AU673006B2 - Morphogen-enriched dietary composition - Google Patents

Description

q 49 SS /13 PCe ANNOUNCEMENT OF THE LATER PUBLICATION OFINTERNATIONAL SEARCH REPORTS INTERNATIONAL APPLICATION PUi3nBIHT RNDERTHE 'PATL"frr-ouPERAT

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N TREATY (PCT) (51) International Patent Classification 5 (11) International Publication Number: WO 94/03075 A23L 1/305, A61K 37/02 A3 (43) International Publication Date: 17 February 1994 (17.02.94) (21) International Application Number: PCT/US93/07190 (74) Agent: KELLEY. Robin. Testa, Hurwitz Thibeault.

Exchange Place. 53 State Street, Boston. MA 02109 (22) International Filing Date: 29 July 1993 (29.07.93) (US).

Priority data: (81) Designated States: AT, AU, BB, BG, BR, CA, CH-, CZ, 923,780 31 July 1992(31.07.92) US DE, DK, ES, FI, GB, HU, JP, KP, KR, LK, LU, MG, -946,235 16 Scpembe- 199 MN, MW, NL, NO, NZ, PL, PT, RO, RU, SD, SE, SK, -029,35 14 March -993-( .0 3.93) S UA. European patent (AT, BE. CH, DE, DK, ES. FR, -,040,510- 3 I-Ma-e.h-+993 GB, GR, IE, IT, LU, MC, NL, PT, SE), OAPI patent (BF, BJ, CF. CG, CI, CM, GA, GN, ML, MR, NE, SN, TD, TG).

(71)Applicant: CREATIVE BIOMOLECULES, INC. [US/ US]; 45 South Street, Hopkinton, MA 01748 (US).

Published (72) Inventors: KUBERASAMPATH, Thangavel Six Spring With international search report Street, Medway, MA 02053 COHEN, Charles, M. Before the expiration of the time limit for amending the 98 Winthrop Street, Mi:dway, MA 02053 RUE- clains and to be republished in the event of the receipt of GER, David, C. 19 Downey Street, Hopkinton, MA amendments.

01748 OPPERMANN, Hermann; 25 Summer Hill Road, Medway, MA 02053 PANG, Roy, L. (88) Date of publication of the international search report: Partridge Road, Etna, NH 03750 10 November 1994 (10.11.94)

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(54)Title: MORPHOGEN-ENRICHED DIETARY COMPOSITION 01 V (57) Abstract Disclosed are methods and compositions useful in dietary applications and capable of enhancing tissue morphogenesis, including tissue development and viability in a mammal, particularly a human. The methods and compositions include a morphogen which, when provided to an individual as a food formulation or supplement, is capable of enhancing tissue development and viability in the individual.

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WO 94/03075 PCT/US93/07190 MORPHOGEN-ENRICHED DIETARY COMPOSITION Field of the Invention This invention relates generally to the field of dietary compositions and supplements.

Background of the Invention The present invention relates to compositions useful as mammalian dietary compositions and supplements. In particular, the invention relates to food additives and dietary supplements capable of enhancing tissue morphogenesis and development, particularly in individuals at risk for normal tissue development and viability. Examples of such individuals include infants, particularly prematurelyborn ("preterm") and low birth weight infants, and juveniles; aged individuals; and individuals experiencing altered metabolic function and/or suffering from metabolic dysfunctions and other disorders that threaten organ or tissue function or viability, such as can result from malnutrition or starvation, autoimmune diseases, organ cirrhosis and other tissue necrotizing dysfunctions, or disorders associated with aging cells (cell senescence.) Mammalian infants are nourished by mother's milk until such time as they can digest food solids. Infant formulas now exist for humans and other mammals which can supplant or supplement mother's milk. The formulas may be milk based cow milk) or non-milk-based WO094/03075 PCT/US93/07190 2 soy). Particularly at risk are prematurely born infants whose tissues and organs are at an earlier stage of development, and whose nutritional requirements may differ from those of full term infants. Formula development is an ongoing endeavor to more accurately mimic the beneficial aspects of mother's milk. Nevertheless, despite the efforts of many researchers, infant formulas still differ in a number of significant ways from human milk. In part this is due because human milk has many substances,such as immunoglobulins, free amino acids, polyamines, nucleotides and polyunsaturated fatty acids not present, for example, in cow's milk. In addition, while infant formulas try to mimic the protein quantity found in human milk, the foreign proteins typically are present in the formula as hydrolysates to avoid rejection or reaction by the infant's digestive system.

The proteins are present primarily as amino acid sources rather than as functional proteins as might normally be transmitted by the nursing mother to the infant. In addition, human milk may contain unidentified growth and differentiation factors that are important for overall tissue and skeletal development.

Another group of individuals with potentially unique nutritional requirements are individuals undergoing metabolic changes which may result from periods of intense growth or stress, including, for example, pregnant women and drowning victims. Other sources of stress to the body may result from WO 94/03075 PC/US93/07190 3 malnutrition or starvation, or from metabolic disorders that affect organ viability, such as autoimmune disease and organ cirrhosis. Aged individuals, and postmenopausal women also have altered or slower metabolic function. All of these individuals are at risk for tissue damage or loss of tissue function due to altered metabolic function.

Reduced or lost tissue function due to malnutrition also is found in many patients admitted to hospitals (protein energy malnutrition, Proper nutritional support for such patients, while not a primary mode of treatment is, nevertheless, an important factor for therapy and recovery. It is, 1L therefore important to administer a nutritionally balanced diet given orally, enterally or parenterally, adequate to the needs of the patient. This is especially true for those patients where conventional feeding is contraindicated in dehydrated or gastroenterological patients) or is insufficient in hypercatabolic patients). The enteral or oral mode of administration of foods typically is preferable to parenteral modes because of the lower morbidity, trophic effect upon the intestinal mucosa, reduced dependency on instrumentation and lower costs.

It is an object of this invention to provide dietary compositions and supplements for enhancing tissue morphogenesis, including tissue growth, development, maintenance and viability in a mmmual, particularly a human. Another object of the invention is to provide an infant formula capable of enhancing tissue development in an infant or juvenile. Still another object is to provide an an infant formula that WO 94/03075 PCT/US93/07190 4 more closely mimics a nursing mother's milk. Another object of the invention is to provide dietary supplements for individuals at risk for normal tissue development, growth, maintenance and viability, including p-emature infants, aged individuals and individuals with altered metabolic function and/or suffering from disorders or metabolic dysfunctions which threaten organ viability and function. These and other objects and features of the invention will be apparent from the description, drawings, and claims which follow.

Summary of the Invention The present invention provides compositions and methods useful in dietary applications and capable of enhancing tissue morphogenesis, including tissue growth, development, maintenance and viability in a mammal, particularly a human. The dietary compositions and supplements of this invention comprise a morphogenic protein ("morphogen"), as described herein, which, when provided to an individual as a food formulation or supplement, is capable of enhancing tissue development, growth, maintenance and/or viability in the individual. The compositions and processes provided herein are suitable for both infants and adults, and as part of clinical nutrition.

As used herein, "enhancing tissue viability" is understood to mean protecting tissue from lost or reduced tissue function due to cell damage or cell senescence, including inducing cells to maintain their differentiated phenotype, inducing regeneration of damaged tissue, and/or inhibiting additional damage WO 94/03075 PCT/US93/07190 5 thereto. "Morphogenically effective concentration" is understood to mean a concentration sufficient to enhance tissue development and tissue viability in an individual at risk for tissue damage and/or reduced or lost tissue function due to insufficient nutritional considerations, tissue damage associated therewith, and/or incomplete tissue development, regardless of etiology. The ability of morphogens to repair, regenerate and protect various disparate tissues, including but not limited to, tissues of the gastrointestinal tract, including the oral mucosa, liver tissue, dentin tissue, periodontal tissue, nerve tissue, bone tissue, and any tissue at risk of damage due to immune response-mediated tissue destruction, including ischemia-reperfusion related tissue damage are disclosed in international applications US 92/01968 (WO 92/15323), US 92/07358 (WO 93/04692) and US 92/07232 (WO 93/05751) respectively, the disclosures of which are incorporated herein by reference.

"Morphogen-solubilizing molecule" is understood to mean a molecule capable of maintaining a morphogen in soluble form in physiologically buffered solutions.

"Food formulation" is understood to mean a dietary composition normally ingested by an individual to satisfy the body's fundamental nutritional requirements; "dietary supplement" is understood to mean supplemental compositions ingested by an individual in addition to the food formulations ingested to satisfy the fundamental nutritional requirements. Multivitamin and iron tablets are examples of commmon dietary supplements. "Dietary composition" is understood to include both food formulations and dietary supplements. As used herein, the term "infant formula" is understood to refer to the I ~ol- L WO 94/03075 PCT/US93/07190 6 well established infant compositions as defined by the American Academy of Pediatrics (AAP) and the AAP Committee on Nutrition ((1985) Pediatrics 75:976, the European Society of Pediatric Gastroenterology and Nutrition (ESPGAN) and the ESPGAN Committee on Nutrition ((1987) Acta Paed Scan Suppl:330), including recent updates published by these committees on infant formula nutritional guidelines.

The dietary composition or supplement preferably is administered orally, and may be provided in liquid form or as a powder to be dissolved in a beverage.

Alternatively, the dietary supplement may be provided as a solid, in a capsular, tablet, troche or lozenge form; or, the supplement may be provided as an aerosol, for oral or nasal administration. Where oral administration is not possible or desirable, other administration routes are envisioned. For example, for some premature infants, or for intibated patients, parenteral administration may be required, via an enteral feeding tube.

The morphogen may be provided alone or in association with one or more suitable excipients or carriers, and/or in combination with other beneficial molecules such as vitamins, minerals, lipids, fiber sources and the like. The dietary supplements also may include pharmaceutically acceptable inert materials for use as binders or stabilizers, including magnesium stearate or calcium carbonate. The morphogen may be CL q_ IL_ -11611 WO 94/03075 PCT/US93/07190 7 formulated together with one or more normal food ingredients, as part of a food formulation.

Alternatively or, in addition, the morphogen may be provided as a dietary supplement in, for example, tablet or syrup form.

The mature form of the morphogen, or active truncated forms thereof which may be formulated in the composition, further may be provided in association with a morphogen precursor "pro" domain, which is known to enhance the solubility of the protein in physiologically buffered solutions. Other useful molecules known to enhance protein solubility include casein, including derivatives, salts and analogs thereof, as well as other milk components, and various serum and milk serum proteins. Additional useful molecules which may be associated with the morphogen include tissue targeting molecules capable of directing the morphogen to a desired target tissue. Tissue targeting molecules envisioned to be useful in the treatment protocols of this invention include antibodies, antibody fragments or other binding proteins which interact specifically with surface molecules on the target tissue cells.

Still another useful tissue targeting molecule may be part or all of a morphogen precursor "pro" domain.

Morphogens may be synthesized in one tissue and secreted and transported to another tissue. 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, lungs and WO 94/03075 PCT/US93/07190 8 gastrointestinal tract (GI tract, 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. As described below, morphogen species comprising the pro domain may be obtained from the culture medium of morphogensecreting mammalian cells. Alternatively, a tissuetargeting species may be formulated by complexing the mature dimer (or an active fragment thereof) with part or all of a pro domain.

Associated tissue targeting or solubility-enhancing molecules also may be covalently linked to the morphogen using standard chemical means.

In one preferred embodiment, the morphogen comprises part of an infant formula. The infant formula may be milk-based or nonmilk-based, soy-based. A typical ready-to-feed morphogen-enriched formulation for infants, when diluted to feeding concentrations, comprises, in addition to the morphogen added to the formula, from about 1-5% by weight fat, from about 0.01 to about 0.5% by weight immunoglobulins as appropriate, from about 4-10% by weight carbohydrate in a quantity substantially to mimic the carbohydrate content of human mother's milk, from about 0.5 to 4% by weight ~1~1~ WO 94/03075 PCT/US93/07190 9 protein in a quantity substantially to mimic the protein content of human mother's milk, optional vitamins and minerals as required, a total solids content of from about 8 to 17% by weight, and the remainder water.

In another preferred embodiment, the dietary composition is formulated for individuals at risk for reduced or lost tissue function, such as postmenopausal women, elderly individuals, undernourished or malnourished individuals, dehydrated individuals, drowning victims, individuals suffering from metabolic disorders including an endocrine imbalance, gastrointestinal disorders, or immune-compromised individuals. Undernourished or malnourished individuals include those suffering from a lack of food (starvation) and/or eating disorders anorexia nervosa), and/or suffering from a maladsorption syndrome individuals afflicted with digestive or intestinal fistulas, shortened bowel, or hypercatabolism.) Individuals receiving a medical therapy, including radiotherapy, chemotherapy or a surgical procedure also are at risk for reduced or lost tissue function as a result of a therapy-related malabsorption-malnutrition dysfunction. In another embodiment, the dietary supplement is formulated for individuals undergoing periods of increased growth or stress, such as infants and juveniles, or pregnant or lactating women. In another embodiment, the dietary supplement is formulated for individuals at risk for reduced or lost organ function as results from tissue cirrhosis or an autoimmune disease.

WO 94/03075 PCT/US93/07190 10 Morphogen-enriched nutritional products, particularly clinical nutrition products for use in hospital or other clinical settings, in addition to comprising a morphogen preferably are based on the utilization of diverse other protein sources (casein, sodium and calcium caseinate, isolated soy protein, protein hydrolyzates and/or crystalline amino acids) mixtures of vegetable and animal fats, carbohydrates (basically glucose polymers), vitamins and minerals to meet, at least, the dietary intakes recommended for healthy individuals (see, for example, Committee on Dietary Allowances, Food and Nutrition Board, Nat Acad Sci, 9th Ed, 1980).

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-3 super-family of proteins, share substantial amino acid sequence homology in their C-terminal regions. The proteins are translated as a precursor, having an N-terminal signal peptide sequence, typically less than about 30 residues, iollowed 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 domnain, and forms a soluble species that appears to be the primary WO 94/03075 PCT/US93/07190 11 form secreted from cultured mammalian cells. The signal peptide is cleaved rapidly upon translation, at a cleavage site that can be predicted in a given sequence using the method of Von Heijne ((1986) Nucleic Acids Research 14:4683-4691.) Table I, below, describes the various morphogens identified to date, including their nomenclature Ps 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.

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 WO 94/03075 P4rUS93/07190 12 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 (hOPi) and residues 30-291 (mOPi).

"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 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 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 18-2'- (mOP2). (Another cleavage site also occurs 21 residues upstream for both OP-2 proteins.) WO 94/03075 PCT/US93/07190 13 "CBMP2" "DPP(fx)" refers generically to the morphogenically active proteins expressed from a DNA sequence encoding the CBMP2 proteins, including allelic and species variants 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 BMP4 (BMP2B) likely includes residues 25-256 or 25-292; the mature protein, residues 257-408 or 293-408.

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 "Vgl(fx) i _~ls _I_ WO 94/03075 P(T/US93/07190 "Vgr-l(fx)" 14 full length protein appears in Weeks (1987) Cell 51: 861-867. The prodomain 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 prodomain 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 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 prodomain likely extends from the signal peptide clavage 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 "GDF-1(fx)" "60A" ssa~r II WO 94/03075 PCT/US93/07190 "BMP3 fx)" 15 amino acid sequence for the full length p~otein 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 prodomain 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.

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 tulx 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.

I a II WO 94/03075 PCT/US93/07190 16 "'BMP6(fx)" 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 includes extends from the signal peptide cleavage site to residue 37V; the mature sequence likely includes residues 375-513.

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 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.

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 11-11 I WO 94/03075 PCT/US93/07190 17 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 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 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 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 IY- I WO 94/03075 PCT/US93/07190 18 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 :3eq. 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 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 Table 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.

WO 94/03075 WO 9403075PCT'/US93/07 190 19 Generic Sequence 3 Leu Tyr Val Xaa Phe 1 Xaa Xaa Xaa Ala Xaa Gly Trp Xaa Xaa Trp Pro Xaa Gly Xaa Xaa Ala Cys Xaa Gly Xaa Cys Xaa Xaa Xaa Tyr Xaa Pro Xaa Xaa Xaa Xaa Xaa Asn His Ala Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Leu Xaa Xaa Xaa Xaa Xaa Xaa Xaa Pro Xaa Xaa Xaa Xaa Xaa Cy s Xaa Xaa Xaa Xaa Leu Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Val Xaa Leu Xaa Met Xaa Val Xaa WO 94/03075 WO 9403075PCT/US93/07 190 20 Xaa Cys Gly Cys Xaa 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, Gin, 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); 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 (Glu, 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 (Glu, His, Tyr, Asp or Gin); Xaa at res.28= (Glu, 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 (Val 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 Val); Xaa at res.53 (Asn, Lys, Ala or Giu); Xaa at res.54 (Pro or Ser); Xaa at res.55 (Glu, Asp, Asn, or Gly); 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 WO 94/03075 WO 943075PT/US93/07 190 21 (Thr or Ala); Xaa at res.66 (Gin, Lys, Arg or Giu); Xaa at res.67 (Lau, Met or Val); Xaa at res.68 (Asn, Ser or Asp); Xaa at res.69 (Ala, Pro or Ser); Xaa at res.70 (Ile, Thr or Val); Xaa at res.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 Val); Xaa at res.84 (Lys or Arg); Xaa at res.85 (Lys, Asn, Gin or His),- Xaa at res.86 (Tyr or His); Xaa at res.87 (Arg, Gin or Giu); Xaa at res.8B (Asn, Giu or Asp); Xaa at res.90 (Vai, Thr or Ala); Xaa at res.92 =(Arg, Lys, Val, Asp or Giu); 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 Giy Xaa Xaa Ala Xaa Tyr Cys Xaa Gly Xaa Cys Xaa Xaa Pro Xaa Xaa Xaa Xaa Xaa WO 94/03075 WO 9403075PCT/US93/07 190 22 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 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 Glu); Xaa at resell (Arg, Gln, Ser or Lys); Xaa at res.12 (Asp or Glu); Xaa at res.13 (Leu or Val); Xaa at res.16= (Gln, 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 (Glu, His, Tyr, Asp or Gln); Xaa at res.33 Glu, Lys, Asp or Gln); Xaa at (Ala, Ser 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, Glu, WO 94/03075 WO 9403075PCr/US93/07 190 23 Leu or Ala); Xaa at res.41 (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 res.45 (Thr, Leu or Ser); Xaa at res.49 (Ile or Val); Xaa at (Val or Leu); Xaa at res.51 (Gin or Arg); Xaa at res.52 (Thr, Ala or Ser); Xaa at res.54 (Val or Met); Xaa at res.55 (His or Asn); Xaa at res.5'6 (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 (Giu, Asp, or Gly); Xaa at 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 res.65 (Pro or Ala); Xaa at res.68 (Ala or Val); Xaa at res.70 (Thr or Ala); Xaa at res.71 (Gin, Lys, Arg or Glu); Xaa at res.72- (Leu, Met or Val); Xaa at 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, Giu, Asn or Scr); Xaa at res.83 (Ser, Gin, Asn or Tyr); Xaa at res.84 (Ser, Asn, Asp or Giu); Xaa at res.85 (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 Giu); Xaa at res.93 (Asn, Giu or Asp); Xaa at (Val, Thr or Ala); Xaa at res.97 (Arg, Lys, Val, Asp or Glu); Xaa at res.98 (Ala, Gly or Giu); and Xaa at res.102 (His or Arg).

I aL--r I P WO 94/03075 P@/US93/07190 24 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 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), and GDF-1 (from mouse, Seq. ID No. 14), human BMP3 (Seq. ID No. 26), human BMP5 (Seq. ID No. 27), human 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 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.

WO 94/03075 WO 9403075PCT/US93/07 190 25 Generic Sequence Leu Xaa Xaa Xaa Phe Xaa Xaa Xaa Gly Trp Xaa Xaa Pro Xaa Xaa Xaa Xaa Trp Xaa Xaa Xaa Ala Xaa Cys Xaa Xaa Tyr Cys Xaa Gly Xaa Pro Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asn Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Pro Xaa Xaa Xaa Xaa Leu His Ala Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa WO 94/03075 WO 9403075PCI'/US93/07190 26 Xaa Xaa Xaa Xaa Val Xaa Leu Xaa Xaa Xaa Xaa Xaa Met Xaa Val Xaa Xaa Cys Xaa Cys Xaa 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 oi. Lys); Xaa at res.3 Val or Ile); Xaa at res.4 (Ser, Asp or Glu); Xaa at res.6 (Arg, Cln, Ser, Lys or Ala); Xaa at res.7 (Asp, Glu or Lys); Xaa at res.8 (Leu, Val or le); Xaa at res.11 (Gln, Leu, Asp, His, Asn or Ser); Xaa at res.12 (Asp, Arg, Asn or Glu); Xaa at res.14 (Ile or Val); Xaa at res.15 (Ile or Val); Xaa at res.16 (Ala or Ser); Xaa at res.18 (Glu, Gln, 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, Gln, Leu or Gly); Xaa at res.23 (Tyr, Asn or Phe); Xaa at res.26 (Glu, His, Tyr, Asp, Gln or Ser); Xaa at res.28 (Glu, Lys, Asp, Gln or Ala); Xaa at res.30 (Ala, Ser, Pro, Gln or Asn); Xaa at res.31 (Phe, Leu or Tyr); Xaa at res.33 (Leu, Val or Met); Xaa at res.34 (Asn, Asp, Ala, Thr or Pro); Xaa at res.35 (Ser, Asp, Glu, 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); Xac at res.40 (Thr, Leu or Ser); Xaa at res.44 (Ile, Val or Thr); Xaa at res.45 (Val, Leu or Ile); Xaa at res.46 (Gln or Arg); Xaa at res.47= (Thr, Ala or Ser); Xaa at res.48 (Leu or Ile); Xaa at WO 94/03075 WO 9403075PCT/US93/071 27 res.49 (Val or Met); Xaa at res.50 (His, Asn or Arg); Xaa at res.51 (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, Glu, Gly or Phe); Xaa at res.54 (Pro, Ser or Val); Xaa at res.55 (Giu, Asp, Asn, Gly, Val or Lys); Xaa at res.56 (Thr, Ala, Val, Lys, Asp, Tyr, Ser, Ala, Pro or His); Xaa at res.57 (Val, Ala or Ile); Xaa at res.58 =(Pro or Asp); Xaa at res.59 (Lys, Leu or Giu); Xaa at res.60 (Pro or Ala); Xaa at res.63 (Ala or Val); Xaa at res.65 (Thr, Ala or Giu); 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 (Phe, Tyr, Leu or His); Xaa at res.76 (Asp, Asn or Leu); Xaa at res.77 (Asp, Giu, 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 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, Giu or Asp Xaa at res.90 (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).

WO 94/03075 WO 9403075PCT/US93/07 190 28 Generic Sequence 6 Cys 1a Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Leu Xaa Xaa Xaa Phe Xaa Xaa Gly Xaa Pro Xaa Tyr Cys Xaa 30 Pro Xaa Xaa Xaa Xaa Asn 45 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Pro Xaa Xaa Xaa Leu Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys 100 Trp Xaa Gly Xaa His Xaa Xaa Xaa Xaa Val.

Met Xaa Xaa Xaa Trp Xaa Xaa Xaa Ala Xaa Cys Xaa Xaa Xaa Ala Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Leu Xaa Xaa Val Xaa 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 In= WO 94/03075 WO 9403075PCT/US93/07190 29 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 (Vai or Ile); .Xaa at res.9 (Ser, Asp or C2ku); Xaa at res.11 =(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, Asip, His, Asn or Ser); Xaa at res.17= (Asp, Arg, Asn or Giu); 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 (Giu, Gin, Leu, Lys, Pro or Arg); Xaa at res.24 (Gly or Ser); Xaa at res.25 (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 (Giu, His, Tyr, Asp, Gin or Ser); Xaa at res.33 Giu, Lys, Asp, Gin or Ala); Xaa at (Ala, Ser, Pro, Gin or Asn); Xaa at res.36 (Phe, Leu 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 ree,.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, A-la or Ser); Xaa at res.53 (Leu or Ile); Xaa at res.54 (Val or Met); Xaa at (His, Asn or Arj); Xaa at res.56 (Phe, Leu, Asn, Ser, Ala or Val); %aa 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 (Glu, Asr, Gly, Val or Lys); Xaa at res.61 (Thr, Ala, Val, Lys, Asp, TLyr, 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 Glu); Xaa at LL It WO 94/03075 PCT/US93/07190 30 (Pro or Ala); Xaa at res.68 (Ala or Val); Xaa at res.70 (Thr, Ala or Glu); Xaa at res.71 (Gln, Lys, Arg or Glu); 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 res.75 (Ile, Thr, Val or Leu); 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 Leu); Xaa at res.82 (Asp, Glu, Asn or Ser); Xaa at res.83 (Ser, Gin, Asn, Tyr or Asp); Xaa at res.84 (Ser, Asn, Asp, Glu 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 (Lys, Asn, Gin, His or Val); Xaa at res.91 (Tyr or His); Xaa at res.92 (Arg, Gln, Glu or Pro); Xaa at res.93 (Asn, Glu or Asp); Xaa at res.95 (Val, Thr, Ala or Ile); Xaa at res.97 (Arg, 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 b-BI1 LI~ C9~J~L Is WO 94/03075 PCT/US93/07190 31 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 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 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 a reference sequence requires that, following alignment of the candidate sequence with the reference sequence, 70% 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 I ii L~L 11I~- WO 94/03075 PCT/US93/07190 32 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-1 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 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 hOP1 residues 43-139 of Seq. ID No. These most preferred sequences include both allelic and species variants of the OP-1 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 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).

I---L114- -I -d WO 94/03075 PCT/US93/07190 33 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 th& 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 370C overnight, and washing in 0.1 X SSPE, 0.1% SDS at 50 0

C.

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 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 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.

I i, WO 94/03075 PCT/US93/07190 34 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 description of the morphogens useful in the methods, compositions and devices of this invention is disclosed in international application US92/01968 (WO 92/15323) the disclosure of which is incorporated herein by reference.

Thus, in view of this disclosure, skilled genetic engineers can isolate genes from cDNA or genomic libraries of various different species which encode appropriate amino acid sequences, or construct DNAs from oligonucleotides, and then can express them in various types of host cells, including both procaryotes and eucaryotes, to produce large quantities of active proteins useful as dietary compositions for enhancing tissue morphogenesis, including enhancing tissue development and tisssue viability in a variety of mammals, including humans.

L- L -1 WO 94/03075 PCT/US93/07190 35 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.

_C I CI WO 94/03075 PCT/US93/07190 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. IA and B shows relative amounts of protein present in mammary gland extract eluate fractions of a C-18 reverse phase chromatography column and the corresponding results of a Western Blot FIG. 2A and B shows relative amounts of protein present in bovine colostrum eluate fractions from purification scheme A of a C-18 reverse phase chromatography column and the corresponding results of a Western blot under reduced and oxidized conditions FIG. 3A and B shows relative amounts of protein present in bovine colostrum eluate fractions from purification scheme B of a C-18 reverse phase chromatography column and the corresponding results of a Western Blot under reduced conditions FIG. 4A and B shows relative amounts of protein present in bovine 57 day milk eluate fractions of a C- 18 reverse phase chromatography column and the corresponding results of a Western Blot under reduced and oxidized conditions I 5-1 L I lII 4 WVO 94/03075 PCT/US93/07190 37 FIG. 5 shows Western Blot analysis of bovine colostrum using OP-1 and BMP2-specific antibodies; FIG. 6A and B show results of in vivo and in vitro activity assays, respectively, for the corresponding fractions shown in Fig. 1; FIG. 7 is a photomicrograph of an immunoblot showing the presence of hOP-1 in serum; and FIG. 8A is a dose response curve for the induction of the 180 kDa and 140 kDa N-CAM isoforms in morphogentreated NG108-15 cells; FIG. 8B is a photomicrograph of a Western blot of whole cell extracts from morphogen-treated NG108-15 cells with an N-CAM-specific antibody; and FIG. 9 (A and B) are photomicrographs showing the effect of morphogen-specific antibody on mouse development (9B) compared to untreated, control mice (9A).

WO 94/03075 PCT/US93/07190 38 Detailed Description of the Invention It now has been discovered that the proteins described herein are found in nursing mother's milk and are useful as components of a dietary composition for enhancing tissue morphogenesis in a mammal, particularly in an individual at risk for normal tissue development and vibility. As described herein, these proteins ("morphogens") are capable of enhancing tissue development in growing mammals, stimulating CAM expression and maintaining the normal tissue function in adult tissue.

Provided below are detailed descriptions of suitable morphogens useful in the compositions and methods 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 active components of a dietary composition for a mammal; and 2) provide assays with which to test candidate morphogens for their efficacy. Specifically, examples are provided which demonstrate the presence of endogenous morphogen in milk and human serum (Examples 1 and 2), demonstrate the ability of morphogens to induce CAM expression in a mammal (Example demonstrate the ability of morphogens to enhance tissue development in developing embryos (Example 4) and juveniles (Example demonstrate the ability of morphogens to reduce an osteoporotic condition in a mammal (Example demonstrate the presence of morphogens in developing tissues and adult stomach and gut tissue, demonstrate the ability of parenterally provided ~I e I ~L 'a rL ldl WO 94/03075 PCT/US93/07190 39 morphogen to localize to stomach tissue, and describe protocols for identifying morphogen-synthesizing tissue (Example 7) and describe protocols for obtaining morphogen-specific antibodies and measuring phogens in solution (Example 8).

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 or its functional equivalent (see supra).

Specifi "lly, 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 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 sequences disclosed therein. Alternatively, novel morphogenic sequences may be identified following the procedures disclosed therein.

c- -L I LI~- lll~_sPII WO 94/03075 PCT/US93/07190 40 Particularly useful proteins include those which comprise the naturally derived sequences disclosed in Table II. Other useful sequences include 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-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 include, at their N-terminus, the sequence Cys Xaa Xaa Xaa Xaa (Seq. ID No. 1 Table II, set forth below, compares the amino eaid 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), CBMP2B (Seq. ID No. 10), BMP3 (Seq. ID No. 26), DPP (from Drosophila, Seq. ID No. 11), Vgl, (from Xenopus, Seq. ID No. 12), Vgr-1 (from mouse, Seq. ID No. 13), L- II WO 94/03075 PCT/US93/07190 41 GDF-1 (from mouse, Seq. ID Nos. 14, 32 and 33), protein (from Drosophila, Seq. ID Nos. 24 and 25), (Seq. ID No. 27) and BMP6 (Seq. ID No. 28). 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, 59), with CBMP-2A then comprising Lys and Ile, whereas CBMP-2L comprises Ser and Ile.

TABLE II hOP-1 mOP-1 hOP-2 m0P-2

DPP

Vgl Vgr-1 CBHP-2A CBMP-2B Cys Lys Lys His Glu Leu Tyr Val Arg Arg Arg Arg Arg Arg Lys Ser Arg His Gly Pro Ser Arg Arg Arg

I

WO 94/03075 WO 9403075PCT/US93/07 190 42 BMP3 GDF- 1 BHP6 Ala Arg Arg Tyr Arg Ala Arg Arg Gin Met Giu Thr Arg Lys hOP-1 MOP-i hOP-2 mOP -2

DPP

Vgi Vgr- 1 CBMP-2A CBMP-2B BiP 3 GDF- 1 60A BliP 6 hOP-i MOP-i hOP -2 mOP -2

DPP

Vgi Vgr-i1 CBMP-2A Ser Ser Asp Glu Asp Asp Asp Asp Trp Phe Ile Val Val Vai Asp Giu Ala Leu Val Val Vai Val Val Ile Val Pro Giy Trp Gin Asp Asn Giu Arg Aia Ser Ser Asp Met His Giu Gin Gin Leu Gin Lys Pro WO 94/03075 WO 9403075PCT/US93/07190 43 CBMP-2B BMP3 GDF- 1

BP

BMP 6 Val Val Pro Ser Lys Ser Phe Arg Phe Lys hOP-i mOP-1 hOP -2 mOP -2

DPP

Vgl Vgr-1 CBHP-2A CBMP-2B BMP3 GDF-1 BiP 6 Tyr Asn Asn Phe Phe Asn Phe Phe Asn Cys Glu His Tyr Asp His His Ser Gin Ser Asp Asp Asn Ser Asp Asp Ala Asp Thr Glu Ala Ala Asp Ala Asp Gly Glu Cys Lys Glu Asp Ala Gin Ala Ser Pro Pro Ser Pro Pro Gin Asn Se r Ser Ala Ser Gly Ser Ser hOP-i mOP-i hOP -2 [fop -2

DPP

Vgl Vgr-1 CBMP-2A CBMP-2B Phe Tyr Mlet Asn Phe Leu Leu Leu WO 94/03075 WO 9403075PCr/US93/07190 44 GDF- 1 BMP3 BMP6 Val Met hOP-1 mOP-i h0P-2 mOP-2

DPP

Vgl Vgr- 1 CBMP-2A CBMP-2B BMP3 GDF-i1 BMP6 Thr Ser Ser Leu Leu Lys Ala Ala Ala Ile Val Thr Val Ser Ser His His His Gly Leu Met Met Ser** Lys Pro hOP-i1 MOP-i hOP-2 mOP-2

DPP

Vgl Vgr-l1 CBHP-2A CBHP-2B BHP 3 His Phe Ile Asn His Leu Met Lys His Leu Met Lys Asn Asn Asn Ser Glu Val Met Asn Ser Val Asn Ser Val Arg Ala** Gly Val Glu Thr Asp Asn Ala Asp Val Gly Lys Asp Tyr Lys Ser Pro Gly Val Ile Ile Ile Ile WO 94/03075 WO 9403075PCT/US93/07 190 45 GDF- 1 BMP 6 Met Ala Ala Ala Gly Leu Leu Giu Lys Leu Met Phe Asp Leu Met Ala Ala Lys His Tyr hOP-i MOP-i hOP-2 niOP-2

DPP

Vgi Vgr-1 CBMP-2A CBMP-2B BMP3 GDF- 1 BHP5 BMP 6 hOP-i1 MOP-i hOP -2 niOP-2 Vgl Vgr-1

DPP

CBHP-2A CBMP-2B BHP3 GDF- I Pro Lys Pro Cys Cys Ala Pro Thr Gin Asp Leu Met Val Met Ala Ala Leu Ala Ala Glu Leu Asn Ala Ile Ser Thr Ser Thr Ser Pro Asp Ser Val Ser Ser Ser Ser Leu Ser Pro Val Val Val Val Val Val Vai Met Met Met Met Ile Lys Lys Lys Lys Giu Giu Glu Lys Ala Arg Arg Lys Lys Tyr Phe Tyr Tyr Phe Tyr Leu Leu Leu Phe Tyr Phe Ser Ala WO 94/03075 WO 9403075PCT/US93/07 190 46 BMP6 hOP -1 mOP-i hOP-2 mOP-2

DPP

Vgi Vgr-i CBMP-2A CBHP-2B BMP3 GDF- 1 BiP 6 Asp, Asn Leu Leu Ser Ser Asn Asn Gin Asn Asp Asn Asn Glu Tyr Asp Asn Lys Asp Asp Giu Asn Ile Val Vai Val Vai Val Val Asn Val His Lys Arg Arg Arg Arg hop-i MOP-i hOP-2 mOP-2

DPP

Vgi Vgr-i CBMP-2A CBIP- 2B Lys Asn His Asn Asn Arg Asn Gin Giu Giu Gin Asp Gin Giu Arg Lys Lys Vai Asp Giu Giu WO 94/03075 WO 9403075PCr/US93/07 190 47 BMP3 GDF- i BMP6 Val Pro Gin Glu Asp Trp Thr Ile Glu Asp Lys hOP-1 MOP-i1 hOP-2 nOP -2

DPP

Vgl Vgr-1 CBHP-2A CBMP-2B B14P3 GDF- 1 60A BHP 6 Aia Cys Gly Cys His Giy Glu Gly Giy Ser Giu Se r Se r ~idues Arg Arg Arg Arg Aia Arg Arg 100 56 and 57 **Between res of BMP3 is a Val residue; between residues 43 and the amino acid sequence 44 of GDF-l lies 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 I I WO 94/03075 PCT/US93/07190 48 the GDF-1 protein sequence depicted in Table II shares only about 50% amino acid identity with the hOP1 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'1 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 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 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).

C 9__ WO 94/03075 PCT/US93/07190 49 II. Formulations and Methods for Administering Therapeutic Agents A. General Considerations The morphogens may be provided to an individual by any suitable means, most preferably orally, or, alternatively, parenterally. Where the morphogen is to be provided parenterally, such as intravenously or by enteral feeding tube, the morphogen preferably comprises part of an aqueous solution. The solution is 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 physiologic saline (0.85% NaCl, 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 physiologically buffered 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 obtained from the culture medium of morphogen-secreting mammalian cells. Alternatively, a I- lm11 WO 94/03075 PCT/US93/07190 50 soluble species may be formulated by complexing via non-covalent interaction) the mature dimer (or an active fragment thereof) with part or all of one or, preferably, two pro domain peptides (see Section A.1, below). Another molecule capable of enhancing solubility and particularly useful for oral administrations, is casein, including derivatives and analogs thereof. For example, addition of 0.2% casein increases solubility of the mature active form of OP-1 in physiologically buffered solutions by 80%. Other components found in milk and/or 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. Biocompatible, preferably bioresorbable, polymers, including, for example, hyaluronic acid, collagen, polybutyrate, tricalcium phosphate, lactide and lactide/glycolide copolymers, may be useful excipients to control the release of the morphogen in vivo.

As described above, the dietary supplements comprising the morphogens described herein preferably are provided orally. 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 O C-L, y WO 94/03075 PCT/US93/07190 51 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, has been identified in mammary gland extract, colostrum and 57 1ay milk (see Example 1, below). 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, the morphogen also is detected in the bloodstream (see Example 2, below). These findings indicate that oral and parenteral administration are 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 (and mammary gland extract and colostrum) is readily soluble, probably by association of the mature, morphogenically active form with part or all of at least one pro domain peptide and/or by association with one or more milk components. Accordingly, the compounds provided herein also may be associated with molecules capable of enhancing their solubility in vitro or in vivo.

The dietary compositions for oral administration may be formu? ted as a liquid, for example, as part of an aqueous medium as described above for parenteral administration, and which further may contain flavoring and coloring agents. The formulation also may be combined with a beverage or may be provided in a syrup. The dietary composition also may be provided as s L- I L WO 94/03075 PCT/US93/07190 52 an aerosol for oral or nasal administi. "on.

Formulations for inhalation administrat 3n contain as excipients, for example, lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9lauryl ether, glycocholate and deoxycholate, or oily solutions for administration in the form of nasal drops, or as a gel to be applied intranasally.

Alternatively, the dietary composition may be provided as a solid, for example as a tablet, capsule or lozenge. As for parenteral administration, formulations for oral administration also may include molecules to enhance a controlled release of the morphogen in vivo.

As will be appreciated by those skilled in the art, the concentration of the compounds described in a given dietary supplement composition will vary depending upon a number of factors, including the dosage number to be administered, the chemical characteristics hydrophobicity) of the compounds employed, and the route of administration. The preferred dosage to be administered also is likely to depend on such variables as the type and extent of tissue development enhancement desired, the type and extent of any tissue damage present to De repaired, the overall health status of the particular individual, the relative biological efficacy of 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 a formulation containing about 0.001 to 10% w/v of morphogen to formulation. Typical dose ranges are from about ng/kg to about 1 g/kg of body weight per day; a preferred dose range is from about 0.1 pg/kg to I U L L~ WO 94/03075 PCT/US93/07190 53 100 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 individual. No obvious morphogen induced pathological lesions are induced when mature morphogen OP-1, 20 pg) is administered daily 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 parenterally 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. In all cases administration dosages then can be monitored by measuring at intervals the levels of the morphogen in the blood.

A.1 Soluble Morphogen Complexes A currently preferred form of the morphogen useful in therapeutic formulations, having improved solubility 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 I- I a WO 94/03075 PCT/US93/07190 54 also preferably comprise at least the N-terminal eighteen amino acids that define a given morphogen 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 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 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 I L~IIL_ _II sil WO 94/03075 PCT/US93/07190 55 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 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 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 encoding 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 respectively.

A.1A Isolation of Soluble morphogen complex from conditioned media or body fluid Morphogens are expressed from mammalian cells as soluble complexes. Typically, however the complex is 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 I- L ~ae WO 94/03075 PCT/US93/07190 56 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 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.

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.)

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).

Ib aLi IL 4 1~~ WO 94/03075 PCT/US93/07190 57 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 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 applied to a Sephacryl S-200HR column equilibrated in 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 HEFES (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 buffer. The soluble OP-1 complex then is eluted with mM imidazole (pH 8.0) in 20 mM HEPES and 500 mM NaCl.

P LI II I WO 94/03075 PCT/US93/07190 58 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 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 mL/minute collecting 10 mL fractions. The apparent 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 one mature OP-1 dimer (35-36 kDa) associated with two a, WO 94/03075 PCT/US93/07190 59 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 ad 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 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.

I s WO 94/03075 PCT/US93/07190 60 A.1B. 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 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 (GuHCl), 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.

L l L WO 94/03075 PCT/US93/07190 61 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 proteins.

A.1C. Stability of Soluble Morphogen Complexes The stability of the highly purified soluble morphogen complex in a physiological buffer, e.g., tris-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 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.

91-~11~ Osll Ir 1 I WO 94/03075 PCT/US93/07190 62 B. Considerations for Infant and Other Formulas 1. Infant Formulas In all cases the morphogens of this invention preferably are added to an infant formula that complies with the nutritional guidelines provided by the 'AAP and ESPGAN. Basic ingredients for infant formulas include cow's milk, protein, whey proteins, casein and its salts calcium caseinate). Soy protein isolates may be substituted for milk-derived proteins, and preferably are used in the produk made for infants with lactose intolerance and/or cow's protein intolerance. Protein hydrolyzates casein and lactalbumin hydrolyzates) with low molecular weight, also may be used for these products.

The proportions of the diverse component nutrients preferably are similar to those of human milk. Thus, the ratio of whey proteins to casein preferably varies from 60:40 to 70:30 in infant formulas based on milk.

The mixture of fats employed is made up of edible fats to provide an essential fatty acid profile. Lactose preferably is used as the carbohydrate source for at-term newborns infants, and dextrinmaltose preferably is employed in products used for the treatment of lactone intolerance and malabsorption syndromes in infancy.

Infant formulas according to the invention also preferably contain minerals (including calcium, phosphorus, sodium, potassium, chloride, magnesium, iron, zinc, copper, manganese and iodine) and vitamins (including vitamin A, vitamin D3, vitamin C, vitamin I- IPIL-4b~41 -IP11 WO 94/03075 PCT/US93/07190 63 Bl, vitamin B2, vitamin B6, vitamin B12, pantothenic acid, vitamin E, vitamin Kl, folic acid, biotin) adequate for the infants' requirements. Also, in the products whose source of proteins is derived from soy or protein isolates or hydrolyzates, carnitine preferably is included to satisfy the nutritional requirements for this compound in infants with malabsorptive syndromes.

A typical ready-to-feed morphogen-enriched formulation for infants, when diluted to feeding concentrations, preferably comprises in addition to the added morphogen, from about 1-5% by weight fat, from about 0.01 to about 0.5% by weight immunoglobulins as appropriate, from about 4-10% by weight carbohydrate in a quantity substantially to mimic the carbohydrate content of human mother's milk, from about 0.5 to 4% by weight protein in a quantity substantially to mimic the protein content of human mother's milk, optional vitamins and minerals as required, a total solids content of from about 8 to 17% by weight, and the remainder water.

A typical protein source for use in infant formula is electrodialyzed whey or electrodialyzed skim milk or milk whey, although other protein sources are also available and may be used. Preferred sugars include food grade substances such as glucose, dextrose, sucrose, or edible lactose. The following vitamins and minerals may also be incorporated in the infant formula: calcium, phosphorus, potassium, sodium, chloride, magnesium, manganese, iron, copper, zinc, selenium, iodine, and vitamins A, E, D, and B complex.

WO 94/03075 PCT/US93/07190 64 These micronutrients are added in the form of commonly accepted nutritional compounds in amounts equivalent to those present in human milk on a per calories basis.

The infant formula according to the present invention also preferably is sterilized and subsequently used on a ready-to-feed basis, or can be stored as a concentrate. The concentrate can be prepared using standard procedures known in the art, and the formula can be reconstituted by rehydrating the concentrate. The infant formula preferably is a stable liquid and has a suitable shelf life. A more detailed description of infant formula considerations, including preferred formulations for newborn, preterm and low birth-weight infants, lactose-intolerant infants, may be found, for example, in US Pat. No. 5,066,500 to Gil et al., the disclosure of which is incorporated herein by reference.

2. Other Nutritional Products The morphogen-enriched dietary products for balanced nutrition dietary food formulations) according to the present invention, preferably have, in addition to added morphogen, a composition of nutrients adequate to the specific requirements of not only healthy human in need of a balanced nutritional product, but also those individuals at risk for lost or reduced tissue function due malnutrition-maladsorption disorder, and/or altered metabolism. Individuals particularly affected by an altered metabolic function include postmenopausal women or aged individuals, hypercatabolic individuals, and individuals undergoing periods of rapid growth or physical stress, such as I- LL~~_ WO 94/031075 PCT/US93/07190 65 developing juveniles, and pregnant, lactating and nursing mothers. Other individuals at risk are those suffering from malnutrition, induced, for example, by starvation and/or an eating disorder, and individuals affected with energy-protein malnutrition and in hypercatablic states derived from traumatic, septic, surgical processes and other clinically-derived malabsorption syndromes.

Morphogen-enriched nutritional products according to the present invention preferably also provide mineral elements which include trace elements and vitamins in adequate proportions to satisfy the specific requirements of normal healthy individuals as well as individuals at risk, such as those suffering malabsorption-malnutrition processes and in a hypercatabolic state. The nutritional products also preferably are enriched with amino acids sources, vitamins, nucleosides and/or nucleotides in similar amounts to those present in ordinary foods.

As described above for infant formulas, liquid products may be formulated ready for consumption or as concentrates to be diluted before use. Preferably, liquid dietary compositions have pH values generally ranging from about 6.0 to about 8.0, most preferbly 6.8-7.5.

I I ler~l WO 94/03075 PCT/US93/07190 66 Useful dietary compositions and considerations for their formulation are well described in the medical and nutritional arts. Useful compositions for clinical nutrition, also are described in detail in US Pat.No. 5,066,500.

III. Examples Example 1. Determination of the Presence of Morphogen in Milk Morphogenically active OP-1 was demonstrated to be present in mammary gland extract, colostrum, and milk, as described below. The discovery that the morphogen naturally is present in milk, together with the known observation that mature, active OP-1 is acid-stable and protease-resistant, indicate that oral administration is a 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, including colostrum, indicates that the protein plays a significant role in tissue development, including skeletal development of juveniles.

Rat mammary gland extract and bovine colostrum and 57 day milk were subjected to purification procedures designed to partially purify OP-1. The 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.

I WO 94/03075 PCT/US93/07190 67 1.1 Purification The purification protocol for all three "milk" forms mammary gland extract, colostrum and 57day milk), involved three chromatography steps: (1) cation-exchange chromatography (S-Sepharose and followed by Phenyl-Sepharose chromatography); (2) Copper-Immobilized Metal Affinity chromatography (Cu++-IMAC); and finally, C-18 reverse phase chromatography. Fractions were sampled at each step for the presence of OP-1. Fraction samples for testing were dialyzed versus water/0.1% TFA, then against acetonitrile/0.1% TFA for analysis on SDSpolyacrylamide gels and immunoblots, using standard methodologies well described in the art. Unless otherwise stated, the primary antibody used for the immunoblots was made against full length OP-1 produced in E.coli using standard recombinant DNA and antibody production techniques (see, for example, Example 8, below for a general description for producing morphogen-specific antibodies.) Fractions found to contain the morphogen then were applied to the next column step or used in the immunoreactivity or activity assays described below.

Essentially the same protocol was followed for all three milk sources, except that two alternative cationexchange methodologies were employed for colostrum purification, described in detail below. Unless -n _L1 dl WO 94/03075 PCT/US93/07190 68 otherwise indicated, all chemicals referenced are standard, commercially available reagents, readily available from a number of sources, including Sigma Chemical, Co., St. Louis; Calbiochem, Corp., San Diego and Aldrich Chemcial Co., Milwaukee.

step 1. Cation-Exchange Chromatography The S-Sepharose purification step was performed as follows. 200ml of cation exchanger (S-Sepharose, Sigma Chemical Corp.) were equilibrated with equilibration buffer (6M urea, 20mM MEb, 70mM NaCl, pH The supernatant from the centrifuged extract was diluted to final concentration of 6M urea, 20mM MES, 70mM NaCI, pH 6.5. After loading, the column was washed to baseline using equilibration buffer, and the bound components were eluted stepwise from the column with 6M urea, MES, 100mM and 500mM NaCl, pH 6.5. The more tightly bound components then were eluted with 4M guanidine, 20mM sodium phosphate, pH The Phenyl-Sepharose purification step was performed as follows. 15ml of Phenyl-Sepharose CL-4B (Sigma) were equilibrated with 6M urea, 20mM HEPES, 1M ammonium sulfate, 300mM NaCl, pH 7.0. The 500mM NaCl eluate from the S-Sepharose step was diluted with 6M urea, 20mM HEPES, 3M ammonium sulfate, 300mM NaC1, pH to a final concentration of 1M ammonium sulfate, pH 7.0. After loading, the column was washed to baseline with equilibration buffer. The column was eluted with 6M t:rea, 20mM HEPES, 0.6M ammonium sulfate, 300mM NaC1, pH 7.0, and then with 4M guanidine, sodium phosphate, pH I- _q ~b WO 94/03075 PCT/US93/07190 69 Two alternative cation-exchange chromatography schemes (A and B) were exploited in the purification of OP-1 from colostrum, as follows. For both schemes, 200 ml of S-Sepharose (Sigma) was poured into a 5 X 10 cm Bio-Rad econocolumn (Bio-Rad, Inc. Cambridge.) Scheme A: The colostrum, which had been diluted to 6M urea, 20mM sodium phosphate, pH 7.0, was loaded onto a column equilibrated with 6M urea, 20mM sodium phosphate, 50mM NaC1, pH 7.0. Elution was stepwise, with 6M urea, 20mM sodium phosphate, 100mM and then 500mM NaC1, pH 7.0; and the final wash was with 4M guanidine, 20mM sodium phosphate, pH 7.0. The Phenyl- Sepharose column was run as described above, except that sodium phosphate was used as the running buffer instead of HEPES. The Phenyl-Sepharose bound fraction (0.OM ammonium sulfate eluate) from scheme A then was dialyzed into 6M urea, 20mM Hepes, 500mM NaC1, pH before it was applied to the Cu++-IMAC column, which was run as described below.

Scheme B: The alternative S-Sepharose purification was performed as follows. Ethanol-precipitated protein was loaded onto an S-Sepharose column equilibrated in 6M urea, 20mM MES, 50mM NaC1, pH 6.5. Elution was stepwise with 6M urea, 20mM MES, 100mM NaCl and then 500mM NaC1, and finally 4M guanidine, 20mM sodium phosphate, pH 7.0. The Phenyl-Sepharose column was run as described above, with the 0.OM ammonium sulfate eluate then applied to a Cu++-IMAC column.

e~-qLI- -C WO 94/03075 PCT/US93/07190 70 step 2. Cu++IMAC Chromatography The Cu++IMAC purification step was performed as follows. 10ml of Pharmacia Fast Flow Chelating Resin were charged with 0.2M cupric sulfate, and equilibrated with 6M urea, 20mM HEPES, 0.5M NaC1, pH 7.0. After loading, the column was washed to baseline with equilibration buffer. Elution from the column was stepwise, using equilibration buffer containing 1mM, 5mM, or 10mM imidazole. The column then was stripped with equilibration buffer containing 10mM EDTA. The imidazole elution was dialyzed against water/0.1% TFA, then against 30% acetonitirile/0.1% TFA.

step 3. Reverse Phase Chromatography The C-18 reverse phase chromatography purification step was performed as follows. A HPLC C-18 semi-prep column was used for the final purification step. The gradient used was 30-50% acetonitrile/0.1% TFA over minutes at 3ml/minutes. After the sample was loaded, the column was washed to baseline with acetonitrile/0.1% TFA before the gradient is started.

Fractions collected were 3ml in size. Chromatograms were read at 214 nm.

OP-1 from Rat Mammary Gland Extract Mammary glands were obtained from 2 female Long Evan rats (Charles River Labs, Wilmington, MA) one week post-partum. The excised glands were mildly homogenized in 6M urea, 20mM methylethansulfonate ~arlllllI -L WO 94/03075 PCT/US93/07190 71 (MES), 0.5M NaC1, pH 6.5 using a polytron homogenizer.

The suspension then was centrifuged for 20 minutes at 8,000 RPM, and the supernatant removed for further purification.

Following S-Sepharose chromatography, fractions containing 6M urea, 20mM MES containing 500mM NaCI, also appeared to contain OP-1 as determined by SDS and immunoblot, and were applied to the Phenyl-Sepharose column. The eluate from the 6M urea, 20mM HEPES, 300mM NaCl, pH 7.0 elution step from this column were found to contain OP-1. This .uate then was applied to a Cu++-IMAC column. Eluata fractions found to contain OP-1 were then applied to the C-18 column and chromatographed as described.

Figure 1(A) shows the chromatogram and 1(B) the corresponding Western blot for fractions from the C-18 reverse phase chromatography step run under reducing conditions. Lane S of the Western blot is a standard, containing reduced, purified, recombinantly-produced OP-1. The arrows show molecular weight markers corresponding to 17, 27, and 39 Kd. The reduced monomer run at approximately 16-18 Kd; the oxidized homodimer at approximately 36 Kd. Lanes 13-30 represent the corresponding fractions of the C-18 reverse phase column as numbered in Fig.l(A). As can be seen in Fig.l(B), mammary extract OP-1 elutes primarily in fractions 21-25 from this final chromatography step.

IL WO 94/03075 PCT/US93/07190 72 OP-1 from Bovine Colostrum Colostrum is the first milk to be produced by the mother following birth. Approximately 5 gallons of bovine colostrum were obtained from a local dairy farm and delipidated by centrifugation (8000 rpm for approximately 10 min. at 4 0 The supernatant then was filtered through cheese cloth. The filtered supernatant was stored in 500ml aliquots at 70 0

C.

ml of colostrum were diluted with 100ml of 9M urea, 30mM sodium phosphate, pH 7.0. Alternately, of colostrum was added to 50ml of 8M guanidine-HC1, Tris, pH 7.2 and precipitated with 40%, then ice cold ethanol. The pellet was washed with 90% cold ethanol and lyophilized overnight. The lyophilized pellet was resuspended in 6M urea, 20mM MES, 500mM NaC1, pH 6.5, stirred overnight at 4°C, and centrifuged at 9,000 RPM for 10 minutes to clarify the suspension before loading onto the column as described in schemes A and B, above.

Following S-Sepharose chromatography by scheme A, both the 100mM and the 500 mM eluate fractions were found to contain OP-1, with the 100mM fraction containing relatively more morphogen. This fraction then was loaded onto the Phenyl-Sepharose column following dilution with an equal volume of 6M urea, mM sodium phosphate, 2M ammonium sulfate, and 300mM NaC1.

W'O 94/03075 PCT/US93/07190 73 Following S-Sepharose chromatography by scheme B, the 500mM NaCI eluate was found to contain OP-1 and was loaded onto a Phenyl-Sepharose column as described above, following dilution with 6M urea, 40mM HEPES, 2M ammonium sulfate, pH Following Cu++IMAC chromatography OP-1 was identified in the 5mM and 10mM imidazole eluates for both purification schemes, and was dialyzed for further purification on the C-18 column.

Both purification schemes produce purified OP-1, as determined by immunoblot. Figure 2 shows the chromatogram and corresponding Western blot for results of purification scheme A (Fig. 2B-1, reduced and Fig. 2B-2, oxidized); and Figure 3 shows the chromatogram and Western blot reduced) for C-18-purified protein from scheme B. As for Fig. 1B, lane S in Figs. 2B and 3B is a stand-rd, containing purified, recombinantly produced OP-1; 17, 27 and 39 are molecular weight markers, and lane numbers correspond to fraction numbers in the corresponding chromatograms. OP-1 purified by scheme A appears predominantly in fractions 18-27, and OP-1 purified by scheme B appears predominantly in fractions 18-25.

OP-1 from 57-day milk Milk was obtained from the same cow from which the colostrum came, 57 days after the birth of the calf.

The milk was delipidated by centrifugation at 10,000 RPM for 15 minutes, and the milk was poured off and away from the fat layer.

WO 94/03075 PCr/US93/7190 74 100ml of milk then were diluted with 200ml of 9M urea, 30mM MES, pH 6.5 and loaded onto a 200ml S- Sepharose column which had been equilibrated with 6M urea, 20mM MES, 50mM NaC1, pH 6.5. Elution was with 6M urea, 20mM MES, 100mM and 500mM NaC1, and 4M guanidine, sodium phosphate, pH 7.0. The 500mM elution was put over a Phenyl-Sepharose column after being diluted with an equal volume of 6M urea, 20mM MES, 2M ammonium sulfate, pH The Phenyl-Sepharose column then was run as described above. The Phenyl-Sepharose-bound sample was eluted and applied to a Cu++IMAC column, prepared and run as described above. The 10mM imidazole eluate was found to contain OP-1 and was dialyzed for further purification on the C-18 column.

The C-18 reverse phase chromatography column and gradient were performed as described above. The results are presented in Fig. 4A (chromatogram) and 4B (immunoblot, 10B-1, oxidized; 4B-2, reduced.) As above, lane S is a standard, containing purified, recombinantly produced OP-1; 17, 27, and 39 are molecular weight markers, and the lane numbers correspond to the fractions numbers in Fig. 4A. OP-1 purified from 57-day milk appears predominantly in fractions 18-26.

1.2 OP-1 Characterization by immunoreactivity OP-1 purified from the different milk sources as described above also were characterized by Western blotting using antibodies raised against OP-1 and BMP2.

Antibodies were prepared using standard immunology _I WO 94/03075 PCT/US93/07190 75 protocols well known in the art, and as described in Example 8, below using full-length E. coli-produced OP- 1 and BMP2 as the immunogens.

As shown in Fig. 5 OP-1 purified from colostrum reacts with the anti-OP-1 antibody, but not with anti-BMP2 antibody. In Fig. 5A and B, lane 1 contains reduced, purified, recombinantly-produced OP-1; lane 2 contains C-18 purified bovine colostrum, and lane 3 contains reduced COP-16, a biosynthetic construct having morphogenic activity and an amino acid sequence modeled on th: proteins described herein, but having highest amino acid sequence homology with BMP2 (see US Pat. No. 5,011,691 for the COP-16 amino acid sequence.) In Fig. 5A the gel was probed with anti-OP-1 antibody; in Fig. 5B, the gel was probed with anti-BMP2 antibody.

As can be seen in the figure, anti-OP-1 antibody hybridizes with protein in lanes 1 and 2, but not 3; while anti-BMP2 antibody hybridizes with lane 3 only.

C-18 purified mammary gland extract and 57-day milk also were shown to renct with anti-OP-1 antibodies, including antibody raised against the full length E.

coli OP-1, full length mammalian-produced OP-1, and the OP-1 Ser-17-Cys peptide the OP-1 N-terminal 17 amino acids).

1.3 OP-1 Characterization by Activity The morphogenic activity of OP-1 purified from mammary gland extract was evaluated in vivo as follows. 33% of each OP-1 immunoreactive fraction of C-18-purified mammary gland extract was lyophilized and resuspended in 220pl of 50% acetonitrile/0.1% TFA. After

I

WO 94/03075 PCT/US93/07190 76 vortexing, 25 mg of collagen matrix was added. The samples were lyophilized overnight, and implanted in 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.

The results are presented in Fig. 6A, where activity" refers to the of bone formation/total area covered by bone in the histology sample. In the figure, solid bars represent implants using mammary extract-derived OP-1, where the fraction numbers correspond to the related fractions eluted from the C-18 reverse phase column (see Fig. 1B), and the hatched bar represents implants using recombinantly produced OP-1 (600 ng). The results demonstrate that the peak bone forming activity of C-18-purified mammary gland extract corresponds with the immunoreactive fraction peaks of Fig. 1B (compare Fig. 6A and 1B.) Similarly, the morphogenic activity of OP-1 purified from mammary gland extract was evaluated in vitro by measuring alkaline phosphatase activity in vitro using the following assay. Test samples were prepared using 15-20% of individual immunoreactive fractions from the C-18 run which were precipitated and resuspended in a smaller volume of acetonitrile/0.1% TFA. Alkaline phosphatase activity was tested using ROS 17/2.8 cells (Rat Osteosarcoma, obtained, for example, from Dr. Robert J.

Majeska, Mt. Sinai Medical Center, New York, NY, in a

_I

WO 94/03075 PCF/US93/07190 77 standard alkaline phosphatase activity assay (see, for example, U.S. Pat. No. 4,968,590). The results, presented in Fig. 6B, indicate that the immunoreactive fractions obtained from C-18-purified mammary gland extract correspond with alkaline phosphatase activity in vitro (compare Fig. 6B and Fig. IB.) In Fig. 6B solid bars represent assays performed with mammary gland-purified OP-1, where the fraction numbers correspond to the related fractions eluted from the C-18 reverse phase column (see Fig. 1B), the hatched bar represents the assay performed with purified, recombinantly-produced OP-1 (100ng/ml), and the cross-hatched bar represents background. As for Fig. 6A, alkaline phosphatase activity corresponds with immunoreactivity of the C-18-purified extract (compare Fig. 6B and lB.) Example 2. Morphogen Identification in Human Serum OP-1 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 8, was immobilized by passing the antibody over an activated agarose gel Affi-GelT 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 PO 4 pH 7.0, applied to a C8 HPLC column, and eluted with a 20 minute, 25-50% acetonitrile/0.1% TFA gradient. Mature, recombinantly produced OP-1 homodimers elute between 20-22 minutes.

WO 94/03075 PCT/US93/07190 78 Fractions then were collected and tested for the presence of OP-1 by standard immunoblot. Fig. 7 is an immunoblot showing OP-1 in human sera under reducing and oxidized conditions. In the figure, lanes 1 and 4 are OP-1 standards, run under oxidized (lane 1) and reduced (lane 4) conditions. Lane 5 shows molecular weight markers at 17, 27 and 39 kDa. Lanes 2 and 3 are human sera OP-1, run under oxidized (lane 2) and reduced (lane 3) conditions.

Example 3. Morphogen-Induced CAM Expression The morphogens described herein induce CAM expression as part of their induction of morphogenesis.

CAMs are morphoregulatory molecules identified in all tissues as an essential step in tissue development. N- CAMs, which comprise at least 3 isoforms (N-CAM-180, N- CAM-140 and N-CAM-120, where "180", "140" and "120" indicate the apparent molecular weights of the isoforms as measured by polyacrylamide gel electrophoresis) are expressed at least transiently in developing tissues, and permanently in nerve tissue. Both the N-CAM-180 and N-CAM-140 isoforms are expressed in both developing and adult tissue. The N-CAM-120 isoform is found only in adult tissue. Another neural CAM is L1.

CAMs are implicated in normal tissue development; N-CAMs are implicated in appropriate neural development, including appropriate neurulation, neuronal migration, fasciculation, and synaptogenesis.

Inhibition of N-CAM production, as by complexing the molecule with an N-CAM-specific antibody, inhibits retina organization, including retinal axon migration, and axon regeneration in the peripheral nervous system, WO 94/03075 PCT/US93/07190 79 as well as axon synapsis with target muscle cells.

CAMs also have been postulated as part of a morphoregulatory pathway whose activity is induced by a to date unidentified molecule (See, for example, Edelman, G.M. (1986) Ann. Rev. Cell Biol. 2:81-116).

Without being limited to any given theory, the morphogens described herein may act as the induced of this pathway.

The morphogens described herein can stimulate CAM production. As described below, the morphogens stimulate L1 and N-CAM production, including all three isoforms of the N-CAM molecule, i. nerve tissue.

In this example NG108-15 cells were cultured for 4 days in the presence of increasing concentrations of OP-1 and standard Western blots performed on whole cells extracts. The NG10875 cell line is a hybrid cell line (neuroblastoma x glioma, American Type Culture Collection, Rockville, MD). N-CAM isoforms were detected with an antibody which crossreacts with all three isoforms, mAb H28.123, obtained from Sigma Chemical Co., St. Louis, the different isoforms being distinguishable by their different mobilities on an electrophoresis gel. Control NG108-15 cells (untreated) express both the 140 kDa and the 180 kDa isoforms, but not the 120 kDa, as determined by western blot analyses using up to 100 pg of protein. As shownin Fig.8, treatment of NG108-15 cells with OP-1 resulted in a dose-dependent increase in the expression of the 180 kDa and 140 kDa isoforms, as well as the induction of the 120 kDa isoform. Fig. 8B is a Western blot of OPl-treated NG108-15 cell extracts, probed with mAb H28.123, showing th- induction of all three isoforms.

WO 94/03075 PCT/US93/07190 80 Fig. 8A is a dose response curve of N-CAM-180 and N- CAM-140 induction as a function of morphogen concentration. N-CAM-120 is not shown in the graph as it could not be quantitated in control cells. However, as is clearly evident from the Western blot in Fig. 8A, N-CAM-120 is induced in response to morphogen treatment. The induction of the 120 isoform also indicates that morphogen-induced redifferentiation of transformed cells stimulates not only redifferentiation of these cells from a transformed phenotype, but also differentiation to a p ~.'.otype corresponding to a developed cell. The dit,. ential induction of N-CAM 180 and 140 isoforms seen may be because constitutive expression of the 140 isoform is close to maximum. In addition, the increase in N-CAM expression corresponded in a dose-dependent manner with the morphogen induction of multicellular aggregates.

In addition, the cell aggregation effects of OP-1 on NG108-15 cells can be inhibited with anti-N-CAM antibodies or antisense N-CAM oligonucleotides.

Antisense oligonucleotides can be made synthetically on a nucleotide synthesizer, using standard means known in the art. Preferably, phosphorothioate oligonucleotides ("S-oligos") are prepared, to enhance transport of the nucleotides across cell membranes. Concentrations of both N-CAM antibodies and N-CAM antisense oliognucleotides sufficient to inhibit N-CAM induction also inhibited formation of multilayered cell aggregates. Specifically, incubation of morphogentreated NG108-115 cells with 0.3-3 pM N-CAM antisense S-oligos, 5-500 pM unmodified N-CAM antisense oligos, I L s 1 WO 94/03075 PCT/US93/07190 81 or 10 pg/ml mAb H28.123 significantly inhibits cell aggregation. It is likely that morphogen treatment also stimulates other CAMs, as inhibition is not complete.

The experiments also have been performed with soluble morphogen mature OP-1 associated with its pro domain) which also specifically induced CAM expression.

Example 4. Effect of Morphogen Neutralization on Embryogenesis As described in Example 7, below, at least one morphogen, OP2, is found principally in early developing embryos (8-day embryos). As described below, morphogen neutralization with morphogen-specific antibodies inhibits embryogenesis.

Morphogen inhibition in developing embryos inhibits tissue and organ development. Specifically, 9-day mouse embryo cells, cultured in vitro under standard culturing conditions, were incubated in the presence and absence of an OP-1-specific monoclonal antibody prepared using recombinantly produced, purified mature OP-1 as the immunogen. The antibody was prepared using standard antibody production means well known in the art and essentially as described for Example 9, below.

After two days, the effect of the antibody on the developing embryo was evaluated by histology using standard histology procedures well known in the art.

As determined by histological examination, the OP-1-specific antibody specifically inhibits eye lobe formation in the developing embryo. In particular, the ~llslss_ II __I WO 94/03075 PCT/US93/07190 82 diencephalon outgrowth does not develop. In addition, the heart is malformed and enlarged. Moreover, in separate immunolocalization studies on embryo sections with labelled OP-1 specific antibody, the OP-1-specific antibody localizes to neural epithelia.

Similarly, morphogen activity may be demonstated in fetal development in the mouse model using the following assay. Single lip injections comprising 10-100pg/injection of morphogen-specific antibody are administered to pregnant female mici during each day of the gestation period and tissue development bone development) in treated and control new mice evaluated by standard histomorphometric analysis at birth.

Finally, stimulation of endogenous morphogen antibody production in egg-laying hens interferes with shell formation in the developing eggs.

All of these data demonstate tnat inhibition of morphogen activity significantly interferes with tissue development during embryogenesis.

Example 5. Effect of Morphogen Neutralization on Juvenile Tissue Development The effect of the morphogens described herein on tissue development in developing mammals also may be demonstrated using neutralizing antibodies specific for particular morphogens and assessing the effect of these antibodies on tissue development as described below.

Specifically, anti-morphogen monoclonal and/or polyclonal antibodies may be prepared using standard methodologies including, for example, the protocol YIP13aL- gl I WO 94/03075 PCT/US93/07190 83 provided in Example 8, below, and provided to juveniles to inhibit the activity of endogenous morphogens.

Generally, purified antibodies are provided regularly to new born mice, lOOg/injection/day for 10-15 days. At 10 or 21 days, the mice are sacrificed and the effect of morphogen on bone development assessed by body weight, gross visual examination and histology. In this example, anti-OP-1 antibodies were used in 10pg injections/day for 14 days, and the mice were sacrificed at 21 days. As is dramatically demonstrated in Fig. 9, mice treated with OP-1 specific antibody show consistent and significant stunted growth, including reduced body length and body weight, (9B) as compared with untreated mice (9A).

Histological examination showed reduced bone growth as evidenced by reduced bone size in the treated mice.

In a variation on this protocol, single lip injections also may be provided to older juveniles and adult mice 10-100 pg) over a prolonged time 10-15 days) to evaluate the effect or morphogen neutralization on bone growth and bone integrity and to evaluate the onset of osteoporosis.

Example 6. Morphogen Treatment of Osteoporsis 6.1 Effect of Morphogen on Trabecular Bone in Ovariectomized (OVX) Rats Aged individuals, and particularly postmenopausal women are particularly at risk for osteoporosis.

Provided below is an animal osteoposis model demonstrating the ability of morphogens to subtantially I s~ WO 94/03075 PCT/US93/07190 84 inhibit and/or reduce the tissue damage effects associated with osteoporosis, wherein osteoporosis is induced by ovary removal in rats. Bone growth is evaluated in these animals by measuring serum alkaline phosphataseans osteocalcin levels in treated an untreated rats.

Forty Long-Evans rats (Charles River Laboratories, Wilmington) weighing about 200g each are ovariectomized (OVX) using standard surgical procedures, and ten rats are sham-operated. The ovariectomization of the rats produces an osteoporotic condition within the rats as a result of decreased estrogen production. Food and water are provided ad libitum. Eight days after ovariectomy, the rats, prepared as described above, were divided into five groups: 10 sham-operated rats; 10 ovariectomized rats receiving 1 ml of phosphate-buffered saline (PBS) i.v. in the tail vein; 10 ovariectomized rats receiving about 1 mg of 17E 2 (17-p-estradiol E 2 by intravenous injection through the tail vein; 9 ovariectomized rats receiving daily injections of approximately 2pg of morphogen by tail vein for 22 days; and 9 ovariectomized rats receiving daily injections of approximately 20 pg of morphogen by tail vein for 22 days. In this example, OP-1 was the morphogen tested.

On the 15th and 21st day of the study, e.-h rat was injected with 5 mg of tetracycline, and on day 22, the rats were sacrificed. The body weights, uterine WO 94/03075 PCT/US93/O7 190 weights, serum alkaline phosphate levels, serum calcium levels and serum osteocalcin levels then were determined for each Tables III and IV.

rat. The results are shown in Table III Body geights. Uterine Veivhts and Alkaline Phosnhatase Group A-SHAl

B-OVX+PBS

C-OVX+E2 D-OVX+OP- 1 (2p.g) E-OVX+OP- 1 p.g) Body Weights (g) 250.90 17.04 273.40 16.81 241.66 21.5k 266.67 10.43 272.40 20.48 Uterine Weights (g) 0.4192 0.10 0.1650 0.04 0.3C'81 0.03 0.1416 0.03 0. 1481 0.05 Alk. Phosphatase

(U/L)

43.25 6.11 56.22 6.21 62.66 4.11 58.09 12.97 66.24 15.74 TABLE IV Serum Calcium and Serum Oste'ocalcin Levels Group A-SHAMt

B-OVX+PBS

C-OVX+E2 D-OVX+OP-1 (2pg) E-OVX+OP- 1 (2011g) Serum Calcium (ng/dl) 8.82 1.65 8.95 1.25 9.20 1.39 8.77 0.95 8.67 1.94 Serum Osteocalcin (ng/ml) 64.66 14.77 69.01 10.20 67.13 17.33 148.50 84.11 182.42 52.11 L- C WO 94/03075 PCT/US93/07190 86 The results presented in Table III and IV show that intravenous injection of morphogen into ovariectomized rats produces a significant increase in serum alkaline phosphatase and serum osteocalcin levels and demonstrates that systemic administration of the morphogen stimulates bone formation in osteoporotic bone.

6.2 Histomorphometric Analysis of Morphogen on the Tibia Diaphysis in Ovariectomized(OVX) Rats Fifteen female Long-Evans rats weighing about 160 g were ovariectomized (OVX) to produce an osteoporotic condition and five rats were sham operated (Charles River Laboratories, Wilmington, MA.) as described for Example 8. Food and water were provided ad libitum.

Twenty-two days after ovariectomy, the rats were divided into four groups: sham-operated (1 ml of PBS by intravenous injection through tail vein rats); OVX, into which nothing was injected rats); OVX, receiving about 1 mg of 17pE 2 by intravenous injection through the tail vein rats), and OVX, receiving about 1 pg of morphogen by intravenous injection through the tail vein (5 rats). In this example, OP-1 was morphogen tested.

The rats were injected daily as described for seven days, except no injections were given on the thirteenth.

day. The rats then were sacrificed on the nineteenth day. The tibial diaphyseal long bones then were removed and fixed in ethanol and histomorphometric analysis was carried out using standard procedures well known in the art. The results are shown in Table V.

WO 94/03075 PCT/US93/07190 87

(A)

CONTROL MEASUREMENT Table V

(B)

OVX

19.4 0.2

(C)

OVX

E

2 4.9 0.5

(D)

OVX OP-1 17.9 0.9 Longitudi-,al Growth 20.2 0.3 Rate (.m/day) Cancellous Bone 20.2 1.5 Volume (BV/TV, bone vol/total vol) Cancellous Bone 16.2 1.8 Perimeter (mm) Labeled Cancellous 35.5 1.5 Perimeter Hineral Apposition 1.76 0.14 Rate (ur/day) 13.0 1.6 9.6 0.9 13.7 2.1 11.5 1.1 16.6 1.8 12.2 0.7 51.9 5.6 2.25 0.16 58.0 4.2 1.87 0.08 39.2 1.9 1.86 0.20 The results presented in Table V confirm the results of Example 6.1, namely that intravenous injection of OP-1 into ovariectomized rats stimulates bone growth for bone which had been lost due to the drop in estrogen within the individual rat.

Specifically, the inhibition of cancellous bone volume in OVX rats is repaired by the systemically provided morphogen. In addition, in morphogen-treated rats the labelled cancellous perimeter and mineral apposition rate now return to levels measured in the control, sham-operated rats. Moreover, morphogen treatment does not inhibit longitudinal bone growth, unlike estrogen I _1 WO 94/03075 PCT/US93/07190 88 treatment, which appears to inhibit bone growth significantly. Accordingly, systemic administration of a morphogen in therapeutically effective concentations effectively inhibits loss of bone mass in a mammal without inhibiting natural bone formation.

Example 7. Identification of Morphogen-Expressing Tissue Determining the tissue distribution of morphogens 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 morphogenstimulating agents. The morphogens (or their mRNA 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 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 sebaence homology in their active, C-terminal domains, the tissue distribution of a specific morphogen transcript may best be determined using a probe _ICI_ C~ -L ILI WO 94/03075 PCT/US93/07190 89 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 the morphogens of this invention, and accordingly, are specific for each protein. For example, a particularly useful Vgr-1-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 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 BstX1-BglI fragment, a 0.68 Kb sequence that covers approximately two-thirds of the mOP-1 pro region; a StuI-Stul fragment, a 0.2 Kb 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 d I WO 94/03075 PCT/US93/07190 90 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 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 2 Prior to hybridization, the appropriate probe is denatured by heating. The hybridization is carried out in a lucite cylinder rotating in a roller bottle apparatus at approximately 1 rev/min for approximately hours at 37 0 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 50 0

C.

Examples demonstrating the tissue distribution of various morphogens, including Vgr-.1, OP-1, BMP2, BMP3, BMP4, BMP5, GDF-1, and OP-2 in developing and adult tissue are disclosed in international application US92/01968 (W092/15323), and in Ozkaynak, et al., (1991) Biochem. Biophys. Res. Commn. 179:116-123, and Ozkaynak, et al. (1992) 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. Lung tissue appears to be the primary tissue expression source for Vgr-1, BMP5, BMP4 and BMP3. Lower

~PIPI

WO 94/03075 PCT/US93/07190 91 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.

Of particular relevance to the present application, OP-1 also is detected in adult rat stomach and gut tissue. Moreover, OP-2 appears to be expressed primarily in early embryonic tissue. Specifically, northern blots of murine embryos and 6-day post-natal 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.

In addition, labelled soluble OP-1 (iodinated with 1251, using standard labelling procedures well known in the art) and injected into the rat tail vein also is localized to the stomach tissue within 30 minutes of injection.

Example 8. Detecting Morphogenic Protein in Solution by Immunoassay Morphogens are readily detected in solution with a standard immunoassay, using a polyclonal or monoclonal antibody specific for that protein and standard Western blot, ELISA (enzyme-linked immunoabsorbant assay) or other immunoassay technique well known in the art. A currently preferred, exemplary protocol for an ELISA assay, as well as means for generating morphogenspecific antibody are presented below. Standard protocols for antibody production, Western blot and other immunoassays also are described, for example, in I L WO 94/03075 PCT/US93/07190 92 Molecular Cloning A Laboratory Manual, Sambrook et al., eds. 1989, Cold Spring Harbor Press, Cold Spring Harbor, NY. Standard ELISA technique is described, for example, by Engvall (1980) Methods Enzymol. 70:419-439.

8.1 Morphogen-Specific Antiserum Polyclonal antibody was prepared as follows. Each rabbit was 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 pl Complete Freund's Adjuvant. The antigen was injected subcutaneously at multiple sites on the back and flanks of the animal. The rabbit was boosted after a month in the same manner using incomplete Freund's Adjuvant.

Test bleeds are taken from the ear vein seven days later. Two additional boosts and test bleeds were performed at monthly intervals until antibody against OP-1 was detected in the serum using an ELISA assay.

Then, the rabbit was boosted monthly with 100 pg of antigen and bled (15 ml per bleed) at days seven and ten after boosting.

8.2 Morphogen-Specific Antibody Monoclonal antibody specific for a given morphogen was prepared as follows. A mouse was given two injections of E. coli produced OP-1 monomer. The first injection contains 100pg of OP-1 in complete Freund's adjuvant and was given subcutaneously. The second injection contained 50 pg of OP-1 in incomplete adjuvant and was given intraperitoneally. The mouse then received a total of 230 pg of OP-1 (amino acids 307-431 in SEQ ID NO:5) in four intraperitoneal ~I I_ WO 94/03075 PCT/US93/07190 93 injections at various times over an eight month period.

One week prior to fusion, both mice were boosted intraperitoneally with 100 pg of OP-1 (307-431) and pg of the N-terminal peptide (Ser293-Asn309-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 then were fused to commercially available myeloma cells at a ratio of 1:1 using PEG 1500 (Boeringer Mannheim, Germany), and the cell fusion plated and screened for OP-1-specific antibodies using OP-1 (307-431) as antigen. The cell fusion and monoclonal screening then were according to standard procedures well described in standard texts widely available in the art Maniatis et al. Molecular Cloning A Laboratory Manual, Cold Spring Harbor Press.

8.3 Morphogen ELISA 1 pg/100 pl of affinity-purified polyclonal rabbit IgG specific for OP-1 was added to each well of a 96-well plate and incubated at 37°C for an hour. The wells were 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 BSD containing 0.1% Tween 20. A 100 pl aliquot of an appropriate dilution of each of the test samples of cell culture supernatant was added to each well in triplicate and incubated at 37 0 C for 30 min. After incubation, 100 pl biotinylated rabbit anti-OP-1 serum I ~azram WO 94/03075 PCT/US93/07190 94 (stock solution is about 1 mg/ml and diluted 1:400 in BSB containing 1% BSA before use) are added to each well and incubated at 37 0 C for 30 min. The wells were then washed four times with BSB containing 0.1% Tween 20. 100 pl strepavidin-alkaline (Southern Biotechnology Associates, Inc. Birmingham, Alabama, diluted 1:2000 in BSB containing 0.1% Tween 20 before use) was added to each well and incubated ot 37 0 C for min. The plates were washed four times with Tris buffered Saline (TBS), pH 7.2. 50pl substrate (ELISA Amplification System Kit, Life Technologies, Inc., Bethesda, MD) was 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 was stopped by the addition of 50 pl 0.3 M sulphuric acid. The OD at 490 nm of the solution in each well was recorded. To quantitate OP-1 in culture media, an OP-1 standard curve was performed in parallel with the test samples.

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 therein.

_~A

WO 94/03075 PC/US93/07190 95 SEQUENCE LISTING GENERAL INFORMATION:

APPLICANT:

NAME: CREATIVE BIOMOLECULES, INC.

STREET: 35 SOUTH STREET CITY: HOPKINTON STATE: HA COUNTRY: USA POSTAL CODE (ZIP): 01748 TELEPHONE: 1-508-435-9001 TELEFAX: 1-508-435-0454

TELEX:

(ii) TITLE OF INVENTION: MORPHOGEN-ENRICHED DIETARY COMPOSITION (iii) NUMBER OF SEQUENCES: 33 (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: CREATIVE BIOMOLECULES, INC.

STREET: 35 SOUTH STREET CITY: HOPKINTON STATE: KA COUNTRY: USA ZIP: 01748 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: Patentln Release Version #1.25 (viii) ATTORNEY/AGENT INFORMATION: NAME: KELLEY, ROBIN D.

REGISTRATION NUMBER: 34,637 REFERENCE/DOCKET NUMBER: CRP-071 (ix) TELECOMMUNICATION INFORMATION: TELEPHONE: 617/248-7000 TELEFAX: 617/248-7100 INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 97 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear WO 94/03075 WO 9403075PCT/US93/07 190 96 (ii) MOLECULE TYPE: protein (ix) FEATURE: NAME/KEY: Protein LOCATION: 1. .97 OTHER INFORMATION: /label= GENERIC-SEQi /note= "'WHEREIN EACH XAA INDEPENDENTLY INDICATES ONE OF THE 20 NATURALLY-OCCURING L-ISOMER, A-AMIN ACIDS, OR A DERIVATIVE THEREOF.- 1 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa X~ 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Cys Xaa Xaa X 25 30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa X~ 40 45 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Cys Xaa X 50 55 60 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa X 70 75 8 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa C 90 aa aa aa aa 0 ys Xaa INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 97 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein WO 94/03075 PCTF/US93/07 190 -97 (ix) FEATURE: NAME/KEY: Protein LOCATION: 1._97 OTHER INFORMATION: /label= GENERIC-SEQ2 /note= "'WHEREIN EACH XAA INDEPENDENTLY INDICATES ONE OF THE 20 NATURALLY OCCURING L-ISOMER A-AMINO ACIDS, OR A DERIVATIVE THEREOF." 1 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 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 Cys Xi.a Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 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 Xaa 65 70 75 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys 90 Xaa INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 97 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein WO 94/03075 W094/3075PCT/US93/07 190 98 (ix) FEATURE: NAME/KiY: Protein LOCATION: 1.-97 OTHER INFORKATION: /note= "'WHEREIN FROM A GROUP OF /label= GENERIC-SEQ3 EACH XAA IS INDEPENDENTLY SELECTED ONE OR MORE SPECIFIED AMINO ACIDS AS DEFINED IN THE SPECIFICATION." (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: Leu Tyr Val Xaa 1 Xaa Xaa Xaa Gly Trp 10 Cys Xaa 25 Xaa Xaa Trp Xaa Xaa Ala Pro Xaa Gly Xaa Xaa Ala Xaa Tyr 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 Cys Cys Xaa Pro Xaa Xaa Xaa Xaa Xaa Val, Xaa Leu Xaa Xaa Xaa Xaa Leu Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Met Xaa Val Xaa Xaa Cys Gly Cys Xaa INFORMATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS! LENGTH: 102 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein I I~B WO 94/03075 PCT/US93/07190 99 (ix) FEATURE: NAME/KEY: Protein LOCATION: 1..102 OTHER INFORMATION: /note= "WHEREIN FROM A GROUP OF /label= GENERIC-SEQ4 EACH XAA IS INDEPENDENTLY SELECTED ONE OR HORE SPECIFIED AMINO ACIDS AS DEFINED IN THE SPECIFICATION." (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: Cys Xaa Xaa Xaa Xaa Leu Tyr Val Xaa Xaa Xaa Xaa Gly Trp Xaa Xaa Trp Xaa Xaa Ala Pro Xaa Gly Xaa 25 Xaa Ala Xaa Tyr Cys Xaa Gly Xaa Cys Xaa Xaa Pro Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asn His Ala Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Leu Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Cys Xaa Pro Xaa Xaa Xaa Xaa Xaa Xaa Xaa Leu Xaa Xaa Xaa Xaa Xaa Val Xaa Leu Xaa Xaa Xaa Xaa Met Xaa Val 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 I I WO 94/03075 PCT/US93/07190 100 (ix) FEATURE: NAME/KEY: Protein LOCATION: 1..139 OTHER INFORMATION: /label= hOPI-MATURE (xi) SEQUENCE DESCRIPTION: SEQ ID Ser Thr Gly Ser Gln Arg Ser Gin Asn Arg Ser Lys Thr Pro Lys Asn Asp Asp Tyr Ala Glu Ser Met Lys Trp Cys Val Cys Asp Ala Lys Ile Ala Gin Cys Ser 120 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 WO 94/03075 PCT/US93/07190 101 (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 GlI Arg Ser Gin 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 25 Asp Gln Arg Gin Ala Cys Lys Lys His Glu Leu Tyr Val Ser Phe Arg 40 Asp Leu Gly Trp Gln Asp Trp Ile Ile Ala 7ro Glu Gly Tyr Ala Ala 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 INFORMATION FOR SEQ ID NO:7: SEQUENCE CHARACTERISTICS: LENGTH: 139 amiho acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE: ORGANISM: HOMO SAPIENS TISSUE TYPE: HIPPOCAHPUS LU I WO 94/03075 PCT/US93/07190 102 (ix) FEATURE: NAME/KEY: Protein LOCATION: 1..139 OTHER INFORMATION: /label= HOP2-MATURE (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: Ala Val Arg Pro Leu Arg Arg Arg Gin 1 5 Pro Gin Ala Asn Arg Leu Pro Gly Ile His Gly Arg Gin Val Cys Arg Arg His 40 Asp Leu Gly Trp Leu Asp Trp Val Ile Tyr Tyr Cys Glu Gly Glu Cys Ser Phe 70 Ala Thr Asn His Ala Ile Leu Gln Ser Asn Ala Val Pro Lys Ala Cys Cys Ala 100 105 Ser Val Leu Tyr Tyr Asp Ser Ser Asn 115 120 Pro Lys Lys Ser Asn Glu Leu 10 Asp Leu Pro Leu Val Thr Val Val Val Gly Ser Leu Leu Leu 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 (ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE: ORGANISM: MURIDAE TISSUE TYPE: EMBRYO WO 94/03075 WO 9403075PCT/US93/07 190 103 (ix) FEATURE: NAME/KEY: Protein LOCATION: 139 OTHER INFORMATION: /label= MOP2-MATURE (xi) SEDUENCE DESCRIPTION: SEQ ID NO:8: Ala Ala Arg Pro Leu Lys Arg Arg Gin Pro Lys Lys Thr Asn Glu Leu Pro Arg Asp Tyr Ala Asp Ser Pro Arg Gly Cys Asn Val Leu 115 Gly Arg 40 Val Ala Gin Cys Set 120 Phe Glu Ala Pro Leu Pro Asn Gly Gly Ser Leu Leu Leu 125 Arg Asn Met Val Val Lys 130 Cys Gly Cys His INFORMATION FOR SEQ ID NO:9: SEQUENCE CHARACTERISTICS: LENGTH: 101 amino acids TYPE: amino acid STRAN'DEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE: ORGANISM: bovinae clo.91.

WO 94/03075 PCT/US93/07190 104 (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 40 Ile Val Gln Thr Leu Val Asn Ser Val Asn Ser Lys Ile Pro Lyp 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 85 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: ORGANISM: HOMO SAPIENS TISSUE TYPE: hippocampus (ix) FEATURE: NAME/KEY: Protein LOCATION: 1..101 OTHER INFORMATION: /label= CBMP-2B-FX ~s WO 94/03075 PCT/US93/07190 105 (xi) SEQUENCE DESCRIPTION: SEQ ID 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 Asp 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 Ser Ile Pro Lys Ala 55 Cys Cys Val Pro Thr Glu Leu Ser Ala Ile Ser Het Leu Tyr Leu Asp 70 75 Glu Tyr Asp Lys Val Val Leu Lys Asn Tyr Gln 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 (ix) FEATURE: NAME/KEY: Protein LOCATION: 1..101 OTHER INFORMATION: /label= DPP-FX (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 Asp Trp Ile Val Ala Pro Leu Gly Tyr Asp Ala Tyr Tyr Cys His Gly 25 WO 94/03075 PCT/US93/07190 106 Lys Cys Pro Phe Pro Leu Ala Asp His Phe Asn Ser Thr Asn His Ala 40 Val Val Gin Thr Leu Val Asn Asn Asn Asn Pro Gly Lys Val Pro Lys 50 55 Ala Cys Cys Val Pro Thr Gin Leu Asp Ser Val Ala Met Leu Tyr Leu 70 75 Asn Asp Gin Ser Thr Val Val Leu Lys Asn Tyr Gln Glu Met Thr Val 90 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 Gin 1 5 10 Asn Trp Val lie Ala Pro Gin Gly Tyr Met Ala Asn Tyr Cys Tyr Gly 25 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 65 70 75 I I WO 94/03075 PCT/US93/07190 107 Asp Asn Asn Asp Asn Val Val Leu Arg His Tyr Glu Asn Met Ala Val 90 Asp Glu Cys Gly Cys Arg 100 INFORMATION FOR SEQ ID NO:13: SEQUENCE CHARACTERISTICS: LENGTH: 102 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE: ORGANISM: HURIDAE (ix) FEATURE: NAME/KEY: Protein LOCATION: 1..102 OTHER INFORMATION: /label= VGR-1-FX (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: Cys Lys Lys His Glu Leu Tyr Val Ser Phe Gin Asp Val Gly Trp Gln 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 35 40 Ile Val Gin Thr Leu Val His Val Met Asn Pro Glu Tyr Val Pro Lys 55 Pro Cys Cys Ala Pro Thr Lys Val Asn Ala Ile Ser Val Leu Tyr Phe 70 75 Asp Asp Asn Ser Asn Val Ile Leu Lys Lys Tyr Arg Asn Met Val Val 90 Arg Ala Cys Gly Cys His 100 WO 94/03075 PCT/US93/07190 108 INFORMATION FOR SEQ ID NO:14: SEQUENCE CHARACTERISTICS: LENGTH: 106 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens TISSUE TYPE: brain (ix) FEATURE: NAME/KEY: Protein LOCATION: 1..106 OTHER INFORMATION: /note= "GDF-1 (f (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: Cys Arg Ala Arg Arg Leu Tyr Val Ser Phe Arg Glu Val Gly Trp His 1 5 10 Arg Trp Val Ile Ala Pro Arg Gly Phe Leu Ala Asn Tyr Cys Gin Gly 25 Gin Cys Ala Leu Pro Val Ala Leu Ser Gly Ser Gly Gly Pro Pro Ala 40 Leu Asn His Ala Val Leu Arg Ala Leu Met His Ala Ala Ala Pro Gly 55 Ala Ala Asp Leu Pro Cys Cys Val Pro Ala Arg Leu Ser Pro Ile Ser 65 70 75 Val Leu Phe Phe Asp Asn Ser Asp Asn Val Val Leu Arg Gin Tyr Glu 90 Asp Met Val Val Asp Glu Cys Gly Cys Arg 100 105 WO 94/03075 PCT/US93/07190 109 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 5 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID Cys Xaa Xaa Xaa Xaa 1 INFORMATION FOR SEQ ID NO:16: SEQUENCE CHARACTERISTICS: LENGTH: 1822 base pairs 1 PE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: HOMO SAPIENS TISSUE TYPE: HIPPOCAMPUS (ix) FEATURE: NAME/KEY: CDS LOCATION: 49..1341 IDENTIFICATION METHOD: experimental OTHER INFORMATION: /function= "OSTEOGENIC PROTEIN" /product= "OP1" /evidence= EXPERIMENTAL /standard name= "OP1" (xi) SEQUEN-E DESCRIPTION: SEQ ID NO:16: WO 94/03075 WO 9403075PC1'/US93/07 190 110 GGTGCGGGCC CGGAGCCCGG AGCCCGGGTA GCGCGTAGAG CCGGCGCG ATG CAC GTG Met His Val 1 CCC TCA Arg Ser CTG CGA CCT GCG Leu Arg Ala Ala CCG CAC AGC TTC Pro His Ser Phe GCC CTC TGG GCA Ala Leu Trp Ala

CCC

Pro CIC TTC CTC CTG Leu Phe Leu Leu TCC GCC CTG GCC Ser Ala Leu Ala TTC AGC CTG GAC AAC Phe Ser Leu Asp Asn GAG CTG CAC TCG Glu Val His Ser TTC ATC CAC CCC Phe Ile His Arg CTC CGC AGC CAG Leu Arg Ser Gin GAG CCC Glu Arg CCC GAG ATG Arg Glu Met CCG CGC CCG Pro Arg Pro CGC GAG ATC CTC Arg Glu Ile Leu ATT TTG GGC TTG Ile Leu Gly Leu CCC CAC CC Pro His Arg ATG TTC ATG Met Phe Met CAC CTC CAG GCC His Leu Gin Gly CAC AAC TCG GCA His Asn Ser Ala CTG fCAC Leu Asp CTG TAC AAC GCC Leu Tyr Asn Ala GCG GTG GAG GAG Ala Val Clu Glu CCC CCC CCC GC Gly Gly Pro Gly

GCC

Gly 100 GAG GGC TTC TCC Gin Gly Phe r CCC TAC AAG GCC Pro Tyr Lys Ala TTC ACT ACC CAG Phe Ser Thr Gin CCC CCT CTC CC Pro Pro Leu Ala CTG CAA GAT AGC Leu Gin Asp Ser TTC CTC ACC GAG Phe Leu Thr Asp CCC CAC Ala Asp 130 ATC GTC ATG Met Val Met CAC CCA CC His Pro Arg 150 TTC CTC MAC CTC GTC CPA CAT GAC AAC Phe Val Asn Leu Val Ciu ils Asp Lys GAA TTC TTC Ciu Phe Phe 145 TCC MCG ATC Ser Lys Ile TAC CAC CAT CGA Tyr His His Arg GAG TTC CCC TTT CAT Ciu Phe Arg Phe Asp 155 GCA CCC CPA TTC CCC Ala Ala Giu Plie Arg 175 CCA CMA Pro Ciu 165 CCC CMA CCT GTC Gly Ciu Ala Val ATC TAC MAG CAC Ile Tyr Lys Asp WO 94/03075 WO 9403075PCT/US93/07 190 ill TAC ATC CGG Tyr 180

CAG

Gin

GAO

Asp

ATO

Ile

GGC

Gly

MAG

Lys 260

TTC

Phe

CGG

Arg

MAG

Lys

AGC

Ser

CGA

Arg 340 Ile

GTG

Val

AGO

Ser

ACA

Thr

CTG

Leu 245

TG

Leu

ATG

Met

TOO

Ser

MAC

Asn

GAC

Asp 325

GAC

Asp Arg

CTC

Leu

CGT

Arg

GCC

Ala 230

CAG

Gin

GCG

Ala

GTG

Val

ACG

Thr

CAG

Gin 310

CAG

Gin

CTG

Leu

GMA

Giu

CAG

Gin

ACC

Thr 215

ACO

Thr

CTC

Leu

GGC

Gly

GCT

Ala

GGG

Giy 295

GMA

Giu

AGG

Arg

GGO

Gly CGO TTC GAC MAT GAG Arg

GAG

Giu 200

CTC

Leu

AGO

Ser

TCG

Ser

CTG

Leu

TTC

Phe 280

AGO

Ser

GCC

Ala

CAG

Gin

TGG

Trp Phe 185

CAO

His

TGG

Trp

AAO

Asn

GTG

Val

ATT

Ile 265

TTO

Phe

AMA

Lys

GTG

Leu

GGO

Ala

CAG

Gin 345

ACG

Thr

GMA

Giu 205

GAG

Giu

GTO

Val

GAT

Asp

GGG

Giy

GAG

Giu 285

GAG

Gin

MOC

Asn

GAO

His

ATO

Ile

TC

Phe 190

TOG

Ser

GGO

Gly

AAT

As n

GGG

Gly

COO

Pro 270

GTO

Val

MAC

Asn

GTG

Val

GAG

Giu

GOG

Ala 350 633 681 729 777 825 873 921 969 1017 1065 1113 1161 GOC TAO TAO TGT GAG GGG GAG TGT GCC TTC COT OTG MAC TCO TAO ATG Ala Tyr Tyr Gys Glu Gly Glu Cys Ala Phe Pro Leu Asn Ser Tyr Met 360 '365 370 WO 94/03075 WO 9403075PCT/US93/07 190 112 MAC GCC ACC Asn Ala Thr CCG GMA ACG Pro Giu Thr 390 ATC TCC GTC Ile Ser Val 405 TAC AGA MAC Tyr Arg Asn 420

GAGMATTCAGA

GMACCAGCAGA

TGTGAGAGTA

ATCCMATGAA

GCATAAAGA

CGTTTCCAGA

GGCGTGGCMA

CTGTMATAAA

MAC

Asn 375

GTG

Val CAC GCC ATC GTG His Ala Ile Val CCC MAG CCC Pro Lys Pro CTC TAC TTC Leu Tyr Phe

GAT

Asp 410 ATG GTG Met Val kCCCTTTGC kCMACTGC

L'TAGGAMAC

:AMGATCCI

kIAATGGCCC

;GTAATTAI

;GGGTGGGC

rGTCACMAI GTC COG GCC Val Arg Ala 425 ;G GCCMAGTTT] :C .rrrrGTGAG1 :A TGAGCAGCA] 'A CMAGCTGTGC ~G GCCAGGTCA] 'G AGCGCCTACC :A CATTGGTGTC 'A MAACGMTGI CAG ACG CTG Gin Thr Leu 380 TGT CC CCC Cys Ala Pro AGC TCC AAC Ser Ser Asn TGT CCC C Cys Gly Cys 430

TCTGGATCCT

CCTTCCCCTC

ATGGCTTTTG

AGGCAAMACC

TGGCTGGGMA

AGCCAGGCCA

TGT I(2GAAAG

ATGAAAAAAA

415

CAC

His GIC CAC TIC ATC MAC Val His Phe Ile Asn 385 ACG CAG CTC MAT CC Thr Gin Leu Asn Ala 400 GTC ATC CTG MAG AAA Val Ile Leu Lys Lys

TAGCTCCTCC

1209 1257 1305 1351 1411 14 11 1531 1591 1651 1711 1771 1822

CCATTGCTCG

C CTATC C CCA

ATCAGTTTT

TAGCAGGAAA

GTCTCAGCCA

CCCAGCCGTG

GAAAATTGAC

CCTTGGCCAC

ACTTTAAAGG

CAGTGGCAGC

AAAAAACAAC

TGCACGGACT

GGAGGMAGGG

CCGGAAGTTC

MAAAAAAAA A INFORMATION FOR SEQ ID NC: 17: SEQUENCE CHARACTER13TICS: LENGTH: 431 amwino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DE9CRIPTION: SEQ ID NO:17: Met His Val Arg Ser Leu Arg Ala Ala Ala Pro His Ser Phe Val Ala 1 510 Leu Irp Ala Pro Leu Phe Leu Leu Arg Ser Ala Leu Ala Asp Phe Ser 25 WO 94/03075 WO 9403075PCT/US93/07 190 113 Leu Asp Asn Giu Val His Ser Ser Phe Ile His Gin Pro Met Gly Thr Asp Giu 145 Ser Tyr Ser Phe Val 225 His Ile Lys Giu His Phe Pro Gin Ala 130 Phe Lys Lys Vai Leu 210 Phe Asn Asn Gin Arg Arg Met Gly Gly 115 Asp Plie Ile Asp Tyr 195 Leu As p Leu Pro Pro 275 Arg Pro Leu Giy 100 Pro Met His Pro Tyr 180 Gin Asp Ile Gly Lys 260 Phe Giu Arg Asp Gin Pro Val Pro Giu 165 Ile Val Ser Thr Leu 245 Leu Met Arg Glu Leu Gin Asn Ala Ser Tyr 105 Ser Leu 120 Phe Val His His Ala Val Arg Phe 185 Giu His 200 Leu Trp Ser Asn Ser Vai L2u Ile 265 Phe Phe 280 Arg Ser 290 Ile Arg Ser Thr Ser Lys Gin Arg Ser Gin Asn Arg Ser C_ WO 94/03075 PCT/US93/07190 1!4 Lys Thr Pro Lys Asn Gin Glu Ala Leu Arg Met Ala Asn Val Ala Glu 305 310 315 320 Asn Ser Ser Ser Asp Gin Arg Gin Ala Cys Lys Lys His Glu Leu Tyr 325 330 335 Val Ser Phe Arg Asp Leu Gly Trp Gin Asp Trp Ile Ile Ala Pro Glu 340 345 350 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 Gin 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 I1e 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: MURIDAE TISSUE TYPE: EMBRYO (ix) FEATURE: NAME/KEY: CDS LOCATION: 104..1393 OTHER INFORMATION: /function= "OSTEOGENIC PROTEIN" /product= "HOP1" /note= "MOP1 (CDNA)" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: WO 94/03075 WO 943075P/1JS93/07 190 .115 CTGCAGCAAG TGACCTCCCG TCGTGGACCG CTGCCCTGCC CGGCGCGGGC CCGGTGCCCC GGATCGCGCG TAGAGCCGC CCCTCCGCTG CCACCTGGGG GCG ATG CAC CTG CC Met His Val Arg

TCG

Ser

CTG

Leu

GTG

Val

GAG

Glu

CGC

Arg

GAC

Asp

GGC

Gly

TTA

Leu

ATG

Met

CGA

Arg

GCT

Ala

CTG

Leu

AGC

Ser

CGG

Arg

CTC

Leu

AAC

Asn

TAC

Tyr

CTG

Leu 120

GTC

Val

CAT

His

GCG

Ala 10

TCC

Ser

ATC

Ile

ATC

Ile

GGA

Gly

ATG

Met 90

TAC

Tyr

GAC

As p

CTA

Leu

GAG

Glu

GCG

Ala

AAC

Asn

CGG

Arg

CC

Arg

ATG

Met

GGA

Gly

CCC

Pro 115

ATG

Met

CAC

His

CCC

Pro

CCT

Pro

GAG

Glu

CCC

Arg

CCC

Pro

TTC

Leu

CAC

Gin 100

CCT

Pro

GTC

Val

CCT

Pro

GAG

Glu 163 211 259 307 355 403 451 499 547 595 GCC GAA CCC GTC ACC GCA CCC GAA TTC ACC ATC TAT MAG GAC TAC ATC Glu Arg Val Thr Ala Ala Giu Phe Arg 170 Tyr Lys Asp Tyr WO 94/03075 WO 9403075PCT/US93/07 190 116 CGG GAG CGA TTT Arg Giu Arg Phe AAC GAG ACC TTC Asn Giu Thr Phe ATC ACA GTC TAT Ile Thr Val Tyr CAG GTG Gin Val 195 CTC CAG GAG Leu Gin Giu

CAC

His 200 TCA GGC AGG GAG Ser Gly Arg Giu GAG CTC TTC TIG Asp Leu Phe Leu CTG GAC AGC Leu Asp Ser 210 CGC ACC ATC TGG GCT TCT GAG Arg Thr Ile Trp Ala Ser Giu 215 GGC TGG TTG GTG Giy Trp Leu Val TTT GAT ATC ACA 787 Phe Asp Ile Thr 225 MAC CTG GGC TTA 835 Asn Leu Giy Leu GCC ACC Ala Thr 230 AGC MAC CAC TGG Ser Asn His Trp

GTG

Val 235 GTC MAC CCT CGG Val Asn Pro Arg

CAG

Gin 245 CIC TCT GIG GAG Leu Ser Val Giu

ACC

Thr 250 CTG GAT GGG CAG AGC ATC MAC CCC MAG Leu Asp Gly Gin Ser Ile Asn Pro Lys GCA GGC CTG ATT Aia Gly Leu Ile

GGA

Gly 265 CGG CAT. GGA CCC Arg His Gly Pro MAC MAG CMA CCC Asn Lys Gin Pro TTC ATG Phe Met 275 GTG GCC TTC TTC Val Ala Phe Phe 280 ACG GGG GGC MAG Thr Gly Giy Lys 295 CMA GAG GCC CTG Gin Giu Aia Leu 310 MAG GCC ACG GMA Lys Ala Thr Giu CAT CTC CGT AGT His Leu Arg Ser ATC CGG TCC Ile Arg Ser 290 CCA MAG MAC Pro Lys Asn CAG CGC AGC Gin Arg Ser AGG ATG GCC Arg Met Aia 315 MAT CGC ICC MAG Asn Arg Ser Lys

ACG

Thr 305 AGT GIG GCA GMA Ser Val Ala Giu

MAC

Asn 320 AGC AGC AGT GAC Ser Ser Ser Asp CAG AGO CAG GCC TGC Gin Arg Gin Ala Cys 325 CTT GGC TGG CAG GAC Leu Gly Trp Gin Asp 345 MAA CAT GAG CTG Lys His Giu Leu GTC AGC TTC CGA Val Ser Phe Arg 1027 1075 1123 1171 1219 TGG ATC ATT GCA Trp Ile Ile Ala GMA GGC TAT GCT Giu Gly Tyr Ala GCC TAC Ala Tyr 355 TAC TGT GAG Tyr Cys Giu GAG TGC GCC TTC Giu Cys Aia Phe CTG MAC ICC TAC Leu Asn Ser Tyr ATG MAC GCC Met Asn Ala 370 WO 94/03075 WO 9403075PCr/US93/07 190 1147 ACC MAC CAC Thr Asn His 375 &CA GTA CCC Thr Val P~ro GCC ATC GTC Ala Ile Val MAG CCC TGC Lys Pro Cys 390 CAG ACA CTG GTT CAC TTC ATC MAC CCA GAC Gin Thr Leu Val His Phe Ile Asn Pro Asp 380 385 TGT GCG CCC ACC CAG CTC MAC GCC ATC TCT Cys Ala Pro Thr Gin Leu Asn Ala Ile Ser 395 400 AGC TCT MAT GTC GAC CTG MAG MAG TAC AGA Ser Ser Asn Val Asp Leu Lys Lys Tyr Arg 415 420 TGT GOC TGC CAC TAGCTCTTCC TGAGACCCTG Cys Gly Cys His 430

GTC

Val 405

MAC

Asn

CTC

Leu TAC TTC GAC Tyr Phe Asp ATG GTG GTC Met Val Val 1267 1315 1363 14, 13 1473 1533 1593 1653 1713 1773 1833 1873

ACCTTTGCGG

CCCACCTTGG

MAGCATGTMA

GGCACGTGAC

GTCTGCCAGG

MTCGCAAGC

TCTGTGTTGA

GAATGAMMA

GGCCACACCT

CGAGGAGMAC

GGGTTCCAGA

GGACMAGATC

AAAGTGTCCA

CTCGTTCAGC

AGGGAAMCCA

TTCCAAMTCT

AGACCAACCT

MACCTGAGCG

CTACCAGCTA

GTGTCCACAT

TGCAGCAGAA

AGCAGAAGCC

TCGATGTCTC

CTCCTGAGCC

TGCAGCAGCT

CCACAGCAAM

GGCCCCTGGC

GGAAGGGCTT

ACTGTMATGA

AAAAGMATTC

ACCATCTAAG

TTCCCTCACC

GATGAGCGCC

CGCCTAAGAG

GCTCTGAGTC

AGCCAGGGTG

TATGTCACAA

TCTCTCACTG

TCCCMACCGG

CTTTCCTTCT

CAGGAAAAAT

"TTTGAGGAGT

GGCGCTGGCG

TMM AC CCAT AAAAAAAA AMAAAAAAAA INFORMIATION FOR SEQ ID NO:19: SEQUENCE CH.ARACTERISTICS: LENGTH: 430 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: Met His Val Arg Ser Leu Arg Ala Ala Ala Pro His Ser Phe Val Ala 1 5 10 Leu Trp Ala Pro Leu Phe Leu Leu Arg Ser Ala Leu Ala Asp Phe Ser 25

~~I

WO 94/03075 PCT/US93/07190 118 Leu Asp Asn Glu Val His Ser Ser Phe Ile His Arg Arg Leu Arg Ser 40 Gin Glu Arg Arg Glu Het Gln Arg Glu Ile Leu Ser Ile Leu Gly Leu 50 55 Pro His Arg Pro Arg Pro His Leu Gin Gly Lys His Asn Ser Ala Pro 70 75 Met Phe Met Leu Asp Leu Tyr Asn Ala Met Ala Val Glu Glu Ser G'y 90 Pro Asp Gly Gin Gly Phe Ser Tyr Pro Tyr Lys Ala Val Phe Ser Thr 100 105 110 Gin Gly Pro Pro Leu Ala Ser Leu Gin Asp Ser His Phe Leu Thr Asp 115 120 125 Ala Asp Met Val Met Ser Phe Val Asn Leu Val Glu His Asp Lys Glu 130 135 140 Phe Phe His Pro Arg Tyr His His Arg Glu Phe Arg Phe Asp Leu Ser 145 150 155 160 Lys Ile Pro Glu Gly Glu Arg Val Thr Ala Ala Glu Phe Arg Ile Tyr 165 170 175 Lys Asp Tyr lie Arg Glu Arg Phe Asp Asn Glu Thr Phe Gin lie Thr 180 185 190 Val Tyr Gin Val Leu Gin Glu His Ser Gly Arg Glu Ser Asp Leu Phe 195 200 205 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 Gin Ser Ile 245 250 255 Asn Pro Lys Leu Ala Gly Leu Ile Gly Arg His Gly Pro Gin Asn Lys 260 265 270 Gin Pro Phe Met Val Ala Phe Phe Lys Ala Thr Glu Val His Leu Arg 275 280 285 WO 94/03075 PCT/US93/07190 119 Ser Ile Arg Ser Thr Gly Gly Lys Gin Arg Ser Gln 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 Gln 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 355 360 365 Tyr Het Asn Ala Thr Asn His Ala Ile Val Gin Thr Leu Val His Phe 370 375 380 Ile Asn Pro Asp Thr Val Pro Lys Pro Cys Cys Ala Pro Thr Gin Leu 385 390 395 400 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: ORGANISH: Homo sapiens TISSUE TYPE: HIPPOCAMPUS (ix) FEATURE: NAME/KEY: CDS LOCATION: 49C..1696 OTHER INFORMATION: /function= "OSTEOGENIC PROTEIN" /product= "hOP2-PP" /note= "hOP2 (cDNA)" (xi) SEQUENCE DESCRIPTION: SEQ ID N0:20: WO 94/03075 WO 9403075PCI'/US93/07 190 120 GGCGCCGGCA GAGCAGGAGT GGGCTGGAGG GOTCTATG CCACAOOGCA CCAAGCGGTG GCGGCCACAG CCGGACTGGC CCGCAGAGTA GCCCCGGCCT GACAGGTGTC GCGCGGCGGG CGCCCCGCOO CGCCGCCGC AGGCCTGGG TCGGCCGCGG CGGCCTGCC ATG ACC GCG GGCTGGAGGA GCTGTGGTTG GAGCAGGAGG TGGCACGGCA AGTGGCGGAG ACGGCCOAGG AGGCGCTGGA GCAACAGCTC GCTGCAGGAG OTOGCOATC GCCCOTGCGC TGCTCGGACC GGGTACGGCG GCGACAGAGG CATTGGCCGA GAGTCCCAGT OGAGGCGGTG GCGTCCCGGT CCTCTCCGTC CAGGAGCCAG GCTCCAGGGA COGOGCOTGA GGCCGGCTGC CCGOCCGTCC CGCOOGCCGA GCCCAGCCTC CTTGCCGTCG GGGCGTOCC AGCCGATGCG CGOOCGCTGA GCGCCCCAGC TGAGCGCOCCC OTC CCC GGC CCG CTC TGG CTC CTG GGC CTG Leu Pro Gly Pro Leu Trp Leu Leu Gly Leu GOG CTA Ala Leu GGC TGT Gly Cys Met Thr Ala 1 TGC GOG CTG GG Cys Ala Leu Gl CCC CAG OGA CC Pro Gin Arg Ai

C

y GGC GGC CCC GGC Gly Gly Pro Gly CGA CCC COG CCC Arg Pro Pro Pro CTG GGC GOG OGC Leu Gly Ala Arg OGG GAC GTG Arg Asp Val

CGC

Arg GAG ATO CTG Glu Ile Leu OTC GGG CTG Leu Gly Leu CGG 000 CGO Arg Pro Arg CCC CGC Pro Arg GCG CCA COO Ala Pro Pro CTG GAO CTG Leu Asp Leu

CC

Ala

TAO

Tyr TOOCC C TG Ser Arg Leu TOO GOG OCG Ser Ala Pro OTO TTC ATG Leu Phe Met GAC GGC C Asp Gly Ala CAC CO ATG His Ala Met GAO GAC GAO Asp Asp Asp 000 CG Pro Ala AAO ATG Asn Met 110 GAG CCC OGO OTG Glu Arg Arg Leu OGO GOC GAO OTG Arg Ala Asp Leu ATG AGC TTO GTT Met Ser Phe Val GTG GAG OGA Val Glu Arg

GAO

Asp 115 GOC OTC GGO Ala Leu Gly GAG COO OAT Glu Pro His WO 94/03075 WO 9403075PCr/US93/07 190 121

MAG

Lys

ACA

Thr

AAC

Asn

MAC

Asn

GGA

Gly 190

TGG

Trp

ACT

Thr

CMA

Gin

GCC

Ala

AGO

Arg 270

CCA

Pro

GAG

Glu

GCT

Ala

AGO

Arg

AGO

Arg 175

GAC

Asp

TTG

Leu

GAG

Glu

CGG

Arg

AGT

Ser 255

AGO

Arg

G

Gly

TTT

Phe 130

TTC

Phe

CAC

His

GAC

Asp

TGG

Trp

CGT

Arg 210

CAC

His

CGC

Arg

CCC

Pro

MAG

Lys

GAT

Asp 290 CAG ATC CCG Gin Ile Pro 135 MAG GTG CCC Lys Val Pro 150 TTC CAG GTG Phe Gin Val TTG GAT CTT Leu Asp Leu GAT GTC ACA Asp Val Thr 200 CTG GGA CTC Leu Gly Leu 215 CCT GGC CGG Pro Gly Leu 230 CCT TTC GTG Pro Phe Val CCT CGG, GCA Pro Arg Ala GAG CIG CCG Glu Leu Pro 280 GGC ICC CAC Gly Ser His 295

GCT

Ala

AGC

Ser

GC

Val

CAG

Gin 185

GCA

Ala

CGC

Arg

GCC

Ala

GIG

Val

GIG

Val 265

CAG

Gin

GC

Gly

GG

Gly

ATC

Ile

CAG

Gin 170

ACG

Ihr

GCC

Ala

CTC

Leu

GC

Gly

ACT

Thr 250

AGG

Arg

GCC

Aia

CGG

Arg

GC

GAG GCG jTC Giu Ala Val 140 CAC CTG CTC His Leu Leu 155 GAG CAG ICC Glu Gin Ser CTC CGA GCT Leu Arg Ala AGT GAC TGC Ser Asp Cys 205 TAT GTG GAG Tyr Val Glu 220 CIG CTG GGT Leu Leu Gly 235 TTC TTC AGG Phe Phe Arg CCA CTG AGO Pro Leu Arg MAC CGA CIC Asn Arg Leu 285 CAG GTC TGC Gin Val Cys 300 TGG CTG GAC 912 960 1008 1056 1104 1152 1200 1248 1296 1344 1392 1440 CGT CGG CAC Arg Arg His CTC TAC GIC AGC TTC CAG GAC CIC Leu Tyr Val Ser Phe Gin Asp Leu Gly Irp 310 315 Leu Asp I WO 94/03075 PCT/US93/07190 122 TGG GTC ATC GCT CCC CAA GGC TAC TCG GCC TAT TAC TGT GAG GGG GAG 1488 Trp Val Ile Ala Pro Gin Gly Tyr Ser Ala Tyr Tyr Cys Glu Gly Glu 320 325 330 TGC TCC TTC CCA CTG GAC TCC TGC ATG AAT GCC ACC CAC GCC ATC 1536 Cys Ser Phe Pro Leu Asp Ser Cys Met Asn Ala Thr s Ala Ile 335 340 345 CTG CAG TCC CTG GTG CAC CTG ATG AAG CCA AAC GCA GTC CCC AAG GCG 1584 Leu Gin Ser Leu Val His Leu Met Lys Pro Asn Ala Val Pro Lys Ala 350 355 360 365 TGC TGT GCA CCC ACC AAG CTG AGC GCC ACC TCT GTG CTC TAC TAT GAC 1632 Cys Cys Ala Pro Thr Lys Leu Ser Ala Thr Ser Val Leu Tyr Tyr Asp 370 375 380 AGC AGC AAC AAC GTC ATC CTG CGC AAA GCC CGC AAC ATG GTG GTC AAG 1680 Ser Ser Asn Asn Val Ile Leu Arg Lys Ala Arg Asn Met Val Val Lys 385 390 395 GCC TGC GGC TGC CAC T GAGTCAGCCC GCCCAGCCCT ACTGCAG 1723 Ala C 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 IL NO:21: Met Thr Ala Leu Pro Gly Pro Leu Trp Leu Leu Gly Leu Ala Leu Cys 1 5 10 Ala Leu Gly Gly Gly Gly Pro Gly Leu Arg Pro Pro Pro Gly Cys Pro 25 Gin Arg Arg Leu Gly Ala Arg Glu Arg Arg Asp Val Gin Arg Glu Ile 40 Leu Ala Val Leu Gly Leu Pro Gly Arg Pro Arg Pro Arg Ala Pro Pro 55 WO 94/03075 WO 9403075PCT/US93/07 190 123 Leu Pro Ala Ser Ala Pro Leu Phe Met Leu Asp Leu Ala Tyr Arg Glu Arg Glu 145 Leu Ser Gly Lys Gly 225 1.

Ser Pro Phe Glu 305 Ala His Arg Arg Phe 130 Phe His Asp Trp Arg 210 His Arg Pro Lys Asp 290 Leu Ser Ala Leu Asp 115 Asp Arg Val Leu Leu 195 His Ser Ser Ile Lys 275 Asp Tyr Arg Met Gly 100 Arg Leu Ile Ser Phe 180 Val Lys Val Gln Arg 260 Ser Val Val Ala Gly Asp Asp Asp Arg Ala Asp Leu Val 105 Ala Leu Gly His Gin 120 Thr Gin Ile Pro Ala 135 Tyr Lys Val Pro Ser 150 Met Phe Gin Val Val 165 Phe Leu Asp Leu Gin 185 Leu Asp Val Thr Ala 200 Asp Leu Gly Leu Arg 215 Asp Pro Gly Leu Ala 230 Gin Pro Phe Val Val 245 Thr Pro Arg Ala Val 265 Asn Glu Leu Pro Gin 280 His Gly Ser His Gly 295 Ser Phe Gin Asp Lelu 310 Tyr Ser Ala Tyr Tyr 325 Gly Phe His Ala 140 Leu Gin Arg Asp Val 220 Leu Phe Leu Arg Val 300 Leu Ala Pro Gin Gly Glu Gly Glu Cys Ser Phe WO 94/03075 PCT/US93/07190 124 Pro Leu Asp Ser Cys Met Asn Ala Thr Asn His Ala Ile Leu Gin Ser 340 345 350 Leu Val His Leu Het Lys Pro Asn Ala Val Pro Lys Ala Cys Cys Ala 355 360 365 Pro Thr Lys Leu Ser Ala Thr Ser Val Leu Tyr Tyr Asp Ser Ser Asn 370 375 380 Asn Val Ile Leu Arg Lys Ala Arg Asn Met Val Val Lys Ala Cys Gly 385 390 395 400 Cys His 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= "mOP2-PP" /note= "m0P2 cDNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: GCCAGGCACA GGTGCGCCGT CTGGTCCTCC CCGTCTGGCG TCAGCCGAGC CCGACCAGCT ACCAGTGGAT GCGCGCCGGC TGAAAGTCCG AG ATG GCT ATG CGT CCC GGG CCA 113 Met Ala Met Arg Pro Gly Pro 1 CTC TGG CTA TTG GGC CTT GCT CTG TGC GCG CTG GGA GGC GGC CAC GGT 161 Leu Trp Leu Leu Gly Leu Ala Leu Cys Ala Leu Gly Gly Gly His Gly 15 CCG CGT CCC CCG CAC ACC TGT CCC CAG CGT CGC CTG GGA GCG CGC GAG 209 Pro Arg Pro Pro His Thr Cys Pro Gln Arg Arg Leu Gly Ala Arg Glu 25 30 L- -L WO 94/03075 PCT/US93/07190 125 CGC CGC GAC ATG CAG CGT GAA ATC CTG GCG GTG CTC GGG CTA CCG GGA 257 Arg Arg Asp Met Gin Arg Glu Ile Leu Ala Val Leu Gly Leu Pro Gly 45 50 CGG CCC CGA CCC CGT GCA CAA CCC GCC GCT GCC CGG CAG CCA GCG TCC 305 Arg Pro Arg Pro Arg Ala Gin Pro Ala Ala Ala Arg Gin Pro Ala Ser 65 GCG CCC CTC TTC ATG TTG GAC CTA TAC CAC GCC ATG ACC GAT GAC GAC 353 Ala Pro Leu Phe Met Leu Asp Leu Tyr His Ala Met Thr Asp Asp Asp 80 GAC GGC GGG CCA CCA CAG GCT CAC TTA GGC CGT GCC GAC CTG GTC ATG 401 Asp Gly Gly Pro Pro Gin Ala His Leu Gly Arg Ala Asp Leu Val Met 90 95 100 AGC TTC GTC AAC ATG GTG GAA CGC GAC CGT ACC CTG GGC TAC CAG GAG 449 Ser Phe Val Asn Met Val Glu Arg Asp Arg Thr Leu Gly Tyr Gin Glu 105 110 115 CCA CAC TGG AAG GAA TTC CAC TTT GAC CTA ACC CAG ATC CCT GCT GGG 497 Pro His Trp Lys Glu Phe His Phe Asp Leu Thr Gin lie Pro Ala Gly 120 125 130 135 GAG GCT GTC ICA GCT GCT GAG TTC CGG ATC TAC AAA GAA CCC AGC ACC 545 Glu Ala Val Thr Ala Ala Glu Phe Arg Ile Tyr Lys Glu Pro Ser Thr 140 145 150 CAC CCG CTC AAC ACA ACC CTC CAC ATC AGC ATG TTC GAA GTG GTC CAA 593 His Pro Leu Asn Thr Thr Leu His Ile Ser Met Phe Glu Val Val Gn 155 160 165 GAG CAC TCC AAC AGG GAG TCT GAC TTG TTC TTT TTG GAT CTT CAG ACG 641 Glu His Ser Asn Arg Glu Ser Asp Leu Phe Phe Leu Asp Leu Gin Thr 170 175 180 CTC CGA TCT GGG GAC GAG GGC TGG CTG GTG CTG GAC ATC ACA GCA GCC 689 Lu Arg Ser Gly Asp Glu Gly Trp Leu Val Leu Asp Ile Thr Ala Ala 185 190 195 AGT GAC CGA TGG CTG CTG AAC CAT CAC AAG GAC CTG GGA CTC CGC CTC 737 Ser Asp Arg Trp Leu Leu Asn His His Lys Asp Leu Gly Leu Arg Leu 200 205 210 215 TAT GTG GAA ACC GCG GAT GGG CAC AGC ATG GAT CCT GGC CTG GCT GGT 785 Tyr Val Glu Thr Ala Asp Gly His Ser Met Asp Pro Gly Leu Ala Gly 220 225 230 WO 94/030775 W094/3075PCTIUS93/07 190 126 CTC CTT GGA Leu Leu Cfly TTC TTC AGG Phe Phe Arg 250 CCA CTG AAG Pro Leu Lys 265

CGA

Arg 235 CAA GCA CCA CC Gin Ala Pro Arg AGA CAG CCT TTC Arg Gin Pro Phe ATG GTA ACC Met Val Thr 245 GCA GCG AGA Ala Ala Arg 7,CC AGC CAG AGT Ala Ser Gin Ser GTG CCC CCC CCT Val Arg Ala Pro

CGG

Arg 260 AGC AGC CAG Arg Atg Gin AAG AAA ACG AAC Lys Lys Thr Asn

GAG

Giu 275 CTT CCG CAC CCC Leu Pro His Pro AkAC Asn 280 AAA CTC CCA GG Lys Leu Pro Gly TTT CAT GAT GGC Phe Asp Asp Gly

CAC

His 290 GGT TCC CCC GCC Cly Ser Arg Cly GAC CTT TCC CC Ciu Val Cys Arg

ACC

Arg 300 CAT CAC CTC TAC His Clu Leu Tyr ACC TTC CCT CAC Ser Phe Arg Asp CTT GC Leu Cly 310 TGC CTC CAC Trp Leu Asp

TGG

Trp 315 CTC ATC CCC CCC Val Ile Ala Pro CCC T!,C TCT GCC Cly 7yr Ser Ala TAT TAC TCT Tyr Tyr Cys 325 CCC ACC AAC Ala Thr Asn GAG GGG GAG Clu Gly Glu 330 CAT CCC ATC His Ala Ile 345 TGT GCT TTC CCA Cys Ala Phe Pro CTG CAC TCC TCT ATC Leu Asp Ser Cys Met 335 GTG CAC CTC ATG MCG Val His Leu Met Lys 355

MAC

Asn 340 TTG CAC TCT Leu Gin Ser

CTC

Leu 350 CCA CAT CTT CTC Pro Asp Val Val 977 1025 1073 1121 1169 1217 1265 1319 1379 1439 1499

CCC

Pro 360 MCG GCA TCC TCT Lys Ala Cys Cys

GCA

Ala 365 CCC ACC AMA CTC Pro Thr Lys Lpu CCC ACC TCT CTC Ala Thr Ser Val TAC TAT CAC AC Tyr Tyr Asp Ser MAC MAT GTC ATC Asn Asn Val Ile CCT AMA CAC CCT Arg Lys His Arg MAC ATC Asn Met 390 GTG GTC MCG Val Val Lys TOT CCC TCC CAC Cys Gly Cys His TGACCCCCC CCCACCATCC TCCTTCTACT ACCTTACCAT CTCGCCGGGC CCCTCTCCAC AGCCAGAMAC CCTTCTATCT TATCATACCT CACACAGGGG CAATGOAGG CCCTTCACTT CCCCTGCCA CTTCCTCCTA AAMTTCTGCT CTTTCCCACT TCCTCTGTCC TTCATCGGGT TTCCCCCCTA TCACCCCCC CTCTCCATCC WO 94/03075 WO 9403075PCT/US93/07 190 127

TCCTACCCCA

CTGGGGTCAG

AATGGCAAAT

CTCTGCACCA

GATCAATGCA

CCAGGTATAG

CTGTGAGTTC

GGAATTC

AGCATAGACT

CACTGAAGGC

TCTGGATG-GT

TTCATTGTGG

TCGCTGTACT

CGGTGCATGT

AAGGCCACAT

GAATGCACAC

CCACATGAGG

CTAAGAAGGC

CAGTTGGGAC

CCTTGAAATC

CATTAATCCC

AGAAAGAGCC

AGCATCCCAG

AAGACTGATC

CCTGGAATTC

ATTTTTAGGT

AGAGCTAGCT

AGCGCTAAAG

TGTCTCGGGA

AGCTATGCTA

CTTGGCCATC

TAAACTAGAT

ATAACAGACA

TGTTAGAAAA

AGACAGAGAC

GCAGGAAAAA

ACTGAGAGGT

CTCAGCCCAC

GATCTGGGCT

CATACACTTA

AGAATCAGAG

AGGAGAATCT

AAAAAAAAAC

1559 1619 1679 1739 1799 1859 1919 1926 INFORH1ATION FOR SEQ ID NO:23: SEQUENCE CHARACTERISTICS: LENGTH: 399 amino acids TYPE: amino acid TOPOLOGY: linear (ii) (xi) MOLECULE TYPE: protein SEQUENCE DESCRIPTION: SEQ ID NO:23: Met Ala Met Arg 1 Pro 5 Gly Pro Leu Trp Leu Gly Leu Ala Leu Cys Ala Leu Gly Arg Arg Leu Gly His Gly Pro Pro His Thr Glu Ile Leu Gly Ala Arg Glu Arg Asp Met Gin Ala Val Leu Gly Leu Pro Arg Pro Arg Pro Ala Gin Pro Ala Ala His Ala Arg Gin Pro Ser Ala Pro Leu Met Leu Asp Leu Ala Met 1hr Asp Asp Asp Gly Pro Pro Gin Ala His Leu Gly Arg Ala Val Met Ser Asn Met Val Glu Arg Asp 110 WO 94/03075 WO 9403075PCI'/US93/07 190 128 Arg Leu Ile 145 Ser Phe Val Lys Met 225 Arg Arg Thr Gly Val 305 Gly Thr Leu Giy Tyr Gin Giu Pro His Trp Lys Giu Phe His Phe Asp 115 Gin Lys Phe Leu Asp 195 Leu Pro Pro Pro Giu 275 Gly Phe Ser Ile Glu Giu As p 180 Ile Gly Giy Phe Arg 260 Leu Ser Arg Ala Pro Pro Val 165 Leu Thr Leu Leu Met 245 Ala Pro Arg Asp Tyr 325 Giy 135 Thr Gin Thr Ala Leu 215 Gly Thr Arg Pro Arg 295 Gly Cys Ala Val Pro Leu His Ser 170 Arg Ser 185 Asp Arg Val Giu Leu Gly Phe Arg 250 Leu Lys 265 Lys Leu Val Cys Leu Asp Gly Glu 330 Arg Ile 160 Leu Leu His Se r Ser 240 Val Lys Asp Tyr Gin 320 Asp Ser Cys Met Asn Ala Thr Asn H~is Ala Ile Leu Gin Ser Leu Val His 340 345 350 I~LLI I I_ WO 94/03075 PCT/US93/07190 129 Leu Met Lys Pro Asp Val Val Pro Lys Ala Cys Cys Ala Pro Thr Lys 355 360 365 Leu Ser Ala Thr Ser Val Leu Tyr Tyr Asp Ser Ser Asn Asn Val Ile 370 375 380 Leu Arg Lys His Arg Asn Met Val Val Lys Ala Cys Gly Cys His 385 390 395 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 AAC ACC TCG GAG GCC GTT GCA GTG CTC GCC TCC 48 Met Cer Gly Leu Arg Asn Thr Ser Glu Ala Val Ala Val Leu Ala Ser 1 5 10 CTG GGA CTC GGA ATG GTT CTG CTC ATG TTC GTG GCG ACC ACG CCG CCG 96 Leu Gly Leu Gly Met Val Leu Leu Met Phe Val Ala Thr Thr Pro Pro 25 GCC GTT GAG GCC ACC CAG TCG GGG ATT TAC ATA GAC AAC GGC AAG GAC 144 Ala Val Glu Ala Thr Gln Ser Gly Ile Tyr Ile Asp Asn Gly Lys Asp 40 CAG ACG ATC ATG CAC AGA GTG CTG AGC GAG GAC GAC AAG CTG GAC GTC 192 Gin Thr Ile Met His Arg Val Leu Ser Glu Asp Asp Lys Leu Asp Val 55 TCG TAC GAG ATC CTC GAG TTC CTG GGC ATC GCC GAA CGG CCG ACG CAC 240 Ser Tyr Glu Ile Leu Glu Phe Leu Gly Ile Ala Glu Arg Pro Thr His 70 75 WO 94/03075 WO 9403075PCr/US93/07 190 130

CTO

Leu

CTG

Leu

GAT

Asp

GAC

Asp

CTG

Leu 145

AAC

Asn

CGC

Arg

ATG

het

ACC

Thr

ACG

Thr 225

GGG

Gly

AGC

Ser

GTC

Val

GAC

Asp 115

GAG

Giu

MAG

Lys

CGC

Arg

TGG

Trp

GAG

Giu 195

MAC

Asn

GGC

Gly

TAC

TEyr

CAC

His

TAC

Tyr 100

GAC

Asp

GAG

Giu

CGG

Arg

CAC

His

TTC

Phe 180

CTG

Leu

AGG

Arg

CAG

Gin

GTG

Val

MAG

Lys

GAG

Giu

CAT

His

CAG

Gin

GAC

Asp

CTG

Leu 170

CCC

Pro

GCC

Ala

GTA

Val

CTG

Leu

MAC

Asn 250

TCG

Ser

GAG

Glu

CGG

Arg

MAG

Lys

ATC

Ile 155

CGT

Arg

AAC

Asn

AAC

Asn

TAC

Tyr

TCC

Ser 235

GTG

Val

MAG

Lys

AGC

Ser 110

AGG

Arg

ATC

Ile

ACC

Thr

CAC

His

TAC

Tyr 190

MAG

Lys

GGC

Gly

MAC

Asn

GGC

Gly

TTC

Phe

GAT

Asp

AGC

Ser

ACC

Thr

TTC

Phe

GGC

Giy 175

CTG

Leu

TGG

Trp

ACC

Thr

ACC

Thr

CTG

Leu 255

CTG

Leu

GAG

Gin

GCC

Ala

GAC

Asp

CTG

Leu 160

CGT

Arg

GTG

Val

CTG

Leu

GGC

Gly

ACC

Thr 240

CAC

His GAG TGG CTG GTC Giu Trp Lea Vai MAG TCG MAG GAC MAT CAT GGC ATC TAC ATT GGA GCA Lys Ser Lys Asp Asn His Gly Ile Tyr Ile Gly Ala I -e a- WO 94/03075 PCT/US93/07190 131 CAC GCT GTC Ala Val 275 AAC CGA CCC GAC Asn Arg Pro Asp GAG GTG AAG CTG Glu Val Lys Leu

GAC

Asp 285 GAC ATT GGA Asp Ile Gly CTG ATC Leu lie 290 CAC CGC AAG GTG His Arg Lys Val GAC GAG TTC CAG Asp Glu Phe Gln TTC ATG ATC GCC Phe Met Ile Gly

TTC

Phe 305 TTC CGC GGA CCG Phe Arg Gly Pro AGG AGC MAG CGA Arg Ser Lys Arg 325

GAG

Glu 310 CTG ATC AAG GCG Leu Ile Lys Ala GCC CAC AGC AGC Ala His Ser Ser

CAC

His AGC GCC AGC CAT Ser Ala Ser His CGC AAG CGC AAG Arg Lys Arg Lys AAG TCG Lys Ser 335 GTG TCG CCC Val Ser Pro AGC TGC CAG Ser Cys Gin 355

AAC

Asn 340 AAC GTG CCG CTG Asn Val Pro Leu

CTG

Leu 345 GA CCG ATG GAG Glu Pro Met Glu AGC ACG CGC Ser Thr Arg 350 CTG GGC TGG Leu Gly Trp ATG CAG ACC CTG Met Gin Thr Leu

TAC

Tyr 360 ATA GAC TTC AAG Ile Asp Phe Lys CAT GAC His Asp 370 TGG ATC ATC GCA Trp lie Ile Ala GAG GGC TAT GGC Glu Giy Tyr Gly TTC TAC TGC AGC Phe Tyr Cys Ser 1008 1056 1104 1152 1200 1248 1296 1344

GGC

Gly 385 GAG TGC AAT TTC Glu Cys Asn Phe CTC AAT GCG CAC Leu Asn Ala His

ATG

Met 395 AAC GCC ACG AC Asn Ala Thr Asn GCG ATC GTC CAG Ala lie Val Gin CTG GTC CAC CTG Leu Val His Leu GAG CCC MAG MG GTG CCC Giu Pro Lys Lys Val Pro 415 AAG CCC TGC Lys Pro Cys CAC CTG AAC His Leu Asn 435 GCT CCG ACC AGG Ala Pro Thr Arg GGA GCA CTA CCC Gly Aia Leu Pro GTT CTG TAC Val Leu Tyr 430 AAC ATG ATT Asn Met lie GAC GAG MAT GTG AAC CTG AAA AAG TAT Asp Giu Asn Vai Asn Leu Lys Lys Tyr 440 GTG AAA Val Lys 450 TCC TGC GGG TGC Ser Cys Giy Cys CAT TGA 1368 His 455 WO 94/03075 WO 9403075PCT/US93/07 190 132 INFORMATION FOR SEQ ID SEQUENCE CHl.&~CTERISTICS: LENGTH: 455 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein Met 1 Leu Ala Gin Ser 65 Leu Leu Asp Asp Leu 145 Asn Arg (xi) SEQUENCE Ser Gly Leu Arg 5 Gly Leu Gly Met Val Giu Ala Thr Thr Ile Met His Tyr Giu Ile Leu Ser Ser His Gin Asp Val Tyr His 100 Glu Asp Asp Asp 115 Leu Glu Glu Asp 130 Asp Lys Arg Ala Lys Arg His His 165 Leu Trp Phe Asp 180 DESCRIPTIOR: SEQ ID Asn Thr Ser Giu Ala Val Ala Val Leu Ala Ser Leu Gly Leu Leu Leu Thr Arg 120 Glu Giu Asp Asn 10 Phe Val Tyr Ile Glu Asp Ile Ala Lys Ser 90 Giu Glu His Arg Gin Lys Asp Ile 155 Leu Arg 170 Pro Asn Thr Gly Leu Pro Lys Se r 110 Arg Ile Thr His Tyr 190 Pro Lys Asp Thr Phe Asp Ser Thr Phe Gly 175 Leu Met Ala Glu Leu Arg Ile Tyr Gin Asn Ala Asn Giu Gly 205 Lys Trp Leu WO 94/03075 WO 9403075PCr/US93/07190 133 Thr Ala Asn Arg Giu Phe Thr Ile Thr Val Tyr Ala Ile Gly Thr Gly 210 215 220 Thr Leu Gly Gin His Thr Met Giu Pro Leu Ser Ser Val Asn Thr Thr 225 230 235 240 Gly Asp Tyr Val Giy Trp Leu Giu Leu Asn Val Thr Giu Gly Leu His 245 250 255 Giu Trp Leu Val Lys Ser Lys Asp Asn His Gly Ile Tyr Ile Gly Ala 260 265 270 His Ala Val Asn Arg Pro Asp Arg Glu Val Lys Leu Asp Asp Ile Gly 275 280 285 Leu Ile His Arg Lys Val Asp Asp Giu Phe Gin Pro Phe Met Ile Gly 290 295 300 Phe Phe Arg Gly Pro Giu Leu Ile Lys Ala Thr Ala His Ser Ser His 305 310 315 320 His Arg Ser Lys Arg Ser Aia Ser His Pro Arg Lys Arg Lys Lys Ser 325 330 335 Val Ser Pro Asn Asn Val Pro Leu Leu Glu Pro Met Giu Ser Thr Arg 340 345 350 Ser Cys Gin Met Gin Thr Leu Tyr Ile Asp Phe Lys Asp Leu Gly Trp 355 360 365 His Asp Trp Ile Ile Ala Pro Glu Gly Tyr Gly Ala Phe Tyr Cys Ser 370 375 380 Gly Giu Cy& Asn Phe Pro Leu Asn Aia His Met Asn Aia Thr Asn His 385 390 395 400 Aia Ile Val Gin Thr Leu Val His Leu Leu Giu Pro Lys Lys Val Pro 405 410 415 Lys Pro Cys Cys Ala Pro Thr Arg Leu Gly Ala Leu Pro Val Leu Tyr 420 425 430 His Leu Asn Asp Glu Asn Val Asn Leu Lys Lys Tyr Arg Asn Met Ile 435 440 445 Val Lys Ser Cys Gly Cys His 450 455

I

WO 94/03075 PCT/US93/07190 134 INFORMATION FOR SEQ ID NO:26: SEQUENCE CHARACTERISTICS: LENGTH: 104 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (ix) FEATURE: NAME/KEY: Protein LOCATION: 1..104 OTHER INFORMATION: /note= "BMP3" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: Cys Ala Arg Arg Tyr Leu Lys Val Asp Phe Ala Asp Ile Gly Trp Ser 1 5 10 Glu Trp Ile Ile Ser Pro Lys Ser Phe Asp Ala Tyr Tyr Cys Ser Gly 25 Ala Cys Gln Phe Pro Met Pro Lys Ser Leu Lys Pro Ser Asn His Ala 40 Thr Ile Gin Ser Ile Val Ala Arg Ala Val Gly Val Val Pro Gly Ile 50 55 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: SEQU'! CE CHARACTERISTICS: LENGTH: 102 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein I _I I WO 94/03075 PCT/US93/07190 135 (vi) ORIGINAL SOURCE: ORGANISM: HOMO SAPIENS (ix) FEATURE: NAME/KEY: Protein LOCATION: 1..102 OTHER INFORMATION: /note= (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 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 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 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" WO 94/03075 PCT/US93/07190 136 (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 35 40 Ile Val Gin Thr Leu Val His Leu Met Asn Pro Glu Tyr Val Pro Lys 55 Pro Cys Cys Ala Pro Thr Lys Leu Asn Ala Ile Ser Val Leu Tyr Phe 70 75 Asp Asp Asn Ser Asn Val Ile Leu Lys Lys Tyr Arg Trp Met Val Val 90 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 (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

II

WO 94/03075 PCT/US93/07190 137 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 50 55 Xaa Cys Cys Ala Pro Thr Xaa Leu Xaa Ala Xaa Ser Val Leu Tyr Xaa 70 75 Asp Xaa Ser Xaa Asn Val Xaa Leu Xaa Lys Xaa Arg Asn Met Val Val 90 Xaa Ala Cys Gly Cys His 100 INFORMATION FOR SEQ ID 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= /note= "WHEREIN EACH XAA IS INDEPENDENTLY SELECTED FROM A GROUP OF ONE OR MORE SPECIFIED AMINO ACIDS AS DEFIiED 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 Pro Xaa Xaa Xaa Xaa Ala Xaa Tyr Cys Xaa Gly Xaa Cys Xaa Xaa Pro 25 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asn His Ala Xaa Xaa Xaa Xaa Xaa 35 40 asl 81 WO 94/03075 PCT/US93/07190 138 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 65 70 75 Val Xaa Leu Xaa Xaa Xaa Xaa Xaa Met Xaa Val Xaa Xaa Cys Xaa Cys 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 40 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55 -I -L ~-dd II WO 94/03075 PCT/US93/07190 139 Xaa Cys Cys Xaa Pro Xaa Xaa Xaa Xaa Xaa Xaa 70 Xaa Xaa Xaa Xaa Xaa Val Xaa Leu Xaa Xaa Xaa 90 Xaa Xaa Leu Xaa Xaa Xaa Met Xaa Val 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

GGC

Gly CAC CAC CTC CTC His His Leu Leu CTC CTG GCC CTG Leu Leu Ala Leu CTG CCC TCG CTG Leu Pro Ser Leu CTG ACC CGC GCC Leu Thr Arg Ala GTG CCC CCA GGC Val Pro Pro Gly GCC GCC GCC CTG Ala Ala Ala Leu CTC CAG Leu Gin GCT CTA GGA Ala Leu Gly CGC GAT GAG CCC Arg Asp Glu Pro GGT GCC CCC AGG Gly Ala Pro Arg CTC CGG CCG 254 Leu Arg Pro WO 94/03075 WO 9403075PCr/US93/07 190 140 OTT CCC CCO GTC ATO TGC CGC CTG TTT CGA CGC Val Pro Pro Val Met Trp Arg

ACC

Thr

TGC

Cys

CC

Pro

GGG

Gly ccc Pro

GCG

Ala

GCG

Ala 170

TTG

Leu

OCT

Ala

GCG

Ala

AGG

Arg

CAC

His

OAC

Asp

CAT

His

OCT

Ala 000 Ala 155 000 Gly

GTG

Val

TG

Trp

CTA

Leu Phe

TCO

Ser 0CC Ala 0CC Ala.

OTC

Val 130

COO

Arg 000 Gly

CCC

Pro

GTO

Val

CCO

Pro 210 0CC Ala

COO

Arg

OTO

Val

ATO

Ile

CCT

Pro

CTG

Leu

OTO

Leu

CTO

Leu 165

OTO

Val

GAO

Olu

CTC

Leu

CGC

Arg

CAG

Gin

CAA

Gln

CAC

His 0CC Al a 120

GTG

Val

GOO

Ala 000 Ala

COC

Arg 000 Oly 200

OCO

Ala

GAG

Glu

GAG

Olu

COO

Pro

ATC

Ile 105

GCG

Ala

GMA

Glu 000 Ala

CAA

Gln

CAG

Gln 185 0CC Al a

CTG

Leu 000 Ala TOO CTG CTO CTG Ser Leu Leu Leu GTO ACC CTC GAO COG CGO OTG TOO CAC COO OTO 000 Val Thr Leu Asp Pro Arg LeL His Pro Leu Ala WO 94/03075 WO 9403075PCT/US93/07 190 141 CCG CGG GGC GAC Pro Arg Arg Asp GAA CCC GTG TTG Glu Pro Val Leu GGC GGC CCC GGG Gly Gly Pro Gly

GGC

Gly 265 GCT TGT CGC GCG Ala Cys Arg Ala CGG CTG TAG GTG Arg Leu Tyr Val TTC CGC GAG GTG Phe Arg Glu Val Go1. TGG Gly Trp 2 8 P CAC CGC TGG His Aig Trp GGT CAG TGC Gly Gin Cys 300 ATC CCG CGC Ile Ala Pro Arg TTC CTG GCC AAC Phe Leu Ala Asn TAG TGC CAG Tyr Cys Gin 295 GGG CCG CCG Gly Pro Pro C CTG CCC GTC Ala Leu Pro Val

GG

Ala 305 GTG TGG COO TCG Leu Ser Cly Ser GCG GTC Ala Leu 315 AAC CAC GGT CTG Asn His Ala Val

CTG

Leu 320 CCC GCG CTC AIG Arg Ala Leu Met CG GCCC GC Ala Ala Ala CCC GGG GAC CTG Ala Ala Asp Leu TGC TC GTG CCC Cys Cys Val Pro GGC GIG TCG CCC ATG Arg Leu Ser Pro Ile 1022 1070 1118 1166 1219 1247 TCC GTG GTG TTC Ser Val Leu Phe GAG AAC AGC GAG Asp Asn Ser Asp GTG GTG GTG CGG Val Val Leu Arg CAG TAT Gin Tyr 360 GAG GAG ATG Glu Asp Met GTG GAG GAG TGC Val Asp Clu Gys GGC TGC CGG Gly Gys Arg 370 TAAGGCGGGG CrGGGCAGGGA CCCGGGCCGA ACAATAAATG CCGGGTGG INFORMATION FOR SEQ ID NO:33: SEQUENCE CHARACTERISTICS: LENGTH: 372 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33: Met Pro Pro Pro Gin Gin Cly Pro Gys Cly His His Leu Leu Leu Leu 1 5 10 WO 94/03075 WO 9403075PC17/US93/07190 142 Leu Pro Pro Leu Thr Val Arg Val Ala 145 Gly Asp Pro Trp Ala 225 As p Ala Gly Gin Phe Ser Ala Ala Val 130 Arg Gly Pro Val Pro 210 Ala Pro Leu Pro Gly Arg Pro Gly Ser 115 Phe Leu Trp Gly Arg 195 Arg Cys Arg Leu Ala Ala Arg Gly As n 100 Glu Asp Glo Glu Pro 180 Ala Ser Ala Leu Gly 260 Pro Ala Arg Asp 70 Thr Val Val Ser Arg 150 Ser Leu Leu Arg Leu 230 His Ser Leu Leu 55 Pro Leu Arg Ser Ala 135 Phe Val Leu Leu Leu 215 Ala Pro Leu Leu 40 Arg Gin Gin His Ala 120 Val Ala Ala Arg Gly 200 Ala Glu Leu Pro 25 Gin Pro Glu Pro Ile 105 Ala Glu Ala Gin Gin 185 Ala Leu Ala Al a Leu Ala Val Thr Cys Pro Gly Pro Al.a Ala 170 Leu Ala Ala Ser Arg 250 Thr Leu Pro Arg 75 His Asp His Al a Ala 155 Gly Val Trp Leu Leu 235 Pro Arg Gly Pro Ser Val Arg Cys Glu 140 Ala Gin Pro Ala Arg 220 Leu Arg Ala Pro Leu Arg Val Met Gly Ser Glu Glu Gly Ala 110 Pro Glu 125 Arg Pro Ala Ala Gly Ala Ala Leu 190 Arg Asn 205 Pro Arg Leu Val Arg Asp Val Asp Trp Ar.

Leu Pro Trp Ser Pro Gly 175 Gly Ala Ala Thr Ala 255 Pro Glu Arg Arg Gly Thr Thr Arg Glu 160 Ala Pro Ser Pro Leu 240 Glu Pro Val Leu Gly Gly Pro Gly Gly Ala Cys Arg Ala 5 Arg Arg Leu 270 L-a I Is a__ WO 94/03075 PCT/US93/07190 143 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 Gin Gly Gin Cys Ala Leu Pro Val 290 295 300 Ala Leu Ser Gly Ser Gly Gly Pro Pro Ala Leu Asn His Ala Val Leu 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 Gin Tyr Glu Asp Met Val Val Asp Glu 355 360 365 Cys Gly Cys Arg 370

Claims (11)

1. An enriched infant formula composition, comprising a morphogen solubilized in a nutrient formulation substantially mimetic of the nutrient content of human milk, said morphogen comprising an acid-stable, protease-resistant dimeric protein that 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 defined by Generic Sequence 6, Seq. ID No. 31; or (iii) a sequence defined by OPX, Seq. ID No. 29.

2. The morphogen-enriched infant formula of Claim I wherein said formula is milk-based.

3. The morphogen-enriched infant formula of Claim 1 wherein said formula is soy-based.

4. A composition comprising: a nutrient, trace mineral, or vitamin required for normal metabolism in mammalian tissue; and a morphogen comprising an acid-stable, protease-resistant dimeric protein comprising 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 defined by Generic Sequence 6, Seq. ID No. 31; or (iii) a sequence defined by OPX, Seq. ID No. 29, said morphogen being dispersed in said composition to a concentration sufficient to protect mammalian tissue from tissue damage or reduced tissue function due to metabolic stress or dysfunction. f'f .Vi 145 A composition comprising: a nutrient, trace mineral, or vitamin required for normal metabolism in mammalian tissue; and a morphogen comprising an acid-stable, protease-resistant dimeric protein comprising 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 ,esidues 38.139 of Seq. ID No. (ii) a sequence defined by Generic Sequence 6, Seq. ID No. 31; or (iii) a sequence defined by OPX, S ID No. 29, said morphogen being dispersed in said composition to a concentration sufficient to restore the function of senescent or quiescent mammalian tissue or mammalian tissue afflicted with tissue damage or reduced tissue function due to metabolic stress or dysfunction.

6. The composition of Claim 4 or 5 wherein said nutrient is selected from proteins, amino acids, carbohydrates, lipids, fatty acids, sugars, nucleosides, or nucleotides.

7. The composition of Claim 4 or 5 wherein said mineral is selected from calcium, 0 phosphorus, sodium, potassium, chloride, magnesium, iron, zinc, copper, manganese, or iodine.

8. The composition of Claim 4 or 5 wherein said vitamin is selected from vitamin A, vitamin D3, vitamin C, vitamin Bl, vitamin B2, vitamin B6, vitamin B 12, pantothenic acid, vitamin E, vitamin KI, folic acid or biotin.

9. The composition of Claim 1, 4, or 5 wherein said morphogen is associated with a Scontrolled release component, adapted such that the morphogen is released in a I- L--IIIQ I

146- controlled manner in the gastrointestinal tract subsequent to passage through the stomach. The composition of Claim 1, 4, or 5 adapted for enteral administration. 11. The composition of Claim 4 or 5 formulated as a solid. 12. The composition of Claim I11 wherein said solid is a tablet, troche or lozenge. 13. The composition of Claim 1, 4, or 5 formulated as a liquid. 14. The composition of Claim 13 wherein said liquid is a beverage or a syrup. The composition of Claim 1, 4, or 5 wherein said morphogen is associated with a morphogen-solubilizing molecule. 16. The composition of Claim 15 wherein said molecule is casein or a derivative, salt or analog thereof. 17. The composition of Claim 15 wherein said morphogen-solubilizing molecule is a prodomain polypeptide of a member of the morphogen family, or an allelic, species or biosynthetic sequence variant thereof. 18. The composition of Claim 17 wherein said morphogen is noncovalently associated with said prodomain polypeptide. 19. The composition of Claim 17 wherein said morphogen-solubilizing molecule comprises one or more prodomain selected from the prodomain of human OP-1, mouse OP-1, human OP-2, mouse OP-2, 60A, GDF-1, BMP2A, BMP2B, DPP, Vgl, Vgr-1, BMP3, BMP5, BMP6, or a naturally-occurring or biosynthetic variant of any thereof. S a a a a a a *r a. a a a a a a a a aa a *ir w LS I

147- The composition of Claim 1, 4, or 5 wherein said morphogen comprises an acid-stable, protease-resistant dimeric protein comprising a pair of folded polypeptides, the amino acid sequence of each of which comprises a sequence sharing at least 80% homology with said C-terminal seven cysteine domain of human OP-1. 21. The composition of Claim 1, 4, or 5 wherein said morphogen comprises an acid-stable, protease-resistant dimeric protein comprising a pair of folded polypeptides, the amino acid sequence of each of which comprises a sequence having greater than 60% amino acid sequence identity with said C-terminal seven cysteine domain of human OP-1. 22. The composition of Claim 21 wherein said morphogen comprises an acid-stable, protease-resistant dimeric protein comprising a pair of folded polypeptides, the amino acid sequence of each of which comprises a sequence having greater than 65% amino acid sequence identity with said C-terminal seven cysteine domain of human OP-1. 23. The composition of Claim 22 wherein said morphogen comprises an acid-stable, protease-resistant dimeric protein comprising a pair of folded polypeptides, the amino acid sequence of each of which comprises said C-terminal seven cysteine domain of human OP-1, or a naturally-occurring or biosynthetic variant thereof. S' 24. A method for improving the human milk mimetic characteristics of an infant formula, comprising the step of adding a morphogen to said formula prior to providing said formula to an infant, said morphogen comprising an acid-stable, protease-resistant dimeric protein comprising 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 defined by Generic Sequence 6, Seq. ID No. 31; or V' (iii) a sequence defined by OPX, Seq. ID No. 29. I- -I s_ II 148 The method of Claim 24 wherein said infant is a preterm or low birth weight infant. 26. A method for prophylactically protecting mammalian tissue at risk of incurring tissue damage or reduced tissue function, or for treating mammalian tissue afflicted with tissue damage or reduced tissue function due to metabolic stress or dysfunction, the method comprising the step of administering the composition of Claim 1 to a premature or low birth weight newborn, or juvenile mammal. 27. A method for prophylactically protecting mammalian tissue at risk of incurring tissue damage or reduced tissue function, or for treating mammalian tissue afflicted with tissue damage or reduced tissue function due to metabolic stress, the method comprising the step of administering the composition of Claim 4 or 5 to a pregnant, lactating or post- menopausal female mammal, or to a premature or low birth weight newborn, juvenile, or aged mammal. 28. A method for prophylactically protecting mammal at risk of incurring tissue damage or reduced tissue function, or for treating mammalian tissue afflicted with tissue damage or reduced tissue function due to metabolic dysfunction, the method comprising the step of administering the composition of Claim 1, 4, or 5 to a mammal at risk of or afflicted with: protein-energy malnutrition resulting from starvation, dehydration, anorexia nervosa, or trauma-induced tissue damage caused by radiotherapy, chemotherapy. or surgery; or malabsorption-malnutrition resulting from a digestive or intestinal fistula, short bowel, gastrointestinal disorder, or hypercatabolism. 29. A method for restoring the function of senescent or quiescent mammalian tissue or mammalian tissue afflicted with tissue damage or reduced tissue function due to metabolic stress or dysfunction, the method comprising the step of administering the YIL- n7 96

149- composition of Claim I to a premature or low birth weight newborn, or juvenile mammal. A method for restoring the function of senescent or quiescent mammalian tissue or mammalian tissue afflicted with tissue damage or reduced tissue function due to metabolic stress, the method comprising the step of administering the composition of Claim 4 or 5 to a pregnant, lactating or post-menopausal female mammal, or to a premature or low birth weight newborn. ;,wenile, or aged mammal. 31. A method for restoring the function of senescent or quiescent mammalian tissue or mammalian tissue afflicted with tissue damage or reduced tissue function due to metabolic dysfunction, the method comprising the step of administering the composition of Claim 1, 4, or 5 to a mammal at risk of or afflicted with: protein-energy malnutrition resulting from starvation, dehydration, anorexia nervosa, or trauma-induced tissue damage caused by radiotherapy, chemotherapy, or surgery; or malabsorption-malnutrition resulting from a digestive or intestinal fistula, short bowel, gastrointestinal disorder, or hypercatabolism. B B 32. The method of Claim 26, 27, 28, 29, 30, or 31 wherein said morphogen is associated with a morphogen-solubilizing molecule. 33. The method of Claim 32 wherein said morphogen-solubilizing molecule is casein or a derivative, salt or analog thereof. S"34. The method of Claim 32 wherein said morphogen-solubilizing molecule is a prodomain polypeptide of a member of the morphogen family, or an allelic, species or biosynthetic sequence variant thereof. ,1 Im~-s ~i b H I e' 150 The method of Claim 34 wherein said morphogen is noncovalently associated with said prodomain poiypeptide. 36. The method of Claim 34 wherein said morphogen-solubilizing molecule comprises one or more prodomains selected from the prodomains of human OP-1, mouse OP-1, human OP-2, mouse OP-2, BMP2A, BM/P2B, BMP3, BMP5, BMP6, Vgl, Vgr-l, DPP, GDF-1, or a naturally-occurring or biosynthetic variant of any thereof. 37. The method of Claim 26, 27, 28, 29, 30, or 31 wherein said morphogen comprises an acid-stable, protease-resistant dimeric protein comprising a pair of folded polypeptides, the amino acid sequence of each of which comprises a sequence sharing at least homology with said C-terminal seven cysteine domain of human OP-1. 38. The method of Claim 26, 27, 28, 29, 30, or 31 wherein said morphogen comprises an acid-stable, protease-resistant dimeric protein comprising a pair of folded polypeptides, the amino acid sequence of each of which comprises a sequence having greater than amino acid sequence identity with said C-terminal seven cysteine domain of human OP-1. t* 39. The method of Claim 38 wherein said morphogen comprises an acid-stable, protease- resistant dimeric comprising a pair of folded polypeptides, the amino acid sequence of 9 each of which comprises a sequence having greater than 65% amino acid sequence identity with said C-terminal seven cysteine domain of human OP-1. 40. The method of Claim 39 wherein said morphogen comprises an acid-stable, protease- resistant dimeric protein comprising a pair of folded polypeptides, the amino acid sequence of each of which comprises said C-terminal seven cysteine domain of human OP-1, or a naturally-occurring or biosynthetic variant thereof. Dated this 8th day of July, 1996 Creative Biomolecules, Inc. By DAVIES COLLISON CAVE Patent Attorneys for the Applicant(s)