AU719153B2 - Morphogen treatment of gastrointestinal ulcers - Google Patents

Description

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AUSTRALIA

PATENTS ACT 1990 DIVISIONAL APPLICATION

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NAME OF APPLICANT(S): Creative Biomolecules, Inc.

ADDRESS FOR SERVICE: DAVIES COLLISON CAVE Patent Attorneys 1 Little Collins Street Melbourne, 3000.

INVENTION TITLE: Morphogen Treatment of Gastrointestinal Ulcers The following statement is a full description of this invention, including the best method of performing it known to us: QA0PF'RNJiMS\2008D1V.204 22f7/07 -la- MORPHOGEN TREATMENT OF GASTROINTESTINAL ULCERS Field of the Invention The invention relates generally to the treatment of gastrointestinal (GI) disorders and the tissue damage associated therewith. In particular, the invention relates to the treatment of ulcerative diseases within the gastrointestinal tract of a mammal.

10 Background of the Invention The luminal lining of the mammalian gastrointestinal tract (GI tract), which extends from the mouth cavity to the rectum, includes a protective layer of continually proliferating basal epithelial cells overlying a mucosal layer. Together, the basal epithelium and mucosa create the protective "gastrointestinal barrier." Disruption of this barrier results in lesions that can become infected and/or 20 expose underlying tissue to the corrosive effect of gastric juices. Gastrointestinal ulcerations can cause oral mucositis, gastric ulcers, necrotizing enterocolitis, regional ileitis, ulcerative colitis, regional enteritis (Crohn's disease), proctitis, and other forms of inflammatory bowel disease (IBD).

Ulcerative oral mucositis is a serious and doselimiting toxic side effect of many forms of cancer therapies, including chemotherapy and radiation therapy. Oral mucositis accounts for significant pain and discomfort for these patients, and ranges in 2 severity from redness and swelling to frank ulcerative lesions. Chemotherapeutic agents and radiation can kill or damage the epithelial cells lining the oral cavity. Such damage includes the inhibitory effect that chemotherapeutic agents may have on mitoses of the rapidly dividing cells of the oral basal epithelium.

The severity of damage is related to the type and dose of chemotherapeutic agent(s) and concomitant therapy such as radiotherapy. Further, ulceration is hastened if sources of chronic irritation such as defective dental restorations, fractured teeth or ill-fitting dental prostheses are present. Oral mucositis most often affects the nonkeratinized mucosa of the cheeks, e 15 lips, soft palate, ventral surface of the tongue and floor of the mouth, approximately one to two weeks after cancer therapy. The lesions often become secondarily infected and become much harder to heal.

The disruption in the oral mucosa results in a systemic portal of entry for the numerous microorganisms found in the mouth. Consequently, the oral cavity is the most frequently identifiable source of sepsis in the granulocytopenic cancer patient. Of primary concern are those patients undergoing: chemotherapy for cancer ;such as leukemia, breast cancer or as an adjuvant to tumor removal; radiotherapy for head and neck cancer; and combined chemotherapy and radiotherapy for bone marrow transplants.

One source of oral mucositis can result from xerostomia, or chronic mouth dryness, which typically results from diminished or arrested salivary secretion or asialism. Salivary gland dysfunction or atrophy may result from tissue senescence in aged individuals, or from an organic disorder. Most frequently, xerostomia is an undesired side effect of a clinical or 3 pharmaceutical therapy. Normally, saliva moistens the oral mucosal membrane, allowing for the dissolution and limited absorption of exogenous substances introduced into the oral cavity. In xerostomaic individuals irritating exogenous substances, including foods and medications, remain exposed to the mucosa and can cause inflammation and ulceration. A description of xerostomia-causing medications is described in Gallager, et al. (1991) Current Opinion in Dentistry 10 1:777-782.

Current therapy for mucositis is limited to either local or systemic palliation or topical antibacterial S1 therapy. At present there is no effective treatment 15 for mucositis. Therapy typically is limited to pain medications and treatment of secondary infection. In Sparticular, recommendations have included treatment with topical anesthetics such as xylocaine, benzocaine and cocaine, treatment with solutions which coat the 20 ulcerative lesions with a polysaccharide gel and use of antiseptic solutions such as Chlorhexadine. While all these treatments do provide some relief, none are directed to the actual healing of oral mucositis, which entails directly healing the mucosal epithelium cells.

Recently, certain local-acting growth factors, such as TGF-a have been shown to have some effect on ulcerative mucositis lesions at low concentrations, but less effect at higher concentrations (see US Pat.

No. 5,102870, issued April 7, 1992 to Florine et al.) The biphasic effect exhibited by such factors may limit their clinical utility. There remains a need for a therapy that inhibits ulcerative mucositis lesion formation and significantly enhances healing of lesions following their formation.

-4 Gastointestinal ulcer disease, in particular, peptic ulcers, affect 5-15% of the United States population. Peptic ulcers include gastric ulcers, which occur as lesions in the wall of the stomach, and duodenal ulcers, which are deep lesions that occur in the wall of the duodenum, the upper portion of the small intestine. Another ulcer disease, particularly worrisome to pediatricians, occurs in the premature infants. This condition, known as 10 necrotizing enterocolitis, affects 10-15% of newborns having a birth weight of under 1.5 kg and results in severe ulceration of the small intestine, which frequently requires surgery. Gastric ulcers can result from an imbalance in factors which maintain the natural gasatrointestinal barrier, including factors which neutralize corrosive gastric juices, such as the mucous bicarbonate, and other factors which protect the body from luminal damaging agents. Although current antiulcer therapeutics, including antisecretory *20 products such as cimetidine and ranitidine, appear to be effective in healing duodenal ulcers, it is generally believed that they are effective because they reduce normal gastric acid secretion. While the reduction in acidity aids in the closure of the ulcer, it also interferes with normal digestion. Accordingly, a high percentage of ulcers healed with current therapies recur within one year of therapy. The high rate of ulcer recurrence is thought to be at least partially attributable to the reduced number of mucusproducing cells in the scar tissue which is left at the site of the healed ulcer, rendering the area more vulnerable to rupture when the gastointestinal acidity returns to normal.

PCT Application No. PCT/US89/03467 discloses the use of an acid-resistant local-acting fibroblast grown factor to treat GI ulcers. US Pat. No. 5,043,329 discloses the use of phospholipids to treat ulcers of the gastrointestinal tract. PCT Publ. No. W089/10409 discloses cDNAs encoding bovine and human BMP3, a bone morphogenetic protein shown to induce formation of "cartilage-like nodules" and said to be useful for inducing formation of bone and/or cartilage. W089/10409 speculates that BMP3 also may be useful in the repair of damage in other tissues, including wounds such as "incisions, burns and ulcers".

Severe ulceration of the gastrointestinal mucosa also can spontaneously occur in the lower bowel (distal ileum and colon) in a spectrum of clinical disorders called inflammatory bowel disease (IBD). The two major diseases in this classification are ulcerative colitis and regional enteritis (Crohn's Disease) which are associated with severe mucosal ulceration (frequently penetrating the wall of the bowel and forming strictures and fistulas), severe mucosal and submucosal inflammation and edema, and fibrosis. Other forms of IBD include regional ileitis and proctitis. Clinically, patients with fulminant IBD can be severely ill with massive diarrhea, blood loss, dehydration, weight loss and fever.

The prognosis of the disease is not good and frequently requires resection of the disease tissue.

It is an object of this invention to provide methods and compositions for maintaining the integrity of the gastrointestinal luminal lining in a mammal. Another 5/1 object is to provide methods and compositions for regenerating basal epithelium and mucosa in ulcerated gastrointestinal tract barrier tissue, including the oral mucosa. Another object of the invention is to provide tissue protective methods and compositions that allow extension or enhancement of a chemical or radiotherapy.

Another object is to provide methods and compositions capable of limiting the proliferation of epithelial cells, particularly the a t O• •o 6 basal epithelial cels of the gastrointestinal tract.

Still antoher object is to provide methods and compositions for substantially inhibiting inflammation normally associated with ulcerative diseases. Another object is to provide methods and compositions for protecting mucosal tissue from the tissue desctructive effects associated with xerostomia. Yet another object is to provide methods and compositions for the treatment of oral mucositis, peptic ulcers, ulcerative colitis, regional enteritis, necrotizing enterocolitis, proctitis and other ulcerative diseases of the gastrointestinal tract.

These and other objects and features of the invention will be apparent from the description, drawings, and claims which follow.

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7 Summary of the Invention It now has been discovered that morphogenic proteins ("morphogen"), as defined herein, are useful as therapeutic methods and compositions for protecting the luminal lining of the gastrointestinal tract from ulceration, particularly in individuals at risk for ulcer formation. Specifically, the morphogens decribed herein can limit the proliferation of epithelial cells, 10 inhibit the inflammation normally associated with ulcerative disease, inhibit scar tissue formation, and/or induce repair and regeneration of the ulcerated tissue.

9a In one aspect, the invention features compositions and therapeutic treatment methods that comprise the step of administering to a mammal a therapeutically effective amount of a morphogenic protein ("morphogen"), as defined herein, upon injury to all or 20 a portion of the GI tract luminal lining, or in anticipation of such injury, for a time and at a concentration sufficient to maintain the integrity of the GI tract luminal lining, including repairing ulcerated tissue, and/or inhibiting damage thereto.

In another aspect, the invention features compositions and therapeutic treatment methods for maintaining the integrity of the GI tract luminal lining in a mammal which include administering to the mammal, upon injury to all or a portion of the GI tract luminal lining, or in anticipation of such injury, a compound that stimulates in vivo a therapeutically effective concentration of an endogenous morphogen within the body of the mammal sufficient to maintain the integrity of the luminal lining, including regenerating ulcerated tissue and/or inhibiting damage thereto. These compounds are referred to herein as morphogen-stimulating agents, and are understood to include substances which, when administered to a mammal, act on cells in tissue(s) or organ(s) that normally are responsible for, or capable of, producing a morphogen and/or secreting a morphogen, and which cause the endogenous level of the morphogen to be altered. The agent may act, for example, by stimulating expression and/or secretion of an 10 endogenous morphogen.

a As used herein, "gastrointestinal tract" means the entire gastrointestinal tract of a mammal, from the 'mouth to the rectum, inclusive, including the mouth cavity, esophagus, stomach, upper and lower intestines, and colon. As used herein, "ulcer" refers to an open lesion or break of the integrity of the epithelial lining of the gastrointestinal tract, resulting in erosion of the underlying mucosa. "Maintaining the 20 integrity of the luminal lining" means providing an effective morphogen concentration to the cells of the gasatrointestinal tract luminal lining, the concentration being sufficient to substantially inhibit lesion formation in the basal epithelium of the gastrointestinal barrier, including stimulating the regeneration of damaged tissue and/or inhibiting additional damage thereto. "Protecting" mucosal tissue means providing a therapeutically effective morphogen concentration to the cells of the gastrointestinal tract luminal lining sufficient to inhibit the tissue damage associated with tissue ulceration, including stimulating regeneration of damaged tissue and/or inhibiting additional damage thereto. "Symptomalleviating cofactor" refers to one or more pharmaceuticals which may be administered together with 9 the therapeutic agents of this invention and which alleviate or mitigate one or more of the symptoms typically associated with periodontal tissue loss.

Exemplary cofactors include antibiotics, antiseptics, anti-viral and anti-fungal agents, non-steroidal antiinflammatory agents, anesthetics and analgesics, and antisecretory agents.

In preferred embodiments of the invention, the mammal is a human and ulcers treatable according to the invention include those found in the ileum which cause regional ileitis, those found in the colon which cause ulcerative colitis, regional enteritis (Crohn's disease), proctitis and other forms of inflammatory bowel disease (IBD), gastric ulcers such as those found in the stomach, small intestines, duodenum and esophagus; and ulcers found in the mouth. The compositions and methods described herein are particularly useful in treating mucositis lesions "20 caused by chemotherapy or radiation therapy.

a .e Because the morphogens described herein inhibit ulceration of the oral mucosa that typically results from cancer therapies, in another aspect, the invention provides cancer treatment methods and compositions that significantly reduce or inhibit the onset of oral mucositis in a patient. In addition, the morphogens described herein may be used in conjunction with existing chemical or radiation therapies to enhance 30 their efficacy. Cancer chemical and radiation therapies currently in use often are limited in dose or duration by the onset of severe oral mucositis and/or the sepsis which often follows lesion formation. The morphogens described herein can inhibit lesion formation and, accordingly, their administration to a 10 patient as part of a cancer therapy may allow significant enhancement of current therapy doses and/or treatment times.

The morphogens described herein can limit cell proliferation in a proliferating epithelial cell population, thereby protecting these cells from the cytotoxic effects of chemotherapeutic and radiotherapeutic treatments. Accordingly, in another aspect, the invention provides methods and compositions for limiting the mitogenic activity of epithelial cells. This activity of the morphogens also has application for other diseases associated with proliferating epithelial cells, including psoriasis and other such skin tissue disorders. In addition, this activity of morphogens also may be useful to limit hair loss typically associated with cancer therapies.

The morphogens described also herein inhibit 20 inflammation. Accordingly, in another aspect, the invention provides methods and compositions for inhibiting the inflammation associated with ulcerative disease.

The morphogens described herein also stimulate tissue morphogenesis at a site of tissue damage, inhibiting scar tissue formation at a lesion site.

Accordingly, another aspect of the invention includes methods and compositions for inhibiting scar tissue 30 formation at a lesion site.

In another aspect of the invention, the morphogens S* described herein are useful in protecting the mucosal membrane from the tissue destructive effects associated with xerostomia. The xerostomaic condition may be S- 11 induced by a clinical therapy, including a cancer therapy, medication, diet or result from tissue senescence or an organic disorder of the salivary glands.

In one preferred embodiment, the morphogen or morphogen-stimulating agent is administered directly to the individual by topical administration, by coating the desired surface to be treated with the morphogen or morphogen-stimulating agent. For example, the therapeutic agent may be provided to the desired site by consuming a formulation containing the therapeutic agent in association with a compound capable of coating or adhering to the luminal lining surface. Such compounds include pectin-containing or sucralfate solutions such as are used in Milk of Magnesia and Kaopectate. For oral mucositis treatments, the agent may be provided in an oral rinse similar to a mouth wash that is swished around the mouth to coat the affected tissue, or disposed in a slow-dissolving lozenge or troche. Alternatively, the therapeutic agent may be provided to the site by physically applying or painting a formulation containing the morphogen or morphogen-stimulating agent to the site.

25 Compositions for topical administration also may include a liquid adhesive to adhere the morphogen or morphogen-stimulating agent to the tissue surface.

Useful adhesives include Zilactin, as is used in Orabase, hydroxypropylcellulose, and fibrinogen/thrombin solutions. Another potentially useful adhesive is the bioadhesive described in U.S.

Patent No. 5,197,973. The liquid adhesive may be painted onto the tissue surface, or formulated into an aerosol that is sprayed onto the affected tissue. For treatment of the lower bowel, the therapeutic agent 12 also may be provided rectally, by suppository, foam, liquid ointment or cream, particularly for the treatment of ulcerations of the ileum and colon. In another embodiment of the invention, the morphogen or morphogen-stimulating agent is provided systemically, by parenteral administration.

In any treatment method of the invention, "administration of morphogen" refers to the administration of the morphogen, either alone or in combination with other molecules. For example, the mature form of the morphogen may be provided in association with its precursor "pro" domain, which is known to enhance the solubility of the protein in physiological solutions. Other useful molecules known to enhance protein solubility include casein and other milk components, as well as various serum proteins.

Additional useful molecules which may be associated with the morphogen or morphogen-stimulating agent 20 include tissue targeting molecules capable of directing the morphogen or morphogen-stimulating agent to epithelial mucosa tissue. Tissue targeting molecules envisioned to be useful in the treatment protocols of this invention include antibodies, antibody fragments 25 or other binding proteins which interact specifically with surface molecules on GI barrier tissue cells.

Non-steroidal anti-inflammatory agents which typically are targeted to inflamed tissue also may be used.

Still another useful tissue targeting molecule may comprise part or all of the morphogen precursor "pro" S. domain. Under naturally occurring conditions, the endogenous morphogens described herein may be synthesized in other tissues and transported to target tissue after secretion from the synthesizing tissue.

S-13- For example, while the protein has been shown to be active in bone tissue, the primary source of OP-1 synthesis appears to be the tissue of the urogenic system renal and bladder tissue), with secondary expression levels occurring in the brain, heart and lungs (see below.) Moreover, the protein has been identified in serum, saliva and various milk forms. In addition, the secreted form of the protein comprises the mature dimer in association with the pro domain of the intact morphogen sequence. Accordingly, the associated morphogen pro domains may act to target specific morphogens to different tissues in vivo.

Associated tissue targeting or solubility-enhancing molecules also may be covalently linked to the morphogen using standard chemical means, including acid-labile linkages, which likely will be preferentially cleaved in the acidic environment of the GI tract.

Finally, the morphogens or morphogen-stimulating agents provided herein also may be administered in combination with other molecules ("cofactors"), known to be beneficial in ulcer treatments, particularly 25 cofactors capable of mitigating or alleviating symptoms typically associated with ulcerated tissue damage and/or loss. Examples of such cofactors include, analgesics/anesthetics such as xylocaine, and benzocaine; antiseptics such as chlorohexidine; antibacterial, anti-viral and anti-fungal agents, including aminoglycosides, macrolides, penicillins, and cephalosporins; and antacids or antisecretory agents such as cimetidine or ramitidine.

3-00;15:08 PATENT OFFICE CBR 7/ 32 -14 Among the morphogens useful in this invention are proteins originally identified as osteogenic proteins, such as the OP-1, OP-2 and CBMP2 proteins, as well as amino acid sequence-related proteins such as DPP (from Drosophila), vgl (from Xenopus), Vgr-1 (from mouse, see U.S. 5,011,691 to Oppermann et GDF-1 (from mouse, see Lee (1991) PNAS 88:4250-4254), all of which are presented in Table II and Seq. ID Nos.5-14), and the recently identified 60A protein (from Drosophila, Seq.

ID No. 24, see Wharton et al. (1991) PNAS 88:9214-9218.) The members of this family, which include members of the TGF-A super-family of proteins, share substantial amino acid sequence homology in their S. *C-terminal regions. The proteins are translated as a 15 precursor, having an N-terminal signal peptide sequence, typically less than about 30 residues, followed by a "pro" domain that is cleaved to yield the mature sequence. The "pro" form of the protein includes the pro domain and the mature domain, and forms a soluble species that appears to be the primary form secreted from cultured mammalian cells. The r signal peptide is cleaved rapidly upon translation, at a cleavage site that can be predicted in a given 2 sequence using the method of Von Heijne ((1986) Nucleic 25 Acids Research 14:4683-4691.) Table I, below, describes the various morphogens identified to date, including their nomenclature as used herein, their Seq.

ID references, and publication sources for the amino acid sequences for the full length proteins not included in the Seq. Listing. The disclosure of these publications is incorporated herein by reference.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

10/03 '00 FRI 15:09 [TX/RX NO 7835] 15 TABLE I "OP-1" Refers generically to the group of morphogenically active proteins expressed from part or all of a DNA sequence encoding OP-1 protein, including allelic and species variants thereof, human OP-1 Seq. ID No. 5, mature protein amino acid sequence), or mouse OP-1 Seq. ID No. 6, mature protein amino acid sequence.) The conserved seven cysteine skeleton is defined by residues 38 to 139 of Seq. ID Nos. 5 and 6. The cDNA sequences and the amino acids encoding the full length proteins are provided in Seq. Id Nos. 16 and 17 (hOP1) and Seq. ID Nos. 18 and 19 (mOPl.) The mature proteins are defined S:by residues 293-431 (hOP1) and 292-430 (mOP1). The "pro" regions of the proteins, cleaved to yield the mature, morphogenically active proteins are defined essentially by residues 30-292 (hOP1) and residues 30-291 (mOPl).

"OP-2" refers generically to the group of active proteins expressed from part or all of a DNA sequence encoding OP-2 protein, including allelic and species variants thereof, human OP-2 Seq.

ID No. 7, mature protein amino acid O-f:i sequence) or mouse OP-2 Seq.- ID No. 8, mature protein amino acid sequence). The conserved seven cysteine skeleton is defined by residues 38 to 139 16 of Seq. ID Nos. 7 and 8. The cDNA sequences and the amino acids encoding the full length proteins are provided in Seq.

ID Nos. 20 and 21 (hOP2) and Seq. ID Nos.

22 and 23 (mOP2.) The mature proteins are defined essentially by residues 264-402 (hOP2) and 261-399 (mOP2). The "pro" regions of the proteins, cleaved to yield the mature, morphogenically active proteins likely are defined essentially by residues 18-263 (hOP2) and residues 18-260 (mOP2). (Another cleavage site also occurs 21 residues upstream for hOP-2 protein.) "CBMP2" refers generically to the morphogenically active proteins expressed from a DNA sequence encoding the CBMP2 proteins, including allelic and species variants 20 thereof, human CBMP2A ("CBMP2A(fx)", Seq ID No. 9) or human CBMP2B DNA ("CBMP2B(fx)", Seq. ID No. 10). The amino acid sequence for the full length proteins, referred to in the literature as BMP2A and BMP2B, or BMP2 and BMP4, appear in Wozney, et al. (1988) Science 242:1528-1534. The pro domain for BMP2 (BMP2A) likely includes residues 25-248 or 25-282; the mature protein, residues 249-396 or 283-396. The pro domain for S" BMP4 (BMP2B) likely includes residues 256 or 25-292; the mature protein, residues 257-408 or 293-408.

0 "DPP(fx)" "Vgl(fx)" 17 refers to protein sequences encoded by the Drosophila DPP gene and defining the conserved seven cysteine skeleton (Seq. ID No. 11). The amino acid sequence for the full length protein appears in Padgett, et al (1987) Nature 325: 81-84. The pro domain likely extends from the signal peptide cleavage site to residue 456; the mature protein likely is defined by residues 457-588.

refers to protein sequences encoded by the Xenopus Vgl gene and defining the conserved seven cysteine skeleton (Seq. ID No. 12). The amino acid sequence for the full length protein appears in Weeks (1987) Cell 51: 861-867. The pro domain likely extends from the signal peptide cleavage site to residue 246; the mature protein likely is defined by residues 247-360.

refers to protein sequences encoded by the murine Vgr-1 gene and defining the conserved seven cysteine skeleton (Seq. ID No. 13). The amino acid sequence for the full length protein appears in Lyons, et al, (1989) PNAS 86: 4554-4558. The pro domain likely extends from the signal peptide cleavage site to residue 299; the mature protein likely is defined by residues 300-438.

refers to protein sequences encoded by the human GDF-1 gene and defining the *te.

C. C C. C C C

C

*C

C S. S "Vgr-l(fx)" "GDF-l(fx)" C C C. C

S

C CC 18 q. a a a. a. a a 44 a.

a.

"60A" 20 "BMP3(fx)" 30 conserved seven cysteine skeleton (Seq. ID No. 14). The cDNA and encoded amino sequence for the full length protein is provided in Seq. ID. No. 32. The pro domain likely extends from the signal peptide cleavage site to residue 214; the mature protein likely is defined by residues 215-372.

refers generically to the morphogenically active proteins expressed from part or all of a DNA sequence (from the Drosophila gene) encoding the 60A proteins (see Seq.

ID No. 24 wherein the cDNA and encoded amino acid sequence for the full length protein is provided). "60A(fx)" refers to the protein sequences defining the conserved seven cysteine skeleton (residues 354 to 455 of Seq. ID No. 24.) The pro domain likely extends from the signal peptide cleavage site to residue 324; the mature protein likely is defined by residues 325-455.

refers to protein sequences encoded by the human BMP3 gene and defining the conserved seven cysteine skeleton (Seq. ID No. 26).

The amino acid sequence for the full length protein appears in Wozney et al.

(1988) Science 242: 1528-1534. The pro domain likely extends from the signal peptide cleavage site to residue 290; the mature protein likely is defined by residues 291-472.

a.

a a ;t o 19 refers to protein sequences encoded by the human BMP5 gene and defining the conserved seven cysteine skeleton (Seq. ID No. 27).

The amino acid sequence for the full length protein appears in Celeste, et al.

(1991) PNAS 87: 9843-9847. The pro domain likely extends from the signal peptide cleavage site to residue 316; the mature protein likely is defined by residues 317-454.

refers to protein sequences encoded by the human BMP6 gene and defining the conserved seven cysteine skeleton (Seq. ID No. 28).

The amino acid sequence for the full length protein appears in Celeste, et al.

(1990) PNAS 87: 9843-5847. The pro domain likely extends from the signal peptide cleavage site to residue 374; the mature sequence likely includes residues 375-513.

"BMP6(fx)" 0.

The OP-2 proteins have an additional cysteine residue in this region see residue 41 of Seq. ID Nos. 7 and in addition to the conserved cysteine skeleton in common with the other proteins in this family. The GDF-1 protein has a four amino acid insert within the conserved skeleton (residues 44-47 of Seq.

ID No. 14) but this insert likely does not interfere with the relationship of the cysteines in the folded 30 structure. In addition, the CBMP2 proteins are missing one amino acid residue within the cysteine skeleton.

The morphogens are inactive when reduced, but are active as oxidized homodimers and when oxidized in combination with other morphogens of this invention.

a a a S- 20 Thus, as defined herein, a morphogen is a dimeric protein comprising a pair of polypeptide chains, wherein each polypeptide chain comprises at least the C-terminal six cysteine skeleton defined by residues 43-139 of Seq. ID No. 5, including functionally equivalent arrangements of these cysteines amino acid insertions or deletions which alter the linear arrangement of the cysteines in the sequence but not their relationship in the folded structure), such that, when the polypeptide chains are folded, the dimeric protein species comprising the pair of polypeptide chains has the appropriate three-dimensional structure, including the appropriate intra- or inter-chain disulfide bonds such that the protein is capable of acting as a morphogen as defined herein. Specifically, the morphogens generally are capable of all of the following biological functions in a morphogenically permissive environment: stimulating proliferation of progenitor cells; stimulating the differentiation of 20 progenitor cells; stimulating the proliferation of differentiated cells; and supporting the growth and maintenance of differentiated cells. In addition, it is also anticipated that these morphogens are capable of inducing redifferentiation of committed cells under appropriate environmental conditions.

In one preferred aspect, the morphogens of this invention comprise one of two species of generic amino acid sequences: Generic Sequence 1 (Seq. ID No. 1) or 30 Generic Sequence 2 (Seq. ID No. where each Xaa indicates one of the 20 naturally-occurring L-isomer, a-amino acids or a derivative thereof. Generic Sequence 1 comprises the conserved six cysteine skeleton and Generic Sequence 2 comprises the conserved six cysteine skeleton plus the additional cysteine 21 identified in OP-2 (see residue 36, Seq. ID No. In another preferred aspect, these sequences further comprise the following additional sequence at their Nterminus: Cys Xaa Xaa Xaa Xaa (Seq. ID No. 1 Preferred amino acid sequences within the foregoing generic sequences include: Generic Sequence 3 (Seq. ID No. Generic Sequence 4 (Seq. ID No. Generic Sequence 5 (Seq. ID No. 30) and Generic Sequence 6 (Seq. ID No. 31), listed below. These Generic Sequences accommodate the homologies shared among the various preferred members of this morphogen family identified in Table II, as well as the amino acid sequence variation among them. Specifically, Generic Sequences 3 and 4 are composite amino acid sequences of the following proteins presented in Table II and 20 identified in Seq. ID Nos. 5-14: human OP-1 (hOP-1, Seq. ID Nos. 5 and 16-17), mouse OP-1 (mOP-1, Seq. ID Nos. 6 and 18-19), human and mouse OP-2 (Seq. ID Nos. 7, 8, and 20-22), CBMP2A (Seq. ID No. CBMP2B (Seq. ID No. 10), DPP (from Drosophila, Seq. ID No. 11), Vgl, (from Xenopus, Seq. ID No. 12), Vgr-1 (from mouse, Seq. ID No. 13), and GDF-1 (from mouse, Seq. ID No. 14.) The generic sequences include both the amino acid identity shared by the sequences in STable II, as well as alternative residues for the variable positions within the sequence. Note that these generic sequences allow for an additional cysteine at position 41 or 46 in Generic Sequences -3 or 4, respectively, providing an appropriate cysteine skeleton where inter- or intramolecular disulfide bonds can form, and contain certain critical amino acids which influence the tertiary structure of the proteins.

22 Generic Seq~uence 3 Leu Tyr Val Xaa Phe 1 Xaa Xaa Xaa Gly Trp Xaa Xaa Trp Xaa Xaa Ala Pro Xaa Gly Xaa Xaa Ala Gly Xaa Cys Xaa Xaa Tyr Cys Xaa Xaa Pro Xaa Xaa Xaa Xaa Xaa Asn Xaa Xaa Leu Xaa Xaa Xaa His Ala Xaa Xaa Xaa Xaa Xaa Xaa Xaa *0 0* 0 *0 00 0 if. 0 0* 0000 Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Pro Xaa Xaa Xaa Leu Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Val Xaa Leu Xaa Met Xaa Val Xaa 25 A Xaa 85 Xaa Cys Gly Cys Xaa a.

wherein each Xaa is independently selected from a group of one or more specified amino acids defined as follows: "Res." means "residue" and Xaa at res.4= (Ser, Asp or Glu); Xaa at res.6 (Arg, Gln, Ser or Lys); Xaa at res.7 (Asp or Glu); Xaa at res.8 (Leu or Val); Xaa at res.11 (Gin, Leu, Asp, His or Asn); 0 -23 Xaa at res.12 (Asp, Arg or Asn); Xaa at res.14 (Ile or Val); Xaa at res.15 (Ile or Val); Xaa at res.18 (Giu, Gin, Leu, Lys, Pro or Arg); Xaa at res.20 (Tyr or Phe); Xaa at res.21 (Ala, Ser, Asp, Met, His, Leu or Gin); Xaa at res.23 (Tyr, Asn or Phe); Xaa at res.26 (Giu, His, Tyr, Asp or Gin); Xaa at res.28 (Giu, Lys, Asp or Gin); Xaa at res.30 (Ala, Ser, Pro or Gin); Xaa at res.31 (Phe, Leu or Tyr); Xaa at res.33 (Leu or Val); Xaa at res.34 (Asn, Asp, Ala or Thr); Xaa at res.35 (Ser, Asp, Giu, Leu or Ala); Xaa at res.36 (Tyr, Cys, His, Ser or Ile); Xaa at res.37 (Met, Phe, Gly or Leu); Xaa at res.38 (Asn or Ser); Xaa at res.39 (Ala, Ser or Gly); Xaa at (Thr, Leu or Ser); Xaa at res.44 (Ile or Val); Xaa at res.45 (Val or Leu); Xaa at res.46 (Gin or Arg); Xaa at res.47 (Thr, Ala or Ser); Xaa at res.49 (Vai or Met); Xaa at res.50 (His or Asn); Xaa at res.51 (Phe, Leu, Asn, Ser, Ala or Val); Xaa at res.52 (Ile, Met, Asn, Ala or Vai); Xaa at .:20 res.53 (Asn, Lys, Ala or Giu); Xaa at res.54 (Pro or Ser); Xaa at res.55 (Glu, Asp, Asn, or Giy); Xaa at res.56 (Thr, Ala, Val, Lys, Asp, Tyr, Ser or Ala); Xaa at res.57 (Val, Ala or Ile); Xaa at res.58 (Pro or Asp); Xaa at res.59 (Lys or Leu); Xaa at (Pro or Ala); Xaa at res.63 (Ala or Val); Xaa at (Thr or Ala); Xaa at res.66 (Gin, Lys, Arg or Giu); Xaa at res.67 (Leu, Met or Vai); Xaa at res.68 (Asn, Ser or Asp); Xaa at res.69 (Ala, Pro Ser); Xaa at res.70 (Ile, Thr or Val); Xaa at 30res.71 (Ser or Ala); Xaa at res.72 (Val or Met); Xaa at res.74 (Tyr or Phe); Xaa at res.75 (Phe, Tyr or Leu); Xaa at res.76 (Asp or Asn); Xaa at res.77 (Asp, Giu, Asn or Ser); Xaa at res.78 (Ser, Gin, Asn or Tyr); Xaa at res.79 (Ser, Asn, Asp or Giu); Xaa at res.80 (Asn, Thr or Lys); Xaa at res.82 (Ile or 0 -24 Val); Xaa at res.84 (Lys or Arg); Xaa at (Lys, Asn, Gin or His); Xaa at res.86 (Tyr or His); Xaa at res.87 (Arg, Gin or Glu); Xaa at res.88 (Asn, Glu or Asp); Xaa at res.90 (Val, Thr or Ala); Xaa at res.92 (Arg, Lys, Val, Asp or Glu); Xaa at res.93 (Ala, Gly or Glu); and Xaa at res.97 =(His or Arg); Generic Sequence 4 Cys Xaa Xaa Xaa Xaa Leu Tyr Val Xaa Phe 1 5 Xaa Xaa Xaa Gly Trp Xaa Xaa Trp Xaa Xaa Ala Pro Xaa Gly Xaa Xaa Ala .20 Xaa Tyr Cys Xaa Gly Xaa Cys Xaa *.Xaa Pro Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asn His Ala Xaa Xaa Xaa Xaa Leu Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Cys Xaa Pro Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Leu Xaa Xaa Xaa 75 Xaa Xaa Xaa Xaa Val Xaa Leu Xaa Xaa Xaa Xaa Xaa Met Xaa Val Xaa Xaa Cys Gly Cys Xaa 100 -25 wherein each Xaa is independently selected from a group of one or more specified amino acids as defined by the following: "Res." means "residue" and Xaa at res.2- (Lys or Arg); Xaa at res.3 (Lys or Arg); Xaa at res.4 (His or Arg); Xaa at res.5 (Glu, Ser, His, Gly, Arg or Pro); Xaa at res.9 (Ser, Asp or Giu); Xaa at res.11 (Arg, Gin, Ser or Lys); Xaa at res.12 (Asp or Giu); Xaa at res.13 (Leu or Val); Xaa at res.16 (Gin, Leu, Asp, His or Asn); Xaa at res.17= (Asp, Arg, or Asn); Xaa at res.19 (Ile or Val); Xaa at res.20 (Ile or Val); Xaa at res.23 (Glu, Gln, Leu, Lys, Pro or Arg); Xaa at res.25 (Tyr or Phe); Xaa at res.26 (Ala, Ser, Asp, Met, His, Leu, or Gln); Xaa at res.28 (Tyr, Asn or Phe); Xaa at res.31 (Giu, His, Tyr, Asp or Gin); Xaa at res.33 Giu, Lys, Asp or Gn;Xaa at res.35 (lSer or Pro); Xaa at res.36 (Phe, Leu or Tyr); Xaa at res.38 (Leu or Val); Xaa at res.39 (Asn, Asp, Ala or Thr); Xaa at res.40 (Ser, Asp, Giu, Leu or Aia); Xaa at res.41- :20 (Tyr, Cys, His, Ser or Ile); Xaa at res.42 (Met, Phe, Gly or Leu); Xaa at res.44 (Ala, Ser or Gly); Xaa at (Thr, Leu or Ser); Xaa at res.49 (Ile or Val); Xaa at res.50 (Val or Leu); xaa at res.51 (Gin or Arg); Xaa at res.52 (Thr, Ala or Ser); Xaa at res.54 (Vai or Met); Xaa at res.55 (His or Asn); Xaa at res.56 (Phe, Leu, Asn, Ser, Ala or Val); Xaa at res.57 (Ile, Met, Asn, Ala or Val); Xaa at res.58 (Asn, Lys, Ala or Giu); Xaa at res.59 (Pro or Ser); Xaa at res.60 (Glu, Asp, or Gly); Xaa at .30 res.61 (Thr, Ala, Val, Lys, Asp, Tyr, Ser or Ala); Xaa at res.62 (Val, Ala or Ile); Xaa at res.63 (Pro or Asp); Xaa at res.64 (Lys or Leu); Xaa at (Pro or Ala); Xaa at res.68 (Ala or Val); Xaa at (Thr or Ala); Xaa at res.71 (Gin, Lys, Arg or Glu); Xaa at res.72 (Leu, Met or Val); Xaa at 26 res.73 (Asn, Ser or Asp); Xaa at res.74 (Ala, Pro or Ser); Xaa at res.75 (Ile, Thr or Val); Xaa at res.76 (Ser or Ala); Xaa at res.77 (Val or Met); Xaa at res.79 (Tyr or Phe); Xaa at res.80 (Phe, Tyr or Leu); Xaa at res.81 (Asp or Asn); Xaa at res.82 (Asp, Glu, Asn or Ser); Xaa at res.83 (Ser, Gin, Asn or Tyr); Xaa at res.84 (Ser, Asn, Asp or Glu); Xaa at (Asn, Thr or Lys); Xaa at res.87 (Ile or Val); Xaa at res.89 (Lys or Arg); Xaa at res.90 (Lys, Asn, Gin or His); Xaa at res.91 (Tyr or His); Xaa at res.92 (Arg, Gin or Glu); Xaa at res.93 (Asn, Glu or Asp); Xaa at res.95 (Val, Thr or Ala); Xaa at res.97 (Arg, Lys, Val, Asp or Glu); Xaa at res.98 (Ala, Gly or Glu); and Xaa at res.102 (His or Arg).

Similarly, Generic Sequence 5 (Seq. ID No. 30) and Generic Sequence 6 (Seq. ID No. 31) accommodate the homologies shared among all the morphogen protein 20 family members identified in Table II. Specifically, Generic Sequences 5 and 6 are composite amino acid sequences of human OP-1 (hOP-1, Seq. ID Nos. 5 and 16- 17), mouse OP-1 (mOP-1, Seq. ID Nos. 6 and 18-19), human and mouse OP-2 (Seq. ID Nos. 7, 8, and 20-22), CBMP2A (Seq. ID No. CBMP2B (Seq. ID No. 10), DPP (from Drosophila, Seq. ID No. 11), Vgl, (from Xenopus, Seq. ID No. 12), Vgr-1 (from mouse, Seq. ID No. 13), Sand GDF-1 (from mouse, Seq. ID No. 14), human BMP3 (Seq. ID No. 26), human BMP5 (Seq. ID No. 27), human 30 BMP6 (Seq. ID No. 28) and 60(A) (from Drosophila, Seq.

ID Nos. 24-25). The generic sequences include both the amino acid identity shared by these sequences in the C-terminal domain, defined by the six and seven cysteine skeletons (Generic Sequences 5 and 6, respectively), as well as alternative residues for the 0 27 variable positions within the sequence. As for Generic Sequences 3 and 4, Generic Sequences 5 and 6 allow for an additional cysteine at position 41 (Generic Sequence or position 46 (Generic Sequence providing an appropriate cysteine skeleton where inter- or intramolecular disulfide bonds can form, and containing certain critical amino acids which influence the tertiary structure of the proteins.

0 Generic Sequence Leu Xaa Xaa Xaa Phe 1 Xaa Xaa Xaa Gly Trp Xaa Xaa Trp Xaa

S.

9*S* *4*S C. C.

C*

C.

C

4 Xaa 15 Xaa Pro Xaa Xaa Tyr Cys Xaa 25 Xaa Pro Xaa Xaa Xaa Xaa Xaa Asn 40 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ala Gly Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa

S.

S

C. C

CC

Cys Xaa Xaa Xaa Xaa Xaa Pro Xaa Xaa Xaa Leu Xaa Xaa Xaa Xaa Xaa Xaa His Ala Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Val Xaa Leu Xaa Met Xaa Val Xaa Cys Xaa Cys Xaa 28 wherein each Xaa is independently selected from a group of one or more specified amino acids defined as follows: "Res." means "residue" and Xaa at res.2 (Tyr or Lys); Xaa at res.3 Val or Ile); Xaa at res.4 (Ser, Asp or Glu); Xaa at res.6 (Arg, Gin, Ser, Lys or Ala); Xaa at res.7 (Asp, Giu or Lys); Xaa at res.8 (Leu, Val or Ile); Xaa at res.ii (Gin, Leu, Asp, His, Asn or Ser); Xaa at res.12 (Asp, Arg, Asn or Giu); Xaa at res.14 (Ile or Val); Xaa at (Ilie or Val); Xaa at res.16 (Ala or Ser); Xaa at res.18 (Glu, Gin, Leu, Lys, Pro or Arg); Xaa at res.19 (Gly or Ser); Xaa at res.20 (Tyr or Phe); Xaa at res.21 (Ala, Ser, Asp, Met, His, Gin, Leu or Gly); Xaa at res.23 (Tyr, Asn or Phe); Xaa at res.26 (Giu, His, Tyr, Asp, Gin or Ser); Xaa at res.28 (Giu, Lys, Asp, Gin or Ala); Xaa at (Ala, Ser, Pro, Gin or Asn); Xaa at res.31 (Phe, Leu or Tyr); Xaa at res.33 (Leu, Val or Met); Xaa at 20 res.34 (Asn, Asp, Ala, Thr or Pro); Xaa at (Ser, Asp, Giu, Leu, Ala or Lys); Xaa at res.36 (Tyr, Cys, His, Ser or Ile); Xaa at res.37 (Met, Phe, Gly or Leu); Xaa at res.38 (Asn, Ser or Lys); Xaa at res.39 (Ala, Ser, Gly or Pro); Xaa at res.40 (Thr, Leu or Ser)'; Xaa at res.44 (Ilie, Val or Thr); Xaa at (Vai, Leu or Ile); Xaa at res.46 (Gin or Arg); Xaa at res.47 (Thr, Ala or Ser); Xaa at res.48 (Leu or Ilie); Xaa at res.49 (Val or Met); Xaa at res.50 (His, Asn or Arg); Xaa at res.Si (Phe, Leu, Asn, Ser, Ala or Val); Xaa at res.52 (Ile, Met, Asn, Ala, Val or Leu); xaa at res.53 (Asn, Lys, Ala, Giu, Gly or Phe); Xaa at res.54 (Pro, Ser or Val); Xaa at res.55 (Giu, Asp, Asn, Giy, Val or Lys); Xaa at res.56 (Thr, Ala, Val, Lys, Asp, Tyr, Ser, Ala, Pro or His); Xaa at res.57 (Vai, Ala or Ile); -29 Xaa at res.58 (Pro or Asp); Xaa at res.59 (Lys, Leu or Glu); Xaa at res.60 (Pro or Ala); Xaa at res.63 (Ala or Val); Xaa at res.65 (Thr, Ala or Glu); Xaa at res.66 (Gin, Lys, Arg or Glu); Xaa at res.67 (Leu, Met or Val); Xaa at res.68 (Asn, Ser, Asp or Gly); Xaa at res.69 (Ala, Pro or Ser); Xaa at res.70 (Ile, Thr, Val or Leu); Xaa at res.71 (Ser, Ala or Pro); Xaa at res.72 (Val, Met or Ile); Xaa at res.74 (Tyr or Phe); Xaa at res.75 (Phe, Tyr, Leu or His); Xaa at res.76 (Asp, Asn or Leu); Xaa at res.77 (Asp, Glu, Asn or Ser); Xaa at res.78 (Ser, Gin, Asn, Tyr or Asp); Xaa at res.79 (Ser, Asn, Asp, Glu or Lys); Xaa at res.80 (Asn, Thr or Lys); Xaa at res.82 (Ile, Val or Asn); Xaa at res.84 (Lys or Arg); Xaa at res.85 (Lys, Asn, Gin, His or Val); Xaa at res.86 (Tyr or His); Xaa at res.87 (Arg, Gin, Glu or Pro); Xaa at res.88 (Asn, Glu or Asp); Xaa at (Val, Thr, Ala or Ile); Xaa at res.92 (Arg, Lys, Val, Asp or Glu); Xaa at res.93 =(Ala, Gly, Glu or Ser); Xaa at res.95 (Gly or Ala) and Xaa at res.97 (His or Arg).

Generic Sequence 6 Cys Xaa Xaa Xaa Xaa Leu Xaa Xaa Xaa Phe *1 5 Xaa Xaa Xaa Gly Trp Xaa Xaa Trp Xaa *Xaa Xaa Pro Xaa Xaa Xaa Xaa Ala 20 Xaa Tyr Cys Xaa Gly Xaa Cys Xaa Xaa Pro Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asn His Ala Xaa Xaa 30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Cys Xaa Pro Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Leu Xaa Xaa Xaa Xaa Xaa Xaa Xaa Val Xaa Leu Xaa Xaa Xaa Xaa Xaa Met Xaa Val Xaa Xaa Cys Xaa Cys Xaa 100 wherein each Xaa is independently selected from a group of one or more specified amino acids as defined by the following: "Res." means "residue" and Xaa at res.2 (Lys, Arg, Ala or Gln); Xaa at res.3 (Lys, Arg or *.20 Met); Xaa at res.4 (His, Arg or Gin); Xaa at (Glu, Ser, His, Gly, Arg, Pro, Thr, or Tyr); Xaa at res.7 (Tyr or Lys); Xaa at res.8 (Val or Ilie); Xaa at res.9 (Ser, Asp or Giu); Xaa at res.l1 (Arg, Gin, Ser, Lys or Ala); Xaa at res.12 (Asp, Giu, or Lys); Xaa at res.13 (Leu, Val or Ile); Xaa at res.16 (Gin, Leu, Asp, His, Asn or Ser); Xaa at res.17 (Asp, Arg, Asn or Glu); Xaa at res.19 (Ile or Val); Xaa at res.20 (Ile or Val); Xaa at res.21= (Ala or Ser); Xaa at res.23 (Glu, Gin, Leu, Lys, Pro 30 or Arg); Xaa at res.24 (Gly or Ser); Xaa at (Tyr or Phe); Xaa at res.26 (Ala, Ser, Asp, Met, His, Gin, Leu, or Gly); Xaa at res.28 (Tyr, Asn or Phe); Xaa at res.31 (Glu, His, Tyr, Asp, Gin or Ser); Xaa at res.33 Glu, Lys, Asp, Gin or Ala); Xaa at (Ala, Ser, Pro, Gln or Asn); Xaa at res.36 (Phe, Leu 31 or Tyr); Xaa at res.38 (Leu, Val or Met); Xaa at res.39 (Asn, Asp, Ala, Thr or Pro); Xaa at (Ser, Asp, Glu, Leu, Ala or Lys); Xaa at res.41 (Tyr, Cys, His, Ser or Ile); Xaa at res.42 (Met, Phe, Gly or Leu); Xaa at res.43 (Asn, Ser or Lys); Xaa at res.44 (Ala, Ser, Gly or Pro); Xaa at res.45 (Thr, Leu or Ser); Xaa at res.49 (Ile, Val or Thr); Xaa at (Val, Leu or Ile); Xaa at res.51 (Gin or Arg); Xaa at res.52 (Thr, Ala or Ser); Xaa at res.53 (Leu or Ile); Xaa at res.54 (Val or Met); Xaa at res.55 (His, Asn or Arg); Xaa at res.56 (Phe, Leu, Asn, Ser, Ala or Val); Xaa at res.57 (Ile, Met, Asn, Ala, Val or Leu); Xaa at res.58 (Asn, Lys, Ala, Giu, Gly or Phe); Xaa at res.59 (Pro, Ser or Val); Xaa at res.60 (Giu, Asp, Gly, Val or Lys); Xaa at res.61 (Thr, Ala, Val, Lys, Asp, Tyr, Ser, Ala, *.:Pro or His); Xaa at res.62 (Val, Ala or Ile); Xaa at res.63 (Pro or Asp); Xaa at res.64 (Lys, Leu or Giu); Xaa at res.65 (Pro or Ala); Xaa at res.68 :20 (Ala or Val); Xaa at res.70 (Thr, Ala or Giu); Xaa at res.71 (Gin, Lys, Arg or Giu); Xaa at res.72 (Leu, Met or Val); Xaa at res.73 (Asn, Ser, Asp or Gly); Xaa at res.74 (Ala, Pro or Ser); Xaa at (Ile, Thr, Val or Leu);6 Xaa at res.76 (Ser, Ala or Pro); Xaa at res.77 (Val, Met or Ile); Xaa at res.79 (Tyr or Phe); Xaa at res.80 (Phe, Tyr, Leu or His); Xaa at res.81 (Asp, Asn or Lbu); Xaa at res.82 (Asp, Giu, Asn or Ser); Xaa at res.83 (Ser, Asn, Tyr or Asp); Xaa at res.84 (Ser, Asn, Asp, Giu or Lys); Xaa at res.85 (Asn, Thr or Lys); Xaa at res.87 (Ile, Val or Asn); Xaa at res.89 (Lys or Arg); Xaa at res.90 (Lys, Asn, Gin, His or Val); Xaa at res.91 (Tyr or His); Xaa at res.92 (Arg, Gin, Giu or Pro); Xaa at res.93 (Asn, Giu or Asp); Xaa at res.95 (Val, Thr, Ala or Ile); Xaa at res.97 (Arg, 32 Lys, Val, Asp or Glu); Xaa at res.98 (Ala, Gly, Glu or Ser); Xaa at res.100 (Gly or Ala); and Xaa at res.102 (His or Arg).

Particularly useful sequences for use as morphogens in this invention include the C-terminal domains, e.g., the C-terminal 96-102 amino acid residues of Vgl, Vgr-1, DPP, OP-1, OP-2, CBMP-2A, CBMP-2B, GDF-1 (see Table II, below, and Seq. ID Nos. 5-14), as well as proteins comprising the C-terminal domains of BMP3, BMP5 and BMP6 (see Seq. ID Nos. 24-28), all of which include at least the conserved six or seven cysteine skeleton. In addition, biosynthetic constructs designed from the generic sequences, such as COP-1, 3-5, 7, 16, disclosed in U.S. Pat. No.

5,011,691, also are useful. Other sequences include the inhibins/activin proteins (see, for example, U.S.

Pat. Nos. 4,968,590 and 5,011,691). Accordingly, other '.useful sequences are those sharing at least 70% amino acid sequence homology or "similarity", and preferably homology or similarity with any of the sequences above. These are anticipated to include allelic, species variants and other sequence variants including "muteins" or "mutant proteins"), whether 25 naturally-occurring or biosynthetically produced, as well as novel members of this morphogenic family of proteins. As used herein, "amino acid sequence homology" is understood to mean amino acid sequence similarity, and homologous sequences share identical or S* 30 similar amino acids, where similar amino acids are conserved amino acids as defined by Dayoff et al., Atlas of Protein Sequence and Structure; Suppl.3, pp.345-362 Dayoff, ed., Nat'l BioMed.

Research Fdn., Washington D.C. 1978.) Thus, a candidate sequence sharing 70% amino acid homology with 33 a reference sequence requires that, following alignment of the candidate sequence with the reference sequence, 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 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 homologies and identities calculated use OP-i as the reference sequence. Also as used herein, sequences are aligned for homology and identity calculations using the method of Needleman et al. (1970) J.Mol. Biol. 48:443-453 and identities 20 calculated by the Align program (DNAstar, Inc.) In all "cases, internal gaps and amino acid insertions in the candidate sequence as aligned are ignored when making the homology/identity calculation.

The currently most preferred protein sequences useful as morphogens in this invention include those having greater than 60% identity, preferably greater than 65% identity, with the amino acid sequence defining the conserved six cysteine skeleton of hOPi 30 residues 43-139 of Seq. ID No. These most preferred sequences include both allelic and species variants of the OP-i and OP-2 proteins, including the Drosophila 60A protein. Accordingly, in another preferred aspect of the invention, useful morphogens include active proteins comprising species of 34 polypeptide chains having the generic amino acid sequence herein referred to as "OPX", which accommodates the homologies between the various identified species of OP1 and OP2 (Seq. ID No. 29).

In still another preferred aspect of the invention, useful morphogens include dimeric proteins comprising amino acid sequences encoded by nucleic acids that hybridize to DNA or RNA sequences encoding the Cterminal sequences defining the conserved seven cysteine domain of OP1 or OP2, nucleotides 1036- 1341 and nucleotides 1390-1695 of Seq. ID No. 16 and respectively, under stringent hybridization conditions. As used herein, stringent hybridization conditions are defined as hybridization in formamide, 5 X SSPE, 5 X Denhardt's Solution, and 0.1% SDS at 37°C overnight, and washing in 0.1 X SSPE, 0.1% SDS at 50 0

C.

20 The morphogens useful in the methods, composition and devices of this invention include proteins comprising any of the polypeptide chains described above, whether isolated from naturally-occurring sources, or produced by recombinant DNA or other synthetic techniques, and includes allelic and species S" ~variants of these proteins, naturally-occurring or biosynthetic mutants thereof, as well as various truncated and fusion constructs. Deletion or addition mutants also are envisioned to be active, including 30 those which may alter the conserved C-terminal cysteine skeleton, provided that the alteration does not functionally disrupt the relationship of these cysteines in the folded structure. Accordingly, such active forms are considered the equivalent of the specifically described constructs disclosed herein.

35 The proteins may include forms having varying glycosylation patterns, varying N-termini, a family of related proteins having regions of amino acid sequence homology, and active truncated or mutated forms of native or biosynthetic proteins, produced by expression of recombinant DNA in host cells.

The morphogenic proteins can be expressed from intact or truncated cDNA or from synthetic DNAs in procaryotic or eucaryotic host cells, and purified, cleaved, refolded, and dimerized to form morphogenically active compositions. Currently preferred host cells include E. coli or mammalian cells, such as CHO, COS or BSC cells. A detailed 15 description of the morphogens useful in the methods, compositions and devices of this invention is disclosed in international application US92/01908 (W092/15323).

A method for their recombinant production is provided in Sampath et al. (1992) J. Biol. Chem. 267: 20352- 20362.

Thus, in view of this disclosure, skilled genetic engineers can isolate genes from cDNA or genomic libraries of various different species which encode 25 appropriate amino acid sequences, or construct DNAs from oligonucleotides, and then can express them in S. various types of host cells, including both procaryotes and eucaryotes, to produce large quantities of active proteins capable of maintaining the integrity of the gastrointestinal tract luminal lining in individuals at risk for ulcer formation.

The foregoing and other objects, features and advantages of the present invention will be made more apparent from the following detailed description of the invention.

S-36 Brief Description of the Drawings The foregoing and other objects and features of this invention, as well as the invention itself, may be more fully understood from the following description, when read together with the accompanying drawings, in which: Fig. 1 graphs the effect of a morphogen OP- 1) and a placebo control on mucositic lesion formation; Fig. 2(A and B) are photomicrographs illustrating the ability of morphogens to inhibit lesion formation in an oral mucositis animal model, where (2A) shows lesion formation in untreated hamster cheek pouches; and (2B) shows the significantly reduced effect on morphogen treated cheek pouches; Fig. 3(A and B) graphs the antiproliferative effect of morphogens on mink lung cells; and Fig. 4(A-D) graphs the effects of a morphogen (eg., OP-1, Figs. 4A and 4C) and TGF-A (Fig. 4B and 4D) on collagen (4A and 4B) and hyaluronic acid (4C and 4D) production in primary fibroblast cultures.

37 Detailed Description of the Invention It now has been discovered that the proteins described herein are effective agents for maintaining the integrity of the gastrointestinal tract luminal lining in a mammal. As described herein, these proteins ("morphogens") are capable of substantially inhibiting lesion formation or associated tissue damage in the basal epithelium, and/or stimulating the repair and regeneration the barrier tissue following ulceration. The proteins are capable of inhibiting epithelial cell proliferation and/or protecting the barrier tissue from damage. The proteins also are capable of inhibiting scar tissue formation that 15 typically follows lesion formation in a mammal. In 'addition, the morphogens also can inhibit the inflammation normally associated with ulcerative diseases. The proteins may be used to treat ulcerative .i diseases of the gastrointestinal tract, including oral mucositis, peptic ulcers, ulcerative colitis, proctitis, and regional enteritis. The proteins also may be used to protect and/or treat GI tissue subject to damage in a xerostomatic individual. Finally, the morphogens may be administered as part of a chemical or radiotherapy to inhibit lesion formation in a patient undergoing cancer therapy and enhance the efficacy of the therapy thereby.

Provided below are detailed descriptions of suitable morphogens useful in the methods and compositions of this invention, as well as methods for their administration and application, and numerous, nonlimiting examples which demonstrate the suitability of the morphogens described herein as therapeutic agents for maintaining the integrity of the gastrointestinal tract luminal lining, and provide 38 assays with which to test candidate morphogens and morphogen-stimulating agents for their efficacy.

Specifically, the examples provide models for demonstrating the utility of morphogens in the treatment of oral mucositis, duodenal ulcers, peptic ulcers, and ulcerative colitis (Examples and demonstrate the ability of morphogens to inhibit epithelial cell proliferation (Example inhibit inflammation (Example 8) and inhibit scar tissue formation (Example The Examples also describe methods for identifying morphogen-expressing tissue and screening for candidate morphogen stimulating agents (Examples 1, 2 and S, 15 I. Useful Morphogens As defined herein a protein is morphogenic if it is capable of inducing the developmental cascade of cellular and molecular events that culminate in the formation of new, organ-specific tissue and comprises at least the conserved C-terminal six cysteine skeleton S"or its functional equivalent (see supra).

Specifically, the morphogens generally are capable of all of the following biological functions in a 25 morphogenically permissive environment: stimulating proliferation of progenitor cells; stimulating the differentiation of progenitor cells; stimulating the proliferation of differentiated cells; and supporting the growth and maintenance of differentiated cells.

Details of how the morphogens useful in the method of this invention first were identified, as well as a description on how to make, use and test them for morphogenic activity, are disclosed in international application US92/01968 (WO 92/15323). As disclosed therein, the morphogens may be purified from naturallysourced material or recombinantly produced from procaryotic or eucaryotic host cells, using the genetic 39 sequences disclosed therein. Alternatively, novel morphogenic sequences may be identified following the procedures disclosed therein.

Particularly useful proteins include those which comprise the naturally derived sequences disclosed in Table II. Other useful sequences include 60A, BMP6, BMP3, and biosynthetic constructs such as those disclosed in U.S. Pat. 5,011,691, the disclosure of which is incorporated herein by reference COP-1, COP-3, COP-4, COP-5, COP-7, and COP-16).

Accordingly, the morphogens useful in the methods and compositions of this invention also may be described by morphogenically active proteins having amino acid sequences sharing 70% or, preferably, homology (similarity) with any of the sequences described above, where "homology" is as defined herein above.

~The morphogens useful in the method of this invention also can be described by any of the 6 generic sequences described herein (Generic Sequences 1, 2, 3, 4, 5 and Generic sequences 1 and 2 also may 25 include, at their N-terminus, the sequence Cys Xaa Xaa Xaa Xaa (Seq. ID No. 1 Table II, set forth below, compares the amino acid sequences of the active regions of native proteins that have been identified as morphogens, including human OP-1 (hOP-1, Seq. ID Nos. 5 and 16-17), mouse OP-1 (mOP-1, Seq. ID Nos. 6 and 18-19), human and mouse OP-2 (Seq. ID Nos. 7, 8, and 20-23), CBMP2A (Seq. ID No. 9), t.

40 CBMP2B (Seq. ID No. 10), DPP (from Drosophila, Seq. ID No. 11), Vgl, (from Xenopus, Seq. ID No. 12), Vgr-1 (from mouse, Seq. ID No. 13), and GDF-1 (from mouse, Seq. ID No. 14.) The sequences are aligned essentially following the method of Needleman et al. (1970) J. Mol.

Biol., 48:443-453, calculated using the Align Program (DNAstar, Inc.) In the table, three dots indicates that the amino acid in that position is the same as the amino acid in hOP-1. Three dashes indicates that no amino acid is present in that position, and are included for purposes of illustrating homologies. For example, amino acid residue 60 of CBMP-2A and CBMP-2B is "missing". Of course, both these amino acid sequences in this region comprise Asn-Ser (residues 58, 15 59), with CBMP-2A then comprising Lys and Ile, whereas CBMP-2B comprises Ser and Ile.

TABLE II a o hOP-1 mOP-1 hOP-2 mOP-2 25 DPP Vgl Vgr-1 CBMP-2A CBMP-2B GDF-1 Cys Lys Lys His Glu Leu Tyr Val Arg Arg Arg Arg Arg Arg Lys Arg Arg Arg Arg Ala Ser Arg His Gly Pro Ser Arg Arg hOP-1 mOP-1 Ser Phe Arg Asp Leu Gly Trp Gin Asp 0 41 hOP -2 mOP-2

DPP

Vgi Vgr-1 CBHP-2A CBMP-2B GDF- 1 Ser As p Giu Asp Asp Gin Ser Lys Gin Ser Ser Giu Val Val Val Val Val Val Giu Gly Gin Gin Leu Gin Lys Pro Pro Leu Leu Asp Asn Asn His Asn Arg hOP-1 MOP-i hOP-2 mOP-2

DPP

Vgi Vgr- 1 CBHP-2A CBHP-2B GDF- 1 Trp Ile Val Vai Val Val Ile Val Val Vai Ala Pro Tyr Aia Se r Ser Asp Met His Gin Arg Phe Leu a.

a a hOP-i 25 MOP-i hOP-2 mOP-2

DPP

Vgi Vgr- 1 CBHP-2A CBMP-2B GDF-i1 Ala Tyr Tyr Asn Asn Phe Phe Asn Cys Giu Gly Giu His Lys Tyr Asp His Giu His Asp Gin Gin 42 hOP-1 MOP-i1 hOP-2 mOP -2

DPP

Vgl Vgr-1 CBMP-2A CBMP-2B GDF- 1 Phe Tyr Leu Pro Leu Val1 As n Asp Asp Ala Thr Ala Ala Ala Ser Tyr Met Asn Ala

B

B.

B

BBS.

B

B. B. B B

B

B

B

B.

hOP-i mOP-i 15 hOP-2 mOP-2

DPP

Vgl Vgr-1 CBMP-2A CBMP-2B GDF- 1 hOP-i mOP-i hOP -2 mOP -2

DPP

Vgi Vgr-1 CBMP-2A CBMP-2B GOF- 1 Thr Se r Leu Val Met Asn His His His Asn Asn Asn 55 His Phe Leu Leu Asn Ser Val Se r Ser Ala Ala Ile Met Met Asn Met Val Val Ala Asp Glu Ala Asp Asp Leu Ile Val Val Asn Lys Lys Glu Ala Cys Cys His Ile His His His Ser Phe Leu Leu Leu Gly Ser** Val Leu Leu Leu Leu Pro Ser Ser Gin Arg Glu Asp Asn Asp Gly Gly Th r Ser Ser Ala Thr Ala Val Lys Asp Tyr Lys Se r Ala Leu Val Ile Ile Ile Ala Ser Gly Ser Ser 0 43 a* hOP-i1 MOP-i hOP-2 mOP-2

DPP

Vgl Vgr-i1 CBMP-2A CBHP-2B GDF- 1 hOP-i 15 MOP-i1 hOP-2 mOP-2 Vgl Vgr- 1

DPP

CBMP-2A CBMP-2B GDF-i1 hOP-i MOP-i hOP-2 DOP -2

DPP

Vgl Vgr-i1 CBMP-2A CBHP-2B GDF- 1 Lys Pro Ala Ala Ala Leu Ala Ala Leu Ala Pro Val Val Val Val Val Leu Asn Ala Ile Ser Ser Thr Ser Thr Met Ser Pro Val. Asp Ser Val Ala Ser Ser Ser Pro Asp Asp Ser Ser Asn Ser .sn. Ser Asn Asn Gin Thr Asn Asn Asp Asn Glu Asn Glu Lys Glu Tyr Asp Lys Asn Asp Val Leu Tyr Met Phe Met Met Met Phe Val Ile Leu Val Val Val Val Val Thr Ala Gln Lys Lys Lys Lys Glu Glu Arg Phe Tyr Tyr Tyr Leu Leu Leu Lys Arg Arg Arg Ar g 44 hop-i MOP-1 hOP -2 mOP -2

DPP

Vgl Vgr- 1 CBHP-2A CBMP-2B GDF- 1 Tyr Arg Asn Met Val Val Arg His His Asn Asn Gin Lys Lys Gin Glu Thr Val Glu Ala Asp Gin Asp Glu Gin Giu Giu Glu Asp Asp a. 0@ a a a a a a.

hOP-i 15 MOP-i hOP-2 mOP-2

DPP

Vgi Vgr- 1 CBHP-2A CBMP-2B GDF- 1 Ala Cys Gly Cys Arg Arg Arg Arg Arg 100 a. a.

a. e a a a **Between residues 43 and 44 of GDF-l lies the amino acid sequence Gly-Gly-Pro-Pro.

As is apparent from the foregoing amino acid sequence comparisons, significant amino acid changes can be made within the generic sequences while retaining the morphogenic activity. For example, while the GDF-1 protein sequence depicted in Table II shares only about 50% amino acid identity with the hoPi sequence described therein, the GDF-1 sequence shares greater than 70% amino acid sequence homology (or "similarity") with the hOP1 sequence, where "homology" or "similarity" includes allowed conservative amino acid changes within the sequence as defined by Dayoff, et al., Atlas of Protein Sequence and Structure supp.3, pp.345-362, Dayoff, ed., Nat'l BioMed.

Res. Fd'n, Washington D.C. 1979.) The currently most preferred protein sequences useful as morphogens in this invention include those having greater than 60% identity, preferably greater than 65% identity, with the amino acid sequence defining the conserved six cysteine skeleton of hOP1 residues 43-139 of Seq. ID No. These most preferred sequences include both allelic and species 15 variants of the OP-1 and OP-2 proteins, including the Drosophila 60A protein. Accordingly, in still another preferred aspect, the invention includes morphogens comprising species of polypeptide chains having the generic amino acid sequence referred to herein as "OPX", which defines the seven cysteine skeleton and accommodates the identities between the various identified mouse and human OP1 and OP2 proteins. OPX is presented in Seq. ID No. 29. As described therein, each Xaa at a given position independently is selected 25 from the residues occurring at the corresponding position in the C-terminal sequence of mouse or human OP1 or OP2 (see Seq. ID Nos. 5-8 and/or Seq. ID Nos.

16-23).

II. Formulations and Methods for Administering Therapeutic Agents The morphogens or morphogen-stimulating agents may be provided to an individual by any suitable means, preferably by oral, rectal or other direct 46 administration or, alternatively, by systemic administration.

The suitability of systemic administration is demonstrated by the detection of endogenous morphogen in milk and human serum described, for example, in international application US92/07432 (W093/05751) and in Example 2, below. Where the morphogen is to be provided parenterally, such as by intravenous, subcutaneous, intramuscular, intraorbital, intraventricular, intracapsular, intraspinal, intracisternal, intraperitoneal or vaginal administration, the morphogen preferably comprises part of an aqueous solution. The solution is 15 physiologically acceptable so that in addition to delivery of the desired morphogen to the patient, the solution does not otherwise adversely affect the patient's electrolyte and volume balance. The aqueous medium for the morphogen thus may comprise normal 20 physiologic saline (0.85% NaC1, 0.15M), pH 7-7.4. The aqueous solution containing the morphogen can be made, for example, by dissolving the protein in 50% ethanol containing acetonitrile in 0.1% trifluoroacetic acid (TFA) or 0.1% HC1, or equivalent solvents. One volume of the resultant solution then is added, for example, *to ten volumes of phosphate buffered saline (PBS), which further may include 0.1-0.2% human serum albumin (HSA). The resultant solution preferably is vortexed extensively. If desired, a given morphogen may be made more soluble by association with a suitable molecule.

For example, the pro form of the morphogenic protein comprises a species that is soluble in physiological solutions. In fact, the endogenous protein is thought to be transported secreted and circulated) in this form. This soluble form of the protein may be 47 obtained from the culture medium of morphogen-secreting cells. Alternatively, a soluble species may be formulated by complexing the mature dimer, (or an active fragment thereof) with part or all of a pro domain as described herein below (see Section II.A.).

Other components, including various serum proteins, also may be useful.

Useful solutions for parenteral administration may be prepared by any of the methods well known in the pharmaceutical art, described, for example, in Remington's Pharmaceutical Sciences (Gennaro, ed.), Mack Pub., 1990. Formulations may include, for example, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, hydrogenated naphthalenes, and the like. Other potentially useful parenteral delivery systems for these morphogens include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and 20 liposomes. Formulations for parenteral administration may also include cutric acid for vaginal administration.

Preferably, the morphogens described herein are administered directly topically, for example, by oral or rectal administration, or by directly applying the therapeutic formulation onto the desired tissue.

Oral administration of proteins as therapeutics generally is not practiced as most proteins are readily degraded by digestive enzymes and acids in the mammalian digestive system before they can be absorbed into the bloodstream. However, the morphogens described herein typically are acid stable and protease-resistant (see, for example, U.S. Pat.No.

4,968,590.) In addition, at least one morphogen, OP-1, 48 has been identified in mammary gland extract, colostrum and 57-day milk. Moreover, the OP-1 purified from mammary gland extract is morphogenically active.

Specifically, this protein induces endochondral bone formation in mammals when implanted subcutaneously in association with a suitable matrix material, using a standard in vivo bone assay, such as is disclosed in U.S. Pat.No. 4,968,590. Moreover, as described above, the morphogen also is detected in the bloodstream.

These findings indicate that oral administration is a viable means for administering morphogens to an individual. In addition, while the mature forms of certain morphogens described herein typically are sparingly soluble, the morphogen form found in milk 15 (and mammary gland extract and colostrum) is readily soluble, probably by association of the mature, morphogenically active form with part or all of the pro domain of the intact sequence and/or by association with one or more milk components. Accordingly, the 20 compounds provided herein also may be associated with molecules capable of enhancing their solubility in vitro or in vivo.

For oral mucositis treatments the morphogens or morphogen-stimulating agents (herein below referred to collectively as "therapeutic agent") may be formulated S. into an oral rinse similar to a mouthwash, where the liquid is swished around in the mouth so that the therapeutic agent is brought in contact with the oral mucosa to maximize treatment of lesions.

Alternatively, the therapeutic agent may be formulated as part of a slow dissolving troche or lozenge, or dispersed in a gum base suitable for a chewing gum, such that the agent is released with mastication.

49 Longer contact with the mucosal surface of the mouth cavity or elsewhere in the G.I. tract can be attained by direct topical administration, using a suitable vehicle which is capable of coating mucosa.

Typical examples are pectin-containing formulations or sucralfate suspensions, such as are found in Kaopectate and Milk of Magnesia. Formulations for direct administration also may include glycerol and other compositions of high viscosity. Tissue adhesives capable of adhering to the mucosal tissue surface and maintaining the therapeutic agent at the tissue locus also may be used. Useful adhesive compositions include hydroxypropyl-cellulose-containing solutions, such as is found in OrabaseR (Colgate-Hoyt Laboratories, 15 Norwood, MA), or fibrinogen/thrombin-containing solutions. Another useful adhesive is the bio-adhesive described in US Patent No. 5,197,973. Preferably these formulations are painted onto the tissue surface or formulated as an aerosol and sprayed onto the tissue i* 20 surface. As for parenteral administration, the therapeutic agent may be associated with a molecule that enhances solubility. For example, addition of 0.2% casein increases solubility of the mature active form of OP-1 by 80%. Another useful molecule is a morphogen pro domain.

For all treatments of the gastrointestinal tract, the therapeutic agent also may be formulated into a solid or liquid to be consumed or as an inhalant. For treatments of the lower bowel, formulations for rectal administration may be preferable, and may include suppositories, creams, gels, lotions and the like.

In all applications, biocompatible, preferably bioresorbable, polymers, including, for example, S- 50 hyaluronic acid, collagen, polybutyrate, tricalcium phosphate, glycolide, lactide and lactide/glycolide copolymers, also may be useful excipients to control the release of the morphogen in vivo. Tablets or capsules may be prepared by employing additives such as pharmaceutically acceptable carriers lactose, corn starch, light silicic anhydride, microcrystalline cellulose, sucrose), binders alpha-form starch, methylcellulose, carboxymethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone), disintegrating agents carboxymethylcellulose calcium, starch, low substituted hydroxypropylcellulose), surfactants Tween Kao-Atlas), Pluronic F68 (Asahi Denka, Japan); 15 polyoxyethylene-polyoxypropylene copolymer)], antioxidants L-cysteine, sodium sulfite, sodium *..ascorbate), lubricants magnesium stearate, talc), and the like.

20 Formulations for inhalation administration may acontain as excipients, for example, lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or oily solutions for administration in the form of nasal drops, or as a gel to be applied intranasally. Formulations for rectal administration also may include methoxy salicylate. The formulations for rectal administration also can be a spreadable cream, gel, suppository, foam, lotion or ointment having a pharmaceutically acceptable nontoxic vehicle or carrier. Biocompatible, preferably bioresorbable, polymers, including, for example, hyaluronic acid, collagen, polybutyrate, tricalcium phosphate, lactide and lactide/glycolide copolymers, also may be useful excipients to control the release of the morphogen in vivo.

0 -51 The compounds provided herein also may be associated with molecules capable of targeting the morphogen or morphogen-stimulating agent to the gastrointestinal barrier tissue. For example, an antibody, antibody fragment, or other binding protein that interacts specifically with a surface molecule on basal epithelial cells, may be used. Useful targeting molecules may be designed, for example, using the single chain binding site technology disclosed, for example, in U.S. Pat. No. 5,091,513.

As described above, the morphogens provided herein share significant sequence homology in the C-terminal 1 active domains. By contrast, the sequences typically diverge significantly in the sequences which define the pro domain. Accordingly, the pro domain is thought to be morphogen-specific. As described above, it is also known that the various morphogens identified to date are differentially expressed in different tissues.

20 Accordingly, without being limited to any given theory, it is likely that, under natural conditions in the body, selected morphogens typically act on a given tissue. Accordingly, part or all of the pro domains which have been identified associated with the active form of the morphogen in solution, may serve as targeting molecules for the morphogens described herein. For example, the pro domains may interact specifically with one or more molecules at the target tissue to direct the morphogen associated with the pro domain to that tissue. Accordingly, another useful targeting molecule for targeting a morphogen to gastrointestinal barrier tissues may include part or all of a morphogen pro domain, particularly part or all of a pro domain normally associated with an endogenous morphogen known to act on GI tract tissue. As 52 described above, morphogen species comprising the pro domain may be obtained from the culture medium of morphogen-secreting mammalian cells. Alternatively, a tissue-targeting species may be formulated by complexing the mature dimer (or an active fragment thereof) with part or all of a pro domain. Example 1 describes a protocol for identifying morphogen-expressing tissue and/or morphogen target tissue.

Finally, the morphogens or morphogen-stimulating agents provided herein may be administered alone or in combination with other molecules known to be beneficial S" in treating gastrointestinal tract ulcers, particularly 15 symptom-alleviating cofactors. Useful pharmaceutical cofactors include analgesics and anesthetics such as xylocaine, benzocaine and the like; antiseptics such as chlorohexidine; anti-viral and anti-fungal agents; and antibiotics, including aminoglycosides, macrolides, 20 penicillins, and cephalosporins. Other potentially useful cofactors include antisecretory agents such as H2-receptor antagonists cimetidine, ranitidine, famotidine, roxatidine acetate), muscarine receptor antagonists Pirenzepine), and antacids such as aluminum hydroxide gel, magnesium hydroxide and sodium bicarbonate. Such agents may be administered either S: separately or as components of the therapeutic composition containing morphogens or morphogenstimulating agents.

The compositions can be formulated for parenteral or direct administration to humans or other mammals in therapeutically effective amounts, amounts which provide appropriate concentrations for a time sufficient to protect the patient's gastrointestinal 0 -53 luminal lining from lesion formation, including amounts which limit the proliferation of epithelial cells, particularly the basal epithelial cells of the G.I.

tract, amounts which limit the inflammation associated with the ulcerative diseases and disorders described above, and amounts sufficient to stimulate lesion repair and tissue regeneration.

As will be appreciated by those skilled in the art, the concentration of the compounds described in a therapeutic composition will vary depending upon a number of factors, including the dosage of the drug to be administered, the chemical characteristics hydrophobicity) of the compounds employed, and the route of administration. The preferred dosage of drug to be administered also is likely to depend on such variables as the type and extent of progression of the ulcerative disease, the overall health status of the particular patient, the relative biological efficacy of 20 the compound selected, the formulation of the compound excipients, and its route of administration. In general terms, the compounds of this invention may be provided in an aqueous physiological buffer solution containing about 0.001 to 10% w/v compound for parenteral administration. Typical dose ranges are from about ng/kg to about 1 g/kg of body weight per day; a S. preferred dose range is from about 0.1 pg/kg to 100 mg/kg of body weight per day. Optimally, the morphogen dosage given is between 0.1-100 pg of protein per kilogram weight of the patient. Administration may be a single dose per day, or may include multiple doses, such as multiple rinsings with a mouthwash, a 1 minute rinse three or four times daily. No obvious induced pathological lesions are induced when mature morphogen OP-i, 20 pg) is administered S-54daily to normal growing rats for 21 consecutive days.

Moreover, 10 pg systemic injections of morphogen OP-1) injected daily for 10 days into normal newborn mice does not produce any gross abnormalities.

In administering morphogens systemically in the methods of the present invention, preferably a large volume loading dose is used at the start of the treatment. The treatment then is continued with a maintenance dose. Further administration then can be determined by monitoring at intervals the levels of the morphogen in the blood using, for example, a morphogenspecific antibody and standard immunoassay procedures.

9* 15 Where injury to the mucosa is induced deliberately or incidentally, as part of, for example, a chemical or radiation therapy, the morphogen preferably is provided just prior to, or concomitant with induction of the treatment. Preferably, the morphogen is administered 20 prophylactically in a clinical setting. Optimally, the morphogen dosage given is between 0.1-100 pg of protein per kilogram weight of the patient. Similarly, the morphogen may be administered prophylactically to individuals at risk for ulcer formation, including xerostomatic or immune-compromised individuals, regardless of etiology.

An effective amount of an agent capable of stimulating endogenous morphogen levels also may be administered by any of the routes described above. For example, an agent capable of stimulating morphogen production in and/or secretion to G.I. tract tissue cells may be provided to a mammal. A method for identifying and testing agents capable of modulating the levels of endogenous morphogens in a given tissue 55 is described generally herein in Example 10, and in detail in international application US 92/07358 (W093/04692). In addition, Example 1 describes a protocol for determining morphogen-expressing tissue.

Briefly, candidate compounds can be identified and tested by incubating the compound in vitro with a test tissue or cells thereof, for a time sufficient to allow the compound to affect the production, the expression and/or secretion, of a morphogen produced by the cells of that tissue. Here, suitable tissue or cultured cells of a tissue preferably would include cells of the G.I. tract barrier. For example, suitable tissue for testing may include cultured cells isolated from the basal epithelium and mucosa, and the like.

A currently preferred detection means for evaluating the level of the morphogen in culture upon exposure to the candidate compound comprises an immunoassay utilizing an antibody or other suitable 20 binding protein capable of reacting specifically with a morphogen and being detected as part of a complex with the morphogen. Immunoassays may be performed using standard techniques known in the art and antibodies raised against a morphogen and specific for that morphogen. As described herein, morphogens may be isolated from natural-sourced material or they may be recombinantly produced. Agents capable of stimulating endogenous morphogens then may formulated into pharmaceutical preparations and administered as described herein.

II.A. Soluble Morphogen Complexes A currently preferred form of the morphogen useful in therapeutic formulations, having improved solubility S- 56 in aqueous solutions and consisting essentially of amino acids, is a dimeric morphogenic protein comprising at least the 100 amino acid peptide sequence having the pattern of seven or more cysteine residues characteristic of the morphogen family complexed with a peptide comprising part or all of a pro region of a member of the morphogen family, or an allelic, species or other sequence variant thereof. Preferably, the dimeric morphogenic protein is complexed with two peptides. Also, the dimeric morphogenic protein preferably is noncovalently complexed with the pro region peptide or peptides. The pro region peptides also preferably comprise at least the N-terminal eighteen amino acids that define a given morphogen 15 pro region. In a most preferred embodiment, peptides defining substantially the full length pro region are *'.*used.

Other soluble forms of morphogens include dimers of 20 the uncleaved pro forms of these proteins, as well as "hemi-dimers" wherein one subunit of the dimer is an uncleaved pro form of the protein, and the other subunit comprises the mature form of the protein, including truncated forms thereof, preferably noncovalently associated with a cleaved pro domain peptide.

As described above, useful pro domains include the full length pro regions, as well as various truncated forms hereof, particularly truncated forms cleaved at proteolytic Arg-Xaa-Xaa-Arg cleavage sites. For example, in OP-1, possible pro sequences include sequences defined by residues 30-292 (full length form); 48-292; and 158-292. Soluble OP-1 complex stability is enhanced when the pro region comprises the S- 57 full length form rather than a truncated form, such as the 48-292 truncated form, in that residues 30-47 show sequence homology to the N-terminal portions of other morphogens, and are believed to have particular utility in enhancing complex stability for all morphogens.

Accordingly, currently preferred pro sequences are those encoding the full length form of the pro region for a given morphogen. Other pro sequences contemplated to have utility include biosynthetic pro sequences, particularly those that incorporate a sequence derived from the N-terminal portion of one or more morphogen pro sequences.

As will be appreciated by those having ordinary 15 skill in the art, useful sequences encoding the pro region may be obtained from genetic sequences encoding known morphogens. Alternatively, chimeric pro regions can be constructed from the sequences of one or more known morphogens. Still another option is to create a 20 synthetic sequence variant of one or more known pro region sequences.

In another preferred aspect, useful pro region peptides include polypeptide chains comprising an amino acid sequence encoded by a nucleic acid that hybridizes under stringent conditions with a DNA or RNA sequence Sencoding at least the N-terminal eighteen amino acids of the pro region sequence for OP1 or OP2, e.g., nucleotides 136-192 and 152-211 of Seq. ID No. 16 and 20, respectively.

II.A1. Isolation of Soluble morphoqen complex from conditioned media or body fluid Morphogens are expressed from mammalian cells as soluble complexes. Typically, however the complex is 58 disassociated during purification, generally by exposure to denaturants often added to the purification solutions, such as detergents, alcohols, organic solvents, chaotropic agents and compounds added to reduce the pH of the solution. Provided below is a currently preferred protocol for purifying the soluble proteins from conditioned media (or, optionally, a body fluid such as serum, cerebro-spinal or peritoneal fluid), under non-denaturing conditions. The method is rapid, reproducible and yields isolated soluble morphogen complexes in substantially pure form.

Soluble morphogen complexes can be isolated from conditioned media using a simple, three step 15 chromatographic protocol performed in the absence of denaturants. The protocol involves running the media (or body fluid) over an affinity column, followed by ion exchange and gel filtration chromatographies. The affinity column described below is a Zn-IMAC column.

20 The present protocol has general applicability to the purification of a variety of morphogens, all of which are anticipated to be isolatable using only minor modifications of the protocol described below. An alternative protocol also envisioned to have utility an immunoaffinity column, created using standard procedures and, for example, using antibody specific for a given morphogen pro domain (complexed, for example, to a protein A-conjugated Sepharose column.) Protocols for developing immunoaffinity columns are well described in the art, (see, for example, Guide to Protein Purification, M. Deutscher, ed., Academic Press, San Diego, 1990, particularly sections VII and

XI.)

59 In this experiment OP-1 was expressed in mammalian CHO (chinese hamster ovary) cells as described in the art (see, for example, international application US90/05903 (W091/05802).) The CHO cell conditioned media containing 0.5% FBS was initially purified using Immobilized Metal-Ion Affinity Chromatography (IMAC).

The soluble OP-1 complex from conditioned media binds very selectively to the Zn-IMAC resin and a high concentration of imidazole (50 mM imidazole, pH 8.0) is required for the effective elution of the bound complex. The Zn-IMAC step separates the soluble OP-1 from the bulk of the contaminating serum proteins that elute in the flow through and 35 mM imidazole wash fractions. The Zn-IMAC purified soluble OP-1 is next S. 15 applied to an S-Sepharose cation-exchange column equilibrated in 20 mM NaPO 4 (pH 7.0) with 50 mM NaCl.

This S-Sepharose step serves to further purify and .concentrate the soluble OP-1 complex in preparation for the following gel filtration step. The protein was 20 applied to a Sephacryl S-200HR column equilibrated in t TBS. Using substantially the same protocol, soluble morphogens also may be isolated from one or more body fluids, including serum, cerebro-spinal fluid or peritoneal fluid.

IMAC was performed using Chelating-Sepharose (Pharmacia) that had been charged with three column volumes of 0.2 M ZnSO 4 The conditioned media was titrated to pH 7.0 and applied directly to the ZN-IMAC resin equilibrated in 20 mM HEPES (pH 7.0) with 500 mM NaCl. The Zn-IMAC resin was loaded with 80 mL of starting conditioned media per mL of resin. After loading, the column was washed with equilibration buffer and most of the contaminating proteins were eluted with 35 mM imidazole (pH 7.0) in equilibration 60 buffer. The soluble OP-1 complex then is eluted with mM imidazole (pH 8.0) in 20 mM HEPES and 500 mM NaCl.

The 50 mM imidazole eluate containing the soluble OP-1 complex was diluted with nine volumes of 20 mM NaPO 4 (pH 7.0) and applied to an S-Sepharose (Pharmacia) column equilibrated in 20 mM NaPO 4 (pH with 50 mM NaCl. The S-Sepharose resin was loaded with an equivalent of 800 mL of starting conditioned media per mL of resin. After loading the S-Sepharose column was washed with equilibration buffer and eluted with 100 mM NaCl followed by 300 mM and 500 mM NaCl in 20 mM NaPO 4 (pH The 300 mM NaCl pool was further 15 purified using gel filtration chromatography. Fifty mls of the 300 mm NaCl eluate was applied to a 5.0 X cm Sephacryl S-200HR (Pharmacia) equilibrated in Tris buffered saline (TBS), 50 mM Tris, 150 mM NaCl (pH The column was eluted at a flow rate of 20 mL/minute collecting 10 mL fractions. The apparent o* molecular of the soluble OP-1 was determined by comparison to protein molecular weight standards (alcohol dehydrogenase (ADH, 150 kDa), bovine serum albumin (BSA, 68 kDa), carbonic anhydrase (CA, 30 kDa) and cytochrome C (cyt C, 12.5 kDa). The purity of the S-200 column fractions was determined by separation on standard 15% polyacrylamide SDS gels stained with coomassie blue. The identity of the mature OP-1 and the pro-domain was determined by N-terminal sequence analysis after separation of the mature OP-1 from the pro-domain using standard reverse phase C18 HPLC.

The soluble OP-1 complex elutes with an apparent molecular weight of 110 kDa. This agrees well with the predicted composition of the soluble OP-1 complex with 61one mature OP-1 dimer (35-36 kDa) associated with two pro-domains (39 kDa each). Purity of the final complex can be verified by running the appropriate fraction in a reduced 15% polyacrylamide gel.

The complex components can be verified by running the complex-containing fraction from the S-200 or S- 200HR columns over a reverse phase C18 HPLC column and eluting in an acetonitrile gradient (in 0.1% TFA), using standard procedures. The complex is dissociated by this step, and the pro domain and mature species elute as separate species. These separate species then can be subjected to N-terminal sequencing using standard procedures (see, for example, Guide to Protein Purification, M. Deutscher, ed., Academic Press, San Diego, 1990, particularly pp. 602-613), and the identity of the isolated 36kD, 39kDa proteins confirmed as mature morphogen and isolated, cleaved pro domain, respectively. N-terminal sequencing of the isolated pro domain from mammalian cell produced OP-1 revealed 2 forms of the pro region, the intact form (beginning at residue 30 of Seq. ID No. 16) and a truncated form, (beginning at residue 48 of Seq. ID No.

16.) N-terminal sequencing of the polypeptide subunit 25 of the isolated mature species reveals a range of N-termini for the mature sequence, beginning at residues 293, 300, 313, 315, 316, and 318, of Seq. ID No. 16, all of which are active as demonstrated by the standard bone induction assay.

II.A2. In Vitro Soluble Morphogen Complex Formation As an alternative to purifying soluble complexes from culture media or a body fluid, soluble complexes may be formulated from purified pro domains and mature *.i 62 dimeric species. Successful complex formation apparently requires association of the components under denaturing conditions sufficient to relax the folded structure of these molecules, without affecting disulfide bonds. Preferably, the denaturing conditions mimic the environment of an intracellular vesicle sufficiently such that the cleaved pro domain has an opportunity to associate with the mature dimeric species under relaxed folding conditions. The concentration of denaturant in the solution then is decreased in a controlled, preferably step-wise manner, so as to allow proper refolding of the dimer and pro regions while maintaining the association of the pro domain with the dimer. Useful denaturants include 4-6M urea or guanidine hydrochloride (GuHC1), in buffered solutions of pH 4-10, preferably pH 6-8. The soluble complex then is formed by controlled dialysis or dilution into a solution having a final denaturant concentration of less than 0.1-2M urea or GuHC1, preferably 1-2 M urea of GuHC1, which then preferably can be diluted into a physiological buffer. Protein purification/renaturing procedures and considerations are well described in the art, and details for developing a suitable renaturing protocol readily can be determined by one having ordinary skill in the art.

One useful text one the subject is Guide to Protein Purification, M. Deutscher, ed., Academic Press, San Diego, 1990, particularly section V. Complex formation also may be aided by addition of one or more chaperone 30 proteins.

II.A3. Stability of Soluble Morphogen Complexes The stability of the highly purified soluble 35 morphogen complex in a physiological buffer, e.g., S-63tris-buffered saline (TBS) and phosphate-buffered saline (PBS), can be enhanced by any of a number of means. Currently preferred is by means of a pro region that comprises at least the first 18 amino acids of the pro sequence residues 30-47 of Seq. ID NO. 16 for OP-1), and preferably is the full length pro region. Residues 30-47 show sequence homology to the N-terminal portion of other morphogens and are believed to have particular utility in enhancing complex stability for all morphogens. Other useful means for enhancing the stability of soluble morphogen complexes include three classes of additives. These additives include basic amino acids L-arginine, lysine and betaine); nonionic detergents Tween 80 or NonIdet P-120); and carrier proteins serum albumin and casein). Useful concentrations of these additives include 1-100 mM, preferably 10-70 mM, including 50 mM, basic amino acid;, 0.01-1.0%, preferably 0.05-0.2%, including 0.1% nonionic detergent;, and 0.01-1.0%, preferably 0.05-0.2%, including 0.1% carrier protein.

III. Examples Example 1. Identification of Morphogen-Expressing S" Tissue Determining the tissue distribution of morphogens 30 may be used to identify different morphogens expressed in a given tissue, as well as to identify new, related morphogens. Tissue distribution also may be used to identify useful morphogen-producing tissue for use in screening and identifying candidate morphogen- 35 stimulating agents. The morphogens (or their mRNA 64 transcripts) readily are identified in different tissues using standard methodologies and minor modifications thereof in tissues where expression may be low. For example, protein distribution may be determined using standard Western blot analysis or immunofluorescent techniques, and antibodies specific to the morphogen or morphogens of interest. Similarly, the distribution of morphogen transcripts may be determined using standard Northern hybridization protocols and transcript-specific oligonucleotide probes.

Any probe capable of hybridizing specifically to a transcript, and distinguishing the transcript of interest from other, related transcripts may be used.

Because the morphogens described herein share such high sequence homology in their active, C-terminal domains, the tissue distribution of a specific morphogen transcript may best be determined using a probe specific for the pro region of the immature protein and/or the N-terminal region of the mature protein.

Another useful sequence is the 3' non-coding region '*flanking and immediately following the stop codon.

These portions of the sequence vary substantially among 25 the morphogens of this invention, and accordingly, are specific for each protein. For example, a particularly useful Vgr-l-specific probe sequence is the PvuII-SacI fragment, a 265 bp fragment encoding both a portion of the untranslated pro region and the N-terminus of the 30 mature sequence (see Lyons et al. (1989) PNAS 86:4554- 4558 for a description of the cDNA sequence).

Similarly, particularly useful mOP-1-specific probe sequences are the BstXl-BglI fragment, a 0.68 Kb sequence that covers approximately two-thirds of the 35 mOP-1 pro region; a StuI-StuI fragment, a 0.2 Kb *oo 65 sequence immediately upstream of the 7-cysteine domain; and the Earl-Pstl fragment, an 0.3 Kb fragment containing a portion of the 3'untranslated sequence (See Seq. ID No. 18, where the pro region is defined essentially by residues 30-291.) Similar approaches may be used, for example, with hOP-1 (Seq. ID No. 16) or human or mouse OP-2 (Seq. ID Nos. 20 and 22.) Using these morphogen-specific probes, which may be synthetically engineered or obtained from cloned sequences, morphogen transcripts can be identified in mammalian tissue, using standard methodologies well known to those having ordinary skill in the art.

Briefly, total RNA is prepared from various adult murine tissues liver, kidney, testis, heart, brain, thymus and stomach) by a standard methodology such as by the method of Chomczyaski et al. ((1987) Anal. Biochem 162:156-159) and described below. Poly RNA is prepared by using oligo (dT)-cellulose chromatography Type 7, from Pharmacia LKB Biotechnology, Inc.). Poly RNA (generally 15 pg) from each tissue is fractionated on a 1% agarose/formaldehyde gel and transferred onto a Nytran S" membrane (Schleicher Schuell). Following the transfer, the membrane is baked at 80 0 C and the RNA is cross-linked under UV light (generally 30 seconds at 1 mW/cm Prior to hybridization, the appropriate probe is denatured by heating. The hybridization is carried out in a lucite cylinder rotating in a roller bottle 30 apparatus at approximately 1 rev/min for approximately hours at 37°C using a hybridization mix of formamide, 5 x Denhardts, 5 x SSPE, and 0.1% SDS.

Following hybridization, the non-specific counts are washed off the filters in 0.1 x SSPE, 0.1% SDS at 66 Examples demonstrating the tissue distribution of various morphogens, including Vgr-l, OP-1, BMP2, BMP3, BMP4, BMP5, GDF-1, and OP-2 in developing and adult tissue are disclosed in US92/01968 (W092/15323), and in Ozkaynak, et al., (1991) Biochem. Biophys. Res. Commn.

179:116-123, and Ozkaynak, et al. (1992) J. Biol. Chem.

267:25220-25227, the disclosures of which are incorporated herein by reference. Using the general probing methodology described herein, Northern blot hybridizations using probes specific for these morphogens to probe brain, spleen, lung, heart, liver and kidney tissue indicate that kidney-related tissue appears to be the primary expression source for OP-1, with brain, heart and lung tissues being secondary sources. OP-1 mRNA also was identified in salivary glands, specifically rat parotid glands, using this probingmethodology. Lung tissue appears to be the primary tissue expression source for Vgr-1, BMP5, BMP4 and BMP3. Lower levels of Vgr-1 also are seen in kidney and heart tissue, while the liver appears to be a secondary expression source for BMP5, and the spleen appears to be a secondary expression source for BMP4.

GDF-1 appears to be expressed primarily in brain tissue. To date, OP-2 appears to be expressed primarily in early embryonic tissue. Specifically, SNorthern blots of murine embryos and 6-day post-natal S" animals shows abundant OP2 expression in 8-day embryos.

Expression is reduced significantly in 17-day embryos and is not detected in post-natal animals.

Immunolocalization studies using OP-1 specific antibodies also localize the morphogen to both the inner circular and outer longitudinal coats of smooth muscles in the tubular organs of the digestive system 35 during early embryo development (gestation: weeks o 67 13), suggesting the endogenous morphogen also plays a role in tissue morphogenesis of the digestive tract.

Moreover, Northern blot analysis on rat tissue (probed with an mOP-1-specific labelled nucleotide fragment, as described above) identifies OP-1 mRNA in the gastrointestinal tract tissues of growing rats, including the stomach, duodenal and intestine tissues.

These data demonstrate that morphogens are both expressed in, and act on, tissues of the GI tract.

Example 2. Active Morphogens in Body Fluids OP-1 expression has been identified in saliva (specifically, the rat parotid gland, see Example 1), human blood serum, and various milk forms, including mammary gland extract, colostrum, and 57-day bovine milk. Moreover, and as described in international application US92/07432 (W093/05751), the body fluidextracted protein is morphogenically active. The discovery that the morphogen naturally is present in milk and saliva, together with the known observation that mature, active OP-1 is acid-stable and proteaseresistant, indicate that oral administration is a 25 useful route for therapeutic administration of morphogen to a mammal. Oral administration typically is the preferred mode of delivery for extended or prophylactic therapies. In addition, the identification of morphogen in all milk forms, 30 including colostrum, suggests that the protein may play a significant role in tissue development, including skeletal development, of juveniles.

*9 9 99 68 2.1 Morphogen Detection in Milk OP-1 was partially purified from rat mammary gland extract and bovine colostrum and 57 day milk by passing these fluids over a series of chromatography columns: cation-exchange, affinity and reverse phase). At each step the eluant was collected in fractions and these were tested for the presence of OP-1 by standard immunoblot. Immunoreactive fractions then were combined and purified further. The final, partially purified product then was examined for the presence of OP-1 by Western blot analysis using OP-1-specific antisera, and tested for in vivo and in vitro activity.

OP-1 purified from the different milk sources were characterized by Western blotting using antibodies raised against OP-1 and BMP2. Antibodies were prepared using standard immunology protocols well known in the art, and as described generally in Example 15, below, using full-length E. coli-produced OP-1 and BMP2 as the immunogens. In all cases, the purified OP-1 reacted only with the anti-OP-1 antibody, and not with anti-BMP2 antibody.

25 The morphogenic activity of OP-1 purified from mammary gland extract was evaluated in vivo essentially following the rat model assay described in U.S. Pat.

No. 4,968,590, hereby incorporated by reference.

Briefly, a sample was prepared from each OP-1 30 immunoreactive fraction of the mammary gland extract-derived OP-1 final product by lyophilizing a portion of the fraction and resuspending the protein in 2201p of 50% acetonitrile/0.1% TFA. After vortexing, 25 mg of collagen matrix was added. The 35 samples were lyophilized overnight, and implanted in 44 e 5.

S- 69 Long Evans rats (Charles River Laboratories, Wilmington, MA, 28-35 days old). Each fraction was implanted in duplicate. For details of the collagen matrix implantation procedure, see, for example, U.S.

Pat. No. 4,968,590, hereby incorporated by reference.

After 12 days, the implants were removed and evaluated for new bone formation by histological observation as described in U.S. Patent No. 4,968,590. In all cases, the immunoreactive fractions were osteogenically active.

2.2 Morphogen Detection in Serum Morphogen may be detected in serum using morphogen- 15 specific antibodies. The assay may be performed using any standard immunoassay, such as Western blot (immunoblot) and the like. Preferably, the assay is performed using an affinity column to which the morphogen-specific antibody is bound and through which 20 the sample serum then is poured, to selectively extract the morphogen of interest. The morphogen then is eluted. A suitable elution buffer may be determined empirically by determining appropriate binding and elution conditions first with a control 25 purified, recombinantly-produced morphogen.) Fractions then are tested for the presence of the morphogen by standard immunoblot, and the results confirmed by N-terminal sequencing. Preferably, the affinity column is prepared using monoclonal antibodies. Morphogen concentrations in serum or other fluid samples then may be determined using standard protein quantification techniques, including by spectrophotometric absorbance or by quantitation of conjugated antibody.

70 Presented below is a sample protocol for identifying OP-1 in serum. Following.this general methodology other morphogens may be detected in body fluids, including serum. The identification of morphogen in serum further indicates that systemic administration is a suitable means for providing therapeutic concentrations of a morphogen to an individual, and that morphogens likely behave systemically as endocrine-like factors. Finally, using this protocol, fluctuations in endogenous morphogen levels can be detected, and these altered levels may be used as an indicator of tissue dysfunction.

Alternatively, fluctuations in morphogen levels may be assessed by monitoring morphogen transcription levels, 15 either by standard Northern blot analysis as described in Example 1, or by in situ hybridization, using a labelled probe capable of hybridizing specifically to morphogen mRNA, and standard RNA hybridization protocols well described in the art and described generally in Example 1.

SOP- was detected in human serum using the following assay. A monoclonal antibody raised against mammalian, recombinantly produced OP-1 using standard immunology techniques well described in the art and described generally in Example 15, was immobilized by passing the antibody over an agarose-activated gel Affi-GelTM, from Bio-Rad Laboratories, Richmond, CA, prepared following manufacturer's instructions) and used to purify OP-1 from serum. Human serum then was passed over the column and eluted with 3M K-thiocyanate. K-thiocyanante fractions then were dialyzed in 6M urea, 20mM PO4, pH 7.0, applied to a C8 HPLC column, and eluted with a 20 minute, 25-50% acetonitrile/0.1% TFA gradient. Since mature, S-71recombinantly produced OP-1 homodimers elute between 20-22 minutes, these fractions then were collected and tested for the presence of OP-1 by standard immunoblot using an OP-1 specific antibody as for Example 2.A.

Administered or endogenous morphogen levels may be monitored in the therapies described herein by comparing the quantity of morphogen present in a body fluid sample with a predetermined reference value, for example, to evaluate the efficiency of a therapeutic protocol, and the like. In addition, fluctuations in the level of endogenous morphogen antibodies may be detected by this method, most likely in serum, using an antibody or other binding protein capable of 15 interacting specifically with the endogenous morphogen antibody. Detected fluctuations in the levels of the morphogen or endogenous antibody may be used, for example, as indicators of a change in tissue status.

:For example, as damaged tissue is regenerated and the tissue or organ's function returns to "normal" and, in the absence of additional tissue damage, lower doses of morphogen may be required, and a higher level of circulating morphogen antibody may be measured.

25 Example 3. Morphoqen Treatment of Oral Mucositis Oral mucositis involves ulcerations of the mouth as a consequence of, radiation therapy or chemotherapy. The course of ulcerative mucositis may be divided into a destructive phase and a healing phase. Since the cells of the basal layer of the oral epithelium divide at a rapid rate, they are susceptible to the antimitogenic and toxic effects of chemotherapy.

As a result, atrophic changes occur which then are followed by ulceration. This constitutes the 72 destructive phase. Following ulcer formation, the lesions slowly resolve during the healing phase.

The example below demonstrates morphogen efficacy in protecting the oral mucosa from oral mucositis in a hamster model, including both inhibiting ulceration and enhancing regeneration of ulcerated tissue. Details of the protocol can be found in Sonis, et al., (1990) Oral Surq. Oral Med. Oral Pathol 69: 437-443, the disclosure of which is incorporated herein by reference. Briefly, golden syrian hamsters (6-8 wks old, Charles River Laboratories, Wilmington, MA) were divided into 3 test groups: Group 1, a placebo saline) control, and a morphogen low dose group (100 ng) and a morphogen 15 high dose group (1 pg), Groups 2 and 3, respectively.

Morphogen dosages were provided in 30% ethanol. Each group contained 12 animals.

Beginning on day 0 and continuing through day Groups 2 and 3 received twice daily morphogen applications. On day 3, all groups began the mucositis-induction procedure. 5-fluorouracil was injected intraperitoneally on days 3 (60 mg/kg) and mg/kg). On day 7, the right buccal pouch mucosa 25 was superficially irritated with a calibrated 18 gauge needle. In untreated animals, severe ulcerative mucositis was induced in at least 80% of the animals by day 4..

For each administration of the vehicle control (placebo) or morphogen, administration was performed by first gently drying the cheek pouch mucosa, then providing an even application over the mucosal surface of the vehicle or morphogen material. A hydroxypropylcellulose-based coating was used to 73 maintain contact of the morphogen with the mucosa.

This coating provided at least 4 hours of contact time.

On day 12, two animals in each group were sacrificed for histological studies. The right buccal pouch mucosa and underlying connective tissue were dissected and fixed in 10% formalin using standard dissection and histology procedures. The specimens were mounted in paraffin and prepared for histologic examination. Sections then were stained with hematoxylin and eosin and were examined blindly by three oral pathologists with expertise in hamster histology and scored blind against a standard mucositis panel. The extent of atrophy, cellular infiltration, 15 connective tissue breakdown, degree of ulceration and epithelialization were assessed.

The mean mucositis score for each group was determined daily for each experimental group for a 20 period of 21 days by photography and visual examination of the right buccal cheek pouch. Differences between groups were determined using the Students' test.

In addition, data was evaluated between groups by comparing the numbers of animals with severe mucositis 25 using Chi Square statistical analysis. The significance of differences in mean daily weights also was determined.

The experimental results are presented in Figs. 1 and 2. Figure 1 graphs the effect of morphogen (high dose, squares; low dose, diamonds) and placebo (circles) on mean mucositis scores. Both low and high morphogen doses inhibit lesion formation significantly in a dose-dependent manner. Fig. 2 (A and B) are photomicrographs of a buccal cheek pouch on day 14, 74 pretreated with morphogen, high dose or saline alone Significant tissue necrosis, indicated by the dark regions in the tissue, and ulceration, indicated by the light globular areas in the tissue, is evident in the untreated pouch in Fig. 2A. By contrast, the morphogen-treated tissue in Fig. 2B shows healthy tissue with no necrosis and little or no ulceration. In addition, histology results consistently showed significantly reduced amounts of tissue atrophy, cellular debris, and immune effector cells, including activated macrophages and neutrophils, in the morphogen-treated animals, as compared with the untreated, control animals.

15 In a variation on this protocol, morphogen also may be administered daily for several days before mucositis-induction and/or for longer periods following treatments.

Example 4. Morphogen Treatment of Duodenal Ulcer Formation The following example provides a rat model for demonstrating morphogen efficacy in treating duodenal 25 ulcers. A detailed description of the protocol is provided in Pilan et al., (1985) Digestive Diseases and Sciences 30: 240-246, the disclosure of which is incorporated herein by reference.

Briefly, Sprague-Dawley female rats Charles River Laboratories, 150-200 grams) receive the duodenal ulcerogen cysteamine-HC1 at a dose of 25-28 milligrams (mg) per 100 grams (gm) of body weight orally by intragastric gavage 3 times on the same day.

Additionally, cortisol is administered subcutaneously 75 to each rat at a single dose of 5mg of cortisol to 100 gm of body weight to decrease the mortality resulting from the administration of the cysteamine- HC1.

Three days after administration of the cysteamine- HC1, rats having penetrating and perforating duodenal ulcers are identified by standard laparotomy and randomized into control and morphogen-treated groups.

The rats of Group 1, all of which have ulcers, receive no morphogen and are treated only with saline.

The rats of Group 2 each of which also have ulcers, receive 50-100 ng of morphogen per 100 gm of body weight. Group 3 rats, all of which have ulcers, receive 200-500 ng of morphogen per 100 gm of body :i weight. All treatments are by gavage twice daily until autopsy on day 21, when the ulcers are measured and histologic sections taken.

Histology of duodenal sections from morphogentreated animals is anticipated to show reduced tissue damage associated with duodenal ulcers and/or healed ulcers. Moreover, treatment with morphogen before or concomitantly with ulceration also is anticipated to inhibit ulcer formation and/or to reduce associated tissue damage.

Example 5. Gastric acid and Pepsin Secretion of Morphogen-Treated Rats The following example demonstrates morphogen efficacy as determined by gastric acid and pepsin secretion. A detailed description of the protocol is S- 76 provided in Pilan et al., disclosed above. Briefly, 18-20 rats are divided into 2 groups, a control group (Group 1) and a morphogen treated group (Group 2).

All rats are fasted for 24 hours and given either saline vehicle alone (Group 1) or morphogen 500 ng/ml, Group The stomachs of the rats then are constricted with a pyloric ligature for one hour.

Gastric juice is collected from each rat in groups 1 and 2, centrifuged and aliquots processed for acid titration to calculate gastric acid output and pepsin determination. Gastric acid is measured by the acidity of the gastric juices, and pepsin levels are determined 15 according to standard protease assays well-known in the art. Since pepsin is the most abundant protease in the stomach, the total protease level is a good measurement of the pepsin level. The gastric juice aliquots are spectrophotometrically analyzed using albumin as a a substrate. (Szabo, S. et al. (1977) Res. Comm. Chem.

Pathol. Pharmacol. 16: 311-323, hereby incorporated by reference).

In both control and morphogen-treated rats normal 25 levels of gastric pepsin output and gastric juice volume can be measured. Morphogen treatment of ulcers of the GI tract is anticipated not to affect significantly the normal levels of gastric acid or pepsin in the GI tract.

Example 6. Morphoqen Treatment of Ulcerative colitis Ulcerative colitis involves ulcers of the colon.

The example provided below demonstrates morphogen efficacy in treating ulcerative colitis using a guinea 77 pig model. A detailed description of the protocol is provided in Onderdonk et al. (1979) Amer. J. Clin.

Nutr. 32: 258-265, the disclosure of which is incorporated herein by reference.

Briefly, guinea pigs, 500-550 gms, Charles River Laboratories) are divided into 3 experimental groups, each group containing multiple animals: a control, Group 1, which receives distilled water to drink; Group 2, which receives distilled water containing 1% degraded carrageenin; and Group 3, which receives distilled water containing 5% degraded carrageenin to drink. Degraded carrageenin is a polysaccharide derived from red seaweeds, (Glaxo 15 Laboratories, Paris, France), and is a known inducer of ulcerative colitis in guinea pigs.

The development of colitis is determined using several criteria: 1) presence of loose and/or bloody feces by visual inspection, 2) detection of occult blood in the feces using Coloscreen III with hemocult developer (Helena Labs, Bumont, TX), and 3) weight loss.

At day 25, each animal is anesthetized with Ketamine (3-5 mg/kg) administered intramuscularly and a 3 mm colorectal mucosa biopsy taken using a small nasal :'scope. All of the specimens are fixed in .formaldehyde and examined histologically using hematoxylin and eosin. The pathologic diagnosis of ulcerative colitis is established by the presence of crypt abscesses, lymphocytic infiltration, capillary congestion of the lamina propria and ulceration of the colon mucosa (Onderdonk, (1985) Digestive Disease Science 30:40(s), hereby incorporated by reference).

78 The severity of ulcerative colitis is graded on a scale of 0 to 3 and expressed as the pathological index according to the standard scoring system (Onderdonk et al. (1979), Amer. J. Clin. Nutrition 32:258.) At day 30, 25% of the guinea pigs in which ulcerative colitis was demonstrated histologically are treated with morphogen and the remaining 25% receive distilled water as a control. Morphogen is administered both at a low dose 100 ng/100 gm) in one half of the guinea pigs; and at a high dose 500-1000 ng/100 gm), administered orally through a 3 mm bulbed needle, twice per day for a period of days (days 28-37).

S During treatment, the animals are evaluated clinically and improvements in body weight, stool consistency and reduction or absence of blood in stools recorded. At day 37, all animals are sacrificed with an overdose of pentobarbital (>200 mg/kg) and the entire colon removed for histological evaluation.

Tissue damage associated with colon ulcers in morphogen treated animals is anticipated to be reduced and/or the ulcers to be significantly repaired and healed as 25 compared with untreated ulcers.

Example 7. Morphogen Inhibition of Epithelial Cell S: Proliferation This example demonstrates the ability of morphogens to inhibit epithelial cell proliferation in vitro, as determined by 3 H-thymidine uptake using culture cells from a mink lung epithelial cell line (ATCC No. CCL 64, Rockville, MD), and standard mammalian cell culturing procedures. Briefly, cells were grown to confluency in Eagle's minimum essential medium (EMEM) supplemented 79 with 10% fetal bovine serum (FBS), 200 units/ml penicillin, and 200 pg/ml streptomycin, and used to seed a 48-well cell culture plate at a cell density of 200,000 cells per well. When this culture became confluent, the media was replaced with 0.5 ml of EMEM containing 1% FBS and penicillin/streptomycin and the culture incubated for 24 hours at 37 C. Morphogen test samples in EMEM containing 5% FBS then were added to the wells, and the cells incubated for another 18 hours. After incubation, 1.0 pCi of 'H-thymidine in pl was added to each well, and the cells incubated for four hours at 37 C. The media then was removed and the cells washed once with ice-cold phosphate-buffer saline and DNA precipitated by adding 0.5 ml of 10% TCA to 15 each well and incubating at room temperature of minutes. The cells then were washed three times with ice-cold distilled water, lysed with 0.5 ml 0.4 M NaOH, and the lysate from each well then transferred to a scintillation vial and the radioactivity recorded using a scintillation counter (Smith-Kline Beckman).

*SS.

The results are presented in Fig. 3A and 3B. The anti-proliferative effect of the various morphogens tested was expressed as the counts of 3H-thymidine (x 25 1000) integrated into DNA. In this example, the biosynthetic constructs COP-5 and COP-7 were tested in duplicate: COP-7-1 (10 ng) and COP-7-2 (3 ng, Fig.

3A), and COP-5-1 (66 ng) and COP-5-2 (164 ng, Fig. 3B.) Morphogens were compared with untreated cells (negative control) and TGF-A (1 ng), a local-acting factor also known to inhibit epithelial cell proliferation. and COP-7 previously have been shown to have osteogenic activity, capable of inducing the complete cascade resulting in endochondral bone formation in a standard rat bone assay (see U.S. Pat. No. 5,011,691.) As is 80 evident in the figure, the morphogens significantly inhibit cell epithelial cell proliferation. Similar experiments, performed with the morphogens COP-16 and bOP (bone-purified osteogenic protein, a dimeric protein comprising CBMP2 and OP-1) and recombinant OP-1 also inhibit cell proliferation. bOP and COP-16 also induce endochondral bone formation (see US Pat. No.

4,968,590 and 5,011,691.) Example 8. Morphogen Inhibition of Cellular and Humoral Inflammatory Response Morphogens described herein inhibit multinucleation of mononuclear phagocytic cells under conditions where 15 these cells normally would be activated, in response to a tissue injury or the presence of a foreign substance. For example, and as described in international application US92/07358 (W093/04692) in the absence of morphogen, an implanted substrate material implanted subcutaneously) composed of, for example, mineralized bone, a ceramic such as titanium oxide or any other substrate that provokes multinucleated giant cell formation, rapidly becomes surrounded by multinucleated giant cells, e.g., activated phagocytes stimulated to respond and destroy the foreign object. In the presence of morphogen however, the recruited cells remain in their mononuclear precursor form and the matrix material is undisturbed. Accordingly, the morphogens' effect in maintaining the integrity of the GI tract luminal lining also may include inhibiting activation of these immune effector cells.

In addition, the morphogens described herein also suppress antibody production stimulated in response to S- 81 a foreign antigen in a mammal. Specifically, when bovine bone collagen matrix alone was implanted in a bony site in a rat, a standard antibody response to the collagen was stimulated in the rat as determined by standard anti-bovine collagen ELISA experiments performed on blood samples taken at four week intervals following implantation between 12 and 20 weeks.) Serum anti-collagen antibody titers, measured by ELISA essentially following the procedure described by Nagler-Anderson et al, (1986) PNAS 83:7443-7446, the disclosure of which is incorporated herein by reference, increased consistently throughout the experiment. However, when the matrix was implanted together with a morphogen OP-1, dispersed in the 15 matrix and adsorbed thereto, essentially as described in U.S. Pat. No. 4,968,590) anti-bovine collagen antibody production was suppressed significantly. This ability of morphogen to suppress the humoral response is further evidence of morphogen utility in alleviating 20 tissue damage associated with GI tract ulceration.

*oo Example 9. Morphogen Effect on Fibrogenesis and Scar Tissue Formation 25 The morphogens described herein induce tissue morphogenesis of damaged or lost tissue. The ability of these proteins to regenerate new tissue enhances the anti-inflammatory effect of these proteins. Provided below are a series of in vitro experiments demonstrating the ability of morphogens to induce migration and accumulation of mesenchymal cells. In addition, the experiments demonstrate that morphogens, unlike TGF-f, do not stimulate fibrogenesis or scar tissue formation. Specifically, morphogens do not stimulate production of collagen, hyaluronic acid (HA) 82 or metalloproteinases in primary fibroblasts, all of which are associated with fibrogenesis or scar tissue formation. By contrast, TGF-P, a known inducer of fibrosis, but not of tissue morphogenesis as defined herein, does stimulate production of these markers of fibrosis.

Chemotaxis and migration of mesenchymal progenitor cells were measured in modified Boyden chambers essentially as described by Fava, R.A. et al (1991) J.

Exp. Med. 173: 1121-1132, the disclosure of which is incorporated herein by reference, using polycarbonate filters of 2, 3 and 8 micron ports to measure migration of progenitor neutrophils, monocytes and fibroblasts.

15 Chemotaxis was measured over a range of morphogen concentrations, 10- 2 0 M to 10- 1 2 M OP-1. For progenitor neutrophils and monocytes, 10 8-10 M OP-1 consistently induced maximal migration, and 10 1 to 101 M OP-1 maximally induced migration of progenitor 20 fibroblasts. In all cases the chemotactic activity could be inhibited with anti-OP-1 antibody. Similar migration activities also were measured and observed with TGF-A.

25 The effect of morphogen on fibrogenesis was determined by evaluating fibroblast production of hyaluronic acid collagen, collagenase and tissue inhibitor of metalloproteinases (TIMP).

Human fibroblasts were established from explants of infant foreskins and maintained in monolayer culture using standard culturing procedures. (See, for example, (1976) J. Exp. Med. 144: 1188-1203.) Briefly, fibroblasts were grown in maintenance medium consisting of Eagle's MEM, supplemented with nonessential amino 83 acids, ascorbic acid (50 pg/ml), NaHCO 3 and HEPES buffers (pH penicillin (100 U/ml), streptomycin (100 pg/ml), amphotericin B (1 pg/ml) and 9% heat inactivated FCS. Fibroblasts used as target cells to measure chemotaxis were maintained in 150 mm diameter glass petri dishes. Fibroblasts used in assays to measure synthesis of collagen, hyaluronic acid, collagenase and tissue inhibitors of metalloproteinases (TIMP) were grown in 100 mm diameter plastic tissue culture petri dishes.

The effects of morphogen on fibroblast production of hyaluronic acid, collagens, collagenase and TIMP were determined by standard assays (See, for example, 15 Posttethwaite et al. (1989) J. Clin. Invest. 83: 629- 636, Posttethwaithe (1988) Cell Biol. 106: 311-318 and Clark et al (1985) Arch. Bio-chem Biophys. 241: 36- 44, the disclosures of which are incorporated by reference.) For these assays, fibroblasts were transferred to 24-well tissue culture plates at a density of 8 x 10 4 cells per well. Fibroblasts were grown confluency in maintenance medium containing 9% FCS for 72 h and then grown in serum-free maintenance medium for 24 h. Medium was then removed from each 25 well and various concentrations of OP-1 (recombinantly produced mature or soluble form) or TGF--1 (R&D Systems, Minneapolis) in 50 pl PBS were added to S: triplicate wells containing the confluent fibroblast monolayers. For experiments that measured production of collagenase and TIMP, maintenance medium (450 pi) containing 5% FCS was added to each well, and culture supernatants were harvested from each well 48 h later and stored at -70 0 C until assayed. For experiments that assessed HA production, maintenance medium (450 pi) containing 2.5% FCS was added to each well, and 84 cultures grown for 48 h. For experiments that measured fibroblast production of collagens, serum-free maintenance medium (450 pl) without non-essential amino acids was added to each well and cultures grown for 72 h. Fibroblast production of HA was measured by labeling newly synthesized glycosaminoglycans (GAG) with H]-acetate the last 24 h of culture and quantitating released radioactivity after incubation with hyaluronidase from Streptomyces hyalurolyticus (ICN Biochemicals, Cleveland, OH) which specifically degrades hyaluronic acid. Production of total collagen by fibroblasts was measured using a collagenasesensitive protein assay that reflects [3H]-proline incorporation the last 24 h of culture into newly synthesized collagens. Collagenase and TIMP protein levels in fibroblast cultures supernatants was measured by specific ELISAs.

*i As shown in Fig. 4, OP1 does not stimulate significant collagen or HA production, as compared with TGF-p. In the figure, panel A shows OP-1 efect on collagen production, panel B shows TGF-A effect on collagen production, and panels C and D show OP-1 (panel C) and TGF-A (panel D) effect on HA production.

25 The morphogen results were the same whether the soluble or mature form of OP1 was used. By contrast, the latent form of TGF-p pro domain-associated form of TGF-p) was not active.

Example 10. Screening Assay for Candidate Compounds which Alter Endogenous Morphogen Levels Candidate compound(s) which may be administered to affect the level of a given morphogen may be found using the following screening assay, in which the level 85 of morphogen production by a cell type which produces measurable levels of the morphogen is determined with and without incubating the cell in culture with the compound, in order to assess the effects of the compound on the cell. This can be accomplished by detection of the morphogen either at the protein or RNA level. A more detailed description also may be found in international application US92/07359 (W093/05172).

10.1 Growth of Cells in Culture Cell cultures of kidney, adrenals, urinary bladder, brain, or other organs, may be prepared as described widely in the literature. For example, kidneys may be explanted from neonatal or new born or young or adult rodents (mouse or rat) and used in organ culture as whole or sliced (1-4 mm) tissues. Primary tissue cultures and established cell lines, also derived from kidney, adrenals, urinary, bladder, brain, mammary, or other tissues may be established in multiwell plates (6 S" well or 24 well) according to conventional cell culture techniques, and are cultured in the absence or presence S. of serum for a period of time (1-7 days). Cells may be cultured, for example, in Dulbecco's Modified Eagle medium (Gibco, Long Island, NY) containing serum fetal calf serum at Gibco) or in serum-deprived medium, as desired, or in defined medium containing insulin, transferrin, glucose, albumin, or other growth factors).

Samples for testing the level of morphogen production includes culture supernatants or cell lysates, collected periodically and evaluated for OP-1 production by immunoblot analysis (Sambrook et al., eds., 1989, Molecular Cloning, Cold Spring Harbor 86 Press, Cold Spring Harbor, NY), or a portion of the cell culture itself, collected periodically and used to prepare polyA+ RNA for RNA analysis. To monitor de novo OP-1 synthesis, some cultures are labeled according to conventional procedures with an "S-methionine/ 3 SS-cysteine mixture for 6-24 hours and then evaluated to OP-1 synthesis by conventional immunoprecipitation methods.

10.2 Determination of Level of Morphogenic Protein In order to quantitate the production of a morphogenic protein by a cell type, an immunoassay may be performed to detect the morphogen using a polyclonal or monoclonal antibody specific for that protein. For example, OP-1 may be detected using a polyclonal antibody specific for OP-1 in an ELISA, as follows.

1 pg/100 pl of affinity-purified polyclonal rabbit 20 IgG specific for OP-1 is added to each well of a 96-well plate and incubated at 37 0 C for an hour. The .:wells are washed four times with 0.167M sodium borate buffer with 0.15 M NaCl (BSB), pH 8.2, containing 0.1% Tween 20. To minimize non-specific binding, the wells are blocked by filling completely with 1% bovine serum albumin (BSA) in BSB and incubating for 1 hour at 37 0

C.

The wells are then washed four times with BSB containing 0.1% Tween 20. A 100 pl aliquot of an appropriate dilution of each of the test samples of 30 cell culture supernatant is added to each well in triplicate and incubated at 37°C for 30 min. After incubation, 100 pl biotinylated rabbit anti-OP-1 serum (stock solution is about 1 mg/ml and diluted 1:400 in BSB containing 1% BSA before use) is added to each well and incubated at 37 0 C for 30 min. The wells are then 87 washed four times with BSB containing 0.1% Tween 100 pl strepavidin-alkaline phosphatase (Southern Biotechnology Associates, Inc. Birmingham, Alabama, diluted 1:2000 in BSB containing 0.1% Tween 20 before use) is added to each well and incubated at 37 0 C for min. The plates are washed four times with 0.5M Tris buffered Saline (TBS), pH 7.2. 50pl substrate (ELISA Amplification System Kit, Life Technologies, Inc., Bethesda, MD) is added to each well incubated at room temperature for 15 min. Then, 50 pl amplifier (from the same amplification system kit) is added and incubated for another 15 min at room temperature. The reaction is stopped by the addition of 50 pl 0.3 M sulphuric acid. The OD at 490 nm of the solution in each well is recorded. To quantitate OP-1 in culture media, a OP-1 standard curve is performed in parallel with the test samples.

Polyclonal antibody may be prepared as follows.

20 Each rabbit is given a primary immunization of 100 ug/500 pl E. coli produced OP-1 monomer (amino acids 328-431 in SEQ ID NO:5) in 0.1% SDS mixed with 500 pi Complete Freund's Adjuvant. The antigen is injected :subcutaneously at multiple sites on the back and flanks of the animal. The rabbit is boosted after a month in the same manner using incomplete Freund's Adjuvant.

Test bleeds are taken from the ear vein seven days later. Additional boosts and test bleeds are performed at monthly intervals until antibody against OP-1 is 30 detected in the serum using an ELISA assay. Then, the rabbit is boosted with 100 pg of antigen and bled ml per bleed) at days seven and ten after boosting.- Monoclonal antibody specific for a given morphogen may be prepared as follows. A mouse is given two S- 88 injections of E. coli produced OP-1 monomer. The first injection contains 100pg of OP-1 in complete Freund's adjuvant and is given subcutaneously. The second injection contains 50 pg of OP-1 in incomplete adjuvant and is given intraperitoneally. The mouse then receives a total of 230 pg of OP-1 (amino acids 307-431 in SEQ ID NO:5) in four intraperitoneal injections at various times over an eight month period. One week prior to fusion, the mouse is boosted intraperitoneally with 100 pg of OP-1 (307-431) and 30 pg of the Nterminal peptide (Ser 293 -Asn 309 -Cys) conjugated through the added cysteine to bovine serum albumin with SMCC crosslinking agent. This boost was repeated five days four days three days (IP) and one day (IV) prior to fusion. The mouse spleen cells are then fused to myeloma 653) cells at a ratio of 1:1 using PEG 1500 (Boeringer Mannheim), and the cell fusion is plated and screened for OP-1-specific antibodies using OP-1 (307-431) as antigen. The cell fusion and 20 monoclonal screening then are according to standard procedures well described in standard texts widely available in the art.

The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced S: therein.

89 SEQUENCE LISTING GENERAL INFORMATION:

APPLICANT:

NAME: CREATIVE BIOMOLECULES,

INC.

STREET: 45 SOUTH STREET CITY: HOPKINTON STATE: MA COUNTRY: USA POSTAL CODE (ZIP): 01748 TELEPHONE: 1-508-435-9001 TELEFAX: 1-508-435-0454

TELEX:

(ii) TITLE OF INVENTION: MORPHOGEN TREATMENT OF GASTROINTESTINAL

ULCERS

(iii) NUMBER OF SEQUENCES: 33 (iv) CORRESPONDENCE

ADDRESS:

ADDRESSEE: CREATIVE BIOMOLECULES, INC.

STREET: 45 SOUTH STREET CITY: HOPKINTON STATE: MA COUNTRY: USA ZIP: 01748 S: 30 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.25 (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: FILING DATE:

CLASSIFICATION:

:(vii) PRIOR APPLICATION DATA: APPLICATION NUMBER: FILING DATE: 45 (viii) ATTORNEY/AGENT INFORMATION: NAME: KELLEY ESQ, ROBIN D.

REGISTRATION NUMBER: 34,637 REFERENCE/DOCKET NUMBER: CRP-074 :50 (ix) TELECOMMUNICATION INFORMATION: TELEPHONE: 617/248-7477 TELEFAX: 617/248-7100 90 INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 97 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (11) MOLECULE TYPE: protein (ix) FEATURE: NAME/KEY: Protein LOCATION: 1. .97 OTHER INFORMATION: /label= GENERIC-SEQi /note= WUHEREIN EACH XAA INDEPENDENTLY INDICATES ONE OF THE 20 NATURALLY-OCCURING L-ISOMER, A-AMINO ACIDS,, OR A DERIVATIVE THEREOF." (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Cys Xaa Xaa Xaa 25 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35 40 *.:Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Cys Xaa Xaa 55 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa *:65 70 75 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys 85 90 Xaa INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 97 amino acids TYPE: amino acid STRAINDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein 91 (ix) FEATURE: NAME/KEY: Protein LOCATION: 1._97 OTHER INFORMATION: /label= GENERIC-SEQ2 /note= WUHEREIN EACH XAA INDEPENDENTLY INDICATES ONE OF THE 20 NATURALLY OCCURING L-ISOMER A-AMINO ACIDS, OR A DERIVATIVE THEREOF.- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Xaa Xaa Xaa Xaa Xaa Xaa 1 5 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys 25 Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Cys Xaa Xaa Xaa 65 Xaa Xaa Xaa Xaa Xaa 70 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys 9* S. Xaa INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 97 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (ix) FEATURE: NAME/KEY: Protein LOCATION: 1- 97 OTHER INFORMATION: /label= GENERIC-SE03 /note= "WHEREIN EACH XAA IS INDEPENDENTLY SELECTED FROM A GROUP OF ONE OR MORE SPECIFIED AMINO ACIDS AS DEFINED IN THE SPECIFICATION." 92 xi) SEQUENCE Leu Tyr

I

Pro Xaa Xaa Xaa Xaa Xaa Xaa Xaa Val Xaa DESCRIPTION: SEQ ID Val Xaa Phe Xaa Xaa 5 Gly Xaa Xaa Ala Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 55 Xaa Xaa Xaa Xaa Xaa 70 Leu Xaa Xaa Xaa Xaa NO: 3: Xaa Gly Trp 10 Tyr Cys Xaa 25 Xaa Asn His 40 Xaa Xaa Xaa Xaa Leu Xaa Xaa Met Xaa 90 Xaa Xaa Trp Gly Xaa Cys Ala Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa 75 Val Zaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Cys Gly Ala Pro Leu Pro Xaa Cys a a a a a a.

a S a S. a Xaa INFORMATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 102 amino acids TYPE: amino acid STRANDEDNESS: single 30 TOPOLOGY: linear (ii) MOLECULE TYPE: protein 35 (ix) FEATURE: NAME/KEY: Protein LOCATION: 1..102 OTHER INFORMATION: /label= GENERIC-SEQ4 /note= "WHEREIN EACH XAA IS INDEPENDENTLY SELECTED FROM A GROUP OF ONE OR MORE SPECIFIED AMINO ACIDS AS DEFINED IN THE SPECIFICATION." (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: Cys Xaa Xaa Xaa Xaa Leu Tyr Val Xaa Phe Xaa Xaa Xaa Gly Trp Xaa 1 5 10 Xaa Trp Xaa Xaa Ala Pro Xaa Gly Xaa Xaa Ala Xaa Tyr Cys Xaa Gly 50 20 25 Xaa Cys Xaa Xaa Pro Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asn His Ala 40 Xaa Xaa Xaa Xaa Leu Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 55 93 Xaa Cys Cys Xaa Pro Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Leu Xaa Xaa 70 75 Xaa Xaa Xaa Xaa Xaa Val Xaa Leu Xaa Xaa Xaa Xaa Xaa Met Xaa Val 90 Xaa Xaa Cys Gly Cys Xaa 100 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 139 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens TISSUE TYPE: HIPPOCAMPUS (ix) FEATURE: NAME/KEY: Protein LOCATION: 1..139 OTHER INFORMATION: /label= hOPI-MATURE (xi) SEQUENCE DESCRIPTION: SEQ ID i Ser Thr Gly Ser Lys Gin Arg Ser Gin Asn Arg Ser Lys Thr Pro Lys 1 5 10 35 Asn Gin Glu Ala Leu Arg Met Ala Asn Val Ala Glu Asn Ser Ser Ser 20 25 Asp Gin Arg Gin Ala Cys Lys Lys His Glu Leu Tyr Val Ser Phe Arg 35 40 Asp Leu Gly Trp Gin Asp Trp Ile Ile Ala Pro Glu Gly Tyr Ala Ala 50 55 Tyr Tyr Cys Glu Gly Glu Cys Ala Phe Pro Leu Asn Ser Tyr Met Asn 65 70 75 Ala Thr Asn His Ala Ile Val Gin Thr Leu Val His Phe Ile Asn Pro 90 Glu Thr Val Pro Lys Pro Cys Cys Ala Pro Thr Gin Leu Asn Ala Ile 100 105 110 Ser Val Leu Tyr Phe Asp Asp Ser Ser Asn Val lie Leu Lys Lys Tyr 115 120 125 S- 94 Arg Asn Met Val Val Arg Ala Cys Gly Cys His 130 135 INFORMATION FOR SEQ ID NO:6: SEQUENCE

CHARACTERISTICS:

LENGTH: 139 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE: ORGANISM: MURIDAE TISSUE TYPE: EMBRYO (ix) FEATURE: NAME/KEY: Protein LOCATION: 1..139 OTHER INFORMATION: /label= MOP1-MATURE (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: Ser Thr Gly Gly Lys Gln Arg Ser Gln Asn Arg Ser Lys Thr Pro Lys 1 5 10 Asn Gln Glu Ala Leu Arg Met Ala Ser Val Ala Glu Asn Ser Ser Ser 20 25 :e Asp Gln Arg Gin Ala Cys Lys Lys His Glu Leu Tyr Val Ser Phe Arg 35 40 Asp Leu Gly Trp Gln Asp Trp Ile Ile Ala Pro Glu Gly Tyr Ala Ala 50 55 Tyr Tyr Cys Glu Gly Glu Cys Ala Phe Pro Leu Asn Ser Tyr Met Asn 70 75 Ala Thr Asn His Ala Ile Val Gln Thr Leu Val His Phe Ile Asn Pro 85 90 Asp Thr Val Pro Lys Pro Cys Cys Ala Pro Thr Gin Leu Asn Ala Ile 100 105 110 Ser Val Leu Tyr Phe Asp Asp Ser Ser Asn Val Ile Leu Lys Lys Tyr 115 120 125 Arg Asn Met Val Val Arg Ala Cys Gly Cys His 130 135 95 INFORMATION FOR SEQ ID NO:7: SEQUENCE CHARACTERISTICS: LENGTH: 139 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE: ORGANISM: HOMO SAPIENS TISSUE TYPE: HIPPOCAMPUS (ix) FEATURE: NAME/KEY: Protein LOCATION: 1..139 OTHER INFORMATION: /label= HOP2-KATURE (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: Ala Val Arg Pro Leu Arg Arg Arg Gin Pro Lys Lys Ser Asn Glu Leu 1 5 10 Pro Gin Ala Asn Arg Leu Pro Gly Ile Phe Asp Asp Val His Gly Ser 25 His Gly Arg Gin Val Cys Arg Arg His Glu Leu Tyr Val Ser Phe Gin 40 55 Tyr Tyr Cys Glu Gly Glu Cys Ser Phe Pro Leu Asp Ser Cys Met Asn 35 65 70 75 Ala Thr Asn His Ala lie Leu Gin Ser Leu Val His Leu Met Lys Pro 85 90 Asn Ala Val Pro Lys Ala Cys Cys Ala Pro Thr Lys Leu Ser Ala Thr 1. 00 105 110 Ser Val Leu Tyr Tyr Asp Ser Ser Asn Asn Val Ile Leu Arg Lys His 115 120 125 Arg Asn Met Val Val Lys Ala Cys Gly Cys His 130 135 INFORMATION FOR SEQ ID NO:8: SEQUENCE CHARACTERISTICS: LENGTH: 139 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear 96 (ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE: ORGANISM: MURIDAE TISSUE TYPE: EMBRYO (ix) FEATURE: NAME/KEY: Protein LOCATION: 1..139 OTHER INFORMATION: /label= MOP2-MATURE (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: Ala Ala Arg Pro Leu Lys Arg Arg Gin Pro Lys Lys Thr Asn Glu Leu 1 5 10 Pro His Pro Asn Lys Leu Pro Gly Ile Phe Asp Asp Gly His Gly Ser 25 Arg Gly Arg Glu Val Cys Arg Arg His Glu Leu Tyr Val Ser Phe Arg 40 Asp Leu Gly Trp Leu Asp Trp Val Ile Ala Pro Gin Gly Tyr Ser Ala 50 55 Tyr Tyr Cys Glu Gly Glu Cys Ala Phe Pro Leu Asp Ser Cys Met Asn 70 75 Ala Thr Asn His Ala Ile Leu Gin Ser Leu Val His Leu Met Lys Pro 85 90 Asp Val Val Pro Lys Ala Cys Cys Ala Pro Thr Lys Leu Ser Ala Thr 100 105 110 35 Ser Val Leu Tyr Tyr Asp Ser Ser Asn Asn Val Ile Leu Arg Lys His 115 120 125 Arg Asn Met Val Val Lys Ala Cys Gly Cys His 130 135 INFORMATION FOR SEQ ID NO:9: SEQUENCE CHARACTERISTICS: LENGTH: 101 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE: ORGANISM: bovinae 97 (ix) FEATURE: NAME/KEY: Protein LOCATION: 1..101 OTHER INFORMATION: /label= CBMP-2A-FX (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: Cys Lys Arg His Pro Leu Tyr Val Asp Phe Ser Asp Val Gly Trp Asn 1 5 10 Asp Trp Ile Val Ala Pro Pro Gly Tyr His Ala Phe Tyr Cys His Gly 25 Glu Cys Pro Phe Pro Leu Ala Asp His Leu Asn Ser Thr Asn His Ala 35 40 Ile Val Gin Thr Leu Val Asn Ser Val Asn Ser Lys Ile Pro Lys Ala 55 Cys Cys Val Pro Thr Glu Leu Ser Ala Ile Ser Met Leu Tyr Leu Asp 70 75 Glu Asn Glu Lys Val Val Leu Lys Asn Tyr Gin Asp Met Val Val Glu 90 Gly Cys Gly Cys Arg 100 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 101 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE: 40 ORGANISM: HOMO SAPIENS TISSUE TYPE: hippocampus (ix) FEATURE: NAME/KEY: Protein 45 LOCATION: 1..101 OTHER INFORMATION: /label= CBMP-2B-FX (xi) SEQUENCE DESCRIPTION: SEQ ID 50 Cys Arg Arg His Ser Leu Tyr Val Asp Phe Ser Asp Val Gly Trp Asn 1 5 10 Asp Trp Ile Val Ala Pro Pro Gly Tyr Gin Ala Phe Tyr Cys His Gly 25 98 Asp Cys Pro Phe Pro Leu Ala Asp His Leu Asn Ser Thr Asn His Ala 40 Ile Val Gin Thr Leu Val Asn Ser Val Asn Ser Ser lie Pro Lys Ala 55 Cys Cys Val Pro Thr Glu Leu Ser Ala Ile Ser Met Leu Tyr Leu Asp 70 75 Glu Tyr Asp Lys Val Val Leu Lys Asn Tyr Gin Glu Met Val Val Glu 90 Gly Cys Gly Cys Arg 100 INFORMATION FOR SEQ ID NO:11: SEQUENCE CHARACTERISTICS: LENGTH: 102 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE: ORGANISM: DROSOPHILA MELANOGASTER 30 (ix) FEATURE: NAME/KEY: Protein LOCATION: 1..101 OTHER INFORMATION: /label= DPP-FX 35 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: Cys Arg Arg His Ser Leu Tyr Val Asp Phe Ser Asp Val Gly Trp Asp 1 5 10 40 Asp Trp Ile Val Ala Pro Leu Gly Tyr Asp Ala Tyr Tyr Cys His Gly 25 Lys Cys Pro Phe Pro Leu Ala Asp His Phe Asn Ser Thr Asn His Ala 40 Val Val Gln Thr Leu Val Asn Asn Asn Asn Pro Gly Lys Val Pro Lys 55 :Ala Cys Cys Val Pro Thr Gin Leu Asp Ser Val Ala Met Leu Tyr Leu 50 65 70 75 Asn Asp Gin Ser Thr Val Val Leu Lys Asn Tyr Gin Glu Met Thr Val 90 99 Val Gly Cys Gly Cys Arg 100 INFORMATION FOR SEQ ID NO:12: SEQUENCE CHARACTERISTICS: LENGTH: 102 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE: ORGANISM: XENOPUS (ix) FEATURE: NAME/KEY: Protein LOCATION: 1..102 OTHER INFORMATION: /label= VGL-FX (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: Cys Lys Lys Arg His Leu Tyr Val Glu Phe Lys Asp Val Gly Trp Gln 1 5 10 Asn Trp Val Ile Ala Pro Gln Gly Tyr Met Ala Asn Tyr Cys Tyr Gly 20 25 30 Glu Cys Pro Tyr Pro Leu Thr Glu Ile Leu Asn Gly Ser Asn His Ala 40 Ile Leu Gln Thr Leu Val His Ser Ile Glu Pro Glu Asp Ile Pro Leu 55 Pro Cys Cys Val Pro Thr Lys Met Ser Pro Ile Ser Met Leu Phe Tyr 70 75

S**

0. Asp Asn Asn Asp Asn Val Val Leu Arg His Tyr Glu Asn Met Ala Val 85 90 Asp Glu Cys Gly Cys Arg 100 45 INFORMATION FOR SEQ ID NO:13: SEQUENCE CHARACTERISTICS: LENGTH: 102 amino acids TYPE: amino acid 50 STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein 100 (vi) ORIGINAL SOURCE: ORGANISM: KURIDAE (ix) FEATURE: NAME/KEY: Protein LOCATION: 102 OTHER INFORMATION: /label= VGR-1-FX (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: Cys Lys Lys His Giu Leu Tyr Val Ser Phe 1 5 10 Asp Trp Ile Ile Ala Pro Lys Gly Tyr Ala 20 25 Giu Cys Ser Phe Pro Leu Asn Ala His Met 40 Ile Val Gin Thr Leu Val His Val Met Asn 55 Pro Cys Cys Ala Pro Thr Lys Val Asn Ala 70 Asp Asp Asn Ser Asn Val Ile Leu Lys Lys 90 Arg Aia Cys Giy Cys His 30 100 INFORMATION FOR SEQ ID NO:14: SEQUENCE CHARACTERISTICS: 35 LENGTH: 106 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear 40 (1i) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens TISSUE TYPE: brain 50 (ix) FEATURE: NAME/KEY: Protein LOCATION: 1..106 OTHER INFORMATION: /note= "GDF-1 (fx)" Gin Ala Asn Pro Ile 75 Tyr Asp Asn Ala Glu Se r Arg Val Giy Trp Tyr Cys Asp Thr Asn His Tyr Val Pro Vai Leu Tyr Asn Met Vai Gin Gly Ala Lys Phe Val *0 a. a.

da a a.

a a a.

101 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: Arg Ala Arg Arg Trp Val Ile Gln Cys Ala Leu Leu Asn His Ala Ala Ala Asp Leu Val Leu Phe Phe Arg Leu Tyr Val Ser Phe Arg Glu Val Gly Trp His 10 Ala Pro Arg Gly Phe Leu Ala Asn Tyr Cys Gin Gly 25 Pro Val Ala Leu Ser Gly Ser Gly Gly Pro Pro Ala 40 Val Leu Arg Ala Leu Met His Ala Ala Ala Pro Gly 55 Pro Cys Cys Val Pro Ala Arg Leu Ser Pro Ile Ser 70 75 Asp Asn Ser Asp Asn Val Val Leu Arg Gln Tyr Glu 90 a.

a *5* a Asp Met Val Val Asp Glu Cys Gly Cys Arg 100 105 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 5 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide 35 (xi) SEQUENCE DESCRIPTION: SEQ ID Cys Xaa Xaa Xaa Xaa 1 40 INFORMATION FOR SEQ ID NO:16: SEQUENCE CHARACTERISTICS: LENGTH: 1822 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO 102 (vi) ORIGINAL SOURCE: ORGANISM: HOMO SAPIENS TISSUE TYPE: HIPPOCAMPUS (ix) FEATURE: NAME/KEY: CUS LOCATION: 49. .1341 IDENTIFICATION METHOD: experimental OTHER INFORMATION: /function= "OSTEOGENIC PROTEIN" /product. "OPi" /evidence= EXPERIMENTAL /standard-name= "OPl" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: GGTGCGGGCC CGGAGCCCGG AGCCCGCGTA GCGCCTAGAG CCGCCGCG ATG CAC GTG Met His Val 1 CGC TCA CIG CGA GCT Arg Ser Leu Arg Ala CCC CTG TIC CTG CTG Pro Leu Phe Leu Leu GCG GCG Ala Ala 10 CCG CAC AGC Pro His Ser TTC GTG Phe Val GCG CTC TGG GCA Ala Leu Trp Ala TCC GCC CTG GCC Ser Ala Leu Ala TTC AGC CTG GAC MAC Phe Ser Leu Asp Asn GAG GTG CAC Glu Val His CGG GAG ATG Arg Giu Met CCG CGC CCG Pro Arg Pro 70 TCG AGC Ser Ser 40 TIC ATC CAC CGG Phe Ile His Arg CTC CGC AGC CAG Leu Arg Ser Gin GAG CGG Glu Arg CGC GAG ATC CTC Arg Glu Ile Leu ATI TIG GCC Ile Leu Gly CAC CTC CAG GGC His Leu Gin Gly MAG CAC MAC TCG Lys His Asn Ser 75 GCG GTG GAG GAG Ala Val Giu Glu

GCA

Ala TIG CCC CAC CC Leu Pro His Arg CCC ATG TIC ATG Pro Met Phe Met GGC GGG CCC GCC Gly Gly Pro Gly CTG GAC Leu Asp CTG TAC MAC GCC Leu Tyr Asn Ala CAG GGC TIC TCC Gin Gly Phe Ser CCC TAC MAG GCC Pro Tyr Lys Ala TIC AGT ACC Phe Ser Thr CAG GGC Gin Gly 115 GCC GAC Ala Asp 130 CCC CCT CTG GCC Pro Pro Leu Ala CTG CMA GAT AGC Leu Gin Asp Ser TIC CTC ACC GAC Phe Leu Thr Asp ATG GTC ATG Met Val Met TIC GTC MAC CTC GTG Phe Val Asn Leu Val 140 GM& CAT GAC MAG Giu His Asp Lys GMA TIC TIC Giu Phe Phe 145 103 CAC CCA CGC His Pro Arg 150 TAC CAC CAT CGA Tyr His His Arg TTC CGG Trr OAT Phe Arg Phe Asp TCC AAG ATC Ser Lys Ile CCA GMA Pro Giu 165 0CC GMA GCT GTC Gly Glu Ala Val OCA GCC GMA T1TC Ala Ala Giu Phe ATC TAC MAG GAC Ile Tyr Lys Asp ATC CGG GMA CGC Ile Arg Giu Arg GAC MAT GAG ACG Asp Asn Giu Thr CGG ATC AGC Arg Ile Ser GTT TAT Val Tyr 195 CTG CTC Leu Leu 210 CAG GTG CTC CAG Gin Val Leu Gin CAC rro GCC AGG His Leu Gly Arg TCG GAT CTC TTC Ser Asp Leu Phe GAC AGC CGT Asp Ser Arg ATC ACA GCC Ile Thr Aia 230 CTC TOG GCC TCG Leu Trp Aia Ser GAG GGC TGG CIG Giu Gly Trp Leu GTG i-rr GAC Val Phe Asp 225 CAC MAC CTG His Asn Leu ACC AGC MAC CAC Thr Set Asn His GTG GTC MAT CCG Val Val Asn Pro 0CC CTG Gly Leu 245 CAG CTC TCG GTG Gin Leu Ser Val GAG ACG CTG OAT GCC Oiu Thr Leu Asp Giy 250 0CC CGG CAC 0CC CCC Gly Arg His Giy Pro 270 AGC ATC MAC CCC Se Ile Asn Pro TTG GCG C Leu Ala Gly CTG ATT Leu Ile 265 CAG MAC MAG CAG Gin Asn Lys Gin TTC ATO GTG OCT Phe Met Val Ala TTC MAG GCC ACG Phe Lys Ala Thr GTC CAC TTC CGC Val His Phe Arg AGC ATC Ser Ile 290 Sess

S.

S. S CGG TCC ACG Arg Set Thr MOG MC CAG Lys Asn Gin 310 GCC AGC AAM Gly Set Lys 295 GMA GCC CTG Giu Ala Leu CAG CGC Gin Arg CGG ATG Arg Met 315 CAG MAC CGC TCC Gin Asn Arg Ser MAG ACG CCC Lys Thr Pro 305 MAC AGC AGC Asn Set Set GCC MAC GTG OCA Ala Asn Val Ala AGC GAC CAG AGG CAG 0CC TOT MAG MAG CAC GAG CTG Set Asp Giri Arg Gin Ala Cys Lys Lys His Oiu Leu 325 330 335 TAT GTC AGC TTC Tyr Val Set Phe 969 1017 1065 1113 1161

S

S

S

*5 CGA GAC CTG GGC TG Arg Asp Leu Gly Trp 340 0CC TAC TAC TOT GAG Ala Tyr Tyr Cys Giu 360 GAC TOG ATC ATC Asp Trp Ile Ile OCO CCT GMA C Ala Pro Giu Gly 350 CCT CTG MAC TCC Pro Leu Asn Set

TAC

Tyr 0CC GAG TOT 0CC Giy Giu Cys Ala TAC ATO Tyr Met 370 104 AAC GCC ACC MAC CAC GCC AIC GTG CAG ACG CTG GTC CAC TTC AIC MAC Asn Ala Thr Asn His Ala Ile Val Gin Thr Leu Val His Phe Ile Asn 375 380 385 CCG GMA ACG GTG CCC MAG CCC TGC TGT GCG CCC ACG CAG CTC MAT GCC Pro Giu Thr Val Pro Lys Pro Cys Cys Ala Pro Thr Gin Leu Asn Ala 390 395 400 ATC TCC GTC CTC TAC TIC GAT GAC AGC TCC MAC GTC ATC CTG MAG AAA Ile Ser Val Leu Tyr Phe Asp Asp Ser Ser Asn Val Ile Leu Lys Lys 405 410 415 TAC AGA MAC ATG GTG GTC CGG GCC TGT GGC TGC CAC TAGCTCCTCC Tyr Arg Asn Met Val Val Arg Ala Cys Gly Cys His 420 425 430 1209 1257 1305 1351 1411 1471 1531 1591 1651 1711 1771 1822

GAGMATTCAG

GMACCAGCAG

TGTGAGAGTA

ATCCMATGAA

GCATAAAGMA

CGTITCCAGA

GGCGTGGCMA

CTGTMATAAM

ACCCTfl'GGG GCCMAGTTTT ACCMACTGCC TTrIGTGAGA TTAGGAAACA TGAGCAGCAT CMAGATCCTA CMAGCTGTGC AMATGGCCGG GCCAGGTCAT GGTMATTATG AGCGCCTACC GGGGTGGGCA CATTGGTGTC TGTCACMATA MAACGMTGA TCTGCATCCT CCATTGCTCG CCTICCCCTC CCTATCCCCA ATGGCTTG ATCAGT=rr AGGCAAMCC TAGCAGGAAA TGGCTGGGMA GTCTCAGCCA AGCCAGGCCA CCCAGCCGTG TGTGCGAAAG GAAAATTGAC ATGAAAAAAA AAAAAAAAAA

CCTTGGCCAG

AC'rTAMAGG

CAGTGGCAGC

AAAALACMAC

TGCACGGACI'

GGAGGCMGGG

CCGGMAGTTC

A

35 INFORMATION FOR SEQ ID NO:17: SEQUENCE CHARACTERISTICS: LENGTH: 431 amino acids TYPE: amino acid 40 TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: Met His Val Arg Ser Leu Arg Ala Ala Ala 1 5 10 Pro His Ser Phe Val Ala Leu Trp Ala Pro Leu Phe Leu Leu Arg Ser Ala Leu Ala Asp Phe Ser 20 25 Leu Asp Asn Giu Val His Ser Ser Phe Ile His Arg Arg Leu Arg Ser 40 Gin Glu Arg Arg Glu Met Gin Arg Giu Ile Leu Ser Ile Leu Gly Leu s0 55 105 Pro His Arg Pro Arg Pro His Leu Gin Gly His Asn Ser Ala Pro

C

CCC.

C. C C9

*C

Met Gly Thr Asp Giu 145 Ser Tyr Se r Phe Val 225 His Ile Lys Arg Lys 305 Asn Val Phe Pro Gin Al a 130 Phe Lys Lys Val Leu 210 Phe Asn Asn Gin Ser 290 Thr Ser Se r Met Gly Gly 115 Asp Phe Ile Asp Tyr 195 Leu Asp Leu Pro Pro 275 Ile Pro Ser Phe Leu Gly 100 Pro Met His Pro Tyr 180 Gin Asp Ile Giy Lys 260 Phe Arg Lys Ser Arg 340 Asp Gin Pro Vai Pro Glu 165 Ile Vai Ser Thr Leu 245 Leu Met Ser Aksn Asp 325 Asp Leu Gly Leu Met Arg 150 Giy Arg Leu Arg Aia 230 Gin Aia Val Thr Gin 310 Gin Leu Tyr Phe Ala Se r 135 Tyr Giu Giu Gin Thr 215 Thr Leu Giy Ala Gly 295 Giu Arg Gly Asn Ser Ser 120 Phe His Ala Arg Giu 200 Leu Ser Ser Leu Phe 280 Ser Ala Gin Trp Ala Tyr 105 Leu Val1 His Vai Phe 185 His Trp Asn Vai Ile 265 Phe Lys Leu Ala Gin 345 Met 90 Pro Gin Asn Arg Thr 170 Asp Leu Ala His Giu 250 Gly Lys Gin Arg Cys 330 Asp Ala Val Giu Glu Tyr Lys Ala Val Phe Ser Asp Le u Giu 155 Ala Asn Gly Ser Trp 235 Thr Arg Ala Arg Met 315 Lys Trp Ser Val 140 Phe Ala Glu Arg Giu 220 Val Leu His Thr Se r 300 Ala Lys Ile His 125 Giu Arg Glu Thr Giu 205 Glu Val Asp Gly Giu 285 Gin Asn His Ile 110 Phe His Ph e Phe Phe 190 Ser Gly Asn Gly Pro 270 Vai Asn Vai Giu Ala 350 Leu Asp Asp Arg 175 Arg Asp Trp Pro Gin 255 Gin His Arg Ala Leu 335 Pro Thr Lys Le u 160 Ile Ile Leu Leu Arg 240 Se r Asn Phe Ser Giu 320 Tyr Glu Gly Gly 0 -106 Gly Tyr Ala Ala Tyr Tyr Cys Glu Gly Glu Cys Ala Phe Pro Leu Asn 355 360 365 Ser Tyr Met Asn Ala Thr Asn His Ala Ile Val Gln Thr Leu Val His 370 375 380 Phe Ile Asn Pro Glu Thr Val Pro Lys Pro Cys Cys Ala Pro Thr Gln 385 390 395 400 Leu Asn Ala Ile Ser Val Leu Tyr Phe Asp Asp Ser Ser Asn Val Ile 405 410 415 Leu Lys Lys Tyr Arg Asn Met Val Val Arg Ala Cys Gly Cys His 420 425 430 INFORMATION FOR SEQ ID NO:18: SEQUENCE CHARACTERISTICS: LENGTH: 1873 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: KURIDAE TISSUE TYPE: EMBRYO

FEATURE:

35* NAME/KEY: CDS LOCATION: 104. .1393 OTHER INFORMATION: /function= "OSTEOGENIC PROTEIN" /product= "HOW1 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18: CTGCAGCAAG TGACCTCGGG TCGTGGACCG CTGCCCTGCC CCCTCCGCTG, CCACCTGGGG 045 CGGCGCGGGC CCGGTGCCCC GGATCGCGCG TAGAGCCGGC GCG ATG CAC GTG CGC 115 Met His Val Arg 1 *TCG CTG CGC GCT GCG GCG CCA CAC AGC TTC GTG GCG CTC TGG GCG CCT 163 Ser Leu Arg Ala Ala Ala Pro His Ser Phe Val Ala Leu Trp Ala Pro .5 10 15 CTG TTC TTG CTG CGC TCC GCC CTG GCC GAT TTC AGC CTG GAC AAC GAG 211 Leu Phe Leu Leu Arg Ser Ala Leu Ala Asp Phe Ser Leu Asp Asn Glu 25 30 107 GTG CAC TCC AGC TTC ATC CAC CGG Val His Ser Ser Phe Ile His Arg CTC CGC AGC CAG Leu Arg Ser Gin GAG CCC CGC Ciu Arg Arg GAG ATG CAG Glu Met Gin CCC GAG ATC CTC TCC ATC TTA GGG TTG CCC CAT CGC CCG Arg Giu Ile Leu Ser Ile Leu Gly Leu Pro His Arg Pro 60 CGC CCG Arg Pro CAC CTC CAG GCA His Leu Gin Gly TAC MAC CCC ATG Tyr Asn Aia Met CAT MAT TCG GC His Asn Ser Aia ATG TTC ATC TTC Met Phe Met Leu

CTG

Leu C GTG GAG GAG AGC CCC CCC CAC GGA Aia Vai Ciu Giu Ser Giy Pro Asp Ciy CCC TTC TCC TAC Giy Phe Ser Tyr TAC M.G CCC GTC Tyr Lys Ala Vai ACT ACC CAC CCC CCC CCT Ser Thr Gin Giy Pro Pro 115 TTA CC Leu Ala ATG AG C Met Ser CGA TAC Arg Tyr 150

AC

Se r CAG GAC AGC CAT Gin Asp Ser His CTC ACT GAC CC Leu Thr Asp Aia GAC ATG GTC Asp Met Val 130 9* S. S S. S S

S.

TTC GTC MAC CTA Phe Vai Asn Leu 135 CAC CAT CCC GAG His His Arg Giu

GTG

Val CAT CAC MAA CMA TTC TIC CAC CCT His Asp Lys Ciu Phe Phe His Pro TTC CCC TTT CAT CTT TCC MCG ATC CCC GAG Phe Arg Phe Asp Leu Ser Lys Ile Pro Giu 155 160

GCC

Ciy 165 GMA CCC GTG ACC Ciu Arg Val Thr CCC GAA TTC AG Ala Ciu Phe Arg TAT MCG GAC TAC ATC Tyr Lys Asp Tyr Ile 180 CGG GAG CGA TTT Arg Giu Arg Phe GAC AAC Asp Asn 185 GAG ACC TTC Ciu Thr Phe ATC ACA GTC TAT Ile Thr Vai Tyr CAC CTG Gin Vai 195 CTC CAG Leu Gin CCC ACC Arg Thr GCC ACC Ala Thr 230

GAG

Giu TCA CCC AGG GAG Ser Giy Arg Glu GAC CTC TTC TTG, Asp Leu Phe Leu CTG GAC AGC Leu Asp Ser 210 CAT ATC ACA Asp Ile Thr Si

S

S

S.

S.

*S~

ATC TGG CCT TCT GAG Ile Trp Aia Ser Giu 215 AGC MAC CAC TCC GTG Ser Asn His Trp Val 235 CCC TCC TIC CTG Ciy Trp Leu Val CTC MAC CCT CGG Val Asn Pro Arg MAC CTC CCC TIA Asn Leu Giy Leu CTC TCT GTG GAG Leu Ser Vai Giu ACC CTG Thr Leu 250 CAT GGG CAG Asp Giy Gin ATC MAC CCC MAG Ile Asn Pro Lys 108 GCA GGC CTG ATT Ala Gly Leu Ile CGG CAT GGA CCC Arg His Giy Pro CAG MAC MKG Gin Asn Lys 270 CAT CTC CGT His Leu Arg CMA CCC TTC ATG Gin Pro Phe Met 275 AGT AIC CGG TCC Ser Ile Arg Ser 290 GTG CCC TTC Val Ala Phe MAG CCC ACG GAA Lys Aia Thr Ciu ACG COG COC MAG CAG CGC AGC CAG MNT CCC TCC MAG ACG CCA MAG M.C Thr Gly Cly Lys Gin Arg Ser Gin Asn Arg Ser Lys Thr Pro Lys Asn 295 300 305 1027 CMA GAG Gin Giu 310 CCC CTG AGG Ala Leu Arg AIG CC Met Ala 315 MAG A LYS LYS 330 ACT GTG GCA Ser Val Ala CAT GAG CTG His Clii Leu GMA MC Clu Asn 320 TAC GTC Tyr Val 335 AGC AGC ACT GAC Ser Ser Ser Asp AGO CAG CCC TGC Arg Gin Ala Cys AGC TIC CGA Ser Phe Arg CTT GGC TGO CAG Leu Gly Trp Gin TCG ATC ATT GCA Trp Ile Ile Ala CCT GMA GGC TAT Pro Ciu Gly Tyr 350 CTG MAC TCC TAC Leu Asn Ser Tyr

S

55 5 I S *5 55@S S S. 5 S. S S

S.

S.

5.5S

S

655.

S S

S

TAC TGT GAG Tyr Cys Ciu ACC MAC CAC Thr Asn His 375 GAG TCC CCC TIC Glu Cys Ala Phe GCT CCC TAC Ala Ala Tyr 355 ATG MAC CC Met Asn Ala 370 MAC CCA GAC Asn Pro Asp CCC ATC GTC CAG Ala Ile Val Gin CTG GTT CAC TIC Leu Vai His Phe ACA CIA Thr Val 390 CCC MAG CCC TGC Pro Lys Pro Cys CC CCC ACC CAG Ala Pro Thr Gin MAC CCC ATC TCT Asn Ala Ile Ser 1075 1123 1171 1219 1267 1315 1363 1413 1473 1533 1593 1653 1713 CTC TAC TIC GAC Leu Tyr Phe Asp AGC TCT MAT GTC Ser Ser Asn Val GAC CTC MAG MAG TAC AGA Asp Leu Lys Lys Tyr Arg 415 420 TAGCTCTTCC TGAGACCCTG MAC ATG CTG GTC Asn Met Val Val CCC TGT GGC Ala Cys Cly TCC CAC Cys His 430 *5 S S

S

Sb S *5 *5 ACCTTTGCGG COCCACACCT TICCAMITCT TCGATGTCTC ACCATCTMAG TCTCTCACTG CCCACCTT GG CGAGGAGMAC AGACCMACCT CTCCTGAGCC TTCCCTCACC TCCCMACCGG MAGCATGTMA CGGTTCCAGA MACCTGAGCG TGCAGCAGCT CATCAGCGCC CTTTCCTTCT GGCACCIGAC GGACMAGATC CTACCAGCTA CCACAGCAMA CGCCTMGAG CAGGAAAAAT GTCTGCCAGG AAAGTGTCCA GTGTCCACAT GGCCCCTGGC CCTCTGAGTC TTTGAGGAGT 109 AATCGCAAGC CTCGTTCAGC TGCAGCAGAA GG AGGGCTI' AGCCAGGGTG GCCGCTGGCG TCTGTGTTGA ACGGAAACCA AGCAGAAGCC ACTGTAATGA TATGTCACAA TAAAACCCAT GAATGAAAAA AAAAAAAAAA AAAAAAAAAA AAAAGAATTC INFORMATION FOR SEQ ID NO:19: SEQUENCE CHARACTERISTICS: LENGTH: 430 amino acids TYPE: amino acid TOPOLOGY: linear 1773 1833 1873 (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: Met His Val Arg Se 9 a. a 9 a Leu Leu Gin Pro 65 Met Pro Gin Ala Phe 145 Lys Lys r Leu Arg u Phe Leu J1 His Ser .u Met Gin -g Pro His 70 p Leu Tyr .y Phe Ser !u Ala Ser !t Ser Phe 135 rg Tyr His 150 Ly Glu Arg rg Glu Arg Arg Phe Glu Gin Ala Pro 105 Gin Asn Arg Thr Asp 185 Ala Ala Ala Pro His Ser Phe Val Ala 10 Ser Ala Ile His Ile Leu Gly Lys Met Ala Tyr Lys Asp Ser Leu Val Giu Phe 155 Ala Ala 170 Asn Glu Val Tyr Gin 195 Val Leu Gin Giu His 200 Ser Gly Arg Glu Asp Leu Phe 110 Leu Leu Asp Ser Arg Thr Ile Trp Ala Ser Glu Glu Gly Trp Leu Val 210 215 220 Phe Asp Ile Thr Ala Thr Ser Asn His Trp Val Val Asn Pro Arg His 225 230 235 240 Asn Leu Gly Leu Gin Leu Ser Val Glu Thr Leu Asp Gly Gln Ser Ile 245 250 255 Asn Pro Lys Leu Ala Gly Leu Ile Gly Arg His Gly Pro Gln Asn Lys 260 265 270 Gin Pro Phe Met Val Ala Phe Phe Lys Ala Thr Glu Val His Leu Arg 275 280 285 Ser Ile Arg Ser Thr Gly Gly Lys Gin Arg Ser Gin Asn Arg Ser Lys 290 295 300 Thr Pro Lys Asn Gin Glu Ala Leu Arg Met Ala Ser Val Ala Glu Asn 305 310 315 320 Ser Ser Ser Asp Gin Arg Gin Ala Cys Lys Lys His Glu Leu Tyr Val 325 330 335 Ser Phe Arg Asp Leu Gly Trp Gin Asp Trp Ile Ile Ala Pro Glu Gly 340 345 350 Tyr Ala Ala Tyr Tyr Cys Glu Gly Glu Cys Ala Phe Pro Leu Asn Ser 30 355 360 365 Tyr Met Asn Ala Thr Asn His Ala Ile Val Gin Thr Leu Val His Phe 370 375 380 *oo 35 Ile Asn Pro Asp Thr Val Pro Lys Pro Cys Cys Ala Pro Thr Gin Leu 390 395 400 Asn Ala Ile Ser Val Leu Tyr Phe Asp Asp Ser Ser Asn Val Asp Leu 405 410 415 Lys Lys Tyr Arg Asn Met Val Val Arg Ala Cys Gly Cys His 420 425 430 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 1723 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens TISSUE TYPE: HIPPOCAMPUS ill (ix) FEATURE: NAME/KEY: CDS LOCATION: 490.-.1696 OTHER INFORMjATION: /function= "OSTEOGENIC

PROTEIN"

/product= "hOP2-PP" /note= "hOP2 (cDNA)" (xi) SEQUENCE DESCRIPTION: SEQ ID GCCGCCGGCA GAGCAGCAGT GGGCTGCAGG GCTCCCTATG CCACACCGCA CCAAGCGGTG GCGGCCACAG CCGGACTGGC CCGCAGAGTA GCCCCGGCCCI GACAGGTGTC GCGCGCGCG CGCCCCGCCC CGCCGCCCGC AGGCCCTGGG TCGGCCGCGG CGGCCTGCC ATG ACC GCG Met Thr Ala

GGCTGCAGGA

AGTGCGCGGAG

GCTGCAGGAG

GGGTACGGCG

CGAGGCGGTG

GCTCCAGGGA

CGCCCGCCGA

AGCCGATGCG

GCTGTGGTTG,

ACGGCCCAGG

CTCGCCCATC

GCGACAGAGG

GCGTCCCGGT

CCGCGCCTGA

GCCCAGCCTC

CGCCCGCTGA

GAGCAGGAGG

AGGCGCTGGA

GCCCCTGCGC

CATTGGCCGA

CCTCTCCGTC

GCCGGCTGC

CTTGCCGTCG

GCGCCCCAGC

TGGCACGCCA

GCAACAGCTC

TGCTCGCACC

GAGTCCCAGT

CAGGAGCCAG

CCGCCCGTCC

GGCCGTCCCC

TGAGCGCCCC

CTC CCC GGC CCG CTC TOG CTC CTG GCC CTG Leu Pro Gly Pro Leu Trp Leu Leu Gly Leu 5 0* GCG CTA TG C CTG 0CC CCC GGC GCC CCC 0CC CTC CGA CCC CCG CCC Ala Leu Cys Ala Leu Gly Gly Gly Gly Pro Gly Leu Arg Pro Pro Pro 20 0CC TGT CCC CAG CGA CGT CTG GGC GCG CGC GAG CGC CG GAC GTG CAG Gly Cys Pro Gin Arg Arg Leu Gly Ala Arg Glu Arg Arg Asp Val Gln 35 40 CG GAG ATC CTG, GG GTG CTC GGG CTG CCT GGC COG CCC COG CCC CGC Arg Glu Ile Leu Ala Val Leu Gly Leu Pro Gly Arg Pro Arg Pro Arg 50 55 GG CCA CCC GCC GCC TCC CGG CIG CCC C TCC GG CCG CIC TTC ATG Ala Pro Pro Ala Ala Ser Arg Leu Pro Ala Ser Ala Pro Leu Phe Met 65 70 CTG GAC CTG TAC CAC GCC ATG GCC GGC GAC GAC GAC GAG GAC 0CC GCG Leu Asp Leu Tyr His Ala Met Ala Gly Asp Asp Asp Glu Asp Gly Ala 85 CCC GC GAG COG CCC CTC 0CC CGC GCC GAC CrC GTC ATG AGC TTC GTT Pro Ala Glu Arg Arg Leu Gly Arg Ala Asp Leu Val Met Ser Phe Val 100 105 112 M.C ATG GTG GAG CGA GAC Asn Met Val Giu Arg Asp 110 115 CGT GCC CTG GCC Arg Ala Leu Gly CAG GAG CCC CAT Gin Giu Pro His MAG GAG TIC CGC ITI GAC CTG ACC CAG ATC CCG GCC G GAG GCG GTC Lys Giu Phe Arg Phe Asp Leu Thr Gin Ile Pro Ala Gly Ciu Ala Val 130 135 140 912 ACA GCT C Thr Ala Ala MAC AGG ACC Asn Arg Thr 160 TIC CCC ATI TAC Phe Arg Ile Tyr GTG CCC AGC ATC CAC CTG CTC Val Pro Ser Ile His Leu Leu CTC CAC GTC AGC ATG TIC CAG GTG GCC Leu His Val Ser Met Phe Gin Val Val GAG CAG TCC Giu Gin Ser M.C AGG Asn Arg 175 GAG TCT GAC TIG Giu Ser Asp Leu Mir TIC CAT CT Phe Leu Asp Leu ACG CTC CGA GCT Thr Leu Arg Ala GGA CAC GAG CCC TCC CTG CTG CTC CAT GTC ACA GCA GCC ACT GAC TGC Cly Asp Ciu Gly Trp Leu Val Leu Asp Val Thr Ala Ala Ser Asp Cys 190 195 200 205 TCG TIC CTC MAG CGT CAC MCG CAC CTC CGA CTC CCC CTC TAT GTG GAG Trp Leu Leu Lys Arg His Lys Asp Leu Ciy Leu Arg Leu Tyr Val Ciu 210 215 220 a. a ACT GAG CAC Thr Clu Asp CMA CC CC Gin Arg Ala 240 CCC ACT CCC Ala Ser Pro 255 CAC ACC GIG CAT His Ser Val Asp CCC CTG CCC GCC Cly Leu Ala Cly CTG CTG CCI Leu Leu Gly 235 TIC TIC AG Phe Phe Arg 960 1008 1056 1104 1152 1200 1248 1296 1344 1392 1440 1488 CCA CCC TCC CAA Pro Arg Ser Gin CCT TIC CTG CTC Pro Phe Val Val a. a.

a ACT CCC ATC Ser Pro Ile ACC CCT CCC CCA Thr Pro Arg Ala ACC CCA CTC AGG Arg Pro Leu Arg ACC ACC CAG CCC MCG AAA Arg Arg Gin Pro Lys Lys 270 275 ACC MAC GAC CTG Ser Asn Giu Leu CAG CCC MAC CGA Gin Ala Asn Arg CCA CCC ATC TI Pro Cly Ile Phe GAC GTC CAC Asp Val His GCC ICC Cly Set 295 CAC CCC CCC CAC His Gly Arg Gin GTC TC Val Cys 300 CCI CCC CAC Arg Arg His TGG GTC ATC Trp Val Ile 320 CTC TAC GTC AC Leu Tyr Val Ser CAG GAC CTC GCC Gin Asp Leu Gly TCC CTG GAC Irp Leu Asp 315 GAG CCC GAG Ciu Cly Ciu CCI CCC CMA GCC Ala Pro Gin Cly TCC CCC TAT TAC Set Ala Tyr Tyr 113 a a.

TGC TCC TrC CCA CTG GAG TCC TGC Cys Ser Phe Pro Let' Asp Ser Cys 335 340 CTG GAG TCC CTG GIG CAC CTG ATG Leu Gin Ser Leu Val His Leu Met 350 355 TGC TGT GCA CCC ACC AAG CTG AGC Cys Cys Ala Pro Thr Lys Leu Ser 370 AGC AGC AAC MAC GTC ATC CTG GGC Ser Ser Asn Asn Val Ile Let' Arg 385 GCC TGC GOC TGC CAC T GAGTCAGC( Ala Cys Gly Cys His 400 INFORMATION FOR SEQ ID NO:21: SEQUENCE CHARACTERISTICS: LENGTH: 402 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID1 Met Thr Ala Leu Pro Gly Pro Leu 1 14 5 Ala Leu Gly Gly Gly Gly Pro Gly Gin Arg Arg Leu Gly Ala Arg Giu 40 Leu Ala Val Leu Gly Leu Pro Gly 50 55 Ala Ala Ser Arg Leu Pro Ala Ser 70 Tyr His Ala Met Ala Gly Asp Asp Arg Arg Leu Gly Arg Ala Asp Leu 100 Glu Arg Asp Arg Ala Leu Gly His 115 120 ATG MAT GCC Met Asn Ala MAG CCA MAC Lys Pro Asn 360 GCC ACC TCT Ala Thr Ser 375 AAA GCC CGC Lys Ala Arg 390

CGCCCAGCCCT

ACC AAC Thr Asn 345 GCA GTG Ala Val GTG CTC Val Let' AAC ATG Asn Met

ACTGCAG

GAG GCC ATC His Ala Ile CCC MAG GCG Pro Lys Ala 365 TAG TAT GAG Tyr Tyr Asp 380 GTG GIG MAG Val Val Lys 395 1536 1584 1632 1680 1723 NO: 2 1: Trp Let' Leu 10 Leu Arg Pro 25 Arg Arg Asp Arg Pro Arg Ala Pro Leu 75 Asp Glu Asp 90 Val Met Ser 105 Gin Glu Pro Gly Leu Ala Leu Cys 15 Pro Pro Gly Cys Pro Val Gin Arg Giu Ile Pro Arg Ala Pro Pro Phe Met Leu Asp Leu Gly Ala Pro Ala Giu Phe Val Asn Met Val 110 His Trp Lys Glu Phe 125 114

U

S

Arg Glu 145 Leu Ser Gly Lys Gly 225 Pro 25 Ser Pro 30 Phe 35 Glu 305 Ala 40 Pro Leu 45 Pro Asn 385 Cys Phe 130 Phe His Asp Trp Arg 210 His Arg Pro Lys Asp 290 Leu Pro Leu Val Thr 370 Val His As p Arg Val Leu Leu 195 His Ser Ser Ile Lys 275 Asp Tyr Gin Asp His 355 Lys Leu Ile Ser Phe 180 Val Lys Val Gin Arg 260 Ser Val Val Gly Se r 340 Leu Leu Thr Tyr Met 165 Phe Leu Asp Asp Gin 245 Thr Asn His Ser Tyr 325 Cys Met Ser Gin Lys 150 Phe Leu Asp Leu Pro 230 Pro Pro Giu Gly Phe 310 Ser Met Lys Ala Lys 390 Ile 135 Val Gln As p Val Giy 215 Gly Phe Arg Leu Ser 295 Gin Ala Asn Pro Thr 375 Pro Pro Val Leu Th r 200 Leu Leu Vai Ala Pro 280 His Asp Tyr Ala Asn 360 Ser Ala Se r Val Gin 185 Al a Arg Al a Val Val 265 Gin Gly Leu Tyr Thr 345 Ala Val Gly Ile Gin 170 Thr Ala Leu Gly Thr 250 Arg Ala Arg Gly Cys 330 Asn Vai Leu Glu His 155 Giu Leu Ser Tyr Leu 235 Phe Pro Asn Gin Trp 315 Glu His Pro Tyr Val 395 Ala 140 Leu Gin Arg Asp Vai 220 Leu Phe Leu Arg Val 300 Leu Gly Ala Lys Tyr 380 Val Leu Ser Ala Cys 205 Giu Gly Arg Arg Leu 285 Cys Asp Giu Ile Ala 365 Asp Thr Asn Asn Giy 190 Trp Thr Gin Ala Arg 270 Pro Arg Trp Cys Leu 350 Cys Ser Ala Arg Arg 175 Asp Leu Glu Arg Se r 255 Arg Gly Arg Val Ser 335 Gin Cys Ser Al a Th r 160 Glu Giu Le u Asp Ala 240 Pro Gin Ile His Ile 320 Phe Ser Ala Asn Gly 400 Ile Leu Arg Ala Arg Asn Met Val Lys Ala Cys 115 INFORMATION FOR SEQ ID NO:22: SEQUENCE CHARACTERISTICS: LENGTH: 1926 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (vi) ORIGINAL SOURCE: ORGANISM: HURIDAE TISSUE TYPE: EMBRYO (ix) FEATURE: NAME/KEY: CDS LOCATION: 93.-.1289 OTHER INFORMATION: /function= "OSTEOGENIC PROTEIN" /product= "wOP2-PP" /note= "mOP2 cDNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: GCCAGGCACA GGTGCGCCGT CTGGTCCTCC CCGTCTGGCG TCAGCCGAGC CCCACCAGCT ACCAGTGGAT GCGCGCCGGC TGAAAGTCCG, AG ATG GCC ATG CCI Met Ala Met Arg 1 CCC GGG CCA 113 Pro Gly Pro

S

**SS

CTC TGG CIA Leu Trp Leu TTG GGC CTT GCT Leu Gly Leu Ala ICC GCG CIG GGA Cys Ala Leu Gly GGC CAC GGT Cly His Gly CCG CGT Pro Arg CGC CGC Arg Arg 40 CCC CCG CAC ACC Pro Pro His Thr GAC ATG CAG CCI Asp Met Gin Arg CCC CAG CCI CC Pro Gin Arg Arg GGA GCG CCC GAG Gly Ala Arg Giu GAA AIC CIG GCG GTC CTC CCC CTA CCG Giu Ile Leu Ala Val Leu Gly Leu Pro CCC CCC CGA CCC Arg Pro Arg Pro CGT GCA Arg Ala CAA CCC CC Gin Pro Ala CCC CCC CAG CCA Ala Arg Gin Pro C ICC Ala Ser

S

*S *S S *S

SS

*S

C CCC Ala Pro CAC GCC Asp Gly AGC TTC Ser Phe 105

C

Leu ATG TTC GCd CIA Met Leu Asp Leu CAC CCC ATG ACC His Ala Met Thr CAT GAd GAd Asp Asp Asp CTG GIC AIC Leu Val Met CCC, CCA CCA CAG GCI Gly Pro Pro Gin Ala CIC MAC ATG GIG GMA Val Asn Met Val Glu 110 TTA GCC CGI CC Leu Gly Arg Ala CCC GAC CGT ACC CTG Arg Asp Arg Ihr Leu 115 GGC TAC CAC GAG Gly Tyr Gin Glu 116 CAC TGG MAG GMA TIC CAC T17 GAC His Irp Lys Glu Phe His Phe Asp CIA ACC CAG ATC CCT Leu Ihr Gin Ile Pro GCI GGG Ala Giy GAG GCT GTC ACA Giu Ala Val Thr GCC GAG TIC CGG Ala Giu Phe Arg ATC TAC AAA GMA CCC AGC ACC Ile Tyr Lys Giu Pro Ser Thr 145 150 CAC CCG CTC His Pro Leu GAG CAC TCC Giu His Ser 170 ACA ACC CIC Thr Thr Leu CAC ATC His Ile 160 AGC ATG TIC CMA Ser Met Phe Giu GIG GTC CAA Val Val Gin 165 CTT CAG ACG Leu Gin Thr MAC AGG GAG TCT Asn Arg Giu Ser TTG TIC TTT TIC Leu Phe Phe Leu CTC CGA Leu Arg 185 TCI COG GAC GAG Ser Ciy Asp Giu ICC CTG GIG CIG Trp Leu Vai Leu ATC ACA GCA GCC 689 Ile Thr Ala Ala ACT GAC CGA TGG CTG CTG MAC CAT CAC Ser Asp Arg Trp Leu Leu Asn His His 200 205 MAG GAC Lys Asp 210 ATC GAT Met Asp 225 CIC GGA CIC CC Leu Giy Leu Arg

S

TAT GIG GMA ACC Tyr Val Ciu Thr GAT CCC CAC AC Asp Ciy His Ser CCT CCC CTC Pro Gly Leu C GGT Ala Gly 230 CIG CTI GGA Leu Leu Gly TIC TIC AG Phe Phe Arg 250 CAA GCA CCA Gin Ala Pro CCC TCC Arg Ser 240 CCT CTC Pro Val 255 AGA GAG CCT TIC Arg Gin Pro Phe ATG CIA ACC Het Val Thr 245 CCA CC AGA Ala Ala Arg CCC AGC CAC ACT Ala Ser Gin Ser

CGC

Arg CCC CCT Ala Pro MAC GAG Asn Giu 275 CCA CTG Pro Leu 265 MAG ACG AGG GAG Lys Arg Arg Gin CCA MAG MAA ACG Pro Lys Lys Ihr 270 Ti-r GAT CAT GCC Phe Asp Asp Gly CTT CCC CAC CCC Leu Pro His Pro MAA CIC CCA GGG Lys Leu Pro Gly CAC CCI ICC CCC CCC AGA His Giy Ser Arg Gly Arg 290 295 GAG GTT TGC CC Giu Val Cys Arg CAT GAG CTC TAC His Glu Leu Tyr AGC TIC CCI GAC Ser Phe Arg Asp CTT GCC Leu Gly 310 b S. 977 1025 1073 1121 ICC GIG GAO Trp Leu Asp GAG CCC GAG Giu Gly Ciu 330 GIG AIC CC Val Ile Ala CCC GAG Pro Gin 320 CCC TAC ICI CCC TAT TAC TCI Cly Tyr Ser Ala Tyr Tyr Cys 325 IGI GCT TIC CGA Cys Ala Phe Pro GAO ICC IGT AIG Asp Ser Cys Met MAC CCC ACC M-C Asn Ala Ihr Asn 340 117 CAT GCC ATC TTG CAG TCT CTG GTG CAC CTG ATO MAG CCA GAT GTT CTC His Ala Ile Leu Gin Ser Leu Val His Leu Met Lys Pro Asp Val Val 345 350 355 CCC MAG GCA TGC TGT GCA CCC ACC AAA CTG AGT GCC ACC TCT GTG CTG Pro Lys Ala Cys Cys Ala Pro Thr Lys Leu Ser Ala Thr Ser Val Leu 360 365 370 375 TAC TAT GAC AGC AGC MAC MAT GTC ATC CIG CGT MAA CAC CGT AAC ATG Tyr Tyr Asp Ser Ser Asn Asn Val Ile Leu Arg Lys His Arg Asn Met 380 385 390 GTG CTC AAG GCC TGT CCC TGC CAC TGAGGCCCCG CCCACCATCC TGCTTCTACT Val Val Lys Ala Cys Cly Cys His 395

C

C S C. .C CC

C.

C C ACCTTACCAT CTCGCCGGC CAGACACGGG CMATGCGAGG CTTTCCCAGT TCCTCTGTCC TCCTACCCCA ACCATAGACT CTGGGGTCAG CACTCMAGGC MTGGCAAAT TCTCGATGGT CTCTGCACCA TTCATYGTGG GATCMATGCA TCGCTGTACT CCAGGTATAG CGGTGCATGT CTGTGAGTTC MAGGCCACAT

CCCTCTCCAG

CCCTTCACTT

TTCATGGGGT

GA.ATGCACAC

CCACATGAGG

CTMAGAAGGC

CAGTTGGGAC

CCTTCAAATC

CATTAATCCC

AGAAACAGCC

ACCCAGAAAC

CCCCTGGCCA

TTCGGGGCTA

ACCATCCCAG

AAGACTGATC

CCTGGAATTC

A YTAGGT

AGAGCTAGCT

AGCGCTAAAG

TGTCTCCGGA

CCTTCTATCT

CMCCTGCTA

TCACCCCGCC

ACCTATGC TA

CTTGGCCATC

TMACTACAT

ATAACAGACA

TCTTAGAAAA

AGACAGAGAC

GCAGGAAAAA

TATCATAGCT

AAATTCTGGT

CTCTCCATCC

ACTGAGAGGT

CTCAGCCCAC

GATCTGGGCT

CATACACTTA

ACMATCAGAG

AGGAGAATCT

AAAAAAAAAC

1169 1217 1265 1319 1379 1439 1499 1559 1619 1679 1739 1799 1859 1919 1926

GGMATTC

INFORMATION FOR SEQ ID NO:23: SEQUENCE CHARACTERISTICS: LENGTH: 399 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: Met Ala Met Arg Pro Gly Pro Leu Trp Leu Leu Cly Leu Ala Leu Cys 1 5 10 Ala Leu Gly Gly Gly His Gly Pro Arg Pro Pro His Thr Cys Pro Gin 25 118 0 0 Arg Arg Ala Val Ala Ala His Ala Gly Arg Arg Thr Leu Thr 130 Ile Tyr 145 25 Ser Met Phe Phe 30 Val Leu 35 Lys Asp 210 Met Asp 225 40 Arg Gin Arg Ala 45 Thr Asn Gly His 290 Leu Leu Arg Met Ala Leu 115 Gin Lys Phe Leu Asp 195 Leu Pro Pro Pro Glu 275 Gly Giy Gly Gin Thr Asp 100 Giy Ile Giu Giu Asp 180 Ile Gly Gly Phe Arg 260- Leu Ser Al a Leu Pro Asp Leu Tyr Pro Pro Val 165 Leu Thr Leu Leu Met 245 Al a Pro Arg Arg Pro Ala 70 Asp Vai Gin Ala Ser 150 Val Gin Ala Arg Ala 230 Val Ala His Gly Glu Giy Se r Asp Met Giu Gly 135 Thr Gin Thr Ala Leu 215 Giy Thr Arg Pro Arg 295 Arg 40 Arg Al a Asp Ser Pro 120 Glu His Giu LeLI Ser 200 Tyr Leu Phe Pro Asn 280 Glu Arg Pro Pro Gly Phe 105 His Ala Pro His Arg 185 Asp Val Leu Phe Leu 265 Lys Val Asp Arg Leu Gly 90 Vai Trp Val Leu Ser 170 Se r Arg Giu Giy Arg 250 Lys Leu Cys Met Pro Phe 75 Pro Asn Lys Thr Asn 155 Asn Gly Trp Thr Arg 235 Ala Arg Pro Arg Trp 315 Arg Ala Leu Gin Val Phe 125 Ala Thr Giu Giu Leu 205 Asp Ala Gin Gin Ile 285 His Glu Gin Asp Ala Giu 110 His Giu Leu Ser Gly 190 Asn Gly Pro Ser Pro 270 Phe Glu Ile Pro Leu His Arg Phe Phe His Asp 175 Trp His His Arg Pro 255 Lys Asp Leu Leu Ala Tyr Leu Asp Asp Arg Ile 160 Leu Leu His Ser Ser 240 Val Lys Asp Tyr Val 305 Ser Phe Arg Asp Leu Gly Trp, Leu Asp 310 Vai Ile Ala Pro Gin 320 119 S S *St* S S S. S S. S

S

*CS

S S S. S

S

S.

S

*5 S 5* .5 Gly Tyr Ser Ala Tyr Tyr Cys Glu Gly Glu 325 330 Ser Cys Met Asn Ala Thr Asn His Ala Ile 340 345 Leu Met Lys Pro Asp Val Val Pro Lys Ala 355 360 Leu Ser Ala Thr Ser Val Leu Tyr Tyr Asp 370 375 Leu Arg Lys His Arg Asn Met Val Val Lys 385 390 INFORMATION FOR SEQ ID NO:24: SEQUENCE CHARACTERISTICS: LENGTH: 1368 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 1- 1368 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: ATG TCG GGA CTG CGA MAC ACC TCG GAG GCC Met Ser Gly Leu Arg Asn Thr Ser Glu Ala 35 1 5 10 CTG GG.A CTC GGA ATG GTT CTG CTC ATG TIC Leu Gly Leu Gly Met Val Leu Leu Met Phe 20 25 GCC GTT GAG GCC ACC CAG TCG GGG, ATT TAC Ala Val Glu Ala Thr Gin Ser Gly Ile Tyr 35 45 CAG ACG ATC ATG CAC AGA GTG CTG AGC GAG Gln Thr Ile Met His Arg Val Leu Ser Giu 50 55 TCG TAC GAG ATC CTC GAG TTC CTG GGC ATC 50 Ser Tyr Glu Ile Leu Glu Phe Leu Gly Ile 70 CTG AGC AGC CAC CAG TTG TCG, CTJG AGG MAG Leu Ser Ser His Gin Leu Ser Leu Arg Lys 85 90 Cys Ala Phe Pro Leu Asp 335 Leu Gin Ser Leu Val His 350 Cys Cys Ala Pro Thr Lys 365 Ser Ser Asn Asn Val Ile 380 Ala Cys Gly Cys His 395 GTT GCA Val Ala GTG GCG Val Ala ATA GAC Ile Asp GAC GAC Asp Asp GCC GMA Ala Giu TCG GCT Ser Ala CTC GCC Leu Ala ACO CCG Thr Pro GGC MAG Gly Lys CTG GAC Leu Asp CCG ACG Pro Thr MAG TTC Lys Phe 120 CIG GAC GTC Leu Asp Val CAT GAG CAC Asp Giu Asp 115 CAC CGC ATC ACG His Arg Ile Thr GAG GAG CCT CTC Giu Giu Giy Leu AGC GAT CAG Ser Asp Gin 110 AGG AGC GCC Arg Ser Ala GAC GAC TAC CMA Asp Asp Tyr Giu CCC CAT CCC TCC Gly His Arg Ser GAC CTC Asp Leu 130 GAG GAG GAT GAG Ciu Giu Asp Giu GAG CAG CAC MCG Giu Gin Gin Lys TTC ATC ACC GAC Phe Ile Thr Asp CTC GAC MAG CCC CCC ATC CAC GAG ACC GAC ATC Leu Asp Lys Arg Ala Ile Asp Giu Ser Asp Ile 145 150 155 ATC ATG ACC TTC Ile Met Thr Phe MAC MAG CCC CAC Asn Lys Arg His MAT GTG GAC CMA Asn Vai Asp Giu CGT CAC GAG CAC Arg His Ciu His GGC CCT Gly Arg 175 CCC CTG Arg Leu ATG CC Met Aia ACC CC Thr Aia 210 TCC TTC GAC GTC Trp Phe Asp Vai 180 GAG CTG CGC ATC Clu Leu Arg Ile 195 MAC AGG GAG TTC Asn Arg Ciu Phe TCC AAC Ser Asn TAT CAG Tyr Gin 200 CCC MAC GAC MAC Pro Asn Asp Asn TAC CTG GTC Tyr Leu Val 190 MCG TGC CTG Lys Trp Leu MAC CCC MAC GAG Asn Ala Asn Ciu

S

S

S

*5 S S *5 S

S.

09

S..S

5 ATC ACG GTA TAC Ile Thr Vai Tyr ATT CCC ACC GCC Ile Giy Thr Cly CTG CCC CAC CAC Leu Gly Gin His ATG GAG CCC CTC Met Ciu Pro Leu TCC GTG MAC ACC Ser Val Asn Thr CCC GAC TAC Ciy Asp Tyr GAG TGG CTC Giu Trp Leu CAC.GCT GTC His Aia Val 275 GTG CCC TCG TTG GAG CTC M.C GTG ACC GAG CCC CTG CAC Vai Ciy Trp Leu Ciu Leu Asn Vai Thr Giu Gly Leu His 245 250 255 GTC MCG TCG MCG GAC MAT CAT CCC ATC TAC ATT GGA GCA Vai Lys Ser Lys Asp Asn His Giy Ile Tyr Ile Gly Ala 260 265 270 MAC CGA CCC Asn Arg Pro CCC MCG GTG Arg Lys Vai 4* S S

S

S. 0* CTG ATC Leu Ile 290

CAC

His CAC CC Asp Arg 280 GAC GAC Asp Asp 295 GAG CTC MCG CTC Giu Val Lys Leu CAC ATT CCA Asp Ile Ciy GAG TTC CAG Giu Phe Gin TIC ATG ATC GC Phe Met Ile Giy 768 816 864 912 960 TTC CCC GGA CCG Phe Arg Gly Pro GAG CTG ATC MCG C ACG Giu Leu Ile Lys Ala Thr 310 315 CCC CAC AGC AC Ala His Ser Ser 121 CAC AGO AGC His Arg Ser MAG CGA Lys Arg 325 AGC GCC AGC CAT Ser Ala Ser His CCA CCC MAG CGC Pro Arg Lys Arg 330 GMA CCG ATG GAG Glu Pro Met Glu GTG TCG CCC MAC MAC Val Ser Pro Asn Asn 340 GTG CCC CTG Val Pro Leu MAG MAG TCG Lys Lys Ser 335 AGC ACO CGC Ser Thr Arg 350 CTG CCC TGG Leu Gly Trp AGC TGC CAG ATG CAG ACC Ser Cys Gin 355 CAT GAC TGG His Asp Trp 370 Met Gin Thr ATC ATC GCA Ile Ile Ala CTG TAC Leu Tyr 360 CCA GAG Pro Glu 375 ATA GAC TTC MAG Ile Asp Phe Lys CCC TAT GGC Gly Tyr Gly TTC TAC TGC AGC Phe Tyr Cys Ser 1008 1056 1104 1152 1200 1248 1296 1344 GAG TGC MAT TTC Glu Cys Asn Phe CTC MAT CG CAC Leu Asn Ala His MAC CCC ACG AAC Asn Ala Thr Asn GCG ATC GTC CAG Ala Ile Val Gin CTG GTC CAC CTG Leu Val His Leu GAG CCC CG MAG Glu Pro Lys Lys GTG CCC Val Pro 415 MCG CCC TGC Lys Pro Cys CAC CTG MAC His Leu Asn 435 GCT CCC ACC AGO Ala Pro Thr Arg GGA GCA CTA CCC Gly Ala Leu Pro GTT CTC TAC Val Leu Tyr 430 MAC ATG ATT Asn'Met Ile A A.

A

A

A A

A

GAC GAG MAT GTC Asp Glu Asn Val CTG AAA MCG Leu Lys Lys TAT AGA Tyr Arg 445 1368 GTG AAA Val Lys 450 TCC TGC COG TGC Ser Cyq Gly Cys CAT TGA His 455 INFORHATION. FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 455 amino acids TYPE: amino acid (12) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID 110:25: Met Ser Cly Leu Arg Asn Thr Ser Clu Ala Val Ala Val Leu Ala Ser 1 5 10 Leu Cly Leu Gly Met Val Leu Leu Met Phe Val Ala Thr Thr Pro Pro 25 122 Ala Val Glu Ala Thr Gin Ser U C U

U

U

U

Gln Ser Leu Leu Asp Asp Leu 145 Asn Arg Met Thr Thr 225 40 Gly Glu His Leu Phe 305 Thr Ile 50 Tyr Glu Ser Ser Asp Val Giu Asp 115 Leu Glu 130 Asp Lys Lys Arg Leu Trp Ala Glu 195 Ala Asn 210 Leu Gly Asp Tyr Trp Leu Ala Val 275 Ile His 290 Phe Arg Met Ile His Tyr 100 Asp Giu Arg His Phe 180 Leu Arg Gln Val Val 260 Asn Arg Gly His Leu Gin His Asp Asp Ala His 165 Asp Arg Glu His Gly 245 Lys Arg Lys Pro Arg Glu 70 Leu Arg Tyr Glu Ile 150 Asn Val Ile Phe Thr 230 Trp Ser Pro Val Glu 310 Val 55 Phe Ser Ile Glu Gly 135 Asp Val Ser Tyr Thr 215 Met Leu Lys Asp Asp 295 Leu Gly Ile Leu Ser Leu Gly Leu Arg Thr Ala 105 Arg Gly 120 Glu Gin Glu Ser Asp Glu Asn Val 185 Gin Asn 200 Ile Thr Giu Pro Giu Leu Asp Asn 265 Arg Glu 280 Asp Glu Ile Lys Ile Asp Ala 75 Ser Glu Arg Lys Ile 155 Arg Asn Asn Tyr Ser 235 Val Gly Lys Gin Thr 315 Asp Asp Glu Ala Gly Ser Asn 140 lie His Asp Glu Ala 220 Ser Thr Ile Leu Pro 300 Ala Asn Lys Arg Pro Leu Arg 125 Phe Met Glu Asn Gly 205 Ile Val Glu Tyr Asp 285 Phe His Gly Leu Pro Lys Ser 110 Arg Ile Thr His Tyr 190 Lys Gly Asn Gly lie 270 Asp Met Ser Lys Asp Thr Phe Asp Ser Thr Phe Gly 175 Leu Trp Thr Thr Leu 255 Gly Ile Ile Ser Asp Val His Leu Gin Ala Asp Leu 160 Arg Val Leu Gly Thr 240 His Ala Gly Gly His 320 His Arg Ser Lys Arg 325 Ser Ala Ser His Pro Arg Lys Arg Lys Lys Ser 335 123 Val Ser Pro Asn Asn Val Pro Leu Leu Glu Pro Me 340 345 Ser Cys Gin Met Gin Thr Leu Tyr Ile Asp Phe Ly 355 360 His Asp Trp Ile Ile Ala Pro Glu Gly Tyr Gly Al 370 375 38 Gly Glu Cys Asn Phe Pro Leu Asn Ala His Met As 385 390 395 Ala Ile Val Gin Thr Leu Val His Leu Leu Giu Pr 405 410 Lys Pro Cys Cys Ala Pro Thr Arg Leu Gly Ala Lei 420 425 His Leu Asn Asp Giu Asn Val Asn Leu Lys Lys Ty: 435 440 Val Lys Ser Cys Gly Cys His 450 455 INFORMATION FOR SEQ ID NO:26: SEQUENCE CHARACTERISTICS: LENGTH: 104 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear *35 (ii) MOLECULE TYPE: protein (ix) FEATURE: NAME/KEY: Protein LOCATION: 104 OTHER INFORMATION: /note= "BMP3" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: Cys Ala Arg Arg Tyr Leu Lys Val Asp Phe A 1 5 10 Trp Ile Ile Ser Pro Lys Ser Phe Asp A 20 25 Ala Cys Gin Phe Pro Met Pro Lys Ser Leu L 40 Thr Ile Gin Ser Ile Val Ala Arg Ala Val G 55 Giu Asp 365 Phe Ala Lys Pro Arg 445 Ser 350 Leu Tyr Thr Lys Vai 430 Asn Th r Giy Cys Asn Val 415 Leu Met Arg Trp Ser His 400 Pro Tyr Ile la Asp Ile Gly Trp Ser la Tyr Tyr Cys Ser Gly ys Pro Ser Asn His Ala ly Val Val Pro Gly Ile 124 Pro Glu Pro Cys Cys Val Pro Glu Lys Met Ser Ser Leu Ser Ile Leu 70 75 Phe Phe Asp Glu Asn Lys Asn Val Val Leu Lys Val Tyr Pro Asn Met 90 Thr Val Glu Ser Cys Ala Cys Arg 100 INFORMATION FOR SEQ ID NO:27: SEQUENCE

CHARACTERISTICS:

LENGTH: 102 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE: ORGANISM: HOMO SAPIENS (ix) FEATURE: NAME/KEY: Protein LOCATION: 1..102 OTHER INFORMATION: /note= 30 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: Cys Lys Lys His Glu Leu Tyr Val Ser Phe Arg Asp Leu Gly Trp Gin 1 5 10 35 Asp Trp Ile Ile Ala Pro Glu Gly Tyr Ala Ala Phe Tyr Cys Asp Gly 25 Glu Cys Ser Phe Pro Leu Asn Ala His Met Asn Ala Thr Asn His Ala S* 35 40 Ile Val Gin Thr Leu Val His Leu Met Phe Pro Asp His Val Pro Lys 55 Pro Cys Cys Ala Pro Thr Lys Leu Asn Ala Ile Ser Val Leu Tyr Phe 65 70 75 Asp Asp Ser Ser Asn Val Ile Leu Lys Lys Tyr Arg Asn Met Val Val 90 Arg Ser Cys Gly Cys His 100 125 INFORMATION FOR SEQ ID NO:28: SEQUENCE

CHARACTERISTICS:

LENGTH: 102 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE: ORGANISM: HOMO SAPIENS (ix) FEATURE: NAME/KEY: Protein LOCATION: 1..102 OTHER INFORMATION: /note= "BMP6" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: Cys Arg Lys His Glu Leu Tyr Val Ser Phe Gin Asp Leu Gly Trp Gin 1 5 10 Asp Trp Ile Ile Ala Pro Lys Gly Tyr Ala Ala Asn Tyr Cys Asp Gly 25 Glu Cys Ser Phe Pro Leu Asn Ala His Met Asn Ala Thr Asn His Ala 40 30 Ile Val Gin Thr Leu Val His Leu Met Asn Pro Glu Tyr Val Pro Lys 50 55 *o° Pro Cys Cys Ala Pro Thr Lys Leu Asn Ala Ile Ser Val Leu Tyr Phe 35 65 70 75 Asp Asp Asn Ser Asn Val Ile Leu Lys Lys Tyr Arg Trp Met Val Val 85 90 40 Arg Ala Cys Gly Cys His 100 INFORMATION FOR SEQ ID NO:29: SEQUENCE CHARACTERISTICS: LENGTH: 102 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein 126 (ix) FEATURE: NAME/KEY: Protein LOCATION: 1..102 OTHER INFORMATION: /label=

OPX

/note= "WHEREIN EACH XAA IS INDEPENDENTLY

SELECTED

FROM A GROUP OF ONE OR MORE SPECIFIED AMINO ACIDS AS DEFINED IN THE SPECIFICATION (SECTION II.B.2.)" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: Cys Xaa Xaa His Glu Leu Tyr Val Xaa Phe Xaa Asp Leu Gly Trp Xaa 1 5 10 Asp Trp Xaa Ile Ala Pro Xaa Gly Tyr Xaa Ala Tyr Tyr Cys Glu Gly 25 Glu Cys Xaa Phe Pro Leu Xaa Ser Xaa Met Asn Ala Thr Asn His Ala 40 Ile Xaa Gin Xaa Leu Val His Xaa Xaa Xaa Pro Xaa Xaa Val Pro Lys 55 Xaa Cys Cys Ala Pro Thr Xaa Leu Xaa Ala Xaa Ser Val Leu Tyr Xaa 65 70 75 Asp Xaa Ser Xaa Asn Val Xaa Leu Xaa Lys Xaa Arg Asn Met Val Val 85 90 30 Xaa Ala Cys Gly Cys His 100 INFORMATION FOR SEQ ID 35 SEQUENCE CHARACTERISTICS: LENGTH: 97 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein 0 (ix) FEATURE: NAME/KEY: Protein LOCATION: 1..97 OTHER INFORMATION: /label= /note= "WHEREIN EACH XAA IS INDEPENDENTLY SELECTED FROM A GROUP OF ONE OR MORE SPECIFIED AMINO ACIDS AS DEFINED IN THE SPECIFICATION." (xi) SEQUENCE DESCRIPTION: SEQ ID Leu Xaa Xaa Xaa Phe Xaa Xaa Xaa Gly Trp Xaa Xaa Trp Xaa Xaa Xaa 1 5 10 127 Pro Xaa Xaa Xaa Xaa Ala Xaa Tyr Cys Xaa Gly Xaa Cys Xaa Xaa Pro 25 Xaa Zaa Xaa Xaa Xaa Xaa Xaa Xaa Asn His Ala Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Cys Xaa Pro 55 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Leu Xaa Xaa Xaa Xaa Xaa Xaa Xaa 70 75 Val Xaa Leu Xaa Xaa Xaa Xaa Xaa Met Xaa Val Xaa Xaa Cys Xaa Cys 85 90 Xaa INFORMATION FOR SEQ ID NO:31: SEQUENCE CHARACTERISTICS: LENGTH: 102 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (ix) FEATURE: NAME/KEY: Protein LOCATION: 1..102 OTHER INFORMATION: /label= GENERIC-SEQ6 /note= "WHEREIN EACH XAA IS INDEPENDENTLY SELECTED FROM A GROUP OF ONE OR MORE SPECIFIED AMINO ACIDS AS DEFINED IN THE SPECIFICATION.

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:31: Cys Xaa Xaa Xaa Xaa Leu Xaa Xaa Xaa Phe Xaa Xaa Xaa Gly Trp Xaa *1 5 10 Xaa Trp Xaa Xaa Xaa Pro Xaa Xaa Xaa Xaa Ala Xaa Tyr Cys Xaa Gly 25 Xaa Cys Xaa Xaa Pro Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asn His Ala -35 40 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa, Xaa 55 Xaa Cys Cys Xaa Pro Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Leu Xaa Xaa 65 70 75 128 Xaa Xaa Xaa Xaa Zaa Val Xaa Leu Xaa Xaa Iaa Xaa Xaa Met Xaa Val 90 Xaa Xaa Cys Xaa Cys Xaa 100 INFORMATION FOR SEQ ID NO:32: SEQUENCE CHARACTERISTICS: LENGTH: 1247 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: ORGANISM: HOMO SAPIENS TISSUE TYPE: BRAIN (ix) FEATURE: NAME/KEY: CDS LOCATION: 84. .1199 OTHER INFORMATION: /product. "GDF-1" /note= "GDF-1 CDNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32: GGGGACACCG GCCCCGCCCT CAGCCCACTG GTCCCGGGCC GCCGCGGACC CTGCGCACTC TCTGGTCATC GCCTGGGAGG AAG ATG CCA CCG CCG CAG CAA GGT CCC TGC Met Pro Pro Pro Gin Gin Gly Pro Cys 1 4 GGC CAC CAC CTC Gly His His Leu CTG ACC CCC CC Leu Thr Arg Ala CTC CTC CTC CTG GCC CTG CTG CTG CCC TCG CTG Leu Leu Leu Leu Ala Leu Leu Leu Pro Ser Leu GTG CCC CCA GCC Val Pro Pro Gly GCC GCC GCC CTG Ala Ala Ala Leu CTC CAG Leu Gin CCT CTA CGA Ala Leu Cly GTT CCC CCG Val Pro Pro CGC CAT GAG CCC Arg Asp Ciu Pro CCT CCC CCC AGG Gly Ala Pro Arg CTC CCC CCC Leu Arg Pro CCC CAG GAG Pro Gin Glu GTC ATG TOG CC Val Met Trp Arg rrr CGA CCC CGG Phe Arg Arg Arg 4. 4 9 a 9* i ACC AGG TCT CCC TCG CCC CCC Thr Arg Ser Cly Ser Arg Arg 80 ACG TCC CCA CCC Thr Ser Pro Gly GTC ACC CTG CAA CCC Val Thr Leu Gln Pro 129

TGC

Cys

CCC

Pro

G

Gly ccc Pro

C

Ala

C

Ala 170 Leu

OCT

Ala

GCC

Ala

TCG,

Ser

CG

Arg 250

OCT

Ala

CAC

His CAC GTG GAG GAG CTO His Vai Giu Giu Leu GGG GTC GCC CCA MAC ATC GTG CGC CAC ATC Cly Val Ala Gly Asn Ile Val Arg His Ile OAC CGC Asp Arg CAT TOC His Cys GCT GAG Ala Olu 140 OCO OCO Ala Ala 155 GCC GO Oly Gin OTO CCC Val Pro TOG OCT Trp Ala CTA CC Leu Arg 220 CTO, CTC Leu Leu 235 CCC CG Pro Arg TOT COC Cys Arg CCC TG Arg Trp 0CC CCC Ala Pro 110 GAO TG Clu Trp CCC AC Pro Ser 0CC CCG Ala Pro 0CC OGC Ala Cly 175 CTC 000 Leu Cly 190 MAC 0CC Asn Ala COG 0CC Arg Ala GTO ACC Val Thr CAC 0CC Asp Ala 255 COG CG Arg Arg 270 ATC C Ile Ala

ACC

Thr

ACA

Thr

CG

Arg

GAG

Clu 160 0,CC Ala

CCO

Pro

TCA

Ser

CCT

Pro

CTC

Leu 240

CMA

Clu

CTC

Leu

CCC

Pro

CG

Arg

GTC

Val

GCC

Ala 145

GCC

Cly

GAC

Asp

CCA

Pro

TG

Trp 0CC Ala 225

CAC

Asp

CCC

Pro

TAC

Tyr

CC

Arg

GCC

Ala

GTC

Val 130

CC

Arg

GCC

Cly

CCC

Pro

OTG

Vai

CCG

Pro 210 0CC Ala

CCG

Pro

GTO

Val

GTG

Val

GC

Cly 290

TCC

Ser 115 GAG CCI GTC Ciu Pro Val 9*

F

*0 *F

P

'F F F F

F.

TIC GAC CTO Phe Asp Leu CTC GAG CTC, Leu Clu Leu TOG GAG CTO Trp Clu Leu 165 COO CCC OTG Oly Pro Val 180 CCC CC GAO Arg Ala Olu 195 CCC AOC CTC Arg Ser Leu TCC 0CC CC Cys Aia Arg CCC CTC T0C Arg Leu Cys 245 rro CCC GCC Leu Cly Cly 260 ACC TIC CC Ser Phe Arg 275 TIC CT, CC Phe Leu Ala 446 494 542 590 638 686 734 782 830 878 926 974 1022 OGT CAC TCC CC CTC, CCC Cly Cmn Cys Ala Leu Pro 300 CIC C Val Ala 305 CTC, TCC COG TCC COG COG CCC CCC Leu Ser Cly Ser Cly Cly Pro Pro 310 -130 GCG CTC AAC CAC GCT GTG CTG CGC GCG CTC ATG CAC GCG GCC GCC CCG 1070 Ala Leu Asn His Ala Val Leu Arg Ala Leu Met His Ala Ala Ala Pro 315 320 325 GGA GCC GCC GAC CTG CCC TGC TGC GTG CCC GCG CGC CTG TCG CCC ATC 1118 Gly Ala Ala Asp Leu Pro Cys Cys Val Pro Ala Arg Leu Ser Pro Ile 330 335 340 345 TCC GTG CTC TTC TTT GAC AAC AGC GAC AAC GTG GTG CTG CGG CAG TAT 1166 Ser Val Leu Phe Phe Asp Asn Ser Asp Asn Val Val Leu Arg Gln Tyr 350 355 360 GAG GAC ATG GTG GTG GAC GAG TGC GGC TGC CGC TAACCCGGGG CGGGCAGGGA 1219 Glu Asp Met Val Val Asp Glu Cys Gly Cys Arg 365 370 CCCGGGCCCA ACAATAAATG CCGCGTGG 1247 INFORMATION FOR SEQ ID NO:33: SEQUENCE CHARACTERISTICS: LENGTH: 372 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein 30 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33: Met Pro Pro Pro Gln Gin Gly Pro Cys Gly His His Leu Leu Leu Leu 1 5 10 35 Leu Ala Leu Leu Leu Pro Ser Leu Pro Leu Thr Arg Ala Pro Val Pro 20 25 Pro Gly Pro Ala Ala Ala Leu Leu Gin Ala Leu Gly Leu Arg Asp Glu 40 Pro Gin Gly Ala Pro Arg Leu Arg Pro Val Pro Pro Val Met Trp Arg 50 55 Leu Phe Arg Arg Arg Asp Pro Gin Glu Thr Arg Ser Gly Ser Arg Arg 65 70 75 Thr Ser Pro Gly Val Thr Leu Gin Pro Cys His Val Glu Glu Leu Gly 85 90 Val Ala Gly Asn Ile Val Arg His Ile Pro Asp Arg Gly Ala Pro Thr 100 105 110 Arg Ala Ser Glu Pro Val Ser Ala Ala Gly His Cys Pro Glu Trp Thr 115 120 125 131 Val Val Phe Asp Leu Ser Ala Val Glu Pro Ala Glu Arg Pro Ser Arg 130 135 140 Ala Arg Leu Glu Leu Arg Phe Ala Ala Ala Ala Ala Ala Ala Pro Glu 145 150 155 160 Gly Gly Trp Glu Leu Ser Val Ala Gln Ala Gly Gln Gly Ala Gly Ala 165 170 175 Asp Pro Gly Pro Val Leu Leu Arg Gln Leu Val Pro Ala Leu Gly Pro 180 185 190 Pro Val Arg Ala Glu Leu Leu Gly Ala Ala Trp Ala Arg Asn Ala Ser 195 200 205 Trp Pro Arg Ser Leu Arg Leu Ala Leu Ala Leu Arg Pro Arg Ala Pro 210 215 220 Ala Ala Cys Ala Arg Leu Ala Glu Ala Ser Leu Leu Leu Val Thr Leu 225 230 235 240 Asp Pro Arg Leu Cys His Pro Leu Ala Arg Pro Arg Arg Asp Ala Glu 245 250 255 Pro Val Leu Gly Gly Gly Pro Gly Gly Ala Cys Arg Ala Arg Arg Leu 260 265 270 Tyr Val Ser Phe Arg Glu Val Gly Trp His Arg Trp Val Ile Ala Pro 275 280 285 Arg Gly Phe Leu Ala Asn Tyr Cys Gln Gly Gln Cys Ala Leu Pro Val 290 295 300 Ala Leu Ser Gly Ser Gly Gly Pro Pro Ala Leu Asn His Ala Val Leu 3.I 305 310 315 320 Arg Ala Leu Met His Ala Ala Ala Pro Gly Ala Ala Asp Leu Pro Cys 325 330 335 Cys Val Pro Ala Arg Leu Ser Pro Ile Ser Val Leu Phe Phe Asp Asn 340 345 350 Ser Asp Asn Val Val Leu Arg Gln Tyr Glu Asp Met Val Val Asp Glu 355 360 365 Cys Gly Cys Arg 370

Claims (22)

1. 3-00;15:08 PATENT OFFICE CBR B/ 32 132 THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS: 1. A method for maintaining integrity of the gastrointestinal tract luminal lining in a mammal, comprising the step of administering a morphogen to tissue of said luminal lining, at a concentration sufficient to substantially inhibit ulceration or ulcer-associated tissue damage therein, said morphogen comprising a dimeric protein that induces morphogenesis of mammalian gastrointestinal barrier tissue and comprises a pair of folded polypeptides, the amino acid sequence of each of which comprises a sequence sharing at least 70% homology with the C-terminal seven cysteine domain of human OP-1, residues 38-139 of SEQ ID NO: (ii) a sequence having greater than 60% identity with the C-terminal seven cysteine domain of human OP-1, residues 38-139 of SEQ ID 9 NO: S(iii) a sequence defined by Generic Sequence 6, SEQ ID NO: 31;or (iv) a sequence encoded by a nucleic acid that hybridizes under stringent conditions with nucleic acid having the complementary sequence of nucleotide residues 1036-1341 of SEQ ID NO: 16, wherein said gastrointestinal tract luminal lining is afflicted with a non- inflammatory or non-fibrogenic response-associated disease. 2. A method for inducing regeneration of lost or damaged gastrointestinal tract t barrier tissue in a mammal afflicted with an ulcerative disease, comprising the step of administering a morphogen to ulcerated gastrointestinal tissue in said mammal, at a concentration sufficient to induce regeneration thereof, said morphogen comprising a dimeric protein that induces morphogenesis of mammalian gastrointestinal barrier tissue and comprises a pair of folded polypeptides, the amino acid sequence of each of which comprises a sequence sharing at least 70% homology with the C-terminal seven cysteine domain of human OP-1, residues 38-139 of SEQ ID NO: cysteine domain of human OP-i, residues 38-139 of SEQ ID NO: 10/03 '00 FRI 15:09 [TX/RX NO 7835] 3-00 15:0 PATENT OFFICE CBR 9/ 32 133 (ii) a sequence having greater than 60% identity with the C-terminal seven cysteine domain of human OP-1, residues 38-139 of SEQ ID NO: (iii) a sequence defined by Generic Sequence 6, SEQ ID NO: 31 ;or (iv) a sequence encoded by a nucleic acid that hybridizes under stringent conditions with nucleic acid having the complementary sequence of nucleotide residues 1036-1341 of SEQ ID NO: 16, wherein said gastrointestinal tract luminal lining is afflicted with a non- inflammatory or non-fibrogenic response-associated disease. 3. The method of claim 1 or 2 wherein said mammal is a human.
2. 4. The method of claim 3 wherein said human is at risk for a condition selected from the group consisting of: xerostomia, oral mucositis, gastric or duodenal ulcer, ulcerative colitis, proctitis, regional enteritis, or necrotizing enterocolitis.
3. 5. The method of claim 3 wherein said human is afflicted with a condition Sselected from the group consisting of: xerostomia, oral mucositis, gastric or Sduodenal ulcer, ulcerative colitis, proctitis, regional enteritis, or necrotizing :.enterocolitis. U
4. 6. The method of claim 1 wherein said morphogen is administered systemically.
5. 7. The method of claim 1 wherein said morphogen is administered orally, rectally or parenterally.
6. 8. The method of claim 7 wherein said morphogen administered in an ingestible controlled-release delivery vehicle.
7. 9. The method of claim I wherein said morphogen is administered locally. 10/03 '00 FRI 15:09 [TX/RX NO 7835]
8. 10-3-0fl5fl2PATENT OFFICE CBR 10/ J2 The method of claim 9 wherein said rnorphogen is administered in a mouthwash solution.
9. 11. The method of claim 9 wherein said morphogen is administered topically.
10. 12. The method of claim I11 wherein said morphogen is dispersed in a tissue adhesive composition-
11. 13. The method of claim I wherein said morphogen is administered to said mammial at a concentration of less thian about 100 jig morphogcn/kg mamrmalian body weight.
12. 14. The method of claim 13 wherein said concentration is less than about 30 jig morphogenlkg body weight. :0 9:15. The method of claim 14 wherein said concentration is less than about 10 j ig morphogen/kg body weight, :16. The method of claim I or 2 wherein the amino acid sequences of said a morphogen polypeptides comprise a sequence defned by OPX, SEQ ID NO:
13. 29. The method of claim I or 2 wherein the amino acid sequence of at least one of said morphogen polypeptides comprises the sequence of the C-terminal seven cysteine domain of human OP-1, residues 38-139 of SEQ ED NO: 5, or an allelic or species variant thereof. 19- The method of claim I or 2 wherein said morphogen is obtained from milk, serun, or culture supernatant of miorphogen-secreting mammalian cells. 19. The method of claim 1 or 2 wherein said morphogen is solubilized by association with one or more prodomain polypeptides of a member of the 10/03 '00 FRI 15:09 1TX/RX NO 7835] 3-00;15 08 PATENT OFFICE CBR 11/ 32 135 morphogen family, or an. allelic, species or biosynthetic sequence variant thereof. The method of claim 1 or 2 wherein said administering step is performed by administering to said mammal an agent that stimulates in vivo production of an endogenous morphogen by cells of said mammal competent to produce said endogenous morphogen. 21. A therapeutic composition for treating ulcerations of the mammalian gastrointestinal tract, comprising: a morphogen, said morphogen comprising a dimeric protein that :induces morphogenesis of gastrointestinal barrier tissue in a mammal and comprises a pair of folded polypeptides, the amino acid sequence of each of which comprises a sequence sharing at least 70% homology with the C-terminal seven cysteine domain of human OP-1, residues 38-139 of SEQ ID NO: (ii) a sequence having greater than 60% identity with the C- terminal seven cysteine domain of human OP-1, residues 38- 139 of SEQ ID NO: (iii) a sequence defined by Generic Sequence 6, SEQ ID NO: 31;or S: (iv) a sequence encoded by a nucleic acid that hybridizes under stringent conditions with nucleic acid having the complementary sequence of nucleotide residues 1036-1341 of SEQ ID NO: 16, and a biocompatible compound capable of coating the gastrointestinal tract luminal lining, said morphogen being admixed with said biocompatible compound at a concentration sufficient to substantially inhibit ulceration or ulcer-associated tissue damage in mammalian gastrointestinal barrier tissue, and wherein said gastrointestinal barrier tissue is afflicted with a non-inflammatory or non- fibrogenic response-associated disease. 10/03 '00 FRI 15:09 [TX/RX NO 7835] 3-00;15:08 PATENT OFFICE CBR 12/ 32 136 22. The composition of claim 21 wherein said biocompatible compound comprises a tissue adhesive. 23. The composition of claim 21 wherein said compound comprises hydroxypropylellulose, pectin, glycerol, a sucralfate suspension, or a combination of fibrinogen and thrombin. 24. A therapeutic composition for treating ulcerations of the gastrointestinal tract comprising; a morphogen, said morphogen comprising a dimeric protein that induces morphogenesis of gastrointestinal barrier tissue in a mammal and comprises a pair of folded polypeptides, the amino acid sequence of each of which comprises: a sequence sharing at least 70% homology with the C-terminal seven cysteine domain of human OP-1, residues 38-139 of SEQ ID NO: (ii) a sequence having greater than 60% identity with the C- terminal seven cysteine domain of human OP-1, residues 38- 139 of SEQ ID NO: (iii) a sequence defined by Generic Sequence 6, SEQ ID NO: 31;or (iv) a sequence encoded by a nucleic acid that hybridizes under stringent conditions with nucleic acid having the complementary sequence of nucleotide residues 1036-1341 of SEQ ID NO: 16, and a biocompatible ulcer-symptom alleviating cofactor, said morphogen being admixed with said biocompatible cofactor at a concentration sufficient to substantially inhibit ulceration or ulcer-associated tissue damage in mammalian gastrointestinal barrier tissue, and wherein said gastrointestinal barrier tissue is afflicted with a non-inflammatory or non- fibrogenic response-associated disease. 10/03 '00 FRI 15:09 [TX/RX NO 7835] 3-00 15:08 PATENT OFFICE CBR 137 The composition of claim 24 wherein said cofactor comprises a biocompatible analgesic, anesthetic, antiseptic, antibiotic, or antiviral or antifungal agent. 26. The composition of claim 24 wherein said cofactor comprises a biocompatible antisecretory agent. 27. An oral rinse for treating oral mucositis in a mammal, comprising a morphogen, said morphogen comprising a dimeric protein that induces morphogenesis of gastrointestinal barrier tissue in a mammal and comprises a pair of folded polypeptides, the amino acid sequence of each of which S* comprises a sequence sharing at least 70% homology with the C-terminal seven cysteine domain of human OP-1, residues 38-139 of SEQ ID NO: (ii) a sequence having greater than 60% identity with the C-terminal seven cysteine domain of human OP-1, residues 38-139 of SEQ ID NO: (iii) a sequence defined by Generic Sequence 6, SEQ ID NO: 31 ;or (iv) a sequence encoded by a nucleic acid that hybridizes under stringent conditions with nucleic acid having the complementary sequence of nucleotide residues 1036-1341 of SEQ ID NO: 16, said morphogen being solubilized in a mouthwash solution at a concentration sufficient to substantially inhibit ulceration or ulcer-associated tissue damage in mammalian gastrointestinal barrier tissue, and wherein said gastrointestinal barrier tissue is afflicted with a non-inflammatory or non- fibrogenic response-associated disease. 28. A therapeutic composition for treating ulcerations of the mammalian gastrointestinal tract, comprising a morphogen dispersed in an ingestible controlled release delivery vehicle, said morphogen comprising a dimeric protein that induces morphogenesis of gastrointestinal barrier tissue in a 13/ 32 10/03 '00 FRI 15:09 [TX/RX NO 7835] 3-O0U15!C8 PATENT OFFICE CBR 14/ 32 138 mammal and comprises a pair of folded polypeptides, the amino acid sequence of each of which comprises a sequence sharing at least 70% homology with the C-terminal seven cysteine domain of human OP-1, residues 38-139 of SEQ ID NO: (ii) a sequence having greater than 60% identity with the C-terminal seven cysteine domain of human OP-1, residues 38-139 of SEQ ID NO: (iii) a sequence defined by Generic Sequence 6, SEQ ID NO: 31 ;or (iv) a sequence encoded by a nucleic acid that hybridizes under stringent conditions with nucleic acid having the complementary sequence of nucleotide residues 1036-1341 of SEQ ID NO: 16, said morphogen being dispersed in said vehicle at a concentration sufficient to substantially inhibit ulceration or ulcer-associated tissue damage in mammalian gastrointestinal barrier tissue, and wherein said gastrointestinal barrier tissue is afflicted with a non-inflammatory or non-fibrogenic response-associated disease. 29. The composition of claim 28 wherein said ingestible delivery vehicle is a tablet, troche, lozenge or chewing gum.
14. 30. A therapeutic composition for treating ulcerations for the mammalian gastrointestinal tract, comprising a morphogen dispersed in a tissue adhesive Icomposition, said morphogen comprising a dimeric protein that induces morphogenesis of gastrointestinal barrier tissue in a mammal and comprises a pair of folded polypeptides, the amino acid sequence of each of which comprises a sequence sharing at least 70% homology with the C-terminal seven cysteine domain of human OP-1, residues 38-139 of SEQ ID NO: (ii) a sequence having greater than 60% identity with the C-terminal seven cysteine domain of human OP-1, residues 38-139 of SEQ ID NO: (iii) a sequence defined by Generic Sequence 6, SEQ ID NO: 31 ;or 10/03 '00 FRI 15:09 [TX/RX NO 7835] 3-00 15:08 PATENT OFFICE CBR 15/ 32 139 (iv) a sequence encoded by a nucleic acid that hybridizes under stringent conditions with nucleic acid having the complementary sequence of nucleotide residues 1036-1341 of SEQ ID NO: 16, said morphogen being dispersed in said adhesive composition at a concentration sufficient to substantially inhibit ulceration or ulcer-associated tissue damage in mammalian gastrointestinal barrier tissue, and wherein said gastrointestinal barrier tissue lining is afflicted with a non-inflammatory or non-fibrogenic response-associated disease.
15. 31. The composition of any one of claim 21, 24, 27, 28 or 30 wherein the amino acid sequences of said morphogen polypeptides comprise a sequence sharing at least 80% homology with the C-terminal seven cysteine domain of human OP-1, residues 38-139 of SEQ ID NO: S:32. The composition of any one of claims 21, 24, 27, 28 or 30, wherein the amino acid sequences of said morphogen polypeptides comprise a sequence having greater than 65% amino acid identity with the C-terminal seven cysteine domain of human OP-i, residues 38-139 of SEQ ID NO:
16. 33. The composition of any one of claims 21, 24, 27, 28 or 30, wherein the amino acid sequences of said morphogen polypeptides comprise a sequence .defined by OPX, SEQ ID NO: 29.
17. 34. The composition of any one of claims 21, 24, 28, 29 or 30 wherein the aino The composition of any one of claims 21, 24, 27, 28 or 30 wherein said morphogen is obtained fom milk, serum, or culture supenatant of morphogen-secreting mammalian cells. morphogen-secreting mammalian cells. 10/03 '00 FRI 15:09 [TX/RX NO 7835] 10-3-U;15Q8PATENT OFFICE C8R 1 B/ 32 140
18. 36. The composition of any one of claims 21, 24, 27, 28, Or 30 wherein xnorphogen is solubiliZed by association with one or more prodoai polypeptides of a member of the morphogen family, or an allelic, species or biosynthetic sequence variant thereof.
19. 37. The composition of claim 36 wherein said morphogen is noncovalently associated with said polypeptides. 38 The composition of any one of claims 21, 24, 27, 28 or 3 0 wherein the amino acid sequence of at least one of said morphogen polypeptides comprises the pro form of human OP-i, residues 30-43 1 of SEQ ID NO: 16, or an allelic or :%we, to species variant thereof 39 The composition of any one ofclajims 21, 24, 27, 28 or 3 0 further comprising a basic amino acid, a detergent or a carrier protein. A method for maintaining integrity of the gastrointestinal tract luminal lining in a mammal, comprising the step of administering a morphogen to tissue of said luminal lining, at a concentration sufficient to substantially inhibit ulceration or ulcer-associated tissue damage therein, wherein said morphogen .**.*comprises the C-terminal seven-cysteine skeleton of a sequence selected from the group consisting of- human OP-i (residues 38-139 of SEQ ID Mott.: NO:5), mouse OP- I (residues 3 8-139 of SEQ ID NO:6), human OP-2 (residues 38-139 of SEQ ID NO:7), mousc OP-2 (residues 38-139 of SEQ ID NO:8), DPP (SEQ ID NO: 11), Vgl (SEQ ID NO: 12), Vgr-I1 (SEQ ID NO: 13), CBMP2A (SEQ.1IDNO:9), CBMP2B (SEQ ID NO: 10), BMP3 (SEQ ID NO:26), GDF-1 (SEQ ID NO: 14), 60A (residues 354-455 of SEQ ID NO:24), BMP5 (SEQ ID NO:27) and BML6 (SEQ ID NO:28), or a conservative amino acid substitution variant of any of the foregoing, and wherein said gastrointestinal tract lurninal lining is afflicted with a non- inflammatory or non-fibrogenic response-associated disease. 10/03 '00 FRI 15:09 [TX/RX No 78351 3-00;fl5!08 PAETOFC CR#1/3 PATENT OFFICE CBR 17/ 32
20. 41. The mnethod of claim 40., wherein said morphogen is human OP-i, SEQ ID) NO:
21. 42. A methiod for inducing regeneration of lost or damaged gastrointestinal tract barrier tissue in a mammal afflicted with an ulcerative disease, comprising the step of administering a inorphogen to ulcerated gastrointestinal tissue in said mammal, at a concentration sufficient to induce regeneration thereof~, wherein said morphogen comprises the C-terminal seven-cysteine skeleton of a sequence selected from the group consisting of: human OP- I (residues 38-139 of SEQ ID NO:5), mouse OP-i (residues 38-139 of SEQ IDNWO:6), human OP-2 (residues 38-139 of SEQ ID NO-7), mouse OP-2 (residues
22. 389-139 of SEQ lID NO:8), DPP (SEQ ID NQO- 11), Vgl (SEQ ID NO: 12), Vgr-1 (SEQ ID NO:13), CBMP2A (SEQ lID NO:9), CBMF2B3 (SEQ ID BMP3 (SEQ ID NO:26), GDF- I (SEQ ID NO: 14), 60A (residues 354-455 of SEQ ID) NO:24), BMP5 (SEQ ID NO;27) and BMP6 (SEQ ID NO:28), or a conservative amino acid substitution variant of any of the foregoing, and wherein said gastrointestinal tract luminal lining is afflicted with a non-inflammatory or non-fibrogenic response-associated disease. 43. The method of claim 42, wherein said morphogen is human OP-i, SEQ ID NO: 44 h ehdo cam4.hri ai amli uani.trs o The method of claim 42 wherein said mammal is a human is affte rstfo a condition selected from the group consisting of: xerostomia, oral mucositis, gastric or duodenal ulcer, ulcerative colitis, proctitis, regional enteritis, or necrotizing enterocolitis. 10/03 '00 FRI 15:09 [TX/RX NO 7835] 1 0 3 0 0 15 0 8 10- 3-00;15~08PATENT OFFICE CBR 8/3 1 S/ 32 142 46. The method of claim 42 wherein said morphogen is administered systemically. 47. The method of claim 42 wherein said morphogen is administered orally, rectally or parenterally. 48. The method of claim 44 wherein said morphogen administered in an ingestible controlled-release delivery vehicle. 49, The method of claim 42 wherein said morphogen is administered locally. The method of claim 49 wherein said morphogen is administered in a 0, mouthwash solution. 0 *53. The method of cli 42 wner-em said morpnogen is administeredi topsiady 52. The method of claim 53 wherein said conrtion is disese ihn au tissue T ahesieo copclsiion. en adcnenrto s esta aot1i *opog k boywegt 53. The method of claim40 42 wherein said morphogen is dmtnineed tomi maimal sat a cncetrato oulesatan abut10f morphogen /kgeig amlanr 10/03 '00 FRI 15:09 [TX/RX NO 78351 in- 3-00:15!08 iC- 3-OU15~O8PATENT OFFICE CBR 8/3 19/ 32 143 57. The method of claim 40 or 42 wherein said morphogen is solubilized by association with one- or more prodomnain polypeptides of a member of the rnorphogen family, or an allelic, species or biosynthetic sequence variant thereof 58. The method of claim 40 or 42 whexein said administering step is performed by administering to said mammnal an agent that stimulates in vivo production of an endogenous; zorphogen by cells of said mammal competent to produce said endageno-us morphogen. 59. A therapeutic composition for treating ulcerations of the mammalian gastrointestinal tract, comprising: a morphogen, wherein said morphogen comprises the C-terminal seven-cysteine skeleton of a sequence selected from the group consisting of: human OP-i (residues 38-139 of SEQ ID NO:5), mouse (residues 38-139 of SEQ ID NO:6), human OP-2 (residues 38-139 of SEQ ID NO:7), mouse OP-2 (residues 38-139 of SEQ ID NO: DPP' (SEQ ID NO: 11), VgI (SEQ ID NO: 12), Vgr-1 (SEQ ID .NO: 13), CBMP2A (SEQ ID NO:9), CBMP2B3 (SEQ ID NO: BM3 (SEQ ID NO:26), GDF-l (SEQ ID NO: 14), 60A (residues 354-455 of SEQ ID) NO:24), BMP5 (SEQ ID NO:27) and BMvP6 (SEQ ID NO:28), or a conservative amino acid substitution variant of any of the foregoing, and a biocompatible compound capable of coating the gastrointestinal tract lumninal lining, said morphogen being admixed with said biocompatible compound at a concentration sufficient to substantially inhibit ulceration or ulcer-associated tissue damage in mammalian gastrointestinal barrier tissue, and wherein said gastrointestinal barrier tissue is afflicted with a non-inflam-matory or- non- fibrogenic response-associated disease. 10/03 '00 FRI 15:09 [TX/RX NO 7835] 3 0 ;15~U PAEN OFC CBR PATENT OFFICE CBR 20/ 32 9* C C. C. *e C C C. CC C. C C C C CC.. C CC.. C CC.. CCC. C 144 The composition of claim 59, wherein said morphogen is human OP-i, SEQ ID NO: 61. The composition of claim 59 wherein said biocompatible compound comprises a tissue adhesive. 62. The composition of claim 59 wherein said compound comprises hydroxypropylcellulose, pectin, glycerol, a sucralfate suspension, or a combination of fibrinogen and thrombin. 63. A therapeutic composition for treating ulcerations of the gastrointestinal tract comprising: a rnorphogcn, wherein said inorphogen comprises the C-terminal seven-cystemne skeleton of a sequence selected from the group consisting of- human OP-i (residues 38-139 of SEQ ID NO:5), mouse OP-I (residues 38-139 of SEQ ID NO:6), human OP-2 (residues 38-139 of SEQ ID NO:7), mouse OP-2 (residues 38-139 of SEQ ID NO:8 DPP (SEQ ID NO: 11), Vgl (SEQ ID NO: 12), Vgr-1I (SEQ ID NO: 13), CBMP2A (SEQ ID NO:9), CBMP2B (SEQ ID NO: BMT3 (SEQ ID NO:26), GDF-1I (SEQ ID NO: 14), 60A (residues 354-455 of SEQ ID NO:24), BMP5 (SEQ ID NO:27) and BMP6 (SEQ ID NO:28), or a conservative amino acid substitution variant of any of the foregoing, and a biocompatible ulcer-symptomn alleviating cofactor, said morphogen being admixed with said bilocomnpatible cofactor at a concentration sufficient to substantially inhibit ulceration or ulcer-associated tissue damage in mammalian gastrointestinal barrier tissue, and wherein said gastrointestinal barrier tissue is afflicted with a non-inflatmmatory or non- fibrogenic response-associated disease- 64 *The composition of claim 63, wherein said morphogen is human OP-I, SEQ ID NO: 10/03 'DO FRI 15:09 [TX/RX NO 78351 in-3-Q;15ORPATENT OFFICE CBR 21/ 32 145 The Composition of claim 63 wherein said cofactor comprises a biocompatible analgesic, anesthetic, antiseptic, antibiotic, or antiviral or antifungal agent. 66. The composition of claim 63 wherein said cofactor comprises a biocompatible antisecretory agent. 67. An oral rinse for treating oral mucositis in a mammal, comprising a morphogen, wherein said mrnophogen comprises the C-terminal seven-cysteine skeleton of a sequence selected from the group consisting of: human OP- I (residues 39-139 of SEQ ID NO:5), mouse OP-i (residues 38-139 of SEQ ID NO:6), human OP-2 (residues 38-139 of SEQ 11) NO:7), mouse OP-2 (residues 38-139 of SEQID NO.8). DPI'(SEQ ID NO.11),Vgl (SEQ ID NO: 12), Vgr-1I (SEQ ID NO: 13), CBMP2A (SEQ ID NO:9), CBMP2B (SEQ ID NOb), BMP3 (SEQ ID NO:26), GDF-1 (SEQ ID NO: 14), 60A (residues 3 54-455 of SEQ ID NO:24), BMPS (SEQ ID NO:27) and BMP6 (SEQ ID NO:28), or a conservative amino acid substitution variant of any of the foregoing, said morphogen being solubilized in a mouthwash solution at a concentration sufficient to substantially inhibit ulceration or ulcer-associated tissue damage in mammalian gastrointestinal barrier tissue, and wherein said gastrointestinal barrier tissue is afflicted with a non-inflammatory or non-fibrogenic response-associated disease. 68. The oral rinse of claim 67, wherein said morphogen is human OP-i, SEQ ID NO: 69. A therapeutic composition for treating ulcerations of the mammalian gastrointestinal tract, comprising a morphogen dispersed in an ingestible controlled release delivery vehicle, said morphogen wherein said morphogen comprises the C-terminal seven-cysteine skeleton of a sequence selected from the group consisting of: human OP- I (residues 38-139 of SEQ ID 10/03 '00 FRI 15:09 ITX/RX NO 7835] 10-3-O;15O8PATENT OFFICE C.BR 22/ 32 146 mouse OP-I (residues 38-139 of SEQ ID NO:6), human OP-2 (residues 38-139 of SEQ ID NO.7). mouse OP-2 (residues 38-139 of SEQ ID NO:8), DPP (SEQ ID NO: 11), Vgl (SEQ ID NO: 12), Vgr-1 (SEQ ID NO:13), CBMP2A (SEQ ID NO:9), CEMP2B (SEQ ID NO.:1O), BMP3 (SEQ TD NO:26), GDF- 1 (SEQ ID NO: 14), 60A (residues 354-455 of SEQ ID NO:24),13MP5 (SEQ ID NO:27) and BMP6 (SEQ ID NO:28), or a conservative am~ino acid substitution variant of any of the foregoing, said mnorphogen being dispersed in said vehicle at a concentration sufficient to substantially inhibit ulceration or ulcer-associated tissue damage in mammalian gastrointestinal barrier tissue, and wherein said gastrointestinal barrier tissue is afflicted with a non-inflammatory or non-fibrogenic response-associated disease. The composition of claim 69, wherein said morphogen is human OP-i, SEQ ID NO: 71. The composition of claim 69 wherein said ingestible delivery vehicle is a tablet, troche, lozenge or chewing gum. :72. A therapeutic composition for treating ulcerations for the mammalian gastrointestinal tract, comprising a morphogen dispersed in a tissue adhesive composition, wherein said morphogen comprises the C-terminal seven-cysteine skeleton of a sequence selected from the group consisting of: human OP-I (residues 38-139 of SEQ ID NO:5), mouse OP-1 (residues 38-139 of SFEQ ID NO:6), human OP-2 (residues 38-139 of SEQ ID NO:7), mouse OP-2 (residues 3 8-139 of SEQ ID NO: DPP (SEQ ID NO: 11), Vgl (SEQ ID NO: 12), Vgr-1I (SEQ ID NO: 13), CBMP2A (SEQ ID NO:9), CBMP2B (SEQ ID NO: 10), BMP3 (SEQ ID NO:26), GDF-1 (SEQ ID NO:14), 60A (residues 354-455 of SEQ ID NO:24), BMP5 (SEQ ID NO:27) and BMP6 (SEQ ID NO:.28), or a conservative amino acid substitution variant of any of the foregoing, said morphogen being dispersed in said adhesive composition at a concentration sufficient to substantially inhibit 10/03 '00 FRI 15:09 [TX/RX NO 78351 3-00;15!08 :PATENT OFFICE CBR 2.1/ 32 147 ulceration or ulcer-associated tissue damage in miammalian gastrointestinal barrier tissue, and wherein said gastrointestinal barrer tissue lining is afflicted with a non-inflammatory or non-fibrogenic response-associated disease. 73. The composition of claim 72, wherein said mnorphogen is human OP-i, SEQ ID NO: S. 74. Thc composition of any one of claims 59, 63, 67, 69 or 72 wherein said morphogen is obtained frorn milk, serum, or culture supenatant of morphogen-secreting mammalian cells. The composition of any one of claims 59, 63, 67, 69 or 72 wherein morphogen is solubilized by association with one or more prodomain polypeptides of a member of the morphogen family, or an allelic, species or biosynthetic sequence variant thereof. :76. The composition of claim 75 wherein said morphogen is noncovalently a associated with said polypepticles. 77. The composition of any one of claims 59, 63, 67, 69 or 72 wherein the amino acid sequence of at least one of said morphogen polypeptides comprises the pro form of human OP-i1, residues 3 0-431 of SEQ ID NO: 16, or an allelic, or species variant thereof. 78- The composition of any one of claims 59, 63, 67, 69 or 72 further comprising a basic amino acid, a detergent or a carrier protein. Dated this I OP' day of March 2000 Creative Biomolecules, Inc. By its Patent Attorneys Davies Collhson Cave 10/03 '00 FRI 15:09 [TX/RX NO 78351