Chemically defined non-polymeric valency platform molecules and conjugates thereof

• US 7,351,855 B2
• Filed: 07/30/2003
• Issued: 04/01/2008
• Est. Priority Date: 07/15/1992
• Status: Expired due to Fees

• Current Assignee: La Jolla Pharmaceutical Company (Innoviva, Inc.)
• Original Assignee: La Jolla Pharmaceutical Company (Innoviva, Inc.)
• Inventors: Yu, Lin, Livingston, Douglas Alan, Coutts, Stephen M., Jones, David S.
• Primary Examiner(s): Lukton; David
• Application Number:
  US10/631,388
• Publication Number:
  US 20050026856A1
• Time in Patent Office: 1,707 Days
• Field of Search: 514/529, 514/44, 530/345, 530/326, 530/327, 530/328, 530/329, 554/103, 562/400, 562/450
• US Class Current: 562/400
• CPC Class Codes: A61K 2039/6093 Synthetic polymers, e.g. polyethyleneglycol [PEG], Polymers or copolymers of (D) glutamate and (D) lysine
• CPC Class Codes: A61K 39/0008 Antigens related to auto-immune diseases; Preparations to induce self-tolerance
• CPC Class Codes: A61K 39/35 Allergens
• CPC Class Codes: A61K 47/54 the modifying agent being an organic compound

Abstract

Chemically-defined, non-polymeric valency platform molecules and conjugates comprising chemically-defined valency platform molecules and biological or chemical molecules including polynucleotide duplexes of at least 20 base pairs that have significant binding activity for human lupus anti-dsDNA autoantibodies.

Claims

1. A conjugate formable by the conjugation of at least one biologically active molecule and a valency platform having the formula:

Q₁-(CH₂CH₂O)_nCH₂CH₂-Q₁

wherein each Q₁ is a chemical moiety independently selected from the group consisting of;

2. A conjugate according to claim 1, wherein each Q₁ is:

3. A conjugate according to claim 1, wherein each Q₁ is:

4. A conjugate according to any one of claims 1, 2 or 3, wherein each Q₃ is a chemical moiety of the formula:

5. A conjugate according to any one of claims 1, 2 or 3, wherein each Q₃ is a chemical moiety of the formula:

6. A conjugate according to claim 1, 2 or 3, wherein each T is independently selected from the group consisting of:

7. A conjugate according to claims 1, 2 or 3, wherein each T is independently selected from the group consisting of:

—

SH

—

R^ALKX.

8. A conjugate according to any one of claims 1, 2 or 3, wherein each said biologically active molecule is selected from the group consisting of a carbohydrate, a drug, a lipid, a lipopolysaccharide, a peptide, a protein, a glycoprotein, a single-stranded oligonucleotide, a double-stranded oligonucleotide, an analog of an immunogen, a hapten, a mimotope, an aptamer, a lipoprotein, a nucleic acid or a combination thereof.

9. A conjugate according to claim 6, wherein each said biologically active molecule is selected from the group consisting of a carbohydrate, a drug, a lipid, a lipopolysaccharide, a peptide, a protein, a glycoprotein, a single-stranded oligonucleotide, a double-stranded oligonucleotide, an analog of an immunogen, a hapten, a mimotope, an aptamer, a lipoprotein, a nucleic acid or a combination thereof.

10. A conjugate according to claim 7, wherein each said biologically active molecule is selected from the group consisting of a carbohydrate, a drug, a lipid, a lipopolysaccharide, a peptide, a protein, a glycoprotein, a single-stranded oligonucleotide, a double-stranded oligonucleotide, an analog of an immunogen, a hapten, a mimotope, an aptamer, a lipoprotein, a nucleic acid or a combination thereof.

11. A conjugate according to any one of claims 1, 2 or 3, wherein each said biologically active molecule is an analog of an immunogen wherein (a) said analog binds specifically to B cells to which said immunogen binds specifically and (b) said conjugate lacks a T cell epitope.

12. A conjugate according to claim 6, wherein each said biologically active molecule is an analog of an immunogen wherein (a) said analog binds specifically to B cells to which said immunogen binds specifically and (b) said conjugate lacks a T cell epitope.

13. A conjugate according to claim 7, wherein each said biologically active molecule is an analog of an immunogen wherein (a) said analog binds specifically to B cells to which said immunogen binds specifically and (b) said conjugate lacks a T cell epitope.

14. A conjugate according to any one of claims 1, 2 or 3, wherein the biologically active molecule, comprises a polynucleotide duplex, that has significant binding activity for human systemic lupus erythematosus anti-double stranded DNA autoantibodies.

15. A conjugate according to claim 14, wherein the conjugate comprises the optional linker moiety.

16. A conjugate according to claim 15 wherein the linker moiety is selected from a thio-6 carbon chain phosphate or a thio-6 carbon chain phosphorothioate.

17. A conjugate according to claim 14, wherein said duplexes are substantially homogeneous in length.

18. A conjugate according to claim 14, wherein said duplexes are substantially homogeneous in nucleotide composition.

19. A conjugate according to claim 14, wherein said duplexes are 20 to 50 base pairs in length.

20. A conjugate according to claim 14, wherein said duplexes are covalently bonded to said valency platform at or proximate to one of their ends.

21. A conjugate according to claim 14, wherein said conjugate suppresses antibody production associated with human systemic lupus erythematosus.

22. A conjugate according to claim 14, wherein said duplexes have a B-DNA helical structure.

23. A composition comprising a conjugate according to claim 14 formulated with a pharmaceutically acceptable injectable vehicle.

24. A composition comprising a conjugate according to claim 11 formulated with a pharmaceutically acceptable injectable vehicle.

25. A method of suppressing antibody production in an individual in need thereof comprising administering the composition according to claim 21 to the individual in an effective amount such that antibody production is suppressed.

26. A method of suppressing antibody production in an individual in need thereof, said method comprising administering the composition according to claim 13 to the individual in an effective amount such that antibody production contributing to thyroiditis, stroke, or myasthenia gravis is suppressed.

27. A method of suppressing antibody production in an individual in need thereof comprising administering the composition according to claim 14 to the individual in an effective amount such that antibody production contributing to human systemic lupus erythematosus is suppressed.

28. A method of making a conjugate according to claim 14, said method comprising the steps of:

(a) reacting a plurality of first single-stranded polynucleotides to said valency platform for a time and under conditions effective to form a conjugate between the valency platform and the plurality of first single-stranded polynucleotides;

(b) annealing a complementary single-stranded polynucleotide to said first single-stranded polynucleotides for a time and under conditions effective to form the conjugate according to claim 16; and

(c) isolating the conjugate of step (b).

29. A conjugate according to claim 14, wherein the polynucleotide duplex is (CA)₁₀:

(TG)₁₀ and n of Q₁-(CH₂CH₂O)_nCH₂CH₂-Q₁ of the valency platform molecule is equal to 2.

30. A conjugate according to claim 29, wherein the molar ratio of duplex to valency platform molecule is about 2:

1 or about 4;

1.

31. A conjugate according to claim 29, wherein the molar ratio of duplex to valency platform molecule is 4:

1.

32. A conjugate according to claim 1, wherein the molar ratio of duplex to valency platform molecule is about 2:

1 or about 4;

1.

33. A conjugate according to claim 1, wherein the molar ratio of duplex to valency platform molecule is 4:

1.

34. A conjugate according to claim 11, wherein the immunogen is an external immunogen.

35. A conjugate according to claim 34, wherein the external immunogen is a biological drug, an allergen or a Rhesus (Rh/D) immunogen associated with Rh hemolytic disease, α

-sperm associated with male infertility or the carbohydrate complex associated with rheumatic fever.

36. A conjugate according to claim 11, wherein the immunogen is a self-immunogen.

37. A conjugate according to claim 36, wherein the self-immunogen contributes to thyroiditis, diabetes, stroke or myasthenia gravis.

38. A conjugate according to claim 11, wherein the immunogen and the analog are polypeptides.

39. A composition comprising a conjugate according to claim 11 formulated with a pharmaceutically acceptable carrier.

40. A composition comprising a conjugate according to claim 29, formulated with a pharmaceutically acceptable carrier.

41. The conjugate according to claim 14 wherein said polynucleotide duplex contains at least 20 base pairs.

42. A conjugate according to any one of claims 1, 2 or 3, wherein each Q₃ is a chemical moiety of the formula:

43. A conjugate according to any one of claims 1, 2 or 3, wherein each Q₃ is a chemical moiety of the formula:

44. A conjugate according to any one of claims 1, 2 or 3, wherein each Q₃ is a chemical moiety of the formula:

45. A conjugate according to any one of claims 1, 2 or 3, wherein each Q₃ is a chemical moiety of the formula:

46. A conjugate according to claim 4, wherein each T is independently selected from the group consisting of:

—

SH

—

R^ALKX.

47. A conjugate according to claim 5, wherein each T is independently selected from the group consisting of:

—

SH

—

R^ALKX.

48. A conjugate according to claim 42, wherein each T is independently selected from the group consisting of:

—

SH

—

R^ALKX.

49. A conjugate according to claim 43, wherein each T is independently selected from the group consisting of:

—

SH

—

R^ALKX.

50. A conjugate according to claim 44, wherein each T is independently selected from the group consisting of:

—

SH

—

R^ALKX.

51. A conjugate according to claim 45, wherein each T is independently selected from the group consisting of:

—

SH

—

R^ALKX.

52. A conjugate according to claim 4, wherein each said biologically active molecule is selected from the group consisting of a carbohydrate, a drug, a lipid, a lipopolysaccharide, a peptide, a protein, a glycoprotein, a single-stranded oligonucleotide, a double-stranded oligonucleotide, an analog of an immunogen, a hapten, a mimotope, an aptamer, a lipoprotein, a nucleic acid or a combination thereof.

53. A conjugate according to claim 5, wherein each said biologically active molecule is selected from the group consisting of a carbohydrate, a drug, a lipid, a lipopolysaccharide, a peptide, a protein, a glycoprotein, a single-stranded oligonucleotide, a double-stranded oligonucleotide, an analog of an immunogen, a hapten, a mimotope, an aptamer, a lipoprotein, a nucleic acid or a combination thereof.

54. A conjugate according to claim 42, wherein each said biologically active molecule is selected from the group consisting of a carbohydrate, a drug, a lipid, a lipopolysaccharide, a peptide, a protein, a glycoprotein, a single-stranded oligonucleotide, a double-stranded oligonucleotide, an analog of an immunogen, a hapten, a mimotope, an aptamer, a lipoprotein, a nucleic acid or a combination thereof.

55. A conjugate according to claim 43, wherein each said biologically active molecule is selected from the group consisting of a carbohydrate, a drug, a lipid, a lipopolysaccharide, a peptide, a protein, a glycoprotein, a single-stranded oligonucleotide, a double-stranded oligonucleotide, an analog of an immunogen, a hapten, a mimotope, an aptamer, a lipoprotein, a nucleic acid or a combination thereof.

56. A conjugate according to claim 44, wherein each said biologically active molecule is selected from the group consisting of a carbohydrate, a drug, a lipid, a lipopolysaccharide, a peptide, a protein, a glycoprotein, a single-stranded oligonucleotide, a double-stranded oligonucleotide, an analog of an immunogen, a hapten, a mimotope, an aptamer, a lipoprotein, a nucleic acid or a combination thereof.

57. A conjugate according to claim 45, wherein each said biologically active molecule is selected from the group consisting of a carbohydrate, a drug, a lipid, a lipopolysaccharide, a peptide, a protein, a glycoprotein, a single-stranded oligonucleotide, a double-stranded oligonucleotide, an analog of an immunogen, a hapten, a mimotope, an aptamer, a lipoprotein, a nucleic acid or a combination thereof.

58. A conjugate according to claim 4, wherein each said biologically active molecule is an analog of an immunogen wherein (a) said analog binds specifically to B cells to which said immunogen binds specifically and (b) said conjugate lacks a T cell epitope.

59. A conjugate according to claim 5, wherein each said biologically active molecule is an analog of an immunogen wherein (a) said analog binds specifically to B cells to which said immunogen binds specifically and (b) said conjugate lacks a T cell epitope.

60. A conjugate according to claim 42, wherein each said biologically active molecule is an analog of an immunogen wherein (a) said analog binds specifically to B cells to which said immunogen binds specifically and (b) said conjugate lacks a T cell epitope.

61. A conjugate according to claim 43, wherein each said biologically active molecule is an analog of an immunogen wherein (a) said analog binds specifically to B cells to which said immunogen binds specifically and (b) said conjugate lacks a T cell epitope.

62. A conjugate according to claim 44, wherein each said biologically active molecule is an analog of an immunogen wherein (a) said analog binds specifically to B cells to which said immunogen binds specifically and (b) said conjugate lacks a T cell epitope.

63. A conjugate according to claim 45, wherein each said biologically active molecule is an analog of an immunogen wherein (a) said analog binds specifically to B cells to which said immunogen binds specifically and (b) said conjugate lacks a T cell epitope.

64. A conjugate according to claim 1 wherein the biologically active molecule is a polynucleotide.

65. A conjugate according to claim 45 wherein the biologically active molecule is a polynucleotide.

66. A conjugate according to claim 64 wherein the polynucleotide is a double stranded polynucleotide.

67. A conjugate according to claim 65 wherein the polynucleotide is a double stranded polynucleotide.

68. A conjugate according to claim 64 wherein the polynucleotide is a single stranded polynucleotide.

69. A conjugate according to claim 65 wherein the polynucleotide is a single stranded polynucleotide.

70. A conjugate according to claim 66 wherein the polynucleotide is DNA.

71. A conjugate according to claim 67 wherein the polynucleotide is DNA.

72. A conjugate according to claim 68 wherein the polynucleotide is DNA.

73. A conjugate according to claim 69 wherein the polynucleotide is DNA.

74. A conjugate according to claim 64 wherein the polynucleotide comprises at least 20 nucleotide base pairs.

75. A conjugate according to claim 65 wherein the polynucleotide comprises at least 20 nucleotide base pairs.

76. A conjugate according to claim 64 wherein the polynucleotide is (CA)₁₀.(TG)₁₀.

77. A conjugate according to claim 65 wherein the polynucleotide is (CA)₁₀.(TG)₁₀.

78. A conjugate according to claim 76 wherein the polynucleotide is bound via the 5′

end of the polynucleotide.

79. A conjugate according to claim 77 wherein the polynucleotide is bound via the 5′

end of the polynucleotide.

80. A conjugate according to claim 65 wherein the polynucleotide has significant binding activity for human systemic lupus erythematosus anti-double stranded DNA autoantibodies.

81. A conjugate according to claim 80 wherein the conjugate comprises the optional linker moiety and the linker moiety is selected from the group consisting of a thio-6 carbon chain phosphate and a thio-6 carbon chain phosphorothioate.

82. A conjugate according to claim 68 wherein the single stranded polynucleotide is (CA)₁₀.

83. A conjugate according to claim 69 wherein the single stranded polynucleotide is (CA)₁₀.

84. A conjugate according to claim 66 wherein the polynucleotide is a 20 base pair polynucleotide.

85. A conjugate according to claim 67 wherein the polynucleotide is a 20 base pair polynucleotide.

86. A conjugate according to claim 66 wherein the polynucleotide is about 20 nucleotide base pairs.

87. A conjugate according to claim 67 wherein the polynucleotide is about 20 nucleotide base pairs.

88. A conjugate according to claim 76 wherein the conjugate comprises the optional linker.

89. A conjugate according to claim 77 wherein the conjugate comprises the optional linker.

90. A conjugate according to claim 88 wherein the linker moiety is selected from the group consisting of a thio-6 carbon chain phosphate and a thio-6 carbon chain phosphorothioate.

91. A conjugate according to claim 89 wherein the linker moiety is selected from the group consisting of a thio-6 carbon chain phosphate and a thio-6 carbon chain phosphorothioate.

92. A method of suppressing antibody production in an individual in need thereof, the method comprising administering the conjugate according to claim 80 to the individual in an effective amount such that antibody production is suppressed.

93. A method of suppressing antibody production in an individual in need thereof, the method comprising administering the conjugate according to claim 81 to the individual in an effective amount such that antibody production is suppressed.

94. A conjugate according to claim 66 wherein the polynucleotide is a polynucleotide duplex that is 20 to 50 base pairs in length.

95. A conjugate according to claim 67 wherein the polynucleotide is a polynucleotide duplex that is 20 to 50 base pairs in length.

96. A conjugate according to claim 90 formulated with a pharmaceutically acceptable injectable vehicle.

97. A conjugate according to claim 91 formulated with a pharmaceutically acceptable injectable vehicle.

100. A method of making a conjugate according to claim 66, said method comprising the steps of:

(a) reacting a plurality of first single-stranded polynucleotides to said valency platform for a time and under conditions effective to form a conjugate between the valency platform and the plurality of first single-stranded polynucleotides; and

(b) annealing a complementary single-stranded polynucleotide to said first single-stranded polynucleotides for a time and under conditions effective to form the conjugate according to claim 66.

101. The method of claim 100 wherein the method comprises the step of reacting the valency platform molecule or the single stranded polynucleotide with a linker moiety for a time and under conditions effective to form a valency platform molecule-linker moiety or a single stranded polynucleotide-linker moiety such that the conjugate comprises a linker moiety linking the polynucleotide and the valency platform molecule.

103. The conjugate of claim 70 wherein the DNA is 20 nucleotide base pairs in length.

104. The conjugate of claim 71 wherein the DNA is 20 nucleotide base pairs in length.

105. The conjugate of claim 70 wherein the DNA is 20 to 50 base pairs in length.

106. The conjugate of claim 71 wherein the DNA is 20 to 50 base pairs in length.

107. A conjugate according to claim 1 formulated with a pharmaceutically acceptable injectable vehicle.

108. A conjugate according to claim 45 formulated with a pharmaceutically acceptable injectable vehicle.

109. A conjugate according to claim 65 formulated with a pharmaceutically acceptable injectable vehicle.

142. The method of claim 100 wherein the polynucleotide is (CA)₁₀.(TG)₁₀.

143. The method of claim 101 wherein the polynucleotide is (CA)₁₀.(TG)₁₀.

98. A valency platform molecule of the formula:

Q₁-(CH₂CH₂O)_nCH₂CH₂-Q₁

wherein each Q₁ is a chemical moiety independently selected from the group consisting of;

99. A method of making a conjugate of a valency platform molecule and a biologically active molecule, wherein the method comprises:

bonding a biologically active molecule to the valency platform molecule of claim 98.

102. The method of claim 99 wherein the method comprises the step of reacting the valency platform molecule or the biologically active molecule with a linker moiety for a time and under conditions effective to form a valency platform molecule-linker moiety or a biologically active molecule-linker moiety such that the conjugate comprises a linker moiety linking the biologically active molecule and the valency platform molecule.

110. A valency platform molecule according to claim 98, wherein each Q₁ is:

111. A valency platform molecule according to claim 98, wherein each Q₁ is:

112. A valency platform molecule according to claim 98, 110 or 111, wherein each Q₃ is a chemical moiety of the formula:

113. A valency platform molecule according to claim 98, 110 or 111, wherein each Q₃ is a chemical moiety of the formula:

114. A valency platform molecule according to claim 98, 110 or 111, wherein each Q₃ is a chemical moiety of the formula:

115. A valency platform molecule according to claim 98, 110 or 111, wherein each Q₃ is a chemical moiety of the formula:

116. A valency platform molecule according to claim 98, 110 or 111, wherein each Q₃ is a chemical moiety of the formula:

117. A valency platform molecule according to claim 98, 110 or 111, wherein each Q₃ is a chemical moiety of the formula:

118. A valency platform molecule of claim 112 wherein T is independently selected from the group consisting of:

119. A valency platform molecule of claim 112 wherein T is independently selected from the group consisting of:

—

SH

—

R^ALKX.

120. A valency platform molecule of claim 113 wherein T is independently selected from the group consisting of:

121. A valency platform molecule of claim 113 wherein T is independently selected from the group consisting of:

—

SH

—

R^ALKX.

122. A valency platform molecule of claim 114 wherein T is independently selected from the group consisting of:

123. A valency platform molecule of claim 114 wherein T is independently selected from the group consisting of:

—

SH

—

R^ALKX.

124. A valency platform molecule of claim 115 wherein T is independently selected from the group consisting of:

125. A valency platform molecule of claim 115 wherein T is independently selected from the group consisting of:

—

SH

—

R^ALKX.

126. A valency platform molecule of claim 116 wherein T is independently selected from the group consisting of:

127. A valency platform molecule of claim 116 wherein T is independently selected from the group consisting of:

—

SH

—

R^ALKX.

128. A valency platform molecule of claim 117 wherein T is independently selected from the group consisting of:

129. A valency platform molecule of claim 117 wherein T is independently selected from the group consisting of:

—

SH

—

R^ALKX.

130. A method of making a conjugate of a valency platform molecule and a biologically active molecule, wherein the method comprises:

bonding a biologically active molecule to the valency platform molecule of claim 118 wherein the conjugate optionally comprises a linker moiety linking the biologically active molecule and the valency platform molecule.

131. A method of making a conjugate of a valency platform molecule and a biologically active molecule, wherein the method comprises:

bonding a biologically active molecule to the valency platform molecule of claim 120 wherein the conjugate optionally comprises a linker moiety linking the biologically active molecule and the valency platform molecule.

132. A method of making a conjugate of a valency platform molecule and a biologically active molecule, wherein the method comprises:

bonding a biologically active molecule to the valency platform molecule of claim 122 wherein the conjugate optionally comprises a linker moiety linking the biologically active molecule and the valency platform molecule.

133. A method of making a conjugate of a valency platform molecule and a biologically active molecule, wherein the method comprises:

bonding a biologically active molecule to the valency platform molecule of claim 124 wherein the conjugate optionally comprises a linker moiety linking the biologically active molecule and the valency platform molecule.

134. A method of making a conjugate of a valency platform molecule and a biologically active molecule, wherein the method comprises:

bonding a biologically active molecule to the valency platform molecule of claim 126 wherein the conjugate optionally comprises a linker moiety linking the biologically active molecule and the valency platform molecule.

135. A method of making a conjugate of a valency platform molecule and a biologically active molecule, wherein the method comprises:

bonding a biologically active molecule to the valency platform molecule of claim 128 wherein the conjugate optionally comprises a linker moiety linking the biologically active molecule and the valency platform molecule.

136. A method of making a conjugate of a valency platform molecule and a biologically active molecule, wherein the method comprises:

bonding a biologically active molecule to the valency platform molecule of claim 127 wherein the conjugate optionally comprises a linker moiety linking the biologically active molecule and the valency platform molecule.

137. The method of claim 134 wherein the biologically active molecule is a polynucleotide.

138. The method of claim 136 wherein the biologically active molecule is a polynucleotide.

139. The method of claim 137 wherein the biologically active molecule is DNA.

140. The method of claim 99 wherein the biologically active molecule is DNA.

141. The method of claim 102 wherein the biologically active molecule is DNA.