Recombinant bone morphogenetic protein heterodimers

• US 5,866,364 A
• Filed: 11/27/1992
• Issued: 02/02/1999
• Est. Priority Date: 11/04/1991
• Status: Expired due to Term

• Current Assignee: Genetics Institute LLC (Pfizer Inc.)
• Original Assignee: Genetics Institute LLC (Pfizer Inc.)
• Inventors: Wolfman, Neil M., Israel, David
• Primary Examiner(s): Allen, Marianne P.
• Application Number:
  US07/989,847
• Time in Patent Office: 2,258 Days
• Field of Search: 435/69.1, 435/69.4, 435/70.2, 435/240.1, 435/240.2, 435/252.3, 435/252.33, 435/255, 435/256, 435/320.1, 536/23.5, 536/23.51, 530/350, 530/399
• US Class Current: 435/69.1
• 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: A61L 27/227 Other specific proteins or polypeptides not covered by A61L27/222, A61L27/225 or A61L27/24
• CPC Class Codes: A61P 17/02 for treating wounds, ulcers, burns, scars, keloids, or the like
• CPC Class Codes: A61P 19/08 for bone diseases, e.g. rachitism, Paget's disease
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C07K 14/51 Bone morphogenetic factor; Osteogenins; Osteogenic factor; Bone-inducing factor

Abstract

The present invention relates to a methods for producing recombinant heterodimeric BMP proteins useful in the field of treating bone defects, healing bone injury and in wound healing in general. The invention also relates to the recombinant heterodimers and compositions containing them.

Claims

1. A method for producing a heterodimeric protein having bone stimulating activity comprising

(a) culturing under suitable conditions to produce said protein a selected host cell containing a first DNA sequence encoding BMP-2 and a second DNA sequence encoding BMP-6 said sequences each being under the control of a suitable regulatory sequence capable of directing co-expression of said proteins;

and

(b) isolating said heterodimeric protein from the culture medium.

2. The method according to claim 1 wherein said BMP-2 sequence is present on a first vector transfected into said host cell and said BMP-6 sequence is present on a second vector transfected into said host cell.

3. The method according to claim 2 wherein more than a single copy of the gene encoding each said BMP is present on each vector.

4. The method according to claim 1 wherein both said BMP sequences are integrated into a chromosome of said host cell.

5. The method according to claim 1 wherein both BMP sequences are present on a single vector.

6. The method according to claim 1 wherein said host cell is a mammalian cell.

7. The method according to claim 1 wherein said host cell is a yeast cell.

8. A method for producing a heterodimeric protein having bone stimulating activity comprising

(a) culturing under suitable conditions to produce said protein a selected host cell containing a first BMP DNA sequence encoding a BMP selected from the group consisting of BMP-2 and BMP-4 and a second BMP DNA sequence encoding BMP-6 wherein said host cell is a hybrid cell prepared by culturing two fused selected, stable host cells, each host cell transfected with a DNA sequence encoding said selected first or second BMP, said sequences under the control of a suitable regulatory sequence capable of directing expression of each protein;

and

(b) isolating said heterodimer protein from the culture medium.

9. A method for producing heterodimeric protein having bone stimulating activity in a bacterial cell comprising

(a) culturing a selected host cell containing a DNA sequence encoding BMP-2 under the control of a suitable regulatory sequence capable of directing expression of the protein under conditions suitable for the formation of a soluble, monomeric protein isolating said monomeric protein from inclusion bodies and/or said culture medium;

(b) culturing a selected host cell containing a second DNA sequence encoding BMP-6 under the control of a suitable regulatory sequence capable of directing expression of the protein or protein fragment under said conditions to form a second soluble, monomeric protein;

(c) isolating said monomeric protein from inclusion bodies and/or said culture medium;

(d) mixing said soluble monomeric proteins under conditions permitting the formation of dimeric proteins associated by at least one covalent disulfide bond; and

(e) isolating from the mixture a heterodimeric protein.

10. The method according to claim 9 wherein said host cell is E. coli.

11. The method according to claim 9 wherein said conditions comprise treating said protein with a solubilizing agent.

12. An isolated recombinant heterodimeric protein having bone stimulating activity comprising a BMP-2 protein subunit associated by at least one disulfide bond with a BMP-6 protein subunit.

13. A cell line comprising a nucleotide sequence encoding BMP-2 under control of a suitable expression regulatory system and a second nucleotide sequence encoding BMP-6 under control of a suitable expression regulatory system, said regulatory systems capable of directing the co-expression of said BMPs and the formation of heterodimeric protein.

14. The cell line according to claim 13 wherein said nucleotide sequences encoding said BMP-2 and BMP-6 proteins are present in a single DNA molecule.

15. The cell line according to claim 14 wherein said single DNA molecule comprises a first transcription unit containing a gene encoding said BMP-2 and a second transcription unit containing a gene encoding said BMP-6.

16. The cell line according to claim 14 wherein said single DNA molecule comprises a single transcription unit containing multiple copies of said gene encoding said BMP-2 and multiple copies of said gene encoding said BMP-6.

17. The cell line according to claim 13 wherein said nucleotide sequence encoding said BMP-2 is present on a first DNA molecule and said nucleotide sequence encoding said BMP-6 is present on a second DNA molecule.

18. A DNA molecule comprising a sequence encoding BMP-2 and a second sequence encoding BMP-6 said sequences under the control of at least one suitable regulatory sequence capable of directing co-expression of each BMP.

19. The molecule according to claim 18 comprising a first transcription unit containing a gene encoding said BMP-2 and said second transcription unit containing a gene encoding BMP-6.

20. The molecule according to claim 18 comprising a single transcription unit containing multiple copies of said gene encoding said BMP-2 and multiple copies of said gene encoding said BMP-6.