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WORLD INTELLECTUAL PROPERTY ORGANIZATION 
Internationa] Bureau 




INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) 



(51) International Patent Classification ^ : 




(11) International Publication Number: 


WO 93/04692 


A61K 37/02, AOIN 1/02 


Al 


(43) International Publication Date: 


18 March 1993 (18.03.93) 



(21) International Application Number: PCT/US92/07358 

(22) International Filing Date: 28 August 1992 (28.08.92) 



(30) Priority data: 
752,764 
752.861 
753,059 



30 August 1991 (30.08.91) US 
30 August 1991 (30.08.91) US 
30 August 1991 (30.08.91) US 



(71) Applicant: CREATIVE BIOMOLECULES, INC. [US/ 

US]; 35 South Street, Hopkinton. MA 01748 (US). 

(72) Inventors: KUBERASAMPATH, Thangavel ; 6 Spring 

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



(74) Agents: KELLEY, Robin, D.; Testa, Hurwitz & Thibeault, 
Exchange Place, 53 State Street, (US) et al. 



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



Published 

With international search report. 
With amended claims. 



(54) Title: MORPHOGEN-INDUCED MODULATION OF INFLAMMATORY RESPONSE 



(57) Abstract 

The present invention is directed to methods and compositions for alleviating tissue destructive effects associated with the 
inflammatory response to tissue injury in a mammal. The methods and compositions include administering a therapeutically ef- 
fective concentration of a morphogen or morphogen-stimulating agent sufficient to alleviate immune <;ell-mediated tissue destruc- 
tion. 



FOR THE PURPOSES OF INFORMATION ONLY 



Codes used to identify Stares party to the PCI- on the front pages of pamphlets publishing international 
applications under the PCT. 



AT 


Ausura 


Fl 


AU 


Auslralla 


FR 


BB 


Baiiuulus 


GA 


BE 


Bcl^uitt 


GB 


fiF 


Burkina Faso 


GN 


BC 


Bulgaria 


GR 


BJ 


Benin 


HU 


BR 


Brazil 


IE 


CA 


C^hitaila 


IT 


CF 


C^icntral African Republic 


JP 


CC 




KF 


CH 


Swiucrbnd 


KR 


CI 


Cote d'lwirc 


CM 


Cameroon 


U 


CS 


OccchoslovaLU 


LK 


CZ 


(>4xh Republic 


LU 


DE 


Germany 


MC 


DK 


Uenniark 


MG 


ES 


Spjiu 


Ml 



Finland 
Fmnce 
Gabon 

United Kingdom 

Guinea 

Graxe 

Hungary 

Ireland 

Italy 

Japan 

Dcmucrulic People^s Republic 
of Korea 

Republic or Korea 

licchtenstein 

Sri tanka 

loixemhourg 

Monaco 

Madagascar 

M^ilt 



MN 


Mongolia 


MR 


Maurilanb 


MW 


Malawi 


NL 


Netherlands 


NO 


Norway 


NZ 


New Zealand 


PL 


Puloiid 


PT 


Portugal 


RO 


Romania 


RU 


Russian Federation 


SO 


Sudan 


SE 


Sweden 


SK 


Slovak Republic 


SN 


Senegal 


5U 


Soviet Union 


TD 


Crhad 


TG 


Togo 


UA 


Ukraine 


US 


United States uf America 



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HORPHOGEN- INDUCED MODULATION OF 
5 INFLAMMATORY RESPONSE 

Field of thB Invention 

10 The present invention relates generally to a method 

for modulating the inflammatory response induced in a 
mammal following tissue injury. More particularly, 
this invention relates to a method for alleviating 
immune-cell mediated tissue destruction associated with 

15 the inflammatory response. 

Background of the Invention 

The hody's inflammatory response to tissue injury 

20 can cause significant tissue destruction, leading to 
loss of tissue function. Damage to cells resulting 
from the effects of inflammatory response e.g., by 
immune-cell mediated tissue destruction, has been 
implicated as the cause of reduced tissue function or 

25 loss of tissue function in diseases of the joints 
(e.g., rheumatoid and osteo-arthritis ) and of many 
organs, including the kidney, pancreas, skin, lung and 
heart. For example, glomular nephritis, diabetes, 
inflammatory bowel disease, vascular diseases such as 

30 atheroclerosis and vasculitis, and skin diseases such 
as psoriasis and dermatitis are believed to result in 
large part from unwanted acute inflammatory reaction 
and fibrosis. A number of these diseases, including 
arthritis, psoriasis and inflammatory bowel disease are 

35 considered to be chronic inflammatory diseases. The 



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damaged tissue also often is replaced by f ibrotic 
tissue, e.g., scar tissue, which further reduces tissue 
function. Graft and transplanted organ rejection also 
is believed to be primarily due to the action of the 
5 body's immune/inflammatory .response system. 

The immune-cell mediated tissue destruction often 
follows an initial tissue injury or insult. The 
secondary damage, resulting from the inflammatory 

10 response, often is the source of significant tissue 
damage. Among the factors thought to mediate these 
damaging effects are those associated with modulating 
the body's inflammatory response following tissue 
injury, e.g., cytokines such as interleukin-1 (IL-1) 

15 and tumor necrosis factor (TNF), and oxygen-derived 

free radicals such as superoxide anions. These humoral 
agents are produced by adhering neutrophilic leukocytes 
or by endothelial cells and have been identified at 
ischemic sites upon reperfusion. Moreover, TNF 

20 concentrations are increased in humans after myocardial 
infarction. 

A variety of lung diseases are characterized by 
airway inflammation, including chronic bronchitis, 

25 emphysema, idiopathic pulmonary fibrosis and asthma. 
Another type of lung-related inflammation disorders are 
inflammatory diseases characterized by a generalized, 
wide-spread acute inflammatory response such as adult 
respiratory distress syndrome. Another dysfunction 

30 associated with the inflammatory response is that 
mounted in response to injury caused by hyperoxia, 
e.g., prolonged exposure to lethally high 
concentrations of O2 (95-100% O^)- Similarly, reduced 



I 

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blood flow to a tissue {and^ therefore reduced or lack 
of oxygen to tissues)^ as described below, also can 
induce a primary tissue injury that stimulates the 
inflammatory response. 

5 

It is well known that damage occurs to cells in 
mammals which have been deprived of oxygen. In fact, 
the interruption of blood flow, whether partial 
(hypoxia) or complete (ischemia) and the ensuing 

10 inflammatory responses may be the most important cause 
of coagulative necrosis or cell death in human disease. 
The complications of atherosclerosis, for example, are 
generally the result of ischemic cell injury in the 
brain, heart, small intestines, kidneys, and lower 

15 extremities. Highly differentiated cells, such as the 
proximal tubular cells of the kidney, cardiac myocytes, 
and the neurons of the central nervous system, all 
depend on aerobic respiration to produce ATP, the 
energy necessary to carry out their specialized 

20 functions. When ischemia limits the oxygen supply and 
ATP is depleted, the affected cells may become 
irreversibly injured. The ensuing inflammatory 
responses to this initial injury provide additional 
insult to the affected tissue. Examples of such 

25 hypoxia or ischemia are the partial or total loss of 
blood supply to the body as a whole, an organ within 
the body, or a region within an organ, such as occurs 
in cardiac arrest, pulmonary embolus, renal artery 
occlusion, coronary occlusion or occlusive stroke. 

30 

The tissue damage associated with ischemia- 
reperfusion injury is believed to comprise both the 
initial cell damage induced by the deprivation of 
oxygen to the cell and its subsequent recirculation, as 
35 well as the damage caused by the body's response to 



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PCTAJS92/07^8 



10 



this initial damage. It is thought that reperfusion 
injury may result in dysfunction to the endothelium of 
the vasculature as well as injury to the surrounding 
tissue. In idiopathic pulmonary fibrosis, for example, 
scar tissue accumulates on the lung tissue lining, 
inhibiting the tissue's elasticity. The tissue damage 
associated with hyperoxia injury is believed to follow 
a similar mechanism, where the initial damage is 
mediated primarily through the presence of toxic oxygen 
metabolites followed by an inflammatory response to 
this initial injury. 



Similarly, tissues and organs for transplantation 
also are subject to the tissue destructive effects 

15 associated with the recipient host body's inflammatory 
response following transplantation. It is currently 
believed that the initial destructive response is due 
in large part to reperfusion injury to the transplanted 
organ after it has been transplanted to the organ 

20 recipient. 

Accordingly, the success of organ or tissue 
transplantation depends greatly on the preservation of 
the tissue activity (e.g., tissue or organ viability) 

25 at the harvest of the organ, during storage of the 
harvested organ, and at transplantation. To date, 
preservation of organs such as lungs, pancreas, heart 
and liver remains a significant stumbling block to the 
successful transplantation of these organs. U.S. 

30 Patent No. 4,952,409 describes a superoxide dismutase- 
containing liposome to inhibit reperfusion injury. 
U.S. Patent NO. 5,002,965 describes the use of 
ginkolides, known platelet activating factor 
antagonists, to inhibit reperfusion injury. Both of 

35 these factors are described working primarily by 



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inhibiting the release of and/or inhibiting the 
damaging effects of free oxygen radicals. A number of 
patents also have issued on the use of 
immunosuppressants for inhibiting graft rejection. A 
5 representative listing includes U.S. Patent Nos. 
5,104,858, 5,008,246 and 5,068,323. A significant 
problem with many immunosuppressants is their low 
therapeutic index, requiring the administration of high 
doses that can have significant toxic side effects. 

10 

Rheumatoid and osteoarthritis are prevalent 
diseases characterized by chronic inflammation of the 
synovial membrane lining the afflicted joint. A major 
consequence of chronic inflammatory joint disease 

15 (e.g., rheumatoid arthritis) and degenerative arthritis 
(e.g., osteoarthritis) is loss of function of those 
affected joints. This loss of function is due 
primarily to destruction of the major structural 
components of the joint, cartilage and bone, and 

20 subsequent loss of the proper joint anatomy. As a 
consequence of chronic disease, joint destruction 
ensues and can lead to irreversible and permanent 
damage to the joint and loss of function. Current 
treatment methods for severe cases of rheumatoid 

25 arthritis typically include the removal of the synovial 
membrane, e.g., synovectomy. Surgical synovectomy has 
many limitations, including the risk of the surgical 
procedure itself, and the fact that a surgeon often 
cannot remove all of the diseased membrane. The 

30 diseased tissue remaining typically regenerates, 

causing the same symptoms which the surgery was meant 
to alleviate. 



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10 



Psoriasis is a chronic, recurrent, scaling skin 
disease of unknown etiology characterized by chronic 
inflammation of the skin. Erythematous eruptions, 
often in papules or plaques, and usually having a white 
silvery scale, can affect any part of the skin, but 
most commonly affect the scalp, elbows, knees and lower 
back. The disease usually occurs in adults, but 
children may also be affected. Patients with psoriasis 
have a much greater incidence of arthritis (psoraitic 
arthritis), and generalized exfoliation and even death 
can threaten afflicted individuals. 



Current therapeutic regimens include topical or 
intralesional application of corticosteroids, topical 

15 administration of keratolytics , and use of tar and UV 
light on affected areas. No single therapy is ideal, 
and it is rare for a patient not to be treated with 
several alternatives during the relapsing and remitting 
course of the disease. Whereas systematic treatment 

20 can induce prompt resolution of psoriatic lesions, 
suppression often requires ever- increasing doses, 
sometimes with toxic side effect, and tapering of 
therapy may result in rebound phenomena with extensions 
of lesions, possibly to exfoliation. 

25 

Inflammatory bowel disease (IBD) describes a class 
of clinical disorders of the gastrointestinal mucosa 
characterized by chronic inflammation and severe 
ulceration of the mucosa. The two major diseases in 

30 this classification are ulcerative colitis and regional 
enteritis (Crohn's Disease). Like oral mucositis, the 
diseases classified as IBD are associated with severe 
mucosal ulceration (frequently penetrating the wall of 
the bowel and forming strictures and fistulas), severe 

35 mucosal and submucosal inflammation and edema, and 



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fibrosis (e.g., scar tissue formation which interferes 
with the acid protective function of the 
gastrointestinal lining.) Other forms of IBD include 
regional ileitis and proctitis. Clinically, patients 
5 with fulminant IBD can be severely ill with massive 
diarrhea, blood loss, dehydration, weight loss and 
fever. The prognosis of the disease is not good and 
frequently requires resection of the diseased tissue. 

10 Therefore, an object of the present invention is to 

provide a method for protecting mammalian tissue, 
particularly human tissue, from the damage associated 
with the inflammatory response following a tissue 
injury. The inflammatory reaction may be in response 

15 to an initial tissue injury or insult. The original 

injury may be chemically, mechanically, biologically or 
immunologically related. Another object is to provide 
methods and compositions for protecting tissue from the 
tissue destructive effects associated with chronic 

20 inflammatory diseases, including arthritis (e.g., 

reheumatoid or osteoarthritis), psoriatic arthritis, 
psoriasis and dermatitis, inflammatory bowel disease 
and other autoimmune diseases. Yet another object is 
to provide methods and compositions for enhancing the 

25 viability of mammalian tissues and organs to be 

transplanted, including protecting the transplanted 
organs from immune cell-mediated tissue destruction, 
such as the tissue damage associated with ischemia- 
reperfusion injury. This tissue damage may occur 

30 during donor tissue or organ harvesting and transport, 
as well as following initiation of blood flow after 
transplantation of the organ or tissue in the recipient 
host. 



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Another object of the invention is to provide a 
method for alleviating tissue damage associated with 
ischemic-reperfusion injury in a mammal following a 
deprivation of oxygen to a tissue in the mammal. Other 
5 objects of the present invention include providing a 
method for alleviating tissue damage associated with 
ischemic-reperfusion injury in a human which has 
suffered from hypoxia or ischemia following cardiac 
arrest, pulmonary embolus, renal artery occlusion, 
10 coronary occlusion or occlusive stroke. A further 
object is to provide a method for alleviating tissue 
damage associated with hyperoxia-induced tissue injury, 
e.g., lethally high oxygen concentrations. 

15 Still another object of the invention is to provide 

a method for modulating inflammatory responses in 
general, particularly those induced in a human 
following tissue injury. 

20 These and other objects and features of the 

invention will be apparent from the description, 
drawings and claims which follow. 



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Summary of the Invention 

The present invention provides a method for 
alleviating the tissue destructive effects associated 
5 with activation of the inflammatory response following 
tissue injury. The method comprises the step of 
providing to the affected tissue a therapeutically 
effective concentration of a morphogenic protein 
("morphogen"^ as defined herein) upon tissue injury or 
10 in anticipation of tissue injury, sufficient to 

substantially inhibit or reduce the tissue destructive 
effects of the inflammatoiy response* 

In one aspect, the invention features compositions 
15 and therapeutic treatment methods that comprise the 
step of administering to a mammal a therapeutically 
effective amount of a morphogenic protein 
( "morphogen" ) , as defined herein, upon injury to a 
tissue, or in anticipation of such injury, for a time 
20 and at a concentration sufficient to inhibit the tissue 
destructive effects associated with the body's 
inflammatory response, including repairing damaged 
tissue, and/or inhibiting additional damage thereto. 

25 In another aspect, the invention features 

compositions and therapeutic treatment methods for 
protecting tissues and organs from the tissue 
destructive effects of the inflammatory response which 
include administering to the mammal, upon injury to a 

30 tissue or in anticipation of such injury, a compound 
that stimulates in vivo a therapeutically effective 
concentration of an endogenous morphogen within the 
body of the mammal sufficient to protect the tissue 
from the tissue destructive effects associated with the 

35 inflammatory response, including repairing damaged 



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10 



30 



tissue and/or inhibiting additional damage thereto. 
These compounds are referred to herein as morphogen- 
stimulating agents, and are understood to include 
substances which, when administered to a mammal, act on 
cells of 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. 



As embodied herein, the term " is chemic-reper fusion 
injury" refers to the initial damage associated with 
oxygen deprivation of a cell and the subsequent damage 
15 associated with the inflammatory response when the cell 
is resupplied with oxygen. As embodied herein, the 
term "hyperoxia- induced injury" refers to the tissue 
damage associated with prolonged exposure to lethally 
high doses of oxygen, e.g., greater than 95% Oj, 
20 including the tissue damage associated with the 
inflammatory response to the toxically high oxygen 
dose. Accordingly, as used herein, "toxic oxygen 
concentrations" refers to the tissue damage associated 
withthe injury induced by both lethally low oxygen 
25 concentrations of oxygen (including a complete lack of 
oxygen), and by lethally high oxygen concentrations. 
The expression "alleviating" means the protection from, 
reduction of and/or elimination of undesired tissue 
destruction, particularly immune cell-mediated tissue 
destruction. The tissue destruction may be in response 
to an initial tissue injury, which may be mechanical, 
chemical or immunological in origin. The expression 
"enhance the viability of" living tissues or organs, as 
used herein, means protection from, reduction of and/or 
elimination of reduced or lost tissue or organ function 



35 



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as a result of tissue deaths particularly immune cell- 
mediated tissue death* "Transplanted" living tissue 
encompasses both tissue transplants (e.g.^ as in the 
case of bone marrow transplants) and tissue grafts. 
5 Finally, a "free oxygen' radical inhibiting agent" means 
a molecule capable of inhibiting the release of and/or 
inhibiting tissue damaging effects of free oxygen 
radicals • 

10 In one embodiment of the invention, the invention 

provides methods and compositions for alleviating the 
ischemic-reperfusion injury in mammalian tissue 
resulting from a deprivation of, and subsequent 
reperfusion of, oxygen to the tissue. In another 

15 embodiment, the invention provides a method for 

alleviating the tissue-destructive effects associated 
with hyperoxia. In still another embodiment of the 
invention, the invention provides methods and 
compositions for maintaining the viability of tissues 

20 and organs, particularly living tissues and organs to 
be transplanted, including protecting them from 
ischemia-reperfusion injury. In still another 
embodiment, the invention provides methods for 
protecting tissues and organs from the tissue 

25 destructive effects of chronic inflammatory diseases, 
such as arthritis, psoriasis, dermatitis, including 
contact dermatitis, IBD and other chronic inflammatory 
diseases of the gastrointestinal tract, as well as the 
tissue destructive effects associated with other, known 

30 autoimmune diseases, such as diabetes, multiple 

sclerosis, amyotrophic lateral sclerosis (ALS), and 
other autoimmune neurodegenerative diseases. 



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In one aspect of the invention, the raorphogen is 
provided to the damaged tissue following an initial 
injury to the tissue. The morphogen may be provided 
directly to the tissue, as by injection to the damaged 
5 tissue site or by topical administration, or may be 
provided indirectly, e.g., systemically by oral or 
parenteral means. Alternatively, as described above, 
an agent capable of stimulating endogenous morphogen 
expression and/or secretion may be administered to the 

10 mammal. Preferably, the agent can stimulate an 
endogenous morphogen in cells associated with the 
damaged tissue. Alternatively, morphogen expression 
and/or secretion may be stimulated in a distant tissue 
and the morphogen transported to the damaged tissue by 

15 the circulatory system. 

In another aspect of the invention, the morphogen 
is provided to tissue at risk of damage due to immune 
cell-mediated tissue destruction. Examples of such 

20 tissues include tissue grafts and tissue or organ 
transplants, as well as any tissue or organ about to 
undergo a surgical procedure or other clinical 
procedure likely to either inhibit blood fltjw to the 
tissue or otherwise induce an inflammatory response. 

25 Here the morphogen or morphogen- stimulating agent 
preferably is provided to the patient prior to 
induction of the injury, e.g., as a prophylactic, to 
provide a cyto-protective effect to the tissue at risk. 



30 



Where the tissue at risk comprises a tissue or 
organ to be transplanted, the tissue or organ to be 
transplanted preferably is exposed to a morphogen prior 
to transplantation. Most preferably, the tissue or 
organ is exposed to the morphogen prior to its removal 
35 from the donor, by providing the donor with a 



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- 13 - 

composition comprising a morphogen or morphogen- 
stimulating agent. Alternatively or, in addition, once 
removed from the donor, the organ or tissue is placed 
in a preservation solution containing a morphogen or 
5 morphogen-stimulating agent. In addition, the 

recipient also preferably is provided with a morphogen 
or morphogen-stimulating agent just prior to, or 
concommitant with, transplantation. In all cases, the 
morphogen or morphogen-stimulating agent may be 
10 administered directly to the tissue at risk, as by 
injection or topical administration to the tissue, or 
it may be provided systemically, either by oral or 
parenteral administration. 

15 The morphogens described herein are envisioned to 

be useful in enhancing viability of any organ or living 
tissue to be transplanted. The morphogens may be used 
to particular advantage in lung, heart, liver, kidney 
or pancreas transplants, as well as in transplantation 

20 and/or grafting of bone marrow, skin, gastrointestinal 
mucosa, and other living tissues. 

Where the patient suffers from a chronic 
inflammatory disease, such as diabetes, arthritis, 

25 psoriasis, IBD, and the like, the morphogen or 

morphogen-stimulating agent preferably is administered 
at regular intervals as a prophylactic/ to prevent 
and/or inhibit the tissue damage normally associated 
with the disease during flare periods. As above, the 

30 morphogen or morphogen-stimulating agent may be 

provided directly to the tissue at risk, for example by 
injection or by topical administration, or indirectly, 
as by systemic e.g., oral or parenteral administration. 



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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 
5 Drosophila), Vgl (from 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. 

10 ID No. 24, see Wharton et al. (1991) PNAS 

88:9214-9218.) The members of this family, which 
Ii[clude 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 

15 precursor, having an N-terminal signal peptide 
sequence, typically less than about 30 residues, 
followed by a "pro" domain that is cleaved to yield the 
mature sequence. The signal peptide is cleaved rapidly 
upon translation, at a cleavage site that can be 

20 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 

25 sources for the amino acid sequences for the full 

length proteins not included in the Seq. Listing. The 
disclosure of these publications is incorporated herein 
by reference. 

TABLE I 

30 

"OP-1" Refers generically to the group ot 

morphogenically active proteins expressed 
from part or all of a DNA sequence 
encoding OP-1 protein, including allelic 
35 and species variants thereof, e.g., human 



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OP-1 {"hOP-1"/ Seq. ID No. 5, mature 
protein amino acid sequence), or mouse 
OP-1 ("mOP-1", Seq. ID No. 6, mature 
protein amino acid sequence.) The 
5 conserved seven cysteine skeleton is 

defined by residues 38 to 139 of Seq. ID 
Nos. 5 and 6. The cDNA sequences and the 
amino acids encoding the full length 
proteins are provided in Seq. Id Nos. 16 

10 and 17 (hOPl) and Seq. ID Nos. 18 and 19 

(mOPl.) The mature proteins are defined 
by residues 293-431 (hOPl) and 292-430 
(mOPl). The "pro" regions of the 
proteins, cleaved to yield the mature, 

15 morphogenically active proteins are 

defined essentially by residues 30-292 
(hOPl) and residues 30-291 (mOPl). 

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

20 proteins expressed from part or all of a 

DNA sequence encoding OP-2 protein, 
including allelic and species variants 
thereof, e.g., human OP-2 {"hOP-2", Seq. 
ID No. 7, mature protein amino acid 

25 sequence) or mouse OP-2 ("mOP-2", Seq. ID 

No. 8, mature protein amino acid 
sequence). The conserved seven cysteine 
skeleton is defined by residues 38 to 139 
of Seq. ID Nos. 7 and 8. The cDNA 

30 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 

35 (hOP2) and 261-399 (mOP2). The "pro" 



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regions of the proteins, cleaved to yield 
the mature, nrarphogenically active 
proteins likely are defined essentially by 
residues 18-263 (hOP2) and residues 18-260 
5 (mOP2). (Another cleavage site also 

occurs 21 residues upstream for both OP-2 
proteins . ) 

"CBMP2" refers generically to the morphogenically 

IQ active proteins expressed from a DNA 

sequence encoding the CBMP2 proteins, 
including allelic and species variants 
thereof, e.g., human CBMI'2A {"CBMP2A(fx) 
Seq ID No. 9) or human CBMP2B DNA 

15 ("CBMP2B(fx)", Seq. ID No. 10). The amino 

acid sequence for the full length 
proteins, referred to in the literature as 
BMP2A and BMP2B, or BMP2 and BMP4, appear 
in Wozney, et al. (1988) Science 242: 1528- 

20 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; 

25 the mature protein, residues 257-408 or 

293-408. 

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

No. 11). The amino acid sequence for the 
full length protein appears in Padgett, et 



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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 
5 residues 457-588. 

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

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

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

15 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 

20 conserved seven cysteine skeleton (Seq. ID 

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

25 peptide cleavage site to residue 299; the 

mature protein likely is defined by 
residues 300-438. 

"GDF-l(fx)" refers to protein sequences encoded by the 
30 human GDF-1 gene and defining the 

conserved seven cysteine skeleton (Seq. ID 
No. 14). The cDNA and encoded amino 
sequence for the full length protein is 



wo 93/04692 



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PCrAJS92/07358 



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

••60A" refers generically to the morphogenically 

active proteins expressed from part or all 
of a DNA sequence (from the Drosophila^ 6 OA 

10 gene) encoding the 60A proteins (see Seq. 

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

15 conserved seven cysteine skeleton 

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

20 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). 

25 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 

30 mature protein likely is defined by 

residues 291-472. 



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PCr/US92/07358 



- 19 - 

"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 
10 317-454. 

refers to protein sequences encoded by the 
human BMP6 gene and defining the conserved 
seven cysteine skeleton (Seq. ID No. 28). 
The amino acid sequence for the full 
length protein appears in Celeste, et al. 
(1990) PNAS 87: 9843-5847. The pro domain 
likely includes extends from the signal 
peptide cleavage site to residue 374; the 
mature sequence likely includes 
residues 375-513. 



"BMP6(fx)" 

15 



20 



The OP-2 proteins have an additional cysteine 
25 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. 
30 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. 



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PCr/US92/07358 



The morphogens are inactive when reduced, but are 
active as oxidized homodimers and when oxidized in 
combination with other morphogens of this invention 
(e.g., as heterodimers ) . Thus, as defined herein, a 
5 morphogen is a dimeric protein comprising a pair of 
polypeptide chains, wherein each polypeptide chain 
comprises at least the C-terminal six cysteine skeleton 
defined by residues 43-139 of Seq. ID No. 5, including 
functionally equivalent arrangements of these cysteines 

10 (e.g., amino acid insertions or deletions which alter 
the linear arrangement of the cysteines in the sequence 
but not their relationship in the folded structure), 
such that, when the polypeptide chains are folded, the 
dimeric protein species comprising the pair of 

15 polypeptide chains has the appropriate three- 
dimensional structure, including the appropriate intra- 
or inter-chain disulfide bonds such that the protein is 
capable of acting as a morphogen as defined herein. 
Specifically, the morphogens generally are capable of 

20 all of the following biological functions in a 

morphogenically permissive environment: stimulating 
proliferation of progenitor cells? stimulating the 
differentiation of progenitor cells? stimulating the 
proliferation of differentiated cells; and supporting 

25 the growth and maintenance of differentiated cells, 

including the "redifferentiation" of transformed cells, 
in addition, it is also anticipated that these 
morphogens are capable of inducing redifferentiation of 
committed cells under appropriate environmental 

30 conditions. 

In one preferred aspect, the morphogens of this 
invention comprise one of two species of generic amino 
acid sequences: Generic Sequence 1 (Seq. ID No. 1) or 
35 Generic Sequence 2 (Seq. ID No. 2); where each Xaa 



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PCr/US92/073S8 



indicates one of the 20 naturally-occurring L-isomer, 
a-amino acids or a derivative thereof. Generic 
Sequence 1 comprises the conserved six cysteine 
skeleton and Generic Sequence 2 comprises the conserved 
5 six cysteine skeleton plus the additional cysteine 

identified in OP-2 (see residue 36, Seq. ID No. 2). In 
another preferred aspect, these sequences further 
comprise the following additional sequence at their N- 
terminus: 

10 

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



Preferred amino acid sequences within the 

15 foregoing generic sequences include: Generic Sequence 
3 (Seq. ID No. 3), Generic Sequence 4 (Seq. ID No. 4), 
Generic Sequence 5 (Seq. ID No. 30) and Generic 
Sequence 6 (Seq. ID No. 31), listed below. These 
Generic Sequences accommodate the homologies shared 

20 among the various preferred members of this morphogen 
family identified in Table II, as well as the amino 
acid sequence variation among them. Specifically, 
Generic Sequences 3 and 4 are composite amino acid 
sequences of the following proteins presented in 

25 Table II and identified in Seq. ID Nos. 5-14: human 
OP-1 (hOP-1, Seq. ID Nos. 5 and 16-17), mouse OP-1 
(mOP-1, Seq. ID Nos. 6 and 18-19), human and mouse OP-2 
(Seq. ID Nos. 7, 8, and 20-22), CBMP2A (Seq. ID No. 9), 
CBMP2B (Seq. ID No. 10), DPP (from Drosophila, Seq. ID 

30 No. 11), Vgl, (from Xenopus, Seq. ID No. 12), Vgr-1 
(from mouse, Seq. ID No. 13), and GDF-1 (from mouse, 
Seq. ID No. 14.) The generic sequences include both 
the amino acid identity shared by the sequences in 
Table II, as well as alternative residues for the 

35 variable positions within the sequence. Note that 



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PCr/US92/07358 



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 form, and contain certain critical amino acids 

which influence the tertiary structure of the proteins. 

Generic Sequence 3 
Leu Tyr Val Xaa Phe 
10 1 5 

Xaa Xaa Xaa Gly Trp Xaa Xaa Trp Xaa 

10 

Xaa Ala Pro Xaa Gly Xaa Xaa Ala 
15 20 
15 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 
20 40 45 

Xaa xaa Leu Xaa Xaa Xaa Xaa Xaa 

50 

Xaa xaa Xaa Xaa Xaa Xaa Xaa Cys 
55 60 
25 Cys Xaa Pro Xaa Xaa Xaa Xaa Xaa 



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- 23 - 

Xaa Xaa Xaa Leu Xaa Xaa Xaa 

70 75 
Xaa Xaa Xaa Xaa Val Xaa Leu Xaa 
80 

5 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 

10 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); 

15 Xaa at res. 12 = (Asp, Arg or Asn); Xaa at res. 14 « (lie 
or Val); Xaa at res. 15 - (lie 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. 2 3 = (Tyr, Asn or Phe); Xaa at 

20 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); 

25 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 = (He or 
Val); Xaa at res. 45 - (Val or Leu); Xaa at res. 46 = 

30 (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 



wo 93/04692 



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10 



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.56 
= (Thr, Ala, Val, Lys, Asp, Tyr, Ser or Ala); Xaa at 
res. 57 = (Val, Ala or lie); 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 
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 - 
15 (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); 
20 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); 

25 

Generic Sequence 4 

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

30 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 
35 30 35 



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PCr/US92/07358 



Xaa Pro Xaa Xaa Xaa Xaa Xaa 

40 

Xaa Xaa Xaa Asn His Ala Xaa Xaa 
45 50 
5 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 Xaia 
10 70 

Xaa Xaa Xaa Leu Xaa Xaa Xaa 

75 80 
Xaa Xaa Xaa Xaa Val Xaa Leu Xaa 
85 

15 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 

20 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 

25 res. 11 = (Arg, <31n, 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 = (He or Val); Xaa at res. 20 = 
(He or Val); Xaa at res. 23 = {<31u. Gin, Leu, Lys, Pro 

30 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 

35 or Tyr); Xaa at res. 38 = (Leu or Val); Xaa at res. 39 = 



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PCr/US92/07358 



10 



15 



(Asn, Asp, Ala or Thr); Xaa at res. 40 » (Ser, Asp, Glu, 
Leu or Ala); Xaa at re&.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 
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, 
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 = 
(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)f Xaa at res. 73 = (Asn, Ser or Asp); 
Xaa at res. 74 = (Ala, Pro or Ser); Xaa at res. 75 = 
(He, Thr or Val); Xaa at res. 7 6 = (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 
= (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). 



20 



30 



wo 93/04692 PCT/US92/07358 

- 27 - 



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-1, Seq. ID Nos. 6 and 18-19), 
human and mouse OP-2 (Seq. ID Nos. 7, 8, and 20-22), 
CBMP2A (Seq. ID No. 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), human BMP3 
(Seq. ID No. 26), human BMP5 (Seq. ID No. 27), human 
BMP6 (Seq. ID No. 28) and 60(A) (from Drosophila, Seq. 

15 ID Nos. 24-25). The generic sequences include both the 
amino acid identity shared by these sequences in the 
C-terminal domain, defined by the six and seven 
cysteine skeletons (Generic Sequences 5 and 6, 
respectively), as well as alternative residues for the 

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/US92/073S8 



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 He); 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 



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PCrAJS92/073S8 



= (Leu/ Val or He); Xaa at res. 11 = (<31n, Leu^ Asp, 
His, Asn or Ser); Xaa at res. 12 - (Asp, Arg, Asn or 
Glu); Xaa at res. 14 = (He 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. 35 = (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 = (He, 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 93/04692 



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PCr/US92/07358 



10 



or lie); 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. 93 = (Ala, Gly, Glu or Ser); Xaa at res. 95 = (Gly 
or Ala) and Xaa at res. 97 = (His or Arg). 

Caneric Sequence 6 





Cys 


xaa 


Xaa 


Xaa 


Xaa 


Leu 


Xaa 


Xaa 




1 








5 










Xaa 


Xaa 


Xaa 


Gly 


Trp 


xaa 


Xaa 


Trp 


20 










15 










Xaa 


xaa 


Pro 


Xaa 


xaa 


Xaa 


Xaa 


Ala 




20 










25 








Xaa 


Tyr 


cys 
30 


Xaa 


Gly 


xaa 


Cys 


Xaa 
35 


25 


Xaa 


Pro 


Xaa 


Xaa 


Xaa 
40 


Xaa 


xaa 






Xaa 


Xaa 


Xaa 
45 


Asn 


His 


Ala 


xaa 


xaa 
50 




Xaa 


Xaa 


Xaa 


Xaa 


Xaa 


Xaa 


Xaa 


Xaa 


30 










55 










Xaa 


xaa 
60 


xaa 


xaa 


Xaa 


Xaa 


Xaa 
65 


Cys 




Cys 


Xaa 


Pro 


Xaa 


Xaa 


Xaa 


Xaa 


Xaa 



10 



70 



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- 31 - 



PCr/US92/073S8 



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 



10 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 = 

15 (Glu, Ser, His, Gly, Arg, Pro, Thr, or Tyr); Xaa at 
res. 7 = (Tyr or Lys); Xaa at res. 8 = (Val or lie); 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 

20 = (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 

25 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 

30 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 

35 res. 44 = (Ala, Ser, Gly or Pro); Xaa at res. 45 = (Thr, 



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PCr/US92/073S8 



Leu or Ser); Xaa at res.49 = (He, Val or Thr); Xaa at 
res.50 = (Val, Leu or He)? Xaa at res. 51 = (Gin or 
Arg); Xaa at res. 52 = (Thr, Ala or Ser); Xaa at res. 53 
= (Leu or He); 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 = (He, 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. 9 5 = 
(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. 100 = (Gly or Ala); and Xaa at res. 102 * 
(His or Arg) . 



wo 93/04692 



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PCT/US92/073S8 



Particularly useful sequences for use as xnorphogens 
in this invention include the C-terminal domains/ e.g., 
the C-terminal 96-102 amino acid residues of Vgl, 
Vgr-1, DPP, OP-1, OP-2, CBMP-2A, CBMP-2B, GDF-1 (see 
5 Table II/ below, and Seq. ID Nos. 5-14)/ as well as 
proteins comprising the C-terminal domains of SOA, 
BMP3, BMP5 and BMP6 (see Seq. ID Nos. 24-28), all of 
which include at least the conserved six or seven 
cysteine skeleton. In addition/ biosynthetic 

10 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 

15 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, 

20 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 

25 changes, e.g., those as defined by Dayoff et al.. Atlas 
of Protein Secruence 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 

30 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 

35 al. 



wo 93/04692 



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PCrAJS92/073S8 



The currently most preferred protein sequences 
useful as morphogens in this invention include those 
having greater than 60% identity, preferably greater 
than 65% identity, with the amino acid sequence 
5 defining the conserved six cysteine skeleton of feOPl 
(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 described 

20 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. 



wo 93/04692 



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PCr/US92/073S8 



The proteins may include forms having varying 
glycosylation patterns, varying N-termini, a family of 
related proteins having regions of amino acid sequence 
homology, and active truncated or mutated forms of 
5 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 

10 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 

15 description of the morphogens useful in the methods, 
compositions and devices 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 disclosure of which are incorporated herein 

20 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 

25 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 protecting tissues and organs from 

30 immune cell-mediated tissue destruction, including 
substantially inhibiting such damage and/or 
regenerating the damaged tissue in a variety of 
mammals, including humans. 



wo 93/04692 



- 36 - 



PCr/US92/07358 



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



wo 93/04692 PCT/US92/073S8 

- 37 - 



Brief Description of the Drawings 

FIG 1 shows the cardioprotective effects of 

morphogen (hOPl) in a rat myocardial ischemia- 
5 reperfusion model/ as evidenced by the smaller 

loss of myocardial creatine kinase in hOPl- 
treated rats; 

FIG 2 shows the effects of 20 /jg of morphogen (hOPl 
10 given 24 hours prior to isolation of rat heart 

on endothelial-dependent vasorelaxation to 
acetycholine following induced ischemia- 
reperfusion injury; 

15 FIG 3 shows the effect of morphogen (hOPl) on 
neutrophil adherence to LTB^ -stimulated 
mesenteric artery endothelium in neutrophil- 
activated rats; 

20 FIG 4 (A and B) are schematic representations of 
morphogen inhibition of early mononuclear 
phagocytic multinuclearization in vivo ; 



FIG 5 graphs the effect of a morphogen [e.g., OP-1) 
25 and a placebo control on mucositic lesion 

formation; and 



FIG 6 (A-D) graphs the effects of a morphogen (eg., 
OP-1/ Figs. 6A and 6C) and TGF-p (Fig. 6B and 
30 6D) on collagen {6A and 6B) and hyaluronic 

acid (6C and 6D) production in primary 
fibroblast cultures. 



wo 93/04692 



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PCr/US92/07358 



10 



Detailed Description of the Invention 

It now has been surprisingly discovered that the 
morphogens defined herein are effective agents in 
alleviating the tissue destructive effects associated 
with the body's inflammatory response to tissue injury. 
In particular, as disclosed herein, the morphogens are 
capable of alleviating the necrotic tissue effects 
associated with the ensuing inflammatory responses that 
occur following an initial tissue injury. 



When tissue injury occurs, whether caused by 
bacteria, trauma, chemicals, heat, or any other 
phenomenon, the body's inflammatory response is 

15 stimulated. In response to signals released from the 
damaged cells (e.g., cytokines), extravascularization 
of immune effector cells is induced. Under ordinary 
circumstances these invading immune effector cells kill 
the infectious agent and/or infected or damaged cells 

20 (through the release of killing substances such as 

superoxides, perforins, and other antimicrobial agents 
stored in granules), remove the dead tissues and 
organisms (through phagocytosis), release various 
biological response modifiers that promote rapid 

25 healing and covering of the wound (quite often 

resulting in the formation of fibrotic scar tissue), 
and then, after the area is successfully healed, exit 
from the site of the initial insult. Once the site is 
perceived to be normal, the local release of 

30 inflammatory cytokines ceases and the display of 

adhesion molecules on the vessel endothelium returns to 
basal levels. In some cases, however, the zeal of 
these interacting signals and cellular systems, which 
are designed to capture and contain very rapidly 

35 multiplying infectious agents, act to the detriment of 



wo 93/04692 



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PCr/US92/073S8 



the body/ killing additional, otherwise healthy, 
surrounding tissue. This additional unnecessary tissue 

, death further compromises organ function and sometimes 

results in death of the individual. In addition, the 

^ 5 resulting scar tissue that often forms can interfere 

with normal tissue function as occurs, for example, in 
idiopathic pulmonary fibrosis, IBD and organ cirrhosis. 

The vascular endothelium constitutes the first 

10 barrier between circulating immune effector cells and 
extravascular tissues. Extravasation of these 
circulating cells requires that they bind to the 
vascular endothelial cells, cross the basement 
membrane, and enter insulted tissues e.g, by 

15 phagocytosis or protease-mediated extracellular matrix 
degradation. Without being limited to a particular 
theory, it is believed that the morphogens of this 
invention may modulate the inflammatory response in 
part by modulating the attachment of immune effector 

20 cells to the luminal side of the endothelium of blood 
vessels at or near sites of tissue damage and/or 
inflammatory lesions. Because the method reduces or 
prevents the attachment of immune effector cells at 
these sites, it also prevents the subsequent release of 

25 tissue destructive agents by these same immune effector 
cells at sites of tissue damage and/or inflammatory 
lesions. Because attachment of immune effector cells 
to the endothelium must precede their 
extravascular izat ion, the method also prevents the 

30 initial or continued entry of these cells into 

extravascular sites of tissue destruction or ongoing 
inflammatory lesions. Therefore, the invention not 
only relates to a method to reduce or prevent the 
immune cell-mediated cellular destruction at 

35 extravascular sites of recent tissue destruction, but 



wo 93/04692 



- 40 - 



PCr/US92/07358 



10 



also relates to a method to prevent or reduce the 
continued entry of immune effector cells into 
extravascular sites of ongoing inflammatory cascades. 
As will be appreciated by those skilled in the art, the 
morphogens of this invention also may be contemplated 
in mechanisms for disrupting the functional interaction 
of immune effector cells with endothelium where the 
adhesion molecules are induced by means other than in 
response to tissue injury. 



One source of tissue injury is induced by cell 
exposure to toxic oxygen concentrations, such as 
ischemic-reperfusion tissue injury (oxygen 
deprivation), and following hyperoxia injury (lethally 
15 high oxygen concentrations). Accordingly, the process 
of the present invention provides a method for 
alleviating the tissue damage induced by ischemic- 
reperfusion injury or hyperoxia-induced injury 
comprising the step of administering to the afflicted 
20 individual a therapeutic amount of a morphogen prior 
to, during, or after damage to the affected tissue. 
Where the toxic oxygen concentrations may be 
deliberately induced, as by a surgical or clinical 
procedure, the morphogen preferably is administered 
25 prior to induction. 

In addition, the morphogens described herein, in 
contrast to fibrogenic growth factors such as TGF-p, 
stimulate tissue morphogenesis and do not stimulate 

30 fibrosis or scar tissue formation (see Example 9, 
below.) Accordingly, in addition to inhibiting the 
tissue destructive effects associated with the 
inflammatory response, the morphogens further enhance 
the viability of damaged tissue and/or organs by 

35 stimulating the regeneration of the damaged tissue and 
preventing fibrogenesis. 



wo 93/04692 



PCr/US92/07358 



- 41 - 

The morphogens described herein also can inhibit 
epithelial cell proliferation (see Example 10, below.) 
This activity of the morphogens also may be 
particularly useful in the treatment of psoriasis and 
5 other inflammatory diseases that involve epithelial 
cell populations. 

Provided below are detailed descriptions of 
suitable morphogens useful in the methods and 

10 compositions of this invention, as well as methods for 
their administration and application, and numerous, 
nonlimiting examples which 1) illustrate the 
suitability of the morphogens and morphogen-stimulating 
agents described herein as therapeutic agents for 

15 protecting tissue from the tissue destructive effects 
associated with the body's inflammatory response; and 
2) provide assays with which to test candidate 
morphogens and morphogen-stimulating agents for their 
efficacy. 

20 

I. Useful Morphogens 

As defined herein a protein is morphogenic if it is 
25 capable of inducing the developmental cascade of 
cellular and molecular events that culminate in the 
formation of new, organ-specific tissue and comprises 
at least the conserved C-terminal six cysteine skeleton 
or its functional equivalent (see supra). 
30 Specifically, the morphogens generally are capable of 
all of the following biological functions in a 
morphogenically permissive environment: stimulating 
proliferation of progenitor cells; stimulating the 
differentiation of progenitor cells; stimulating the 
35 proliferation of differentiated cells; and supporting 



wo 93/04692 



- 42 - 



PCT/US92/07358 



the growth and maintenance of differentiated cells, 
including the "redif ferentiation" of transformed cells. 
Details of . how the morphogens useful in the method of 
this invention first were identified, as well as a 
5 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 hereinabove 
incorporated by reference. As disclosed therein, the 

10 morphogens may be purified from naturally-sourced 

material or recombinantly produced from procaryotic or 
eucaryotic host cells, using the genetic sequences 
disclosed therein. Alternatively, novel morphogenic 
sequences may be identified following the procedures 

15 disclosed therein. 

Particularly useful proteins include those which 
comprise the naturally derived sequences disclosed in 
Table II. Other useful sequences include biosynthetic 
20 constructs such as those disclosed in U.S. Pat. 

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). 

25 Accordingly, the morphogens useful in the methods 

and compositions of this invention also may be 
described by morphogenically active proteins having 
amino acid sequences sharing 70% or, preferably, 80% 
homology (similarity) with any of the sequences 

30 described above, where "homology" is as defined herein 
above. 



wo 93/04692 



- 43 - 



PCr/US92/07358 



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, 
A, 5 and 6). Generic sequences 1 and 2 also may 
5 include, at their N-terminus, the sequence 

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

10 Table II, set forth below, compares the amino acid 

sequences of the active regions of native proteins that 
have been identified as morphogens, including human 
OP-1 (hOP-1, Seq. ID Nos. 5 and 16-17), mouse OP-1 
(mOP-1, Seq. ID Nos. 6 and 18-19), human and mouse OP-2 

15 (Seq. ID Nos. 7, 8, and 20-23), CBMP2A (Seq. ID No. 9), 
CBMP2B (Seq. ID No. 10), BMP3 (Seq. ID No. 26), DPP 
(from Drosophila, Seq. ID No. 11), Vgl, (from Xenopus, 
Seq. ID No. 12), Vgr-1 (from mouse, Seq. ID No. 13), 
GDF-1 (from mouse, Seq. ID Nos. 14, 32 and 33), 60A 

20 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, 
calculated using the Align Program (DNAstar, Inc.) In 

25 the table, three dots indicates that the amino acid in 
that position is the same as the amino acid in hOP-1. 
Three dashes indicates that no amino acid is present in 
that position, and are included for purposes of 
illustrating homologies. For example, amino acid 

30 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 
and lie. 



PCr/US92/07358 

WO 93/04692 

- 44 - 



TABLE II 



10 



15 



20 



25 



30 



hOP-1 


cys 


Lys 


Lys 


His 


Glu 


Leu 


Tyr 


Val 




mOP-I 




• « 


• • • 


• • • - 


• • • 




• • • 






hOP-2 




Arg 


Arg 


• • • 


• • • 










mOP-2 




Arg 


Arg 


• ■ • 


• • • 




« • • 






DPP 




Arg 


Arg 


• • • 


Ser 




... 










• • • 


Lys 


Arg 


His 










Vgr-l 




• « « 


♦ • • 


* * * . 


Gly 
















Arg 


• • * 


Pro 




« • • 






CBMP-2B 




Arg 


Arg 


• • • 


Ser 










BMPS 




Ala 


Arg 


Arg 


Tyr 




Lys 










Are 


Ala 


Arg 


Arg 




• • * 






An A 




Gin 


Met 


Glu 


Thr 




• • • 






BHP5 




*. • * 


• • • 


■ • • 


• • • 




• • • 






BHP6 




Arg 


• • • 


• • • 


• • • 




• • • 








1 








5 










hOP-1 


Ser 


Phe 


Arg 


Asp 


Leu 


Gly 


Trp 


Gin 


Asp 


mOP-1 






• • • 




« •- • 






-• • • 


* • « 


hOP-2 






Gin 


• • « 


• * • 






Leu 


• • • 




Ser 














Leu 


• • 


mOP-2 




• • • 












DPP 


Asp 




Ser 




Val 






Asp 


Asn 


Vel 


Glu 




Lys 




Val 






♦ • 




• * • 




Gin 




Val 






• « 

Asn 


• • • 


CBMP-2A 


Asp 




Ser 




Val 






• • • 


CBHP-2B 


Asp 




Ser 




Val 






Asn 




BMP3 


Asp 




Ala 


• » • 


He 






Ser 


Glu 


GDF-1 


.• • • 




• » * 


Glu 


Val 






His 


Arg 


60A 


Asp 




Lys 


• •' • 


" • • • 






His 


• • • 


B1IP5 


♦ •■ • 




• • • 


«' 








• •■ • 


• • • 


BMP6 


• •■ • 




Gin 


* • « 








« • • 








10 










15 







35 



wo 93/04692 



- 45 " 



PCT/US92/07358 



hOP-1 

mOF-l 

hOP-2 

idOP-2 

DPP 

fgl 

Vgr-1 

CBHP-2A 

CBMP-2B 

BHP3 

GDF-1 

60A 

BIIP5 

BHP6 



Trp lie He Ala Pro Glu Gly Tyr Ala 



Val 
Val 

• « • 

Val 



Val 



Val 



Val 
Val 



Ser 



Lys 



Gin ... ... Ser 

Gin Ser 

Leu Asp 

Gin Met 

Lys ••• ... 

Fro His 

Fro Gin 

Lys Ser Fhe Asp 

Arg ... Fhe Leu 

... ... ... Gly' 



... ... 



20 



25 



hOP-1 

mOP-1 

hOP-2 

mOP-2 

DPP 

Vgl 

Vgr-l 

CBHP-2A 

CBHP-2B 

BHP3 

GDF-1 

60A 

BHP5 

BHP6 



Ala Tyr Tyr Cys Glu Gly Glu Cys Ala 



Asn 
Asn 
Pbe 
Phe 

... 

Asn 
Phe 
Phe 
Asn 



His 
Tyr 
Asp 
His 
His 
Ser 
Gin 
Ser 
Asp 
Asp 



Lys 



Glu 
Asp 
Ala 
Gin 



30 



Ser 

... 

Pro 
Pro 
Ser 
Pro 
Pro 
Gin 

... 

Asn 
Ser 
Ser 
35 



hOP-1 



Phe Pro Leu Asn Ser lyr Met Asn Ala 



wo 93/04692 



46 - 



PCr/US92/»7358 



10 



hOP-2 

inOP-2 

DPP 

Vgl 

Vgr-1 

CB1IP-2A 

CBMP-2B 

GDF-1 

BHP3 

60A 

BUPS 

BHF6 



Leu 



Val 
Met 



Asp 
Asp 
Ala 
Thr 

• • • 

Ala 
Ala 
Ala 
Pra 



Asp 
Glu 
Ala 
Asp 
Asp 
Leu 
Lys 
Ala 
Ala 
Ala 
40 



Cys 
Cys 
His 
He 
His 
His 
His 
Ser 
Ser 
His 
His 
His 



Phe 
Leu 
• * • 
Leu 
Leu 
Gly 
Leu 
• « • 
Het 
Het 



Ser** 
Lys 



Ser 
Gly 

Ser 
Ser 

• • •- 

Pro 



15 



20 



25 



30 



hOP-1 

mOP-l 

hOP-2 

inOP-2 

DPP 

Vgl 

Vgr-1 

CBMP-2A 

CBMP-2B 

BHP3 

GDF-1 

60A 

BUPS 

BHF6 



35 



hOP-1 
mOP-1 
hOP-2 
mOP-2 



Thr Asn His Ala He Val Gin Thr Leu 



Ser 



Ser 
Leu 



45 



Leu 
Leu 

• « * 

Leu 

r • • • • 



Val 



Thr He 
Val Leu 



His 
His 



Leu 
Leu 



Het 
Het 



Lys 
Lys 



50 



Val His Phe He Asn Pro 



Glu 
Asp 
Asn 
Asp 



Ser 
Ser 



Ser He 
Arg Ala 



Thr Val 
• « • • • • 
Ala 
Val 



wo 93/04692 



- 47 - 



PCT/US92/073S8 





DPP 


... 


Asn 


Asn 


Asn 


• . . 


... 


Gly 


Lys 


-• • 




Vgl 


... 


... 


Ser 


... 


Glu 


... 


... 


Asp 


He 




Vgr-1 


... 


... 


Val 


Het 


... 


... 


... 


Tyr 


... 




CBHP-2A 


... 


Asn 


Ser 


Val 


... 


Ser 


— 


Lys 


lie 


5 


CBHP-2B 


• • . 


Asn 


Ser 


Val 




Ser 




Ser 


He 




BHF3 




Arg 


Ala:llr* 


Gly 


Val 


Val 


Pro 


Gly 


lie 




GDF-1 


Het 


... 


Ala 


Ala 


Ala 


... 


Gly 


Ala 


Ala 




60A 






Leu 


Leu 


Glu 




Lys 


Lys 


... 




BHP5 


... 


... 


Leu 


Het 


Phe 


... 


Asp 


His 


... 


10 


BHP6 






Leu 


Het 


... 


... 


. * * * 


Tyr 


... 








55 










60 








hOP-1 


Pro 


Lys 


Pro 


Cys 


Cys 


Ala 


Pro 


Thr 


Gin 


15 


mOP-l 


. • * 


... 


... 




... 












hOP-2 


... 


• • • 


Ala 




• • • 


... 






Lys 




inOP-2 


• • • 


• • • 


Ala 




... 


w * • 






Lys 




DPP 


... 




Ala 




... 


Val 






... 




Vgl 


... 


Leu 






... 


Val 






Lys 


20 


Vgr-1 


• • • 


• • • 






... 








Lys 




CB1IP-2A 


... 




Ala 






Val 






Glu 




CBMP-2B 






Ala 




... 


Val 






Glu 








uXU 












Glu 


jujr o 




GDF-1 


Asp 


Leu 






• * • 


Val 




Ala 


Arg 


25 


60A 










... 


... 




... 


Arg 




BMP5 


... 


... 








... 




... 


Lys 




BHP6 


... 


... 






... 


... 




... 


Lys 










65 










70 




30 


hOP-1 


Leu 


Asn 


Ala 


He 


Ser 


Val 


Leu 


Tyr 


Phe 




mOP-1 


... 


. • « 














• * «. 




hOP-2 




Ser 




Thr 




... 






Tyr 




mOP-2 




Ser 




Thr 


... 


... 






Tyr 




Vgl 


Met 


Ser 


Pro 






Het 









35 Vgr-1 Val ... 



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- 48 - 



10 



15 



20 



DPP 

CBMP-2A 

CBHP-2B 

B1IP3 

GDF-1 

60A 

B1IP5 

B1IP6 



hOP-1 

mOP-1 

hOP-2 

mOP-2 

DPP 

Vgl 

Vgr-1 

CBHP-2A 

CBHP-2B 

BMP3 

GDF-1 

60A 

BHP5 

BVB6 



Het 



Asn 



Leu 



Asp 
Ser 
Ser 
Ser 
Ser 
Gly 



Ser Val Ala 



Ser 
Pro 



Leu 



Leu Pro 



75 



Het 
Het 
Het 
lie 



Leu 
Leu 
Leu 

Phe Tyr 

Phe 

His 



80 



Asp Asp Ser Ser Asn Val He Leu 



Ser 
Ser 

• • 

Asn 

Glu 
Glu 
Glu 
Asn 
Asn 



Gin 
Asn 
Asn 
Asn 
Tyx 
Asn 

Asp 
- • • « 
Asn 



Asn 
Asn 

Asp 
» ■ • 

Glu 
Asp 
Lys 
Asp 
Glu 



Thr 



Lys 
Lys 



Val 
Val 
• • • 
Val 
Val 
Val 
Val 
Asn 



Lys 

*. * * 

Arg 
Arg 

Arg 



Arg 



25 



85 



30 



35 



hOP-1 

mOP-l 

hOP-2 

inOP-2 

DPP 

Vgl 

Vgr-1 

CBHP-2A 



Lys Tyr Arg Asn Het Val Val Arg 



Asn 
His 

• • • 

Asn 



His 
Bis 



Gin 
Glu 



Glu 



Gin Asp 



Ala 



Lys 
Lys 
Val 
Asp 

Glu 



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PCT/US92/073S8 



CBHP-2B 

BHP3 

GDF-l 

60A 

BHP5 

BHP6 



Asn 
Val 
Gin 



Gin Glu 
Fro 

Glu Asp 



Tip 



90 



Thr 

• • « 

He 

« *■ 

• • ■ 

95 



€lu 
Glu 
Asp 
Lys 



10 



15 



20 



25 



hOP-1 

mOP-1 

hOP-2 

inOP-2 

DPP 

Vgl 

Vgr-l 

CBMP-2A 

CBMP-2B 

BHP3 

GDF-l 

60A 

BMP5 

BHP6 



Ala Cys Gly Cys His 



Gly 
Glu 

Gly 
Gly 
Ser 
Glu 
Ser 
Ser 



Ala 



100 



Arg 
Arg 

Arg 
Arg 
Arg 
Arg 



it^Betveen residues 56 and 57 of BHPS is a Val residue; 

between residues A3 and 44 of GDF-l lies 
the amino acid sequence Gly-Gly-Pro-Pro. 



30 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 

35 only about 50% amino acid identity with the hOPl 



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10 



15 



sequence described therein, the GDF-1 sequence shares 
greater than 70% amino acid sequence homology (or 
"similarity") with the hOPl sequence, where "homology" 
or "similarity" includes allowed conservative amino 
acid changes within the sequence as defined by Dayoff , 
et al.. Atlas of Protein Sequ ence and Structure vol.5, 
supp.3, pp.345-362, (M.O. Dayoff, ed., Nat'l BioMed. 
Res. Fd'n, Washington D.C. 1979.) 



The currently most preferred protein sequences 
useful as morphogens in this invention include those 
having greater than 60% identity, preferably greater 
than 65% identity, with the amino acid sequence 
defining the conserved six cysteine skeleton of 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 still another 
preferred aspect, the invention includes morphogens 
20 comprising species of polypeptide chains having the 
generic amino acid sequence referred to herein as 
"OPX", which defines the seven cysteine skeleton and 
accommodates the identities between the various 
identified mouse and human OPl and 0P2 proteins. OPX 
25 is presented in Seq. ID No. 29. As described therein, 
each Xaa at a given position independently is selected 
from the residues occurring at the corresponding 
position in the C-terminal sequence of mouse or human 
OPl or 0P2 (see Seq. ID Nos. 5-8 and/or Seq. ID Nos. 
30 16-23). 



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II. Formulations and Methods for Administering 
Therapeutic Agents 

The morphogens may be provided to an individual by 
5 any suitable means / preferably directly (e.g., locally/ 
as by injection or topical administration to a tissue 
locus) or systemically (e.g./ parenterally or orally). 
Where the morphogen is to be provided parenterally / 
such as by intravenous, subcutaneous , intramuscular/ 

10 intraorbital/ ophthalmic, intraventricular/ 
intracranial , intracapsular / intraspinal , 
intracisternal/ intraperitoneal, buccal, rectal/ 
vaginal, intranasal or by aerosol administration, the 
morphogen preferably comprises part of an aq\ieous 

15 solution. The solution is physiologically acceptable 
so that in addition to delivery of the desired 
morphogen to the patient, the solution does not 
otherwise adversely affect the patient's electrolyte 
and volume balance. The aqueous medium for the 

20 morphogen thus may comprise noarmal physiologic saline 
(9.85% NaCl, 0.15M)/ pH 7-7.4. The aqtieous 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% 

25 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 

30 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 keeps the morphogen soluble 
in physiological buffers. In fact, the endogenous 

35 protein is thought to be transported in this form. 



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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%. 
5 Other components found in milk and/or various serum 
proteins also may be useful. 

Useful solutions for parenteral administration may 
be prepared by any of the methods well known in the 

10 pharmaceutical art, described, for example, in 

Remington's Pharmaceutical Sciences (Gennaro, A., ed.). 
Mack Pub., 1990. Formulations may include, for 
example, polyalkylene glycols such as polyethylene 
glycol, oils of vegetable origin, hydrogenated 

15 naphthalenes, and the like. Formulations for direct 
administration, in particular, may include glycerol and 
other compositions of high viscosity to help maintain 
the morphogen at the desired locus. Biocompatible, 
preferably bioresorbable, polymers, including, for 

20 example, hyaluronic acid, collagen, tricalcium 
phosphate, polybutyrate, lactide and glycolide 
polymers, and lactide/glycolide copolymers, may be 
useful excipients to control the release of the 
morphogen in vivo. Other potentially useful parenteral 

25 delivery systems for these morphogens include ethylene- 
vinyl acetate copolymer particles, osmotic pumps, 
implantable infusion systems, and liposomes. 
Formulations for inhalation administration contain as 
excipients, for example, lactose, or may be aqueous 

30 solutions containing, for example, polyoxyethylene-9- 
lauryl ether, glycocholate and deoxycholate , or oily 
solutions for administration in the form of nasal 
drops, or as a gel to be applied intranasally. 



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Formulations for parenteral administration may also 
include glycocholate for buccal administration/ 
methoxy salicylate for rectal administration , or cutric 
acid for vaginal administration. 

5 

Suppositories for rectal administration also may be 
prepared by mixing the morphogen or morphogeri- 
stimulating agent with a non- irritating excipient such 
as cocoa butter or other compositions which are solid 
10 at room temperature and liquid at body temperatures. 

Formulations for topical administration to the skin 
surface may be prepared by dispersing the morphogen or 
morphogen- stimulating agent with a dermally acceptable 

15 carrier such as a lotion, cream, ointment or soap. 

Particularly useful are carriers capable of forming a 
film or layer over the skin to localize application and 
inhibit removal. For topical administration to 
internal tissue surfaces, the morphogen may be 

20 dispersed in a liquid tissue adhesive or other 

substance known to enhance adsorption to a tissue 
surface. For example, hydroxypropylcellulose or 
fibrinogen/ thrombin solutions may be used to advantage. 
Alternatively, tissue-coating solutions, such as 

25 pectin-containing formulations, may be used. 

Mtematively, the morphogens described herein may 
be administered orally • Oral administration of 
proteins as therapeutics generally is not practiced as 

30 most proteins are readily degraded by digestive enzymes 
and acids in the mammalian digestive system before they 
can be absorbed into the bloodstream. However, the 
morphogens described herein typically are acid stable 
and protease-resistant (see, for example, U.S. Pat. No. 

35 4,968,590.) In addition, at least one morphogen, OP-1, 



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has been identified in mammary gland extract, colostrum 
and 57-day milk. Moreover, the OP-1 purified from 
mammary gland extract is morphogenically active. 
Specifically, this protein induces endochondral bone 
5 formation in mammals when implanted subcutaneous ly in 
association with a suitable matrix material, using a 
standard in vivo bone assay, such as is disclosed in 
U.S. Pat. No. 4,968,590. Moreover, the morphogen also 
is detected in the bloodstream. Finally, soluble form 

10 morphogen, e.g., mature morphogen associated with the 
pro domain, is morphogenically active. These findings 
indicate that oral and parenteral administration are 
viable means for administering morphogens to an 
individual. In addition, while the mature forms of 

15 certain morphogens described herein typically are 
sparingly soluble, the morphogen form found in milk 
(and mammary gland extract and colostrum) is readily 
soluble, probably by association of the mature, 
morphogenically active form with part or all of the pro 

20 domain of the intact sequence and/or by association 
with one or more milk components. Accordingly, the 
compounds provided herein also may be associated with 
molecules capable of enhancing their solubility in 
vitro or in vivo . 

25 

Where the morphogen or morphogen-stimulating agent 
comprises part of a tissue or organ preservation 
solution, any commercially available preservation 
solution may be used to advantage. For example, useful 

30 solutions known in the art include Collins solution, 
Wisconsin solution, Belzer solution, Eurocollins 
solution and lactated Ringer's solution. Generally, an 
organ preservation solution usually possesses one or 
more of the following properties: (a) an osmotic 

35 pressure substantially equal to that of the inside of a 



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PCT/US92/073S8 



mammalian cell, (solutions typically are hyperosmolar 
and have K+ and/or Mg++ ions present in an amount 
sufficient to produce an osmotic pressure slightly 
higher than the inside of a mammalian cell; (b) the 
5 solution typically is capable of maintaining 

substantially normal ATP levels in the cells; and (c) 
the solution usually allows optimum maintenance of 
glucose metabolism in the cells. Organ preservation 
solutions also may contain anticoagulants^ energy 

10 sources such as glucose/ fructose and other sugars, 
metabolites, heavy metal chelators, glycerol and other 
materials of high viscosity to enhance survival at low 
temperatures, free oxygen radical inhibiting agents and 
a pH indicator. A detailed description of 

15 preservation solutions and useful components may be 
found, for example, in US Patent No. 5,002,965, the 
disclosure of which is incorporated herein by 
reference. 

20 The compounds provided herein also may be 

associated with molecules capable of targeting the 
morphogen or morphogen-stimulating agent to the desired 
tissue. For example, an antibody, antibody fragment, 
or other binding protein that interacts specifically 

25 with a surface molecule on cells of the desired tissue, 
may be used. Useful targeting molecules may be 
designed, for example, using the single chain binding 
site technology disclosed, for example, in U.S. Pat. 
No. 5,091,513. 

30 

As described above, the morphogens provided herein 
share significant sequence homology in the C-terminal 
active domains. By contrast, the sequences typically 
diverge significantly in the sequences which define the 
35 pro domain. Accordingly, the pro domain is thought to 



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be morphogen- specific. As described above, it is also 
known that the various inorphogens identified to date 
are differentially expressed in the different tissues. 
Accordingly, without being limited to any given theory, 
5 it is likely that, under natural conditions in the 
body, selected morphogens typically act on a given 
tissue. Accordingly, part or all of the pro domains 
which have been identified associated with the active 
form of the morphogen in solution, may serve as 

10 targeting molecules for the morphogens described 
herein. For example, the pro domains may interact 
specifically with one or more molecules at the target 
tissue to direct the morphogen associated with the pro 
domain to that tissue. Accordingly, another useful 

15 targeting molecule for targeting morphogen to a tissue 
of interest is part or all of a morphogen pro domain. 
For example, part or all of the pro domain of GDF-1 may 
be used to target a morphogen to nerve tissue. 
Alternatively, part or all of the pro domain of OP-1 or 

20 CBMP2 may be used to target a morphogen to bone tissue, 
both of which proteins are found naturally associated 
with bone tissue. 

The morphogens described herein are useful for 
25 providing neuroprotective effects to alleviate neural 
pathway damage associated with the body's 
immune/inflammatory response to an initial injury to 
nerve tissue. As used herein, a "neural pathway" 
describes a nerve circuit for the passage of electric 
30 signals from a source to a target cell site and 
includes both the central nervous system (CNS) and 
peripheral nervous system (PNS). The pathway includes 
the neurons through which the electric impulse is 
transported, including groups of interconnecting 
35 neurons, the nerve fibers formed by bundled neuronal 



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PCr/US92/07358 



axons, and the glial cells surrounding and associated 
with the neurons. An inflanimatory response to nerve 
tissue injury may follow trauma to nerve tissue , 
caused, for example, by an autoimmune (including 
5 autoantibody) dysfunction, neoplastic lesion, 

infection, chemical or mechanical trauma, or other 
disease. An exemplary nerve-related inflammatory 
disease is multiple sclerosis. Neural pathway damage 
also can result from a reduction or interruption, e.g., 

10 occlusion, of a neural blood supply, as in an embolic 
stroke, (e.g, ischemia or hypoxia-induced injury), or 
by other trauma to the nerve or surrounding material. 
In addition, at least part of the damage associated 
with a number of primary brain tumors also appears to 

15 be immunologically related. Application of the 
morphogen directly to the cells to be treated, or 
providing the morphogen to the mammal systemically, for 
example, intravenously or indirectly by oral 
administration, may be used to alleviate and/or inhibit 

20 the immunologically related response to a neural 
injury. Alternatively, administration of an agent 
capable of stimulating morphogen expression and/or 
secretion in vivo, preferably at the site of injury, 
also may be used. Where the injury is to be induced, 

25 as during surgery or other aggressive clinical 

treatment, the morphogen or agent may be provided prior 
to induction of the injury to provide a neuroprotective 
effect to the nerve tissue at risk. 

30 Where the morphogen is intended for use as a 

therapeutic to alleviate tissue damage associated with 
an immune/inflammatory condition of the CNS, an 
additional problem must be addressed: overcoming the 
so-called "blood-brain barrier", the brain capillary 

35 wall structure that effectively screens out all but 



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selected categories of molecules present in the blood, 
preventing their passage into the brain. The 
blood-brain barrier may be bypassed effectively by 
direct infusion of the morphogen or morphogen- 
5 stimulating agent into the brain. Alternatively, the 
morphogen or morphogen-stimulating agent may be 
modified to enhance its transport across the 
blood-brain barrier. For example, truncated forms of 
the morphogen or a morphogen-stimulating agent may be 

10 most successful. Alternatively, the morphogen or 

morphogen-stimulating agent may be modified to render 
it more lipophilic, or it may be conjugated to another 
molecule which is naturally transported across the 
barrier, using standard means known to those skilled in 

15 the art, as, for example, described in Pardridge, 
Endocrine Reviews 7 ; 314-330 (1986) and U.S. Pat. 
No. 4,801,575. 

Finally, the morphogens or morphogen-stimulating 
20 agents provided herein may be administered alone or in 
combination with other molecules known to be beneficial 
in the treatment compositions and methods described 
herein, including, but not limited to anticoagulants, 
free oxygen radical inhibiting agents, salicylic acid, 
25 vitamin D, and other antiinflammatory agents. Psoriais 
treatments also may include ultra-violet light 
treatment, zinc oxide and retinoids. 

The compounds provided herein can be formulated 
30 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 



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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. 

5 

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 for a time 
10 sufficient to alleivate the tissue destructive effects 
associated with the inflammatory response, including 
protecting tissue in anticipation of tissue damage. 

As will be appreciated by those skilled in the art, 

15 the concentration of the compounds described in a 
therapeutic composition will vary depending upon a 
number of factors, including the dosage of the drug to 
be administered, the chemical characteristics (e.g., 
hydrophobicity ) of the compounds employed, and the 

20 route of administration. The preferred dosage of drug 
to be administered also is likely to depend on such 
variables as the type and extent of progression of the 
tissue damage, the overall health status of the 
particular patient, the relative biological efficacy of 

25 the compound selected, the formulation of the compound 
excipients, and its route of administration. In general 
terms, the compounds of this invention may be provided 
in an aqueous physiological buffer solution containing 
about 0.001% to 10% w/v compound for parenteral 

30 administration. Typical dose ranges are from about 10 
ng/kg to about 1 g/kg of body weight per day; a 
preferred dose range is from about 0.1 fjg/Kg to 
100 mg/kg of body weight per day. Optimally, the 
morphogen dosage given is between 0.1-100 fjg of protein 

35 per kilogram weight of the patient. No obvious 



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PCT/US92/073S8 



10 



15 



20 



30 



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

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



Where tissue injury is induced deliberately as part 
of, for example, a surgical procedure, the morphogen 
preferably is provided just prior to, or concomitant 
with induction of the trauma. Preferably, the morphogen 
is administered prophylactically in a surgical setting. 

Alternatively, an effective amount of an agent 
capable of stimulating endogenous morphogen levels may 
be administered by any of the routes described above. 
For example, an agent capable of stimulating morphogen 
25 production and/or secretion from cells of affected 
tissue and/or transplant tissue may be provided to a 
mammal, e.g., by direct administration of the agent to 
the tissue to be treated. A method for identifying and 
testing agents capable of modulating the levels of 
endogenous morphogens in a given tissue is described 
generally herein in Example 15, and in detail in 
copending USSN 752,859, filed August 30, 1991, the 
disclosure of which is incorporated herein by 
reference. Briefly, candidate compounds can be 



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PCTAIS92/07358 



identified and tested by incubating the compound in 
vitro with a test tissue or cells thereof/ for a time 
sufficient to allow the compound to affect the 
production/ i.e., the expression and/or secretion, of a 
5 morphogen produced by the cells of that tissue. 

For purposes of the present invention, the above- 
described morphogens effective in alleviating tissue 
damage associated with ischemic-reper fusion injury (or 

10 the agents that stimulate them, referred to 

collectively herein as "therapeutic agent") are 
administered prior to or during the restoration of 
oxygen (e.g., restoration of blood flow, reperfusion. ) 
Where treatment is to follow an existing injury, the 

15 therapeutic agent preferably is administered as an 
intravenous infusion provided acutely after the 
hypoxic or ischemic condition occurs. For example, the 
therapeutic agent can be administered by intravenous 
infusion immediately after a cerebral infarction, a 

20 myocardial infarction, asphyxia, or a cardiopulmonary 
arrest. Where ischemia or hypoxia injury is 
deliberately and/or unavoidably induced as part of, for 
example, a surgical procedure where circulation to an 
organ or organ system is deliberately and/or 

25 transiently interrupted, e.g., in carotid enterectomy, 
coronary artery bypass, grafting, organ transplanting, 
fibrinolytic therapy, etc., the therapeutic agent 
preferably is provided just prior to, or concomitant 
with, reduction of oxygen to the tissue- Preferably/ 

30 the morphogen is administered prophylactically in a 
surgical setting. 



35 



Similarly, where hyper oxia- induced injury already 
has occurred, the morphogen is administered upon 
diagnosis. Where hyperoxia injury may be induced as. 



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for example, during treatment of prematurely newborn 
babies, or patients suffering from pulmonary diseases 
such as emphysema, the therapeutic agent preferably is 
administered prior to administration of oxygen e.g., 
prophy lactically . 



III. Examples 

10 Example 1. Identification of Mor phngen-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 increscent 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 immediately following the stop codon. 
These portions of the sequence vary substantially among 
the morphogens of this invention, and accordingly, are 
specific for each protein. For example, a particularly 

10 useful Vgr-l-specif ic probe sequence is the PvuII-SacI 
fragment, a 265 bp fragment encoding both a portion of 
the untranslated pro region and the N-teiminus 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-1- specific 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 iiranediately 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 

30 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 

35 such as by the method of Chomczyaski et al. ((1987) 



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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 /ig) 
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 DV light (generally 30 seconds at 1 

10 mW/cm' ) . Prior to hybridization, the appropriate probe 
is denatured by heating. The hybridization is carried 
out in a Incite 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% 

15 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 

20 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 

25 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, liver 

30 and kidney tissue indicate that kidney-related tissue 
appears to be the primary expression source for OP-1, 
with brain, heart and lung tissues being secondary 
sources. OP-1 mRNA also was identified in salivary 
glands, specifically rat parotid glands, using this 

35 probing methodology. Lung tissue appears to be the 



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PCrAJS92/07358 



primary tissue expression source for Vgr-1, BMP5, BMP4 
and BMP3. Lower levels of Vgr-1 also are seen in kidney 
and heart tissue, while the liver appears to be a 
secondary expression source for BMP5/ and the spleen 
5 appears to be a secondary expression source for BMP4. 
GDF-1 appears to be expressed primarily in brain 
tissue. To date, OP-2 appears to be expressed 
primarily in early embryonic tissue. Specifically, 
northern blots of murine embryos and 6-day post-natal 
10 animals shows abundant 0P2 expression in 8-day embryos. 
Expression is reduced significantly in 17 -day embryos 
and is not detected in post-natal animals. 

Example 2. Active Morphogens in Body Fluids 

15 

OP-1 expression has been identified in saliva 
(specifically, the rat parotid gland, see Example 1), 
h^lman blood sertam, and various milk forms, including 
mammary gland extract, colostrum, and 57-day bovine 

20 milk. Moreover, and as described in USSN 923,780, the 
disclosure of which is incorporated herein by 
reference, the body fluid-extracted protein is 
morphogenically active. The discovery that the 
morphogen naturally is present in milk and saliva, 

25 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 

30 delivery for extended or prophylactic therapies. In 
addition, the identification of morphogen in all milk 
forms , including colostrum, suggests that the protein 
may play a significant role in tissue development, 
including skeletal development, of juveniles. 

35 



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2.1 Morphogen Detection in Milk 

GP-1 was partially purified from rat manunary gland 
extract and bovine colostrum and 57 day milk by passing 
5 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 
imraunoblot. Immunoreactive fractions then were 
10 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. 

15 OP-1 purified from the different milk sources were 

characterized by Western blotting using antibodies 
raised against OP-1 and BMP2. Antibodies were prepared 
using standard immunology protocols well known in the 
art, and as described generally in Example 15, below, 

20 using full-length E. coli- produced OP-1 and BMP2 as the 
immunogens. In all cases, the purified OP-1 reacted 
only with the anti-OP-1 antibody, and not with 
anti-BMP2 antibody. 

25 The morphogenic activity of OP-1 purified from 

mammary gland extract was evaluated in vivo essentially 
following the rat model assay described in U.S. Pat. 
No. 4,968,590, hereby incorporated by reference. 
Briefly, a sample was prepared from each OP-1 

30 immunoreactive fraction of the mammary gland 

extract-derived OP-1 final product by lyophilizing a 
portion (33%) of the fraction and resuspending the 
protein in 220,il of 50% acetonitrile/0 . 1% TFA. After 
vortexing, 25 mg of collagen matrix was added. The 

35 samples were lyophilized overnight, and implanted in 



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

2.2 Morphogen Detection in Serum 

Morphogen may be detected in serum using morphogen- 

15 specific antibodies. The assay may be performed using 
any standard immunoassay, such as Western blot 
(immunoblot) and the like. Preferably, the assay is 
performed using an affinity column to which the 
morphogen-specif ic antibody is bound and through which 

20 the sample serum then is poured, to selectively extract 
the morphogen of interest. The morphogen then is 
eluted. A suitable elution buffer may be determined 
empirically by determining appropriate binding and 
elution conditions first with a control (e.g., 

25 purified, recombinantly-produced morphogen . ) Fractions 
then are tested for the presence of the morphogen by 
standard immunoblot, and the results confirmed by 
N-terminal sequencing. Preferably, the affinity column 
is prepared using monoclonal antibodies. Morphogen 

30 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. 



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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 
5 morphogen in serum further indicates that systemic 
administration is a suitable means for providing 
therapeutic concentrations of a morphogen to an 
individual, and that morphogens likely behave 
systemically as endocrine-like factors. Finally, using 

10 this protocol, fluctuations in endogenous morphogen 

levels can be detected, and these altered levels may be 
used as an indicator of tissue dysfunction. 
Alternatively, fluctuations in morphogen levels may be 
assessed by monitoring morphogen transcription levels, 

15 either by standard northern blot analysis as described 
in Example 1, or by in situ hybridization, using a 
labelled probe capable of hybridizing specifically to 
morphogen mRNA, and standard RNA hybridization 
protocols well described in the art and described 

20 generally in Example 1. 

OP-1 was detected in human serum using the 
following assay. A monoclonal antibody raised against 
mammalian, recombinantly produced OP-1 using standard 

25 immunology techniques well described in the art and 
described generally in Example 15, was immobilized by 
passing the antibody over an agarose-activated gel 
(e.g., Affi-Gel'"', f rom Bio-Rad Laboratories, Richmond, 
CA, prepared following manufacturer's instructions) and 

30 used to purify OP-1 from serum. Human serum then was 
passed over the column and eluted with 3M 
K-thiocyanate. K-thiocyanante fractions then were 
dialyzed in 6M urea, 20mM PO^, pH 7.0, applied to a C8 
HPLC column, and eluted with a 20 minute, 25-50% 

35 acetonitrile/0.1% TFA gradient. Mature, recombinantly 



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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 2. A. 

5 

Administered or endogenous morphogen levels may be 
monitored in the therapies described herein by 
comparing the quantity of morphogen present in a body 
fluid sample with a predetermined reference value, for 

10 example, to evaluate the efficiency of a therapeutic 
protocol, and the like. In addition, fluctuations in 
the level of endogenous morphogen antibodies may be 
detected by this method, most likely in serum, using an 
antibody or other binding protein capable of 

15 interacting specifically with the endogenous morphogen 
antibody. Detected fluctuations in the levels of the 
mo3rphogen or endogenous antibody may be used, for 
example, as indicators of a change in tissue status. 
For example, as damaged tissue is regenerated and the 

20 tissue or organ's function returns to "normal" and, in 
the absence of additional tissue damage, lower doses of 
morphogen may be required, and a higher level of 
circulating morphogen antibody may be measured. 

25 Example 3. Effect of Morphogen after the Onset of 

the Ischemic Process 

The cardioprotective effect of morphogens following 
ischemic-reperfusion injury in a mammal can readily be 

30 assessed in a rat model. In this example, morphogen 

(e.g., OP-1) is administered just prior to the onset of 
the ischemic process in experimentally-induced 
myocardial infracted rats, essentially following the 
method of Lefer, et al. (1990) Science 249:61-64 and 

35 (1992) J. Mol. Cell. Cardiol. 24: 385-393, the 



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disclosures of which are hereby incorporated by 
reference. Briefly, loss of myocardial tissue function 
following ischemia and reperf usion is assayed by 
measuring loss of myocardial creatine kinease activity 
5 (CK) and loss of endothelium-dependent vasorelaxation 
function (see Example 4, below). 

In a first group of ether-anesthetized rats, the 
left coronary artery was occluded just proximal to the 

10 first main branch with a silk ligature to induce a 
myocardial infarction (MI). The ligature was removed 
10 minutes after occlusion to allow for coronary 
reperfusion. This first group is referred to herein as 
the "myocardial infarcted" (MI) group. A second group 

15 of rats underwent the same procedure except that the 
coronary artery was not occluded, and thus no 
myocardial infarction occurred. The second group of 
rats is referred to herein as the "sham myocardial 
infarcted group" (SHAM MI). 



20 



The first group of rats, the MI group of rats, 
further was divided into three sup-groups. 2pg of 
morphogen (OP-1) were injected intravenously into the 
first sub-group of MI rats 10 minutes after ligature, 
25 immediately before reperfusion? into the second sub- 
group of MI rats 20 fjg of OP-1 were injected 
intravenously 10 minutes after ligature and immediately 
before reperfusion; and into the third sub-group of MI 
rats (control) was injected vehicle only, e.g., 0.9% 
NaCl, as for the OP-1 treated rats. 



30 



Twenty-four hours later, the hearts were removed 
from all of the rats and the levels of creatine kinase 
(CK) from the left ventricle (the infarcted region) and 
35 from the interventricular septum (the control 



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nonischemic region) were determined by standard means. 
By comparing the difference in CK activities in both 
regions, the amount of CK activity lost from the 
infarcted region was used as an index of cardiac 
5 cellular injury to the infarcted region. 

As shown in Figure 1, the data indicate that 
morphogens (e.g., OP-1) can provide significant 
cardioprotective effect when provided to ischemic 
10 tissue. In the figure, CK loss is graphed as the 
difference in specific CK activity between the 
interventricular septum and the left ventricle. 

The loss of CK activity by the subgroup of MI rats 
15 which received 2 /jg of OP-1 just before reperfusion 
showed some protection as compared with the control MI 
rats which received injections of vehicle alone, when 
the levels from both subgroups are measured against, 
and compared to, the levels obtained for the SHAM MI 
20 control. Significant cardioprotection was observed in 
the subgroup of MI rats which received 20 ^g of OP-1 
immediately before reperfusion as compared with the 
control MI rats which received injections of vehicle 
alone, when the levels from both subgroups are measured 
25 against, and compared to, the levels contained within 
the SHAM MI control. 

These data indicate that OP-1 offers significant 
cardiac protection when administered after ischemia and 
30 before reperfusion. 



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A variation of this example also may be performed 
providing morphogen to the animal prior to induction of 
ischemia. The experiments may be performed both in 
normal and immune-compromised rats to assess the 
5 cardioprotective effects of morphogen administered 
prior to ischemia. 

Example 4. Vasodilation of Myocardial I nfarcted 
Cardiac Tissue Treated with Morphogen 

10 

Certain vasodilators like acetylcholine (ACh) and 
adenosine diphosphate (ADP, an immune mediator) exert 
their vasodilation activity only in the presence of 
intact endothelium, which is stimulated to release a 

15 substance termed endothelium-derived relaxing factor 
(EDRF). If the endothelium is injured so that EDRF is 
not released, no vasodilation occurs in response to 
these endothelium-dependent agents. In contrast, 
several other vasodilators including nitroglycerine 

20 (NTG) and nitroprusside, are endothelium- independent 
dilators, as they dilate blood vessels directly. 

The present example demonstrates the ability of OP- 
1 to prevent the loss of cardioendothelium-dependent 

25 relaxation (EDR) activity in the coronary 

microvasculature following reperfusion of ischemic 
myocardium, and their ability to reduce myocardial 
injury 24 hours after morphogen treatment. Briefly, 2 
or 24 hours after morphogen treatment ischemia- 

30 reperfusion injury is induced in isolated rat hearts, 
the reperfused hearts are are vasodilated with either 
ACh or NTG. In the absence of morphogen treatment, 
injured tissue should inhibit ACh-induced vasodilation, 
but not NTG-induced vasodilation. Morphogen treatment 

35 in expected to enhance ACh-induced vasodilation in the 
reperfused hearts. 



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Accordingly^ 48 adult male Sprague-Dawley rats 
(250-330 g) were divided into eight groups of 6 rats 
each. Twelve rats were subjected to sham myocardial 
infarcts (SHAM MI) as described in Example 3. The 
5 hearts of the remaining 36 rats were isolated as 
follows: one set of twelve rats was injected 
intravenously with OP-1 24 hours prior to isolation of 
the heart; another set of rats was injected 
intravenously with 20pg of OP-1 2 hours prior to 

10 isolation of the heart; the final group of rats was 

injected with vehicle only (e.g., 0.9% NaCl.). The rats 
then were anesthetized with pentobarbital sodium 
(35.mg/kg, intraperitonial ) ; their hearts were isolated 
and perfused by the Langendorff method at a constant 

15 flow (15 ml/min) with oxygenated Krebs-Henseleit 
solution (Aoki et al. (1988) J. Pharmacol . 95:35). 

Each group of rats then were divided into two 
subgroups of six rats each. Twenty minutes before 
reperfusion, coronary vasodilator response was measured 

20 by inducing constri<:tion with 0.05 pmol U-44619 (9,11- 
methanoepoxyprostaglandin Hj) followed by a 
vasodilating agent 3 minutes later: subgroup one - 
15 nmol ACh; subgroup 2-15 nmol NTG and the increase 
in coronary perfusion pressure (GPP) level measured as 

25 an indication of vasodilation. When CPP levels 
returned to normal, the hearts were subjected to 
ischemia by reducing coronary infusion to 15% of 
control flow for 30 minutes, then reestablishing normal 
flow, i.e., reperfusion, for an additional 20 minutes. 

30 

The vasodilator reponse then was remeasured by 
constriction and administration of vasodilating agent 
as described above. 



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The results of these experiments are shown in FIG 
2. Before the ischemic event, both Ach and NT6 gave 
normal vasorelaxant results in all events. The hearts 
which received OP-1 24 hours prior to ischemia showed 
5 an approximately 70% response to ACh while the hearts 
which received OP-1 2 hours prior to ischemia showed a 
55% response to ACh. The group which received vehicle 
alone showed a 40% response to ACh. Finally, the 
control group which was not subjected to ischemia 

10 showed an ACh response of approximately 95%. This 
shows that endotheliura-dependent vasodilators exert a 
reduced vasodilator response following ischemia and 
reperfusion in the rat heart. Moreover, OP-1 
significantly preserved endothelium-dependent dilation 

15 when provided 24 hours prior to induction of myocardial 
ischemia. Ho defect in vasodilation occurred in 
response to the direct vasodilator (NTG) ; NTG-induced 
vasodilation activities were 95% of initial in hearts 
subject to ischemia and 100% of initial nonischemic 

20 hearts. 

Example 5. Effect of Mo mhoaen on Neutrophil Adherence 

The role of neutrophU adherence in endothelium 
25 dysfunction and the cardioprotective effects of 

morphogens in modulating this activity can be assessed 
using a standard polymorphonuclear neutrophil (PMN) 
adherence assay such as described in Lefer et al., 
(1992) J. Mol. Cell. Cardiol. 24 ; 385-393, disclosed 
30 hereinabove by reference. Briefly, segments of 
superior mesenteric artery were isolated from rats 
which had either been treated with morphogen (OP-1, 20 
Ijg) or 0.9% NaCl, 24 h prior to isolation of the 
artery. The segments were cleaned, cut into transverse 
35 rings of l-2mm in length, and these were subsequently 



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cut open and incubated in K-H solution at 37 ^C, pH 7.4. 
Neutrophils were prepared and fluorescently labelled 
using standard procedures (e.g./ leukocytes were 
isolated from rats essentially following the procedure 
5 of Pertroft et. al. (1968) Exp Cell Res 50; 355-368, 
washed in phosphate buffered saline (PBS), purified by 
gradient centrifugation; and labelled by the method of 
Yuan et. al. (1990) Microvasc Res 40 : 218-229.. 

10 Labelled neutrophils then were added to open ring 

baths and activated with lOOnM leukotriene (LTB^)* 
Rings were incubated for 20 minutes and the number of 
neutrophils adhering to the endothelial surface then 
determined visually by fluorescent microscopy. 

15 

As shown in Figure 3, unstimulated PMNs (i.e., PMNs 
alone) added to the baths did not significantly adhere 
to the vascular endothelium. In rings taken from rats 
injected with 0.9% NaCl, activation of neutrophils with 
20 LTB^ (100 nM) greatly increased the number of PMNs 
adherent to the endothelium (P<0.001). OP-1 (20 pg 
administered 24 h prior) significantly inhibited 
adherence of PMNs activated by LTB^ (P<0.01 from 
control ) . 

25 

Example 6. In Vivo Models for Ischemic-Reperfusion 
Protection in Lung, Nerve and Renal 
Tissue . 

30 Other tissues seriously affected by ischemic- 

reperfusion injury include neural tissue, r^nal tissue 
and lung tissue. The effect of morphogens on 
alleviating the ischemic-reperfusion injury in these 
tissues may be assessed using methodologies and models 

35 known to those skilled in the art, and disclosed below. 



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Similarly, a methodology also is provided for assessing 
the tissue-protective effects of a morphogen on damaged 
lung tissue following hyperoxia injury. 

5 For example, the rabbit embolic stroke model 

provides a useful method for assessing the effect of 
morphogens on tissue injury following cerebral 
ischemia-reperfusion. The protocol disclosed below is 
essentially that of Phillips et al. (1989) Annals of 

10 Neurology 25:281-285, the disclosure of which is herein 
incorporated by reference. Briefly, white New England 
rabbits (2-3kg) are anesthesized and placed on a 
respirator. The intracranial circulation then is 
selectively catheterized by the Seldinger technique. 

15 Baseline cerebral angiography then is performed, 
employing a digital substration unit. The distal 
internal carotid artery or its branches then is 
selectively embolized with 0.035 ml of 18-hour-aged 
autologous thrombus. Arterial occlusion is documented 

20 by repeat angiography immediately after embolization. 
After a time sufficient to induce cerebral infarcts (15 
minutes or 90 minutes), reperfusion is induced by 
administering a bolus of a reperfusion agent such as 
the TPA analogue Fb-FB-CF (e.g., 0.8 mg/kg over 2 

25 minutes). 

The effect of morphogen on cerebral infarcts can be- 
assessed by administering varying concentrations of 
morphogens, e.g., OPl, at different times preceding or 
30 following embolization and/or reperfusion. The rabbxts 
are sacrificed 3-14 days post embolization and their 
brains prepared for neuropathological examination by 
fixing- by immersion in 10% neutral buffered formalin 



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for at least 2 weeks. The brains then are sectioned in 
a coronal plane at 2-3 mm interval s^ numbered and 
submitted for standard histological processing in 
paraffin, and the degree of neutral tissue necrosis 
5 determined visually. 

The renal-protective effects of morphogens on renal 
is chemia-reper fusion injury readily can be assessed 
using the mouse model disclosed by Oueliette, et al. 

10 (1990), J. Clin. Invest. 85;766-771, the disclosure of 
which is hereby incorporated by reference. Briefly, 
renal ischemia is induced surgically in 35-45 days old 
out-bred Swiss male mice by performing a standard right 
nephrectomy, and occluding the artery to the left 

15 kidney with a microaneurism clamp for 10-30 minutes. 
Morphogen then may be provided parentally, at various 
times prior to or following occulsion and/or 
reperfusion. The effects of morphogen then may be 
assessed by biological evaluation and histological 

20 evaluation using standard techniques well known in the 
art. 

The tissue protective effects of morphogen on 
tissue exposed to lethally high oxygen concentrations 

25 may be assessed by the following procedure. Adult rats 
(275-300 gms) first are provided with morphogen (e.g., 
hOPl) or vehicle only, and then are exposed to 96-98% 
oxygen essentially as described by Rinaldo et al (1983) 
Am. Rev. Respir. Pis. 130; 1065 , to induce hyperoxia. 

30 Animals are housed in plastic cages (38 cm x 48 xm x 21 
cm) . A cage containing 4-5 animals is placed in a 75 
liter water-sealed plexiglass chamber. An atmosphere 
of 96-98% oxygen then is maintained by delivery of Oj 
gas (liquid Oj). Gas flow through the chamber is 

35 adjusted to maintain at least 10 air changes/hr.. 



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temperature at 22 + l^C, minimal levels of condensation 
within the cage, and carbon dioxide concentration of < 
0.5% as measured with a mass spetrophotometric medical 
gas analyzer. 

5 

At the end of 72 hours all survivors are observed 
at room air for 1.5 hours and at longer time periods to 
assess degree of respiratory distress and cyanosis 
induced by the initial insult and subsequent immune 

10 cell-mediated damage. The number of survivors at the 
end of the challenge is recorded and the treated groups 
compared with the untreated control group by chi-square 
test of proportions. Several of the surviving animals 
for each group are randomly chosen for histological 

15 processing of lung tissue. 

Lung tissue for histological processing is fixed by 
infusion of 10% buffered formalin through a tracheal 
cannula at a constant pressure of 20 cm HjO. After 

20 fixation for 24-48 hours, sections from each lobe are 
cut and subsequently stained with hematoxylin and 
eosin. Coded slides then are examined, preferably in a 
double-blind fashion for evidence of pathological 
changes such as edema, interstitial cellularity, and 

25 inflammatory response. 

Example 7. Morohogen Inhibition of Cellular and 
Humoral Inflammatorv Response 

30 Morphogens described herein inhibit multinucleation 

of mononuclear phagocytic cells under conditions where 
these cells normally would be activated, e.g., in 
response to a tissue injury or the presence of a 
foreign substance. For example, in the absence of 

35 morphogen, an implanted substrate material (e.g.. 



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implanted subcutaneous ly) composed of, for example/ 
mineralized bone, a ceramic such as titanium oxide or 
any other substrate that provokes multinucleated giant 
cell formation, rapidly becomes surrounded by 
5 multinucleated giant cells, e.g., activated phagocytes 
stimulated to respond and destroy the foreign object. 
In the presence of morphogen however, the recruited 
cells remain in their mononuclear precursor form and 
the matrix material is undisturbed. Figure 4 

10 illustrates this effect of morphogens, in a schematic 
representation of histology results of a titanium oxide 
substrate implanted subcutaneously . In the figure, 
"mg" means mononuclear giant cells and "ob" means 
osteoblasts. The substrate represented in Fig. 4B was 

15 implanted together with morphogen (OP-1) and newly 
formed osteoblasts are evident surrounding the 
substrate. By contrast, the substrate represented in 
Fig. 4A was implanted without morphogen and extensive 
multinucleated giant cell formation is evident 

20 surrounding the substrate. Accordingly, the 

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

25 In addition, the morphogens described herein also 

suppress antibody production stimulated in response to 
a foreign antigen in a mammal. Specifically, when 
bovine bone collagen matrix alone was implanted in a 
bony site in a rat, a standard antibody response to the 

30 collagen is stimulated in the rat as determined by 
standard anti-bovine collagen ELISA experiments 
performed on blood samples taken at four week intervals 
following implantation (e.g., between 12 and 20 weeks.) 
Serum anti-collagen antibody titers, measured by ELISA 

35 essentially following the procedure described by 



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Nagler-Anderson et al, (1986) PTO^ 83-7443-7446, the 
disclosure of which is incorporated herein by 
reference, increased consistently throughout the 
experiment. However, when the matrix was implanted 
5 together with a morphogen (e.g., OP-1, dispersed in the 
matrix and adsorbed thereto, essentially as described 
in U.S. Pat. No. 4,968,590) anti-bovine collagen 
antibody production was suppressed significantly. This 
ability of morphogen to suppress the humoral response 
10 is further evidence of morphogen utility in alleviating 
tissue damage associated with autoimmune diseases, 
including autoantibody diseases, such as rheumatoid 
arthritis . 

15 Example 8. Morphogen protection of Gastrointestinal 

Tract Mucosa from alceration and 
Inflammation - 



20 



Oral mucositis is a gastrointestinal tract 
inflammatory disease which involves ulcerations of the 
mouth mucosa as a consequence of, e.g., radiation 
therapy or chemotherapy. While not typically a chronic 
disease, the tissue destructive effects of oral 
mucositis mirror those of chronic inflammatory diseases 
25 such as IBD. The example below demonstrates morphogen 
efficacy in protecting the oral mucosa from oral 
mucositis in a hamster model, including both inhibiting 
inflammatory ulceration and enhancing regeneration of 
ulcerated tissue. Details of the protocol can be found 
in Sonis, et al., (1990) Oral Surg. Oral Med. Oral 
Pathol 69: 437-443, the disclosure of which is 
incorporated herein by reference. Based on these data, 



30 



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the morphogens described herein should be efficacious 
in treating chronic inflammatory diseases including 
IBD, arthritis / psoriasis and psoriatic arthritis, 
multiple sclerosis, and the like. 

5 

Golden Syrian hamsters (6-8 wks old, Charles River 
Laboratories, Wilmington, MA) were divided into 3 test 
groups: Group 1, a placebo (e.g., saline) control, and 
a morphogen low dose group (100 ng) and a raorphogen 
10 high dose group (1 /ug). Groups 2 and 3, respectively. 
Morphogen dosages were provided in 30% ethanol. Each 
group contained 12 animals. 

Beginning on day 0 and continuing through day 5, 
15 Groups 2 and 3 received twice daily morphogen 
applications. On day 3, all groups began the 
mucositis-induction procedure. 5-f luorouracil (60 
mg/kg) was injected intraperitoneally on days 3 and 5, 
On day 7, the right buccal pouch mucosa was 
20 superficially irritated with a calibrated 18 gauge 
needle. In untreated animals, severe ulcerative 
mucositis was induced in at least 80% of the animals by 
day 10. 

25 For each administration of the vehicle control 

(placebo) or morphogen, administration was performed by 
first gently drying the cheek pouch mucosa, then 
providing an even application over the mucosal surface 
of the vehicle or raorphogen material. A 

30 hydroxypropylcellulose-based coating was used to 
maintain contact of the morphogen with the mucosa. 
This coating provided at least 4 hours of contact time. 



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

15 The mean mucositis score for each group was 

determined daily for each experimental group for a 
period of 21^ days by photography and visual examination 
of the right buccal cheek pouch. Differences between 
groups were determined using a standard 't' test, e.g., 

20 the Students' 't' test. In addition, data was 

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

25 

The experimental results are presented in Fig. 5, 
which graphs the effect of morphogen (high dose, 
squares; low dose, diamonds) and placebo (circles) on 
mean mucositis scores. Both low and high morphogen 
30 doses inhibit lesion formation significantly in a dose- 
dependent manner. In addition, histology results 
consistently showed significantly reduced amounts of 



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tissue atrophy^ cellular debris, and immune effector 
cells, including macrophages and activated neutrophils, 
in the morphogen-treated animals, as compared with the 
untreated, control animals. 

5 

Example 9. Morphoqen Effect on Fibrogenesis and Scar 
Tissue Formation 

The morphogens described herein induce tissue 
10 morphogenesis of damaged or lost tissue- The ability 
of these proteins to regenerate new tissue enhances the 
anti-inflammatory effect of these proteins. Provided 
below are a series of in vitro experiments 
demonstrating the ability of morphogens to induce 
15 migration and accumulation of mesenchymal cells. In 
addition, the experiments demonstrate that morphogens, 
unlike TGF-P/ do not stimulate fibrogenesis or scar 
tissue formation. Specifically, morphogens do not 
stimulate production of collagen, hyaluronic acid <HA) 
20 or metalloproteinases in primary fibroblasts, all of 
which are required for fibrogenesis or scar tissue 
formation. By contrast, TGF-p, a known inducer of 
fibrosis, but not of tissue morphogenesis, does 
stimulate production of these fibrosis markers. 

25 

Chemotaxis and migration of mesenchymal progenitor 
cells were measured in modified Boyden chambers 
essentially as described by Fava, R.A. et al (1991) 
Exp. Med. 173 ; 1121-1132, the disclosure of which is 

30 incorporated herein by reference, using polycarbonate 
filters of 2, 3 and 8 micron ports to measure migration 
of progenitor neutrophils, monocytes and fibroblasts. 
Chemotaxis was measured over a range of morphogen 
concentrations, e.g., 10"^°M to lO'^M OP-1. For 

35 progenitor neutrophils and monocytes, 10'^®-10"^^M OP-1 



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consistently induced maximal migration, and 10" to 
10" ^^M OP-1 maximally induced migration of progenitor 
fibroblasts. In all cases the chemotactic activity 
could be inhibited with anti-OP-1 antibody. Similar 
5 migration activities also were measured and observed 
with TGF-p. 

The effect of morphogen on fibrogenesis was 
determined by evaluating fibroblast production of 
10 hyaluronic acid (HA), collagen, collagenese and tissue 
inhibitor of metalloproteinases (TIME). 

Human fibroblasts were established from explants of 
infant foreskins and maintained in monolayer culture 

15 using standard culturing procedures. (See, for 

example, (1976) J. Exp. Med . 14i- 1188-1203.) Briefly, 
fibroblasts were grown in maintenance medium consisting 
of Eagle's MEM, supplemented with nonessential amino 
acids, ascorbic acid (50 fig/mL) , J^aBCO^ and HEPES 

20 buffers (pH 7.2), penicillin (100 U/ml), streptomycin 
(100 pg/ml), amphotericin B (1 //g/ml) and 9% heat 
inactivated FCS. Fibroblasts used as target cells to 
measure chemotaxis were maintained in 150 mm diameter 
glass petri dishes. Fibroblasts used in assays to 

25 measure synthesis of collagen, hyaluronic acid, 

collagenase and tissue inhibitors of metalloproteinases 
(TIMP) were grown in lOO mm diameter plastic tissue 
culture petri dishes. 



30 



The effects of morphogen on fibroblast production 
of hyaluronic acid, collagens, collagenase and TIMP 
were determined by standard assays (See, for example, 
Posttethwaite et al. (1989) J. Clin. Invest. 83: 629- 
636, posttethwaithe (1988) J./ Cell Biol. 106: 311-318 
35 and Clark et al (1985) Arch. Bio-chem Biophys. 241: 36- 



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PCT/US92/07358 



44, the disclosures of which are incorporated by 
reference.) For these assays, fibroblasts were 
transferred to 2 4 -well tissue culture plates at a 
density of 8 x 10^ cells per well. Fibroblasts were 
5 grown confluency in maintenance medium containing 9% 
FCS for 72 h and then grown in serum-free maintenance 
medium for 24 h. Medium was then removed from each 
well and various concentrations of OP-1 ( recombinantly 
produced mature or soluble form) or TGF-p-1 (R&D 

10 Systems, Minneapolis) in 50 pi PBS were added to 

triplicate wells containing the confluent fibroblast 
monolayers. For experiments that measured production 
of collagenase and TIMP, maintenance medium (450 fjl) 
containing 5% FCS was added to each well, and culture 

15 supernatants were harvested from each well 48 h later 
and stored at -70° C until assayed. For experiments 
that assessed HA production, maintenance medium (450 
/il) containing 2.5% FCS was added to each well, and 
cultures grown for 48 h. For experiments that measured 

20 fibroblast production of collagens, serum-free 

maintenance medium (450 fjl) without non-essential amino 
acids was added to each well and cultures grown for 72 
h. Fibroblast production of HA was measured by 
labeling newly synthesized glycosaminoglycans (GAG) 

25 with [^H]-acetate the last 24 h of culture and 

quantitating released radioactivity after incubation 
with hyaluronidase from Streptomyces hyalurolyticus 
(ICN Biochemicals, Cleveland, OH) which specifically 
degrades hyaluronic acid. Production of total collagen 

30 by fibroblasts was measured using a collagenase- 
sensitive protein assay that reflects [^H] -proline 
incorporation the last 24 h of culture into newly 
synthesized collagens. Collagenase and TIMP protein 
levels in- fibroblast cultures supernatants was measure 

35 by specific ELISAs. 



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As shown in Fig. 6, OPl does not stimulate 
significant collagen or HA production, as compared with 
TGF-p. In the figure, panel A shows OP-1 efect on 
collagen production, panel B shows TGF-p effect on 
5 collagen production, and panels C and D show OP-1 

(panel C} and TGF-p (panel D) effect on HA production. 
The morphogen results were the same whether the soluble 
or mature form of OPl was used. By contrast, the 
latent form of TGF-p (e.g., pro domain-associated form 
10 of TGF-p ) was not active. 

Example 10. Morphooen I nhibition of Epithelial Cell 
Proliferation 

15 This example demonstrates the ability of morphogens 

to inhibit epithelial cell proliferation in 2ritro, as 
determined by ^ H-thymidine uptake using culture cells 
from a mink lung epithelial cell line (ATCC No. CCL 
64), and standard mammalian cell culturing procedures. 

20 Briefly, cells were grown to confluency in Eagle's 
minimum essential medium (EMEM) supplemented with 10% 
fetal bovine serum (FBS), 200 units/ml penicillin, and 
200 pg/ml streptomycin, and used to seed a 48-well cell 
culture plate at a cell density of 200,000 cells per 

25 well. When this culture became confluent, the media 
was replaced with 0.5 ml of EMEM containing 1% FBS and 
penicillin/streptomycin and the culture incubated for 
24 hours at 37 C. Morphogen test samples in EMEM 
containing 5% FBS then were added to the wells, and the 

30 cells incubated for another 18 hours. After 

incubation, 1.0 ^Ci of H-thymidine in 10 /il was added 
to each well, and the cells incubated for four hours at 
37 c. The media then was removed and the cells washed 
once with ice-cold phosphate-buffer saline and DNA 

35 precipitated by adding 0.5 ml of 10% TCA to each well 



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and incubating at room temperature of 15 minutes. The 
cells then were washed three times with ice-cold 
distilled water, lysed with 0.5 ml 0.4 M NaOH, and the 
lysate from each well then transferred to a 
5 scintillation vial and the radioactivity recorded using 
a scintillation counter (Smith-Kline Beckman). 

The results are presented in Table III/ below. The 
anti-proliferative effect of the various morphogens 

10 tested was expressed as the counts of 3H-thymidine (x 
1000) integrated into DNA, and were compared with 
untreated cells (negative control) and TGF-p (1 ng), a 
local-acting factor also known to inhibit epithelial 
cell proliferation. COP-5 and COP-7 are biosynthetic 

15 constructs that previously have been shown to have 
osteogenic activity, capable of inducing the complete 
cascade resulting in endochondral bone formation in a 
standard rat bone assay (see U.S. Pat. No. 5,011,691.) 
The morphogens significantly inhibit epithelial cell 

20 proliferation. Similar experiments, performed with the 
morphogens COP-16, bOP (bone-purified osteogenic 
protein, a dimeric protein comprising CBMP2 and OP-1), 
and recombinant OP-1, also inhibit cell proliferation. 
bOP and COP-16 also induce endochondral bone formation 

25 (see US Pat. No. 4,968,590 and 5,011,691.) 

TABLE III 



30 control 

COP-7-1 (10 ng) 
COP-7-2 (3 ng) 
COP-5-1 (66 ng) 
COP-5-2 (164 ng) 

35 TGF-p (1 ng) 



Thymidine uptake (x 1000) 
50.048, 53.692 
11.874 
11.136 
16.094 
14.43 
1.86, 1.478 



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Example 11. Morphogen Treatment of a Systemic 
Inflammatory Disease 

5 

The following example provides a rat adjuvant- 
induced arthritis model for demonstrating morphogen 
efficacy in treating arthritis and other systemic 
inflammatory diseases. Rat adjuvant- induced arthritis 

10 induces a systemic inflammatory disease with bone and 
cartilage changes similar to those observed in 
rhematoid arthritis, but in an accelerated time span 
(see, for example, Pearson (1964) Arth. Rheum. 7:80). 
A detailed description of the protocol is provided in 

15 Walz, et al., (1971) J. Pharmac . Exp. Ther. 178: 223- 
231, the disclosure of which is incorporated herein by 
reference. 



20 



25 



30 



Briefly, Sprague-Dawley female rats (e.g., Charles 
River Laboratories, Wilmington, MA) are randomized into 
3 groups: control; morphogen, low dose (e.g., 1- 
10 pg/kg weight per day) and morphogen, high dose 
(e.g., 10-20 pg/kg weight per day), referred to as 
Groups 1, 2, and 3, respectively. 



Adjuvant arthritis is induced in all three groups 
by injection of 0.05 ml of a suspension of 1.5% dead 
Mycobacterium butyricum in mineral oil into the 
subplantar surface of the right hand paw. On Day 18 
after adjuvant injection, the limb volumes of both hind 
limb are determined. In the absence of morphogen 
treatment, a systemic arthritic condition is induced in 
adjuvant-injected rats by this time, as determined by 
significant swelling of the uninjected hind limbs (< 
35 2.3 ml, volume measured by mercury displacement). 



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Subsequent determinations of paw edema and x-ray scores 
are made on the uninjected hind limb. Rats in Group 2 
and 3 also are dosed orally daily, beginning on Day 1, 
with morphogen. Limb volumes are recorded on Days 29 
5 and 50 after adjuvant injection and edema determined by 
volume difference compared to Day 18. The uninjected 
hind limb on each rat is x-rayed on Day 50 and the 
joint damage assayed on an arbitrary scale of 1 to 10 
(1-no damage, 10=^aximum damage). Data on differences 

10 between control and treated groups (Day 29 edema. Day 
50 edema and Day 50 x-ray scores) are analyzed by using 
a standard "t-test. Morphogen-treated rats show 
consistently reduced joint damage (e.g., decreased in 
edema and in x-ray scores) as compared with untreated 

15 control rats. 

As another, alternative example. Groups 2 and 3 are 
dosed daily with morphogen beginning on Day 18 and 
continuing through Day 50 to demonstrate the efficacy 
20 of morphogens in arthritic animals. 

Example 12. Morphogen Inhibition of Localized Edema 

The following example demonstrates morphogen 
25 efficacy in inhibiting a localized inflammatory 

response in a standard rat edema model. Experimental 
rats (e.g., Long-Evans from Charles River Laboratories, 
Wilmington, MA) are divided into three groups: Group 
1, a negative control, which receives vehicle alone; 
30 Group 2, a positive control, to which is administered a 
well-known characterized anti-inflammatory agent 
(e.g., indomethacin) , and Group 3, to which morphogen 
is provided. 



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Groups 2 and 3 may be further subdivided to test 
low, medium and high doses (e.g.. Group 2: 1.0 mg/kg, 
3.0 mg/kg and 9.0 mg/kg indomethacin; Group 3: 0.1-5//gj 
5-20/ig, and 20-50;ig of morphogen). Sixty minutes after 
5 indomethacin or morphogen is provided to the rats of 
Group 2 or 3 (e.g., as by injection into the tail vein, 
or by oral gavage) inflammation is induced in all rats 
by a sub-plantar injection of a 1% carrageenin solution 
(SOpl) into the right hind paw. Three hours after 
10 carrageenin administration paw thickness is measured as 
an indication of edema (e.g., swelling) and induced 
inflammatory response to the injected carrageenin 
solution. 

15 Significant swelling is evident in untreated rats 

by three hours after carrageenin injection, 
inflammation also is measured by histology by standard 
means, following euthanasia e.g.: the right hind paw 
from each animal is removed at the ankle joint and 

20 weighed and foot pad tissue is fixed in 10% neutral 
buffered formalin, and slides prepared for visual 
examination by staining the prepared tissue with 
hematoxylin and eosin. 

25 The morphogen-treated rats show substantially 

reduced edema induction following carrageenin injection 
as compared with the untreated rats. 



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Example 13. Morphogen Treatment of Allergic 
Encephalomyelitis 

The following example demonstrates morphogen 
5 efficacy in treating experimental allergic 
encephalomyelitis (EAE) in a rat. EAE is a 
well-characterized animal model for multiple sclerosis^ 
an autoimmune disease. A detailed description of the 
protocol is disclosed in Kuruvilla, et al.^ (1991) PNAS 
10 88:2918-2921, the disclosure of which is incorporated 
herein by reference. 

Briefly, EAE is induced in rats (e.g./ Long-Evans, 
Charles River Laboratories, Wilmington, MA) by 

15 injection of a CNS tissue (e.g., spinal cord) 

homogenate in complete Freund's adjuvant (CPA) on days 
-44, -30 and 0 (last day of immunization), by 
subcutaneous injection to three sites on the animal's 
back. Morphogen is administered daily by 

20 interperitoneal injection beginning on day -31. 
Preferably, a series of morphogen dose ranges is 
evaluated (e.g., low, medium and high) as for 
Example 12, above.) Control rats receive morphogen 
vehicle only (e.g. 0.9% NaCl or buffered saline). Rats 

25 are examined daily for signs of disease and graded on 
an increasing severity scale of 0-4. 

In the absence of morphogen treatment, significant 
neurological dysfunction (e.g., hind and fore limb 
30 weakness, progressing to total hind limb paralysis) is 
evident by day +7 to +10. Hematology, serum chemistry 
profiles and histology are performed to evaluate the 



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10 



degree of tissue necropsy using standard procedures. 
Morphogen treatment significantly inhibits the 
neurological dysfunction normally evident in an EAE 
animal. In addition, the histopathological markers 
typically associated with EAE are absent in the 
morphogen-treated animals. 

Example 14. Morphogen Treatment of Collagen-Induced 
Arthritis 



The following example demonstrates the efficacy of 
morphogens in inhibiting the inflammatory response in a 
collagen-induced arthritis (CIA) in a rat. CIA is a 
well-characterized animal model for rheumatoid 
15 arthritis, an autoimmune disease. The protocol 

disclosed is essentially that disclosed in Kuruvilla et 
al., (1991) PNAS 88:2918-2921, incorporated by 
reference hereinabove. Briefly, CIA is induced in 
experimental rats (e.g., Long-Evans, Charles River 
20 Laboratories, Wilmington) , by multiple intradermal 
injection of bovine Type II collagen (e.g., lOOpg) in 
CFA (0.2 ml) on Day 1. Animals are divided into two 
groups: Group 1, control animals, which receive vehicle 
alone, and Group 2: morphogen-treated animals, which, 
25 preferably, are subdivided into low, medium and high 
dose ranges, as described for Example 13, above. 
Morphogen is administered daily (e.g., by tail vein 
injection) beginning at different times following 
collagen injection, e.g., beginning on day 7, 14, 28, 
30 35 and 42. Animals are evaluated visually and paw 
thickness and body weight is monitored throughout the 
experiment. Animals are sacrificed on day 60 and the 
proximal and distal limb joints, and ear, tail and 
spinal cord prepared for histological evaluation as 
35 described for Examples 12 and 13, above. In a 



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variation of the experiment/ morphogen may be 
administered for prescribed periods, e.g., five day 
periods, beginning at different times following 
collagen injection (e.g., on days 0-4, 7-11, 14-18, 28- 
5 32.) 

In the absence of morphogen treatment, an arthritic 
condition typically is induced by 30 days post collagen 
injection. In morphogen-treated animals, CIA is 

10 suppressed and the histopathological changes typically 
evidenced in control CIA- induced animals are absent: 
e.g., accumulations of activated mononuclear 
inflammatory cells and fibrous connective tissue. In 
addition, consistent with the results in Example 7, 

15 above, serum anti-collagen antibody titers are 
suppressed significantly in the morphogen-treated 
animals. 

Example 15. Screening Assay for Candidate Compounds 
20 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 

25 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 

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



PCrAJS92/07^8 

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15.1 Growth of Cells in Culture 

Cell cultures of kidney, adrenals, urinary bladder, 
brain, or other organs, may be prepared as described 
5 widely in the literature. For example, kidneys may be 
explanted from neonatal or new born or young or adult 
rodents (mouse or rat) and used in organ culture as 
whole or sliced (1-4 mm) tissues. Primary tissue 
cultures and established cell lines, also derived from 

10 kidney, adrenals, urinary, bladder, brain, mammary, or 
other tissues may be established in multiwell plates (6 
well or 24 well) according to conventional cell culture 
techniques, and are cultured in the absence or presence 
of serum for a period of time (1-7 days). Cells may be 

15 cultured, for example, in Dulbecco's Modified Eagle 
medium (Gibco, Long Island, NY) containing serum (e.g., 
fetal calf serum at 1%-10%, Gibco) or in serum-deprived 
medium, as desired, or in defined medium (e.g., 
containing insulin, transferrin, glucose, albumin, or 

20 other growth factors)* 

Samples for testing the level of morphogen 
production includes culture supernatants or cell 
lysates, collected periodically and evaluated for OP-1 

25 production by immunoblot analysis (Sambrook et al., 
eds., 1989, Molecular Cloning, Cold Spring 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 

30 novo OP-1 synthesis, some cultures are labeled 
according to conventional procedures with an 
^^S-methionine/^'s-cysteine mixture for 6-24 hours and 
then evaluated to OP-1 synthesis by conventional 
immunoprecipitation methods. 



35 



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15.2 Determination of Level of Morphogenic Protein 

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

10 1 //g/lOO 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 
wells are washed four times with 0.167M sodium borate 
buffer with 0.15 M NaCl (BSB), pH 8.2, containing 0.1% 

15 Tween 20. To minimize non-specific binding, the wells 
are blocked by filling completely with 1% bovine serum 
albumin (BSA) in BSB and incubating for 1 hour at 37**C. 
The wells are then washed four times with BSB 
containing 0.1% Tween 20. A 100 fjl aliquot of an 

20 appropriate dilution of each of the test samples of 
cell culture supernatant is added to each well in 
triplicate and incubated at 37**C for 30 min. After 
incubation, 100 ^Jl biotinylated rabbit anti-OP-1 serum 
(stock solution is about 1 mg/ml and diluted 1:400 in 

25 BSB containing 1% BSA before use) is added to each well 
and incubated at 37**C for 30 min. The wells are then 
washed four times with BSB containing 0.1% Tween 20. 
100 pi strepavidin-alkaline (Southern Biotechnology 
Associates, Inc. Birmingham, Alabama, diluted 1:2000 in 

30 BSB containing 0.1% Tween 20 before use) is added to 
each well and incubated at 37 *C for 30 min. The plates 
are washed four times with 0.5M Tris buffered Saline 
(TBS), pH 7.2. 50;il substrate (ELISA Amplification 
System Kit, Life Technologies, Inc., Bethesda, MD) is 

35 added to each well incubated at room temperature for 15 



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PCr/US92/07358 



min. Then, 50 pi amplifier (from the same 
amplification system kit) is added and incubated for 
another 15 min at room temperature. The reaction is 
stopped by the addition of 50 /il 0.3 M sulphuric acid. 
5 The OD at 490 nm of the solution in each well is 

recorded. To quant itate OP-1 in culture media, a OP-1 
standard curve is performed in parallel with the test 
samples . 

10 Polyclonal antibody may be prepared as follows. 

Each rabbit is given a primary immunization of 100 
ug/500. fjl E. coli produced OP-1 monomer (amino acids 
328-431 in SEQ ID N0:5) in 0-1% SDS mixed with 500 /il 
Complete Freund's Adjuvant. The antigen is injected 

15 subcutaneously at multiple sites on the back and flanks 
of the animal. The rabbit is boosted after a month in 
the same manner using incomplete Freund's Adjuvant. 
Test bleeds are taken from the ear vein seven days 
later. Two additional boosts and test bleeds are 

20 performed at monthly intervals until antibody against 
OP-1 is detected in the serum using an ELISA assay. 
Then, the rabbit is boosted monthly with 100 fjg of 
antigen and bled (15 ml per bleed) at days seven and 
ten after boosting. 

25 

Monoclonal antibody specific for a given morphogen 
may be prepared as follows. A mouse is given two 
injections of E. coli produced OP-1 monomer. The first 
injection contains 100//g of OP-1 in complete Freund's 

30 adjuvant and is given subcutaneously. The second 

injection contains 50 pg of OP-1 in incomplete adjuvant 
and is given intraperitoneally. The mouse then 
receives a total of 230 pg of OP-1 (amino acids 307-431 
in SEQ ID NO: 5) in four intraperitoneal injections at 

35 various times over an eight month period. One week 



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PCr/US92/07358 



prior to fusion, both mice are boosted 
intraperitoneally with 100 pg of OP-1 (307-431) and 30 
fjg of the N-terminal peptide (Ser2g3-Asn3pg-Cys) 
conjugated through the added cysteine to bovine serum 
5 albumin with SMCC crosslinking agent. This boost was 
repeated five days (IP), four days (IP), three days 
(IP) and one day (IV) prior to fusion. The mouse 
spleen cells are then fused to myeloma (e.g./ 653) 
cells at a ratio of 1:1 using PEG 1500 (Boeringer 
10 Maimheim), and the cell fusion is plated and screened 
for OP-l-specific antibodies using OP-1 (307-431) as 
antigen. The cell fusion and monoclonal screening then 
are according to standard procedures well described in 
standard texts widely available in the art. 

15 

The invention may be embodied in other specific 
forms without departing from the spirit or essential 
characteristics thereof. The present embodiments are 
therefore to be considered in all respects as 

20 illustrative and not restrictive, the scope of the 

invention being indicated by the appended claims rather 
than by the foregoing description, and all changes 
which come within the meaning and range of equivalency 
of the claims are therefore intended to be embraced 

25 therein. 



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SEQUENCE LISTING 



(1) GENERAL INFORMATION: 



( i) APPLICANT: KUBERASAHPATH, THANGAVEL 
PANG, ROY H.L. 
OPFERHANN, HERHANN 
RHEGER, DAVID C. 
IQ COHEN, CHARLES K. 

OZKAYNAK, ENGIN 
SHART, JOHN 

(ii) TITLE OF INVENTION: MORPHOGEN-DTOUCED MODULATION OF 
15 INFLAMMATORY RESPONSE 

(iii) NUMBER OF SEQUENCES: 33 

(iv) CORRESPONDENCE ADDRESS: 

20 (A) ADDRESSEE: CREATIVE BIOMOLECULES 

(B) STREET: 35 SOUTH STREET 

(C) CITY: HOPKINTON 

(D) STATE: MASSACHUSETTS 

(E) COUNTRY: U.S.A. 
25 (F) ZIP: 

(V) COMPUTER READABLE FORM: 

(A) MEDIUM TYPE: Floppy disk 

(B) COMPUTER: IBM PC compatible 

30 (C) OPERATING SYSTEM: PC-DOS/MS-DOS 

(D) SOFTPARE: Patent In Release n.O, Version #1.25 

(vii) PRIOR APPLICATION DATA: 

(A) APPLICATION NUMBER: US 667,274 
35 (B) FILING DATE: lL-MAR-1991 

fvii) PRIOR APPLICATION DATA: 

(A) APPLICATION NUMBER: US 753,059 

(B) FILING DATE; 30-AUG-1991 



40 



45 



50 



fvii) PRIOR APPLICATION DATA: 

(A) APPLICATION NUMBER: US 752,764 
(Bl FILING DATE: 30-AUG-1991 

(2) INFORMATION FOR SEQ ID NO:l! 

{ i ) SEQUENCE CHARACTERISTICS : 

(A) LENGTH: 97 amino acids 

(B) TYPE: amino acids 

(C) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 



SUBSTITUTE SH^ET 



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(ix) FEATURE: 

(A) NAME: Generic Sequence 1 
(D) OTHER INFORMATION: Each Xaa 

indicates one of the 20 naturally- 
5 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 
10 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 
15 30 35 

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

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys 

55 60 
20 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 
25 85 90 

Xaa Cys Xaa 
95 

(2) INFORMATION FOR SEQ ID NO: 2: 

30 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 97 amino acids 

(B) TYPE: amino acids 

(C) TOPOLOGY: linear 
35 (ii) MOLECULE TYPE: protein 

( ix ) FEATURE : 

(A) NAME: Generic Sequence 2 

(D) OTHER INFORMATION: Each Xaa 
indicates one of the 20 naturally- 

40 occurring L- isomer, a-amino acids 

or a derivative thereof, 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: 

Xaa Xaa Xaa Xaa Xaa Xaa 
45 15 

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 

10 IS 
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa 
20 25 
50 Cys Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa 

30 35 
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 
40 45 50 



SUBSTITUTE SHEET 



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30 



50 



_ 100 - 

PCr/US92/07358 



Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys 



55 

Cvs Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 
65 70 
5 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 
10 95 

(2) INFORMATION FOR SEQ ID NO: 3: 

(i) SEQUENCE CHARACTERISTICS: 
15 (A) LENGTH: 97 amino acids 

(B) TYPE: amino acids 

(C) TOPOLOGY: linear 

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

20 (A) NAME: Generic Sequence 3 

(D) OTHER INFORMATION: wherein each 
Xaa is independently selected from 
a group of one or more specified 
amino acids as defined in the 

25 specification. 

( xi ) SEQUENCE DESCRIPTION : SEQ ID NO : 3 : 

Leu Tyr Val Xaa Phe 
1 5 
xaa Xaa Xaa Gly Trp Xaa Xaa Trp Xaa 

10 

Xaa Ala Pro Gly Xaa Xaa Xaa Ala 
15 20 
35 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 40 45 

Xaa Xaa Leu Xaa Xaa Xaa Xaa Xaa 

50 

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys 
55 60 
45 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 Gly Cys Xaa 
95 



SUBSTITUTE SH^ET 



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(2) INFORMATION FOR SEQ ID NO: 4: 

(i) SEQUENCE CHARACTERISTICS: 

5 (A) LENGTH: 102 amino acids 

(B) TYPE: amino acids 

(C) TOPOLOGY: linear 

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

10 (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 

15 specification. 



20 



25 



30 



35 



40 



45 



(xi) SEQUENCE DESCRIPTION: 


SEQ 


Cys 


Xaa 


Xaa 


Xaa 


Xaa 
5 


Leu 


Tyr 


Val 


1 

Xaa 


Xaa 


Xaa 


Gly 


Trp 


Xaa 


Xaa 


Trp ; 








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 


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 









10 



(2) INFORMATION FOR SEQ ID NO: 5: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 139 amino acids 

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

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

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



SUBSTITUTE SH^ET 



wo 93/04692 



25 



30 



- 102 - 

PCr/US92/073S8 

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

Ser Thr Gly Ser 
1 

Asn Arg Ser Lys 
10 

Glu Ala Leu Arg 
20 



10 30 

Ala Cys Lys Lys 
40 

Ser Phe Arg Asp 

X5 Trp He He Ala 
55 

Ala Tyr Tyr Cys 
65 

Phe Pro Leu Asn 

20 _?5 



Lys 


Gin 


Arg 


Ser 


Gin 


5 

Thr 


Pro 


Lys 


Asn 


Gin 




10 








Met 


Ala 


Asn 


Val 


Ala 






25 






Ser 


Asp 


Gin 


Arg 


Gin 






35 




His 


Glu 


Leu 


Tyr 


val 
45 


Leu 
50 


GlV 


Trp 


Gin 


Asp 


Pro 


Glu 


Gly 


Tyr 


Ala 




60 








Glu 


Gly 


Glu 


Cys 


Ala 




70 






Ser 


Tyr 


Met 


Asn 


Ala 






80 




He 


Val 


Gin 


Thr 


Leu 








90 


Asn 


Pro 


Glu 


Thr 


Val 










Gin 


Cys 


Ala 


Pro 


Thr 


105 






Phe 


Ser 


Val 


Leu 


Tyr 






115 






Asn 


Val 


He 


Leu 


Lys 






125 




Met 


Val 


Val 


Arg 


Ala 






135 



85 

Val His Phe He 

Pro Lys Pro Cys 
100 

Leu Asn Ala He 
110 

Asp Asp Ser Ser 
120 

Lys Tyr Arg Asn 
130 

Cys Gly cys His 

35 (2) INFORMATION FOR SEQ ID NO: 6: 

^ ' (i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 139 amino acids 

(B) TYPE: amino acids 

(C) TOPOLOGY: linear 
40 (ii) MOLECULE TYPE: protein 

(ix) FEATURE: 

(A) NAME: mOP-1 (mature form) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: 

45 Ser Thr Gly Gly Lys Gin Arg Ser Gin 

Asn Arg Ser Lys Thr Pro Lys Asn Gin 

GlS Ala Leu Arg Met Ala Ser Val Ala 

^° Glu AsS Ser Ser Ser Asp Gin Arg Gin 

30 35 



SUBSTITUTE SH^ET 



- 103 - 

WO 93/04692 PCT/US92/07358 



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 
85 


He 


Val 


Gin 


Thr 


Leu 
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 


Tyr 


Arg 


Asn 


Met 


Val 


Val 


Arg 


Ala 


130 










135 


Cys 


Gly 


Cys 


His 













10 



15 



20 



25 (2) INFORMATION FOR SEQ ID NO: 7: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 139 amino acids 

(B) TYPE: amino acids 

(C) TOPOLOGY: linear 
30 (ii) MOLECULE TYPE: protein 

(ix) FEATURE: 

(A) NAME: hOP-2 (mature form) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: 



35 


Ala 
1 


Val 


Arg 


Pro 


Leu 
5 


Arg 


Arg 


Arg 


Gin 




Pro 


Lys 


Lys 


Ser 


Asn 


Glu 


Leu 


Pro 


Gin 




10 






15 










Ala 


Asn 


Arg 


Leu 


Pro 


Gly 


He 


Phe 


Asp 


40 




20 






25 








Asp 


Val 


His 


Gly 


Ser 


His 


Gly 


Arg 


Gin 






30 








35 






Val 


Cys 


Arg 


Arg 


His 


Glu 


Leu 


Tyr 


Val 






40 










45 


45 


Ser 


Phe 


Gin 


Asp 


Leu 


Gly 


Trp 


Leu 


Asp 










50 










Trp 


Val 


He 


Ala 


Pro 


Gin 


Gly 


Tyr 


Ser 




55 










60 










Ala 


Tyr 


Tyr 


Cys 


Glu 


Gly 


Glu 


Cys 


Ser 


50 




65 










70 








Phe 


Pro 


Leu 


Asp 


Ser 


Cys 


Met 


Asn 


Ala 








75 






80 





SUBSTITUTE SH^ET 



wo 93/04692 



30 



35 



45 



Asn 


pXS 


Ala 








nxs 




Mot- 


T.vc 


Ala 




Ser 


Ala 


Thr 


110 






Ser 


Ser 


Asn 




120 




His 


Arg 


Asn 




130 


Gly 


Cys 


His 



- 104 - 

PCT/US92/07358 



90 



lie 


Leu 


Gin 


Ser 


Lvs 


Pro 


Asn 


Ala 


95 






Thr 


Cys 


Ala 


Pro 


105 






Ser 


Val 


Leu 


Tyr 






115 




Asn 


Val 


He 


Leu 








125 


Met 


val 


val 


Lys 



loo 

Leu 
Asp 

^° Lys His Arg Asn Met Val Val lys Ala 

Tin 1j3 

Cys 

15 (2) INFORMATION FOR SEQ ID NO:B: 

^ ' (i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 139 amino acids 

(B) TYPE: amino acids 

(C) TOPOLOGY r linear 
20 (ii) MOLECULE TYPE: protein 

(ix) FEATURE: 

(A) NAME: mOP-2 (mature form) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: 

25 Ala Ala Arg Pro Leu Lys Arg Arg Gin 

1 5 
Pro Lys Lys Thr Asn Glu Leu Pro Hxs 

Pro Asn Lys Leu Pro Gly lie Phe Asp 

20 25 

ASP Gly His Gly Ser Arg Gly Arg Glu 
30 , 

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 



40 65 



Ala Tyr Tyr Cys Glu Gly Glu Cys Ala 



Phe Pro Leu Asp Ser Cys Met Asn Ala 



Thr Asn Hil Ala He Leu Gin Ser Leu 
85 

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 

cn 110 

Asp ser Ser Asn Asn Val He Leu Arg 



SUBSTITUTE SH^ET 



- 105 - 

WO 93/04692 PCT/US92/073S8 

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

5 (2) INFORMATION FOR SEQ ID NO: 9: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 96 amino acids 

(B) TYPE: amino acids 

(C) TOPOLOGY: linear 
10 (ii) MOLECULE TYPE: protein 

(ix) FEATURE: 

(A) NAME: CBMP-2A(£z) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: 

15 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 
20 25 30 

Cys Pro Phe Pro Leu Ala Asp Bis Leu Asn Ser 

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

25 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 
30 80 85 

Asn Tyr Gin Asp Met Val Val Glu Gly Cys Gly 

90 95 
Cys Arg 
100 

35 

(2) INFORMATION FOR SEQ ID NO: 10: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 101 amino acids 

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

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

(A) NAME: CBHP-2B(£x) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: 

45 

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

10 15 
50 Asp Trp He Val Ala Pro Pro Gly Tyr Gin Ala 

20 25 



SUBSTITUTE SH^ET 



- 106 - 



PCr/US92/07358 



40 

7&1 Gin 
50 



Cys 


His 


Glv 




Cys 










Thr 






Asn 


Ser 








45 


Thr 


Leu 


Val 


Asn 


Ser 








55 






Ala 


Cys 


Cvs 


Val 










He 


Ser 


Met 


Leu 


Tyr 




75 








Val 


Val 


Leu 


Lys 


Asn 


85 










Glu 


Gly 


Cys 


Gly 


Cys 






100 



Pro 


Phe 


Pro 


Leu 


35 








Asn 


His 


Ala 


He 


Val 


Asn 


Ser 


Ser 








60 


Pro 


Thr 


Glu 


Leu 






70 




Leu 


Asp 


Glu 


Tyr 




80 






Tyr 


Gin 


Glu 


Met 


90 








Arg 









95 

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 

\sv Phe 
10 

/al Ala 
20 

Asp Ala Tyr Tyr Cys i 

25 

?ro Phe 
35 



His 


Ser 
5 


Leu 


Tyr 


Val 


Trp 


Asp 


Asp 


Trp 


He 


15 










Asp 


Ala 


Tyr 


Tyr 


Cys 








30 


Pro 


Leu 


Ala 


Asp 


His 








40 




Ala 


Val 


Val 


Gin 


Thr 






50 






Pro 


Gly 


Lys 


Val 


Pro 




60 








Thr 


Gin 


Leu 


Asp 


Ser 


70 










Asn 


Asp 


Gin 


Ser 


Thr 








85 


Gin 


Glu 


Met 


Thr 


Val 






95 





45 50 55 

tst-n fiiv T.v«! Val Pro Lys 

65 

Tal Al* 
75 

1 Asn Asp Gin Ser Thr ^ 

' 80 
Lys Asn Tyr 
90 

Gly Cys Arg 
100 

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 



SUBSTITUTE SH^ET 



wo 93/04692 



- 107 - 



PCr/US92/073S8 



(iz) FEATURE: 

(A) NAME: Vgl(fx) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: 

5 Cys Lys Lys Arg His Leu Tyr Val Glu Phe Lys 

1 5 10 

Asp Val Gly Trp Gin Asn Trp Val lie Ala Pro 

15 20 
Gin Gly Tyr Met Ala Asn Tyr Cys Tyr Gly Glu 
10 25 30 

Cys Pro Tyr Pro Leu Thr Glu lie Leu Asn Gly 

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

15 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 
20 80 85 

Arg His Tyr Glu Asn Met Ala Val Asp Glii Cys 

90 95 
Gly Cys Arg 
100 

25 

(2) INFORMATION FOR SEQ ID NO: 13: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 102 amino acids 

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

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

(A) NAME: Vgr-l(£x) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: 

35 

Cys Lys Lys His Glu Leu Tyr Val Ser Phe Gin 

15 10 
Asp Val Gly Trp Gin Asp Trp He He Ala Pro 
15 20 
40 Xaa Gly Tyr Ala Ala Asn Tyr Cys Asp Gly Glu 

25 30 
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 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 



SUBSTITUTE SH^ET 



- 108 - 

PCr/US92/07358 

WO 93/04692 

Leu Tyr Phe Asp Asp Asn Ser Asn Val He Leu 

80 , ^ 

Lys Lys Tyr Arg Asn Met Val Val Arg Ala Cys 
90 95 
5 Gly Cys His 

100 

(2) INFORMATION FOR SEQ ID NO: 14: 

10 (i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 106 amino acids 

(B) TYPE: protein 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 

15 (ii) MOLECULE TYPE: protein 

(vl) ORIGINAL SOURCE: 
(A) ORGANISH: human 
(F) TISSUE TYPE: BRAIN 



0 



0 



5 



(ix) FEATURE: 
(D) OTHER INFORMATION: 

/products "GDF-1 (fx)' 



5 (xi) SEQUENCE DESCRIPTION: SEQ ID N0il4: 

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

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

Cys Gin Gly Gin Cys Ala Leu Pro Val Ala Leu Ser Gly Ser Gly 
30 35 *0 

Glv Pro Pro Ala Uu Asn His Ala Val Leu Arg Ala Leu Met His 

45 50 55 

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

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

5 Val Val Leu Arg Gin lyr Glu Asp Met Val Val Asp Glu Cys Gly 



3 



90 

Cys Arg 
105 



SUBSTITUTE SH^ET 



wo 93/04692 



- 109 - 



PCr/US92/073S8 



10 



15 



25 



(2) IN?OIUfAII(m FOR SEQ ID N0:15: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 5 amino acids 

(B) TYPE: anino acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 

(ii) HOLECDLE TYPE: peptide 

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

Cys Xaa Zaa Xaa Zaa 
1 5 

(2) INFORHATION FOR SEQ ID NO: 16: 



(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 1822 base pairs 
20 (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 

30 (ix) FEATURE: 

(A) NAME/KEY: CDS 

(B) LOCATION: 49.. 1341 

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

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

GGTGCGGGCC CGGAGCCCGG AGCCCGGGTA GCGCGTAGAG CCGGCGCG ATG CAC GTG 57 

Met His Val 
1 

40 

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 

45 CCC CTG TTC CTG CTG CGC TCC 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 
50 Glu Val His Ser Ser Phe lie His Arg Arg Leu Arg Ser Gin Glu Arg 

40 45 50 



SUBSTITUTE SH^ET 



wo 93/04692 



- 110 - 

PCr/US92/073S8 



10 



30 



CGG GAG ATG CAG CGC GAG ATC CTC TCC ATT TIG GGC TTG CCC CAC CGC 
Are Glu Met Gin Arg Glu He Leu Ser lie Leu Gly Leu Pro His Arg 
55 60 W 

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



249 



297 



CTG GAC CTG TAC AAC GCC ATG GCC GTG GAG GAG GGC GGC GGG CCC GGC 345 
Leu ASP Uu Tyv 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 
Sy Sn ^ Phe Ser lyr Pro Tyr Lys Ala Val Phe Ser Thr Gin Gly 
15 100 105 110 

CCC CCT CTG GCC AGC CTG CAA GAT AGC CAT TTC CTC ACC GAC GCC GAC 441 
Pro Pro Su Sa 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 
Met Val Met Ser Phe Val Asn Leu Val Glu His Asp Lys Glu Phe Phe 
135 140 1" 

25 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 loO 

CCA GAA GGG GAA GCT GTC ACG GCA GCC GAA TTC CCG 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 CGG ATC AGC GTT TAT 633 
^ ne Arg Glu Arg Phe Asp Asn Glu Thr Phe Arg He Ser Val Tyr 
35 180 185 190 

CAG GTG CTC CAG GAG CAC TTG GGC 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 

GAC AGC CGT ACC CTC TGG GCC TCG GAG GAG GGC TGG CTG GTG TTT GAC 729 
ter Arg Thr Leu Trp Ala Ser Glu Glu Gly Trp Leu Val Phe Asp 
*^ 215 220 223 



45 ATC ACA GCC ACC AGC AAC CAC TGG GTG GTC AAT CCG CGG CAC AAC CTG 777 
lie ar Sa 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 
50 Gly Leu Gin Leu Ser Val Glu Thr Leu Asp Gly Gin Ser He Asn Pro 
245 250 255 



SUBSTITUTE SH^ET 



wo 93/04692 



- Ill - 



PCr/US92/07358 



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

5 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 
10 Arg Ser Thr Gly Ser Lys Gin Arg Ser Gin Asn Arg Sef 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 
15 310 315 320 

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 

20 

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

25 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 
30 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 
35 390 395 400 

ATC TCC GTC CTC TAC TTC GAT GAC AGC TCC AAC GTC ATC CTG AAG AAA 1305 
He Ser Val Leu Tyr Phe Asp Asp Ser Ser Asn Val He Leu Lys Lys 
405 410 415 

40 

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

45 GAGAATTCAG ACCCTTTGGG GCCAAGTTTT TCTGGATCCT CCATTGCTCG CCTTGGCCAG 1411 

GAACCAGCAG ACCAACTGCC TTTTGTGAGA CCTTCCCCTC CCTATCCCCA ACTTTAAACG 1471 

TGTGAGAGTA TTAGGAAACA TGAGCAGCAT ATGGCTTTTG ATCAGTTTTT CAGTGGCAGC 1531 

50 

ATCCAATGAA CAAGATCCTA CAAGCTGTGC AGGCAAAACC TAGCAGGAAA AAAAAACAAC 1591 



SUBSTITUTE SH^ET 



- 112 - 

PCr/US92/073S8 

WO93/04692 

GCATAAAGAA MATGGCCGG GCCAGGTCAT TGGCTCGG&& GTCTC&GCCA TGCACGGACT 1651 
CGTTTCCAG& GGTAATTATG AGCGCCTACC AGCCAGGCCA CCCAGCCGTG GGAGGAAGGG 1711 
5 GGCGTGGCAA GGGGTGGGCA CATTGGTGTC TGTGCGAAAG GAAAATTCAC CCGGAAGTTC 1771 
CTGTAATAAA TGTCACAATA AAACGAATGA ATGAAAAAAA AAAAAAAAAA A 1822 
(2) INFORKATIOM FOR SEQ ID N0:17i 

10 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 431 amino acids 

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

15 . 

(ii) MOLECULE TYPE: protein 

(ix) FEATURE: 

(D) OTHER INFORKATION: /Product»"OPl-PP" 

20 

(xi) SEQUENCE DESCRIPTIONr SEQ ID NO: 17: 

Met His Val Arg Ser Leu Arg Ala Ala Ala Pro His Ser Phe Val Ala 
1 5 10 .15 

25 

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 
30 35 40 « 

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

35 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 

Gly Pro Gly Gly Gin Gly Phe Ser Tyr 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 
45 115 120 125 

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

50 Glu Phe Phe His Pro Arg Tyr His His Arg Glu Phe Arg Phe Asp Leu 
145 150 155 



SUBSTITUTE SHEET 



- 113 - 

WO 93/04692 PCT/US92/073S8 

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

Tyr Lys Asp Tyr He Arg Glu Arg Phe Asp Asn Glu Thr Phe Arg He 
5 180 185 190 

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

10 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 Arg 
225 230 235 240 

15 

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 
20 260 265 270 

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

25 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 

30 

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

Val Ser Phe Arg Asp Leu Gly Trp Gin Asp Trp He He Ala Pro Glu 
35 340 345 350 

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

40 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 

45 

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 
50 420 425 430 



SUBSTITUTE SHEET 



wo 93/04692 



- 114 - 



PCr/US92/073S8 



10 



25 



40 



45 



(2) INFOmTIfiH FOR SEQ ID NO: 18: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 1873 base pairs 

(B) TYPE: nucleic acid 

(C) STRANDEONESS: single 

(D) TOPOLOGYr linear 

(ii) HOLECULE TYPE: cDNA 

(vi) ORIGINAL SOURCE: 

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



15 (ix) FEATURE: 

(A) NAHE/KEY: CDS 

(B) LOCATION: 104.. 1393 

(D) OTHER INFORMATIONt /note= "MOPl (CDNA)" 

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

CTGCAGCAA6 TGACCTCGGG TCGTGGACCG CTGCCCTGCC CCCTCCGCTG CCACCTGGGG 
CGGCGCGGGC CCGGTGCCCC GGATCGCGCG TAGAGCCGGC GCG ATG CAC GTC CGC 



1 



60 
115 



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 
30 5 10 15 20 

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

GAG ATG CAG CGG GAG ATC CTG TCC ATC TTA GGG TTG CCC CAT CGC CCG 307 
Glu Met Gin Arg Glu lie Leu Ser He Uu Gly Leu Pro Hxs Arg Pro 
55 60 o5 

CGC CCG CAC CTC CAG GGA AAG CAT AAT TCG GCG CCC ATG TTC ATG m 355 
Are Pro His Leu Gin GI7 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 CGA CAG 403 
Sp Su ^ Asn Ala Met Ala Val Glu Glu Ser Gly Pro Asp Gly Gin 
50 85 90 95 100 



SUBSTITUTE SH^ET 



wo 93/04692 



- 115 - 



PCT/US92/073S8 





GGC TTC TCC TAC 


CCC TAC 


AAG 


GCC 


GTC 


TTC 


AGT 


ACC 


CAG 


GGC 


CCC 


CCT 


/CI 

451 




Gly Phe Ser Tyr 


Pro Tyr 


Lys 


Ala 




P(lO 

irnc 




Thr 


Gin 


Glv 


Pro 


Pro 






105 






no 










115 






5 


TTA GCC AGC CTG 


CAG GAC 


AGC 


CAT 


TTC 


CTC 


ACT 


GAC 


GCC 


GAC 


ATG 


GTC 


499 




Leu Ala Ser Leu 


Gin Asp 


Ser 


His 


Phe 


Leu 


Thr 


Asp 


Ala 


Asp 


Met 


Val 






120 






125 










130 










A TP A PP TTP PTP 

Alb AbL JliL blL 


A AP PTA 


uiu 




PAT 


GAC 


AAA 


GAA 


TTC 


TTC 


CAC 


CCT 


547 


10 


Met Ser Phe Val 


Asn Leu 


Val 


Glu 


His 


Asp 


Lys 


Glu 


Phe 


Phe 


His 


Pro 






135 






140 










145 








595 




CGA TAC CAC CAT 


CGG GAG 


TTC 


CGG 


TTT 


GAT 


CTT 


TCC 


AAG 


ATC 


CCC 


GAG 




Arg Tyr his his 


A <T>rT Pill 

Arg uiu 


rne 


Arg 


rne 


A cn 


AiCtA 




Lvfi 

A4jr o 


He 


Pro 


Glu 






150 


155 










160 












15 


GGC GAA CGG GIG 


ACC Ga 


GCC 


GAA 


TTC 


AGG 


ATC 


TAT 


AAG 


GAC 


TAC 


ATC 


643 




O^.a Ol.a AwM VTnl 

Gly Glu Arg val 


a.nr Aj.a 


AlE 


Pi « 

ulU 


rne 


Arg 


Tip 








Tvr 


He 






165 


170 










175 










180 




20 


CGG GAG CGA TTT 


GAC AAC 


GAG 


ACC 


TTC 


CAG 


ATC 


ACA 


GTC 


TAT 


CAG 


GTG 


691 


Arg Glu Arg rne 


Asp Asn 


Pill 


inr 


rne 




Tip 


Thr 


Val 




Gin 


Val 






185 








190 










195 








CTC CAG GAG CAC 


TCA GGC 


AGG 


GAG 


TCG 


GAC 


CTC 


TTC 


TTG 


CTG 


GAC 


AGC 


739 


25 


Leu Gin Glu His 


Ser Gly 


Arg 


Pi 19 

blU 


ber 


Asp 


Leu 


Php 


T Alt 


T oil 


Acn 
nap 


Ser 






200 




205 










210 










CGC ACC ATC TGG 


GCT TCT 


GAG 


GAG 


GGC 


TGG 


TTG 


GTG 


TTT 


GAT 


ATC 


ACA 


787 




Arg Thr lie Trp 


Axa ber 


Pi 11 

UXU 


pi 11 

uXU 


uxy 


irp 


T oil 


Val 




Asp 


lie 


Thr 




30 


215 






220 










225 












GCC ACC AGC AAC 


CAC TGG 


GTG 


GTC 


AAC 


CCT 


CGG 


CAC 


AAC 


CTG 


GGC 


TTA 


835 




Ala Thr Ser Asn 


nxs irp 


Vdi. 


Vai. 


A en 


Pro 
r X u 




His 


Asn 


Leu 


Glv 


Leu 






230 


235 










240 












35 


CAG CTC TCT GTG 


GAG ACC 


CTG 


GAT 


GGG 


CAG 


AGC 


ATC 


AAC 


CCC 


AAG 


TTG 


883 




Gin Leu Ser vai 


ulu inr 


Leu 


Asp 






OCA 


Hp 




Pro 


Lys 


Leu 






245 


250 






255 










2^0 




40 


GCA GGC CTG ATT 


GGA CGG 


CAT 


GGA 


CCC 


CAG 


AAC 


AAG 


CAA 


CCC 


TTC 


ATG 


931 




Ala Gly Leu lie 


Gly Arg 


ills 


Pi W 

bxy 


rru 




Acn 




Gin 


Pro 


Phe 


Met 






265 








270 










275 








GTG GCC TTC TTC 


AAG GCC 


ACG 


GAA 


GTC 


CAT 


CTC 


CGT 


AGT 


ATC 


CGG 


TCC 


9/9 


45 


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 


CCC 


TCC 


AAG 


ACG 


CCA 


AAG 


AAC 


1027 




Thr Gly Gly Lys 


Gin Arg 


Ser 


Gin 


Asn 


Arg 


Ser 


Lys 


Thr 


Pro 


Lys 


Asn 




50 


295 






300 










305 











SUBSTITUTE SH^ET 



- 116 - 

PCr/US92/073S8 

WO 93/04692 

CAA GAG GCC CTG AGG ATG GCC AGT GTG GCA GAA AAC AGC AGC AGT GAC 1075 
Gin Glu Ala Uu Arg Met Ala Ser Val Ala Glu Asn Ser Ser Ser Asp 
310 315 320 

5 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 IGG CAG GAC TGG ATC ATT GCA CCT GAA GGC TAT GCT GCC TAC 1171 
10 Leu Gly Trp Gin Asp Trp He He Ala Pro Glu Giy Tyr Ala Ala Tyr 

345 350 

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 
15 360 365 370 

ACC 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 «00 

25 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 *15 420 

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

425 «0 

ACCTTTGCGG GGCCACACCT TTCCAAATCT TCGATGTCTC ACCATCTAAG TCTCTCACTG 1473 

35 CCCACCTTGG CGAGGAGAAC AGACCAACCT CTCCTGAGCC TTCCCTCACC TCCCAACCGG 1533 

AAGCATGTAA GGGTTCCAGA AACCTGAGCG TGCAGCAGCT GATGAGCGCC CTTTCCTTCT 1593 

GGCACGTGAC GGACAAGATC CTACCAGCTA CCACAGCAAA CGCCTAAGAG CAGGAAAAAT 1653 

GTCTGCCAGG AAAGTGTCCA GTGTCCACAT GGCCCCTGGC GCTCTGAGTC TTTGAGGAGT 1713 

AATCGCAAGC CTCGTTCAGC TGCAGCAGAA GGAAGGGCTT AGCCAGGGTG GGCGCKKCG 1773 

45 TCTGTGTTGA AGGGAAACCA AGCAGAAGCC ACTGTAATGA TATGTCACAA TAAAACCCAT 1833 

GAATGAAAAA AAAAAAAAAA AAAAAAAAAA AAAA6AATTC 1873 



SUBSTITUTE SH^ET 



wo 93/04692 



- 117 - 



PCr/US92/07358 



(2) INFORHATION FOR SEQ ID NO: 19: 

(1) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 430 amino acids 
5 (B) TYPE: amino acid 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: protein 

10 (ix) FEATURE: 

(D) OTHER INFORMATION: /producto "mOPl-PP" 

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

15 Met His Val Arg Ser Leu Arg Ala Ala Ala Pro His Ser Phe Val Ala 
1 5 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 Uu Gly Leu 
25 50 55 60 

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

30 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 

35 

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 
40 130 135 140 

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

45 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 

50 

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



SUBSTITUTE SH^ET 



~ ' PCr/US92/07358 

WO 93/04692 

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

Phe Asp He Thr Ala Thr Ser Asn His Trp Val Val Asn Pro Arg His 

230 235 ^"^^ 



5 225 



Asn Leu Gly Leu Gin Leu Ser Val Glu Thr Leu Asp Gly Gin Ser He 
' 250 



245 



10 Asn Pro Lys Leu Ala Gly Leu He Gly Arg His Gly Pro Gin Asn Lys 
260 265 2/u 

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 Uu Arg Met Ala Ser Val Ala Glu Asn 
20 305 310 315 320 

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

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

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

^° Tvr 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 
35 385 390 395 400 

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

40 Lys Lys Tyr Arg Asn Met Val Val Arg Ala Cys Gly Cys His 
420 *25 

(2) INPORMATIOK FOR SEQ ID N0:20: 

45 (i)SEQUEMCE CHARACTERISTICS: 

(A) LENGTH: 1723 base pairs 

(B) TYPE: nucleic acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 

50 

(ii) MOLECULE TYPE: cDNA 



SUBSTITUTE SHI^ET 



wo 93/04692 



- 119 - 



PCT/US92/073S8 



(vi) ORIGINAL SOURCE; 

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

5 (ix) FEATURE: 

(A) NAME/KEY: CDS 

(B) LOCATION: 490.. 1696 

(D) OTHER INFORMATION: /note= "hOP2 (cDNA)" 
10 (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 2A0 
20 CCGCAGAGTA GCCCCGGCCT CGAGGCGGTG GCGTCCCGGT CCTCTCCGTC CAGGAGCCAG 300 
GACAGGTGTC GCGCGGCGGG GCTCCAGGGA CCGCGCCTGA GGCCGGCTGC CCGCCCGTCC 360 
CGCCCCGCCC CGCCGCCCGC CGCCCGCCGA GCCCAGCCTC CTTGCCGTCG GGGCGTCCCC 420 
AGGCCCTGGG TCGGCCGCGG AGCCGATGCG CGCCCGCTGA GCGCCCCAGC TGAGCGCCCC 480 



25 



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 
30 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 

35 

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 AO 45 

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

GCG CCA CCC GCC GCC TCC CGG CTG CCC GCG TCC GCG CCG CTC TTC ATG 720 
45 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 
50 80 85 90 



SUBSTITUTE SH^ET 



wo 93/04692 



- 120 - 



PCr/US92/073S8 



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

5 AAC ATG GT6 GAG CGA GAC CGT GCC CTG GGC CAC CAG GAG CCC CAT TGG 
Asn Met Val Glu Arg Asp Arg Ala Leu Gly His Gin Glu Pro Hxs Trp 
110 115 120 1-^3 

AAG GAG TTC CGC TIT GAC CTG ACC CAG ATC CCG GGT GGG GAG GCG GTC 
10 lys Glu Phe Arg Phe Asp Leu Thr Gin Ue Pro Ala Gly Glu Ala Val 



15 



20 



25 



ACA GCT GCG GAG TTC CGG ATT TAC AAG GTG CCC AGC ATC CAC CTG CTC 

Thr Ala Ala Glu Phe Arg He Tyv 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 

Arg Thr Su 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 

Asn Are 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 

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 

30 Trp Leu Leu Lys Arg His Lys Asp Leu Gly Leu Arg Leu Tyr Val Glu 

210 215 



ACT GAG GAC GGG CAC AGC GTG GAT CCT GGC CTG GCC GGC CTG CTG GGT 
ar Glu Asp Gly His Ser Val Asp Pro Gly Leu Ala Gly Leu Uu Gly 
35 225 230 235 

CAA CGG GCC CCA CGC TCC CAA CAG CCT TTC GTG GTC ACT TTC TTC AGG 
Gli 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 
Ala Ser Pro Ser Pro He Arg Thr Pro Arg Ala Val Arg Pro Leu Arg 
255 260 265 

45 AGG AGG CAG CCG AAG AAA AGC AAC GAG CTG CCG CAG GCC AAC CGA CTC 
aS S Sn iro Lys Lys Ser Asn Glu Leu Pro Gin Ala Asn Arg Leu 
270 275 280 

CCA GGG ATC TTT GAT GAC GTC CAC GGC TCC CAC GGC CGG CAG GTC TGC 
50 P« ^ He Asp Asp Val His Gly Ser His Gly Arg Gin Val Cys 



816 



864 



912 



960 



1008 



1056 



1104 



1152 



1200 



1248 



1296 



1344 



1392 



SUBSTITUTE SH^ET 



wo 93/04692 



- 121 - 



PCT/US92/073S8 



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

5 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 Tyr Cys Glu Gly Glu 
320 325 330 

TGC TCC TTC CCA CTG GAC TCC TGC ATG AAT GCC ACC AAC CAC CCC ATC 1536 
10 Cys Ser Phe Pro Leu Asp Ser Cys Met Asn Ala Thr Asn His Ala lie 
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 
15 350 355 360 365 

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 

20 

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 

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



30 



35 



40 



45 



(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= "h0P2-PP'' 

(xi) SEQUENCE DESCRIPTION: SEQ ID N0j21: 

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

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 
50 35 «0 45 



SUBSTITUTE SWET 



- 122 - 

PCT/US92/073S8 

WO 93/04692 

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 
5 65 70 75 80 

Tyr His Ala Met Ala Gly Asp Asp Asp Glu Asp Gly Ala Pro Ala Glu 
85 90 '5 

10 Arg Arg Leu Gly Arg Ala Asp Leu Val Met Ser Phe Val Asn Met Val 
100 105 11" 

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

^ Are Phe Asp Leu Thr Gin He Pro Ala Gly Glu Ala Val Ihr Ala Ala 
130 135 140 

Gin Phe Arg He Tyr Lys Val Pro Ser He His Leu Leu Asn Arg Thr 
20 145 150 155 160 

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

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

Glv Tm Leu Val Leu Asp Val Thr Ala Ala Ser Asp Cys Trp Leu Leu 
^ 195 200 205 

Lys Are 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 
35 225 230 235 240 

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

40 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 krg His 
290 295 300 

Glu Leu Tyr Val Ser Phe Gin Asp Leu Gly Trp Leu Asp Trp Val He 
50 305 310 315 320 



SUBSTITUTE SH^ET 



wo 93/04692 



- 123 - 



PCr/US92/073S8 



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

Pro Leu Asp Ser Cys Het Asn Ala Thr Asn His Ala lie Leu Gin Ser 
5 340 345 350 

Leu Val His Uu Het Lys Fro Asn Ala Val Pro Lys Ala Cys Cys Ala 
355 360 365 

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



15 



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

Cys His 

(2) INFORHATIGN FOR SEQ ZD NO: 22: 

20 (i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 1926 base pairs 

(B) TYPE: nucleic acid 

(C) STRANDEONESS: single 
(D TOPOLOGY: linear 



25 



(ii) HOLECULE TYPE: cDNA 



(vi) ORIGINAL SOURCE: 

(A) ORGANISH: HURIDAE 
30 (F) TISSUE TYPE: EHBRYO 

(ix) FEATURE: 

(A) NAME/KEY: COS 

(B) LOCATION; 93.. 1289 

35 (D) OTHER INFORHATION: /note- "inOP2 cDNA" 

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:22: 

GCCAGGCACA GGTGCGCCGT CTGGTCCTCC CCGTCTGGCG TCAGCCGAGC 50 



40 



CCGACCAGCT ACCAGTGGAT GCGCGCCGGC TGAAAGTCCG AG ATG GCT ATG CGT 104 

Net Ala Het Arg 
1 



45 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 

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

25 30 35 



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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 lie Leu Ala Val Leu Gly 
40 *5 

5 CXA 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 
10 Pro Ala Ser Ala Pro Leu Phe Het Leu Asp Leu Tyr His Ala Met Thr 
70 75 80 

GAT GAC GAC GAC GGC GGG CCA CCA CAG GCT CAC TIA GGC CGT GCC GAC 392 
Asp Asp Asp Asp Gly Gly Pro Pro Gin Ala His Leu Gly Arg Ala Asp 
15 85 90 95 AW" 

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 

25 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 
30 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 
35 165 170 175 "0 

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 
185 150 195 

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 

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

CTG GCT GGT CTG CTT GGA CGA CAA GCA CCA CGC TCC AGA CAG CCT TTC 824 
50 Leu Ala Gly Leu Leu Gly Arg Gin Ala Pro Arg Ser Arg Gin Pro Phe 
230 235 240 



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ATG GTA ACC TTC TTC AGG GCC AGC CAG ACT CCT GTG CGG GCC CCT CGG 872 
Met Val Thr Phe Phe Arg Ala Ser Gin Ser Pro Val Arg Ala Pro Arg 
245 250 255 260 

5 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 
10 Pro His Pro Asn Lys Leu Pro Gly He 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 Tyr Val Ser Phe Arg 
15 295 300 305 

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 

20 

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

25 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 Leu Val His Leu Met Lys Pro 
345 350 355 

GAT GTT GTC CCC AAG GCA TGC TGT CCA CCC ACC AAA CTG AGT GCC ACC 1208 
30 Asp Val Val Pro Lys Ala Cys Cys Ala Pro Thr Lys Uu 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 lyr lyr Asp Ser Ser Asn Asn Val He Leu Arg Lys His 
35 375 380 385 

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

40 

TGCTTCTACT ACCTTACCAT CTGGCCGGGC CCCTCTCCAG AGGCAGAAAC CCTTCTATCT 1369 
TATCATAGCT CAGACAGGGG CAATGGGAGG CCCTTCACTT CCCCTGGCCA CTTCCTGCTA 1429 
45 AAATTCTGGT CTTTCCCAGT TCCTCTCTCC TTCATGGGCT TTCGGGGCTA TCACCCCGCC 1489 
CTCTCCATCC TCCTACCCCA AGCATAGACT GAATGCACAC AGCATCCCAG AGCTATGCTA 1549 
ACTGAGAGGT CTGGGGTCAG CACTGAAGGC CCACATGAGG AAGACTGATC CTTGGCCATC 1609 

50 

CTCAGCCCAC AATGGCAAAT TCTGGATGGT CTAAGAAGGC CGTGGAATTC TAAACTAGAT 1669 



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GATCT(WGCT CTCTGCACCA TTCATTGTGG CAGTTG05AC Aim "29 
CATACACTTA GATCAATGCA TCGCTGTACT CCTTGAAATC AGAGCTAGCT TGTTAGAAAA 1789 
5 AG&ATCAGAG CCAGGTATAG CGGTGCATGT CATTAATCCC AGCGCTAAAG AGACAGAGAC 1849 
AGGAGAATCT CTGTGAGTTC AAGGCCACAT AGAAAGAGCC TGTCTCGGGA CaCGAAAAA 1909 
AAAAAAAAAC GGMITC 

10 

(2) INFOWIATION FOR SEQ ID NO: 23: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 399 amino acids 
15 (B) TYPE: amino acid 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: protein 

20 (ix) FEATURE: 

(D) OTHER INFORHATION: /product= "mOP2-PP" 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23: 

25 Het Ala Het 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 He Leu Ala 
35 40 *5 

Val Leu Gly Uu Pro Gly Arg Pro Arg Pro Arg Ala Gin Pro Ala Ala 
35 50 55 60 65 

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

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

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

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

Gin He Pro Ala Gly Glu Ala Val Thr Ala Ala Glu Phe Arg lie Tyr 
50 135 1*0 1*5 



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

Phe Glu Val Val Gin Glu His Ser Asn Arg Glu Ser Asp Leu Phe Phe 
5 165 170 175 

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

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



15 



30 



45 



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 
20 245 250 255 

Pro Arg Ala Ala Arg Pro Leu Lys Arg Arg Gin Pro Lys Lys Thr Asn 

260 265 270 

25 Glu Leu Pro His Pro Asn Lys Leu Pro Gly He 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 
35 325 330 335 

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

40 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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10 



15 



20 



25 



(2) INFORMATION FOR SEQ ID NO: 24: 

(1) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 1368 base pairs 

(B) TYPE: nucleic acid 

(C) STRANDEONESS: single 

(D) TOPOLOGY: linear 

(ii) HOLECOLE TYPE: cDNA 

(ix) FEATURE: 

(A) NAME/KEY: CDS 

(E) LOCATION: 1..1368 

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

fxl 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 

(E) ReSvANT^RESIDUES IN SEQ ID N0:3: FROM 1 TO 1368 

(F) PAGES: 9214-9218 

(G) DATE: OCT - 1991 

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



48 



96 



ATG TCG GGA CTG CGA AAC ACC TCG GAG GCC GTT GCA GTG CTC GCC TCC 
Je? Ser G^ 2u Arg Asn Thr Ser Glu Ala Val Ala Val Leu Ala Ser 
30 1 5 10 15 

rPG GGA CTC GGA ATG GTT CTG CTC ATG TTC GTG GCG ACC ACG CCG CCG 
^ ^ Mei ?al Leu Leu Met Phe Val Ala Thr Thr Pro Pro 

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 T^r lie Asp Asn Gly Lys Asp 
35 40 *5 

40 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 GAA CGG CCG ACG CAC 240 
45 Ser ^r Su lie Leu Glu Phe Leu Gly He Ala Glu Arg Pro Thr His 
65 70 75 80 

rrr acc aGC CAC CAG TTG TCG CTG AGG AAG TCG GCT CCC AAG TTC CTG 288 
S ter ser Ms Sn 2u Ser Leu Arg Lys Ser Ala Pro Lys Phe Leu 
50 85 50 95 



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

5 GAT GAG GAC GAC GAC TAC GAA CGC GGC CAT CGG TCC AGG AGG AGC GCC 
Asp Glu Asp Asp Asp Tyx 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 
10 Asp Leu Glu Glu Asp Glu Gly Glu Gin Gin Lys Asn Phe He Thr Asp 
130 



15 



20 



35 



40 



45 



135 



140 



CTG GAC AAG CGG GCC ATC GAC GAG AGC GAC ATC ATC ATG ACC TTC CTG 
Leu Asp Lys Arg Ala He Asp Glu Ser Asp He He Met Thr Phe Leu 
U5 150 155 160 

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

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

25 ATG GCC GAG CTG CGC ATC TAT CAG AAC GCC AAC GAG CGC AAG TGG CTG 
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 
30 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 

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 

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



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



336 



384 



432 



480 



528 



576 



624 



672 



720 



768 



816 



864 



912 



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TTC TTC CGC GGA CCG GAG CTG ATC AAG GCG ACG GCC CAC AGC AGC CAC 
?S Se 2i Gly Fro Glu Leu He Lys Ala Thr Ala His Ser Ser His 
305 

5 CAC AGG AGC AAG CGA AGC GCC AGC CAT CCA CGC AAG CGC AAG AAG ICG 
His £S ser Lys Arg Ser Ala Ser His Pro Arg Lys Arg Lys Lys Ser 

325 330 



S !Si in iks et A3„ Ala Tto Asn His 

385 350 395 

25 GCG ATC GTC CAG ACC CTG GTC CAC CTG CTG GAG CCC AAG AAG GTG CCC 
Ala lie v5 Gin Thr Leu Val His Leu Leu Glu Pro Lys Lys Val Pro 



40 



CAC CTG AAC GAC Aax uxi» iuiv u*« -y- -- — -— - 

His Leu Asn Asp Glu Asn Val Asn Leu Lys Lys Tyr Arg Asn Met He 

440 



35 435 

GIG AAA TCC TGC GGG TGC CAT TGA 
Val Lys Ser Cys Gly Cys His 
450 *55 



(2) INFORMATION FOR SEQ ID N0:25r 



(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 455 amino acids 
45 (B) TYPE: amino acid 

(D) TOPOLOGY: linear 

(ii) HOLECULE TYPE: protein 

50 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25: 



960 



1008 



PTT TCC CCC AAC AAC GTG CCG CTG aG GAA CCG ATG GAG AGC ACG CGC 1056 
10 5S ser PrS Z Z SS Pro Leu Leu Glu Pro Met Glu Ser Thr Arg 
340 345 J-*" 



1104 



Arr TCC CAG ATG CAG ACC CTG TAC ATA GAC TTC AAG GAT CTG GGC TGG 
ser gJS Gin ihr Su Tyr lie Asp Phe Lys Asp Leu Gly Trp 
15 355 360 365 

PAT CAC TGG ATC ATC GCA CCA GAG GGC TAT GGC GCC TTC TAC TGC AGC 1152 
SI Sp ne nl Ait 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 



1248 



AAG CCC TGC TGC GCT CCG ACC AGG CTG GGA GCA CTA CCC GTT CTG TAC 1296 
30 Pro Ala Pro Thr Arg Leu Gly Ala Leu Pro Val Leu lyr 

420 ^25 

CAC CTG AAC GAC GAG AAT GTG AAC CTG AAA AAG TAT AGA AAC ATG ATT 1344 



1368 



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Met Ser Gly Uu 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 
5 20 25 30 

Ala Val Glu Ala Thr Gin Ser Gly He Tyr He Asp Asn Gly Lys Asp 
35 40 A5 

10 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 
100 105 110 

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

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



15 



20 



Leu Asp Lys Arg Ala He Asp Glu Ser Asp He He Met Thr Phe Leu 
145 150 155 160 

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 Asri Asp Asn Tyr Leu Val 
180 185 190 

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

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



30 



35 



45 



50 



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 



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

Leu lie His Arg Lys Val Asp Asp Glu Phe Gin Pro Phe Met He Gly 
5 290 295 300 

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

10 His Arg Ser Lys Arg Ser Ala Ser His Pro Arg Lys Arg Lys Lys Ser 

325 330 •3-3-> 

Val Ser Pro Asn Asn Val Pro Leu Leu Glu Pro Met Glu Ser Thr Arg 
340 345 350 

Ser Cys Gin Met Gin Thr Leu Tyr He Asp Phe Lys Asp Uu Gly Trp 
355 360 365 

His Asp Trp lie He Ala Pro Glu Gly lyr Gly Ala Phe Tyr Cys Ser 
20 370 375 380 

Gly Glu Cys Asn Phe Pro Leu Asn Ala His Met Asn Ala Thr Asn His 
385 390 395 *00 

25 Ala He Val Gin Thr Leu Val His Leu Leu Glu Pro Lys Lys Val Pro 

405 *10 



30 



Lys Pro Cys Cys Ala Pro Thr Arg Leu Gly Ala Leu Pro Val Leu l^r 
420 *25 *30 

His Leu Asn Asp Glu Asn Val Asn Leu Lys Lys Tyr Arg Asn Met He 
435 440 



Val Lys Ser Cys Gly Cys His 
35 450 *55 

(2) INFOBMATIOH FOR SEQ ID N0:26: 

(i) SEQUENCE CHARACTERISTICS: 
40 (A) LENGTH: amino acids 

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

(ii) MOLECULE TYPE: protein 

(iii) ORIGINAL SOURCE: 

(A) ORGANISM: Homo Sapiens 



45 



(ix) FEATURE: 
50 (A) NAME/KEY: Protein 

(B) LOCATION: 1..102 
(D) OTHER INFORMATION: /note="BMP3' 



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10 



15 



20 



35 



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

(1) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 104 amino acids 

(B) TYPE: amino acid 

(C) SIRANDEDNESS: single 

(D) TOPOLOGY: linear 

(ii)H0L£CULE TY?E: protein 

(ix)FEATUR£: 

(A) NAHE/KEYt Protein 

(B) LOCATION: 1..104 

(D) OTHER INFORHATION: /note="BHP3" 

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

Cys Ala Arg Arg Tyr Leu Lys Val Asp Phe Ala Asp He 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 

25 35 40 45 

Thr He Gin Ser He Val Ala Arg Ala Val Gly Val Val Pro Gly He 

50 55 60 

30 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: 

40 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 102 amino acids 

(B) TYPE: amino acid 

(C) STRANDEDNESS: single 
45 (D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: protein 

(vi) ORIGINAL SOURCE: 
50 (A) ORGANISM: HOMO SAPIENS 



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(ix) FEATOBE: 

(A) NAME/KEY: Protein 

(B) LOCATION: 1..I02 

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

(xi) SEQUENCE DESCRIPTIONt SEQ ID N0:27: 

Glu Leu Tyr Val Ser Phe Arg Asp Leu Gly Trp Gin 
5 10 15 

Ala Pro Glu Gly Tyr Ala Ala Phe Tyr Cys Asp Gly 
25 30 

Pro Leu Asn Ala His Het Asn Ala Thr Asn His Ala 

40 . 45 . 





(xi) s: 


Cys Lys 


Lys His 


1 




Asp Trp 


He He 


20 


Glu Cys 


Ser Phe 


35 


lie Val 


Gin Thr 



50 55 60 

20 Pro cys Cys Ala Pro Thr Lys Leu Asn Ala lie Ser Val Leu Tyr Phe 
65 70 75 80 

Asp Asp ser Ser Asn Val He Leu Lys Lys lyr Arg Asn Met Val Val 
85 90 '-^ 



25 



30 



Arg Ser Cys Gly Cys His 
100 

(2) INFOBHATION FOR SEQ ID NO: 28: 



(i) SEQOHJCE CHAEACTERISTICS: 

(A) LENGTH: 102 amino acids 

(B) TYPE: amino acid 

(C) STRANDEDNESS: single 
35 (D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: protein 

fvi) ORIGINAL SODRCE: 
40 (A) ORGANISM: HOMO SAPIENS 

(ix) FEATURE: 

(A) NAME/KEY: Protein 

(B) LOCATION: 1..102 

45 (D) OTHER INFORMATION: /note= "BHP6" 

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

Cys Arg Lys His Glu Leu Tyr Val Ser Phe Gin Asp Leu Gly Trp Gin 
50 1 5 10 



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Asp Trp He He Ala Pro Lys Gly Tjrr 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 
5 35 40 45 

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

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

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

15 

Arg Ala Cys Gly Cys His 
100 

(2) INFORMATION FOR SEQ ID NO: 29: 

20 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 102 amino acids 

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



25 



(ii) MOLECULE TYPE: protein 



(ix) FEATURE: 

(A) NAME/KEY: Protein 
30 (B) LOCATION: 1..102 

(0) OTHER INFORMATION: /labelo DPI 

/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 
35 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 NO: 29: 

40 Cys Xaa Xaa His Glu Leu Tyr Val Xaa Phe Xaa Asp Leu Gly Trp Xaa 
1 5 10 15 

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

45 

Glu Cys Xaa Phe Pro Leu Xaa Ser Xaa Met Asn Ala Thr Asn His Ala 
35 40 45 

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



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Xaa Cys Cys Ala Pro Thr Xaa Leu Xaa Ala Xaa Ser Val Leu lyr Xaa 
65 70 75 

Asp Xaa Ser Xaa Asn Val Xaa Leu Xaa Lys Xaa Arg Asn Met - Val Val 
5 85 90 95 

Xaa Ala Cys Gly Cys His 
100 

10 (2) INFOBHATION FOR SEQ ID N0:30i 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 97 amino acids 

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

(ii) HOLEaiLE TYPE: protein 
(ix)FEATORE: 

(A) NAME: Generic Sequence 5 . . j j 

fD) OTHER INFORMATION: wherein each Xaa as independently 
20 selected from a group of one or more specified amino acids as 

defined in the specification. 



25 



30 



35 



40 



45 



50 



(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 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 


Fro 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 




Cys 


Xaa Pro 


Xaa 


Xaa Xaa 


Xaa 


Xaa 


Xaa 


65 
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 











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(2) INFORMATION FOR SEQ ID N0:31: 

(1) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 102 amino acids 
5 (B) TYPE: amino acids 

(C) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(ix) FEATURE: 

(A) NAME: Generic Sequence 6 
10 (D) OTHER INFORMATION: wherein each Xaa is independently 

selected from a group of one or more specified amino acids as 
defined in the specification. 



15 



(xi) SEQUENCE DESCRIPTION: SEQ ID N0:31: 

a Phe 
10 





Cys 


Xaa 


Xaa 


Xaa 


Xaa 


Leu 


Xaa 


Xaa 




1 








5 










Xaa 


Xaa 


Xaa 


Gly 


Trp 


Xaa 


Xaa 


Trp 










15 








20 


Xaa 
20 


Xaa 


Pro 


Xaa 


Xaa 


Xaa 
25 


Xaa 


Ala 




Xaa 


Tyr 


Cys 


Xaa 


Gly 


Xaa 


Cys 


Xaa 






30 










35 




Xaa 


Pro 


Xaa 


Xaa 


Xaa 


Xaa 


Xaa 




25 










40 










Xaa 


Xaa 


Xaa 
45 


Asn 


His 


Ala 


Xaa 


Xaa 
50 




Xaa 


Xaa 


Xaa 


Xaa 


Xaa 
55 


Xaa 


Xaa 


Xaa 


30 


Xaa 


Xaa 
60 


Xaa 


Xaa 


Xaa 


Xaa 


Xaa 
65 


Cys 




Cys 


Xaa 


Pro 


Xaa 


Xaa 


Xaa 


Xaa 


Xaa 








70 












Xaa 


Xaa 


Xaa 


Leu 


Xaa 


Xaa 


Xaa 




35 


75 










80 








Xaa 


Xaa 


Xaa 


Xaa 
85 


Val 


Xaa 


Leu 


Xaa 




Xaa 


Xaa 


Xaa 


Xaa 


Met 


Xaa 


Val 


Xaa 




90 










95 






40 


Xaa 


Cys 


Xaa 


Cys 


Xaa 












100 













(2) INFORMATION FOR SEQ ID NO: 32: 

45 (1) SEQUENCE CHARACTERISTICS: 

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

(B) TYPE: nucleic acid, amino acid 

(C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 



50 



(ii) MOLECULE TYPE: cDNA 



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10 



(ill) ORIGINAL SOURCE: 
(A) ORGANISH: human 
(F) TISSUE TIFE: BRAIN 

(iv) FEATURE: 

(A) NAME/KEY: CDS 

(B) LOCATION: 

(D) OTHER INFORHATION: 

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



25 



30 



(X) PUBLICATION INFORMATION: 

(A) AUTHORS: Lee, Se-Jin . . ^ i 

(B) TITTLE: Expression of Growth/Differentiation factor l 
15 (C) JOURNAL: Proc. Nat'l Acad. Sci. 

(D) VOLUME: 88 

(E) RELEVANT RESIDUES: 1-1238 

(F) PAGES: 4250-4254 

(G) DATE: Hay- 1991 

20 (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 

15 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 

CAG GCT CTA GGA CTG GGC 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 5-> 

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

GCC CAG GAG ACC AGG TCT GGC TCG CGG CGG ACG TCC CCA 6GG 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 €TC GCC GGA AAC 383 
Thr Leu Gin Pro Cyc 
50 90 



40 



45 



Thr Leu Gin Pro Cyc His Val Glu Glu Leu Gly Val Ala Gly Asn 



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10 



15 



20 



25 



Ate GTG CGC CAC ATC CCG GAC CGC GGT GCG CCC ACC CGG GCC TCG 428 
lie Val Arg His He Pro Asp Arg Gly Ala Pr6 Thr Arg Ala Ser 
105 110 115 

GAG CCT GTC TCG GCC GCG GGG CAT TGC CCT GAG 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 1*0 145 

CGC CTG GAG CTG CGT TTC GCG GCG GCG GCG GCG GCA GCC CCG GAG 563 
Are 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 



30 



35 



40 



45 



50 



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 <XC 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 Are 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 Are Trp Val He Arg Pro Arg Gly Phe Leu Ala Asn Tyr 
285 290 295 



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TGC CAG GGT CAG TGC GCG CTG CCC GTC GCG CTG TCG GGG TCC GGG 1013 
^ Sn Gin Cys Ala Leu Pro Val Ala Leu Ser Gly Ser Gly. 
^ 300 

5 GGG CCG CCG GCG CTC AAC CAC GCT GTG CTG CGC GCG CTC ATG CAC 1058 

gS Pro Pro Ala Leu Asn His Ala Val Leu Arg Ala Leu Met Hxs 
315 320 

GCG GCC GCC CCG GGA GCC GCC GAC CTG CCC TGC TGC GTG CCC GCG 1103 
10 Ala Ala Ala Pro Gly Ala Ala Asp Leu Pro Cys Cys Val Pro Ala 

330 335 •J*" 

CGC CTG TCG CCC ATC TCC GTG CTC TTC TTI GAC AAC AGC GAC AAC 1148 
Are Leu Ser Pro He Ser Val Leu Phe Phe Asp Asn Ser Asp Asn 
15 345 350 353 

GTG GTG CTG CGG CAG TAT GAG GAC ATG GTG GTG GAC GAG TGC GGC 1193 



20 



(jIU UIIj UlU i^UU WIVJ AXIA w«w - 

Val Val Leu Arg Gin Tyr Glu Asp Met Val Val Asp Glu Cys Gly 

365 



360 



TGC CGC TAACCCGGGG CGGGCAGGGA CCCGGGCCCA ACAATAAATG CCGCGTGG 1238 



Cys Arg 
372 



25 (2) INFORMATION FOR SEQ ID N0:33: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTR: 372 amino acids 

(B) TYPE: amino acid 

30 (C) STRANDEDNESS: single 

(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: cDNA 
35 (iii) HYPOTHETICAL: NO 

(iv) ANTI-SENSE: NO 

(vi) ORIGINAL SOURCE: 
40 (A) ORGANISM: human 

(F) TISSUE TYPE: BRAIN 

(ix) FEATURE: 

(A) NAME/KEY: CDS 
45 (B) LOCATION^ 

(D) OTHER INFORMATION: /function:^ 
/product- "GDF-l" 

50 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33: 

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



SUBSTITUTE SH^ET 



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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 Uu Leu 

5 30 35 *0 

Gin Ala Leu Gly Leu Arg Asp Glu Pro Gin Gly Ala Pro Arg Leu 

45 50 55 

10 Arg Pro Val Pro Pro Val Met Trp Arg Leu Phe Arg Arg Arg Asp 

60 65 70 



15 



30 



45 



Pro Gin Glu Thr 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 



lie Val Arg His He Pro Asp Arg Gly Ala Pro Thr Arg Ala Ser 
20 105 110 115 

Glu Pro Val Ser Ala Ala Gly His Cys Pro Glu Trp Thr Val Val 
120 125 130 

25 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 
35 180 185 190 

Gly Pro Pro Val Arg Ala Glu Leu Leu Gly Ala Ala Trp Ala Arg 
195 200 205 

40 Asn Ala Ser Trp Pro Arg Ser Leu Arg Leu Ala Leu Ala Leu Arg 

210 215 220 



Pro Arg 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 Are Asp Ala Glu Pro Val Leu Gly Gly Gly Pro Gly Gly 
50 255 260 265 



SUBSTITUTE SH^ET 



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Ala Cys Arg Ala Arg Arg Leu Tyr Val Ser Phe Arg Glu Val Gly 
270 275 280 

Trp His Arg Trp Val He Arg Pro Arg Gly Phe Leu Ala Asn Tyr 
5 285 290 295 

Cys Gin Gly Gin Cys Ala Leu Pro Val Ala Leu Ser Gly Ser Gly 
' 300 305 310 

10 Gly Pro Pro Ala Leu Asn His Ala Val Leu Arg Ala Leu Met His 

315 320 325 



15 



20 



Ala Ala Ala Pro Gly Ala Ala Asp Leu Pro Cys Cys Val Pro Ala 
330 335 340 

Are Leu Ser Pro He Ser Val Leu Phe Phe Asp Asn Ser Asp Asn 
. 345 350 355 

Val Val Leu Arg Gin Tyr Glu Asp Met Val Val Asp Glu Cys Gly 
360 365 370 

Cys Arg 
372 



SUBSTITUTE SH^ET 



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What is claimed is: 

1. A method for alleviating the tissue destructive 
effects associated vith the inflammatory response 
to tissue injury in a mammal, the method 
comprising the step of: 

providing to the injured tissue a therapeutically 
effective concentration of a morphogen sufficient 
to substantially inhibit or reduce the tissue 
damage resulting from said inflammatory response. 

2. The method of claim 1 where said step of providing 
a therapeutically effective morphogen 
concentration to said injured tissue comprises the 
step of administering a therapeutically effective 
concentration of a morphogen to said mammal. 

3. The method of claim 1 where said step of providing 
a therapeutically effective morphogen 
concentration to said injured tissue comprises the 
step of administering to said mammal an agent that 
stimulates in vivo a therapeutically effective 
concentration of an endogenous morphogen. 

4. The method of claim 1 wherein said step of 
providing a therapeutically effective 
concentration of a morphogen is conducted prior to 
reduction or interruption of blood flow to the 
tissue . 



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5. The method of claim 1 wherein said step of 
providing a therapeutically effective 
concentration of a morphogen is conducted after 
reduction or interruption of blood flow to the 
tissue and before reperfusion. 

6 The method of claim 1 wherein said step of 

administering a therapeutically effective amount 
of a morphogen is conducted following isChemia- 
reperfusion injury. 



7. 



10. 



11. 



The method of claim 1 wherein said said step of 
administering a therapeutically effective amount 
of a morphogen is conducted following hyperopia 
injury. 

The method of claim 1 wherein said morphogen is 
provided to said tissue prior to said tissue 
injury. 

The method of claim 1 wherein said step of 
providing a therapeutically effective 
concentration of a morphogen is conducted prior to 
ischemia-reperfusion injury. 

The method of claim 1 wherein said tissue damage 
results from an abnormal immune response in said 
mammal . 

The method of claim 1 wherein said tissue damage 
is associated with an inflammatory disease. 



12. The method of claim 11 wherein said inflammatory 
disease is an autoimmune disease. 



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13. The method of claim 11 wherein said inflammatory 
disease comprises arthritis, psoriasis, dermatitis 
or diabetes. 

14. The method of claim 13 wherein said arthritis is 
rhematoid, degenerative or psoriatic arthritis. 

15. The method of claim 11 wherein said inflammatory 
disease comprises an airway inflammation in a 
mammal • 

16. The method of claim 15 wherein said airway 
inflammation comprises chronic bronchitis, 
emphysema, idiopathic pulmonary fibrosis or 
asthma. 

17. The method of claim 11 wherein inflammatory 
disease comprises a generalized acute inflammatory 
response. 

18. The method of claim 17 wherein said inflammatory 
disease comprises adult respiratory distress 
syndrome • 

19. The method of claim 1 wherein said tissue damage 
is to a transplanted organ or tissue. 

20. A method for reducing tissue damage associated 
with ischemia-reperfusion injury in a human, the 
method comprising the step of: 

providing to the injured tissue a therapeutic 
concentration of a morphogen sufficient to 
alleviate the damage associated with said injury. 



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21. A method for reducing the tissue damage associated 
vith hyperoxia injury in a human, the method 
comprising the step of: 

providing to the injured tissue a therapeutic 
concentration of a morphogen sufficient to 
alleviate the damage associated vlth said injury. 



22, 



23, 



The method of claim 20 or 21 wherein said step of 
providing a therapeutically effective morphogen 
concentration to said injured tissue comprises the 
step of administering a therapeutically effective 
concentration of a morphogen to said mammal. 

The method of claim 20 or 21 wherein said step of 
providing a therapeutically effective morphogen 
concentration to said injured tissue comprises the 
step of administering to said mammal an agent that 
stimulates in vivo a therapeutically effective 
concentration of an endogenous morphogen. 

24. The method of claim 1, 20 or 21 wherein said 
tissue is lung tissue, cardiac tissue, hepatic 
tissue or renal tissue. 

25. The method of claim 6, 9 or 20 wherein said 
ischemic-reperfusion injury results from cardiac 
arrest, preliminary occlusion, arterial occlusion, 
coronary occlusion or occlusive stroke. 



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26. The method of claim 1, 20 or 21 wherein said 
morphogen comprises an amino acid sequence sharing 

^ at least 70X homology with one of the sequences 

selected from the group consisting of: OP-1, OP-2, 
CBMP2, Vgl(fx), Vgr(fx), DPP(fx)i GDF-l(fx) and 
60A(fx). 

27. The method of claim 26 wherein said morphogen 
comprises an amino acid sequence sharing a last 
80% homology with one of the sequences selected 
from the group consisting of: OP-1^ OP-2, CBMP2, 
BMP3(fx), BMP5(fx), BMP6(fx), Vgl(fx), Vgr<fx), 
DPP(fx), GDF-l(fx) and 60A(fx). 

28- The method of claim 1, 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). 

29. The method of claim 28 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). 

30. The method of claim 29 wherein said morphogen 
comprises an amino acid sequence defined by 
residues 43rl39 of Seq. ID No. 5 (hOPl), including 
allelic and species variants thereof. 

31. The method of claim 1, 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). 



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32. The method of claim 1, 20 or 21 wherein said 
morphogen comprises an amino acid sequence defined 
by OPX (Seq. ID No. 29). 

33. A method for reducing the ischemic-reperf usion 
injury associated with the interruption of blood 
flow to an organ in a clinical procedure, the 
method comprising the step of providing a 
therapeutic concentration of a morphogen to said 
organ prior to the interruption of blood flow. 



34 



A method for reducing the tissue injury associated 
with the reduction or interruption of blood flow 
to an organ or tissue in a clinical procedure, the 
method comprising the step of providing a 
therapeutic concentration of a morphogen to said 
organ or tissue after the reduction or 
interruption of blood flow to said organ or 
tissue. 

The method of claim 33 or 34 wherein said clinical 
procedure is a carotid enterectomy, a coronary 
artery bypass, a tissue grafting procedure, an 
organ transpiant, or a fibrinolytic therapy. 

36. The method of claim 1, 33 or 34 wherein said 
morphogen is administered parenterally. 

37. The method of claim 1, 33 or 34 wherein said 
morphogen is administered prophylactically. 



35, 



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- 149 - 

38* A pharraaceutical composition for use in 

alleviating the injury associated with tissue 
» exposure to toxic oxygen concentrations comprising 

a therapeutically effective amount of a morphogen 
in admixture with a free oxygen radical inhibiting 
agent or an anticoagulent . 

39. A pharmaceutical composition for topical 

administration comprising a therapeutically 
effective concentration of a morphogen in 
admixture with a dermatologically acceptable 
carrier. 



40. A pharmaceutical composition for topical 
administration to a tissue comprising a 
therapeutically effective concentration of a 
morphogen dispersed in a biocompatible , non- 
irritating tissue surface adhesive. 

41. The composition of claim 40 wherein said adhesieve 
comprises hydroxypropylcellulose . 

- 42. The composition of claim 38, 39 or 40 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) and 
60A(fx). 

43. The composition of claim 42 wherein said morphogen 
comprises an amino acid sequence sharing a last 
80Z homology with one of the sequences selected 
from the group consisting of: OP^l, OP-2, CBMP2, 
BMP3(fx), BMP5(fx), BMP6(fx), Vgl(fx), Vgr(fx), 
DPP(fx), GDF-l(fx) and 60A(fx). 



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44. The composition of clai» 38. 39 or 40 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)- 

45. The composition of claim 44 vherein said morphogen 
comprises an amino acid sequence having greater 
than 65Z amino acid identity with the sequence 
defined by residues 43-139 of Seq. ID No. 5 
(hOPl). 

46. The method of claim 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. 

47. The composition of claim 38, 39 or 40 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). 

48. The composition of claim 38, 39 or 40 wherein said 
morphogen comprises an amino acid sequence defined 
by OPX (Seq. ID Ho. 29). 



49. 



A method of enhancing the viability of an organ or 
tissue to be transplanted in a mammal, the method 
of comprising the step of: 

providing a therapeutically effective 
concentration of a morphogen to said tissue or 
organ to be transplanted. 



SUBSTITUTE SH^ET 



wo 93/04692 PCr/US92/073S8 

- 151 - 



50- The method of claim 49 wherein said 

therapeutically effective concentration is 
sufficient to substantially inhibit reperfusion 
injury to said tissue or organ. 

51. The method of claim 49 wherein said morphogen is 
provided to said tissue or organ prior to 
reperfusion injury. 

52. The method of claim 49 wherein said morphogen is 
provided to said tissue or organ prior to removal 
of said tissue or organ from the donor. 

53. The method of claim 49 wherein said organ is 
placed in an organ preservation solution 
containing said morphogen or a morphogen- 
stimulating agent after removal of said organ from 
the donor and prior to transplantation in the 
recipient. 

54. The method of claim 49 wherein said organ is 
selected from the group consisting of lung, heart, 
kidney, liver or pancreas. 

55. The method of claim 49 wherein said living tissue 
comprises skin, bone marrow or gastrointestinal 
mucosa tissue. 



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56. A method for protecting a living tissue or 
transplant organ from the tissue destructive 
effects associated vith the inflammatory response 
in a mammal, the method comprising the step of: 

providing to said tissue or organ a 
therapeutically effective concentration of a 
morphogen. 

57. A method of protecting a living tissue or 
transplanted organ from ischemia-reperf usion 
injury in a mammal, the method comprising the step 
ofj 

providing to said tissue or organ a 
therapeutically effective concentration of a 
morphogen, said concentration being sufficient to 
substantially inhibit or reduce the tissue damage 
associated vith ischemia-reperfusion injury. 

58. The method of claim 49, 56 or 57 wherein said step 
of providing a therapeutically effective morphogen 
concentration to said injured tissue comprises the 
step of administering a therapeutically effective 

)ncentration of a morphogen to said mammal. 



59. 



coi 

The method of claim 49, 56 or 57 wherein said step 
of providing a therapeutically effective morphogen 
concentration to said injured tissue comprises the 
step of administering to said mammal an agent that 
stimulates in vivo a therapeutically effective 
concentration of an endogenous morphogen. 



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60. A composition useful as a living cell or living 
tissue preservation solution comprising: 

a fluid formulation having as osmotic pressure 
substantially equivalent to the osmotic pressure 
of living mammalian cells in admixture with 

a therapeutically effective concentration of a 
roorphogen or morphogen-stimulating agent, said 
concentration being sufficient to protect living 
cell or tissue from the tissue destructive effects 
associated with the inflammatory response in a 
mammal when exposed to said cells or tissue. 

61. The preservation solution of claim 60 wherein said 
therapeutically effective concentration is 
sufficient to substantially inhibit or reduce the 
tissue damage associated with ischemia-reperfusion 
injury. 

62. The preservation solution of claim 60 wherein said 
formulation further comprises a sugar. 

63. The preservation solution of claim 60 wherein said 
formulation further comprises an anticoagulant or 
a free oxygen radical inhibiting agent. 

64. The invention of claim 49, 56, 57 or 60 wherein 
said morphogen comprises an amino acid sequence 
having greater than 60Z amino acid identity with 

, the sequence defined by residues 43-139 of Seq. ID 
. No. 5 (hOPl). 



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65. 



A composition useful in a treatment method to 
alleviate tissue damage associated with the 
inflammatory response in a mammal, the composition 
comprising a therapeutically effective 
concentration of a morphogen or morphogen- 
stimulating agent. 

The composition of claim 65 wherein said tissue 
damage is associated with iscbemia-reperf usion 
injury or hyperoxia injury. 

67. The composition of claim 65 wherein said tissue 
damage is to lung, cardiac, renalor hepatic 
tissue. 



66. 



68. 



The composition of claim 65 wherein said tissue 
damage is to a transplanted organ or tissue. 



SUBSTITUTE SH^ET 



wo 93/04692 



- 155 - 



PCr/US92/07358 



AMEM)ED CLAIMS 

[received by the International Bureau 
on 10 F^ruary 1993 (10.02.93); 
original claims 46 and 49 amended; 
remaining claims unchanged (1 page)] 



44. The composition of claim 38, 39 or 40 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). 

45. The composition of claim 44 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). 

46. The composition of claim 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. 

47. The composition of claim 38, 39 or 40 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). 

48. The composition of claim 38, 39 or 40 wherein said 
morphogen comprises an amino acid sequence defined 
by OPX (Seq. ID No. 29). 

49. A method of enhancing the viability of an organ or 
tissue to be transplanted in a mammal, the method 
comprising the step of: 



providing a therapeutically effective 
concentration of a morphogen to said tissue or 
organ to be transplanted. 



wo 93/04692 PCr/US92/073S8 

1/6. 



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1 7.5 ^ 



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CO 



u -2.5 - 



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VEHICLE 0P-l{2Mg) OP-1 (20 ^ig) 



Rg. 1 



SUBSTITUTE SH^ET 



wo 93/04692 



2/6 



I>Cr/US92/073S8 




SUBSTITUTE SH^ET 



wo 93/04692 



3/6 



PCT/US92/07358 




CONTROL OP-1 

|20jig/RAT) 



Rg.3 



SUBSTITUTE SHEET 



wo 93/04692 



4/6 



PCT/US92/07358 




SUBSTITUTE SH^ET 



wo 93/04692 



5/6 



PCr/US92/073S8 




> 

r 1 \ 1 \ r 1 r 

3aODSSIilSODnWN\;GW 



SUBSTITUTE SHiET 



wo 93/04692 



PCT/US92/073a 



Rg. 6A 



u 
o 

UJ 

a. 

i 



2000 

1750 H 

1500 

1250 

1000 

750 

500 H 

250 

0 
10 



6/6 



■rmr 



16 10-1510-1410-1310-12 IO-H 10-10 10*9 
OP-1 [AAI 



Fig. 6B 



Fig. 6C 



Fig. 6D 




u 

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DC 



b 



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U 



-16 10-15 10-14 10-13 10-12 lo-HlO-lOlO-' 
TGF-BETAHAAl 



5000] 
4500 
4000: 
3500: 
3000 
2500 
2000 
1500: 
1000: 
500: 
0 
10 



5000 ] 
4500 
4000- 
3500- 
3000- 
2500: 
2000- 
1500: 
1000 
500 
0 
10 



16 ib-15 16-14 10-13 10-12 io-lllO-10io-9 
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TGF-BETA1[M] 



SUBSTITUTE SH^ET 



aNTCEi^ATnONAL SEAKCIHI EEPOOT PCT/US 92/07358 



B, CLASSIFSCAnON OF SUaJECT MATSTSa ( tf sGvcml dasstterica syiBfcoto apply, todicato c 
AcoDJdias to ilnioniational PatQK OassiflcaJion (EPQ or to Mi Nndoaal Oasslflcasloa ond IDPC 

Int. CI. 5 AS1K37/02; AOlNl/02 




DocoBiQattitioa Scorebod otbc? limn MlniisQia IDccamoteSioa 
to tho ISsctGat tbas such iDcaiooazs aro Bncludod ia the Fid& Soarchod^ 



Enu DoanwEms conshdesed to m kelevap^t^ 



Cftaitea cJ ©oanQcaa, " tadicotaa, ^ao oppjppgfato, cf tfao goicwnat passagas ^ 



SCIENCE 

vol. 249, no. 4964, 6 July 1990, 
LANCASTER, PA US 
pages 61 ° 64 

LEFER A.M. ET AL 'Mediation of 
cardioprotcction by Transfonaing growth 
f actor^beta • 

cited in the application 
see the vfhole document 

PROCEEDINGS OF THE NATIONAL ACADEMY OF 
SCIENCES OF USA. 

vol. 88, no. 7, April 1991, VfASHINGTON US 
pages 2918 - 2921 

KURUVILLA, A.P, 'Protective effect of 
transforming growth factor betal on 
experimental autoimmune diseases in mice 
cited in the application 
see the whole document ' 



1-2,4-9, 

20-22, 

25, 

33=37, 
57-58, 
65-68 



1=2, 
10=19, 
36-37, 
56,58,65 



.10 



" Spocaol caSG^araos dtod dooiiDcats : 
''A" docaiDaatdi^iiJn8«ho||20G2alstatoirftboQrtvj5^ 
asazidcaad to Iks of paramlar rotovaooD 

ffllagdoSQ 

If doamGat yblcb ma? thra^ doato on B?to^ 

ubicSi Is dtod to GstnUisb tbo psbUcaxtoa doto tS tmasGS 
ctoxto or cxbcr spcaal rcisea (as specified) 

^'iy docQEitaa Ft^sTiag to aa oral dbdosGro, bsq, cmfef Mtica or 
otbc7 Ennpp 

<^ docoEDcat pnbUsbcd prior to ttie tntGrnastonal fiHag dato to3 
last? «£ma tSio priority dato daiiocd 



btc? docQsacat po&lisSicd oftc? tbQ imcniadmia] fiUng doso 
or ariority dato and ao8 tn eonfilct tbo OBsilcation tot 
dtod to ondcEStaad tbo prindpla or tbowy onacsl^s tao 



<^ docaiacit partfcalar retcvaQcc; tbo daia(s] lnvcnttto 
ennst bs oosisidf^^ novol or caasot bo considcTCd to 
favoSvo aa iavsaitivc stc^ 

^ doanacat €S ^Eistlcotor idctrDaco; tbo dsiiacj lavcatioa 
caaaot ba oonsidcrcd to iovolvQ aa iavativo step ^^btai tbo 
doGQEiGnt is comtinod ^tb ono or store oths sndi dosE>- 
BicaSz, SBcb combinatEOD bQBQ o&vioos to a psTSoa shiUod 
b tboort. 

''A'' dOGOOGnt QQSBbor 0? tbo samo patent fosUy 



OV. CEEtlOTCATHOW 



Dato cf tbo Acton! Cosnplaifia ^ tbo OotosnatiiKiiQi Scorch 

19 NOVEMBER 1992 



Oatonatiosai Scsrduao Aixibsst^ 

EUROPEAN PATENT OFFDCE 



Dato of MaiUas of tbis Aatcraational Soardi 



Peso 1?CT/ISA/210 <K3C±J ot=:l> (Jc=:iV tOS) 




Intmttenal AppUotion No 



PCT/US 92/07358 



m, DOCUMENTS COWSIDEREP TO BEKELEVANT CCONTINUED FROM THE SECOND SHEET) 



Catcgny*' 



atatloD of DoaiiacDt, irith todlottoiu wtog appnyriitt, of the wtevint pamgg 



ttoCXaiBNa 



X.P 



EP.A.O 269 408 (GENENTECH INC.) 
I June 1988 



see the whole document 

WO, A, 9 000 900 (AMGEN INC.) 
8 February 1990 

see the whole document 

W0,A,9 215 323 (CREATIVE BIOMOLECULES) 
17 September 1992 



see page 6, Tine 1 - page 7, line 27 
see page 77 - page 119 

WO, A, 9 105 802 (CREATiyE BIOMOLECULES)- 
2 Nay 1991 



see page 20, Tine 19 - Tine 31 
see page 59 - page 80 

WO, A, 9 207 073 (CREATIVE BIOMOLECULES) 
30 ApriT 1992 



see page 11, Tine 3 - Tine 15 
see page 41 - page 60 

W0,A,8 909 788 (CREATIVE BIOMOLECULES) 

19 October 1989 

cited in the appTi cation 



see page 6 - page 15 
EMBO JOURNAL 

vol. 9, no. 7, 1990, EYNSHAM, OXFORD GB 
pages 2085 - 2093 

eZKAYNAK, E. ET AL 'OP-l cDNA encodes an 
osteogenic protein in the TGF-faeta famiTy' 
see the whole document 



1-2, 
10-19, 
36-37, 
56,58,65 



1-2. 
10-19, 
36-37, 
56,58,65 



1-6, 

8-12. 

15-17, 

''20r — 

22-37, 
56-59, 
64-67 



10-13, 
26-32, 
36-37,65 



1-2, 

10-13. 

26-29, 

31-32, 

36-37,65 



1-2, 

10-13, 

26-29. 

31-32, 

36-37.65 



1-68 



rm PCTASA/aiO (son iMI liMt 1W> 



INTERNATIONAL SEARCH REPORT 



It Ational ippBcatioo No. 

PCT/US 92/ 07358 




Tto internatioma «arch itpon h» not been established in respect 



of certain daims under Article 17(2)(a) for the following reasons: 



^ S^'e'i'^reU.e to subject nutter not required to be searched by this Authority, namely: 



because they 

see annex 



see annex 



• n SS^'S^^^e dependent daims «d«^^^ 

BOX II Observations where unhy of invention is lacking (Continuation of item 2 of fir^^ 



This Intemiuional Searching Authority found multiple inventions 



in this international appUcation, as follows: 



1. I I As all required additional search fees 
' — * searchable daims. 



timdy paid by the applicant, this intcmatiomd seardi report covers aU 



2. I 1 As ail searchable daims could 
" — * of any addhionai fee. 



I — ' covers only those claims for whidi fees were pam, specurcauy 



Remark on Protect 



Q The additional search fees «ft «xompanied by the appBcaf s protest 
Q No protest accompanied the paymBit of «Jditional search Sees. 



Form PCr/ISA/MO (continuation of first sheet (1)) (July 1992) 



International Applicatian No. PCT/US92/ 07358 



FURTHER INFORMATION CONTINUED FROM PCT/ISA/210 

Remark: Although claims 1-37,49-52 (partially, when the method 
is carried cut in vivo), 54-57 (partially, when the method 
is carried out in vivo)> 58 - 59.64 (partially, according 
to the method of claims 49,56 or 57) are directed to a method 
of treatment of the human or animal body the search has 
been carried out and based on the alleged effects of the 
composition . 

OBSCURITIES, INCONSISTENCIES, CONTRADICTIONS, LACK OF 
CONCISENESS; LACK OF READY COMPREHENSIBILITY) 

(ART. 6 PCT) 
REASON: 

1. Claim 46 has been understood as being dependant of claim 45. 
Therefore claim 46 should read: "The composition of claim 45, 
wherein said morphogen comprises an amino acid sequence defined 
by residues 43-139 of Seq. ID No. 5 (hOPI), 

including allelic and species variants thereof. 

2. In view of the extremely large number of compounds used 

in the methods and compositions of claims 26-29, 31 (in as 
far as seq. ID 1 to 4 and 30-31), 42-45,47 (in as far as 
seq. ID 1 to 4 and 30-31), 64, the search division considers 
that it is not economically reasonable to draw up a ^ 
search report for the methods using, or the compositions 
comprising all the compounds defined in the claims* 
The search has therefore been limited, on the basis of the^ 
examples ana claims, to the methods using, or the compositions 
comprising the seq. ID no. 5 to 29, 32 and 33 (Art. 17 (2) 
(a){ii| and (b) PCT. 

3. The term "morphogen" is not concise. 

Therefor, and for the same reaons as given in paragraph 2 
above, it has been understood as being one of the proteins 
defines in seq* ID 5 to 29^ 32 or 33. 
(Art. 6 PCT and Art. 17/2) (a) (ii) and (b) PCT) 



ANNEX TO THE INTERNATIONAL SEARCH REPORT^ 
ON INTERNATIONAL PATENT APPLICATION NO. 9207358 

SA 64364 



This uncx lists the patent funQy members relatiiig to the pitcnt documents dted Jo the aboveincntioDed internitiona] much report. 
The members are as contained in the European Patent Office EDP file on 

The European Patent Office is in no way liable for these partkulars wfatcfa are merely eiven for the purpose of information. 19/1 1/92 



Patent document 
dted in search 



Publication 
date 



Patent funily 
membcr(8) 



PublicatioD 



EP-A-0269408 



WO-A-9000900 
WO-A-9215323 
WO-A-9105802 



01-06-88 
08-02-90 



17-09-92 
02-05-91 



WO-A-9207073 
WO-A-8909788 



30-04-92 
19-10-89 



JP-A- 



AU-A- 
None 



63211234 
4056089 



AU-A- 
CA-A- 
EP-A- 
JP-T- 
CA-A- 



6648190 
2042577 
0448704 
4502336 
2027259 



AU-A- 



8900091 



US-A- 
US-A- 
AU-B- 
AU-A- 
AU-B- 
AU-A- 
EP-A- 
EP-A- 
JP-T- 
JP-T- 
WO-A- 
US-A- 
AU-B- 
AU-A- 
EP-A- 
JP-T- 
WO-A- 
US-A- 



4968590 
5011691 

628050 
3444989 

618357 
3530589 
0372031 
0362367 
3500655 
3502579 
8909787 
5108753 

627850 
5174790 
0411105 
3504736 
9010018 
4975526 



02-09-88 
19-02-90 



16-05-91 
18-04-91 
02-10-91 
23-04-92 
18-04-91 



20-05-92 



06-11-90 
30-04-91 

10- 09-92 
03-11-89 
19-12-91 
03-11-89 

13- 06-90 

11- 04-90 

14- 02-91 
13-06-91 
19-10-89 
28-04-92 

03- 09-92 
26-09-90 

06- 02-91 
17-10-91 

07- 09-90 

04- 12-90 



f 

z 
8 

O 

ta For more detMis about this annei : see Official Jooraal of the Enropeao Patent Office, No. 12^2 



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