Methods for reducing the mitogenicity of lectin compositions

• US 20070110811A1
• Filed: 10/19/2006
• Published: 05/17/2007
• Est. Priority Date: 10/19/2005
• Status: Active Grant

• Current Assignee: SmartCells, Inc. (Merck & Co., Inc.)
• Original Assignee: SmartCells, Inc. (Merck & Co., Inc.)
• Inventors: Cai, Wenyi, Pecukonis, Matthew, Faucette, Ryan, Zion, Todd, Lancaster, Thomas
• Granted Patent: US 7,687,608 B2
• Time in Patent Office: Days
• US Class Current: 424/486
• CPC Class Codes: A61K 38/25 Growth hormone-releasing factor [GH-RF], i.e. somatoliberin
• CPC Class Codes: A61K 38/26 Glucagons
• CPC Class Codes: A61K 38/28 Insulins
• CPC Class Codes: A61K 47/60 the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
• CPC Class Codes: A61K 47/61 the organic macromolecular compound being a polysaccharide or a derivative thereof
• CPC Class Codes: A61K 47/6903 the form being semi-solid, e.g. an ointment, a gel, a hydrogel or a solidifying gel
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: C08B 30/18 Dextrin , e.g. yellow canari, white dextrin, amylodextrin or maltodextrin; Methods of depolymerisation, e.g. by irradiation or mechanically
• CPC Class Codes: C08B 37/00 Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof cellulose D21; microbiological processes C12P
• CPC Class Codes: C08B 37/003 Chitin, i.e. 2-acetamido-2-deoxy-(beta-1,4)-D-glucan or N-acetyl-beta-1,4-D-glucosamine; Chitosan, i.e. deacetylated product of chitin or (beta-1,4)-D-glucosamine; Derivatives thereof
• CPC Class Codes: C08B 37/0069 Chondroitin-4-sulfate, i.e. chondroitin sulfate A; Dermatan sulfate, i.e. chondroitin sulfate B or beta-heparin; Chondroitin-6-sulfate, i.e. chondroitin sulfate C; Derivatives thereof
• CPC Class Codes: C08B 37/0072 Hyaluronic acid, i.e. HA or hyaluronan; Derivatives thereof, e.g. crosslinked hyaluronic acid (hylan) or hyaluronates
• CPC Class Codes: C08B 37/0084 Guluromannuronans, e.g. alginic acid, i.e. D-mannuronic acid and D-guluronic acid units linked with alternating alpha- and beta-1,4-glycosidic bonds; Derivatives thereof, e.g. alginates
• CPC Class Codes: Y10S 436/827 Lectins

Abstract

Methods for reducing the T-cell mitogenicity of lectin compositions are provided. In one aspect this is achieved by chemically modifying mitogenic lectin compositions under optimized conditions. Additionally or alternatively, the reduction in T-cell mitogenicity is achieved by removing unmodified subunits chemically modified mixtures. Modified lectin compositions with reduced T-cell mitogenicity are also provided as are uses of the inventive compositions.

Claims

1. A method comprising steps of:

providing a chemically modified lectin composition that includes a mixture of modified and unmodified lectin subunits; and

removing at least a portion of the unmodified lectin subunits from the chemically modified lectin composition, wherein the T-cell mitogenicity of the chemically modified lectin composition is reduced as a result of the step of removing.

2. The method of claim 1 further comprising the steps of:

providing a mitogenic lectin composition that includes a plurality of mitogenic lectins, wherein the mitogenic lectins are each comprised of one or more lectin subunits; and

reacting the mitogenic lectin composition with one or more chemical compounds to produce the chemically modified lectin composition, wherein the one or more chemical compounds react with at least some of the lectin subunits to produce modified lectin subunits.

3. The method of claim 2, wherein the T-cell mitogenicity of the chemically modified lectin composition is reduced as compared to the mitogenic lectin composition.

4. The method of claim 2, wherein in the step of reacting, the mitogenic lectin composition is reacted with a monovalent chemical compound.

5. The method of claim 2, wherein in the step of reacting, the mitogenic lectin composition is reacted with a multivalent chemical compound that covalently crosslinks native lectin subunits.

6. The method of claim 5, wherein in the step of reacting, the mitogenic lectin composition is further reacted with a monovalent chemical compound.

7. The method of claim 2, wherein the mitogenic lectin composition includes a mitogenic lectin that is multimeric and comprised of identical subunits in its native form.

8. The method of claim 7, wherein the mitogenic lectin is tetrameric in its native form and the mitogenic lectin composition comprises monomers, dimers, trimers, or tetramers of the mitogenic lectin, or any mixture thereof.

9. The method of claim 2, wherein the mitogenic lectin composition includes a mitogenic lectin selected from the group consisting of Concanavalin A, Pisum sativum agglutinin, Lens culinaris lectin, Vicia faba lectin, Glycine max lectin, Wheat germ agglutinin, and combinations thereof.

10. The method of claim 9, wherein the mitogenic lectin composition includes Concanavalin A.

11. The method of claim 2, wherein the one or more chemical compounds react with a group on a lectin subunit selected from the group consisting of amines, carboxyls, hydroxyls, and sulfhydryls.

12. The method of claim 11, wherein said group was introduced into the lectin subunit by site-directed mutagenesis.

13. The method of claim 11, wherein said group is an amine.

14. The method of claim 12, wherein said group is an amine.

15. The method of claim 2, wherein the one or more chemical compounds react with at least some of the lectin subunits to form a covalent linkage selected from the group consisting of amide bonds, carbamate bonds, ester bonds, thioether bonds, ether bonds, and disulfide bonds.

16. The method of claim 15, wherein said covalent linkage is an amide bond.

17. The method of claim 4 or 6, wherein the monovalent chemical compound is a polymer.

18. The method of claim 17, wherein the polymer is a pegylation agent.

19. The method of claim 18, wherein the molecular weight of the pegylation agent is greater than 1,000 daltons.

20. The method of claim 18, wherein the molecular weight of the pegylation agent is greater than 2,000 daltons.

21. The method of claim 18, wherein the molecular weight of the pegylation agent is greater than 5,000 daltons.

22. The method of claim 18, wherein the molecular weight of the pegylation agent is greater than 10,000 daltons.

23. The method of claim 18, wherein the pegylation agent is selected from the group consisting of N-hydroxysuccinimide activated PEG, succinimidyl ester of PEG propionic acid, succinimidyl ester of PEG butanoic acid, and succinimidyl ester of PEG alpha-methylbutanoate.

24. The method of claim 17, wherein the polymer is chemically activated dextran.

25. The method of claim 17, wherein the polymer is a polyvinyl alcohol.

26. The method of claim 5, wherein the multivalent chemical compound is ethylene glycol-bis(succinic acid-N-hydroxy-succinimide ester).

27. The method of claim 2, wherein the step of reacting is performed at a temperature in the range of 10 to 36°

C.

28. The method of claim 27, wherein the step of reacting is performed at a temperature of 23 to 27°

C.

29. The method of claim 2, wherein the step of reacting is performed at a pH of less than 11.

30. The method of claim 29, wherein the step of reacting is performed at a pH of less than 10.

31. The method of claim 29, wherein the step of reacting is performed at a pH in the range of 8.5 to 10.

32. The method of claim 29, wherein the step of reacting is performed at a pH of less than 8.5.

33. The method of claim 1, wherein in the step of removing, at least a portion of the unmodified lectin subunits are isolated from the remainder of the chemically modified lectin composition based on their differential electrical charges.

34. The method of claim 33, wherein at least a portion of the unmodified lectin subunits are isolated from the remainder of the chemically modified lectin composition by ion-exchange chromatography.

35. The method of claim 1, wherein in the step of removing, at least a portion of the unmodified lectin subunits are isolated from the remainder of the chemically modified lectin composition based on their differential molecular weights.

36. The method of claim 35, wherein at least a portion of the unmodified lectin subunits are isolated from the remainder of the chemically modified lectin composition by gel-filtration chromatography.

37. The method of claim 1, wherein in the step of removing, at least a portion of the unmodified lectin subunits are isolated from the remainder of the chemically modified lectin composition based on their differential binding affinities for a ligand.

38. The method of claim 37, wherein at least a portion of the unmodified lectin subunits are isolated from the remainder of the chemically modified lectin composition by affinity chromatography.

39. The method of claim 38, wherein the affinity chromatography is performed with an affinity chromatography resin comprising a carbohydrate that preferentially binds the unmodified lectin subunits over the modified lectin subunits.

40. The method of claim 39, wherein the carbohydrate is dextran.

41. The method of claim 38, wherein the affinity chromatography is performed with an affinity chromatography resin comprising a carbohydrate that preferentially binds the modified lectin subunits over the unmodified lectin subunits.

42. The method of claim 41, wherein the carbohydrate includes mannose.

43. The method of claim 1, wherein in the step of removing, substantially all of the unmodified lectin subunits are removed.

44. A composition prepared according to the method of claim 1.

45-47. -47. (canceled)

48. A composition comprising a plurality of modified lectins, wherein each modified lectin is comprised of one or more subunits that have been modified by reaction with a chemical compound, wherein said composition is substantially free of unmodified subunits, and wherein the T-cell mitogenicity of said composition is reduced as compared to an otherwise equivalent composition that comprises a plurality of unmodified lectins.

49. The composition of claim 48, wherein the one or more subunits have been modified by reaction with a monovalent chemical compound.

50. The composition of claim 48, wherein the one or more subunits have been crosslinked by reaction with a multivalent chemical compound.

51. The composition of claim 50, wherein the crosslinked subunits have been further modified by reaction with a monovalent chemical compound.

52. The composition of claim 48, wherein the composition includes a modified lectin that is multimeric and comprised of identical subunits in its native form.

53. The composition of claim 52, wherein the composition includes a modified lectin that is tetrameric in its native form.

54. The composition of claim 48, wherein the composition includes modified Concanavalin A, modified Pisum sativum agglutinin, modified Lens culinaris lectin, modified Vicia faba lectin, modified Glycine max lectin, modified Wheat germ agglutinin, or a combination thereof.

55. The composition of claim 54, wherein the composition includes modified Concanavalin A.

56. The composition of claim 55, wherein the chemical compound reacted with a group on a lectin subunit selected from the group consisting of amines, carboxyls, hydroxyls, and sulfhydryls.

57. The composition of claim 56, wherein said group was introduced into the lectin subunit by site-directed mutagenesis.

58. The composition of claim 56 or 57, wherein said group is an amine.

59. The composition of claim 48, wherein the chemical compound reacted with at least some of the lectin subunits to form a covalent linkage selected from the group consisting of amide bonds, carbamate bonds, ester bonds, thioether bonds, ether bonds, and disulfide bonds.

60. The composition of claim 59, wherein said covalent linkage is an amide bond.

61. The composition of claim 49 or 51, wherein the monovalent chemical compound is a polymer.

62. The composition of claim 61, wherein the polymer is a pegylation agent.

63. The composition of claim 62, wherein the molecular weight of the pegylation agent is greater than 1,000 daltons.

64. The composition of claim 62, wherein the molecular weight of the pegylation agent is greater than 2,000 daltons.

65. The composition of claim 62, wherein the molecular weight of the pegylation agent is greater than 5,000 daltons.

66. The composition of claim 62, wherein the molecular weight of the pegylation agent is greater than 10,000 daltons.

67. The composition of claim 62, wherein the pegylation agent is selected from the group consisting of N-hydroxysuccinimide activated PEG, succinimidyl ester of PEG propionic acid, succinimidyl ester of PEG butanoic acid, and succinimidyl ester of PEG alpha-methylbutanoate.

69. The composition of claim 61, wherein the polymer is chemically activated dextran.

70. The composition of claim 61, wherein the polymer is a polyvinyl alcohol.

71. The composition of claim 50, wherein the multivalent chemical compound is ethylene glycol-bis(succinic acid-N-hydroxy-succinimide ester).

72. The composition of claim 48, wherein fewer than 50% of the subunits in the composition are unmodified as determined by denaturing gel electrophoresis.

73. The composition of claim 48, wherein fewer than 25% of the subunits in the composition are unmodified as determined by denaturing gel electrophoresis.

74. The composition of claim 48, wherein fewer than 10% of the subunits in the composition are unmodified as determined by denaturing gel electrophoresis.

75. The composition of claim 48, wherein the T-cell mitogenicity of said composition is less than 50% the T-cell mitogenicity of an otherwise equivalent composition that comprises a plurality of unmodified lectins.

76. The composition of claim 75, wherein the T-cell mitogenicity is measured by a radioactive thymidine uptake assay using peripheral blood mononuclear cells (PBMCs) from one or more healthy human patients.

77. The composition of claim 76, wherein the ratio of the maximum CPM of said composition to the maximum CPM of the composition of unmodified lectins is less than 50% in the thymidine uptake assay when assayed at lectin concentrations of 0.01, 0.1, 1, 10, 100 and 1000 ug/ml at a PBMC concentration of 500,000 cells/ml.

78. The composition of claim 77, wherein the ratio of the maximum CPM of said composition to the maximum CPM of the composition of unmodified lectins is less than 25%.

79. The composition of claim 77, wherein the ratio of the maximum CPM of said composition to the maximum CPM of the composition of unmodified lectins is less than 10%.

80. The composition of claim 48, wherein said composition exhibits a binding affinity for a cognate saccharide ligand that is substantially equal to or greater than an otherwise equivalent composition that comprises a plurality of unmodified lectins.

81. The composition of claim 48, wherein said composition binds detectably with a cognate polysaccharide ligand.

82. The composition of claim 80 or 81, wherein the binding affinity is determined by isothermal titration microcalorimtery.

83. The composition of claim 80, wherein said composition is incapable of precipitating glycogen comprised entirely of glucose units.

84. The composition of claim 80, wherein said composition is incapable of precipitating glycogen comprised entirely of glucose units under conditions that would cause an otherwise equivalent composition that comprises a plurality of unmodified lectins to precipitate.

85-120. -120. (canceled)