα相关疾病的治疗
MXPA05003889A (es) 2002-10-15 2005-06-22 Celgene Corp Farmacos inhibidores de citosina selectiva para tratar sindrome mielodisplastico.
US7101978B2 (en) 2003-01-08 2006-09-05 Applied Molecular Evolution TNF-α binding molecules
JP2007504122A (ja) 2003-08-29 2007-03-01 ファイザー・インク 新規抗血管形成剤として有用なチエノピリジン−フェニルアセトアミドおよびその誘導体
WO2005041877A2 (en) * 2003-10-29 2005-05-12 Children's Medical Center Corporation Method of inhibiting rejection following organ transplantation
AP2006003619A0 (en) 2003-12-23 2006-06-30 Pfizer Novel quinoline derivatives
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BRPI0610058A2 (pt) 2005-05-16 2010-05-25 Abbott Biotech Ltd uso de inibidor de tnf para tratamento da poliartrite erosiva
JP2008120722A (ja) * 2006-11-10 2008-05-29 Benesis Corp 生物由来製品の偽薬
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