Vertebrate tissue pattern-inducing proteins, and uses related thereto

• US 6,576,237 B1
• Filed: 08/16/2000
• Issued: 06/10/2003
• Est. Priority Date: 12/30/1993
• Status: Expired due to Fees

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation), Imperial Cancer Research Technology, Ltd.
• Original Assignee: President and Fellows of Harvard College (Harvard Corporation), Imperial Cancer Research Technology, Ltd.
• Inventors: Bumcrot, David A., Marti-Gorostiza, Elisa, Ingham, Philip W., McMahon, Andrew P., Tabin, Clifford J.
• Primary Examiner(s): Eyler, Yvonne
• Assistant Examiner(s): Andres, Janet L.
• Application Number:
  US09/639,695
• Time in Patent Office: 1,028 Days
• Field of Search: 530/388.1, 530/389.1, 530/350
• US Class Current: 424/158.1
• CPC Class Codes: A01K 2217/05 Animals comprising random inserted nucleic acids (transgenic)
• CPC Class Codes: A01K 2227/105 Murine
• CPC Class Codes: A01K 2227/30 Bird
• CPC Class Codes: A01K 2227/50 Amphibians, e.g. Xenopus
• CPC Class Codes: A01K 2267/03 Animal model, e.g. for test or diseases
• CPC Class Codes: A01K 2267/0318 Animal model for neurodegenerative disease, e.g. non- Alzheimer's
• CPC Class Codes: A01K 2267/0368 Animal model for inflammation
• CPC Class Codes: A01K 67/0271 Chimeric vertebrates, e.g. comprising exogenous cells
• CPC Class Codes: A01K 67/0275 Genetically modified vertebrates, e.g. transgenic
• CPC Class Codes: A01K 67/033 Rearing or breeding invertebrates; New breeds of invertebrates
• CPC Class Codes: A01K 67/0339 Genetically modified insects, e.g. Drosophila melanogaster, medfly
• 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: C07K 14/46 from vertebrates
• CPC Class Codes: C07K 14/475 Growth factors; Growth regulators
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C12N 15/8509 for producing genetically modified animals, e.g. transgenic
• CPC Class Codes: C12N 2799/027 where the vector is derived from a retrovirus

Abstract

The present invention concerns the discovery that proteins encoded by a family of vertebrate genes, termed here hedgehog-related genes, comprise morphogenic signals produced by tissue patterning centers, and are involved in the formation of ordered spatial arrangements of differentiated tissues in vertebrates. The present invention makes available compositions and methods that can be utilized, for example to generate and/or maintain an array of different vetebrate tissue both in vitro and in vivo.

Claims

1. An isolated antibody, or fragment thereof, wherein said antibody or fragment is specifically immunoreactive with a hedgehog polypeptide that binds to a patched receptor, and which polypeptide is encoded by a nucleic acid sequence which hybridizes under stringent conditions, including a wash step of 0.2×

SSC at 65°

C., to a nucleotide sequence of SEQ ID Nos;

1-7, and wherein further said antibody does not substantially cross-react with a polypeptide less than 85% identical to an amino acid sequence as set forth in any of SEQ ID Nos;

8-14.

3. An isolated antibody of claim 1, wherein said antibody is specifically immunoreactive with a hedgehog polypeptide that binds to a patched receptor, and which hedgehog polypeptide is encoded by a nucleic acid sequence of any of SEQ ID Nos:

1-7.

7. The antibody of any one of claims 1-2, wherein said antibody is a monoclonal antibody.

8. The antibody or fragment of any one of claims 1-2, wherein said fragment is a Fab fragment.

9. The antibody or fragment of any one of claims 1-2, wherein said fragment is a F(ab′

)₂ fragment.

10. The antibody of any one of claims 1-2, wherein said antibody is labeled with a detectable label.

11. The antibody of any one of claims 1-2, wherein said antibody is specifically immunoreactive with a hedgehog polypeptide as set forth in any of SEQ ID Nos:

8, 11, 12 and 13.

12. The antibody of any one of claims 1-2, wherein said antibody is immuno-selective for a hedgehog polypeptide as set forth in SEQ ID No:

9.

13. The antibody of any one of claims 1-2, wherein said antibody is immuno-selective for a hedgehog polypeptide as set forth in SEQ ID Nos:

10 and 14.

2. An isolated antibody, or fragment thereof, wherein said antibody or fragment is specifically immunoreactive with a hedgehog polypeptide comprising an amino acid sequence at least 85% identical to an amino acid sequence as set forth in any of SEQ ID Nos:

8-14 and which antibody does not substantially cross-react with a polypeptide less than 85% identical to an amino acid sequence as set forth in any of SEQ ID Nos;

8-14.

4. An isolated antibody of claim 2, wherein said antibody is specifically immunoreactive with a hedgehog polypeptide comprising an amino acid sequence at least 90% identical to an amino acid sequence as set forth in any of SEQ ID Nos:

8-14.

5. An isolated antibody of claim 2, wherein said antibody is specifically immunoreactive with a hedgehog polypeptide comprising an amino acid sequence at least 95% identical to an amino acid sequence as set forth in any of SEQ ID Nos:

8-14.

6. An isolated antibody of claim 2, wherein said antibody is specifically immunoreactive with a hedgehog polypeptide comprising an amino acid sequence as set forth in any of SEQ ID Nos:

8-14.

14. A purified preparation of polyclonal antibodies, or fragments thereof, wherein said antibodies or fragments are specifically immunoreactive with a hedgehog polypeptide that binds to a patched receptor, and which hedgehog polypeptide is encoded by a nucleic acid sequence which hybridizes under stringent conditions, including a wash step of 0.2×

SSC at 65°

C. to a nucleotide sequence of any of SEQ ID Nos;

1-7, and wherein further said antibodies do not substantially cross-react with a polypeptide less than 85% identical to an amino acid sequence as set forth in any of SEQ ID Nos;

8-14.

15. A purified preparation of polyclonal antibodies, or fragments thereof, wherein said antibodies or fragments are specifically immunoreactive with a hedgehog polypeptide comprising an amino acid sequence at least 85% identical to an amino acid sequence as set forth in any of SEQ ID Nos:

8-14 and which antibodies do not substantially cross-react with a polypeptide less than 85% identical to an amino acid sequence as set forth in any of SEQ ID Nos;

8-14.

16. A kit for detecting a hedgehog polypeptide protein comprising (i) isolated anti-hedgehog antibodies, or fragment thereof, specifically immunoreactive with a vertebrate hedgehog polypeptide comprising an amino acid sequence at least 85% identical to an amino acid sequence as set forth in any of SEQ ID NOs:

8-14 and which antibodies do not substantially cross-react with a polypeptide less than 85% identical to any of SEQ ID Nos;

8-14, (ii) a detectable label for detecting said anti-hedgehog antibody in immunocomplexes with said hedgehog polypeptide.

17. The kit of claim 16, wherein means for detecting said anti-hedgehog antibody is a second antibody immunoreactive with the anti-antibody.

18. The kit of claim 16, wherein the anti-hedgehog polypeptide antibody is specifically immunoreactive with a polypeptide comprising an amino acid sequence selected from the group comprising SEQ ID Nos. 8-14.

19. The kit of claim 16, wherein said antibody is a monoclonal antibody.

20. The kit of claim 16, wherein said antibody comprises a purified preparation of polyclonal antibodies.

21. The kit of claim 16, wherein said antibody is a is a Fab fragment.

22. The kit of claim 16, wherein said antibody is a F(ab′

)₂ fragment.

23. The kit of claim 16, wherein said antibody is provided in a form suitable for detecting the polypeptide in samples in cells.

24. An isolated antibody, or fragment thereof, wherein said antibody or fragment is specifically immunoreactive with a hedgehog polypeptide, and which polypeptide is encoded by a nucleic acid sequence which hybridizes under stringent conditions, including a wash step of 0.2×

SSC at 65°

C., to a nucleotide sequence of any of SEQ ID Nos;

1-7.

26. An isolated antibody of claim 24, wherein said antibody is specifically immunoreactive with a hedgehog polypeptide, and which polypeptide is encoded by a nucleic acid sequence of any of SEQ ID Nos:

1-7.

25. An isolated antibody, or fragment thereof, wherein said antibody or fragment is specifically immunoreactive with a hedgehog polypeptide comprising an amino acid sequence at least 85% identical to an amino acid sequence as set forth in any of SEQ ID Nos:

8-14.

27. An isolated antibody of claim 25, wherein said antibody is specifically immunoreactive with a hedgehog polypeptide comprising an amino acid sequence at least 90% identical to an amino acid sequence as set forth in any of SEQ ID Nos:

8-14.

28. An isolated antibody of claim 25, wherein said antibody is specifically immunoreactive with a hedgehog polypeptide comprising an amino acid sequence at least 95% identical to an amino acid sequence as set forth in any of SEQ ID Nos:

8-14.

29. An isolated antibody of claim 25, wherein said antibody is specifically immunoreactive with a hedgehog polypeptide comprising an amino acid sequence as set forth in any of SEQ ID Nos:

8-14.

30. A purified preparation of polyclonal antibodies, or fragments thereof, wherein said antibodies or fragments are specifically immunoreactive with a hedgehog polypeptide, and which hedgehog polypeptide is encoded by a nucleic acid sequence which hybridizes under stringent conditions, including a wash step of 0.2×

SSC at 65°

C. to a nucleotide sequence of any of SEQ ID Nos;

1-7.

31. A purified preparation of polyclonal antibodies, or fragments thereof, wherein said antibodies or fragments are specifically immunoreactive with a hedgehog polypeptide comprising an amino acid sequence at least 85% identical to an amino acid sequence as set forth in any of SEQ ID Nos:

8-14.