Active modified hedgehog proteins

• US 20060073566A1
• Filed: 11/15/2005
• Published: 04/06/2006
• Est. Priority Date: 11/28/1997
• Status: Abandoned Application

• Current Assignee: Curis, Inc.
• Original Assignee: Curis, Inc.
• Inventors: Lang, Kurt, Leser, Ulrike, Seytter, Tilmann
• Application Number:
  US11/273,989
• Publication Number:
  US 20060073566A1
• Time in Patent Office: Days
• US Class Current: 435/69.100
• 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: A61P 19/04 for non-specific disorders of the connective tissue
• CPC Class Codes: A61P 19/08 for bone diseases, e.g. rachitism, Paget's disease
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: C07K 14/46 from vertebrates
• CPC Class Codes: C07K 2319/02 containing a signal sequence
• CPC Class Codes: C12N 2799/026 where the vector is derived from a baculovirus

Abstract

The present invention provides isolated highly active hedgehog proteins esterified with a fatty acid having from 14 to 20 carbon atoms at the N-terminal domain of the protein. The highly active hh proteins are particularly useful therapeutic agents for treating bone disorders and neurodegenerative diseases. Methods for obtaining the highly active modified hedgehog proteins are also provided.

Claims

1. A protein in isolated form wherein said protein comprises a human hedgehog protein which is esterified at the N-terminal domain with a fatty acid having from 14 to 20 carbon atoms, said protein having a molecular weight of from about 19 to about 26 kD.

2. The protein of claim 1, wherein the fatty acid is palmitic acid.

3. The protein of claim 2, wherein the amino acid sequence of said protein is at least 80% homologous with the amino acid sequence of EMBL database sequence No. L38518.

4. The protein of claim 3, wherein said protein contains the amino acid sequence of EMBL database sequence No. L38518.

5. The protein of claim 3, wherein the protein is esterified at a cysteine residue at the N-terminal domain of said sequence.

6. A method for producing a hedgehog protein esterified with a fatty acid having from 14 to 20 carbon atoms comprising:

a) providing an insect cell in a medium, said medium and said insect cell being capable upon fermentation of producing a fatty acid having from 14 to 20 carbon atoms, said insect cell containing a baculovirus vector having a gene inserted therein capable of expressing a hedgehog protein;

b) fermenting said insect cell in said medium for a period of about 30 hours or less to produce the fatty acid and-express the hedgehog protein esterified with said fatty acid; and

c) isolating said esterified hedgehog protein from the protein produced during said fermentation.

7. The method of claim 6,.wherein the fermentation period of said host cell is from about 24 hours to about 27 hours.

8. The method of claim 6, further comprising isolating said esterified hedgehog protein in the presence of a protease inhibitor and a non-ionic detergent.

9. The method of claim 6, further comprising isolating said esterified hedgehog protein in the presence of suramin.

10. Post-translationally processed hedgehog protein mutant which is obtainable by expressing a gene which codes for a hedgehog protein in a baculovirus expression system in a fermentation for a period of up to 30 hours, purifying the cell supernatant in the presence of a protease inhibitor and a non-ionic detergent and isolating the hh mutant which binds to heparin-Sepharose and hydroxylapatite and is characterized in that this hh mutant

exhibits a molecular weight of 22±

2 kDa under alkylating conditions,

exhibits a molecular weight of 24±

2 kD under reducing conditions,

is stabilized with respect to its activity by suramin

is inactivated when 8 or more amino acids are cleaved N-terminally

is inactivated by 90% or more when incubated with 10 mmol/l DTE for 2.5 hours at 37°

C.,

induces an activity for alkaline phosphatase of ca. 90 nmol pNP/min/mg at a concentration of 5 nmol/l in the presence of suramin,

is not modified by cholesterol and

has an at least 50-fold activity compared to the recombinant hh protein isolated from the cytoplasm of E. coli.

14. Pharmaceutical composition containing a hh mutant as claimed in claim 10.

15. Pharmaceutical composition as claimed in claim 14, containing suramin, serum-albumin, a biocompatible matrix and/or a sequestering agent.

16. Process for the production of a pharmaceutical composition by combination of a hh mutant as claimed in claim 10 with a pharmaceutical auxiliary substance or with suramin.

17. Process for the production of a pharmaceutical composition by combination of a hh mutant as claimed in claim 10 with a biocompatible matrix and/or a sequestering agent.

11. Process for the production of a post-translationally processed hedgehog protein mutant by expressing a gene which codes for a hedgehog protein in a baculovirus expression system in a fermentation for a period of 24 to 27 hours, purifying the cell supernatant in the presence of a protease inhibitor and a non-ionic detergent and isolating the hh mutant which binds to heparin-Sepharose and hydroxylapatite and characterized in that this hh mutant

exhibits a molecular weight of 22±

2 kDa under alkylating conditions,

exhibits a molecular weight of 24±

2 kD under reducing conditions,

is stabilized with respect to its activity by suramin

is inactivated when 8 or more amino acids are cleaved N-terminally

is inactivated by 90% or more when incubated with 10 mmol/l DTE for 2.5 hours at 37°

C.,

induces an activity for alkaline phosphatase of ca. 90 nmol pNP/min/mg at a concentration of 5 nmol/l in the presence of suramin,

is not modified by cholesterol and

has an at least 50-fold activity compared to the recombinant hh protein isolated from the cytoplasm of E. coli.

12. Process as claimed in claim 11, wherein, after chromatography on heparin-Sepharose, it is dialysed against lower ionic strengths.

13. Process as claimed in claim 12, wherein the dialysis is carried out in the presence of 10-100 mmol/l sodium chloride.

18. Post-translationally processed hedgehog protein with an at least 50-fold higher activity than the hedgehog protein expressed cytoplasmically in E. coli.

19. Pharmaceutical composition containing a hedgehog protein as claimed in claim 18.

20. Process for the production of a pharmaceutical composition, wherein a hedgehog protein as claimed in claim 18 is used as an essential component of this pharmaceutical composition.