Immunoglobulin chimeric monomer-dimer hybrids

• US 20050032174A1
• Filed: 05/06/2004
• Published: 02/10/2005
• Est. Priority Date: 05/06/2003
• Status: Active Grant

• Current Assignee: Bioverativ Therapeutics Inc. (Sanofi )
• Original Assignee: Syntonix Pharmaceuticals, Inc.
• Inventors: Peters, Robert T., Mezo, Adam R., Low, Susan C., Bitonti, Alan J., Rivera, Daniel S., Stattel, James
• Granted Patent: US 7,404,956 B2
• Time in Patent Office: Days
• US Class Current: 435/69.700
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• 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 47/642 the peptide or protein in the drug conjugate being a cytokine, e.g. IL2, chemokine, growth factors or interferons being the inactive part of the conjugate
• CPC Class Codes: A61K 47/68 the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
• CPC Class Codes: A61K 47/6803 Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
• CPC Class Codes: A61K 47/6811 the drug being a protein or peptide, e.g. transferrin or bleomycin
• CPC Class Codes: A61K 47/6813 the drug being a peptidic cytokine, e.g. an interleukin or interferon
• CPC Class Codes: A61K 47/6835 the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
• CPC Class Codes: A61P 31/00 Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• CPC Class Codes: A61P 31/12 Antivirals
• CPC Class Codes: A61P 31/18 for HIV
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 7/00 Drugs for disorders of the blood or the extracellular fluid
• CPC Class Codes: A61P 7/04 Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
• CPC Class Codes: A61P 7/06 Antianaemics
• CPC Class Codes: A61P 9/00 Drugs for disorders of the cardiovascular system
• CPC Class Codes: C07K 14/475 Growth factors; Growth regulators
• CPC Class Codes: C07K 14/505 Erythropoietin [EPO]
• CPC Class Codes: C07K 14/555 Interferons [IFN]
• CPC Class Codes: C07K 14/56 IFN-alpha
• CPC Class Codes: C07K 14/565 : IFN-beta
• CPC Class Codes: C07K 14/59 : Follicle-stimulating hormone [FSH]; Chorionic gonadotropins, e.g.hCG [human chorionic gonadotropin]; Luteinising hormone [LH]; Thyroid-stimulating hormone [TSH]
• CPC Class Codes: C07K 14/61 : Growth hormone [GH], i.e. somatotropin
• CPC Class Codes: C07K 14/70503 : Immunoglobulin superfamily
• CPC Class Codes: C07K 14/745 : Blood coagulation or fibrinolysis factors
• CPC Class Codes: C07K 14/755 : Factors VIII , e.g. factor VIII C (AHF), factor VIII Ag (VWF)
• CPC Class Codes: C07K 16/00 : Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies antibodies with enzymatic activity, e.g. abzymes C12N9/0002
• CPC Class Codes: C07K 2317/52 : Constant or Fc region; Isotype
• CPC Class Codes: C07K 2319/00 : Fusion polypeptide
• CPC Class Codes: C07K 2319/30 : Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
• CPC Class Codes: C12N 9/6437 : Coagulation factor VIIa (3.4.21.21)
• CPC Class Codes: C12N 9/644 : Coagulation factor IXa (3.4.21.22)
• CPC Class Codes: C12N 9/647 : Blood coagulation factors not provided for in a preceding group or according to more than one of the proceeding groups
• CPC Class Codes: C12N 9/96 : Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
• CPC Class Codes: C12Y 304/21021 : Coagulation factor VIIa (3.4.21.21)
• CPC Class Codes: C12Y 304/21022 : Coagulation factor IXa (3.4.21.22)

Abstract

The invention relates to a chimeric monomer-dimer hybrid protein wherein said protein comprises a first and a second polypeptide chain, said first polypeptide chain comprising at least a portion of an immunoglobulin constant region and a biologically active molecule, and said second polypeptide chain comprising at least a portion of an immunoglobulin constant region without the biologically active molecule of the first chain. The invention also relates to methods of using and methods of making the chimeric monomer-dimer hybrid protein of the invention.

Claims

1. A chimeric protein comprising a first and second polypeptide chain, wherein said first chain comprises a biologically active molecule, and at least a portion of an immunoglobulin constant region and

wherein said second chain comprises at least a portion of an immunoglobulin constant region without a biologically active molecule or immunoglobulin variable region.

2. The chimeric protein of claim 1, wherein said second chain further comprises an affinity tag.

3. The chimeric protein of claim 2, wherein the affinity tag is a FLAG tag.

4. The chimeric protein of claim 1, wherein the portion of an immunoglobulin is an Fc fragment.

5. The chimeric protein of claim 4, wherein the portion of an immunoglobulin is an FcRn binding partner.

6. The chimeric protein of claim 5, wherein the FcRn binding partner is a peptide mimetic of an Fc fragment of an immunoglobulin.

7. The chimeric protein of claim 1, wherein the immunoglobulin is IgG.

8. The chimeric protein of claim 1, wherein the biologically active molecule is a polypeptide.

9. The chimeric protein of claim 7, wherein the IgG is an IgG1 or an IgG2.

10. The chimeric protein of claim 1, wherein the biologically active molecule is a viral fusion inhibitor.

11. The chimeric protein of claim 10, wherein the viral fusion inhibitor is an HIV fusion inhibitor.

12. The chimeric protein of claim 11, wherein the HIV fusion inhibitor is T20 (SEQ ID NO:

1), T21 (SEQ ID NO;

2), or T1249 (SEQ ID NO;

3).

13. The chimeric protein of claim 1, wherein the biologically active molecule is a clotting factor.

14. The chimeric protein of claim 13, wherein the clotting factor is Factor VII or VIIa.

15. The chimeric protein of claim 13, wherein the clotting factor is Factor IX.

16. The chimeric protein of claim 1, wherein the biologically active molecule is a small molecule.

17. The chimeric protein of claim 16, wherein the biologically active molecule is leuprolide.

18. The chimeric protein of claim 1, wherein the biologically active molecule is interferon.

19. The chimeric protein of claim 18, wherein the interferon is interferon α

and has a linker of 15-25 amino acids.

20. The chimeric protein of claim 19, wherein the interferon α

has a linker of 15-20 amino acids.

21. The chimeric protein of claim 1, wherein the biologically active molecule is a nucleic acid.

22. The chimeric protein of claim 21, wherein the nucleic acid is DNA or RNA.

23. The chimeric protein of claim 21, wherein the nucleic acid is an antisense molecule.

24. The chimeric protein of claim 21, wherein the nucleic acid is a ribozyme.

25. The chimeric protein of claim 1, wherein the biologically active molecule is a growth factor.

26. The chimeric protein of claim 25, wherein the growth factor is erythropoietin.

27. The chimeric protein of claim 16, wherein the small molecule is a VLA4 antagonist.

60. A pharmaceutical composition comprising the chimeric protein of claim 1 and a pharmaceutically acceptable excipient.

131. A method of treating a disease or condition in a subject comprising administering the chimeric protein of claim 1 to said subject.

132. The method of claim 131, wherein the disease or condition is a viral infection.

133. The method of claim 131, wherein the biologically active molecule is interferon.

134. The method of claim 133, wherein the interferon is interferon α

and has a linker of 15-25 amino acids.

135. The method of claim 134, wherein the interferon α

has a linker of 15-20 amino acids.

136. The method of claim 132, wherein the viral infection is HIV.

137. The method of claim 131, wherein the disease or condition is a bleeding disorder.

138. The method of claim 137, wherein the bleeding disorder is hemophilia A.

139. The method of claim 137, wherein the bleeding disorder is hemophilia B.

140. The method of claim 131, wherein the disease or condition is anemia.

141. The method of claim 131, wherein the chimeric protein is administered intravenously, intramuscularly, subcutaneously, orally, buccally, sublingually, nasally, rectally, vaginally, via an aerosol, or via a pulmonary route.

142. The method of claim 141, wherein the chimeric protein is administered via a pulmonary route.

143. The method of claim 141, wherein the chimeric protein is administered orally.

144. The method of claim 131, wherein the immunoglobulin is IgG.

145. The method of claim 131, wherein the portion of an immunoglobulin is an Fc fragment.

185. The method of claim 133, wherein the biologically active molecule is interferon α

.

186. The method of claim 133, wherein the biologically active molecule is interferon β

.

195. The pharmaceutical composition of claim 60, wherein the portion of an immunoglobulin is an FcRn binding partner.

196. The pharmaceutical composition of claim 60, wherein the first chain of the chimeric protein further comprises a first domain having at least one specific binding partner and wherein the second chain of the chimeric protein further comprises a second domain said second domain being a specific binding partner of said first domain.

197. The pharmaceutical composition of claim 196, wherein the portion of an immunoglobulin is an FcRn binding partner.

198. The method of claim 131, wherein the portion of an immunoglobulin constant region is an FcRn binding partner.

199. The method of claim 131, wherein the first chain of the chimeric protein further comprises a first domain having at least one specific binding partner and wherein the second chain of the chimeric protein further comprises a second domain said second domain being a specific binding partner of said first domain.

200. The method of claim 199, wherein the portion of an immunoglobulin constant region is an FcRn binding partner.

28. A chimeric protein comprising a first and second polypeptide chain, wherein said first chain comprises a biologically active molecule, and at least a portion of an immunoglobulin constant region and

wherein said second chain consists of at least a portion of an immunoglobulin constant region and optionally an affinity tag.

29. The chimeric protein of 28, wherein the affinity tag is a FLAG tag.

30. A chimeric protein comprising a first and second polypeptide chain, wherein said first chain comprises a biologically active molecule, and at least a portion of an immunoglobulin constant region and

wherein said second chain consists essentially of at least a portion of an immunoglobulin constant region and optionally an affinity tag.

31. The chimeric protein of 30, wherein the affinity tag is a FLAG tag.

32. A chimeric protein comprising a first and second polypeptide chain

a) wherein said first chain comprises a biologically active molecule, at least a portion of an immunoglobulin constant region, and a first domain having at least one specific binding partner;

and

b) wherein said second chain comprises at least a portion of an immunoglobulin without a biologically active molecule or immunoglobulin variable region and further comprising a second domain said second domain being a specific binding partner of said first domain.

33. The chimeric protein of claim 32, wherein said second chain further comprises an affinity tag.

34. The chimeric protein of claim 33, wherein the affinity tag is a FLAG tag.

35. The chimeric protein of claim 32, wherein the portion of an immunoglobulin is an Fc fragment.

36. The chimeric protein of claim 32, wherein the immunoglobulin is IgG.

37. The chimeric protein of claim 35, wherein the portion of an immunoglobulin is an FcRn binding partner.

38. The chimeric protein of claim 37, wherein the FcRn binding partner is a peptide mimetic of an Fc fragment of an immunoglobulin.

39. The chimeric protein of claim 32, wherein the first domain binds with the second domain non-covalently.

40. The chimeric protein of claim 32, wherein the first domain is one half of a leucine zipper coiled coil and said second domain is the complementary binding partner of said leucine zipper coiled coil.

41. The chimeric protein of claim 32, wherein the biologically active molecule is a peptide.

42. The chimeric protein of claim 32, wherein the biologically active molecule is interferon.

43. The chimeric protein of claim 42, wherein the interferon is interferon α

and has a linker of 15-25 amino acids.

44. The chimeric protein of claim 43, wherein the interferon α

has a linker of 15-20 amino acids.

45. The chimeric protein of claim 41, wherein the biologically active molecule is leuprolide.

46. The chimeric protein of claim 32, wherein the biologically active molecule is a viral fusion inhibitor.

47. The chimeric protein of claim 46, wherein the viral fusion inhibitor is an HIV fusion inhibitor.

48. The chimeric protein of claim 47, wherein the HIV fusion inhibitor is T20 (SEQ ID NO:

1), or T21 (SEQ ID NO;

2), or T1249 (SEQ ID NO;

3).

49. The chimeric protein of claim 32, wherein the biologically active molecule is a clotting factor.

50. The chimeric protein of claim 49, wherein the clotting factor is Factor VII or Factor VIIa.

51. The chimeric protein of claim 49, wherein the clotting factor is Factor IX.

52. The chimeric protein of claim 32, wherein the biologically active molecule is a small molecule.

53. The chimeric protein of claim 52, wherein the small molecule is a VLA4 antagonist.

54. The chimeric protein of claim 32, wherein the biologically active molecule comprises a nucleic acid.

55. The chimeric protein of claim 54, wherein the nucleic acid is DNA or RNA.

56. The chimeric protein of claim 54, wherein the nucleic acid is an antisense nucleic acid.

57. The chimeric protein of claim 54, wherein the nucleic acid is a ribozyme.

58. The chimeric protein of claim 32, wherein the biologically active molecule is a growth factor or hormone.

59. The chimeric protein of claim 58, wherein the growth factor is erythropoietin.

179. The chimeric protein of claim 42, wherein the biologically active molecule is interferon α

.

180. The chimeric protein of claim 42, wherein the biologically active molecule is interferon β

.

61. A chimeric protein comprising a first and second polypeptide chain

a) wherein said first chain comprises a biologically active molecule, at least a portion of an immunoglobulin constant region, and a first domain having at least one specific binding partner;

and

b) wherein said second chain consists of at least a portion of an immunoglobulin, a second domain said second domain being a specific binding partner of said first domain and optionally an affinity tag.

62. The chimeric protein of 61, wherein the affinity tag is a FLAG tag.

63. A chimeric protein comprising a first and second polypeptide chain

a) wherein said first chain comprises a biologically active molecule, at least a portion of an immunoglobulin constant region, and a first domain having at least one specific binding partner;

and

b) wherein said second chain consists essentially of at least a portion of an immunoglobulin, and a second domain said second domain being a specific binding partner of said first domain and optionally an affinity tag.

64. The chimeric protein of 63, wherein the affinity tag is a FLAG tag.

65-89. -89. (Canceled)

90. A method of treating a subject with a disease or condition comprising administering a chimeric protein to the subject, such that said disease or condition is treated, wherein said chimeric protein comprises a first and second polypeptide chain,

a) said first chain comprising an FcRn binding partner, and a biologically active molecule and

b) said second chain comprising an FcRn binding partner without a biologically active molecule or a variable region of an immunoglobulin.

91. The method of claim 90, wherein said chimeric protein is administered intravenously, subcutaneously, orally, buccally, sublingually, nasally, parenterally, rectally, vaginally or via a pulmonary route.

92. The method of claim 90, wherein said disease or condition is a viral infection.

93. The method of claim 90, wherein the biologically active molecule is interferon.

94. The method of claim 93, wherein the interferon is interferon α

and has a linker of 15-25 amino acids.

95. The method of claim 94, wherein the interferon α

has a linker of 15-20 amino acids.

96. The method of claim 90, wherein said disease or condition is HIV.

97. The method of claim 90, wherein said biologically active molecule is a viral fusion inhibitor.

98. The method of claim 97, wherein said viral fusion inhibitor is T20, T21, or T1249.

99. The method of claim 90, wherein said disease or condition is a hemostatic disorder.

100. The method of claim 90, wherein said disease or condition is hemophilia A.

101. The method of claim 90, wherein said disease or condition is hemophilia B.

102. The method of claim 90, wherein said biologically active molecule is Factor VII or Factor VIIa.

103. The method of claim 90, wherein said biologically active molecule is Factor IX.

104. The method of claim 90, wherein said disease or condition is anemia.

105. The method of claim 90, wherein said biologically active molecule is erythropoietin.

183. The method of claim 93, wherein the biologically active molecule is interferon α

.

184. The method of claim 93, wherein the biologically active molecule is interferon β

.

106. A chimeric protein of the formula

X-L_a-F:

F or F;

F-L_a-X

wherein X is a biologically active molecule, L is a linker, F is at least a portion of an immunoglobulin constant region and, a is any integer or zero.

109. The chimeric protein of claim 106, wherein each F is chemically associated with the other F.

110. The method of claim 109, wherein the chemical association is a non-covalent interaction.

111. The method of claim 109, wherein the chemical bond association is a covalent bond.

112. The method of claim 109, wherein the chemical bond association is a disulfide bond.

113. The chimeric protein of claim 106, wherein F is linked to F by a bond that is not a disulfide bond.

114. The chimeric protein of claim 106, wherein F is an IgG immunoglobulin constant region.

115. The chimeric protein of claim 106, wherein F is an IgG1.

116. The chimeric protein of claim 106, wherein F is an Fc fragment.

117. The chimeric protein of claim 106, wherein X is a polypeptide.

118. The chimeric protein of claim 106, wherein X is leuprolide.

119. The chimeric protein of claim 106, wherein X is a small molecule.

121. The chimeric protein of claim 106, wherein X is a viral fusion inhibitor.

122. The chimeric protein of claim 121, wherein the viral fusion inhibitor is an HIV fusion inhibitor.

123. The chimeric protein of claim 122, wherein the HIV fusion inhibitor is T20 (SEQ ID NO:

1), or T21 (SEQ ID NO;

2), or T1249 (SEQ ID NO;

3).

124. The chimeric protein of claim 106, wherein X is a clotting Factor.

125. The chimeric protein of claim 124, wherein the clotting factor is Factor VII or VIIa.

126. The chimeric protein of claim 124, wherein the clotting factor is Factor IX.

127. The chimeric protein of claim 106, wherein X is a nucleic acid.

128. The chimeric protein of claim 127, wherein the nucleic acid is a DNA or an RNA molecule.

129. The chimeric protein of claim 106, wherein X is a growth factor.

130. The chimeric protein of claim 129, wherein the growth factor is erythropoietin.

201. A method of treating a disease or condition in a subject comprising administering the chimeric protein of claim 106.

202. The method of claim 201, wherein F is an FcRN binding partner.

107. The chimeric protein of 106, wherein F is an FcRn binding partner.

108. The chimeric protein of 106, wherein the FcRn is a peptide mimetic of an Fc fragment of an immunoglobulin.

120. The chimeric protein of 119, wherein the small molecule is a VLA4 antagonist.

146. A chimeric protein comprising a first and a second polypeptide chain linked together, wherein said first chain comprises a biologically active molecule and at least a portion of an immunoglobulin constant region, and said second chain comprises at least a portion of an immunoglobulin constant region without the biologically active molecule of the first chain and wherein said second chain is not covalently bonded to any molecule having a molecular weight greater than 2 kD.

147. The chimeric protein of claim 146, wherein the portion of an immunoglobulin constant region is an FcRn binding partner.

148. A chimeric protein comprising a first and a second polypeptide chain linked together, wherein said first chain comprises a biologically active molecule and at least a portion of an immunoglobulin constant region, and said second chain comprises at least a portion of an immunoglobulin constant region not covalently linked to any other molecule except the portion of an immunoglobulin of said first polypeptide chain.

149. The chimeric protein of claim 148, wherein the portion of an immunoglobulin constant region is an FcRn binding partner.

150. A chimeric protein comprising a first and a second polypeptide chain linked together, wherein said first chain comprises a biologically active molecule and at least a portion of an immunoglobulin constant region, and said second chain consists of at least a portion of an immunoglobulin constant region.

151. The chimeric protein of claim 150, wherein the portion of an immunoglobulin constant region is an FcRn binding partner.

152. A chimeric protein comprising a first and a second polypeptide chain linked together, wherein said first chain comprises a biologically active molecule and at least a portion of an immunoglobulin constant region, and said second chain comprises at least a portion of an immunoglobulin constant region without the biologically active molecule of the first chain and a molecule with a molecular weight less than 2 kD covalently attached.

153. The chimeric protein of claim 152, wherein the portion of an immunoglobulin constant region is an FcRn binding partner.

154-178. -178. (Canceled)

189. A chimeric protein comprising a first and second polypeptide chain, wherein said first chain comprises EPO, an eight amino acid linker having the amino acid sequence EFAGAAAV, and an Fc fragment of an immunoglobulin constant region comprising a mutation of aspargine to alanine at position 297;

and

wherein said second chain comprises an Fc fragment of an immunoglobulin constant region comprising a mutation of aspargine to alanine at position 297.

190. The chimeric protein of claim 189, further comprising an affinity tag.

191. A chimeric protein comprising a first and second polypeptide chain, wherein said first chain comprises IFNβ

, an eight amino acid linker having the amino acid sequence EFAGAAAV, and an Fc fragment of an immunoglobulin constant region comprising a mutation of aspargine to alanine at position 297; and

wherein said second chain comprises an Fc fragment of an immunoglobulin constant region comprising a mutation of aspargine to alanine at position 297.

192. The chimeric protein of claim 191, further comprising an affinity tag.

193. A chimeric protein comprising a first and second polypeptide chain, wherein said first chain comprises factor IX, an eight amino acid linker having the amino acid sequence EFAGAAAV, and an Fc fragment of an immunoglobulin constant region comprising a mutation of aspargine to alanine at position 297;

and

wherein said second chain comprises an Fc fragment of an immunoglobulin constant region comprising a mutation of aspargine to alanine at position 297.

194. The chimeric protein of claim 193, further comprising an affinity tag.