PEGylated tyrosyl-tRNA synthetase polypeptides

Patent metadata

Field	Value
Publication number	US-9714419-B2
Application number	US-201214237544-A
Country	US
Kind code	B2
Filing date	Aug 8, 2012
Priority date	Aug 9, 2011
Publication date	Jul 25, 2017
Grant date	Jul 25, 2017

Abstract

The present invention provides PEGylated tyrosyl-tRNA synthetase (YRS) polypeptides, compositions comprising the same, and methods of using such polypeptides and compositions for treating or diagnosing a variety of conditions. The PEGylated YRS polypeptides of the invention have improved controlled release properties, stability, half-life, and other pharmacokinetic properties compared to non-PEGylated YRS polypeptides.

First claim

The invention claimed is:

1. A PEGylated tyrosyl-tRNA synthetase (YRS) polypeptide selected from,
(a) a YRS polypeptide consisting of a sequence that is at least 95% identical to the sequence set forth in SEQ ID NO:7 YRS(1-353), which is modified by A4C, C67S, and C250S substitutions, where a maleimide monomethoxy polyethylene glycol (mPEG) derivative of about 40,000 Daltons is covalently attached via a thio ether linkage to A4C;
(b) a YRS polypeptide consisting of a sequence that is at least 98% identical to the sequence set forth in SEQ ID NO:7 YRS(1-353), which is modified by A4C, C67S, and C250S substitutions, where a maleimide monomethoxy polyethylene glycol (mPEG) derivative of about 40,000 Daltons is covalently attached via a thio ether linkage to A4C;
(c) a YRS polypeptide consisting of the sequence set forth in SEQ ID NO:7 YRS(1-353), which is modified by A4C, C67S, and C250S substitutions, where a maleimide monomethoxy polyethylene glycol (mPEG) derivative of about 40,000 Daltons is covalently attached via a thio ether linkage to A4C;
(d) a YRS polypeptide consisting of a sequence that is at least 95% identical to the sequence set forth in SEQ ID NO:7 YRS(1-353), which is modified by C67S, C250S, and A351C substitutions, where a maleimide monomethoxy polyethylene glycol (mPEG) derivative of about 40,000 Daltons is covalently attached via a thio ether linkage to A351C;
(e) a YRS polypeptide consisting of a sequence that is at least 98% identical to the sequence set forth in SEQ ID NO:7 YRS(1-353), which is modified by C67S, C250S, and A351C substitutions, where a maleimide monomethoxy polyethylene glycol (mPEG) derivative of about 40,000 Daltons is covalently attached via a thio ether linkage to A351; and
(f) a YRS polypeptide consisting of the sequence set forth in SEQ ID NO:7 YRS(1-353), which is modified by C67S, C250S, and A351C substitutions, where a maleimide monomethoxy polyethylene glycol (mPEG) derivative of about 40,000 Daltons is covalently attached via a thio ether linkage to A351C.

2. The PEGylated YRS polypeptide of claim 1 , wherein the PEGylated product exhibits a higher specific activity in a charging assay compared to the non PEGylated protein.

3. The PEGylated YRS polypeptide of claim 1 , comprising the structure:

4. The PEGylated YRS polypeptide of claim 1 , comprising the structure:

5. The PEGylated YRS polypeptide of claim 1 :

6. The PEGylated YRS polypeptide of claim 1 :

7. The PEGylated YRS polypeptide of claim 1 , wherein the YRS polypeptide has substantially the same secondary structure as unmodified YRS polypeptide, as determined via UV circular dichroism analysis.

8. The PEGylated YRS polypeptide of claim 1 , wherein the PEGylated YRS polypeptide has a plasma or sera pharmacokinetic AUC profile at least 5-fold greater than unmodified YRS polypeptide when administered to rats.

9. The PEGylated YRS polypeptide of claim 1 , wherein the PEGylated YRS polypeptide has greater than 2 fold the activity of the unPEGylated protein in a charging assay.

10. The PEGylated YRS polypeptide of claim 1 , wherein the PEGylated YRS polypeptide has greater than 3 fold the activity of the unPEGylated protein in a charging assay.

11. The PEGylated YRS polypeptide of claim 1 , wherein the PEGylated YRS polypeptide has greater than 4 fold the activity of the unPEGylated protein in a charging assay.

12. A dosing regimen which maintains an average steady-state concentration of YRS polypeptide in the subjects's plasma of between about 0.3 μg/ml and about 3 μg/ml when using a dosing interval of 3 days or longer, comprising administering to the patient a therapeutic dose of PEGylated YRS polypeptide of any of claim 1 .

13. A method for maintaining YRS polypeptide levels above the minimum effective therapeutic level in a subject in need thereof, comprising administering to the subject a therapeutic dose of PEGylated YRS polypeptide of claim 1 .

14. A method for treating a hematopoiesis related disease in a subject in need thereof, comprising administering to the subject a therapeutic dose of PEGylated YRS polypeptide of claim 1 .

15. The method of claim 14 , wherein the hematopoiesis related disease is selected from thrombocytopenia, lymphocytopenia, neutropenia, basopenia, eosinopenia, anemias, polycythemia, neutrophilia, eosinophilia, or basophilia.

16. A method for treating a subject with a myelodysplastic syndrome comprising administering to the subject a therapeutic dose of PEGylated YRS polypeptide of claim 1 and a pharmaceutically acceptable carrier.

17. The method of claim 16 , wherein the myelodysplastic syndrome is selected from Refractory Anemia (RA)(ICD-O code M9980/3), Refactory cytopenia with unilineage dysplasia (Refactory anemia, Refactory neutropenia, and Refractory thrombocytopenia), Refractory Anemia with Ring Sideroblasts (RARS) (ICD-O code M9982/3), Refractory Anemia with Ring Sideroblasts thrombocytosis, Refractory cytopenia with multilineage dysplasia (RCMD), Refractory Anemia with Excess Blasts (RAEB) (ICD-O code M9983/3), Refractory Anemia with Excess Blasts I or II, Refractory Anemia with Excess Blasts in Transformation (RAEB-T) (ICD-O code M9984/3), Chronic Myelomoncytic Leukemia (CMML) (ICD-O code M9945/3), 5q-syndrome, myelodysplastic-myeloproliferative overlap syndromes, Myelodysplasia unclassifiable, and refractory cytopenia of childhood.

18. A pharmaceutical composition comprising a PEGylated tyrosyl-tRNA synthetase (YRS) polypeptide of claim 1 and a pharmaceutically acceptable carrier or excipient.

19. The pharmaceutical composition of claim 18 , wherein the composition is buffered to a pH of about 5.5 to about 6.5.

20. The pharmaceutical composition of claim 18 , wherein the composition is buffered to a pH of about 6.0.

21. The pharmaceutical composition of claim 18 , wherein the composition is buffered with a phosphate buffer at a concentration of about 10 to 20 mM.

22. The pharmaceutical composition of claim 18 , wherein the composition is characterized by decreased aggregation of the PEGylated YRS polypeptides of any of claims 3 to 6 compared to a composition incubated under identical conditions but at pH 7.0 or higher.

Assignees

• Watkins Jeffrey D
• Buechler Ying Ji
• Wu Chi-Fang
• Do Minh-Ha
• Vasserot Alain P
• Mendlein John D
• Atyr Pharma Inc

Inventors

• Watkins Jeffrey D
• Buechler Ying Ji
• Wu Chi-Fang
• Do Minh-Ha
• Vasserot Alain P
• Mendlein John D

Classifications

• C12N9/93 Primary

  Ligases (6) · CPC title

• C12Y601/01001

  Tyrosine-tRNA ligase (6.1.1.1) · CPC title

• A61K38/00

  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 title

• A61P7/00

  Drugs for disorders of the blood or the extracellular fluid · CPC title

• A61K47/60

  the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol · CPC title