AU7142000A - Morphogen treatment for chronic renal failure - Google Patents

Description

AUSTRALIA

PATENTS ACT 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT

(ORIGINAL)

S.

Name of Applicant: Actual Inventors: Address for Service: Invention Title: Creative Biomolecules, Inc.

Kuber SAMPATH AND Charles COHEN DAVIES COLLISON CAVE, Patent Attorneys, 1 Little Collins Street, Melbourne, 3000 Morphogen treatment for chronic renal failure The following statement is a full description of this invention, including the best method of performing it known to Q:\OPER\PDB\CREATIVEDIV.DOC 3/I1100

A-

MORPHOGEN TREATMENT FOR CHRONIC RENAL FAILURE This application is a divisional application derived from Australian Patent Application No.

28322/97, the entire contents of which are incorporated herein by reference.

Field of the Invention The present invention relates generally to methods of treatment for renal disease. In particular, the invention relates to methods of treatment for conditions which place mammals, including humans, in, or at risk of, chronic renal failure. The methods preferably involve the 5 administration of certain proteins of the osteogenic protein/bone morphogenetic protein (OP/BMP) family within the TGF-P superfamily of proteins. More generally, the methods involve the administration of certain morphogens, inducers of those morphogens, or agonists of the corresponding morphogen receptors, or implantation of renal cells induced with those morphogens.

10 Background of the Invention The mammalian renal system serves primary roles both in the removal of catabolic waste products from the bloodstream and in the maintenance of fluid and electrolyte balances in the body. Renal failures are, therefore, life-threatening conditions in which the build-up of catabolites and other toxins, and/or the development of significant imbalances in electrolytes or fluids, may 15 lead to the failure of other major organs systems and death. As a general matter, renal failure is classified as "acute" or "chronic." As de:;iled below, the differences between these two conditions are not merely a matter of severity or rapidity but, rather, reflect differences in etiology, prognosis, and treatment.

Acute Renal Failure Acute renal failure is defined as an abrupt cessation or substantial reduction of renal function and, in as many as 90-95% of cases, may be secondary to trauma, surgery or another acute medical condition. Acute renal failure may be due to pre-renal causes decreased cardiac output, hypovolemia, altered vascular resistance) or to post-renal causes obstructions or constrictions of the ureters, bladder or urethra) which do not directly involve the kidneys and which, if treated quickly, will not entail significant loss ofnephrons or other damage to the kidneys. Alternatively, acute renal failure may be due to intrinsic renal causes which involve a more direct insult or injury to the kidneys, and which may entail permanent damage to WO 97/41881 PCT/US97/07816 -2the nephrons or other kidney structures. Intrinsic causes of acute renal failure include but are not limited to infectious diseases various bacterial, viral or parasitic infections), inflammatory diseases glomerulonephritis, systemic lupus erythematosus), ischemia renal artery occlusion), toxic syndromes heavy metal poisoning, side-effects of antimicrobial treatments or chemotherapy), and direct traumas.

The diagnosis and treatment of acute renal failure is as varied as its causes. In human patients, oliguria (urine output 400 ml/day) or anuria (urine output 50 ml/day) may be present in 50-70% of cases, BUN levels may climb 10-20 mg/dL/day or faster, plasma creatinine levels may climb 0.5-1.0 mg/dL/day, and metabolic acidosis is almost always present. If not treated, the electrolyte and fluid imbalances hyperkalemia, acidosis, edema) associated with acute renal failure may lead to life-threatening arrhythmia, congestive heart failure, or multiple organ system failures. Present therapies are typically directed at the underlying causes of the acute renal failure pre-renal, post-renal, or infectious causes) and management of the complications. Due to severity of acute renal failure, episodes rarely last longer than several weeks without mortality and are treated on an in-patient basis.

Chronic Renal Failure Chronic renal failure may be defined as a progressive, permanent and significant reduction of the glomerular filtration rate (GFR) due to a significant and continuing loss of nephrons.

Chronic renal failure typically begins from a point at which a chronic renal insufficiency a permanent decrease in renal function of at least 50-60%) has resulted from some insult to the renal tissues which has caused a significant loss of nephron units. The initial insult may or may not have been associated with an episode of acute renal failure. Irrespective of the nature of the initial insult, chronic renal failure manifests a "final common path" of signs and symptoms as nephrons are progressively lost and GFR progressively declines. This progressive deterioration in renal function is slow, typically spanning many years or decades in human patients, but seemingly inevitable.

The early stage of chronic renal failure typically begins when GFR has been reduced to approximately one-third of normal 30-40 ml/min for an average human adult). As a result of the significant nephron loss, and in an apparent "attempt" to maintain the overall GFR with fewer nephrons, the average single nephron GFR (SNGFR) is increased by adaptations of the remaining nephrons at both the structural and functional level. One structural manifestation of this adaptation, readily detectable by microscopic examination of biopsy samples, is a WO 97/41881 PCT/US97/07816 -3- "compensatory hypertrophy" of both the glomeruli and the tubules of the kidney, a process which literally increases the volume of filtrate which can be produced by each remaining nephron by literal enlargement of the glomeruli and tubules. Indeed, as a result of the hypertrophy or dilation of the collecting ducts, the urine of subjects with chronic renal failure often contains broad "casts," typically 2-6 times normal diameter, which aid in diagnosis and have also been referred to as "renal failure casts." At the same time, there are functional changes in the remaining nephrons, such as decreased absorption or increased secretion of normally excreted solutes, which may be responses to hormonal or paracrine changes elsewhere in the body increasing levels of parathyroid hormone (PTH) in response to changes in serum levels of calcium and phosphate).

These adaptations in early stage chronic renal failure are not successful in completely restoring GFR or other parameters of renal function and, in fact, subject the remaining nephrons to increased risk of loss. For example, the increased SNGFR is associated with mechanical stresses on the glomerulus due to hypertension and hyperperfusion. The loss of integrity of podocyte junctures leads to increased permeability of the glomerulus to macromolecules or 15 "leakiness" of the glomerular capsule. Proliferative effects are also observed in mesangial, epithelial and endothelial cells, as well as increases in the deposition of collagen and other matrix proteins. Sclerosis of both the glomeruli and tubules is another common symptom of the hypertrophied nephrons and the risk of coagulation in the glomerulus is increased. In particular, 0. "these adaptations of the remaining nephrons, by pushing the SNGFR well beyond its normal level, 20 actually decrease the capacity of the remaining nephrons to respond to acute changes in water, solute, or acid loads and, therefore, actually increase the probability of additional nephron loss.

As chronic renal failure progresses, and GFR continues to decline to less than 10% of normal 5-10 ml/min), the subject enters end-stage renal disease (ESRD). During this phase, the inability of the remaining nephrons to adequately remove waste products from the blood, while retaining useful products and maintaining fluid and electrolyte balance, leads to a rapid decline in which many organ systems, and particularly the cardiovascular system, may begin to fail. For example, BUN and creatinine levels may be expected to rise and, at BUN levels of 100 mg/dL and serum creatinine levels of 8-12 mg/dL, a uremic syndrome will typically develop in which the kidneys can no longer remove the end products of nitrogen metabolism. At this point, renal failure will rapidly progress to death unless the subject receives renal replacement therapy chronic hemodialysis, continuous peritoneal dialysis, or kidney transplantation).

WO 97/41881 PCT/US97/07816 -4- Approximately 600 patients per million receive chronic dialysis each year in the United States, at an average cost approaching $60,000-$80,000 per patient per year. Of the new cases of end-stage renal disease each year, approximately 28-33% are due to diabetic nephropathy (or diabetic glomerulopathy or diabetic renal hypertrophy), 24-29% are due to hypertensive nephrosclerosis (or hypertensive glomerulosclerosis), and 15-22% are due to glomerulonephritis.

The 5-year survival rate for all chronic dialysis patients is approximately 40%, but for patients over 65, the rate drops to approximately Morphogens and Growth Factors A great many proteins have now been identified which appear to act as morphogenetic or growth factors, regulating cell proliferation or differentiation. Typically these growth factors exert their effects on specific sets or subsets of cells or tissues. Thus, for example, epidermal growth factors, nerve growth factors, fibroblast growth factors, various hormones, and many other proteins inducing or inhibiting cell proliferation or differentiation have been identified and shown to affect some subgroup of cells or tissues.

15 One group of morphogenetic proteins, referred to herein as "morphogens," includes members of the family of osteogenic proteins/bone morphogenetic proteins (OP/BMPs) which were initially identified by their ability to induce ectopic, endochondral bone morphogenesis.

Subsequent characterization of the nucleic acid and amino acid sequences of the BMPs has shown them to be a subgroup of the TGF-P superfamily of growth factors. Members of this morphogen family have now been shown to include the mammalian osteogenic protein-1 (OP-1, also known as BMP-7), osteogenic protein-2 osteogenic protein-3 BMP-2 (also known as BMP-2A or CBMP-2A), BMP-3, BMP-4 (also known as BMP-2B or CBMP-2B), BMP-6, Vgr-1, and GDF-1, as well as the Xenopus homologue Vgl and the Drosophila S homologues DPP and 60A. Members of this family encode secreted polypeptides that share common structural features and that are similarly processed from pro-proteins to yield carboxy terminal mature proteins having a conserved pattern of cysteines. The active forms of these proteins are either disulfide-bonded homodimers of a single family member, or heterodimers of two different members (see, Massague (1990) Annu. Rev. Cell Biol. 6:597; Sampath, et al.

(1990) J. Biol. Chem. 265:13198).

The members of the morphogen family of proteins are expressed in a variety of tissues during development. BMP-3 for, example, has been shown to be expressed in developing human lung and kidney (Vukicevic et al. (1994) J. Histochem. Cvtochem. 42:869-875), BMP-4 has been WO 97/41881 PCT/US97/07816 shown to be expressed in the developing limbs, heart, facial processes and condensed mesenchyme associated with early whisker follicles in embryonic mice (Jones, et al. (1991) Development 111:531-542), and OP-1 (BMP-7) has been shown immunohistochemically to be associated with basement membranes in human embryos, including those of the developing lungs, pancreas, skin, and convoluted tubules of kidneys (Vukicevic, et al. (1994) Biochem. Biophvs Res. Commun. 198:693-700). Some of the morphogens OP-2 and BMP-2) were not detected in analyses of adult tissues, suggesting only an early developmental role for these morphogens (Ozkaynak, et al. (1992) J. Biol. Chem. 267:25220-25227). In contrast, high levels of murine OP-1 expression have been observed in adult mouse kidneys (Ozkaynak, et al. (1991) Biochem. Biophvs. Res. Commun. 179:116-123). This suggests a possible role for OP-1 synthesized in the kidney as a paracrine regulator of bone growth, and would be consistent with the role of the kidneys in both calcium regulation and bone homeostasis.

A great variety of growth factors have been considered which may participate in the regulation of the growth and repair of renal tissues (reviewed in, Toback (1992) Kidney Intl.

15 41:226-246). For example, EGF, TGF-a, TGF-P, IGF-I, IGF-II, PDGF, FGF, Renin/Angiotensin I, L-1 and OP-I have all been found to be expressed by various adult renal cells or tissues and to have effects on renal cell proliferation or differentiation (see, Toback (1992) supra, Ozkaynak, et al. (1991) supra). In addition, several of these have been found to be expressed in the developing kidney, including IGF-I, TGF-P and OP-1 (reviewed in, Bard, et al. (1994) Mech. Develop.

48:3-11).

\Interestingly, TGF-P has been shown in a murine metanephric organ culture system to retard overall growth and segmental differentiation of all segments of developing nephrons except the thick ascending limb-early distal tubules (Avner and Sweeney (1990) Pediatr. Nephrol. 4:372- S 377). In addition, TGF-P expression has been found to be increased in several models of renal disease, suggesting that TGF-P mediated increases in the synthesis of extracellular matrix components may be involved in the etiology of diabetic nephropathy (or diabetic glomerulopathy or diabetic renal hypertrophy), renal fibrosis, glomerulosclerosis and glomerulonephritis, interstitial fibrosis, and hypertensive nephrosclerosis (Shankland, et al. (1994) Kidney Intl.

46:430-442; Yamamoto, et al. (1994) Kidney Intl. 45:916-927; Yamamoto, et al. (1993) PNAS 90:1814-1818; Tamaki, et al. (1994) Kidney Intl. 45:525-536; Border, et al. (1990) Nature 346:371-374; Hamaguchi, et al. (1995) Hypertension 26:199-207).

WO 97/41881 PCT/US97/07816 -6- Also of interest is the fact that serum levels of human growth hormone (GH) are elevated in subjects with chronic renal failure (Wright et al. (1968) Lancet 2:798; Samaan and Freeman (1970) Metabolism 19:102). Recombinant GH has been shown to help maintain protein balance in malnourished chronic renal failure patients, and to promote "catch-up" growth in children with chronic renal failure. It has been suggested that these effects are mediated by IGF-I (see, e.g., Kopple (1992) Miner. Electrolyte Metab. 18:269-275). Although some studies have found that the administration of IGF-I increases renal plasma flow and GFR in chronic renal failure patients Guler, et al. (1989) PNAS 86:2868-2872; Hirschberg, et al. (1993) Kidney Intl. 43:387- 397), other studies have found that this effect is merely transient (Miller, et al. (1994) Kidney Intl.

46:201-207).

Thus, although some growth factors have been shown to be expressed in both developing and adult renal tissues, and although at least one has been shown to increase renal function in the short term, none has yet been shown to be of therapeutic benefit in preventing, inhibiting, or delaying the progressive loss of renal function that characterizes chronic renal failure. A need 15 remains, therefore, for treatments which will prevent the progressive loss of renal function which •causes hundreds of thousand of patients to become dependent upon chronic dialysis, and which results in the premature deaths of tens of thousands each year.

~Summary of the Invention The present invention is directed to methods of treatment, and pharmaceutical 20 preparations for use in the treatment, of mammalian subjects in, or at risk of, chronic renal failure, or at risk of the need for renal replacement therapy. Such subjects include subjects already afflicted with chronic renal failure, or which have already received renal replacement therapy, as well as any subject reasonably expected to suffer a progressive loss of renal function associated with progressive loss of functioning nephron units. Whether a particular subject is at risk is a determination which may routinely be made by one of ordinary skill in the relevant medical or veterinary art. Subjects in, or at risk of, chronic renal failure, or at risk of the need for renal replacement therapy, include but are not limited to the following: subjects which may be regarded as afflicted with chronic renal failure, end-stage renal disease, chronic diabetic nephropathy, hypertensive nephrosclerosis, chronic glomerulonephritis, hereditary nephritis, and/or renal dysplasia; subjects having a biopsy indicating glomerular hypertrophy, tubular hypertrophy, chronic glomerulosclerosis, and/or chronic tubulointerstitial sclerosis; subjects having an WO 97/41881 PCTIUS97/07816 -7ultrasound, MRI, CAT scan, or other non-invasive examination indicating renal fibrosis; subjects having an unusual number of broad casts present in urinary sediment; subjects having a GFR which is chronically less than about 50%, and more particularly less than about 40%, 30% or of the expected GFR for the subject; human male subjects weighing at least about 50 kg and having a GFR which is chronically less than about 50 ml/min, and more particularly less than about 40 ml/min, 30 ml/min or 20 ml/min; human female subjects weighing at least about 40 kg and having a GFR which is chronically less than about 40 ml/min, and more particularly less than about 30 ml/min, 20 ml/min or 10 ml/min; subjects possessing a number of functional nephron units which is less than about 50%, and more particularly less than about 40%, 30% or 20%, of the number of functional nephron units possessed by a healthy but otherwise similar subject; subjects which have a single kidney; and subjects which are kidney transplant recipients.

The methods and compositions of this invention capitalize in part upon the discovery that certain proteins of eukaryotic origin may be used as renal therapeutic agents in the treatment of subjects at risk, as defined herein, of chronic renal failure or the need for renal replacement S 15 therapy. Generally, these renal therapeutic agents are proteins, or are based upon proteins, which are members of the osteogenic protein/bone morphogenetic protein (OP/BMP) family of proteins.

Thus, useful OP/BMP renal therapeutic agents of the invention include polypeptides, or functional variants of polypeptides, comprising at least the C-terminal six- or seven-cysteine domain of a mammalian protein selected from the group consisting of OP-1, OP-2, OP-3, BMP2, BMP3, 20 BMP4, BMP5, BMP6, BMP9, and proteins which exhibit at least 70% or, more preferably. or 80% amino acid sequence homology with the amino acid sequence of the seven-cysteine domain of human OP-1; and which are capable of inducing chondrogenesis in the Reddi- Sampath ectopic bone assay (Sampath and Reddi (1981), Proc. Natl. Acad. Sci. (USA) 78:7599- 7603) or a substantially equivalent assay, capable of significantly preventing, inhibiting, delaying or alleviating the progressive loss of renal function in a standard animal model of chronic renal failure, or capable of causing a clinically significant improvement in a standard marker of renal function when administered to a mammal in, or at risk of, chronic renal failure. More generally speaking, the invention provides for the use of"morphogens" which are dimeric proteins that induce morphogenesis of one or more eukaryotic mammalian) cells, tissues or organs.

Of particular interest herein are morphogens that induce morphogenesis at least of mammalian renal tissue, including formation of functional renal epithelium and, in particular, functional glomerular and tubular epithelium. Morphogens comprise a pair of polypeptides that, when WO 97/41881 PCT/US97/07816 -8folded, adopt a configuration sufficient for the resulting dimeric protein to elicit morphogenetic responses in cells and tissues displaying receptors specific for said morphogen. That is, morphogens generally induce 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. "Progenitor" cells are uncommitted cells that are competent to differentiate into one or more specific types of differentiated cells, depending on their genomic repertoire and the tissue specificity of the permissive environment in which morphogenesis is induced. Morphogens further can delay or mitigate the onset of senescence- or quiescence-associated loss of phenotype and/or tissue function. Morphogens still further can stimulate phenotypic expression of differentiated cells, :including expression of metabolic and/or functional, secretory, properties thereof In addition, morphogens can induce redifferentiation of committed cells under appropriate environmental conditions. As noted above, morphogens that induce proliferation and/or e 15 differentiation at least of mammalian renal tissue, and/or support the growth, maintenance and/or functional properties of mammalian nephrons, are of particular interest herein.

In preferred embodiments, the pair of morphogen polypeptides have amino acid sequences each comprising a sequence that shares a defined relationship with an amino acid sequence of a reference morphogen. Herein, preferred morphogen polypeptides share a defined relationship 20 with a sequence present in morphogenically active human OP-1, SEQ ID NO: 4. However, any one or more of the naturally occurring or biosynthetic sequences disclosed herein similarly could be used as a reference sequence. Preferred morphogen polypeptides share a defined relationship with at least the C-terminal six cysteine domain of human OP-1, residues 43-139 of SEQ ID NO: 4. Preferably, morphogen polypeptides share a defined relationship with at least the Cterminal seven cysteine domain of human OP-1, residues 38-139 of SEQ ID NO: 4. That is, preferred morphogen polypeptides in a dimeric protein with morphogenic activity each comprise a sequence that corresponds to a reference sequence or is functionally equivalent thereto.

Functionally equivalent sequences include functionally equivalent arrangements of cysteine residues disposed within the reference sequence, including amino acid insertions or deletions which alter the linear arrangement of these cysteines, but do not materially impair their relationship in the folded structure of the dimeric morphogen protein, including their ability to form such intra- or inter-chain disulfide bonds as may be necessary for morphogenic activity.

WO 97/41881 PCTIUS97/07816 -9- Functionally equivalent sequences further include those wherein one or more amino acid residues differs from the corresponding residue of a reference morphogen sequence, the C-terminal seven cysteine domain (or "skeleton") of human OP-1, provided that this difference does not destroy morphogenic activity. Accordingly, conservative substitutions of corresponding amino acids in the reference sequence are preferred. Amino acid residues that are "conservative substitutions" for corresponding residues in a reference sequence are those that are physically or functionally similar to the corresponding reference residues, that have similar size, shape, electric charge, chemical properties including the ability to form covalent or hydrogen bonds, or the like. Particularly preferred conservative substitutions are those fulfilling the criteria defined for an "accepted point mutation" in Dayhoff et al. (1978), 5 Atlas of Protein Sequence and Structure, Suppl. 3, ch. 22 (pp. 354-352), Natl. Biomed. Res. Found., Washington, D.C. 20007, the teachings of which are incorporated by reference herein.

In certain embodiments, a polypeptide suspected of being functionally equivalent to a reference morphogen polypeptide is aligned therewith using the method ofNeedleman, et al.

15 (1970), J. Mol. Biol. 48:443-453, implemented conveniently by computer programs such as the Align program (DNAstar, Inc.). As noted above, internal gaps and amino acid insertions in the candidate sequence are ignored for purposes of calculating the defined relationship, conventionally expressed as a level of amino acid sequence homology or identity, between the candidate and reference sequences. "Amino acid sequence homology" is understood herein to 20 mean amino acid sequence similarity. Homologous sequences share identical or similar amino acid residues, where similar residues are conservative substitutions for, or "allowed point mutations" of, corresponding amino acid residues in an aligned reference sequence. Thus, a "candidate polypeptide sequence that shares 70% amino acid homology with a reference sequence is one in which any 70% of the aligned residues are either identical to or are conservative substitutions of the corresponding residues in a reference sequence.

Of particular interest herein are morphogens, which, when provided to the kidney of a mammal, induce or maintain the normal state of differentiation and growth of nephron units. Of still more particular interest herein are morphogens which, when administered to a mammal, prevent, inhibit or delay the development of compensatory hypertrophy, including glomerular hypertrophy and/or tubular hypertrophy. Such morphogens can be used to treat a mammal in, or at risk of, chronic renal failure by preventing, inhibiting or delaying the progressive loss of functional nephron units and the consequent progressive loss of renal function.

WO 97/41881 PCT/US97/07816 The present invention alternatively can be practiced with methods and compositions comprising a morphogen stimulating agent or morphogen inducer in lieu of a morphogen. A "morphogen inducer" is a compound that stimulates in vivo production, expression, of a therapeutically effective concentration of an endogenous morphogen in the body of a mammal sufficient to regenerate or maintain renal tissue and/or to inhibit additional loss thereof Such compounds are understood to include substances which, when administered to a mammal, act on cells of tissue(s) or organ(s) that normally are competent to produce and/or secrete a morphogen encoded within the genome of the mammal, and which cause the endogenous level of the morphogen in the mammal's body to be altered. Endogenous or administered morphogens can act as endocrine, paracrine or autocrine factors. That is, endogenous morphogens can be synthesized by the cells in which morphogenetic responses are induced, by neighboring cells, or by *cells of a distant tissue, in which circumstances the secreted endogenous morphogen is transported to the site of morphogenesis, by the individual's bloodstream. In preferred embodiments, the agent stimulates expression and/or secretion of an endogenous morphogen so 15 as to increase amounts thereof in renal tissues.

In still other embodiments, an agent which acts as an agonist of a morphogen receptor may be administered instead of the morphogen itself. An "agonist" of a receptor means a compound which binds to the receptor and for which such binding has a similar functional result as binding of the natural, endogenous ligand of the receptor. That is, the compound must, upon 20 interaction with the receptor, produce the same or substantially similar transmembrane and/or intracellular effects as the endogenous ligand. Thus, an agonist of a morphogen receptor binds to the receptor and such binding has the same or a similar functional result as morphogen binding induction of morphogenesis). The activity or potency of an agonist can be less than that of \the natural ligand, in which case the agonist is said to be a "partial agonist," or it can be equal to or greater than that of the natural ligand, in which case it is said to be a "full agonist." Thus, for example, a small peptide or other molecule which can mimic the activity of a morphogen in binding to and activating the morphogen's receptor may be employed as an equivalent of the morphogen. Preferably the agonist is a full agonist, but partial morphogen receptor agonists may also be advantageously employed. Methods of identifying such agonists are known in the art and include assays for compounds which induce morphogen-mediated responses induction of differentiation of metanephric mesenchyme, induction of endochondral bone formation, and the like). Such an agent may also be referred to as a morphogen "mimic," "mimetic," or "analog." WO 97/41881 PCTIUS97/07816 -11- The OP/BMP renal therapeutic agents of the invention, or the morphogens, morphogen inducers and agonists of morphogen receptors of the invention, may be administered by any route of administration which is compatible with the selected agent, and may be formulated with any pharmaceutically acceptable carrier appropriate to the route of administration. Preferred routes of administration are parenteral and, in particular, intravenous, intraperitoneal, and renal intracapsular. Treatments are also preferably conducted over an extended period on an outpatient basis. Daily dosages of the renal therapeutic agents are expected to be in the range of about 0.01- 1000 tg/kg body weight, and more preferably about 10-300 pg/kg body weight, although precise dosages will vary depending upon the particular renal therapeutic agent employed and the particular subject's medical condition and history.

Finally, in yet further embodiments, renal cells may be implanted into the kidney of a subject in, or at risk, chronic renal failure, or at risk of needing renal replacement therapy, in order to serve as a source of morphogen and/or to provide a source of additional functional renal tissue.

i These cells may be renal mesenchymal progenitor cells, or renal mesenchymal progenitor cells 15 which have been induced to undergo metanephric differentiation. The cells may be derived from a donor a tissue-type matched donor, sibling, identical twin), may be derived from a tissue culture undifferentiated renal mesenchyme culture, fetal renal tissue culture), or may be explanted from the subject and then be re-implanted after proliferation and/or differentiation.

Preferably, the cells are induced to undergo metanephric differentiation by treatment with a 20 morphogen OP-1) either before or after implantation.

S The treatments of the present invention are useful in preventing, inhibiting or delaying the progressive loss of functional nephron units, and the consequent progressive loss of renal S..function, which typify chronic renal failure. As such they are of great value in preventing or delaying the need for chronic dialysis or renal replacement therapy in subjects with chronic renal insufficiency, or reducing the necessary frequency of chronic renal dialysis in subjects with endstage renal disease. As such, they are useful in prolonging the lives, and in maintaining the quality of life, of subjects at risk of, or already afflicted with, chronic renal failure.

Brief Description of the Figures Figure 1. This figure is a bar graph showing average serum creatinine levels for groups of sham-operated ("SHAM") or partially nephrectomized ("Nx Contr" and rats. 5-6 months WO 97/41881 PCT/US97/07816 -12post-surgery, rats received injections of vehicle only ("Nx control" and "SHAM") or 1, 3, 10 or pg/kg body weight of soluble OP-1 three times a week for 4-8 weeks.

Figure 2. This figure is a bar graph showing average serum urea levels for groups of sham-operated ("SHAM") or partially nephrectomized ("Nx Contr" and rats. 5-6 months post-surgery, rats received injections of vehicle only ("Nx control" and "SHAM") or 1, 3, 10 or pg/kg body weight of soluble OP-1 three times a week for 4-8 weeks.

Figure 3. Panels A-C of this figure are micrographs of renal tissue from rats at 1Ox magnification. Tissue from sham-operated rat. Tissue from rat in chronic renal failure after 5/6 nephrectomy (Nx control). Tissue from rat treated with OP-I after 5/6 nephrectomy.

Figure 4. Panels A-C of this figure are micrographs of renal tissue from rats at magnification. Tissue from sham-operated rat. Tissue from rat in chronic renal failure after 5/6 nephrectomy (Nx control). Tissue from rat treated with OP-I after 5/6 nephrectomy.

15 Figure 5. This figure is a line graph showing average serum creatinine levels over 9 weeks for groups of partially nephrectomized rats. 2-3 weeks post-surgery, rats received injections of vehicle only ("Control") or 10 lg/kg body weight of soluble OP-I 3 times per week.

Figure 6. This figure is a line graph showing average creatinine clearance rates as a measure of GFR over 8 weeks for groups of partially nephrectomized rats. 2-3 weeks post- 20 surgery, rats received injections of vehicle only ("Control") or 10 pg/kg body weight of soluble OP-1 3 times per week.

Figure 7. Panels 7-1 through 7-12 of this figure are a tabular alignment of the amino acid sequences of various naturally occurring morphogens with a preferred reference sequence of human OP-1, residues 38-139 of SEQ ID NO: 4. Morphogen polypeptides shown in this figure also are identified in the Sequence Listing.

WO 97/41881 PCT/US97/07816 -13- Detailed Description of the Invention I. Definitions In order to more clearly and concisely point out the subject matter of the claimed invention, the following definitions are provided for specific terms used in the following written description and appended claims.

Renal therapeutic acent. As used herein, the term "renal therapeutic agent" means a polypeptide, or a functional variant of a polypeptide, comprising at least the C-terminal six- or seven-cysteine domain of a mammalian protein selected from the group consisting of OP-1, OP-2, OP-3, BMP2, BMP3, BMP4, BMP5, BMP6, BMP9, and proteins which exhibit at least 70% or, more preferably, 75% or 80% amino acid sequence homology with the amino acid sequence of the seven-cysteine domain of human OP-1; and which is capable of inducing chondrogenesis in the Reddi-Sampath ectopic bone assay (Sampath and Reddi (1981), Proc. Natl. Acad. Sci.

°U.SA) 78:7599-7603) or a substantially equivalent assay, capable of significantly preventing, inhibiting, delaying or alleviating the progressive loss of renal function in a standard animal model 15 of chronic renal failure, or capable of causing a clinically significant improvement in a standard marker of renal function when administered to a mammal in, or at risk of, chronic renal failure. As used herein, a percentage "homology" between two amino acid sequences indicates o. the percentage of amino acid residues which are identical or similar between the sequences and, as used herein, "similar" residues are "conservative substitutions" which fulfill the criteria defined for 20 an "accepted point mutation" in Dayhoffet al. (1978), Atlas of Protein Seuence and Structure *e *e Vol. 5 (Suppl. pp. 354-352, Nail. Biomed. Res. Found., Washington,

D.C.

Therapeutic efficacy. As used herein, a renal therapeutic agent of the invention is said to have "therapeutic efficacy," and an amount of the agent is said to be "therapeutically effective," if administration of that amount of the agent is sufficient to cause a clinically significant improvement in a standard marker of renal function when administered to a mammalian subject a human patient) in, or at risk of, chronic renal failure. Such markers of renal function are well known in the medical literature and include, without being limited to, rates of increase in BUN levels, rates of increase in serum creatinine, static measurements of BUN, static measurements of serum creatinine, glomerular filtration rates (GFR), ratios of BUN/creatinine, serum concentrations of sodium urine/plasma ratios for creatinine, urine/plasma ratios for urea, urine osmolality, daily urine output, and the like (see, for example, Brenner and Lazarus (1994), in Harrison's Principles of Internal Medicine, 13th edition, Isselbacher et al., eds., WO 97/41881 PCT/US97/07816 -14- McGraw Hill Text, New York; Luke and Strom (1994), in Internal Medicine, 4th Edition, J.H.

Stein, ed., Mosby-Year Book, Inc. St. Louis.) Glomerular Filtration Rate (GFR). The "glomerular filtration rate" or "GFR" is proportional to the rate of clearance into urine of a plasma-borne substance which is not bound by serum proteins, is freely filtered across glomeruli, and is neither secreted nor reabsorbed by the renal tubules. Thus, as used herein, GFR preferably is defined by the following equation: GFR= U c o. xV conc where Uc,, is the urine concentration of the marker, is the plasma concentration of the marker, and Vis the urine flow rate in ml/min. Optionally, GFR is corrected for body surface area. Thus, the GFR values used herein may be regarded as being in units of ml/min/1.73m 2 The preferred measure of GFR is the clearance of inulin but, because of the difficulty of o* measuring the concentrations of this substance, the clearance of creatinine is typically used in clinical settings. For example, for an average size, healthy human male (70 kg, 20-40 yrs), a i. typical GFR measured by creatinine clearance is expected to be approximately 125 ml/min with plasma concentrations of creatinine of 0.7-1.5 mg/dL. For a comparable, average size woman, a S. typical GFR measured by creatinine clearance is expected to be approximately 115 ml/min with creatinine levels of 0.5-1.3 mg/dL. During times of good health, human GFR values are relatively stable until about age 40, when GFR typically begins to decrease with age. For subjects surviving *to age 85 or 90, GFR may be reduced to 50% of the comparable values at age 20 Expected Glomerular Filtration Rate An estimate of the "expected GFR" or "GFR.xp" may be provided based upon considerations of a subject's age, weight, sex, body surface area, and degree of musculature, and the plasma concentration of some marker compound creatinine) as determined by a blood test. Thus, as an example, an expected GFR or GFRxp may be estimated as: GFR (140-age) x weight (kg) 72 x Poe (mg/dl) This estimate does not take into consideration such factors as surface area, degree of musculature, or percentage body fat. Nonetheless, using plasma creatinine levels as the marker, this formula has been employed for human males as an inexpensive means of estimating GFR. Because creatinine is produced by striated muscle, the expected GFR or GFRp, of human female subjects WO 97/41881 PCT/US97/07816 is estimated by the same equation multiplied by 0.85 to account for expected differences in muscle mass. (See Lemann, et al. (1990) Am. J. Kidney Dis. 16(3):236-243.) Broad Cast. Microscopic examination of urinary sediment for the presence of formed elements is a standard procedure in urinalysis. Amongst the formed elements which may be present in urine are cylindrical masses of agglutinated materials that typically represent a mold or "cast" of the lumen of a distal convoluted tubule or collecting tubule. In healthy human subjects, such casts typically have a diameter of 15-25 pLm. In subjects with chronic renal failure, however, hypertrophy of the tubules may result in the presence of "broad casts" or "renal failure casts" which are 2-6 times the diameter of normal casts and often have a homogeneous waxy appearance. Thus, as used herein, a "broad cast" means a urinary sediment cast having a diameter of 2-6 times normal, or about 30-150 plm for human casts.

:Chronic. As used herein with respect to clinical indications such as urinary casts, measured GFR, or other markers of renal function, "chronic" means persisting for a period of at l..east three, and more preferably, at least six months. Thus, for example, a subject with a 15 measured GFR chronically below 50% of GFRL,, is a subject in which the GFR has been measured and found to be below 50% of GFR,,X in at least two measurements separated by at least three, and more preferably, by at least six months, and for which there is no medically sound reason to believe that GFR was substantially 10%) higher during the intervening period.

Subjects in, or at risk of, chronic renal failure As used herein, a subject is said to be in, or 20 at risk of, chronic renal failure, or at risk of the need for renal replacement therapy, if the subject is reasonably expected to suffer a progressive loss of renal function associated with progressive loss of functioning nephron units. Whether a particular subject is in, or at risk of, chronic renal failure is a determination which may routinely be made by one of ordinary skill in the relevant medical or veterinary art. Subjects in, or at risk of, chronic renal failure, or at risk of the need for renal replacement therapy, include but are not limited to the following: subjects which may be regarded as afflicted with chronic renal failure, end-stage renal disease, chronic diabetic nephropathy, hypertensive nephrosclerosis, chronic glomerulonephritis, hereditary nephritis, and/or renal dysplasia; subjects having a biopsy indicating glomerular hypertrophy, tubular hypertrophy, chronic glomerulosclerosis, and/or chronic tubulointerstitial sclerosis; subjects having an ultrasound, MRI, CAT scan, or other non-invasive examination indicating renal fibrosis; subjects having an unusual number of broad casts present in urinary sediment; subjects having a GFR which is chronically less than about 50%, and more particularly less than about 40%, 30% or WO 97/41881 PCT/US97/07816 -16of the expected GFR for the subject; human male subjects weighing at least about 50 kg and having a GFR which is chronically less than about 50 ml/min, and more particularly less than about 40 ml/min, 30 ml/min or 20 ml/min; human female subjects weighing at least about 40 kg and having a GFR which is chronically less than about 40 ml/min, and more particularly less than about 30 ml/min, 20 ml/min or 10 ml/min; subjects possessing a number of functional nephron units which is less than about 50%, and more particularly less than about 40%, 30% or 20%, of the number of functional nephron units possessed by a healthy but otherwise similar subject; subjects which have a single kidney; and subjects which are kidney transplant recipients.

II. Description of the Preferred Embodiments A. General The present invention depends, in part, upon the surprising discovery that administration of certain protein-based renal therapeutic agents to subjects in, or at risk of, chronic renal failure, S*can reduce mortality and/or morbidity rates, and prevent, inhibit, delay or alleviate the progressive loss of renal function which characterizes chronic renal failure. Alternatively, or in addition, 15 administration of the renal therapeutic agents of the present invention can prevent, inhibit or delay the progressive loss of functional nephron units and the progressive decline in glomerular filtration rate (GFR) which slowly but inevitably leads to the need for renal replacement therapy renal S transplant or chronic dialysis) or death. In preferred embodiments, the therapeutic agents of the invention are members of the osteogenic protein/bone morphogenetic protein (OP/BMP) family 20 within the TGF-P superfamily of proteins.

B. Renal Therapeutic Agents The renal therapeutic agents of the present invention are naturally occurring proteins, or functional variants of naturally occurring proteins, in the osteogenic protein/bone morphogenetic protein (OP/BMP) family within the TGF-P superfamily of proteins. That is, these proteins form a distinct subgroup, referred to herein as the "OP/BMP family," within the loose evolutionary grouping of sequence-related proteins known as the TGF-P superfamily. Members of this protein family comprise secreted polypeptides that share common structural features, and that are similarly processed from a pro-protein to yield a carboxy-terminal mature protein. Within the mature protein, all members share a conserved pattern of six or seven cysteine residues defining a 97-106 amino acid domain, and the active form of these proteins is either a disulfide-bonded homodimer of a single family member, or a heterodimer of two different members (see, e.g., Massague (1990), Annu. Rev. Cell Biol. 6:597; Sampath et al. (1990), J. Biol. Chem.

WO 97/41881 PCTIUS97/07816 -17- 265:13198). For example, in its mature, native form, natural-sourced human OP-I is a glycosylated dimer typically having an apparent molecular weight of about 30-36 kDa as determined by SDS-PAGE. When reduced, the 30 kDa protein gives rise to two glycosylated peptide subunits having apparent molecular weights of about 16 kDa and 18 kDa. The unglycosylated protein has an apparent molecular weight of about 27 kDa. When reduced, the 27 kDa protein gives rise to two unglycosylated polypeptide chains, having molecular weights of about 14 kDa to 16 kDa.

Typically, the naturally occurring OP/BMP proteins are translated as a precursor, having an N-terminal signal peptide sequence, a "pro" domain, and a "mature" protein domain. The signal peptide is typically less than 30 residues, and is cleaved rapidly upon translation at a cleavage site that can be predicted using the method of Von Heijne (1986), Nucleic Acids Research 14:4683-4691. The "pro" domain is variable both in sequence and in length, ranging from approximately 200 to over 400 residues. The pro domain is cleaved to yield the "mature" C-terminal domain of approximately 115-180 residues, which includes the conserved six- or 15 seven-cysteine C-terminal domain of 97-106 residues. As used herein, the "pro form" of an OP/BMP family member refers to a protein comprising a folded pair of polypeptides, each comprising a pro domain in either covalent or noncovalent association with the mature domains of the OP/BMP polypeptide. Typically, the pro form of the protein is more soluble than the mature form under physiological conditions. The pro form appears to be the primary form secreted from 20 cultured mammalian cells. The "mature form" of the protein refers to mature C-terminal domain which is not associated, either covalently or noncovalently, with the pro domain. Any preparation of OP-I is considered to contain mature form when the amount of pro domain in the preparation is no more than 5% of the amount of "mature" C-terminal domain.

OP/BMP family members useful herein include any of the known naturally-occurring native proteins including allelic, phylogenetic counterpart and other variants thereof, whether naturally-sourced or biosynthetically produced including "muteins" or "mutant proteins"), as well as new, active members of the OP/BMP family of proteins.

Particularly useful sequences include those comprising the C-terminal seven cysteine domains of mammalian, preferably human, human OP-1, OP-2, OP-3, BMP2, BMP3, BMP4, BMP5, BMP6, BMP8 and BMP9. Other proteins useful in the practice of the invention include active forms of GDF-5, GDF-6, GDF-7, DPP, Vgl, Vgr-1, 60A, GDF-1, GDF-3, GDF-6, GDF-7, BMP 10, BMP11, BMP 13, BMP 15, UNVIN, NODAL, SCREW, ADMP or WO 97/41881 PCT/US97/07816 -18- NURAL and amino acid sequence variants thereof. In one currently preferred embodiment, the renal therapeutic agents of the invention are selected from any one of: OP-1, OP-2, OP-3, BMP2, BMP3, BMP4, BMP5, BMP6, and BMP9.

Publications disclosing these sequences, as well as their chemical and physical properties, include: OP-1 and OP-2: U.S. Pat. No. 5,011,691, U.S. Pat. No. 5,266,683, and Ozkaynak et al.

(1990), EMBO J. 9:2085-2093; OP-3: W094/10203; BMP2, BMP3, and BMP4: U.S. Pat.

No. 5,013,649, W091/18098, W088/00205, and Wozney et al. (1988), Science 242:1528-1534; and BMP6: W090/11366 and Celeste et al. (1991), Proc. Natl. Acad. Sci. (USA) 87:9843-9847; Vgr-1: Lyons et al. (1989), Proc. Natl. Acad. Sci. (USA) 86: 4554-4558; DPP: Padgett et al. (1987), Nature 325:81-84; Vgl: Weeks (1987), Cell 51:861-867; BMP-9: W095/33830; BMP10: W094/26893; BMP-11: W094/26892; BMP12: W095/16035; BN' BMP-13: W095/16035; GDF-1: W092/00382 and Lee et al. (1991), Proc. Natl. Acad. Sci.

S(USA) 88:4250-4254; GDF-8: WO94/21681; GDF-9: W094/15966; GDF-10: W095/10539; GDF-11: W096/01845; BMP-15: W096/36710; MP121: W096/01316; GDF-5 (CDMP-1, 15 MP52): W094/15949, W096/14335, WO93/16099 and Storm et al. (1994), Nature 368:639- 643; GDF-6 (CDMP-2, BMP13): W095/01801, W096/14335 and W095/10635; GDF-7 (CDMP-3, BMP12): W095/10802 and WO95/10635; BMP-3b: Takao, et al. (1996), Biochem.

Biophys. Res. Comm. 219:656-662; GDF-3: WO94/15965; 60A: Blaster et al. (1993), Cell 73:687-702 and GenBank accession number L12032. In another embodiment, useful proteins 20 include biologically active biosynthetic constructs, including novel biosynthetic proteins and chimeric proteins designed using sequences from two or more known OP/BMP family proteins.

See also the biosynthetic constructs disclosed in U.S. Pat. No. 5,011,691, the disclosure of which is incorporated herein by reference COP-1, COP-3, COP-4, COP-5, COP-7, and COP-16).

In other preferred embodiments, the renal therapeutic agents useful herein include therapeutically effective proteins in which the amino acid sequences comprise a sequence sharing at least 70% amino acid sequence "homology" and, preferably, 75% or 80% homology with the C-terminal seven cysteine domain present in the active forms of human OP-1 residues 330-431, as shown in SEQ ID NO: 2 of U.S. Pat. No. 5,266,683). In other preferred embodiments, the renal therapeutic agents useful herein include therapeutically effective proteins in which the amino acid sequences comprise a sequence sharing at least 60% amino acid sequence identity and, preferably, 65% or 70% identity with the C-terminal seven cysteine domain present in the active forms of human OP-1. Thus, a candidate amino acid sequence thought to have WO 97/41881 PCT/US97/07816 -19therapeutic efficacy in the present invention can be aligned with the amino acid sequence of the C-terminal seven cysteine domain of human OP-I using the method of Needleman et al. (1970), J Mol. Biol. 48:443-453, implemented conveniently by computer programs such as the Align program (DNAstar, Inc.). As will be understood by those skilled in the art, homologous or functionally equivalent sequences include functionally equivalent arrangements of the cysteine residues within the conserved cysteine domain, including amino acid insertions or deletions which alter the linear arrangement of these cysteines, but do not materially impair their relationship in the folded structure of the dimeric protein, including their ability to form such intra- or inter-chain disulfide bonds as may be necessary for biological activity. Therefore, internal gaps and amino acid insertions in the candidate sequence are ignored for purposes of calculating the level of amino acid sequence homology or identity between the candidate and reference sequences.

"Amino acid sequence homology" is understood herein to include both amino acid sequence identity and similarity. Thus, as used herein, a percentage "homology" between two amino acid sequences indicates the percentage of amino acid residues which are identical or 15 similar between the sequences. "Similar" residues are "conservative substitutions" which fulfill the criteria defined for an "accepted point mutation" in Dayhoff et al. (1978), Atlas of Protein Sequence and Structure Vol. 5 (Suppl. pp. 354-352, Natl. Biomed. Res. Found., Washington, D.C. Thus, "conservative substitutions" are residues that are physically or functionally similar to the corresponding reference residues, having similar size, shape, electric charge, and/or chemical 20 properties such as the ability to form covalent or hydrogen bonds, or the like. Examples of conservative substitutions include the substitution of one amino acid for another with similar characteristics, substitutions within the following groups: valine, glycine; glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine, methionine; and phenylalanine, tyrosine. The term "conservative substitution" or "conservative variation" also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid in a given polypeptide chain, provided that the resulting substituted polypeptide chain also has therapeutic efficacy in the present invention.

The renal therapeutic agents of the invention are also characterized by biological activities which may be readily ascertained by those of ordinary skill in the art. Specifically, a renal therapeutic agent of the present invention is capable of inducing chondrogenesis in the Reddi-Sampath ectopic bone assay (Sampath and Reddi (1981), Proc. Natl. Acad. Sci. (USA) WO 97/41881 PCT/US97/07816 78:7599-7603) or a substantially equivalent assay, capable of significantly preventing, inhibiting, delaying or alleviating the progressive loss of renal function in a standard animal model of chronic renal failure, or capable of causing a clinically significant improvement in a standard marker of renal function when administered to a mammal in, or at risk of, chronic renal failure.

The Reddi-Sampath ectopic bone assay is well known in the art as an assay of chondrogenic activity. The assay, which can be easily performed, is described and discussed in, for example, Sampath and Reddi (1981), Proc. Natl. Acad. Sci. (USA) 78:7599-7603; and Wozney (1989), "Bone Morphogenetic Proteins," Progress in Growth Factor Research 1:267- 280. Many equivalent assays, using other animals and tissue sites, may be employed or developed by those of skill in the art to evaluate the biological activity of the renal therapeutic agents of the present invention. See, for example, the bioassays described in U.S. Pat. No. 5,226,683.

The renal therapeutic agents of the present invention also may be tested in animal models 00*0 of chronic renal failure. Mammalian models of chronic renal failure in, for example, mice, rats, 15 guinea pigs, cats, dogs, sheep, goats, pigs, cows, horses, and non-human primates, may be created by causing an appropriate direct or indirect injury or insult to the renal tissues of the animal.

Animal models of chronic renal failure may, for example, be created by performing a partial 5/6) nephrectomy which reduces the number of functioning nephron units to a level which initiates compensatory renal hypertrophy, further nephron loss, and the progressive decline in renal 20 function which characterizes chronic renal failure.

Finally, the renal therapeutic agents of the present invention may be evaluated for their therapeutic efficacy in causing a clinically significant improvement in a standard marker of renal function when administered to a mammalian subject a human patient) in, or at risk of, chronic renal failure. Such markers of renal function are well known in the medical literature and include, without being limited to, rates of increase in BUN levels, rates of increase in serum creatinine, static measurements of BUN, static measurements of serum creatinine, glomerular filtration rates (GFR), ratios of BUN/creatinine, serum concentrations of sodium urine/plasma ratios for creatinine, urine/plasma ratios for urea, urine osmolality, daily urine output, and the like (see, for example, Brenner and Lazarus (1994), in Harrison's Principles of Internal Medicine, 13th edition, Isselbacher et al., eds., McGraw Hill Text, New York; Luke and Strom (1994), in Internal Medicine, 4th Edition, J.H. Stein, ed., Mosby-Year Book, Inc. St.

Louis.) WO 97/41881 PCT/US97/07816 -21 The renal therapeutic agents contemplated herein can be expressed from intact or truncated genomic or cDNA or from synthetic DNAs in prokaryotic or eukaryotic host cells. The dimeric proteins can be isolated from the culture media and/or refolded and dimerized in vitro to form biologically active compositions. Heterodimers can be formed in vitro by combining separate, distinct polypeptide chains. Alternatively, heterodimers can be formed in a single cell by co-expressing nucleic acids encoding separate, distinct polypeptide chains. See, for example, W093/09229, or U.S. Pat. No. 5,411,941, for several exemplary recombinant heterodimer protein production protocols. Currently preferred host cells include, without limitation, prokaryotes including E. coli, or eukaryotes including yeast, Saccharomyces, insect cells, or mammalian cells, such as CHO, COS or BSC cells. One of ordinary skill in the art will appreciate that other host cells can be used to advantage. Detailed descriptions of the proteins useful in the practice of this invention, including how to make, use and test them for chondrogenic activity, are disclosed in numerous publications, including U.S. Pat. Nos. 5,266,683 and 5,011,691, the *o disclosures of which are herein incorporated by reference.

15 C. Morphogens. Inducers and Agonists Table 1, below, summarizes various naturally occurring members of the OP/BMP family identified to date, including their nomenclature as used herein, their Sequence Listing references, and publication sources for the amino acid sequences for the full length proteins not included in e the Sequence Listing. Each of the generic terms set forth in Table 1 is intended and should be understood to embrace the therapeutically effective proteins expressed from nucleic acids encoding the identified sequence menuoned below and set fonh in the Sequence Listing, or an active fragment or precursor thereof, or a functional equivalent thereof such as a naturally occurring or biosynthetic variant. Naturally occurring variants include allelic variant forms i isolated from other individuals of a single biological species, as well as species variants 25 (homologues) isolated from phylogenetically distinct biological species.

TABLE 1 "OP-I" Refers generically to mammalian proteins equivalent to the human OP-1 protein disclosed in SEQ ID NO: 4 and includes at least mouse OP-1, SEQ ID NO: 5 In each of human and mouse OP-1, SEQ ID NOs: 4 and 5, the conserved C-terminal seven cysteine domain is defined by residues 38 to 139.

cDNA sequences and corresponding amino acid sequences for the full length WO 97/41881 PCTIUS97/07816 -22proteins are provided in SEQ ID NOs: 15 and 16 (hOP-1) and SEQ ID NOs: 17 and 18 (mOP-1.) The mature proteins are defined by residues 293-431 (hOP-1) and 292-430 (mOP-1). The "pro" regions of the proteins, cleaved to yield the mature proteins are defined essentially by residues 30-292 (hOP-1) and residues 30-291 (mOP-1).

"OP-2" Refers generically to mammalian proteins equivalent to the human OP-2 protein disclosed in SEQ ID NO: 6 and includes at least mouse OP-2 ("mOP- SEQ ID NO: In each of human and mouse OP-2, the conserved C-terminal seven domain is defined by residues 38 to 139 of SEQ ID NOs: 6 and 7. cDNA sequences and corresponding amino acid sequences for the full length proteins are provided in SEQ ID NOs: 19 and 20 (hOP-2) and SEQ ID NOs: 21 and 22 (mOP- The mature proteins are defined essentially by residues 264-402 (hOP-2) and 261-399 (mOP-2). The "pro" regions of the proteins, cleaved to yield the mature proteins are defined essentially by residues 18-263 (hOP-2) and residues 18-260 15 (mOP-1).

"OP-3" Refers generically to mammalian proteins equivalent to the mouse OP-3 protein disclosed in SEQ ID NO: 26 The conserved C-terminal seven domain is defined by residues 298 to 399 of SEQ ID NO: 26, which shares greater than 79% amino acid identity with the corresponding mOP-2 and hOP-2 sequences, and 20 greater than 66% identity with the corresponding OP-1 sequences. A cDNA °i sequence encoding the above-mentioned amino acid sequence is provided in SEQ ID NO: 25. OP-3 is unique among the morphogens identified to date in that the Sresidue at position 9 in the conserved C-terminal seven domain residue 315 of SEQ ID NO: 26) is a serine, whereas other morphogens typically have a tryptophan at this location.

"BMP-2" Refers generically to mammalian proteins equivalent to the BMP-2 proteins, including at least human BMP-2 (or CBMP-2A, SEQ ID NO: The amino acid sequence for the full length proteins, referred to in the literature as BMP-2 or BMP-2A, appear in Wozney, et al. (1988) Science 242:1528-1534. The pro WO 97/41881 PCT/US97/07816 -23domain for BMP-2 (BMP-2A) likely includes residues 25-248; the mature protein, residues 249-396.

"BMP-4" Refers generically to mammalian proteins equivalent to the CBMP-4 proteins, including at least human BMP-4 (or BMP-2B, SEQ ID NO: The amino acid sequence for the full length proteins, referred to in the literature as BMP-4 and BMP-2B, appear in Wozney, et al. (1988) Science 242:1528-1534. The pro domain for BMP-4 (BMP-2B) likely includes residues 25-256; the mature protein, residues 257-408.

"DPP" refers to proteins encoded by a Drosophila DPP gene and defining a conserved Cterminal seven domain (SEQ ID NO: 10). 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.

"Vgl" refers to proteins encoded by a Xenopus Vgl gene and defining a conserved C- 15 terminal seven domain (SEQ ID NO: I The amino acid sequence for the 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.

"Vgr-1" refers to proteins encoded by a murine Vgr-1 gene and defining a conserved C- *i 20 terminal seven domain (SEQ ID NO: 12). 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.

"GDF-I" refers to proteins encoded by a human GDF-1 gene and defining a conserved Cterminal seven domain (SEQ ID NO: 13). The cDNA and encoded amino sequence for the full length protein are provided in SEQ ID NOs: 30 and 31. The prodomain likely extends from the signal peptide cleavage site to residue 214; the mature protein likely is defined by residues 215-372.

WO 97/41881 PCT/US97/07816 24 "BMP-3" 15 "BMP-5" "BMP-6" 0 6 00..

.00..

V..0.

0 r refers generically to proteins expressed from a nucleic acid the Drosophila gene) encoding a 60A protein or active fragments thereof (see SEQ ID NOs: 23 and 24 wherein the cDNA and encoded amino acid sequence for the full length protein are provided). The conserved C-terminal seven domain is defined by 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. The 60A protein is considered likely to be a phylogenetic counterpart of the human and mouse OP-1 genes; Sampath, et al. (1993) PNAS 90:6004-6008.

refers to proteins encoded by a human BMP-3 gene and defining a conserved Cterminal seven domain (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 proteins encoded by a human BMP-5 gene and defining a conserved Cterminal seven domain (SEQ ID NO: 27). The amino acid sequence for the full length protein appears in Celeste, et al. (1991) PNAS 87:9843-9847. The pro domain likely extends from the signal peptide cleavage site to residue 316; the mature protein likely is defined by residues 317-454.

refers to proteins encoded by a human BMP-6 gene and defining a conserved Cterminal seven domain (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 374; the mature sequence likely includes residues 375-513.

As shown in Figure 7, the OP-2 and OP-3 proteins have an additional cysteine residue in the conserved C-terminal region see residue 41 of SEQ ID NOs: 6 and The GDF-1 protein has a four amino acid insert within the conserved C-terminal cysteine domain (residues 44-47 of SEQ ID NO: 13). Further, the BMP-2 and BMP-4 proteins are missing one amino acid residue within the cysteine domain. Thus, the alignment of these amino acid WO 97/41881 PCT/US97/07816 sequences in Figure 7 illustrates the principles of alignment used herein with respect to the preferred reference sequence of human OP-1, residues 38-139 of SEQ ID NO: 4.

In addition to the OP/BMP renal therapeutic agents described in the previous section, the present invention may be practiced using "morphogens," as defined herein. Morphogens useful in the present invention include those in which the amino acid sequences ofmorphogen polypeptides comprise a sequence sharing at least 70% amino acid sequence homology or "similarity", and preferably 80% homology or similarity with a reference sequence selected from the foregoing naturally OP/BMP family members. Preferably, the reference protein is human OP-I, and the reference sequence thereof is the C-terminal seven cysteine domain present in active forms of human OP-1, residues 38-139 of SEQ ID NO: 4. Morphogens useful herein accordingly include allelic, phylogenetic counterpart and other variants of the preferred reference sequence, whether naturally-occurring or biosynthetically produced including "muteins" or "mutant proteins"), S' as well as novel members of the OP/BMP family of proteins set forth and identified above, in connection with Table 1. Certain particularly preferred morphogen polypeptides share at least 15 60% amino acid identity with the preferred reference sequence of human OP-1, still more preferably at least 65% amino acid identity therewith.

In other preferred embodiments, the morphogen polypeptides useful in the present invention are defined by a generic amino acid sequence. For example, Generic Sequence 7 (SEQ ID NO: 1) and Generic Sequence 8 (SEQ ID NO: 2) disclosed below, accommodate the homologies shared among preferred OP/BMP protein family members identified to date, including at least OP-1, OP-2, OP-3, BMP-2, BMP-3, BMP-4, 60A, DPP, Vgl, BMP-5, BMP-6, Vgr-1, and GDF-I (SEQ ID NOs: 4-15, 24, and 26-29). 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 domains (Generic Sequences 7 and 8, respectively), as well as alternative residues for the 25 variable positions within the sequence. The generic sequences provide an appropriate cysteine domain where inter- or intramolecular disulfide bonds can form, and contain certain critical amino acids likely to influence the tertiary structure of the folded proteins. In addition, the generic sequences allow for an additional cysteine at position 41 (Generic Sequence 7) or position 46 (Generic Sequence thereby encompassing the active sequences of OP-2 and OP-3.

Generic Sequence 7 Leu Xaa Xaa Xaa Phe Xaa Xaa 1 WO 97/41881 WO 971881PCTIUS97/07816 Xaa Gly Trp Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Mez: Xaa 90 Xaa Xaa Xaa Xaa Xaa Xaa Ala Xaa Tyr Cys Xaa Pro Xaa Xaa Xaa Asn His Ala Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Cys Xaa Pro Xaa Xaa Xaa Leu Xaa Xaa Val Xaa Leu Xaa Xaa Val Xaa Xaa Cys Xaa Xaa Pro Xaa Gly Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa

S*S

wherein each Xaa independently is selected from a group of one or more specified amnino acids defined as follows: "Res." means "residue" and Xaa at res.2 (Tyr or Lys); Xaa at res.3 Val or lie); Xaa at res.4 (Ser, Asp or Giu); Xaa at res.6 =(Arg, Gin, Ser, Lys or Ala); Xaa at res.7 (Asp or Giu); Xaa at res.8 (Leu, Val or Ilie); Xaa at res. I I (Gin, Leu, Asp, His, Asn or Ser); Xaa at res. 12 (Asp, Arg, Asn or Giu); Xaa at res. 13 (Trp or Ser); Xaa at res. 14 =(lie or Val); Xaa at res. 15 (lie or Val); Xaa at res. 16 (Ala or Ser); Xaa at res. 18 =(Giu, Gin, Leu, Lys, Pro or Mrg);- Xaa at res. 19 (Gly or Ser); Xaa at res.20 =(Tyr or Phe); Xaa at res.21 =(Ala, Ser, Asp, Met, His, Gin, Leu or Giy); Xaa at res.23 (Tyr, Asn or Phe); Xaa at res.26 (Glu, His, Tyr, Asp, Gin, Ala or Ser); Xaa at res.28 (Glu, Lys, Asp, Gin or Ala); Xaa at res.30 (Ala, Ser, Pro, Gin, Ilie or Asn); Xaa at res.3 I (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 Ilie); Xaa at res.37 (Met, Phe, Gly or Leu); Xaa at res.38 (Asn, Ser or Lys), Xaa at res.39 (Ala, Ser, Gly or Pro); Xaa at res.40 (Thr, Leu or Ser); Xaa at res.44 (lie, Val or Thr); Xaa at res.45 (Val, Leu, Met or lie); Xaa at res.46 (Gin or Arg); Xaa at res.47 (Thr, Ala or Ser); Xaa at res.48 (Leu or lie); Xaa at res.49 (Val or Met); Xaa at res.5O (Hfis, Asn or Mg); Xaa at res.5 I (Phe, Leu, Asn, Ser, Ala or Val); Xaa at res.52 (le, Met, Asn, Ala, Vai, Gly 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, Giy, Val, Pro or Lys); Xaa at res.56 WO 97/41881 PCT/US97/07816 -27- (Thr, Ala, Val, Lys, Asp, Tyr, Ser, Gly, Ile or His); Xaa at res.57 (Val, Ala or Ile); Xaa at res.58 (Pro or Asp); Xaa at res.59 (Lys, Leu or Glu); Xaa at res.60 (Pro, Val or Ala); Xaa at res.63 (Ala or Val); Xaa at res.65 (Thr, Ala or Glu); Xaa at res.66 (Gin, Lys, Arg or Glu); Xaa at res.67 (Leu, Met or Val); Xaa at res.68 (Asn, Ser, Asp or Gly); Xaa at res.69 (Ala, Pro or Ser); Xaa at res.70 (Ile, Thr, Val or Leu); Xaa at res.71 (Ser, Ala or Pro); Xaa at res.72 (Val, Leu, Met or Ile); Xaa at res.74 (Tyr or Phe); Xaa at res.75 (Phe, Tyr, Leu or His); Xaa at res.76 (Asp, Asn or Leu); Xaa at res.77 (Asp, Glu, Asn, Arg or Ser); Xaa at res.78 (Ser, Gin, Asn, Tyr or Asp); Xaa at res.79 (Ser, Asn, Asp, Glu or Lys); Xaa at res.80 (Asn, Thr or Lys); Xaa at res.82 (Ile, Val or Asn); Xaa at res.84 (Lys or Arg); Xaa at (Lys, Asn, Gin, His, Arg or Val); Xaa at res.86 (Tyr, Glu or His); Xaa at res.87 (Arg, Gin, Glu or Pro); Xaa at res.88 (Asn, Glu, Trp or Asp); Xaa at res.90 (Val, Thr, Ala or Ile); Xaa at res.92 (Arg, Lys, Val, Asp, Gin or Glu); Xaa at res.93 (Ala, Gly, Glu or Ser); Xaa at res.95 (Gly or Ala) and Xaa at res.97 (His or Arg).

Generic Sequence 8 (SEQ ID NO: 2) includes all of Generic Sequence 7 and in addition 15 includes the following sequence (SEQ ID NO: 14) at its N-terminus: Cys Xaa Xaa Xaa Xaa 1 Accordingly, beginning with residue 7, each "Xaa" in Generic Sequence 8 is a specified amino acid defined as for Generic Sequence 7, with the distinction that each residue number described for Generic Sequence 7 is shifted by five in Generic Sequence 8. Thus, "Xaa at res.2 or Lys)" in Generic Sequence 7 refers to Xaa at res. 7 in Generic Sequence 8. In Generic Sequence 20 8, Xaa at res.2 (Lys, Arg, Ala or Gin); Xaa at res.3 (Lys, Arg or Met); Xaa at res.4 (His, Arg or Gin); and Xaa at res.5 (Glu, Ser, His, Gly, Arg, Pro, Thr, or Tyr).

"As noted above, certain currently preferred morphogen polypeptide sequences useful in this invention have greater than 60% identity, preferably greater than 65% identity, with the amino acid sequence defining the preferred reference sequence of hOP-1. These particularly preferred sequences include allelic and phylogenetic counterpart variants of the OP-I and OP-2 proteins, including the Drosophila 60A protein. Accordingly, in certain particularly preferred embodiments, useful morphogens include active proteins comprising pairs of polypeptide chains within the generic amino acid sequence herein referred to as "OPX" (SEQ ID NO: which defines the seven cysteine domain and accommodates the homologies between several identified WO 97/41881 PCT/US97/07816 -28variants of OP-1 and OP-2. 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 OP-1 or OP-2 (see SEQ ID NOs: 4-7 and/or SEQ ID NOs: 15-22).

In still other preferred embodiments, useful morphogen polypeptides have amino acid sequences comprising a sequence encoded by a nucleic acid that hybridizes, under stringent hybridization conditions, to DNA or RNA encoding reference morphogen sequences, Cterminal sequences defining the conserved C-terminal seven domains ofOP-1 or OP-2, e.g., nucleotides 1036-1341 and nucleotides 1390-1695 of SEQ ID NO: 15 and 19, respectively. As used herein, stringent hybridization conditions are defined as hybridization according to known techniques in 40% formamide, 5 X SSPE, 5 X Denhardt's Solution, and 0.1% SDS at 37 C overnight, and washing in 0.1 X SSPE, 0.1% SDS at 50 0

C.

As noted above, morphogens useful in the present invention generally are dimeric proteins comprising a folded pair of the above polypeptides. Morphogens are inactive when reduced, but *i are active as oxidized homodimers and when oxidized in combination with other morphogens of 15 this invention to produce heterodimers. Thus, members of a folded pair of morphogen Spolypeptides in a morphogenically active protein can be selected independently from any of the specific morphogen polypeptides mentioned above. As noted above, a protein is morphogenic herein generally if it induces the developmental cascade of cellular and molecular events that culminate in the formation of new, organ-specific tissue. The morphogens generally are competent to induce 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.

The morphogens useful in the methods, compositions and devices of this invention include 25 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 phylogenetic counterpart variants of these proteins, as well as biosynthetic variants (muteins) thereof, and various truncated and fusion constructs. Deletion or addition mutants also are envisioned to be active, including those which may alter the conserved Cterminal six or seven cysteine domain, 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. The proteins WO 97/41881 PCT/US97/07816 -29may 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.

Figure 7 herein sets forth an alignment of the amino acid sequences of the active regions of naturally occurring proteins that have been identified or appreciated herein as OP/BMP renal therapeutic agents, including human OP-1 (hOP-1, SEQ ID NOs: 4 and 15-16), mouse OP-I (mOP-1, SEQ ID NOs: 5 and 17-18), human and mouse OP-2 (SEQ ID NOs: 6, 7, and 19-22), mouse OP-3 (SEQ ID NOs: 25-26), BMP-2 (SEQ ID NO: BMP-4 (SEQ ID NO: BMP-3 (SEQ ID NO: 27), DPP (from Drosophila, SEQ ID NO: 10), Vgl, (from Xenopus, SEQ ID NO: 11), Vgr-1 (from mouse, SEQ ID NO: 12), GDF-1 (from mouse and/or human, SEQ ID NOs: 13, 30 and 31), 60A protein (from Drosophila, SEQ ID NOs: 23 and 24), BMP-5 (SEQ ID NO: 28) and BMP-6 (SEQ ID NO: 29). The sequences are aligned essentially following the method ofNeedleman, et al. (1970) J. Mol. Biol., 48:443-453, calculated using the Align Program (DNAstar, Inc.). In Figure 7, three dots indicates that the amino acid in that position is 15 the same as the corresponding 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 ofBMP-2 (CBMP-2A) and BMP-4 (CBMP-2B) is "missing." Of course, both of these amino acid sequences in this region comprise Asn-Ser (residues 58, 59), with BMP- 2 then comprising Lys and Ile, whereas BMP-4 comprises Ser and Ile. Figure 7 also illustrates the handling of insertions in the morphogen amino acid sequence: between residues 56 and 57 of BMP-3 is an inserted Val residue; between residues 43 and 44 of GDF-1 is inserted the amino acid sequence, Gly-Gly-Pro-Pro. Such deviations from the reference morphogen sequence are ignored for purposes of calculating the defined relationship between, GDF-1 and hOP-1. As :.is apparent from the amino acid sequence comparisons set forth in Figure 7, significant amino acid 25 changes can be made from the reference sequence while retaining activity. For example, while the GDF-1 protein sequence depicted in Figure 7 shares only about 50% amino acid identity with the hOP-1 sequence described therein, the GDF-1 sequence shares greater than 70% amino acid sequence homology (or "similarity") with the hOP-1 sequence, where "homology" or "similarity" includes allowed conservative amino acid substitutions within the aligned sequence, as defined by Dayhoff, et al. (1979) 5 Atlas of Protein Sequence and Structure Suppl. 3, pp. 345- 362, Dayhoff, ed., Natl. BioMed. Res. Found., Washington WO 97/41881 PCT/US97/07816 Accordingly, in still another preferred aspect, the invention includes morphogens comprising species ofpolypeptide chains having the generic amino acid sequence referred to herein as "OPX", which defines the seven cysteine domain and accommodates the identities and homologies between the various identified OP-I and OP-2 proteins. OPX is presented in SEQ ID NO: 3. 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 OP-I or OP-2 (see Figure 7 and SEQ ID NOs: 4-7 and/or SEQ ID NOs: 15-22).

In another set of embodiments, an effective amount of an agent competent to stimulate or induce increased endogenous expression of an OP/BMP renal therapeutic agent or morphogen in a mammal may be administered. For example, an agent competent to stimulate or induce OP-1 production and/or secretion from renal tissue may be provided to a mammal, by systemic administration to the mammal or by direct administration of the morphogen-stimulating agent to renal tissue. Alternatively, the morphogen-stimulating agent or "morphogen inducer" may induce morphogen expression and/or secretion at a distant site at a tissue locus other than renal 15 tissue), with the expressed morphogen circulating to renal tissue. A method for identifying and testing agents competent to modulate the levels of endogenous morphogens in a given tissue is described in detail in published applications WO93/05172 and W093/05751, the teachings of which are incorporated herein by reference. Briefly, candidate compounds can be identified and tested by incubation in vitro with a test tissue or cells thereof, or a cultured cell line derived therefrom, for a time sufficient to allow the compound to affect the production, the expression and/or secretion, of a morphogen produced by the cells of that tissue. Here, suitable tissue, or cultured cells of a suitable tissue, preferably can be selected from renal epithelium, fibroblasts, and osteoblasts.

In another series of embodiments, an agent which acts as an agonist of an OP/BMP renal 25 therapeutic agent or morphogen receptor may be administered instead of the OP/BMP renal therapeutic agent or morphogen itself. Such an agent may also be referred to an a morphogen "mimic," "mimetic," or "analog." Thus, for example, a small peptide or other molecule which can mimic the activity of an OP/BMP renal therapeutic agent or morphogen in binding to and activating the OP/BMP renal therapeutic agent or morphogen's receptor may be employed as an equivalent of the OP/BMP renal therapeutic agent or morphogen. Preferably the agonist is a full agonist, but partial receptor agonists may also be advantageously employed. Methods of identifying such agonists are known in the art and include assays for compounds which induce WO 97/41881 PCT/US97/07816 -31 morphogen-mediated responses induction of differentiation of metanephric mesenchyme, induction of endochondral bone formation). For example, methods of identifying morphogen inducers or agonists of morphogen receptors may be found in U.S. Ser. No. 08/478,097 filed June 7, 1995 and U.S. Ser. No. 08/507,598 filed July 26, 1995, the disclosures of which are incorporated herein by reference.

Finally, in other embodiments cells may be implanted into the kidney of a subject in, or at risk of, chronic renal failure, or at risk of needing renal replacement therapy, in order to serve as a source of an OP/BMP renal therapeutic agent or morphogen and/or to provide a source of additional functional renal tissue. Such cells may be host or donor cells which normally express OP/BMP renal therapeutic agents or morphogens, which have been transformed so as to express OP/BMP renal therapeutic agents or morphogens, or which have been treated with OP/BMP renal therapeutic agents or morphogens.

D. Subjects for Treatment As a general matter, the methods of the present invention may be utilized for any 15 mammalian subject in, or at risk of, chronic renal failure, or at risk of the need for renal Sreplacement therapy chronic dialysis or renal transplant). Mammalian subjects which may be treated according to the methods of the invention include, but are not limited to, human subjects or patients. In addition, however, the invention may be employed in the treatment of domesticated mammals which are maintained as human companions dogs, cats, horses), which have significant commercial value dairy cows, beef cattle, sporting animals), which have significant scientific vaiue captive or free specimens of endangered species), or which otherwise have value. In addition, as a general matter, the subjects for treatment with the methods of the present invention need not present indications for treatment with an OP/BMP renal therapeutic agent or morphogen other than those indications associated with risk of chronic 25 renal failure. That is, the subjects for treatment are expected to be otherwise free of indications for treatment according to the present invention. In some number of cases, however, the subjects may present with other symptoms osteodystrophy) for which treatment with an OP/BMP renal therapeutic agent or morphogen would be indicated. In such cases, the treatment should be adjusted accordingly so to avoid excessive dosing.

One of ordinary skill in the medical or veterinary arts is trained to recognize subjects which may be at a substantial risk of chronic renal failure, or at substantial risk of the need for renal replacement therapy. In particular, clinical and non-clinical trials, as well as accumulated WO 97/41881 PCT/US97/07816 -32experience, relating to the presently disclosed and other methods of treatment, are expected to inform the skilled practitioner in deciding whether a given subject is in, or at risk of, chronic renal failure, or at risk of needing renal replacement therapy, and whether any particular treatment is best suited to the subject's needs, including treatment according to the present invention.

As a general matter, a mammalian subject may be regarded as being in, or at risk of, chronic renal failure, or at risk of needing renal replacement therapy, if that subject has already been diagnosed as afflicted with, or would be regarded as being afflicted with, a condition which typically leads to progressive loss of renal function associated with progressive loss of functioning nephron units. Such conditions include, but are not limited to, chronic renal failure, end-stage renal disease, chronic diabetic nephropathy, hypertensive nephrosclerosis, chronic glomerulonephritis, hereditary nephritis, renal dysplasia and the like. These, and other diseases and conditions known in the art, typically lead to a progressive loss of functioning nephrons and oooto the onset of chronic renal failure.

.o 0 Frequently, one of skill in the medical or veterinary arts may base a prognosis, diagnosis 15 or treatment decision upon an examination of a renal biopsy sample. Such biopsies provide a o *wealth of information useful in diagnosing disorders of the kidney but, due to the invasiveness of the procedure, and the additional trauma to a presumably unhealthy kidney, may not be appropriate for all subjects. Nonetheless, subjects in, or at risk of, chronic renal failure, or at risk of needing renal replacement therapy, may be recognized by histological indications from renal biopsies including, but not limited to, glomerular hypertrophy, tubular hypertrophy, glomerulosclerosis, tubulointerstitial sclerosis, and the like.

Less invasive techniques for assessing kidney morphology include MIRI, CAT and ultrasound scans. Scanning techniques are also available which employ contrasting or imaging agents radioactive dyes) but, it should be noted, some of these are particularly toxic to renal tissues and structures and, therefore, their use may be ill-advised in subjects in, or at risk of, chronic renal failure. Such non-invasive scanning techniques may be employed to detect conditions such as renal fibrosis or sclerosis, focal renal necrosis, renal cysts, and renal gross hypertrophy which will place a subject in, or at risk of, chronic renal failure, or at risk of needing renal replacement therapy.

Quite frequently, prognosis, diagnosis and/or treatment decisions are based upon clinical indications of renal function. One such indication is the presence in urinary sediment of an unusual number of "broad" or "renal failure" casts, which is indicative of tubular hypertrophy and WO 97/41881 PCT/US97/07816 -33suggests the compensatory renal hypertrophy which typifies chronic renal failure. A better indication of renal function is the glomerular flow rate (GFR), which can be measured directly by quantifying the rate of clearance of particular markers, or which may be inferred from indirect measurements.

It should be noted that the present invention is not directed to the measurement of GFR or to the diagnosis of chronic renal failure. The methods of treatment of the present invention need not, therefore, be restricted to subjects presenting with any particular measures of GFR, or any other particular marker of renal function. Indeed, it is not necessary that the GFR of a subject, or any other particular marker of renal function, be determined before practicing the treatments of the present invention. Nonetheless, the measurement of GFR is considered to be a preferred means of assessing renal function.

As is well known in the art, GFR reflects the rate of clearance of a reference or marker '"compound from the plasma to the urine. The marker compound to be considered is typically one which is freely filtered by the glomeruli, but which is not actively secreted or reabsorbed by the renal tubules, and which is not significantly bound by circulating proteins. The rate of clearance is oo typically defined by the formula, presented above, which relates the volume of urine produced in a twenty-four period, and the relative concentrations of the marker in the urine and plasma. To be more accurate, the GFR should also be corrected for body surface area. The "gold standard" reference compound is inulin because of its filtration properties and lack of serum-binding. The *•go concentration of this compound is, however, difficult to quantify in blood or urine. The clearance !rates of other compounds, including p-aminohippurate (PAN) and creatinine, are therefore often ]used instead of inulin. In addition, various formulas are often employed which seek to simplify the estimation of actual GFR by omitting considerations of actual urine concentrations of the marker, actual daily volumes of urine produced, or actual body surface area. These values may be 25 replaced by estimates based on other factors, by baseline values established for the same subject, or by standard values for similar subjects. These estimates should be used with caution, however, as they may entail inappropriate assumptions based upon the renal function of normal or healthy subjects.

Various methods and formulas have been developed in the art which describe an expected value of GFR for a healthy subject with certain characteristics. In particular, formulas are available which provide an expected value of the GFR based upon plasma creatinine levels, age, weight and sex. One such formula for an expected GFR is presented above. Other formulas may, WO 97/41881 PCT/US97/07816 -34of course, be employed and tables of standard values may be produced for subjects of a given age, weight, sex, and/or plasma creatinine concentration. Newer methods of measuring or estimating GFR using NMR or MRI technologies) are also now available in the art and may be used in accordance with the present invention (see, U.S. Pat. Nos. 5,100,646 and 5,335,660).

As a general matter, irrespective of the manner in which GFR is measured or estimated, a subject may be considered to be in, or at risk of, chronic renal failure, or at risk of needing renal replacement therapy, when the subject has a GFR which is chronically less than about 50% of the expected GFR for that subject. The risk is considered greater as the GFR falls lower. Thus, a subject is increasingly considered at risk if the subject has a GFR which is chronically less than about 40%, 30% or 20% of the expected GFR.

As a general matter, irrespective of the manner in which GFR is measured or estimated, a human male subject weighing at least about 50 kg may be considered to be in, or at risk of, "..chronic renal failure, or at risk of needing renal replacement therapy, when the subject has a GFR 0. which is chronically less than about 50 mi/min. The risk is considered greater as the GFR falls 15 lower. Thus, a subject is increasingly considered at risk if the subject has a GFR which is chronically less than about 40, 30 or 20 ml/min.

As a general matter, irrespective of the manner in which GFR is measured or estimated, a human female subject weighing at least about 40 kg may be considered to be in, or at risk of, chronic renal failure, or at risk of needing renal replacement therapy, when the subject has a GFR which is chronically less than about 40 ml/min. The risk is considered greater as the GFR falls lower. Thus, a subject is increasingly considered at risk if the subject has a GFR which is chronically less than about 30, 20 or 10 ml/min.

By a employing a variety of methods, including the histological examinations, non-invasive scanning procedures, evaluations of clinical indicators, and other techniques described above and 25 known in the art, those in the medical and veterinary arts may provide estimates of either the number of functioning nephron units which a subject possesses, or the percentage of functioning nephron units which a subject possesses relative to a healthy but otherwise similar subject a conspecific subject of approximately the same age, weight, and sex). Thus, for example, a biopsy may reveal a decrease in the density of functional nephrons, or imaging with filtered agents may indicate losses of functional renal tissue and/or filtering capacity. Such measures or estimates provide another means of expressing when a subject is in, or at risk of, chronic renal failure, or at risk of needing renal replacement therapy. Thus, as a general matter, a subject may be regarded WO 97/41881 PCT/US97/07816 to be in, or at risk of, chronic renal failure, or at risk of needing renal replacement therapy, if that subject possesses a number of functional nephron units which is less than about 50% of the number of functional nephron units of a healthy, but otherwise similar, subject. As above, the risk is considered greater as the number of functional nephrons decreases further. Thus, a subject is increasingly considered at risk if the subject has a number of functional nephrons which is less than about 40, 30 or 20% of the number for a similar but healthy subject.

Finally, it should be noted that subjects possessing a single kidney, irrespective of the manner of loss of the other kidney physical trauma, surgical removal, birth defect), may be considered to be prima facie at risk of chronic renal failure, or the need for renal replacement therapy. This is particularly true for those subjects in which one kidney has been lost due to a disease or condition which may afflict the remaining kidney. Similarly, subjects which are already recipients of a renal transplant, or which are already receiving chronic dialysis chronic hemodialysis or continuous ambulatory peritoneal dialysis) may be considered prima facie to be at risk of chronic renal failure, or the need for further renal replacement therapy.

15 E. Formulations and Methods of Treatment The OP/BMP renal therapeutic agents, morphogens, morphogen inducers, or agonists of .*ee morphogen receptors of the present invention may be administered by any route which is compatible with the particular morphogen, inducer, or agonist employed. Thus, as appropriate, administration may be oral or parenteral, including intravenous, intraperitoneal, and renal intracapsular routes of administration. In addition, administration may be by periodic injections of a bolus of the agent, or may be made more continuous by intravenous or intraperitoneal administration from a reservoir which is external an i.v. bag) or internal a bioerodable implant).

S* The therapeutic agents of the invention may be provided to an individual by any suitable 25 means, preferably directly locally, as by injection or topical administration to a tissue locus) or systemically parenterally or orally). Where the agent is to be provided parenterally, such as by intravenous, subcutaneous, intramuscular, intraorbital, ophthalmic, intraventricular, intracranial, intracapsular, intraspinal, intracisternal, intraperitoneal, buccal, rectal, vaginal, intranasal or by aerosol administration, the agent preferably comprises part of an aqueous solution. The solution is physiologically acceptable so that in addition to delivery of the desired agent to the patient, the solution does not otherwise adversely affect the patient's electrolyte and/or volume balance. The aqueous medium for the agent thus may comprise normal WO 97/41881 PCT/US97/07816 -36physiologic saline 9.85% NaCI, 0.15M, pH Such an aqueous solution containing the agent can be made, for example, by dissolving the agent in 50% ethanol containing acetonitrile in 0.1% trifluoroacetic acid (TFA) or 0.1% HCI, 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, an agent may be made more soluble by association with a suitable molecule.

For example, association of the mature OP/BMP or morphogen dimer with the pro domain results in the pro form of the protein which typically is more soluble or dispersible in physiological solutions than the corresponding mature form. In fact, endogenous OP/BMP proteins are thought to be transported secreted and circulated) in the mammalian body in this form. This soluble form of the protein can be obtained from culture medium of mammalian cells, cells transfected with nucleic acid encoding and competent to express the OP/BMP protein or morphogen. Alternatively, a soluble species can be formulated by complexing the mature dimer 15 (or an active fragment thereof) with a pro domain or a solubility-enhancing fragment thereof o* *(described more fully below). Another molecule capable of enhancing solubility and particularly useful for oral administrations, is casein. For example, addition of 0.2% casein increases solubility of the mature active form of OP-I by 80%. Other components found in milk and/or various serum proteins also may be useful.

Finally, as noted above, in another series of embodiments renal cells may be implanted into the kidney of a subject in, or at risk of, chronic renal failure, or at risk of needing renal replacement therapy, in order to serve as a source of an OP/BMP renal therapeutic agent or morphogen and/or to provide a source of additional functional renal tissue. These cells may be any compatible mammalian cells, including renal mesenchymal progenitor cells, or renal 25 mesenchymal progenitor cells which have been induced to undergo metanephric differentiation.

The cells may be derived from a donor a tissue-type matched donor, sibling, identical twin), may be derived from a tissue culture undifferentiated renal mesenchyme culture, fetal renal tissue culture), or may be explanted from the subject and then be re-implanted after proliferation and/or differentiation. Preferably, the cells are induced to undergo metanephric differentiation by treatment with an OP/BMP renal therapeutic agent or morphogen OP-1) either before or after implantation. Thus, for example, renal mesenchymal progenitor cells may be explanted from a subject, allowed or caused to proliferate in vitro, be induced to undergo metanephric WO 97/41881 PCT/US97/07816 -37differentiation by morphogen treatment, and be re-implanted where they may provide a source of morphogen and/or differentiate further into functional renal tissue.

Practice of the invention, including additional preferred aspects and embodiments thereof, will be still more fully understood from the following examples, which are presented herein for illustration only and should not be construed as limiting the invention in any way.

Examples Rat Remnant Kidney Model A rat partial nephrectomy or rat remnant kidney model (RRKM) model was employed essentially as described (Vukicevic, et al. (1987) J. Bone Mineral Res. 2:533). Male rats (2-3 months old, weighing about 150-200 g) were subjected to unilateral nephrectomy (either left or right kidney). After approximately one week, 2/3 of the remaining kidney was surgically removed. Immediately following surgery, plasma creatinine and BUN levels rise dramatically due to the loss of renal mass and function. Over the next several weeks of this "acute" failure phase, plasma creatinine and BUN levels of surviving animals decline somewhat toward normal values 15 but remain elevated. Renal function then appears to remain relatively constant or stable for a period of variable duration. After this point, the animals enter a period of chronic renal failure in which there is an essentially linear decline in renal function ending in death.

"As surgical controls, additional rats were subjected to a "sham" operation in which the kidneys were decapsulated but no renal tissue was removed.

Intervention Model for Chronic Renal Failure In this model, both nephrectomized and sham-operated rats were maintained for approximately 5-6 months after surgery. At this point, surviving nephrectomized animals were past the stable phase and had entered chronic renal failure.

Rats were divided into 8 groups with 12 rats in each group. Two groups of nephrectomized rats were used as controls (Nx controls), with one of those groups receiving no treatment at all, while the other received injections of only the vehicle buffer. In addition, two groups of sham-operated rats were used as controls (sham controls), with one group receiving only the vehicle buffer, while the other received soluble OP-1 (sOP-1) at 10 pg/kg body weight.

Four experimental groups of nephrectomized rats were employed, receiving sOP-1 at 1, 3, 10 or 50 gg/kg body weight by intraperitoneal injection (OP-I Nx animals). OP-1 treated and vehicleonly rats received three injections per week for 4-8 weeks. Total injection volume was 300 pl.

WO 97/41881 PCT/US97/07816 -38- No statistically significant differences were observed between the two Nx control groups or between the two sham control groups.

Compared to the sham group receiving only vehicle, the Nx control receiving only vehicle demonstrated significantly (p 0.01) elevated serum creatinine (Figure 1) at the end of the study, indicating a significant loss of renal function. Although nephrectomized rats treated with either 1 or 3 pg/kg body weight sOP-1 did not show significantly reduced serum creatinine when compared to the Nx control, nephrectomized rats treated with sOP-1 at doses of 10 or 50 pg/kg body weight showed significant (p 0.05) reductions in creatinine values (Figure Similar results were observed for serum urea levels: Although nephrectomized rats treated with either 1 or 3 pg/kg body weight sOP-1 did not show significantly reduced serum urea when compared to the Nx control, nephrectomized rats treated with sOP-I at doses of 10 or 50 pg/kg body weight showed significant (p 0.01) reductions in serum urea values (Figure All nephrectomized rats showed significantly (p 0.01) higher serum urea when compared to the sham-operated rats (Figure 2).

15 Histological observations indicate that, in contrast to the vehicle treated Nx control group, OP-1 treated nephrectomized rats exhibit relatively normal glomerular histology,. Figure 3, for example, shows typical renal samples from normal rat kidney, untreated Nx control animals, and OP-1 treated nephrectomized rats under low magnification (1 Ox). Figure 4 shows similar samples under higher magnification (40x). Histomorphometric analysis indicates that OP-1 Nx rats showed reduced incidence of glomerular sclerosis and loop collapse, relatively scattered sclerosis and microaneurysms, and more viable glomeruli compared to Nx control rats •(Table 2).

None of the rats died in any group during this study.

Prophylactic Model for Chronic Renal Failure S" 25 Rats were subjected to partial nephrectomies or sham-operated as described above. In this model, in order to test the ability of OP/BMP renal therapeutic agents to prevent, inhibit or delay the initiation of chronic renal failure, the rats were allowed to recover for approximately two weeks after surgery before initiation of OP-1 therapy. At this point, surviving animals were past the acute renal failure phase and had not yet entered chronic renal failure.

Rats were divided into two groups of 15-20 rats. One group received only vehicle buffer (Nx control) whereas the other received OP-l treatment at 10 gg/kg body weight given WO 97/41881 PCT/US97/07816 -39intraperitoneally three times per week. Administration of OP-1 or vehicle continued for a period of approximately 8-9 weeks.

During weeks 1-5 of treatment, both groups showed elevated serum creatinine 100 pmol/L) relative to sham-operated controls (35 7 4mol/L). At about 5 weeks, both groups began to show a rise in serum creatinine suggesting the onset of progressive or chronic renal failure. The rise in serum creatinine was, however, markedly less rapid in the OP-1 treated group and was significantly lower than in the Nx controls (Figure 5: p 0.02 at weeks 6 and 8; p 0.01 at weeks 7 and Similar results were observed in serum BUN values as well.

More important, measurements of GFR, based on serum and urine creatinine values, showed a highly significant decrease in both groups of nephrectomized rats 1.8 ml/min) relative to sham-operated controls (4.7 1.1 ml/min). The GFR in both groups continued to decline during weeks 1-3 of treatment. At approximately three weeks, however, GFR in the OP-1 treated group stabilized whereas the decline in renal function continued in the Nx controls. By week the difference in GFR values between OP-1 treated and Nx control rats had become statistically significant (p 0.02). This difference in GFR continued to increase over time (p 0.01 at week 6; p 0.001 at weeks 7 and as the Nx controls continued to decline but the OP-1 treated rats remained stable (Figure By the end of 9 weeks, 40% of the Nx control rats were dead whereas none of the OP-1 treated rats had died.

Histological evaluation of tissue sections confirmed that OP-1 treated rats showed greater preservation or maintenance of lomeruli, as well as proximal and distal tubule structures. There were also signs in the OP-1 treated rats of nephrogenic mesenchymal condensations and the appearance of developmental nephrogenic structures. Table 2 reports results of several standard quantitative PAS-staining of extracellular matrix) and semi-quantitative visual ranking) histomorphometric measures obtained for tissue slices from Nx control and OP-1 treated 25 Nx rats. These results indicate that OP-1 treatment of nephrectomized rates resulted in overall improvement (or reduced degeneration) of kidney tissue morphology, increased mesangial or perivascular thickening, decreased glomerular sclerosis and loop collapse, decreased presence of "scattered" sclerosis and microaneurysms, and an increase in viable glomeruli.

WO 97/41881 PCT/US97/07816 Group Control OP-1 Significance TABLE 2 Glomerular Mesangial Sclerosis Thickening Loop Collapse Normal Histology Scattered Absence of Sclerosis Viable Microaneurysms Glomeruli 2.58 +0.22 27.3 ±2.4 26.5±3.5 34.7±4.2 8.9±0.7 11.41±1.1 58.6±3.2 14.7±1.3 11.8±1.1 2.5±0.2 p <0.01 p <0.01 p <0.02 Equivalents p <0.0lP <0.0I

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The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof The foregoing embodiments are therefore to be considered in 5 all respects illustrative rather than limiting on the invention described herein. Scope of the invention is thus 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 intended to be embraced therein.

5 0

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Page(s) are claims pages they appear after the sequence listing WO 97/41881 PCT/US97/07816 -41- SEQUENCE LISTING GENERAL INFORMATION:

APPLICANT:

NAME: CREATIVE BIOMOLECULES,

INC.

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

TELEX:

(ii) TITLE OF INVENTION: MORPHOGEN TREATMENT FOR CHRONIC RENAL FAILURE (iii) NUMBER OF SEQUENCES: 31 (iv) CORRESPONDENCE

ADDRESS:

ADDRESSEE: CREATIVE BIOMOLECULES,

INC.

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

DATA:

APPLICATION NUMBER: FILING

DATE:

CLASSIFICATION:

(vii) PRIOR APPLICATION

DATA:

APPLICATION NUMBER: US 08/643,321 FILING DATE: 06-MA--1996 (viii) ATTORNEY/AGENT

INFORMATION:

NAME: TWOMEY, MICHAEL J REGISTRATION NUMBER: 38,349 REFERENCE/DOCKET NUMBER: CRP-118PC (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 (ii) MOLECULE TYPE: protein WO 97/41881 PCT/US97/07816 -42- (ix) FEATURE: NAME/KEY: Protein LOCATION: 1..97 OTHER INFORMATION: /label= Generic-Seq-7 /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:1: Leu Xaa Xaa Xaa Phe Xaa Xaa Xaa Gly Trp Xaa Xaa Xaa 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 40 S 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 85 90 Xaa INFORMATION FOR SEQ ID NO:2: 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-Seq-8 /note= "wherin 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:2: Cys Xaa Xaa Xaa Xaa Leu Xaa Xaa Xaa Phe Xaa Xaa Xaa Gly Trp Xaa 1 5 10 Xaa Xaa Xaa Xaa Xaa Pro Xaa Xaa Xaa Xaa Ala Xaa Tyr Cys Xaa Gly WO 97/41881 WO 9741881PCT/US97/07816 43 Xaa Cys Xaa Xaa Pro Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asn His Al 40 Xaa Xaa xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa 55 60 Xaa Cys Cys xaa Pro Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Leu Xaa Xa 70 75 Xaa Xaa Xaa Xaa Xaa Val Xaa Leu Xaa Xaa Xaa Xaa Xaa Iv~et Xaa Va 90 95 Xaa Xaa Cys xaa Cys Xaa 100 INFORMATION FOR SEQ ID NO:3: 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' (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: Cys Xaa xaa His Giu 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 G1 25 Glu Cys Xaa Phe Pro Leu Xaa Ser Xaa Met Asn Ala Thr Asn His Ala 40 Ile Xaa Gln Xaa Leu Val His Xaa Xaa Xaa Pro Xaa Xaa Val Pro Lys 55 Xaa Cys Cys Ala Pro Thr Xaa Leu Xaa Ala Xaa Ser Val Leu Tyr Xaa 70 75 Asp Xaa Ser Xaa Asn Val Xaa Leu Xaa Lys Xaa Arg Asn Met Val Val 90 a 1 WO 97/41881 PCT/US97/07816 -44- Xaa Ala Cys Gly Cys His 100 INFORMATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 139 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens TISSUE TYPE: HIPPOCAMPUS (ix) FEATURE: NAME/KEY: Protein LOCATION: 1..139 OTHER INFORMATION: /label= hOPI-MATURE (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: Ser Thr Gly Ser Lys Gln Arg Ser Gln Asn Arg Ser Lys Thr Pro Lys 1 5 10 Asn Gln Glu Ala Leu Arg Met Ala Asn Val Ala Glu Asn Ser Ser Ser 0* 20 25 Asp Gln Arg Gln Ala Cys Lys Lys His Glu Leu Tyr Val Ser Phe Arg 35 40 Asp Leu Gly Trp Gln Asp Trp Ile Ile Ala Pro Glu Gly Tyr Ala Ala 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 90 Glu Thr Val Pro Lys Pro Cys Cys Ala Pro Thr Gln 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 SEQUENCE CHARACTERISTICS: WO 97/41881 PCT/US97/07816 LENGTH: 139 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE: ORGANISM: MURIDAE TISSUE TYPE: EMBRYO (ix) FEATURE: NAME/KEY: Protein LOCATION: 1..139 OTHER INFORMATION: /label= MOP1-MATURE (xi) SEQUENCE DESCRIPTION: SEQ ID Ser Thr Gly Gly Lys Gin 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 20 25 0**0 Asp Gln Arg Gln Ala Cys Lys Lys His Glu Leu Tyr Val Ser Phe Arg 35 40 Asp Leu Gly Trp Gin Asp Trp Ile Ile Ala Pro Glu Gly Tyr Ala Ala 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 Gin Thr Leu Val His Phe Ile Asn Pro 85 9C 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:6: SEQUENCE

CHARACTERISTICS:

LENGTH: 139 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE: WO 97/41881 PCT/US97/07816 -46- ORGANISM: HOMO SAPIENS TISSUE TYPE: HIPPOCAMPUS (ix) FEATURE: NAME/KEY: Protein LOCATION: 1..139 OTHER INFORMATION: /label= HOP2-MATURE (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: Ala Val Arg Pro Leu Arg Arg Arg Gin Pro Lys Lys Ser Asn Glu Leu 1 5 10 Pro Gin Ala Asn Arg Leu Pro Gly Ile Phe Asp Asp Val His Gly Ser 25 His Gly Arg Gin Val Cys Arg Arg His Glu Leu Tyr Val Ser Phe Gin 40 S Asp Leu Gly Trp Leu Asp Trp Val Ile Ala Pro Gin Gly Tyr Ser Ala S50 55 Tyr Tyr Cys Glu Gly Glu Cys Ser Phe Pro Leu Asp Ser Cys Met Asn 70 75 Ala Thr Asn His Ala Ile Leu Gin Ser Leu Val His Leu Met Lys Pro 85 90 Asn Ala Val Pro Lys Ala Cys Cys Ala Pro Thr Lys Leu Ser Ala Thr 100 105 110 Ser Val Leu Tyr Tyr Asp Ser Ser Asn Asn Val Ile Leu Arg Lys His 115 120 125 Arg Asn Met Val Val Lys Ala Cys Gly Cys His 130 135 INFORMATION FOR SEQ ID NO:7: SEQUENCE CHARACTERISTICS: LENGTH: 139 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE: ORGANISM: MURIDAE TISSUE TYPE: EMBRYO (ix) FEATURE: NAME/KEY: Protein LOCATION: 1..139 OTHER INFORMATION: /label= MOP2-MATURE WO 97/41881 PCT/US97/07816 -47- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: Ala Ala Arg Pro Leu Lys Arg Arg Gin Pro Lys Lys Thr Asn Glu Leu 1 5 10 Pro His Pro Asn Lys Leu Pro Gly Ile Phe Asp Asp Gly His Gly Ser 25 Arg Gly Arg Glu Val Cys Arg Arg His Glu Leu Tyr Val Ser Phe Arg 40 Asp Leu Gly Trp Leu Asp Trp Val Ile Ala Pro Gin Gly Tyr Ser Ala 55 Tyr Tyr Cys Glu Gly Glu Cys Ala Phe Pro Leu Asp Ser Cys Met Asn 70 75 Ala Thr Asn His Ala Ile Leu Gin Ser Leu Val His Leu Met Lys Pro 90 Asp Val Val Pro Lys Ala Cys Cys Ala Pro Thr Lys Leu Ser Ala Thr 100 105 110 er Val Leu Tyr Tyr Asp Ser Ser Asn Asn Val Ile Leu Arg Lys His 115 120 125 Arg Asn Met Val Val Lys Ala Cys Gly Cys His 130 135 INFORMATION FOR SEQ ID NO:8: SEQUENCE CHARACTERISTICS: LENGTH: 101 amino acids TYPE: amino acid C STRA;:DEDNESS: cincle o TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE: ORGANISM: bovinae (ix) FEATURE: NAME/KEY: Protein LOCATION: 1..101 OTHER INFORMATION: /label= CBMP-2A-FX (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: 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 WO 97/41881 PCT/US97/07816 -48- Glu Cys Pro Phe Pro Leu Ala Asp His Leu Asn Ser Thr Asn His Ala 40 Ile Val Gin Thr Leu Val Asn Ser Val Asn Ser Lys Ile Pro Lys Ala 55 Cys Cys Val Pro Thr Glu Leu Ser Ala Ile Ser Met Leu Tyr Leu Asp 70 75 Glu Asn Glu Lys Val Val Leu Lys Asn Tyr Gin Asp Met Val Val Glu 90 Gly Cys Gly Cys Arg 100 INFORMATION FOR SEQ ID NO:9: SEQUENCE CHARACTERISTICS: LENGTH: 101 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein S(vi) ORIGINAL SOURCE: ORGANISM: HOMO SAPIENS TISSUE TYPE: hippocampus (ix) FEATURE: NAME/KEY: Protein LOCATION: 1..101 OTHER INFORMATION: /label= CBMP-2B-FX (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: 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 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 Met Leu Tyr Leu Asp 70 75 Glu Tyr Asp Lys Val Val Leu Lys Asn Tyr Gin Glu Met Val Val Glu 90 Gly Cys Gly Cys Arg 100 WO 97/41881 PCT/US97/07816 -49- INFORMATION FOR SEQ ID 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 Cys Arg Arg His Ser Leu Tyr Val Asp Phe Ser Asp Val Gly Trp Asp S1 5 10 Asp Trp Ile Val Ala Pro Leu Gly Tyr Asp Ala Tyr Tyr Cys His Gly 20 25 Lys Cys Pro Phe Pro Leu Ala Asp His Phe Asn Ser Thr Asn His Ala 35 40 Val Val Gln Thr Leu Val Asn Asn Asn Asn Pro Gly Lys Val Pro Lys 55 Ala Cys Cys Val Pro Thr Gln Leu Asp Ser Val Ala Met Leu Tyr Leu 65 70 75 Asn Asp Gln Ser Thr Val Val Leu Lys Asn Tyr Gin Glu Met Thr Val 90 Val 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: XENOPUS (ix) FEATURE: WO 97/41881 PCT/US97/07816 NAME/KEY: Protein LOCATION: 1..102 OTHER INFORMATION: /label= VGL-FX (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: Cys Lys Lys Arg His Leu Tyr Val Glu Phe Lys Asp Val Gly Trp Gin 1 5 10 Asn Trp Val Ile 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 Gin 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 Asp Asn Asn Asp Asn Val Val Leu Arg His Tyr Glu Asn Met Ala Val 85 90 Asp Glu Cys Gly Cys Arg 100 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: MURIDAE (ix) FEATURE: NAME/KEY: Protein LOCATION: 1..102 OTHER INFORMATION: /label= VGR-1-FX (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: Cys Lys Lys His Glu Leu Tyr Val Ser Phe Gin Asp Val Gly Trp Gin 1 5 10 Asp Trp Ile Ile Ala Pro Lys Gly Tyr Ala Ala Asn Tyr Cys Asp Gly 25 Glu Cys Ser Phe Pro Leu Asn Ala His Met Asn Ala Thr Asn His Ala 40 WO 97/41881 ~'/O97/488 1PCTIUS97/07816 51 Ile Val Gin Thr Leu Val His Val Met Asn Pro Giu Tyr Val Pro Lys 55 Pro Cys Cys Ala Pro Thr Lys Val Asn Ala Ilie 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 INFORMATION FOR SEQ ID NO:13: 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 INFORI4-ATION: /note= 'GDF-1 (fx)" (xi SEUEC DESCRIPTION: SEQ ID 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 Asm 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 70 75 Val Leu Phe Phe Asp Asn Ser Asp Asn Val Val Leu Arg Gin Tyr Glu 90 WO 97/41881 PCT/US97/07816 -52- Asp Met Val Val Asp Glu Cys Gly Cys Arg 100 105 INFORMATION FOR SEQ ID NO:14: SEQUENCE CHARACTERISTICS: LENGTH: 5 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: Cys Xaa Xaa Xaa Xaa 1 INFORMATION FOR SEQ ID SEQUENCE

CHARACTERISTICS:

LENGTH: 1822 base pairs TYPE: 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: :0.0 NAME/KEY: CDS LOCATION: 49..1341 IDENTIFICATION METHOD: experimental OTHER INFORMATION: /function= "OSTEOGENIC PROTEIN" /product= "OPl" /evidence= EXPERIMENTAL /standard_name= "OP1" (xi) SEQUENCE DESCRIPTION: SEQ ID GGTGCGGGCC CGGAGCCCGG AGCCCGGGTA GCGCGTAGAG CCGGCGCG ATG CAC GTG 57 Met His Val 1 CGC TCA CTG CGA GCT GCG GCG CCG CAC AGC TTC GTG GCG CTC TGG GCA 105 Arg Ser Leu Arg Ala Ala Ala Pro His Ser Phe Val Ala Leu Trp Ala WO 97/41881 ~VO 9741881PCTJUS97/07816 53 CCC CTG T'rC CTG CTG CGC TCC GCC CTG Pro Leu Phe Leu Leu Arg Ser Ala Leu GAC TTC AGC CTG GAC AAC Asp Phe Ser Leu Asp Asn

GAG

Giu

CGG

Arg

CCG

Pro

CTG

Leu

GGC

Gly 100

CCC

Pro

ATG

Met

CAC

His

CCA

Pro

TAC

Tyr 180

CAG

Gin

GAC

Asp

GTG

Val1

GAG

Giu

CGC

Arg

GAC

Asp 85

CAG

Gin

CCT

Pro

GTC

Val1

CCA

Pro

CAA

Glu 165

ATC

Ile

GTG

Val1

AGC

Ser

CAC

His

ATG

Met

CCG

Pro

CTG

Leu

GCC

Gly

CTC

Leu

ATG

Met

CGC

Arg 150

GGG

Gly

CGG

Arg

CTC

Leu

CGT

Arg

AGC

Ser

CGC

Arg

CTC

Leu

AAC

Asn

TCC

Ser

AC

Ser 120

TTC

Phe

CAC

His

GCT

Ala

CC

Arg

GAG

Ciu 200

CTC

Leu

TTC

Phe

GAG

G iu

CAG

Gin

GCC

Al a

TAC

Tyr 105

CTC

Leu

CTC

Val1

CAT

His

GTC

Vali

TTC

Phe 185

CAC

His

TG

Trp,

ATC

Ile

ATC

Ile

GC

Gly AT G Met 90

CCC

Pro

CAA

Gin

AAC

Asn

CGA

Arg

ACG

Thr 170

GAC

Asp

TTC

Leu

CC

Ala

CAC

His

CTC

Leu

AAC

Lys 75

GC

Aila

TAC

Tyr

GAT

Asp

CTC

Leu

GAG

Ciu 155

GCA

Aila

AAT

As n

GGC

Gly

TCG

Ser

CGG

Arg

TCC

Ser

CAC

His

CTG

Val1

AAG

Lys

AGC

Ser

GTG

Vali 140

TTC

Phe

GCC

Al a

GAG

Giu

AGG

Arg

GAG

Ciu 220

CC

Arg

GGC

G ly

GCA

Ala

GC

Gly

TTC

Phe

CTC

Leu

GAC

Asp

GAT

Asp

CGG

Arg 175

CGG

Arg

GAT

Asp

TGG

Trp

AGC

Ser

TTG

Leu

CCC

Pro

GGC

Gly

AGT

Ser

ACC

Th r

AAG

Lys

CTT

Leu 160

ATC

Ile

ATC

Ile

CTC

Leu

CTG

Leu

CAG

Gin

CCC

Pro

ATG

Me t

GGG

Giy

ACC

Thr

GAC

Asp,

GAA

Ciu 145

TCC

Ser

TAC

Tyr

AC

Ser

TTC

Phe

GTG

Val1 225 153 201 249 297 345 393 441 489 537 585 633 681 729 ATC ACA GCC Ile Thr Ala 230 ACC AGC AAC CAC Thr Ser Asn His GTG GTC AAT CCG Val Val Asn Pro CAC AAC CTC His Asn Leu WO 97/41881 WO 971881PCTIUS97/07816 54 GGC CTG CAG CTC TCG GTG GAG ACG CTG G CAG AGC Giy Leu Gin Leu Ser Val Giu Thr Leu Asp Giy Gin Ser ATC AAC CCC Ile Asn Pro a.

AAG

Lys 260

TTC

Phe

CGG

Arg

AAG

Lys

AGC

Ser

CGA

Arg 340

GCC

Aia

AAC

Asn

CCG

Pro

ATC

Ile

TAC

245 TTG GCG Leu Aia ATG GTG Met Vai TCC ACG Ser Thr A.AC CAG Asn Gin 310 GAC CAG Asp Gin 325 GAC CTG Asp Leu TAC TAC Tyr Tyr GCC ACC Aia Thr GAA ACG Glu Thr 390 TCC GTC Ser Vai 405 AGA AAC

GGC

Giy

GCT

Ala

GGG

Giy 295

GAA

G iu

AGG

Arg

GGC

Gly

TGT

Cys

AAC

As n 375

GTG

Val1

CTC

Leu

ATG

CTG

Leu

TTC

Phe 280

AGC

Ser

GCC

Aila

CAG

Gin

TGG

Trp

GAG

Giu 360

CAC

His

CCC

Pro

TAC

T'yr

GTG

250 ATT GGG Ile Gly 265 TTC AAG Phe Lys AAA CAG Lys Gin CTG CG Leu Arg GCC TGT Aia Cys 330 CAG GAC Gin Asp 345 GGG GAG Gly Glu GCC ATC Aia Ile AAG CCC Lys Pro TTC GAT Phe Asp 410 GTC CGG

CGG

Arg

GC

Aia

CGC

Arg

ATG

Met 315

AAG

Lys

TGG

Trp

TGT

Cys

GTG

Val1

TGC

Cys 395

GAC

Asp

GCC

CAC

His

ACG

Thr

AGC

Ser 300

GCC

Ala

AAG

Lys

ATC

Ile

GCC

Al a

CAG

Gin 380

TOT

Cys

AGC

S er GGG CCC CAG AAC AAG CAG CCC Gly Pro Gin Asn Lys Gin Pro 270 275 GAG GTC CAC TTC CGC AOC ATC Giu Val His Phe Arg Ser Ile 285 290 CAG AAC CGC TCC AAG ACG CCC Gin Asn Arg Ser Lys Thr Pro 305 AAC GTG OCA GAG AAC AGC AGC Asn Val Ala Giu Asn Ser Ser .320 CAC GAG CTG TAT GTC AGC TTC His Giu Leu Tyr Vai Ser Phe 335 ATC GCG CCT GAA GGC TAC GCC Ile Aia Pro Giu Gly Tyr Ala 350 355 TTC CCT CTG AAC TCC TAC ATG Phe Pro Leu Asn Ser Tyr Met 365 370 ACG CTG GTC CAC TTC ATC AAC Thr Leu Val His Phe Ile Asn 385 OCO CCC ACG CAG CTC AAT GCC Ala Pro Thr Gin Leu Asn Aia 400 TCC AAC OTC ATC CTG MAG AAA Ser Asn Val Ile Leu Lys Lys 415 0CC TGC CAC TAGCTCCTCC Giy Cys His 430 'GGATCCT CCATTGCTCG CCTTGGCCAG 'TCCCCTC CCTATCCCCA ACTTTAAAGG ;OCT'TTTG ATCAGTTTTT CAGTGGCAGC 3CAAAACC TAGCAGGAAA AAAAAACAAC 825 873 921 969 1017 1065 1113 1161 1209 1257 1305 1351 1411 1471 1531 1591 1651 Tyr Arg Asn Met Val Vai Arg Ala Cys

GAGAATTCAG

GAACCAGCAG

TGTGAGAGTA

ATCCAATGA

ACCCTTTGGG GCCAAGTTTT

TC']

ACCAACTOCC TTTTGTGAGA

CC']

TTAGGAAACA TGAGCAGCAT

ATC

CAAGATCCTA CAAGCTGTGC AGC GCATAAAGAA AAATGGCCGG GCCAGGTCAT TGGCTGGGAA GTCTCAGCCA TGCACGGACT WO 97/41881 WO 9741881PCTIUS97/07816 55 CGTTTCCAGA GGTAATTATG AGCGCCTACC AGCCAGGCCA CCCAGCCGTG

GGAGGAAGGG

GGCGTGGCAA GGGGTGGGCA CATTGGTGTC TGTGCGAAAG GAAAATTGAC CCGGAAGTTC CTGTAATAAA TGTCACAATA AAACGAATGA ATGAAAAAAA AAAAAAAAA A INFORMATION FOR SEQ ID NO:16: SEQUENCE CHARACTERISTICS: LENGTH: 431 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein 1711 1771 1822 (xiJ) SEQUENCE Met His Val Arg Ser 1 Leu Trp Ala Pro Leu Leu Asp Asn Glu Val 35 Gin Glu Arg Arg Glu Pro His Arg Pro Arg Met Phe Met Leu Asp Gly Pro Gly Gly Gin 100 Thr Gin Gly Pro Pro 115 Asp Ala Asp Met Val 130 Giu Phe Phe His Pro 145 Ser Lys Ile Pro Glu 165 Tyr Lys Asp Tyr Ile 180 Ser Val Tyr Gin Val 195 DESCRIPTION: SEQ ID N0:16: Leu Arg Ala Ala Ala Pro His Ser ?he Val Ala Ala Arg Ile Asn Glu Ala His 125 G iu Arg Glu Thr Giu 205 WO 97/41881 PCT/US97/07816 -56- Phe Leu Leu Asp Ser Arg Thr Leu Trp 210 215 u Val 225 His Ile Lys Arg Lys 305 Asn Val Gly Ser Phe 385 Leu Leu Asp Ile Leu Gly Pro Lys 260 Pro Phe 275 Ile Arg Pro Lys Ser Ser Phe Arg 340 Ala Ala 355 Met Asn Asn Pro Ala Ile Lys Tyr 420 Asn Val Ile 265 Phe Lys Leu Ala Gin 345 Gly Ala Lys Phe Val 425 Glu Gly Trp Val Asn Pro Asp Gly Gin 255 Gly Pro Gin 270 Glu Val His 285 Gin Asn Arg Asn Val Ala His Glu Leu 335 Ile Ala Pro 350 Phe Pro Leu 365 Thr Leu Val Ala Pro Thr Ser Asn Val 415 Gly Cys His 430 INFORMATION FOR SEQ ID NO:17: SEQUENCE CHARACTERISTICS: LENGTH: 1873 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO WO 97/41881 PCT/US97/07816 7 (vi) ORIGINAL SOURCE: ORGANISM: MURIDAE TISSUE TYPE: EMBRYO (ix) FEATURE: NAME/KEY: CDS LOCATION: 104. .1393 OTHER INFORMATION: /function= "OSTEOGENIC PROTEIN" /product= "mop'", /note= "1MOPi (CDNA)" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: CTGCAGC1AAG TGACCTCGGG TCGTGGACCG CTGCCCTGCC CCCTCCGCTG CCACCTGGGG CGGCGCGGGC CCGGTGCCCC GGATCGCGCG TAGAGCCGGC GC- ATG CAC GTG CGC 115 Met His Val Arg

TCG

Ser

CTG

Leu

GTG

Val1

GAG

Glu

CGC

Arg

GAC

Asp

GGC

Gly

TTA

Leu

ATG

Met TTC GTG Phe Val GAT TTC Asp Phe CTC CGC Leu Arg TTA GGG Leu Gly TCG GCG Ser Ala GAG AGC Glu Ser TTC AGT Phe Ser 110 CTC ACT Leu Thr GAC AAA Asp Lys 1

CTC

Leu

CTG

Leu

CAG

Gin

CCC

Pro

ATS

Met

CCG

Pro

CAG

Gin

GCC

Al a

'ITC

Phe 135 140 145 CGA TAC CAC CAT CGG GAG TTC CGG TTT GAT CTT TCC AAG ATC CCC GAG WO 97/41881 PCT/US97/07816 -58- Arg Tyr His His Arg Glu Phe Arg Phe Asp Leu Ser Lys Ile Pro Glu 150 155 160 GGC GAA CGG GTG ACC GCA GCC GAA TTC AGG ATC TAT AAG GAC TAC ATC 643 Gly Glu Arg Val Thr Ala Ala Glu Phe Arg Ile Tyr Lys Asp Tyr Ile 165 170 175 180 CGG GAG CGA TTT GAC AAC GAG ACC TTC CAG ATC ACA GTC TAT CAG GTG 691 Arg Giu Arg Phe Asp Asn Glu Thr Phe Gin Ile Thr Val Tyr Gin Val 185 190 195 CTC CAG GAG CAC TCA GGC AGG GAG TCG GAC CTC TTC TTG CTG GAC AGC 739 Leu Gin Glu His Ser Gly Arg Glu Ser Asp Leu Phe Leu Leu Asp Ser 200 205 210 CGC ACC ATC TGG GCT TCT GAG GAG GGC TGG TTG GTG TTT GAT ATC ACA 787 Arg Thr Ile Trp Ala Ser Glu Glu Gly Trp Leu Val Phe Asp Ile Thr 215 220 225 GCC ACC AGC AAC CAC TGG GTG GTC AAC CCT CGG CAC AAC CTG GGC TTA 835 Ala Thr Ser Asn His Trp Val Val Asn Pro Arg His Asn Leu Gly Leu S* 230 235 240 CAG CTC TCT GTG GAG ACC CTG GAT GGG CAG AGC ATC AAC CCC AAG TTG 883 Gin Leu Ser Val Glu Thr Leu Asp Gly Gin Ser Ile Asn Pro Lys Leu 245 250 255 260 GCA GGC CTG ATT GGA CGG CAT GGA CCC CAG AAC AAG CAA CCC TTC ATG 931 Ala Gly Leu Ile Gly Arg His Gly Pro Gin Asn Lys Gin Pro Phe Met 265 270 275 GTG GCC TTC TTC AAG GCC ACG GAA GTC CAT CTC CGT AGT ATC CGG TCC 979 Val Ala Phe Phe Lys Ala Thr Glu Val His Leu Arg Ser Ile Arg Ser 280 285 290 ACG GGG GGC AAG CAG CGC AGC CAG AAT CGC TCC AAG ACG CCA AAG AAC 1027 Thr Gly Gly Lys Gin Arg Ser Gin Asn Arg Ser Lys Thr Pro Lys Asn 295 300 305 CAA GAG GCC CTG AGG ATG GCC AGT GTG GCA GAA AAC AGC AGC AGT GAC 1075 SGin Glu Ala Leu Arg Met Ala Ser Val Ala Glu Asn Ser Ser Ser Asp S* 310 315 320 CAG AGG CAG GCC TGC AAG AAA CAT GAG CTG TAC GTC AGC TTC CGA GAC 1123 Gin Arg Gin Ala Cys Lys Lys His Glu Leu Tyr Val Ser Phe Arg Asp 325 330 335 340 CTT GGC TGG CAG GAC TGG ATC ATT GCA CCT GAA GGC TAT GCT GCC TAC 1171 Leu Gly Trp Gin Asp Trp Ile Ile Ala Pro Glu Gly Tyr Ala Ala Tyr 345 350 355 TAC TGT GAG GGA GAG TGC GCC TTC CCT CTG AAC TCC TAC ATG AAC GCC 1219 Tyr Cys Glu Gly Glu Cys Ala Phe Pro Leu Asn Ser Tyr Met Asn Ala 360 365 370 ACC AAC CAC GCC ATC GTC CAG ACA CTG GTT CAC TTC ATC AAC CCA GAC 1267 Thr Asn His Ala Ile Val Gin Thr Leu Val His Phe Ile Asn Pro Asp WO 97/41881 WO 971881PCTIUS97/07816 59 375 ACA GTA CCC AAG Thr Val Pro Lys 390 GTC CTC TAC TTC Val Leu Tyr Phe 405 380 CCC TGC TGT GCG CCC Pro Cys Cys Ala Pro 395 385 ACC CAG CTC AAC GCC ATC TCT Thr Gin Leu Asn Ala Ile Ser GAC G1 Asp As 41 AAC ATG GTC Asn Met Val

ACCTTTGCGG

CCCACCTTGG

AAGCATGTAA

GGCACGTGAC

GTCTGCCAGG

AATCGCAAGC

TCTGTGTT-A

GAATGAAAAA

GTC CGG G -Val Arg A.

425

GGCCACACCT

CGAGGAGAAC

GGGTTCCAGA

GGACAAGATC

AAAGTGTCCA

CTCGTTCAGC

AGC-GAAACCA

1 .C AGC TCT AAT GTC ATC CTG AAG AAG TAC AGA ;p Ser Ser Asn Val Ile Leu Lys Lys Tyr Arg .0 4315 420 C TGT GGC TGC CAC TAGCTCTTCC TGAGACCCTG .a Cys Gly Cys His 430 TTCCAAATCT TCGATGTCTC ACCATCTAAG TCTCTCACT AGACCAACCT CTCCTGAGCC TTCCCTCACC TCCCAACCG AACCTGAGCG TGCAGCAGCT GATGAGCGCC CTTTCCTTC CTACCAGCTA CCACAGCAAA CGCCTAAGAG CAGGAAAAP GTGTCCACAT GGCCCCTGGC GCTCTGAGTC TTTGAGGAG TGCAGCAGAA GGAAGGGCTT AGCCAGGGTG GGCGCTGGC AGCAGAAGCC ACTGTAATGA TATGTCACAA TAAAACCCA AAAAAAAAAA AAAAGAATTC

G

G

1315 1363 1413 1473 1533 1593 1653 1713 1773 183 3 1873 INFORMATION FOR SEQ ID NO:18: SEQUENCE CHARACTERISTICS: LENGTH: 430 amino acids 1' TY-P7E: ~ticaci, TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID Met His Vai Arg Ser Leu Arg Ala Ala Ala 1 5 10 Leu Trp Ala Pro Leu Phe Leu Leu Arq Ser NO: 18: Pro His Ala Leu Ser Phe Val Ala Al a Leu Asp Asn Giu Gin Giu Arg Arg Pro His Arg Pro Val His Ser Giu Met Gin Arg Pro His Ile His Arg Asp Phe Ser Leu Arg Ser Leu Gly Leu Glu Ile Leu Leu Gin Gly Asn Ser Ala WO 97/41881 PCTIUS97/07816 Met Phe Met Leu Pro Asp Gly Gin 100 Gin Gly Pro Pro 115 Asp Leu. Tyr Asn Gly Phe Ser Tyr Leu Ala Ser Leu 120 Ala Pro 105 Gin 60 Met A: 90 Tyr L Asp SE Giu Giu Val Phe 110 Gly Thr 125 Ala Asp Met Val Met Ser Phe Val Asn Leu Val Glu His Asp Lys Phe 145 Lys Lys Val1 Leu Phe 225 Asn Asn Gin Ser Thr 305 Ser Ser Tyr Ile Asp Tyr Leu 210 Asp Leu Pro Pro Ile 290 Pro Ser Phe Al a Pro Tyr Gin 195 Asp Ile Giy Lys Phe 275 Arg Lys Ser Arg2 Ala 355 Giu Ile 180 Val1 Ser Thr Leu Leu 260 Me t Ser Asn Esp O'sp 340 ['yr Gly 165 Arg Leu Arg Ala Gin 245 Ala Val1 Thr Gin Gin 325 Leu Tyr Glu Giu Gin Thr Thr 230 Leu Gly Ala G ly G iu 310 Arg Gly Cys Arg Arg Giu Ile 215 Ser Ser Leu Phe Gly 295 Ala Gin Trp Glu Thr Asp 185 Ser Al a 1-,i s Glu Giy 265 Lys Gin Arg Cys Asp 345 Glu Al a 170 As n Gly Ser Trp Thr 250 Arg Al a Arg Me t Lys 330 rrp Cys 155 Ala Giu Arg Glu Val1 235 Leu His Thr Ser Ala 315 Lys Ile Al a Phe His Pro Arg Tyr His His Arg Giu Phe 14( Arc Thr Glu Glu 220 Val1 Asp G ly Giu Gin 300 Ser His Ile Phe 0h Phe Phe Ser 205 G ly As n Gly Pro Val1 285 Asn Val Giu Aia Pro 365 Asp Arg Gin 190 Asp Trp Pro Gin Gin 270 His Ara Ala Leu Pro 350 Ueu2 Leu Ile 175 Ile Leu Leu Arg Ser 255 Asn Leu Ser Glu T'yr 335 Glu %sn Glu Ser 160 Tyr Thr Phe Val1 His 240 Ile Lys Arg Lys Asn 320 Val1 Gly Ser Tyr Met 370 Asn Ala Thr Asn Ala Ile Val Gln Ala le Va Gin Leu Val His Phe WO 97/41881 PCT/US97/07816 -61- 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 Ile 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 NO:19: SEQUENCE CHARACTERISTICS: LENGTH: 1723 base pairs TYPE: nucleic acid STRA.NDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens TISSUE TYPE: HIPPOCAMPUS (ix) FEATURE: NAME/KEY: CDS LOCATION: 490..1696 OTHER INFORMATION: /fu-iction= "OSTEOGENIC

PROTEIN"

/product= "hOP2-PP" /note= 1hOP2 (cDNA)" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: GGCGCCGGCA GAGCAGGAGT GGCTGGAGGA GCTGTGGTTG GAGCAGGAGG TGGCACGGCA GCGCTG2AC- GCTCCCT1TG AGTGT.GG ACCCCAGG AGGCGCTG-2A,: GC.L:-.CAGCTC 2C CCACACCGCA CCAAGCGGTG GCTGCAGGAG CTCGCCCATC GCCCCTGCGC TGCTCGGACC 180 GCGGCCACAG CCGGACTGGC GGGTACGGCG GCGACAGAGG CATTGGCCGA GAGTCCCAGT 240 CCGCAGAGTA GCCCCGGCCT CGAGGCGGTG GCGTCCCGGT CCTCTCCGTC CAGGAGCCAG 300 GACAGGTGTC GCGCGGCGGG GCTCCAGGGA CCGCGCCTGA GGCCGGCTGC CCGCCCGTCC 360 CGCCCCGCCC CGCCGCCCGC CGCCCGCCGA GCCCAGCCTC CTTGCCGTCG GGGCGTCCCC 420 AGGCCCTGGG TCGGCCGCGG AGCCGATGCG CGCCCGCTGA GCGCCCCAGC TGAGCGCCCC 480 CGGCCTGCC ATG ACC GCG CTC CCC GGC CCG CTC TGG CTC CTG GGC CTG 528 Met Thr Ala Leu Pro Gly Pro Leu Trp Leu Leu Gly Leu 1 5 GCG CTA TGC GCG CTG GOC GGG GGC GGC CCC GGC CTG CGA CCC CCG CCC 576 Ala Leu Cys Ala Leu Gly Gly Gly Gly Pro Gly Leu Arg Pro Pro Pro WO 97/41881 PCT/IUS97/07816 -62- GGC TGT CCC CAG CGA CGT CTG GGC CCG CGC GAG CGC CGG GAC GTG CAG Gly Cys Pro Gin Arg Arg Leu Gly Ala Arg Glu Arg Arg Asp Val Gin CGC GAG ATC Arg Giu Ile GCG CCA CCC Ala Pro Pro CTG GAC CTG Leu Asp Leu CCC GCG GAG Pro Ala Glu AAC ATG GTG Asn Met Val 110 AAG GAG TTC Lys Glu Phe ACA GCT GCG Thr Ala Ala AAC AGG ACC Asn Arg Thr 160 AAC AGG GAG Asn Arg Giu 175 GGA GAC GAG Gly Asp Glu 190 TGG TTG CTG Trp Leu Leu ACT GAG GAC Thr Giu Asp CAA CGG GCC Gin Arg Ala 240 CTG GCG GTG CTC GGG Leu Ala Val Leu Gly GCC GCC TCC CGG CTG Ala Ala Ser Arg Leu TAC CAC GCC ATG GCC Tyr His Ala Met Ala 85 CGG CGC CTG GGC CGC Arg Arg Leu Gly Arg 100 GAG CGA GAC CGT GCC Glu Arg Asp Arg Ala 115 CGC TTT GAC CTG ACC Arg Phe Asp Leu Thr 130 GAG TTC CGG ATT TAC Glu Phe Arg Ile Tyr 145 CTC CAC GTC AGC ATG Leu His Val Ser Met 165 TCT GAC TTG TTC TTT Ser Asp Leu Phe Phe 180 GGC TGG CTG GTG CTG Gly Trp Leu Val Leu 195 AAG CGT CAC AAG GAC Lys Arg His Lys Asp 210 GGG CAC AGC GTG GAT Gly His Ser Val Asp 225 CCA CGC TCC CAA CAG Pro Arg Ser Gin Gin 245

CTG

Leu

CCC

Pro

GGC

Gly

GCC

Ala

CTG

Leu

CAG

Gin

AAG

Lys 150

TTC

Phe

TTG

Leu

GAT

Asp

CTG

Leu

CCT

Pro 230

CCT

Pro CCT GGG Pro Gly GCG TCC Ala Ser GAC GAC Asp Asp GAC CTG Asp Leu GGC CAC Gly His 120 ATC CCG Ile Pro 135 GTG CCC Val Pro CAG GTG Gin Val GAT CTT Asp Leu GTC ACA Val Thr 200 GGA CTC Gly Leu 215 GGC CTG Gly Leu TTC GTG Phe Val

CGG

Arg

GCG

Ala

GAC

Asp

GTC

Val 105

CAG

Gin

GCT

Ala

AGC

Ser

GTC

Val

CAG

Gin 185

GCA

Ala

CGC

Arg

GCC

Ala

GTC

Val

CCC

Pro

CCG

Pro

GAG

Glu

ATG

Met

GAG

Glu

GGG

Gly

ATC

Ile

CAG

Gin 170

ACG

Thr

GCC

Ala

CTC

Leu

GGC

Gly

ACT

Thr 250 624 672 720 768 816 864 912 960 1008 1056 1104 1152 1200 1248 1296 GCC AGT CCG AGT CCC ATC CGC ACC CCT CGG GCA GTG AGG CCA CTG AGG WO 97/41881 WO 9741881PCT[US97/07816 63 Ala Ser Pro Ser Pro Ile Arg Thr Pro Arg Ala Val Arg Pro Leu Arg 260

AGG

Arg 270

CCA

Pro

COT

Arg

TG

Trp

TGC

Cys

CTG

Leu 350

TGC

Cys

AGC

Ser 0-CC Al a

AGO

Arg

G

Gly

CG

Arg

GTC

Val1

TCC

Ser 335

CAG

Gln

TOT

Cys

AGO

Ser Cys

CAG

Gin

ATC

Ile

CAC

His

ATC

Ile 320

TTC

Phe

TCC

Ser

OCA

Ala

AAC

As n 000 dz y 400

C

Pro

TTT

Phe

GAG

Glu 305

GOT

Ala

CCA

Pro

CTG

Leu

CCC

Pro

AAC

As n 385

TC

Cys Lys Lys 275 OAT GAC Asp Asp 290 CTC TAC Leu Tyr CCC CAA Pro Gin CTG GAC Leu Asp GTO CAC Val His 355 ACC AAO Thr Lys 370 GTC ATC Val ile C;IC T GA ,:is Ser Asn Giu AAG AAA AGC AAC GAG CAC GOC His Gly AGC TTC Ser Phe 310 TAC TCG Tyr Ser 325 TIC ATG Cys met ATG AAG Met Lys AGO 0CC Ser Ala CGC AAA

CTO

Leu

TCC

Ser 295

CAG

Gin 0CC Al a

AAT

Asn

CCA

Pro

ACC

Thr 375

CAC

OCG

Pro 280

CAC

His

GAC

Asp

TAT

Tyr 0CC Ala

A-AC

As n 360

TCT

Ser

COO

265

CAG

Gin 000 Gly

CTC

Leu

TAO

Tyr

ACC

Thr 345

GCA

Ala

GTG

Val1

AAC

As n 0CC AAO OGA CTC Ala Asn Arg Leu 285 COG CAG OTO TOO Arg Gin Val Cys 300 000C TOG OTG GAO Gly Trp Leu Asp 315 TOT GAO 000 GAG Cys Olu Gly Giu 330 AAC CAC 000 ATC Asn His Ala Ile GTC CCC AAG 000 Val Pro Lys Ala 365 OTC TAC TAT GAO Leu Tyr Tyr Asp 380 ATO OTO OTO AAG Met Val Val Lys 1344 1392 1440 1488 1536 1584 1632 1680 1723 Leu Arg Lys His Arg .OTC.7.SGC.- ;,CO00 CTO CAS INFORMATION FOR SEQ ID Wi SEQUENCE CHARACTERISTICS: LENGTH: 402 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID Met Thr Ala Leu Pro Oly Pro Leu Trp Leu Leu Gly Leu Ala Leu Cys 1 5 10 Ala Leu Gly Gly Gly Oly Pro Gly Leu Arg Pro Pro Pro Oly Cys Pro 25 WO 97/41881 WO 971881PCT/US97/07816 Gin Arg Arg Leu Giy Ala Arg Giu Arg 40 **so* Leu Ala Tyr Arg Giu Arg G lu 145 Leu Ser Gly Lys C ly 225 Pro Ser Pro Phe G lu 305 Leu Gly Arg Leu Met Ala Gly Arg 100 Arg Ala Leu Thr Ile Tyr Ser Met 165 Phe Phe 180 Val Leu Lys Asp Val Aso Gin Gln 245 Arg Thr 260 Ser Asn Val His Val Ser 64 Arg Asp Val Gin Arg Glu Ile Pro Arg Pro Arg Ala Pro Pro Pro Leu Phe Met Leu Asp Leu 75 Glu Asp Gly Ala Pro Ala Glu 90 Met Ser Phe Val Asn Met Val 110 Glu Pro His Trp Lys Glu Phe 125 Gly Giu Ala Val Thr Ala Ala 140 Ile His Leu Leu Asn Arg Thr 155 160 Gin Giu Gin Ser Asn Ary Giu 170 175 rhr Leu Arg Ala Gly Asp Glu 190 kla Ser Asp Cys Trp Leu Leu 205 Leu Tyr Val Glu Thr Giu Asp 220 Gly Leu Leu Gly Gin Ara Ala 235 240 Thr Phe Phe Ary Ala Ser Pro 250 255 Arg Pro Leu Arg Arg Arg Gin 270 Ala Asn Arg Leu Pro Gly Ile 285 Arg Gin Val Cys Arg Arg His 300 Gly Trp Leu Asp Trp Val le 315 320 Ala Pro Gin Gly Tyr Ser Ala Tyr Tyr Cys Giu Gly Glu Cys Ser Phe 325 330 335 WVO 97/41881 PCTIUS97/07816 65 Pro Leu Asp Leu Val His 355 Cys Met Asn Ala Thr Asn His Ala 345 Leu Met Lys Pro Asn 360 Thr Ser 375 Ala Val Pro Lys Ile Leu Gin Ser 350 Ala Cys Cys Ala 365 Asp Ser Ser Asn Pro Thr 370 Lys Leu Ser Ala Val Leu Tyr Val Ile Leu Arg His Arg Asn Met Val Val Lys Ala Cys 00 Cys His INFORMATION FOR SEQ ID NO;21: SEQUENCE CHARACTERISTICS: LENGTH: 1926 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (vi) ORIGINAL SOURCE: ORGANISM: MURIDAE TISSUE TYPE: EMBRYO (ix) FEATURE: NAME/KEY: CDS LOCATION: 93. .1289 OTHER INFORMATION: /.'unction= /product= 'rnOP2-PP"1 /note= "mOP2 cDNA" "OSTEOGENIC PROTEIN" SEQUENl"CE LESCRIFTION: SEQ 1D 1,0:21: OCCAGOCACA GGTGCGCCGT CTGGTCCTCC CCGTCTGGCG TCAGCCOAGC CCGACCAGCT ACCAGTGGAT GCOCGCCGGC TGAAAGTCCG AG 00 55 4 S

S

.5.5 ATG GCT ATG COT CCC GGG CCA Met Ala Met Arg Pro Oly Pro CTC TOG CTA TTO GGC CTT GCT CTO TGC GCG CTG GGA GGC GGC CAC GGT Leu Trp Leu Leu Oly Leu Ala Leu Cys Ala Leu Gly Gly Oly His Gly CCG COT Pro Arg CCC CCG CAC Pro Pro His ACC TGT Thr Cys 30 CCC CAG CGT CGC CTG GGA GCG CGC GAG Pro Gin Arg Arg Leu Oly Ala Arg Glu CGC GAC ATO CAG Arg Asp Met Gin GAA ATC CTG OCG Giu Ile Leu Ala CAA CCC GCC GCT CTC GGG CTA CCG Leu Gly Leu Pro CGG CCC COA CCC COT OCA Arg Pro Arg Pro Arg Ala Gln 0CC COG CAG CCA OCO TCC Ala Arg Gin Pro Ala Ser GPro Ala Ala WO 97/41881 PCT/US97/07816 66 GCG CCC CTC TTC ATG TTG GAC CTA TAC Ala Pro Leu Phe Met Leu Asp Leu Tyr A 65 CAC GCC ATG ACC His Ala Met Thr Asp Asp Asp

GAC

Asp

AGC

Ser

CCA

Pro 120

GAG

Glu

CAC

His

GAG

Glu

CTC

Leu

AGT

Ser 200

TAT

Tyr

CTG

Leu

TTC

Phe

CCA

Pro

AAC

Asn 280

GGC

Gly

TTC

Phe 105

CAC

His

GCT

Ala

CCG

Pro

CAC

His

CGA

Arg 185

GAC

Asp

GTG

Val1

CTT

Leu rTC Phe

CTG

Leu 265

~AA

Lys

GGG

Gly

GTC

Val1

TGG

Trp

GTC

Val1

CTC

Leu

TCC

Ser 170

TCT

Ser

CGA

Arg

GAA

G lu

GGA

G ly

AGG

Arg 250

AAG

Lys

CTC

Leu

CCA

Pro

AAC

As n

AAG

Lys

ACA

Thr

AAC

As n 155

AAC

As n

GGG

Gly

TGG

Trp

ACC

Thr

CGA

Arg 235

GCC

Ala

AGG.

Arg

CCA

Pro cCC Prc

ATG

Met

GAA

G lu

GCT

Ala 140

ACA

Thr

AGG

Arg

GAC

Asp

CTG

Leu

GCG

Al a 220

CAA

Gln

A.GC

Ser

AGG

TArg

GGG

Gly

CAG

Gln

GTG

Val1

TTC

Phe 125

GCT

Al a

ACC

Thr

GAG

Glu

GAG

Glu

CTG

Leu 205

GAT

Asp

GCA

Al a

CAG

Gln

CAG

Gln

ATC

I le 285 GCT CAC TTA Ala His Leu 95 GAA CGC GAC Glu Arg Asp 110 CAC TTT GAC His Phe Asp GAG TTC CGG Glu Phe Arg CTC CAC ATC Leu His Ile 160 TCT GAC TTG Ser Asp Leu 175 GGC TGG CTG Gly Trp Leu 190 AAC CAT CAC Asn His His GGG CAC AGC Gly His Ser CCA CGC TCC Pro Arg Ser 240 AGT CCT GTG Ser Pro Val 255 CCA AAG AAA Pro Lys Lys 270 TTT GAT GAT Phe Asp Asp

GGC

Gly

CGT

Arg

CTA

Leu

ATC

Ile 145

AGC

Ser

TTC

Phe

GTG

Val.

AAG

Lys

ATG

Me t 225

A~GA

Arg

CGG

Arg

ACG

Thr

GGC

Gly

CGT

*Arg

ACC

Thr

ACC

Thr 130

TAC

Tyr

ATG

Met

TTT

Phe

CTG

Leu

GAC

Asp 210

GAT

Asp

CAG

Gln

GCC

Al a

AAC

As n

CAC

His 290

GCC

Al a

CTG

Leu 115

CAG

Gl n

AAA

Lys

TTC

Phe

TTG

Leu

GAC

Asp 195

CTG

Leu

CCT

Pro

CCT

Pro

CCT

Pro

GAG

Glu 275

GGT

Gly

GAC

Asp 100

GGC

G ly

ATC

Ile

GAA

Glu

GAA

Glu

GAT

Asp 180

ATC

Ile

GGA

Gly

GGC

Gly

TTC

Phe I

CGG

Arg 260

CTT

Leu

TCC

Ser AGC ACC Ser Thr 150 GTC CAA Val. Gln CAG ACG Gln Thr GCA GCC Ala Ala CGC CTC Arg Leu 215 GCT GGT Ala Gly 230 GTA ACC Val Thr GCG AGA Ala Arg CAC CCC His Pro GGC AGA Gly Arg 295 Pro Ala Gly WO 97/41881 WO 9741881PCTIUS97/07816 67 GAG GTT TGC CGC Giu Val Cys Arg AGG CAT GAG CTC TAC GTC AGC TTC CGT GAC CTT GGC Arg His Glu Leu Tyr Val Ser Phe Arg Asp Leu Gly TGG CTG GAC Trp Leu Asp GAG GGG GAG Giu Gly Glu 330 CAT GCC ATC His Ala Ile ATC GCC CCC Ile Ala Pro GGC TAC TCT GCC Gly Tyr Ser Ala GCT TTC CCA Ala Phe Pro CTG GAC TCC TGT ATG Leu Asp Ser Cys Met 335 GTG CAC CTG ATG AAG Val His Leu met Lys 355

AAC

Asn 340

CCA

Pro TAT TAC TGT Tyr Tyr Cys 325 GCC ACC AAC Ala Thr Asn GAT GTT GTC Asp Val Val TTG CAG TCT Leu Gin Ser 345 CCC AAG Pro Lys GCA TGC TGT Ala Cys Cys TAT GAC AGC Tyr Asp Ser GCA CCC Ala Pro 365 AAC AAT Asn Asn GGC TGC Gly Cys ACC AAA CTG AGT GCC ACC TCT GTG CTG Thr Lys Leu Ser Ala Thr Ser Val Leu 370 375 GTC ATC CTG CGT AAA CAC CGT AAC ATG Val Ile Leu Arg Lys His Arg Asn Met 385 390 CAC TGAGGCCCG CCCAGCATCC TGCTTCTACT His GTG GTC AAG GCC Val Val Lys Ala 395 1025 1073 1121 1169 1217 1265 1319 1379 1439 1499 1619 1679 1739 1799 1859 1919 1926

ACCTTACCAT

CAGACAGGGG

CTTTCCCAGT

TC'V CCC

CTGGGGTCAG

AATGGCAAAT

CTCTGCACCA

GATCAATGCA

CCAGGTATAG

CTGTGAGTTC

GGAATTC

CTGGCCGGGC

CAATGGGAGG

TCCTCTGTCC

A~AGAC:-

CACTGAAGGC

TCTGGATGGT

TTCATTGTGG

TCGCTGTACT

CGGTGCATGT

AAGGCCACAT

CCCTCTCCAG

CCCTTCACTT

TTCATGGGGT

GC- -TG C A C I

CCACATGAGG

CTAAGAAGGC

CAGTTGGGAC

CCTTGAAATC

CATTAATCCC

AGAAAGAGCC

AGGCAGAAAC CCTTCTATGT TATCATAGCT CCCCTGGCCA CTTCCTGCTA AAATTCTGGT TTCGGGGCTA. TCACCCCGCC CTCTCCATCC AGCATCCCAG '.=7A7:;CTA ACTGAGAGGT AAGACTGATC CTTGGCCATC

CTCAGCCCAC

CCTGGAATTC TAA.ACTAGAT GATCTGGGCT ATTTTTAGGT ATAACAGACA

CATACACTTA

AGAGCTAGCT TGTTAGAAAA AGAATCAGAG AGCGCTAA.AG AGACAGAGAC AGGAGAATCT TGTCTCGGGA GCAGGAAAAA

AAAAAAAAAC

0..

INFORMATION FOR SEQ ID NO:22: SEQUENCE CHARACTERISTICS: LENGTH: 399 amino acids TYPE: amino acid WO 97/41881 PCTIUS97/07816 -68- TOPOLOGY: linear (ii) MOLECULE TYPE: protein Met 1 Ala Arg Ala Ala His Gly Arg Leu Ile 145 Ser Phe Val Lys Met 225 Arg Arg (xi) SEQUENCE Ala Met Arg Pro 5 Leu Gly Gly Gly Arg Leu Gly Ala Val Leu Gly Leu Ala Arg Gin Pro Ala Met Thr Asp Arg Ala Asp Leu 100 Thr Leu Gly Tyr 115 Thr Gin Ile Pro 130 Tyr Lys Glu Pro Met Phe Glu Val 165 Phe Leu Asp Leu 180 Leu Asp Ile Thr 195 Asp Leu Gly Leu 210 Asp Pro Gly Leu Gin Pro Phe Met 245 Ala Pro Arg Ala 260 His Arg Pro Ala 70 Asp Val Gin Ala Ser 150 Val Gin Ala Arg Ala 230 Val Ala Gly Glu Gly 55 Ser Asp Met Glu Gly 135 Thr Gin Thr Ala Leu 215 Gly Thr Arg Pro Arg Arg Ala Asp Ser Pro 120 Glu His Glu Leu Ser 200 Tyr Leu Phe Pro Arg 25 Arg Pro Pro Gly Phe 105 His Ala Pro His Arg 185 Asp Val Leu Phe Leu 265 Pro His Met Gin Pro Arg Phe Met 75 Pro Pro Asn Met Lys Glu Thr Ala 140 Asn Thr 155 Asn Arg Gly Asp Trp Leu Thr Ala 220 Arg Gin 235 Ala Ser Arg Arg Cys Glu Gin Asp Ala Glu 110 His Glu Leu Ser Gly 190 Asn Gly Pro Ser Pro 270 Pro Ile Pro Leu His Arg Phe Phe His Asp 175 Trp His His Arg Pro 255 Lys Gin Leu Ala Tyr Leu Asp Asp Arg Ile 160 Leu Leu His Ser Ser 240 Val Lys DESCRIPTION: SEQ ID NO:22: Gly Pro Leu Trp Leu Leu Gly Leu Ala Leu Cys 10 WO 97/41881 PCT/US97/07816 -69- Thr Asn Glu Leu Pro His Pro Asn Lys Leu Pro Gly Ile Phe Asp Asp 275 280 285 Gly His Gly Ser Arg Gly Arg Glu Val Cys Arg Arg His Glu Leu Tyr 290 295 300 Val Ser Phe Arg Asp Leu Gly Trp Leu Asp Trp Val Ile Ala Pro Gin 305 310 315 320 Gly Tyr Ser Ala Tyr Tyr Cys Glu Gly Glu Cys Ala Phe Pro Leu Asp 325 330 335 Ser Cys Met Asn Ala Thr Asn His Ala Ile Leu Gin Ser Leu Val His 340 345 350 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:23: S(i) SEQUENCE CHARACTERISTICS: LENGTH: 1368 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY:

CDS

o LOCATION: 1..1368 OTHER INFORMATION: /label= (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: ATG TCG GGA CTG CGA AAC ACC TCG GAG GCC GTT GCA GTG CTC GCC TCC 48 Met Ser 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 Gin 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 WO 97/41881 PCTIUS97/07816 70 TCG TAC GAG ATC CTC GAG TTC CTG GGC ATC GCC GAA CGG CCG ACG CAC Ser Tyr Giu Ile Leu Glu Phe Leu Gly Ile Ala Glu Arg Pro Thr His CTG AGC AGC CAC C Leu Ser Ser His G

CTG

Leu

GAT

Asp

GAC

Asp

CTG

Leu 145

AAC

Asn

CGC

Arg

ATG

Met

ACC

Thr

ACG

Thr 225

GGG

Gly.

GAG

Glu

CAC

His

GAC

Asp

GAG

Giu

CTC

Leu 130

GAC

Asp

AAG

Lys

CTG

Leu

GCC

Al a

GCC

Al a 210

CTG

Leu

GAC

Asp

TGG

rrp

GCT

Al a

GTC

Val1

GAC

Asp 115

GAG

Giu

AAG

Lys

CGC

Arg

TGG

Trp

GAG

Giu 195

AAC

Asn

GGC

Gly

TAC

Tyr

CTG

Leu

GTC

Val1 275

TAC

Tyr 100

GAC

Asp

GAG

Glu

CGG

Arg

CAC

His

TTC

Phe 180

CTG

Leu

AGG

Arg

CAG

Gin

GTG

Val1

GTC

Val1 260

AAC

Asn

C

G

A

G

A

G

A

C.

H

A:

A:

HG

G(

AG TTG TCG CTG 1n Leu Ser Leu AC CGC ATC ACG is Arg Ile Thr AC TAC GAA CGC sp Tyr Glu Arg 120 AT GAG GGC GAG sp Giu Gly Giu 135 CC ATC GAC GAG la Ile Asp Giu 150 AC AAT GTG GAC is Asn Val Asp AC GTC TCC AAC sp Vai Ser Asn GC ATC TAT CAG rg Ile Tyr Gin 200 AG TTC ACC ATC lu Phe Thr Ile 215 bC ACC ATG GAG is Thr Met Giu 230 GC TGG TTG GAG ly Trp Leu Glu ~G TCG AAG GAC s Ser Lys Asp GA CCC GAC CC rg Pro Asp Arg 280

AGG

Arg

GCG

Al a 105

GGC

Gly

CAG

Gin

AGC

Ser

GAA

Giu

GTG

Val1 185

AAC

Asn

ACG

Thr

CCG

Pro

CTC

Leu

AAT

Asn 265

GAG

Glu

AAG

Lys 90

GAG

Giu

CAT

His

CAG

Gin

GAC

Asp

CTG

Leu 170

CCC

Pro

GCC

Al a

GTA

Val1

CTG

Leu

AAC

Asn 250

CAT

His

GTG

Val1

TCG

Ser

GAG

Giu

CGG

Arg

AAG

Lys

ATC

Ile 155

CGT

Arg

AAC

As n

AAC

As n

TAC

Tyr

TCC

Ser 235

GTG

Val1

GGC

Gly

AAG

Lys

GCT

Al a

GGT

Gly

TCC

Ser

AAC

Asn 140

ATC

Ile

CAC

His

GAC

Asp

GAG

Giu

GCC

Ala 220

TCG

Ser

ACC

Thr

ATC

Ile

CTG

Leu Pro

CTC

Leu

AGG

Arg 125

TTC

Phe

ATG

Met

GAG

Glu

AAC

Asn

GGC

C ly 205

ATT

Ile

GTG

Val1

GAG

G iu

TAC

GAC

Asp 285 Lys

AGC

S er 110

AGG

Arg

ATC

Ile

ACC

Thr

CAC

His

TAC

Tyr 190

AAG

Lys

GGC

Gly

AAC

As n

GGC

G ly

ATT

Ile 270

GAC

Asp Phe

GAT

Asp

AGC

Ser

ACC

Thr

TTC

Phe

GC

Gly 175

CTG

Leu

TGG

Trp

ACC

Thr

ACC

Thr

CTG

Leu 255

GGA

Gly

ATT

Ile Leu

CAG

Gin

GCC

Al a

GAC

Asp

CTG

Leu 160

CGT

Arg

GTG

Val1

CTG

Leu

GGC

G ly

ACC

Thr 240

CAC

His

GCA

Ala

GGA

Gly CCC AAG TTC CTG 288 336 384 432 480 528 576 624 672 720 768 816 864 WO 97/41881 PCT/US97/07816 CTG ATC CAC CGC AAG GTG GAC GAC GAG TTC CAG Phe Gin Leu Ile 290 His Arg Lys Val Asp Asp Glu 295 CCC TTC ATG ATC GGC Pro Phe Met Ile Gly TTC CGC GGA CCG Phe Arg Gly Pro CTG ATC AAG GCG Leu Ile Lys Ala GCC CAC AGC AGC Ala His Ser Ser CAC AGG AGC AAG His Arg Ser Lys AGC GCC AGC CAT Ser Ala Ser His CGC AAG CGC Arg Lys Arg GTG TCG CCC Val Ser Pro AGC TGC CAG Ser Cys Gin 355

AAC

Asn 340 AAC GTG CCG CTG Asn Val Pro Leu GAA CCG ATG GAG Glu Pro Met Glu AAG AAG TCG Lys Lys Ser 335 AGC ACG CGC Ser Thr Arg 350 CTG GGC TGG Leu Gly Trp ATG CAG ACC CTG Met Gin Thr Leu ATA GAC TTC AAG Ile Asp Phe Lys

GAT

Asp 365 CAT GAC His Asp 370 TGG ATC ATC GCA Trp Ile Ile Ala GAG GGC TAT GGC GCC TTC TAC TGC AGC Glu Gly Tyr Gly Ala Phe Tyr Cys Ser GAG TGC AAT TTC Glu Cys Asn Phe CTC AAT GCG CAC Leu Asn Ala His AAC GCC ACG AAC Asn Ala Thr Asn 1008 1056 1104 1152 1200 1248 1296 1344 1368 GCG ATC GTC CAG Ala Ile Val Gin CTG GTC CAC CTG CTG GAG CCC AAG Leu Val His Leu Leu Glu Pro Lys 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 Ala Leu Pro AAG GTG CCC Lys Val Pro 415 GTT CTG TAC Val Leu Tyr 430 AAC ATG ATT Asn Met Ile GAC GAG AAT GTG Asp Glu Asn Val CTG AAA AAG TAT Leu Lys Lys Tyr r GTG AAA Val Lys 450 TCC TGC GGG TGC CAT TGA Ser Cys Gly Cys His INFORMATION FOR SEQ ID NO:24: SEQUENCE CHARACTERISTICS: LENGTH: 455 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: WO 97/41881 WO 971881PCT/US97/07816 72 Ala Val Met Ser Gly Leu Arg Asn Thr Ser Glu 1 5 Ala Val Leu Ala Ser Leu Ala Gin Ser Leu Leu Asp Asp Leu 145 Asn Arg Met Thr Thr 225 Gly Giu His Leu Giy Val1 Thr Tyr Ser Asp Glu Leu 130 Asp Lys Leu Ala Ala 210 Leu Asp Trp Ala Ile 290 Leu G iu Ile Glu Ser Val1 Asp 115 C ha Lys Ary Trp G'lu 195 Asn G ly Tyr Leu Val1 275 His Gly Al a Met Ile His Tyr 100 Asp G lu Arg His Phe 180 Leu Arg Gin Val1 Val1 260 Asn Arg Met Thr His Leu Gin His Asp Asp Ala His 165 Asp Arg G lu His Gly 245 Lys Arg Lys Val1 Gin Arg Giu 70 Leu Arg Tyr Giu Ile 150 Asn Val1 Ile Phe Thr 230 Trp Ser Pro Val1 Leu Ser Val1 55 Phe Ser Ile Giu G ly 135 Asp Val1 Ser Tyr Thr 215 Met Leu Lys Asp Asp 295 Leu Gly 40 Leu Leu Leu Thr Arg 120 Giu Giu Asp As n Gin 200 Ile Giu Giu Asp Arg 280 Asp Met 25 Ile Ser Giy Arg Al a 105 Giy Gin Ser Giu Val1 185 As n Thr Pro Leu As n 265 Giu Glu Val1 Ile Asp Ala 75 Ser Glu Arg Lys Ile 155 Arg As n As n Tyr Ser 235 Val1 Gly Lys Gin Al a Asp Asp Giu Al a Gly Se r Asn 140 Ile His Asp Giu Al a 220 Ser Thr Ile Leu Pro 300 Thr Asn Lys Arg Pro Leu Arg 125 Phe Met Giu Asn Gly 205 Ile Val1 Giu Tyr Asp 285 Phe Thr Gly Leu Pro Lys Ser 110 Arg lie Thr His Tyr 190 Lys Gly Asn G iy Ile 270 Asp Met Pro Lys Asp Thr Phe Asp Ser Thr Phe G ly 175 Leu Trp Thr Thr Leu 255 Gly Ile Ile Pro Asp Val1 His Leu Gin Ala Asp Leu 160 Arg Val1 Leu Giy Thr 240 His Ala Gly Gly WO 97/41881 PCTIUS97/07816 -73- Phe Phe Arg Gly Pro Glu Leu Ile Lys Ala Thr Ala His Ser Ser His 305 310 315 320 His Arg Ser Lys Arg Ser Ala Ser His Pro Arg Lys Arg Lys Lys Ser 325 330 335 Val Ser Pro Asn Asn Val Pro Leu Leu Glu Pro Met Glu 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 Glu Cys Asn Phe Pro Leu Asn Ala His Met Asn Ala Thr Asn His 385 390 395 400 Ala Ile Val Gin Thr Leu Val His Leu Leu Glu 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 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 1674 base pairs =TY!E: nucleic acid STRANDEDNESS: single S* TOPOLOGY: linear (ii) MOLECULE TYPE: protein (ix)

FEATURE:

NAME/KEY: CDS LOCATION: 69..1268 OTHER INFORMATION: /note= "mOP3-PP" (xi) SEQUENCE DESCRIPTION: SEQ ID GGATCCGCGG CGCTGTCCCA TCCTTGTCGT CGAGGCGTCG CTGGATGCGA GTCCGCTAAA CGTCCGAG ATG GCT GCG CGT CCG GGA CTC CTA TGG CTA CTG GGC CTG GCT 110 Met Ala Ala Arg Pro Gly Leu Leu Trp Leu Leu Gly Leu Ala 1 5 CTG TGC GTG TTG GGC GGC GGT CAC CTC TCG CAT CCC CCG CAC GTC TTT 158 WO 97/41881 WO9481PCTfUS97/07816 ATT CGG GAG GTG C

C

C. *C Ile

GTC

Val1

CTG

Leu

CAC

His

CC

Arg

TTT

Phe

TTC

Phe

CAC

His

GAC

Asp 175

TGG

Trp

CAT

His

CAC

His

CGC

Arg S lu

GCI

Ala

CGT

Arg

GAC

Asp

CGT

Arg

CTA

Leu

ATC

Ile 145

AGC

Ser

TTC

Phe

GTG

Val1

AAG

Lys

ATA

Ile 225

AGA

Arg Val

GCC

Ala

GCC

Ala

CGT

Arg

ACC

Thr

ACC

Thr 130

TAC

T1yr ATC3 Met

TTT

Phe

CTG

Leu

GAC

Asp 210

GAT

Asp

CAG

Gin

L

G

A

M

G

A

C,

Li P1

C:

CC

Cc ily Sly Sly 'TA GGA GTA ,eu Gly Val TG GGG CTA eu Sly Leu AG CAG CCA in Gin Pro TG ACG GAT et Thr Asp CT GAC CTG la Asp Leu 100 TGS GGC TAC eu Sly Tyr AG ATC CCT in Ile Pro .A GAA CCC {s Giu Pro PC GAA GTG ae Glu Val 165 PG GAT CTT u Asp Leu 180 kC ATC ACA 5p Ile Thr CA GGA CTC u Gly Leu 'T 5CC CTA -o Gly Leu 'T TTC ATG :o Phe Met His

CGC

Arg

GCC

Al a

GCG

Ala 70

SAC

Asp

ATT

Ile

CAG

Gin

CCT

Al a

ACT

Ser I50

GTC

Val1

CAS

Gin

GCA

Al a

CGC

Arg

GCT

Al a 230

GTT

Val1 Leu

GAS

Giu

SG

G ly 55

TCT

Ser

AST

Ser

ATG

Met

GAG

Glu

GSG

Gly 135

ACC

Thr

CAA

Gin

ACS

Thr

GCC

Ala

CTC

Leu 215

GST

Sly

GGT

Sly 74 Ser H CCC C Pro A 40 CSS CC Arg P2 GCG CC Ala Pr SGC CC Sly Gi AGC TT Ser Ph 10 CCA CA Pro Hi 120 GAG SC Slu Al CAC CC His Pr SAG CA Giu Hi CTC CS.

Leu Ar 18 AST GA Ser As 200 TAT ST Tyr Va CTC CT Leu Le TTC TT Phe Ph 25 To y

T

5

T

a

G

0

C

s

A

5

C

p

G

I

C

e Prc

SAC

Asp

CGA

Arg

CTC

Leu

GGG

Sly

STC

Val1

TGS

Trp,

STC

Val1

CTC

Leu

TCC

Ser 170

TCT

Ser

CGA

Arg

SAA

S lu

GA

Sly

ASS

Arg Pro His -ATS3 CAG Met Sln TCC CSA Ser Arg TTT ATG Phe Met ACC CCC Thr Pro AAC ATA Asn Ile AAS GAA Lys Giu ACA GCT Thr Ala 140 AAC ACA.

Asn Thr 155 AAC ASS Asn Arg GGG SAC Sly Asp TSG CTG Trp Leu ACC SAG Thr Siu 220 CSA CAA Arg Gin 235 GCC AAC Ala Asn Val1

CSC

Arg

SCA

Ala

TTG

Leu

CAG

Sln

STS

Val1

TTC

Phe 125

SCT

Aila

ACC

Thr

GAG

Clu

GAS

Clu

CTS

Leu 205

GAT

Asp

GCA

Al a

CAS

Gin Phe

SAC

Siu

CCS

Pro

SAC

Asp

CCT

Pro

CAA

Slu 110

CAC

His

GAS

S lu

CTC

Leu

TCT

Ser

GGC

Sly 190

AAC

Asn

GGG

Siy

CCA

Pro

AST

Ser 206 254 302 350 398 446 494 542 590 638 686 734 782 830 WO 97/41881 WO 971881PCT/US97/07816 75 240 GTG CGG GCC 245 CCT CGA ACA GCA AGA CCA CTG AAG AAG CAG r'TA Val Arg Ala Pro Arg Thr Ala Arg Pro Leu Lys Lys Lys Gin Leu 260 265 270 AAT CAA ATC AAC CAG CTG CCG CAC TCC AAC AAA CAC CTA GGA ATC CTT

S

S

*5S*

S

*5bS iAsn Gin Ile ASf l Jn Leu Pro His Ser Asn Lys His Leu 275 280 GAT GAT GGC CAC GGT TCT CAC GGC AGA GAA GTT TGC CGC Asp Asp Gly His Gly Ser His Gly Arg Glu Val Cys Arg 290 295 CTC TAT GTC AGC TTC CGT GAC CTT GCC TGG CTG GAC TCT Leu Tyr Val Ser Phe Arg Asp Leu Gly Trp Leu Asp Ser 305 310 315 CCC CAG GCC TAC TCC GCC TAT TAC TGT GCT GGG GAG TGC Pro Gin Gly Tyr Ser Ala T'yr Tyr Cys Ala Gly Ciu Cys 320 325 330 CTG AAC TCC TGT ATG AAC TCC ACC AAC CAC GCC ACT ATG Leu Asn Ser Cys Met Asn Ser Thr Asn His Ala Thr Met 335 340 345 GTA CAT CTG ATG AAG CCA CAT ATC ATC CCC AAG CTG C Vai His Leu Met Lys Pro Asp Ile Ile Pro Lys Val Cys 355 360 ACT GAG CTC ACT CCC ATT TCT CTC CTC TAC TAT CAT AGA Thr Ciu Leu Ser Ala Ile Ser Leu Leu Tyr Tyr Asp Arg 370 375 GTC ATC CTC CCC AGO GAG CCC AAC ATC GTA CTC CAG GCC Val I2e Ara Arc 31u zA-c Ac a. Val. 0'7.A 385 390 395 CAC TGACTCCCTG CCCAACAGCC TCCTCCCATC CCATCTATCT

ACTC

His 400 CTCTTCCAAC GCAGGAAACC AACAAAGAGG GAAGGCAGTG

CTTTCAAC

TTCACACTCT TCCCCCTCTC TCTTCTTTTT GCCAAGCCTG

AGAAGATG

ACCCTCGTCA CCTCACTACC CCCATCTCTC ATCTCCCCAA

ACTCCCCA

CCATCTATCT CCTTTGCCAT TGCGCACACA AGTCCAATTT

ACCAACTT

CTACTCCCCC ACCCTCGACT TGAACCTGGA ACACACGGTA

CACCTCAG

CCATCACAAG ATTTAGGTGT CTCCAGACAT GACCACACTC

CCCCTAGC

INFORMATION FOR SEQ ID NO:26:

ACC

Arg 300

GTC

Val1

ATC

Ile

CAC

TGT

Cys

AAC

Asn 380

CAT

His

ATT

Ile

TAC

Tvr

CC

Ala

GTG

Val1 365

AAT

As n

GAG

Ciu

CC

Ala

CCA

Pro

CTC

Leu 350

CCT

Pro

AAT

As n 974 1022 1070 1118 1166 1214 1262 1315 1375 1435 1495 1555 1615 1674 Cly Ile Leu 285 Cs GyCys 5*

S

S

.55.

S

AGCCCT

TC

CT

AT

AT

C

AC

CATGTCCACA

CCTACTTATA

CCACCCAGCG

TCATCACTCA

TCTTCACTAT

TCCATACC

WO 97/41881 PCT/US97/07816 -76- SEQUENCE CHARACTERISTICS: LENGTH: 399 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: Met Ala Ala Arg Pro Gly Leu Leu Trp Leu Leu Gly Leu Ala Leu Cys 1 5 10 Val Leu Gly Gly Gly His Leu Ser His Pro Pro His Val Phe Pro Gin 25 Arg Arg Leu Gly Val Arg Glu Pro Arg Asp Met Gin Arg Glu Ile Arg 40 Glu Val Leu Gly Leu Ala Gly Arg Pro Arg Ser Arg Ala Pro Val Gly 55 Ala Ala Gln Gln Pro Ala Ser Ala Pro Leu Phe Met Leu Asp Leu Tyr 70 75 Arg Ala Met Thr Asp Asp Ser Gly Gly Gly Thr Pro Gin Pro His Leu 85 90 Asp Arg Ala Asp Leu Ile Met Ser Phe Val Asn Ile Val Glu Arg Asp 100 105 110 Arg Thr Leu Gly Tyr Gin Glu Pro His Trp Lys Glu Phe His Phe Asp 115 120 125 Leu Thr Gin Ile Pro Ala Gly Glu Ala Val Thr Ala Ala Glu Phe Arg 130 135 140 Ile Tyr Lys Glu Pro Ser Thr His Pro Leu Asn Thr Thr Leu His Ile 145 150 155 160 **oo Ser Met Phe Glu Val Val Gin Glu His Ser Asn Arg Glu Ser Asp Leu 165 170 175 Phe Phe Leu Asp Leu Gin Thr Leu Arg Ser Gly Asp Glu Gly Trp Leu 180 185 190 Val Leu Asp Ile Thr Ala Ala Ser Asp Arg Trp Leu Leu Asn His His 195 200 205 Lys Asp Leu Gly Leu Arg Leu Tyr Val Glu Thr Glu Asp Gly His Ser 210 215 220 Ile Asp Pro Gly Leu Ala Gly Leu Leu Gly Arg Gin Ala Pro Arg Ser 225 230 235 240 Arg Gin Pro Phe Met Val Gly Phe Phe Arg Ala Asn Gin Ser Pro Val 245 250 255 WO 97/41881 PCTIS97/07816 -77- Arg Ile Gly Val 305 Gly Ser Leu Leu Leu 385 (2) Ala Pro Arg Thr Ala Arg Pro Leu Lys Lys Lys Gin Leu Asn Gin 260 265 270 Asn Gin Leu Pro His Ser Asn Lys His Leu Gly Ile Leu Asp Asp 275 280 285 His Gly Ser His Gly Arg Glu Val Cys Arg Arg His Glu Leu Tyr 290 295 300 Ser Phe Arg Asp Leu Gly Trp Leu Asp Ser Val Ile Ala Pro Gin 310 315 320 Tyr Ser Ala Tyr Tyr Cys Ala Gly Glu Cys Ile Tyr Pro Leu Asn 325 330 335 Cys Met Asn Ser Thr Asn His Ala Thr Met Gin Ala Leu Val His 340 345 350 Met Lys Pro Asp Ile Ile Pro Lys Val Cys Cys Val Pro Thr Glu 355 360 365 Ser Ala Ile Ser Leu Leu Tyr Tyr Asp Arg Asn Asn Asn Val Ile 370 375 380 Arg Arg Glu Arg Asn Met Val Val Gin Ala Cys Gly Cys His 390 395 INFORMATION FOR SEQ ID NO:27: 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:27: 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 Gin 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 WO 97/41881 PCT/US97/07816 -78- 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:28: SEQUENCE CHARACTERISTICS: LENGTH: 102 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein o (vi) ORIGINAL SOURCE: ORGANISM: HOMO SAPIENS (ix) FEATURE: NAME/KEY: Protein LOCATION: 1..102 OTHER INFORMATION: /note= (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: 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 20 25 Glu Cys Ser Phe Pro Leu Asn Ala His Met Asn Ala Thr Asn His Ala 35 40 *Ile Val Gln 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 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 INFORMIATION FOR SEQ ID NO:29: SEQUENCE CHARACTERISTICS: LENGTH: 102 amino acids WO 97/41881 PCT/US97/07816 -79- TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE: ORGANISM: HOMO SAPIENS (ix) FEATURE: NAME/KEY: Protein LOCATION: 1..102 OTHER INFORMATION: /note= "BMP6" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: Cys Arg Lys His Glu Leu Tyr Val Ser Phe Gin Asp Leu Gly Trp Gin 1 5 10 Asp Trp Ile I l e Ala Pro Lys Gly Tyr Ala Ala Asn Tyr Cys Asp Gly 20 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 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 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" WO 97/41881 PCTIUS97/07816 80 (xi) SEQUENCE DESCRIPTION: SEQ ID 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

GGC

Gly

CTG

Leu

GCT

Ala

GTT

Val.

ACC

Thr

TGC

Cys

CCG

Pro

GGG

Gly

CCC

Pro

GCG

Ala

GCG

Al a 17 0

TTG

Leu

CAC

His

ACC

Thr

CTA

Leu

CCC

Pro

AGG

Arg

CAC

His

GAC

Asp

CAT

His

GCT

Ala

GCG

Al a 155

GGC

Gly

GTG

Val1

CAC

His

CGC

Arg

GGA

Gly

CCG

Pro

TCT

Ser

GTG

Val1

CGC

Arg

TGC

Cys

GAG

Glu 140

GCG

Ala

CAG

Gln

CCC

Pro

CTC

Leu

GCC

Ala

CTG

Leu

GTC

Val1

GGC

Gly

GAG

Glu

GGT

Gly

CCT

Pro 125

CGC

Arg

GCA

Ala

GGC

Gly

GCC

Ala

*CTC

Leu

CCC

Pro

CGC

Arg

ATG

Met

TCG

Ser

GAG

Glu

GCG

Al a 110

GAG

G lu

CCG

Pro

GCC

Al a

GCG

Al a

CTG

Leu 190 CTC CTC Leu Leu 1s GTG CCC Val Pro GAT GAG Asp Glu TGG CGC Trp Arg CGG CCGG Arg Arg 80 CTG GG Leu Gly 95 CCC ACC Pro Thr TGG ACA Trp Thr AGC CGG Ser Arg CCG GAG Pro Giu 160 GGC GCG Gly Ala 175 GGG CCG Gly Pro Leu Ala Leu

CCA

Pro

CCC

Pro

CTG

Leu 65

ACG

Thr

GTC

Val1

CGG

Arg

GTC

Val1

GCC

Ala 145

GGC

Gly

GAC

Asp

CCA

Pro

CCA

Pro 35

GGT

Gly

CGA

Arg

CCA

Pro

GGA

Gly

TCG

Ser 115

TTC

Phe

CTG

Leu

TG

Trp

GGG

G ly

CGC

Arg 195 Leu Leu Pro 20 GCC GCC GCC Ala Ala Ala GCC CCC AGG Ala Pro Arg COC COG GAC Arg Arg Asp GGG GTC ACC Gly Val Thr AAC ATC GTG Asn Ile Val 100 GAG CCT GTC Oiu Pro Val GAC CTG TCG Asp Leu Ser GAG CTG CGT Giu Leu Arg 150 GAG CTO AGC Glu Leu Ser 165 CCG GTO CTG Pro Val Leu 180 OCO GAG CTG Ala Glu Leu Ser

CTG

Leu

CTC

Leu

CCC

Pro

CTG

Leu

CGC

Arg

TCG

Se r

GCT

Al a 135

TTC

Phe

GTG

Val1

CTC

Leu

CTG

Leu Leu

CTC

Leu

CGG

Arg

CAG

0 ln

CAA

Gln

CAC

His 0CC Ala 120

GTG

Val1

GCG

Al a

GCG

Ala

CGC

Arg

GOC

Gly 200 Pro

CAG

Gin

CCG

Pro

GAG

Glu

CCG

Pro

ATC

Ile 105

GCG

Ala

GAA

G iu

GCG

Al a

CAA

Gin

CAG

Oln 185

GCC

Ala CTG GCC CTO CTG CTG CCC TCG CTG CCC 158 206 254 302 350 398 446 494 542 590 638 686 WO 97/41881 WO 971881PCT[US97/07816 81 GCT TGG GCT CGC AAC GCC TCA TGG CCG Ala Trp Ala Arg Asn Ala Ser Trp Pro 205 210 CGC AGC CTC CGC CTG GCG CTG Arg Ser Leu Arg Leu Ala Leu 0t*.

GCG

Al a

TCG

Ser

CGG

Arg 250

GCT

Ala

CAC

His

GGT

Gly

GCG

Ala

GGA

Gly 330

TCC

Ser

GAG

CTA

Leu

CTG

Leu 235

CCG

Pro

TGT

Cys

CGC

Arg

CAG

Gin

CTC

Leu 315

GCC

Al.a

GTG

Val1

GAC

CGC

Arg 220

CTG

Leu

CGG

Arg

CGC

Arg

TGG

Trp

TGC

Cys 300

AAC

Asn

GCC

Ala

CTC

Leu

ATG

CCC

Pro

CTG

Leu

CGC

Arg

GCG

Al a

GTC

Val1 285

GCG

Ala

CAC

His

GAC

Asp

TTC

Phe

GTG

Arg

GTG

Val1

GAC

Asp

CGG

Arg 270

ATC

Ile

CTG

Leu

GCT

Al a

CTG

Leu

TTT

Phe 350

GTG

Ala Pro

ACC

Thr

GCC

Al a 255

CGG

Arg

GCG

Al a

CCC

Pro

GTG

Val1

CCC

Pro '335

GAC

Asp

GAC

CTC

Leu 240

GAA

Glu

CTG

Leu

CCG

Pro

GTC

Val1

CTG

Leu 320

TOC

Cys

AAC

Asn

GAG

CGG GCC CCT GCC GCC TGC Ala Ala Cys 225 GAC CCG CGC Asp Pro Arg CCC GTG TTG Pro Val Leu TAC GTG AGC Tyr Val Ser 275 CGC GGC TTC Arg Gly Phe 290 GCG CTG TCG Ala Leu Ser 305 CGC GCG CTC Arg Ala Leu TGC GTG CCC Cys Val Pro AGC GAC AAC Ser Asp Asn 355 TGC GGC TGC(

GCG

Ala

CTG

Leu

GGC

Gly 260

TTC

Phe

CTG

Leu

GGG

Gly

ATG

iMe t

GCG

Al a 340

GTG

Val1

CGC

Arg

TGC

Cys 245

GGC

G ly

CGC

Arg

GCC

Ala

TCC

Ser

CAC

His 325

CGC

Arg

GTG

CTG

Leo 230

CAC

His

GGC

Gly

GAG

Glu

AAC

As n

GGG

Gly 310

GCG

Ala

CTG

Leu

GCC

Al a

CCC

Pro

CCC

Pro GT 0 Val

TAC

Tyr 295

GGG

G 1y

GCC

Al a

TCG

Se r

GAG

Glu

CTG

Leu

GGG

Gly

GGC

Gly 280

TGC

Cy s

CCG

Pro

GCC

Ala

CCC

Pro

GCC

Al a

GCC

Ala

GGC

Gly 265

TG

Trp

CAG

Gin

CCG

Pro

CCG

Pro

ATC

Ile 345 782 830 878 926 974 1022 1070 1118 1166 1219 1247 Val Leu Arg Gln Tyr 360 TAACCCGGGG CGGGCAGGGA Glu Asp met Val Val Asp Glu Cys 365 rrzr Gly Cys Arg 370 CCCGGGCCCA ACAATAAATG CCGCGTGG INFORMATION FOR SEQ ID NO:31: SEQUENCE CHARACTERISTICS: LENGTH: 372 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein WO 97/41881 WO 971881PCTIUS97/07816 (xi) SEQUENCE Met Pro Pro Pro Gin 82 DESCRIPTION: SEQ ID NC Gin Gly Pro CYS Gly Hi :31: .5 His Leu Leu Leu Leu Leu Ala Leu Leu Leu Pro Ser Leu Pro Leu Thr Arg Ala Pro Val Pro Pro Pro Leu Thr Val1 Ara Val1 Al a 145 Gly Asp Pro Trp Ala 225 Asp Pro Tyr Arg Gly Gin Phe Ser Al a Ala Val1 130 Arg Gly Pro Val Pro 210 Ala Pro Val Val Gly Pro Gly Arg Pro Gly Ser 115 Phe Leu rrp Gly Arg 1.95 krg :ys %rg eu 3er 275 Phe Ala Ala Arg Gly As n 100 Glu Asp Giu Glu Pro 180 Al a Ser Al a Leu Gly 260 Phe Leu Ala Pro Arg Val Ile Pro Leu Leu Leu 165 Val1 Glu Leu Arg Cys 245 G ly Arg Ala Ala Arg Asp 70 Thr Val1 Val1 Ser Arg 150 Ser Leu Leu Arg Leu 230 His Gly Glu Asn 25 Leu Leu 55 Pro Leu Arg Ser Ala 135 Phe Val1 Leu Leu Leu 215 Al a Pro Pro Val1 Tyr Gin Pro Glu Pro Ile 105 Al a Giu Ala Gin Gin 185 Ala Leu Ala Ala Gly 265 Trp Gin Al a Val1 Thr Cys Pro Gly Pro Al a Al a 170 Leu Al a Al a Ser Arg 250 Al a His Gly Leu Pro Arg 75 Pis Asp His Ala Ala 155 Gly Val1 Trp Leu Leu 235 Pro Cys Arg Gin Gly Pro Ser Val1 Arg Cys G iu 140 Ala Gin Pro Ala Arg 220 Leu Arg Arg Trp Cys Leu Val1 Gly Glu Gly Pro 125 Arg Al a Gly Ala Arg.

205 Pro Leu Arg Ala Val1 285 Ala Arg Met Ser 0 lu Ala 110 G lu Pro Al a Al a Leu 190 As n Arg Val1 Asp Arg 270 Ile Leu Glu Arg Arg Gly Thr Thr Arg Giu 160 Ala Pro Ser Pro Leu 240 Glu Leu Pro Val WO 97/41881 PCT/US97/07816 83 290 295 300 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 Gln Tyr Glu Asp Met Val Val Asp Glu 355 360 365 Cys Cly Cys Arg 370

Claims (1)

1. 89- PCT/US97/07816 1 57. A method as in claim 53 wherein said polypeptide has at least 65% identity with an amino 2 acid sequence of a C-terminal seven-cysteine domain of human OP-1. 1 58. A method as in claim 53 wherein said polypeptide has at least 70% identity with an amino 2 acid sequence of a C-terminal seven-cysteine domain of human OP-i. 1 59. A method as in any one of claims 51-58 wherein said renal therapeutic agent 2 induces chondrogenesis in an ectopic bone assay; 3 prevents, inhibits, delays or alleviates loss of renal function in an animal model of 4 chronic renal failure; or causes a clinically significant improvement in a standard marker of renal function when 6 administered to a mammal in, or at risk of, chronic renal failure. 1 60. A method as in claim 1 wherein said renal therapeutic agent is selected from the group 2 consisting of human osteogenic proteins and human bone morphogenetic proteins. *e *o o 1,0 e ooo o go o•