MODIFIED AND STABILIZED GDF PROPEPTIDES AND USES THEREOF

• US 20070149455A1
• Filed: 12/21/2006
• Published: 06/28/2007
• Est. Priority Date: 02/08/2001
• Status: Active Grant

• Current Assignee: Wyeth LLC (Pfizer Inc.)
• Original Assignee: Wyeth (Pfizer Inc.)
• Inventors: Khor, Soo-Peang, Wolfman, Neil, Tomkinson, Kathleen
• Granted Patent: US 7,737,116 B2
• Time in Patent Office: Days
• US Class Current: 514/4.800
• CPC Class Codes: A61P 11/00 Drugs for disorders of the respiratory system
• CPC Class Codes: A61P 19/00 Drugs for skeletal disorders
• CPC Class Codes: A61P 19/02 for joint disorders, e.g. arthritis, arthrosis
• CPC Class Codes: A61P 19/08 for bone diseases, e.g. rachitism, Paget's disease
• CPC Class Codes: A61P 19/10 for osteoporosis
• CPC Class Codes: A61P 21/00 Drugs for disorders of the muscular or neuromuscular system
• CPC Class Codes: A61P 21/04 for myasthenia gravis
• CPC Class Codes: A61P 21/06 Anabolic agents androgens A61P5/26
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 3/00 Drugs for disorders of the metabolism of the blood or the extracellular fluid A61P7/00
• CPC Class Codes: A61P 3/04 Anorexiants; Antiobesity agents
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: C07K 14/475 Growth factors; Growth regulators
• CPC Class Codes: C07K 14/51 Bone morphogenetic factor; Osteogenins; Osteogenic factor; Bone-inducing factor
• CPC Class Codes: C07K 16/18 against material from animals or humans
• CPC Class Codes: C07K 2319/00 Fusion polypeptide

Abstract

Modified and stabilized propeptides of Growth Differentiation Factor proteins, such as GDF-8 and Bone Morphogenetic Protein-11, are disclosed. Also disclosed are methods for making and using the modified propeptides to prevent or treat human or animal disorders in which an increase in muscle tissue would be therapeutically beneficial. Such disorders include muscle or neuromuscular disorders (such as amyotrophic lateral sclerosis, muscular dystrophy, muscle atrophy, congestive obstructive pulmonary disease, muscle wasting syndrome, sarcopenia, or cachexia), metabolic diseases or disorders (such as such as type 2 diabetes, noninsulin-dependent diabetes mellitus, hyperglycemia, or obesity), adipose tissue disorders (such as obesity), and bone degenerative diseases (such as osteoporosis).

Claims

1-118. -118. (canceled)

119. A BMP-11 propeptide comprising an amino acid sequence that is at least 95% identical to SEQ ID NO:

11 and has a mutation that modifies the aspartate residue at position 98 of SEQ ID NO;

11, wherein the propeptide inhibits one or more BMP-11 or GDF-8 activities.

120. The BMP-11 of claim 119, wherein the BMP-11 or GDF-8 activity is chosen from BMP-11 or GDF-8 propeptide binding, negative regulation of skeletal muscle mass, modulation of preadipocyte differentiation, inhibition of muscle formation, inhibition of muscle cell growth, inhibition of muscle development, regulation of muscle-specific enzymes, inhibition of myoblast cell proliferation, modulation of preadipocyte differentiation to adipocytes, increasing sensitivity to insulin, regulation of glucose uptake, glucose hemostasis, and modulation of neuronal cell development and maintenance.

121. The BMP-11 propeptide of claim 119, wherein the BMP-11 propeptide has an increased in vivo or in vitro half-life relative to a corresponding unmodified BMP-11 propeptide.

122. The BMP-11 propeptide of claim 119, wherein the mutation that modifies the aspartate residue at position 98 of SEQ ID NO:

11 is a substitution mutation.

123. The BMP-11 propeptide of claim 119, wherein the mutation that modifies the aspartate residue at position 98 of SEQ ID NO:

11 is an insertion mutation.

124. The BMP-11 propeptide of claim 119, wherein the mutation that modifies the aspartate residue at position 98 of SEQ ID NO:

11 is a deletion mutation.

125. The BMP-11 propeptide of claim 119, wherein the mutation results in the expression of an alanine at position 98 of SEQ ID NO:

11.

126. The BMP-11 propeptide of claim 119 further comprising an Fc region of an IgG molecule.

127. The BMP-11 propeptide of claim 126, wherein the IgG molecule is IgG1 or IgG4.

128. The BMP-11 propeptide of claim 126, wherein the amino acid sequence of the IgG molecule is at least 95% identical to SEQ ID NO:

16.

129. The BMP-11 propeptide of claim 126, wherein the BMP-11 propeptide is fused to the Fc region of the IgG molecule via a linker peptide.

130. The BMP-11 propeptide of claim 119, further comprising an altered glycosylation site.

131. The BMP-11 propeptide of claim 119, further comprising at least one carbohydrate moiety.

132. The BMP-11 propeptide of claim 119, further comprising albumin.

133. The BMP-11 propeptide of claim 119, further comprising a nonproteinaceous polymer.

134. The BMP-11 propeptide of claim 119, further comprising a second moiety.

135. The BMP-11 propeptide of claim 134, wherein the second moiety is chosen from a protein, polypeptide, carbohydrate, and nonproteinaceous polymer.

136. The BMP-11 propeptide of claim 119, wherein the BMP-11 propeptide further comprises an immunoglobulin molecule, or a fragment thereof.

137. A pharmaceutical composition comprising the BMP-11 propeptide of claim 119 and a pharmaceutically acceptable excipient.

140. The BMP-11 propeptide according to any one of claims 119, 138, and 139, wherein the mutation inactivates a proteolytic cleavage site.

138. A BMP-11 propeptide comprising a non-human BMP-11 propeptide homolog of SEQ ID NO:

11 chosen from bovine, chicken, murine, rat, porcine, ovine, turkey, baboon, and fish, wherein the propeptide has a mutation that modifies an aspartate residue at position 98 of SEQ ID NO;

11, wherein the BMP-11 propeptide has an increased in vivo or in vitro half-life; and

wherein the modified BMP-11 propeptide inhibits one or more BMP-11 or GDF-8 activities chosen from BMP-11 propeptide binding, negative regulation of skeletal muscle mass, modulation of preadipocyte differentiation, inhibition of muscle formation, inhibition of muscle cell growth, inhibition of muscle development, regulation of muscle-specific enzymes, inhibition of myoblast cell proliferation, modulation of preadipocyte differentiation to adipocytes, increasing sensitivity to insulin, regulation of glucose uptake, glucose hemostasis, and modulation of neuronal cell development and maintenance.

139. A BMP-11 propeptide comprising an amino acid sequence that is at least 95% identical to SEQ ID NO:

11, wherein the BMP-11 propeptide has a mutation that modifies the aspartate residue at position 98 of SEQ ID NO;

11, wherein the mutation increases the in vivo or in vitro half-life of the BMP-11 propeptide, and wherein the BMP-11 propeptide inhibits a BMP-11 activity associated with negative regulation of skeletal muscle mass.

141. A nucleic acid molecule encoding a BMP-11 propeptide comprising a nucleic acid sequence that encodes a BMP-11 propeptide that is at least 95% identical to SEQ ID NO:

11 and has a mutation that modifies an aspartate residue corresponding to aspartate 98 of SEQ ID NO;

11, wherein the encoded propeptide inhibits at least one BMP-11 or GDF-8 biological activity.

142. The nucleic acid of claim 141, wherein the BMP-11 propeptide has an increased in vivo or in vitro half life relative to a corresponding unmodified BMP-11 propeptide.

143. The nucleic acid of claim 141, wherein the BMP-11 propeptide inhibits one or more BMP-11 or GDF-8 activities chosen from BMP-11 or GDF-8 propeptide binding, negative regulation of skeletal muscle mass, modulation of preadipocyte differentiation, inhibition of muscle formation, inhibition of muscle cell growth, inhibition of muscle development, regulation of muscle-specific enzymes, inhibition of myoblast cell proliferation, modulation of preadipocyte differentiation to adipocytes, increasing sensitivity to insulin, regulation of glucose uptake, glucose hemostasis, and modulation of neuronal cell development and maintenance.

144. The nucleic acid of claim 141, wherein the nucleic acid encodes an alanine residue at the position corresponding to aspartate 98 of SEQ ID NO:

11.

145. The nucleic acid of claim 141 further comprising a nucleic acid sequence encoding an Fc region of an IgG molecule.

146. The nucleic acid of claim 145, wherein the IgG molecule is IgG1 or IgG4.

147. The nucleic acid of claim 145, wherein the nucleic acid encodes an amino acid sequence that is at least 95% identical to SEQ ID NO:

16.

148. The nucleic acid of claim 141, further comprising a nucleic acid sequence encoding albumin or an albumin derivative.

149. The nucleic acid of claim 141, wherein the nucleic acid encodes a modified BMP-11 propeptide with an altered glycosylation site.

150. The nucleic acid of claim 141, wherein the nucleic acid encodes a second moiety.

151. The nucleic acid of claim 150, wherein the second moiety is a protein or a polypeptide.

152. The nucleic acid of claim 141, wherein the BMP-11 propeptide further comprises an immunoglobulin molecule, or a fragment thereof.

156. A pharmaceutical composition comprising the nucleic acid of any one of claims 141, 153, or 154 and a pharmaceutically acceptable carrier or vector.

157. A vector comprising the nucleic acid of any one of claims 141, 153, or 154.

158. The vector of claim 157 further comprising a nucleic acid that encodes an Fc region of an IgG molecule.

159. The vector of claim 158 further comprising a linker peptide between the nucleotides encoding the BMP-11 propeptide and the IgG Fc region.

160. The vector of claim 159, wherein the linker peptide comprises nucleotides encoding for the amino acid sequence consisting of glycine-serine-glycine-serine (GSGS).

161. A host cell comprising the vector of claim 157.

162. A method of making a BMP-11 propeptide that has been modified at the aspartate residue corresponding to aspartate 98 of SEQ ID NO:

11 comprising;

(a) preparing a nucleic acid molecule according to claim 141, 153, or 154;

(b) preparing a nucleic acid molecule encoding the Fc region of the IgG molecule; and

(c) fusing the a nucleic acid molecules from steps (a) and (b) to produce a modified BMP-11 propeptide.

163. The method of claim 162, further comprising preparing a double stranded oligonucleotide encoding a linker peptide and fusing the nucleic acid molecules from steps (a) and (b) to either end of the double stranded oligonucleotide encoding the linker peptide.

164. The method of claim 163, wherein the linker peptide comprises the amino acid sequence consisting of glycine-serine-glycine-serine (GSGS).

180. A host cell comprising the vector of claim 158.

181. A host cell comprising the vector of claim 159.

182. A host cell comprising the vector of claim 160.

153. A nucleic acid molecule encoding a BMP-11 propeptide comprising a nucleic acid sequence that is at least 95% identical to SEQ ID NO:

12 and encodes a propeptide that is modified at the aspartate residue corresponding to aspartate 98 of SEQ ID NO;

11, wherein the encoded propeptide inhibits at least one BMP-11 or GDF-8 biological activity.

155. The nucleic acid according to claims 153 or 154, wherein the BMP-11 propeptide negatively regulates skeletal muscle mass.

154. A nucleic acid molecule encoding a BMP-11 propeptide comprising a nucleic acid that hybridizes under stringent hybridization conditions (hybridization at 65°

C. in 6×

SSC) to the complement of SEQ ID NO;

12 and encodes a propeptide that has a mutation that modifies the aspartate residue corresponding to aspartate 98 of SEQ ID NO;

11, wherein the encoded propeptide inhibits at least one BMP-11 biological activity.

165. A method of treating a patient suffering from a medical disease or disorder comprising:

administering a therapeutically effective amount of a BMP-11 propeptide that has a mutation that modifies the aspartate residue corresponding to aspartate 98 of SEQ ID NO;

11 and a pharmaceutically acceptable excipient to said patient, wherein the BMP-11 propeptide comprises an amino acid sequence that is at least 95% identical to SEQ ID NO;

11 and has a mutation that modifies the aspartate residue at position 98 of SEQ ID NO;

11, and wherein the BMP-11 propeptide inhibits one or more BMP-11 or GDF-8 activities chosen from BMP-11 or GDF-8 propeptide binding, negative regulation of skeletal muscle mass, modulation of preadipocyte differentiation, inhibition of muscle formation, inhibition of muscle cell growth, inhibition of muscle development, regulation of muscle-specific enzymes, inhibition of myoblast cell proliferation, modulation of preadipocyte differentiation to adipocytes, increasing sensitivity to insulin, regulation of glucose uptake, glucose hemostasis, and modulation of neuronal cell development and maintenance thereby treating a patient suffering from the medical disease or disorder.

166. The method of claim 165, wherein the BMP-11 propeptide has an increased in vivo or in vitro half life relative to a corresponding unmodified BMP-11 propeptide.

167. The method of claim 165, wherein the BMP-11 propeptide inhibits negative regulation of skeletal muscle mass.

168. The method of claim 165, wherein the BMP-11 propeptide further comprises an Fc region of an IgG molecule.

169. The method of claim 168, wherein the IgG molecule is IgG1 or IgG4, or a derivative of IgG1 or IgG4.

170. The method of claim 168, wherein the Fc region of an IgG molecule is at least 95% identical to SEQ ID NO:

16.

171. The method of claim 165, wherein the medical disorder is a muscular or neuromuscular disorder.

172. The method of claim 171, wherein the muscular or neuromuscular disorder is muscular dystrophy.

173. The method of claim 171, wherein the muscular or neuromuscular disorder is amyotrophic lateral sclerosis.

174. The method of claim 171, wherein the muscular or neuromuscular disorder is selected from the group consisting of muscle atrophy, congestive obstructive pulmonary disease, muscle wasting syndrome, sarcopenia, and cachexia.

175. The method of claim 165, wherein the medical disorder is a metabolic disorder.

176. The method of claim 175, wherein the metabolic disorder is selected from the group consisting of type 2 diabetes, noninsulin-dependent diabetes mellitus, hyperglycemia, and obesity.

177. The method of claim 165, wherein the medical disorder is a bone degenerative disorder.

178. The method of claim 177, wherein the bone degenerative disorder is osteoporosis.

179. The method of claim 165, wherein the medical disorder is an adipose tissue disorder.