U.S. patent application number 12/964284 was filed with the patent office on 2011-03-31 for bmp-1 procollagen c-proteinase for diagnosis and treatment of bone and soft tissue defects and disorders.
This patent application is currently assigned to GENERA ISTRAZIVANJA d.o.o. Invention is credited to Lovorka Grgurevic, Boris Macek, Slobodan Vukicevic.
Application Number | 20110076281 12/964284 |
Document ID | / |
Family ID | 38957430 |
Filed Date | 2011-03-31 |
United States Patent
Application |
20110076281 |
Kind Code |
A1 |
Vukicevic; Slobodan ; et
al. |
March 31, 2011 |
BMP-1 PROCOLLAGEN C-PROTEINASE FOR DIAGNOSIS AND TREATMENT OF BONE
AND SOFT TISSUE DEFECTS AND DISORDERS
Abstract
Uses of BMP-1 isoforms for diagnosing and treating defects and
disorders of bone and soft tissues are described. Also described is
a newly isolated variant of the BMP-1 isoform BMP-1-3.
Inventors: |
Vukicevic; Slobodan;
(Zagreb, HR) ; Grgurevic; Lovorka; (Zagreb,
HR) ; Macek; Boris; (Zagreb, HR) |
Assignee: |
GENERA ISTRAZIVANJA d.o.o
Kalinovica
HR
|
Family ID: |
38957430 |
Appl. No.: |
12/964284 |
Filed: |
December 9, 2010 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
12309510 |
Jan 21, 2009 |
7850964 |
|
|
PCT/US2007/016605 |
Jul 23, 2007 |
|
|
|
12964284 |
|
|
|
|
60832325 |
Jul 21, 2006 |
|
|
|
Current U.S.
Class: |
424/158.1 ;
435/7.1; 514/8.8 |
Current CPC
Class: |
A61K 38/4886 20130101;
G01N 2333/96486 20130101; A61P 13/12 20180101; C07K 16/22 20130101;
G01N 33/6887 20130101; G01N 33/573 20130101; A61K 9/0024 20130101;
C07K 2317/76 20130101; A61P 7/02 20180101; A61P 43/00 20180101;
A61K 38/1875 20130101; G01N 2800/10 20130101; A61P 1/18 20180101;
A61M 5/31596 20130101; A61P 9/10 20180101; A61K 39/3955 20130101;
A61K 45/06 20130101; A61P 19/00 20180101; A61P 19/08 20180101; C12N
9/6491 20130101; A61K 2039/505 20130101 |
Class at
Publication: |
424/158.1 ;
435/7.1; 514/8.8 |
International
Class: |
A61K 39/395 20060101
A61K039/395; G01N 33/68 20060101 G01N033/68; A61K 38/18 20060101
A61K038/18; A61P 13/12 20060101 A61P013/12 |
Claims
1. A method for diagnosing acute pancreatitis in an individual
comprising: testing a blood sample from an individual to determine
the presence in the sample of the BMP-1 isoform BMP-1-7, wherein
the presence of said BMP-1 isoform in circulating blood of said
individual is indicative of acute pancreatitis in the
individual.
2. A method for diagnosing chronic renal failure in an individual
comprising: testing a blood sample from an individual to determine
the presence in the sample of BMP-1 isoforms BMP-1-3 and BMP-1-5,
wherein the presence of both said BMP-1 isoforms in circulating
blood of said individual is indicative of chronic renal failure in
said individual.
3. A method of treating ischemic acute renal failure in an
individual comprising administering a BMP-1 isoform systemically to
the individual after diagnosis of renal injury.
4. A method of treating ischemia/reperfusion damage to a kidney in
an individual comprising: administering to the individual one or
more antibody molecules specific for one or more BMP-1 isoforms in
an amount effective to inhibit ischemia/reperfusion injury in said
individual.
5. The method according to claim 4, wherein the one or more
antibody molecules to one or more BMP-1 isoforms is administered
systemically to the individual prior to an ischemia/reperfusion
event.
6. The method according to claim 4, wherein an antibody molecule to
BMP-1-1, an antibody molecule to BMP-1-3, or a combination of such
antibody molecules is administered to the individual.
Description
RELATED APPLICATIONS
[0001] This application is a divisional application of U.S. Ser.
No. 12/309,510, filed Jan. 21, 2009, which is a United States
national stage filing under 35 U.S.C. .sctn.371 of international
application No. PCT/US2007/016605, filed Jul. 23, 2007, designating
the U.S., which claims priority to U.S. Provisional Application No.
60/832,325, filed Jul. 21, 2006.
FIELD OF THE INVENTION
[0002] This invention is in the field of diagnosis and regeneration
of tissue defects and disorders. In particular, the invention
provides compositions and methods comprising isoforms of BMP-1 to
diagnose and treat tissue defects and disorders.
BACKGROUND
[0003] Bone morphogenetic proteins (BMPs) are bone-inducing
(osteogenic, osteoinductive) molecules that have been purified and
characterized from bone (Sampath and Reddi, Proc. Natl. Acad. Sci.
USA, 78: 7599 (1981)). The term "bone morphogenetic protein",
"BMP", and "morphogen" are synonymous and refer to members of a
particular subclass (i.e., the BMP family) of the transforming
growth factor-.beta. (TGF-.beta.) superfamily of proteins (see,
e.g., Hoffmann et al., Appl. Microbiol. Biotechnol., 57: 294-308
(2001); Reddi, J. Bone Joint Surg., 83-A(Supp. 1): S1-S6 (2001);
U.S. Pat. Nos. 4,968,590; 5,011,691; 5,674,844; 6,333,312). All
such BMPs have a signal peptide, prodomain, and a carboxy-terminal
(mature) domain. The carboxy-terminal domain is the mature form of
the BMP monomer and contains a highly conserved region
characterized by seven cysteines that form a cysteine knot (see,
Griffith et al., Proc. Natl. Acad. Sci. USA, 93: 878-883 (1996)).
BMPs were originally isolated from mammalian bone using protein
purification methods (see, e.g., Urist et al., Proc. Soc. Exp.
Biol. Med., 173: 194-199 (1983); Urist et al., Proc. Natl. Acad.
Sci. USA, 81: 371-375 (1987); Sampath et al., Proc. Natl. Acad.
Sci. USA, 84: 7109-7113 (1987); U.S. Pat. No. 5,496,552). However,
BMPs have also been detected in or isolated from a variety of other
mammalian tissues and organs such as kidney, liver, lung, brain,
muscle, teeth, and gut. Most BMPs (including BMP-2, BMP-4, BMP-6,
BMP-7, BMP-9, BMP-12, BMP-13) also stimulate cartilage and bone
formation as demonstrated in a standard ectopic assay for bone
formation (see, e.g., Sampath and Reddi, Proc. Natl. Acad. Sci.
USA, 80: 6591-6595 (1983)). Accordingly, such authentic BMPs are
also referred to as "osteogenic" even though they may also promote
soft tissue regeneration.
[0004] The protein referred to routinely as BMP-1 is not an
authentic member of the BMP family of osteogenic, tissue
regenerative proteins. BMP-1 was originally isolated from highly
purified BMP bovine bone extracts and was originally reported to
induce the formation of cartilage in vivo in a subcutaneous
(ectopic) bone formation assay (Wozney et al., Science, 242: 1528
(1988)). However, BMP-1 does not share significant amino acid
sequence homology with other BMPs, nor does BMP-1 exhibit the
characteristic signal peptide, prodomain, carboxy-terminal (mature
domain), or cysteine knot found in other BMPs. In fact, BMP-1 was
shown to be identical to procollagen C-proteinase, an enzyme
essential for the proper assembly of collagen within the
extracellular matrix (ECM) (Kessler et al., Science, 271: 360-362
(1996)). The erroneous status of BMP-1 within the TGF-.beta. family
resulted from flaws in the original bioassay for osteogenesis
(Wozney et al., op. cit.) in which the cartilage observed in the
bioassay appears to have been old growth plate cartilage
contaminating the insoluble bone matrix that was misidentified as
newly formed tissue (see, Reddi, Science, 271: 463 (1996)). As
shown herein, unlike authentic osteogenic BMPs, the BMP-1-1 isoform
does not induce cartilage or bone formation in a standard ectopic
bone formation assay.
[0005] The BMP-1 gene is related to the Drosophila gene tolloid
(TLD), which is implicated in the patterning controlled by the
decapentaplegic (DPP) gene by virtue of its ability to activate
TGF-.beta.-like morphogens. The BMP-1 protein is now known to be an
essential control point of morphogenesis during the cascade of
pattern formation (Ge and Greenspan, Birth Defect Res., 78: 47-68
(2006)).
[0006] BMP-1 is the prototype of a small subgroup of
metalloproteinases found in a broad range of species. In mammals,
there are four BMP-1/TLD-related (or BMP-1/TLD-like)
metalloproteinases. The gene encoding BMP-1 also encodes a second,
longer proteinase that is encoded by alternatively spliced mRNA.
With a domain structure that is essentially identical to TLD, this
proteinase was designated mammalian Tolloid (mTLD) (Takahara et
al., J. Biol. Chem., 269: 32572-32578 (1994)). In addition, there
are two genetically distinct mammalian BMP-1/TLD-related
proteinases, designated mammalian Tolloid-like 1 and 2 (mTLL1 and
mTLL2). The prodomains of BMP-1/TLD-like proteinases must be
proteolytically removed by subtilisin-like proprotein convertases
(SPCs) (Leighton and Kadler, J. Biol. Chem., 278: 18478-18484
(2003)) to achieve full activity of these proteinases. The role of
the prodomain of BMP-1/TLD-like proteinases appears to be in
maintaining the BMP-1/TLD-like proteinases in a latent form
(Marques et al., Cell, 91: 417-426 (1997); Sieron et al., Biochem.,
39: 3231-3239 (2000); Leighton and Kadler, op. cit.).
[0007] BMP-1/TLD-related metalloproteinases are responsible for the
proteolytic maturation of a number of extracellular proteins
related to formation of the extracellular matrix (ECM). These
include various collagens, small leucine-rich proteoglycans,
SIBLING proteins, and the enzyme lysyl oxidases, laminin-5, and an
anti-angiogenic factor from the basement membrane proteoglycan
perlecan (Iozzo, Nat. Rev. Mol. Cell. Biol., 6: 646-656 (2005);
Greenspan, Top. Curr. Chem., 247: 149-183 (2005); Ge and Greenspan
Birth Defect Res., op. cit.). BMP-1 is also involved in releasing
BMPs from extracellular matrix or in activating latent TGF-.beta.
family members, such as BMP-4, BMP-11 and GDF-8 (Wolfman et al.,
Proc. Natl. Acad. Sci. USA, 100: 15842-15846 (2003); Ge et al, Mol.
Cell. Biol., 25: 5846-5858 (2005)).
[0008] The originally discovered form of BMP-1 is designated as
BMP-1-1, and other BMP-1 isoforms encoded by splice variant RNA
transcripts have been described on the transcriptional level and
designated with sequential suffixes: BMP-1-2, BMP-1-3, BMP-1-4,
BMP-1-5, BMP-1-6, and BMP-1-7 (Li et al., Proc. Natl. Acad. Sci.
USA, 93: 5127-5131 (1996); Wozney et al., Science, 242: 1528
(1988); Janitz et al., J. Mol. Med., 76:141 (1998); Takahara et al
J. Biol. Chem., 269: 32572 (1994); Hillman et al., Genome Biol., 5:
16 (2004). As expected, the BMP-1 isoforms encoded by the splice
variant transcripts share a number of domains, including leader
peptide, proregion, and protease (catalytic) region. Only the
original BMP-1, i.e., BMP-1-1, has previously been confirmed on the
protein level following its isolation from bone. The sequences for
BMP-1-2 and other BMP-1 isoforms were deduced from nucleotide
sequences of the splice variant transcripts, but have not been
described at the protein level.
[0009] Despite the correction in the literature of the identity of
BMP-1-1, whether this protein or other BMP-1 isoforms have any role
of therapeutic relevance remains to be elucidated.
SUMMARY OF THE INVENTION
[0010] The present invention provides new methods of diagnosis and
therapy based on discoveries relating to the circulation of BMP-1
isoforms in the blood of individuals. The differential appearance
of particular isoforms in the circulating blood of individuals has
now been associated with particular bone defects or disorders of
soft tissue. Accordingly, it is now possible for early diagnosis of
particular disorders such as acute bone fracture, chronic renal
failure, fibrodysplasia ossificans progressive, osteogenesis
imperfecta, acute pancreatitis, and liver cirrhosis using a simple
blood test to detect the presence of one or more BMP-1 isoforms in
a sample of blood. Moreover, the discoveries disclosed herein have
led to the development of new treatment methods which enhance the
effects of osteogenic bone morphogenic proteins (BMPs) in
individuals suffering from particular bone defects. (See, Example
14, below.)
[0011] One embodiment of the present invention involves a method of
diagnosing a defect or disorder in a bone or soft tissue of an
individual comprising determining the profile of BMP-1 isoforms in
the blood of the individual and comparing the profile to a standard
blood profile of BMP-1 isoforms associated with various defects and
disorders. Such a standard blood profile based on pooled blood from
healthy individuals and individuals undergoing treatment for
various bone and soft tissue disorders is presented in Table 1
(infra).
[0012] The diagnostic methods of the present invention are
advantageously carried out using detector molecules capable of
binding to one or more BMP-1 isoforms. Suitable such detector
molecules include antibody molecules (including polyclonal
antibodies and monoclonal antibodies, and binding fragments of
antibodies such as Fab fragments, F(ab').sub.2 fragments, and the
like) and aptamers (i.e., nucleic acid molecules that have a
specific binding affinity for particular proteins).
[0013] Thus, in a particular embodiment for diagnostic methods of
the invention, a blood isoform profile for an individual is made,
using one or more detector molecules to assay a sample of blood
from the individual for the presence of one or more BMP-1 isoforms.
Circulating BMP-1 isoforms, or the complete absence of any
circulating isoforms, is demonstrated herein to be indicative of
particular disorders. The ability to detect these defects or
disorders from a blood sample is advantageous because a positive
diagnosis can be achieved much earlier in the course of the
disorder. Acute pancreatitis, for example, may be detected from the
presence of circulating BMP-1-7 and may be diagnosed prior to the
manifestation of more overt symptoms of the disease. Similarly, an
acute bone fracture such as a hairline fracture or crack that is
not easily detectable (or not detectable without expensive x-rays)
may be deduced in the first instance using a blood test and
observing the complete absence of BMP-1 isoforms. In particular
embodiments, detector molecules such as antibody molecules or
aptamers specific for one or more BMP-1 isoforms are used in an
assay to detect the presence of one or more BMP-1 isoforms in a
sample of blood, and the presence of certain isoforms (or the
complete absence of isoforms) is indicative of a disorder
associated with such presence (or absence) herein.
[0014] Preferred detector molecules for the diagnostic methods of
this invention are monoclonal antibody molecules. A suitable
anti-BMP-1 isoform antibody molecule for use herein may be an
immunoglobulin, a Fab fragment, a F(ab').sub.2 molecule, a single
chain antibody molecule (scFv), a double scFv molecule, a single
domain antibody molecule (dAb), a Fd molecule, a diabody molecule,
a fusion protein comprising any of said antibody molecules, or
combinations of one or more of the foregoing.
[0015] In a particular method according to the present invention, a
method is provided for diagnosing liver cirrhosis in an individual
comprising: testing a blood sample from an individual to determine
the presence in the sample of the BMP-1 isoforms BMP-1-1, BMP-1-3,
BMP-1-5, and BMP-1-7, wherein the absence of said BMP-1 isoforms in
the sample is indicative of liver cirrhosis in the individual.
[0016] Another particular embodiment of the present invention is a
method for diagnosing acute bone fracture in an individual
comprising: testing a blood sample from an individual to determine
the presence in the sample of the BMP-1 isoforms BMP-1-1, BMP-1-3,
BMP-1-5, and BMP-1-7, wherein the absence of said BMP-1 isoforms in
the sample is indicative of an acute bone fracture in the
individual.
[0017] A further embodiment of the present invention is a method
for diagnosing acute pancreatitis in an individual comprising:
testing a blood sample from an individual to determine the presence
in the sample of the BMP-1 isoform BMP-1-7, wherein the presence of
said BMP-1 isoform in circulating blood of said individual is
indicative of acute pancreatitis in the individual.
[0018] A further embodiment of the present invention is a method
for diagnosing chronic renal failure in an individual comprising:
testing a blood sample from an individual to determine the presence
in the sample of the BMP-1 isoforms BMP-1-3 and BMP-1-5, wherein
the presence of both said BMP-1 isoforms in circulating blood of
said individual is indicative of chronic renal failure in said
individual.
[0019] A particularly advantageous method disclosed herein is a
method for diagnosing fibrodysplasia ossificans progressive in an
individual comprising: testing a blood sample from an individual to
determine the presence in the sample of the BMP-1 isoform BMP-1-3,
wherein elevated levels (for example at least 5 times) of said
BMP-1 isoform in comparison with levels of the same isoform in a
healthy individual is indicative of fibrodysplasia ossificans
progressive in said individual.
[0020] Another particularly advantageous embodiment of the present
invention is a method for diagnosing osteogenesis imperfecta in an
individual comprising: testing a blood sample from an individual to
determine the presence in the sample of the BMP-1 isoform
[0021] BMP-1-3, wherein elevated levels (for example, at least 5
times) of said BMP-1 isoform in comparison with levels of the same
isoform in a healthy individual is indicative of osteogenesis
imperfecta in said individual.
[0022] A further embodiment of the present invention is a method of
treating an individual for a defect or disorder in bone or soft
tissue of an individual comprising: [0023] (a) diagnosing a defect
or disorder in a bone or soft tissue in an individual by steps
comprising: [0024] (i) determining the profile of BMP-1 isoforms in
the blood and [0025] (ii) comparing the profile to a standard blood
profile of BMP-1 isoforms associated with various defects and
disorders, [0026] (b) administering to the individual an amount of
at least one BMP-1 isoform effective to enhance the therapeutic
effect of an osteogenic BMP toward the diagnosed defect or
disorder, or administering to the individual an amount of one or
more antibody molecules specific for one or more BMP-1 isoforms
effective to inhibit the effects of said one or more BMP-1 isoforms
in the progression of the diagnosed defect or disorder.
[0027] The diagnosing step (a) of the foregoing method may be
performed by comparing the patient's blood BMP-1 isoform profile
with, for example, the standard blood isoform association table
shown in Table 1, below. The therapeutic step (b) of the foregoing
method may be accomplished via systemic or local administration of
the therapeutic agent. In treating bone defects in particular,
local administration to the area of the defect is preferred. Local
administration of BMP-1 isoform BMP-1-1, for instance, is shown
herein to accelerate bone repair in in vivo fracture models. (See,
Examples 12 and 14, below.) Local administration of a BMP-1 isoform
and/or an authentic, osteogenic BMP such as BMP-7 may
advantageously be effected using a whole blood coagulum as a
carrier/matrix for localized delivery of those agents to the bone
defect. A whole blood-derived coagulum device is described herein
which provides a mechanically stable (self-supporting) therapeutic
with the consistency of a gel, which in turn is easily applied to
bone ends or in a gap between bone sections where rebridgement of
bone is desired.
[0028] In particular embodiments of the foregoing diagnostic
methods, the detection step will be directed toward detecting one
or more of BMP-1-1, BMP-1-3, BMP-1-5, and BMP-1-7, having the amino
acid sequences shown in SEQ ID NO:1, SEQ ID NOS:2 or 4, SEQ ID
NO:6, and SEQ ID NO:7, respectively, or detecting an epitope or a
detectable fragment (such as a tryptic fragment) of said amino acid
sequences.
[0029] In a particular embodiment, the present invention provides
an osteogenic whole blood-derived coagulum device (WBCD) for
treating a bone defect in an individual prepared by mixing together
in an aliquot of whole blood a substance providing calcium ions
(Ca.sup.++), such as calcium chloride; at least one BMP-1 isoform
and optionally at least one osteogenic BMP; and optionally a
composition comprising fibrin and thrombin. The mixture is
incubated until a coagulum having the consistency of a mechanically
stable gel forms, and thereafter the coagulum is easily applied as
a matrix to the site where bone rebridgement or repair is desired.
Such mechanically stable gel will preferably be homogenous,
cohesive, syringeable, injectable and malleable. The consistency of
the coagulum ensures that the mixture, entraining the therapeutic
BMP (if present) and BMP-1 isoform, will remain in place adjacent
the bone defect to be repaired.
[0030] The proportions of the ingredients of the coagulum may be
varied, but the amount of calcium ion substance should be such that
the concentration of calcium ion provides a coagulum gel having the
desired features mentioned above. A preferred concentration of
calcium ions in the coagulum will fall in the range of 1-2.5 mM.
Calcium chloride is a preferred exogenous Ca.sup.++-supplying
substance. When calcium chloride is used in a WBCD of the
invention, the concentration is advantageously in the range of 5-15
mM.
[0031] The amount of BMP-1 isoform in a coagulum according to the
invention is advantageously in the range of 1-500 .mu.g/mL,
preferably 2-200 .mu.g/mL, more preferably 5-20 .mu.g/mL, although
lesser or greater amounts may also be used: it is a basic discovery
disclosed herein that the presence of BMP-1 isoforms is helpful to
catalyze the activity of authentic, osteogenic BMPs locally, e.g.,
in repairing bone defects and rebridging bone fractures. Thus, any
amount of a BMP-1 isoform effective to enhance the osteogenic
activity of BMP (whether activated from the extracellular matrix or
supplied exogenously, e.g., as a component of a whole blood-derived
coagulum device) may be used. Similarly, if one or more BMP is used
as a component of a coagulum device according to the invention, the
amount may advantageously be adjusted to fall in the range of
50-500 .mu.g/mL, preferably 100-200 .mu.g/mL. As with the BMP-1
isoform component, however, lesser or greater amounts are
contemplated, and any amount of a BMP effective to promote
osteogenesis at the intended site of the bone defect may be used.
Alternatively, the amounts of a BMP-1 isoform or a BMP used in a
coagulum may be adjusted to provide an overall dose of isoform or
BMP based on the overall weight of the individual, considering the
amount of coagulum to be used. For example, an amount of BMP-1
isoform to provide 2-200 .mu.g/kg, preferably 5-20 .mu.g/kg, more
preferably 8-12 .mu.g/kg patient weight, may be used; and an amount
of a BMP to provide, e.g., 1-1000 .mu.g/kg, preferably 2-500
.mu.g/kg, more preferably 50-200 .mu.g/kg, most preferably 100
.mu.g/kg patient weight, may be used. In determining the amounts of
ingredients for use in a WBCD, it will be understood that the
amounts or volumes of the ingredients cannot be so much (or so
little) as to adversely affect the desired features of the coagulum
gel.
[0032] Accordingly, in a particular embodiment of the invention, an
osteogenic whole blood-derived coagulum device (WBCD) for treating
a bone defect in an individual is prepared by the steps comprising:
[0033] (a) mixing together: [0034] (i) whole blood, [0035] (ii)
2-200 .mu.g/mL of at least one BMP-1 isoform, [0036] (iii) 5-15
millimoles/L calcium chloride, [0037] (iv) optionally, a mixture
comprising 5-10 mg/mL fibrin and 0.5-5 mg/mL thrombin; and [0038]
(b) incubating the mixture of step (a) until a mechanically stable
gel is formed.
[0039] If desired, an amount of a BMP, preferably in the range of
50-500 .mu.g/mL, may be added to the mixture of (a) in the
foregoing embodiment, to take advantage of the synergistic effect
of the combination of BMP-1 isoform and BMP disclosed herein.
[0040] Many suitable substances for providing calcium ions are
known. Calcium chloride is preferred.
[0041] Fibrin-thrombin mixtures useful in a WBCD described herein
may be made by simply mixing fibrin and thrombin in with the other
ingredients of the WBCD. Alternatively, fibrin and thrombin may be
premixed or purchased as a mixture and the mixture then added to
the other ingredients. Fibrin-thrombin mixtures useful in a WBCD
include those known in the art as "fibrin glue" or "fibrin
sealant". Commercial preparations of fibrin-thrombin mixtures,
fibrin glues, and fibrin sealants are readily available. Fibrin and
thrombin used in preparing a WBCD as described herein are of
pharmaceutically acceptable quality and are not a source of
significant immunogenicity that would normally elicit an immune
response in most individuals.
[0042] An exogenously provided fibrin-thrombin mixture may enhance
one or more of the properties provided to the coagulum gel by
calcium ion as mentioned above. In addition, a fibrin-thrombin
mixture can also be used to entrap the BMP-1 isoform (and optional
BMP) component(s) of a WBCD. Such entrapment of such active
ingredients enhances retention by the WBCD and thereby decreases
the rate of migration of the active ingredients from the WBCD and
the local defect site to which the WBCD has been applied.
Preferably, the exogenously provided fibrin-thrombin mixture used
in a WBCD described herein provides fibrin in the range of 5 mg/mL
to 10 mg/mL, inclusive, and provides thrombin in the range of 0.5
mg/mL to 5 mg/mL.
[0043] In preparing the osteogenic WBCD according to the invention,
the whole blood is most preferably autologous whole blood drawn
from the individual. Thus, it is contemplated that the WBCD will be
prepared for use in bone repair surgery, in the operating theater,
immediately prior to use, and employing the patient's own whole
blood to make the WBCD. This has the obvious advantage of avoiding
the necessity of typing and cross-matching donor blood for
administration to a particular patient. Nevertheless, it is
recognized that in some situations, crossmatched whole blood may be
used as, e.g., when a patient may already have lost a significant
amount of blood or may already be receiving a blood transfusion. In
such situations, the use of crossmatched whole blood in a WBCD
introduces the same or similar risks of serum sickness associated
with any transfusion employing crossmatched whole blood.
[0044] In a particular embodiment, the osteogenic WBCD according to
the invention may be prepared by first combining any
fibrin/thrombin composition, the calcium ion substance, and the
BMP-1 isoform and optionally BMP to form a first mixture, then
adding whole blood to the first mixture to form a second mixture,
and incubating the second mixture until a mechanically stable
(self-supporting) gel is formed.
[0045] In another embodiment, all the components necessary for
preparation of a WBCD except the whole blood component may be
conveniently and advantageously collected in a kit. The kit may be
opened and used in the operating room at the moment it is needed,
to form a WBCD using autologous blood obtained from the patient.
Such a kit could include, for example, the following items: [0046]
(a) a vial containing one or more lyophilized BMP-1 isoform, [0047]
(b) a buffer for reconstituting the lyophilized BMP-1 isoforms(s),
[0048] (c) a syringe for reconstituting the lyophilized BMP-1
isoform(s) in the buffer, [0049] (d) a vaccutainer for collecting a
patient's blood, [0050] (e) a sterile solution of 1 M calcium
chloride, [0051] (f) a fibrin-thrombin mixture, [0052] (g) a
container for mixing whole blood with the reconstituted BMP-1
isoform(s) and other ingredients, [0053] (h) a spatula or syringe
(or both) suitable for applying an osteogenic coagulum to bone ends
during open bone repair surgery, and [0054] (i) instructions for
the preparation and use of a WBCD comprised of whole blood mixed
with one or more BMP-1 isoforms, calcium chloride and, optionally,
a mixture comprising fibrin and thrombin, to form a mechanically
stable gel suitable for application to a bone defect.
[0055] The discoveries disclosed herein provide new approaches to
therapy of bone defects and soft tissue disorders, based on the
discovered role of BMP-1 isoforms and their presence in circulating
blood.
[0056] In a particular embodiment, a method is provided for
treating ischemic acute renal failure in an individual comprising
administering a BMP-1 isoform systemically to the individual after
diagnosis of renal injury. (See, Example 8, below.) In a related
embodiment, a method is provided for treating chronic renal failure
in an individual comprising administering systemically to the
individual one or more antibody molecules specific for one or more
BMP-1 isoforms. (See, Example 9, below.) In a particular embodiment
of this method, the antibody molecule is an antibody molecule
specific for the BMP-1-1 isoform, an antibody molecule specific for
the BMP-1-3 isoform, or a combination of such antibody
molecules.
[0057] A further embodiment of the invention provides a method of
treating ischemia/reperfusion damage to a kidney in an individual
comprising: administering to the individual one or more antibody
molecules specific for one or more BMP-1 isoforms in an amount
effective to inhibit ischemia/reperfusion injury in said
individual. In particular embodiments, one or more antibody
molecules recognizing one or more BMP-1 isoforms is administered
systemically to the individual prior to an ischemia/reperfusion
event. In particular, an antibody molecule binding to BMP-1-1, an
antibody molecule binding to BMP-1-3, or a combination of such
antibody molecules may be administered to the individual.
[0058] The present invention also provides a method of pretreating
an individual to resolve clots that may occur during thoracic or
abdominal surgery comprising administering a BMP-1 isoform to the
individual prior to surgery in an amount effective to resolve clots
that occur.
[0059] A further embodiment of the present invention provides a
method of treating acute pancreatitis in an individual comprising
administering to the individual a therapeutically effective amount
of at least one antibody molecule specific for a BMP-1 isoform. In
particular, in this embodiment, an anti-BMP-1-7 antibody molecule
may be used.
[0060] A further embodiment of the present invention provides a
method of treating pancreatitis in an individual comprising
administering to an individual suffering from pancreatitis, after
the acute phase of the inflammatory process, an amount of a BMP-1
isoform in an amount effective to promote pancreatic regeneration.
In particular, in this embodiment, the BMP-1-7 isoform may be
administered.
[0061] In the course of our investigation of circulating BMP-1
isoforms, we also isolated, from a placental cDNA library, a
polynucleotide encoding a previously unreported variant of BMP-1
isoform BMP-1-3. The coding sequence for this isoform is shown in
SEQ ID NO:5; the amino acid sequence for this variant isoform is
shown in SEQ ID NO:4. The BMP-1-3 isoform expressed from the
isolated placental cDNA exhibits some additional properties as
compared to the previously reported BMP-1-3 isoform (SEQ ID NO:2).
(See, Example 5, below.) Accordingly, an additional aspect of the
present invention is to provide an isolated polynucleotide encoding
the polypeptide having the amino acid sequence of SEQ ID NO:4. One
such polynucleotide has the sequence of SEQ ID NO:5.
[0062] In its broadest aspects, the present invention relates to
the use of a detector molecule that specifically binds a BMP-1
isoform in an in vitro diagnostic method to test for the presence
of one or more BMP-1 isoforms in circulating blood of an
individual, for diagnosing a defect or disorder in bone or soft
tissue in said individual. In preferred embodiments such a detector
molecule is an antibody molecule or an aptamer. Advantageously,
such detector molecules are detectably labeled.
[0063] The present invention, in its therapeutic aspects, provides
for the use of a BMP-1 isoform in the manufacture of a medicament
for the treatment of bone defects. Also, the present invention
provides for the use of an antibody molecule that binds a BMP-1
isoform in the manufacture of a medicament for treatment of soft
tissue disorders as herein described.
BRIEF DESCRIPTION OF THE DRAWINGS
[0064] FIG. 1 shows a graph of the concentration (mg/dL) of
creatinine versus time (days) in blood of rats subjected to
ischemic acute renal failure. Diagonal line bars show levels of
creatinine in the blood of rats of the control group (ischemia, no
treatment) at indicated times after the ischemic event. Stippled
bars show levels of creatinine in blood of rats treated
systemically with antibodies to BMP-1-1 and to BMP-1-3 prior to
ischemia and for five days thereafter. Asterisks indicate
significant (P<0.01) difference between creatinine levels
between animals treated with antibodies and those of the untreated
control group. The results indicate that systemic administration of
BMP-1-1 and BMP-1-3 neutralizing antibodies prevented loss of
kidney function in rats with ischemia/reperfusion acute renal
failure if administered prior to injury. See Example 7, below, for
details.
[0065] FIG. 2 shows histological sections of kidney tissues from
rats subjected to ischemia/reperfusion acute renal failure as
described for FIG. 1, above, and in Example 7, below. Panel 2A
shows a representative histological section of kidney tissue from a
rat of the control group that was subjected to acute
ischemia/reperfusion injury without antibody therapy (physiological
saline vehicle, pH 7.2, only). Significant loss of structural
integrity of kidney tissue is evident in Panel 2A. Panel 2B shows a
representative histological section of kidney tissue from a rat of
the prophylactic therapy group that was systemically administered
antibodies to BMP-1-1 and BMP-1-3 prior to acute
ischemia/reperfusion injury and for five days thereafter. Tissue in
Panel 2B indicates significant preservation of kidney structures,
as compared to the untreated tissues depicted in Panel 2A. See,
Example 7, below, for details.
[0066] FIG. 3 shows a graph of the percent survival of rats over
time (days) after ischemic acute renal failure injury as described
in Example 8, below. Diamonds (.diamond-solid., "control") show
survival of rats in the negative control group that did not receive
therapy after ischemia/reperfusion injury. Squares (.box-solid.,
"BMP-7") show survival of rats in the positive control group that
received BMP-7, a known therapeutic agent for treatment of
ischemia/reperfusion injury in kidney. Triangles (.tangle-solidup.,
"BMP-1") show survival of rats that received BMP-1-1 after injury.
Diagonal crosses (x, "BMP-1 Ab") show survival of rats that
received antibody to BMP-1-1 after injury. The results indicate
that administration of BMP-1-1 isoform after injury increased the
survival rate of rats with ischemia/reperfusion acute renal
failure. See, Example 8, for details.
[0067] FIG. 4 shows fractures in femurs after 8 weeks from rats
treated systemically with BMP-1-1 (bones 4A and 4D), BMP-7 (bones
4B, 4C, and 4E), and antibody to BMP-1-1 (bone 4F). Systemic
administration of BMP-1-1 to rats resulted in accelerated healing
of fractures as compared to systemic administration of BMP-7 to
rats. Systemic administration of BMP-1-1 neutralizing antibody
delayed the fracture healing due inhibition of BMP-1-1 activity at
the fracture site.
[0068] FIGS. 5A and 5B show ulnar defect in representative bone
after 6 weeks from rabbits of a control group treated locally with
a whole blood-derived coagulum device (WBCD) only, without BMP-1
isoform or BMP-7, as described in Example 14, below.
[0069] FIGS. 6A and 6B show ulnar defect in representative bone
after 6 weeks from rabbits treated locally with a WBCD containing
BMP-1-1 as described in Example 14, below.
[0070] FIGS. 7A and 7B show ulnar defect in representative bone
after 6 weeks from rabbits treated locally with a WBCD containing
BMP-7 as described in Example 14, below.
[0071] FIGS. 8A and 8B show ulnar defect in representative bone
after 6 weeks from rabbits treated locally with a WBCD containing
BMP-1-1 and BMP-7 as described in Example 14, below.
DETAILED DESCRIPTION OF THE INVENTION
[0072] In order that the invention may be fully understood the
following terms are defined.
[0073] "Antibody" or "antibody molecule", as used and understood
herein, refers to a specific binding member that is a protein
molecule or portion thereof or any other molecule, whether produced
naturally, synthetically, or semi-synthetically, which possesses an
antigenic binding domain formed by an immunoglobulin variable light
chain region or domain (V.sub.L) or portion thereof, an
immunoglobulin variable heavy chain region or domain (V.sub.H) or
portion thereof, or a combination thereof. The term "antibody" also
covers any polypeptide or protein molecule that has an
antigen-binding domain that is identical, or homologous to, an
antigen-binding domain of an immunoglobulin. Antibodies may be
"polyclonal", i.e., a population of antigen-binding molecules that
bind to different sites on the antigen, or "monoclonal", i.e., a
population of identical antigen-binding molecules that bind to only
one site on an antigen. Examples of an antibody molecule, as used
and understood herein, include any of the well known classes of
immunoglobulins (e.g., IgG, IgM, IgA, IgE, IgD) and their isotypes;
fragments of immunoglobulins that comprise an antigen binding
domain, such as Fab or F(ab').sub.2 molecules; single chain
antibody (scFv) molecules; double scFv molecules; single domain
antibody (dAb) molecules; Fd molecules; diabody molecules; and
fusion proteins comprising such molecules. Diabodies are formed by
association of two diabody monomers, which form a dimer that
contains two complete antigen binding domains wherein each binding
domain is itself formed by the intermolecular association of a
region from each of the two monomers (see, e.g., Holliger et al.,
Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993)). Use of such
antibody molecules offers the vast array of antibody detection
systems and formats available in the art that may be adapted to
selectively detect particular BMP-1 isoforms in mixtures, including
whole blood, plasma, serum, and various tissue extracts. Examples
of formats for using antibody molecules to detect BMP-1 isoforms
may include, but are not limited to, immunoblotting (e.g., Western
blots, dot blots), immunoprecipitations, affinity methods,
immunochips, and the like. Any of a variety methods known in the
art may be employed to produce antibody molecules to a specific
BMP-1 isoform or a portion thereof comprising at least one epitope
(antibody binding site) of the BMP-1 isoform.
[0074] "Circulate" and "circulating" describe anything that travels
or is otherwise transported through the vascular system of an
individual.
[0075] The terms "disorder" and "disease" are synonymous and refer
to any pathological condition, irrespective of cause or etiological
agent. A "defect" in a tissue refers to a site of abnormal or
deficient tissue growth. A "disease" or "disorder" may be
characterized by one or more "defects" in one or more tissues.
[0076] As used herein, the terms "treatment" and "treating" refer
to any regimen that alleviates one or more symptoms or
manifestations of a disease or disorder, that inhibits progression
of a disease or disorder, that arrests progression or reverses
progression (causes regression) of a disease or disorder, or that
prevents onset of a disease or disorder. Treatment includes
prophylaxis and includes but does not require cure of a disease or
disorder.
[0077] A "therapeutically effective amount" is an amount of a
compound (e.g., a BMP-1 isoform or a BMP-1 isoform binding molecule
when used therapeutically) which inhibits, totally or partially,
the progression of the condition, which alleviates, at least
partially, one or more symptoms of the disorder, or which enhances
or catalyzes the therapeutic or otherwise beneficial effects of
another compound (e.g., an osteogenic BMP). A therapeutically
effective amount can also be an amount which is prophylactically
effective. The amount which is therapeutically effective will
depend upon the patient's size and gender, the condition to be
treated, the severity of the condition and the result sought. For a
given patient, a therapeutically effective amount can be determined
by methods known to those of skill in the art.
[0078] The term "isolated" when used to describe the various
polypeptides disclosed herein, means a polypeptide that has been
identified and separated and/or recovered from a component of its
natural environment. Contaminant components of its natural
environment are materials that would typically interfere with
diagnostic or therapeutic uses for the polypeptide, and may include
enzymes, hormones, and other proteinaceous or non-proteinaceous
solutes. Isolated polypeptide includes polypeptide in situ within
recombinant cells engineered to express it, since at least one
component of the polypeptide's natural environment will not be
present. Ordinarily, however, isolated polypeptide will be prepared
by at least one purification step. An "isolated polynucleotide" or
isolated polypeptide-encoding nucleic acid is a nucleic acid
molecule that is identified and separated from at least one
contaminant nucleic acid molecule with which it is ordinarily
associated in the natural source of such nucleic acid, e.g., the
human genome. An isolated polynucleotide is other than in the form
or setting in which it is found in nature. Isolated polynucleotides
therefore are distinguished from the specific polypeptide-encoding
nucleic acid molecule as it exists in natural cells. However, an
isolated polynucleotide includes polypeptide-encoding nucleic acid
molecules contained in cells that ordinarily express the
polypeptide but where, for example, the nucleic acid molecule is in
a chromosomal location different from that of natural cells.
[0079] "Gel" means a semi-solid jelly-like material.
[0080] "Homogenous", as applied to a coagulum gel, means that the
coagulum gel has a uniform consistency as opposed to a nonuniform
fibrous network connecting clumps of clots.
[0081] "Syringeable" as used herein to describe a coagulum gel
means that the coagulum gel can be drawn up into a syringe with a
needle in the range of 18 to 23 gauge, inclusive, without clogging
the needle or breaking up into clumps.
[0082] "Injectable" as used herein to describe a coagulum gel means
that the coagulum gel can be expelled from a syringe through the
aperture of the syringe or through a needle in the range of 18 to
23 gauge, inclusive, without clogging the aperture or needle and
without breaking up into clumps.
[0083] "Malleable" as used herein to describe a coagulum gel means
that the coagulum gel is capable of being shaped or formed to fill
or cover a bone defect. A malleable coagulum gel is self-supporting
(or mechanically stable) and will subtantially retain the shape
into which it was formed.
[0084] A composition or method described herein as "comprising" one
or more named elements or steps is open-ended, meaning that the
named elements or steps are essential, but other elements or steps
may be added within the scope of the composition or method. To
avoid prolixity, it is also understood that any composition or
method described herein as "comprising" (or "which comprises") one
or more named elements or steps also describes the corresponding,
more limited, composition or method "consisting essentially of" (or
"which consists essentially of") the same named elements or steps,
meaning that the composition or method includes the named essential
elements or steps and may also include additional elements or steps
that do not materially affect the basic and novel characteristic(s)
of the composition or method. It is also understood that any
composition or method described herein as "comprising" or
"consisting essentially of" one or more named elements or steps
also describes the corresponding, more limited, and close-ended
composition or method "consisting of" (or "which consists of") the
named elements or steps to the exclusion of any other unnamed
element or step. In any composition or method disclosed herein,
known or disclosed equivalents of any named essential element or
step may be substituted for that element or step.
[0085] Unless indicated otherwise, the meaning of other terms is
the same as understood and used by persons in the art, including
the fields of medicine, biochemistry, molecular biology, and tissue
regeneration.
[0086] The invention is based on the discovery that BMP-1 isoforms
in the blood of an adult individual (human or other mammal) are
useful as biological markers (biomarkers) for the state or
condition of the tissues of the individual. In particular, the
presence or absence of one or more isoforms of BMP-1 in the blood,
i.e., the BMP-1 isoform blood profile, of an adult individual is
indicative of the health or a particular pathological state of bone
and various soft tissues of the individual. BMP-1-1, which is
identical to the metalloproteinase procollagen C-proteinase (also
referred to as BMP-1 procollagen C-proteinase) was originally
discovered in the bone matrix. However, the BMP-1-1 isoform is not
found circulating in the blood of the healthy adult individual, nor
in patients with various diseases. Previously, the existence of
isoforms other than BMP-1-1 was inferred only at the level of
tissue RNA transcripts.
[0087] Table 1, below, provides profiles of circulating BMP-1
isoforms associated with normal health and with several disorders,
i.e., an acute bone fracture, chronic renal failure, fibrodysplasia
ossificans progressive (FOP), osteogenesis imperfecta (IO), acute
pancreatitis, and cirrhosis of the liver. A description of the
study that generated the diagnostic profiles in Table 1 is provided
in Example 6 (below).
TABLE-US-00001 TABLE 1 BMP-1 isoforms in various tissue defects and
disorders BMP Isoform Pathology of Patient BMP-1-1 BMP-1-3 BMP-1-5
BMP-1-7 healthy (normal) -- + -- -- acute bone fracture -- -- -- --
chronic renal failure -- + + -- FOP -- ++ -- -- OI -- ++ -- --
acute pancreatitis -- -- -- + liver cirrhosis -- -- -- -- FOP =
fibrodysplasia ossificans progressive; IO = osteogenesis imperfecta
++ indicates much higher than normal levels (i.e., at least 5-fold
higher than in healthy individuals)
[0088] Blood obtained from an individual can be easily analyzed for
the presence of various BMP-1 isoforms, e.g., using
isoform-specific antibodies or other isoform detector molecules.
The profile of BMP-1 isoforms in the blood sample can then be
compared to the profiles in Table 1 to diagnose any of the
indicated pathological states.
[0089] Table 1 shows that circulating BMP-1 isoforms are useful as
biological markers (i.e., biomarkers) of a broad spectrum of
diseases. The use of the BMP-1 isoform blood profiles to diagnose
the pathologies in Table 1 is not dependent on an understanding of
the mechanism by which such profiles are generated. Nevertheless,
there are implications to the data presented herein beyond
providing a convenient method of diagnosing various disorders. In
particular, data presented herein demonstrate for the first time
the existence of circulating enzymes that are variant products of a
single gene, BMP-1. Moreover, without wishing to be bound by any
particular mechanism or theory of operation, the data in Table 1
dispel a long-held model for the action of authentic osteogenic
BMPs in which each tissue or organ was assumed to release a
particular authentic BMP (e.g., BMP-4, BMP-5, BMP-6) into the
circulation during injury and in the process of regeneration of
that tissue or organ. On the contrary, as shown in Table 1, in
healthy individuals only the BMP-1-3 isoform circulates, and no
authentic osteogenic BMPs have been found in the blood of healthy
individuals (see, Example 1, below). Moreover, as shown herein, as
much as 80% of intravenously administered BMP-1-3 becomes localized
at the orthotopic site of fractured femurs in rats and results in
an accelerated rate of bone healing compared to untreated control
animals (see, Example 7, below). In addition, in cultures of rat
calvariae, which are rich in ECM, exogenously provided BMP-1-3
promotes release into the culture medium of authentic osteogenic
BMP-4 and BMP-7 (see, Example 9, below). These data are more
consistent with the tissue repair model shown herein, that
circulating BMP-1 isoforms can act catalytically as key processing
enzymes of the ECM (which is a repository of authentic osteogenic
BMP molecules (see, e.g., Martinovic et al., Arch. Cytol. Histol.,
1: 23-36 (2006))) to effect a local release of one or more
authentic osteogenic BMPs. In bone repair, for example, BMP-1
isoform-catalyzed release of authentic BMP locally acts in turn
locally to promote bone regeneration and repair during the
formation of callus during the rebridgement of fractured bone
ends.
[0090] As shown in Table 1, a number of pathological conditions are
characterized by a disappearance of the BMP-1-3 isoform from the
blood, i.e., acute bone fracture, acute pancreatitis, and liver
cirrhosis. If in addition to BMP-1-3, the BMP-1-5 isoform is also
present in the blood of an individual, then the isoform profile is
diagnostic of chronic renal failure (CRF). If BMP-1-3 is found in
the blood at much higher concentrations than in a normal individual
(i.e., at least 5 times the normal level), then the isoform profile
is diagnostic of FOP or OI. If BMP-1-7 is the only isoform present,
then the profile is diagnostic of acute pancreatitis.
[0091] With respect to soft tissue organs, an absence of BMP-1-3 in
the blood may indicate a condition in which the BMP-1-3 accumulates
in a parenchymal organ to facilitate processing of the
extracellular matrix (ECM), which in turn stimulates fibrosis. A
common feature of the soft tissue pathologies in Table 1 is a
progressing fibrosis of the tissue, which untreated can lead to
organ failure. Such fibrosis is characteristic of cirrhosis of the
liver and acute pancreatitis. Accordingly, when a blood profile
indicates the absence of BMP-1-3, and there is no evidence of bone
fracture, chronic renal failure, FOP, or OI, then Table 1 directs
the diagnosis to the specified pathologies of parenchyma organs,
such as liver or pancreas. In such situations, the healthcare
professional is alerted to perform additional tests for pathology
in such organs. Accordingly, such additional tests may include
determining whether one or more parenchyma organs exhibits
increased fibrosis as evidenced by performing standard tests for an
accumulation of collagen, laminin, fibronectin, and other
extracellular molecules leading to increased fibrosis.
[0092] For Table 1, the sera from patients with acute pancreatitis
were collected at an early stage of the disease, i.e., prior to
robust serum elevation of the pancreatic enzymes such as pancreatic
amylase and lipase. Surprisingly, the blood of these patients
contained the BMP-1-7 isoform, which has not been previously
detected at the protein level (that is, as an expressed protein
rather than a theoretical BMP variant deduced from detection of
mRNA transcripts). The appearance in the blood of BMP-1-7 is useful
as an early diagnostic marker for acute injury of the pancreas.
[0093] The BMP-1-3 and BMP-1-5 isoforms were found in patients with
chronic kidney failure on dialysis and suggest a specific function
of these isoforms in the disorder, e.g., involvement in the
fibrotic processes in bone called renal osteodystrophy. The BMP-1-5
isoform has also been detected in the circulation of rats with
chronic renal failure reflecting the severity of the disease. Our
detection of BMP-1-5 in the blood of patients is also the first
demonstration of the BMP-1-5 isoform on the protein level.
[0094] According to the profiles in Table 1, a BMP-1 isoform
profile that indicates there are no BMP-1 isoforms circulating in
the blood of a patient is evidence that the individual has an acute
bone fracture and/or has liver cirrhosis. Both of these conditions
involve fibrosis. Such fibrosis may be beneficial as part of callus
formation in the healing of an acute bone fraction, whereas in soft
tissue, fibrosis is destructive and is characteristic of liver
cirrhosis.
[0095] Determining the circulating BMP-1 isoform profile may be
used not only when an individual presents symptoms of a tissue
defect or disease, but also as part of an individual's routine
blood test conducted by an attending healthcare professional, e.g.,
as part of an annual physical examination. BMP-1 isoforms are
readily detected in samples of blood obtained from an individual
using any of a variety of methods and compositions known in the
art. Such methods include, but are not limited to, high performance
liquid chromatography (HPLC), mass spectrometry (MS) of tryptic
peptides of BMP-1 isoforms, and affinity methods, particularly
those that employ affinity molecules that specifically bind a
particular BMP-1 isoform to the exclusion of other isoforms. Such
affinity molecules include, but are not limited to, antibody
molecules and aptamers. Antibody molecules specific for each BMP-1
isoform are particularly preferred as there is a wide variety of
assay formats available in the art that can employ an antibody
molecule to detect or isolate a target protein present in the blood
of an individual. Such formats include, but are not limited to,
filter paper (e.g., nitrocellulose, cellulose acetate), microtiter
plates, polymeric particles (e.g., agarose, polyacrylamide),
silicon chips, etc. It is understood that for any particular method
used to detect or isolate a BMP-1 isoform from the blood of an
individual, it may be preferred to make such detection or isolation
from the plasma or serum portion of whole blood.
[0096] Recombinant BMP-1 isoforms described herein were cloned and
expressed in eukaryotic and prokaryotic host cells. Such
recombinant cells may be employed to produce sufficient amounts of
the isoforms for use in the methods described herein. The specific
coding sequences for each of the BMP-1 isoforms discussed herein
are known, and the encoded amino acid sequences have been deduced.
See, e.g., EMBL Nucleotide Sequence Database (worldwide
web.ebi.ac.uk/embl). For convenience, the amino acid sequence for
BMP-1-1 is included herein as SEQ ID NO:1. The amino acid sequence
for BPM-1 isoform BMP-1-3 is shown in SEQ ID NO:2, and a cDNA
sequence coding for BMP-1-3 is shown in SEQ ID NO:3. The amino acid
sequence for BMP-1 isoform BMP-1-5 is shown in SEQ ID NO:6. The
amino acid sequence for BMP-1 isoform BMP-1-7 is shown in SEQ ID
NO:7. A new variant form of BMP-1-3 derived from human placenta and
having properties that differ from the previously known form of
BMP-1-3 has been discovered, having the amino acid sequence of SEQ
ID NO:4 and a coding sequence shown in SEQ ID NO:5.
[0097] BMP-1 isoforms and peptides thereof may be produced by
standard recombinant, synthetic, or semi-synthetic methods
available in the art. BMP-1 isoforms and peptides thereof may also
be used to produce various affinity molecules, including polyclonal
and monoclonal antibody molecules, using standard methods available
in the art.
[0098] All or a portion of a nucleotide sequence encoding the
isoforms of SEQ ID NOS:1, 2, 4, 6, and 7 may be incorporated into
the nucleotide sequence of any of a variety of nucleic acid
molecules, such as vectors, primers, nucleic acid probes for
hybridization, and the like. Such recombinant nucleic acid
molecules may be used to clone nucleic acid molecules encoding a
BMP-1 isoform of interest, to identify or detect BMP-1 isoform
nucleotide sequences (e.g., by various hybridization methods),
and/or to amplify a nucleic acid molecule encoding a BMP-1 isoform
of interest (e.g., using a polymerase chain reaction (PCR)
protocol). Nucleic acid molecules may be synthesized chemically
(e.g., using an automated nucleic acid synthesizer), produced by
PCR, and/or produced by various recombinant nucleic acid methods
known in the art. Nucleic acid molecules may be synthesized with
various modifications known in the art to provide molecules that
resist cleavage by various nucleases and chemicals, such as
replacing phosphodiester linkages with thiol linkages. Methods of
detecting a specific nucleotide sequence (DNA, cDNA, or RNA)
encoding all or a portion of a BMP-1 isoform are well known in the
art and include, without limitation, Southern blots (for DNA and
cDNA), Northern blots (for RNA), polymerase chain reaction (PCR)
methods, dot blots, colony blots, and in vitro transcription of DNA
or cDNA molecules. Nucleic acid molecules as described herein may
also be immobilized by standard methods to any of a variety
surfaces including but not limited to a cellulose-containing paper
(e.g., nitrocellulose, cellulose acetate), nylon, a well of a
plastic microtiter dish, polymeric particles (e.g., agarose
particle, acrylamide particles), and a silicon chip.
[0099] The profiles in Table 1 also suggest possible targets for
drug discovery and new methods of treating defects and disorders.
For example, as noted above, BMP-1 isoforms are implicated as key
enzymes to promote fibrosis. Accordingly, fibrotic diseases may be
treated by inhibiting or inactivating one or more BMP-1 isoforms
that are implicated in tissue fibrosis. A preferred method of
treating a fibrotic disease comprises administering to a patient an
antibody to a BMP-1 isoform associated with tissue fibrosis. Such
fibrotic diseases include, without limitation, fibrotic kidney
disease, liver cirrhosis, acute pancreatitis, and FOP. For example,
in a method of treating a patient with chronic renal failure and on
dialysis therapy, an antibody to a BMP-1 isoform(s) may be
administered to the patient to delay the kidney failure and prevent
the development of renal osteodystrophy, which leads to fragile
bones and fibrotic bone marrow that inhibits the regenerative
process. In patients with FOP, an antibody molecule may be
administered to inhibit a BMP-1 isoform to prevent or inhibit
ectopic ossifications, which depend on the fibrotic process to
develop the characteristic "second skeleton" of FOP patients.
Preferably, an antibody molecule useful in methods described herein
is an antibody molecule that has very low or, most preferably, no
immunogenicity, so that the antibody molecule may be administered
in multiple doses to a patient without invoking an immune response
in the patient that would inactivate the antibody molecule. It is
also understood that administration of a therapeutic agent, such as
an antibody, to inhibit or inactivate a BMP-1 isoform, may also
inhibit healing of bone fractures, which depends on fibrosis in the
formation of a bone callus in normal healing of fractures.
Accordingly, it will be appreciated by the healthcare professional
that a therapy described herein to inhibit a BMP-1 isoform(s) is
not recommended until any bone fractures that may be present in a
patient have healed or unless the healing of any fractures in the
patient is outweighed by a more critical need for therapy to
inhibit or inactivate a BMP-1 isoform(s).
[0100] Another method of treatment of the invention comprises
administering a recombinant BMP-1 isoform to a patient lacking a
particular BMP-1 isoform that could accelerate tissue repair or
that could prevent a disease. As shown herein, BMP-1-3 disappears
from circulation and becomes localized in the orthotopic site of
acute bone fracture.
[0101] Administration of recombinant BMP-1-1 to an individual that
has sustained an acute form of a disease can accelerate bone repair
whether the BMP-1 isoform is administered systemically (see,
Example 7, below) or locally (see, Example 8, below).
Administration of a BMP-1 isoform may also be employed
therapeutically to resolve blood clots that can occur in patients
following an ischemic acute renal failure during major open
surgery, such as thoracic or abdominal surgery. In such cases, a
BMP-1 isoform is preferably administered prior to surgery as a
preventative therapy for resolving clots that might form during the
surgery.
[0102] In patients with acute pancreatitis, inhibition of the
BMP-1-7 isoform may be used prophylactically to prevent or to
inhibit progression of the disease, while systemic administration
of BMP-1-7 following the acute phase of the inflammatory process
may be used to promote pancreatic regeneration. The dual function
of BMP-1 isoforms was shown in acute renal failure in rats, where
BMP-1-1 and BMP-1-3 antibodies injected prior to kidney ischemia
preserved the kidney function, while systemic administration of
BMP-1-1 isoform following the ischemia resulted in a significantly
greater survival of rats (see, Example 11, below). Thus, a dual
function of BMP-1-1 isoform in an acute ischemic disease suggests
two treatment methods, i.e., a preventative (prophylactic)
treatment and a therapeutic (regenerative) treatment. Accordingly,
a method of preventing acute kidney ischemic disease may comprise
administering (e.g., parenterally) to an individual an antibody to
one or more BMP-1 isoforms, e.g., antibody to circulating BMP-1-3
isoform and an antibody to circulating BMP-1-1 isoform, to prevent
fibrosis or to prevent substantial progression of fibrosis. In
contrast, a method of treating acute ischemic kidney disease may
comprise administering (e.g., parenterally) to an individual one or
more recombinant BMP-1 isoforms to support better regeneration of
the kidney(s) in a subacute stage of the disease. A method of
treating chronic renal failure may comprise administering (e.g.,
parenterally) to an individual an antibody to one or more BMP-1
isoforms (e.g., antibody molecules to BMP-1-1 and to BMP-1-3) to
inhibit fibrosis and progression of the disease. A healthcare
professional is able to assess the condition of an individual's
kidneys to determine whether the individual is at risk of acute
ischemia and, therefore, is a candidate for preventative treatment
(e.g., antibody molecules to inhibit BMP-1-3 and BMP-1-1 isoforms),
or whether the individual already suffers from significant acute
ischemic kidney disease, so as to be a candidate for the
therapeutic (regenerative) treatment (administration of BMP-1
isoform(s)).
[0103] An important aspect of the findings described herein (see,
Examples, below) is that contrary to the teachings and assumptions
of the prior art, an osteogenic BMP of the BMP family (e.g., BMP-2,
BMP-4, BMP-6, BMP-7, and the like) should not be administered
systemically to provide therapeutic treatment for local repair of
bone fractures or disorders since any compromise in the wall of a
blood vessel may release the osteogenic BMP locally thereby
potentially inducing ossification of local soft tissue. Such
compromise of blood vessels readily occurs at injection sites,
bruises, and wounds where the combination of locally available stem
cells and an osteogenic BMP can result in undesired ossification of
soft tissue (e.g., muscle tissue). In contrast, BMP-1 isoforms such
as BMP-1-3 or BMP-1-1 may be administered systemically to release
an osteogenic BMP from extracellular matrix at a local site of bone
fracture. BMP-1-1 and its isoforms are not authentic BMPs but are
enzymes.
[0104] A BMP-1 isoform may be employed as an active ingredient in a
whole blood-derived coagulum device (WBCD) to treat a bone defect,
such as a fracture or a bone that is characterized by inadequate
bone growth (e.g., as occurs in various metabolic bone disorders),
in an individual. Such WBCDs comprising one or more BMP-1 isoforms
(e.g., BMP-1-1, BMP-1-3) may be implanted or injected into a site
of fracture or other defect characterized by inadequate bone growth
to promote bone regeneration. WBCDs prepared for the delivery of
one or more BMPs are described in detail in commonly assigned,
copending international application no. PCT/US07/016,601, filed 23
Jul. 2007 (PCT Publication No. WO 2008/011192). The disclosure of
that application is hereby incorporated by reference. The discovery
as part of this invention that BMP-1 isoforms catalyze authentic,
osteogenic BMPs from EMC (or introduced from exogenous sources) to
enhance bone repair activity provides a basis for describing herein
improved WBCDs which include at least one BMP-1 isoform or a
combination of at least one BMP-1 isoform with at least one
osteogenic BMP.
[0105] Thus, in a preferred embodiment, this invention provides an
osteogenic WBCD for treating a bone fracture or other bone defect
that is characterized by inadequate bone growth in an individual
comprising:
[0106] (a) whole blood;
[0107] (b) a BMP-1 isoform in the amount of 1-500 .mu.g/mL,
preferably 2-200 .mu.g/mL, more preferably 5-20 .mu.g/mL, and
optionally an authentic BMP in the amount of 50-500 .mu.g/mL;
[0108] (c) an exogenous substance to supply calcium ions
(Ca.sup.++) at a concentration of 1-2.5 mM; and
[0109] (d) optionally, a mixture of 5-10 mg/mL fibrin and 0.5-5
mg/mL thrombin.
[0110] A whole blood-derived coagulum device described herein is
preferably prepared by the steps comprising: [0111] (a) mixing
together: [0112] (1) whole blood, [0113] (2) 1-500 .mu.g/mL,
preferably 2-200 .mu.g/mL, more preferably 5-20 .mu.g/mL, of at
least one BMP-1 isoform, [0114] (3) 5-15 millimoles/L calcium
chloride, and [0115] (4) optionally, a mixture of 5-10 mg/mL fibrin
and 0.5-5 mg/mL thrombin; [0116] (b) incubating the mixture of step
(a) until a mechanically stable (i.e., a non-fluid,
self-supporting, adherent) coagulum gel is formed.
[0117] In the foregoing embodiment, one or more authentic,
osteogenic BMPs, preferably in an amount of 50-500 .mu.g/mL, may
also be added to the mixing step (a).
[0118] In a preferred embodiment, the coagulum device is prepared
by first combining the fibrin-thrombin mixture, calcium ion, and
BMP-1 isoform or BMP components to form a first mixture; followed
by combining said first mixture with whole blood until the
concentrations of the ingredients fall within the ranges set forth
above and a mechanically stable coagulum of gel consistency is
formed.
[0119] Preferably, the whole blood used in the preparation of a
WBCD described herein is the autologous whole blood drawn from the
individual who is to receive the WBCD, as autologous whole blood
will not be immunogenic, that is, will not be rejected as non-self
tissue by the immune system of the recipient. Nevertheless, it is
recognized that in some situations, crossmatched whole blood may be
used as, e.g., when a patient may already have lost a significant
amount of blood or may already be receiving a blood transfusion. In
such situations, the use of crossmatched whole blood in the WBCD
introduces the same or similar risks of serum sickness associated
with any transfusion employing crossmatched whole blood.
[0120] The invention also provides kits for preparing an osteogenic
whole blood-derived coagulum device (WBCD) containing one or more
BMP-1 isoforms for treating a bone defect. For example, in a
preferred embodiment, such a kit may be comprised of: [0121] (a) a
vial containing lyophilized BMP-1 isoform(s), [0122] (b) a buffer
for reconstituting the lyophilized BMP-1 isoforms(s) powder, [0123]
(c) a syringe and a needle for reconstituting the lyophilized BMP-1
isoform(s) in the buffer, [0124] (d) a vaccutainer for collecting a
patient's blood, [0125] (e) a sterile solution of 1 M calcium
chloride, [0126] (f) a fibrin-thrombin mixture, [0127] (g) a
container for mixing whole blood with the reconstituted BMP-1
isoform(s) and other ingredients, [0128] (h) a spatula or syringe
(with or without a needle) (or both) for applying an osteogenic
coagulum to bone ends during open bone repair surgery, and [0129]
(i) instructions for the preparation and use of the WBCD containing
BMP-1 isoform(s) using autologous or crossmatched whole blood.
EXAMPLES
Example 1
[0130] Purification of BMP-1 isoform, but not authentic osteogenic
BMPs, from human blood plasma by heparin Sepharose affinity
chromatography, and protein identification using liquid
chromatography-mass spectrometry (LC-MS).
[0131] This study was originally made to determine whether any
osteogenic BMPs could be detected and isolated from human blood
plasma.
Plasma Collection
[0132] Blood samples from 50 healthy adult humans (21-50 years of
age) were drawn into syringes containing 3.8% sodium citrate to
form an anticoagulant-to-blood ratio (v/v) of 1:9. Plasma was
obtained by centrifugation (15 min. at 3000.times.g), and aliquots
of each adult blood sample were used to make a pooled plasma stock.
Aliquot samples were stored at -80.degree. C. prior to
analysis.
Affinity Column Purification
[0133] Pooled human plasma (80 ml) was diluted 2-fold with 10 mM
sodium phosphate buffer (pH 7), and applied to a 5 ml heparin
Sepharose column (Amersham Pharmacia Biotech) previously
equilibrated with 10 mM sodium phosphate buffer (pH 7). Bound
proteins were eluted from the column with 10 mM sodium phosphate
buffer (pH 7) containing 1.0 M and 2.0 M NaCl.
Ammonium Sulfate Precipitation
[0134] Saturated ammonium sulfate (SAS) was added into the protein
eluate drop-by-drop with mixing on a vortex to a final
concentration of 35% (w/v). Samples were kept on ice for 10
minutes, and centrifuged for 5 minutes at 12,000.times.g. The
supernatant was discarded, and the pellet was prepared for
subsequent analysis by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE).
SDS-PAGE and Western Blot Analysis of the Purified Protein
[0135] The pellet was run on standard SDS-PAGE using a 10% gel
according to the method of Laemmli After electrophoresis, one part
of the SDS-PAGE gel was transferred to nitrocellulose and the other
was directly stained with Coomassie Brilliant Blue (CBB).
Nitrocellulose membrane was first incubated with mouse monoclonal
antibody specific for BMP-7 (Genera Research Laboratory), and kept
overnight at 4.degree. C. Alkaline phosphatase-conjugated goat
anti-mouse antibody was used as secondary antibody for 1 hour at
room temperature. The membrane was developed with 5 ml of a
chromogenic substrate. The other part of the gel was stained with
Coomassie Brilliant Blue (CBB) under standard staining procedure
(0.1% CBB in 45% methanol, 10% acetic acid; 30 minutes at room
temperature).
[0136] The gel was cut into slices corresponding to each protein
band as revealed by staining with CBB. The gel slices were then
processed to determine what proteins were present in each slice
using a method of analyzing tryptic peptides released from each
protein band by HPLC and mass spectrometry (MS) using a
nanoelectrospray LC-MS interface as described by Olsen and Mann
(Proc. Natl. Acad. Sci. USA, 101: 13417-13422 (2004) as modified by
Grgurevic et al. (J. Nephrol., 20: 311-319 (2007)). Aspects of the
steps of this method that are specifically related to this study
are indicated below.
In-Gel Trypsin Digestion Protocol
[0137] Bands in the gel were excised from CBB stained gels and
digested with trypsin. Briefly, gel pieces were shrunk with 100
.mu.l of acetonitrile for 8 minutes. Liquid was removed and gel
pieces were re-swelled with 100 .mu.l of ammonium hydrogencarbonate
for 12 minutes and then dried in SpeedVac for 10 minutes.
Dithiothreitol (DTT, 100 .mu.l) was added and incubated for 45
minutes at 57.degree. C. Gel pieces were shrunk with 100 .mu.l of
acetonitrile for 8 minutes at 57.degree. C., spun down, and liquid
was removed. Iodoacetamide (100 .mu.l) was added to each gel piece
and incubated for 45 minutes at room temperature in the dark
without agitation. Trypsin (10 .mu.l) was added per gel piece. Then
the gel pieces were spun down and re-swelled for 10 minutes.
Samples were incubated overnight at 37.degree. C. in a
thermo-mixer.
Peptide Extraction Protocol
[0138] Samples were removed from the 37.degree. C. thermo-mixer. A
solution (50 .mu.l) containing acetonitrile, water, and formic acid
was added. Samples were sonicated for 15 minutes. Supernatant was
transferred to the reserve tube and 50 .mu.l of acetonitrile were
added. Extracts were dried under vacuum in the SpeedVac to complete
dryness (about 40 minutes). Peptides were re-dissolved with 10
.mu.l of solution containing water, methanol, and formic acid.
Samples were sonicated for 5 minutes, and stored at -20.degree. C.
until analysis.
Mass Spectrometry
[0139] Tryptic peptides were analyzed by liquid chromatography-mass
spectrometry (LC-MS) as follows. Agilent 1100 nanoflow HPLC system
(Agilent Technologies, Palo Alto, Calif.) was coupled to a 7-Tesla
LTQ-FT mass spectrometer (Thermo Electron, Bremen, Germany) using a
nano-electrospray LC-MS interface (Proxeon Biosystems, Odense,
Denmark). Peptides were separated on a home-made 75 .mu.m C.sub.18
HPLC column and mass-analyzed on-the-fly in the positive ion mode.
Each measurement cycle consisted of a full mass spectrometry (MS)
scan, followed by selected ion monitoring (SIM) scan, MS/MS, and
MS/MS/MS scans of the three most intense ions. This provided a
typical peptide mass accuracy of 2 ppm, as well as additional
sequence information from the MS/MS and MS/MS/MS fragment ions.
Resulting spectra were centroided, and searched against NCBInr
database using Mascot search engine (Matrix Science). Searches were
done with tryptic specificity, carboxyamidomethylation as fixed
modification, and oxidized methionine as variable modification.
Mass tolerance of 5 ppm and 0.6 Da was used for MS and MS/MS
spectra, respectively.
Results
[0140] The LS-MS and immunoblotting analyses revealed twelve (12)
tryptic peptides that were compared with the NCBInr database. The
12 peptides were found not to belong to any known osteogenic BMP,
but to the splice isoform 3 of the precursor of BMP-1-3
(Swiss-Prot: P13497-2; SEQ ID NO:2), i.e., procollagen
C-proteinase. The amino acid sequences of each of the 12 peptides
are:
TABLE-US-00002 (amino acids 193-203 of SEQ ID NO: 2) GGGPQAISIGK,
(amino acids 233-238 of SEQ ID NO: 2) HVSIVR, (amino acids 308-314
of SEQ ID NO: 2) GDIAQAR, (amino acids 352-359 of SEQ ID NO: 2)
ISVTPGEK (amino acids 401-411 of SEQ ID NO: 2) LPEPIVSTDSR (amino
acids 497-519 of SEQ ID NO: 2) DGHSESSTLIGRYCGYEKPDDIK (amino acids
529-537 of SEQ ID NO: 2) FVSDGSINK, (amino acids 572-584 of SEQ ID
NO: 2) CSCDPGYELAPDK, (amino acids 653-660 of SEQ ID NO: 2)
SGLTADSK, (amino acids 826-836 of SEQ ID NO: 2) KPEPVLATGSR, (amino
acids 841-849 of SEQ ID NO: 2) FYSDNSVQR, (amino acids 958-966 of
SEQ ID NO: 2) FHSDDTITK.
The 12 peptides had a combined Mascot score of 190, which presents
10.sup.-19 probability of random (false) identification. No other
protein in the NCBInr database matched the same set of peptides. No
authentic osteogenic BMP proteins were detected at molecular weight
of 100 kDa and 35 kDa by LS-MS or by immunoblotting.
[0141] The results indicate that authentic osteogenic BMPs do not
normally circulate in the blood of healthy adult humans, whereas
BMP-1-3, i.e., procollagen C-proteinase, is a soluble protein
component of normal human blood.
Example 2
[0142] Osteogenic BMP cannot be isolated from human blood plasma or
24-hour urine rat sample as determined by heparin Sepharose
affinity chromatography and subsequent protein identification using
mass spectrometry (MS).
Plasma Collection
[0143] Blood samples from 17 healthy adults (21-50 years) were
drawn into syringes containing 3.8% sodium citrate to form an
anticoagulant-to-blood ratio (v/v) of 1:9 Plasma was obtained by
centrifugation (15 min at 3,000.times.g), and aliquots of each
adult sample were used to make a pooled plasma stock. Aliquot
samples were stored at -80.degree. C. prior to analysis.
Urine Collection
[0144] A 24 hour urine sample from healthy rats (Sprague-Dawley, 5
months old, Harlan Winkelmann, Borchen, Germany) was collected in
metabolic cages. Prior to purification, the urine was filtrated
through Whatmann filter paper (large pore size) to remove big
particles. Samples were stored at -80.degree. C. until studied.
Affinity Column Purification of Plasma Samples
[0145] Pooled human plasma (35 ml) was diluted 2-fold with 10 mM
sodium phosphate buffer (pH 7) and applied to a 5 ml heparin
Sepharose column (Amersham Pharmacia Biotech), previously
equilibrated with 10 mM sodium phosphate buffer (pH 7). Bound
proteins were eluted from the column 10 mM sodium phosphate buffer
(pH 7) containing 1.0 M and 2.0 M NaCl.
Affinity Column Purification of Urine Rat Samples
[0146] A 24 hour urine rat sample (20 ml) was diluted 2-fold with
10 mM sodium phosphate buffer (pH 7), and applied to a 1 ml heparin
Sepharose column (Amersham Pharmacia Biotech), previously
equilibrated with 10 mM sodium phosphate buffer (pH 7). Bound
proteins were eluted with 10 mM sodium phosphate buffer (pH 7)
containing 1.0 M and 2.0 M NaCl.
Ammonium Sulfate Precipitation
[0147] Saturated ammonium sulfate (SAS) was added into the protein
eluate drop-by-drop on the vortex until the final concentration of
35%. Samples were kept on ice for 10 minutes, and centrifuged for 5
minutes at 12,000.times.g. Supernatant was discarded, and pellet
was prepared for subsequent SDS-PAGE analysis. The pellet was run
on SDS-PAGE, and proteins in the gel analyzed as described
below.
SDS-Polyacrylamide Gel Electrophoresis and Western Blot Analysis of
the Purified Protein
[0148] The pellet was run on standard SDS-PAGE using a 10% gel
according to the method of Laemmli as described above. After
electrophoresis, one part of the SDS-PAGE gel was then transferred
to nitrocellulose and the other was directly stained with CBB.
Nitrocellulose membrane was first incubated with mouse monoclonal
antibody specific for BMP-7 (Genera Research Laboratory), and kept
overnight at 4.degree. C. Alkaline phosphatase-conjugated goat
anti-mouse was used as the secondary antibody for 1 hour at room
temperature. The membrane was developed with 5 ml chromogenic
substrate. The other part of the gel was stained with CBB under
standard staining procedure (0.1% CBB in 45% methanol, 10% acetic
acid; 30 minutes at room temperature).
[0149] The gel was cut into slices corresponding to each protein
band as revealed by staining with CBB. The gel slices were then
processed to determine what proteins were present in each slice
using the method of analyzing tryptic peptides as described above.
Aspects of the steps of this method that are specifically related
to this study are indicated below.
In-Gel Trypsin Digestion Protocol
[0150] Comparing the molecular weight position of bands on the gel
stained with CBB with their position on the nitrocellulose
membrane, bands 39 kDa, 35 kDa, and 50 kDa from the urine sample
and bands 39 of kDa and 35 kDa from plasma sample were excised from
CBB stained gel. Gel pieces were shrunk with 100 .mu.l of
acetonitrile for 8 minutes. Liquid was removed and gel pieces were
re-swelled with 100 .mu.l of ammonium hydrogencarbonate for 12
minutes and then dried in a SpeedVac for 10 minutes. DTT (100
.mu.l) was added and incubated for 45 minutes at 57.degree. C. Gel
pieces were shrunk with 100 .mu.l of acetonitrile for 8 minutes at
57.degree. C., spin down and liquid were removed. Iodoacetamide
(100 .mu.l) was added to each gel piece and incubated for 45
minutes at room temperature in the dark without agitation. Trypsin
(10 .mu.l) was added per gel piece. Then the pieces were spun down,
and re-swelled for 10 minutes. Samples were incubated overnight at
37.degree. C. in a thermo-mixer.
Peptide Extraction Protocol
[0151] Samples were removed from the 37.degree. C. thermo-mixer. A
solution (50 .mu.l) containing acetonitrile, water, and formic acid
was added. Samples were sonicated for 15 minutes.
[0152] Supernatant was transferred into the reserve tube, and
acetonitrile (50 .mu.l) was added. Extracts were dried in the
SpeedVac to complete dryness (about 40 min.). Peptides were
re-dissolved with 10 .mu.l of a solution containing water,
methanol, and formic acid. Samples were sonicated for 5 minutes,
and stored at -20.degree. C. until analysis.
Mass Spectrometry (MS)
[0153] Tryptic peptides were analyzed by liquid chromatography-mass
spectrometry (LC-MS) as follows: Agilent 1100 nanoflow HPLC system
(Agilent Technologies, Palo Alto, Calif.) was coupled to a 7-Tesla
LTQ-FT mass spectrometer (Thermo Electron, Bremen, Germany) using a
nano-electrospray LC-MS interface (Proxeon Biosystems, Odense,
Denmark). Peptides were separated on a home-made 75 .mu.m C.sub.18
HPLC column and mass-analyzed on-the-fly in the positive ion mode.
Each measurement cycle consisted of a full MS scan, followed by
selected ion monitoring (SIM) scan, MS/MS and MS/MS/MS scans of the
three most intense ions. This has resulted in a typical peptide
mass accuracy of 2 ppm, as well as additional sequence information
from the MS/MS and MS/MS/MS fragment ions.
[0154] Resulting spectra were centroided, and searched against
NCBInr database using Mascot search engine (Matrix Science).
Searches were done with tryptic specificity,
carboxyamidomethylation as fixed modification, and oxidized
methionine as variable modification. Mass tolerance of 5 ppm and
0.6 Da was used for MS and MS/MS spectra, respectively.
Results
[0155] No authentic, osteogenic BMPs were detected in any of the
proteins isolated from the entire molecular range of purified sera
from normal healthy individuals or from urine of rats by mass
spectrometry or by Western blotting.
Example 3
[0156] Lack of ectopic bone formation by implantation of
lyophilized human blood samples into nude mice and autologous rat
lyophilized blood samples into rat.
Blood Collection
[0157] Blood (50 ml) was collected from 10 healthy human
individuals. The blood was centrifuged to remove cells, and the
serum was stored at -20.degree. C. until analyzed. Autologous blood
(5 ml) was collected from ten 6-months old male Sprague Dawley rats
at five time intervals in a period of two weeks. Samples were
centrifuged and the serum was stored at -20.degree. C. until
analyzed.
Implantation into Nude Mice and Rats
[0158] One bone pellet was formed by mixing 100 mg of human
lyophilized blood with 200 mg of demineralized rat bone matrix
(DBM) and implanted into the back area of nude mice. In addition,
20 mg of autologous rat lyophilized blood was mixed with 100 mg of
DBM and implanted subcutaneously into the axillar area of the same
rats from which the blood had been drawn. Pellets were removed
three weeks following implantation, fixed and processed for
histology.
Results
[0159] Tested blood samples implanted under the skin of nude mice
were negative for bone formation, indicating that blood does not
contain authentic osteogenic BMPs in an amount that could induce
ectopic bone formation in mice and rats.
Example 4
[0160] Unlike recombinant human BMP-7, systemically administered
BMP-1-1 does not induce bone formation in an ectopic bone formation
assay.
[0161] Bone pellets consisting of demineralized bone matrix (100
.mu.g) were implanted subcutaneously (ectopic site) into 20 adult
Sprague Dawley rats in the axillar region as described previously
(Simic et al, J. Biol. Chem., 281:13514 (2006)). Ten rats were then
injected intravenously with 20 .mu.g of recombinant human BMP-7
from days 2 to 7 following implantation, while another ten rats
were injected on a similar schedule with recombinant human BMP-1-1.
Two weeks following implantation, the pellets were removed and
processed for histological evaluation.
Results
[0162] In pellets of rats injected with the BMP-7, cartilage and
bone were formed via a mechanism which involved binding of BMP-7 to
the implanted DBM and induction of endochondral bone formation
cascade as previously described (Simic et al, supra). In contrast,
in the pellets of rats treated systemically with BMP-1-1, there was
no cartilage or bone detected, indicating that BMP-1-1 cannot
induce bone at an ectopic site.
[0163] The results indicate that unlike authentic osteogenic BMP-7,
systemically administered BMP-1-1 cannot induce bone formation in
an ectopic bone formation assay.
Example 5
[0164] Cloning and sequence analysis of cDNA encoding BMP-isoforms
from human placental cDNA library.
[0165] The cDNA comprising the coding sequences for BMP-1-1,
BMP-1-3, BMP-1-4, and BMP-1-7 were cloned from a human placental
cDNA library using the GATEWAY.RTM. recombination cloning and
expression system (Invitrogen, Carlsbad, Calif.). The correctness
of clones was confirmed by standard colony PCR and restriction
enzyme analysis.
[0166] The nucleotide base sequences of the cDNA clones were
determined and the corresponding amino acid sequences deduced. The
amino acid sequence for the 83 kDa BMP-1-1 is shown in SEQ ID NO:1.
The nucleotide base sequence of the cDNA clone encoding the BMP-1-3
isoform is shown in SEQ ID NO:3 and the corresponding amino acid
sequence for the 111 kDa BMP-1-3 isoform is shown in SEQ ID NO:2.
The amino acid sequence for the 91 kDa BMP-1-7 isoform is shown in
SEQ ID NO:7.
[0167] The nucleotide base and corresponding amino acid sequences
as determined for the cDNA clone in this study for the BMP-1-1 and
BMP-1-7 isoforms were found to be identical to those present in the
EMBL and Swiss-Prot databases. However, the cDNA sequence for the
BMP-1-3 clone as determined herein differs at a single nucleotide
base from that in the EMBL database. In particular, the EMBL
reference sequence (SEQ ID NO:3) has a thymine (T) base at position
1487, whereas the sequence of cloned BMP-1-3 cDNA (SEQ ID NO:5) has
an adenine (A), which in turn results in a codon change of a CTG
(leucine) in the EMBL sequence to a CAG (glutamine) in the
placental BMP-1-3 cDNA sequence isolated by us. Thus, the amino
acid sequence of the Swiss-Prot database for BMP-1-3 (SEQ ID NO:2)
contains a leucine residue at position 493, whereas the amino acid
sequence of the placental BMP-1-3 protein (SEQ ID NO:4) encoded by
the isolated cDNA clone contains glutamine at position 493.
[0168] Site-directed mutagenesis was performed on the placental
BMP-1-3 protein of the isolated cDNA clone to convert base 1478 of
its reported sequence (SEQ ID NO:3), i.e., a switch from adenine
(A) to thymine (T). On expression, this yielded a "converted"
protein of BMP-1-3 having the amino acid sequence of SEQ ID NO:
2.
Results
[0169] The placental BMP-1-3 protein, which has the amino acid
sequence of SEQ ID NO:4 when expressed from the library-isolated
cDNA clone, and the "converted" BMP-1-3 protein, which has the
amino acid sequence as reported in the Swiss-Prot database (SEQ ID
NO:2), were both active in processing in vitro procollagen type I,
II, and III, with the "converted" BMP-1-3 protein being more active
at lower concentrations. However, the placental BMP-1-3 expressed
from the isolated cDNA clone processed calmodulin and type IV
collagen, which properties were not exhibited with the "converted"
BMP-1-3 protein. Accordingly, the BMP-1-3 isoform expressed from
the cloned cDNA of the placental library differs in both amino acid
sequence and functional enzymatic properties from the BMP-1-3
protein reported in the Swiss-Prot database.
Example 6
[0170] Several specific BMP-1 isoforms circulate in human blood in
different diseases.
Plasma Collection
[0171] Blood samples were drawn from 10 healthy adults, from 10
patients each who were diagnosed and undergoing treatment for
diseases including acute pancreatitis, cirrhosis, acute bone
fracture, chronic renal failure on dialysis, and from 4 patients
with rare bone diseases, namely fibrodysplasia ossificians
progressive (FOP) and osteogenesis imperfecta (OI). The blood
samples were drawn into syringes containing 3.8% sodium citrate to
form an anticoagulant-to-blood ratio (v/v) of 1:9. Plasma was
obtained by centrifugation (15 minutes at 3000.times.g), and
aliquots of each blood sample were used to make a pooled plasma
stock to represent each of the listed normal or pathological cases.
Aliquot samples were stored at -80.degree. C. prior to
analysis.
Affinity Column Purification
[0172] 80 ml of pooled human plasma from each group of patients was
diluted 2-fold with 10 mM sodium phosphate buffer (pH 7), and
applied to a 5 ml heparin Sepharose column (Amersham Pharmacia
Biotech), previously equilibrated with 10 mM sodium phosphate
buffer (pH 7). Bound proteins were eluted from the column with 10
mM sodium phosphate buffer (pH 7) containing 1.0 M and 2.0 M
NaCl.
Ammonium Sulfate Precipitation
[0173] Saturated ammonium sulfate (SAS) was added into the protein
eluate drop-by-drop on the vortex until the final concentration of
35%. Samples were kept on ice for 10 minutes, and centrifuged for 5
minutes at 12,000.times.g. Supernatant was discarded, and pellet
was prepared for subsequent SDS-PAGE analysis.
SDS-PAGE and Western Blot Analysis of the Purified Protein
[0174] The pellet was run on standard SDS-PAGE on a 10% gel
according to the method of Laemmli After electrophoresis, one part
of the SDS-PAGE gel was then transferred to nitrocellulose and the
other was directly stained with Coomassie Brilliant Blue (CBB).
[0175] Nitrocellulose membrane was first incubated with rabbit
polyclonal antibody specific for the BMP-1 carboxyl terminal domain
(Sigma-Aldrich, Chemie GmbH, Germany), and kept overnight at
4.degree. C. Alkaline phosphatase-conjugated anti-rabbit antibody
(Invitrogen Corporation Carlsbad, SAD) was used as secondary
antibody for 1 hour at room temperature. The membrane was developed
with 5 ml chromogenic substrate.
[0176] The other part of the gel was stained under standard
staining procedure (0.1% CBB in 45% methanol, 10% acetic acid; 30
minutes at room temperature).
[0177] The gel was cut into slices corresponding to each protein
band as revealed by staining with CBB. The gel slices were then
processed to determine what proteins were present in each slice
using a method of analyzing tryptic peptides as described above.
Aspects of the steps of this method that are specifically related
to this study are indicated below.
In-Gel Trypsin Digestion Protocol
[0178] Gel pieces were shrunk with 100 .mu.l of acetonitrile for 8
minutes at 57.degree. C., spun down, and liquid was removed. Gel
pieces were re-swelled with 100 .mu.l of ammonium hydrogencarbonate
for 12 minutes and then dried in a SpeedVac for 10 minutes. DTT
(100 .mu.l) was added and incubated for 45 minutes at 57.degree. C.
Iodoacetamide (100 .mu.l) was added to each gel piece and incubated
for 45 minutes at room temperature in the dark without agitation.
Trypsin (10 .mu.l) was added per gel piece, spun down, and gel
pieces were re-swelled for 10 minutes. Samples were incubated
overnight at 37.degree. C. on a thermo-mixer.
Peptide Extraction Protocol
[0179] Samples were removed from the 37.degree. C. thermo-mixer. A
solution (50 .mu.l) containing acetonitrile, water, and formic acid
was added. Samples were sonicated for 15 minutes. Supernatant was
transferred into the reserve tube, and acetonitrile (50 .mu.l) was
added. Extracts were dried in the SpeedVac to complete dryness
(about 40 minutes). Peptides were re-dissolved with 10 .mu.l of a
solution containing water, methanol and formic acid. Samples were
sonicated for 5 minutes, and stored at -20.degree. C. until
analysis.
Mass Spectrometry
[0180] Tryptic peptides were analyzed by liquid chromatography-mass
spectrometry (LC-MS) as follows: Agilent 1100 nanoflow HPLC system
(Agilent Technologies, Palo Alto, Calif.) was coupled to a 7-Tesla
LTQ-FT mass spectrometer (Thermo Electron, Bremen, Germany) using a
nano-electrospray LC-MS interface (Proxeon Biosystems, Odense,
Denmark). Peptides were separated on a home-made 75 .mu.m C.sub.18
HPLC column and mass-analyzed on-the-fly in the positive ion mode.
Each measurement cycle consisted of a full MS scan, followed by
selected ion monitoring (SIM) scan, MS/MS and MS/MS/MS scans of the
three most intense ions. This resulted in a typical peptide mass
accuracy of 2 ppm, as well as additional sequence information from
the MS/MS and MS/MS/MS fragment ions.
[0181] Resulting spectra were centroided, and searched against
NCBInr database using Mascot search engine (Matrix Science).
Searches were done with tryptic specificity,
carboxyamidomethylation as fixed modification, and oxidized
methionine as variable modification. Mass tolerance of 5 ppm and
0.6 Da was used for MS and MS/MS spectra, respectively.
Results
[0182] The results of this study are shown in Table 1 (supra),
which provides profiles of circulating BMP-1 isoforms associated
with normal health and the indicated disorders. The results
indicate that the BMP-1-3 isoform is normally present in the blood
of healthy individuals but disappears from circulation in patients
with acute bone fracture, cirrhosis, and acute pancreatitis. It was
surprisingly noted that in FOP and OI patients BMP-1-3 isoform was
still present, but present at more than ten times the level
observed in the blood of healthy individuals.
[0183] Disappearance of the BMP-1-3 isoform from the circulation of
patients with acute bone fracture confirms the potential function
of BMP-1 isoforms in processing the ECM proteins in bone
regeneration and repair during the formation of callus during the
rebridgement of fractured bone ends. Disappearance of BMP-1-3 from
circulation in patients with cirrhosis suggests its involvement in
processes related to fibrotic changes in the liver. In acute
pancreatitis, several ECM molecules involved in the pathophysiology
of the disease eventually require the BMP-1-3 for processing of ECM
molecules.
[0184] The sera from patients with acute pancreatitis were
collected at an early stage of the disease, i.e., prior to robust
serum elevation of the pancreatic enzymes such as pancreatic
amylase and lipase. Surprisingly, the blood of these patients
contained the BMP-1-7 isoform, which has not been previously
detected at the protein level.
[0185] The BMP-1-5 isoform was found only in patients with chronic
kidney failure on dialysis, which suggests a specific function for
this enzyme isoform, e.g., involvement in the fibrotic processes in
bone called renal osteodystrophy. Interestingly, this is also the
first demonstration of BMP-1-5 isoform on the protein level.
Previously, the BMP-1-5 isoform was inferred only at the level of
tissue mRNA transcripts.
[0186] The presence of BMP-1-3 isoform in circulation was further
confirmed by Western blot using a specific BMP-1-3 antibody
developed by Genera (data not shown).
Example 7
[0187] Protection of kidney function in ischemic acute renal
failure in rats by inhibiting circulating BMP-1-1 and BMP-1-3 prior
to ischemia/reperfusion.
Animals
[0188] Female Sprague-Dawley rats weighting about 350-400 g were
housed and allowed free access to water and food.
Ischemia/Reperfusion Model
[0189] Rats were anesthetized with 100 mg/kg ketamine, 10 mg/kg
xylazine, and 1 mg/kg acepromazine (intramuscularly, im) and placed
on a heating table kept at 37.degree. C. A midline incision was
made and both renal pedicles were clamped for 60 minutes. After
removal of the clamp, 5 ml of prewarmed normal saline were
instilled into the peritoneal cavity, and the incision was sutured.
A total of 24 animals were assigned to two different experimental
groups: [0190] Group 1. Control group (n=12); ischemia/reperfusion
model without therapy (administered physiological saline vehicle,
pH 7.2, only) [0191] Group 2. Antibody treatment group (n=12);
ischemia/reperfusion model+16 .mu.g of anti-BMP-1-1 antibody (c=1
.mu.g/.mu.l) and 16 .mu.g of anti-BMP-1-3 antibody (c=1
.mu.g/.mu.l) prior to ischemia/reperfusion and then for 5 days
after ischemia/reperfusion.
[0192] Blood samples were obtained before occlusion and at 0, 24,
72, 96, 120, and 168 hours after reperfusion. The plasma was
separated by centrifugation renal function parameters were
measured. Rats were killed at day 7 after reperfusion and kidneys
were harvested for histological analysis. Therapy was applied in a
prophylactic mode at 2 hours prior to clamping and then following
the release of the clamps for five days thereafter.
Assessment of Renal Function
[0193] Blood samples (0.5 ml) were obtained from the orbital venous
plexus at 0, 24, 72, 96, 120, and 168 hours after ischemia. Serum
creatinine was measured by Jaffe method (alkaline picrate) and
blood urea nitrogen (BUN) by enzymatic glutamate dehydrogenase-UV
procedure as previously described (Vukicevic et al., J. Clin.
Invest., 102: 202-214 (1998)). The cumulative survival rate was
observed and recorded for both control and experimental rats.
Renal Morphology
[0194] Kidneys for histological examination were fixed in 2%
paraformaldehyde, and 7 .mu.m paraffin sections were cut and
stained with haematoxylin and eosin. Tubulointestinal injury,
defined as tubular dilatation and/or atrophy, interstitial fibrosis
and inflammatory cell infiltrate, as well as glomerular damage were
graded using a semi-quantitative scale from 0 to 4 according to the
following criteria: 0=no changes; 1=focal changes involving 1-25%
of the samples; 2=changes affecting 26-50% of the sample; 3=changes
involving 51-75% of the sample; and 4=lesions affecting more than
75% of the sample as previously described (Vukicevic et al., J.
Clin. Invest., id.). Two independent observers performed histologic
studies in a blinded fashion.
Results
[0195] Creatinine levels in blood from rats of the untreated
control group (Group 1, no antibody therapy) and from rats of the
treatment group (Group 2, antibodies against BMP-1-1 and BMP-1-3)
are shown in FIG. 1. In control rats, following a 60-minute
clamping of both kidneys followed by reperfusion, the creatinine
(FIG. 1, diagonal line bars) and BUN (not shown) rose sharply and
remained high at 24 hours (1 day) and 72 hours (3 days) following
ischemia, then showed normalization at day 7 in animals that
survived the procedure. When antibodies to BMP-1-1 and BMP-1-3 were
administered (Group 2) prior to ischemia and then for five days
following ischemia, both the creatinine (FIG. 1, stippled bars) and
BUN (not shown) values remained low. The survival rate was 35% in
rats of the control group (no antibody therapy) and 55% in rats
treated with antibodies to BMP-1-1 and BMP-1-3 prior to and
following ischemia/reperfusion (data not shown). As observed on the
histology slides (FIG. 2), kidneys of rats of the control group
that were exposed to ischemia/reperfusion injury without antibody
therapy had lost the structural integrity in more than 75% of the
kidney area with dilated proximal and distal tubules, had lost the
tubular epithelium, and about 30% of the entire kidney area was
undergoing fibrotic healing due to necrosis (see, FIG. 2, Panel
2A). In contrast, sections of kidney tissue from rats that received
antibodies to BMP-1-1 and BMP-1-3 prior to ischemia/reperfusion
injury indicated significant preservation of kidney structures
(see, FIG. 2, Panel 2B).
[0196] These results show that the severity of damage to kidney
structure that would otherwise occur as the result of an
ischemic/reperfusion event can be prevented by a regimen of
systemic administration of neutralizing antibodies to the BMP-1-1
and BMP-1-3 isoforms prior to the ischemia/reperfusion event.
Example 8
[0197] Enhancing survival by systemic administration of BMP-1
isoform following ischemic acute renal failure in rats.
Animals
[0198] Female Sprague-Dawley rats weighting about 300 g-400 g were
housed and allowed free access to water and food.
Ischemia/Reperfusion Model
[0199] Rats were anesthetized with 100 mg/kg ketamine, 10 mg/kg
xylazine, and 1 mg/kg acepromazine (im) and placed on a heating
table kept at 37.degree. C. A midline incision was made, and both
renal pedicles were clamped for 60 min. After removal of the clamp,
5 ml of normal saline were instilled into the peritoneal cavity and
the incision was sutured. A total of 24 animals were assigned to
four different experimental groups:
[0200] Group 1. Negative control group (n=12); ischemia/reperfusion
model without therapy.
[0201] Group 2. Positive control group ("BMP-7") (n=8); 100
.mu.g/kg BMP-7 for five days.
[0202] Group 3. BMP-1-1 treatment group ("BMP-1-1") (n=8); 4 .mu.g
of BMP1-1 (c=0.2 .mu.g/.mu.l) for five days.
[0203] Group 4. BMP-1-1 antibody treatment group ("BMP-1 Ab")
(n=8); 16 .mu.g of anti-BMP-1-1 antibody (c=1 .mu.g/.mu.l) for five
days after release of clamps (post ischemia/reperfusion event).
[0204] Blood samples were obtained before occlusion and at 0, 24,
72, 96, 120, and 168 hours after reperfusion. The plasma was
separated by centrifugation. These samples were used for
measurement of renal function parameters. Rats were killed at day 7
after reperfusion, and kidneys were harvested for histological
analysis. Therapy was applied following clamping and for five days
thereafter.
Assessment of Renal Function
[0205] Blood samples (0.5 ml) were obtained from the orbital venous
plexus at 0, 24, 72, 96, 120, and 168 hours after ischemia. Serum
creatinine was measured by Jaffe method (alkaline picrate) and
blood urea nitrogen (BUN) by enzymatic glutamate dehydrogenase-UV
procedure as previously described. The cumulative survival rate was
observed and recorded for both control and experimental rats.
Results
[0206] Survival of rats in the various treatment groups is shown in
FIG. 3. In negative control rats (Group 1, no therapy) following a
60-minute clamping of both kidneys followed by a reperfusion,
levels of creatinine and BUN rose sharply (not shown), and greater
than 60% of the animals did not survive (see, FIG. 3, diamond data
points). Administering BMP-1-1 immediately following reperfusion
("BMP-1-1" group) significantly decreased the mortality and
maintained the survival rate at 80% compared to the 40% survival
rate of untreated negative control rats (see, FIG. 3, triangle data
points).
[0207] Although higher at days 2 and 3 in BMP-1-1 treated rats,
serum creatinine levels sharply declined on day 4 (data not shown),
probably due to a rapid processing of extracellular matrix in the
thrombotic area and a relatively fast recovery of the structural
elements that prevented significant necrosis due to accumulation of
the fibrotic post-necrotic tissue. Administration of the BMP-1-1
antibody ("BMP-1 Ab") for five days following the removal of the
clamps (see, FIG. 3, cross data points) was not effective in
preventing a high mortality rate (i.e., as low as 40% survival rate
at day 7 as seen also in the untreated control group).
[0208] The results of this experiment indicate that the
administration of a recombinant BMP-1 isoform following ischemic
acute renal failure is effective to reduce structural damage to the
kidney and to increase survival rate of the affected
individual.
Example 9
[0209] Delaying progression of chronic renal failure (CRF) in rats
by inhibiting BMP-1 isoforms.
Animals
[0210] Female Sprague-Dawley rats weighting about 350-400 g were
housed and allowed free access to water and food.
5/6 Nephrectomy (Nx) Model of CRF
[0211] Rats were anesthetized with 100 mg/kg ketamine, 10 mg/kg
xylazine, and 1 mg/kg acepromazine (im) and placed on a heating
table kept at 37.degree. C. A midline incision was made, and both
renal pedicles were clamped for 60 min. The left kidney was
removed, and the rats were left for a week to recover. Then, 5/6 of
the right kidney mass was removed, and rats were left to recover
for a period of two weeks. A total of 88 animals were assigned to 4
different experimental groups:
[0212] Group 1. Control group (n=12); 5/6 Nx rats receiving the
physiological vehicle solution.
[0213] Group 2. BMP-1-1 antibody group (n=12); Nx+16 .mu.g of
BMP-1-1 antibody (c=1 .mu.g/.mu.l) weekly for a period of 12
weeks
[0214] Group 3. BMP-1-3 antibody group (n=12); Nx+16 .mu.g of
BMP-1-3 antibody (c=1 .mu.g/.mu.l) weekly for a period of 12
weeks
[0215] Group 4. BMP-1-1+BMP-1-3 antibody group (n=12); Nx+16 .mu.g
of BMP1-1 antibody (c=1 .mu.g/.mu.l) weekly for a period of 12
weeks and 16 .mu.g of BMP-1-3 antibody (c=1 .mu.g/.mu.l) weekly for
a period of 12 weeks.
[0216] Blood samples were obtained before surgery and then weekly
throughout the duration of the experiment. Rats were killed at 12
weeks following the removal of the right kidney mass. Therapy was
applied intravenously (iv) weekly for a period of 12 weeks.
Assessment of Renal Function
[0217] Blood samples (0.5 ml) were obtained from the orbital venous
plexus weekly. Serum creatinine was measured by Jaffe method
(alkaline picrate) and blood urea nitrogen (BUN) by enzymatic
glutamate dehydrogenase-UV procedure as previously described
(Vukicevic et al., J. Clin. Invest., op. cit.). The cumulative
survival rate was observed and recorded for both control and
experimental rats.
Renal Morphology
[0218] Kidneys for histological examination were fixed in 2%
paraformaldehyde, and 7 .mu.m paraffin sections were cut and
stained with haematoxylin and eosin. Kidney damage was graded as
described (Borovecki et al., in Bone morphogenetic proteins--Bone
regeneration and beyond, edited by Vukicevic S. and Sampath K. T.,
2002). Briefly, the structure of glomeruli, kidney tubules, and the
amount of interstitial fibrosis were measured on the kidney area
using an automated computer program. The measured parameters were
expressed as a number of vital versus damaged glomeruli and as a
percent of fibrotically altered kidney area. Two independent
observers performed histologic studies in a blinded fashion.
Results
[0219] Following 12 weeks of therapy, control rats (Group 1), which
received only the vehicle solution, had creatinine values above 300
mEq/L Animals treated with a single antibody, i.e., antibody to
BMP-1-1 (Group 2) or antibody to BMP-1-3 (Group 3), or with a
combination of both antibodies (Group 4) had significantly lower
creatinine serum values as compared to control rats. In particular,
rats treated with anti-BMP-1-1 antibody (Group 2) or with
anti-BMP-1-3 antibody (Group 3) had, respectively, 36% and 39%
lower creatinine serum values than control rats. Creatinine serum
values were 54% lower in rats treated with a combination of both
anti-BMP1-1 and anti-BMP-1-3 antibodies than in the control rats.
In animals treated with a combination of both antibodies (Group 4),
the fibrotic area was reduced by 57% relative to control rats,
while in rats treated with only the anti-BMP-1-1 antibody (Group 2)
or with only the anti-BMP-1-3 antibody (Group 3), the fibrotic area
was reduce by 23% and 16%, respectively. In addition, the fibrotic
area was reduced by 43% in rats treated with a combination of both
antibodies as compared to rats treated with BMP-7, a positive
control.
[0220] These results indicate that inhibition of BMP-1-1 and
BMP-1-3 in a model of a chronic renal failure (CRF) delayed the
progression of the disease by maintaining the structural integrity
of glomeruli and preventing accumulation of fibrotic tissues, thus,
improving the kidney function by about 50% in a period of 12 weeks
following CRF. This relates to increasing a human life span by
about 120 months or about 10 years.
Example 10
[0221] Acceleration of fracture repair with systemically
administered BMP-1-1 and localization of BMP-1-1 at orthotopic site
of bone fraction.
Animals and Experimental Protocol
[0222] Fifty (50) 4-month old Sprague-Dawley female rats were used
in this study. Animals weighed approximately 300 grams (g). They
were kept in standard conditions (24.degree. C., 12 hour/12 hour
light/dark cycle) in 20.times.32.times.20 cm cages during the study
and were allowed free access to water and pelleted commercial diet
(Harlan Teklad, Borchen, Germany). Rats were divided into three
treatment groups and two control groups:
[0223] Group 1. Control rats (10) were treated with a Kirschner
wire following surgically produced fracture and then treated
systemically with a vehicle solution (physiological saline, pH 7.2)
only.
[0224] Group 2. Rats treated with BMP-1-1 (10 .mu.g/kg) for a
period of one week. Ten rats were treated with Kirschner wire
following fracture of the femur and then intravenously treated with
BMP-1-1.
[0225] Group 3. Rats treated with BMP-1-1 (10 .mu.g/kg) for a
period of three weeks. Ten rats were treated with Kirschner wire
following fracture of the femur and then intravenously treated with
BMP-1-1.
[0226] Group 4: Rats treated with BMP-1-1 (10 .mu.g/kg) for a
period of five weeks. Ten rats were treated with Kirschner wire
following fracture of the femur and then intravenously treated with
BMP-1-1.
[0227] Group 5: Positive control. Ten rats were treated with a
Kirschner wire following fracture of the femur and then injected
systemically with 100 .mu.g/kg of BMP-7 for a period of five (5)
weeks.
[0228] Anesthetized rats were prepared for surgery by shaving and
cleaning the lower extremities. With a medial peripatellar
incision, the patella was dislocated laterally exposing the femoral
condyle. A Kirschner wire (1.1 mm in diameter and 2.7 cm long) was
introduced into the intramedullary canal through the intercondylar
notch. The Kirschner wire did not protrude into the knee joint or
interfere with motion of the patella. After closing the knee joint,
the mid-diaphysis of the pinned right femur was fractured by
applying a bending force, as described by Bonarens and Einhom (J.
Orthop. Res., 97:101 (1984)). Radiographs were obtained immediately
after surgery, and rats with proximal or distal fractures were
excluded from this experiment so that only mid-diaphyseal fractures
were included in this study.
[0229] All animals were sacrificed following seven weeks of
therapy. Radiographs were taken at week one and seven following
surgery in two planes: AP (anterior-posterior) and LL
(latero-lateral).
Biodistribution and Pharmacokinetics of .sup.125I-labeled BMP-1-1
(.sup.125I-BMP-1-1)
[0230] Recombinant human BMP-1-1 was radioiodinated with 5 mCi of
carrier-free Na .sup.125I using a modification of the
lactoperoxidase method. Gel filtration on a Sephadex G-25 column
was used to separate radioiodinated BMP-1-1 (.sup.125I-BMP-1-1)
from the free iodide. The column was eluted with 20 mM sodium
acetate buffer, pH 4.5 containing 0.2% Tween-80 and 0.1% ovalbumin.
The specific activity of the .sup.1251-BMP-1-1 preparation used in
this study was 0.273 mCi/mg. Rats (n=10) received a single
injection of .sup.125I-BMP-1-1 at a dose level of 10 .mu.g/kg with
the activity of 20 .mu.Ci. Injection volume was 500 .mu.l Animals
were sacrificed 30 minutes, 1, 3, 6 and 24 hours following
injection. Tissues were removed, weighed, and radioactivity was
measured in a gamma counter. The relative uptake of .sup.125I-BMP-1
by tissues during time was expressed as nanograms (ng) of
.sup.125I-BMP-1 per gram (g) wet tissue weight. The experiments
were also performed in five rats with acutely fractured femurs on
day five following surgical osteotomy of the femur.
In Vivo and Ex Vivo Bone Mineral Density (BMD) Measurement by
DXA
[0231] At two-week intervals (in period of 10 weeks), the animals
were scanned for bone density measurements by dual-energy X-ray
absorptiometry (DXA; Hologic QDR-4000, Hologic, Waltham, Mass.). At
the end of the experiment, animals were anesthetized, weighed, and
euthanized. The right femur was removed and fixed in 70% ethanol
and was used for determination of the bone mineral content (BMC)
and BMD by DXA equipped with Regional High Resolution Scan
software. The scan field size was 5.08.times.1.902 cm, resolution
was 0.0254.times.0.0127 cm, and the speed was 7.25 mm/s. The scan
images were analyzed and the bone area, bone mineral content, and
bone density of whole bone.
PQCT
[0232] Isolated femurs were scanned by a peripheral quantitative
computerized tomography (PQCT) X-ray machine (Stratec XCT Research
M; Norland Medical Systems, Fort Atkinson, Wis.) with software
version 5.40. Volumetric content, density, and area of the total
bone, trabecular, and cortical regions were determined.
MicroCT
[0233] The microcomputerized tomography (MicroCT) apparatus (.mu.CT
40) and the analyzing software used in these experiments were
obtained from SCANCO Medical AG (Bassersdorf, Switzerland). The
right femur was scanned in 250 slices, each 13 .mu.m thick in the
dorsoventral direction. Three-dimensional reconstruction of bone
was performed using the triangulation algorithm. The trabecular
bone volume (BV, mm.sup.3), trabecular number (Tb. N, 1/mm), the
trabecular thickness (Tb. Th, .mu.m), and the trabecular separation
(Tb. Sp, .mu.m) were directly measured on 3-dimensional (3D) images
using the method described by Hildebrand et al. (Comp. Meth.
Biochem. Biomed. Eng., 1: 15 (1999)). The trabecular bone pattern
factor (TBPf) and the structure model index (SMI) were computed
using software provided with the microCT machine.
Histology
[0234] The femur was removed for histologic analyses, embedded in
paraffin, cut in 10 .mu.m thick sections, stained with
hemalaun-eosin and toluidine blue.
Results
[0235] Radioactively labeled BMP-1-1 was injected intravenously
into healthy rats and into rats with fractured femurs. In healthy
animals, radioactive BMP-1-1 accumulated predominantly in the liver
(23%), bones (31%), and muscles (9%). In rats with a fracture, 80%
of injected BMP-1-1 accumulated at the fracture site.
[0236] Rats treated with BMP-1-1 for one week with daily
intravenous injections showed 43% accelerated bone regeneration,
which was calculated based on a scoring system of bone repair as
previously described (Paralkar et al., Proc. Natl. Acad. Sci. USA,
100: 6736 (2003)). The formed callus was bigger by 43% in rats
treated with BMP-1 for one week, and it was increased by 63% and
71% in rats treated with BMP-1 for three to five weeks,
respectively. The bone healing was accelerated by 40-80% in rats
treated with BMP-1-1 for a period of one or five weeks,
respectively, as evidenced by full rebridgement of the three or
four cortices of rat femurs.
[0237] In vivo bone mineral density measurement showed increased
accumulation of mineral in the formed callus, while PQCT analyses
showed increased mineral accumulation on the cortical bone of
fractured femurs. MicroCT measurement showed increased accumulation
of newly formed trabeculi in the regenerating fracture at seven
weeks following surgical osteotomy.
[0238] These results of this study of acute femur fracture in rats
collectively indicate that the vast majority (e.g., about 80%) of
systemically administered BMP-1-1 becomes localized in the
orthotopic site of a bone fracture and that systemically
administered BMP-1-1 is effective at accelerating healing of such
acute fractured femurs.
Example 11
[0239] Systemically administered BMP-1-1 into rats with fractured
femur
[0240] Employing similar procedures as in Example 10, above, a
study was made to compare the effect of systemic administration of
BMP-1-1 isoform, BMP-7, and antibody to the BMP-1-1 isoform on
healing of fractured femurs in rats.
[0241] At 4 weeks following fracture, the callus at the fracture
site in rats treated systemically with BMP-1 isoform was about 20%
bigger than that in untreated control rats and about 90% bigger
than in rats treated systemically with BMP-7.
[0242] Results at 8 weeks following fracture are shown in FIG. 4.
The area of the fracture is encircled in each of the pictured
femurs FIGS. 4A-4F. Systemic administration of BMP-1-1 to rats with
a fractured femur resulted in accelerated healing as compared to
systemic administration of BMP-7. The fracture line had almost
disappeared, and the cortical bone had rebridged in rats treated
systemically with BMP-1-1 (see, bones 4A and 4D in FIG. 4), whereas
the fracture line was still visible in rats treated systemically
with BMP-7 (see, bones 4B, 4C, and 4E in FIG. 4). Systemic
administration of neutralizing antibody to BMP-1-1 delayed fracture
healing (see, bone 4F in FIG. 4).
[0243] The results indicate that systemic administration of a BMP-1
isoform is an effective method for treating bone defects.
Example 12
[0244] Locally administered BMP-1-1 into rats with fractured
femur.
Animal Model of Fracture
[0245] Twenty four (24) 3-month old Sprague-Dawley male rats (350
g) were treated with Kirschner wire following fracture of the
femur. Rats were divided into the following three treatment
groups:
[0246] Group 1. Control rats (8) were treated with a whole
(autologous) blood-derived coagulum device containing vehicle
solution only (physiological solution; no BMP-1-1, no BMP-7).
[0247] Group 2. Rats treated locally with whole blood-derived
coagulum device containing BMP-1 (10 .mu.g/kg of BMP-1-1).
[0248] Group 3. Rats (8) treated with whole blood-derived coagulum
device containing BMP-7 (10 .mu.g/kg).
[0249] All animals were sacrificed seven weeks after surgery.
Radiographs were taken at week 1, 4, and 7 in two planes, i.e., AP
(anterior-posterior) and LL (latero-lateral).
[0250] Anesthetized rats were prepared for surgery by shaving and
cleaning the lower extremities. With a medial peripatellar
incision, the patella was dislocated laterally exposing the femoral
condyle. A Kirschner wire (1.1 mm in diameter and 2.7 cm long) was
introduced into the intramedullary canal through the intercondylar
notch. The Kirschner wire did not protrude into the knee joint or
interfere with motion of the patella. After closing the knee joint,
the mid-diaphysis of the pinned right femur was fractured by
applying a bending force, as described by Bonarens and Einhom (J.
Orthop. Res., 97: 101 (1984)). Radiographs were obtained
immediately after surgery, and rats with proximal or distal
fractures were excluded from this experiment, so that the only
mid-diaphyseal fractures were included in this study.
Preparation of Whole Blood-Derived Coagulum Device (WBCD)
Containing BMP-1
[0251] Whole blood-derived coagulum devices (WBCDs) for treating
bone fractures were prepared to treat bone fractures in rat femurs.
Briefly, 1 ml of autologous whole blood was drawn from the orbital
plexus of each rat. The whole blood was then combined with a
thrombin-fibrin reagent, 1 M exogenous calcium chloride, and the
indicated amount of BMP-1-1 or BMP-7, and then incubated at room
temperature for 30-45 minutes to permit coagulum formation prior to
implantation into the fractured femur of the rat that provided the
corresponding autologous blood.
Biomechanical Testing
[0252] Femurs from both sides were removed for biomechanical
testing, which included three-point bending as previously described
(Simic et al., J. Biol. Chem., 281: 13472 (2006)). The healthy
bones from the contra-lateral side were used as positive controls.
Both three-point bending test and the indentation test were used
for measuring biomechanical characteristics of both the cortical
and the trabecular bone.
Results
[0253] Radiographic analysis of X-rays showed that in rats treated
with a WBCD containing only the vehicle solution (no BMP-1-1, no
BMP-7) as a control at 4 weeks following surgery, 0.6.+-.0.03
cortices healed, while at seven weeks following surgery 1.8.+-.0.4
cortices healed. The callus area was 24.3.+-.7.8 mm.sup.2 at four
weeks and 18.7.+-.6 4 mm.sup.2 at seven weeks.
[0254] In rats treated with a whole blood-derived coagulum
device+BMP-1-1 at four weeks 1.3.+-.0.5 (t-test, P>0.01 vs
control) cortices healed, while at seven weeks 2.9.+-.0.9 (t-test,
P>0.01) cortices healed. The callus area was 13.4.+-.4.7
mm.sup.2 (t-test, P>0.01 vs control), and at seven weeks it was
7.6.+-.3.8 mm.sup.2 (t-test, P>0.05 vs control).
[0255] In rats treated with WBCD+BMP-7 at four weeks 1.7.+-.0.7
(t-test, P>0.01 vs control and P>0.1 vs BMP-1) cortices
healed, while at seven weeks 3.2.+-.1.4 (t-test, P>0.01 vs
control and P>0.1 vs BMP-1) cortices healed. The callus area was
11.3.+-.3.9 mm.sup.2 (t-test, P>0.01 vs control and P>0.1 vs
BMP-1), and at seven weeks it was 6.7.+-.2.9 mm.sup.2 (t-test,
P>0.05 vs control and P>0.1 vs BMP-1).
[0256] These results indicate that locally administered BMP-1-1 at
an orthotopic site (defect site) in a model of femoral fracture
repair significantly accelerated the bone fracture healing as
compared to control rats. Surprisingly, when BMP-7 was used in a
composition with WBCD, femurs healed faster than in control rats,
but were not different from animals treated with BMP-1-1, which is
an ECM processing enzyme. BMP-7 is commercially used with bovine
collagen as a carrier. Bovine collagen implanted alone in a similar
model of bone repair in a rat inhibits bone repair as compared to
untreated control rats.
Biomechanical Testing
[0257] Three point bending test indicated that BMP-1-1 treated
femurs needed a significantly greater maximal load to re-fracture
as compared to control femurs treated only with the whole
blood-derived coagulum device (no BMP-1-1) (see, Table 2, below).
As compared with the femur from the opposite leg (contralateral
femur), bones treated with BMP-1-1 required 26% less load to cause
re-fracture; whereas control bones needed 51% less load to
re-fracture than the normal contralateral bones (see Table 2).
[0258] The maximal load needed to break BMP-7 treated bones was not
statistically different from those treated with BMP-1-1 (see, Table
2, below). These results confirmed the radiographic findings
collectively indicating that BMP-1-1 accelerates bone repair and
regeneration of acute fractures in a rat model, and that it is
equally as effective as BMP-7 when used with the whole
blood-derived coagulum device. Indentation test of trabecular bone
indicates that BMP-1-1 treated bones had more trabecular bone than
control animals (see, Table 3).
TABLE-US-00003 TABLE 2 Results of three point bending test on rat
femurs after therapy BMP-1 BMP-7 Parameter Control BMP-1-1 BMP-7
contralateral contralateral F.mu. (N) 119.99 .+-. 19.77 175.32 .+-.
24.87* 189.12 .+-. 28.69* 212.33 .+-. 37.82 234.56 .+-. 24.59 S
(N/mm) 266.84 .+-. 48.81 356.12 .+-. 53.09 377.40 .+-. 39.94 390.27
.+-. 43.30 402.75 .+-. 40.13 W (mJ) 91.67 .+-. 23.35 106.08 .+-.
15.54 116.06 .+-. 17.80 122.25 .+-. 18.16 131.15 .+-. 32.65 T
(MJ/m.sup.3) 8.65 .+-. 2.49 11.84 .+-. 1.7 11.33 .+-. 1.5 12.12
.+-. 1.61 12.36 .+-. 3.89 *P < 0.01 vs control, one way
ANOVA-Dunnett test
TABLE-US-00004 TABLE 3 Results of indentation test on rat femurs
after therapy BMP-1 BMP-7 Parameter Control BMP-1-1 BMP-7
contralateral contralateral F.mu. (N) 67.47 .+-. 25.7 84.30 .+-.
13* 104.95 .+-. 31* 101.31 .+-. 32.73 129.13 .+-. 19.5* S (N/mm)
93.25 .+-. 44.33 118.03 .+-. 14.34 132.11 .+-. 32.68* 180.36 .+-.
38.6* 170.54 .+-. 32.6* W (mJ) 54.62 .+-. 14.2 83.89 .+-. 15.1*
93.65 .+-. 16.5* 104.21 .+-. 25.2* 106.24 .+-. 16.8 .sigma.
(N/mm.sup.2) 21.49 .+-. 11.3 31.37 .+-. 1.19 43.68 .+-. 9.8* 51.61
.+-. 10.42* 59.28 .+-. 6.2* *P < 0.01 vs control, one way
ANOVA-Dunnett test
Example 13
[0259] The release of BMP-4 and BMP-7 into the medium of in vitro
cultured rat calvariae explant cultures treated with BMP-1-1 and
BMP-1-3.
[0260] Rat fetuses that were 18 days old were obtained from
pregnant rats and their calvariae were isolated, cleaned, equally
sized, and placed into cultures containing bone specific medium as
previously described (Vukicevic et al., Proc. Natl. Acad. Sci. USA,
86: 8793 (1989)). Such calvariae explant cultures produce bone
cells as well as extracellular matrix (ECM). At 48 hours following
culture, the explanted calvariae were treated with 100 ng/ml
BMP-1-1 or 100 ng/ml BMP-1-3 daily for a period of 3 days. The
medium was collected daily, stored at -20.degree. C., and on day 4
purified over a heparin column. Following purification over a
heparin column, the protein concentration was determined and BMP-2,
BMP-4, BMP-6, and BMP-7 were detected by immunoblotting as
previously described (Simic et al., J. Biol. Chem., 286: 13472
(2006)).
[0261] The results indicated that in the medium of control cultures
there were no detectable amounts of authentic osteogenic BMPs
found, while in the medium of calvariae treated with BMP-1-1, the
mature domain of BMP-4 was detected, whereas BMP-2, BMP-6 and BMP-7
were not detected. These results indicate that BMP-1-1 has an
effect on the release of BMP-4 from culture explants consisting of
fetal calvariae rich in bone cells and ECM, which appears to act as
a repository of stored authentic BMP molecules (see, also,
Martinovic et al., Arch. Cytol. Histol., 1: 23 (2006)). In the
medium of cultures treated with BMP-1-3 in addition to BMP-4, BMP-7
was detected, indicating that BMP-1-3 releases more authentic BMPs
from ECM than BMP-1-1.
Example 14
[0262] Synergistic acceleration of bone defect healing in rabbits
treated locally with BMP-1-1 and BMP-7.
Animals
[0263] An ulnar segmental-defect model was used to evaluate bone
healing in adult male New Zealand White rabbits (3 kg to 4 kg in
weight). The implants consisted of blood coagulum as a carrier to
which different amounts of recombinant human BMP-1-1 and
recombinant human mature BMP-7 were added (Genera Research
Laboratory). These animals were compared with animals receiving
blood coagulum implant alone (negative control). Rabbits were
treated with anti-parasitics one week before surgery Animals were
also given enrofloxacin, by intramuscular injection, at one day
before operation and then ten days following surgery.
[0264] With the rabbit under anesthesia and analgesia, one forelimb
was shaved and then prepared and draped in a sterile fashion. A
lateral incision, approximately 2.5 centimeters in length, was
made, and the tissues overlying the ulna were dissected. A
1.5-centimeter segmental osteoperiostal defect was created in the
middle of the ulna with an oscillating saw. The radius was left
intact for mechanical stability, and no internal or external
fixation devices were used. After copious irrigation with saline
solution to remove bone debris and spilled marrow cells, the
implant was packed carefully into place to fill the defect.
Coagulum was then overlaid with serum. The soft tissues were closed
meticulously in layers to contain the implant. The animals were
allowed full weight-bearing activity, water, and rabbit chow.
WBCD Preparation
[0265] Blood samples were collected from rabbit marginal ear veins
into tubes without any anticoagulants substance in a volume of 1.5
mL, one day before surgery. BMP-1-1 and BMP-7 were added into blood
in amounts of 14 .mu.g and 100 .mu.g, respectively. Blood samples
were left at 4.degree. C. to coagulate. The next day, samples were
centrifuged at 8000.times.g for 5 minutes. Liquid part (serum) was
removed and saved, and coagulum was ready to use.
[0266] The rabbits were devided into one of the groups listed below
and defects have been treated as follows:
[0267] Group 1. Control rabbits treated with the whole blood
coagulum device (WBCD) without BMP or BMP-1 isoform only (n=8).
[0268] Group 2. Rabbits treated with WBCD containing 14 .mu.g/1.5
mL of BMP-1-1.
[0269] Group 3. Rabbits treated with WBCD containing 100 .mu.g/1.5
mL of BMP-7.
[0270] Group 4. Rabbits treated with WBCD containing 14 .mu.g/1.5
mL of BMP-1+100 .mu.g of BMP-7/1.5 mL.
Results
[0271] The results are shown in FIGS. 5-8. Rabbit ulna defects did
not heal in the control rabbits (Group 1) treated with WBCD only
(no BMP-1-1, no BMP-7), as observed by X-ray biweekly follow up.
The unhealed defect in a representative bone after 6 weeks from the
control group is shown in FIGS. 5A and 5B (two views of the same
bone).
[0272] Results after 6 weeks in a representative bone from rabbits
treated locally with a WBCD having BMP-1-1 (Group 2) are shown in
FIGS. 6A and 6B. Results after 6 weeks in a representative bone
from rabbits treated locally with WBCD having BMP-7 (Group 3) are
shown in FIGS. 7A and 7B. Results after 6 weeks in a representative
bone from rabbits treated locally with WBCD having BMP-1-1 and
BMP-7 (Group 3) are shown in FIGS. 8A and 8B. Rabbits treated with
BMP-7-containing WBCD (Group 3) rebridged the bone defect at 8
weeks following surgery, while rabbits treated with
BMP-1-1-containing WBCD (Group 2) showed initial bone formation as
early as two weeks and advanced healing at 8 weeks following
surgery. However, rabbits treated locally with a WBCD having a
combination of both BMP-1-1 and BMP-7 (Group 4), had a synergistic
healing of the ulnar defect with a complete rebridgmenet of the
defect and formation of the new cortex with a pronounced
remodelling of newly formed bone as early as 6 weeks (see, FIGS. 8A
and 8B).
[0273] These results indicate that BMP-1-1 and BMP-7 applied
locally at an orthotopic site of a fracture act synergistically to
accelerate bone regeneration.
[0274] All patents, applications, and publications cited in the
above text are incorporated herein by reference.
[0275] Other variations and embodiments of the invention described
herein will now be apparent to those of skill in the art without
departing from the disclosure of the invention or the claims below.
Sequence CWU 1
1
71730PRTHomo sapiens 1Met Pro Gly Val Ala Arg Leu Pro Leu Leu Leu
Gly Leu Leu Leu Leu1 5 10 15Pro Arg Pro Gly Arg Pro Leu Asp Leu Ala
Asp Tyr Thr Tyr Asp Leu 20 25 30Ala Glu Glu Asp Asp Ser Glu Pro Leu
Asn Tyr Lys Asp Pro Cys Lys 35 40 45Ala Ala Ala Phe Leu Gly Asp Ile
Ala Leu Asp Glu Glu Asp Leu Arg 50 55 60Ala Phe Gln Val Gln Gln Ala
Val Asp Leu Arg Arg His Thr Ala Arg65 70 75 80Lys Ser Ser Ile Lys
Ala Ala Val Pro Gly Asn Thr Ser Thr Pro Ser 85 90 95Cys Gln Ser Thr
Asn Gly Gln Pro Gln Arg Gly Ala Cys Gly Arg Trp 100 105 110Arg Gly
Arg Ser Arg Ser Arg Arg Ala Ala Thr Ser Arg Pro Glu Arg 115 120
125Val Trp Pro Asp Gly Val Ile Pro Phe Val Ile Gly Gly Asn Phe Thr
130 135 140Gly Ser Gln Arg Ala Val Phe Arg Gln Ala Met Arg His Trp
Glu Lys145 150 155 160His Thr Cys Val Thr Phe Leu Glu Arg Thr Asp
Glu Asp Ser Tyr Ile 165 170 175Val Phe Thr Tyr Arg Pro Cys Gly Cys
Cys Ser Tyr Val Gly Arg Arg 180 185 190Gly Gly Gly Pro Gln Ala Ile
Ser Ile Gly Lys Asn Cys Asp Lys Phe 195 200 205Gly Ile Val Val His
Glu Leu Gly His Val Val Gly Phe Trp His Glu 210 215 220His Thr Arg
Pro Asp Arg Asp Arg His Val Ser Ile Val Arg Glu Asn225 230 235
240Ile Gln Pro Gly Gln Glu Tyr Asn Phe Leu Lys Met Glu Pro Gln Glu
245 250 255Val Glu Ser Leu Gly Glu Thr Tyr Asp Phe Asp Ser Ile Met
His Tyr 260 265 270Ala Arg Asn Thr Phe Ser Arg Gly Ile Phe Leu Asp
Thr Ile Val Pro 275 280 285Lys Tyr Glu Val Asn Gly Val Lys Pro Pro
Ile Gly Gln Arg Thr Arg 290 295 300Leu Ser Lys Gly Asp Ile Ala Gln
Ala Arg Lys Leu Tyr Lys Cys Pro305 310 315 320Ala Cys Gly Glu Thr
Leu Gln Asp Ser Thr Gly Asn Phe Ser Ser Pro 325 330 335Glu Tyr Pro
Asn Gly Tyr Ser Ala His Met His Cys Val Trp Arg Ile 340 345 350Ser
Val Thr Pro Gly Glu Lys Ile Ile Leu Asn Phe Thr Ser Leu Asp 355 360
365Leu Tyr Arg Ser Arg Leu Cys Trp Tyr Asp Tyr Val Glu Val Arg Asp
370 375 380Gly Phe Trp Arg Lys Ala Pro Leu Arg Gly Arg Phe Cys Gly
Ser Lys385 390 395 400Leu Pro Glu Pro Ile Val Ser Thr Asp Ser Arg
Leu Trp Val Glu Phe 405 410 415Arg Ser Ser Ser Asn Trp Val Gly Lys
Gly Phe Phe Ala Val Tyr Glu 420 425 430Ala Ile Cys Gly Gly Asp Val
Lys Lys Asp Tyr Gly His Ile Gln Ser 435 440 445Pro Asn Tyr Pro Asp
Asp Tyr Arg Pro Ser Lys Val Cys Ile Trp Arg 450 455 460Ile Gln Val
Ser Glu Gly Phe His Val Gly Leu Thr Phe Gln Ser Phe465 470 475
480Glu Ile Glu Arg His Asp Ser Cys Ala Tyr Asp Tyr Leu Glu Val Arg
485 490 495Asp Gly His Ser Glu Ser Ser Thr Leu Ile Gly Arg Tyr Cys
Gly Tyr 500 505 510Glu Lys Pro Asp Asp Ile Lys Ser Thr Ser Ser Arg
Leu Trp Leu Lys 515 520 525Phe Val Ser Asp Gly Ser Ile Asn Lys Ala
Gly Phe Ala Val Asn Phe 530 535 540Phe Lys Glu Val Asp Glu Cys Ser
Arg Pro Asn Arg Gly Gly Cys Glu545 550 555 560Gln Arg Cys Leu Asn
Thr Leu Gly Ser Tyr Lys Cys Ser Cys Asp Pro 565 570 575Gly Tyr Glu
Leu Ala Pro Asp Lys Arg Arg Cys Glu Ala Ala Cys Gly 580 585 590Gly
Phe Leu Thr Lys Leu Asn Gly Ser Ile Thr Ser Pro Gly Trp Pro 595 600
605Lys Glu Tyr Pro Pro Asn Lys Asn Cys Ile Trp Gln Leu Val Ala Pro
610 615 620Thr Gln Tyr Arg Ile Ser Leu Gln Phe Asp Phe Phe Glu Thr
Glu Gly625 630 635 640Asn Asp Val Cys Lys Tyr Asp Phe Val Glu Val
Arg Ser Gly Leu Thr 645 650 655Ala Asp Ser Lys Leu His Gly Lys Phe
Cys Gly Ser Glu Lys Pro Glu 660 665 670Val Ile Thr Ser Gln Tyr Asn
Asn Met Arg Val Glu Phe Lys Ser Asp 675 680 685Asn Thr Val Ser Lys
Lys Gly Phe Lys Ala His Phe Phe Ser Glu Lys 690 695 700Arg Pro Ala
Leu Gln Pro Pro Arg Gly Arg Pro His Gln Leu Lys Phe705 710 715
720Arg Val Gln Lys Arg Asn Arg Thr Pro Gln 725 7302986PRTHomo
sapiens 2Met Pro Gly Val Ala Arg Leu Pro Leu Leu Leu Gly Leu Leu
Leu Leu1 5 10 15Pro Arg Pro Gly Arg Pro Leu Asp Leu Ala Asp Tyr Thr
Tyr Asp Leu 20 25 30Ala Glu Glu Asp Asp Ser Glu Pro Leu Asn Tyr Lys
Asp Pro Cys Lys 35 40 45Ala Ala Ala Phe Leu Gly Asp Ile Ala Leu Asp
Glu Glu Asp Leu Arg 50 55 60Ala Phe Gln Val Gln Gln Ala Val Asp Leu
Arg Arg His Thr Ala Arg65 70 75 80Lys Ser Ser Ile Lys Ala Ala Val
Pro Gly Asn Thr Ser Thr Pro Ser 85 90 95Cys Gln Ser Thr Asn Gly Gln
Pro Gln Arg Gly Ala Cys Gly Arg Trp 100 105 110Arg Gly Arg Ser Arg
Ser Arg Arg Ala Ala Thr Ser Arg Pro Glu Arg 115 120 125Val Trp Pro
Asp Gly Val Ile Pro Phe Val Ile Gly Gly Asn Phe Thr 130 135 140Gly
Ser Gln Arg Ala Val Phe Arg Gln Ala Met Arg His Trp Glu Lys145 150
155 160His Thr Cys Val Thr Phe Leu Glu Arg Thr Asp Glu Asp Ser Tyr
Ile 165 170 175Val Phe Thr Tyr Arg Pro Cys Gly Cys Cys Ser Tyr Val
Gly Arg Arg 180 185 190Gly Gly Gly Pro Gln Ala Ile Ser Ile Gly Lys
Asn Cys Asp Lys Phe 195 200 205Gly Ile Val Val His Glu Leu Gly His
Val Val Gly Phe Trp His Glu 210 215 220His Thr Arg Pro Asp Arg Asp
Arg His Val Ser Ile Val Arg Glu Asn225 230 235 240Ile Gln Pro Gly
Gln Glu Tyr Asn Phe Leu Lys Met Glu Pro Gln Glu 245 250 255Val Glu
Ser Leu Gly Glu Thr Tyr Asp Phe Asp Ser Ile Met His Tyr 260 265
270Ala Arg Asn Thr Phe Ser Arg Gly Ile Phe Leu Asp Thr Ile Val Pro
275 280 285Lys Tyr Glu Val Asn Gly Val Lys Pro Pro Ile Gly Gln Arg
Thr Arg 290 295 300Leu Ser Lys Gly Asp Ile Ala Gln Ala Arg Lys Leu
Tyr Lys Cys Pro305 310 315 320Ala Cys Gly Glu Thr Leu Gln Asp Ser
Thr Gly Asn Phe Ser Ser Pro 325 330 335Glu Tyr Pro Asn Gly Tyr Ser
Ala His Met His Cys Val Trp Arg Ile 340 345 350Ser Val Thr Pro Gly
Glu Lys Ile Ile Leu Asn Phe Thr Ser Leu Asp 355 360 365Leu Tyr Arg
Ser Arg Leu Cys Trp Tyr Asp Tyr Val Glu Val Arg Asp 370 375 380Gly
Phe Trp Arg Lys Ala Pro Leu Arg Gly Arg Phe Cys Gly Ser Lys385 390
395 400Leu Pro Glu Pro Ile Val Ser Thr Asp Ser Arg Leu Trp Val Glu
Phe 405 410 415Arg Ser Ser Ser Asn Trp Val Gly Lys Gly Phe Phe Ala
Val Tyr Glu 420 425 430Ala Ile Cys Gly Gly Asp Val Lys Lys Asp Tyr
Gly His Ile Gln Ser 435 440 445Pro Asn Tyr Pro Asp Asp Tyr Arg Pro
Ser Lys Val Cys Ile Trp Arg 450 455 460Ile Gln Val Ser Glu Gly Phe
His Val Gly Leu Thr Phe Gln Ser Phe465 470 475 480Glu Ile Glu Arg
His Asp Ser Cys Ala Tyr Asp Tyr Leu Glu Val Arg 485 490 495Asp Gly
His Ser Glu Ser Ser Thr Leu Ile Gly Arg Tyr Cys Gly Tyr 500 505
510Glu Lys Pro Asp Asp Ile Lys Ser Thr Ser Ser Arg Leu Trp Leu Lys
515 520 525Phe Val Ser Asp Gly Ser Ile Asn Lys Ala Gly Phe Ala Val
Asn Phe 530 535 540Phe Lys Glu Val Asp Glu Cys Ser Arg Pro Asn Arg
Gly Gly Cys Glu545 550 555 560Gln Arg Cys Leu Asn Thr Leu Gly Ser
Tyr Lys Cys Ser Cys Asp Pro 565 570 575Gly Tyr Glu Leu Ala Pro Asp
Lys Arg Arg Cys Glu Ala Ala Cys Gly 580 585 590Gly Phe Leu Thr Lys
Leu Asn Gly Ser Ile Thr Ser Pro Gly Trp Pro 595 600 605Lys Glu Tyr
Pro Pro Asn Lys Asn Cys Ile Trp Gln Leu Val Ala Pro 610 615 620Thr
Gln Tyr Arg Ile Ser Leu Gln Phe Asp Phe Phe Glu Thr Glu Gly625 630
635 640Asn Asp Val Cys Lys Tyr Asp Phe Val Glu Val Arg Ser Gly Leu
Thr 645 650 655Ala Asp Ser Lys Leu His Gly Lys Phe Cys Gly Ser Glu
Lys Pro Glu 660 665 670Val Ile Thr Ser Gln Tyr Asn Asn Met Arg Val
Glu Phe Lys Ser Asp 675 680 685Asn Thr Val Ser Lys Lys Gly Phe Lys
Ala His Phe Phe Ser Asp Lys 690 695 700Asp Glu Cys Ser Lys Asp Asn
Gly Gly Cys Gln Gln Asp Cys Val Asn705 710 715 720Thr Phe Gly Ser
Tyr Glu Cys Gln Cys Arg Ser Gly Phe Val Leu His 725 730 735Asp Asn
Lys His Asp Cys Lys Glu Ala Gly Cys Asp His Lys Val Thr 740 745
750Ser Thr Ser Gly Thr Ile Thr Ser Pro Asn Trp Pro Asp Lys Tyr Pro
755 760 765Ser Lys Lys Glu Cys Thr Trp Ala Ile Ser Ser Thr Pro Gly
His Arg 770 775 780Val Lys Leu Thr Phe Met Glu Met Asp Ile Glu Ser
Gln Pro Glu Cys785 790 795 800Ala Tyr Asp His Leu Glu Val Phe Asp
Gly Arg Asp Ala Lys Ala Pro 805 810 815Val Leu Gly Arg Phe Cys Gly
Ser Lys Lys Pro Glu Pro Val Leu Ala 820 825 830Thr Gly Ser Arg Met
Phe Leu Arg Phe Tyr Ser Asp Asn Ser Val Gln 835 840 845Arg Lys Gly
Phe Gln Ala Ser His Ala Thr Glu Cys Gly Gly Gln Val 850 855 860Arg
Ala Asp Val Lys Thr Lys Asp Leu Tyr Ser His Ala Gln Phe Gly865 870
875 880Asp Asn Asn Tyr Pro Gly Gly Val Asp Cys Glu Trp Val Ile Val
Ala 885 890 895Glu Glu Gly Tyr Gly Val Glu Leu Val Phe Gln Thr Phe
Glu Val Glu 900 905 910Glu Glu Thr Asp Cys Gly Tyr Asp Tyr Met Glu
Leu Phe Asp Gly Tyr 915 920 925Asp Ser Thr Ala Pro Arg Leu Gly Arg
Tyr Cys Gly Ser Gly Pro Pro 930 935 940Glu Glu Val Tyr Ser Ala Gly
Asp Ser Val Leu Val Lys Phe His Ser945 950 955 960Asp Asp Thr Ile
Thr Lys Lys Gly Phe His Leu Arg Tyr Thr Ser Thr 965 970 975Lys Phe
Gln Asp Thr Leu His Ser Arg Lys 980 98532961DNAHomo sapiens
3atgcccggcg tggcccgcct gccgctgctg ctcgggctgc tgctgctccc gcgtcccggc
60cggccgctgg acttggccga ctacacctat gacctggcgg aggaggacga ctcggagccc
120ctcaactaca aagacccctg caaggcggct gcctttcttg gggacattgc
cctggacgaa 180gaggacctga gggccttcca ggtacagcag gctgtggatc
tcagacggca cacagctcgt 240aagtcctcca tcaaagctgc agttccagga
aacacttcta cccccagctg ccagagcacc 300aacgggcagc ctcagagggg
agcctgtggg agatggagag gtagatcccg tagccggcgg 360gcggcgacgt
cccgaccaga gcgtgtgtgg cccgatgggg tcatcccctt tgtcattggg
420ggaaacttca ctggtagcca gagggcagtc ttccggcagg ccatgaggca
ctgggagaag 480cacacctgtg tcaccttcct ggagcgcact gacgaggaca
gctatattgt gttcacctat 540cgaccttgcg ggtgctgctc ctacgtgggt
cgccgcggcg ggggccccca ggccatctcc 600atcggcaaga actgtgacaa
gttcggcatt gtggtccacg agctgggcca cgtcgtcggc 660ttctggcacg
aacacactcg gccagaccgg gaccgccacg tttccatcgt tcgtgagaac
720atccagccag ggcaggagta taacttcctg aagatggagc ctcaggaggt
ggagtccctg 780ggggagacct atgacttcga cagcatcatg cattacgctc
ggaacacatt ctccaggggc 840atcttcctgg ataccattgt ccccaagtat
gaggtgaacg gggtgaaacc tcccattggc 900caaaggacac ggctcagcaa
gggggacatt gcccaagccc gcaagcttta caagtgccca 960gcctgtggag
agaccctgca agacagcaca ggcaacttct cctcccctga ataccccaat
1020ggctactctg ctcacatgca ctgcgtgtgg cgcatctctg tcacacccgg
ggagaagatc 1080atcctgaact tcacgtccct ggacctgtac cgcagccgcc
tgtgctggta cgactatgtg 1140gaggtccgag atggcttctg gaggaaggcg
cccctccgag gccgcttctg cgggtccaaa 1200ctccctgagc ctatcgtctc
cactgacagc cgcctctggg ttgaattccg cagcagcagc 1260aattgggttg
gaaagggctt ctttgcagtc tacgaagcca tctgcggggg tgatgtgaaa
1320aaggactatg gccacattca atcgcccaac tacccagacg attaccggcc
cagcaaagtc 1380tgcatctggc ggatccaggt gtctgagggc ttccacgtgg
gcctcacatt ccagtccttt 1440gagattgagc gccacgacag ctgtgcctac
gactatctgg aggtgcgcga cgggcacagt 1500gagagcagca ccctcatcgg
gcgctactgt ggctatgaga agcctgatga catcaagagc 1560acgtccagcc
gcctctggct caagttcgtc tctgacgggt ccattaacaa agcgggcttt
1620gccgtcaact ttttcaaaga ggtggacgag tgctctcggc ccaaccgcgg
gggctgtgag 1680cagcggtgcc tcaacaccct gggcagctac aagtgcagct
gtgaccccgg gtacgagctg 1740gccccagaca agcgccgctg tgaggctgct
tgtggcggat tcctcaccaa gctcaacggc 1800tccatcacca gcccgggctg
gcccaaggag taccccccca acaagaactg catctggcag 1860ctggtggccc
ccacccagta ccgcatctcc ctgcagtttg acttctttga gacagagggc
1920aatgatgtgt gcaagtacga cttcgtggag gtgcgcagtg gactcacagc
tgactccaag 1980ctgcatggca agttctgtgg ttctgagaag cccgaggtca
tcacctccca gtacaacaac 2040atgcgcgtgg agttcaagtc cgacaacacc
gtgtccaaaa agggcttcaa ggcccacttc 2100ttctcagaca aggacgagtg
ctccaaggat aacggcggct gccagcagga ctgcgtcaac 2160acgttcggca
gttatgagtg ccaatgccgc agtggcttcg tcctccatga caacaagcac
2220gactgcaaag aagccggctg tgaccacaag gtgacatcca ccagtggtac
catcaccagc 2280cccaactggc ctgacaagta tcccagcaag aaggagtgca
cgtgggccat ctccagcacc 2340cccgggcacc gggtcaagct gaccttcatg
gagatggaca tcgagtccca gcctgagtgt 2400gcctacgacc acctagaggt
gttcgacggg cgagacgcca aggcccccgt cctcggccgc 2460ttctgtggga
gcaagaagcc cgagcccgtc ctggccacag gcagccgcat gttcctgcgc
2520ttctactcag ataactcggt ccagcgaaag ggcttccagg cctcccacgc
cacagagtgc 2580gggggccagg tacgggcaga cgtgaagacc aaggaccttt
actcccacgc ccagtttggc 2640gacaacaact accctggggg tgtggactgt
gagtgggtca ttgtggccga ggaaggctac 2700ggcgtggagc tcgtgttcca
gacctttgag gtggaggagg agaccgactg cggctatgac 2760tacatggagc
tcttcgacgg ctacgacagc acagccccca ggctggggcg ctactgtggc
2820tcagggcctc ctgaggaggt gtactcggcg ggagattctg tcctggtgaa
gttccactcg 2880gatgacacca tcaccaaaaa aggtttccac ctgcgataca
ccagcaccaa gttccaggac 2940acactccaca gcaggaagtg a 29614986PRTHomo
sapiens 4Met Pro Gly Val Ala Arg Leu Pro Leu Leu Leu Gly Leu Leu
Leu Leu1 5 10 15Pro Arg Pro Gly Arg Pro Leu Asp Leu Ala Asp Tyr Thr
Tyr Asp Leu 20 25 30Ala Glu Glu Asp Asp Ser Glu Pro Leu Asn Tyr Lys
Asp Pro Cys Lys 35 40 45Ala Ala Ala Phe Leu Gly Asp Ile Ala Leu Asp
Glu Glu Asp Leu Arg 50 55 60Ala Phe Gln Val Gln Gln Ala Val Asp Leu
Arg Arg His Thr Ala Arg65 70 75 80Lys Ser Ser Ile Lys Ala Ala Val
Pro Gly Asn Thr Ser Thr Pro Ser 85 90 95Cys Gln Ser Thr Asn Gly Gln
Pro Gln Arg Gly Ala Cys Gly Arg Trp 100 105 110Arg Gly Arg Ser Arg
Ser Arg Arg Ala Ala Thr Ser Arg Pro Glu Arg 115 120 125Val Trp Pro
Asp Gly Val Ile Pro Phe Val Ile Gly Gly Asn Phe Thr 130 135 140Gly
Ser Gln Arg Ala Val Phe Arg Gln Ala Met Arg His Trp Glu Lys145 150
155 160His Thr Cys Val Thr Phe Leu Glu Arg Thr Asp Glu Asp Ser Tyr
Ile 165 170 175Val Phe Thr Tyr Arg Pro Cys Gly Cys Cys Ser Tyr Val
Gly Arg Arg 180 185 190Gly Gly Gly Pro Gln Ala Ile Ser Ile Gly Lys
Asn Cys Asp Lys Phe 195 200 205Gly Ile Val Val His Glu Leu Gly His
Val Val Gly Phe Trp His Glu 210 215 220His Thr Arg Pro Asp Arg Asp
Arg His Val Ser Ile Val Arg Glu Asn225 230 235 240Ile Gln Pro Gly
Gln Glu Tyr Asn Phe Leu Lys Met Glu Pro Gln Glu 245
250 255Val Glu Ser Leu Gly Glu Thr Tyr Asp Phe Asp Ser Ile Met His
Tyr 260 265 270Ala Arg Asn Thr Phe Ser Arg Gly Ile Phe Leu Asp Thr
Ile Val Pro 275 280 285Lys Tyr Glu Val Asn Gly Val Lys Pro Pro Ile
Gly Gln Arg Thr Arg 290 295 300Leu Ser Lys Gly Asp Ile Ala Gln Ala
Arg Lys Leu Tyr Lys Cys Pro305 310 315 320Ala Cys Gly Glu Thr Leu
Gln Asp Ser Thr Gly Asn Phe Ser Ser Pro 325 330 335Glu Tyr Pro Asn
Gly Tyr Ser Ala His Met His Cys Val Trp Arg Ile 340 345 350Ser Val
Thr Pro Gly Glu Lys Ile Ile Leu Asn Phe Thr Ser Leu Asp 355 360
365Leu Tyr Arg Ser Arg Leu Cys Trp Tyr Asp Tyr Val Glu Val Arg Asp
370 375 380Gly Phe Trp Arg Lys Ala Pro Leu Arg Gly Arg Phe Cys Gly
Ser Lys385 390 395 400Leu Pro Glu Pro Ile Val Ser Thr Asp Ser Arg
Leu Trp Val Glu Phe 405 410 415Arg Ser Ser Ser Asn Trp Val Gly Lys
Gly Phe Phe Ala Val Tyr Glu 420 425 430Ala Ile Cys Gly Gly Asp Val
Lys Lys Asp Tyr Gly His Ile Gln Ser 435 440 445Pro Asn Tyr Pro Asp
Asp Tyr Arg Pro Ser Lys Val Cys Ile Trp Arg 450 455 460Ile Gln Val
Ser Glu Gly Phe His Val Gly Leu Thr Phe Gln Ser Phe465 470 475
480Glu Ile Glu Arg His Asp Ser Cys Ala Tyr Asp Tyr Gln Glu Val Arg
485 490 495Asp Gly His Ser Glu Ser Ser Thr Leu Ile Gly Arg Tyr Cys
Gly Tyr 500 505 510Glu Lys Pro Asp Asp Ile Lys Ser Thr Ser Ser Arg
Leu Trp Leu Lys 515 520 525Phe Val Ser Asp Gly Ser Ile Asn Lys Ala
Gly Phe Ala Val Asn Phe 530 535 540Phe Lys Glu Val Asp Glu Cys Ser
Arg Pro Asn Arg Gly Gly Cys Glu545 550 555 560Gln Arg Cys Leu Asn
Thr Leu Gly Ser Tyr Lys Cys Ser Cys Asp Pro 565 570 575Gly Tyr Glu
Leu Ala Pro Asp Lys Arg Arg Cys Glu Ala Ala Cys Gly 580 585 590Gly
Phe Leu Thr Lys Leu Asn Gly Ser Ile Thr Ser Pro Gly Trp Pro 595 600
605Lys Glu Tyr Pro Pro Asn Lys Asn Cys Ile Trp Gln Leu Val Ala Pro
610 615 620Thr Gln Tyr Arg Ile Ser Leu Gln Phe Asp Phe Phe Glu Thr
Glu Gly625 630 635 640Asn Asp Val Cys Lys Tyr Asp Phe Val Glu Val
Arg Ser Gly Leu Thr 645 650 655Ala Asp Ser Lys Leu His Gly Lys Phe
Cys Gly Ser Glu Lys Pro Glu 660 665 670Val Ile Thr Ser Gln Tyr Asn
Asn Met Arg Val Glu Phe Lys Ser Asp 675 680 685Asn Thr Val Ser Lys
Lys Gly Phe Lys Ala His Phe Phe Ser Asp Lys 690 695 700Asp Glu Cys
Ser Lys Asp Asn Gly Gly Cys Gln Gln Asp Cys Val Asn705 710 715
720Thr Phe Gly Ser Tyr Glu Cys Gln Cys Arg Ser Gly Phe Val Leu His
725 730 735Asp Asn Lys His Asp Cys Lys Glu Ala Gly Cys Asp His Lys
Val Thr 740 745 750Ser Thr Ser Gly Thr Ile Thr Ser Pro Asn Trp Pro
Asp Lys Tyr Pro 755 760 765Ser Lys Lys Glu Cys Thr Trp Ala Ile Ser
Ser Thr Pro Gly His Arg 770 775 780Val Lys Leu Thr Phe Met Glu Met
Asp Ile Glu Ser Gln Pro Glu Cys785 790 795 800Ala Tyr Asp His Leu
Glu Val Phe Asp Gly Arg Asp Ala Lys Ala Pro 805 810 815Val Leu Gly
Arg Phe Cys Gly Ser Lys Lys Pro Glu Pro Val Leu Ala 820 825 830Thr
Gly Ser Arg Met Phe Leu Arg Phe Tyr Ser Asp Asn Ser Val Gln 835 840
845Arg Lys Gly Phe Gln Ala Ser His Ala Thr Glu Cys Gly Gly Gln Val
850 855 860Arg Ala Asp Val Lys Thr Lys Asp Leu Tyr Ser His Ala Gln
Phe Gly865 870 875 880Asp Asn Asn Tyr Pro Gly Gly Val Asp Cys Glu
Trp Val Ile Val Ala 885 890 895Glu Glu Gly Tyr Gly Val Glu Leu Val
Phe Gln Thr Phe Glu Val Glu 900 905 910Glu Glu Thr Asp Cys Gly Tyr
Asp Tyr Met Glu Leu Phe Asp Gly Tyr 915 920 925Asp Ser Thr Ala Pro
Arg Leu Gly Arg Tyr Cys Gly Ser Gly Pro Pro 930 935 940Glu Glu Val
Tyr Ser Ala Gly Asp Ser Val Leu Val Lys Phe His Ser945 950 955
960Asp Asp Thr Ile Thr Lys Lys Gly Phe His Leu Arg Tyr Thr Ser Thr
965 970 975Lys Phe Gln Asp Thr Leu His Ser Arg Lys 980
98552961DNAHomo sapiens 5atgcccggcg tggcccgcct gccgctgctg
ctcgggctgc tgctgctccc gcgtcccggc 60cggccgctgg acttggccga ctacacctat
gacctggcgg aggaggacga ctcggagccc 120ctcaactaca aagacccctg
caaggcggct gcctttcttg gggacattgc cctggacgaa 180gaggacctga
gggccttcca ggtacagcag gctgtggatc tcagacggca cacagctcgt
240aagtcctcca tcaaagctgc agttccagga aacacttcta cccccagctg
ccagagcacc 300aacgggcagc ctcagagggg agcctgtggg agatggagag
gtagatcccg tagccggcgg 360gcggcgacgt cccgaccaga gcgtgtgtgg
cccgatgggg tcatcccctt tgtcattggg 420ggaaacttca ctggtagcca
gagggcagtc ttccggcagg ccatgaggca ctgggagaag 480cacacctgtg
tcaccttcct ggagcgcact gacgaggaca gctatattgt gttcacctat
540cgaccttgcg ggtgctgctc ctacgtgggt cgccgcggcg ggggccccca
ggccatctcc 600atcggcaaga actgtgacaa gttcggcatt gtggtccacg
agctgggcca cgtcgtcggc 660ttctggcacg aacacactcg gccagaccgg
gaccgccacg tttccatcgt tcgtgagaac 720atccagccag ggcaggagta
taacttcctg aagatggagc ctcaggaggt ggagtccctg 780ggggagacct
atgacttcga cagcatcatg cattacgctc ggaacacatt ctccaggggc
840atcttcctgg ataccattgt ccccaagtat gaggtgaacg gggtgaaacc
tcccattggc 900caaaggacac ggctcagcaa gggggacatt gcccaagccc
gcaagcttta caagtgccca 960gcctgtggag agaccctgca agacagcaca
ggcaacttct cctcccctga ataccccaat 1020ggctactctg ctcacatgca
ctgcgtgtgg cgcatctctg tcacacccgg ggagaagatc 1080atcctgaact
tcacgtccct ggacctgtac cgcagccgcc tgtgctggta cgactatgtg
1140gaggtccgag atggcttctg gaggaaggcg cccctccgag gccgcttctg
cgggtccaaa 1200ctccctgagc ctatcgtctc cactgacagc cgcctctggg
ttgaattccg cagcagcagc 1260aattgggttg gaaagggctt ctttgcagtc
tacgaagcca tctgcggggg tgatgtgaaa 1320aaggactatg gccacattca
atcgcccaac tacccagacg attaccggcc cagcaaagtc 1380tgcatctggc
ggatccaggt gtctgagggc ttccacgtgg gcctcacatt ccagtccttt
1440gagattgagc gccacgacag ctgtgcctac gactatcagg aggtgcgcga
cgggcacagt 1500gagagcagca ccctcatcgg gcgctactgt ggctatgaga
agcctgatga catcaagagc 1560acgtccagcc gcctctggct caagttcgtc
tctgacgggt ccattaacaa agcgggcttt 1620gccgtcaact ttttcaaaga
ggtggacgag tgctctcggc ccaaccgcgg gggctgtgag 1680cagcggtgcc
tcaacaccct gggcagctac aagtgcagct gtgaccccgg gtacgagctg
1740gccccagaca agcgccgctg tgaggctgct tgtggcggat tcctcaccaa
gctcaacggc 1800tccatcacca gcccgggctg gcccaaggag taccccccca
acaagaactg catctggcag 1860ctggtggccc ccacccagta ccgcatctcc
ctgcagtttg acttctttga gacagagggc 1920aatgatgtgt gcaagtacga
cttcgtggag gtgcgcagtg gactcacagc tgactccaag 1980ctgcatggca
agttctgtgg ttctgagaag cccgaggtca tcacctccca gtacaacaac
2040atgcgcgtgg agttcaagtc cgacaacacc gtgtccaaaa agggcttcaa
ggcccacttc 2100ttctcagaca aggacgagtg ctccaaggat aacggcggct
gccagcagga ctgcgtcaac 2160acgttcggca gttatgagtg ccaatgccgc
agtggcttcg tcctccatga caacaagcac 2220gactgcaaag aagccggctg
tgaccacaag gtgacatcca ccagtggtac catcaccagc 2280cccaactggc
ctgacaagta tcccagcaag aaggagtgca cgtgggccat ctccagcacc
2340cccgggcacc gggtcaagct gaccttcatg gagatggaca tcgagtccca
gcctgagtgt 2400gcctacgacc acctagaggt gttcgacggg cgagacgcca
aggcccccgt cctcggccgc 2460ttctgtggga gcaagaagcc cgagcccgtc
ctggccacag gcagccgcat gttcctgcgc 2520ttctactcag ataactcggt
ccagcgaaag ggcttccagg cctcccacgc cacagagtgc 2580gggggccagg
tacgggcaga cgtgaagacc aaggaccttt actcccacgc ccagtttggc
2640gacaacaact accctggggg tgtggactgt gagtgggtca ttgtggccga
ggaaggctac 2700ggcgtggagc tcgtgttcca gacctttgag gtggaggagg
agaccgactg cggctatgac 2760tacatggagc tcttcgacgg ctacgacagc
acagccccca ggctggggcg ctactgtggc 2820tcagggcctc ctgaggaggt
gtactcggcg ggagattctg tcctggtgaa gttccactcg 2880gatgacacca
tcaccaaaaa aggtttccac ctgcgataca ccagcaccaa gttccaggac
2940acactccaca gcaggaagtg a 29616622PRTHomo sapiens 6Met Pro Gly
Val Ala Arg Leu Pro Leu Leu Leu Gly Leu Leu Leu Leu1 5 10 15Pro Arg
Pro Gly Arg Pro Leu Asp Leu Ala Asp Tyr Thr Tyr Asp Leu 20 25 30Ala
Glu Glu Asp Asp Ser Glu Pro Leu Asn Tyr Lys Asp Pro Cys Lys 35 40
45Ala Ala Ala Phe Leu Gly Asp Ile Ala Leu Asp Glu Glu Asp Leu Arg
50 55 60Ala Phe Gln Val Gln Gln Ala Val Asp Leu Arg Arg His Thr Ala
Arg65 70 75 80Lys Ser Ser Ile Lys Ala Ala Val Pro Gly Asn Thr Ser
Thr Pro Ser 85 90 95Cys Gln Ser Thr Asn Gly Gln Pro Gln Arg Gly Ala
Cys Gly Arg Trp 100 105 110Arg Gly Arg Ser Arg Ser Arg Arg Ala Ala
Thr Ser Arg Pro Glu Arg 115 120 125Val Trp Pro Asp Gly Val Ile Pro
Phe Val Ile Gly Gly Asn Phe Thr 130 135 140Gly Ser Gln Arg Ala Val
Phe Arg Gln Ala Met Arg His Trp Glu Lys145 150 155 160His Thr Cys
Val Thr Phe Leu Glu Arg Thr Asp Glu Asp Ser Tyr Ile 165 170 175Val
Phe Thr Tyr Arg Pro Cys Gly Cys Cys Ser Tyr Val Gly Arg Arg 180 185
190Gly Gly Gly Pro Gln Ala Ile Ser Ile Gly Lys Asn Cys Asp Lys Phe
195 200 205Gly Ile Val Val His Glu Leu Gly His Val Val Gly Phe Trp
His Glu 210 215 220His Thr Arg Pro Asp Arg Asp Arg His Val Ser Ile
Val Arg Glu Asn225 230 235 240Ile Gln Pro Gly Gln Glu Tyr Asn Phe
Leu Lys Met Glu Pro Gln Glu 245 250 255Val Glu Ser Leu Gly Glu Thr
Tyr Asp Phe Asp Ser Ile Met His Tyr 260 265 270Ala Arg Asn Thr Phe
Ser Arg Gly Ile Phe Leu Asp Thr Ile Val Pro 275 280 285Lys Tyr Glu
Val Asn Gly Val Lys Pro Pro Ile Gly Gln Arg Thr Arg 290 295 300Leu
Ser Lys Gly Asp Ile Ala Gln Ala Arg Lys Leu Tyr Lys Cys Pro305 310
315 320Ala Cys Gly Glu Thr Leu Gln Asp Ser Thr Gly Asn Phe Ser Ser
Pro 325 330 335Glu Tyr Pro Asn Gly Tyr Ser Ala His Met His Cys Val
Trp Arg Ile 340 345 350Ser Val Thr Pro Gly Glu Lys Ile Ile Leu Asn
Phe Thr Ser Leu Asp 355 360 365Leu Tyr Arg Ser Arg Leu Cys Trp Tyr
Asp Tyr Val Glu Val Arg Asp 370 375 380Gly Phe Trp Arg Lys Ala Pro
Leu Arg Gly Arg Phe Cys Gly Ser Lys385 390 395 400Leu Pro Glu Pro
Ile Val Ser Thr Asp Ser Arg Leu Trp Val Glu Phe 405 410 415Arg Ser
Ser Ser Asn Trp Val Gly Lys Gly Phe Phe Ala Val Tyr Glu 420 425
430Ala Ile Cys Gly Gly Asp Val Lys Lys Asp Tyr Gly His Ile Gln Ser
435 440 445Pro Asn Tyr Pro Asp Asp Tyr Arg Pro Ser Lys Val Cys Ile
Trp Arg 450 455 460Ile Gln Val Ser Glu Gly Phe His Val Gly Leu Thr
Phe Gln Ser Phe465 470 475 480Glu Ile Glu Arg His Asp Ser Cys Ala
Tyr Asp Tyr Leu Glu Val Arg 485 490 495Asp Gly His Ser Glu Ser Ser
Thr Leu Ile Gly Arg Tyr Cys Gly Tyr 500 505 510Glu Lys Pro Asp Asp
Ile Lys Ser Thr Ser Ser Arg Leu Trp Leu Lys 515 520 525Phe Val Ser
Asp Gly Ser Ile Asn Lys Ala Gly Phe Ala Val Asn Phe 530 535 540Phe
Lys Glu Val Asp Glu Cys Ser Arg Pro Asn Arg Gly Gly Cys Glu545 550
555 560Gln Arg Cys Leu Asn Thr Leu Gly Ser Tyr Lys Cys Ser Cys Asp
Pro 565 570 575Gly Tyr Glu Leu Ala Pro Asp Lys Arg Arg Cys Glu Gly
Cys Tyr Asp 580 585 590Leu Gln Val Gly Lys Pro Leu Leu Trp Asp Arg
His Cys Phe Arg Leu 595 600 605Ser Thr His Gly Pro Glu Met Leu Gly
Thr Ala Leu Arg Gly 610 615 6207823PRTHomo sapiens 7Met Pro Gly Val
Ala Arg Leu Pro Leu Leu Leu Gly Leu Leu Leu Leu1 5 10 15Pro Arg Pro
Gly Arg Pro Leu Asp Leu Ala Asp Tyr Thr Tyr Asp Leu 20 25 30Ala Glu
Glu Asp Asp Ser Glu Pro Leu Asn Tyr Lys Asp Pro Cys Lys 35 40 45Ala
Ala Ala Phe Leu Gly Asp Ile Ala Leu Asp Glu Glu Asp Leu Arg 50 55
60Ala Phe Gln Val Gln Gln Ala Val Asp Leu Arg Arg His Thr Ala Arg65
70 75 80Lys Ser Ser Ile Lys Ala Ala Val Pro Gly Asn Thr Ser Thr Pro
Ser 85 90 95Cys Gln Ser Thr Asn Gly Gln Pro Gln Arg Gly Ala Cys Gly
Arg Trp 100 105 110Arg Gly Arg Ser Arg Ser Arg Arg Ala Ala Thr Ser
Arg Pro Glu Arg 115 120 125Val Trp Pro Asp Gly Val Ile Pro Phe Val
Ile Gly Gly Asn Phe Thr 130 135 140Gly Ser Gln Arg Ala Val Phe Arg
Gln Ala Met Arg His Trp Glu Lys145 150 155 160His Thr Cys Val Thr
Phe Leu Glu Arg Thr Asp Glu Asp Ser Tyr Ile 165 170 175Val Phe Thr
Tyr Arg Pro Cys Gly Cys Cys Ser Tyr Val Gly Arg Arg 180 185 190Gly
Gly Gly Pro Gln Ala Ile Ser Ile Gly Lys Asn Cys Asp Lys Phe 195 200
205Gly Ile Val Val His Glu Leu Gly His Val Val Gly Phe Trp His Glu
210 215 220His Thr Arg Pro Asp Arg Asp Arg His Val Ser Ile Val Arg
Glu Asn225 230 235 240Ile Gln Pro Gly Gln Glu Tyr Asn Phe Leu Lys
Met Glu Pro Gln Glu 245 250 255Val Glu Ser Leu Gly Glu Thr Tyr Asp
Phe Asp Ser Ile Met His Tyr 260 265 270Ala Arg Asn Thr Phe Ser Arg
Gly Ile Phe Leu Asp Thr Ile Val Pro 275 280 285Lys Tyr Glu Val Asn
Gly Val Lys Pro Pro Ile Gly Gln Arg Thr Arg 290 295 300Leu Ser Lys
Gly Asp Ile Ala Gln Ala Arg Lys Leu Tyr Lys Cys Pro305 310 315
320Ala Cys Gly Glu Thr Leu Gln Asp Ser Thr Gly Asn Phe Ser Ser Pro
325 330 335Glu Tyr Pro Asn Gly Tyr Ser Ala His Met His Cys Val Trp
Arg Ile 340 345 350Ser Val Thr Pro Gly Glu Lys Ile Ile Leu Asn Phe
Thr Ser Leu Asp 355 360 365Leu Tyr Arg Ser Arg Leu Cys Trp Tyr Asp
Tyr Val Glu Val Arg Asp 370 375 380Gly Phe Trp Arg Lys Ala Pro Leu
Arg Gly Arg Phe Cys Gly Ser Lys385 390 395 400Leu Pro Glu Pro Ile
Val Ser Thr Asp Ser Arg Leu Trp Val Glu Phe 405 410 415Arg Ser Ser
Ser Asn Trp Val Gly Lys Gly Phe Phe Ala Val Tyr Glu 420 425 430Ala
Ile Cys Gly Gly Asp Val Lys Lys Asp Tyr Gly His Ile Gln Ser 435 440
445Pro Asn Tyr Pro Asp Asp Tyr Arg Pro Ser Lys Val Cys Ile Trp Arg
450 455 460Ile Gln Val Ser Glu Gly Phe His Val Gly Leu Thr Phe Gln
Ser Phe465 470 475 480Glu Ile Glu Arg His Asp Ser Cys Ala Tyr Asp
Tyr Leu Glu Val Arg 485 490 495Asp Gly His Ser Glu Ser Ser Thr Leu
Ile Gly Arg Tyr Cys Gly Tyr 500 505 510Glu Lys Pro Asp Asp Ile Lys
Ser Thr Ser Ser Arg Leu Trp Leu Lys 515 520 525Phe Val Ser Asp Gly
Ser Ile Asn Lys Ala Gly Phe Ala Val Asn Phe 530 535 540Phe Lys Glu
Val Asp Glu Cys Ser Arg Pro Asn Arg Gly Gly Cys Glu545 550 555
560Gln Arg Cys Leu Asn Thr Leu Gly Ser Tyr Lys Cys Ser Cys Asp Pro
565 570 575Gly Tyr Glu Leu Ala Pro Asp Lys Arg Arg Cys Glu Ala Ala
Cys Gly 580 585 590Gly Phe Leu Thr Lys Leu Asn Gly Ser Ile Thr Ser
Pro Gly Trp Pro 595 600 605Lys Glu Tyr Pro Pro Asn Lys Asn Cys Ile
Trp Gln Leu Val
Ala Pro 610 615 620Thr Gln Tyr Arg Ile Ser Leu Gln Phe Asp Phe Phe
Glu Thr Glu Gly625 630 635 640Asn Asp Val Cys Lys Tyr Asp Phe Val
Glu Val Arg Ser Gly Leu Thr 645 650 655Ala Asp Ser Lys Leu His Gly
Lys Phe Cys Gly Ser Glu Lys Pro Glu 660 665 670Val Ile Thr Ser Gln
Tyr Asn Asn Met Arg Val Glu Phe Lys Ser Asp 675 680 685Asn Thr Val
Ser Lys Lys Gly Phe Lys Ala His Phe Phe Ser Val Leu 690 695 700Glu
Gly Ala Gly Asp Arg His Ser His Leu Ser Gly Leu Glu Leu Leu705 710
715 720Leu Cys Pro His Ala Leu Val Asp Thr Val Pro Ala Pro Pro Ser
Ala 725 730 735Leu His Gly Asp Thr His Ala His Thr His Thr His Val
His Thr His 740 745 750Cys Pro Ile Ala Gln Glu Thr Cys Arg Gly Pro
Pro Leu Gly Ala Ser 755 760 765Arg Leu Ser Pro Gln Gly Pro Gly His
Leu Thr Leu Ala Pro Gln Glu 770 775 780Gly Ser Tyr Leu Asp Phe Trp
Asp Thr His Arg Gly Asp Pro Lys Pro785 790 795 800Arg Arg Arg Arg
Lys Ser Leu Lys Thr Phe Ser Leu Thr Pro Ala Thr 805 810 815Phe Arg
Gly Ile Trp Ala Leu 820
* * * * *