MODIFIED AND STABILIZED GDF PROPEPTIDES AND USES THEREOF

• US 20100305194A1
• Filed: 05/04/2009
• Published: 12/02/2010
• Est. Priority Date: 02/08/2001
• Status: Active Grant

• Current Assignee: Wyeth LLC (Pfizer Inc.)
• Original Assignee: Wyeth (Pfizer Inc.)
• Inventors: Khor, Soo-Peang, Tomkinson, Kathleen N., Wolfman, Neil M.
• Granted Patent: US 8,222,384 B2
• Time in Patent Office: Days
• US Class Current: 514/44R
• 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 nucleic acid molecule encoding a GDF-8 propeptide comprising a nucleic acid sequence that encodes a GDF-8 propeptide that is at least 95% identical to SEQ ID NO:

5 and that is modified at the aspartate residue corresponding to aspartate 76 of SEQ ID NO;

5.

120. The nucleic acid of claim 119, wherein the GDF-8 propeptide has an increased in vivo or in vitro half life relative to a corresponding unmodified GDF-8 propeptide.

121. The nucleic acid of claim 119, wherein the GDF-8 propeptide inhibits one or more GDF-8 activities chosen from 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.

122. The nucleic acid of claim 119, wherein the nucleic acid encodes an alanine residue at the position corresponding to aspartate 76 of SEQ ID NO:

5.

123. The nucleic acid of claim 119 further comprising a nucleic acid sequence encoding an Fc region of an IgG molecule.

124. The nucleic acid of claim 123, wherein the IgG molecule is IgG1 or IgG4.

125. The nucleic acid of claim 123, wherein the nucleic acid encodes an amino acid sequence that is at least 95% identical to SEQ ID NO:

16.

126. The nucleic acid of claim 119, further comprising a nucleic acid sequence encoding albumin or an albumin derivative.

127. The nucleic acid of claim 119, wherein the nucleic acid encodes a modified GDF-8 propeptide with an altered glycosylation site.

128. The nucleic acid of claim 119, wherein the nucleic acid encodes a second moiety.

129. The nucleic acid of claim 128, wherein the second moiety is a protein or a polypeptide.

130. The nucleic acid of claim 119, wherein the GDF-8 propeptide further comprises an immunoglobulin molecule, or a fragment thereof.

134. A pharmaceutical composition comprising the nucleic acid of claim 119 and a pharmaceutically acceptable carrier or vector.

135. A vector comprising the nucleic acid of claim 119.

137. The vector of claim 135 further comprising a nucleic acid that encodes an Fc region of an IgG molecule.

138. The vector of claim 137 further comprising a linker peptide between the nucleotides encoding the GDF-8 propeptide and the IgG Fc region.

139. The vector of claim 138, wherein the linker peptide comprises nucleotides encoding for the amino acid sequence consisting of glycine-serine-glycine-serine (GSGS) (SEQ ID NO:

17).

140. A host cell comprising the vector of any one of claims 135, 136, 137, 138, and 139.

141. The method of making a GDF-8 propeptide that has been modified at the aspartate residue corresponding to aspartate 76 of SEQ ID NO:

5 comprising;

(a) preparing a nucleic acid molecule according to claim 119, 131, or 132;

(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 GDF-8 propeptide.

142. The method of claim 141, further comprising preparing a double stranded oligonucleotide encoding a linker peptide and fusing the cDNA molecules from steps (a) and (b) to either end of the double stranded oligonucleotide encoding the linker peptide.

143. The method of claim 142, wherein the linker peptide comprises the amino acid sequence consisting of glycine-serine-glycine-serine (GSGS).

144. The pharmaceutical composition of claim 134, wherein the vector is a viral vector.

145. The vector of claim 135 or 136, wherein the vector is a viral vector.

146. The nucleic acid molecule of claim 119, further comprising a nucleic acid sequence encoding a nonproteineous polymer.

131. A nucleic acid molecule encoding a GDF-8 propeptide comprising a nucleic acid sequence that is at least 95% identical to SEQ ID NO:

6 and encodes a propeptide that is modified at the aspartate residue corresponding to aspartate 76 of SEQ ID NO;

5.

132. A nucleic acid molecule encoding a GDF-8 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;

6 and encodes a propeptide that is modified at the aspartate residue corresponding to aspartate 76 of SEQ ID NO;

5, wherein the encoded propeptide inhibits at least one GDF-8 biological activity.

133. The nucleic acid of claim 132, wherein the GDF-8 propeptide negatively regulates skeletal muscle mass.

136. A vector comprising the nucleic acid of claim 132.

147. A nucleic acid molecule encoding a GDF-8 propeptide comprising a nucleic acid sequence that encodes a GDF-8 propeptide that is at least 95% identical to SEQ ID NO:

5 and that is modified at the aspartate residue corresponding to aspartate 76 of SEQ ID NO;

5, wherein the encoded propeptide inhibits at least one GDF-8 biological activity. 148 (New) A nucleic acid molecule encoding a GDF-8 propeptide comprising a nucleic acid sequence that is at least 95% identical to SEQ ID NO;

6 and encodes a propeptide that is modified at the aspartate residue corresponding to aspartate 76 of SEQ ID NO;

5, wherein the encoded propeptide inhibits at least one GDF-8 biological activity.