Claims
- 1. A Neutrokine-alpha conjugate having the formula NA-(Chel)n, wherein
NA is a Neutrokine-alpha protein; Chel is said chelator; and n is an integer from 1 to about 30; wherein said Neutrokine-alpha protein comprises an amino acid sequence selected from the group consisting of: (a) the amino acid sequence of amino acid residues 134 to 285 of SEQ ID NO:2; (b) the amino acid sequence of amino acid residues n to 285 of SEQ ID NO:2, where n is an integer in the range of 2-190; (c) the amino acid sequence of amino acid residues 1 to m of SEQ ID NO:2, where m is an integer in the range of 274-284; (d) the amino acid sequence of amino acid residues n to m of SEQ ID NO:2, where n is an integer in the range of 2-190 and m is an integer in the range of 274-284; and (e) an amino acid sequence which has at least 80% identity to any of the proteins described in (a), (b), (c), and (d); wherein said Neutrokine-alpha protein binds a Neutrokine-alpha receptor.
- 2. The conjugate according to claim 1, wherein said Neutrokine-alpha protein comprises an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of amino acid residues n to 285 of SEQ ID NO:2, where n is an integer in the range of 2-190; (b) the amino acid sequence of amino acid residues 1 to m of SEQ ID NO:2, where m is an integer in the range of 274-284; and (c) the amino acid sequence of amino acid residues n to m of SEQ ID NO:2, where n is an integer in the range of 2-190 and m is an integer in the range of 274-284; wherein said Neutrokine-alpha protein binds a Neutrokine-alpha receptor.
- 3. The conjugate of claim 1, wherein n is 1, 2, 3, 4, 5, or 6.
- 4. The conjugate according to claim 3, wherein n is 3.
- 5. The conjugate according to claim 3, wherein n is 1.
- 6. The conjugate according to claim 4, wherein said Neutrokine-alpha protein is a mature, soluble Neutrokine-alpha protein.
- 7. The conjugate according to claim 6, wherein said protein comprises a sequence that is at least 85% identical to amino acids 134-285 of SEQ ID NO:2.
- 8. The conjugate according to claim 7, wherein said Neutrokine-alpha consists of a trimer of Neutrokine-alpha monomeric subunits and wherein each subunit consists of amino acids 134-285 of SEQ ID NO:2.
- 9. The conjugate according to claim 8, wherein at least one or more lysine residues or N-terminal alanine residues of said Neutrokine-alpha protein forms the covalent bond with said chelator.
- 10. The conjugate according to claim 9, wherein at least one or more N-terminal alanine residues of said Neutrokine-alpha protein forms the covalent bond with said chelator.
- 11. The conjugate according to claim 1, wherein said chelator is DOTA, a DOTA derivative, or a DOTA analogue, optionally containing a linker moiety.
- 12. The conjugate according to claim 11, having the formula
- 13. The conjugate according to claim 12, having the formula
- 14. The conjugate according to claim 11, wherein NA is a human, mature, soluble Neutrokine-alpha protein.
- 15. The conjugate according to claim 14, wherein said NA has a sequence that is at least 85% identical to amino acids 134-285 of SEQ ID NO:2.
- 16. The conjugate according to claim 15, wherein said Neutrokine-alpha consists of a trimer of Neutrokine-alpha monomeric subunits and wherein each subunit consists of amino acids 134-285 of SEQ ID NO:2.
- 17. The conjugate according to claim 16, wherein at least one or more lysine residues or N-terminal alanine residues of said Neutrokine-alpha protein forms the covalent bond with (Chel)n.
- 18. The conjugate according to claim 17, wherein at least one or more N-terminal alanine residues of said Neutrokine-alpha protein forms the covalent bond with (Chel)n.
- 19. The conjugate according to claim 18, wherein n is 1.
- 20. A Neutrokine-alpha conjugate having the formula:
- 21. A Neutrokine-alpha complex comprising a Neutrokine-alpha conjugate and a metal ion wherein said metal ion is associated with the chelator moiety of said Neutrokine-alpha conjugate.
- 22. The complex of claim 21, wherein said Neutrokine-alpha conjugate has the formula NA-(Chel)n, wherein
NA is a Neutrokine-alpha protein; Chel is said chelator; and n is an integer from 1 to about 10; wherein said Neutrokine-alpha protein comprises an amino acid sequence selected from the group consisting of: (a) the amino acid sequence of amino acid residues n to 285 of SEQ ID NO:2, where n is an integer in the range of 2-190; (b) the amino acid sequence of amino acid residues 1 to m of SEQ ID NO:2, where m is an integer in the range of 274-284; (c) the amino acid sequence of amino acid residues n to m of SEQ ID NO:2, where n is an integer in the range of 2-190 and m is an integer in the range of 274-284; and (d) an amino acid sequence which has at least 80% identity to any of the proteins described in (a), (b), and (c); wherein said Neutrokine-alpha protein binds a Neutrokine-alpha receptor.
- 23. The complex according to claim 22 wherein said Neutrokine-alpha protein comprises an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of amino acid residues n to 285 of SEQ ID NO:2, where n is an integer in the range of 2-190; (b) the amino acid sequence of amino acid residues 1 to m of SEQ ID NO:2, where m is an integer in the range of 274-284; and (c) the amino acid sequence of amino acid residues n to m of SEQ ID NO:2, where n is an integer in the range of 2-190 and m is an integer in the range of 274-284; wherein said Neutrokine-alpha protein binds a Neutrokine-alpha receptor.
- 24. The complex of claim 22, wherein n is 1, 2, 3, 4, 5, or 6.
- 25. The complex according to claim 24, wherein n is 3.
- 26. The complex according to claim 25, wherein n is 1.
- 27. The complex according to claim 25, wherein said Neutrokine-alpha protein is a mature, soluble Neutrokine-alpha protein.
- 28. The complex according to claim 27, wherein said Neutrokine-alpha protein has a sequence that is at least 85% identical to amino acids 134-285 of SEQ ID NO:2.
- 29. The complex according to claim 28, wherein said Neutrokine-alpha consists of amino acids 134-285 of SEQ ID NO:2.
- 30. The complex according to claim 29, wherein at least one or more lysine residues or N-terminal alanine residues of said Neutrokine-alpha protein forms the covalent bond with said chelator.
- 31. The complex according to claim 30, wherein at least one or more N-terminal alanine residues of said Neutrokine-alpha protein forms the covalent bond with said chelator.
- 32. The complex according to claim 22, wherein the chelator is DOTA, a DOTA derivative, or a DOTA analog, optionally containing a linker moiety.
- 33. The complex according to claim 32, wherein the conjugate has the formula
- 34. The complex according to claim 33, wherein the conjugate has the formula
- 35. The complex according to claim 34, wherein NA is a human, mature, soluble Neutrokine-alpha protein.
- 36. The complex according to claim 35, wherein said NA has a sequence that is at least 85% identical to amino acids 134-285 of SEQ ID NO:2.
- 37. The complex according to claim 36, wherein said Neutrokine-alpha consists of a trimer of Neutrokine-alpha monomeric subunits and wherein each subunit consists of amino acids 134-285 of SEQ ID NO:2.
- 38. The complex according to claim 37, wherein at least one or more lysine residues or N-terminal alanine residues of said Neutrokine-alpha protein forms the covalent bond with said chelator.
- 39. The complex according to claim 38, wherein at least one or more N-terminal alanine residues of said Neutrokine-alpha protein forms the covalent bond with said chelator.
- 40. A Neutrokine-alpha complex comprising a Neutrokine-alpha conjugate and a metal ion wherein said metal ion is associated with the chelator moiety of said Neutrokine-alpha conjugate, wherein said conjugate has the formula
- 41. The complex according to claim 40, wherein said metal ion is selected from the group consisting of 90Y, 111In, 177Lu, 166Ho, 215Bi, and 225Ac.
- 42. The complex according to claim 41, wherein said metal ion is 90 Y.
- 43. The complex according to claim 41, wherein said metal ion is 111In.
- 44. The complex according to claim 41, wherein said metal ion is 177Lu.
- 45. The complex according to claim 41, wherein said metal ion is 166Ho.
- 46. The complex according to claim 41, wherein said metal ion is 215Bi.
- 47. The complex according to claim 41, wherein said metal ion is 225Ac.
- 48. A composition comprising a Neutrokine-alpha conjugate according to claim 1 and a suitable carrier.
- 49. A composition comprising a Neutrokine-alpha conjugate according to claim 20 and a pharmaceutically acceptable carrier.
- 50. The composition according to claim 49, wherein said carrier is sterile water.
- 51. The composition according to claim 50 further comprising a buffer.
- 52. The composition according to claim 51 wherein said buffer is an acetate buffer having a concentration of from about 10 mM to about 200 mM.
- 53. The composition according to claim 49 further comprising a metal ion selected from the group consisting of 90Y, 111In, 177Lu, 166Ho, 215Bi, and 225Ac.
- 54. A composition comprising a Neutrokine-alpha complex according to claim 22 and a suitable carrier.
- 55. A composition comprising a Neutrokine-alpha complex according to claim 40 and a pharmaceutically acceptable carrier.
- 56. The composition according to claim 55 wherein said carrier is sterile water.
- 57. The composition according to claim 56 further comprising a buffer.
- 58. The composition according to claim 57 wherein said buffer is an acetate buffer having a concentration of from about 10 mM to about 200 mM.
- 59. A method of preparing the conjugate of claim 1 comprising reacting a Neutrokine-alpha protein with a chelator.
- 60. The method according to claim 59, wherein said chelator is an activated chelator.
- 61. The method according to claim 60, wherein said activated chelator is a DOTA derivative or a DOTA analogue.
- 62. The method according to claim 61, wherein said chelator has the formula activated chelator having the formula:
- 63. The method according to claim 62, wherein said activated chelator is α-(5-isothiocyanato-2-methoxyphenyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid.
- 64. The method according to claim 63, wherein the Neutrokine-alpha protein is a human, mature, soluble Neutrokine-alpha protein.
- 65. The method according to claim 64, wherein the Neutrokine-alpha protein has a sequence that is at least 85% identical to amino acids 134-285 of SEQ ID NO:2.
- 66. The method according to claim 65, wherein said Neutrokine-alpha consists of a trimer of Neutrokine-alpha monomeric subunits and wherein each subunit consists of amino acids 134-285 of SEQ ID NO:2.
- 67. The method according to claim 66, wherein at least one or more lysine residues or N-terminal alanine residues of said Neutrokine-alpha protein forms the covalent bond with the chelator.
- 68. The conjugate according to claim 67, wherein at least one or more N-terminal alanine residues of said Neutrokine-alpha protein forms the covalent bond with said chelator.
- 69. The method according to claim 68, comprising mixing, agitating, or preparing a solution comprising said Neutrokine-alpha protein and said chelator at a temperature of about 0° C. to about 50° C. for about 0.5 hours to about 10 hours, wherein said solution has a pH of about 8.0 to about 9.0.
- 70. The method according to claim 69, wherein said mixing, agitating, or preparing occurs at a temperature of about 20° C. to about 30° C. for about 3 hours to about 5 hours.
- 71. The method according to claim 70, further comprising adding a quenching agent after from about 3 hours to about 5 hours.
- 72. The method according to claim 71, wherein said pH is about 8.5.
- 73. The method according to claim 72, wherein the molar ratio of chelator to chelator bonding sites in the Neutrokine-alpha protein is from about 10:1 to about 12:1.
- 74. The method according to claim 73, wherein said quenching agent is glycine or glycine hydrochloride.
- 75. The method according to claim 74, wherein said solution further comprises citrate buffer and HEPES.
- 76. The method according to claim 75, wherein the concentration of said Neutrokine-alpha protein is from about 1.5 to about 3.0 mg/mL, and wherein the molar ratio of said chelator to chelator bonding sites in the Neutrokine-alpha protein is from about 10:1 to about 12:1.
- 77. The method according to claim 76, further comprising purifying said Neutrokine-alpha conjugate.
- 78. The method according to claim 77, wherein said purifying comprises a diafiltration method.
- 79. A method of preparing a Neutrokine complex according to claim 22, comprising mixing, agitating, or preparing a solution comprising a Neutrokine-alpha conjugate and a metal ion capable of complexing with a Neutrokine-alpha conjugate;
wherein said Neutrokine-alpha conjugate having the formula NA-(Chel)n, wherein (a) NA is a Neutrokine-alpha protein; (b) Chel is said chelator; and (c) n is an integer from 1 to about 10; wherein said Neutrokine-alpha protein comprises an amino acid sequence selected from the group consisting of: (a) the amino acid sequence of amino acid residues n to 285 of SEQ ID NO:2, where n is an integer in the range of 2-190; (b) the amino acid sequence of amino acid residues 1 to m of SEQ ID NO:2, where m is an integer in the range of 274-284; (c) the amino acid sequence of amino acid residues n to m of SEQ ID NO:2, where n is an integer in the range of 2-190 and m is an integer in the range of 274-284; and (d) an amino acid sequence which has at least 80% identity to any of the proteins described in (a), (b), and (c); wherein said Neutrokine-alpha protein binds a Neutrokine-alpha receptor
- 80. A method of preparing the complex according to claim 40, comprising mixing, agitating, or preparing a solution comprising a Neutrokine-alpha conjugate and a metal ion capable of associating with a Neutrokine-alpha conjugate;
wherein said conjugate has the formula 14or a pharmaceutically acceptable salt thereof, wherein NA is a Neutrokine-alpha protein that consists of a trimer of Neutrokine-alpha monomeric subunits and wherein each subunit consists of amino acids 134-285 of SEQ ID NO:2; n1 is 1; N′ is a nitrogen from the amino terminus or from a lysine residue of said Neutrokine-alpha protein.
- 81. The method according to claim 80, further comprising removing excess metal ion.
- 82. The method according to claim 81, wherein said removing comprises adding a chelating agent selected from the group consisting of DPTA, EDTA, and MeO-DOTA-glycine.
- 83. The method according to claim 80, wherein said solution further comprises an acetate buffer having a concentration of from about 1 mM to about 20 mM and NaCl having a concentration of about 100 mM to about 200 mM.
- 84. The method according to claim 80, wherein the metal ion is selected from the group consisting of 90Y, 111In, 177Lu, 166Ho, 25Bi, and 225Ac.
- 85. The method according to claim 81, wherein the solution further comprises an acetate buffer having a concentration of from about 1 mM to about 20 mM; and
further comprising allowing the solution to mix, agitate, or stand for from about 5 minutes to about 60 minutes at a temperature from about 20° C. to about 30° C.; and adding a second solution, said second solution comprising an acetate buffer having a concentration of about 10 mM, NaCl having a concentration of about 140 mM, HSA having a concentration of about 7.5%, and DPTA having a concentration of about 1 mM, wherein said second solution has a pH of about 6.
- 86. A method of administering radiotherapy to a subject in need thereof, comprising administering to said subject an effective amount of a Neutrokine-alpha complex according to claim 22.
- 87. A method of administering radiotherapy to a subject in need thereof, comprising administering to said subject an effective amount of a Neutrokine-alpha complex according to claim 40.
- 88. The method of claim 87, wherein said Neutrokine-alpha complex is administered as an injectable solution.
- 89. The method according to claim 88, wherein said solution is administered intravenously.
- 90. The method according to claim 87, wherein a dosage of radioactivity from about 5 mCi to about 200 mCi is administered.
- 91. The method of claim 90, wherein said subject is a human.
- 92. The method of claim 90, wherein said subject has a B-cell mediated disease.
- 93. The method of claim 90, wherein said subject has a condition selected from the group consisting of non-Hodgkin's lymphoma, chronic lymphocytic leukemia, multiple myeloma, systemic lupus erythrematosus, rheumatoid arthritis, multiple sclerosis, Crohn's disease, diabetes, Wegener's granulomatous, myasthenia gravis, and asthma.
- 94. The method of claim 93, wherein said subject has non-Hodgkin's lymphoma.
- 95. A method of treating cancer comprising administering to a subject with cancer, an effective amount of a Neutrokine-alpha complex according to claim 40.
- 96. The method of claim 95 wherein a cell of said cancer expresses a Neutrokine-alpha receptor on its surface.
- 97. The method of claim 95 wherein said cancer is a B cell cancer.
- 98. The method of claim 97 wherein said B cell cancer is selected from the group consisting of:
(a) Non-Hodgkin's Lymphoma; (b) Multiple Myeloma; and (c) Chronic Lymphocytic Leukemia.
- 99. A method of treating an autoimmune disease or disorder comprising administering to a subject with an autoimmune disease or disorder, an effective amount of a Neutrokine-alpha complex according to claim 40.
- 100. The method of claim 99 wherein said autoimmune disease or disorder is selected from the group consisting of:
(a) Systemic Lupus Erythematosus; (b) Rheumatoid Arthritis; and (c) Sjögren's Syndrome.
- 101. A method of killing a cell selected from the group consisting of:
(a) a cell bearing a Neutrokine-alpha receptor; and (b) a cell in close proximity to a cell bearing Neutrokine-alpha receptors; wherein said method comprises contacting said cell with a composition according to claim 22 in an amount effective to kill a said cell.
- 102. The method of claim 101 wherein said cell is (a).
- 103. The method of claim 101 wherein said cell is (b).
- 104. The method of claim 101 wherein said cell is a lymphocyte.
- 105. The method of claim 104 wherein said cell is B cell.
- 106. The method of claim 101 wherein said cell is cancerous cell that has metastasized into the lymphatic system.
- 107. A method of diagnostic imaging, comprising administering a Neutrokine-alpha complex according to claim 22.
- 108. A kit comprising a first vial containing a Neutrokine-alpha conjugate.
- 109. The kit according to claim 108, wherein
said first vial contains a Neutrokine-alpha conjugate in a solution of acetate buffer (10 mM sodium acetate, 140 mM sodium chloride, pH 6.0) said conjugate having the formula 15or a pharmaceutically acceptable salt thereof, wherein NA is a Neutrokine-alpha protein that consists of a trimer of Neutrokine-alpha monomeric subunits wherein each subunit consists of amino acids 134-285 of SEQ ID NO:2; n1 is 1, 2, 3, 4, 5 or 6; and N′ is a nitrogen from the amino terminus or from a lysine residue of said Neutrokine-alpha protein.
- 110. The kit of claim 108 comprising
(a) a second vial containing a radionuclide; and (b) a third vial containing a buffer solution.
- 111. The kit according to claim 110 which further comprises a fourth vial wherein said vial is empty.
- 112. The kit according to claim 110, wherein said first vial contains a Neutrokine-alpha conjugate in a solution of acetate buffer (10 mM sodium acetate, 140 mM sodium chloride, pH 6.0) said conjugate having the formula
- 113. The kit of claim 108 comprising
(c) a second vial containing buffer solution; and (d) a third vial containing a diluent.
- 114. The kit according to claim 113 which further comprises a fourth vial wherein said vial is empty.
- 115. The kit according to claim 113, wherein said first vial contains a Neutrokine-alpha conjugate in a solution comprising acetate buffer having a concentration of from about 1 mM to about 20 mM and NaCl having a concentration of about 100 mM to about 200 mM and having a pH of about 6, said conjugate having the formula
- 116. The kit according to claim 115, wherein said solution in said first vial comprises 10 mM sodium acetate, 140 mM NaCl.
- 117. The kit according to claim 115, wherein said buffer solution in said second vial comprises 220 mM sodium acetate and 75 mM sodium bicarbonate.
- 118. The kit according to claim 115, wherein said diluent in said third vial comprises 140 mM NaCl, 2 mM DPTA and 10% sodium ascorbate.
- 119. The method according to claim 80, further comprising adding to the solution a second solution to form a mixture, said second solution comprising about 220 mM sodium acetate and about 75 mM sodium bicarbonate and allowing the mixture of the solution containing the Neutrokine-alpha conjugate, the metal ion, and the second solution to mix, agitate, or stand for about 5 minutes to about 60 minutes at a temperature from about 20° C. to about 30° C.
- 120. The method according to claim 113, further comprising adding, after the mixture has mixed, agitated, or stood, a third solution comprising about 140 mM NaCl, about 2 mM DPTA, and about 10% sodium ascorbate.
- 121. A composition comprising a Neutrokine-alpha conjugate according to claim 20 and a suitable carrier.
- 122. A composition comprising a Neutrokine-alpha complex according to claim 40 and a suitable carrier.
- 123. A method of diagnostic imaging, comprising administering a Neutrokine-alpha complex according to claim 40.
Parent Case Info
[0001] This application claims the benefit of U.S. Provisional Application No. 60/467,198, filed May 2, 2003, and U.S. Provisional Application No. 60/435,262, filed Dec. 23, 2002.
Provisional Applications (2)
|
Number |
Date |
Country |
|
60435262 |
Dec 2002 |
US |
|
60467198 |
May 2003 |
US |