U.S. patent application number 10/865514 was filed with the patent office on 2005-05-05 for morphogen-induced modulation of inflammatory response.
Invention is credited to Cohen, Charles M., Kuberasampath, Thangavel, Oppermann, Hermann, Ozkaynak, Engin, Pang, Roy H. L., Rueger, David C., Smart, John E..
Application Number | 20050096339 10/865514 |
Document ID | / |
Family ID | 27538725 |
Filed Date | 2005-05-05 |
United States Patent
Application |
20050096339 |
Kind Code |
A1 |
Kuberasampath, Thangavel ;
et al. |
May 5, 2005 |
Morphogen-induced modulation of inflammatory response
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 effective
concentration of a morphogen or morphogen-stimulating agent
sufficient to alleviate immune cell-mediated tissue
destruction.
Inventors: |
Kuberasampath, Thangavel;
(Medway, MA) ; Pang, Roy H. L.; (Etna, NH)
; Oppermann, Hermann; (Medway, MA) ; Rueger, David
C.; (Hopkinton, MA) ; Cohen, Charles M.;
(Medway, MA) ; Ozkaynak, Engin; (Milford, MA)
; Smart, John E.; (Weston, MA) |
Correspondence
Address: |
FISH & NEAVE IP GROUP
ROPES & GRAY LLP
ONE INTERNATIONAL PLACE
BOSTON
MA
02110-2624
US
|
Family ID: |
27538725 |
Appl. No.: |
10/865514 |
Filed: |
June 9, 2004 |
Related U.S. Patent Documents
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Application
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Filing Date |
Patent Number |
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10865514 |
Jun 9, 2004 |
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09597517 |
Jun 20, 2000 |
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09597517 |
Jun 20, 2000 |
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08445467 |
May 22, 1995 |
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6077823 |
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08445467 |
May 22, 1995 |
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08165511 |
Dec 9, 1993 |
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08165511 |
Dec 9, 1993 |
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07938336 |
Aug 28, 1992 |
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07938336 |
Aug 28, 1992 |
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07753059 |
Aug 30, 1991 |
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07753059 |
Aug 30, 1991 |
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07667274 |
Mar 11, 1991 |
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07938336 |
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07752764 |
Aug 30, 1991 |
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07752764 |
Aug 30, 1991 |
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07667274 |
Mar 11, 1991 |
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Current U.S.
Class: |
514/282 |
Current CPC
Class: |
A61K 2300/00 20130101;
A61F 2310/00365 20130101; A61K 38/1875 20130101; C07K 14/51
20130101; C07K 16/22 20130101; A61K 38/1703 20130101; A61L 27/227
20130101; G01N 2500/10 20130101; A61K 38/17 20130101; A61K 38/1875
20130101; A01N 1/0226 20130101; C07K 14/495 20130101; A61L 27/24
20130101 |
Class at
Publication: |
514/282 |
International
Class: |
A61K 031/485 |
Claims
1-48. (canceled)
49. A method for enhancing recovery of central nervous system
function in a mammal, comprising administering an effective amount
of a morphogen to a mammal afflicted with a central nervous system
injury selected from ischemia or trauma, wherein said morphogen
induces tissue-specific morphogenesis in said mammal and comprises
a pair of folded polypeptides, each having an amino acid sequence
having at least 70% homology with or 60% identity to the C-terminal
seven-cysteine domain of human OP-1, residues 38-139 of SEQ ID NO:
5.
50. A method for enhancing recovery of central nervous system
function in a mammal, comprising administering an effective amount
of a morphogen to a mammal afflicted with a central nervous system
injury selected from ischemia or trauma, wherein said morphogen
induces tissue-specific morphogenesis in said mammal and comprises
a pair of folded polypeptides, each having an amino acid sequence
selected from: (a) Generic Sequence 3 defined by SEQ ID NO: 3; (b)
Generic Sequence 4 defined by SEQ ID NO: 4; (c) Generic Sequence 5
defined by SEQ ID NO: 30; or, (d) Generic Sequence 6 defined by SEQ
ID NO: 31.
51. A method for enhancing recovery of central nervous system
function in a mammal, comprising administering an effective amount
of a morphogen to a mammal afflicted with a central nervous system
injury selected from ischemia or trauma, wherein said morphogen is
human OP-1, mouse OP-1, human OP-2, mouse OP-2, 60A, GDF-1, BMP-2A,
BMP-2B, DPP, Vgl, Vgr-1, BMP-3, BMP-5, or BMP-6.
52. The method claim 49, wherein said amino acid sequence is that
of the C-terminal seven-cysteine domain of human OP-1, residues
38-139 of SEQ ID NO: 5.
53. The method of claim 49, wherein said morphogen is complexed
with at least one pro-domain peptide comprising an N-terminal 18
amino acid peptide selected from N-termini of the pro domains of
OP-1, OP-2, 60A, GDF-1, BMP-2A, BMP-2B, DPP, Vgl, Vgr-1, BMP-3,
BMP-5, or BMP-6.
54. The method claim 49, wherein said morphogen is noncovalently
complexed with at least one solubility-enhancing fragment of a
pro-domain polypeptide selected from the pro-domains of
naturally-occurring morphogens.
55. The method of claim 54, wherein said morphogen is complexed
with a pair of said fragments.
56. The method of claim 49, wherein said morphogen comprises the
amino acid sequence of SEQ ID NO: 5.
Description
CROSS REFERENCE RELATIONSHIP TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of (1) U.S. Ser.
No. 753,059, filed Aug. 30, 1991, which is a continuation-in-part
of U.S. Ser. No. 667,274, filed Mar. 11, 1991, (2) U.S. Ser. No.
752,764, filed Aug. 30, 1991, which is a continuation-in-part of
U.S. Ser. No. 667,274 and [Atty. Docket No. CRP-068] filed on even
date herewith.
FIELD OF THE INVENTION
[0002] 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 the inflammatory response.
BACKGROUND OF THE INVENTION
[0003] The body's inflammatory response to tissue injury 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 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
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 considered to be chronic inflammatory diseases. The
damaged tissue also often is replaced by fibrotic 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 body's immune/inflammatory response
system.
[0004] The immune-cell mediated tissue destruction often follows an
initial tissue injury or insult. The secondary damage, resulting
from the inflammatory 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) 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 concentrations are increased in
humans after myocardial infarction.
[0005] A variety of lung diseases are characterized by airway
inflammation, including chronic bronchitis, 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 associated with
the inflammatory response is that mounted in response to injury
caused by hyperoxia, e.g., prolonged exposure to lethally high
concentrations of O.sub.2-(95-100% O.sub.2). Similarly, reduced
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.
[0006] 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 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 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 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 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.
[0007] 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 well as the damage caused by the body's response
to 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.
[0008] Similarly, tissues and organs for transplantation also are
subject to the tissue destructive effects 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
recipient.
[0009] Accordingly, the success of organ or tissue transplantation
depends greatly on the preservation of the tissue activity (e.g.,
tissue or organ viability) 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. Pat. No. 4,952,409 describes
a superoxide dismutase-containing liposome to inhibit reperfusion
injury. U.S. Pat. No. 5,002,965 describes the use of ginkolides,
known platelet activating factor antagonists, to inhibit
reperfusion injury. Both of these factors are described as working
primarily by 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 representative listing includes U.S. Pat. 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
effect.
[0010] 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 (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 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 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 diseased tissue remaining typically regenerates,
causing the same symptoms which the surgery was meant to
alleviate.
[0011] 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.
[0012] Current therapeutic regimens include topical or
intralesional application of corticosteroids, topical
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 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.
[0013] 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 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 mucosal and submucosal
inflammation and edema, and 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 with fulminant IBD can
be severely ill with massive diarrhea, blood loss, dehydration,
weight loss and ever. The prognosis of the disease is not good and
frequently requires resection of the diseased tissue.
[0014] 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 to an initial tissue injury or insult. The original injury
may be chemically, mechanically, immunologically or biologically
related. Another object is to provide methods and compositions for
protecting tissue from the tissue destructive effects associated
with chronic inflammatory diseases, including arthritis (e.g.,
reheumatoid or osteoarthritis), psoriatic arthritis, psoriasis and
dermatitis, inflammatory bowel disease and other
autoimmune-diseases.
[0015] Another object of the invention is to provide methods and
compositions for enhancing the 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, such as
can occur upon initiation of blood flow after transplantation of
the organ in the recipient host.
[0016] 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 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, coronary occlusion or occlusive stroke, as
well as tissue damage associated with a surgical or other
aggressive clinical procedure. Still another object is to provide a
method for alleviating tissue damage associated with
hyperoxia-induced injury in a human following exposure to lethally
high oxygen concentrations.
[0017] 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.
[0018] These and other objects and features of the invention will
be apparent from the description, drawings and claims which
follow.
SUMMARY OF THE INVENTION
[0019] The present invention provides a method for alleviating the
tissue destructive effects associated 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 in anticipation of tissue
injury, sufficient to substantially inhibit or reduce the tissue
destructive effects of the inflammatory response.
[0020] In one aspect, the invention features compositions and
therapeutic treatment methods that comprise the step of
administering to a mammal a therapeutically effective amount of a
morphogenic protein ("morphogen"), as defined herein, upon injury
to a tissue, or in anticipation of such injury, for a time 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.
[0021] 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 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
inflammatory response, including repairing damaged 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.
[0022] As embodied herein, the term "ischemic-reperfusion injury"
refers to the initial damage associated with oxygen deprivation of
a cell and the subsequent damage 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% O.sub.2, including the tissue damage
associated with the inflammatory response to the initial toxically
high oxygen concentration. Accordingly, as used herein, "toxic
oxygen concentrations" refers to the tissue damage associated with
the injury induced by both lethally low oxygen concentrations of
oxygen (including the 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"
tissues or organs, as used herein, means protection from, reduction
of and/or elimination of reduced or lost tissue or organ function
as a result of tissue death, particularly immune cell-mediated
tissue death. "Transplanted" living tissue includes both tissue
transplants, (e.g., as in the case of bone marrow transplants, for
example), and tissue grafts. Finally, a "free oxygen radical
inhibiting agent" means a molecule capable of inhibiting the
release of and/or inhibiting the tissue damaging effects of free
oxygen radicals.
[0023] 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
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 and
organs, particularly transplanted tissues and organs, including
protecting these tissues and organs from ischemia-reperfusion
injury. In still another embodiment, the invention provides methods
for protecting tissues and organs from the tissue 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
autoimmune diseases, such as diabetes, multiple sclerosis,
amyotrophic lateral sclerosis (ALS), and other autoimmune
neurodegenerative diseases.
[0024] In one aspect of the invention, the morphogen 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 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 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 the circulatory system.
[0025] 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 tissues include tissue grafts
and transplanted tissue or organs, as well as any tissue or organ
about to undergo a surgical procedure or other clinical procedure
likely to either inhibit blood flow to the tissue or otherwise
induce an inflammatory response. 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.
[0026] 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, kidney, liver and pancreas transplants, as well as in
transplantation and/or grafting of skin, gastrointestinal mucosa,
bone marrow and other living tissues.
[0027] Where the patient suffers from a chronic inflammatory
disease, such as diabetes, arthritis, 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 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.
[0028] Among the morphogens useful in this invention are proteins
originally identified as osteogenic proteins, such as the OP-1,
OP-2 and CBMP2 proteins, as well as amino acid sequence-related
proteins such as DPP (from Drosophila), Vgl (from Xenopus), Vgr-1
(from mouse, see U.S. Pat. No. 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. ID No. 24, see
Wharton et al. (1991) PNAS 88:9214-9218.) The members of this
family, which include members of the TGF-.beta. super-family, of
proteins, share substantial-amino acid sequence homology in their
C-terminal regions. The proteins are translated as a precursor,
having an N-terminal signal peptide sequence, typically less than
about 30 residues, 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 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 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.
1TABLE I "OP-1" Refers generically to the group of morphogenically
active proteins expressed from part or all of a DNA sequence
encoding OP-1 protein, including allelic and species variants
thereof, e.g., human 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 conserved seven cysteine skeleton
is defined by residues 38 to 139 of Seq. ID Nos. 5 and 6. The cDNA
sequences and the amino acids encoding the full length proteins are
provided in Seq. Id Nos. 16 and 17 (hOP1) and Seq. ID Nos. 18 and
19 (mOP1.) The mature proteins are defined by residues 293-431
(hOP1) and 292-430 (mOP1). The "pro" regions of the proteins,
cleaved to yield the mature, morphogenically active proteins are
defined essentially by residues 30-292 (hOP1) and residues 30-291
(mOP1). "OP-2" refers generically to the group of active proteins
expressed from part or all of a DNA sequence encoding OP-2 protein,
including allelic and species variants thereof, e.g., human OP-2
("hOP-2", Seq. ID No. 7, mature protein amino acid sequence) or
mouse OP-2 ("mOP-2", Seq. ID No. 8, mature protein amino acid
sequence). The conserved seven cysteine skeleton is defined by
residues 38 to 139 of Seq. ID Nos. 7 and 8. The cDNA sequences and
the amino acids encoding the full length proteins are provided in
Seq. ID Nos. 20 and 21 (hOP2) and Seq. ID Nos. 22 and 23 (mOP2.)
The mature proteins are defined essentially by residues 264-402
(hOP2) and 261-399 (mOP2). The "pro" regions of the proteins,
cleaved to yield the mature, morphogenically active proteins likely
are defined essentially by residues 18-263 (hOP2) and residues
18-260 (mOP2). (Another cleavage site also occurs 21 residues
upstream for both OP-2 proteins.) "CBMP2" refers generically to the
morphogenically active proteins expressed from a DNA sequence
encoding the CBMP2 proteins, including allelic and species variants
thereof, e.g., human CBMP2A ("CBMP2A(fx)", Seq ID No. 9) or human
CBMP2B DNA ("CBMP2B(fx)", Seq. ID No. 10). The amino acid sequence
for the full length proteins, referred to in the literature as
BMP2A and BMP2B, or BMP2 and BMP4, appear in Wozney, et al. (1988)
Science 242: 1528-1534. The pro domain for BMP2 (BMP2A) likely
includes residues 25-248 or 25-282; the mature protein, residues
249-396 or 283-396. The pro domain for BMP4 (BMP2B) likely includes
residues 25-256 or 25-292; the mature protein, residues 257-408 or
293-408. "DPP(fx)" refers to protein sequences encoded by the
Drosophila DPP gene and defining the conserved seven cysteine
skeleton (Seq. ID No. 11). The amino acid sequence for the full
length protein appears in Padgett, et al (1987) Nature 325: 81-84.
The pro domain likely extends from the signal peptide cleavage site
to residue 456; the mature protein likely is defined by residues
457-588. "Vgl(fx)" refers to protein sequences encoded by the
Xenopus Vgl gene and defining the conserved seven cysteine skeleton
(Seq. ID No. 12). The amino acid sequence for the full length
protein appears in Weeks (1987) Cell 51: 861-867. The prodomain
likely extends from the signal peptide cleavage site to residue
246; the mature protein likely is defined by residues 247-360.
"Vgr-1(fx)" refers to protein sequences encoded by the murine Vgr-1
gene and defining the conserved seven cysteine skeleton (Seq. ID
No. 13). The amino acid sequence for the full length protein
appears in Lyons, et al, (1989) PNAS 86: 4554-4558. The prodomain
likely extends from the signal peptide cleavage site to residue
299; the mature protein likely is defined by residues 300-438.
"GDF-1(fx)" refers to protein sequences encoded by the human GDF-1
gene and defining the conserved seven cysteine skeleton (Seq. ID
No. 14). The cDNA and encoded amino sequence for the full length
protein is provided in Seq. ID. No. 32. The prodomain likely
extends from the signal peptide clavage site to residue 214; the
mature protein likely is defined by residues 215-372. "60A" refers
generically to the morphogenically active proteins expressed from
part or all of a DNA sequence (from the Drosophila 60A gene)
encoding the 60A proteins (see Seq. ID No. 24 wherein the cDNA and
encoded amino acid sequence for the full length protein is
provided). "60A(fx)" refers to the protein sequences defining the
conserved seven cysteine skeleton (residues 354 to 455 of Seq. ID
No. 24.) The prodomain likely extends from the signal peptide
cleavage site to residue 324; the mature protein likely is defined
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). The amino acid sequence for the full
length protein appears in Wozney et al. (1988) Science 242:
1528-1534. The pro domain likely extends from the signal peptide
cleavage site to residue 290; the mature protein likely is defined
by residues 291-472. "BMP5(fx)" refer to protein sequences encoded
by the human BMP5 gene and defining the conserved seven cysteine
skeleton (Seq. ID No. 27). The amino acid sequence for the full
length protein appears in Celeste, et al. (1991) PNAS 87:
9843-9847. The pro domain likely extends from the signal peptide
cleavage site to residue 316; the mature protein likely is defined
by residues 317-454. "BMP6(fx)" refers to protein sequences encoded
by the human BMP6 gene and defining the conserved seven cysteine
skeleton (Seq. ID No. 28). The amino acid sequence for the full
length protein appears in Celeste, et al. (1990) PNAS 87:
9843-5847. The pro domain likely includes extends from the signal
peptide cleavage site to residue 374; the mature sequence likely
includes residues 375-513.
[0029] The OP-2 proteins have an additional cysteine residue in
this region (e.g., see residue 41 of Seq. ID Nos. 7 and 8), in
addition to the conserved cysteine skeleton in common with the
other proteins in this family. The GDF-1 protein, has a four amino
acid insert within the conserved skeleton (residues 44-47 of Seq.
ID No. 14) but this insert likely does not interfere with the
relationship of the cysteines in the folded structure. In addition,
the CBMP2 proteins are missing one amino acid residue within the
cysteine skeleton.
[0030] 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., asheterodimers). Thus, as
defined herein, a morphogen is a dimeric protein comprising a pair
of polypeptide chains, wherein each polypeptide chain comprises at
least the C-terminal six cysteine skeleton defined by residues
43-139 of Seq. ID No. 5, including functionally equivalent
arrangements of these cysteines (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 polypeptide chains has the
appropriate three-dimensional structure, including the appropriate
intra-or inter-chain disulfide bonds such that the protein is
capable of acting as a morphogen as defined herein. Specifically,
the morphogens generally are capable of all of the following
biological functions in a morphogenically permissive environment:
stimulating proliferation of progenitor cells; stimulating the
differentiation of progenitor cells; stimulating the proliferation
of differentiated cells; and supporting the growth and maintenance
of differentiated cells, 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 conditions.
[0031] In one preferred aspect, the morphogens of this invention
comprise one of two species of generic amino acid sequences:
Generic Sequence 1 (Seq. ID No. 1) or Generic Sequence 2 (Seq. ID
No. 2); where each Xaa indicates one of the 20 naturally-occurring
L-isomer, .alpha.-amino acids or a derivative thereof. Generic
Sequence 1 comprises the conserved six cysteine skeleton and
Generic Sequence 2 comprises the conserved six cysteine skeleton
plus the additional cysteine identified in OP-2 (see residue 36,
Seq. ID No. 2). In another preferred aspect, these sequences
further comprise the following additional sequence at their
N-terminus:
2 Cys Xaa Xaa Xaa Xaa (Seq. ID No. 15) 1 5
[0032] Preferred amino acid sequences within the 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 among the various
preferred members of this morphogen family identified in Table II,
as well as the amino acid sequence variation among them.
Specifically, Generic Sequences 3 and 4 are composite amino acid
sequences of the following proteins presented in Table II and
identified in Seq. ID Nos. 5-14: human OP-1 (hOP-1, Seq. ID Nos. 5
and 0.16-17), 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 No. 11),
Vgl, (from Xenopus, Seq. ID No. 12), Vgr-1 (from mouse, Seq. ID No.
13), and GDF-1 (from mouse, Seq. ID No. 14.) The generic sequences
include both the amino acid identity shared by the sequences in
Table II, as well as alternative residues for the variable
positions within the sequence. Note that these generic sequences
allow for an additional cysteine at position 41 or 46 in Generic
Sequences 3 or 4, respectively, providing an appropriate cysteine
skeleton where inter- or intramolecular disulfide bonds can form,
and contain certain critical amino acids which influence the
tertiary structure of the proteins.
3 Generic Sequence 3 Leu Tyr Val Xaa Phe 1 5 Xaa Xaa Xaa Gly Trp
Xaa Xaa Trp Xaa 10 Xaa Ala Pro Xaa Gly Xaa Xaa Ala 15 20 Xaa Tyr
Cys Xaa Gly Xaa Cys Xaa 25 30 Xaa Pro Xaa Xaa Xaa Xaa Xaa 35 Xaa
Xaa Xaa Asn His Ala Xaa Xaa 40 45 Xaa Xaa Leu Xaa Xaa Xaa Xaa Xaa
50 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys 55 60 Cys Xaa Pro Xaa Xaa Xaa
Xaa Xaa 65 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
[0033] wherein each Xaa is independently selected from a group of
one or more specified amino acids defined as follows: "Res." means
"residue" and Xaa at res.4=(Ser, Asp or Glu); Xaa at res.6=(Arg,
Gln, Ser or Lys); Xaa at res.7=(Asp or Glu); Xaa at res.8=(Leu or
Val); Xaa at res.11=(Gln, Leu, Asp, His or Asn); Xaa at
res.12=(Asp, Arg or Asn); Xaa at res.14=(Ile or Val); Xaa at
res.15=(Ile or Val); Xaa at res.18=(Glu, Gln, Leu, Lys, Pro or
Arg); Xaa at res.20=(Tyr or Phe); Xaa at res.21=(Ala, Ser, Asp,
Met, His, Leu or Gln); Xaa at res.23=(Tyr, Asn or Phe); Xaa at
res.26=(Glu, His, Tyr, Asp or Gln); Xaa at res.28=(Glu, Lys, Asp or
Gln); Xaa at res.30=(Ala, Ser, Pro or Gln); Xaa at res.31=(Phe,
Leu- or Tyr); Xaa at res.33=(Leu or Val); Xaa at res.34=(Asn, Asp,
Ala or Thr); Xaa at res.35=(Ser, Asp, Glu, Leu or Ala); Xaa at
res.36=(Tyr, Cys, His, Ser or Ile); Xaa at res.37=(Met, Phe, Gly or
Leu); Xaa at res.38=(Asn or Ser); Xaa at res.39=(Ala, Ser or Gly);
Xaa at res.40=(Thr, Leu or Ser); Xaa at res.44=(Ile or Val); Xaa at
res.45=(Val or Leu); Xaa at res.46=(Gln or Arg); Xaa at
res.47=(Thr, Ala or Ser); Xaa at res.49=(Val or Met); Xaa at res.50
(His or Asn); Xaa at res.51=(Phe, Leu, Asn, Ser, Ala or Val); Xaa
at res.52=(Ile, Met, Asn, Ala or Val); Xaa at res.53=(Asn, Lys, Ala
or 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 Ile); 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=(Gln, 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=(Ile, Thr
or Val); Xaa at res.71=(Ser or Ala); Xaa at res.72=(Val or Met);
Xaa at res.74=(Tyr or Phe); Xaa at res.75=(Phe, Tyr or Leu); Xaa at
res.76=(Asp or Asn); Xaa at res.77=(Asp, Glu, Asn or Ser); Xaa at
res.78=(Ser, Gln, Asn or Tyr); Xaa at res.79=(Ser, Asn, Asp or
Glu); Xaa at res.80=(Asn, Thr or Lys); Xaa at res.82=(Ile or Val);
Xaa at res.84=(Lys or Arg); Xaa at res.85=(Lys, Asn, Gln or His);
Xaa at res.86=(Tyr or His); Xaa at res.87=(Arg, Gln 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);
4 Generic Sequence 4 Cys Xaa Xaa Xaa Xaa Leu Tyr Val Xaa Phe 1 5 10
Xaa Xaa Xaa Gly Trp Xaa Xaa Trp Xaa 15 Xaa Ala Pro Xaa Gly Xaa Xaa
Ala 20 25 Xaa Tyr Cys Xaa Gly Xaa Cys Xaa 30 35 Xaa Pro Xaa Xaa Xaa
Xaa Xaa 40 Xaa Xaa Xaa Asn His Ala Xaa Xaa 45 50 Xaa Xaa Leu Xaa
Xaa Xaa Xaa Xaa 55 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys 60 65 Cys Xaa
Pro Xaa Xaa Xaa Xaa Xaa 70 Xaa Xaa Xaa Leu Xaa Xaa Xaa 75 80 Xaa
Xaa Xaa Xaa Val Xaa Leu Xaa 85 Xaa Xaa Xaa Xaa Met Xaa Val Xaa 90
95 Xaa Cys Gly Cys Xaa 100
[0034] wherein each Xaa is independently selected from a group of
one or more specified amino acids as defined by the, following:
"Res." means "residue" and Xaa at res.2=(Lys or Arg); Xaa at
res.3=(Lys or Arg); Xaa at res.4=(His or Arg); Xaa at res.5=(Glu,
Ser, His, Gly, Arg or Pro); Xaa at res.9=(Ser, Asp or Glu); Xaa at
res.11=(Arg, Gln, Ser or Lys); Xaa at res.12=(Asp or Glu); Xaa at
res.13=(Leu or Val); Xaa at res.16=(Gln, Leu, Asp, His or Asn); Xaa
at res.17=(Asp, Arg, or Asn); Xaa at res.19=(Ile or Val); Xaa at
res.20=(Ile or Val); Xaa at res.23=(Glu, Gln, Leu, Lys, Pro or
Arg); Xaa at res.25=(Tyr or Phe); Xaa at res.26 (Ala, Ser, Asp,
Met, His, Leu, or Gln); Xaa at res.28=(Tyr, Asn or Phe); Xaa at
res.31=(Glu, His, Tyr, Asp or Gln); Xaa at res.33=Glu, Lys, Asp or
Gln); Xaa at res.35=(Ala, Ser or Pro); Xaa at res.36=(Phe, Leu or
Tyr); Xaa at res.38=(Leu or Val); Xaa at res.39=(Asn, Asp, Ala or
Thr); Xaa at res.40=(Ser, Asp, Glu, Leu or Ala); Xaa at
res.41=(Tyr, Cys, His, Ser or Ile); Xaa at res.42=(Met, Phe, Gly or
Leu); Xaa at res.44=(Ala, Ser or Gly); Xaa at res.45=(Thr, Leu or
Ser); Xaa at res.49=(Ile or Val); Xaa at res.50=(Val or Leu); Xaa
at res.51=(Gln 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=(Ile, 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 Ile); Xaa at res.63=(Pro or Asp); Xaa at
res.64=(Lys or Leu); Xaa at res.65=(Pro or Ala); Xaa at res.68=(Ala
or Val); Xaa at res.70=(Thr or Ala); Xaa at res.71=(Gln, Lys, Arg
or Glu); Xaa at res.72=(Leu, Met or Val); Xaa at res.73=(Asn, Ser
or Asp); Xaa at res.74=(Ala, Pro or Ser); Xaa at res.75=(Ile, Thr
or Val); Xaa at res.76=(Ser or Ala); Xaa at res.77=(Val or Met);
Xaa at res.79=(Tyr or Phe); Xaa at res.80=(Phe, Tyr or Leu); Xaa at
res.81=(Asp or Asn); Xaa at res.82=(Asp, Glu, Asn or Ser); Xaa at
res.83=(Ser, Gln, Asn or Tyr); Xaa at res.84=(Ser, Asn, Asp or
Glu); Xaa at res.85=(Asn, Thr or Lys); Xaa at res.87=(Ile or Val);
Xaa at res.89=(Lys or Arg); Xaa at res.90=(Lys, Asn, Gln or His);
Xaa at res.91=(Tyr or. His); Xaa at res.92=(Arg, Gln 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).
[0035] Similarly, Generic Sequence 5 (Seq. ID No. 30) and Generic
Sequence 6 (Seq. ID No. 31) accommodate the homologies shared among
all the morphogen protein family members identified in Table II.
Specifically, Generic Sequences 5 and 6 are composite amino acid
sequences of human OP-1 (hOP-1, Seq. ID Nos. 5 and 16-17), mouse
OP-1 (mOP-1, Seq. ID Nos. 6 and 18-19), human and mouse OP-2 (Seq.
ID Nos. 7, 8, and 20-22), CBMP2A (Seq. ID No. 9), CBMP2B (Seq. ID
No. 10), DPP (from Drosophila, Seq. ID No. 11), Vgl, (from Xenopus,
Seq. ID No. 12), Vgr-1 (from mouse, Seq. ID No. 13), and GDF-1
(from mouse, Seq. ID No. 14), human BMP3, (Seq. ID No. 26),
human-BMP5 (Seq. ID No. 27), human BMP6 (Seq. ID No. 28) and 60(A).
(from Drosophila, Seq. ID Nos. 24-25), The generic sequences
include both the amino acid identity shared by these sequences in
the C-terminal domain, defined by the six and seven cysteine
skeletons (Generic Sequences 5 and 6, respectively), as well as
alternative residues for the variable positions within the
sequence. As for Generic Sequences 3 and 4, Generic Sequences 5 and
6 allow for an additional cysteine at position 41 (Generic Sequence
5) or position 46, (Generic Sequence 6), providing an appropriate
cysteine skeleton where inter- or intramolecular disulfide bonds
can form, and containing certain critical amino acids which
influence the tertiary structure of the proteins.
5 Generic Sequence 5 Leu Xaa Xaa Xaa Phe 1 5 Xaa Xaa Xaa Gly Trp
Xaa Xaa Trp Xaa 10 Xaa Xaa Pro Xaa Xaa Xaa Xaa Ala 15 20 Xaa Tyr
Cys Xaa Gly Xaa Cys Xaa 25 30 Xaa Pro Xaa Xaa Xaa Xaa Xaa 35 Xaa
Xaa Xaa Asn His Ala Xaa Xaa 40 45 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
50 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys 55 60 Cys Xaa Pro Xaa Xaa Xaa
Xaa Xaa 65 Xaa Xaa Xaa Leu Xaa Xaa Xaa 70 75 Xaa Xaa Xaa Xaa Val
Xaa Leu Xaa 80 Xaa Xaa Xaa Xaa Met Xaa Val Xaa 85 90 Xaa Cys Xaa
Cys Xaa 95
[0036] wherein each Xaa is independently selected from a group of
one or more specified amino acids defined as follows: "Res." means
"residue" and Xaa at res.2=(Tyr or Lys); Xaa at res.3 Val or Ile);
Xaa at res.4=(Ser, Asp-or Glu); Xaa at res.6=(Arg, Gln, Ser, Lys or
Ala); Xaa at res.7=(Asp, Glu or Lys); Xaa at res.8=(Leu, Val or
Ile); Xaa at res.11=(Gln, Leu, Asp, His, Asn or Ser); Xaa at res.12
(Asp, Arg, Asn or Glu); Xaa at res.14=(Ile or Val); Xaa at res.15
(Ile or Val); Xaa at res.16 (Ala or Ser); Xaa at res.18=(Glu, Gln,
Leu, Lys, Pro or Arg); Xaa at res.19 (Gly or Ser), Xaa at
res.20=(Tyr or Phe); Xaa at res.21=(Ala, Ser, Asp, Met, His, Gln,
Leu or Gly); Xaa at res.2-3 (Tyr, Asn or Phe); Xaa at res.26=(Glu,
His, Tyr, Asp, Gln or Ser); Xaa at res.28 (Glu, Lys, Asp, Gln or
Ala); Xaa at res.30=(Ala, Ser, Pro, Gln or Asn); Xaa at
res.31=(Phe, Leu or Tyr); Xaa at res.33=(Leu, Val or Met); Xaa at
res.34=(Asn, Asp, Ala, Thr or Pro); Xaa at res.35=(Ser, Asp, Glu,
Leu, Ala or Lys); Xaa at res.36=(Tyr, Cys, His, Ser or Ile); Xaa at
res.37=(Met, Phe, Gly or Leu); Xaa at res.38=(Asn, Ser or Lys); Xaa
at res.39=(Ala, Ser, Gly or Pro); Xaa at res.40=(Thr, Leu or Ser);
Xaa at res.44=(Ile, Val or Thr); Xaa at res.45=(Val, Leu or Ile);
Xaa at res.46=(Gln or Arg); Xaa at res.47=(Thr, Ala or Ser); Xaa at
res.48=(Leu or Ile); Xaa at res.49=(Val or Met); Xaa at
res.50=(His, Asn or Arg); Xaa at res.51=(Phe, Leu, Asn, Ser, Ala or
Val); Xaa at res.52=(Ile, Met, Asn, Ala, Val or Leu); Xaa at
res.53=(Asn, Lys, Ala, Glu, Gly or Phe); Xaa at res.54=(Pro, Ser or
Val); Xaa at res.55=(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 Ile); Xaa at res.58 (Pro or Asp); Xaa at
res.59=(Lys, Leu or Glu); Xaa at res.60=(Pro or Ala); Xaa at res.63
(Ala or Val); Xaa at res.65=(Thr, Ala or Glu); Xaa at res.66=(Gln,
Lys, Arg or Glu); Xaa at res.67=(Leu, Met or Val); Xaa at
res.68=(Asn, Ser, Asp or Gly); Xaa at res.69 (Ala, Pro or Ser); Xaa
at res.70=(Ile, Thr, Val or Leu); Xaa at res.71=(Ser, Ala or Pro);
Xaa at res.72=(Val, Met or Ile); Xaa at res.74=(Tyr or Phe); Xaa at
res.75=(Phe, Tyr, Leu or His); Xaa at res.76=(Asp, Asn or Leu); Xaa
at res.77=(Asp, Glu, Asn or Ser); Xaa at res.78=(Ser, Gln, Asn, Tyr
or Asp); Xaa at res.79=(Ser, Asn, Asp, Glu or Lys); Xaa at
res.80=(Asn, Thr or Lys); Xaa at res.82=(Ile, Val or Asn); Xaa at
res.84=(Lys or Arg); Xaa at res.85=(Lys, Asn, Gln, His or Val); Xaa
at res.86=(Tyr-or His); Xaa at res.87=(Arg, Gln, Glu or Pro); Xaa
at res.88=(Asn, Glu or Asp); Xaa at res.90=(Val, Thr, Ala or Ile);
Xaa at res.92=(Arg, Lys, Val, Asp or Glu); Xaa at res.93=(Ala, Gly,
Glu or Ser); Xaa at res.95=(Gly or Ala) and Xaa at res.97 (His or
Arg).
6 Generic Sequence 6 Cys Xaa Xaa Xaa Xaa Leu Xaa Xaa Xaa Phe 1 5 10
Xaa Xaa Xaa Gly Trp Xaa Xaa Trp Xaa 15 Xaa Xaa Pro Xaa Xaa Xaa Xaa
Ala 20 25 Xaa Tyr Cys Xaa Gly Xaa Cys Xaa 30 35 Xaa Pro Xaa Xaa Xaa
Xaa Xaa 40 Xaa Xaa Xaa Asn His Ala Xaa Xaa 45 50 Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa 55 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys 60 65 Cys Xaa
Pro Xaa Xaa Xaa Xaa Xaa 70 Xaa Xaa Xaa Leu Xaa Xaa Xaa 75 80 Xaa
Xaa Xaa Xaa Val Xaa Leu Xaa 85 Xaa Xaa Xaa Xaa Met Xaa Val Xaa 90
95 Xaa Cys Xaa Cys Xaa 100
[0037] wherein each Xaa is independently selected from a group of
one or more specified amino acids as defined by the following:
"Res." means "residue" and Xaa at res.2=(Lys, Arg, Ala or Gln); Xaa
at res.3=(Lys, Arg or Met); Xaa at res.4=(His, Arg or Gln); Xaa at
res.5=(Glu, Ser, His, Gly, Arg, Pro, Thr, or Tyr); Xaa at
res.7=(Tyr or Lys); Xaa at res.8=(Val or Ile); Xaa at res.9=(Ser,
Asp or Glu); Xaa at res.11=(Arg, Gln, Ser, Lys or Ala); Xaa at
res.12=(Asp, Glu, or Lys); Xaa at res.13=(Leu, Val or Ile); Xaa at
res.16=(Gln, Leu, Asp, His, Asn or Ser); Xaa at res.17=(Asp, Arg,
Asn or Glu); Xaa at res.19 (Ile or Val); Xaa at res.20=(Ile or
Val); Xaa at res.21=(Ala or Ser); Xaa at res.23=(Glu, Gln, Leu,
Lys, Pro or Arg); Xaa at res.24=(Gly or Ser); Xaa at res.25=(Tyr or
Phe); Xaa at res.26=(Ala, Ser, Asp, Met, His, Gln, Leu, or Gly);
Xaa at res.28=(Tyr, Asn or Phe); Xaa at res.31=(Glu, His, Tyr, Asp,
Gln or Ser); Xaa at res.33=Glu, Lys, Asp, Gln or Ala); Xaa at
res.35=(Ala, Ser. Pro, Gln or Asn); Xaa at res.36=(Phe, Leu or
Tyr); Xaa at res.38=(Leu, Val or Met); Xaa at res.39=(Asn, Asp,
Ala, Thr or Pro); Xaa at res.40=(Ser, Asp, Glu, Leu, Ala or Lys);
Xaa at res.41=(Tyr, Cys, His, Ser or Ile); Xaa at res.42=(Met, Phe,
Gly or Leu); Xaa at res.43=(Asn, Ser or Lys); Xaa at res.44=(Ala,
Ser. Gly or Pro); Xaa at res.45=(Thr, Leu or Ser); Xaa at
res.49=(Ile, Val or Thr); Xaa at res.50=(Val, Leu or Ile); Xaa at
res.51=(Gln or Arg); Xaa at res.52=(Thr, Ala or Ser); Xaa at
res.53=(Leu or Ile); Xaa at res.54=(Val or Met); Xaa at
res.55=(His, Asn or Arg); Xaa at res.56=(Phe, Leu, Asn, Ser, Ala or
Val); Xaa at res.57=(Ile, Met, Asn, Ala, Val or Leu); Xaa at
res.58=(Asn, Lys, Ala, 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=(Thr, Ala, Val, Lys, Asp, Tyr, Ser, Ala, Pro or His); Xaa at
res.62=(Val, Ala or Ile); Xaa at res.63=(Pro or Asp); Xaa at
res.64=(Lys, Leu or 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=(Gln, Lys, Arg or Glu); Xaa at res.72=(Leu, Met or Val); Xaa
at res.73=(Asn, Ser, Asp or Gly); Xaa at res.74=(Ala, Pro or Ser);
Xaa at res.75=(Ile, Thr, Val or Leu); Xaa at res.76=(Ser, Ala or
Pro); Xaa at res.77=(Val, Met or Ile); Xaa at res.79=(Tyr or Phe);
Xaa at res.80=(Phe, Tyr, Leu or His); Xaa at res.81=(Asp, Asn or
Leu); Xaa at res.82=(Asp, Glu, Asn or Ser); Xaa at res.83=(Ser,
Gln, Asn, Tyr or Asp); Xaa at res.84=(Ser, Asn, Asp, Glu or Lys);
Xaa at res.85=(Asn, Thr or Lys); Xaa at res.87=(Ile, Val or Asn);
Xaa at res.89=(Lys or Arg); Xaa at res.90=(Lys, Asn, Gln, His or
Val); Xaa at res.91=(Tyr or His); Xaa at res.92=(Arg, Gln, Glu or
Pro); Xaa at res.93=(Asn, Glu or Asp); Xaa at res.95=(Val, Thr, Ala
or Ile); Xaa at res.97-=(Arg, Lys, Val, Asp or Glu); Xaa at
res.98=(Ala, Gly, Glu or Ser); Xaa at res.100=(Gly or Ala); and Xaa
at res.102=(His or Arg).
[0038] Particularly-useful sequences for use as morphogens in this
invention include the C-terminal domains, e.g., the C-terminal
96-102-amino acid residues of Vgl, Vgr-1, DPP, OP-1, OP-2, CBMP-2A,
CBMP-2B, GDF-1 (see Table II, below, and Seq. ID Nos. 5-14), as
well as proteins comprising the C-terminal domains of 60A, BMP3,
BMP5 and BMP6 (see Seq. ID Nos. 24-28), all of which include at
least the conserved six or seven cysteine skeleton. In addition,
biosynthetic constructs designed from the generic sequences, such
as COP-1,3-5, 7, 16; disclosed in U.S. Pat. No. 5,011,691, also are
useful. Other sequences include the inhibins/activin proteins (see,
for example, U.S. Pat. Nos. 4,968,590 and 5,011,691). Accordingly,
other useful sequences are those sharing at least 70% amino acid
sequence homology or "similarity", and preferably 80% homology or
similarity with any of the sequences above. These are anticipated
to include allelic and species variants and mutants, and
biosynthetic muteins, as well as novel members of this morphogenic
family of proteins. Particularly envisioned in the family of
related proteins are those proteins exhibiting morphogenic activity
and wherein the amino acid changes from the preferred sequences
include conservative changes, e.g., those as defined by Dayoff et
al., Atlas of Protein Sequence and Structure; vol. 5 Suppl. 3, pp.
345-362, (M. O. Dayoff, ed., Nat'l BioMed. Research Fdn.,
Washington, D.C. 1979). As used herein, potentially useful
sequences are aligned with a known morphogen sequence using the
method of Needleman et al. ((1970) J. Mol. Biol. 48:443-453) and
identities calculated by the Align program (DNAstar, Inc.).
"Homology" or "similarity" as used herein includes allowed
conservative changes as defined by Dayoff et al.
[0039] The currently most preferred protein sequences useful as
morphogens in this invention include those having greater than 60%
identity, preferably greater than 65% identity, with the amino acid
sequence defining the conserved six cysteine skeleton of hOP1
(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 preferred aspect of the invention, useful morphogens
include active proteins comprising species of polypeptide chains
having the generic amino acid sequence herein referred to as "OPX",
which accommodates the homologies between the various identified
species of OP1 and OP2 (Seq. ID No. 29).
[0040] The morphogens useful in the methods, composition and
devices of this invention include proteins comprising any of the
polypeptide chains described above, whether isolated from naturally
occurring sources, or produced by recombinant DNA or other
synthetic techniques, and includes allelic and species variants of
these proteins, naturally occurring or biosynthetic mutants
thereof, as well as various truncated and fusion constructs.
Deletion or addition mutants also are envisioned to be active,
including those which may alter the conserved C-terminal cysteine
skeleton, provided that the alteration does not functionally
disrupt the relationship of these cysteines in the folded
structure. Accordingly, such active forms are considered the
equivalent of the specifically described constructs disclosed
herein. The proteins may include forms having varying glycosylation
patterns, varying N-terminal family of related proteins having
regions of amino acid sequence homology, and active truncated or
mutated forms of native or biosynthetic proteins, produced by
expression of recombinant DNA in host cells.
[0041] The morphogenic proteins can be expressed from intact or
truncated cDNA or from synthetic DNAs in procaryotic or eucaryotic
host cells, and purified, cleaved, refolded, and dimerized to form
morphogenically active compositions. Currently preferred host cells
include E. coli or mammalian cells, such as CHO, COS or BSC cells.
A detailed description of the morphogens useful in the methods,
compositions and devices of this invention is disclosed in
copending U.S. patent application Ser. No. 752,764, filed Aug. 30,
1991, and Ser. No. 667,274, filed Mar. 11, 1991, the disclosure of
which are incorporated herein by reference.
[0042] Thus, in view of this disclosure, skilled genetic engineers
can isolate genes from cDNA or genomic libraries of various
different species which encode appropriate amino acid sequences, or
construct DNAs from oligonucleotides, and then can express them in
various types of host cells, including both procaryotes and
eucaryotes, to produce large quantities of active proteins capable
of protecting tissues and organs from immune cell-mediated tissue
destruction, including substantially inhibiting such damage and/or
regenerating the damaged tissue in a variety of mammals, including
humans.
[0043] The foregoing and other objects, features and advantages of
the present invention will be made more apparent from the following
detailed description of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0044] FIG. 1 shows the cardioprotective effects of morphogen
(hOP1) in a rat myocardial ischemia-reperfusion model, as evidenced
by the smaller loss of myocardial creatine kinase in hOP1-treated
rats;
[0045] FIG. 2 shows the effects of 20 .mu.g of morphogen (hOP1
given 24 hours prior to isolation of rat heart on
endothelial-dependent vasorelaxation to acetycholine following
induced ischemia-reperfusion injury;
[0046] FIG. 3 shows the effect of morphogen (hOP1) on neutrophil
adherence to LTB.sub.4-stimulated mesenteric artery endothelium in
neutrophil-activated rats;
[0047] FIGS. 4(A and B) are schematic representations of morphogen
inhibition of early mononuclear phagocytic multinuclearization in
vivo;
[0048] FIG. 5 graphs the effect of a morphogen (e.g., OP-1) and a
placebo control on mucositic lesion formation; and
[0049] FIG. 6(A-D) graphs the effects of a morphogen (eg., OP-1,
FIGS. 6A and 6C) and TGF-.beta. (FIGS. 6B and 6D) on collagen (6A
and 6B) and hyaluronic acid (6C and 6D) production in primary
fibroblast cultures.
DETAILED DESCRIPTION OF THE INVENTION
[0050] 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.
[0051] When tissue injury occurs, whether caused by bacteria,
trauma, chemicals, heat, or any other phenomenon, the body's
inflammatory response is 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 (through the
release of killing substances such as superoxides, performs, and
other antimicrobial agents stored in granules), remove the dead
tissues and organisms (through phagocytosis), release various
biological response modifiers that promote rapid 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 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 multiplying infectious
agents, act to the detriment of 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 resulting scar
tissue that often forms can interfere with normal tissue function
as occurs, for example, in idiopathic pulmonary fibrosis, IBD and
organ cirrhosis.
[0052] The vascular endothelium constitutes the first 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 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 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 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 extravascularization,
the method also prevents the 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 extravascular sites of recent tissue destruction,
but 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.
[0053] One source of tissue damage follows cell exposure to toxic
oxygen concentrations, such as the tissue damage following
ischemic-reperfusion tissue injury (oxygen deprivation), and
following hyperoxia injury (lethally 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 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 or
unavoidably induced, as by a surgical or clinical procedure, the
morphogen preferably is administered prior to induction.
[0054] In addition, the morphogens described herein, in contrast to
fibrogenic growth factors such as TGF-.beta., stimulate tissue
morphogenesis and do not stimulate 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 stimulating the regeneration of
the damaged tissue and preventing fibrogenesis.
[0055] 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 other inflammatory diseases that involve epithelial
cell populations.
[0056] Provided below are detailed descriptions of suitable
morphogens useful in the methods and compositions of this
invention, as well as methods for their administration and
application, and numerous, nonlimiting examples which 1) illustrate
the suitability of the morphogens and morphogen-stimulating agents
described herein as therapeutic agents for 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.
[0057] I. Useful Morphogens
[0058] As defined herein a protein is morphogenic if it is capable
of inducing the developmental cascade of cellular and molecular
events that culminate in the formation of new, organ-specific
tissue and comprises at least the conserved C-terminal six cysteine
skeleton or its functional equivalent (see supra).
[0059] Specifically, the morphogens generally are capable of all of
the following biological functions in a morphogenically permissive
environment: stimulating proliferation of progenitor cells;
stimulating the differentiation of progenitor cells; stimulating
the proliferation of differentiated cells; and supporting the
growth and maintenance of differentiated cells, including the
"redifferentiation" of transformed cells. Details of how the
morphogens useful in the method of this invention first were
identified, as well as a description on how to make, use and test
them for morphogenic activity are disclosed in U.S. Ser. No.
667,274, filed Mar. 11, 1991 and U.S. Ser. No. 752,764, filed Aug.
30, 1991, the disclosures of which are hereinabove incorporated by
reference. As disclosed therein, the 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 disclosed therein.
[0060] Particularly useful proteins include those which comprise
the naturally derived sequences disclosed in Table II. Other useful
sequences include biosynthetic constructs such as those disclosed
in U.S. Pat. No. 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).
[0061] 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 described above, where "homology" is as defined herein
above.
[0062] The morphogens useful in the method of this invention also
can be described by any of the 6 generic sequences described herein
(Generic Sequences 1, 2, 3, 4, 5 and 6). Generic sequences 1 and 2
also may include, at their N-terminus, the sequence
7 Cys Xaa Xaa Xaa Xaa (Seq. ID No. 15) 1 5
[0063] Table II, set forth below, compares the amino acid sequences
of the active regions of native proteins that have been identified
as morphogens, including human OP-1 (hOP-1, Seq. ID Nos. 5 and
16-17), mouse OP-1 (mOP-1, Seq. ID Nos. 6 and 18-19), human and
mouse OP-2 (Seq. ID Nos. 7, 8, and 20-23), CBMP2A (Seq. ID No. 9),
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 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 the table, three dots indicates that the
amino acid in that position is the same as the amino acid in hOP-1.
Three dashes indicates that no amino acid is present in that
position, and are included for purposes of illustrating homologies.
For example, amino acid residue 60 of CBMP-2A and CBMP-2B is
"missing". Of course, both these amino acid sequences in this
region comprise Asn-Ser (residues 58, 59), with CBMP-2A then
comprising Lys and Ile, whereas CBMP-2B comprises Ser and Ile.
8TABLE II hOP-1 Cys Lys Lys His Glu Leu Tyr Val mOP-1 ... ... ...
... ... ... ... ... hOP-2 ... Arg Arg ... ... ... ... ... mOP-2 ...
Arg Arg ... ... ... ... ... DPP ... Arg Arg ... Ser ... ... ... Vgl
... ... Lys Arg His ... ... ... Vgr-1 ... ... ... ... Gly ... ...
... CBMP-2A ... ... Arg ... Pro ... ... ... CBMP-2B ... Arg Arg ...
Ser ... ... ... BMP3 ... Ala Arg Arg Tyr ... Lys ... GDF-1 ... Arg
Ala Arg Arg ... ... ... 60A ... Gln Met Glu Thr ... ... ... BMP5
... ... ... ... ... ... ... ... BMP6 ... Arg ... ... ... ... ...
... 1 5 hOP-1 Ser Phe Arg Asp Leu Gly Trp Gln Asp mOP-1 ... ... ...
... ... ... ... ... ... hOP-2 ... ... Gln ... ... ... ... Leu ...
mOP-2 Ser ... ... ... ... ... ... Leu ... DPP Asp ... Ser ... Val
... ... Asp ... Vgl Glu ... Lys ... Val ... ... ... Asn Vgr-1 ...
... Gln ... Val ... ... ... ... CBMP-2A Asp ... Ser ... Val ... ...
Asn ... CBMP-2B Asp ... Ser ... Val ... ... Asn ... BMP3 Asp ...
Ala ... Ile ... ... Ser Glu GDF-1 ... ... ... Glu Val ... ... His
Arg 60A Asp ... Lys ... ... ... ... His ... BMP5 ... ... ... ...
... ... ... ... ... BMP6 ... ... Gln ... ... ... ... ... ... 10 15
hOP-1 Trp Ile Ile Ala Pro Glu Gly Tyr Ala mOP-1 ... ... ... ... ...
... ... ... ... hOP-2 ... Val ... ... ... Gln ... ... Ser mOP-2 ...
Val ... ... ... Gln ... ... Ser DPP ... ... Val ... ... Leu ... ...
Asp Vgl ... Val ... ... ... Gln ... ... Met Vgr-1 ... ... ... ...
... Lys ... ... ... CBMP-2A ... ... Val ... ... Pro ... ... His
CBMP-2B ... ... Val ... ... Pro ... ... Gln BMP3 ... ... ... Ser
... Lys Ser Phe Asp GDF-1 ... Val ... ... ... Arg ... Phe Leu 60A
... ... ... ... ... ... ... ... Gly BMP5 ... ... ... ... ... ...
... ... ... BMP6 ... ... ... ... ... Lys ... ... ... 20 25 hOP-1
Ala Tyr Tyr Cys Glu Gly Glu Cys Ala mOP-1 ... ... ... ... ... ...
... ... ... hOP-2 ... ... ... ... ... ... ... ... Ser mOP-2 ... ...
... ... ... ... ... ... ... DPP ... ... ... ... His ... Lys ... Pro
Vgl ... Asn ... ... Tyr ... ... ... Pro Vgr-1 ... Asn ... ... Asp
... ... ... Ser CBMP-2A ... Phe ... ... His ... Glu ... Pro CBMP-2B
... Phe ... ... His ... Asp ... Pro BMP3 ... ... ... ... Ser ...
Ala ... Gln GDF-1 ... Asn ... ... Gln ... Gln ... ... 60A ... Phe
... ... Ser ... ... ... Asn BMP5 ... Phe ... ... Asp ... ... ...
Ser BMP6 ... Asn ... ... Asp ... ... ... Ser 30 35 hOP-1 Phe Pro
Leu Asn Ser Tyr Met Asn Ala mOP-1 ... ... ... ... ... ... ... ...
... hOP-2 ... ... ... Asp ... Cys ... ... ... mOP-2 ... ... ... Asp
... Cys ... ... ... DPP ... ... ... Ala Asp His Phe ... Ser Vgl Tyr
... ... Thr Glu Ile Leu ... Gly Vgr-1 ... ... ... ... Ala His ...
... ... CBMP-2A ... ... ... Ala Asp His Leu ... Ser CBMP-2B ... ...
... Ala Asp His Leu ... Ser GDF-1 Leu ... Val Ala Leu Ser Gly Ser**
... BMP3 ... ... Met Pro Lys Ser Leu Lys Pro 60A ... ... ... ...
Ala His ... ... ... BMP5 ... ... ... ... Ala His Met ... ... BMP6
... ... ... ... Ala His Met ... ... 40 hOP-1 Thr Asn His Ala Ile
Val Gln Thr Leu mOP-1 ... ... ... ... ... ... ... ... ... hOP-2 ...
... ... ... ... Leu ... Ser ... mOP-2 ... ... ... ... ... Leu ...
Ser ... DPP ... ... ... ... Val ... ... ... ... Vgl Ser ... ... ...
... Leu ... ... ... Vgr-1 ... ... ... ... ... ... ... ... ...
CBMP-2A ... ... ... ... ... ... ... ... ... CBMP-2B ... ... ... ...
... ... ... ... ... BMP3 Ser ... ... ... Thr Ile ... Ser Ile GDF-1
Leu ... ... ... Val Leu Arg Ala ... 60A ... ... ... ... ... ... ...
... ... BMP5 ... ... ... ... ... ... ... ... ... BMP6 ... ... ...
... ... ... ... ... ... 45 50 hOP-1 Val His Phe Ile Asn Pro Glu Thr
Val mOP-1 ... ... ... ... ... ... Asp ... ... hOP-2 ... His Leu Met
Lys ... Asn Ala ... mOP-2 ... His Leu Met Lys ... Asp Val ... DPP
... Asn Asn Asn ... ... Gly Lys ... Vgl ... ... Ser ... Glu ... ...
Asp Ile Vgr-1 ... ... Val Met ... ... ... Tyr ... CBMP-2A ... Asn
Ser Val ... Ser ... Lys Ile CBMP-2B ... Asn Ser Val ... Ser ... Ser
Ile BMP3 ... Arg Ala** Gly Val Val Pro Gly Ile GDF-1 Met ... Ala
Ala Ala ... Gly Ala Ala 60A ... ... Leu Leu Glu ... Lys Lys ...
BMP5 ... ... Leu Met Phe ... Asp His ... BMP6 ... ... Leu Met ...
... ... Tyr ... 55 60 hOP-1 Pro Lys Pro Cys Cys Ala Pro Thr Gln
mOP-1 ... ... ... ... ... ... ... ... ... hOP-2 ... ... Ala ... ...
... ... ... Lys mOP-2 ... ... Ala ... ... ... ... ... Lys DPP ...
... Ala ... ... Val ... ... ... Vgl ... Leu ... ... ... Val ... ...
Lys Vgr-1 ... ... ... ... ... ... ... ... Lys CBMP-2A ... ... Ala
... ... Val ... ... Glu CBMP-2B ... ... Ala ... ... Val ... ... Glu
BMP3 ... Glu ... ... ... Val ... Glu Lys GDF-1 Asp Leu ... ... ...
Val ... Ala Arg 60A ... ... ... ... ... ... ... ... Arg BMP5 ...
... ... ... ... ... ... ... Lys BMP6 ... ... ... ... ... ... ...
... Lys 65 70 hOP-1 Leu Asn Ala Ile Ser Val Leu Tyr Phe mOP-1 ...
... ... ... ... ... ... ... ... hOP-2 ... Ser ... Thr ... ... ...
... Tyr mOP-2 ... Ser ... Thr ... ... ... ... Tyr Vgl Met Ser Pro
... ... Met ... Phe Tyr Vgr-1 Val ... ... ... ... ... ... ... ...
DPP ... Asp Ser Val Ala Met ... ... Leu CBMP-2A ... Ser ... ... ...
Met ... ... Leu CBMP-2B ... Ser ... ... ... Met ... ... Leu BMP3
Met Ser Ser Leu ... Ile ... Phe Tyr GDF-1 ... Ser Pro ... ... ...
... Phe ... 60A ... Gly ... Leu Pro ... ... ... His BMP5 ... ...
... ... ... ... ... ... ... BMP6 ... ... ... ... ... ... ... ...
... 75 80 hOP-1 Asp Asp Ser Ser Asn Val Ile Leu Lys mOP-1 ... ...
... ... ... ... ... ... ... hOP-2 ... Ser ... Asn ... ... ... ...
Arg mOP-2 ... Ser ... Asn ... ... ... ... Arg DPP Asn ... Gln ...
Thr ... Val ... ... Vgl ... Asn Asn Asp ... ... Val ... Arg Vgr-1
... ... Asn ... ... ... ... ... ... CBMP-2A ... Glu Asn Glu Lys ...
Val ... ... CBMP-2B ... Glu Tyr Asp Lys ... Val ... ... BMP3 ...
Glu Asn Lys ... ... Val ... ... GDF-1 ... Asn ... Asp ... ... Val
... Arg 60A Leu Asn Asp Glu ... ... Asn ... ... BMP5 ... ... ...
... ... ... ... ... ... BMP6 ... ... Asn ... ... ... ... ... ... 85
hOP-1 Lys Tyr Arg Asn Met Val Val Arg mOP-1 ... ... ... ... ... ...
... ... hOP-2 ... His ... ... ... ... ... Lys mOP-2 ... His ... ...
... ... ... Lys DPP Asn ... Gln Glu ... Thr ... Val Vgl His ... Glu
... ... Ala ... Asp Vgr-1 ... ... ... ... ... ... ... ... CBMP-2A
Asn ... Gln Asp ... ... ... Glu CBMP-2B Asn ... Gln Glu ... ... ...
Glu BMP3 Val ... Pro ... ... Thr ... Glu GDF-1 Gln ... Glu Asp ...
... ... Asp 60A ... ... ... ... ... Ile ... Lys BMP5 ... ... ...
... ... ... ... ... BMP6 ... ... ... Trp ... ... ... ... 90 95
hOP-1 Ala Cys Gly Cys His mOP-1 ... ... ... ... ... hOP-2 ... ...
... ... ... mOP-2 ... ... ... ... ... DPP Gly ... ... ... Arg Vgl
Glu ... ... ... Arg Vgr-1 ... ... ... ... ... CBMP-2A Gly ... ...
... Arg CBMP-2B Gly ... ... ... Arg BMP3 Ser ... Ala ... Arg GDF-1
Glu ... ... ... Arg 60A Ser ... ... ... ... BMP5 Ser ... ... ...
... BMP6 ... ... ... ... ... 100 **Between residues 56 and 57 of
BMP3 is a Val residue; between residues 43 and 44 of GDF-1 lies the
amino acid sequence Gly--Gly-Pro--Pro.
[0064] As is apparent from the foregoing amino acid sequence
comparisons, significant amino acid changes can be made within the
generic sequences while retaining the morphogenic activity. For
example, while the GDF-1 protein sequence depicted in Table II
shares only about 50% amino acid identity with the hOP1 sequence
described therein, the GDF-1 sequence shares greater than 70% amino
acid sequence homology (or "similarity") with the hOP1 sequence,
where "homology" or "similarity" includes allowed conservative
amino acid changes within the sequence as defined by Dayoff, et
al., Atlas of Protein Sequence and Structure vol.5, supp.3, pp.
345-362, (M. O. Dayoff, ed., Nat'l BioMed. Res. Fd'n, Washington
D.C. 1979.)
[0065] The currently most preferred protein sequences useful as
morphogens in this invention include those having greater than 60%
identity, preferably greater than 65% identity, with the amino acid
sequence defining the conserved six cysteine skeleton of hOP1
(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 comprising species of polypeptide chains having the
generic amino acid sequence referred to herein as "OPX", which
defines the seven cysteine skeleton and accommodates the identities
between the various identified mouse and human OP1 and OP2
proteins. OPX is presented in Seq. ID No. 29. As described therein,
each Xaa at a given position independently is selected from the
residues occurring at the corresponding position in the C-terminal
sequence of mouse or human OP1 or OP2 (see Seq. ID Nos. 5-8 and/or
Seq. ID Nos. 16-23).
[0066] II. Formulations and Methods for Administering Therapeutic
Agents
[0067] The morphogens may be provided to an individual by 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,
intraorbital, ophthalmic, intraventricular, intracranial,
intracapsular, intraspinal, intracisternal, intraperitoneal,
buccal, rectal, vaginal, intranasal or by aerosol administration,
the morphogen preferably comprises part of an aqueous solution. The
solution is physiologically acceptable so that in addition to
delivery of the desired morphogen to the patient, the solution does
not otherwise adversely affect the patient's electrolyte and volume
balance. The aqueous medium for the morphogen thus may comprise
normal physiologic saline (9.85% NaCl, 0.15M), pH 7-7.4. The
aqueous solution containing the morphogen can be made, for example,
by dissolving the protein in 50% ethanol containing acetonitrile in
0.1% trifluoroacetic acid (TFA) or 0.1% HCl, or equivalent
solvents. One volume of the resultant solution then is added, for
example, to ten volumes of phosphate buffered saline (PBS), which
further may include 0.1-0.2% human serum albumin (HSA). The
resultant solution preferably is vortexed extensively. If desired,
a given morphogen may be made more soluble by association with a
suitable molecule. For example, association of the mature dimer
with the pro domain of the morphogen keeps the morphogen soluble in
physiological buffers. In fact, the endogenous protein is thought
to be transported in this form. Another molecule capable of
enhancing solubility and particularly useful for oral
administrations, is casein. For example, addition of 0.2% casein
increases solubility of the mature active form of OP-1 by 80%.
Other components found in milk and/or various serum proteins also
may be useful.
[0068] Useful solutions for parenteral administration may be
prepared by any of the methods well known in the pharmaceutical
art, described, for example, in Remington's Pharmaceutical Sciences
(Gennaro, A., ed.), Mack Pub., 1990. Formulations may include, for
example, polyalkylene glycols such as polyethylene glycol, oils of
vegetable origin, hydrogenated naphthalenes, and the like.
Formulations for direct administration, in particular, may include
glycerol and other compositions of high viscosity to help maintain
the morphogen at the desired locus. Biocompatible, preferably
bioresorbable, polymers, including, for 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 delivery systems for these morphogens
include ethylene-vinyl acetate copolymer particles, osmotic pumps,
implant able infusion systems, and liposomes. Formulations for
inhalation administration contain as excipients, for example,
lactose, or may be aqueous solutions containing, for example,
polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or
oily solutions for administration in the form of nasal drops, or as
a gel to be applied intranasally. Formulations for parenteral
administration may also include glycocholate for buccal
administration, methoxysalicylate for rectal administration, or
cutric acid for vaginal administration.
[0069] Suppositories for rectal administration also may be prepared
by mixing the morphogen or morphogen-stimulating agent with a
non-irritating excipient such as cocoa butter or other compositions
which are-solid at room temperature and liquid at body
temperatures.
[0070] Formulations for topical administration to the skin surface
may be prepared by dispersing the morphogen or
morphogen-stimulating agent with a dermatologically acceptable
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
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
pectin-containing formulations may be used.
[0071] Alternatively, the morphogens described herein may be
administered orally. Oral administration of proteins as
therapeutics generally is not practiced as most proteins are
readily degraded by digestive enzymes and acids in the mammalian
digestive system before they can be absorbed into the bloodstream.
However, the morphogens described herein typically are acid stable
and protease-resistant (see, for example, U.S. Pat. No. 4,968,590.)
In addition, at least one morphogen, OP-1, has been identified in
mammary-gland extract, colostrum and 57-day milk. Moreover, the
OP-1 purified from mammary gland extract is morphogenically active.
Specifically, this protein induces endochondral bone formation in
mammals when implanted subcutaneously in association with a
suitable matrix material, using a standard in vivo bone assay, such
as is disclosed in U.S. Pat. No. 4,968,590. Moreover, the morphogen
also is detected in the bloodstream. Finally, soluble form
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
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 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.
[0072] 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 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.
[0073] 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 pro domain. Accordingly, the pro domain
is thought to be morphogen-specific. As described above, it is also
known that the various morphogens identified to date are
differentially expressed in the different tissues. Accordingly,
without being limited to any given theory, it is likely that, under
natural conditions in the body, selected morphogens typically act
on a given tissue. Accordingly, part or all of the pro domains
which have been identified associated with the active form of the
morphogen in solution, may serve as targeting molecules for the
morphogens described herein. For example, the pro domains may
interact specifically with one or more molecules at the target
tissue to direct the morphogen associated with the pro domain to
that tissue. Accordingly, another useful targeting molecule for
targeting 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 domains of OP-1 or CBMP2 may
be used to target a morphogen to bone tissue, both of which
proteins are found naturally associated with bone tissue.
[0074] The morphogens described herein are useful for 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 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 neurons, the nerve
fibers formed by bundled neuronal axons, and the glial cells
surrounding and associated with the neurons. An inflammatory
response to nerve tissue injury may follow trauma to nerve tissue,
caused, for example, by an autoimmune (including 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., 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 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 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, as
during surgery or other aggressive clinical treatment, the
morphogen or agent may be provided prior to induction of the injury
to provide a neuraprotective effect to the nerve tissue, at
risk.
[0075] Where the morphogen is intended for use as a therapeutic to
alleviate tissue damage associated with an immune/inflammatory
condition of the central nervous system (CNS) an additional problem
must be addressed: overcoming the so-called "blood-brain barrier",
the brain capillary wall structure that effectively screens out all
but 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-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 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 the art, as, for example, described in
Pardridge, Endocrine Reviews: 7:314-330 (1986) and U.S. Pat. No.
4,801,575. A more detailed description of morphogens for use in
treating inflammatory, conditions in nerve tissue, including a
model for evaluating morphogen transport across the blood brain
barrier is disclosed in U.S. Ser. No. 922,813, the disclosure of
which is incorporated herein by reference.
[0076] Finally, the morphogens or morphogen-stimulating 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, vitamin D, and other antiinflammatory agents.
Psoriais treatments also may include ultra-violet light treatment,
zinc oxide and retinoids.
[0077] The compounds provided herein can be formulated into
pharmaceutical compositions by admixture with pharmaceutically
acceptable nontoxic excipients and carriers. As noted above, such
compositions may be prepared for parenteral administration,
particularly in the form of liquid solutions or suspensions; for
oral administration; particularly in the form of tablets or
capsules; or intranasally, particularly in the form of powders,
nasal drops, or aerosols.
[0078] 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 sufficient to alleivate the tissue
destructive effects associated with the inflammatory response,
including protecting tissue in anticipation of tissue damage.
[0079] As will be appreciated by those skilled in the art, the
concentration of the compounds described in a therapeutic
composition will vary depending upon a number of factors, including
the dosage of the drug to be administered, the chemical
characteristics (e.g., hydrophobicity) of the compounds employed,
and the route of administration. The preferred dosage of drug to be
administered also is likely to depend on such variables as the type
and extent of progression of the tissue damage, the overall health
status of the particular patient, the relative biological efficacy
of the compound selected, the formulation of the compound
excipients, and its route of administration. In general terms, the
compounds of this invention may be provided in an aqueous
physiological buffer solution containing about 0.001% to 10% w/v
compound for parenteral 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 .mu.g/kg to 100 mg/kg of
body weight per day. Optimally, the morphogen dosage given in most
cases is between 0.1-100 .mu.g of protein per kilogram weight of
the patient. No obvious morphogen induced pathological lesions are
induced when mature morphogen (e.g., OP-1, 20 .mu.g) is
administered daily to normal growing rats for 21 consecutive days.
Moreover, 10 .mu.g systemic injections of morphogen (e.g., OP-1)
injected daily for 10 days into normal newborn mice does not
produce any gross abnormalities.
[0080] 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.
[0081] 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.
[0082] 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 production and/or secretion from cells of
affected tissue or a transplanted organ may be provided to a
mammal, e.g., by direct administration of the morphogen to the
tissue or organ. 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 U.S. Ser. No. 752,859, filed Aug. 30, 1991, the
disclosure of which is incorporated herein by reference. Briefly,
candidate compounds can be identified and tested by incubating the
compound in vitro with a test tissue or cells thereof, for a time
sufficient to allow the compound to affect the production, i.e.,
the expression and/or secretion, of a morphogen produced by the
cells of that tissue.
[0083] For purposes of the present invention, the above-described
morphogens effective in alleviating ischemic-reperfusion injury (or
the agents that stimulate them, referred to herein collectively 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
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
myocardial infarction, asphyxia, or a cardiopulmonary arrest. Where
ischemia or hypoxia is deliberately induced as part of, for
example, a surgical procedure where circulation to an organ or
organ system is deliberately and/or 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, the therapeutic
agent is administered prophylactically in a surgical setting.
[0084] Similarly, where hyperoxia induced-injury already has
occurred, the morphogen is administered upon diagnosis. Where
hyperoxia may be induced an, 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.,
prophylactically).
III. EXAMPLES
Example 1
Identification of Morphogen-Expressing Tissue
[0085] Determining the tissue distribution of morphogens may be
used to identify different morphogens expressed in a given tissue,
as well as to identify new, related morphogens. Tissue distribution
also may be used to identify useful morphogen-producing tissue for
use in screening and identifying candidate morphogen-stimulating
agents. The morphogens (or their mRNA transcripts) readily are
identified in different tissues using standard methodologies and
minor modifications thereof in tissues where expression may be low.
For example, protein distribution may be determined using standard
Western blot analysis or immunofluorescent techniques, and
antibodies specific to the morphogen or morphogens of interest.
Similarly, the distribution of morphogen transcripts may be
determined using standard Northern hybridization protocols and
transcript-specific-probes.
[0086] Any probe capable of hybridizing specifically to a
transcript, and distinguishing the transcript of interest from
other, related transcripts may be used. Because the morphogens
described herein share such high sequence homology in their active,
C-terminal domains, the tissue distribution of a specific morphogen
transcript may best be determined using a probe specific for the
pro region of the immature protein and/or the N-terminal region of
the mature protein. Another useful sequence is the 3' non-coding
region flanking and immediately following the stop codon. These
portions of the sequence vary substantially among the morphogens of
this invention, and accordingly, are specific for each protein. For
example, a particularly useful Vgr-1-specific probe sequence is the
PvuII-SacI fragment, a 265 bp fragment encoding both a portion of
the untranslated pro region and the N-terminus of the mature
sequence (see Lyons et al. (1989) PNAS 86:4554-4558 for a
description of the cDNA sequence). Similarly, particularly useful
mOP-1-specific probe sequences are the BstX1-BglI fragment, a 0.68
Kb sequence that covers approximately two-thirds of the mOP-1 pro
region; a StuI-StuI fragment, a 0.2 Kb sequence immediately
upstream of the 7-cysteine domain; and the Ear1-Pst1 fragment, an
0.3 Kb fragment containing a portion of the 3'untranslated sequence
(See Seq. ID No. 18, where the pro region is defined essentially by
residues 30-291.) Similar approaches may be used, for example, with
hOP-1 (Seq. ID No. 16) or human or mouse OP-2 (Seq. ID Nos. 20 and
22.)
[0087] Using these morphogen-specific probe's, which may be
synthetically engineered or obtained from cloned sequences,
morphogen transcripts can be identified in mammalian tissue, using
standard methodologies well known to those having ordinary skill in
the art. Briefly, total RNA is prepared from various adult murine
tissues (e.g., liver, kidney, testis, heart, brain, thymus and
stomach) by a standard methodology such as by the method of
Chomczyaski et al. ((1987) 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 .mu.g) 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.degree. C. and the RNA is
cross-linked under UV light (generally 30 seconds at 1
mW/cm.sup.2). Prior to hybridization, the appropriate probe is
denatured by heating. The hybridization is carried out in a lucite
cylinder rotating in a roller bottle apparatus at approximately 1
rev/min for approximately 15 hours at 37.degree. C. using a
hybridization mix of 40% formamide, 5.times. Denhardts,
5.times.SSPE, and 0.1% SDS. Following hybridization, the
non-specific counts are washed off the filters in 0.1.times.SSPE,
0.1% SDS at 50.degree. C.
[0088] Examples demonstrating the tissue distribution of various
morphogens, including Vgr-1, OP-1, BMP2, BMP3, BMP4, BMP5, GDF-1,
and OP-2 in developing and adult tissue are disclosed in co-pending
U.S. Ser. No. 752,764, and in Ozkaynak, et al., (1991) Biochem.
Biophys. Res. Commn. 179:116-123, and Ozkaynak, et al. (1992) (JBC,
in press), the disclosures of which are incorporated herein by
reference. Using the general probing methodology described herein,
northern blot hybridizations using probes specific for these
morphogens to probe brain, spleen, lung, heart, liver and kidney
tissue indicate that kidney-related tissue appears to be the
primary expression source for OP-1, with brain, heart and lung
tissues being secondary sources. OP-1 RNA also was identified in
salivary glands, specifically rat parotid glands, using this
probing methodology. Lung tissue appears to be the primary tissue
expression source for Vgr-1, BMP5, BMP4 and BMP3. Lower levels of
Vgr-1 also are seen in kidney and heart tissue, while the liver
appears to be a secondary expression source for BMP5, and the
spleen appears to be a secondary expression source: for BMP4. GDF-1
appears to be expressed primarily in brain tissue. To date, OP-2
appears to be expressed primarily in early embryonic tissue.
Specifically, northern blots of murine embryos and 6-day post-natal
animals shows abundant OP2 expression in 8-day embryos. Expression
is reduced significantly in 17-day embryos and is not detected in
post-natal animals.
Example 2
Active Morphogens in Body Fluids
[0089] OP-1 expression has been identified in saliva (specifically,
the rat parotid gland, see Example-1), human blood serum, and
various milk forms, including mammary gland extract, colostrum, and
57-day bovine milk. Moreover, and as described in U.S. Ser. No.
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, together with the known observation that mature,
active OP-1 is acid-stable and protease-resistant, indicate that
oral administration is a useful route for therapeutic
administration of morphogen to a mammal. Oral administration
typically is the preferred mode of delivery for extended or
prophylactic therapies. In addition, the identification of
morphogen in all milk forms, including colostrum, suggests that the
protein may play a significant role in tissue development,
including skeletal development, of juveniles.
[0090] 2.1 Morphogen Detection in Milk
[0091] OP-1 was partially purified from rat mammary gland extract
and bovine colostrum and 57 day milk by passing these fluids over a
series of chromatography columns: (e.g., cation-exchange, affinity
and reverse phase). At each step the eluant was collected in
fractions and these were tested for the presence of OP-1 by
standard immunoblot. Immunoreactive fractions then were combined
and purified further. The final, partially purified product then
was examined for the presence of OP-1 by Western blot analysis
using OP-1-specific antisera, and tested for in vivo and in vitro
activity.
[0092] OP-1 purified from the different milk sources were
characterized by Western blotting using antibodies raised against
OP-1 and BMP2. Antibodies were prepared using standard immunology
protocols well known in the art, and as described generally in
Example 15, below, using full-length E. coli-produced OP-1 and BMP2
as the immunogens. In all cases, the purified OP-1 reacted only
with the anti-OP-1 antibody, and not with anti-BMP2 antibody.
[0093] 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
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 .mu.l of 50% acetonitrile/0.1% TFA.
After vortexing, 25 mg of collagen matrix was added. The samples
were lyophilized overnight, and implanted in Long Evans rats
(Charles River Laboratories, Wilmington, Mass., 28-35 days old).
Each fraction was implanted in duplicate. For details of the
collagen matrix implantation procedure, see, for example, U.S. Pat.
No. 4,968,590, hereby incorporated by reference. After 12 days, the
implants were removed and evaluated for new bone formation by
histological observation as described in U.S. Pat. No. 4,968,590.
In all cases, the immunoreactive: fractions-were osteogenically
active.
[0094] 2.2 Morphogen Detection in Serum
[0095] Morphogen may be detected in serum using morphogen-specific
antibodies. The assay may be performed using any standard
immunoassay, such as Western blot (immunoblot) and the like.
Preferably, the assay is performed using an affinity column to
which the morphogen-specific antibody is bound and through which
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., purified, recombinantly-produced morphogen.) Fractions then
are tested for the presence of the morphogen by standard
immunoblot, and the results confirmed by N-terminal sequencing.
Preferably, the affinity column is prepared using monoclonal
antibodies. Morphogen concentrations in serum or other fluid
samples then may be determined using standard protein
quantification techniques, including by spectrophotometric
absorbance or by quantitation of conjugated antibody.
[0096] Presented below is a sample protocol for identifying OP-1 in
serum. Following this general methodology other morphogens may be
detected in body fluids, including serum. The identification of
morphogen in serum further indicates that systemic administration
is a suitable means for providing therapeutic concentrations of a
morphogen to an individual, and that morphogens likely behave
systemically as endocrine-like factors. Finally, using this
protocol, fluctuations in endogenous morphogen levels can be
detected, and these altered levels may be used as an indicator of
tissue dysfunction. Alternatively, fluctuations in morphogen levels
may be assessed by monitoring morphogen transcription levels,
either by standard northern blot analysis as described in Example
1, or by in situ hybridization, using a labelled probe capable of
hybridizing specifically to morphogen "mRNA", and standard RNA
hybridization protocols-well-described in the art and described
generally in Example 1.
[0097] OP-1 was detected in human serum using the following assay.
A monoclonal antibody raised against mammalian, recombinantly
produced OP-1 using standard immunology techniques well described
in the art and described generally in Example 15, was immobilized
by passing the antibody over an agarose-activated gel (e.g.,
Affi-Gel.TM., from Bio-Rad Laboratories, Richmond, Calif., prepared
following-manufacturer's instructions) and used to purify OP-1 from
serum. Human serum then was passed over the column and eluted with
3M K-thiocyanate. K-thiocyanante fractions then were dialyzed in 6M
urea, 20 mM PO.sub.4, pH 7.0, applied to a C8 HPLC column, and
eluted with a 20 minute, 25-50% acetonitrile/0.1% TFA gradient.
Mature, recombinantly produced OP-1 homodimers elute between 20-22
minutes. 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.
[0098] Administered or endogenous morphogen levels may be monitored
in the therapies described herein by comparing the quantity of
morphogen present in a body fluid sample with a predetermined
reference value, for example, to evaluate the efficiency of a
therapeutic protocol, and the like. In addition, fluctuations in
the level of endogenous morphogen antibodies may be detected by
this method, most likely in serum, using an antibody or other
binding protein capable of interacting specifically with the
endogenous morphogen antibody. Detected fluctuations in the levels
of the morphogen or endogenous antibody may be used, for example,
as indicators of a change in tissue status. For example, as damaged
tissue is regenerated and the tissue or organ's function returns to
"normal" and, in the absence of additional tissue damage, lower
doses of morphogen may be required, and a higher level of
circulating morphogen antibody may be measured.
Example 3
Effect of Morphogen after the Onset of the Ischemic Process
[0099] The cardioprotective effect of morphogens following
ischemic-reperfusion injury in a mammal can readily be 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
(1992) J. Mol. Cell. Cardiol. 24: 385-393, the disclosures of which
are hereby incorporated by reference. Briefly, loss of myocardial
tissue function following ischemia and reperfusion is assayed by
measuring loss of myocardial creatine kinease activity (CK) and
loss of endothelium-dependent vasorelaxation function (see Example
4, below).
[0100] In a first group of ether-anesthetized rats, the left
coronary artery was occluded just proximal to the 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 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).
[0101] The first group of rats, the MI group of rats, further was
divided into three sup-groups. 2 .mu.g of morphogen (OP-1) were
injected intravenously into the first sub-group of MI rats 10
minutes after ligature, immediately before reperfusion; into the
second sub-group of MI rats 20 .mu.g 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.
[0102] 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 from the interventricular
septum (the control 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 cellular injury to the infarcted
region.
[0103] As shown in FIG. 1, the data indicate that morphogens (e.g.,
OP-1) can-provide significant cardioprotective effect when provided
to ischemic tissue. In the figure, CK loss is graphed as the
difference in specific CK activity between the interventricular
septum and the left ventricle.
[0104] The loss of CK activity by the subgroup of MI rats which
received 2 .mu.g 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 control. Significant cardioprotection was observed in the
subgroup of MI rats which received 20 .mu.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 against, and compared to, the levels
contained within the SHAM MI control.
[0105] These data indicate that OP-1 offers significant cardiac
protection when administered after ischemia and before
reperfusion.
[0106] 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 cardioprotective effects of morphogen
administered prior to ischemia.
Example 4
Vasodilation of Myocardial Infarcted Cardiac Tissue Treated with
Morphogen
[0107] 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 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 (NTG) and nitroprusside, are
endothelium-independent dilators, as they dilate blood vessels
directly.
[0108] The present example demonstrates the ability of OP-1 to
prevent the loss of cardioendothelium-dependent 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-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 in expected to enhance
ACh-induced vasodilation in the reperfused hearts.
[0109] 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 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 20 .mu.g of OP-0.1 2 hours prior to
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
flow (15 ml/min) with oxygenated Krebs-Henseleit solution (Aoki et
al. (1988) J. Pharmacol. 95:35).
[0110] Each group of rats then were divided into two subgroups of
six rats each. Twenty minutes before reperfusion, coronary
vasodilator response was measured by inducing constriction with
0.05 .mu.mol U-44619 (9,11-methanoepoxyprostaglandin H.sub.2)
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 (CPP) level measured as 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.
[0111] The vasodilator reponse then was remeasured by constriction
and administration of vasodilating agent as described above.
[0112] The results of these experiments are shown in FIG. 2. Before
the ischemic event, both Ach and NTG gave normal vasorelaxant
results in all events. The hearts which received OP-1 24 hours
prior to ischemia showed 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 showed an ACh response of approximately 95%.
This shows that endothelium-dependent vasodilators exert a reduced
vasodilator response following ischemia and reperfusion in the rat
heart. Moreover, OP-1 significantly preserved endothelium-dependent
dilation when provided 24 hours prior to induction of myocardial
ischemia. No 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 hearts.
Example 5
Effect of Morphogen on Neutrophil Adherence
[0113] The role of neutrophil adherence in endothelium 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 hereinabove by
reference. Briefly, segments of superior mesenteric artery were
isolated from rats which had either been treated with morphogen
(OP-1, 20 .mu.g) or 0.9% NaCl, 24 h prior to isolation of the
artery. The segments were cleaned, cut into transverse rings of 1-2
mm in length, and these were subsequently cut open and incubated in
K-H solution at 37.degree. C., pH 7.4. Neutrophils were prepared
and fluorescently labelled using standard procedures (e.g.,
leukocytes were isolated from rats essentially following the
procedure 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.
[0114] Labelled neutrophils then were added to open ring baths and
activated with 100 nM leukotriene B.sub.4 (LTB.sub.4). Rings were
incubated for 20 minutes and the number of neutrophils adhering to
the endothelial surface then determined visually by fluorescent
microscopy.
[0115] As shown in FIG. 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 LTB.sub.4 (100 nM) greatly increased
the number of PMNS adherent to the endothelium (P<0.001). OP-1
(20 .mu.g administered 24 h prior) significantly inhibited
adherence of PMNs activated by LTB.sub.4 (P<0.01 from
control).
Example 6
In Vivo Models for Ischemic-Reperfusion Protection in Lung, Nerve
and Renal Tissue
[0116] Other tissues seriously affected by ischemic-reperfusion
injury-include neural tissue, renal tissue and lung tissue. The
effect of morphogens on alleviating the ischemic-reperfusion injury
in these tissues may be assessed using methodologies and models
known to those skilled in the art, and disclosed below. Similarly,
a methodology also is provided for assessing the tissue-protective
effects of a morphogen on damaged lung tissue following hyperoxia
injury.
[0117] 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 Neurology 25:281-285, the disclosure of which is herein
incorporated by reference. Briefly, white New England rabbits (2-3
kg) are anesthesized and placed on a respirator. The intracranial
circulation then is selectively catheterized by the Seldinger
technique. 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 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 minutes).
[0118] The effect of morphogen on cerebral infarcts can be assessed
by administering varying concentrations of morphogens, e.g., OP1,
at different times preceding or following embolization and/or
reperfusion. The rabbits are sacrificed 3-14 days post embolization
and their brains prepared for neuropathological examination by
fixing by immersion in 10% neutral buffered formalin or at least 2
weeks. The brains then are sectioned in coronal plane at 2-3 mm
intervals, numbered and submitted for standard histological
processing in paraffin, and the degree of neutral tissue necrosis
determined visually.
[0119] The renal-protective effects of morphogens on renal
ischemia-reperfusion injury readily can be assessed using the mouse
model disclosed by Oueliette, et al. (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 kidney with a
microaneurism clamp for 10-30 minutes. Morphogen then may be
provided parentally at various times prior to or following,
occlusion and/or reperfusion. The effects of morphogen then may be
assessed by biological and histological evaluation using standard
techniuques well known in the art.
[0120] The tissue protective effects of morphogen on tissue exposed
to lethally high oxygen concentrations may be assessed by the
following procedure. Adult rats (275-300 gms) first are provided
with morphogen (e.g., hOP1) or vehicle only, and then are exposed
to 0.96-98% oxygen essentially as described by Rinaldo et al (1983)
Am. Rev. Respir. Dis. 130:1065, to induce hyperoxia. Animals are
housed in plastic cages (38 cm.times.48 xm.times.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 O.sub.2 gas (liquid O.sub.2). Gas flow
through the chamber is adjusted to maintain at least 10 air
changes/hr., temperature at 22.+-.1.degree. C., minimal levels of
condensation within the cage, and carbon dioxide concentration of
<0.5% as measured with a mass spetrophotometric medical as
analyzer.
[0121] 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 insult and subsequent immune 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
processing of lung tissue.
[0122] Lung tissue for histological processing is fixed by infusion
of 10% buffered formalin through a tracheal cannula at a constant
pressure of 20 cm H.sub.2O. After 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 inflammatory response.
Example 7
Morphogen Inhibition of Cellular and Humoral Inflammatory
Response
[0123] 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 morphogen, an implanted substrate material (e.g., implanted
subcutaneously) composed of, for example, mineralized bone, a
ceramic such as titanium oxide or any other substrate that provokes
multinucleated giant cell formation, rapidly becomes surrounded by
multinucleated giant cells, e.g., activated-phagocytes stimulated
to respond and destroy the foreign object. In the presence of
morphogen however, the recruited cells remain in their mononuclear
precursor form and the matrix material is undisturbed. FIG. 4
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 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 surrounding the substrate. Accordingly, the morphogens'
effect in inhibiting excessive bone mass loss in a mammal also may
include inhibiting activation of these cells.
[0124] 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 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 essentially following the procedure described by
Nagler-Anderson et al, (1986) PNAS 83:7443-7446, the disclosure of
which is incorporated herein by reference, increased consistently
throughout the experiment. However, when the matrix was implanted
together with a morphogen (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 is further evidence of morphogen utility in alleviating
tissue damage associated with autoimmune diseases, including
autoantibody diseases, such as rheumatoid arthritis.
Example 8
Morphogen protection of Gastrointestinal Tract Mucosa from
Ulceration and Inflammation
[0125] 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 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, the morphogens described herein
should be efficacious in treating chronic inflammatory diseases
including IBD, arthritis, psoriasis and psoriatic arthritis,
multiple sclerosis, and the like.
[0126] Golden syrian hamsters (6-8 wks old, Charles River
Laboratories, Wilmington, Mass.) were divided into 3 test groups:
Group 1, a placebo (e.g., saline) control, and a morphogen-low dose
group (100 ng) and a morphogen high dose group (1 .mu.g), Groups 2
and 3, respectively. Morphogen dosages were provided in 30%
ethanol. Each group contained 12 animals.
[0127] Beginning on day 0 and continuing through day 5, Groups 2
and 3 received twice daily morphogen applications. On day 3, all
groups began the mucositis-induction procedure. 5-fluorouracil (60
mg/kg) was injected intraperitoneally on days 3 and 5. On day 7,
the right buccal pouch mucosa was superficially irritated with a
calibrated 18 gauge needle. In untreated animals, severe ulcerative
mucositis was induced in at least 80% of the animals by day 10.
[0128] For each administration of the vehicle control (placebo) or
morphogen, administration was performed by first gently drying the
cheek pouch mucosa, then providing an even application over the
mucosal surface of the vehicle or morphogen material. A
hydroxypropylcellulose-based coating was used to maintain contact
of the morphogen with the mucosa. This coating provided at least 4
hours of contact time.
[0129] On day 12, two animals in each group were sacrificed for
histological studies. The right buccal pouch mucosa and underlying
connective tissue were dissected and fixed in 10% formalin using
standard dissection and histology procedures. The specimens were
mounted in paraffin and prepared for histologic examination.
Sections then were stained with hematoxylin and eosin and were
examined blindly by three oral pathologists with expertise in
hamster histology and scored blind against a standard mucositis
panel. The extent of atrophy, cellular infiltration, connective
tissue breakdown, degree of ulceration and epithelialization were
assessed.
[0130] 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., 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.
[0131] 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 doses inhibit lesion formation significantly in
a dose-dependent manner. In addition, histology results
consistently showed significantly reduced amounts of 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.
Example 9
Morphogen Effect on Fibrogenesis and Scar Tissue Formation
[0132] The morphogens described herein induce tissue morphogenesis
of damaged or lost tissue. The ability of these proteins to
regenerate new tissue enhances the anti-inflammatory effect of
these proteins. Provided below-are a series of in vitro experiments
demonstrating the ability of morphogens to induce migration and
accumulation of mesenchymal cells. In addition, the experiments
demonstrate that morphogens, unlike TGF-.beta., do not stimulate
fibrogenesis or scar tissue formation. Specifically, morphogens do
not stimulate production of collagen, hyaluronic acid (HA), or
metalloproteinases in primary fibroblasts, all of which are
required for fibrogenesis or scar tissue formation. By contrast,
TGF-.beta., a known-inducer of fibrosis, but not of tissue
morphogenesis, does stimulate production of these fibrosis
markers.
[0133] Chemotaxis and migration of mesenchymal progenitor cells
were measured in modified Boyden chambers essentially as described
by Fava, R. A. et al (1991) J. Exp. Med. 173: 1121-1132, the
disclosure of which is incorporated herein by reference, using
polycarbonate filters of 2, 3 and 0.8 micron ports to measure
migration of progenitor neutrophils, monocytes and fibroblasts
Chemotaxis was measured over a range of morphogen concentrations,
e.g., 10.sup.-20 M to 10.sup.-12M OP-1. For progenitor neutrophils
and monocytes, 10.sup.-18-10.sup.-17M OP-1 consistently induced
maximal, migration, and 10.sup.-14 to 10.sup.-13M OP-1 maximally
induced migration of progenitor fibroblasts. In all cases the
chemotactic activity could be inhibited with anti-OP-1 antibody.
Similar migration activities also were measured and observed with
TGF-.beta..
[0134] The effect of morphogen on fibrogenesis was determined by
evaluating fibroblast production of hyaluronic acid (HA), collagen,
collagenese and tissue inhibitor of metalloproteinases (TIMP).
[0135] Human fibroblasts were established from explants of infant
foreskins and maintained in monolayer culture using standard
culturing procedures. (See, for example, (1976) J. Exp. Med. 144:
1188-1203.) Briefly, fibroblasts were grown in maintenance medium
consisting of Eagle's MEM, supplemented with nonessential amino
acids, ascorbic acid (50 .mu.g/ml), NaHCO.sub.3 and HEPES buffers
(pH 7.2), penicillin (100 U/ml), streptomycin (100 .mu.g/ml),
amphotericin B (1 .mu.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 measure synthesis of collagen, hyaluronic acid,
collagenase and tissue inhibitors of metalloproteinases (TIMP) were
grown in 100 mm diameter plastic tissue culture petri dishes.
[0136] 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 and Clark et al (1985) Arch. Bio-chem Biophys. 241:
36-44, the disclosures of which are incorporated by reference.) For
these assays, fibroblasts were transferred to 24-well tissue
culture plates at a density of 8.times.10.sup.4 cells per well.
Fibroblasts were grown confluency in maintenance medium containing
9% FCS for 72 h and then grown in serum-free-maintenance medium for
24 h. Medium was then removed from each well and various
concentrations of OP-1 (recombinantly produced mature or soluble
form) or TGF-.beta.-1 (R&D Systems, Minneapolis) in 50 .mu.l
PBS were added to triplicate wells containing the confluent
fibroblast monolayers. For experiments that measured production of
collagenase and TIMP, maintenance medium (450 .mu.l) containing 5%
FCS was added to each well, and culture supernatants were harvested
from each well 48 h later and stored at -70.degree. C. until
assayed. For experiments that assessed HA production, maintenance
medium (450 .mu.l) containing 2.5% FCS was added to each well, and
cultures grown for 48 h. For experiments that measured fibroblast
production of collagens, serum-free maintenance medium (450 .mu.l)
without non-essential amino acids was added to each well and
cultures grown for 72 h. Fibroblast production of HA was measured
by labeling newly synthesized glycosaminoglycans (GAG) with
[.sup.3H]-acetate the last 24 h of culture and quantitating
released radioactivity after incubation with hyaluronidase from
Streptomyces hyalurolyticus (ICN Biochemicals, Cleveland, Ohio)
which specifically degrades hyaluronic acid. Production of total
collagen by fibroblasts was measured using a collagenase-sensitive
protein assay that reflects [.sup.3H]-proline incorporation the
last 24 h of culture into newly synthesized collagens. Collagenase
and TIMP protein levels in fibroblast cultures supernatants was
measured by specific ELISAs.
[0137] As shown in FIG. 6, OP1 does not stimulate significant
collagen or HA production, as compared with TGF-.beta.. In the
figure, panel A shows OP-1 efect on collagen production, panel B
shows TGF-.beta. effect on collagen production, and panels C and D
show OP-1 (panel C) and TGF-.beta. (panel D) effect on HA
production. The morphogen results were the same whether the soluble
or mature form of OP1 was used. By contrast, the latent form of
TGF-.beta. (e.g., pro domain-associated form of TGF-.beta.) was not
active.
Example 10
Morphogen Inhibition of Epithelial Cell Proliferation
[0138] This example demonstrates the ability of morphogens to
inhibit epithelial cell proliferation in vitro, as determined by
.sup.3H-thymidine uptake using culture cells from a mink lung
epithelial cell line (ATCC No., CCL 64), and standard mammalian
cell culturing procedures. Briefly, cells were grown to confluency
in Eagle's minimum essential medium (EMEM) supplemented with 10%
fetal bovine serum (FBS), 0.200 units/ml penicillin, and 200
.mu.g/ml streptomycin, and used to seed a 48-well cell culture
plate at a cell density of 200,000 cells per well. When this
culture became confluent, the media was replaced with 0.5 ml of
EMEM containing 1% FBS and penicillin/streptomycin and the culture
incubated for 24 hours at 37 C. Morphogen test samples in EMEM
containing 5% FBS then were added to the wells, and the cells
incubated for another 18 hours. After incubation, 1.0 .mu.Ci of
.sup.3H-thymidine in 10 .mu.l was added to each well, and the cells
incubated for four hours at 37 C. The media then was removed and
the cells washed once with ice-cold phosphate-buffer saline and DNA
precipitated by adding 0.5 ml of 10% TCA to each well 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
scintillation vial and the radioactivity recorded using a
scintillation counter (Smith-Kline Beckman).
[0139] The results are presented in Table-III, below. The
anti-proliferative effect of the various morphogens tested was
expressed as the counts of 3H-thymidine (x 1000) integrated into
DNA, and were compared with untreated cells (negative control) and
TGF-.beta. (1 ng), a local-acting factor also known to inhibit
epithelial cell proliferation. COP-5 and COP-7 are biosynthetic
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 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 (see U.S. Pat. No. 4,968,590 and
5,011,691.)
9 TABLE III Thymidine uptake (.times.1000) control 50.048, 53.692
COP-7-1 (10 ng) 11.874 COP-7-2 (3 ng) 11.136 COP-5-1 (66 ng) 16.094
COP-5-2 (164 ng) 14.43 TGF-.beta. (1 ng) 1.86, 1.478
Example 11
Morphogen Treatment of a Systemic Inflammatory Disease
[0140] 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 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 Walz, et al., (1971) J. Pharmac.
Exp. Ther. 178: 223-231, the disclosure of which is incorporated
herein by reference.
[0141] Briefly, Sprague-Dawley female rats (e.g., Charles River
Laboratories, Wilmington, Mass.) are randomized into 3 groups:
control; morphogen, low dose (e.g., 1-10 .mu.g/kg weight per day)
and morphogen, high dose (e.g., 10-20 .mu.g/kg weight per day),
referred to as Groups 1, 2, and 3, respectively.
[0142] 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 limbs are determined. In the absence of morphogen
treatment, a systemic arthritic condition is induced in the rats by
this time, as determined by rats with significant swelling of the
uninjected hind limbs (<2.3 ml, volume measured by mercury
displacement). 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 and 50 after adjuvant
injection and edema determined by volume difference compared to Day
18. The uninjected hind limb on each-rat was x-rayed on Day 50 and
the joint damage assayed on an arbitrary scale of 1 to 10 (1=no
damage, 10=maximum damage). Data on differences 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 control rats.
[0143] 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 of morphogens in arthritic
animals.
Example 12
Morphogen Inhibition of Localized Edema
[0144] The following example demonstrates morphogen efficacy in
inhibiting a localized inflammatory response in a standard rat
edema model. Experimental rats (e.g., Long-Evans from Charles River
Laboratories, Wilmington, Mass.) are divided into three groups:
Group 1, a negative control, which receives vehicle alone; 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.
[0145] 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 .mu.g; 5-20 .mu.g, and 20-50 .mu.g of
morphogen). Sixty minutes after 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 (50
.mu.l) into the right hind paw. Three hours after carrageenin
administration paw thickness is measured as an indication of edema
(e.g., swelling) and induced inflammatory response to the injected
carrageenin solution.
[0146] 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 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.
[0147] The morphogen-treated rats show substantially reduced edema
induction following carrageenin injection as compared with the
untreated rats.
Example 13
Morphogen Treatment of Allergic Encephalomyelitis
[0148] The following example demonstrates morphogen 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 88:2918-2921, the
disclosure of which is incorporated herein by reference.
[0149] Briefly, EAE is induced in rats (e.g., Long-Evans, Charles
River Laboratories, Wilmington, Mass.) by injection of a CNS tissue
(e.g., spinal cord) homogenate in complete Freund's adjuvant (CFA)
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 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 are examined daily for signs of disease and graded on
an increasing severity scale of 0-4.
[0150] In the absence of morphogen treatment, significant
neurological dysfunction (e.g., hind and fore limb 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 degree of tissue necropsy using standard
procedures. Morphogen treatment significantly inhibits the
neurological dysfunction normally evident iii 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
[0151] 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 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 Laboratories,
Wilmington), by multiple intradermal injection of bovine Type II
collagen (e.g., 100 .mu.g) 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,
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, 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
described for Examples 12 and 13, above. In a 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, 28-32.)
[0152] In the absence of morphogen treatment, an arthritic
condition typically is induced by 30 days post collagen injection.
In morphogen-treated animals, CIA is 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, above, serum
anti-collagen antibody titers are suppressed significantly in the
morphogen-treated animals.
Example 15
Screening Assay for Candidate Compounds which Alter Endogenous
Morphogen Levels
[0153] 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 of morphogen production by a
cell type which produces measurable levels of the morphogen is
determined with and without incubating the cell in culture with the
compound, in order to assess the effects of the compound on the
cell. This can be accomplished by detection of the morphogen either
at the protein or RNA level. A more detailed description also may
be found in U.S. Ser. No. 752,861, incorporated hereinabove by
reference.
[0154] 15.1 Growth of Cells in Culture
[0155] Cell cultures of kidney, adrenals, urinary bladder, brain,
or other organs, may be prepared as described widely in the
literature. For example, kidneys may be explanted from neonatal or
new born or young or adult rodents (mouse or rat) and used in organ
culture as whole or sliced (1-4 mm) tissues. Primary tissue
cultures and established cell lines, also derived from kidney,
adrenals, urinary, bladder, brain, mammary, or other tissues may be
established in multiwell plates (6 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 cultured, for example, in Dulbecco's Modified Eagle medium
(Gibco, Long Island, N.Y.) 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., transferring glucose,
albumin, or other growth factors).
[0156] Samples for testing the level of morphogen production
includes culture supernatants or cell lysates, collected
periodically and evaluated for OP-1 production by immunoblot
analysis (Sambrook et al., eds., 1989, Molecular Cloning, Cold
Spring Harbor Press, Cold Spring Harbor, N.Y.), or a portion of the
cell culture itself, collected periodically and used to prepare
polyA+ RNA for RNA analysis. To monitor de novo OP-1 synthesis,
some cultures are labeled according to conventional procedures with
an .sup.35S-methionine/.sup.35S-cysteine mixture for 6-24 hours and
then evaluated to OP-1 synthesis by conventional
immunoprecipitation methods.
[0157] 15.2 Determination of Level of Morphogenic Protein
[0158] In order to quantitate the production of a morphogenic
protein by a cell type, an immunoassay may be performed to detect
the morphogen using a polyclonal or monoclonal antibody specific
for that protein. For example, OP-1 may be detected using a
polyclonal antibody specific for OP-1 in an ELISA, as follows.
[0159] 1 .mu.g/100 .mu.l of affinity-purified polyclonal-rabbit IgG
specific for OP-1 is added to each well of a 96-well plate and
incubated at 37.degree. C. for an hour. The wells are washed four
times with 0.167M sodium borate buffer with 0.15 M NaCl (BSB), pH
8.2, containing 0.1% Tween 20. To minimize non-specific binding,
the wells are blocked by filling completely with 1% bovine serum
albumin (BSA) in BSB and incubating for 1 hour at 37.degree. C. The
wells are then washed four times with BSB containing 0.1% Tween 20.
A 100 .mu.l aliquot of an appropriate dilution of each of the test
samples of cell culture supernatant is added to each well in
triplicate and incubated at 37.degree. C. for 30 min. After
incubation, 100 .mu.l biotinylated rabbit anti-OP-1 serum (stock
solution is about 1 mg/ml and diluted 1:400 in BSB containing 1%
BSA before use) is added to each well and incubated at 37.degree.
C. for 30 min. The wells are then washed four times with BSB
containing 0.1% Tween 20. 100 .mu.l strepavidin-alkaline (Southern
Biotechnology Associates, Inc. Birmingham, Ala., diluted 1:2000 in
BSB containing 0.1% Tween 20 before use) is added to each well and
incubated at 37.degree. C. for 30 min. The plates are washed four
times with 0.5M Tris buffered Saline (TBS), pH 7.2. 50 .mu.l
substrate (ELISA Amplification System Kit, Life Technologies, Inc.,
Bethesda, Md.) is added to each well incubated at room temperature
for 15 min. Then, 50 .mu.l 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 .mu.l
0.3 M sulphuric acid. The OD at 490 nm of the solution in each well
is recorded. To quantitate OP-1 in culture media, a OP-1 standard
curve is performed in parallel with the test samples.
[0160] Polyclonal antibody may be prepared as follows. Each rabbit
is given a primary immunization of 100 ug/500 .mu.l E. coli
produced OP-1 monomer (amino acids 328-431 in SEQ ID NO:5) in 0.1%
SDS mixed with 500 .mu.l Complete Freund's Adjuvant. The antigen is
injected subcutaneously at multiple sites on the back and flanks of
the animal. The rabbit is boosted after a month in the same manner
using incomplete Freund's Adjuvant. Test bleeds are taken from the
ear vein seven days later. Two additional boosts and test bleeds
are performed at monthly intervals until antibody against OP-1 is
detected in the serum using an ELISA assay. Then, the rabbit is
boosted monthly with 100 .mu.g of antigen and bled (15 ml per
bleed) at days seven and ten after boosting.
[0161] 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 .mu.g of
OP-1 in complete Freund's adjuvant and is given subcutaneously. The
second injection contains 50 .mu.g of OP-1 in incomplete adjuvant
and is given intraperitoneally. The mouse then receives a total of
230 .mu.g of OP-1 (amino acids 307-431 in SEQ ID NO:5) in four
intraperitoneal injections at various times over an eight month
period. One week prior to fusion, both mice are boosted
intraperitoneally with 100 .mu.g of OP-1 (307-431) and 30 .mu.g of
the N-terminal peptide (Ser.sub.293-Asn.sub.309-Cys) conjugated
through the added-cysteine to bovine serum 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 Mannheim), and the cell
fusion is plated and screened for OP-1-specific antibodies using
OP-1 (307-431) as antigen. The cell fusion and monoclonal screening
then are according to standard procedures well described in
standard texts widely available in the art.
[0162] The invention may be embodied in other specific forms
without departing from the spirit or essential characteristics
thereof. The present embodiments are therefore to be considered in
all respects as illustrative and not restrictive, the scope of the
invention being indicated by the appended claims rather than by the
foregoing description, and all changes which come within the
meaning and range of equivalency of the claims are therefore
intended to be embraced therein.
Sequence CWU 1
1
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