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WO2003088991A1 - Methodes de traitement d'une maladie auto-immune au moyen d'une molecule ctla4 soluble et d'un armm ou d'un ains - Google Patents

Methodes de traitement d'une maladie auto-immune au moyen d'une molecule ctla4 soluble et d'un armm ou d'un ains Download PDF

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Publication number
WO2003088991A1
WO2003088991A1 PCT/US2003/012356 US0312356W WO03088991A1 WO 2003088991 A1 WO2003088991 A1 WO 2003088991A1 US 0312356 W US0312356 W US 0312356W WO 03088991 A1 WO03088991 A1 WO 03088991A1
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subject
weight
day
ctla4
molecule
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PCT/US2003/012356
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WO2003088991A9 (fr
Inventor
Robert Cohen
Suzette Carr
David Hagerty
Robert J. Peach
Jean-Claude Becker
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Bristol-Myers Squibb Company
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Priority to CA002482042A priority Critical patent/CA2482042A1/fr
Priority to AU2003243152A priority patent/AU2003243152A1/en
Priority to EP03747047A priority patent/EP1496931A4/fr
Publication of WO2003088991A1 publication Critical patent/WO2003088991A1/fr
Publication of WO2003088991A9 publication Critical patent/WO2003088991A9/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A61K38/1793—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A61K38/1774—Immunoglobulin superfamily (e.g. CD2, CD4, CD8, ICAM molecules, B7 molecules, Fc-receptors, MHC-molecules)

Definitions

  • the present invention relates generally to the field of immune system diseases, e.g., rheumatic diseases.
  • the invention relates to methods and compositions for treating immune system diseases, e.g., rheumatic diseases, such as rheumatoid arthritis, by administering to a subject an effective amount of soluble CTLA4 molecules alone, or in conjunction with a Disease Modifying Anti-Rheumatic Drug (DMARD).
  • DMARD Disease Modifying Anti-Rheumatic Drug
  • Rheumatic diseases encompass a group of diseases that affect the musculo-skeletal and connective tissues of the body. These diseases are characterized by chronic inflammation that often leads to permanent tissue damage, deformity, atrophy and disability. Rheumatic diseases affect the joints, bone, soft tissue, or spinal cord (Mathies, H. 1983 Rheuma) and are classified as inflammatory rheumatism, degenerative rheumatism, extra- articular rheumatism, or collagen diseases. Some rheumatic diseases are known to be autoirnmune diseases caused by a subject's altered immune response.
  • Rheumatoid arthritis is a progressive rheumatic disease, affecting approximately 2% of the adult population of developed countries (Utsinger, P. D., et al, 1985 Rheumatoid Arthritis, p. 140). This disease is characterized by persistent inflammatory synovitis that causes destruction of cartilage and bone erosion, leading to structural deformities in the peripheral joints.
  • the symptoms associated with rheumatoid arthritis include joint swelling, joint tenderness, inflammation, morning stiffness, and pain, especially upon flexing.
  • Subjects having advanced stages of arthritis suffer from structural damage, including joint destruction with bone erosion (in: "Principals of Internal Medicine, Harrison, 13 th edition, pages 1648-1655).
  • patients can present other clinical symptoms of various organic lesions, including lesions of tlie skin, kidney, heart, lung, central nervous system, and eyes due to vasculitis related to the autoimmune process.
  • erythrocyte sedimentation rates include elevated erythrocyte sedimentation rates, and elevated levels of serum C-reactive protein (CRP) and/or soluble IL-2 receptor (IL-2r).
  • CRP serum C-reactive protein
  • IL-2r soluble IL-2 receptor
  • the erythrocyte sedimentation rate is increased in nearly all patients with active rheumatoid arthritis.
  • the level of serum C-reactive protein is also elevated and correlates with disease activity and the likelihood of progressive joint damage.
  • the level of soluble LL-2r a product of activated T-cells, is elevated in bloo serum and synovial fluid of patients with active rheumatoid arthritis (see: "Principals of Internal Medicine, Harrison, 13 th edition, page 1650).
  • Rheumatoid arthritis is believed to be a T-cell-mediated autoirnmune disease, involving antigen-nonspecific intercellular interactions between T-lymphocytes and antigen- presenting cells.
  • the magnitude of tlie T-cell response is determined by the co-stimulatory response elicited by the interaction between T-cell surface molecules and their ligands (Mueller, et al., 1989 Ann. Rev. Immunol. 7:445-480).
  • T-cell surface receptors CD28 and CTLA4, and their ligands, such as B7-related molecules CD80 (i.e., B7-1) and CD86 (i.e., B7-2), on antigen presenting cells (Linsley, P. and Ledbetter, J. 1993 Ann. Rev. Immunol. 11:191-212).
  • T-cell activation in the absence of co-stimulation results in anergic T-cell response (Schwartz, R. H., 1992 Cell 71:1065-1068) wherein the immune system becomes nonresponsive to stimulation.
  • rheumatoid arthritis is thought to be a T-cell-mediated immune system disease
  • one strategy to develop new agents to treat rheumatoid arthritis is to identify molecules that block co-stimulatory signals between T-lymphocytes and antigen presenting cells, by blocking the interaction between endogenous CD28 or CTLA4 and B7.
  • Potential molecules include soluble CTLA4 molecules that are modified (i.e. CTLA4 mutant molecules) to bind to B7 with higher avidity than wildtype CTLA4 (the sequence of which is shown in Figure 23) or CD28, thereby blocking the co-stimulatory signals.
  • Soluble forms of CD28 and CTLA4 have been constructed by fusing variable (V)-like extracellular domains of CD28 and CTLA4 to immunoglobulin (Ig) constant domains resulting in CD28Ig and CTLA4Ig.
  • V variable
  • Ig immunoglobulin
  • a nucleotide and amino acid sequence of CTLA4Ig is shown in Figure 24 with the protein beginning with methionine at position +1 or alanine at position -1 and ending with lysine at position +357.
  • CTLA4Ig binds both CD80-positive and CD86-positive cells more strongly than CD28Ig (Linsley, P., et al., 1994 Immunity 1:793-80).
  • CTLA4Ig fusion molecules were modified by mutation of amino acids in the CTLA4 portion of the molecules.
  • Regions of CTLA4 that, when mutated, alter the binding affinity or avidity for B7 ligands include the complementarity determining region 1 (CDR-1 as described in U.S. Patents 6,090,914, 5,773,253, 5,844,095; in copending U.S. Patent Application Serial Number 60/214,065; and by Peach et al, 1994. J. Exp.
  • CDR-3 complementarity determining region 3
  • CDR-3 is the conserved region of the CTLA4 extracellular domain as described in U.S. Patents 6,090,914, 5,773,253 and 5,844,095; in copending U.S. Patent Application Serial Number 60/214,065; and by Peach, R.J., et al J Exp Med 1994 180:2049-2058; the CDR-3-like region encompasses the CDR-3 region and extends, by several amino acids, upstream and/or downstream of the CDR-3 motif).
  • the CDR-3-like region includes a hexapeptide motif MYPPPY (SEQ LD NO.: 20) that is highly conserved in all CD28 and CTLA4 family members. Alanine scanning mutagenesis through the hexapeptide motif in CTLA4, and at selected residues in CD28Ig, reduced or abolished binding to CD80 (Peach, R.J., et al J Exp Med 1994 180:2049-2058; U.S. Patent No. 5,434,131; U.S. Patent No. 6,090,914; U.S. Patent No. 5,773,253.
  • soluble CTLA4Ig molecules by interchanging homologous regions of CTLA4 and CD28.
  • These chimeric CTLA4/CD28 homologue mutant molecules identified the MYPPPY hexapeptide motif common to CTLA4 and CD28, as well as certain non-conserved amino acid residues in the CDR-1- and CDR-3- like regions of CTLA4, as regions responsible for increasing the binding avidity of CTLA4 with CD80 (Peach, R. J., et ⁇ l., 1994 JExp Med 180:2049-2058).
  • Soluble CTLA4 molecules such as CTLA4Ig, CTLA4 mutant molecules or chimeric CTLA4/CD28 homologue mutants as described supra, introduce a new group of therapeutic drags to treat rheumatic diseases.
  • rheumatic diseases such as rheumatoid arthritis
  • DMARDs Disease Modifying Anti-Rheumatic Drags
  • DMARDs Disease Modifying Anti-Rheumatic Drags
  • TNF ⁇ tumor necrosis factor-alpha
  • these drugs merely slow down the progress of the rheumatoid arthritis, which resumes at an accelerated pace after the therapy is discontinued. Additionally, prolonged therapy with these nonspecific drugs produces toxic side effects, including a tendency towards development of certain malignancies, kidney failure, bone marrow suppression, pulmonary fibrosis, malignancy, diabetes, and liver function disorders. These drags may also gradually cease being effective after about 2-5 years (Kelley's Textbook of Rheumatology, 6 th Edition, pages 1001-1022). Newer, biologically based, DMARDs such as cytokine blockers may be more potent and may have longer lasting effects than older DMARDS such as hydrochloroquine, however, the long term safety of these newer drugs is still unknown. Reports of multiple sclerosis and lupus exist with the use of TNF blockers.
  • NSALDS Non- Steroidal Anti-mflarnmatory Drugs
  • steroid compounds e.g., corticosteroids or glucocorticoids
  • prednisone and methylprednisolone e.g., prednisone and methylprednisolone.
  • Steroids also have significant toxic side effects associated with their long-term use. (Kelley's Textbook of Rheumatology, 6 Edition, pages 829-833).
  • the present invention provides compositions and methods for treating immune system diseases, by administering to a subject soluble CTLA4 molecules, which bind to B7 molecules on B7-positive cells, thereby inhibiting endogenous B7 molecules from binding CTLA4 and/or CD28 on T-cells.
  • Soluble CTLA4 molecules used in the methods of the invention include CTLA4Ig and soluble CTLA4 mutant molecule L104EA29YIg.
  • the present invention provides compositions and methods for treating immune system diseases, by administering to a subject a combination of a DMARD and a molecule that blocks B7 interaction with CTLA4 and/or CD28.
  • the present invention also provides methods for inhibiting T-cell function, but not causing T-cell depletion, in a human by contacting B7-positive cells in the human with soluble CTLA4.
  • soluble CTLA4 include CTLA4Ig and soluble CTLA4 mutant molecules, such as L104EA29YIg.
  • the present invention also provides methods for treating (e.g. reducing symptoms of) rheumatic diseases, such as rheumatoid arthritis, by administering to a subject suffering from symptoms of arthritis, soluble CTLA4 molecules such as CTLA4Ig and/or soluble CTLA4 mutant molecule L104EA29YIg and/or a mix of any soluble CTLA molecule.
  • soluble CTLA4 molecules such as CTLA4Ig and/or soluble CTLA4 mutant molecule L104EA29YIg and/or a mix of any soluble CTLA molecule.
  • the CTLA4 mutant molecule L104EA29YIg e.g. beginning with methionine at position +1 or alanine at position -1 and ending with lysine at position +357, as shown in Figure 19, is preferred for use in the methods of the invention.
  • the present invention also provides methods for treating (e.g. reducing symptoms of) rheumatic diseases, such as rheumatoid arthritis, by administering to the subject a combination of 1) a DMARD, such as methotrexate or a molecule that blocks TNF interactions, and 2) soluble CTLA4 molecules, such as CTLA4Ig.
  • a DMARD such as methotrexate or a molecule that blocks TNF interactions
  • CTLA4 molecules such as CTLA4Ig.
  • the present invention also provides methods for reducing pathophysiological changes associated with an immune system disease (e.g., rheumatic disease), such as stractural damage, by administering to the subject diagnosed with the immune system disease (e.g., rheumatoid arthritis), soluble CTLA4 molecules alone or in conjunction with other therapeutic drugs, such as a DMARD.
  • the present invention also provides a pharmaceutical composition for treatmg immune system diseases, such as rheumatic diseases, comprising a pharmaceutically acceptable carrier and a biologically effective agent, such as soluble CTLA4 molecules, alone or in conjunction with other therapeutic drags, such as a DMARD, a NSAID, a corticosteroid and/or a glucocorticoid.
  • a pharmaceutical composition for treatmg immune system diseases such as rheumatic diseases
  • a pharmaceutically acceptable carrier and a biologically effective agent, such as soluble CTLA4 molecules, alone or in conjunction with other therapeutic drags, such as a DMARD, a NSAID, a corticosteroid and/or a glucocorticoid.
  • Kits comprising pharmaceutical compositions therapeutic for irnrnune system disease are also encompassed by the invention.
  • a kit comprising one or more of the pharmaceutical compositions of the invention is used to treat an immune system disease, e.g. rheumatoid arthritis.
  • the pharmaceutical composition comprises an effective amount of soluble CTLA4 molecules that bind to B7 molecules on B7-positive cells, thereby blocking the B7 molecules from binding CTLA4 and/or CD28 on T-cells.
  • the kit may contain one or more immunosuppressive agents used in conjunction with the pharmaceutical compositions of the invention.
  • Potential immunosuppressive agents include, but are not limited to, corticosteroids, nonsteroidal antiinflammatory drugs (e.g.
  • Cox-2 inhibitors prednisone, cyclosporine, cyclosporin A, azathioprine, methotrexate, TNF blockers or antagonists, hydroxychloroquine, sulphasalazopyrine (sulfasalazine), gold salts, infliximab, etanercept, anakinra and any biological agent targeting an inflammatory cytokine.
  • the present invention also provides methods for reducing the erythrocyte sedimentation rate that is associated with rheumatoid arthritis. Additionally, the present invention provides methods for reducing the levels of certain components of blood serum which are associated with rheumatoid arthritis, including C- reactive protein, LL-6, TNF- ⁇ , soluble ICAM-1, soluble E-selectin and/or soluble IL-2r.
  • Figure 1 A Demographic data of patient cohorts. Demographic data including gender, race, and disease duration as described in Example 3, infra.
  • Figure IB Demographic data of patient cohorts. Demographic data including gender, age, weight, and disease activity, evaluated by the patient and by the physician, as described in Example 3, infra.
  • Figure IC Demographic data of patient cohorts as described in Example 3, infra. Demographic data including disease activity, erythrocyte sedimentation rate (ESR), physical function (disability evaluated by health questionnaire), and C-reactive protein (CRP).
  • ESR erythrocyte sedimentation rate
  • CRP C-reactive protein
  • Figure ID Demographic data of patient cohorts as described in Example 3, infra. Demographic data including joint swelling, joint tenderness, morning stiffness, and pain.
  • Figure IE Demographic data of patient cohorts as described in Example 3, infra. Demographic data including prior treatments.
  • Figure 2 Summary of 'discontinuations at day 85 by reason as described in Example 3, infra.
  • Figure 3A ACR responses at Day 85 as described in Example 3, infra: ACR-20, -50, and —70 responses.
  • Figure 3B ACR-20 responses at Day 85, including placebo response, as described in Example 3, infra: ACR-20 response with 95% confidence limits.
  • Figure 3C ACR-20 responses at Day 85 as described in Example 3, infra: Difference in ACR-20 response with respect to 95% confidence intervals.
  • Figure 4A Basic (20% improvement) clinical responses in swollen and tender joint count in percentage of patients at Day 85 as described in Example 3, infra: basic clinical response, ACR-20.
  • Figure 4B Clinical responses (in percentage improvement) in swollen and tender joint count in percentage of patients at Day 85 as described in Example 3, infra: change in clinical response in percentage improvement.
  • Figure 5 A Pain response (by Likert scale by mean unit change from baseline) in percentage of patients at Day 85 as described in Example 3, infra: pain score changes from baseline.
  • Figure 5B Patient global disease changes (by Likert scale by mean unit change from baseline) in percentage of patients at Day 85 as described in Example 3, infra: patient global disease activity changes.
  • Figure 5C Physician global disease changes (by Likert scale by mean unit change from baseline) in percentage of patients at Day 85 as described in Example 3, infra: physician global disease activity changes.
  • Figure 5D Pain (by Likert scale by mean unit change from baseline) in percentage of patients at Day 85 as described in Example 3, infra: pain changes from baseline.
  • Figure 6 A Patient global assessment of disease activity change from baseline by range of 2 imits at Day 85 as described in Example 3, infra; disease activity improvement.
  • Figure 6B Physician global assessment of disease activity change from baseline by range of 2 units at Day 85 as described in Example 3, infra; disease activity improvement.
  • FIG. 7 A Percent reduction in C-reactive protein (CRP) levels at Day 85 as described in Example 3, infra: percentage reduction in CRP levels from baseline.
  • Figure 7B Difference in reduction in C-reactive protein (CRP) levels at Day 85 as described in Example 3, infra: percent reduction difference in CRP levels with 95% confidence intervals. '
  • Figure 7C Mean reduction in C-reactive protein (CRP) levels at Day 85 as described in Example 3, infra: mean change from baseline.
  • CRP C-reactive protein
  • Figure 8 Reduction in soluble IL-2 receptor levels mean change from baseline at Day 85 as described in Example 3, infra.
  • Figure 9A The effect of CTLA4Ig on tender joints over time as described in Example 3, infra: median difference from baseline.
  • Figure 9B The effect of CTLA4Ig on tender joints over time as described in Example 3, infra: mean difference from baseline.
  • Figure 10 A The effect of CTLA4Ig on swollen joints over time as described in Example 3, infi-a: median difference from baseline.
  • Figure 10B The effect of CTLA4Ig on swollen joints over time as described in Example 3, infra: mean difference from baseline.
  • Figure 11 The effect of CTLA4Ig on pain assessment mean difference from baseline over time as described in Example 3, infra.
  • Figure 12A The effect of CTLA4Ig on patient assessment of disease activity mean difference from baseline over time as described in Example 3, infra.
  • Figure 12B The effect of CTLA4Ig on physician assessment of disease activity mean difference from baseline over time as described in Example 3, infra.
  • Figure 13A The effect of L104EA29YIg on tender joints over time as described in Example 3, infra: median difference from baseline.
  • Figure 13B The effect of L104EA29YIg on tender joints over time as described in Example 3, infra: mean change from baseline.
  • Figure 14A The effect of L104EA29YIg on swollen joints over time as described in Example 3, infra: median difference from baseline.
  • Figure 14B The effect of L104EA29YIg on swollen joints over time as described in Example 3, infra: mean change from baseline.
  • Figure 15 The effect of L104EA29YIg on pain assessment over time as described in Example 3, infra: mean change from baseline over time.
  • Figure 16A The effect of L104EA29YIg on patient assessment of disease activity mean difference from baseline over time as described in Example 3, infra.
  • Figure 16B The effect of L104EA29YIg on physician assessment of disease activity mean difference from baseline over time as described in Example 3, infra.
  • Figure 17 Percent improvement in patient disability assessed by Health Assessment Questionnaire (HAQ) compared to the baseline at Day 85 with CTLA4Ig and L104EA29YIg treatment as described in Example 3, infra.
  • Figure 18 Nucleotide and amino acid sequence of L104EIg (SEQ ID NOs: 6-7) as described in Example 1, infra.
  • Figure 19 Nucleotide and amino acid sequence of L104EA29YIg (SEQ ID NOs: 8-9) as described in Example 1, infra.
  • Figure 20 Nucleotide and amino acid sequence of L104EA29LIg (SEQ ID NOs: 10-11) as described in Example 1, infra.
  • Figure 21 Nucleotide and amino acid sequence of L104EA29TIg (SEQ ID NOs: 12-13) as described in Example 1, infra.
  • Figure 22 Nucleotide and amino acid sequence of L104EA29WIg (SEQ LD NOs: 14-15) as described in Example 1, infra.
  • Figure 23 Nucleotide and amino acid sequence of CTLA4 receptor (SEQ ID NOs: 16- 17).
  • Figure 24 Nucleotide and amino acid sequence of CTLA4Ig (SEQ ID NOs: 18-19).
  • FIG. 25 SDS gel (FIG. 25 A) for CTLAttg (lane 1), L104EIg (lane 2), and L104EA29YIg (lane 3 A); and size exclusion chromatographs of CTLA4Ig (FIG. 25B) and L104EA29YIg (FIG. 25C).
  • Figures 26 (left and right depictions): A ribbon diagram of the CTLA4 extracellular Ig V-like fold generated from the solution structure determined by NMR spectroscopy.
  • FIG. 26 (right depiction) shows an expanded view of the CDR-1 (S25-R33) region and tlie MYPPPY region indicating the location and side-chain orientation of the avidity enhancing mutations, LI 04 and A29.
  • Figures 27 A & 27B FACS assays showing binding of L104EA29YIg, L104EIg, and CTLA4Ig to human CD 80- or CD86-transfected CHO cells as described in Example 2, infra.
  • Figures 28A & 28B Graphs showing inhibition of proliferation of CD80-positive and CD86-positive CHO cells as described in Example 2, infra.
  • Figures 29A & 29B Graphs showing that L104EA29YIg is more effective than CTLA4Ig at inhibiting proliferation of primary and secondary allostimulated T cells as described in Example 2, infra.
  • Figures 30 A-C Graphs illustrating that L104EA29YIg is more effective than CTLA4Ig at inhibiting IL-2 (FIG. 30A), IL-4 (FIG. 30B), and gamma ( ⁇ )-interferon (FIG. 30C) cytokine production of allostimulated human T cells as described in Example 2, infra.
  • Figure 31 A graph demonstrating that L104EA29YIg is more effective than CTLA4Ig at inhibiting proliferation of phytohemaglutinin- (PHA) stimulated monkey T cells as described in Example 2, infra.
  • PHA phytohemaglutinin-
  • Figure 32 A graph showing the equilibrium binding analysis of L104EA29YIg, L104EIg, and wild-type CTLA4Ig to CD86Ig.
  • Figures 33A & B Reduction in soluble ICAM-1 and soluble E-selectin levels mean change from baseline at Day 85 as described in Example 3, infra.
  • Figure 34 A graph showing the summary of ACR20 response by visit day in response to methotrexate and CTLA4Ig (2 and 10 mg/kg) therapy, as described in Example 5, infra.
  • Figure 35 A graph showing the summary of ACR50 response by visit day in response to methotrexate alone or methotrexate and CTLA4Ig (2 and 10 mg/kg) therapy, as described in Example 5, infra.
  • Figure 36 A graph showing the summary of ACR70 response by visit day in response to methotrexate alone or methotrexate and CTLA4Ig (2 and 10 mg/kg) therapy, as described in Example 5, infra.
  • Figure 37 A graph showing the mean ACR-N over time in response to methotrexate alone or methotrexate and CTLA4Ig (2 and 10 mg/kg) therapy, as described in Example 5, infra.
  • Figure 38 A bar graph showing the ACR response in response to methotrexate alone or methotrexate and CTLA4Ig (2 and 10 mg/kg) therapy on day 180 with a 95% confidence interval, as described in Example 5, infra.
  • Figure 39 A bar graph showing the proportion of New Active Joints in response to methotrexate alone or methotrexate and CTLA4Ig (2 and 10 mg/kg) therapy on day 180, as described in Example 5, infra.
  • Figure 40 A bar graph showing ACR response after therapy with methotrexate alone or methofrexate and CTLA4Ig (2 and 10 mg/kg) on day 180, as described in Example 5, infra.
  • Figure 41 A graph showing percent improvement in tender joints after therapy with methotrexate alone or methotrexate and CTLA4Ig (2 and 10 mg/kg) - mean percent improvement from baseline, as described in Example 5, infra.
  • Figure 42 A graph showing percent improvement in swollen joints after therapy with methotrexate alone or methotrexate and CTLA4Ig (2 and 10 mg/kg) - mean percent improvement from baseline, as described in Example 5, infra.
  • Figure 43 A graph showing percent improvement in pain after therapy with methotrexate alone or methotrexate and CTLA4Ig (2 and 10 mg/kg) - mean percent improvement from baseline, as described in Example 5, infra.
  • Figure 44 A graph showing percent improvement in regard to disease activity as reported by the subject after therapy with methofrexate alone or methotrexate and CTLA4Ig (2 and 10 mg/kg) - mean percent improvement from baseline, as described in Example 5, infra.
  • Figure 45 A graph showing percent improvement in regard to disease activity as reported by the physician after therapy with methotrexate alone or methotrexate and CTLA4Ig (2 and 10 mg/kg) - mean percent improvement from baseline, as described in Example 5, infra.
  • Figure 46 A graph showing percent improvement regarding physical function after therapy with methotrexate alone or methotrexate and CTLA4Ig (2 and 10 mg/kg) - mean percent improvement from baseline as measured by HAQ, as described in Example 5, infra.
  • Figure 47 A graph showing percent improvement in CRP levels function after therapy with methotrexate alone or methotrexate and CTLA4Ig (2 and 10 mg/kg) - mean percent improvement from baseline, as described in Example 5, infra.
  • Figure 48 A graph showing percent improvement in CRP levels fimction after therapy with methotrexate alone or methotrexate and CTLA4Ig (2 and 10 mg/kg) - median percent improvement from baseline, as described in Example 5, infra.
  • Figure 49 A graph showing the difference in ACR response rate on day 180 in two groups after therapy with CTLA4Ig (2 and 10 mg/kg) in comparison to a group treated with methotrexate (MTX) only (95% Confidence Limits), as described in Example 5, infra.
  • Figure 50 A graph showing the change from baseline for SF-36 Physical Health Component on day 180, in two groups after therapy with CTLA4Ig (2 and 10 mg/kg) compared to a group treated with methotrexate only (95% Confidence Limits), as described in Example 5, infra.
  • Figure 51 A graph showing the change from baseline for SF-36 Mental Health Component on Day 180, in two groups after therapy with CTLA4Ig (2 and 10 mg/kg) compared to a group treated with methotrexate only (95% Confidence Limits), as described in Example 5, infra.
  • Figure 52 A bar graph showing CRP levels at day 180 after therapy with methotrexate alone or methotrexate and CTLA4Ig (2 and 10 mg/kg), as described in Example 5, infra.
  • Figure 53 A bar graph showing Rheumatoid Factor levels on day 180 after therapy with methotrexate alone or methotrexate and CTLA4Ig (2 and 10 mg/kg), as described in Example 5, infra.
  • Figure 54 A bar graph showing LL-2r levels on day 180 after therapy with methotrexate alone or methotrexate and CTLA4Ig (2 and 10 mg/kg), as described in Example 5, infra.
  • Figure 55 A bar graph showing IL-6 levels on day 180 after therapy with methotrexate alone or methotrexate and CTLA4Ig (2 and 10 mg/kg), as described in Example 5, infra.
  • Figure 56 A bar graph showing TNF ⁇ levels on day 180 after therapy with methotrexate alone or methotrexate and CTLA4Ig (2 and 10 mg/kg), as described in Example 5, infra.
  • Figure 57 A table of the univariate methotrexate dose at screening/emollment for treatment group BMS 10 - freated with CTLA4Ig at 10 mg/kg body weight as described in Example 5, infra.
  • Figure 58 A table of the univariate methotrexate dose at screening/enrollment for treatment group BMS 2 - treated with CTLA4Ig at 2 mg/kg body weight as described in Example 5, infra.
  • Figure 59 A table of the univariate methotrexate dose at screening/enrollment for the placebo group, as described in Example 5, infi'a.
  • Figure 60 A table of tlie univariate methofrexate dose up to and including day 180 of the study for treatment group BMS 10 - freated with CTLA4Ig at 10 mg/kg body weight as described in Example 5, infra.
  • Figure 61 A table of the univariate methofrexate dose up to and including day 180 of the study for treatment group BMS 2 - treated with CTLA4Ig at 2 mg/kg body weight as described in Example 5, infra.
  • Figure 62 A table of the univariate methofrexate dose up to and including day 180 of the study for the placebo group, as described in Example 5, infra.
  • Figure 63 A bar graph showing the difference in modified ACR response rates on day 180 in two groups after therapy with etanercept alone (25 mg twice weekly) or in combination with CTLA4Ig (2mg/kg), as described in Example 6, infra.
  • Figure 64 A-C Graphs showing percentage improvement of individual components of the modified ACR criteria as assessed on each visit day after therapy with etanercept alone (25 mg twice weekly) or in combination with CTLA4Ig (2 mg/kg) as described in Example 6, infra.
  • Figure 65 A graph showing the change from baseline for SF-36 Physical Health
  • Component on day 180 in two groups after therapy with etanercept (25 mg biweekly) alone or in combination with CTLA4Ig (2 mg/kg) (95 % Confidence Limits), as described in Example 6, infra.
  • B A graph showing the change from baseline for SF-36 Mental Health Component on day 180, in two groups after therapy with etanercept (25 mg biweekly) alone or in combination with CTLA4Ig (2 mg/kg) (95% Confidence Limits), as described in Example 6, infra.
  • Figure 66 Nucleotide sequence of a CTLA4Ig encoding a signal peptide; a wild type amino acid sequence of the extracellular domain of CTLA4 starting at methionine at position +1 to aspartic acid at position +124, or starting at alanine at position -1 to aspartic acid at position +124; and an Ig region (SEQ ID NO.: 21).
  • Figure 67 Amino acid sequence of a CTLA4Ig having a signal peptide; a wild type amino acid sequence of the extracellular domain of CTLA4 starting at methionine at position +1 to aspartic acid at position +124, or starting at alanine at position -1 to aspartic acid at position +124; and an Ig region (SEQ ID NO.: 22).
  • Figure 68 A schematic diagram showing the disposition of subjects into three cohorts as described in Example 7, infra.
  • Figure 69 A Kaplan-Meier plot of the cumulative proportion of subjects who discontinued for any reason during the first 12 months of the study, as described in Example 7, infra.
  • Figure 70 A Kaplan-Meier plot of the cumulative proportion of subjects who discontinued due to lack of efficacy during the first 12 months of study, as described in Example 7, infra.
  • Figure 71 A A graph showing the ACR Responses on Day 180 for patients administered methofrexate alone or methotrexate and CTLA4Ig (2 or 10 mg/kg body weight) as described in Example 7, infra.
  • Figure 7 IB A graph showing the 95 Percent Confidence Intervals for Differences in ACR Responses on Day 180 for patients administered methofrexate alone or methotrexate and CTLA4Ig (2 or 10 mg/kg body weight) as described in Example 7, infra.
  • Figure 72 A A graph showing tlie ACR Responses on Day 360 for patients admimstered methotrexate alone or methotrexate and CTLA4Ig (2 or 10 mg/kg body weight) as described in Example 7, infra.
  • Figure 72B A graph showing the 95 Percent Confidence Intervals for Differences in ACR Responses on Day 360 for patients administered methotrexate alone or methotrexate and CTLA4Ig (2 or 10 mg/kg body weight) as described in Example 7, infra.
  • Figure 73A A graph summarizing the ACR 20 Response by Nisit during a one year interval for patients administered methotrexate alone or methotrexate and CTLA4Ig (2 or 10 mg/kg body weight) as described in Example 7, infra.
  • Figure 73B A graph summarizing the ACR 50 Response by Nisit during a one year interval for patients administered methotrexate alone or methotrexate and CTLA4Ig (2 or 10 mg/kg body weight) as described in Example 7, infra.
  • Figure 73 C A graph summarizing the ACR 70 Response by Nisit during a one year interval for patients administered methotrexate alone or methotrexate and CTLA4Ig (2 or 10 mg/kg body weight) as described in Example 7, infra.
  • Figure 74 A graph showing the Mean ACR- ⁇ over a one year time interval for patients administered methotrexate alone or methotrexate and CTLA4Ig (2 or 10 mg/kg body weight) as described in Example 7, infra.
  • Figure 75 A graph showing the Proportion of New Active Joints at Day 180 for patients administered methotrexate alone or methotrexate and CTLA4Ig (2 or 10 mg/kg body weight) as described in Example 7, infra.
  • Figure 76A A graph showing the Frequency of New Tender Joints per Subject at Day 180 for patients administered methotrexate alone or methofrexate and CTLA4Ig (2 or 10 mg/kg body weight) as described in Example 7, infra.
  • Figure 76B A graph showing tlie Frequency of New Tender Joints per Subject at Day 360 for patients administered methotrexate alone or methotrexate and CTLA4Ig (2 or 10 mg/kg body weight) as described in Example 7, infi'a.
  • Figure 77 A A graph showing the Frequency of New Swollen Joints per Subject at Day 180 for patients administered methotrexate alone or methotrexate and CTLA4Ig (2 or 10 mg/kg body weight) as described in Example 7, infra.
  • Figure 77B A graph showing the Frequency of New Swollen Joints per Subject at Day 360 for patients administered methotrexate alone or methofrexate and CTLA4Ig (2 or 10 mg/kg body weight) as described in Example 7, infra.
  • Figure 78 A graph showing the Proportion of New Active Joints at Day 360 for patients administered methotrexate alone or methotrexate and CTLA4Ig (2 or 10 mg/kg body weight) as described in Example 7, infra.
  • Figure 79 Graphs showing the: A) Change from Baseline in the Physical Health Domains on Day 180, and B) Change from Baseline in the Mental Health Domains on 180, for patients administered methotrexate alone or methotrexate and CTLA4Ig (2 or 10 mg/kg body weight) as described in Example 7, infra.
  • Figure 80 Graphs showing the: A) Change from Baseline in the Physical Health Domains on Day 360, and B) Change from Baseline in the Mental Health Domains on Day 360, for patients administered methotrexate alone or methotrexate and CTLA4Ig (2 or 10 mg/kg body weight) as described in Example 7, infra.
  • Figure 81 A graph showing the Soluble IL-2r Levels at Baseline, Days 180 and 360 for patients administered methotrexate alone or methotrexate and CTLA4Ig (2 or 10 mg/kg body weight) as described in Example 7, infra.
  • Figure 82 A graph showing the Rlieumatoid Factor Levels at Baseline, Days 180 and 360 for patients administered methotrexate alone or methotrexate and CTLA4Ig (2 or 10 mg/kg body weight) as described in Example 7, infra.
  • Figure 83 A graph showing the ICAM-1 Levels at Baseline, Days 180 and 360 for patients administered methotrexate alone or methotrexate and CTLA4Ig (2 or 10 mg/kg body weight) as described in Example 7, infi-a.
  • Figure 84 A graph showing the e-Selectin Levels at Baseline, Days 180 and 360 for patients administered methotrexate alone or methotrexate and CTLA4Ig (2 or 10 mg/kg body weight) as described in Example 7, infra.
  • Figure 85 A graph showing the Serum IL-6 at Baseline, Days 180 and 360 for patients administered methotrexate alone or methofrexate and CTLA4Ig (2 or 10 mg/kg body weight) as described in Example 7, infra.
  • Figure 86A A graph showing the CRP Levels at Baseline, Days 180 and 360 for patients admimstered methotrexate alone or methotrexate and CTLA4Ig (2 or 10 mg/kg body weight) as described in Example 7, infi-a.
  • Figure 86B A graph showing the TNF ⁇ Levels at Baseline, Days 180 and 360 for patients administered methotrexate alone or methofrexate and CTLA4Ig (2 or 10 mg/kg body weight) as described in Example 7, infra. DETAILED DESCRIPTION OF THE INVENTION
  • DMARD refers to a Disease Modifying Anti-Rheumatic Drag.
  • a DMARD is any agent that modifies the symptoms and/or progression associated with an immune system disease, including autoimmune diseases (e.g. rheumatic diseases),, graft- related disorders and immunoproliferative diseases. DMARDs modify one or more of the symptoms and/or disease progression associated with rheumatic disease.
  • Symptoms of rheumatic diseases include the following: joint swelling, pain, tenderness, morning stiffness, structural damage, an elevated level of serum C-reactive protein (CRP), an elevated level of soluble IL-2r, an elevated level of soluble ICAM-1, an elevated level of soluble E-selectin, an elevated level of rheumatoid factor, an elevated level of IL-6 or an elevated erythrocyte sedimentation rate (ESR).
  • CRP serum C-reactive protein
  • ICAM-1 an elevated level of soluble E-selectin
  • ESR erythrocyte sedimentation rate
  • DMARDs include, but are not limited to, dihydrofolic acid reductase inhibitors e.g., methotrexate; cyclophosphamide; cyclosporine; cyclosporin A; chloroquine; hydroxychloroquine; sulfasalazine (sulphasalazopyrine) gold salts D-penicillamine; leflunomide; azathioprine; anakinra; TNF blockers e.g., infliximab (REMICADE R ) or etanercept; and a biological agent that targets an inflammatory cytokine.
  • dihydrofolic acid reductase inhibitors e.g., methotrexate; cyclophosphamide; cyclosporine; cyclosporin A; chloroquine; hydroxychloroquine; sulfasalazine (sulphasalazopyrine) gold salts D-pen
  • NSALO refers to a Non-Steroidal Anti-mflarnmatory Drag.
  • NSAIDs reduce inflamniatory reactions in a subject.
  • NSAIDs include, but are not limited to acetyl salicylic acid, choline magnesium salicylate, difiunisal, magnesium salicylate, salsalate, sodium salicylate, diclofenac, etodolac, fenoprofen, flurbiprofen, indomethacin, ketoprofen, ketorolac, meclofenamate, naproxen, nabumetone, phenylbutazone, piroxicam, sulindac, tolmetin, acetaminophen, ibuprofen, Cox-2 inhibitors, meloxicam and tramadol.
  • ligand refers to a molecule that specifically recognizes and binds another molecule
  • a ligand for CTLA4 is a B7 molecule.
  • a ligand for the B7 molecule is a CTLA4 and/or CD28 molecule.
  • CTLA4 interaction with its ligand B7 can be blocked by administration of CTLA4Ig molecules.
  • Tumor Necrosis Factor (TNF) interaction with its ligand, TNF receptor (TNFR) can be blocked by administration of etanercept or other TNF/TNFR blocking molecules.
  • TNF Tumor Necrosis Factor
  • wild type CTLA4 or non-mutated CTLA4 has the amino acid sequence of naturally occurring, full length CTLA4 as shown in Figure 23 (also as described in U.S. Patent Nos. 5,434,131, 5,844,095, and 5,851,795 herein incorporated by reference in their entirety), or any portion or derivative thereof, that recognizes and binds a B7 or interferes with a B7 so that it blocks bmding to CD28 and/or CTLA4 (e.g., endogenous CD28 and/or CTLA4).
  • CD28 and/or CTLA4 e.g., endogenous CD28 and/or CTLA4
  • the extracellular domain of wild type CTLA4 begins with metliionine at position +1 and ends at aspartic acid at position +124, or the extracellular domain of wild type CTLA4 begins with alanine at position -1 and ends at aspartic acid at position +124 as shown in Figure 23.
  • Wild type CTLA4 is a cell surface protein, having an N-terminal extracellular domain, a transmembrane domain, and a C-terminal cytoplasmic domain.
  • the exfracellular domain b ids to target molecules, such as a B7 molecule.
  • the naturally occurring, wild type CTLA4 protein is translated as an immature polypeptide, which includes a signal peptide at the N-terminal end.
  • the immature polypeptide undergoes post-translational processing, which includes cleavage and removal of the signal peptide to generate a CTLA4 cleavage product having a newly generated N-terminal end that differs from the N-terminal end in the immature form.
  • post-translational processing includes cleavage and removal of the signal peptide to generate a CTLA4 cleavage product having a newly generated N-terminal end that differs from the N-terminal end in the immature form.
  • CTLA4 cleavage product removes one or more of the amino acids from the newly generated N-terminal end of the CTLA4 cleavage product.
  • the signal peptide may not be removed completely, generating molecules that begin before the common starting amino acid methionine.
  • the mature CTLA4 protein may start at methionine at position +1 or alanine at position —1.
  • the mature form of the CTLA4 molecule includes the extracellular domain or any portion thereof, which binds to B7.
  • CTLA4 mutant molecule means wildtype CTLA4 as shown in Figure 23 or any portion or derivative thereof, that has a mutation or multiple mutations
  • a CTLA4 mutant molecule has a sequence that it is similar but not identical to the sequence of wild type CTLA4 molecule, but still binds a B7.
  • the mutations may include one or more amino acid residues substituted with an amino acid having conservative (e.g., substitute a leucine with an isoleucine) or non-conservative (e.g., substitute a glycine with a tryptophan) structure or chemical properties, amino acid deletions, additions, frameshifts, or truncations.
  • CTLA4 mutant molecules may include a non-CTLA4 molecule therein or attached thereto. The mutant molecules may be soluble (i.e., circulating) or bound to a cell surface. Additional
  • CTLA4 mutant molecules include those described in U.S. Patent Application Serial Numbers 09/865,321, 60/214,065 and 60/287,576; in U.S. Patent Numbers 6,090,914
  • CTLA4 mutant molecules can be made synthetically or recombinantly.
  • CTLA4Ig is a soluble fusion protein comprising an extracellular domain of wildtype CTLA4 that binds B7, or a portion thereof, joined to an immunoglobulin constant region (Ig) , or a portion thereof .
  • a particular embodiment comprises the extracellular domain of wild type CTLA4 (as shown in Figure 23) starting at methionine at position +1 and ending at aspartic acid at position +124, or starting at alanine at position -1 to aspartic acid at position +124; a junction amino acid residue glutamine at position +125; and an immunoglobulin portion encompassing glutamic acid at position +126 through lysine at position +357 (DNA encoding CTLA4Ig was deposited on May 31, 1991 with the American Type Culture Collection (ATCC), 10801 University Boulevard., Manassas, VA 20110-2209 under the provisions of the Budapest Treaty, and has been accorded ATCC accession number ATCC 68629; Linsley, P., et al., 1994 Immunity
  • CTLA4Ig- 24 a Chinese Hamster Ovary (CHO) cell line expressing CTLA4Ig was deposited on May 31, 1991 with ATCC identification number CRL-10762).
  • the soluble CTLA4Ig molecules used in the methods and/or kits of the invention may or may not include a signal (leader) peptide sequence. Typically, in the methods and/or kits of the invention, the molecules do not include a signal peptide sequence.
  • L104EA29YIg is a fusion protein that is a soluble CTLA4 mutant molecule comprising an extracellular domain of wildtype CTLA4 with amino acid changes A29Y (a tyrosine amino acid residue substituting for an alanine at position 29) and L104E (a glutamic acid amino acid residue substituting for a leucine at position +104), or a portion thereof that binds a B7 molecule, joined to an Ig tail (included in Figure 19; DNA encoding L104EA29YIg was deposited on June 20, 2000 with ATCC number PTA-2104; copending in U.S. Patent Application Serial Numbers 09/579,927, 60/287,576 and 60/214,065, incorporated by reference herein).
  • the soluble L104EA29YIg molecules used in the methods and/or kits of the invention may or may not include a signal (leader) peptide sequence. Typically, in the methods and/or kits of the invention, the molecules do not include a signal peptide sequence.
  • soluble refers to any molecule, or fragments and derivatives thereof, not boimd or attached to a cell, i.e., circulating.
  • CTLA4, B7 or CD28 can be made soluble by attaching an immunoglobulin (Ig) moiety to the extracellular domain of CTLA4, B7 or CD28, respectively.
  • a molecule such as CTLA4 can be rendered soluble by removing its transmembrane domain.
  • the soluble molecules used in the methods, compositions and/or kits of the invention do not include a signal (or leader) sequence.
  • soluble CTLA4 molecules means non-cell-surface-bound (i.e. circulating) CTLA4 molecules or any functional portion of a CTLA4 molecule that binds B7 including, but not limited to: CTLA4Ig fusion proteins (e.g.
  • CTLA4 immunoglobulin
  • Ig immunoglobulin
  • IgC ⁇ l immunoglobulin
  • IgCgammal IgC ⁇ 2
  • IgC ⁇ 3 IgCgamma3
  • IgC ⁇ 4 IgCgamma4
  • IgC ⁇ IgCmu
  • IgC ⁇ l IgCalphal
  • IgC ⁇ 2 IgCalpha2
  • IgCdelta IgC ⁇ (IgCepsilon
  • the soluble CTLA4 molecules used in the methods of the invention may or may not include a signal (leader) peptide sequence. Typically, in the methods, compositions and/or kits of the invention, the molecules do not include a signal peptide sequence.
  • the extracellular domain of CTLA4 is the portion of CTLA4 that recognizes and binds CTLA4 ligands, such as B7 molecules.
  • an extracellular domain of CTLA4 comprises methionine at position +1 to aspartic acid at position +124 ( Figure 23).
  • an extracellular domain of CTLA4 comprises alanine at position —1 to aspartic acid at position +124 ( Figure 23).
  • the extracellular domain includes fragments or derivatives of CTLA4 that bind a B7 molecule.
  • the extracellular domain of CTLA4 as shown in Figure 23 may also include mutations that change the binding avidity of the CTLA4 molecule for a B7 molecule.
  • the te ⁇ n “mutation” means a change in the nucleotide or amino acid sequence of a wildtype molecule, for example, a change in the DNA and/or amino acid sequences of the wild-type CTLA4 exfracellular domain.
  • a mutation in DNA may change a codon leading to a change in the amino acid sequence.
  • a DNA change may include substitutions, deletions, insertions, alternative splicing, or trancations.
  • An amino acid change may include substitutions, deletions, insertions, additions, truncations, or processing or cleavage errors of the protein.
  • mutations in a nucleotide sequence may result in a silent mutation in the amino acid sequence as is well understood in the art.
  • nucleotide codons encode the same amino acid.
  • examples include nucleotide codons CGU, CGG, CGC, and CGA encoding the amino acid, arginine (R); or codons GAU, and GAC encoding the amino acid, aspartic acid (D).
  • R arginine
  • GAU codons GAU
  • GAC GAC encoding the amino acid, aspartic acid
  • a protein can be encoded by one or more nucleic acid molecules that differ in their specific nucleotide sequence, but still encode protein molecules having identical sequences.
  • the amino acid coding sequence is as follows:
  • the mutant molecule may have one or more mutations.
  • a "non-CTLA4 protein sequence" or "non-CTLA4 molecule” means any protein molecule that does not bind B7 and does not interfere with the binding of CTLA4 to its target.
  • the non-CTLA4 molecule, attached to the extracellular domain of a CTLA4 molecule can alter the solubility or affinity of the CTLA4 molecule.
  • An example includes, but is not limited to, an immunoglobulin (Ig) constant region or portion thereof.
  • the Ig constant region is a human or monkey Ig constant region, e.g., human C(gamma)l, including the hinge, CH2 and CH3 regions.
  • the Ig constant region can be mutated to reduce its effector functions (U.S. Patents 5,637,481, 5,844,095 and 5,434,131).
  • a "fragment” or “portion” is any part or segment of a molecule e.g. CTLA4 or CD28, preferably the extracellular domain of CTLA4 or CD28 or a part or segment thereof, that recognizes and binds its target, e.g., a B7 molecule.
  • B7 refers to the B7 family of molecules including, but not limited to, B7-1 (CD80) (Freeman et al, 1989, J Immunol. 143:2714-2722, herein incorporated by reference in its entirety), B7-2 (CD86) (Freeman et al, 1993, Science 262:909-911 herein incorporated by reference in its entirety; Azuma et al, 1993, Nature 366:76-79 herein mcorporated by reference in its entirety) that may recognize and bind CTLA4 and/or CD28.
  • a B7 molecule can be expressed on an activated B cell.
  • CD28 refers to the molecule that recognizes and binds B7 as described in U.S. Serial No. 5,580,756 and 5,521,288 (herein incorporated by reference in their entirety).
  • B7-positive cells are any cells with one or more types of B7 molecules expressed on the cell surface.
  • a "derivative" is a molecule that shares sequence similarity and activity of its parent molecule.
  • a derivative of CTLA4 includes a soluble CTLA4 molecule having an amino acid sequence at least 70% similar to the extracellular domain of wildtype CTLA4, and which recognizes and binds B7 e.g. CTLA4Ig or soluble CTLA4 mutant molecule L104EA29YIg.
  • a derivative means any change to the amino acid sequence and/or chemical quality of the amino acid e.g., amino acid analogs.
  • an immune response is to activate, stimulate, up-regulate, inhibit, block, down-regulate or modify the immune response.
  • the auto-immune diseases described herein may be treated by regulating an immune response e.g., by regulating functional CTLA4- and/or CD28- positive cell interactions with B7-positive cells.
  • a method for regulating an immune response comprises contacting the B7- positive cells with a soluble CTLA4 molecule of the invention so as to form soluble CTLA4/B7 complexes, the soluble CTLA4 molecule interfering with reaction of an endogenous CTLA4 and/or CD28 molecule with said B7 molecule.
  • a receptor, signal or molecule means to interfere with the activation of the receptor, signal or molecule, as detected by an art-recognized test. For example, blockage of a cell-mediated immune response can be detected by determining reduction of Rheumatic Disease associated symptoms. Blockage or inhibition may be partial or total.
  • blocking B7 interaction means to interfere with the binding of B7 to its ligands, such as CD28 and/or CTLA4, thereby obstructing T-cell and B7-positive cell interactions.
  • agents that block B7 interactions include, but are not limited to, molecules such as an antibody (or portion or derivative thereof) that recognizes and binds to the any of CTLA4, CD28 or B7 molecules (e.g. B7-1, B7-2); a soluble form (or portion or derivative thereof) of the molecules such as soluble CTLA4; a peptide fragment or other small molecule designed to interfere with the cell signal through the CTLA4/CD28/B7-mediated interaction.
  • the blocking agent is a soluble CTLA4 molecule, such as CTLA4Ig (ATCC 68629) or L104EA29YIg (ATCC PTA-2104), a soluble CD28 molecule such as CD28Ig (ATCC 68628), a soluble B7 molecule such as B7Ig (ATCC 68627), an anti-B7 monoclonal antibody (e.g. ATCC HB- 253, ATCC CRL-2223, ATCC CRL-2226, ATCC HB-301, ATCC HB-11341 and monoclonal antibodies as described in by Anderson et al in U.S. Patent 6,113,898 or Yokochi et al., 1982. J.
  • CTLA4Ig ATCC 68629
  • L104EA29YIg ATCC PTA-2104
  • a soluble CD28 molecule such as CD28Ig (ATCC 68628)
  • a soluble B7 molecule such as B7Ig (ATCC 68627)
  • Immun., 128(2)823-827 an anti-CTLA4 monoclonal antibody (e.g. ATCC HB-304, and monoclonal antibodies as described in references 82-83) and/or an anti-CD28 monoclonal antibody (e.g. ATCC HB 11944 and mAb 9.3 as described by Hansen (Hansen et al, 1980. Immunogenetics 10: 247-260) or Martin (Martin et al., 1984. J. Clin. Immun., 4(1): 18-22)).
  • an anti-CTLA4 monoclonal antibody e.g. ATCC HB-304, and monoclonal antibodies as described in references 82-83
  • an anti-CD28 monoclonal antibody e.g. ATCC HB 11944 and mAb 9.3 as described by Hansen (Hansen et al, 1980. Immunogenetics 10: 247-260) or Martin (Martin et al., 1984. J. Clin. Immun., 4(1): 18-22)
  • Blocking B7 interactions can be detected by art- recognized tests such as determining reduction of immune disease (e.g., rheumatic disease) associated symptoms, by determining reduction in T-cell/B7-cell interactions or by determining reduction in B7 interaction with CTLA4 and/or CD28. Blockage may be partial or total.
  • immune disease e.g., rheumatic disease
  • Blockage may be partial or total.
  • an "effective amount" of a molecule is defined as an amount that blocks the interaction of the molecule with its ligand.
  • an effective amount of a molecule that blocks B7 interaction with CTLA4 and/or CD28 may be defined as the amount of the molecule that, when bound to B7 molecules on B7-positive cells, inhibit B7 molecules from binding endogenous hgands such as CTLA4 and CD28.
  • an effective amount of a molecule that blocks B7 interaction with CTLA4 and/or CD28 may be defined as the amount of the molecule that, when bound to CTLA4 and/or CD28 molecules on T cells, inhibit B7 molecules from binding endogenous ligands such as CTLA4 and CD28.
  • treating a disease means to manage a disease by medicinal or other therapies. Treatment of a disease may ameliorate the symptoms of a disease, reduce the severity of a disease, alter the course of disease progression and/or ameliorate or cure the basic disease problem.
  • to treat an auto-immune disease may be accomplished by regulating an immune response e.g., by regulating functional CTLA4- and/or CD28- positive cell interactions with B7-positive cells.
  • treating an auto-immune disease may be accomplished by preventing the disease from occurring or progressing through the use of the compositions described herein.
  • immune system disease means any disease mediated by T-cell interactions with B7-positive cells including, but not limited to, autoimmune diseases, graft related disorders and immunoproliferative diseases.
  • immune system diseases include graft versus host disease (GVHD) (e.g., such as may result from bone marrow transplantation, or in the induction of tolerance), immune disorders associated with graft transplantation rejection, chronic rejection, and tissue or cell allo- or xenografts, including solid organs (e.g., kidney transplants), skin, islets, muscles, hepatocytes, neurons.
  • GVHD graft versus host disease
  • immunoproliferative diseases include, but are not limited to, psoriasis, T-cell lymphoma, T-cell acute lymphoblastic leukemia, testicular angiocentric T-cell lymphoma, benign lymphocytic angiitis, lupus (e.g. lupus erythematosus, lupus nephritis), Hashimoto's thyroiditis, primary myxedema, Graves' disease, pernicious anemia, autoirnmune afrophic gastritis, Addison's disease, diabetes (e.g.
  • insulin dependent diabetes mellitis type I diabetes mellitis, type LI diabetes mellitis
  • good pasture's syndrome myasthenia gravis, pemphigus, Crohn's disease, sympathetic ophthalmia, autoimmune uveitis, multiple sclerosis, autoimmune hemolytic anemia, idiopathic thrombocytopenia, primary biliary cirrhosis, chronic action hepatitis, ulceratis colitis, Sjogren's syndrome, rheumatic diseases (e.g. rheumatoid arthritis), polymyositis, scleroderma, and mixed connective tissue disease.
  • rheumatic diseases means any disease that affects the joints, bone, soft tissue, or spinal cord (Mamies, H. 1983 Rheuma) and comprises inflammatory rheumatism, degenerative rheumatism, extra-articular rheumatism, and collagen diseases.
  • rheumatic diseases include, but are not limited to, chronic polyarthritis, psoriasis arthropathica, ankylosing spondylitis, rheumatoid arthritis, panarteriitis nodosa, systemic lupus erythemat ⁇ sus, progressive systemic seleroderma, periarthritis humeroscapularis, arthritis uratica, chondrocalcinosis, dermatomyositis, muscular rheumatism, myositis, and myogelosis.
  • Some rheumatic diseases are known to be autoimmune diseases caused by a subject's altered immune response.
  • Gene therapy is a process to treat a disease by genetic manipulation.
  • Gene therapy involves introducing a nucleic acid molecule into a cell and the cell expressing a gene product encoded by the nucleic acid molecule.
  • introducing the nucleic acid molecule into a cell may be performed by introducing an expression vector containing the nucleic acid molecule of interest into cells ex vivo or in vitro by a variety of methods including, for example, calcium phosphate precipitation, diethyaminoethyl dextran, polyethylene glycol (PEG), electroporation, direct injection, lipofection or viral infection (Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press 1989); Kriegler M.
  • nucleotide sequences of interest may be introduced into a cell in vivo using a variety of vectors and by a variety of methods including, for example: direct administration of the nucleic acid into a subject (Williams et al, 1991 PNAS 88:2726-2730); or insertion of the nucleic acid molecule into a viral vector, production of the recombinant virus or viral particle, and infection of the subject with the recombinant virus (Battleman et al, 1993 JNeurosci 13:94-951; Carroll et al, 1993 J Cell Biochem 17E.241; Lebkowski et al, U.S.
  • HAQs Health Questionnaire Assessments
  • ACR refers to clinical response studies based on criteria established by the American College of Rheumatology. A subject satisfied the "ACR20" criterion if there was about a 20 percent improvement in tender and swollen joint counts and 20 percent improvement in three of five remaining symptoms measured, such as patient and physician global disease changes, pain, physical disability, and an acute phase reactant such as CRP or ESR (Felson, D. T., et al, 1993 Arthritis and Rheumatism 36:729-740; Felson, D. T., et al., 1995 Arthritis and Rheumatism 38:1-9).
  • a subject satisfied the "ACR50" or "ACR70” criterion if there was about a 50 or 70 percent improvement, respectively, in tender and swollen joint counts and about 50 or 70 percent improvement, respectively, in three of five rem ning symptoms measured, such as patient and physician global disease changes, pain, physical disability, and an acute phase reactant such as CRP or ESR.
  • SF-36 Medical Outcomes Study Short Form-36
  • DMARD e.g., methotrexate or etanercept
  • CTLA4Ig therapy on health-related quality of life
  • the SF-36 consists of 36 items which covers four physical and four mental domains (physical function, role- physical, bodily pain, general health, vitality, social function, role emotional, and mental health). These individual domains are used to derive the physical and mental component summary scores which range from about 0 to 100, with higher scores indicating better quality of life. Absolute differences of 5 or more in the SF-36 scores were considered clinically meaningful.
  • an immune disease e.g., rheumatic disease
  • an immune disease including, but not limited to, joint swelling, pain, tenderness, morning stiffness, stractural damage, an elevated level of serum C-reactive protein (CRP), an elevated level of soluble J-L-2r, an elevated level of soluble ICAM-1, an elevated level of soluble E-selectin, an elevated level of rheumatoid factor, an elevated level of IL-6 or an elevated erythrocyte sedimentation rate.
  • the present invention provides compositions and methods for treating immune system diseases, such as rheumatic diseases, by administering to a subject an effective amount of a ligand that blocks B7 interactions with CTLA4 and/or CD28.
  • ligands include: soluble CTLA4 molecules (such as CTLA4Ig, CTLA4-E7, CTLA4-p97, CTLA4-env g ⁇ l20, and mutant CTLA4 molecules such as, CTLA4/CD28Ig, L104EA29YIg, L104EA29LIg, L104EA29TIg and or L104EA29WIg), soluble CD28 molecules, soluble B7-1 molecules, soluble B7-2 molecules, and monoclonal antibodies that recognize and bind B7, CD28 and/or CTLA4 (e.g., an anti-CTLA4 monoclonal antibody, an anti-CD28 monoclonal antibody, an anti-B7-l monoclonal antibody or an anti-B7-2 monoclonal antibody.
  • compositions and methods for treating immune system diseases such as rheumatic diseases, by administering to a subject a combination of an effective amount of 1) a DMARD (such as methotrexate or a molecule that blocks TNF interactions, e.g., blocks TNF interactions with its ligand) or other therapeutic agent, plus 2) an effective amount of a molecule that blocks B7 interaction with CTLA4 and/or CD28 such as soluble CTLA4 molecules (e.g., CTLA4Ig, CTLA4Ig/CD28Ig, CTLA4- E7, CTLA4-p97, CTLA4-env gpl20, L104EA29YIg, L104EA29LIg, L104EA29TIg and/or L104EA29WIg), soluble CD28 molecules, soluble B7-1 molecules, soluble B7-2 molecules, and monoclonal antibodies that recognize and bind B7, CD28 and/or CTLA4 (e.g., an anti-CTLA4)
  • An effective amount of a molecule that blocks B7 interaction with CTLA4 and/or CD28 may be defined as the amount of anti-B7 monoclonal antibodies, soluble CTLA4 and/or soluble CD28 molecules that, when bound to B7 molecules on B7-positive cells, inhibit B7 molecules from binding endogenous ligands such as CTLA4 and CD28.
  • the inhibition maybe partial or complete.
  • an effective amount of a molecule that blocks B7 interaction with CTLA4 and/or CD28 may be defined as the amount of anti-CTLA4 monoclonal antibody, anti- CD28 monoclonal antibody or soluble B7 (B7-1 or B7-2) molecules that, when bound to CTLA4 and/or CD28 molecules on T cells, inhibit B7 molecules from binding endogenous ligands such as CTLA4 and CD28.
  • the inhibition may be partial or complete.
  • an effective amount of a molecule that blocks B7 interaction with CTLA4 and/or CD28 is an amount about 0.1 to 100 mg/kg weight of a subject.
  • the effective amount is an amount about 0.5 to 5 mg/kg weight of a subject, 0.1 to 5 mg/kg weight of a subject, about 5 to 10 mg/kg weight of a subject, about 10 to 15 mg/kg weight of a subject, about 15 to 20 mg kg weight of a subject, about 20 to 25 mg/kg weight of a subject, about 25 to 30 mg/kg weight of a subject, about 30 to 35 mg/kg weight of a subject, about 35 to 40 mg/kg weight of a subject, about 40 to 45 mg/kg of a subject, about 45 to 50 mg/kg weight of a subject, about 50 to 55 mg/kg weight of a subject, about 55 to 60 mg/kg weight of a subject, about 60 to 65 mg/kg weight of a subject, about 65 to 70 mg/kg weight of a subject, about 70 to 75 mg/kg weight of a subject
  • the effective amoimt of a molecule that blocks B7 interaction with CTLA4 and/or CD28 is an amount about 2 mg/kg to about 10 mg/kg weight of a subject.
  • the preferred amount is 10 mg/kg weight of a subject.
  • the effective amount is an amount about 0.1 to 4 mg/kg weight of a subject.
  • the effective amount is an amount about 0.1 to 0.5 mg/kg weight of a subject, about 0.5 to 1.0 mg/kg weight of a subject, about 1.0 to 1.5 mg/kg weight of a subject, about 1.5 to 2.0 mg/kg weight of a subject, about 2.0 to 2.5 mg/kg weight of a subject, about 2.5 to 3.0 mg/kg weight of a subject, about 3.0 to 3.5 mg/kg weight of a subject, about 3.5 to 4.0 mg/kg weight of a subject, about 4.0 to 4.5 mg/kg weight of a subject, about 4.5 to 5.0 mg/kg weight of a subject, about 5.0 to 5.5 mg/kg weight of a subject, about 5.5 to 6.0 mg/kg weight of a subject, about 6.0 to 6.5 mg/kg weight of a subject, about 6.5 to 7.0 mg/kg weight of a subject, about 7.0 to 7.5 mg/kg weight of a subject, about 7.5 to 8.0 mg/kg weight of a subject, about 8.0 to 8.5 mg/kg weight of
  • the effective amount is an amount about 0.1 to 20 mg/kg weight of a subject. In another embodiment, the effective amount is an amount about 0.1 to 2 mg kg weight of a subject, about 2 to 4 mg/kg weight of a subject, about 4 to 6 mg/kg weight of a subject, about 6 to 8 mg/kg weight of a subject, about 8 to 10 mg/kg weight of a subject, about 10 to 12 mg/kg weight of a subject, about 12 to 14 mg/kg weight of a subject, about 14 to 16 mg/kg weight of a subject, about 16 to 18 mg/kg weight of a subject or about 18 to 20 mg/kg weight of a subject.
  • the effective amount is about 2 mg/kg weight of a subject. In yet another embodiment, the effective amount is about 10 mg/kg weight of a subject.
  • CD28 is soluble CTLA4 and the effective amount of a soluble CTLA4 molecule is about 2 mg/kg weight of a subject. In another specific embodiment, the effective amoimt of a soluble CTLA4 molecule is about 10 mg/kg weight of a subject. In another specific embodiment, an effective amount of a soluble CTLA4 is 500 mg for a subject weighing less than 60 kg, 750 mg for a subject weighing between 60-100 kg and 1000 mg for a subject weighing more than 100 kg.
  • An effective amount of the molecule that blocks B7 interaction with CTLA4 and/or CD28 is soluble CTLA4 may be administered to a subject daily, weekly, monthly and/or yearly, in single or multiple times per hour/day/week/month/year, depending on need.
  • the molecule may initially be administered once every two weeks for a month, and then once every month thereafter.
  • the immune disease is a rheumatic disease.
  • Rheumatic diseases are any diseases which are characterized by (i) inflammation or degeneration of musculo-skeletal or connective tissue structures of the body, particularly the joints, and including muscles, tendons, cartilage, synovial and fibrous tissues, (ii) accompanied by joint swelling, joint tenderness, inflammation, morning stiffness, and/or pain, or impairment of locomotion or function of those structures and, in some cases, (iii) often accompanied by serological evidence of rheumatoid factor and other inflammatory surrogate markers.
  • Rheumatic diseases include, but are not limited to, rheumatoid arthritis.
  • the symptoms of rheumatoid arthritis include joint swelling, joint tenderness, inflammation, morning stiffness, and pain leading to physical disability.
  • Subjects afflicted with the advanced stages of arthritis suffer from symptoms of structural damage and debihtating pain.
  • Other organs also can be impaired by the autoirnmune mechanism.
  • the DMARD is methotrexate or a molecule that blocks TNF interactions such as etanercept
  • the molecule that blocks B7 interaction with CTLA4 and/or CD28 is a soluble CTLA4.
  • the methods of the invention comprise administering to a subject an effective amoimt of methotrexate or a molecule that blocks TNF interactions in combination with an effective amount of soluble CTLA4 in order to treat rheumatic diseases such as rheumatoid arthritis.
  • Effective amounts of methotrexate range about 0.1 to 40 mg/week.
  • the effective amount includes ranges of about 0.1 to 5 mg/week, about 5 to 10 mg/week, about 10 to 15 mg/week, about 15 to 20 mg/week, about 20 to 25 mg/week, about 25 to 30 mg/week, about 30 to 35 mg/week, or about 35 to 40 mg/ week.
  • methotrexate is administered in an amount ranging about 5 to 30 mg/week.
  • the effective amount of a soluble CTLA4 molecule is about 2 mg/kg weight subject and the effective amount of methotrexate is about 10 to 30 mg week.
  • the effective amount of a soluble CTLA4 molecule is about 10 mg/kg weight subject and the effective amount of methotrexate is about 10 to 30 mg week.
  • the DMARD is etanercept and the molecule that blocks B7 interaction with CTLA4 and or CD28 is a soluble CTLA4.
  • the methods of the invention comprise administering to a subject an effective amount of etanercept in combination with an effective amount of soluble CTLA4 in order to treat rheumatic diseases such as rheumatoid -arthritis.
  • Effective amounts of etanercept range about 0.1 to 100 mgweek.
  • the effective amount includes ranges of about 10 to 100 mk/week, about 0.1 to 50 mg/week, about 0.1 to 5 mg/week, about 5 to 10 mg/week, about 10 to 15 mg/week, about 15 to 20 mg/week, about 20 to 25 mg/week, about 25 to 30 mg/week, about 30 to 35 mg/week, about 35 to 40 mg/week, about 40 to 45 mg/week, about 45 to 50 mg/week, about 50 to 55 mg/week, about 55 to 60 mg/week, about 60 to 65 mg/week, about 65 to 70 mg/week, about 70 to 75 mg/week, about 75 to 80 mg/week, about 80 to 85 mg/week, about 85 to 90 mg/week, about 90 to 95 mg/week or about 95 to 100 mg/week.
  • etanercept is admimstered in an amount of about 50 mg/week, alternatively etanercept may be administered in an amount of about 25 mg twice weekly.
  • the effective amount of a soluble CTLA4 molecule is about 2 mg/kg weight subject and the effective amount of etanercept is about 25 mg twice a week.
  • the effective amount of a soluble CTLA4 molecule is about 10 mg/kg weight subject and the effective amount of etanercept is about 25 mg twice a week.
  • the invention also provides compositions and methods for treating immune system diseases, such as rheumatic diseases, by administering to a subject a combination of an effective amount of an NSAJ-D and/or other therapeutic agent plus an effective amount of a molecule that blocks B7 interaction with CTLA4 and/or CD28.
  • immune system diseases such as rheumatic diseases
  • the invention also provides compositions and methods for treating immune system diseases, such as rheumatic diseases, by administering to a subject a an effective amount of a glucocorticoid, corticosteroid and/or other therapeutic agent plus an effective amount of a molecule that blocks B7 interaction with CTLA4 and/or CD28.
  • immune system diseases such as rheumatic diseases
  • the present invention provides compositions for treating immune diseases, such as rheumatic diseases, comprising soluble CTLA4 molecules. Further, the present invention provides compositions comprising a biological agent that inhibits T-cell function but not T-cell depletion in a human by contacting B7-positive cells in the human with a soluble CTLA4.
  • soluble CTLA4 include CTLA4Ig and soluble CTLA4 mutant molecules such as L104EA29YIg ( Figure 19), L104EA29LIg ( Figure 20), L104EA29Tig ( Figure 21), and L104EA29WIg ( Figure 22).
  • CTLA4 molecules, with mutant or wildtype sequences may be rendered soluble by deleting the CTLA4 transmembrane segment (Oaks, M. K., et al., 2000 Cellular Immunology 201:144-153).
  • soluble CTLA4 molecules, with mutant or wildtype sequences may be fusion proteins, wherein the CTLA4 molecules are fused to non-CTLA4 moieties such as immunoglobulin (Ig) molecules that render the CTLA4 molecules soluble.
  • a CTLA4 fusion protein may include the extracellular domain of CTLA4 fused to an immunoglobulin constant domain, resulting in the CTLA4Ig molecule ( Figure 24) (Linsley, P.
  • immunoglobulin domains that may be fused to CTLA4 include, but are not limited to IgC ⁇ l (IgCgarnmal), IgO ⁇ 2 (IgCgamma2), IgC ⁇ 3 (IgCgamma3), IgC ⁇ 4 (IgCgamma4), IgC ⁇ (IgCmu), IgC ⁇ l (IgCalphal), IgC ⁇ 2 (IgCalpha2), IgC ⁇ (IgCdelta) or lgC ⁇ (IgCepsilon).
  • IgC ⁇ l IgCgarnmal
  • IgO ⁇ 2 IgCgamma2
  • IgC ⁇ 3 IgCgamma3
  • IgC ⁇ 4 IgCgamma4
  • IgC ⁇ IgCmu
  • IgC ⁇ l IgCalphal
  • IgC ⁇ 2 IgCalpha2
  • IgC ⁇ IgCdelta
  • lgC ⁇ IgCepsilon
  • the immunoglobulin moiety does not elicit a detrimental immune response in a subject.
  • the preferred moiety is the immunoglobulin constant region, including the human or monkey immunoglobulin constant regions.
  • One example of a suitable immunoglobulin region is human C ⁇ l, including the hinge, CH2 and CH3 regions which can mediate effector functions such as binding to Fc receptors, mediating complement-dependent cytotoxicity (CDC), or mediate antibody-dependent cell-mediated cytotoxicity (ADCC).
  • the immunoglobulin moiety may have one or more mutations therein, (e.g., in the CH2 domain, to reduce effector functions such as CDC or ADCC) where the mutation modulates the binding capability of the immunoglobulin to its ligand, by increasing or decreasing the binding capability of the immunoglobulin to Fc receptors.
  • mutations in the immunoglobulin moiety may include changes in any or all its cysteine residues within the hinge domain, for example, the cysteines at positions +130, +136, and +139 are substituted with serine ( Figure 24).
  • the immunoglobulin moiety may also include the proline at position +148 substituted with a serine, as shown in Figure 24.
  • the mutations in the immunoglobulin moiety may include having the leucine at position +144 substituted with phenylalanine, leucine at position +145 substituted with glutamic acid, or glycine at position +147 substituted with alanine.
  • Additional non-CTLA4 moieties for use in the soluble CTLA4 molecules or soluble CTLA4 mutant molecules include, but are not limited to, p97 molecule, env gpl20 molecule, E7 molecule, and ova molecule (Dash, B. et al. 1994 J. Gen. Virol. 75 (Pt 6):1389-97; Ikeda, T., et al. 1994 Gene 138(1-2): 193-6; Falk, K., et al. 1993 Cell. Immunol 150(2):447-52; Fujisaka, K. et al. 1994 Virology 204(2):789-93).
  • Other molecules are also possible (Gerard, C. et al. 1994 Neuroscience 62(3):721; Byrn, R. et al. 1989 63(10):4370; Smith, D. et al. 1987 Science 238:1704; Lasky, L. 1996 Science 233:209).
  • the soluble CTLA4 molecule of the invention can include a signal peptide sequence linked to the N-terminal end of the extracellular domain of the CTLA4 portion of the molecule.
  • the signal peptide can be any sequence that will permit secretion of the molecule, mcluding the signal peptide from oncostatin M (Malik, et al, (1989) Molec. Cell. Biol. 9: 2847-2853), or CD5 (Jones, N. H. et al., (1986) Nature 323:346-349), or the signal peptide from any extracellular protein.
  • the soluble CTLA4 molecule of the invention can include the oncostatin M signal peptide linked at the N-terminal end of the extracellular domain of CTLA4, and the human immunoglobulin molecule (e.g., hinge, CH2 and CH3) linked to the C-terminal end of the extracellular domain (wildtype or mutated) of CTLA4.
  • This molecule includes the oncostatin M signal peptide encompassing an amino acid sequence having methionine at position -26 through alanine at position -1, the CTLA4 portion encompassing an amino acid sequence having mettaonine at position +1 through aspartic acid at position +124, a junction amino acid residue glutamine at position +125, and the immunoglobulin portion encompassing an amino acid sequence having glutamic acid at position +126 through lysine at position +357.
  • the soluble CTLA4 mutant molecules of the invention comprising the mutated CTLA4 sequences described infi-a, can be fusion molecules comprising human Ig, e.g., IgC(gamma)l (i.e. IgC ⁇ l) moieties fused to the mutated CTLA4 fragments.
  • human Ig e.g., IgC(gamma)l (i.e. IgC ⁇ l) moieties fused to the mutated CTLA4 fragments.
  • the soluble CTLA4 mutant molecules comprise IgC ⁇ l (IgCgarnmal) fused to an extracellular domain of CTLA4 comprising a single-site mutation in the extracellular domain.
  • the extracellular domain of CTLA4 comprises metliionine at position +1 through aspartic acid at position +124 (e.g., Figure 23).
  • the extracellular domain of the CTLA4 can comprise alanine at position -1 tlirough aspartic acid at position +124 (e.g., Figure 23).
  • single-site mutations include the following wherein the leucine at position +104 is changed to any other amino acid:
  • mutant molecules having the extracellular domain of CTLA4 with two mutations, fused to an Ig C ⁇ l (IgCgammal) moiety include the following wherein the leucine at position +104 is changed to another amino acid (e.g. glutamic acid) and the glycine at position +105, the serine at position +25, the threonine at position +30 or the alanine at position +29 is changed to any other ai ino acid:
  • mutant molecules having the extracellular domain of CTLA4 comprising three mutations, fused to an IgC ⁇ l (IgCgammal ) moiety.
  • IgC ⁇ l IgCgammal
  • Examples include the following wherem the leucine at position +104 is changed to another amino acid (e.g. glutamic acid), the alanine at position +29 is changed to another amino acid (e.g. tyrosine) and the serine at position +25 is changed to another amino acid:
  • Soluble CTLA4 mutant molecules may have a junction amino acid residue which is located between the CTLA4 portion and the Ig portion of the molecule.
  • the junction amino acid can be any amino acid, including glutamine.
  • the junction amino acid can be introduced by molecular or chemical synthesis methods known in the art.
  • the soluble CTLA4 proteins of the invention can be generated by chemical synthesis methods.
  • the principles of solid phase chemical synthesis of polypeptides are well known in the art and may be found in general texts relating to this area (Dugas, H. and Penney, C. 1981 Bioorganic Chemistry, pp 54-92, Springer-Verlag, New York).
  • the soluble CTLA4 proteins may be synthesized by solid-phase methodology utilizing an Applied Biosystems 430 A peptide synthesizer (Applied Biosystems, Foster City, Calif.) and synthesis cycles supplied by Applied Biosystems.
  • Protected amino acids such as t-butoxycarbonyl-protected amino acids, and other reagents are commercially available from many chemical supply houses.
  • the present invention provides CTLA4 mutant molecules mcluding a signal peptide sequence linked to the N-terminal end of the extracellular domain of the CTLA4 portion of the mutant molecule.
  • the signal peptide can be any sequence that will permit secretion of the mutant molecule, including the signal peptide from oncostatin M (Malik, et al., 1989 Molec. Cell. Biol. 9: 2847-2853), or CD5 (Jones, N. H. et al., 1986 Nature 323:346-349), or the signal peptide from any extracellular protein.
  • the invention provides soluble CTLA4 mutant molecules comprising a single-site mutation in the extracellular domain of CTLA4 such as L104EIg (as included in Figure 18) or LI 04SIg, wherein LI 04EIg and L 104SIg are mutated in their CTLA4 sequences so that leucine at position +104 is substituted with glutamic acid or serine, respectively.
  • the single-site mutant molecules further include CTLA4 portions encompassing methionine at position +1 through aspartic acid at position +124, a junction amino acid residue glutamine at position +125, and an immunoglobulin portion encompassing glutamic acid at position +126 through lysine at position +357.
  • the immunoglobulin portion of the mutant molecule may also be mutated so that the cysteines at positions +130, +136, and +139 are substituted with serine, and the proline at position +148 is substituted with serine.
  • the single-site soluble CTLA4 mutant molecule may have a CTLA4 portion encompassing alanine at position -1 through aspartic acid at position +124.
  • the invention provides soluble CTLA4 mutant molecules comprising a double-site mutation in the extracellular domain of CTLA4, such as L104EA29YIg, L104EA29LIg, L104EA29TIg or L104EA29WIg, wherein leucine at position +104 is substituted with a glutamic acid and alanine at position +29 is changed to tyrosine, leucine, threonine and tryptophan, respectively.
  • soluble CTLA4 mutant molecules comprising a double-site mutation in the extracellular domain of CTLA4, such as L104EA29YIg, L104EA29LIg, L104EA29TIg or L104EA29WIg, wherein leucine at position +104 is substituted with a glutamic acid and alanine at position +29 is changed to tyrosine, leucine, threonine and tryptophan, respectively.
  • L104EA29YIg, L104EA29LIg, L104EA29TIg and L104EA29WIg The sequences for L104EA29YIg, L104EA29LIg, L104EA29TIg and L104EA29WIg, starting at methionine at position +1 and ending with lysine at position +357, plus a signal (leader) peptide sequence are shown in Figures 19- 22 respectively.
  • the double-site mutant molecules further comprise CTLA4 portions encompassing methionine at position +1 tlirough aspartic acid at position +124, a junction amino acid residue glutamine at position +125, and an immunoglobulin portion encompassing glutamic acid at position +126 through lysine at position +357.
  • the inrmunoglobulin portion of the mutant molecule may also be mutated, so that the cysteines at positions +130, +136, and +139 are substituted with serine, and the proline at position +148 is substituted with serine.
  • these mutant molecules can have a CTLA4 portion encompassing alanine at position -1 through aspartic acid at position +124.
  • the invention provides soluble CTLA4 mutant molecules comprising a double-site mutation in tlie exfracellular domain of CTLA4, such as L104EG105FIg, L104EG105WIg and L104EG105LIg, wherein leucine at position +104 is substituted with a glutamic acid and glycine at position +105 is substituted with phenylalanine, tryptophan and leucine, respectively.
  • the double-site mutant molecules further comprise CTLA4 portions encompassing methionine at position +1 through aspartic acid at position +124, a junction amino acid residue glutamine at position +125, and an immunoglobulin portion encompassing glutamic acid at position +126 through lysine at position +357.
  • the immunoglobulin portion of the may also be mutated, so that the cysteines at positions +130, +136, and +139 are substituted with serine, and the proline at position +148 is substituted with serine.
  • these mutant molecules can have a CTLA4 portion encompassing alanine at position -1 through aspartic acid at position +124.
  • L104ES25RIg which is a double-site mutant molecule comprising a CTLA4 portion encompassing methionine at position +1 through aspartic acid at position +124, a junction amino acid residue glutamine at position +125, and the immunoglobulin portion encompassing glutamic acid at position +126 through lysine at position +357.
  • the portion having the extracellular domain of CTLA4 is mutated so that serine at position +25 is substituted with arginine, and leucine at position +104 is substituted with glutamic acid.
  • L104ES25RIg can have a CTLA4 portion encompassing alanine at position -1 through aspartic acid at position +124.
  • the invention provides soluble CTLA4 mutant molecules comprising a double-site , mutation in the extracellular domain of CTLA4, such as L104ET30GIg and L104ET30NIg, wherein leucine at position +104 is substituted with a glutamic acid and threonine at position +30 is substituted with glycine and asparagine, respectively.
  • the double-site mutant molecules further comprise CTLA4 portions encompassing methionine at position +1 through aspartic acid at position +124, a junction amino acid residue glutamine at position +125, and an immunoglobulin portion encompassing glutamic acid at position +126 through lysine at position +357.
  • the immunoglobulin portion of the mutant molecule may also be mutated, so that the cysteines at positions +130, +136, and +139 are substituted with serine, and the proline at position +148 is substituted with serine.
  • these mutant molecules can have a CTLA4 portion encompassing alanine at position -1 through aspartic acid at position +124.
  • the invention provides soluble CTLA4 mutant molecules comprising a triple-site mutation in the extracellular domain of CTLA4, such as L104EA29YS25KIg, L104EA29YS25NIg, L104EA29YS25RIg, wherein leucine at position +104 is substituted with a glutamic acid, alanine at position +29 is substituted with tyrosine, and serine at position +25 is substituted with lysine, asparagine and arginine, respectively.
  • a triple-site mutation in the extracellular domain of CTLA4 such as L104EA29YS25KIg, L104EA29YS25NIg, L104EA29YS25RIg, wherein leucine at position +104 is substituted with a glutamic acid, alanine at position +29 is substituted with tyrosine, and serine at position +25 is substituted with lysine, asparagine and arginine, respectively.
  • the triple- site mutant molecules further comprise CTLA4 portions encompassing methionine at position +1 through aspartic acid at position +124, a junction amino acid residue glutamine at position +125, and an immunoglobulin portion encompassing glutamic acid at position +126 through lysine at position +357.
  • the immunoglobulin portion of the mutant molecule may also be mutated, so that the cysteines at positions +130, +136, and +139 are substituted with serine, and the proline at position +148 is substituted with serine.
  • these mutant molecules can have a CTLA4 portion encompassing alanine at position -1 through aspartic acid at position +124.
  • soluble CTLA4 mutant molecules include chimeric CTLA4/CD28 homologue mutant molecules that bind a B7 (Peach, R. J., et al, 1994 J Exp Med 180:2049-2058).
  • Examples of these chimeric CTLA4/CD28 mutant molecules include HS1, HS2, HS3, HS4, HS5, HS6, HS4A, HS4B, HS7, HS8, HS9, HS10, HSU, HS12, HS13 and HS14 (U.S.
  • Preferred embodiments of the invention are soluble CTLA4 molecules such as CTLA4Ig (as shown in Figure 24, starting at methionine at position +1 and ending at lysine at position +357) and soluble CTLA4 mutant L104EA29YIg (as shown in Figure 19, starting at methionine at position +1 and ending at lysine at position +357).
  • CTLA4Ig as shown in Figure 24, starting at methionine at position +1 and ending at lysine at position +357
  • L104EA29YIg as shown in Figure 19, starting at methionine at position +1 and ending at lysine at position +357.
  • the invention further provides nucleic acid molecules comprising nucleotide sequences encoding the amino acid sequences corresponding to the soluble CTLA4 molecules of the invention.
  • the nucleic acid molecule is a DNA (e.g., cDNA) or a hybrid thereof.
  • a CTLA4Ig molecule can comprise a GCT or GCC codon, encoding alanine, at nucleotide position +49 to +51 as shown in Figure 24.
  • a CTLA4Ig molecule can comprise a GGT or GGG codon, encoding glycine, at nucleotide position +436 to +438 as shown in Figure 24.
  • a CTLA4Ig molecule can comprise a CGG or CGT codon, encoding arginine, at nucleotide position +631 to +633 as shown in Figure 24.
  • DNA encoding CTLA4Ig ( Figure 24) was deposited on May 31, 1991 with tlie American Type Culture Collection (ATCC), 10801 University Boulevard., Manassas, NA 20110-2209 and has been accorded ATCC accession number ATCC 68629.
  • D ⁇ A encoding L104EA29YIg (sequence included in Figure 19) was deposited on June 19, 2000 with ATCC and has been accorded ATCC accession number PTA-2104.
  • the nucleic acid molecules are R ⁇ A or a hybrid thereof.
  • the nucleic acid molecules of the invention also include derivative nucleic acid molecules which differ from D ⁇ A or R ⁇ A molecules, and anti-sense molecules.
  • Derivative molecules include peptide nucleic acids (P ⁇ As), and non-nucleic acid molecules including phosphorothioate, phosphotriester, phosphoramidate, and methylphosphonate molecules, that bind to single-stranded D ⁇ A or R ⁇ A in a base pair- dependent manner (Zamecnik, P. C, et al, 1978 Proc. Natl. Acad. Sci. 75:280284; Goodchild, P. C, et al, 1986 Proc Natl. Acad. Sci. 83:4143-4146).
  • Peptide nucleic acid molecules comprise a nucleic acid oligomer to which an amino acid residue, such as lysine, and an amino group have been added. These small molecules, also designated anti-gene agents, stop franscript elongation by binding to their complementary (template) strand of nucleic acid (Nielsen, P. E., et al., 1993 Anticancer Drug Des 8:53-63). Reviews of methods for synthesis of DNA, RNA, and their analogues can be found in: Oligonucleotides and Analogues, eds. F. Eckstein, 1991, IRL Press, New York; Oligonucleotide Synthesis, ed. M. J. Gait, 1984, IRL Press, Oxford, England.
  • the invention provides a vector, which comprises the nucleotide sequences of the invention.
  • the term vector includes, but is not limited to, plasmids, cosmids, and phagemids.
  • the vector can be an autonomously replicating vector comprising a replicon that directs the replication of the rDNA within the appropriate host cell.
  • the vector can direct integration of the recombinant vector into the host cell.
  • Various viral vectors may also be used, such as, for example, a number of well known refroviral and adenoviral vectors (Berkner 1988 Biotechniques 6:616-629).
  • the vectors can permit expression of the soluble CTLA4 transcript or polypeptide sequences in prokaryotic or eukaryotic host cells.
  • the vectors include expression vectors, comprising an expression control element, such as a promoter sequence, which enables transcription of the inserted soluble CTLA4 nucleic acid sequences and can be used for regulating the expression (e.g., transcription and/or translation) of an operably linked soluble CTLA4 sequence i an appropriate host cell.
  • Expression control elements are known in the art and include, but are not limited to, inducible promoters, constitutive promoters, secretion signals, enhancers, transcription te ⁇ ninators, and other transcriptional regulatory elements.
  • initiation and termination codons include the Shine-Dalgamo sequence (e.g., prokaryotic host cells), and initiation and termination codons.
  • Specific initiation signals may also be required for efficient translation of a soluble CTLA4 sequence. These signals include the ATG-initiation codon and adjacent sequences. In cases where the soluble CTLA4 initiation codon and upstream sequences are inserted into the appropriate expression vector, no additional translational control signals may be needed. However, in cases where only the coding sequence, or a portion thereof, is inserted, exogenous transcriptional control signals including the ATG- hiitiation codon may be provided.
  • the initiation codon should be in the co ⁇ ect reading- frame to ensure translation of the entire insert.
  • Exogenous transcriptional elements and initiation codons can be of various origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of enhancers appropriate to the cell system in use (Scharf, D., et al, 1994 Results Probl. Cell. Differ. 20:125-62; Bittner, et al., 1987 Methods in Enzymol 153:516-544).
  • the prefe ⁇ ed vectors for expression of the soluble CTLA4 sequences in eukaryote host cells include expression control elements, such as the baculovirus polyhedrin promoter for expression in insect cells.
  • Other expression control elements include promoters or enhancers derived from the genomes of plant cells (e. g., heat shock, RUBISCO, storage protein genes), viral promoters or leader sequences or from plant viruses, and promoters or enhancers from the mammalian genes or from mammalian viruses.
  • the preferred vector includes at least one selectable marker gene that encodes a gene product that confers drug resistance such as resistance to ampicillin or tetracyline.
  • the vector also comprises multiple endonuclease restriction sites that enable convenient insertion of exogenous DNA sequences.
  • the prefe ⁇ ed vectors for generating soluble CTLA4 transcripts and/or the encoded soluble CTLA4 polypeptides are expression vectors which are compatible with prokaryotic host cells.
  • Prokaryotic cell expression vectors are well known in the art and are available from several commercial sources.
  • pET vectors e.g., pET-21, Novagen Corp.
  • BLUESCR ⁇ PT phagemid (Stratagene, LaJolla, CA)
  • pSPORT Gibco BRL, Rockville, MD
  • ptrp-lac hybrids may be used to express soluble CTLA4 polypeptides in bacterial host cells.
  • the preferred expression vectors for generating soluble CTLA4 transcripts and/or the encoded soluble CTLA4 polypeptides are expression vectors which are compatible with eukaryotic host cells. Tlie more prefe ⁇ ed vectors are those compatible with vertebrate cells. Eukaryotic cell expression vectors are well known in the art and are available from several commercial sources. Typically, such vectors are provided containing convenient restriction sites for insertion of the desired DNA segment. Typical of such vectors are PSVL and pKSV-10 (Pharmacia), pBPV-l/pML2d (International Biotechnologies, Inc.), pTDTl (ATCC, #31255), and similar eukaryotic expression vectors.
  • expression vectors examples include, but are not limited to, vectors for mammalian host cells (e.g., BPV-1, pHyg, pRSV, pSV2, pTK2 (Maniatis); pLRES (Clontech); pRc/CMV2, pRc/RSV, pSFVl (Life Technologies); pVPakc Vectors, pCMV vectors, pSG5 vectors (Stratagene)), retroviral vectors (e.g., pFB vectors (Stratagene)), pCDNA-3 (Invitrogen) or modified forms thereof, adenoviral vectors; adeno-associated virus vectors, baculovirus vectors, yeast vectors (e.g., pESC vectors (Stratagene)).
  • vectors for mammalian host cells e.g., BPV-1, pHyg, pRSV, pSV2, pTK2 (Maniatis); pLRES (
  • a host vector system comprises the vector of the invention in a suitable host cell.
  • suitable host cells include, but are not limited to, prokaryotic and eukaryotic cells.
  • eukaryotic cells are also suitable host cells.
  • eukaryotic cells include any animal cell, whether primary or immortalized, yeast (e.g., Saccharomyces cerevisiae. Schizosaccharomyces pombe. and Pichia pastoris . and plant cells.
  • Exemplary animal cells include cells from bovine, ovine, porcine, murine, equine, monkey and ape. Myeloma, COS and CHO cells are examples of animal cells that may be used as hosts.
  • CHO cells include, but are not limited to, DG44 (Chasin, et la., 1986 Som. Cell. Molec. Genet. 12:555-556; Kolkekar 1997 Biochemistry 36:10901- 10909), CHO-K1 (ATCC No.
  • CHO-K1 Tet-On cell line CCL-61
  • CHO-K1 Tet-On cell line Clontech
  • CHO designated ECACC 85050302 (CAMR, Salisbury, Wiltshire, UK)
  • CHO ' clone 13 GELVIG, Genova, IT
  • CHO clone B GEJMG, Genova, IT
  • CHO-K1/SF designated ECACC 93061607 (CAMR, Salisbury, Wiltshire, UK
  • RR-CHOKl designated ECACC 92052129 (CAMR, Salisbury, Wiltshire, UK).
  • Exemplary plant cells include whole plants, cell culture, or callus, from tobacco, com, soybean, and rice cells. Com, soybean, and rice seeds are also acceptable.
  • the CTLA4 mutant molecules of the invention may be isolated as natiirally-occurring polypeptides, or from any source whether natural, synthetic, semi-synthetic or recombinant. Accordingly, the CTLA4 mutant polypeptide molecules may be isolated as naturally- occurring proteins from any species, particularly mammalian, including bovine, ovine, porcine, murine, equine, and preferably human. Alternatively, the CTLA4 mutant polypeptide molecules may be isolated as recombinant polypeptides that are expressed in prokaryote or eukaryote host cells,. or isolated as a chemically synthesized polypeptide.
  • CTLA4 mutant molecules and fragments or derivatives thereof can be produced by recombinant methods. Accordingly, an isolated nucleotide sequence encoding wild-type CTLA4 molecules may be manipulated to introduce mutations, resulting in nucleotide sequences that encode the CTLA4 mutant polypeptide molecules.
  • the nucleotide sequences encoding the CTLA4 mutant molecules may be generated by site- directed mutagenesis methods, using primers and PCR amplification.
  • the primers can include specific sequences designed to introduce desired mutations. Alternatively, the primers can be designed to include randomized or semi-randomized sequences to introduce random mutations.
  • the invention includes pharmaceutical compositions comprising pharmaceutically effective amounts of a molecule that blocks B7 interaction with CTLA4 and or CD28 such as soluble CTLA4 molecules, CD28 molecules, B7 (B7-1 or B7-2) molecules, anti- CTLA4 monoclonal antibodies, anti-CD28 monoclonal antibodies or anti-B7 (B7-1 or B7-2) monoclonal antibodies.
  • a molecule that blocks B7 interaction with CTLA4 and or CD28 such as soluble CTLA4 molecules, CD28 molecules, B7 (B7-1 or B7-2) molecules, anti- CTLA4 monoclonal antibodies, anti-CD28 monoclonal antibodies or anti-B7 (B7-1 or B7-2) monoclonal antibodies.
  • the pharmaceutical compositions of the invention are useful for treatment of immune system diseases.
  • immune system diseases are mediated by CD28/CTLA4/B7 interactions.
  • the soluble CTLA4 molecules are preferably soluble CTLA4 molecules with wildtype sequence and/or soluble CTLA4 molecules having one or more
  • the pharmaceutical composition can include soluble CTLA4 protein molecules and/or nucleic acid molecules, and/or vectors encoding the molecules.
  • the soluble CTLA4 molecules have the amino acid sequence of the extracellular domain of CTLA4 as shown in either Figures 24 or 19 (CTLA4Ig or L104EA29Y, respectively). Even more preferably, the soluble CTLA4 mutant molecule is L104EA29YIg as disclosed herein.
  • the compositions may additionally include other therapeutic agents, including, but not limited to, DMARDs, NSAIDs, corticosteroids, glucocorticoids, drag toxins, alkylating agents, anti-neoplastic drugs, enzymes, antibodies, or conjugates.
  • An embodiment of the pharmaceutical composition of the invention comprises an effective amount of a molecule that blocks B7 interaction with CTLA4 and or CD28, such as the molecules and the suitable amounts of the molecules described supra, and an effective amount of a DMARD.
  • the amoimt of DMARDS administered to a subject varies depending on several factors including the efficacy of the drug on a specific subject and the toxicity (i.e. the tolerability) of a drug to a specific subject (Guidelines for the Management of Rheumatoid Arthritis, Arthritis and Rheumatism Vol. 39, No. 5, May 1996, pages 713- 711; Physician's Desk Reference 2002, Medical Economics Company, Inc. Montvale, NJ 07645). The following provides a range of drug dosages for each DMARD. An attending physician will determine specific dosages for each subject.
  • an effective amount can be in a range of about 1 to about 5000 mg/day. This range can be modified to an amount of about 1 to 10 mg/day, about 10 to 50 mg/day, about 50 to 100 mg/day, ab ut 100 to 150 mg/day, about 150 to 200 mg/day, about 200 to 250 mg/day, about 250 to 300 mg/day, about 300 to 350 mg/day, about 350 to 400 mg/day, about 400 to 450 mg/ day, about 450 to 500 mg/day, about 500 to 550 mg/day, about 550 to 600 mg/day, about 600 to 650 mg/day, about 650 to 700 mg/day, about 700 to 750 mg/day, about 750 to 800 mg/day, about 800 to 850 mg/day, about 850 to 900 mg/day, about 900 to 950 mg/day, about 950 to 1000 mg/day, about 1000 to 1100 mg/day, about 1100 to 1200 mg/day, about 1200 to 1300 mg/day, about
  • an effective amount of a DMARD can be in a range of about 0.1 mg/week to 40 mg/week; 0.1 mg/week to 5 mg/week; 5 mg/week to 10 mg/week; 10 mg/week to 30 mg/week; 30 mg/week to 35 mg/week; 0.1 mg/week to 100 mg/week; or 30 mg/week to 50 mg/week.
  • a DMARD can be administered in an amount of about 50 mg/week or 25 mg twice weekly. It would be clear to one skilled in the art that dosage range will vary depending on the particular DMARD being used, for example see below. Methotrexate is an antimetabolite molecule that interferes with DNA synthesis, repair and cellular replication.
  • Methotrexate functions as an inhibitor of dihydrofolic acid reductase i.e. it is a folic acid antagonist.
  • Methofrexate is commonly administered in an amount about 0.1 to 40 mg per week with a common dosage ranging about 5 to 30 mg per week.
  • Methotrexate may be administered to a subject in various increments: about 0.1 to 5 mg/week, about 5 to 10 mg/week, about 10 to 15 mg/week, about 15 to 20 mg/week, about 20 to 25 mg/week, about 25 to 30 mg/week, about 30 to 35 mg/week, or about 35 to 40 mg/ week.
  • an effective amount of a DMARD, including methotrexate is an amount about 10 to 30 mg/week.
  • Cyclophosphamide an alkylating agent
  • Cyclophosphamide may be administered in dosages ranging about 1 to 10 mg/kg body weight per day.
  • Cyclosporine e.g. NEORAL R
  • Cyclosporin A is commonly administered in dosages ranging from about 1 to 10 mg/kg body weight per day. Dosages ranging about 2.5 to 4 mg per body weight per day are commonly used.
  • Chloroquine or hydroxychloroquine is commonly administered in dosages ranging about 100 to 1000 mg daily. Preferred dosages range about 200-600 mg administered daily.
  • Sulfasalazine e.g., AZULF1OINE EN-tabs R
  • Sulfasalazine is commonly administered in amounts ranging about 50 to 5000 mg per day, with a common dosage of about 2000 to 3000 mg per day for adults. Dosages for children are commonly about 5 to 100 mg/kg of body weight, up to 2 grams per day.
  • Gold salts are formulated for two types of administration: injection or oral. Injectable gold salts are commonly prescribed in dosages about 5 to 100 mg doses every two to four weeks. Orally administered gold salts are commonly prescribed in doses ranging about 1 to 10 mg per day. D-penicillamine or penicillamine (CUPRIMINE R ) is commonly administered in dosages about 50 to 2000 mg per day, with preferred dosages about 125 mg per day up to 1500 mg per day.
  • Azathioprine is commonly administered in dosages of about 10 to 250 mg per day. Preferred dosages range about 25 to 200 mg per day. •
  • Analdnra (e.g. KINERET R ) is an interleukin-1 receptor antagonist.
  • a common dosage range for anakinra is about 10 to 250 mg per day, with a recommended dosage of about 100 mg per day.
  • Infliximab (REMICADE R ) is a chimeric monoclonal antibody that binds to tumor necrosis factor alpha (TNF ⁇ ) and inhibits the activity of TNF ⁇ . Infliximab is commonly administered in dosages about 1 to 20 mg/kg body weight every four to eight weeks. Dosages of about 3 to 10 mg/kg body weight may be administered every four to eight weeks depending on the subject.
  • Etanercept is a dimeric fusion protein that binds the tumor necrosis factor (TNF) and blocks its interactions with TNF receptors.
  • TNF tumor necrosis factor
  • Commonly administered dosages of etanercept are about 10 to 100 mg per week for adults with a preferred dosage of about 50 mg per week. Dosages for juvenile subjects range about 0.1 to 50 mg/kg body weight per week with a maximum of about 50 mg per week. For adult patients, etanercept is commonly admimstered e.g., injected, in 25 mg doses twice weekly e.g., 72- 96 hours apart in time.
  • Leflimomide (ARAVA R ) is commonly administered at dosages about 1 and 100 mg per day. A common daily dosage is about 10 to 20 mg per day.
  • a flirther embodiment of the invention is a pharmaceutical composition comprising an effective amount of a soluble CTLA4, such as CTLA4Ig, and an effective amount of a DMARD, such as methotrexate or etanercept.
  • a pharmaceutical composition comprising soluble CTLA4 can be used for methods for blocking B7 interaction with CTLA4 and/or CD28; or for treating immune system diseases. Effective amounts of soluble CTLA4 in the pharmaceutical composition range about 0.1 to 100 mg/kg weight of the subject.
  • the effective amount is an amount about 0.5 to 5 mg/kg weight of a subject, about 5 to 10 mg/kg weight of a subject, about 10 to 15 mg/kg weight of a subject, about 15 to 20 mg/kg weight of a subject, about 20 to 25 mg/kg weight of a subject, about 25 to 30 mg/kg weight of a subject, about 30 to 35 mg/kg weight of a subject, about 35 to 40 mg/kg weight of a subject, about 40 to 45 mg/kg of a subject, about 45 to 50 mg/kg weight of a subject, about 50 to 55 mg/kg weight of a subject, about 55 to 60 mg/kg weight of a subject, about 60 to 65 mg/kg weight of a subject, about 65 to 70 mg/kg weight of a subject, about 70 to 75 mg/kg weight of a subject, about 75 to 80 mg/kg weight of a subject, about 80 to 85 mg/kg weight of a subject, about 85 to 90 mg/kg weight of a subject, about 90 to 95 mg/kg weight of a subject, about
  • the effective amount of soluble CTLA4 is an amount about 2 mg/kg to about 10 mg/kg weight of a subject. In another embodiment, the effective amount is an amount about 0.1 to 4 mg/kg weight of a subject. In another embodiment the effective amount is an amount about 0.1 to 0.5 mg/kg weight of a subject, about 0.5 to 1.0 mg/kg weight of a subject, about 1.0 to 1.5 mg/kg weight of a subject, about 1.5 to 2.0 mg/kg weight of a subject, about 2.0 to 2.5 mg/kg weight of a subject, about 2.5 to 3.0 mg/kg weight of a subject, about 3.0 to 3.5 mg/kg weight of a subject or about 3.5 to 4.0 mg/kg weight of a subject.
  • the effective amount is an amount about 0.1 to 20 mg/kg weight of a subject
  • the effective amount is an amount about 0.1 to 2 mg/kg weight of a subject, about 2 to 4 mg/kg weight of a subject, about 4 to 6 mg/kg weight of a subject, about 6 to 8 mg/kg weight of a subject, about 8 to 10 mg/kg weight of a subject, about 10 to 12 mg/kg weight of a subject, about 12 to 14 mg/kg weight of a subject, about 14 to 16 mg/kg weight of a subject, about 16 to 18 mg/kg weight of a subject or about 18 to 20 mg/kg weight of a subject.
  • the effective amount is 2 mg/kg weight of a subject.
  • the effective amount is about 10 mg/kg weight of a subject.
  • an effective amount of soluble CTLA4 is 500 mg for a subject weighing less than 60 kg, 750 mg for a subject weighing between 60-100 kg and 1000 mg for a subject weighing more than 100 kg.
  • Effective amounts of methotrexate in the pharmaceutical composition range about 0.1 to 40 mg/week.
  • the effective amount is an amount about 0.1 to 5 mg/week, about 5 to 10 mg/week, about 10 to 15 mg/week, about 15 to 20 mg/week, about 20 to 25 mg/week, about 25 to 30 mg/week, about 30 to 35 mg/week, or about 35 to 40 mg/ week.
  • an effective amount of a DMARD, including methotrexate is an amount about 10 to 30 mg/week.
  • the effective amount of a soluble CTLA4 molecule is about 2 mg/kg weight subject and the effective amount of methofrexate is about 10 to 30 mg/week. In another embodiment, the effective amount of a soluble CTLA4 molecule is about 10 mg/kg weight subject and the effective amount of methofrexate is about 10 to 30 mg week..
  • Effective amounts of etanercept in the pharmaceutical composition range about 0.1 to 100 mg/week. In one embodiment, the effective amount is ranges about 0.1 to 5 mg/week, about 5 to 10 mg/week, about 10 to 15 mg/week, about 15 to 20 mg/week, about 20 to 25 mg/week, about 25 to 30 mg/week, about 30 to 35 mg/week, about 35 to 40 mg/week, about 40 to 45 mg/week, about 45 to 50 mg/week, about 50 to 55 mg/week, about 55 to 60 mg/week, about 60 to 65 mg/week, about 65 to 70 mg/week, about 70 to 75 mg/week, about 75 to 80 mg/week, about 80 to 85 mg/week, about 85 to 90 mg/week, about 90 to 95 mg/week or about 95 to 100 mg/week. In one embodiment, etanercept is administered in an amount ranging about 50 mg/week e.g., 25 mg administered twice weekly.
  • the effective amount of a soluble CTLA4 molecule is about 2 mg/kg weight subject and the effective amount of etanercept is about 25 mg twice a week, hi another embodiment, the effective amount of a soluble CTLA4 molecule is about 10 mg/kg weight subject and the effective amount of etanercept is about 25 mg twice a week.
  • compositions of the invention further encompass a pharmaceutical composition comprising soluble CTLA4 in combination with other treatments for rheumatic disease including, but not limited to: collagen, dnaJ, molecules that block TNF function (e.g., pegsunercept), molecules that block cytokine function (e.g., AMG719), molecules that block LFA-1 function (e.g., efalizumab) and stem cell transplants.
  • TNF function e.g., pegsunercept
  • cytokine function e.g., AMG719
  • LFA-1 function e.g., efalizumab
  • Collagen for example in the form of bovine II collagen, may be orally administered to a patient suffering from rheumatoid arthritis in order to alleviate one or more symptoms of rheumatoid arthritis.
  • DnaJ is a small peptide which mimics a protein contained in a gene in many patients with rheumatoid arthritis.
  • the peptide is derived from E. coli bacteria heat shock protein.
  • DnaJ may be orally administered to a patient suffering from rheumatoid arthritis in order to alleviate one or more symptoms of rheumatoid artiiritis.
  • TNF is a molecule involved in the inflammatory response of patients with rheumatoid arthritis.
  • any molecule that blocks TNF function e.g., by blocking TNF binding to the TNF receptor (TNFR)
  • TNF receptor TNF receptor
  • TNF blockers such as infliximab and etanercept, have been shown to be efficacious in treating rheumatoid arthritis.
  • Other TNF blockers such as pegsunercept are being developed and tested (Phase IT clinical trial) for their efficacy in treating rheumatoid arthritis.
  • Cytokines e.g., Interleukin-1 (IL-1), are cell secreted molecules involved in mediating immune responses. Conceivably, any molecule that blocks cytokine function e.g., by blocking IL-1 interaction with its receptor, may help modify the progression of rheumatoid arthritis and alleviate one or more of its symptoms.
  • IL-1R Interleukin-1 receptor
  • AMG719 An IL-1 inhibitor, AMG719, is being developed and tested (Phase ⁇ clinical trial) for its efficacy in treating rheumatoid arthritis.
  • Lymphocyte function associated molecule 1 is a molecule composed of two subunits, CDlla and CD18, which functions by mediating lymphocyte adhesion to various cell types such as endothelium. Conceivably, interference of LFA-1 function may help modify the progression of rheumatoid arthritis and alleviate one or more of its symptoms.
  • An anti-LFA-1 antibody, efalizumab is being developed and tested (Phase II clinical trial) for its efficacy in treating rheumatoid arthritis.
  • Blockage of TNF, cytokine or LFA-1 interaction to their ligands by a potentially therapeutic molecule can be determined by any number of assays known to those skilled in the art.
  • competition assays may be used to test blockage by the molecule of interest e.g., a molecule can be exposed to a TNF/TNFR binding pair in order to compete with TNF to bind to TNFR.
  • functional assays can be performed to test blockage e.g., a molecule can be tested for its ability to inhibit an inflammatory cascade, or any part of an inflammatory reaction such as swelling, redness or pain, caused by a cytokine.
  • the present invention also provides pharmaceutical compositions comprising the rnolecules of the inventon e.g., CTLA4Ig and an acceptable carrier or adjuvant which is known to those of skill of the art.
  • the pharmaceutical compositions preferably include suitable carriers and adjuvants which include any material which when combined with the molecules of the invention (e.g., a soluble CTLA4 molecule, such as, CTLA4Ig or L104EA29Y) retain the molecule's activity, and is non-reactive with the subject's immune system.
  • carriers and adjuvants include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, phosphate buffered saline solution, water, emulsions (e.g. oil/water emulsion), salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances and polyethylene glycol.
  • buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, phosphate buffered saline solution, water, emulsions (e.g
  • compositions comprising such carriers are formulated by well known conventional methods. Such compositions may also be formulated within various lipid compositions, such as, for example, liposomes as well as in various polymeric compositions, such as polymer microspheres.
  • kits i.e., a packaged combination of reagents with instructions
  • the molecules of the invention useful for blocking B7 interactions with its ligands and/or for treating an immune system disease.
  • the kit can contain a pharmaceutical composition that includes one or more agents, for example, a soluble CTLA4 molecule alone, or with a second agent, and an acceptable carrier or adjuvant, e.g., pharmaceutically acceptable buffer, such as phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.
  • the agents may be provided as dry powders, usually lyophilized, including excipients that upon dissolving will provide a reagent solution having the appropriate concentration.
  • Second agents can include the following: steroids, glucocorticoids, drug toxins, alkylating agents, anti-neoplastic drags, enzymes, antibodies, conjugates, immunosuppressive agents, corticosteroids, DMARDs, nonsteroidal antiinflammatory drags (NSATDs), prednisone, azathioprine, methotrexate, TNF ⁇ blockers or antagonists, infliximab, any biological agent targeting an mflammatory cytokine, chloroquine, hydroxychloroquine, sulfasalazine (sulphasalazopryine), gold salts, etanercept, anakinra, cyclophosphamide, leflunomide, collagen, dnaJ, a molecule that blocks TNF receptors (e.g., pegsunercept), a molecule that blocks cytokine function(e.g., AMG719), a molecule that blocks LFA-1 function (e.g.
  • the kit comprises a container with a label and/or instructions.
  • Suitable containers include, for example, bottles, vials, and test tubes.
  • the containers can be formed from a variety of materials such as glass or plastic.
  • the container can have a sterile access port (for example the container can be an intravenous solution bag or a vial having a stopper pierceable by a needle such as a hypodermic injection needle).
  • the container can hold a pharmaceutical composition such as a pharmaceutical composition having an agent that is effective for blocking B7 interactions with its ligand and/or treating an immune system disease.
  • the kit can also comprise a second container comprising one or more second agents as described herein (e.g., any of the DMARDS or NSAIDS) and/or a pharmaceutically acceptable buffer, such as phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.
  • the kit may also suitably include a label and/or instructions on, or associated with the container.
  • the label can provide directions for carrying out the preparation of the agents for example, dissolving of the dry powders, and/or treatment for a specific immune system disease.
  • the label and/or the instructions can indicate directions for either in vivo or in vitro use of the pharmaceutical composition.
  • the label and/or the instructions can indicate that the pharmaceutical composition is used alone, or in combination with a second agent.
  • the label can indicate appropriate dosages for the molecules of the invention.
  • the label can indicate that dosages for a molecule that is effective for blocking B7 interactions with its ligand and/or treating an immune system disease is about 0.1 to 100 mg/kg weight of the subject, about 0.5 to 5 mg/kg weight of a subject, about 5 to 10 mg/kg weight of a subject, about 10 to 15 mg/kg weight of a subject, about 15 to 20 mg/kg weight of a subject, about 20 to 25 mg/kg weight of a subject, about 25 to 30 mg/kg weight of a subject, about 30 to 35 mg/kg weight of a subject, about 35 to 40 mg/kg weight of a subject, about 40 to 45 mg/kg of a subject, about 45 to 50 mg/kg weight of a subject, about 50 to 55 mg/kg weight of a subject, about 55 to 60 mg/kg weight of a subject, about 60 to 65 mg/kg weight of a subject, about 65 to 70 mg/kg weight of a subject, about 70 to 75 mg/kg weight of
  • the label and or instructions can also indicate dosages for a second agent, such as a DMARD, is about 1 to about 5000 mg/day, about 1 to 10 mg/day, about 10 to 50 mg/day, about 50 to 100 mg/day, about 100 to 150 mg/day, about 150 to 200 mg/day, about 200 to
  • the label and/or the instructions can also indicate that the pharmaceutical composition can be used alone, or in combination, with a second agent to treat a condition of choice e.g., immune system diseases, autoimmune diseases, immunoproliferative diseases, graft- related disorders, graft versus host disease (GNHD) (e.g., such as may result from bone marrow fransplantation, or in tlie induction of tolerance), immune disorders associated with graft transplantation rejection, immune disorders associated with chronic rejection, immune disorders associated with tissue or cell allo- or xenografts (e.g., kidneys, skin, islets, muscles, hepatocytes, neurons, solid organs and the like), psoriasis, T cell lymphoma, T cell acute lymphoblastic leukemia, testicular angiocentric T cell lymphoma, benign lymphocytic angiitis, as lupus (e.g., lupus erythematosus, lupus nep
  • insulin dependent diabetes mellitus type I diabetes mellitus, type IT diabetes mellitus
  • good pasture's syndrome myasthenia gravis, pemphigus, Crohn's disease, sympathetic ophthalmia, autohmnune uveitis, multiple sclerosis, autoimmune hemolytic anemia, idiopathic thrombocytopenia, primary biliary cirrhosis, chronic action hepatitis, ulcerative colitis, Sjogren's syndrome, rheumatic diseases (e.g., rheumatoid arthritis), polymyositis, scleroderma, mixed connective tissue disease, and the like.
  • the kit comprises a pharmaceutical composition comprising a pharmaceutically acceptable carrier and an effective amount of a first agent, wherein the first agent is a molecule that blocks B7 interaction with CTLA4 and/or CD28 such as soluble CTLA4 molecules, CD28 molecules, B7 (B7-1 or B7-2) molecules, anti-CTLA4 monoclonal antibodies, anti-CD28 monoclonal antibodies or anti-B7 (B7-l ' or B7-2) monoclonal antibodies.
  • the soluble CTLA4 molecules have the amino acid sequence of the exfracellular domain of CTLA4 as shown in either Figures 24 or 19 (CTLA4Ig or L104EA29Y, respectively).
  • the invention provides methods for regulating functional CTLA4- and CD28- positive cell interactions with B7-positive cells.
  • the methods comprise contacting the B7- positive cells with a soluble CTLA4 molecule of the invention so as to regulate iiuictional
  • CTLA4- and CD2S- positive cell interactions with B7-positive cells e.g., by interfering with reaction of an endogenous CTLA4 and/or CD28 molecule with a B7 molecule.
  • Suitable amounts of soluble CTLA4 for use in the methods of the invention are described supra.
  • the present invention also provides methods for inhibiting T-cell function but not T-cell depletion in a human by contacting B7-positive cells in the human with a soluble CTLA4.
  • soluble CTLA4 include CTLA4Ig and soluble CTLA4 mutant molecule e.g. L104EA29YIg.
  • the present invention further provides methods for treating immune system diseases and auto- nmune diseases such as rheumatic diseases.
  • the methods comprise administering a therapeutic composition, comprising soluble CTLA4 molecules of the invention, to a subject in an amount effective to relieve at least one of the symptoms associated with immune system diseases.
  • the invention may provide long-term therapy for immune system diseases by blocking the T- cell/B7-positive cell interactions, thereby blocking T-cell activation/stimulation by co- stimulatory signals such as B7 binding to CD28, leading to induction of T-cell anergy or tolerance.
  • Immune system diseases include, but are not limited to, autoimmune diseases, immunoproliferative diseases, and graft-related disorders. Examples of graft-related diseases include graft versus host disease (GVHD) (e.g., such as may result from bone ma ⁇ ow transplantation, or in the induction of tolerance), .
  • GVHD graft versus host disease
  • graft transplantation rejection chronic rejection, and tissue or cell allo- or xenografts
  • tissue or cell allo- or xenografts cluding allo- or xenografts solid organs (e.g., kidneys), skin, islets, muscles, hepatocytes, neurons.
  • immunoproliferative diseases include, but are not limited to, psoriasis; T cell lymphoma; T cell acute lymphoblastic leukemia; testicular angiocentric T cell lymphoma; benign lymphocytic angiitis; and autoimmune diseases such as lupus (e.g., lupus erythematosus, lupus nephritis), Hashimoto's thyroiditis, primary myxedema, Graves' disease, pernicious anemia, autoimmune afrophic gastritis, Addison's disease, diabetes (e.g.
  • insulin dependent diabetes mellitus type I diabetes mellitus, type II diabetes mellitus
  • good pasture's syndrome myasthenia gravis, pemphigus, Crohn's disease, sympathetic ophthalmia, autoiimnune uveitis, multiple sclerosis, autoimmune hemolytic anemia, idiopathic tlirombocytopenia, primary biliary ci ⁇ hosis.
  • chronic action hepatitis ulcerative colitis, Sjogren's syndrome, rheumatic diseases (e.g., rheumatoid arthritis), polymyositis, scleroderma, and mixed connective tissue disease.
  • the soluble CTLA4 molecules of the invention exhibit inhibitory properties in vivo. Under conditions where T-cell/B7-positive cell interactions, for example T cell/B cell interactions, are occurring as a result of contact between T cells and B7-positive cells, binding of introduced CTLA4 molecules to react to B7-positive cells, for example B cells, may interfere, i.e., inhibit, the T cell B7-positive cell interactions resulting in regulation of immune responses.
  • the invention provides methods for regulating immune responses.
  • Immune responses downregulated (reduced) by the soluble CTLA4 molecules of the invention may be by way of inhibiting or blocking an immune response already in progress or may involve preventing the induction of an immune response.
  • the soluble CTLA4 molecules of the invention may inhibit the functions of activated T cells, such as T lymphocyte proliferation, cytokine secretion and/or cytokine production, by suppressing T cell responses or by inducing specific tolerance in T cells, or both.
  • the soluble CTLA4 molecules of this invention, interfering with the CTLA4/CD28/B7 pathway may inhibit T-cell proliferation and/or cytokine secretion, and thus result in reduced tissue destruction and induction of T-cell unresponsiveness or anergy.
  • a preferred embodiment of the invention comprises use of the soluble CTLA4 mutant molecule L104EA29YIg to regulate functional CTLA4- and CD28- positive cell interactions with B7-positive cells, to treat immune system diseases such as rheumatic diseases and/or to downregulate immune responses.
  • the L104EA29YIg of the invention is a soluble CTLA4 mutant molecule comprising at least the two amino acid changes, the leucine (L) to glutamic acid (E) at position +104 and the alanine (A) to tyrosine (Y) change at position +29 ( Figure 19).
  • the L104EA29YIg molecule may encompass further mutations beyond the two specified herein.
  • a preferred embodiment of the mvention comprises use of a molecule to block the interaction of B7 with CTLA4 and/or CD28 in conjunction with a DMARD to regulate an immune response in order to treat an immune system disease such as a rheumatic disease.
  • a molecule to block the interaction of B7 with CTLA4 and/or CD28 in conjunction with a DMARD to regulate an immune response in order to treat an immune system disease such as a rheumatic disease.
  • Suitable amounts of the molecule used to block the B7 interaction with CTLA4 and/or CD28 are described supra.
  • the molecule used to block tlie B7/CTLA4 interaction may be a soluble CTLA4 such as CTLA4Ig, CTLA4Ig/CD28Ig or L104EA29YIg, a soluble CD28 such as CD28Ig, a soluble B7 (B7-1 or B7-2) such as B7Ig, anti-CTLA4 monoclonal antibodies, anti-CD28 monoclonal antibodies or anti-B7 monoclonal antibodies.
  • a soluble CTLA4 such as CTLA4Ig, CTLA4Ig/CD28Ig or L104EA29YIg
  • a soluble CD28 such as CD28Ig
  • a soluble B7 B7-1 or B7-2
  • B7Ig anti-CTLA4 monoclonal antibodies, anti-CD28 monoclonal antibodies or anti-B7 monoclonal antibodies.
  • the DMARD may be a dihydrofolic acid reductase inhibitor such as methotrexate, cyclophosphamide, cyclosporine, cyclosporin A, chloroquine, hydroxychloroquine, sulfasalazine, sulphasalazopyrine, leflunomide, gold salts, D- penicillamine, azathioprine, anakinra, infliximab, etanercept, TNF ⁇ blockers or a biological agent that targets an inflammatory cytokine.
  • a dihydrofolic acid reductase inhibitor such as methotrexate, cyclophosphamide, cyclosporine, cyclosporin A, chloroquine, hydroxychloroquine, sulfasalazine, sulphasalazopyrine, leflunomide, gold salts, D- penicillamine, azathioprine, anakinr
  • a prefe ⁇ ed embodiment includes methods for treating a rheumatic disease, such as rheumatoid arthritis, by adrninistering an effective amount of soluble CTLA4 molecules alone, or in conjunction with an effective amount of methotrexate or a molecule that blocks TNF interactions, to a subject.
  • Administration of an effective amount of the therapeutic composition ⁇ thereby relieving the subject of at least one of the symptoms associated with the disease, including reducing: joint swelling, joint tenderness, inflammation, morning stiffness, and pain, and structural damage subsequently decreasing the physical disability.
  • the methods of the invention also may be used to reduce at least one symptom associated with rheumatoid arthritis, mcluding reducing erythrocyte sedimentation rates, serum levels of C-reactive protein, soluble ICAM-1, soluble E-selectin and/or soluble IL-2r.
  • the amount of symptom relief provided by the present invention can be measured using any of the accepted criteria established to measure and document symptom relief in a clinical setting.
  • Acceptable criteria for measuring symptom relief may include scores based on the criteria established by the American College of Rhei iatology (e.g., ACR 20), the four measures of symptom relief (in: "CDER Guideline for the Clinical Evaluation of Anti-mfiammatory and Antirheumatic Drags — FDA 1988), and the Health Assessment Questionnaire (HAQ) (Fries, J. F., et al., 1982 J. of Rheumatology 9:789- 793).
  • HAQ Health Assessment Questionnaire
  • the present invention provides improving ACR response rates using the methods of the invention.
  • the embodiments of the invention include improving ACR response rates of ACR 20, 50, and/or 70, using the methods of the invention.
  • the subjects treated by the present invention include mammalian subjects, including, human, monkey, ape, dog, cat, cow, horse, goat, pig, rabbit, mouse and rat.
  • the present invention provides various methods, local or systemic, for administering the therapeutic compositions of the invention such as soluble CTLA4 molecule alone or in conjunction with a DMARD, such as methotrexate, a molecule that blocks TNF interactions and or other therapeutic drug.
  • the methods include intravenous, intramuscular, intraperitoneal, oral, inhalation and subcutaneous methods, as well as implantable pump, continuous infusion, gene therapy, liposomes, suppositories, topical contact, vesicles, capsules, biodegradable polymers, hydrogels, controlled release patch. and injection methods.
  • the therapeutic agent, compounded with a carrier is commonly lyophilized for storage and is reconstituted with water or a buffered solution with a neutral pH (about pH 7-8, e.g., pH 7.5) prior to adminisfration.
  • compositions of the invention may be administered to the subject in any pharmaceutically acceptable form.
  • tlie methods comprise administering to a subject the soluble CTLA4 molecules of the invention to regulate CD28- and/or CTLA4- positive cell interactions with B7-positive cells.
  • the B7-positive cells are contacted with an effective amoimt of the soluble CTLA4 molecules of the invention, or fragments or derivatives thereof, so as to fonn soluble CTLA4/B7 complexes. Suitable amounts of soluble CTLA4 are described supra.
  • the complexes interfere with interaction between endogenous CTLA4 and CD28 molecules with B7 family molecules.
  • the soluble CTLA4 molecules may be administered to a subject in an amount and for a time (e.g. length of time and/or multiple times) sufficient to block endogenous B7 molecules from binding their respective ligands, in the subject. Blockage of endogenous B7/ligand binding thereby inliibiting interactions between B7-positive cells with CD28- and/or CTLA4- ⁇ ositive cells, hi an embodiment, soluble CTLA4 may be administered to a subject daily, weeldy, monthly and/or yearly, in single or multiple times per day/week/month/year, depending on need. For example, in one embodiment, the molecule may initially be administered once every two weeks for a month, and then once every month thereafter.
  • a time e.g. length of time and/or multiple times
  • soluble CTLA4 molecules may be administered in an amount from about 0.1 to 100 mg/kg weight of the patient/day. Suitable amounts of soluble CTLA4 are described supra. Methotrexate may be admimstered to a subject in an amount from about 0.1 to 100 mg/week. Suitable amounts of soluble methotrexate are described supra. A molecule that blocks TNF interactions e.g., etanercept, may be admimstered to a subject in an amount from about 0.1 to 100 mg week. Suitable amounts of TNF blockers are described supra.
  • the invention also encompasses the use of the compositions of the invention together with other pharmaceutical agents to treat immune system diseases.
  • rheumatic diseases may be treated with molecules of the invention in conjunction with, but not limited to, immunosuppressants such as corticosteroids, cyclosporin (Mathiesen 1989 Cancer Lett. 44(2):151-156), prednisone, azathioprine, (R. Handschumacher, in: "Drugs Used for hnmunosuppression" pages 1264-1276), TNF ⁇ blockers or antagonists (New England Journal of Medicine, vol. 340: 253-259, 1999; The Lancet vol. 354: 1932- 39, 1999, Annals of Internal Medicine, vol.
  • immunosuppressants such as corticosteroids, cyclosporin (Mathiesen 1989 Cancer Lett. 44(2):151-156), prednisone, azathioprine, (R. Handschumacher, in: "Drugs Used for hnmunos
  • the soluble CTLA4 molecules can also be used in combination with one or more of the following agents to regulate an immune response: soluble gp39 (also known as CD40 ligand (CD40L), CD154, T-BAM, TRAP), soluble CD29, soluble CD40, soluble CD80 (e.g. ATCC 68627), soluble CD86, soluble CD28 (e.g. ATCC accession number 68628), soluble CD56, soluble Thy-1, soluble CD3, soluble TCR, soluble VLA-4, soluble VCAM-1, soluble LECAM-1, soluble ELAM-1, soluble CD44, antibodies reactive with gp39 (e.g.
  • ATCC HB-10916, ATCC HB-12055 and ATCC HB-12056 antibodies reactive with CD40 (e.g. ATCC HB-9110), antibodies reactive with B7 (e.g. ATCC HB-253, ATCC CRL-2223, ATCC CRL-2226, ATCC HB- 301, ATCC HB-11341, etc), antibodies reactive with CD28 (e.g. ATCC HB-11944 or mAb 9.3 as described by Martin et al (J. Clin. Immun. 4(1): 18-22, 1980), antibodies reactive with LFA-1 (e.g.
  • antibodies reactive with LFA-2 antibodies reactive with EL-2, antibodies reactive with IL-12, antibodies reactive with IFN-gamma, antibodies reactive with CD2, antibodies reactive with CD48, antibodies reactive with any ICAM (e.g., ICAM-1 (ATCC CRL-2252), ICAM-2 and ICAM-3), antibodies reactive with CTLA4 (e.g. ATCC HB-304),, antibodies reactive with Thy-1, antibodies reactive with CD56, antibodies reactive with CD3, antibodies reactive with CD29, antibodies reactive with TCR, antibodies reactive with VLA-4, antibodies reactive with VCAM-1, antibodies reactive with LECAM-1, antibodies reactive with ELAM-1, antibodies reactive with CD44.
  • monoclonal antibodies are prefe ⁇ ed.
  • antibody fragments are preferred.
  • the combination can include: the soluble CTLA4 molecules of the invention and one other immunosuppressive agent; the soluble CTLA4 molecules with two other immunosuppressive agents; the soluble CTLA4 molecules with three other immunosuppressive agents; and the like.
  • the determination of tire optimal combination and dosages can be dete ⁇ nined and optimized using methods well known in the art.
  • L104EA29YIg and CD80 monoclonal antibodies mAbs
  • L104EA29YIg and CD86 mAbs L104EA29YI& CD80 mAbs, and CD86 mAbs
  • L104EA29YIg and gp39 mAbs L104EA29YIg and CD40 mAbs
  • L104EA29YIg and CD28 mAbs L104EA29YIg, CD80 and CD86 mAbs, and gp39 mAbs
  • L104EA29YIg, CD80 and CD86 mAbs and CD40 mAbs and L104EA29YIg, anti-LFAl mAb, and anti-gp39 mAb.
  • a specific example of a gp39 mAb is MR1.
  • Other combinations will be readily appreciated and understood by persons skilled in the art.
  • the soluble CTLA4 molecules of the invention may be administered as the sole active ingredient or together with other drags in inmiunomodulating regimens or other anti-inflammatory agents such as DMARDs e.g. for the treatment or prevention of allo- or xeno graft acute or chronic rejection or inflammatory or autoimmune disorders, or to induce tolerance.
  • DMARDs e.g. for the treatment or prevention of allo- or xeno graft acute or chronic rejection or inflammatory or autoimmune disorders, or to induce tolerance.
  • DMARDs e.g. for the treatment or prevention of allo- or xeno graft acute or chronic rejection or inflammatory or autoimmune disorders, or to induce tolerance.
  • a calcineurin inhibitor e.g. cyclosporin A or FK506
  • an immunosuppressive macrolide e.g. rapamycine or a derivative thereof (e.g.
  • lymphocyte homing agent e.g. FTY720 or an analog thereof
  • corticosteroids cyclophosphamide
  • azathioprene a dihydrofolic acid reductase inhibitor such as methotrexate; lefliuiomide or an analog thereof; mizoribine
  • mycophenolic acid mycophenolate mofetil
  • 15-deoxyspergualine or an analog thereof immunosuppressive monoclonal antibodies, e.g., monoclonal antibodies to leukocyte receptors, e.g., MHC, CD2, CD3, CD4, CD 11 a/CD 18, CD7, CD25, CD 27, B7, CD40, CD45, CD58, CD 137, ICOS, CD150 (SLAM), OX40, 4-1BB or their ligands; or other immunomodulatory compounds, e.g.
  • CTLA4/CD28-Ig or other adhesion molecule inliibitors, e.g. mAbs or low molecular weight inliibitors including LFA-1 antagonists, Selectin antagonists and VLA-4 antagonists.
  • the compound is particularly useful in combination with a compound that interferes with CD40 and its ligand, e.g. antibodies to CD40 and antibodies to CD40-L.
  • soluble CTLA4 mutant molecules of the invention are administered in conjunction with other inununosuppressive/immimomodulatory or anti-im ammatory therapy, e.g. as hereinabove specified, dosages of the co-administered immunosuppressant, immunomodulatory or anti-inflammatory compound will of course vary depending on the type of co-drug employed, e.g. whether it is a steroid or a cyclosporin, on the specific drug employed, on the condition being treated and so forth.
  • the present invention provides in a yet further aspect methods as defined above comprising co-administration, e.g. concomitantly or in sequence, of a therapeutically effective amount of soluble CTLA4 molecules of the invention, e.g. CTLA4Ig and/or L104EA29YIg, in free form or in pharmaceutically acceptable salt form, and a second drag substance, said second drag substance being an immunosuppressant, immunomodulatory or anti-inflammatory drag, e.g. as indicated above.
  • kits of tlie invention can be used in any method of the present invention.
  • the invention also provides methods for producing the soluble CTLA4 mutant molecules of the invention.
  • Expression of soluble CTLA4 mutant molecules can be in prokaryotic cells or eukaryotic cells. Prokaryotes most frequently are represented by various strains of bacteria. The bacteria may be a gram positive or a gram negative. Typically, gram-negative bacteria such as E. coli are prefe ⁇ ed. Other microbial strains may also be used. Sequences encoding soluble CTLA4 mutant molecules can be inserted into a vector designed for expressing foreign sequences in prokaryotic cells such as E. coli.
  • These vectors can include commonly used prokaryotic control sequences which are defined herein to include promoters for transcription initiation, optionally with an operator, along with ribosome binding site sequences, including such commonly used promoters as the beta-lactamase (penicillinase) and lactose (lac) promoter systems (Chang, et al., (1977) Nature 198:1056), the tryptophan (trp) promoter system (Goeddel, et al, (1980) Nucleic Acids Res. 8:4057) "and the lambda derived P promoter and N-gene ribosome binding site (Shimatake, et al., (1981) Nature 292:128).
  • promoters as the beta-lactamase (penicillinase) and lactose (lac) promoter systems (Chang, et al., (1977) Nature 198:1056)
  • trp tryptophan
  • trp lambda derived
  • Such expression vectors will also include origins of replication and selectable markers, such as a beta-lactamase or neomycin phospho transferase gene conferring resistance to antibiotics, so that the vectors can replicate in bacteria and cells ca ⁇ ying the plasmids can be selected for when grown in the presence of antibiotics, such as ampicillin or kanamycin.
  • selectable markers such as a beta-lactamase or neomycin phospho transferase gene conferring resistance to antibiotics, so that the vectors can replicate in bacteria and cells ca ⁇ ying the plasmids can be selected for when grown in the presence of antibiotics, such as ampicillin or kanamycin.
  • the expression plasmid can be introduced into prokaryotic cells via a variety of standard methods, including but not limited to CaCl -shock (Cohen, (1972) Proc. Natl. Acad. Sci. USA 69:2110, and Sambrook et al. (eds.), "Molecular Cloning:: A Laboratory Manual", 2nd Edition, Cold Spring Harbor Press, (1989)) and electroporation.
  • eulcaryotic cells are also suitable host cells.
  • eukaryotic cells include any animal cell, whether primary or immortalized, yeast (e.g., Saccharomyces cerevisiae, Schizosaccharomvces pombe, and Pichia pastoris), and plant cells.
  • yeast e.g., Saccharomyces cerevisiae, Schizosaccharomvces pombe, and Pichia pastoris
  • Myeloma, COS and CHO cells are examples of animal cells that may be used as hosts.
  • Particular CHO cells include, but are not limited to, DG44 (Chasin, et la., 1986 Som. Cell. Molec. Genet. 12:555-556; Kolkekar 1997 Biochemistry 36:10901-10909), CHO-K1 (ATCC No.
  • CHO-K1 Tet-On cell line CCL-61
  • CHO designated ECACC 85050302 (CAMR, Salisbury, Wiltshire, UK)
  • CHO clone 13 (GETMG, Genova, IT)
  • CHO clone B (GEIMG, Genova, IT)
  • CHO-K1/SF designated ECACC 93061607 (CAMR, Salisbury, Wiltshire, UK)
  • RR-CHOK1 designated ECACC 92052129 (CAMR, Salisbury, Wiltshire, UK).
  • Exemplary plant cells include tobacco (whole plants, cell culture, or callus), com, soybean, and rice cells. Com, soybean, and rice seeds are also acceptable.
  • Nucleic acid sequences encoding the CTLA4 mutant molecules can also be inserted into a vector designed for expressing foreign sequences in an eukaryotic host.
  • the regulatory elements of the vector can vary according to the particular eukaryotic host.
  • Commonly used eukaryotic control sequences for use in expression vectors include promoters and control sequences compatible with mammalian cells such as, for example, CMV promoter (CDM8 vector) and avian sarcoma virus (ASV) (7rLN vector).
  • CMV promoter CDM8 vector
  • ASV avian sarcoma virus
  • Other commonly used promoters include the early and late promoters from Simian Virus 40 (SV40) (Fiers, et al., (1973) Nature 273:113), or other viral promoters such as those derived from polyoma, Adenovirus 2, and bovine papilloma virus.
  • An inducible promoter such as hMTII (Karin, et al., (1982) Nature 299:797-802) may also be used.
  • Vectors for expressing CTLA4 mutant molecules in eukaryotes may also carry sequences called enhancer regions. These are important in optimizing gene expression and are found either upstream or downstream of the promoter region.
  • expression vectors for eukaryotic host cells include, but are not hmited to, vectors for mammalian host cells (e.g., BPV-1, pHyg, pRSV, pSV2, pTK2 (Maniatis); pIRES (Clontech); pRc/CMV2, pRc/RSN, pSFNl (Life Technologies); pVPakc Vectors, pCMN vectors, pSG5 vectors (Stratagene)), retroviral vectors (e.g., pFB vectors (Stratagene)), pCD ⁇ A-3 (Invitrogen) or modified forms thereof, adenoviral vectors; Adeno-associated vims vectors, baculovrms vectors, yeast vectors (e.g., pESC vectors (Stratagene)).
  • mammalian host cells e.g., BPV-1, pHyg, pRSV, pSV2, pTK2
  • Nucleic acid sequences encoding CTLA4 mutant molecules can integrate into the genome of the eukaryotic host cell and replicate as the host genome replicates.
  • the vector carrying CTLA4 mutant molecules can contain origins of replication allowing for extrachromosomal replication.
  • the origin of replication from the endogenous yeast plasmid can be used.
  • the 2 ⁇ circle can be used.
  • sequences from the yeast genome capable of promoting autonomous replication can be used (see, for example, Stinchcomb et al., (1979) Nature 282:39); Tschemper et al., (1980) Gene 10:157; and Clarke et al, (1983) Meth. Enz. 101:300).
  • Transcriptional control sequences for yeast vectors include promoters for the synthesis of glycolytic enzymes (Hess et al., (1968) J. Adv. Enzyme Reg. 7:149; Holland et al, (1978) Biochemistry 17:4900). Additional promoters known in the art include the CMV promoter provided in the CDM8 vector (Toyama and Okayama, (1990) FEBS 268:217- 221); the promoter for 3-phosphoglycerate kinase (Hitzeman et al., (1980) J. Biol. Chem. 255:2073), and those for other glycolytic enzymes.
  • promoters are inducible because they can be regulated by environmental stimuh or by the growth medium of the cells.
  • These inducible promoters include those from the genes for heat shock proteins, alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, enzymes associated with nitrogen catabolism, and enzymes responsible for maltose and galactose utilization.
  • Regulatory sequences may also be placed at the 3' end of the coding sequences. These sequences may act to stabilize messenger RNA. Such terminators are found in the 3' untranslated region following the coding sequences in several yeast-derived and mammalian genes.
  • Exemplary vectors for plants and plant cells include, but are not limited to, Agrobacterium Tj plasmids, cauliflower mosaic virus (CaMV), and tomato golden mosaic- virus (TGMV).
  • Mammalian cells can be transformed by methods including but not limited to, transfection in the presence of calcium phosphate, microinjection, electroporation, or via transduction with viral vectors.
  • Methods for introducing foreign DNA sequences into eukaryote genomes, including plant and yeast genomes include; (1) mechanical methods, such as microinjection of DNA into single cells or protoplasts, vortexing cells with glass beads in the presence of DNA, or shooting DNA-coated tungsten or gold spheres into cells or protoplasts; (2) introducing DNA by making cell membranes permeable to macromolecules through polyethylene glycol treatment or subjection to high voltage electrical pulses (electroporation); or (3) the use of liposomes (cont-iining cDNA) which fuse to cell membranes.
  • CTLA4 mutant molecules of the inventions can be harvested by methods well known in the art such as cell lysis (e.g. sonication, lysozyme and/or detergents) and protein recovery performed using standard protein purification means, e.g., affmity chromatography or ion-exchange chromatography, to yield substantially pure product (R. Scopes in: "Protein Purification, Principles and Practice", Third Edition, Springer-Nerlag (1994); Sambrook et al. (eds.), “Molecular Cloning: A Laboratory Manual", 2nd Edition, Cold Spring Harbor Press, (1989)).
  • Expression of CTLA4 mutant molecules can be detected by methods known in the art. For example, the mutant molecules can be detected by Coomassie staining SDS-PAGE gels and immimoblotting using antibodies that bind CTLA4.
  • a CTLA4Ig encoding plasmid was first constructed, and shown to express CTLA4Ig molecules as described in U.S. Patent Nos. 5,434,131, 5,885,579 and 5,851,795. Then single-site mutant molecules (e.g., L104EIg) were generated from the CTLA4Ig encoding sequence, expressed and tested for binding kinetics for various B7 molecules.
  • the L104EIg nucleotide sequence (as included in the sequence shown in Figure 18) was used as a template to generate the double-site CTLA4 mutant sequences (as included in the sequences shown in Figures 19-22) which were expressed as proteins and tested for bmding kinetics.
  • the double-site CTLA4 mutant sequences include: L104EA29YIg, L104EA29LIg, L104EA29TIg, and L104EA29WIg. Triple-site mutants were also generated.
  • CTLA4Ig comprising the extracellular domain of CTLA4 and an IgCgammal domain was constructed as described in U.S. Patents 5,434,131, 5,844,095 and 5,851,795, the contents of which are incorporated by reference herein.
  • the extracellular domain of the CTLA4 gene was cloned by PCR using synthetic oligonucleotides co ⁇ esponding to the published sequence (Dariavach et al., Eur. Journ. hrimixnol. 18:1901- 1905 (1988)).
  • the N- terminus of the predicted sequence of CTLA4 was fused to the signal peptide of oncostatin M (Malik et al., Mol. and Cell. Biol. 9:2847 (1989)) in two steps using overlapping oligonucleotides.
  • the oligonucleotide For the first step, the oligonucleotide,
  • the template for this step was cDNA synthesized from 1 micro g of total RNA from H38 cells (an HTLN IT infected T-cell leukemic cell line provided by Drs. Salahudin and Gallo, ⁇ CI, Bethesda, MD).
  • a portion of the PCR product from the first step was reamplified, using an overlapping forward primer, encoding the ⁇ terminal portion of the oncostatin M signal peptide and containing a Hind flT restriction endonuclease site, CTAGCCACTGAAGCTTCACCAATGGGTGTACTGCTCACACA- GAGGACGCTGCTCAGTCTGGTCCTTGCACTC (SEQ ID NO: 3) and the same reverse primer.
  • the product of the PCR reaction was digested with Hind HI and Bel I and ligated together with a Bel 1/Xba I cleaved cDNA fragment encoding the amino acid sequences co ⁇ esponding to the hinge, CH2 and CH3 regions of IgC(gamma)l into the Hind IH/Xba I cleaved expression vector, CDM8 or Hind Hl/Xba I cleaved expression vector piLN (also known as ⁇ LN).
  • a mutagenesis and screening sfrategy was developed to identify mutant CTLA4Ig molecules that had slower rates of dissociation ("off rates) from CD80 and or CD86 molecules i.e. improved binding ability, hi this embodiment, mutations were carried out in and/or about the residues in the CDR-1, CDR-2 (also known as the C strand) and/or CDR-3 regions of the extracellular domain of CTLA4 (as described in U.S. Patents U.S. Patents 6,090,914, 5,773,253 and 5,844,095; in copending U.S. Patent Application Serial Number 60/214,065; and by Peach, R.J., et al J Exp Med 1994 180:2049-2058.
  • a CDR- like region encompasses the each CDR region and extends, by several amino acids, upstream and/or downstream of the CDR. motif). These sites were chosen based on studies of chimeric CD28/CTLA4 fusion proteins (Peach et al, J. Exp. Med., 1994, 180:2049-2058), and on a model predicting which amino acid residue side chains would be solvent exposed, and a lack of amino acid residue identity or homology at certain positions between CD28 and CTLA4. Also, any residue which is spatially in close proximity (5 to 20 Angstrom Units) to the identified residues is considered part of the present invention.
  • a two-step strategy was . adopted.
  • the experiments entailed first generating a library of mutations at a specific codon of an extracellular portion of CTLA4 and then screening these by BIAcore analysis to identify mutants with altered reactivity to B7.
  • the Biacore assay system (Pharmacia, Piscataway, NJ.) uses a surface plasmon resonance detector system that essentially involves covalent binding of either CD80Ig or CD86Ig to a dextran-coated sensor chip which is located in a detector.
  • test molecule can then be injected into the chamber containing the sensor chip and the amount of complementary protein that binds can be assessed based on the change in molecular mass which is physically associated with the dextran-coated side of the sensor chip; the change in molecular mass can be measured by the detector system.
  • single-site mutant nucleotide sequences were generated using non-mutated (e.g., wild-type) DNA encoding CTLA4Ig (U.S. Patent Nos: 5,434,131, 5,844,095; 5,851,795; and 5,885,796; ATCC Accession No. 68629) as a template.
  • Mutagenic oligonucleotide PCR primers were designed for random mutagenesis of a specific codon by allowing any base at positions 1 and 2 of the codon, but only guanine or thymine at position 3 (XXG/T or also noted as NNG/T). In this manner, a specific codon encoding an amino acid could be randomly mutated to code for each of the 20 amino acids.
  • XXG/T mutagenesis yields 32 potential codons encoding each of the 20 amino acids.
  • PCR products encoding mutations in close proximity to the CDR3-like loop of CTLA4Ig (MYPPPY) were digested with Sacl/Xbal and subcloned into similarly cut CTLA4Ig (as mcluded in Figure 24) ⁇ LN expression vector. This method was used to generate the single-site CTLA4 mutant molecule L104EIg (as included in Figure 18).
  • a silent Nhel restriction site was first introduced 5' to this loop, by PCR primer-directed mutagenesis. PCR products were digested with Nhel/Xbal and subcloned into similarly cut CTLA4Ig or L104EIg expression vectors. This method was used to generate the double-site CTLA4 mutant molecule L104EA29YIg (as included in Figure 19). In particular, the nucleic acid molecule encoding the single-site CTLA4 mutant molecule, L104EIg, was used as a template to generate the double-site CTLA4 mutant molecule, L104EA29YIg.
  • CTLA4 mutant molecules such as L104EA29YIg (deposited on June 19, 2000 with the American Type Culture Collection (ATCC), 10801 University Boulevard., Manassas, VA 20110-2209 and accorded ATCC accession number PTA-2104), were generated by repeating the mutagenesis procedure described above using L104EIg as a template.
  • This method was used to generate numerous double-site mutants nucleotide sequences such as those encoding CTLA4 molecules L104EA29YIg (as included in the sequence shown in Figure 19), L104EA29LIg (as included in the sequence shown in Figure 20), L104EA29TIg (as included in the sequence shown in Figure 21), and L104EA29WIg (as included in the sequence shown in Figure 22).
  • Triple-site mutants such as those encoding L104EA29YS25KIg, L104EA29YS25NIg and L104EA29YS25RIg, were also generated
  • the soluble CTLA4 molecules were expressed from the nucleotide sequences and used in the phase II clinical studies described in Example 3, infra.
  • nucleotide changes do not necessarily translate into amino acid changes as ' some codons redimdantly encode the same amino acid. Any changes of nucleotide from the original or wildtype sequence, silent (i.e. causing no change in the translated amino acid) or otherwise, while not explicitly described herein, are encompassed within the scope of the invention.
  • the following example provides a description of the screening methods used to identify the single- and double-site mutant CTLA polypeptides, expressed from the constructs described in Example 1, that exhibited a higher binding avidity for B7 molecules, compared to non-mutated CTLA4Ig molecules.
  • the soluble CTLA4 mutant molecules described in Example 1 above were screened using a novel screening procedure to identify several mutations in the extracellular domain of CTLA4 that improve binding avidity for CD80 and CD86.
  • This screening strategy provided an effective method to directly identify mutants with apparently slower "off” rates without the need for protein purification or quantitation since "off rate determination is concentration independent (O'Shannessy et al., (1993) Anal. Biochem., 212:457-468).
  • COS cells were transfected with individual miniprep purified plasmid DNA and propagated for several days.
  • Ligands were immobilized on research grade NCM5 sensor chips (Pharmacia) using standard N-ethyl-N'-(dimethylaminopropyl) carbodiimidN-hyffroxysuccimmide coupling (Johnsson, B., et al. (1991) Anal. Biochem. 198: 268-277; Khilko, S.N., et al.(T993) J. Biol. Chem 268:5425-15434).
  • Conditioned COS cell culture media was allowed to flow over BIAcore biosensor chips derivitized with CD86Ig or CD80Ig (as described in Greene et al., 1996 J. Biol. Chem. 271:26762-26771), and mutant molecules were identified with off-rates slower than that observed for wild type CTLA4Ig.
  • the DNAs corresponding to selected media samples were sequenced and more DNA prepared to perform larger scale COS cell transient transfection, from which CTLA4Ig mutant protein was prepared following protein A purification of culture media.
  • Senosorgram baselines were normalized to zero response units (RU) prior to analysis. Samples were nm over mock-derivatized flow cells to determine background RU values due to bulk refractive index differences between solutions. Equilibrium dissociation constants (K d ) were calculated from plots of R eq versus C, where R eq is the steady-state response minus the response on a mock-derivatized chip, and C is the molar concentration of analyte. Binding curves were analyzed using commercial nonlinear curve-fitting software (Prism, GraphPAD Software).
  • Murine mAb L307.4 (anti-CD80) was purchased from Becton Dickinson (San Jose, California) and IT2.2 (anti-B7-0 [also known as CD86]), from Pharmingen (San Diego, California).
  • CD80-positive and/or CD86-positive CHO cells were removed from their culture vessels by incubation in phosphate-buffered saline (PBS) containing lOmM EDTA.
  • PBS phosphate-buffered saline
  • CHO cells (1-10 x 10 5 ) were first incubated with mAbs or immunoglobulin fusion proteins in DMEM containing 10% fetal bovine serum (FBS), then washed and incubated with fluorescein isothiocyanate-conjugated goat anti-mouse or anti-human immunoglobulin second step reagents (Tago, Burlingame, CaHfornia). Cells were given a final wash and analyzed on aFACScan (Becton Dickinson).
  • FBS fetal bovine serum
  • CTLA4Xri ns and L104EA29YXri ns were prepared as previously described (Linsley et al, (1995) J. Biol. Chem., 270:15417-15424).
  • an oncostatin M CTLA4 (OMCTLA4) expression plasmid was used as a template, the forward primer, GAGGTGATAAAGCTTCACCAATGGGTGTACTGCTCACACAG (SEQ TD NO: 4) was chosen to match sequences in the vector; and the reverse primer, GTGGTGTATTGGTCTAGATCAATCAGAATCTGGGCACGGTTC (SEQ ID NO: 5) co ⁇ esponded to the last seven amino acids (i.e. amino acids 118-124) in the extracellular domain of CTLA4, and contained a restriction enzyme site, and a stop codon (TGA).
  • the reverse primer specified a C120S (cysteine to serine at position 120) mutation.
  • nucleotide sequence GCA (nucleotides 34-36) of the reverse primer shown above is replaced with one of the following nucleotide sequences: AGA, GGA, TGA, CGA, ACT, or GCT.
  • nucleotide sequence GCA is a reversed complementary sequence of the codon TGC for cysteine.
  • nucleotide sequences AGA, GGA, TGA, CGA, ACT, or GCT are the reversed complementary sequences of the codons for serine.
  • L104EA29YIg ( ⁇ 1 ⁇ g; lane 3) and L104EIg ( ⁇ 1 ⁇ g; lane 2) apparently had tlie same electrophoretic mobility as CTLA4Ig ( ⁇ 1 ⁇ g; lane 1) under reducing ( ⁇ 50kDa; + ⁇ ME; plus 2-mercaptoethanol) and non-reducing (-lOOkDa; -BME) conditions (FIG. 25A).
  • Size exclusion chromatography demonstrated that L104EA29YIg (FIG. 25C) apparently had the same mobility as dimeric CTLA4Ig (FIG.
  • L104EA29YIg binds more strongly to CD86Ig than does L104EIg or CTLA4Ig.
  • the lower K d of L104EA29YIg (3.21 nM) than L104EIg (6.06 nM) or CTLA4Ig (13.9 nM) indicates higher binding avidity of L104EA29YIg to CD86Ig.
  • L104EA29YIg (3.66 nM) than L104EIg (4.47 nM) or CTLA4Ig (6.51 nM) indicates higher binding avidity of L104EA29YTg to CD80Ig.
  • L104EA29YIg had approximately 2-fold slower "off rate from
  • CD80Ig and approximately 4-fold slower “off rate from CD86Ig.
  • L104E had "off rates intermediate between L104EA29YIg and CTLA4Ig. Since the introduction of these mutations did not significantly affect "on” rates, the increase in avidity for CD80Ig and
  • L104EA29YXci2 ⁇ s bound with approximately 2-fold higher affinity than CTLA4X C ⁇ 2 os to both CD80Ig and CD86Ig.
  • the increased affinity was due to approximately 3-fold slower rate of dissociation from both Hgands. Therefore, stronger ligand binding by L104EA29Y was most likely due to avidity enhancing structural changes that had been introduced into each monomeric chain rather than alterations in which the molecule dimerized.
  • FIG. 27 FACS analysis (Fig. 27) of CTLA4Ig and mutant molecules binding to stably transfected CD80+ and CD86+CHO cells was performed as described herein.
  • CD80-positive and CD86-positive CHO cells were incubated with increasing concentrations of CTLA4Ig, L104EA29YTg, or L104EIg, and then washed.
  • Bound immunoglobulin fusion protein was detected using fluorescein isothiocyanate-conjugated goat anti-human immuno globulin.
  • CD80-positive or CD86-positive CHO cells (1.5xl0 5 ) were incubated with the indicated concenfrations of CTLA4Ig (closed squares), L104EA29YIg (circles), or L104EIg (triangles) for 2 hr. at 23°C, washed, and incubated with fluorescein isothiocyanate-conjugated goat anti-human immunoglobulin antibody. Binding on a total of 5,000 viable cells was analyzed (single determination) on a FACScan, and mean fluorescence intensity (MFI) was dete ⁇ nined from data histograms using PC-LYSYS.
  • CTLA4Ig closed squares
  • L104EA29YIg circles
  • L104EIg triangles
  • CD4-positive T cells Human CD4-positive T cells were isolated by immunomagnetic negative selection (Linsley et al., (1992) J. Exp. Med. 176:1595-1604). Isolated CD4-positive T cells were stimulated with phorbal myristate acetate (PMA) plus CD80-positive or CD86-positive CHO cells in the presence of titrating concenfrations of inhibitor. CD4-positive T cells (8-10 x 10 4 /well) were cultured in the presence of 1 nM PMA with or without irradiated CHO cell stimulators. Proliferative responses were measured by the addition of 1 ⁇ Ci/well of [3H]thymidine during the final 7 hours of a 72 hour culture.
  • PMA phorbal myristate acetate
  • CD80-positive or CD86-positive CHO cells in the presence of titrating concenfrations of inhibitor.
  • CD4-positive T cells (8-10 x 10 4 /well)
  • L104EA29YIg inhibits proHferation of CD80-positive PMA treated CHO cells more than CTLA4Ig (FIG. 28 A).
  • L104EA29YIg is also more effective than CTLA4Ig at inhibiting proliferation of CD86-positive PMA freated CHO cells (FIG. 28B). Therefore, L104EA29YIg is a more potent inhibitor of both CD80- and CD86-mediated costimulation of T cells.
  • Figure 29 shows inhibition by L104EA29YIg and CTLA4Ig of allostimulated human T cells prepared above, and further allostimulated with a human B lymphoblastoid cell line (LCL) called PM that expressed CD80 and CD86 (T cells at 3.0x10 4 /well and PM at 8.0xl0 3 /well). Primary allostimulation occurred for 6 days, then the cells were pulsed with H-thymidine for 7 hours, before incorporation of radiolabel was determined.
  • LCL human B lymphoblastoid cell line
  • L104EA29YIg and CTLA4Ig are shown in Figure 31.
  • Peripheral blood mononuclear cells (PBMCS; 3.5xl0 4 cells/well from each monkey) from 2 monkeys were purified over lymphocyte separation medium (LSM) and mixed with 2 ⁇ g/ml phytohemaglutinin (PHA). The cells were stimulated 3 days then pulsed with radiolabel 16 hours before harvesting.
  • LSM lymphocyte separation medium
  • PHA phytohemaglutinin
  • the cells were stimulated 3 days then pulsed with radiolabel 16 hours before harvesting.
  • L104EA29YIg inhibited monkey T cell proliferation better than CTLA4Ig.
  • CTLA4 mutant molecule L104EA29YIg also known as LEA29Y or LEA
  • CTLA4Ig to relieve at least one symptom associated with rheumatoid arthritis, including reducing: joint swelling, joint tenderness, inflammation, mormng stiffness, and pain.
  • the CTLA4Ig molecule used herein begins with methionine at position +1 (or alternatively with alanine at position -1) and ends with lysine at position +357 as shown in Figure 24. DNA encoding an embodiment of the CTLA4Ig molecule has been deposited as ATCC 68629.
  • the L104EA29YIg molecule used herein begins with methionine at position +1 (or alternatively with alanine at position -1) and ends with lysine at position +357 as shown in Figure 19.
  • DNA encoding an embodiment of the L104EA29YIg molecule has been deposited as ATCC PTA2104.
  • the following provides a description of human patients administered L104EA29YTg or CTLA4Ig to reheve at least one biological surrogate marker associated with rheumatoid arthritis, including reducing erythrocyte sedimentation rates, and serum levels of C-reactive protein and/or IL2 receptor.
  • the doses administered included 0.5, 2.0, or 10.0 mg/kg of L104EA29YIg (denoted as LEA.5, LEA2 and LEA10, respectively in Figures 1A-1E) or of CTLA4Ig (denoted as CTLA.5, CTLA2 and CTLA10, respectively in Figures 1A-1E).
  • CTLA4Ig and L104EA29YIg were supplied in single-use glass vials containing 200 mg/vial of CTLA4Ig or 100 mg/vial of L104EA29YIg, respectively. Prior to infusion, the CTLA4Ig and L104EA29YIg were diluted to a final concentration of 25 mg/ml with sterile water for injection (SWFI).
  • SWFI sterile water for injection
  • Group 1 32 patients, CTLA4Ig or L104EA29YIg matching placebo.
  • Group 2 26 patients; dosage 0.5 mg/kg of CTLA4Ig.
  • Group 3 32 patients; dosage 2.0 mg/kg of CTLA4Ig.
  • Group 4 32 patients; dosage 10.0 mg/kg of CTLA4Ig.
  • Group 5 32 patients; dosage 0.5 mg/kg of L104EA29YIg.
  • Group 6 29 patients; dosage 2.0 mg/kg of L104EA29YIg.
  • Group 7 31 patients; dosage 10.0 mg/kg of L104EA29YIg.
  • ACR American College of Rheumatology
  • a subject satisfied the ACR50 or ACR70 criterion if there was a 50 or 70 percent improvement, respectively, in tender and swollen joint counts and 50 or 70 percent improvement, respectively, in tliree of the five remaining symptoms measured, such as patient and physician global disease changes, pam, physical disability, and an acute phase reactant such as CRP or ESR.
  • EIA enzyme irnmunoassay
  • IL-2sR IL-2sR
  • sICAM-1 IL-2sR
  • sE-selectin were measured using commercially available colorimetric EIA kits from R&D Systems, Inc. (Minneapolis, MN).
  • the lower and upper Hmits of quantitation were 312-20,000 pg mL, 40-907 ng/mL and 10-206 ng/mL, respectively.
  • the inter-assay coefficient of variation ranged from 4.48-8.4%, 3.8-5.0%) and 5.5-9.0% respectively.
  • normal seram values range from 676-2,132 pg/mL, respectively.
  • MMP-3 was measured using a commercially available colorimetric EIA kit from Amersham Pharmacia Biotech (Piscataway, NJ). The lower and upper limits of quantitation were 30-7,680 ng/mL. The inter-assay coefficient of variation ranged from 6.3-10.6%). According to the kit manufacturer, normal serum values range from 28-99 ng/mL.
  • IL-6 and TNF ⁇ were measured using commercially available chenfrluminescent EIA kits from R&D Systems, Inc. (Minneapolis, MN).
  • the lower and upper Hmits of quantitation were 0.3-3,000 pg/mL and 0.7-7,000 pg/mL, respectively.
  • the inter-assay coefficient of variation ranged from 3.1-5.7%) and 6.4-20.7%, respectively.
  • normal seram values range from ⁇ 0.3-12 pg/mL and ⁇ 0.7-7.5 pg/mL.
  • Serum samples were obtained for assessment of drag-specific antibodies prior to dosing on day 1, and approximately on days 15, 29, 57, 85 and 169. Due to high, preexisting titers directed to the immimoglobulin (Ig) portion of the molecule, specific antibody formation against CTLA4Ig and LEA29Y without Ig constant regions was also assessed.
  • Ig immimoglobulin
  • EPT endpoint titer
  • CTLA4Ig and L104EA29YIg were generally well-tolerated at all dose-levels. Peri- infusional adverse events were similar across all dose groups, with the exception of headaches. Headache response of patients on day 85 increased dose-dependently 23%, 44%, and 53% in CTLA4Ig-treated patients, and 34%, 45%, and 61% in L104EA29YIg- treated patients, at 0.5, 2.0, and 10.0 mg kg respectively. In contrast, 31% of tlie patients administered placebos experienced headaches.
  • FIG. 3 A The ACR-20, -50, and -70 responses of patients treated with CTLA4Ig, L104EA29YIg, or placebo at day 85 are summarized in Figure 3 A.
  • Figures 3B and C describe the ACR-20 responses with 95% confidence limits. The responses appear to be dose- dependent with a clear significant response at 10 mg/kg per body weight of the patient.
  • FIGs 5 A, B, C, and D The therapeutic responses, as monitored by the Likert scale, appear to be dose-dependent in favor of the active treatment groups as compared to placebo on day 85.
  • the Likert scale is a validated verbal rating scale using adjectives to rank the symptoms (The American College of Rheumatology Preliminary Core Set of Disease Activity Measures for Rheumatoid Arthritis Clinical Trials: Arthritis and Rheumatism, June 1993, 36(6):729-740).
  • CRP C-reactive protein
  • the median and mean tender joint counts in patients freated with CTLA4Ig or placebo over time are shown in Figures 9A and B.
  • the change from baseline e.g., reduction in tender joints
  • the median and mean swollen joint counts in patients treated with CTLA4Ig or placebo over time are shown in Figures 10A and B.
  • the change from baseline e.g., reduction in swollen joints
  • the mean disease activity assessment scores evaluated by patient or physician in patients treated with L104EA29YIg (denoted as LEA in the figures) or placebo over time are shown in Figures 16A and B.
  • the change from baseline e.g., reduction in disease activity
  • the percent improvement of physical disability assessed by HAQ at day 85 for patients treated with CTLA4Ig, L104EA29YIg, or placebo are shown in Figure 17 (Health Assessment Questionnaire (HAQ); Fries, J. F., et al., 1982 J. of Rheumatology 9:789- 793). There is a clear dose dependent improvement with this parameter.
  • HAQ Health Assessment Questionnaire
  • soluble IL-2r and C-reactive protein levels were dose- dependent in both treatment groups.
  • soluble IL-2r levels were -2%, - 10%, and -22% for CTLA4Ig and -4%, -18%, and -32% for L104EA29YIg at 0.5, 2.0, and 10.0 mg/kg respectively, compared to +3% for the placebo.
  • C-reactive protein levels were +12%, -15%, and -32% for CTLA4Ig and +47%, -33%, and -47% for L104EA29YIg at 0.5, 2.0, and 10.0 mg/kg respectively, compared to +20% for the placebo (Figure 7 A).
  • phase II clinical studies of human patients that will be administered L104EA29YIg, to reduce or prevent structural damage, including bone or joint erosion using validated radiographic scales. This improvement in reducing or preventing structural damage is parallel to the clinical improvement measured by the cHnical parameters.
  • the status of the bone structure is monitored in some of the human patients prior to freatment with CTLA4Ig or L104EA29YIg. These patients are administered from 0.5 to 20 mg/kg of CTLA4Ig or L104EA29YIg chronically every two to twelve weeks (alone or in combination with other agents) to maintain their therapeutic improvement over time. Radiographs of patients' hands and feet are taken at predefined intervals: 6 months, and then yearly, as recommended by the FDA guidelines. These patients are monitored in long-term extension after 6 and 12 months to determine if treatment with CTLA4Ig or L104EA29YIg reduces the progression of bone deterioration, and then yearly.
  • the patients are monitored by radiographic methods, including X-ray and/or magnetic resonance imaging (MRI), according to standard practice in the art (Larsen, A. K. and M. Eek 1977 Acta. Radiol Diag. 18:481-491; Sharp, J. T., et al., 1985 Arthritis and Rheumatism 28:1326-1335).
  • the results of the radiographic data are evaluated for prevention of structural damage, including slowing the progression of bone erosion and cartilage damage, with joint space narrowing and/or prevention of new erosions.
  • RA Rheumatoid Arthritis
  • Methofrexate remains the cornerstone of the RA freatment. It was the first agent that demonstrated early onset of action, superior efficacy and tolerability compared to the classical DMARDs (e.g. gold, hydroxychloroquine, sulfasalazine) used to treat RA. Clinical benefit may be seen as early as 3 weeks after initiating freatment, and the maximal improvement is generally achieved by 6 months.
  • methotrexate has a number of limitations. For example, despite its increased tolerability, the window between efficacy and liver toxicity is quite narrow. Subjects treated with methotrexate require careful monitoring and unacceptable toxicity is often the reason for discontinuation of treatment.
  • Methotrexate also does not appear .to efficiently control disease progression or joint deterioration. For some subjects, practitioners feel compelled to add a second DMARD with the hope of increasing efficacy despite the risk of increased toxicity.
  • co-treatment with methotrexate and a costimulator blocker e.g. CD80 and CD86 blockers such as CTLA4Ig
  • a costimulator blocker e.g. CD80 and CD86 blockers such as CTLA4Ig
  • Example 3 As noted in Example 3, above, significant clinical responses and reductions in su ⁇ ogate markers of disease activity were observed for CTLA4Ig at doses of 2 and 10 mg/kg with a good tolerability profile. It has also been confirmed that the composition CTLA4Ig, used in Example 3 above, did not induce any side effects. As a result, it was decided to continue the cHnical development of CTLA4Ig for rheumatoid arthritis in Phase 1TB.
  • This example describes a twelve month study in which primary efficacy was assessed after all subjects completed six months of treatment or discontinue therapy. Efficacy, safety, and disease progression were also assessed throughout the duration of the study.
  • the study utilized a randomized, double blind, placebo controlled, parallel dosing design.
  • the study was designed to evaluate the safety, clinical activity, immunogenicity and pharmacokinetics of two doses of CTLA4Ig: 2 or 10 mg/kg.
  • CTLA4Ig or a placebo were also administered on Day 15.
  • Each dose of study medication was infused intravenously over approximately 30 minutes.
  • the primary efficacy endpoint was the ACR 20 response rate after 6 months.
  • Analgesics that did not contain ASA or NSAIDs were permitted in subjects experiencing pain not adequately controlled by the baseline and study medications, except for 12 hours before a joint evaluation. Decreases in NSAIDs were permitted but only if due to adverse events such as gastrointestinal toxicity.
  • CTLA4Ig at 2 mg/kg or 10 mg/kg was infused every two weeks for the first month, and monthly thereafter for 12 months.
  • the primary endpoint of the first stage of the study was the proportion of subjects meeting the American College of Rheumatology criteria for 20% improvement (ACR 20) on Day 180 (month six).
  • the ACR 20 definition of improvement is a 20% improvement from baseline in the number of tender and swollen joint counts, and a 20% improvement from baseline in 3 of the following 5 core set measurements: subject global assessment of pain, subject global assessment of disease activity, physician global assessment of disease activity, subject assessment of physical function and acute phase reactant value (C- reactive protein (CRP)).
  • C- reactive protein (CRP) C- reactive protein
  • the evaluation for 50% improvement (ACR 50) and 70% improvement (ACR 70) follow similarly. Subjects who discontinued the study due to lack of efficacy (i.e. worsening RA) were considered as ACR non-responders from that time on. For all subjects who dropped out for other reasons, their ACR response at the time of discontinuation was carried forward.
  • CTLA4Ig Two doses of CTLA4Ig (2 mg/kg and 10 mg/kg) were compared with the placebo control group. All subjects were maintained at the same stable entry doses of methotrexate. The primary analysis was the comparison of CTLA4Ig 10 mg/kg with placebo. Sample sizes were based on a 5% level (2-tailed) of significance. Based on pubhshed studies, the placebo plus methotrexate control ACR 20 response rate at 6 months is about 25%. A sample of 107 subjects (adjusted for a possible 15% dropout) per freatment arm was determined to yield a 94% power to detect a difference of 25%) at the 5% level (two- tailed).
  • the sample was determined to yield a power of 95% and 90% > to detect differences of 20% and 14%) in ACR 50 and ACR 70, respectively. If the comparison between CTLA4Ig 10 mg/kg and placebo was significant with regards to ACR 20, then the comparison between CTLA4Ig 2 mg/kg and placebo was carried out. This second testing should have a power of 88%>. This sequentially rejective procedure based on Chi- square tests was also used to test for differences in ACR 50 and ACR 70 responses.
  • Demographic and baseline clinical characteristics were similar among the treatment groups. Sixty three to 75 percent of subjects were female, 87%) were Caucasian. The mean duration of the disease at entry was 9.7 ⁇ 9.8, 9.7 ⁇ 8.1, and 8.9 ⁇ 8.3 years respectively in the 10, 2 mg/kg and the control group. The mean weight in kg was very similar between 77.8 and 79.9 kg with a range of 40.1 to 186.8 kg (Table m).
  • Morning Stiffness (in min.) 97.9 + 63.1 104.1 + 63.9 106.0 + 64.2
  • the mean number of tender and swollen joints at baseline was comparable among the three treatment groups.
  • the mean number of tender joints and swollen joints in the 10 mg group was 30.8 + 12.2 and 21.3 + 8.4, respectively.
  • the mean number of tender joints and swollen joints in the 2 mg group was 28.2 + 12.0, and 20.2 ⁇ 8.9, respectively.
  • the mean number of tender joints and swollen joints in the control group was 29.2 + 13.0, and 21.8 ⁇ 8.8, respectively.
  • Figures 34-37 presents the ACR response rates from Day 1 to Day 180.
  • Figures 38 and 40 presents the ACR20, -50 and -70 response rates on day 180 for the various treatment groups.
  • the ACR 50 and ACR 70 response rates suggest the possibility that maximal efficacy may not have been achieved at 10 mg/kg.
  • Figure 39 shows the proportion of new tender and swollen joints at day 180 of the study after therapy with methotrexate alone or in combination with CTLA4Ig (admimstered at 2 or 10 mg/kg body weight of subject).
  • Figure 46 shows the mean percent improvement in physical function from baseline as measured by HAQ.
  • Table NI Individual ACR Components at Day 180
  • the 2 and 10 mg/kg dose groups demonstrated some degree of efficacy among all clinical components of the ACR response criteria (Table NI; Figures 41-45, 47-48); the subject's global assessment in the 2 mg/kg dose group being the only exception.
  • the reduction of tender and swollen joints appears dose-dependent.
  • the number of tender joints was decreased by 59.9%, 43.3% and 32.1%> in the 10 mg/kg, 2 mg/kg and control groups, respectively.
  • a similar pattern was observed for the swollen joint counts with a decrease of 54.9%>, 45.1% and 33.4%o in the 10 mg/kg, 2 mg/kg and control groups, respectively.
  • CTLA4Ig health-related quality of life
  • HRQOL health-related quality of life
  • the SF-36 was administered to all subjects at basefine, 90 and 180 days.
  • the SF-36 consists of 36 items which covers eight domains (physical fimction, role-physical, bodily pain, general health, vitality, social fimction, role emotional, and mental health). These individual domains are used to derive the physical and mental component summary scores which range from 0 to 100, with
  • Baseline SF-36 scores were comparable between the three treatment groups. Improvements in quality of life show a clear dose-response trend after 6 months of treatment. Subjects in the CTLA4Ig 10 mg/kg treatment group demonstrated clinically and statistically significant improvements from baseline in all 8 domains of the SF-36. The greatest effects were shown in the role-physical, bodily pain, and role-emotional domains. This positive finding was consistent with the efficacy results. For subjects treated with CTLA4Ig 2 mg/kg, improvements from baseline were also statistically significant for all domains except mental health.
  • CTLA4Ig The pharmacokinetics of CTLA4Ig were derived from serum concenfration versus time data between dosing days 60 and 90. Samples were collected prior to dosing on day 60, at
  • CTLA4Ig CTLA4Ig Methotrexate +
  • Serum levels of pharmacodynamic biomarkers were analyzed at various times during the study. Baseline values are shown in Table NIT. The values on Day 180 relative to basehne are shown in the Figures 52-56.
  • CRP levels decreased from baseline in both CTLA4Ig-treated groups more than in the control group, with greater reduction observed in the 10 mg/kg dosing group (see Figures 47, 48 and 52).
  • Rheumatoid factor levels decreased from baseline in both CTLA4Ig-freated groups more than in the control group, with greater reduction observed in the 10 mg/kg dosing group (see Figure 53).
  • Soluble TL-2r levels decreased from baseline in both CTLA4Ig-treated groups more than in the control group, with greater reduction observed in the 10 mg kg dosing group (see Figure 54).
  • Serum IL-6 levels decreased from in both CTLA4Ig-treated groups more than in the control group (see Figure 55).
  • CTLA4Ig The effects of CTLA4Ig on serum TNF ⁇ levels were inconclusive. The 2 mg/kg group increased and the 10 mg/kg group decreased relative to the control group (see Figure 56).
  • CTLA4Ig was well tolerated at all doses. There were no deaths, malignancies or opportunistic infections in any subjects receiving CTLA4Ig. Serious adverse events (SAEs) and non-serious adverse events (NSAEs) were similar or less frequent in the active-treatment groups compared to the control group.
  • SAEs Serious adverse events
  • NSAEs non-serious adverse events
  • CTLA4Ig significantly reduced the signs and symptoms of rheumatoid artiiritis in subjects receiving methotrexate as assessed by ACR response criteria.
  • the effects of CTLA4Ig appear to increase in proportion to dose level.
  • the improvement from baseline in all ACR core components is higher in the 10 mg/kg group than the 2 mg/kg group.
  • CTLA4Ig at 10 mg/kg doses demonstrated climcally and statistically significant improvements in all 8 domains of the SF-36. All pharmacodynamic biomarkers assayed appeared to decrease in proportion to CTLA4Ig dose level except for TNF ⁇ .
  • CTLA4Ig was safe and well tolerated in subjects with rheumatoid arthritis receiving methotrexate.
  • the adverse event profile for both CTLA4Ig doses was similar to the control group.
  • CTLA4Ig Given Monthly In Combination with Etanercept to Patients with Active Rheumatoid Arthritis.
  • CTLA4Ig in combination with etanercept, to treat patients with active Rheumatoid Arthritis.
  • Etanercept along with infliximab, comprises a new generation of Rheumatoid Arthritis drugs which targets Tumor Necrosis Factor (TNF.
  • Etanercept is a dimeric fusion protein having an extracellular portion of the TNF receptor linked to the Fc portion of human immunoglobulin (IgGl). This fusion protein binds to TNF, blocks its interactions with cell surface TNF receptors and render TNF molecules biologically inactive.
  • This example describes a twelve month study in which efficacy was assessed after all subjects completed six months of treatment or discontinued therapy. Efficacy, safety and disease progression were also assessed throughout the duration of the study.
  • CTLA4Ig Infusions of CTLA4Ig were given on Days 1, 15, 30, and monthly thereafter, for 6 months (primary treatment phase). Each dose of study medication was infused intravenously for approximately 30 minutes.
  • the primary treatment phase of the study took place during the first six months of treatment. During this period, subjects were required to remain on stable doses of etanercept (25 mg twice weekly). DMARDs other than etanercept were not permitted. Low-dose stable corticosteroid (at 10 mg daily or less) and/or stable non-steroidal anti- infl-immatory drug (NSAID), including acetyl salicylic acid (ASA), use was allowed. Analgesics (that do not contain ASA or NSAIDs) were permitted in subjects experiencing pain that was not adequately controlled by the baseline and study medications, except for 12 hours before a j oint evaluation.
  • NSAID stable non-steroidal anti- infl-immatory drug
  • Analgesics that do not contain ASA or NSAIDs were permitted in subjects experiencing pain that was not adequately controlled by the baseline and study medications, except for 12 hours before a j oint evaluation.
  • the primary endpoint of this study was to collect, data regarding the proportion of subjects meeting modified American College of Rheumatology (ACR) criteria for 20% improvement (ACR 20) after six months.
  • the modified ACR 20 criteria were used to accommodate the low CRP levels in this study's subject population.
  • the modified ACR criteria were defined as 1) a greater than 20% improvement in tender and swollen joint count and 2) a greater than 20% improvement in 2 of the remaining 4 core data set measures (global pain, physician, subject, functional assessment).
  • CRP which is normally a part of the standard ACR core data sets, was not included in the modified ACR criteria due to the low levels of CRP in subjects using TNF blockers, such as etanercept.
  • the standard ACR criteria, and two alternative criteria were also evaluated as secondary endpoints.
  • sample was determined to yield a power of 91% and 83% to detect differences of 30 and 25% in ACR 50 and 70, respectively.
  • due to slow enrollment only 122 subjects were randomized and 121 treated and analyzed (one subj ect was randomized but never received treatment) .
  • Table 1 Subject Disposition at Day 180
  • Demographic characteristics were similar between treatment groups. The majority of subjects were female and Caucasian. The mean duration of the disease was 13 years and the mean age was 52 years (Table 2).
  • CTLA4Ig and etanercept Two subjects receiving CTLA4Ig and etanercept had a dermatological malignancy.
  • ACR 70 10.6 % d 0% 16.5 % d 10.5 % 1.7 % a) Modified ACR. See criteria for evaluation b) Standard ACR criteria c) Placebo + Background therapy (etanercept or methotrexate) d) p ⁇ 0.05 for the difference in ACR response vs placebo + background therapy
  • CTLA4Ig plus etanercept at 2 mg/kg was similar to that observed in subjects receiving the same dose of CTLA4Ig plus methotrexate therapy (Example 5).
  • the criteria for evaluation in the methotrexate (Example 5) trial was the standard ACR, that includes CRP among the core components, while in the etanercept trial (Example 6) the criteria for evaluation was the modified ACR, that excludes CRP.
  • CTLA4Ig (2 mg/kg) in combination with etanercept reduced the signs and symptoms of rheumatoid arthritis, as compared with etanercept alone.
  • the increases in the modified ACR20 and ACR 70 assays were statistically significant.
  • Efficacy of CTLA4Ig plus etanercept therapy was observed within one month of the start of treatment.
  • CTLA4Ig was generally safe and well tolerated when admimstered in combination with etanercept with the safety profile similar to etanercept therapy alone.
  • CTLA4Ig was not irnmunogenic during the six month trial period. Additionally, the efficacy of CTLA4Ig therapy in combination with
  • etanercept (Example 6) was similar to the same dose of CTLA4Ig with methotrexate (Example 5).
  • the following example provides the one-year results from a Phase IIB, multi-center, randomized, double-blind, placebo controlled clinical study to evaluate the safety and clinical efficacy of administering two different doses of CTLA4Ig in combination with methotrexate to treat patients with active Rheumatoid Arthritis (RA).
  • the study presented in this example is a continuance of the six-month study presented in Example 5.
  • results presented in this clinical study report are based on data from an analysis perfo ⁇ ned after all subjects completed 6 months of treatment and again after all subjects completed 12 months of freatment.
  • the 10 mg/kg CTLA4Ig plus MTX group may be referred to as the 10 mg/kg group, the 2 mg/kg plus MTX group is refe ⁇ ed to as the 2 mg/kg group, and the CTLA4Ig (BMS-188667) placebo plus MTX group is refe ⁇ ed to as the placebo group.
  • This study compared the clinical efficacy of two different doses (10 and 2 mg/kg) of CTLA4Ig (BMS- 188667) combined with methotrexate (MTX) or with MTX plus placebo in subjects with active RA as assessed by ACR at 6 month and 12 month intervals.
  • This study enrolled adult subjects with active RA who had had an inadequate response to MTX.
  • Treatment Groups Subjects were randomized 1:1:1 to one of three treatment groups:
  • Group 3 CTLA4Ig (BMS-188667) placebo by intravenous infusion
  • Infusion doses were based upon the subject's body weight from the pre-treatment visit immediately prior to the Day 1 visit (for a subject on MTX monotherapy, the weight was obtained at the screening visit; for a subject on MTX combination therapy [in combination with other DMARDs], the weight was obtained from the washout visit, Day -2).
  • the infusion doses were not modified during Day 1 to Day 360. Infusions were to occur at approximately the same time of day throughout the study. All doses of study medication were administered in a fixed volume of 75 mL at a constant rate over approximately 30 minutes.
  • the intravenous bag and line were flushed with 25 mL of dextrose 5% in water (D5W) solution at the end of each infiision. All intravenous infusions were administered with the subject in the seated position. Subjects were observed for Adverse Events (Aes) and changes in vital signs (blood pressure, heart rate, body temperature) from the start of each infusion (pre-dose, 15, 30, 45, 60, 75, 90, 120 minutes) and for a minimum of 2 hours after the start of the infusion. The observation period could be extended, if clinically indicated.
  • Adverse Events Adverse Events
  • vital signs blood pressure, heart rate, body temperature
  • Systemic (non-topical) corticosteroids Provided that the dose was stable and the total dose was less than or equal to the equivalent of prednisone 10 mg/day. hitra-articular injections were to be avoided; however, if necessary, up to two infra-articular injections were permitted. NOTE: A joint that received an infra-articular injection was counted as "active" in ALL subsequent assessments/evaluations .
  • NSAIDs, including ASA Provided the dose was stable
  • Acetaminophen, combination products including acetaminophen and narcotic analgesics i.e., acetaminophen with codeine phosphate, acetaminophen with propoxyphene napsylate, acetaminophen with oxycodone hydrochloride, acetaminophen with oxycodone bitartrate, etc.
  • tramadol For subjects experiencing pain not adequately controlled by baseline or study medication (except for 12 hours before a joint evaluation)
  • Table 1 is a schedule of study procedures and evaluations. Table 1: Schedule of Study Procedures and Evaluations
  • a C est X-ray an EC was per orme not per orme w t n 6 mont s or not on e.
  • the DMARDs must have been washed out for at least 28 days prior to Day 1.
  • This visit was required only if the subject was on MTX therapy, d Urine or serum pregnancy test performed within 48 hours prior to dosing, for all women of child bearing potential. Serum pregnancy test was to be processed locally, e Subjects who discontinued must have had an "early termination" visit. Assessments at this visit were identical to assessments performed on Day 360. The assessments for this visit replaced what might have been scheduled on the day of discontinuation.
  • Day 1 required a follow-up mammogram on the one year anniversary of the mammogram that was performed during the screening period, m Subject's body weight was provided to central randomization system, n No radiographic assessments were required at the termination visit for subjects who discontinued within the first nine months of treatment, o Subjects who were terminated early had adverse events and concomitant medications recorded 30 and 60 days after the last dose of study medication.
  • ACR American College of Rheumatology
  • ACR 70 ACR 20 definition of response corresponds to a 20%, 50%, or 70% improvement, respectively, over baseline in tender and swollen joints (components 1 and 2) and a 20%, 50%, and 70% improvement, respectively, in three of the five remaining core data set measures (components 3 to 7).
  • a Major Clinical Response is defined as maintenance of an ACR 70 response over a continuous 6-month period. See Table 1 for the days that data for each component was collected.
  • a sequential testing procedure was employed. First, a Chi-square test was used to compare the data for the 10 mg/kg CTLA4Ig group with the data for the placebo group at the 0.05 level of significance. If this was significant, the data for the 2 mg/kg CTLA4Ig group was compared with the placebo group at the 0.05 level. This testing procedure preserved the overall alpha level at 5%. Similar analyses were carried out for the ACR 50 and ACR 70 responses at 6 months.
  • ACR 20, ACR 50, and ACR 70 response rates on Day 360 were compared between each CTLA4Ig (BMS-188667) treatment group and placebo at the Dunnett-adjusted 0.027 (two-tailed) level of significance.
  • ACR 20, ACR 50, and ACR 70 responses on Days 15, 30, 60, 90, 120, 150, 180, 240, 300, and 360 were also presented for the two CTLA4Ig (BMS-188667) groups and the placebo group.
  • the differences in ACR responses between the CTLA4Ig (BMS-188667) groups and placebo group were summarized using 95% confidence intervals.
  • the ACR data plotted over time were used to assess onset-of-action and to determine time to maximal response.
  • a Major Clinical Response was defined as the maintenance of an ACR 70 response over a continuous 6-month period. At the 12-month analysis, the proportion of subjects who achieved a Major Clinical Response among the three groups was summarized.
  • the cumulative index, ACR-N was evaluated at each follow-up assessment, and the AUC was assessed for up to 6 months and up to 12 months.
  • the trapezoidal rule was used to compute the AUC.
  • the ACR-N AUC was compared between the two CTLA4Ig (BMS-188667) treatment groups and the placebo group using an analysis of variance (ANON A) for 6- and 12-month data. This allowed for the assessment of subject response throughout the study. These analyses were carried out on the LOCF data sets.
  • Biomarkers for immunomodulation or inflammation in RA include CRP, soluble LL-2r, RF, soluble ICAM-1, E-selectin, serum LL-6, and TNF ⁇ . These parameters were summarized by treatment group, using frequencies and mean change from baseline to Day 180 and Day 360.
  • An Adverse Event was defined as any new or worsening illness, sign symptom or clinically significant laboratory test abnormality noted by the Investigator during the course of the study, regardless of causality.
  • a serious adverse event was defined as an AE that met any of the following criteria: was fatal; was life-threatening; resulted in or prolonged hospitalization; resulted in persistent or significant disability or incapacity, was cancer, was a congenital anomaly/birth defect, resulted in an overdose, resulted in the development of drug dependency or drug abuse, or was an important medical event.
  • Vital sign measurements were obtained at screening and at each study visit during and following study drug administration. Vital sign measurements (seated blood pressure, heart rate, and body temperature) were summarized by treatment group using means.
  • the two CTLA4Ig (BMS-188667) treatment groups (10 and 2 mg/kg) were compared with the placebo group.
  • the primary analysis was the comparison of 6-month ACR response rate for 10 mg/kg and placebo groups, to be followed by the comparison of 2 mg/kg with placebo only if the former was significant. Sample sizes were based on a 5% level (two-tailed) of significance.
  • the ACR 20 response rate for a placebo group at 6 months was estimated to be about 25% (Weinblatt M, Kremer JM, Bankhurst AD et. al. A trial of etanercept, a recombinant TNF:Tc fusion protein in patients with RA receiving methotrexate. NEJM 1999; 340: 253-259).
  • the power of the test would be at least 0.88, 0.90, and 0.81 for the comparison involving ACR 20, ACR 50, and ACR 70 responses at 6 months, respectively (Koch DD, Gansky SA. Statistical considerations for multiplicity in confirmatory protocols. Drug Info Journal 1996; 30: 523-534).
  • Subject IM101100-32-5 in the 10 mg/kg CTLA4Ig group reported an AE that was recorded as having resulted in discontinuation from the study; however, this subject was not included in this table.
  • CTLA4Ig group were higher than those for 10 mg/kg.
  • baseline demographic characteristics and baseline clinical RA characteristics were generally comparable across the three treatment groups and were typical of relatively advanced RA encountered in clinical practice (Table 5 and Table 6).
  • the majority of subjects were white females approximately 55 years old with a mean duration of RA of approximately 9 to 10 years, a relatively large number of active joints (approximately 29 tender and 21 swollen joints) and visual analogue scores (VAS) approximately 59-65 mm (100 mm scale).
  • VAS visual analogue scores
  • CTLA4Ig (BMS-188667; I >
  • Subject Global Assess (VAS 100 mm), n 113 105 119 Mean ⁇ SD 60.1 ⁇ 20.7 59.4 ⁇ 23.7 62.8 ⁇ 21.6 Range 10.0, 100.0 8.0, 99.0 4.0, 94.0
  • Morning Stiffness (in minutes), n 115 103 119 Mean ⁇ SD 97.9 ⁇ 63.1 104.1 ⁇ 63.9 106.0 ⁇ 64.2 Range 0.0, 180.0 0.0, 180.0 0.0, 180.0
  • Baseline demographics and RA characteristics of the overall population of subjects who had at least one dose of study drug and discontinued due to lack of efficacy were generally comparable to the entire study population, however, a greater proportion of subjects in this subpopulation had been diagnosed with RA for >10 years (45%) compared to the overall study population (34%).
  • Medical history findings for subjects in this study were consistent with relatively advanced RA and were generally similar among treatment groups. The most frequently occurring findings (in >40% of the subjects) were musculoskeletal findings (not including RA symptoms; 59.3%), gastrointestinal findings (45.1%), and genitourinary findings (42.2%). Other important medical history findings included cardiovascular disease in approximately 39% of subjects in all treatment groups and endocrine/metabolic findings in approximately 29% of all subjects.
  • Chloroquine 1 (0.9) 0 0
  • the 10 mg/kg CTLA4Ig group had the longest mean duration of exposure for both study phases and the placebo group had the shortest mean duration of exposure for both study phases (Day 180: 163 days, 156 days, 140 days; Day 360: 286 days, 268 days, and 234 days; 10 mg/kg, 2 mg/kg, and placebo, respectively).
  • Subjects were to have been treated with a "stable" dose of MTX (10-30 mg weekly) for at least 6 months, for 28 days prior to Day 1. With the exception of 4 subjects , all subjects received between 10 and 30 mg of MTX weekly in addition to CTLA4Ig (BMS-188667) during the primary phase (Day 1-180). During the secondary phase (Day 181-360), the dose of MTX could have been adjusted provided it remained between 10 and 30 mg weekly.
  • Table 11 Subjects Who Received Clinically Relevant Concomitant Medications During Both Study Phases
  • Drug catego ⁇ es were not mutually exclusive. Note: Subject IM101100-83-3 (10 mg/kg CTLA4Ig) took mefloquine and subject IM101100-28-7 (placebo) took quinine between Days 1 and 180; subject IM101100-18-11 (10 mg/kg CTLA4Ig) took quinine between Days 181 and 360 as an antimalarial, and was not considered a significant protocol violation. EFFICACY RESULTS
  • CTLA4Ig (BMS-188667) 10 mg/kg group had superior efficacy compared to the placebo group at Day 180 and Day 360.
  • results for some efficacy parameters were significantly better compared to the placebo group, results for most other efficacy parameters were numerically higher compared to placebo.
  • the ACR50 and ACR70 responses at Day 180 for the 10 mg/kg CTLA4Ig group were also significantly higher compared to the placebo group (Table 12, Figure 71 A and Figure 7 IB).
  • the ACR50 and the ACR70 responses at Day 180 for the 2 mg/kg CTLA4Ig group were significantly higher compared to the placebo group.
  • the ACR20 response at Day 180 for the 2 mg/kg CTLA4Ig group was slightly higher compared to the placebo group; however, no statistically significant differences were observed.
  • CTLA4Ig group vs placebo.
  • Major Clinical Response was defined as maintenance of an ACR 70 response over a continuous 6-month period.
  • Table 14 Summary of Major Clinical Response by Day 360
  • ACR-N Mean Numeric ACR
  • ACR-N-AUC ACR-N Area Under the Curve
  • ACR-N mean numeric ACR for all treatment groups increased over time during the first 6 months of the study ( Figure 74).
  • mean ACR-N increased slightly with 10 mg/kg CTLA4Ig, but remained relatively unchanged with 2 mg/kg CTLA4Ig and placebo.
  • the ACR-N was consistently higher for the 10 mg/kg CTLA4Ig group compared to the 2 mg/kg CTLA4Ig and placebo groups.
  • CTLA4Ig For the 2 mg/kg CTLA4Ig group, statistically significant improvements compared to the placebo group were observed in physician global assessment and CRP at Day 180. Furthermore, CRP levels in the placebo group actually worsened at Day 180. Change from baseline in mean duration of morning stiffness was comparable among the three treatment groups at Day 180.
  • Table 15 Mean Percentage Improvement from Baseline at Day 180 (Individual Components of ACR Criteria)
  • CTLA4Ig BMS-188667
  • Table 16 Mean Percentage Improvement from Baseline at Day 360 (Individual Components of ACR Criteria)

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Abstract

La présente invention concerne des compositions et des méthodes permettant de traiter des maladies du système immunitaire, telles que du rhumatisme, par administration, à un sujet, de molécules CTLA4 solubles empêchant les molécules endogènes B7 molécules de se lier à leurs ligands, seules ou conjointement avec d'autres agents parmi lesquels, les antirhumatismaux modificateurs de la maladie (ARMM).
PCT/US2003/012356 2002-04-19 2003-04-18 Methodes de traitement d'une maladie auto-immune au moyen d'une molecule ctla4 soluble et d'un armm ou d'un ains WO2003088991A1 (fr)

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AU2003243152A AU2003243152A1 (en) 2002-04-19 2003-04-18 Methods for treating an autoimmune disease using a soluble ctla4 molecule and a dmard or nsaid
EP03747047A EP1496931A4 (fr) 2002-04-19 2003-04-18 Methodes de traitement d'une maladie auto-immune au moyen d'une molecule ctla4 soluble et d'un armm ou d'un ains

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WO2006108035A1 (fr) * 2005-04-06 2006-10-12 Bristol-Myers Squibb Company Methodes de traitement de troubles immunologiques associes a des transplantations de greffes, a l'aide de molecules solubles de ctla4 mutant
EP1954836A2 (fr) * 2005-11-08 2008-08-13 Avi Biopharma, Inc. Compose destine a l'immunodepression et procede de traitement
EP2032151A2 (fr) * 2006-06-01 2009-03-11 The Regents of the University of California Procédé de traitement combiné et formulation
US8475790B2 (en) 2008-10-06 2013-07-02 Bristol-Myers Squibb Company Combination of CD137 antibody and CTLA-4 antibody for the treatment of proliferative diseases
US8501704B2 (en) 2005-11-08 2013-08-06 Sarepta Therapeutics, Inc. Immunosuppression compound and treatment method
EP2699258A2 (fr) * 2011-04-15 2014-02-26 The United States of America As Represented by the Secretary Department of Health and Human Services Transfert du gène ctla-4 médié par un aav pour traiter le syndrome de sjögren
US9492444B2 (en) 2013-12-17 2016-11-15 Pharmaceutical Manufacturing Research Services, Inc. Extruded extended release abuse deterrent pill
US9707184B2 (en) 2014-07-17 2017-07-18 Pharmaceutical Manufacturing Research Services, Inc. Immediate release abuse deterrent liquid fill dosage form
EP3273958A4 (fr) * 2015-03-25 2018-12-26 Taiwanj Pharmaceuticals Co., Ltd. Antagonistes du récepteur de type toll 4 et son utilisation dans des maladies hépatiques auto-immunes
US10166299B2 (en) 2012-08-31 2019-01-01 The United States Of America As Represented By The Secretary Dept. Of Health And Human Services National Institutes Of Health AAV mediated aquaporin gene transfer to treat Sjogren's syndrome
US10172797B2 (en) 2013-12-17 2019-01-08 Pharmaceutical Manufacturing Research Services, Inc. Extruded extended release abuse deterrent pill
US10195153B2 (en) 2013-08-12 2019-02-05 Pharmaceutical Manufacturing Research Services, Inc. Extruded immediate release abuse deterrent pill
US10300095B2 (en) 2011-04-20 2019-05-28 The United States Of America, As Represented By The Secretary, Dept. Of Health And Human Services AAV mediated exendin-4 gene transfer to salivary glands to protect subjects from diabetes or obesity
US10508144B2 (en) 2005-12-20 2019-12-17 Bristol-Myers Squibb Company Carbohydrate content of CTLA4 molecules
US10851150B2 (en) 2005-12-20 2020-12-01 Bristol-Myers Squibb Company Carbohydrate content of CTLA4 molecules
US10959958B2 (en) 2014-10-20 2021-03-30 Pharmaceutical Manufacturing Research Services, Inc. Extended release abuse deterrent liquid fill dosage form

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NO342966B1 (no) * 2005-04-06 2018-09-10 Bristol Myers Squibb Co Fremgangsmåter for behandling av immunforstyrrelser assosiert med organtransplantasjon med løselige CTLA4-mutantmolekyler
JP2008535841A (ja) * 2005-04-06 2008-09-04 ブリストル−マイヤーズ スクイブ カンパニー 可溶性ｃｔｌａ４変異分子によるグラフト移植に関連する免疫不全の治療方法
US7482327B2 (en) 2005-04-06 2009-01-27 Bristol-Myers Squibb Company Methods for treating immune disorders associated with graft transplantation with soluble CTLA4 mutant molecules
WO2006108035A1 (fr) * 2005-04-06 2006-10-12 Bristol-Myers Squibb Company Methodes de traitement de troubles immunologiques associes a des transplantations de greffes, a l'aide de molecules solubles de ctla4 mutant
KR101235484B1 (ko) 2005-04-06 2013-02-22 브리스톨-마이어스 스큅 컴퍼니 가용성 ｃｔｌａ4 돌연변이체 분자를 이용하여 이식편이식과 연관된 면역 장애를 치료하는 방법
EA013122B1 (ru) * 2005-04-06 2010-02-26 Бристол-Маерс Сквибб Компани Способы лечения иммунных нарушений, связанных с пересадкой трансплантата, растворимым мутантным ctla4
US9487786B2 (en) 2005-11-08 2016-11-08 Sarepta Therapeutics, Inc. Immunosuppression compound and treatment method
US8501704B2 (en) 2005-11-08 2013-08-06 Sarepta Therapeutics, Inc. Immunosuppression compound and treatment method
EP1954836A4 (fr) * 2005-11-08 2010-09-22 Avi Biopharma Inc Compose destine a l'immunodepression et procede de traitement
EP1954836A2 (fr) * 2005-11-08 2008-08-13 Avi Biopharma, Inc. Compose destine a l'immunodepression et procede de traitement
US8933216B2 (en) 2005-11-08 2015-01-13 Sarepta Therapeutics, Inc. Immunosuppression compound and treatment method
US10508144B2 (en) 2005-12-20 2019-12-17 Bristol-Myers Squibb Company Carbohydrate content of CTLA4 molecules
US10941189B2 (en) 2005-12-20 2021-03-09 Bristol-Myers Squibb Company Carbohydrate content of CTLA4 molecules
US10851150B2 (en) 2005-12-20 2020-12-01 Bristol-Myers Squibb Company Carbohydrate content of CTLA4 molecules
US10808021B2 (en) 2005-12-20 2020-10-20 Bristol-Myers Squibb Company Compositions and methods for producing a composition
US12030923B2 (en) 2005-12-20 2024-07-09 Bristol-Myers Squibb Company Compositions and methods for producing a composition
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US9707284B2 (en) 2006-06-01 2017-07-18 The Regents Of The University Of California Formulations of peptides and chloroquines for the treatment of pathogenic immune responses in immune mediated diseases
JP2009538922A (ja) * 2006-06-01 2009-11-12 ザ リージェンツ オブ ザ ユニバーシティ オブ カリフォルニア 併用療法および製剤
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US8475790B2 (en) 2008-10-06 2013-07-02 Bristol-Myers Squibb Company Combination of CD137 antibody and CTLA-4 antibody for the treatment of proliferative diseases
EP2699258A2 (fr) * 2011-04-15 2014-02-26 The United States of America As Represented by the Secretary Department of Health and Human Services Transfert du gène ctla-4 médié par un aav pour traiter le syndrome de sjögren
EP2699258A4 (fr) * 2011-04-15 2014-10-29 Us Sec Dep Of Health And Human Services Transfert du gène ctla-4 médié par un aav pour traiter le syndrome de sjögren
US11207361B2 (en) 2011-04-20 2021-12-28 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services AAV mediated exendin-4 gene transfer to salivary glands to protect subjects from diabetes or obesity
US10300095B2 (en) 2011-04-20 2019-05-28 The United States Of America, As Represented By The Secretary, Dept. Of Health And Human Services AAV mediated exendin-4 gene transfer to salivary glands to protect subjects from diabetes or obesity
US10166299B2 (en) 2012-08-31 2019-01-01 The United States Of America As Represented By The Secretary Dept. Of Health And Human Services National Institutes Of Health AAV mediated aquaporin gene transfer to treat Sjogren's syndrome
US10195153B2 (en) 2013-08-12 2019-02-05 Pharmaceutical Manufacturing Research Services, Inc. Extruded immediate release abuse deterrent pill
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US10172797B2 (en) 2013-12-17 2019-01-08 Pharmaceutical Manufacturing Research Services, Inc. Extruded extended release abuse deterrent pill
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US9492444B2 (en) 2013-12-17 2016-11-15 Pharmaceutical Manufacturing Research Services, Inc. Extruded extended release abuse deterrent pill
US9707184B2 (en) 2014-07-17 2017-07-18 Pharmaceutical Manufacturing Research Services, Inc. Immediate release abuse deterrent liquid fill dosage form
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