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WORLD INTELLECTUAL PROPERTY ORGANIZATION 
International Bureau 




INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) 



(51) iDternational Patent Gassification ^ : 
A61K 



A2 



(11) International Publication Number: 
(43) International Publication Date: 



WO 93/05751 

I April 1993(01.04.93) 



1^ 



(21) International Application Number: PCT/US92/07432 

(22) International Filing Date: 28 August 1 992 (28.08.92) 



(30) Priority data: 

752,857 
752,764 
752,861 
923,780 



30 August 1991 (30.08.91) US 

30 August 1991 (30.08.91) US 

30 August 1991 (30.08.91) US 

31 July 1992(31.07.92) US 



(71) Appllcant: CREATIVE BIOMOLECULES, INC. [US/ 
US]; 35 South Street, Hopkinton, MA 01748 (US). 

(72) Inventors: KUBERASAMPATH, Thangavel ; 6 Spring 
Street, Medway, MA 02053 (US). COHEN, Charles, M. ; 
98 Winthrop Street, Medway, MA 02053 (US). OPPER- 
MANN, Hermann ; 25 Summer Hill Road, Medway, 
MA 02053 (US). 02KAYNAK, Engin ; 44 Purdue 
Drive, Milford, MA 01757 (US). RUEGER, David, C ; 
19 Downey Street, Hopkinton. MA 01748 (US). PANG, 
Roy, H., L. ; 15 Partridge Road, Etna, NH 03750 (US). 
SMART, John, E. ; 50 Meadow Brook Road, Weston, 
MA 02193 (US). 



(74) Agent: KELLEY, Robin, D.; Testa, Hunvitz & Thibeault, 
Exchange Place, 53 State Street, Boston, MA 02109-2809 
(US). 

(81) Designated States: AU, CA, JP, European patent (AT, BE, 
CH, DE, DK, ES, FR, GB, GR, IE, IT, LU, MC, NL, 
SE). 



Published 

Without international search report and to be republished 
upon receipt of that report. 



(54) Title: TREATMENT TO PREVENT LOSS OF AND/OR INCREASE BONE MASS IN METABOLIC BONE DISEAS- 



(57) Abstract 

TTie invention is a treatment for increasing the bone mass or preventing bone loss in an individual afflicted with a bone dis- 
ease which mcludes admmistenng to the individual a morphogen in a therapeutically effective amount so as to maintain or stimu- 

late Done lormation. 



9 



FOR THE PURPOSES OF INFORMATION ONLY 



Codes used to identify Scares pany lo the PCTon the front pages of |jamphleLs publishing internation«il 
applications under the PCX. 



AT 


Ausirfd 


Fl 


Finbnd 


MN 


Mongolia 


AU 


Australia 


FR 


France 


MR 


Mauritania 


BB 


BarhaJub 


CA 


Gabon 


MW 


Malawi 


BE 


Belgium 


CB 


United Kingdom 


NL 


Neiberland:* 


BF 


Burkina Fa^o 


CN 


Guinea 


NO 


Norway 


EC 


Bulgaria 


GR 


Greece 


NZ 


New Zealand 


BJ 


Bunin 


HU 


Hungary 


PL 


Pubnd 


BR 


Bra/tl 


IE 


Ireland 


FT 


Poriugal 


CA 


Cllanaiia 


IT 


lialy 


RO 


Romania 


CF 


Central African Republic 


JP 


Japan 


RU 


Ru£iian Federation 


CC 


f*ongo 


KP 


llcntocratic People** Republic 


SO 


Sudan 


CH 


Swit/rrlanU 




ur Korea 


SE 


Sweden 


CI 


Cfilc a*I»oirc 


KR 


Republic of Korea 


SK 


Sluvak Republic 


CM 


C^'amcroon 


U 


IJechienstein 


SN 


Senegal 


CS 


OccbcKilovaLij 


UC 


Siri Lanka 


5U 


Soviet Union 


CZ 


Occh Rcpubltc 


LU 


Ufxembourg 


TO 


(niad 


OE 


Gurmany 


MC 


Muiuico 


TC 


Togo 


DK 


Denmark 


MG 


Maduga^icar 


UA 


Ukraine 


ES 


Spain 


Ml 


Mali 


US 


United Slates uf Americii 



wo 93/05751 



PCr/lJS92/07432 



TREATMENT TO PREVENT LOSS OF 
AND/OR INCREASE BONE MASS IN METABOLIC BONE DISEASES 

This invention relates to means for increasing the 
bone mass and/or preventing the loss of bone mass in a 
mammal • 

Background of the Invention 

Throughout adult life, bone is continually 
undergoing remodeling through the interactive cycles of 
bone formation and resorption (bone turnover) • Bone 
resorption typically is rapid, and is mediated by 
osteoclasts (bone resorbing cells), formed by 
mononuclear phagocytic precursor cells at bone 
remodeling sites • This process then is followed by the 
appearance of osteoblasts (bone forming cells) which 
form bone slowly to replace the lost bone. The 
activities of the various cell types that participate 
in the remodeling process are controlled by interacting 
systemic (e.g., hormones, lymphokines, growth factors, 
vitamins) and local factors (e.g., cytokines, adhesion 
molecules, lymphokines and growth factors). The fact 
that completion of this process normally leads to 



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balanced replacement and renewal of bone indicates that 
the molecular signals and events that influence bone 
remodeling are tightly controlled • 

5 A number of bone growth disorders are known which 

cause an imbalance in the bone remodeling cycle. Chief 
among these are metabolic bone diseases / such as 
osteoporosis, osteoplasia (osteomalacia), chronic renal 
failure and hyperparathyroidism, which result in 
10 abnormal or excessive loss of bone mass (osteopenia). 
Other bone diseases, such as Paget' s disease, also 
cause excessive loss of bone mass at localized sites. 

Osteoporosis is a structural deterioration of the 
skeleton caused by loss of bone mass resulting from an 
imbalance in bone formation, bone resorption, or both, 
such that the resorption dominates the bone formation 
phase, thereby reducing the weight-bearing capacity of 
the affected bone. In a healthy adult, the rate at 
which bone is formed and resorbed is tightly 
coordinated so as to maintain the renewal of skeletal 
bone. However, in osteoporotic individuals an 
imbalance in these bone remodeling cycles develops 
which results in both loss of bone mass and in 
formation of microarchitectural defects in the 
continuity of the skeleton. These skeletal defects, 
created by perturbation in the remodeling sequence, 
accumulate and finally reach a point at which the 
structural integrity of the skeleton is severely 
compromised and bone fracture is likely. Although this 
imbalance occurs gradually in most individuals as they 



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age ("senile osteoporosis"), it is much more severe and 
occurs at a rapid rate in postmenopausal women. In 
addition/ osteoporosis also may result from nutritional 
and endocrine imbalances, hereditary disorders and a 
number of malignant transformations. 

Patients suffering from chronic renal (kidney) 
failure almost universally suffer loss of skeletal bone 
mass (renal osteodystrophy). While it is khbvm that 
kidney malfunction causes a calcium and phosphate 
imbalance in the blood, to date replenishment of 
calcium and phosphate by dialysis does not 
significantly inhibit osteodystrophy in patients 
suffering from chronic renal failure. In adults, 
OS teodys trophic symptoms often are a significant cause 
of morbidity. In children, renal failure often results 
in a failure to grow, due to the failure to maintain 
and/or to increase bone mass- 

Osteoplasia, also known as osteomalacia ("soft 
bones"), is a defect in bone mineralization (e.g., 
incomplete mineralization), and classically is related 
to vitamin D deficiency ( 1 ,25-dihydroxy vitamin D^). 
The defect can cause compression fractures in bone, and 
a decrease in bone mass, as well as extended zones of 
hypertrophy and proliferative cartilage in place of 
bone tissue. The deficiency may result from a 
nutritional deficiency (e.g., rickets in children), 
malabsorption of vitamin D or calcium, and/or impaired 
metabolism of the vitamin. 



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Hyperparathyroidism (overproduction of the 
parathyroid hormone) is known to cause malabsorption of 
calcium,^ leading to abnormal bone loss. In children, 
hyperparathyroidism can inhibit grovrtih, in adults the 
5 skeleton integrity is compromised and fracture of the 
ribs and vertebrae are characteristic. The parathyroid 
hormone imbalance typically may result from thyroid 
adenomas or gland hyperplasia, or may result from 
prolonged pharmacological use of a steroid. Secondary 
10 hyperparathyroidism also may result from renal 

osteodystrophy. In the early stages of the disease 
osteoclasts are stimulated to resorb bone in response 
to the excess hormone present. As the disease 
progresses, the trabecular bone ultimately is resorbed 
15 and marrow is replaced with fibrosis, macrophages and 
areas of hemorrhage as a consequence of microfractures. 
This condition is referred to clinically as osteitis 
fibrosa. 

20 Paget 's disease (osteitis deformans) is a disorder 

currently thought to have a viral etiology and is 
characterized by excessive bone resorption at localized 
sites which flare and heal but which ultimately are 
chronic and progressive, and may lead to malignant 

25 transformation. The disease typically affects adults 
over the age of 25- 

To date, osteopenia treatments are based on 
inhibiting further bone resorption, e.g., by 1) 
30 inhibiting the differentiation of hemopoietic 

mononuclear cells into mature osteoclasts, 2) by 
directly preventing osteoclast-mediated bone 
resorption, or 3 ) by affecting the hormonal control of 
bone resorption. Drug regimens used for the treatment 
of osteoporosis include calcium supplements, estrogen. 



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calcitonin and diphosphonates . Vitamin and its 
metabolites, known to enhance calcium and phosphate 
absorption, also are being tried. None of the current 
therapies stimulate regeneration of new bone tissue. 
In addition, all of these agents have only a transient 
efffect on bone remodeling. Thus, while in some cases 
the progression of the disease may be halted or slowed, 
patients with significant bone deterioration remain 
actively at risk. This is particularly prevalent in 
disorders such as osteoporosis where early diagnosis is 
difficult and/or rare and significant structural 
deterioration of the bone already may have occurred. 

It is an object of the present invention to develop 
methods and compositions for inhibiting or preventing 
the loss of bone mass and/or for increasing bone 
formation in an individual who, for example, is 
afflicted with a disease which decreases skeletal bone 
mass, particularly where the disease causes an 
imbalance in bone remodeling. Another object is to 
enhance bone growth in children suffering from bone 
disorders, including metabolic bone diseases • Still 
another object is to prevent or inhibit bone 
deterioration in individuals at risk for loss of bone 
mass, including postmenopausal women, aged individuals, 
and patients undergoing dialysis. Yet another object 
is to provide methods and compositions for repairing 
defects in the microstructure of structurally 
compromised bone, including repairing bone fractures. 
Thus, the invention is aimed at stimulating bone 



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formation and increasing bone mass, optionally over 
prolonged periods of time, and particularly to decrease 
the occurrence of new fractures resulting from 
structural deterioration of the skeleton. These and 
other objects and features of the invention will be 
apparent from the description, drawings, and claims 
which follow* 



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Siimmary of the Invention 

The present invention provides methods and 
compositions for inhibiting loss of bone mass, and/or 
for stimulating bone formation in mammals, particularly 
humans. 

In one aspect, the invention features a therapeutic 
treatment method and composition for preventing loss of 
bone mass and/or for increasing bone mass in a mammal 
which includes administering to the individual a 
therapeutically effective morphogen in an amount and 
for a time sufficient to inhibit the loss of bone mass, 
and/or to increase bone mass in the individual. 

In another aspect, the invention features a 
therapeutic treatment method and composition for 
preventing loss of bone mass and/or for increasing bone 
mass in a mammal which includes administering to the 
mammal a compound that stimulates in vivo a 
therapeutically effective concentration of an 
endogenous morphogen in the body of the mammal 
sufficient to prevent loss of and/or to increase bone 
mass in the individual. These compounds are referred 
to herein as morphogen- stimulating agents, and are 
understood to include substances which, when 
administered to a mammal, act on tissue{s) or organ(s) 
that normally are responsible for, or capable of, 
producing a morphogen and/or secreting a morphogen, and 
which cause the endogenous level of the morphogen to be 
altered. The agent may act, for example, by 
stimulating expression and/or secretion of an 
endogenous morphogen. 



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PCr/US92/07432 



The morphogens described herein are believed to 
play a significant role in maintaining appropriate bone 
mass in an individual. Thus, a morphogen may be 
administered according to the invention to any 
5 individual who requires assistance in maintaining 
appropriate bone mass and/or who suffers from a bone 
remodeling imbalance • For example, the morphogen or 
morphogen- stimulating agent may be administered 
according to the invention to an adult suffering from 

10 renal failure to prevent bone deterioration which is 
associated with that disease, e.g., to correct bone 
loss due to late stage kidney failure. Similarly, the 
administration of a morphogen to a child suffering from 
renal failure is expected not only to alleviate loss of 

15 bone mass in the child, but also to stimulate bone 

formation and thus growth. In addition, administration 
of a morphogen or morphogen-stimulating agent to an 
individual suffering from defects in skeletal 
microstructure is expected to result in repair of that 

20 defect, and to enhance the weight-bearing capacity of 
the treated bone. 

Accordingly, in another aspect of the invention, 
the treatment methods and compositions of the invention 

25 may be used to treat a bone fracture or any disease 
which causes or results in bone fractures or other 
defects in skeletal microstructure, including loss of 
bone mass, and which compromise the weight-bearing 
capacity of bone. Such diseases include, for example, 

30 chronic renal failure and other kidney diseases, 

particularly those requiring dialysis; osteomalacia; 
vitamin D deficiency-induced osteopenia or 
osteoporosis; postmenopausal or senile osteoporosis; 
hyperparathyroidism and Paget 's disease. 



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In still another aspect, the invention provides 
methods and compositions for protecting an individual 
at risk for the loss or deterioration of skeletal bone 
mass by prophylactic administration of a morphogen or 
5 morphogen-stimulating agent. Individuals at risk 

include postmenopausal females, aged individuals/ and 
individuals undergoing dialysis, particularly prolonged 
or chronic dialysis. 

10 In one preferred embodiment of the invention, the 

morphogen or morphogen-stimulating agent is 
administered systemically to the individual, e.g., 
orally or parenterally . In another embodiment of the 
invention, the morphogen may be provided directly to 

15 the bone, e.g., by injection to the bone periosteum or 
endosteum. Direct injection is particularly useful for 
repairing defects in the microstructure of the bone, 
including bone fractures . 

20 In any treatment method of the invention, 

"administration of morphogen" refers to the 
administration of the morphogen, either alone or in 
combination with other molecules. For example, the 
mature form of the morphogen may be provided in 

25 association with its precursor "pro" domain, which is 
known to enhance the solubility of the protein. Other 
useful molecules known to enhance protein solubility 
include casein and other milk components, as well as 
various serum proteins. Additional useful molecules 

30 which may be associated with the morphogen or 

morphogen-stimulating agent include tissue targeting 
molecules capable of directing the morphogen or 
morphogen-stimulating agent to bone. Tissue targeting 
molecules envisioned to be useful in the treatment 

35 protocols of this invention include tetracycline. 



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PCr/US92/07432 



diphosphonates / and antibodies or other binding 
proteins which interact specifically with surface 
molecules on bone tissue cells. 

Still another useful tissue targeting molecule is 
the morphogen precursor "pro" domain, particularly that 
of OP-1/ BMP2 or BMP4. These proteins are found 
naturally associated with bone tissue but likely are 
synthesized in other tissues and targeted to bone 
tissue after secretion from the synthesizing tissue. 
For example, the primary source of OP-1 synthesis 
appears to be the tissue of the urinary tract (e.g., 
renal tissue), while the protein has been shown to be 
active in bone tissue (see below.) Moreover, the 
protein has been identified in serum, saliva and 
various milk forms. In addition, the secreted form of 
the protein comprises the mature dimer in association 
with the pro domain of the intact morphogen sequence. 
Accordingly, the associated morphogen pro domains may 
act to target specific morphogens to different tissues 
in vivo. 

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

The morphogens or morphogen-stimulating agents also 
may be administered together with other "co-factors" 
known to have a beneficial effect on bone remodeling, 
including parathyroid hormone, vitamin D^f 
prostaglandins, dexamethasone, IGF (I, II) and their 



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PCT/US92/07432 



binding proteins, and other agents known to enhance 
osteoblast activity. Other useful confactors include 
calcitonin and estrogen and other agents which inhibit 
bone resorption. 

5 

Among the morphogens useful in this invention are 
proteins originally identified as osteogenic proteins, 
such as the OP-1, OP-2 and CBMP2 proteins, as well as 
amino acid sequence-related proteins such as DPP (from 
10 Drosophila), Vgl ( f rom Xenopus ) , Vgr-1 (from mouse, see 
U.S. 5,011,691 to Oppermann et al.), GDF-1 (from mouse, 
see Lee (1991) PNAS 88:4250-4254), all of which are 
presented in Table II and Seq. ID Nos.5-14), and the 
recently identified 60A protein (from Drosophila, Seq. 
15 ID No. 24, see Wharton et al. (1991) PNAS 

88:9214-9218.) The members of this family, which 
include members of the TGF-p super-family of proteins, 
share substantial amino acid sequence homology in their 
C-terminal regions. The proteins are translated as a 
20 precursor, having an N-terminal signal peptide 
sequence, typically less than about 30 residues, 
followed by a "pro" domain that is cleaved to yield the 
mature sequence. The signal peptide is cleaved rapidly 
upon translation, at a cleavage site that can be 
25 predicted in a given sequence using the method of Von 
Heijne ((1986) Nucleic Acids Research 14 ; 4683-4691 . ) 
Table I, below, describes the various morphogens 
identified to date, including their nomenclature as 
used herein, their Seq. ID references, and publication 
30 sources for the amino acid sequences of the full length 
proteins not included in the Seq. Listing. The 
disclosure of these publications is incorporated herein 
by reference. 



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TABLE I 

"OP-1" Refers generically to the group of 

morphogenically active proteins expressed 
5 from part or all of a DNA sequence 

encoding OP-1 protein^ including allelic 
and species variants thereof, e.g., human 
OP-1 ("hOP-l"r Seq. ID No. 5, mature 
protein amino acid sequence), or mouse 

10 OP-1 ("mOP-1", Seq. ID No. 6, mature 

protein amino acid sequence. ) The 
conserved seven cysteine skeleton is 
defined by residues 38 to 139 of Seq. ID 
Nos. 5 and 6. The cDNA sequences and the 

15 amino acids encoding the full length 

proteins are provided in Seq- Id Nos. 16 
and 17 (hOPl) and Seq. ID Nos. 18 and 19 
(mOPl.) The mature proteins are defined 
by residues 293-431 (hOPl) and 292-430 

20 (mOPl). The "pro" regions of the 

proteins, cleaved to yield the mature, 
morphogenically active proteins are 
defined essentially by residues 30-292 
(hOPl) and residues 30-291 (mOPl). 

25 

"OP-2" refers generically to the group of active 

proteins expressed from part or all of a 
DNA sequence encoding OP-2 protein, 
including allelic and species variants 

30 thereof, e.g., human OP-2 ("hOP-2", Seq. 

ID No. 7, mature protein amino acid 
sequence) or mouse OP-2 {"mOP-2", Seq. ID 
No. 8, mature protein amino acid 
sequence). The conserved seven cysteine 

35 skeleton is defined by residues 38 to 139 



- 13 - 



Of Seq. ID Nos. 7 and 8. The cDNA 
sequences and the amino acids encoding the 
full length proteins are provided in Seq. 
ID Nos. 20 and 21 (hOP2) and Seq. ID Nos. 
22 and 23 (mOP2.) The mature proteins are 
defined essentially by residues 264-402 
(hOP2) and 261-399 {mOP2). The "pro" 
regions of the proteins^ cleaved to yield 
the mature, morphogenically active 
proteins are defined essentially by 
residues 18-263 (hOP2) and residues 18-260 
{mOP2). (Another cleavage site also 
occurs 21 residues further upstream for 
both OP-2 proteins.) 

refers generically to the morphogenically 
active proteins expressed from a DNA 
sequence encoding the CBMP2 proteins, 
including allelic and species variants 
thereof, e.g., human CBMP2A ( "CBMP2A(fx) 
Seq ID No. 9) or human CBMP2B DNA 
("CBMP2B{fx)", Seq. ID No. 10). The amino 
acid sequence for the full length 
proteins, referred to in the literature 
collectively as BMP2A and BMP2B, or BMP2 
and BMP4, appear in Wozney, et al. (1988) 
Science 242:1528-1534. The pro domain for 
BMP2 (BMP2A) likely includes residues 25- 
248 or 25-282; the mature protein, 
residues 249-396 or 283-396. The pro 
domain for BMP4 (BMP2B) likely includes 
residues 25-256 or 25-292; the mature 
protein, residues 257-408 or 293-408. 



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"DPP(fx)" refers to protein sequences encoded by the 
Drosophila DPP gene and defining the 
conserved seven cysteine skeleton (Seq* ID 
No. 11) • The amino acid sequence for the 
5 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 
10 residues 457-588. 



"Vgl(fx)" refers to protein sequences encoded by the 
Xenopus Vgl gene and defining the 
conserved seven cysteine skeleton (Seq. ID 

15 No. 12). The amino acid sequence for the 

full length protein appears in 
Weeks (1987) Cell 51: 861-867. The pro 
domain likely extends from the signal 
peptide cleavage site to residue 246; the 

20 mature protein likely is defined by 

residues 247-360. 



"Vgr-l(fx)" refers to protein sequences encoded by the 
murine Vgr-1 gene and defining the 

25 conserved seven cysteine skeleton (Seq. ID 

No. 13). The amino acid sequence for the 
full length protein appears in Lyons, et 
al, (1989) PNAS 86 : 4554-4558. The pro 
domain likely extends from the signal 

30 peptide cleavage site to residue 299; the 

mature protein likely is defined by 
residues 300-438. 



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"GDF-l(fx)" refers to protein sequences encoded by the 
human GDF-1 gene and defining the 
conserved seven cysteine skeleton (Seg, ID 
No. 14). The cDNA and encoded amino 
5 sequence for the full length protein is 

provided in Seq. ID. No. 32. The pro 
domain likely extends from the signal 
peptide clavage site to residue 214; the 
mature protein likely is defined by 
10 residues 215-372. 



"6 OA" refers generically to the morphogenically 

active proteins expressed from part or all 
of a DNA sequence (from the Drosophila 60A 

15 gene) encoding the 6 OA proteins (see Seq. 

ID No. 24 wherein the cDNA and encoded 
amino acid sequence for the full length 
protein is provided). "60A(fx)" refers to 
the protein sequences defining the 

20 conserved seven cysteine skeleton 

(residues 354 to 455 of Seq. ID No. 24.) 
The pro domain likely extends from the 
signal peptide cleavage site to residue 
324; the mature protein likely is defined 

25 by residues 325-455. 

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

30 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 

35 mature protein likely is defined by 

residues 291-472. 



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"BMP5(fx)" refers to protein sequences encoded by the 
human BMP5 gene and defining the conserved 
seven cysteine skeleton (Seq. ID No. 27). 
The amino acid sequence for the full 
5 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. 

"BMP6(fx)" refers to protein sequences encoded by the 
human BMP6 gene and defining the conserved 
seven cysteine skeleton (Seq. ID No. 28). 
The amino acid sequence for the full 
length protein appear sin 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. 

The OP-2 proteins have an additional cysteine 
residue in this region (e.g., see residue 41 of Seq. ID 
Nos. 7 and 8), in addition to the conserved cysteine 
skeleton in common with the other proteins in this 
family. The GDF-1 protein has a four amino acid insert 
within the conserved skeleton (residues 44-47 of Seq. 
ID No. 14) but this insert likely does not interfere 
with the relationship of the cysteines in the folded 
structure. In addition, the CBMP2 proteins are missing 
one amino acid residue within the cysteine skeleton. 

The morphogens are inactive when reduced, but are 
active as oxidized homodimers and when oxidized in 



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combination with other morphogens of this invention. 
ThuS/ as defined herein, a morphogen is a dimeric 
protein comprising a pair of polypeptide chains, 
wherein each polypeptide chain comprises at least the 
5 C- terminal six cysteine skeleton defined by residues 
43-139 of Seq. ID No. 5, including functionally 
equivalent arrangements of these cysteines (e.g., amino 
acid insertions or deletions which alter the linear 
arrangement of the cysteines in the sequence but not 
10 their relationship in the folded structure), such that, 
when the polypeptide chains are folded, the dimeric 
protein species comprising the pair of polypeptide 
chains has the appropriate three-dimensional structure, 
including the appropriate intra- or inter-chain 
15 disulfide bonds such that the protein is capable of 

acting as a morphogen as defined herein. Specifically, 
the morphogens generally are capable of all of the 
following biological functions in a raorphogenically 
permissive environment: stimulating proliferation of 
20 progenitor cells; stimulating the differentiation of 
progenitor cells; stimulating the proliferation of 
differentiated cells; and supporting the growth and 
maintenance of differentiated cells, including the 
"redif ferentiation" of transformed cells. In addition, 
25 it also is anticipated that these morphogens are 
capable of inducing redif f erentiation of committed 
cells under appropriate environmental conditions. 

In one preferred aspect, the morphogens of 
30 this invention comprise one of two species of generic 
amino acid sequences: Generic Sequence 1 (Seq. ID 
No. 1) or Generic Sequence 2 (Seq. ID No. 2); where 
each Xaa indicates one of the 20 naturally-occurring 
L-isomer, a-amino acids or a derivative thereof. 
35 Generic Sequence 1 comprises the conserved six cysteine 



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PCr/US92/07432 



skeleton and Generic Sequence 2 comprises the conserved 
six cysteine skeleton plus the additional cysteine 
identified in 0P*2 (see residue 36, Seq. ID No. 2). In 
another preferred aspect, these sequences further 
5 comprise the following additional sequence at their N- 
terminus t 



Cys Xaa Xaa Xaa Xaa (Seq. ID No. 15) 
1 5 



10 



Preferred amino acid sequences within the 
foregoing generic sequences include: Generic Sequence 
3 (Seq. ID No. 3), Generic Sequence 4 (Seq. ID No. A), 
Generic Sequence 5 (Seq. ID No. 30) and Generic 

15 Sequence 6 (Seq. ID No. 31), listed below. These 
Generic Sequences accommodate the homologies shared 
among the various preferred members of this raorphogen 
family identified in Table 11, as well as the amino 
acid sequence variation among them. Specifically, 

20 Generic Sequences 3 and 4 are composite amino acid 
sequences of the following proteins presented in 
Table II and identified in Seq. ID Nos. 5-14: human 
OP-1 (hOP"l, Seq. ID Nos. 5 and 16-17), mouse OP-1 
(mOP-1, Seq. ID Nos. 6 and 18-19), human and mouse OP-2 

25 (Seq. ID Nos. 7, 8, and 20-22), CBMP2A (Seq. ID No. 9), 
CBMP2B (Seq. ID No. 10), DPP (from Drosophila, Seq. ID 
No. 11), Vgl, (from Xenopus, Seq. ID No. 12), Vgr-1 
(from mouse, Seq. ID No. 13), and GDF-1 (from mouse, 
Seq. ID No. 14.) The generic sequences include both 

30 the amino acid identity shared by the sequences in 
Table II, as well as alternative residues for the 
variable positions within the sequence- Note that 



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these generic sequences allow for an additional 
cysteine at position 41 or 46 in Generic Sequences 3 or 
4, respectively, providing an appropriate cysteine 
skeleton where inter- or intramolecular disulfide bonds 
5 can formy and contain certain critical amino acids 

which influence the tertiary structure of the proteins. 

Generic Sequence 3 
Leu Tyr Val Xaa Phe 

1 5 

Xaa Xaa Xaa Gly Trp Xaa Xaa Trp Xaa 

10 

Xaa Ala Pro Xaa Gly Xaa Xaa Ala 

15 20 
Xaa Tyr Cys Xaa Gly Xaa Cys Xaa 

25 30 
Xaa Pro Xaa Xaa Xaa Xaa Xaa 

35 

Xaa Xaa Xaa Asn His Ala Xaa Xaa 

40 45 
Xaa Xaa Leu Xaa Xaa Xaa Xaa Xaa 

50 

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys 

55 60 
Cys Xaa Pro Xaa Xaa Xaa Xaa Xaa 
65 



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Xaa Xaa Xaa Leu Xaa Xaa Xaa 

70 75 
Xaa Xaa Xaa Xaa Val Xaa Leu Xaa 
80 

Xaa Xaa Xaa Xaa Met Xaa Val Xaa 

85 90 
Xaa Cys Gly Cys Xaa 
95 

wherein each Xaa is independently selected from a group 
of one or more specified amino acids defined as 
follows: "Res." means "residue" and Xaa at res. 4 = 
(Ser, Asp or Glu); Xaa at res -6 = (Arg, Gin, Ser or 
Lys); Xaa at res.7 = (Asp or Glu); Xaa at res. 8 = (Leu 
or Val); Xaa at res. 11 ^ (Gin, Leu, Asp, His or Asn) ; 
Xaa at res. 12 = (Asp, Arg or Asn); Xaa at res. 14 = (lie 
or Val); Xaa at res. 15 = (He or Val); Xaa at res. 18 = 
(Glu, Gin, Leu, Lys, Pro or Arg); Xaa at res. 20 (Tyr 
or Phe); Xaa at res. 21 = (Ala, Ser, Asp, Met, His, Leu 
or Gin); Xaa at res. 23 = (Tyr, Asn or Phe); Xaa at 
res. 26 = (Glu, His, Tyr, Asp or Gin); Xaa at res. 28 = 
(Glu, Lys, Asp or Gin); Xaa at res. 30 = (Ala, Ser, Pro 
or Gin); Xaa at res. 31 = (Phe, Leu or Tyr); Xaa at 
res. 33 = (Leu or Val); Xaa at res. 34 = (Asn, Asp, Ala 
or Thr); Xaa at res. 35 = (Ser, Asp, Glu, Leu or Ala); 
Xaa at res. 36 = (Tyr, Cys, His, Ser or He); Xaa at 
res. 37 = (Met, Phe, Gly or Leu); Xaa at res. 38 = (Asn 
or Ser); Xaa at res.39 = (Ala, Ser or Gly); Xaa at 
res. 40 = (Thr, Leu or Ser); Xaa at res. 44 = (lie or 
Val); Xaa at res. 45 = (Val or Leu); Xaa at res-46 = 
(Gin or Arg); Xaa at res. 47 = (Thr, Ala or Ser); Xaa at 
res. 49 = (Val or Met); Xaa at res. 50 « (His or Asn); 
Xaa at res. 51 = (Phe, Leu, Asn, Ser, Ala or Val); Xaa 



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PCr/US92/07432 



at res. 52 = (He, Met, Asn, Ala or Val); Xaa at res. 53 
= (Asn, Lys, Ala or Glu); Xaa at res. 54 = (Pro or Ser); 
Xaa at res. 55 = (Glu, Asp, Asn, or Gly); Xaa at res. 5 6 
« (Thr, Ala, Val, Lys, Asp, Tyr, Ser or Ala); Xaa at 
5 res. 57 = (Val, Ala or He); Xaa at res. 58 = (Pro or 
Asp); Xaa at res. 59 - (Lys or Leu); Xaa at res. 60 = 
(Pro or Ala); Xaa at res. 63 = (Ala or Val); Xaa at 
res. 65 = (Thr or Ala); Xaa at res. 66 = (Gin, Lys, Arg 
or Glu); Xaa at res. 67 = (Leu, Met or Val); Xaa at 
10 res. 68 = (Asn, Ser or Asp); Xaa at res. 69 = (Ala, Pro 
or Ser); Xaa at res. 70 = (He, Thr or Val); Xaa at 
res. 71 = (Ser or Ala); Xaa at res. 72 = (Val or Met); 
Xaa at res. 74 = (Tyr or Phe); Xaa at res. 75 = (Phe, Tyr 
or Leu); Xaa at res. 76 = (Asp or Asn); Xaa at res. 77 = 
L5 (Asp, Glu, Asn or Ser); Xaa at res. 78 = (Ser, Gin, Asn 
or Tyr); Xaa at res. 79 = (Ser, Asn, Asp or Glu); Xaa at 
res. 80 = (Asn, Thr or Lys); Xaa at res. 82 = (He or 
Val); Xaa at res. 84 = (Lys or Arg); Xaa at res. 85 = 
(Lys, Asn, Gin or His); Xaa at res. 86 = (Tyr or His); 
0 Xaa at res. 87 = (Arg, Gin or Glu); Xaa at res. 88 = 
(Asn, Glu or Asp); Xaa at res. 90 = (Val, Thr or Ala); 
Xaa at res. 92 = (Arg, Lys, Val, Asp or Glu); Xaa at 
res. 93 = (Ala, Gly or Glu); and Xaa at res. 97 = (His or 
Arg); 

5 

Generic Sequence 4 

Cys Xaa Xaa Xaa Xaa Leu Tyr Val Xaa Phe 
1 5 10 

3 Xaa Xaa Xaa Gly Trp Xaa Xaa Trp Xaa 

15 

Xaa Ala Pro Xaa Gly Xaa Xaa Ala 

20 25 
Xaa Tyr Cys Xaa Gly Xaa Cys Xaa 
•30 35 



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PCT/US92/07432 



Xaa Pro Xaa Xaa Xaa Xaa Xaa 

40 

Xaa Xaa Xaa Asn His Ala Xaa Xaa 
45 50 
Xaa Xaa Leu Xaa Xaa Xaa Xaa Xaa 

55 

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys 

60 65 
Cys Xaa Pro Xaa Xaa Xaa Xaa Xaa 
70 

Xaa Xaa Xaa Leu Xaa Xaa Xaa 

75 80 
Xaa Xaa Xaa Xaa Val Xaa Leu Xaa 
85 

Xaa Xaa Xaa Xaa Met Xaa Val Xaa 

90 95 
Xaa Cys Gly Cys Xaa 
100 

wherein each Xaa is independently selected from a group 
of one or more specified amino acids as defined by the 
following: "Res." means "residue" and Xaa at res. 2 - 
(Lys or Arg); Xaa at res. 3 = (Lys or Arg); Xaa at res. 4 
= (His or Arg); Xaa at res. 5 = (Glu, Ser, His, Gly, Arg 
or Pro); Xaa at res. 9 = (Ser, Asp or Glu); Xaa at 
res. 11 = (Arg, Gin, Ser or Lys); Xaa at res. 12 = (Asp 
or Glu); Xaa at res. 13 = (Leu or Val); Xaa at res. 16 = 
(Gin, Leu, Asp, His or Asn); Xaa at res. 17 = (Asp, Arg, 
or Asn); Xaa at res. 19 = (lie or Val); Xaa at res. 20 = 
(lie or Val); Xaa at res. 23 = (Glu, Gin, Leu, Lys, Pro 
or Arg); Xaa at res. 25 = (Tyr or Phe); Xaa at res. 26 = 
(Ala, Ser, Asp, Met, His, Leu, or Gin); Xaa at res. 28 = 
(Tyr, Asn or Phe); Xaa at res. 31 = (Glu, His, Tyr, Asp 
or Gin); Xaa at res. 33 = Glu, Lys, Asp or Gin); Xaa at 
res. 35 = (Ala, Ser or Pro); Xaa at res. 36 = (Phe, Leu 
or Tyr); Xaa at res. 38 = (Leu or Val); Xaa at res. 39 = 



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(Asn, Asp, Ala or Thr); Xaa at res. 40 = (Ser, Asp, Glu, 
Leu or Ala); Xaa at res. 41 = (Tyr, Cys, His, Ser or 
He); Xaa at res. 42 = (Met, Phe, Gly or Leu); Xaa at 
res. 44 = (Ala, Ser or Gly); Xaa at res. 45 = (Thr, Leu 
5 or Ser); Xaa at res. 49 = (He or Val); Xaa at res. 50 = 
(Val or Leu); Xaa at res. 51 = (Gin or Arg); Xaa at 
res. 52 = (Thr, Ala or Ser); Xaa at res. 54 = (Val or 
Met); Xaa at res. 55 = (His or Asn); Xaa at res. 56 = 
(Phe, Leu, Asn, Ser, Ala or Val); Xaa at res. 57 «= (He, 
10 Met, Asn, Ala or Val); Xaa at res. 58 = (Asn, Lys, Ala 
or Glu); Xaa at res. 59 = (Pro or Ser); Xaa at res. 60 = 
(Glu, Asp, or Gly); Xaa at res. 61 = (Thr, Ala, Val, 
Lys, Asp, Tyr, Ser or Ala); Xaa at res. 62 = (Val, Ala 
or He); Xaa at res. 63 = (Pro or Asp); Xaa at res. 64 = 
15 (Lys or Leu); Xaa at res. 65 = (Pro or Ala); Xaa at 
res. 68 = (Ala or Val); Xaa at res. 70 = (Thr or Ala); 
Xaa at res. 71 = (Gin, Lys, Arg or Glu); Xaa at res. 72 = 
(Leu, Met or Val); Xaa at res. 73 = (Asn, Ser or Asp); 
Xaa at res. 74 = (Ala, Pro or Ser); Xaa at res. 75 = 
20 (He, Thr or Val); Xaa at res. 76 = (Ser or Ala); Xaa at 
res. 77 = (Val or Met); Xaa at res. 79 = (Tyr or Phe); 
Xaa at res. 80 = (Phe, Tyr or Leu); Xaa at res. 81 = (Asp 
or Asn); Xaa at res. 82 = (Asp, Glu, Asn or Ser); Xaa at 
res. 83 = (Ser, Gin, Asn or Tyr); Xaa at res. 84 = (Ser, 
25 Asn, Asp or Glu); Xaa at res. 85 = (Asn, Thr or Lys); 
Xaa at res. 87 = (He or Val); Xaa at res. 89 = (Lys or 
Arg); Xaa at res. 90 = (Lys, Asn, Gin or His); Xaa at 
res. 91 = (Tyr or His); Xaa at res. 92 = (Arg, Gin or 
Glu); Xaa at res. 93 = (Asn, Glu or Asp); Xaa at res. 95 
30 = (Val, Thr or Ala); Xaa at res. 97 = (Arg, Lys, Val, 
Asp or Glu); Xaa at res. 98 = (Ala, Gly or Glu); and Xaa 
at res. 102 = (His or Arg). 



wo 93/05751 



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Similarly, Generic Sequence 5 (Seq. ID No. 30) and 
Generic Sequence 6 (Seq. ID No. 31) accommodate the 
homologies shared among all the morphogen protein 
family members identified in Table II. Specifically/ 
5 Generic Sequences 5 and 6 are composite amino acid 
sequences of human OP-1 (hOP-l, Seq. ID Nos. 5 and 16- 
17), mouse OP-1 (mOP-l, Seq. ID Nos. 6 and 18-19), 
human and mouse OP-2 (Seq. ID Nos. 7, 8, and 20-22), 
CBMP2A (Seq. ID No. 9), CBMP2B (Seq. ID No. 10), DPP 
10 (from Drosophila, Seq. ID No. 11), Vgl, (from Xenopus, 
Seq. ID No. 12), Vgr-1 (from mouse, Seq. ID No. 13), 
and GDF-1 (from mouse, Seq. ID No. 14 and 32), human 
BMP3 (Seq. ID No. 26), human BMP5 (Seq. ID No. 27), 
human BMP6 (Seq. ID No. 28) and 60A (from Drosophila, 
15 Seq. ID No. 24). The generic sequences include both 
the amino acid identity shared by these sequences in 
the C-terminal domain, defined by the six and seven 
cysteine skeletons (Generic Sequences 5 and 6, 
respectively), as well as alternative residues for the 
20 variable positions within the sequence. As for Generic 
Sequences 3 and 4, Generic Sequences 5 and 6 allow for 
an additional cysteine at position 41 (Generic Sequence 
5) or position 46 (Generic Sequence 6), providing an 
appropriate cysteine skeleton where inter- or 
25 intramolecular disulfide bonds can form, and containing 
certain critical amino acids which influence the 
tertiary structure of the proteins. 



30 



Generic Sequence 5 

Leu Xaa Xaa Xaa Phe 
1 5 
Xaa Xaa Xaa Gly Trp Xaa Xaa Trp Xaa 

10 



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PCr/US92/07432 



Xaa Xaa Pro Xaa Xaa Xaa Xaa Ala 

15 20 
Xaa Tyr Cys Xaa Gly Xaa Cys Xaa 
25 30 
5 Xaa Pro Xaa Xaa Xaa Xaa Xaa 

35 

Xaa Xaa Xaa Asn His Ala Xaa Xaa 

40 45 
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 
10 50 

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys 

55 60 . 

Cys Xaa Pro Xaa Xaa Xaa Xaa Xaa 
65 ' 

15 Xaa Xaa Xaa Leu Xaa Xaa Xaa 

70 75 
Xaa Xaa Xaa Xaa Val Xaa Leu Xaa 
80 

Xaa Xaa Xaa Xaa Met Xaa Val Xaa 
20 85 90 

Xaa Cys Xaa Cys Xaa 
95 

wherein each Xaa is independently selected from a group 
of one or more specified amino acids defined as 
25 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 Glu); Xaa at res. 6 = (Arg, Gin, Ser, Lys 
or Ala); Xaa at res. 7 = (Asp, Glu or Lys); Xaa at res. 8 



wo 93/0S751 



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PCT/US92/07432 



(Leu, Val or He); Xaa at res. 11 « (Gin, Leu, Asp, 
His, Asn or Ser); Xaa at res. 12 = (Asp, Arg, Asn or 
Glu); Xaa at res. 14 = (lie or Val); Xaa at res. 15 = 
(He or Val); Xaa at res. 16 (Ala or Ser); Xaa at res. 18 
5 = (Glu, Gin, Leu, Lys, Pro or Arg); Xaa at res. 19 = 
(Gly or Ser); Xaa at res. 20 = (Tyr or Phe); Xaa at 
res. 21 = (Ala, Ser, Asp, Met, His, Gin, Leu or Gly); 
Xaa at res. 23 = (Tyr, Asn or Phe); Xaa at res. 26 = 
(Glu, His, Tyr, Asp, Gin or Ser); Xaa at res. 28 = (Glu, 
10 Lys, Asp, Gin or Ala); Xaa at res. 30 = (Ala, Ser, Pro, 
Gin or Asn); Xaa at res. 31 = (Phe, Leu or Tyr); Xaa at 
res. 33 = (Leu, Val or Met); Xaa at res. 34 = (Asn, Asp, 
Ala, Thr or Pro); Xaa at res. 3 5 = (Ser, Asp, Glu, Leu, 
Ala or Lys); Xaa at res. 36 = (Tyr, Cys, His, Ser or 
15 He); 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 
or He); Xaa at res. 46 = (Gin or Arg); Xaa at res.47 = 
20 (Thr, Ala or Ser); Xaa at res. 48 = (Leu or He); Xaa at 
res. 49 = (Val or Met); Xaa at res.50 = (His, Asn or 
Arg); Xaa at res. 51 = (Phe, Leu, Asn, Ser, Ala or Val); 
Xaa at res. 52 = (He, Met, Asn, Ala, Val or Leu); Xaa 
at res -53 = (Asn, Lys, Ala, Glu, Gly or Phe); Xaa at 
25 res. 54 - (Pro, Ser or Val); Xaa at res. 55 = (Glu, Asp, 
Asn, Gly, Val or Lys); Xaa at res. 56 = (Thr, Ala, Val, 
Lys, Asp, Tyr, Ser, Ala, Pro or His); Xaa at res. 57 = 
(Val, Ala or He); Xaa at res. 58 = (Pro or Asp); Xaa at 
res. 59 = (Lys, Leu or Glu); Xaa at res. 60 = (Pro or 
30 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 = (He, Thr, Val or Leu); Xaa at 
35 res. 71 = (Ser, Ala or Pro); Xaa at res. 72 = (Val, Met 



wo 93/05751 



27 - 



PCr/US92/07432 



or He); Xaa at res. 74 = (Tyr or Phe); Xaa at res. 75 = 
(Phe, Tyr, Leu or His); Xaa at res. 76 = (Asp, Asn or 
Leu); Xaa at res. 77 « (Asp, Glu, Asn or Ser); Xaa at 
res. 78 » (Ser, Gin, Asn, Tyr or Asp); Xaa at res. 79 = 
(Ser, Asn, Asp, Glu or Lys); Xaa at res. 80 » (Asn, Thr 
or Lys); Xaa at res. 82 = (He, Val or Asn); Xaa at 
res. 84 = (Lys or Arg); Xaa at res. 85 = (Lys, Asn, Gin, 
His or Val); Xaa at res. 86 = (Tyr or His); Xaa at 
res. 87 = (Arg, Gin, Glu or Pro); Xaa at res. 88 = (Asn, 
Glu or Asp); Xaa at res. 90 = (Val, Thr, Ala or He); 
Xaa at res. 92 = (Arg, Lys, Val, Asp or Glu); Xaa at 
res. 9 3 = (Ala, Gly, Glu or Ser); Xaa at res. 95 = (Gly 
or Ala) and Xaa at res. 97 = (His or Arg). 

Generic Sequence 6 

Cys Xaa Xaa Xaa Xaa Leu Xaa Xaa Xaa Phe 
1 5 10 

Xaa Xaa Xaa Gly Trp Xaa Xaa Tit> Xaa 

15 

Xaa Xaa Pro Xaa Xaa Xaa Xaa Ala 

20 25 
Xaa Tyr Cys Xaa Gly Xaa Cys Xaa 
30 35 
Xaa Pro Xaa Xaa Xaa Xaa Xaa 

40 

Xaa Xaa Xaa Asn His Ala Xaa Xaa 
4 5 50 
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 

55 

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys 

60 65 
Cys Xaa Pro Xaa Xaa Xaa Xaa Xaa 
70 



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PCr/US92/07432 



Xaa Xaa Xaa Leu Xaa Xaa Xaa 

75 80 
Xaa Xaa Xaa Xaa Val Xaa Leu Xaa 
85 

Xaa Xaa Xaa Xaa Met Xaa Val Xaa 

90 95 
Xaa Cys Xaa Cys Xaa 
100 



wherein each Xaa is independently selected from a group 
of one or more specified amino acids as defined by the 
following: "Res." means "residue" and Xaa at res. 2 = 
(Lys, Arg/ Ala or Gin); Xaa at res. 3 = (Lys, Arg or 
Met); Xaa at res. 4 = (His, Arg or Gin); Xaa at res. 5 = 
(Glu/ Ser, His, Gly, Arg, Pro, Thr, or Tyr); Xaa at 
res. 7 (Tyr or Lys); Xaa at res. 8 = (Val or He); Xaa 
at res. 9 = (Ser, Asp or Glu); Xaa at res .11 = (Arg, 
Gin, Ser, Lys or Ala); Xaa at res. 12 = (Asp, Glu, or 
Lys); Xaa at res. 13 = (Leu, Val or He); Xaa at res. 16 
= (Gin, Leu, Asp, His, Asn or Ser); Xaa at res. 17 = 
(Asp, Arg, Asn or Glu); Xaa at res. 19 = (He or Val); 
Xaa at res. 20 = (He or Val); Xaa at res. 21 - (Ala or 
Ser); Xaa at res-23 = (Glu, Gin, Leu, Lys, Pro or Arg); 
Xaa at res. 24 = (Gly or Ser); Xaa at res. 25 = (Tyr or 
Phe); Xaa at res,26 = (Ala, Ser, Asp, Met, His, Gin, 
Leu, or Gly); Xaa at res. 28 = (Tyr, Asn or Phe); Xaa at 
res. 31 = (Glu, His, Tyr, Asp, Gin or Ser); Xaa at 
res. 33 = Glu, Lys, Asp, Gin or Ala); Xaa at res. 35 = 
(Ala, Ser, Pro, Gin or Asn); Xaa at res. 36 = (Phe, Leu 
or Tyr); Xaa at res. 38 = (Leu, Val or Met); Xaa at 
res. 39 = (Asn, Asp, Ala, Thr or Pro); Xaa at res. 40 = 
(Ser, Asp, Glu, Leu, Ala or Lys); Xaa at res. 41 = (Tyr, 
Cys, His, Ser or He); Xaa at res. 42 = (Met, Phe, Gly 
or Leu); Xaa at res. 43 = (Asn, Ser or Lys); Xaa at 
res. 44 = (Ala, Ser, Gly or Pro); Xaa at res. 45 = (Thr, 



wo 93/05751 PCT/US92/07432 

- 29 " 



Leu or Ser); Xaa at res. 4 9 = (He, Val or Thr); Xaa at 
res. 50 = (Val^ Leu or lie); Xaa at res. 51 = (Gin or 
Arg); Xaa at res. 52 «= (Thr, Ala or Ser); Xaa at res. 53 
= (Leu or lie); Xaa at res. 54 = (Val or Met); Xaa at 
5 res. 55 = (His, Asn or Arg); Xaa at res. 56 = (Phe, Leu, 
Asn, Ser, Ala or Val); Xaa at res. 57 = (lie, Met^ Asn, 
Ala, Val or Leu); Xaa at res. 58 = (Asn, Lys, Ala, Glu, 
Gly or Phe); Xaa at res. 59 = (Pro, Ser or Val); Xaa at 
res. 60 = (Glu, Asp, Gly, Val or Lys); Xaa at res. 61 
10 (Thr, Ala, Val, Lys, Asp, Tyr, Ser, Ala, Pro or His); 
Xaa at res. 62 = (Val, Ala or He); Xaa at res. 63 = (Pro 
or Asp); Xaa at res. 64 = (Lys, Leu or Glu); Xaa at 
res. 65 = (Pro or Ala); Xaa at res. 68 = (Ala or Val); 
Xaa at res. 70 = (Thr, Ala or Glu); Xaa at res. 71 = 
15 (Gin, Lys, Arg or Glu); Xaa at res. 72 = (Leu, Met or 
Val); Xaa at res. 73 = (Asn, Ser, Asp or Gly); Xaa at 
res. 74 = (Ala, Pro or Ser); Xaa at res. 75 = (He, Thr, 
Val or Leu); Xaa at res. 76 = (Ser, Ala or Pro); Xaa at 
res.77 = (Val, Met or He); Xaa at res. 79 = (Tyr or 
20 Phe); Xaa at res. 80 = (Phe, Tyr, Leu or His); Xaa at 
res. 81 = (Asp, Asn or Leu); Xaa at res. 82 = (Asp, Glu, 
Asn or Ser); Xaa at res. 83 = (Ser, Gin, Asn, Tyr or 
Asp); Xaa at res. 84 = (Ser, Asn, Asp, Glu or Lys); Xaa 
at res. 85 = (Asn, Thr or Lys); Xaa at res. 87 = (He, 
25 Val or Asn); Xaa at res. 89 = (Lys or Arg); Xaa at 

res. 90 = (Lys, Asn, Gin, His or Val); Xaa at res. 91 = 
(Tyr or His); Xaa at res. 92 = (Arg, Gin, Glu or Pro); 
Xaa at res. 93 = (Asn, Glu or Asp); Xaa at res. 95 = 
(Val, Thr, Ala or He); Xaa at res. 97 ~ (Arg, Lys, Val, 
30 Asp or Glu); Xaa at res. 98 = (Ala, Gly, Glu or Ser); 
Xaa at res.iOO = (Gly or Ala); and Xaa at r^s.102 = 
(His or Arg) . 



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Particularly useful sequences for use as morphogens 
in this invention include the C-terminal domains, e.g./ 
the C-terminal 96-102 amino acid residues of Vgl, 
Vgr-1, DPP, OP-1, OP-2/ CBMP-2A, CBMP-2Br GDF-1 (see 
Table II/ below, and Seq. ID Nos. 5-14), as well as 
proteins comprising the C-terminal domains of 60A, 
BMP3, BMP5 and BMP6 (see Table 11, below,, and Seq. ID 
Nos. 24-28)/ all of which include at least the 
conserved six or seven cysteine skeleton. In addition, 
biosynthetic constructs designed from the generic 
sequences, such as COP-1, 3-5, 7, 16, disclosed in U.S. 
Pat. No. 5,011,691, also are useful. Other sequences 
include the inhibins/activin proteins (see, for 
example, U.S. Pat. Nos. 4,968,590 and 5,011,691). 
Accordingly, other useful sequences are those sharing 
at least 70% amino acid sequence homology or 
"similarity", and preferably 80% homology or similarity 
with any of the sequences above. These are anticipated 
to' include allelic and species variants and mutants , 
and biosynthetic muteins, as well as novel members of 
this morphogenic family of proteins. Particularly 
envisioned in the family of related proteins are those 
proteins exhibiting morphogenic activity and wherein 
the amino acid changes from the preferred sequences 
include conservative changes, e.g., those as defined by 
Dayoff et al.. Atlas of Protein Sequence and Structure ; 
vol. 5, Suppl. 3, pp. 345-362, (M.O. Dayoff, ed., Nat'l 
BioMed. Research Fdn., Washington, D.C. 1979). As used 
herein, potentially useful sequences are aligned with a 
known morphogen sequence using the method of Needleman 
et al. {(1970) J,Mol.Biol. 48;443-453) and identities 
calculated by the Align program (DNAstar, Inc.). 
"Homology" or "similarity" as used herein includes 
allowed conservative changes as defined by Dayoff et 
al. 



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- 31 - 



The currently most preferred protein sequences 
useful as raorphogens in this invention include those 
having greater than 60% identity, preferably greater 
than 65% identity, with the amino acid sequence 
5 defining the conserved six cysteine skeleton of hOPl 
(e.g., residues 43-139 of Seq. ID No. 5). These most 
preferred sequences include both allelic and species 
variants of the OP-1 and OP-2 proteins, including the 
Drosophila 60A protein. Accordingly, in another 

10 preferred aspect of the invention, useful morphogens 
include active proteins comprising species of 
polypeptide chains having the generic amino acid 
sequence herein referred to as "OPX", which 
accommodates the homologies between the various 

15 identified species of OPl and 0P2 (Seq. ID No. 29). 

The morphogens useful in the methods, 
composition and devices of this invention include 
proteins comprising any of the polypeptide chains 

20 described above, whether isolated from naturally- 
occurring sources, or produced by recombinant DNA or 
other synthetic techniques, and includes allelic and 
species variants of these proteins, naturally-occurring 
or biosynthetic mutants thereof, as well as various 

25 truncated and fusion constructs. Deletion or addition 
mutants also are envisioned to be active, including 
those which may alter the conserved C-terminal cysteine 
skeleton, provided that the alteration does not 
functionally disrupt the relationship of these 

30 cysteines in the folded structure. Accordingly, such 
active forms are considered the equivalent of the 
specifically described constructs disclosed herein. 
The proteins may include forms having varying 
glycosylation patterns, varying N- termini, a family of 

35 related proteins having regions of amino acid sequence 



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PCr/US92/07432 



homology/ and active truncated or mutated forms of 
native or biosynthetic proteins, produced by expression 
of recombinant DNA in host cells. 

The morphogenic proteins can be expressed from 
intact or truncated cDNA or from synthetic DNAs in 
procaryotic or eucaryotic host cells, and purified, 
cleaved, refolded, and dimerized to form 
morphogenically active compositions. Currently 
preferred host cells include coli or mammalian 
cells/ such as CHO, COS or BSC cells. A detailed 
description of the morphogens useful in the methods and 
compositions of this invention is disclosed in 
copending US patent application Serial Nos. 752,764, 
filed August 30 ^ 1991, and 667,274, filed March 11, 
1991, the disclosures of which are incorporated herein 
by reference. 

Thus, in view of this disclosure, skilled 
genetic engineers can isolate genes from cDNA or 
genomic libraries of various different species which 
encode appropriate amino acid sequences, or construct 
DNAs from oligonucleotides, and then can express them 
in various types of host cells, including both 
procaryotes and eucaryotes, to produce large quantities 
of active proteins capable of enhancing bone formation 
and/or inhibiting abnormal bone deterioration in a 
variety of mammals, including humans, for use in 
maintaining appropriate bone mass and bone remodeling 
in developing and adult bone tissue. 



wo 93/05751 PCr/US92/07432 

- 33 - 



Brief Description of the Drawings 

The foregoing and other objects and features of 
this invention, as well as the invention itself, may be 
more fully understood from the following description/ 
when read together with the accompanying drawings, in 
which: 

FIG. 1 compares the mitogenic effect of hOP-1 and 
TGF-B on rat osteoblasts; 

FIG. 2 illustrates the effect of human osteogenic 
protein-1 (hOP-1) on the collagen synthesis of 
osteoblasts; 

FIG. 3 compares the alkaline phosphatase induction 
effect of hOP-1 and TGF-B on rat osteoblasts; 

FIG. 4 shows the long-term effect of hOP-1 on the 
production of alkaline posphatase by rat osteoblasts; 

FIG. 5 shows the effect of hOP-1 on parathyroid 
hormone-mediated cAMP production using rat osteoblasts 
in culture; 

FIG. 6A and B graphs the effect of morphogen on 
osteoclacin synthesis (A), and the effect of morphogen 
on the rate of mineralization (B); 

FIG- 7 shows Western Blot analysis of bovine 
colostrum using OP-1 and BMP2-specif ic antibodies; 



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PCr/US92/07432 



FIG. 8A and B show results of in vivo and in vitro 
activity assays, respectively/ for manunary extract 
purified OP-1; 

5 FIG. 9 is a photomicrograph of an immunoblot 

showing the presence of hOP-1 in serum; and 

FIG. 10 (A and B) are photomicrographs showing new 
endosteum bone formation following morphogen injection 
10 onto the endosteal surface (A) , and new periosteum bone 
formation following morphogen injection onto the 
periosteal surface (B); 

FIG. 11 is a graphic representation of the dose- 
15 dependent effect of morphogen on bone resorption; and 

FIG. 12 (A and B) are schematic representations of 
morphogen inhibition of early mononuclear phagocytic 
cell multinuclearization in vivo ; 



wo 93/05751 



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PCT/IIS92/07432 



Detailed Description of the Invention 

It now has been discovered that the proteins 
described herein are effective agents for preventing 
5 loss of bone mass and/or for stimulating bone formation 
when provided systemically or injected directed into 
bone tissue in a mammal. As described herein, these 
proteins ( "morphogens " ) may be used in the treatment of 
metabolic bone diseases and other disorders that cause 
10 an imbalance of the bone remodeling cycle, and/or which 
cause deterioration of the skeletal microstructure. 

The invention is based on the discovery of a family 
of morphogenic proteins capable of inducing tissue 
15 morphogenesis in a mammal. More particularly, the 

invention is based on the discovery that these proteins 
play an important role, not only in embryogenesis, but 
also in the growth, maintenance and repair of bone 
tissue in juvenile and adult mammals. 

20 

It has been shown that implantation of a morphogen 
(including OP" 1, CBMP2, DPP and 60A protein, and 
various biosynthetic constructs, such as C0P5 and C0P7) 
together with a suitable matrix in subcutaneous sites 

25 in mammals induces a sequence of cellular events which 
leads to the formation of fully functional new bone, as 
determined by the specific activity of alkaline 
phosphatase, calcium content and histology of day 12 
implants (see, for example, U.S. Pat. Nos. 4,968,590 

30 and 5,011,691, and USSN 667,274 and 752,857, the 
disclosures of which are incorporated herein by 
reference. ) The morphogen-containing implants recruit 
nearby mesenchymal stem cells and trigger their 
differentiation into chondrocytes within 5-7 days. 

35 Upon capillary invasion, the chondrocytes hypertrophy. 



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PCr/US92/07432 



become calcified and subsequently are replaced by newly 
formed bone within 9-12 days. The mineralized bone 
then is remodeled extensively and becomes occupied by 
ossicles filled with functional bone marrow elements by 
5 14-21 days* 

As described herein^ the morphogens provided herein 
stimulate the proliferation, growth and differentiation 
of osteoblasts in vitro (see Examples 2-7, below), and 

10 can induce bone formation in osteoporotic bone tissue 
in vivo when provided systemically to a mammal, or 
directly to bone tissue, without an associated matrix 
carrier (see Examples 8, 9, below.) In addition, the 
morphogens inhibit multinucleation of activated early 

15 mononuclear phagocytic cells (see Example 12, below). 
Moreover, inhibition of endogenous morphogen activity 
can inhibit normal skeleton development in a mammal 
(see Example 13, below.) 

20 As described in Example 1 and in detail in 

copending USSN 752,764 and 752,861, the disclosures of 
which are incorporated herein by reference, the 
naturally-occurring morphogens are widely distributed 
in the different tissues of the body. For example, as 

25 determined by northern blot hybridization, OP-1 is 
expressed primarily in the tissue of the urogental 
tract (e.g., renal and bladder tissues). By contrast, 
Vgr-1, BMP3, BMP4 and BMP5 appear to be expressed 
primarily in the heart and lung. BMP5 also appears to 

30 be expressed significantly in liver tissue. GDF-1 
appears to be expressed primarily in brain tissue. 
(See, for example, Ozkaynak et al. (1992) JBC, in 
publication.) Moreover, the tissue of synthesis may 
differ from the natural site of action of specific 

35 morphogens. For example, although OP-1 appears to be 



wo 93/05751 PCr/US92/07432 

- 37 - 



primarily synthesized in renal tissue , the protein is 
active in bone tissue. In addition, at least one 
morphogen, OP-1, is present in a number of body fluids, 
including saliva, milk (including mammary gland 
5 extract/ colostnim and 5 7 -day milk) and serum (see 

Example 11, below.) Accordingly, without being limited 
to a given theory, the morphogens described herein may 
behave as endocrine factors, e.g., proteins secreted 
from a factor-producing tissue in response to 

10 particular stimuli, and capable of being transported 
to, and acting on, a distant tissue. These findings 
further distinguish morphogens from other members of 
the TGF"P superfamily of proteins, including TGF-p, 
which act as local or autocrine factors produced by the 

15 tissue on which they act. 

The pro domain may function to enhance protein 
solubility and/or to assist in tissue targeting of 
morphogens to particular tissues. For example, the 

20 mature, active form of OP-1 appears to be secreted from 
cells in association with the pro domain of the intact 
sequence. Accordingly, while, as explained herein, the 
morphogens useful in this invention have significant 
amino acid sequence homologies within the active 

25 domains and are similar in their ability to induce 
tissue morphogenesis, without being limited to any 
theory, it is hypothesized that the sequence variation 
within the morphogenic protein family members may 
reflect the different specific roles each morphogen 

30 plays in specific tissues under natural occurring 
conditions. For example, the significant sequence 
variation within the pro domains may mean that these 
regions of the protein sequence are important for 
targeting specific morphogens to different tissues for 

35 morphogenic activity therein. 



wo 93/05751 PCr/US92/07432 

- 38 - 



Accordingly/ the present invention comprises two 
fundamental aspects* In one aspect/ the methods and 
compositions of this invention comprise a morphogen 
which^ when administered to an individual/ is capable 
5 of inhibiting loss of bone mass and/or stimulating bone 
formation in the individual. In another aspect, the 
methods and compositions of the invention comprise a 
morphogen-stimulating agent which/ when administered to 
an individual, is capable of inducing the expression 
10 and/or secretion of sufficient endogenous morphogen 
within the individual to provide therapeutically 
effective concentrations capable of inhibiting loss of 
bone mass and/or stimulating bone formation dLn the 
individual . 

15 

Example 14 describes an assay for screening 
compounds to identify candidate morphogen-stimulating 
agents. A detailed description of useful screening 
assays for identifying candidate morphogen-stimulating 
20 agents also is provided in USSN 752/861/ the disclosure 
of which is incorporated herein by reference. 
Candidate agents then may be tested for their efficacy 
in vivo using, for example/ the osteoporosis model 
described in Examples 8 and 9 below • 

25 

Provided below are detailed descriptions of 
suitable morphogens useful in the methods and 
compositions of this invention, as well as methods for 
the administration and application of these morphogens 
30 and/or of morphogen-stimulating agents. Also provided 
are numerous, nonlimiting examples which 1) illustrate 
the suitability of the morphogens and morphogen- 



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PCT/US92/07432 



stimulating agents described herein as therapeutic 
agents for inhibiting abnormal bone loss and/or for 
enhancing bone formation in a human, and 2) provide 
assays with which to test candidate morphogens and 
5 morphogen-stiraulating agents for their efficacy* 

I. Useful Morphogens 

10 As defined herein a protein is morphogenic if 

it is capable of inducing the developmental cascade of 
cellular and molecular events that culminate in the 
formation of new, organ-specific tissue and comprises 
at least the conserved C-terminal six cysteine skeleton 
15 or its functional equivalent (see supra). 

Specifically, the morphogens generally are capable of 
all of the following biological functions in a 
morphogenically permissive environment: stimulating 
proliferation of progenitor cells; stimulating the 
20 differentiation of progenitor cells; stimulating the 
proliferation of differentiated cells; and supporting 
the growth and maintenance of differentiated cells, 
including the "redif ferentiation*' of transformed cells. 
Details of how the morphogens useful in the method of 
25 this invention first were identified, as well as a 
description on how to make, use and test them for 
morphogenic activity are disclosed in USSN 667,274, 
filed March 11, 1991 and USSN 752,764, filed August 30, 
1991, the disclosures of which are hereby incorporated 
30 by reference. As disclosed therein, the morphogens may 
be purified from naturally-sourced material or 
recombinantly produced from procaryotic or eucaryotic 



wo 93/05751 



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PCr/US92/07432 



host cells, using the genetic sequences disclosed 
therein. Alternatively, novel morphogenic sequences 
may be identified following the procedures disclosed 
therein. 

5 

Particularly useful proteins include those which 
comprise the naturally derived sequences disclosed in 
Table II. Other useful sequences include biosynthetic 
constructs such as those disclosed in U.S. Pat. 
10 5,011,691, the disclosure of which is incorporated 

herein by reference (e.g., COP-1, COP-3, COP-4, COP-5, 
COP-7, and COP-16) . 

Accordingly, the morphogens useful in the methods 
15 and compositions of this invention also may be 

described by morphogenically active proteins having 
amino acid sequences sharing 70% or, preferably, 80% 
homology (similarity) with any of the sequences 
described above, where "homology" is as defined herein 
20 above. 



The morphogens useful in the method of this 
invention also can be described by any of the 6 generic 
sequences described herein (Generic Sequences 1, 2, 3, 
25 4, 5 and 6) . Generic sequences 1 and 2 also may 
include, at their N-terminus, the sequence 



Cys Xaa Xaa Xaa Xaa (Seq. ID No. 15) 
1 5 

30 

Table II, set forth below, compares the amino acid 
sequences of the active regions of native proteins that r 
have been identified as morphogens, including human 
OP-I (hOP-1, Seq. ID Nos. 5 and 16-17), mouse OP-1 
35 (mOP"l, Seq. ID Nos. 6 and 18-19), human and mouse OP-2 



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PCT/US92/07432 



(Seq. ID Nos. 1, 8, and 20-23), CBMP2A (Seq. ID No. 9), 
CBMP2B (Seq. ID No. 10), BMP3 (Seq. ID No. 26), DPP 
(from Drosophila, Seq. ID No. 11), Vgl, (from Xenopus, 
Seq. ID No. 12), Vgr-1 (from mouse, Seq. ID No. 13), 
5 GDF-1 (from mouse, Seq. ID No. 14, 32 and 33), 60A 

protein (from Drosophila, Seq. ID Nos. 24 and 25), BMP5 
(Seq. ID No. 27) and BMP6 (Seq. ID No. 28). The 
sequences are aligned essentially following the method 
of Needleman et al. (1970) J. Mol. Biol. . 48:443-453, 

10 calculated using the Align Program (DNAstar, Inc.) In 
the table, three dots indicates that the amino acid in 
that position is the same as the amino acid in hOP-1. 
Three dashes indicates that no amino acid is present in 
that position, and are included for purposes of 

15 illustrating homologies. For example, amino acid 
residue 60 of CBMP-2A and CBMP-2B is "missing". Of 
course, both these amino acid sequences in this region 
comprise Asn-Ser (residues 58, 59), with CBMP-2A then 
comprising Lys and lie, whereas CBMP-2B comprises Ser 

20 and lie. 



TABLE II 



25 



30 



35 



hOP-1 

mOP-l 

hOP-2 

inOP-2 

DPP 

Vgl 

Vgr-1 

CBMP-2A 

CBMP-2B 

BMP3 



Cys Lys Lys His Glu Leu Tyr Val 



Arg Arg 
Arg Arg 
Arg Arg 

Lys Arg 

Arg 

Arg Arg 

Ala Arg Arg 



Ser 
His 
Gly 
Pro 
Ser 
Tyr 



Lys 



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PCr/US92/07432 



20 



25 



GDF-1 


* • • 


Arg 


Ala 


Arg 


Arg 






... 




60A 




Gin 


Met 


Glu 


Thr 


::: 




... 




BMP5 


W m m 




• . • 






... 




... 




BMP6 




Arg 






... 


... 


... 








1 








5 










hOP-1 


Ser 


Phe 


Arg 


Asp 


Leu 


Gly 


Trp 


Gin 


Asp 


mOP-l 


* • • 






— 








... 


... 


hOP-2 






Gin 


... 




— 




Leu 




raOP-2 


Ser 




... 


... 


... 






Leu 




DPP 


Asp 




Ser 




Val 




... 


Asp 




Vgl 


Glu 




Lys 


... 


Val 




... 




Ash 


Vgr-1 






Gin 




Val 










CBHP-2A 


Asp 


... 


Ser 


... 


Val 






Asn 


— 


CBMP-2B 


Asp 




Ser 




Val 






Asn 






Asp 




Ala 




He 






Ser 


Glu 


GDF-1 








Glu 


Val 


.... 


... 


His 


Arg 


60A 


Asp 


... 


Lys 










His 


— 


BMP5 




... 
















BMP6 






Gin 










... 


— 






10 










15 






hOP-1 


Trp 


He 


He 


Ala 


Pro 


Glu 


Gly 


Tyr 


Ala 


mOP-1 










— 










hOP-2 




Val 




... 


... 


Gin 






Ser 


mOP-2 




Val 






... 


Gin 






Ser 


DPP 






Val 






Leu 






Asp 


Vgl 




Val 








Gin 






Met 


Vgr-1 












Lys 








CBMP-2A 






Val 






Pro 






His 


CBHP-2B 






Val 






Pro 






Gin 


BMP3 








Ser 




Lys 


Ser 


Phe 


Asp 



WO93/0S751 



- 43 - 



PCr/US92/07432 



GDF-l 
60A 
BMP5 
BMP6 



Val 



Arg 



Lys 



Phe Leu 
Gly 









ZU 










25 




nur- 1 


Ala 


Tyr 


Tyr 


Cys 


Glu 


Gly 


Glu 


Cys 


Ala 


mur- i 






• • * 


" " • 


* • • 


• • . 


... 






nur— z 




« « • 


• • • 


• • • 


• • " 


• . • 


. • * 




Ser 


niUi — z 






• • • 


• « * 




• • • 


. • • 






npp 










His 




Lys 




Pro 


Vgl 




Asn 


* « • 




Tyr 




■ • ♦ ♦ 


• • • 


Fro 


Vgr-l 




Asn 


... 




Asp 


... 






Ser 


Li5nr~ZA 


• • a 


Phe 


... 


• ♦ • 


His 




Glu 


* • • 


Pro 


CBMP-2B 


" ■ * 


Phe 


• • . 


• « • 


His 




Asp 




Pro 








... 


... 


Ser 


... 


Ala 


... 


Gin 


GDF-l 




Asn 




... 


bin 


• • ♦ 


Gin 


• • • 


• • • 






PKa 


"■ 


• * • 


Ser 


• • • 


" • • 


• • • 


Asn 


RMP^ 




rne 




• • * 


Asp 


... 


... 




Ser 


RHP A 




Asn 






Asp 


... 


. • • 


... 


Ser 










OA 










35 


nur — 1 


rne 


rro 


Leu 


Asn 


Ser 


Tyr 


Met 


Asn 


Ala 


mOP-l 




















hop- 2 








Asp 




Cys 




... 




niOP-2 








Asp 




Cys 








DPP 








Ala 


Asp 


His 


Phe 




Ser 


Vgl 


Tyr 






Thr 


Glu 


lie 


Leu 




Gly 


Vgr-l 










Ala 


His 








CBMP-2A 








Ala 


Asp 


His 


Leu 




Ser 


CBHP-2B 








Ala 


Asp 


His 


Leu 




Ser 


GDF-l 


Leu 




Val 


Ala 


Leu 


Ser 


Gly 


Ser** 





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- 44 - 



PCr/US92/07432 





BHP3 




... 


Met 


Pro 


Lys 


Ser 


Leu 


Lys 


Pro 






60A 


• • * 


• • • 


... 




A J. a 














BHP5 


• • • 


■ • • 


• • * 


. • • 


Ala 

Axa 


fT*! c 

nis 


Mot* 










BMP 6 


* • • 


• * • 


... 


• . « 


Ala 


nis 


nex. 








5 












An 

Hi) 














nur— i 


XXI XT 


A cn 






lie 


Val 


Gin 


Thr 


Leu 






mUr-l 
























hOP-2 


• " • 


• • • 


* • » 


* * * 




Leu 




OCX 






10 


0OP-2 


• • • 


• • ♦ 


.* * ' 


• . • 




Leu 


• * • 










DPP 


• • • 


• ' • 


» • • 


• • • 


Val 




... 










Vgl 


Ser 


• • ■ 


• • . 






Leu 












Vgr-1 






... 


... 
















CBMP-2A 


• • * 


... 


... 


... 














15 


CBHP-2B 


















« * • 






BMP3 


Ser 


... 


... 




Thr 


He 




Ser 


He 






GDF-1 


Leu 








Val 


Leu 


Arg 


A 1 ^ 

Axa 








60A 






... 


... 
















BMP5 


• • • 


" • ■ 


. • • 




... 












20 


BMP6 






... 


... 


















45 






















hOP-1 


1 

vai 


nlS 


rne 




Asn 


r ro 




1,111. 


V ox 




25 


raOP-1 


* • * 












A cr\ 
AO p 










hOP-2 


• • • 


His 


Leu 


net 


Lys 


... 


Asn 


Ala 

A. J. a 








raOP-2 


• * • 


His 


Leu 


Met 


Lys 




Asp 


vax 








DPP 




Asn 


Asn 


Asn 






Giy 


Lys 








Vgl 






Ser 




Glu 






Asp 


He 




30 


Vgr-1 






Val 


Met 








Tyr 








CBMP-2A 




Asn 


Ser 


Val 




Ser 




Lys 


He 






CBMP-2B 




Asn 


Ser 


Val 




Ser 




Ser 


He 






BMP3 




Arg 


Ala** 


Gly 


Val 


Val 


Pro 


Gly 


He 





wo 93/0S751 



- 45 - 



PCr/US92/07432 



GDF-1 


Met 


Ala 


Ala 


Ala 


Gly 


Ala Ala 


60A 




Leu 


Leu 


Glu 


Lys 


Lys 


BHP5 




Leu 


Met 


Phe 


Asp 


His 


BMF6 


• • • • « 


Leu 


Met 


« • • • < 




Ty r 



55 60 



hOP-1 

mOP-1 

hOP-2 

mOP.2 

DPP 

Vgl 

Vgr-1 

CBMP-2A 

CBHP-2B 

BMP3 

GDF-1 

60A 

BMP5 

BMP6 



Pro Lys Pro Cys Cys Ala Pro Thr Gin 



Leu 



Glu 

Asp Leu 



Ala 
Ala 
Ala 



Ala 
Ala 



Val 
Val 

Val 
Val 
Val 
Val 



65 



Lys 
Lys 

— Lys 
Lys 
Glu 
Glu 

Glu Lys 
Ala Arg 
Arg 
Lys 
Lys 

70 



hOP-1 

mOP-1 

hOP-2 

niOP-2 

Vgl 

Vgr-1 

DPP 

CBHP-2A 
CBMP-2B 
BMP3 



Leu Asn Ala He Ser Val Leu Tyr Phe 



Ser 
Ser 

Met Ser 



Val 



Asp 
Ser 
Ser 



Thr 
Thr 



Pro 



Met 



Ser Val Ala Met 
Met 

... ... ... Met 



Met Ser Ser Leu 



He 



Tyr 
Tyr 
Phe Tyr 
... • * • 
Leu 

— Leu 
Leu 

Phe Tyr 



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iPCr/US92/07432 



GDF-1 
60A 
BMP5 
BMP6 



Ser Pro 

Gly - . . Leu Pro 



75 



IPhe 



His 



80 



10 



15 



20 



hOP-1 

mOP-l 

hOP-2 

mOP-2 

DPP 

Vgl 

Vgr-1 

CBMP-2A 

CBMP-2B 

BMP3 

GDF-1 

60A 

BMP5 

BMP6 



Asp Asp Ser Ser Asn Val lie Leu Lys 



Asn 



Leu 



Ser 
Ser 

Asn 

Glu 
Glu 
Glu 
Asn 
Asn 



Gin 
Asn 
Asn 
Asn 
Tyr 
Asn 

Asp 

Asn 



Asn 
Asn 

Asp 

Glu 
Asp 
Lys 
Asp 
Glu 



Thr 



Lys 
Lys 



Val 
Val 

Val 
Val 
Val 
Val 
Asn 



Arg 
Arg 

Arg 



Arg 



85 



25 



30 



hOP-1 

mOP-1 

hOP-2 

mOP-2 

DPP 

Vgl 

Vgr-1 

CBMP-2A 

CBMP-2B 

BHP3 



Lys Tyr Arg Asn Met Val Val Arg 



Asn 
His 

Asn 
Asn 
Val 



His 
His 



Gin 
Glu 

Gin 
Gin 
Pro 



Glu 



Asp 
Glu 



Thr 
Ala 



Thr 



Lys 
Lys 
Val 
Asp 

Glu 
Glu 
Glu 



WO93/0S7S1 



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PCT/US92/07432 



GDF-l 


Gin 


Glu 


Asp 




60A 










RMP^ 

pnr J 


* • * 


• • " • • • 


• • r 


• • • 


BMP 6 







Trp 






90 








flUr-l 


Ala 


Cys Gly 


Cys 


His 


mOP-l 


. • • 


... ... 




• • • 


nOr-2 


• • ♦ 


• • • • • • 






inOP-2 


• • • 


• . • • • • 




• • • 


DPP 


Gly 


• • • • . • 


— 


Arg 


Vgl 


Glu 


• . * • . • 


• ' ' 


Arg 


Vgr-1 









. • * 


CBMP-2A 


Gly 


• . • • • • 




Arg 


CBMP-2B 


Gly 


• • • * • • 




Arg 


BMP3 


Ser 


Ala 




Arg 


GDF-l 


Glu 






Arg 


60A 


Ser 


• • . • • • 






BMP5 


Ser 


• . . * ■ « 






BMP6 




• « a • • 










100 






★★Between 


residues 


56 and 57 of 


BMP3 


is a 



• • • ... Asp 
He ... Lys 



95 



between residues 43 and 44 of GDF-l lies 
the amino acid sequence Gly-Gly-Pro-Pro. 

As is apparent from the foregoing amino acid 
sequence comparisons, significant amino acid changes 
can be made within the generic sequences while 
retaining the morphogenic activity. For example, while 
the GDF-l protein sequence depicted in Table II shares 
only about 50% amino acid identity with the hOP-1 
sequence described therein, the GDF-l sequence shares 
greater than 70% amino acid sequence homology (or 
"similarity") with the hOP-1 sequence, where "homology" 



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or "similarity" includes allowed conservative amino 
acid changes within the sequence as defined by Dayoff , 
et al . r Atlas of Protein Sepuence and Structure vol • 5 , 
supp.3, pp. 345-362, (M.O. Dayoff, ed., Nat'l BioMed. 
5 Res* Fd'n, Washington D.C. 1979.) 

The currently most preferred protein sequences 
useful as morphogens in this invention include those 
having greater than 60% identity, preferably greater 

10 than 65% identity, with the amino acid sequence 

defining the conserved six cysteine skeleton of hOP-1 
(e.g., residues 43-139 of Seq. ID No. 5). These most 
preferred sequences include both allelic and species 
variants of the OP-1 and OP-2 proteins, including the 

15 Drosophila 6 OA protein. Accordingly, in still another 
preferred aspect, the invention includes moiphogens 
comprising species of polypeptide chains having the 
generic amino acid sequence referred to herein as 
"OPX", which defines the seven cysteine skeleton and 

20 accommodates the identities between the various 

identified mouse and human OPl and 0P2 proteins- OPX 
is presented in Seq. ID No. 29. As described therein, 
each Xaa at a given position independently is selected 
from the residues occurring at the corresponding 
25 position in the C- terminal sequence of mouse or human 
OPl or 0P2 (see Seq. ID Nos. 5-8 and/or Seq. ID Nos. 
16-23) . 

30 II. Formulations and Methods for Administering 
Therapeutic Agents 



The morphogens may be provided to an individual by 
any suitable means, preferably directly, parenterally 
35 or orally. Where the morphogen is to be provided 



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directly (e,g., locally, as by injection, to a bone 
tissue site), or parenterally, such as by intravenous, 
subcutaneous, intramuscular, intraorbital, ophthalmic, 
intraventricular, intracranial, intracapsular, 
5 intraspinal, intracisternal, intraperitoneal, buccal, 
rectal, vaginal, intranasal or by aerosol 
administration, the morphogen preferably comprises part 
of an aqueous solution. The solution is 
physiologically acceptable so that in addition to 
0 delivery of the desired morphogen to the patient, the 
solution does not otherwise adversely affect the 
patient's electrolyte and volume balance. The aqueous 
medium for the morphogen thus may comprise normal 
physiologic saline (9.85% NaCl, O.ISM)^ pH 7-7.4 • The 
5 aqueous solution containing the morphogen can be made, 
for example, by dissolving the protein in 50% ethanol 
containing acetonitrile in 0.1% trif luoroacetic acid 
(TFA) or 0.1% HCl, or equivalent solvents. One volume 
of the resultant solution then is added, for example, 
to ten volumes of phosphate buffered saline (PBS), 
which further may include 0.1-0.2% human serum albumin 
(HSA). The resultant solution preferably is vortexed 
extensively. If desired, a given morphogen may be made 
more soluble by association with a suitable molecule. 
For example, association of the mature dimer with the 
pro domain of the morphogen increases solubility of the 
protein significantly. In fact, the endogenous protein 
is thought to be transported in this form. 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-1 by 80%. 
Other components found in milk and/or various serum 
proteins also may be useful. 



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Useful solutions for oral or parenteral 
administration may be prepared by any of the methods 
well known in the pharmaceutical art, described/ for 
example, in Remington's Pharmaceutical Sciences , 
5 (Gennaro, A. ^ ed. } r Mack Pub., 1990. Formulations may 
include, for example, polyalkylene glycols such as 
polyethylene glycol, oils of vegetable origin, 
hydrogenated naphthalenes, and the like. Formulations 
for direct administration, in particular, may include 
10 glycerol and other compositions of high viscocity. 
Biocompatible, preferablly bioresorbable polymers, 
including, for example, hyaluronic acid, collagen, 
tricalcium phosphate, polybutyrate, lactide and 
lactide/glycolide copolymers, may be useful excipients 
15 to control the release of the morphogen in vivo . Other 
potentially useful parenteral delivery systems for 
these morphogehs include ethylene-vinyl acetate 
copolymer particles , osmotic pumps, implantable 
infusion systems, and liposomes. Formulations for 
20 inhalation administration contain as excipients, for 
example, lactose, or may be aqueous solutions 
containing, for example, polyoxyethylene-9-lauryl 
ether, glycocholate and deoxycholate, or oily solutions 
for administration in the form of nasal drops, or as a 
25 gel to be applied intranasally. Formulations for 

parenteral administration may also include glycocholate 
for buccal administration, methoxy sal icy late for rectal 
administration, or cutric acid for vaginal 
administration . 

30 

Alternatively, the morphogens described herein may 
be administered orally. Oral administration of 
proteins as therapeutics generally is not practiced as 
most proteins readily are degraded by digestive enzymes 
35 and acids in the mammalian digestive system before they 



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can be absorbed into the bloodstream. However, the 
morphogens described herein typically are acid-stable 
and protease-resistant (see, for example, U.S. Pat. No. 
4,968,590.) In addition, at least one morphogen, OP-1, 
5 has been identified in bovine mammary gland extract, 
colostrum and milk (see Example 10, below) as well as 
saliva. Moreover, the OP-1 purified from mammary gland 
extract has been shown to be morphogenically active. 
Specifically, this protein has been shown to induce 
10 endochondral bone formation in mammals when implanted 
subcutaneously in association with a suitable matrix 
material, using a standard in vivo bone assay, such as 
is disclosed in U.S. Pat. No. 4,968,590. In addition, 
endogenous morphogen also has been detected in the 
15 bloodstream (see Example 11). These findings indicate 
that oral and parenteral administration are viable 
means for administering morphogens to an individual. 
In addition, while the mature forms of certain 
morphogens described herein typically are sparingly 
0 soluble, the morphogen form found in milk (and mammary 
gland extract and colostrum) is readily soluble, 
probably by association of the mature, morphogenically 
active form with the pro domain of the intact sequence 
and/or by association with one or more milk components. 
5 Accordingly, the compounds provided herein also may be 
associated with molecules capable of enhancing their 
solubility in vitro or in vivo , including, for example, 
part or all of a morphogen pro domain, and casein, as 
described above. 
0 . 

The compounds provided herein also may be 
associated with molecules capable of targeting the 
morphogen or morphogen-stimulating agent to bone 
tissue. For example, tetracycline and diphosphonates 
) are known to bind to bone mineral , particularly at 



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zones of bone remodeling, when they are provided 
systemically in a mammal- Alternatively, an antibody 
or other binding protein that interacts specifically 
with a surface molecule on bone tissue cells also may 
5 be used. Such targeting molecules further may be 
covalently associated to the morphogen or morphogen- 
stimulating agent with, for example, an acid labile 
bond such as an Asp-Pro linkage, using standard 
chemical means well known in the art. Because the 

10 local environment at bone remodeling sites is acidic, 
acid-labile linkages are expected to be preferentially 
cleaved at these sites, yielding active morphogen or 
morphogen-stimulating agent at the desired site. 
Useful targeting molecules may be designed, for 

15 example, using the single chain binding site technology 
disclosed^ for example, in U.S. Pat. No. 5,091,513. 

As described above, the morphogens provided herein 
share significant sequence homology in the C-terminal 

20 active domains. By contrast, the sequences diverge 
significantly in the sequences which define the pro 
domain. Accordingly, the pro domain may be 
morphogen-specif ic. As described above, it is also 
known that the various morphogens identified to date 

25 are differentially expressed in the different tissues. 
Accordingly, without being limited to any given theory, 
it is likely that^ under natural conditions in the 
body, selected morphogens typically act on a given 
tissue. Accordingly, part or all of pro domains, which 

30 have been identified associated with the active form of 
the morphogen in solution, may serve as targeting 
molecules for the morphogens described herein. For 
example, the pro domains may interact specifically with 
one or more molecules at the target tissue to direct 

35 the morphogen associated with the pro domain to that 



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tissue. Accordingly, another useful targeting molecule 
for targeting morphogen to bone tissue is part or all 
of a morphogen pro domain, particularly part or all of 
the pro domains of OP-1, BMP2 or BMP4, all of which 
proteins are found naturally associated with bone 
tissue. 

Finally, the raorphogens or morphogen-stiraulating 
agents provided herein may be administered alone or in 
combination with other molecules known to have a 
beneficial effect on maintaining appropriate bone 
remodeling cycles in an individual at risk for 
excessive bone loss. Examples of useful cof actors 
include vitamin D^, calcitonin, prostaglandins, 
parathyroid hormone, dexamethasone, estrogen and IGF. 

The compounds provided herein can be formulated 
into pharmaceutical compositions by admixture with 
pharmaceutically acceptable nontoxic excipients and 
carriers. As noted above, such compositions may be 
prepared for parenteral administration, particularly in 
the form of liquid solutions or suspensions; for oral 
administration, particularly in the form of tablets or 
capsules; or intranasally, particularly in the form of 
powders, nasal drops, or aerosols. 

The compositions can be formulated for parenteral 
or oral administration to humans or other mammals in 
therapeutically effective amounts, e.g., amounts which 
provide appropriate concentrations of a morphogen to 
bone tissue for a time sufficient to inhibit loss of 
bone mass and/or to stimulate bone formation in 
individuals suffering from metabolic bone diseases and 
other bone remodeling disorders as described above. 



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Therapeutic concentrations also are sufficient to 
repair fractures and other defects in skeletal 
micros tructure^ and to enhance maintenance of 
appropriate bone mass in developing juveniles and 
5 adults, including protecting individuals at risk for 
bone mass deterioration* 

As will be appreciated by those skilled in the art, 
the concentration of the compounds described in a 

10 therapeutic composition will vary depending upon a 

number of factors, including the dosage of the drug to 
be administered, the chemical characteristics (e.g., 
hydrophobicity) of the compounds employed, and the 
route of administration • The preferred dosage of drug 

15 to be administered also is likely to depend on such 
variables as the type and extent of bone loss or 
defect, the overall health status of the particular 
patient, the relative biological efficacy of the 
compound selected, the formulation of the compound 

20 excipients, and its route of administration. In general 
terms, the compounds of this invention may be provided 
in an aqueous physiological buffer solution containing 
about 0.1 to 10% w/v compound for parenteral 
administration. Typical dose ranges are from about 10 

25 ng/kg to about 1 g/kg of body weight per day; a 
preferred dose range is from about 0.1 A^g/kg to 
100 mg/kg of body weight per day. Optimally, the 
morphogen dosage given in all cases is between 2-20 fjg 
of protein per kilogram weight of the patient per day. 

30 Currently preferred dose ranges for local injection of 
soluble morphogen to bone tissue are 0.1-50 /jg 
morphogen/injection. No obvious morphogen-induced 



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pathological lesions are induced when mature morphogen 
(e.g., OP-1, 20 /jg) is administered daily to normal 
growing rats for 21 consecutive days- Moreover, 10 fjg 
systemic injections of morphogen (e.g., OP-1) injected 
5 daily for 10 days into normal newborn mice does not 
produce any gross abnormalties . 

III. Examples 

10 Example 1. Identification of Morphogen-Expressinq 
Tissue 

Determining the tissue distribution of morphogens 
may be used to identify different morphogens expressed 
15 in a given tissue, as well as to identify new, related 
morphogens. Tissue distribution also may be used to 
identify useful morphogen-producing tissue for use in 
screening and identifying candidate morphogen- • 
stimulating agents. The morphogens (or their mRNA 
20 transcripts) readily are identified in different 
tissues using standard methodologies and minor 
modifications thereof in tissues where expression may 
be low. For example, protein distribution may be 
determined using standard Western blot analysis or 
25 immunof luorescent techniques, and antibodies specific 
to the morphogen or morphogens of interest. Similarly, 
the distribution of morphogen transcripts may be 
determined using standard Northern hybridization 
protocols and transcript-specific probes. 

30 

Any probe capable of hybridizing specifically to a 
transcript, and distinguishing the transcript of 
interest from other, related transcripts may be used. 
Because the morphogens described herein share such high 
35 sequence homology in their active, C-terminal domains. 



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the tissue distribution of a specific morphogen 
transcript may best be determined using a probe 
specific for the pro region of the immature protein 
and/or the N- terminal region of the mature protein. 
5 Another useful sequence is the 3 ' non-coding region 
flanking and iimnediately following the stop codon. 
These portions of the sequence vary substantially among 
the morphogens of this invention, and accordingly, are 
specific for each protein- For example, a particularly 
10 useful Vgr-l-specif ic probe sequence is the PvuII-SacI 
fragment, a 265 bp fragment encoding both a poartion of 
the untranslated pro region and the N-terminus of the 
mature sequence (see Lyons et al. (1989) PNA S 86:4554- 
4558 for a description of the cDNA sequence). 
15 Similarly, particularly useful mOP-l-specif ic probe 
sequences are the BstXl-Bgll fragment, a 0.68 Kb 
sequence that covers approximately two-thirds of the 
mOP-1 pro region; a StuI-StuI fragment, a 0.2 Kb 
sequence immediately upstream of the 7-cysteine domain; 
20 and the Earl-Pstl fragment, an 0.3 Kb fragment 

containing a portion of the 3 'untranslated sequence 
(See Seq- ID No. 18, where the pro region is defined 
essentially by residues 30-291.) Similar approaches 
may be used, for example, with hOP-1 (Seq. ID No. 16) 
25 or human or mouse OP-2 (Seq. ID Nos. 20 and 22.) 

Using these morphogen-specif ic probes, which 
may be synthetically engineered or obtained from cloned 
sequences, morphogen transcripts can be identified in 
mammalian tissue, using standard methodologies well 
known to those having ordinary skill in the art- 
Briefly, total RNA is prepared from various adult 
murine tissues (e.g., liver, kidney, testis, heart, 
brain, thymus and stomach) by a standard methodology 
such as by the method of Chomczyaski et al. ((1987) 



30 



35 



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Anal. Biochem 162:156-159) and described below. Poly 
(A)+ RNA is prepared by using oligo (dT) -cellulose 
chromatography (e.g.. Type 7, from Pharmacia LKB 
Biotechnology, Inc.). Poly (A)+ rna (generally 15 fjg) 
5 from each tissue is fractionated on a 1% 

agarose/formaldehyde gel and transferred onto a Nytran 
membrane (Schleicher & Schuell). Following the 
transfer, the membrane is baked at 80*'C and the RNA is 
cross-linked under UV light (generally 30 seconds at 1 
0 mW/cm ) . Prior to hybridization, the appropriate probe 
is denatured by heating. The hybridization is carried 
out in a lucite cylinder rotating in a roller bottle 
apparatus at approximately 1 rev/min for approximately 
15 hours at 37**C using a hybridization mix of 40% 
5 formamide, 5 x Denhardts, 5 x SSPE, and 0.1% SDS. 
Following hybridization, the non-specific counts are 
washed off the filters in 0.1 x SSPE, 0.1% SDS at 50**C. 

Examples demonstrating the tissue distribution of 
various morphogens, including Vgr-1, OP-1, BMP2, BMP3, 
BMP4, BMP5, GDF-1, and OP-2 in developing and adult 
tissue are disclosed in co-pending USSN 752,764, and in 
Ozkaynak, et al., (1991) Biochem. Biophys. Res. Commn . 
179:116-123, and Ozkaynak, et al. (1992) (JBC, in 
press), the disclosures of which are incorporated 
herein by reference. Using the general probing 
methodology described herein, northern blot 
hybridizations using probes specific for these 
morphogens to probe brain, spleen, lung, heart, liv^r 
and kidney tissue indicate that kidney-related tissue 
appears to be the primary expression source for OP-1, 
with brain, heart and lung tissues being secondary 
sources. Lung tissue appears to be the primary tissue 
expression source for Vgr-1, BMP5, BMP4 and.BMP3. Low^r 
levels of Vgr-1 also are seen in kidney and heart 



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tissue, while the liver appears to be a secondary 
expression source for BMP5, and the spleen appears to 
be a secondary expression source for BMP4. GDF-1 
appears to be expressed primarily in brain tissue. To 
5 date, OP-2 appears to be expressed primarily in early 
embryonic tissue. Specifically, northern blots of 
murine embryos and 6-day post-natal animals shows 
abundant 0P2 expression in 8-day embryos. Expression 
is reduced significantly in 17-day embryos and is not 
10 detected in post-natal animals. 

Example 2 . Mitogenic Effect of Morphogen on Rat and 
Human Osteoblasts 

15 The ability of a morphogen to induce proliferation 

of osteoblasts may be determined in vitro using the 
following assay. In this and all examples involving 
osteoblast cultures, rat osteoblast-enriched primary 
cultures preferably are used. Although these cultures 

20 are heterogeneous in that the individual cells are at 
different stages of differentiation, the culture is 
believed to more accurately reflect the metabolism and 
function of osteoblasts in vivo than osteoblast culture 
obtained from established cell lines. Unless otherwise 

25 indicated, all chemicals referenced are standard, 

commercially available reagents, readily available from 
a number of sources, including Sigma Ch-emical, Co., St. 
Louis; Calbiochem, Corp., San Diego, and Aldrich 
Chemical Co., Milwaukee. 

30 

Rat osteoblast-enriched primary cultures were 
prepared by sequential collagenase digestion of newborn 
suture-free rat calvaria (e.g., from 1-2 day-old 
animals, Long-Evans strain, Charles River Laboratories, 
35 Wilmington, MA), following standard procedures, such as 



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are described, for example, in Wong et al., (1975 J PNAS 
72:3167-3171. Rat osteoblast single cell suspensions 
then were plated onto a multi-well plate (e.g., a 
4 8 well plate) at a concentration of 50,000 osteoblasts 
5 per well in alpha MEM (modified Eagle's medium, Gibco, 
Inc., Long Island) containing 10% FBS (fetal bovine 
serum), L-glutamine and penicillin/streptomycin. The 
cells were incubated for 24 hours at 37 °C, at which 
time the growth medium was replaced with alpha MEM 
0 containing 1% FBS and the cells incubated for an 
additional 24 hours so that cells were in serum- 
deprived growth medium at the time of the experiment. 

The cell culture then was divided into three 
5 groups: (1) wells which received 0.1, 1.0, 10.0, 40 
and 80-0 ng of morphogen; (2) wells which received 0.1, 
1.0, 10.0 and 4 0 ng of a local-acting growth factor; 
and (3) the control group, which received no growth 
factors. In this example, OP-1 was the morphogen 
tested, and TGF-p was the local-acting growth factor. 
The cells then were incubated for an additional 
18 hours after which the wells were pulsed with 
2pCi/well of ^H-thymidine and incubated for six more 
hours. The excess label then was washed off with a 
cold solution of 0.15 M NaCl, 250 /jl of 10% 
tricholoracetic acid then was added to each well and 
the wells incubated at room temperature for 30 minutes. 
The cells then were washed three times with cold 
distilled water, and lysed by the addition of 250 ^1 of 
1% sodium dodecyl sulfate (SDS) for a period of 
30 minutes at 37 °C. The cell lysates then were 
harvested using standard means well known in the art, 
and the incorporation of ^H-thymidine into cellular DNA 
was determined by liquid scintillation as an indication 
of mitogenic activity of the cells. The results, shown 



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in FIG. 1/ demonstrate that OP-1 (identified in the 
figure by squares ) stimulates ^ H-thymidine 
incorporation into DNA, and thus promotes osteoblast 
cell proliferation. The mitogenesis stimulated by 
5 40 ng of OP-1 in serum- free medium was equivalent to 
the mitogenic effect of 10% fresh serum alone. By 
contrast^ the effect of TGF-p (indicated by diamonds in 
Fig. 1) is transient and biphasic. At high 
concentrations, TGF-p has no significant effect on 
10 osteoblast cell proliferation. This system may be used 
to test other morphogens for their effect on cell 
proliferation. 

The in vitro effect of a morphogen on osteoblast 
15 proliferation also was tested on human primary 

osteoblasts (obtained from bone tissue of a normal 
adult patient and prepared as described above) and on 
osteosarcoma-derived cells, and in all cases induced 
cell proliferation. In addition, similar experiments, 
20 performed using BMP4 (BMP2B) and BMP3 shows these 

morphogens also can stimulate osteoblast proliferation 
and growth. (See Chen et al., (1991) J. Bone and Min. 
Res. 6 ; 1387-1393, and Vukicevic, (1989) PNAS 86; 8793- 
8797.) 

25 

The effect of a given morphogen on bone cell growth 
and/or development also may be tested using a variety 
of bone cell markers: e.g., collagen synthesis, 
alkaline phosphatase activity, parathyroid 

30 hormone-mediated cyclic AMP (cAMP) production, 

osteocalcin synthesis, and by assessing the rate of 
mineralization in osteoblasts. Of these, alkaline 
phophatase activity, parathyroid hormone-mediated cAMP 
production, osteocalcin synthesis and mineralization 

35 promotion are specific markers for the differentiated 



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osteoblast phenotype. Experimental systems for testing 
these parameters as well as collagen synthesis are 
described below in Examples 3-7. In all cases 
morphogen alone stimulated expression of these 
phenotype-specif ic markers* In Examples 3-7 OP-1 was 
the morphogen tested. Similar experiments ^ performed 
using BMP4 (BMP2B) shows that this morphogen also 
induces osteoblast differentiation. (See Chen, et al. 
(1991) T. Bone and Min. Res. 6: 1387-1392, and 
Vukicevic, (1989) PNAS 86: 8793-8797.) 

Example 3. Effect of Morphogen on Collagen Synthesis 
in Rat Osteoblasts 

The effect of a morphogen on collagen production in 
rat osteoblasts in vitro may be determined as follows. 

Rat osteoblasts were prepared and cultured in a 
multi-well plate as described for Example 2. In this 
example a 24-well plate was used. The cultured cells 
then were divided into three groups: (1) wells which 
received 1, 10 or 4 0 ng of morphogen per ml of medium; 
(2) wells which received 1, 10 or 40 ng of a 
local-acting growth factor per ml of medium; and (3) a 
control group which received no growth factors. In 
this example, OP-1 was the morphogen tested, and TGF-^ 
was the local-acting growth factor. 

The samples were incubated for 68 hours at 37 °C with 
5% CO2 in a, humidified incubator. Twenty-five (25) pCi 
of proline were added into each well and incubated 
for six additional hours. The cells then were frozen 
at -20°C until the collagen assay was performed. The 
cells then were assayed for collagen production by 



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detecting incorporation of H-proline into total 
collagenase-digestible protein (CDP). The results, 
shown in FIG. 2, demonstrate that OP-1 stimulates 
type-I collagen synthesis, as measured by ^H-proline 
incorporation into total CDP. Thus, OP-1 promotes 
collagen synthesis in vitro by preosteoblasts and 
mature osteoblasts. 

Example 4. Alkaline Phosphatase Induction of 
Osteoblasts by Morphogen 

4.1 Morphogen-specific Alkaline Phosphatase 
Induction 

Since alkaline phosphatase production is an 
indicator of bone formation by differentiated, 
functional osteoblasts, a morphogen may be assessed for 
its potential osteogenic effects using this osteoblast 
marker in the following in vitro test system. 

Rat osteoblasts were prepared and cultured in a 
multi-well plate as described for Example 2. In this 
example a 24-well plate was used. The cultured cells 
then were divided into three groups: (1) wells which 
received varying concentrations of morphogen; (2) wells 
which received varying concentrations of a local-acting 
growth factor; and (3) a control group which received 
no growth factors. In this example OP-1 was the 
morphogen tested at the following concentrations : 0.1, 
1.0, 10.0, 40.0 or 80.0 ng/ml medium; and TGF-p was the 
local-acting growth factor, tested at 0.1, 1.0, 10.0, 
40.0 or 80.0 ng/ml medium. The cells then were 
incubated for 72 hours. After the incubation period 
the cell layer was extracted with 0.5 ml of 1% Triton 



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X-100. The resultant cell extract was centrifuged, 
100 fjl of the extract was added to 90 pi of 
paranitrosophenylphospate (PNPP) /glycerine mixture and 
incubated for 30 minutes in a 37**C water bath and the 
5 reaction stopped with 100 /j1 NaOH. The samples then 
were run through a plate reader (e.g., Dynatech MR700 
plate reader, and absorbance measured at 400 nm, using 
p-nitrophenol as a standard ) to determine the presence 
and amount of alkaline phosphate activity. Protein 
0 concentrations were determined by the Biorad method. 
Alkaline phosphatase activity was calculated in 
units//ig protein, where 1 unit=l nmpl p-nitrophenol 
liberated/30 minutes at 37 °C. 

The results, shown in FIG. 3, illustrate that 
morphogen alone stimulates the production of alkaline 
phosphatase in osteoblasts, and thus promotes the 
growth and expression of the osteoblast differentiated 
phenotype. In the figure, squares represent OP-1 
concentrations, and diamonds represent TGF-p 
concentrations . 



4.2. Long Term Effect of Morphogen on the 

Production of Alkaline Phosphatase by Rat 
Osteoblasts 

In order to determine the long term effect of a 
morphogen on the production of alkaline phosphatase toy 
rat osteoblasts, the following assay may be performed. 

Rat osteoblasts were prepared and cultured in 
multi-well plates as described in Example 2, In this 
example six sets of 24 well plates are plated with 
50,000 rat osteoblasts per well. The wells in each 



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plate, prepared as described above, then were divided 
into three groups: (1) those which received 1 ng of 
morphogen per ml of medium; (2) those which received 
40 ng of morphogen/ml of medium; and (3) those which 
received 80 ng of raorphogen/ml of medium. Each plate 
then was incubated for different lengths of time: 
0 hours (control time), 24 hours, 48 hours, 96 hours, 
120 hours and 144 hours. After each incubation period, 
the cell layer was extracted with 0.5 ml of 1% Triton 
X-100. The resultant cell extract was centrifuged, and 
alkaline phosphatase activity determined as for Example 
4, using paranitrosophenylphosphate (PNPP). The 
results, shown in FIG. 4, illustrate that morphogen 
alone stimulates the production of alkaline phosphatase 
in osteoblasts, that increasing doses of OP-1 further 
increase the level of alkaline phosphatase production, 
and that the morphogen-stimulated elevated levels of 
alkaline phosphatase in the treated osteoblasts lasts 
for an extended period of time. In the figure, circles 
represent 1 ng OP-1; squares, 40 ng OP-1; and diamonds, 
80 ng OP-1. 

Example 5. Morphogen- Induced Parathyroid Hormone 

Mediated cAMP Production in Rat Osteoblasts 

The effect of a morphogen on parathyroid hormone- 
mediated cAMP production in rat osteoblasts in vitro 
may be determined as follows. 

Rat osteoblasts were prepared and cultured in a 
multiwell plate as described for Example 2 above. In 
this example a 24 -well plate was used. The cultured 
cells then were divided into three groups: (1) wells 
which received varying concentrations of morphogen (in 



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this example, OP-1, at 1.0, 10.0 and 40.0 ng/ml 
medium); (2) wells which received varying 
concentrations of a local-acting growth factor (in this 
example, TGF-p, at 0.1, 1.0, and 5.6 ng/ml medium); and 
(3) a control group which received no growth factors. 
The plate was then incubated for another 72 hours. At 
the end of the 72 hours the cells were treated with 
medium containing 0.5% bovine serum albumin (BSA) and 
ImM 3-isobutyl-l-methyl xanthine for 20 minutes 
followed by the addition into half of the wells of 
human recombinant parathyroid hormone (hPTH, Sigma, 
St. Louis) at a concentration of 200ng/ml for 
10 minutes. The cell layer was extracted from each 
well with 0.5 ml of 1% Triton X-100. The cAMP levels 
were then determined using a radioimmunoassay kit 
(Araersham, Arlington Heights, Illinois). The results, 
shown in FIG. 5, demonstrate that morphogen alone 
stimulates an increase in the PTH-mediated cAMP 
response, and thus promotes the growth and expression 
of the osteoblast differentiated phenotype- 

Example 6. Effect of Morphogen on Osteocalcin 

Synthesis and the Rate of Mineralization by 
Osteoblasts in Culture 

Osteocalcin is a bone-specific protein synthesized 
by osteoblasts which plays an integral role in the rate 
of bone mineralization in vivo . Circulating levels of 
osteocalcin in serum are used as a marker for 
osteoblast activity and bone formation in vivo . 
Induction of osteocalcin synthesis in 
osteoblast-enriched cultures can be used to assay 
morphogen efficacy in vitro . 



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Rat osteoblasts are prepared and cultured in a 
multi-well plate as for Example 2. In this example 
cells were cultured in a 24 -well plate. In this 
experiment the medium was supplemented with 10%FBS, and 
5 on day 2, cells were fed with fresh medium supplemented 
with fresh 10 mM ^-glycerophosphate (Sigma, Inc*)* 
Beginning on day 5 and twice weekly thereafter, cells 
were fed with a complete mineralization medium 
containing all of the above components plus fresh L{+)- 

10 ascorbate, at a final concentration of SOji/g/ml medium. 
Morphogen then was added to the wells directly. In 
this example, OP-1 in 50% acetonitrile (or 50% ethanol) 
containing 0.1% trif luoroacetic acid (TFA) was added at 
no moire than 5/il morphogen/ml medium. Control wells 

15 received solvent vehicle only. The cells then were 

re-fed and the conditioned medium sample diluted 1:1 in 
standard radioimmunoassay buffer containing standard 
protease inhibitors and stored at -20** C until assayed 
for osteocalcin. Osteocalcin synthesis then was 

20 measured by standard radioimmoassay using a 

commercially available rat osteocalcin-specif ic 
antibody. 

Mineralization was determined on long term cultures (13 
25 day) using a modified von Kossa staining technique on 
fixed cell layers: cells were fixed in fresh 4% 
paraformaldehyde at 23° C for 10 mn, following rinsing 
cold 0.9% NaCl. Fixed cells then were stained for 
endogenous alkaline phosphatase at pH 9.5 for 10 min, 
30 using a commercially available kit (Sigma, Inc.) 

Purple stained cells then were dehydrated with methanol 
and air dried, after 30 min incubation in 3% AgNO^ in 



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the dark, HjO-rinsed samples were exposed for 30 sec to 
254 nm UV light to develop the black silver-stained 
phosphate nodules. Individual mineralized foci (at 
least 20 /jm in size) were counted under a dissecting 
5 microscope and expressed as nodules/culture (see Fig. 
6B) • 

As can be seen in Fig. 6A OP-1 stimulates 
osteocalcin synthesis in oseoblast cultures. The 

10 increased osteocalcin synthesis in response to OP-1 is 
dose dependent and showed a 5-fold increase over the 
basal level using 25 ng of OP- 1/10 ml medium after 13 
days of incubation. The enhanced osteocalcin synthesis 
also can be confirmed by detecting the elevated 

15 osteocalcin mRNA message (20-fold increase) using a rat 
osteocalcin-specif ic probe. In addition, the increase 
in osteoclacin synthesis correlates with increased 
mineralization in long term osteoblast cultures as 
determined by the appearance of mineral nodules 

20 (compare Fig, 6A and 6B.) OP-1 increases the initial 
mineralization rate about 20-fold compared to untreated 
cultures. Similar experiments performed using TGF-^ 
indicate that TGF-p does not induce osteocalcin 
synthesis or promote the mineralization process. Thus, 

25 morphogen alone promotes the growth and expression of 
the osteoblast differentiated phenotype. 

Example 7. Effect of Morphogen on Bone Derived Growth 
30 Factor Induction in vitro 



IGF-I and IGF-II are bone-derived growth factors 
involved in coupling bone formation with bone 
resorption in the bone remodeling cycle. The effect of 



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morphogen on the production of these and other bone- 
derived growth factors, including TGF-p, may be 
evaluated using the following procedure. 

Rat or human osteoblasts were prepared and cultured 
in a multiwell plate as for Example 2. The wells of 
the plate were divided in to groups in which different 
concentrations of morphogen were added (e.g., 0, 1, 10, 
and 100 ng). In this example, OP- 1 was the morphogen 
used. The plate then was incubated for a prescribed 
period of time, e.g., 72 hours, and the level of IGF 
detected, e.g., by iramunolocalization, using a 
commercially available antibody specific for IGFs. 
OP-1 induced the level of both IGF-I and IGF-II 
significantly. Greater than six fold IGF-I and two 
fold IGF-II were induced following exposure to 100 ng 
OP-l/ml. In addition, OP-1 stimulated production of 
the IGF-I stimulating factor, BP3 (IGF-I binding 
protein 3 ) . 

Example 8. Effect of Morphogen on Trabecular Bone 
in Ovariectomized (OVX) Rats 

As indicated above, serum alkaline phosphatase and 
osteocalcin levels are indicators of bone formation 
within an individual. In order to determine the effect 
of a morphogen on bone production in vivo , these 
parameters are measured under conditions which promote 
osteoporosis, e.g., wherein osteoporosis is induced by 
ovary removal in rats • 

Forty Long-Evans rats (Charles River Laboratories, 
Wilmington) weighing about 20 Og each are ovariectomized 
(OVX) using standard surgical procedures, and ten rats 
are sham-operated. The ovariectomization of the rats 



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produces an osteoporotic condition within the rats as a 
result of decreased estrogen production. Food and 
water are provided ad libitum * Eight days after 
ovariectomy, the rats, prepared as described above , 
5 were divided into five groups: (A), 10 sham-operated 
rats; (B), 10 ovariectomized rats receiving 1 ml of 
phosphate-buffered saline (PBS) i.v. in the tail vein; 
(C) 10 ovariectomized rats receiving about 1 mg of 
17PE2 ( 17-p-estradiol Ej) by intravenous injection 

10 through the tail vein; (D) 9 ovariectomized rats 
receiving daily injections of approximately 2fjg of 
morphogen by tail vein for 22 days; and (E) 9 
ovariectomized rats receiving daily injections of 
approximately 20 fjg of morphogen by tail vein for 

15 22 days. In this example, OP-1 was the morphogen 
tested. 

On the 15th and 21st day of the study, each rat was 
injected with 5 mg of tetracycline, and on day 22, the 

20 rats were sacrificed. The body weights, uterine 

weights, serum alkaline phosphate levels, senm calcium 
levels and serum osteocalcin levels then were 
determined for each rat. The results are shown in 
Tables III and IV. 

25 Table III 



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Body Weights, Uterine ffeights and Alkaline Phosphatase 



Group Body Weights Uterine Weights Alk. Phosphatase 

(g) (g) (o/i-) 



A-SHAH 


250. 


.90 


+ 


17. 


.04 


0- 


4192 


+ 


0. 


10 


43, 


.25 


+ 


6. 


11 


B-OVX+PBS 


273. 


.40 


+ 


16. 


81 


0. 


1650 


+ 


0. 


04 


56. 


.22 


+ 


6. 


21 


C-0VX+E2 


241. 


.66 


+ 


21. 


54 


0. 


3081 


+ 


0. 


03 


62. 


.66 


+ 


4. 


11 


D-OVX+OP-1 


266. 


.67 


+ 


10. 


43 


0. 


1416 


+ 


0. 


03 


58. 


.09 


+ 


12 


.97 


{2Mg) 
































E-OVX+OP-1 


272. 


,40 


+ 


20. 


48 


0. 


1481 


+ 


0. 


05 


66. 


.24 


+ 


15 


.74 



(20 pg) 

TABLE IV 

15 Serum Calcium and Serum Osteocalcin Levels 

Group Serum Calcium Serum Osteocalcin 

(ng/dl) (ng/ml) 



20 A-SHAM 8-82 + 1-65 64.66 + 14.77 

B-OVX+PBS 8.95+1.25 69.01+10.20 

C-0VX+E2 9.20 + 1.39 67.13 + 17.33 

D-OVX+OP-1 8.77 + 0.95 148.50 + 84.11 

(2yg) 

25 E-OVX+OP-1 8.67 + 1.94 182.42 + 52.11 

(20yg) 

The results presented in Table III and IV show that 
intravenous injection of morphogen into ovariectomized 
30 rats produces a significant increase in serum alkaline 
phosphatase and serum osteocalcin levels and 
demonstrates that systemic administration of the 
morphogen stimulates bone formation in osteoporotic 
bone. 



35 



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Example 9 . Histomorphometric Analysis of Morphoqen 
on the Tibia Diaphysis in Ovariectomized 
(OVX) Rats 

Fifteen female Long-Evans rats weighing about 160 g 
were ovariectomized (OVX) to produce an osteoporotic 
condition and five rats were sham operated (Charles 
River Laboratories, Wilmington, MA.) as described for 
Example 8. Food and water were provided ad libitum . 
Twenty-two days after ovariectomy, the rats were 
divided into four groups: (A) sham-operated ( 1 ml of 
PBS by intravenous injection through tail vein (5 
rats); (B) OVX, into which nothing was injected (5 
rats); (C) OVX, receiving about 1 mg of ll^E^ by 
intravenous injection through the tail vein 
(5 rats), and (D) OVX, receiving about 1 pg of 
morphogen by intravenous injection through the tail 
vein (5 rats). In this example, OP-1 was morphogen 
tested. 

The rats were injected daily as described for seven 
days, except no injections were given on the thirteenth 
day. The rats then were sacrificed on the nineteenth 
day. The tibial diaphyseal long bones then were 
removed and fixed in ethanol and histomorphometric 
analysis was carried out using standard procedures well 
known in the art. The results are shown in Table V. 



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Table V 

(A) (B) (C) (D) 

MEASUREMENT CONTROL OVX OVX + E^ OVX + OP-1 

5 Longitudinal Growth 20.2 + 0.3 19.4 + 0.2 4.9 + 0.5 17.9 + 0.9 

Rate (ym/day) 

Cancellous Bone 20.2 + 1.5 13.0 + 1.6 13.7 + 2.1 16.6 +_1.8 

Volume (BV/TV, 
10 bone vol/total vol) 

Cancellous Bone 16.2 + 1.8 9.6 + 0.9 11.5 +1.1 12.2 + 0.7 

Perimeter (mm) 

15 Labeled Cancellous 35.5 + 1.5 51.9 + 5.6 58.0 + 4.2 39.2 + 1.9 

Perimeter (%) 

Mineral Apposition 1.76 + 0.14 2.25 + 0.16 1.87 + 0.08 1.86 + 0.20 
Rate (ym/day) 

20 

The results presented in Table V confirm the 
results of Example 8^ that intravenous injection of 
OP-1 into ovariectomized rats stimulates bone growth 
for bone which had been lost due to the drop in 

25 estrogen within the individual rat. Specif ically/ the 
inhibition of cancellous bone volume in OVX rats is 
repaired by the systemically provided morphogen. In 
addition, in morphogen- treated rats the labelled 
cancellous perimeter and mineral apposition rate now 

30 return to levels measured in the control, sham-operated 
rats. Moreover, morphogen treatment does not inhibit 
longitudinal bone growth, unlike estrogen treatment, 
which appears to inhibit bone growth significantly- 



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Accordingly, systemic administration of a morphogen in 
therapeutically effective concentations effectively 
inhibits loss of bone mass in a mammal without 
inhibiting natural bone formation. 

Example 10. Determination of the Presence of 
Morphogen in Body Fluids 

OP-1 has been identified in saliva, human blood 
serum, and various milk forms, including mammary gland 
extract, colostrum, and 57-day bovine milk. Moreoever, 
as described below, the body fluid extracted protein is 
morphogenically active. The discovery that the 
morphogen naturally is present in milk, together with 
the known observation that mature, active OP-1 is acid- 
stable and protease-resistant, indicate that oral 
administration is a useful route for therapeutic 
administration of morphogen to a mammal. Oral 
administration typically is the preferred mode of 
delivery for extended or prophylactic therapies. In 
addition, the identification of morphogen in all milk 
forms, including colostrum, indicates that the protein 
plays a significant role in tissue development, 
including skeletal development of juveniles (see 
Example 13, below). 

10.1 Morphogen Detection in Milk 

OP-1 was partially purified from rat mammary gland 
extract and bovine colostrum and 57 day milk by passing 
these fluids over a series of chromatography columns: 
(e.g., cation-exchange, affinity and reverse phase). At 
each step the eluant was collected in fractions and 
these were tested for the presence of OP-1 by standard 
immunoblot. Immunoreactive fractions then were 



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combined and purified further. The final, partially 
purified product then was examined for the presence of 
OP-1 by Western blot analysis using OP-l-specif ic 
antisera, and tested for in vivo and in vitro activity. 

5 

OP-1 purified from the different milk sources were 
characterized by Western blotting using antibodies 
raised against OP-1 and BMP2. Antibodies were prepared 
using standard immunology protocols well known in the 
10 art/ and as described generally in Example 14, below, 
using full-length E . coli -produced OP-1 and BMP2 as the 
immunogens • 

As shown in Fig- 7 OP-1 purified from colostrum 
15 reacts with the anti-OP-1 antibody, but not with 

anti-BMP2 antibody. In Fig. 7 lane 1 contains reduced, 
purified, recombinantly-produced OP-1; lane 2 contains 
purified bovine colostrum, and lane 3 contains reduced 
COP-16, a biosynthetic contruct having morphogenic 
20 activity and an amino acid sequence modeled on the 
proteins described herein, but having highest amino 
acid sequence homology with BMP2 (see US Pat. No. 
5,011,691 for the COP-16 amino acid sequence.) In 
Fig. 7A the gel was probed with anti-OP-1 antibody; in 
25 Fig. 17B, the gel was probed with anti-BMP2 antibody. 
As can be seen in the figure, anti-OP-1 antibody 
hybridizes only with protein in lanes 1 and 2, but not 
3; while anti-BMP2 antibody hybridizes with lane 3 
only . 



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Column-purified manunary gland extract and 57-day 
milk also reacts specifically with anti-OP-1 
antibodies, including antibody raised against the full 
length coli OP-1, full length mammalian-produced 
5 OP-1, and the OP-1 Ser-17-Cys peptide (e.g., the OP-1 
N-terminal 17 amino acids). 

The morphogenic activity of OP-1 purified from 
mammary gland extract was evaluated in vivo as follows. 
0 A sample was prepared from each OP-1 immunoreactive 
fraction of the mammary gland extract-derived OP-1 
final product by lyophilizing a portion (33%) of the 
fraction and resuspending the protein in 220^1 of 50% 
acetonitrile/0.1% tfa. After vortexing, 25 mg of 
5 collagen matrix was added. The samples were 

lyophilized overnight, and implanted in Long Evans rats 
(Charles River Laboratories, Wilmington, MA, 28-35 days 
old). Each fraction was implanted in duplicate. For 
details of the collagen matrix implantation procedure, 
see, for example, U.S. Pat. No. 4,968,590, hereby 
incorporated by reference. After 12 days, the implants 
were removed and evaluated for new bone formation by 
histological observation. 

The results are presented in Fig.SA, where "% 
activity" refers to the percent of bone formation/total 
area covered by bone in the histology sample. In the 
figure, solid bars represent implants using maimnary 
extract-derived OP-1, each bar corresponding to an 
immunoreactive fraction of the purified product, the 
fraction number being indicated on the x-axis. The 
hatched bar represents an implant using r^combinantly 
produced OP-1 (600 ng). As can be seen in the figure, 
all immunoreactive fractions are osteogenically active. 



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Similarly, the morphogenic activity of OP-1 
purified from mammary gland extract was evaluated in 
vitro by measuring alkaline phosphatase activity in 
vitro using the following assay. Test samples were 
5 prepared as for the in vivo assay, using 15-20% of 

individual immunoreactive fractions collected from the 
final product. Alkaline phosphatase activity was 
tested as described above in Example 4 • The results / 
presented in Fig. 8B, indicate that the immunoreactive 
10 fractions can stimulate alkaline phosphatase activity 
in vitro. Moreover, the activity correlates well with 
that produced by highly purified, recombinant ly 
produced, OP-1. In Fig. 8B solid bars represent assays 
performed with mammary gland-purified OP-1, each bar 
15 corresponding to an immunoreactive fraction of column- 
purified OP-1, the fraction numbers being indicated on 
the X-axis; the hatched bar represents the assay 
performed with purified, recombinantly-produced OP-1 
(100 ng ml); and the cross-hatched bar represents 
20 background. 

10.2 Morphogen Detection in Serum 

Morphogen may be detected in serum using morphogen- 
25 specific antibodies. The assay may be performed using 
any standard immunoassay, such as Western blot 
(immunoblot) and the like. Preferably, the assay is 
performed using an affinity column to which the 
morphogen-specific antibody is bound and through which 
30 the sample serum then is poured, to selectively extract 
the morphogen of interest. The morphogen then is 
eluted. A suitable elution buffer may be determined 
empirically by determining appropriate binding and 
elution conditions first with a control (e.g., 
35 purified, recombinantly-produced morphogen.) Fractions 



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then are tested for the presence of the morphogen by 
standard immunoblot, and the results confirmed by 
N-terminal sequencing. Preferably, the affinity column 
is prepared using monoclonal antibodies. Morphogen 
5 concentrations in serum or other fluid samples then may 
be determined using standard protein quantification 
techniques, including by spectrophotometric absorbance 
or by quantitation of conjugated antibody. 

10 Presented below is a sample protocol for 

identifying OP-1 in serum. Following this general 
methodology other morphogens may be detected in body 
fluids, including serum. The identification of 
morphogen in serum further indicates that systemic 
15 administratrion is a suitable means for providing 
therapeutic concentrations of a morphogen to an 
individual, and that morphogens likely behave 
systemically as endocrine-like factors. Finally, using 
this protocol, fluctuations in endogenous morphogen 
20 levels can be detected, and these altered levels may be 
used as an indicator of bone tissue dysfunction. 
Alternatively, fluctuations in morphogen levels may be 
assessed by monitoring morphogen transcription levels, 
either by standard northern blot analysis as described 
25 in Example 1, or by in situ hybridization, using a 

labelled probe capable of hybridizing specifically to 
morphogen RNA, and standard RNA hybridization protocols 
well described in the art and described generally in 
Example 1. 

30 

OP-1 was detected in human serum using the 
following assay- A monoclonal antibody raised against 
mammalian, recombinantly produced OP-1 using standard 
immunology techniques well described in the art and 
35 described generally in Example 14, was immobilized by 



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passing the antibody over an agarose-activated gel 
(e.g./ Affi-Gel^", from Bio-Rad Laboratories, Richmond, 
CA, prepared following manufacturer's instructions) and 
used to purify OP-1 from serum. Human serum then was 
5 passed over the column and eluted with 3M 

K-thiocyanate - K-thiocyanante fractions then were 
dialyzed in 6M urea, 20mM PO^, pH 7.0, applied to a C8 
HPLC column, and eluted with a 20 minute, 25-50% 
acetonitrile/0.1% TFA gradient. Mature, recombinantly 

10 produced OP-1 homodimers elute between 20-22 minutes. 
Fractions then were collected and tested for the 
presence of OP-1 by standard immunoblot using an OP-1 
specific antibody as for Example 10. A. Fig. 9 is an 
immunoblot showing OP-1 in human sera under reducing 

15 and oxidized conditions. In the figure, lanes 1 and 4 
are OP-1 standards, run under oxidized (lane 1) and 
reduced (lane 4) conditions. Lane 5 shows molecular 
weight markers at 17, 27 and 39 kDa. Lanes 2 and 3 are 
human sera OP-1, run under oxidized (lane 2) and 

20 reduced (lane 3) conditions. 

Morphogens may be used in diagnostic applications 
by comparing the quantity of morphogen present in a 
body fluid sample with a predetermined reference value, 

25 with fluctuations in fluid morphogen levels indicating 
a change in the status of bone tissue. Alternatively, 
fluctuations in the level of endogenous morphogen 
antibodies may be detected by this method, most likely 
in serum, using an antibody or other binding protein 

30 capable of interacting specifically with the endogenous 
morphogen antibody. Detected fluctuations in the 
levels of the endogenous antibody may be used as 
indicators of a change in tissue status. 



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Example 11 • Morphoqen-induced Periosteal and 
Endosteal Bone Formation 

Osteoclast-induced bone resorption occurs primarily 
at the endosteal surface of bone tissue. Accordingly, 
in bone remodeling disorders the marrow cavity is 
enlarged unnaturally , weakening the weight bearing 
capacity of the remaining bone. The following example 
provides means for evaluating the ability of the 
morphogens decribed herein to increase endosteal and 
preiosteal bone mass in a mammal, in this example, 
both periosteal and endosteal bone formation are 
induced by direct injection of a morphogen in a 
biocompatible solution directly to the bone tissue. As 
demonstrated below, morphogens can induce new bone 
formation and increase bone mass at both surfaces when 
provided to the bone by direct injection. Direct 
injection may be a preferred mode of administration for 
providing therapeutically effective concentrations to 
reduce an enlarged marrow cavity, and/or to repair 
fractures and other damage to bone tissue 
microstructure . 

Morphogen was provided to either the periosteum 
(outer or peripheral bone surface) and endosteum 
(interior bone surface, e.g., that surface lining the 
marrow cavity) of a rat femur by a single injection in 
each case. Specifically, morphogen (e.g., 0P--1, 2-20 
fJ9) was provided to the bone tissue as an insoluble 
colloidal suspension in phosphate-buffered saline. 
Endosteal injection was performed through a microhole 
made with a hand-held orthopedic drill. After 7 days, 
the treated bones were removed and prepared for 
histological evaluation as described in U.S. Pat. 
No. 4 ,968,590. As little as 2 (jg morphogen is 



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sufficient to induce new bone formation at the site of 
injection within 4-7 days. In addition, bone induction 
is dose-dependent- Photomicrographs of the histology 
are presented in Fig. 10. In the figure, "ob" means 
5 old bone, "bm" means bone marrow, "nb" means new bone, 
and "m" means muscle. Fig.lOA shows new bone formed 
following injection of morphogen to the endosteal 
surface. As can be seen in the figure, new bone has 
formed within the bone marrow cavity, filling in the 
10 periphery of the cavity. Fig lOB shows new bone formed 
following injection of morphogen to the periosteal 
surface, replacing the muscle- normally present. 

15 Example 12. Effect of Morphogen on Bone Resorption 

The effect of morphogen on bone resorption may be 
evaluated using rat osteoclasts on bovine bone slices, 
in the presence and absence of morphogen, and the 

20 effect of morphogen on pit formation {resorption index) 
determined. Under standard conditions rat osteoclasts 
begin resorbing the bone tissue, causing pit formation 
on the bone slice surface. In this experiment OP-1 was 
the morphogen tested, at concentrations of 0, 5, 10, 

25 20, 40, 50, and 100 ng/ml. 

The results are presented in figure 11, where the 
resorption index is calculated as a percent of the 
control (e.g., bone resorption in the absence of 
30 morphogen), calculated as the number of pits per a 

given slice surface area. Below 40 ng bone resorption 
is enhanced; above 40 ng, OP-1 has no apparent effect 
on bone resorption. The results highlight the integral 
role the morphogen plays in bone remodeling. OP-1 is 



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stored in bone tissue in vivo * In a normal bone 
remodeling cycle, the local concentration of OP-1 at 
the surface likely is low when osteoclasts begin 
res orbing bone, and the low concentration may enhance 
5 and/or stimulate bone resorption. As resorption 
continues, the local concentration of OP-1 at the 
surface likely increases, to a concentration that no 
longer has an effect on osteoclasts, but continues to 
affect osteoblast growth and activity (see 
10 Examples 2-7), stimulating bone growth. 

In addition, morphogens can inhibit multinucleation 
of mononuclear phagocytic cells under conditions where 
these cells normally would be activated. For example, 
15 in the absence of morphogen, an implanted substrate 
material (e.g., implanted subcutaneously) composed of, 
for example, mineralized bone, a ceramic such as 
titanium oxide or any other substrate that provokes 
multinucleated giant cell formation, rapidly becomes 
20 surrounded by multinucleated giant cells, e.g., 

activated phagocytes stimulated to respond and destroy 
the foreign object. In the presence of morphogen 
however, the recruited cells remain in their 
mononuclear precursor form and the matrix material is 
25 undisturbed. Figure 12 illustrates this effect of 

morphogens, in a schematic representation of histology 
results of a titanium oxide substrate implanted 
subcutaneously. In the figure, "mg" means multinucleated 
giant cells and "ob" means osteoblasts. The substrate 
10 represented in Fig. 12B was implanted together with 
morphogen (OP-1) and newly formed osteoblasts are 
evident surrounding the substrate. By contrast, the 
substrate represented in Fig. 12A was implanted without 
morphogen and extensive multinucleated giant cell 
^ formation is evident surrounding the substrate. 



SUBSTITUTE SHEET 



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Accordingly, the morphogens' effect in inhibiting 
excessive bone mass loss in a mammal also may include 
inhibiting activation of these cells. 

5 Example 13. Effect of Morphogen Neutralization on 

Bone Growth 

The effect of the morphogens described herein on 
bone growth in developing mammals also may be evaluated 

10 using neutralizing antibodies specific for particular 
morphogens and assessing the effect of these antibodies 
on bone development. Specifically/ anti-morphogen 
monoclonal and/or polyclonal antibodies may be prepared 
using standard methodologies including, for example, 

15 the protocol provided in Example 14, below. 

Purified antibodies then are provided regularly to 
new born mice, e.g., 10-100//g/injection/day for 10- 
15 days. At 10 or 21 days, the mice are sacrificed and 

20 the effect of morphogen on bone development assessed by 
body veight/ gross visual examination and histology. 
In this example, anti-OP-1 antibodies were used. 
Morphogen neutralization significantly stunted body 
growth/ including bone growth, as indicated by the 

25 reduced body weight and reduced bone length of the 
treated mammals. 

Similarly, morphogen activity may be assessed in 
fetal development in the mouse model using the 

30 following assay. Single lip injections comprising 

10-lOO^yg/injection of morphogen-specif ic antibody are 
administered to pregnant female mice during each day of 
the gestation period and bone development in treated 
and control new mice evaluated by standard 

35 histomorphometric analysis at birth. Similarly, single 



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lip injections also may be provided to juvenile and 
adult mice (e.g., 10-100 fjg] over a prolonged time 
(e.g., 10-15 days) to evaluate the effect on bone 
growth and bone integrity and to evaluate the onset of 
5 osteoporosis. The antibodies are anticipated to 

inhibit tissue morphogenesis, including bone growth and 
bone development, in the developing embryos. 

Example 14. Screening Assay for Candidate Compounds 
10 which Alter Endogenous Morphogen Levels 

Candidate compound(s) which may be administered to 
affect the level of a given morphogen may be found 
using the following screening assay, in which the level 

15 of morphogen production by a cell type which produces 
measurable levels of the morphogen is determined with 
and without incubating the cell in culture with the 
compound, in order to assess the effects of the 
compound on the cell. This can be accomplished by 

20 detection of the morphogen either at the protein or RNA 
level. A detailed description also may be found in 
USSN 752,861, incorporated hereinabove by reference. 

14.1 Growth of Cells in Culture 

25 

Cell cultures of kidney, adrenals, urinary bladder, 
brain, or other organs, may be prepared as described 
widely in the literature. For example, kidneys may be 
explanted from neonatal or new born or young or adult 

30 rodents (mouse or rat) and used in organ culture as 
whole or sliced (1-4 mm) tissues. Primary tissue 
cultures and established cell lines, also derived from 
kidney, adrenals, urinary, bladder, brain, mammary, or 
other tissues may be established in multiwell plates (6 

35 well or 24 well) according to conventional cell culture 



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techniques, and are cultured in the absence or presence 
of serum for a period of time (1-7 days). Cells may be 
cultured, for example, in Dulbecco's Modified Eagle 
medium (Gibco, Long Island, NY) containing serum (e.g., 
5 fetal calf serum at 1%-10%, Gibco) or in serum-deprived 
meditim, as desired, or in defined medium (e.g., 
containing insulin, transferrin, glucose, albumin, or 
other growth factors ) . 

10 Samples for testing the level of morphogen 

production includes culture supernatants or cell 
lysateS/ collected periodically and evaluated for 
morphogen production by immunoblot analysis (Sambrook 
et al., eds., 1989, Molecular Cloning, Cold Spring 

15 Harbor Press, Cold Spring Harbor, NY), or a portion of 
the cell culture itself, collected periodically and 
used to prepare polyA+ RNA for RNA analysis. To 
monitor de novo morphogen synthesis, some cultures are 
labeled according to conventional procedures with an 
20 ^^S-methionine/^^S-cysteine mixture for 6-24 hours and 
then evaluated for morphogenic protein synthesis by 
conventional immunoprecipitation methods. 

14.2 Determination of Level of Morphogenic Protein 

25 

In order to quantitate the production of a 
morphogenic protein by a cell type, an immunoassay may 
be performed to detect the morphogen using a polyclonal 
or monoclonal antibody specific for that protein. For 
30 example, QP-1 may be detected using a polyclonal 

antibody specific for OP-1 in an ELISA, as follows. 



35 



1 /;g/100 fjl of affinity-purified polyclonal rabbit 
IgG specific for OP-1 is added to each well of a 
96-well plate and incubated at 37 °C for an hour. The 



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wells are washed four times with 0.167M sodium borate 
buffer with O.IS M NaCl (BSB), pH 8.2, containing 0.1% 
Tween 20. To minimize non-specific binding, the wells 
are blocked by filling completely with 1% bovine serum 
5 albumin (BSA) in BSB and incubating for 1 hour at 37 *C. 
The wells are then washed four times with BSB 
containing 0.1% Tween 20. A 100 pi aliquot of an 
appropriate dilution of each of the test samples of 
cell culture supernatant is added to each well in 
10 triplicate and incubated at 37*^0 for 30 min. After 

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



35 



Polyclonal antibody may be prepared as follows. 
Each rabbit is given a primary immunization of 100 
ug/500 /j1 £. coli -produced OP-1 monomer (amino acids 



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PCT/US92/07432 



328-431 in SEQ ID NO: 5) in 0.1% SDS mixed with 500 fjl 
Complete Freund's Adjuvant. The antigen is injected 
subcutaneous ly at multiple sites on the back and flanks 
of the animal. The rabbit is boosted after a month in 
5 the same manner using incomplete Freund's Adjuvant. 
Test bleeds are taken from the ear vein seven days 
later. Two additional boosts and test bleeds are 
performed at monthly intervals until antibody against 
OP-1 is detected in the serum using an ELISA assay. 
10 Then, the rabbit is boosted monthly with 100 fjg of 
antigen and bled (15 ml per bleed) at days seven and 
ten after boosting. 

Monoclonal antibody specific for a given morphogen 
15 may be prepared as follows. A mouse is given two 

injections of E . coli produced OP-1 monomer. The first 
injection contains lOOpg of OP-1 in complete Freund's 
adjuvant and is given subcutaneous ly. The second 
injection contains 50 /L;g of OP-1 in incomplete adjuveoit 

20 and is given intraperitoneally. The mouse then 

receives a total of 230 /yg of OP-1 (amino acids 307-431 
in SEQ ID NO: 5) in four intraperitoneal injections at 
various times over an eight month period. One week 
prior to fusion, both mice are boosted 

25 intraperitoneally with 100 pg of OP-1 (307-431) and 30 
fjg of the N-terminal peptide (Ser2g3-Asn2Qg-Cys ) 
conjugated through the added cysteine to bovine serum 
albumin with SMCC cross linking agent. This boost was 
repeated five days (IP)/ four days (IP), three days 

30 (IP) and one day (IV) prior to fusion. The mouse 

spleen cells are then fused to commercially available 
myeloma cells at a ratio of 1:1 using PEG 1500 
(Boeringer Mannheim, Germany), and the cell fusion is 
plated and screened for OP-l-specif ic antibodies using 

35 OP-1 (307-431) as antigen. The cell fusion and 



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PCr/US92/07432 



monoclonal screening then are according to standard 
procedures well described in standard texts widely 
available in the art, 

5 Other Embodiments 

The invention may be embodied in other specific 
forms without departing from the spirit or essential 
characteristics thereof. The present embodiments are 

10 therefore to be considered in all respects as 

illustrative and not restrictive, the scope of the 
invention being indicated by the appended claims rather 
than by the foregoing description, and all changes 
which come within the meaning and range of equivalency 

15 of the claims are therefore intended to be embraced 
therein. 

Other embodiments of the invention are within the 
following claims. 



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SEQUENCE LISTING 

(1) GENERAL INFORMATION: 

(i) APPLICANTS: Thangavel Kuberasampath 
Charles Cohen 
Hermann Opperinann 
Engin Ozkayanak 
David C* Rueger 
Roy H.L. Pang 

(ii) TITLE OF INVENTION: TREATMENT TO 
PREVENT LOSS OF AND/OR INCREASE 
BONE MASS IN METABOLIC BONE DISEASE 

(iii) NUMBER OF SEQUENCES: 33 

(iv) CORRESPONDENCE ADDRESS: 

(A) ADDRESSEE: Testa, Hurwitz & Thib 
eault 

(B) STREET: 53 State Street 

(C) CITY: Boston 

(D) STATE: Massachusetts 

(E) COUNTRY: U.S.A. 

(F) ZIP: 02109 

(V) COMPUTER READABLE FORM: 

(A) MEDIUM TYPE: Floppy Disk 

(B) COMPUTER: IBM XT 

(C) OPERATING SYSTEM: DOS 3.30 

(D) SOFTWARE: PatentIn Release 1.0, 
Version 1.25 

(vi) CURRENT APPLICATION DATA: 
(B) FILING DATE: 



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(vii) PRIOR APPLICATION DATA: 

(A) APPLICATION NUMBER: US 752,857 

(B) FILING DATE: 30-AUG-1991 

(viii) PRIOR APPLICATION DATA: 

(A) APPLICATION NUMBER: US 667,274 

(B) FILING DATA: ll-MAR-1991 

(2) INFORMATION FOR SEQ ID N0:1: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 97 amino acids 

(B) TYPE: amino acids 

(C) TOPOLOGY: linear 

(ii) MOLECULE TYPE: protein 
(ix) FEATURE: 

(A) NAME: Generic Sequence 1 

(D) OTHER INFORMATION: Each Xaa 
indicates one of the 20 naturally- 
occurring L-isomer, a- amino acids 
or a derivative thereof. 

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:1: 
Xaa Xaa Xaa Xaa Xaa Xaa 
1 5 
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 

10 15 
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa 

20 25 
Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 

30 35 
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 
AO 45 50 

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys 

55 60 
Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 
65 70 



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PCTAJS92/07432 



Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 

75 80 
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys 

85 90 
Xaa Cys Xaa 
95 

(2) INFORHATION FOR SEQ ID N0:2: 



(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 97 amino acids 

(B) TYPE: amino acids 

(C) TOPOLOGY: linear 

(ii) MOLECULE TYPE: protein 

(ix) FEATURE: 

(A) NAME: Generic Sequence 2 

(D) OTHER INFORMATION: Each Xaa 
indicates one of the 20 naturally- 
occurring L-isomer, a-amino acids 
or a derivative thereof. 



(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: 

Xaa Xaa Xaa Xaa Xaa Xaa 
1 5 
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 

10 15 
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa 

20 25 
Cys Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa 
30 35 



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Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 
^0 45 50 

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys 

55 60 
Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 

65 70 
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 

75 80 
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys 

85 90 
Xaa Cys Xaa 
95 

(2) INFORMATION FOR SEQ ID NO: 3: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 97 amino acids 

(B) TYPE: amino acids 
(C> TOPOLOGY: linear 

(ii) MOLECULE TYPE: protein 

(ix) FEATURE: 

(A) NAME: Generic Sequence 3 
(D) OTHER INFORMATION: vherein 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: 



Leu Tyr Val Xaa Phe 
1 5 



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Xaa Xaa Xaa Gly Trp Xaa Xaa Trp Xaa 
10 

Xaa Ala Fro Gly Xaa Xaa Xaa Ala 

15 20 
Xaa Tyr Cys Xaa Gly Xaa Cys Xaa 
25 30 
Xaa Pro Xaa Xaa Xaa Xaa Xaa 
35 

Xaa Xaa Xaa Asn His Ala Xaa Xaa 
40 45 
Xaa Xaa Leu Xaa Xaa Xaa Xaa Xaa 
50 

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys 

55 60 
Cys Xaa Pro Xaa Xaa Xaa Xaa Xaa 
65 

Xaa Xaa Xaa Leu Xaa Xaa Xaa 

70 75 
Xaa Xaa Xaa Xaa Val Xaa Leu Xaa 
80 

Xaa Xaa Xaa Xaa Het Xaa Val Xaa 

85 90 
Xaa Cys Gly Cys Xaa 
95 



(2) INFORHATION FOR SEQ ID NO: 4: 



(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 102 amino acids 

(B) TYPE: amino acids 

(C) TOPOLOGY: linear 

(ii) MOLECULE TYPE: protein 



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PCT/US92/07432 



(ix) FEATURE: 

(A) NAME: Generic Sequence 4 
(D) OTHER INFORMATION: wherein each 
Xaa is independently selected from 
a group of one or more specified 
amino acids as defined in the 
specification. 

<xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: 



Cys Xaa Xaa Xaa Xaa Leu Tyr Val Xaa Phe 
1 5 10 

Xaa Xaa Xaa Gly Trp Xaa Xaa Trp Xaa 
15 

Xaa Ala Pro Xaa Gly Xaa Xaa Ala 

20 25 
Xaa Tyr Cys Xaa Gly Xaa Cys Xaa 
30 35 
Xaa Pro Xaa Xaa Xaa Xaa Xaa 
40 

Asn Xaa Xaa Asn His Ala ^aa Xaa 
45 50 
Xaa Xaa Leu Xaa Xaa Xaa Xaa Xaa 
55 

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys 

60 65 
Cys Xaa Pro Xaa Xaa Xaa Xaa Xaa 
70 

Xaa Xaa Xaa Leu Xaa Xaa Xaa 

75 80 
Xaa Xaa Xaa Xaa Val Xaa Leu Xaa 
85 

Xaa Xaa Xaa Xaa Met Xaa Val Xaa 

90 95 
Xaa Cys Gly Cys Xaa 
100 



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PCr/US92/07432 



(2) INFORMATION FOR SEQ ID N0:5: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 139 amino acids 

(B) TYPE: amino acids 

(C) TOPOLOGY: linear 



(ii) MOLECOLE TYPE: protein 

(ix) FEATDRE: 

(A) NAME: hOP-1 (mature form) 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: 

Ser Thr Gly Ser Lys Gin Arg Ser Gin 

1 5 
Asn Arg Ser Lys Thr Pro Lys Asn Gin 

10 15 
Glu Ala Leu Arg Met Ala Asn Val Ala 

20 25 
Glu Asn Ser Ser Ser Asp Gin Arg Gin 

30 35 
Ala Cys Lys Lys His Glu Leu Tyr Val 
40 45 
Ser Phe Arg Asp Leu Gly Trp Gin Asp 

50 

Trp lie lie Ala Pro Glu Gly Tyr Ala 
55 60 
Ala Tyr !iyr Cys Glu Gly Glu Cys Ala 

65 70 
Phe Pro Leu Asn Ser Tyr Met Asn Ala 

75 80 
Thr Asn His Ala He Val Gin Thr Leu 
85 90 



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Val His Phe He Asn Pro Glu Thr Val 

95 

Pro Lys Pro Cys Cys Ala Pro Thr Gin 

100 105 

Leu Asn Ala He Ser Val Leu Tyr Phe 

110 115 
Asp Asp Ser Ser Asn Val He Leu Lys 

120 125 
Lys Tyr Arg Asn Met Val Val Arg Ala 
130 135 
Cys Gly Cys His 

(2) INFORMATION FOR SEQ ID NO: 6: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 139 amino acids 

(B) TYPE: amino acids 

(C) TOPOLOGY: linear 

(ii) MOLECULE TYPE: protein 

(ix) FEATURE: 

(A) NAME: mOP-1 (mature form) 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: 

Ser Thr Gly Gly Lys Gin Arg Ser Gin 

1 5 
Asn Arg Ser Lys Thr Pro Lys Asn Gin 

10 15 
Glu Ala Leu Arg Met Ala Ser Val Ala 

20 25 
Glu Asn Ser Ser Ser Asp Gin Arg Gin 
30 35 



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Ala Cys Lys Lys His Glu Leu Tyr Val 

40 45 
Ser Phe Arg Asp Leu Gly Trp Gin Asp 

50 

Trp He He Ala Pro Glu Gly Tyr Ala 

55 60 
Ala Tyr Tyr Cys Glu Gly Glu Cys Ala 

65 70 
Phe Pro Leu Asn Ser Tyr Met Asn Ala 

75 80 
Thr Asn His Ala He Val Gin Thr Leu 
85 90 
Val His Phe He Asn Pro Asp Thr Val 

95 

Pro Lys Pro Cys Cys Ala Pro Thr Gin 

100 105 

Leu Asn Ala He Ser Val Leu Tyr Phe 

110 115 
Asp Asp Ser Ser Asn Val He Leu Lys 

120 125 
Lys lyr Arg Asn Het Val Val Arg Ala 
130 135 
Cys Gly Cys His 



(2) INFORHATION FOR SEQ ID NO: 7: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 139 amino acids 

(B) TYPE: amino acids 

(C) TOPOLOGY: linear 

(ii) MOLECULE TYPE: protein 



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PCT/US92/07432 



(ix) FEATURE: 

(A) NAME: hOP-2 (mature form) 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: 

Ala Val Arg Pro Leu Arg Arg Arg Gin 

1 5 
Pro Lys Lys Ser Asn Glu Leu Pro Gin 

10 15 
Ala Asn Arg Leu Pro Gly He Phe Asp 

20 25 
Asp Val His Gly Ser His Gly Arg Gin 

30 35 
Val Cys Arg Arg His Glu Leu Tyr Val 
40 45 
Ser Phe Gin Asp Leu Gly Trp Leu Asp 

50 

Trp Val He Ala Pro Gin Gly Tyr Ser 

55 60 
Ala Tyr lyr Cys Glu Gly Glu Cys Ser 

65 70 
Phe Pro Leu Asp Ser Cys Met Asn Ala 

75 80 
Thr Asn His Ala He Leu Gin Ser Leu 
85 90 
Val His Leu Met Lys Pro Asn Ala Val 

95 

Pro Lys Ala Cys Cys Ala Pro Thr Lys 

100 105 

Leu Ser Ala Thr Ser Val Leu Tyr Tyr 

110 115 
Asp Ser Ser Asn Asn Val He Leu Arg 

120 125 
Lys His Arg Asn Met Val Val Lys Ala 
130 135 
Cys Gly Cys His 



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(2) INFORMATION FOR SEQ ID NO: 8: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 139 amino acids 

(B) TYPE: amino acids 

(C) TOPOLOGY: linear 

(ii) MOLECULE TYPE: protein 

(ix) FEATURE: 

(A) NAME: mOP-2 (mature form) 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: 

Ala Ala Arg Pro Leu Lys Arg Arg Gin 

1 5 
Pro Lys Lys Thr Asn Glu Leu Pro His 

10 15 
Pro Asn Lys Leu Pro Gly lie Phe Asp 

20 25 
Asp Gly His Gly Ser Arg Gly Arg Glu 

30 35 
Val Cys Arg Arg His Glu Leu Tyr Val 
40 45 
Ser Phe Arg Asp Leu Gly Trp Leu Asp 

50 

Trp Val He Ala Pro Gin Gly Tyr Ser 

55 60 
Ala Tyr Tyv Cys Glu Gly Glu Cys Ala 

65 70 
Phe Pro Leu Asp Ser Cys Met Asn Ala 

75 80 
Thr Asn His Ala He Leu Gin Ser Leu 
85 90 



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Val His Leu Met Lys Pro Asp Val Val 

95 

Pro Lys Ala Cys Cys Ala Pro Thr Lys 

100 105 

Leu Ser Ala Thr Ser Val Leu Tyr Tyr 

110 115 
Asp Ser Ser Asn Asn Val He Leu Arg 

120 125 
Lys His Arg Asn Met Val Val Lys Ala 
130 135 
Cys Gly Cys His 
(2) INFORMATION FOR SEQ ID NO: 9: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 96 amino acids 

(B) TYPE: amino acids 

(C) TOPOLOGY: linear 

(ii) MOLECULE TYPE: protein 

(ix) FEATURE: 

(A) NAME: CBMP2A(fx) 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: 

Cys Lys Arg His Pro Leu Tyr Val Asp Phe Ser 

1 5 10 

Asp Val Gly Trp Asn Asp Trp He Val Ala Pro 

15 20 
Pro Gly Tyr His Ala Phe Tyr Cys His Gly Glu 

25 30 
Cys Pro Phe Pro Leu Ala Asp His Leu Asn Ser 
35 40 



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Thr Asn His Ala lie Val Gin Thr Leu Val Asn 
45 50 55 

Ser Val Asn Ser Lys He Pro Lys Ala Cys Cys 

60 65 
Val Pro Thr Glu Leu Ser Ala He Ser Met Leu 

70 75 
Tyr Leu Asp Glu Asn Glu Lys Val Val Leu Lys 

80 85 
Asn Tyv Gin Asp Met Val Val Glu Gly Cys Gly 

90 95 
Cys Arg 
100 

(2) INFOEMATION FOR SEQ ID NO: 10: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 101 amino acids 

(B) TYPE: amino acids 

(C) TOPOLOGY: linear 

(ii) MOLECULE TYPE: protein 

(ix) FEATURE: 

(A) NAME: CBMP2B(fx) 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: 

Cys Arg Arg His Ser 
1 5 
Leu Tyr Val Asp Phe Ser Asp Val Gly Trp Asn 

10 15 
Asp Trp He Val Ala Pro Pro Gly Tjrr Gin Ala 

20 25 
Phe Tyr Cys His Gly Asp Cys Pro Phe Pro Leu 
30 35 



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Ala Asp His Leu Asn Ser Thr Asn His Ala lie 

40 45 
Val Gin Thr Leu Val Asn Ser Val Asn Ser Ser 
50 55 60 

He Pro Lys Ala Cys Cys Val Pro Thr Glu Leu 

65 70 
Ser Ala He Ser Met Leu Tyr Leu Asp Glu Tyr 

75 80 
Asp Lys Val Val Leu Lys Asn Tyr Gin Glu Met 

85 90 
Val Val Glu Gly Cys Gly Cys Arg 
95 100 

(2) INFORMATION FOR SEQ ID NO: 11: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 102 amino acids 

(B) TYPE: amino acids 

(C) TOPOLOGY: linear 

(ii) MOLECULE TYPE: protein 

(ix) FEATURE: 

(A) NAME: DPP(fx) 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: 

Cys Arg Arg His Ser Leu Tyr Val Asp Phe Ser 

1 5 10 

Asp Val Gly Trp Asp Asp Trp He Val Ala Pro 

15 20 
Leu Gly Tyr Asp Ala Tyr Tyr Cys His Gly Lys 
25 30 



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Cys Pro Phe Pro Leu Ala Asp His Phe Asn Ser 

35 40 
Thr Ash His Ala Val Val Gin Thr Leu Val Asn 
45 50 55 

Asn Asn Asn Pro Gly Lys Val Pro Lys Ala Cys 

60 65 
Cys Val Pro Thr Gin Leu Asp Ser Val Ala Met 

70 75 
Leu lyr Leu Asn Asp Gin Ser Thr Val Val Leu 

80 85 
Lys Asn Tyr Gin Glu Met Thr Val Val Gly Cys 

90 95 
Gly Cys Arg 
100 

(2) INFORMATION FOR SEQ ID NO: 12: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 102 amino acids 

(B) TYPE: amino acids 

(C) TOPOLOGY: linear 

(ii) MOLECULE TYPE: protein 

(ix) FEATURE: 

(A) NAME: Vgl{fx) 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: 

Cys Lys Lys Arg His Leu Tyr Val Glu Phe Lys 

1 5 10 

Asp Val Gly Trp Gin Asn Trp Val He Ala Pro 

15 20 
Gin Gly Tyr Met Ala Asn Tyr Cys Tyr Gly Glu 
25 30 



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Cys Pro Tyr Pro Leu Thr Glu lie Leu Asn Gly 

35 40 
Ser Asn His Ala He Leu Gin Thr Leu Val His 
45 50 55 

Ser He Glu Pro Glu Asp He Pro Leu Pro Cys 

60 65 
Cys Val Pro Thr Lys Met Ser Pro He Ser Met 

70 75 
Leu Phe Tyr Asp Asn Asn Asp Asn Val Val Leu 

80 85 
Arg His Tyr Glu Asn Met Ala Val Asp Glu Cys 

90 95 
Gly Cys Arg 
100. 

(2) INFORMATION FOR SEQ ID NO: 13: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 102 amino acids 

(B) TYPE: amino acids 

(C) TOPOLOGY: linear 

(ii) MOLECULE TYPE: protein 

(ix) FEATURE: 

(A) NAME: Vgr-l(fx) 

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: 

Cys Lys Lys His Glu Leu Tyr Val Ser Phe Gin 

1 5 10 

Asp Val Gly Trp Gin Asp Trp He He Ala Pro 

15 20 
Xaa Gly Tyr Ala Ala Asn Tyr Cys Asp Gly Glu 
25 30 



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PCr/US92/07432 



Cys Ser Phe Pro Leu Asn Ala His Met Asn Ala 

35 40 
Thr Asn His Ala He Val Gin Thr Leu Val His 
45 50 55 

Val Met Asn Pro Glu Tyr Val Pro Lys Pro Cys 

60 65 
Cys Ala Pro Thr Lys Val Asn Ala He Ser Val 

70 75 
Leu Tyr Phe Asp Asp Asn Ser Asn Veil He Leu 

80 85 
Lys Lys Tyr Arg Asn Met Val Val Arg Ala Cys 

90 95 
Gly Cys His 
100 

(2) INFORMATION FOR SEQ ID NO: 14: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 106 amino acids 

(B) TYPE: protein 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: protein 

(vi) ORIGINAL SOURCE: 
(A) ORGANISM: human 
(F) TISSUE TYPE: BRAIN 

(ix) FEATURE: 
(D) OTHER INFORMATION: 

/product^ "GDF-1 (fx)" 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: 

Cys Arg Ala Arg Arg Leu Tyr Val Ser Phe Arg Glu Val Gly 
1 5 10 

Trp His Arg Trp Val He Ala Pro Arg Gly Phe Leu Ala Asn Tyr 
15 20 25 

Cys Gin Gly Gin Cys Ala Leu Pro Val Ala Leu Ser Gly Ser Gly 
30 35 40 



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PCr/US92/07432 



Gly Pro Pro Ala Leu Asn His Ala Val Leu Arg Ala Leu Met His 
« 50 55 

Ala Ala Ala Pro Gly Ala Ala Asp Leu Pro Cys Cys Val Pro Ala 
60 65 70 

Arg Leu Ser Pro He Ser Val Leu Phe Phe Asp Asn Ser Asp Asn 
75 80 85 

Val Val Leu Arg Gin Tyr Glu Asp Met Val Val Asp Glu Cys Gly 
90 95 100 

Cys Arg 
105 

(2) INFORMATION FOR SEQ ID NO: 15: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 5 amino acids 

(B) TYPE: amino acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: peptide 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15: 

Cys Xaa Xaa Xaa Xaa 
1 5 
(2) INFORMATION FOR SEQ ID NO: 16: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 1822 base pairs 

(B) TYPE: nucleic acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: cDNA 

(vi) ORIGINAL SOURCE: 

(A) ORGANISM: HOMO SAPIENS - 
(F) TISSUE TYPE: HIPPOCAMPUS 

(ix) FEATURE: 

(A) NAME/KEY: CDS 

(B) LOCATION: A9..1341 

(D) OTHER INFORMATION: /standard_name= "hOPl" 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16: 

GGTGCGGGCC CGGAGCCCGG AGCCCGGGTA GCGCGTAGAG CCGGCGCG ATG CAC GTG 57 

Met His Val 
1 



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PCr/US92/07432 



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 
5 10 15 

CCC CTG TTC CTG CTG CGC TGC GCC CTG GCC GAC TTC AGC CTG GAC AAC 153 
Pro Leu Phe Leu Leu Arg Ser Ala Leu Ala Asp Phe Ser Leu Asp Asn 
20 25 30 35 

GAG GTG CAC TCG AGC TTC ATC CAC CGG CGC CTC CGC AGC CAG GAG CGG 201 
Glu Val His Ser Ser Phe He His Arg Arg Leu Arg Ser Gin Glu Arg 
40 45 50 

CGG GAG ATG CAG CGC GAG ATC CTC TCC ATT TTG GGC TTG CCC CAC CGC 249 
Arg Glu Het Gin Arg Glu He Leu Ser He Leu Gly Leu Pro His Arg 
55 60 65 

CCG CGC CCG CAC CTC CAG GGC AAG CAC AAC TCG GCA CCC ATG TTC ATG 297 
PrBo Arg Pro His Leu Gin Gly Lys His Asn Ser Ala Pro Met Phe Met 
70 75 80 

CTG GAC CTG TAC AAC GCC ATG GCG GTG GAG GAG GGC GGC GGG CCC GGC 345 
Leu Asp Leu lyr Asn Ala Met Ala Val Glu Glu Gly Gly Gly Pro Gly 
85 90 95 

GGC CAG GGC TTC TCC TAC CCC TAC AAG GCC GTC TTC AGT ACC CAG GGC 393 
Gly Gin Gly Phe Ser Tyr Pro Tyr Lys Ala Val Phe Ser Thr Gin Gly 
100 105 110 115 

CCC CCT CTG GCC AGC CTG CAA GAT AGC CAT TTC CTC ACC GAC GCC GAC 441 
Pro Pro Leu Ala Ser Leu Gin Asp Ser His Phe Leu Thr Asp Ala Asp 
120 125 130 

ATG GTC ATG AGC TTC GTC AAC CTC GTG GAA CAT GAC AAG GAA TTC TTC 489 
Het Val Met Ser Phe Val Asn Leu Val Glu His Asp Lys Glu Phe Phe 
135 140 145 

CAC CCA CGC TAC CAC CAT CGA GAG TTC CGG TTT GAT CTT TCC AAG ATC 537 
His Pro Arg Tyr His His Arg Glu Phe Arg Phe Asp Leu Ser Lys He 
150 155 160 

CCA GAA GGG GAA GCT GTC ACG GCA GCC GAA TTC C€G ATC TAC AAG GAC 585 
Pro Glu Gly Glu Ala Val Thr Ala Ala Glu Phe Arg He Tyr Lys Asp 
165 170 175 

TAC ATC CGG GAA CGC TTC GAC AAT GAG ACG TTC C-GG ATC AGC GTT TAT 633 
Tyr He Arg Glu Arg Phe Asp Asn Glu Thr Phe Arg He Ser Val Tyr 
180 185 190 195 

CAG GTG CTC CAG GAG CAC TTG €GC AGG GAA TCG GAT CTC TTC CTG CTC 681 
Gin Val Leu Gin Glu His Leu Gly Arg Glu Ser Asp Leu Phe Leu Leu 
200 205 210 



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GAC AGC CGT ACC CTC TGG GCC TCG GAG GAG GGC TGG CTG GTG TTT GAC 729 
Asp Ser Arg Thr Leu Trp Ala Ser Glu Glu Gly Trp Leu Val Phe Asp 
215 220 225 

ATC ACA GCC ACC AGC AAC CAC TGG GTG GTC AAT CCG CGG CAC AAC CTG 777 
lie Thr Ala Thr Ser Asn His Trp Val Val Asn Pro Arg His Asn Leu 
230 235 240 

GGC CTG CAG CTC TCG GTG GAG ACG CTG GAT GGG CAG AGC ATC AAC CCC 825 
Gly Leu Gin Leu Ser Val Glu Thr Leu Asp Gly Gin Ser He Asn Pro 
245 250 255 

AAG TTG GCG GGC CTG ATT GGG CGG CAC GGG CCC CAG AAC AAG GAG CCC 873 
Lys Leu Ala Gly Leu He Gly Arg His Gly Pro Gin Asn Lys Gin Pro 
260 265 270 275 

TTC ATG GTG GCT TTC TTC AAG GCC ACG GAG GTC CAC TTC CGC AGC ATC 921 
Phe Met Val Ala Phe Phe Lys Ala Thr Glu Val His Phe Arg Ser He 
280 285 290 

CGG TCC ACG GGG AGC AAA CAG CGC AGC CAG AAC CGC TCC AAG ACG CCC 969 
Arg Ser Thr Gly Ser Lys Gin Arg Ser Gin Asn Arg Ser Lys Thr Pro 
295 300 305 

AAG AAC CAG GAA GCC CTG CGG ATG GCC AAC GTG GCA GAG AAC AGC AGC 1017 
Lys Asn Gin Glu Ala Leu Arg Met Ala Asn Val Ala Glu Asn Ser Ser 
310 315 320 

AGC GAC CAG AGG CAG GCC TGT AAG AAG CAC GAG CTG TAT GTC AGC TTC 1065 
Ser Asp Gin Arg Gin Ala Cys Lys Lys His Glu Leu Tyr Val Ser Phe 
325 330 335 

CGA GAC CTG GGC TGG CAG GAC TGG ATC ATC GCG CCT GAA GGC TAC GCC 1113 
Arg Asp Leu Gly Trp Gin Asp Trp He He Ala Pro Glu Gly Tyr Ala 
340 345 350 355 

GCC TAC TAC TGT GAG GGG GAG TGT GCC TTC CCT CTG AAC TCC TAC ATG 1161 
Ala Tyr Tyr Cys Glu Gly Glu Cys Ala Phe Pro Leu Asn Ser Tyr Met 
360 365 370 

AAC GCC ACC AAC CAC GCC ATC GTG CAG ACG CTG GTC CAC TTC ATC AAC 1209 
Asn Ala Thr Asn His Ala He Val Gin Thr Leu Val His Phe He Asn 
375 380 385 

CCG GAA ACG GTG CCC AAG CCC TGC TGT GCG CCC ACG CAG CTC AAT GCC 1257 
Pro Glu Thr Val Pro Lys Pro Cys Cys Ala Pro Thr Gin Leu Asn Ala 
390 395 400 

ATC TCC GTC CTC TAC TTC GAT GAC AGC TCC AAC GTC ATC CTG AAG AAA 13G5 
He Ser Val Leu Tyr Phe Asp Asp Ser Ser Asn Val He Leu Lys Lys 
405 410 415 



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TAC AGA AAC ATG GTG GTC CGG GCC TGT GGC TGC CAC TAGCTCCTCC 1351 
Tyr Arg Asn Met Val Val Arg Ala Cys Gly Cys His 
420 425 430 

GAGAATTCAG ACCCTTTGGG GCCAAGTTTT TCTGGATCCT CCATTGCTCG CCTTGGCCAG 1411 

GAACCAGCAG ACCAACTGCC TTTTGTGAGA CCTTCCCCTC CCTATCCCCA ACTTTAAAGG 1471 

TGTGAGAGTA TTAGGAAACA TGAGCAGCAT ATGGCTTTTG ATCAGTTTTT CAGTGGCAGC 1531 

ATCCAATGAA CAAGATCCTA CAAGCTGTGC AGGCAAAACC TAGCAGGAAA AAAAAACAAC 1591 

GCATAAAGAA AAATGGCCGG GCCAGGTCAT TGGCTGGGAA GTCTCAGCCA TGCACGGACT 1651 

CGTTTCCAGA GGTAATTATG AGCGCCTACG AGCCAGGCCA CCCAGCCGTG GGAGGAAGGG 1711 

GGCGTGGCAA GGGGTGGGCA CATTGGTGTC TGTGCGAAAG GAAAATTGAC CCGGAAGTTC 1771 

CTGTAATAAA TGTCACAATA AAACGAATGA ATGAAAAAAA AAAAAAAAAA A 1822 
(2) INFORMATION FOR SEQ ID NO: 17: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 431 amino acids 

(B) TYPE: amino acid 
(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: protein 

(ix) FEATURE: 

(D) OTHER INFORMATION: /Product="OPl-PP" 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17: 

Met His Val Arg Ser Leu Arg Ala Ala Ala Pro His Ser Phe Val Ala 
15 10 15 

Leu Trp Ala Pro Leu Phe Leu Leu Arg Ser Ala Leu Ala Asp Phe Ser 
20 25 30 

Leu Asp Asn Glu Val His Ser Ser Phe He His Arg Arg Leu Arg Ser 
35 40 45 

Gin Glu Arg Arg Glu Met Gin Arg Glu He Leu Ser He Leu Gly Leu 
50 55 60 

Pro His Arg Pro Arg Pro His Leu Gin Gly Lys His Asn Ser Ala Pro 
65 70 75 80 

Met Phe Met Leu Asp Leu Tyr Asn Ala Met Ala Val Glu Glu Gly Gly 
85 90 95 



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Gly Pro Gly Gly Gin Gly Phe Set Tyx Pro Tyr Lys Ala Val Phe Ser 
100 105 110 

Thr Gin Gly Pro Pro Leu Ala Ser Leu Gin Asp Ser His Phe Leu Thr 
115 120 125 

Asp Ala Asp Met Val Het Ser Phe Val Asn Leu Val Glu His Asp Lys 
130 135 140 

Glu Phe Phe His Pro Arg Tyr His His Arg Glu Phe Arg Phe Asp Leu 
1« 150 155 160 

Ser Lys He Pro Glu Gly Glu Ala Val Thr Ala Ala Glu Phe Arg He 
165 170 175 

Tyr Lys Asp Tyr He Arg Glu Arg Phe Asp Asn Glu Thr Phe Arg He 
180 185 190 

Ser Val Tyr Gin Val Leu Gin Glu His Leu Gly Arg Glu Ser Asp Leu 
195 200 205 

Phe Leu Leu Asp Ser Arg Thr Leu Trp Ala Ser Glu Glu Gly Trp Leu 
210 215 220 

Val Phe Asp He Thr Ala Thr Ser Asn His Trp Val Val Asn Pro Are 
225 230 235 240 

His Asn Leu Gly Leu Gin Leu Ser Val Glu Thr Leu Asp Gly Gin Ser 
245 250 255 

He Asn Pro Lys Leu Ala Gly Leu He Gly Arg His Gly Pro Gin Asn 
260 265 270 

Lys Gin Pro Phe Met Val Ala Phe Phe Lys Ala Thr Glu Val His Phe 
275 280 285 

Arg Ser He Arg Ser Thr Gly Ser Lys Gin Arg Ser Gin Asn Arg Ser 
290 295 300 

Lys Thr Pro Lys Asn Gin Glu Ala Leu Arg Met Ala Asn Val Ala Glu 
305 310 315 320 

Asn Ser Ser Ser Asp Gin Arg Gin Ala Cys Lys Lys His Glu Leu Tyr 
325 330 335 

VIBal Ser Phe Arg Asp Leu Gly Trp Gin Asp Trp He He Ala Pro Glu 
340 345 350 

Gly Tyr Ala Ala Tyr Tyr Cys Glu Gly Glu Cys Ala Phe Pro Leu Asn 
355 360 365 



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Ser Tyr Met Asn Ala Thr Asn His Ala He Val Gin Thr Leu Val His 
370 375 380 

Phe He Asn Pro Glu Thr Val Pro Lys Pro Cys Cys Ala Pro Thr Gin 
385 390 395 400 

Leu Asn Ala He Ser Val Leu Tyr Phe Asp Asp Ser Ser Asn Val He 
405 410 415 

Leu Lys Lys Tyr Arg Asn Met Val Val Arg Ala Cys Gly Cys His 
420 425 430 



(2) INFORMATION FOR SEQ ID NO: 18: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 1873 base pairs 

(B) TYPE: nucleic acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: cDNA 

(vi) ORIGINAL SOURCE: 

(A) ORGANISM: MURIDAE 
(F) TISSUE TYPE: EMBRYO 

(ix) FEATURE: 

(A) NAME/KEY: CDS 

(B) LOCATION: 104.. 1393 

(D) OTHER INFORMATION: /note= "MOPl (CDNA)" 



(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18: 

CTGCAGCAAG TGACCTCGGG TCGTGGACCG CTGCCCTGCC CCCTCCGCTG CCACCTGGGG 60 

CGGCGCGGGC CCGGTGCCCC GGATCGCGCG TAGAGCCGGC GCG ATG CAC GTG CGC 115 

Met His Val Arg 
1 

TCG CTG CGC GCT GCG GCG CCA CAC AGC TTC GTG GCG CTC TGG GCG CCT 163 
Ser Leu Arg Ala Ala Ala Pro His Ser Phe Val Ala Leu Trp Ala Pro 
5 10 15 20 

CTG TTC TTG CTG CGC TCC GCC CTG GCC GAT TTC AGC CT-G GAC AAC GAG 211 
Leu Phe Leu Leu Arg Ser Ala Leu Ala Asp Phe Ser Leu Asp Asn Glu 
25 30 35 

GTG CAC TCC AGC TTC ATC CAC CGG CGC CTC CGC AGC CAG GAG CGG CGG 259 
Val His Ser Ser Phe He His Arg Arg Leu Arg Ser Gin Glu Arg Arg 
40 45 50 



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GAG ATG CAG CGG GAG ATC CTG TCC ATC TTA GGG TTG CCC CAT CGC CCG 
Glu Met Gin Arg Glu He Leu Ser He Leu Gly Leu Pro His Ate Pro 
55 60 65 

CGC CCG CAC CTC CAG GGA AAG CAT AAT TCG GCG CCC ATG TTC ATG TTG 
Arg Pro His Leu Gin Gly Lys His Asn Ser Ala Pro Met Phe Met Leu 
70 75 80 

GAC CTG TAC AAC GCC ATG GCG GTG GAG GAG AGC GGG CCG GAC GGA CAG 
Asp Leu Tyr Asn Ala Met Ala Val Glu Glu Ser Gly Pro Asp Gly Gin 
85 90 95 100 

GGC TTC TCC TAC CCC TAC AAG GCC GTC TTC AGT ACC CAG GGC CCC CCT 
Gly Phe Ser Tyr Pro Tyr Lys Ala Val Phe Ser Thr Gin Gly Pro Pro 
105 110 115 

TTA GCC AGC CTG CAG GAC AGC CAT TTC CTC ACT GAC GCC GAC ATG GTC 
Leu Ala Ser Leu Gin Asp Ser His Phe Leu Thr Asp Ala Asp Met Val 
120 125 130 

ATG AGC TTC GTC AAC CTA GTG GAA CAT GAC AAA GAA TTC TTC CAC CCT 
Met Ser Phe Val Asn Leu Val Glu His Asp Lys Glu Phe Phe His Pro 

135 140 145 

CGA TAC CAC CAT CGG GAG TTC CGG TTT GAT CTT TCC AAG ATC CCC GAG 
Arg Tyr His His Arg Glu Phe Arg Phe Asp Leu Ser Lys He Pro Glu 
150 155 160 

GGC GAA CGG GTG ACC GCA GCC GAA TTC AGG ATC TAT AAG GAC TAC ATC 
Gly Glu Arg Val Thr Ala Ala Glu Phe Arg He Tyr Lys Asp Tyr He 
165 170 175 *^ 

CGG GAG CGA TTT GAC AAC GAG ACC TTC CAG ATC ACA GTC TAT CAG GTG 
Arg Glu Arg Phe Asp Asn Glu Thr Phe Gin He Thr Val Tyr Gin Val 
185 190 195 

CTC CAG GAG CAC TCA GGC AGG GAG TCG GAC CTC TTC TTG CTG GAC AGC 
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 
Arg Thr He Trp Ala Ser Glu Glu Gly Trp Leu Val Phe Asp He Thr 
215 220 225 

GCC ACC AGC AAC CAC TGG GTG GTC AAC CCT CGG CAC AAC CTG GGC TTA 
Ala Thr Ser Asn His Trp Val Val Asn Pro Arg His Asn Leu Gly Leu 
230 235 240 

CAG CTC TCT GTG GAG ACC CTG GAT CGG CAG AGC ATC AAC CCC AAG TTG 
Gin Leu Ser Val Glu Thr Leu Asp Gly Gin Ser He Asn Pro Lys Leu 
2^5 250 255 260 



307 

355 

403 

451 

499 

547 
595 

643 

691 

739 
787 
835 
883 



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GCA GGC CTG ATT GGA CGG CAT GGA CCC CAG AAC AAG CAA CCC TTC ATG 931 
Ala Gly Leu He 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 He 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 
Gin Glu Ala Leu Arg Met Ala Ser Val Ala Glu Asn Ser Ser Ser Asp 
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 He He 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 

ACCB AAC CAC GCC ATC GTC CAG ACA CTG GTT CAC TTC ATC AAC CCA GAC 1267 
Thr Asn His Ala He Val Gin Thr Leu Val His Phe He Asn Pro Asp 
375 380 385 

ACA GTA CCC AAG CCC TGC TGT GCG CCC ACC CAG CTC AAC GCC ATC TCT 1315 
Thr Val Pro Lys Pro Cys Cys Ala Pro Thr Gin Leu Asn Ala He Ser 
390 395 400 

GTC CTC TAC TTC GAC GAC AGC TCT AAT GTC ATC CTG AAG AAG TAC AGA 1363 
Val Leu Tyr Phe Asp Asp Ser Ser Asn Val He Leu Lys Lys Tyr Arg 
405 410 415 420 



AAC ATG GTG GTC CGG GCC TGT GGC TGC CAC TAGCTCTTCC TGAGACCCTG 1413 
Asn Met Val Val Arg Ala Cys Gly Cys His 
425 430 

ACCTTTGCGG GGCCACACCT TTCCAAATCT TCGATGTCTC ACCATCTAAG TCTCTCACTG 1473 

CCCACCTTGG CGAGGAGAAC AGACCAACCT CTCCTGAGCC TTCCCTCACC TCCCAACCGG 1533 

AAGCATGTAA GGGTTCCAGA AACCTGAGCG TGCAGCAGCT GATGAGGGCC CTTTCCTTCT 1593 

GGCACGTGAC GGACAAGATC CTACCAGCTA CCACAGCAAA CGCCTAAGAG CAGGAAAAAT 1653 



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GTCTGCCAGG AAAGTGTCCA GTGTCCACAT GGCCCCTGGC GCTCTGAGTC TTTGAGGAGT 1713 

AATCGCAAGC CTCGTTCAGC TGCAGCAGAA GGAAGGGCTT AGCCAGGGTG GGCGCTGGCG 1773 

TCTGTGTTGA AGGGAAACCA AGCAGAAGCC ACTGTAATGA TATGTCACAA TAAAACCCAT 1833 

GAATGAAAAA AAAAAAAAAA AAAAAAAAAA AAAAGAATTC 1873 

(2) INFORMATION FOR SEQ ID NO: 19: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: A30 amino acids 

(B) TYPE: amino acid 
(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: protein 

(ix) FEATURE: 

(D) OTHER INFORMATION: /product= "mOPl-PP" 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19: 

Met His Val Arg Ser Leu Arg Ala Ala Ala Pro His Ser Phe Val Ala 
15 10 15 

Leu Trp Ala Pro Leu Phe Leu Leu Arg Ser Ala Leu Ala Asp Phe Ser 
20 25 30 

Leu Asp Asn Glu Val His Ser Ser Phe He His Arg Arg Leu Arg Ser 
35 40 45 

Gin Glu Arg Arg Glu Met Gin Arg Glu He Leu Ser He Leu Gly Leu 
50 55 60 

Pro His Arg Pro Arg Pro His Leu Gin Gly Lys His Asn Ser Ala Pro 
65 70 75 80 

Met Phe Met Leu Asp Leu Tyr Asn Ala Met Ala Val Glu Glu Ser Gly 
85 90 95 

Pro Asp Gly Gin Gly Phe Ser Tyr Pro Tyr Lys Ala Val Phe Ser Thr 
100 105 110 

Gin Gly Pro Pro Leu Ala Ser Leu Gin Asp Ser His Phe Leu Thr Asp 
115 120 125 

Ala Asp Met Val Met Ser Phe Val Asn Leu Val Glu His Asp Lys Glu 
130 135 140 

Phe Phe His Pro Arg Tyr His His Arg Glu Phe Arg Phe Asp Leu Ser 
145 150 155 160 



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Lys He Pro Glu Gly Glu Arg Val Thr Ala Ala Glu Phe Arg He Tyr 
165 170 175 

Lys Asp Tyr He Arg Glu Arg Phe Asp Asn Glu Thr Phe Gin He Thr 
180 185 190 

Val Tyr Gin Val Leu Gin Glu His Ser Gly Arg Glu Ser Asp Leu Phe 
195 200 205 

Leu Leu Asp Ser Arg Thr He Trp Ala Ser Glu Glu Gly Trp Leu Val 
210 215 220 

Phe Asp He Thr Ala Thr Ser Asn His Trp Val Val Asn Pro Arg His 
225 230 235 240 

Asn Leu Gly Leu Gin Leu Ser Val Glu Thr Leu Asp Gly Gin Ser He 
245 250 255 

Asn Pro Lys Leu Ala Gly Leu He Gly Arg His Gly Pro Gin Asn Lys 
260 265 270 

Gin Pro Phe Met Val Ala Phe Phe Lys Ala Thr Glu Val His Leu Arg 
275 280 285 

Ser He Arg Ser Thr Gly Gly Lys Gin Arg Ser Gin Asn Arg Ser Lys 
290 295 300 

Thr Pro Lys Asn Gin Glu Ala Leu Arg Met Ala Ser Val Ala Glu Asn 
305 310 315 320 

Ser Ser Ser Asp Gin Arg Gin Ala Cys Lys Lys His Glu Leu Tyr Val 
325 330 335 

Ser Phe Arg Asp Leu Gly Trp Gin Asp Trp He He Ala Pro Glu Gly 
340 345 350 

Tyr Ala Ala Tyr Tyr Cys Glu Gly Glu Cys Ala Phe Pro Leu Asn Ser 
355 360 365 

Tyr Met Asn Ala Thr Asn His Ala He Val Gin Thr Leu Val His Phe 
370 375 380 

He 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 He Leu 
405 410 415 



Lys Lys Tyr Arg Asn Met Val Val Arg Ala Cys Gly Cys His 
420 425 430 



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(2) INFORMATION FOR SEQ ID NO: 20: 

(1) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 1723 base pairs 

(B) TYPE: nucleic acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 

(ii)MOLECULE TYPE: cDNA 

(vi) ORIGINAL SOURCE: 

(A) ORGANISM: Homo sapiens 
(F) TISSUE TYPE: HIPPOCAMPUS 

(ix) FEATURE: 

(A) NAME/KEY: CDS 

(B) LOCATION: 490.. 1696 

(D) OTHER INFORMATION: /note= "hOP2 (cDNA)" 

(xi)SEQUENCE DESCRIPTION: SEQ ID N0:20: 

GGCGCCGGCA GAGCAGGAGT GGCTGGAGGA GCTGTGGTTG GAGCAGGAGG TGGCACGGCA 60 

GGGCTGGAGG GCTCCCTATG AGTGGCGGAG ACGGCCCAGG AGGCGCTGGA GCAACAGCTC 120 

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 G<5GCGTCCCC 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 10 

GCG CTA TGC GCG CTG GGC 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 
15 20 25 

GGC TGT CCC CAG CGA CGT CTG GGC GCG CGC GAG CGC CGG GAC GTG CAG 624 
Gly Cys Pro Gin Arg Arg Leu Gly Ala Arg Glu Arg Arg Asp Val Gin 
30 35 1,0 45 

CGC GAG ATC CTG GCG GTG CTC GGG CTG CCT GGG CGG CCC CGG CCC CGC 672 
Arg Glu He Leu Ala Val Leu Gly Leu Pro Gly Arg Pro Arg Pro Arg 
50 55 60 



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GCG CCA CCC GCC GCC TCC CGG CTG CCC GCG TCC GCG CCG CTC TTC ATG 720 
Ala Pro Pro Ala Ala Ser Arg Leu Pro Ala Ser Ala Pro Leu Phe Met 
65 70 75 

CTG GAC CTG TAC CAC GCC ATG GCC GGC GAC GAC GAC GAG GAC GGC GCG 768 
Leu Asp Leu Tyr His Ala Met Ala Gly Asp Asp Asp Glu Asp Gly Ala 
80 85 90 

CCC GCG GAG CGG CGC CTG GGC CGC GCC GAC CTG GTC ATG AGC TTC GTT 816 
Pro Ala Glu Arg Arg Leu Gly Arg Ala Asp Leu Val Met Ser Phe Val 
95 100 105 

AAC ATG GTG GAG CGA GAC CGT GCC CTG GGC CAC CAG GAG CCC CAT TGG 864 
Asn Met Val Glu Arg Asp Arg Ala Leu Gly His Gin Glu Pro His Trp 
110 115 120 125 

AAG GAG TTC CGC TTT GAC CTG ACC CAG ATC CCG GCT GGG GAG GCG GTC 912 
Lys Glu Phe Arg Phe Asp Leu Thr Gin He Pro Ala Gly Glu Ala Val 
130 135 140 

ACA GCT GCG GAG TTC CGG ATT TAC AAG GTG CCC AGC ATC CAC CTG CTC 960 
Thr Ala Ala Glu Phe Arg He Tyr Lys Val Pro Ser He His Leu Leu 
145 150 155 

AAC AGG ACC CTC CAC GTC AGC ATG TTC CAG GTG GTC CAG GAG CAG TCC 1008 
Asn Arg Thr Leu His Val Ser Met Phe Gin Val Val Gin Glu Gin Ser 
160 165 170 

AAC AGG GAG TCT GAC TTG TTC TTT TTG GAT CTT CAG ACG CTC CGA GCT 1056 
Asn Arg Glu Ser Asp Leu Phe Phe Leu Asp Leu Gin Thr Leu Arg Ala 
175 180 185 

GGA GAC GAG GGC TGG CTG GTG CTG GAT GTC ACA GCA GCC AGT GAC TGC 1104 
Gly Asp Glu Gly Trp Leu Val Leu Asp Val Thr Ala Ala Ser Asp Cys 
190 195 200 205 

TGG TTG CTG AAG CGT CAC AAG GAC CTG GGA CTC CGC CTC TAT GTG GAG 1152 
Trp Leu Leu Lys Arg His Lys Asp Leu Gly Leu Arg Leu Tyr Val Glu 
210 215 220 

ACT GAG GAC GGG CAC AGC GTG CAT CCT GGC CTG GCC GGC CTG CTG GGT 1200 
Thr Glu Asp Gly His Ser Val Asp Pro Gly Leu Ala Gly Leu Leu Gly 
225 230 235 

CAA CGG GCC CCA CGC TCC CAA CAG CCT TTC GTG GTC ACT TTC TTC AGG 1248 
Gin Arg Ala Pro Arg Ser Gin Gin Pro Phe Val Val Thr Phe Phe Arg 
240 245 250 

GCC AGT CCG AGT CCC ATC CGC ACC CCT CGG GCA GTG AGG CCA CTG AGG 1296 
Ala Ser Pro Ser Pro He Arg Thr Pro Arg Ala Val Arg Pro Leu Arg 
255 260 265 



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AGG AGG CAG COG AAG AAA AGC AAC GAG CTG CCG CAG GCC AAC CGA CTC 1344 
Arg Arg Gin Pro Lys Lys Ser Asn Glu Leu Pro Gin Ala Asn Are Leu 
270 275 280 285 

CCA GGG ATC TTT GAT GAC GTC CAC GGC TCC CAC GGC CGG CAG GTC TGC 1392 
Pro Gly He Phe Asp Asp Val His Gly Ser His Gly Arg Gin Val Cys 
290 295 300 

CGT CGG CAC GAG CTC TAC GTC AGC TTC CAG GAC CTC GGC XCG CTG GAC 1440 
Arg Arg His Glu Leu Tyr Val Ser Phe Gin Asp Leu Gly Trp Leu Asp 
305 310 315 

TGG GTC ATC GCT CCC CAA GGC TAC TCG GCC TAT TAC TGT GAG GGG GAG 1488 
Trp Val He Ala Pro Gin Gly Tyr Ser Ala Tyr TVr Cys Glu Gly Glu 
320 325 330 

TGC TCC TTC CCA CTG GAC TCC TGC ATG AAT GCC ACC AAC CAC GCC ATC 1536 
Cys Ser Phe Pro Leu Asp Ser Cys Met Asn Ala Thr Asn His Ala He 
335 340 345 

CTG CAG TCC CTG GTG CAC CTG ATG AAG CCA AAC GCA GTC CCC AAG GCG 1584 
Leu Gin Ser Leu Val His Leu Met Lys Pro Asn Ala Val Pro Lys Ala 
350 355 360 365 

TGC TGT GCA CCC ACC AAG CTG AGC GCC ACC TCT GTG CTC TAC TAT GAC 1632 
Cys Cys Ala Pro Thr Lys Leu Ser Ala Thr Ser Val Leu Tyr Tyr Asp 
370 375 380 

AGC AGC AAC AAC GTC ATC CTG CGC AAA CAC CGC AAC ATG GTG GTC AAG 1680 
Ser Ser Asn Asn Val He Leu Arg Lys His Arg Asn Met Val Val Lys 
385 390 395 

GCC TGC GGC TGC CAC T GAGTCAGCCC GCCCAGCCCT ACTGCAG 1723 
Ala Cys Gly Cys His 
400 

(2) INFORMATION FOR SEQ ID NO: 21: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 402 amino acids 

(B) TYPE: amino acid 
(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: protein 

(ix) FEATURE: 

(A)OTHER INFORMATION: /product= "hOP2-PP'' 

(xi)SEQUENCE DESCRIPTION: SEQ ID N0:21: 

Met Thr Ala Leu Pro Gly Pro Leu Trp Leu Leu Gly Leu Ala Leu Cys 
1 5 10 15 



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Ala Leu Gly Gly Gly Gly Pro Gly Leu Arg Pro Pro Pro Gly Cys Pro 
20 25 30 

Gin Arg Arg Leu Gly Ala Arg Glu Arg Arg Asp Val Gin Arg Glu He 
35 40 45 

Leu Ala Val Leu Gly Leu Pro Gly Arg Pro Arg Pro Arg Ala Pro Pro 
50 55 60 

Ala Ala Ser Arg Leu Pro Ala Ser Ala Pro Leu Phe Met Leu Asp Leu 
65 70 75 80 

Tyr His Ala Met Ala Gly Asp Asp Asp Glu Asp Gly Ala Pro Ala Glu 
85 90 95 

Arg Arg Leu Gly Arg Ala Asp Leu Val Met Ser Phe Val Asn Met Val 
100 105 110 

Glu Arg Asp Arg Ala Leu Gly His Gin Glu Pro His Trp Lys Glu Phe 
115 120 125 

Arg Phe Asp Leu Thr Gin He Pro Ala Gly Glu Ala Val Thr Ala Ala 
130 135 140 

Glu Phe Arg He Tyr Lys Val Pro Ser He His Leu Leu Asn Arg Thr 
145 150 155 160 

Leu His Val Ser Met Phe Gin Val Val Gin Glu Gin Ser Asn Arg Glu 
165 170 175 

Ser Asp Leu Phe Phe Leu Asp Leu Gin Thr Leu Arg Ala Gly Asp Glu 
180 185 190 

Gly Trp Leu Val Leu Asp Val Thr Ala Ala Ser Asp Cys Trp Leu Leu 
195 200 205 

Lys Arg His Lys Asp Leu Gly Leu Arg Leu Tyr Val Glu Thr Glu Asp 
210 215 220 

Gly His Ser Val Asp Pro Gly Leu Ala Gly Leu Leu Gly Gin Arg Ala 
225 230 235 240 

Pro Arg Ser Gin Gin Pro Phe Val Val Thr Phe Phe Arg Ala Ser Pro 
245 250 255 

Ser Pro He Arg Thr Pro Arg Ala Val Arg Pro Leu Arg Arg Arg Gin 
260 265 270 

Pro Lys Lys Ser Asn Glu Leu Pro Gin Ala Asn Arg Leu Pro Gly He 
275 280 285 

Phe Asp Asp Val His Gly Ser His Gly Arg Gin Val Cys Arg Arg His 
290 295 300 



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Glu Leu lyr Val Set Phe Gin Asp Leu Gly Trp Leu Asp Trp Val lie 
305 310 315 320 

Ala Pro Gin Gly Tyr Ser Ala Tyr Tyr Cys Glu Gly Glu Cys Ser Phe 
325 330 335 

Pro Leu Asp Ser Cys Met Asn Ala Thr Asn His Ala lie Leu Gin Ser 
340 345 350 

Leu Val His Leu Met Lys Pro Asn Ala Val Pro Lys Ala Cys Cys Ala 
355 360 365 

Pro Thr Lys Leu Ser Ala Thr Ser Val Leu Tyr Tyr Asp Ser Ser Asn 
370 375 380 

Asn Val He Leu Arg Lys His Arg Asn Met Val Val Lys Ala Cys Gly 
385 390 395 400 

Cys His 

(2) INFORMATION FOR SEQ ID N0:22: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 1926 base pairs 

(B) TYPE; nucleic acid 

(C) STRANDEDNESS: single 
(D TOPOLOGY: linear 

(ii) MOLECULE TYPE: cDNA 

(vi) ORIGINAL SOURCE: 

(A) ORGANISM: MURIDAE 
(F) TISSUE TYPE: EMBRYO 

(ix) FEATURE: 

(A) NAME/KEY: CDS 

(B) LOCATION: 93.. 1289 

(D) OTHER INFORMATION: /note= "mOP2 cDNA" 
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:22: 

GCCAGGCACA GGTGCGCCGT CTGGTCCTCC CCGTCTGGCG TCAGCCGAGC 50 

CCGACCAGCT ACCAGTGGAT GCGCGCCGGC TGAAAGTCCG AG ATG GCT ATG CGT 104 

Met Ala Met Arg 
1 

CCC GGG CCA CTC TGG CTA TTG GGC CTT GCT CTG TGC GCG CTG GGA GGC 152 
Pro Gly Pro Leu Trp Leu Leu Gly Leu Ala Leu Cys Ala Leu Gly Gly 
5 10 15 20 



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GGC CAC GGT CCG CGT CCC CCG CAC ACC TGT CCC CAG CGT CGC CTG GGA 200 
Gly His Gly Pro Arg Pro Pro His Thr Cys Pro Gin Arg Arg Leu Gly 
25 30 35 

GCG CGC GAG CGC CGC GAC ATG CAG CGT GAA ATC CTG GCG GTG CTC GGG 248 
Ala Arg Glu Arg Arg Asp Met Gin Arg Glu He Leu Ala Val Leu Gly 
40 45 50 

CTA CCG GGA CGG CCC CGA CCC CGT GCA CAA CCC GCG GCT GCC CGG CAG 296 
Leu Pro Gly Arg Pro Arg Pro Arg Ala Gin Pro Ala Ala Ala Arg Gin 
55 60 65 

CCA GCG TCC GCG CCC CTC TTC ATG TTG GAC CTA TAC CAC GCC ATG ACC 344 
Pro Ala Ser Ala Pro Leu Phe Met Leu Asp Leu Tyr His Ala Met Thr 
70 75 80 

GAT GAC GAC GAC GGC GGG CCA CCA CAG GCT CAC TTA GGC CGT GCC GAC 392 
Asp Asp Asp Asp Gly Gly Pro Pro Gin Ala His Leu Gly Arg Ala Asp 
85 90 95 100 

CTG GTC ATG AGC TTC GTC AAC ATG GTG GAA CGC GAC CGT ACC CTG GGC 440 
Leu Val Met Ser Phe Val Asn Met Val Glu Arg Asp Arg Thr Leu Gly 
105 110 115 

TAC CAG GAG CCA CAC TGG AAG GAA TTC CAC TTT GAC CTA ACC CAG ATC 488 
Tyr Gin Glu Pro His Trp Lys Glu Phe His Phe Asp Leu Thr Gin He 
120 125 130 

CCT GCT GGG GAG GCT GTC ACA GCT GCT GAG TTC CGG ATC TAC AAA GAA 536 
Pro Ala Gly Glu Ala Val Thr Ala Ala Glu Phe Arg He Tyr Lys Glu 
135 140 145 

CCC AGC ACC CAC CCG CTC AAC ACA ACC CTC CAC ATC AGC ATG TTC GAA 584 
Pro Ser Thr His Pro Leu Asn Thr Thr Leu His He Ser Met Phe Glu 
150 155 160 

GTG GTC CAA GAG CAC TCC AAC AGG GAG TCT GAC TTG TTC TTT TTG GAT 632 
Val Val Gin Glu His Ser Asn Arg Glu Ser Asp Leu Phe Phe Leu Asp 
165 170 175 180 

CTT CAG ACG CTC CGA TCT GGG GAC GAG GGC TGG CTG GTG CTG GAC ATC 680 
Leu Gin Thr Leu Arg Ser Gly Asp Glu Gly Trp Leu Val Leu Asp He 
185 190 195 

ACA GCA GCC AGT GAC CGA TGG CTG CTG AAC CAT CAC AAG GAC CTG GGA 728 
Thr Ala Ala Ser Asp Arg Trp Leu Leu Asn His His Lys Asp Leu Gly 
200 205 210 

CTC CGC CTC TAT GTG GAA ACC GCG GAT GGG CAC AGC ATG GAT CCT GGC 776 
Leu Arg Leu Tyr Val Glu Thr Ala Asp Gly His Ser Met Asp Pro Gly 
215 220 225 



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CTG GCT GGT CTG CTT GGA CGA CAA GCA CCA CGC TCC AGA CAG CCT TTC 
Leu Ala Gly Leu Leu Gly Arg Gin Ala Pro Arg Ser Are Gin Pro Phe 
230 235 240 



824 



ATG GTA ACC TTC TTC AGG GCC AGC CAG AGT CCT GTG CGG GCC CCT CGG 872 
Met Val Thr Phe Phe Arg Ala Ser Gin Ser Pro Val Arg Ala Pro Are 
2A5 250 255 260 

GCA GCG AGA CCA CTG AAG AGG AGG CAG CCA AAG AAA ACG AAC GAG CTT 920 
Ala Ala Arg Pro Leu Lys Arg Arg Gin Pro Lys Lys Thr Asn Glu Leu 
265 270 275 

CCG CAC CCC AAC AAA CTC CCA GGG ATC TTT GAT GAT GGC CAC GGT TCC 968 
Pro His Pro Asn Lys Leu Pro Gly lie Phe Asp Asp Gly His Gly Ser 
280 285 290 

CGC GGC AGA GAG GTT TGC CGC AGG CAT GAG CTC TAC GTC AGC TTC CGT 1016 
Arg Gly Arg Glu Val Cys Arg Arg His Glu Leu TVr Val Ser Phe Arc 
295 300 305 

GAC CTT GGC TGG CTG GAC TGG GTC ATC GCC CCC CAG GGC TAC TCT GCC 1064 
Asp Leu Gly Trp Leu Asp Trp Val He Ala Pro Gin Gly Tyr Ser Ala 
310 315 320 

TAT TAC TGT GAG GGG GAG TGT GCT TTC CCA CTG GAC TCC TGT ATG AAC 1112 
Tyr Tyr Cys Glu Gly Glu Cys Ala Phe Pro Leu Asp Ser Cys Met Asn 
325 330 335 340 

GCC ACC AAC CAT GCC ATC TTG CAG TCT CTG GTG CAC CTG ATG AAG CCA 1160 
Ala Thr Asn His Ala He Leu Gin Ser Uu Val His Leu Met Lys Pro 
345 350 355 

GAT GTT GTC CCC AAG GCA TGC TGT GCA CCC ACC AAA CTG AGT GCC ACC 1208 
Asp Val Val Pro Lys Ala Cys Cys Ala Pro Thr Lys Leu Ser Ala Thr 
360 365 370 

TCT GTG CTG TAC TAT GAC AGC AGC AAC AAT GTC ATC CTG CGT AAA CAC 1256 
Ser Val Leu Tyr Tyr Asp Ser Ser Asn Asn Val He Leu Arg Lys His 
375 380 385 

CGT AAC ATG GTG GTC AAG GCC TGT «;C TGC CAC TGAGGCCCCG CCCAGCATCC 1309 
Arg Asn Met Val Val Lys Ala Cys Gly Cys His 
390 395 

TGCTTCTACT ACCTTACCAT CTGGCCGGGC CCCTCTCCAG AGGCAGAAAC CCTTCTATGT 1369 

TATCATAGCT CAGACAGGGG CAATGGGAGG CCCTTCACTT CCCCTGGCCA CTTCCTGCTA 1429 

AAATTCTGGT CTTTCCCAGT TCCTCTGTCC TTCATGGGGT TTCGGGGCTA TCACCCCGCC 1489 

CTCTCCATCC TCCTACCCCA AGCATAGACT GAATCCACAC AGCATCCCAG ACCTATGCTA 1549 



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ACTGAGAGGT CTGGGGTCAG CACTGAAGGC CCACATGAGG AAGACTGATC CTTGGCCATC 1609 

CTCAGCCCAC AATGGCAAAT TCTGGATGGT CTAAGAAGGC CGTGGAATTC TAAACTAGAT 1669 

GATCTGGGCT CTCTGCACCA TTCATTGTGG CAGTTGGGAC ATTTTTAGGT ATAACAGACA 1729 

CATACACTTA GATCAATGCA TCGCTGTACT CCTTGAAATC AGAGCTAGCT TGTTAGAAAA 1789 

AGAATCAGAG CCAGGTATAG CGGTGCATGT CATTAATCCC AGCGCTAAAG AGACAGAGAC 1849 

AGGAGAATCT CTGTGAGTTC AAGGCCACAT AGAAAGAGCC TGTCTCGGGA GCAGGAAAAA 1909 

AAAAAAAAAC GGAATTC 1926 

(2) INFORMATION FOR SEQ ID NO: 23; 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 399 amino acids 

(B) TYPE: amino acid 
(D) TOPOWGY: linear 

(ii) MOLECULE TYPE: protein 

(ix) FEATURE: 

(D) OTHER INFORMATION: /product^ "mOP2-PP" 

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:23: 

Met Ala Met Arg Pro Gly Pro Leu Trp Leu Leu Gly Leu Ala Leu Cys 
1 5 10 15 

Ala Leu Gly Gly Gly His Gly Pro Arg Pro Pro His Thr Cys Pro Gin 
20 25 30 

Arg Arg Leu Gly Ala Arg Glu Arg Arg Asp Met Gin Arg Glu lie Leu Ala 
35 40 45 

Val Leu Gly Leu Pro Gly Arg Pro Arg Pro Arg Ala Gin Pro Ala Ala 
50 55 60 65 

Ala Arg Gin Pro Ala Ser Ala Pro Leu Phe Met Leu Asp Leu Tyr His Ala 
70 75 80 

Met Thr Asp Asp Asp Asp Gly Gly Pro Pro Gin Ala His Leu Gly Arg 
85 90 95 

Ala Asp Leu Val Met Ser Phe Val Asn Met Val Glu Arg Asp Arg Thr 
100 105 110 

Leu Gly Tyr Gin Glu Pro His Trp Lys Glu Phe His Phe Asp Leu Thr 
115 120 125 130 



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Gin lie Pro Ala Gly Glu Ala Val Thr Ala Ala Glu Phe Are lie Tyr 
135 140 145 

Lys Glu Pro Ser Thr His Pro Leu Asn Thr Thr Leu His He Ser Met 
150 155 160 

Phe Glu Val Val Gin Glu His Ser Asn Arg Glu Ser Asp Leu Phe Phe 
165 170 175 

Leu Asp Leu Gin Thr Leu Arg Ser Gly Asp Glu Gly Trp Leu Val Leu 
180 185 190 

Asp He Thr Ala Ala Ser Asp Arg Trp Leu Leu Asn His His Lys Asp 
195 200 205 210 

Leu Gly Leu Arg Leu Tyr Val Glu Thr Ala Asp Gly His Ser Met Asp 
215 220 225 

Pro Gly Leu Ala Gly Leu Leu Gly Arg Gin Ala Pro Arg Ser Arg Gin 
230 235 240 

Pro Phe Met Val Thr Phe Phe Arg Ala Ser Gin Ser Pro Val Arg Ala 
245 250 255 

Pro Arg Ala Ala Arg Pro Leu Lys Arg Arg Gin Pro Lys Lys Thr Asn 
260 265 270 

Glu Leu Pro His Pro Asn Lys Leu Pro Gly lie Phe Asp Asp Gly His 
275 280 285 290 

Gly Ser Arg Gly Arg Glu Val Cys Arg Arg His Glu Leu Tyr Val Ser 
295 300 305 

Phe Arg Asp Leu Gly Trp Leu Asp Trp Val He Ala Pro Gin Gly Tyr 
310 315 320 

Ser Ala Tyr Tyr Cys Glu Gly Glu Cys Ala Phe Pro Leu Asp Ser Cys 
325 330 335 



Met Asn Ala Thr Asn His Ala He Leu Gin Ser Leu Val His Leu Met 
340 345 350 

Lys Pro Asp Val Val Pro Lys Ala Cys Cys Ala Pro Thr Lys Leu Ser 
355 360 365 370 

Ala Thr Ser Val Leu Tyr Tyr Asp Ser Ser Asn Asn Val He Leu Arg 
375 380 385 

Lys His Arg Asn Met Val Val Lys Ala Cys Gly Cys His 
390 395 



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(2) INFORMATION FOR SEQ ID NO: 24: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 1368 base pairs 

(B) TYPE: nucleic acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: cDNA 
(ix) FEATURE: 

(A) NAME/KEY: CDS 

(B) LOCATION: 1..1368 

(D) OTHER INFORMATION: /STANDARD NAME=»»60A" 

(X) PUBLICATION INFORMATION: 

(A) AUTHORS: WHARTON, KRISTI A.; THOHSEN, GERALD H.; 
GELBERT, WILLIAM M. 

(B) TITLE: DROSOPHILA 60A GENE. . . 

(C) JOURNAL: PROC. NAT'L ACAD, SCI, USA 

(D) VOLUME: 88 

(E) RELEVANT RESIDUES IN SEQ ID NO: 3: FROM 1 TO 1368 

(F) PAGES: 9214-9218 

(G) DATE: OCT - 1991 

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:24: 

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 
15 10 15 

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 
20 25 30 

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 He Tyr He Asp Asn Gly Lys Asp 
35 40 45 

CAG ACG ATC ATG CAC AGA GTG CTG AGC GAG GAC GAC AAG CTG GAC GTC 192 
Gin Thr He Met His Arg Val Leu Ser Glu Asp Asp Lys Leu Asp Val 
50 55 60 

TCG TAC GAG ATC CTC GAG TTC CTG GGC ATC GCC XSAA CGG CCG ACG CAC 240 
Ser Tyx Glu He Leu Glu Phe Leu Gly He Ala Glu Arg Pro Thr His 
65 70 75 80 

CTG AGC AGC CAC CAG TTG TCG CTG AGG AAG TCG GCT CCC AAG TTC CTG 288 
Leu Ser Ser His Gin Leu Ser Leu Arg Lys Ser Ala Pro Lys Phe Leu 
85 90 95 

CTG GAC GTC TAC CAC CGC ATC ACG GCG GAG GAG GGT CTC AGC GAT CAG 336 
Leu Asp Val Tyr His Arg He Thr Ala Glu Glu Gly Leu Ser Asp Gin 
100 105 110 



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GAT GAG GAC GAC GAC TAG GAA CGC GGC CAT CGG TCC AGG AGG AGC GCC 384 
Asp Glu Asp Asp Asp Tyr Glu Arg Gly His Arg Ser Arg Arg Ser Ala 
115 120 125 

GAC CTC GAG GAG GAT GAG GGC GAG CAG CAG AAG AAC TTC ATC ACC GAC 432 
Asp Leu Glu Glu Asp Glu Gly Glu Gin Gin Lys Asn Phe He Thr Asp 
130 135 140 

CTG GAC AAG CGG GCC ATC GAC GAG AGC GAC ATC ATC ATG ACC TTC CTG 480 
Leu Asp Lys Arg Ala He Asp Glu Ser Asp He He Met Thr Phe Leu 
145 150 155 160 



AAC AAG CGC CAC CAC AAT GTG GAC GAA CTG CGT CAC GAG CAC GGC CGT 
Asn Lys Arg His His Asn Val Asp Glu Leu Arg His Glu His Gly Arg 
165 170 175 



528 



CGC CTG TGG TTC GAC GTC TCC AAC GTG CCC AAC GAC AAC TAC CTG GTG 576 
Arg Leu Trp Phe Asp Val Ser Asn Val Pro Asn Asp Asn Tyr Leu Val 
180 185 190 

ATG GCC GAG CTG CGC ATC TAT CAG AAC GCC AAC GAG GGC AAG TGG CTG 624 
Met Ala Glu Leu Arg He Tyr Gin Asn Ala Asn Glu Gly Lys Trp Leu 
195 200 205 

ACC GCC AAC AGG GAG TTC ACC ATC ACG GTA TAC GCC ATT GGC ACC GGC 672 
Thr Ala Asn Arg Glu Phe Thr He Thr Val Tyr Ala He Gly Thr Gly 
210 215 220 



ACG CTG GGC CAG CAC ACC ATG GAG CCG CTG TCC TCG GTG AAC ACC ACC 
Thr Leu Gly Gin His Thr Met Glu Pro Leu Ser Ser Val Asn Thr Thr 
225 230 235 240 

GGG GAC TAC GTG GGC TGG TTG GAG CTC AAC GTG ACC GAG GGC CTG CAC 
Gly Asp Tyr Val Gly Trp Leu Glu Leu Asn Val Thr Glu Gly Leu His 
245 250 255 



720 



768 



GAG TGG CTG GTC AAG TCG AAG GAC AAT CAT GGC ATC TAC ATT GGA GCA 
Glu Trp Leu Val Lys Ser Lys Asp Asn His Gly He Tyr He Gly Ala 
260 265 270 



816 



CAC GCT GTC AAC CGA CCC GAC CGC GAG GTG AAG CTG GAC GAC ATT ^GA 
His Ala Val Asn Arg Pro Asp Arg Glu Val Lys Leu Asp Asp He Gly 
275 280 285 



864 



CTG ATC CAC CGC AAG GTG GAC GAC GAG TTC CAG CCC TTC ATG ATC GGC 
Leu He His Arg Lys Val Asp Asp Glu Phe Gin Pro Phe Met He Gly 
290 295 300 



912 



TTC TTC CGC GGA CCG GAG CTG ATC AAG GCG ACG GCC CAC AGC AGC CAC 
Phe Phe Arg Gly Pro Glu Leu He Lys Ala Thr Ala His Ser Ser His 
305 310 315 320 



960 



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CAC AGG AGC AAG CGA AGC GCC AGC CAT CCA CGC AAG CGC AAG AAG TCG 1008 
His Arg Ser Lys Arg Ser Ala Ser His Pro Arg Lys Arg Lys Lys Ser 
325 330 335 

GTG TCG CCC AAC AAC GTG CCG CTG CTG GAA CCG ATG GAG AGC ACG CGC 1056 
Val Ser Pro Asn Asn Val Pro Leu Leu Glu Pro Met Glu Ser Thr Arg 
340 345 350 

AGC TGC CAG ATG CAG ACC CTG TAC ATA GAC TTC AAG GAT CTG GGC TGG 1104 
Ser Cys Gin Met Gin Thr Leu Tyr He Asp Phe Lys Asp Leu Gly Trp 
355 360 365 

CAT GAC TGG ATC ATC GCA CCA GAG GGC TAT GGC GCC TTC TAC TGC AGC 1152 
His Asp Trp He He Ala Pro Glu Gly Tyr Gly Ala Phe Tyr Cys Ser 
370 375 380 

GGC GAG TGC AAT TTC CCG CTC AAT GCG CAC ATG AAC GCC ACG AAC CAT 1200 
Gly Glu Cys Asn Phe Pro Leu Asn Ala His Met Asn Ala Thr Asn His 
385 390 395 400 

GCG ATC GTC CAG ACC CTG GTC CAC CTG CTG GAG CCC AAG AAG GTG CCC 1248 
Ala He Val Gin Thr Leu Val His Leu Leu Glu Pro Lys Lys Val Pro 
405 410 415 

AAG CCC TGC TGC GCT CCG ACC AGG CTG GGA GCA CTA CCC GTT CTG TAC 1296 
Lys Pro Cys Cys Ala Pro Thr Arg Leu Gly Ala Leu Pro Val Leu Tyr 
420 425 430 

CAC CTG AAC GAC GAG AAT GTG AAC CTG AAA AAG TAT AGA AAC ATG ATT 1344 
His Leu Asn Asp Glu Asn Val Asn Leu Lys Lys Tyr Arg Asn Met He 
435 440 445 

GTG AAA TCC TGC GGG TGC CAT TGA 1368 
Val Lys Ser Cys Gly Cys His 
450 455 

(2) INFORMATION FOR SEQ ID NO: 25: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 455 amino acids 

(B) TYPE: amino acid 
(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: protein 

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:25: 

Met Ser Gly Leu Arg Asn Thr Ser Glu Ala Val Ala Val Leu Ala Ser 
15 10 15 



Leu Gly Leu Gly Met Val Leu Leu Met Phe Val Ala Thr Thr Pro Pro 
20 25 30 



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Ala Val Glu Ala Thr Gin Ser Gly lie Tyr He Asp Asn Gly Lys Asp 
35 40 45 

Gin Thr He Met His Arg Val Leu Ser Glu Asp Asp Lys Leu Asp Val 
50 55 60 

Ser Tyr Glu He Leu Glu Phe Leu Gly He Ala Glu Arg Pro Thr His 
65 70 75 80 

Leu Ser Ser His Gin Leu Ser Leu Arg Lys Ser Ala Pro Lys Phe Leu 
85 90 95 

Leu Asp Val Tyr His Arg He Thr Ala Glu Glu Gly Leu Ser Asp Gin 
100 105 HO 

Asp Glu Asp Asp Asp Tyr Glu Arg Gly His Arg Ser Arg Arg Ser Ala 
115 120 125 

Asp Leu Glu Glu Asp Glu Gly Glu Gin Gin Lys Asn Phe He Thr Asp 
130 135 140 

Leu Asp Lys Arg Ala He Asp Glu Ser Asp He He Met Thr Phe Leu 
1« 150 155 160 

Asn Lys Arg His His Asn Val Asp Glu Leu Arg His Glu His Gly Arg 
165 170 175 

Arg Leu Trp Phe Asp Val Ser Asn Val Pro Asn Asp Asn Tyr Leu Val 
180 185 190 

Met Ala Glu Leu Arg He Tyr Gin Asn Ala Asn Glu Gly Lys Trn Leu 
195 200 205 

Thr Ala Asn Arg Glu Phe Thr He Thr Val Tyr Ala He Gly Thr Gly 
210 215 220 

Thr Leu Gly Gin His Thr Met Glu Pro Leu Ser Ser Val Asn Thr Thr 
225 230 235 240 

Gly Asp Tyr Val Gly Trp Leu Glu Leu Asn Val Thr Glu Gly Leu His 
245 250 255 

Glu Trp Leu Val Lys Ser Lys Asp Asn His Gly He Tyr He Gly Ala 
260 265 270 

His Ala Val Asn Arg Pro Asp Arg Glu Val Lys Leu Asp Asp He Gly 
275 280 285 

Leu He His Arg Lys Val Asp Asp Glu Phe Gin Pro Phe Met He Gly 
290 295 300 

Phe Phe Arg Gly Pro Glu Leu He Lys Ala Thr Ala His Ser Ser His 
305 310 315 320 



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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 Tjrr lie Asp Phe Lys Asp Leu Gly Trp 
355 360 365 

His Asp Trp lie lie 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 He 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 He 
435 440 445 

Val Lys Ser Cys Gly Cys His 
450 455 

(2) INFORMATION FOR SEQ ID N0:26: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: amino acids 

(B) TYPE: amino acid 
(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: protein 

(iii) ORIGINAL SOURCE: 

(A) ORGANISM: Homo Sapiens 

(ix) FEATURE: 

(A) NAME/KEY: Protein 

(B) LOCATION: 1..102 

(D) OTHER INFORMATION: /note="BMP3" 

(2) INFORMATION FOR SEQ ID N0:26: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 104 amino acids 

(B) TYPE: amino acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 



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{ii)MOLECULE TYPE: protein 

(ix) FEATURE: 

(A) NAME/KEY: Protein 

(B) LOCATION: 1..104 

(D) OTHER INFORMATION: /note="BMP3" 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26: 

Cys Ala Arg Arg Tyr Leu Lys Val Asp Phe Ala Asp lie Gly Trp Ser 
1 5 10 15 

Glu Trp He He Ser Pro Lys Ser Phe Asp Ala Tyr Try Cys Ser Gly 
20 25 30 

Ala Cys Gin Phe Pro Met Pro Lys Ser Leu Lys Pro Ser Asn His Ala 
35 40 45 

Thr He Gin Ser He Val Ala Arg Ala Val Gly Val Val Pro Gly He 
50 55 60 . 

Pro Glu Pro Cys Cys Val Pro Glu Lys Met Ser Ser Leu Ser He Leu 
65 70 75 80 

Phe Phe Asp Glu Asn Lys Asn Val Val Leu Lys Val Tyr Pro Asn Met 
85 90 95 

Thr Val Glu Ser Cys Ala Cys Arg 
100 

(2) INFORMATION FOR SEQ ID NO: 27: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 102 amino acids 

(B) TYPE: amino acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: protein 

(vi) ORIGINAL SOURCE: 

(A) ORGANISM: HOMO SAPIENS 

(ix) FEATURE: 

(A) NAME/KEY: Protein 

(B) LOCATION: 1..102 

(D) OTHER INFORMATION: /note= "BMP5" 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: 

Cys Lys Lys His Glu Leu Tyr Val Ser Phe Arg Asp Leu Gly Trp Gin 
15 10 15 



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Asp Trp He He Ala Pro Glu Gly Tyr Ala Ala Phe Tyr Cys Asp Gly 
20 25 30 

Glu Cys Ser Phe Pro Leu Asn Ala His Met Asn Ala Thr Asn His Ala 
35 40 A5 

He Val Gin Thr Leu Val His Leu Met Phe Pro Asp His Val Pro Lys 
50 55 60 

Pro Cys Cys Ala Pro Thr Lys Leu Asn Ala He Ser Val Leu Tyr Phe 
65 70 75 80 

Asp Asp Ser Ser Asn Val He Leu Lys Lys Tyr Arg Asn Met Val Val 
85 90 95 

Arg Ser Cys Gly Cys His 
100 

(2) INFORMATION FOR SEQ ID NO: 28: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 102 amino acids 

(B) TYPE: amino acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: protein 

(vi) ORIGINAL SOURCE: 

(A) ORGANISM: HOMO SAPIENS 

(ix) FEATURE: 

(A) NAME/KEY: Protein 

(B) LOCATION: 1..102 

(D) OTHER INFORMATION: /note= "BMP6" 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28: 

Cys Arg Lys His Glu Leu Tyr Val Ser Phe Gin Asp Leu Gly Trp Gin 
15 10 15 

Asp Trp He He Ala Pro Lys Gly Tyr Ala Ala Asn Tyr Cys Asp Gly 
20 25 30 

Glu Cys Ser Phe Pro Leu Asn Ala His Met Asn Ala Thr Asn His Ala 
35 AO 45 

He Val Gin Thr Leu Val His Leu Met Asn Pro Glu Tyr Val Pro Lys 
50 55 60 

Pro Cys Cys Ala Pro Thr Lys Leu Asn Ala He Ser Val Leu Tyr Phe 
65 70 75 80 



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Asp Asp Asn Ser Asn Val He Leu Lys Lys Tyr Arg Trp Met Val Val 
85 90 95 

Arg Ala Cys Gly Cys His 
100 



(2) INFORMATION FOR SEQ ID N0:29: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 102 amino acids 

(B) TYPE: amino acid 
(D) TOPOLOGY: linear 



(ii) MOLECULE TYPE: protein 

(ix) FEATURE: 

(A) NAME/KEY: Protein 

(B) LOCATION: 1..102 

(D) OTHER INFORMATION: /label= OPX 

/note= "WHEREIN XAA AT EACH POS'N IS INDEPENDENTLY 
SELECTED FROM THE RESIDUES OCCURRING AT THE 
CORRESPONDING POS'N IN THE C-TERMINAL SEQUENCE OF MOUSE 
OR HUMAN OPl OR 0P2 (SEE SEQ. ID NOS. 5,6,7 and 8 or 
16,18,20 and 22.)" 

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:29: 



Cys Xaa Xaa His Glu Leu Tyr Val Xaa Phe Xaa Asp Leu Gly Trp Xaa 
15 10 15 

Asp Trp Xaa He Ala Pro Xaa Gly Tyr Xaa Ala Tyr Tyr Cys Glu Gly 
20 25 30 

Glu Cys Xaa Phe Pro Leu Xaa Ser Xaa Met Asn Ala Thr Asn His Ala 
35 40 A5 

He Xaa Gin Xaa Leu Val His Xaa Xaa Xaa Pro Xaa Xaa Val Pro Lys 
50 55 60 

Xaa Cys Cys Ala Pro Thr Xaa Leu Xaa Ala Xaa Ser Val Leu Tyr Xaa 
65 70 75 80 

Asp Xaa Ser Xaa Asn Val Xaa Leu Xaa Lys Xaa Arg Asn Met Val Val 
85 90 95 



Xaa Ala Cys Gly Cys His 
100 



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(2) INFORMATION FOR SEQ ID N0:30: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 97 amino acids 

(B) TYPE: amino acids 

(C) TOPOLOGY: linear 

(ii) MOLECULE TYPE: protein 

(ix) FEATURE: 

(A) NAME: Generic Sequence 5 

(D) OTHER INFORMATION: 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 N0:30: 

Leu Xaa Xaa Xaa Phe 
1 5 
Xaa Xaa Xaa Gly Trp Xaa Xaa Trp Xaa 
10 

Xaa Xaa Pro Xaa Xaa Xaa Xaa Ala 

15 20 
Xaa Tyr Cys Xaa Gly Xaa Cys Xaa 

25 30 
Xaa Pro Xaa Xaa Xaa Xaa Xaa 
35 

Xaa Xaa Xaa Asn His Ala Xaa Xaa 
40 45 
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 
50 

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys 
55 60 



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Cys Xaa Pro Xaa Xaa Xaa Xaa Xaa 
65 

Xaa Xaa Xaa Leu Xaa Xaa Xaa 

70 75 
Xaa Xaa Xaa Xaa Val Xaa Leu Xaa 
80 

Xaa Xaa Xaa Xaa Met Xaa Val Xaa 

85 90 
Xaa Cys Xaa Cys Xaa 
95 

(2) INFORMATION FOR SEQ ID NO: 31: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 102 amino acids 

(B) TYPE: amino acids 

(C) TOPOLOGY: linear 

(ii) MOLECULE TYPE: protein 

(ix) FEATURE: 

(A) NAME: Generic Sequence 6 

(D) OTHER INFORMATION: wherein each Xaa is 
independently selected from a group of one 

or more specified amino acids as defined in 
the specif ication- 

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31: 

Cys Xaa Xaa Xaa Xaa Leu Xaa Xaa Xaa Phe 
15 10 
Xaa Xaa Xaa Gly Trp Xaa Xaa Trp Xaa 
15 

Xaa Xaa Pro Xaa Xaa Xaa Xaa Ala 
20 25 



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Xaa lyr Cys Xaa Gly Xaa Cys Xaa 
30 35 
Xaa Fro Xaa Xaa Xaa Xaa Xaa 
40 

Xaa Xaa Xaa Asn His Ala Xaa Xaa 
45 50 
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 
55 

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys 

60 65 
Cys Xaa Pro Xaa Xaa Xaa Xaa Xaa 
70 

Xaa Xaa Xaa Leu Xaa Xaa Xaa 

75 80 
Xaa Xaa Xaa Xaa Val Xaa Leu Xaa 
85 

Xaa Xaa Xaa Xaa Met Xaa Val Xaa 

90 95 
Xaa Cys Xaa Cys Xaa 
100 



(2) INFORMATION FOR SEQ ID NO: 32: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 1238 base pairs, 372 amino acids 

(B) TYPE: nucleic acid, amino acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: cDNA 



(iii) ORIGINAL SOURCE: 
(A) ORGANISM: human 
(F) TISSUE TYPE: BRAIN 

(iv) FEATURE: 

(A) NAME/KEY: CDS 

(B) LOCATION: 

(D) OTHER INFORMATION: 

/product= "GDF-1" 
/note= "GDF-1 CDNA" 



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(X) PUBLICATION INFORMATION: 

(A) AUTHORS: Lee, Se-Jin 

(B) TITTLE: Expression of Growth/Differentiation Factor 1 

(C) JOURNAL: Free. Nat'l Acad. Sci. 

(D) VOLUME: 88 

(E) RELEVANT RESIDUES: 1-1238 

(F) PAGES: 4250-4254 

(G) DATE: May- 1991 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32: 

GGGGACACCG GCCCCGCCCT CAGCCCACTG GTCCCGGGCC GCCGCGGACC CTGCGCACTC 60 

TCTGGTCATC GCCTGGGAGG AAG ATG CCA CCG CCG CAG CAA GGT CCC TGC GGC 113 

Met Pro Pro Pro Gin Gin Gly Pro Cys Gly 
1 5 10 

CAC CAC CTC CTC CTC CTC CTG GCC CTG CTG CTG CCC TCG CTG CCC 158 
His His Leu Leu Leu Leu Leu Ala Leu Leu Leu Pro Ser Leu Pro 
15 20 25 

CTG ACC CGC GCC CCC GTG CCC CCA GGC CCA GCC GCC GCC CTG CTC 203 
Leu Thr Arg Ala Pro Val Pro Pro Gly Pro Ala Ala Ala Leu Leu 
30 35 40 

CAG GCT CTA GGA CTG CGC GAT GAG CCC CAG GGT GCC CCC AGG CTC 248 
Gin Ala Leu Gly Leu Arg Asp Glu Pro Gin Gly Ala Pro Arg Leu 
45 50 55 

CGG CCG GTT CCC CCG GTC ATG TGG CGC CTG TTT CGA CGC CGG GAC 293 
Arg Pro Val Pro Pro Val Met Trp Arg Leu Phe Arg Arg Arg Asp 
60 65 70 

CCC CAG GAG ACC AGG TCT GGC TCG CGG CGG ACG TCC CCA GGG GTC 338 
Pro Gin Glu Thr Arg Ser Gly Ser Arg Arg Thr Ser Pro Gly Val 
75 80 85 

ACC CTG CAA CCG TGC CAC GTG GAG GAG CTG GGG GTC GCC GGA AAC 383 
Thr Leu Gin Pro Cyc His Val Glu Glu Leu Gly Val Ala Gly Asn 
90 95 100 

ATC GTG CGC CAC ATC CCG GAC CGC GGT GCG CCC ACC CGG GCC TCG 428 
He Val Arg His He Pro Asp Arg Gly Ala Pro Thr Arg Ala Ser 
105 110 115 

GAG CCT GTC TCG GCC GCG GGG CAT TGC CCT <;aG TGG ACA GTC GTC 473 
Glu Pro Val Ser Ala Ala Gly His Cys Pro Glu Trp Thr Val Val 
120 125 130 

TTC GAC CTG TCG GCT GTG GAA CCC GCT GAG CGC CCG AGC CGG GCC 518 
Phe Asp Leu Ser Ala Val Glu Pro Ala Glu Arg Pro Ser Arg Ala 
135 lAO 145 



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CGC CTG GAG CTG CGT TTC GCG GCG GCG GCG GCG GCA GCC CCG GAG 563 
Arg Leu Glu Leu Arg Phe Ala Ala Ala Ala Ala Ala Ala Pro Glu 
150 155 160 

GGC GGC TGG GAG CTG AGC GTG GCG CAA GCG GGC CAG GGC GCG GGC 608 
Gly Gly Trp Glu Leu Ser Val Ala Gin Ala Gly Gin Gly Ala Gly 
165 170 175 

GCG GAC CCC GGG CCG GTG CTG CTC CGC CAG TTG GTG CCC GCC CTG 653 
Ala Asp Pro Gly Pro Val Leu Leu Arg Gin Leu Val Pro Ala Leu 
180 185 190 

GGG CCG CCA GTG CGC GCG GAG CTG CTG GGC GCC GCT TGG GCT CGC 698 
Gly Pro Pro Val Arg Ala Glu Leu Leu Gly Ala Ala Trp Ala Arg 
195 200 205 

AAC GCC TCA TGG CCG CGC AGC CTC CGC CTG GCG CTG GCG CTA CGC 743 
Asn Ala Ser Trp Pro Arg Ser Leu Arg Leu Ala Leu Ala Leu Arg 
210 215 220 

CCC CGG GCC CCT GCC GCC TGC GCG CGC CTG GCC GAG GCC TCG CTG 788 
Pro Arg Ala Pro Ala Ala Cys Ala Arg Leu Ala Glu Ala Ser Leu 
225 230 235 

CTG CTG GTG ACC CTC GAC CCG CGC CTG TGC CAC CCC CTG GCC CGG 833 
Leu Leu Val Thr Leu Asp Pro Arg Leu Cys His Pro Leu Ala Arg 
240 245 250 

CCG CGG CGC GAC GCC GAA CCC GTG TTG GGC GGC GGC CCC GGG GGC 878 
Pro Arg Arg Asp Ala Glu Pro Val Leu Gly Gly Gly Pro Gly Gly 
255 260 265 

GCT TGT CGC GCG CGG CGG CTG TAC GTG AGC TTC CGC CAG GTG GGC 923 
Ala Cys Arg Ala Arg Arg Leu Tyr Val Ser Phe Arg Glu Val Gly 
270 275 280 

TGG CAC CGC TGG GTC ATC GCG CCG CGC CCC TTC CTG GCC AAC TAC 968 
Trp His Arg Trp Val lie Arg Pro Arg Gly Phe Leu Ala Asn Tyr 
285 290 295 

TGC CAG GGT CAG TGC GCG CTG CCC GTC GCG CTG TCG GGG TCC CGG 1013 
Cys Gin Gly Gin Cys Ala Leu Pro Val Ala Leu Ser Gly Ser Gly 
300 305 310 

GGG CCG CCG GCG CTC AAC CAC GCT GTG CTG CGC GCG CTC ATG CAC 1058 
Gly Pro Pro Ala Leu Asn His Ala Val Leu Arg Ala Leu Met His 
315 320 325 

GCG GCC GCC CCG GGA GCC GCC GAC CTG CCC TGC TGC GTG CCC -GCG 1103 
Ala Ala Ala Pro Gly Ala Ala Asp Leu Pro Cys Cys Val Pro Ala 
330 335 340 



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CGC CTG TCG CCC ATC TCC GTG CTC TTC TTT GAC AAC AGC GAC AAC 1148 
Arg Leu Ser Pro He Ser Val Leu Phe Phe Asp Asn Ser Asp Asn 
345 350 355 

GTG GTG CTG CGG CAG TAT GAG GAC ATG GTG GTG GAC GAG TGC GGC 1193 
Val Val Leu Arg Gin Tyr Glu Asp Met Val Val Asp Glu Cys Gly 
360 365 370 

TGC CGC TAACCCGGGG CGGGCAGGGA CCCGGGCCCA ACAATAAATG CCGCGTGG 1238 
Cys Arg 
372 

(34) INFORMATION FOR SEQ ID NO: 33: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 372 amino acids 

(B) TYPE: amino acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: cDNA 

(iii) HYPOTHETICAL: NO 

(iv) ANTI-SENSE: NO 

(vi) ORIGINAL SOURCE: 

(A) ORGANISM: human 
(F) TISSUE TYPE: BRAIN 

(ix) FEATURE: 

(A) NAME/KEY: CDS 

(B) LOCATION: 

(D) OTHER INFORMATION: /function= 
/products "GDF-1" 



(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33: 

Met Pro Pro Pro Gin Gin Gly Pro Cys Gly 
1 5 10 

His His Leu Leu Leu Leu Leu Ala Leu Leu Leu Pro Ser Leu Pro 
15 20 25 

Leu Thr Arg Ala Pro Val Pro Pro Gly Pro Ala Ala Ala Leu Leu 
30 35 40 

Gin Ala Leu Gly Leu Arg Asp Glu Pro Gin Gly Ala Pro Arg Leu 
45 50 55 



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Arg Pro Val Pro Pro Val Met Trp Arg Leu Phe Arg Arg Arg Asp 
60 65 70 

Pro Gin Glu Thv Arg Ser Gly Ser Arg Arg Thr Ser Pro Gly Val 
75 80 85 

Thr Leu Gin Pro Cyc His Val Glu Glu Leu Gly Val Ala Gly Asn 
90 95 100 

He Val Arg His He Pro Asp Arg Gly Ala Pro Thr Arg Ala Ser 
105 110 115 

Glu Pro Val Ser Ala Ala Gly His Cys Pro Glu Trp Thr Val Val 
120 125 130 

Phe Asp Leu Ser Ala Val Glu Pro Ala Glu Arg Pro Ser Arg Ala 
135 140 145 

Are Leu Glu Leu Arg Phe Ala Ala Ala Ala Ala Ala Ala Pro Glu 
150 155 160 

Gly Gly Trp Glu Leu Ser Val Ala Gin Ala Gly Gin Gly Ala Gly 
165 170 175 

Ala Asp Pro Gly Pro Val Leu Leu Arg Gin Leu Val Pro Ala Leu 
180 185 190 

Gly Pro Pro Val Arg Ala Glu Leu Leu Gly Ala Ala Trp Ala Arg 
195 200 205 

Asn Ala Ser Trp Pro Arg Ser Leu Arg Leu Ala Leu Ala Leu Arg 
210 215 220 

Pro Are Ala Pro Ala Ala Cys Ala Arg Leu Ala Glu Ala Ser Leu 
225 230 235 

Leu Leu Val Thr Leu Asp Pro Arg Leu Cys His Pro Leu Ala Arg 
240 245 250 

Pro Arg Arg Asp Ala Glu Pro Val Leu Gly Gly Gly Pro Gly Gly 
255 260 265 

Ala Cys Arg Ala Arg Arg Leu Tyr Val Ser Phe Arg Glu Val Gly 
270 275 280 

Ttd His Arg Trp Val He Arg Pro Arg Gly Phe Leu Ala Asn Tyr 
•^ 285 290 295 

Cys Gin Gly Gin Cys Ala Leu Pro Val Ala Leu Ser Gly Ser Gly 
^ 300 305 310 

Gly Pro Pro Ala Leu Asn His Ala Val Leu Arg Ala Leu Met His 
315 320 325 



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Ala Ala Ala Pro Gly Ala Ala Asp Leu Pro Cys Cys Val Pro Ala 

330 335 340 

Arg Leu Ser Pro He Ser Val Leu Phe Phe Asp Asn Ser Asp Asn 

345 350 355 

Val Val Leu Arg Gin lyr Glu Asp Met Val Val Asp Glu Cys Gly 

360 365 370 

Cys Arg 
372 



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140 

What is claimed is: 

1. A therapeutic treatment , method for preventing 
loss of bone mass or increasing bone mass in an 
individual, the method comprising: 

administering to the individual a 
therapeutically effective morphogen in an amount 
and for a time sufficient to prevent loss of or to 
increase bone mass in said individual. 

2. A therapeutic treatment method for preventing 
loss of bone mass or for increasing bone mass in an 
individual / compris ing : 

administering an agent that stimulates in vivo 
the effective concentration of a naturally 
occurring morphogen in said individual sufficient 
to prevent loss of or to increase bone mass in said 
individual. 

3. The method of claim 1 or 2 wherein said loss 
of bone mass results from a metabolic bone disease. 

4. The method of claim 3 wherein said metabolic 
bone disease comprises osteoporosis or 
osteomalacia. 

5. The method of claim 1 or 2 wherein said loss 
of bone mass results from an imbalance in bone 
resorption or bone formation- 



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141 

6. The method of claim 1 or 2 wherein said loss 
of bone mass results from an imbalance of calcium 
or phosphate metabolism* 

7. The method of claim 1 or 2 wherein said loss 
of bone mass results from a vitamin D imbalance in 
the individual. 

8. The method of claim 1 or 2 wherein said loss 
of bone mass is nutritionally or hormonally 
induced. 

9. The method of claim 4 wherein said 
osteoporosis is postmenopausal or senile 
osteoporosis. 

10. The method of claim 1 or 2 wherein said morphogen 
comprises an amino acid sequence sharing at least 
70% homology with one of the sequences selected 
from the group consisting of: OP-1^ OP-2, CBMP2, 
Vgl(fx), Vgr(fx), DPP(fx), GDF-l(fx), BMP3(fx), 
BMP5(fx), BMP6{fx) and 60A{fx). 

11. The method of claim 10 wherein said morphogen 
comprises an amino acid sequence sharing at least 
80% homology with one of the sequences selected 
from the group consisting of: 0P--1, OP-2, CBMP2^ 
Vgl{fx), Vgr{fx), DPP(fx), GDF-l(fx), BMP3{fx), 
BMP5(fx), BMP6(fx) and 60A(fx). 

2. The method of claim 1 or 2 wherein said morphogen 
comprises an amino acid sequence having greater 
than 60% amino acid identity with the sequence 
defined by residues 43-139 of Seq. ID No. 5 (hOPl). 



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142 

13. The method of claim 12 wherein said morphogen 
comprises an amino acid sequence having greater 
than 65% amino acid identity with the sequence 
defined by residues 43-139 of Seq. ID No. 5 (hOPl). 

14. The method of claim 1 or 2 wherein said morphogen 
comprises an amino acid sequence defined by 
residues 43-139 of Seq. ID No. 5 (hOPl), including 
allelic and species variants thereof. 

15. The method of claim 1 or 2 wherein said morphogen 
comprises an amino acid sequence defined by Generic 
Sequences 1, 2, 3, 4, 5 or 6 (Seq. ID Nos. 1, 2, 3, 
A, 30 or 31} . 

16. The method of claim 1 or 2 wherein said morphogen 
comprises an amino acid sequence defined by OPX 
(Seq. ID No. 29)- 

17. A method for repairing defects in bone tissue 
microstmicture resulting from a metabolic bone 
disease, the method comprising: 

administering to an individual a 
therapeutically effective morphogen in an amount 
and for a time sufficient to repair defects in said 
microstructure . 

18. A method for repairing defects in bone tissue 
microstructure resulting from a metabolic bone 
disease, the method comprising: 

administering an agent capable of stimulating 
in vivo the effective concentration of a naturally 
occurring morphogen sufficient to repair said 
defects - 



wo 93/0S7S1 PCT/US92/07432 

143 

19. The method of claim 17 or 18 wherein said metabolic 
bone disease comprises osteoporosis or 
osteomalacia. 

20. A method for protecting an individual at risk for 
loss of bone mass, the method comprising: 

providing to the individual a therapeutically 
effective morphogen in an amount and for a time 
sufficient to protect said individual from loss of 
bone mass. 

21. A method for protecting an individual at risk for 
loss of bone mass, the method comprising: 

providing to the individual an agent that 
stimulates in vivo a therapeutically effective 
concentration of a naturally occurring morphogen 
sufficient to protect said individual from loss of 
bone mass. 

22. The method of claim 20 or 21 wherein said 
individual is a postmenopausal female or is 
undergoing dialysis. 

23. The method of claim 20 or 21 wherein said 
individual is at risk for loss of bone mass as a 
result of senile osteoporosis. 

24. The method of claim 17, 18, 20 or 21 wherein said 
morphogen comprises an amino acid sequence sharing 
at least 70% homology with one of the sequences 
selected from the group consisting of: OP-1, OP-2, 
CBMP2, Vgl(fx), Vgr(fx), DPP{fx), GDF"l(fx), 
BMP3(fx), BMP5(fx), BMP6(fx) and 60A(fx). 



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25* The method of claim 24 wherein said morphogen 

comprises an amino acid sequence sharing at least 
80% homology with one of the sequences selected 
from the group consisting of: OP-1, OP-2, CBMP2, 
Vgl{fx), Vgr(fx), DPP(fx), GDF-l(fx), BMP3(fx), 
BMP5(fx), BMP6(fx) and 60A(fx)« 

26* The method of claim 17, 18, 20 or 21 wherein said 
morphogen comprises an amino acid sequence having 
greater than 60% amino acid identity with the 
sequence defined by residues 43-139 of Seq. ID No. 
5 (hOPl)* 

27. The method of claim 26 wherein said morphogen 
comprises an amino acid sequence having greater 
than 65% amino acid identity with the sequence 
defined by residues 43-139 of Seq. ID No. 5 (hOPl). 

28. The method of claim 17, 18, 20 or 21 wherein said 
morphogen comprises an amino acid sequence defined 
by residues 43-139 of Seq. ID No. 5 (hOPl), 
including allelic and species variants thereof. 

29. The method of claim 17, 18, 20 or 21 wherein said 
morphogen comprises an amino acid sequence defined 
by Generic Sequences 1, 2, 3, 4, 5 or 6 (Seq. ID 
Nos. 1, 2, 3, 4, 30 or 31) . 

30. The method of claim 17, 18, 20 or 21 wherein said 
morphogen comprises an amino acid sequence defined 
by OPX (Seq. ID No. 29). 



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31. The method of claim 1, 2, 17, 18, 20 or 21, wherein 
said morphogen or said morphogen-stimulating agent 
is provided to the individual by oral 
administration . 

32. The method of claim 1, 2, 17, 18, 20 or 21, wherein 
said morphogen or morphogen-stimulating agent is 
provided to the individual by parenteral 
administration. 

33. The method of claim 1, 17, or 20 wherein said 
morphogen is provided to said individual in 
association with a molecule capable of enhancing 
the solubility of said morphogen. 

34. The method of claim 33 wherein said molecule 
comprises casein or part or all of the pro domain 
of a morphogen . 

35. The method of claim 34 wherein said pro domain 
comprises part or all of the sequence described by 
residues 30 to 292 of Seq. ID No. 16. 

36. The method of claim 1, 17, or 20 wherein said 
morphogen is provided to the individual in 
association with a molecule capable of targeting 
said morphogen to bone tissue. 

37. The method of claim 36 wherein said targeting 
molecule comprises tetracycline, diphosphonates, or 
an antibody that binds specifically to a molecule 
on the surface of bone tissue cells. 



wo 93/05751 

146 

38. The method of claim 2, 18 or 21 wherein said 
morphogen-stimulating agent is provided to the 
individual in association with a molecule capable 
of targeting said agent to morphogen-producing or 
morphogen-secreting tissue. 

39. A morphogen useful in the manufacture of a 
pharmaceutical for use in a treatment for 
preventing loss of bone mass or for increasing bone 
mass in an individual. 

40. A therapeutic agent useful in the manufacture of a 
pharmaceutical for use in a treatment for 
preventing loss of bone mass or for increasing bone 
mass in an individual, the agent being capable of 
stimulating in vivo an effective concentration of a 
naturally occurring morphogen, sufficient to 
prevent loss of or to increase bone mass in the 
individual . 

41. The pharmaceutical of claim 39 or 40 for use in a 
treatment to prevent bone loss due to a metabolic 
bone disease. 

42. The pharmaceutical of claim 39 or 40 for use in a 
treatment to prevent bone loss due to an imbalance 
in bone resorption or bone formation. 

43. The pharmaceutical of claim 41 for use in treating 
osteomalacia or osteoporosis. 

44. A morphogen useful in the manufacture of a 
pharmaceutical for use in a treatment for 
protecting an individual at risk for loss of bone 
mass. 



wo 93/05751 PCr/US92/07432 

147 

45. A therapeutic agent useful in the manufacture of a 
pharmaceutical for use in a treatment for 
protecting an individual at risk for loss of bone 
mass, the agent being capable of stimulating in 
vivo an effective concentration of a naturally 
occurring morphogen sufficient to protect said 
individual from loss of bone mass. 

46. The pharmaceutical of claim 44 or 45 for use in a 
treatment to protect an individual at risk for loss 
of bone mass as a result of senile osteoporosis. 

47. The pharmaceutical of claim 44 or 45 for use in a 
treatment of an individual at risk for loss af bone 
mass as a result of undergoing dialysis. 

48. The pharmaceutical of claim 39, 40, 44 or 45 
wherein said morphogen comprises an amino acid 
sequence sharing at least 70% homology with a 
sequence selected from the group consisting of OP- 
1, OP-2, CBMP2, Vgl(fx), Vgr(fx), DPP(fx), GDF- 
l(fx), BMP3{fx), BMP5(fx), BMP6(fx) and 60A(fx). 

49. The pharmaceutical of claim 48 wherein said 
morphogen comprises an amino acid sequence sharing 
at least 80% homology with one of the sequences 
selected from the group consisting of: OP-1, OP-2, 
CBMP2, Vgl(fx), Vgr{fx), DPP(fx), GDF-l(fx), 
BMP3(fx), BMP5(fx), BMP6(fx) and 60A(fx). 

50. The pharmaceutical of claim 39, 40, 44 or 45 
wherein said morphogen comprises an amino acid 
sequence having greater than 60% amino acid 
identity with the sequence defined by residues 
43-139 of Seq. ID No. 5 (hOPl). 



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148 



51. The pharmaceutical of claim 50 wherein said 
morphogen comprises an amino acid sequence having 
greater than 65% amino acid identity with the 
sequence defined by residues 43-139 of Seq. ID No. 
5 (hOPl). 

52. The pharmaceutical of claim 39^ 40, 44 or 45 
wherein said morphogen comprises an amino acid 
sequence defined by residues 43-139 of Seq. ID 
No. 5 (hOPl), including allelic and species 
variants thereof. 

53. The pharmaceutical of claim 39, 40, 44 or 45 
wherein said morphogen comprises an amino acid 
sequence defined by Generic Sequences 1, 2, 3, 4, 5 
or 6 (Seq. ID Nos. 1, 2, 3, 4, 30 or 31). 

54. The pharmaceutical of claim 39, 40, 44 or 45 
wherein said morphogen comprises an amino acid 
sequence defined by OPX (Seq. ID No. 29). 

55. A composition useful in a treatment to prevent 
loss of bone mass or to increase bone mass in an 
individual, the composition comprising a morphogen 
or morphogen-stimulating agent in association with 
a bone tissue targeting molecule. 

56. The composition of claim 55 wherein said bone 
tissue targeting molecule comprises tetracycline, a 
diphosphonate, or an antibody or antibody fragment 
that binds specifically to a molecule on the 
surface of bone tissue cells. 



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149 

57 • A composition useful in a treatment to prevent 
loss of bone mass or to increase bone mass in an 
individual, the composition comprising a raorphogen 
or morphogen stimulating agent in association with 
a cofactor. 

58. The composition of claim 57 wherein said cofactor 
is selected from the group consisting of vitamin 
D^/ calcitonin, a prostaglandin, parathyroid 
hormone, dexamethasone, estrogen and IGF» 

59. The composition of claim 55 wherein said 
composition is provided to said individual in 
association with a molecule capable of enhancing 
the solubility of said morphogen. 



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• Protein Concentration {(iQ/mi) 
-«"0>-1 -O- TQF-8 



Figure 1 



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PCr/US92/07432 



0} 

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£ 
o 



CL 



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CVJ 



in o in 
c6 corf 



CM lO CO 

TT CU CD 
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CM CO CJ 



in CO CO 

CT) CO CD 

o o o 



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PCr/US92/07432 




wo 93/05751 



PCr/US92/07432 




Protein Concentration (ng/ml) 



Figure 6A 



0) 800 




Protein Concentration (ng/ml) 



Figure 



6B 



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I "Z- 3 



39-* 



/ : f 



B 



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PCT/US92/07432 



% activity 
80| - 




17 16 to 20 21 22 * 23 24 26 26 27 GoO( 



OD 
1.2 1 




Fractions 



l-iG- 86 



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PCr/US92/07432 




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PCT/US92/07432 




wo 93/05751 



PCr/US92/07432 




WO93/0S7S1 



PCr/US92/07432 




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PCr/US92/07432 




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