Methods of identifying morphogen receptor-binding analogs Number:7,067,260 from the United States Patent and Trademark Office (PTO) owispatent

Title: Methods of identifying morphogen receptor-binding analogs

Abstract: Disclosed are (1) nucleic acid sequences, amino acid sequences, homologies, structural features and various other data characterizing a morphogen cell surface receptors particularly OP-1-binding cell surface receptors; (2) methods for producing receptor proteins, including fragments thereof, using recombinant DNA technology; (3) methods for identifying novel morphogen receptors and their encoding DNAs; (4) methods and compositions for identifying compounds capable of modulating endogenous morphogen receptor levels; and (5) methods and compositions for identifying morphogen receptor binding analogs useful in the design of morphogen agonists and antagonists for therapeutic, diagnostic and experimental uses.

Patent Number: 7,067,260 Issued on 06/27/2006 to Dijke,   et al.

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Inventors:	Dijke; Peter ten (Uppsala, SE); Heldin; Carl-Henrik (Uppsala, SE); Miyazono; Kohei (Saitama, JP); Sampath; Kuber T. (Holliston, MA)
Assignee:	Curis, Inc. (Cambridge, MA)
Appl. No.:	982543
Filed:	October 18, 2001

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Current U.S. Class:	435/7.1 ; 435/7.2
Current International Class:	G01N 33/53 (20060101); G01N 33/567 (20060101)
Field of Search:	530/350 435/7.1,7.2

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References Cited [Referenced By]

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U.S. Patent Documents

	5434067		July 1995		Michaelis et al.
	5538892		July 1996		Donahoe et al.

Foreign Patent Documents

	WO 90/05802		May., 1991		WO
	WO 91/05802		May., 1991		WO
	WO 93/05172		Mar., 1993		WO
	WO 93/19177		Sep., 1993		WO
	WO 94/11502		May., 1994		WO
	WO 95/07982		Mar., 1995		WO
	WO 95/14778		Jun., 1995		WO

Other References

Stratagene Catalog, p. 39, 1988. cited by examiner . Attisano et al. Identification of Human Activin & TGF-.beta. . . . with Type II Receptors. Cell 75, 671-680 (1993). cited by other . Bassing et al. A Transforming Growth Factor .beta. Type I Receptor . . . Gene Expression. Science 263, 87-89 (1994). cited by other . Cagan et al. The role of induction in cell choice and cell cycle in the developing drosophila retina. Molecular Basis of Morphogenesis. M. Bernfield, ed. Wiley-Liss, N.Y., 109-133 (1993). cited by other . Childs et al. Identification of a Drosophila Activin Receptor. PNAS 90. 9475-9479 (1993). cited by other . Ebner et al. Cloning of a Type 1 TGF-.beta. Receptor and Its Effect on TGF-.beta. Binding to the Type II Receptor. Science 260, 1344-1348 (1993). cited by other . Ebner et al. Determination of Type 1 Receptor Specificity by the Type II Receptors for the TGF-.beta. or Activin. Science 262, 900-902 (1993). cit- ed by other . Estevez et al. The daf-4 Gene Encodes a Bone Morphogenetic . . . Dauer Larva Development. Nature 365, 644-649 (1993). cited by other . Frazen et al. Cloning of a TGF-.beta. Type I Receptor that Forms a Heteromeric Complex with the TGF-62 Type II Receptor. Cell 75. 681-692 (1993). cited by other . Gibbs, W. More fun than a root canal. Scientific Am. 269, 106 (Nov. 1993). cited by other . He et al. Development Expression of Four Novel Serine/Threonine Kinase . . . Type II Receptor Family. Development Dynamics 196, 133-142 (1993). cite- d by other . Inagaki et al. Growth Inhibition by Transforming Growth Factor .beta.Type I . . . TGF-.beta. Receptor Type II cDNA. PNAS 90, 5359-5363 (1993). cite- d by other . Kawabata et al. Cloning of a Novel Type II Serine/Threonine Kinase Receptor Through Interaction with the Type I Transforming Growth Factor-.beta. Receptor. J. Biol. Chem. 270, 5625-5630 (1995). cited by other . Koenig et al. Characterization and Cloning of a Receptor for BMP-2 and BMP-4 from NIH 3T3 Cells. Mol. Cell. Biol. 14, 5961-5974 (1994). cited by other . Lin et al. Receptors for the TGF-.beta. Superfamily: Multiple Polypeptides and Serine/Threonine Kinases. Cell 3, 14-25 (1993). cited by other . Massague. Receptors for the TGF-.beta. Family. Cell 69, 1067-1070 (1992). cited by other . Mathews et al. Cloning of a Second Type of Activin Receptor and Functional Characterization in Xenopus Embryos. Science 255, 1702-1705 (1992). cited by other . Matsuzaki et al. A Widely Expressed Transmembrane Serine/Threonine Kinase . . . Bone Morphogenic Factor. J. Biol. Chem. 268, 12719-12722 (1993). cited by other . Paralkar et al. Identification and Characterization of Cellular Binding Proteins . . . Bone Differentiation Cascade. PNAS 88, 3397-3401 (1991). cited by other . Sampath et al. Recombinant human ostegenic protein-1 (hOP-1) induces new bone formation in vivo with a specific activity comparable with natural bovine ostogenic protein and stimulates osteoblast proliferation and differentiation in vitro. J. Biol. Chem. (Oct. 1992), 267(28): 20352-20362. cited by other . Short Protocols in Molecular Biology. Ausubel et al., eds. John Wiley & Sons, N.Y. 185-191 (1989). cited by other . Strader et al. Structural basis of beta-adrenergic receptor function. FASEB J. 3, 1825-1832 (1989). cited by other . ten Dijke et al. Activin Receptor-Like Kinases: A Novel Subclass of Cell Surface Receptors with Predicted Serine/Threonine Kinase Activity. Oncogene 8, 2879-2887 (1993). cited by other . ten Dijke et al. Characterization of Type I Receptors for Transforming Growth Factor-.beta. and Activin. Science 264, 101-103 (1994). cited by other . ten Dijke et al. Identification of Type I Receptors for Osteogenic Protein-1 and Bone Morphogenetic Protein-4. J. Biol. Chem. 269, 16985-16988 (1994). cited by other . Thompson et al. Vargula hilgendorfii luciferase: a secreted reporter enzyme for monitoring gene expression in mammalian cells. Gene 96, 257-262 (1990). cited by other . Tsuchida et al. Cloning and Characterization of a Transmembrane Serine Kinase . . . Type I Receptor. PNAS 90, 11242-11246 (1993). cited by other . Vukicevic et al. Localization of Ostogenic Protein-1 (Bone Morphogenetic Protein-7) During Human Embryonic Development: High Affinity Binding to Basement Membranes. Biochem. & Biophys. Res. Comm. 198, 693-700 (1994). cited by other . Wrana et al. TGF-.beta. Signals Through a Heteromeric Protein Kinase Receptor Complex. Cell 71, 1003-1014 (1992). cited by other . Yamaji et al. The molecular cloning of bone morphogenic protein receptors. J. Bone & Min. Res. 8, S145 (1993). cited by other.

Primary Examiner: Brumback; Brenda
Assistant Examiner: Landsman; Robert S.
Attorney, Agent or Firm: Ropes & Gray, LLP

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Parent Case Text

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RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No. 08/448,371, filed Jun. 2, 1995 now abandoned, which is a continuation of International Application No. PCT/US95/05467, filed Apr. 28, 1995, which is a continuation-in-part of U.S. application Ser. No. 08/236,428, filed Apr. 29, 1994. International Application No. PCT/US95/05467 was published under PCT Article 21(2) in English.

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Claims

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What is claimed is:

1. A method for identifying a binding analog for a morphogen receptor, said morphogen sharing at least 60% amino acid sequence identity or at least 70% amino acid sequence homology with the sequence of the C-terminal 102 amino acid of SEQ ID NO: 7, and being able to substitute for OP-1 in binding to a protein comprising SEQ ID NOs. 4, 6, or 8, said binding analog having substantially the same binding affinity for said morphogen receptor as said morphogen, the method comprising: (a) providing a sample without a Type II serine/threonine kinase morphogen receptor but containing a protein selected from: (i) a polypeptide comprising an amino acid sequence defined by residues 16-123 of SEQ ID NO: 4 (ALK-2); (ii) a polypeptide comprising an amino acid sequence defined by residues 24-152 of SEQ ID NO: 6 (ALK-3); (iii) a polypeptide comprising an amino acid sequence defined by residues 23-122 of SEQ ID NO: 8 (ALK-6); or (iv) a polypeptide having binding affinity for OP-1 and encoded by a first nucleic acid capable of hybridizing under stringent conditions with a second nucleic acid comprising the sequence defined by nucleotides 256-552 of SEQ ID NO: 7 (ALK-6), the stringent conditions being hybridization in 40% formamide 5.times.SSPE, 5.times.Denhardt's Solution, 0.1% SDS at 37.degree. C. overnight, then washing in 0.1.times.SSPE, 0.1% SDS at 50.degree. C.; (b) contacting said sample with a candidate morphogen receptor-binding analog; and (c) detecting specific binding between said candidate morphogen receptor-binding analog and said protein; wherein binding of said candidate morphogen receptor-binding analog to said protein is indicative that said candidate analog is a morphogen receptor-binding analog.

2. A method for identifying a binding analog of an OP-1 receptor, said analog being characterized as having substantially the same binding affinity for a cell surface receptor protein as OP-1, the method comprising: (a) providing a cell that expresses a surface receptor protein having binding specificity for OP-1 selected from: (i) a polypeptide comprising an amino acid sequence defined by residues 16-123 of SEQ ID NO: 4 (ALK-2); (ii) a polypeptide comprising an amino acid sequence defined by residues 24-152 of SEQ ID NO: 6 (ALK-3); (iii) a polypeptide comprising an amino acid sequence defined by residues 23-122 of SEQ ID NO: 8 (ALK-6); or (iv) a polypeptide having binding affinity for OP-1 and encoded by a first nucleic acid capable of hybridizing under stringent conditions with a second nucleic acid comprising the sequence defined by nucleotides 256-552 of SEQ ID NO: 7 (ALK-6), the stringent conditions being hybridization in 40% formamide 5.times.SSPE, 5.times.Denhardt's Solution, 0.1% SDS at 37.degree. C. overnight, then washing in 0.1.times.SSPE, 0.1% SDS at 50.degree. C.; (b) contacting said cell with a candidate OP-1 receptor-binding analog; and (c) detecting induction of an OP-1-mediated cellular response; wherein detection of induction of said OP-1-mediated cellular response is indicative that said candidate analog is an OP-1 receptor-binding analog.

3. The method of claim 2 wherein said OP-1 mediated cellular response detected in step (c) is induction of threonine or serine-specific phosphorylation, inhibition of epithelial cell growth, or induction of a cell differentiation marker.

4. The method of claim 2 or 3 wherein said cell comprises a transfected nucleic acid comprising a reporter gene in operative association with a control element derived from an OP-1 inducible protein, and wherein the activity of said reporter gene can be detected as said OP-1-mediated cellular response upon stimulation by OP-1 or analog thereof in said cell.

5. The method of claim 2 or 3, wherein said surface receptor protein further comprises part or all of a Type II serine/threonine kinase receptor protein having binding affinity for OP-1, activin or BMP-4.

6. A kit for identifying OP-1 or a candidate OP-1 receptor binding analog in a sample, the kit comprising: (a) a receptacle adapted to receive said sample, said receptacle containing a protein selected from: (i) a polypeptide comprising an amino acid sequence defined by residues 16-123 of SEQ ID NO: 4 (ALK-2); (ii) a polypeptide comprising an amino acid sequence defined by residues 24-152 of SEQ ID NO: 6 (ALK-3); (iii) a polypeptide comprising an amino acid sequence defined by residues 23-122 of SEQ ID NO: 8 (ALK-6); or (iv) a polypeptide having binding affinity for OP-1 and encoded by a first nucleic acid capable of hybridizing under stringent conditions with a second nucleic acid comprising the sequence defined by nucleotides 256-552 of SEQ ID NO: 7 (ALK-6), the stringent conditions being hybridization in 40% formamide 5.times.SSPE, 5.times.Denhardt's Solution, 0.1% SDS at 37.degree. C. overnight, then washing in 0.1.times.SSPE, 0.1% SDS at 50.degree. C.; and (b) means for detecting induction of an OP-1-mediated cellular response as a means for detecting interaction of OP-1 or a candidate OP-1 receptor-binding analog with said protein of part (a), said OP-1 or candidate analog comprising part of said sample provided to said receptacle.

7. The kit of claim 6, further comprising a serine/threonine Type II receptor having binding specificity for OP-1, activin or BMP-4.

8. The method of claim 1, wherein said morphogen is OP-1.

9. The method of claim 1, wherein said morphogen is 60A, DPP, OP-2, OP-3, BMP-2, BMP-4, BMP-5, BMP-6, Vg1, GDF-1, or Vgr-1.

10. The method of claim 4, wherein said surface receptor protein further comprises part or all of a Type II serine/threonine kinase receptor protein having binding affinity for OP-1, activin or BMP-4.

11. A kit for identifying a binding analog for a morphogen receptor in a sample, said morphogen being characterized as sharing at least 60% amino acid sequence identity or at least 70% amino acid sequence homology with the sequence of the C-terminal 102 amino acids of SEQ ID NO: 7, and being able to substitute for OP-1 in binding to a protein comprising SEQ ID NOs. 4, 6, or 8, the kit comprising: (a) a receptacle adapted to receive said sample, said receptacle does not contain a Type II serine/threonine kinase morphogen receptor but contains protein selected from: (i) a polypeptide comprising an amino acid sequence defined by residues 16-123 of SEQ ID NO: 4 (ALK-2); (ii) a polypeptide comprising an amino acid sequence defined by residues 24-152 of SEQ ID NO: 6 (ALK-3); (iii) a polypeptide comprising an amino acid sequence defined by residues 23-122 of SEQ ID NO: 8 (ALK-6); or (iv) a polypeptide having binding affinity for OP-1 and encoded by a first nucleic acid capable of hybridizing under stringent conditions with a second nucleic acid comprising the sequence defined by nucleotides 256-552 of SEQ ID NO: 7 (ALK-6), the stringent conditions being hybridization in 40% formamide 5.times.SSPE, 5.times.Denhardt's Solution, 0.1% SDS at 37.degree. C. overnight, then washing in 0.1.times.SSPE, 0.1% SDS at 50.degree. C.; and (b) means for detecting specific binding interaction of OP-1 or said candidate analog with said protein of part (a), said OP-1 or candidate analog comprising part of said sample provided to said receptacle.

12. The method of claim 1, wherein said protein is a polypeptide comprising an amino acid sequence defined by residues 16-123 of SEQ ID NO: 4 (ALK-2).

13. The method of claim 1, wherein said protein is a polypeptide comprising an amino acid sequence defined by residues 24-152 of SEQ ID NO: 6 (ALK-3).

14. The method of claim 1, wherein said protein is a polypeptide comprising an amino acid sequence defined by residues 23-122 of SEQ ID NO: 8 (ALK-6).

15. The method of claim 2, wherein said surface receptor protein is a polypeptide comprising an amino acid sequence defined by residues 16-123 of SEQ ID NO: 4 (ALK-2).

16. The method of claim 2, wherein said surface receptor protein is a polypeptide comprising an amino acid sequence defined by residues 24-152 of SEQ ID NO: 6 (ALK-3).

17. The method of claim 2, wherein said surface receptor protein is a polypeptide comprising an amino acid sequence defined by residues 23-122 of SEQ ID NO: 8 (ALK-6).

18. The kit of claim 6 or claim 11, wherein said protein is a polypeptide comprising an amino acid sequence defined by residues 16-123 of SEQ ID NO: 4 (ALK-2).

19. The kit of claim 6 or claim 11, wherein said protein is a polypeptide comprising an amino acid sequence defined by residues 24-152 of SEQ ID NO: 6 (ALK-3).

20. The kit of claim 6 or claim 11, wherein said protein is a polypeptide comprising an amino acid sequence defined by residues 23-122 of SEQ ID NO: 8 (ALK-6).

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Description

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FIELD OF THE INVENTION

This invention relates generally to the field of tissue morphogenesis and more particularly to morphogenic protein-specific cell surface receptors.

BACKGROUND OF THE INVENTION

Cell differentiation is the central characteristic of tissue morphogenesis which initiates during embryogenesis, and continues to various degrees throughout the life of an organism in adult tissue repair and regeneration mechanisms. The degree of morphogenesis in adult tissue varies among different tissues and is related, among other things, to the degree of cell turnover in a given tissue.

The cellular and molecular events which govern the stimulus for differentiation of cells is an area of intensive research. In the medical and veterinary fields, it is anticipated that the discovery of the factor or factors which control cell differentiation and tissue morphogenesis will advance significantly medicine's ability to repair and regenerate diseased or damaged mammalian tissues and organs. Particularly useful areas for human and veterinary therapeutics include reconstructive surgery and in the treatment of tissue degenerative diseases including arthritis, emphysema, osteoporosis, cardiomyopathy, cirrhosis, degenerative nerve diseases, inflammatory diseases, and cancer, and in the regeneration of tissues, organs and limbs. (In this and related applications, the terms "morphogenetic" and "morphogenic" are used interchangeably.)

A number of different factors have been isolated in recent years which appear to play a role in cell differentiation. Recently, a distinct subfamily of the "superfamily" of structurally related proteins referred to in the art as the "transfoming growth factor-b (TGF-.beta.) superfamily of proteins" have been identified as true tissue morphogens.

The members of this distinct "subfamily" of true tissue morphogenic proteins share substantial amino acid sequence homology within their morphogenetically active C-terminal domains (at least 50% identity in the C-terminal 102 amino acid sequence), including a conserved six or seven cysteine skeleton, and share the in vivo activity of inducing tissue-specific morphogenesis in a variety of organs and tissues. The proteins apparently contact and interact with progenitor cells e.g., by binding suitable cell surface molecules, predisposing or otherwise stimulating the cells to proliferate and differentiate in a morphogenetically permissive environment. These morphogenic proteins are capable of inducing the developmental cascade of cellular and molecular events that culminate in the formation of new organ-specific tissue, including any vascularization, connective tissue formation, and nerve innervation as required by the naturally occurring tissue. The proteins have been shown to induce morphogenesis of both bone cartilage and bone, as well as periodontal tissues, dentin, liver, and neural tissue, including retinal tissue.

The true tissue morphogenic proteins identified to date include proteins originally identified as bone inductive proteins. These include OP-1, (osteogenic protein-1, also referred to in related applications as "OP1"), its Drosophila homolog, 60A, with which it shares 69% identity in the C-terminal "seven cysteine" domain, and the related proteins OP-2 (also referred to in related applications as "OP2") and OP-3, both of which share approximately 70-75% identity with OP-1 in the C-terminal seven cysteine domain, as well as BMP5, BMP6 and its murine homolog, Vgr-1, all of which share greater than 85% identity with OP-1 in the C-terminal seven cysteine domain, and the BMP6 Xenopus homolog, Vg1, which shares approximately 57% identity with OP-1 in the C-terminal seven cysteine domain. Other bone inductive proteins include the CBMP2 proteins (also referred to in the art as BMP2 and BMP4) and their Drosophila homolog, DPP. Another tissue morphogenic protein is GDF-1 (from mouse). See, for example, PCT documents US92/01968 and US92/07358, the disclosures of which are incorporated herein by reference.

As stated above, these true tissue morphogenic proteins are recognized in the art as a distinct subfamily of proteins different from other members of the TGF-.beta. superfamily in that they share a high degree of sequence identity in the C-terminal domain and in that the true tissue morphogenic proteins are able to induce, on their own, the full cascade of events that result in formation of functional tissue rather than merely inducing formation of fibrotic (scar) tissue. Specifically, members of the family of morphogenic proteins are capable of all of the following in a morphogenetically permissive environment: stimulating cell proliferation and cell differentiation, and supporting the growth and maintenance of differentiated cells. The morphogenic proteins apparently may act as endocrine, paracrine or autocrine factors.

The morphogenic proteins are capable of significant species "crosstalk." That is, xenogenic (foreign species) homologs of these proteins can substitute for one another in functional activity. For example, DPP and 60A, two Drosophila proteins, can substitute for their mammalian homologs, BMP2/4 and OP-1, respectively, and induce endochondral bone formation at a non-bony site in a standard rat bone formation assay. Similarly, BMP2 has been shown to rescue a dpp mutation in Drosophila. In their native form, however, the proteins appear to be tissue-specific, each protein typically being expressed in or provided to one or only a few tissues or, alternatively, expressed only at particular times during development. For example, GDF-1 appears to be expressed primarily in neural tissue, while OP-2 appears to be expressed at relatively high levels in early (e.g., 8-day) mouse embryos. The endogenous morphogens may be synthesized by the cells on which they act, by neighboring cells, or by cells of a distant tissue, the secreted protein being transported to the cells to be acted on.

A particularly potent tissue morphogenic protein is OP-1. This protein, and its xenogenic homologs, are expressed in a number of tissues, primarily in tissues of urogenital origin, as well as in bone, mammary and salivary gland tissue, reproductive tissues, and gastrointestinal tract tissue. It is also expressed in different tissues during embryogenesis, its presence coincident with the onset of morphogenesis of that tissue.

The morphogenic protein signal transduction across a cell membrane appears to occur as a result of specific binding interaction with one or more cell surface receptors. Recent studies on cell surface receptor binding of various members of the TGF-.beta. protein superfamily suggests that the ligands can mediate their activity by interaction with two different receptors, referred to as Type I and Type II receptors to form a hetero-complex. A cell surface bound beta-glycan also may enhance the binding interaction. The Type I and Type II receptors are both serine/threonine kinases, and share similar structures: an intracellular domain that consists essentially of the kinase, a short, extended hydrophobic sequence sufficient to span the membrane one time, and an extracellular domain characterized by a high concentration of conserved cysteines.

A number of Type II receptor sequences recently have been identified. These include "TGF-.beta.R II", a TGF-.beta. Type II receptor (Lin et al. (1992) Cell 68:775-785); and numerous activin-binding receptors. See, for example, Mathews et al. (1991) Cell 65:973-982 and international patent application WO 92/20793, published Nov. 26, 1992, disclosing the "ActR II" sequence; Attisano et al., (1992) Cell 68:97-108, disclosing the "ActR-IIB" sequence; and Legerski et al. (1992) Biochem Biophys. Res. Commun 183:672-679. A different Type II receptor shown to have affinity for activin is Atr-II (Childs et al. (1993) PNAS 90:9475-9479.) Two Type II receptors have been identified in C. elegans, the daf-1 gene, (Georgi et al. (1990) Cell 61:635-645), having no known ligand to date, and daf-4, which has been shown to bind BMP4, but not activin or TGF-.beta. (Estevez, et al. (1993) Nature 365:644-649.)

Ten Dijke et al. disclose the cloning of six different Type I cell surface receptors from murine and human cDNA libraries. ((1993) Oncogene 8:2879-2887, and Science (1994) 264:101-104. These receptors, referenced as ALK-1 to ALK-6 ("activin receptor-like kinases"), share significant sequence identities (60-79%) and several have been identified as TGF-.beta. binding (ALK-5) or activin binding (ALK-2, ALK-4) receptors. Xie et al. also report a Drosophila Type I receptor encoded by the sax gene (Science (1994) 263:1756-1759). The authors suggest that the protein binds DPP.

To date, the Type I receptors with which the morphogenic proteins described herein interact on the cell surface have not yet been identified, and no Type II receptor has been described as having binding affinity for OP-1 and its related sequences. Identification of these cell surface molecules, with which the morphogens interact and through which they may mediate their biological effect, is anticipated to enhance elucidation of the molecular mechanism of tissue morphogenesis and to enable development of morphogen receptor binding "analogs", e.g., compounds (which may or may not be amino acid-based macromolecules) capable of mimicing the binding affinity of a morphogen for its receptor sufficiently to act either as a receptor binding agonist or antagonist. These "analogs" have particular utility in therapeutic, diagnostic and experimental research applications.

It is an object of this invention to provide nucleic acid molecules and amino acid sequences encoding morphogenic protein binding cell surface receptors, particularly OP-1-specific binding receptor sequences. Another object is to provide methods for identifying genes in a variety of species and/or tissues, and in a variety of nucleic acid libraries encoding morphogenic protein binding receptors, particularly receptors that bind OP-1. Still another object is to provide means for designing biosynthetic receptor-binding ligand analogs, particularly OP-1 analogs, and/or for identifying natural-occurring ligand analogs, including agonists and antagonists, using the receptor molecules described herein, and analogs thereof. Another object is to provide antagonists, including soluble receptor constructs comprising the extracellular ligand-binding domain, which can modulate the availability of OP1 for receptor binding in vivo. Another obect is to provide means and compositions for competing with activin-receptor and BMP2/4-receptor interactions. Yet another object is to provide means and compositions for ligand affinity purification and for diagnostic detection and quantification of ligands in a body fluid using OP1-specific cell surface receptors and ligand-binding fragments thereof. Still another object is to provides means and compositions for modulating the endogenous expression or concentration of these receptor molecules. Yet another object is to provide ligand-receptor complexes and analog sequences thereof, as well as antibodies capable of identifying and distinguishing the complex from its component proteins. Still another object is to provide means and compositions for modulating a morphogenesis in a mammal. These and other objects and features of the invention will be apparent from the description, drawings and claims which follow.

SUMMARY OF THE INVENTION

Type I and Type II cell surface receptor molecules capable of specific binding affinity with true tissue morphogenic proteins, particularly OP-1-related proteins, now have been identified. Accordingly, the invention provides ligand-receptor complexes comprising at least the ligand binding domain of these receptors and OP-1 or an OP-1 receptor-binding analog as the ligand; means for identifying and/or designing useful OP-1 receptor-binding analogs and OP-1-binding-receptor analogs; and means for modulating the tissue morphogenesis capability of a cell.

The morphogen cell surface receptors useful in this invention are referred to in the art as Type I or Type II serine/threonine kinase receptors. They share a conserved structure, including an extracellular, ligand-binding domain generally composed of about 100-130 amino acids (Type I receptors; up to about 196 amino acids for Type II receptors), a transmembrane domain sufficient to span a cellular membrane one time, and an intracellular (cytoplasmic) domain having serine/threonine kinase activity. The intact receptor is a single polypeptide chain of about 500-550 amino acids and having an apparent molecular weight of about 50-55 kDa.

Of particular utility in the methods and compositions of the invention are the Type I cell surface receptors referenced herein and in the literature as, ALK-2, ALK-3 and ALK-6, whose nucleic acids and encoded amino acid sequences are represented by the sequences in Seq. ID Nos. 3, 5 and 7 respectively, and which, as demonstrated herein below, have specific binding affinity for OP1 and OP1-related analogs. Accordingly, in one embodiment, the receptor sequences contemplated herein include OP-1 binding analogs of the ALK-2, ALK-3 and ALK-6 proteins described herein.

As used herein, ligand-receptor binding specificity is understood to mean a specific, saturable noncovalent interaction between the ligand and the receptor, and which is subject to competitive inhibition by a suitable competitor molecule. Preferred binding affinities (defined as the amount of ligand required to fill one-half (50%) of available receptor binding sites) are described herein by dissociation constant (Kd). In one embodiment, preferred binding affinities of the ligand-receptor complexes described herein have a Kd of less than 10.sup.-7M, preferably less than 5.times.10.sup.-7M, more preferably less than 10.sup.-8M. In another preferred embodiment, the receptor molecules have little or no substantial binding affinity for TGF-.beta..

As used herein, an "OP1-specific receptor analog" is understood to mean a sequence variant of the ALK-2, ALK-3 or ALK-6 sequences which shares at least 40%, preferably at least 45%, and most preferably at least 50%, amino acid identity in the extracellular ligand binding domain with the sequence defined by residues 23-122 of Seq. ID No. 7 (ALK-6), and which has substantially the same binding affinity for OP1 as ALK-2, ALK-3 or ALK-6. ALK-6 and ALK-3 share 46% amino acid sequence identity in their ligand binding domains. Accordingly, in one preferred embodiment, the OP1-specific receptor analogs share at least 46% amino acid sequence identity with the extracellular, ligand binding domains of ALK-6 or ALK-3.

As will be appreciated by those having ordinary skill in the art, OP1-specific receptor analogs also can have binding affinity for other, related morphogenic proteins. As used herein, an OP1-specific receptor analog is understood to have substantially the same binding affinity for OP-1 as ALK-2, ALK-3 or ALK-6 if it can be competed successfully for OP-1 binding in a standard competition assay with a known OP-1 binding receptor, e.g., with ALK-2, ALK-3 or ALK-6. In one preferred embodiment, OP1-specific receptor analogs have a binding affinity for OP-1 defined by a dissociation constant of less than about 10.sup.-7 M, preferably less than about 5.times.10.sup.-7M or 10.sup.-8 M. It is anticipated however, that analogs having lower binding affinities, e.g., on the order of 10.sup.-6M also will be useful. For example, such analogs may be provided to an animal to modulate availability of serum-soluble OP1 for receptor binding in vivo. Similarly, where tight binding interaction is desired, for example as part of a cancer therapy wherein the analog acts as a ligand-receptor antagonist, preferred binding affinities may be on the order of 5.times.10.sup.-8M.

In another embodiment, the OP-1 binding receptor analogs contemplated by the invention include proteins encoded by nucleic acids which hybridize with the DNA sequence encoding the extracellular, ligand binding domain of ALK-2, ALK-3 or ALK-6 under stringent hybridization conditions, and which have substantially the same OP-1 binding affinity as ALK-2, ALK-3 or ALK-6. As used herein, stringent hybridization conditions are as defined in the art, (see, for example, Molecular Cloning: A Laboratory Manual, Maniatis et al., eds. 2d.ed., Cold Spring Harbor Press, Cold Spring Harbor, 1989.) An exemplary set of conditions is defined as: hybridization in 40% formamide, 5.times.SSPE, 5.times. Denhardt's Solution, and 0.1% SDS at 37.degree. C. overnight, and washing in 0.1.times.SSPE, 0.1% SDS at 50.degree. C.

In still another embodiment, the OP-1 binding receptor analogs contemplated by the invention include part or all of a serine/threonine kinase receptor encoded by a nucleic acid that can be amplified with one or more primers derived from ALK-1 (Seq. ID No. 1), ALK-2, ALK-3 or ALK-6 sequence in a standard PCR (polymerase chain reaction) amplification scheme. In particular, a primer or, most preferably, a pair of primers represented by any of the sequences of SEQ ID Nos. 12-15 are envisioned to be particularly useful. Use of primer pairs (e.g., SEQ. ID No. 12/15; 13/15; 14/15) are described in WO94/11502 (PCT/GB93/02367).

Useful OP1-specific receptor analogs include xenogenic (foreign species) homologs of the murine and human ALK sequences described herein, including those obtained from other mammalian species, as well as other, eukaryotic, non-mammalian xenogenic homologs. Also contemplated are biosynthetic constructs and naturally-occurring sequence variants of ALK-2, ALK-3 and ALK-6, provided these molecules, in all cases, share the appropriate identity in the ligand binding domain, and bind OP-1 specifically as defined herein. In one embodiment, sequence variants include receptor analogs which have substantially the same binding affinity for OP1 as ALK-2, ALK-3 or ALK-6 and which are recognized by an antibody having binding specificity for ALK-2, ALK-3 or ALK-6.

In another embodiment the receptors and OP-1 binding receptor analogs contemplated herein provide the means by whichla morphogen, e.g., OP-1, can mediate a cellular response. In one embodiment these receptors include ALK-2, ALK-3, or ALK-6, or sequence variants or OP-1 binding analogs thereof. In another embodiment, ALK-1, including sequence variants thereof is contemplated to participate in an OP-1 mediated cellular response.

OP1-specific receptor analogs may be used as OP1 antagonists. For example, a soluble form of a receptor, e.g., consisting essentially of only is the extracellular ligand-binding domain, may be provided systemically to a mammal to bind to soluble ligand, effectively competing with ligand binding to a cell surface receptor, thereby modulating (reducing) the availability of free ligand in vivo for cell surface binding.

The true tissue morphogenic proteins contemplated as useful receptor ligands in the invention include OP-1 and OP-1 receptor-binding analogs. As used herein, an "OP-1 analog" or "OP-1 receptor-binding analog" is understood to include all molecules able to functionally substitute for OP-1 in Type I receptor binding, e.g., are able to successfully compete with OP-1 for receptor binding in a standard competition assay. In one embodiment, useful OP-1 receptor-binding analogs include molecules whose binding affinity is defined by a dissociation constant of less than about 5.times.10.sup.-6M, preferably less than about 10.sup.-7 or 5.times.10.sup.-7M. As for the OP-specific receptor analogs above, both stronger and weaker binding affinities are contemplated to be useful in particular applications. In one preferred embodiment, these receptor-binding OP-1 analogs also bind OP-1 specific Type II serine/kinase receptors.

The OP-1 analogs contemplated herein, all of which mimic the binding activity of OP-1 or an OP-1-related protein sufficiently to act as a substitute for OP-1 in receptor binding, can act as OP-1 agonists, capable of mimicking OP-1 both in receptor binding and in inducing a transmembrane effect e.g., inducing threonine or serine-specific phosphorylation following binding. Alternatively, the OP-1 analog can act as an OP-1 antagonist, capable of mimicking OP-1 in receptor binding only, but unable to induce a transmembrane effect, thereby blocking the natural ligand from interacting with its receptor, for example. Useful applications for antagonists include their use as therapeutics to modulate uncontrolled differentiated tissue growth, such as occurs in malignant transformations such as in osteosarcomas or Paget's disease.

OP-1 analogs contemplated by the invention can be amino acid-based, e.g., sequence variants of OP-1, or antibody-derived sequences capable of functionally mimicking OP-1 binding to an OP-1-specific receptor. Examples of such antibodies may include anti-idiotypic antibodies. In a specific embodiment, the anti-idiotypic antibody mimics OP1 both in receptor binding and in ability to induce a transmembrane effect. Alternatively, the OP-1 analogs can be composed in part or in whole of other chemical structures, e.g., the analogs can be comprised in part or in whole of nonproteinaceous molecules. In addition, the OP-1 analogs contemplated can be naturally sourced or synthetically produced.

As used herein, OP-1 related sequences include sequences sharing at least 60%, preferably greater than 65% or even 70% identity with the C-terminal 102 amino acid sequence of OP-1 as defined in Seq ID NO.7, and which are able to substitute for OP-1 in ligand binding to ALK-2, ALK-3 or ALK-6, (e.g., able to compete successfully with OP-1 for binding to one or more of these receptors.) OP-1 related sequences contemplated by the invention include xenogenic homologs (e.g., the Drosophila homolog 60A), and the related sequences referenced herein and in the literature as OP-2, OP-3, BMP5, BMP6 (and its xenogenic homolog Vgr-1.) OP-1 related sequences also include sequence variants encoded by a nucleic acid which hybridizes with a DNA sequence comprising the C-terminal 102 amino acids of Seq. ID No. 9 under stringent hybridization conditions and which can substitute for OP1 in an OP1-receptor binding assay. In another embodiment, an OP1 sequence variant includes a protein which can substitute for OP1 in a ligand-receptor binding assay and which is recognized by an antibody having binding specificity for OP1.

As used herein, "amino acid sequence homology" is understood to mean amino acid sequence similarity, and homologous sequences sharing identical or similar amino acids, where similar amino acids are conserved amino acids as defined by Dayoff et al., Atlas of Protein Sequence and Structure; vol.5, Suppl.3, pp.345-362 (M. O. Dayoff, ed., Nat'l BioMed. Research Fdn., Washington D.C. 1978.) Thus, a candidate sequence sharing 60% amino acid homology with a reference sequence requires that, following alignment of the candidate sequence with the reference sequence, 60% of the amino acids in the candidate sequence are identical to the corresponding amino acid in the reference sequence, or constitute a conserved amino acid change thereto. "Amino acid sequence identity" is understood to require identical amino acids between two aligned sequences. Thus, a candidate sequence sharing 60% amino acid identity with a reference sequence requires that, following alignment of the candidate sequence with the reference sequence, 60% of the amino acids in the candidate sequence are identical to the corresponding amino acid in the reference sequence.

As used herein, all receptor homologies and identities calculated use ALK-6 as the reference sequence, with the extracellular domain reference sequence constituting residues 23-122 of Seq. ID No.7; and the intracellular serine/threonine kinase domain reference sequence constituting residues 206-495 of Seq. ID No.7. Similarly, all OP-1 related protein homologies and identities use OP-1 as the reference sequence, with the C-terminal 102 amino acids described in Seq. ID No. 10 constituting the seven cysteine domain.

Also as used herein, sequences are aligned for homology and identity calculations as follows: Sequences are aligned by eye to maximize sequence identity. Where receptor amino acid extracellular domain sequences are compared, the alignment first maximizes alignment of the cysteines present in the two sequences, then modifies the alignment as necessary to maximize amino acid identity and similarity between the two sequences. Where amino acid intracellular domain sequences are compared, sequences are aligned to maximize alignment of conserved amino acids in the kinase domain, where conserved amino acids are those identified by boxes in FIG. 3. The alignment then is modified as necessary to maximize amino acid identity and similarity. In all cases, internal gaps and amino acid insertions in the candidate sequence as aligned are ignored when making the homology/identity calculation. Exemplary alignments are illustrated in FIGS. 2 and 3 where the amino acid sequences for the extracellular and intracellular domains, respectively are presented in single letter format. In the figures "gaps" created by sequence alignment are indicated by dashes.

In one aspect, the invention contemplates isolated ligand-receptor complexes comprising OP-1 or an OP-1 analog as the ligand in specific binding interaction with an OP-1 binding Type I receptor or receptor analog, as defined herein. In another aspect, the invention contemplates the ligand-receptor complex comprises part or all of an OP-1 binding Type II receptor. Type II receptors contemplated to be useful include Type II receptors defined in the literature (referenced hereinabove) as having binding specificity for activin or a bone morphogenic protein such as BMP-4. Such Type II receptors include daf4, ActRII and AtrII. In still another aspect, the ligand-receptor complex comprises both a Type I and a Type II receptor and OP1, or an OP1 analog as the ligand. In all complexes, the bound receptor can comprise just the extracellular, ligand binding domain, or can also include part or all of the transmembrane sequence, and/or the intracellular kinase domain. Similarly, the OP-1 ligand may comprise just the receptor binding sequence, longer sequences, including the mature dimeric species or any soluble form of the protein or protein analog.

The OP-1 and OP-1 analogs described herein can interact specifically with Type I and Type II receptors also known to interact with other morphogenic proteins (e.g., BMP2/BMP4) and activin. Thus invention also contemplates the use of OP-1 and OP-1 receptor-binding analogs as competitors of specific BMP-receptor and activin-receptor interactions. As will be appreciated by those having ordinary skill in the art, these binding competitors may act as either agonists or antagonists (e.g., to inhibit an activin or BMP-mediated cellular response).

In another aspect, the invention contemplates binding partners having specific binding affinity for an epitope on the ligand-receptor complex. In a preferred embodiment, the binding partner can discriminate between the complex and the uncomplexed ligand or receptor. In another embodiment, the binding partner has little or no substantial binding affinity for the uncomplexed ligand or receptor. In another preferred embodiment, the binding partner is a binding protein, more preferably an antibody. These antibodies may be monoclonal or polyclonal, may be intact molecules or fragments thereof (e.g., Fab, Fab', (Fab)'.sub.2), or may be biosynthetic derivatives, including, but not limited to, for example, monoclonal fragments, such as single chain F.sub.v fragments, referred to in the literature as sF.sub.vs, BABs and SCAs, and chimeric monoclonals, in which portions of the monoclonals are humanized (excluding those portions involved in antigen recognition (e.g., complementarity determining regions, "CDRs".) See, for example, U.S. Pat. Nos. 5,091,513 and 5,132,405, the disclosures of which are incorporated herein by reference. Biosynthetic chimeras, fragments and other antibody derivatives may be synthesized using standard recombinant DNA methodology and/or automated chemical nucleic acid synthesis methodology well described in the art and as described below.

In still another aspect, the invention provides molecules useful in the design and/or identification of receptor-binding morphogenic protein analogs as described below, as well as kits and methods, e,g., screening assays, for identifying these analogs. The molecules useful in these assays can include part or all of the receptor sequence of SEQ ID NO. 3, 5 or 7, including amino acid sequence variants and OP-1 binding analogs and amino acid sequence variants thereof.

As described above, sequence variants are contemplated to have substantially the same binding affinity for OP-1 as the receptors represented by the sequences in SEQ. ID Nos. 3-7. OP-1 binding receptor analogs include other, known or novel Type I or Type II serine/threonine kinase receptors having binding affinity and specificity for OP-1 as defined herein and which (1) share at least 40% amino acid identity with residues 23-122 of Seq. ID No. 7, (2) are encoded by a nucleic acid that hybridizes under stringent conditions with a nucleic acid comprising the sequence defined by nucleotides 256-552 of Seq ID No. 7; or (3) are encoded by a nucleic acid obtainable by amplification with one or more primer sequences defined by Seq. ID Nos. 12-15. Currently preferred for the assays of the invention are receptor sequences comprising at least the sequence which defines the extracellular, ligand binding domains of these proteins. The kits and assays may include just Type I receptors or both Type I and Type II receptors. Similarly, the kits and screening assays can be used in the design and/or identification of OP1-specific receptor analogs. The OP-1 receptor-binding analogs and OP-1-binding receptor analogs thus identified then can be produced in reasonable quantities using standard recombinant expression or chemical synthesis technology well known and characterized in the art. Alternatively, promising candidates can be modified using standard biological or chemical methodologies to, for example, enhance the binding affinity of the candidate analog as described in Example 10, below, and the preferred candidate derivative then produced in quantity.

In still another aspect, the receptor and/or OP1-specific receptor analogs can be used in standard methodologies for affinity purifying and/or quantifying OP1 and OP1 analogs. For example, the receptor's ligand binding domain first may be immobilized on a surface of a well or a chromatographic column; ligand in a sample fluid then may be provided to the receptor under conditions to allow specific binding; non-specific binding molecules then removed, e.g., by washing, and the bound ligand then selectively isolated and/or quantitated. Similarly, OP1 and OP1 analogs can be used for affinity purifying and/or quantifying OP1-specific receptors and receptor analogs. In one embodiment, the method is useful in kits and assays for diagnostic purposes which detect the presence and/or concentration of OP1 protein or related morphogen in a body fluid sample including, without limitation, serum, peritoneal fluid, spinal fluid, and breast exudate. The kits and assays also can be used for detecting and/or quantitating OP-1-specific receptors in a sample.

In still another aspect the invention comprises OP1-specific receptors and OP-1-binding receptor analogs useful in screening assays to identify organs, tissues and cell lines which express OP1-specific receptors. These cells then can be used in screening assays to identify ligands that modulate endogenous morphogen receptor expression levels, including the density of receptors expressed on a cell surface. Useful assay methodologies may be modeled on those described in PCT US92/07359, and as described below.

The invention thus relates to compositions and methods for the use of morphogen-specific receptor sequences in diagnostic, therapeutic and experimental procedures. Active receptors useful in the compositions and methods of this invention can include truncated or full length forms, as well as forms having varying glycosylation patterns. Active receptors useful in the invention also include chimeric constructs as described below. Active OP1-specific receptors/analogs can be expressed from intact or truncated genomic or cDNA, or from synthetic DNAs in procaryotic or eucaryotic host cells, and purified, cleaved, refolded and oxidized as necessary to form active molecules. Useful host cells include prokaryotes, including E. coli and B. subtilis, and eukaryotic cells, including mammalian cells, such as fibroblast 3T3 cells, CHO, COS, melanoma or BSC cells, Hela and other human cells, the insect/baculovirus system, as well as yeast and other microbial host cell systems.

Thus, in view of this disclosure, skilled genetic engineers now can, for example, identify and produce OP1-specific cell surface receptors or analogs thereof; create and perform assays for screening candidate OP1 receptor-binding analogs and evaluate promising candidates and their progency in therapeutic regimes and preclinical studies; modulate the availability of endogenous morphogen for cell surface interactions; modulate endogenous morphogen-specific cell surface receptor levels; elucidate the signal transduction pathway induced by morphogen-cell surface receptor binding; and modulate tissue morphogenesis in vivo.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of the encoded ALK-2, ALK-3, ALK-6 amino acid sequences, showing the signal sequence 10, the transmembrane domain 12, the extracellular ligand binding domain 14, and the intracellular serine/threonine kinase domain 16;

FIG. 2 is a homology alignment of the extracellular domains of ALK-2, ALK-3, and ALK-6, aligned to maximize amino acid identity, wherein conserved amino acids are identified by boxes and conserved cysteines are indicated by asterisks; and

FIG. 3 is a homology alignment of the intracellular domain of ALK-2, ALK-3 and ALK-6, aligned to maximize amino acid identity, wherein conserved amino acids are boxed and the serine/threonine kinase domain is indicated by arrows.

DETAILED DESCRIPTION

Disclosed herein are Type I and Type II receptors having binding specificity for true tissue morphogenic proteins, particularly OP1 and OP1-related proteins. It further has been determined that OP1 binds to a broader range of receptors than other known tissue morphogens or TGF-.beta. family members. The Type I receptors disclosed herein, can be used together with OP1 and OP1 analogs for therapeutic, diagnostic and experimental uses as described herein below. Moreover, soluble forms of the OP1-binding receptor proteins, e.g., forms consisting essentially of the extracellular domain or a fragment thereof sufficient to bind OP1 with specificity, can be used as a soluble therapeutic morphogen antagonist, as described below.

Following this disclosure, related OP1-specific receptors are available, as are high and medium flux screening assays for identifying OP1 analogs and OP1-specific receptor analogs. These analogs can be naturally occurring molecules, or they can be designed and biosynthetically created using a rational drug design and an established structure/function analysis. The analogs can be amino acid-based or can be composed in part or whole of non-proteinaceous synthetic organic molecules. Useful analogs also can include antibodies, preferably monoclonal antibodies (including fragments thereof, e.g., Fab, Fab', and (Fab)'.sub.2), or synthetic derivatives thereof, such as monoclonal single chain F.sub.v fragments known in the art as sF.sub.vs, BABs, and SCAs (see below), and bispecific antibodies or derivatives thereof. When these antibodies mimic the binding activity of OP-1 to a cell surface receptor without inducing the biological response OP-1 does upon binding, the antibody can compete for OP-1 binding and act as an antagonist. These antibodies or derivatives thereof also can mimic OP-1 both in receptor binding and signal transduction, in which case the antibody acts as an OP-1 agonist. The antibodies and derivatives also can be used for inducing the morphogenic cellular response by crosslinking receptors to morphogenic proteins, particularly OP1 and OP1-related proteins to form either homo- or hetero-complexes of the Type I and Type II receptors.

The OP1-binding receptor sequences described herein (ALK2, ALK3, ALK6) also can be used to create chimeric sequences, wherein, for example, part or all of either the extracellular domain or the intracellular domain is a non-ALK sequence or is created from two or more ALK sequences. These chimeric receptors can be synthesized using standard recombinant DNA methodology and/or automated chemical nucleic acid synthesis methodology well described in the art and as disclosed below. Chimerics can be used, for example, in OP1 analog assays, wherein the OP1-binding extracellular domain is coupled to a non-ALK intracellular domain that is well characterized and/or readily detectable as a second messenger response system, as described below. Chimerics also can be used, for example, in high flux OP1 analogs screens and as part of purification protocols, wherein a soluble ligand binding domain of an OP1-specific receptor is immobilized onto a support e.g., by covalent or non-covalent interactions, with a chromatographic matrix or the well surface of a 96-well plate. When immobilized onto a chromatographic matrix surface, the receptor fragment can be used in a protocol to isolate OP1 or OP1 analogs. When immobilized on a well surface the receptor fragment is particularly useful in a screening assay to identify receptor-binding OP1 analogs in a standard competition assay.

The true tissue morphogenic proteins contemplated to be useful in the methods and compositions of the invention include forms having varying glycosylation patterns and varying N-termini. The proteins can be naturally occurring or biosynthetically derived, and can be produced by expression of recombinant DNA in prokaryotic or eukaryotic host cells. The proteins are active as a single species (e.g., as homodimers), or combined as a mixed species. Useful sequences and eucaryotic and procaryotic expression systems are well described in the art. See, for example, U.S. Pat. Nos. 5,061,911 and 5,266,683 for useful expression systems.

Particularly contemplated herein are OP1 and OP1-related sequences. Useful OP1 sequences are recited in U.S. Pat. Nos. 5,011,691; 5,018,753 and 5,266,683; in Ozkaynak et al. (1990) EMBO J 9:2085-2093; and Sampath et al. (1993) PNAS 90: 6004-6008. OP-1 related sequences include xenogenic homologs, e.g.; 60A, from Drosophila, Wharton et al. (1991) PNAS 88:9214-9218; and proteins sharing greater than 60% identity with OP1 in the C-terminal seven cysteine domain, preferably at least 65% identity. Examples of OP-1 related sequences include BMP5, BMP6 (and its species homolog Vgr-1, Lyons et al. (1989) PNAS 86:4554-4558), Celeste, et al. (1990) PNAS 87:9843-9847 and PCT international application WO93/00432; OP-2 (Ozkaynak et al. (1992) J.Biol.Chem. 267:13198-13205) and OP-3 (PCT international application WO94/06447). As will be appreciated by those having ordinary skill in the art, chimeric constructs readily can be created using standard molecular biology and mutagenesis techniques combining various portions of different morphogenic protein sequences to create a novel sequence, and these forms of the protein also are contemplated herein.

A particularly preferred embodiment of the proteins contemplated by the invention includes proteins whose amino acid sequence in the cysteine-rich C-terminal domain has greater than 60% identity, and preferably greater than 65% identity with the amino acid sequence of OPS (OP-1 sequence defining the C-terminal conserved six cysteines, e.g., residues 335-431 of Seq. ID No. 9).

In another preferred aspect, the invention contemplates osteogenic proteins comprising species of polypeptide chains having the generic amino acid sequence herein referred to as "OPX" which accommodates the homologies between the various identified species of the osteogenic OP1 and OP2 proteins, and which is described by the amino acid sequence presented below and in Sequence ID No. 11.

TABLE-US-00001 Cys Xaa Xaa His Glu Leu Tyr Val Ser Phe 1 5 10 Xaa Asp Leu Gly Trp Xaa Asp Trp Xaa Ile 15 20 Ala Pro Xaa Gly Tyr Xaa Ala Tyr Tyr Cys 25 30 Glu Gly Glu Cys Xaa Phe Pro Leu Xaa Ser 35 40 Xaa Met Asn Ala Thr Asn His Ala Ile Xaa 45 50 Gln Xaa Leu Val His Xaa Xaa Xaa Pro Xaa 55 60 Xaa Val Pro Lys Xaa Cys Cys Ala Pro Thr 65 70 Xaa Leu Xaa Ala Xaa Ser Val Leu Tyr Xaa 75 80 Asp Xaa Ser Xaa Asn Val Ile Leu Xaa Lys 85 90 Xaa Arg Asn Met Val Val Xaa Ala Cys Gly 95 100 Cys His,

and wherein Xaa at res. 2=(Lys or Arg); Xaa at res. 3=(Lys or Arg); Xaa at res. 11=(Arg or Gln); Xaa at res. 16=(Gln or Leu); Xaa at res. 19=(Ile or Val); Xaa at res. 23=(Glu or Gln); Xaa at res. 26=(Ala or Ser); Xaa at res. 35=(Ala or Ser); Xaa at res. 39=(Asn or Asp); Xaa at res. 41=(Tyr or Cys); Xaa at res. 50=(Val or Leu); Xaa at res. 52=(Ser or Thr); Xaa at res. 56=(Phe or Leu); Xaa at res. 57=(Ile or Met); Xaa at res. 58=(Asn or Lys); Xaa at res. 60=(Glu, Asp or Asn); Xaa at res. 61=(Thr, Ala or Val); Xaa at res. 65=(Pro or Ala); Xaa at res. 71=(Gln or Lys); Xaa at res. 73=(Asn or Ser); Xaa at res. 75=(Ile or Thr); Xaa at res. 80=(Phe or Tyr); Xaa at res. 82=(Asp or Ser); Xaa at res. 84=(Ser or Asn); Xaa at res. 89=(Lys or Arg); Xaa at res. 91=(Tyr or His); and Xaa at res. 97=(Arg or Lys).

In still another preferred aspect, the invention contemplates osteogenic proteins encoded by nucleic acids which hybridize to DNA or RNA sequences encoding the C-terminal seven cysteine domain of OP1 or OP2 under stringent hybridization conditions.

A brief description of the various terms of OP-1 useful in the invention is described below:

OP1--Refers generically to the family of osteogenically active proteins produced by expression of part or all of the hOP1 gene. Also referred to in related applications as "OPI" and "OP-1".

OP1-PP--Amino acid sequence of human OP1 protein (prepro form), Seq. ID No. 9, residues 1-431. Also referred to in related applications as "OP1-PP" and "OPP".

OP1-18Ser--Amino acid sequence of mature human OP1 protein, Seq. ID No. 9, residues 293-431. N-terminal amino acid is serine. Originally identified as migrating at 18 kDa on SDS-PAGE in COS cells. Depending on protein glycosylation pattern in different host cells, also migrates at 23 kDa, 19 kDa and 17 kDa on SDS-PAGE. Also referred to in related applications as "OP1-18."

OP1-16Ser; OP1-16Ala; OP1-16 Met; OP1-16 leu; OP1-16Val--N-terminally truncated mature human OP1 protein species defined, respectively, by residues 300-431; 316-431; 315-431; 313-431 and 318-431.

OPS--Amino acid sequence defining the C-terminal six cysteine domain, residues 335-431 of Seq. ID No. 9.

OP7--Amino acid sequence defining the C-terminal seven cysteine domain, residues 330-431 of Seq. ID No. 9.

Soluble form OP1--mature dimeric OP1 species having one or, preferably two copies of pro domain, e.g., at least residues 158-292 of Seq. ID No. 9, preferably residues 48-292 or 30-292, non-covalently complexed with the dimer.

The cloning procedure for obtaining OP1-binding ALK nucleic acid sequences, means for expressing receptor sequences, as well as other material aspects concerning the nature and utility of these sequences, including how to make and how to use the subject matter claimed, will be further understood from the following, which constitutes the best mode currently contemplated for practicing the invention.

EXAMPLE 1

Identification of ALK-1, ALK-2, ALK-3 and ALK-6

The cloning and characterization of ALK-1, -2, -3, and -6 receptors are described in detail in ten Dijke et al. (1993) Oncogene 8:2879-2887; and (1994) Science 264:101-104. The general structures of these proteins is described in FIG. 1, and the sequence alignments between the ALK genes are shown in FIGS. 2 and 3. These molecules have similar domain structures: an N-terminal predicted hydrophobic signal sequence (von Heijne (1986) Nucl. Acids Res. 14: 4683-4690) is followed by a relatively small extracellular cysteine-rich ligand binding domain, a single hydrophobic transmembrane region (Kyte & Doolittle (1982) J. Mol. Biol. 157, 105-132) and a C-terminal intracellular portion, which consists almost entirely of a kinase domain (FIG. 3).

The extracellular domains of these receptors, defined essentially by residues 22-118 (SEQ. ID No. 1 ) for ALK-1; residues 16-123 (SEQ ID No. 3) for ALK-2; residues 24-152 (SEQ. ID No. 5) for ALK-3; and residues 23-122 (SEQ ID No. 7) for ALK-6, have cysteine-rich regions, but sequence similarity varies among the proteins. For example, ALK-3 and ALK-6 share a high degree of sequence similarity in their extracellular domains (46% identity) whereas ALK-2 shows less similarity with ALK 3 or A