Treatment of paroxysmal nocturnal hemoglobinuria patients by inhibitor of complement

Abstract

To provide treatment of paroxysmal nocturnal hemoglobinuria patients by an inhibitor of a complement.SOLUTION: Eculizumab, a humanized monoclonal antibody against C5 that inhibits terminal complement activation, showed activity in a preliminary 12-week open-label trial in a small cohort of patients with paroxysmal nocturnal hemoglobinuria (PNH). The present study has examined whether chronic eculizumab therapy can reduce intravascular hemolysis, stabilize hemoglobin levels, reduce transfusion requirements, and improve quality of life in a double-blind, randomized, placebo-controlled, multi-center global Phase III trial. It is found that eculizumab has stabilized hemoglobin levels, decreased the need for transfusions, and improved quality of life in PNH patients via reduced intravascular hemolysis.SELECTED DRAWING: None

Description

[Background technology]

[0001] Paroxysmal nocturnal hemoglobinuria (PNH) is a condition characterized by PIG-A 1,2 PNH is an acquired blood disorder resulting from the clonal expansion of hematopoietic stem cells that harbor somatic mutations in an X-linked gene called PIG-A. Mutations in PIG-A result in a premature block in the synthesis of glycosylphosphatidylinositol (GPI) anchors, which are required for the attachment of many proteins to the cell surface. As a result, PNH blood cells have a partial (type II) or complete (type III) deficiency of GPI-anchored proteins.

[0002] Intravascular hemolysis is a hallmark of PNH and is associated with the expression of the GPI-anchored complement regulatory protein CD59. 3,4 Under normal conditions, CD59 blocks the formation of the terminal complement complex (also called the membrane attack complex) on the cell surface, thereby preventing erythrocyte lysis and platelet activation. 5~8 Excessive or persistent intravascular hemolysis in PNH patients can lead not only to anemia (normal hemoglobin ranges from 14 to 18 g / dL in men and 12 to 16 g / dL in women; low values are considered anemic) but also to hemoglobinuria and the clinical sequelae associated with the release of red blood cell contents into the circulation: fatigue, thrombosis, abdominal pain, dysphagia, erectile dysfunction, and pulmonary hypertension. 9,10,2l,22 Indeed, the impairment of quality of life in PNH is disproportionate to the degree of anemia. Many PNH patients are transfusion-dependent to maintain adequate red blood cell hemoglobin levels. No treatment effectively reduces intravascular hemolysis and improves clinical morbidity associated with PNH.

[0003] Eculizumab is a humanized monoclonal antibody directed against the terminal complement protein C5. 11 (Non-Patent Document 1). In a preliminary 12-week open-label clinical trial in 11 PNH patients, eculizumab was shown to reduce intravascular hemolysis and the need for blood transfusions. 12(Non-Patent Document 2) However, this open-label study included patients without a control group and without transfusion criteria based on the clinical trial protocol. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Thomas TC et al., Mol Immunol (1996) 33;1389-401 [Non-patent document 2] Hillmen P et al. N Engl J Med (2004)350;552~9 Summary of the Invention [Means for solving the problem]

[0005] (Abstract) This pivotal Phase 3 trial (TRIUMPH: Multicenter, Double-Blind, Randomized, Placebo-Controlled Trial of Transfusion Reduction Efficacy and Safety with Eculizumab in Paroxysmal Nocturnal Hemoglobinuria) evaluated the effect of eculizumab on hemoglobin stability and transfusion requirements during 6 months of treatment in a cohort of 87 transfusion-dependent patients with PNH. It also evaluated the management of intravascular hemolysis and quality of life. This is the first placebo-controlled trial in a PNH patient population to control hemolysis and distinguish between effects due to hemolysis and anemia.

[0006] It has now been discovered that administration of eculizumab to patients with PNH unexpectedly improves certain aspects of quality of life. Furthermore, these improvements in quality of life are independent of blood transfusions. Improved aspects include, for example, overall health, physical functioning, emotional functioning, cognitive functioning, role functioning, social functioning, fatigue, pain, dyspnea, loss of appetite, and insomnia. Improvements were also observed in nausea and vomiting, diarrhea, constipation, and financial difficulties, although these improvements did not reach statistical significance. Because the treated patients remained anemic throughout treatment, all of these improvements were unexpected, as they were thought to be a result of the patients' anemia. Without wishing to be bound by any theory, it is believed that some of the symptoms are due, or at least partially due, to hemolysis and the release of hemoglobin into the bloodstream, and not simply because the patient is anemic. Treatment with eculizumab reduces the amount of lysis, thereby limiting the release of hemoglobin into the bloodstream, which clearly results in an improvement in the quality of life of the treated patient. The results presented herein demonstrate that any treatment that reduces hemolysis in patients will improve the quality of life of the patient.

[0007] In certain aspects, the present application provides methods for improving at least one aspect of quality of life in a patient suffering from paroxysmal nocturnal hemoglobinuria, the method comprising administering to the patient in need thereof a compound that inhibits complement or inhibits the formation of C5b-9.

[0008] In certain aspects, the present application provides a method for improving at least one aspect of quality of life in a patient suffering from paroxysmal nocturnal hemoglobinuria, comprising administering to the patient in need thereof a compound that inhibits intravascular hemolysis, and in certain embodiments, the method reduces LDH in the patient by more than 30%.

[0009] In certain aspects, the present application provides a method for improving at least one aspect of the quality of life of a patient with anemia at least partially caused by hemolysis, comprising administering to the patient in need thereof a compound that inhibits intravascular hemolysis, wherein the patient remains anemic. In certain embodiments, the method reduces the patient's LDH by more than 30%.

[0010] In certain aspects, the present application provides a method of extending the health-adjusted life expectancy of a patient, comprising administering to the patient in need thereof a compound that inhibits the formation of C5b-9. In certain embodiments, the patient is anemic. In certain embodiments, the patient remains anemic after treatment. In certain embodiments, the patient has a hemoglobin level of i) less than 14 g / dL for men, or ii) less than 12 g / dL for women. In certain embodiments, the patient has a hemoglobin level of i) less than 13 g / dL for men, or ii) less than 11 g / dL for women. In certain embodiments, the patient has a hemoglobin level of i) less than 12 g / dL for men, or ii) less than 10 g / dL for women. In certain embodiments, the patient suffers from paroxysmal nocturnal hemoglobinuria.

[0011] In certain aspects, the present application provides a pharmaceutical composition comprising an antibody or an active antibody fragment thereof that binds to C5. In certain embodiments, the antibody or an active antibody fragment thereof that binds to C5 is eculizumab. In certain embodiments, the antibody or an active antibody fragment thereof that binds to C5 is pexelizumab. In certain embodiments, the pharmaceutical formulation of the present application can be administered to a subject, particularly a subject with PNH.

[0012] In certain aspects, the present application provides methods for treating patients suffering from paroxysmal nocturnal hemoglobinuria by administering a pharmaceutical composition comprising an antibody or an active antibody fragment thereof that binds to C5. In certain embodiments, the antibody or an active antibody fragment thereof that binds to C5 is eculizumab. In certain embodiments, the antibody or an active antibody fragment thereof that binds to C5 is pexelizumab. In certain embodiments, the pharmaceutical formulations of the present application can be administered to subjects, particularly subjects with PNH.

[0013] In certain aspects, the present application provides kits comprising the pharmaceutical compositions of the present application. In some embodiments, the kits further comprise at least one component of a closed sterile system. Components of a closed sterile system include, but are not limited to, needles, syringes, catheter-type syringes, needle-type injection devices, needleless injection devices, filters, tubing, valves, and cannulas. In related embodiments, the kits include components for removing preservatives from the composition. Such components include filters, syringes, vials, containers, tubing, and the like.

[0014] In certain embodiments, the quality of life is measured by the FACIT-Fatigue score. In certain embodiments, the FACIT-Fatigue score increases by at least 3 points. In certain embodiments, the FACIT-Fatigue score increases by 4 points or more.

[0015] In certain embodiments, the quality of life is measured by the EORTC QLQ-C30 score. In certain embodiments, the EORTC QLQ-C30 score improves by 10% or more compared to the pre-treatment score. In certain embodiments, the aspect of quality of life measured by the EORTC QLQ-C30 score is selected from the group consisting of a) overall health, b) physical function, c) emotional function, d) cognitive function, e) role function, f) social function, g) fatigue, h) pain, i) dyspnea, j) loss of appetite, and k) insomnia. In certain embodiments, the aspect of quality of life is fatigue.

[0016] In certain embodiments, the compound is selected from the group consisting of CR1, LEX-CR1, MCP, DAF, CD59, Factor H, cobra venom factor, FUT-175, complestatin, and K76 COOH. In certain embodiments, the compound is a steroid that inhibits complement.

[0017] In certain embodiments, the compound is selected from the group consisting of an antibody, an active antibody fragment, a soluble complement inhibitory compound, a protein, a soluble complement inhibitor with a lipid tail, a protein fragment, a peptide, a small organic compound, an RNA aptamer, an L-RNA aptamer, a spiegelmer, an antisense compound, a serine protease inhibitor, a double-stranded RNA, a small interfering RNA, a locked nucleic acid inhibitor, and a peptide nucleic acid inhibitor. In certain embodiments, the compound is an antibody or an active antibody fragment. In certain embodiments, the antibody or active antibody fragment is selected from the group consisting of a) a polyclonal antibody, b) a monoclonal antibody, c) a single-chain antibody, d) a chimeric antibody, e) a humanized antibody, f) a Fab, g) a F(ab'), h) a F(ab')2, i) a Fv, j) a diabody, and k) a human antibody.

[0018] In certain embodiments, the antibody or active antibody fragment thereof binds to C5. In certain embodiments, the antibody or active antibody fragment blocks C5 cleavage. In certain embodiments, the antibody or active antibody fragment inhibits the formation of C5b-9. In certain embodiments, the antibody is eculizumab. In certain embodiments, the antibody or active antibody fragment is administered for at least 6 months. In certain embodiments, the patient has aplastic anemia or myelodysplastic syndrome.

[0019] In certain embodiments, the antibody or active antibody fragment thereof that binds to C5 is administered in a single unit dosage form. In certain embodiments, the single unit dosage form is a 300 mg unit dosage form. In certain embodiments, the single unit dosage form is lyophilized. In certain embodiments, the single unit dosage form is a sterile solution. In certain embodiments, the single unit dosage form is a preservative-free formulation. In certain embodiments, a 300 mg single-use dosage form contains 30 mL of a 10 mg / mL sterile, preservative-free solution.

[0020] In certain embodiments, the antibody or active antibody fragment thereof that binds to C5 comprises an altered constant region, wherein the antibody or antigen-binding fragment exhibits reduced effector function compared to an anti-CDCP1 antibody having a native constant region. In certain embodiments, the reduced effector function comprises one or more properties from the following group: a) reduced antibody-dependent cell-mediated cytotoxicity (ADCC), and b) reduced complement-dependent cytotoxicity (CDC) compared to an anti-CDCP1 antibody having a native constant region. In certain embodiments, the altered constant region comprises a G2 / G4 construct instead of a G1 domain.

[0021] In certain embodiments, the antibody or active antibody fragment thereof that binds to C5 comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises one or more CDR regions having an amino acid sequence selected from the group consisting of SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, and wherein the light chain variable region comprises a CDR region having an amino acid sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10. In certain embodiments, the antibody or active antibody fragment thereof that binds to C5 comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region consists of SEQ ID NO:1 and the light chain variable region consists of SEQ ID NO:3. In certain embodiments, the pharmaceutical composition comprises eculizumab. In certain embodiments, the pharmaceutical composition comprises pexelizumab. In certain embodiments, the antibody or active antibody fragment thereof that binds to C5 comprises a heavy chain and a light chain, wherein the heavy chain consists of SEQ ID NO:2 and the light chain consists of SEQ ID NO:4.

[0022] In certain embodiments, the patient is anemic. In certain embodiments, the patient remains anemic after treatment. In certain embodiments, the patient's hemoglobin level is i) less than 14 g / dL for men, or ii) less than 12 g / dL for women. In certain embodiments, the patient's hemoglobin level is i) less than 13 g / dL for men, or ii) less than 11 g / dL for women. In certain embodiments, the patient's hemoglobin level is i) less than 12 g / dL for men, or ii) less than 10 g / dL for women.

[0023] In certain embodiments, the health-adjusted life expectancy is measured according to units selected from the group consisting of Years of potential life lost, Disability-free life expectancy, Health-adjusted life year, Quality adjusted life year, Healthy years equivalents, Healthy days gained, Episode-free day, Q-TWiST, Health Utilities Index, or Years of healthy life.

[0024] In certain embodiments, the subject's health-adjusted life expectancy is increased by at least one day. In certain embodiments, the subject's health-adjusted life expectancy is increased by at least one week. In certain embodiments, the subject's health-adjusted life expectancy is increased by at least one month. In certain embodiments, the subject's health-adjusted life expectancy is increased by at least one year.

[0025] In certain embodiments, the pharmaceutical composition is in a single unit dosage form. In certain embodiments, the single unit dosage form is a 300 mg unit dosage form. In certain embodiments, the pharmaceutical composition is lyophilized. In certain embodiments, the pharmaceutical composition is a sterile solution. In certain embodiments, the pharmaceutical composition is a preservative-free formulation. In certain embodiments, the pharmaceutical composition comprises a 300 mg single-use formulation of 30 mL of a 10 mg / mL sterile, preservative-free solution. In certain embodiments, the pharmaceutical composition comprises an antibody or active antibody fragment thereof that binds to C5. For example, the present invention provides the following items: (Item 1) A method for improving at least one aspect of the quality of life of a patient suffering from paroxysmal nocturnal hemoglobinuria, comprising administering to the patient in need thereof a compound that inhibits complement or inhibits the formation of C5b-9. (Item 2) Item 10. The method of item 1, wherein the quality of life is measured by the FACIT-Fatigue score. (Item 3) 3. The method of item 2, wherein the FACIT-Fatigue score increases by at least 3 points. (Item 4) 3. The method of item 2, wherein the FACIT-Fatigue score increases by 4 or more points. (Item 5) Item 10. The method of item 1, wherein the quality of life is measured by EORTC QLQ-C30 score. (Item 6) 6. The method of item 5, wherein the EORTC QLQ-C30 score improves by 10% or more from the pre-treatment score. (Item 7) 6. The method of claim 5, wherein the aspect of quality of life as measured by EORTC QLQ-C30 score is selected from the group consisting of a) general health status, b) physical functioning, c) emotional functioning, d) cognitive functioning, e) role functioning, f) social functioning, g) fatigue, h) pain, i) dyspnea, j) loss of appetite, and k) insomnia. (Item 8) 8. The method of claim 7, wherein said aspect of quality of life is fatigue. (Item 9) 2. The method of claim 1, wherein the compound is selected from the group consisting of CR1, LEX-CR1, MCP, DAF, CD59, Factor H, cobra venom factor, FUT-175, complestatin, and K76 COOH. (Item 10) 2. The method of claim 1, wherein the compound is a steroid that inhibits complement. (Item 11) 2. The method of claim 1, wherein the compound is selected from the group consisting of an antibody, an active antibody fragment, a soluble complement inhibitory compound, a protein, a soluble complement inhibitor with a lipid tail, a protein fragment, a peptide, a small organic compound, an RNA aptamer, an L-RNA aptamer, a spiegelmer, an antisense compound, a serine protease inhibitor, a double-stranded RNA, a small interfering RNA, a locked nucleic acid inhibitor, and a peptide nucleic acid inhibitor. (Item 12) 12. The method of claim 11, wherein the compound is an antibody or an active antibody fragment. (Item 13) 13. The method of claim 12, wherein the antibody or active antibody fragment is selected from the group consisting of a) a polyclonal antibody, b) a monoclonal antibody, c) a single-chain antibody, d) a chimeric antibody, e) a humanized antibody, f) a Fab, g) a F(ab'), h) a F(ab')2, i) a Fv, j) a diabody, and k) a human antibody. (Item 14) 13. The method of claim 12, wherein the antibody or active antibody fragment blocks C5 cleavage. (Item 15) 13. The method of claim 12, wherein the antibody or active antibody fragment inhibits the formation of C5b-9. (Item 16) 13. The method of item 12, wherein the antibody is eculizumab. (Item 17) 13. The method of claim 12, wherein the antibody or active antibody fragment is administered for at least 6 months. (Item 18) 2. The method of item 1, wherein the patient has aplastic anemia or myelodysplastic syndrome. (Item 19) Item 10. The method of item 1, wherein the patient is anemic. (Item 20) 20. The method of claim 19, wherein the patient remains anemic after treatment. (Item 21) 20. The method of item 19, wherein the patient has a hemoglobin level of i) less than 14 g / dL for men, or ii) less than 12 g / dL for women. (Item 22) 20. The method of item 19, wherein the patient has a hemoglobin level of i) less than 13 g / dL for men, or ii) less than 11 g / dL for women. (Item 23) 20. The method of item 19, wherein the patient has a hemoglobin level of i) less than 12 g / dL for men, or ii) less than 10 g / dL for women. (Item 24) A method for improving at least one aspect of the quality of life of a patient suffering from paroxysmal nocturnal hemoglobinuria, comprising administering to the patient in need thereof a compound that inhibits intravascular hemolysis. (Item 25) 25. The method of claim 24, wherein the method reduces LDH in the patient by more than 30%. (Item 26) 25. The method of item 24, wherein the quality of life is measured by the FACIT-Fatigue score. (Item 27) 27. The method of item 26, wherein the FACIT-Fatigue score increases by at least 3 points. (Item 28) 27. The method of item 26, wherein the FACIT-Fatigue score increases by 4 or more points. (Item 29) 25. The method of item 24, wherein the quality of life is measured by the EORTC QLQ-C30 score. (Item 30) 30. The method of item 29, wherein the EORTC QLQ-C30 score improves by 10% or more from the pre-treatment score. (Item 31) 30. The method of claim 29, wherein said aspect of quality of life is selected from the group consisting of a) overall health status, b) physical function, c) emotional function, d) cognitive function, e) role function, f) social function, g) fatigue, h) pain, i) dyspnea, j) loss of appetite, and k) insomnia. (Item 32) 32. The method of claim 31, wherein said aspect of quality of life is fatigue. (Item 33) 25. The method of claim 24, wherein the compound is selected from the group consisting of CR1, LEX-CR1, MCP, DAF, CD59, Factor H, cobra venom factor, FUT-175, complestatin, and K76 COOH. (Item 34) 25. The method of claim 24, wherein the compound is a steroid that inhibits complement. (Item 35) 25. The method of claim 24, wherein the compound is selected from the group consisting of an antibody, an active antibody fragment, a soluble complement inhibitory compound, a protein, a soluble complement inhibitor with a lipid tail, a protein fragment, a peptide, a small organic compound, an RNA aptamer, an L-RNA aptamer, a spiegelmer, an antisense compound, a serine protease inhibitor, a double-stranded RNA, a small interfering RNA, a locked nucleic acid inhibitor, and a peptide nucleic acid inhibitor. (Item 36) 36. The method of claim 35, wherein the compound is an antibody or an active antibody fragment. (Item 37) 37. The method of claim 36, wherein the antibody or active antibody fragment is selected from the group consisting of a) a polyclonal antibody, b) a monoclonal antibody, c) a single-chain antibody, d) a chimeric antibody, e) a humanized antibody, f) a Fab, g) a F(ab'), h) a F(ab')2, i) a Fv, j) a diabody, and k) a human antibody. (Item 38) 37. The method of claim 36, wherein the antibody or active antibody fragment blocks C5 cleavage. (Item 39) 37. The method of item 36, wherein the antibody or active antibody fragment is administered for at least 6 months. (Item 40) 25. The method of claim 24, wherein the compound inhibits complement or inhibits the formation of C5b-9. (Item 41) 41. The method of claim 40, wherein the compound is an antibody or an active antibody fragment. (Item 42) 42. The method of claim 41, wherein the antibody is eculizumab. (Item 43) 25. The method of item 24, wherein the patient has aplastic anemia or myelodysplastic syndrome. (Item 44) 1. A method for improving at least one aspect of the quality of life of a patient with anemia whose anemia is at least partially caused by hemolysis, comprising administering to said patient in need thereof a compound that inhibits intravascular hemolysis, wherein said patient remains anemic. (Item 45) 45. The method of claim 44, wherein the method reduces LDH in the patient by more than 30%. (Item 46) 45. The method of item 44, wherein the patient has a hemoglobin level of i) less than 14 g / dL for men, or ii) less than 12 g / dL for women. (Item 47) 45. The method of item 44, wherein the patient has a hemoglobin level of i) less than 13 g / dL for men, or ii) less than 11 g / dL for women. (Item 48) 45. The method of item 44, wherein the patient has a hemoglobin level of i) less than 12 g / dL for men, or ii) less than 10 g / dL for women. (Item 49) 45. The method of item 44, wherein the patient suffers from paroxysmal nocturnal hemoglobinuria. (Item 50) Item 45. The method of item 44, wherein the quality of life is measured by the FACIT-Fatigue score. (Item 51) 51. The method of item 50, wherein the FACIT-Fatigue score increases by at least 3 points. (Item 52) 51. The method of item 50, wherein the FACIT-Fatigue score increases by 4 or more points. (Item 53) Item 45. The method of item 44, wherein the quality of life is measured by EORTC QLQ-C30 score. (Item 54) 54. The method of item 53, wherein the EORTC QLQ-C30 score improves by 10% or more from the pre-treatment score. (Item 55) 54. The method of claim 53, wherein said aspect of quality of life is selected from the group consisting of a) overall health status, b) physical function, c) emotional function, d) cognitive function, e) role function, f) social function, g) fatigue, h) pain, i) dyspnea, j) loss of appetite, and k) insomnia. (Item 56) 56. The method of item 55, wherein said aspect of quality of life is fatigue. (Item 57) 45. The method of claim 44, wherein the compound is selected from the group consisting of CR1, LEX-CR1, MCP, DAF, CD59, Factor H, cobra venom factor, FUT-175, complestatin, and K76 COOH. (Item 58) 45. The method of claim 44, wherein the compound is a steroid that inhibits complement. (Item 59) 45. The method of claim 44, wherein the compound is selected from the group consisting of an antibody, an active antibody fragment, a soluble complement inhibitory compound, a protein, a soluble complement inhibitor with a lipid tail, a protein fragment, a peptide, a small organic compound, an RNA aptamer, an L-RNA aptamer, a spiegelmer, an antisense compound, a serine protease inhibitor, a double-stranded RNA, a small interfering RNA, a locked nucleic acid inhibitor, and a peptide nucleic acid inhibitor. (Item 60) 60. The method of claim 59, wherein the compound is an antibody or an active antibody fragment. (Item 61) 61. The method of claim 60, wherein the antibody or active antibody fragment is selected from the group consisting of a) a polyclonal antibody, b) a monoclonal antibody, c) a single-chain antibody, d) a chimeric antibody, e) a humanized antibody, f) a Fab, g) a F(ab'), h) a F(ab')2, i) a Fv, j) a diabody, and k) a human antibody. (Item 62) 61. The method of item 60, wherein the antibody or active antibody fragment blocks C5 cleavage. (Item 63) 61. The method of item 60, wherein the antibody or active antibody fragment is administered for at least 6 months. (Item 64) 45. The method of claim 44, wherein the compound inhibits complement or inhibits the formation of C5b-9. (Item 65) 65. The method of claim 64, wherein the compound is an antibody or an active antibody fragment. (Item 66) 66. The method of item 65, wherein the antibody is eculizumab. (Item 67) 45. The method of item 44, wherein the patient has aplastic anemia or myelodysplastic syndrome. (Item 68) 1. A method of extending health-adjusted life expectancy in a patient, comprising administering to said patient in need thereof a compound that inhibits the formation of C5b-9. (Item 69) 69. The method of item 68, wherein the patient is anemic. (Item 70) 70. The method of claim 69, wherein the patient remains anemic after treatment. (Item 71) 69. The method of item 68, wherein the patient has a hemoglobin level of i) less than 14 g / dL for men, or ii) less than 12 g / dL for women. (Item 72) 69. The method of item 68, wherein the patient has a hemoglobin level of i) less than 13 g / dL for men, or ii) less than 11 g / dL for women. (Item 73) 69. The method of item 68, wherein the patient has a hemoglobin level of i) less than 12 g / dL for men, or ii) less than 10 g / dL for women. (Item 74) 69. The method of item 68, wherein the patient suffers from paroxysmal nocturnal hemoglobinuria. (Item 75) 69. The method of item 68, wherein the health-adjusted life expectancy is measured according to units selected from the group consisting of years of life lost, disability-free life expectancy, health-adjusted life years, quality-adjusted life years, health equivalent years, gain in healthy days, episode-free days, Q-TWiST, health utility index, or healthy life years. (Item 76) 76. The method of item 75, wherein the subject's health-adjusted life expectancy is extended by at least 1 day. (Item 77) 76. The method of item 75, wherein the subject's health-adjusted life expectancy is increased by at least 1 week. (Item 78) The method according to Item 75, wherein the subject's health-adjusted life expectancy is extended by at least one month. (Item 79) 76. The method of item 75, wherein the subject's health-adjusted life expectancy is increased by at least 1 year. (Item 80) Item 69. The method of item 68, wherein the compound is selected from the group consisting of CR1, LEX-CR1, MCP, DAF, CD59, Factor H, cobra venom factor, FUT-175, complestatin, and K76 COOH. (Item 81) 69. The method of claim 68, wherein the compound is a steroid that inhibits complement. (Item 82) 69. The method of item 68, wherein the compound is selected from the group consisting of an antibody, an active antibody fragment, a soluble complement inhibitory compound, a protein, a soluble complement inhibitor with a lipid tail, a protein fragment, a peptide, a small organic compound, an RNA aptamer, an L-RNA aptamer, a spiegelmer, an antisense compound, a serine protease inhibitor, a double-stranded RNA, a small interfering RNA, a locked nucleic acid inhibitor, and a peptide nucleic acid inhibitor. (Item 83) 83. The method of claim 82, wherein the compound is an antibody or an active antibody fragment. (Item 84) 84. The method of claim 83, wherein the antibody or active antibody fragment is selected from the group consisting of a) a polyclonal antibody, b) a monoclonal antibody, c) a single-chain antibody, d) a chimeric antibody, e) a humanized antibody, f) a Fab, g) a F(ab'), h) a F(ab')2, i) a Fv, j) a diabody, and k) a human antibody. (Item 85) 84. The method of item 83, wherein the antibody or active antibody fragment blocks C5 cleavage. (Item 86) 84. The method of item 83, wherein the antibody or active antibody fragment is administered for at least 6 months. (Item 87) 69. The method of claim 68, wherein the compound inhibits complement or inhibits the formation of C5b-9. (Item 88) 88. The method of item 87, wherein the compound is an antibody or an active antibody fragment. (Item 89) 89. The method of item 88, wherein the antibody is eculizumab. (Item 90) 69. The method of item 68, wherein the patient has aplastic anemia or myelodysplastic syndrome. [Brief explanation of the drawings]

[0026] [Figure 1A] Figures 1A-B show that eculizumab administration reduces intravascular hemolysis and increases PNH type III red blood cell counts. Figure 1A shows the degree of intravascular hemolysis in PNH patients as indicated by mean lactate dehydrogenase (LDH) levels. [Figure 1B] Figures 1A-B show that eculizumab treatment reduces intravascular hemolysis and increases PNH type III red blood cells. Figure 1B shows the mean percentage of PNH type III red blood cells assessed for placebo-treated and eculizumab-treated patients. [Figure 2] Figure 1 shows the effect of eculizumab treatment on transfusion requirements in patients with PNH. This is a Kaplan-Meier plot of time to first transfusion for patients receiving eculizumab and placebo from baseline to week 26. [Figure 3] Figure 1 shows the effect of eculizumab on fatigue as assessed by the FACIT-Fatigue instrument. Quality of life scores were assessed using the Functional Assessment of Chronic Illness Therapy-Fatigue (FACIT-Fatigue) instrument. Change values from baseline to week 26 represent least-squares means. A positive change indicates improvement in the FACIT-Fatigue measure of quality of life, and a negative change indicates deterioration. DETAILED DESCRIPTION OF THE INVENTION

[0027] I. Definition The term "derived from" means "obtained from" or "made by" or "derived from."

[0028] The term "genetically engineered antibody" refers to an antibody whose amino acid sequence has been altered from that of a naturally occurring antibody. Because this application involves recombinant DNA technology, there is no need to be limited to the sequence of amino acids found in natural antibodies; antibodies can be redesigned to obtain desired properties. The possible variations are many and range from changing just one or a few amino acids to complete redesign of, for example, the variable or constant regions. Generally, changes in the constant region are made to improve or alter properties such as complement fixation, membrane interaction, or other effector functions. Changes in the variable region are made to improve antigen-binding characteristics.

[0029] The term "antigen-binding fragment of an antibody" refers to any portion of an antibody that retains its ability to bind to an antigen. Exemplary antigen-binding fragments of an antibody are the heavy and / or light chain CDRs or the heavy and / or light chain variable regions.

[0030] The term "homologous," in the context of two nucleic acids or polypeptides, refers to two or more sequences or subsequences that have at least about 85%, at least 90%, at least 95% or more nucleotide or amino acid residue identity when compared and aligned for maximum correspondence as determined using the sequence pair comparison method described below and / or by visual inspection. In specific embodiments, "homologs" exist over a sequence region that is about 50 residues in length, at least about 100 residues, at least about 150 residues, or across the full length of the two sequences being compared.

[0031] Methods for determining percent identity are known in the art. "Percent (%) sequence identity" with respect to a designated subject sequence or a designated portion thereof can be defined as the percentage of nucleotides or amino acids in a candidate derivative sequence that are identical to those in the subject sequence (or a designated portion thereof), after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, as generated by the WU-BLAST-2.0a19 program (Altschul et al., J. Mol. Biol. 215:403-410 (1997); http: / / blastwustl.edu / blast / README.htm-l) with search parameters set to default values. The HSP S and HSP S2 parameters are dynamic values established by the program itself, depending on the composition of the particular sequence and the particular database against which the subject sequence is being searched. The "% identity value" is determined by the number of matching identical nucleotides or amino acids divided by the sequence length for which the percent identity is being reported.

[0032] II. Overview The present disclosure relates to methods of treating paroxysmal nocturnal hemoglobinuria ("PNH"), and more particularly to improving certain aspects of impaired quality of life in patients with PNH and other hemolytic diseases in mammals. Specifically, the methods of treating hemolytic diseases described herein involve the use of compounds that bind to or block the production and / or activity of one or more complement components. This method has been shown to provide impressive results. For example, when a compound that binds to or blocks the production and / or activity of one or more complement components is administered, hemolysis rapidly ceases with a significant reduction in hemoglobinuria. Furthermore, hemolytic patients can become largely or completely transfusion-independent for extended periods (12 months or more), successfully exceeding the 120-day life cycle of red blood cells. Furthermore, type III red blood cell counts can rapidly increase during other mechanisms of red blood cell lysis (non-complement-mediated and / or early complement component-mediated (e.g., Cb3)). Another example of a striking result was the disappearance of symptoms, indicating that serum levels did not increase significantly even if other mechanisms of red blood cell lysis were present. These and other results reported herein were unexpected and could not have been predicted from previous treatments for hemolytic diseases.

[0033] III. Complement system The complement system, useful complement inhibitors, and the use of these inhibitors to treat PNH and other patients are more fully described in PCT Patent Application No. PCT / US2005 / 003225, filed February 3, 2005, and published as International Publication No. WO2005 / 074607(A2) on August 18, 2005, the entire disclosure of which is incorporated herein by reference.

[0034] The complement system works with the rest of the body's immune system to defend against invading cellular and viral pathogens. There are at least 25 complement proteins, which are found as a complex collection of plasma proteins and membrane cofactors. Plasma proteins make up approximately 10% of the globulins in vertebrate serum. Complement components achieve their immune defense function by interacting in a complex but precise series of enzymatic cleavage and membrane binding events. The resulting complement cascade leads to the production of products with opsonic, immunoregulatory, and lytic functions.

[0035] The complement cascade progresses via either the classical or alternative pathway, which share many components and differ in their initial steps but converge on and share the same "terminal complement" components (C5-C9) that are responsible for target cell activation and destruction.

[0036] The classical complement pathway is usually initiated by antibody recognition of and binding to an antigenic site on a target cell. The alternative pathway is usually antibody-independent and can be initiated by specific molecules on the surface of pathogens. Both pathways converge at the point where complement component C3 is cleaved by an active protease (which is different in each pathway) to generate C3a and C3b. Other pathways that activate complement attack can act later in the sequence of events leading to various aspects of complement function.

[0037] C3a is an anaphylatoxin. C3b binds to bacteria and other cells, as well as certain viruses and immune complexes, and tags them for removal from the circulation. In this role, C3b is known as an opsonin. The opsonic function of C3b is considered to be the most important anti-infective action of the complement system. Patients with genetic lesions that block C3b function are susceptible to infection with a wide variety of pathogens, while patients with lesions later in the complement cascade (i.e., those with lesions that block C5 function) have been found to be more prone to, and somewhat more prone to, Neisserial infections (Fearon, 1983).

[0038] C3b also forms complexes with other components specific to each pathway to form the C5 convertase of either the classical or alternative pathway, which cleaves C5 into C5a and C5b. Therefore, C3 is considered a central protein in the complement sequence because it is essential for both the alternative and classical pathways (Wurzner et al., 1991). This property of C3b is regulated by the serum protease factor I, which acts on C3b to produce iC3b. iC3b further functions as an opsonin but is unable to form active C5 convertase.

[0039] C5 is a 190 kDa β-globulin found in normal serum at approximately 75 μg / mL (0.4 μM). C5 is glycosylated to approximately 1.5–3% of its mass with carbohydrate. Mature C5 is a heterodimer of a 115 kDa α-chain at 999 amino acids that forms a disulfide bond with a 75 kDa β-chain at 656 amino acids. C5 is synthesized as a single-chain precursor protein product of a single-copy gene (Haviland et al., 1991). The cDNA sequence of the transcript of this gene predicts a secreted pro-C5 precursor of 1659 amino acids with an 18-amino acid leader sequence.

[0040] The pro-C5 precursor is cleaved after amino acids 655 and 659, yielding the beta chain as an amino-terminal fragment (amino acid residues +1 to 655) and the alpha chain as a carboxy-terminal fragment (amino acid residues 660 to 1658), with four amino acids missing between them.

[0041] C5a is cleaved from the C5 α-chain by either the alternative or classical pathway C5 convertase as an amino-terminal fragment containing the first 74 amino acids of the α-chain (i.e., amino acid residues 660-733). Approximately 20 percent of C5a's 11 kDa mass is due to carbohydrate. The cleavage site for convertase action is at or immediately adjacent to amino acid residue 733. Compounds that bind at or adjacent to this cleavage site can block access of the C5 convertase enzyme to the cleavage site and thereby act as complement inhibitors.

[0042] C5 can also be activated by means other than C5 convertase activity: limited trypsin digestion (Minta and Man, 1977; Wetsel and Kolb, 1982) and acid treatment (Yamamoto and Gewurz, 1978; Vogt et al., 1989) can also cleave C5 to produce active C5b.

[0043] C5a is another anaphylatoxin. C5b binds with C6, C7, and C8 to form the C5b-8 complex on the surface of target cells. Upon binding of several C9 molecules, the membrane attack complex (MAC, C5b-9, terminal complement complex - TCC) is formed. When sufficient numbers of MACs insert into the target cell membrane, the openings they create (MAC pores) mediate rapid osmotic lysis of the target cell. If the non-lytic concentration of MAC becomes low, other effects can occur. In particular, intramembrane insertion of small numbers of C5b-9 complexes into endothelial cells and platelets can result in deleterious cell activation. In some cases, activation can precede cell lysis.

[0044] As mentioned above, C3a and C5a are anaphylatoxins. These activated complement components can induce mast cell degranulation, which releases histamine and other mediators of inflammation, resulting in other inflammatory phenomena such as smooth muscle contraction, increased vascular permeability, leukocyte activation, and cell proliferation resulting in cellular hyperplasia. C5a also functions as a chemotactic peptide that acts to attract proinflammatory granulocytes to the site of complement activation.

[0045] The beneficial effects of anti-C5 mAbs have already been reported in several experimental models, such as myocardial reperfusion (Vakeva et al., 1998), systemic lupus erythematosus (Wang et al., 1996), and rheumatoid arthritis (Wang et al., 1995); as well as in human clinical trials of autoimmune diseases, cardiopulmonary bypass, and acute myocardial infarction (Kirschfink, 2001).

[0046] IV. Measuring Quality of Life Various measures exist to assess quality of life and the effects of medical interventions on quality of life, such as the Mini-Mental State Examination (MMSE), the Short Test of Mental Status, the European Organization for Research and Treatment of Cancer (EORTC) Quality of Life Questionnaire, the FACIT questionnaire and subscales for fatigue and anemia, the Likert scale, and the Borg scale (Tombaugh et al., J. Am. Geriatr. Soc. 40:922, 1992; Cummings, JAMA. 269(18):2420, 1993; Crum et al., JAMA. 269(18):2386, 1993; Folstein et al., J. Psychiat. Res. 12:189, 1975; Kokmen et al., Mayo (Clin. Proc. 62:281, 1987; Tang-Wai et al., Arch. Neurol. 60:1777, 2003; Tamburini, Ann. Oncol. 12(Suppl. 3):S7, 2001; Webster et al., Health and Quality of Life Outcomes. 1:79, 2003, www.hqlo.com / content / I / I / 79; Grant et al., Chest. 116:1208, 1999; and www.qolid.org). Any of these assays can be used to assess changes in quality of life due to administration of compounds that inhibit complement or inhibit the formation of C5b-9.

[0047] In certain embodiments, the improvement in quality of life due to administration of a compound that inhibits complement or inhibits the formation of C5b-9 is measured by the Functional Assessment of Chronic Illness Therapy (FACIT) Measurement System. In certain embodiments, the improvement in quality of life is measured by a) full scale; b) independent subscale; and c) symptom index.

[0048] In certain embodiments, the improvement in quality of life due to administration of a compound that inhibits complement or inhibits the formation of C5b-9 is measured by the European Organisation for Research and Treatment of Cancer (EORTC) Quality of Life questionnaire. In certain embodiments, the EORTC questionnaire is the QLQ-C30.

[0049] In certain embodiments, the improvement in quality of life is measured by the health-adjusted life expectancy (HALE) index as described in Wilkins, R. and Adams, O.B., Am J Public Health, 73:1073-1080 (1983). Health-adjusted life expectancy is the average quality-adjusted life years (QALYs) for a particular population and can be used to evaluate the therapeutic effectiveness of medical interventions. Quality-adjusted life years are a health index that scales each life year on a scale from 1 to 0 (Weinstein M.C. and Stason W.B., N Engl J Med, 296:716-721 (1977)). Perfect health is rated 1, death is rated 0, and disability and pain are rated based on severity. QALYs are determined by multiplying the number of years of each health state.

[0050] In certain embodiments, improvement in quality of life is measured by the following instruments: years of life lost, disability-free life expectancy, health-adjusted life years, quality-adjusted life years, health equivalent years, healthy days gained, episode-free days, Q-TWiST, Health Utility Index, and years of healthy life. These measures account for both changes in mortality and changes in morbidity and disability. Any of these measures can be used to assess changes in quality of life due to administration of a compound that inhibits complement or inhibits the formation of C5b-9.

[0051] In one embodiment, the disclosed methods improve the quality of life of a patient for at least 1 day, at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month, at least 2 months, at least 3 months, at least 6 months, at least 1 year, at least 18 months, at least 2 years, at least 30 months, or at least 3 years, or the duration of treatment.

[0052] In certain embodiments, the symptoms used to measure quality of life are measured on an intensity scale. In certain embodiments, the symptoms are measured on a frequency scale. In certain embodiments, the symptoms are measured on an intensity and frequency scale.

[0053] In certain aspects, the present application provides methods for increasing health-adjusted life expectancy in a subject, comprising administering to the subject a compound that inhibits complement or inhibits the formation of C5b-9. The above assays account for both changes in mortality and changes in morbidity and disability. Any of these assays can be used to assess changes in health-adjusted life expectancy due to administration of a compound that inhibits complement or inhibits the formation of C5b-9.

[0054] In one embodiment, the disclosed method extends a subject's health-adjusted life expectancy by at least 1 day, at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month, at least 2 months, at least 3 months, at least 6 months, at least 1 year, at least 18 months, at least 2 years, at least 30 months, or at least 3 years, as measured by the health-adjusted life expectancy (HALE) index as described in Wilkins et al., Am J Public Health, 73:1073-1080 (1983). Health-adjusted life expectancy is the average quality-adjusted life years (QALYs) for a particular population and can be used to evaluate the therapeutic effectiveness of medical interventions. Quality-adjusted life years are a health index that scales each life year on a scale from 1 to 0 (Weinstein et al., N Engl J Med, 296:716-721 (1977)). Perfect health is rated 1, death is rated 0, disability and pain are rated based on severity, and QALYs are determined by multiplying the number of years of each health state.

[0055] V. Inhibitors of the Complement Cascade In certain embodiments, any compound that binds to one or more complement components or blocks the production and / or activity of one or more complement components can be used in the present methods. In certain embodiments, the complement inhibitor can be a small molecule (molecular weight up to 6,000 Da), nucleic acid or nucleic acid analog, peptidomimetic or non-nucleic acid macromolecule, serine protease inhibitor, or protein. These agents include, but are not limited to, small organic molecules, RNA aptamers (such as ARC187, commercially available from Archemix Corp., Cambridge, Massachusetts, USA), L-RNA aptamers, spiegelmers, antisense compounds, molecules that can be used in RNA interference (RNAi) (e.g., double-stranded RNA, including small interfering RNA (siRNA)), locked nucleic acid (LNA) inhibitors, and peptide nucleic acid (PNA) inhibitors.

[0056] In certain embodiments, the complement inhibitor can be a protein or protein fragment. Proteins that inhibit the complement cascade, such as CD59 and other inhibitors of CD55, CD46, C8, and C9, are known (see U.S. Patent No. 6,100,443). Proteins that bind complement, known as complement receptors, are also known (see PCT Patent Application Publication No. WO 92 / 10205 and U.S. Patent No. 6,057,131). The use of soluble forms of complement receptors (e.g., soluble CR1) can inhibit the consequences of complement activation (e.g., neutrophil oxidative destruction, complement-mediated neuronal damage, C3a and C5a production). In certain embodiments, the complement inhibitor can be a naturally occurring or soluble form of a complement inhibitory compound (e.g., CR1, LEX-CR1, MCP, DAF, CD59, Factor H, cobra venom factor, FUT-175, complestatin, K76 COOH). Those skilled in the art will recognize that the above are some, but not all, of the known methods of inhibiting complement and its activation.

[0057] In certain embodiments, the complement inhibitor can be an antibody capable of inhibiting complement (e.g., an antibody capable of blocking the formation of MAC). For example, an antibody complement inhibitor can include an antibody that binds to C5. Such an anti-C5 antibody can interact directly with C5 and / or C5b to inhibit the formation and / or physiological function of C5b.

[0058] Suitable anti-C5 antibodies are known to those skilled in the art. Antibodies can be directed to individual components of activated complement (e.g., antibodies to C7, C9, etc.) (see, e.g., U.S. Pat. No. 6,534,058; U.S. Patent Application Publication No. US2003 / 0129187; and U.S. Pat. No. 5,660,825). U.S. Pat. No. 6,355,245 teaches antibodies that bind to C5 and inhibit its cleavage into C5a and C5b, thereby reducing the formation of not only C5a but also downstream complement components.

[0059] The concentrations and / or physiological activities of C5a and C5b in body fluids can be measured by methods well known in the art. For C5a, such methods include chemotaxis assays, RIAs, or ELISAs (see, e.g., Ward and Zvaifler, J Clin Invest. 1971 Mar;50(3):606-16; Wurzner, et al., Complement Inflamm. 8:328-340, 1991). For C5b, hemolytic assays or assays for soluble C5b-9 as discussed herein can be used. Other assays known in the art can also be used. These or other suitable types of assays can be used to screen currently known or subsequently identified candidate antibodies capable of inhibiting complement (e.g., anti-C5 antibodies) to 1) identify compounds useful in the practice of the present application and 2) determine appropriate dosage levels for such compounds.

[0060] Preferably, an antibody capable of inhibiting complement (e.g., an antibody that binds to C5, which affects C5b) is used at a concentration that results in a substantial reduction (i.e., at least about 25% reduction) of C5b levels present in at least one blood-derived body fluid of the patient following complement activation in that body fluid, compared to the absence of the antibody that binds C5. Such a concentration can be conveniently determined by measuring the cytolytic capacity (e.g., hemolytic activity) of complement present in the body fluid or the level of soluble C5b-9 present in the body fluid. Thus, a particular concentration for an antibody that affects C5b is one that results in a substantial reduction (i.e., at least about 25% reduction) of the cytolytic capacity of complement present in at least one blood-derived body fluid of the patient. The reduction in the cytolytic ability of complement present in a patient's body fluid can be measured by methods well known in the art (e.g., by conventional hemolytic assays such as the hemolytic assay described in Kabat and Mayer (eds), "Experimental Immunochemistry, 2nd Edition," 135-240, Springfield, IL, CC Thomas (1961), pages 135-139, or conventional variations of that assay, such as the chicken erythrocyte hemolysis method described below).

[0061] Specific antibodies that can inhibit complement (e.g., antibodies that bind C5) are relatively specific and do not block the function of early complement components. In particular, such specific agents do not substantially impair the opsonization function associated with complement component C3b, which provides a means for removing foreign particles and substances from the body.

[0062] C3b is produced by the cleavage of C3. C3 cleavage is accomplished by C3 convertases of the classical and / or alternative pathways, resulting in the production of both C3a and C3b. Therefore, to avoid impairing the opsonization function associated with C3b, specific antibodies capable of inhibiting complement (e.g., antibodies that bind C5) do not substantially interfere with the cleavage of complement component C3 into C3a and C3b in a patient's body fluid (e.g., serum). Such interference with C3 cleavage can be detected by measuring the fluid levels of C3a and / or C3b, which are produced in equimolar ratios by the action of C3 convertase. Such measurements are beneficial because, if cleavage is interfered with by an antibody capable of inhibiting complement (e.g., an antibody that binds C5), C3a and C3b levels will be reduced (compared to matched samples that do not contain an antibody capable of inhibiting complement (e.g., an antibody that binds C5)).

[0063] In practice, quantitative measurement of such cleavage is generally more accurate when performed by measuring body fluid C3a levels rather than body fluid C3b levels, because C3a remains in the fluid phase while C3b is rapidly cleared. C3a levels in body fluids can be measured by methods well known in the art, for example, by using commercially available C3a EIA kits, such as those sold by Quidel Corporation (San Diego, California, USA), according to the manufacturer's specifications. In particular, specific antibodies capable of inhibiting complement (e.g., antibodies that bind to C5) do not essentially cause a decrease in C3a levels in body fluids after complement activation when tested in such an assay.

[0064] Certain disclosed antibodies block the cleavage of C5 to form C5a and C5b, thus blocking the generation of anaphylatoxin activity associated with C5a and the assembly of the membrane attack complex associated with C5b. As noted above, in certain embodiments, these anti-C5 antibodies do not impair the opsonization function associated with the action of C3b.

[0065] A preferred method of inhibiting complement activity is to use a monoclonal antibody that binds to and inhibits the cleavage of complement C5. This reduces the formation of both C5a and C5b while allowing the formation of C3a and C3b, which are beneficial to the recipient. Such antibodies specific for human complement are known (U.S. Patent No. 6,355,245). These antibodies disclosed in U.S. Patent No. 6,355,245 include all preferred antibodies (eculizumab being mentioned herein). A similar antibody against mouse C5 is called BB5.1 (Frei et al., Mol. Cell. Probes. 1:141-149 (1987)). Antibodies that inhibit complement activity need not be monoclonal antibodies. They can be, for example, polyclonal antibodies. They may also be antibody fragments. Antibody fragments include, but are not limited to, Fab, F(ab'), F(ab')2, single-chain antibodies, and Fv. Furthermore, it is well known to those skilled in the art that antibodies can be humanized (Jones et al., Nature 321:522-5 (1986)), chimerized, or deimmunized. The antibodies used in the present disclosure may be any of these. It is preferable to use humanized antibodies.

[0066] In certain embodiments, the disclosed therapeutic agents include antibodies or antibody fragments. Antibodies and fragments thereof can be produced by any conventional method, such as those described herein. Antibodies are found in multiple forms (e.g., IgA, IgG, IgM, etc.). Furthermore, antibodies can be engineered in numerous ways. They can be produced as single-chain antibodies (such as small modular immunopharmaceuticals or SMIPs), Fab and F(ab')2 fragments, etc. Antibodies can be humanized, chimerized, deimmunized, or fully human. Numerous publications describe the many types of antibodies and methods for engineering such antibodies. See, for example, U.S. Patent Nos. 6,355,245; 6,180,370; 5,693,762; 6,407,213; 6,548,640; 5,565,332; 5,225,539; 6,103,889; and 5,260,203.

[0067] The present invention provides fragments of anti-C5 antibodies, which may contain a portion of the intact antibody, preferably the antigen-binding or variable region of the intact antibody. Examples of antibody fragments include Fab fragments, Fab' fragments, F(ab')2 fragments, and Fv fragments; diabodies; linear antibodies (Zapata et al., Protein Eng. 8:1057-1062 (1995)); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[0068] Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, the name of which denotes the ability to crystallize readily. Pepsin digestion of antibodies produces an F(ab')2 fragment that has two antigen-binding sites and is still capable of cross-linking antigen.

[0069] "Fv" refers to the minimum antibody fragment that contains a complete antigen-recognition and antigen-binding site. This region consists of a dimer of one heavy-chain and one light-chain variable domain tightly linked by non-covalent bonds. In this structure, the three CDRs of each variable domain interact to form a V H -V L The six CDRs define an antigen-binding site on the surface of the dimer. Collectively, the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although likely with lower affinity than the entire binding site.

[0070] Fab fragments also contain the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region. Fab'-SH, as used herein, refers to Fab' in which the cysteine residues in the constant domains bear a free thiol group. F(ab')2 antibody fragments are naturally produced as pairs of Fab' fragments with hinge cysteines between them. Other chemical linkages of antibody fragments are also known.

[0071] "Single-chain Fv" or "scFv" antibody fragments are fragments of the V chain of an antibody. H Domain and V L Preferably, the Fv polypeptide further comprises a V domain, and these domains are present in a single polypeptide chain. H Domains and V L The scFv domains contain a polypeptide linker between them, which enables the scFv to form the desired structure for antigen binding. For a general overview of scFvs, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore, eds. (Springer-Verlag: New York, 1994), pp. 269-315.

[0072] SMIPs are a class of single-chain peptides engineered to contain a target-binding region (effector domains (CH2 and CH3 domains)). See U.S. Patent Application Publication No. 20050238646. The target-binding region can be derived from the variable region or CDRs of an antibody (e.g., an antibody that binds to C5 of the present application). Alternatively, the target-binding region can be derived from a protein that binds to C5.

[0073] The term "diabody" refers to a small antibody fragment with two antigen-binding sites, which are bound to the same polypeptide chain (V H -V L ) in the light chain variable domain (V L ) linked to a heavy chain variable domain (V H (The term "diabodies" refers to a diabody that contains a constant domain of a given antigen.) If a linker is used that is too short to connect the two domains in the same chain, the domains will pair with the constant domains of the other chain to form two antigen-binding sites. Diabodies are described more fully in, for example, EP 404,097; WO 93 / 11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

[0074] It is well known that the binding of antibodies with an Fc region to molecules (or pathogens) promotes the processing and elimination of the molecules (or pathogens). The Fc portion of an antibody is recognized by specific receptors expressed by immune effector cells. The Fc portions of IgG1 and IgG3 antibodies are recognized by Fc receptors present on the surface of phagocytes, such as macrophages and neutrophils, allowing these phagocytes to bind to and engulf molecules or pathogens coated with antibodies of these isotypes (C.A. Janeway et al., Immunobiology, 5th edition, page 147, Garland Publishing (New York, 2001)).

[0075] The present disclosure also provides monoclonal anti-C5 antibodies. Monoclonal antibodies are obtained from a substantially homogeneous antibody population, i.e., the individual antibodies comprising the population are homogeneous except for minor mutations that may occur naturally. Monoclonal antibodies are highly specific, acting against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations, which typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they are synthesized by a hybridoma culture uncontaminated by other immunoglobulins. Monoclonal antibodies can also be produced in transfected cells, such as CHO cells or NS0 cells. The modifier "monoclonal" indicates the character of the antibody as obtained from a substantially homogeneous antibody population and does not require that the antibody be produced by any particular method. For example, the monoclonal antibodies used in this disclosure may be made by the hybridoma method first described by Kohler et al., Nature 256:495-497 (1975), or may be made by recombinant DNA methods (see, e.g., U.S. Pat. Nos. 4,816,567 and 6,331,415). The "monoclonal antibodies" may also be isolated from phage antibody libraries using, for example, the techniques described in Clackson et al., Nature 352:624-628 (1991) and Marks et al., J. Mol. Biol. 222:581-597 (1991).

[0076] A description of mouse anti-human C5 monoclonal antibody preparations with specific binding properties is given in U.S. Patent Application Publication No. 20050226870. Wurzner et al., Complement Inflamm. 8:328-340 (1991) describe other mouse anti-human C5 monoclonal antibody preparations designated N19-8 and N20-9.

[0077] Another specifically contemplated antibody is an "oligoclonal" antibody. As used herein, the term "oligoclonal antibody" refers to a predetermined mixture of different monoclonal antibodies. See PCT International Publication No. WO 95 / 20401; U.S. Patent Nos. 5,789,208 and 6,335,163. In one embodiment, oligoclonal antibodies, consisting of a predetermined mixture of antibodies directed against one or more epitopes, are produced within a single cell. In other embodiments, oligoclonal antibodies contain multiple heavy chains that can pair with a common light chain to produce antibodies with multiple specificities (e.g., PCT International Publication No. WO 04 / 009618). Oligoclonal antibodies are particularly useful when multiple epitopes of a target on a single target molecule (e.g., C5) are required. In light of the assays and epitopes disclosed herein, one skilled in the art can generate or select an antibody or mixture of antibodies applicable to the intended use and desired needs.

[0078] In certain embodiments, including humanized and / or chimeric antibodies, one or more of the CDRs are derived from an anti-human C5 antibody. In certain embodiments, all of the CDRs are derived from an anti-human C5 antibody. In another specific embodiment, CDRs from two or more anti-human C5 antibodies are mixed and matched in a chimeric antibody. For example, a chimeric antibody can include CDR1 from the light chain of a first anti-human C5 antibody combined with CDR2 and CDR3 from the light chain of a second anti-human C5 antibody, and the CDRs from the heavy chain can be derived from a third anti-human C5 antibody. Furthermore, the framework regions can be derived from one of the same anti-human C5 antibodies or from one or more different antibodies, such as a human antibody or a humanized antibody. Human or humanized antibodies are specific for administration to human patients.

[0079] Also included in this disclosure as antigen-binding fragments of antibodies are, in certain embodiments, single-chain antibodies, chimeric antibodies, humanized antibodies, or primatized (CDR-grafted) antibodies, as well as chimeric or CDR-grafted single-chain antibodies, which contain portions derived from different species. The various portions of these antibodies can be chemically coupled by conventional techniques or prepared as a contiguous protein using genetic engineering techniques. For example, nucleic acids encoding chimeric or humanized chains can be expressed to produce a contiguous protein. See, e.g., U.S. Pat. Nos. 4,816,567 and 6,331,415; U.S. Pat. No. 4,816,397; European Patent No. 0120694; International Publication No. WO 86 / 01533; European Patent No. 0194276(B1); U.S. Pat. No. 5,225,539; and European Patent No. 0239400(B1). See also, Newman et al., BioTechnology 10:1455-1460 (1992) regarding primatized antibodies. For example, regarding single chain antibodies, see Ladner et al., U.S. Patent No. 4,946,778; and Bird et al., Science, 242:423-426 (1988).

[0080] Furthermore, functional fragments of antibodies, including fragments of chimeric, humanized, primatized, or single-chain antibodies, can also be produced. The subject functional fragments of antibodies retain at least one binding and / or regulatory function of the full-length antibody from which they are derived. Preferred functional fragments retain the antigen-binding function of the corresponding full-length antibody (e.g., the ability of an antibody to bind C5).

[0081] General methods for immunizing animals (in this case, with C5 and / or C5b, etc.), isolating antibody-producing cells, fusing such cells with immortalized cells (e.g., myeloma cells) to produce hybridomas that secrete monoclonal antibodies, screening hybridoma supernatants for reactivity of the secreted monoclonal antibodies with a desired antigen (in this case, an immunogen, or a molecule that contains an immunogen), preparing large quantities of such antibodies in hybridoma supernatants or ascites fluid, and purifying and storing such monoclonal antibodies can be found in numerous publications, including: Coligan et al., eds. Current Protocols In Immunology, John Wiley & Sons, New York, 1992; Harlow and Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1994; Laboratory,New York,1988;Liddell and Cryer,A Practical Guide To Monoclonal Antibodies,John Wiley & Sons,Chichester,West Sussex, England, 1991; Montz et al., Cellular Immunol. 127:337-351 (1990); Wurzner et al., Complement Inflamm. 8:328-340 (1991); and Mollnes et al., Scand. J. Immunol. 28:307-312 (1988).

[0082] VI. Treatment Methods The methods of the present application can be used to treat symptoms associated with paroxysmal nocturnal hemoglobinuria. The methods of the present application can be used to treat symptoms associated with anemia. Treatment for paroxysmal nocturnal hemoglobinuria and / or anemia can be administered by standard means. The treatments of the present application can be used in combination with other treatments of the present application or known treatments for paroxysmal nocturnal hemoglobinuria and / or anemia. The treatments of the present application can be administered simultaneously with other treatments that treat symptoms of paroxysmal nocturnal hemoglobinuria and / or anemia.

[0083] VII. Pharmaceutical Formulations and Uses Methods for administering small molecules, proteins, and nucleic acids are well known to those skilled in the art. Methods for administering antibodies are well known to those skilled in the art. Antibodies can be administered in various unit dosage forms to achieve the desired inhibition. Dosages vary depending on the specific antibody. For example, different antibodies can have different masses and / or affinities, thus requiring different dosage levels. Antibodies prepared as Fab fragments also require different dosages than equivalent intact immunoglobulins because they are significantly smaller in mass than intact immunoglobulins, and therefore require smaller dosages to achieve the same molar levels in the patient's blood. Dosages will also vary depending on the mode of administration, the specific condition of the patient being treated, the patient's overall health, condition, size, and age, and the judgment of the prescribing physician. Dosage levels of antibodies for human subjects are generally about 1 mg to about 100 mg per kg per administration per patient, preferably about 5 mg to about 50 mg per kg per administration per patient. In terms of plasma concentration, antibody concentrations are preferably in the range of about 25 μg / mL to about 500 μg / mL, although larger amounts may be required in extreme cases, and smaller amounts may be sufficient in milder cases.

[0084] In certain embodiments, the pharmaceutical composition is a single unit dosage form. In certain embodiments, the single unit dosage form is a 300 mg unit dosage form. In certain embodiments, the pharmaceutical composition is lyophilized. In certain embodiments, the pharmaceutical composition is a sterile solution. In certain embodiments, the pharmaceutical composition is a preservative-free formulation. In certain embodiments, the pharmaceutical composition comprises a 300 mg single-use formulation in 30 mL of a 10 mg / mL sterile, preservative-free solution. In certain embodiments, the antibody is administered according to the following protocol: 600 mg intravenous infusion over 25-45 minutes every 7±2 days for the first 4 weeks, followed by a fifth dose of 900 mg 7±2 days later, and then 900 mg every 14±2 days thereafter. The antibody is administered by intravenous infusion over 25-45 minutes.

[0085] Generally, administration of anti-C5 antibodies is via an intravenous route (e.g., intravenous infusion by injection). Other routes of administration can be used if desired, but the intravenous route is most preferred. Suitable formulations for injection can be found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, Pa., 17th ed. (1985). Such formulations must be sterile and nonpyrogenic and generally contain a pharmaceutically effective carrier (e.g., saline, buffered (e.g., phosphate-buffered) saline, Hank's solution, Ringer's solution, dextrose / saline, glucose solution, etc.). The formulation may optionally contain pharmaceutically acceptable adjuvants (e.g., isotonicity agents, wetting agents, bactericides, preservatives, stabilizers, etc.). In a specific embodiment, a complement inhibitor such as eculizumab can be administered via intravenous injection and diluted to a final concentration of 5 mg / mL prior to administration.

[0086] Generally, administration of antibodies capable of inhibiting complement (e.g., antibodies that bind C5) is carried out parenterally, usually by injection, such as intraarticular, intravascular (e.g., intravenous), or intramuscular. Other routes of administration, such as oral (po), can be used if desired and may be practiced for the particular complement-inhibiting antibody being administered. Antibodies capable of inhibiting complement (e.g., antibodies that bind C5) can also be administered in various unit dosage forms, with the dosage varying depending on the size, potency, and in vivo half-life of the particular complement-inhibiting antibody being administered. The dosage of antibodies capable of inhibiting complement (e.g., antibodies that bind C5) will vary depending on the mode of administration, the particular condition of the patient being treated, the patient's overall health, condition, size, and age, and the judgment of the prescribing physician.

[0087] In certain embodiments, typical therapeutic administration involves a series of doses typically administered with monitoring of clinical endpoints, with dose levels adjusted as needed to achieve the desired clinical outcome. In certain embodiments, treatment is administered in multiple doses over at least one week. In certain embodiments, treatment is administered in multiple doses over at least one month. In certain embodiments, treatment is administered in multiple doses over at least one year. In certain embodiments, treatment is administered in multiple doses for the remaining lifespan of the patient.

[0088] The frequency of administration can also be adjusted based on various parameters, including clinical efficacy, plasma half-life of the therapeutic agent of the present disclosure, and antibody levels in bodily fluids (e.g., blood, plasma, serum, synovial fluid). The levels of the therapeutic agent of the present disclosure in bodily fluids can be monitored during the course of treatment to guide adjustments to the frequency of administration.

[0089] In certain embodiments, the frequency of administration can be adjusted by an assay that measures the cytolytic potential of complement present in one or more of the patient's body fluids. The cytolytic potential can be measured as percent hemolysis in a hemolytic assay of the type described herein. A 10%, 25%, or 50% reduction in the cytolytic potential of complement present in a body fluid after administration of an antibody capable of inhibiting complement used in the practice of this application means that the percent hemolysis after administration is 90, 75, or 50%, respectively, of the percent hemolysis before administration.

[0090] For the treatment of hemolytic diseases such as PNH by systemic administration (as opposed to local administration) of antibodies capable of inhibiting complement (e.g., antibodies that bind C5), it is specific to administer a large initial dose, i.e., a single initial dose sufficient to produce a substantial reduction, more preferably at least about a 50% reduction, in the patient's serum hemolytic activity. Preferably, such a large initial dose is administered before periodic repeated administration of decreasing doses necessary to maintain a substantial reduction in serum hemolytic titer. In another embodiment, the initial dose is given by both local and systemic routes, followed by repeated systemic administration of decreasing doses as described above.

[0091] Formulations particularly useful for antibody-based therapeutics are also described in U.S. Patent Application Publication Nos. 20030202972, 20040091490, and 20050158316. In certain embodiments, the liquid formulations of the present application are substantially free of surfactants and / or inorganic salts. In another specific embodiment, the liquid formulation has a pH ranging from about 5.0 to about 7.0. In yet another specific embodiment, the liquid formulation comprises histidine at a concentration ranging from about 1 mM to about 100 mM. In yet another specific embodiment, the liquid formulation comprises histidine at a concentration ranging from 1 mM to 100 mM. It is also contemplated that the liquid formulation can further comprise one or more excipients, such as a saccharide, an amino acid (e.g., arginine, lysine, methionine), or a polyol. Further descriptions and methods of preparing and characterizing liquid formulations can be found, for example, in PCT Publication Nos. WO03 / 106644, WO04 / 066957, and WO04 / 091658.

[0092] Wetting agents, emulsifying agents and lubricating agents (e.g., sodium lauryl sulfate and magnesium stearate), as well as coloring agents, release agents, coating agents, sweetening agents, flavoring agents, perfuming agents, preservatives and antioxidants can also be present in the pharmaceutical compositions of the present application.

[0093] In certain embodiments, the subject antibody formulations are pyrogen-free, meaning they are substantially free of endotoxins and / or related pyrogens. Endotoxins include toxins trapped inside microorganisms and released upon the breakdown or death of the microorganisms. Pyrogens also include heat-resistant pyrogens (glycoproteins) from the outer membranes of bacteria and other microorganisms. Both of these substances can cause fever, hypotension, and shock when administered to humans. Due to the potential adverse effects, it is advantageous to remove even small amounts of endotoxin from intravenously administered pharmaceutical solutions. The Food & Drug Administration ("FDA") has set a limit of 5 endotoxin units (EU) per kilogram of body weight per administration during a single hourly period for intravenous drug use (The United States Pharmacopeial Convention, Pharmacopeial Forum 26(1):223 (2000)). When therapeutic proteins are administered in amounts of hundreds or thousands of milligrams per kilogram of body weight, as may be the case with monoclonal antibodies, it is advantageous to remove even trace amounts of endotoxin.

[0094] Formulations of the subject antibodies include those suitable for oral, alimentary, topical, parenteral (e.g., intravenous, intraarterial, intramuscular, or subcutaneous) administration, ophthalmic (e.g., topical or intraocular), inhalation (e.g., intrabronchial, intranasal, oral, or nasal drops), rectal, and / or intravaginal administration. Other suitable methods of administration can also include rechargeable or biodegradable devices and slow-release polymer devices. In particular, stents can be coated with slow-release polymers mixed with the agents of the present application. The pharmaceutical compositions of the present disclosure can also be administered as part of a combination therapy with other agents (either in the same formulation or in separate formulations).

[0095] The therapeutically effective amount of a formulation can be determined by standard clinical techniques. Additionally, in vitro assays can optionally be used to help identify optimal dosage ranges. The precise dosage to be employed in a formulation will also depend on the route of administration and the severity of the disease or disorder, and must be decided according to the judgment of the practitioner and each patient's circumstances. Effective amounts can be extrapolated from dose-response curves derived from in vitro or animal model test systems. The dosage of a composition to be administered can be determined by one of ordinary skill in the art without undue experimentation, using standard dose-response studies. Relevant circumstances to consider in this determination include the condition(s) being treated, the choice of composition to be administered, the age, weight, and response of the individual patient, and the severity of the patient's symptoms. For example, the actual patient weight can be used to calculate the dosage in milliliters (mL) of the formulation to be administered. Downward adjustment to "ideal" body weight cannot be made. In such circumstances, the appropriate dosage can be calculated using the following formula: dosage (mL) = [patient weight (kg) × dosage (mg / kg) / drug concentration (mg / mL)].

[0096] To achieve the desired therapeutic result, anti-C5 antibodies can be administered in various unit dosage forms. The dosage varies depending on the specific antibody. For example, different antibodies can have different masses and / or affinities, and therefore require different dosage levels. Antibodies prepared as Fab' fragments or single-chain antibodies also require different dosages than the equivalent native immunoglobulins because they have much smaller masses than native immunoglobulins, and therefore require smaller dosages to reach the same molar levels in the patient's blood.

[0097] Other therapeutic agents of the present disclosure may also be administered in a variety of unit dosage forms, the dosage also varying depending on the size, potency and in vivo half-life of the particular therapeutic agent to be administered.

[0098] The dosage of the therapeutic agents of the disclosure will also vary depending on the mode of administration, the particular condition of the patient being treated, the patient's overall health, condition, size and age, and the judgment of the prescribing physician.

[0099] The formulations of the present application can be distributed as a product comprising a pharmaceutical product and packaging materials containing an antibody capable of inhibiting complement and a pharmaceutically acceptable carrier, appropriate for the mode of administration. The packaging materials can include a label indicating that the formulation is for treating a hemolytic disease such as PNH. While antibodies, particularly anti-C5 antibodies that have been shown to be safe and effective in reducing the accumulation of downstream complement components in humans, are preferred, the use of other complement inhibitors is also contemplated by the present disclosure. The pharmaceutical formulations and uses of the present disclosure can be used in conjunction with any known complement inhibitor or hemolytic disease treatment known in the art.

[0100] In certain aspects, the present application provides kits comprising the pharmaceutical compositions of the present application. In some embodiments, the kits further comprise at least one component of a closed sterile system. Components of a closed sterile system include, but are not limited to, needles, syringes, catheter-type syringes, needle-type injection devices, needleless injection devices, filters, tubing, valves, and cannulas. In related embodiments, the kits include components for removing preservatives from the composition. Such components include filters, syringes, vials, containers, tubing, and the like. [Example]

[0101] method Patient Selection The TRIUMPH trial consisted of a 2-week screening period, an observation period of up to 3 months, and a 26-week treatment period.

[0102] During the screening period, patients were assessed for inclusion and exclusion criteria. Subjects were men and women aged 18 years or older, diagnosed with PNH with a type III red blood cell population of 10% or greater, and who had received at least four blood transfusions in the past 12 months. Concomitant use of erythropoietin, immunosuppressants, corticosteroids, Coumadin, low-molecular-weight heparin, iron supplements, and folic acid was not considered a reason for exclusion, as long as the dosage was consistent before the first visit and throughout the study period. Because of the increased frequency of Neisserial infections in individuals with genetic deficiencies in terminal complement proteins, all patients received Neisseria meningitides vaccination. Patients used contraception. The study protocol was approved by the institutional review board at each study site, and written informed consent was obtained from all enrolled patients.

[0103] Patients transfused with a mean pretransfusion hemoglobin level greater than 10.5 g / dL over the past 12 months and those with evidence of a suppressed immune response, complement deficiency, or active bacterial infection (including any history of meningococcal disease) were excluded from the study. Patients were also excluded if they had previously undergone a bone marrow transplant, participated in another clinical trial, or received another investigational drug within 30 days of their first visit. An individualized transfusion algorithm was calculated for each patient based on their transfusion history over the past 12 months. A written algorithm recorded the units of packed red blood cells (PRBCs) transfused for a given hemoglobin level and served as a predetermined guide for transfusions during the observation and treatment periods.

[0104] Each eligible patient participated in a maximum 13-week observation period to confirm PBRC transfusion dependence. According to the transfusion algorithm provided for each patient, at least one transfusion during the 13-week observation period with a hemoglobin level of 9 g / dL or less if symptomatic or 7 g / dL or less, referred to as an "eligible" transfusion, was required to proceed to randomization. The hemoglobin level at which each individual's eligible transfusion was administered was defined as that individual's hemoglobin "target value" for the primary efficacy variable. A platelet count of 100,000 cells / mL or greater and an LDH level 1.5 times the upper limit of the normal range were also required, either at screening for eligibility or during the observation period.

[0105] Study design Patients were randomized (1:1) to receive either placebo or eculizumab (Soliris®, Alexion Pharmaceuticals, Inc.) within 10 days of eligible transfusion. Study drug was administered, maintaining blinding, as follows: 600 mg of eculizumab for patients randomized to active drug or placebo for patients randomized to placebo was administered intravenously every 7 ± 1 days for four doses; 7 ± 1 days later, 900 mg of eculizumab or placebo, respectively, was administered intravenously; thereafter, maintenance doses of 900 mg of eculizumab or placebo, respectively, were administered intravenously every 14 ± 2 days for a total of 26 weeks of administration.

[0106] Clinical efficacy measures The study had two common primary endpoints: (1) hemoglobin stabilization, defined as hemoglobin levels maintained above an individual transfusion-free hemoglobin target for the entire 26-week treatment period, and (2) a reduction in units of PRBCs transfused during the 26-week treatment phase of the study. Compared with treatment prior to study entry, triggers for transfusion remained unchanged for each patient during the study. Patients received transfusions when they had symptoms resulting from anemia and reached their individual predefined "target." Prespecified secondary endpoints included transfusion avoidance, hemolysis measured by area under the LDH curve from baseline to week 26, and the Functional Assessment of Chronic Illness Therapy-Fatigue (FACIT-Fatigue) instrument. 13 Prespecified exploratory analyses included changes in QoL measured from baseline to week 26 using the EORTC QLQ-C30 instrument. 14 The study included assessment of PNH type III blood cell counts, change in LDH from baseline to week 26, and thrombosis. Other prespecified measures included the pharmacokinetics, pharmacodynamics, and immunogenicity of eculizumab. Time to first transfusion during the 26-week treatment phase and percentage of PNH type III blood cells were also assessed.

[0107] Safety evaluation Treatment-emergent adverse events, clinical laboratory tests (e.g., serum chemistry and complete blood count), electrocardiogram data, and vital signs were assessed. Adverse events were defined using MedDRA preferred terms and tabulated as incidence rates per treatment group.

[0108] statistical analysis For the co-primary endpoint, analyses were performed by intention to treat using data from all patients who were randomized and received study drug. Hemoglobin stabilization was analyzed using Fisher's exact test, and total PRBC units transfused were analyzed using the Wilcoxon rank-sum test. To compare the effect of treatment on transfusion avoidance, Fisher's exact test was used for incidence rates and the log-rank test for time to first transfusion. The Wilcoxon rank-sum test was used for area under the LDH blood concentration curve.

[0109] For measuring fatigue quality of life, the FACIT-Fatigue instrument 15 The quality of life measure based on the EORTC QLQ-C30 instrument was assessed using the appropriate scoring guidelines. 16 Analysis was performed according to the guidelines. Changes in FACIT-Fatigue and EORTC QLQ-C30 scores from baseline to week 26 were analyzed using mixed models with baseline as a covariate, treatment and time as fixed effects, and patient as a random effect. Changes in LDH levels and PNH type III red blood cells from baseline to week 26 were analyzed using the same mixed models. All analyses used two-sided tests. Adverse events and the long-term safety checklist were tabulated separately, and comparisons between treatments were performed using Fisher's exact test. A p value of ≤0.05 was considered statistically significant.

[0110] result Patient characteristics A total of 115 patients with PNH were screened. Six patients did not meet the inclusion / exclusion criteria during the screening period. Twenty-one other patients did not receive eligible transfusions and were not randomized to the treatment phase. One patient who did not meet the inclusion criteria was inadvertently randomized but did not receive the study drug. Thus, 87 patients with hemolytic PNH (35 men and 52 women) were included and randomized to receive either eculizumab (N = 43) or placebo (N = 44), exceeding the original goal of randomizing 75 patients.

[0111] Patient characteristics were similar in the eculizumab and placebo cohorts (median age: 41 years (range: 20-85 years) and 35 years (range: 18-78 years); median duration of PNH: 4.2 years (range: 0.8-29.7 years) and 9.2 years (range: 0.4-38.3 years); patients with a history of aplastic anemia: 4 and 11; history of myelodysplastic syndrome: 1 and 0; and history of thrombosis: 9 (16 events) and 8 (11 events)). Stable use of concomitant medications at baseline in the eculizumab and placebo groups included the following: erythropoietin (3 patients and 0 patients); cyclosporine (1 and 1 patient); anticoagulants (coumarin or heparin) (21 and 11 patients); and steroids (glucocorticoids or androgenic steroids) (12 and 12 patients), respectively.

[0112] Of the 87 randomized patients, 85 completed the study. Two patients who did not complete the study were randomized to the eculizumab group; one patient withdrew due to travel difficulties to the study site, and the other became pregnant. All 10 patients in the placebo group discontinued the infusion due to lack of efficacy but remained in the study for monitoring purposes.

[0113] Pharmacokinetics / Pharmacodynamics In 42 of the 43 eculizumab-treated patients, drug levels during the maintenance period (900 mg every 2 weeks ± 2 days) were sufficient to completely block serum hemolytic activity (mean trough level of 101.8 μg / mL at week 26). One patient did not sustain therapeutic trough levels of eculizumab and showed changes in complement blockade during the last few days of each dosing interval. These changes were clinically manageable and resolved rapidly after the next dose.

[0114] Hemolytic efficacy variables The effect of terminal complement inhibition by eculizumab on chronic intravascular hemolysis in patients with PNH was demonstrated in this study by an immediate (1-week) and sustained decrease in mean LDH levels (Figure 1A). The median LDH area under the curve decreased by 85.8% in patients treated with eculizumab compared with patients treated with placebo over the 26-week study period (p<0.001). In patients treated with eculizumab, mean LDH levels decreased from 2199.7±157.7 IU / L at baseline to 327.3±67.6 IU / L by week 26, whereas levels in patients treated with placebo remained consistently elevated, from 2259.0±158.5 IU / L at baseline to 2418.9±140.3 IU / L at week 26 (p<0.001 for eculizumab vs. placebo). Additionally, a second biochemical measure of hemolysis (serum aspartate aminotransferase (AST)) showed a statistically significant improvement after eculizumab administration compared with placebo (data not shown). Haptoglobin levels were statistically significantly increased in eculizumab-treated patients compared with placebo-treated patients, but mean haptoglobin levels were further below normal in eculizumab-treated patients (data not shown).

[0115] Figure 1A shows the extent of intravascular hemolysis in patients with PNH, as indicated by the mean lactate dehydrogenase (LDH) levels (± standard error) from baseline (the first week of treatment) to week 26 for both eculizumab-treated and placebo-treated patients. Screening occurred up to 3 months prior to the first week of treatment. The upper limit of the normal range for LDH (103–223 IU / L) is indicated by the dashed line. For eculizumab-treated patients, LDH decreased to mean levels just above the upper limit of normal at week 26, and 15 of the 41 patients who completed the study had LDH levels within the normal range. All placebo-treated patients remained at least 5-fold above the upper limit of normal at week 26. p values are based on mixed-model analyses from baseline to week 26. Figure 1B shows the mean percentage of PNH type III red blood cells (± standard error) assessed for placebo-treated and eculizumab-treated patients. The screening visit occurred up to 3 months before the first week of treatment. p values are based on mixed model analysis from baseline to week 26.

[0116] A corollary of the reduction in intravascular hemolysis during eculizumab treatment was the observed increase in the PNH type III red blood cell population (Figure IB). For eculizumab-treated patients, the mean percentage of type III red blood cells increased from 28.1 ± 2.0% at baseline to 56.9 ± 3.6% by week 26, whereas the percentage in the placebo group remained constant from a mean of 35.7 ± 2.8% before treatment to 35.5 ± 2.8% at week 26 (p < 0.001 for eculizumab vs. placebo). In contrast, the percentage of PNH type III granulocytes and monocytes did not change significantly between treatment groups during treatment and remained above 90% at week 26.

[0117] Clinical efficacy Common primary endpoint The co-primary endpoints of the TRIUMPH trial were stabilization of hemoglobin levels and reduction in units of transfused PRBCs. At the end of the treatment period, 48.8% of patients receiving eculizumab maintained hemoglobin levels above the prespecified target (median target for both treatment groups was 7.7 g / dL) in the absence of transfusions, whereas hemoglobin stabilization did not occur in any patients in the placebo group (p<0.001; Table 1). By week 26, the median units of transfused PRBCs per patient were 0 in the eculizumab group and 10.0 in the placebo cohort (p<0.001), whereas the mean units of transfused PRBCs were 3.0 and 11.0 in the eculizumab and placebo cohorts, respectively. During the 6-month period of the study, the median units of PRBCs transfused per patient was 9.0 in the eculizumab cohort and 8.5 in placebo patients, while the mean units of PRBCs transfused were 9.6 ± 0.6 and 9.7 ± 0.7, respectively. Mean hemoglobin levels at baseline were similar between treatment groups (10.0 ± 1.8 g / dL for eculizumab-treated patients and 9.7 ± 1.8 g / dL for placebo-treated patients) and remained virtually unchanged by week 26 (10.1 ± 2.5 g / dL and 8.9 ± 2.2 g / dL for the eculizumab and placebo cohorts, respectively).

[0118] The median time to first transfusion was not reached in patients receiving eculizumab during the study period (>26 weeks), whereas the placebo group reached the median time to first transfusion in only 4 weeks (p<0.001; Figure 2). Transfusion avoidance was achieved in 51.2% and 0% of the eculizumab and placebo cohorts, respectively (p<0.001). By the end of the 26-week treatment period, the total number of PRBC units transfused was 131 in eculizumab-treated patients and 482 in the placebo group (Table 1). In contrast, during the 6-month period prior to the study, the total number of PRBC units transfused in the eculizumab and placebo cohorts was 413 and 417, respectively.

[0119] (Table 1) Stabilization of hemoglobin levels and reduction in transfusion requirements during eculizumab administration.

[0120] [Table 1] Improving quality of life measurements Quality of life assessment of PNH patients receiving eculizumab was performed using two different instruments (FACIT-Fatigue and EORTC QLQ-C30). Patients receiving eculizumab demonstrated a mean increase (improvement) in FACIT-Fatigue scores of 6.4 ± 1.2 points from baseline to week 26, whereas placebo patients' mean scores decreased by 4.0 ± 1.7 points, for an overall difference of 10.4 points between treatment groups (Figure 3). A mixed covariance model analysis demonstrated statistically significant differences between treatment groups (p < 0.001).

[0121] For the EORTC instruments, improvements with eculizumab were observed in each subscale. Compared with the placebo group, statistically significant improvements were observed in the following quality of life subscales with eculizumab (Table 2): global health (p<0.001), physical function (p<0.001), emotional function (p=0.008), cognitive function (p=0.002), role function (p<0.001), social function (p<0.003), fatigue (p<0.001), pain (p<0.002), dyspnea (p<0.001), loss of appetite (p<0.001), and insomnia (p=0.0014). Improvements with eculizumab in other scales (including nausea and vomiting, diarrhea, constipation, and financial difficulties) did not reach statistical significance.

[0122] (Table 2) Effect of eculizumab administration on quality of life assessed by EORTC QLQ-C30 instrument

[0123] [Table 2] Relationship Between FACIT-Fatigue Quality of Life and Intravascular Hemolysis To determine whether there was a treatment-independent relationship between the FACIT-Fatigue quality of life instrument and intravascular hemolysis, an analysis was performed whereby the mean LDH level (throughout the 26-week study period) for each TRIUMPH patient was analyzed as a function of the patient's mean percent change from baseline (throughout the 26-week study period) in their respective FACIT-Fatigue scores (see Table 3). For this analysis, mean LDH levels were categorized into four groups: normal levels, 1-2 times the upper limit of normal (ULN), 2-10 times the upper limit of normal, and >10 times the upper limit of normal. This analysis showed that patients who maintained normal LDH levels throughout the study had significantly more improvement in fatigue than patients whose LDH levels increased more significantly throughout the study (p=0.0048). These data established a clear relationship between increased intravascular hemolysis, as measured by LDH levels, and decreased quality of life, as measured by the FACIT-Fatigue Quality of Life Instrument.

[0124] (Table 3) Relationship between FACIT-Fatigue and intravascular hemolysis

[0125] [Table 3] safety There were no deaths during the study. Serious adverse events were reported in 13 patients, four in the eculizumab cohort and nine in the placebo cohort (see Table 4). All patients recovered without sequelae.

[0126] The most commonly reported AEs in patients receiving eculizumab were headache, nasopharyngitis, back pain, and upper respiratory tract infection. Headache and back pain occurred more frequently in the eculizumab group than in the placebo group. However, the increase in headache was limited to the first 2 weeks of treatment and was mild to moderate in severity. There were no statistically significant differences in incidence between treatment groups for any of the reported AEs.

[0127] One episode of thrombosis (Budd-Chiari) occurred in a patient receiving placebo. No thrombosis occurred in patients receiving eculizumab.

[0128] In the eculizumab-treated cohort, only one patient developed detectable levels of anti-eculizumab antibodies, but this response was weak (not titrated), occurred at only one time point, and did not result in disruption of complement blockade.

[0129] (Table 4 Adverse Event Reports)

[0130] [Table 4] Consideration Chronic intravascular hemolysis with periods of acute exacerbations is a classic manifestation of PNH, resulting in anemia, the need for transfusions to maintain hemoglobin levels, and a worsening quality of life. In this pivotal phase III trial (TRIUMPH), we investigated the effect of terminal complement inhibition with eculizumab on hemoglobin levels and transfusion requirements in patients with PNH. Over a 6-month period, 49 percent of patients receiving eculizumab achieved hemoglobin stabilization without transfusions, compared with none in the placebo group. Compared to no patients in the placebo group, over 50% of patients receiving eculizumab remained transfusion-independent throughout the entire study, resulting in a 73% reduction in overall mean transfusion rates. Furthermore, even in patients who did not achieve transfusion independence, eculizumab administration was associated with a 44% reduction in transfusion rates (data presented here).

[0131] Lactate dehydrogenase (a biochemical marker of hemolysis in PNH) 9 LDH levels decreased immediately and consistently in all eculizumab-treated patients, whereas patients in the placebo cohort continued to experience hemolysis at the end of the study, with all patients experiencing LDH levels exceeding five times the upper limit of the normal range. LDH levels decreased to the normal range in approximately one-third of eculizumab-treated patients, while the remainder stabilized at levels just above the upper limit of normal, suggesting low residual hemolysis in some patients. Haptoglobin levels (a more sensitive marker for the presence of cell-free hemoglobin in the circulation) were undetectable in the majority of patients. The low levels of hemolysis in a subset of eculizumab-treated patients are likely due to an intrinsic decrease in survival of these cells or C3b-mediated extravascular clearance of PNH red blood cells by the reticuloendothelial system. 17 .

[0132] Before eculizumab administration, the hemoglobin levels of study patients were artificially maintained by frequent blood transfusions. Therefore, stabilization of hemoglobin levels, accompanied by the cessation or reduction of transfusions, represents a net increase in endogenous hemoglobin levels. Our data suggest that the elimination of hemolysis by eculizumab results in a new steady-state hemoglobin level determined by the balance between the degree of underlying bone marrow dysfunction, the number of PNH red blood cells maintained by eculizumab treatment, and the new level (if any) of transfusion requirements.

[0133] Generally, patients with PNH experience a significantly impaired quality of life characterized by fatigue, anemia, thrombosis and pulmonary hypertension, and smooth muscle dystonia, including abdominal pain, dysphagia, and erectile dysfunction. 9、10、19These symptoms were attributed to both excessive intravascular hemolysis and downstream scavenging of nitric oxide by cell-free hemoglobin in plasma. The reduction in intravascular hemolysis in eculizumab-treated patients in this study was associated with a significantly greater improvement in the fatigue component of quality of life, as assessed by the FACIT-Fatigue instrument, compared with placebo-treated patients. Furthermore, eculizumab administration was associated with a median increase of 6.4 points above the baseline value established before treatment. A 3-point or greater increase from baseline has previously been shown to represent a clinically important difference on this quality of life instrument. 19 Additionally, patients receiving eculizumab experienced significant improvements in most domains of the EORTC QLQ-30 (including global health status, physical function, emotional function, cognitive function, role function, social function, fatigue, pain, dyspnea, loss of appetite, and insomnia) compared with the placebo-treated cohort. The improvement in the fatigue component of the EORTC QLQ-30 supports the improvement noted in the FACIT-Fatigue instrument during eculizumab treatment. Importantly, these improvements in quality of life for eculizumab-treated patients occurred despite similar red blood cell hemoglobin levels in the two treatment groups, which in itself further supports the contribution of hemolysis, as opposed to anemia, in mediating the decline in quality of life in patients with PNH. Clinical assessment of additional PNH quality-of-life symptoms (e.g., abdominal pain, dysphagia, and erectile dysfunction) has also been reported to improve during eculizumab treatment. 20 .

[0134] Eculizumab was safe and well tolerated. There were no deaths during the study, and only one thrombotic event occurred in a placebo patient at a site typical of thrombosis in PNH (the hepatic vein). The relatively short duration of this study was insufficient to address the question of possible thrombosis prevention by terminal complement inhibition with eculizumab.

[0135] Adverse events were generally mild, with a high incidence of headache in eculizumab-treated patients, but this incidence did not persist after the first two doses of treatment. Four SAEs occurred in the eculizumab group and nine in the placebo group. There was no evidence of an increased risk of infection in eculizumab-treated patients during the study. One eculizumab-treated patient demonstrated low levels of anti-eculizumab antibodies at some point during the study, but these low levels did not persist and did not result in disruption of complement blockade. No AEs were associated with discontinuation of eculizumab in the two eculizumab-treated patients who did not complete the study. Further safety and efficacy assessments are being investigated in an ongoing, multicenter, open-label, phase 3 safety study of eculizumab (SHEPHERD) in approximately 95 patients with PNH.

[0136] The results of this double-blind, randomized, placebo-controlled, international study demonstrate that terminal complement inhibition with eculizumab appears to be a safe and effective treatment for patients with the rare disease PNH. Eculizumab administration reduced intravascular hemolysis and stabilized hemoglobin levels to the point where most PNH patients became transfusion-independent, despite the need for transfusions. Substantial and clinically meaningful improvements in fatigue and other key quality-of-life parameters were also observed. All 85 patients who completed the study elected to receive eculizumab in an open-label extension study, and all are currently continuing the drug. The results of the TRIUMPH trial demonstrate that terminal complement inhibition with eculizumab safely and effectively addresses an important consequence of the underlying genetic defect in PNH hematopoietic stem cells by providing a therapeutic alternative to the deficiency of terminal complement inhibitors.

[0137] The present invention provides, inter alia, for the administration of inhibitors of complement. Many variations of the present invention will become apparent to those skilled in the art upon review of this specification. The full scope of the invention should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations.

[0138] All publications and patents mentioned herein, including those listed below, are herein fully incorporated by reference to the same extent as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In case of conflict, the present specification, including all definitions, will control.

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Claims

[Claim 1] The invention described in the present specification.

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